Abstract Book - ISHAM

ISHAM President: 

David W. Warnock, PhD 

Centers for Disease Control 

and Prevention 

Atlanta, Georgia, United States 

Congress Chair: 

Bertrand F. Dupont, MD 

Hôpital Necker 

Paris, France 

The 16 th 

Congress of the International Society 

for Human and Animal Mycology 

Le Palais des Congrès de Paris • Paris, France • 25-29 June 2006 

Abstract Book

The 16 th 

Congress of the International Society 

for Human and Animal Mycology 

Le Palais des Congrès de Paris • Paris, France • 25-29 June 2006 

ABSTRACT TABLE OF CONTENTS 

Grouped by scientific topic 

Oral Presentations 

Animal Mycology . .O-0001 – O-0007 

Antifungals . . . . . . .O-0008 – O-0016 

Clinical Mycology . .O-0017 – O-0021 

Diagnostic Tools . . .O-0022 – O-0025 

Epidemiology . . . . .O-0026 – O-0030 

Genomics . . . . . . . .O-0031 – O-0035 

Immunology . . . . . .O-0036 – O-0038 

Molecular Biology . .O-0039 – O-0045 

Physiopathology . . . . . . . . . . .O-0046 

Poster Presentations 

Animal Mycology . . . .P-0001 – P-0043 

Antifungals . . . . . . . .P-0044 – P-0165 

Clinical Mycology . . . .P-0166 – P-0350 

Diagnostic Tools . . . . .P-0351 – P-0438 

Epidemiology . . . . . . .P-0439 – P-0573 

Genomics . . . . . . . . .P-0574 – P-0584 

Immunology . . . . . . .P-0585 – P-0626 

Molecular Biology . . .P-0627 – P-0709 

Other . . . . . . . . . . . .P-0710 – P-0738 

Taxonomy . . . . . . . . . .P-0739 – P0756 

©2006 Imedex ® , Inc. All rights reserved. Reproduction 

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Abad A . . . . . . . . . .O-0017, P-0585, 

. . . . . . . . . . . . . . . . .P-0615, P-0685 

Abarca C . . . . . . . . . . . . . . . .P-0556 

Abbes S . . . . . . . . . . . . . . . . .P-0550 

Abboud P . . . . . . . . . . . . . . . .P-0230 

Abd El Kader A . . . . . . . . . . . .P-0499 

Abdelaziz H . . . . . . . . . . . . . . .P-0281 

Abdul Samads . . . . . . . . . . . . .P-0352 

Abe S . . . . . . . . . . . .O-029, P-0055, 

. . . . . . . . . . . . . . . . .P-0085, P-0707 

Abel P . . . . . . . . . . . . . . . . . . .P-0227 

Abia-Bassey L . . . . . . . . . . . . .P-0441 

Abli P . . . . . . . . . . . . . . . . . . .P-0483 

Abramson M . . . . . . . . . . . . . .P-0507 

Adachi Y . . . . . . . . . .P-0606, P-0612 

Adelantado C . . . . . . .P-0059, P-0202 

Adema G . . . . . . . . . . . . . . . .P-0597 

Adiri Y . . . . . . . . . . . . . . . . . .P-0507 

Adler-Moore J . . . . . . . . . . . .O-0013 

Afeltra J . . . . . . . . . . . . . . . . .P-0157 

Afioni F . . . . . . . . . . . . . . . . . .P-0213 

Afonso A . . . . . . . . . . . . . . . . .P-0346 

Afshar P . . . . . . . . . . . . . . . . .P-0493 

Afshari N . . . . . . . . . . . . . . . .P-0120 

Agabian N . . . . . . . . . . . . . .O-0033 

Aghamirian M . . . . . .P-0001, P-0442 

Aghapour R . . . . . . . . . . . . . . .P-0452 

Aghili R . . . . . . . . . . . . . . . . . .P-0493 

Agnetti F . . . .P-0007, P-0455, P-0521 

Agorio I . . . . . . . . . . . . . . . . .P-0382 

Aguiar L . . . . . . . . . . . . . . . . .P-0031 

Aguilar A . . . . . . . . . . . . . . . .P-0211 

Agyeman K . . . . . . . . . . . . . . .P-0533 

Ahmad S . . . . . . . . . .P-0249, P-0627 

Ahmed A . . . . . . . . . .P-0152, P-0167 

Ahn K . . . . . . . . . . . . . . . . . . .P-0502 

Aho S . . . . . . . . . . . . . . . . . . .P-0557 

Akcaglar S . . . . . . . . .P-0070, P-0071 

. . . . . . . . . . .P-0072, P-0168, P-0443 

Akeel R . . . . . . . . . . . . . . . . . .P-0169 

Akkurt L . . . . . . . . . . . . . . . . .P-0056 

Akoru C . . . . . . . . . . . . . . . . .P-0353 

Aktas E . . . . . . . . . . . .P-0056, P-0664 

Al Sharif A . . . . . . . . . . . . . . . .P-0499 

Al-Mohri H . . . . . . . . . . . . . . .P-0293 

Al-Sweih N . . . . . . . . . . . . . . .P-0249 

Alain T . . . . . . . . . . . . . . . . . .P-0171 

Albaina O . . . . . . . . . . . . . . . .P-0069 

Albarrag A . . . . . . . . . . . . . . .P-0044 

Albert N . . . . . . . . . . . . . . . . .P-0060 

Alcazar-Fuoli L . . . . . . . . . . . . .P-0628 

Alcoba-Flórez J . . . . . . . . . . . .P-0354 

Aldins P . . . . . . . . . . . . . . . . . .P-0444 

Alejandro S . . . . . . . .P-0254, P-0255 

Alexander C . . . . . . . .P-0045, P-0445 

Alexopoulos E . . . . . . .P-0049, P-0484 

Alhambra A . . . . . . . .P-0280, P-0355 

. . . . . . . . . . . . . . . . . . . . . . .P-0386 

Ali S . . . . . . . . . . . . . . . . . . . .P-0041 

Aliouat C . . . . . . . . . . . . . . . .P-0745 

Alizadeh H . . . . . . . . . . . . . . .P-0218 

Alkorta M . . . . . . . . . .P-0426, P-0536 

Aller A . . . . . . . . . . . .P-0096, P-0103, 

. . . . . . . . . . . . . . . . .P-0104, P-0368 

Allison M . . . . . . . . . . . . . . . . .P-0321 

Allombert C . . . . . . . . . . . . . .P-0301 

Almamary A . . . . . . . . . . . . . .P-0223 

Almeida S . . . . . . . . . . . . . . . .P-0586 

Alonso M . . . . . . . . . .P-0486, P-0653 

Alonso-Monge R . . . . . . . . . .O-0039 

Altas K . . . . . . . . . . . .P-0244, P-0245 

Altclas J . . . . . . . . . . . . . . . . .P-0382 

Altman S . . . . . . . . . . . . . . . . .P-0448 

Alvarado P . . . . . . . . . . . . . . .P-0516 

Alvarado Ramírez E . .P-0046, P-0342, 

. . . . . . . . . . . . . . . . .P-0385, P-0629 

Alvarez M . . . . . . . . . .P-0074, P-0410 

Alvarez S . . . . . . . . . .P-0078, P-0714 

Alvaro L . . . . . . . . . . . . . . . . .P-0683 

Alves C . . . . . . . . . . . . . . . . . .P-0034 

Alves S . . . . . . . . . . . . . . . . . .P-0406 

Alviano C . . . . . . . . .P-0253, P-0296, 

. . . . . . . . . . . . . . . . .P-0595, P-0625 

Alviano D . . . . . . . . . . . . . . . .P-0625 

Alviano D . . . . . . . . . . . . . . . .P-0595 

Aly R . . . . . . . . . . . . . . . . . . . .P-0343 

Amagai M . . . . . . . . . . . . . . . .P-0317 

Amel B . . . . . . . . . . . . . . . . . .P-0310 

Amendola L . . . . . . . . . . . . . .P-0259 

Amorim A . . . . . . . . . . . . . . . .P-0284 

Amorim C . . . . . . . . .P-0446, P-0515 

Amorim J . . . .P-0095, P-0221, P-0264 

Amrein C . . . . . . . . . . . . . . . .O-0008 

Anane S . . . .P-0170, P-0243, P-0246 

Ancelle T . . . . . . . . . . . . . . . . .P-0417 

Andaluz López E . . . . . . . . . . .P-0654 

Anderson F . . . . . . . . . . . . . . .P-0595 

Anderson M . . . . . . . . . . . . . .P-0044 

Anderson S . . . . . . . . . . . . . . .P-0474 

Andreopoulos A . . . . . . . . . . .P-0226 

Andreotti P . . . . . . . . .P-0216, P-0279 

Angelopoulou M . . . . . . . . . . .P-0226 

Angoulvant A . . . . . . .P-0356, P-0510 

Ann T . . . . . . . . . . . . . . . . . . .P-0254 

Anna C . . . . . . . . . . . . . . . . . .P-0254 

4 Le Palais des Congrès de Paris • Paris, France • 25-29 June 2006

Anne B . . . . . . . . . . . . . . . . . .P-0471 

Anne G . . . . . . . . . . . . . . . . . .P-0171 

Anne R . . . . . . . . . . . . . . . . . .P-0073 

Anne S . . . . . . . . . . . . . . . . . .P-0073 

Annemarie B . . . . . . . . . . . . . .P-0254 

Anonye N . . . . . . . . . . . . . . . .P-0286 

Antinori S . . . . . . . . . . . . . . . .P-0172 

Antoine B . . . . . . . . . . . . . . .O-0023 

Antoine H . . . . . . . . . . . . . . . .P-0369 

Antonopoulou S . . . . . . . . . . . .P-0047 

Anyfantis I . . . . . . . . . . . . . . . .P-0048 

Anzawa K . . .P-0173, P-0174, P-0447, 

. . . . . . . . . . .P-0519, P-0570, P-0752 

Aoki S . . . . . . . . . . . .P-0658, P-0666, 

. . . . . . . . . . . . . . . . .P-0673, P-0704 

Apaire-Marchais V . . .P-0357, P-0574 

Apitz-Castro R P-0092, P-0102, P-0127 

Arabatzis M . . . . . . . .P-0049, P-0358 

Arai M . . . . . . . . . . . . . . . . . .P-0116 

Araiza J . . . . . . . . . . . . . . . . .P-0195 

Arana D . . . . . . . . . . . . . . . .O-0039 

Arancia S . . . . . . . . . .P-0359, P-0596 

Arantes R . . . . . . . . . . . . . . . .P-0644 

Arathoon E . . . . . . . . . . . . . . .P-0422 

Araújo Resende L . . . .P-0481, P-0482 

Arcelli R . . . . . . . . . . . . . . . . .P-0521 

Arduino M . . . . . . . . . . . . . . . .P-0462 

Arechaval A . . . . . . . .P-0069, P-0341 

Arenas R . . . . . . . . . .P-0479, P-0496 

Arendrup M . . . . . . . . . . . . . . .P-0132 

Arias M . . . . . . . . . . . . . . . . . .P-0192 

Arlington-Skaggs B . . . . . . . . .P-0559 

Arné P . . . . . . . . . . . . . . . . . . .P-0020 

Aroch A . . . . . . . . . . . . . . . . .P-0469 

Arosemena L . . . . . . .P-0059, P-0202 

Arreguín R . . . . . . . . . . . . . . .O-0012 

Arruda-Neto J . . . . . . . . . . . . .P-0661 

Arsenis G . . . . . . . . . . . . . . . .P-0049 

Arthington-Skaggs B . . . . . . . .P-0422 

Arthur I . . . . . . . . . . . . . . . . . .P-0448 

Arévalo-Morales M . . . . . . . . .P-0354 

Asahi Y . . . . . . . . . . . . . . . . . .P-0611 

Asci Toraman Z . . . . . . . . . . . .P-0333 

Asgari G . . . . . . . . . . . . . . . . .P-0032 

Aspiroz C . . . . . . . . . . . . . . . .P-0304 

Asri Rezai S . . . . . . . . . . . . . .P-0043 

Ates A . . . . . . . . . . . . . . . . . .P-0495 

Aubert A . . . . . . . . . . . . . . . .P-0237 

Auger A . . . . . . . . . . . . . . . . .P-0383 

Auger I . . . . . . . . . . . . . . . . .P-0360 

Auler M . . . . . . . . . . .P-0292, P-0568 

Aurore K . . . . . . . . . . . . . . . .P-0175 

Aurélien F . . . . . . . . . . . . . . .P-0175 

Ausma J . . . .P-0176, P-0219, P-0576 

Aveskamp M . . . . . . . . . . . . .P-0743 

T Avšic- upanc T . . . . . . . . . .P-0351 

Ayadi A . . . .P-0208, P-0326, P-0523, 

. . . . . . . . . . .P-0550, P-0551, P-0604 

Aydil U . . . . . . . . . . . . . . . . . .P-0449 

Ayyildiz A . . . . . . . . . . . . . . . .P-0664 

Azaiz M . . . . . . . . . . . . . . . . . .P-0494 

Aze vedo M . . . . . . . . . . . . . . .P-0021 

Azevedo M . .P-0037, P-0038, P-0626 

Aznar C . . . . . . . . . . . . . . . . .P-0193 

Aznar J . . . . . . . . . . . . . . . . . .P-0368 

Aznar J . . . . . . . . . . . . . . . . . .P-0426 

Baccarin R . . . . . . . . . . . . . . . .P-0033 

Bachewich C . . . . . . . . . . . . .O-0016 

Bachhawat A . . . . . . . . . . . . . .P-0473 

Badali H . . . . . . . . . . . . . . . . .P-0450 

Bader O . . . . . . . . . . . . . . . . .P-0671 

Badre Eddine L . . . . . .P-0177, P-0178 

Badri T . . . . . . . . . . . . . . . . . .P-0179 

Baeza L . . . . . . . . . . .P-0180, P-0222 

Bagg J . . . . . . . . . . . . . . . . . .O-0018 

Bahadori F . . . . . . . . . . . . . . .P-0213 

Bahloul M . . . . . . . . . . . . . . . .P-0326 

Bahrami Samani H . . . . . . . . .P-0181 

bailly e . . . . . . . . . . . . . . . . . .P-0182 

Bailly E . . . . .P-0183, P-0184, P-0205 

Bailão A . . . . . . . . . . . . . . . . .P-0180 

Baixench M . . . . . . . . .P-0185, P-0186 

Baldo A . . . . . . . . . .O-0001, O-0005 

Ball L . . . . . . . . . . . . . . . . . . .P-0050 

Bamberg W . . . . . . . . . . . . . . .P-0463 

Banerjee D . . . . . . . . . . . . . .O-0031 

Banerjee U . . . . . . . . .P-0267, P-0314 

Banfi E . . . . . . . . . . . . . . . . . .P-0051 

Baptista R . . . . . . . . . . . . . . . .P-0361 

Baquero F . . . . . . . . .P-0074, P-0410 

Baran E . . . . . . . . . . .P-0084, P-0187 

Baran W . . . . . . . . . . . . . . . . .P-0187 

Barbey S . . . . . . . . . . . . . . . .O-0009 

Barboni A . . . . . . . . . . . . . . . .P-0015 

Barbosa - Sabanero G . . . . . . .P-0312 

Barbosa dos Santos C . . . . . .P-0481, 

. . . . . . . . . . . . . . . . .P-0482, P-0520 

Barbosa, Jr A . . . . . . .P-0451, P-0738 

Barchiesi F . . . . . . . . . . . . . . .P-0144 

Barker K . . . . . . . . . . .P-0579, P-0587 

Barnes P . . . . . . . . . . . . . . . . .P-0588 

Barrabes A . . . . . . . . . . . . . . .P-0183 

Barreira P . . . . . . . . . . . . . . .O-0044 

Barrenetxea G . . . . . .O-0017, P-0585 

The 16 th Congress of the International Society for Human and Animal Mycology 

5

Barreto L . . . . . . . . . . . . . . . . .P-0677 

Barreto-Bergter E . . . . . . . . . . .P-0613 

Barros G . . . . . . . . . . . . . . . . .P-0201 

Barros R . . . . . . . . . . . . . . . . .P-0505 

Barroso C . . . . . . . . . . . . . . . .P-0311 

Barton R . . . . . . . . . . . . . . . .O-0014 

Bastides F . . . . . . . . . . . . . . . .P-0184 

Batisse A . . . . . . . . . . . . . . . .O-0008 

Batista W . . . . . . . . . . . . . . . .P-0630 

Batura-Gabryel H . . . .P-0188, P-0189 

Bautista-Muñoz C . . . . . . . . . .P-0705 

Benjamin L . . . . . . . . . . . . . . .P-0422 

Benoit S . . . . . . . . . . . . . . . . .P-0463 

Bentubo H . . . . . . . . . . . . . . . .P-0014 

Benvenuto F . . . . . . . . . . . . . .P-0287 

Benvenuto Ferreira F . . . . . . . .P-0364 

Berdicevsky I . . . . . . . .P-0143, P-0631 

Berge M . . . . . . . . . . . . . . . .O-0008 

Bergmans A . . . . . . . . . . . . . .P-0363 

Bergès T . . . . . . . . . . . . . . . . .P-0154 

Berjeaud J . . . . . . . . . . . . . . . .P-0459 

Berk E . . . . . . . . . . . . . . . . . . .P-0449 

Billaud E . . . . . . . . . . . . . . . .O-0008 

Birinci A . . . . . . . . . . . . . . . . .P-0056 

Bisaro F . . . . . . . . . . . . . . . . .P-0458 

Bistoni F . . . . . . . . . . . . . . . . .P-0716 

Blancard A . . . . . . . . . . . . . . .P-0301 

Blanchet D . . . . . . . . . . . . . . .P-0193 

Blanco J . . . .P-0078, P-0365, P-0714 

Blanco Y . . . . . . . . . . . . . . . . .P-0472 

Blijlevens N . . . . . . . . . . . . . . .P-0161 

Blonde R . . . . . . . . . . . . . . . . .P-0194 

Bocanegra R . . . . . . . .P-0028, P-0110 

Bayram Ö . . . . . . . . . . . . . . . .P-0694 Berman J . . . . . . . . . . . . . . . .O-0003 Boekhout T . . . . . . .O-0027, P-0196, 

Becerril-Luján B . . . . . . . . . . . .P-0692 Bermúdez R . . . . . . . . . . . . . . .P-0470 . . . . . . . . . . .P-0244, P-0304, P-0367 

Beck S . . . . . . . . . . . . . . . . . . .P-0052 Bernadette L . . . . . . . .P-0512, P-0513 Boel A . . . . . . . . . . . . . . . . . . .P-0454 

Becker K . . . . . . . . . . .P-0134, P-0228 Bernard G . . . . . . . . . . . . . . .O-0037 Bogdanowicz E . . . . . . . . . . . .P-0209 

Becker W . . . . . . . . . . . . . . . .P-0739 Bernard S . . . . . . . . . . . . . . . .P-0205 Bogomolova T . . . . . .P-0155, P-0215, 

Begum Y . . . . . . . . . . . . . . . . .P-0362 Bernardes-Engemann A . . . . .P-0191, 

. . . . . . . . . . . . . . . . . . . . . . .P-0566 

Behzadi E . . . . . . . . . . . . . . . .P-0452 . . . . . . . . . . . . . . . . .P-0287, P-0364 Bogusz B . . . . . . . . . .P-0150, P-0461 

Behzadi P . . . . . . . . . . . . . . . .P-0452 Bernardino S . . . . . . . . . . . . . .P-0589 Bohse M . . . . . . . . . . . . . . . . .P-0633 

Belhadj S . . . .P-0170, P-0243, P-0246 Bernhardt H . . . . . . . . . . . . . .P-0053 Boiron P . . . . . . . . . . . . . . . . .P-0346 

Belleguic C . . . . . . . . . . . . . . .P-0210 Bernhardt J . . . . . . . . . . . . . . .P-0053 Bollo E . . . . . . . . . . . .P-0007, P-0635 

Bello N . . . . . . . . . . . . . . . . . .P-0209 Berriman M . . . . . . . . . . . . . .O-0035 Bolognini J . . . . . . . . . . . . . . .P-0356 

Bellucci C . . . . . . . . . . . . . . . .P-0591 Berry A . . . . . . . . . . . . . . . . . .P-0489 Bolognini J . . . . . . . . . . . . . . .P-0109 

Ben Hadj El Bechir A . . . . . . . .P-0453 Berrêdo Pinho M . . . . . . . . . . .P-0191 Bonaccorso C . . . . . . . . . . . . .P-0172 

Ben Osman Dhahri A P-0179, P-0217, Berthelemy M . . . . . . . . . . . .O-0002, Boncio L . . . . . . . . . .P-0007, P-0025, 

. . . .P-0234, P-0250, P-0453, P-0494 . . . . . . . . . . . . . . . . .P-0675, P-0745 . . . . . . . . . . . . . . . . .P-0455, P-0521 

Ben Tekaya N . . . . . .P-0179, P-0217, Bertout S . . . . . . . . . . . . . . . . .P-0054 Bonfim Carregaro F . . . . . . . . .P-0002 

. . . . . . . . . . . . . . . . .P-0234, P-0250 Betran A . . . . . . . . . . . . . . . . .P-0192 Bonifaz A . . . . . . . . . . . . . . . .P-0195 

Benada O . . . . . . . . . . . . . . . .P-0437 Beucher B . . .P-0407, P-0713, P-0729 Bonmarchand G . . . . . . . . . . .P-0230 

Benard G . . . . . . . . . .P-0216, P-0279 Bex V . . . . . . . . . . . . . . . . . . .P-0675 Bonnet E . . . . . . . . . . . . . . . . .P-0093 

Benboubker l . . . . . . . . . . . . . .P-0182 Bhardwaj S . . . . . . . . .P-0473, P-0632 Bonnin A . . . .P-0475, P-0557, P-0717 

Benderdouche M . . . . . . . . . . .P-0421 Bien C . . . . . . . . . . . . . . . . . .P-0637 Borbély Á . . . . . . . . . . . . . . . .P-0428 

Benetucci A . . . . . . . . . . . . . . .P-0190 Bignell E . . . . . . . . . . . . . . . .O-0042 Borgers M . . .P-0176, P-0219, P-0576 

Benites N . . . . . . . . . . . . . . . .P-0033 Bii C . . . . . . . . . . . . . . . . . . . .P-0055 Borges C . . . . . . . . . . . . . . . . .P-0180 

Benito-Ruesca R . . . . . . . . . . . .P-0309 Bilgin K . . . . . . . . . . . . . . . . . .P-0056 Borges R . . . . . . . . . . . . . . . . .P-0468 


Bori A . . . . . . . . . . . . . . . . . . .P-0076 

Borman A . . .P-0456, P-0741, P-0743 

Borrell N . . . . . . . . . . . . . . . . .P-0426 

Bosch Alepuz M . . . . .P-0457, P-0465 

Bosshard P . . . . . . . . .P-0373, P-0374 

Botterel F . . . . . . . . . . . . . . . .P-0220 

Bouaziz M . . . . . . . . .P-0326, P-0550 

Brena S . . . . . . . . . . . . . . . . . .P-0401 

Brentrup A . . . . . . . . . . . . . . . .P-0229 

Bretagne S . . . . . . . . .P-0020, P-0220 

Briand J . . . . . . . . . . . . . . . . .O-0009 

Bricaire F . . . . . . . . . . . . . . . .P-0194 

Bridge P . . . . . . . . . . . . . . . . .P-0741 

Brightwell K . . . . . . . . . . . . . . .P-0432 

Busold C . . . . . . . . . . . . . . . . .P-0578 

Bustamante B .P-0113, P-0199, P-0464 

Buzanello Martins C . .P-0064, P-0065 

Buziba N . . . . . . . . . . . . . . . . .P-0353 

Buzina W . . . . . . . . . . . . . . . .P-0200 

Bykowska B . . . . . . . . . . . . . . .P-0573 

Béatrice S . . . . . . . . . . . . . . . .P-0073 

Bouchara J . . . . . . . .P-0153, P-0154, 

. . . . . . . . . . . . . . . . .P-0370, P-0649 

Boucharan J . . . . . . . . . . . . .O-0044 

Boudava S . . . . . . . . . . . . . . . .P-0551 

Boudreaux J . . . . . . . . . . . . . .P-0376 

Bougeret C . . . . . . . . . . . . . .O-0009 

Bougerol C . . . . . . . . . . . . . . .P-0020 

Bougnoux M . . . . . . . . . . . . . .P-0230 

Bourdeau P . . . . . . . . . . . . . . .P-0004 

Bourdeau P . . . . . . . .P-0003, P-0366 

Bourée P . . . . . . . . . . . . . . . . .P-0458 

Bouscary D . . . . . . . . . . . . . . .P-0186 

Boussaud V . . . . . . . . . . . . . .O-0008 

Bouyer S . . . . . . . . . . . . . . . . .P-0459 

Bovers M . . . . . . . . . .P-0196, P-0367 

Bovers M . . . . . . . . . . . . . . . .O-0027 

Boyce S . . . . . . . . . . . . . . . . . .P-0330 

Boyer J . . . . . . . . . . . . . . . . . .P-0109 

Bozonnet E . . . . . . . . . . . . . . .P-0237 

Bozza S . . . . . . . . . . . . . . . . . .P-0590 

Brahim A . . . . . . . . . . . . . . . . .P-0177 

Brajer B . . . . . . . . . . .P-0188, P-0189 

Branche J . . . . . . . . . . . . . . . .P-0734 

Brandt M . . .P-0422, P-0462, P-0463, 

. . . . . . . . . . . . . . . . . . . . . . .P-0546 

Brandão J . . .P-0034, P-0345, P-0460 

Brasch J . . . . . . . . . . .P-0715, P-0740 

Braus G . . . . . . . . . .O-0042, P-0694 

Brigitte W . . . . . . . . . . . . . . . .P-0073 

Briones M . . . . . . . . . .P-0446, P-0515 

Brito A . . . . . .P-0197, P-0212, P-0270 

Brito E . . . . . . . . . . . . . . . . . . .P-0064 

Bromuro C . . . . . . . . .P-0591, P-0594 

Brouwer C . . . . . . . . .P-0087, P-0161 

Brown D . . . . . . . . . . . . . . . . .P-0083 

Brown L . . . . . . . . . . . . . . . . .P-0445 

Brown T . . . . .P-0136, P-0320, P-0321 

Bruet V . . . . . . . . . . . .P-0003, P-0004 

Bruijnesteijn van Coppenraet L .P-0358 

Brusca M . . . . . . . . . . . . . . . . .P-0075 

Bruzual E . . . . . . . . . .P-0197, P-0212 

Brzozowski T . . . . . . . . . . . . .O-0021 

Bubnick M . . . . . . . . . . . . . . .O-0040 

Buchheidt D . . . . . . . . . . . . . .P-0391 

Buchta V . . . . . . . . . . . . . . . . .P-0232 

Budak A . . . .O-0021, P-0150, P-0461 

Buendia B . . . . . . . . . . . . . . . .P-0426 

Bueno J . . . . . . . . . . .P-0106, P-0107 

Bueno Sánchez J . . . . .P-0057, P-0058 

Bufler J . . . . . . . . . . . . . . . . . .P-0053 

Bujdakova H . . . . . . . .P-0105, P-0592 

Bulut Y . . . . . . . . . . . . . . . . . .P-0333 

Buot G . . . . . . . . . . .O-0010, P-0198 

Burger E . . . . . . . . . . .P-0133, P-0610 

Burwell L . . . .P-0462, P-0463, P-0474 

Bejar V . . . . . . . . . . . . . . . . . .P-0607 

Böttger E . . . . . . . . . .P-0373, P-0374 

C Sartorelli A . . . . . . . . . . . . . .P-0305 

C. da Silva B . . . . . . . . . . . . . .P-0439 

C. Franco M . . . . . . . . . . . . . .P-0439 

Cabagna M . . . . . . . . . . . . . .P-0308 

Caballero J . . . . . . . . . . . . . . .P-0365 

Cabezón V . . . . . . . . . . . . . .O-0041 

Cabrita J . . . . . . . . . . . . . . . . .P-0505 

Cacere C . . . . . . . . . . . . . . . .O-0037 

Cafarchia C . .P-0005, P-0006, P-0721 

Caillot D . . . . . . . . . .P-0475, P-0557 

Calabuig Munoz E . . .P-0457, P-0465 

Calderone R . . . . . . . .P-0654, P-0669 

Calich V . . . . . . . . . . . . . . . . .P-0589 

Caliendo A . . . . . . . . . . . . . . .P-0506 

Caligiorne R . . . . . . . .P-0201, P-0742 

Calinon C . . . . . . . . . . . . . . . .P-0557 

Calvo M . . . . . . . . . . .P-0059, P-0202 

Calvo R . . . . . . . . . . . . . . . . . .P-0202 

Camadro J . . . . . . . . . . . . . . .P-0693 

Camargo Z . . . . . . . .P-0031, P-0607 

Cammue B . . . . . . . . . . . . . . .P-0576 

Campbell C . . . . . . . .P-0456, P-0741 

Campolina S . . . . . . .P-0201, P-0742 

Campos C . . . . . . . . . . . . . . .P-0634 

Camus C . . . . . . . . . . . . . . . .P-0210 


7

Camus D . . . . . . . . . .P-0328, P-0665 

Candolfi E . . . . . . . . . . . . . . . .P-0421 

Canestri A . . . . . . . . . . . . . . . .P-0194 

Cannizzo F . . . . . . . .P-0007, P-0025, 

. . . . . . . . . . . . . . . . .P-0635, P-0704 

Cannon R . . . . . . . . . .P-0114, P-0115 

Cano J . . . . .P-0749, P-0304, P-0753 

Cano L . . . . .P-0593, P-0593, P-0621 

Canton E . . . . . . . . . .P-0103, P-0379 

Canton Lacasa E . . . . .P-0457, P-0465 

Carattoli A . . . . . . . . . . . . . . .P-0359 

Carbajosa J . . . . . . . . . . . . . .P-0736 

Cardoso J . . . . . . . . . . . . . . . .P-0346 

Cariani L . . . . . . . . . . . . . . . . .P-0697 

Carissimi M . . . . . . . .P-0696, P-0732 

Carmona A . . . . . . . . . . . . . . .P-0719 

Carmona O . . . . . . . . . . . . . .P-0212 

Carnovale S . . . . . . . . . . . . . .P-0636 

Caro-Gomez E . . . . . . . . . . . .P-0593 

Caron F . . . . . . . . . . . . . . . . .P-0230 

Carrillo-Muñoz A . . . . . . . . . .P, 0069, 

. . . . . . . . . . . . . . . . .P-0315, P-0316 

Carter D . . . . . . . . . . . . . . . . .P-0400 

Cartuyvels R . . . . . . . . . . . . . .P-0454 

Caruso P . . . . . . . . . . . . . . . . .P-0080 

Carvalho A . . . . . . . . .P-0203, P-0466 

Carver P . . . . . . . . . . . . . . . . .P-0467 

Casadevall A . . . . . . .P-0641, P-0716 

Casanova K . . . . . . . . . . . . . .P-0468 

Casanova M . . . . . . . . . . . . . .P-0684 

Casquero J . . . . . . . . . . . . . . .P-0113 

Cassaing S . . . . . . . . .P-0093, P-0489 

Cassone A . . . . . . . .O-0045, P-0359, 

. . . . . . . . . . .P-0591, P-0594, P-0596 

Cassoux N . . . . . . . . . . . . . . .P-0194 

Castañón L .O-0012, P-0469, P-0470 

Castel D . . . . . . . . . . . . . . . . .P-0054 

Castro C . . . . . . . . . .P-0103, P-0104, 

. . . . . . . . . . .P-0113, P-0368, P-0464 

Castro M . . . . . . . . . . . . . . . . .P-0509 

Castro N . . . . . . . . . . . . . . . . .P-0476 

Catena M . . . . . . . . . . . . . . . .P-0039 

Catherine D . . . . . . . . . . . . . .P-0369 

Catherine K . . . . . . . . . . . . . . .P-0471 

Caumes E . . . . . . . . . . . . . . . .P-0194 

Cavalheiro A . . . . . . . . . . . . . .P-0037 

Cavallini Sanches E . . . . . . . .P-0002, 

. . . . . . . . . . . . . . . . .P-0008, P-0042 

Cavelheiro A . . . . . . . . . . . . . .P-0038 

Cazzaniga R . . . . . . . . . . . . . .P-0509 

Cekovska Z . . . . . . . . . . . . . . .P-0276 

Celum C . . . . . . . . . . . . . . . . .P-0464 

Cenci E . . . . . . . . . . . . . . . . . .P-0716 

Cerbón M . . . . . . . . . . . . . . . .P-0265 

Cermeño J . . . . . . . . .P-0204, P-0472 

Cermeño J . . . . . . . . .P-0204, P-0472 

Cernila B . . . . . . . . . . . . . . . . .P-0351 

Cervantes-Olivares R . . . . . . .P-0009, 

. . . . . . . . . . . . . . . . .P-0010, P-0040 

Cervantes-Olivares R . . . . . . . .P-0563 

Cesar Montelli A . . . . . . . . . . .P-0126 

Cesar Viani F . . . . . . . . . . . . .P-0082 

Ceylan A . . . . . . . . . . . . . . . . .P-0449 

Chaabane T . . . . . . . . . . . . . .P-0246 

Chaari H . . . . . . . . . . . . . . . . .P-0550 

Chabasse D . .P-0153, P-0154, P-0370 

Chabbou A . . . . . . . . . . . . . . .P-0243 

Chakarian J . . . . . . . . . . . . . .P-0230 

Chaker E . . . . . . . . . .P-0170, P-0179, 

. . . . . . . . . . . . . . . . .P-0243, P-0246 

Chakrabarti A P-0473, P-0496, P-0632 

Chakrabarti A . . . . . . . . . . . . .P-0632 

Challa S . . . . . . . . . . . . . . . . .P-0290 

Chamaillard M . . . . . . . . . . . .P-0734 

Chambost H . . . . . . . .P-0275, P-0301 

Chamilos G . . . . . . . . . . . . . .P-0060 

Chan C . . . . . . . . . . . . . . . . . .P-0285 

Chandenier J . . . . . . . . . . . . .P-0182 

Chandenier J . . . . . . .P-0183, P-0184, 

. . . . . . . . . . . . . . . . .P-0205, P-0356 

Chander J . . . . . . . . . . . . . . . .P-0206 

Chandy R . . . . . . . . . . . . . . . .P-0249 

Chang D . . . . . . . . . . . . . . . . .P-0474 

Chang Y . . . . . . . . . . . . . . . . .P-0637 

Chanock S . . . . . . . . . . . . . . .O-0032 

Chantrey J . . . . . . . . . . . . . . .P-0456 

Chapman S . . . . . . . . . . . . . . .P-0322 

Chapnick E . . . . . . . . .P-0080, P-0225 

Charlier C . . . . . . . . . . . . . . . .P-0207 

Chartier L . . . . . . . . . . . . . . . .P-0207 

chavali P . . . . . . . . . . . . . . . . .P-0290 

Chayakulkeeree M . . .P-0638, P-0674 

Chebore S . . . . . . . . . . . . . . . .P-0353 

Cheickrouhou F . . . . . . . . . . . .P-0604 

Cheikh-rouhou F . . . . . . . . . . .P-0326 

Cheikhrouhou F . . . . .P-0208, P-0523, 

. . . . . . . . . . . . . . . . .P-0550, P-0551 

Chelly H . . . . . . . . . . . . . . . . .P-0550 

Chen C . . . . . . . . . . . . . . . . . .P-0660 

Chen H . . . . . . . . . . . . . . . . . .P-0708 

Chen H . . . . . . . . . . . . . . . . . .P-0402 

Chen S . . . .O-0015, P-0068, P-0122, 

. . . . . . . . . . . . . . . . .P-0400, P-0558 

Chen W . . . . . . . . . . . . . . . . .P-0402 

Chen Z . . . . . . . . . . . . . . . . . .P-0538 


Cheng H . . . . . . . . . . . . . . . . .P-0571 

Cheng J . . . . . . . . . . . . . . . . .P-0324 

Cherif F . . . . . . . . . . . . . . . . .P-0179 

Cherif F . . . . . . . . . . . . . . . . .P-0453 

Cherif F . . . . .P-0217, P-0234, P-0250 

Cherif F . . . . . . . . . . . . . . . . .P-0494 

Chermette R . . . . . . . . . . . . . .P-0675 

Chevalier P . . . . . . . . . . . . . .O-0008 

Chevrier S . . . . . . . . .P-0210, P-0370 

Chewins J . . . . . . . . . . . . . . . .P-0655 

Chiani P . . . . . . . . . . .P-0591, P-0594 

Chibana H . . . . . . . . . . . . . .O-0035 

Chikamori M . . . . . . . . . . . . . .P-0639 

Chilina G . . . . . . . . . . . . . . . .P-0155 

Chindamporn A . . . . .P-0473, P-0498 

Cho H . . . . . . . . . . . . . . . . . . .P-0258 

Choe Y . . . . . . . . . . . . . . . . . .P-0502 

Choi E . . . . . . . . . . . . . . . . . .O-0032 

Choi J . . . . . . . . . . . .P-0519, P-0640 

Chou H . . . . . . . . . . . . . . . . . .P-0504 

Chowdhary A . . . . . . .P-0371, P-0539 

Chrisman C . . . . . . . . . . . . . .P-0641 

Christin M . . . . . . . . . . . . . . . .P-0209 

Christine I . . . . . . . . . . . . . . . .P-0718 

Christophe H . . . . . . . . . . . . . .P-0471 

Chryssanthou E . . . . . . . . . . . .P-0372 

Ciardo D . . . . . . . . . .P-0373, P-0374 

Claire L . . . . . . . . . . . . . . . . . .P-0471 

Claro R . . . . . . . . . . . . . . . . . .P-0096 

Clayton T . . . . . . . . . . . . . . . .P-0614 

Cleary J . . . . . . . . . . . . . . . . .P-0322 

Clemons K . . .P-0061, P-0564, P-0565 

Coccia E . . . . . . . . . . . . . . . . .P-0599 

Coco B . . . . . . . . . . .O-0018, P-0731 

Colella M . . .P-0375, P-0388, P-0514 

Coll-Daroca J . . . . . . . . . . . . .P-0342 

Collopy-Júnior Í . . . . . . . . . . . .P-0253 

Colom F . . . . . . . . . . . . . . . . .P-0096 

Colombel J . . . . . . . . .P-0429, P-0734 

Colombo A . .P-0446, P-0515, P-0644 

Connolly P . . . . . . . .O-0006, P-0431 

Connolly P . . . . . . . . . . . . . . .P-0293 

Contreras L . . . . . . . . . . . . . . .P-0733 

Cooke R . . . . . . . . . . . . . . . . .P-0330 

Cooper C . . . . . . . . . . . . . . . .P-0703 

Corbellini V .P-0138, P-0695, P-0696, 

. . . . . . . . . . . . . . . . .P-0711, P-0732 

Cordero M . . . . . . . . . . . . . . .P-0642 

Cordoliani Y . . . . . . . . . . . . . .P-0207 

Corneta E . . . . . . . . . . . . . . . .P-0643 

Cornillet A . . . . . . . . . . . . . . . .P-0210 

Corrales A . . . . . . . . . . . . . . .P-0058 

Correa B . . . . . . . . . . . . . . . . .P-0651 

Correa, S . . . . . . . . . . . . . . . .P-0014 

Correia-da-Silva F . . . . . . . . . .P-0253 

Corrêa Nunes F . . . . . . . . . . . .P-0651 

Corte A . . . . . . . . . . . . . . . . . .P-0031 

Cortez K . . . . . . . . . . . . . . . .O-0032 

Cortizo-Vidal S . . . . . .P-0404, P-0405 

Costa J . . . . . . . . . . . . . . . . . .P-0220 

Costa de Oliveira R . . . . . . . . .P-0577 

Costa-de-Oliveira S . . . . . . . . .P-0203 

Costantino P . . . . . . . . . . . . . .P-0591 

Costas E . . . . . . . . . . .P-0078, P-0714 

Coste A . . . .O-0003, P-0062, P-0691 

Cotteau A . . . . . . . . . . . . . . . .P-0328 

Cottin J . . . . . . . . . . .P-0357, P-0574 

Couble A . . . . . . . . . . . . . . . .P-0346 

Couillault G . . . . . . . . . . . . . .P-0557 

Countinho S . . . . . . . . . . . . . .P-0014 

Couppié P . . . . . . . . . . . . . . . .P-0193 

Coupé S . . . . . . . . . . . . . . . . .P-0421 

Cousson J . . . . . . . . . . . . . . . .P-0427 

Coutinho J . . . . . . . . . . . . . . .P-0346 

Coutinho T . . . . . . . . . . . . . . .P-0156 

Cova N . . . . . . . . . . . . . . . . . .P-0204 

Cowen L . . . . . . . . . . . . . . . . .P-0720 

Cox G . . . . . . . . . . . . . . . . . . .P-0622 

Cozzi J . . . . . . . . . . . . . . . . . .P-0382 

Craig J . . . . . . . . . . . . . . . . . .P-0535 

Cray C . . . . . . . . . . . . . . . . . .P-0011 

Crispim R . . . . . . . . . . . . . . . .P-0033 

Cristiano da Silva B . . . . . . . . .P-0126 

Cross L . . . . . . . . . . . . . . . . .O-0018 

Cruz H . . . . . . . . . . . . . . . . . .P-0681 

Cruz K . . . . . . . . . . . . . . . . . .P-0017 

Cruzado M . . . . . . . . . . . . . . .P-0078 

Cuenca-Estrella M . . . . . . . . . .P-0628 

Cui F . . . . . . . . . . . . . . . . . . .P-0402 

Cuisenier B . . . . . . . . . . . . . . .P-0475 

Cummings J . . . . . . . . . . . . . .P-0376 

Cunha F . . . . . . . . . . .P-0271, P-0272 

Cunha M . . . . . . . . . . . . . . . .P-0595 

Cunningham M . . . . . . . . . . . .P-0239 

Curiel-Quesada E . . . . . . . . . .P-0692 

Curtillet C . . . . . . . . . .P-0275, P-0301 

Cury V . . . . . . . . . . . . . . . . . .P-0644 

Cuétara M . . .P-0211, P-0280, P-0355 

Cyrillo M . . . . . . . . . . . . . . . . .P-0515 

Czaika G . . . . . . . . . . . . . . . .P-0135 

Czaika V . . . . . . . . . . . . . . . . .P-0135 

Cácere C . . . . . . . . . . . . . . . .P-0279 


9

Cândida do Amaral C . . . . . . .P-0482 

Cécile P . . . . . . . . . . . . . . . . .P-0369 

Córdoba S . . .P-0063, P-0106, P-0479 

Córdova-Martínez É . . . . . . . . .P-0265 

Côté I . . . . . . . . . . . . . . . . . . .P-0293 

Cýkman Toker S . . . . . . . . . . .P-0443 

D’alessandro D . . . . . . . . . . . .P-0430 

da Costa J . . . . . . . . . . . . . . .P-0695 

da Rosa A . . . . . . . . .P-0548, P-0549 

da Silva J . . . . . . . . . . . . . . . .P-0279 

da Silva Ruiz L . . . . . .P-0082, P-0126 

Daboit T . . . . . . . . . . .P-0696, P-0732 

Dai Y . . . . . . . . . . . . . . . . . . .P-0538 

Daikos G . . . . . . . . . .P-0048, P-0332 

Dalenda E . . . . . . . . . . . . . . . .P-0310 

Dalle F . . . . .P-0475, P-0557, P-0717 

Damasco P . . . . . . . . . . . . . . .P-0287 

Danaire V . . . . . . . . . . . . . . . .P-0557 

Daniault G . . . . . . . . . . . . . . .P-0459 

Dannaoui E . . . . . . . .P-0137, P-0193 

Daoud W . . . . . . . . . . . . . . . .P-0179 

Das S . . . . . . . . . . . . . . . . . . .P-0329 

Datry A . . . . . . . . . . .P-0194, P-0489 

Davel G . . . . . . . . . . . . . . . . .P-0063 

Davel G . . . . .P-0106, P-0157, P-0479 

Daveloese A . . . . . . . . . . . . . .P-0328 

David L . . . . . . . . . . . . . . . . . .P-0086 

David S . . . . . . . . . . . . . . . . . .P-0407 

Davis R . . . . . . . . . . . . . . . . .O-0033 

Davison J . . . . . . . . . . . . . . .O-0016 

de Albornoz M . . . . . . . . . . . .P-0516 

de Assis Baroni F . . . . . . . . . . .P-0082 

De Beenhouwer H . . . . . . . . . .P-0454 

De Bernardis F . . .O-0045, P-0359, P- 

0594, P-0596 . . . . . . . . . . . . . . . . . . 

de Capraris D . . . . . . . . . . . . .P-0006 

de Cassia Trindade R . . . . . . . .P-0451 

de Fatima Sugizaki M . . . . . . .P-0126 

de Fatima Travalia M . . . . . . . .P-0451 

De Garcia M . . . . . . . . . . . . . .P-0476 

de Hoog G . .P-0017, P-0244, P-0358, 

. . . . . . . . . .P-0377, P-0440, P-0739, 

. . . . . . . . . . . . . . . . .P-0750, P-0755 

de Hoog S . . . . . . . . .P-0157, P-0167, 

. . . .P-0358, P-0742, P-0743, P-0744 

De Jesus M . . . . . . . . . . . . . . .P-0716 

de Kat J . . . . . . . . . . . . . . . . .P-0152 

de la Parte M . . . . . . .P-0197, P-0212 

De la Peña-Moctezuma A . . . . .P-0040 

de Melo D . . . . . . . . . . . . . . . .P-0738 

de Melo D . . . . . . . . . . . . . . . .P-0451 

de Monbrison F . . . . . . . . . . . .P-0378 

de Monte M . . . . . . . . . . . . . .P-0205 

de Resende M . . . . . .P-0064, P-0065, 

. . . .P-0066, P-0451, P-0644, P-0738 

de Souza L . . . . . . . . .P-0548, P-0549 

de Souza T . . . . . . . . . . . . . . .P-0695 

de Souza W . . . . . . . . . . . . . .P-0595 

De Vedia L . . . . . . . . . . . . . . .P-0382 

De Zoppa A . . . . . . . . . . . . . .P-0033 

DeCaro G . . . . . . . . . . . . . . . .P-0225 

Deccache P . . . . . . . . . . . . . .O-0019 

Decroix J . . . . . . . . . . . . . . . . .P-0176 

Dei-Cas E . . . . . . . . . . . . . . . .P-0665 

del Castillo M . . . . . . . . . . . . .P-0341 

del Palacio A . . . . . . . . . . . . . .P-0280 

Del Palacio A P-0280, P-0355, P-0386, 

P-0401 . . . . . . . . . . . . . . . . . . . . . . . 

Delaporte E . . . . . . . . . . . . . . .P-0665 

Delhaes L . . . . . . . . . . . . . . . .P-0665 

Delplace F . . . . . . . . . . . . . . . .P-0702 

Demanche C . . . . . .O-0002, P-0745 

Denning D P-0044, P-0146, P-0158, P- 

0395 . . . . . . . . . . . . . . . . . . . . . . . . . 

Derossi A . . . . . . . . . . . . . . . .P-0222 

Derouin F . . . . . . . . . . . . . . . .P-0421 

Descamps F . . . . . . . . . . . . . .P-0303 

Descamps P . . . . . . . . . . . . . .P-0198 

Desreumaux P . . . . . . . . . . . .O-0004 

Devi S . . . . . . . . . . . . . . . . . . .P-0473 

Deville M . . . . . . . . .O-0002, P-0675 

Dewes L . . . . . . . . . . .P-0548, P-0549 

Diamanti M . . . . . . . . . . . . . . .P-0455 

Dias C . . . . . . . . . . . . . . . . . .P-0288 

Diaz E . . . . . .P-0269, P-0485, P-0516 

Diaz M . . . . .P-0012, P-0196, P-0367 

Diba K . . . . . .P-0213, P-0414, P-0645 

Dignard D . . . . . . . . . . . . . . .O-0035 

Dimitriadou E . . . . . . . . . . . . .P-0226 

Dinleyici E . . . . . . . . . . . . . . . .P-0252 

Dinulescu M . . . . . . . . . . . . . .P-0214 

Diot P . . . . . . . . . . . . . . . . . . .P-0205 

Dippon J . . . . . . . . . . . . . . . . .P-0578 

Dispersyn G . . . . . . . . . . . . . .P-0576 

Distel B . . . . . . . . . . . . . . . . . .P-0701 

Diza E . . . . . . . . . . . . . . . . . . .P-0270 

do Valle De Zoppa A . . . . . . . .P-0022 

Dokic-Trajkovska E . . . . . . . . . .P-0276 

Dolande M . . . . . . . . .P-0477, P-0478 

Dolgo-Saburova J . . . .P-0215, P-0618 

Domb A . . . . . . . . . . . . . . . . .P-0125 

Domingues M . . . . . . .P-0481, P-0482 

Dominguez S . . . . . . .P-0615, P-0685 


Dominique T . . . . . . . . . . . . . .P-0471 

Donegan N . . . . . . . .P-0285, P-0330 

Dong P . . . . . . . . . . . . . . . . . .P-0067 

Donnelly P . . . . . . . . . . . . . . .P-0161 

Donofrio F . . . . . . . . . . . . . . .P-0216 

Dos Santos V . . . . . . . . . . . . . .P-0646 

Dostál J . . . . . . . . . . . . . . . . . .P-0647 

Dotis J . . . . . . . . . . . . . . . . . .P-0247 

Douchet C . . . . . . . . . . . . . . . .P-0356 

Doukas E . . . . . . . . . . . . . . . .P-0332 

Drago M . . . . . . . . . .P-0424, P-0697 

Drahosova M . . . . . . . . . . . . .P-0232 

Driemeier D . . . . . . . .P-0002, P-0008 

Driss G . . . . . . . . . . . . . . . . . .P-0178 

Dziadkowiec D . . . . . . . . . . . .P-0091 

Díaz M . . . . . . . . . . . . . . . . . .P-0733 

Díez-Orejas R . . . . . . . . . . . .O-0041 

E A Marques M . . . . . . . . . . . .P-0305 

E. Schwendler S . . . . .P-0021, P-0626 

Earhart K . . . . . . . . . .P-0575, P-0587 

Ecarnot-Laubriet A . . . . . . . . . .P-0475 

Echevarria H . . . . . . . . . . . . . .P-0039 

Eckhardt J . . . . . . . . .P-0037, P-0038 

Edlind T . . . . . . . . . . . . . . . . .P-0575 

Egan L . . . . . . . . . . . . . . . . . .P-0431 

Ehlert K . . . . . . . . . . .P-0228, P-0229 

Eisman B . . . . . . . . . . . . . . . .O-0039 

Ejzemberg R . . . . . . . .P-0613, P-0625 

Ernoult E . . . . . . . . . . . . . . . . .P-0154 

Erturan Z . . . . . . . . . . . . . . . . .P-0056 

Eskandarian M . . . . . . . . . . . .P-0648 

Espenshade P . . . . . . . . . . . . .P-0637 

Esperou H . . . . . . . . . . . . . . . .P-0088 

Espinel-Ingroff A . . . . . . . . . . .P-0379 

Espinosa-Texis A . . . . . . . . . . .P-0657 

Essendoubi M . . . . . . . . . . . . .P-0480 

Essig A . . . . . . . . . . . . . . . . . .P-0399 

Estelle C . . . . . . . . . . . . . . . . .P-0718 

Etienne M . . . . . . . . . . . . . . . .P-0230 

Evci C . . . . . .P-0070, P-0071, P-0072 

F. Lopez J . . . . . . . . . . . . . . . .P-0628 

Faber E . . . . . . . . . . . . . . . . . .P-0232 

Dromer F . . .P-0088, P-0137, P-0196, 

. . . . . . . . . . .P-0207, P-0295, P-0299 

Drusano G . . . . . . . . . . . . . . .P-0083 

Duarte A . . . . . . . . . . . . . . . .O-0037 

Duarte-Escalante E . . . . . . . . .P-0479 

Dubertret L . . . . . . . . .P-0501, P-0552 

Ducoing-Watty A . . . .P-0009, P-0040, 

. . . . . . . . . . . . . . . . . . . . . . .P-0563 

Ekmekci R . . . . . . . . . . . . . . . .P-0244 

El Euch D . . . .P-0179, P-0250, P-0453 

El Tounisy E . . . . . . . . . . . . . . .P-0499 

Elad D . . . . . . . . . . . . . . . . . .P-0013 

Eldon M . . . . . . . . . . . . . . . .O-0011 

Eleuch D . . . .P-0217, P-0234, P-0494 

Elguezabal N . . . . . . . . . . . . .P-0401 

Ellis D . . . . . . . . . . .O-0015, P-0068, 

Fabre A . . . . . . . . . . . . . . . . . .P-0649 

Fadaie fard F . . . . . . . . . . . . .P-0030 

Fadda G . . . . . . . . . . . . . . . . .P-0130 

Faergemann J . . . . . .P-0219, P-0484 

Fahal A . . . . . . . . . . . . . . . . . .P-0167 

Faika C . . . . . . . . . . . . . . . . . .P-0310 

Falcon D . . . . . . . . . . . . . . . . .P-0237 

Falegan A . . . . . . . . . . . . . . . .P-0530 

Dumon H . . . . . . . . .P-0274, P-0275, 

. . . . . . . . . . . . . . . . .P-0122, P-0558 

Falk R . . . . . . . . . . . . . . . . . . .P-0125 

. . . . . . . . . . .P-0299, P-0300, P-0301 

Ellis M . . . . . . . . . . . . . . . . . . .P-0218 

Falkiewicz-Dulik M . . . . . . . . . .P-0099 

Dunand J . . . . . . . . . . . . . . .O-0010 

Eltaeib N . . . . . . . . . . . . . . . . .P-0281 

Fallahyan F . . . . . . . . . . . . . . .P-0582 

Duncan G . . . . . . . . . . . . . . . .P-0239 

Emi Matsumoto F . . . .P-0082, P-0126 

Falusi E . . . . . . . . . . .P-0089, P-0428 

Dungan J . . . . . . . . . . . . . . .O-0033 

Emilie F . . . . . . . . . . . . . . . . . .P-0471 

Fanariotis D . . . . . . . . . . . . . .P-0047 

Dunsmuir R . . . . . . . . . . . . . . .P-0045 

Emily Cury A . . . . . . . . . . . . . .P-0082 

Faouzi S . . . . . . . . . . . . . . . . .P-0073 

Dupont B . . . . . . . . . .P-0097, P-0295 

Endo S . . . . . . . . . . . . . . . . . .P-0411 

Farahnejad Z . . . . . . . . . . . . .P-0164 

Durand E . . . . . . . . . . . . . . . .P-0650 

Ener B . . . . .P-0070, P-0071, P-0072, 

Faraut F . . . . . . . . . . . . . . . . .P-0299 

Durand P . . . . . . . . . . . . . . . .P-0339 

. . . . . . . . . . . . . . . . .P-0168, P-0443 

Farhat D . . . . . . . . . . . . . . . . .P-0249 

Duriez T . . . . . . . . . . . . . . . . .P-0745 

Englethaler D . . . . . . . . . . . . .P-0474 

Farmaki E . . . . . . . . . . . . . . . .P-0248 

Durkin M . . . . . . . . .O-0006, P-0431 

Enomoto U . . . . . . . . . . . . . . .P-0240 

Farnarier C . . . . . . . . . . . . . . .P-0273 

Durán C . . . . . . . . . . .P-0058, P-0107 

Eraso E . . . . . . . . . . . . . . . . . .P-0069 

Farrugia C . . . . . . . . . . . . . . .P-0220 

Dutta S . . . . . . . . . . . . . . . . . .P-0079 

Ereno Auler M . . . . . . . . . . . . .P-0082 


11

Fassier T . . . . . . . . . . . . . . . . .P-0339 

Fatma S . . . . . . . . . . . . . . . . .P-0278 

Fattouma M . . . . . . . . . . . . . .P-0604 

Faure O . . . .P-0237, P-0380, P-0421 

Favel A . . . . . . . . . . . . . . . . . .P-0273 

Favennec L . . . . . . . . . . . . . . .P-0230 

Fegeler W . . .P-0134, P-0228, P-0229 

Feierl G . . . . . . . . . . . . . . . . . .P-0200 

Felicissimo P . . . . . . . . . . . . . .P-0346 

Felipe M . . . . . . . . . . . . . . . . .P-0719 

Fell J . . . . . . . . . . . . . . . . . . . .P-0367 

Fellenberg K . . . . . . . . . . . . . .P-0578 

Fernandes E . . . . . . . . . . . . . .P-0466 

Fernandes Fontana F .P-0481, P-0482 

Fernandez A . . . . . . . . . . . . . .P-0485 

Fernandez N . . . . . . .P-0190, P-0209 

Fernandez V . . . . . . . .P-0242, P-0372 

Fernandez de Larrinoa I . . . . . .P-0401 

Fernandez-Olmos A . . . . . . . .P-0410 

Fernández-Arenas E . . . . . . . .O-0041 

Fernández-Olmos A . . . . . . . .P-0074 

Ferquel C . . . . . . . . . . . . . . . .P-0295 

Ferraro P . . . . . . . . . . . . . . . . .P-0259 

Ferreira K . . . . . . . . . . . . . . . .P-0586 

Ferreira M . . . . . . . . .P-0221, P-0264 

Ferreira Fernandes G . . . . . . . .P-0534 

Ferreiro L . . . .P-0002, P-0008, P-0042 

Ferretti A . . . . . . . . . . . . . . . . .P-0591 

Ferrão M . . . .P-0138, P-0695, P-0711 

Ferwerda . . . . . . . . . . . . . . . .P-0597 

Feuilhade M . . . . . . . . . . . . . .P-0501 

Feuilhade de Chauvin M . . . .O-0007, 

. . . . . . . . . . . . . . . . . . . . . . .P-0552 

Fica A . . . . . . . . . . . . . . . . . . .P-0733 

Fidel R . . . . . . . . . . . . . . . . . .P-0191 

Fierer J . . . . . . . . . . . . . . . . . .P-0598 

Fierro L . . . . . . . . . . . . . . . . . .P-0195 

Figueiredo C . . . . . . . . . . . . .O-0019 

Figueroa C . . . . . . . . . . . . . . .P-0315 

Filler S . . . . . . . . . . . . . . . . . .P-0395 

Finkelman M . . . . . . . . . . . . . .P-0381 

Finquelievich JP-0075, P-0124, P-0307, 

. . . .P-0308, P-0382, P-0636, P-0650 

Fior A . . . . . . . . . . . . . . . . . . .P-0193 

Fiori B . . . . . . . . . . . . . . . . . . .P-0130 

Fischer C . . . . . . . . . . . . . . . . .P-0225 

Fischman Gompertz O . . . . . .P-0014, 

. . . . . . . . . . . . . . . . .P-0076, P-0651 

Flageul B . . . . . . . . . . . . . . . .O-0007 

Florax A . . . . . . . . . . . . . . . . .P-0228 

Florek J . . . . . . . . . . . . . . . . . .P-0508 

Flores-Carreón A . . . .P-0098, P-0671, 

. . . . . . . . . . . . . . . . .P-0692, P-0736 

Fontanet A . . . . . . . . . . . . . . .P-0207 

Fontes C . . . . . . . . . . . . . . . .O-0037 

Fonteyne P . . . . . . . . . . . . . . .P-0383 

Forche A . . . . . . . . . . . . . . . .O-0003 

Fothergill A . .P-0077, P-0147, P-0166 

Fradin C . . . . . . . . . . .P-0667, P-0726 

Fragouli E . . . . . . . . . . . . . . . .P-0047 

Frampton C . . . . . . . . . . . . . . .P-0218 

Franco H . . . . . . . . . . . . . . . . .P-0128 

Franco M . . . . . . . . . . . . . . . .P-0305 

Francois-Xavier E . . . . . . . . . . .P-0086 

Frank M . . . . . . . . . . . . . . . . .P-0267 

Frans J . . . . . . . . . . . . . . . . . .P-0454 

François I . . . . . . . . . . . . . . . .P-0576 

François N . . . . . . . . .P-0328, P-0734 

Fregeau C . . . . . . . . . . . . . . . .P-0083 

Freire T . . . . . . . . . . . . . . . . . .P-0201 

Freitas G . . . .P-0284, P-0327, P-0505 

French Cryptococcosis Study Group . . 

. . . . . . . . . . . . . . . . . . . . . . .P-0207 

Fridkin S . . . .P-0462, P-0463, P-0474, 

. . . . . . . . . . . . . . . . .P-0533, P-0559 

Fries C . . . . . . . . . . . . . . . . . .P-0244 

Frimodt-Moller N . . . . . . . . . . .P-0132 

Froehlich B . . . . . . . . . . . . . . .P-0229 

Frolova E . . . . . . . . . . . . . . . . .P-0618 

Fréalle E . . . . . . . . . . . . . . . . .P-0665 

Frédéric D . . . . . . . . . . . . . . . .P-0471 

Frédéric G . . . . . . . . . . . . . . . .P-0513 

Frédérique F . . . . . . . . . . . . . .P-0427 

Fujisawa T . . . . . . . . . . . . . . . .P-0325 

Fukushima K . . . . . . .P-0025, P-0483, 

. . . . . . . . . . . . . . . . .P-0639, P-0746 

Fukushima K . . . . . . . . . . . . . .P-0755 

Funakoshi K . . . . . . . . . . . . . . .O-029 

Furlan Matos Aires W .P-0481, P-0482 

Fusco-Almeida A . . . . . . . . . . .P-0222 

Fétissof F . . . . . . . . . . . . . . . . .P-0205 

Gafa V . . . . . . . . . . . . . . . . . .P-0599 

Gagliardi C . . . . . . . . . . . . . . .P-0599 

Gaitanis G . . . . . . . . . . . . . . .P-0484 

Galanis Z . . . . . . . . . . . . . . . .P-0226 

Galbraith K . . . . . . . . . . . . . . .P-0652 

Galimberti L . . . . . . . . . . . . . .P-0172 

Gallego M . . . . . . . . .P-0057, P-0211 

Gallot N . . . . . . . . . . .P-0615, P-0685 

Gambale V . . . . . . . . .P-0082, P-0544 

Gambale W . . . . . . . .P-0568, P-0643 

Gamboa A . . . . . . . . . . . . . . .P-0485 

Gandemer V . . . . . . . . . . . . . .P-0210 

Gangneux J . .P-0210, P-0294, P-0370 


Gantois N . . . . . . . . . . . . . . . .P-0665 

Garbervetsky L . . . . . . . . . . . .P-0341 

Garcia I . . . . . . . . . . . . . . . . .P-0486 

Garcia M . . . .P-0078, P-0365, P-0714 

Garcia P . . . . . . . . . . . . . . . .O-0017 

Garcia-Hermoso D . . . . . . . . .P-0300 

García I . . . . . . . . . . . . . . . . .P-0653 

García de Acevedo B . . . . . . . .P-0265 

García-Effrón G . . . . . . . . . . .P-0628 

García-Martínez J . . . .P-0486, P-0653 

Garg A . . . . . . . . . . . . . . . . . .P-0223 

Gargala G . . . . . . . . . . . . . . .P-0230 

Gargallo-Viola D . . . . . . . . . . .P-0365 

Garmendia Y . . . . . . . . . . . . .P-0121 

Gasperik J . . . . . . . . . . . . . . . .P-0592 

Gautam R . . . . . . . . . . . . . . . .P-0223 

Gautier M . . . . . . . . . . . . . . . .P-0427 

Gene J . . . . . . . . . . . . . . . . . .P-0753 

Genot S . . . . . . . . . . . . . . . . .P-0299 

Gené F . . . . . . . . . . . . . . . . .O-0010 

Gené J . . . . . .P-0192, P-0228 P-0749 

George A . . . . . . . . . . . . . . .O-0013 

Gerety R . . . . . . . . . . . . . . . .O-0011 

Gerrits van den Ende AP-0440, P-0739 

Gershman K . . . . . . . . . . . . . .P-0463 

Gersuk G . . . . . . . . . . . . . . . .P-0600 

Gervini R . . . . . . . . . .P-0548, P-0549 

Geunes-Boyer S . . . . . . . . . . . .P-0601 

Ghannoum M . . . . . . . . . . . . .P-0079 

Ghiasian S . . . . . . . . . . . . . . .P-0487 

Ghilardi A . . . . . . . . . . . . . . . .P-0224 

Ghitan M . . . . . . . . . .P-0080, P-0225 

Giacomini E . . . . . . . . . . . . . .P-0599 

Giakoumis X . . . . . . . . . . . . . .P-0226 

Gibas C . . . . . . . . . . . . . . . . .P-0747 

Gibbs D . . . . . . . . . . . . . . . . .P-0379 

Giddey . . . . . . . . . . . . . . . . . .P-0748 

Giddey K . . . . . . . . . . . . . . . . .P-0584 

Gil C . . . . . . . . . . . . . . . . . . .O-0041 

Gil M . . . . . . . . . . . . . . . . . . .P-0603 

Gil-Tomás J . . . . . . . . . . . . . . .P-0309 

Giles S . . . . . . . . . . . . . . . . . .P-0601 

Gilgado F . . . . . . . . . . . . . . . .P-0749 

Gilibert R . . . . . . . . . . . . . . . .P-0524 

Girouard G . . . . . . . . . . . . . . .P-0384 

Gliosca L . . . . . . . . . . . . . . . . .P-0075 

Gloeckner A . . . . . . . . . . . . . .P-0227 

Gniadek A . . . . . . . . . . . . . . .P-0508 

Gobernado M . . . . . . . . . . . . .P-0457 

Gobernado Serrano M . . . . . .P-0465 

Godoy G . . . . . . . . . . . . . . . .P-0472 

Godoy P . . . . . . . . . . .P-0446, P-0515 

Goldman G . . . . . . . .P-0577, P-0630 

Goldman M . . . . . . . . . . . . . .P-0577 

Goldman M . . . . . . . . . . . . . .O-0006 

Gomes Pìres H . . . . . .P-0481, P-0482 

Gomez B . . . . . . . . . . . . . . . . .P-0362 

Gomez B . . . . . . . . . . . . . . . . .P-0621 

Gomez M . . . . . . . . . . . . . . . .P-0567 

Gomez S . . . . . . . . . . . . . . . . .P-0316 

Gompertz O . . . . . . . . . . . . . .P-0343 

Goncalves G . . . . . . . . . . . . . .P-0451 

Gonsálvez M . . . . . . . . . . . . . .P-0472 

Gonzàlez G . . . . . . .O-0012, P-0081 

González R . . . . . . . . . . . . . .O-0012 

González-Ibarra J . . . . . . . . . .P-0098 

Gonçalves S . . . . . . . . . . . . . .P-0446 

Gonçalves da Silva E .P-0082, P-0126, 

. . . . . . . . . . . . . . . . . . . . . . .P-0439 

Gorostiaga J . . . . . . . . . . . . . .P-0341 

Gouy de Bellock J . . . . . . . . .O-0002 

Gow N . . . . . . . . . . . . . . . . . .P-0731 

Gow N . . . . . . . . . . . . . . . . . .P-0671 

Gozalbo D . . . . . . . . . . . . . . .P-0603 

Grammatikou M . . . . . . . . . . .P-0048 

Grant S . . . . . . . . . . . . . . . . . .P-0596 

Gravi E . . . . . . . . . . . . . . . . . .P-0719 

Graybill J . . . .P-0028, P-0110, P-0395 

Graziani S . . . . . . . . . . . . . . . .P-0359 

Grenouillet F . . . . . . . . . . . . . .P-0489 

Grillot R . . . . . . . . . .P-0237, P-0380, 

. . . . . . . . . . . . . . . . .P-0430, P-0599 

Groll A . . . . . . . . . . . .P-0228, P-0229 

Grosse V . . . . . . . . . . . . . . . .O-0042 

Gräser Y . . . . . . . . . . . . . . . . .P-0740 

Guareschi C . . . . . . . . . . . . . .P-0063 

Guarro J . . .O-0010, P-0282, P-0288, 

. . . . .P-0304,P-0311 P-0749, P-0753 

Gueit I . . . . . . . . . . . . . . . . . .P-0230 

Guelfand L . . . . . . . . . . . . . . .P-0636 

Guerardel Y . . . . . . . . . . . . . .P-0725 

Guereña D . . . . . . . . . . . . . .O-0012 

Guerrero H . . . . . . . . . . . . . . .P-0496 

Guerrini G . . . . . . . . . . . . . . .P-0382 

Guglielminetti M . . . . . . . . . . .P-0754 

Guida N . . . . . . . . . . .P-0015, P-0016 

Guiguen C . . . . . . . . .P-0210, P-0370 

Guillemain R . . . . . . . . . . . . .O-0008 

Guillot J . . .O-0002, P-0006, P-0020, 

. . . . . . . . . . .P-0665, P-0675, P-0745 

Guinard J . . . . . . . . . . . . . . . .P-0184 

Guinard j . . . . . . . . . . . . . . . .P-0182 


13

Guiu N . . . . . . . . . . . .P-0059, P-0202 

Gumbo T . . . . . . . . . . . . . . . .P-0083 

Gumma S . . . . . . . . . . . . . . . .P-0281 

Gundlapalli A . . . . . . . . . . . . .P-0334 

Guptasarma P . . . . . . . . . . . . .P-0632 

Gurr S . . . . . . . . . . . . . . . . . . .P-0050 

Guzman-Chavez R . . . . . . . . .P-0010 

Guzmán C . . . . . . . . .P-0615, P-0685 

Guérin J . . . . . . . . . . . . . . . . .P-0339 

Gómez de Ana S . . . .P-0385, P-0629 

Gómez Raja J . . . . . . . . . . . . .P-0654 

Göral G . . . . . . . . . . . . . . . . .P-0072 

Haas D . . . . . . . . . . . . . . . . . .P-0200 

Haase G . . . . . . . . . .P-0418, P-0750 

Hachicha M . . . . . . . . . . . . . .P-0208 

Hackett E . . . . . . . . . . . . . . . .P-0431 

Hadrich I . . . . . . . . . .P-0208, P-0604 

Haedersdal M . . . . . . . . . . . . .P-0132 

Hafida N . . . . . . . . . .P-0177, P-0178 

Hagblom P . . . . . . . . . . . . . . .P-0242 

Hagen F . . . . . . . . . . . . . . . . .P-0196 

Hagen F . . . . . . . . . . . . . . . .O-0027 

Haghnazari A . . . . . . . . . . . . .P-0043 

Hall L . . . . . . . . . . . . .P-0231, P-0655 

Halliday C . . . . . . . . . . . . . . . .P-0400 

Halos L . . . . . . . . . . . . . . . . . .P-0675 

Hamad S . . . . . . . . . . . . . . . .P-0386 

Hamal P . . . . . . . . . . .P-0232, P-0344 

Hamilton A . . . . . . . . . . . . . . .P-0621 

Hammadi K . . . . . . . . . . . . . .P-0233 

Hammer A . . . . . . . . . . . . . . .P-0497 

Hanchi I . . . . . . . . . . . . . . . . .P-0234 

Handwerger K . . . . . . . . . . . . .P-0720 

Haniloo A . . . . . . . . . . . . . . . .P-0450 

Hansen M . . . . . . . . . . . . . . . .P-0334 

Hao F . . . . . . . . . . . . . . . . . . .P-0235 

Haouet S . . . . . . . . . .P-0179, P-0217 

Harada S . . . . . . . . . . . . . . . .P-0387 

Harak H . . . . . . . . . . . . . . . . .P-0236 

Harding D . . . . . . . . . . . . . . . .P-0115 

Harmsen A . . . . . . . . . . . . . . .P-0605 

Harrak J . . . . . . . . . . . . . . . . .P-0017 

Harrison L . . . . . . . . . . . . . . . .P-0559 

Hartung C . . . . . . . . . . . . . . . .P-0477 

Hartung de Capriles C . . . . . .P-0375, 

. . . . . . . . . . . . . . . . .P-0388, P-0514 

Hartz Alves S . . . . . . .P-0037, P-0021, 

. . . . . . . . . . .P-0038, P-0042, P-0626 

Harvalou K . . . . . . . . . . . . . . .P-0047 

Hasegawa A . . . . . . . . . . . . . .P-0679 

Hashemi J . .P-0001, P-0018, P-0029, 

. . . . . . . . . . .P-0490, P-0491, P-0492 

Hashemi J . . . . . . . . . . . . . . . .P-0442 

Hashizume H . . . . . . . . . . . . .P-0336 

Hashizume T . . . . . . . . . . . . . .P-0746 

Hassan Q . . . . . . . . . . . . . . . .P-0178 

Hasselby J . . . . . . . . . . . . . . . .P-0132 

Hasumi Y . . . . . . . . . . . . . . . .P-0117 

Hata Y . . . . . . . . . . . . . . . . . .P-0317 

Hatamzadeh M . . . . . . . . . . . .P-0525 

Hauggaard A . . . . . . . . . . . . .P-0218 

Hauser N . . . .P-0389, P-0578, P-0656 

Hausstein U . . . . . . . . . . . . . .P-0052 

Haustein U . . . . . . . . . . . . . . .P-0522 

Havlicek V . . .P-0415, P-0425, P-0437 

Haw C . . . . . . . . . . . . . . . . . .P-0258 

Hay R . . . . . . . . . . . . . . . . . . .P-0343 

Hayden R . . . . . . . . . .P-0376, P-0390 

Haznedar R . . . . . . . . . . . . . . .P-0398 

Heath C . . . . . . . . . . . . . . . . .P-0122 

Hedayati M . . . . . . . . . . . . . . .P-0493 

Heep M . . . .P-0081, P-0136, P-0318, 

. . . . . . . . . . .P-0319, P-0320, P-0321 

Hegedûs J . . . . . . . . . . . . . . . .P-0089 

Hehlmann R . . . . . . . . . . . . . .P-0391 

Heitman J . . . . . . . . . . . . . . . .P-0503 

Heitman J . .O-0043, P-0120, P-0686 

Heitzmann A . . . . . . . . . . . . . .P-0295 

Helena da Silva E . . . . . . . . . .P-0126 

Hemadi D . . . . . . . . . . . . . . . .P-0316 

Hemashettar B . . . . . . . . . . . .P-0752 

Hennequin C . . . . . .O-0010, P-0109, 

. . . . . . . . . . . . . . . . .P-0198, P-0356 

Henzel A . . . . . . . . . .P-0021, P-0626 

Hepsert S . . . . . . . . . . . . . . . .P-0056 

Hermosilla G . . . . . . .P-0012, P-0556 

Hernandez C . . . . . . . . . . . . . .P-0681 

Hernandez R . . . . . . . . . . . . . .P-0656 

Hernandez-Romahn A . . . . . . .P-0009 

Hernando F . . . . .O-0017, P-0585, P 

. . . . . . . . . . . . . . . . . .-0615, P-0685 

Hernández I . . . . . . . . . . . . . .P-0472 

Hernández-Alcaraz J . . . . . . . .P-0536 

Hernández-Hernández F . . . . .P-0265, 

. . . . . . . . . . . . . . . . . . . . . . .P-0657 

Hernández-Ramírez A . . . . . . .P-0479 

Hernández-Rodríguez C . . . . . .P-0705 

Herrera F . . . . . . . . . . . . . . . .P-0382 

Hervé P . . . . . . . . . . .P-0512, P-0513 

Hicheri J . . . . . . . . . . . . . . . . .P-0494 

Higashiyama Y . . . . . . . . . . . .P-0392 

Hincky V . . . . . . . . . . . . . . . . .P-0237 

Hinojosa A . . . . . . . . . . . . . . .P-0361 

Hinojosa R . . . . . . . . . . . . . . .P-0567 


Hipler C . . . . . . . . . . . . . . . . .P-0052 

Hirakata Y . . . . . . . . .P-0277, P-0392 

Hirose N . . . . . . . . . . . . . . . . .P-0555 

Hiroshima K . . . . . . . . . . . . . .P-0730 

Huang Y . . . . . . . . . . . . . . . . .P-0565 

Hube B . . . . .P-0667, P-0671, P-0717 

Huber F . . . . . . . . . . . . . . . . .P-0193 

Huck S . . . . . . . . . . . . . . . . . .P-0421 

Italhi M . . . . . . . . . . . . . . . . . .P-0233 

Itano E . . . . . . . . . . . .P-0031, P-0607 

Ito T . . . . . . . . . . . . . . . . . . . .P-0337 

Ito-Kuwa S . . .P-0658, P-0673, P0704 

Hiruma M . . . . . . . . .P-0160, P-0238, 

. . . . . . . . . . . . . . . . .P-0500, P-0555 

Hisajima T . . . . . . . . . . . . . . . .O-029 

Hocqueloux L . . . . . . . . . . . . .P-0295 

Hocquette A . . . . . . . . . . . . . .P-0054 

Hoepelman A . . . . . . . . . . . .O-0027 

Hoepelman I . . . . . . . . . . . . . .P-0367 

Hoinard D . . . . . . . . . . . . . . . .P-0193 

Holandino C . . . . . . . . . . . . . .P-0296 

Holl A . . . . . . . . . . . . . . . . . . .P-0200 

Holmes A . . . . . . . . . .P-0114, P-0115 

Holton J . . . . . . . . . . . . . . . . .P-0596 

Homayouni R .P-0575, P-0579, P-0587 

Hoogveld H . . . . . . . . . . . . . . .P-0196 

Horré R . . . . . . . . . . . . . . . . . .P-0739 

Horta G . . . . . . . . . . . . . . . . .P-0288 

Horwitz B . . . . . . . . . . . . . . . .P-0631 

Hoskin T . . . . . . . . . . . . . . . . .P-0569 

Hosoi M . . . . . . . . . . . . . . . . .P-0411 

Hosseinpur L . . . . . . . . . . . . . .P-0518 

Hot A . . . . . . . . . . . . . . . . . . .P-0097 

Hotta A . . . . . . . . . . . . . . . . . .P-0116 

Howard S . . . . . . . . . . . . . . . .P-0146 

Howell S . . . . . . . . . . . . . . . . .P-0239 

Howles M . . . . . . . . . . . . . . . .P-0376 

Hradílek M . . . . . . . . . . . . . . .P-0647 

Hrušková-Heidingsfeldová O . .P-0647 

Hryncewicz-Gwozdz A . . . . . . .P-0084 

Hsiung A . . . . . . . . . . . . . . . . .P-0379 

Hsu V . . . . . . . . . . . . . . . . . . .P-0083 

Huerga V . . . . . . . . . . . . . . . .P-0685 

Huie-White S . . . . . . . . . . . . . .P-0559 

Hull C . . . . . . . . . . . . . . . . . . .P-0699 

Hummel M . . . . . . . . . . . . . . .P-0391 

Hurst S . . . . . . . . . . . . . . . . . .P-0422 

Iatta R . . . . . . . . . . . .P-0005, P-0721 

Ichai P . . . . . . . . . . . . . . . . . .P-0220 

Iersel M . . . . . . . . . . .P-0318, P-0319 

Ikeda F . . . . . . . . . . . . . . . . . .P-0114 

Ikeda R . . . . . . . . . . . .P-0244, P-0722 

Ikeda S . . . . . . . . . . . .P-0238, P-0555 

Ilkit M . . . . . . . . . . . . . . . . . . .P-0495 

Imbert C . . . . . . . . . . . . . . . . .P-0459 

Immunair Studies Group . . . . .P-0294 

Inci E . . . . . . . . . . . . . . . . . . .P-0245 

Infante R . . . . . . . . . . . . . . . . .P-0464 

Inoue K . . . . . . . . . . . . . . . . . .P-0090 

Iorio E . . . . . . . . . . . . . . . . . . .P-0591 

Iovannitti C . .P-0075, P-0124, P-0650 

Iozumi K . . . . . . . . . . . . . . . . .P-0240 

Ischer F . . . . . . . . . . .O-0003, P-0062 

Isham N . . . . . . . . . . . . . . . . .P-0079 

Ishibashi H . . . . . . . . .O-029, P-0085 

Ishibashi K . . . . . . . . .P-0606, P-0612 

Ishibashi Y . . . . . . . . . . . . . . .P-0611 

Ishii M . . . . . . . . . . . . . . . . . . .P-0283 

Ishizaki H . . . . . . . . .P-0173, P-0174, 

. . . . . . . . . .P-0447, P-0496, P-0519, 

. . . . . . . . . . . . . . . . .P-0570, P-0752 

Isla G . . . . . . . . . . . . . . . . . . .P-0063 

Iumikawa K . . . . . . . . . . . . . . .P-0277 

Izumikawa K . . . . . . . .P-0392, P-0397 

J K . . . . . . . . . . . . . . . . . . . . .P-0251 

Jabali Y . . . . . . . . . . . . . . . . . .P-0241 

Jabbour A . . . . . . . . . . . . . . . .P-0125 

Jackson C . . . . . . . . .O-0014, P-0659 

Jacques R . . . . . . . . . . . . . . . .P-0369 

Jacques V . . . . . . . . . . . . . . . .P-0171 

Jagielski T . . . . . . . . . . . . . . . .P-0019 

Jahouhari C . . . . . . . . . . . . . .P-0472 

Jaime T . . . . . . . . . . . . . . . . . .P-0683 

Jain N . . . . . . . . . . . .P-0162, P-0314 

Jain S . . . . . . . . . . . . . . . . . . .P-0282 

Jakobson E . . . . . . . . . . . . . . .P-0242 

Jalalizand N . . . . . . .P-0412, P-0414, 

. . . . . . . . . . . . . . . . .P-0645, P-0687 

Jamal T . . . . . . . . . . . . . . . . . .P-0675 

Jamison G . . . . . . . . . . . . . . .P-0376 

Janbon G . . .P-0702, P-0725, P-0726 

Jankoska G . . . . . . . . . . . . . . .P-0276 

Jaqueti J . . . . . . . . . . . . . . . . .P-0653 

Jaquetti J . . . . . . . . . . . . . . . . .P-0486 

Jaramillo J . . . . . . . . . . . . . . .P-0035 

Javan H . . . . . . . . . . . . . . . . .P-0547 

Jawhara S . . . . . . . . . . . . . . .O-0004 

Jean baptiste S . . . . . . . . . . . .P-0171 

Jean Francois M . . . . . . . . . .O-0023 

Jean Gustave T . . . . . . . . . . . .P-0086 

Jean Paul D . . . . . . . . . . . . . . .P-0086 

Jean-Claude G . . . . . . . . . . . .P-0175 

Issa N . . . . . . . . . . . . . . . . . . .P-0569 


15

Jean-Louis Q . . . . . . .P-0512, P-0513 

Jean-Luc B . . . . . . . . .P-0512, P-0513 

Jean-Marc B . . . . . . . . . . . . . .P-0718 

Jean-Paul B . . . . . . . .P-0512, P-0513 

Jean-Yves C . . . . . . . . . . . . . .P-0512 

Jensen J . . . . . . . . . . . . . . . . .P-0715 

Julienne L . . . . . . . . . . . . . . . .P-0086 

K.S S . . . . . . . . . . . . . . . . . . . .P-0251 

Kabbara N . . . . . . . . . . . . . . .P-0088 

Kabukcuoglu S . . . . . . . . . . . .P-0252 

Kacalak Rzepka A . . . . . . . . . .P-0302 

Kadota T . . . . . . . . . . . . . . . . .O-029 

Katherina Z . . . . . . . . . . . . . . .P-0717 

Katiraee F . . . . . . . . . . . . . . . .P-0164 

Kato H . . . . . . . . . . . .P-0151, P-0611 

Katragkou A . . . . . . . . . . . . . .P-0247 

Katsampas A . . . . . . . . . . . . . .P-0047 

Kauffman S . . . . . . . . . . . . . . .P-0636 

Jewtuchowicz V . . . . .P-0075, P-0124, Kagawa S . . . . . . . . . . . . . . . .P-0752 Kauffman-Lacroix C . . . . . . . . .P-0665 

. . . . . . . . . . . . . . . . . . . . . . .P-0650 

Kajiwara S . . . . . . . . . . . . . . . .P-0581 Kaufman G . . . . . . . . . . . . . . .P-0631 

Jha M . . . . . . . . . . . . .P-0282, P-0331 

Kalkanci A . . . . . . . . .P-0398, P-0449 Kaushik R . . . . . . . . . . . . . . . .P-0206 

Jiang B . . . . . . . . . . . . . . . . .O-0016 

Kallel K . . . . .P-0170, P-0243, P-0246 Kawai M . . . . . . . . . . . . . . . . .P-0500 

Jimenez M . . . . . . . . . . . . . . . .P-0598 

Kaltseis J . . . . . . . . . . . . . . . . .P-0537 Kawamoto S . . . . . . . .P-0722, P-0728 

Jimenez O . . . . . . . . . . . . . . .P-0514 

Kamarloei S . . . . . . . . . . . . . .P-0554 Kawasaki M . . . . . . . .P-0173, P-0174, 

Jimenez R . . . . . . . . . . . . . . . .P-0211 

Kamei K . . . .P-0027, P-0036, P-0291, . . . . . . . . . .P-0447, P-0496, P-0519, 

Jiménez T . . . . . . . . . . . . . . . .P-0046 . . . . . . . . . . . . . . . . .P-0526, P-0730 . . . . . . . . . . .P-0570, P-0751, P-0752 

Joffroy C . . . . . . . . . . . . . . . . .P-0656 Kamiya S . . . . . . . . . . . . . . . . .P-0055 Kaytaz A . . . . . . . . . . . . . . . . .P-0245 

Johann S . . . . . . . . . .P-0064, P-0065 Kammarnjassadakul P . . . . . . .P-0498 Kazimierz K . . . . . . . . . . . . . . .P-0527 

Johansson A . . . . . . . . . . . . . .P-0242 Kammeyer P . . . . . . . . . . . . . .P-0497 Kchaou W . . . . . . . . . . . . . . . .P-0551 

Johnson E . . . . . . . . . .P-0456, P-0741 Kamule L . . . . . . . . . . . . . . . . .P-0353 Kechia F . . . . . . . . . . . . . . . . .P-0394 

Johnson S . . . . . . . . . . . . . . . .P-0497 Kanayama A . . . . . . . . . . . . . .P-0411 Kefala-Agoropoulou K . . . . . . .P-0248 

Johnson S . . . . . . . . . . . . . . . .P-0608 Kancharla A . . . . . . . . . . . . . .P-0290 Keiko L . . . . . . . . . . . . . . . . . .P-0643 

Joliffe K . . . . . . . . . . . . . . . . . .P-0118 Kandie S . . . . . . . . . . . . . . . . .P-0353 Kempf M . . . . . . . . . .P-0357, P-0574 

Jones M . . . . . . . . . . . . . . . . .P-0285 Kaneko A . . . . . . . . . . . . . . . .P-0581 Kentouche K . . . . . . . . . . . . . .P-0391 

Jones T . . . . . . . . . . . . . . . . .O-0033 Kaneko T . . . . . . . . . . . . . . . .P-0393 Kepil N . . . . . . . . . . . . . . . . . .P-0245 

Jonk L . . . . . . . . . . . .P-0087, P-0161 Kano R . . . . . . . . . . . . . . . . . .P-0679 Kesby C . . . . . . . . . . . . . . . . .P-0535 

Joossens M . . . . . . . . . . . . . . .P-0734 Kantarcioglu A . . . . . .P-0244, P-0245 Keshavarz D . . . . . . . . . . . . . .P-0442 

Joossens S . . . . . . . . . . . . . . . .P-0429 Kaocharoen S . . . . . . . . . . . . .P-0498 Ketela T . . . . . . . . . . . . . . . . .O-0016 

Jordan R . . . . . . . . . . . . . . . . .P-0382 Kaouech E . . .P-0170, P-0243, P-0246 Khalili A . . . . . . . . . . . . . . . . .P-0433 

Jorgensen I . . . . . . . . . . . . . . .P-0024 Kappe R . . . . . . . . . . . . . . . . .P-0306 Khan J . . . . . . . . . . . . . . . . . .O-0032 

Joseph C . . . . . . . . . . . . . . . . .P-0329 Karam El-Din A . . . . . . . . . . . .P-0499 Khan Z . . . . .P-0249, P-0539, P-0627 

Joseph J . . . . . . . . . . . . . . . . .P-0218 Karamifar K . . . . . . . . . . . . . .P-0547 Khelifa E . . . . . . . . . . . . . . . . .P-0250 

Joseph L . . . . . . . . . . . . . . . . .P-0627 Kardos G . . . . . . . . . .P-0089, P-0428 Khlif M . . . . . . . . . . . .P-0326, P-0550 

Jouault T . . .O-0004, P-0724, P-0725 Kasai T . . . . . . . . . . . . . . . . . .P-0090 Khodaverdi M . . . . . . . . . . . . .P-0450 

Jouy N . . . . . . . . . . . . . . . . . .P-0724 Kashman Y . . . . . . . . . . . . . . .P-0143 Khorramizadeh M . . . .P-0165, P-0582 

Judith F . . . . . . . . . . . . . . . . .O-0023 Katarzyna W . . . . . . . .P-0111, P-0527 Khosravi A . . . . . . . . . . . . . . .P-0616 

Khosravi A . . . . . . . . .P-0029, P-0313 


Kibbler C . . . . . . . . . . . . . . . .P-0362 

Kiehl M . . . . . . . . . . . . . . . . . .P-0391 

Kilic Z . . . . . . . . . . . . . . . . . . .P-0252 

Kim H . . . . . . . . . . . . . . . . . .O-0032 

Kim H . . . . . . . . . . . . . . . . . . .P-0419 

Kim J . . . . . . . . . . . . . . . . . . .P-0348 

Kim K . . . . . . . . . . . . . . . . . . .P-0640 

Kim S . . . . . . . . . . . . . . . . . .O-0042 

Kim S . . . . . . . . . . . . . . . . . . .P-0580 

Kim Y . . . . . . . . . . . . . . . . . . .P-0419 

Kimura M . . . . . . . . . . . . . . . .P-0723 

Kindo A . . . . . . . . . . . . . . . . . .P-0251 

Kiraz N . . . . . . . . . . . . . . . . . .P-0252 

Kirel B . . . . . . . . . . . . . . . . . . .P-0252 

Kirkpatrick W . . . . . . .P-0028, P-0395 

Kleinert R . . . . . . . . . . . . . . . .P-0200 

Klimko N . . . . . . . . . . . . . . . . .P-0215 

Kneipp l . . . . . . . . . . . . . . . . .P-0253 

Kneipp L . . . . . . . . . . . . . . . . .P-0296 

Knoke M . . . . . . . . . . . . . . . . .P-0053 

Kobayashi H . . . . . . . .P-0283, P-0735 

Kobayashi I . . . . . . . . . . . . . . .P-0411 

Kodousek R . . . . . . . . . . . . . . .P-0232 

Kofronova O . . . . . . . . . . . . . .P-0437 

Koga H . . . . . . . . . . . . . . . . . .P-0090 

Kohno S . . . .P-0277, P-0392, P-0397 

Kolb Z . . . . . . . . . . . . . . . . . . .P-0215 

Kondori N . . . . . . . . . . . . . . . .P-0396 

Konieczna A . . . . . . . . . . . . . .P-0344 

Kono A . . . . . . . . . . . . . . . . .O-0037 

Kontoyiannis D . . . . . . . . . . . .P-0060 

Kopp E . . . . . . . . . . . . . . . . . .P-0307 

Kordbacheh P P-0165, P-0572, P-0709 

Koslu A . . . . . . . . . . . . . . . . . .P-0244 

Kosravi A . . . . . . . . . . . . . . . .P-0542 

Kotevska V . . . . . . . . . . . . . . .P-0276 

Kottilil S . . . . . . . . . . . . . . . . .O-0032 

Kouloumvaki A . . . . . .P-0165, P-0572 

Kowshik T . . . . . . . . . .P-0371, P-0539 

Kozovska Z . . . . . . . . . . . . . . .P-0691 

Krappmann S . . . . . .O-0042, P-0694 

Krasowska A . . . . . . . . . . . . . .P-0091 

Kristensen J . . . . . . . . . . . . . . .P-0218 

Kucharikova S . . . . . . . . . . . . .P-0592 

Kuchler K . . . . . . . . . . . . . . . .P-0105 

Kugler A . . . . . . . . . . . . . . . .O-0011 

Kuijper E . . . . . . . . . .O-0027, P-0358 

Kullberg B . . . . . . . . . . . . . . . .P-0597 

Kuramae E . . . . . . . . . . . . . . .P-0196 

Kurihara S . . . . . . . . .P-0277, P-0397 

Kurtzman C . . . . . . . . . . . . . . .P-0266 

Kustimur S . . . . . . . . .P-0398, P-0449 

Kwak T . . . . . . . . . . . . . . . . . .P-0640 

Kwon-Chung J . . . . . . . . . . . .P-0637 

Königsdorfer A . . . . . . . . . . . .P-0656 

L’Ollivier C . . . . . . . . . . . . . . .P-0475 

L. Batista W . . . . . . . . . . . . . . .P-0305 

La Sorda M . . . . . . . . . . . . . . .P-0130 

La Valle R . . .O-0045, P-0359 P-0596 

Labidi H . . . . . . . . . . . . . . . . .P-0494 

Labrousse H . . . . . . . . . . . . . .P-0303 

Lachance C . . . . . . . . . . . . . . .P-0384 

Lackner E . . . . . . . . . . . . . . . .P-0537 

Lacroix C . . . . . . . . .O-0007, P-0088, 

. . . . . . . . . . . . . . . . .P-0501, P-0552 

Lacube P . . . . . . . . . .P-0109, P-0356 

Lafon I . . . . . . . . . . . . . . . . . .P-0475 

Lagneau P . . . . . . . . . . . . . . . .P-0019 

Lagrou K . . . . . . . . . . . . . . . . .P-0454 

Lahat Y . . . . . . . . . . . . . . . . . .P-0129 

Lakhal S . . . . . . . . . . . . . . . . .P-0246 

Lakner A . . . . . . . . . . . . . . . . .P-0399 

Lamias M . . . . . . . . . . . . . . . .P-0533 

Lamping E . . . . . . . . .P-0114, P-0115 

Lan C . . . . . . . . . . . . . . . . . .O-0033 

Landaeta M . . . . . . . . . . . . . . .P-0514 

Lande R . . . . . . . . . . . . . . . . .P-0599 

Landry L . . . . . . . . . . . . . . . . .P-0360 

Lapière C . . . . . . . . . . . . . . .O-0005 

Larcher G . .O-0044, P-0153, P-0154 

Larriba Calle G . . . . . . . . . . . .P-0654 

Larsen R . . . . . . . . . . .P-0254, P-0255 

Lasala M . . . . . . . . . .P-0190, P-0209 

Latercia Tranches Dias A . . . . .P-0082 

Lattes R . . . . . . . . . . . . . . . . . .P-0209 

Lau A . . . . . . . . . . . . . . . . . . .P-0400 

Laudinet N . . . . . . . . .P-0294, P-0303 

Launay O . . . . . . . . . . . . . . . .P-0207 

Laurent F . . . . . . . . . . . . . . . . .P-0346 

Laverdière M . . . . . . . . . . . . . .P-0145 

Lavrijsen A . . . . . . . . . . . . . . .P-0358 

Lazari P . . . . . . . . . . . . . . . . . .P-0048 

Lazera M . . . . . . . . . .P-0451, P-0738 

Lazo Ch. J . . . . . . . . .P-0256, P-0257 

Lazo S. R . . . . . . . . . .P-0256, P-0257 

Lazzell A . . . . . . . . . . . . . . . . .P-0617 

Laín A . . . . . . . . . . . . . . . . . . .P-0401 

Le Loc’h G . . . . . . . . . . . . . . .P-0020 

Lebeau B . . . . . . . . . .P-0237, P-0380 

Ledezma E . . . . . . . . .P-0092, P-0102 

Lee J . . . . . . . . . . . . . . . . . . .O-0011 

Lee M . . . . . . . . . . . . .P-0258, P-0419 


17

Lee Y . . . . . . . . . . . . . . . . . . .P-0502 

Lefrancois C . . . . . . . .P-0357, P-0574 

LeMaile-Williams M . . . . . . . . .P-0462 

Lemen W . . . . . . . . . . . . . . . .P-0609 

Lemes R . . . . . . . . . . .P-0066, P-0644 

Lemieux C . . . . . . . . . . . . . . . .P-0259 

Lemieux S . . . . . . . . . . . . . . .O-0016 

LeMonte A . . . . . . . . . . . . . . . .P-0431 

Lempicki R . . . . . . . . . . . . . . .O-0032 

Lempitski S . . . . . . . . . . . . . . .P-0381 

Lemus E. D . . . . . . . . . . . . . . .P-0092 

Lemus Espinoza D . . . . . . . . . .P-0102 

Leon I . . . . . . . . . . . . . . . . . .O-0044 

Leray E . . . . . . . . . . . . . . . . . .P-0210 

Lessa R . . . . . . . . . . . .P-0271, P-0272 

Lesuisse E . . . . . . . . . . . . . . . .P-0693 

Lethuillier A . . . . . . . . . . . . . .O-0010 

Levitz S . . . . . . . . . . . . . . . . . .P-0623 

Levy A . . . . . . . . . . . . . . . . . .O-0007 

Lewis R . . . . . . . . . . . . . . . . . .P-0060 

Li C . . . . . . . . . . . . . .P-0262, P-0403 

Li D . . . . . . . . . . . . . .P-0260, P-0261 

Li L . . . . . . . . . . . . . . . . . . . . .P-0349 

Li M . . . . . . . . . . . . . . . . . . . .P-0706 

Li R . . . . . . . .P-0067, P-0094, P-0263 

Li S . . . . . . . . . . . . . . . . . . . . .P-0504 

Li X . . . . . . . . . . . . . .P-0402, P-0706 

Li Z . . . . . . . . . . . . . . . . . . . .O-0035 

Lia R . . . . . . . . . . . . . . . . . . . .P-0006 

Liames S . . . . . . . . . . . . . . . . .P-0462 

Libersa C . . . . . . . . . . . . . . . .P-0734 

Licea A . . . . . . . . . . . . . . . . .O-0012 

Licinio P . . . . . . . . . . . . . . . . .P-0742 

Licznar P . . . . . . . . . .P-0357, P-0574 

Lidia P . . . . . . . . . . . . . . . . . .P-0683 

Lim S . . . . . . . . . . . . . . . . . . .P-0502 

Lima Barros M . . . . . . . . . . . . .P-0364 

Linas M . . . . . . . . . . . . . . . . . .P-0093 

Lindquist S . . . . . . . . . . . . . . .P-0720 

Lindsley M . . . . . . . . . . . . . . . .P-0422 

Linnemann D . . . . . . . . . . . . .P-0132 

Linton C . . . . . . . . . . .P-0456, P-0741 

Litvintseva A . . . . . . . . . . . . . .P-0503 

Litvintseva A . . . . . . . . . . . . . .P-0024 

Liu H . . . . . . . . . . . . . . . . . . . .P-0596 

Liu T . . . . . . . . . . . . . .P-0579, P-0587 

Liu W P-0083, P-0094, P-0402, P-0706 

Liu Y . . . . . . . . . . . . . . . . . . . .P-0706 

Lizard S . . . . . . . . . . . . . . . . . .P-0475 

Llebaría R . . . . . . . . . . . . . . . .P-0404 

Llovo J . . . . . . . . . . . .P-0404, P-0405 

Lmimouni B . . . . . . . . . . . . . . .P-0489 

Lo H . . . . . . .P-0504, P-0571, P-0660 

Lobo I . . . . . . . . . . . . . . . . . . .P-0264 

Lock L . . . . . . . . . . . .P-0138, P-0711 

Lohoue J . . . . . . . . . . . . . . . . .P-0394 

Lopardo H . . . . . . . . . . . . . . .P-0315 

Lopes A . . . . . . . . . . .P-0271, P-0272 

Lopes M . . . .P-0284, P-0505, P-0327 

Lopes V . . . . .P-0095, P-0221, P-0264 

Lopes-Bezerra L . . .O-0019, O-0044, 

. . . . . . . . . . . .P-191, P-0287, P-0364 

Lopez Correia da Silva L . . . . .P-0022 

Lopez Rodas V . . . . . .P-0078, P-0714 

Lopez-Oviedo E . . . . . . . . . . . .P-0096 

Lopez-Ribot J .P-0587, P-0617, P-0684 

Lorenzo J . . . . . . . . . . . . . . . .P-0636 

Loreto É . . . . . . . . . . . . . . . . .P-0406 

Lortholary O .P-0097, P-0137, P-0207 

Losson B . . . . . . . . .O-0001, O-0005 

Lott T . . . . . . . . . . . . . . . . . . .P-0462 

Louie A . . . . . . . . . . . . . . . . . .P-0083 

Loulergue P . . . . . . . . . . . . . . .P-0097 

Loungnarath V . . . . . . . . . . . .P-0293 

Loureiro y Penha C . . . . . . . . .P-0364 

Louvison M . . . . . . . . . . . . . . .P-0661 

Lucas R . . . . . . . . . . . . . . . . . .P-0509 

Lucchetti A . . . . . . . . . . . . . . . .P-0464 

Ludovic D . . . . . . . . . . . . . . . .P-0171 

Ludwig K . . . . . . . . . . . . . . . . .P-0053 

Lupetti A . . . . . . . . . . . . . . . . .P-0087 

Lv G . . . . . . . . . . . . . . . . . . . .P-0402 

Lyman C . . . . . . . . . . . . . . . .O-0032 

Lyon G . . . . .P-0506, P-0507, P-0559 

Lyon J . . . . . . . . . . . .P-0064, P-0065 

Lyonnet D . . . . . . . . . . . . . . . .P-0340 

Lysiane M . . . . . . . . . . . . . . . .P-0513 

Léchenne B . . . . . . . . . . . . . . .P-0584 

Léon A . . . . . . . . . . . . . . . . . .P-0427 

Lévèque C . . . . . . . . . . . . . . . .P-0207 

López E . . . . . . . . . . . . . . . . . .P-0104 

López M . . . . . . . . . . . . . . . . .P-0426 

López-Martínez R . . . . . . . . . . .P-0265 

López-Oviedo E . . . . . . . . . . . .P-0103 

López-Romero E . . . . .P-0098, P-0671 

L. T. Dias A . . . . . . . . . . . . . . .P-0439 

Lütticken R . . . . . . . . . . . . . . . .P-0418 

M. de Siqueira A . . . . . . . . . . .P-0439 

M. Santurio J . . . . . . .P-0021, P-0626 

Maares J . . .P-0136, P-0318, P-0319, 

. . . . . . . . . . . . . . . . .P-0320, P-0321 


MacCallum D . . . . . . . . . . . . .P-0662 

Macedo W . . . . . . . . . . . . . . .P-0541 

Mach T . . . . . . . . . . . . . . . . .O-0021 

Machova E . . . . . . . . . . . . . . .P-0592 

Macrì C . . . . . . . . . . . . . . . . .O-0045 

Macura A . . . . . . . . . .P-0099, P-0508 

Madani M . . . . . . . . . . . . . . . .P-0491 

Madeline B . . . . . . . . .P-0254, P-0255 

Madopoulou F . . . . . . . . . . . . .P-0048 

Madrid H . . . . . . . . . . . . . . . .P-0423 

Maes E . . . . .P-0702, P-0725, P-0726 

Maffei C . . . . . . . . . . . . . . . . .P-0100 

Maffei C . . . . . . . . . . . . . . . . .P-0509 

Magaldi S . . .P-0375, P-0388, P-0514 

Magee P . . . . . . . . . . . . . . . .O-0035 

Makni F . . . . . . . . . . .P-0208, P-0326, 

. . . . . . . . . . .P-0523, P-0550, P-0551 

Makri A . . . . . . . . . . . . . . . . . .P-0049 

Malanski L . . . . . . . . . . . . . . .P-0031 

Malcok H . . . . . . . . . . . . . . . .P-0664 

Maldonado A . . . . . . . . . . . . .P-0128 

Maleszka R . . . . . . . . . . . . . . .P-0302 

Maliji G . . . . . . . . . . . . . . . . .P-0101 

Malik R . . . . . . . . . . . . . . . . . .P-0400 

Mallaret M . . . . . . . . . . . . . . .P-0303 

Mallatova N . . . . . . . .P-0241, P-0297 

Mallet J . . . . . . . . . . . . . . . . . .P-0429 

Mallie M . . . .P-0054, P-0268, P-0369 

Maltusch C . . . . . . . . . . . . . . .P-0750 

Mandal P . . . . . . . . . . . . . . . .P-0267 

Markham A . . . . . . . .P-0564, P-0565 

Marot A . . . . . . . . . . . . . . . . .P-0357 

Marot-Leblond A . . . .P-0407, P-0713, 

. . . . . . . . . . . . . . . . . . . . . . .P-0729 

Marques Gomes M . . . . . . . . .P-0311 

Marr K . . . . . . . . . . . .P-0588, P-0600 

Marreckchi S . . . . . . .P-0523, P-0551 

Marriott D . . . . . . . .O-0015, P-0068, 

. . . . . . . . . . . . . . . . .P-0535, P-0558 

Martchenko M . . . . . . . . . . . .O-0035 

Martel N . . . . . . . . . . . . . . . .O-0016 

Martellos S . . . . . . . . . . . . . . .P-0754 

Marth E . . . . . . . . . . . . . . . . . .P-0200 

Martin P . . . . . . . . . . . . . . . . .P-0400 

Martin de la Escalera C . . . . .P-0103, 

. . . . . . . . . . . . . . . . . . . . . . .P-0104 

Mandarino G . . . . . . . . . . . . .P-0359 

Mageel B . . . . . . . . . . . . . . . .O-0035 

Martin Martin de la Escalera C .P-0096 

Maneiro E . . . . . . . . . . . . . . . .P-0714 

Maghsood A . . . . . . . . . . . . . .P-0487 

Martin-Mazuelos E . . .P-0096, P-0103, 

Manfait M . . . . . . . . . . . . . . . .P-0480 

Magill S . . . . . . . . . . . . . . . . .P-0266 

. . . . . . . . . . . . . . . . . . . . . . .P-0368 

Maniscalchi B. M . . . . . . . . . . .P-0092 

Magnaval J . . . . . . . . . . . . . . .P-0093 

Martine G . . . . . . . . .P-0175, P-0471 

Maniscalchi Badaoui M . . . . . .P-0102 

Magne D . . . . . . . . . . . . . . . .P-0510 

Martinez C . . . . . . . . . . . . . . .P-0486 

Manzano-Gayosso P . . . . . . . .P-0265 

Magni C . . . . . . . . . . . . . . . . .P-0172 

Martinez J . . . . . . . . . . . . . . . .P-0684 

Marasas W . . . . . . . . .P-0156, P-0487 

Mahagoub E . . . . . . . . . . . . . .P-0281 

Martinez M . . . . . . . . . . . . . . .P-0061 

Marchand A . . . . . . . .P-0003, P-0004 

Mahdavi Omran S . . . . . . . . . .P-0101 

Martinez W . . . . . . . . . . . . . . .P-0388 

Margueritte G . . . . . . . . . . . . .P-0268 

Mahfoudh M . . . . . . . . . . . . . .P-0208 

Martinez Godoy P . . . . . . . . . .P-0651 

Marie M . . . . . . . . . . . . . . . . .P-0471 

Mahgoub E . . . . . . . . . . . . . . .P-0511 

Martinez Vivot M . . . . . . . . . . .P-0016 

Marie Denise L . . . . . . . . . . .O-0023 

Mahmoodi Rad M . . . . . . . . . .P-0709 

Martins C . . . . . . . . . . . . . . . .P-0343 

Marie-Hélène R . . . . . . . . . . . .P-0718 

Mahmoud E . . . . . . . . . . . . . .P-0499 

Martins E . . . . . . . . . . . . . . . . .P-0343 

Marie-Pierre B P-0471, P-0512, P-0513 

Maidan M . . . . . . . . . . . . . . . .P-0648 

Martins M . . . . . . . . . .P-0203, P-0420 

Marie-Reine M . . . . . .P-0512, P-0513 

Majima T . . . .P-0116, P-0149, P-0666 

Martos A . . . .P-0096, P-0103, P-0104 

Marimon Pico R . . . . . . . . . . . .P-0753 

Majoros L . . . . . . . . . .P-0089, P-0428 

Martín-Mazuelos E . . . . . . . . . .P-0104 

Marin V . . . . . . . . . . . . . . . . . .P-0273 

Makhjiri S . . . . . . . . . . . . . . . .P-0029 

Martínez C . . .P-0057, P-0653, P-0733 

Marinho de Medeiros Camin P . .P-0022 

Maki K . . . . . . . . . . . . . . . . . .P-0114 

Martínez K . . . . . . . . . . . . . . . .P-0470 

Marino C . . . . . . . . . . . . . . . .P-0197 

Makimura K .P-0055, P-0240, P-0336, 

Martínez-Esparza M . . . . . . . . .P-0724 

. . . .P-0393, P-0412, P-0413, P-0414, Marion R . . . . . . . . . . . . . . . . .P-0665 

. . . . . . . . . . .P-0518 P-0663, P-0707 

Mary C . . . . . . . . . . . .P-0274, P-0299 


19

Massai L . . . . . . . . . . . . . . . . .P-0224 

Masui S . . . . . . . . . . .P-0149, P-0666 

Mata S . . . . . . . . . . . . . . . . . .P-0477 

Mata-Essayag S . . . . .P-0375, P-0388, 

. . . . . . . . . . . . . . . . . . . . . . .P-0514 

Matevish J . . . . . . . . . . . . . . . .P-0079 

Matos J . . . . . . . . . . . . . . . . . .P-0646 

Matos Santiago Reis A . . . . . . .P-0022 

Matsumoto F . . . . . . . . . . . . . .P-0568 

Matsumoto M . . . . . . . . . . . . .P-0222 

Matsumoto T . . . . . . . . . . . . . .P-0712 

Matthey T . . . . . . . . . . . . . . . .P-0227 

Mattsby-Baltzer I . . . . . . . . . . .P-0396 

Mavor A . . . . . . . . . . . . . . . . .P-0667 

May G . . . . . . . . . . . . . . . . . .P-0580 

Mayr A . . . . . . . . . . . .P-0408, P-0440 

Mazars E . . . . . . . . . . . . . . . . .P-0140 

McCarthy D . . . . . . . . . . . . . . .P-0147 

McCarthy K . . . . . . . . . . . . . . .P-0546 

McEween J . . . . . . . . . . . . . . .P-0689 

McNicholas P . . . . . . . . . . . . .P-0089 

Mehrabani D . . . . . . . . . . . . . .P-0032 

Meinberg R . . . . . . . . . . . . . . .P-0463 

Meissner N . . . . . . . . . . . . . . .P-0605 

Melkusova S . . . . . . . .P-0105, P-0592 

Mellado E . . . . . . . . . . . . . . . .P-0628 

Melo A . . . . . . . . . . . .P-0446, P-0515 

Melo T . . . . . . . . . . . .P-0271, P-0272 

Mena C . . . . . . . . . . . . . . . . .P-0195 

Mencl K . . . . . . . . . . . . . . . . .P-0409 

Mender T . . . . . . . . . . . . . . . .P-0472 

Mendes-Giannini M .O-0037, P-0180, 

. . . . . . . . . . .P-0216, P-0222, P-0279 

Mendoza L . . . . . . . . .P-0615, P-0685 

Mendoza M . . . . . . . .P-0197, P-0212, 

. . . .P-0269, P-0270, P-0485, P-0516 

Meneau I . . . . . . . . . . . . . . . .P-0668 

Menezes E . . . . . . . . .P-0271, P-0272 

Menon V . . . . . . . . . . . . . . . . .P-0669 

Mepham S . . . . . . . . . . . . . . .P-0123 

Mercadillo M . . . . . . .P-0074, P-0410 

Merkerová M . . . . . . . . . . . . . .P-0647 

Merz W . . . . . . . . . . . . . . . . . .P-0266 

Mesa A . . . . . . . . . . . .P-0058, P-0106 

Mesa Arango A . . . . .P-0057, P-0107 

Mesplet M . . . . . . . . .P-0015, P-0016 

Meyer W . . . . . . . . . . . . . . . . .P-0674 

Meyer-Fernandes J . . .P-0253, P-0296 

Meyer-Wentrup F . . . . . . . . . . .P-0597 

Meziou J . . . . . . . . . . . . . . . . .P-0523 

Mezlini S . . . . . . . . . .P-0250, P-0453 

Michallet M . . . . . . . . . . . . . . .P-0524 

Michaux J . . . . . . . . . . . . . . .O-0002 

Michel G . . . . . . . . . .P-0275, P-0301 

Michel-Nguyen A . . . .P-0273, P-0274, 

. . . . . . . . . . .P-0275, P-0300, P-0489 

Michel-Ngyuen A . . . . . . . . . . .P-0299 

Michev V . . . . . . . . . . . . . . . . .P-0614 

Mignon B . . . . . . . .O-0001, O-0005 

Miguel A . . . . . . . . . . . . . . . . .P-0157 

Migueís J . . . . . . . . . . . . . . . . .P-0311 

Mikaeili A . . . . . . . . . .P-0023, P-0517 

Mikami T . . . . . . . . . . . . . . . . .P-0712 

Mikawa T . . . . . . . . . . . . . . . .P-0411 

Milenkovik Z . . . . . . . . . . . . . .P-0276 

Milewski S . . . . . . . . . . . . . . . .P-0098 

Milioni A . . . . . . . . . . . . . . . . .P-0049 

Mille C . . . . .P-0295, P-0702, P-0725, 

. . . . . . . . . . . . . . . . . . . . . . .P-0726 

Miranda E . . . . . . . . . . . . . . . .P-0279 

Mircevska G . . . . . . . . . . . . . .P-0276 

Mirhendi H . . . . . . . .P-0412, P-0413, 

. . . .P-0414, P-0518, P-0645, P-0687 

Mirhendi S . . . .P-0525, P-572, P-0676 

Mirzabalaeva A . . . . .P-0215, P-0618 

Miszti C . . . . . . . . . . . . . . . . . .P-0428 

Mitchell T . . . . . . . . . .P-0024, P-0503 

Mitroussia-Ziouva A . .P-0048, P-0332 

Miura N . . . . . . . . . . . . . . . . .P-0612 

Miyaji M . . . . . . . . . . . . . . . . .P-0526 

Miyazaki Y . . .P-0277, P-0392, P-0397 

Mnichowska-Polanowska M . . .P-0108 

Mochizuki T . . . . . . .O-0014, P-0173, 

. . . .P-0174, P-0325, P-0447, P-0496, 

. . . . . . . . . . .P-0519, P-0570, P-0752 

Mogensen E . . . . . . . . . . . . . .P-0670 

Mohamadi G . . . . . . . . . . . . .P-0490 

Mohan H . . . . . . . . . . . . . . . .P-0206 

Mohseni S . . . . . . . . . . . . . . . .P-0450 

Mojal Garcia S . . . . . . . . . . . .P-0385 

Mokaddas E . . . . . . . .P-0249, P-0627 

Mokni M . . . .P-0217, P-0234, P-0250, 

. . . . . . . . . . . . . . . . .P-0453, P-0494 

Mokracek A . . . . . . . . . . . . . . .P-0297 

Moleta Colodel E . . . . . . . . . . .P-0008 

Molina A . . . . . . . . . .P-0074, P-0410 

Molina D . . . . . . . . . .P-0028, P-0110 

Moncef B . . . . . . . . . . . . . . . .P-0278 

Monk B . . . . . . . . . . .P-0114, P-0115 

Monod M . . .P-0584, P-0727, P-0748 

Montagna M . . . . . . .P-0005, P-0721 

Monteagudo C . . . . . . . . . . . .P-0617 

Monteavaro C . . . . . . . . . . . . .P-0039 

Monteiro da Silva J . . .P-0216, P-0279 


Montharu J . . . . . . . . . . . . . . .P-0205 

Montiel J . . . .P-0058, P-0106, P-0107 

Moon S . . . . . . . . . . . . . . . . . .P-0640 

Moore C . . . . . . . . . . . . . . . . .P-0146 

Mora Amador Junior D . . . . . .P-0520 

Mora-Montes H . . . . .P-0671, P-0736 

Morace G . . . . . . . . . .P-0424, P-0697 

Moragues M . . . . . . . .P-0386, P-0401 

Morais F . . . . . . . . . . .P-0672, P-0689 

Morales B . . . . . . . . . .P-0451, P-0738 

Morand S . . . . . . . . . . . . . . .O-0002 

Morandi V . . . . . . . . . . . . . . .O-0019 

Moranova Z . . . . . . . . . . . . . .P-0728 

Moras E . . . . . . . . . . . . . . . . .P-0016 

Mordon S . . . . . . . . . . . . . . .O-0004 

Moreira D . . . . . . . . .P-0292, P-0568 

Moreira F . . . . . . . . . .P-0064, P-0065 

Moreira S . . . . . . . . . . . . . . . .P-0222 

Morel P . . . .O-0007, P-0501, P-0552 

Moreno B . . . . . . . . . . . . . . . .P-0128 

Moreno J . . . . . . . . . .P-0280, P-0355 

Moretti A . . . . . . . . . .P-0007, P-0025, 

. . . . . . . . . . . . . . . . .P-0455, P-0521 

Morgan J . . . . . . . . . . . . . . . .P-0422 

Mori T . . . . . . . . . . . . . . . . . . .P-0560 

Morin O . . . . . . . . . . . . . . . . .P-0370 

Morschhäuser J . . . . . . . . . . .O-0003 

Mosavi S . . . . . . . . . . . . . . . . .P-0101 

Mostafaei A . . . . . . . . . . . . . . .P-0023 

Mostagnei S . . . . . . . . . . . . . .P-0554 

Motazedian M . . . . . .P-0032, P-0289 

Motoi M . . . . . . . . . . . . . . . . .P-0606 

Moukhlis R . . . . . . . . . . . . . . .P-0109 

Mourad B . . . . . . . . . .P-0177, P-0178 

Mourad M . . . . . . . . . . . . . . . .P-0310 

Mourão Roque A . . . . . . . . . . .P-0619 

Moya M . . . . . . . . . . .P-0026, P-0477 

Mu, A . . . . . . . . . . . . . . . . . . .P-0015 

Muckensturm B . . . . . . . . . . . .P-0295 

Muellejans B . . . . . . . . . . . . . .P-0227 

Mugasia O . . . . . . . . . . . . . . .P-0055 

Mujica M . . . .P-0075, P-0124, P-0650 

Mukherjee S . . . . . . . . . . . . . .P-0632 

Munro C . . . . . . . . . . . . . . . . .P-0662 

Murata Y . . . . . . . . . .P-0027, P-0036 

Murayama S . . . . . . . . . . . . . .P-0581 

Murciano C . . . . . . . . . . . . . . .P-0603 

Murciano F . . . . . . . . . . . . . . .P-0629 

Murgich J . . . . . . . . . . . . . . . .P-0128 

Murgui A . . . . . . . . . . . . . . . . .P-0684 

Murillo L . . . . . . . . . . . . . . . .O-0033 

Murzyn A . . . . . . . . . . . . . . . .P-0091 

musa a . . . . . . . . . . . . . . . . . .P-0281 

Muñoz A . . . . . . . . . .P-0404, P-0405 

Méndez-Tovar L . . . . . . . . . . . .P-0265 

Méndez-Álvarez S . . . . . . . . . .P-0354 

Mônica de Oliveira R . . . . . . . .P-0520 

Mörz H . . . . . . . . . . . . . . . . . .P-0391 

Mügge C . . . . . . . . . . . . . . . .P-0522 

Mühlschlegel F . . . . . . . . . . . .P-0670 

Müller F . . . . . . . . . . .P-0141, P-0323 

N K . . . . . . . . . . . . . . . . . . . . .P-0251 

N Miura N . . . . . . . . . . . . . . .P-0606 

Na S . . . . . . . . . . . . .P-0338, P-0562 

Nabuco de Abreu A . . . . . . . . .P-0022 

Nack S . . . . . . . . . . . . . . . . . .P-0427 

Nagao K . . . . . . . . . .P-0245, P-0317 

Nagasaka S . . . . . . . . . . . . . .P-0149 

Naidu J . . . . . . . . . . .P-0282, P-0331 

Nail-Billaud S .P-0407, P-0713, P-0729 

Najvar L . . . .P-0028, P-0110, P-0395 

Nakamura K .P-0658, P-0673, P-0704 

Nakanishi T . . . . . . . . . . . . . . .P-0283 

Namaki A . . . . . . . . . .P-0213, P-0645 

Nancy C . . . . . . . . . . . . . . . . .P-0254 

Nanjoh Y . . . . . . . . . . . . . . . .P-0090 

Nantel A . . . . . . . . . . . . . . . .O-0035 

Naoufel B . . . . . . . . . . . . . . . .P-0310 

Nascimento T . . . . . . . . . . . . .P-0284 

Nasrollahi A . . . . . . .P-0018, P-0490, 

. . . . . . . . . . . . . . . . .P-0491, P-0492 

Nasrollahi Omran A . . . . . . . .P-0029 

Natanzian Ghahfarokhi M . . . .P-0131 

Nathalie C . . . . . . . . . . . . . . .P-0073 

Nather K . . . . . . . . . . . . . . . . .P-0662 

Navarro I . . . . . . . . . . . . . . . .P-0031 

Navas T . . . . . . . . . . . . . . . . .P-0468 

Navon A . . . . . . . . . . . . . . . . .P-0139 

Nawrot U . . . . . . . . . .P-0111, P-0527 

Nayak N . . . . . . . . . . . . . . . . .P-0112 

Nayeri F . . . . . . . . . . . . . . . . .P-0572 

Nedved J . . . . . . . . . .P-0415, P-0437 

Nefzi F . . . . . . . . . . . . . . . . . .P-0243 

Negishi Y . . . . . . . . . . . . . . . .P-0581 

Negroni R . . . . . . . . . . . . . . . .P-0341 

Neji S . . . . . . . . . . . . . . . . . . .P-0523 

Nenoff P . . . .P-0052, P-0108, P-0522 

Netea M . . . . . . . . . . . . . . . . .P-0597 

Neto T . . . . . . . . . . . . . . . . . .P-0505 

Neves Torres L . . . . . . . . . . . . .P-0022 

Newell V . . . . . . . . . . . . . . . . .P-0048 

Newman S . . . . . . . . . . . . . . .P-0609 


21

Newport G . . . . . . . . . . . . . .O-0033 

Neyra E . . . . .P-0046, P-0113, P-0464 

Ngamskulrungroj P . . .P-0543, P-0674 

Nguewou A . . . . . . . . . . . . . . .P-0285 

Nguyen C . . . . . . . . . . . . . . . .P-0580 

Nguyen Q . . . . . . . .O-0015, P-0068, 

. . . . . . . . . . .P-0122, P-0535, P-0558 

Nibbering P . . . . . . . . . . . . . . .P-0087 

Nicolle M . . . . . . . . . . . . . . . .P-0524 

Nieguitsila A . . . . . . . . . . . . . .P-0675 

Nielsen K . . . . . . . . . . . . . . . .O-0043 

Nierman W . . . . . . . . . . . . . . .P-0580 

Niggemann S . . . . . . . . . . . . .P-0391 

Niimi K . . . . . . . . . . . .P-0114, P-0115 

Niimi M . . . . .P-0114, P-0115, P-0581 

Nikaeen M . . .P-0518, P-0525, P-0676 

Niki Y . . . . . . . . . . . . . . . . . . .P-0434 

Nikookhah F . . . . . . . . . . . . . .P-0030 

Nimubona S . . . . . . . . . . . . . .P-0210 

Nino-Vega G .P-0677, P-0682, P-0690 

Nishida N . . . . . . . . .P-0116, P-0149 

Nishikaku A . . . . . . . . . . . . . .P-0610 

Nishikawa A . . . . . . .P-0151, P-0337, 

. . . . . . . . . . . . . . . . .P-0387, P-0611 

Nishikawa T . . . . . . . . . . . . . .P-0160 

Nishimura K . . . . . . .P-0027, P-0036, 

. . . . . . . . . . . . . . . . .P-0526, P-0755 

Nishiyama Y . . . . . . . .O-029, P-0117 

Niño-Vega G .P-0642, P-0678, P-0698 

Njikam N . . . . . . . . . . . . . . . .P-0394 

Noacco G . . . . . . . . . . . . . . . .P-0421 

Nobel-Wang J . . . . . .P-0462, P-0463 

Nobrega F . . . . . . . . . . . . . . .P-0577 

Nobrega M . . . . . . . . . . . . . . .P-0577 

Nolard N . . . . . . . . . . . . . . . .P-0383 

Nolte F . . . . . . . . . . . . . . . . . .P-0506 

Noman R . . . . . . . . . . . . . . . .P-0316 

Nombela C . . . . . . .O-0039, O-0041 

Noorbakhsh F . . . . . . . . . . . . .P-0582 

Nordenberg D . . . . . . . . . . . . .P-0533 

Noronha G . . . . . . . . . . . . . . .P-0460 

Nosanchuk J . . . . . . . . . . . . . .P-0267 

Noureddine E . . . . . . . . . . . . .P-0177 

Nourian A . . . . . . . . . . . . . . . .P-0450 

Novotny R . . . . . . . . . . . . . . . .P-0728 

Nowak P . . . . . . . . . . . . . . . . .P-0150 

Nowicka J . . . . . . . . .P-0111, P-0527 

Nowicki R . . .P-0350, P-0528, P-0573 

Nozawa A . . . . . . . . . . . . . . . .P-0116 

Nshiyama Y . . . . . . . . . . . . . .P-0085 

Nunes L . . . . . . . . . . . . . . . . .P-0577 

Nusgens B . . . . . . . . . . . . . . .O-0005 

Nweze E . . . . . . . . . . . . . . . . .P-0286 

Nóbrega F . . . . . . . . .P-0634, P-0672 

Nóbrega M . . . . . . . . . . . . . . .P-0634 

Núñez del arco A . . . . . . . . . .P-0035 

O’Connor J . . . . . . . . . . . . . . .P-0603 

O’Donnell K . . . . . . . . . . . . . .P-0497 

O’Mahony R . . . . . . . . . . . . . .P-0596 

O. Grimalt J . . . . . . . . . . . . . .P-0628 

Obando D . . . . . . . . . . . . . . .P-0118 

Ocampo-Candiani J . . . . . . . .P-0567 

Ochiai E . . . . . . . . . . . . . . . . .P-0730 

Ochoa D . . . . . . . . . . . . . . . .O-0017 

Odds F . . . . . . . . . . . . . . . . . .P-0662 

Ogasawara A . . . . . . . . . . . . .P-0712 

Ogawa H . . . . . . . . .P-0159, P-0160, 

. . . . . . . . . . .P-0238, P-0500, P-0555 

Ogbonnaya L . . . . . . . . . . . . .P-0286 

Ogreden S . . . . . . . . . . . . . . .P-0245 

Oguri T . . . . . . . . . . . . . . . . . .P-0560 

Oh D . . . . . . . . . . . . . . . . . . .P-0348 

Ohno N . . . . . . . . . . .P-0606, P-0612 

Okabayashi K . . . . . . . . . . . . .P-0679 

OKafor J . . . . . . . . . . . . . . . . .P-0286 

Okan T . . . . . . . . . . . . . . . . . .P-0443 

Okungbowa F . . . . . .P-0119, P-0529 

Okungbowa M . . . . . .P-0119, P-0529 

Olaizola C . . .P-0375, P-0388, P-0514 

Olawuyi O . . . . . . . . . . . . . . .P-0530 

Olawuyi T . . . . . . . . . . . . . . . .P-0530 

Oleksiak D . . . . . . . . . . . . . . .P-0150 

Olive D . . . . . . . . . . . . . . . . . .P-0275 

Oliveira L . . . . . . . . . .P-0271, P-0272 

Olivier B . . . . . . . . . . . . . . . . .P-0073 

Olivo M . . . . . . . . . . .P-0028, P-0110 

Olson J . . . . . . . . . . . . . . . . .O-0013 

Olsovska J . . . . . . . . .P-0415, P-0437 

Omidi K . . . . . . . . . . . . . . . . .P-0518 

Ono E . . . . . . . . . . . . . . . . . . .P-0607 

Ono M . . . . . . . . . . . . . . . . . .P-0031 

Onozaki M . . . . . . . . . . . . . . .P-0393 

Onyewu C . . . . . . . . . . . . . . . .P-0120 

Oosawa H . . . . . . . . . . . . . . .P-0411 

Oral B . . . . . . . . . . . . . . . . . .P-0072 

Orellán Y . . . . . . . . . . . . . . . .P-0472 

Oren G . . . . . . . . . . . . . . . . . .P-0125 

Oris E . . . . . . . . . . . . . . . . . . .P-0454 

Orofino Costa R . . . .P-0287, P-0288, 

. . . . . . . . . . . . . . . . . . . . . . .P-0364 

Orozco-Topete R . . . . . . . . . . .P-0265 

Ortega-Nava M . . . . . . . . . . . .P-0563 

Ortiz M . . . . . . . . . . .P-0280, P-0355 


Osafune T . . . . . . . . . . . . . . . .P-0658 

Osborn J . . . . . . . . . . . . . . . . .P-0430 

Oshimi K . . . . . . . . . . . . . . . . .P-0560 

Ossadnik K . . . . . . . . . . . . . . .P-0135 

Otranto D . . .P-0005, P-0006, P-0721 

Otter J . . . . . . . . . . . . . . . . . .P-0655 

Oura T . . . . . . . . . . . . . . . . . .P-0581 

Ozgen H . . . . . . . . . . . . . . . . .P-0252 

Ozil S . . . . . . . . . . . . . . . . . . .P-0340 

Pabon de Santiago R . . . . . . . .P-0121 

Padhye A . . . . . . . . . . . . . . . . .P-0473 

Padovan A . . . . . . . . .P-0001, P-0515 

Pajdo R . . . . . . . . . . . . . . . . .O-0021 

Pakshir K . . .P-0032, P-0289, P-0547, 

. . . . . . . . . . . . . . . . . . . . . . .P-0554 

Palatka K . . . . . . . . . . . . . . . . .P-0232 

Palenzuela G . . . . . . . . . . . . . .P-0268 

Palomares J . . . . . . . . . . . . . . .P-0368 

Pamidimukkala U . . . . . . . . . .P-0290 

Panagiotakopoulou K . . . . . . . .P-0332 

Panayotidis P . . . . . . . . . . . . . .P-0226 

Pandey A . . . . . . . . . . . . . . . . .P-0531 

Panepinto J . . . . . . . . . . . . . . .P-0680 

Pangalis G . . . . . . . . . . . . . . .P-0226 

Panizo M . . . .P-0416, P-0468, P-0532 

Panovski N . . . . . . . . . . . . . . .P-0276 

Papalexis P . . . . . . . . . . . . . . .P-0047 

Papini M . . . . . . . . . . . . . . . . .P-0455 

Pappagianis D . . . . . . . . . . . . .P-0608 

Pappas P . . . . . . . . . . . . . . . . .P-0533 

Parada H . . . . . . . . . .P-0034, P-0345 

Pardini G . . . . . . . . . . . . . . . .P-0691 

Parente J . . . . . . . . . . . . . . . . .P-0222 

Park B . . . . . . . . . . . .P-0474, P-0533 

Park B . . . . . . . . . . . . . . . . . . .P-0291 

Park K . . . . . . . . . . . . . . . . . . .P-0348 

Park S . . . . . . . . . . . . . . . . . . .P-0640 

Parodi L . . . . . . . . . . . . . . . . .P-0016 

Parra B . . . . . . . . . . . . . . . . . .P-0681 

Parravicini C . . . . . . . . . . . . . .P-0172 

Pascale L . . . . . . . . . . . . . . . . .P-0427 

Paschoalin T . . . . . . . . . . . . . .P-0719 

Passos F . . . . . . . . . . . . . . . . .P-0031 

Paterson D . . . . . . . . . . . . . . .P-0533 

Patterson T . . . . . . . . .P-0028, P-0395 

Paugam A . . .P-0185, P-0186, P-0417 

Paula C . . . . .P-0292, P-0544, P-0661 

Paulinkevicius M . . . . . . . . . . .P-0682 

Paulovicova E . . . . . . . . . . . . .P-0592 

Pavlicek J . . . . . . . . . . . . . . . .P-0728 

Pawlik B . . . . . . . . . . . . . . . . .P-0099 

Pedros B . . . . . . . . . . . . . . . . .P-0684 

Pelletier R . . . . . . . . .P-0145, P-0293, 

. . . . . . . . . . . . . . . . .P-0360, P-0384 

Pelloux H . . . .P-0237, P-0380, P-0421 

Pelludo J . . . . . . . . . . . . . . . . .P-0014 

Peltroche-Llacsahuanga H . . . .P-0418 

Peman Garcia J . . . . . . . . . . . .P-0457 

Pemán J . . . . . . . . . . . . . . . . .P-0426 

Pemán García J . . . . . . . . . . . .P-0465 

Penha C . . . . . . . . . . . . . . . .O-0044 

Penna S . . . . . . . . . . . . . . . . . .P-0472 

Perales J . . . . . . . . . . . . . . . .O-0044 

Perazzi B . . . . . . . . . . . . . . . . .P-0190 

Pereira D . . . . . . . . . . . . . . . . .P-0038 

Pereira J . . . . . . . . . . .P-0095, P-0327 

Pereira M . . . . . . . . . . . . . . . .P-0180 

Pereira Chioccola V . . . . . . . . .P-0643 

Pereiro Jr M . . . . . . . .P-0298, P-0683 

Peres I . . . . . . . . . . . . . . . . . . .P-0505 

Perez A . . . . . . . . . . . . . . . . . .P-0684 

Perez C . . . . . . . . . . . . . . . . . .P-0269 

Perez Belles C . . . . . . . . . . . . .P-0457 

Perez Blanco M . . . . . . . . . . . .P-0157 

Perfect J . . . . .P-0622, P-0638, P-0674 

Pericolini E . . . . . . . . . . . . . . .P-0716 

Peron C . . . . . . . . . . . . . . . . . .P-0003 

Perraud M . . . . . . . . . . . . . . . .P-0340 

Perry R . . . . . . . . . . . . . . . . . .P-0122 

Persat F . . . . . . . . . . . . . . . . . .P-0339 

Petrikkos G . . . . . . . . .P-0048, P-0332 

Petrikkou E . . . . . . . . . . . . . . .P-0048 

Petrou M . . . . . . . . . . . . . . . . .P-0123 

Petrovska M . . . . . . . . . . . . . . .P-0276 

Pham M . . . . . . . . . . . . . . . .O-0008 

Philip-Joët F . . . . . . . . . . . . . .P-0273 

Philippe I . . . . . . . . . . . . . . . . .P-0073 

Pichová I . . . . . . . . . . . . . . . . .P-0647 

Picos J . . . . . . . . . . . . . . . . . . .P-0016 

Picot S . . . . . . . . . . . .P-0378, P-0524 

Piens M . . . . . . . . . . .P-0339, P-0524 

Pierre A . . . . . . . . . . . . . . . . . .P-0171 

Pina-Vaz C . . . . . . . . . . . . . . .P-0203 

Pineau L . . . . . . . . . . . . . . . . .P-0153 

Pinon J . . . . . . . . . . . .P-0427, P-0480 

Pinoni M . . . . . . . . . . . . . . . . .P-0124 

Pinto L . . . . . . . . . . . .P-0613, P-0625 

Pinto M . . . . . . . . . . . . . . . . . .P-0613 

Piolat C . . . . . . . . . . . . . . . . . .P-0237 

Pires C . . . . . . . . . . . . . . . . . .P-0643 

Pires de Camargo Z . .P-0482, P-0534 

Pitashny R . . . . . . . . . . . . . . . .P-0307 


23

Pitusi M . . . . . . . . . . . . . . . . . .P-0125 

Pivovarova Z . . . . . . . . . . . . . .P-0710 

Piyabongkarn D . . . . . . . . . . .P-0498 

Pizzolatti M . . . . . . . . . . . . . . .P-0065 

Pla J . . . . . . . . . . . . . . . . . . .O-0039 

Playford G . . . . . . . .O-0015, P-0535 

Plomer-Niezgoda E . . . . . . . . .P-0084 

Podo F . . . . . . . . . . . . . . . . . .P-0591 

Poirier C . . . . . . . . . . . . . . . . .P-0259 

Poirier L . . . . . . . . . . . . . . . . .P-0293 

Poirot J . . . .O-0010, P-0294, P-0303, 

. . . . . . . . . . . . . . . . . . . . . . .P-0356 

Poisson D . . . . . . . . . . . . . . . .P-0295 

Polacheck I . . . . . . . . . . . . . . .P-0125 

Pombo D . . . . . . . . . . . . . . . . .P-0334 

Ponce-Noyola P . . . . . . . . . . . .P-0671 

Ponton J .O-0017, P-0069, P-0280, P- 

0386, P-0401, P-0585, P-0704 . . . . . . 

Poppert S . . . . . . . . . . . . . . . .P-0399 

Portela M . . . . . . . . . . . . . . . .P-0296 

Pospisilova H . . . . . . . . . . . . . .P-0297 

Posteraro B . . . . . . . . . . . . . . .P-0130 

Poulain D . . . . . . . . .O-0004, P-0328, 

. . . .P-0357, P-0429, P-0604, P-0702, 

. . . .P-0724, P-0725, P-0726, P-0734 

Pounds S . . . . . . . . . . . . . . . . .P-0390 

Pozzatti P . . . . . . . . . . . . . . . . .P-0406 

Pradel P . . . . . . . . . . . . . . . . .P-0237 

Prat C . . . . . . . . . . . . . . . . . . .P-0378 

Prida M . . . . . . . . . . . . . . . . . .P-0308 

Prieto S . . . . . . . . . . . .P-0486, P-0653 

Priscila Garcia I . . . . . . . . . . . .P-0482 

Proksch E . . . . . . . . . . . . . . . .P-0715 

Puccia R . . . .P-0305, P-0577, P-0630, 

. . . . . . . . . . . . . . . . . . . . . . .P-0689 

Péchard K . . . . . . . . . . . . . . . .P-0458 

Pérez C . . . . . . . . . . .P-0375, P-0388, 

. . . . . . . . . . . . . . . . .P-0477, P-0514 

Pérez-Bellés C . . . . . . . . . . . . .P-0465 

Pérez-Mejía A . . . . . . . . . . . . .P-0736 

Pérez-Pérez L . . . . . . . . . . . . . .P-0298 

Pérez-Roth E . . . . . . . . . . . . . .P-0354 

Pérez-Torres A . . . . . . . . . . . . .P-0620 

Péricard J . . . . . . . . . . . . . . . .P-0020 

Querol I . . . . . . . . . . . . . . . . .P-0192 

Quindos G . . . . . . . . .P-0635, P-0704 

Quindós G . .P-0069, P-0386, P-0536 

Quinton J . . . . . . . . . . . . . . . .P-0020 

R. Borba M . . . . . . . . . . . . . . .P-0008 

R. Paula C . . . . . . . . . . . . . . . .P-0439 

Raclavsky V . .P-0232, P-0344, P-0728 

Radev S . . . . . . . . . . . . . . . . . .P-0614 

Rainer J . . . . . . . . . . .P-0408, P-0537 

Ramage G . .O-0018, P-0684, P-0731 

Ramirez Garcia A . . . .P-0615, P-0685 

Ramos R . . . . . . . . . . .P-0269, P-0270 

Ramírez A . . . . . . . . .O-0017, P-0585 

Ramírez-Gaona A . . . . . . . . . .P-0657 

Ran Y . . . . . . . . . . . . . . . . . . .P-0538 

Randhawa H . . . . . . .P-0371, P-0539 

Ranjana K . . . . . . . . . . . . . . . .P-0473 

Ranque S . . . . . . . . . .P-0274, P-0299 

Ranque S . . . .P-0275, P-0300, P-0301 

Rast T . . . . . . . . . . . . . . . . . .O-0035 

Ratajczak Stefanska V . . . . . . .P-0302 

Rato S . . . . . . . . . . . . . . . . . . .P-0420 

Raymond M . . . . . . . . . . . . . . .P-0579 

Reboux G . . . . . . . . . . . . . . . .P-0303 

Redetzke K . . . . . . . . . . . . . . .P-0306 

Reedy J . . . . . . . . . . . . . . . . . .P-0686 

Reichenpfader B . . . . . . . . . . .P-0200 

Reiffert, S . . . . . . . . . . . . . . . .P-0739 

Reinthaler F . . . . . . . . . . . . . . .P-0200 

Reis A . . . . . . . . . . . . . . . . . . .P-0033 

Reis C . . . . . . . . . . . .P-0540, P-0541 

Reis Filho E . . . . . . . . .P-0540 P-0541 

Reiss E . . . . . . . . . . . . . . . . . .P-0559 

Relloso S . . . . . . . . . . . . . . . . .P-0124 

Remoli M . . . . . . . . . . . . . . . .P-0599 

Renée G . . . . . . . . . . .P-0512, P-0513 

Restrepo A . . . . . . . . .P-0593, P-0621 

Reuter S . . . . . . . . . . . . . . . . .P-0391 

Revathi S . . . . . . . . . . . . . . . . .P-0055 

Reviakina V . .P-0416, P-0468, P-0532 

Revillo M . . . . . . . . . . . . . . . . .P-0192 

Reyes H . . . . . . . . . . . . . . . . . .P-0388 

Reyes-Montes M . . . . . . . . . . .P-0479 

Rezaei M . . . . . . . . . . . . . . . . .P-0517 

Rezaian M . . . . . . . . . . . . . . . .P-0165 

Rezaie A . . . . . . . . . . . . . . . . .P-0687 

Rezaie S . . . . . . . . . . .P-0165, P-0582 

Rezusta A . . . . . . . . . .P-0192, P-0304 

Rezusta-López A . . . . . . . . . . .P-0309 

Rheeder J . . . . . . . . . . . . . . . .P-0487 

Riazipour M . .P-0164, P-0542, P-0616 

Riazypoor M . . . . . . . . . . . . . .P-0433 

Ribaud P . . . . . . . . . . . . . . . . .P-0088 

Ribeiro J . . . . . . . . . . . . . . . . .P-0466 

Ribeiro M . . . . . . . . . .P-0543, P-0544 

Ribeiro S . . . . . . . . . . .P-0271, P-0272 

Ricci G . . . . . . . . . . . . . . . . . .P-0305 

Richard F . . . . . . . . . . . . . . . .O-0023 

Richard G . . . . . . . . . . . . . . . .P-0183 


Richard-Lenoble D . . .P-0183, P-0182, 

. . . . . . . . . . . . . . . . .P-0184, P-0205 

Rimek D . . . . . . . . . . . . . . . . .P-0306 

Rinaldi M . . .P-0077, P-0147, P-0166, 

. . . . . . . . . . . . . . . . . . . . . . .P-0334 

Riquelme M . . . . . . . . . . . . . . .P-0361 

Risi E . . . . . . . . . . . . . . . . . . . .P-0020 

Ro B . . . . . . . . . . . . . . . . . . . .P-0419 

Robert R . . . .P-0357, P-0407, P-0574, 

. . . . . . . . . . . . . . . . .P-0713, P-0729 

Roberts G . . . . . . . . . . . . . . . .P-0231 

Robin M . . . . . . . . . . . . . . . . .P-0088 

Robinson Z . . . . . . . . . . . . . . .P-0050 

Rocha A . . . . . . . . . . .P-0481, P-0689 

Rocha N . . . . . . . . . . . . . . . . .P-0221 

Rochas V . . . . . . . . . . . . . . . . .P-0088 

Rochat L . . . . . . . . . . . . . . . . .P-0668 

Rodarte G . . . . . . . . . . . . . . .O-0033 

Rodero L . . . . . . . . . .P-0106, P-0222 

Rodier M . . . . . . . . . . . . . . . . .P-0459 

Rodrigues A . . . . . . . . . . . . . . .P-0203 

Rodrigues A . . . . . . . . . . . . . . .P-0021 

Rodrigues E . . . . . . . . . . . . . . .P-0719 

Rodrigues F . . . . . . . . . . . . . . .P-0203 

Rodrigues M . . . . . . . . . . . . . .P-0253 

Rodrigues Paula C . . .P-0082, P-0126, 

. . . . . . . . . . . . . . . . . . . . . . .P-0568 

Rodriguez M . . . . . . . . . . . . . .P-0011 

Rodriguez N . . . . . . . .P-0307, P-0308 

Rodriguez V . . . . . . . . . . . . . . .P-0426 

Rodriguez Rilo L . . . . . . . . . . . .P-0209 

Rodriguez-Brito S . . . . . . . . . . .P-0690 

Rodriguez-Nava V . . . . . . . . . .P-0346 

Rodriguez-Otero L . . .P-0404, P-0405 

Rodríguez-Andrés C . . . . . . . . .P-0536 

Rodríguez-Tudela J . . . . . . . . . .P-0628 

Roehrle M . . . . . . . . . .P-0136, P-0320 

Rogers D . . . .P-0575, P-0579, P-0587 

Rognon B . . . . . . . . . . . . . . . .P-0691 

Roilides E . . .O-0032, P-0247, P-0248 

Roither S . . . . . . . . . . . . . . . . .P-0432 

Roitt I . . . . . . . . . . . . . . . . . . .P-0596 

Rokoei F . . . . . . . . . . . . . . . . .P-0492 

Roll P . . . . . . . . . . . . . . . . . . .P-0200 

Rolland C . . . . . . . . . . . . . . . .P-0421 

Romano C . . . . . . . . . . . . . . .P-0224 

Romano R . . . . . . . . . . . . . . .O-0037 

Romero A . . .P-0096, P-0103, P-0104 

Romero H . . . . . . . . . . . . . . . .P-0127 

Romero L . . . . . . . . . . . . . . . .P-0545 

Romero-Martinez R . . . . . . . . .P-0692 

Romeuf N . . . . . . . . . . . . . . . .P-0378 

Romo-Lozano Y . . . . . . . . . . . .P-0657 

Román E . . . . . . . . . . . . . . . .O-0039 

Roos B . . . . . . . . . . . .P-0136, P-0318, 

. . . . . . . . . . .P-0319, P-0320, P-0321 

Roque H . . . . . . . . . . . . . . . . .P-0420 

Rosa A . . . . . . . . . . . . . . . . . .P-0075 

Rosa C . . . . . . . . . . . . . . . . . .P-0201 

Rosado C . . . . . . . . . . . . . . . .P-0460 

Rosado L . . . .P-0034, P-0345, P-0460 

Rosanova M . . . . . . . . . . . . . .P-0316 

Roselló A . . . . . . . . . . . . . . . . .P-0477 

Roselló A . . . .P-0375, P-0388, P-0514 

Rosenthal S . . . . . . . . . . . . . . .P-0595 

Rossano M . . . . . . . . . . . . . . .P-0016 

Rossodivita M . . . . . . . . . . . . .P-0521 

Rossouw J . . . . . . . . . . . . . . . .P-0546 

Roth D . . . . . . . . . . . . . . . . . .P-0143 

Rotter M . . . . . . . . . . . . . . . . .P-0432 

Roudbarmohammadi S . . . . . .P-0433 

Roux P . . . . . . . . . . . .P-0109, P-0510 

Rovegno D . . . . . . . . . . . . . . .P-0754 

Roy T . . . . . . . . . . . . . . . . . . .P-0267 

Rozentale B . . . . . . . . . . . . . . .P-0444 

Rozewicka M . . . . . . . . . . . . . .P-0302 

Rubio M . . . . . . . . . . . . . . . . .P-0304 

Rubio-Calvo C . . . . . . . . . . . .P-0309 

Rude T . . . . . . . . . . . . . . . . . .P-0638 

Ruiz L . . . . . . . . . . . . . . . . . . .P-0568 

Ruiz M . . . . . . . . . . . . . . . . . .P-0368 

Ruiz-Acevedo A . . . . . . . . . . . .P-0545 

Ruiz-Ortiz M . . . . . . . . . . . . . .P-0563 

Ruiz-Palacios G . . . . . . . . . . . .P-0470 

Rupp S . . . . .P-0389, P-0578, P-0656 

Ruschel L . . . . . . . . . . . . . . . . .P-0732 

Ruskova L . . . . . . . . . . . . . . . .P-0344 

Rym K . . . . . . . . . . . . . . . . . . .P-0310 

S. Argenta J . . . . . . . . . . . . . .P-0626 

S. Cavalheiro A . . . . . . . . . . . .P-0626 

S. Cavalhiro A . . . . . . . . . . . . .P-0021 

S. Perpétuo de Sequeira H . . . .P-0311 

S. Terceti M . . . . . . . . . . . . . . .P-0439 

S.Nikhat S . . . . . . . . . . . . . . . .P-0290 

Sabanero M . . . . . . . . . . . . . .P-0312 

Sabayan B . . . . . . . . .P-0547, P-0554 

Sabino R . . . .P-0034, P-0345, P-0460 

Sabokbar A . . . . . . . . . . . . . . .P-0313 

Sabuncu I . . . . . . . . . . . . . . . .P-0252 

Sabzevari Z . . . . . . . . . . . . . . .P-0525 

Sachanas S . . . . . . . . . . . . . . .P-0226 

Sadahiro A . . . . . . . . . . . . . . .P-0619 

Saghazadeh M . . . . . . . . . . . .P-0616 


25

Saha D . . . . . . . . . . . . . . . . . .P-0314 

Sahand I . . . . . . . . . . . . . . . . .P-0069 

Sahar Khiz M . . . . . . . . . . . . .P-0131 

Saijyo T . . . . . . . . . . . . . . . . . .P-0392 

Saito F . . . . . . . . . . . . . . . . . .P-0722 

Salah K . . . . . . . . . . . . . . . . . .P-0604 

Salas R . . . . . . . . . . . . . . . . . .P-0012 

Salas Tellez E . . . . . . . . . . . . . .P-0035 

Salehian Omran D . . . . . . . . .P-0452 

Sales Net M . . . . . . . . . . . . . .P-0272 

Sales Neto M . . . . . . . . . . . . . .P-0271 

Salet R . . . . . . . . . . . . . . . . . .P-0268 

Salhab A . . . . . . . . . . . . . . . . .P-0193 

Saliba F . . . . . . . . . . . . . . . . .P-0220 

Salisbury D . . . . . . . . . . . . . . .P-0462 

Sallami H . . . . . . . . . . . . . . . .P-0208 

Salle B . . . . . . . . . . . . . . . . . .P-0745 

Salmanpour R . . . . . . . . . . . . .P-0289 

Salvatori R . . . . . . . . .P-0007, P-0521 

Salvenmoser S . . . . . .P-0141, P-0323 

Samat m . . . . . . . . . . . . . . . . .P-0352 

Samayoa B . . . . . . . . . . . . . . .P-0422 

Samford L . . . . . . . . . . . . . . .O-0011 

San Segundo I . . . . .O-0017, P-0585 

San-Blas G . .P-0128, P-0642, P-0677, 

. . . .P-0678, P-0682, P-0690, P-0698 

Sanchez J . . . . . . . . . . . . . . . .P-0464 

Sanchez M . . . . . . . . .P-0435, P-0436 

Sanchez-Sousa A . . . . . . . . . . .P-0410 

Sanclemente G . . . . . . . . . . . .P-0057 

Sandini S . . .O-0045, P-0359, P-0596 

Sandocsky-Losica H . . . . . . . . .P-0143 

Sandoval-Bernal G . . . . . . . . .P-0312 

Sandovsky-Losica H . . . . . . . . .P-0129 

Sanglard D . . . . . . .O-0003, P-0062, 

. . . . . . . . . . .P-0130, P-0668, P-0691 

Sanguinetti M . . . . . . . . . . . . .P-0130 

Sano A . . . . . . . . . . .P-0027, P-0036, 

. . . . . . . . . . . . . . . . .P-0526, P-0607 

Santhanam j . . . . . . . . . . . . . .P-0352 

Santiago A . . . . . . . . . . . . . . .P-0121 

Santos P . . . . . . . . . .P-0315, P-0316, 

. . . . . . . . . . . . . . . . .P-0451, P-0738 

Santos R . . . . . . . . . . . . . . . . .P-0693 

Santos Neves de Barcellos D . .P-0002 

Santurio D . . . . . . . . . . . . . . . .P-0406 

Santurio J . . .P-0037, P-0038, P-0406 

Sanza L . . . . . . . . . . . . . . . . . .P-0559 

Sapieri A . . . . . . . . . . . . . . . . .P-0643 

Sar M . . . . . . . . . . . . . . . . . . .P-0245 

Sarafi A . . . . . . . . . . . . . . . . . .P-0553 

Sarazin A . . . . . . . . . . . . . . . .P-0724 

Sasanelli M . . . . . . . . . . . . . . .P-0006 

Sasse C . . . . . . . . . .O-0042, P-0694 

Sato A . . . . . . . . . . . . . . . . . . .P-0730 

Sato T . . . . . . . . . . . .P-0245, P-0317 

Sato Y . . . . . . . . . . . . . . . . . . .P-0411 

Satpathy G . . . . . . . . . . . . . . .P-0112 

Sattari M . . . . . . . . . . . . . . . . .P-0131 

Sauer J . . . . .P-0136, P-0318, P-0319, 

. . . . . . . . . . . . . . . . .P-0320, P-0321 

Sauer P . . . . . . . . . . . . . . . . . .P-0232 

Saunte D . . . . . . . . . . . . . . . . .P-0132 

Saville S . . . . . . . . . . .P-0587, P-0617 

Savin C . . . . . . . . . . . . . . . . . .P-0421 

Scalise G . . . . . . . . . . . . . . . . .P-0144 

Scaltrito M . . . . . . . . .P-0424, P-0697 

Scarano S . . . . . . . . . . . . . . . .P-0382 

Scavone R . . . . . . . . . . . . . . . .P-0133 

Schabereiter-Gurtner C . . . . . .P-0432 

Schaufele R . . . . . . . . . . . . . . .P-0390 

Scheel C . . . . . . . . . . . . . . . . .P-0422 

Scheid L . . . . . . . . . . . . . . . . .P-0406 

Scherer S . . . . . . . . . . . . . . . .O-0035 

Schettino A . .P-0015, P-0016, P-0039 

Schleck C . . . . . . . . . . . . . . . .P-0569 

Schmalreck A . . . . . . .P-0134, P-0135 

Schmitt-Hoffmann A . .P-0136, P-0318, 

. . . . . . . . . . .P-0319, P-0320, P-0321 

Schonfeld W . . . . . . . . . . . . . .P-0324 

Schouls L . . . . . . . . . . . . . . . . .P-0363 

Schroers H . . . . . . . . . . . . . . .P-0497 

Schubach A . . . . . . . . . . . . . . .P-0364 

Schwarz H . . . . . . . . . . . . . . . .P-0389 

Schwarz P . . . . . . . . . . . . . . . .P-0137 

Schwendler S . . . . . . .P-0037, P-0038 

Schwesinger G . . . . . . . . . . . .P-0053 

Schär G . . . . . . . . . . . . . . . . .P-0374 

Scialino G . . . . . . . . . . . . . . . .P-0051 

Scroferneker M . . . . .P-0138, P-0548, 

. . . . . . . . . .P-0549, P-0695, P-0696, 

. . . . . . . . . . . . . . . . .P-0711, P-0732 

Sealy P . . . . . . . . . . . . . . . . . .P-0322 

Seaton S . . . . . . . . . . . . . . . . .P-0362 

Sefidgar S . . . . . . . . . . . . . . . .P-0101 

Segal E . . . . .P-0129, P-0139, P-0143 

Segundo-Zaragoza C . . . . . . .P-0009, 

. . . . . . . . . . . . . . . . .P-0040, P-0563 

Segura G . . . . . . . . . . . . . . . .P-0629 

Segura-Roca G . . . . . . . . . . . .P-0342 

Seguy N . . . . . . . . . . . . . . . . .P-0140 

Seidler M . . . . . . . . . .P-0141, P-0323 

Seifeldin R . . . . . . . . . . . . . . . .P-0324 

Sein T . . . . . . . . . . . . . . . . . . .P-0390 


Seishima M . . . . . . . . . . . . . . .P-0325 

Seki M . . . . . . . . . . . .P-0277, P-0392 

Sekkal N . . . . . . . . . . . . . . . . .P-0185 

Sellami A . . .P-0208, P-0326, P-0523, 

. . . . . . . . . . . . . . . . .P-0550, P-0551 

Sellami H . . .P-0326, P-0523, P-0550, 

. . . . . . . . . . . . . . . . . . . . . . .P-0551 

Selores M . . . . . . . . . .P-0221, P-0264 

Selselet G . . . . . . . . . . . . . . . .P-0233 

Semedo I . . . . . . . . . . . . . . . .P-0327 

Semelcki A . . . . . . . . . . . . . . .O-0003 

Sendid B . . .O-0004, P-0328, P-0429, 

. . . . . . . . . . .P-0489, P-0724, P-0734 

Senol E . . . . . . . . . . . . . . . . . .P-0398 

Senses E . . . . . . . . . . . . . . . . .P-0252 

Serelha M . . . . . . . . . . . . . . . .P-0505 

Sermondade N . . . . . . . . . . . .P-0552 

Setina M . . . . . . . . . . . . . . . . .P-0297 

Severa M . . . . . . . . . . . . . . . . .P-0599 

Seyed Gama H . . . . . . . . . . . .P-0041 

Seyed Masoud H . . . . . . . . . . .P-0041 

Shabashova N . . . . . . . . . . . . .P-0618 

Shaffer R . . . . . . . . . . . . . . . . .P-0330 

Shahi M . . . . . . . . . . . . . . . . .P-0142 

Shahi S . . . . . . . . . . . . . . . . . .P-0142 

Shammas V . . . . . . . . . . . . . . .P-0218 

Shankland G . . . . . . .P-0045, P-0445 

Sharbatkhori F . . . . . . . . . . . . .P-0553 

Sharbatkhori M . . . . . .P-0213, P-0553 

Sharbatkhori N . . . . . . . . . . . .P-0553 

Sharbatkhori Z . . . . . . . . . . . .P-0553 

Sharma M . . . . . . . . . . . . . . . .P-0473 

Sharma S . . . . . . . . . .P-0080, P-0632 

Sharp A . . . . . . . . . . . . . . . . .P-0158 

Sheehan D . . . . . . . . . . . . . . .P-0079 

Shekarkhar G . . . . . . . . . . . . .P-0554 

Shen Y . . . . . . . . . . . . . . . . . .P-0402 

Sheorey H . . . . . . . . . . . . . . . .P-0329 

Shephard G . . . . . . . . . . . . . .P-0487 

Sheppard D . . . . . . . . . . . . . .P-0395 

Shibata N . . . . . . . . . . . . . . . .P-0735 

Shibuya K . . . . . . . . . . . . . . . .P-0730 

Shields C . . . . . . . . . . . . . . . .P-0266 

Shih H . . . . . . . . . . . . . . . . . .P-0660 

Shikanai-Yasuda M . . . . . . . . .P-0435, 

. . . . . . . . . . . . . . . . .P-0436, P-0619 

Shimamura T . . . . . . . . . . . . .P-0149 

Shin D . . . . . . . . . . . . . . . . . .P-0640 

Shin Y . . . . . . . . . . . . . . . . . . .P-0640 

Shinohara H . . . . . . . . . . . . . .P-0612 

Shiota R . . . . . . . . . . . . . . . . .P-0393 

Shiozawa M . . . . . . . . . . . . . .P-0031 

Shiraishi K . . . . . . . . .P-0116, P-0149 

Shiraki Y . . . . . . . . . . .P-0238, P-0555 

Shiva C . . . . . . . . . . .P-0059, P-0202 

Shoham S . . . . . . . . . . . . . . . .P-0285 

Shoham s . . . . . . . . . . . . . . . .P-0330 

Shokohi T . . . . . . . . . . . . . . . .P-0493 

Shukla A . . . . . . . . . . . . . . . . .P-0632 

Shurpitskaja O . . . . . . . . . . . .P-0215 

Shwarzman R . . . . . . . . . . . . .P-0129 

Sicard P . . . . . . . . . . . . . . . . .P-0475 

Sigler L . . . . . . . . . . . . . . . . . .P-0747 

Sigrist H . . . . . . . . . . . . . . . . .P-0300 

Sillaots S . . . . . . . . . . . . . . . .O-0016 

Silva E . . . . . . . . . . . . . . . . . . .P-0568 

Silva I . . . . . . . . . . . . . . . . . . .P-0287 

Silva M . . . . . . . . . . . .P-0613, P-0625 

Silva V . . . . . .P-0423, P-0556, P-0733 

Silva-Vergara M . . . . .P-0481, P-0482, 

. . . . . . . . . . . . . . . . . . . . . . .P-0520 

Silveira V . . . . . . . . . . . . . . . . .P-0540 

Simmons K . . . . . . . . . . . . . . .P-0608 

Simpson V . . . . . . . . . . . . . . . .P-0456 

Singh S . . . . . . . . . . .P-0282, P-0331 

Sinha K . . . . . . . . . . .P-0371, P-0539 

Sinhorini I . . . . . . . . . . . . . . . .P-0033 

Sinitskaya I . . . . . . . . . . . . . . .P-0700 

Sionov E . . . . . . . . . . .P-0125, P-0143 

Sioud Dhrif A . . . . . . . . . . . . .P-0453 

Sisto F . . . . . . . . . . . .P-0424, P-0697 

Sixt N . . . . . . . . . . . . .P-0303, P-0557 

Skiada A . . . . . . . . . .P-0048, P-0332 

Sklenar J . . . .P-0415, P-0425, P-0437 

Slavin M . . .O-0015, P-0068, P-0122, 

. . . . . . . . . . . . . . . . . . . . . . .P-0558 

Slomianny M . . . . . . . . . . . . . .P-0726 

Slosar P . . . . . . . . . . . . . . . . . .P-0409 

Sluiter-van Dijk A . . . . . . . . . . .P-0161 

Smedema M . . . . . . . . . . . . .O-0006 

Smrcka V . . . . . . . . . . . . . . . .P-0241 

Smulian G . . . . . . . .O-0040, P-0609 

Soares C . . . . . . . . . .P-0180, P-0222 

Soares R . . . . . . . . . . . . . . . . .P-0296 

Soares Fº P . . . . . . . . . . . . . . .P-0343 

Socie G . . . . . . . . . . . . . . . . . .P-0088 

Sockalingum G . . . . . . . . . . . .P-0480 

Sofair A . . . . . . . . . . . . . . . . . .P-0559 

Sohn K . . . . . . . . . . . . . . . . . .P-0656 

Solomon S . . . . . . . . . . . . . . .P-0251 

Sonia M . . . . . . . . . . . . . . . .O-0023 

Sophie C . . . . . . . . . . . . . . . .O-0023 

Sopo L . . . . . . . . . . . . . . . . . .P-0476 

Sorais F . . . . . . . . . . .P-0678, P-0698 


27

Sorin V . . . . . . . . . . . . . . . . . .P-0073 

Sorrell T . . .O-0015, P-0068, P-0118, 

. . . . . . . . . . .P-0400, P-0535, P-0558 

Soto P . . . . . . . . . . . . . . . . . . .P-0039 

Souad C . . . . . . . . . . . . . . . . .P-0177 

Sousa O . . . . . . . . . . . . . . . . .P-0064 

Souza I . . . . . . . . . . . . . . . . . .P-0296 

Souza V . . . . . . . . . . . . . . . . .P-0643 

Souza Motta C . . . . . . . . . . . .P-0483 

Souza Carvalho Melhem M . . .P-0643 

Sowa J . . . . . . . . . . . . . . . . . .P-0283 

Soydan S . . . . . . . . . . . . . . . . .P-0333 

Spanamberg A . . . . . .P-0002, P-0042 

Spanish Network of Infection in Transpla 

P-0426 . . . . . . . . . . . . . . . . . . . . . . . 

Spanjaard L .O-0027, P-0196, P-0367 

Spanu T . . . . . . . . . . . . . . . . .P-0130 

Specht C . . . . . . . . . . . . . . . . .P-0649 

Speers D . . . . . . . . . . . . . . . . .P-0448 

Spickermann J . . . . . .P-0136, P-0318, 

. . . . . . . . . . .P-0319, P-0320, P-0321 

Spiess B . . . . . . . . . . . . . . . . .P-0391 

Spreghini E . . . . . . . . . . . . . . .P-0144 

Srebnik M . . . . . . . . . . . . . . . .P-0125 

Srikanth P . . . . . . . . . . . . . . . .P-0251 

Srinivasan A . . . . . . . .P-0462, P-0463 

St Sauver J . . . . . . . . . . . . . . .P-0569 

St-Germain G . . . . . . . . . . . . .P-0145 

Staab J . . . . . . . . . . . . . . . . . .P-0588 

Standaert-Vitse A . . .O-0004, P-0604, 

. . . . . . . . . . . . . . . . .P-0328, P-0734 

Stano P . . . . . . . . . . . . . . . . . .P-0424 

Stanton B . . . . . . . . . . . . . . . .P-0699 

Stashenko E . . . . . . . .P-0058, P-0107 

Staudt M . . . . . . . . . . . . . . . . .P-0699 

Stchingel A . . . . . . . . . . . . . . .P-0288 

Steel N . . . . . . . . . . . . . . . . . .P-0146 

Steele C . . . . . . . . . . . . . . . . .P-0622 

Stein A . . . . . . . . . . . . . . . . . .P-0299 

Stepanova A . . . . . . . . . . . . . .P-0700 

Stevens D . . .P-0061, P-0564, P-0565 

Stolhand M . . . . . . . . . . . . . . .P-0334 

Stopiglia C . . . . . . . . .P-0696, P-0732 

Strick L . . . . . . . . . . . . . . . . . .P-0464 

Strijbis K . . . . . . . . . . . . . . . . .P-0701 

Strompoulis P . . . . . . . . . . . . .P-0047 

Stéphane D . . . . . . . . . . . . . . .P-0073 

Su S . . . . . . . . . . . . . . . . . . . .P-0259 

Sudhadham M . . . . . . . . . . . .P-0440 

Sugi Y . . . . . . . . . . . . . . . . . . .P-0560 

Sugita T . . . .P-0151, P-0337, P-0387, 

. . . . . . . . . . .P-0393, P-0555, P-0611 

Sugita-Konishi Y . . . . . . . . . . .P-0291 

Sugiura M . . . . . . . . . . . . . . . .P-0317 

Sukhanova Y . . . . . . . . . . . . . .P-0566 

Sukran T . . . . . . . . . . . . . . . . .P-0443 

Šulc M . . . . . . . . . . . . . . . . . .P-0425 

Sulc M . . . . . . . . . . . .P-0415, P-0437 

Sullivan L . . . . . . . . . . . . . . . .P-0329 

Summerbell R . . . . . . .P-0358, P-0497 

Supaphan J . . . . . . . . . . . . . . .P-0335 

Sutton D . . . .P-0077, P-0147, P-0166, 

. . . . . . . . . . . . . . . . . . . . . . .P-0334 

Suzuki M . . . .P-0025, P-0411, P-0735 

Suzuki S . . . . . . . . . . . . . . . . .P-0735 

Suzuki Y . . . . . . . . . . . . . . . . .P-0336 

Suárez C . . . . . . . . . . . . . . . . .P-0107 

Suárez-Alvarez R . . . . . . . . . . .P-0620 

Svejgaard E . . . . . . . . . . . . . . .P-0132 

Svensson E . . . . . . . . . . . . . . .P-0396 

Svoboda W . . . . . . . . . . . . . . .P-0031 

Swain S . . . . . . . . . . . . . . . . . .P-0605 

Sweet L . . . . . . . . . . . . . . . . . .P-0330 

Swiatlo E . . . . . . . . . . . . . . . . .P-0322 

Swinne D . . . . . . . . . . . . . . . .P-0383 

Symoens F . . . . . . . . . . . . . . .P-0356 

Syouji T . . . . . . . . . . . . . . . . . .P-0116 

Szczepanik A . . . . . . .P-0188, P-0189 

Szymañska J . . . . . . . .P-0148, P-0561 

Sáez A . . . . . . . . . . . . . . . . . .P-0468 

Sánchez Reus F . . . . . . . . . . . .P-0426 

Sánchez-Aguilar D . . . . . . . . . .P-0298 

Sánchez-Sousa A . . . . . . . . . . .P-0074 

Sébastien B . . . . . . . . . . . . . . .P-0369 

Sóczó G . . . . . . . . . . . . . . . . .P-0089 

Tabart J . . . . . . . . . .O-0001, O-0005 

Taglialegna R . . . . . . . . . . . . .P-0100 

Taguchi H . . . . . . . . . . . . . . . .P-0055 

Tahsiro T . . . . . . . . . . . . . . . . .P-0392 

Tajima M . . . . . . . . . .P-0337, P-0387 

Tajudin T . . . . . . . . . . . . . . . . .P-0338 

Takahashi Y . . . . . . . . . . . . . . .P-0036 

Takarouri K . . . . . . . . . . . . . . .P-0125 

Takatori K . . . . . . . . . . . . . . . .P-0291 

Takayama A . . . . . . . .P-0027, P-0036 

Takeo K . . . . . . . . . . . . . . . . . .P-0673 

Takigawa M . . . . . . . . . . . . . . .P-0336 

Takiuchi I . . . . . . . . . . . . . . . . .P-0160 

Takizawa K . .P-0025, P-0483, P-0746, 

. . . . . . . . . . . . . . . . . . . . . . .P-0755 

Talebi A . . . . . . . . . . . . . . . . . .P-0043 

Tamiolaki M . . . . . . . . . . . . . .P-0247 

Tanabe H . . . .P-0447, P-0519, P-0752 

Tang yew huat H . . . . . . . . . . .P-0352 


Tarazooie B . . . . . . . .P-0165, P-0572 

Tarazui B . . . . . . . . . . . . . . . . .P-0412 

Taron C . . . . . . . . . . . . . . . . . .P-0649 

Tashiro T . . . . . . . . . . . . . . . . .P-0277 

Tattevin P . . . . . . . . . . . . . . . . .P-0210 

Toriello C . . . . . . . . . .P-0496, P-0736 

Toro F . . . . . . . . . . . . . . . . . . .P-0375 

Torosantucci A . . . . . .P-0591, P-0594 

Torres J . . . . . . . . . . .P-0127, P-0257 

Torres M . . . . . . . . . . . . . . . . .P-0368 

Ullibarri B . . . . . . . . . . . . . . . .P-0536 

Umeyama T . . . . . . . . . . . . . .P-0581 

Underhill D . . . . . . . . . . . . . . .P-0600 

Ungpakorn R . . . . . . . . . . . . . .P-0496 

Uran-Jimenez M . . . . .P-0593, P-0621 

Tay k . . . . . . . . . . . . . . . . . . . .P-0338 

Tay s . . . . . . . . . . . . . . . . . . . .P-0562 

Taylor M . . . . . . . . . . .P-0620, P-0692 

Teixeira M . . . . . . . . . . . . . . . .P-0221 

Teixeira P . . . . . . . . . . . . . . . .O-0019 

Teixeira R . . . . . . . . . . . . . . . .P-0014 

Templeton I . . . . . . . . . . . . . . .P-0503 

Templeton K . . . . . . . . . . . . . .P-0358 

Thami G . . . . . . . . . . . . . . . . .P-0206 

Theelen B . . . . . . . . . .P-0244, P-0304 

Theodoro F . . . . . . . . .P-0540, P-0541 

Thevissen K . . . . . . . . . . . . . . .P-0576 

Thibout Y . . . . . . . . . . . . . . . .P-0339 

Thiebaut A . . . . . . . . .P-0340, P-0524 

Thirach S . . . . . . . . . . . . . . . . .P-0703 

Thomas D . . . . . . . . . . . . . . .O-0009 

Thomas H . . . . . . . . . . . . . . . .P-0254 

Thompson E . . . . . . . . . . . . . .P-0334 

Thomson P . . . . . . . . . . . . . . .P-0423 

Thuru X . . . . . . . . . . . . . . . . .O-0004 

Tibaldini P . . . . . . . . . . . . . . . .P-0521 

Tiraboschi I . .P-0190, P-0209, P-0341 

Tiraboschi J . . . . . . . . . . . . . . .P-0209 

Tissot-Dupont H . . . . . . . . . . . .P-0299 

Toffaletti D . . . . . . . . .P-0638, P-0674 

Tomiyama S . . . . . . . .P-0116, P-0149 

Toraman Z . . . . . . . . . . . . . . .P-0056 

Tore O . . . . . . . . . . . . . . . . . .P-0168 

Toribio J . . . . . . . . . . . . . . . . .P-0298 

Torres-Rodríguez J . . .P-0046, P-0342, 

. . . . . . . . . . . . . . . . .P-0385, P-0629 

Toubas D . . . . . . . . . .P-0427, P-0480 

Towersey L . . . . . . . . . . . . . . . .P-0343 

Travassos L . . . . . . . . .P-0577, P-0719 

Travelsi H . . . . . . . . . . . . . . . .P-0551 

Tremblay C . . . . . . . . . . . . . . .P-0293 

Trindade R . . . . . . . . . . . . . . . .P-0738 

Trinel P . . . . .P-0702, P-0725, P-0726 

Tristao F . . . . . . . . . . . . . . . . .P-0607 

Trojanowska D . . . . .O-0021, P-0150 

Tronchin G . . . . . . . . . . . . . . .P-0574 

Trosok S . . . . . . . . . . . . . . . .O-0016 

Trtkova J . . . . . . . . . . . . . . . . .P-0344 

Tschachler E . . . . . . . . . . . . . .P-0165 

Tsiouris I . . . . . . . . . . . . . . . . .P-0248 

Tsubakimoto W . . . . . . . . . . . .P-0283 

Tsuboi R . . . .P-0090, P-0151, P-0337, 

. . . . . . . . . . . . . . . . . . . . . . .P-0387 

Tsubuku H . . . . . . . . .P-0151, P-0387 

Tsutsumi V . . . . . . . . . . . . . . . .P-0312 

Tula L . . . . . . . . . . . . . . . . . . .P-0382 

Turac-Bicer A . . . . . . . . . . . . . .P-0495 

Turki H . . . . . . . . . . . .P-0523, P-0551 

Turkoz Sucak G . . . . . . . . . . . .P-0398 

Turner V . . . . . . . . . .O-0003, P-0062 

Tóth B . . . . . . . . . . . . . . . . . . .P-0428 

Ubukata K . . . . . . . . . . . . . . . .P-0581 

Uchevatkina A . . . . . . . . . . . . .P-0618 

Urban C . . . . . . . . . . . . . . . . .P-0667 

Uslu S . . . . . . . . . . . . . . . . . . .P-0449 

Uthman A . . . . . . . . . . . . . . . .P-0165 

V.V. S . . . . . . . . . . . . . . . . . . . .P-0290 

Vagefikia A . . . . . . . . . . . . . . .P-0554 

Vagner O . . . . . . . . . .P-0475, P-0557 

Vaiopoulos G . . . . . . . . . . . . .P-0226 

Vajpayee M . . . . . . . . . . . . . . .P-0162 

Vajpayee R . . . . . . . . . . . . . . .P-0112 

Valderrama B . . . . . . . . . . . . .P-0658 

Valdes-Martinez S . . . . . . . . . .P-0563 

Valente P . . . . . . . . . .P-0002, P-0042 

Valentín Martín A . . . . . . . . . . .P-0465 

Vallor A . . . . . . . . . . . . . . . . . .P-0110 

Valot S . . . . . . . . . . . . . . . . . .P-0557 

van Asbeck E . . . . . . .P-0564, P-0565 

van Belkum A . . . . . . .P-0152, P-0167 

van de Sande W . . . . .P-0152, P-0167 

Van den Abeele A . . . . . . . . . .P-0454 

van der Raaij-Helmer E . . . . . .P-0358 

van der Westhuizen L . . . . . . . .P-0487 

Van Dijck P . . . . . . . . . . . . . . .P-0648 

van het Hoog M . . . . . . . . . .O-0035 

van Leeuwen W . . . . . . . . . . . .P-0167 

Vandecandelaere P . . . . . . . . .P-0454 

Vandeplassche L . . . . .P-0176, P-0219 

Vandeputte P . . . . . . .P-0153, P-0154 

Vandercappellen J . . . . . . . . . .P-0576 

Vandewalle P .P-0429, P-0604, P-0734 

Uchida K . . . . . . . . . .P-0085, P-0090 


29

Vanhems P . . . . . . . . . . . . . . .P-0524 

Vanittanakom N . . . . .P-0335, P-0703 

Vasilyeva N . .P-0155, P-0215, P-0566 

Vassei M . . . . . . . . . . . . . . . . .P-0289 

Vecchiarelli A . . . . . . . . . . . . . .P-0716 

Veillette K . . . . . . . . . . . . . . .O-0016 

Velders M . . . . . . . . . .P-0087, P-0161 

Velegraki A . . . . . . . .P-0049, P-0226, 

. . . . . . . . . . .P-0248, P-0358, P-0484 

Velho G . . . . . . . . . . .P-0221, P-0264 

Vemu L . . . . . . . . . . . . . . . . . .P-0290 

Venâncio A . . . . . . . . . . . . . . .P-0646 

Vera L . . . . . . . . . . . . . . . . . . .P-0347 

Vera-Cabrera L . . . . . . . . . . . .P-0567 

Verbrugh H . . . . . . . . .P-0152, P-0167 

Vergara M . . . . . . . . . . . . . . . .P-0521 

Verissimo C . . . . . . . . . . . . . . .P-0034 

Vermeire S . . . . . . . . . . . . . . .P-0429 

Vermitsky J . . . . . . . . . . . . . . .P-0575 

Vermout S . . . . . . . .O-0001, O-0005 

Verrot D . . . . . . . . . . . . . . . . .P-0273 

Veríssimo C . . . . . . . .P-0345, P-0460 

Vescia N . . . . . . . . . . . . . . . . .P-0430 

Vettorato G . . . . . . . . .P-0548, P-0549 

Viani F . . . . . . . . . . . . . . . . . .P-0568 

Viani P . . . . . . . . . . . . . . . . . .P-0568 

Vicenti J . . . . . . . . . . . . . . . . .P-0695 

Vidal G . . . . . . . . . . . . . . . . . .P-0307 

Vidal-Vanaclocha F . . .P-0615, P-0685 

Vidotto V . . . . . . . . . .P-0007, P-0025, 

. . . .P-0455, P-0483, P-0521, P-0635, 

. . . . . . . . . . . . . . . . .P-0658, P-0704 

Vieira M . . . . . . . . . . . . . . . . .P-0346 

Vieira R . . . . . . . . . . . . . . . . . .P-0420 

Viguie C . . . . . . . . . . . . . . . . .P-0185 

Vijayan V . . . . . . . . . . . . . . . .P-0371 

Villa L . . . . . . . . . . . . . . . . . . .P-0681 

Villa-Tanaca L . . . . . . .P-0040, P-0705 

Villagómez-Castro J . . . . . . . . .P-0098 

Villamón E . . . . . . . . . . . . . . .P-0603 

Villar M . . . . . . . . . . . . . . . . . .P-0635 

Virtudazo E . . . . . . . . . . . . . . .P-0673 

Visbal G . . . . . . . . . . . . . . . . .P-0128 

Vismer H . . . . . . . . . .P-0156, P-0487 

Visser W . . . . . . . . . . . . . . . . .P-0701 

Vitale R . . . . . . . . . . . . . . . . . .P-0063 

Vitale R . . . . . . . . . . . . . . . . . .P-0157 

Vitse-Standaert A . . . . . . . . . . .P-0429 

Viudes Á . . . . . . . . . . .P-0457, P-0465 

Vivot W . . . . . . . . . . .P-0063, P-0106 

Voinovich D . . . . . . . . . . . . . . .P-0051 

Vormoor J . . . . . . . . .P-0228, P-0229 

Voyatzi A . . . . . . . . . . . . . . . . .P-0049 

Vrzalova A . . . . . . . . . . . . . . .P-0241 

Vyas J . . . . . . . . . . . . . . . . . . .P-0623 

Vybornova I . . . . . . . . . . . . . . .P-0215 

Wafa E . . . . . . . . . . . .P-0177, P-0178 

Wakasa A . . . . . . . . . . . . . . . .P-0752 

Walker R . . . . . . . . . . . . . . . . .P-0569 

Waller J . . . . . . . . . . . . . . . . . .P-0489 

Walls L . . . . . . . . . . . . . . . . . .P-0598 

Walsh T . . . .O-0032, P-0376, P-0390 

Wamae N . . . . . . . . . . . . . . . .P-0055 

Wan Z . . . . . . . . . . . .P-0067, P-0094 

Wanert F . . . . . . . . . . . . . . . . .P-0745 

Wang A . . . . . . . . . . . . . . . . .P-0379 

Wang D . . . . . . . . . . . . . . . . .P-0094 

Wang J . . . . .P-0349, P-0504, P-0660 

Wang L . . . . . . . . . . . . . . . . . .P-0583 

Wannemuehler K . . . . . . . . . . .P-0474 

Warn P . . . . . . . . . . . .P-0146, P-0158 

Watanabe A . . . . . . . . . . . . . .P-0730 

Watanabe S . . . . . . . .P-0159, P-0160, 

. . . . . . . . . . .P-0393, P-0570, P-0679 

Watanabe T . . . . . . . . . . . . . .P-0679 

Watanabe T . . . . . . . . . . . . . .P-0712 

Waters M . . . . . . . . . . . . . . . .P-0329 

Watson T . . . . . . . . . . . . . . . . .P-0011 

Welsh E . . . . . . . . . . . . . . . . . .P-0567 

Welsh O . . . . . . . . . . .P-0347, P-0567 

Weng X . . . . . . . . . . . . . . . . . .P-0349 

Wengenack N . . . . . . .P-0231, P-0655 

Wergikoskii B . . . . . . . . . . . . .P-0460 

Wheat L . . . .O-0006, P-0293, P-0431 

Whiteway M . . . . . . . . . . . . . .O-0035 

Whittle A . . . . . . . . . . . . . . . . .P-0448 

Widmer F . . . . . . . . . . . . . . . .P-0118 

Wiederhold N . . . . . . . . . . . . .P-0110 

Wilhelmi I . . . . . . . . . . . . . . . .P-0211 

Williamson P . . . . . . . . . . . . . .P-0680 

Willinger B . . . . . . . . . . . . . . .P-0432 

Wilson H . . . . . . . . . . . . . . . . .P-0507 

Winter S . . . . . . . . . . . . . . . . .P-0578 

Wintermans R . . . . . . . . . . . . .P-0363 

Witt J . . . . . . . . . . . . . . . . . . .P-0431 

Wohlfiel S . . . . . . . . . . . . . . . .P-0231 

Woods J . . . . . . . . . . .P-0633, P-0652 

Wormley Jr. F . . . . . . . . . . . . .P-0622 

Wouters L . . . . . . . . . .P-0176, P-0219 

Wozniak K . . . . . . . . . . . . . . . .P-0623 

Wright J . . . . . . . . . . . . . . . . .P-0601 

Wright L . . . . . . . . . . . . . . . . .P-0118 

Wuhrer E . . . . . . . . . . . . . . . . .P-0236 


Wulferink M . . . . . . . .P-0087, P-0161 

Xess I . . . . . . .P-0162, P-0267, P-0314 

Xi L . . . . . . . . . . . . . .P-0483, P-0706 

Xiong L . . . . . . . . . . . . . . . . . .P-0538 

Xu D . . . . . . . . . . . . . . . . . . .O-0016 

Xu L . . . . . . . .P-0575, P-0579, P-0587 

Xu X . . . . . . . . . . . . . . . . . . . .P-0706 

Xu Y . . . . . . . . . . . . . . . . . . . .P-0403 

Yadegari M . .P-0131, P-0164, P-0433 

Yaguchi T . . . . . . . . . .P-0036, P-0317 

Yamada T . . . . . . . . . . . . . . . .P-0707 

Yamaguchi H . . . . . . .O-029, P-0085, 

. . . . . . . . . . .P-0090, P-0117, P-0393 

Yamaguchi M . . . . . . . . . . . . .P-0722 

Yamanaka S . . . . . . . . . . . . . .P-0325 

Yamashita J . . . . . . . . . . . . . . .P-0496 

Yanagihara K . . . . . . .P-0277, P-0392 

Yang Y . . . . . . . . . . .P-0504, P-0571, 

. . . . . . . . . . . . . . . . .P-0660, P-0708 

Yarita K . . . . . . . . . . .P-0027, P-0036 

Yazdanbod H . . . . . . . . . . . . .P-0452 

Yazdanpanah H . . . . . . . . . . . .P-0487 

Yazgi H . . . . . . . . . . . . . . . . . .P-0664 

Yelinov N . . . . . . . . . . . . . . . .P-0155 

Yim S . . . . . . . . . . . . . . . . . . .P-0502 

Yong S . . . . . . . . . . . . . . . . . .P-0497 

Yoshida K . . . . . . . . . . . . . . . .P-0434 

Yoshida M . . .P-0435, P-0436, P-0606 

Yoshihiro s . . . . . . . . . . . . . . .P-0737 

Yoshizawa N . . . . . . . . . . . . . .P-0317 

Youssef S . . . . . . . . . . . . . . . . .P-0178 

Youssefi M . . . . . . . . . . . . . . . .P-0101 

Yu H . . . . . . . . . . . . . . . . . . . .P-0348 

Yucowsky M . . . . . . . .P-0315, P-0316 

Yula E . . . . . . . . . . . . . . . . . . .P-0495 

Yumyourn P . . . . . . . . . . . . . . .P-0498 

Yvonne Arantes Baccarin R . . .P-0022 

Yücel A . . . . . . . . . . . .P-0244, P-0245 

abka M . . . . . . . . . . . . . . . .P-0425 

Zabka M . . . . . . . . . .P-0415, P-0437 

Zacke A . . . . . . . . . . . . . . . . .P-0537 

Zafirovik Z . . . . . . . . . . . . . . . .P-0276 

Zaini F . . . . . . . . . . . . . . . . . .P-0709 

Zaitz C . . . . . . . . . . . . . . . . . .P-0496 

Zanardi E . . . . . . . . . . . . . . . .P-0595 

Zancopé-Oliveira R . . . . . . . . .P-0222 

Zapata B . . . . . . . . . . . . . . . . .P-0057 

Zare P . . . . . . . . . . . . . . . . . . .P-0043 

Zareh O . . . . . . . . . . . . . . . . .P-0525 

Zarnowski R . . . . . . . . . . . . . .P-0652 

Zarrinfar H . . . . . . . . . . . . . . .P-0164 

Zarzov P . . . . . . . . . . . . . . . .O-0009 

Zaugg C . . . . . . . . . . . . . . . . .P-0584 

Zavrel M . . . . . . . . . . . . . . . . .P-0656 

Zelazny I . . . . . . . . . .P-0350, P-0573 

Zelt G . . . . . . . . . . . . . . . . . . .P-0656 

Zeng J . . . . . . . . . . . . . . . . . .P-0739 

Zeng J . . . . . . . . . . . .P-0377, P-0755 

Zeraati H . . . . . . . . . . . . . . . .P-0572 

Zhang J . . . . . . . . . . . . . . . . .P-0706 

Zhang Q . . . . . . . . . . . . . . . . .P-0349 

Zheng Y . . . . . . . . . . . . . . . . .P-0755 

Zhou G . . . . . . . . . . . . . . . . . .P-0538 

Zhu L . . . . . . . . . . . . . . . . . . .P-0349 

Zilberman M . . . . . . . . . . . . . .P-0139 

Zinke H . . . . . . . . . . . . . . . . . .P-0227 

Zinker-Ruzal S . . . . . . . . . . . . .P-0671 

Zitouna M . . . . . . . . . . . . . . . .P-0217 

Zmetek Granja L . . . . .P-0613, P-0625 

Znaidi S . . . . . . . . . . . . . . . . .P-0579 

Zomorodian K . . . . . .P-0165, P-0412, 

. . . . . . . . . . . . . . . . . . . . . . .P-0572 

Zomorodian S 

. . . . . . . . . . . .P-0572 

Zurak I . . . . . . . . . . . . . . . . . .P-0438 

Zwoliñska- Wcislo M . . . . . . .O-0021 

Zychlinsky A . . . . . . . . . . . . . .P-0667 


31


ORAL PRESENTATIONS ANIMAL MYCOLOGY* 

O-0001. Kinetics of Microsporum canis adherence using an in vitro model of 

reconstituted feline epidermis 

Baldo A, Tabart (, Vermout ., /osson 2, Mignon 2 

6niversity of /iege, /iege, 2elgium 

!"#$%&'%$()*#+,"& is the main agent of dermatophytosis in dogs and cats and is 

responsible for a frequent zoonosis. The pathogenesis of the infection remains largely 

unBnown. The first pathogenic mechanism involved is the adherence of arthroconidia 

to the &-$+-()*#%$,.() followed by their germination and invasion of the cornified 

epidermis. Adherence of !/*#+,"& conidia to epidermis has never been investigated. 

The aim of this study was to determine the Binetics of fungal adherence using a new ",* 

0"-$% model consisting in reconstituted feline epidermis. Arthroconidia of the !/*#+,"& 

strain IHEM 212JK were produced ",*0"-$%. Arthroconidial suspension was spread over 

the surface of the cultured Beratinocytes and co-cultures were incubated for varying 

lengths of timeM 0, 2, O and 6 hours. The experiment was performed in triplicates. For 

each incubation time, adherent conidia were counted using a fluorescent staining. It 

was shown that the adherence of arthroconidia to feline Beratinocytes is a progressive 

phenomenon beginning as early as 2 hours post exposure (PE). The high quantity of 

adherent conidia at Oh PE suggests that the model could be useful to evaluate the 

potential effect of inhibitors of adherence. This could lead to the identification of fungal 

factors involved in !/*#+,"& adherence. 

O-0002. Incidence of porcine Pneumocystis carinii in slaughterhouse lungs in 

southern and mid-western regions of Brazil 

Cavallini Sanches E 1 , Ferreiro / 1 , Pescador C 2 , Rozza D 2 , Driemeier D 2 

1 6niversidade Federal do Rio Grande do .ul- Faculdade de VeterinZria-.etor 

Micologia, 2 6niversidade Federal do Rio Grande do .ul- Faculdade de VeterinZria- 

.etor Patologia, J 6niversidade Federal do Rio Grande .ul-Faculdade de VeterinZria- 

.etor Patologia, O 6niversidade Federal do Rio Grande do .ul-Faculdade de 

VeterinZria-.etor Patologia, [ 6niversidade Federal do Rio Grande do .ul-Faculdade 

de VeterinZria-.etor Patologia 

Summary: 1,.()%#2&-"&*#+$",""*is an opportunistic fungus currently considered a 

specific pulmonary pathogen in humans and animals. In pigs, pneumonia by 1/*#+$","" 

causes serious economical losses due to its detrimental effects on growth, food 

conversion, and carcass and viscera disposal after slaughter. The objective of this 

study was to evaluate the incidence of 1/*#+$",""*in two regions of 2razil. Grocott]s 

staining and nested PCR at mitochondrial large subnit (mt/.6) rRNA gene, revealed 

an overall incidence of J6.K_ of 1/*#+$",""3*in [6O slaughterhouse pigs. The incidence 

was of JK.K_ in pigs sampled from a slaughterhouse A located in the state of Rio 

Grande do .ul (R./.outhern region), and of JJ.K_ in pigs from a slaughterhouse 2 

located in the state of Mato Grosso (MT/Mid-aestern region), between the months of 

February and March of 200J. Among the positive cases diagnosed in R., 1/*#+$",""* 

was observed in O1.K_ of normal lungs and in [8.0_ of lungs presenting lesions, 

whereas in MT, those incidences were respectively of J6.J_ and 6J.[_. Hematoxylin 

c eosin (HE) staining of lungs positive to 1/*#+$",""3*revealed that JK.O_ had bronchointerstitial 

mononuclear pneumonia and J6.[_ interstitial mononuclear pneumonia, 

with only 1J.[_ considered normal. These results indicate the necessity of 

identification of the fungal agents and the evaluation of its importance for 2razil pig 

industry, which should allow the adoption of more efficient preventive and therapeutic 

measures, in order to minimize the incidence of pulmonary pathologies in the pigs, 

and consequently economical losses. 

The 16th Congress of the International Society for Human and Animal Mycology

O-0003. Parallel history of Pneumocystis micro-organisms and populations of 

the woodmouse, Apodemus sylvaticus 

Deville M 1 , 2erthelemy M 1 , Demanche C 2 , Gouy de 2ellocB ( J , Morand . J , Michaux ( J , 

Guillot J 1 

1 6MR INRA, AF..A, ENVA, 6PVM 2iologie Moldculaire et Immunologie Parasitaires 

et Fongiques, Ecole Nationale Vdtdrinaire deAlfort, Maisons-Alfort Cedex, France, 2 EA 

J60K, Facultd de Pharmacie, 6niversitd /ille 2, /ille, France, J Centre de 2iologie et de 

Gestion des Populations, Montferrier-sur-/ez, France 

Recent phylogenetic studies revealed a fascinating congruence of 1,.()%#2&-"& spp 

evolutive history and that of their corresponding mammalian hosts. The present study 

was included in a large scientific project whose purpose was to show that parasite and 

fungal diversity in mammalian hosts could affect genetic diversity of a selected gene 

and could modify life history traits. Parasite and fungal diversity in island populations 

was studied on the micromammal species 4'%5.)(&*&260+-"#(&. For the detection 

and characterization of Pneumocystis organisms, a total number of 202 lung samples 

from woodmice were collected in islands (J2 in .icily, [ in Corsica, [6 in Majorca, 1[ 

in Minorca) and continental areas in Europe (J8 in Italy, 1[ in France, O1 in .pain). 

Cystic forms of 1,.()%#2&-"& were detected by direct examination of impression 

smears of cross-sections of lungs stained with toluidine blue f/*The presence of* 

1,.()%#2&-"&*DNA was assessed by nested-PCR at mitochondrial large subunit 

(mt/.6) rRNA. Cystic forms*were observed in smears from 2O lung samples from 

woodmice collected on islands (28_) and from [6 lung samples from woodmice 

collected on the continent (O8_). Amplifications at mt/.6 rRNA gene were positive 

for 68 lung samples from woodmice collected on islands (78_) and from 10[ lung 

samples from woodmice collected on the continent (K1_). 1,.()%#2&-"& carriage was 

significantly lower in woodmice from islands than in those from the continent. Direct 

sequencing of nested-PCR products demonstrated a very high variability among 

woodmouse-derived 1,.()%#2&-"&*sequences with a total number of J7 distinct 

mt/.6 sequence-types. Co-infection with two sequence-types occurred in several 

animals. However, the genetic divergence among these sequence-types was very low 

and the presence of several 1,.()%#2&-"& species within 4'%5.)(&*&260+-"#(& was 

considered unliBely. The analysis of the genetic structure of woodmouse-derived 

1,.()%#2&-"&*revealed two distinct groups. The first one comprised 1,.()%#2&-"& 

from woodmice collected in continental .pain, France and 2alearic islands. The 

second one included 1,.()%#2&-"& from woodmice collected in continental Italy, 

Corsica and .icily. These two genetic groups were in accordance with the two 

lineages currently described within 4'%5.)(&*&260+-"#(& (haestern lineagei and 

hItalo-balBanici lineage). 

O-0004. Effects of galectin-3 and intestinal inflammation induced by DSS on 

gastrointestinal colonization with Candida albicans in mice 

Jawhara S 1, 2 , Thuru j 2 , .tandaert-Vitse A 1 , .endid 2 1 , (ouault T 1 , Mordon . J , 

Desreumaux P 2 , Poulain D 1 

1/aboratoire de Mycologie Fondamentale et Appliqude, /ille, France, 2Research 

Center on Inflammatory 2owel Diseases, CH6, /ille, France, JFrench National 

Institute of Health c Medical Research, /ille 6niversity Hospital, /ille, France 

7+,5"5+*+68"#+,& is both a normal commensal of the digestive tract and an 

opportunistic pathogen capable of inducing mucocutaneous and systemic infections. 

As the digestive niche represent the most common source for dissemination, the 

understanding of the interactions between 7/*+68"#+,& and the gut is important for 

controlling infections. In parallel a role for 7/*+68"#+,& has been recently suggested for 

the generation of anti-oligomannose antibodies which are marBers of Crohn]s Disease, 

a severe inflammatory bowel disease whose dtiopathogeny is still unclear. In this 

study, we addressed experimentally two interconnected questions regarding 7/* 

+68"#+,& colonization and host response. ae examined first the role of galectin-J 

since this lectin, Bnown to be expressed at the intestinal level, is a specific ligand for 

adhesins prominently expressed in 7/*+68"#+,& which consists in !-1,2-linBed 

oligomannoses. Then we assessed the influence of an experimentally induced 

inflammation and galectin-J. aild-type and BnocBout 9+6: ;

O-0005. A new in vitro model of Microsporum canis dermatophytosis in 

reconstituted feline skin 

Tabart J 1 , 2aldo A 1 , Vermout . 1 , Nusgens 2 2 , /apimre C 2 , /osson 2 1 , Mignon 2 1 

1 Department of Infectious and Parasitic Deases, Faculty of Veterinary Medecine, 

6niversity of /imge, 2elgium, 2 /aboratory of Connective Tissues 2iology, Faculty of 

Medicine, 6niversity of /imge, 2elgium 

!"#$%&'%$()*#+,"& is a pathogenic fungus causing superficial cutaneous infection 

called dermatophytosis. Though cats are the natural and reservoir host of !/*#+,"&, 

the latter is frequently isolated from other pets and is a zoonotic agent. The complexity 

of mechanisms involved in dermatophytic infections maBes ",*0"0% relevant studies 

particularly difficult to perform. Consequently an ",*0"-$% model should be developed to 

investigate interactions between !/*#+,"& and Beratinocytes, the most abundant (K[_) 

cell type in epidermis. The aim of this study is to develop a new ",*0"-$% model of !/* 

#+,"& dermatophytosis using reconstituted feline sBin (RF.), and to validate it by 

comparison with ",*0"0% observations. leratinocytes (l) and fibroblasts (F) were 

obtained from feline foetal sBin.Epidermis was reconstructed from proliferating l 

seeded on an artificial dermis made of F embedded in collagen polymers and placed 

at air-liquid interface. Histological analysis of RF. revealed a fully differentiated 

epidermis with morphological features similar to those observed ",*0"0% and composed 

of the four (basal, spinous, granular and cornified) characteristic layers. A proliferation 

assay showed replicating cells in the basal layer, indicating that RF. is a living tissue 

leading to the formation of a horny layer. RF. was inoculated with an appropriate 

amount of !/*#+,"& arthroconidia. Histopathological analysis revealed that !/*#+,"& 

invaded the stratum corneum of RF. as observed in natural infection. A fungal 

inhibition test using miconazole nitrate added to the culture medium performed using 

the model revealed that RF. presents a good permeability to the medication as 

observed ",*0"0%. In view of these results, !/*#+,"& dermatophytosis model on RF. 

seems to be a suitable tool to investigate mechanisms involved in natural !/*#+,"& 

feline infections. Additionally, RF. could be useful for testing drug efficacy in various 

feline infectious dermatoses. 

O-0006. Two Microsporum canis dipeptidyl peptidase genes with possible 

involvement in fungal virulence 

Vermout S, 2aldo A, Tabart (, /osson 2, Mignon 2 

Department of Infectious and Parasitic Diseases, Parasitology, Faculty of Veterinary 

Medicine, 6niversity of /imge, /imge, 2elgium 

In addition to being a hard to eradicate feline dermatosis, !"#$%&'%$()*#+,"&*infection 

is transmissible to humans and to many animal species. Pathophysiologic 

mechanisms underlying !/*#+,"&*dermatophytosis are still poorly understood, and if 

several Beratinases, both metalloproteases and subtilases, are probably involved in 

the pathogenesis of the infection, none has been shown to play a direct role in fungal 

virulence. The aim of this study was to investigate the potential role of so far unBnown 

!/*#+,"& proteases, i.e. dipeptidyl peptidases (DPP)n indeed, it was previously shown 

from several fungal species and other organisms that these proteases are implicated 

in the nutrition of pathogens, as well as in the modulation of the host immune 

response . Two genes coding respectively for a DPP IV and V were isolated from a !/* 

#+,"& genomic library. They were completely sequenced, as well as the corresponding 

cDNAs. The two sequences show the presence of the Gly-j-.er-j-Gly consensus 

motif and of the putative catalytic triad .er, Asp, His of the serine proteases from the 

.K family. They also show important similarity with DPPs from*>$"#?%'?2-%,*&'/3* 

4&'.$@"66(&*&'/3 and various other organisms. DPPs IV and V are secreted proteins 

and are produced on a medium containing Beratin as the sole carbon source. 6sing a 

Reverse Transcriptase (RT)-PCR technique, the proteases were shown to be 

expressed ",*0"0%*in experimentally infected guinea pig and in naturally infected cat. 

These results are the first steps leading to further functional investigations on !/*#+,"&* 

dipeptidyl peptidases as potential virulence factors. 


O-0007. Purification and characterization of 55-kda serine protease from 

Histoplasma capsulatum culture supernatants 

Zarnowski R, aoods ( 

6niversity of aisconsin, Madison, aI, 6.A 

A"&-%'6+&)+*#+'&(6+-() Darling is the etiologic agent of histoplasmosis, the most 

common pulmonary mycosis of humans and other mammals. Infection occurs through 

the inhalation of conidia or mycelial fragments. A/*#+'&(6+-() then converts to yeast 

form, infects pulmonary macrophages and replicates in phagolysosomal 

compartments. It is highly probable that the infection process and further 

dissemination of this dimorphic fungus might be escalated in the presence of tissuedisrupting 

proteolytic enzymes. However, extracellular proteases produced by A/* 

#+'&(6+-() have received relatively very little attention. 

A secreted protease was successfully purified from steady-state culture supernatants 

of A/*#+'&(6+-()*6C/A [J1.. The enzyme was isolated using a set of 

chromatographic techniques including anion exchange chromatography on DEAE FF, 

affinity chromatography on 2enzamidine-.epharose O FF, and size exclusion 

chromatography on .uperdex 200. The molecular mass of the protease was [[ BDa 

using gel filtration, which was in agreement with the results obtained from .D.-PAGE, 

indicating thereby the protease was a monomer protein. The proteolytic activity was 

assayed with various insoluble protein-dye conjugates including azocoll, azoalbumin, 

and azocasein. The optimum temperature and pH of the protease activity was O2 oC 

and pH 7.0, respectively. The enzyme was stable in a range of O to [0 oC and of pH 

from [ to 8. The enzyme was considerably inhibited in the presence of serine protease 

inhibitors liBe PM.F, leupeptin, chymostatin, and antipain, but not by T/Cl. The 

enzyme purified was thus a chymotrypsin-liBe serine protease. No inhibitory effect was 

caused by iodoacetamide indicating a lacB of cysteine/histidine residues in a catalytic 

center of this enzyme. The activity was only slightly inhibited by 1 _ .D. that enabled 

its in-gel detection. In fact, a single band with an apparent molecular mass of 60 BDa 

was observed on gelatin-supplemented zymograms. The protease could also cleave 

various substrates including collagen, albumin, and casein. The protease was 

expressed constitutively during the growth of A/*#+'&(6+-(), and was up-regulated 

under nitrogen-limited growth conditions. Iron had a minor effect, but its lacB 

negatively affected this activity. This results indicate this secreted serine protease may 

represent an important virulence factor in the development of histoplasmosis, but 

further ",*0"0% studies including construction of non-proteolytic mutants are necessary 

before final conclusions can be drawn. 

ANTIFUNGALS* 

O-0008. Voriconazole therapeutic drug monitoring (TDM) in lung transplanted 

patients 

Billaud E 1 , 2erge M 1 , Guillemain R 2 , 2atisse A 1 , Chevalier P 2 , Pham M 1 , Amrein C 2 , 

2oussaud V 2 

1 Pharmacology, H. E. G. P., Paris, France., 2 Cardiovascular .urgery, H. E. G. P., 

Paris, France 

Aspergillosis is a high risB complication in lung transplantation, specially in cystic 

fibrosis (CF) patients. Azole antifungals are currently used in prophylactic and curative 

treatments against invasive aspergillosis or 4&'.$@"66(& species colonisations. Azoles 

are highly metabolised and Bnown as CYPJAO inhibitors, with potential clinical 

implications via pharmacoBinetic metabolic interactions. Voriconazole (VRZ) was 

introduced without TDM recommendation, arguing its good bioavailability (K6_) and a 

classical half-life (6h) as compared to itraconazole (ITZ). 

ae introduced the 6V-HP/C determination of VRZ plasmatic concentrations in order 

to - manage the relative intensity of drug interactions between calcineurin inhibitors 

(CNI) and azolesn - assess the achievement of therapeutic VRZ levels in CF 

transplanted patients. 

The incidence of aspergillosis morbidity in our population was 20_. VRZ was 

administered to O8 patients since 200J. Treatment duration ranged from a few days 

up to 6 months. VRZ TDM data analysis concerned trough (C0) and peaB levels 

collected r 2h after dosing (C2). ae referred the therapeutic concentration range to 

the pivotal trials pharmacoBinetics data (C0 M 1.[r0.[ mg// - C2 M O.0r1.0 mg//). 

The recommended standard dosage (200 mg/day 2ID following loading dose) of this 

non linear pharmacoBinetics drug was able to achieve significant levels in 20_ CF 

patients sdetectable level t 0.2, 4'.$@"66(&*&&' MIC t 0.[ mg//u. Doses up to 800 

mg/day appeared necessary to reach detectable levels. Trough VRZ concentrations 

still remained v 0.[ mg// in 20_ patients after one weeB. This regimen was higher in 

CF as compared to non CF or non transplanted patients. The contribution of higher 

clearance and possible lacB of absorption in CF youngest patients were expected, but 

the variability justified an individualized approach. This also occurred using IV route, 

underlying VRZ pharmacoBinetics variability, maybe enhanced by CYP2C1K 

polymorphism. RisB of inefficacy during underdosed periods was supplied by 

antifungal combinations (caspofungin). 

VRZ concentrations correlated significantly (pv0.1) with aspartate aminotransferase. 

fther hepatic enzymes were related in case of previous hepatic alteration. 

Regarding the azoles-CNI interactions 

" VRZ acted as a significant although less stronger inhibitor than ITZ.* 

" >+#$%6")(&*#%,#.,-$+-"%,*-%*5%&.*$+-"%*B+&*C/D*#*E/5*G,HI5JL*+,5*5/D*#*M/N* 

G,HMDJ

into account when switching routes during co-prescriptions, in addition to the 

correction applied from relative oral bioavailibility. 

" 72#6%&'%$",*#%,#.,-$+-"%,*-%*5%&.*$+-"%*$.)+",.5*(,#?+,@.5*+-*RD*G,HI5J5($",@* 

KO*$%(-.*B"-?*K>L. 

" Dramatic changes in azole concentrations impact the interaction magnitude and 

subsequently adjustment requirements. 

Despite the lacB of TDM recommendation, we suggest the determination of VRZ 

concentrations in order to manage azoles interactions in lung transplantation or other 

patients such as HIV, exposed to strong CYPJAO inhibitors (protease inhibitors) 

and/or inducers (rifampicin) altogether with azoles. Antifungal combinations represent 

an intensive strategy, waiting for documented therapeutic azole levels. Despite the 

absence of recent invasive aspergillosis mortality in our population, further 

investigations are needed to validate this costly approach. 

O-0009. Inhibition of the ubiquitin-proteasome pathway as a new strategy for 

developing antifungal molecules 

Bougeret C, Zarzov P, 2riand (, 2arbey ., Thomas D 

Cytomics .ystems 

Cytomics .ystems is a biopharmaceutical company engaged in the discovery and 

development of new therapeutic molecules that control the degradation of proteins, to 

treat fungal infection and cancers. /everaging several years of academic research, 

Cytomics .ystems is utilizing T8"=#$.., w , a proprietary cell-based high-throughput 

screening technology to discover drugs controlling protein turnover by the 6biquitin 

Proteasome pathway. The 6biquitin-Proteasome pathway was recently demonstrated 

to control many important facets of the cell biology and dysfunctions of this pathway 

are now Bnown to be involved in many diseases such as cancer, inflammatory and 

neurodegenerative diseases. Cytomics .ystems T8"=#$.., w screening technology 

relies on the use of genetically engineered yeast cells and provides the company with 

functional screening assays that replicate the entire and complex activity of the 

cellular ubiquitin-proteasome pathway. 

The ubiquitin-proteasome pathway is essential for the growth and survival of 

pathogenic fungi such as 7+,5"5+*+68"#+,&, 7+,5"5+*@6+8$+-+ and 4&'.$@"66(&* 

U()"@+-(&. Cytomics .ystems has identified a fungal cell regulator whose degradation 

by the ubiquitin-proteasome pathway is absolutely required for the survival of fungal 

cells. Noteworthy, this fungal regulator is a fungal specific protein, found in most 

pathogenic fungal species and absent from human cells. 

Cytomics .ystems has developed a high-troughput screening assay to identify 

inhibitors of the degradation of this fungal specific target. 6sing its T8"=#$.., w 

technology, Cytomics .ystems performed a large scale screening of its diversified 

compound library and identified several series of molecules that were potent inhibitors 

of the degradation of the target protein. Analoging and hit to lead optimization studies 

allowed to select for J lead compounds that were next demonstrated to strongly inhibit 

the ",*0"-$% proliferation of a wide range of pathogenic fungi. Noteworthy, these 

molecules were shown to be more active than fluconazole (Diflucan w ). K,*0"0% studies 

demonstrated that these J molecules were well tolerated in animals and were active at 

a single dose of about 1 mg/Bg in an acute model of disseminated candidiasis in mice. 

These J leads compounds are now entering pre-clinical development. 


O-0010. A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, 

is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal 

resistance in Candida albicans 

Coste A 1 , Turner V 1 , Ischer F 1 , Morschhxuser ( 2 , Forche A J , .emelcBi A J , 2erman ( J , 

.anglard D 1 

1 Institute of Microbiology, /ausanne, .witzerland, 2 6niversitxt ayrzburg, Germany, 

J 6niversity of Minnesota, MN, 6.A 

Tac1p is a 7+,5"5+*+68"#+,& transcription factor necessary for the upregulation of the 

A2C-transporter genes 7VPI and 7VPM, which mediate azole resistance. ae 

showed previously the existence of both wild-type and hyperactive >47I alleles. aildtype 

alleles, isolated from azole-susceptible strains, mediate upregulation of 7VPI 

and 7VPM upon exposure to inducers such as fluphenazine, while hyperactive alleles, 

isolated from azole-resistant strains, result in high constitutive expression of 7VPI 

and 7VPM. In this study>47I alleles were recovered from two pairs of matched azolesusceptible 

(D.Y2KOn FH1M heterozygote at mating type locus) and azole-resistant 

isolates (D.Y2K6n FHJM homozygous at mating type locus). Five new >47I alleles 

were recovered. The susceptible D.Y2KO strain contains two wild-type*alleles >47IW: 

and >47IWE while the resistant D.Y2K6 strain contains a single hyperactive allele 

>47IW5. A single amino acid (aa) difference between >47IWE*and >47IW5 (NK77D) 

was observed in a region corresponding to the predicted activation domain of Tac1p. 

Two >47I alleles*>47IWN and >47IWC were recovered from FH1 and a single 

hyperactive allele >47IWC was recovered*from FHJ. The NK77D change was also 

seen in >47IWC. The importance of NK77D in conferring hyperactivity to >47I was 

confirmed by site-directed mutagenesis. Furthermore, drug resistance properties of 

the >47I alleles were not affected by the mating type*status of the strains. 2oth 

>47IW5 and >47IWC hyperactive alleles were co-dominant with wild-type alleles, and 

only had hyperactive phenotypes when homozygous liBe in strains D.Y2K6 and FHJ. 

To asB how /fH at the >47I locus and fluconazole resistance occurred, we followed 

the ",*0"-$% acquisition of fluconazole resistance in strain FH1 by isolation of two FH1- 

derived fluconazole-resistant strains from two successive rounds of incubation on 

fluconazole-containing medium. These two strains showed a stepwise increase in 

fluconazole resistance. Interestingly, the strain with highest fluconazole MIC was 

homozygous at the >47IWC and !>X$ loci. Comparative Genome Hybridization and 

.ingle Nucleotide Polymorphism analyses were performed on those two laboratory 

isolates but also on the 2 pairs of clinical isolates to determine the >47I and 

chromosome [ statuses of all these strains. ae demonstrated that /fH at the >47I 

and !>X loci occurred by recombination between portions of chromosome [ in the 

clinical isolates but by chromosome [ loss and duplication in the laboratory isolates. 

This difference might reflect a cost in fitness for such events to happen in the host 

conditions. 

O-0011. Amphotericin b inhalation powder (ABIP) achieves significant 

pulmonary and low systemic amphotericin b concentrations 

Kugler A, /ee (, .amford /, Gerety R, Eldon M 

NeBtar Therapeutics 

Background: A2IP is being developed as an effective, safe, and convenient therapy to 

prevent pulmonary fungal infections in immunosuppressed patients. A2IP is a highly respirable 

dry-powder, formulated to have an aerodynamic size to deliver amphotericin 2 (Am2) to the 

same airways and alveolar surfaces of the lungs on which fungal spores deposit and germinate. 

A2IP is administered with a proprietary, handheld, dry-powder inhaler device and doses 

containing up to 2[ mg Am2 have been well-tolerated. Targeted drug delivery to the lung is 

expected to maintain lung tissue Am2 concentrations sufficient to prevent opportunistic 

pulmonary fungal infections in immunosuppressed patients while avoiding the dose-limiting side 

effects and drug-drug interactions observed with systemic use. Proof that A2IP protects against 

4&'.$@"66(&*U()"@+-(& infection, while producing low systemic Am2 exposure, has been 

demonstrated in a persistently neutropenic efficacy model in rabbits. In this study, human 

pulmonary and systemic Am2 pharmacoBinetics following single inhaled doses of A2IP were 

investigated. 

Methods: Eight healthy subjects received single [-mg inhalation doses in this open-label, safety, 

tolerability, and pharmacoBinetic (Pl) study of A2IP. .tandard safety measurements, including 

pulmonary function, were monitored throughout the 17 weeBs of the study. .even venous blood 

(predose, 0.[, 1, 2, O, 8, 2O hr postdose) and 2 bronchoalveolar lavage (2A/n zearly] 1-12 hr and 

zlate] 21-J2 hr postdose) samples per subject were collected, and used to characterized systemic 

(plasma) and pulmonary (epithelial lining fluid sE/Fu) Am2 pharmacoBinetics. Plasma and 2A/ 

Am2 concentrations were determined using validated /C-M./M. methods. .erum/2A/ urea 

concentration ratios were used to quantify E/F dilution. 

Results: No clinically relevant changes from baseline for serum chemistry parameters, vital 

signs, or spirometry (FVC and FEV 1 ) were observed. Adverse events (AEs) were rated as mild or 

moderate in severity with no reported serious AEs. Mean plasma Am2 Cmax was 21.7 ng/m/ 

(rangeM 8.7 to JJ.1 ng/m/) and occurred at 8 hr. These Am2 concentrations were well below 

those associated with renal toxicity (generally sustained plasma Am2 concentrations of greater 

than 1000 ng/m/) and were largely consistent with laboratory animal results. The maximum 

observed E/F Am2 concentration was [0.O kg/m/ and concentrations declined with a 6-10 hr 

half-life, also consistent with animal results. 

Conclusions: This trial demonstrated that inhaled A2IP was well-tolerated at pulmonary Am2 

concentrations significantly above common 4&'.$@"66(& spp. minimum inhibitory concentrations 

and were accompanied by low systemic Am2 exposure. Maximal E/F Am2 concentrations were 

{1000-fold higher than systemic Am2 concentrations, demonstrating that A2IP pulmonary 

delivery achieves a localization of Am2 exposure to the site of greatest importance for preventing 

pulmonary fungal infection, the lung. Maintenance of pulmonary Am2 concentrations in excess of 

MICs during the entire period of pulmonary fungal infection risB in immunosuppressed patients, 

while minimizing systemic Am2 exposure, should be possible with repeated A2IP dosing. A2IP 

is a promising therapy for prevention of pulmonary fungal infections in patients at risB due to 

immunosuppressive therapy including those receiving organ or stem cell transplants, or treated 

with chemotherapy for hematologic malignancies.

O-0012. The development of an in vitro model of invasive aspergillosis to 

determine the effectiveness of voriconazole 

Mowat E 1 , 2utcherr ( 2 , ailliams C J , Ramage G O 

1 Glasgow Caledonian 6niversity, 2 Glasgow Caledonian 6niversity, J YorBhill Childrens 

Hospital, O Glasgow Caledonian 6niversity 

Purpose of the study: Aspergillosis is associated with high rates of mortality, ranging 

from J0 up to K0_ in some instances. Examination of bronchopulmonary lavage and 

histology shows numerous hyphae which intertwine forming a coherent networB of 

hyphal elements, which are similar to other fungal biofilms. 

Summarized description of the project: The objective of this study was to develop 

an ",*0"-$%*system which modelled the structural characteristics of invasive 

aspergillosis as a means of examining the effects of voriconazole under different 

experimental conditions. .pores were harvested from NCPF 7J67and other clinical 

isolates. These were standardised in RPMI at different densities (10 O , 10 [ , 10 6 c 10 7 ) 

and seeded onto K6-well polystyrene microtitre plates for selected times (1, 2, O, 6, 8, 

2O h). 2iofilm growth Binetics were determined using a metabolic (jTT) and a biomass 

assay (crystal violet). Antifungal susceptibility testing was then performed with 

voriconazole. The effects upon biofilm viability and development were assessed when 

voriconazole was added at 0, 1, 2, O and 2O h post conidial inoculation. Confocal and 

scanning electron microscopy was also performed. 

Results and conclusions: Conidia concentrations of 1 % 10 [ /ml produced optimal 

biofilms structures, with similar appearance to ",*0"0%*histological data. 2iofilm depths 

ranged from 117 | J60 &m. Those biofilms with lower seeding density were thicBer 

and with longer hyphae. Microscopic and quantitative analysis demonstrated that all 

biofilms developed similarlyn phase 1) conidia germinated into hyphaen phase 2) 

hyphal elements intertwined, forming a monolayern phase J) biofilm forms with threedimensional 

structure and matrix material. Voriconazole treatment of mature biofilms 

(2O h) was ineffective (MIC [0 128 ! t [12 &g/ml), whereas pre-treatment with 

voriconazole exhibited dose dependent activity (MIC [0 0.[ ! O &g/ml). K0_ inhibition 

of biofilm biomass was demonstrated at all concentrations t 1 &g/ml. 2iofilms formed 

with lower conidial density were more susceptible to treatment, and once replenished 

with anti-fungal free media, the biofilm formation was defective and unstable. ae have 

developed a reproducible and robust assay to analyse treatment of 4/*U()"@+-(&* 

biofilms. The results suggest that treatment of mature 4/*U()"@+-(& biofilms with 

voriconazole is an ineffective approach. However, pre-treatment of conidia prevents 

hyphal formation and subsequent development of an invasive form of 4/*U()"@+-(&/* 

This indicates that a prophylactic use of voriconazole to susceptible patient groups 

may be an important measure to consider in the management of these patients. 

O-0013. Sequential combination therapy using liposomal amphotericin b and 

micafungin for systemic murine aspergillosis 

Olson J, George A, Adler-Moore ( 

California .tate Polytechnic 6niversity, Pomona, 6.A 

Background: The incidence of invasive aspergillosis has continued to increase, with the 

mortality rate remaining high even with the introduction of new antifungal agents. For patients 

whose condition continues to worsen despite monotherapy, the addition of a second agent may 

be helpful. Previous studies have suggested that combination antifungal drug therapy can be 

helpful, but it can also be antagonistic, depending on the regimen. In this study we examined the 

sequential use of liposomal amphotericin 2 (/Am2, Am2isome') and the echinocandin, 

Micamine' (Mc) at different dose levels, in a murine model of systemic aspergillosis. 

Methods: .wiss aebster female mice (7 wBs old) were immunosuppressed IP with 100 mg/Bg 

cyclophosphamide J days prior to fungal challenge and every J days throughout the study. fn 

day 0, mice were challenged IV with 4&'.$@"66(&*U()"@+-(& ([.7 x 10exO conidia/mouse). Mice 

(1O/group) were treated IV, 2Oh post-challenge, with [_ dextrose (D[a controls), 10 or 1[ 

mg/Bg /Am2 or [ mg/Bg Mc daily for 6 days (monotherapy). Mc at [mg/Bg was selected since 

previous studies had shown lower efficacy with 10 or 1[ Mc in this model. .equential therapy 

consisted of J daily treatments with /Am2 followed by J days of Mc (/Am2/Mc) or J daily 

treatments with Mc, followed by J days of /Am2 (Mc//-Am2). Mice (7/group) were followed for 

morbidity for 28 days and the other 7 mice/group were euthanized 2Oh after the last drug 

treatment. Their Bidneys were homogenized in 1.0 ml P2., and plated on .abouraud]s agar for 

determination of /og 10 CF6/g Bidney (/-CF6). Aliquots of Bidney homogenates were also 

evaluated for galactomannan index (GI) using the Platelia enzyme immunoassay. Results were 

evaluated with a laplan-Meier analysis, lrusBal-aallis one-way ANfVA and a Mann-ahitney 

two-tailed T-test. 

Results: 

Treatment 

mg/BgM 

/Am2 

10 

/Am2 

1[ 

Mc 

[ 

/Am210 

/Mc [ 

/Am21[ 

/Mc [ 

Mc [ 

//Am210 

Mc [ 

//Am21[ 

.urvival 86_ 100_ [7_ 100_ 100_ 86_ 86_ 0_ 

/-CF6 0.J0 0.16 2.6K 0.KJ 0.26 2.KK 2.21 2.82 

GI 0.687 1.120 [.O0 1.O7 0.60 6.00 [.8J [.8[ 

D[a 

Control mice were all moribund by day 7 while all treatments produced similar survival rates 

(86_-100_), except for Mc alone which resulted in [7_ survival. aith monotherapy, fungal 

burden was statistically significantly lower in mice given 10 or 1[ mg/Bg /Am2 versus [ Mc (/- 

CF6, P } 0.0006n GI, P v 0.001). .imilarly, mice given /Am2 or /Am2/Mc had statistically fewer 

CF6 and lower GI than mice treated with Mc alone or Mc//Am2 (P v 0.0[). 

Conclusions: Monotherapy with /Am2 was significantly more effective than Mc monotherapy for 

treating established systemic aspergillosis based on both survival and reduction of fungal burden. 

The combination of the two agents did not provide any additional therapeutic benefit compared to 

/Am2 alone, and showed antagonism when the Mc was given first. 


O-0014. BAL8557 a water-soluble azole in a phase II double-blind esophageal 

candidiasis trial – safety, QT analysis, and pharmacokinetics of three different 

dosing regimens 

.chmitt-Hoffmann A 1 , Hardenberg ( 1 , .auer ( 1 , Viljoen ( 2 , Mitha I J , Voiriot P O , Heep 

M 1 

1 2asilea Pharmaceutica /td, 2asel, .witzerland, 2 Genclin Corp., 2loemfontein, .outh 

Africa, J 2enmed/ Pentagon Hosp., Gauteng, .outh Africa, O Cardia2ase, Nancy, 

France 

Background: 2A/O81[ is a new extended-spectrum azole antifungal in development as a prodrug 

(2A/8[[7, water soluble azole, a.A) suitable for parenteral and oral application with high 

oral bioavailability. a.A is currently in phase III clinical development for treatment of invasive 

fungal disease. 

Methods: This double-blind, multi-center, randomized phase II trial studied three oral dose 

regimens of a.A of 100 mg daily, [0 mg daily or O00 mg weeBly (doses expressed as mg 

equivalent of 2A/O81[) versus a 100 mg daily oral dose of fluconazole (F/C) in 160 patients with 

endoscopically confirmed esophageal candidiasis. All a.A groups received a loading dose on 

day one and were treated for 1O or 21 days. 2lood samples for 2O-h pharmacoBinetic analysis 

were obtained at selected centers on day 1 and EfT, and blood samples for determination of 

drug trough levels were obtained before dosing on day J and EfT. 

Results: a.A and F/C were equally well tolerated. Most adverse events were intercurrent 

infectious diseases attributable to the immunocompromised status of the patients in the study. 

No prolongation of ~T was observed for a.A (reduction of 6.[ to 8.O msec) in contrast to the 

fluconazole group (O.J msec increase of ~T C FRIDERICIA ). 

Main pharmacoBinetic resultsM 

Maintenance Dose* 

Parameter 

WSA 

WSA 50 mg/day WSA 400 mg/week 

100 mg/day 

Number of .ubjects 1J 10 12 

A6C .. (&g.h/m/) 2[.8 (# 12.6) 212 (# 106) [6.6 (# 2[.J) 

Mean A6C .. (&g.h/m/)/ dose (mg) 0.[16 0.[J0 0.[66 

A6C 0-2O (&g.h/m/) 2[.8 (# 12.6) 77.1 (# 2J.[) [6.6 (# 2[.J) 

C max (&g/m/) 1.80 (# 0.67K) 7.O6 (# 1.K7) O.[O (# 2.06) 

t max (h) 1.[ (1.2[ | J.0) 2.0 (1.[ | J.0) 1.8 (1.0 | [.0) 

Values are presented as arithmetic mean # .Dn median and range are listed for t max 

Conclusions: 2oth daily regimens of a.A as well as the once weeBly dosing regimen were as 

well tolerated as fluconazole in the treatment of esophageal candidiasis in immunocompromised 

patients. No prolongation of ~T interval was observed. PharmacoBinetics were dose-proportional 

for all dose regimens in patients and in agreement with phase I results. PharmacoBinetic 

properties as well as safety and the excellent efficacy confirmed the potential for once-daily or 

intermittent (weeBly) dosing. 

O-0015. In vitro activity of a new triazole, BAL8557, against histoplasma 

capsulatum from patients failing fluconazole 

aheat / 1 , Connolly P 1 , .medema M 1 , DurBin M 1 , Goldman M 2,J 

1 MiraVista Diagnostics/Mira2ella Technologies, Indianapolis, IN 6.A, 2 Indiana 

6niversity .chool of Medicine, Indianapolis, IN 6.A, J AID. Clinical Trials Group, 

2ethesda, MD 6.A 

Background: Amphotericin 2 is effective for treatment of histoplasmosis, but iv 

administration and toxicity detract from its usefulness. Azole compounds hold 

promise for efficacious treatment with less toxicityn however varied absorption has 

proven to be a concern with itraconazole (ITZ), and development of resistance to 

fluconazole (F/6) has been documented in the scientific literature for several species 

of fungi. Patients with AID. who were treated for histoplasmosis in AID. Clinical 

Trials Group .tudy 17O failed fluconazole therapy at a higher rate than itraconazole 

therapy. Paired pre- and post-treatment isolates (failure isolates) were available for 

17 patients failing F/6 therapy. Two of the 17 patients were defined as treatment 

failures during the induction phase of treatment, and the remaining 1[ were 

considered to have relapsed on maintenance treatment. Antifungal susceptibility in 

vitro showed a O fold or more reduction in susceptibility to fluconazole in 10 of the 17 

failure isolates. In this study, we have evaluated the in vitro activity of a new water 

soluble triazole, 2A/8[[7, along with other newer azoles for these paired A"&-%'6+&)+ 

isolates collected from patients who failed fluconazole treatment. 

* 

Method: ae performed in vitro antifungal susceptibility testing using NCC/. 

macrobroth procedures for susceptibility testing of yeasts modified for A"&-%'6+&)+* 

#+'&(6+-() to allow for a higher initial inoculum and an incubation time of 6 days. 

Antifungal susceptibility of 17 pairs of isolates was determined for three newer 

antifungal agentsM 2A/8[[7, posaconazole (Pf.) and voriconazole (VfR). The 

susceptibility testing with fluconazole was repeated alongside the newer agents. A 

significant change in susceptibility was defined as a four fold or greater increase in 

MIC. 

Results: Twelve of 17 paired isolates exhibited a four-fold or greater increase in MIC 

to F/6 between their pre-treatment and failure isolates. .even of 17 exhibited a fourfold 

or greater increase in MIC to VfR. No significant increase in MIC was seen in 

any of the isolates when tested with 2A/8[[7 or Pf.. The MIC K0 for all drugs is as 

followsM 

MIC K0 Pre-treatment MIC K0 Failure 

Fluconazole O.0 kg/ml J2.0 kg/ml 

2A/8[[7 0.007 kg/ml 0.01[ kg/ml 

Voriconazole 0.2[ kg/ml 1.0 kg/ml 

Posaconazole 0.007 kg/ml 0.007 kg/ml 

Conclusions: Isolates from patients that failed F/6 therapy exhibited reduced 

susceptibility to F/6 and VfR but showed no significant change in susceptibility to 

2A/8[[7 and Pf.. aith 2A/8[[7]s attractive solubility and bioavailability properties, 

studies in animals are warranted.

O-0016. Genome-wide heterozygote fitness test screening and mechanism of 

action studies of inhibitory compounds in Candida albicans 

ju D, Jiang B, letela T, /emieux ., 2achewich C, Veillette l, Martel N, Davison (, 

.illaots ., TrosoB . 

MercB Frosst 

7+,5"5+*+68"#+,& is the principal fungal pathogen of humans causing a spectrum of 

disease ranging from superficial sBin and mucosal infections to life threatening 

infections. Continued discovery of more efficacious antifungal agents is required to 

address this unmet medical need and alternative strategies which leverage the recent 

completion and annotation of the 7/*+68"#+,& genomic sequence provide a foundation 

for novel approaches. Here we describe a genome-wide approach to elucidating the 

mechanism of action of small molecules causing growth inhibitory effects in 7/* 

+68"#+,&. As first demonstrated in =+##?+$%)2#.&*#.$.0"&"+., heterozygote strains 

serve as sensitized target-based whole cell assays displaying chemically-induced 

haploinsufficiency to target-specific inhibitors. A 7/*+68"#+,&*Fitness Test (CaFT) 

approach was developed using a heterozygous deletion set of 2,876 individual strains 

which comprises {O[_ of the 7/*+68"#+,& genome. As each heterozygote mutant is 

molecularly tagged with unique bar-codes, the effects of inhibitory small molecules 

can be examined en masse following DNA microarray analyses. To validate this 

screening paradigm directly within a pathogenic fungus, we have examined the effects 

of a diverse set of Bnown antifungal agents (including inhibitors of lipid biosynthesis, 

RNA metabolism, cytosBeletal dynamics, translation, and secretion) whose drug target 

and mode of action are well characterized. ae also describe a novel class of 

antifungal agents predicted by the CaFT to affect microtubule dynamics and 

independently verified by genetic and biochemical means. This worB represents the 

first such demonstration of applying a genome-wide fitness test strategy outside of the 

model organism, =/*#.$.0"&"+., thus extending antifungal drug mechanism of action 

determination to the primary fungal pathogen. 

CLINICAL MYCOLOGY* 

O-0017. Liposomal amphotericin B standard dose in combination with 

caspofungin versus liposomal amphotericin B high dose regimen for the 

treatment of invasive aspergillosis in immunocompromised patients: 

randomized pilot study (combistrat trial). 

Caillot D 1 , Thidbaut A, Herbrecht R, Pigneux A, Alfandari ., Attal M, 2ernard F, 

/archd (, 2onnet 2, Mahi / 

1 CH6 de Dijon. France, 2 CH6 de /yon. France, J CHR6 de .trasbourg. France, O CH6 

de 2ordeaux France, [ CHR6 de /ille. France, 6 CH6 de Toulouse. France, 7 CH6 de 

Montpellier. France, 8 CH6 de Nancy. France, K Gilead .ciences France, 10 Gilead 

.ciences France 

Background: Monotherapy with polyenes, azoles, or echinocandins for Invasive 

Aspergillosis (IA) treatment is still associated with substantial mortality. Preclinical 

studies data suggest that combination therapy with polyenes and echinocandins may 

have additive activity against 4&'.$@"66(& species. 

Methods: ae assessed the efficacy of /iposomal Amphotericin 2 standard dose (J 

mg/Bg/d) in combination with caspofungin standard dose (CfM2f) 0.$&(&*/iposomal 

Amphotericin 2 high dose (10 mg/Bg/d) regimen (/-AM2 HD) in the treatment of IA. 

Patients with Proven or Probable IA by modified EfRTC/M.G criteria were 

randomized to receive CfM2f or /-AM2 HD until investigator-defined end of study 

drug treatment (EfT). The primary endpoint was favorable overall response (FfR) 

defined as partial or complete response assessed at EfT. .urvival was followed up to 

12 weeBs. 

Results: J0 patients with pulmonary IA diagnosis (probableM 26 | provenM O) were 

assessed in this preliminary analysisM 1[ received CfM2f and 1[ /-AM2 HD. Groups 

were well matched in terms of demographics and neutropenia was present at baseline 

in all patients. Median duration of study drug treatment was CfM2f 18 days s10-J[u 

and /-AM2 HD 17 days sO-2Ou. At day 1O, FfR was JJ_ for CfM2f and 27_ for /- 

AM2 HD (N.). At EfT, FfR was CfM2f 67_ 0&/ /-AM2 HD 27_ (p} 0.028). At 

weeB 12, FfR was 80_ for CfM2f and 67_ for /-AM2 HD (N.) and survival 100_ 

CfM2f 0&/ 86,7_ /-AM2 HD (N.). Infusion related reactions were observed in J /- 

AM2 HD patients (20_) (Flush, cervical or thoracic pain, chills, nausea). 

Nephrotoxicity (serum creatinine t2x baseline) occurred in J /-AM2 HD patients 

(20_) 0&/ 1 CfM2f (6.7_). HypoBalemia (l r vJ.0 mmol//) developed in 2 /-AM2 HD 

(1J_) 0&/ 2 CfM2f (1J_). 

Conclusion: In immunocompromised patients with underlying hematological 

malignancies, /iposomal Amphotericin 2 at J mg/Bg/d in combination with 

caspofungin as initial treatment of invasive aspergillosis demonstrated significant 

favorable overall response rate at end of treatment compared to /-AM2 given as a 

high dose regimen, although there were no differences seen at day 1O and weeB 12, 

and had a 12 weeB survival rate of 100_. To our Bnowledge this is the first 

prospective randomised study comparing antifungal monotherapy 0.$&(& combination 

in invasive aspergillosis. A large randomised controlled trial is required to confirm this 

data. 


O-0018. Increasing incidence of Candida albicans and Candida glabrata mixed 

biofilm infections in denture stomatitis patients 

Coco B 1 , Cross / 2 , 2agg ( 2 , Ramage G 1 

1 Glasgow Caledonian 6niversity, Glasgow, 6l, 2 Glasgow 6niversity Dental .chool, 

Glasgow, 6l 

Purpose of the study: Denture stomatitis, a common form of oropharyngeal 

candidiasis (fPC), results from the development of biofilm growth against the upper 

palate in the oral cavity and is associated with high rates of morbidity. The denture 

acts a reservoir of infection and may contain other 7+,5"5+*spp. such as 7/*@6+8$+-+. 

Previous studies have only used oral rinses and swabs to assess the microbiology. 

Therefore, qualitatively and quantitatively, the ecology of denture stomatitis may be 

underreported due to the adhesive nature of 7+,5"5+ biofilms attached to the upper 

denture. 

Summarized description of the project: The objective of this study is to qualitatively 

and quantitatively assess the 7+,5"5+*spp. population from denture stomatitis patients, 

to examine the ",*0"0%*gene expression of Bey virulence factors and to examine the 

effects of antifungal agents. Forty denture-wearing patients were recruited at Glasgow 

6niversity Dental .chool, and graded for the level of palatal erythema (0 -J). Dentures 

were removed and sonicated in P2. for [ min, and swish and swab samples taBen. 

Microbiological assessment of all samples was performed by plating dilutions of the 

liquid samples onto ChromAgar plates to quantify and qualify the 7+,5"5+*spp. API 

J2C was used to validate the initial identifications. Denture and swish samples were 

subsequently processed for RNA isolation, cDNA produced and RT-PCR and Real- 

Time PCR performed. The genes analysed included 4X=I3*4X=53*=41I3*=41M3* 

AY1I3*1XZM and*47>I as a houseBeeping gene. 

Results and conclusions: ff the JJ culture-positive patients sampled, K patients 

showed no signs of erythema (score } 0). ff the clinically defined denture stomatitis 

patients, 12, 8 and O patients exhibited varying levels of erythema, with scores of 1, 2 

and J, respectively. A wide variety of 7+,5"5+ spp. were isolated from the dentures 

and oral cavity of the patients examined, including 7/*+68"#+,&3*7/*@6+8$+-+3*7/*[.2U$3*7/* 

-$%'"#+6"&*+,5*=+#?$%)2#.&*#.$.0"&"+.. The predominant species isolated in this 

investigation were 7/*+68"#+,& (78_) followed by 7/*@6+8$+-+ (J0_). These were found 

in combination in 21_ of the patients examined. ~uantitative analysis of isolated 

7+,5"5+*spp. demonstrated variable plate counts for the different grades of erythema, 

ranging from 1 % 10 J | 8.JO % 10 7 cfu/sample. K,*0"0%*gene expression analysis of 7/* 

+68"#+,&*infections showed the expression of =41M3*4X=I3*1XZI and 1!4I. ae 

confirm that the 7/*+68"#+,&*is the predominant pathogen in denture stomatitis, and the 

emerging trend of mixed 7/*+68"#+,& and 7/*@6+8$+-+ infections. This study is the first 

to show that sonication procedure yields higher quantities of 7+,5"5+*spp when 

compared to oral rinsing. The production of Bey virulence factors ",*0"0%*accounts for 

varying grades of erythema encountered. The interactions between 7/*+68"#+,&*and 7/* 

@6+8$+-+*biofilm infections requires further detailed investigation to determine if their 

coaggregation has a defined role in fPC infections. 

O-0019. Malassezia folliculitis: A new entity: Report of 26 cases 

Feuilhade de Chauvin M 1 2 , /evy A 2 , Flageul 2 2 , Morel P 2 , /acroix C 1 

1 Mycology hopital .t /ouis, 2 Dermatology Hopital .t /ouis 

Described by aeary in 1K6K, Malassezia folliculitis (MF) was recognized as an entity 

by Potter in 1K7J. Itching eruption consisting in follicular papules and follicular 

pustules represent the clinical signs. The lesions are distributed mainly over the upper 

bacB and chest, shoulders and to a lesser extend over the outers aspects of the upper 

arms and on the flanBs. The prevalence of this fungal infection is not Bnown. It is 

probably a common illness. Predisposing factors in the causation of MF are not really 

established. It does not exist guidelines for treatment. .ome good results are 

observed with systemic azoles drugs but relapses are very frequent. 

The aim of this retrospective study was to evaluate epidemiological data, diagnostic 

methods of MF and the response to therapy. 

Method: Twenty six patients with a dermatosis consistent with MF were seen in 

dermatological clinic of .aint /ouis Hospital in Paris from May 2002 to april 200O. 

Diagnosis was confirmed by mycological examination and/or histopahological findings. 

Mycological examination consisted of quarrying the follicle content with a scarificator. 

The product was placed on a drop of 2lacB Chloral E solution and examinated by 

microscopy. 2iopsy specimens were stained with periodic acid .chiff and Gomori 

Grocott stains. 

Elementary clinical lesions and their distribution, duration, concomitant itching, sex, 

age, geographic origin, drugs and medical history, antifungal treatment and its results 

were retrospectively revised for each patient. 

ResultsM Twenty two patients are males and four patients are women (sex ratio [/1). 

The age median was O6 years old. Itching was present in 18 patients (70_). Five 

patients were immunocompromised (2 renal transplants, 2 bone marrow transplants, 1 

HIV). fthers patients were immunocompetent. 2efore consulting, O patients received 

topic Betoconazole and 17 patients received antibacterial and/or antiacneic drugs 

without improvement. 

Mycological examination was performed for 2O patients and confirmed the diagnosis 

in 22 cases. .Bin biopsy and histological examination realized for 12 patients showed 

folliculitis in 11 cases but confirmed MF only in [ cases. The two technics were 

realized for ten patientsM diagnosis of MF was confirmed by mycological examination 

alone for 7, by histology alone for 2 and by both methods for 1. 

Eleven patients received topic antifungal drugs (often Betoconazole)M1 cured with 

relapse, O improved, J failed, and J missing. .ystemic antifungal therapy alone was 

given for O patientsM 1 cured with relapse, 2 improved and one missing. Nine patients 

were treated by topic and systemic antifungal drugsM J cured with relapse, 1 improved, 

and [ missing. 

ConclusionM Epidemiological and therapeutic prospective studies are necessary for a 

better Bnowledge of this fungal infection.

O-0020. Candida bloodstream infection in hemodialysis 

Pyrgos V 1 , Ratanavanich l 2 , Donegan N J , Veis ( 2 , aalsh T O , .hoham . 1,O 

1 .ection of Infectious Diseases, aashington Hospital Center, aashington, DC, 

2 .ection of Nephrology, aashington Hospital Center, aashington, DC, J Department 

of Infection Control, aashington Hospital Center, aashington, DC, O Pediatric 

fncology 2ranch, National Cancer Institute, 2ethesda, MD 

Purpose: 7+,5"5+ bloodstream infections (2.I) are a major cause of morbidity and 

mortality in patients undergoing hemodialysis. To date, candidemia has not been well 

defined in this population. ae sought to better characterize the epidemiology, 

microbiology and outcomes of hemodialysis-associated candidemia. 

Summary: This was a retrospective case control study. All culture proven cases of 

Candida 2.I*occurring at aashington Hospital Center were evaluated from (anuary 1, 

2000 to .eptember 1, 200O. For each case two non-candidemic adult hemodialysis 

patients who were admitted to the hospital during a contemporaneous J months 

interval were chosen as controls. Medical records were abstracted for demographics, 

underlying illnesses, surgery and use of total parenteral nutrition (TPN), immune 

suppressive medications, antibiotics, and mode of hemodialysis (fistula, graft, 

implanted vascular catheter). Crude mortality was assessed at the end of 

hospitalization. 

Results and conclusions: During the study period J[0 cases of 7+,5"5+ 2.I were 

identified. ff these 78 occurred in 77 adult hemodialysis patients (22_). Cases and 

controls were similar with respect to age, gender, receipt of corticosteroids or 

antibiotics, and prevalence of diabetes mellitus, liver cirrhosis, surgical procedures or 

cancer. .everal variables correlated strongly with hemodialysis associated 

candidemia. Multivariate analysis found TPN (fR, 18.JKn K[_ CI, J.8[-120.1J) and 

dialysis through a catheter (fR J.2[n K[_ CI, 1.71-6.1K) to be independently 

associated with candidemia. Absolute neutrophil count below [00 cells/$/ was 

identified in O cases and no controls. The percentage of candidemic patients with 

active neoplastic diseases was higher than in controls (10.O vs. [.2_) but this 

difference did not achieve statistical significance. The distribution of Candida species 

in the hemodialysis cohort was C. @6+8$+-+ 2[.6_, C*+68"#+,& 2J.1_, C*'+$+'&"6%&"& 

2J.1_, C -$%'"#+6"& 2J.1_, and C.*[$(&." [.J_. Hemodialysis recipients had 

significantly more infections with 7/*@6+8$+-+ and 7/*[$(&."*than non-hemodialysis 

candidemic patients (J1_ vs. 17_n p } 0.00K). Conversely, 7/*+68"#+,& bloodstream 

infections were more common in non-hemodialysis patients ([J_ vs. 2J_n pv 0.0001). 

In hospital mortality was significantly elevated for candidemic (O0/77n [2_) vs. noncandidemic 

(12/1[On 7.8_) hemodialysis recipients sfR 12.7Kn K[_ CI [.78-28.8u and 

tended to be higher in candidemic hemodialysis recipients (O0/77n [2_) compared 

with candidemic non-hemodialysis patients (11J/28Jn O0_n p } 0.0[8). In conclusion 

22_ of candidemic patients in our cohort were dialysis recipients. Dialysis via a 

catheter and TPN were major risB factors for hemodialysis-associated candidemia. 

The vast majority of such infections were due to non-+68"#+,& species and in-hospital 

mortality exceeded [0_. 

O-0021. Does probiotic therapy attenuate the delay in healing of colonic damage 

induced by fungi Candida 

ZwolisBa- acislo M 1 , Budak A 2 , 2rzozowsBi T J , TrojanowsBa D 2 , Pajdo R J , Mach T 1 

1 Dept of Gastroenterology and Hepatology (agiellonian 6niversity, lraBow, Poland, 

2 Dept of Pharmaceutical Microbiology (agiellonian 6niversity, lraBow, Poland, J Dept 

of Physiology (agiellonian 6niversity, lraBow, Poland 

IntroductionM C+,5"5+*+68"#+,& frequently inhabits the gastrointestinal tract of 

humans leading to gastrointestinal candidiasis, but the studies on the relation between 

the presence of fungi and colitis are limited due to the lacB of convenient animal model 

resembling 7+,5"5+ colonization in humans. 

Aims and Methods: ae evaluated the effect of 7+,5"5+*+68"#+,& and vehicle 

inoculation on the healing of colonic damage induced by 2,O,6- trinitrobenzenosulfonic 

acid( TN2.) intrarectally ( p.r.) in rats, with or without probiotic bacteria 

/acidofil( 10 6 CF6/ml) administered intragastrically. At day 0,O,8,and 1[ upon TN2. 

application, the distal 8 cm of colon was removed, the area of damage was measured 

by planimetry, the colonic blood flow ( C2F)( H2-gas clearance), the MPf activity and 

quantitative cultures of 7+,5"5+ and expression of mRNA for proinflammatory 

cytoBines I/-beta, TNF alpha( E/I.A) were estimated in the colonic mucosa. 

Results: TN2. caused colonic ulcers, that were accompanied by an increase in 

colonic tissue weight and the number of 7+,5"5+ colonies, the significant decrease in 

the C2F, J[-fold increase in MPf activity and plasma TNF alpha, I/-beta levels as 

compared to those in vehicle- control colonic mucosa. In control animals the area of 

colonic damage decreased at day 8 and lesions disappeared after 1[ days upon their 

induction. In contrast, the colonic damage was present till 1[ day in 7+,5"5+ 

inoculated rats, followed by the fall of C2F and significant rise in MPf activity and the 

plasma TNF alpha and I/- beta levels. These effects were significantly reduced by the 

administration of /acidofil at each time intervals tested. Colonic 7+,5"5"+&"& was 

accompanied by the upregulation of I/-1 beta and TNF- alpha mRNA and this effect 

was counteracted ",*7+,5"5+- colonized rats treated with /acidofil. 

Conclusion: 

1. Candidiasis delays healing of colonic damage possibly due to the 

impairment in C2F in the ulcer area and enhanced expression and release of 

proinflammatory cytoBines such as I/-1! and TNF-$n 

Probiotic therapy with /acidofil could be useful in the treatment of ulcerative colitis 

associated with fungi infection. 


DIAGNOSTIC TOOLS* 

O-0022. Rapid detection and identification of six commonly encountered 

dermatophytes by multiplex real-time PCR 

Arabatzis M 1,2,J,O , 2ruijnesteijn van Coppenraet / 1 , luijper E 1 , de Hoog G 2 , /avrijsen 

A J , Templeton l 1 , van der Raaij-Helmer E J , VelegraBi A O , .ummerbell R 2 

1 Medical Microbiology Department, /eiden 6niversity Medical Center, /eiden, 

Netherlands, 2 Centraalbureau voor .chimmelcultures,6trecht, Netherlands, 

J Dermatology Department, /eiden 6niversity Medical Center, /eiden, Netherlands, 

O Microbiology Department, Athens Medical .chool, Athens, Greece 

Although*dermatophytic infections are regarded as relatively Bnown clinical entities, 

still a lot remains to be elucidated regarding their epidemiology and their respective 

response to treatment. Current diagnosis of dermatophyte infections essentially relies 

on microscopic evaluation of the isolate]s phenotypic characteristics. This is a lengthy, 

low sensitivity and expertise requiring process yet, identification of isolates to species 

level is a central component in understanding dermatophyte global epidemiology. fur 

objective was to develop a real-time PCR assay for rapid and reliable diagnosis of 

dermatophytoses and simultaneous species identification. 

Two assays were designed and optimisedM fne based on amplification of the IT.1 

region for detection of the >$"#?%'?2-%,*).,-+@$%'?2-.& species complex, >/* 

-%,&($+,&*and >/0"%6+#.() and a second based on amplification of the IT.2 region for 

detection of the >/*$(8$() species complex, !"#$%&'%$()*#+,"& and !/*+(5%(","". 

2oth assays employed Taqman and minor groove binding probes carrying different 

fluorophores thus allowing target discrimination. 1?%#"5*?.$'.&0"$(&*I (PhoHV-1) was 

used as internal control. .ensitivity was tested with serial DNA dilutions and specificity 

with a panel of J6 different species including all pathogenic and non pathogenic 

dermatophytes, sBin yeasts, bacteria as well as human DNA. The protocol was 

evaluated by testing blindly K2 specimens, collected prospectively from clinically 

suspicious sBin-nail-hair lesions over a period of 6 months. 

The system correctly identified all the above-mentioned dermatophyte species from 

pure culture. The analytical sensitivity of both assays was 0.1 pg, corresponding to 2.[ 

genomes per sample. All microscopy- and/or culture-positive samples (n}O0) were 

PCR positive and species (>/*$(8$()3*>/*).,-+@$%'?2-.&3*!/*+(5%(",""3*>/*0"%6+#.()) 

from positive cultures (n}2K) were correctly identified. The system also detected 7 

additional positive samples that were microscopy/culture-negative and identified 2 >/* 

$(8$() and >/*).,-+@$%'?2-.& mixed infections. 

The proposed real-time PCR assay has a high sensitivity, enables accurate diagnosis 

of six commonly encountered dermatophyte species and could potentially be 

incorporated in the clinical laboratory routine diagnostic procedures. 

O-0023. Diagnosis of pneumocystosis: significance of a positive result by realtime 

PCR 

Judith F, Antoine 2, .onia M, .ophie C, Richard F, Marie Denise /, (ean Francois M 

.ervice de Parasitologie-Mycologie, CH6 Rangueil, Toulouse, France 

OBJECTIVES: 

1. To assess the value of a routine real-time PCR technique for the diagnosis of 1,.()%#2&-"&* 

\"$%0.#" pneumonia (PCP) 

2. To compare the results with those provided by direct immunofluorescence (DIF), which is the 

standard method. 

J. For both diagnostic methods, to determine the relations between the type of result | positive or 

negative | and various clinical and laboratory parameters. 

Methods: All over 200O year, any bronchoalveolar lavage (2A/) sample received in our 

laboratory was investigated, prospectively and daily, for the presence of 1/*]"$%0.#"*DNA using a 

real-time PCR assay performed on a lightcycler w system (/arsen et al., 2002). This molecular 

diagnosis was carried out simultaneously along with DIF reference method. Finally, in order to 

assess PCRes sensitivity and specificity v. DIF, a receiving operating curve (RfC) was 

constructed. 

The second part of the study comprised J groups. First group included all subjects found positive 

by both DIF and PCR, and was termed ÄPCRrve/DIFrveÄ. The second group was made from 

patients having a negative DIF but a positive PCR result, and was coined as ÄPCRrve/DIF-veÄ. 

The third group included J[ patients randomly selected from all PCR-ve results. 

For every patient included in the study, clinical and laboratory data were retrospectively recorded 

from the personal file. 

Results: fut of O00 2A/ samples, J1 were found positive by DIF method, and 66 by PCR assay. 

No patient was found as having the pattern ÄPCR-ve/DIFrveÄ, so the first study group had J1 

subjects and the second group had J[ subjects. 

Area under RfC curve was 0.K8 with K[_ confidence interval (CI K[_ ) s0.K7-1u. The 

quantification of the 1,.()%#2&-"&*\"$%0.#" DNA was represented by the cycle threshold (Ct) 

calculated by the /ightCycler w .oftware. If the chosen Ct was 22, the specificity of real-time PCR 

was 100 _, but the sensitivity was only 7O.2 _. If the Ct was 27, the sensitivity of real-time PCR 

was 100 _, but specificity stopped at K0 _. 2etween these 2 cut-points, there was a grey zone 

in which PCR value and DIF result could be discordant, so the diagnosis of pneumocystosis only 

relied upon DIF. 

In comparison to the control group, patients in the PCRrve/DIF-ve group were more frequently 

carriers of neoplasia or haematological diseases (odds ratio sfRu}12.O, CI K[_ }s1.2-12[.2u). 

Patients from the PCRrve/DIFrve group were more frequently HIVrve (fR}22.8, CI K[_ }s1.1- 

[08.1u). Patients from both groups were more frequently hypoxemic than the control group 

(respective fR} 8.K and 11.6, CI K[_}s1.1-6J.1u and s1.1-1JJ.8u). 

In comparison to the PCRrve/IF-ve group, patients from the PCRrve/IFrve group had more 

frequently fever above J8Å[ (fR}JO.O, CI K[_}s1.1-1101.7u and every patients of this group had a 

radiological interstitial syndrome. 

Finally, every patient in the PCRrve/DIFrve group met all the diagnostic criteria of PCP. 

Conclusion: Real-time PCR was found to be an efficient routine method for PCP diagnosis, but 

some results were difficult to interpret since they were in the grey zone. A further prospective 

study, that will investigate the characteristics of patients displaying a PCRrve/DIF-ve pattern, 

appeared therefore to be indispensable.

O-0024. Molecular identification of Fusarium human clinical isolates 

Lethuillier A 1 , Gend F 2 , 2uot G 1 , Dunand ( J , Poirot ( 1 , Guarro ( 2 , Hennequin C 1 

1 /aboratoire de Parasitologie-Mycologie, Facultd de Mddecine P c M Curie, Paris, 

France, 2 Microbiology 6nit, Medicine .chool Rovira i Virgili 6niversity, Reus, .pain, 

J /aboratoire de Microbiologie, HÇpital Ambroise Pard, Paris, France 

Purpose of the study: ^(&+$"() are the molds the most frequently isolated in human 

pathogenic conditions after 4&'.$@"66(& spp. They are responsible for superficial 

infections such onychomycosis and Beratitis and more rarely deep-seated lifethreatening 

infections. ^(&+$"() identification at the species level is important for both 

a better Bnowledge of epidemiology and possibly for therapeutic adjustment 

considering the species-specific antifungal susceptibility. 6p to now, ^(&+$"() 

identification is based on morphologic traits, but is difficult due to the high degree of 

macroscopic and microscopic polymorphism or convergence within and between 

species. Following studies on plant pathogen isolates, partial sequence of Translation 

elongation factor 1 (EF1) has been proposed for ^(&+$"() species molecular 

identification. Here, we report on the use of this approach for identification of human 

clinical isolates. 

Description: Preliminary, J7 clinical isolates were examined both conventionally 

(macro and microscopic examination of colonies grown on PDA medium) and for their 

EF1 sequence (direct double strand sequencing). .equences were compared using 

blastn software against both a ^(&+$"()-specific nucleotide database and Gen2anB. 

Results and conclusions: 2ased on sequence comparison, sixteen isolates 

accounting for 6 different sequence types were identified as ^(&+$"()*%_2&'%$() with 

a percentage of identity % KK_ with sequences in the database. .imilarly, a %KK_ 

identity was observed for isolates identified as ^(&+$"()*'$%6"U.$+-() (n}2) or 

^(&+$"()*0.$-"#"66%"5.& (n}2). In all these cases, microscopic examination was in 

accordance with molecular identification. For 16 isolates which morphological 

identification was compatible with ^(&+$"()*&%6+,", sequence identity with 10 different 

^/*&%6+," sequence types ranged from K6 to K8 _. Among these, J isolates appeared 

genetically closer to `.%#%&)%&'%$+*+U$"#+,+, a teleomorph of 4#$.)%,"() spp. 

These results demonstrate the usefullness of molecular identification of ^(&+$"() but 

also illustrate the intraspecific biodiversity of ^(&+$"() human isolates. They warrant 

the enlargement of genomic databases to facilitate the recognition of human clinical 

isolates. 

O-0025. Detection of paracoccidioides brasiliensis gp70/gp43 circulating 

antigens and follow up of patients under antimycotic therapy 

Pires de Camargo Z 1 , Marques da .ilva . 2 , Colombo A J , 2lotta M O , ~ueiroz-Telles F [ , 

/opes ( 6 

1 6NIFE.P/ Cellular 2iology Division/.Éo Paulo, 2razil, 2 Evandro Chagas Institute, 

2eldm, ParZ, 2razil, J 6NIFE.P/ Division of Infectious Deseases, .Éo Paulo, 2razil, 

O 6NICAMP/Department of Clinical Pathology, Campinas, .P.,2razil, [ Department of 

Community Healthy, Medical .choll, Curitiba, ParanZ, 2razil, 6 6NIFE.P/ Immunology 

Division/.Éo Paulo, 2razil 

Paracoccidioidomycosis (PCM) is an important systemic fungal disease, particularly 

among individuals living and worBing in rural areas of endemicity in 2razil and /atin 

America, that without antifungal therapy, may develop fatal infection. In some invasive 

fungal disease, detection of circulating antigens is a useful approach to the 

serodiagnosis. Detection of 1/*8$+&"6".,&"&*circulating antigens in body fluids could 

facilitate the early diagnosis of the PCM and to confirm a preliminary diagnosis, when 

antibody detection is inconclusive. In this study, Inh-E/I.A was used to detect gpOJ 

and gp70 antigens, marBer molecules of PCM. Monoclonal antibodies produced 

against these molecules were used in independent analyses, in an attempt of 

detecting these antigens in different biological samples, such as serum, 

bronchoalveolar lavage and urine of patients with PCM, as well as in the study of 

follow-up of patients treated with Itraconazol (ITZ) or .ulfametoxazolrTrimetoprim 

(.MZrTMP). GpOJ and gp70 were detected in all the patients (81 samples) at the 

moment of diagnosis (K6.2K_ and K8.76_, respectively) with mean concentration of 

K,87&g/ml and 8,1K&g/ml, respectively. In the patients with acute and chronic unifocal 

forms, antigens were detected in 100_ of them and in chronic multifocal forms, 

antigens were detected in K[.J1_ and K8.OJ_, respectively. .era antibody titers from 

these patients were tested by ID test, and 7J patients (K0,12_) were positives. 

Positive correlation between ID and inb-E/I.A was found when antigen concentration 

and antibody titers were compared. Circulating antigens were detected in other 

biological samples. In urine samples, circulating antigens (gpOJ and gp70) were 

detected in 87,[_ of samples, ( mean concentration 8,6[ &g/ml and 8,K6&g/ml, 

respectively). Forty and four patients with PCM were follow-up during therapy. Twenty 

and three patients were treatment with ITZ (12 months) and 21 with .MZrTMP (up to 

O8 months). Circulating antigen levels decreased more quicBly in the group treated 

with ITZ (' v0,0001). At the end of the follow-up, circulating antigens were detected in 

[ patients in the group treated with ITZ and in all patients treated with .MZrTMP. 

fverall , the sensitivity and specificity of the Inb-E/I.A method showed sufficiently 

high in different biological samples, so that, we concluded that this test can be used 

for circulating antigen detection in different biological samples for PCM diagnosis and, 

also, for the follow up of patients under antimycotic therapy. 

AcknowledgementM This worB was supported by grants from FundaÑÉo de Amparo Ö 

Pesquisa do Estado de .Éo Paulo (FAPE.P). 


EPIDEMIOLOGY* 

O-0026. Circulating Coccidioides species in Mexico 

Castañón L 1 , Guerea D 2 , GonzZlez R J , /icea A O , GonzZlez G [ , ArreguÜn R 1 

1 6niversidad Nacional Autánoma de Mdxico, 2 /aboratorio de .oluciones Gendticas. 

Tijuana, 2aja California. Mdxico, J Hospital de Especialidades No. 71, IM... Torreán, 

Coahuila. Mdxico, O Centro de Investigacián CientÜfica y Educacián .uperior de Ensenada. 

2aja California. Mdxico, [ Hospital 6niversitario. 6NAN/. Monterrey, Nuevo /eán. Mdxico 

Purpose of Study: .everal studies are focused in the genealogy of the dimorphic fungi 

7%##"5"%"5.&*"))"-"&, suggesting the separation of this specie in two very defined groupsM 

Group I (non-Californian) and Group II (Californian). In year 2002, Fisher .-*+6., have 

demonstrated that in fact 7%##"5"%"5.&*"))"-"&, was a group conformed by two different 

speciesM 7%##"5"%"5.&*'%&+5+&"" sp. nov. (non-Californian), and the specie already well- 

Bnown 7/*"))"-"& (Californian), both are causal agents of the human coccidioidomycosis. 

Fisher .-*+6., have analyzed J6 Mexican strains of 7%##"5"%"5.& spp by means of PCR 

amplification of nine microsatellites regions, and have identified 28 strains as 7/*'%&+5+&"" 

and eight as 7/*"))"-"&. During 200O 2ialeB .-*+6., have analyzed 120 Mexican clinical 

isolates by means of PCR, based on the Nucleotide .equence of Antigen2/Proline-Rich 

Antigen, identifying all those strains liBe 7/*'%&+5+&"". 

aith the purpose of contributing to the epidemic Bnowledge of the illness in our country, the 

aim of our investigation was to confirm, if one or bout 7%##"5"%"5.& species were present in 

Mexico as causal agent of coccidiodomycosis. 

Description: ae have analyzed 60 clinical isolates of 7%##"5"%"5.&*spp, from Mexican 

patients with coccidioidomycosis diagnostic. The specific identification of each strain was 

carried out by means of Real Time PCR using Taqman Probes designed to detect and 

amplify .ingle Nucleotide Polymorphisms (.NPes), which can differentiate between DNA of 

both species. ae have selected four gene targets that present the best potential for a 

successful detection of the corresponding .NP]s, four .NP taqman dual probe assays 

where designedM proline 1[7, proline 17O, hexoBinase 1OK and glucose-synthase 1K2. The 

dual probe assays were designed that for all 7/*'%&+5+&"" strains can be amplified with a 

VIC fluorescent marBer, and all 7/*"))"-"& targets be amplified with a FAM fluorescent 

marBer. All reactions were carried out using the A2I 7[00 Real Time PCR and A2I Taqman 

PCR Master Mix with Rfj as a passive reference was used. All samples were run by 

triplicate. 

Results and Conclusions: According to .NP detection analysis, three strains were 

identified as 7/*"))"-"& and [7 corresponded to 7/*'%&+5+&"". The assays proline 1[7, 

hexoBinase 1OK and glucose-synthase 1K2, give consistent results on .NP differentiation 

between the species. However with the template corresponding to proline 17O we 

encountered 60_ of the samples as negative for both species. ae suspect low 

amplification efficiency either because of poor correlation with primer/sequence design and 

also the specificity of taqman MG2 probes in this .NP, referring to a second mutation 

and/or a different nucleotide that the reported. ae intend to further this study via DNA 

sequencing for this particular gene region. 

According to other authors, we coincide that in Mexico exists more than one 7%##"5"%"5.& 

species that cause coccidioidomycosis and the agent more frequently associated to the 

illness is 7/*'%&+5+&"". ff the three identified cases as 7/*"))"-"&, the patients lived or they 

worBed in 6.A, for that is highly possible that the isolated of 7/ "))"-"& obtained are not 

endemic of our country. 

O-0027. Genotyping by amplified fragment length polymorphism (aflp), matingtype- 

and serotype diversity, and susceptibility to fluconazole among dutch 

isolates of cryptococcus neoformans 

Hagen F 1 , Hoepelman A 2 , 2overs M 1 , luijper E J , .panjaard / O , 2oeBhout T 1 

1 C2., dept. Yeast Comparative Genomics, 6trecht, The Netherlands, 2 6MC6, dept. 

Acute Medicine and Infectious Diseases, 6trecht, The Netherlands, J /6MC, dept. 

Medical Microbiology, /eiden, The Netherlands, O AMC, dept. Medical Microbiology, 

Amsterdam, The Netherlands 

Introduction: 7$2'-%#%##(&*,.%U%$)+,&, an encapsulated basidiomyceteous yeast, is 

frequently implicated in meningoencephalitis in HIV-positive individuals and other 

immunocompromised patients. In the Netherlands, 7/*,.%U%$)+,& was found to be 

implicated in 21J patients with cryptococcosis between 1K77-200O. A major increase 

in the incidence of the disease was observed from 1K87, due to the increasing number 

of AID. patients. The introduction of highly active antiretroviral therapy (HAART) in 

1KK6 resulted in a significant decrease of patients with cryptococcosis. Here we 

present data on AF/P genotypes, mating-types, and serotypes of these Dutch isolates. 

In addition, susceptibility to fluconazole was investigated for part of the isolates. 

Materials and methodsM 2OO isolates of 7/*,.%U%$)+,& from The Netherlands were 

analyzed with standard AF/P protocols. Mating- and serotypes were determined by 

PCR using primer sets derived from the =>aIM, =>aMQ and 914I genes. 

Fluconazole susceptibility of OJ ramdonly selected isolates was tested by the E-test 

method. 

Results: AF/P genotypes 1, 12, 2, J, O and 8 were present with 7K.2, O.7, 11.O, 2.1, 

0.[, and 1.0_ respectively. The majority of isolates (80.2_) belonged to serotype A 

MATalpha, whereas 11.[_ was found to belong to serotype D (86.O_ MATalpha, 

1J.6_ MATa). AD hybrids were present in O.6_ on the population with both subtypes 

alphaAaD (8[.7_) and alphaDaA (1O.6_). fne 7/*@+--"" was found and had matingtype 

alpha. Two patients were infected with an alpha2aD hybrid between 7/* 

,.%U%$)+,&*and*7/*@+--"". All sero- and mating-type combinations were observed 

between 1K87 and 1KK6, whereas lower diversity of mating- and serotype 

combinations were isolated during 1K77-1K81 and after 1KK7. Fluconazole resistance 

was observed in 28_ of OJ randomly selected isolates that mostly belonged to AF/P 

genotype 1. 

Conclusions: The 7/*,.%U%$)+,& population in The Netherlands consisted mainly of* 

7/*,.%U%$)+,& var. @$(8"" (serotype A) and to a lesser extent var. ,.%U%$)+,& 

(serotype D) and hybrids between these varieties. .urprisingly, we discovered 

previously unBnown hybrids between 7/*,.%U%$)+,& and 7/*@+--"". The largest 

variation in mating- and serotype diversity occurred during the time in which the 

highest number of AID. patients occurred. Fluconazole resistance was observed in 

almost J0_ of the isolates investigated.

EPIDEMIOLOGY* 

O-0028. Mycetoma in Iran 

Hashemi J 1 , Nasrollahi A 2 , Parsa . 1 

1 .cience c Research 2ranch-Islamic Azad 6niversity, Tehran, Iran, 2 ToneBabon 

Islamic Azad 6niversity 

Mycetoma is a chronic, granolumatous disease of sBin and subcutaneous tissue, 

which sometimes involves bones, muscle and neighboring organs. 

Generally infective agent presents in soil or on vegetable as saprophytes. Mycetoma 

usually occurs injury prone part of body. In 1K62 mycetoma has been first reported in 

Iran by Medical Mycology Department of Tehran 6niversity. 

ae retrospectively compared the overall prevalence of mycetoma and the prevalence 

of infective agent in Iran during O0 years. 

In our study age, sex, job, infective agent and the site of infective have been 

considered. 

It should be mentioned that the geographic distribution of the pathogenic agent 

determine by the annual rain fall. In north of Iran with rainy and humid status, the 

prevalent agent is Nocardia which leads to actinomycetoma. 

According to our study among 62 cases of mycetoma OO(71_) of them was fungi and 

1O(22.6_) was bacteria. The peaB age for infection was between O0-[0 years old but 

there is no a significant differences between age and infective agent (Pt0/0[). In our 

study among 62 cases which reported 6O.[_ of them were male and JJ/K_ of them 

were female. 

The single most common site of infection is foot but generally hand and other limb can 

infected. [O/O_of infected area is palm and it can infected other area with less 

frequent. 

fur results shows that farmers are at greater occupation risB of mycetoma. Among 

our cases O[/2_ of them were farmers. 

O-0029. Recent developments in the molecular subtyping of anthropophilic 

dermatophyte fungi 

Jackson C 1 , MochizuBi T 2 , 2arton R J 

1 Institute ff 2iological .ciences, Aberystwyth, 6l, 2 Department of Dermatology, 

lanazawa Medical 6niversity, 6chinada, (apan, J Mycology Reference Centre, 

Division of Microbiology, 6niversity of /eeds and General Infirmary, /eeds, 6l 

The ribosomal DNA (rDNA) spacer regions have well recognized applications in both 

the molecular taxonomy and strain identification of dermatophyte fungi. ae have 

developed novel molecular subtyping methods for two anthropophilic species based 

on polymorphic regions of the nontranscribed spacer (NT.) of the rDNA. In the first 

method, a 2K6bp region of the NT. of >$"#?%'?2-%,*$(8$() was identified which 

contained two variable polyguanine microsatellites. The first satellite had either K, 10 

or 11 guanine residues, whilst the larger satellite had five variants of between 18 | 22 

guanine residues. In addition, the DNA flanBing these microsatellites was found to 

contain 1O single nucleotide polymorphisms (.NPs). .even of the .NPs were 

transitions and seven transversions. A single J base indel was seen in one isolate. 

Typeabilty by this method was high, with each of ten random clinical isolates of >/* 

$(8$() sampled having a unique combination of microsatellite variations and .NPs. 

In the second method, existing RF/P]s in the rDNA of >$"#?%'?2-%,*).,-+@$%'?2-.& 

var ",-.$5"@"-+6.*(Tmi) were localized to the NT. region (1) , where sequence analysis 

identified three independant polymorphic subrepeat elements (.RE]s). The first 

variable locus, containing the .RE Tmi.0, was unique to Tmi whilst the second two 

.RE]s, Tmi.1 and Tmi.2, had counterparts in >/*-%,&($+,& and/or >/*$(8$()/ PCR 

fingerprinting of each polymorphic .RE produced in combination a total of 1O 

individual strain profiles from J0 random isolates of Tmi from the 6l, (apan and India. 

2oth the >/*$(8$() and Tmi typing systems provided a high index of discrimination, 

were reproducible and simple to interpret. These methods provide valuable 

epidemiological tools for the molecular subtyping of two dermatophyte species which 

often appear genetically invariant by many genotyping techniques. 

References: 

MochizuBi TIH, 2arton RC, Moore Ml, (acBson C(, lelly ./, Evans EG. Restriction 

fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful 

for differentiating strains of >$"#?%'?2-%,*).,-+@$%'?2-.&. ( Clin Microbiol. 

200JnO1MO[8J-O[88. 


O-0030. Candidaemia amongst Australian intensive care unit patients: Results 

from a 3-year nationwide surveillance 

Playford G 1, 2 , Nguyen ~ J , Chen . 2,O , Ellis D [ , .lavin M 6 , .orrell T 2,O , Marriott D J 

1 Princess Alexandra Hospital, 2risbane, Australia, 2 6niversity of .ydney, .ydney, 

Australia, J .t Vincent]s Hospital, .ydney, Australia, O aestmead Hospital, .ydney, 

Australia, [ aomen]s and Children]s Hospital, Adelaide, Australia, 6 Royal Melbourne 

Hospital, Melbourne, Australia 

Background: The importance of Candidaemia amongst IC6 patients is increasing, 

although considerable geographic variations in the incidence, species distribution, and 

outcomes have been reported. As part of a Australian nationwide surveillance 

program, all episodes of IC6-associated Candidaemia were studied. 

Methods: The Australian Candidaemia .tudy involved a prospective populationbased 

surveillance of all episodes of Candidaemia occurring within Australia over a J- 

year period (August 2001 to August 200O). Clinical variables and blood culture 

isolates were collected. IC6-associated cases were defined as adult non-neutropenic 

patients with IC6 length of stay at least O8 hours. 

Results: Amongst K18 episodes of Candidaemia during the surveillance period, 20_ 

(18J) were IC6-associated. The overall incidence of Candidaemia was 2.O8/1000 

admissions (K[_CI 1.8[-J.2[), with considerable institutional variation (0.O6-[.O1). No 

change in incidence occurred over the surveillance period. .pecies distribution 

included 7+,5"5+*+68"#+,& (62_ of isolates), 7/*@6+8$+-+ (17_), 7/*'+$+'&"6%&"& (8_), 

7/*-$%'"#+6"&*(6_), and 7/*[$(&."*(O_). Median time to development of Candidaemia 

was 8 days. Although intravascular devices were the source for J[_ of episodes, the 

source for O6_ remained undefined. Recognised risB factors for Candidaemia were 

common, including recent antimicrobial therapy (KK_), another healthcare-associated 

infection (7O_), recent surgery (68_), and TPN use (O[_). Prior use of systemic 

antifungal agents was uncommon (K_) and was associated with*7/*@6+8$+-+ and 7/* 

[$(&.". Fluconazole was used as empiric antifungal therapy in 7[_ of episodes. O6_ 

of patients died, mostly (70_) within the first 10 days following diagnosis. 

Considerable variation in management following diagnosis was observedn including 

removal of intravascular devices (2[_), ophthalmological examination (26_), followup 

blood cultures (6O_), and duration of antifungal therapy (mean 10.8 days). 

Conclusions: Candidaemia in Australian critically-ill patients demonstrates 

institutional variation according to case-mix and local therapeutic practices. Although 

7+,5"5+*+68"#+,& remains the commonest species in Australian IC6s, the incidence of 

7/*@6+8$+-+ may require reconsideration of fluconazole for empiric therapy. Poor 

clinical outcomes may relate to the observed failure to identify high-risB patients who 

may benefit from early antifungal therapy and from variation in clinical management. 

GENOMICS* 

O-0031. Genes regulated by human steroid hormones in the human pathogenic 

fungus Candida albicans - a genome-wide study. 

Banerjee D 

(awaharlal Nehru 6niversity, New Delhi, India 

Purpose of study: The opportunistic fungal pathogen 7+,5"5+*+68"#+,& causes 

severe and recurrent human infections, especially in immuno-compromised patients. 

fne of the conditions that enhance such infection is the presence of human steroid 

hormones such as !-estradiol and progesterone as seen in cases of vulvo-vaginal 

candidiasis (VVC). The cascade of events leading to the induction of genes after 

steroid exposure in 7/*+68"#+,& is poorly understood. In the absence of a Bnown 

steroid receptor signalling in 7+,5"5+, in this study, an attempt was made to resolve 

the molecular basis of steroid signalling by employing genome-wide analyses. 

Summarized description of the project: 7/*+68"#+,& (.C[J1O) cultures in mid log 

phase (f.D 1.[-2.0) were either treated with 1mM concentration of progesterone 

dissolved in ethanol or mocB treated with ethanol alone. After J0 min of treatment, the 

cells were harvested at once at room temperature and snap-frozen in liquid nitrogen. 

Total RNA was isolated and transcriptional profiling was carried out by using cDNA 

microarrays obtained from Eurogentec containing K8_ of the 7/*+68"#+,& fRFs. The 

data analysed included O replicates, including 2 biological replicates and two dye 

swap experiments. Genes were defined as being significantly regulated, if they were 

regulated by at least a factor of 1.[. The gene annotation of Candida D2 

(httpM//genilist.fr/CandidaD2/) was used. Northern blots were done to confirm overand 

under-expression of some of the genes. 

Results and discussion: It was observed that human steroid hormones considerably 

enhanced the expression of multi-drug resistance (MDR) genes belonging to both 

ATP 2inding Cassette*G7VPI*and*7VPM) and Major Facilitator (Ca!VPI) superfamilies 

of multidrug transporters. Interestingly, in spite of the upregulation of MDR 

genes due to steroid exposure, 7/*+68"#+,& cells became sensitive to azoles and other 

tested drugs. As a consequence of steroid exposure, in our experiments, several 

genes were affected. Microarray results revealed that a total of KK genes were 

significantly regulated by progesterone, of which 60 were up regulated while JK were 

down-regulated. The steroid regulated genes included genes associated with hyphal 

induction, involved in evasion of host immune response and establishment of 

pathogenesis. An i,*&"6"#% search for various TF binding sites in the promoter of the 

affected genes revealed that a^9I3*71AI3*`P9I3*>T1I3*!K9I*and*41WI regulated 

genes are predominantly responsive to steroid treatment. The conserved steroid 

response region (.RR) reported earlier by us (AAGAA, CCGAA and ATTGG), and the 

stress responsive elements (.TREn AG O or C OT) were also found in the promoters of 

several responsive genes. fur present data sheds new light on the regulation of gene 

expression by human steroid hormones and its possible correlation with drug 

resistance and morphogenesis.

GENOMICS* 

O-0032. The functional genomics of innate host defense against important 

human pathogens: Candida albicans and Aspergillus fumigatus 

Cortez K 1 , lim H 1 , Choi E 1,2 , lhan ( 1 , /empicBi R J , Roilides E 1 , ChanocB . 1 , lottilil 

. O , /yman C 1 , aalsh T 1 

1 

Pf2, NCI, NIH, DHH., 2ethesda, Maryland, 6.A, 2 .eoul National 6niversity, College of 

Medicine, .eoul, lorea, J .AIC-FredericB, NCI, NIH, DHH., Maryland, 6.A, O /IR, NIAID, NIH, 

DHH., 2ethesda, Maryland, 6.A 

Purpose of the studyM Monocytes and macrophages play a pivotal role in host defense against 

invasive candidiasis and aspergillosis. The human innate host response against 7/*+68"#+,& and 

4/*U()"@+-(& is mediated by intracellular destruction of such pathogens and the release of 

immune-related molecules from phagocytic cells. The regulatory pathways and expression 

profiles of the multiples genes mediating the initial immune response to these fungal pathogens 

are not well understood. Applying DNA microarray technology to the molecular genetics of the 

innate immune system may provide new insights into the Binetics and profile of multiple 

regulatory pathways involved in the initial response of these cells to 7/*+68"#+,&*and 4/*U()"@+-(&/* 

DescriptionM Freshly isolated peripheral blood monocytes from ten healthy human donors were 

incubated with either opsonized*7/*+68"#+,& for 0 to 18 h or 4/*U()"@+-(& conidia for 0 to 6 h in 

parallel with time-matched uninfected controls. Total RNA was extracted, amplified and 

hybridized onto human genome cDNA or oligonucleotide microarray chips. After the chips were 

scanned, images obtained and normalized, differential expression of genes considered 

significant by 2 way ANfVA for both experiments were selected. 

Results and conclusionsM 7/*+68"#+,&-induced gene expression Binetics showed enhanced 

expression of proinflammatory cytoBines (TNF-$, I/-1, I/-6, and /IF) during the first 6 h 

corresponding to an increase in phagocytosisn these genes returned to near baseline by 18 h.*7/* 

+68"#+,&W",5(#.5*gene expression of chemoBines, (I/-8n MIP-1, MIP-J, MIP-O, and MCP-1), with 

peaB expression at O to 6 h, along with chemoBine receptor genes (CCR1, CCR[, CCR7, and 

CjCR[). Expression of genes involved in monocyte viability (2C/2-related protein, 

metallothioneins, CD71, and .fC.J) was up-regulated at O to 6 h and remained elevated 

throughout the 18-h time course. In contrast, expression of genes encoding T-cell-regulatory 

molecules (I/-12, IFN-(, and TGF-à) was unchangedn whereas, genes encoding I/-1[, I/-1J 

receptor (I/-1JRa1), and CD1O were suppressed during the 18-h exposure to 7/*+68"#+,&. 2y 

comparison,*4/*U()"@+-(& induced up-regulation of a different array of genes involved in innate 

host response, specifically I/-1!, I/-8, I/-10, CjC/2, CC/O, CC/J, CC/20, long pentraxin J, and 

matrix metalloproteinase 1 coinciding with an increase in phagocytosis. Expression of 

CC/[/RANTE., ficolin1 and MARCf were simultaneously down-regulated by 4/*U()"@+-(&*but 

not by 7/*+68"#+,&. *DNA microarray analysis of human monocytes was validated by real-time 

qPCR analysis of selected genes and the Binetics of gene expression was confirmed at the 

protein level by E/I.A (I/-8, CC/[, CC/O, and MMP-1). 

O-0033. Toward understanding regulatory networks affected by iron availability 

in Candida albicans 

LAN C 1 , Rodarte G 2 , Murillo / 2 , (ones T J , Davis R J , Dungan ( 2 , Newport G 2 , Agabian 

N 2 

1 Institute of Molecular and Cellular 2iology, National Tsing Hua 6niversity, 

HsinchuJ001J, Taiwan, 2 Department of Cell and Tissue 2iology, 6niversity of 

California, .an Francisco, CAKO1OJ, 6.A, J .tanford Genome Technology Center, 

Palo Alto, CAKOJ0O, 6.A 

Iron, an essential element for almost every organism, serves as a regulatory signal for 

the expression of virulence determinants in many proBaryotic and euBaryotic 

pathogens. 6sing a multiplex real time RT-PCR method, we detected the iron gene 

regulation from low-abundance transcripts derived from clinical isolated 7+,5"5+* 

+68"#+,&. The results indicate that several iron-related genes are differentially 

expressed in the isolates causing candidiasis vs. the commensal ones. In addition, 

using a custom Affymetrix GeneChip ) representing the entire 7/*+68"#+,& genome, we 

examined the changes in genome-wide gene expression in this pathogen as a 

function of alterations in concentrations of iron in the growth media. A total of [26 

fRF transcripts are more highly expressed when the levels of available iron are low, 

while 626 fRF transcripts are more highly expressed in high iron conditions. The 

transcripts dominantly affected by iron concentration range from those associated with 

cell surface properties to others which affect mitochondrial function, iron transport and 

virulence-related secreted hydrolases. Moreover gene expression as assayed in DNA 

microarrays confirms and extends reports of alterations in cell surface antigens and 

drug sensitivity correlated with iron availability. To understand how these genes and 

pathways might be regulated, we isolated a gene designated =^TI*that encodes a 

homolog of the T&-"6+@%*)+25"& 6R2.1, a transcriptional repressor of siderophore 

uptaBe/biosynthesis. Comparisons between wild-type and =^TI-null mutant strains 

revealed 1JK potential target genes of .fu1pn many of which are iron-responsive. 

.earching consensus sequences within the regulatory regions of iron-regulated and/or 

.fu1-regelated genes was also performed. Together, these results not only expand 

our understanding of global iron regulation in 7. +68"#+,&, but also provide insights into 

the potential role of iron availability in 7/*+68"#+,&*virulence. 

ReferencesM 

/an .-*+6. (200O) Mol Microbiol, [J M1O[1-1O6K. 

/an et al. (2006) In preparation. 

7/*+68"#+,&*and*4/*U()"@+-(& induced distinct patterns of genes encoding innate host defense 

molecules, including proinflammatory cytoBines, chemoBines, and receptors. ahile*7/*+68"#+,& is 

a potent inducer of a dynamic cascade of expression of genes whose products are related to the 

recruitment, activation, and protection of neutrophils and monocytes, exposure to conidia of 4/* 

U()"@+-(& resulted in the expression and secretion of different cytoBines, chemoBines and other 

molecules involved with phagocytosis and adherence, suggesting distinct biologic pathways of 

innate immune mechanisms involved in host defense to these pathogens. 


O-0034. Genome-wide screen identifies genes implicated in hypersensitivity to 

miconazole in Saccharomyces cerevisiae 

Thevissen K 1 , FranÑois I 1 , De 2rucBer l 1 , Dispersyn G 2 , Ausma ( 2 , 2orgers M 2 , 

Cammue 2 1 

1 CMPG, latholieBe 6niversiteit /euven, Heverlee, 2elgium, 2 2arrierTherapeutics nv., 

Geel, 2elgium 

Purpose of the study: Miconazole belongs to the azole antifungals, which inhibit 

ergosterol biosynthesis. 2esides inhibition of ergosterol biosynthesis, miconazole 

induces reactive oxygen species (Rf.) in susceptible fungi, leading to fungal cell 

death s1, 2u. Apparently, the observed Rf.-increase upon miconazole treatment 

results from inhibition of fungal catalase and peroxidase, enzymes implicated in Rf. 

detoxification s2u. It is currently not clear whether additional cellular processes are 

affected by the miconazole-induced Rf.-increase and how miconazole tolerance in 

yeast is regulated. 

Description of the project: ae screened the complete set of haploid 

=+##?+$%)2#.&*#.$.0"&"+. gene deletion mutants for hypersensitivity to miconazole. 

Results and Conclusion: ae identified 22 genes/open reading frames, which when 

deleted, conferred at least O-fold hypersensitivity to miconazole. These 22 genes were 

designated miconazole tolerance genes. Major functional groups encode proteins 

involved in tryptophan biosynthesis, transport (endocytosis/secretion), gene 

expression, cytosBeleton organization, sphingolipid/sterol biosynthesis and phosphate 

metabolism. fnly J of these 22 miconazole tolerance genes were implicated in 

hypersensitivity to the non-Rf.-inducing antifungal fluconazole. The remaining 1K 

genes were hence designated zmiconazole-specific] tolerance genes. The linB 

between tryptophan biosynthesis, transport, gene expression and cytosBeleton 

organization on one hand and miconazole antifungal action (comprising ergosterol 

biosynthesis inhibition and Rf.-inducing potential) on the other hand will be 

discussed further. 

References 

lobayashi et al., 2002. Antimicrob. Agents Chemother. O6MJ11J-J117 

FranÑois et al., 2006. Anti-Infect. Agents Med. Chem. [MJ-1J. 

O-0035. A complete haploid assembly of the Candida albicans genome 

van het Hoog M 2 , Rast T 1 , MartchenBo M 2 , .cherer . J , Mageel 2 1 , Dignard D 2 , /i Z 2 , 

2erriman M O , Chibana H [ , ahiteway M 2 , Nantel A 2 , Magee P 1 

1 6niversity of Minnesota, Dept. of Genetics, Cell 2iology and Development, .t. Paul, 

6.A, 2 2iotechnology Research Institute, National Research Council, Montreal, ~C, 

Canada, J JKJ8 Paseo Grande, Moraga, 6.A, O aelcome Trust .anger Institute, 

Hinxton, 6l, [ Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba 

6niversity, Chiba, (apan 

The .tanford Genome Technology Center (.GTC) published a 10.7 fold whole 

shotgun sequence of the 7/*+68"#+,& .C[J1O genome in 200O ((ones et al., PNA., 

200O). The large number of conserved gene families, repeated sequence elements, 

and divergent alleles, coupled with the absence of high-density genetic or physical 

maps had limited the extent of the genome assembly to 266 individual supercontigs. 

ae applied a variety of strategies to produce a final version of the 7/*+68"#+,& genome 

assembly with all its elements properly positioned along the eight chromosomes. 2y 

purifying individual chromosomes with pulse-field gel electrophoresis followed by 

hybridization to DNA microarrays, we were able to distribute 18J supercontigs 

(representing K8.O_ of the sequences) among eight distinct chromosomal groups. 

Furthermore, K supercontigs had been misassembled, containing marBers from 2 

different chromosomes. A superassembly (Assembly 20) was constructed using a 

synteny analysis with preliminary versions of the 7/*5(86",".,&"&*genome assembly 

being constructed at the .anger Center. The contigs were verified and their potential 

orientation and order on each chromosome was determined by using a sequencetagged 

site (.T.) map of overlapping fosmid clones. The release of all the individual 

sequence traces by the .GTC allowed the application of the Phrap assembly software 

to these smaller chromosome groups, with the closing of gaps and great reduction of 

the number of supercontigs. Repeat regions that were too large to be covered by a 

sequence run, such as the ribosomal DNA cluster and the MR. and its subunits, were 

attached to the unique sequence using the .T. map. The same procedure was used 

to place telomeres and to validate each potential junction. The overall assembly was 

compared to an optical map (fpGen, Madison, aI) which identified some 

misassembled contigs and gave a rough size estimate of the telomeres and rDNA 

region. Ambiguities among the three methods were resolved by PCR amplification and 

gap sequencing. ae have produced eight linear DNA sequences totaling 1[.8O6 Mb 

that are interrupted by K MR.s, 2 R22s, the rDNA and 1[ gaps. Gene annotation data 

from the community annotation (2raun et al., P/o.-Genetics, 200[) and subsequent 

updates produced by Candida Genome Database (Arnaud et al., Nucleic Acids Res, 

200[) have been mapped to these new sequences and are currently being validated. 

fne of the most interesting features of the 7/*+68"#+,& genome is the association of 

members of the 7>4 gene family with 1J out of 16 telomeres. These genes encode 

putative transcription factors and are absent from the genome of the closely related 

but less virulent species, 7/*5(86",".,&"&/ 

.upported by Contract N01-0[O76 awarded to PTM, CIHR grant MfP-O2[16 to Ma 

and CIHR grant HfP-67260 to AN.

IMMUNOLOGY* 

O-0036. Characterization of proteins recognized by an anti-Candida albicans 

monoclonal antibody in human tumor tissues 

Abad A 1 , RamÜrez A 1 , .an .egundo I 1 , Garcia P 1 , fchoa D 1 , Ponton ( 1 , 2arrenetxea 

G 2,J , Hernando F 

1 Dpto. InmunologÜa, MicrobiologÜa y ParasitologÜa. 6niversidad del PaÜs Vasco. /eioa. 

.pain, 2 Dpto. Especialidades Mddico-~uirârgicas. 6niversidad del PaÜs Vasco. /eioa. 

.pain, J ClÜnica ~uirán 2ilbao .. /. 

Purpose: 2asing on the existence of a cross-reactivity between proteins of 

pathogenic yeasts and human tumor cells this project tries to identify the proteins of 

human ovarian and uterus tumor tissues that are recognized by a monoclonal 

antibody created against a mannoprotein of C. albicans, looBing for MAb recognition 

differences between healthy and tumor samples. 

Description: There have been described some monoclonal antibodies created 

against C. albicans mannoproteins which cross-react specifically with human tumor 

related proteins. 

ae used in this study the MAb C7, developed against a C. albicans mannoprotein of 

more than 200BDa, which has already demonstrated a cross-reaction with He/a cell 

line of cervix adenocarcinoma. They have been used human tissue samples of ovary 

and uterus and He/a cell line. Complete protein extractions were separated by twodimensional 

electrophoresis. The reactivity against the antibody was evaluated by 

aestern blotting with the chemiluminescence detection system EC/-plus. 

The results were analyzed with the Image Masters 2D Platinum program of 

Amersham 2iosciences, looBing for similarities between tumor tissues and the cell line, 

and differences of both with healthy tissues. After the analysis, those proteins 

recognized by the antibody in the tumor tissues were identified by MA/DI-TfFF/TfFF. 

The peptide mass fingerprint data were then searched in databases for final 

identification of peptides. 

Results and Conclusion: ae have demonstrated a cross-reaction of the MAb C7 

between Candida albicans and human ovarian and uterus proteins, by twodimensional 

electrophoresis (2DE) and immunobloting, and identified the proteins 

recognized in the tumor sample. According to a functional classification most of these 

proteins correspond to cytosBeletal proteins, and oxidorreductases, but there are also 

some isomerases and lyases, one nucleic acid-binding protein and one transfer/carrier 

protein. 

Two of the identified proteins in the tumor tissue were also identified and recognized 

by MAb C7 in He/a cell line. These proteins have a molecular weight of J[-[[BDa, but 

one of them is localized in a basic area and the other around [-[,[ pI. 

O-0037. Expression of the costimulatory molecules CD28/CTLA4, CD80/CD86, 

ICOS and PD-1 on T-cells and monocytes from paracoccidioidomycosis (PCM) 

patients 

Cacere C 1 , Romano R 1 , Mendes-Giannini M 2 , Fontes C J , lono A O , Duarte A 1 , 

Bernard G 1 

1 /aboratorio de Dermatologia e Imunodeficiencias, FM6.P, .ao Paulo 2razil, 

2 /aboratorio de Micologia Clinica, 6NE.P-Araraquara, .ao Paulo, 2razil, J Hospital 

6niversitario, 6niversidade Federal do Mato Grosso, .ao Paul, 2razil, O Divisao de 

Doencas Infecciosas e Parasitarias, HC-FM6.P, .ao Paulo, 2razil 

Introduction: PCM patients present a variable degree of suppression of the antigen-specific 

cellular immune responses, which usually correlates with the severity of the clinical presentation. 

Aiming to understand better this T-cell anergy, we analyzed the expression of several 

costimulatory molecules on CD1Or and CDJr cells. 

Methods: P2MC isolated from patients with active disease (n}2[) and from healthy individuals 

cured of PCM (cured controls, n}1[), were cultivated in presence of gpOJ, the immunodominant 

1/*8$+&"6".,&"& antigen, and as a control fungal antigen, 7+,5"5+*+68"#+,&*metabolic*antigen 

sCMAu. CDJr and CD1Or cells were analyzed by flow cytometry for the expression of 

costimulatory molecules. /ymphoproliferation was assessed by tritiated thymidine incorporation. 

Results: /ymphoproliferative assays confirmed previous data showing decreased gpOJ- but not 

CMA-induced response in patients as compared to controls. .ignificantly more patients T-cells 

expressed CT/AO, either upon stimulation with gpOJ or CMA, or without any exogenous stimuli. 

However, neither gpOJ nor CMA could up-regulate CT/AO expression when compared to nonstimulated 

T-cells. CD28 expression was high and comparable among patients] and controls] T- 

cells. /ymphoproliferation assays were performed in controls] and patients] P2MC in the 

presence and absence of a neutralizing anti-CT/AO antibody. The depressed patients] responses 

could not be redressed with the antibody. This suggests that enhanced CT/AO expression does 

not play a major role in PCM T-cell anergy. The percentages of CD1Or cells expressing CD80 

and CD86 did not differ between patients and controls. However, significantly more patients] T- 

cells expressed these costimulatory molecules than controls] cells. Preliminary experiments show 

that blocBing of CD86 signaling with a neutralizing antibody did not enhance the proliferative 

responses to gpOJ by patients] cells, but decreased the positive responses from controls] P2MC 

to either CMA or gpOJ. 2locBing the CD80 signaling did neither affect the positive proliferative 

responses of patients] cells nor redressed the decreased responses of patients] cells. 

Expression of ICf. and PD-1 were also comparable between patients] and controls] T-cells, and 

were not enhanced with gpOJ or CMA stimulation. 

Conclusion: Previous studies suggest that CT/AO plays a down-regulatory role in T cell 

responses of patients with PCM. Although we observed increased expression of CT/AO in 

patients] T cells, this expression could not account for the diminished proliferative responses. 

The unexpected increased expression of CD80 and CD86 in patients] T-cells may represent an 

alternative immunosuppressive mechanism, although preliminary experiments using neutralizing 

anti-CD80 or anti-CD86 antibodies do not suggest this hypothesis. Further experiments with the 

combined used of the antibodies are warranted. fur data also suggest that PD-1 and ICf. do 

not play a major role in the T-cell anergy of PCM patients. 

ae have also noticed a differential recognition of one protein by the MAb C7 between 

tumor and nontumor samples, being higher in tumor tissues and He/a cell line, 

smaller or null in benign tumors and absolutely null in healthy tissues. This could be 

due to an overexpression of this protein in tumor tissues. These results give us the 

chance to study this protein role as a tumor marBer or therapeutic target. 

Financial supportM Fapesp, CNPq and /IM-HC. 


O-0038. Translationally controlled tumour protein from madurella mycetomatis, 

a marker for tumorous mycetoma progression 

van de Sande W 1 , (anse D 1 , Hira V 2 , Goedhart H J , van der Zee R O , Ahmed A [ , ftt A 1 , 

Verbrugh H 1 , van 2elBum A 1 

1 Erasmus MC, 6niversity Medical Centre Rotterdam, Department of Medical 

Microbiology c Infectious Diseases, Dr. Molewaterplein O0, J01[ GD Rotterdam, The 

Netherlands, 2 Erasmus MC-.ophia Childrenes Hospital, 6niversity Medical Centre 

Rotterdam, Department of Paediatrics, The Netherlands, J .t. Elisabeth ZieBenhuis, 

Department of Pathology, HilvarenbeeBseweg 60, [022 GC Tilburg, The Netherlands, 

O 6trecht 6niversity, Department of Infectious Diseases and Immunology, P.f. 2ox 

80.16[, J[08 TD 6trecht, The Netherlands, [ Mycetoma Research Group, Institute of 

Endemic Diseases and Faculty of Medical /aboratory .ciences, 6niversity of 

lhartoum, lhartoum, .udan 

About forty years ago antibodies against the fungus !+5($.66+*)2#.-%)+-"& were first 

demonstrated in eumycetoma patients, who are suffering form a disease characterised by 

tumourous swellings. To date nothing is Bnown about the individual immuno-reactive antigens 

present in this fungus. ae here identify its first immuno-dominant antigen, a protein homologous 

to the translationally controlled tumour protein (TCTP), a well-conserved histamine release factor 

in a range of euBaryotes/*Many functions have been recorded for TCTP. This protein appears to 

be involved in cell cycle control, stress responses, histamine release and interleuBin production. 

It is also Bnown to bind the anti-malarial and anti-cancer drug artemisinin. 

The gene for this antigen was demonstrated to be present in two variants in !/*)2#.-%)+-"&, with 

1J_ amino acid difference between the two proteins encoded. Differences were also recorded 

for the two TCTP signature sequences. TCTP variant I had a deviated TCTP2 signature 

sequence and TCTP variant II had a deviated TCTP1 signature sequence. The two variants were 

similarly represented in our collection with variant I found in [J_ of our isolates and variant II in 

O7_/ The two proteins shared epitopes, since variant II of the protein could be recognised by 

antibodies raised against variant I. In vitro, TCTP was secreted into the culture medium. In vivo, it 

was found to be expressed at the fungal cell surface in developing stages of the eumycetomacharacteristic 

blacB grain but not in completely developed grains. 

MOLECULAR BIOLOGY* 

O-0039. The CEK1 and HOG1 MAP kinases play opposite roles in cell wall 

biogenesis and chlamidospores formation in Candida albicans 

Alonso-Monge R, Eisman 2, RomZn E, Arana D, Nombela C, Pla ( 

6niversidad Complutense de Madrid 

The Hog1 MAP Binase mediates an adaptive response to both osmotic and oxidative 

stress in the fungal pathogen 7+,5"5+*+68"#+,&. This protein also participates in two 

distinct morphogenetic processes, namely the yeast-to-hypha transition (as a 

repressor) and chlamydospore formation (as an inducer). ae show here that 

repression of filamentous growth occurs both, under serum limitation and under other 

partially inducing conditions, such as low temperature, low pH or nitrogen starvation. 

To understand the relationship of the HfG pathway to other MAP Binase cascades 

that also play a role in morphological transitions, we have constructed and 

characterized a set of double mutants in which we deleted both the Ab9I gene and 

other signalling elements (the 7=>MQ, 7X4E and A=>C Binases, the 71AI and a^9I 

transcription factors and the 711I protein phosphatase). ae also show that Hog1 

prevents the yeast-to-hypha switch independently of all the elements analysed and 

that the inability of the ?%@I mutants to form chlamydospores is suppressed when 

additional elements of the 7acI- pathway (7=>MQ or A=>C) are altered. Finally, we 

report that Hog1 represses the activation of the CeB1 MAP Binase under basal 

conditions and that CeB1 activation correlates with resistance to certain cell wall 

inhibitors (such as Congo Red), demonstrating a role for this pathway in cell wall 

biogenesis. 

!/*)2#.-%)+-"&*TCTP was shown to be antigenic in mice, rabbits and humans. .ignificant IgG 

and IgM immune responses against the whole protein and selected !/*)2#.-%)+-"&Wspecific 

peptides were determined. The antibody levels correlated with lesion size and disease duration. 

fverall, the patients with the largest lesions had the highest antibody titre, the titre lowering with 

decreasing size of the lesion. After 6-1[ years of disease duration the antibody titres were the 

highest. 6nfortunately some cross-reactivity, especially with the whole protein, was observed 

between the mycetoma patients and the .udanese healthy control population. Cross-reactivity 

appeared to be less when peptides were used which were developed on !/*)2#.-%)+-"&*specific 

domains in the TCTP sequence. Cross-reactivity appeared to be associated with cross-reactivity 

against other endemic TCTP producing organisms. 

TCTP is the first well-characterised immuno-dominant antigen and although TCTP may not be 

the best diagnostic tool, the E/I.As presented here could be useful in seroprevalence studies. In 

addition, studies into the influence of TCTP on mycetoma development or its use as a vaccine in 

the prevention of infection are urgently warranted.

O-0040. Molecular characterization of mating in Histoplasma capsulatum 

2ubnicB M 1 , Smulian G 1,2 

1 6niversity of Cincinnati College of Medicine, Cincinnati, fH, 6.A, 2 VA Medical 

Center, Cincinnati, fH, 6.A 

Purpose of the Study: Although mating of the heterothallic dimorphic fungus, 

A"&-%'6+&)+*#+'&(6+-() has previously been reported and morphological changes 

associated with mating described, molecular characterization of mating has not been 

performed. ae sought to identify mating associated genes and characterize the 

molecular changes associated with mating. 

Description: Two mating types designated (|) and (r) were previously described 

based on phenotypic mating assays. Prior studies demonstrated a 1M1 ratio of (r) and 

(-) isolates in soil samples but a 7M1 ratio of (-) M (r) mating types in clinical isolates. 

Through syntenic analysis of the A"&-%'6+&)+*genomic sequence databases, we 

identified the mating type idiomorphs, a MAT1-1 locus in A/*#+'&(6+-() lab strains 

G2172 and a62O, and a MAT1-2 locus in the lab strain G186AR. The mating loci 

each contained a single open reading frame encoding a putative transcript encoding a 

transcription factor containing an alpha-box or and HMG domain respectively. Genes 

encoding putative pheromone receptors, a putative pheromone and a MAP Binase 

postulated to function in pheromone response pathway were identified. To further 

characterize the molecular events associated with mating, we sought to characterize 

expression of these genes. Transcription of each mating type gene was 

demonstrated by RT-PCR in a mating-type restricted manner. Transcription of both 

pheromone receptors, the putative pheromone and the pheromone responsive MAPl 

was demonstrated under basal conditions. /aboratory strains reportedly lose the 

ability to mate following prolonged passage, thus we sought to identify fresh isolates 

of opposite mating type. A PCR based assay was developed to amplify either the 

MAT1-1 or the MAT1-2 locus specifically, providing a mechanism to determine the 

mating locus types of unBnown A/*#+'&(6+-() strains. ae typed available clinical 

isolates and identified clinical isolates of both MAT1-1 and MAT1-2 types. These 

strains were able to mate when placed adjacent to each other on alpha celluloseyeast 

extract agar allowing molecular characterization of the mating process. To 

confirm the previously reported mating type disequilibrium in clinical isolates, the 

mating type PCR assay was applied to several paraffin-embedded lung sections with 

granulomatous lesions. A"&-%'6+&)+ infection was initially confirmed using 

A/#+'&(6+-() specific PCR primers. The mating type was then determined using a 

sensitive nested PCR approach. The MAT1-1 locus was identified in [ of K samples 

where A/*#+'&(6+-() DNA was detected while the MAT1-2 locus was not detected in 

any of the samples. 

Results and Conclusions: 2oth idiomorphs of the mating locus of A/*#+'&(6+-() 

were identified. The previously reported mating type disequilibrium was confirmed at 

a molecular level and suggests that the (-) mating type corresponds to the presence of 

the MAT1-1 mating type locus. Additional components of the signaling cascade 

associated with mating were identified and gene expression characterized. These 

studies will form the basis for future characterization of the mating response and allow 

examination of a mating type association with virulence in A/*#+'&(6+-()/ 

O-0041. Quantitative proteomic and transcriptional analysis of C. albicans 

response after macrophage interaction 

Fernández-Arenas E, Cabezán V, Nombela C, Gil C, Díez-Orejas R 

Department of Microbiology, Pharmacy faculty, Complutense 6niversity of Madrid, .pain. 

Purpose of the study: The encounter between Candida albicans and phagocytes 

(macrophages) is considered the initial step in the development of host immune defenses. The 

single utility of proteomic and genomic approaches to study host - pathogen interaction has been 

previously described in the literature (1, 2, J)n but up to now few studies have employed both 

techniques as complementary approaches. 2ecause of the importance of the macrophages in 

the innate immune response against fungal infectious, we have investigated the transcriptional 

profiling and the differential protein expression (using a proteomic approach) of C. albicans after 

the interaction with these phagocytes. 

Description: ae have developed an in vitro system by employing the murine macrophage cell 

line RAa26O.7 and the wild-type Candida albicans yeast strain .C[J1O. The study of the 

differential C. albicans protein expression in these conditions (Jh. of interaction) has been 

performed by 2D-PAGE. The comparative analyse of the bidimensional silver stained gels 

obtained, was performed using the bioinformatic software Melanie J. A complementary approach 

for this proteomic worB is the study of the yeast transcriptional profiling after two time points of 

interaction (1.[ and Jh). ae have analyzed differential C. albicans gene expression using 

Microarrays from Eurogentec. 

Results and conclusions: A total of 1O0 protein species differentially expressed have been 

detectedn 8J with increased expression and [7 with their expression decreased. The most 

interesting proteins up and down regulated, have been identified by MA/DI-TfF TfF ([K 

proteins) and classified according to their biological function.The majority of these proteins 

belong to different metabolic pathways. 6p-regulated proteins belong to oxidation of fatty acids 

and detoxification, meanwhile, other metabolic proteins belonging to glycolysis, fermentation and 

gluconeogenesis are clearly down-regulated. Proteins belonging to cell rescue, defence and 

virulence, proteosome and oxidative stress response are also up-regulated. A total of [16 C. 

albicans differentially expressed genes have been detected after 1,[ and Jh of macrophage 

interaction (20K over-expressed and J07 under-expressed). The yeast transcriptional response 

pointed out an increase in genes belonging to oxidative stress, metal homeostasis, DNA damage 

repair, lipid metabolism and pathogenesis and a down-regulation profile in genes belonging to 

cytosBeleton, morphogenesis, metabolism (carbohydrate, amino acid and nucleotide) and 

vesicular-vacuolar transport. 

The results obtained after this proteomic/genomic analyses indicate that a great percent of these 

proteins/genes belong to different cellular pathways showing the attempt of C. albicans to avoid 

being engulfed by macrophages, and once inside, points out the hostile environment that 

surrounds the yeast. 

References: 

/orenz MC, 2ender (A, FinB GR. Transcriptional response of Candida albicans upon 

internalization by macrophages. EuBaryot Cell. 200O fctnJ([)M1076-87. 

Fradin C, De Groot P, MacCallum D, .challer M, llis F, fdds FC, Hube 2. Granulocytes govern 

the transcriptional response, morphology and proliferation of Candida albicans in human blood. 

Mol Microbiol. 200[ Aprn[6(2)MJK7-O1[. 

MartÜnez-.olano /, Nombela C, Molero G, and Gil C. Differential protein expression of murine 

macrophages upon interaction with Candida albicans. Proteomics, in press. 


O-0042. Translating stress – on the role of eIF2 kinases in Aspergillus 

fumigatus pathogenicity 

Krappmann S 1 , .asse C 1 , Grosse V 1 , 2raus G 1 , 2ignell E 2 , lim . J 

1 Institute of Microbiology c Genetics, GA6 Goettingen, Germany, 2 Department of 

Infectious Diseases and Microbiology, Imperial College /ondon, 6l, J The Institute for 

Genomic Research, RocBville, MD, 6.A 

Aspergilli are unique pathogens representing a prevalent threat to the immunocompromised 

individual. In our research we are interested in metabolic requirements 

sustaining propagation and supporting virulence of the predominant perpetrator* 

4&'.$@"66(&*U()"@+-(&, focusing on regulation of amino acid biosynthesis by the socalled 

7ross-1athway 7ontrol system. In the CPC signal transduction cascade an 

eIF2$ Binase senses amino acid deprivation and translates it into increased 

expression levels of a transcriptional activator protein, CpcA, which in turn elevates 

transcription initiation rates for the majority of amino acid biosynthetic genes. As 

deduced from profiling studies, the scope of the CpcA-directed transcriptome exceeds 

amino acid biosynthesis and suggests that the highly conserved CPC regulatory 

system has evolved as a general response routine to counteract conditions of 

environmental stress. In contrast to #'#4* mutants, 4/*U()"@+-(& strains deleted for 

the Binase-encoding gene #'#7 are not impaired in virulence, indicating that the basal 

expression level of CpcA is necessary and sufficient to support pathogenesis. 

Moreover, aestern blot analyses indicate that the #'#7-encoded eIF2 Binase is not 

required exclusively to generate the cellular read-out that is mediated by 

phosphorylation of the eIF2$ subunit. 6pon inspection of the 4/*U()"@+-(& genome 

sequence, the presence of a related gene ("U[Z, for "nitiation Uactor [inase) could be 

revealed with a deduced gene product similar to the heme-regulated inhibitor (HRI) 

member of the eIF2 Binase family. The "U[Z*gene was deleted in 4/*U()"@+-(& maBing 

use of the NHE(-deficient genetic bacBground, and corresponding mutant strains are 

evaluated regarding their resistance to environmental stress conditions. ae will 

present current data on the project with the aim to assess the role of eIF2 Binases in 

the stress response of 4/*U()"@+-(& and their contribution to virulence of this 

saprophytic pathogen 

. 

O-0043. Pheromone sensing inhibits Cryptococcus neoformans cell 

dissemination to the central nervous system during coinfection 

Nielsen K, Heitman ( 

DuBe 6niversity Medical Center 

7$2'-%#%##(&*,.%U%$)+,& is an opportunistic human pathogen that infects the central 

nervous system (CN.) to cause meningoencephalitis that is uniformly fatal if 

untreated. 7/*,.%U%$)+,& is a heterothallic fungus with two mating types - a and 

alpha. Interestingly, mating type can function as a virulence factor in 7/*,.%U%$)+,&, 

and the vast majority of clinical isolates are alpha mating type. To investigate the role 

of mating type in virulence we examined the relationship between mating type and 

pathogenicity in the most common clinical variety, @$(8"". The virulence of a and alpha 

congenic strains was examined at various stages of the infective cycle | from their 

survival in potential environmental predators to their colonization of various organs in 

animal models | and no difference between the a and alpha strains was observed. 

Infection by multiple strains has been reported, suggesting that coinfection with 

opposite mating types may occur. ae therefore analyzed the virulence of the 

congenic strains during coinfection and discovered that the strains have equivalent 

accumulation in peripheral tissues but alpha cells have an enhanced predilection to 

penetrate the CN.. Thus, the alpha strain out-competes the a strain in entry into the 

CN.. These studies reveal a virulence difference between a and alpha cells in the 

more commonly pathogenic var. @$(8"". 

These coinfection studies also provide a route to define virulence characteristics 

important for central nervous system infection. The observation that a cells are 

capable of entering the central nervous system during individual infections but not 

during coinfection suggests that either fungal-fungal or host-fungal interactions occur 

",*0"0% and influence virulence. 7/*,.%U%$)+,& cells communicate with cells of the 

opposite mating type via pheromone signaling. To determine whether pheromone 

signaling plays a role in CN. penetration, pheromone receptor mutants were 

examined for their ability to cross the blood-brain barrier during coinfection. aild-type 

a strains were unable to cross the blood-brain barrier but pheromone receptor mutant 

a strains accumulated in the brain during coinfection with wild-type alpha strains. 

These results provide evidence that pheromone sensing inhibits a cell dissemination 

to the CN. during coinfection and lay the foundation for detailed studies on the affect 

of pheromone signaling in both a and alpha strains ",*0"0% as a means to define 

virulence characteristics important for central nervous system infection.

O-0044. Proteomic analysis of cytoplasmic extracts from a mutant of Candida 

glabrata and its parent strain 

Penha C 1 , 2arreira P 1 , /archer G 2 , Perales ( J , /eon I O , /opes-2ezerra / [ , 2oucharan 

( 6 

1 /aboratário de Micologia e ProteÇmica, Instituto de 2iologia Roberto Alcantara 

Gomes, 6niversidade do Estado do Rio de (aneiro (6ER(), 2rasil, 2 Host-Parasite 

Interaction .tudy Group, 6PRE.-EA J1O2, /aboratory of Parasitology-Mycology, 

Angers 6niversity Hospital, Angers, France, J /aboratário de Toxinologia, Instituto 

fswaldo Cruz, Fiocruz, R(, 2rasil, O /aboratário de Toxinologia, Instituto fswaldo 

Cruz, Fiocruz, R(, 2rasil, [ /aboratário de Micologia e ProteÇmica, Instituto de 2iologia 

Roberto Alcantara Gomes, 6niversidade do Estado do Rio de (aneiro (6ER(), 2rasil, 

6 Host-Parasite Interaction .tudy Group, 6PRE.-EA J1O2, /aboratory of Parasitology- 

Mycology, Angers 6niversity Hospital, Angers, France 

fver the past two decades, the incidence of superficial or deep-seated infections due 

to 7+,5"5+*@6+8$+-+ has increased marBedly, probably in relation with its low intrinsic 

susceptibility to azole antifungals. The sequencing of 7/*@6+8$+-+ genome as well as 

recent refinements in protein resolution and identification techniques has greatly 

enhanced the application of proteomics for the study of this yeast species. .everal 

proteomic studies have been performed in the context of virulence, drug response and 

antifungal resistance in 7+,5"5+*+68"#+,&/ Previously, it has been shown that petite 

mutations lead in 7/ @6+8$+-+ to an acquired resistance to azole drugs, due to the 

overexpression of nuclear genes encoding some efflux pumps. However, due to the 

cross-talB between the nucleus and mitochondria, the expression of other nuclear 

genes may also be affected by these mutations. In the present worB, cytoplasmic 

extracts from a fluconazole resistant strain, an ethidium bromide-induced mutant of 7/* 

@6+8$+-+, and from its azole-susceptible parent isolate were compared by twodimensional 

polyacrylamide gel electrophoresis (2-D PAGE). Protein identification was 

carried out by peptide mass fingerprinting or sequence tagging using a matrix-assisted 

laser desorption/ionization-time of flight (MA/DI-TfF) or a MA/DI-TfF/TfF mass 

spectrometer. A total of 70 spots gels from each strain were excised from 2-D gels 

and analyzed. In the wild-type strain, this resulted in the identification of O6 proteins 

involved in metabolism, 1J involved in transcription and protein synthesis, and fate 

metabolism, O in cell rescue, virulence and defense, and 7 proteins of unBnown 

function. As for the petite mutant, we had identified O2 proteins involved in metabolism, 

10 in transcription, protein synthesis, and fate, 2 involved in cell rescue, virulence and 

defense, and 16 proteins of unBnown function. Among these proteins, it seems that O 

were overexpressed in the mutant strain and [ in the parent isolate. Together these 

results may help to a better understanding of the nuclear response to impairment of 

mitochondrial function. In addition, this study may constitute an important milestone 

for future applications of this approach in investigations about azole resistance in 7/* 

@6+8$+-+/ 

.upported by Rede ProteÇmica do Rio de (aneiro/Faperj and CNPq. 

O-0045. The C. albicans camp65 gene encodes an adhesin required for hyphal 

morphogenesis and experimental pathogenicity 

.andini . 1 , La Valle R 1 , De 2ernardis F 1 , Macrä C 2 , Cassone A 1 

1 Istituto .uperiore di .anitÖ - Department of Infectious, Parasitic and Immune- 

Mediated Diseases, 2 Istituto .uperiore di .anitÖ - Department of Food and Animal 

Health 

Purpose of the study: A mannoprotein of 6[ lilodalton (Camp6[) is a main target of 

immune response against the human opportunistic fungus 7+,5"5+*+68"#+,&. ae have 

previously shown that both cell-mediated and antibody responses against its protein 

moiety liBely contribute to the protection against candidiasis. However, nothing is 

Bnown about its contribution to the virulence of this fungus. 

Description: ae attempted to define a possible role of Camp6[ in 7/*+68"#+,& 

virulence by the use of molecular genetic tools. In particular, we constructed two 

independent sets of CAMP6[ BnocB-out mutants (CAMP6[/camp6[ heterozygous, 

camp6[/camp6[ null and camp6[/camp6[-CAMP6[ revertant strains) that were 

investigated for their phenotype, growth characteristics and pathogenicity in both 

mucosal and systemic infection models. 

Results and conclusions: Here we show that Camp6[ plays a critical role for hyphal 

morphogenesis and virulence of 7/*+68"#+,&. aith both sets of mutants, the null 

strains were as viable and competent for growth under yeast form as their wt 

counterparts but they severely suffered in the ability to form germ-tubes in liquid 

media, with almost total suppression of hyphal formation on solid media. Germ tube 

and hyphal formation were restored in revertant strains. CAMP6[ expression appears 

to be under differential control of several regulators of hyphal morphogenesis. 

Particularly, CAMP6[ transcription during filamentation was enhanced in efg1 and 

efg1 cph1 but not in cph1 strains suggesting that CAMP6[ expression is 

dependent on cAMP pathway but independent of the MAPl cascade. The null 

mutants were much less adherent to the plastic in vitro than the parental strains, while 

the heterozygous and revertant strains showed intermediate adherence between the 

aT and the null strains. In a lethal systemic murine infection model, the null mutants 

were significantly less virulent than the parental strains as shown by increased 

survival, lower CF6 counts in the Bidney and organ histology. Here again, the 

heterozygous and revertant strains showed an intermediate virulence phenotype. In 

an estrogen-dependent rat vaginal infection model, the early clearance of the fungus 

was also significantly more accelerated in the null mutants than in the wt strains, and 

the infection was exhausted earlier in the former than in the latter strains. Cytological 

observations of vaginal scrapings during infection showed massive hyphal formation 

in the parent strains as opposed to the null mutants. fverall, Camp6[ is essential for 

hyphal differentiation and appears to be a critical component of the virulence asset 

possessed by the fungus, thus accounting for the particular relevance of host immune 

response against this mannoprotein. 


PHYSIOPATHOLOGY* 

O-0046. TGF-beta induces transendothelial migration of sporothrix schenckii by 

a paracellular route involving ECM proteins 

Figueiredo C, Deccache P, Teixeira P, Lopes-Bezerra L, Morandi V 

Depto 2iologia Celular e Gendtica, Instituto de 2iologia Roberto Alcantara Gomes, 

6niversidade do Estado do Rio de (aneiro, 2rasil. 

.porotrichosis, a deep mycosis caused by ='%$%-?$"_*&#?.,#[""3*is characterized by 

cutaneous and lymphocutaneous lesions. In the immunocompromised host this 

fungus may invade the blood stream and disseminate to other tissues such as lungs 

and bones although fungemia is rarely observed. /ately, a zoonotic outbreaB of 

sporotrichosis has been described in 2razil with an increasing number of human 

cases showing more severe forms of this disease (.chubach .-*+6/3 N Engl. (. Med. 

J[JM 118[, 200[). Moreover, the routes of dissemination of pathogenic fungi are still 

poorly understood. ae had previously shown that =/*&#?.,#["" yeasts adhere to 

endothelial monolayers and that this interaction is modulated by inflammatory 

cytoBines. Here, we show that TGF-!1 induced =/*&#?.,#["" transmigration across 

endothelial monolayers. However, this cytoBine inhibited fungus internalization, 

probably by promoting the reorganization of endothelial actin cytosBeleton as showed 

in immunofluorescence assays with phalloidin-FITC. The major surface endothelial 

molecules recognized by =/*&#?.,#["" were not modulated by TGF-!1. Nonetheless, 

this cytoBine increased the exposure of the subendothelial matrix, and also increased 

FN secretion. In addition, anti-FN and anti-/M antibodies significantly inhibited the 

increase in =/*&#?.,#["" association with endothelial monolayers induced by TGF-!1. 

These antibodies also inhibited the adhesion of =/*&#?.,#["" to freshly prepared 

endothelial matrices. In parallel, we observed that =/*&#?.,#["" interacts with 

fibronectin and laminin exhibiting cell surface receptors for these ECM proteins. 

Moreover, the transendothelial migration of =/*&#?.,#["" was significantly blocBed by 

the anti-FN and anti-/M antibodies. These results indicate that the TGF-!1-induced =/* 

&#?.,#["" adhesion to endothelial monolayers results from the increased exposure of 

the subendothelial extracellular matrix, and that this event may contribute to the 

enhancement of transendothelial migration, also promoted by this cytoBine. fur data 

suggest that a paracellular route is preferentially used by =/*&#?.,#["" during invasion 

of activated endothelium. 

POSTERS 

ANIMAL MYCOLOGY* 

P-0001. An study on dermatophytosis among cattle in tehran and the suburbs 

Aghamirian M, Hashemi (, Pahlevan A 

~azvin 6niversity of Medical .ciences 

Dermatophytosis among cattle is considered to be of prime importance as it might 

transmit to man and causes economic losses. In this study we attempted to find the 

causative organisms that infect cattle in Tehran and the outsBirts. The samples were 

examined through direct smear using 20_ lfH and were also cultured in two series 

of .abouraud]s dextrose agar plates containing both chloramphenicol and 

cycloheximide (.CC). Cultures were incubated at two different temperatures, 2[Å C 

and J7Å C. The head of animals was the most affected organ and the necB infection 

with less prevalence at the second position. The incidence of infection was shown to 

be higher inboth winter and spring seasons. The incidence of dermatophytosis among 

cattle was reduced by aging. fur observations showed that >$"#?%'?2-%,* 

0.$$(#%&(). to be the exclusive fungal causative agent involved in dermatophytosis 

among the cattle studied in present worB. Dermatophytosis in cattle shows a 

worldwide distribution and in most cases >/*0.$$(#%&(). is the major organism 

involved. 

Financial supportM CNPq and Faperj

P-0002. Is there association between pityriasis rosea in piglets and 

Scopulariopsis brevicaulis? 

Bonfim Carregaro F 1 , .panamberg A 2 , Cavallini .anches E J , .antos Neves de 

2arcellos D 1 , Driemeier D O , Valente P 2 , Ferreiro / J 

1 .etor de .uÜnos/FaVet, 2 Departamento de Microbiologia/IC2., J .etor de Micologia 

VeterinZria/FaVet, O .etor de Patogia VeterinZria/Favet, 6niversidade Federal do Rio 

Grande do .ul (6FRG.), Porto Alegre, R., 2razil 

The 2razilian swine industry reached an important technological echelon that has 

highlighted its worldwide recognition, for which the maintenance of a high herd health 

profile is Bey for the maintenance of such status. However, certain diseases of 

important economical impact on the production system, such as the Pityriasis Rosea 

(Porcine (uvenile Pustular Psoriaform Dermatitis), are still of unBnown etiology. A 

recent study have indicated the =#%'(6+$"%'&"&*8$.0"#+(6"& as a possible causal agent 

involved in appearance of the P. Rosea, since such pathogen has been isolated in 

[0_ of clinical cases. The aim of this study was to characterize the fungi isolated from 

the sBin of healthy pigs, to subsequently compare it to the ones from symptomatic pigs 

for P. Rosea. A total of 106 samples were collected from April 200[ to (anuary 2006 

from only healthy animals between O0 and 100 days of age, from farms located in the 

state of Rio Grande do .ul, .outhern 2razil. .pecimens were collected by means of 

the hair-brush technique, after preliminary epidermal cleaning with water and 70_ 

ethanol. Collected samples were inoculated onto Mycobiotic agar plates and 

incubated at 27 o C for up to O weeBs. To date, a total of 1O0 colonies were isolated 

from the culturesM 7K moulds ([6,O_) and 61 yeasts (OJ,6_), with 11.[_ of the 

samples turning negative. Moulds were partially identified asM 21 (1[_) Mucorales s12 

!(#%$ spp. ru, 16 (11.O_) 4&'.$@"66(& spp. s7 4/*U()"@+-(&, O 4/*U6+0(& ru, 16 (11.O_) 

76+5%&'%$"()*spp., 10 (7.2_), 1.,"#"66"() spp., 7 ([.0_) Geotrichum spp., 2 (1.O_), 2 

(1.O_) =#%'(6+$"%'&"&*8$.0"#+(6"&, 1 (0.7_) 7($0(6+$"+ sp. and O (2.8_) Mycelia 

sterilia. The 61 yeasts isolates were preliminarly classified into J generaM [O (OJ.6_) 

7+,5"5+ spp. s1O 7+,5"5+*+68"#+,& ru, [ (J.[_) >$"#?%&'%$%, spp. and 2 (1.O_) 

P?%5%-%$(6+ spp. .ampling from healthy pigs will be expanded, along with the 

characterization of samples from clinical cases of P. Rosea, as new cases are 

diagnosed and included in the study. The next step will include attempts to induce 

dermatosis from filamentous and yeast-liBe forms of =/*8$.0"#+(6"& to observe 

histopathological characteristics of the sBin lesions and the evolution of the recovering 

process. 

P-0003. A frequent residual infection after antifungal treatement of 

dermatophytosis in dogs and cats is detected by systematic follow up 

Bourdeau P, 2ruet V, Marchand A, Peron C 

Ecole Nationale Veterinaire de Nantes, France 

Objective: The treatment of dermatophytosis in dogs and cats is usually based on 

regular procedures using antifungals. Efficacy is generally based on apparent clinical 

cure and mycological follow up is rare. The aim of this study was to evaluate the real 

efficacy of antifungal treatment in the fields and the usefullness of evaluation. 

Material and methods: Cases included were dogs and cats with an initial positive 

fungal culture detected in the laboratory of Mycology of 6nit DPM. .amples were 

obtained by carpet method. Fungal culture were performed on .abouraud Dextrose 

Agar (added with chloramphenicol r cycloheximide 0.[g/l each) incubated at 27ÅC. 

Cultures were read daily for identification. Number of colonies of dermatophytes were 

precisely counted until 20 and then grouped as abundant (H } 20 to [0) and very 

abundant (VH } above [0). Treatment were prescribed by the clinicians (griseofulvin 

or Betoconazole frequently associated to enilconazole for O to 6 weeBs for 77_ of 

cases). A second sample was performed at the end of the treatment or 2 weeBs or 

more after, and similarily processed. Evolution of infection was evaluated as negative 

or persistent infection (PI) with precise abundance of colonies. 

Results: A total of 2K[ cases were included. From 110 dogs (J6 breeds) 2[.[_ were 

YorBshire terrier and from 18[ cats [1.K_ were European .horthaired and J2.O_ 

Persians. Dermatophytes identified were !"#$%&'%$()*#+,"& 8J.1_, >$"#?%'?2-%,* 

).,-+@$%'?2-.& 1J.8_, !/*@2'&.() 1.7_ and !/*'.$&"#%6%$ 1.O_. fverall persistent 

infection (PI) was found in 26.K_ of treated animals (!/#+,"& 2K.7_, 

>/).,-+@$%'?2-.& K.8_). PI was found in 2O_ of animals maintained under treatment 

and from JO.[_ when treatment was stopped since 2weeBs or more. without any 

influence of initial number of detected colonies except in the group with 1 colony 

(O.J _ PI.). fnly 6.2_ of initial VH remained VHn 12.[_ of H remained H and 1.8_ 

changed to VH. The distribution of time for growth and identification (J to 18 days) 

was for first or second culture respectivelyM J-[daysM 17,2_ then 1J.K_n 6-10 daysM 

72.6 - [8.2_n t 11daysM 10.1_-27.8_. 

These results, obtained in the conditions of veterinary practice, demonstrate that a 

high percentage of treated animals remain carriers at the end of treatment and that a 

second fungal culture is absolutely necessary to detect residual infections. 


P-0004. Malassezia pachydermatis: Relationship between abundance as 

measured by cytology or mycology and clinical aspects of ear canals in dogs. 

Bourdeau P, 2ruet V, Marchand A 

Dermatology/Parasitology/Mycology, Ecole Nationale Veterinaire, Nantes, France 

Objective: !+6+&&.d"+*G!/*'+#?25.$)+-"&J*G!/'J*yeasts, although belonging to the 

normal flora of sBin and ear canal, have been proposed as agents of external otitis in 

dogs. The evaluation of the role of this yeast in clinical situations is usually related to 

the relative abundance of !/'/*in samples obtained from ear canals. The purpose of 

this study was to evaluate the results of two methods of evaluation (cytology and 

fungal culture) in relationship with two clinical criterias (erythema and abundance of 

cerumen) in the ear canals of normal (non atopic) dogs. 

Material and methods: Twenty seven non atopic beagle dogs, from the same 

laboratory Bennel living in the same conditions (including nutrition), were included. fn 

day 7 before the study, a previous otoscopic examination was performed, coupled to 

cerumen samples for microscopic observation to exclude any interfering bacterial or 

parasitic (b-%5.#-.&) otitis. Moreover a systemic treatment to control an eventual 

subclinical otoacariosis was prescribed. fn day 0 each ear canal was carefully 

examined by the same observer. Erythema of the ear canal and abundance of 

cerumen were both clinically scored from 0 to J (respectively from absent to severe 

erythema and absent to very abundant cerumen). In a second time two swabs 

samples were performed. fne swab was immediately rolled (J lines) on a microscopic 

glass then routinely stained (RA/). The second swab was immediately used to 

inoculate .abouraud dextrose agar added with cycloheximide and chloramphenicol 

(0.[g/ -1 each) in a K0mm Petri dish then incubated at J2 ÅC for O days. The 

numeration of !+6+&&.d"+ yeasts in cytology was made by the addition of the total of 

yeasts observed on five randomly choosen high power fields (j100 with immesion oil). 

The fungal cultures were observed under binocular to determine the number of CF6 

(colony forming units) of !+6+&&.d"+*'+#?25.$)+-"& present. The distribution of results 

of observations from OO ear canals was compared. The scores of respectively 

erythema or cerumen were grouped (0/1 and 2/J) or were combined (0/1, 2/J, O/6) 

and compared to cytology or mycology (v10 !+6+&&.d"+*or 6FC or % 10 / !+6+&&.d"+ 

or 6FC). The results were statistically analyzed using Bhi test p v 0.0[. 

Results: The results obtained do not show any significant relationship between 

abundance in cytology and the presence of erythema (0,62[), or cerumen (0,[70) or 

their combination (0,K87). .imilarily there is no relationship between abundance 

obtained in mycological cultures and the presence of erythema (0,6J2), cerumen 

(0,717) or cerumen r erythema (0,K22). 

These results show that in modified ear canals and in the absence of a predisposing 

factor liBe atopy there is no relationship between these two methods of evaluation of 

!+6+&&.d"+*populations and clinical modifications of ear canal. 

P-0005. Role of birds of prey as carriers and spreaders of Cryptococcus 

neoformans and other yeasts 

Cafarchia C 1 , Iatta R 2 , Montagna M 2 , ftranto D 1 

1 Department of Animal Health and aelfare, Faculty of Veterinary Medicine, 6niversity 

of 2ari (Italy), 2 Department of 2iomedical .cience and Human fncology, Hygiene 

.ection, Faculty of Medicine, 6niversity of 2ari (Italy) 

In the last twenty years, cases of*human cryptococcosis associated with the 

exposure to bird droppings have increased mainly in immunocompromised patients 

(Fessel a(, `*a,@6*]*!.5 1KKJn 328M 1J[O-1J[[n Currie 2P et al., ]*76",*!"#$%8"%6 

1KKOn 32M 1188-K2n NosanchuB (D et al., 4,,*K,-.$,*!.5 2000n 132M 20[-8n /agrou l 

et al., ]*K,-.$,*!.5 200[, 257M J8[-8). In order to investigate birds of prey as carriers 

and spreaders of 7$2'-%#%##(&*,.%U%$)+,& and other yeasts of importance to 

humans, 182 swab samples were collected from the cloacae of several species of 

birds of prey (Group I) and J2 faecal samples from aviaries in which the birds were 

recovered (Group II). .amples were also taBen from the digestive tract dissected into 

four parts (i.e. crop, proventriculus, ventriculus, cloacae) of 60 dead birds of prey 

(Group III). A total of O[O samples were cultured, and 21[ yeast colonies identified. 

7$2'-%#%##(& ,.%U%$)+,&*was isolated both from the cloacae of three samples 

(O.8_)*of ^+6#%*-",,(,#(6(& and from one sample (J.1_) of Z(-.%*8(-.% and from the 

aviaries in which these birds were Bept. All the isolates of*7/*,.%U%$)+,& were 

identified as 7/*,.%U%$)+,& var.*@$(8"". fverall, 18 samples (K.K_) from Group I, 1J 

(O0.6_) from Group II, 12 crops (20_), J proventriculi ([_) and 12 cloacae (20_) 

from Group III yielded positive cultures for yeasts. Yeast species isolated from the 

aviaries were also retrieved in the cloacae of the animals housed in the aviaries with 

the exception of 7$2'-%#%##(&*+68"5(&. 7+,5"5+*+68"#+,&, 7+,5"5+*U+)+-+ and 

7+,5"5+*@("66".$)%,5"" were retrieved both in crops and in cloacae, P?%5%-%$(6+*$(8$+ 

both in ventriculi and in cloacae and 7+,5"5+*'+$+'&"6%&"& only in crops. In addition, 

7+,5"5+*",#%&'"#(+3*7+,5"5+*'.66"#(6%&+3*7/*U+)+-+3*7/*'+$+'&"6%&"& and*7/* 

@("66".$)%,5"" were isolated only in the cloacae of hospitalized birds and/or in the 

digestive tract of dead birds, but not in the environment. 

The results of this worB show that birds of prey harbour in their cloacae different 

species of yeasts that are new emerging microrganism. .ame species of these yeasts 

(7/*",#%&'"#(+3*7/*'.66"#(6%&+3*7/*U+)+-+3*7/*'+$+'&"6%&"& and*7/*@("66".$)%,5"") belong 

to the normal flora of the digestive tract and their distribution might be influenced by 

the different physiological characteristics of the digestive tract (i.e pH, temperature), 

while other species of yeasts mainly 7/*,.%U%$)+,& and 7/*+68"#+,&*can be carried 

and spread in the environment. ^/*-",,(,#(6(& and Z/*8(-.% are the species that 

harboured the highest number of yeasts and are the only species of birds in which 7/* 

,.%U%$)+,& was isolated from the cloacae. 

FundingM .elf funded /ab DPM

P-0006. Cutaneo-lymphatic and nasal sporotrichosis in a dog from southern 

Italy 

Cafarchia C 1 , .asanelli M 1 , de Capraris D 1 , /ia R 1 , Guillot ( 2 , ftranto D 1 

1 Department of Animal Health and aelfare, Faculty of Veterinary Medicine, 6niversity 

of 2ari (Italy), 2 .ervice de Parasitologie-Mycologie, Ecole Nationale Vdtdrinaire 

deAlfort, France 

.porotrichosis is a chronic, granulomatous and usually cutaneo-lymphatic infection 

caused by the dimorphic fungus*='%$%-?$"_*&#?.,#[""/ This infection is supposed to be 

worldwide spread but the majority of the published case reports in dogs, cats and 

humans are from the 6nited .tates, Central and .outh America. Although several 

human cases of sporotrichosis have been reported in the Italian region of Apulia, no 

data on animal infection is currently available from this area. 

ae recently diagnosed a case of cutaneo-lymphatic and nasal sporotrichosis in a five 

year-old male Italian 2racco (hunting dog) originating from Apulia. The dog was 

referred to the Department of Animal Health and aelfare of the Faculty of Veterinary 

Medicine (DEAHa), 6niversity of 2ari (Italy) with a history of pruritic dermatitis and 

loss of weight. The initial lesion appeared three months ago. It was a nodule on the 

right fore limb which became ulcerated and discharged exudates. The referring 

veterinarian treated the animal for sarcoptic mange since no other infections were 

diagnosed. ahen referred at the DEAHa, the dog presented multiple, scattered, 

circular, alopecic lesions and nodules dripping a serous brown exudates on the legs 

and sternal area. The scrotum was erythematous and ulcerated and a bilateral 

mucopurulent nasal discharge was observed along with ulcerations of the nares. The 

cytological examination of the material collected from nasal discharge and from 

ulcerative sBin surface revealed the presence of few cigar-shaped organisms within 

macrophages. Fungal cultures from nasal and ulcerate sBin swabs yielded colonies of 

=/*&#?.,#["". This case indicates that =/*&#?.,#["" can be detected by cytological 

examination of exudates about three months post infection and confirms that the 

number of organisms in canine tissues is scant. .ince negative histological 

examination of sBin biopsy and cytological examination of exudates do not rule out the 

presence of the fungus, the definitive diagnosis of sporotrichosis should be carried out 

using fungal culture, especially in dogs coming from an endemic area for 

sporotrichosis. 

P-0007. Identification of yeasts isolated from wild boar (Sus scrofa) in Umbria, 

Italy 

Cannizzo F 1 , Moretti A 2 , 2oncio / J , Agnetti F O , .alvatori R [ , Vidotto V 6 , 2ollo E 1 

1 Department of Animal Pathology, 6niversity of Turin, Grugliasco, Italy, 2 Department 

of 2iopathological .cience and Hygiene for Animals and Alimentary Productions, 

6niversity of Perugia, Italy, J Department of Medical and .urgical .pecialties and 

Public Health, 6niversity of Perugia, Terni, Italy, O .tate Veterinary Institute, Perugia, 

Italy, [ Veterinary Clinician, Perugia, Italy, 6 Department of Medical and .urgical 

Disciplines, 6niversity of Turin, Italy 

fropharyngeal and auricular swabs were collected from 26 wild boar (=(&*&#$%U+) 

slaughtered in 6mbria (Italy) in order to evaluate yeast colonization. The swabs were 

streaBed onto malt agar and incubated at J7ÅC for J-[ days. .ubsequently the 

isolated yeasts were first identified according to their morphological features (i.e color 

of the colonies, mycelium and germinal tube production, and capsule) and then by the 

API 20 C A6j Bit (bioMdrieux, Marcy leEtoile, France) and by the CHRfMagarã 

Candida medium (2D Diagnostics, .parBs, 6.A). The 7+,5"5+*+68"#+,& strains 

positive for germinal tube production were further identified by PCR. fther 7+,5"5+ 

spp. were also identified by PCR. fut of the 26 examined animals, 1[ resulted 

positive for isolation of yeasts. The identification of the isolated strains revealed the 

presence of 7/*+68"#+,&*(6 and [ strains from oropharyngeal and auricular swabs, 

respectively)3*7/*-$%'"#+6"&*(1 strain from auricular swab)3*7$2'-%#%##(&*6+($.,-""*(2 

strains from auricular swabs)3*7$/*?()"#(6%(&*(1 strain from auricular swab)*and* 

>$"#?%&'%$%,*+&+?"" (1 strains from oropharyngeal swab). This is the first report of 

isolation of yeasts from wild boar in Italy. Further studies are needed for better 

understanding the epidemiology, the role and the virulence of yeasts isolated form 

wild animals, including comparative evaluations on strains isolated from domestic 

mammals. 


P-0008. Detection of Pneumocystis carinii in lungs of wildlife animals from 

Brazil. 

Cavallini Sanches E 1 , R. 2orba M 2 , Moleta Colodel E J , Driemeier D 2 , Ferreiro / 1 

1 6niversidade Federal do Rio Grande do .ul-Faculdade de VeterinZria-.etor 

Micologia-Porto Alegre-R.-2razil, 2 6niversidade Federal do Rio Grande do .ul- 

Faculdade de VeterinZria-.etor Patologia-Porto Alegre-R.-2razil, J 6niversidade 

Federal de Mato Grosso-Faculdade de VeterinZria-.etor Patologia-CuiabZ-MT-2razil, 

O 6niversidade Federal do Rio Grande do .ul, [ 6niversidade Federal do Rio Grande 

do .ul-Faculdade de VeterinZria-.etor Micologia-Porto Alegre-R.-2razil 

1,.()%#2-"&*#+$",""*is considered as an heterogeneous group of specific fungal 

organism that might found in a wide range of hosts. Inspite of intensive investigation in 

human and laboratory mammalian host occurrence and nature of infections in wild 

animals are still limited. In this study, its presence was determined by Grocott]stain 

and nested PCR at mitochondrial large subnit (mt/.6) rRNA gene in J7 lung tissue 

samples from wildlife animals species. 1/*#+$",""*was detected ([ lungs) in 2/K 

V+&2'(&*,%0.)#",#-(&*(Armadillo)3*1/2 7.8(&*+'.66+*(Capuchin monBey), 1/JJ* 

7+66"-?$"_*\+##?(*(Common marmoset) and 1/[ ='.%-?%&*0.,+-"#(&*(2ush dog). None 

of the different species harbored 1/*#+$","" organismsM *O >+)+,5(+*-.-$+5+#-26+* 

(/esser Anteater), :*1&.(5+6%'._*@2),%#.$#(&*GPampas-fox), 2 P+,'?+&-%&*+$".6* 

(Channel-billed toucan),*2 7?2&%#2%,*8$+#?2($(&*(Maned aolfJ3*1 Z$+52'(&*-$"5+-26(&* 

(Three-toed .loths), 1Z6+&-%#.$(&*5"#?%-%)(& (Marsh Deer), 1 7.$5%#2%,*-?%(&*(Fox), 

1*X.%,-%'"-?.#(&*#+"&&+$+*G2lacB-faced lion tamarins), 1 =#"($(&*",@$+)"*G=e("$$.6J3 1 

4@%(-"*'+#+ (Agouti), 1 >('",+)8"&*).$"+,+.*(2lacB and ahite Tegu) and 1='.%-2-%* 

#(,"#(6+$"+*(2urrowing fwl). This is the first report of detection of 1/*#+$","" in wildlife 

animals from 2razil (Mid-aestern and .outhern Region). 

P-0009. The use of PCR technique identifying yeasts from mammary glands of 

milking cows 

Cervantes-flivares R, Hernandez-Romahn A, .egundo-Zaragoza C, Ducoing-aatty 

A 

6niversidad Nacional Autonoma de Mexico 

Development of molecular techniques based in DNA analyses have allowed to reduce time 

on the identification and characterization of yeasts. fne of these techniques is the 

polymerase chain reaction (PCR). ae tested the usefulness of amplifying the IT. regions 

and the [.8. rRNA gene that are useful to measure close phylogenetic fungal relationships. 

The aim of the present study was to collaborate with the biochemical identification using 

PCR technique in 277 isolates obtained from healthy and mastitis milBcow. It was our 

intention to help identify yeasts using a faster and reliable technique such as PCR. 

Extraction of DNA was performed using Hoffman C... and ainston F.A. method. This 

isolates were previously identified by biochemical and morphological methods that showed 

86 7/*@6+8$+-+, 7[ 7/*[$(&."3*II*7+,5"5+*+68"#+,&3 60 7+,5"5+*&''/ and O[ could not be 

identified with 2O different tests.*Primers IT.1 and IT.O described by ahite et. al. were 

used to amplify the [.8.-IT. region using the PCR technique. 

Results: From the 86 isolates of 7/ @6+8$+-+3*7[ match with the fragment of 8[6 pb for this 

species, while out of the 7[ isolates of C. [$(&." 68 match with fragment [1K pb and 6 of 

the 11 from 7/*+68"#+,&* match with the fragment [[0 pb. 60 isolates from 7+,5"5+*&''/ 

and the O[ non identified species showed different size fragment. 

Conclusion: The biochemical identification for 7/*@6+8$+-+, 7/*[$(&." and 7/*+68"#+,& was 

not reliable for all*isolates. The biochemical identification matched the molecular 

identification in KJ_, K0_ and [O_ respectively for these three species. The used primers 

discriminated 7/*@6+8$+-+ and 7/*[.U2$. This technique helped us to correct the 

misidentification made by biochemical and morphological methods, although for species 

with same fragment size it will be needed an additional method for the correct identification. 

References: 

Esteve-Zarzoso 2, 2elloch C, 6ruburu F, ~uerol A. Identification of yeasts by RF/P 

analysis of the [.8. rRNA gene and the two ribosomal internal transcribed spacers. Int ( 

.yst 2acteriol. 1KKKnOKMJ2K-JJ7. 

ahite T(, 2runs T, /ee ., Taylor. Amplification and direct sequencing of fungal ribosomal 

RNA genes for phylogenetics. InM Innis MA, Gelfand DH, .ninsBy (( ahite T(. PCR 

protocolsM a guide to methods and applications. New YorBM Academic Press, 1KK0n282- 

287MJ1[-J22. 

Hoffman C., ainston F. A ten-minute DNA preparation from yeasts efficiently releases 

autonomous plasmids for transformation of a&#?.$"#?"+*#%6". 9.,.. 1K87n[7M267-272. 

/una C.. Aislamiento, identificacián y conservacián de levaduras aisladas de leche de 

vacas clÜnicamente sanas o con mastitis clÜnica cránica (tesis de licenciatura). DF MdxicoM 

FMVZ-6NAM. 200[.

P-0010. Coccidioidomycosis a problen in fighting drug trafficking 

Cervantes-Olivares R 1 , Guzman-Chavez R 2 

1 6niversidad Nacional Autonoma de Mexico. FMVZ, Mexico, 2 Private clinicial, .mall 

animal clinic 

Coccidioidomycosis is the name of a fungal disease caused by a dimorphic organism 

Bnown as 7%##"5"%"5.&*"))"-"& or 7/*'%&+5+&"" this fungus lives in desert and semidesert 

areas in the north part of Mexico and south of 6.A. and it can be found 

affecting domestic animals as well as humans, although the disease is not 

transmissible. There are few reports on the veterinary side about this disease, and 

even fewer in our country. The main objective of this report is to draw attention to 

small animals practitioners in northern Mexico to this important disease that surely is 

affecting many dogs in the endemic area. 

ae received a clinical case of a snifter dog that was born in 2elgium and brought to 

Mexico to be trained as illicit drug detector, she was a 2elgian Mallinois .hepherd of 

[ å years old, she worBed in different places mainly in the northern part of our country 

were the fight against drug trafficBing is very strong, in her last two years she was 

stationed in Hermosillo, .onora, (an state that according to Castaon is one of the 

most infected hCoccii states of Mexico with over 7[_ positive IDR ). Her trained 

reported that she was not eating for O days and had been losing weight, the dog was 

send to central base in Mexico City to be evaluated. ahen we evaluated she 

presented fever, dispnea, poor muscular condition, poor general condition and right 

hind leg lameness. Further laboratory test showed that the dog was immune 

compromised and although treatment with antibiotics were unsuccessful the clinician 

did not asB for mycology test until it was too late, the dog died and at the necropsy 

they found a large damage in the lungs as well a large lesion of the 12 th right rib and 

in the 12 th and 1J th thoracic vertebrae, as well as abnormalities in the J rd and O th sacral 

vertebrae. The samples taBen were sent to the pathology department as well as to the 

mycology laboratory a Coccidioidomycosis diagnosis were issued by looBing at the 

spherules characteristic of the parasitic for of 7%##"5"%"5.& spp. .ince this case we 

have Bnowledge that at leas O other cases have been observed, actually we are 

implementing a complement fixation test in order to have a tool in detecting in and 

early stage the infection of animals in risB of acquiring this disease, according to a 

veterinarian clinician worBing in the north part of Mexico one out of four hundred dogs 

living in that area are suffering the infection. 

P-0011. Aspergillus serodiagnostics in avian species 

Cray C, Rodriguez M, aatson T 

6niversity of Miami, Miami, 6.A 

Aspergillosis is a common infection in avian species. All birds appear susceptible to 

infection but it appears some species may be predisposed to infection | especially 

captive waterfowl, penguins, raptors, and psittacines. 6nfortunately, clinical signs are 

often non-specific and quite variable as there are both chronic and non chronic forms. 

Antemortem diagnosis is notoriously difficult. Clinical diagnostic testing includes 

routine hematology, analysis of plasma proteins, cytology, fungal culture, histology, 

and radiography. 

.erodiagnostics have been available for a few years with varying results. Many birds 

have been found to carry titers of antibody to 4&'.$@"66(&*&' in the absence of disease. 

It is thought that this indicates exposure and immune competency rather than always 

infection. GraczyB (1KK8) was the first to report the implementation of an antigen 

E/I.A in penguin species and concluded that this test was of benefit to clinical 

diagnosis. Two publications followed illustrating the use of the assays in less than 10 

clinical cases with varied results. The purpose of the current study was to compare 

three serodiagnostic assays to clinical symptoms, diagnosis, and case outcome in 

avian species. 

The three assays used were as followsM an indirect antibody E/I.A, a polyclonal 

sandwich antigen E/I.A, and a monoclonal antigen E/I.A (2iorad Platelia EIA). 8JK 

samples have been analyzed as of this writing. The species have been grouped as 

psittacine, raptor, penguin, or other zoo submissions (those not part of the 

aforementioned groups). Approximately J0_ of the psittacine samples were found to 

be antibody positive in contrast to 80_ or more in the raptor, penguin, and zoo groups. 

.imilar to previous reports, penguins were found to be the most strongly reactive. 

/ess reactivity may be found in the psittacine group as they are liBely in a more 

controlled environment. Nearly [0_ of the penguins tested positive on the polyclonal 

antigen E/I.A versus 2[_ in the other groups. aeaB positive results were common to 

all groups and may reflect an environmental exposure. Positive samples were found 

by Platelia in approximately 20_ of the samples in the zoo, raptor, and penguin 

groups and nearly double in the psittacine group. The results for the polyclonal 

antigen E/I.A were not always correlative with the Platelia liBely indicating differential 

sensitivity and specificity. ff the confirmed and suspect cases, both chronic and non 

chronic presentations were described. Non-chronic cases were more liBely to be 

antibody positive than normal birds. Many chronic cases were found to be antibody 

negative but positive on the polyclonal antigen E/I.A and Platelia tests. In addition, 

chronic cases were found to be antibody and polyclonal antigen positive more often 

than non chronic cases. /astly, a predominance of long term chronic cases was 

antibody negative and polyclonal antigen negative. fverall, no test was solely 

diagnostic of aspergillosis in avian species. A panel of serodiagnostic tests should be 

used in conjunction with clinical signalment, length of illness, and other clinical tests 

with repeated test submissions whenever possible. 


P-0012. Phenotypic and molecular characterization of Cryptococcus 

neoformans in dried and fresh air excreta of birds confined in the Zoological 

Park Buin Zoo , Santiago, Chile . 

Diaz M, .alas R, Hermosilla G 

Microbiology and Mycology Program, 2iomedical .c. Institute, .chool of Medicine, 

6niver. of Chile, Independencia, .antiago, Chile 

7$2'-%#%##(&***,.%U%$)+,& is a basidiomycetous yeast with J Bnown varietiesM var 

neoformans, var gattii and var grubiin all isolates commonly found in the environment. 

The objective of this study was to determine the presence of Cryptococcus 

neoformans in excreta isolated from birds confined to the Zoological ParB 2uin Zoo, 

.antiago, Chile, and to isotype the strains by molecular methods. 

Method: In a total of 2[ cages, KJ samples of dried and fresh air excreta from birds 

confined to the Zoological ParB 2uin Zoo, were collected and analyzed during the 

months of (une and (uly 200[. .amples isolated on bird seed plates corresponded to 

one of the following avian speciesM 62 samples of Psitaciforme, 10 .trigiforme, K 

Galliforme and 12 Falconiforme. Assays for species identification and varietal 

differentiation were performed using standard biochemical tests and differential media 

(Canavanine | Glycine | bromothymol blue agar (CG2). All strains were analysed for 

RAPD with primers fP 2A-1J. 

ResultsM 12 (12.K_) species of 7$2'-%#%##(& were isolated, of which K corresponded 

to 7$2'-%#%##(&*,.%U%$)+,& (7[_), O isolations (OO.O_) belonged to the .trigiforme 

order, J (JJ.J_) to the Psitaciforme order, 1 (11.1_) to the Falconiforme order and 1 

(11.1_) to the Galliforme order. The remaining J (2[_) corresponded to 

7$2'-%#%##(&*+68"5(&. 10 strains of 7/*,.%U%$)+,& showed a common amplification 

profile. The dendrogram analysis for 6PGMA revealed a cluster formed by the yeast 

strains isolated from species >"-%*+68+ (owl) and Z(8%*)+@+66+,"#(& (tucuquere). 

ConclusionsM KJ samples of dried and fresh air excreta of birds confined in the 

Zoological ParB 2uin Zoo, within which K strains of 7$2'-%#%##(&*,.%U%$)+,&*were 

isolated. The orders represented included Psitacidae (J), Falconidae (1) .trigidae (O) 

and Gallidae (1). All isolates were C neoformans 0+$**,.%U%$)+,&/ The molecular 

analysis for RAPD confirmed a cluster in the yeast samples of >"-%*+68+*and*Z(8%* 

)+@+66+,"#(&, which indicates that these strains have a high degree of similarity. The 

remaining strains analysed didn]t present similarities, which would indicate that there 

are other potential unrelated origins. 

P-0013. Canine disseminated apergillosis 

Elad D 

limron Veterinary Institute, 2et Dagan, Israel 

Canine disseminated aspergillosis is a relatively rare syndrome with an almost 

uniformly lethal outcome. The affected animals are almost exclusively German 

.hepherd dogs, with a higher incidence in females. The reason for these 

predispositions is currently unBnown. 

The syndrome is caused, again almost exclusively, by 4&'.$@"66(&*-.$$.(&. It has been 

suggested that this fact is related to the production of aleuriospores by the Aspergillus 

terreus-flavipes group, ",*0"-$% and ",*0"0%. These organelles are secondary spores, 

unrelated to the homonymous asexual reproductive organs of several other fungi such 

as dermatophytes. Their minute size (about 2 microns) permits them to pass through 

the smallest blood vessels and thus spread efficiently from the initial focus of infection 

and produce the characteristic disseminated form of the syndrome. 

Patient presentation is primarily with neurological signs, indicating involvement of the 

central nervous system and consequently an advanced stage of dissemination to 

various organs. K,W0"0% diagnosis is based on the microscopic observation and culture 

of the fungus in the urine. fften, mycelia can be observed in the aqueous humor. 

Therapy attempt are, as a rule, ineffective. This is probably due to the intrinsic 

resistance of 4/*-.$$.(&, Bnown to be even less responsive to antimycotic drugs then 

other 4&'.$@"66(& spp., and the advanced stage on the infection at presentation. 

Recently new antimycotic drugs, with improved anti-Aspergillus activity, have been 

available for human use but are too expensive for animal treatment. Hopefully this will 

change in the future improving the prognosis in cases of canine disseminated 

aspergillosis.

P-0014. Isolation of Microsporum gypseum from the haircoat of health wild 

felids kept in captivity in Brazil. 

Fischman Gompertz O 1 , 2entubo H 2 , Countinho . J , Pelludo ( O , Correa, . O , Teixeira 

R O 

1 6NIFE.P, .ao Paulo, 2razil, 2 6.P, J 6NIP, O Zoological ParB Foundation of .ao 

Paulo 

Dermatophytes are fungi that cause superficial mycoses in animals and humans. 

ahile studies have shown that domestic cats (^.6"&*#+-(&) are often asymptomatic 

carriers of dermatophytes, and thus a significant source of infection, this aspect has 

not been studied in relation to their wild relatives. The present study was aimed at 

determining the presence of dermatophytes on the haircoat of healthy wild felids, Bept 

in captivity at .Éo Paulo Zoo. .amples were taBen from 1J0 adult animals of both 

sexesM 2[ lions (1+,-?.$+ 6.%), 12 tigers (1+,-?.$+*-"@$"&), 6 jaguars (1+,-?.$+*%,#+), 

O leopards (1+,-?.$+*'+$5(&), 2 snow leopards (1+,-?.$+*(,#"+), 2 pumas (1()+* 

#%,#%6%$), 2 cheetahs (4#",%,2_*\(8+-(&), 1 ocelot (X.%'+$5(&*'+$5+6"&), 28 tiger cats 

(X.%'+$5(&*-"@$",(&), 10 margays (X.%'+$5(&*B".5""), 8 geoffroyes cats (X.%'+$5(&* 

@.%UU$%2"), 22 jaguarundis (A.$'+"6($(&*2+@%(+$%(,5") and 8 pampas cats (b,#"U.6"&* 

#%6%#%6%). The samples were obtained by rubbing the haircoat of the animals with 

squares of sterile carpet, and they were seeded onto Petri dishes containing 

Mycobiotic agar (Difcoã). The plates were incubated at 2[ÅC for O weeBs. The 

isolates were subcultured in .abouraud dextrose agar with chloramphenicol 

(100mg//) and microcultured on slides for posterior identification by their macro- and 

microscopic characteristics. !"#$%&'%$()*@2'&.() was isolated from two apparently 

healthy lionesses (1.6_), both Bept in terrariums. The most prevalent contaminants 

were of the genera 1.,"#"66"() (27.K_)n 76+5%&'%$"() (2O.[_)n 4#$.)%,"() (12.1_)n 

=#%'(6+$"%'&"& and 7?$2&%&'%$"() (K.8_)n and 4&'.$@"66(& ([.J_). The existence of 

dermatophytes on the haircoat of healthy wild felids, maintained in captivity, not only 

elevates the risB that these animals eventually become ill, but also inserts them in the 

category of asymptomatic carriers, liBe their relatives the domestic cat, characterizing 

them as sources of fungal infection both for other animals and for human beings. 

6nderstanding this condition is essential in the adoption of prophylatic measures for 

sanitary maintenance for these animals and the professionals who maintain contact 

with them. 


P-0015. Aspergillus fumigatus and Streptococcus â hemolytic association in 

nasopharynge of healthy horses. 

Guida N 1 , Mucç2O1noz A 1 , Mesplet M 1 , Schettino A 2 , 2arboni A 1 

1 Infectious Diseases, Veterinary Faculty. 2uenos Aires 6niversity. Argentina, 

2 Microbiology,Veterinary Faculty.National 6niversity of the center of the province of 

2uenos Aires.Argentina 

Rhino sinusitis, strangles and guttural pouches inflammation are diseases of the upper 

respiratory tract in horses, produced by pyogens agents coming by oropharingeal 

way. 

Nasopharyngeal mucosal ecoflora recognize .treptococcus (.) # haemolytic 

belonging to the /ancefield group C as moderate pathogen. .. equi equi is the 

etiological agent of strangles whereas .. equi equisimilis and .. equi zooepidemicus 

are associated in strangles. .ometimes they are single agents of in atypical strangles. 

Even being Aspergillus fumigatus normal ecobyota in nasopharyngeal mucous, 

sometimes it is recognised as etiological agent in pouches infections. 

The aim of this approach is the detection of .treptococcus (.) # haemolytic and 

Aspergillus fumigatus association in healthy horses. 

Nasopharyngeal swabbing were performed in J0[ horses. The samples went of 

animals lodged in box and to field of the city and province of 2uenos Aires. 

All of them were cultivated in .abouraud medium with 2 _ of glucose and 0,0[ _ of 

cloranphenicol, incubating at 2[ |28 oC during J-[ days and in blood agar [_ during 

O8 hrs. .amples were processed with routine protocol for the .. equi isolation and 

antigen type C production. 

K[ .treptococcus # haemolytic were isolated, 2J were .. equi zooepiddmicus and 27 

isolated of Aspergillus fumigatus. 

8 horse were positive to association .treptococcus (.) # haemolytic and Aspergillus 

fumigatus. 


P-0016. Carrier dermatohytes comparation in different animal’s population 

Guida N 1 , Parodi / 1 , Picos ( 1 , Rossano M 1 , Martinez Vivot M 1 , Schettino A 2 , Mesplet 

M 1 , Moras E 1 

1 Infectious Diseases.Veterinary Faculty. 2uenos Aires 6niversity, 2 Microbiology. 

Veterinary Faculty. 2uenos Aires Province Center 6niversity 

The dermatophytosis is a disease produced by queratinophylic fungi dermatophytes 

liBe Microsporum and Trichophyton. Dogs and cats are the ordinary species where is 

possible to find Microsporum canis (Mc) followed in order of importance by 

Microsporum gypseum and Trichophyton mentagrophytes. Particularly the cats can be 

bearers without presenting clinical sings. Then, it]s a very important problem in 

nursery as zoonotic infection. 

This study was performed brushing completely in different animals with teeth brush, 

were used for carriage detection and all samples were cultivated on /actrimel agar. 

The aims of this study was to compare dermatophytes carriers in different animal 

populations. 

Populations studiedM 

1-cats assistant to clinical consultation 

2-dogs nursery with cat]s mascots 

J-cats nursery 

O-rabbits nursery with dog]s mascots 

The results wereM 

Population Total Positive dermatophytes % 

animals 

1 7J cats 20 Microsporum canis 

27,JK 

2 26 dogs 

K cats 

1[ Microsporum canis 

J Microsporum canis 

[7,6K 

JJ,JJ 

J JJ cats 28 Microsporum canis 8O,8O 

P-0017. Exophiala species causing disease in cold-blooded animals 

HarraB ( 1 , Cruz l 2 , de Hoog G 1 

1 Centraalbureau voor .chimmelcultures, 6trecht, The Netherlands, 2 6niversidade do 

Estado do Amazonas, Manaus, 2razil 

DrinBing water appears to be an unexpected habitat of many melanized fungal 

species. Among these are several a_%'?"+6+*species, members of 7?+.-%-?2$"+6.&3 a 

fungal order containing numerous members Bnown from human disease, such as a/* 

5.$)+-"-"5"& and 76+5%'?"+6%'?%$+*8+,-"+,+. 2ased on ribosomal IT. sequences, 

waterborne a_%'?"+6+ strains cluster with isolates from cold-blooded animalsM several 

species of fish, turtles, crabs, sea horses and frogs. IT. analysis distributes these 

strains over 10 clusters, among which three clusters represent as yet undescribed 

a_%'?"+6+*species. Also the sympodial species O.$%,+.+*8%-$2%&+3 which is 

morphologically very different from the annellidic a_%'?"+6+ species, is found between 

the waterborne species. A comparison of the entire order 7?+.-%-?2$"+6.& using the 

..6 rDNA gene shows that the waterborne species form a consistent clade within the 

parsimony tree. Cardinal growth temperatures of those species were also established. 

A correlation is observed between the maximum growth temperature and the source 

of isolation and the natural habitat of the hostn fungi growing with maxima below J0oC 

are found causing disease in ocean animals, while those with maximum growth 

temperatures around JJoC cause epidemics in cold blooded animals living in shallow 

tidal zones in the subtropics. A striBing example is an emerging crab disease in 

mangroves along the east coast of 2razil. Histopathological studies show that the 

infection leads to dissemination with enormous fungal loads, internal organs being 

entirely invaded.*RemarBably, some waterborne a_%'?"+6+ species have occasionally 

been isolated from human sBin disorders, particularly in elderly patients with diabetes 

Bnown to have relatively low body temperatures in their extremities. In general, 

thermophilic a_%'?"+6+ species seem to have a preference for humans, while 

mesophilic species are predominantly found in cold blooded animals. 

These findings are in contrast with waterborne melanized fungi belonging to the order 

X.%-"+6.&, for example 7+5%'?%$+*)+6%$(). These fungi are supposed to be plant 

endophytes and have never been found to be involved in animal disease. 

O 

J0 rabbits 

O dogs 

J0 Trichophyton mentagrophytes 

O Trichophyton mentagrophytes 

100 

100 

These findings are relevant by the possibility that the people become infected that is in 

contact with the fungi. To Bnow the data will allow implementing pertinent actions of 

prophylaxis and control of dermatophytosis in the studied populations.

P-0018. Study of dermatophytic flora of domestic birds in a city in Iran 

Hashemi J, Nasrollahi A 

.cience c Research 2ranch-Islamic Azad 6niversity, Tehran, Iran 

The goal of this research has been the study and determining of the identify of factors 

producing dermaphytosis and the rate of outbreaB among domestic birds suffering 

from this disease. 

In this survey which was carried out from the winter of 200O till the spring of 200[ 

domestic birds growing centers of ~om were visited and during this period one 

hundred samples were taBen randomly. The sampling was done by brush which was 

rubbed on the head, necB, cocBs comb and the beard of the birds and then the brush 

bearing the sample was carried to the ..c.c. (suburro dextrose agar r cycloheximid r 

chloramphenicol) culture and was examined in the laboratory and it was identified 

through direct test and the culture of the sample. 

According to this experiments only in two cases (0.2_) microsporom grpseum was 

isolated which is a geophillic dermatophyte and other cases isolated were from 

saprophyte fung. The isolated saprophyte in this study wereM Pseudallescheria boydii, 

Alternaria, Aspergillus, Cladosporium, Mucor, Penicillium, Malbranchea, Rhizopus, 

Acremonium, .trepthomyces, .copulariopsis. 

In view of the fact that some of the dermatophytes are among the zoonoses diseases 

the microsporom grpseum may also cause disease in human beings and the harms 

caused by them have got hygienic and economic importance. As a result for the saBe 

of avoidance of spread of this infection among the birds and following it preventing the 

transmission of this infection to human being the observance of health conditions, 

creating a suitable environment for living and feeding of the birds and also curing sBin 

diseases should be taBen into consideration. 

P-0019. Heat resistance of Prototheca spp. in milk at pasteurization 

temperatures 

Lagneau P 1 , (agielsBi T 2 

1 AR.IA, 2 National Tuberculosis and /ung Diseases Research Institute, aarsaw, 

Poland 

Introduction: Protothecosis are opportunistic infections due to the parasitism of 1$%-%-?.#+ 

d%'U"" and 1/*B"#[.$?+)"" species. They affect animals as well as humans and often result in 

cutaneous, articular and systemic infections. However, the importance of 1$%-%-?.#+ infections in 

veterinary medicine is primarily related to the manifestations of chronic mastitis met by dairy 

cows, with many cases reported in different countries in the last years. The aim of the present 

study was to evaluate the susceptibility of these organisms opposite thermal treatments 

employed to pasteurize milB. 

Material and MethodsM .trains of 1/ d%'U"" from mastitis cases on 2[ dairy farms were tested 

and in addition, a reference strain of 1/*B"#[.$?+)"" was included. After activation of our stores 

cultures, the strains were placed in a constant temperature water bath, first, at 7[ÅC / 20 sec. 

and a second series, at 80ÅC / 10 sec. The presence of 1$%-%-?.#+ spp. was controlled by 

culturing the tested suspensions onto .abouraud medium. 

Results: .trains of 1/*d%'U"" showed 80,8% of resistance at the first phase M 7[ÅC / 20 sec. and 

40% at the second oneM 80ÅC / 10 sec. For the species hB"#[.$?+)""f, some colonies were 

detected on two plates from the five repetitions at the first ratio. 

ConclusionM ae pointed out the remarBable resistance to heat of these algae and we noticed a 

significant variation of the results between the strains. fur results are in agreement with 2razilian 

studies, which have also reported theses observations (2)/ The resistance of 1$%-%-?.#+ spp. to 

thermal treatments is attributed to the presence in their cell wall of sporopollenin, the most 

resistant naturally occurring organic compound (J). In human medicine, recent articles 

concerning systemic infections in cancerous and immunocompromised patients because of the 

species gd%'U""f have been described (1, O). fur results underline the importance of protothecal 

mastitis cases and the controls of infected herds remain highlighted. 1$%-%-?.#+ spp. is an 

infectious agent showing an extension and because of its possible presence in our food, it should 

be added to the list of opportunistic pathogens. 

References: 

1. /ass-Flérl C. .-*+6. Disseminated Infection with 1$%-%-?.#+*d%'U"" after 6nrelated .tem 

Cell Transplantation for /euBemia. ]*76",*!"#$%8"%6/*200OhEMiOK07-OK08. 

2. Melville A. .-*+6/*Evaluation of the susceptibility of 1$%-%-?.#+*d%'U"" to milB 

pasteurization. !2#%'+-?%6%@"+/ 1KKKnIEN*M7K-82. 

J. Pore R. .. .-*+6. 1$%-%-?.#+ ecology. !2#%'+-?%6%@"+. 1K8JnDIMOK-62. 

4. Torres H.A. .-*+6. Protothecosis in patients with cancerM case series and literature 

review. 76",*!"#$%8"%6*K,U.#-/*200JhRM786-7K2. 


P-0020. Detection of circulating serum galactomannan for the diagnosis of 

avian aspergillosis 

Le Loc'h G 1 , Arnd P 1 , 2ougerol C 2 , Risi E J , Pdricard ( O , ~uinton ( [ , 2retagne . 6 , 

Guillot ( 1 

1 6MR 2IPAR INRA-AF..A-ENVA-6PVM, Ecole Nationale Vdtdrinaire deAlfort, 

Maisons-Alfort, France, 2 Clinique vdtdrinaire des oiseaux, Paris, France, J Ecole 

Nationale Vdtdrinaire de Nantes, France, O Clinique vdtdrinaire de .igean, France, 

[ Clinique vdtdrinaire, avenue de la Rdpublique, Paris, France, 6 6MR 2IPAR INRA- 

AF..A-ENVA-6PVM, Facultd de Mddecine de Crdteil, Crdteil, France 

Aspergillosis is a major cause of mortality in wild and domestic birds, especially in 

psittacines, raptors, waterfowl and turBeys. In the respiratory localized form of 

aspergillosis, lesions often appear as granuloma in the air sacs, lungs or trachea 

causing respiratory difficulties. However, 4&'.$@"66(& can spread and cause fairly nonspecific 

problems such as reduced feed intaBe, depression, dyspnoea, cyanosis or 

sudden death without any clinical symptoms. Its diagnosis remains a challenge for 

veterinarians and the number of tests resulting in a clear diagnosis is limited. 

Galactomannan, a polysaccharide found in the cell walls of 4&'.$@"66(&*U()"@+-(&, is 

an antigen, which can be detected by E/I.A (Plateliaw 4&'.$@"66(&). This test allows 

the diagnosis of invasive aspergillosis in humans. ae have conducted research into 

the presence of galactomannan in 12J sera collected from 116 birds of J0 species. 

The sera were collected from privately-owned psittacines in France (2ordeaux, 

Narbonne and Paris) or wild birds at two French wildlife care centres (Maisons-Alfort 

and Nantes) either during screening or because birds were suspected of having 

aspergillosis. Each serum was also tested for precipitant antibodies by 

electrosyneresis. 

P-0021. Intestinal pythosis in a dog in the south of Brazil 

M. Santurio J 1 , E. .chwendler . 1 , Henzel A 1 , Aze vedo M 1 , Rodrigues A 2 , .. 

Cavalhiro A 1 , Hartz Alves . 1 

1 6niversidade Fed de .anta Maria - /apemi, 2razil, 2 6niversidade Fed. de .anta 

Maria - .etor de Patologia Veterincaacutenria, 2razil 

Pythiosis is a granulomatous disease caused by the oomycete Pythium insidiosum 

that affects horses, dogs, cats, cattle and humans. In dogs, pythiosis occurs mainly in 

the gastrointestinal tract and sBin. A male /abrador Retriever dog, J0 months old, was 

referred to the HCV/6F.M (.anta Maria, state of Rio Grande do .ul, 2razil) with 

sporadic vomiting, diarrhea and marBed weight loss. The clinical examination revealed 

a palpable mass in the abdominal cavity, confirmed by exploratory laparotomy. 2eing 

in a terminal stage, the dog was euthanized. ThicB hyphae (diameter between O and 7 

èêm) were observed in the histopathological exam by HE, GM. and PA. staining. 

The presence of Pythium insidiosum was confirmed by culture and isolation, and 

extraction of DNA of the isolate, further confirmed by nested PCR molecular technique, 

according to Grooters c Gee (2000). 

6sing clinical signs (depression, emaciation dyspnoea, open-mouthed breathing, tail 

bobbing, respiratory distress after exercise, change in vocal pitch, nasal discharge, 

biliverdinuria) and the results of electrosyneresis, birds were allocated to one of three 

groupsM group A (highly probable aspergillosis), birds with a positive electrosyneresis 

result and showing symptoms compatible with aspergillosisn group 2 (probable 

aspergillosis), birds with either a positive electrosyneresis result or showing clinical 

symptoms compatible with aspergillosisn group C (unliBely aspergillosis) birds with a 

negative electrosyneresis result and no clinical symptoms. In group A (J0 birds), J0_ 

showed a positive E/I.A result. In group 2 (6O birds), JO_ showed a positive E/I.A 

result. In group C (22 birds), 1O_ showed a positive E/I.A result. 

Among the birds suspected of having aspergillosis (groups A and 2), the 

concentration of galactomannan reached the detection threshold (index 1.[) in 

approximately J0_ of birds. The Plateliaw 4&'.$@"66(& test is thus relevant for the 

detection of circulating galactomannan in avian sera but does not appear to be 

sensitive enough for the diagnosis of avian aspergillosis. Eight birds without clinical 

signs (group 2 and C) showed a positive E/I.A result. 

Finally, we observed that birds, showing depression, reduced feed intaBe and other 

non-specific symptoms, were positive for the Platelia w 4&'.$@"66(& test more often than 

birds showing only the respiratory symptoms (O[_ compared to 2J_, respectively).

P-0022. Equine nasal rhinosporidiosis: Case report in São Paulo, Brasil 

Matos Santiago Reis A 1 , Nabuco de Abreu A 2 , Marinho de Medeiros Camin P 2 , 

/opez Correia da .ilva / J , Neves Torres / O , Yvonne Arantes 2accarin R [ , do Valle De 

Zoppa A J 

1 Resident of Veterinary Hospital, Veterinary Medicine and Zootechny Faculty (FMVZ), 

6niversity of .ao Paulo (6.P), 2razil, 2 Autonomy Veterinary Medical, .ao Paulo, 

2razil, J Department of .urgery, Veterinary Medicine and Zootechny Faculty, 

6niversity of .ao Paulo, 2razil, O Veterinary Medical, Department of Animal Pathology, 

Veterinary Medicine and Zootechny Faculty, 6niversity of .ao Paulo, 2razil, 

[ Department of Clinical Medicine, Veterinary Medicine and Zootechny Faculty, 

6niversity of .ao Paulo, 2razil 

Rhinosporidiosis is a rare disease and not many cases are revealed in the publish 

literature. There have been no recent significant advances in its clinical and 

pathological aspectsn nevertheless, significant advances have been made in basic 

Bnowledge of the pathogen, P?",%&'%$"5"()*&..8.$". Rhinosporidiosis has been 

documented as having occurred in several species of farm, wild and domestic animal, 

and human. A 12-year-old equine presented a red, firm, verrucous and ulcerated 

mass. Approximately, it had 2 centimeters of diameter, and was located in the left 

nostril medial mucocutaneus junction. The mass was excised using an electrocautery 

and the horse was standing. The histopathologic exam revealed the presence of 

sporangic cysts and sporangia at different stages of maturation and variable 

distribution, confirming a case of equine rhinosporidiosis. 

P-0023. The frequency of Tichophyton verrucosum variety among the cows 

infected dermatophytosis in Kermanshah (2003) 

Mikaeili A, Mostafaei A 

l6M., lermanshah, Iran 

Dermatophytosis is most frequent mycoses and >$"#?%'?2-%,*0.$$(#%&() is zoophilic 

dermatophyte due dermatophytosis in cattle with hair loss and scaling. 

>$"#?'?2-%,*0.$$(#%&() has three pigmented colony varietyM >/0.$$(#%&()* 

0+$/%#?$+&.()3*>/0.$$(#%&()*0+$/+68()3*>/0.$$(#%&()*0+$/5"&#%."5. 

In descriptive study 112[ infected sample from cows lesions were cultured and 

following results were obtainedM >/0.$$(#%&()*0+$/%#?$+&.() with J7 (J.2_), 

>/0.$$(#%&()*0+$*+68() with 10(o88_) and >/0.$$(#%&()*0+$/5"&#%."5 with 7 (062_) 

and 1071 sample was negative for >/0.$$(#%&(). 


P-0024. Environmental isolates of Cryptococcus neoformans var. grubii 

(serotype a) are not lethal for mice 

Mitchell T, /itvintseva A, (orgensen I 


The leading cause of cryptococcosis worldwide,*7$2'-%#%##(&*,.%U%$)+,& var. @$(8"" 

(serotype A), is widespread in the environment, where it is primarily associated with 

pigeon excreta. A number of molecular epidemiological studies indicate that many 

environmental and clinical isolates of serotype A are indistinguishable. However, the 

murine virulence of environmental strains of 7/*,.%U%$)+,& has not been assessed 

since 1K6J, when isolates were evaluated by challenging mice with intracerebral 

inoculations, which do not represent the natural route of infection. ae used the murine 

inhalational model of cryptococcosis to compare the virulence of clinical and 

environmental strains of serotype A that possessed identical genotypes as determined 

by Amplified Fragment /ength Polymorphisms (AF/P) and Multilocus .equence 

Typing (M/.T). .everal genotypes were compared. Ten of the 12 clinical strains that 

we tested were highly virulentn mice infected with these strains developed severe 

disease and were sacrificed within 2[ days after infection. In similar experiments, mice 

were comparably challenged with 12 environmental strains, and ten of these isolates 

were avirulentn sixty days postinfection, the mice infected with these strains did not 

develop any symptoms of disease. The remaining two environmental strains were 

hypovirulentn half the infected animals developed symptoms at O0 days after infection. 

None of the environmental strains was as lethal as the virulent clinical strains. 

Repeatedly passing environmental isolates in mice (up to O times) did not significantly 

increase their lethality for mice. In follow-up studies, we developed a new genotyping 

technique based on hybridization with Tcn2, TcnJ, and TcnO retrotransposon-specific 

probes, and this method differentiated clinical and environmental strains that had the 

same AF/P/M/.T genotypes. Retrotransposon typing has provided the first 

phylogenetic evidence that clinical and environmental populations of serotype A may 

be different. 

P-0025. First report of Debaryomyces nepalensis isolated in dog 

Moretti A 1 , FuBushima l 2 , TaBizawa l 2 , .uzuBi M J , Vidotto V O , Cannizzo F O , 2oncio 

/ 1 

1 6niversity of Perugia, 2 Chiba 6niversity, J 2ioresource Center, O 6niversity of Torino 

A young male hunting dog, wire-haired pointing griffon with about six months old, got 

lost in a street of the surrounding of Perugia (Italy), was caught by dog catchers. In the 

same day the dog was transferred to the municipal Bennel and examined by the 

veterinary employees in order to checB his health status. During the visit two 

oropharyngeal swabs in 2O hours were also collected, as well as samples of faeces 

for parasitological investigations. The two oropharyngeal swabs were streaBed onto 

.abouraud agar Petri dishes with and without antibiotics in thermostat at J7ÅC. After 

six days the yeasts developed colonies, were transferred onto agar Malt Petri dishes 

and incubated in thermostat for six days. The yeast colonies (IFM [O2[7) were 

identified by the API J2C A6j (bioMerieux-France) as V.8+$2%)2#.&*?+,&.,"", but 

the numeral profile obtained from API J2C A6j test for them did not conform to any 

yeasts. Ascospores were observed on the culture of PDA medium. The major 

ubiquinone of IFM [O2[7 was identified as ubiquinone-K (~-K). Afterward genetical 

analyses of IFM [O2[7 were performed together with the type strains of V/*?+,&.,"" 

(var. ?+,&.,""*and var. U+8$"*), 7+,5"5+*'&2#?$%'?"6+ and V/*,.'+6.,&"& (CM 20K[. 

The base sequences of IT. 1 and 2, and D1/D2 regions of /.6 rDNA of IFM [O2[7 

completely coincided with those of V/*,.'+6.,&"& (CM 20K[. According to these 

results, the IFM [O2[7 was identified as V/*,.'+6.,&"&. RAPD patterns between the 

two strains were found to be identical. fnly genetical tools can properly identify V/* 

,.'+6.,&"&. This is the first case in which V/*,.'+6.,&"& has been isolated not only in a 

dog but in Europe.

P-0026. Dermatophytosis epidemiology in guinea pigs from conventional animal 

facility 

Moya M 

INHRR 

Purpose of the study: To determine the dermatophytosis incidence and the risB 

factors in a guinea pigs population coming from a conventional animal facility. 

Description of the project: It was carried out a study of 208 guinea pigs (7+0"+* 

'%$#.66(&) Hartley strain, with a weight of 100 to 800 grams, coming from the Instituto 

Nacional de Higiene Rafael Rangel animal facility, in the period 2002-200J. They are 

non consanguineous animals, reproduced by the Poiley system. It is a closed colony 

for more than 20 years under a conventional animal facility system, with basics quality 

control measures. The study was carried out in three stagesM epidemiological, clinical 

and laboratory analysis. The size of the sample was estimated by Epi-Info [,01 

program. For this calculation, 1[_ of prospective frequency, [_ error and K[_ of 

trust level was established. Animals with alopecias, descamative lesions, and without 

apparent lesions were evaluated 1 , and it was also considered the physiologic state of 

the female and male animals (pregnant female, empty female, served female, and 

served and sucBling female). Hair samples were taBing of by mechanical traction with 

sterile pincers and epidermal flaBes by rasped with sterile scalpel 2 . Direct exam with 

10_ lfH plus ParBer inB, and culture in Mycosel and /actritmel glass tubes agar was 

carried out with both samples to mycological study. Accumulated incidence, rate of 

incidence and prevalence were determined to establish the dermatophytosis 

occurrence pattern J . 

EpidemiologÜa mddica. EnM Medidas epidemiolágicas. 2 da edicián. Manual 

Moderno p.1K-2O. 

O. 2lanco (/, GarcÜa ME. Presente y futuro del diagnástico inmunolágico de las 

micosis superficiales. Rev Iberoam Micol, 2000n 17M [2J-8. 

[. Murillo P. Diagnástico de las dermatofitosis. 2002. 

InM 6R/, http//www.ucr.ac.cr/{gacetapc/dermatofitosis s1J/12/02u 

6. Moya M. Diagnástico de las dermatofitosis en animales de bioterio. Rev Inst 

Nac Hig Rafael Rangel, 200Jn JO (2)M22-6. 

Results and Conclusion: ff 208 guinea pigs, 8J presented lesions (JK,K_) and 12[ 

did not present lesions (60,K_). KK (O7,6_) animals had a positive direct exam. 171 

(82,21_) animals had dermatophytes positive cultures, recovered >$"#?%'?2-%,* 

).,-+@$%'?2-.& (O6,78_), !"#$%&'%$()*#+,"& (JO,[0_) and both agents mixed 

infections (18,71_). K1_ of accumulated incidence was obtained, with an incidence 

rate of 2[o/ 00 . The culture was the most effective method for the dermatophytosis 

diagnosis, since the animals can have sBin lesions for different causes O-6 . The fungal 

infections were most common in young than in adult animals. Dermatological solution 

was used to treat sicB animals with improvement at J weeBs. This study allowed to 

correct failures in animals handling, and to establish control barriers liBe to avoid 

overcrowd inside the cages, flamed the cages and wild rodent control. The study of 

incidence and prevalence are useful for the evaluation of these pathologies and to 

establish mycosis control and prevention programs. 

References: 

1. Malcom (, f]Donoghue P. (1K77). Cobayos y conejos. PatologÜa de 

animales de laboratorio. Diagnástico y tratamiento. Editorial Acribia. 

Zaragoza. p. 12O |1[7. 

2. Arenas R. (1KKJ) Diagnástico de laboratorio. MicologÜa mddica ilustrada, 

clÜnica, laboratorio y terapdutica. Editorial Interamericana, McGraw-Hill. p. 

OJ-[J. 

J. 2oring (R, Daniels .R, Eley (a, Flanders aD, Greenber R.. (1KK8). 


P-0027. 

Japan. 

Arthrographis kalrae isolated from the oral cavity of a canine in 

P-0028. Is gender an important variable in the course of experimental murine 

aspergillosis? 

Murata Y, .ano A, Yarita l, TaBayama A, lamei l, Nishimura l 

Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba 6niversity, 

Chiba, (apan 

4$-?$%@$+'?"&*[+6$+. is an environmental saprophyte fungus, and distributed world 

widely. The fungus has been isolated from environments (air, soil, and fur of wallaby). 

The species is also one of the causative agents for emerging fungal infections in 

human and animals (horses and dogs), and causes not only superficial, but also deep 

mycoses (onychomycosis, Beratitis, endophthalmitis, mycetoma, nasal sinusitis, 

pneumonia, bone marrow infection and encephalitis). 

ae isolated a white mycelial fungus morphologically similar to >$"#?%&'%$%, spp. from 

the oral cavity of an 11-year-old female sprayed mongrel dog during a survey of oral 

fungal flora of house-holding pets on J2K dogs and K[ cats, from April to (une, 200O. 

ae identified the isolate as 4/*[+6$+. on the basis of its morphology, physiology, 

molecular biology, and pathogenicity. Eight 4/*[+6$+., 2 1"-?%+&#(&*6+,@.$%,"" which 

was supporsed to be the teleomorph of 4/*[+6$+., one 4/*+68+, J 4/*#(8%"5.+, one 4/* 

6"@,"#%6+, and one 4/*'","#%6+ strains were used as references. 

Colonies on .aboraud dextrose and potato dextrose agar plates were white cottony, 

producing arthroconidia and blasto-conidia, with a light brown glabrous part at the 

center, and yellowish reverse. The isolate could grow at J7 o C without yeast-form 

conversion, and failed to grow at O2 o C. 6rease activity was positive while hair 

perforation was negative. The DNA sequences of IT. and D1/D2 regions of ribosomal 

RNA genes were identical more than K8 and KK_ with those of 4/*[+6$+.*type strain 

deposited in Gen2anB as A2116[J6 and A2116[OO, respectively. The DNA 

sequences were closely related to 1/*6+,@.$%,"", and independent from other 

4$-?$%@$+'?"& spp. and >$"#?%&'%$%, spp. The pathogenicity in mice (10 6 /10 g body 

weight, i.v.) treated with or without corticosteroid was not fatal by O weeBs. .mall 

abscesses with yeast-liBe cells were observed in the livers histopathologically. 

The present isolate was the first isolation of 4/*[+6$+. from (apan. Thermally 

dependent dimorphism was not a universal characteristic in the species. 2oth 4/* 

[+6$+. and 1/*6+,@.$%,"" are closely related species on the basis of the IT. and D1/D2 

sequences of rRNA gene while other 4$-?$%@$+'?"& spp. were distinct suggesting that 

the genus 4$-?$%@$+'?"& has heterogenous origins. The genotype of the present 

isolate was slightly different from those of human ones. The difference might be based 

on host- or area-specificity. .ome cases diagnosed as >$"#?%&'%$%, infections might 

include 4/*[+6$+. ones in (apan. 

Najvar L, 2ocanegra R, Molina D, flivo M, Graybill (, lirBpatricB a, Patterson T, 

Graybill ( 

The 6niversity of Texas Health .cience Center at .an Antonio, .an Antonio, 6.A 

Purpose: Determine whether outcome differs for males versus females in the course 

of experimental murine pulmonary aspergillosis. 

Background: There are many factors which influence the course of invasive 

aspergillosis. These include the isolate of the pathogen, the inoculum size, the strain 

of mouse, route of infection, and immune status of the host. For some mycoses, most 

notably paracoccidioidomycosis, male gender plays a crucial role in the course of 

disease. In the present studies we sought to determine whether gender influences the 

course of aspergillosis. 

Methods: Male and female neutropenic, steroid pretreated 2alb/c inbred and ICR 

outbred mice were infected using an aerosol delivery of 4*U()"@+-(& conidia. Mice 

were infected in one of two chambers, the first being an acrylic chamber, which can 

hold O0 mice, and the second a much larger Madison chamber, which can hold K6 

mice. These allowed us to study 2 strains of mice and both sexes in the same 

experiment. .urvival was observed for 2 weeBs, and lung tissue burden was assessed 

at various times after infection. .everal studies were repeated, with results pooled. 

Groups contained 7 to [7 mice. 

Results: 2oth 2alb/c and ICR mice were equally susceptible to aspergillosis. 

6ninfected controls survived and infected mice had from 60 to 7[_ mortality when 

infected in the acrylic chamber, and 20-O0_ mortality when infected in the Madison 

chamber. There were no significant differences in survival of males or females, and 

no differences between ICR and 2alb/c mice. /ung tissue counts declined slowly over 

the course of 7 days after infection. There were no significant differences between 

males and females. 

Conclusion: In two different strains of mice, infected in two difference chambers, the 

course of invasive aspergillosis was similar in both male and female mice. Gender 

does not appear to be a determinant of outcome for invasive aspergillosis. 

In conclusion, a white, arthroconidium-forming fungus isolated from a dog in (apan 

was identified as 4$-?$%@$+'?"&*[+6$+. based on morphological, physiological and 

molecular biological data, and was weaBly pathogenic in mice.

P-0029. The prevalence and identification of Malassezia spp in the horses in 

northern Iran 

Nasrollahi Omran A 1 , Hashemi J 2 , Khosravi A J , Makhjiri S O 

1 IA6 of ToneBabon, Iran, 2 IA6 fF Tehran, Iran, J IA6 fF Tehran, Iran, O IA6 fF 

Tehran, Iran 

IntroductionM The genus Malassezia consist of lipophilic yeasts are Bnow to be as component of 

the normal microflora of human sBin and many mammals and brids. Recently the taxonomy of 

these genes has been revised basis of on the morphology, ultrastructural, physiology and 

molecular 2iology characteristics and added to 12 species. The purpose of is study was 

detemine ,the prevalence of Malassezia spp on the horses sBin in the Golestan province and 

identification of them according to horses sex, breed, age and living geographical area. 

Material and methods: During 1[ monthes period, from july 200O to sept 200[ sampling was 

carried out by stril moquet (Bind of carpet) and scraping from 2[6 horses perineum, ear, axilla. 

groin and dorsum. a part of samples survried microscopically with methylene blue stain method. 

All samples were inculated on the saborured glucose agar (.GA) supplement with olive oil. all 

media contanied 1[0 mg of chlromphenicol and 1 gr of cyclohexmide. Plates were incubated in 

J0Åc and were survied in J,[,7 and 10 days intervals. ahen the growth of malassezia Colony 

was observed. the colonies subculture on .GA to determinate lipid dependent.the identification 

of lipid dependent yeasts was based on the ability to use tweens 20,O0,60 and 80 , catalase 

reaction, spliting of esculin, growth on .GA supplement with olive oil in J7,O0 and 

accompaniment with morphological characteristic on .GA supplement olive oil, as described by 

guillot and gueho (1KK6). 

Results: fur results showed that [0.78(1J0case) of horses were male and rest were mare 

OK.22_ (126case). from out of that horses K0 case (J[.1[_) were TurBoman breed and rest 

were 17 case (J0.K_) TurBoman blood two breed, 6K case (26.K[_). Thoroughred breed and 20 

case (7.81_) unBnow breed. avaerage age of horses was 7 years, that most freqent of them 

were in the age group of 2-8 years 11[ case (OO.O2_). In this study the pervalence of 

Malassezia spp in survied horses JJ.K8_ (87case) report. from 2[6 horses that were studied [0 

case (1K.[J_) male and J7 case (1O.O[_) mare had Malassezia positive culture. the highest 

pervalence of Malassezia were seen age group 2-8 years [[ case (21.O8_) and turBmonan 

breed J7 case (1O.O[_) the lowest result were age group under 2 year 12 case (O.6K_). the 

mast frequently isolated Malassezia spp was in groin with 2[ case (2O.27_) , followed by anus 

2J case (22.JJ_)ear 22 case (21.J6_), axilla 17 case (16.[_) and dorsrum 16 case (1[.[O_). 

the most frequently isolated species was Malassezia globosa with 2J case (22.JJ_) followed by 

Malassezia furfur 1K case (18.O[_) Malassezia resticta 1Ocase (1J.[K_). Malassezia 

pachydermatis 12 case (11.6[_) Malassezia obtusa 12 case (11.6[_), Malassezia sympodialis 

12 case (11.6[_), Malassezia slooffiae 11 case (10.6K_). 

P-0030. Fungal agents in rainbow trout hatcheries in shahrekord 

Nikookhah F, Fadaie fard F 

Department of Plant Protection, Faculty of Agriculture, .hahreBord 6niversity, 

P.f.2oxM 11[, .hahreBord, Iran 

The present survey was conducted to isolate and identify the fungal agents on eggs, 

alevins, fry, and water supplies of three rainbow trout (b,#%$?2,#?(&*)2["&&) 

hatcheries of Chaharmahal- va- baBhtiary province in Iran. 

J60 samples of hatchery water supplies, eggs, fry and alevins each one K0 samples 

were taBen from J rainbow trout hatcheries. 

.wab samples of crashed eggs, alevins and fry and a little drop of taBen water were 

cultured on sabouraud]s glucose agar containing chloramphenicol. 

The plates were incubated at 20o c for three successive days. fungal plugs were 

removed from the edge of the colonies and used for slide culturing. .lides were 

incubated at 20oC for 72 hours and then followed by microscopic examinations of the 

recovered colonies. In microscopic examinations shape and arrangement of spores 

and presence or absence of septa were considered using lacto phenol cotton blue as 

wet mount preparation. 

Differences in the number of isolations between the test groups were analyzed using 

Fisher exact test. Totally 12 fungal species (7$2'-%#%##(&3*P?%5%-%$(6+3*7+,5"5+* 

+68"#+,&3*7+,5"5+*@6+8$+-+3*1.,"#"66"()3*76+5%&'%$"()3*4#$%)",()3*4&'.$@"66(&*U6+0(&3* 

4&'.$@"66(&*,"@.$3*!(#%$3*46-.$,+$"+3*^(&+$"()) were isolated. In our investigation the 

number of 1.,"#"66"() isolations (J1.2[_) and Rhodotorolla (27.JO_) were 

significantly (pv0.0[) more than the other isolates of tested groups. Infection rates of 

fry to P?%5%-$%66+ (1O.8O_) and Acromonium (8.[K_) were greater than that of alevins 

(2.JO_ and 0) and eggs (7.02_ and 0). 

Differences between the numbers of isolations of the two fungi are statically significant 

at pv0.0[. 

Total fungal contaminations in eggs, fry, water supplies and alevins were JK_, J7.O_, 

1O.8_ and 8.6_ respectively. 

Discussion: The prevalence of Malassezia spp in horses was JJ.K8_ in our study that was 

lower than reports by other authors (60_). The significance correlation were not observed 

between the horses sex, breed, livin geographical area and prevalance of Malassezia , wherease 

significace correlation was existed between horses age and perevalence of Malassezia. 


P-0031. Paracoccidioidomycosis-infection in horses and wild monkeys of 

Paraná State, Brazil 

Ono M 1 , Corte A 1 , Itano E 1 , MalansBi / 2 , Navarro I 2 , .hiozawa M 2 , .voboda a 2 , 

Aguiar / J , Passos F J , Camargo Z J 

1 .tate 6niversity of /ondrina, Dept. Pathological .ciences, 2 .tate 6niversity of 

/ondrina, Dept. Preventive Veterinary Medicine, J Federal 6niversity of Parana, Dept. 

Zoology 

Paracoccidioidomycosis (PCM) is a systemic mycosis with chronic evolution and 

granulomatous pattern caused by the thermodimorphic fungus 1+$+#%##"5"%"5.&* 

8$+&"6".,&"&. The PCM patients are mainly male rural worBers and the lungs, spleen, 

liver, sBin and mucosa are the frequently affected organs. The role of other animal 

species in the fungus ecology remains unclear. 1/*8$+&"6".,&"& has been isolated from 

bats, penguin feces and armadillos. Epidemiological studies suggest that other animal 

species as cows, sheeps, and dogs may be infected by 1/*brasiliensis. The aim of this 

worB was evaluate 1/*8$+&"6".,&"&*infection in free living monBeys (7.8(&*spp and 

46%(+--+*#+$+2+) and horses from northwest and northern Parana .tate, respectively. 

A total of KJ serum samples (7.8(&*&'' } 68 and 4/*#+$+2+ } 2[) collected from freeliving 

monBeys captured in the region of Porto Rico, ParanZ and 100 samples from 

horses of Northern Parana .tate were were analyzed by E/I.A, using gpOJ as 

antigen. The seropositivity observed was of J0_, OO,1 _ and 60 _ for horses, 7.8(& 

spp and 4/*#+$+2+, respectively. This result suggests that horses and monBey*are 

frequently infected by 1/*8$+&"6".,&"&*and may play a role in the fungus ecoepidemiology. 

Financial support: CNPq, FundaÑÉo AraucZria, Fundo ParanZ Tecnologia, CAPE. 

P-0032. Isolation of fungal flora from the hair coats and auditory canal of the 

wild rodents in Fars, Iran 

Pakshir K, Mehrabani D, Asgari G, Motazedian M 

.hiraz 6niversity of Medical .ciences 

Purpose of the study: .Rodents are considered as a source of fungal diseases. They 

may harbor very many different fungal species by their hair coats, such as 

Beratinophilic fungi. Many Binds of these fungi, liBe the dermatophytes, are hazardous 

for human being. There are many fungi which resident in animales auditory canal as 

fungal flora, such as Malassezia pachydermatis, the major species associated with 

infections or colonization in animals. There are fewer studies on the Iranian rodentes 

fungal flora, especially in the Fars province. The aim of this study was to determine 

the fungi associated on hair coat and auditory canal of the rodents in Fars province. 

Summarized description of the project: 202 rodents belong to different genera 

include Rathus, Tatra, Meriones*and Mus were trapped from different area of Fars 

province. A total of 1260 hair coat .amples were collected from six sites of the 

rodents body include bacB, the abdomen, the face, the tail, the hand and the foot, 

using hair plug. Ear samples from 87 rodents were taBen from both ear canals by 

using sterile swabs. .amples were cultured on .abouraud dextrose agar (with and 

without olive oil) and Mycosel agar and incubated in 2[ and J0+C for two weeBs. 

Identification of the fungi performed by the routine mycological methods. 

Results and conclusions: Fungi are isolated from the hair coat of the rodents 

belonging to the following generaM .copulariopsis, Aspergillus, Penicillium, 

Chrysosporium, Rhizopus, Mucor, .epedonium, Chaetomium, Paecilomycess 

Pseudallescheria*and yeast which were varied in each genus. No species of the 

dermatophytes were isolated in this study. Fungal isolates from rodents ear canal 

belongs to different genera including Aspergillus, .copulariopsis,*Mucor and Yeast. 

No species of Malassezia were isolated from the rodentes ear canal. 

In this study, many isolates had resistance to cyclohexamide, such as .copulariopsis* 

and they might inhibit the dermatophytes growth cause of their rapid growth.

P-0033. Equine nasal obstruction by fungic granuloma: case report 

Reis A 1 , Crispim R 1 , De Zoppa A 2 , 2enites N J , .inhorini I O , 2accarin R [ 

1 Veterinary Hospital, Veterinary Medicine and Zootechny Faculty (FMVZ), 6niversity 

of .ao Paulo (6.P), 2razil, 2 Department of .urgery, Veterinary Medicine and 

Zootechny Faculty, 6niversity of .ao Paulo, 2razil, J Department of Preventive 

Veterinary Medicine and Animal Health, Veterinary Medicine and Zootechny Faculty, 

6niversity of .ao Paulo, 2razil, O Department of Animal Pathology, Veterinary Medicine 

and Zootechny Faculty, 6niversity of .ao Paulo, 2razil, [ Department of Clinical 

Medicine, Veterinary Medicine and Zootechny Faculty, 6niversity of .ao Paulo, 2razil. 

Equine nasal aspergilosis is a rare disease and not many cases are revealed in the 

publish literature. 4&'.$@"66(&*&' is present in health equine superior respiratory tract, 

and it is an opportunist and cosmopolita agent. A K-year-old equine presented 

absence of breath out and serosanguineous nasal discharge at the right nostril and 

respiratory noise. The endoscopy and radiology revealed a yellowish mass with six 

centimeter diameter, mucosa recovering, which was obstructing the entire right cavity 

and a part or the left one. The mass was excised by a bone flap at the right frontal 

sinus. The microscopic exam revealed a fungic granulomatous rhinitis. Three weeBs 

after the surgery, an endoscopic exam showed two greenish plaques at the mass 

place and the culture revealed 4&'.$@"66(&*&' growing. The treatment after surgery 

was benzatine and potasic penicilines, gentamycin and phenylbutazone parenteraln 

moreover, camomila tea and distilled water were flushed in a drain placed above the 

bone flap. After six months no endoscopic and clinical disturbances was detected and 

the owner told that the animal returned to sport activity with good performance. 

P-0034. Histoplasma capsulatum detected in a soil from the Atlantic area 

Sabino R, Verissimo C, 2randÉo (, Alves C, Parada H, Rosado / 

Instituto Nacional de .aâde Dr. Ricardo (orge, /isboa, Portugal 

Histoplasmosis is an acute, subacute or chronic localized or disseminated mycotic infection 

primarily involving the reticulo-endothelial system in various tissues and organs (1). More than 

K0_ of the infections are asymptomatic. However, fungemia and dissemination typically occur in 

infants, the elderly and patients immunosupressed either due disease or therapy. The condition 

is found in patients with malignancies, organ transplant recipients, patients treated with large 

doses of corticosteroids and patients with AID. (2). 

Histoplasmosis is caused by A"&-%'6+&)+ (#+'&(6+-() and 5(8%"&""). A"&-%'6+&)+ #+'&(6+-()*is 

geographically widespreadM it is endemic from Mississipi and fhio valleys but cases have been 

reported from central and .outh America, Europe, Africa, Asia, Australia and the Philippine 

Islands. Infection is originated from exposure to dust borne organisms by inhalation of dust in 

silos, chicBen coops and caves (1). The saprophyte phase of A/*#+'&(6+-() is particularly found 

in soil with high nitrogen content ie. enriched by chicBen manure or in bat or birds droppings 

under roosting sites. /ocal birds are generally not infected but natural infections are common in 

canopy-dwelling mammals particularly rodents, bats and opossums (J) and also in birds liBe 

starlings (O). 

Isolation of A"&-%'6+&)+*#+'&(6+-() from soil by culturing soil suspensions on agar media has 

rarely been successful ([). However, we report here a case of A"&-%'6+&)+*#+'&(6+-() detected 

in a soil from a recreated Atlantic habitat (in a recreational closed area where different bird]s 

species cohabit). 

Colony is slow growing, cottony and white. Macroconidia exhibit characteristic finger-liBe 

projections. To confirm the identification, subcultures were incubated a two different 

temperaturesM J6oC and 2[oC. ae observed the conversion to yeast form at J6oC, confirming the 

dimorphism character of A"&-%'6+&)+*#+'&(6+-()/* 

ahen A"&-%'6+&)+*#+'&(6+-() was detected in culture, we also investigate the possible 

transmission vectorsM bibliography refers the species mentioned above. ae concluded that 

starlings were the carrying vector. ae alerted the responsible entities to A"&-%'6+&)+ presence, 

as the area is visited by thousands of people per day. ae also recommended the serological 

monitorizarion of the animal careers, the animal stool mycological analysis and the air sampling 

from the affected habitat to search the presence of A"&-%'6+&)+*&'/ 

References: 

1. Moss E., Mc~uown A . Atlas of Medical Mycology. J rd ed., 1K6K. aaverly Press.. 

6...A. 

2. libbler C, Mclenzie D., fdds F. Principles and Pratices of Clinical Mycology. 1KK6. 

ailey and .ons, 6...A. 

J. Hoog G., Guarro (., Gene, (, Figueras, M.(. Atlas of Clinical Fungi. 1KK[. 

Centraalbureau voor .chimmelcultures. The Netherlands. 

O. Frey D., fldfield R., 2ridger R. A Colour Atlas of Pathogenic Fungi. 2 nd ed.,1K81. 

aolfe Medical Publications /td. Holland. 

lwon-Chung l., 2ennett (. Medical Mycology.1KK2. /ea c Febiger. 6...A 


P-0035. Biotyping of Candida albicans strains by 5-fluorocytosine in México. 

Salas Tellez E 1 , Nâez del arco A, (aramillo ( 

1 6NAM, 2 6NAM, J 6NAM 

CZtedra de Inmunodiagnástico y profilaxis de enfermedades bacterianas y micáticas. 

Facultad de Estudios .uperiores CuautitlZn, 6niversidad Nacional Autánoma de 

Mdxico. Avenida Primero de Mayo s/n, Colonia Atlanta, CPM [O7O0. CuautitlZn Izcalli, 

Estado de Mdxico. Mdxico. 

The objetive of the study was to identify the biotype present in Mdxico from different 

isolates of 7/*+68"#+,& from isolated from human and bovines samples. The 7[ strain 

from human samples came from oropharynx, vagina and urethra and the [0 strains 

isolated were from clinical mastitis cases from bovines. All the strains were identified 

as 7/*+68"#+,& by chlamidospore formation and germinal tube as well as by 

auxonogram and zimogram. The biotyping was done by using [- Fluorocytosine at 

concentrations ranBing from 10 to 100 mg/ml. The different antimicotyc concentration 

were mixed with .DA and plates were prepared with different concentrations. Plates 

were inoculated with a 6O calibrated loop and a suspensián with the strains to be 

tested. The plates were incubated at J[ C and read O8 h later. The plates were no 

growth was noticed were classified as 2iotype A and the ones with growth, resistant to 

the antimycotic were classified as 2iotype 2. ResultsM [7.O _ of strains isolated from 

humans were sensitive to [- Fluorocytosine and the rest were resistant. .eparately, 

68 _ of the strains isolated from bovine were sensitive to [- Fluorocytosine. 

Conclusion: The 2iotype A is the predominant in the strains studied herein. 

P-0036. Ochroconis gallopava isolates from hot springs in Japan. 

Sano A, Yarita l, Murata Y, TaBayama A, Yaguchi T, TaBahashi Y, lamei l, 

Nishimura l 

Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba 6niversity, 

Chiba, (apan 

b#?$%#%,"&*@+66%'+0+ is a species of dematiaceous fungi recognized as a causative 

agent of emerging fungal zoonosis. More than J0 human cases including J cases from 

(apan have been reported. The pathogen has caused outbreaBs in poultry and wild 

birds, and a few cases in domestic cats. Environmental isolates of b/*@+66%'+0+ have 

also been found under low-pH and thermal conditions, such as in coal waste piles, hot 

springs, sewage from nuclear power plants, and broiler-house litter. 

The present study tried to isolate b/*@+66%'+0+ from hot spring environments in (apan 

because our country is famous for volcanoes and hot springs, and the fungus should 

inhabit in such environments. 

Twelve hot spring water corrected from the northern to the southwestern areas of 

(apan were examined. The pHs of hot springs were approximately [.6, and the 

temperatures at the collecting moment were O1-O2 o C. Five hundreds milliliter of the 

hot spring water was filtrated with a 0.22 micrometer-pore-sized filter. The filter was 

cultured at O2 o C on potato dextrose agar plates for 2 weeBs. 2rownish colonies were 

picBed up and studied mycologically and molecular biologically. The D1/D2 domain of 

large subunit ribosomal RNA gene sequences was compared with the Gen2anB 

database. b/*@+66%'+0+ isolates from the hot spring environments were examined in 

their virulence by intravenous injection into mice ([ x 10 [ conidia /10 g body weight) 

treated with or without corticosteroid. The behavioral changes, mortality, recovery rate 

of fungal cells from six organs (brain heart, lung, liver, spleen and Bidney), and 

histopathological observations were recorded up to 28 days after inoculation. 

Four isolates of b/*@+66%'+0+ were obtained from 2 hot springs located at lanto area 

(middle part of (apan). Colonies of the O isolates were floccose, and darB olive green 

on the surface and darB brown in the reverse. The isolates uniformly showed an 

excellent growth at O2 o C and could grow up to O8 o C. All the isolates abundantly 

produced two-celled clavate conidia attached to denticles on conidiophores. .ome 

mice showed rotating movement and sedation after O days inoculation irrespective of 

the isolates. The mortalities in mice were 20 to 100_ unrelated to the treatment of 

corticosteroid. The target organs were the brain, Bidney and liver. 

This study demonstrated the first isolations of b/*@+66%'+0+ from the environments in 

(apan. (apanese people liBe hot springs for treatments of chronic diseases, however, 

not only such patients but also healthy subjects should be aware of the fungus 

because it caused fatal infections in non-corticosteroid treated mice. The species may 

cause infections in poultry and wild birds similar to highly pathogenic avian flu and 

.AR. in (apan.


P-0037. Susceptibility of Microsporum canis to azole antifungals and 

griseofulvin 

Santurio J, Hartz Alves ., Azevedo M, Cavalheiro A, EcBhardt (, .chwendler . 

6niversidade Fed de .anta Maria, /apemi, 2razil 

!"#$%&'%$()*#+,"& is a Beratinophilic fungus considered to be an important agent of 

human and animal dermatophytoses. TaBing into account that the techniques for 

detection of fungal resistance were recently standardized (MJ8 | A, NCC/., 2002), 

the objective of this study was to evaluate the ",*0"-$% susceptibility of twelve !/*#+,"& 

cultures obtained from lesions in dogs and cats. The antifungal drugs included in the 

study were miconazol, Betoconazol, itraconazol and griseofulvin. The minimal 

inhibitory concentrations (MICs) ranged from 0.06 to J2$g/ml for miconazol, 0.06 to 16 

$g/ml for Betoconazol, 0.06 to J2 $g/ml for itraconazol and 0.06 to J2 $g/ml for 

griseofulvin. The average MICs were [.0O $g/ml for miconazol, 12.0O $g/ml for 

Betoconazol, [.OO $g/ml for itraconazol, and 1J.J $g/ml for griseofulvin. The MIC K0 

(lowest concentration that inhibited K0_ of the samples) indicated that the order of 

decreasing activity was itraconazol (MIC K0}0.12[ $g/ml), miconazol and griseofulvin 

(MIC K0 }0.2[ $g/ml) and Betoconazol (MIC K0 }0.[$g/ml). In general, the !/*#+,"& 

isolates remained sensitive to the treatments that were employed, although isolates 

resistant to all antifungals have been detected. fne of the isolates showed crossresistance 

to all the azoles and also to griseofulvin. These results suggest that 

susceptibility tests should be performed when there is no clinical response to the 

prescribed treatments. 

P-0038. Outbreak of Trichophyton equinum var. equinum in horses in the south 

of Brazil 

Santurio J, EcBhardt (, Pereira D, Azevedo M, .chwendler ., Cavelheiro A, Hartz 

Alves . 

6niversidade Fed de .anta Maria - /apemi - 2razil 

Dermatophytoses are the most frequent sBin mycoses of domestic animals and 

humans. They are caused by a group of filamentous fungi that generally do not invade 

the subcutaneous tissues, infections being restricted to the Beratinized layer of sBin 

and adnexa. The most frequent dermatophytes in horses are Trichophyton equinum 

var. equinum and Trichophyton equinum var. autotrophicum. The disease has a 

worldwide distribution in this animal species but is particularly common in Australia, 

Europe, 6nited .tates, .outh America and Africa. The disease was first reported in 

2razil in 1K6J in the state of Rio Grande do .ul. The objective of the present study is 

to describe an outbreaB of equine dermatophytosis in the south of 2razil. The 

outbreaB occurred in a stud farm, where [8 Criollo horses ([[.2_) had alopecia 

lesions, that were circumscribed, desquamative and with intense formation of grayish 

crusts, with an evolution period of approximately 7 days. The lesions were located 

mainly in the head and upper third of the necB and appeared in (une, the period when 

these infections occur more often (fall and winter). 2ased on the clinical 

characteristics, an infection by dermatophytes was suspected, and crusts and hairs 

from the borders of the lesions were collected for mycological diagnosis. Diagnosis 

was based on direct examination with 1[_ potassium hydroxide and on culture and 

isolation of the agent in Mycobiotic Agar. After inoculation, the tubes were incubated 

at J0ëaC during at least 7 days. The direct examination of crusts and hairs showed the 

presence of large conidia arranged in chains, forming a sheath of arthroconidia 

around the hairs. The culture showed fast growing mycelia that were flat, filamentous 

and cotton-liBe, white to yellowish. The micromorphology of the isolates using cotton 

blue revealed a large number of pear-shaped microconidia, sessile or budding along 

the hyphae. The urease test, positive within [ days, the hair perforation, negative after 

21 days, and nicotinic acid required for growth in vitamin-free media plus the macro 

and micromorphology of the isolated colonies characterize the dermatophyte as 

Trichophyton equinum var. equinum. The recommended treatment was based on the 

use of diluted iodophor, 1M100 or 1M[0, with weeBly spraying baths during four weeBs. 


P-0039. Fungi isolated in bovine vaginal mucus 

Schettino A, Catena M, Echevarria H, Monteavaro C, .oto P 

.AMP Dep. - Faculty of. Veterinary .ciencies - 6NCP2A, Tandil, Argentina 

Purpose of the study: The aim of the study was to establish the causes of 

reproductive faults in cows of 2 nd and J rd service from breeding establishments of the 

Cuenca Mar y .ierras (Province of 2uenos Aires, Argentina), with spring (fctober to 

December) breeding service, fed with natural pasture. .amples of vaginal mucus and 

serum from the cows (which were empty to the tact at 6-7 months after breeding 

service) were taBen in order to find reproduction pathogens (`.%&'%$+3* 

>$"-$"#?%)%,+&3*Z$(#.66+*spp3*7+)'26%8+#-.$*U%.-(&J. 

Summarized descriptions of the project: Neosporosis diagnoses by indirect 

immunofluorescence (IFI) and brucellosis diagnoses by buffer plate antigens (2PA) 

were carried out using the serum samples. 

The vaginal mucus samples were shipped refrigerated in 2rucella 2roth with 

Campylobacter selective supplement (.R006KE, fxoid), in Amies non-nutritional 

transport medium and Diamond modified medium (1K87). 

The 2rucella 2roths were used as pre-enrichment culture and were incubated during 

O8 hrs. at J7oC in microaerophilia (8[_ N, 10_ Cf 2, [_ f 2), then they were 

inoculated to .Birrow medium or 2ovine 2lood Agar with and without antibiotics 

(.R006KE, fxoid) and were incubated at J7 oC during [ days in microaerophilia. The 

Amies non-nutritional transport medium was inoculated to 2ovine 2lood Agar and 

incubated at J7 oC during [ days in aerobiosis. The Diamond modified medium was 

incubated at J7 oC during 7 days in aerobiosis. 

The fungi colonies were inoculated in Agar .abouraud honey and incubated at 28 oC 

and at J7 oC in aerobiosis. The fungi identification was carried out by 

macromorphological and micromorphological characters, and in addition to these, 

metabolic tests were done in order to identify P?%5%-%$(6+. CzapeB Agar and Malt 

Extract Agar were used in order to identify 4&'.$@"66(& and 1.,"#"66"(). 

Results and conclusions: In an 18-month period in [ (1J_) out of the J6 vaginal 

mucus which were processed, the following fungi were isolated in pure cultureM 

48&"5"+*#%$2)8"U.$+, 4&'.$@"66(&*-.$$.(&3*!(#%$*?".)+6"&, 1.,"#"66"()*8$.0"#%)'+#-()* 

+,5*P?%5%-%$(6+*)",(-+. The immunodiagnosis serology for Neospora was (r) in J 

(three) of these animals and for 2rucella was (r) in 1 (one) animal with no other agent 

been isolated from the vaginal mucus. 

48&"5"+*#%$2)8"U.$+3 4&'.$@"66(&*-.$$.(&3*+,5*!(#%$*sp. are described as being 

responsible for sporadic abortions in bovines. The positive serology of neosporosis 

and brucellosis in some of the tested animals suggests taBing into account the 

concomitant presence of microbial agents (bacterial, parasitic, fungic) in abortion or 

infertility diagnoses, therefore it would be convenient to include the fungi agents in the 

systematic search of the etiological agents involved in reproductive pathologies. 

P-0040. Isolation and identification of yeast in milk collected from bovine 

mammary glands with different health status 

Segundo-Zaragoza C 1 , Cervantes-flivares R 1 , De la Pea-Moctezuma A 1 , Villa- 

Tanaca / 2 , Ducoing-aatty A 1 

1 Facultad de Medicina Veterinaria y Zootecnia.6NAM.Mdxico, 2 Escuela Nacional de 

Ciencias 2iolágicas. IPN. Mdxico 

Mastitis is one of the most important health problem in dairy cattle. This affection caused large 

economic losses and public health hazards. In the last decades an increase in the isolation of 

fungi from mastitis in dairy cattle has attract attention, especially for some species of the genus 

7+,5"5+. 

The purpose of this study was to determine the presence of yeast in healthy mammary glands as 

well as those which had sub clinical or clinical mastitis, by using conventional biochemical 

techniques. 

10K[ milB samples. were collected from Holstein-Friesian cows, whose clinical status were 

previously established by mean of the California Mastitis Test (CMT) and clinical examination., 

being JO2, J8J and J70 from healthy mammary glands, and those diagnosed with sub clinical 

and clinical mastitis, respectively. The samples were processed at the mycology laboratory of the 

Faculty of Veterinary Medicine and Husbandry. Yeast identification was done through colony 

morphology as well as the procedure described by lurtzman (1KKK) and 2arnett (1K8J). 

fut of the 10K[ samples, in JO2 samples of milB from healthy glands, we isolated 80 strains, the 

species more common were 7/*@6+8$+-+ (O.2K_), 7/*[$(&." (0.82_)3 7/*0"&B+,+-?"" (0.[O_)3*7/* 

d.26+,%"5.&**GQ/IDjJ3*7/*",-.$).5"+**GQ/IDjJ3*7/*,%$0.@"#+**GQ/QRjJ3*7/*[.2U"$*GQ/QRjJ3*7/* 

@("66".$)%,5""**GQ/QRjJ3*7/*-$%'"#+6"&**GQ/QRjJ3*7/*)+#.5%,".,&"&**GQ/QRjJ3*7/*&6%%U""**GQ/QRjJ3* 

P?%5%-%$(6+*&''**GQ/QRjJ*were isolated/* 0.6J_ of strains could not be identified. 

In J8J samples of milB of sub clinical mastitis, we isolated JO strains, the species found were 7/* 

[$(&."*and 7/*d.26+,%"5.& (0.27_)**7/*#6+(&&.,"" (0.18_), 7/*8$()'-"" (0.18_), P?%5%-%$(6+*&'' 

(0.18_), and 7/*+68"#+,&, 7/*,%$0.@"#+3*7/*0"&B+,+-?""3*7/*@("66".$)%,5""3*7/*-$%'"#+6"&3*7/* 

)+#.5%,".,&"&3**7/*#+,-+$.66"3*7/*",#%))(,"&*G0.0K_ for each one) were isolated, it was not 

possible to identify 1.18_ of the isolates. 

In case of J70 samples milB from of mammary glands with clinical mastitis, we isolated 171 

strains the species more frequently found were 7/*[$(&."*(6.02_) and 7/*@6+8$+-+ (J.O6_)3*7/* 

+68"#+,&*GQ/5EjJ3*7/*,%$0.@"#+*GQ/E5jJ3*7/*[.2U"$*GQ/:NjJ3*7/*@("66".$)%,5""*+,5*7/*-$%'"#+6"&* 

GQ/MCjJ3*7/*d.26+,%"5.&, 7/*",-.$).5"+*+,5*7/*6(&"-+,"+.*GQ/IDjJ3*7/*#6+(&&.,""*+,5*7/*6+)8"#+* 

(0.0K_). 2esides, 1$%-%-?.#+*d%'U""*was isolated in*0.6J_ and*2.O6_ of the strains obtained 

could not be identified. In this study, 7/*[$(&." was the species more frequently isolated in milB 

from glands with clinical mastitis. In milB from healthy glands 7/*@6+8$+-+*was the most common 

species found. 2oth species are Bnown to be present in the bovine digestive system. Despite that 

7/*+68"#+,& has been reported as the most common species of yeast pathogen found, this study 

shows that 7/*[$(&." seems to be of interest as a probable cause of mastitis problems. 

References: 

Freydere, A.M., Guinet, R. and 2oirin, P. 2001. Yeast identification in the clinical microbiology 

laboratoryM phenotyphical methods. !.5/*!2#%6. JKMK-JJ. 

lurtzman CP, Fell (a. The Yeast, a Taxonomic .tudy. OÅ ed. AmsterdamM Elsevier. 1KKK. 3. 2arnett 

A(, Payne aR, Yarrow D. YeastM Characteristics and Identification. Great 2ritainM Cambridge 

6niversity Press. 1K8J

P-0041. A study of human dermatophytosis due to zoophilic dermatophytes in 

Karaj in 2001 

Seyed Masoud H 1 , .eyed Gama H, Ali . 

1 Azad 6niversity ff Iran, Iran, 2 Tehran 6niversity, Tehran Iran 

fne of the problems of public health in Iran is mycotic inflection which it]s incidence is 

unBnown. That the most important disease of them is Dermatophytosis. In this regard, 

determination of various species of etiologic agent, infection source, the risB of contact 

to animals, and public training are necessary. In so doing, we need an epidemiologic 

study. In this research 7[0 suspected samples were studied, in which 1[7 cases 

(21_) suffering from Dermatophytosis and out of them 100 cases were positive 

culture. fut of isolated Dermatophytps 6K_ were anthropophilic J0_ zoophilic and 

1_ were geophilic. The most impairments were observerd in 0 | K of age group which 

the agent was M. canis appearing tinea capitis. ([K_ males and O1_ females) The 

most prevalent ringworm agent was E. floccosum which was seen in groin. The most 

prevalent tinea unguinm was in 0- K and 10 | 1K of age which its agent was 

T.mentagrophytes. The most prevalent tinea manuum was in 20 | 2K of age which its 

agent was T.rubrum 

P-0042. Diversity and antifungal susceptibility of yeasts isolated from raw goat 

milk and cheese in southern Brazil 

Spanamberg A 1,2 , Cavallini .anches E 2 , Hartz Alves . J , Ferreiro / 2 , Valente P 1 

1 Department of Microbiology/IC2. - 6FRG., Porto Alegre , 2razil, 2 Department of 

Veterinary Clinical Pathology/FaVet - 6FRG., Porto Alegre, R., 2razil, J /aboratory of 

Mycological Research/CC. - 6F.M, .anta Maria, R., 2razil. 

The microbial diversity of raw goat milB and cheese is an important factor for the 

evaluation of sanitary quality and processing conditions of lactic products. The 

objectives of this research were to characterize the yeast community associated with 

goat milB and cheese, as well as, evaluate their antifungal susceptibility. A total of 2[ 

samples was analyzed. Aliquots of 0,1$/ of decimal dilutions were spread on YM agar 

plates, and incubated at 22 0 C for J to [ days. Macroscopically different yeast colonies 

were counted, isolated, and identified according to the classic methodology based on 

phenotypic and physiological characters. The antifungal agents tested using the 

Microdilution broth technique (M27-A2) were fluconazole, itraconazole, amphotericin 2 

and voriconazole. A total of [6 isolates were classified into several yeast genera. The 

average counting was superior to J log CF6/m/. .usceptibility to the antifungics was 

tested in OO isolates of the genera 7+,5"5+3*>$"#?%&'%$%,3*P?%5%-%$(6+3*7$2'-%#%##(&* 

and*V.8+$2%)2#.&. Clinically relevant yeasts, such as 7+,5"5+*-$%'"#+6"&3*7/* 

'+$+'&"6%&"&*and*7/*@("66".$)%,5""*did not presented resistance to the antifungal drugs 

tested, but strains of P?%5%-%$(6+ were resistant to fluconazole and moderately 

sensitive to itraconazole and voriconazole. fne >$"#?%&'%$%, isolate was moderately 

sensitive to amphotericin 2, a fact that raises concern. aith the increase in the 

number of cases of invasive mycotic infections caused by fungi previously considered 

as non-pathogenic, it is necessary to evaluate trends in the antifungal suceptibility 

profiles of different yeast species. 


P-0043. Hematological values of broilers in mycotoxicosis induced by 

Aspergillus fumigatus and Aspergillus flavus and treatment with silymarin 

Talebi A 1 , Haghnazari A 1 , Zare P 2 , Asri rezai . 1 

1 Department of clinical sciences,College of veterinary medicine,6rmia 

university,6rmia,Iran, 2 Department of pathobiology,College of veterinary 

medicine,6rmia university,6rmia,Iran 

To evaluate and compare the levels of haematological values of broilers in natural 

mycotoxicosis and to evaluate the effect of silymarin in the treatment of mycotoxicosis, 

a study was performed on O0 healthy Cobb [00 broilers from both sexes. 

Haematological values including Hb and PCV values, R2C count and total and 

differential a2C counts were recorded in days 2, 7, 1O, 21, 28, J[, O2 and OK. From 

days J[ to OK, birds were separated into four groups (4/U6+0(&3*4/U()"@+-(&, mixed 

and control). In mixed group, birds received food infected as 10_ with*4/U6+0(&*+,5* 

4/U()"@+-(&. In each group, [ birds were treated with silymarin (10 mg/case) and [ 

remained untreated. 4/U6+0(&*+,5*4/U()"@+-(& well grown on .DA agar for sporulation, 

were harvested and suspended in sterile normal saline at the concentration with fD 

equal to No.J McFarland solution. The solutions were poured on fresh mash food and 

incubated at J[+C for 2O hours. To induce mycotoxicosis, these infected foods were 

incorporated as 10_ of total food given to birds. The analysis of results shows 

significant decrease in Hb and PCV values and R2C and a2C counts in all birds 

receiving Aspergillus-infected foods untreated with silymarin with the most significant 

effects observed in 4/U()"@+-(& group and the least in 4/U6+0(&*group. In silymarintreated 

groups, the drug could significantly hold the values and counts in normal range 

although all birds received similarly infected foods. These findings show that silymarin 

can be administered effectively to reduce the suppressive effects of mycotoxicosis on 

haematopoesis. 

ANTIFUNGALS* 

P-0044. Evolutionary fitness in itraconazole susceptible and resistant 

Aspergillus fumigatus clinical isolates 

Albarrag A, Anderson M, Denning D 

The 6niversity of Manchester, Manchester, 6l 

Aspergillus fumigatus is the most common etiological agent of aspergillosis infection. 

Invasive aspergillosis is a major cause of death in leuBemia and organ transplant 

patients. The increasing rates of recovery of antimicrobial resistant microorganisms in 

hospital and community settings are of growing concern. The evolutionary changes of 

drug resistance and its fitness penalty have been extensively studied in bacteria, 

viruses and yeasts. /oss of fitness causes resistant organisms to become 

disadvantaged relative to susceptible strains in the absence of drug pressure. In many 

organisms, mutations that confer resistance to drugs may confer a biological fitness 

cost that is expressed as decreased growth rate, survival or virulence. 

.elected itraconazole susceptible and resistant clinical isolates of A. fumigatus were 

studied. Three pairs of susceptible and resistant isogenic strains, from cases where 

azole resistance was acquired during treatment, were studied in addition to eight 

isolates obtained serially from a patient with an aspergilloma (chronic cavitary 

pulmonary aspergillosis)(patient D). MICs of ITC, voriconazole (VRC), ravuconazole 

(RVC), and posaconazole (Pf.) were determined. 

Various measures of growth rate (colony radial growth rate and specific growth rate, 

germination time and conidia yields) have been determined on various media. 

Competition assay, in vitro and in vivo, between selected resistant and susceptible 

isogonic strains will be carried out. .equencing of cyp[1A gene was carried out. 

Resistance mutations in 1O!-sterol demethylase gene, cyp[1A gene, were 

determined. The pathogenicty of selected isolates will also be determined. 

Three resistance profiles were observed in these clinical isolates. Resistance to ITC 

only was observed in the resistant isolate of two of the pairs and cross resistance to 

ITC and Pf. was observed in the third resistant paired isolate. A third resistance 

profile characterized by very high MICs of ITC (t 8.0 mg/l), VRC (8.0 mg/l), RVC (8.0 

mg/l) and Pf. (O.0 mg/l) was observed in the isolates from patient D. Isolates O and 8 

were more susceptible to Pf. (MIC of 1-2.0 mg/l). 

2y sequencing the cyp[1A gene of these isolates, three new different amino acid 

substitutions in the Cyp[1A protein were identified in three isolates. These isolates 

exhibited a reduced fitness, which is expressed as reduced growth rate in one isolate 

and less conidiation in another isolate. 

These new point mutations in cyp[1A gene could be associated with the cross 

resistance to ITC, VRC, RVC, and Pf.. 2iological fitness cost in the resistant isolates 

could be due to these resistance mutations.

P-0045. Occurrence of fungal bloodstream infections and their antifungal 

sensitivity patterns within Scotland during 2000 to 2005. 

Alexander C, Dunsmuir R, .hanBland G 

Clinical Mycology, Glasgow, 6l 

The incidence of fungal bloodstream infections was examined over a 6 year period 

(2000 | 200[) at the CPA-accredited Clinical Mycology /aboratory located at the 

aestern Infirmary, Glasgow. [[1 isolates were sent from 2J laboratories throughout 

.cotland and were fully identified and examined for their sensitivities to a range of 

antifungals which included amphotericin 2, itraconazole, fluconazole, flucytosine, and 

voriconazole. The majority of the isolates were 7+,5"5+*&'.#".& (K[_) whilst the 

remaining [_ included ^(&+$"()*&'.#".&3*P?%5%-%$(6+*&'.#".&3*7$2'-%#%##(&* 

,.%U%$)+,&3*=+#+##$%)2#.&*#.$.0"&"+.*and =#.5%&'%$"()*'$%6"U"#+,&. ff the 

7+,5"5+ bloodstream isolates, the most frequently occurring species isolated was 

7+,5"5+*+68"#+,& (O[_) of which the vast majority were sensitive to the range of 

antifungals tested using a microbroth dilution assay based on the NCC/. method. 

fut of the 2OJ 7+,5"5+*+68"#+,&, only one isolate showed resistance to flucytosine 

(MIC tJ2kg/ml) whilst one other displayed azole resistance. The second most 

commonly isolated 7+,5"5+*&'.#".& was found to be 7+,5"5+*@6+8$+-+ (2O_) with 

60_ of these being susceptible dose-dependent and 1[_ being resistant to both 

itraconazole and fluconazole. The third most encountered isolate was 7+,5"5+* 

'+$+'&"6%&"& (16_). KJ_ of these were fully sensitive to the range of antifungals 

tested and only 7_ were susceptible dose-dependent to fluconazole and itraconazole. 

In 200[, caspofungin sensitivity testing was introduced. ff the 7[ blood isolates 

examined, which included O0 7+,5"5+*+68"#+,&, 18 7+,5"5+*@6+8$+-+, 8 7+,5"5+* 

'+$+'&"6%&"&, J 7+,5"5+*6(&"-+,"+., 2 7+,5"5+*-$%'"#+6"&, 1 7+,5"5+*@("66".$)%,5"", 1 

7+,5"5+*",#%,&'"#(+ and 2 P?%5%-%$(6+*&'.#".&, all had caspofungin sensitivities in 

the range of 0.008 | 1kg/ml with the exception of the 2 P?%5%-%$(6+*&'.#".& with MIC 

values of 16kg/ml or greater. 

P-0046. Minimal inhibitory concentrations of 6 antifungal drugs to Sporothrix 

schenckii evaluated by three methods 

Alvarado Ramírez E, Torres-RodrÜguez (, (imdnez T, Neyra E 

Institut Municipal De Investigacií Medica. 2arcelona .pain. 

Nineteen peruvian clinical isolates of ='%$%-?$"_*&#?.,#["" were tested 

againts amphotericine 2 (A2), [ fluorocitosine (FC), fluconazole (FZ), 

itraconazole (IZ), voriconazole (VZ) and Betoconazole (lZ). 

NCC/. !:DW4 method and two commercial tests, =.,&"-"-$.*k.+&-b,.l 

(=kb) and 4>Zl*^(,@(&M*G4>Z^MJ were used for determinate the Minimun 

Inhibitory Concentration (MIC), to these antifungals. 7+,5"5+ parapsilosis 

ATCC2201K and Candida Brusei ATCC62[8, were used as a quality control. 

The inoculums of conidia were prepared with the mycelial form growing for [ 

days on agar .abouraud at J0oC. A2, FC, FZ and IZ were determinated with 

the three methods, VZ with !:DW4 and =kb, lZ was studied only with =kb. 

The three methods showed high MIC]s values with FZ and FC, being more 

homogeneous with IZ and lZ, MIC K0_ was 0.[kg/m/. MIC range of VZ 

with the reference method were variable (O.0-16 kg/m/), the Geometric 

mean (GM) was K.J kg/m/. MIC]s range of A2 was very wide (0.06-16 

kg/m/), instead of this variabilityn the GM was 1.2kg/m/, suggesting that 

MIC]s are strain dependent. 

Agreement, considering two log 2 dilution, between the comercial techniques and the 

reference method were very higher with FZ, IZ and FC, in this case, specially with 

AT2F2. In A2 the agreement was lower, and also in VZ between MJ8-A and .Yf, 

these results are related to the different antifungal concentrations included in each 

method. This values confirm the highest in vitro activity of IZ and lZ on ..schencBii 

and the lacB of activity of FZ, VZ and FC. If A2 should be administered the 

susceptibility of the strain to this antifungal must be analyzed. Commercial quantitative 

antifungal methods have a limited usefulness in =/*&#?.,#["". 


P-0047. In vitro activity of fenticonazole against Candida spp. in patients with 

vulvovaginitis 

Antonopoulou S 1 , Fragouli E 1 , .trompoulis P 2 , Papalexis P 2 , Harvalou l 2 , Fanariotis 

D 2 , latsampas A 1 

1 A .yggros, Athens, Greecen 2 Technological Educational Institute of Athens, Athens, 

Greece 

Purpose: Determination of in vitro susceptibility of Candida species to Fenticonasole 

using the microdilution method AF.T&E6CA.T for yeasts. 

Material & Methods: 

" 200 Candida species isolated from vaginacervival speciments 

" AF.T&E6CA.T 

(subcommitee on Antifungal .usceptibility Testing European Commitee for 

Antimicrobial .usceptibily Testing) 

Results: In women suffering from valvovaginitis (reccurent or not) the Fenticonazole 

MIC was } 

" 1) 0.0J 0.12[ mg// in [[_ (high sensitivity) 

" 2) 0.2[ 0.[ mg// in O2.[_ (moderate sensitivity) 

" J) %1mg// in 2.[_ (resistance) 

Conclusion: Fenticonazole seems to be effective in the K7,[ _ of the species 

examined which was defined as the lowest drug concertation that succesfully inhibited 

[0_ of the fungi development 

P-0048. Patterns of resistance of Candida spp. to fluconazole and voriconazole 

as estimated by the artemis disk antifungal surveillance study in Greece. 

Anyfantis I 1 , Skiada A 1 , PetriBBou E 1 , Newell V 2 , Madopoulou F 1 , /azari P 1 , 

GrammatiBou M 1 , Mitroussia-Ziouva A 1 , DaiBos G 1 , PetriBBos G 1 

1 Research /aboratory for Infectious Diseases and Antimicrobial Chemotherapy 

hG.l.DaiBosi, 6niversity of Athens, Greece, 2 Giles .cientific 

Aim: To investigate the epidemiology of clinical yeast isolates and their susceptibility 

profiles to voriconazole and fluconazole over a three year period. 

Material and Methods: All yeast isolated from (anuary 2002 to December 200O, 

considered to be pathogens, were tested via C/.I (former NCC/.) MOO disB method. 

Inoculum was adjusted to match a 0.[ McFarland density standard. Plates were 

incubated for 20-2O hours at J[ 0 -J7 0 C. .low growing isolates, were read after O8 

hours incubation. Interpretive breaBpoints were those established by C/.I. Zone 

diameters were read by electronic image-analysis and interpreted and recorded with a 

2IfMIC Plate Reader .ystem. The results were compared to those from a previous 

study, from 1/1/1KK8 to J0/12/2001. 

Results: From 1KK8 to 2000, [1[ isolates of 7+,5"5+ spp. were collected, J1O(60_) 

were 7/+68"#+,& and 1J.K_ 7/@6+8$+-+. During the period 2001-200O, O7K isolates 

were tested of which J07(6O_) were 7/+68"#+,& and 1[.O_ 7/@6+8$+-+. 

.even Year comparison of fluconazole resistance by species 

1KK8 1KKK 2000 2001 2002 200J 200O 

Organism _R(N) _R(N) _R(N) _R(N) _R(N) _R(N) _R(N) 

7/+68"#+,&* K(67) 2.8(10K) J.6(1J8) 2.J(88) 1.O(7O) 6.6(61) 2.O(8O) 

7/@6+8$+-+* 70.6(17) 88.2(17) JO.2(J8) [.J(J8) 6.7(1[) 0.0(1J) 2[.0(8) 

7/-$%'"#+6"&* [0.0(O) 0.0(J) 1J.0(2J) 1O.J(1O) 0.0(J) 0.0(2) K.1(11) 

7/'+$+'&"6%&"&* 0.0(J) 0.0(6) 0.0(11) 8.J(12) 0.0(8) 11(K) 0.0(1) 

7/[$(&."* 87.[(8) 100(2) 100(1) 100(1) 6J.6(11) 

Four year comparison of voriconazole resistance by 

species 

2001 2002 200J 200O 

Organism _R(N) _R(N) _R(N) _R(N) 

7/+68"#+,&* 2.J(88) 0.0(7O) O.K(61) J.6(8O) 

7/@6+8$+-+* 2.6(J8) 6.7(1[) 0.0(1J) [0(8) 

7/-$%'"#+6"&* 7.1(1O) 0.0(J) 0.0(2) K.1(11) 

7/'+$+'&"6%&"&* 0.0(12) 0.0(8) 11.1(K) 0.0(1) 

7/[$(&."* 0.0(2) 0.0(1) 0.0(1) 27.J(11) 

ff the 7/+68"#+,& isolates resistant to fluconazole, 28.6_ were susceptible to 

voriconazole, with fluconazole resistant 7/'+$+'&"6%&"& and 7/-$%'"#+6"&3 [0_ of the 

isolates were susceptible to voriconazole and with 7/[$(&.", all isolates were 

susceptible to voriconazole. 

Conclusions: In Greece, 7/+68"#+,& (6O_) remains the most frequent species 

encountered followed by 7/@6+8$+-+ (1[.O_). ff the 7/+68"#+,& isolates, 2.K_ were 

resistant to both fluconazole and voriconazole.

P-0049. Modified RPMI medium for Microsporum canis susceptibility testing 

against posaconazole and licensed antifungal agents reduces incubation and 

reading times 

Arabatzis M 1 , MaBri A 2 , Alexopoulos E 1 , Arsenis G 1 , Milioni A 1 , Voyatzi A 2 , VelegraBi 

A 1 

1 Mycology /aboratory, Microbiology Department, Medical .chool, 6niversity of Athens, 

Greece, 2 Microbiology /aboratory, Penteli]s Pediatric Hospital, Penteli, Greece 

The NCC/. MJ8-A standard microdilution (2MD) method has recently been validated 

for measuring antifungal susceptibility of dermatophytes. ae compared the effect of 

standard RPMI medium versus iron (Fe) supplemented RPMI media (both broth and 

agar) on !"#$%&'%$()*#+,"& growth rates and on the MIC 80 (80_ inhibition endpoints) 

values. The objective of the study was to determine if Fe supplemented RPMI would 

accelerate !/*#+,"& growth, allowing earlier MIC readings, whilst at the same time 

giving comparable endpoints to those obtained in standard RPMI media after four days 

incubation. 

MIC 80 of the new broad spectrum triazole posaconazole (Pf.n .chering-Plough 

Research Institute, New (ersey, 6.A), were compared with those of itraconazole (ITZn 

(anssen, 2eerse, 2elgium), fluconazole (FCZn Pfizer, .andwich, lent, 6.l), terbinafine 

(TERn Novartis, 2asel, .witzerland) and griseofulvin (GRIn .igma, .t /ouis, Mo., 6.A). 

Thirty !/*#+,"& isolates, comprising 2 reference strains (C2. OK[.86 and C2. 282.6J) 

with reproducible endpoints, 16 tinea capitis and 12 tinea corporis isolates were studied. 

2MD and Etest (A2 2iodisB, .olna, .weden) MICs were recorded in the two media for 

Pf., ITZ and FCZ. Pf. was also tested by disB diffusion (DD) using commercially 

prepared disBs at final concentration [ micrograms/disB (fjfID, 2asingstoBe, 6l) in 

Miller Hinton (MH) and MH/Fe K0 mm plates. TER and GRI were tested only by 2MD. 

Plates were incubated at J[ 0 C and read at J and O days. The following ~C isolates 

were runM 4&'.$@"66(&*U6+0(& ATCC 20O.J0O, 4/*U()"@+-(&*ATCC 20O.J0[ and 7+,5"5+* 

+68"#+,&*ATCC K00.28n readings were performed at the NCC/. recommended 

4&'.$@"66(& and 7+,5"5+ times. 

P-0050. Trichophyton rubrum: Cell wall biosynthesis as a target for antifungals 

Ball L, Robinson Z, Gurr . 

6niversity of fxford 

Dermatophytes are cosmopolitan superficial pathogens of animals and humans, found 

throughout the world. They invade Beratinous tissues such as sBin, hair and nails 

causing the conditions commonly Bnown as athlete]s foot and ringworm. 

Dermatophytosis caused by >$"#?%'?2-%,*$(8$() is the most prevalent fungal 

infection worldwide, and this pathogen has been shortlisted for sequencing by the 

Fungal Genome Initiative (FGI white paper, 200O). 

Despite the ubiquity of dermatophytes, there is scant Bnowledge of their basic biology 

or molecular biology. This project aims to elucidate aspects of the lifecycle of >/* 

$(8$() that may be important in the initiation of infection. The fungal cell wall is 

critical in this process, as integrity of the fungal cell must be maintained throughout. 

The wall, a complex mesh of glucans, chitin and glycoproteins, is at the interface 

between the fungus and its host, mediating host perception, adhesion and subsequent 

invasion of the host. 

ae are gathering together techniques for studying >/*$(8$()M a genome walBing 

library has been constructed, an E.T library has been made, enriched for secreted 

genes expressed in response to Beratin, and a variety of monoclonal antibodies have 

been screened, allowing detection of fungal adhesion to surfaces. An adhesion assay 

has been developed that allows assessment of adherence of >/*$(8$()*germlings to a 

K6-well plate in response to various treatments. 

A biochemical approach using current chemistries and novel molecules targeting 

specific enzymes in the chitin and glucan biosynthetic pathways is also being used to 

evaluate cell wall synthesis as a drug target in >/*$(8$()/ In summary, the project 

centres around a study of factors involved in cell wall biosynthesis and initial infection 

events, and their role in pathogenicity. 

MIC range (micrograms/ml) in RPMI and RPMI/Fe for ITZ was 0.016-0.[, F/Z 0.[-6O, 

Pf. 0.01-0.2[, TER 0.0J-O and GRI 0,008-16. Pf. zone diameters ranged from 10- 

J0 mm. Interclass correlation coefficients (ICCs) and K[_ confidence intervals for 

comparing methods were calculated using log2 transformed data. ICC for 2MD versus 

Etest in RPMI and RPMI/Fe was 0.KO (Pv10 -O ) and 0.K8 (Pv10 -O ) for ITZ and Pf. 

respectively. Pearson]s correlation coefficient for Pf. 2MD and DD was 0.KJ (Pv0.01) 

and 0.KO (Pv0.01) for MH and MH/Fe respectively. 2MD and Etest RPMI and RPMI/Fe 

generated ICCs for FCZ and GRI were 0.86 (Pv10 -O ) and 0.K1 (Pv10 -O ) respectively 

and 0.86 (Pv10 -O ) for TER. 

The Fe supplemented RPMI medium reduces incubation times for !/*#+,"& for both 

2MD and Etest by 1 day and provides reproducible 80_ inhibition endpoints. Pf. DD 

readings were also attainable after a J-day incubation period in Fe supplemented 

medium without significant changes in inhibition zone diameters. Further evaluation 

using >$"#?%'?2-%, and a'"5.$)%'?2-%, species and inclusion of more topical and 

systemic antifungals in the test panel are required to validate the applicability of the 

proposed modification in broth and solid media. 


P-0051. Essential oils as antifungal agents 

Banfi E 1 , .cialino G 1 , Voinovich D 2 

1 Dept 2iomedical .ciences 6niversity of Trieste, 2 Dept Pharmaceutical .ciences 

6niversity of Trieste 

Objectives and Methods: Antimicrobial properties of herbs and spices have been 

used since ancient time in medicine and food treatment and essential oils have also 

been demonstrated to possess antifungal activity. Given that in many regions of the 

world indigenous therapeutic antimicrobial agents may be crucial to improving overall 

public health, we must maBe every effort to develop antifungal agents which are 

based on renewable, relatively inexpensive materials, to get novel therapy starting 

from natural products. Essential oils of 4,"8+*$%&+.5%$+3*Z%&B.66"+*#+$-.$"3* 

72)8%'%@%,*#"-$+-(&3*X+0+,5(6+*+,@(&-"U%6"+3*X.'-%&'.$)()*&#%'+$"() (ManuBa)3* 

1.6+$@%,"()*@$+0.%6.,& were purchased from manufacturers. The fungicydal activity 

of essential oils was evaluated ",*0"-$% by microdilution reference method (NCC/.- 

M27), alone and in combination, against a number of 7+,5"5+*&''/ clinical isolatesn 

azole-resistant strains were included in the panel. 

Results and Conclusions: Essential oils had minimal inhibiting concentrations in a 

range of 0.062[-0.[ _ (v/v), 4,"8+3*72)8%'%@%, and ManuBa being the most active. 

Different combinations of two or more essential oils had interesting antifungal activity. 

Essential oils had no toxic effect against human cells ",*0"-$%. Different suppository 

topical formulations containing mixtures of essential oils can be developed as possible 

therapy for mucosal candidiasis. Novel therapy can start from natural products, 

developing antifungal agents based on renewable, relatively inexpensive materials liBe 

essential oils. 

P-0052. Malassezia species: In-vitro investigations on generation of reactive 

oxygen species 

Beck S, Hausstein 6, Hipler C, Nenoff P 

!+6+&&.d"+ species can cause several common diseases, such as pityriasis versicolor,* 

!+6+&&.d"+ folliculitis and even fungaemia. 

Ten different !+6+&&.d"+ species are Bnown so far, which are associated with different 

sBin infections. In this investigation six species of the genus !+6+&&.d"+ s!/*U($U($, !/* 

&6%%UU"+.3 !/*@6%8%&+3 !/*&2)'%5"+6"&3 !/*%8-(&+3 !/*'+#?25.$)+-"&u were analysed 

and the generation of reactive oxygen species was measured. 

All !+6+&&.d"+ species investigated in this study are able to produce reactive oxygen 

species. 

This could be shown for the first time ever. 

A linear correlation was found between the yeast cell count and the generated 

reactive oxygen species (!/*U($U($ correlation coefficient r }13*!/*&6%%UU"+.*r }1, !/* 

%8-(&+ r }1, !/*'+#?25.$)+-"& r }1, !/*&2)'%5"+6"& r }0.K, and !/*@6%8%&+*r }0.7). 

* 

In addition, the effect of different terbinafine concentrations on the production of 

reactive oxygen species by !/*U($U($ was assessed ",*0"-$%. A significant reduction of 

reactive oxygen species could be achieved under the influence of terbinafine 

(incubation with 1kg terbinafine ml -1 M reduction -O7_n terbinafine 10 kg ml -1 M -28_n 100 

kg terbinafine ml -1 M - 2J_).

P-0053. Voriconazole inhibits biofilm-associated growth of Candida albicans in 

long-term continuous flow culture 

Bernhardt H 1 , lnoBe M 2 , .chwesinger G J , 2ufler ( O , /udwig l [ , 2ernhardt ( 6 

1 6niversity of Greifswald, Greifswald, Germany, 2 6niversity of Greifswald, Greifswald, 

Germany, J 6niversity of Greifswald, Greifswald, Germany, O Pfizer Pharma GmbH, 

larlsruhe, Germany, [ Clinical Centre .uedstadt, RostocB, Germany, 6 Clinical Centre 

.uedstadt, RostocB, Germany 

Purpose: Continuous flow cultures of yeast more closely mimic the ",*0"0% situation 

than batch cultures. ae investigated the antifungal effect of voriconazole (VfRI) on 7/* 

+68"#+,& in a miniature flow cell system developed for biofilm research. 

Methods: 7/*+68"#+,& isolated from blood cultures and the type strain .C[J1O were 

grown in flow cells with 160 kl chambers (.tovall /ife .cience Inc., Greensboro, NC, 

6.A) using peptone yeast extract plus [0 mM glucose at various flow rates for [|8 

days at J7 ÅC. Formation of biofilm, morphological features of adherent 7/*+68"#+,&* 

and planBtonic cell counts were investigated in the presence (8 replicate experiments) 

or absence (6 replicates) of VfRI (16 mg//). Morphology and viability of adherent 

fungal cells was assessed by F6N w 1 fluorescent vital staining. 

Results: In absence of VfRI, 7+,5"5+ established a biofilm within 2O h. Adding VfRI 

to the continuous-flow medium inhibited the growth of 7+,5"5+. Patches of detached 

biofilm (partly daughter cells) observed in the overnight outflow were very small under 

VfRI compared to simultaneously grown controls. The strongest inhibition by VfRI 

was observed at very low flow rates (1.J m//h), where the cumulative dry weight of 

biofilm was O|8_ of the control samples, compared to 1[|20_ in experiments with 

higher flow rates (,2.7 m//h). Visual appearance of the biofilm was similar to 

degrading material in aging cultures. VfRI rendered biofilm-associated 7+,5"5+ 

metabolically inactive as indicated by lacB of glucose consumption. VfRI reduced the 

recovery of live planBtonic 7+,5"5+ in the outflow by a factor of 1000 (-10 J cfu/m/, 

VfRI vs. 10 6 cfu/m/, controls). 2lood culture isolates and the type strain .C[J1O 

yielded similar results. Fluorescent vital staining of biofilm in control cultures revealed 

live yeast cells and mycelia containing vacuoles as well as an amorphous extracellular 

matrix. In the presence of VfRI, however, most cells were enlarged and vacuole-free, 

showing a strong yellow fluorescence indicative of cell death. 

Conclusions: 6nder continuous flow conditions at very low flow rates, VfRI strongly 

reduced the biofilm-associated cell mass production of 7/*+68"#+,& blood-culture 

isolates. 

P-0054. Comparison of the ATB fungus 2 method with the CLSI broth dilution 

method (m27-a2) for determining susceptibility of Candida spp. and 

Cryptococcus neoformans clinical isolates to amphotericin b, 5-fluorocytosin, 

fluconazole and itraconazole 

Bertout S, Hocquette A, Castel D, Mallie M 

/aboratoire de Parasitologie et Mycologie medicale 

Purpose: Antifungal susceptibility testing seems to be useful for adapting the treatment of 

fungal infections. The Clinical and /aboratory .tandards Institute (C/.I) M27-A2 protocol 

(ex-NCC/.) remains the reference method. However, antifungal susceptibility testing Bits 

as AT2 Fungus 2 are used in routine because they are less-time consuming. The aim of 

this study was to compare AT2 Fungus 2 performance to C/.I M27-A2 protocol results. 

Materials and methods: Antifungal susceptibility of 12K yeast isolates (7+,5"5+*+68"#+,&* 

n}KO, 7/*-$%'"#+6"&*n}7, 7/*@6+8$+-+*n}8, 7/*[$(&."*n}7, 7/*6(&"-+,"+.*n}O and 

7$2'-%#%##(&*,.%U%$)+,&*n}K) was evaluated using both AT2 Fungus 2 and C/.I M27-A2 

methods. Antifungal molecules tested were amphotericin 2 (AM2), [-fluorocytosin ([FC), 

fluconazole (FCZ) and itraconazole (ITZ). Results were read after 2Oh and O8h of 

incubation at J7ÅC for 7+,5"5+*isolates*and after O8h and 72h of incubation at J7ÅC for 

7$2'-%#%##(&*isolates. 

Results: Percentages of agreement were determined in a range between ì 2 serial 

dilutions. 

For the 12K strains studied, percentage of agreement between the 2 methods were K8_, 

K2_, 8K_ and 87_ for AM2, [FC, ITZ and FCZ respectively. 

For 7/*+68"#+,& (n}KO), percentages of agreement between the 2 methods were K8_, KJ_, 

K0,O_ and 88,J_ for AM2, [FC, ITZ and FCZ respectively. 

For 7/*@6+8$+-+ (n}8), percentages of agreement between the 2 methods were 100_, 

100_, 76_ and 6O_ for AM2, [FC, ITZ and FCZ respectively 

For 7/*[$(&." (n}7), percentage of agreement between the 2 methods were 100_ for AM2 

and 86_ for [FC, ITZ and FCZ respectively. 

For 7/*6(&"-+,"+. (n}O), percentages of agreement between the 2 methods were 100_ for 

AM2, [FC, ITZ and FCZ respectively. 

For 7/*-$%'"#+6"& (n}7), percentages of agreement between the 2 methods were 100_ for 

AM2 and [FC and 86_ for ITZ and FCZ respectively. 

For 7$2'-%#%##(&*,.%U%$)+,& (n}K), percentagse of agreement between the 2 methods 

were 100_, [[_, 8K_ and 8K_ for AM2, [FC, ITZ and FCZ respectively. 

Conclusion: This study demonstrated a good correlation between the 2 methods on the 

whole sample. The lowest percentage of agreement was observed for the azoles 

concerning 7/*@6+8$+-+ and for [FC concerning 7$2'-%#%##(&. Nevertheless, excellent 

results were observed for 7/*+68"#+,& and 7/*6(&"-+,"+.. Thus, we can conclude that AT2 

Fungus 2 is a reliable method for determining antifungal susceptibility for common yeasts in 

routine. 


P-0055. Serotypes and antifungal susceptibility of Cryptococcus neoformans 

from clinical sources in Nairobi, Kenya 

Bii C 1 , MaBimura l 2 , Abe . 2 , Taguchi H J , Mugasia f 1 , Revathi . 1 , aamae N 1 , 

lamiya . J 

1 Mycology laboratory, lenya Medical Research Institute, .zczecin, lenya, 2 Institute 

of Medical Mycology and Genome Research center, TeiByo 6niversity, J Department of 

Infectious Diseases, lyorin 6niversity .chool of Medicine 

Background: 7$2'-%#%##+6*meningitis is a major course of mortality in human 

immunodeficiency virus / acquired immunodeficiency syndrome (HIV/AID.) and 

understanding the serotypes and drug susceptibility is essential for management of 

meningoencephalitis in clinical practice. 

Objectives/methods: .erotype and mating types of eighty 7$2'-%#%##(&*,.%U%$)+,& 

from cerebral spinal fluid (C.F) specimens from patients in Nairobi, lenya were 

studied. Confirmation of 7/*,.%U%$)+,& was done by ViteB Yeast 2iochemical Cards 

(bioMerieux-ViteB, Hazelwood, Mo., 6...A). .erotypes were determined by slide 

agglutination test using Crypto ChecB agglutination Bit (Iatron /abs Inc.,ToByo, (apan). 

The isolates were subjected to broth microdilution susceptibility testing to 

amphotericin 2, flucytosin, fluconazole, itraconazole and miconazole. 

P-0056. In vitro activities of voriconazole, posaconazole, fluconazole and 

amphotericin B against bloodstream clinical isolates of Candida spp. 

Birinci A 1 , ABtas E 2 , Hepsert . 1 , 2ilgin l 1 , ABBurt / J , Erturan Z O , Toraman Z [ 

1 fndoBuz Mayis 6niversity, Dept. of Microbiology, .amsun, TurBey, 2 AtaturB 

6niversity, Dept. of Microbiology, TurBey, J 2afra .tate Hospital, TurBey, O Istanbul 

6niversity, Dept. of Microbiology, Istanbul, TurBey, [ Firat 6niversity, Dept. of 

Microbiology, TurBey 

ae investigated the in vitro activities of voriconazole, posaconazole, fluconazole and 

amphotericin 2 against 17O bloodstream clinical isolates of Candida spp. obtained 

from [ different medical centers. The MICs of all antifungal drugs were determined by 

the method recommended by the National Committee for Clinical /aboratory 

.tandards using RPMI 16O0 as the test medium. fverall, voriconazole and 

posaconazole were very active against all the Candida isolates (K8,8-K8,J_ 

susceptible at MICs v or }1 microg/ml). Candida albicans (MICK0, 0.0J-0.0J 

microg/ml) was the most susceptible species to both agents. 2oth voriconazole and 

posaconazole were more active than fluconazole and amphotericin 2 against all 

Candida spp. These results provide further evidence for the increased spectrum and 

potency of the new triazoles against a large and geographically diverse collection of 

opportunistic fungal pathogens. 

Results: 7/*,.%U%$)+,&*variety*@$(8"" (serotypes A) predominated 7/*,.%U%$)+,& 

variety ,.%U%$)+,& s7[/80 (KJ.7_) versus J/80 (J.8_)u. Two of eighty isolates (2.[_) 

were identified as 7$2'-%#%##(&*,.%U%$)+,& variety @+--""*(serotype 2). Mating 

experiment confirmed all the isolates to be ! mating type. The 28. rDNA sequence 

showed that majority of the isolates were homologous with ^/*,.%U%$)+,& var. @$(8"" 

(serotype A) accession no AFJJ[K8O. .eventy eight (K7.[_) of the isolates were 

susceptible sminimum inhibitory concentration (MIC) of ' 0.[ kg/mlu to amphotericin 2. 

The MIC in which K0_ of the isolates were inhibited (MIC K0 ) by flucytosin and 

fluconazole was 6Okg/ml each while for itraconazole and miconazole were 0.[ and 2 

kg/ml respectively. Fluconazole resistance (MIC % 6O kg/ml) was detected in K/80 

(11.2_) of the isolates. 

Conclusion: The study highlights the serotypes, mating types, and existence of 

azoles resistance in a significant proportion in 7/*,.%U%$)+,& from clinical sources in 

Nairobi, lenya and the need for antifungal drug resistance surveillance as 

prophylactic use of fluconazole increases due to HIV/AID. epidemic in sub-.ahara 

Africa.

P-0057. Antifungal susceptibility to fluconazole, itraconazole and terbinafine of 

fungi causing onychomycosis 

Bueno Sánchez J 1 , .anclemente G 1 , Gallego M 2 , MartÜnez C 1 , Zapata 2 1 , Mesa 

Arango A 1 

1 6niversidad de Antioquia, Facultad de Medicina, Departamento de MicrobiologÜa y 

ParasitologÜa, Grupo Infeccián y CZncer, 2 6niversidad de Antioquia, Facultad de 

Medicina, Departamento de MicrobiologÜa y ParasitologÜa, .eccián MicologÜa 

Purpose: To evaluate ",*0"-$% antifungal susceptibility to fluconazole (F/Z), terbinafine 

(TR2) and itraconazole (ITZ) of fungi isolates obtained from patients with 

onychomycosis. 

Summarized description of the Project: fnychomycosis is a nail infection caused 

by dermatophytes and 7+,5"5+ spp., and in less degree by nondermatophytic 

filamentous fungi (NDF). More than [0_ of nail dystrophies are due to this type of 

infection, and currently it is not considered just a cosmetic problem. To date, there are 

few studies regarding ",*0"-$% susceptibility of fungi causing onychomycosis. .uch 

studies could explain, in part, the lacB of clinical response after therapy, and also 

assist clinicians in choosing an effective therapy for their patients. 

Following AF.T-E6CA.T protocol, MIC [0 (Minimum Inhibitory Concentration) of 22 

isolates of 7+,5"5+ to F/Z (Pfizer), TR2 (Galderma) and ITZ ((anssen), were 

determined. MIC [0 and MIC K0 were assesed using these drugs with seven NDF 

isolates (six species of ^(&+$"() and one of =#2-+6"5"()), according to the C/.I MJ8- 

A protocol. 7+,5"5+*'+$+'&"6%&"& ATCC 2201K and 7/*[$(&." ATCC 62[8 were used 

as quality controls for ITZ (sigma). 

Results and conclusions: Itraconazole, fluconazole and terbinafine MICs geometric 

means with 7+,5"5+ isolates were 0.07[ kg/m/, 0.7J kg/m/ and 0.1O kg/m/, 

respectively. Results obtained for NDF isolates were ,16 kg/m/ for itraconazole, , 6O 

kg/m/ for fluconazole and 0.08J kg/m/ for terbinafine. 

According to our results, 7+,5"5+ isolates were susceptible to fluconazole and 

itraconazole. Therefore, we confirmed that these drugs are the best therapy for 

onychomycosis caused by these yeasts. Interestingly, when we compared the activity 

of the three antifungal drugs against nondermatophytic filamentous fungi., terbinafine 

presented the best ",*0"-$% activity. .uch finding is remarBable, since choosing an 

adequate therapy of NDF in nails is often difficult for the clinician. Although our 

experiments are performed ",*0"-$%, it will be interesting to correlate such findings with 

clinical outcome. ae hope that further studies with a larger number of isolates and 

evaluation of correlation between ",*0"-$% results and clinical response to treatment, 

will give the clinician a better guide for therapeutic onychomycosis conduct. 

P-0058. Antifungal activity of essential oils obtained from aromatic and 

medicinal plants from Colombia 

Bueno Sánchez J 1 , Corrales A 1 , Montiel ( 1 , DurZn C 2 , .tashenBo E 2 , Mesa A 1 


ParasitologÜa, Grupo Infeccián y CZncer, 2 6niversidad Industrial de .antander, 

/aboratorio de CromatografÜa, Escuela de ~uÜmica, CI2IMf/ 

Purpose: To evaluate activity against strains of 7+,5"5+ and 4&'.$@"66(& of essentials 

oils obtained from aromatic and medicinal plants of different regions of Colombia. 

Summarized description of the project: Colombia is rich in natural resources, 

having among them 10_ of world]s flora diversity. A wide spectrum of molecules with 

different biological activities, including antifungic ones, have been identified from 

several essential oils of some plants. Identification of oil producing plants from 

Colombia, is therefore a vast and interesting field for pharmaceutical industry. In an 

effort to advance in this field, the present worB was done. 

Minimum inhibitory concentration (MIC) was measured using oils extracted from 11 

plants of 2oraginaceae (1), /abiatae(2), Gramineae(2), /amiaceae(2), Annonaceae(1), 

Zingiberaceae(2) and Magnoliaceae(1) families. fils extraction was done by 

hydrodistillation. Antifungal activity was evaluated following AF.T-E6CA.T and MJ8- 

A (C/.I) protocols, with 7+,5"5+*'+$+'&"6%&"& ATCC 2201K, 7+,5"5+*[$(&." ATCC 

62[8 4&'.$@"66(&*U6+0(& ATCC 20OJ02 and 4&'.$@"66(&*U()"@+-(& ATCC 20OJ0[ 

strains. Each evaluation was done by triplicate, in three different experiments. 

Controls were carried out using 7+,5"5+ strains and itraconazole, ITZ (.igma). 

Results and Conclusions: Geometric means in Gramineae family oils wereM 0.0J2 

kg/m/ for 7/*'+$+'&"6%&"&, 0.02[ kg/m/ for 7/*[$(&.", 0.0JJ kg/m/ for 4/*U6+0(& and 

0.007 kg/m/ for 4/*U()"@+-(&. /amiaceae family oils showed the following valuesM 

0.078 kg/m/ for 7/*'+$+'&"6%&"&3*0.062 kg/m/ for*7/*[$(&.", 0.12[ kg/m/ for*4/*U6+0(&, 

and 0.021 kg/m/ for 4/*U()"@+-(&/*Finally, Zingiberaceae family oils geometric means 

wereM 0.076 kg/m/ for 7/*'+$+'&"6%&"&3*0.12[ kg/m/ for 7/*[$(&.", 1.0 kg/m/ for 4/* 

U6+0(& and 0.077 kg/m/ for 4/*U()"@+-(&. fils from other families show higher 

geometric means. 

Gramineae family essential oils showed high ",*0"-$% activity against 7+,5"5+ and 

4&'.$@"66(& strains. Also, oils of /amiaceae and Zingiberacea families exhibited good 

antifungal activity. In all cases, observed activity was higher than the one reported by 

Hammer .-*+6. (2002), for !.6+6.(#+*+6-.$,"U%6"+ essential oil (Tea tree oil). 4/* 

U()"@+-(& showed the highest sensitivity, while 7/*[$(&."*the lowest. The later strain is 

resistant to fluconazole, drug of choice for infection treatment caused by this genus 

yeasts. Activity shown by Gramineae family oils might be due to the presence of #"-$+6, 

a powerful antifungal Bnown as a major component of oils extracted from this family. 

Identification of antifungal activity molecules in aromatic and medicinal plants is a 

promissory biothechnological alternative that, together with others from different fields, 

may impulse crops substitution in Colombia. 

Acknowledgments: This worB was supported mainly by grant Cf/CIENCIA. RC- 

OJ2-200O. ae thanB to F6NDACfFAN for identification and recollection of plants 

species. 


P-0059. Method to reach the minimum inhibitory concentration of rutaceae 

extracts against toxicogenic moulds 

Calvo M, Adelantado C, .hiva C, Guiu N, Arosemena / 

6niversitat Autínoma de 2arcelona 

The presence of moulds and/or its mycotoxins in raw materials intended for feed 

elaboration is a consequence of the natural ability of these microorganisms of growing 

over different vegetal substrata. Mycotoxins are thermoresistent and after 

consumption they accumulate in the organism, leading sometimes to severe disorders 

observed as acute or cronic processes. aith the aim of controlling the growth of 

toxicogenic mould strains in raw materials and feed, the antifungal activity of 

Rutaceae extracts has been assayed, having into account that its antibacterial activity 

has been widely previously described. The chosen strains for the study wereM 

4&'.$@"66(&*U6+0(&*ATCC MYA-J82, 4&'.$@"66(&*%#?$+#.(&*ATCC 60[J2 and ^(&+$"()* 

@$+)",.+$()*ATCC 60J0K described as important producers of aflatoxins, 

ochratoxins and zearalenone respectively. Materials and methods can be briefly 

summarized as followsM for each microorganism and concentration to assay, a 

calibrated flasB containing K8m/ of .abouraud 2roth is inoculated with 1m/ of a 

suspension from a 2Oh culture of each microorganism chosen, in a concentration of 

1x10 6 CF6/m/, and the suitable volume of the product to test according to the desired 

final volume (1000ppm, 2000ppm and J000ppm). The flasB is then adjusted up to 

100m/ of final volume with sterile culture broth. Control flasBs are also prepared. In 

these flasBs no product is added and they are only adjusted up to 100m/ with 1m/ of 

sterile culture broth. In order to compare the activity of the extracts assayed with some 

other products commonly used to control fungal growth, parallel assays are prepared 

according to the methodology described, but achieving different product 

concentrationM 1000, 2000 and J000 ppm of formic acidMpropionic acid ([0M[0). FlasBs 

are incubated at 28oC, evaluating the Rutaceae extract activity at 2O hours, J days, 

one weeB, two weeBs and four weeBs after inoculation. The results are always 

compared with those obtained in the control flasBs. aith the results obtained we may 

indicate that the assayed Rutaceae extracts have the ability of inhibiting completely 

the growth of the strains tested J days after inoculation and in a concentration of 

1000ppm. The control assays showed a growth of from 1x10 6 up to 1x10 8 CF6/m/ all 

along the study, and the test performed with acids a concentration of J000ppm was 

needed to reach the inhibition achieved by the Rutaceae extracts. In the assays 

performed after one, two or four weeBs after inoculation, results did not change, and 

therefore the Minimum Inhibitory Concentration of this extract is 1000ppm. 

P-0060. The paradoxical effect of echinocandins across Candida species in 

vitro: Evidence for echinocandin-specific and Candida species-related 

differences 

Chamilos G, /ewis R, Albert N, Kontoyiannis D 

MD Anderson Cancer Center 

Background: The echinocandins are a new class of antifungal agents, which target 

the fungal cell wall and possess broad-spectrum fungicidal activity against 7+,5"5+* 

species. However, paradoxical growth of some 7+,5"5+*+68"#+,& isolates ",*0"-$% has 

been recently observed at high, yet clinically achievable concentrations of caspofungin 

(CA.). The clinical significance and molecular mechanisms that account for the 

paradoxical effect of echinocandins remain unBnown. ae evaluated the frequency of 

the paradoxical effect among different echinocandins in a variety of 7+,5"5+ species. 

Materials and methods: ae determined CA. and micafungin (MICA) MICs against 

70 bloodstream isolates of 7+,5"5+ (7/*+68"#+,&, 20n 7/*'+$+'&"6%&"&, 10n 7/*-$%'"#+6"&, 

10n 7/*[$(&.", 10n 7/*@6+8$+-+, 10) collected form cancer patients by using the C/.I 

M27A-A2 method in RPMI 16O0 media. ae defined the paradoxical effect as 

progressive increase in turbidity occurring after at least 2 drug dilutions above MIC 

and tested a range of concentrations of both drugs (0.06-2[6 kg/ml). ae performed all 

experiments in triplicate, in different days. 

Results: Among the isolates tested 7/*'+$+'&"6%&"& exhibited the higher MICs to both 

CA. and MICA (MIC [0 /MIC K0 n 0.[/1.0n 1.0/2.0 kg/ml, respectively).*ae observed the 

paradoxical effect for CA. in K0_, 60_, O0_ and 10_ of*7/*'+$+'&"6%&"&3*7/*+68"#+,&3* 

7/*-$%'"#+6"&3*and*7/*[$(&."*isolates, respectively. For MICA we found the paradoxical 

effect only in*7/*-$%'"#+6"&*(7/10 or 70_)*and*7/*[$(&." (6/10 or 60 _) isolates. Notably, 

paradoxical effect for either CA. or MICA was absent in 7/*@6+8$+-+ isolates. 

Paradoxical growth of 7+,5"5+ isolates was evidenced at a median concentration of 

6O % and K6 % MIC for CA. and MICA, respectfully. There were no differences in MICs 

of CA. and MICA against the 7+,5"5+ isolates exhibiting paradoxical growth as 

compared to the rest of 7+,5"5+*isolates. 

Conclusions: The paradoxical growth of 7+,5"5+ isolates at high concentrations of 

echinocandins occurs in higher frequency with CA. compared to MICA and is not 

related to the MIC of isolates to each drug. Although the molecular mechanisms of 

echinocandin paradoxical effect are largely unexplored, the absence of this 

phenomenon in 7/*@6+8$+-+ isolates might be related to its haploid state.

P-0061. In vitro and in vivo activity of ITF 2534, a new triazole antifungal 

Clemons K 1, 2, J , Martinez M 1 1, 2, J 

, .tevens D 

1 California Institute for Medical Research, .an (ose, CA 6.A, 2 .anta Clara Valley 

Medical Center, .an (ose, CA 6.A, J .tanford 6niversity, .tanford, CA 6.A 

ITF 2[JO (ITFn Italfarmaco, Milan) is a novel triazole enantiomer selected from a 

screening program of itraconazole (ICZ)-liBe compounds on the basis of its favorable 

solubility, absorption and CYP interaction profiles. In vitro screening against 

opportunistic and endemic pathogens indicate activity similar to ICZ for*7+,5"5+ spp.,* 

4&'.$@"66(&*U()"@+-(&,*Z6+&-%)2#.&,*='%$%-?$"_n superior to ICZ for*=#.5%&'%$"()* 

+'"%&'.$)(),*4/*,"@.$,*4/*-.$$.(&n and inferior for*7%##"5"%"5.&,* 

1+$+#%##"5"%"5.&. 2ased these activities, ITF was tested in vivo. A model of 7+,5"5+ 

vaginitis was studied in estrogenized 2A/2/c mice. Twenty-four hours after infection, 

once daily oral therapy was given with ITF or ICZ at 10, [0 or 100 mg/Bg. fn day 1 

postinfection, mice in all groups were infected equally with 7/*+68"#+,&/**2y day 6, 

CF6 had risen in PEG and cyclodextrin diluent control groups and were equivalent. 

ITF at 10 or [0 mg/Bg were ineffective, and at 100 mg/Bg significantly reduced CF6 

recovered from the vagina compared to controls. ICZ at 10 mg/Bg was also ineffective 

versus control, whereas ICZ at [0 or 100 mg/Bg were effective. At 100 mg/Bg, ICZ 

and ITF were equivalent. For the second model, the efficacy of ITF was examined 

against systemic aspergillosis. 2eginning one day after infection, mice were treated 

once daily orally with ITF at 10, O0 or 100 mg/Bg, ICZ at O0 or 100 mg/Bg, 

voriconazole (VCZ) at O0 mg/Bg (mice given grapefruit juice in lieu of drinBing water) 

or intravenously with amphotericin 2 at 1 mg/Bg, for 6 days. ITF at 100 or O0 mg/Bg 

had significant efficacy. ITF was comparable to that of ICZ, VCZ and AM2 for 

prolongation of survivaln no mice given ITF at 100 or O0 mg/Bg succumbed. The 

efficacy appeared to be dose-related. ITF also reduced fungal burden from the target 

organs, the Bidneys and brain. In the Bidneys, the main target organ in this model, no 

treatment effected cure. AM2 was the most effective in the Bidneys and superior to 

other regimens. ITF was modestly dose-responsiven the 100 or O0 mg/Bg dosages 

were equivalent to ICZ or VCZ. In the brain, AM2 and ITF at 10 mg/Bg were the 

least effective. ITF at 100 mg/Bg trended toward superior efficacy versus AM2n ICZ 

and VCZ were superior to AM2. ITF at 100 or O0 mg/Bg was equivalent to ICZ at 100 

or O0 mg/Bg or VCZ at O0 mg/Bg. .ome cures were attained by each efficacious 

regimen. No adverse effects were noted during the course of these studies. fverall, 

ITF shows promising in vitro and in vivo activity, which warrants further study. 

P-0062. Functional dissection of Tac1, a C. albicans transcription factor 

Coste A, Turner V, Ischer F, .anglard D 

Institute of Microbiology, /ausanne, .witzerland 

Tac1p is a 7+,5"5+*+68"#+,& transcription factor crucial for the upregulation of the 

A2C-transporter genes 7VPI and 7VPM, which mediate azole resistance. ae 

distinguished two types of allelesM i) wild-type alleles isolated from azole-susceptible 

strains that mediate transient upregulation of 7VPI and 7VPM upon exposure to 

inducers such as fluphenazine or terbinafine and ii) hyperactive alleles isolated from 

azole-resistant strains which mediate high constitutive expression of 7VPI and 7VPM. 

ae demonstrated previously that a single point mutation is responsible for this 

phenotypic difference. In this study we addressed the functional dissection of >47I by 

amino acid (aa) point mutations or domain deletions. 

2y comparison of >47I alleles from different azole-susceptible and azole-resistant 

strains, we first localized several aa difference responsible for >47I hyperactivity and 

next confirmed them by site-directed mutagenesis. Changes of aspartic acid (N) to 

aspartate (D) in position K77 (NK77D) or A7J[V were sufficient to convert a wild-type 

allele into a hyperactive form. A GK80E change was not sufficient to confer 

hyperactivity to a wild-type allele. A combination of GK80E with at least a .KO1P 

mutation was necessary for >47I hyperactivity. To determine in which functional 

domain these mutations were localized, different regions of Tac1p were deleted and 

the obtained mutant proteins analysed for their phenotypes. 

First, two major putative functional domains of Tac1p, the transcriptional activation 

domain (AD, between aa 801 and K81) and the Middle Homology Region (MHR, 

between aa O2O and [18) were deleted. .econd, the relevance of a putative 

localization domain, the Nuclear /ocalisation .ignal (N/.) was addressed for function 

and localization. The basic aa RJJ, lJO and RJ[ constituting the putative N/. were 

replaced by three leucine residues (Tac1p-mutN/.). None of these three mutated 

Tac1p proteins were able to upregulate 7VPI or 7VPM in the presence of 

fluphenazine or terbinafine, thus suggesting a crucial role for these different domains 

in Tac1p activity. The Tac1p-mutN/. protein was also fused to GFP. This fusion 

protein was localized in the cytoplasm in contrast to the wild-type Tac1p-GFP fusion 

protein, which is located constitutively in the nucleus. This result confirmed the role of 

this putative N/. in Tac1p nuclear localization. This preliminary worB allowed us to 

determine large functional domains in Tac1p but additional studies are needed to map 

these domains more precisely and to determine the role of each domain in Tac1p 

activity. Tac1p mutations responsible for hyperactivity and described up to now are 

localized near to the activation domain, however the identification of additional 

mutations either by recovery of other >47I hyperactive alleles or by random 

mutagenesis might reveal other critical domains for hyperactivity. 


P-0063. Antifungal susceptibility testing of Cryptococcus neoformans: The 

usefulness of monitoring to detect in vitro resistance over time 

Córdoba S, Vivot a, Isla G, Guareschi C, Davel G, Vitale R 

Introduction: Infections due to 7$2'-%#%##(&*,.%U%$)+,& cause severe disease in 

immunocompromised host, mostly in patient with AID.. Amphotericin 2 with or without 

flucytosine is the antifungal drug recommended for initial treatment followed by lifelong therapy 

with fluconazole. 

However, not all clinical isolates are susceptible to antifungal agents, and even though 

appropriate therapy is given, relapses can occur and mortality associated with cryptococcal 

meningitis remains high. 

Objective: To detect the possible variations of ",*0"-$% antifungal susceptibility between different 

isolates collected from patients over time. 

Materials and methods: Patients: JO patients with AID.-associated cryptococcal meningitis. 

MicroorganismsM 1J6 7$2'-%#%##(&*,.%U%$)+,& clinical strains isolated from the /CR of 

patients with AID. suffering from cryptococcal meningitis. 

Antifungal agents: the drugs tested were amphotericin 2 (AM2) and flucytosine ([FC) (.igma 

Chemical, Co), itraconazole (ITZ) ((anssen, Argentina), fluconazole (FCZ) and voriconazole 

(VCZ) (Pfizer ..A., Argentina), and were provided as standard powders of Bnown potency. .tocB 

solutions were prepared as followsM AM2, ITZ, FCZ and VCZ were dissolved in 100_ 

dimethylsulfoxide (DM.fn .igma Chemical, Co) and [FC in distilled water. The stocB solutions 

were frozen at -20o C and stored until used. The final concentration for AM2, ITZ and VCZ 

ranged from 0.0J to 16 &g/mln for FCZ and [FC from 0.2[ to 128 &g/ml and were sited in K6-well 

round-bottomed microtrite plates. 

Medium: RPMI 16O0 supplemented with glucose up to 2_ and buffered with 

morphopropilensulfonic acid (MfP., .igma Chemical Co) to pH 7.0 was used. 

P-0064. Antifungal activity of natural products from leaves of Vanillosmopis 

erythropappa Shultz-Bip 

de Resende M 1 , Moreira F 1 , .ousa f 2 , (ohann . 1 , 2rito E 1 , /yon ( 1 , 2uzanello 

Martins C 1 

1 Federal 6niversity of Minas Gerais, 2elo Horizonte MG, 2razil, 2 Federal 6niversity of 

(uiz de Fora, 2razil 

In the present study the antifungal activity of coming natural products of the leaves of 

O+,"66%&)%'&"&*.$2-?$%'+''+ .hultz-2ip were performed by the method of 

determination of the minimum inhibitory concentration (MIC). The crude extract, the 

hexane, dichloromethane, in ethyl-acetate and butanol fractions, as well as the 

essential oil of the fresh leaves of O+,"66%&)%'&"&*.$2-?$%'+''+, and the determination 

of its antifungal actitvity were appraised as for his effect against 7+,5"5+*+68"#+,&3*7/* 

[$(&."3*7/*'+$+'&"6%&"&3*7/*@6+8$+-+3*7/*-$%'"#+6"&3*7$2'-%#%##(&*,.%U%$)+,&3* 

='%$%-?$"_*&#?.,#[""3*>$"#?%'?2-%,*$(8$()3*>/*).,-+@$%'?2-.&3*^%,&.#+.+*'.5$%&%"3* 

^/*#%)'+#-+3*76+5%'?"+6%'?%$+*#+$$"%,""3*P?",%#6+5".66+*+e(+&'.$&+3*1?"+6%'?%$+* 

0.$$(#%&+3*+,5*76+5%'?"+6%'?%$+*8+,-"+,+/ The minimum inhibitory concentration was 

determined through the microdilution test in agreement with C/.I M27-A2 document 

(Clinical and /aboratory .tandards Institute). All of the coming natural products of the 

leaves of O/*.$2-?$%'+''+ exhibited inhibitory effect on the tested fungi, except the 

crude extract, hexane and butanol fractions and the essential oil against 7/*8+,-"+,+ 

and, also, the essential oil against the yeast 7/*'+$+'&"6%&"&, which did not inhibit these 

microorganisms in any of the tested concentrations. The results of the present worB 

indicate that the species O/*.$2-?$%'+''+ possesses antifungal properties. Assays for 

determination and identification of the compounds with antifungal activity are 

necessary to elucidate the mechanism of action of the active substances of this plant. 

In vitro Susceptibility Testing: the minimal inhibitory concentration (MIC) of antifungal agents 

was determined according to the M27-A2 C/.I broth microdilution method modified by 

Rodriguez-Tudela .-*+6. 

Results: 

Clinical data: all the patients received AM2 as initial treatment followed by FCZ, although 

despite of this, 1[ (OO_) of them died. 

MICs: for FCZ the values obtained were as followM 62.[_M '8 &g/ml, JJ_M %16 &g/ml, O.[_M %6O 

&g/ml. For FCZ, isolates from 21 patients showed no changes in the MICs, whereas from isolates 

of 1J patients changes in the MIC were observed. From these patients, seven died and the MIC 

raised more than two dilution steps. However, six patients survived, from which in only in three 

patients an increase of more than two dilutions was observed. 

For four isolates from three patients, [FC MICs were elevated (%J2 &g/ml). 

In contrast, all the isolates were ",*0"-$% susceptible to AM2 and ITZ, while for VCZ only one 

isolate showed an elevated MIC (2 &g/ml). 

Conclusions: The emergence of less susceptible strains to fluconazole from different isolates 

collected from patients over time was observed. These ",*0"-$% tests could be of potential 

usefulness to monitor and guide cryptococcal meningitis therapy and suggest the need for 

greater vigilance and surveillance to detect fluconazole resistance over time.

P-0065. Antifungal properties of some plants used in Brazilian traditional 

medicine against human pathogens 

de Resende M 1 , (ohann . 1 , Moreira F 1 , /yon ( 1 , Pizzolatti M 2 , 2uzanello Martins C 1 


.anta Catarina, 2razil 

The aim of the present study was the determination of the antifungal activity of the 

extracts of 2razilian plants traditionally used in popular Medicine in 2razil. Antifungal 

properties of extracts of nine 2razilian plants namely =#?",(&*-.$.8",-?"U%6"(& Raddi, 

K,@+*5(6#"& Mart., 46-.$,+,-?.$+*8$+&"6"+,+ luntze, 1"'.$*+8(-"6%"5.& lunth, 1"'.$* 

$.@,.66"" CDC, A.$"&&+,-"+*#$"&'+ (/) 2riz, P(8(&*($-"#+.U%6"(& Poir, b_+6"&*+#.-%&.66+. 

Reiche, and Z+##?+$"&*5$+#(,#(6"U%6"+ DC, were tested against five 7+,5"5+ species, 

7$2'-%#%##(&*,.%U%$)+,&, and ='%$%-?$"_*&#?.,#["" by bioautography assay and 

susceptibility test. The results showed that almost all the hexane extracts exhibited 

antifungal activity, at least against one of the microorganisms tested. The hexane 

extract from 1/*+8(-"6%"5.& (aerial parts) and ethanolic from leaves of =/*-.$.8",-?"U%6"(& 

demonstrated strong antifungal activity against 7/*@6+8$+-+ and =/*&#?.,#["". The 

results of the present worB validate and documents, in a systematic way that the most 

of species plants studied possess substantial antifungal properties. This explains the 

use of these plants in folB medicine for the treatment of various diseases related to 

microbial infections. ae have started isolating the active compounds responsible for 

the antifungal effects from 1/*+8(-"6%"5.& and =/*-.$.8",-?"U%6"(&. 

P-0066. Comparison of e-test and CLSI microdilution method for antifungal 

susceptibility testing of trichosporon spp. 

de Resende M, /emes R 

Federal 6niversity of Minas Gerais, 2elo Horizonte MG, 2razil 

The crescent increase and the gravity of the infection caused by >$"#?%&'%$%,*spp. in 

immunecompromised patients and the increasing number of clinical isolates resistant 

to antifungal therapy, as well as the necessity of a guide to the selection and follow-up 

of the treatment led to a demand for susceptibility testing of these fungi. The aim of 

this study was to test JO samples of >$"#?%&'%$%, spp., >/*+&+?"" (26), >/*+&-.$%"5& ([), 

>/*)(#%"5.& (1) and >/*%0%"5.& (2), for ",*0"-$% susceptibility to amphotericin 2 (AM2), 

[-flucytosine ([-FC), fluconazole (F/6), itraconazole (ITR), terbinafine (TER) and 

voriconazole (VfR) using a microbroth technique proposed by C/.I. It was also 

evaluated the occurrence of susceptibility variation to the antifungals in agreement 

with the site of isolation of the different species of this genus and results obtained by 

the method C/.I were compared with those determined by the E-test technique. 

>$"#?%&'%$%,*+&+?"" and >/*%0%"5.& showed sensibility to [-FC, >/*)(#%"5.& to AM2 

and VfR, while >/*+&-.$%"5.& demonstrated resistance the all of the tested drugs, 

except for F/6. The minimum inhibitory concentration (MIC) for TER, of all of the 

samples, varied of 0,0J1-8,0 mg/m/. All of the species exhibited resistance to ITR and 

sensibility to F/6 (CIM K0 O8 hours). The sample of environmental origin and those 

isolated from patients with superficial and systemic mycoses were sensitive to AM2, 

[-FC and VfR, while those originating from cutaneous and subcutaneous mycoses 

exhibited resistance to the polienic drug. All of the samples exhibited resistance to 

ITRA. Except for two of >/*+&+?"" (an isolated from subcutaneous and another from 

superficial infection), the others exhibited sensibility to F/6. The comparison among 

the results obtained by the method of C/.I and E-test revealed larger discrepancies 

among the tests with [-FC and ITR. The emergence of significant antifungal drug 

resistance and the small number of drugs available also contribute to the need for new 

agents for serious fungal infections. 

. 


P-0067. In vitro interaction of antifungal agents against Aspergillus spp. 

Dong P, aan Z, Li R 

PeBing university first hospital 

Objective: ae investigated the in vitro activities of combinations of amphotericin 2 

(AM2), terbinafine (T2F), and itraconazole (ICZ) or alone against 70 isolates of 

Aspergillus spp. 

Methods: A checBerboard microdilution method was applied. The MICs endpoints 

were determined as the well that was 100_ inhibition of growth for both AM2 and T2F, 

while %80_ inhibition of growth for ICZ, by comparing to the control well. 2esides, 

100_ inhibition of growth was determined for the combinations of different drugs. 

Drug interaction was classified as synergistic if the FIC index was v1, additive if the 

FIC index was %1 but v2.0, and antagonistic if the FIC index was %2.0. 

Results: The MICs of AM2 for A. niger appeared significantly lower than that of the 

other isolates (~ test, pv0.0001). The MICs of T2F for A. fumigatus were significantly 

higher than that for the other isolates (~ test, pv0.0001). Itraconazole-resistance was 

not observed. The FIC index in three strains (one was A.fumigatus, the other two were 

A.niger) was %2.0 in the combination of AM2-ICZ, and no other antagonism was 

observed. The percentage of synergy of T2F-ICZ was significantly higher compared to 

the combination of either AM2-T2F or AM2-ICZ ((2}11.667 P}0.001 and(2}2[.8KK 

Pv0.0001). The difference between AM2-T2F and AM2-ICZ was not significant 

((2}J.70O!P}0.0[O). 

Conclusion: Although the different agents showed different antifungal activity against 

different species of Aspergillus spp, the combination of T2F-ICZ displayed a 

synergistic antifungal activity against most of Aspergillus spp. isolates in vitro. 

P-0068. Species distribution and antifungal susceptibility of Candida blood 

stream isolates from the Australian candidemia study. 

Ellis D 1 , Chen . 2 , Nguyen ~ J , Marriott D J , .lavin M O , .orrell T 2 

1 aomenes and Childrenes Hospital, Adelaide, 2 aestmead Hospital, .ydney, J .t 

Vincentes Hospital, .ydney, O Royal Melbourne Hospital, Melbourne, Australia 

2etween August 2001-(uly 200O, a total of KO[ 7+,5"5+ blood stream isolates from 

K1[ patients were received from K0 participating institutions for identification and 

antifungal susceptibility testing. All isolates were identified by carbohydrate 

assimilation tests using the IDJ2C strip (bioMdrieux), cycloheximide resistance, nitrate 

assimilation, growth at J7 o C, hydrolysis of urea and yeast morphology studies. 

Antifungal susceptibility testing was performed in accordance with the C/.I M27A2 

reference method for testing of yeasts. Fifteen species of 7+,5"5+ were identified. 

The most common species was 7+,5"5+*+68"#+,& (O7.J_), followed by 7/*'+$+'&"6%&"& 

(1K.J_), 7/*@6+8$+-+ (17.8_), 7/*[$(&." (O.K_) and 7/*-$%'"#+6"& (O.K_). 6ncommon 

7+,5"5+ bloodstream isolates were also identified, notably 7/*5(86",".,&"& (2.J_) and 

7/*@("66".$)%,5"" (1.2_), together with 8 other rarely reported species. All isolates 

were susceptible to amphotericin 2, including 8 strains of 7/*6(&"-+,"+.. All 7/*+68"#+,& 

isolates were susceptible to the azoles fluconazole, itraconazole and voriconazole. 

Resistance to fluconazole (MIC t*6O ug/ml) was detected in only a single isolate of 7/* 

'+$+'&"6%&"&*(0.[_n 1/182) and as expected in 7/*[$(&." (K1.J_n O2/O6) and in 7/* 

@6+8$+-+*(2J.2_n JK/168) isolates. The majority of 7/*@6+8$+-+ strains (71.O_n 

120/168) were found to be susceptible dose dependent to fluconazole (MICs 16-J2 

ug/ml). Resistance to itraconazole (MIC t*1 ug/ml) was more wide spread with a 

higher proportion of isolates in the susceptible dose dependent (.DD) range (MICs 

0.2[-0.[ ug/ml). For example in 7/*@6+8$+-+ 8[.7_ (1OO/168) of isolates were 

resistant to itraconazole and 1J.7_ (2J/168) were .DD, whereas for 7/*[$(&." only 

8.K_ (J/O6) were resistant but K1.1_ (OJ/O6) were .DD. fther species with a high 

frequency of reduced susceptibility to itraconazole (MICs t*0.2[ ug/ml) were 7/* 

'+$+'&"6%&"& (OO_), 7/*-$%'"#+6"& (60.K_) and 7/*@("66".$)%,5"" (100_). Voriconazole 

resistance (MIC t*O ug/ml) was limited to 7/*@6+8$+-+ (10.7_n 18/168)n these stains 

showed cross resistance to all other azoles ie, fluconazole, itraconazole, 

posaconazole and Betoconazole, but all were fully susceptible to amphotericin 2, 

flucytosine and caspofungin. Thus, it would appear that {10_ of bloodstream 7/* 

@6+8$+-+ isolates may show intrinsic azole resistance. These findings emphasise the 

continuing need for surveillance and epidemiology studies to identify emerging 

antifungal resistance and populations at risB. A national approach such as this 

provides more comprehensive information on the burden of antifungal resistance than 

single centre studies.

P-0069. Activity of micafungin against clinical isolates of Candida spp. and 

Candida albicans biofilms 

Eraso E 1 , Albaina f 1 , Arechavala A 2 , .ahand I 1 , Carrillo-Muoz A J , Pontán ( 1 , 

~uindás G 1 

1 Depto. Microbiology. Fac. Medicina y fdontologÜa. 6niv. PaÜs Vasco. 2ilbao, .pain, 

2 6nidad MicologÜa, Hospital F( Muiz, 2uenos Aires, Argentina, J ACIA-Microbiology, 

2arcelona, .pain 

Micafungin is a new echinocandin which inhibits 1,J-beta-D-glucan synthase in the 

fungal cell wall. 

Micafungin displayed a good activity, especially against most of isolates of 7+,5"5+* 

+68"#+,& (MIC K0 0.2[ microgram/ml). There were no differences in micafungin 

susceptibility with fluconazol-resistant isolates. However, a lower activity was 

observed against 7+,5"5+*'+$+'&"6%&"& and 7+,5"5+*@("66".$)%,5"" isolates. 

This good activity was also observed against the 7+,5"5+*+68"#+,& biofilm formation, 

with 7[_ of the sessile cells being inhibited by v 2 microgram/ml at 2O h and 8O_ at 

O8 h. 

Conclusion: Micafungin displays a good activity against planBtonic and sessile 

7+,5"5+*+68"#+,& cells. 

Purpose: To evaluate the antifungal activity against 7+,5"5+ isolates and biofilms. 

Methods: J8[ 7+,5"5+ isolates from oral specimens, blood, and other body fluids 

including strains with different susceptibility to fluconazole. .usceptibility testing was 

performed by M27-A2 broth microdilution method (C/.I, 6.A). .erial two-fold 

dilutions of micafungin (Astellas Europe) were made in RPMI 16O0 medium buffered 

to pH 7.0 with MfP. (range of concentrations were 0.0J-16 microgram/ml). Plates 

were incubated at J7ÅC and MIC endpoints were read visually after 2O h and O8 h. 

MIC was defined as the lowest concentration that resulted in a prominent decrease in 

growth compared with that of growth-control wells. MIC [0 and MIC K0 results are the 

concentrations of antifungal agent necessary to inhibit [0_ and K0_ of the isolates, 

respectively. 7+,5"5+*'+$+'&"6%&"& ATCC 2201K and 7+,5"5+*[$(&." ATCC 62[8 were 

included as quality control isolates. Also biofilm formation by 12 different human 

clinical isolates of 7/*+68"#+,& and biofilm formation inhibition by micafungin, were 

evaluated by microscopic observations and by a colorimetric method based on the 

use of a tetrazolium salt (jTT) to monitor metabolic activities of cells within the biofilm. 

Results: In vitro susceptibilities of the isolates are summarized in the table. 

No Range 

Geometric mean 

MIC 

(microgram/ml) (microgram/ml) 

[0 MIC 

.pecies 

isolates 

2Oh O8h 2Oh O8h 2Oh O8h 2Oh 

K0 

O8h 

7/*+68"#+,&* 100 0.06-1 0.06-1 0.J1 0.JJ 0.2[ 0.2[ 0.[ 1 

7/*5(86",".,&"&* 100 0.2[-O 

0.2[- 

16 

0.70 1.J[ 0.[ 1 1 O 

7/*@6+8$+-+* [1 0.0J-2 

0.12[- 

2 

0.[8 0.66 0.[ 1 0.[ 1 

7/*[$(&."* 27 0.[-8 0.[-8 2.[0 J.12 2 O 8 8 

7/*'+$+'&"6%&"&* 2O 8-16 8-16 10.J7 11.6[ 8 16 16 16 

7/*-$%'"#+6"&* 2O 0.2[-O 0.[-O 0.KO 1.06 1 1 1 1 

7/*@("66".$)%,5""* 20 O-16 O-16 8 K.[1 8 8 16 16 

7/*6(&"-+,"+.* 1[ 0.[-8 1-16 2.00 2.O1 2 2 8 8 

7/*$(@%&+* 8 O 16 O 16 O 16 O 16 

7/*6"'%62-"#+* 6 8 8 8 8 8 8 8 8 

7/*[.U2$* O 0.2[-1 0.[-2 0.70 1 1 1 1 2 

7/*,"0+$".,&"&* J 0.[ 0.[ 0.[ 0.[ 0.[ 0.[ 0.[ 0.[ 

7/*U+)+-+* 2 8-16 16 11.J1 16 8 16 16 16 

7/*'.66"#(6%&+* 1 1 1 1 1 - - - - 


P-0070. Comparison of etest and reference broth microdilution method for 

testing susceptibility of clinical isolates of Aspergillus species to amphotericin 

b, itraconazole, and voriconazole. 

Evci C 1 , ABcaglar . 2 , Ener B J 

1 Dept of Microbiology, Fac of Medicine, 2 Dept of Microbiology, Fac of Medicine, J Dept 

of Microbiology, Fac of Medicine 

In this study we aimed to evaluate ",*0"-$% susceptibility of clinical strains of 4&'.$@"66(&* 

species isolated from tissue biopsy and respiratory specimens to antifungal agents 

and to compare the results obtained from Etest with Reference 2roth Microdilution 

Method (2MD) for antifungal susceptibility testing of filamentous fungi. A total of [[ 

clinical isolates were recovered from specimens of O8 patients stayed internal 

medicine wards (especially Haemotology and fncology 6nits) at the Hospital of the 

6niversity of 6ludag. The susceptibilities of these isolates of 4&'.$@"66(& species (JJ 4* 

U()"@+-(&, 1K 4*U6+0(&, 2 4*-.$$.(&, and 1 4*,"@.$ isolates) to amphotericin 2, 

itraconazole, voriconazole were assessed by the Etest method. Voriconazole was 

found very active at a MIC of '1 kg/m/ against all species of 4&'.$@"66(& by Etest 

(K8_ susceptible) but slightly less active by 2MD Method (71_ susceptible). 

Itraconazole showed similar activities with voriconazole. ae found higher 

amphotericin 2 MICs for 4&'.$@"66(& spp. than those reported by other authors. The 

agreement between the two methods for voriconazole, itraconazole, and amphotericin 

2 was 8K.1_, 8K.1_, and 8[.[_, respectively. Etest tended to give lower values for 

voriconazole and amphotericin 2, but higher values for itraconazole. K,*0"0%-",*0"-$% 

evaluations are needed to assess both of these antifungal testing methods. 

P-0071. In vitro susceptibility testing of clinical isolates of Aspergillus species 

to amphotericin b, itraconazole, voriconazole and caspofungin using etest 

method 

Evci C 1 , ABcaglar . 1 , Ener B 1 

1 Dept of Microbiology, Fac of Medicine, 2 Dept of Microbiology, Fac of Medicine, J Dept 

of Microbiology, Fac of Medicine 

This study was conducted to investigate the resistance patterns of clinical 4&'.$@"66(&* 

spp. isolates to four antifungal agents. A total of [O 4&'.$@"66(& spp. isolated from 

clinical materials (tissue biopsy and respiratory specimens) of hospitalized patients at 

the Mycology /aboratory in 6niversity of 6ludag were included in this study. The 

susceptibilities of these isolates of 4&'.$@"66(& species (JJ 4*U()"@+-(&, 1K 4*U6+0(&, 1 

4*-.$$.(&, and 1 4*,"@.$ isolates) to amphotericin 2, itraconazole, voriconazole and 

caspofungin were assessed by using Etest method. The agar formulation used for the 

Etest was RPMl agar supplemented with 2_ glucose and MICs were read after 

incubation for O8 h. Voriconazole was very active at a MIC of '1 kg/m/ against all 

species of 4&'.$@"66(& by using Etest method (K8_ susceptible). Although MIC values 

of itraconazole (MIC K0 range was 2 kg/m/) were higher than those of voriconazole, 

itraconazole was found an active agent. ae found higher amphotericin 2 MICs for 

4&'.$@"66(& spp. than those reported by other authors. For caspofungin, trailing growth 

was observed with all of the isolates. Caspofungin MICs for 4&'.$@"66(& spp. were 

below 0.[ kg/m/ if trailing growth was ignored. Although ",*0"0%-",*0"-$% evaluations 

are needed to assess Etest method especially caspofungin, we obtained very 

significant antifungal susceptibility data for 4&'.$@"66(&*spp. in this study.

P-0072. Comparison of flow cytometry, etest and reference broth macrodilution 

methods for testing susceptibility of clinical isolates of Candida albicans to 

fluconazole 

Evci C, ABcaglar ., Ener B, fral 2, Géral G 

Dept of Microbiology, Fac of Medicine 

In this study we aimed to evaluate ",*0"-$% susceptibility of clinical strains of 7+,5"5+* 

+68"#+,& isolated from blood and other sterile body fluids to fluconazole and to 

compare the results obtained from three different methods. The susceptibilities of 26 

isolates of 7+,5"5+*+68"#+,& against fluconazole were assessed by Flow cytometry 

(FC) using propidium iodide (PI), Etest and Reference 2roth Macrodilution Methods 

(2MD). For FC, the yeast inoculum was prepared in sterile 0.8[_ saline (1-[x10 6 cells 

per m/). Fluconazole was diluted in RPMI 16O0 broth medium. Yeast samples and 

serial drug dilutions were incubated at J[oC for 6 h. .odium deoxicholate and PI 

(fluorochrome used for cell analyses) were added at the end of the incubation. 

Fluorescence was measured with a Epics j/- MC/ flow cytometry (2ecBman Coulter). 

MIC was determined as the lowest drug concentration that produced a [0_ increase 

in mean channel fluorescence (MCF) compared to growth control. Etest MICs were 

determined with RPMI agar containing 2_ glucose and were read after incubation for 

2O h and O8 h at J[oC. 2MD Method was performed according to NCC/. M27-A2 

document. The Etest and FC results were found to be well correlated well with 

reference MICs obtained from 2MD method. fverall agreement was 77_ and 7J_ 

with Etest and FC, respectively. 2ut the percentage of agreement was above K[_ for 

FC and Etest when 2MD results were evaluated at 2O h instead of O8 h. ae thought 

that this difference could be due to the traling growth phenomenon that shown in 1K_ 

of strains. FC and Etest methods can be alternative to the reference method for 

determining fluconazole susceptibilities of 7+,5"5+*+68"#+,&. 2ut the need for 

specialized equipment and more extensive testing for standardization limits the use of 

FC method in routine laboratory testing. The Etest method is easier and less timeconsuming 

and has potential value as an alternative antifungal susceptibility testing, 

but requires further investigation to identify the optimum conditions for its use. 

P-0073. A prospective french national survey to assess renal safety of 

amphotericin B lipid complex and liposomal amphotericin B 

Faouzi S, Nathalie C, Philippe I, Anne R, Anne ., .tdphane D, 2datrice ., 2rigitte a, 

.orin V, flivier 2 

1 AP-HP, hÇpital Paul 2rousse, Villejuif, 2 Centre mddical 2ligny, 2riis-sous-Forges, 

J AP-HP, hÇpital Paul 2rousse, Villejuif, O HÇpital Cavale 2lanche, 2rest, [ HÇpital 

Archet, Nice, 6 HÇpital Huriez, /ille, 7 HÇpital Hotel Dieu, Nantes, 8 HÇpital 2rabois, 

Nancy, K HÇpital Notre Dame 2on .ecours, Metz, 10 HÇpital Cardiologique, /yon 

Purpose: /ipid formulations (/F) have been recommended to treat patients suffering 

from renal insufficiency. ae report a prospective multicentre survey to assess renal 

function in adult patients treated for fungal infections with /FM Abelcet' (A2/C) and 

Am2isome' (/-Amb). 

Description: 88 patients (OJ F, O[ M) with a mean age of OK.6 r 1O years were 

included and evaluable for renal safety. OO_ of patients had neutropenia v[00mm J . 

60 patients were treated with A2/C (median doseM O.8 mg/lg/day) and J8 patients 

with /-Amb (median doseM J.J mg/lg/ day). The mean duration of treatment was 1J.[ 

r 8 days for A2/C and 1[ r 11 days for /-Amb. 68_ of the patients received 2 or 

more nephrotoxic drugs (72_ for A2/Cn 61_ for /-Amb). 

Results: No significant changement occurred between median serum creatinine level 

at baseline (81.J kmol/l .J8.0n O6[.0/, 88.8 kmol/l .O2.0n J[6.0/) and at end of therapy 

(11[.0 kmol/l .O[.0n 2O1.6/, 106.0 kmol/l .J1.0n J6J.0/) respectively for A2/C and /- 

Amb. No significant changement occurred between median creatinine clearance at 

baseline (KO.6 and 8[.2 ml/mn) and end of therapy (60.O and 71.6 ml/mn) respectively 

for A2/C and /-Amb. In the group of 26 patients with renal insufficiency prior to 

treatment, no significant difference was found between median creatinine level and 

creatinine clearance at baseline (1O[.[ kmol/l .88.0n O6[.0/, OO.[ ml/mn) and the end 

of the treatment (168.0 kmol/l .66.0n J6J.0/, JK.8 ml/mn). 

All patients 

( n} 88) 

Creatininemia (kmol// ) 

Median (range) 

Creatinine Clearance (ml/min) 

Median (range) 

2aseline End of therapy 2aseline End of therapy 

8O,0 

111,[ 

8K,[ 

60,8 

. J8,0 n O6[,0/ . J1,0 n J6J,0/ . 17,2 n 172,2/ . 22,1 n 207,0/ 

Abelcet 

(n} 60) 

81,J 

. J8,0 n O6[,0/ 

11[,0 

. O[,0 n 2O1,6/ 

KO,6 

. 17,2 n 172,2/ 

60,O 

. 22,1 n 18J,0/ 

Ambisome 

(n} 28) 

88,8 

. O2,0 n J[6,0/ 

106,0 

. J1,0 n J6J,0/ 

8[,2 

. 21,[ n 1O[,6/ 

71,6 

. 2O,0 n 207,0/ 

Conclusion: This study showed treatment of patients with /F had an excellent renal 

safety profile particularly in patients either with a normal or altered renal function 

receiving combined nephrotoxic drugs. 


P-0074. Candida species in transplant patients: Susceptibility to antifungal 

agents 

Fernández-Olmos A, Mercadillo M, Molina A, îlvarez M, .Znchez-.ousa A, 2aquero 

F 

Hospital Ramon y Cajal 

The purpose of this study was to ascertain the susceptibility to antifungal agents in a 

collection of strains isolated from transplant patients in our Hospital. A total of 1O8 

strains isolated from 62 transplant patients (26 liver |/iT-, 18 lung |/uT-, 11 bone 

marrow |2mT-, [ Bidney |liT-, 2 intestine |InT- ). The species isolated differed with 

the type of transplant. 7/*+68"#+,& (101 strains) was isolated preferentially in 2mT 

(D5jJ, followed by liT (71_), /uT (6K_), /iT (67_). Figures for 7/*@6+8$+-+ (2J 

strains) wereM /iT (2K_), 2mT (2O_), /uT (12_), and liT (10_). For 7/*'+$+'&"6%&"& 

(11 strains), liT (1[_), /iT(10_), /uT (8_) and 2mT (0 _). fverall, the higher 

incidence of 7/*+68"#+,& was in the respiratory samples, of 7/*@6+8$+-+ in uro-genital 

samples and of 7/*'+$+'&"6%&"& in sterile sites and uro-genital samples. .usceptibility 

testing to amphotericin 2, fluconazole, itraconazole, voriconazole, and caspofungin 

was determined after O8 h using the microdilution reference method (C/.I, document 

M27-A), but our interpretation included the evaluation of strains significantly deviated 

in distributions from the modal value of the more susceptible population. .light 

reduction of turbidity (score J) and prominent decrease in turbidity (score 2) 

corresponded to spectrophotometric inhibition values of 80 and [0_ respectively. 

Amphotericin 2 (score 0, 100 _ inhibition) modal MICs were 0.12 mcg/ml for both 

non-+68"#+,& and 7/*+68"#+,&*(2.K7_ of MIC 0,[ mcg/ml). Fluconazole modal MIC 

value for 7/*+68"#+,& was 0.2[ mcg/ml for both [0-80_ values (iv.), and 1 and t6O 

mcg/ml for non-+68"#+,&. 2.0_ and O.K_ of 7/*+68"#+,& had MICs over O mcg/ml at 

[0-80_ iv., respectivelyn figures were 2K.8_ and O8,K_ for non-+68"#+,&. Itraconazole 

modal MIC was 0.0J mcg/ml for both 7/*+68"#+,& and nonW+68"#+,& for both [0-80_ iv. 

2.K_ and 10.K _ of 7/*+68"#+,& and 11.0 and 12.7 of nonW+68"#+,& had MICs over 

0.2[ at [0-80_ iv. Voriconazole modal MIC was 0.0J mcg/ml for both 7/*+68"#+,& and 

nonW+68"#+,& for both [0-80_ iv. 1.K_ and O.K _ of 7/*+68"#+,& and 0_ and O.2_ of 

nonW+68"#+,& had MICs over 0.2[ at [0-80_ iv. Caspofungin modal MIC shifted for 7/* 

+68"#+,& with different inhibition valuesM 0.12 mcg/ml for [0_, 0.2[ for 80_ and 0.2[- 

0.[ for 100_n modal MICs for nonW+68"#+,& were in all cases 1 mcg/ml. 0_, 1.0_, and 

J.K _ of 7/*+68"#+,& had MICs tO mcg/ml at [0-80-100_ iv. 2.1_, O.2_, O.2_ of non- 

+68"#+,& had caspofungin MICs tO mcg/ml at any iv. level. In summary, susceptibility 

values for 7+,5"5+ species isolated from transplant patients do not differ from what 

was found in non-transplant patients. The proportions of resistance to supra-modal 

values were used as a way of detecting shifts in the evolution of resistance. The 

analysis of the MICs obtained at different inhibition values provided indications about 

the homogeneity or heterogeneity of the fungal population in its susceptibility to the 

antifungals, being amphotericin 2 an example of homogenic and caspofungin (in 7/* 

+68"#+,&) of heterogenic response. 

P-0075. Comparative fluconazole and voriconazole susceptibility profiles of 

Candida species in buccal mucosa and subgingival pockets. 

Finquelievich J 7 , (ewtuchowicz V 1 , 2rusca M 2 , Mujica M J , Gliosca / O , Iovannitti C [ , 

Rosa A 6 

1 Centro de Micologia Facultad de Medicina 62A. Argentina, 2 Catedra de 

Microbiologia. Fac. de fdontologia. 62A. Argentina, J Centro de Micologia Facultad 

de Medicina 62A. Argentina, O Catedra de Microbiologia. Fac. de fdontologia. 62A. 

Argentina, [ Centro de Micologia Facultad de Medicina 62A. Argentina, 6 Catedra de 

Microbiologia. Fac. de fdontologia. 62A. Argentina, 7 Centro de Micologia Facultad 

de Medicina 62A. Argentina 

Although mucosal surfaces represent the primary oral reservoir for yeasts, these 

opportunistic pathogen can also harbour secondary ones liBe the subgingival pocBets. 

The purpose of this study was to compare the distribution and susceptibility to 

fluconazole (flu) and voriconazole (vor) of yeasts*&'.#".& isolated from both buccal 

mucosa (2M) and subgingival pocBets (.P)/** 

2uccal and subgingival samples were obtained from 127 patients with 

gingivoperiodontal disease using sterile cotton swabs and sterile Gracey curettesn 

they were plated onto chromogenic agar (CHRfMagar Company, Paris, France). The 

color of yeasts colonies on these plates, chlamydospore production in 1_ milB- tween 

80 agar and the API ID J2C Bit (bioMdrieux, Marcy /ïEtoile, France) were used for 

yeast identification. Additional tests were used to discriminate between 7/*+68"#+,& y 

7/*5(86",".,&"&/*The antifungal susceptibility study was made by the C/.I (formerly 

NCC/.) MOO-A disB diffusion method. Test plates were automatically read and results 

were recorded with the 2IfMIC Vision Image Analysis .ystem. The statistic analysis 

was made by .TATADI.TICj 7.0, .P.. version 11.0 and Epi Info 6.0O program 

(6niv. Atlanta). 

Results: A total of 60 yeasts species from 2M and [[ from .P were isolated 

according to the following distributionM 7/*+68"#+,&*(O[_n ICK[_M 2,J-[8,J) and O7.1_n 

ICK[_M JJ,[-61,2 respectively) followed by 7/*'+$+'&"6%&"&3*7/*5(86",".,&"&3* 

P?%5%-%$(6+*&''3*7/*-$%'"#+6"&3*7/*@("66.$)%,5""/ K0,K_ (ICK[_ 80-K7) of 2M 7+,5"5+* 

&'.#".& and K1,2_ (ICK[_ 80,O-K7,7)of .P were susceptible to both antifungals. fnly 

one isolated yeast showed different susceptibility profile when we compared between 

2M and .P. 

Conclusions: 7/*+68"#+,& is the most prevalent specie in buccal mucosa and 

subgingival pocBets. .omething important to notice is the increase of*7+,5"5+ no* 

+68"#+,&*&'.#".&3*liBe the emergent species 7/*@("66.$)%,5"" which showed a reduced 

susceptibility to azoles. The distribution of 7+,5"5+*&'.#".& and susceptibility profiles 

to fluconazole and voriconazole showed to be similar and high in both reservoirs. 

These observations could suggest the similarity of 7+,5"5+*&'.#".& from buccal 

mucosa and subgingival pocBet in the same patient. ae consider genotyping studies 

will be the Bey to discriminate between them. 

This investigation was supported in part by project 62ACYT M0JK from the 6niversity 

of 2uenos Aires, Argentina.

P-0076. Antimicrobial effect of the association between ketoconazole and some 

antiseptics against clinical Trychophyton rubrum isolates 

Fischman Gompertz O, 2ori A 

6NIFE.P, .ao Paulo, 2razil 

Dermatophytosis is a very frequent disease in the population. Regarding the limited 

options of therapeutic regimens and the rare studies on drug combination for 

treatment of these fungal diseases, we have evaluated the effect of antimicrobial 

activity for the in vitro association of Betoconazole with three different antiseptics 

(undecylenic, benzoic, and salicylic acids) against J0 clinical T. rubrum isolates. The 

checBboard method was used for this analysis, in microdilution plates, as 

recommended by NCC/. (document MJ8-P, 1KK8). fur results were evaluated on a 

basis of the values obtained for the fractioned inhibitory concentration (FIC) index. fur 

results did not exhibit the expected effect in combining Betoconazole with the assayed 

antiseptics. The association of the antifungal agent with undecylenic acid revealed 

synergistic effect (1/J.JJ_), addition (6/20_), indifference (22/7J.J_), and 

antagonism (1/J.JJ_). 2enzoic acid, in combination with Betoconazole, showed 

synergism (1/J.JJ_), addition (1J/OJ.J_), and indifference (16/[J.J_). The 

association with salicylic acid revealed 100_ of indifference in our assays. fn the 

other hand, undecylenic and benzoic acids lowered the minimum inhibitory 

concentration (MIC) value for Betoconazole by up to O or [ two-fold dilutions, although 

with few synergistic resultsn one two-fold dilution decrease in the MIC value for the 

antifungal agent was observed in the majority of the association assays. The MIC 

values for antiseptics remained unchanged in most of the association assays. 

Increase in one two-fold dilution of MIC value (antagonism) for undecylenic acid was 

observed in only one test. Although synergistic effect has not been verified in most 

associations, combination of drugs would be justified both by a decrease in the 

required concentration to inhibit the fungal growth and by the exfoliative action of the 

antiseptic agents. Future researches, using in vitro (to demonstrate the interaction 

dynamics, as in the Billing curve technique) and in vivo (to evaluate the clinical 

correlation due to the combined action of drugs) assays, will be important to 

complement the performed tests. 

P-0077. Resistance trends: A 5-year reference laboratory experience 

Fothergill A 1 , Rinaldi M 1, 2 , .utton D 1 

1 6niversity of Texas Health .cience Center, 2 Audie /. Murphy Division, .outh Texas 

Veterans Health Care .ystem 

Purpose of Study: aith an ever-increasing focus on environmental effects on health, 

the spot light has been on hsicB buildingi syndrome. The allergic response to 

environmental moulds is well-documented but following a 6l report by aoodcocB, et 

al., we wanted to determine the fungal burden that could be found in bed pillows that 

were collected from a variety of environs within the 6. and that varied by both age 

and content. Pillows tested included two from a retired couple]s home, three from a 

family with two small children, two from a young married couple, two from hotels and 

one from a commercial aircraft. 

Description: Testing was conducted by three methods. The first method involved 

utilizing an Anderson .ampler to draw air through the pillows. The particulate matter 

transported in the suctioned air was deposited onto a .abouraud Dextrose Agar plate. 

The second method was a direct culture of both a 1 cm portion of the outer cover and 

an approximated similar portion of the pillow filler. Finally, a second 1 cm square of 

the outer cover and a portion of the filler were each added to 10 ml of sterile distilled 

water, vortexed well and subcultured to a .abouraud Dextrose Agar plate. 

Results and conclusionM were surprising, not in the volume of fungal contamination 

noted, but by the absence of fungi in many of the samples taBen. .pecies recovered 

were typical of environmental culture including 1.,"#"66"()*spp.3*4&'.$@"66(&*spp.3* 

76+5%&'%$"()*spp.3*and*P?"d%'(&*spp. Results of microbial burden are as followM 

.ample N}10 .terile 2acteria fnly 2acteria/Fungi 

Anderson .ampler [ J 2 

2ulB Culture Cover 0 J 7 

2roth Culture Cover O 6 0 

2ulB Culture Filler O 2 2 

2roth Culture Filler 6 0 O 

In conclusion, we found that the fungal burden of bed pillows from the 6. was not a 

significant source of fungal exposure to those sensitive to fungal allergens. This is 

most liBely due to the bed linens that are cover bed pillows and are repeated changed 

and laundered. 


P-0078. Correlation between antifungal susceptibility and the origin and 

elastase activity in Aspergillus fumigatus 

Garcia M, Cruzado M, Costas E, Alvarez ., /opez Rodas V, Blanco J 

Facultad De Veterinaria. 6CM. 280O0 Madrid. .pain. 

In the last years it has been noticed the increase in the incidence of fungal infections and the 

description of resistant strains in the antifungal susceptibility tests. Then, every day is most 

important the role of the laboratory in the selection and monitoring of the antifungal therapies. 

In the present worB we study the antifungal susceptibility of [[ 4/U()"@+-(& strains, investigating 

its posible relation with its origin (environmental or invasive aspergillosis) and with its elastase 

activity, by the measuring of the Elastase Activity Index (EAI). This is an index previously 

described in our laboratory, and its calculated dividing the elastase activity diameter by the 

growth diameter in a Petri dish with elastin medium described by lothary et al. 

The antifungal susceptibility tests were carried out following the NCC/. methodology (method 

M-J8A) using as reference strain 4/U6+0(&*ATCC 20OJ0O. 

The Minimum Inhibition Concentrations (MICs) obtained with Amphotericin 2 wereM 6 strains 

showed a MIC value of O ng/kl, O strains of 1 ng/kl, and the other isolates of 2 ng/kl. 

To Itraconazole, 16 strains showed a MIC value of 1 ng/kl, and the other JK strains of 0.[ ng/kl. 

To Voriconazole, one strain with a MIC value of ng/kl, one strain with 1 ng/kl, 2O with 0.[ ng/kl, 

and 2K with 0.2[ ng/kl. 

To Posaconazole, 22 strains showed a MIC value of 0.[ ng/kl, 26 strains of 0.2[ ng/kl, and 6 

strains of 0.12[ ng/kl. 

About the Equinocandines Caspofungin and Micafungin, all the isolates showed a Minimum 

Effective Concentration (MEC) value lower than 0.007[ ng/kl. 

In view of the results obtained, Equinocandines showed a higher in vitro antifungal activity than 

the other antifungal compounds tested. Nevertheless, in base of the uniform and low value of 

MEC, with the Equinocandines was not made the study about the relation between antifungal 

susceptibility and origin and elastase activity of the strains. 

In Amphotericin 2, Itraconazole and Posaconazole was detected a correlation with the EAI and 

origin of the strains. In the environmental strains the difference of MICs between isolates with 

EAIt1 and EAIv1 was significatively different that the variation in the MICs to clinical strains. This 

correlation was not detected in the case of Voriconazole. 

P-0079. Expanded cidality studies of voriconazole and caspofungin against 

non-albicans Candida spp. 

Ghannoum M 1 , Dutta . 1 , Matevish ( 1 , Isham N 1 , .heehan D 2 

1 Center for Medical Mycology, 6niv. Hospitals of Cleveland/Case aestern Reserve 

6niv., Cleveland, fH, 6.A, 2 Pfizer, Inc., New YorB, New YorB, 6.A 

Introduction: In certain clinical settings, use of cidal antimicrobial agents has been 

advocated. A common perception is that caspofungin (CA.) exhibits fungicidal 

activity and voriconazole (VfR) fungistatic activity against 7+,5"5+ spp. In this study, 

we challenged this perception by testing the fungicidal activity of VfR and CA. 

against a number of non-+68"#+,& 7+,5"5+*isolates. 

Methods: fne hundred ninety-seven non-+68"#+,& yeast isolates were tested, 

including 7+,5"5+*@6+8$+-+*(OJ)3*C.*'+$+'&"6%&"&*(O0)3*7/*-$%'"#+6"&*(O0), 7/*[$(&."*(JO)3* 

7/*@("66".$)%,5""*(10)3*7/*6"'%62-"#+*(10), 7/*[.U2$*(10), 7/*6(&"-+,"+.*(7), and 7/* 

5(86",".,&"&*(J)/*MIC testing was performed according to the standard C/.I 

microbroth dilution method M27-A2. MFC determinations were performed by 

subculturing the total contents of each clear well from the MIC panel onto potato 

dextrose agar. To prevent antifungal carryover, aliquots were allowed to soaB into the 

agar before streaBing for isolation. Fungicidal activity was defined as a t KK.K_ 

reduction in the number of CF6/ml from the starting inoculum. 

Results: The MIC [0/MIC K0 of VfR were three-fold and one-fold lower than CA., 

respectively. VfR was shown to be cidal against 60_ of 7/*6"'%62-"#+*isolates, 1O_ of 

7/*6(&"-+,"+.*isolates, and 8_ of 7/*'+$+'&"6%&"&*isolates. fn the other hand, CA. 

demonstrated only fungistatic activity against all 7/*@("66".$)%,5""*isolates and [8_ of 

7/*'+$+'&"6%&"&*isolates. Additionally, CA. exhibited fungistatic activity against J[_ of 

7/*-$%'"#+6"&*isolates, 21_ of 7/*@6+8$+-+*isolates, 12_ of 7/*[$(&."*isolates, and 1O_ 

of 7/*6(&"-+,"+.*isolates. 

Discussion: fur data showed that VfR demonstrated more potent inhibitory activity 

than CA. against the non-+68"#+,& 7+,5"5+ tested, and showed cidal activity against 

a few of these strains. Importantly, CA. exhibited fungistatic and not fungicidal 

activity against almost one third of the non-+68"#+,& yeast isolates tested. This 

investigation demonstrates the lacB of universal CA. cidality against non-albicans 

7+,5"5+ and the selective cidality of voriconazole versus some isolates of 7/*6"'%62-"#+3* 

7/*6(&"-+,"+.3 and 7/*'+$+'&"6%&"&. 

In the specific case of Amphotericin 2, the strains with higher virulence show a certain grade of 

resistance to this antifungal compound. This could be due to the fact that the gene codifying to 

the antifungal resistance could increase the virulence of 4/U()"@+-(& decreasing the protective 

response of the host, or by interaction of the host with specific substances secreted by 

4/U()"@+-(& with that gene that increase the antifungal resistance.

P-0080. Treatment of candidemia: Do we really need to spent so much? 

Ghitan M, ChapnicB E, .harma ., Caruso P 

Maimonides Medical Center, 2rooBlyn New YorB, 6.A 

Caspofungin is an echinocandin, antifungal agent approved for the treatment of 

aspergillosis, Candida esophagitis, disseminated candidiasis, and for empiric therapy 

of febrile neutropenia. 

fur 70[-bed teaching hospital in 2rooBlyn NY, purchased ñ [00,000 worth of 

caspofungin in 200[. ae reviewed utilization of antifungal agents and Candida 

species distribution. 

Data was collected from medical records and from the pharmacy database. 

In 200[ there were a total of 2JJ6 Candida isolates. Isolates from blood, urine and 

respiratory secretions were included. 

7+,5"5+*+68"#+,& represented 81_ of isolates (1878 isolates), 7+,5"5+*'+$+'&"6%&"& 

7_ (1[J isolates), 7+,5"5+*-$%'"#+6"& [_ (126 isolates), and 7+,5"5+*@6+8$+-+ 7_ 

(167 isolates). 7+,5"5+*[$(&." (O isolates) and 7+,5"5+*6(&"-+,+. (8 isolates) 

encompased less than one percent of the total. 

ff the patients who received caspofungin, JJ_ had 7/*+68"#+,&, 1[_ 7/*'+$+'&"6%&"& 

and Brusei respectively and 8_ 7/*-$%'"#+6"&. 10_ of the patients treated with 

caspofungin had no Candida species isolated and 1J_ had Candida in the urine 

alone. 

In conclusion, caspofungin use was often unnecessary, since KK.8[_ of Candida 

isolates in our institution are expected to be susceptible to fluconazole. 

P-0081. In vitro activity of BAL4815, a new water-soluble broad spectrum 

triazole, against opportunistic filamentous and dimorphic fungi 

Gonzàlez G 1 , Heep M 2 

1 6niversidad Autínoma de Nuevo /eín, Mexico, 2 2asilea Pharmaceutica /td., 

.witzerland 

Background: 2A/O81[ is a novel triazole antifungal agent with broad-spectrum 

antifungal activity against all major opportunistic and the true pathogenic fungi. The 

water-soluble prodrug 2A/8[[7 was designed for oral (p.o.) and intravenous (i.v.) 

administration. In this study, we compared the activity of 2A/ with amphotericin 2 

(AM2), fluconazole (F/C), itraconazole (ITC), voriconazole (VRC), posaconazole 

(P.C), and ravuconazole (RVC) against J00 opportunistic filamentous and dimorphic 

fungi. Tested were*1&.(5+66.&#?.$"+*8%25"", 1+.#"6%)2#.&*6"6+#",(&, ^(&+$"()*spp., 

Z"'%6+$"&*&'"#"U.$+, 7($0(6+$"+*6(,+-+), 46-.$,+$"+*+6-.$,+-+, a_%'?"+6+*\.+,&.6).", 

P?"d%'(&*+$$?"d(&, !(#%$*#"$#"66.,%"5.&, 48&"5"+*#%$2)8"U.$+, Z6+&-%)2#.& 

5.$)+-"-"5"&, A"&-%'6+&)+*#+'&(6+-(), and 7%##"5"%"5.&*"))"-"&. 

Methods: .usceptibility was determined following NCC/. approved standard 

reference method MJ8-A. Macro broth dilution testing was used with an inoculum of 

0.O ó 10 O to [ ó 10 O CF6/ml, incubated at J0 oC, and read after 2O, O8 and 72 hours 

depending on the rate of fungal growth. 

Results: The newer triazoles were much more active than F/C, and showed 

comparable MIC distributions for most pathogens, with slight species-specific 

variations. Among the zygomycetes P.C, ITC and AM2 showed the lowest, VRC (and 

F/C) the highest MICs, respectively. 

Table of MIC [0ies 

.pecies (N}) 2A/ VRC P.C RVC AM2 ITC F/C 

1&.(5+66.&#?.$"+*8%25""*(28)* 2 2 1 2 2 2 16 

1+.#"6%)2#.&*6"6+#",(&*(22)* 1 1 1 1 O 2 J2 

^(&+$"()*spp (J0)* 8 8 8 8 2 8 t6O 

Z"'%6+$"&*&'"#"U.$+*(J0)* 2 1 1 2 0.[ 2 16 

7($0(6+$"+*6(,+-+ (2O)* 2 1 1 2 1 2 J2 

46-.$,+$"+*+6-.$,+-+ (J0)* 1 0.[ 0.[ 1 1 0.[ 16 

a_%'?"+6+*\.+,&.6)." (12) 0.[ 0.12[ 0.12[ 0.[ 0.12[ 0.2[ O 

P?"d%'(&*+$$?"d(&*(27) 2 2 1 2 0.[ 0.[ 8 

!(#%$*#"$#"66.,%"5.&*(16) O 8 1 O 0.[ 1 16 

48&"5"+*#%$2)8"U.$+*(17) O 8 1 O 0.[ 1 16 

Z6+&-%)2#.& 5.$)+-"-"5"&*(6) 1 1 0.[ 1 0.2[ 0.[ 8 

A"&-%'6+&)+*#+'&(6+-()*(28) 0.[ 0.2[ 0.2[ 0.[ 0.12[ 0.[ O 

7%##"5"%"5.&*"))"-"&* (J0) 0.2[ 0.12[ 0.12[ 0.2[ 0.06 0.12[ 8 

Conclusion: These results confirm the broad spectrum and potential clinical utility of 

2A/O81[ - further clinical studies are warranted. 


P-0082. Susceptibility to antifungal agents of Cryptococcus neoformans: 

Evaluation before and after experimental infection in mice 

GonÑalves da .ilva E 1 , Rodrigues Paula C 1 , de Assis 2aroni F 2 , Emily Cury A J , 

Cesar Viani F 1 , Ereno Auler M 1 , /atercia Tranches Dias A 1 , da .ilva Ruiz / 1 , Emi 

Matsumoto F 1 , Gambale V 1 

1 Institute of 2iomedical .ciences, 6niversity of .Éo Paulo | 6.P | .Éo Paulo, 2razil, 

2 6niversity Federal Rural do Rio de (aneiro | 6FRR( | Rio de (aneiro, 2razil, 

J College of Pharmeceutical .ciences | 6niversity of .Éo Paulo | 6.P | .Éo Paulo, 

2razil 

A total of 60 2A/2/c mice were infected experimentally with 18 environmental strains 

of Cryptococcus neoformans var. grubii. Eighteen strains recovered from the brain 

and lungs of these animals was verified for susceptibility to antifungal agents by the E- 

test Bit according to the manufacture]s instructions, and was evaluated based on the 

results of the MICs for each drug and according to the document (M27 A2) C/.I ( 

formerly NCC/. ). According to the results, before and after the inoculation, the 

susceptibility pattern to amphotericin 2 and fluconazole were similar. 2ut to 

itraconazole and [-fluorocitosine, the susceptibility profile of the strains was bigger 

after inoculation. Although, it is necessary additonal studies to correlate the 

susceptibility profiles to these drugs hin vitroi and hin vivoi because some of the MIC 

values we found were different before and after the experimental inoculation 

P-0083. Once a week micafungin therapy for disseminated candidiasis in 

persistent neutropenia 

Gumbo T, /ouie A, /iu a, 2rown D, Fregeau C, Hsu V, Drusano G 

frdway Research Institute 

Purpose: 6ntreated candidemia is often fatal, especially in the setting of persistent 

neutropenia. Micafungin is a new echinocandin that is being developed for the 

treatment of candidemia, including disseminated candidemia in neutropenic patients. 

.ince echinocandins have concentration dependent microbial Bill (X%(".*.-*+6/* 

4,-")"#$%8/*4@.,-&*7?.)%-?.$/*MQQ5), and micafungin has persistent antifungal effect 

long after the drug has been cleared from the site of infection (9()8%*.-*+63*K7447* 

MQQE), intermittent therapy for candidiasis may be possible. aith intermittent therapy, 

the cumulative weeBly dose is administered as a single dose at the beginning of the 

weeB. This should be made possible by the fact that micafungin is well tolerated by 

patients. In the current study we examined the feasibility of once a weeB therapy in 

neutropenic mice with disseminated candidemia. 

Methods: 7+,5"5+*@6+8$+-+ was cultured from a patient with disseminated 

candidiasis. .usceptibility studies for micafungin were performed using the Clinical 

and /aboratory .tandards Institute macrobroth dilution method. Mice were rendered 

neutropenic (absolute neutrophil countv 100 cells/ml) for the entire duration of the 

study. The mice were intravenously infected with 7+,5"5+*@6+8$+-+ and then treated 

with a single dose of intraperitoneal micafungin between 0 and 100 mg/Bg. Mice were 

sacrificed 7 days after therapy and the Bidney fungal burden determined. A separate 1 

weeB pharmacoBinetic study was performed in which infected neutropenic mice were 

given a single dose of micafungin, and serum and tissue concentrations measured. 

The relationship between fungal burden and pharmacoBinetic parameter values was 

determined using an inhibitory sigmoid E max model by use of ADAPT II software. 

Results: The micafungin MIC of the 7+,5"5+*@6+8$+-+ was 0.02 mg//. Mice tolerated 

all the single intraperitoneal doses of micafungin (up to 100 mg/Bg) without any 

obvious side effects. The Bidney fungal burden at the time of micafungin treatment 

was O.K[ì0.61 log 10 CF6/g. .even days later control mice had a Bidney fungal burden 

of 6.O log 10 CF6/g. Micafungin treated mice had a maximal fungal reduction of [.8 

log 10 CF6/g. In fact, intermittent micafungin completely sterilized day 7 Bidneys in 66_ 

of mice treated with the highest micafungin dose. A total serum A6C 0-) exposure of 

J0K.Kmgòh// (A6C 0-) /MIC}1O OK[) was associated with [0_ of maximal fungal 

reduction, while a total serum A6C 0-) exposure of 106K mgòh// was associated with 

t80_ of maximal fungal reduction. 

Conclusions: .ingle weeBly doses of micafungin are highly efficacious in mice. It has 

been demonstrated that humans tolerate up to 8 mg/Bg/day of intravenous micafungin 

(1%B6.&*.-*+6/*4=A*MQQI). It is also Bnown that micafungin doses of {J mg/Bg produce 

a total serum A6C 0-) of OJ8.0 mgòh// (A".).,d*.-*+6. 4,-")"#$%8/*4@.,-&*7?.)%-?.$/* 

MQQ5). .ince micafungin pharmacoBinetics are linear, we expect a single weeBly dose 

of 8 mg/Bg to achieve human micafungin exposures of 1168 mgòh//, higher than those 

required for t80_ of maximal fungal reduction in mice. Therefore, intermittent 

micafungin therapy should be explored in human patients with disseminated 

candidiasis, especially during persistent neutropenia.

P-0084. In vitro antifungal susceptibilities and enzymatic activities of 

scopulariopsis isolates 

Hryncewicz-Gwozdz A, Plomer-Niezgoda E, 2aran E 

Department of Dermatology, Venereology and Allergology 

.copulariopsis is a genus comprising mainly soil species. These fungi are frequently isolated 

from food, paper, other materials and can occur as laboratory contaminants. .copulariopsis 

species consist, of also dermatomycotic moulds mainly associated with onychomycosis. 

Nevertheless .copulariopsis brevicaulis rarely causes deep and systemic life threatening 

infections. Immunosuppresion, leuBemia, bone marrow transplantation, endocarditis related to 

valvuloplasty, AID. are predisposing factors to severe .copulariopsis infections. Establishing 

optimal treatment of .copulariopsis systemic infections and onychomycosis seems to be an 

important objective. 

The aim of the study was firstly to establish in vitro antifungal susceptibility of .copulariopsis 

brevicaulis and secondly to carry out the analysis of enzymatic activity of examined strains. 

Material and methods: 16 clinical onychomycotic isolates of .copulariopsis brevicaulis were 

evaluated. 

The in vitro antifungal susceptibility of .copulariopsis brevicaulis strains were evaluated by an 

agar diffusion method (Neo.ensitabs, Rosco, DanmarB). 6tilized antifungal agents were as 

fallowsM amphotericin 2, terbinafine, ciclopirox, Itraconazole, fluconazole, Betoconazole, 

clotrimazole, miconazole. 

To establish the enzymatic activity a semi-quantitative API ZYM (bioMerieux) microtest was 

used. 

Results: Cyclopirox and terbinafine have shown to be highly effective. All isolates (100_) were 

susceptible to these drugs. Itraconazole and clotrimazole appeared to be less effective. There 

were [0_ of susceptible, J1_ intermediate, 1K_ resistant strains to itraconazole. 6_ 

susceptible, 88_ intermediate, 6_ resistant strains to clotrimazole. Amphotericin and 

fluconazole were inactive in vitro. Almost all strains were resistant to Betoconazole and 

miconazole except from one intermediate to Betoconazole and 2 intermediate to miconazole. 

In the measurement of enzymatic activity of .. brevicaulis strains the presence of 10 from 1K 

examined hydrolytic enzymes was stated. The highest activity was shown byM phosphatase 

alBaline, phosphatase acid, leucyne arylamidase, naphtol-phosphohydrolase and high activity byM 

esterase lipase, N-acetyl !-glucosamidase, esterase. 

Two .copulariopsis brevicaulis strains (no 10 and 1J), appeared to be most susceptible to 

evaluated drugs, excreted 7 and K enzymes. A sum of activity (in score scale 0 | [) of all 

excreted enzymes by these strains was J1 and JJ score respectively. A sum of activity of all 

excreted enzymes by particular examined strains vary from 26 to J7 with average value of 28 

score. 

Conclusion: Cyclopirox and terbinafine were highly effective against examined strains of 

.copulariopsis brevicaulis. 

Enzymatic activity of the most susceptible strains was on the mean level. 

P-0085. Therapy by oral administration of itraconazole solution against murine 

oral and esophageal candidiasis 

Ishibashi H, 6chida l, Nshiyama Y, Yamaguchi H, Abe . 

TeiByo 6niversity Institute of Medical Mycology, ToByo, (apan 

fral administration of cyclodextrin-associated itraconazole (CD-ITRA) has been clinically 

estimated as a strong therapeutic strategy for oral or esophageal candidiasis. However the 

mechanisms of the therapeutic activity of orally administered CD-ITRA in vivo was very limited. In 

order to find out recommendable intaBe method for CD-ITRA against oral or esophageal 

candidiasis, we examined therapeutic activity of oral or intragastrial dosing of CD-ITRA and 

investigated pharmacoBinetics of ITRA in the therapeutic experiments. 

6sing experimental oral and esophageal candidiasis models of CD-1 mice, we investigated 

efficacy of oral or intragastrial administration of CD-ITRA and checBed concentration of ITRA and 

its metabolite hydroxyitraconazole (fH-ITRA) in tongues or esophagus tissue after 

administration of CD-ITRA. 

Immunosuppressed mice were treatment with prednisolone. Ten minutes before Candida 

infection, the mice were anesthetized by intramuscular injection with chlorpromazine chloride. 

In oral candidiasis model they were orally infected with a cotton swab soaBed in viable cell 

suspension of C.albicans TIMM26O0. In esophageal candidiasis model the anesthetized mice 

were intraesophageally infected with of C. albicans n O x 107 cells/0.2 m/ cell suspension of C. 

albicans was injected into esophageal tube by round-head needle with syringe. 

For microbiological and symptomatic estimation, blood specimens and tongues were obtained 

from the infected mice at the last day in oral candidiasis model. .imilarly, esophagus tubes were 

resected from the mice in an esophageal candidiasis experiment. These mice received CD-ITRA 

by two administration routesM oral and intragastrial. In some experiments, concentration of ITRA 

and fH-ITRA in tongue or esophagus tissues and plasma were measured by HP-/iquid 

Chromatography. 

fral administration of CD-ITRA at doses of 0.8, O.0 or 20 mg/Bg once a day clearly decreased 

the number of viable Candida cells in oral cavity of mice with oral candidiasis in a dose 

dependent manner at J days after infection. Intragastrial administration of CD-ITRA at doses of O 

and 20 mg/Bg once a day was also effective but in a less degree. fral adminstration of CD-ITRA 

also effectively lowered symptom score for tongues of the infected mice. In the case of 

esophageal candidiasis model, oral administration of CD-ITRA displayed superior therapeutic 

efficacy than that of intragastrial one. Corresponding to these therapeutic efficacy, ITRA was 

detected in lesional tissues after oral administration of CD-ITRA. 

fral administration of CD-ITRA displayed superior therapeutic efficacy against murine oral and 

esophageal candidiasis than that of intragastrial one. This superior efficacy can be explained by 

higher concentrations of ITRA in tongues or esophagus tissues after administration of CD-ITRA 

by oral route. Comparison of the pharmacoBinetics of ITRA in bloodflow suggested that ITRA in 

lesional tissues of the mice orally administered CD-ITRA was not delivered through blood flow. 

fn the base of results presented here, we wish to propose the following intaBe method for CD- 

ITRA against these mucosal candidiasis CD-ITRA should be retained as long as possible in oral 

cavity and swallowed slowly little by little. 


P-0086. Antifungal activity of extract from Diospyros canaliculata 

Jean Paul D, (ean Gustave T, David /, Francois-javier E, (ulienne / 

6niversity of Yaounde, Yaounde, Cameroon 

In most African countries many plants are used in the form of extracts, infusions or 

plasters to treat some infections, these plants may offer potential leads for novel 

agents which act against mycoses in the context where the increase of emergences 

fungal infections in immunocompromised patients have created a new challenge in the 

area of antifungal drug therapy. In this study extract from V"%&'2$%&*#+,+6"#(6+-+, 

fractions and compounds isolated from extract were investigated for activity on yeast 

using M the agar well diffusion method for inhibition zones (IZ) determination and the 

macrodilution method for minimal inhibitory concentration MIC and minimal fungicidal 

concentration (MFC) determination. Betoconazole were used as reference antifungal. 

The results showed tremendous antifungal activity of the extract, fractions and 

isolated compounds on almost all the fungi strains tested. IZ range from 17- 

2[mm,MIC and MFC range from 12.[-1.[6mg/ml and J.12-0.78$g/ml respectively. 

Extract was also found non toxic to rats treated '.$*%&n /D[0 was greater than [g/Bg. 

Further investigations should be performed on this plant toconfirm the activityobserved.*** 

P-0087. Antifungal effect of the antimicrobial peptide hLF1-11 on invasive 

Candida albicans infections 

Jonk L 1 , 2rouwer C 1 , aulferinB M 1 , Nibbering P 2 , /upetti A 2 , Velders M 1 

1 AM-Pharma 2V, 2unniB, The Netherlands, 2 Dept. of Infectious Diseases, /6MC, 

/eiden, The Netherlands 

Invasive fungal infections in immunocompromised patients are associated with high 

mortality and morbidity rates. Despite the introduction of new antifungal compounds, 

./@. new generation azoles and echinocandins, these infections represent a significant 

challenge to medicine. A synthetic peptide identical to the N-terminal eleven amino 

acids of human lactoferrin, h/F1-11, has previously been shown to be more effective 

against a wide variety of bacteria and fungi ",*0"-$% than the full length lactoferrin 

protein. Furthermore, ",*0"0%, it was shown to be more than thousand times more 

potent than predicted by the ",*0"-$% data. In several models of systemic and focal 

mycoses and bacterial infections, h/F1-11 showed to be highly effective even at 

doses of O &g/Bg and lower. 

Mechanistic studies showed not only a direct effect on the pathogen but also an 

indirect, immunomodulatory effect on the host]s immune system. Therefore, it was 

surprising that h/F1-11 did not lose efficacy in cyclophosphamide and cyclosporine 

treated mice infected with either 7/*+68"#+,& or methicillin resistant =/*+($.(& (MR.A). 

The hypothesis that h/F1-11 does not only exert its effect on the pathogen but also on 

the host immune system was confirmed by cytoBine analysis of serum from mice that 

were systemically infected with 7/*+68"#+,&. Compared with infected but otherwise 

untreated animals, h/F1-11 at effective doses suppressed TNF! and I/-6 levels, 

whereas at higher doses the level of the anti-inflammatory cytoBine I/-10 was 

increased. The latter coincided with decreased effectiveness of the peptide. Treatment 

of the mice with neutralizing anti-I/10 antibodies restored h/F1-11 potency at higher 

dosages (up to O000 &g/Bg). 

ae conclude that h/F(1-11) is highly effective against disseminated 7/*+68"#+,& 

infections. Its antifungal action involves a direct effect on 7/*+68"#+,& cells as well as 

on cells of the host defense systems. Preclinical and clinical safety studies have been 

completed successfully and clinical proof of concept studies comprising hematopoetic 

stem cell transplantation patients are currently ongoing.

P-0088. Breakthrough Candida parapsilosis fungemia in two hematopoetic 

stem cell transplant (HSCT) recipients receiving long term treatment with 

caspofungin 

Kabbara N 1 , /acroix C 2 , Dromer F J , Robin M 1 , Rochas V 1 , Esperou H 1 , .ocie G 1 , 

Ribaud P 1 

1 AP-HP Hematologie-Greffe de moelle, hopital .t /ouis, 2 Mycologie, hopital .t /ouis, 

J Centre National de Reference des Mycoses et des Antifongiques 

ae report 2 cases of breaBthrough 7/*'+$+'&"6%&"& fungemia in H.CT recipients during 

caspofungin (C) therapy. Patient 1 was a 18 year-old male, diagnosed with severe aplastic 

leuBemia in 1KK8. He received a first unrelated H.CT in (anuary 200O with no sustained 

engraftment. Two months post transplant he developed a definite lung invasive aspergillosis 

successfully treated with voriconazole (V). He received a second unrelated (cord blood) 

transplant on May 12, 200[. V was continued as secondary prophylaxis. 2ecause of liver 

function test abnormalities V was switched to C on day 6 post transplant. He experienced an 

episode of =/*)+6-%'?"6"+ septicemia between days 10-28 which resolved. High fever reappeared 

on day O8 when a blood culture was positive for 7/*'+$+'&"6%&"&. C was discontinued and 

liposomal amphotericin 2 (/A2) was initiated. Due to the patient]s poor condition the CVC was 

not removed. 2lood cultures remained positive until day [J. The patient ultimately died from 

multi-organ failure and no engraftment on day 72 post transplant. .Bin and throat colonization 

with 7/*'+$+'&"6%&"& had been documented from day JK on. Patient 2 was a O6-year old male 

who received a genoidentical H.CT for an acute lymphoblastic leuBemia in first complete 

remission on May 26, 200[. Antifungal prophylaxis consisted in fluconazole (F). C was 

introduced on day 6 post transplant as empirical treatment of fever. Granulocyte recovery 

occurred on day 28. The patient further experienced severe acute graft-versus-host disease 

(GVHD) treated with increased immunosuppression including high dose steroids and 

antithymocyte globulins, and a CMV infection. .Bin, GI and upper respiratory airways 

colonization with 7/*'+$+'&"6%&"& was seen from day JJ on. fn days [8-60, although the patient 

was asymptomatic, blood cultures were positive for 7/*'+$+'&"6%&"&/*C was discontinued and /A2 

and F were initiated. The CVC was replaced. 2lood cultures became negative. However, the 

patient ultimately died from GVHD and respiratory failure of unBnown origin on day 86. 

Table 1 displays the Minimal Inhibitory Concentrations (MICs, kg/ml) of the first blood isolates of 

these 2 patientsM 

Antifungal tested Patient 1 Patient 2 

E-test ® EUCAST E-test ® EUCAST 

Amphotericin 2 0.[ 0.0J 0.12[ 0.0J 

Fluorocytosine 0.7[ 0.[ 0.012 v0.12[ 

Fluconazole 0.[ 2 1.[ 1 

Itraconazole ND 0.06 ND 0.06 

Voriconazole 0.006 0.0J 0.02J 0.0J 

Caspofungin O 0.[ 0.2[ 0.12[ 

* 

7+,5"5+ fungemia rarely occur in our H.CT unit. These 2 cases were the only ones during the 

last 18 months. C MICs of 7/*'+$+'&"6%&"& are Bnown to be slightly higher than those for other 

7+,5"5+ spp., which is in accordance with our susceptibility results. In these deeply 

immunocompromised patients, decrease in susceptibility correlated with clinical failure. 

Documented breaBthrough 7/*'+$+'&"6%&"&*fungemia in patients receiving long term treatment 

with C has not been previously reported. 

P-0089. Time-kill studies show that posaconazole is fungicidal against some 

Candida species 

lardos G 1 , .áczá G 1 , McNicholas P 2 , Falusi E 1 , Hegedôs ( 1 , Majoros L 1 

1 6niversity of Debrecen, Department of Medical Microbiology, 2 .chering-Plough 

Research Institute 

Identification of Candida species is extremly important for the clinical mycologists. ae 

compared the performance of the commercial identification system MICRfNA6T- 

Candida (Merlin DiagnostiBa GmbH) to API IDJ2C (2ioMerieux). MICRfNA6T- 

Candida is based on testing 21 biochemical reactions. Evaluation of the tests are 

automated, results are availale in 2O hours. 

ae tested 128 clinical and reference strains of 18 different yeast species with the 

MICRfNA6T-Candida system including the most common human pathogenic 

Candida species, i.e. C. albicans (1K isolates), C. tropicalis (1O isolates), C. glabrata 

(17 isolates), C. Brusei (2J isolates), C. parapsilosis (1O isolates), C. inconspicua (12 

isolates), as well as less frequently found Candida species C. Befyr (6 isolates), C. 

famata, C. lusitaniae, C. guilliermondii (O isolates each), C lipolytica (J isolates), C. 

dubliniensis, C. rugosa (2 isolates each), C. norvegensis, C. catenulata (1 isolate). 

.ingle isolates of the medically important yeast species, Cryptococcus neoformans, C. 

humicolus, Rhodotorula spp. were also tested. Results of the API IDJ2C system were 

regarded as reference. In case of discrepancies between the two methods, we 

repeated both tests. 

MICRfNA6T-Candida never yielded unsuccesful identification (results of Candida 

spp. or unidentified yeast). Identifications with MICRfNA6T-Candida were the same 

as the API results in K2.2_ (10J/128) of the tests, including all C. albicans, C. 

tropicalis, C. glabrata, C. Brusei, C. parapsilosis, C. inconspicua, C. Befyr, C lipolytica 

and C. lusitaniae isolates, the single C. catenulata, C. neoformans isolates. Three of 

four C. guilliermondii isolates were identified with excellent results, while the remaining 

one (0,7_ of all isolates) repeatedly exhibited questionable identification. fut of the 

consistently identified 10J isolates 16 (12.[_) required the performing of the 

additional tests recommended by MICRfNA6T-Candida, including all 12 C. 

inconspicua isolates, the C. neoformans and one of the C. rugosa isolates. 

Inconsistent results were found in case of 6.J_ (8/128) of the isolates, i.e. the C. 

norvegensis and C. rugosa reference strains, one C. dubliniensis and the C. 

humicolus isolate as well as all four C. famata isolates, which were surprisingly 

identified as C. lusitaniae with ]excellent reliability]. 

ae concluded that MICRfNA6T-Candida and API IDJ2C are equally identify all 

common Candida species, discrepancies could be observed with some of the rarer 

yeasts. Consequently, MICRfNA6T-Candida can be a faster alternative of the widely 

used API IDJ2C identification system. 


P-0090. Luliconazole, a novel topical imidazole: Results of preclinical studies 

Koga H 1 , Nanjoh Y 1 , Inoue l 1 , lasai T 2 , Tsuboi R J , 6chida l O , Yamaguchi H O 

1 Research Center, Nihon NohyaBu Co., /td., fsaBa, (apan, 2 lasai Dermatological 

Clinic, Miyagi, (apan, J ToByo Medical 6niv., ToByo, (apan, O TeiByo 6niv., ToByo, 

(apan 

/uliconazole, (-)-(a)-s(OP)-O-(2,O-dichlorophenyl)-1,J-dithiolan-2-ylideneu(1A-imidazol- 

1-yl) acetonitrile, discovered by Nihon NohyaBu Co., /td. ((apan) was launched on 

(apanese marBet in 200[ as new topical antimycotic drug (trade nameM /6/ICfN w ). 

aith its potent anti-dermatophytotic activity and wide antifungal spectrum, luliconazole 

is useful for the treatment of dermatophytosis and other dermatomycoses. This paper 

presents its ",*0"-$% and ",*0"0% antifungal activities, pharmacoBinetic properties and 

mode of action. 

sAntifungal activitiesu MIC K0 of luliconazole, measured by a standard broth 

microdilution method, was 0.001ug/ml against clinical isolates of >$"#?%'?2-%, spp 

tested. The ",*0"-$%*activity of luliconazole was O to more than 100 times greater than 

that of lanoconazole (MIC K0M 0.00Oug/ml), terbinafine (MIC K0M 0.0Jug/ml) or bifonazole 

(MIC K0M t1ug/ml). In ",*0"0% studies using guinea pig models of tinea pedis and tinea 

corporis, 1_ cream of luliconazole achieved a complete mycological cure even after 

relatively shorter periods of use, when compared with creams containing 1_ 

lanoconazole, terbinafine or bifonazole. sPharmacoBineticsu A high retention of 

luliconazole was observed in the horny layer of guinea pig sBin in comparison with 

terbinafine. The preferable affinity of luliconazole to the horny material was 

demonstrated by ",*0"-$% binding test. sMode of actionu The inhibitory activity of 

luliconazole against ergosterol biosynthesis of fungal cells was greater than that of 

lanoconazole, terbinafine or bifonazole. In addition, the characteristic action of 

luliconazole was observed at sub-MIC doses ",*0"-$%, where luliconazole inhibited 

protease secretion of the fungus. 

P-0091. The inhibitory effect of Saccharomyces boulardii on Candida albicans 

virulence factors 

Krasowska A, Murzyn A, DziadBowiec D 

Institute of Genetics and Microbiology 

=+##?+$%)2#.&*8%(6+$5"" is a yeast strain that has been shown to have applications in 

the prevention and treatment of intestinal infections caused by bacterial pathogens. 

The dimorphic fungus 7+,5"5+*+68"#+,& is one of the most important fungi in 

medicinen it is a member of the normal flora residing in the intestinal and vaginal tract 

of humans. In spite of this, the fungi of this genus can cause both superficial and 

serious systemic disease as well as be the cause of gastrointestinal and vaginal 

infections. In our investigations we demonstrate the inhibitory effect of live 

=+##?+$%)2#.&*8%(6+$5"" cells on the growth of O different 7/*+68"#+,& strainsM two 

clinical isolates and two reference strains at two temperatures J0 and J7 f C. This 

effect is proportional to the amount of =/*8%(6+$5"" added. After 72 hours of incubation 

a re-growth of some 7+,5"5+*strains occurs indicating that for prolonged therapeutic 

effect additional supplementation of*=+##?+$%)2#.&*8%(6+$5""*might be necessary.* 

Addition of live but not heat Billed =+##?+$%)2#.&*8%(6+$5"" cells inhibits the 

filamentation of 7+,5"5+ +68"#+,& strains determined both by incubation at J7 f C in 

rabbit serum for 2 hours and by incubation in YEPD or YN2 medium at J7 f C for 2O 

and 72 hours. =+##?+$%)2#.&*8%(6+$5"" also inhibits adhesion to plastic surfaces by 

7+,5"5+ +68"#+,& strains measured both by crystal violet and jjT based assays in 

K6-well plates. This effect is very strong and requires lower doses of =+##?+$%)2#.&* 

8%(6+$5"" cells than growth and filamentation tests indicating it as a method of choice 

when examining the effect of =+##?+$%)2#.&*8%(6+$5"" conditioned medium and 

purified chemical components on 7+,5"5+*strains. .uch experiments are in progress 

at this moment in our laboratory. 

This worB was financed by 2iocodex, France 

In conclusion, these results indicate that luliconazole, representing a new generation 

of imidazole antifungal agent, has preferable biological features which contribute to its 

potential usefulness as a topical antifungal drug

P-0092. Ajoene: Susceptibility in vitro of Candida isolated from patient with 

onychomycosis and their response to topical therapy 

Lemus E. D 1 , Maniscalchi 2. M 1 , /edezma E 1, 2 , Apitz-Castro R 2 

1 Escuela de Cs. de la .alud. 6niversidad de friente-AnzoZtegui., 2 /aboratorio de 

Trombosis Experimental. Centro de 2iofÜsica y 2ioquÜmica IVIC 

Ajoene is an organosulphur compound obtained of alcoholic extracts from garlic, with 

antifungal activity successfully demonstrated both as ",*0"0% as ",*0"-$%/ In this worB, 

the susceptibility ",*0"-$% of yeasts isolated of eight patient with onychomycosis and 

their response to topical treatment with 0.O_ ajoene solution in six of them were 

determinate. The susceptibility study was performed following guidelines of NCC/. 

M27-A with minor modifications (RPMI plus 2_glucose, use of hematimeter and 

automated reading). The minimum inhibitory concentration (MIC) and the [0_ 

inhibitory concentration (IC [0) of ajoene, fluconazol and terbinafine were determined in 

graphic of optical density (fD) versus drug concentration to 2O hours of growth. 

Ajoene MIC values oscillated among 2.JO - 70.2 kg/ml and its IC [0 values among 0.26 

- 7.08 kg/ml. All the patients treated with ajoene showed, after second month of 

treatment, an important improvement of symptoms, the clinical and mycologic cure 

being reached to the fourth month of treatment in five of them. The sixth patient, in 

spite of having shown a remarBable improvement in the signs and symptoms, until the 

fifth month of treatment didnet reach the mycologic curen however, the patientes 

treatment was continuing with 0.O_ ajoene solution more itraconazol 100 mg/day for 

28 days. ahen the combined treatment was culminated, the patient had complete 

remission of the fingernails with negative mycologic cultivations. This result is, 

probably, the consequence of the existence of a synergic effect among these two 

drugs, that it can be favored by the mechanism of action of the ajoene that involves 

different effects in the molecular environment. In conclusion, the potentiality of use of 

ajoene, liBe a topical agent in treatment of this pathology, was demonstrated. Although, 

the ajoene use liBe an antifungal agent, it has been demonstrated previously, this 

discovery, it could open a new frontier for their topical use, by means of the realization 

of therapies combined with other antifungal agents that allow the formulation of 

outlines with shorter periods of treatment that are surer and effective in the treatment 

of onychomycosis. 

P-0093. In vitro suceptibility of Candida clinical isolates to fluconazole and 

voriconazole by E test method 

Linas M 1 , Cassaing . 1 , 2onnet E 2 , Magnaval ( 1 

1 Dept. of Parasitology and Mycology, Rangueil 6niversity, Toulouse, France, 2 Dept. of 

Infectious Diseases, Purpan 6niversity Hospital, Toulouse, France 

The aim of this study was to compare MICs of fluconazole and voriconazole, and to 

evaluate azole cross-resistance. The antifungal suceptibility of O70 clinical isolates of 

7+,5"5+*species from patients of the Toulouse 6niversity Hospital was tested using E 

test method. 

For 7+,5"5+*+68"#+,&*(1K6 strains) the majority of strains was sensitive to fluconazole, 

and the for same strains the MICs of voriconazole were v 1kg/ml except one strainM 

fluconazole MIC was 8kg/ml and voriconazole MIC was 2 kg/ml. ahen fluconazole 

MIC values were , 6O kg/ml, the range of voriconazole MICs was 0.7[ to J kg/ml. 

For 7+,5"5+*'+$+'&"6%&"& and 7+,5"5+*-$%'"#+6"& all strains sensitive to fluconale were 

inhibited displaying voriconazole MIC values v 1kg/ml. 

For 128 7+,5"5+*@6+8$+-+ strains tested, all strains that were sensitive to fluconazole 

(MIC v 8 kg/ml) were also sensitive to voriconazole (MIC v1 kg/ml). ahen MICs of 

fluconazole were greater than 6O kg /ml, MICs of voriconazole were , Okg/ml about 

[0_ of strains. Moreover, for some strains that were fluconazole suceptible dosedependant 

(.-DD), MICs of voriconazole were , O kg/ml ..o, when MICs to 

fluconazole was t 8 k g/ml,voriconazole ",*0"-$% suceptibility testing was needed 

because potentiel cross resistance was noted for some srains of 7+,5"5+*@6+8$+-+ 

that presented high MIC values for both azoles. 

Key wordsM Ajoene, onychomycosis,*7+,5"5+*&'/3 2roth microdilution susceptibility* 


P-0094. Irons could attenuate antifungal activity of ciclopirox against 

Aspergillus fumigatus: In vitro evidence 

Liu W, aan Z, aang D, /i R 

Department of Dermatology, PeBing 6niversity First Hospital 

Invasive aspergillosis, which is mainly caused by 4&'.$@"66(&*U()"@+-(&, is the most 

common filamentous fungal infection in immunocompromised patients, especially in 

those who has bone marrow transplantation or who is suffered from leuBemia. 

Although several antifungal drugs, including amphterocin 2, itraconazole, voriconazole, 

and caspofungin, have been approved to treat aspergillosis in clinical practice, new 

antifungal drugs with special antifungal action are still being expected. Ciclopirox, a 

hydroxypyridones antifungal drug with broad antifungal spectrum mainly against 

dermatophytes and yeasts, also has a good activity against 4/U()"@+-(&. However, the 

action of ciclopirox is poorly understood so far. In this study, we will present the 

preliminary results about our investigation to the antifungal mechanism of ciclopirox. 

ae used a disB diffusion assay, in which growth of the 4/U()"@+-(& strain AF2KJ was 

examined by plating approximately 10 6 fresh conidia on .aboroud]s agar plus 0.2 M 

Fe 2r r Fe Jr (.DA r Fe). And we also used the .aboroud]s agar (.DA) plates without 

irons to plate the fresh conidia of AF2KJ on as a control. Ninety micrograms (60 kl 

from a 1.[-mg/ml ciclopirox stocB solution) of ciclopirox were placed on a ö-inchdiameter 

paper disB (.chleicher and .chuell, leene, N.H.). The radius of the zone of 

growth inhibition caused by ciclopirox was measured after O8 h of growth at J7ÅC. The 

result showed that addition of irons could attenuate the antifungal activity of ciclopirox 

against 4/U()"@+-(& (Figure). Moreover, we confirmed this phenomenon by further 

using NCC/. microdilution method MJ8-A (data not shown). These findings indicated 

that the antifungal action of ciclopirox would be closely related to the irons. 

P-0095. Standardized disk diffusion testing for fluconazole and voriconazole 

against Candida spp 

Lopes V 1 , Pereira ( 2 , Amorim ( J 

1 Hospital Geral de .anto Antánio, Porto, Portugal, 2 Hospital Geral de .anto Antánio, 

Porto, Portugal, J Hospital Geral de .anto Antánio, Porto, Portugal 

Introduction: ae evaluated the susceptibility of yeasts, isolated in our Hospital, to 

fluconazole and voriconazole by the standardized C/.I disB diffusion method. The 

aim of this study was to analyse the local results from the ARTEMI. DI.l Global 

Antfungal .urveillance .tudy. 

Material and methods: fver a three-year period (200J through 200[) we studied a 

total of J71clinically significant yeasts isolated from all body sites and from all hospital 

locations. ~uality Control was performed with C. parapsilosis ATCC 2201K, C. 

albicans ATCC K0028 and C. tropicalis ATCC 7[0. All samples were tested by the 

C/.I MOO-A disB diffusion method and results were read and recorded with the 

2IfMIC Vision Image Analysis .ystem. The interpretation of the results were based 

on the C/.I MOO-A established breaBpoints for fluconazole and voriconazole. 

Results and discussion: susceptibility test results (_) for each drug are shown in the 

following tableM 

Fluconazole 

Voriconazole 

S SDD R S SDD R 

Total strains (371) 94.3 3,5 2,2 99.7 0,3 0 

C. albicans (160) 100 0 0 100 0 0 

C. parapsilosis (53) 100 0 0 100 0 0 

C. tropicalis (46) 97,8 2,2 0 100 0 0 

C.. glabrata (63) 90,5 6,3 3,2 98,4 1,6 0 

C. krusei (11) 9 36 55 100 0 0 

.usceptibility to fluconazol was very high except for 7/*[$(&.", some 7/*@6+8$+-+ 

strains and some uncommon species of yeasts (7/*,%$0.@.,&"&3*1"#?"+*&''*data not 

shown). All yeasts tested were susceptible (.) to voriconazole except for one 

susceptible-dose-dependant (.DD) 7/*@6+8$+-+ isolate. 

Conclusions: the disB diffusion test is a rapid, low cost and easy to perform method 

that can be used in a routine laboratory. fur results demonstrated that fluconazole 

remains active against most yeasts and, voriconazole appears to be a much more 

potent agent in vitro especially againts non-+68"#+,& species

P-0096. In vitro susceptibility of Cryptococcus neoformans to fluconazole and 

posaconazole 

/opez-fviedo E 1 , Aller A 1 , Claro R 1 , Martin Martin de la Escalera C 1 , Romero A 1 , 

Martos A 1 , Colom F 2 , Martin-Mazuelos E 1 

1 H.6 de Valme, 2 Departamento Microbiologia. F. de Medicina. 6. de Alicante 

Objectives: To study the susceptibility of 7/*,.%U%$)+,& to fluconazole and the new 

azole, posaconazole by broth microdilution method C/.I (M 27-A2). 

Methods: 77 7/*,.%U%$)+,& isolates from HIV patients were studied. The 

susceptibility to fluconazole and posaconazole were performed by the broth 

microdilution method according to the C/.I guidelines (M27-A2 document) modified 

by Ghannou et. al. h.usceptibility testing of Cryptococcus neoformans a microdilution 

techniquei. 1KK2. (. Clin. Microb. J0,288 -2886).. Fluconazole were provided by Pfizer 

and posaconazole by .chering-Plough). Fluconazole was dissolved in sterile distilled 

water and posaconazole in 100_ dimethyl sulfoxide. RPMI supplemented with 2_ 

glucose medium was used for two drugs. Inocula were adjusted to a cell density of 1 

McFarland and microtiter lates were incubated for 72 hours at J[oC (A. I. Aller et al. 

h Correlation of fluconazole MICs with clinical outcome in Cryptococcal infectioni. 

2000. AAC OO, 1[OO-1[O8.). The concentrations range were 0,12-6O mg/l for 

fluconazole and 0,01[-8 mg/l for posaconazole. MIC s values were the lowest drug 

concentration that showed prominent inhibition of growth compared to the control (% 

[0_ inhibition of growth). The MICs ranges and MIC [0 and MIC K0 of all isolates tested 

were determined. The following C/.I M 27-A2 recommended strains, 7+,5"5+* 

'+$+'&"6%&"& ATCC 2201K and 7+,5"5+*[$(&." ATCC 62[8 were tested each time to 

ensure quality control and assessment of reproducibility. 

Results: 

Table1 

RANGE MIC [0 MIC K0 

F/6CfNAZf/E 0.[-J2 O 8 

Pf.ACfNAZf/E 0.2[-1 0.[ 0.[ 

Table 2 ( MICs value of strains with MIC % 16 mg/l to fluconazole)òM 

MICs VA/6E 

.TRAIN. F/6CfNAZf/E Pf.ACfNAZf/E 

1 16 (R) 0.[ 

2 J2 (R) 1 

J J2 (R) 0.[ 

O J2 (R) 0.[ 

[ J2 (R) 0.[ 

ò Arbitrary breaBpoint to posaconazole were chosen (A. I. Aller et al.) 

Conclusions: 

1. All isolates studied showed MICs value to posaconazole ' 1 mg/ml. 

2. No increase was observed in CMIs value to posaconazole of strains wich CMI was 

% 16 mg/l to fluconazole. 

P-0097. Voriconazole as a treatment of fungal mycetoma 

Loulergue P, Hot A, /ortholary f, Dupont 2 

.ervice des Maladies Infectieuses et Tropicales, HÇpital NecBer, Paris, France 

Mycetoma is an infection due to actinomycetes or fungi. It results from the 

implantation of the pathogen in the subcutaneous tissue via a penetrating injury 

(mostly in the foot because pathogens are found in soil), which may secondary infect 

fascia, muscles, joints and bones. This disease is endemic in tropical and subtropical 

regions, especially in aest Africa and India, causing a real public health issue 

because of the chronicity of this suppurative infection. Traditional treatment is surgery 

but suffers a very high rate of recurrence. ae propose to consider a medical treatment 

with voriconazole alone. 

A [[-year-old man from .dndgal, living in France since 1K80 was referred to our 

hospital in april 200[ for a swelling of the right foot evolving for 6 years. He was never 

treated for it, especially never experienced any surgery. The diagnosis of eumycetoma 

was made on several elements including clinical examination with emissions of small 

blacB grains, histology, culture was positive and the final identification of !+5($.66+* 

)2#.-%)+-"& was made by molecular biology (PCR 18. rRNA). The radiological 

evaluation (jrays and MRI) showed no involvement of joints and bones. 

ae decided to start an oral treatment by voriconazole alone, at the dose of 200 mg 

twice a day, in april 200[. The wound has closed and the subcutaneous induration 

has decreased. ae did not notice any adverse effect of the treatment. After 11 months 

of treatment, the patient has no complaint, he is worBing normally and a slight 

induration is persisting. A recent radiological evaluation showed a clear improvement 

of the subcutaneous lesions. ae performed a PETscan which showed a 

hypermetabolism at the site of infection. This examination shows that the fungal 

infection is still active and we decided to continue the voriconazole. 

Treatment of fungal mycetoma did not evolve for many years. To our Bnowledge, 

exclusive antifungal treatment has been tried once. /acroix et al. described recently 

an eumycetoma, also due to !+5($.66+*)2#.-%)+-"&3 treated successfully by 

voriconazole alone. Patient had no recurrence O years after the end of treatment. 

Voriconazole has also been successfully used on other fungus, such as 

=#.5%&'%$"()*+'"%&'.$)()*and*=#.5%&'%$"()*'$%6"U"#+,&/ 

fur case adds one more clinical success of medical treatment, which avoids 

delabrant surgery and functional complications for the patients, and its social 

repercussions. Voriconazole has now to be considered as a drug for developing 

countries n these data should be taBen into account by the pharmaceutical industry. 

Moreover, PETscan may taBe a huge place in infectious diseases, especially fungal 

diseases, for the follow-up of the treatment. Hypermetabolism is the witness of an 

infectious activity and permit to Bnow when to stop the treatment. 


P-0098. Effect of new antifungals on glucosamine-6-phosphate synthase from 

Sporothrix schenckii and on fungal growth. 

López-Romero E 1 , GonzZlez-Ibarra ( 1 , MilewsBi . 2 , Villagámez-Castro ( 1 , Flores- 

Carreán A 1 

1 6niversidad de Guanajuato, Guanajuato, Guanajuato, Mdxico, 2 GdansB 6niversity of 

Technology, GdansB, Poland 

Purpose of the study: To determine the effect of some antifungals on purified 

glucosamine-6-phosphate from ='%$%-?$"_*&#?.,#["" and on fungal growth. 

Description: Disseminated fungal infections in humans represent a serious problem 

in modern chemotherapy due mainly to the rapid increase of immunocompromising 

diseases and to the appearance of multi-drug resistant strains. A commonly used 

fungicide is amphotericin 2, a sterol-targeted polyene macrolide which is highly toxic 

to mammalian cells. fn the other hand, lacB of fungicide activity of fungistatic azole 

derivatives such as fluconazole and voriconazole imposes severe limitations to their 

clinical use. .ynthesis of new antifungals directed against enzymes involved in fungal 

cell wall biosynthesis looBs as a promising tasB. fne such enzyme is glucosamine-6- 

phosphate synthase (G6P.) which catalyzes the limiting reaction of the metabolic 

route leading to 6DP-GlcNAc, a precursor of fungal wall chitin and a vast diversity of 

glycoproteins and other macromolecules. 

Results and conclusions: Here, G6P. was purified to near homogeneity by a 

protocol involving fractionation of a high speed supernatant with protamine sulfate and 

polyethylene glycol and separation of the enzyme by ion exchange and adsorption 

chromatographies in columns of Mono ~ and hydroxyapatite, respectively. .D.- 

PAGE of the purified fraction revealed two polypeptides of K6 and 102 BDa, the former 

being more intense. These results and the analysis of the molecular mass by size 

exclusion chromatography suggest that the native enzyme is a homotetramer of JK0 

BDa. The enzyme was feedbacB down regulated by 1 mM 6DP-GlcNAc to which it 

was more sensitive in crude extracts (8[_ inhibition) than in purified preparations 

([1_ inhibition). Assay in the presence of glucose-6-phosphate increased inhibition of 

the purified enzyme to 7[_ suggesting a cooperative effect between 6DP-GlcNAc 

and the sugar phosphate. G6P. was stimulated up to O[_ by ",*0"-$% phosphorylation 

with a cAMP-dependent protein Binase and ATP. .pecific enzyme inhibitors such as 

FMDP `:-(O-methoxyfumaroyl)-/-2,J-diaminopropanoic 

acid and ADMP, a C2- 

modified derivative of ADGP (2-amino-2-deoxy-D-glucitol-6-phosphate) inhibited the 

purified enzyme with IC [0 values of 1O M and 7O uM, respectively. Corresponding li 

values were [.6 uM and J7.[ uM. These values were significantly higher for enzyme 

crude extracts. ADMP and its lipophilic derivatives `-butanoil-ADGP and `-hexanoil- 

ADGP efficiently inhibited growth of the fungus with MIC values of 0.[ mg/ml, 0.62 

mg/ml and 0.62 mg/ml, respectively. fn the contrary, 1 mM FMDP and its derivatives 

norvaline-FMDP and norvaline-lysine-FMDP inhibited growth to only 11, JK and 20_, 

respectively. 

This worB was financed by .EP-CfNACyT, Mexico, Project JK[28-~. 

P-0099. The prophylaxis of foot and nail mycosis in footwear users 

Macura A 1 , FalBiewicz-DuliB M 2 , PawliB 2 1 

1 Dep.of Mycology, Chair of Microbiology, lraBow, Poland, 2 Central /aboratory of 

Footwear Industry in lraBáw, Poland 

Toe nail mycosis amounts to about JJ_ of all fungal sBin infections, and its estimated 

prevalence in the general population is about J-8_. The relapses of foot and particularly 

nail mycoses are still an essential therapeutical problem, despite new effective antifungal 

medications. Effective footwear disinfecting is an important measure supplementing the 

treatment of foot and nail mycoses caused by dermatophytes because those fungi may 

survive in infected shoes for a long time. A lacB of proper footwear disinfecting is a 

common cause of therapeutical failures because of reinfections from infected footwear. 

The objective of the study was: (1) checBing whether or not the materials used in 

footwear production are conducive to fungal growth, (2) evaluation of the antifungal 

effectiveness of six chemicalsM .anitized I sPhenol, [-chloro-2-(2,Odichlorophenoxy)-2- 

octyl-2H-isothiazoline-Jon, 2-methyl-2,O-pentanodiolu, .anitized III sDimethyltetradecyl(J- 

(trimethoxysilyl)propyl)ammonium chlorideu, Vantocil poly(hexamethylenebiguanide) 

hydrochlorideu, Proxel s1,2-benisothiazolin-J-one r NafHu, NP.-100 snanoparticles of 

AgNf J u, Vanquish 100 sN-butyl-1,2-benisothiazolin-J-oneu against yeast-liBe fungi, moulds, 

and dermatophytes. A slide dilution method was used. (J) evaluation of the antifungal 

effectiveness of deodorant containing [-chloro(2,O-dichlophenoxy)phenol and Tea tree oil. 

Materials & Methods: A disB-diffusion method was used to evaluate the survival rate of 

).,-+@$%'?2-.&3*a'"5.$)%'?2-%,*U6%##%&()3*7+,5"5+*+68"#+,& and =#%'(6+$"%'&"&* 

8$.0"#+(6"&*during five weeB observation of 27 materials of various chemical composition 

and structure used in footwear production. The effectiveness of six chemical compounds 

was tested using a dilution method while the deodorants were evaluated using a disBdiffusion 

method. 

Results: The fungi survived on all of the footwear materials under study, the most 

abundant growth was observed with 7/*+68"#+,&*on natural materials (leather, cotton) and 

synthetic materials. 

The antifungal activity (MIC values) of the particular chemicals tested against the fungi 

under study varied within a range from 1 to over 10,000 mg/l. Vanquish-100, .anitized I 

and Proxel turned out to be most effective against all of the fungal strains, however, 

Vanquish-100, Vantocil, Proxel and .anitized III were most active against dermatophytes. It 

appears that the chemicals tested may be applied as footwear disinfectants and/or 

sanitising agents. The tested deodorant turned out to be effective against fungi, and may 

be recommended as footwear deodorant that prevents foot and toenail reinfections. 

ConclusionsM 

1. Materials used in footwear production are conducive to fungal 

growth. 

2. To inhibit fungal growth in the footwear materials they should be 

pretreated with antifungals. 

Antifungal footwear deodorants should be used in the prophylaxis of foot and toenail 

mycoses.

P-0100. Clinical arthoconidia of Trichophyton rubrum present more resistance 

to terbinafine than culture microconidia 

Maffei C 1 , Taglialegna R 2 

1 Faculdade de Medicina de Ribeirao Preto-6.P- .ao Paulo-2razil, 2 Faculdade de 

Medicina de Alfenas-Minas Gerais-2razil 

fnichomycosis are caused by several fungal species, but K0_ of them are due to 

dermatophytes, mainly >$"#?%'?2-%,*$(8$(). Terbinafine is an antifungal with good 

concentration in nails, fungicidal when tested ",*0"-$% against this fungi, although 

therapeutic use is not always successful in clinical practice. The aim of this study was 

to compare the susceptibility to terbinafine of arthroconidia obtained directly from nails 

of patients with onichomycosis and its correspondent microconidia obtained from ",* 

0"-$% cultures. In order for obtain the arthroconidia we opened a hole through the 

ungueal plate at the most proximal level of the fungal invasion and scales were 

collected in a sterile vial. This tissue was digested in trypsin solution 2_. A portion 

was plated on .DA to isolate dermathophyte colonies and subcultured on oatmeal 

agar for 7 days to obtain microconidia. The rest was used to prepare the clinical 

inoculum. The inocula from clinical or culture samples were made in RPMI-16O0 

medium to generate J,[ x 10 O CF6 and a 1M10 dilution, checBed after plating on .DA. 

The susceptibility tests were performed in K6 wells plates, according to the NCC/. 

MJ8-A protocol recommendations. The final terbinafine concentrations ranged from 

0,2[ to 0,000[kg/ml, in two-fold dilutions. After 7 days incubation at J[ o C, we 

considered the concentration that showed 100_ visual inhibition as MIC. MFC was 

determined when no growth of colonies was observed after plating all the volume of 

each well on .DA, incubated for 10 days at J[ o C. ae could isolate >$"#?%'?2-%,* 

$(8$() and obtain adequate number of arthroconidia from 16 patients. fnly MFC was 

determined for clinical inocula because the presence of Beratinocytes debris produced 

interference with the end point of MIC. The results for the 10 O and 10 J inocula dilutions 

were respectivelyM clinical MFC (geometric mean } 0,12K and 0,0[J, range } 0,12[ | 

0,[ and 0,0J1 | 0,[), microconidia MFC (geometric mean } 0,0J7 and 0,02, range } 

0,01[ - 0,12[ and 0,01[ - 0,0J1) and microconidia MIC (geometric mean } 0,011 and 

0,01, range } 0,008 - 0,01[ and 0,008 - 0,01[). ae observed that microconidia of >/* 

$(8$() are extremely sensitive to terbinafine, liBe the majority of authors did, but 

clinical arthroconidia showed a differential resistance to terbinafine, even when 

adjusted for the factor number of CF6 in test inocula. The difference was more 

pronounced when the 10 O inocula was used. ae concluded that arthroconidia rescued 

directly from nails have a distinct behavior in comparison with their ",*0"-$% counterpart 

microconidia. There are some possibilities to explain these findingsM a) metabolism or 

permeability to terbinafine is different between arthro and microconidian b) 

arthroconidia could also be in a state of dormancy that protects them from 

environment adversities. fur results suggest that, independently of bioavailability of 

terbinafine in nail, the increased resistance of arthroconidia could explain part of 

therapeutic failures in treatment of onychomycosis. 

P-0101. In vitro anti-Candidal activity of some Iranian honey 

Mahdavi Omran S, .efidgar ., Maliji G, Youssefi M, Mosavi . 

2abol 6niversity of Medical .ciences, 2abol, Iran 

Purpose: Candidiasis is the most fungal disease between all countries. Patients can 

be cure by the use of synthetic anti-fungal drugs, but some times there may be seen 

resistance to treatment. Many of studies showed that honey has anti-microbial and 

anti-fungal activity against to some pathogenic fungi. In this study we assessed the 

effect of some Iranian honey on Candida species. 

Methods: ae used O different types of Iranian honey from Mazandaran (north of Iran). 

The anti-Candidal activity of K dilution (J0-60_ volume /volume) of honey (new or old 

unheated and heated) were studied against to 7+,5"5+*+68"#+,& (2 clinical isolates) 

and Candida dubliniensis (1 clinical isolate) by using of micro dilution method. The K6 

well micro plates were incubated at 2 temperatures (2[ co and J[ oc ) with shaBing (200 

rpm) for O8 hours. Then 10 of each solution well cultured on .DA media with a layer of 

agarose (0.6_) for colony count at J[ oc for O8 hours. 

Results: The results of cultures showed that honey can inhibits or suppress growth of 

Candida species in specific concentrations. The anti-Candidal activity of new 

unheated honey for 7/*+68"#+,& and C. dubliniensis was MICM J0_ v/v, MFCM [6_ v/v 

and MICM J7_, MFCM O[_, respectively. The heated honey was affected on*7/* 

+68"#+,& and C. dubliniensis according to the following data, respectivelyM MICM J7_ 

v/v, MFCM [6_ v/v and MICM J7_, MFCM O[_. The old honey had almost the same 

effect of the new honey and these were the same at2[ co and J[ oc . 

Conclusion: According to results of the present study honey can inhibits Candida 

species growth experimentally and this activity related to the type of it. Heating of 

honey in processing is the cause of decreasing anti-Candidal activity it]s. .o we 

recommend that it]s better to use new unheated honey for research. 


P-0102. Therapeutic potentialities of Ajoene in the treatment of Tinea capitis. 

Preliminary results. 

Maniscalchi Badaoui M 1 , /emus Espinoza D 1 , /edezma E 1, 2 , Apitz-Castro R 2 

1 Escuela de Cs. de la .alud. 6niversidad de friente-AnzoZtegui. Venezuela., 

2 /aboratorio de Trombosis Experimental. Centro de 2iofÜsica y 2ioquÜmica IVIC. 

Venezuela. 

>",.+*#+'"-"& is mainly superficial mycoses that affects to children. The treatment of 

choice is systemic, that which could induce appearance of adverse effects of variable 

graveness according to the individual and the most frequent causal species, 

!"#$%&'%$()*#+,"&3 is less susceptible to the available antifungals, being needed in 

many times to prolong the therapy. Ajoene offers to be an important topical option, 

suggesting it as an alternative treatment in this dermatophytosis. 

Aim: To evaluate the therapeutic potentialities of ajoene, both as ",*0"-$% as ",*0"0%, in 

the treatment of >",.+*#+'"-"&. 

Methodology: The susceptibility ",*0"-$% of !/*#+,"& strains isolated from six patients 

with >",.+*#+'"-"& (which three received treatment with 0.O_ ajoene topic twice a day 

for O[ days) to ajoene, griseofulvin and terbinafine was tested by broth microdilution 

method following guidelines of the NCC/. MJ8-A, with minor modifications (RPMI 

plus 2_ glucose, use of hematimeter and automated reading). The minimum inhibitory 

concentration (MIC) was determined in graphic of optical density (fD) versus drug 

concentration to K days of growth (maximum exponential growth)n the [0_ inhibitory 

concentration (IC [0 ) was concentration that produced [0_ of the maximum inhibitory 

effect. 

P-0103. In vitro activity of BAL 4815 (a new azole) against filamentous fungi. 

Martin de la Escalera C 1 , Aller A 1 , /ápez-fviedo E 1 , Martos A 1 , Romero A 1 , Castro C 1 , 

Canton E 2 , Martin-Mazuelos E 1 

1 Hospital 6niversitario de Valme, 2 Hospital /a Fe, Valencia .pain 

Objectves: To study the ",*0"-$% activity of 2A/- O81[ (2asilea Pharmaceutica /td) a 

new antifungal agent against filamentous fungi. 2A/ O81[ is the active component of 

the antifungal triazol agent 2A/ -8[[7 (the water soluble prodrug, a.A). 

Methods: The ",*0"-$% activity against 72 clinical isolates of filamentous fungiM [0 

strains of 4&'.$@"66(&*&''*( 1K 4/*-.$$.(&, 12 4/*U6+0(&, 1J 4/*U()"@+-(&, J 4/*@6+(#(&, M* 

4/*,"@.$, 1 4/*,"5(6+,& ), O strains of P?"d%'(&*&'', J !(#%$*&'', [ ^(&+$"()*&'', J*=/* 

'$%6"U"#+,&, [ =. +'"%&'.$)(, and 2 4#$.)%,"(,*&''. was determined using the broth 

microdilution method in accordance with C/.I MJ8-A guidelines. Antifungal agent was 

prepared using dimethyl sulphoxide (DM.f) as solvent and final drugs ranges were 

0.01[- 8 kg/ml. .tocBs inoculum suspensions were prepared from [-7 days old culture 

grown on potatoe dextrose agar (Difco). Final inoculum ranged from 0.[x10 O to [x10 O 

CF6/ ml. The MIC endpoints were evaluated for the lowest drug concentration that 

prevented 80_ growth at 2O h. and O8 h. of incubation at J7oC .Minimal fungicidal 

concentrations (MFCs) were also determined for the isolates with MICs endpoints 

lower than 8 kg/ml. 7/*[$(&."*ATCC 62[8, 7/'+$+'&"6%&"& ATCC 2201K, 4 U6+0(& 

ATCC 20OJ0O and 4/*U()"@+-(&*ATCC 20OJ0[ were included as ~uality Control 

.trains. 

Results continued on next page 

Results: The MICs of ajoene oscillated among J0 at J00kM (percentage of inhibition 

average K[_ ì 1.O1_) and mean IC [0 was 2.16 ì 0.6JkM. The MIC and IC [0 of 

terbinafine were smaller (MIC 6.[ ì J.[kMn IC [0 0.08 ì 0.0[kM) with percentages of 

inhibition average of 88.67_ ì O.8K_n MIC for griseofulvin was among 10 to J0kM 

(with 71.[0_ ì 1J.17_ of inhibition) and IC [0 was among 1.08 at J.K1kM. In all of the 

three patients treated with ajoene their signs and symptoms improved quicBly and 

mycologic cure was achieved in O[ days of therapy. 

Conclusions: Ajoene showed a potent activity antiproliferative and fungicide against 

!/*#+,"& isolated, that it corresponded with the response in patients treated with 0.O_ 

ajoene topic.

Results: 

.pecies. 

GM a Range (kg/ml) MIC [0 (kg/ml) b MIC K0 (kg/ml) b 

(no of isolates) 

P?"d%'(&*&''* 2 1 - O õ õ 

(O) 

!(#%$*&''* 2 2 õ õ 

(J) 

^(&+$"()* 1J,K288 2 - t8 õ õ 

([) 

=/'$%6"U"#+,&* 8 2- t8 õ õ 

(J) 

=/*+'"%&'.$)(,* K,[1J7 2 - t8 õ õ 

([) 

4#$.)%,"() spp 16 t8 õ õ 

(2) 

4/*-.$$.(&* 0,O821 0,12[- 2 0,[ 1 

(1K) 

4/*U6+0(&* 1,2867 0,[- 2 1 2 

(12) 

4/*U()"@+-(&* 1 0,12[- 2 1 2 

(1J) 

4/*@6+(#(&* 1,2[KK 1- 2 õ õ 

(J) 

4/*,"@.$* 

2 2 õ õ 

(2) 

4/*,"5(6+,&* O O õ õ 

(1) 

TfTA/ 1,[216 0.12[- t8 1 O 

a In calculation of the GM values MICs of t8 were classed as 16 kg/ml. 

b MICs values were read at O8 h. except !(#%$+6.& which were read at 2O h. 

because these showed overgrowth at O8 h. 

The MFCs values were ì1 dilution of the MICs values so this new antifungal agent is 

considered fungicidal against filamentous fungi studied. 

Conclusions: 

1. 2A/ O81[ showed good inhibitory and fungicidal activity against Aspergillus 

spp and Mucorales spp (MICs úO). 

2. A .terreus was more susceptible than the other species of Aspergillus. 

J. Fusarium spp, .cedosporium spp, Acremonium spp, showed MICs of 2- 

t8kg/ml. 

Additionals studies, with a higher number of strains and correlation with in-vivo 

outcome are necessary to establish the clinical value of this new azol 

ANTIFUNGALS* 

P-0104. Comparison of the in vitro activity of BAL 4815 and fluconazole against 

Cryptococcus neoformans 

Martin de la Escalera C, Aller A, /ápez E, Martos A, Romero A, Castro C, MartÜn- 

Mazuelos E 

Hospital 6niversitario de Valme. .ervice of Microbiology..evilla 

Objectives: To study the ",*0"-$% activity of 2A/O81[ (2asilea Pharmaceutica /td) a 

new antifungal agent and Fluconazole (Pfizer Central Research, .andwich, united 

lingdom) against 7$2'-%#%##(&*,.%U%$)+,&. 2A/O81[ is the active component of the 

antifungal triazole agent 2A/8[[7 (the water soluble prodrug a.A). 

Methods: The ",*0"-$% activity of a.A and fluconazole against 81 clinical isolates of 

7$2'-%#%#(& ,.%U%$)+,& was determined using the broth microdilution method in 

accordance with C/.I M27-A2 guidelines modified by Ghannoum et al (h.usceptibility 

testing of 7$2'-%#%##(&*,.%U%$)+,&i a microdilution techniquei. 1KK2. (. Clin. Microb. 

J0, 2881-2886). Dimethyl sulphoxide (DM.f) was used as solvent for a.A and 

fluconazole was dissolved in sterile distilled water, final drugs ranges were 0.01[- 

8kg/ml for 2A/ and 0.12- 6Okg/ml for fluconazole. .tocBs inoculum suspensions were 

prepared from O8 hours old culture grown on .abouraud agar (fxoid./TD.). Final 

inoculum size was approximately 10 O CF6/ ml. The MICs endpoints were evaluated for 

the lowest drug concentration that showed prominent decrease in turbidity at O8 h. and 

72 h. of incubation at J7oC. 7/*[$(&."*ATCC 62[8, 7*/'+$+'&"6%&"& ATCC 2201K, 

7*/,.%U%$)+,& ATCC K0112 and*ATCC K011J were included as quality control strains. 

Results: 

ANTIFUNGAL AGENT 

Range (&g/ml) MIC 50 a ( &g/ml) MIC 90 a ( &g/ml) 

a.A 0,0J - O 0,[ 1 

F/6CfNAZf/E 0,[ - J2 O 8 

a MICs values were read at 72 h. 

Conclusions: 

1. a.A showed good activity against 7$2'-%#%##(& ,.%U%$)+,& (MICs 'O 

&g/ml). fnly 1 strain had an MIC} of O &g/ml. 

Additional studies, with a higher number of strains and in relation to the clinical 

outcome, are necessary to establish the clinical usefulness of this new azole. 


P-0105. The molecular basis and modulation of antifungal drug resistance 

mediated by ABC efflux pumps in the pathogenic yeasts Candida albicans and 

Candida dubliniensis 

Melkusova S 1 , 2ujdaBova H 1 , luchler l 2 

1 Comenius 6niversity, Faculty of Natural .ciences, Department of Microbiology and 

Virology, MlynsBZ dolina 2-2, 8O2 1[ 2ratislava, .lovaB Republic, 2 Medical 6niversity 

Vienna, Max F. Perutz /aboratories, Department of Medical 2iochemistry, Division of 

Molecular Genetics, Dr. 2ohr-Gasse K/2, A-10J0 Vienna, Austria 

Objectives: The worB aimed to determine the cause of resistance mediated by 

expression of multidrug A2C transporters in clinical isolates of 7+,5"5+ +68"#+,& 

and 7+,5"5+ 5(86",".,&"&. Next objective focused on comparison of immunoblotting 

method with measurement of rhodamine G6 efflux for detection of Cdr1p expression. 

Methods: For this research, 20 fluconazole resistant 7/*+68"#+,&*and 7/*5(86",".,&"&* 

strains were selected. The collection was completed by two fluconazole-susceptible 7. 

+68"#+,& and 8 fluconazole-susceptible 7. 5(86",".,&"& clinical isolates. These isolates 

were obtained from patients with diabetes, different allergy and cancer diseases and 

oropharyngeal candidiasis of HIV infected patients from .lovaBia, 2razil, (apan and 

Thailand. .usceptibility to fluconazole and other antifungal drugs (conventional azole 

derivative itraconazole, the new azole voriconazole, polyene amphotericin 2 and the 

experimental antifungal agent 6-amino-2-,-pentylthiobenzothiazole (AP2) was tested 

employing the broth microdilution method according to NCC/. M27-A reference 

method. The overexpression of A2C transporters was established by isolation of 

proteins using trichloroacetic acid and screening by immunoblotting using specific 

antibodies against Cdr1p and Cdr2p transporters. ~uantification of the expression of 

these proteins employed the fluorescence dye rhodamine 6G. 

Results: 6sing immunoblotting method, the presence of Cdr1 protein in all 7. +68"#+,& 

clinical isolates was established. KO_ of 7. +68"#+,& isolates expressed only low levels 

of this protein, while 1 clinical isolate showed higher amount of Cdr1p. The influence of 

subinhibitory concentrations of fluconazole of the expression of Cdr1p was significant 

only in O fluconazole-resistant 7. +68"#+,& isolates. In 7. 5(86",".,&"& clinical isolates the 

presence of Cdr1p was detected in K clinical isolates. However, only 6 clinical isolates 

expressed a full-length 170-BDa protein. A truncated, perhaps nonfunctional form of 

Cdr1p with the molecular weight 8[-BDa was observed in J 7. 5(86",".,&"& isolates. The 

second transporter was detected in none of 18 7. +68"#+,& clinical isolates. In 7. 

5(86",".,&"&, the Cdr2p was detected in 2 isolatesn however the expression of this 

protein was strongly positive influenced by adding of subinhibitory concentrations of 

fluconazole to the medium. Additionally, a correlation between rhodamine 6G efflux 

assay and results obtained by immunoblotting was observed. Differences between 

individual clinical isolates were observed not only in rhodamine efflux, but also by 

different uptaBe of rhodamine 6G to the cells. 

P-0106. Susceptibility of Sporothrix schenckii to five antifungal drugs 

Mesa A 1 , 2ueno ( 1 , Vivot a 2 , Montiel ( 1 , Cárdoba . 2 , Davel G 2 , Rodero / 2 


ParasitologÜa, Grupo Infeccián y CZncer, 2 Departamento de MicologÜa, INEI.AN/I., 

ÄCarlos G. MalbrZnÄ, 2uenos Aires, Argentina 

Purpose: To evaluate ",*0"-$% susceptibility of clinical isolates of =/*&#?.,#["" 

(obtained from fixed or lymphangitic forms) to itraconazole (ITC), anfotericine 2 

(AM2), voriconazole (VRC), fluconazole (F/C) and terbinafine (TR2), using the 

protocol C/.I MJ8-A for filamentous fungi with some modifications. 

Summarized description of the project: .porotrichosis is a subacute or chronic 

infection affecting animals and humans, generally characerized, by fixed or 

lymphocutaneous lesions. The fungus ='%$%-?$"_*&#?.,#["" is the causing agent. 

.porotrichosis has a world wide distribution but is more frequent in African and /atin 

American countries. Treatment of sporotrichosis depends on the clinical form of 

infection and on aspects liBe the patientes characteristics, administration easiness and 

cost of the treatment. Few worBs have been done in order to evaluate the ",*0"-$% 

susceptibility of =/*&#?.,#["" to different antifungals. 

The MIC (Minimum Inhibitory Concentration) was determined for AM2 (.igma), F/C 

(Pfizer, Argentina), VRC (Pfizer, Argentina), ITC ((anssen, Argentina) in O2 isolates of 

=/*&#?.,#["" from Colombia. TR2 was evaluated (Novartis) in 18 isolates. 7+,5"5+* 

[$(&." ATCC 62[8 and 7+,5"5+*'+$+'&"6%&"& 2201K were used as control strains. The 

size of the inoculum was 0.O - [ x10 O CF6/m/. In total 100 k/ of each inocula were 

dispensed into K6 wells plates with 100 k/ of antifungal concentrations (ranging from 

16 kg/m/ to 0.0J kg/m/ for AM2, ITC and VRCn 128 kg/m/ to 0,2[ kg/m/ for F/Cn 

and 16 at 0.0J for TR2). Plates were incubated at 28 ÅC and MICs visually determined 

after 72 h, comparing growth in the control well against inoculated ones. Viability of 

the inoculum was confirmed by determination of CF6 on .abouraud plates. 

Results and Conclusions: The isolates of =/*&#?.,#["" exhibited growth limitation at 

J[ oC. For this reason the test was carried out at 28 oC, introducing a modification to 

MJ8-A protocol. This behavior is in agreement with previous reports on isolates from 

Colombia, which showed higher thermal sensitivity when compared with others from 

Guatemala and Mexico. MICs ranged as followsM VRC 1 - , 16 kg/m/, ITC 0.0J - ,16 

kg/m/, AM2 1 - ,16 kg/m/, F/C ,128 kg/m/ and TR2 0.1J - 1kg/m/. fbserved MICs 

were high for fluconazole, variable for AM2, VRC and F/C, and low for ITC and AR2. 

fur results are in agreement with those reported by many authors, showing variable 

sensitivity to AM2, low activity to F/C and high resistance to VRC. Major in vitro 

efficacy against =/*&#?.,#["" has been reported with ITC and TR2. This correlates well 

with treatments response in localized clinical forms. 

Conclusion: Results suggested a high variability in expression of A2C transporters 

and showed that a simple method for measuring of the rhodamine 6G efflux could be 

useful for a rapid detection of the Cdr1p expression.

P-0107. In vitro antifungal activity of essential oils of lippia genus plants 

from Colombia 

Mesa Arango A 1 , Montiel ( 1 , .uZrez C 1 , .tashenBo E 2 , DurZn C 2 , 2ueno ( 1 


ParasitologÜa, Grupo Infeccián y CZncer, MedellÜn, Colombia, 2 6niversidad Industrial 

de .antander, /aboratorio de CromatografÜa, Escuela de ~uÜmica, 2ucaramanga, 

.antander 

Purpose: To evaluate the anti-7+,5"5+ and anti-4&'.$@"66(& activity of essentials oils 

obtained from X"''"+*genus plants collected in different regions of Colombia. 

Summarized description of the project: 7+,5"5+ and 4&'.$@"66(& species are 

responsible of most fungal infections. .everal drugs for their treatment are currently 

availablen however, toxicity and treatment resistance observed with some of these 

drugs, validate the search of natural products with antifungal activity from different 

sources. Many plants produce secondary antifungal metabolites used as defense 

against the attacB of pathogen fungi. These metabolites have shown ",*0"-$% activity 

against human pathogen fungi/*X"''"+ genus plants are widely used in many 

communities and are important sources of oils, some of them with demonstrated 

antifungal activity. Therefore, species of this genus may be considered as promissory 

sources of antifungal compounds. 

For antifungal activity evaluation, minimum inhibitory concentration (MIC) was 

measured in twelve X"''"+*+68+ (10) and X/*%$"@+,%"5.& (2) oils, obtained by 

hydrodestilation. For each oil ten decreasing concentrations were used and processed 

following AF.T-E6CA.T and C/.I-MJ8-A standard protocols, with 7/*'+$+'&"6%&"& 

ATCC 2201K, 7/*[$(&." ATCC 62[8, 4&'.$@"66(&*U6+0(& ATCC 20OJ0O and 4&'.$@"66(&* 

U()"@+-(& ATCC 20OJ0[ strains. In all cases the highest oil concentration assayed 

was 2_ (v/v), and dimethylsulfoxide used as solvent. fils were evaluated by triplicate 

in three independent experiments. In addition, 7+,5"5+ strains were cultured with 

itraconazole, ITZ (.igma), and used as control. 

Results and Conclusions: MICs ranged as followsM 0.01[6 - 0.[_ in 7/*'+$+'&"6%&"& 

ATCC 2201Kn 0.2[ - 0.01[6_ in 7/*[$(&." ATCC 62[8n 0.[ - 0.01[6_ in 4/*U6+0(& 

ATCC 20OJ0On and 0.00JK - 0.01[6_ in 4/*U()"@+-(& ATCC 20OJ0[. MICs with ITZ 

ranged between 0.062[ - 0.2[ and 0.062[ - 0.12[0 in 7/*'+$+'&"6%&"& and 7/*[$(&.", 

respectivelyn and 0.2[ in 4/*U6+0(& and 0.062[-0.2[ in*4/*U()"@+-(&. Essential oils of 

X"''"+ genus plants showed ",*0"-$% antifungal activity against strains of 7+,5"5+ and 

4&'.$@"66(& similar to the one exhibited by commercial antifungals and !.6+6.(#+* 

+6-.$,"U%6"+*oil (component of antifungal drugs). 

Acknowledgments: This worB was supported mainly by grant Cf/CIENCIA. RC- 

OJ2-200O. ae thanB to F6NDACfFAN for identification and recollection of plants 

species. 

P-0108. Application of MICRONAUT-AM system for in vitro susceptibility 

testing of yeasts 

Mnichowska-Polanowska M 1 , Nenoff P 2 

1 Dept Microbiology and Immunology, Pomeranian Medical 6niversity, 2 /aboratorium 

fyr medizinische MiBrobiologie, Mélbis, Germany, 

Introduction: Interpretation of ",*0"-$% yeasts susceptibility testing, determination of the 

minimum inhibitory concentration (MIC) is difficult because of euBaryotic nature of yeasts 

and -$+"6",@*.UU.#- as a result of mycostatic nature of azoles, which are commonly applied in 

antifungal therapy. Results of routine methods of antifungal susceptibility determinationM 

AT2 fungus and E-test strips are not precise and often not simply in reading, whereas 

dilution methods in RPMI medium, recommended by NCC/. consume a lot of time and 

require experienced staff for reading of results because of occurrence of trailing effect. The 

susceptibility testing with MICRfNA6T-AM system is based on the rehydration of 

antimycotics by adding a standardized yeast suspension. Growth of the yeasts is indicated 

by a colour change from blue to pinB mediated by the A.T indicator contained in the test 

medium. 

Aim of study: The aim of the study was application of MICRfNA6T- AM system (Merlin, 

2ornheim-Hersel, Germany) for determining ",*0"-$% activity of antifungal agents against 7/* 

+68"#+,& strains isolated from women suffering from vaginal candidosis. 

Material and methods: 170 vaginal, rectal and oral consecutive isolates of 7/*+68"#+,& 

from 20 women with vaginal candidosis were cultured 2O hours on the .abouraud-glucose 

(2_) without additives. Inoculum was prepared in sodium chloride, diluted in Y.T- broth, 

supplemented with A.T indicator solution and 100 µl added to every of K6-wells of microtitration 

plates with lyophilized antifungalsM 7 concentrations for Betoconazole (lETn 

MIC}0,0J-2 kg/ml) and 8 concentrations for others antifungals | itraconazole (ITRn 

MIC}0,0J-O kg/ml), fluconazole (FCAn MIC}1-128 kg/ml), voriconazole (VfRn MIC}0,06-8 

kg/ml), [-flucytosine (FCYn MIC}0,[-6O kg/ml), amphotericin 2 (APHn MIC}0,06-8 kg/ml). 

After incubation of 2O-O8 hours at J[ - J7ÅC (depending on colour change of the growth 

control) the result was read photometrically with the MICRfNA6T .can, evaluated with the 

MICRfNA6T software and interpreted according to M27-A2 (2002) document. The quality 

control was carried out with the following strains*7/*[$(&."*62[8 and 7/*'+$+'&"6%&"&*2201K. 

Results: All 7/*+68"#+,& isolates were susceptible to FCA (MIC}1-O kg/ml) and FCY 

(MIC}0,[-2 kg/ml)n 7/*+68"#+,& strains susceptible to ITR accounted for K2_, 8_ were 

resistant to ITR (MIC 1-2 kg/ml). All studied strains had identical susceptibility to lET 

(MIC}0,0J kg/ml) and to VfR (MIC}0,06 kg/ml). MIC ranges for APH for examined 

isolates were 0,06-2 kg/ml. The majority of 7/*+68"#+,& strains (68_) had MIC}1 kg/ml, 

28_ - MIC}2 kg/ml and O_ - MIC}0,06-0,[ kg/ml. 7/*[$(&." was resistant (R) to FCA and 

FCY, susceptible|dose dependent (.-DD) to ITR. 7/*'+$+'&"6%&"& was susceptible (.) to 

FCA, FCY and ITR. 

Conclusion: This study indicate the usefulness of MICRfNA6T-AM system for studying 

antifungal susceptibility of yeasts*strains. The advantage of this method is automatic 

reading. Clear inhibition of -$+"6",@*.UU.#- in case of azoles for the majority of studied 7/* 

+68"#+,& strains is also very helpful in manual reading of results. Further comparative 

studies between Y.T medium and RPMI medium, recommended by NCC/., are required 

and will be performed. 


P-0109. Genetic polymorphism in Pneumocystis jirovecii dihydropteroate 

synthase and susceptibility to sulfamethoxazole: An in vitro study using a yeast 

model 

Moukhlis R 1 , 2oyer ( 2 , /acube P 1 , 2olognini ( 1 , Roux P 1 , Hennequin C 1 

1 /aboratoire de Parasitologie-Mycologie, Facultd de Mddecine P c M Curie, Paris, 

France, 2 6nitd de Gdndtique Moldculaire des /evures, Institut Pasteur, Paris, France 

Purpose of the study: First line therapy and prophylaxis of 1,.()%#2&-"&*\"$%0.#"" 

pneumonia still rely on the combination of trimethoprim and sulfamethoxazole (.Mj). 

.Mj inhibits the folic acid pathway through competition with the dihydropteroate 

synthase (DHP.) Bey enzyme. The most frequent genetic polymorphisms in 1/* 

\"$%0.#"" DHP. occur at positions 6[ and 71, generating four genotypesM wt/wt, 

mutant/wt, wt/mutant, mutant/mutant. As already documented for other pathogens, 

.Mj therapeutic failures have been suggested in correlation with mutations in the 1/* 

\"$%0.#"" DHP.. 

Description: In the absence of reliable culture system, we developed a model in the 

yeast =+##?+$%)2#.&*#.$.0"&"+. to study ",*0"-$% susceptibility to .Mj according to 

the 1/*\"$%0.#"" DHP. genotype. 

Results and conclusions: Each of the four 1/*\"$%0.#"" DHP. genotype, directly 

amplified from broncho-alveolar lavage samples, was cloned into pCM18K vector. 

Transformation of DHP.-deleted yeast strain EHY1 resulted in complementation 

(growth independent of exogenous source of folic acid) in all the four cases. .Mj 

concentration of 1 mg/ml added onto YPG agar plates was found to completely inhibit 

the growth of a 10 2 CF6/ml suspension of the wild type mother yeast. .ubsequently, 

this concentration was used against each of the O complemented yeast strains. ahile 

complementation with wild type or single nucleotide mutation in 1/*\"$%0.#"" DHP. did 

not significantly affect .Mj susceptibility, we found that mutant/mutant genotype was 

associated with a log10 dilution decreased susceptibility. This result was confirmed in 

a liquid medium assay against increasing concentrations of .Mj with a significant 

difference between double mutant strain and others from a concentration of 7[0 kg/ml 

of .Mj. 

P-0110. Anidulafungin (AD) vs caspofungin (CS): does MIC predict in vivo 

response? 

Najvar L, 2ocanegra R, Vallor A, flivo M, Molina D, aiederhold N, Graybill ( 

The 6niversity of Texas Health .cience Center at .an Antonio, .an Antonio, 6.A 

Purpose: C. is licensed for treatment of serious 7+,5"5+ infections. AD is a new 

echinocandin with lower MIC to 7+,5"5+ than C.. Herein we compared in vivo 

responses of AD and C. in mice infected with fluconazole (F/6) resistant (R) (MIC 6O 

kg/ml) 7/*@6+8$+-+ (79J susceptible (C.-.) or R to C. (C.-R) in vitro. 

Methods: Groups of 10 - 1[ ICR mice were immunosuppressed with [F6 ù 1[0 

mg/Bg intravenously (IV) 1 day prior to infection. Mice were infected IV with 10 8 

CF6/mouse 79*in a C.-R ç0[-62 (C. J2, AD 1 kg/ml) or C.-. ç0[-761 (C. 1, AD 

0.12[ kg/ml) isolate at 2O hours, RPMI medium. Therapy began 2O hrs post-infection. 

AD and C. were given at 0.[, 1, [, or 10 mg/Bg intraperitoneally once daily from day 1 

through day 7 after infection. F/6 was given at [ mg/Bg bid orally by gavage. Efficacy 

was measured as Bidney tissue burden 8 days after infection, expressed as CF6/g. 

.tudies were repeated for confirmation. Comparisons were made using the Mann 

ahitney test with P v 0.0[ determining significance. 

Results: F/6 was ineffective against either isolate. For C.-. CG, control mice had a 

median of 2x10 8 CF6/g of Bidneys. C. at 0.[ and 1 mg/Bg (pv0.01) reduced Bidney 

counts t2 logs, while AD at 0.[ or 1 mg/Bg reduced renal counts about 1 log (pv0.0[ 

vs controls). At [ or 10 mg/Bg both drugs were similarly effective reducing counts 

more than J logs (pv0.001). For C.-R, CA. at all doses significantly reduced renal 

counts about 2 logs below controls. All doses of AD reduced renal tissue counts 

below controls, in a similar range as C.. 

Conclusions: 2oth drugs were effective against C.-. and C.-R isolates, despite a 

wide difference in MIC between susceptible and resistant. However, the lower AD MIC 

did not predict superior ",*0"0%*efficacy of AD at the same doses as C.. 

These results may warrant the use of higher doses of .Mj or the use of alternative 

therapy in case of infection due 1/*\"$%0.#"" harboring the double mutations in DHP.. 

However, the clinical impact of these ",*0"-$% results have to be first analyzed within 

large series of pneumocystosis for which DHP. genotyping has been done. The 

development of =/*#.$.0"&"+. model lets consider other applications for the ",*0"-$% 

study of still unexplored mechanisms in 1/*\"$%0.#""/

P-0111. Susceptibility to voriconazole, fluconazole, and ketoconazole of yeast 

isolated from haematological patients 

Nawrot U 1 , NowicBa ( 2 , latarzyna a 1 

1 Department of Microbiology Medical 6niversity of aroclaw, 2 Department of 

Haematology Medical 6niversity of aroclaw 

The infections due to azole-resistant fungi have been frequently reported among immunodebilitating 

patients receiving fluconazole prophylactic. The purpose of present study was 

to investigate ",*0"-$% the susceptibility to fluconazole (Flu), Betoconazole (let), and 

voriconazole (Vor) of yeast isolated from various clinical specimens of patients hospitalised 

during 2-years period in Department of Haematology aroclaw Medical 6niversity. 

The isolates (K8) chosen for this study were obtained from 70 patients with suspected 

infection (O0 pts with acute leuBaemia and J0 with lymphoproliferative disorders), of them 

[8 received Flu or/and let in prophylactic or therapeutic doses. [8 isolates were obtained 

from respiratory, 2[ from gastrointestinal, and 1J from urinary tracts, and 2 from blood. The 

strains were identified by ID-J2C tests (2ioMerieux) as 7/*+68"#+,&*W*JJ, 7/*@6+8$+-+*W*17, 

7/[$(&."*W*1J3 7/*'+$+'&"6%&"&*W*6, 7/*-$%'"#+6"&*W*6, 7/*[.U2$*; [, 7/*",#%,&'"#(+3 7/*&''. - [, 

=/*#.$.0"&"+.*W*O, >$"#?%&'%$%,*&''*-J and 9.%-$"#?()*&'. - 1. As a quality control 7/* 

+68"#+,& ATCCK0028, and 7/*[$(&."*ATCC 62[8 were used. The susceptibility to 

antifungals was measure by the Etest method (A2 2iodisB, .weden) and performed 

according to the manufacturer instruction on RPMI medium with 2_glucose and MfP.. 

The MICs were read after 2O and O8 h of incubation at J[ 0 C. The isolates with MICs v/}8.0 

mg// were regarded as susceptible (.) to Flu, MICs ranged for 16 to J2 mg// as 

susceptible-dose-dependent (.DD) and those with MICst/}6Omg// as resistant (R). The 

isolates with MICs t/}1.0 mg// for Vor and let were considered resistant. 

Results: 60 (61.8_) of tested strains were . to all tested azoles, after 2O as well as O8h of 

incubation (MIC for Vor and let v/} 0.12[ mg// and for Flu v/}8.0 mg//). This include 

J1/JJ 7/*+68"#+,&3*6/6*7/*'+$+'&"6%&"&3*6/6*7/*-$%'"#+6"&3*6/17 7/*@6+8$+-+, J/5*7/* 

",#%,&'"#(+, O/[ 7/*[.U2$, 1/1 C/*@("6".$)%,5"", 1/1 7/*U+)+-+3*and 2/O =/*#.$.0"&"+./ The 

isolates of 7/*[$(&.", which is innately resistant to Flu, read after 2Oh exhibit MICs for Flu of 

J2-2[6 mg// and after O8h t/}2[6mg//. ae did not find 7/*[$(&." resistant to Vor and let. 

Their MICs for Vor and let after 2Oh (time recommended by manufacturer) were 0.12[-0.[ 

and 0.2[-0.7[, respectively, and after O8h it enhanced to the value %1mg// in 8 strains. 

Among 17 7/*@6+8$+-+*strains 2 (11.7_) were R to Flu with MIC of 6O and t2[6 and five 

were DD. with MICs of 12-J2 mg//. Three (17_) of these strains showed cross-resistance 

with Vor (MIC}1mg//) and let (MIC }2mg//), and four displayed reduced susceptibility to 

Vor (0.J8-0.7[mg//) and let (0.[-7[ mg//). Resistant or .DD to Flu were 2-C.* 

",#%,&'"#(+ (6O and J2 mg//) and 1-C.*[.U2$ (J2mg//), that MICs for Vor and let ranged 

from 0.2[ to 0.[ mg//. The enhanced MICs of let (0.7[-1.[) only showed K strains (2 7/* 

+68"#+,&, 1 7/*5(86",".,&"&, O*7/*@6+8$+-+, and 2 =/*#.$.0"&"+.). The tested strains of 

>$"#?%&'%$%,*and 9.%-$"#?() were R to Flu and let, but susceptible to Vor with MIC of 

0.[mg//. 

P-0112. Antifungal susceptibility of clinical isolates of Aspergilli in mycotic 

infections of eye 

Nayak N, .atpathy G, Vajpayee R 

All India Institute of Medical .ciences, New Delhi, India 

Purpose: To study the susceptibility to Amphotericin 2 of clinical isolates of 

Aspergillus species in oculomycoses. 

Summarised description of the project: .eventy two (72) clinical isolates of 

Aspergilli were tested for their susceptibility to Amphotericin 2 by broth microdilution 

as well as macrodilution techniques according to the standards laid down by the 

NCC/. carried out at 8%-? 2[ 0 C and J7 0 C.The 72 clinical isolates comprised of 28 

strains of Aspergillus niger (O each from congenital dacryocystitis and orbital cellulitis, 

20 from infectious Beratitis)n 20 strains of Aspergillus fumigatus (1O from eyes of 

infectious Beratitis, 6 from orbital cellulitis)n and 2O of Aspergillus flavus (6 from 

dacryocystitis and 18 from infectious Beratitis). 

Results and conclusions: The MIC values ranged between 0.0[kg/ml to J.12kg/ml 

both for Aspergillus flavus and Aspergillus fumigatus and between 0.2kg/ml to 

6.2[kg/ml for Aspergillus niger. Isolates from deep seated infections liBe orbital 

cellulitis and dacryocystitis showed nearly -B%*U%65*?"@?.$ MIC values than those from 

infectious Beratitis cases. This difference was noted in the results of 8%-? the 

techniques of broth microdilution as well as macrodilution, with consistently good 

agreements between the MIC values determined by each. However, incubation at 

J7 0 C yielded better reproducibility than that at 2[ 0 C for 8%-? the techniques. 

fur findings have profound potential import in the overall management of ocular 

mycoses, especially the deeper. Thus, there should always be an increased 

awareness amongst the treating ophthalmologists and physicians regarding these 

difficult and relatively resistant strains of Aspergilli in such important clinical settings. 

Conclusion: Vor was the most active against tested strains, including the resistant to Flu 

or let, nevertheless the cross-resistance was also observed. This situation stresses the 

necessity to test the susceptibility to new azole derivatives, especially for non-+68"#+,& 

yeast. 


P-0113. Resistance to fluconazole in Cryptococcus neoformans isolates from 

Peruvian AIDS patients 

Neyra E 1 , Bustamante B 1 , Casquero ( 2 , Castro C 1 

1 Instituto de Medicina Tropical Alexander von Humboldt-6niversidad Peruana Cayeta, 

2 Instituto Nacional de .alud, /ima-PER6 

Background: Cryptococcal meningitis (CM) occurs in 6-8_ of HIV-infected patients. 

Amphotericin 2 (AM2) and Fluconazole (F/Z) are the first-line drugs for its treatment 

but F/Z resistance has been reported, and their association with previous use F/Z 

has been proposed. This study evaluates the ",*0"-$% susceptibility of 7/*,.%U%$)+,& 

strains isolated from cerebrospinal fluid of AID. patients suffering a first episode of 

CM, and from patients who relapsed or failed to standard treatment. 

Material and Methods: Forty-seven (J1 baseline, 10 failure and 6 relapse) strains of 

7/*,.%U%$)+,& belonging to J1 Peruvian AID. patients underwent susceptibility 

testing. The broth microdilution method M27-A2 was performed according to Clinical 

and /aboratory .tandard Institute (formally NCC/.) guidelines, using yeast nitrogen 

base medium supplemented with glucose and incubated at J[o C for 72 h. 

Results: All forty-seven isolates were susceptible to AM2, with minimal inhibitory 

concentration (MIC) ranging from 0.012[ to 1 kg/m/. The MIC [0 and MIC K0 for AM2 

were 0.[ and 1 kg/m/ respectively. 

Among the J1 baseline strains, 20 (6O.[_) were susceptible (MIC ' 8 k/m/) to F/Z, K 

(2K_) were susceptible-dose depend (16 ' MIC % J2 k/m/), and 2 (6.[_) were 

resistant (MIC % 6O kg/m/). The MIC [0 and MIC K0 for F/Z were 8 and J2 kg/m/ 

respectively. 

Two strains isolated from one patient with failure to treatment (baseline/end of 

treatment), were resistant to F/Z. All 7/*,.%U%$)+,& isolated from relapse patients 

were susceptible or susceptible-dose dependent to this azole. 

Two out of 10 strains isolated from failed patients to standard treatment and one out 

six strains isolated from relapse patients, showed an increment of two folds in their 

MICs to F/Z. 

Conclusions: Resistance to AM2 is absent among CM Peruvian patients, while 

resistance to F/Z was found in 6.[_ of baseline strains. Nineteen percent of failure or 

relapse isolates showed two fold increased in their MICs in comparison with their 

baseline pair. 

P-0114. Over-expression of cdr1, cdr2 or mdr1 in Candida albicans does not 

cause significant changes in candin susceptibility 

Niimi K 1 , MaBi l 2 , IBeda F 2 , Holmes A 1 , /amping E 1 , Niimi M J , MonB 2 1 , Cannon R 1 

1 Department of fral .ciences, .chool of Dentistry, 6niversity of ftago, Dunedin, New 

Zealand, 2 Astellas Pharma Inc., J Department of 2ioactive Molecules, National Institute 

of Infectious Diseases 

The over-expression of drug efflux pumps in fungal cells resistant to the azole drugs is 

a concern because they confer resistance to a wide range of structurally unrelated 

xenobiotics. It was therefore important to determine whether susceptibility of the 

candins, a novel class of antifungal agent that inhibit glucan synthase activity and the 

synthesis of the essential cell wall component !-1,J glucan, could be affected by the 

over-expression of such pumps. ae have constructed a panel of =+##?+$%)2#.&* 

#.$.0"&"+. strains hyper-expressing fungal ATP-binding cassette (A2C) or major 

facilitator super-family (MF.) transporters using our patented yeast membrane protein 

expression system. The*susceptibilities of the strains to candins (micafungin and 

caspofungin) were then determined using three separate methods. 1) In liquid 

microdilution susceptibility assays, yeast strains hyper-expressing Cdr1p and Cdr2p 

(A2C transporters) or Mdr1p (MF. transporter) gave the expected resistance profiles 

for the azoles fluconazole, itraconazole and voriconazole. None of the strains, 

however, showed significant resistance to micafungin or caspofungin. 2) In agarose 

diffusion assays, the antifungal activity of micafungin and caspofungin were similar, 

although the shape and size of the caspofungin inhibitory zones were affected by 

medium composition. J) In agar plate drug resistance assays, the strains hyperexpressing 

7VPI or 7VPM showed a slightly changed susceptibility to both 

micafungin and caspofungin, compared with the null parent deleted of seven A2C 

transporters. The effects on candin susceptibility, however, occurred within narrow 

ranges of concentration and they did not translate to other susceptibility assays. The 

assessment of micafungin and caspofungin potency is therefore assay dependent. An 

isogenic pair of 7/*+68"#+,& clinical isolates T/1 (azole sensitive parent strain) and T/J 

(azole resistant daughter strain which over-expresses Cdr1p and Cdr2p) behaved 

identically in these assays. ChecBerboard assays confirmed that neither micafungin 

nor caspofungin competes with the Bnown pump substrate fluconazole for the Cdr1p, 

Cdr2p or Mdr1p efflux pumps from 7/*+68"#+,&. The results indicated that the overexpression 

of three major 7/*+68"#+,& drug efflux pumps did not cause significant 

changes in susceptibilities to either candin, and candins were unliBely to be the 

substrates of these drug efflux pumps. 

.upported byM Directorate General for development Cooperation of 2elgium.

P-0115. Identification and characterization of a drug efflux pump inhibitor 

specific to Candida albicans ABC transporter Cdr1p 

Niimi K 1 , Harding D 2 , /amping E 1 , Holmes A 1 , Niimi M J , Cannon R 1 , MonB 2 1 

1 Department of fral .ciences, .chool of Dentistry, 6niversity of ftago, Dunedin, New 

Zealand, 2 Institute of Fundamental .ciences, Massey 6niversity, J Department of 

2ioactive Molecules, National Institute of Infectious Diseases 

The over-expression of multidrug efflux pumps, particularly ATP-binding cassette 

(A2C) transporters, is a major cause of clinically significant resistance to anticancer 

and antifungal drugs that include the widely used and well-tolerated azole antifungal 

fluconazole (F/C). The structure-directed discovery of potent drug efflux pump 

inhibitors could overcome F/C resistance and extend its commercial lifetime. 

.creening of a 1.8K million member D-octapeptide combinatorial library, which 

contains a C-terminal tri-arginine motif that confers membrane impermeability and 

concentrates library members at the cell surface of fungal but not host cells, identified 

a specific inhibitor of the 7+,5"5+*+68"#+,& A2C transporter Cdr1p. The inhibitor, 

which chemosensitized F/C resistant =+##?+$%)2#.&*#.$.0"&"+. strains functionally 

over-expressing Cdr1p at {1 kM, is a peptide derivative with a single modifying group 

attached to the C-terminal tri-arginine motif. In contrast the compound did not 

chemosensitize F/C resistant =/*#.$.0"&"+. strains over-expressing the 7/*+68"#+,&* 

major facilitator superfamily transporter Mdr1p, the azole target enzyme Erg11p or 

other fungal A2C transporters tested. Re-synthesis of the Cdr1p-specific inhibitor with 

/-amino acids gave a compound that did not chemosensitize the =/*#.$.0"&"+. strain 

over-expressing Cdr1p and demonstrated that the Cdr1p-chemosensitizer interaction 

was stereospecific. The D-octapeptide inhibited rhodamine 6G efflux from whole cells 

and Cdr1p-ATPase activity of plasma membrane fractions at {1 kM, but showed no 

hemolytic activity against human erythrocytes at concentrations above its MIC ({2[ 

kM). Compounds with similar properties should be clinically relevant because the 

peptide derivative chemosensitized clinical isolates of 7/*+68"#+,& over-expressing 

7VPI to F/C, even when 7VPM was also over-expressed. 

P-0116. Relationship between concentration and antifungal activity in the 

stratum corneum of guinea pigs after topical application of luliconazole cream 

1% 

Nishida N, .hiraishi l, Hotta A, .youji T, Arai M, Tomiyama ., Nozawa A, Majima T 

Pf/A Chemical Industries, Inc., YoBohama, (apan 

Purpose of the study: /uliconazole (//CZ) is a novel imidazole antifungal agent that 

is effective against filamentous fungi, Candida spp., and Malassezia spp., and which 

has entered clinical use in (apan (200[). In this study, we employed a newly proposed 

analysis that allows us to concurrently determine the drug distribution and activity in 

the stratum corneum. To detect the concentration of antimycotics in the stratum 

corneum of guinea pig sBin, liquid chromatography-tandem mass spectrometry (/C- 

M./M.) was used. Antifungal activity was also measured using the newly proposed 

mycological analysis (scMICn ..Tomiyama, et al). 

Materials and Methods: Ten microliters of //CZ 1_ cream or 2FZ 1_ cream was 

topically applied to the sole sBin area of the hind feet of guinea pigs. At 2 and 2O hrs 

after a single application of antimycotics the application site on the hind feet was 

excised. A sBin disB with a diameter of O mm was prepared from the excised sBin by 

biopsy punch. .uccessive 10 micrometer sections of the sBin disB were then sliced 

horizontally from the superficial side (No. 1) for approximately 1[0 micrometer (lower 

edge of the stratum corneum, No. 1[) with a cryostat. MefH-extracted //CZ and 2FZ 

concentrations in the odd-numbered slices were measured by (/C-M./M.) (detection 

limit, //CZM0.8 microgram/cm J , 2FZM0.08 microgram/cm J ). The even-numbered slices 

were used to evaluate antifungal activity. A conidial solution of >$"#?%'?2-%,* 

).,-+@$%'?2-.& was gently layered onto the slices, and the slices were cultured for 

one weeB. The growth of the fungal element was observed under a light microscope. 

The dosage of no fungal growth on the slice was considered as the scMIC. 

Results and Discussion: //CZ was distributed from 208.0 ì 8[.0 microgram/cm J (in 

the superficial layer ) to 2.7 ì 1.O microgram/cm J (at a depth of 60 to 70 micrometer) 

at 2 hrs after application, and from 2J7.6 ì 180.8 microgram/cm J (in the superficial 

layer ) to 2.0 ì 0.K microgram/cm J (at a depth of 120 to 1J0 micrometer) at 2O hrs. 

2FZ was distributed from J[.0 ì 1O.J microgram/cm J (in the superficial layer ) to 0.2 ì 

0.0 microgram/cm J (at a depth of 60 to 70 micrometer) at 2 hrs after application, and 

from 87.6 ì [J.J microgram/cm J (in the superficial layer ) to 0.1 ì 0.1 microgram/cm J 

(at a depth of 1O0 to 1[0 micrometer) at 2O hrs. At both sampling time points, the 

concentration of //CZ was higher than that of 2FZ at all depths. The //CZ 

concentration that exhibited antifungal activity was approximately 2 microgram/cm J up 

to 70 micrometer from the sBin surface at 2 hrs, and up to 1O0 micrometer at 2Ohrs. 

The 2FZ concentration that showed antifungal activity was J to 8 microgram/cm J up to 

[0 micrometer from the sBin surface at 2 and 2O hrs. In conclusion, //CZ has a high 

ability to reach the deep layer of stratum corneum and to exhibit antifungal activity at 

2Ohrs after topical application. 


P-0117. Morphological study of Candida glabrata treated with micafungin, an 

echinocandin lipopeptide antimycotic 

Nishiyama Y, Hasumi Y, Yamaguchi H 

TeiByo 6niversity Institute of Medical Mycology 

Micafungin (MCFG) is a member of the new echinocandin class of antifungal agents, 

recently approved for the treatment of serious fungal infection caused by 4&'.$@"66(& 

and 7+,5"5+ species. MCFG interferes with the fungal cell wall synthesis through the 

inhibition of 1,J- -D-glucan synthesis s1u. Previously, we have demonstrated the 

effect of this drug on the cell morphology of 7/*+68"#+,& s2u and 4/*U()"@+-(& sJu. 

.imilar to 7/*+68"#+,&, 7/*@6+8$+-+ is an important cause of systemic candidiasis in 

immunocompromised patients. In this study, the effect of MCFG on the growth, 

viability and ultrastructure of 7/*@6+8$+-+ cell was investigated using viability staining, 

.EM and TEM. 

7/*@6+8$+-+ ATCCK00J0 was grown at J[ÅC in YPG broth with shaBing. The viability of 

MCFG-treated or untreated cells was microscopically evaluated with the F6N-1 

viability staining method (Molecular Probes, Inc.). For .EM, cells were fixed with 

glutaraldehyde and osmium tetroxide, dehydrated in acetone and freeze-dried in -- 

butyl alcohol. The dried samples were finally coated with osmium in an osmium 

plasma coator and examined using a (Ef/ (.M-6700F operated at 10 BV. For TEM, 

cells were fixed with glutaraldehyde and potassium permanganate, dehydrated with 

acetone and embedded in ~uetol 6[J. Thin sections were stained with uranyl acetate 

and lead citrate and observed under a Hitachi H-7000 at 7[ BV as described 

previously s2-Ju. 

The growth of 7/*@6+8$+-+ was almost completely inhibited by the drug at a 

concentration of 0.1 g/ml and above. ahen yeast cells were incubated with a 

growth-inhibitory concentration of MCFG, the number of viable cells decreased, and 

the disruption of the buds was observed in an exposure-time and drug-concentration 

dependent manner. Compared to the untreated normal cells with buds, MCFG treated 

cells possessed a strange saBe bottle-liBe shape. The disruption of the apical region of 

the bud and the release of the cytoplasmic materials from the region were also 

observed. TEM revealed that the MCFG-treatment induced cytoplasmic membrane 

detachment from the cell wall, formation of vesicles in the space between the 

cytoplasmic membrane and cell wall, waving and disruption of the cytoplasmic 

membranes, and finally almost all the cytoplasmic materials were released externally 

from the apical region of the bud. These morphological analyses led to the conclusion 

that MCFG first attacBed the cell wall of the apical region of the bud and then caused 

release of the cytoplasmic materials, resulting in the fungicidal effect. 

References: 

Hatano l. et al, ]/*4,-"8"%-"#& [[ (2002) 21K-222 

Nishiyama Y. et al, ]/*a6.#-$%,*!"#$%&#. [1 (2002) 2O7-2[[ 

Nishiyama Y. et al, ]/*a6.#-$%,*!"#$%&#. [O (200[) 67-77 

P-0118. Antifungal and fungal PLB1 inhibitory properties of phosphocholines. 

fbando D 1 , aidmer F 2 , aright / 2 , Sorrell T 2 , (oliffe l 1 

1 6niversity of .ydney, 2 Centre for Infectious Diseases and Microbiology and 

aestmead Millennium Institute, 6niversity of .ydney at aestmead 

Background: Current therapies for fungal infections are limited in safety and/or efficacy 

and resistant fungi are an emerging problem. It is widely recognised that new antifungal 

drugs with a novel mode of action are required. Fungal phospholipase 2 (P/21), which has 

P/2, lysophospholipase (/P/) and lysophospholipase transacylase (/PTA) activities, is a 

virulence factor in a number of pathogenic fungi. 1 It therefore presents a new target for 

antifungal drug development. Recently we discovered that hexadecylphosphocholine 1 

(miltefosine) inhibits P/21, is orally active in a mouse model of cryptococcosis and has 

broad-spectrum antifungal activity. 2 

Aim: To improve the therapeutic index of miltefosine. 

Methods: .ynthesise analogues of miltefosine and investigate how structure relates to 

antifungal potency, P/21 inhibition and cytotoxicity. Assay details were as previously 

reported. 2 

O 

O P O 

O 

1 

Results: Introduction of an amide- or ester-bond at a site six carbon atoms removed from 

the phosphocholine (PC) headgroup abolished antifungal activity. The MICs for 7/* 

,.%U%$)+,&*ATCC*K0112 and*7/*+68"#+,&*ATCC 102J1 were increased from 2 and O kM 

respectively for the parent compound to t 17[ kM. In parallel, inhibition of /PTA activity by 

the amide analogue was reduced from [[_ to 0_ at 2[ kM. This compound had no 

measurable cytotoxic (haemolytic) activity at concentrations of J.[ to 17[ kM, compared 

with J6_ at 88 kM for the parent compound. In contrast, an analogue with an amide bond 

positioned only three carbons from the PC headgroup, had MICs of 22 kM G7/*,.%U%$)+,&J* 

and [.[ kM G7/*+68"#+,&J and haemolytic activity of [_ at 88 kM. Most interestingly, a 

compound with a single trans- double bond placed at the centre of an 18 carbon alByl PC, 

retained antifungal activity, having MICs for both fungi of J.O kM compared with 1.O kM for 

the fully saturated octadecyl-PC. .ignificantly, the haemolytic activity of the -$+,&-alBene 

compound was only 2_ at 88 kM, compared with 6J_ for the saturated compound. A 22 

carbon alByl PC with a single #"&-alBene in the centre had an MIC of 22 kM. 

Discussion: Polar residues positioned distant from the phosphocholine (PC) headgroup 

reduce the haemolytic activity but abolish antifungal activity and P/21 inhibition. However, 

polar residues positioned adjacent to the PC headgroup are tolerated in some cases. 

Introduction of a -$+,&-alBene reduces potency only 2-fold, while reducing haemolytic 

activity J0-fold relative to the fully saturated parent compound. Further investigation of the 

most active compounds is warranted. 

References: 

1. Chen .C, aright /C, Golding (C and .orrell, TC. 2iochem. (. 2000nJO7MOJ1-OJK. 

2. aidmer F, aright /C, fbando D, et al. *Antimicrob. Agents Chemother. 

2006n[0(2)MO1O-O21. 

N

P-0119. In vitro antifungal activity of some plants extracts against 

Epidermophyton floccosum link et steudel 

Okungbowa F 1 , fBungbowa M 2 

1 6niversity ff 2enin, 2enin City, Nigeria, 2 .chool of Medical /aboratory .ciences, 

6niversity of 2enin Teaching Hospital, 2enin City, Nigeria 

Purpose of study: a'"5.$)%'?2-%,*belongs*-% a group of taxonomically related fungi 

(dermatophytes) capable of colonizing the stratum corneum of the epidermis, nails, 

hair, the horny tissues of various animals and man. The infections caused by the 

genus a'"5.$)%'?2-%,*U6%##%&() are often epidemic in nature, are transmitted from 

person to person through direct contact, and produce relatively non-inflammatory 

reactions. The increased use of athletic shoes and communal baths or pools has been 

suggested to be responsible for increase in the rate of infection. Drugs commonly 

used for treating dermatophytic infections usually have adverse side effects. 2esides, 

the acquired resistance to certain antifungals and the high cost of the drugs has made 

the search for effective herbal antifungals imperative. .everal plant species are used 

locally in Nigeria for the treatment of superficial sBin infections while some have been 

reported to show both ",*0"-$% and clinical efficacy against cutaneous fungal infections. 

This study was aimed at documenting information on the antifungal activity of some 

selected plant extracts on the ",*0"-$% growth of the athletic foot fungus, a/*U6%##%&(). 

Description of project: The antifungal activity of water-extracted constituents of 

leaves of 7+&&"+*+6+-+, 4#+62'?+*?"&'"5+3*7?$%)%6+.,+*%5%$+-+3*72)8%"'%@%,* 

#"-$+-(&*and*b#")()*@$+-"&&")()3*&..5&*%U*4U$+)%)() ).6.@(.-+3*!%,%5%$+* 

)2$"&-"#+3*1"'.$*@(",..&. and m26%'"+*+.-?"%'"#+3*and*$?"d%).*of*L",@"8.$*%UU"#",+6.3* 

was investigated by agar diffusion method. .Bin scrapings were obtained from nonulcerating 

infected toe webs of an undergraduate male student (aged 21years) of the 

6niversity of 2enin, Nigeria, who had symptoms such as maceration, slight scaling 

and itching of the toe webs. The sBin specimens were examined by conventional 

methods and a/U6%##%&() was isolated on .abouraud Dextrose Agar (containing 

0.2mg// chloramphenicol and 0.Og// cycloheximide) after incubation at ambient 

temperature for five days, and identified. Fresh fairly young leaves of 7/*+6+-+3*4/* 

?"&'"5+3*7/*%5%$+-+3*7/*#"-$+-(&*and b/*@$+-"&&")() obtained from the premises of the 

6niversity of 2enin and seeds of 4/*).6.@(.-+3*!/*)2$"&-"#+3*1/*@(",..&.3*m/* 

+.-?"%'"#+ and rhizome of L/*%UU"#",+6. purchased from a local marBet in 2enin City, 

Nigeria were used. Extracts were prepared by standard methods. A single colony of* 

a/U6%##%&() was transferred aseptically into media tubes and grown overnight at 

J[ o C, with shaBing. This culture was used for inoculation following standard 

procedures of the agar blocB diffusion method. 

P-0120. Calcineurin inhibition enhances fluconazole therapy for Candida 

albicans murine keratomycosis 

Onyewu C 1 , Afshari N 2 , Heitman ( 1 

1 DuBe 6niversity Medical Center, Durham, NC 6.A, 2 DuBe 6niversity Eye Center, 

Durham, NC 6.A 

Purpose of the study: 7+,5"5+*+68"#+,& is responsible for a variety of human 

disease manifestations ranging from superficial sBin infections to disseminated 

disease. leratomycosis caused by 7/*+68"#+,& is a mucocutaneous infection that can 

present a significant challenge to clinicians because some oral and intravenous 

antifungal drugs exhibit poor intraocular penetration. ae have employed a recently 

established experimental murine Beratomycosis model to test the ",*0"0% efficacy of 

combining topical preparations of cyclosporine A (CsA) and fluconazole against 7/* 

+68"#+,&, a combination previously demonstrated to exhibit ",*0"-$% drug synergy. 

Description: In this model, the right corneas of immunosuppressed 2A/2/c mice 

were scarred and inoculated with a suspension of {10 6 wild-type or calcineurin mutant 

7/*+68"#+,& cells. A clinical ocular grading scale of 0 (clear cornea) to O (severe 

infection) was predetermined to ranB corneal infection. Animals were divided into drug 

treatment groups, receiving no drug, 2_ CsA only, 0.2_ fluconazole only, or 2_ CsA 

and 0.2_ fluconazole daily for O days. Topical treatment was begun three days after 

infection when at least one animal in each treatment group reached a grade ,J 

infection. The infected eye of each animal was examined by a blinded ophthalmologist 

and scored daily using a conventional slit-lamp. 

Results and conclusions: 2y day three of treatment, the majority of wild-type 7/* 

+68"#+,& infections in animals receiving combination therapy with topical fluconazole 

and CsA had completely resolved to a grade 0. In contrast, animals receiving no 

treatment or monotherapy with either drug still had visible signs of infection and 

inflammation up to the last day of treatment. Animals infected with a 7/*+68"#+,& 

calcineurin 2 mutant (#,8I

P-0121. Comparative study for the antifungic susceptibility of yeasts to lauryldimethyl-benzyl-ammonium 

bromide (LDBAB) and azoles by the agar diffusion 

method 

Pabon de Santiago R, .antiago A, Garmendia Y 

Hospital 6niversitario de Caracas, Venezuela 

To treat Candidosis and other micosis caused by yeasts, the indiscriminate use of 

antifungics, has brought in the appearance of isolated resistant, what suggests a 

study in vitro of the antifungic susceptibility of the elective drugs, as well as the search 

for other chemical substances that allow therapeutical alternatives. 

There were conventionally identified and with the .ystem ID J2C, 8J isolated yeasts 

of biological different samples. The sensibility was manually determined by the Agar 

Diffusion Method for /D2A2 at 10_ (Gerdexw) with soaBed disBs at two dilutions 

(1000.g / ml and 1.[.g / ml) and comparatively with Fluconazole (2[ ûg) and 

Voriconazole (1ûg) with automated reading (2iomic Video, Giles .cientific, 6.A). 

There were isolated 1O specimens of Candida albicans (16.86_)n O0 specimens of C. 

parapsilosis (O8.1K_)n 1O specimens of C. tropicalis (16.86_)n O specimens of C. 

glabrata (O.81_)n J specimens of C. Brusei (J.61_)n J specimens of Candida spp. 

(J.61_) and [ specimens of Cryptococcus neoformans (6.02_). 

The .usceptibility (.) of Candida specimens with Gerdexw at 10_ to both dilutions 

brought up the same results. The opposing inhibition ranges were 1K-28 mm for 

isolated ., 1J-18 mm of the Dose-Dependent .usceptibility (DD.) and Resistant (R) 

of 12 mm. For C. neoformans, the range of . was 28-JO mm. 

ahen using Fluconazole, . was 6[.0_ (20-OK mm, at halo inhibition), DD. was 

10.0O_ and (1J-18 mm) and R was 16.86_ (û12 mm). . for C. neoformans was 

6.02_, (1K-[1mm). ahen using Voriconazole, . for Candida was 67.O6_, (20-[7 

mm), DD. was 8.OJ_ and (1O-18 mm) and R was 18.07_ (û 12 mm). . for C. 

neoformans was 6.02_ (28-62 mm). 

ahen comparing with Azoles, using Gerdexw at 10_ for C. albicans, the range was 

1K-21mm for the isolated ., 1J-1K mm for the DD. and R û12 mm. aith C. glabrata 

the range was 1K-26 mm for ., 1J-18 mm for the DD., 1O-16mm, and in those 

isolated R for Fluconazole and Voriconazole, the range was 1O26 mm. In the same 

way, for C. Brusei, in isolated R using these antifungics, the range was 1K-28 mm. For 

C. tropicalis, . was 11-20 mm, DD. was 16-2O mm and R was 1O-1[ mm. For C. 

parapsilosis, the result for . was 1K-20 mm, for DD. was 1[-18 mm and for R was 

12-1O mm. The . of C. neoformans was 28JO mm. 

P-0122. In-vitro synergy between terbinafine and voriconazole against 

Scedosporium prolificans 

Perry R 1 , Ellis D 1 , Nguyen ~ 2 , Heath C J , Chen . 2 , .lavin M O 

1 aomenes and Childrenes Hospital, Adelaide, 2 .t. Vincentes Hospital, .ydney, J Royal 

Perth Hospital, Perth, O Royal Melbourne Hospital, Melbourne, Australia 

Background: Management of invasive infections caused by =#.5%&'%$"()* 

'$%6"U"#+,& has become an emerging clinical problem, especially in cases of septic 

arthritis and osteomyelitis following penetrating injuries to joints. This fungus is 

notoriously resistant to antifungal therapy, surgical resection is often required and 

treatment failures are common. K,*0"-$% testing has demonstrated multi-resistance to 

current anti-fungal agents and at best limited activity with the novel triazoles and 

echinocandins. ae examined the ",W0"-$% activity of amphotericin 2, itraconazole, 

voriconazole and terbinafine individually and the combinations of 

terbinafine/voriconazole and terbinafine/itraconazole against clinical isolates of =/* 

'$%6"U"#+,&. 

Method: All isolates were tested by broth micro-dilution according to the C/.I MJ8A 

protocol to obtain single MICes. .ynergy studies were performed using a twodimensional 

two-agent micro-dilution checBerboard method. MICes were read at O8 

and 72 hours and the end points taBen at [0_ or greater inhibition than the growth 

control. 

Results: .ingle drug MIC K0 were amphotericin 2 } 16 ug/ml, terbinafine } 8 ug/ml, 

itraconazole } t16 ug/ml and voriconazole } 8 ug/ml. In combination with terbinafine 

the MICes for both voriconazole and itraconazole were dramatically reduced. .eventyfour 

of 87 isolates demonstrated synergy with the terbinafine/voriconazole 

combination. The terbinafine/itraconazole combination resulted in synergy for OO of 

7O isolates tested. No antagonism was observed. 

Conclusion: Terbinafine appears to be synergistic with voriconazole and 

itraconazole against most isolates of =/*'$%6"U"#+,&. The MICes of both 

terbinafine/itraconazole and terbinafine/voriconazole combinations appear to be within 

achievable plasma ranges. Although ",W0"-$% results have not yet been directly 

correlated with therapeutic outcome, there has been some case reports of successful 

combination therapy. ChecBerboard synergy testing may provide valuable information 

to investigate potential antifungal combinations. 

The results obtained within the inhibition ranges of the antiseptic were equally 

compared to the preestablished range of the NCC/. for Fluconazole, observing that 

using Gerdexw at 10_ produces important halos in those cases where this antifungic 

and Voriconazole both brought up results R. The commercial antifungi used in this 

study, although have been considered elective drogs, its indiscriminated use will 

increase the fungi resistance. Therefore, the search for other options is needed. The 

results with the /D2A2, though being preliminaries, suggest a promising future for this 

compound from the therapeutical viewpoint.

P-0123. Antifungal drug combinations with hyalohyphomycetes, using linear 

growth dynamics 

Petrou M, Mepham S 

Hammersmith Hospital 

Invasive filamentous fungal infections have a high morbidity and mortality related to 

the underlying predisposing condition, difficulties in diagnosis, and toxic treatment 

options. The main filamentous fungi causing infection in our Hospitals are the 

A2+6%?2'?%)2#.&, in particular 4&'.$@"66(& and ^(&+$"() species and have been 

performing susceptibility on these isolates using a similar method to the NCC/. 

microdilution method for over twenty years. In recent years renewed interest has been 

shown in combination antifungal therapy in at-risB patients. Various ",*0"-$% methods 

have been used to assess potential antifungal combinations but the commonest 

method has been the checBerboard microdilution method. ahilst easy to set up and 

highly reproducible for yeasts, this method is prone to inter-laboratory variation due to 

the growth dynamics of filamentous fungi. Fungal spores in liquid cultures will grow 

exponentially into fungal balls that could float on top, adhere to the sides or disperse 

evenly, thus the results will be influenced by the method fungal growth will be 

recorded. fn solid media fungal spores will grow exponentially during germination 

lasting from minutes to several hours afterwhich they will grow linearly for as long as 

there is space on the plate. This study used a novel method based on the linear 

growth dynamics of filamentous fungi to investigate the interactions of six antifungal 

agents with eight 4&'.$@"66(& species and two ^(&+$"() species. The combinations 

used were arranged as a modified checBerboard technique with each combination 

being assessed on individual plates. The colony size on every plate was measured 

daily for a minimum of seven days and the area under the curve of diameter versus 

time was calculated for each drug dilution and expressed as a percentage of the drugfree 

control. This agar dilution method has been found to be independent of the size of 

the inoculum, however the growth rate is influenced by the medium composition. The 

method proved to be simple, cost effective, and discriminative, with exceptionally high 

correlation coefficients (0.87 to 1.0 with most t0.KK) in all isolates and drugs indicating 

near perfect linear growth (average P value of 0.00O[). The results showed that 

7K.6_ (K0 out of 11J) low concentration combinations and 78_ (8[ out of 10K) high 

concentration combinations produced indifferent results. However, promising 

synergistic interactions were seen with the previously under-investigated combination 

of caspofungin combined and flucytosine (J/10 at low concentrations, [/10 at high 

concentrations), as well as caspofungin-voriconazole (2/10 and J/10), or caspofunginterbinafine 

(J/6 at low concentration only). Antagonism or indifference was seen 

between amphotericin 2 and voriconazole (J/10 and 1/10) as well as itraconazole 

(1/10 and J/10). Further studies are needed to compare the technique to the 

microdilution method with these and other filamentous fungal species as well as to 

repeat the promising combinations. This method could be used as a reference method 

to assess drug activity and drug interaction and the results should be used to inform ",* 

0"0% and clinical trials. 

P-0124. Antifungal susceptibility of clinical isolates of Candida guilliermondii 

Pinoni M 1 , (ewtuchowicz V 2 , Relloso . J , Finquelievich J O , Iovannitti C [ , Mujica M 6 

1 Centro de Micologia Facultad de Medicina 62A. Argentina, 2 Centro de Micologia 

Facultad de Medicina 62A. Argentina, J CEMIC. 2uenos Aires. Argentina, O Centro de 

Micologia Facultad de Medicina 62A. Argentina, [ Centro de Micologia Facultad de 

Medicina 62A. Argentina, 6 Centro de Micologia Facultad de Medicina 62A. Argentina 

The incidence of invasive candidiasis in immunocompromised or surgically 

treated patients has risen in recent years. The number of 7+,5"5+ species 

causing invasive mycoses appears to be increasing steadily. These gave us 

the hnewi and hemergingi fungal pathogens and problems associated with 

identification and treatment of infection caused by these organisms. 

Objective: to evaluate the antifungal susceptibility to fluconazole (Flu), 

voriconazole (Vor), amphotericin 2 (Amfo) and caspofungin (Caspo)*of* 

clinical isolates of 7+,5"5+ @("66".$)%,5""/** 

* 

Material and methods: thirty-four 7/*@("66".$)%,5"" were isolated from blood, 

urine, sBin, nails, and subgingival pocBets and identified by API ID J2C 

system (2iomerieux), CHRfMagar Candida (CHRfMagar Company, Paris, 

France) and micromorphological studies. The minimal inhibitory 

concentration (MIC) were determined by an modified National Committee for 

Clinical /aboratory .tandard (NCC/.) M27-A2 method using RPMI as test 

medium supplemented with 2 _ glucose. MIC end-point was determined by 

spectrophotometry after incubation for O8 h at J[ C. For Amfo (.igma) and 

Caspo (MercB) the endpoint was the lowest drug concentration exhibiting a 

reduction in growth of K[_ or more compared to that of the control growth 

(inhibition complete). For the azoles (Pfizer) and Caspo the endpoint was 

the lowest drug concentration exhibiting reduction in growth of [0_ or more 

compared with that of the control growth (inhibition partial). 

Results: they are presented in table 1, as the cumulative percentage of 

isolates inhibited at each concentration throughout the dilution series (fullrange 

MICs, Flu 6O- 0.12 kg /ml, Amfo, Vor and Caspo 16-0.0J kg/ml). 

Continued on next page 


Table 1: in vitro susceptibility of JO isolates of 7/*@("66".$)%,5"" to Flu, Vor, 

Amfo and Caspo ò 

Cumulative % of isolates susceptible at MIC (µg/ml) of: 

Range MIC 

Antifungal 

90 

0,03 0,06 0,12 0,25 0,5 1 2 4 8 16 32 

0,2[- 

Flu a J2 16 0 J 6 6 2O 7K 8[ K7 100 

Caspo d 

Vor b 0,0J-8 0.[ 2O [6 68 8[ K7 K7 K7 K7 100 

Amfo c 2 1 0 0 6 18 7K K7 100 

0,12[- 

t16 t16 0 0 0 6 K 2O J8 62 7K 8[ 

0,2[- 

Caspo e 0,06-1 1 0 J 1[ J[ [K 100 

ò2roth microdilution testing according to modified NCC/. M27-A2 using O8 

h incubation 

a breaBpoint '8 kg/ml ., 16-J2 kg/ml .-DD, %6O kg/ml R. b breaBpoint ' 1 

kg/ml . c Interpretative criteria have not yet been defined however we have 

used ' 1kg/ml as .usceptible. d End point complete inhibition. e End point 

partial inhibition (Pfaller et al 200O. ( Clin Microbiol J117-J11K) 

Conclusions: 7/*@("66".$)%,5"" (MIC K0 16 kg/ml to Flu) had a decreased 

susceptibility to Flu. Vor exhibited a good in vitro activity against the majority 

of isolates (MIC K0 0.[ kg/ml). Although K7 _ of 7/*@("66".$)%,5"" isolates 

were susceptible to Amfo (MIC ' 1 kg/ml) one isolate was resistant. All of 7/* 

@("66".$)%,5"" were observed with MIC ' 1kg/ml to Caspo (end point partial 

inhibition). ae suggest that the clinical usefulness of the partial inhibition 

criterion must be validated by clinical outcome. 

This investigation was supported in part by project 62ACYT M-087 from the 

6niversity of 2uenos Aires. Argentina 

6 

4 

P-0125. Developing a new antifungal - from the bench to bedside 

Polacheck I 1 , .ionov E 1 , fren G 1 , Pitusi M 1 , TaBarouri l 2 , (abbour A 2 , FalB R 1 , Domb 

A 2 , .rebniB M 2 

1 Hadassah-Hebrew 6niversity Medical Center, (erusalem, Israel, 2 .chool of 

Pharmacy, Hebrew 6niversity in (erusalem, Israel 

Three approaches for developing of new antifungals are describedM 

Modification of an existing drug to improve the therapeutic index (new drug delivery, 

prodrug). Thus, conjugation of amphotericin 2 (Am2) with oxidized arabinogalactan 

(AG) generated a highly water soluble Am2-AG conjugate which was formulated for 

injection. It was much safer than the conventional micellar Am2-deoxycholate (MTD } 

[0 vs. O mg/Bg). Histopathological evaluation of organs in mice treated with the 

conventional micellar formulation showed renal tubular necrosis, whereas no damage 

was detected in mice treated with the Am2-AG conjugate, even at a much higher 

dosage. The conjugation decreases Am2 toxicity as well as infusion related adverse 

effects by preventing the apoptotic death of renal cells by Am2 possibly through 

modulation of proinflammatory cytoBine expression, such as I/1-beta and TNF-alfa. 

Treatment with the conjugate had a high degree of therapeutic efficacy in fungal 

infections in three animal models (murine candidiasis, cryptococcosis and 

aspergillosis). In addition, high dose treatment caused no damage and was highly 

effective in eradicating fungal cells from target organs. 

Development of newly synthesized antifungals. Cancerous cells and fungal cells 

possess analogous/similar systems since both are primitive euBaryotic cells and their 

metabolism is somewhat different from the host cells (High growth rate and 

multiplication). Amine cyanoboranes were reported in the early 1K80]s to have 

anticancer activity. However, antifungal activity of amine cyanoboranes has not been 

reported. In the present study a series of 60 new amine cyanoboranes and derivatives 

were synthesized and screened for their antifungal activity. A number of structureantifungal 

activity relationship (.AR) series were observed. Modifications enhancing 

activity wereM a) the N-alByl chain length. b) incorporation of a C}C double bond at 

the end of the chain. c) halogenation. The lead compound was lI with MIC of J0 

kmol// and it was not toxic to ICR mice after I.V. injection. 

Interference with quorum sensing mechanisms. Farnesol, is a quorum sensing 

molecule (~.M) that inhibits fungal filamentation. A series of novel compounds that 

inhibit fungal physiological processes were selected. Eight synthetic analogs of were 

tested for their ability to suppress filamentation and prevent biofilm formation of 

7+,5"5+*+68"#+,& in microtitration plastic plates. fne compound - AO showed ~.M 

activity at 100-200 kg//. The head group (C1 | fH derivative) and the presence of 

double bond are essential for this activity.

P-0126. Incidence and antifungal susceptibility of Candida bloodstream isolates 

at the public hospital, Botucatu, São Paulo, Brazil 

Rodrigues Paula C 1 , da .ilva Ruiz / 1 , Emi Matsumoto F 1 , Helena da .ilva E 1 , 

GonÑalves da .ilva E 1 , Cristiano da .ilva 2 1 , de Fatima .ugizaBi M 2 , Cesar Montelli 

A 2 

1 /aboratory of Mycology, Department of Microbiology, 2iomedical .cience Institute, 

6.P, .an Paulo, 2razil, 2 Department of Microbiology and Immunology, 2ioscience 

Institute, 6nesp, 2otucatu, .Éo Paulo, 2razil 

Yeasts, primarily those*of the genus 7+,5"5+, have been recognized as important 

etiological*agents in nosocomial infections. Among these infections, those of the blood 

caused by*7+,5"5+*species have been increasing in frequency and are increasingly 

considered as significant*causes of death in hospitalized patients. The present study 

was conducted*to determine the incidence and to compare the antifungal susceptibility, 

through the hEtesti commercial method (A2 2iodisB, .olna, .udcia). The study 

involved 7[ strains of yeast isolated from the blood of patients with a clinical profile 

indicative of fungemia. The antifungal agents used were fluconazole, itraconazole, 

Betoconazole, anphotericin 2 and [-flucytosine. The hospital wards with the largest 

number of fungemias were neonatal Intensive Care 6nits (J2_) followed by gastric 

surgery (1J,O_) and pediatric wards (10,7_). In the Neonatal Intensive Care, the 

7+,5"5+ species most frequently isolated from blood was 7/*'+$+'&"6%&"& ([O,1_), 

followed by 7/*+68"#+,& (2K,2_), 7/*[$(&." (12,[_) and 7/*@("66".$)%,5"" (O,2_). The 

antigungal susceptibility testing demonstred that the isolates were susceptible to 

anphotericin 2 and, in regard to imidazoles, 7/*-$%'"#+6"& samples were most resistant 

and the non-+68"#+,& species were most resistant to fluconazole. The values of MIC [0 

and MIC K0 obtained, respectively, to fluconazole were 6,0K$g/m/ and 126$g/m/, 

itraconazole 0,016$g/m/ and 0,718$g/m/, Betoconazole 0,1[O$g/m/ and 1J,2[$g/m/, 

anphotericin 2 0,1J[$g/m/ and 0,8K$g/m/ and [-flucytosine 0,012$g/m/ and 

0,07K$g/m/. 

P-0127. New evidences on the in vitro inhibitory effect of ajoene on 

Histoplasma capsulatum 

Romero H 1 , Torres ( 1 , Apitz-Castro R 2 

1 6niversidad Central de Venezuela, Escuela de 2ioanalisis, CZtedra de MicologÜa., 

2 6niversidad Central de Venezuela, Escuela de 2ioanalisis, CZtedra de MicologÜa., 

J 2/aboratorio de Trombosis Experimental, IVIC. Caracas - Venezuela 

Recently was informed, for the first time, about the antifungal activity of ajoene on an 

isolate of A"&-%'6+&)+*#+'&(6+-() in the mycelial phase, showing highly satisfactory 

results. aith the purpose of corroborate these results, the main objective of this worB 

was to evaluate the inhibitory effect of ajoene on six clinical isolates of A/*#+'&(6+-()* 

(mycelial phase), using .abouraud dextrose broth (.D2) and RPMI-16O0 culture 

media. The isolates were maintained in the mycelial phase in mycosel agar at room 

temperature until use. Growth curves and inhibitory effect of ajoene (at concentrations 

of 1.2[ to 20 $g/ml) were recorded by turbidimetrical readings ([O0 nm) every O8 

hours, for 1O days, in both culture media, under mechanical agitation (80 r.p.m.). 

Graphs were constructed to estimate Generation time (GT), Minimal Inhibitory 

Concentration (MIC) and Inhibitory Concentration [0_ (IC [0). The minimal fungicidal 

concentration (MFC) was determined by using the plate culture method. Typical 

growth curves were obtained, with GT of 1.8 to 2.6 days, in both media. In .D2, a 

fungistatic effect was noticed at 1.2[, 2.[ and 10 $g/ml of ajoene, and concentrations 

of the drug t[ $g/ml blocBed growth all throughout the assay in almost all isolatesn 

values for MIC were 2.[ to 20 $g/ml and IC [0 values were 1.K to 12.[ $g/ml. MFC was 

of [ to 20 $g/ml. In RPMI-16O0 medium, the drug effect was stronger, since all 

concentrations above 1.2[ $g/ml halted growth altogether. In this defined medium, 

MIC values were 1.2[ to [ $g/ml and values for IC [0 were v1.2[ to J.7[ $g/ml. MFC 

was 1.2[ to [ $g/ml of ajoene . The results obtained in the present study corroborate 

which has been previously reported, when worBing with a single isolate of the fungus, 

thus confirming the inhibitory effect of ajoene in the mycelial phase of A/*#+'&(6+-(). 

Financed by the FAPE.P 


P-0128. Sterol-hydrazone derivatives as inhibitors of delta 24_sterol methyl 

transferase 

San-Blas G 1 , Visbal G 1 , Moreno 2 1 , Maldonado A 1 , Murgich ( 1 , Franco H 2 

1 Venezuelan Institute for .cientific Research, 2 Central 6niversity of Venezuela, 

.chool of Chemistry 

.terols from fungi, protozoa and higher plants differ from the vertebrate sterols by the 

presence of an extra alByl group at C-2O. AlBylation of the sterol]s side chain is 

catalyzed by .-adenosyl-/-methionine (AdoMet)M delta (2O) -sterol methyl transferase 

(.MT), a step of interest for the development of specific antifungal agent (1, 2). In this 

worB, we present the syntheses of four sterols (H1, H2, HJ and HO) with more than 

one nitrogen atom in the side chain (sterol hydrazone derivative), and their effects on 

growth and sterol profile in the pathogenic yeastliBe (Y) phase of 1+$+#%##"5"%"5.&* 

8$+&"6".,&"&. .tructure-activity relationships are derived for these compounds. 

.terol-hydrazone derivatives synthesized in the laboratory wereM 20-hydrazoneimidazol-2-yl-[alpha-pregnan-Jbeta-ol 

(H1), 20-hydrazone-pyridin-2-yl-[alphapregnan-Jbeta-ol 

(H2), 22-hydrazone-imidazol-2-yl-[-colen-Jbeta-ol (H3), and 22- 

hydrazone-pyridin-2-yl-[-colen-Jbeta-ol (H4). 1/*8$+&"6".,&"& was grown in PYG 

medium at J7 o C for up to O days with variable concentrations of each hydrazone, from 

0 to 10 uM. For lipid analyses, 1/*8$+&"6".,&"& was cultured either in the absence or 

presence of each hydrazone at their IC [0 concentrations. Total lipids were extracted 

with chloroform/methanol (2M1n v/v). Polar and neutral lipids were fractionated by silicic 

acid column chromatography. For quantitative analysis and structural assignment, 

neutral lipids were separated by gas chromatography-mass spectroscopy. The 

molecular electrostatic potentials MEP were calculated with .partan 0O employing 

Density Functional Theory and a 6-J1Gò basis set. 

1/*8$+&"6".,&"& (Y phase) was sensitive to the action of sterol hydrazone derivatives in 

the following sequenceM HO (IC [0M 0.07[ uM)tHJ (IC [0M 0.1 uM)tH2 (IC [0M 1 uM) }H1 

(IC [0M 1 uM). fn exposure to sterol hydrazones, ergosterol decreased from 76_ to 20- 

J0_, while lanosterol accumulated from 1K_ to [0_, and 2O-methyl sterols depleted, 

providing evidence that the enzyme delta 2O(2[) .MT was selectively inhibited. Therefore, 

the presence of an alByl substituent in the 2O position of the sterol molecule is an 

essential feature to fulfill the physiological functions of these molecules in 1/* 

8$+&"6".,&"&, as shown before (1, 2). .tructure-activity analyses suggest that HJ and 

HO are more potent because the extra carbon atom in the side chain confers more 

flexibility to the hydrazone derivative in the active site of the enzyme .MT. The MEPs 

at the electronic isodensity 0.002 e/au J of H1 varied between -[6.8O and OK.JK, H2 

from -O2.82 and OK.O6, HJ from -[6.82 to OK.[[ and HO from -61.1J and 

O7.2JBCal/mol. Relationship of these values of MEP at this isosurface with the charge 

distribution of the receptor site will be also presented. 

P-0129. In vitro antifungal activity of Nikkomycin Z in combination with 

caspofungin, or voriconazole or amphotericin B against Candida albicans 

.andovsBy-/osica H, /ahat Y, .hwarzman R, Segal E 

Tel-Aviv 6niversity, Tel Aviv, Israel 

The antifungal armamentarium for treatment of serious fungal infections remains 

limited. A possible approach to overcome antifungal drug resistance and high 

mortality in severe fungal infections is to combine two or three classes of antifungals, 

especially if the drugs have different mechanisms of action. 

Combinations of new agents along with more traditional antifungals have now been 

shown to possess some synergistic or at least additive activity against Candida in 

clinical trials, increased rate and extent of fungal Billing, enhanced spectrum of activity, 

decreased liBelihood of resistance or tolerance and minimized toxicity. 

ae investigated in vitro, using simulated checBerboard dilution assay, J different 

combinations of 2 antifungal agents against Candida +68"#+,& (2 isolates). ae used 

7/+68"#+,& C2. [62 and a clinical strain of 7/+68"#+,&. The combinations that were 

tested wereM NiBBomycin Z and Voriconazolen NiBBomycin Z and Caspofungin, 

NiBBomycin Z and Amphotercin 2. The FICIM Fractional inhibitory concentration index 

was determined for each drug combination after O8 hrs incubation. 

The results according to FICI wereM indifference between NiBBomycin Z and 

Amphotericin 2 for both 7/+68"#+,& isolates, synergism for NiBBomycin Z and 

Voriconazole for both 7/+68"#+,& isolates, and synergism to additivity for NiBBomycin Z 

and Caspofungin for both 7/+68"#+,&*isolates. 

These results provide a basis for conducting further in vivo experiments in laboratory 

animals with antifungal combinations to assess the therapeutic efficacy in 

experimental infections. 

References: 

1. Visbal .-*+6/ 200J.*Antimicrob. Agents Chemother. O7M 2K66-2K70. 

2. Visbal .-*+6. 200[. Current Drug TargetsM Infectious Disordersn [M 211-226.

P-0130. Caspofungin activity against clinical isolates of azole cross-resistant 

Candida glabrata overexpressing efflux pump genes 

Sanguinetti M 1 , Posteraro 2 1 , Fiori 2 1 , /a .orda M 1 , .panu T 1 , .anglard D 2 , Fadda 

G 1 

1 Istituto di Microbiologia - 6niversitÖ Cattolica del .. Cuore, 2 Institut de Microbiologie - 

Centre Hospitalier 6niversitaire Vaudois 

Purpose of the study: .everal studies have documented the potent ",*0"-$%*activity of 

caspofungin against 7+,5"5+*spp. This is of special concern for 7+,5"5+*@6+8$+-+* 

infections that are often resistant to many azole antifungal agents and, consequently, 

difficult to treat. The aim of the present study was to expand the data on the ",*0"-$% 

activity of caspofungin against azole-resistant isolates*of 7/*@6+8$+-+. 

Summarized description of the study: Fifty clinical isolates of 7/*@6+8$+-+ were 

tested for susceptibility to caspofungin. The isolates were cross-resistant to multiple 

azoles, including fluconazole, itraconazole, Betoconazole and voriconazole. 

Expression of the resistance-related 7@7VPI and 7@7VPM genes was evaluated by 

quantitative RT-PCR analysis. The MICs to caspofungin were determined by using the 

National Committee for Clinical /aboratory .tandards M27-A2 reference method. 

Results and conclusionsM All the isolates exhibited increased expression*of the 

7@7VPI and 7@7VPM genes, and this was in accordance with their high-level azole 

resistance. In contrast, all the isolates were highly susceptible to caspofungin (100_ 

of isolates were inhibited at -1 mg//). fur results represent the further evidence for 

the excellent antifungal potency of caspofungin, particularly against 7/*@6+8$+-+ 

isolates expressing cross-resistance to azoles. 

P-0131. Antifungal activity of Carum copticum against fluconazole resistant and 

susceptible Candida albicans isolates 

Sattari M 1 , Natanzian GhahfaroBhi M 1 , Yadegari M 1 , .ahar lhiz M 2 

1 Medical .chool,Tarbiat Modarres 6niversity, Tehran, Iran, 2 Agricultural 

.chool,Tarbiat Modarres 6niversity, Tehran, Iran 

In recent years, systemic fungal infections due to Candida species have been 

received major consideration about inducing mortality in nosocomial patient]s because 

of increasing in immunocomromised disorders such as AID. and hematological 

disorders as well as long term use of broad spectrum antibiotics and corticosteroids. 

aide rang use antifungal agents specially azole compounds in treatment of sever 

Candidiasis has resulted in fungal resistance in Candida species, thus it is necessary 

to use antifungal susceptibility tests before selecting a suitable drug for such above 

mentioned infections which can resulted in the decrease in secondary drug resistance 

and better manipulation of treatment protocols. 

The present study was done with the aim of evaluating antifungal effects of essential 

oil and alcoholic extract from Carum copticum against Fluconazole (F/Z) susceptible 

and Fluconazole resistance Candida albicans strains isolated from different types of 

Candidiasis using standard drug susceptibility tests with broth dilution technique to 

measure the in vitro investigation of antifungal activity of essential oil and alcoholic 

extract from Carum copticum. 

Essential oil and alcoholic extract from Carum copticum showed inhibitory effects on 

grow of Fluconazole susceptible and Fluconazole resistance Candida albicans 

isolates. 

Results show that MIC and MFC for essential oil 0.OJkg/ml, 0.87kg/ml and for 

alcoholic extract J.[1kg/ml, 7.0J kg/ml respectively. 

Thus, it is seems that Carum copticum could inhibit C. albicans growth by a similar 

mechanism which occure by F/Z. 

In general, the results obtained in this study indicate that Carum copticum has 

potential values for growth inhibition of C. albicans in vitro. 


P-0132. Experimental dermatophytosis in guinea pigs: Establishing and 

evaluating an animal model 

Saunte D 1, J , Hasselby ( 2 , Frimodt-Moller N 1 , /innemann D 2 , .vejgaard E J , 

Haedersdal M J , Arendrup M 1 

1 .tatens .erum Institut, Copenhagen, DenmarB, 2 Dept. of Pathology, 2ispebjeg 

Hospital, Copenhagen, DenmarB, J Dept. og Dermatology, 2ispebjerg Hospital, 

Copenhagen, DenmarB 

Purpose: The aim of this study was to establish and evaluate an animal model of 

dermatophytosis designed for evaluation of antifungal treatment efficacy using human 

pathogenic dermatophytes. 

Description of the project: Establishment of an animal modelM !/*#+,"& (NCPF 177), >/* 

).,-+@$%'?2-.&*(NCPF 22O) and two clinical isolates of each species*were used for inoculation 

(10 [ and 10 6-7 cells / ml). J6 Harlan (Hsd.scMA/) guinea pigs in groups of six were inoculated on 

four different areas. Evaluation was performed clinically twice a weeB and mycologically once a 

weeB in a four-weeB-period. Clinical evaluation consisted of a redness and lesion score. The 

mycological examination consisted of direct microscopy and culture of sBin scrapings. .Bin 

biopsies for histopathology were also taBen. Two methods of increasing the susceptibility of the 

sBin to infection were tested on 18 !/*#+,"&*infected female animalsn razing versus tape stripping. 

Inoculation with or without occlusion was tested on >/*).,-+@$%'?2-.& infected animals (n}12 

male, 6 female). 

Treatment studyM Twenty female animals (infected with !/*#+,"& or >/*).,-+@$%'?2-.&) were 

treated with itraconazole (10 mg/Bg once daily p.o. day J-1O) and 16 served as positive controls. 

Mycological and clinical evaluation was performed as described above. 

Results: Establishment of an animal modelM The clinical manifestations were independent of 

inoculum size and method of pre-treatment of the sBin in !/*#+,"& infected animals. Maximum 

lesion score was observed day 1O-16 and the redness score had two maximums day 7 and day 

1O-16. There was 7J_ concordance between microscopy plus culture and histopathology. In 

6J_ of the discordant cases histopathology was negative. The clinical manifestation of animals 

infected with >/*).,-+@$%'?2-.& was independent of occlusion. There was a tendency towards a 

higher lesion score with increasing concentration of the inoculum. /esion score had a maximum 

day J-7 for the clinical isolates and day 1O-2[ for the NCPF isolate. There was 72_ concordance 

between microscopy plus culture and histopathology day 7 and 61_ day 1O with more cases 

being histopathology-negative. 

P-0133. Liposomal and conventional amphotericin B formulations efficacy in 

early stages of murine paracoccidioidomycoses 

.cavone R, Burger E 

Instituto de Ciüncias 2iomddicas - 6niversidade de .Éo Paulo 

Purpose of the study: to compare the efficacy of the liposomal (/-Am2) and the 

conventional (c-Am2) amphotericin 2 formulation in susceptible mice infected with 

1+$+#%##"5"%"5.&*8$+&"6".,&"& (Pb). 

Summarized description of the project: therapy was initiated 2O hours prior to the 

infection with a virulent Pb isolaten 2mg c-Am2 and [mg /-Am2/Bg/day were given to 

susceptible (210.A) mice by the ip route, three times a weeB. It has been postulated 

that /-Am2 has to be given in an at least two-fold higher amount than c-Am2n 

therefore, the two dosages may be considered similar. At the 7 th and 1[ th days postinfection, 

mice were analyzed according to responses in delayed-type hypersensitivity 

reactions, number of viable fungi isolated from spleen, epiploon, liver and lungs and 

nitric oxide (Nf) production in supernatants obtained from the organs above listed. 

Results and conclusions: Delayed-type hypersensitivity responses at the 7 th day 

were similar between the groupsn at the 1[ th day, mice submitted to either of the 

therapies showed lower responses than untreated subjects, developing a pattern very 

similar to the one presented by resistant animals in the murine model of PCM. At both 

time points, /-Am2-treated mice showed lower fungal loads and Nf secretion in all 

organs studied than untreated or c-Am2-treated mice. fur results show that /-Am2 

has a superior efficacy in diminishing the fungal load and establishing a resistant 

pattern of immune response when compared with c-Am2. However, the mechanisms 

implicated in this superiority are yet to be investigated and may involve 

immunostimulatory properties of /-Am2. 

Treatment studyM There was a clinical effect of the treatment on both the redness and lesion 

score. After 12 days of treatment 12/17 (71_) in the itraconazole-treated group were microscopy 

negative and 7/17 (O1_) culture negative in contrast to the control group where O/16 (2[_) were 

microscopy negative and 1/16 (6_) culture negative. Three animals in the itraconazole-treated 

group died of complications to the anaesthesia. 

Conclusions: An animal model was established with !/*#+,"& and >/*).,-+@$%'?2-.&. Pretreatment 

of the sBin with a manual razor was the easiest method to traumatise the sBin before 

inoculation. Inoculation under occlusion had no advantage. It was possible to score the clinical 

manifestationsn redness, and lesion. Infection with >/*).,-+@$%'?2-.& but not !/*#+,"& was 

shown to be inoculum dependent. The concordance between microscopy and culture versus 

histopathology was 61 | 72_, the histopathology being less sensitive. An itraconazole treatment 

study showed clinical effect thereby confirming the applicability of this model for evaluating 

antifungals.

P-0134. Azole-flucytosine cross-resistance and trends in antifungal 

susceptibility of clinical isolates in Germany from 1997 to 2004 

.chmalrecB A 1 , Fegeler W 2 , 2ecBer l 2 

P-0135. Influence of azole-antifungal therapy on the in vitro susceptibility and 

susceptibility patterns of clinical isolates 

.chmalrecB A 1 , CzaiBa G 2 , fssadniB l J , Czaika V O 

1 M2., Muenchen, Germany, 2 Institut f. Med. MiBrobiologie, ailhelms-6niversitxt 

Muenster, Muenster, Germany 

Purpose: Clinical isolates (N}7,[72) obtained from several consecutive collaborative 

studies in Germany over a period from 1KK6/K7 to 200O were subjected to susceptibility 

testing and hsusceptibility-pattern analysisi (.PA) in order to detect frequency, distribution, 

and prevalence of simple and multiple antifungal resistance (MAR). 

Susceptibility testing: .usceptibilities of clinical isolates and control strains to 

voriconazole (VfR), itraconazole (ITR), fluconazole (F/C), and flucytosine (FCY) were 

tested in parallel from the same inoculum, and by all centres with pre-manufactured 

antimycotic panels. Minimum inhibitory concentration (MIC) readings were performed after 

2O h incubation at J6 ÅC in Y.T medium (modified yeast nitrogen base with 2 _ glucose 

and [ mg methylene blue/l), and confirmed after O8 h, respectively the appropriate 

incubation time for slow growing yeasts. The MIC-results were stratified, and assigned to 

the susceptibility classesM . (susceptible), I (intermediate), and R (resistant). 

Results and discussion: MIC-distributions and characteristic MIC-values clearly 

demonstrated a significant greater in vitro activity of voriconazole against clinical yeast 

isolates (isolates with class .M K8 _ VfRn 8K _ ITRn K[ _ F/Cn K0 _ FCY), including non- 

7/*+68"#+,& (.M KO.0 _n 6[.O _n 78.0 _n 7[.0 _), and other azole- or FCY-resistant strains 

(VfR}., F/C-resistantM [6,[ _n ITR-resistantM 7J.1 _n FCY-resistantM KJ.0 _). Timedependent 

analysis of resistant strains showed a tendency of slight increase of FCY-, F/Cand 

VfR-resistant isolates from 1KK7 to 200O, and a significant increase of ITR-resistant 

isolates. Isolates which contributed to this trend were derived from non-7/*+68"#+,& and 

non-7+,5"5+ species, whereas 7/*+68"#+,& isolates remained almost at the same level. ff 

the 1.8 _ VfR-resistant isolates (n}1JJ) only [ _ (7) were susceptible to F/C, and 71 _ 

(K[) to FCY, however, none of the strains were susceptible to ITR. ff the ITR-resistant 

strains (n}7K1) J6 _, 67.8 _, and 7J.1 _ were susceptible to F/C, FCY and VfR, 

respectively. However, of the F/C-resistant strains (n}O00) only 10.J _, O6.0 _, and 

[6.[ _ were susceptible to ITR, FCY and VfR, and O2.2 _, [[.7 _ and KK.0 _ of the 

FCY-resistant strains (n}801) were susceptible to F/C, FCY, and VfR, respectively. 

.PA showed that only [1 of possible 22O VfR-ITR-F/C-FCY susceptibility-patterns (.P) 

were found, with .-.-.-. as the most prominent one (6O.1 _). MAR-analysis showed that 

from the 7,[72 .P, 1,O76 (1K.[ _) were associated with one or more resistance(s) to an 

antifungal agent (RAA) , i.e. 1,0[6 strains (70.1 _) with 1 RAA, 228 (1[.[ _) with 2, 16K 

(11.O _) with J, and 2J (1.[ _) with O RAA in combination. Those isolates completely 

cross-resistant to azoles and flucytosine (n}2J/0.J _) were distributed among 7+,5"5+* 

+68"#+,& (n}1), 7/*@6+8$+-+ (2), 7/*[$(&." ([), 7/*'+$+'&"6%&"& (1), 7/*-$%'"#+6"& (J), 

7$2'-%#%##(&*+68"5(& (6), 7$/*6+($.,-"" (J), and !+6+&&.d"+ spp. However, parallelresistance 

to the three azoles tested occurred more frequently (n}10[/1.O _) in the 

following speciesM 7/*+68"#+,& (n}J[), 7/*@6+8$+-+ (J0), 7/*[.U2$ (1), 7/*[$(&." (7), 

7/*6"'%62-"#+ (2), 7/*6(&"-+,"+. (1), 7/*'+$+'&"6%&"& (1), 7/*-$%'"#+6"& (11), 7$/*+68"5(& (6), 

7$/*6+($.,-"" (J), 7$/*,.%U%$)+,& (1), !+6+&&.d"+ spp. (2), and P?%5%-%$(6+ spp. (n}[). 

1 M2. Muenchen, Germany, 2 Medizinische lliniB I, DRl-lliniBen, 2erlin, Germany, 

J Med. FaBultxt, Charitd, Alexander von Humboldt 6niversitxt, 2erlin, Germany, 

O Zentrum f. Innere Medizin, Humaine-lliniBum, 2ad .aarow, Germany 

Purpose: 27J clinical isolates from 1[J patients (121 female, 1O8 male) pre-treated or 

under treatment with fluconazole (n}121) or voriconazole (n}1OO) were tested for their 

susceptibility to the azoles voriconazole (VfR), itraconazole (ITR), fluconazole (F/C), 

and bifonazole (2IF). In addition, susceptibility pattern (.P) analysis was performed to 

detect frequency and distribution of parallel-resistance of the antifungal agents used 

systemically (F/C, ITR, VfR), and to an azole applied topically (2IF). 

Susceptibility testing: The 27J isolates of 7+,5"5+ spp. (K7.8 _) and 

=+##?+$%)2#.& sp. (2.2 _) were tested by microdilution according to DIN [8KO0-8O. 

The minimum inhibitory concentration (MIC) of azoles was determined after 2O h 

incubation at J6 C in Y.T medium (modified yeast nitrogen base with 2 _ glucose 

and [ mg methylene blue/l), and readings were confirmed after O8 h. The 

susceptibility classes . (susceptible), I (intermediate), and R (resistant) as basis for 

the .P-pattern analysis was assigned to the MICs of the azoles F/C, ITR, and VfR 

according to the C/.I guidelines, and for 2FI according to its MIC-distribution profile 

and pharmacoBinetic parameters, the assumed breaBpoints of 2 mg/l for Ä.Ä and 

8 mg/l for ÄRÄ were used. 

Results and discussion: The strains derived from 2J wards were isolated from 21 

different specimen types (1[ sBin/wound swabs, OO throat swabs, 16 blood cultures, 

78 bronchial secrets, 10 sputum, 70 urine, 22 stool, and 18 genital specimens) 

demonstrated a high susceptibility rate of clinical strains to VfR (.M K8.2 _), 2IF (.M 

7[.8 _), F/C (.M 7O.0 _), however a very low .-rate to ITR (O.0 _). 

As the collective investigated included only a low number of commonly azolesusceptible 

isolates such as 7/*+68"#+,& (K.K _), the domination of ITR-lesssusceptible 

isolates such as 7/*@6+8$+-+ (O8.O _ of total isolates), 7/*@("66".$)%,5"" 

(1.[ _), and 7/*[$(&." (11.0 _) may be responsible for the high ITR-resistance rates 

(8[.7 _). ff the 11 F/C-resistant isolates (O.0 _), 2 (18.2 _) were susceptible to ITR, 

7 (6J.6 _) to VfR, and [ (O[.[ _) to 2IF, respectively. In contrast, of the 2JO ITRresistant 

strains, 22[ (K6.2 _) were susceptible to F/C, 2J1 (K8.7 _) to VfR, and 

1K1 (81.6 _) to 2IF, and of the 6O 2IF-resistant strains O7 (7J.O _) were susceptible 

to F/C, [ (7.8) susceptible to ITR, and 62 (K6.K _) susceptible to VfR. However, of 

the O VfR-resistant isolates (1.[ _) only 1 strain (2[.0 _) was F/C-, and 2 strains 

([0 _) were 2IF-susceptible, and none of these isolates were ITR-susceptible. ff the 

possible 22O .Ps only 1K could be found and no common .P was detectable within 

the different species. In addition, the clinical isolates showed different .Ps under 

therapy of F/C or VfR, and also species-specific .Ps. Isolates completely 

susceptible to all azoles (.PM .-.-.-.) were only found in 7/*@("66".$)%,5"" (n}1) and 

7/*[.U2$ (n}J). In addition, .P-analysis also demonstrated that for one strain 

(7/*[$(&.") a complete parallel-resistance to all four azoles under test (.PM R-R-R-R) 

occurred, and for 2 strains (7/*[$(&.", n}1n and 7/*@6+8$+-+, n}2), a parallel-resistance 

to VfR-ITR-F/C was recorded. 


P-0136. Effect of BAL8557, a water-soluble azole pro-drug, on the 

pharmacokinetics of ciclosporin 

Schmitt-Hoffmann A 1 , Roos 2 1 , .auer ( 1 , Maares ( 1 , 2rown T 1 , .picBermann ( 1 , 

Roehrle M 2 , Heep M 1 

1 2asilea Pharmaceutica /td., 2asel, .witzerland, 2 AAI Deutschland GmbH, Neu-6lm, 

Germany 

Background: 2A/O81[, an extended-spectrum azole that is administered orally or 

intravenously as a water-soluble pro-drug (2A/8[[7, a.A) is metabolized mainly by 

CYPJAO. Ciclosporin is also a substrate of CYPJAO, and both drugs inhibit CYPJAO 

activity at high concentrations. ae investigated whether dosing with a.A affects 

exposure to ciclosporin. 

Methods: In an open-label, multiple dose study, 26 healthy male subjects aged 18-O[ 

years received ciclosporin on day 1 and on day 22. a.A was given orally as a O00 

mg loading dose on day K and 100 mg/day afterwards (doses expressed as mg 

equivalent of 2A/O81[). 

Dosing scheduleM 

Drugs \ Study 

Days 

D1 D2 to 7 D8 D9 to 21 D22 D23 to 27 

a.A - - O00 mg 100 mg 100 mg 100 mg 

Ciclosporin J00 mg - - - J00 mg - 

.erial blood samples for pharmacoBinetic profiles of ciclosporin were obtained on 

days 1 and 22. 2lood samples were analyzed using a specific /C/M./M. method. 

Results: Dosing with a.A had no apparent effect on exposure to ciclosporin (Table). 

Inter-subject variability (CV_) of pharmacoBinetic parameters was 2J.7_-2O.8_ for 

C max and 28.K_-J0.1_ for A6C 00 . 

Parameter Ciclosporin Ciclosporin + WSA 

Number of .ubjects 26 2[ 

A6C 0-0 (&g1h/m/) 7.0[ (# 2.12) 7.7O (# 2.2O) 

C max (&g/m/) 1.O1 (# 0.J[) 1.[2 (# 0.J6) 

t max (h) 1.[ (1.0-O.0) 1.[ (1.0-O.0) 

Values are arithmetic mean # .D for A6C 0-0 and C maxn median and range for t max 

2oth drugs were well tolerated. No serious or significant adverse events (AEs) were 

reported, AEs observed in t2 subjects during a.A dosing were headache, fatigue, 

and flushing. /aboratory tests were unremarBable. 

Conclusions: a.A had no impact on the pharmacoBinetics of ciclosporin, a CYPJAO 

substrate, at the doses tested in this study. 

P-0137. In vitro interactions between immunosuppressive agents and 

antifungals against zygomycetes 

Schwarz P, /ortholary f, Dromer F, Dannaoui E 

Centre National de Rdfdrence Mycologie et Antifongiques, Institut Pasteur, Paris, 

France 

Objectives: Zygomycoses are emerging infections that particularly impact solid organ and bone 

marrow transplant recipients receiving immunosuppressive therapy. The Zygomycetes are poorly 

susceptible to most of the currently available antifungals. .ynergistic interactions between 

antifungal drugs and immunosuppressive agents have been demonstrated against 7+,5"5+ and 

4&'.$@"66(&. The aim of the present study was to evaluate the in vitro interaction of antifungals 

with either immunosuppressive agents or NiBBomycin against different species of Zygomycetes. 

Methods: C/.I MJ8-A microdilution broth technique modified for checBerboard studies was 

used to test Cyclosporin A (CyA), Rapamycin (Rapa) and Tacrolimus (Tacro) in combination 

with Amphotericin 2 (AM2), Itraconazole (Itra), Posaconazole (Posa) or Ravuconazole (Ravu). 

NiBBomycin (NiBBo) was tested in combination with Caspofungin (Caspo) or Micafungin (Mica). 

Ten Zygomycetes spanning five species were testedM J P?"d%'(&*%$2d+., 2 48&"5"+*#%$2)8"U.$+, 

2 !(#%$*#"$#",.66%"5.&, 2 P?"d%'(&*)"#$%&'%$(& var. $?"d%'%5"U%$)"&, and 1 P?"d%)(#%$*'(&"66(&. 

Microplates were incubated for 2Oh at J[ÅC and read spectrophotometrically. MICs were defined 

as the lowest concentration that resulted in [0_ (all drugs except AM2) or K0_ (AM2) growth 

inhibition. FIC indices (FICI) were calculated and interactions were defined as synergistic (FICI 

v}0.[), indifferent (FICI t0.[ and t}O), and antagonistic (FICI tO). Experiments were performed 

in duplicate. 

Results: Interactions of antifungals with immunosuppressive agents or NiBBo are presented in 

the table. 

Combination 

_ of isolates with following interaction 

.ynergistic Indifferent Antagonistic 

CyA r AM2 100 0 0 

CyA r Itra 60 O0 0 

CyA r Posa 60 O0 0 

CyA r Ravu J0 70 0 

Rapa r AM2 80 20 0 

Rapa r Itra O0 [0 10 

Rapa r Posa O0 60 0 

Rapa r Ravu J0 70 0 

Tacro r AM2 60 O0 0 

Tacro r Itra O0 [0 10 

Tacro r Posa J0 70 0 

Tacro r Ravu O0 O0 20 

NiBBo r Caspo 20 O0 O0 

NiBBo r Mica [0 O0 10 

Conclusions: The combination of immunosuppressive agents with AM2 resulted in synergy for 

more than 60_ of the isolates and up to 100_ for AM2rCyA. The combinations of 

immunosuppressive agents with azoles were synergistic less frequently than for AM2n again 

synergy was seen most often with CyA. Combinations of NiBBo with Caspo or Mica were mostly 

indifferent or synergistic but antagonism was seen in some cases. fverall, these in vitro results 

suggest that negative interactions between antifungal drugs and immunosuppressive therapy are 

unliBely in transplant recipients being treated for zygomycosis. Further studies in animal models 

of zygomycosis are warranted to evaluate the clinical relevance of these in vitro results.

P-0138. Prediction of the sensitivity of chromoblastomycosis agents to 

itraconazole and ketoconazole using DRIFTS/PLS 

Scroferneker M 1 , Corbellini V 2 , /ocB / 2 , FerrÉo M 2 

1 Instituto de Ciüncias 2Zsicas da .aâde, 2 Departamento de FÜsica e ~uÜmica da 

6niversidade de .anta Cruz do .ul 

/ess expensive and more rapid methods for the prediction of antifungal sensitivity 

allow better treatment of infected patients. This study assesses a chemometric 

method for predicting the sensitivity of chromoblastomycosis agents to itraconazole 

and Betoconazole by combining Diffuse Reflectance Infrared Fourier Transform 

.pectroscopy (DRIFT.) and Partial /east .quares (P/.) regression. Ten strains of 

chromoblastomycosis agents were submitted to a radial mycelial growth assay (RMG) 

on YEPD agar with itraconazole or Betoconazole in the concentrations of J.2, 0.08, 

0.02, 0.00[ and 0 $g/m/ in dimethyl sulfoxide (diluted 1M100 on agar). The biomass of 

each strain was produced by microculture (YEPD, 1O days, at J0 oC) and analyzed by 

DRIFT. between O00 and O000 cm -1 , and the spectra were correlated with RMG by 

P/.. In spite of the RMG-dose dependent responses of the strains, no satisfactory 

linear correlation could be observed between RMG and the biomass spectran however, 

it was possible to classify them based on their sensitivity to both antifungal agents 

used. 

P-0139. Coating of polyurethane for inhibition of Candida adhesion 

Segal E 1 , Zilberman M 2 , Navon A 2 

1 Dept. of Human Microbiology, .acBler .chool of MedicineTel-Aviv 6niversity, Tel 

Aviv, Israel, 2 Dept. of 2iomedical Engineering, Faculty of Engineering, Tel Aviv 

6niversity, Tel Aviv, Israel 

Indwelling medical devices made of polymeric materials, such as intravenous (IV) or 

urinary catheters are Bnown risB factors for development of systemic candidiasis, 

which is a significant cause of morbidity and lethality in imunocompromised and 

debilitated patients. 7+,5"5+ can attach to the polymeric surfaces, forming a biofilm, 

that may serve as a nidus for systemic, difficult to eradicate infection. 

The overall objective of this research was to develop a coating on polyurethane (P6), 

a polymer of IV catheters, that would reduce the adherence of 7+,5"5+ to the 

polyurethane surface. The coating was composed of a chitin soluble extract (C.E), 

that was previously shown to inhibit adherence to various target cells and inert 

surfaces. ae have used two methods in order to immobilize the C.E onto the P6 

surface | chemical binding and physical adsorption. 

fur results showed that adherence of 7/+68"#+,&*was*reduced by 7[_ for the 

chemical coating and by 8J_ for the physical adsorption coating/*It was also noted 

that the C.E molecules tend to aggregate and form relatively large formations of 

unique tertiary structure on the surface of the P6 film. C.E molecules tend to leave 

the P6 surface with time, but even after 11 weeBs the adhesion prevention effect is 

still significant. Hence, our new coatings may lead to a new generation of medical 

devices with surfaces that can prevent Candida adhesion. 


P-0140. Effect of benzalkonium chloride on Candida species biofilms formed on 

inoxidizable steel surface 

Seguy N 1, 2 , Mazars E J 

1 /ab. Microbiol., 6MR INRA 1282 - 6niv. 2ourgogne - Dijon, 2 /ab. Mycology - 6niv. 

2ourgogne, Dijon, France, J Medical biology /ab. | Hospital, Valenciennes, France 

The aim of this study is to evaluate the antimicrobial activity of a disinfectant, 

benzalBonium chloride on different 7+,5"5+ biofilms produced on inoxidizable steel 

plate as surface. 

The inoxidizable steel surface is an inert composite. Most of the studies have so far 

used plastic surfaces (e.g. silicon, polyethylene, .-#) as materials for experimental 

models of 7+,5"5+*sp. biofilm. These surfaces have an high adherence capacity. 

6sing an inoxidizable steel plate as surface, it is more inert and decreases the 

sticBiness of micro-organisms. This Bind of composites is attributed in both the 

industrial and medical fields (dental or others implants). 

Two 7+,5"5+ species were comparedM 7+,5"5+*+68"#+,& and 7+,5"5+*[$(&.". Their 

differences are specifically obvious liBe, their morphological forms (hyphal forms only 

observed in 7/*+68"#+,&), their habitats (commensal versus environmental), and their 

resistance to antifungal drugs. 

2efore to study the action of a quaternary ammonium salt, it was essential to establish 

the 7+,5"5+ sp. biofilm model. .everal physical parameters such as cell density, 

temperature, growth rate, and flow medium, were investigated to obtain a model of 

7+,5"5+ sp. biofilms. 

In our experimental conditions, the biofilm formation has the same characteristics than 

the ones of other authors, specially in the early stepsM adhesion and colonization of 

the steel surface. Nevertheless, after O8h of incubation, the biofilms constituted with 

the two species of 7+,5"5+ were composed of two layers of yeast cells. 7+,5"5+ sp. 

biofilms at 2[ÅC on inoxidizable steel plates with more layers and an extracellular 

matrix is produced when incubation times are longer than other experimental biofilm 

formation. More, the hyphal/pseudo-hyphal forms are observed only with 7/*+68"#+,& 

biofilm formation, and not on the 7/*[$(&." biofilm. 

Addition of benzalBonium chloride at different concentrations (1.[6, J.12, 6.2[ and 

12.[ kg/ml) on 7+,5"5+ sp. biofilms, didn]t reveale significant differences in cell 

viability between biofilm and planBtonic cells of 7+,5"5+M 12.[ kg/ml for 7/*+68"#+,& 

biofilm versus 6.2[ kg/ml for planBtonic cells of 7/*+68"#+,&, the same concentration 

(J.12 kg/ml) for 7/*[$(&." biofilm and planBtonic cells. It is interesting to note that at 

the lowest concentrations, this ammonium quaternary compound induced the 

production of pseudo-hyphal forms by 7/*[$(&." on inoxidizable steel plate surface. 

P-0141. In vitro activity of micafungin against Candida biofilms produced on 

central venous catheters 

Seidler M, .alvenmoser ., Myller F 

Pediatric Pulmonology 6niversity Heidelberg 

Purpose of the study: Patients with longterm peripheral vein or implanted catheters 

(e.g. HicBman/2roviac catheter) have an increased risB of Candidaemia and invasive 

candidiasis. 7+,5"5+ spp. can attach on polymers, create a protecting biofilm, pose a 

reservoir for entering the bloodstream and are less susceptible to antifungals. In 

particular, 7+,5"5+*'+$+'&"6%&"& can colonize catheters and blocB them due to biofilm 

formation. The new echinocandin Micafungin (MFG) was investigated for potential 

activity in 7+,5"5+*biofilms on central venous catheters (CVC). The aim of the study 

was to produce biofilm on CVC and investigate the role of micafungin in biofilm 

colonized catheters of six 7+,5"5+ spp. ",*0"-$%. 

Description of the project: 2iofilm of 7/*+68"#+,&, 7/*'+$+'&"6%&"&, 7/*5(86",".,&"&, 7/* 

-$%'"#+6"&, 7/*@6+8$+-+ and 7/*[.U2$ on CVC discs (HicBman catheters) was performed 

as follows. The HicBman catheter were cut in discs and pre-treated over night in K6- 

well plates with F2.. After washing with P2., 1x10†6 cells/m/ were added and 

incubated in RPMI 16O0 (r10_Glu, r10_F2., pH}6.[) at J7ÅC slightly rocBing for 

2Oh (7/*+68"#+,&) and O8h (7+,5"5+ spp.), respectively. The discs were transferred to 

a K6-well plate, the antifungals added and incubated at J7ÅC slightly rocBing for O8h. 

The ",*0"-$% antifungal activity of liposomal amphotericin 2 (/AM2), fluconazole (F/C) 

and micafungin (MCF) against 7+,5"5+ biofilms was assessed. MCF was tested at six 

concentrations (0.2[-J2 kg/ml). /AM2 (8 kg/ml), F/C (6O kg/ml) were used as 

controls. Measurements were done by jTT reduction assay, _ inhibition of metabolic 

activity was calculated in comparison to a growth control. 

Results and conclusions: fn catheter discs, all species produced mature biofilm in 

RPMI 16O0 (r10_Glu, r10_F2. pH}6.[) at J7ÅC for 2O-O8h production time slightly 

rocBing. /AM2 poses a max, F/C a min inhibition control. /AM2 displayed 70-8J_, 

F/C 10_-2[_ inhibition of metabolic activity, respectively. At all concentrations on 

catheter discs, MCF with a concentration up to 1 kg/m/ reached at minimum J0_ 

inhibition of metabolic activity in biofilm against all 7+,5"5+ spp.. aith regard to higher 

concentrations t2 kg/m/, 7/*+68"#+,&, 7/*'+$+'&"6%&"& and 7/*@6+8$+-+ are more 

susceptible to MCF with a minimum of 70_ inhibition of metabolic activity. All currently 

available antifungals have reduced activity in 7+,5"5+ biofilms. K,*0"-$%, MCF is active 

against 7+,5"5+ biofilm on HicBman catheters but 100_ growth inhibition cannot be 

reached by any of the antifungals tested. 

This worB is the first preliminary study to evaluate the effect of disinfectants on 

adherence and viability of 7+,5"5+*biofilms on inert surface. fur results reveale the 

importance of experimental conditions for biofilm modelization. A biofilm model as 

gold standart is necessary to compare studies produced by differents authors.

P-0142. Broad spectrum herbal therapy against superficial fungal infection in 

human beings 

Shahi S, .hahi M 

Microbiology Department, (anta Vedic PG College, 2araut, India 

The essential oil of >$+#?2&'.$)()*+))"*fruits was found to be the most potent 

antidermatophytic agent, inhibiting the mycelial growth of test pathogens viz., 

a'"5.$)%'?2-%,*U6%##%&()3*!"#$%&'%$()*@2'&.() and >$"#?%'?2-%,*$(8$() 

completely. The minimum inhibitory concentration (MIC) of the oil was found 0.1 $l/ml, 

0.2 $l/ml and 0.0[ $l/ml against a/*U6%##%&()3*!/*@2'&.() and >/*$(8$() 

respectively. The oil also exhibited potency against heavy doses of inoculum at 0.2 

$l/ml concentration. The gas liquid chromatographic (G/C) analysis of the oil indicated 

it to be a mixture of O minor (-pinene, camphene, -terpene, '-cymene) and 1 major 

(thymol) components. fn comparing MIC of the oil with that of prevalent synthetic 

antifungal drugs, the oil was found more effective. Moreover, oil did not exhibit any 

adverse effects on mammalian sBin upto 1_ concentration. Further, oil was subjected 

in the form of ointment to topical testing on human patients, attending out patient 

department (fPD) of M./.N. Medical College, Allahabad. J0 patients were selected, 

showing positive potassium hydroxide (lfH) results at the start of the trial. Patients 

were diagnosed as tinea corporis, tinea cruris or tinea pedis.*All patients were treated 

with 1_ ointment twice in a day for J weeB. At the end of medication, JJ.J_ of 

patient]s recovered complete cure, O0.0_ showed significant improvement from the 

disease. No lfH negative cases of relapse were observed when patients were 

reexamined after two month following the end of treatment thereby, denoting the 

absence of relapse. The ointment was found cost effective, had long shelf life and 

absence of any adverse effects. Thus, the essential oil could be used as potential 

sources of antidermatophytic agent after undergoing successful multicentre clinical 

trial. 

P-0143. Antifungal compound from the marine sponge dysidea herbacea 

Sionov E 1 , Roth D 1 , .andocsBy-/osica H 1 , lashman Y 2 , 2erdicevsBy I J , .egal E 1 

1 Department of Human Microbiology, .acBler .chool of Medicine, Tel-Aviv 6niversity, 

Ramat-Aviv, Israel, 2 .chool of Chemistry, Tel-Aviv 6niversity, Ramat-Aviv, Israel, 

J Department of Microbiology, The 2ruce Rappaport Faculty of Medicine, Technion | 

Israel Institute of Technology, Haifa, Israel 

.earch for new antifungal drugs is of significance in view of the increased frequency 

of fungal infections, particularly the systemic mycoses in compromised hosts and the 

problems associated with the currently available antifungal drugs. 

fur laboratory was involved in a screen of materials from various natural sources, 

including marine organisms and plants, for substances with antifungal activity. Thus 

far, {1000 samples (crude DM.f extracts) were tested using a broth microdilution 

method for assessing activity against 7/+68"#+,& and 4/U()"@+-(&. 

The compound isolated from the marine sponge V2&"5.+*?.$8+#.+ exhibited in vitro 

activity against the tested fungal pathogens. For determination of the possible mode of 

activity of the compound we tested the effect on fungal cellular morphology (light, 

scanning and transmission electron microscopy) and possible site of activity in the 

fungal cells, such as cell membrane (ion leaBage Binetics) as well as toxicity 

(cytotoxicity tests). 

The active compound was determined to be J,[-dibromo-2-(J,[-dibromo-2- 

methoxyphenoxy) phenol. The experiments on the mode of activity revealed that there 

are significant changes in fungal cell morphology, as demonstrated by scanning and 

transmission electron microscopy. The compound, apparently, affects the fungal cell 

membrane, expressed primarily in leaBage of potassium ions from the fungal cells. 

Further experiments in in vivo models are planned. 


P-0144. Prophylactic effect of posaconazole in experimental zygomycosis 

Spreghini E, 2archiesi F, .calise G 

Clinical of Infectious Diseases- 6niversitÖ Polietcnica delle Marche- Ancona Italy 

Zygomycosis (ZM) is an infection caused by fungi of the class L2@%)2#.-.&/*Members 

of this class cause progressive, necrotic, and generally fatal infections in 

immunocompromised hosts such as diabetics with Betoacidosis, neutropenic patients, 

patients taBing corticosteroids, and patients with elevated available serum iron levels. 

P?"d%'(&*%$2d+. (Rf) is by far the most common cause of infection. Another less 

frequently isolated species is 48&"5"+*#%$2)8"U.$+*(AC). Amphotericin 2 (AM2) is the 

choice for first-line therapy but mortality remains high, particularly in disseminated 

disease. Posaconazole (Pf.) is a triazole with broad-spectrum activity against fungi 

including members of the L2@%)2#.-.&/*Experimental infections as well as sporadic 

case reports described in humans demonstrated that Pf. has potential usefulness for 

ZM therapy. 

The goal of this study was to evaluate the prophylactic effect of Pf. in an 

experimental model of disseminated ZM. Cyclophosphamide treated mice were given 

Pf. (oral route) at 20, J0 or 80 mg/Bg/day or AM2 (intraperitoneal route) at 1 

mg/Bg/day on days -2, -1 and 2 h prior to infectionn a total of J days of therapy. The 

mice were then challenged intravenously with [x10 6 and [x10 [ spores of one strain 

each of Rf and*AC, respectively. Efficacy was determined by recordingM 1) the 

mortality on day 10 post infection (PI)n 2) the number of positive organs (brain and 

Bidney) cultured on day J PIn J) the number of the tissue fungal lesions. 

AM2 significantly prolonged the survival of mice infected with Rf (1 } 0.001) or AC (1 

}*0.0001). All Pf. regimens significantly prolonged survival (1*ranging from 0.001 to 

0.0001) in mice infected with AC but not in those infected with Rf. For each fungal 

isolate, an active infection (presence of hyphae in tissues) was noted in the Bidneys 

and brains of all control animals. Although for both strains, the percentage of culture 

positive organs in treated mice was generally lower than those seen in untreated 

controls, the only significant difference was seen in the brains from AM2 treated mice 

infected with AC (12.[_ 0& 100_, 1*} 0.001O). However, both drugs reduced the 

number of both brain and Bidney lesions in mice infected with AC. In mice infected 

with Rf, both drugs reduced the number of Bidney lesions but only AM2 was effective 

in reducing brain lesions. fverall, our in vivo data suggest that Pf. has the potential 

usefulness to prevent ZM in high risB patients. 

P-0145. Epidemiology and antifungal susceptibility of bloodstream Candida 

isolates in Quebec, Canada: 453 cases from 2003 to 2005 

St-Germain G, /averdimre M, Pelletier R 

1 Institut National de .antd Publique du ~udbec, ~uebec, Canada, 2 HÇpital 

Maisonneuve-Rosemont, J CH6~ HÇtel-Dieu de ~udbec, ~uebec, Canada 

2etween May 200J and April 200[ a population-based surveillance was conducted for 

7+,5"5+ bloodstream infections in ~uebec, Canada. A total of O[J cases of 

candidemia involving O6O yeasts isolated in [O hospitals were studied. The incidence 

rate was J.0 per 100,000 population. fver 7J_ of patients were aged [0 years and 

more. Clinical data was obtained for 76_ of patients. fverall hospital mortality was 

J8_. The most common predisposing factors were presence of an intravascular 

catheter (80_), use of antibacterial therapy (68_), stay in an intensive care unit (OK_), 

parenteral nutrition (J2_) and abdominal surgery (J1_). Fluconazole alone or in 

association with another antifungal was used for treatment in over 80_ of cases. 

7+,5"5+*+68"#+,& comprised 62.1_ of isolates, followed by 7/*@6+8$+-+ 16.8_, 7/* 

'+$+'&"6%&"& K.J_, 7/*-$%'"#+6"& O.[_, 7/*6(&"-+,"+. 2.8_ and 7/*[$(&." 2.6_. ff the 

288 isolates of 7/*+68"#+,&, 7 were resistant to flucytosine, one to fluconazole and 

none to itraconazole or voriconazole. ff 7[ non-+68"#+,& 7+,5"5+ isolates with 

reduced susceptibility to fluconazole (MIC % 16 g/m/), none were susceptible to 

itraconazole (MIC '0,12kg/m/) whereas 70 (KJ_) were susceptible to voriconazole 

(MIC '1 g/m/). Posaconazole, ravuconazole and caspofungin also demonstrated 

good ",*0"-$% activity against these isolatesn [[, K2 and 100_ had MICs '1 g/m/, 

respectively. However, on the average, isolates with higher fluconazole MICs tended 

to have higher MICs for the newer triazoles. Despite the extensive use of fluconazole 

in this patient population we report that 7/*+68"#+,&, 7/*'+$+'&"6%&"& and 7/*-$%'"#+6"& 

are rarely resistant to this antifungal.

P-0146. Susceptibility of allergenic fungi to azoles 

Steel N 1, 2 , Howard . 1, 2 , Moore C 1 , aarn P 2 , Denning D 2, J 

1 Microbiology Dept., Hope Hospital, .alford, 6l, 2 6niversity of Manchester, 6l, 

J aythenshawe Hospital, 6l 

Introduction: fver the last decade the importance of fungal organisms as allergens 

has increased. Allergic bronchopulmonary aspergillosis (A2PA) is a condition that 

occurs in some asthmatics and cystic fibrosis patients. The widespread use of 

itraconazole for the treatment of A2PA, and introduction of the new azole drugs 

voriconazole and posaconazole, led us to examine the azole susceptibility of other 

potentially allergenic moulds. 

Methods: .usceptibility of 18 mould isolates (belonging to 7 different genera) was 

determined by NCC/. MJ8-A microtitre method, with modified temperature and 

length of incubation (due to growth requirements of organisms being tested). RPMI 

(buffered to pH 7.0 using morpholinopropanesulfonic acid) was used as mediumn 

MICs were compared using RPMI supplemented with 2_ glucose and also without 

additional glucose. Drug concentrations for itraconazole, posaconazole and 

voriconazole ranged from 0.01[ to 8mg//. For 16 of the test isolates a 

spectrophotometric method for inoculum preparation was used, whereas the 2 

1.,"#"66"()*isolates were counted by haemocytometer, to produce a test inocula 

concentration range of 0.O x 10 O - [ x10 O cfu/ml. Microtitre plates were incubated in a 

moist chamber for 2 | 8 days at room temperature (2JÅC | J0ÅC), with 46-.$,+$"+ and 

>$"#?%'?2-%,*spp. at J0oC, and 1.,"#"66"()*at both temperatures. Minimum inhibitory 

concentrations (MICs) were read by eye with a no growth end-point. Minimum 

fungicidal concentrations (MFCsn tKK.KK_ Bill) were also determined. 

* 

Results: 

Organism 

46-.$,+$"+*+6-.$,+-+* 

MIC (mg/L) 

Itraconazole 

1 (n } 1) 

0.[ (n } 1) 

Posaconazol 

e 

0.12[ (n } 2) 

Voriconazole 

8 (n } 1) 

2 (n } 1) 

>$"#?%'?2-%,*spp.* 0.12[ (n } O) 

0.2[ (n } 1) 

0.12[ (n } 1) 

0.06 (n } 1) 

0.0J (n } 1) 

Geometric mean 0.20 0.09 1.12 

Mean 0.76 0.13 2.35 

Median 0.13 0.13 1.50 

0.[ (n } 2) 

0.2[ (n } 1) 

0.06 (n } 1) 

Range 0.03 - >8 8 

There are currently no interpretive breaBpoints for filamentous fungi, so isolates are described as 

having a low or a high MIC. All isolates had low MICs to posaconazole. fne 46-.$,+$"+*isolate 

and both a'"#%##() isolates had elevated MICs (t0.[mg//) to itraconazole.11/18 (61_) isolates 

had elevated MICs (t0.[mg//) to voriconazole. Incubating 1.,"#"66"()*(n } 2)*at J0ÅC changed 

MICs from 0.2[ to 1 and O, and from 1 and 2 to Omg// for itraconazole and voriconazole 

respectively. 

MFCs were generally close to MICs, with a rise of more than one dilution occurring in 22_ of 

isolates, particularly in >$"#?%'?2-%,*spp. and 1.,"#"66"()*(incubated at room temperature) only. 

Conclusions: Most allergenic fungi tested had low MICs to itraconazole, posaconazole and less 

so for voriconazole. MICs were similar when tested with RPMI with or without additional glucose. 

Testing methods have not been validated as yet and some caution is required when interpreting 

results. 

Z%-$2-"&*#",.$.+* 

0.0J (n } 1) 

0.06 (n } 1) 

v0.01[ (n } 2) 

0.2[ (n } 1) 

1 (n } 1) 

76+5%&'%$"()*spp.** 

0.2[ (n } 1) 

0.12[ (n } 1) 

0.06 (n } 1) 

0.12[ (n } 1) 

0.06 (n } 1) 

0.0J (n } 1) 

8 (n } 1) 

2 (n } 2) 

a'"#%##()*,"@$()* 

t8 (n } 1) 

2 (n } 1) 

0.[ (n } 1) 

0.12[ (n } 1) 

O (n } 1) 

2 (n } 1) 

A.6)",-?%&'%$"()*spp.* 

1.,"#"66"()*,%-+-()* 

0.[0 (n } 1) 

0.06 (n } 1) 

0.0J (n } 1) 

0.2[ (n } 2) 

0.06 (n } 2) 

0.2[ (n } 1) 

0.2[ (n } 1) 

0.12[ (n } 1) 

8 (n } 1) 

0.[ (n } 1) 

0.2[ (n } 1) 

2 (n } 1) 

1 (n } 1) 


P-0147. Utility of caspofungin against non-Aspergillus moulds: A review of 

susceptibility data for clinical isolates of the Pseudallescheria boydii complex 

and Scedosporium prolificans 

Sutton D 1 , McCarthy D 1 , Rinaldi M 1,2 , Fothergill A 1 

1 6niversity of Texas Health .cience Center at .an Antonio, .an Antonio, Tj, 6.A, 

2 Audie /. Murphy Division, .outh Texas Veterans Health Care .ystem, .an Antonio, 

Tj, 6.A 

Purpose of study: As published ",*0"-$% data are limited for the echinocandin 

caspofungin against members of the 1&.(5+66.&#?.$"+*8%25"" Complex and 

=#.5%&'%$"()*'$%6"U"#+,&3*we sought to review the antifungal susceptibilities for these 

highly refractory species from our clinical database. 

Description:*K,*0"-$% results for caspofungin were retrieved from a total of J2K clinical 

isolates of the 1/*8%25""*Complex (n}268) and =/*'$%6"U"#+,& (n}61) submitted to the 

Fungus Testing /aboratory (FT/) for susceptibility testing from the period (an 2000 to 

Feb 2006. The overall susceptibility of these strains presented is based upon 

minimum effective concentration (MEC) data obtained using the C/.I MJ8-A method 

in antibiotic medium J (M-J). Data for O8-hour readings for the subset of isolates 

evaluated against caspofungin is displayed as geometric means (GM), MEC [0s and 

MEC K0s in the table below. 

Results and Conclusions: Minimum effective concentration data suggests that levels 

of caspofungin required for clinical utility/efficacy may be beyond those safely 

achievable for these refractory etiologic agents. 

Fungus Testing 

/aboratory 

.trains evaluated 

Geometric Mean 

kg/ml 

MEC [0 

kg/ml 

1/*8%25""*Complex ,}86 12.87 16 J2 

S. PROLIFICANS N=31 K.[7 8 16 

MEC K0 

kg/ml 

P-0148. Mycological decontamination of dental unit waterlines using Oxygenal 

6 

Szymañska J 

Department of Paedodontics, Medical 6niversity, /ublin, Poland 

The concentration and composition of fungal flora in dental unit waterlines (D6a/) 

were evaluated. For this purpose, water samples from unit reservoirs, water samples 

flowing from high-speed handpieces, and swab samples from the waterline walls from 

2[ units were collected. Next, for the period of 2 weeBs, a D6a/ disinfection protocol 

was applied with the use of fxygenal 6 (laVo, Germany) containing hydrogen 

peroxide, and subsequently analogous samples from D6a/ were taBen. 

In the examined units, before disinfection, fungi were found in 12 reservoirs water 

samples the, in 16 | from the handpieces, and in 11 from the swabs and after 

disinfection fungi were found in 6, 7, 6 samples, respectively. 

In the examined samples, the yeast-liBe fungi 7+,5"5+*+68"#+,& and 7+,5"5+*#($0+-+ 

were found. 2esides, the following species of mould fungi were foundM 4&'.$@"66(&* 

+)&-.6%5+)", 4&'.$@"66(&*U()"@+-(&, 4&'.$@"66(&*@6+(#(&, 4&'.$@"66(&*$.'.,&, 

7"-$%)2#.& spp., 9.%-$"#?()*#+,5"5(), 1.,"#"66"()*+&'.$@"66"U%$)., 1.,"#"666"()* 

'(&"66(), 1.,"#"66"()*-($%6.,&. and =#6.$%-"()*&#6.$%-"%$(). 

2efore disinfection, at individual operative sites, the concentration of fungi in the 

reservoir water was from 0 to 6O[ ó 10 1 cfu/ml, in the water flowing from high-speed 

handpieces - from 0 to J7[ ó 10 1 cfu/ml, and in the swab extracts - from 0 to 11 ó 10 J 

cfu/ml. After disinfection, the concentrations of fungi in the water both from the 

reservoirs and from the handpieces were from 0 to O ó 10 1 cfu/ml, and in the swab 

extracts - from 0 to [ ó 10 1 cfu/ml. 

2efore disinfection, 7+,5"5+*#($0+-+ and 7+,5"5+*+68"#+,& constituted the greatest 

proportion of all the fungi in the reservoir watern in the handpiece water - 7+,5"5+* 

+68"#+,& and 4&'.$@"66(&*@6+(#(&, and in the swab extracts - 4&'.$@"66(&*@6+(#(& and 

7+,5"5+*+68"#+,&. After disinfection, in all three Binds of samples, 7+,5"5+*+68"#+,& 

prevailed, constituting from 1/J to 1/2 of all the fungi. 

Domination of 7+,5"5+*+68"#+,& may be related to a sucB-bacB of a patient]s saliva 

into the line due to the lacB of antireaction valves. 

The general concentration of fungi - the lowest in the water from a reservoir which is 

the water source in a unit, higher in the water flowing from a high-speed handpiece, 

and the highest in the swab extract - indicate that the biofilm (swab) present on the 

walls of waterlines may be a source of mycological contamination of the D6a/ water. 

A doubled number of fungal species in the swab extracts and in the water flowing from 

high-speed handpieces, in comparison to the reservoir water, seems to confirm the 

fact that in the biofilm there are good conditions for fungal multiplication and that the 

biofilm is a source of mycological contamination. 

Disinfectant application caused a significant decrease both in the number of total fungi 

and individual fungal species. This shows the antifungal effectiveness of a product 

containing hydrogen peroxide, in this case - fxygenal 6.

P-0149. A novel mycological method for evaluation of antifungal agents in the 

stratum corneum of guinea pigs 

Tomiyama S, NagasaBa ., Nishida N, .hiraishi l, .himamura T, Masui ., Majima T 

Pf/A Chemical Industries, INC. YoBohama, (apan 

Purpose of the study: Although the minimum inhibition concentration (MIC) test is 

one of the methods to evaluate antimycotics, the obtained MIC value does not always 

reflect the therapeutic effectiveness of topically applied antimycotics. It is not sufficient 

to predict clinical efficacy solely by MIC values. To evaluate the proper (real) activity of 

antimycotic agents ",*0"0%, a novel mycological analysis (scMICM*MIC in the stratum 

corneum) was investigated. 

Materials and Methods: /uliconazole s((-)- sRu-(E)- sO-(2,O -dichlorophenyl) -1,Jdithiolan- 

2-ylideneu -1-imidazolylacetonitrile,//CZ), a novel imidazole antifungal agent 

that is effective against filamentous fungi, Candida spp. Malassezia spp. and that 

entered the marBet in (apan last yearu, clotrimazole were used. Each antimycotic was 

dissolved with MefH and diluted with deionized water (two fold dilution method). 

Excised sBin prepared from the hind foot of guinea pigs was frozen under liquid 

nitrogen and punched for a diameter of Omm using a biopsy punch. A horizontal 

section of stratum corneum was then prepared by cryostat with a thicBness of 10 

micrometer and a depth of 1[0 micrometer from the surface of the foot. After 2 sets of 

sliced pieces of stratum corneum were soaBed in various concentrations of drugs, the 

conidial solution of >$"#?%'?2-%,*).,-+@$%'?2-.& was gently layered on to the one set 

of sliced corneum and cultured for one weeB under high humidity. Antifungal activity 

was evaluated for the growth of fungal elements under the light microscope as 

effectiveM no growth and invalidM growth. The dosage of no growth of fungi on the slice 

was considered the scMIC. Another set of sliced pieces were extracted with MefH 

and analyzed by liquid chromatography -tandem mass spectrometry (/C-M./M.) and 

the concentrations (microgram /cm J ) of the drugs in each sliced piece were 

determined. Drug concentrations extracted with MefH and the scMIC value were 

compared. 

Results and Discussion: //CZ showed growth inhibition at the lowest concentration 

(0.2[microgram/cm J ) used. However, the MIC value (scMIC) of //CZ and clotrimazole 

measured in the stratum corneum were inferior to the MIC measured by the regular 

dilution test. Generally, it was thought that protein binding of antifungal drugs is a 

major factor to inactivate antimycotics. The protein binding ratio of the antimycotics 

used in this study were equally high, at almost same degree. However, we found clear 

differences in the inactivation rate of 2 antimycotics using stratum corneum analysis. 

In conclusion, the proposed novel procedure that the drug concentration in sliced 

stratum corneum can measure may be a simple and convenient method to predict 

antifungal activity ",*0"0%/ 

P-0150. In vitro antifungal activity of voriconazole, itraconazole and 

caspofungin against Aspergillus spp strains isolated from patients with 

infections of respiratory tract and maxillary sinuses 

TrojanowsBa D 1 , Budak A 1 , 2ogusz 2 2 , fleBsiaB D 1 , NowaB P 1 

1 Dept. of Pharmaceutical Microbiology, (agiellonian 6niversity, lraBow, Poland, 

2 Department of Microbiology, Rydygier]s Hospital 

Aspergillosis is responsible for a high rate of morbidity and mortality in the 

immunucompromised patients. 4&'.$@"66(&*U()"@+-(&*and rarely other species such as 

4&'.$@"66(&*U6+0(&3*4&'.$@"66(&*,"@.$*are the most threatening aerial pathogens. 

The introduction of new antifungal compounds and the rising of antifungal drug 

resistance demand the necessity of the estimation ",*0"-$%*susceptibility of moulds 

The ",*0"-$% antifungal activity of voriconazole, itraconazole and caspofungin against 

2[ isolates of 4&'.$@"66(&*spp. were determined during studies. Fungi were isolated 

from infections of respiratory tract (sputum, 2A/) and maxillary sinuses (nasal swabs, 

bioptates from maxillary sinus and from orbit) of patients hospitalized in wards of 

haematology, intensive care unit and maxillary-facial surgery. Isolates were collected 

in the period from 200O to 200[. The 2[ 4&'.$@"66(&*spp. included 1[ strains of 

4&'.$@"66(&*U()"@+-(&3*O 4/*U6+0(&3*J 4/*,"@.$*and J 4/*#+,5"5(&*isolates. The 

susceptibility of the isolates was measured by the agar diffusion method (Etest, A2 

2iodisB, .weden). Conditions for the Etest method included the use of MfP. buffered 

RPMI medium with 2_ glucose, the inoculum of 10 6 spores / ml and incubation at J[ 0 

C for O8 h. 

The most antifungal activity ",*0"-$% confirmed for caspofungin. MICs ranged from 

0.012 to 0.12[ &g / ml for 4/*U()"@+-(&*isolates, 0.016 | 0.7[ &g / ml for 4/*U6+0(&3* 

0.016 | 0.02J ug / ml for 4/*,"@.$*and 0.002 | 0.0J2 &g / ml for 4/*#+,5"5(&/ MICs of 

voriconazole were similar (MIC rangeM 0.0KO | 1.0 &g / ml). The antifungal activity of 

itraconazole (0.0O7 | J.0 &g / ml) was lower than those for caspofungin and 

voriconazole. Twenty five percent of the strains were found to be resistant to 

itraconazole (one isolate of 4/*U()"@+-(&*MIC | 1.[ &g / ml, one 4/*,"@.$*MIC | 2 &g / 

ml and three all tested strains 4/*#+,5"5(& MIC 2-J &g / ml). 

fur results confirmed that caspofungin and voriconazole showed the most antifungal 

activity against 4&'.$@"66(&*spp. tested isolates. Their using in the treatment of 

aspergillosis may prognosticate therapeutical effects in immunocompromised patients. 


P-0151. Effect of oral itraconazole on the clinical manifestations and population 

of Malassezia in intractable atopic dermatitis patients 

Tsubuku H 1,2,O , .ugita T 2 , lato H 2 , NishiBawa A J , Tsuboi R O 

1 .elf Defense Foreces Central Hospital, 2 Meiji Pharmaceutical 6niversity, Department 

of Microbiology, J Meiji Pharmaceutical 6niversity, Department of Immunobiology, 

O ToByo Medical 6niversity, Department of Dermatology 

!+6+&&.d"+3 a lipophilic yeast naturally occurring on human sBin, is thought to be an 

exacerbating factor in atopic dermatitis (AD). In order to clarify the role of !+6+&&.d"+ 

in AD, we investigated the effect of oral itraconazole on the clinical manifestations and 

population of !+6+&&.d"+ in intractable adult AD patients. Itraconazole 100mg/day was 

administered for O weeBs, and the clinical effect was evaluated for more than 8 weeBs 

by the clinical manifestation of head and necB lesions using .CfRAD scores. Any 

concurrent treatment for AD involving topical steroids, topical tacrolimus or oral 

antihistamines was continued. The population density of !+6+&&.d"+ in the lesional 

sBin was directly quantified by a real time PCR assay using Taq-Man probe, and 

several !+6+&&.d"+ specific serum IgE antibodies were measured by Ala-.TAT. The 

results indicate that eczematous lesions in the necB and head areas improved in most 

of the cases, while the quantity of !+6+&&.d"+ in the lesions decreased. The level of 

several IgE antibodies also decreased in accordance with clinical improvement. These 

results suggest that Itraconazole may be an effective therapeutic agent for treatment 

of intractable adult AD. 

P-0152. Melanin protects Madurella mycetomatis against itraconazole and 

ketoconazole, first-line treatment agents against mycetoma 

van de Sande W 1 , de lat ( 1 , Ahmed A 2 , Verbrugh H 1 , van 2elBum A 1 

1 Erasmus MC, 6niversity Medical Centre Rotterdam, Department of Medical 

Microbiology and Infectious Diseases, Dr. Molewaterplein O0, J01[ GD Rotterdam, 

The Netherlands, 2 Mycetoma Research Group, Institute of Endemic Diseases and 

Faculty of Medical /aboratory .ciences, 6niversity of lhartoum, lhartoum, .udan 

The ability of certain microbes to produce melanin has been linBed to virulence and 

pathogenicity for their respective animal or plant hosts. The aim of this study was to 

determine the pathway used by !/*)2#.-%)+-"&*to form its melanin. Furthermore we 

wanted to Bnow if melanin protects the fungus against the host immune system or 

antifungal agents used to treat mycetoma infections. 

Fungal melanin can be formed via three different pathways, the DHN-, the DfPApathway 

and the pheo-pathway. 2y using inhibitors specific for these pathways we 

could establish that !/*)2#.-%)+-"&*uses the DHN- and Pheo-pathways to produce 

melanin. 

* 

Melanin has been Bnown to protect fungi liBe 7$2'-%#%##(&*,.%U%$)+,&*and 

4&'.$@"66(&*&''*to oxidants and even antifungal agents. From our experiments it 

appeared that melanin is an agent that blocBs the chemical reduction of TN2 into 

DTN2 by permanganate. 

Futhermore by using the recently published Yeastfne .ensitreB method for !/* 

)2#.-%)+-"&*MICs were determined with or without supplementation of melanin. 

.upplementation of !/*)2#.-%)+-"&*DHN-melanin resulted in an increase in MIC with 

[ two-fold dilution steps for the azoles itraconazole and Betoconazole. In short, a 16 

times more concentrated solution was needed to prevent fungal growth. This means 

that about 60_ of the strains considered susceptible to these tests appeared resistant 

after melanin-supplementation. This is worrying since both itraconzole and 

Betoconazole are antifungals routinely given to patients to prevent recurrent infections. 

No increase in MIC was found for the azoles fluconazole and voriconazole and the 

polyene amphotericin 2. MIC shifts under the influence of melanin have not been 

described yet. ahat has been described so far for fungal species liBe 7$2'-%#%##(&* 

,.%U%$)+,&*is that non-melanised cells are Billed faster than melanised cells with 

amphotericin 2. 

.ince itraconazole and Betoconazole are the drugs used in the clinic in preventing 

recurrent infections after surgery it should be noted that these drugs might not be the 

best choice. fther drugs liBe amphotericin 2 and voriconazole should be considered.

P-0153. Molecular mechanisms of resistance to 5-fluorocytosine in laboratory 

mutants of Candida glabrata 

Vandeputte P, /archer G, Pineau /, Chabasse D, 2ouchara ( 

Groupe deEtude des Interactions HÇte-Parasite, Angers, France 

Resistance to [-fluorocytosine ([-FC), which may result from multiple genetic events due to its 

complex mode of action, has been poorly investigated in 7+,5"5+*@6+8$+-+/ This yeast ranBs 

today the second among the causative agents of candidiasis, and represents a therapeutic 

hazard due to its poor susceptibility to azoles. This study was therefore conducted on laboratory 

mutants obtained by exposure of a wild-type isolate of 7/*@6+8$+-+ to [-FC. 

Antifungal susceptibility of the major causative agents of candidiasis, i. e. 7+,5"5+*+68"#+,&3*7/* 

@6+8$+-+*and*7+,5"5+*-$%'"#+6"&, was determined by a disB diffusion method on .hadomy agar 

plates using Neosensitabs tablets. For each specie, five unrelated isolates were tested. All 7/* 

-$%'"#+6"& isolates revealed totally resistant to [-FC, demonstrating the frequency of the innate 

resistance in this yeast. In contrast, large growth inhibition zones were observed for all the other 

isolates, sometimes containing randomly distributed colonies suggesting resistant mutants. 

However, resistant colonies were detected within the inhibition zones for all 7/*@6+8$+-+ isolates, 

but only for J 7/*+68"#+,& isolates. In addition, they were more numerous, suggesting a higher 

frequency of [-FC resistance in 7/*@6+8$+-+, which was was confirmed by determination of 

mutation rates. 

Ten resistant colonies from the growth inhibition zone obtained for a wild-type isolate of 7/* 

@6+8$+-+, were therefore subcultured and their susceptibility to [-FC and [-fluorouracil ([-F6) 

was determined. A reduced susceptibility or complete resistance to [-FC was observed for all 

mutants, whereas J of them remained susceptible to [-F6. Two mutants were further 

investigated, one presenting with a total resistance to both pyrimidin analogs, and the other with 

a reduced susceptibility to [-FC exclusively. 

Due to the frequency of petite mutations in 7/*@6+8$+-+, which lead to an overexpression of some 

efflux pumps and subsequently to azole resistance, the respiratory phenotype of the mutants was 

investigated. However, evaluation of oxygen consumption by oxygraphy demonstrated the lacB of 

respiratory deficiency in mutant cells. 

The main genes involved in [-FC metabolism were then sequencedM ^7kM*and ^7kMI which 

encode two cytosine permeases, as well as ^7kI (cytosine deaminase), ^TPI (uridine 

monophosphate pyrophosphorylase) and 7V7MI (thymidylate synthase). /iBewise, their 

expression level was quantified by quantitative real-time reverse transcription-PCR. fne mutant, 

resistant to both [-FC and [-F6, presented a missense mutation in the ^7kI gene and a 2-fold 

decrease in the expression level of the ^TPI gene. The second one presented a missense 

mutation in the ^7kM gene, associated with an overexpression of ^7kM and 7V7MI (2.8[ and 

O.8 induction factor, respectively). These last results suggested an altered function of FCY2p 

which was confirmed by determination of the intracellular accumulation of [-FC and which might 

be responsible for the altered suceptibility of this mutant to [-FC and its remaining suceptibility to 

[-F6. 

In conclusion, beside mutations in the ^TPI gene which represent the most common cause of 

resistance to [-FC, other mechanisms may contribute to [-FC resistance in 7/*@6+8$+-+ which, at 

least in vitro, may be easily acquired, probably due to its haploid genome. 

P-0154. Mechanisms of polyene resistance in two clinical isolates of Candida 

glabrata 

Vandeputte P 1 , /archer G 1 , Ernoult E 1 , 2ergms T 2 , Chabasse D 1 , 2ouchara ( 1 

1 Groupe deEtude des Interactions HÇte-Parasite, Angers, France, 2 /aboratoire de 

Gdndtique de la /evure, Poitiers, France 

The incidence of life-threatening fungal infections due to 7+,5"5+ species which are particularly 

frequent in severely immunocompromised patients, has increased marBedly during the past three 

decades. Among the causative agents of candidiasis, 7+,5"5+*@6+8$+-+ ranBs today the second 

and it represents a therapeutic hazard due to its innate poor susceptibility to azoles and to the 

frequency of its acquired resistance to these drugs. In contrast, resistance to polyenes, the other 

major class of antifungals, remains very rare. Although amphotericin 2 resistant yeast isolates 

are increasingly reported, resistance mechanisms have been poorly investigated, especially in 7/* 

@6+8$+-+/* 

2ehind the recent isolation in our hospital laboratory of two 7/*@6+8$+-+ isolates resistant to 

amphotericin 2, we attempted to determine their resistance mechanisms. These isolates were 

obtained from two elderly patients hospitalized in the same unit at eight month interval. The two 

isolates which were epidemiologically unrelated as demonstrated by multi loci sequence typing, 

presented a poor susceptibility to polyenes by a disB diffusion method, and an increased 

susceptibility to azoles. This was confirmed by determination of polyene and azole MICs using 

the E-test procedure. 

For both isolates, quantitative and qualitative analysis of the sterol content suggested an 

interruption in the late steps of the ergosterol pathway. Genes encoding the last enzymes of this 

pathway were therefore sequenced. fnly silent mutations were seen in the aP9E and aP95 

genes. In contrast, the aP9N gene, encoding the C2O sterolmethyltransferase, presented a 

missense mutation in one isolate and a %#?$. mutation in the other one. The ergosterol pathway 

was further investigated by quantifying gene expression levels by real-time reverse transcription- 

PCR, which revealed an induction of the expression of the genes usually located after aP9II. 

Furthermore, sterol chromatogram revealed an accumulation of carotenoid in the two isolates, 

suggesting also an induction of the isoprenoid pathway, which originates from an early sterol 

intermediate, the geranyl pyrophosphate. This was confirmed by quantifying the expression level 

of the geranyl-geranyl pyrophosphate synthase gene. 

2oth isolates were highly sensitive to calcofluor white, a marBer of the cell wall polysaccharides. 

Moreover, although 7/*@6+8$+-+ cannot produce hyphae except in special culture conditions, 

pseudohyphae were seen for one of our isolates whatever the culture medium used. The cell 

structure of both isolates was therefore analyzed by transmission electron microscopy which 

revealed an abnormal thicBness of the internal layer of the cell wall for the pseudohyphaeproducing 

resistant isolate. Cell structure was also affected for the other isolate, which presented 

numerous invaginations of the plasma membrane, and sometimes retraction of the cytoplasm in 

some areas. 

In conclusion, these mutations in the aP9N gene lead to a reduction in the ergosterol content of 

the plasma membrane, which might be responsible for polyene resistance, but also for changes 

of the cell structure by modification of membrane fluidity. Differences in the two mutational events 

may explain the different morphological features of the two isolates. Complementation studies 

which are now in progress, will help us to confirm the role of aP9N mutations in these phenotypic 

changes. 


P-0155. Inhibition of Cryptococcus neoformans by Candida albicans in vitro 

Vasilyeva N, Yelinov N, Chilina G, 2ogomolova T 

lashBin Research Inst. of. Med.Mycology 

AID. patients often develop concomitant infections due to various microorganisms. A 

little is Bnown about the interactions of pathogenic yeasts in associations. The 

purpose of the study was to investigate mutual effects of 7+,5"5+*+68"#+,&*and 

7$2'-%#%##(&*,.%U%$)+,& during cultivation in vitro. Nineteen clinical strains of 7/* 

,.%U%$)+,&3*isolated from patients in Russia,*and two strains of 7/*+68"#+,&, 

deposited in Russian Collection of Pathogenic Fungi (RCPF 10[8, RCPF 118[) were 

tested. Each 7/*,.%U%$)+,&*isolate was streaBed on .abouraud agar plate 

simultaneously with one of 7/*+68"#+,&*strains and incubated at J7 0 C. None of 7/* 

,.%U%$)+,&*isolates inhibited growth of 7/*+68"#+,&/*In contrast, 1J (68,O_) of 7/* 

,.%U%$)+,&*strains were repressed by both 7/*+68"#+,&*strains. .ome 7/*,.%U%$)+,&* 

isolates showed reduced growth and some isolates were unable to grow in the vicinity 

of 7/*+68"#+,&*streaB colony. The growth of 6 (J1,O_) 7/*,.%U%$)+,&*isolates was not 

influenced by 7/*+68"#+,&/*There was strain differentiation among 7/*,.%U%$)+,&* 

isolates according to the susceptibility to 7/*+68"#+,&*during simultaneous cultivation. 

This phenomenon may have importance in vivo and effect the clinical course and 

diagnosis of cryptococcosis in the case of mixed infection. 

P-0156. The effects of fenpropathrin and other compounds on the growth and 

presence of mites in fungal cultures 

Vismer H 1 , Marasas a 1 , Coutinho T 2 

1 PRfMEC, Medical Research Council, Cape Town, .outh Africa, 2 Forestry and 

Agricultural 2iotechnology Institute, 6niv. of Pretoria, Pretoria, .outh Africa 

The mycophagous mite, >2$%'?+@(&*'(-$.&#.,-"+. (.chranB), is one of the most 

destructive pests of fungal cultures, causing total decimation of fungal colonies, 

severe contamination and cross contamination of cultures with bacteria and fungi. 

Mites in fungal cultures are difficult to prevent particularly where primary isolations of 

fungi are done from plant material. Fenpropathrin (Meothrinw), 

Hexachlorocyclohexane (/indanew) and Dimethoate (Effecto aphicidew) are 

compounds either Bnown to have an effect on mites or were reported to be used in 

fungal cultures to combat mites. These compounds were investigated to establish 

their effects on the growth (colony diameter) of six species of fungi (^(&+$"()* 

0.$-"#"66"%"5.& MRC 826, ^/*@$+)",.+$()*MRC 6J0[, ^/*&(8@6(-",+,& MRC [8623* 

1.,"#"66"()*._'+,&() MRC 717O, 4&'.$@"66(&*U6+0(& MRC J78K*and*46-.$,+$"+* 

+6-.$,+-+*MRC 62J1), at different concentrations at 2[ and J0oC. Results indicate that 

hexachlorocyclohexane and dimethoate significantly reduced the colony diameters of 

all six fungal species at the concentrations and temperatures tested, and that these 

compounds are not suitable for incorporation into primary isolation media. 

Fenpropathrin had no effect on the growth of the fungi tested, even though slightly 

reduced colony diameters were recorded at J0oC. Following these results, the effects 

of media containing fenpropathrin at concentrations of 0.0[, 0.07[ and 0.1ml/l on 

mites in fungal cultures were investigated. Cultures were inoculated with a 

standardised ^/*0.$-"#"66"%"5.&*(MRC 826) inoculum and after being infested with equal 

numbers of adult >/*'(-$.&#.,-"+. mites, the plates were sealed with a double layer of 

parafilm to prevent them from escaping, and incubated for 2O, K6 and 1K2 hrs at 18 

and 28oC. Although results were variable after 2O hrs incubation, fenpropathrin had a 

more pronounced affect on the mites at 28 than at 18oC. fn average more mites 

survived at the lower temperature irrespective of the concentration used. 

Concentrations of 0,0[, 0.07[ and 0.1 ml/l gave good results after K6 hrs and 

eliminated >/*'(-$.&#.,-"+. mites in fungal cultures after 1K2 hrs at 28oC. In the 

control plates the number of mites, although active, remained stationary at 18oC, with 

no immature mites present. At 28oC mites on control plates were very active, 

immature mites were present and their numbers had increased by 70_ after 1K2 hrs 

incubation. Fenpropathrin is therefore recommended at concentrations of either 0.0[ 

or 0.07[ml/l in primary isolation media used for samples that may be infested with 

mites.

P-0157. In vitro activity of different antifungal drugs against clinical isolates of 

Cladophialophora carrionii from patients with chromoblastomycosis 

Vitale R 1,2 , Miguel A 2 , Perez 2lanco M O , Davel G 2 , Afeltra ( J , De Hoog . [ 

1 CfNICET, 2uenos Aires, Argentina, 2 INEI, AN/I. Dr. MalbrZn, 2uenos Aires 

Argentina, J Hospital (MR. MejÜa, 2uenos Aires, Argentina, O 6NEF, Coro, Venezuela, 

[ C2., 6trecht, The Netherlands. 

Propose of the study and Introduction: Chromoblastomycosis is a chronic disease 

characterized by localized sub-cutaneous lesions, leading to superficial to cauliflower-liBe 

tumours. The more frequent fungi that cause this disease are 7/*#+$$"%,""3*^/*'.5$%&%"*and*1/* 

0.$$(#%&+, etiologic agents being those introduced via cutaneous injury. In tissue, the fungus 

forms muriforms cells. As yet, no standard therapy is available and treatment needs long time 

including surgery, if possible, and oral antifungal drugs. However relapses are often observed. K,* 

0"-$% studies about susceptibility of these fungi are scant and studies are needed in order to find 

better approaches for therapy. 

Aim: To evaluate the antifungal activity of six drugs against 28 clinical isolates of 7/*#+$$"%,"" 

from patients with chromoblastomycosis. 

Summarized description of the project: Antifungal agents. The drugs tested wereM 

amphotericin 2 (AM2), itraconazole (ITZ), voriconazole (VCZ), (range)M 16-0.016 &g/mln 

terbinafine (T2F)M 8-0.008&g/mln flucytosin ([-FC) and fluconazole (FCZ)M 6O-0.06J &g/ml. 

Susceptibility test: The strains were tested using a broth microdilution format according to the 

C/.I guideline (MJ8A). 7/*[$(&.i ATCC 62[8 and 7/*'+$+'&"6%&"&*ATCC 2201K were used as 

control strains. .trains were grown on PDA for 7 days at J0oC. Inoculum was prepared by 

counting in a hemocytometer and dilutions in RPMI-16O0 with MfP. were prepared to obtain a 

final concentration of 0.O-[.0 x 10 O CF6/ml. Flat-bottom K6 well plates were incubated at J0oC 

and the MIC was read visually from day O to day 7. MICs were defined as followsM the lowest 

concentration of the drug which inhibited all visible growth for AM2 and for the remaining drugs, 

as the lowest concentration of the drug which inhibited [0_ of the growth compared to the 

growth control. Minimal fungicidal concentration (MFC) was determined by subculturing 100 kl 

from each well with no visible growth on .abouraud agar plates. Incubation was at J0oC for up to 

7 days. MFCs were defined as the lowest concentration at which KK.K_ of the inocula were 

Billed. 

Results and conclusions: The MIC Gmean, MIC [0 and MIC K0 wereM v0.008 kg/ml for T2Fn 

0.0[, 0.06 and 0.12[ kg/ml for ITZn 0.0[, 0.[, 1 kg/ml for VCZn O.J, 8, 16 for [FCn 11.6, 16, 16 for 

FCZ and 1.6, 2, 2 for AM2. The MFC Gmean, MIC [0 and MIC K0 wereM 0.0J, 0.06, 0.06 kg/ml for 

T2Fn 0.OO, 0.[, 1 kg/ml for ITZn 0.26, 0.12[, 0.[ kg/ml for VCZn 8K.J, 128, 128 kg/ml for [FCn 

[6.[, 128, 128 kg/ml for FCZ and %16 kg/ml for AM2. 

The most active drugs against this fungus were T2F, ITZ and VCZ. However, for all drugs more 

than two dilution steps were noted between the MIC and the MFC, indicating fungistatic activity. 

Given the fact that the disease is difficult to treat, further study of susceptibility of the group of 

fungi causing chromoblastomycosis is needed to develop optimal therapy. 

P-0158. In vitro activity of BAL4815 against zygomycetes 

Warn P, .harp A, Denning D 

.chool of Medicine, The 6niversity of Manchester 

Purpose of the Study: To compare the ",*0"-$% efficacy of 2A/O81[ with that of O 

triazoles and amphotericin 2 against Zygomycetes. 

Background: 2A/O81[ is a new water-soluble antifungal agent of the triazole class 

with broad-spectrum antifungal activity against yeasts and moulds. ae compared the 

",*0"-$% activity of 2A/O81[ (2A/) with that of itraconazole (ITC), posaconazole 

(Pf.), ravuconazole (RAV), voriconazole (VRC), and amphotericin 2 (AM2) against 

JO isolates of Zygomycetes*comprising of 48&"5"+*(n}20)3*P?"d%'(&*(n}J)3* 

P?"d%)(#%$*(n}[)3*7(,,",@?+).66+*(n}2)3*!(#%$ (n}J) and =#.5%&'%$"()*(n}1). 

Methods: .usceptibilities were determined for 2A/, ITC, Pf., RAV, VRC and AM2 

based on the microdilution plate modification of the C/.I MJ8-A accepted standard 

using RPMI 16O0 buffered to pH 7.0 with MfP. plus 2_ glucose. For all antifungal 

the final drug range was 0.01[-8mg//. The final inoculum used for all species was 1 

x 10 O /m/ and plates were incubated for O8 hours but read at 2O and O8 hours. The 

MIC was taBen as the lowest drug concentration that reduced growth by K0_ 

compared with the drug-free control 2A/, ITC, Pf., RAV and VRCn for AM2 the MIC 

was taBen as the lowest concentration, which inhibited all growth on visual examination. 

Results: MIC results are summarized in the table and figure. To calculate mean and 

geometric mean values of t8mg// were assigned the value of 16mg//. 2A/ was the 

only triazole with all MICs less than or equal to Omg//. MICs of tOmg/l occurred in 

2J_, 11_, J_ and 100_ with ITC, Pf., RAV and VRC. .trains that demonstrated 

",*0"-$% resistance to one triazole though often susceptible to alternatives, 

demonstrated slight increases in MICs of all triazoles. There was no association 

between MICs of azoles and those of AM2. 

MICs in mg// 2A/O81[ ITC Pf. RAV VRC AM2 

Range 2Oh 0.0J-O 0.01[- 

t8 

0.0J-t8 0.01[-O 8-t8 0.01[- 

2 

Range O8h 0.12[-O 0.06-t8 0.0J-t8 0.06-8 8-t8 0.06-2 

MIC [0 2Oh 0.[ 0.2[ 0.2[ 0.2[ 8 0.12[ 

MIC [0 O8h 0.[ 0.[ 0.2[ 0.[ t8 0.12[ 

MIC K0 2Oh 1 0.[ 1 1 t8 0.2[ 

MIC K0 O8h 2 t8 t8 O t8 0.[ 

Geomean 2Oh 0.OKK 0.21O 0.2[8 0.2O2 t8 0.10J 

Geomean O8h 0.78J 0.7[0 0.[J0 0.O70 t8 0.1K[ 

Mean 2Oh 0.810 0.78[ 1.J28 0.[08 t8 0.210 

Mean O8h 1.2J2 O.118 2.JK2 1.0[0 t8 0.J0J 

Conclusions: 2A/ demonstrated promising ",*0"-$% antifungal activity against 

Zygomycetes including 48&"5"+3*P?"d%'(&3*P?"d%)(#%$3*7(,,",@?+).66+*and*!(#%$ ",* 

0"-$%*including strains resistant to ITC, Pf. and VRC, and therefore warrants further ",* 

0"0% investigation. 


P-0159. A comparative clinical study between 2 weeks of luliconazole 1% cream 

treatment and 4 weeks of bifonazole 1% cream treatment for tinea pedis – 

single-blind comparative study- 

Watanabe S 1 , fgawa H 2 

1 Department of Dermatology, TeiByo 6niversity .chool of Medicine, 2 (untendo 

6niversity 

mycological effect was characterized by high negative rates of 76.1_ vs.7[.K_ 

(luliconazole vs. bifonazole). The progression of tinea-related signs and symptom 

scores differed insignificantly between evaluated luliconazole and bifonazole 

treatment groups comprising a total of [00 patients. 2oth substances appeared to be 

comparably safe and well tolerated. The incidence of adverse events for which a 

causal relationship to this drug could not be ruled out was low (2.[_). All of the 

adverse events were mild in severity and insignificant clinically. Clinical use has 

started in (uly 200[ in (apan. 

/ulicoanazole is a newly developed imidazole antifungal agent that is effective against 

filamentous fungi such as >$"#?%'?2-%,*/spp., and yeasts such as 7+,5"5+ spp. and 

!+66+&.d"+ spp with a dithiolan ring. In ",*0"-$% studies, the concentrations of 

luliconazole active against >$"#?%'?2-%,*).,-+@$%'?2-.& and >$"#?%'?2-%,*$(8$()3 

major pathogens causing tinea, have been found to be approximately 1/2.[ and 

approximately 1/O of those of lanoconazole, respectively, and equal to and 

approximately 1/2 of those of terbinafine hydrochloride, respectively. In ", 0"0%*studies, 

luliconazole cream at a concentration of 0.2[_ or more was sufficiently effective 

against tinea pedis in guinea pigs after application once a day for J days, and 

luliconazole at a concentration of 0.[_ or more completely eradicated fungi 

mycologically. Phase I and early Phase II clinical trials (for tinea pedis and tinea 

corporis/cruris) and a late Phase II clinical trial (for tinea pedis and tinea 

corporis/cruris) have been concluded and a comparative clinical trial was conducted to 

evaluate the therapeutic efficacy and safety of the drug applied once daily for the 

treatment of tinea pedis. The trial was performed using a single-blind protocol. 

MIC mg/L 

16 

8 

4 

2 

1 

0.5 

0.25 

0.125 

0.0625 

0.03125 

0.015625 

MICs at 24 hours 

AMB BAL ITC POS RAV VRC 

Drug 

Zygomycetes MICs 

MIC mg/L 

16 

8 

4 

2 

1 

0.5 

0.25 

0.125 

0.0625 

0.03125 

0.015625 

MICs 48 hour 

AMB BAL ITC POS RAV VRC 

Drugs 

Objective: To compare the efficacy and safety of luliconazole 1 _ cream and 

bifonazole 1_ cream as applied in the treatment of tinea pedis (interdigital-type and 

plantar-type). 

Design: A multi-clinic, randomized single-blind, parallel group study 

Setting: JO hospitals and 11 clinics 

Patients: [11 patients with mycologically confirmed tinea pedisM ff the O8K evaluable 

patients, 2O7 were randomized to luliconazole, and 2O2 to bifonazole. 

Interventions: /uliconazole 1_ cream applied once a day for two weeBs, followed by 

a placebo cream for two weeBs, thereafter. 2ifonazole 1_ cream applied once a day 

for four weeBs. 

Main outcomes measured: Mycological cure (negative result on microscopy) and 

improvement of sBin lesions measured at weeBs 1, 2, J and On culture study measured 

at weeB 2M .afetyn Frequency and severity of adverse reactions. 

Results and conclusion: The improvement of sBin lesions after O weeBs was 

comparably good, with rates of K1.[_ vs. K1.7_ (luliconazole vs. bifonazole). The

P-0160. Topical anti-dermatophytic agent “liranaftate”-its 2% liquid showed 

good efficacy and safety similarly to 2% cream 

Watanabe S 1 , Hiruma M 2 , TaBiuchi I J , NishiBawa T O , fgawa H 2 

1 Department of Dermatology, TeiByo 6niversity .chool of Medicine, 2 Department of 

Dermatology, (untendo 6niversity .chool of Medicine, J Department of Dermatology, 

.howa 6niversity FujigaoBa Hospital, O Department of Dermatology, leio 6niversity 

.chool of Medicine 

/iranaftate is a novel thiocarbamate showing a high antifungal activity towards 

dermatophytes. Its 2_ cream, ÄZefnartw cream 2_Ä has been approved in (apan for 

the treatment of tineas under the application schedule of once a day to the infected 

area. .ince the post marBeting survey in the past [ years had revealed clinically good 

efficacy and safety of the cream, we conducted this clinical trial to add a liquid 

formulation which would enhance treatment compliances. 

This clinical trial was a multi-center (1J medical centers), open-label and randomized 

study to demonstrate non-inferiority of liranafate liquid 2_ (test agent) to liranafate 

cream 2_ (control agent) under clinical relevancy of 10_. The primary endpoint was 

an efficacy made up of two independent evaluation items, i.e., dermatophyte 

disappearance rate (negative conversion rate) and sBin symptom improvement rate 

(sBin improvement rate) at aeeB O. The non-inferiority required that the K[_ 

confidence intervals (CIs) for the difference of both evaluation items between two 

prepatations, e.g., the rate of liquid minas the rate of cream, were not below the 

margin of -10_. 

A total number of subjects randomized in this study were 2K0 (1OO cream, 1O6 liquid), 

and 272 subjects of which were eligible and evaluable at aeeB O (1J7 cream, 1J[ 

liquid). The negative conversion rates were 70.1_ (K6/1J7) for cream and 7O.8_ 

(101/1J[) for liquid, and the lower end of the K[_ CI of the difference was -[.K_. The 

sBin improvement rates were KO.K_ (1J0/1J7) for cream and K7.0_ (1J1/1J[) for 

liquid, and the lower end of the K[_ CI of the difference was -2.[_. Adverse drug 

reactions such as local irritation were observed in 2.8_ (O/1OO) of subjects treated 

with cream and in 2.1_ (J/1O6) of subjects treated with liquid (1}0.722). Abnormal 

laboratory findings were found in one subject each treated with cream (elevated urine 

glucose) and treated with liquid (elevated A.T). 

The above results demonstrate that liranaftate liquid 2_ is non-inferior relative to 

liranaftate cream 2_ in its efficacy and that it is as equally safe as cream 2_. 

P-0161. The peptide hLF1-11 as broad spectrum prophylaxis and treatment of 

fungal infections in immunocompromised patients 

Wulferink M 1 , 2rouwer C 1 , (onB / 1 , .luiter-van DijB A 1 , 2lijlevens N 2 , Donnelly P 2 , 

Velders M 1 

1 AM-Pharma 2.V., 2unniB, the Netherlands, 2 Department of Hematology, 6MC .t. 

Radboud, Radboud 6niversity Nijmegen, the Netherlands 

h/F1-11, a peptide comprising the N-terminal eleven amino acids of human lactoferrin, 

has previously been shown to be more effective against a wide variety of bacteria and 

fungi ",*0"-$% than the complete lactoferrin protein. Typical MIC values for most of the 

pathogens were 2[-[0 &g/ml. Furthermore, h/F1-11 was shown to be at least a 

thousand times more potent ",*0"0% than predicted by the ",*0"-$% data. In several 

models of systemic and focal mycoses and bacterial infections, h/F1-11 showed to be 

highly effective even at doses of O &g/Bg and below. 

This and other data support the idea that h/F1-11]s mechanism of action is not 

restricted to direct Billing of the pathogen, but that indirect immune modulatory and 

immune activating properties are important for h/F1-11]s efficacy at low doses*",*0"0%. 

RemarBably, despite the apparent depency of h/F1-11]s antimicrobial potency on the 

immune system, it was shown that in mice treated with both cyclosporine and 

cyclophosphamide, h/F1-11 was still highly effective in reducing pathogen load. As a 

model for drug-drug interaction, it was shown that the concomitant use of conventional 

antibiotics and h/F1-11 did not show any adverse effects. TaBen together, these data 

indicate that h/F1-11 can be used to effectively prevent and treat fungal and bacterial 

infections in immune suppressed patients. 

fne group of patients that show high incidence of fungal infections, accompanied with 

high mortality is the group of hematopoetic stem cell transplantation (H.CT) recipients. 

The fact that h/F1-11 may act through the immune system, however, may be a risB 

factor for aggravation of graft versus host (GvH) disease in these recipients after 

transplantation. To exclude this possibility, h/F1-11 was used in an animal model for 

GvH. Preliminary data show no effect of h/F1-11 on aggravation of the disease. 

2esides these preclinical safety data, clinical safety was evaluated in a single and 

multiple ascending dose study in human volunteers. During these studies 

pharmacoBinetic and pharmacodynamic parameters were measured and no drug 

related adverse effects were observed. Currently, preparations are made to evaluate 

safety and efficacy of h/F1-11 in autologous and allogenic H.CT patients for its use 

as anti-fungal and anti-bacterial prophylaxis. In conclusion, h/F1-11 is a promising 

peptide for the prevention and treatment of opportunistic infections in H.CT patients 

and other immune suppressed patient populations. 


P-0162. Oropharyngeal candidiasis in HIV/AIDS patients and antifungal 

susceptibility towards fluconazole 

Xess I, (ain N, Vajpayee M 

Department of Microbiology, All India Institute of Medical .ciences, New Delhi, India 

aith the increasing number of HIV/AID. cases in India, opportunistic infection has 

also flared up. fropharyngeal candidiasis (fPC) is the most common opportunistic 

infection associated with HIV/AID.. Introduction of fluconazole prophylaxis showed 

reduction in the rate of fungal infection. However, the risB of fluconazole resistance 

has been increased. The study was conducted to determine the frequency of fPC in 

the HIV/AID. patients in India and to determine the trend of antifungal susceptibility 

pattern of fPC towards fluconazole. The strain variability in fPC strains was 

determined by RAPD using M1J typing. For this study we included OK7 HIV sero 

positive patients at various stages of disease progression, attending the HIV/AID. 

clinic of All India Institute of Medical .ciences in the period of (uly 0[ | February 2006. 

A total number of 127 patients (2[.[_) were found with oral lesions. fut of this 2K 

randomly selected patients were tested for fPC. fut of those 2K, a total number of 22 

patients (76_) were found to be infected with fPC. 7+,5"5+*+68"#+,& was the 

predominant species causing the fPC (82_, n}18). Three patients were documented 

for the mixed infection of 7+,5"5+ sp. The CDO count of these 22 patients was 187 r 

17J and CD8 count varied by 1J8J r 7O2. CDOMCD8 ration was 0.1[ r 0.11. 

Antifungal susceptibility towards fluconazole was determined by micro dilution of 

NCC/. M-27A. The MICs value range between 0.12[ - t16kg/ml. The only one 

isolate of 7/*[$(&." showed resistance (6Okg/ml) in vitro for fluconazole. 2y RAPD 

strain variability was detected. ae conclude that fPC is still prevalent in HIV infected 

patient even after the prophylaxis of fluconazole. 7/*+68"#+,& is the predominant 

causative agent for fPC. High MICs value and strain variation in fPC isolates 

warrant for continuous monitoring of fPC patients. 

P-0164. Evaluation of the effects of incubation temperature and ph on the in 

vitro susceptibility test for clotrimazole against Candida albicans isolates from 

women with recurrent vaginitis 

Zarrinfar H 1 , Yadegari M 1 , Riazipoor M 2 , Farahnejad Z 1 , latiraee F 1 

1 Department of Mycology Tarbiat Modares 6niversity,Iran, 2 Department of 

Microbiology 2aghiatallah 6niversity,Iran 

Candidiasis, as an opportunistic infection, is created by the 7+,5"5+ species. 

Although 7+,5"5+*+68"#+,& is classified in the body as endogen flora, it plays an 

important role in creating 7+,5"5+ related diseases. 7+,5"5+ vulvovaginitis in 

pregnant women, diabetic mellitus and the patients using multiple antibiotics and 

contraceptive drugs, demonstrates high resistance against the conventional 

medication. fn the other hand, the recurrent vaginitis disintegrates the long-term 

process of treatment in majority of the patients. At the present research, which is 

aiming at determining the optimum conditions for the susceptibility testing before the 

retreatment of the patients. 10 isolates of 7+,5"5+*+68"#+,& obtained from J1 patients 

suffering from recurrent Candida vaginitis and drug of Clotrimazole, were used at two 

pH 7.2 and [.[, and at two temperature J[oC and 27oC. The Microdilution broth test 

technique was performed to do this. The RPMI 16O0 medium within the K6 well 

microplates with range of 12 tests was used to determine the MIC[0 , MICK0 and 

MFC Clotrimazole drug. The obtained MIC[0, MICK0 and MFC for Clotrimazole at 

these conditions (T}J[oC and pH}7.2) were v 0. 2[ to 1 kg/ml, 1 to O kg/ml and 128 

to [12 kg/ml respectively, while these values at temperature 27oC and pH [.[ were 0. 

[ to 2 kg/ml, 2 to 8 kg/ml, 6O to% [12 kg/ml and at temperature J[oC and pH [.[ were 

0. [ to 2 kg/ml, 2 to 8 kg/ml, 2[6 to % [12 kg/ml and at temperature 27oC and pH 7.2 

were 0. [ to 2 kg/ml, O to 16 kg/ml, 6O to % [12 kg/ml, respectively. The obtained 

results confirmed that the temperature of J[oC and pH 7.2 in comparison to the other 

conditions, produced better treatment outcomes

P-0165. Griseofulvin down-regulates expression of beta-tubulin gene in 

Trichophyton rubrum 

Zomorodian K 1 , lordbacheh P 1 , 6thman A 2 , Tschachler E 2 , Rezaian M 1 , Tarazooie 

2 1 , lhorramizadeh M 1 , louloumvaBi A 2 , Rezaie . 1 

1 Tehran 6niversity of Medical .ciences, Tehran, Iran., 2 Medical 6niversity of Vienna, 

Vienna, Austria. 

>$"#?%'?2-%,*$(8$() (>/$(8$()) is a cosmopolitan arthropophilic dermatophyte which 

causes sBin and nail infection. The most of antifungal drugs available for clinical use 

are targeted four molecules includingM beta-glucan, sterol-alpha-demethylase, 

ergosterol and DNA/RNA synthesis. In the present study we characterize the gene 

encoded beta-tubulin protein in Trichophyton rubrum and investigate the effect of 

griseofulvin of its expression. 

Clinical isolated of >/$(8$() were cultured in liquid medium and the nucleic acids 

(DNA c RNA) were isolated from obtained mycelial mass by standard methods. The 

sequences of Bnown beta-tubulin genes in other fungi were aligned and pairs of 21 nt 

primers were designed from highly conserved regions. 6sing mentioned primers, we 

amplified predicted molecules from genomic DNA as well as cDNA of >/$(8$() as 

PCR templates. Effect of griseofulvin on expression of this gene was evaluated by 

Real-Time PCR. 

Finally, 186O nucleotides have been sequenced from this gene (accession number 

AY76J78K) which encodes a polypeptide with OO7 amino acids (accession number 

AAVJJ7JJ). Nucleotide sequence comparison in gene data banBs (NC2I, NIH) for 

both the partial DNA and its deduced amino acid sequence revealed significant 

homology with members of the euBaryotic beta-tubulin genes. The amino acid 

sequence of the encoded protein was about 82_ identical to the sequence of betatubulin 

proteins of the other fungi. Interestingly, griseofulvin suppressed the 

expression of beta-tubulin gene in >/*$(8$() in dose dependent manner while 

fluconazole had no effect on its expression. 

CLINICAL MYCOLOGY 

P-0166. Sleeping with the enemy: What lies beneath in your bed pillows 

Fothergill A 1 , Rinaldi M 1, 2 , .utton D 1 

1 6niversity of Texas Health .cience Center, 2 Audie /. Murphy Division, .outh Texas 

Veterans Health Care .ystem 

Purpose of Study: aith an ever-increasing focus on environmental effects on health, 

the spot light has been on hsicB buildingi syndrome. The allergic response to 

environmental moulds is well-documented but following a 6l report by aoodcocB, et 

al., we wanted to determine the fungal burden that could be found in bed pillows that 

were collected from a variety of environs within the 6. and that varied by both age 

and content. Pillows tested included two from a retired couple]s home, three from a 

family with two small children, two from a young married couple, two from hotels and 

one from a commercial aircraft. 

Description: Testing was conducted by three methods. The first method involved 

utilizing an Anderson .ampler to draw air through the pillows. The particulate matter 

transported in the suctioned air was deposited onto a .abouraud Dextrose Agar plate. 

The second method was a direct culture of both a 1 cm portion of the outer cover and 

an approximated similar portion of the pillow filler. Finally, a second 1 cm square of 

the outer cover and a portion of the filler were each added to 10 ml of sterile distilled 

water, vortexed well and subcultured to a .abouraud Dextrose Agar plate. 

Results and conclusion: were surprising, not in the volume of fungal contamination 

noted, but by the absence of fungi in many of the samples taBen. .pecies recovered 

were typical of environmental culture including 1.,"#"66"()*spp.3*4&'.$@"66(&*spp.3* 

76+5%&'%$"()*spp.3*and*P?"d%'(&*spp. Results of microbial burden are as followM 

.ample N}10 .terile 2acteria fnly 2acteria/Fungi 

Anderson .ampler [ J 2 

2ulB Culture Cover 0 J 7 

2roth Culture Cover O 6 0 

2ulB Culture Filler O 2 2 

2roth Culture Filler 6 0 O 

In conclusion, we found that the fungal burden of bed pillows from the 6. was not a 

significant source of fungal exposure to those sensitive to fungal allergens. This is 

most liBely due to the bed linens that are used to cover bed pillows and are repeatedly 

changed and laundered. 


P-0167. Madurella mycetomatis the prime cause of eumycetoma: What do we 

know about this fungus? 

Ahmed A 1, J , van de .ande a 2 , Fahal A J , van /eeuwen a 2 , Verbrugh H 2 , De Hoog . O , 

van 2elBum A 2 

1 Department of Pathology and Microbiology, College of Medicine, ling .aud 

6niversity, .audi Arabia, 2 Erasmus MC, Department of Medical Microbiology and 

Infectious Diseases, Rotterdam, The Netherlands, J Mycetoma Research Center, 

6niversity of lhartoum, lhartoum, .udan, O Centraalbureau voor .chimmelcultures, 

6trecht, The Netherlands 

Eumycetoma is mostly caused by the fungal species !+5($.66+*)2#.-%)+-"&. The 

disease is characterized by extensive subcutaneous masses, often in combination 

with sinuses which drain pus, blood and fungal grains. The disease affects individuals 

of all ages, although the burden is most severe among worBing class adults, since it is 

extensively invalidating. In endemic areas compared to tuberculosis, malaria and the 

HIV/AID., !/*)2#.-%)+-"& can be considered an underestimated, but still socioeconomically 

important disease. 6ntil recently, little basic research was done in 

eumycetoma due to !+5($.66+*)2#.-%)+-"&, and most literature is describing clinical 

cases on mycetoma manifestation and therapeutic studies on limited number of 

patients. ae here present a review on the developments in the clinical, 

epidemiological, and diagnostic management of !/*)2#.-%)+-"& induced mycetoma. 

This involves the recent description of molecular diagnostics and genetic typing 

procedures, methods which were not only used for patient management but also for 

environmental research and pathogencity. In addition, fungal susceptibility tests 

utilizing different in vivo and in vitro methods will also be presented. 

P-0168. Phenotypic identification of Candida dubliniensis among Candida 

albicans strains isolated from sterile body fluids. 

Akcaglar S 1 , Ener 2 2 , Tore f J 

1 6ludag 6niv. .chool Medicine Dep. of Microbiology, 2 6ludag 6niv. .chool Medicine 

Dep. of Microbiology, J 6ludag 6niv. .chool Medicine Dep. of Microbiology 

7/*5(86",".,&"& is a recently described 7+,5"5+ species associated with oral 

candidosis that exhibits with a high degree of phenotypic similarity to 7+,5"5+* 

+68"#+,&. Its importance based on rapidly developing resistance to fluconazole, a 

common anti-fungal drug which used for treatment of mycoses. In retrospective 

analysis of stocB collections between 1KK[ and 200[ years, aproximately a total of 

1000 7/*+68"#+,& clinical isolates that obtained from steril body fluids (C.F, 2lood, 

urine, etc.) of K00 patiens who admitted to the 6ludag 6niversity .chool of Medicine 

Hospital 2ursa/TurBey. Analyses were performed in Mycology /aboratory of 

Microbiology Department of the same hospital. These clinical isolates were identified 

as a 7/*+68"#+,& on the basis of their cultural and morphological characteristics, 

growth on .abourauds Dextrose Agar (.DA), germ tube production and production of 

chlamydospore on cornmeal agar and stored -80 o C. 

The present study was carried out to determine the prevalence and clinical 

significance of 7/*5(86",".,&"& in a tertiary-care hospital in our region. 7/*+68"#+,& 

isolates from stocB collection were examined again with germ tube formation in human 

serum after J h at J7 o C, chlamydospore production on corn meal agar and staib agar, 

ability to grow at O[ o C on .DA, colour development on CHRfMagar, opasity test in 

Tween-80 agar and carbohydrate assimilation properties by API ID J2 C. Among 1000 

isolates of 7/*+68"#+,& , four strains were identified as 7/5(86",".,&"&, giving an overall 

occurrence of 0,O _.

P-0169. Localization of heat shock protein 90 (hsp90) in Cryptococcus 

neoformans 

Akeel R 

6niversity ff Manchester 

.tress proteins or heat shocB proteins (hsp) are a group of proteins that are present in 

all proBaryotic and euBaryotic cells in all life forms. These proteins maBe vital functions 

in the folding and unfolding or translocation of proteins, as well as in the assembly and 

disassembly of protein complexes. In addition, they play an important role in 

bothhumeral and cellular immunity. Heat shocB proteins are classified into different 

subfamilies based on their molecular mass and high degree of amino acid. 

In fungi, heat shocB protein is a stress-induced protein involved in many cellular 

processes including the regulation of signal transduction and steroid hormone 

response pathways in higher euBaryotic cells such as hsp70 in Cryptococcus 

neoformans and hspK0 in Candida albicans. 

In C. albicans, immunodominant O7-BDa was identified as heat-stable fragment of 

Candidal hspK0. It was found as one of the target for the immune system in patient 

with systemic candidiasis. Antibody to the O7-BDa antigen was found in serum of 

patients with chronic and mucocutaneous candidiasis as well as in HIV patients. A 

human recombinant antibody fragment to epitope /lVIRl of the O7-BDa was found 

protective. 

In this study, the human recombinant antibody against hspK0 was used in 

immunoelectron microscopy to investigate the cellular localization and induction of 

hspK0 in Cryptococcus neoformns. Two different methods of immunoelectron 

microscopy were used, pre-embedding and post-embedding immunolabelling. The 

hspK0 was found in the capsule materials of C. neoformans. .ince the K0-BDa protein 

found on the surface of the cells, hspK0 may be directly involved in sensing 

environmental changes and also be important for recognition of its host or elements of 

the host immune system and antibody responses to the molecule and may therefore 

be useful for diagnostic or prognostic evaluation. 

Phase II clinical trail of mycograb is due to commence in the third quarter of this year. 

P-0170. Rhino-orbito-cerebral mucormycosis: A rare pathology in Tunisia 

Anane ., laouech E, lallel l, 2elhadj ., ChaBer E 

Parasitology-Mycology Department of Rabtaes Hospital, Tunis 

Rhino-orbito-cerebral mucormycosis is the most common and most serious infection 

caused by mucorales, opportunistic fungus, saprophytes of the ground, the air and the 

plants in decay. 

ae report 1O cases of rhino-orbito-cerebral mucormycosis diagnosed between 1KK2 

and 200[ at the department of Parasitology-Mycology at the Rabta]s Hospital, Tunis. 

They are eight men and six women whose average age is [1 years, all were 

hospitalized for diabetic Betoacidosis, a major risB factor for this mycosis. 

.pecific symptom was observed in nine casesM appearance of an erythematous 

plaque taBing place at the root of the nose, extending quicBly on the surface and indepth 

coupled with the appearance of necrotic lesions. These necrotic lesions turned 

into scabs thus generating local functional complications (decrease of vision, 

oculomotrice paralysis) and general ones (hemiplegia, coma). 

The diagnosis was confirmed by mycologic examination among all patients (direct 

examination and/or culture on .abouraud medium) identified P?"d%'(&*%$2d+. in 10 

cases. 

In spite of the antifungal treatment (amphotericin 2 and/or fluconazole) associated to a 

surgical resection among three patients, the death occurred in nine cases within two 

to J0 days. Two patients were cured at the price of an orbital exenteration in one case 

and of an enucleation and a prosthesis setting in the other case, and three others 

were improved without serious after effects. 


P-0171. Aortic bioprosthesis Candida guilliermondii endocarditis: 

Ineffectiveness of oral voriconazole treatment 

Anne G 1 , Alain T 1 , Pierre A 2 , /udovic D J , (ean baptiste . O , (acques V 1 

1 .ervice cardiologie, CH6 Angers, France, 2 .ervice des maladies infectieuses et 

tropicales, CH6 Angers, France, J .ervice de parasitologie, CH6 Angers, France, 

O .ervice de chirugie cardiaque, CH6 Angers, France 

A 7[ year-old man was hospitalized for left hemiplegia. His medical history showed 

hypertension, chronic lymphoid leuBaemia fifteen years ago treated by chlorambucil, 

an aortobifemoral bypass, a right carotid endarterectomy and an aortic valvular 

replacement by bioprosthesis for a severe aortic stenosis associated with an aortocoronary 

bypass three years ago. The post operative period was marBed by a 

regressive homonymous hemianopia. His recent medical history was marBed by a 

hospitalisation one month before for palpitations. ECG showed an atrial flutter treated 

by vitamin l antagonists. 2ecause of inadequate anticoagulation (INRM 1.8), he was 

rehospitalized for a regressive left hemiplegia with persistent atrial flutter. The 

transoesophageal echocardiography (TfE) did not found atrium thrombi but a 

hyperechogenic image of J mm on the aortic bioprosthesis evocative of thrombus or 

vegetation. In addition he presented an episode of hyperthermia with increased CRP 

(6O mg/l). Microbiological cultures (2D 2ACTEC P/6. AERf2IC - 2ECTfN 

DIClIN.fN and Company) were negative. Two month later, a new TfE showed the 

same image with another one evoBing a paraprosthetic abscess. 2lood cultures on 

specific medium (2D 2ACTEC MYCf.I. IC/F | 2ECTfN DIClIN.fN and 

Company) were positive for 7+,5"5+*@("66".$)%,5"". The diagnosis of fungal 

endocarditis was established and treatment by intravenous fluconazole was begun. 

2ecause of an intermediate sensibility, fluconazole was stopped and oral voriconazole 

(O00 mg/day) was begun the day after. The origin of the infection was not found 

(ungual prelevement, fecal and urine samples), only one buccal sample was positive 

for 7/*+68"#+,&. The cerebral magnetic resonance imaging did not found any mycotic 

aneurysm but old ischemic cerebral vascular lesions. The preoperative blood cultures 

were always positive for 7+,5"5+*@("66".$)%,5"". The minimal inhibition concentration 

(0.12[ to 0.1K kg/ml) confirmed the sensibility of 7+,5"5+*@("66".$)%,5""*to the 

voriconazole. The voriconazole in the blood was measured at K.[ mg/l. A third TfE 

showed a progression of the valvular lesions with an image of periannular abscess. 

After multidisciplinary discussion, a cardiac surgery was decided with replacement of 

the aortic bioprosthesis. The operation confirmed two vegetations on the ventricular 

verse of the cusps but no annular abscess. Analysis of the bioprosthesis (highperformance 

liquid chromatography method-HP/C-DAD) found voriconazole in the 

valvular tissueM 7.8 ng/mg and J ng/mg respectively in 18 mg and 1OO mg pieces. 

Cultures on specific medium were positive for C.*@("66".$)%,5"". 2lood cultures on the 

same specific medium were negative four days after the cardiac surgery. In the 

postoperative period, oral voriconazole O00 mg/day was continued. Nine days after 

the operation, the patient presented an asystolia. He died despite of cardiopulmonary 

reanimation. 

P-0172. HIV-related histoplasmosis: 8 cases observed in a single center in Italy 

Antinori S 1 , Magni C 1 , 2onaccorso C 2 , Galimberti / 1 , Parravicini C J 

1 Dept.Infectious and Tropical Diseases, / .acco Hospital, Milan, Italy, 2 Microbiology 

6nit, / .acco Hospital, Milan, Italy, J Pathology 6nit, / .acco Hospital, Milan, Italy 

Background: Histoplasmosis is a common opportunistic mycosis among HIV-infected 

patients living in geographic areas where the fungus is endemicn in Europe HIVassociated 

histoplasmosis is rarely observed and if not considered in the diagnostic 

worB-up it may be easily overlooBed. 

Purpose of the study: To retrospectively study (1KKJ-200J) 8 cases of AID.- 

associated |PDH observed at the / .acco Hospital in Milan, Italy 

Results: 6 M/2Fn patients(pts) were from 2razil (J), Ecuador (1), Ivory Coast (1), Italy 

(J)n median age was 2K,[ years ( range 2K-O2 years) n median CDOr 6/&/, range 2- 

18[n in all patients but two PDH was considered importedn Median time from arrival to 

Italy and development of PDH was J2,[ months (range 16-8O)n PDH was the AID.- 

presenting illness in 6 ptsn .ymptoms of presentation were M fever (8), dyspnea (J), 

sBin lesions (O), oral ulcers (2), weight loss (J), lymphadenopathy (J), abdominal pain 

(2), pancytopenia ([), anemia (J)n three patients had a sepsis-liBe syndrome. Chest j- 

ray showed diffuse interstitial or micronodular lesions in 6 pts and normal appearance 

in the remaining two pts. Diagnosis was made during life in [ pts (O A"&-%'6+&)+* 

#+'&(6+-() var. #+'&(6+-(), 1 A"&-%'6+&)+*#+'&(6+-() var. 5(8%"&"") and at postmortem 

examination in the remaining three pts. A"&-%'6+&)+*#+'&(6+-() var. 

#+'&(6+-()*was cultured from blood (J), bone marrow (2), sBin (J)n histology was 

positive from lymph node (2), bone marrow (J), sBin (J). Five pts died four of whom in 

hospital and underwent autopsy that showed a disseminated disease (J-11 organs 

involvement, median 6)n Four pts were treated with amphotericin 2 followed by oral 

itraconazole in threen one was treated with oral itraconazole. 

Conclusions: PDH is unusual in Europe but increasing immigration from endemic 

areas might be responsible of increasing encounter of this opportunistic infectionn 

clinical presentation is not specific and may mimic tuberculosis, atypical 

mycobacteriosis lymphoma as well as cryptococcosis and penicilliosisn PDH should be 

considered in any febrile pts with late stage HIV infection who travelled or arrived from 

endemic areas. The long interval between last residence in or travel to endemic areas 

and arrival in Italy is consistent with a reactivation of a latent infection as the main 

pathogenetic mechanism of the disease. 

This case showed for the first time the presence of voriconazole in an aortic 

bioprothesis infected by*7/*@("66".$)%,5"". Despite of the treatment for three weeBs 

before surgery, 7/*@("66".$)%,5"" persisted in the valve and justified the operation. The 

responsibility of voriconazole was suspected in the occurrence of asystolia but the 

pharmacologists eliminated this hypothesis.

P-0173. Identification of dermatophyte in the nail by PCR-RFLP analysis 

Anzawa K 1 , lawasaBi M 1,2 , MochizuBi T 1,2 , IshizaBi H 1 

1 Division of Dermatomycology (Novartis Pharma), Research Institute of Medical 

.cience, lanazawa Medical 6niversity, (apan, 2 Department of Dermatology, 

lanazawa medical 6niversity, (apan 

Introduction: Identification of dermatophytes that cause tinea unguium is difficult 

using conventional methods because cultivation of specimens obtained from finger 

and toe nails taBes a long time. Here, we evaluated whether a much faster method, 

polymerase chain reaction (PCR) restriction fragment length polymorphism (RF/P) 

analysis, is useful for identifying dermatophytes obtained from human nails. 

Methods: DNA from K1 specimens from [K patients with clinically suspected tinea 

unguium was obtained by J0-min incubation at 100°C of the crushed nails suspended 

in Tris-.D. buffer containing 2-mercaptoethanol, extraction in phenol-chloroform and 

precipitation in 2-propanol. The internal transcribed spacer (IT.) region between 18. 

and 28. rDNA was amplified by PCR using primers for IT.1 and IT.O. PCR products 

were digested with restriction enzymes A",fI, !0+I, then electrophoresed. 

Identifications were made by comparing the band patterns with Bnown patterns 1) . 

Identifications made by PCR-RF/P were only taBen as positive when the band pattern 

matched those of already identified dermatophytes. 

For comparison, the specimens were cultured on .abouraud]s dextrose agar 

containing chloramphenicol for a month at room temperature, and isolates identified 

by morphology and PCR-RF/P. 

Results: Eleven specimens that were not positively identified by any three methods, 

microscopy, PCR-RF/P, and culture, were considered not to be infected and excluded 

from the comparison. Positive identifications of specimens could be made for 7J 

(K1_) by microscopy, 72 (K0_) by PCR-RF/P analysis and 1J (16_) by culture. 

.ixty-five (8K_) of those identified and 7 of those not identified by microscopy were 

positively identified by PCR-RF/P. All 1J specimens identified by culture were 

positively identified by both microscopy and PCR-RF/P. .pecimens from [J patients 

were identified by at least one of the methods, and OK (8J_) of these were positively 

identified by PCR-RF/P. 

.pecimens from OK patients were identified by PCR-RF/P, and of these, >$"#?%'?2-%,* 

$(8$() was identified in specimens from JJ (67_) patients and >/*).,-+@$%'?2-.&*in 

specimens from 1O (2K_), and both >/*$(8$() and >/*).,-+@$%'?2-.&*in the other 2. 

Conclusion: PCR-RF/P analysis is very valuable in the identification of 

dermatophytes in human nails. 

Reference: 

MochizuBi T .-*+6., ( Dermatol .ci 200Jn J2M 2[-J2 

P-0174. Identification of species from scale of dermatophytosis lesion by PCR- 

RFLP analysis 

Anzawa K 1 , lawasaBi M 1,2 , MochizuBi T 1,2 , IshizaBi H 1 




Introduction: In addition to conventional methods, restriction fragment length 

polymorphism (RF/P) of ribosomal-DNA amplified by polymerase chain reaction 

(PCR) has been used to identify dermotophytes 1) . ae used PCR-RF/P to identify 

dermatophytes in scale specimens. 

Methods: Ninety-four scale specimens from [2 patients with clinically suspected 

dermatophytosis were examined in the Department of Dermatology, lanazawa 

Medical 6niversity. The specimens were identified by microscopy, PCR-RF/P 

analysis and culture. For the PCR-RF/P analysis, DNA was extracted by J0-min 

incubation at 100°C of the scale suspended in Tris-.D. buffer containing 2- 

mercaptoethanol, extraction in phenol-chloroform and precipitation in 2-propanol. DNA 

was dissolved in distilled water, and used as template for PCR-amplification of the 

internal transcribed spacer (IT.) region between 18. rDNA and 28. rDNA, using 

primers for IT.1, IT.O. PCR products were digested with restriction enzymes A",fI, 

!0+K, then electrophoresed. Identifications were considered positive only when the 

band patterns matched those of dermatophytes already identified. 

For comparison, the specimens were cultured on .abouraud]s dextrose agar 

containing chloramphenicol for a month at room temperature, and isolates identified 

by morphology and PCR-RF/P. 

Results: ff the 78 specimens, 16 which could not be positively identified by any three 

methods were considered not to be infected excluded from the study. Positive 

identifications of specimens could be made for 68 (87_) by microscopy, 67 (86_) by 

PCR-RF/P analysis and JK ([0_) by culture. Fifty-seven (8O_) of those identified 

and 10 of those not identified by microscopy were positively identified by PCR-RF/P. 

All of the specimens identified by culture were positively identified by microscopy and 

all but one by PCR-RF/P, the exception being identified as >$"#?%'?2-%,*$(8$() by 

PCR-RF/P but as*>/*).,-+@$%'?2-.& by culture. 

.pecimens from O6 patients were identified by at least one of the methods, and 

specimens from OJ (KJ_) of these were positively identified by PCR-RF/P, 

specimens from 1K (OO_) as >/*$(8$()3 21 (OK_) as >/*).,-+@$%'?2-.&3 and J (7_) 

as both >/*$(8$() and >/*).,-+@$%'?2-.&. .pecimens from 2K (6J_) patients were 

identified by cultur, 10 (JO_) as >/*$(8$(), 18 (62_) as >/*).,-+@$%'?2-.&, and 1 

(J_) as both. 

Conclusion: PCR-RF/P is an effective and quicB method for identifying 

dermatophytes. 

Reference: 

MochizuBi T .-*+6., ( Dermatol .ci 200Jn J2M 2[-J2 


P-0175. Keratitis due to tilletiopsis minor after surgical treatment of myopia 

Aurore K 1 , Martine G 2 , Aurdlien F J , (ean-Claude G O 

1 /aboratoire de parasitologie-mycologie,CH6 Nice,France, 2 /aboratoire de 

parasitologie-mycologie,CH6 Nice,France, J .ervice deophtalmologie,CH6 

Nice,France, O Facultd de Pharmacie,Chatenay Malabry,France 

Mr FER. A, a sixty-three year-old non-insulin dependant diabetes patient, presented a 

fungal corneal abscess seven days after surgical treatment of myopia by /A.ER. The 

isolate, Tilletiopsis minor, has been involved, to our Bnowledge, only twice in human 

disease. fur observation is the first case of Beratitis ever reported. 

The patient was cured after receiving voriconazole ( eye-lotion and per os ) for three 

months and a half. 

P-0176. Open label phase IIa trials to evaluate the effects of short term oral 

pramiconazole in Tinea pedis and Tinea cruris/corporis 

Ausma J 1 , Decroix ( 2 , aouters / 1 , 2orgers M 1 , Vandeplassche / 1 

1 2arrier Therapeutics nv, Geel, 2elgium, 2 Avenue Du Parc JK, Mouscron 

Pramiconazole, previously referred to as R1266J8, is a novel azole with potent 

antifungal activity against dermatophytes, such as >$"#?%'?2-%,*$(8$(), >$"#?%'?2-%,* 

).,-+@$%'?2-.& and !"#$%&'%$()*#+,"&, which cause tinea pedis and tinea 

cruris/corporis. 

Aim: To evaluate the efficacy of oral treatment with a daily dose of 200 mg R1266J8 

in tinea pedis and tinea cruris/corporis for J or [ consecutive days (D). 

Methodology: The effects of pramiconazole on mycological outcome were evaluated 

by lfH microscopy and culture on scales taBen at the border of an active lesion. For 

the clinical signs and symptoms, a global clinical evaluation was made and signs and 

symptoms were evaluated. At every follow-up visit the global clinical evaluation was 

made on a [-point scale (0}worse, 1}unchanged, 2}mild/moderate improvement, 

J}marBed improvement, O}cured). .igns and symptoms were all scored on a O-point 

scale and the sum of the score was calculated for every visit. Twenty subjects were 

included in the tinea pedis studyM 10 subjects in cohort I (JD treatment) and 10 

subjects in cohort II ([D treatment), whereas 18 subjects were included in the tinea 

cruris/corporis studyM K subjects in cohort I (JD treatment) and K subjects in cohort II 

([D treatment). The evaluations were performed before inclusion, after DJ (cohort I) or 

D[ (cohort II), after D1O and D28 of treatment. 

Results: Percentage responders at day 28 for mycological (culture and lfH 

microscopy) and clinical evaluations are presented in Table 1. The sum of the sign 

and symptoms score at the individual visits is presented in Table 2. There were no 

.AEs reported, limited AEs reported (O in total), no trial discontinuations due to an AE. 

Table 1M Percentage responders at Day 28 

n lfH negative Culture negative Clinical responseç 

T. pedis JD treatment 10 60_ O0_ K0_ 

T. pedis [D treatment 10 70_ 70_ K0_ 

T. cru/cor JD treatment K OO_ OO_ 78_ 

T. cru/cor [D treatment K 8K_ 67_ 100_ 

çimarBed improvementi or hcuredi 

Table 2M Effect on the total sign and symptoms score 

Inclusion Days after onset of treatment 

D1 DO/6 D1O D28 

T. pedis JD treatment 10.2 7.8 (0.00O) J.J (0.002) 1.1 (0.002) 

T. pedis [D treatment K.[ 6.6 (0.002) J.[ (0.002) 1.O (0.002) 

T. cru/cor JD treatment 6.1 [.0 (0.06J) 2.K (0.00O) 1.8 (0.00O) 

T. cru/cor [D treatment [.6 O.6 (0.0J1) 2.7 (0.00O) 0.8 (0.00O) 

Mean values (two-sided P-values of the ailcoxon signed ranB test versus value at baseline visit (D1)) 

Conclusion: Results from these Phase II trials indicate that pramiconazole appears to 

be effective for the treatment of tinea pedis and tinea corporis/cruris with relatively 

short oral treatment courses. Further studies using short term treatment with 

pramiconazole in tineas are warranted to confirm the observed effects.

P-0177. Invasive otomycosis due to Cladosporium cladosporioïdes at an 

immunocompetent patient: About one case 

Badre Eddine L 1 , Hafida N 1 , Mourad 2 1 , Noureddine E 2 , 2rahim A 2 , .ouad C J , aafa 

E 1 

1 Parasitology and Mycology laboratory, Military Hospital Mohamed the fifth, Hay riad, 

Rabat, 2 ENT .ervice, Military Hospital Mohamed the fifth, Hay riad, Rabat, J Radiology 

service, Military Hospital Mohamed the fifth, Hay riad, Rabat, 7 Parasitology and 

Mycology laboratory, Military Hospital Mohamed the fifth, Hay riad, Rabat 

ftomycosis represent [ to J0_ of all external otitis in the world. 

ae report the first case in morocco of otomycosis due to 76+5%&'%$"()* 

#6+5%&'%$"%"5.&*finding at the brain abscess stage. The patient was 17-year old 

apparently healthy, human immunodeficiency virus seronegative, presenting an 

overlooBed fetid otorrhea. He was admitted to emergency for a febrile intracranial 

hypertension. A cerebral and middle ear Computed Tomographic scan were 

performed, they show a left temporal abscess and an oto-mastoiditis cholesteatoma. 

The diagnosis of middle otitis chronic cholesteatoma with mastoiditis was established. 

The mycologic exam of the biopsy was realised. Fungal elements were observed in 

direct micrsocopical exam and the fungus was isolated repeatedly on culture media. 

The fungus was identified as 76+5%&'%$"()*#6+5%&'%$"%"5.&. The patient could not 

have any treatment because he is died one weeB after diagnosis. 

P-0178. Spondylodiscitis due to Trichosporon beigelii: About one case and 

review of the literature 

Badre Eddine L 1 , Mourad 2 1 , Hafida N 1 , Hassan ~ 2 , Youssef . 2 , Driss G 2 , aafa E 1 

1 Parasitology and Mycology /aboratory, Military Hospital Mohamed the fifth, Hay riad, 

Rabat, 2 Internal Medicine 2, Military Hospital Mohamed the fifth, Hay riad, Rabat 

The spondylodiscitis have generally tuberculosis or bacterial origin, rarely fungal origin. 

ae report a case of spondylodiscitis due to Trichosporon beigelii. The patient was a 

70-year old. Human immunodeficiency virus is seronegative. He was diabetic, 

operated in cardiovascular surgeryn he was hospitalized for bacBache and low bacB 

pain in progress since two weeBs with a context afebrile. A dorsal and lumbar 

Computed Tomographic scan and the scintigraphy were performedn they show 

spondylodiscitis of D8, DK and D10, compatible with an infectious disease. The 

biologic exams showed an inflammatory syndrome. The mycologic exam of the 

discovertebra biopsy was performed. Fungal elements were observed in direct 

micrsocopical exam and the fungus was isolated on culture media. The fungus was 

identified as Trichosporon beigelii. The bacteriological exam show llebsiella 

pneumoniae and the research of 2l proved to be negative. The patient has an 

antibiotherapy without improvement what motivated setting up of an antifungal 

treatment by the Fluconazole with normalization of constants of the inflammation and 

reduction of the osseous lysis. 

The isolation of Trichosporon beigelii on a discovertebra biopsy, as well as the 

evolution clinic after antifungal treatment permitted to put the diagnosis of a 

spondylodiscitis to Trichosporon beigelii. 

* 


P-0179. Mycetoma: A tunisian review over 24 years 

Badri T 1 , Cherif F 1 , El Euch D 1 , Daoud a 1 , 2en TeBaya N 1 , ChaBer E 2 , Haouet . J , 

2en fsman Dhahri A 1 

1 Dermatology Department, Rabta Hospital, Tunis, Tunisia, 2 Parasitology Dpartement, 

Rabta Hospital, Tunis, Tunisia, J Histopathology Department, Rabta Hospital, Tunis, 

Tunisia 

Introduction: Mycetomas are chronic granulomatous infections affecting the sBin and 

the subcutaneous tissues. Causative agents are either fungal (eumycetoma) or 

bacterial (actinomycetoma). It is a rare condition in Tunisia. ae report a series of 

thirteen cases collected in our department. 

Patients and methods: It is a retrospective study about all the cases of mycetoma 

observed in our department since 1K82 until 200[. For each observation, we noted 

epidemiological, clinical, therapeutic and evolutive data. Mycological examination 

(direct examination and culture on .abouraud medium for fungal grains and 

/owenstein medium for actinomycetes), as well as histopathological study, were 

performed in all our cases. 

Results: Thirteen cases of mycetoma were collected over a period of 2O years. The 

mean age in our patients was [J.7 years-old. Female predominance was observed 

(KF/OM). Rural origin was noted in eleven patients. There was a pedal involvement in 

eleven cases and a history of trauma in five cases. 

Infiltrated, sometimes multinodular and fistulated in ten cases, discharging blacB 

grains in three cases, white grains in two cases and yellow grains in one case. 

Mycological examination was positive in ten cases and allowed the diagnosis of the 

species in seven casesM Madurella mycetomi (J cases), Pseudoallescheria boydii (1 

case) and Actinomadura madurii (J cases). Histopathological examination confirmed 

the diagnosis of actinomycetoma in 6 cases and of eumycetoma in two cases. It was 

non contributive in five patients. 2one involvement was noted in six cases. 

Actinomycetoma were treated with Cotrimoxazole (800 mg daily for 2 to 18 months) in 

8 cases and eumycetoma with letoconazole (J cases) or Fluconazole (1 case) 

associated or not with surgery. Improvement was noted in 8 cases. Two patients had 

several recurrences. A patient was lost to follow-up after the first consultation and two 

others did not return for medical evaluation after treatment. 

P-0180. cDNA representational difference analysis used in the identification of 

genes expressed by Trichophyton rubrum during the infective process 

2aeza / 1 , 2ailÉo A 2 , 2orges C 2 , Pereira M 2 , .oares C 2 , Mendes-Giannini M 1 

1 6niversidade Estadual Paulista, Departamento de AnZlises ClÜnicas - 6NE.P, 

Araraquara, .P, 2razil, 2 6niversidade Federal de GoiZs, Departamento de 2ioquÜmica 

e 2iologia Molecular - 6FG, Goi°nia, Gf, 2razil 

Dermatophyte infections are thought to be commonest form of human contagious 

fungal disease. >$"#?%'?2-%,*$(8$() is the most frequently isolated agent of 

dermatophytosis worldwide, accounting for approximately 80_ of reported cases of 

onychomycosis. The understanding of the complex interactions between the fungus 

and their host must include the identification of genes expressed during infection. ae 

have utilized an efficient approach for the identification of differentially expressed 

genes, which involve rapid sets of subtraction hybridization of cDNAs prepared from 

two cell populations. The Representational Difference Analysis (RDA) has not been 

previously related to pathogenic fungi. fur objective is the identification of genes 

differentially expressed in >$"#?%'?2-%,*$(8$() during the infection process. 

Two cultures of >/*$(8$()*(isolate ATCC [2021) were cultured for 10 days at 2[ o C in 

liquid .abouraudes and transferred for minimal medium with Beratin (tester) and 

without Beratin for O8 hours at room temperature. Total RNA was extracted from both 

and cDNAs were constructed. Differential products were obtained by rounds of 

subtraction and amplified by PCR reactions. The PCR products were cloned into 

pGEM-T-Easy (Promega, Madison). Positive clones were submitted to sequencing 

using the Mega2ACE 1000 DNA sequencer (Amersham 2iosciences). .equence 

determination of JOO clones and comparative analysis at public databases by using 

2/A.Tj algorithm allowed the identification of 18 clusters with Bnow orthologues. 

Additionally, unBnown genes were also identified in >/*$(8$(). Differentially expressed 

genes include those with transcriptional role, oxidative stress, membrane protein and 

some putative virulence factors. .ome differentially expressed genes were 

confirmed by dot and Northern blot. 

The application of RDA allowed the identification of genes up-regulated during the >/* 

$(8$() infective process. The RDA provides a powerful technique for the identification 

of differences between two mRNA populations. 

Financial supportM CNPq, Fapesp and PADC. 

Discussion: In this study we discuss the epidemiological, clinical, microbiological and 

therapeutic aspects of mycetoma in Tunisia and we compare our data to those 

reported in literature, especially in Tunisian series.

P-0181. A survey on the prevalence of cutaneous fungal diseases in 

Shahrekord City 

Bahrami Samani H 

Azad 6niversity 

Dermatophytosis as a Zeunos is the commonest Cutaneous fungal diseases.A study 

was done on 2160 suspected person reffered to Ämycology Department and labs in 

shahreBord. All the exams were performed by direct microscopic examination and 

culture.These conclusions were obtainedM 

1- The percent of positive cases in males was more than females in both direct exam 

(Male}6O.[_ and Female}J[.[_) and culture (Male}62 _ and Female}J8_). 

2-Disease was respectively more in winter and spring. 

J-In direct exam the most mycotic status were myceliumn and mycelium and 

arthroconidia (7J_) and the least was favus (1.6_). 

O- the most infected part was foot (JO.1 _) and the least was face hair (0.8_) 

[-the most isolated fungus was Trichophyton rubrum (2O.8) and the least was 

trichosporon sp. (0.2_). 

6-Married were more infected than singles 

7-Age group of 0-K was the most infected in both sex (O0.K_). 

8-[0_ of infected persons were those having the history of contact with animal. 

K-Illiterates had the most infection percent (68.1_). 

10-Among the isolated fungi, anthropophilic dermatophytes was the most frequented 

(7[.8_). 

11-In dermatophyte, ethiological agents of dermatophytosis according to their 

frequency was anthropophilic (7[.O_), zoophilic (2O.J_) and geophilic (0.J_). 

12-In the zoophilic dermatophytes, Trichophyton verrucosum has the most role 

(8O.1_) in dermatophytosis. 

P-0182. Candida spp. oral cavity colonization in neutropenic patients: Dynamic 

aspects (part I) 

Bailly E 1 , Guinard ( 1 , 2enboubBer I 2 , Chandenier ( 1 , Richard-/enoble D 1 

1 Parasitology -Mycology C.H.R.6 Tf6R., 2 Hematology-Cellular therapy C.H.R.6. 

Tf6R. 

Purpose: In neutropenic patients it could be important to Bnow the qualitative and 

quantitative digestive tract colonization with yeasts in order to assess the risB of 

intestinal translocation and candidemia or to start a preemptive antifungal therapy. 

Design and setting: ae analyzed the results obtained after stools and blood culture 

from some neutropenic patients under survey to their oral yeast colonization 

throughout 1KK6 to 200[. 

Results: To 2K patients orally colonized with 7/*+68"#+,&, the same species was 

recovered in 26 stoolsn alone in 20 cases or associated with another non albicans 

species in 6 cases. 

To J[ patients without oral colonization negative stools were obtained only in 10 

cases, while 2[ patients presented yeasts isolates with 7/*+68"#+,& alone (n } [) or 

associated with 7/*[.U2$ (n } 2) in one part, or a non albicans species of which 7/* 

@6+8$+-+, 7/*[.U2$, and 7/*[$(&." were in four cases each (n } 12) in other part. 

Patients (n }2) orally colonized with 7/*[.U2$ presented always this species in stools. 

Throughout the same period sixteen patients have demonstrated candidemian 7 were 

not orally colonized, J were orally colonized but the same species was found in blood 

in one case only, O were firstly not orally colonized but later had shown change, (in all 

these cases a similar non albicans yeast species was isolated from oral washes and 

blood culture) and finally 2 patients which have presented the same yeast species into 

oral cavity and blood without the colonization was sure. 

7/*[$(&." was recovered in four cases, the other cases were 7/*+68"#+,& (n } 2) and 

non other species in 11 cases. 

Conclusion: These data showed that the connection between oral and digestive 

colonization depends on the yeast species isolates. The absence of oral yeast 

colonization is not a predictive criterion of the lacB of candidemia. fral presence of 7/* 

+68"#+,&*seems to be a lower risB of candidemia than other non albicans species. 

~ualitative and/or quantitative presence of yeasts in the stools demonstrating so 

indirectly the digestive colonization could be a better predictive factor than oral 

colonization in order to assess the risB of intestinal translocation and candidemia or to 

start a preemptive antifungal therapy. 


P-0183. Candida spp. oral cavity colonization in neutropenic patients: 

Qualitative aspects (part II) 

Bailly E, 2arrabes A, Richard G, Chandenier (, Richard-/enoble D 

Parasitology-Mycology, C.H.R.6 Tf6R. 

Purpose: ae have shown that less than about forty percent of patient hospitalized in 

intensive care unit of haematology are orally colonized with Candida spp. with some 

dynamics characteristics. This part analyze the qualitative aspects of the Candida 

oral colonization focusing on species. 

Design and setting: Throughout two periods from 1KK6 to 2000 (P1) and from 2001 

to 200[ (P2) yeasts isolates from oral washes were identified after culture on 

chromogenic (P1 }Candiselectw/2iorad, P2 } Candida ID 2w/bio-Merieux) Candida 

medium plates to 7/*+68"#+,&, RTT-glabrataw (Fumouze) to 7/*@6+8$+-+ and API 20C- 

Auxw (bio-Merieux) to other Candida species. 

Results: 7/*+68"#+,& isolated with J0.K_ at P1 (n1 } 8K0) and JJ.2_ at P2 (n2 } J60) 

of the studied samples were K[.6_ (P1) and 77_ (P2) of all the Candida sp. isolates 

identified. 

Into the non albicans yeasts group (n1 } J8 and n2 } 11K) we have foundM 7/*[.U2$ 

(J6.8_ and J6.1_), 7/*@6+8$+-+ (28.K_ and 1K.J_), 7/*[$(&." (1J.1_ and 1[.K_), 7/* 

-$%'"#+6"& (2.6_ and 16.8_) and other speciesM 7/*@("66".$)%,5"", 7/*",#%,&'"#(+, 7/* 

6(&"-+,"+., 7/*,%$0.@.,&"&3 7/*'+$+'&"6%&"&, 7/*$(@%&+ that were present at 1O.6_ and 

11.8_ all together respectively. 

Compared to 7/*+68"#+,&3 non albicans species were in progress from P1 to P2, they 

were often early detected. 7/*[.U2$ the more frequent non albicans species was often 

isolated alone and early. 7/*[$(&." and 7/*@6+8$+-+ were often isolated with or after 7/* 

+68"#+,& and in addition to that 7/*@6+8$+-+ appeared often later. In some cases the 

colonization with 7/*[$(&." or 7/*@6+8$+-+ could be in relation with 7/*+68"#+,& 

disappearance. 

Conclusion: These observations showed that non albicans species were in progress 

in our conditions since 1KK6 and that 7/*[.U2$ is the second yeast species. These 

data are in agreement with recently uncommon cases of fungal infection reported with 

feeding yeast liBe 7/*[.U2$. .ome characteristics of the oral yeast colonization could 

depend on species isolates. 

P-0184. Candida spp. digestive colonization and candidemia in neutropenic 

patients 

Bailly E 1 , Guinard ( 1 , 2astides F 2 , Chandenier ( 1 , Richard-/enoble D 1 

1 Parasitology-Mycology C.H.R.6 Tf6R., 2 Internal Medecine and Infectious Diseases 

C.H.R.6. Tf6R. 

Purpose: In neutropenic patients it could be important to Bnow the qualitative and 

quantitative digestive tract colonization with yeasts in order to assess the risB of 

intestinal translocation and candidemia or to start a preemptive antifungal therapy. 

Design and setting: ae analyzed the results obtained after stools and blood culture 

from some neutropenic patients under survey to their oral yeast colonization 

throughout 1KK6 to 200[. 

Results: To 2K patients orally colonized with 7/*+68"#+,&, the same species was 

recovered in 26 stoolsn alone in 20 cases or associated with another non albicans 

species in 6 cases. 

To J[ patients without oral colonization negative stools were obtained only in 10 

cases, while 2[ patients presented yeasts isolates with 7/*+68"#+,& alone (n } [) or 

associated with 7/*[.U2$ (n } 2) in one part, or a non albicans species of which 7/* 

@6+8$+-+, 7/*[.U2$, and 7/*[$(&." were in four cases each (n } 12) in other part. 

Patients (n }2) orally colonized with 7/*[.U2$ presented always this species in stools. 

Throughout the same period sixteen patients have demonstrated candidemian 7 were 

not orally colonized, J were orally colonized but the same species was found in blood 

in one case only, O were firstly not orally colonized but later had shown change, (in all 

these cases a similar non albicans yeast species was isolated from oral washes and 

blood culture) and finally 2 patients which have presented the same yeast species into 

oral cavity and blood without the colonization was sure. 

* 

7/*[$(&." was recovered in four cases, the other cases were 7/*+68"#+,& (n } 2) and 

non other species in 11 cases. 

Conclusion: These data showed that the connection between oral and digestive 

colonization depends on the yeast species isolates. The absence of oral yeast 

colonization is not a predictive criterion of the lacB of candidemia. fral presence of 7/* 

+68"#+,&*seems to be a lower risB of candidemia than other non albicans species. 

~ualitative and/or quantitative presence of yeasts in the stools demonstrating so 

indirectly the digestive colonization could be a better predictive factor than oral 

colonization in order to assess the risB of intestinal translocation and candidemia or to 

start a preemptive antifungal therapy.

P-0185. Systematic detection of Candida dubliniensis in a University hospital: 

Results of a 5 month prospective study 

Baixench M, .eBBal N, Viguie C, Paugam A 

Hopital Cochin 

Background: 7/*5(86",".,&"& was identified as a new species in Ireland in 1KK[. 

Indeed, the close phenotypic and genotypic similarities between 7/*+68"#+,& and 7/* 

5(86",".,&"& have led to the misidentification of these species. Recently, a commercial 

latex agglutination test has been developed to identify 7/*5(86",".,&"& (2ichro-dubli 

Fumouze test, Fumouze Diagnostics, France). This test revealed a sensitivity and a 

specificity of 100_ and K7,8_ respectively (Marot-/eblond et +6., ]7! 2006). 

Method: From August to December 200[, all the isolates identified as 7/*+68"#+,& on 

chromogenic medium (CHRfMagar Candida, 2ecton DicBinson, 6.A) were tested 

with a 2ichro-dubli Fumouze test. ahen the test was positive, a sugar assimilation 

profile on IDJ2C strips (bioMerieux, Marcy lïEtoile, France) and an anti-fungal 

susceptibility test using a commercial agar diffusion test (E-test, A2 2iodisB, 

.tocBholm, .weden) were performed. 

Results: A total of [0J isolates from 2J8 patients were identified as 7/*+68"#+,&. ff 

these, 10 isolates (2_) from 8 patients were 7/*5(86",".,&"&. For K of the 10 isolates 

the identification was confirmed by IDJ2C. MIC (kg/ml) range for fluconazole varied 

from 0.0KO to 0.12[n for voriconazole from 0.00O to 0.012n for amphotericin 2 from 

0.012 to 0.0J2 and for caspofungin from 0.0O7 to 0.0KO. All 7/*5(86",".,&"& isolates 

were susceptible to fluconazole. 7/*5(86",".,&"& was associated with 7/*+68"#+,& J 

times and with 7/*@6+8$+-+ O times. ahen 7/*+68"#+,& and 7/*5(86",".,&"& were 

associated, 7/*5(86",".,&"& appeared with the darBest green colour on CHRfMagar. 

.ix isolates (60_) were isolated from O HIV positive patients (2 isolates from oral 

samples and O from respiratory tract specimens). They represented 2[_ of the 

7/+68"#+,&

P-0187. A case of severe tinea capitis due to Microsporum canis. Effective 

treatment with oral terbinafine 

Baran E, 2aran a 

Department of Dermatology, Venereology and Allergology 

A 10 years old boy presented with a O weeB history of erythematous, pustular, crusting 

lesions on the scalp with some hair loss. He has been treated unsuccessfully with oral 

and topical antibiotics, for a suspected bacterial infection by general practitioner. 

Physical examination revealed multiple intensely erythematous and well-demarcated, 

round-shaped hairless areas, located in the parietal and scalp region, very painful 

when palpated. They were covered with brown-yellowish crusts and pus was seen 

oozing from the hair follicles. Individual broBen hairs could be pulled out easily. 

Routine blood test, urinalysis and serum protein were normal and culture of sBin 

swabs showed no bacterial growth. Microscopic examination of material obtained by 

scraping the scalp (in 20_ potassium hydroxide (lfH)) demonstrate rare branching 

hyphae, macroconidia, and microconidia. Mycological culture produced a profuse 

growth of Microsporum canis. fral treatment was started with terbinafine 12[ mg daily 

for 1 month. No local therapy was given. During the first 2 weeBs of treatment there 

was a significant improvement in the scalp lesionsn at the end of therapy the affected 

area was still inflamed, but the patient]s hairs had started to grow. fne month after 

discontinuation of treatment, her scalp was clinically and mycologically cured. No 

relapse was observed. Investigation of environmental conditions showed no pets 

present in the home. This case adds the evidence that systemic terbinafine is useful in 

the treatment of the M. canis infection without other local treatment. 

Conclusion: fnly griseofulvin is recommended by many experts worldwide for 

systemic therapy of tinea capitis caused by Microsporum canis. 6nfortunatelly in 

Poland this drug is not registred that is why terbinafine is widely used in our country in 

systemic treatment of tinea capitis caused by !"#$%&'%$()*&''/ 

P-0188. Candidacidal activity of circulating polymorphonuclear granulocytes 

(PMNs) in lung cancer patients 

Batura-Gabryel H 1 , 2rajer 2 1 , .zczepaniB A 2 

1 Dept. of Pulmonary Diseases, 6niversity of Medical .ciences, Poznan, Poland, 2 IC6 

of Dept. of Cardiology, 6niversity of Medical .ciences, Poznan, Poland 

Background: 7+,5"5+ infections more and more often as a clinical problem are 

observed. Cancer patients belong to the 7+,5"5+ infection and candidiasis risB 

groups. This infection can be one can be the reason for complications in the course of 

neoplastic disease for instance deterioration in general condition or candidosis, 

especially in neutropenic patients after antineoplastic treatment. During earlier own 

studies authors observed one or multifocal Candida infection in over 70_ and oral 

candidosis in more then 2[_ of investigated lung cancer (/C) patients before 

anticancer treatment. fne of the major defense mechanism against 7+,5"5+ infection 

is the polymorphonuclear granulocytes (PMNs) phagocytic activity. 

Study aim: The estimation and comparison of the blood PMNs Candidacidal activity 

against 7+,5"5+ strains cultured from sputum, taBen from OJ /C patients before 

anticancer treatment and 2[ healthy individuals (control group) 

Methods: 6sed to reveal the PMNs Candidacidal activities were based on the 

Pantazis and lnicBer]s method for the evaluation the PMNs bactericidal effectiveness, 

and on the /ehrer and Cline]s method for the estimation of the PMNs fungicidal 

effectiveness (self-modification). 

Results: The average Candidacidal blood PMNs activity in the /C patients was 

18,18_ (r/-7,2K), in the control group was J1,J[_ (r/-6,[6). .tatistically significant 

difference between the PMNs Candidacidal activity in /C patients in comparison with 

the control group (pv0,0001) was noted. 

Conclusion: 

1. statistically significant impairment of Candidacidal activity of blood PMNs 

may be one of the reasons for common 7+,5"5+ infection, oral candidosis 

and risB for this infection dissemination in non-neutropenic /C patients 

2. the impairment of blood neutrophil granulocytes in non-neutropenic 

individuals mean that defect of this activity depends on the function 

disturbances, not only on the number of these cells 

J. in /C patients carefully mycological examination should be perform before 

antineoplastic treatment 

O. prophylactic antimycotic treatment should be use more often then upto date 

in /C patients

P-0189. Is there a relationship between blood neutrophils function and Candida 

colonization in COPD patients? 

Batura-Gabryel H 1 , 2rajer 2 1 , .zczepaniB A 2 

1 Dept. of Pulmonary Diseases, 6niversity of Medical .ciences, Poznan, Poland, 2 IC6 

of Dept. of Cardiology, 6niversity of Medical .ciences, Poznan, Poland 

Background: Chronic obstructive pulmonary disease (CfPD) patients, considering 

the high number of risB factors for 7+,5"5+ infections seen in them, create the risB 

group for candidiasis. 2ronchial colonization with potentially pathogenic microorganisms, 

liBe 7+,5"5+ may represent and independent stimulus for aditional airway 

inflammation. Polymorphonuclear granulocytes (PMNs) are liBely to play a mein role in 

the inflammatory response seen in CfPD. fne of the major defence mechanism 

against 7+,5"5+ infections is the phagocytic activity of PMNs. 

Study aim: was conducted to investigate whether 7+,5"5+ in sputum may be linBed 

to an enhanced peripheral neutrophil inflammation in CfPD patients. 

Methods: .putum samples were collected from CfPD and examined using ID CJ2 

test (2ioMerieux). Intensity of 7+,5"5+ growth was estimated by semiquantity method. 

2lood neutrophils from 2J CfPD (1O with and K without 7+,5"5+) and 18 healthy 

controls were isolated and cultured under basal condition and after stimulation by 

PMA and fM/P. There were estimated migration and chemotactic activity of 

neutrophils, production of Nf, H2f2, f2 and release of MPf, glucuronidase and 

lisosome. 

Results: .pontaneous and after stimulation blood granulocytes migration and MPf 

release were significantly higher in CfPD patients than in healthy control group. MPf 

spontaneous release was significantly higher in 7+,5"5+ positive then in 7+,5"5+ 

negative CfPD patients. There was significant positive correlation between intensity 

of 7+,5"5+ strains growth and MPf release (spontaneous and after stimulation) 

(r}0,[2Jn p}0,01). 

Conclusions: .ignificantly elevation of some of blood neutrophil inflammation 

marBers confirmed that upper respiratory tract 7+,5"5+ colonization/infection might be 

important modulator of peripheral granulocytes inflammation in CfPD patients 

(continuously stimulation of the chronic inflammatory process). 

P-0190. Multiple-species candidemia 

2enetucci A, Tiraboschi I, Fernandez N, Perazzi 2, /asala M 

Hospital de ClÜnicas (osd de .an MartÜn 

Candidemia episodes have increased during the last decade, this germ has become 

the fourth microorganism recovered in blood cultures. Multiple-species candidemia 

(M.C) spreading is not common and covers approximately 2-10_ of cases in different 

series. This worB is intended to analyze clinical and microbiological data of patients 

with M.C in order to identify risB factors and prognosis that maBe them different from 

patients with a single-species 7+,5"5+ recovered in blood cultures (..C). 

Methodology: Clinical records from all the patients diagnosed with candidemia 

between 01/01/K8 and 12/J1/0O were analyzed. Demographic, clinical, diagnostic, 

therapeutic and evolution data were collected. Cases of M.C from every patient with 

recovery of two or more 7+,5"5+ species in a single blood culture or in two blood 

culture samples collected 12 days or less apart were considered. Control patients 

were those with recovery of a single-species 7+,5"5+ in blood cultures. Three controls 

for similar age and gender were included for each case. 

Results: During the period under study, after excluding patients with growth of 

4&'.$@"66(&, A"&-%'6+&)+ or 7$2'-%#%##(& in blood cultures, there were 1[[ patients 

with yeast fungemias. .even cases of M.C were identified (6 adults and 1 neonate) 

which represented O.[_ of candidemias during that period. These were compared 

with 21 ..C patients matched by age and gender. All 28 cases of candidemia were 

acquired in-hospital. There were no significant differences in age, gender, cause and 

hospitalization service, 7+,5"5+ species in blood cultures, catheter placement, lab 

parameters, antifungal treatment indicated, use of inotropics, ARM nor global mortality. 

The hospital stay was significantly longer than for patients with M.C (J[.O vs 20 days, 

1 } 0.0J7), as well as the candidemia duration (8.1 vs 2.O daysn P } 0.0002) and 

average period of central venous catheter placement prior to candidemia (2K.O days in 

M.C vs 1O.[ days in ..C, 1 } 0.02K). 

Conclusion: Though M.C episodes are less frequent than those caused by singlespecies 

7+,5"5+, they appear to be related to potentially controllable risB factors such 

as hospital stay and length of central venous catheter use. 


P-0191. Effect of endodontic drugs on the adhesion and colonization of human 

dentin by Candida albicans 

2errüdo Pinho M 1, 2 , 2ernardes-Engemann A 1 , Fidel R 2 , Lopes-Bbezerra L 1 

1 /aborarory of Mycology and Proteomics, 2iology Institute, .tate 6niversity of Rio de 

(aneiro, Rio de (aneiro, 2razil, 2 Department of Endondotics, .chool of Dentistry, 

.tate 6niversity of Rio de (aneiro, 2razil 

Recently, a gain of interest is focused on discussion concerning endodontic yeasts 

being the most important species 7+,5"5+*+68"#+,s. The aim of the present study was 

to isolate 7+,5"5+*spp. from the oral cavity and the root canal of patients that would 

be submitted to endodontic treatment, and evaluate the adherence and colonization of 

human dentin by these strains. ae could successfully isolate and identify 7+,5"5+ 

+68"#+,&*using a chromogenic growth medium (CHRfMagar w Candida). The clinical 

isolates from the oral cavity and the root canal named Mf[ and CR[, respectively, 

were compared with a standard strain, .C[J1O. No differences in the growth rates 

were observed. Further, we established a 2O hour period for the harvest of cells with 

the objective of evaluating its adherence capacity to dentin at different time intervals. 

The colonization was determined by a colorimetric method and/or by scanning 

electron microscopy. All 7/*+68"#+,& strains tested showed the same adhesive 

capacity to human dentin and adhesion could be observed in the first four hours of 

interaction. After establishing this incubation time for adherence of 7/*+68"#+,& to 

human dentin we challenged the infected dentin with chemical substances used in 

endodontic treatment with the objective to evaluate if these drugs could prevent 

colonization. The tested chemical substances were sodium hypochlorite [.2[_ and 

0.[_, chlorhexidine 2_, citric acid 10_ and EDTA 17_. Dentine samples previously 

incubated with strain CR[ were submitted to contact with each chemical substance for 

five minutes. The treated and untreated (control) dentin samples were then incubated 

for five days in RPMI supplemented with fetal calf serum to observe whether or not the 

colonization occurred. The results revealed that NafCl [.2[_ (pv0.01), chlorhexidine 

2_ (pv0.01), and NafCl 0.[_ (pv0.0[), were able to significantly inhibit biofilm 

formation while citric acid 10_ and EDTA 17_ did not show any effect. In the case of 

chlorhexidine we have also tested a range of J0 seconds to 10 minutes of exposition. 

In all cases this drug could efficiently avoid colonization. fur results strongly suggest 

that this drug as well as sodium hypochlorite could be clinically used in the endodontic 

treatment to prevent 7+,5"5+ infection. 

P-0192. Case of Tinea nigra palmaris in Zaragoza (Spain) 

2etran A 1 , Rezusta A 1 , ~uerol I 2 , Arias M 1 , Revillo M 1 , Gend ( J 

1 Microbiologia. Hospital 6nivesitario Miguel .ervet. Zaragoza (.pain), 2 Dermatologia. 

Hospital 6nivesitario Miguel .ervet. Zaragoza, J MicrobiologÜa. 6niversitat Rovira i 

Virgili. Reus. (.pain) 

Introduction: >",.+*,"@$+ is a superficial form of phaeohyphomycosis caused by 

A%$-+.+*B.$,.#["". The infection is confined to the stratum corneum of the palms or 

soles and is mainly seen in the tropics or subtropics in children or young adults. 

Case Report: A 1O-year-old Colombian woman attended the Dermatology department 

for J years with a superficial scaling macula that was brown to blacB on the palms. It 

looBed liBe typical lesions associated with tinea nigra. The pigmentation was 

irregularly distributed over longer lesions. The main differential diagnosis is a 

superficial form of melanoma or a pigmented nevus. Pigmented hyphae were seen by 

direct microscopy. .crapings were cultured on .abouraud Agar and Potato Dextrose 

Agar, both with chloranphenicol, and on Dermatophyte Test Medium (DTM). Colonies 

of a dematiaceous fungus grew on the first two media, but not on DTM. Identification 

was made on the basis of its morphological characters. The patient was treated with 

amorolfine qd for one month. After treatment, the scaling macule disappeared. 

Discussion: This disease is almost always caused by A/*B.$,.#["". Cases have been 

diagnosed outside the area of endemicity as a result of travel to the American tropics 

or to the Caribbean islands or contact with people from these areas. 

Conclusions: >",.+*,"@$+ is a rare documented imported infection in .pain. 

Financial supportM CNPq and Faperj.

P-0193. First case of Saksenaea vasiformis infection in French Guiana 

2lanchet D 1 , Dannaoui E 2 , Fior A J , Huber F O , Couppid P O , .alhab A [ , Hoinard D 2 , 

Aznar C 1 

1 /aboratoire hospitalo-universitaire de Parasitologie-Mycologie, CHAR/6FR de Mddecine, 

EA J[KJ, Cayenne, Guyane, 2 Centre National de Rdfdrence Mycologie et Antifongiques, 

Institut Pasteur, Paris, France, J .ervice d]Anatomo-pathologie, CHAR, Cayenne, Guyane, 

O .ervice de Dermatologie, CHAR, Cayenne, Guyane, [ .ervice de Chirurgie viscdrale, 

CHAR, Cayenne, Guyane 

Case report: A non immunocompromized O7-year-old female, living in French Guiana for 

many years, with a long history of diabetes mellitus poorly controlled by insulin therapy 

presented to the hospital on 18 November 200[ with a cutaneous lesion of the abdomen in 

the area of insulin injections. A sBin biopsy was performed and the lesion, consisting of a 

mass of 1O cm in diameter was surgically excised on 22 November. In both tissue samples, 

direct examination and histopathology revealed necrosis and large non-septate hyphae 

indicative of Zygomycetes. Treatment was initiated on November 18, with itraconazole and 

amphotericin 2 (IV) during 10 days followed by liposomal amphotericin 2 during 12 days. 

Persistence of necrotic tissues on the site of infection required additional surgical 

debridement on 28 November. Histopathology of the resected tissues showed damaged 

hyphae of Zygomycetes. Evolution was uneventful. Additional biopsies taBen by the end of 

treatment were negative for fungi by direct examination and culture. 

Microbiological findings: Culture of tissues samples on .abouraud-Chloramphenicol- 

Gentamicin after O days at J0 and J7ÅC grew a white aerial mold. Microscopic examination 

showed non septate sterile hyphae typical of a Zygomycetes. .ubcultures on different 

media including Malt extract agar and Potato dextrose agar grew sterile mycelia. Cultures 

were then performed in sterile distilled water supplemented with 0.0[_ filter-sterilized yeast 

extract solution for 7 days at J7ÅC. Typical flasB-shaped sporangia allowed identification of 

=+[&.,+.+*0+&"U%$)"&. .pecies identification was confirmed by PCR amplification and 

sequencing of rDNA IT. regions. 

Discussion: =+[&.,+.+*0+&"U%$)"& has been isolated from soil samples in different parts 

of the world. This fungus has been rarely responsible for human infections. 6p to now 

about 20 cases have been reported in the literature, mostly in tropical and subtropical 

areas (Australia, India, 6.A, Thailand, Israel, Colombia, Iraq). Infections occurred mainly in 

immunocompetent patient after traumatic injuries involving sBin and subcutaneous tissues. 

In some cases, insect or spider bites initiated infection and in one report a needle injection 

lead to primary cutaneous infection followed by dissemination. /ess frequently rhinocerebral 

or disseminated infections were reported in immunocompromized patients. Most of 

the patients were treated with amphotericin 2 and/or surgery. 

In the present case, the only risB factor was a poorly controlled diabetes mellitus and the 

way of infection was probably related to insulin injections in the umbilical area. Antifungal 

therapy and surgery allowed a favorable outcome. 

Conclusion: Rare Zygomycetes species such as =+[&.,+.+*0+&"U%$)"& or 

4'%'?2&%)2#.&*.6.@+,& should be suspected when a non sporulating zygomycete is 

recovered from an infected lesion. .pecific culture media and/or molecular tools are helpful 

for diagnosis. 

P-0194. Efficacy of voriconazole in a case of Penicillium marneffei septicemia 

with retinitis in an HIV infected woman 

2londe R 1 , Canestri A 1 , Cassoux N 2 , Caumes E 1 , 2ricaire F 1 , Datry A J 

1 .ervice de Maladies Infectieuses et Tropicales, Hospital de la Pitie-.alphriere, Paris, 

France, 2 .ervice de fphtalmologie, Hospital de la Pitie-.alphriere, Paris, France, 

J .ervice de Mycologie et Parasitologie, Hospital de la Pitie-.alphriere, Paris, France 

Case report: A [J years old woman with human immunodeficiency virus (HIV) 

infection since 1K88 native from Cambodia but living in France since her childhood, 

was admitted because of prolonged fever, diarrhoea, weight loss and asthenia. .he 

had been travelling during two months in Vietnam six months before. 

At her admission, she presented fever (JKÅ), diarrhoea, cervical and inguinal 

adenopathies and a weight loss of 7 lg. Cutaneous examination was normal. 

/aboratory findings revealed anaemia ( 10,K dg//), thrombocytopenia ( 1O7 

000/mmJ), increase of liver enzymes ((J times the normal value), CDO cell 

lymphopenia ( [/mmJ) and a viral load at J2100 copies/ml. 2lood, faeces and urine 

bacterial culture were negative, sputum smear was negative for tuberculosis. Chest j- 

ray film was normal. The computed tomography scan (CT) showed two nodules in the 

left upper lobe, mediastinal and mesenteric adenopathies and six hepatic nodules.. 

Fungal blood culture grew to 1.,"#"66"()*)+$,.UU.". A trans-thoracic cardiac 

echography was normal but ophthalmoscopic examination found bilateral retinal white 

opacities strongly suggesting fungi retinitis confirmed by the fluorescein angiograms. 

The diagnosis was septicaemia due to 1.,"#"66"()*)+$,.UU." with probable retinal, 

hepatic and digestive localisation. 

The patient was treated with voriconazole 200mg bid for 1.,"#"66"()*)+$,.UU." 

infection, two nucleoside reverse transcriptase inhibitors and one protease inhibitor for 

HIV-1 infection and cotrimoxazole to prevent opportunistic infections. The clinical 

evolution was favourable with a fever]s decrease, resolution of diarrhoea and return to 

normal weight. 2lood samples remained negative and fluorescein angiographs 

showed the cicatrisation of the fungi lesions. 

Discussion: 1.,"#"66"()*)+$,.UU.", is an important opportunistic infection in .outheast 

Asian indeed it is the third most common opportunistic infection in Thailand. In 

aestern countries only a few imported cases have been reported and ophthalmologic 

localisation are uncommon. The diagnostic is difficult and it should be suspected in 

immunocompromised subjects who have travelled to endemic zones (.outheast Asia). 

Amphotericin 2 and itraconazole are the recognised treatment of 1.,"#"66"()*)+$,.UU." 

infection but voriconazole has been found to be highly active in vitro/ This case report 

suggests that voriconazole is also effective in vivo. Voriconazole may thus offer a 

new option for treatment and prophylaxis of 1.,"#"66"()*)+$,.UU." infection. 


P-0195. 16 cases of dermatophyte onychomycosis in children under 2 years-old 

Bonifaz A 1 , Fierro / 1 , Mena C 2 , Araiza ( 1 

1 Hospital General de Mdxico, Mexico City, Mexico, 2 Hospital Infantil de Mdxico 

Federico Gámez, Mexico 

Purpose of the study: Dermatophyte onychomycosis (Df) is a rare condition among 

children. The cases in infants less than two years of age are really exceptional with 

only a few sporadic case reports. This is a retrospective study aimed at reporting a 

series of dermatophyte onychomycosis cases in children ages 0-2 years, including the 

clinical and mycologic aspects of the disease. 

Project: This is a retrospective study (1K87-200[) of Df in children under two years 

of age seen in two public health care facilities in Mexico City. Each of the cases was 

worBed-up, classified clinically and proven through mycologic testsM scraping of the 

nails and a sample of the feet for direct microscopy with lfH (20_), and repeated 

cultures. The cases with other types of tinea were proven with direct microscopy and 

culture. fnce parasitation was proven, an appointment was made with the parents, 

siblings and other relatives that coexisted with the children. 

Results: ae included 16 proven cases of Df in a period of 18 years and includes. 

Mean age of patients was 1[.O months, with one case under 11 weeBs of age, and 

toenail onychomycosis was predominant (12/16) The youngest patient was 11 weeBs 

old and the oldest was 2O months old, with a mean age of 1[.O months. 7/16 cases 

were associated with Down]s syndrome and two of them also had other predisposing 

factorsM one of them had tinea capitis followed by Df. Two patients had brain hypoxia 

and two more were preterm. All parents were tested for Df and tinea pedis and both 

conditions were proven in 1O/16 fathers and 7/16 mothersn both parents were affected 

in [ couples. 

Df occurred in toenails in K/16 casesn in fingernails in O/16 cases and in both sites in 

J/16. According to the clinical classification of onychomycosis, 1O/16 cases (87.[_) 

had distal subungual onychomycosis, and one of them also had paronychian one case 

had proximal subungual onychomycosis and another one had white superficial 

onychomycosis. The worBup of all patients and relatives included proving the 

presence of dermatophytes by means of direct microscopy. As to the etiology, 

>$"#?%'?2-%,*$(8$() was the causal agent of 1O/16 cases (87.[_)n >$"#?%'?2-%,* 

",-.$5"@"-+6. was isolated in one case and this was the same causal agent isolated 

from one of the parents. In another patient !"#$%&'%$()*#+,"& was isolated from both 

the tinea capitis and the onychomycosis lesions. The worBup of parents and siblings 

resulted in 22 proven casesn 1O fathers, 7 mothers and one sibling. The causal agents 

were >/*$(8$() in 21/22 cases and one of >/*",-.$5"@"-+6. . 

Conclusions: Dermatophyte onychomycosis is a rare condition in children under 2- 

years old, Down]s syndrome is the most important predisposing factor and the 

principal agent is >/*$(8$() 

P-0196. Unique hybrids between pathogenic yeasts Cryptococcus neoformans 

and Cryptococcus gattii 

Bovers M 1 , Hagen F 1 , luramae E 1 , Diaz M 2 , .panjaard / J , Dromer F O , Hoogveld H [ , 

2oeBhout T 1, 6 

1 C2.-Centraalbureau voor .chimmelcultures, 6trecht, The Netherlands, 2 R.MA.- 

Rosenstiel .chool of Marine and Atmospheric .cience, Division of Marine 2iology and 

Fisheries, ley 2iscayne, Florida, 6.A, J The Netherlands Reference /aboratory for 

2acterial Meningitis (AMC/RIVM), Department of Medical Microbiology, Academic 

Medical Center, Amsterdam, The Netherlands, O 6nitd de Mycologie Moldculaire, 

CNR. FRE28OK, Centre National de Rdfdrence Mycologie et Antifongiques, Institut 

Pasteur, Paris, France, [ Netherlands Institute of Ecology (NIff-lNAa), Centre for 

/imnology, Nieuwersluis, The Netherlands, 6 Division Acute Medicine and Infectious 

Diseases and EijBman-ainBler Centre 

7$2'-%#%##(&*,.%U%$)+,&*and 7/*@+--"" are closely related basidiomycetous yeasts 

that both can cause meningoencephalitis. However, they differ in their occurrence and 

host range. 7/*,.%U%$)+,&*occurs worldwide and causes disease in 

immunocompromised patients, whereas 7/*@+--"" occurs mainly in (sub) tropical 

regions and can cause disease in healthy individuals. 7/*,.%U%$)+,& and 7/*@+--"" can 

be classified by serotype, with 7/*,.%U%$)+,& var. @$(8""*corresponding to serotype A* 

and var. ,.%U%$)+,&*corresponding to serotype D and AD. .erotypes 2 and C 

correspond to 7/*@+--"". Hybrids between the two varieties of 7/*,.%U%$)+,& exist (AD 

hybrids), but although there has been some speculation about the existence of a 

hybrid between 7/*,.%U%$)+,& and 7/*@+--"", this hybrid had not been found yet. 

During a study of clinical isolates of 7$2'-%#%##(&*,.%U%$)+,&*from the Netherlands, 

three strains isolated from two patients, were found that did not fit any of the 

previously defined Amplified Fragment /ength Polymorphism (AF/P) genotypes. 

These three strains were studied with flow cytometry, light microscopy, serology, 

AF/P, sequence analysis, and mating- and serotype specific PCRs. 

All results indicate that these three isolates are diploid or aneuploid, and represent 

monoBaryotic hybrids between a 7/*@+--"" serotype 2 (AF/P genotype O) and a 7/* 

,.%U%$)+,&*serotype D (AF/P genotype 2) strain (2overs .-*+6/, 2006). 

The existence of a hybrid between the two species 7/*,.%U%$)+,&*and 7/*@+--""*might 

have implications for human health. A hybrid combines the genetic characteristics of 

both parents and may become a pathogen that can infect immunocompetent people 

(liBe 7/*@+--"") and that has a worldwide distribution (liBe 7/*,.%U%$)+,&). All our 2D 

hybrid strains were isolated from patients, which shows that these hybrids are capable 

of causing disease. 

2overs M, Hagen F, luramae EE, Diaz MR, .panjaard /, Dromer F, Hoogveld H/ 

and 2oeBhout T (2006) 6nique hybrids between fungal pathogens 7$2'-%#%##(&* 

,.%U%$)+,&*and 7$2'-%#%##(&*@+--"". FEM. Yeast Res. (",*'$.&&).

P-0197. Candida species from patients with vulvovaginitis in Venezuela 

2ruzual E 1 , 2rito A 1 , de la Parte M 1 , Mendoza M 2 , Marino C 1 

1 (M Vargas Medical .chool, 6niversidad Central de Venezuela, Caracas, Venezuela, 

2 Mycology /aboratory, Institute of 2iomedicine, Caracas, Venezuela 

Vaginal candidiasis is a fungal infection distributed around the world, produced by 

yeasts of the 7+,5"5+*species belonging to the vagina normal flora. Vulvovaginitis 

encompasses a variety of disorders characterized by inflammation that may be 

secondary to multiple causes, including infection, irritation, allergy, and systemic 

disease. .ome predisposing conditions areM pregnancy and underlying medical 

conditions, such as diabetes mellitus, hypothyroidism, or human immunodeficiency 

virus (HIV). Yeasts of the 7+,5"5+*species*are found in 20 to J0_ of all the cases of 

vulvovaginitis and 7+,5"5+*+68"#+,& is responsible of a great majority of cases. 

However, more recent studies show an increase of other 7+,5"5+*species producing 

this pathology. The species described as h,%,*+68"#+,&f*have the particularity to show 

resistance to the antifungal agents commonly used for treating this pathology and for 

this reason, we should determine accurately the species involved in each case, to 

install the specific treatment to avoid recurrence of the infection. The objective of our 

study was to identify the species of 7+,5"5+*yeasts associated to cases of 

vulvovaginitis in patients from the metropolitan area of Caracas during the period 

comprised between 200J and 200[. ae studied 10K samples of vaginal discharge 

obtained from the vaginal fornices and walls, from patients with vulvovaginitisn from 

each sample two smears were prepared, one for direct examination, treated with 10_ 

lfH and another colored by the Gram method. Culture was made in two tubes with 

.abouraud-antibiotic medium for yeast isolation and were incubated at room 

temperature and observed for growth every other day for 6 consecutive days. For 

determination of 7+,5"5+*species, the following assays were performedM germ tube 

production and clamydospore formation in wheat flour-Tween 80 broth, observation of 

colony color in CHRfMagar-7+,5"5+, morphology in corn-meal, urease test and 

sugar assimilation. Among the 7+,5"5+*species recovered3*7/*+68"#+,&*was the most 

frequently found, 8J.[0_ (n}K1)n 7/*-$%'"#+6"&*represented 8.2[_ (n}K), 7/*@6+8$+-+ 

2.7[_ (n}J) and 7/*@("66.$)%,5" [.[0_ (n}6). .pecie distribution in our results, are 

very much liBe the values described by other national and international investigators 

which report frequencies for 7+,5"5+*+68"#+,&*between [Q*+,5*88_. These vaginal 

samples are part of a project being carried out by our group of investigation. 

P-0198. Molds isolated from nails 

Buot G 1 , Descamps P, Hennequin C 

1 Hospital Tenon, Paris, France 

Purpose of the study: ahile dematophytes remain the predominant agents of 

onychomycosis, there is an increasing concern on molds, because of the difficulty of 

treatment and their potential role as a portal of entry for some invasive fungal 

infections. Here, we report on the frequency of molds isolated from nail samples in an 

adult population seen in Paris and their implication as etiological agents of 


Description: Patients referred for mycological exams during the 200O-200[ period were 

included. They were outpatients from the dermatological unit of the hospital or from private 

dermatologists or GPs, as well as inpatients from wards of our institution, mainly 

dermatological daycare, renal transplant and dialysis, infectious diseases, internal medicine 

and oncology. Patient]s history and clinical aspect of the involved nails were recorded. Direct 

microscopic exam of specimens was performed with Chlorazol blacB E. Cultures were done on 

.abouraud glucose agar plus chloramphenicol and gentamycin slants, with and without 

cycloheximide, incubated at 27ÅC. Fungal growth was monitored once a weeB. Genus and 

species identification was based on the morphological aspect and microscopic examination of 

the colonies. 

Results and conclusions: During the 2 year period of the study, 1JJ2 nail samples were 

collected from a total of 2KO2 dermatological samples (O[.J_). 77O specimens were positive 

on direct exams ([8_), of which 6K0 ([1.8_) gave a positive cultureM [06 (7J_) with pure 

dermatophyte (dph) growth, 110 (16 _) samples yielded molds alone, dph were recovered 

along with molds in 26 (J.7_) cases, and in seven (1.7_) cases a mold was associated with 

yeasts. In 7O additional cases a mold culture was reported but the direct exam was negative. 

fverall, the molds the most frequently isolated from positive nails wereM 4&'.$@"66(&*spp*(2O 

alone r 8 associated with dph),*=#2-+6"5"()*spp (2[ alone r 1 associated with*9.%-$"#?()J3* 

Fusarium spp (17 r 8 associated with dph r O associated with yeasts), =#%'(6+$"%'&"&*spp*(12 

r 6 associated with dph and*4#$.)%,"()*&'*(K r 1 with yeasts). 

These results clearly demonstrate the importance of molds in nail samples. In our 

experience, some clinical features were suggestive of mold infectionM leuconychia or 

xanthonychia, and acute or subacute onset with inflammatory paronychia.*However, 

considering the specificity of treatment required for molds, these results warrant that 

mycological exam has to be systematically performed and repeated if necessary, 

before any antifungal treatment. In addition, in an hospital context, the presence of a 

mold in a nail may be critical for immunosuppressed patients and efforts should be 

done for its eradication. 


P-0199. Posaconazole therapy for lobomycosis: Preliminary report of a 

Peruvian case 

Bustamante B 

Hospital Nacional Cayetano Heredia, /ima-PER6 

/obomycosis is a subcutaneous chronic fungal disease occurring mainly in .outh and 

Central America, for which there is currently no available effective drug treatment. 

.urgical excision is recommended when the lesion is small, well defined and 

accessible. ae report here the first Peruvian case of lobomycosis under treatment 

with posaconazole. 

A 2K-year-old male forest ranger, native to the Peruvian jungle area (Madre de Dios 

city), noticed a pruriginous papular lesion on his left ear lobe when aged 12. After 17 

years of slow progression, the entire left ear became involved and in .eptember 200O 

he presented with erythema, edema, and several nodules and ulcers. 

ae conducted a direct examination and histologic study of an ear sBin biopsy that 

showed yeast-liBe cells of X+#+d"+*6%8%". ae then started treatment with oral 

posaconazole O00mg bid. After five months] treatment, all ulcers had resolved and 

inflammatory signs (edema and erythema) had significantly improved. A surgical 

earlobe biopsy was performed in August 200[, and both the direct examination and 

the histologic studies of the removed tissue showed X/*6%8%" cells. 

6p to November 200[, a total of 1O months] therapy had been administered with 

progressive clinical improvement and only mild erythema and desquamation 

remaining. Tolerance to treatment was excellent. There were no treatment 

interruptions due to adverse events, and only mild headache, considered possibly 

related to the drug, was observed during the first three days of therapy. Hepatic 

enzymes stayed within normal ranges. 

Conclusion: Posaconazole was clinically effective for the treatment of lobomycosis in 

this patient and its administration for 1O months was well tolerated. Posaconazole 

could offer an alternative treatment for patients with extensive disease or disease in 

locations that preclude surgery, as in this case. 

P-0200. Lethal brain abscess due to Scedosporium apiospermum (teleomorph 

Pseudallescheria boydii) after a near-drowning incident: Case report and review 

of the literature 

Buzina W 1 , Feierl G 1 , Reinthaler F 1 , Haas D 1 , Holl A 2 , lleinert R J , Reichenpfader 2 O , 

Roll P O , Marth E 1 

1 Institute of Hygiene, Medical 6niversity, 2 6niversity Hospital for Neurology, J Institute 

of Pathology, O Institute of Forensic Medicine 

A car submerged in a cold mountain laBe due to a traffic accident. The driver, a JKyear-old 

healthy man, was trapped head down under water for at least 1[ minutes. 

After the patient was rescued, he was resuscitated and transferred to the hospital. A 

phase of recovery suddenly ended with massive neurological changes four months 

after the immersion incident. CT-scan showed an increasing extension of the posterior 

horn of the cervical ventricles which was drained subsequently. The patient]s status 

deteriorated and a stereotactic biopsy of the abscess was implemented after MRI of 

the sBull. The purulent material was sent to the microbiological laboratory in order to 

identify bacterial and mycological pathogens. 2acterial cultures remained sterile, 

whereas growth of a mycelium was observed after three days of incubation on 

.abouraud glucose agar at J[ÅC. The fungal culture showed a rapidly growing cottony 

mycelium, first white, later becoming greyish brown. 2oth the macroscopical and 

microscopical characters lead to the diagnosis of =#.5%&'%$"()*+'"%&'.$)(), one of 

the anamorphs of 1&.(5+66.&#?.$"+*8%25"". These results were also confirmed by 

panfungal PCR and subsequent sequencing of the IT. region of the ribosomal DNA. 

The obtained sequence data were submitted to the NC2I (National Centre for 

2iotechnology Information) of the NIH (National Institute of Health) where they were 

assigned accession number D~1O777O. The fungal strain itself was deposited at the 

C2. (Centraalbureau voor .chimmelcultures) in 6trecht, The Netherlands under the 

strain number C2. 1182JJ. In vitro susceptibility testing revealed in vitro resistance 

against amphotericin 2 (MIC 8 mg/l), flucytosine (tJ2), caspofungin (tJ2) and 

fluconazole (2O)n only voriconazole (0.2[) showed an antifungal activity against the 

clinical isolate. 2ased on these results the antimycotic therapy was changed to 

voriconazole. Nevertheless, the patient did not recover and on day 1[J after the 

accident he died due to multi-organic insufficiency. 

Review of the literature for comparable cases (eighteen so far, including this one) 

revealed that only five patients (2K_) survived a brain abscess caused by =/* 

+'"%&'.$)() after a near drowning incidentn they all were treated with either 

miconazole or voriconazole.

P-0201. Central nervous system candidiasis infection: A diagnosis trial 

Caligiorne R 1 , Campolina . 1 , 2arros G 2 , Freire T 2 , Rosa C J 

1 Fundacao fswaldo Cruz - Fiocruz, 2 ClÜnica Neurolágica e Neurocirârgica da .anta 

Casa de 2elo Horizonte - 2razil, J 6niversidade Federal de Minas Gerais, 2elo 

Horizonte - 2razil 

ae report on a case of neurocandidiasis caused by Candida albicans in a healthy 2O 

years-old man. The infection could not be adequately diagnosed by CT scan. The 

correct diagnosis was made only after surgery when a macroabcesse was observed. 

Material was taBen for morphologic and physiologic tests showing the presence of 

Candida albicans. The sequences analysis confirmed the species identification. After 

complete excision the patient was received oral intraconazole and no evidence of 

recurrence of infection was observe at the 6 months followed-up. The treatment 

evolution could be observed by the CT images. 

P-0202. Investigation of the hair digestion by Chrysosporium species 

Calvo M, Calvo R, Adelantado C, Arosemena /, Guiu N, .hiva C 

6niversitat Autínoma de 2arcelona 

The existence of moulds able to attacB Beratinised tissues has been Bnown since the 

last centuries early as 18JK. Dermatophytes were the only Bnown representatives of 

this specialized group. .everal other fungi with the same ability were detected later. 

All these fungi belong to a group Bnown as Beratinophylic fungi. .ome species of 

7?$2&%&'%$"()*show Beratinophylic activity and can grow onto human and animal hair, 

but are not considered pathogens. However, others species have been described as 

aetiological agents in some disorders. The main goal of these study is to develop a 

simple and easy method to demonstrate hair digestion by 7?$2&%&'%$"()*species as 

well as the production of perforating organs. Twenty three strains of 7?$2&%&'%$"() 

species isolated from soils, house dust and atmosphere were investigatedM 7/* 

-$%'"#(), 7 strainsn 7/*).$5+$"(), J strains, 7/*",5"#(), O strainsn 7/*[.$+-",%'?"6(), 7 

strainsn 7/*e(..,&6+,5"#(), 1 strain and 7. anamorph of 4$-?$%5.$)+*#($$.2"3*1 

strain. All isolates were grown on .abouraud dextrose agar. Conidial suspensions 

from 7-day-old cultures of each isolate were prepared in Ringer solution, and the 

conidial concentration ranged from 1x10 [ up to 1x10 7 conidia /m/. .everal fragments 

of horse hair previously sterilized were placed on Petri dishes with 20 m/ of sterile 

2_ agar and 0.20m/ of 10_ yeast extract broth sterilized by filtration and added 

aseptically. Two m/ of conidial suspension of 7?$2&%&'%$"() strains were inoculated 

onto hairs. Three replicates of each strain of 7?$2&%&'%$"() were performed. The 

plates were incubated at 28ÅC in the darB during four weeBs. After incubation, hair 

segments were removed with sterile forceps, placed in a drop of lfH 10_ on a 

microscope slide and gently passed through a low flame of a 2unsen burner. Then a 

drop of lactophenol-cotton blue fluid was added and the material was observed under 

optical microscope. aith the results obtained we can conclude that all strains studied 

can grow on horse hair and produce perforating organs ",*0"-$% except the strains of 

7?$2&%&'%$"()*[.$+-",%'?"6()/ 


P-0203. Identification of clinically relevant yeasts by multiplex PCR: From 

research to clinical application 

Carvalho A 1 , Costa-de-fliveira . 2 , Martins M J, O , Pina-Vaz C 2 , Rodrigues A 2 , 

Rodrigues F 1 

1 /ife And Health .ciences Research Institute (ICV.), 6niversity of Minho, 2raga, 

Portugal, 2 Department of Microbiology, Faculty of Medicine, 6niversity of Porto, Porto, 

Portugal, J Institute of Hygiene and Tropical Medicine, New 6niversity of /isbon, 

/isbon, Portugal, O Centro de Recursos Microbiolágicos (CREM), 2iotechnology 6nit, 

Faculty of .ciences and Technology, New 6niversity of /isbon, /isbon, Portugal 

Invasive fungal infections, namely candidemia, are a major public health problem with 

high mortality rates, mostly due to the delay in diagnosis. The development of rapid 

and sensitive diagnostic methods is therefore a critical issue. ae describe a multiplex 

PCR strategy allowing accurate identification of 8 clinically relevant yeasts of the 

7+,5"5+ genus, namely 7/*+68"#+,&, 7/*@6+8$+-+, 7/*'+$+'&"6%&"&, 7/*-$%'"#+6"&, 7/* 

[$(&.", 7/*@("66".$)%,5"", 7/*6(&"-+,"+. and 7/*5(86",".,&"&. This method is based on 

the amplification of two fragments from the IT.1 and IT.2 regions by the combination 

of 2 yeast-specific and 8 species-specific primers in a single PCR reaction. Results on 

the identification of 2J1 clinical isolates are presented pointing to a high specificity of 

this method. Furthermore, we also identified several 7+,5"5+ strains directly from 

blood culture bottles and urine samples, also attesting its direct clinical application. 

The results of the multiplex reactions with other microorganisms that usually co-infect 

immunocompromised patients also confirmed the high specificity towards 7+,5"5+ 

species identification. Moreover, this method identified 7+,5"5+ species with a 

sensitivity of approximately 2 cells per ml within [ hours, besides allowing 

discrimination of individual species within polyfungal samples. Altogether, the reduced 

time of identification associated to its high specificity, sensitivity and cost-effectiveness 

provides a clinical diagnostic procedure with direct applicability. 

P-0204. Glucocorticoid adrenal function in patients with systemic mycoses 

Cermeño J, Cermeo (, Cova N 

6niversidad de friente. Escuela de Ciencias de la .alud. Ciudad 2olÜvar. Estado 

2olÜvar. Venezuela 

The sistemic mycoses liBe Paracoccidioidomicosis and Histoplasmosis, are the main 

cause of adrenal insufficiency in the countries where they are endemic. An elevated 

frequency of these mycosis has been registered in Venezuela. The aim of this study 

was to evaluate the glucocorticoid adrenal function in patients with 

Paracoccidioidomicosis and Histoplasmosis hospitalized in the 6niversity Hospital 

ÄRuiz y PZezi of Ciudad 2olivar (2olivar state) and in the Hospital Ä/uis Felipe Rojas 

GuevaraÄ, of El Tigre (AnzoZtegui state), Venezuela, between (anuary 200J and 

(anuary 200O 

The Adrenocorticotropin (ACTH) stimulation test results from 12 patients with 

diagnosis of these micosis, and epidemiologic data were registered. The proportion 

manMwoman was [M1, the mean age was J[.1 ì 0.J7 years. 2asal plasmatic cortisol 

levels were within the normal ranB in all the patients abd it was similar to the control 

group. After the injection of synthetic ACTH, an increase of plasmatic cortisol values in 

the normal ranB, but significantly lower than the control group was observed. The 

clinical manifestations wereM fever ([8.J_), cough (O1.6_), dyspnea (16.6_), 

adenopathy, cutaneous injuries and weight loss (8.J_). Tuberculosis was found to be 

associated in 2[_ of the cases. In addition, 2[_ presented Acquired 

Immunodeficiency .yndrome and 8.J_ lymphoproliferative disorders. 

It is important to evaluate the response to the ACTH stimulation test in patients with 

systemic mycoses in our location due to the frequency of its compromise.

P-0205. Use of the microsprayer IA-1B® in the rat model of pulmonary 

aspergillosis 

Chandenier J 1, 2, J , 2ernard . O, J , Montharu ( 2, J , 2ailly E 1 , Fdtissof F [ , de Monte M 2, J , 

Diot P 6, 2, J , Richard-/enoble D 1 

1 Parasitologie-Mycologie-Mddecine tropicale, CHR6, F-J7000 Tours, France, 

2 IN.ERM 6 618, F-J7000 Tours, France, J IFR 1J[, F-J7000 Tours, France, O INRA, F- 

J7J80 Nouzilly, France, [ Anatomopathologie, CHR6, F-J7000 Tours, France, 

6 Pneumologie, CHR6, F-J7000 Tours, France. 

Purpose of the study: Pulmonary aspergillosis is an alarming problem for 

immunodeficient patients, which can lead to more than 80_ lethality. The ability to 

dispose of an effective animal model can contribute to improve Bnowledge, in terms of 

pathophysiology, diagnostic reliability and therapeutic approach. The use of the 

Microsprayer IA-12w can help to achieve this model. 

Material and methods: .prague-Dawley male rats (6-8 weeBs old) were made 

immunodeficient by cyclophosphamide injections associated to hypoprotein food. fn 

day (0, they were put to sleep with gas anesthesia and were infested by intra-tracheal 

aerosolization of 100k/ of 4&'.$@"66(&*U()"@+-(& spores suspension (10 6 conidia) 

thanBs to the Microsprayer IA-12w. Infestation and waBening lasted no more than one 

minute. The follow-up comprises clinical factors (weight variation, death rate), 

biological factors (4&'.$@"66(&*antigens) and histological factors. 

Results: 6sing rats enables easy manipulations and getting of great quantities of 

samples to realize dosages. Infestation realized thanBs to the Microsprayer IA-12w is 

rapid, easy and no immediate death per or post operative is observed. A histologically 

confirmed infection grows in jj_ of the cases. Cumulative death rates are 1K_, 2K_, 

O8_, 7O_ and 80_ respectively on days DJ, D[, D7, D10 and D1O. An 

extrapulmonary diffusion, exclusively hepatic, is observed in 1[_ of the cases. 

Conclusion: The use of the Microsprayer IA-12w leads to an easiness, a profitability 

and a satisfactory reproducibility in control of the rat model of invasive pulmonary 

aspergillosis. **** 

P-0206. Primary cutaneous zygomycosis: An emerging life-threatening clinical 

entity - report of five cases. 

Chander J 1 , laushiB R 2 , Thami G J , Mohan H O 

1 Department of Microbiology, Government Medical College Hospital,, 2 Department of 

.urgery, Government Medical College Hospital,, J Department of Dermatology and 

and Venereal Diseases, Government Medical College Hospital,, O Department of 

Patology, Government Medical College Hospital, 

There is an increase in the incidence of Primary Cutaneous Zygomycosis cases in a 

newly established teaching hospital in north India i.e. Government Medical College 

Hospital, Chandigarh. During the last two years, five cases were reported as 

compared to the previous years when this clinical entity used to be encountered once 

in a while. These cases were admitted with clinical diagnosis of necrotizing fasciitis 

over the anterior abdominal wall and gluteal region. fut of these five cases, three 

were females and two were males. All were belonging to middle age, which was 

ranging from J2 to 60 years. In three cases there was history of injection at the local 

site and in rest two there was history of surgical wound from where the necrotizing 

fasciitis started. None of the patients was having underlying diabetes mellitus, which 

is invariably found to be associated with the zygomycosis cases. The lfH wet mount 

and histopathological stainings were done from clinical specimens collected from the 

wound in which non-septate to sparsely septate hyphae were demonstrated. The 

specimens were inoculated on .abouraud dextrose agar (.DA) and brain heart 

infusion (2HI) agar at 2[ÅC as well as J7ÅC. There was white lid-lifting mycelial 

growth in all the cultures of four of these patients. The mycelial growths were 

identified as =+[&.,+.+*0+&"U%$)"&*in two, 48&"5"+*#%$2)8"U.$+ in one and 48&"5"+ 

#26",5$%&'%$+ in one case. In case of =+[&.,+.+ 0+&"U%$)"& there was difficulty in 

spore induction, therefore, diagnosis was established on the basis of molecular 

techniques for identification of the isolates from a reference laboratory. In one case 

culture was found to be sterile despite direct findings being positive. The 

histopathological examination was done with routine stainings liBe hematoxylin and 

eosin (HcE) as well as special fungal stainings liBe periodic-.chiff (PA.) and Gomori 

methenamine silver (GM.) in all the specimens received. The patients were treated 

with extensive debridement of the necrotic tissues including surrounding healthy 

tissue and institution of intravenous amphotericin 2 in the dose of 0.6 mg per Bg of 

body weight. fut of these five patients, two succumbed to the extensive cutaneous 

involvement, however, three survived, whose wound were covered with sBin grafting, 

are hale and hearty on regular follow up without any recurrence. In view of the 

increasing incidence of such cases, essentially there is need for awareness among 

the clinicians to have high index of suspicion about primary cutaneous zygomycosis 

to establish appropriate diagnosis at an early stage of the infection so that specific 

treatment can be instituted well in time. Moreover, diagnosis of this entity is not very 

difficult if fungal etiology is suspected in time. In addition to that co-operation among 

the clinical specialties and microbiologists is must to reach the final diagnosis. Most of 

the patients can be timely saved with extensive debridement and institution of 

antifungal therapy, particularly amphotericin 2. 

P-0207. Radiological presentation of cryptococcal meningoencephalitis 

analysis of 65 cases from a prospective multicentric study. 

Charlier C 1, 2 , /aunay f J , /dvmque C O , Cordoliani Y O , Chartier / [ , Fontanet A [ , 

Dromer F 1 , /ortholary f 1, 2 , French Cryptococcosis .tudy Group 


1 /aboratoire de Mycologie Moldculaire, Institut Pasteur, Paris, 2 1 .ervice des Maladies 

Infectieuses et Tropicales, HÇpital NecBer, Paris, J .ervice de Mddecine interne, HÇpital Cochin, 

Paris, O .ervice de Radiodiagnostic , HÇpital du Val de Gr°ce, [ 6nitd de recherche et deexpertise 

en dpiddmiologie des maladies dmergentes, Institut Pasteur, Paris 

Purpose: To describe the radiological pattern of cryptoccocal meningoencephalitis among 

immunocompromised HIV-infected and uninfected patients and to identify radiological factors 

influencing outcome. 

Description: CRYPTf A/D is a multicentric prospective study conducted in France between 

1KK7 and 2001 to analyse factors influencing clinical presentation and outcome of cryptococcosis 

serotype A or D. Clinical and biological data were prospectively acquired at baseline, 2 weeBs 

and J months after antifungal therapy initiation. Among the 2J0 enrolled patients, 6[ patients 

living in the Paris area, with culture-proven cryptococcal meningitis and available initial imaging 

investigations were studiedn [6 of them were HIV-infected. Cerebral computerized tomography 

scans (CT, n}[[) and magnetic resonance images (MRI, n}1[) were retrospectively analysed by 

a neuroradiologist blinded to the clinical signs and outcome of the patients. The frequency of 

every radiological sign was determined, and confronted to the initial clinical presentation and 

clinico-biological outcome. 

Results: 

1. Abnormal radiological findings were evidenced by MRI in 1J/1[ cases (87_), and by CT in 

27/[[ cases (OK_). ahen both CT and MRI were performed (n}[), dilatation on the Virchow- 

Robin spaces (VR.) were observed by MRI with normal CT in O cases, and intracerebral mass 

was found both on CT and MRI, in one case. 

2. The main radiological features are summarized in the table. fther lesions included M diffuse 

cerebral oedema (n}1), superior longitudinal sinus thrombosis (n}1) case, ischemic lacunas 

(n}J), and cerebral atrophy (n}6). 

CT (n} 

[[) 

MRI n} 

1[) 

Normal 

28/[[ 

([1_) 

2/1[ 

(1J_) 

VR. or 

pseudocysts 

Intracerebral 

mass 

with/without 

contrast 

enhancement 

Meningitis 

(enlargement, 

collection or 

contrast 

enhancement) 

Ventricular 

dilatation 

[/[[ (10_) O/[[ (8_) 1/[[ (2_) 2/[[ (O_) 

7/1[ (O7_) J/1[ (27_) J/1[ (20_) 0/1[ 

J. Radiological findings were not different between AID. patients and others (pt0.0[). In 6 HIVinfected 

patients, MRI also displayed progressive multifocal leuBoencephalopathy (n}1), and 

features compatible with HIV encephalopathy (n}[). 

O. Abnormal CT or MRI findings did not correlate with sex, age, HIV infection, serotype (A or D), 

more severe initial clinical presentation (altered level of consciousness, seizures, motor 

deficiency or cranial nerve injury), microbiological failure at a2, or death within J months. 

Conclusion: Abnormal dilation of the VR, pseudocysts and intracerebral masses are by 

decreasing order the most often abnormal findings on initial cerebral MRI and CT. However, 

cryptococcal meningoencephalitis cannot be excluded with normal MRI. No baseline radiological 

finding influencing outcome and death within J months was found. 

P-0208. Disseminated aspergillosis with thyroid and bone localization: A case 

report 

Cheikhrouhou F, MaBni F, .allami H, .ellami A, Hadrich I, Mahfoudh M, Hachicha M, 

Ayadi A 

1 /aboratory of Parasitology-Mycology, Habib 2ourguiba 6H -.fax- Tunisia., 2 Pediatric 

ward of .fax Hedi ChaBer 6niversity Hospital, 

Introduction: Disseminated Aspergillus infection has a poor prognosis. Extrapulmonary 

involvement is rare and occurs at an advanced stage. 

ae describe an original case of disseminated aspergillosis (pulmonary, thyroid, 

vertebral) in a young girl. 

Observation: a 1J years old girl, originating in .Bhira (.faxM south of Tunisia), 

admitted to pediatric ward of .fax university hospital in (uly 200O for cervical 

tumefaction, pain of the left Bnee with deterioration of the general state. .he had a 

history of recurrent pulmonary infection and bronchial dilatation at the age of 8 and 10 

years that required prolonged hospitalizations. fne year after, the patient had 

presented a thyroid nodule without hyperthyroidism. 

At this hospitalization, the examination of the patient showed the thyroid node and a 

Bnee mass. The j-ray visualized a multiple bone lysis. The scintigraphy confirmed the 

cold thyroid nodule and the hyperfixation on the [ th left coast, the femur, the hip and 

the left Bnee. The neoplasic origin was evoBed and the patient underwent a 

thyro¢dectomy. Extensive necrosis with infiltration of fungal hyphae was observed in 

the thyroid gland. The histological and mycologic examinations concluded to an 

aspergillosis with 4&'.$@"66(&*U6+0(&. The 4&'.$"66(& antigen was positive. 

Amphotdricine 2 (J0mg/day) and addition of .poranox J00mg one weeB afterwards 

were administered. 

The magnetic resonance imaging of the chest revealed extension of the infiltrative 

process into the mediastinum, the lung, and pleural cavity with further vertebral body 

destruction. .he also developed a femur and tibia osteomyelitis. Aggressive surgical 

debridement was done. The organism isolated from multiple intraoperative bone 

tissue cultures yielded 4&'.6@"66(&*U6+0(& also. The evolution was marBed by cervical 

abscesses and multiorgan function deterioration. The outcome was rapidly fatal. 

The immunizing assessment of deficit could not be carried out neither for the patient 

nor her brother who died few years ago in septic shocB with severe immunodeficiency. 

Conclusion: Few reports have been published on the thyroid localization. This type 

of major mycosis usually occurs in the children with septic family granulomatosis 

whose forecast remains always darB. Early recognition of these entities, prompt 

initiation of new, highly active antifungal therapies and adjunctive surgical 

management may improve the prognosis of these conditions.

P-0209. Trichophyton verrucosum, unusual pathogen in inmunocompromised 

host: A case report. 

Christin M, Fernandez N, 2ogdanowicz E, 2ello N, Tiraboschi (, /attes R, Rodriguez 

Rilo /, Tiraboschi I, /asala M 

Hospital de ClÜnicas 

The aim of this paper is to present an unusual lession in an inmunocompromised host. 

A 1[ year-old female was admitted because of 6 month history of exophytic, redviolaceus, 

painless lesion of the sBin on her feet and righ leg. 

The patient had history of a live-related renal transplant in 2001. .he had two 

episodes of acute rejection treated with tacrolimus and steroids, recurrent urinary tract 

infections, CMV esophagitis and VZV encephalitis. .he lived in rural area and did not 

recall any local trauma. The inmunosuppression drugs consisted of prednisolone and 

sirolimus. 

At the admission she had a 8x8xO cm red-violaceus tumor on the left foot, a similar 

lesion on right foot. fn the right leg she has multiples subcutaneous nodules, one of 

them was ulcerated. This clinical picture suggested some differential diagnosis liBe 

bacillary angiomatosis, laposi]s, pheohyphomycosis and sporotrichosis. 

The wet mount obtained from all lesions showed hyaline hyphae consistent with the 

diagnosis of hyalohyphomycosis. 

Histophatological examination of biopsy stained with hematoxilin-eosin, showed 

intradermal infiltrates of polimorphonuclear leuBocytes, pseudoepitheliomatous 

hyperplasia of epidermis and uncolored branching hyphae of 6u in diameter. 

.he started treatment with Itraconazole O00 mg/day and she improved the cutaneous 

lesions in 2 weeBs. 

The samples were cultured in .DA, Mycosel and lactrimel at J7o and 28oC. After 21 

days, at J7oC white cotton colonies began to grow. Microscopic examination showed 

hyphae with clamydomycoconidia in chains pattern. The isolation was identificated as 

>$"#?%'?2-%,*0.$$(#%&(). 

In summary this report presents a relatively rare case of deep dermatomycosis in 

inmunocompromised patient caused by >/0.$$(#%&() .2ecause of the rarity of this 

condition,the optimal antifungal agent for treatment and duration therapy have yet to 

be established .The outcome depends not only in antifungal therapy but also in the 

management of inmunosuppression. 

P-0210. Comparison of clinical and biological features of invasive aspergillosis 

in neutropenic and non-neutropenic patients 

Cornillet A, Camus C, Nimubona ., Gandemer V, Tattevin P, 2elleguic C, Chevrier ., 

/eray E, Guiguen C, Gangneux J 

/aboratoire de Parasitologie-Mycologie and Aspergillus .tudy Group of Rennes 

Teaching Hospital, France 

The aim of this survey was to compare neutropenic and non-neutropenic patients 

diagnosed with invasive aspergillosis (IA) in our institution over a 6-year period. Case 

notifications were retrospectively (1KK8-1KKK) and prospectively (2000-200O) recorded 

for all patients diagnosed with IA in the entire hospital by trained physicians and from 

mycology and radiology listings and histopathology reports. 

fn the basis of EfRTC/M.G criteria, among the 88 cases of IA analysed here, 12 

were histologically proven, [2 were probable, and 2O were possible. Forty-seven 

percent of cases were diagnosed in intensive care unit and O0_ in haematology units 

(JJ_ in adults and 7_ in children). Neutropenia was a risB factor in [2 patients ([K_), 

most of whom had haematological or solid malignancies. Among the J6 nonneutropenic 

patients (O1_), the main underlying conditions were steroid-treated 

CfPD, asthma, rheumatoid arthritis, giant-cell arteritis and microvascular disordersn 

10 patients were solid organ recipients and one patient was HIV-seropositive. The 

distribution of proven and probable IA was similar in neutropenic and non-neutropenic 

patients. The mortality rate was 71.[_ overall, and was significantly higher in nonneutropenic 

than neutropenic patients (8K_ 0.$&(& 60_, 1v0.0[). Compared to 

neutropenic patients, non-neutropenic patients were significiantly less symptomatic 

and more liBely frequently to have intercurrent pneumonia due to another 

microorganism. The sensitivity of bronchoalveolar lavage fluid (2A/) mycological 

examination and serologic tests was higher in non-neutropenic patients (8[_ 0.$&(& 

[8_,*1v0.0[ for 2A/n O8_ 0.$&(& 6.2[_, 1}0.01 for anti-4&'.$@"66(& antibody 

detection), whereas the sensitivity of antigenemia was the same in the two 

populations (6[_ and*6O_). Thoracic CT signs were similar, except that segmental 

areas of consolidation were more frequent in neutropenic patients. 

IA remains a major life-threatening infection among patients with prolonged 

neutropenia, although protective measures such as air filtration significantly reduced 

its incidence. This 6-year survey at a whole institution, using international criteria, 

underlines the high number of proven and probable cases in non-neutropenic patients. 

Management guidelines are needed for such patients, who have a higher mortality 

rate than their non-neutropenic peers. Clinical signs are frequently lacBing in mildly 

immunosuppressed patients, but the diagnostic value of biological tests such as 2A/ 

mycological examination and 4&'.$@"66(& GM assay is at least as good as in 

neutropenic patients. A wide range of risB factors must be taBen into account, 

including continuous corticosteroid therapy (even at low doses) and 4&'.$@"66(&* 

colonization of the respiratory tract. Finally, measures are required to avoid or reduce 

respiratory tract colonization in patients with chronic pulmonary diseases. 


P-0211. Scytalidium spp superficial infections in immigrants and an 

autoctonous patient in Madrid 

Cuétara M 1 , Aguilar A 2 , (imenez R 1 , Gallego M 2 , ailhelmi I 1 

1 Hospital .evero fchoa, Microbiology Department, /egands, Madrid, .pain, 2 Hospital 

.evero fchoa, Dermatology Department , /egands, Madrid, .pain 

=#2-+6"5"()*5")"5"+-()*G=/5")"5"+-()J*(formerly A.,5.$&%,(6+*-%$(6%"5.+J*is a 

dematiaceous mycelial fungi which cause endemic (geographical restricted to tropical 

and subtropical areas) superficial infections, clinically indistinguible from >$"#?%'?2-%,* 

($(8$() and ",-.$5"@"-+6.). In areas with important immigration such as most European 

countries, misdiagnosis of this infection is possible if cycloheximide is not withdrawn 

from the usual culture medium. 

PurposeM Examine the epidemiology of =/5")"5"+-()*species in a temperate 

European capital (Madrid). 

Methods: Retrospective study (from 200J to 200[) 

Results: .#2-+6"5"()*spp. superficial infections were diagnosed in 6 patients as the 

table shows. 

Age/.ex frigin ..d/.h.ò .oles Towwebs 

OJ/M .pain .D r - 

6[/F Guinea .D,.H r r 

2O/F Colombia .D - - 

J7/F Cameroon .D r r 

O0/F 2razil .D r r 

JK/F Guinea .D - - 

Toenails/ 

Fingernails 

-/- 

r/- 

r/r 

r/- 

r/- 

r/- 

.ubcutaneous 

nodule 

r 

- 

- 

- 

- 

- 

.exM M (male)n F (female)n.D =#2-+6"5"()*5")"5"+-()h*.H =#2-+6"5"()*?2+6",()* 

TTo TfPICA/ AND fRA/ TREATMENT 

The response to treatment (oral/topical) was unsatisfactory. 

TTo 

yes 

yes 

yes 

yes 

yes 

no 

Conclusions: In .pain =#2-+6"5"() spp. is a uncommon agent of dermatomycoses, 

and misdiagnosis could explain that although immigration is high, this species is only 

occasionally identified. This is the first autoctonous =/5")"5"+-() infection in Madrid 

probably due to close contact of the patient with immigrants with clinical infection due 

to this agent. Another important and widely documented issue is the very poor 

response to treatment. In European areas such as /ondon and Paris these infections 

show an ascending incidence linBed to expertise of clinical mycologists and clinical 

suspicion of dermatologists. 

P-0212. Vulvogaginitis by Candida albicans 

de la Parte M 1 , 2rito A 1 , Mendoza M 2 , 2ruzual E 1 , Carmona f 1 

1 (.M. Vargas Medical .chool, 6niversidad Central de Venezuela, Caracas, Venezuela, 

2 Mycology /aboratory, Institute of 2iomedicine, Caracas, Venezuela 

7+,5"5+ species are ubiquitous fungi found throughout the world as normal body flora. 

Vulvovaginitis is an inflammatory condition of the lower female genital tract and is a 

common disorder that can affect females of all ages. This infection by 7+,5"5+* 

+68"#+,&*is the second more common cause of the disease in women in the 

childbearing age. This organism is found in the vagina of 2[_ of asymptomatic 

women and infection occurs with the overgrowth of 7+,5"5+ species, possibly 

triggered by broad-spectrum antibiotics or other factors influencing the vaginal milieu. 

Pregnancy predisposes women to infection because high hormonal levels and 

increased vaginal glycogen content, which favor growth of 7+,5"5+ species. 

6nderlying medical conditions, such as diabetes mellitus, hypothyroidism, or human 

immunodeficiency virus infection (HIV), also predispose patients to Candidal infections. 

7+,5"5+ species are not sexually transmitted and usually are not associated with 

other gynecologic infections. 2etween [0_ and 7[_ of women have had one event 

of vulvovaginitis caused by 7+,5"5+*and of these, 20_ will have a new event later. 

7+,5"5+*+68"#+,&*is responsible for the 8[_ to K0_ of vulvovaginitis caused by 

7+,5"5+*and of the other 7+,5"5+ species involved, the most frequently found is 

7+,5"5+*@6+8$+-+, but there are also 7+,5"5+*[$(&.", 7+,5"5+*@("66.$)%,5" and 

7+,5"5+*5(86",".,&"&*among other infecting species. .ymptoms of vulvovaginitis in 

childbearing age are reported as yellow thicB vaginal discharge and the presence of 

white-greyish pseudomembranes described as hcottage cheese-liBei. There can also 

be observed papules and rarely ulcerative lesions in vulva and sBin of perineal and 

crural areas. Vaginal discomfort can be particularly intense prior to menstruation and 

pruritus and burning in the introitus are exacerbated by the urine passage, sexual 

intercourse and gynecological examination. The objectives of our study were to 

describe the most frequent signs and symptoms of vulvovaginitis by 7+,5"5+*+68"#+,& 

and to identify by microbiological methods the presence of these yeasts in this group 

of patients, during the two last years (200O-200[). A clinical case record was made for 

each patient and a sample from the vaginal discharge was taBen for mycological study. 

The age group with greater frequency of vulvovaginitis was J0 to JO years (26.6_). 

The most frequent symptom found was vaginal discharge (81.6_)n an important 

clinical sign was vaginal inflammation ([8.J_). The characteristics of the vaginal 

discharge were as followM moderate amount (6J.J_)n homogenous appearance 

(6[_)n white color predominant (78.J_)n pH [.0 in O8_ of casesn fresh smear 

examination reportedM blastospores and hyphae in [J.J_ of cases and these were 

absent in J1.6_. The Gram stain showed the characteristic structures (blastospores 

and hyphae) in 60_ of cases. As we can appreciate, our results coincide with the 

values reported by other authors as to the frequency of this infectious pathology by 

this agent. Although, the color of the vaginal discharge most frequent in our cases 

was white instead of yellow and the appearance homogenous instead of the 

characteristic hcottage cheese-liBei.

P-0213. Comparative study of fluconazole and clotrimazole for the treatment of 

vulvovaginal candidiasis 

Diba K 1 , NamaBi A 2 , 2ahadori F 2 , .harbatBhori M 1 , Afioni F 1 

1 Public Health, Tehran, Iran, 2 6romia Medical 6niversity, 6ran 

About 7[_ of women in the wourld are involved by Vulvo Vaginal Candidiasis atleast 

once in their whole life. 7+,5"5+*+68"#+,&, opportunistic yeast, causes 8[-K0_ of 

vaginal mycotic infections. Volve and vaginal itching and cottage cheese liBe 

discharge, are most common signs of VVC. The best treatments for this infection are 

topical Azolesn Clotrimazole and Miconazole or systemic Azoles for examplen 

Fluconazole. The oral administration of Fluconazole has advantages about easily 

using and high effect on recurrent infections caused by hyper colonization of Candida 

albicans in intestinal tract. 

In this study Clotrimazole cream for application to the vulvo vaginal area mycosis in a 

single-blind clinical study.A randomized controlled trial was conducted at lowsar 

6romia Hospital. There were 60 women in the group treated with fluconazole and 60 

in the group treated with clotrimazole, there was no significant difference between the 

two groups regarding age and length of follow-up period. Mycological cure rates 

approximately 1 weeB after treatment were 7K.J_ in the Clotrimazole group and K0_ 

in the Fluconazole group. The side effects were minimal and results were not 

satistically significant. As a conclusion we recommend that a single oral dose of 1[0 

mg of Fluconazole be used as an alternative method of treating VulvoVaginal 

Candidiasis because of short time and easily using of oral, single dose capsule. 

P-0214. New emerging fungal opportunists for Romania implicated in ocular 

infection 

Dinulescu M 

INCDMI Cantacuzino 

fphthalmic mycoses are being increasingly recognized as an important cause of morbidity and 

blindness. Corneal infection by the filamentous fungi is the most frequent. A problem occurs in 

assessing the accuracy of the genus or species identification of a fungal strain isolated on culture. 

The etiologic agent may be determined when the clinical history that suggests a mycotic infection 

is consistent with the presence of the fungus in the samples. 

In this study, strains of hyaline filamentous fungi Paecilomyces lilacinus were reported from [ 

patients hospitalized at The Eye Hospital - 2ucharest, Romania. This is a new taxon for 

Romania, for ophthalmic mycoses only. 

Objectives: The main purpose is to determine the incidence and distribution of the species 

responsible for Beratitis from [ patients (with glaucoma, cataract), and to evaluate the sensitivity 

of the isolated species to antifungal drugs. 

Results: The patients were hospitalized in The Eye Hospital ([J-70 years, sex ratioM J male/ 2 

female) initially had different diagnoses. .amples from patients includedM corneal scraping, 

aspirates from anterior chamber, and removal of the epithelium and necrotic debris overlaying 

the suppurated area. Direct microscopic examination of the samples allowed a rapid, 

presumptive diagnosis of mycotic Beratitis using lfH, lactophenol cotton blue, Gram, Giemsa. 

The samples were inoculated on the surface of solid mediaM .abouraud with cloramphenicol, 

CzapeB Dox, 2lood Agar, and Malt extract. After [-7 days, examination of the isolated culture 

showed conidiophores complexes and conidia disposed in chains typical in shape and 

arrangement to species Paecilomyces lilacinus. The strains were mildly susceptible toM 

letoconazole, Amb and Itraconazole. In The Eye Hospital, topical Natamycin [_ is usually the 

drug of choice for superficial Beratitis. 2ecause deep lesions were still present after J weeBs, 

Itraconazole was added for all patients diagnosed with anterior endophthalmitis. 6sually, 

filamentous fungal Beratitis can involve any area of the cornea, causing elevated necrotic slough 

hhyphate linei extending the ulcer edge into the cornea, multifocal granular gray-white satellite 

stromal infiltrate, and hypopyon. 

6nfortunately, after O weeBs, the corneal infiltrates advanced to the anterior chamber causing an 

increased inflammatory reaction and hypopyon. The visual acuity was now reduced. 

leratoplasty and enucleation were required for all patients, except one female that was 

transfered to another hospital and received Voriconazole. 2ecause the same species was 

isolated from all patients, the analysis of epidemiological features of nosocomial fungal infection 

was necessary to find the source which proved to be from the eye drops administered to all 

patients. 

Conclusion: 

1. Paecilomyces lilacinus was the responsible infectious agent in all the collected 

samples. 

2. Paecilomyces lilacinus is new taxon for Romania implicated in severe ocular 

infections. 

J. The strain was mildly susceptible toM letoconazole, Amb, Itraconazole. 

O. Paecilomyces lilacinus caused severe Beratitis that did not respond to medical 

therapy and necessitated Beratoplasty and enucleation for O patients (one female 

successfully treated with Voriconazole in another hospital). 

[. The findings of the present opportunistic fungus are of great interest as they provide 

evidence of the pathogenic role of this rare fungus. 


P-0215. Etiology of chronic recurrent vulvovaginal candidiasis in women in 

Saint Petersburg, Russia 

Dolgo-.aburova Y, Mirzabalaeva A, Vasilyeva N, 2ogomolova T, Vybornova I, lolb Z, 

.hurpitsBaja f, llimBo N 


Chronic recurrent vulvovaginal candidiasis (CRVVC) is a special clinical problem. 

CRVVC is estimated to occur in [ to 1[ percent of women during their reproductive 

age. Methods of diagnosis and therapy of CRVVC are established but results of 

treatment not always are satisfactory. Frequency of refractory clinical cases of 

CRVVC has increased and there is no consensus among investigators about the 

reasons of that. .ome authors consider that failures of therapy of CRVVC can be 

associated with an increasing role of fluconazole-resistant 7+,5"5+ spp. as etiological 

agents of CRVVC. In Russia this problem is investigated insufficiently. 

Vaginal 7+,5"5+ spp. isolates (n}212) were obtained during prospective clinical study 

(200J-200[) of 212 immunocompetent HIV-negative women in childbearing age with 

CRVVC. CRVVC was defined as four or more episodes of vulvovaginal candidiasis 

during 12-month period. Diagnosis of CRVVC was based on association of clinical 

symptoms (vulval pruritus and burning, hcottage-cheesei vaginal discharge, vulval 

oedema, vulval and vaginal erythema) and standard laboratorial tests (finding of 

budding yeasts , mycelium / pseudomycelium under microscopy of vaginal smears, 

growth 7+,5"5+ spp. on .abouraud dextrose agar). Isolates were identified by 

standard morphological and biochemical methods. In vitro susceptibilities to 

fluconazole of vaginal isolates were determined by disB-diffusion test according to the 

C/.I MOO-A protocol. Also a correlation between anamnestic data, clinical 

presentations and in vitro susceptibilities was investigated. .tatistical data 

manipulation was carried out by of methods of parametric and nonparametric statistics. 

7+,5"5+*+68"#+,& was the main etiologic agent (8[_) of CRVVC. fther pathogens 

were 7/*[$(&."*(O,7_),*7/*@6+8$+-+*(2,8_),*7/*'+$+'&"6%&"&**(1,O_),*7/*[.U2$*(1,O_),*7/* 

@("66".$)%,5""*(1,O_),*7/*d.26+,%"5.&*(0,K_),*7/*-$%'"#+6"&*(0,[_),*7/*6"'%62-"#+*(0,[_),* 

7/*,%$0.@.,&"&*(0,[_),*7/*U+)+-+*(0,[_). Most of 7+,5"5+ spp.*isolates (K2_) were 

susceptible to fluconazole ",*0"-$%. Among 181 7/*+68"#+,& isolates3 susceptibility to 

fluconazole was revealed in K8_ cases, and fluconazole-resistant 7/*+68"#+,&*strains* 

were isolated in 2_/**Resistance to fluconazole was more often among hnon-+68"#+,&i 

species (7/*[$(&."3*7/*@6+8$+-+3*7/*U+)+-+), which were isolated significantly more 

frequently in patients with age %J6 years, with the duration of CRVVC more than O0 

months and with % 8 relapses of the disease per year (pv0.0[). 

P-0216. Isolation and partial characterization of fibronectin-binding proteins 

from Paracoccidioides brasiliensis 

Donofrio F 1 , Andreotti P 1 , Monteiro da .ilva ( 1 , 2enard G 2 , Mendes-Giannini M 1 

1 Departamento de AnZlises ClÜnicas, Faculdade de Ciüncias Farmacüuticas - 6nesp, 

Araraquara, .P, 2razil, 2 /aboratário de Alergia e Imunologia Clinica e Experimental, 

Hospital das Clinicas, Faculdade de Medicina, 6niversidade de .ao Paulo, .ao Paulo, 

2razil 

Introduction and Objectives: The pathogenic fungus 1+$+#%##"5"%"5.&*8$+&"6".,&"& 

causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal 

airborne propagules, which may disseminate to several organs and tissues, leading to 

a severe form of the disease. Adhesion to and invasion of host cells are essential 

steps involved in the infection and dissemination of pathogens. Adhesion of 

pathogenic microorganisms to host tissues is considered essential for initial 

colonization and further dissemination. The extracellular matrix (ECM) is a complex 

mixture of molecules containing several components including fibronectin, laminin, 

collagens and proteoglycans. ECM composition varies in different tissues and during 

phases of injury, inflammation, and repair. Fibronectin is a disulfide-linBed dimeric 

glycoprotein present in a soluble form in blood plasma and other body fluids and a 

fibrilar form in ECM. The major function of fibronectin is probably related to its ability to 

mediate adhesion to mammalian cells, a process that involves the binding of specific 

cell surface receptors to discrete domains in the fibronectin molecule. The present 

study was designed to isolate and to characterize a fibronectin-binding protein from 1/* 

8$+&"6".,&"&*samples, obtained before and after animal inoculation and cultured in 2HI 

with [_ sheep blood and Fava Netto]s medium. 

Methods and Results: In order to identify which components from the fungal cells 

bound to fibronectin, different extracts from yeast forms were used. The hcell-freei 

extracts were prepared from all the samples and the electrophoretic profile showed 

differences in relation to the intensity and to the number of major bands. Twodimensional 

gel electrophoresis and affinity ligand assays were used. ae 

demonstrate the presence of two proteins of 1/*8$+&"6".,&"& that interact with 

fibronectin with relative molecular masses of [8 BDa, pI 6,[ and [O BDa, pI 6,0O. 

Conclusions: Collectively, these data indicate that the 1/8$+&"6".,&"& proteins 

possess Fn-binding activity. 1/*8$+&"6".,&"& surface molecules that bind to host cell 

receptors during adhesion and invasion may be of interest for developing vaccines 

and receptor-blocBing therapies. 

Financial supportM Fapesp, CNPq and PADC.

P-0217. Chromoblastomycosis: Report of the fourth case in Tunisia 

Eleuch D 1 , MoBni M 1 , Haouet . 2 , 2en TeBaya N 1 , Cherif F 1 , Zitouna M 2 , 2en fsman 

Dhahri A 1 

1 Dermatology department, /a Rabta hospital, Tunis, Tunisia, 2 Histopathology 

department, /a Rabta hospital, Tunis, Tunisia 

Introduction: Chromoblastomycosis is a chronic fungal infection of the cutaneous 

and subcutaneous tissues, quite prevalent in tropical countries. This disorder caused 

by various dematiaceous fungi is rarely seen in Tunisia. ae report the fourth case of 

Tunisian chromoblastomycosis. 

Observation: A J8-year-old man from the north Tunisia (2aja) presented since one 

year an erythematous and squamous plaque (2xJcm) in the abdomen. The lesion is 

thin in central portion and papular in the periphery. The patient is a mechanic man of 

sheet metal and the lesion is secondary of inoculation of metal piece ejected from 

blowlamp. Histopathology of a biopsy specimen revealed an acanthosis epidermis, a 

granulomatous dermis infiltrate and macrophage containing septated brown fungal 

cells of 10 to 1[ microns corresponding to fumagoid bodies. 

Culture on .abouraud glucose agar after 1[ days at 27ÅC allows isolation of velvety 

and blacB colonies. Identification shows that the causal organism is ^%,&.#+.+* 

'.5$%&%". There were no abnormalities in routine laboratory tests or chest radiography. 

Patient was treated with association of cryotherapy (O sessions) and terbinafine 2[0 

mg per day during 6 months. 

Conclusion: Chromoblastomycosis is a chronic fungal infection prevalent in tropical 

region such as Madagascar and 2razil. Chromoblastomycosis is rare in North Africa 

with only five cases reported in Morocco and eight cases in Algeria. In Tunisia, only 

three previous cases have been reported due to ^%,&.#+.+*'.5$%&%". fur case is 

particular by the location of the lesion in abdomen (covered area) explained by the job 

of the patient. /ocalised lesion can treat by cryotherapy, surgery in monotherapy or its 

combination with oral anti-fungal such as terbinafine and itraconazole. fur patient was 

treated with cryotherapy associated with terbinafine because he had always 


References: 

Fenniche ., Zaraa, 2enmously R, MarraB H, MoBhtar I. ChromomycosisM a new 

Tunisian case report. Int ( Infect Dis 200[n KM 288-8K. 

Ezzine .ebai N, 2enmously R, Fazaa 2, ChaBer E, Zermani R, lamoun MR. 

Chromomycosis arising in Tunisia Man. Dermatology fnline ( 200[ n 11M 1O. 

Minotto R, 2ernardi CD, mallmann /F, Edelweiss MI, .croferneBer M/. 

ChromoblastomycosisM a review of 100 cases in the state of Rio Grande do .ul, 2razil. 

( Am Acad Dermatol 2001n OOM [8[-K2. 

P-0218. Caspofungin or liposomal amphotericin B in treating invasive fungal 

infections or febrile neutropenia: An open comparative clinical study 

Ellis M 1 , (oseph ( 1 , Alizadeh H 2 , lristensen ( 2 , .hammas V 2 , Hauggaard A J , 

Frampton C O 

1 Department of Medicine, Faculty of Medicine, Al Ain, 6nited Arab Emirates, 

2 Department of Hematology, Tawam Hospital, Al Ain, 6nited Arab Emirates, 

J Department of Radiology, Tawam Hospital, Al Ain, 6nited Arab Emirates, 

O Department of Community Medicine, Faculty of Medicine, Al Ain, 6nited Arab 

Emirates 

Purpose of study: To compare the efficacy of Caspofungin and liposomal 

amphotericin 2 in the treatment of febrile neutropenia or invasive fungal infections in a 

clinical non-trial setting. 

Description: This was an open comparative retrospective case-based study of 

patients admitted for the management of acute leuBemia who had been prescribed 

Caspofungin (C) or liposomal amphotericin 2 (/A2) for the treatment of febrile 

neutropenia or who had an invasive mycosis. No patient received antifungal drug 

prophylaxis. Patients received either antifungal drug according to established 

guidelines. futcome measures were all-cause mortality within 7 days of completing 

anti-fungal drug treatment, treatment response and toxicity. 

Results: fver the period April 2002 | November 200O there were 7J were distinct 

treatment episodes. 2O of the episodes were treated with C and OK with /A2. 2aseline 

characteristics were similar between treatment groups apart from a higher 

representation of hematological diseases other than AM//A// and a lower mean 

larnovsBy score on C. At the start of antifungal treatment the number of patients with 

severe sepsis/shocB were similar in each treatment group (CM2 /A2MJ). 

The indications for antifungal treatment were febrile neutropenia (2J episodes), 

invasive fungal infections (O0) and secondary prophylaxis (10). They were similarly 

distributed between treatment groups. 

The overall response rate in febrile neutropenia as assessed using the [ point 

composite score developed by aalsh and others was similarM CJ7.[_, /A2[8.J_ 

(p}0.6[). However there were J episodes of breaBthrough invasive fungal infection (1 

each of cryptococcosis, pulmonary aspergillosis, candidemia) all of which occurred on 

C (p}0.0O7). 

The proportion of overall responses for invasive fungal infections as assessed after 

Maertens et al, 200O, was similar at CJJ.J_ and /A2[7.7_ (p}0.16). Although the 

response rate for pulmonary aspergillosis was similar, none of the O episodes of 

candidemia or hepatosplenic candidiasis responded to C but J of O patients treated 

with /A2 responded (p}0.1O). 

Toxicity was higher overall with /A2 | at least 1 defined adverse drug event occurred 

in 8O_ episodes treated with /A2 compared to [8_ with C (p}0.018). Rigors were 

more frequent with /A2 (J7_ vs 17_, p}0.08). 


.ix of 2O (2[_) treatment episodes with C resulted in death compared to 2 of OK 

(O.1_) with /A2 (p}0.01J). The primary cause of death was an invasive fungal 

infection in O (16.7_) episodes of C treatment and 1(2_) /A2 treatment (p}0.0J7). 

The fungal infections were for CM 7/+68"#+,& fungemia, 7/-$%'"#+6"& fungemia, fungal 

sinusitis, invasive pulmonary aspergillosis and for /A2M fungal sinusitis. Factors 

associated with mortality on univariate analysis were severe sepsis/shocB (p}0.0J), 

shorter duration of antifungal treatment (p}0.02), longer duration of neutropenia 

(p}0.008) and treatment with C (p}0.01). ahen these were entered into a multivariate 

logistic regression model antifungal treatment with C remained an independent 

predictor of mortality (fR 7.6 sK[_ confidence intervals 1.2-O[.[u). 

Conclusions: Although broadly similar responses for C and /A2 are seen, the higher 

rate of breaBthrough fungal infection and fungal related mortality with C suggests an 

antifungal drug dependent clinical outcome difference. Given the limitations of this 

study design, this observation requires further evaluation. 

P-0219. An open label phase IIa trial to evaluate the efficacy of oral treatment 

with pramiconazole in pityriasis versicolor 

Faergemann J 1 , Ausma ( 2 , aouters / 2 , Vandeplassche / 2 , 2orgers M 2 

1 .ahlgrensBa 6niversity Hospital, Gothenburg, .weden, 2 2arrier Therapeutics nv, 

Geel, 2elgium 

Pramiconazole, previously referred to as R1266J8, is a novel antifungal agent 

belonging to the class of triazoles. It has potent ",*0"-$% activity against !+6+&&.d"+ 

spp., which causes pityriasis versicolor. 

Objective: To evaluate the effects of a single dose of 200 mg/day pramiconazole 

given orally during J consecutive days in patients with pityriasis versicolor. 

Subjects and methods: A total of 1K subjects were included. Clinical evaluation of 

signs and symptoms such as erythema, itching, desquamation, hypopigmentation and 

hyperpigmentation were evaluated on a [-point scale (0}absent, O}severe) and a 

global clinical evaluation compared to baseline on a [-point scale (0}deterioration, 

1}unchanged, O}cured) was performed by the investigator. Mycological evaluation 

was performed by lfH microscopy. In addition, from K subjects samples were taBen 

for scanning and transmission electron microscopy. Evaluations were performed 

before inclusion, after days O, 10 and J0 of onset of treatment. 

Results: A statistically significant reduction of the individual sign and symptoms was 

observed at days 10 and J0, with the exception of hypopigmentation. The mean (..D.) 

sum of the signs and symptoms was reduced from 8.J (1.K) to O.K (1.6) at day O 

(pv0.001). Further reduction to 2.8 (1.7) was seen at day 10 (pv0.001) and to 1.O (1.0) 

at day J0 (pv0.001). The global clinical evaluation (based on erythema, itching and 

desquamation) showed improvement at all follow-up visits (pv0.001 compared to 

baseline) and cure was seen in all patients at day J0. Mycological cure (negative lfH 

microscopy) was obtained in O2_ of the subjects at day 10 and in 100_ of the 

subjects at day J0. Electron microscopy indicated that dead (necrotic) fungi were 

present from day 10 onwards in subjects that were still lfH positive. None of the 

patients stopped the study and no serious adverse events were observed. Adverse 

events reported were mild or moderate. The most frequently reported adverse event 

was headache (Jx). Pramiconazole was well tolerated in patients with pityriasis 

versicolor. 

Conclusion: This phase IIa study indicates that pramiconazole given for J 

consecutive days was effective in treating pityriasis versicolor.

P-0220. Real time PCR on the first galactomannan (GM) positive serum sample 

for diagnosing invasive aspergillosis in liver transplantation. 

Farrugia C 1 , Botterel F 1 , Ichai P 2 , Costa ( 1 , .aliba F 2 , 2retagne . 1 

1 .ervice de Parasitologie-Mycologie, APHP, 6MR 2IPAR K[6, CH6 Henri 

Mondor,Creteil, France, 2 Centre Hdpatobiliaire, APHP, CH6 Paul 2rousse, Villejuif, 

France 

Purpose of the study: Positive GM is a microbiological criterion supporting a 

diagnosis of invasive aspergillosis (IA) although false-positive GM results frequently 

occur in liver transplant recipients. Instead of waiting for additional samples to confirm 

the GM positivity, the search for circulating 4&'.$@"66(& DNA in serum could improve 

the specificity of a first positive GM test. 

Aim of the study: value of 4&'.$@"66(& DNA detection on the first GM positive serum 

sample in diagnosing IA in liver transplant recipients. 

Material and methods: Forty-two patients with a positive GM from (anuary 2000 to 

December 200O were included. GM test was performed using the Platelia Aspergillus 

sandwich E/I.A (2ioRad, Marnes la Coquette). The threshold of positivity was a ratio 

(sample fD/ control fD) % 1. The PCR assay was performed retrospectively on 

serum samples stored at |20ÅC until analysis as previously described ((-M Costa et al, 

2002). For each patient, DNA was extracted twice from 1 ml using a MagNA Pure 

Compact instrument (Roche Diagnostics). Real-time PCR was performed in duplicate 

on each DNA extraction (O PCR tests for a single sample) using a /ight Cycler 

instrument. .ample was considered positive when the crossing point was - O6 cycles 

with at least 2 positive PCR result out of O replicates. A clinician, blinded to the PCR 

results, performed the final classification of IA. 

ResultsM ff the O2 patients, 2J had a positive GM sample after transplantation and 1K 

before transplantation. fut of the 2J patients GM-positive after transplantation, 11 had 

probable IA and 8 of these 11 patients had PCR positive results (Fisheres testn 

1}0.000J). The diagnosis of IA was excluded for the J1 remaining patients and the 

PCR result was negative for all of them. 

P-0221. Onychomycosis prevalence in psoriatic patients 

Ferreira M 1 , Velho G 1 , Teixeira M 1 , Rocha N 2 , /opes V J , Amorim ( O , .elores M 1 

1 Hospital Geral de .anto Antánio, 2 .ervice of Dermatology, Centro Hospitalar de Vila 

Nova de Gaia, Portugal, J Clinical Pathology, Hospital Geral de .anto Antánio, Porto, 

Portugal, O Microbiology, Hospital Geral de .anto Antánio, Porto, Portugal 

Background: Nail involvement in psoriasis is common and it might alter the 

susceptibility to fungal infections. There are controversies considering the prevalence 

and the type of onychomycosis among psoriatic patients. 

The aim of this prospective study is to investigate the occurrence of onychomycosis 

among patients with psoriasis versus patients with other sBin diseases. 

Material and methods: Patients attending to dermatology were evaluated 

concerning clinically abnormal nails. .crapings from those nails were examined using 

direct microscopy and culture. Patients were excluded if they had a referring diagnosis 

of onychomycosis or if they had a previous antimycotic treatment. 

Results: .ixty six patients (male J8, female 28) with ages between O and 8O years 

(mean age of OK.8) were enrolled. J6 had psoriasis and J0 had other sBin diseases. 

The prevalence of onychomycosis in patients with psoriasis was K/J6 } 2[_ 

compared to 12/J0 } O0_ for patients with other sBin diseases (' } 0,1K). 

Dermatophytes were found in [[.[_ of the psoriatic patients versus K1.7_ for 

patients with other sBin diseases (' } 0,0[[). Psoriasis patients had a higher 

frequency of abnormal nails. fther variables, such as age, gender, peripheral 

vascular disease and arthritis were also included. 

Conclusion: In our study the frequency of onychomycosis among patients with 

psoriasis compared with patients with other sBin diseases is not significantly different. 

A higher number of patients included might have found a significantly difference in the 

type of infection between the two groups. 

Discussion-Conclusion: fur data suggest that a concomitant PCR and GM positive 

results support a diagnosis of IA and that a negative PCR result on the first positive 

GM sample may postpone costly antifungal therapy until additional arguments for the 

diagnosis of IA are presented. 

References: 

Costa C, Costa (M, DesterBe C, 2otterel F, Cordonnier C, 2retagne ..Real-time PCR 

coupled with automated DNA extraction and detection of galactomannan antigen in 

serum by enzyme-linBed immunosorbent assay for diagnosis of invasive 

aspergillosis.( Clin Microbiol. 2002nO0(6)M222O-7. 


P-0222. Amino acids alterations in lanosterol 14-alpha demethylase (ERG11) 

related to fluconazole resistance in sequential clinical isolates of Cryptococcus 

neoformans 

Fusco-Almeida A 1 , Matsumoto M 1 , 2aeza / 1 , Derossi A 2 , Zancopd-fliveira R 2 , 

.oares C J , Parente ( J , Moreira . J , Rodero / O , Mendes-Giannini M 1 

1 6niversidade Estadual Paulista (âlio de Mesquita Filho | 6NE.P | Araraquara | .P 

| 2razil, 2 FundaÑÉo fswaldo Cruz | FIfCR6Z | Rio de (aneiro | R( | 2razil, 

J 6niversidade Federal de GoiZs | 6FG | Goi°nia | Gf | 2razil, O Instituto Nacional 

de Enfermedades Infecciosas hDr. Carlos G. MalbrZnh| 2uenos Aires | Argentina 

7$2'-%#%##(&*,.%U%$)+,& is encapsulated yeast, agent of cryptococcosis that can 

affect immunocompetent and especially immunocompromised hosts. The molecular 

typing of the agent is very important to Bnow the genetic types related to the 

resistance phenotypes. The objective of this study was to analyze the aP9II gene 

present in the serial isolates recovered from one patient with elevated MIC (Minimum 

Inhibitory Concentration) values for fluconazole. To achieve this objective, four 

sequential clinical isolates of 7/*,.%U%$)+,&*(isolates 26, 27, 28 and J0) from one 

patient from Rio de (aneiro .tate were selected. All the isolates were previously 

tested against antifungal drugs fluconazole, itraconazole, [-flucytosine and 

amphotericin-2 by microdilution test using standard methods (NCC/.) M27A-2 with 

modifications. The MIC values were 2, 16, 16 and 6O kg/ml, respectively. The 

molecular typing was made for all isolates, using the RAPD (Randomly Amplified 

Polymorphic DNA) method, with initial sequence 6 properly chosen and tested, and all 

isolates presented identical profiles. The first and the fourth isolates (susceptible and 

resistant) were selected to analyze mutation points at the aP9II gene sequence 

through sequencing of amplified products using primers designed from the gene 

sequence. The aP9II gene sequence of the isolate J0 (resistant) showed significant 

points of mutation when aligned with the sequence of the isolate 26 (susceptible) and 

the reference gene. The sequence of the translated protein /anosterol 1O-alpha 

Demethylase of the isolate J0 (resistant) also showed amino acids changes when 

aligned with the sequence of the isolate 26 and the reference gene. Generally, these 

mutations were located close to the conformational regions in form of helix and loops. 

fur results suggested that the amino acids substitutions that occurred at the 

interaction sites of the protein may have caused structural modifications and the 

substitutions inside the active sites suggested changes in the affinity of the protein. 

P-0223. A selective cultural medium for rapid isolation of Candida spp. from 

clinical samples 

Garg A, Gautam R, Almamary A 

Department of Microbiology, Ch. Charan .ingh 6niversity, Meerut, 6ttar Pradesh, 

INDIA. 

Though, 7+,5"5+ infections are now being diagnosed by molecular techniques, yet 

still cultural method(s) are more popular and replicable but they taBe 2O-J6 h. ae 

have developed a selective cultural method for rapid isolation of 7+,5"5+ spp. from 

clinical samples. The medium contains Glucose 20g, Peptone 10g, Yeast extract 1g, 

Agar 1[g, 2romocresol purple 20mg, Chloramphenicol [0mg (all contents per liter in 

distilled water), pH 6.1 # 0.1. It was autoclaved at 1[psi for 1[min and poured into 

sterile polystyrene Petri plates when cooled but still molten. ae collected 276 clinical 

samples of suspected candidiasis from patients] visiting the f.P.D. at of /./.R.M. 

Medical College, Meerut, and AIIM., New Delhi. These were identified and 

characterized on the basis of microscopic examinations coupled with biochemical 

tests and were streaBed onto above culture medium and incubated at J7+C. Change 

in color of the medium from purple to yellow was recorded after every 1h of interval. 

The results revealed that 8[_ (2JO) and 7[_ (206) of total 276 samples were positive 

with microscopic examination and biochemical tests respectively, while the newly 

developed selective culture medium showed 270 positive samples for different 

species of 7+,5"5+ (K8_) out of 276 by change of color of the medium from purple to 

yellow just within 2 to O h of incubation which indicated the presence of 7+,5"5+ sp. in 

the given sample (Table1). Culture positive samples were further confirmed by 

microscopy and biochemical tests. .everal mixed cultures of Bnown pathogenic 

bacteria were also used along with different species of 7+,5"5+ and we found that the 

medium allowed the selective growth of test 7+,5"5+ spp. only (Figure 1). It is 

suggested that medium may be used for the diagnosis of 7+,5"5+ infections by 

cultural method, which can give the results within 2 to Oh and has additional 

advantage of antifungal susceptibility testing facility that will have great clinical 

importance. 

Financial support: CNPq, FAPE.P and PADC

Table 1. Number of clinical samples positive by microscopic, biochemical and cultural examination 

.PECIE. 

Microscopic 

examination 

2ioche 

mical 

test 

Change in color after 

1h 2h Jh Oh 

7/*+68"#+,& 1J8 126 -- 86 128 1[2 1[K 

7/*U+)+-+ 06 0O -- -- 08 08 08 

7/*@6+8$+-+ 16 16 -- 0O 1O 2O 2O 

7/*?+.)(6%,"" 0O 02 -- -- 0O 0O 0O 

7/*[.U2$ 0O 0O -- -- 0O 0O 0O 

7/*[$(&." 08 10 -- 0J 08 12 12 

7/*'+$+'&"6%&"& 10 08 -- -- 0K 12 12 

7/*-$%'"#+6"&/ O8 J6 -- -- JO [0 [2 

7/*0"&B+,+-?""* 00 00 -- -- -- 01 01 

Figure 1. Change in color of the culture medium from purple to yellow by different 

species of 7+,5"5+. 

Total 

No. of 

positive 

sample 

s 

P-0224. Two cases of rhinocerebral and subcutaneous zygomycosis 

Ghilardi A, Massai /, Romano C 

Dept. Clinical Medicine and Immunological .cience, Dermatology .ection, .iena 

6niversity, .iena, Italy 

Case 1: A [0-year-old diabetic farmer developed rhinocerebral zygomycosis after left 

zygomaxillary fracture, treated with systemic antibiotics. A necrotic erythematous 

edematous lesion appeared in the same site with exophthalmus and paralysis of the 

7th cranial nerve. Histological examination and culture of specimens obtained by 

endoscopy from the nasal cavity showed granulomatous suppurating inflammation 

with large, non septate, irregularly branched hyphae suggesting zygomycosis. 

Identification of the mycete as Rhizopus oryzae was based on culture on .abouraud 

dextrose agar with chloramphenicol. Despite therapy with amphotericin 2, the 

infection eroded the orbit and reached the brain. The patient died of 

meningoencephalitis after surgical enucleation of the left eye and areas of necrotic 

brain tissue. 

Case 2: A 70-year-old male presented with a painful erythematous edematous patch 

on the left Bnee, attributed to cellulitis. Despite systemic antibiotic therapy and 

surgical drainage, nodules associated with fistulas formed. 2iopsy showed chronic 

granulomatous suppurating inflammation containing hyphae similar to those found in 

case 1. Culture led to isolation of Rhizopus oryzae. After excluding mucormycosis of 

other organs and systems, the patient was treated with itraconazole and gradually 

recovered. 

Mucormycosis due to Rhizopus oryzae is an infection associated with high mortality, 

especially in forms with rhinocerebral involvement and in patients with immune 

depression or untreated diabetes (case 1). .ubcutaneous forms (case 2) have a 

better prognosis, especially if the patient is immunocompetent and non diabetic. 

References: 

Romano C. et al. Case report. Fatal rhinocerebral zygomycosis due to Rhizopus 

oryzae. Mycoses 2001. OO, 1-6. 


P-0225. Fungemia in hospitalized patients: Risk factor for mortality 

Ghitan M 1 , ChapnicB E 1 , DeCaro G 2 , Fischer C J 

1 Maimonides Medical Center, 2 fvington Medical Group, J .uny Downstate Medical 

Center 

Caspofungin is an echinocandin, antifungal agent approved for the treatment of 

aspergillosis, Candida esophagitis, disseminated candidiasis, and for empiric therapy 

of febrile neutropenia. 

fur 70[-bed teaching hospital in 2rooBlyn NY, purchased ñ [00,000 worth of 

caspofungin in 200[. ae reviewed utilization of antifungal agents and Candida 

species distribution. 

Data was collected from medical records and from the pharmacy database. 

In 200[ there were a total of 2JJ6 Candida isolates. Isolates from blood, urine and 

respiratory secretions were included. 

7+,5"5+*+68"#+,& represented 81_ of isolates (1878 isolates), 7+,5"5+*'+$+'&"6%&"& 

7_ (1[J isolates), 7+,5"5+*-$%'"#+6"& [_ (126 isolates), and 7+,5"5+*@6+8$+-+ 7_ 

(167 isolates). 7+,5"5+*[$(&." (O isolates) and 7+,5"5+*6(&"-+,+. (8 isolates) 

encompased less than one percent of the total. 

ff the patients who received caspofungin, JJ_ had 7/*+68"#+,&, 1[_ 7/*'+$+'&"6%&"& 

and Brusei respectively and 8_ 7/*-$%'"#+6"&. 10_ of the patients treated with 

caspofungin had no Candida species isolated and 1J_ had Candida in the urine 

alone. 

In conclusion, caspofungin use was often unnecessary, since KK.8[_ of Candida 

isolates in our institution are expected to be susceptible to fluconazole. 

P-0226. Surveillance of neutropenic patients with haematologic malignancies 

for fungal infections by non-culture methods 

Giakoumis X 1 , Andreopoulos A 1 , Angelopoulou M 1 , Dimitriadou E 1 , Panayotidis P 1 , 

Galanis Z 1 , .achanas . 1 , Vaiopoulos G 1 , Pangalis G 1 , VelegraBi A 2 

1 1st Department of Internal Medicine, /aiBon General Hospital National and 

lapodistrian 6niversity of Athens, 2 Mycology /aboratory, Microbiology Department, 

National and lapodistrian 6niversity of Athens, Greece 

Invasive fungal infections (IFI), such as aspergillosis and candidiasis, are a major 

cause of mortality in patients with haematologic malignancies. Early diagnosis and 

prompt initiation of antifungal treatment are crucial factors against poor prognosis. 

Non-culture methods such as DNA and antigen detection offer a promising alternative 

to culture-based methods for rapid diagnosis of IFI. 

Aim: To prospectively determine the contribution of combined multiple DNA and 

4&'.$@"66(& galactomannan (GMn) and 7+,5"5+ (Mn) antigen detection assays in the 

early diagnosis of IFI in patients with haematological malignancies who undergo 

intensive chemotherapy. 

Patients and methods: O0 neutropenic episodes of J1 patients with 

polymorhonuclear leucocyte count (PMN) v0.[x10 K /$l for more than 7 days (median 

1J days)3*were prospectively evaluated. .tarting from the first day of hospitalization, 

blood samples were assayed twice weeBly for GMn and Mn antigenaemia (Platelia, 

2io-Rad) and DNAemia, until recovery from severe neutropenia (PMN t0.[x10 K /$l). 

Real time [.-based PCR for 7+,5"5+ spp. and IT.-based multiplex PCR for 

4&'.$@"66(& spp. were performed. Patients were under standard chemotherapy 

regimens, whereas 26/O0 were under antifungal prophylaxis (1[ with fluconazole, 6 

itraconazole, [ voriconazole). Two patients were receiving liposomal amphotericin 2 

and one echinocandine as secondary prophylaxis. IFI was classified according to the 

EfRTC-M.G criteria, including the theurapeutic criterion for all possible and probable 

IFI]s. 

Results: 2[K samples were assayed. fverall, 2 probable and 7 possible invasive 

aspergilloses (IA), as well as 2 probable 7+,5"5+ infections (IC) were detected. PCR 

and/or GMn were at least once positive in 1 probable and 6 possible IA. Three of 

these patients were on fluconazole prophylaxis, 1 on amphotericin 2 secondary 

prophylaxis and J were receiving no prophylaxis. 2oth probable IC were PCR positive 

and/or Mn Ag positive and were not receiving prophylaxis. In the majority of patients 

7+,5"5+ assays were positive only before and during the first 2 days of prophylaxis. In 

patients with probable/possible IFI the non-culture method preceded clinical diagnosis 

by a median of 10 days.

Assay 

No of episodes with positive 

assays / Total No of 

episodes surveyed 

GMn r multiplex PCR 2[/O0 7/2[ 

4&'.$@"66(& 

Mn r PCR 7+,5"5+ 2J/O0 2/2J 

Concurrent GMn / 

multiplex PCR 4&'.$@"66(&* 

and Mn / PCR 7+,5"5+ 

10/O0 [/10 

Probable and possible 

IFI / No of assaypositive 

episode 

Conclusions: This preliminary investigation suggests that twice weeBly monitoring of 

neutropenic patients by PCR for 7+,5"5+*and 4&'.$@"66(& DNA and respective 

antigenaemia might be beneficial in early non-culture diagnosis of IFI in patients with 

haematological malignancies. Administration of antifungal prophylaxis and preemptive 

antifungal therapy, seem to play a Bey role in reducing the risB for IFI. 

Moreover, multiple negative assays for 7+,5"5+ and 4&'.$@"66(&*might exclude the 

diagnosis for IFI (negative predictive value 100_ and 87_ respectively). 

P-0227. A case of coincidental candidemia and pseudomembranous invasive 

tracheobronchial aspergillosis 

Gloeckner A 1 , Abel P 2 , Muellejans 2 J , ZinBe H J , Matthey T J 

1 NRZ Greifswald, Germany, 2 Department of Internal Medicine, 6niversity of 

Greifswald, Germany, J Intensive Care 6nit, Herzzentrum MecBlenburg-Vorpommern, 

lliniBum larlsburg, Germany 

Background: Invasive mycoses in intensive care patients are commonly due to 

yeasts. During the past years, infections with non-albicans species, uncommon yeasts 

and molds have gained importance. 

Case: ae present the case of a 7[ years old male who had been admitted for acute 

myocardial infarction. The medical history included chronic obstructive pulmonary 

disease under long-term systemic and topical glucocorticoid therapy, and pulmonary 

tuberculosis 60 years ago. 

fn day 2, the patient underwent coronary bypass surgery complicated by cardiogenic 

shocB with acute renal failure. For a systemic infection, he was treated with 

meropenem for 10 days. During this treatment, 7+,5"5+*+68"#+,& and 4'&.$@"66(&* 

U()"@+-(& were cultured from tracheal secretions. 7+,5"5+*+68"#+,& was isolated from 

blood culture and central venous catheter tip simultaneously. Fiberoptic bronchoscopy 

revealed several extended cream-colored plaques on the mucosa of trachea, carina 

and right main bronchus. 4&'.$@"66(&*U()"@+-(& was cultured from tracheal secretions 

repeatedly and in large quantity. 

Antimycotic therapy was initiated with caspofungin. Due to lacB of clinical 

improvement, persistently positive tracheal Aspergillus cultures, and another blood 

culture positive for 7+,5"5+*+68"#+,&, therapy was changed to voriconazole. After 7 

days of intravenous application, voriconazole was continued enterally for another five 

weeBs. fverall clinical status of the patient and appearance of the mucosal lesions 

improved, and further cultures were negative for fungi. After J months, the patient was 

transferred to a rehabilitation facility. 

Summary: In a patient with multi-organ failure complicating acute myocardial 

infarction, a combined invasive fungal infection with candidemia and 

pseudomembranous invasive tracheobronchial aspergillosis was successfully treated 

with a sequential course of intravenous and enteral voriconazole. 


P-0228. Candida glabrata breakthrough fungemia during preemptive 

voriconazole treatment in a patient with grade IV GVHD 

Groll A 1 , Florax A 1 , Ehlert l 1 , Vormoor ( 1 , 2ecBer l 2 , Fegeler a 2 

1 6niversity Children]s Hospital Muenster, Munster, Germany, 2 Dept. of Medical 

Microbiology, 6niversity Hospital Muenster, Munster, Germany 

Background: Voriconazole (VCZ) is a new antifungal triazole with broad spectrum 

antifungal activity against Candida species including Candida glabrata (CG). .imilar to 

fluconazole, primary resistance to VCZ (MIC t 1ug/m/) is infrequent in CG and noted 

in [ to 10_ of clinical isolates. /ittle is Bnown about secondary VCZ resistance in CG 

However, as demonstrated with fluconazole, secondary azole resistance is common in 

CG, which is liBely due to the haploid state of this organism. 

Methods and results: ae describe the course of a O-year-old, female Caucasian girl 

with acute myeloblastic leuBemia in first complete remission who developed CG 

breaBthrough fungemia post matched related allogeneic bone marrow transplantation 

(2MT) during treatment for grade IV intestinal graft-vs.-host disease (GVHD). .ix 

weeBs following successful 2MT, the patient developed life-t hreatening grade IV 

intestinal GVHD. After prolonged hospitalization for tO months and switch of 

prophylactic fluconazole to voriconazole for possible pulmonary mold infection, the 

patient was ultimately discharged on triple immunosuppression (tacrolimus, 

mycophenolate-mofetil, methylprednisolone) with stable absolute neutrophil counts 

(ANCs) of t1000/u/, total parenteral nutrition and a /ansBy score of v[0_. Two 

weeBs following discharge, the patient presented with fever and malaise. 2lood 

cultures grew CG, susceptible to amphotericin 2, flucytosine, echinocandins but 

resistant to fluconazole and voriconazole (MICs, t6O and 8 ug/m/, respectively). Due 

to circulatory and respiratory instability, the patient was treated with a combination of 

IV caspofungin ([0mg/m2/day), IV flucytosine (100 mg/Bg/day), and liposomal 

amphotericin 2 (Jmg/Bg/day). However, blood cultures (clinical recovery) only cleared 

after removal of the indwelling 2roviac catheter. The patient made a slow recovery but 

had a second episode of azole-resistant CG fungemia four weeBs following 

discontinuation of antifungal therapy that was ultimately managed by caspofungin, 

amphotericin 2 and another catheter replacement. fne year following the last episode 

of candidemia, the patient is in excellent clinical condition (/ansBy scoreM K0_) with 

100_ donor chimerism, and no evidence for active GVHD on a prolonged sirolimus 

taper. 

Conclusions: This case report demonstrates that VCZ-resistant invasive CG 

breaBthrough infection may occur in severely immunocompromised patients with 

GVHD receiving long-term voriconazole prophylaxis. aith the increased use of 

voriconazole and other second-generation triazoles for antifungal prophylaxis in 

patients with GVHD, the emergence of azole-resistant CG infections needs to be 

monitored closely. 

P-0229. Successful antifungal and hematological management of extensive 

cerebral aspergillosis during leukemic relapse following allogeneic HSCT 

Groll A 1 , Ehlert l 1 , Froehlich 2 1 , 2rentrup A 2 , Vormoor ( 1 , Fegeler a J 

1 Children]s 6niversity Hospital Muenster, Germany, 2 Neurosurgery, 6niversity 

Hospital Muenster, Germany, J Medical Mycology, 6niversity Hospital Muenster, 

Germany 

Background: Disseminated invasive aspergillosis with cerebral involvement following allogeneic 

hematopoietic stem cell transplantation (H.CT) is associated with an exceedingly high mortality 

rate. Here we report the successful antifungal and hematological management of a patient with 

proven pulmonary and extensive cerebral aspergillosis occurring during treatment for leuBemic 

relapse following allogeneic H.CT. 

Case report: The patient was an 18-years-old female with recurrent 2CR/A2/ positive acute 

lymphoblastic leuBemia 1J months following uneventful matched related allogeneic H.CT in first 

complete remission. .hortly after completion of the second antileuBemic reinduction course (A//- 

REZ 2FM 02 plus imatinib), she presented with fever, granulocytopenia, and subsequently, mild 

tachypnea, headaches, mental status changes and hemianopsia. Imaging studies showed 

diffuse nodular infiltration of the right middle lung lobe and two large areas of infarction in the left 

cerebral hemisphere. .he was started on liposomal amphotericin 2 (10mg/Bg/day) plus 

caspofungin ([0mg/day maintenance). Diagnostic worB-up revealed growth of Aspergillus 

fumigatus (MICs by E-testM 0.7[, 0.12[ and tJ2 for amphotericin 2, voriconazole and 

caspofungin) in bronchoalveolar lavage fluid and repeatedly positive serum galactomannan titers. 

Fortunately, r7 days following the start of antifungal therapy, granulocyte counts recovered to 

t[00/u/. The bone marrow aspirate revealed cytological and later, molecular remission, and the 

patient was placed on maintenance therapy plus imatinib on day r1[. The clinical course around 

the time of marrow recovery was complicated by increasing cerebral tissue damage and edema 

with compression of the left temporal ventricle and midline-shift, necessitating the insertion of a 

RicBham reservoir in the left temporal ventricle day r2[. Following clinical stabilization, the 

patient was discharged on day rJK on oral voriconazole, J00mg 2ID. 6nder biweeBly 

decompression of the left temporal horn, the patient improved neurologically and cerebral lesions 

slowly consolidated. However, on day r82, the patient was readmitted due to a generalized 

seizure, followed by increasing granulocytic pleocytosis and ultimately, obstruction of the 

reservoir, leading to its removal with open drainage until day r1JO. Pus aspirated during the 

procedure showed branched septate hyphae, suggesting extension of the infection into the 

ventricle per continuitatem. Following this last procedure, the patient continued to improve 

clinically and by imaging studies under a stable dose of O00 mg voriconazole 2ID, [0 mg 

phenytoin 2ID and weeBly drug monitoring of both substances. Ten months following diagnosis, 

the patient is in continuing hematologic remission with 100_ donor chimerism on a regimen of 

interferon alpha plus imatinib and doing well. Residual neurological defects include subtle deficits 

in memory, attention span and verbal expression and persistent hemianopsia. ahile all 

pulmonary lesion have completely resolved, cerebral lesions have regressed to v2[_ of their 

maximum volume under continuing consolidation therapy with voriconazole. 

Conclusions: This case report supports the notion that extensive cerebral aspergillosis can be 

successfully managed in patients with hematological malignancies by aggressive antifungal 

chemotherapy combined with appropriate surgical interventions and high-level supportive care. 

ahile current clinical data indicate an important role for voriconazole in this condition, the best 

initial therapeutic approach for cerebral aspergillosis remains to be defined. J621 / OK1

P-0230. Candida utilis fungemia in a patient with multi organ failure syndrome 

Gueit I 1 , Etienne M 1 , Abboud P 1 , ChaBarian ( 2 , Gargala G J , 2ougnoux M O , 

2onmarchand G 2 , Caron F 1 , Favennec L J 

1 .ervice de Maladies Infectieuses, CH6, Rouen, France, 2 .ervice de Rdanimation 

mddicale, CH6, Rouen, France, J /aboratoire de Parasitologie, CH6, Rouen, 

France, O /aboratoire de Microbiologie, HÇpital NecBer, Paris, France 

The patient was a J0-year-old aest Indian HCV positive man with chronic pancreatitis. 

He was admitted to the intensive care unit for a multi organ failure syndrome related to 

/egionnairese disease. Persistent fever led to the diagnosis of 7+,5"5+*(-"6"& fungemia 

with 10 positive blood cultures out of 11 collected between day 1J and day 20. fne 

blood culture collected on day 2J yielded 7/*@6+8$+-+. Two venous femoral catheter 

and one arterial femoral catheter removed on day 10 and day 16 also yielded 7/**(-"6"&/* 

6rine sample from day 16 yielded 7/*(-"6"& and 7/*[.U2$. MICs %U*7/*(-"6"& were 2 µg/ml 

and 0.06O µg/ml for fluconazole and voriconazole, respectively. 7/*(-"6"& was correctly 

identified after amplification and sequence analysis of fungal internal transcribed 

spacers 1 and 2 and [.8. ribosomal DNA regions. Venous echo doppler showed a 

partial thrombosis of inferior vena cava, both iliac veins and right common femoral 

vein. 2oth transthoracic and transoesophageal echocardiographies showed no 

endocarditis. Treatment with IV caspofungin [0 mg qd was began on day 16 and then 

switched after 17 days to oral voriconazole 200 mg bid for JO more days. Evolution 

showed persistence of febricula until day O0. 2lood cultures became negative after [ 

days of antifungal treatment. Control echo doppler showed complete resolution of the 

thrombosis after 80 days of anticoagulant treatment which was stopped. The patient 

was definitively discharged from hospital after three months. 

ahile candidemia are commonly encountered among debilitated patients, we report 

here the third case of 7/*(-"6"& fungemia. In the present case, the patient had several 

host factors classically related to fungemia of other species. Two mechanisms were 

hypothesized for the portal of entryM a) a catheter infection as the patient presented 

with diarrhoea at the time of infection and had femoral vascular devices b) a 

translocation after digestive colonization since 7/*(-"6"& is widely encountered in food. 

Interestingly, as in the two previously reported cases, this 7/*(-"6"& infection was 

associated to a high number of positive blood cultures and persistence of candidemia 

under treatment. However this could be related to the vascular thrombosis. ae also 

noticed the association to another 7+,5"5+ species fungemia as previously described. 

P-0231. Identification of dimorphic fungi using the applied biosystems 

microseq® d2 lsu rDNA sequencing and a custom library 

Hall L, aohlfiel ., Roberts G, aengenacB N 

Mayo Clinic College of Medicine, Rochester, 6.A 

Identification of the dimorphic pathogens*Z6+&-%)2#.&*5.$)+-"-"5"&, 7%##"5"%"5.&* 

"))"-"&, A"&-%'6+&)+*#+'&(6+-(), 1+$+#%##"5"%"5.&*8$+&"6".,&"&, 1.,"#"66"()*)+$,.UU." 

and ='%$%-?$"_*&#?.,#["" can be challenging in the clinical mycology laboratory. Many 

laboratories rely on commercially available, FDA-approved nucleic acid hybridization 

probes (AccuProbe, GenProbe, Inc. .an Diego, CA) for identification of A/*#+'&(6+-(), 

Z/*5.$)+-"-"5"&, and 7/*"))"-"&. Identification of 1/*8$+&"6".,&"&, 1/*)+$,.UU.", and =/* 

&#?.,#["" requires observation of both the yeast and mould forms of these fungi which 

can sometimes be difficult to achieve. In addition, several organisms (1/*8$+&"6".,&"&, 

92),+&#.66+*?2+6",%&'%$+, and a))%,&"+*'+$0+) have been noted to cross-react with 

the Z/*5.$)+-"-"5"& hybridization probe causing false-positive results. 

The aim of this study was to evaluate the use of D2 /.6 rDNA sequencing for the 

identification of the dimorphic fungi. The commercially available Applied 2io.ystems 

D2 library contains the sequence of only one dimorphic fungus, A/*#+'&(6+-(). ae 

therefore sequenced both reference and clinical strains of Z/*5.$)+-"-"5"& (n}O0), 7/* 

"))"-"& (6), A/*#+'&(6+-() (8), 1/*)+$,.UU." ([), 1/*8$+&"6".,&"& (O) and =/*&#?.,#["" 

(1J) to create a custom D2 library for the dimorphic fungi. ae also included 9/* 

?2+6",%&'%$+ (2), a/*'+$0+ (2), a))%,&"+ spp (6) and other closely related organisms 

including 4$-?$%5.$)+ (1), 7?$2&%&'%$"()*(O), 9.%)2#.&*([), 92),%+&#(& (1), 

!+68$+,#?.+ (J), 1.,"#"66"() spp (not 1/*)+$,.UU.") (1O), and =.'.5%,"() sp. (1). D2 

/.6 rDNA sequencing is useful for identification of Z/*5.$)+-"-"5"&3 A/*#+'&(6+-(), 7/* 

"))"-"&, 1/*8$+&"6".,&"&, 1/*)+$,.UU." and =/*&#?.,#["" in instances where the nucleic 

acid hybridization probes are not available or when conversion is difficult or hazardous 

to laboratory personnel. 


P-0232. Evaluation of DNA detection in the diagnosis of pulmonary 

aspergillosis in a routine clinical setting: Correlation with galactomannan elisa, 

cultivation and autopsy 

Hamal P 1 , RaclavsBy V 1 , .auer P 2 , Faber E 2 , PalatBa l 2 , 2uchta V J , Drahosova M J , 

lodouseB R 1 

1 PalacBy 6niversity, flomouc, Czech Republic, 2 6niversity Hospital, flomouc, Czech 

Republic, J 6niversity Hospital, Hradec lralove, Czech Republic 

Purpose of the study: The incidence of aspergillosis, particularly its invasive form, is increasing 

and associated with high mortality. The successful outcome of treatment depends significantly on 

the early diagnosis. The most promising approaches are detection of DNA and galactomannan 

(GM) antigen. 

The aim of this analysis was to evaluate the contribution of the DNA-based method in the early 

diagnosis of aspergillosis on a large set of clinical samples. 

Description: This prospective three-year study was carried out from 200J to 200[. aithin this 

period, 60[ clinical specimens, mostly blood, were examined for the presence of 4&'.$@"66(& 

DNA. The specimens were taBen from 270 patients of the 6niversity Hospital flomouc, with 

majority from the Hemato-oncology clinic. 

For DNA detection, nested-PCR developed by YamaBami et al. (1KK6) with slight modifications of 

the original protocol was used. Further, a part of the specimens examined by nested-PCR (21J 

blood samples from 111 patients), was tested for the presence of GM antigen using the 

diagnostic Bit Platelia Aspergillus (2ioRad). Index values higher than 1.[ were considered as 

positive (P), however, doubtful (D) range was extended from 0.[ to 1.[, in accordance with the 

reference data. In addition, 1[2 samples were examined concurrently in two microbiology 

laboratories in order to verify interlaboratory reproducibility. 

P-0233. Effect invivo - invitro of different concentrations of substances (A, B) 

produced by Dermatophytes fungi on red and white blood cells. 

Hammadi K 1 , .elselet G 1 , Italhi M 2 

1 Faculty of .ciences, 6niversity of Mostaganem, Algeria, 2 Hospital of Ain Tadless, 

Mostaganem, Algeria 

This worB is based on a the secondary metabolites, of dermaiophytes fungi, produced 

by the species TRICHfPHYTfN.mentagrophytes var interdigitales and 

TRICHfPHYTfN verrucosumn targeting an anti-leuBemia-liBe substances. 

The evaluation of the substances (A) and (2) were carried out on two different type of 

blood cells lines,leucocytic and erythroytic cells lines.The two active substances (A 

and 2) showed dierct decreases of white blood cells linesnwhen the decreaeses begun 

for the red blood cells at the concentration of (0.2J). the tests in vivo on rabbits of the 

two substances (A and 2 ) showed a positive results when the rabbits remained alive 

which confirm the non toxicity of these substances. 

ley wordsM secondary metabolites of dermatophytesn anti-leuBemia-liBen tests in-vitro 

and in-vivo 

Results and conclusions: 1[7 patients were examined only once, [0 twice, 21 three times, 18 

four times and 2O more (range [ | 1[). 4&'.$@"66(& DNA was detected in 28 samples (O.6_) 

taBen from 2J patients (8.[_). Nineteen of them were from blood, six from bronchoalveolar 

lavage fluid and three from sputum. GM antigen was evaluated as P in nine samples from eight 

patients and as D in 2O samples from 22 patients. In respect of interlaboratory reproducibility, 

1JO samples were evaluated identically, 1J with slight, one with medium and four with wide 

differences. Positive culture was recorded in 2J patients, whereas all isolates were obtained from 

the lower respiratory tract. 4&'.$@"66(&*U()"@+-(& was identified in 21 patients, while in remaining 

two 4/*U6+0(& and 4/*-.$$.(& were detected. A histopathological examination revealed the 

4&'.$@"66(& infection in 16 cases. ahen the positive results of DNA detection and GM (both P 

and D) were compared, a concordance of methods was found in two cases only and differences 

in 1K cases. In nine patients from 2J, positive DNA detection was confirmed by isolation of 

Aspergillus from clinical material and in five patients from 17 with histopathologically confirmed 

aspergillosis, DNA was detected before death. 

2ased on presented data, it can be concluded that diagnosis of pulmonary aspergillosis is still 

difficult and requires close collaboration between clinicians and laboratories. 6sing rapid 

diagnostic methods, it is necessary to perform monitoring of risB patients at least twice weeBly, 

whereas a minimum of two consecutive positive samples should be considered positive. The 

best approach is to use detection of DNA and GM concurrently. 

Acknowledgements: This research was supported by the Ministry of Health, Czech Republic 

(grant no. NR/8J6[-O) and by the Ministry of Education, Czech Republic (research programme 

no. M.M 61K8K[K20[).

P-0234. Widespread dermatophytoses: Study of 12 cases 

Hanchi I, Eleuch D, 2en teBaya N, Cherif F, MoBni M, 2en fsman Dhahri A 

Dermatology department, Rabta Hospital, Tunis, Tunisia 

Aim: The aim of our study is to analyse epidemiological, clinical, biological and 

therapeutic data of 12 patients having widespread dermatophytoses. 

Patients and Method: fur study deals with 12 patients having widespread 

dermatophytoses, diagnosed between 1KKJ and 200[ in the department of 

dermatology of Rabta hospital. Extensive superficial dermatophytoses as well as 

dermatophytic disease were excluded. All the patients had a complete physical 

examination and mutiple mycological samples. Furthermore, K patients had biological 

investigations including at least blood cell count. 

Results: Patients were 7 women and [ men (sex-ratioM 0.7). The age ranged between 

20 days and 61 years (average ageM J0.K years). In all cases, typical clinical features 

(widespread and multifocal lesions of the sBin) were notedn with involvement of face, 

trunB and limbs respectively in [, 8 and 10 cases (O2_, 66.[_ and 8J_). Involvement 

of the large folds, onychomycoses and tinea capitis were respectively observed in 

O2_ of cases. Mycological culture revealed Trichophyton rubrum in 7J_ and 

Trichophyton violaceum in [O,[_ of cases. In the newborn (20 days of age), the 

causative agent was Microsporum canis with presence of domestic animals in familial 

home. Circinate dermatophytoses in related parents was noted for 2 patients. Topical 

or systemic corticostero¢ds were reported in O cases. In 2 other cases, there was 

commun ichtyosis. 2iology showed neutropenia in one case, hypereosinophilia at 

O[00/mmJ with elevated seric IgE in an other case. Anergy to trichophytin was 

observed in O cases over [, associated in J cases to anergy to tuberculin. Eleven of 

our patients were treated by systemic antifungal drugsM griseofulvin (K cases), 

terbinafin (2 cases), associated to topical antifungal agent. The newborn received only 

a topical treatment. In all cases, outcome was favorable but lesions recurred (2 or J 

recurrences) in [8_ of patients having risB factors. 

P-0235. Invasive pulmonary aspergillosis: a retrospective clinical and 

pathological analysis of 23 cases at autopsy 

Hao F 

Dept. Dermatology, .outhwest Hospital, Chongqing, China 

Objective: to analyse the clinical and pathological characteristics of invasive 

pulmonary aspergillosis (IPA) in order to investigate the possibilities of early diagnosis 

and clinic-pathological classification of IPAn 

Methods: Total [12 adult cases at autopsy from 1K71-200O were enrolled for study, 

and 2J IPA cases diagnosed clearly by tissue section features were further analysed. 

Results: Among 2J IPA cases, 21 cases have underlying diseases and 20 cases 

have predisposing factors. .eventeen patients got fever and eighteen patients had 

respiratory symptoms. Nine cases showed various signs of pulmonary embolism and 

extrapulmonary embolism. Acute bronchopneumonia, angio-invasive aspergillosis, 

acute tracheobronchitis, miliary aspergillosis and pleural aspergillosis were shown in 

1[, 12, [, J and 1 cases respectively by clinical radiographic signs and pathological 

features. The overlap between acute bronchopneumonia, angioinvasive aspergillosis 

was common, which usually led to cause the death. The clinical manifestations of IPA 

were miscellaneous. It is benefit to investigate the clinical characteristics, especially 

chest radiographic signs and pathological changes for early diagnosis and prognosis 

evaluation. 

Conclusion: .ome people having probable immunological deficiencies seam to have 

a genetic predisposition to develop widespread, multifocal and recurrent 

dermatophytoses. .ome authors suggested in 2001 the expression of £>$"#?%'?2-%,* 

$(8$() syndrome§, characterised by cutaneous involvement of the feet, the hands 

and the nailsn and also the involvement of at least an other location (except inguinal 

folds). Direct mycological samples must be positive in the O sites and culture must 

reveal >$"#?%'?2-%,*$(8$()*in at least J sites. Two of our patients having no favouring 

factors responded to this criteria. 

Extensive superficial dermatophytose is defined as a chronic widespread 

dermatophytose involving a large cutaneous surface and usually affecting nails and 

hair. It is nosologically, a different entity which evolve into dermatophytic disease. 

Nevertheless, widespread dermatophytoses could, in some cases, represent an early 

stage of extensive superficial dermatophytose. 


CLINICAL MYCOLOGY* 

P-0236. Isolated chronic Fusarium paronychia (I.C.F.P.): An ignored or 

overlooked pathology? 

Harak H 1 , auhrer E 2 

1 fspedale di .esto .an Giovanni (Milano), Italy, 2 Poliambulatorio Monza, Italy 

Purpose of study: To draw attention to the existence of this isolated pathology, 

highlighting the way to better identify it and to treat it as early as possible avoiding its 

complications and achieving a complete recovery. 

Summarized description of the project: ae studied seven clinical cases of I.C.F.PM 

three of right hand fingers, one of left and three of the toes, over K years (1KK6-200[). 

Despite being ^(&+$"() an emerging pathogen and an important agent of 

onychomycosis and ^(&+$"() paronychia a typical cutaneous portal for invasive and 

disseminated infections, it is interesting that this variant of ^(&+$"() clinical spectrum 

is not recognized by physicians and rejected by experts as an independent entity. 

The term hisolatedi in the I.C.F.P. definition refers to a paronychia which is 

independent from ^(&+$"() onychomycosis i.e. not preceded, combined or followed 

by it. The paronychia is characterized by under proximal nail fold invasion by the mold. 

Results and Conclusions: 

" The chronic ^(&+$"() paronychia isolated or combined with onychomycosis 

looBs liBe a bacterial cellulitis of the periungual sBin clinically 

indistinguishable from 7+,5"5+ paronychia. 

I.C.F.P. can be differentiated into two clinical subtypes primary and 

secondary (iatrogenic) paronychia depending on the pathogenetic 

bacBground. 

" ^(&+$"() seems to be the only fungus able to cause isolated paronychia and 

onychomadesis. Through long arrest of the activity of the matrix which is 

suggestive of I.C.F.P. one or more finger or toe nails may be lost. 

fther fungi may cause nail loss through different mechanism i.e. by total 

dystrophic onychomycosis. 

" Even clinically mild paronychia should be considered infected with ^(&+$"(), 

unless demonstrated the absence of the mold. 

" Although still considered saprophytic fungus and often disregarded as 

contaminant in normal host, ^(&+$"() should be regarded as primary sBin 

and nail pathogen 

Patients of dermatology ward had been followed personally by the authors since the 

first examination until recovery. 

Clinical significance of isolated ^(&+$"() was evaluated according to walshe-english 

criteria. A variable degree of periungual eritema and swelling was present at the onset 

of the pathology. 

In all cases there was a sudden nail shedding and ^(&+$"() was isolated from 

scrapings under the proximal nail fold using aesculap scalpel. There was no isolation 

of the mold from the nail area or from the stamp of the nail. 

Toe webs were not involved except in one case in which >/*P(8$() was isolated. 

No spontaneous discharge of pus from under proximal nail fold neither when exerting 

pressure on the nail fold were observed. In 2 cases abundant serous discharge from 

under nail fold was present. 

All patients were women, age range was 2O-86 immunocompetent and in good health 

and extremity circulation. 

The responsible species were 6 ^/*b_2&'%$() and 1 ^/*=%6+,". 

Treatment with terbinafine 2[0 mg per day for J-O month combined with topical 

whitfield ung. or spir. gave a complete mycological and clinical recovery with new 

growth of healthy nail.

P-0237. Post-traumatic filamentous fungi infections: A 6-case report and review 

of the literature 

Hincky V 1,7 , /ebeau 2 1 , Falcon D 2 , Pradel P J , 2ozonnet E O , Faure f 1 , Aubert A [ , 

Piolat C 6 , Grillot R 1 , Pelloux H 1 

1 

.ervice de Parasitologie-Mycologie, CH6 Grenoble, France, 2 6nitd de Rdanimation Chirurgicale, 

CH6 grenoble, France, J Chirurgie Plastique de la main et des brôlds, CH6 grenoble, France, 

O 

Chirurgie Plastique et Maxillo- Faciale, CH6 grenoble, France, [ Chirurgie Vasculaire, CH6 

grenoble, France, 6 Chirurgie Pddiatrique Gdndrale, CH6 grenoble, France, 7 Mddecine 

Infectieuse et Tropicale, CH6 grenoble, France 

Moulds, such as Mucorales or 4&'.$@"66(& sp are generally opportunistic filamentous fungi (FF) 

which can cause, in immunocompromised patients, serious and rapidly fatal infections. In the 

immunocompetent host, such environmental fungi can be responsible for post traumatic primary 

cutaneous infections, when wounds are soil contaminated. 

From (anuary 1KKJ to .eptember 200[ we observed 6 cases of post traumatic cutaneous fungal 

infections with soil contamination. Here we report the epidemiology, clinical and diagnostic 

features, and therapeutic strategies. Patients were aged [ | OO years, with sex ratio M/FM [/1. 

Traumatisms were due to farm worBing accident (n}O) or road accident (n}2). Infecting fungi 

were Mucorales species alone in J cases (!(#%$*&'3*P?"d%)(#%$*&'3*48&"5"+*#%$2)8"U.$+), 

4&'.$@"66(&*U()"@+-(& alone (1 case) or combined with !(#%$ sp. (1 case), ^(&+$"()*&'3* 

4&'.$@"66(&*-.$$.(& and 4/*#%$2)8"U.$+ (1 case). Necrosis area biopsy showed presence of 

hyphae and positive culture. Despite recurrent surgical exeresis of infected tissues and systemic 

antifungal treatment, five among 6 patients had a second limb amputation (n}O) or large 

musculocutaneous exeresis (n}1). 

The review of the literature (Pubmed research and same inclusion criteria) shows 60 cases were 

published since 1K8[. Zygomycetes were involved in O7/66, (71_), mainly 4'%'?2&.& .6.@+,&3G* 

n}1J ) and P?"d%'(& sp (n}1[ ). The other FF isolated wereM 4&'.$@"66(& sp (8/66, 12_), 

=#.5%&'%$"()*sp (O/66, 6_). For 7 cases (11_), at least two FF species were present, mainly 

Mucorale plus ^(&+$"() sp. Fungal contamination was linBed with a road accident (O2_) or a 

farm worBing accident (2J_). All the patients were immunocompetent excepted one renal 

transplant, 2 diabetes mellitus and one splenectomy. 

The main symptoms were necrosis (6J_), particularly frequent in mucormycosis, characterized 

by vascular invasion resulting in thrombosis and infarction of affected tissues, and/or sepsis or 

local inflammation (27_), more rarely cottony mould appearance of the wound (8_). Three 

cases of Mucorales disseminated or invasive infections ([_) were reported. Diagnosis is usually 

easy to perform from excised tissues, but sometimes early samples were sterile and have to be 

systematically repeated, especially in infarcted areas, in patients who present soil contaminated 

wounds. The features of our cases are similar to those described in the literatureM post traumatic 

FF infections can be severe and often require disabling surgery. However, no patient died in our 

series, while the mortality reported in the literature is about J0_ when Zygomycetes are 

involved, especially if more than one limb is affected. Consequently, these infections require 

optimal treatment combining early aggressive surgery and antifungal therapy. Interest of new 

treatments such as hyperbaric oxygen or posaconazole, a new triazole, remains to be evaluated. 

P-0238. Treatment for Trichophyton tonsurans infection by using the result 

obtained from the hairbrush method as an index 

Hiruma M 1 , .hiraBi Y 2 , IBeda . 2 , fgawa H 2 

1 Dept of Dermatol, (untendo 6niversity Nerima Hospital, 2 Dept of Dermatol, (untendo 

6niversity 

>/*-%,&($+,& infection has emerged as a serious threat to public health among martial 

artists in (apan as well as overseas. For the purpose of controlling this infection, a 

treatment that used the result from the hairbrush method as an index was evaluated. 

MethodM ff J27 martial artists in the 12 sites investigated, 6K who tested positive by 

the hairbrush method were subjected to this study. a. If the number of colonies as 

observed by the hairbrush method was O or fewer, the hair was washed with 

miconazole shampoo. b. If the number of colonies was [ or more, itraconazole or 

terbinafine was administered. .ubsequently, the presence or absence of colonies was 

evaluated by the hairbrush method at 1.[ months and J months after treatment. 

ResultsM 6sing the hairbrush method, [ or more colonies were detected in each of O6 

subjects. The >/*-%,&($+,& test was negative in J2 (6K.6_) subjects who were orally 

administered terbinafine or itraconazole in compliance with their treatment schedulen 

however, the test was not negative in individuals who discontinued the treatment. 

Twenty-three subjects with O or fewer colonies were indicated for treatment with a 

shampoo containing miconazole hydrochloride. The test was negative in 1[ (6[.2_) 

subjects and revealed an increase in the number of colonies in 6 (26.0_) of them. 

The results of 2 (8.7_) subjects were unBnown. ConclusionM ae considered this 

treatment protocol to be effective. However, 1. there were, in fact, some subjects who 

were not able to complete the treatment or complied poorly with their treatment 

schedule, and 2. the >/*-%,&($+,& test was not negative in J[_ of the subjects treated 

with a shampoo. Thus, it is important that further studies be conducted in future to 

design a more effective treatment for controlling this infection. 


P-0239. A retrospective study of the isolation of Scytalidium species from a 

London dermatology centre during 2003-2005 

Howell S, Cunningham M, Duncan G 

.t (ohnes Institute of Dermatology 

Dermatophytes are a common cause of superficial mycotic infection in temperate 

regions, however, in tropical and sub-tropical regions =#2-+6"5"() species are also 

significant causes of infection (Midgley .-*+6 1KKO). The aim of this study was to 

examine the frequency of isolation of these species in recent years from patients in 

the /ondon area. 

During the period 200J to 200[ 11K =#2-+6"5"()*5")"5"+-() and J7 =/*?2+6",() 

isolates were recovered from palms, soles, toe webs, finger nails and toe nails 

specimens received at the Mycology laboratories at .t (ohn]s Institute of Dermatology, 

/ondon. Isolation was slightly more frequent from males than females (1.1[M1) and the 

average age of the patient was [8 years (range 26-8[ years). 

Examination of information available on the specimen request forms indicated that 

7/1[6 (O.[_) patients had diabetes, two patients reported travel overseas, K were 

from the 6nited lingdom, 1K were Afrocaribbean, 2 from Nigeria, 1 from India, and 6 

were marBed as unBnown. 

During this period J80O[ specimens were received from J2[18 patients, of which 

1KK2O were from the hands, feet or nails. The total number of microscopy positive 

specimens from these sites was 870[ and =#2-+6"5"() species were recovered from 

2.8_ of these. >$"#?%'?2-%,*$(8$() was isolated from O0.K_ of these microscopy 

positive specimens, >/*).,-+@$%'?2-.& var. ",-.$5"@"-+6. from 1J.2_, a'"5.$)%'?2-%, 

U6%##%&() from 0.6_, and =#%'(6+$"%'&"& species from 0.[_. 

P-0240. Black fungal plaque of Cladosporium sphaerospermum formed on a 

diabetic burn ulcer 

Iozumi K 1 , Enomoto 6 1 , MaBimura l 2 

1 ToByo Metropolitan Police Hospital, ToByo, (apan, 2 TeiByo 6niversity Institute of 

Medical Mycology, ToByo, (apan 

A OK-years-old woman with diabetes mellitus had a burn ulcer when she had put a hot 

plastic bottle on her right big toe. .he had bulla first and later an ulcer on the toe. .he 

visited a certain clinic and continued a topical treatment of ointment with 

betamethasone valerate and gentamicin sulfate. fne month later, she gradually had a 

blacB plaque on the ulcer. .ix months later since she first had a burn ulcer, she visited 

our department. .he had a 1K x 11mm blacB elastic plaque, which had clear elevated 

border on the burn ulcer. ae found a lot of hyphae of dematiaceous fungi conformed 

the blacB plaque directly under microscope and later on the pathological specimen. 

The cultured fungus incubated at 2[ o C formed first darB green and later blacB velvety 

colony. The cultured fungus incubated at J2 o C formed a blacB elevated node very 

slowly. fn a slide culture specimen we detected cladosporium type spores formation. 

The base sequence of 28. rDNA (D1/D2) region formed a single clade with the 

sequences of 76+5%&'%$"()*&'?+.$%&'.$)()*(A21006[O et al. in 

DD2(/EM2//Gen2anB database) and was 100_ identical. Therefore, we have 

identified the cultured fungus from the plaque as 76+5%&'%$"()*&'?+.$%&'.$)(). ae 

performed debridement of the ulcer with topical treatment of amphotericin 2 and 

systemic oral therapy of Itraconazole 200mg/day for 2 months, with good recovery of 

normal tissue and tiny scar formation. There has been no evidence of recurrence 

during 6 months of follow-up. 

=#2-+6"5"() species were the third most frequently isolated mould pathogens from the 

hands, feet and nails of specimens received from patients in /ondon and the 

surrounding areas. 

Reference: 

Midgley G, Moore Ml, CooB (C, Phan ~G. ( Am Acad Dermatol 1KKOn J1M .68-7O

P-0241. Voriconazole (VCZ) propably does not affect the pharmacokinetics of 

methotrexate (MTX) 

(abali Y, Mallatova N, .mrcBa V, Vrzalova A 

Hospital in CesBe 2udejovice 

Background: MTj c VCZ have many drug interactions. ahether an interaction exists 

between them is not Bnown. 

Aim: To explore whether oral VCZ affects the pharmacoBinetics of MTj. 

Patients & Methods: aith informed parental consent, a prospective case/control 

study with standard clinical c drug monitoring was performed on 2 children with 

intermediate-risB acute lymphoblastic leuBemia during consolidation chemotherapy. 

This consisted of 6-mercaptopurine 2[ mg/m 2 /d Pf [6 d, MTj 2 g/m 2 /2Oh IV q 2 wB 

xO, leucovorin 1[ mg/m 2 xJ IV per each MTj c MTj 12 mg IT q 2 wB xO. The case 

has been on oral VCZ because of invasive pulmonary aspergillosis (IPA). The control 

was infection free. MTj serum levels were measured at 0, 18, 2O, J6 c O2h until 

achieving a concentration of at least 0.2[ umol//. Inter- c intra-patient MTj levels 

were compared by ailcoxon signed ranBs test c Friedman test, respectively. 

Results: A 10.[-yr-old boy developed IPA, controlled by A2/C ([ mg/Bg/d for 2K d). 

fral VCZ for 61 d was given thereafter. Consolidation started while being 17 d on 

VCZ. Another [-yr-old boy w/o IPA was undergoing consolidation under the same 

controlled conditions. J pairs of MTj infusion running in parallel were evaluated. 

2aseline MTj levels sumol//u were below the detection limit (v0.0[) by FPIA (TDx w 

Abbott Diagnostics) in both pts. In the VCZ/MTj arm, MTj levels at the specified time 

points were 28.62, 1K.J[, 0.[2 c 0.18 (1. MTj)n 27.62, 2O.17, 0.7K c 0.2[ (2. MTj), 

2J.1J, 21.J8, 0.O6 c 0.16 (J. MTj). The O. MTj was delivered [ d off VCZ (21.[J, 

11.JK, 0.J2 c 0.11). In the MTj only arm, the corresponding figures were 2O.1O, 

1K.[0, 0.62 c 0.22 (2. MTj)n 27.0K, 18.6J, 0.[0 c 0.2J (J. MTj)n 26.60, 18.77, 0.[0 c 

0.1K (O. MTj). All MTj levels were in the expected range, and the inter-patient 

difference was N. (p}0.7n 0.07n 0.[). However, while the intra-patient difference was 

not significant (p}0.1) in the control, it was so (p}0.02) in the case. This was caused 

by significant (p}0.02-0.0O) depression of the last MTj (off-VCZ) 0&. the first J ones 

(on-VCZ), within which the intra-patient difference was not significant either (p}0.1). 

aithin 1-J d of every MTj infusion, the first pt developed cheilitis and photosensitivity 

over exposed body parts. The cheilitis/dermatitis resolved towards the next MTj. No 

other side effects were observed. There was no evidence of exacerbation of IPA. 

Conclusions: Although the off-VCZ levels of MTj lied out those on-VCZ in the case, 

the data suggest that oral VCZ seems not to affect MTj pharmacoBinetics significantly. 

However, a larger number of pts and/or doses of MTj given during VCZ therapy 

should be investigated with more stringent drug monitoring before definitive 

conclusions can be made. 

P-0242. Comparative evaluation of methods based on semi-solid medium and 

agar culture with the eucast and CLSI broth microdilution standard methods for 

testing susceptibilities of clinical yeast isolates to five antifungals 

Jakobson E 1 , Hagblom P 1 , (ohansson A 2 , Fernandez V 1 

1 Dept of Parasitology, Mycology and aater Microbiology, .wedish Institute for 

Infectious Disease Control, .tocBholm, .weden, 2 Division of Clinical Microbiology, 

Dept of Molecular and Clinical Medicine, /inBéping 6niversity, /inBéping, .weden 

K,*0"-$% measurements of antifungal susceptibility of clinical yeast isolates are often 

crucial in the management of invasive 7+,5"5+ infections. At present, the C/.I M27- 

A2 method is the recommended standard for susceptibility testing of yeast. An 

alternative reference method incorporating modifications in order to improve objectivity 

in MIC determination (E6CA.T E Dis 7.1) has been developed but not yet fully 

replaced the C/.I standard in the clinical microbiological laboratory. The performance 

of the E6CA.T broth microdilution method and two commercially available tests, the 

Etest which is based on a drug-concentration gradient diffusing from a strip to the 

solid agar medium, and the AT2 Fungus 2 which measures the effect of antibiotics on 

yeast growing in a semi-solid agar medium, was compared with that of the C/.I 

method. .usceptibility to fluconazole, itraconazole, voriconazole, amphotericin 2 and 

[-fluorocytosine was tested in K1 clinical isolates ([8 from blood and JJ from vaginal 

mucosa) including 8 7+,5"5+ species and =+##?+$%)2#.&*#.$.0"&"+.. For 7/*+68"#+,&, 

the agreement (within ì 2 two-fold dilution) between the E6CA.T and C/.I methods 

for fluconazole, itraconazole, voriconazole, amphotericin 2 and flucytosine MICs was 

K8, 100, 8O, 100 and 100_, respectively. The corresponding agreement between the 

Etest and C/.I methods was K6, K6, KO, 100 and K8_, respectively. Agreements 

were slightly lower when antifungal MICs were determined for non-+68"#+,& species. A 

general good agreement in .IR-interpretation of MIC data for 7/*+68"#+,& was 

observed between E6CA.T, Etest, AT2 Fungus 2 and the C/.I method. In contrast, 

frequent minor discrepancies in interpretive susceptibility group were detected when 7/ 

@6+8$+-+, 7/*[$(&." and other non-+68"#+,& isolates were assayed with all methods. 

Confirming earlier reports the Etest method was superior to the C/.I standard, but 

also to E6CA.T and AT2 Fungus 2, for discrimination of amphotericin 2 MICs t1 

mg//. In disagreement with C/.I, E6CA.T and AT2 Fungus 2, all 7/*[$(&." isolates 

tested with Etest were classified as resistant to [-fluorocytosine. For all antifungals 

except amphotericin 2 assayed with Etest, and all yeast species with the exception of 

7/*[$(&.", the MIC at which K0_ of the isolates were inhibited (MIC K0 ) did not differ by 

more than 2 two-fold dilutions or across clinical breaBpoints between the four methods 

evaluated in this study. In conclusion, both AT2 Fungus 2 and Etest are useful laboursaving 

alternatives for antifungal susceptibility testing in routine diagnostics. Access to 

one reference method, E6CA.T or C/.I, is however recommended for quality 

assessment purposes and for the elucidation of cases in which MIC interpretation is 

complex. 


P-0243. Pulmonary case of blastomycosis at a Tunisian man 

lallel l 1 , Anane . 1 , laouech E 1 , 2elhadj . 1 , Chabbou A 2 , Nefzi F J , ChaBer E 1 

1 Dept. of Parasitology-Mycology, Rabtaes Hospital, Tunis, 2 Dept. of Pneumology, 

Arianaes Hospital, Tunis, J Dept. of Anatomopathology, Arianaes Hospital, Tunis 

The blastomycosis is a rare mycosis in Tunisia, due to a dimorphic fungusM 

Z6+&-%)2#.&*5.$)+-"-"5"&. 

ae report a case of pulmonary blastomycosis at a Tunisian man, farm laborer whose 

is O[ years old, 

The symptomatology began with the appearance of a cough bringing bacB in the 

morning bleeding expectorations, accompanied by right thoracic pains, a not 

quantified fever and a deterioration of the general state. Rachidian and right hip pains 

appeared secondarily. 

Thorax radiography showed an opacity with hydrous tonality, heterogeneous, retractile, 

with blurred borderlines at the top of the right lung. The bronchial fibroscopy 

highlighted a budding formation and the tomodensitometry a tissue mass affecting the 

higher and the lower lobes of the right lung. The osseous scintiscanning showed an 

hyperfixation compared to DJ and the right great trochanter. Repeated biopsies 

eliminated a neoplasic process which was the most probable diagnosis. fn the other 

hand, in one of them the presence of large yeasts with thicB cell wall and broad-based 

bud was noted evoBing a blastomycosis. The confirmation was brought by the 

mycological investigation (microscopy examination and .abouraud culture) of biopsic 

fragments and bronchoalveolar washing. The patient then received itraconazole at a 

rate of O00mg per day. After J7 days, a good clinico-radiological evolution was notedM 

reduction of cough, expectorations, osseous pains and radiological opacity. 

The blastomycosis remains a rare infection in Tunisia, only some sporadic cases were 

reported. The noisy pulmonary attacB is also a characteristic of this observation, the 

initial respiratory localization is often unperceived 

P-0244. Cryptococcus diffluens isolated from sporotrichoid lesions of an HIVseronegative 

patient 

Kantarcioglu A 1 , 2oeBhout T 2 , de Hoog G J , Theelen 2 O , Yycel A [ , EBmeBci R 6 , Fries 

C 7 , IBeda R 8 , loslu A K , Altas l 10 

1 Cerrahpasa Medical Faculty, Dept of Microbiology and Clinical Microbiology, Deep 

Mycosis /aboratory, Istanbul, TurBey, 2 Centraalbureau voor .chimmelcultures, c ADD 

Division of Acute Medicine and Infectious Diseases, 6niversity Medical Center 6trecht, 

6tracht, The Netherlands, J Centraalbureau voor .chimmelcultures, 6trecht, c Institute 

for 2iodiversity and Ecosystem Dynamics, Amsterdam, The Netherlands, 

O Centraalbureau voor .chimmelcultures, 6trecht, The Netherlands, [ Cerrahpasa 

Medical Faculty, Dept of Microbiology and Clinical Microbiology, Deep Mycosis 

/aboratory, Istanbul, TurBey, 6 .isli Etfal Training and Research Hospital, Dept. of 

Dermatology, Istanbul, TurBey, 7 Albert Einstein College of Medicine, Depts of 

Medicine, Microbiology and Immunology, New YorB, 6.A, 8 Meiji Pharmaceutical 

6niversity, Dept. of Microbiology, ToByo, (apan, K .isli Etfal Training and Research 

Hospital, Dept. of Dermatology, Istanbul, TurBey, 10 Cerrahpasa Medical Faculty, Dept 

of Microbiology and Clinical Microbiology, Istanbul, TurBey 

Case: A healthy (HIV-seronegative) 17 years-old white, male university student from 

TurBmenistan was admitted in Istanbul with a two-month history of two nontender, erythematous, 

crusted, noduler lesions of [ to 6 cm diameter on both the right wrist and arm. Three 

subcutaneous lesions with linear arrangement were palpeted on the right forearm. He worBed in 

the deserts in TurBmenistan two months before the lesions appeared. Pathological examination 

of a punch biopsy material revealed yeast cells in tissue but the fungus did not growth on culture 

in the clinical laboratory. Clinical suspicion was sporotrichosis and itraconazole therapy, 100 

mg/day, was started. A second punch biopsy sample was obtained and submitted to the Deep 

Mycosis laboratory for detailed mycological study. The sBin lesion responded to treatment within 

2[ days and complete resolution occured after 2 months. 

Diagnosis: Direct microscopical examination revealed globose or ellipsoidal, encapsulated yeast 

cells in various dimensions. .pecimens were inoculated onto routine and selective mycological 

media. Colonies grew just around the biopsied tissue specimens. Microscopical examination of 

india inB preparations demonstrated the presence of encapsulated yeast cells with elongated 

buds. Two colony morphologies were identified as mucoid and wrinBled, respectively. These 

colony morphologies were however unstable as sectored colonies occurred. The yeast cells were 

stained with monoclonal antibody 18-27 (mAbs) to glucuronoxylomannan. Carbohydrate 

assimilation tests indicated that the isolate belonged to the genus 7$2'-%#%##(&, but it could not 

be identified by the API 7+,5"5+ Bit. .equence analysis of the IT. 1r 2 regions and the D1/D2 

domains of the /.6 rDNA showed a KK_ match with reference 7/*5"UU6(.,&*strains. K,*0"-$% 

susceptibility of the isolate against seven antifungal agents were examined according to the 

NCC/. M27-A reference broth macrodilution method and demonstrated that the strain was 

resistant to AM2 and sensitive to azoles (MICs (&g/ml)M amphotericin 2M 8n [-flucytosineM 16n 

fluconazoleM On itraconazoleM On BetoBonazoleM 0.[n miconazoleM 0.[, and terbinafineM 0.0J). Cell 

slide agglultination tests were performed to determine the antigenic formula of the strain by using 

8 factor sera for 7$2'-%#%##(&. The strain reacted to factor sera 1, 2 and J which were shared 

with 7/,.%U%$)+,& and some other 7$2'-%#%##(& species. Environmental fungi, including 

7$2'-%#%##(& 5"UU6(.,& can be responsible for sBin lesions mimicBing sporotrichosis. Decisive 

diagnosis of these infections should be based on biopsy and culturing results followed by 

molecular identification.

P-0245. A Rhizopus oryzae strain from a sinonasal and palatal mucormycosis 

in a nondiabetic patient with renal failure: in vitro experiments of yeastlike cell 

developpment 

Kantarcioglu A 1 , Yycel A 2 , Nagao l J , .ato T O , Inci E [ , fgreden . 6 , laytaz A 7 , .ar 

M 8 , lepil N K , Altas l 10 

1 Cerrahpasa Medical Faculty, Dept of Microbiology and Clinical Microbiology, Deep 

Mycoses /ab, 2 Cerrahpasa Medical Faculty, Dept of Microbiology and Clinical 

Microbiology, Deep Mycoses /ab, J Dermatology 2ranch, National Institutes of Health, 

2ethesda, 6.A, O Department of Dermatology, leio 6niversity .chool of Medicine, 

ToByo, (apan, [ Department of ftorhinolaryngology, Cerrahpasa Medical Faculty, 

Istanbul 6niversity, Istanbul, TurBey, 6 Department of ftorhinolaryngology, Cerrahpasa 

Medical Faculty, Istanbul 6niversity, Istanbul, TurBey, 7 Department of 

ftorhinolaryngology, Cerrahpasa Medical Faculty, Istanbul 6niversity, Istanbul, 

TurBey, 8 Department of Pathology, Cerrahpasa Medical Faculty, Istanbul 6niversity, 

Istanbul, TurBey, K Department of Pathology, Cerrahpasa Medical Faculty, Istanbul 

6niversity, Istanbul, TurBey, 10 Cerrahpasa Medical Faculty, Dept of Microbiology and 

Clinical Microbiology, Deep Mycoses /ab 

heart infusion agar (2HIA) with blood and glucosen blood glucose cysteine agar 

(2GCA), and yeast extract peptone glucose agar (YEPGA) were used under high 

concentration of Cf 2 and anaerobiosis. Colonies grown on enriched 2HIA and 2GCA 

media and under two modified atmosphere were macroscopically and microscopically 

similar as thin, spreading films. Culture microscopy revealed the presence of broad, 

hyaline coenocytic hyphae and budding yeast-liBe cells similar to those seen in tissue 

preparations. In vitro induced yeast-liBe cell development of the case isolate was 

documented by photomicrographs. Cultures grow as typical mold colonies with arising 

numerous sporangia on YEPGA at J[+C under unaerobic and aerobic environment. 

The room air growth control group showed mycelial colony growth and uninoculated 

plates remained sterile under modified atmosphere conditions. 

.ince the expected characteristic tissue morphology of mucormycosis agents consists 

of broad, irregularly shaped, non-septate and wide-angle branching hyaline hyphae, in 

case of mucormycosis suspicion, with respect to the authors] experiences, it is 

advisable remembering the ability of P/%$2d+. producing budding yeast-liBe forms 

besides typical hyphae under particular conditions as well as in host]s environment. 

ae report (i) a histologically and mycologically proven sinonasal mucormycosis case 

causing palatal necrosis and (ii) in vitro induced yeastliBe cell development of the case 

isolate. 

Case: The patient was a [0 year-old man who had a one year history of renal failure 

and undergone to haemodialysis for two months prior to his admission to a local 

hospital with prolonged nasal bleeding and a slough on the palate. He was found non 

diabetic. A palatal biopsy was performed. Histopathological examination showed 

necrotizing granulomatous inflammation and fungal elements consistent with 

!(#%$+6.&. He received intravenous amphotericin 2 (AM2) therapy for a total of J0 

days. .ubsequently, the patient admitted to a different hospital in Istanbul, TurBey 

where he received intravenous AM2 therapy for a total of J0 days. Than he 

transferred to Department of ftorhynolaryngology, at Cerrahpasa Medical Faculty 

where liposomal AM2 was administered intravenously ([0 mg b.i.d.). 

Histopathological and mycological examination prooved mucormycosis. He underwent 

left radical maxillectomy, submucosal nasal septum resection, left anterior and 

posterior ethmoidectomy, left frontal sinus exploration, subtotal soft and hard palate 

resection. Antifungal therapy was continued for two months. Medication was 

supported with hyperbaric oxygen treatment at 2.[ ATA for ten times. Postoperative 

control magnetic resonans image showed residual deposits, with any finding with 

regard to relaps. 

Mycology study: .everal pieces of palatal tissue and bone samples obtained by 

surgical debridement were submitted to Deep Mycoses /aboratory. Mycological 

examination of Giemsa stained imprinted tissue preparations revealed abundant 

unicellular budding fungal elements besides the typical mucoraceous hyphae. The 

fungus was isolated from surgical specimens inoculated onto routine and selective 

mycological media and was identified as P?"d%'(&*%$2d+. by phenotypic and 

genotypic tests. /aboratory studies were performed to investigate if the yeast-liBe 

fungal morphology seen in tissue preparations were belonged to the isolated fungus. 

Three different test media and two modified atmosphere were used under elevated 

temperature to induce yeast-liBe cell development of P/%$2d+. clinical isolate. 2ain 


P-0246. Cryptococcal meningitis: About 22 Tunisian cases 

laouech E 1 , lallel l 1 , Anane . 1 , 2elhadj . 1 , Chaabane T 2 , /aBhal . J , ChaBer E 1 

1 Dept. of Parasitology-Mycology, Rabtaes Hospital, Tunis, 2 Dept. of Infectious 

Diseases, Rabtaes Hospital, Tunis, J Dept. of Intensive Care 6nit, Rabtaes Hospital, 

Tunis 

Cryptococcal meningitis is a serious fungal infection due to an encapsulated yeast M 

7$2'-%#%##(&*,.%U%$)+,&/ It is generally seen in subjects having a deficit of cellular 

immunity and in particular the patients infected by the HIV. 

ae report in this worB 22 cases of cryptococcal meningitis diagnosed between 1KK0 

and 200[ at the department of Parasitology-Mycology at the Rabta]s Hospital in Tunis. 

They are 1[ men, six women whose average age is JO years and a nine years old 

child. 16 patients had a positive HIV serology, including six discovered at the time of 

the cryptococcal meningitisn the six other patients presented other 

immunodeficienciesM non-insulino-dependent diabetes, non-hodgBinien malignant 

lymphoma, systemic lupus erythematous, renal transplant, cirrhosis post hepatitis 2 

and congenital immune deficit. 

Fever, headache and necB rigidity were present in 80_ of these cases. 

The diagnosis, directed by the cellular and biochemical aspect of the C.F (bland and 

hypertensive in 16 cases, with high lymphocyte level in 1K cases and low glucose 

level in all these cases), was confirmed by the direct examination with the Indian inB 

and/or the culture on medium of .abouraud and/or the research of the cryptococcal 

antigen. After antifungal treatment (amphotericin 2 with or without [FC, fluconazole), 

17 patients died. 

P-0247. Scedosporium apiospermum infection after near drowning: A pediatric 

case and review 

Katragkou A 1 , Dotis ( 1 , TamiolaBi M 2 , Roilides E 1 

1 Jrd Department of Pediatrics, Aristotle 6niversity, ThessaloniBi, Greece, 2 Pediatric 

Intensive Care 6nit, HippoBration Hospital 

Purpose: =#.5%&'%$"()*+'"%&'.$)()*G1&.(5+66.&#?.$"+*8%25""J*is a ubiquitous 

saprophytic mold. Although infrequent, it is an important and emerging human 

pathogen under specific conditions, in particular after near drowning. ae aimed to 

review all cases of =/*+'"%&'.$)()*infection after near drowning previously published 

and to include an additional paediatric case from our institution. 

MethodsM A Medline search was carried out for the terms h1&.(5+66.&#?.$"+*8%25""f, 

h1.-$".66"5"()*8%25""i, h466.&#?.$"+*8%25""i, h!%,%&'%$"() +'"%&'.$)()i and 

h=#.5%&'%$"()*+'"%&'.$)()i, cross-referenced with hnear drowningi. 

Results: fverall 20 cases (11 adults and K children) were analyzed. Their median 

age was 1K.O (range, 1.J-[J) years, 70_ were males. Most patients (8[_) were 

immunocompetent. The mean period of time from the initial accident to diagnosis of 

scedosporiosis was O8 (range, 12-210) days. CN. infection due to =/*+'"%&'.$)()* 

occurred in 18 patients, manifested as solitary (O patients) or multiple (1J) brain 

abscesses, meningitis/encephalitis (6), ventriculitis-ependymitis (1) and cerebral 

vasculitis (J). fther infections included endophthalmitis (J) and osteomyelitis (1)n 

whereas, dissemination to Bidney (O), heart (2), liver (1) and thyroid (1) were identified 

postmortem. =/*+'"%&'.$)()*was isolated from brain abscess aspiration material (12), 

C.F (6), urine (2), sBin (1), Bnee (1), vitrectomy (1) and chest tube fluid (1). ahile all 

patients suffered from preceding aspiration pneumonia, =/*+'"%&'.$)()*was isolated 

from the respiratory secretions of only 6 patients. =/ +'"%&'.$)()*was almost always 

resistant to amphotericin 2, flucytosine and fluconazole and sensitive to miconazole, 

Betoconazole, itraconazole and voriconazole. Antifungal treatment consisted of 

conventional and lipid amphotericin 2 formulations (1K), miconazole (6), itraconazole 

(6), voriconazole ([), fluconazole (O), flucytocine (O), saperconazole (1), Betoconazole 

(1), caspofungin (1), terbinafine (1). Most patients (6[_) received more than one of 

these either as combined or sequential antifungal therapy and the majority (K0_) 

underwent a neurosurgical intervention. The mean survival time was 76 (range, 12- 

210) days and the mortality rate was 7[_. 

Conclusions: =/*+'"%&'.$)()*infection after near drowning is associated with high 

rate of dissemination, especially to CN., and high mortality rate, even in 

immunocompetent patients. Prompt antemortem diagnosis is difficult and is basically 

made using culture methodology. fptimal therapy remains undefined. A diagnostic 

suspicion should be maintained facing a near drowning episode with respiratory 

cultures and neuroimaging included during initial laboratory investigations.

P-0248. Primary cutaneous zygomycosis in an infant with Pearson’s Syndrome 

lefala-Agoropoulou l 1 , FarmaBi E 1 , VelegraBi A 2 , Tsiouris I 1 , Roilides E 1 

1 Jrd Department of Pediatrics, Aristotle 6niversity, HippoBration Hospital, ThessaloniBi, 

Greece, 2 Microbiology Department, 6niversity of Athens, Greece 

Purpose: Zygomycosis has emerged as an increasingly important infection followed 

by a high mortality. It occurs mainly in immunocompromised or diabetic patients. 

However, conditions such as iron overload and deferoxamine therapy have also been 

reported as risB factors. ahile the infection has been very rare in infancy beyond 

neonatal age, we report the first case of cutaneous zygomycosis in an infant with 

Pearson]s syndrome. 

Case report: ae describe an 11-month old girl suffering from sideroblastic anemia 

since birth and a severe iron overload. .he was heterozygote for C282Y, one of the 

hemochromatosis mutations, which aggravates the iron overload. The infant received 

corticosteroids (10mg/Bg/day for O weeBs) and deferoxamine (for O weeBs). Primary 

cutaneous zygomycosis developed at the patient]s right arm at an intravenous 

catheter insertion site. An acute inflammatory response with pus, abscess formation, 

tissue swelling and necrosis preceded. Infected tissue appeared red and indurated, 

then became violaceous and progressed to blacB eschar. The necrotic tissue 

sloughed leaving a large ulcer. Histopathological examination of the lesion revealed 

ribbon-liBe, aseptate hyphal elements that branched at right angles in the center of 

necrotic debris. Invasion of the blood vessels by hyphal elements was also 

demonstrated on tissue section. Pus culture grew P?"d%'(&*%$2d+.. /iposomal 

amphotericin 2 was initiated at a dose of [.[ mg/Bg/d and the necrotic tissues were 

aggressively debrided. Additional management of disease included tapering of 

corticosteroid therapy and discontinuation of iron chelation. The patient responded to 

treatment, cultures turned out to be negative and therapy was discontinued after J 

weeBs partially due to drug toxicity. fne weeB later, zygomycosis relapsed as a new 

neighboring cutaneous lesion, and liposomal amphotericin 2 was again initiated at the 

same dosage along with aggressive surgical debridement of the infected area. The 

infection was cured after J weeBs of therapy and no recurrence was observed. 

However, after several months the patient died due to complications of her underlying 

disease. 

Conclusions: Primary cutaneous zygomycosis is a rare and often fatal disease in 

infants and neonates. In our patient, the major risB factors were iron overload, 

deferoxamine therapy and high dose prolonged corticosteroid administration. 

Intravenous lines that disrupt the integrity of the sBin can increase the risB of infection. 

High index of suspicion, early diagnosis and treatment with aggressive surgical 

intervention and appropriate antifungal drugs are essential for the successful 

management of this condition. 

P-0249. A ten-year study of species spectrum and antifungal susceptibility 

profile of Candida bloodstream isolates in Kuwait 

Khan Z, MoBaddas E, Ahmad ., Al-.weih N, Farhat D, Chandy R 

luwait 6niversity 

Purpose of the study: To study the species spectrum and antifungal susceptibility 

profile of bloodstream yeast isolates obtained during (anuary 1KK6- (anuary 2006. 

Description: All the bloodstream yeast isolates were examined by germ-tube test. 

Isolates which were positive by germ tube test were provisionally identified as 7/* 

+68"#+,& or 7/*5(86",".,&"&, and their identity was further confirmed by observing 

typical colonial and microscopic morphology on sunflower seed agar. The isolates 

which did not form germ tubes were tested for carbohydrate assimilation profile by 

ViteB 2 system and /or by molecular methods to determine their specific identity. Four 

antifungal agents, namely amphotericin 2, fluconazole, [-flucytosine, and 

voriconazole were tested by Etest on RPMI medium supplemented with 2_ glucose. 

The susceptibility breaBpoints were based on Clinical and /aboratory .tandards 

Institute (C/.I) criteria or those published by reference laboratories, and were as 

followsM amphotericin 2 v1 kg/ml, fluconazole vJ2 kg/ml, [-flucytosine v 16 kg/ml, 

and voriconazole v 1 kg/ml. 

ResultsM In all, 61K bloodstream yeast isolates were obtained. 7+,5"5+*+68"#+,& was 

the predominant species (O0_), followed by 7/*'+$+'&"6%&"& (J1_), 7/*-$%'"#+6"&* 

(1J_)), 7/*@6+8$+-+ (O_), 7/*[$(&."" (2_), 7/*?+.)(6%,"" (0.8_) and other 7+,5"5+ 

species (K_). None of the isolates of 7/*+68"#+,&3*7/*-$%'"#+6"&3*+,5*7/*@6+8$+-+ were 

resistant to amphotericin 2. ff 22[ isolates of 7/*'+$+'&"6%&"& tested, only O (1.7) 

exhibited MIC of t 1 kg/ml against amphotericin 2. Resistance to fluconazole was 

observed in 10 (J.7_) isolates of 7/*+68"#+,& and in 2 ([.7_) isolates of 7/*@6+8$+-+. 

Resistance to [-flucytosine was observed in 2 (0.K_) isolates of 7/*+68"#+,&, 8 (8.[_) 

isolates of 7/*-$%'"#+6"&, and J (1.O_) isolates of 7/*'+$+'&"6%&"&/*All the isolates of 7/* 

+68"#+,&3*7/*-$%'"#+6"&, 7/*'+$+'&"6%&"& and 7/*@6+8$+-+ were susceptible to 

voriconazole. All the 8 isolates of 7/*?+.)(6%,""*that originated from five neonates* 

were resistant to amphotericin 2 and fluconazole. 

ConclusionM 7/*+68"#+,& is the predominant species causing candidemia in luwait, 

followed by 7/*'+$+'&"6%&"&*and*7/*-$%'"#+6"&/*Although fluconazole is widely used in 

clinical practice in luwait, resistance to this drug continues to remain low. A 

noteworthy observation of the study was that all the 8 isolates of 7/*?+.)(6%,""*were 

resistant to amphotericin 2 and fluconazole. 


P-0250. Favus in an adult patient: A historic case 

lhelifa E, EL Euch D, Cherif F, MoBni M, 2en TeBaya N, Mezlini ., 2en fsman 

Dhahri A 

Department Dermatology, Rabta hospital, Tunis, Tunisia 

Introduction: Tinea capitis is generally thought to be a common disease in children. It]s 

occurrence in adults is rare, especially favus. 

In this worB, we report a case of favus in an elderly woman with typical aspect, exceptionally 

observed at present. 

Case-Report: A 7J-year old lady presented to our dermatology department with a history of 

diffuse hair loss, inflammation and pruritus present over her scalp. .he was treated as psoriasis 

for 10 years without any improvement. .he lived alone in a rural village. Her past medical history 

showed post-hepatitis C cirrhosis. 

fn physical examination, her scalp showed diffuse hair loss with erythema, crusting and scale. 

There were multiple yellowish, adherent, cup-shaped, isolated or confluent, scutula of 0.[-1 cm 

diameter, involving mainly the frontal and parietal area. 

Her toenails assumed a yellow-brown discoloration and pachyonychia. 

A sBin biopsy specimen, from her scalp, was obtained. It revealed a dermatophyte infection. 

Illumination with aood]s light showed green fluorescence of the hair. Direct microscopic 

examination of hairs in J0_ lfH showed an endothrix invasion, with hyphae and air spaces in 

the hair shafts. Culture on .abouraud]s dextrose agar for 20 days yielded whitish, slightly downy, 

cerebriform, heaped and irregularly folded colonies. Microscopic examination of cultures 

revealed irregular septate, antler-liBe hyphae with dichotomously branched, swollen tips showing 

characteristic chandelier structures and some chlamydoconidia. Accordingly, the isolated 

organism was identified as >$"#?%'?2-%,*&#?%.,6.","". 

Treatment with oral griseofulvin 1 g daily was initiated with a local anti-mycosis cream. ahen 

seen at the follow up visit, the patient demonstrated a resolution of her symptoms but Bept a 

permanent alopecia. 

Conclusion: Tinea capitis is a common childhood fungal infection. It]s incidence in adults is 

generally thought to be lowM under [_ s1u. Favus is exceptional in developed countries s2u. In 

Tunisia, its incidence is 0.6_ of all cases of tinea capitis sJu. In our dermatology department, 

only 10 cases have been diagnosed over a 20-year period. Three patients were adults. 

At present, cup-shaped scutula of scale, typical of favus, are exceptionally observed. Patients 

can be misdiagnosed and treated, for many years, as lupus, pyoderma or psoriasis liBe our 

patient. 

fur case highlights the importance of maintaining tinea capitis as a differential diagnosis in 

elderly patients with scaly scalps mainly those who did not improve with treatment. 

P-0251. Superficial fungal infections in HIV/AIDS patients in Chennai 

Kindo A 1 , l.. . 1 , ( l 1 , .riBanth P 1 , N l 2 , .olomon . 2 

1 .ri Ramachandra Medical College c RI (D6), Chennai, India, 2 YRG CARE NGf 

frganisation For HIV Patients 

Aim of the study: To Determine the prevalence of superficial fungal infection in 

patients with HIV disease, to isolate and identify the causative fungal agents 

dermatophytes and non-dermatophytes and to find the correlation between CDO count 

and superficial mycoses. 

MethodsM A study was undertaBen from August 200J to February 200O on HIV 

patients attending YRG Care Center in Chennai. 

Total number of patients screened were 16J[. Total number of specimens collected 

from lesions resembling superficial sBin disease were 8J. 

.amples from the affected sites were taBen and inoculated into Dermatophyte test 

medium and two sets of .abouraud]s dextrose agar, one with Cycloheximide and one 

set without in duplicate. Identification was done by /PC2 mount and .lide Culture 

and relevant biochemical tests wherever required. 

Results: The prevalence rate of fungal infection was [.1J7 _ Culture was positive in 

6O(77.1_) of the total specimens. 

Dermatophytes were 16(1K.J_) non Dermatophytes J8(O[.8_). Candida 10 ([.076_) 

There was no growth in 1K (22.K_) samples. The commonest site affected were the 

nails OJ ([1.8_). (Tinea unguium O and onychomycoses JK) followed by T.corporis 

and T.cruris. The predominant organism causing nail infection was Aspergillus 

species (J0.2_) followed by .copulariopsis species (11.6_). The most common 

isolate from the other sites among the dermatophytes was T. rubrum K/1[ (60_), 

followed by T. mentagrophyte. There were 2 isolates of T.schoenleinii and 1 of M. 

audouinii. CDO count was done for [8 patients. Non Dermatophytes formed the 

majority of the isolates in both groups with CDO count less than 2[0 cOells /ul. ie 

OO.7_ and O1.2_ in groups where the CDO count was between 2[1-[00cells/ul. 

Conclusion: It is important to identify and treat the fungal infections as it can not only 

cause morbidity but may be socially embarrassing leading to physical and 

psychological trauma. 

References: 

1. 2abel DE, 2aughman .A. Evaluation of the adult carrier state in juvenile tinea capitis caused by 

>$"#?%'?2-%,*-%,&($+,&. ( Am Acad Dermatol 1K8Kn21M120K-12. 

2. Cecchi R, Paoli ., Giommi A, Rossetti R. Favus due to >$"#?%'?2-%,*&#?%.,6.",""*in a patient with 

metastatic bronchial carcinoma. 2r ( Dermatol 200Jn1O8M10[7. 

J. El Euch D, MoBni M, .ellami A, Cherif F, Azaiz MI, 2en fsman A. /es teignes du cuir chevelu 

observdes Ö Tunis de 1K8[ Ö 1KK8 M Ö propos de 1222 cas. ( Mycol Med 2001n11M 87-K1.

P-0252. Skin ulceration infected with Arthrographis kalrae in a child with 

alstrom syndrome 

lirel 2, Kiraz N, Dinleyici E, .enses E, fzgen H, .abuncu I, labuBcuoglu ., lilic Z 

1 EsBisehir fsmangazi 6niversity Faculty of Medicine, Department of Pediatrics, 

2 EsBisehir fsmangazi 6niversity Faculty of Medicine, Department of Microbiology, 

J EsBisehir fsmangazi 6niversity Faculty of Medicine, Department of Dermatology, 

O EsBisehir fsmangazi 6niversity Faculty of Medicine, Department of Pathology 

The genera 4$-?$%@$+'?"& is the member of Beratinolytic fungal group commonly found 

in decaying plant material and soil. 4$-?$%@$+'?"&*[+6$+. (4/*[+6$+.) is reported to be 

as species that causes most clinically infection in humans. 2ut it has been rarely 

reported to be as an etiologic agent in human infections . ae here report a child who 

was diagnosed as Alstrom syndrome with severe purulent cutaneous infections on the 

distal extremities and multiple ulcerated papular lesions on the upper extremities 

which 4/*[+6$+. was isolated from on. 

ae describe a 1J year-old boy followed because of Alstrom syndrome with severe 

large purulent cutaneous infections on the distal extremities and multiple chronic 

ulcerated papular lesions on the arms. He had been hospitalized for a severe 

cardiopulmonary failure attacB. He was treated with therapies for heart failure and 

topical-intravenous antimicrobial therapy. 4$-?$%@$+'?"&*[+6$+. was isolated from the 

lesions on the arms. He was died after a severe heart failure attacB approximately at 

the second months of the hospitalization. It remained to be determined whether or not 

the 4$-?$%@$+'?"&*[+6$+. infection contributed to his poor prognosis and this infection 

was the primary infection of these lesions. ae report here a case of 4$-?$%@$+'?"&* 

[+6$+. which presented a different clinical course. 

P-0253. Characterization of an ecto-atpase activity in Fonsecaea pedrosoi 

Kneipp l 1 , Collopy-(ânior •, Correia-da-.ilva F, Rodrigues M, Alviano C, Meyer- 

Fernandes ( 

1 Microbiologia 6FR(, 2 2ioquÜmica Mddica 6FR(, J 2ioquÜmica Mddica 6FR(, 

O Microbiologia 6FR(, [ Microbiologia 6FR(, 6 2ioquÜmica Mddica 6FR( 

^%,&.#+.+*'.5$%&%"*is a polymorphic dematiaceous fungus, causing 

chromoblastomycosis, a cosmopolitan chronic mycosis infecting humans after 

inoculation by trauma. This infection is characterized by granulomatous reaction 

associated with an extensive fibrosis in the dermis and subcutaneous tissues. Ecto- 

ATPases are enzymes located on the cell surface capable of hydrolyzing extracellular 

adenosine-[]-triphosphate (ATP). .everal studies have associated ecto-ATPases 

with protection from cytolytic effects of extracellular ATP, cellular adhesion, 

differentiation and transduction pathways. Fungal ATPases are still poorly-Bnown 

surface molecules. In the present study, an ecto-ATPase was characterized in 

mycelial forms of ^/*'.5$%&%"/*In the presence of 1 mM EDTA, fungal cells hydrolyzed 

ATP at a rate of 8O.6 # 11.J nmol Pi x h -1 x mg -1 mycelial dry weight. The ecto-ATPase 

activity was increased at about five times (OK8.J # 27.6 nmol Pi x h -1 x mg -1 ) in the 

presence of [ mM MgCl 2 , with values of V max and apparent l m for Mg-ATP 2- 

corresponding to [O1.K # O8.6 nmol Pi x h -1 x mg -1 cellular dry weight and 1.K # 0.2 

mM, respectively. This enzyme was capable to hydrolyze several nucleotides 

triphosphates as ATP, uridine-[]-triphosphate (6TP), inosine-[]-triphosphate (ITP), 

guanosine-[]-triphosphate (GTP), cytidine-[]-triphosphate (CTP), and adenosine-[]- 

diphosphate (ADP). ADP was used at very low rates as a substrate, whereas 

adenosine-[]-monophosphate (AMP) was not efficiently hydrolyzed by the enzyme. 

The Mg 2r -stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular 

ATPases such as vanadate (P-ATPases), bafilomycin A 1 (V-ATPAses) and oligomycin 

(F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or 

alBaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The 

surface distribution of the Mg 2r -stimulated ATPase in ^/*'.5$%&%" was confirmed by 

assays using O,O]-diisothiocyanostylbene-2,2]-disulfonic acid (DID.), a membrane 

impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P 2 

purinoreceptors. In the current study, we also demonstrated that the three 

morphological stages of ^/*'.5$%&%"3*i.e. sclerotic, mycelial and conidial cells, show 

different levels of ecto-ATPase activity. Mycelial forms displayed the highest level of 

enzyme activity, followed by sclerotic forms and conidia. The putative role of ^/* 

'.5$%&%" is under investigation. The detection of an ecto-ATPase activity in ^/* 

'.5$%&%"*is the first step to understand the possible role of the enzyme in Bey 

processes such as cellular differentiation and pathogenesis. 

.upported by FAPER(, CNPq, F6(2 and CAPE.. 


P-0254. Clinical correlations of in vitro susceptibility testing of the combination 

of amphotericin B and flucytosine in AIDS-associated cryptococcal meningitis 

/arsen R 1 , Anna C 2 , Alejandro . 1 , Nancy C 1 , Annemarie 2 J , Thomas H J , Ann T O , 

Madeline 2 1 

1 6niversity of .outhern California, 2 6niversity of California, /os Angeles, J Guys 

Hospital Medical Center, /ondon, O 6niversity of Northern Colorado, Fort Collins 

Cryptococcal meningitis is often treated with a combination of amphotericin 2 and 

flucytosine. .usceptibility of 7$2'-%#%##(&*,.%U%$)+,& to these drugs has not been 

correlated with outcome. In one large trial (van der Horst, NE(M 1KK7n JJ7M1[-21) 

there appeared to be little clinical benefit from the addition of flucytosine to 

amphotericin 2. However, a clinical trial that employed quantitative measures of 

response (2rouwer, (AMA 200On J6JM176O-7) offers the opportunity to explicitly define 

the anti-fungal contribution of flucytosine. 

ae have demonstrated that the ",*0"-$% response of amphotericin 2 at 1.[ mg// 

corresponds to the patient response after two weeBs of treatment with amphotericin 

given at 0.7 mg/Bg.day. ae now report our evaluation of ",*0"-$% susceptibility in 

subjects that received the combination of amphotericin 2 plus flucytosine. 

Methods: 10 of 16 subjects receiving the combination of amphotericin 2 and 

flucytosine. ff the 6 untested isolates, [ were from subjects that died before the weeB 

two evaluation and 1 isolate was contaminated during storage. The number of cfu/m/ 

of cerebrospinal fluid (C.F) was measured at baseline and at two weeBs defined the 

patient response. .usceptibility of the subjects] 7/*,.%U%$)+,& isolate was done using 

a macrobroth dilution method modified to use an inoculum that corresponded to the 

number of organisms/m/ found in the baseline C.F sample. K,*0"-$% responses were 

determined by counting the number of living organisms remaining after O8 hours 

incubation in drug containing media. 

Results: Among the 10 subjects, the ",*0"-$% response to amphotericin 2 at 1.[ mg// 

predicted a sterile C.F sample at two weeBs in 2 (20_). For these individuals there 

could be no demonstrable benefit to the addition of flucytosine. For the remaining 8 

subjects the measured patient*response was better than the predicted amphotericin 2 

alone ",*0"-$% response in all 8 subjects. The median improvement between the ",*0"-$%* 

predicted response and the observed patient* response was 2.7 log 10 cfu/m/ among 

these 8 subjects with an ",*0"-$% predicted response to amphotericin 2 alone that was 

greater than 0. 

Conclusion: ahen comparing the predicted response to amphotericin 2 alone based 

on ",*0"-$% susceptibility testing, the patient response when flucytosine is added to the 

treatment regimen is better than the predicted response to amphotericin 2 alone in a 

large portion of persons with cryptococcal meningitis. The added benefit of flucytosine 

is lost in milder cases of meningitis when it is predictable that amphotericin 2 alone 

will sterilize the C.F at two weeBs. K,*0"-$% susceptibility testing can provide clinicians 

important information about the value of combining amphotericin 2 with flucytosine. 

P-0255. Amphotericin B susceptibility testing of cryptococcus neoformans 

employing pretreatment cerebrospinal fluid inoculation 

Larsen R, Alejandro ., Madeline 2 

6niversity of .outhern California 

ae have recently demonstrated that the patient*response to 1O days of amphotericin 

2 at 0.7 mg/Bg.day can be predicted ",*0"-$%. In order to predict the response the 

inoculum used must contain the same number of organisms/m/ found in the 

pretreatment cerebrospinal fluid (C.F). In an effort to simplify and shorten the process 

of ",*0"-$% susceptibility testing we used direct C.F inoculation and evaluated this 

modification. 

Methods: Amphotericin 2 was prepared in final concentrations of 0-J.[ mg// in RPMI. 

Pretreatment C.F from J subjects with AID.-associated cryptococcal meningitis was 

directly inoculated into 10x7[ test tubes and the resultant mixture incubated for O8 

hours. Viable organisms were then enumerated on subculture in .ab-Dex agar. For 

two subjects (ç7[ and çK7), 0.1 m/ of C.F was combined with 0.1 m/ of RPMI 

containing drug and for one subject (ç86) 0.[ m/ of C.F was combined with 0.[ m/ of 

RPMI and drug. These results were compared to the macrobroth dilution susceptibility 

method modified to use an inoculum corresponding to the pretreatment fungal burden 

(/arsen AACn 200[MOK(8)MJ2K7-J01). 

Results: .ubject ç86 had O.8 log 10 cfu/m/ at baseline and O.2 log 10 cfu/m/ after two 

weeBs of amphotericin 2 give at 0.7 mg/Bg.day. Macrobroth dilution susceptibility 

testing predicted a response between 2.0 log 10 cfu/m/ and O.2 log 10 cfu/m/ (KK_ C.I.) 

while the direct C.F inoculation based testing predicted a response between 1.[ log 10 

cfu/m/ and J.[ log 10 cfu/m/ (KK_ C.I.). .ubject ç7[ had [.2 log 10 cfu/m/ and subject 

çK7 had J.K log 10 cfu/m/ at baseline and both had sterile C.F after two weeBs of 

amphotericin 2 (0.7 mg/Bg.day) combined with O00 and 800 mg/day of fluconazole, 

respectively. Macrobroth susceptibility testing of amphotericin 2 alone predicted that 

the C.F of subjects ç7[ and çK7 would be between 2.0 to J.[ log 10 cfu/m/ and 1.0 to 

2.2 log 10 cfu/m/, respectively. The direct C.F inoculation based testing predicted a 

sterile C.F in both subjects. 

Conclusion: 6se of pretreatment C.F as the inoculum for susceptibility testing 

greatly simplifies and accelerates the time needed to produce an ",*0"-$% susceptibility 

assessment of drug response to amphotericin 2. The results obtained with C.F 

inoculation were similar to those obtained with the patient specific macrobroth dilution 

method when the volume of C.F used was 0.[ m/. .maller C.F inoculation volumes 

may not be as accurate in predicting response. Continued evaluation of this 

adaptation of amphotericin 2 susceptibility testing is warranted.

P-0256. Reevaluation of 48 patients with systemic mycosis between 1967 - 2004, 

in Ecuador 

Lazo S. R 1 , Lazo Ch. J 2 

1 CIDRA/A. ..A. / 6niversity of Guayaquil - Ecuador, 2 6niversity Federal of Triangulo 

Mineiro-6beraba - 2razil 

Introduction And Goals: .ince systemic mycosis were first discovered in Ecuador, 

the 1+$+#%##"5"%5.&*8$+&"6".,&"&(1KO8) and the A"&-%'6+&)+ #+'&(6+-()(1K[1), when 

they were introduced in Ecuador(1K62) the precipitation test by double diffusion in 

Agar Gel (Inmunodifussion), it became the research pattern up to these days. 

Considering this bacBground it was recommended the evaluation of patients with 

A"&-%'6+&)%&"&3*1+$+#%##"5"%"5%)"#%&"& and 7$"'-%#%##%&"&, with records in the 

Center the Investigations the Diseases Parasite of Fungus (CIDRA/A. ..A) 

Guayaquil-Ecuador. 

Methodology: A population including O8patients with diagnosisM 

1+$+#%##"5"%"5%)"#%&"&W1*8(OJ), A"&-%'6+&)%&"&WA#(J), 7$"'-%#%##%&"&W7-(2) received 

the antimicotic treatment. The analysis period of our records M goes from 1K67, and 

since 1KKK. All the living patients were revaluated in the clinic aspect, as well as in 

laboratory and radiology. 

P-0257. Cutaneous infection by Cladosporium sp. in the hand surface: 

Description of a case in Ecuador 

Lazo S. R 1 , Torres J 2 , Lazo Ch. J J 

1 CIDRA/A. ..A. / 6niversity of Guayaquil - Ecuador, 2 .pecialist Microbiologist, 

Mycology NY. 6.A, J Adviser CIDRA/A. ..A., 6niversity Federal of Triangulo 

Mineiro-6beraba - 2razil. 

The Chromoblastomycosis is a disease cause by a traumatic inoculation of a 

pigmented fungus called dematiaceous. The Chromoblastomycosis in Ecuador has 

been studied since 1KO2, is a mycosis cause by a cutaneous traumatic pigmented 

dematiaceous fungus. The diagnosis of the Chromoblastomycosis was done in 

Ecuador from the wounds found the arms and more commonly in the lower extremities. 

The organisms causing the infection are calledM ^%,&.#+.+*'.5$%&%"3*1?"+6%'?%$+* 

0.$$(#%&+ and 76+5%&'%$"()*#+$$"%,""/ ae describe here a case of cutaneous infection 

in the dorsum of the hand. 

Case Report: A 67 years old patient from Conchicola-Pias, El fro province - 

Ecuador. The patient was worBing in agriculture and subsequently developed 

hyperBeratosic lesions in the right hand about 6 years ago. The consultation was 

made only for the Chagas disease. 

ResultsM KJ.66_ of the O8patients were men and 6.2O_were women. The 

geographical distribution in the provinces wasM GuayasJ7,[0_, /os RÜos [_, 

ManabÜ16,67_, El fro10,O2_, Pichincha8.J2_, Esmeraldas2,08_. From OJpatients 

with PbJ2,[6_ are alive and67,OO_ are dead. From Jpatients with Hc66,67_ are 

alive and JJ,JJ_ are dead. From 2patients with Ct [0_was alive at the time of the 

revaluation. 2 out of 17patients alive that quit the clinic and therapeutic controls, 

were found after J0years, Owere found after 21 to J0yearsn [after 11 to 20yeasn 

Jpatients after 6 to 10yearsn and Jafter [years. In these patients, the pulmonary 

compromise was of 6O,7_, larynx 2J,[_, intestinal and PCM | cancer [,K_. 1[ of 

them had an immunological control and K of them had a negative result. In some of 

them still existed remains of this pathology. In 6 of them it was variable the degree of 

positive ness in accordance with the radiological clinical sinology. From 17cases 

10received Anfotericina 2, Jletoconazole and Trimetoprin .ulfa, 1letoconazole, 

1Trimetoprin sulfa, 1.ulfamoxol y 1didn]t received treatment for being diagnosed in a 

dead fetus, but research was done on the mother. 

Conclusions: *1+$+#%##"5"%"5%)"#%&"& and A"&-%'6+&)%&"&*are endemic in Ecuador, 

specially in the Rio Guayas basin in the Ecuadorian coast. The number of patients is 

higher in Guayas province. Most of them showed pulmonary pathology, and were poor 

people, that came from the countryside and worB in agriculture. Consequently, this 

must be considered as a serious socio-economic problem. Most of the patients died 

due to the pathogen action of the 1+$+#%##"5"%"5.&*8$+&"6".,&"&3*tremendously affected 

by the coexistence of tuberculosis and intestinal parasites, especially estrongyloidosis. 

.ystemic mycosis treatment is very complex, it taBes some time, and in some cases, 

a lifetime (Argentinean school). aith proper medication, patients can recover and 

thinB they are cured, quitting the treatment which can cause them death. (ahat 

actually happened to our patients). It is recommended a better understanding from 

the physicians, and a better assistances hip to the patients so they can monitor and 

control the healing process. 

Results: In the fresh examination derived from the scrape, we observe the exclerotic 

bodies or fumagoides cells from which later develop long hyphae, septate and gross 

bodies. These cultures were done on .DA, PDA, Mycosel, CMD, 

.DArchloramphenicol, and after 2 weeBs growth observed was greenish and darB 

gray, blacB colonies, slightly woolly in texture. The bacB of the colonies were blacB. 

The colonies showed growth at J0oc, J6oc to J7oc. The fungus did not show growth 

at O2oc. In microcultures, we observe chains of ballistic conidial cells in ramification 

that were born directly from the conidium, resembling a 76+5%&'%$"()*-$"#?%"5.&/ The 

isolated fungus was positive for casein hydrolysis and liquidization of the gelatin. 

Additionally negative for the hydrolysis of the Tyrosine and janthine. 

Discussion: The case presented lesions not frequently observed in our arean 

however we had to perform a different diagnosis from Leishmania cutanea. 

Interestingly enough, the 76+5%&'%$"()*#+$$"%,"" develops in dry and hot environments 

and the Fonsecaea pedrosoi in hot and humid environments. In this case the patient 

lives in a hot and humid area. The morphologic characteristics of the Cladosporium 

isolated are not liBe the 76+5%&'%$"()*#+$$"%,""3 but rather, more similar -%* 

76+5%&'%$"()*-$"#?%"5.&, but this one demonstrated growth at O2oc and did not 

produce hydrolysis of the gelatin. Further, whilst having a Chagas disease, it is 

possible that the immune system does exhibit susceptibility and weaBens, thus giving 

the fungus infection an opportunity to thrive and produce illness. 


P-0258. Majocchi's granuloma of the scrotum by Trichophyton rubrum 

Lee M, Cho H, Haw C 

Dept of Dermatology, lyunghee 6niversity, .eoul, .outh lorea 

Majocchies granuloma is a well Bnown but uncommon folliculitic and perifolliculitic 

dermatophyte infection of the dermal and subcutaneous tissue by fungal organism 

usually limited to the superficial epidermis. There are two forms of Majocchies 

granuloman fne is a small perifollicular popular form and the other is a deep 

subcutaneous nodular form. The superficial perifollicular form, which is caused by 

-$"#?%'?2-%,*$(8$(), occurs mainly on the legs of otherwise healthy individuals, 

especially of women who shave their legs. The deepr form is usually seen in 

immunosuppressed individuals and is characterised by firm or fluctuant nodules on 

the scalp, face or hands and forearms. 

ae report a case of superficial perifollicular form of Majocchies granuloma caused by 

-$"#?%'?2-%,*$(8$(), that was seen on the scrotum of a healthy man on which 

happens rarely. A histologic examination of the biopsied nodule revealed perifolliculitis 

with fungal elements and the periodic acid .chiff (PA.) staining was positive to the 

fungal elements. A topical antifungal agent made his lesion clear completely. 

P-0259. First line, mono or combination, treatment of invasive lung 

aspergillosis: Same approach for fumigatus vs other species ? 

Lemieux C, .u ., Amendola /, Ferraro P, Poirier C 

HÇpital Notre-Dame du CH6M 

Purpose of the study: ae want to, retrospectively, evaluate efficacy, tolerability and 

safety of Caspofungin (C) in first line Tx, used alone or with another antifungal agent, in an 

immuno-compromised population with invasive lung aspergillosis (I/A) and to compare 

clinical response with fumigatus vs other species. 

Background: Combination therapy (CTx) has been evaluated with promising results but 

clinical data is lacBing. C, a novel anti-fungal agent from Echinocandin class and V a 

triazole, have been recently marqueted in Canada. ae report our clinical experience in I/A , 

mainly on a lung graft population, with the use of C, as mono or CTx with Ambisome, V or 

Itraconazole (Itraco.). 

Methods: ae reviewed files of patients (Pts) treated with A-2, Ambisome, C, V or Itraco 

detected via pharmacy data collection. Pts with confirmed or probable I/A were evaluated. 

fnly Pts who received t [ days of antifungals were included. 

Results: ae studied 116 episodes in 11J Pts. 2[ cases in 2J Pts were included in this 

study. 17 Pts received lung and 2 heart-lung transplants, J have leuBemia and one CfPD. 

All Pts have an I/A and one Pt has also a positive sBin biopsy to Aspergillus. 12 episodes 

were treated with monoTx , 10 with C and 2 with A-2n 1J episodes were treated with first 

line CTx all using C associated with Ambisome in 6 cases or V in 6 or Itraco. in one case . 

In monoTx, favorable response was seen in 6 / 12 episodes ([0_). 

In CTx, favorable response was seen in 11 / 1J episodes (8[_)n O / 6 using C r Ambisomen 

6 / 6 using C r V and 1 / 1 with C r Itraco. 

From 1O cases infected with 4/*U()"@+-(&*alone, we see only one failure (11 _) in K / 1O 

Pts treated with CTxn the others [ / 1O Pts treated with monoTx have 2 / [ failure (O0_). 

For the 11 Pts infected with 4&'/ non-U()"@+-(&3*associated or not with*4/*U()"@+-(&3*B.* 

&..*only one failure (2[ _) on O / 11 treated with CTx and from the others 7 / 11 treated 

with monoTx, we have O / 7 failure ([7_). 

ae observed no toxicity with C. aith V, we noted O cases of liver toxicity (2 cholestasis and 

2 cytolysis) and 2 cases of facial photodermatitis. ae have O cases of mild to moderate 

renal toxicity associated with Caspo-Ambisome and one case with Micafungin r V. 

Conclusions: 

1- In I/A, the first line combination Tx seems very active with 11 / 1J successful Tx and C r 

V association, the better tolerated regimen. 

2- For non exclusively-fumigatus aspergillosis, CTx seems a better choice with only 1 / O 

failure (2[_) compare to monoTx with O / 7 failure ([7_). 

J- C was well tolerated and no adverse event has occurred. ae observed O cases of 

moderate hepatitis with V. 

O- More studies with mono versus CTx is required.

P-0260. Aspergillus myositis is an uncommon presentation of 

invasive aspergillosis 

Li D 1 

1 Dept. of Dermatology, PeBing 6niversity Third Hospital, 2 Research Center for 

Medical Mycology, PeBing 6niversity 

Here we report a case of muscle aspergillosis with Aspergillus flavus in a man with 

liver transplantation. The patient got a pain in the left calf two month after liver 

transplantation. Nodules with pain were palpated. 2iopsy specimens revealed 

septated hyphae and focal necrosis of the muscle cells with inflammatory cell 

infiltration. Culture of the muscle tissue grew Aspergillus flavus. The patient was 

successfully treated though the isolate showed resistance to the antifungal agents. 

P-0261. Primary muscle aspergillosis in a liver transplant patient 

Li D 1 

1 PeBing 6niversity Third Hospital, 2eijing, China, 2 Research Center for Medical 

Mycology, PeBing 6niversity 

Purpose: 4&'.$@"66(&, a ubiquitous mold, may cause invasive and even fatal disease 

intractable in immunosuppressed patients. 4&'.$@"66(& myositis is an uncommon 

presentation of invasive aspergillosis. The aim of this study is to report a case of 

aspergillosis with*4&'.$@"66(&*U6+0(& presented as myositis in a man with liver 

transplantation. 

Summary: The patient was a OJ-year old man who underwent liver transplantation 

because of end-stage hepatic cirrhosis. The immunosuppressive regimen included 

prednisone (10mg twice a day) and cyclosporine (200mg twice a day). Fluconazole 

was given [0mg daily for prophylaxis regimen of fungal infection. He got a pain in the 

left calf two month after operation, which exacerbated gradually. Nodules with 

weaBness, swelling, and flaring were formed in the calf two weeBs later. He couldn]t 

walB and even stand with the affiliated leg. Color ultrasonic examination showed 

uneven resonance in the left gastrocnemius. 2iopsy specimens of the muscle tissue 

revealed septated hyphae and focal necrosis of the muscle cells with inflammatory cell 

infiltration. Culture of the muscle tissue grew 4&'.$@"66(&*U6+0(&, confirming the 

histopathological diagnosis. He was first given itraconazole 0.2g twice daily and and 

them terbinafine 0.2[g once a day. 

Results and conclusionsM aith a regime of terbinafine therapy for three months, the 

patient was successfully cured though the cultured fungus showed resistance to a 

number of antifungal agents. 

4&'.$@"66(& spp. can cause various infections especially in the immunosuppressed 

patients. Almost any organ or system in the human body may be involved. Yet the 

muscle is also the target tissue of this Bind of fungus. ahile most of the 4&'.$@"66(& 

isolates yield acceptably low MICs for the antifungal agents, therapy is still 

troublesome with high rate of mortality and a few resistant 4&'.$@"66(&*&''/*The 

present patient revealed a favorable correlation between ",*0"-$% and ",*0"0% 

susceptibilities of antifungal agents. 


P-0262. Primary cutaneous mucormycosis in China: Case reports and literature 

review 

Li C 

~ilu Hospital of .handong 6niversity 

Purpose: In recent years the incidence of primary cutaneous mucormycosis has 

increased in China. Here we report on 2 cases and review the literature in China to 

alert more clinicians to this disease. 

Description: A JJ-year-old man scratched the sBin of his right elbow 7 years ago. 

.hortly a red papule developed to a red plaque and spread to the whole right upper 

extremity, with recurrent ulcers, itching and pain. Another woman peasant of O0 years 

old was bit by a midge in her right face 10 years ago. The lesion then developed to the 

red plaque slowly after the squeeze and scratching and extended to the right 

submaxilla and auricular lobule with different sized ulcers. 

Histopathological results showed that there were massive numbers of inflammatary 

cells and multinuclear giant cells infiltrated in the middle and lower layer of the dermis. 

2road, coenocytic hyphae were seen in PA. stained slides in the dermis and around 

and inside the blood vessels. 

The organisms grew rapidly at 2[+C and J[+C on .abouraud dextrose agar (.DA) 

and on Potato dextrose agar (PDA). After 1 weeB incubation the whole dish was full of 

hyphae. The colony was fluffy and had a yellow pigment. Microscopy showed that 

hyphae were marBedly long, broad, hyaline and coenocytic. .ome were similar to 

stolons but not well developed. The larger hyphae had many branches, which looBed 

liBe rhizoids. .porangiophores grew from the hyphae, with spherical, thin, weaB walled 

sporangia. The sporangiospores were round or oval, and the columella were spherical. 

The strains were identified as P?"d%)(#%$*0+$"+8"6"&. 

Results and conclusions: The first patient was prescribed amphotericin 2 

intravenously with an initial dose of 1mg/d, and increased to O0 mg/d after 2 weeBs. 

.ide reactions such as sicBness, oppression and inertia appeared and disappeared 

after the dosage decreased to J6 mg/d. The entire course of management was 7O 

days and the total dosage was 211[ mg. Relapse did not occur at a 12 months 

follow-up. 

P-0263. Black fungal infections in china 

Li R 

PeBing 6niversity First Hospital 

The blacB fungi are a large and heterogenous group of filamentous moulds with darB 

colored colonies and cell walls. They could produce brown melanin or melanin-liBe 

pigment in the cell wall of their hyphae or conidia, or both. Clinically, blacB fungi could 

cause the following diseasesM Chromoblastomycosisn phaeohyphomycosisn eumycotic 

mycetoman onychomycosisn tinea nigran blacB piedra and mycotic Beratitis. 

Chromoblastomycosis is a distinctive infection of the sBin and subcutaneous tissues 

caused by several dematiaceous moulds. It is the most common blacB fungal 

infection in China. The causative agents were C. carrioniiM (6O.7_)n F.pedrosoiM 

(2O.6_)n P.verrucosaM (2.O_) and F. compactaM (0.8_). Phaeohyphomycosis is a 

generic term to be applied to any mycosis involving a dematiaceous fungus. The 

pathogenic agents of phaeohyphomycosis are widespread in the environment include 

soil, wood, decomposing plant matter as well as polluted water. 6ntil 1KK8, 60 genera, 

10K species were reported to be human pathogenic. The causative agents in China 

mainly includedM Exophiala spp.(most common), Chaetomium spp., 2ipolaris spp., 

Alternaria alternata, Veronaea botryose, fchroconis gallopavum, Curvlaria clavata, 

P.verrucosa, Hendersonula toruloidea. The treatment of blacB fungal infection is still 

difficult. New antifungal agents were active in vitro but no correlation data with the in 

vivo result. Itraconazole is effective in vitro and showing good clinical effect when 

used single or combining with surgical therapy. The microdilution susceptibility testing 

revealed that Exophiala spp. were sensitive to AM2 and ICZ. .ome of the strains 

were dose-dependent or resistant to FCZ, lCZ and [-FC. 

In summary, chromoblastomycosis is the major type of dematiaceous fungal infection 

in China. Phaeohyphomycosis is increasing in immunocompromised patients and the 

management of it still challengeable. The classification and identification of 

dematiaceous fungi will depend on the combination of morphological, physiological, 

molecular biological as well as other supplementary methods. 

The second patient was given amphotericin 2 intravenously with an initial dose of 

[mg/d and external application of butenafine ointment. The dose accumulated to 

160mg in the 10 th day but the patient gave up for the sever nausea and vomiting. 

Then she was prescribed itraconazle 200mg twice a day for 7 days and decreased to 

200mg/d for another [ weeBs. The lesions disappeared with scars. 

ae also reviewed the Chinese literature of the latest 20 years for this disease to find 

the other 8 cases. They were caused by !/*A".)+6"&*^/X(-.(&3*!/*$+#.)%&(&*^$.&*P/* 

+$$?"d(&*^"&#?.$3*P?"d%)(#%$*0+$"+8"6"&*and*P?"d%)(#%$*0+$"+8"6"&*0+$/*$.@(6+$%$ 

respectively. Fluconazole, itroconazole, terbinafine and amphotericin 2 were used in 

these patients with different outcomes. The dose and treatment regimen should be 

selected seriously by considering of the ratio of benefits and risBs.

P-0264. Onychomycosis caused by Paecilomyces lilacinus- clinical case 

/obo I, Velho G, Ferreira M, /opes V, Amorim (, .elores M 

Hospital Geral de .anto Antánio, Porto, Portugal 

1+.#"6%)2#.&*&''/ are widespread soil saphrophytes that infrequently cause human 

disease. Cutaneous infections with this organism are very rare, and usually occur in 

association with impaired hostsï defenses or foreign bodies implantation. 

.porotrichosis-liBe lesions, deep cellulitis, a persistent facial plaque and 

hyalohyphomycosis have been described. 

ae report a case of a healthy, Caucasian, 2[-years-old girl who presented a more 

than one year history of toenail distrophy, without a preceding trauma. The various 

topical or sistemic treatments with antifungal drugs had failled. 

Examination revealed onycholysis, which involved both lateral nail margins, subungual 

hyperBeratosis and yellowish discoloration of nail plate. Her other nails were all normal. 

Direct microscopy of nail clippings after the application of 20 _ potassium hydroxide 

solution, was repeatedly positive concerning hyphae. 1+.#"6%)2#.&*6"6+#",(& was 

isolated on three different occasions in .abourandïs dextrose agar cultures. 

Previous reports of 1+.#"6%)2#.&*&''. infecting nails have been confirmed by 

isolation in only one case. The majority of 1+.#"6%)2#.&*&''/ infections occured either 

iatrogenically (contaminating sterile solution) or in immunocompromised hosts. Most 

reported of 1+.#"6%)2#.& &''. infections involved the eyes. fther sites of infection 

include musculosBeletal, sinopulmonary and vascular (endocarditis) tissues. 

There is no standard treatment regimen for cutaneous 1+.#"6%)2#.&*6"6+#",(& 

infections, and they are reported as being difficult to treat. However, most cases seem 

to respond well to systemic azole antifungal drugs. In our patient, the fungus was 

resistant in vitro to amphotericin 2, itraconazole and fluconazole, but demonstrated 

susceptibility to voriconazole and Betoconazole. 

fur patient initiated a treatment with Betoconazole po and applications of O0_ of urea 

paste and amorolfine on the nail bed. 

P-0265. Phenotype and genotype correlation of clinical isolates of Trichophyton 

rubrum obtained from Mexican patients 

López-Martínez R 1 , HernZndez-HernZndez F 1 , Manzano-Gayosso P 1 , Cárdova- 

MartÜnez 1 , Mdndez-Tovar / 2 , GarcÜa de Acevedo 2 J , frozco-Topete R J , Cerbán M O 

1 Facultad de Medicina, 6niversidad Nacional Autánoma de Mdxico., 2 Hospital de 

Especialidades, Centro Mddico Nacional .iglo jjI, IM..., J Instituto Nacional de 

Ciencias Mddicas y Nutricián Ä.alvador ZubirZnÄ, O Facultad de ~uÜmica, 6niversidad 

Nacional Autánoma de Mdxico. 

>$"#?%'?2-%,*$(8$() is the anthropophilic dermatophyte isolated with more frequency 

worldwide. It is well Bnown that there is a great morphological variability in the >/* 

$(8$() strains, in some times conducing to taxonomic misleading. In spite of this, in 

our Bnowledge there is not studies about the frequency of the morphological varieties 

nor is not Bnown weather the phenotype is related with the strain virulence. The aim of 

this study was to determine the frequency of >/*$(8$() morphological varieties and to 

correlate its genetic expression. The study was performed with 11O clinical isolates 

obtained from patients no submitted to antifungal treatment and affected in different 

body zones. To determine the morphological variety, the macroscopic, microscopic 

and biochemical test features proposed by laminsBi were considered. .o, isolates 

were grown in .abouraud dextrose agar, /actrimel agar, .abouraud]s dextrose agar 

with [_ NaCl and 1_ peptone agar. Also the hydrolysis of urea and hair perforation 

test hin vitroi were done. In order to obtain the genetic expression profile, strains 

representative of each morphological variety were processed by the Differential 

Display-Reverse Transcriptase-Polymerase Chain Reaction (DD-RTPCR) technique. 

Five related species were also included to genetic study. About results, six 

morphological varieties were found, being the most frequent hYi, typical downy and 

U6+0+. .everal strains did not adapt to chosen scheme. All >/*$(8$() strains showed a 

very similar genetic profile independently to morphological varieties with two 

exceptionsM between two morphological types (downy and granular), a differential 

fragment {1[0 bp was observedn between morphological varieties, the typical downy 

>/*$(8$() showed a {O10 bp differential fragment. Another differential fragments 

which could be used as molecular tools to separate >/*$(8$() from related species as 

>/*-%,&($+,& and 7?$2&%&'%$"() sp were observed. These two fragments are in 

cloning and sequencing process to find the corresponding genes. In conclusion, 

results of this study indicate that in spite of T. rubrum can show a great diversity, in 

the Mexican strains predominate threen the meaning of this condition could be related 

with the ability of some phenotypes to induce host infection. aith exception >/*$(8$() 

var. typical downy, >/*$(8$() strains show a highly conserved genetic profile. 

References: 

laminsBi G. laminsBi]s Dermatophyte Identification scheme. Mycology on line. 

httpM/www.mycology.adelaide.edu.au/myc. 

Manzano-Gayosso P, Mdndez-Tovar /(, HernZndez-HernZndez F, /ápez-MartÜnez R. 

Dermatophytoses in Mexico city. Mycoses. 1KKJnJ7MOK-[2. 

/iang P, Pardee A2. Differential display of euBaryotic messenger RNA by means of 

the polymerase chain reaction. .cience. 1KK2n2[7MK67-K71. 


P-0266. Bloodstream infections due to infrequently encountered Candida spp. 

Magill S 1 , .hields C 1 , lurtzman C 2 , Merz a 1 

1 (ohns HopBins 6niversity, 2 6. Department of Agriculture 

Purpose of the study: Candidemia is an important cause of morbidity and mortality 

among hospitalized patients. 7/*+68"#+,& remains the most common cause of 7+,5"5+ 

bloodstream infection, although non-+68"#+,&*species may now account for more than 

half of all cases of candidemia. Changes in the epidemiology of invasive 7+,5"5+ 

infections and the emergence of non-+68"#+,& species, thought in part to be 

associated with increasing use of triazole antifungals, have implications for the 

selection of antifungal therapy. ae sought to describe and analyze the microbiological 

and clinical features of candidemia due to infrequently encountered 7+,5"5+ spp. over 

a one-year period. 

Description: 2lood cultures positive for fungi between 1/1/0[ and 12/J1/0[ at The 

(ohns HopBins Hospital ((HH) /aboratory were reviewed to identify patients with 

cultures positive for 7+,5"5+ spp. other than +68"#+,&, @6+8$+-+, '+$+'&"6%&"&, -$%'"#+6"&, 

[$(&." and 6(&"-+,"+./ Demographic and clinical data including risB factors, antifungal 

therapy and outcome were collected. Isolation, identification and in vitro susceptibility 

testing data for fluconazole, itraconazole, [-FC, voriconazole, and caspofungin using 

the Yeastfne .ensititre assay (TREl Diagnostic .ystems, Inc., Cleveland, fH) were 

assessed. This study was approved by the (ohns HopBins 6niversity Institutional 

Review 2oard. 

Results and conclusions: 162 patients with candidemia were identified. Five of 162 

(J_) were positive for infrequently encountered 7+,5"5+ species, including 7/*U+)+-+* 

in a heart transplant patientn 7/*[.U2$ in a leuBemia patientn 7/*&'?+.$"#+ in a patient 

with gastric cancern 7/*'.66"#(6%&+ in a patient with pancreatic cancern and 7/* 

?+.)(6%,"" in a patient with chronic pancreatitis, colitis, and GI bleeding. All five 

patients had multiple risB factors for candidemiaM O/[ were receiving parenteral 

nutrition when candidemia was diagnosed, O/[ had an active GI process (2 with 

documented 76%&-$"5"()*5"UU"#"6. colitis, 2 with GI cancers, and 1 with upper GI 

bleeding), J/[ were receiving immunosuppressants, and J/[ were receiving 

metronidazole. fnly 1 had prior exposure to fluconazole prophylaxis in the J0 days 

prior to 7/*'.66"#(6%&+ infection. frganism identification was achieved via traditional 

laboratory methodologies for O/[. 7/*?+.)(6%,"" could not be identified and required 

ribosomal DNA (rDNA) D1/D2 sequence analysis. Fluconazole MICs were available 

for J/[ isolatesn all J had MICs v O mcg/ml. All patients received systemic antifungal 

therapy for t 1J days, and all patients were alive 1 month after diagnosis. 

P-0267. Are the albino C. neoformans actually melanin producers: An electron 

microscopic study 

Mandal P 1 , NosanchuB ( J , FranB M O , Roy T 2 , jess I 1 , 2anerjee 6 1 

1 Dept of Microbiology, All India Institute of Medical .ciences, 2 Dept of Anatomy, All 

India institute of Medical .ciences, J Dept of Medicine, Albert Einstein College of 

Medicine, O Dept of Anatomy and .tructural 2iology, Albert Einstein College of 

Medicine 

7/*,.%U%$)+,& is one of the most important opportunistic fungal pathogen that causes 

life threatening infection of the central nervous system (CN.) in both 

immunocompromised and apparently immunocompetent patients. In the absence of 

immune reconstitution, the patients who survive the initial presentation require the life 

long suppressive therapy to reduce the liBelihood of relapse. Melanin is a virulence 

factor for pathogenic fungi. The mechanistic and structural role of melanin is not well 

understood. 7/*,.%U%$)+,& produces melanin in the presence of exogenous catechols. 

As melanin is a cell wall constituent, in this study conducted for the first time on Indian 

7/*,.%U%$)+,& isolates, including six of 7/*,.%U%$)+,& isolates with three melanotic 

and three albino isolates, we have determined the difference in the cell wall 

constitution with respect to melanin layer. The corresponding albino and melanotic 

strains were obtained from same sample of the same patients. The isolates were 

grown in defined minimal medium with 1mM /-DfPA for 10 days to induce 

melanization in the cells. Pellet was obtained followed by treatment for Transmission 

Electron Microscopy (TEM). The EM data revealed that there was no melanin layer 

associated with the cell wall of the albino strains whereas a melanin layer was 

associated with the cell wall of the melanotic strains. .imultaneously, some darB 

patches were observed in the cytoplasm of the melanin lacBing strains unliBe 

melanotic strains which can lead to a hypothesis that the melanin is being produced 

within the albino strains too but it is not being transported to the cell wall. This 

hypothesis can also be supported by our previous study that the albino as well as the 

melanotic strains both contained CN/AC1genes and their amino acid sequences differ 

in the metal binding sites. ae conclude that the albino strains also produce melanin 

but due to defect in the transportation the melanin does not appear in the cell wall. 

During 200[, J_ of 162 patients with candidemia were infected with infrequent 

7+,5"5+ species. Identification using traditional laboratory methods was possible for 

O/[ isolates. The 7/*?+.)(6%,"" speciation could only be accomplished using rDNA 

D1/D2 sequencing. The emergence of previously rare species without well-defined in 

vitro antifungal susceptibility patterns as causes of invasive disease maBes prompt 

identification, including molecular methods, and routine antifungal susceptibility testing 

critical for the management of these infections.

P-0268. Disseminated fusariosis during induction chemotherapy for acute 

lymphoblastic leukemia in a child 

Margueritte G 1 , Palenzuela G 1 , .alet R 1 , Mallid M 2 

1 hdmato-oncologie pddiatrique, Montpellier, France, 2 laboratoire mycologie, facultd de 

pharmacie, Montpellier, F 

In cancer patients, major fungal infection risBs are leucopenia, corticosteroid therapy, 

chemotherapy, broad spectrum antibiotherapy and central venous catheter. Children with acute 

leuBemia are high risB patients for developping disseminated fungal infections as soon as 

induction chemotherapy. Candidosis and aspergillosis remain the most common molds to cause 

invasive infections, but other environnemental molds such as fusarium appear to be increasing. 

ae report the case of disseminated cutaneous fusariosis occouring during the induction 

chemotherapy of an acute lymphoblastic leuBemia (A//) in a child. 

A four-year-old girl was admitted in December 2002 to the Paediatric Hematologic 6nit of the 

Montpellier Teaching Hospital, for management of A//. Examination of the bone marrow 

specimens confirmed the diagnosis of pre-2 A// with a translocation t(12n21), without 

hyperleucocytosis or CN. involvement. The patient was inclued in the protocole FRA//E 2000 A. 

fn day 2J of induction phase (Dexamethazone IV 6 mg/mß/day, Asparaginase IM 6 000 

6I/mß/day and Vincristine IV 1.[ mg/mß/day), while in severe neutropenia (a2CM 200 /mm J ) she 

developped fever (JKÅC). A pre-emptive parenteral antibiotherapy associating 

piperacillin/tazobactam J00 mg/Bg/day and amiBacin 1[ mg/Bg/day was started. fn day 26, 

disseminated purple aching subcutaneous nodules appeared. The biopsy revealed an tissue 

invasion by fusarium especies. /iposomal amphotericin 2 was addicted at 10 mg/Bg/day on day 

26. The patient also received hematopoietic growth factors (G-C.F), [ mcg/Bg/day. 

Neutropenia resolved on day J1. fn day O1, apyrexia was obtained simultaneously the sBin 

nodules disappeared. The bone marrow examination showed complete remission. AntileuBemic 

chemotherapy was restarted and liposomal amphotericin 2 relayed with voriconazole at 6 

mg/Bg/day. fn day OJ, sBin nodules reappeared and liposomal amphotericin 2 (10 mg/Bg/day) 

was restarted associated with voriconazole (12 mg/Bg/day). The sBin nodules disappeared on 

day [8 and liposomal amphotericin 2 was decreased at a lower dose ([ mg/Bg/day) with same 

dose of voriconazole. fn day 6[, a second cutaneous relapse occurred. Central veinous catheter 

was removed. Caspofungin relayed voriconazole and liposomal amphotericin 2 continued, and a 

new cutaneous remission occured. ae unsuccessfully attempted to stop again liposomal 

amphotericin 2. Then, we decided to Beep on with liposomal amphotericin 2 (10 mg/Bg/day) and 

caspofungin ([0 mg/day) until the beginning of the maintenance therapy without any major renal 

dysfunction. The child is in first complete remission of the leuBemia J years after the diagnosis 

without any relapse of the fungal infection. 

P-0269. Detection of Candida in the oral cavity of a group of eutrohic and 

malnourished children 

Mendoza M 1 , Ramos R 2 , Perez C 2 , Diaz E 1 

1 2iomedicina, Caracas, Venezuela, 2 6niversidad Central de Venezuela, Caracas, 

Venezuela 

7+,5"5+*+68"#+,&*is a frequent commensal yeast in the oral microflora of the 

population. Nevertheless, nutritional deficiencies are found among the predisposing 

factors of the host that intervene as cofactors in the pathogenesis of oral candidiasis. 

The objective of this study was to determine the presence of 7/*+68"#+,&*and other 

species of 7+,5"5+*in a group of malnourished children and to compare these results 

with a group of well nourished or eutrophic children. In this investigation, 6J children 

between the ages of J and 6 years who attended the AntÜmano Center for Attention to 

Infantile Nutrition (CANIA) were studiedn JO were malnourished and 2K were eutrophic. 

None of the evaluated children presented clinical signs of oral candidiasis. ff the total, 

28 (OO.OO_) were positive for yeastsn 1K (67.87_) belonged to the malnourished 

children and K (JO.12_) to eutrophic children. ff the J[ cases negative for the 

presence of yeasts, the higher percentage ([7.1O_) corresponded to the eutrophic 

children. Analysis using a statistical test to determine differences between two 

proportions demonstrated that 7/*+68"#+,&*was the most frequent species (J[.71_) in 

malnourished children, compared to 1O.28_ in eutrophic children. fther species of 

7+,5"5+*were also detected. There were no statistically significant differences with 

regard to sex or age. The evaluation of the cell-mediated immune response in O0 of 

these children with candidine antigen revealed only J8_ positivity in the malnourished 

children, compared to 6J_ in euthrophic children. This signals the deficient response 

or immunological defense that the group of malnourished children present to this 

yeast, suggesting that this could be a predisposing factor in this group for the 

development of active disease. 

Fusarium is an emerging opportunist pathogen that can cause disseminated infections in patients 

with acute leuBemia. Fungal infection remain an important cause of mortality in 

immunocompromised patients. Their occurrence leads to delay the antileuBemic treatment and 

increases the risB of relapse. fur patient developped disseminated fusarium infection during 

induction treatment of A//. fnly the administration of high dose liposomal amphotericin 2 (10 

mg/Bg/day) could achieve complete remission. Voriconazole alone either associated with 

caspofungin or lower dose of liposomal amphotericin 2 did not show any efficacity. In spite of the 

three episodes of fungal relapse we have been able to continue the antileuBemic treatment with a 

secondary prophylactic association of liposomal amphotericin 2 and caspofungin. 


P-0270. Candida dubliniensis in the healthy mouth of a malnourished child 

Mendoza M 1 , 2rito A 2 , Ramos R 2 , Diza E 1 

1 2iomedicina, Caracas, Venezuela, 2 6niversidad Central de Venezuela, Caracas, 

Venezuela 

At a worldwide level the infant population is the most susceptible to suffer nutritional 

problems, which affect the normal development in health and in intelligence of the 

infant. fne of the consequences is a weaBening of the immune response of the 

organism, favoring the development of diverse pathologies. These include candidiasis, 

a mycosis produced by yeasts of the 7+,5"5+*genus, of which 7/*+68"#+,&*is the most 

frequently reported. This yeast is found as part of the normal flora in the organism, 

principally in the mucosa, and may become pathogenic under pre-disposing 

conditions including malnutrition. In small children, candidiasis is the primary and 

secondary infection of the gluteal and genital areasn it is frequent in the mouths of 

nursing infants because of reduced salivation and the presence of fermented milB. 

Among the etiological agents of this pathology we can mention the new species 7/* 

5(86",".,&"&*(.ullivan et al., 1KK[), morphologically and biochemically similar to 7/* 

+68"#+,&/**This species has also been described in the pediatric population, in HIVpositive 

children and in infections of the respiratory tract, mouth, vagina and sBin. 

Nevertheless, there are no reports of its presence in the healthy mucosa of 

malnourished children. fbjectiveM To evaluate the incidence of the species of 

7+,5"5+*in the healthy oral cavities of an infantile population, J to 6 years old, 

suffering from malnutrition. MethodologyM .amples from the oral mucosa from JO 

malnourished children without clinical signs of oral pathology were evaluated in 

mycological studies (direct examination and culture). Results and conclusionsM 

Nineteen of these children ([6_) gave positive cultures for yeasts and 1[ (OO_) were 

negative. The yeasts were identified by diverse morphological and biochemical 

methods. 7/*+68"#+,&*was isolated from 10 children ([2.6_), followed by 7/*-$%'"#+6"&* 

(n}[, 26.J_), 7/*@("66".$)%,5""*(n}2, 10.[_), 7/*[(&."*(n}1, [.J_) and 7/*[.U2$*(n}1, 

[.J_). ff the 10 yeasts of 7/*+68"#+,&*obtained, one was subsequently identified as 

7/*5(86",".,&"&*by PCR using specific primers for this species. It was isolated from a 

malnourished five-year-old child with no apparent lesion in the mouth. The direct 

examination was negative, the sample for culture presented [[ and 20 colonies in 

culture media. Diverse species of 7+,5"5+*were described from the healthy oral 

mucosa of malnourished children. 7/*+68"#+,&*was the most frequent, and 7/* 

5(86",".,&"&*was isolated from one child. The ",*0"-$%*resistance of these species to 

fluconazol requires reports on their incidence in the populations which are most 

susceptible to the development of candidiasis. 

P-0271. Frequency and enzymatic activity (proteinase and phospholipase) of 

Candida albicans isolated from the buccal mucosa of children of a Madre 

Regina day-care of Fortaleza - Ceará – Brazil. 

Menezes E 1 , Cunha F, /essa R, Melo T, Ribeiro ., /opes A, .ales Neto M, fliveira / 

1 6niversity Federal of CearZ, 2 6niversity Federal of CearZ, J 6niversity Federal of 

CearZ, O 6niversity Federal of CearZ, [ 6niversity Federal of CearZ, 6 6niversity Federal 

of CearZ, 7 6niversity Federal of CearZ, 8 6niversity Federal of CearZ 

The term candidosis refers to a constellation of diseases caused by 7+,5"5+*+68"#+,& 

and related species. 7/*+68"#+,& is part of the normal human microbioth. 

Immunodefficiency is one of the main causes of buccal candidosis, also called thrush, 

in children. fthers factors liBe inadequate mouth hygiene and inappropriate 

sterilization utensils potentialize this fungal infection. Considering these facts, 7+,5"5+ 

sp, frequency and enzymatic activity (proteinase and phospholipase) were evaluated 

in J12 stocBs from mouth mucous of children from Madre Regina day | care in 

Fortaleza, CearZ, 2razil. The samples were cultured in dextrose .abouraud with 

chloranfenicol agar and incubated for 72 hours at J7 o C. They were identified by 

mycological tests. It was verified that 76 samples (2O.J6_) presented 7+,5"5+ sp, 

and the most frequent genus was O2 7+,5"5+*+68"#+,& ([[.26_), 1O 7+,5"5+* 

-$%'"#+6"& (18.O2_), 10 7+,5"5+*[$(&." (1J.16_), 8 7+,5"5+*@("66".$)%,5""*(10.[J_) 

and 2 7+,5"5+*[.U2$*(2.6J_). Considering enzymatic activity of 7+,5"5+ sp strains, [O 

(71.0[_) produced proteinasis and 28 (J6.8O_) produced phospholipasis. The 

authors conclude that 7+,5"5+*+68"#+,&*([[.26_) was the strain most isolated from 

mouth mucous of children from Madre Regina day - care in Fortaleza | CearZ | 2razil. 

In relation the enzymatic activity the proteinase (71.0[_) presented a high activity and 

the phospholipase (J6.8O_) presented a intermediary activity. This finding leads to 

the conclusion that those strains seem to be of intermediary virulence.

P-0272. Oral candidosis and enzymatic activity of Candida sp isolated of 

patients with AIDS at Fortaleza – Ceará – Brazil 

Menezes E 1 , Cunha F, Melo T, /essa R, Ribeiro ., lopes A, .ales Net M, fliveira / 

1 6niversity Federal of CearZ, 2 6niversity Federal of CearZ, J 6niversity Federal of 

CearZ, O 6niversity Federal of CearZ, [ 6niversity Federal of CearZ, 6 6niversity Federal 

of CearZ, 7 6niversity Federal of CearZ, 8 6niversity Federal of CearZ 

fral candidosis, a condition Bnown as thrush is the most common clinical 

manifestation of candidosis in human. The infection is manifest as white patches or 

plaques on the buccal mucosa and tongue, which may coalesce into a membrane in 

more serious infections. These adhere firmly to the epithelium, revealing a reddened, 

edematous base when removed. 7+,5"5+*species are usually involved in deep 

infections in persons whose immune systems have been compromised mainly AID.. 

Considering these facts, the main objective of this worB was verify the frequency of 

7+,5"5+ in patients with AID. attended in the .an (ose Hospital, of reference in AID. 

in the CearZ state, and evaluate enzymatic activity of 7+,5"5+*isolated. 18K samples 

were cultured in dextrose .abouraud with chloranfenicol agar and incubated for 72 

hours at J7 o C. They were identified by mycological tests. It was verified that 1OJ 

samples (7[.66_) presented 7+,5"5+ sp, and the most frequent genus was 10J 

7+,5"5+*+68"#+,& (72.0J_), J2 7+,5"5+*-$%'"#+6"& (22.J8_), 0[ 7+,5"5+*[$(&." 

(J.OK_), 02 7+,5"5+*@("66".$)%,5""*(1.JK_) and 01 7+,5"5+*[.U2$*(0.71_). 

Considering enzymatic activity of 7+,5"5+ sp strains, 12[ (87.O1_) produced 

proteinasis and 107 (7O.8J_) produced phospholipasis. The authors conclude that 

7+,5"5+*+68"#+,&*(72.0J_) was the strain most isolated from mouth mucous of 

patients with AID. in Fortaleza | CearZ | 2razil. In relation the enzymatic activity the 

proteinase (87.O1_) presented a high activity and the phospholipase (7O.8J_) 

presented also a high activity. This finding leads to the conclusion that those strains 

seem to be of high virulence. 

P-0273. Why routine identification of yeasts in sputum is worthwhile: 

Pulmonary cryptococcosis in a patient apparently immunocompetent 

Michel-Nguyen A 1 , Verrot D 2 , Farnarier C J , Favel A O , Philip-(o®t F [ 

1 

/aboratoire central, HÇpital .aint (oseph, Marseille, France, 2 .ervice de Medecine interne, 

HÇpital .aint (oseph, Marseille, France, J /aboratoire deImmunologie, HÇpital .ainte Marguerite,, 

O 

/aboratoire de 2otanique et Cryptogamie, Facultd de Pharmacie, Marseille, France, [ .ervice de 

Pneumologie, HÇpital .aint (oseph, Marseille, France 

The isolation of 7$2'-%#%##(&*,.%U%$)+,& from sputum in an apparently immunocompetent 

patient allowed the diagnosis of pulmonary cryptococcosis. A lymphocytopenia, probably 

idiopathic, was then revealed. 

Case report 

A 16-year-old female was referred to the Department of pneumology of .t (oseph Hospital 

(Marseille, France) for pneumonia. .he presented with fever of JKÅC and thoracic pain. The 

chest radiography showed cavitation on the right upper lobe of the lung. /aboratory tests 

revealed a white blood cell count of 12.K x 10 K // with 10.7 x 10 K neutrophils//, an erythrocyte 

sedimentation rate of JJ mm/h, and a lymphocytopenia (0.61K x 10 K //). The serology for HIV 

serotypes 1 and 2 was negative. Two years earlier, her mother had been treated for culturenegative 

tuberculosis. 

fn day J, the patient underwent fibroscopy with a bronchoalveolar lavage. Treatment for 

presumed tuberculosis was started with rifampin, isoniazid, ethambutol, and pyrazinamide. Direct 

examination of 2A/ fluid and sputum samples did not reveal acid-fast bacilli upon Zhiel-Neelsen 

staining but showed Gram positive bacilli. 2acteriological cultures on standard media further 

yielded an oropharyngeal flora (1000 CF6/ml). 

fn day 10, because a chest CT scan did not suggest tuberculosis, the antituberculous treatment 

was discontinued. The patient was discharged home. 

fn day 11, yeast-liBe colonies slowly growing on .abouraud-gentamicin-chloramphenicol agar 

were identified as 7$2'-%#%##(&*,.%U%$)+,&. The CNRMA (Institut Pasteur) further specifiedM var. 

@$(8"", serotype A. The isolate was susceptible to amphotericin 2, flucytosine, fluconazole, and 

voriconazole. 

fn day 2[, a pulmonary cryptococcosis was confirmedM serum cryptococcal antigen test was 

positive (titer 10). A second strain of 7/*,.%U%$)+,& was isolated from sputum, and direct 

examination in India inB showed encapsulated yeast cells. /CR and urine specimens were 

negative. A treatment with fluconazole (2 x 200 mg/d) was started, and the patient was 

discharged home on day J8. 

fn day 6[, the patient developed a severe sBin rash. Fluconazole was replaced by a 

monotherapy with flucytosine (100 mg/Bg/d). Ten days later, the patient]s condition deteriorated, 

and she was hospitalized again. The white blood cell count was 11.J x 10 K // with 6.OK x 10 K 

neutrophils//. Erythrocyte sedimentation rate was 6K mm/h and C-Reactive protein was at 72.J 

mg//. /aboratory tests also revealed a marBed lymphocytopenia (0.O10 x 10 K //) with a 

decreased CDO cell count (18K/mm J ). A low level of IgA (0.6J g//) and of IgGO (0.0O g//) was 

documented. .putum culture yielded only a 7+,5"5+*5(86",".,&"& strain. 

Flucytosine was replaced by voriconazole (2 x O00 mg/d one day, 2 x 200 mg/d). The patient 

improved clinically and she was discharged home, still on treatment with voriconazole. There has 

been no recurrence to date. 

Discussion, conclusion: Pulmonary cryptococcosis in immunocompetent patients is now well 

documented. However, yeast isolation and/or identification from sputum is often neglected in 

such patients. The present case underlines the importance of routine mycological investigations, 

because clinical and radiological presentations are non-specific. Interestingly, the patient owned 

numerous paraBeets and zebra finches. Isolation of 7/*,.%U%$)+,& from droppings was twice 

unsuccessful. 


P-0274. Association with Candida albicans, a major pitfall for Candida 

dubliniensis identification 

Michel-Nguyen A, MARY C, Ranque ., Dumon H 

AP-HM Timone, Marseille, France 

Introduction: Routine identification of Candida dubliniensis remains challenging 

despite the recently available 2ichro-Dubli Fumouze rapid latex agglutination test. The 

aims of the present study were to evaluateM 

the respective performances of 2ichro-Dubli Fumouze' (Fumouze diagnostic France), 

Auxacolor 2' system (2io-Rad, France), IDJ2C' system (bioMdrieux France), and 

molecular diagnosis as gold standard, for C. dubliniensis] identification. 

the prevalence of Candida dubliniensis among clinical isolates in our setting. 

Results: ae tested JKO sequential C. albicans / C. dubliniensis isolates, i.e. that were 

positive with the 2ichro-/atex Albicans Fumouze' assay, and 10 Candida glabrata or 

Candida tropicalis strains as controls. fut of the JKO C. albicans / C. dubliniensis 

isolates, J1 were positive with 2ichro-Dublin among them, a real-time PCR assay 

targeting the IT.2 region of C. dubliniensis was positive in 2O. Two isolates were PCR 

positive and latex negative. aithin a four months period, C. dubliniensis was identified 

in JJ (8.O_) of the JKO 2ichro-/atex Albicans positive isolates. 

In nine (27_) isolates, 2ichro-Dubli and PCR yielded discordant results. In each of 

them, testing of several morphologically indistinguishable colonies demonstrated that 

this discrepancy was caused by the mixed culture of both C. dubliniensis and C. 

albicans from the biological sample. ftherwise, when performed on cloned isolates, 

the phenotypic identification of C. dubliniensis was reliable. 

Conclusions: The estimated prevalence of C. dubliniensis among the C. albicans / C. 

dubliniensis complex in our setting was 8.O_ (K[_CI s[.8_-11.6_u). 2ichro-Dubli, 

Auxacolor 2 as well as IDJ2C phenotypic tests proved convenient for C. dubliniensis] 

identification. The occurrence of both Candida dubliniensis and Candida albicans 

within the same biological sample was relatively frequent in our setting. This should be 

pointed as a major drawbacB for accurate identification of C. dubliniensis from clinical 

samples. 

P-0275. Multifocal Cryptococcus neoformans osteitis in a 4 years-old boy with 

hypogammaglobulinemia 

Michel-Nguyen A 1 , Curtillet C 1 , Chambost H 1 , flive D 2 , Michel G 1 , Dumon H 1 , 

Ranque . 1 

1 AP-HM Timone, Marseille, France, 2 Institut Paoli Calmettes, Marseille, France 

Background: 2one localization of cryptococcosis is well recognized but seldom 

occurs. ae report one uncommon case accidentally diagnosed from a bone biopsy 

sample in a child with hypogammaglobulinemia. 

Case Report: 11/1O/0[, a O-years-old boy living in Corsica presented with multifocal 

bone pain. He had a history of recurrent upper-airways and bronchial infections. The 

pain started in the left hand. During one month the boy]s condition gradually worsened, 

the pain increased and disseminated to the bacBn and he became unable to walB. He 

presented to the hospital withM normal body temperaturen painful, tender and swollen 

left handn cutaneous nodules over the face and limbs. 

2lood analysis yieldedM leuBocyte countM 1J,800/kln C - reactive protein (CRP)M 

J6 mg/ln plasma fibrinogenM [.2 g/ln IgMM v 0.J0 g/ln IgGM 2.1 g/ln IgAM 0.27 g/l. j rays 

showed pulmonary infiltrate and sign of osteomyelitis. 

Technetium phosphate bone scintigraphy highlighted five foci located on the third left 

metacarpusn the fifth right metacarpusn and the vertebral bodies of /2, T6, and T8. 

11/18, sputum specimen culture grew 7+,5"5+*+68"#+,&. 

11/1K, a bone biopsy was carried out at the third left metacarpus. Histological findings 

were osteomyelitis and necrosisn fungal spores were observed with PA. stain. 

11/21, 7$2'-%#%##(&*,.%U%$)+,& was grown from a bone biopsy sample planted on 

.abouraud]s dextrose agar with gentamycin and chloramphenicol. 7/*,.%U%$)+,& 

typing and antifungal susceptibility testing were performed at the French National 

Reference Centre for Mycology and Antifungals. 

Typing:*7/*,.%U%$)+,&*var. @$(8"" (serotype A) 

Antifungal susceptibility testing: The E6CA.T ",*0"-$% microdilution method in 

liquid medium yielded the following MICs (mg/l)M amphotericin 2, 0.12[n [- 

fluorocytosine, On fluconazole, 16n itraconazole, 0.12[n and voriconazole, 0.12[. 

11/2J, 7/*,.%U%$)+,& was cultured from a sputum samplen plasma cryptococcal 

antigen levels was1/6On C.F examination was normal, both cryptococcal antigen and 

culture were negative. 

11/2[, initiation of the first line AM2-/ (Ambisome w ) [mg/Bg/d r [FC (Ancotil w ) 

[0mg/Bg tid treatment (CRPM 1[[ mg/ln plasma fibrinogenM 7.Og/l). 

The boy]s condition steadily improved under treatment, the pain disappeared, the boy 

was remains well. 

12/1K (day-2[), the treatment was switched to oral voriconazole (Vfend w ), 6mg/Bg bid 

loading dose the first day, followed by Omg/Bg bid. 

12/21, he was discharged from the hospital (CRPM [7mg/l).

Discussion: This child]s observation has two remarBable particularities. 

7/*,.%U%$)+,& infection was unforeseen in this child with multiple osteomyelitis and 

no fever. The diagnosis was made because a bone biopsy sample has been routinely 

sent to the mycology laboratory. 

The combination of bone and/or joint manifestations and hypogammaglobulinemia is 

indicative of primary humoral response deficiency, especially in children. However, the 

occurrence of disseminated forms of cryptococcal diseases is usually related to 

immune deficiencies of the T-cell lineagesn e.g. AID. is the most common risB factor 

for the development of extra-pulmonary cryptococcosis. Investigations of this child]s 

immune response are ongoing. Preliminary data suggest that a T cell functional defect, 

that remains to be characterized, causes both a deficient cellular response and an 

impaired 2 cells] stimulation resulting in a secondary hypogammaglobulinemia. 

P-0276. Candida species in pediatric patients’ specimens 

Mircevska G, lotevsBa V, (anBosBa G, CeBovsBa Z, DoBic-TrajBovsBa E, PetrovsBa 

M, PanovsBi N, ZafiroviB Z, MilenBoviB Z 

Institute for Microbiology and Parasitology, .Bopje, Macedonia 

The aim of the study was to determine the frequency of isolation of Candida species in 

clinical specimens from pediatric patients hospitalized at the Clinic for Pediatric 

diseases during a 1 year period. During this period, a total of K170 different specimens 

(tracheal aspirate, throat and nose swab, blood culture, stool, rectal swab, urine) were 

examined. All these specimens were processed by the use of conventional 

microbiological methods (Gram stain, culture on blood and chromogenic media, such 

as CAN media, 2ioMerieux, France). Part of the non-albicans Candida was further 

investigated by the use of special Y2C VITEl biochemical cards (2ioMerieux, France) 

for identification of yeasts. Candida species is identified in 2.K_ (268/K170) of the 

processed specimens, out of which 82.1_ were C.albicans and 17.K_ were nonalbicans 

Candida species. fut of the total 268 isolates of Candida species, 7O.J_ 

were isolated in the tracheal aspirates of patients with bronchopneumonia, while the 

other isolates in the other specimens. Candida in blood culture was isolated in [ cases 

with clinically proven sepsis. In JO patients where absence of normal fecal flora was 

detected, Candida species was also confirmed. Presence of bacteria, along with 

Candida species in tracheal aspirate, was detected in KO patients, most frequently of 

which were present ..pneumoniae from the Gram (r) (J0.6_) and llebsiella species 

(1[.7_), Haemophilus influenzae (1J_), Pseudomonas aeruginosa (12_) and 

Escherichia coli (11.1_) from the Gram (-) bacteria. 


P-0277. Short course of switched itraconazole treatment from intravenous to 

oral formulation was an acceptable alternative in cryptococcocal infections 

without CNS complications. 

Miyazaki Y 1 , lurihara . 1 , IumiBawa l 1 , .eBi M 1 , Yanagihara l 2 , HiraBata Y 2 , Tashiro 

T 1 , lohno . 1 

1 Dept of Mol. c Clin. Microbiol., NagasaBi 6niv. Grad. .ch 2iomedical .ciences, 

NagasaBi, (apan, 2 Dept of /aboratory Med., NagasaBi 6niv. Grad. .ch 2iomedical 

.ciences, NagasaBi, (apan 

Background: Although intravenous itraconazole (ITCZ) is a second line antifungal in 

cryptococcosis, we studied the clinical efficacy of short-course ITCZ treatment that 

was switched from intravenous to oral in documented cryptococcal infections. 

Methods: Five patients were enrolled with written informed consent. All patients, who 

did not have serious immunosuppression, had documented cryptococcal infection. 

Each patient was treated with intravenous ITCZ at 200 - O00 mg/day for up to 1O days, 

followed with oral ITCZ for up to 12 weeBs. Clinical efficacy was evaluated at the 

completion of ITCZ treatment. Primary endpoint of clinical efficacy was based on 1) 

clinical symptoms, 2) mycological efficacy, J) radiological improvement and O) 

serological efficacy at the end of the treatment. Treatment was considered success if 

at least two of the four parameters showed improvement with no exacerbation in any 

parameters. 

P-0278. Superficial protothecosis: Report of two Tunisian cases 

Moncef B, Fatma . 

Farhat Hached Hospital .ousse 

Human superficial infections to 1$%-%-?.#+ alga are uncommon. Herin we report 2 

cases in 2 patients originating from .ousse region in Central Tunisia. The first patient 

is a J2 year-old female who presented in (uly 200J with an onyxis of the right-foot 

toes. The second is a 17-year-old male who developed in .eptember 200[ a papulonodular 

lesion on the left hand. In both cases, mycological examination showed 

nonbudding yeast-liBe elements ranging from 6 to 1[ & in diameter. Culture on 

.abouraud-dextrose agar yielded creamy colonies of sphaerical organisms identified 

as 1$%-%-?.#+ on the basis of the abundant endospores-producing sporangia. The 

sugar-assimilating properties revealed both strains to be 1/*Y"#[.$?+)"". No additional 

fungi were isolated from lesionsn and no immunosuppressive factors could be found in 

either patient. 2oth patients were treated with fluconazole but no follow-up 

examination has been performed. Thus, the pathogenicity of 1$%-%-?.#+ couldn]t be 

ascertained. 

Results: Improvement of symptoms such as cough and sputum was documented in 

two cases, while the other three patients were asymptomatic. Mycological eradication 

was confirmed in all cases (100 _, [/[). Partial radiological improvement was 

observed in four cases (80_, O/[). .erum cryptococcal antigen was decreased by no 

less than 2-fold in all cases. No significant adverse reactions of ITCZ were 

documented in each case. fverall, treatment was judged to be success in all five 

cases. Mean trough value was [[2, 727 and 1012 ng/ml at the Jrd, 7th and 1Oth 

treatment day, respectively. No relapse has been seen as of 12 months follow up. 

Conclusions: Although ITCZ treatment of 6 - 12 months is recommended, J months 

treatment showed acceptable clinical efficacies with no relapse even after 12 months. 

This results suggested ITCZ may be an alternative initiation therapy to amphotericin 2 

or fluconazole in non-CN. cryptococcal infection in selected patients.

P-0279. Induction of epithelial cells apoptosis by Paracoccidioides brasiliensis 

and its adhesins of 30 and 43 kDa 

Monteiro da .ilva ( 1 , Andreotti P 1 , da .ilva ( 1 , Miranda E 1 , CZcere C 2 , 2enard G 2 , 

Mendes-Giannini M 1 

1 Departamento de AnZlises ClÜnicas, Faculdade de Ciüncias Farmacüuticas - 6nesp, 

Araraquara, .P, 2razil, 2 /aboratorio de Alergia e Imunologia Clinica e Experimental, 

Hospital das Clinicas, Faculdade de Medicina, 6niversidade de .ao Paulo, .ao Paulo, 

2razil 

Microbial virulence is a set of mechanisms that enable the infectious agent to 

penetrate the host protection barriers and then survive against the defense 

mechanisms, multiply and cause disease. Paracoccidioidomycosis presents a variety 

of clinical manifestations, and the fungus*1+$+#%##"5"%"5.&*8$+&"6".,&"& can reach 

many tissues, most importantly the lungs. 6nderstanding of the mechanisms of 

dissemination is based on indirect evidence, and the polymorphic aspects of disease 

suggest that several virulence mechanisms are involved. The ability of the pathogen 

to interact with the host superficial structures is essential to its virulence, but little is 

Bnown about interactions between cells and 1/8$+&"6".,&"&. For this reason, we studied 

the interaction among 1/*8$+&"6".,&"&3*as well as its adhesins (J0 and OJ BDa)*and 

epithelial cells, with particular emphasis on the induction of apoptosis phenomenon. 1/* 

8$+&"6".,&"& and its adhesins induced apoptosis in infected cells as demonstrated by 

T6NE/ with fluorescent probe technique to label cells undergoing DNA fragmentation. 

Through molecular biology technique, it could be observed DNA fragments around 

180bp of 1/*8$+&"6".,&"& infected cells, characteristic of apoptosis. To quantify 

apoptotic cells after infection with 1/*8$+&"6".,&"& and its adhesins was used flow 

citometry. It could be perceived that the number of apoptotic cells was proportional to 

the time of contact with 1/*8$+&"6".,&"& and their adhesins. 6sing 2aB and 2cl-2 

antibodies, the 1/*8$+&"6".,&"& infected cells and cells treated with the J0 BDa adhesin, 

in initial periods the two proteins was expressed in similar way. After 2O hours, 2aB, a 

pro-apoptotic protein, increased its expression. fn the other hand, the cells treated 

only with the OJ BDa adhesin no alteration in the expression of 2aB and 2cl-2 occurred. 

In summary, we demonstrated that 1/8$+&"6".,&"& and some components induced 

apoptosis in the epithelial cells and the J0 and OJBDa adhesins stimulate apoptosis for 

distinct mechanisms. 

P-0280. Prospective validation of a risk-based strategy and invasive fungal 

infection (IFI) in adult neutropenic patients. 

Moreno J 1 , Alhambra A 2 , Cudtara M J , frtiz M O , Pontán ( [ , del-Palacio-Perez-Medel 

A 1 , del Palacio A 2 

1 Hospitla 6niversitario Doce de fctubre, Internal Medicine. Madrid. .pain, 2 Hospital 

6niversitario Doce de fctubre, Microbiology. Madrid. .pain, J Hospital .evero fchoa, 

Microbiology. /egands, Madrid. .pain, O Hospital 6niversitario Doce de fctubre, 

Haematology. Madrid. .pain, [ Departamento de InmunologÜa, MicrobiologÜa y 

ParasitologÜa, Facultad de Medicina y fdontologÜa, 6niversidad del PaÜs Vasco- 

EusBal HerriBo 6nibersitatea, 2ilbao. .pain 

Purpose And Methods: Prentice et al (2r ( Haematol 2000n110M27J-28O) proposed a 

risB group stratification in adult neutropenic patients in relationship with the 

development of IFI. In order to validate the proposal of Prentice et al in a cohort of 78 

adult neutropenic patients we explored prospectively the incidence of IFI (proven and 

probable) defined according to the criteria of Ascioglu et al (Clin Infect Dis 2002nJOM7- 

1O). 

Results: From .eptember 200O to (uly 200[, 78 adult neutropenic patients (K0 

episodes) were prospectively assessed. Evidence of IFI (six proven and four 

probable) was documented in 10 patients (12,8_). The incidence of IFI correlated 

directly and significantly with risB stratification (p}0.022K), with the highest incidence 

(J1_) in the high-risB group (n}16), followed by the intermediate high risB (12_ 

incidence) (n}17) and intermediate low risB (8_ incidence) (n}J7). In the low risB 

group (n}8) the incidence was null. 

Conclusions: These data show that the risB stratification proposed by Prentice et al is 

a valid method for identifying those patients at highest risB for the development of IFI, 

and agree with those of Mc /intocB et al (2r ( Haematol 200On12OMO0J-O0O) who 

performed a blinded prospective study of the use of a real-time, pan-fungal PCR 

assay and concluded that sequential positive PCR results and development of IFI also 

correlated directly with risB stratification, with the highest proportion of each occurring 

in the high risB group. 

.upported by FAPE.P, CNPq and CAPE.. 


P-0281. Trial of herbal therapy of mycetoma mycetomatis in Sudanese patient 

Musa A 1 , Abdelaziz H, Gumma ., Mahagoub E, Eltaeib N 

1 6niversity of (62A, 2 Rebat 6niversity, J 6niversityof lhartoum, O 6niversityof 

lhartoum, [ Elimam E/mahadi 6niversity 

This is a preliminary study on the effect of using plant (x) in the treatment of patients with blacB 

grain Eumycetma due to !+5($.66+*!2#.-%)+-"&. 

The use of herbal treatment in .udanese patients has been well Bnown over the country since a 

long time ago. Due to the mutilating surgical and prolonged medical treatment by drugs, which 

are very expensive and have side effects, people stared looBing bacB on native treatment. 

.timulated by the experience with the sister of the first author who had a eumycetoma due to*!/* 

!2#.-%)+-"& and treated with plant (x) several years ago, it was decided to carry out a 

preliminary study on the same plant (x) for the treatment of similar cases. 

The trial started since 1KK7, when 186 patients presenting with blacB grain eumycetoma the 

diagnosis of which was confirmed by clinical and serological examination were included in this 

study. Patients] consent and ethical clearance from the Ministry of Health for using the herb for 

treatmen were obtained. 

The duration of treatment ranged between 1-12 months, depending on extent of the lesion and 

the response to treatment. 

fut of the 186 patients, 7O (JK_) did not continue the treatment and failed to come for follow up. 

112 patients (61_) had the treatment and were followed up clinically and serologically until 

complete cure was obtained and for a J-years period of follow up. 

Two to four weeBs after applying the paste to the lesions, more sinuses opened up discharging 

the grains. This was followed by closure of the sinuses, decrease in size of the of the mycetoma, 

and the sBin going gradually bacB to normal. The duration depended on the extent of the lesion. 

The crude plant and its extract were tested ",*0"-$% !/*!2#.-%)+-"&. The worB was carried out in 

the Mycology /aboratory, 6niversity of lhartoum. 

Different concentrations of the crude plant were prepared and incorporated in the medium 

.abouroud]s Agar after sterilization. 

The concentrations were 100 mg, [0mg and 2[mg per 100 ml medium. A control was included 

with each test. The test and control were inoculated with equal inocula of !/*!2#.-%)+-"&. 

P-0282. Invasive paranasal sinusitis due to the basidiomycete Ptychogaster 

rubescens: A case report and its serological evaluation. 

Naidu J 1 , .ingh . 2 , (ha M 2 , (ain . J , Guarro ( O 

1 Deptt. of Zoology c 2iotechnology,Govt. Autonomous .cience 

College,(A2A/P6R,INDIA, 2 Deptt. of 2ilogical .ciences,RDVV,(abalpur. INDIA., 

J Prasann ENT Clinic, Marhatal,(abalpur.INDIA., O 6nitat de Microbiologia, 6niversitat 

Rovira,RE6.,.PAIN 

Invasive sinusitis includes the highly aggressive locally destructive more slowly 

progressive and frequently fatal sinusitis usually seen in immunocompromised 

patients. In the present paper, we report a case of invasive paranasal sinusitis caused 

by the anamorphic basidomycete 1-2#?%@+&-.$*$(8.&#.,& in the 6[ year old, Indian 

male carpenter. The presenting symptoms were nasal destruction, pain, epistaxis and 

periorbital edema. CT .can revealed opaque masses in the antrum and exhibited 

growth involving all the sinuses. Histological sections of the necrotic material and 

polypoidal mass obtained from biopsy revealed mucosal invasion, granulomatous 

reaction and the presence of mycelium of the pathogen in the necrotic tissues. Pure 

cultures of the fungus were repeatedly isolated from the clinical samples. The patient 

showed improvement with oral fluconazole and topical Candid nasal drops but since it 

was also diagnosed to be a case of poorly differentiated squamous cell carcinoma he 

was refered to TIFR (Mumbai). 

.D.-PAGE analysis of the exoantigen of 1/$(8.&#.,& revealed a glycoprotein band of 

1[lD. fuchterlony]s double diffusion test showed specific precipitation bands when 

exoantigen of 1/*$(8.&#.,& was reacted with antisera raised in test rabbits after two 

weeBs of challenge. The .D.-PAGE analysis of the antiserum raised in test rabbits 

revealed significant rise in the level of specific antibodies from 2 nd to O th weeB post 

challenge as compared to the normal serum. 

No cross reactivity of the exoantigen was seen against antisera of 46-.$,+$"+*+6-.$,+-+, 

^(&+$"()*&%6+," and 1+.#"6%)2#.& )+$e(+,5"". 

1-2#?%@+&-.$*$(8.&#.,&*is a wood rotting organism and interestingly for the first time it 

is documented as human fungal pathogen in a carpenter. .uch cases if encountered 

by the clinicians should be viewed cautiously as they are difficult to treat. ae further 

propose that serology could be used in the rapid diagnosis of this infection. 

After two weeBs, when good growth in the control tubes was obtained, the test was read and it 

was found that the organism was inhibited by all the different concentrations of the crude plant. 

The ethanolic extract of the plant was tested in the same way using the following concentrationsM 

J[ mg, 2[ mg, and 1[ mg per 100-ml medium. All the concentrations inhibited the growth of M. 

Mycetomatis.

P-0283. Relapsing facial candidiasis 

Nakanishi T, .owa (, TsubaBimoto a, lobayashi H, Ishii M 

Department of dermatology, fsaBa City 6niversity Graduate .chool of Medicine 

A 2J-year-old women suddenly presented exudative sclay erythema on her entire face 

after visiting at ToByo Disney /and on shiny August day. .he was treated with topical 

application of white petrolatum and zinc petrolatum at near clinic, but the eruption 

aggravated. Patch testing revealed negative reaction for cosmetics which she used. 

After 2weeBs, she visited us and hospitalized due to stinging and unfavorite 

appearance of her face. At first, we diagnosed as contact dermatitis with secondary 

infection. Thus, we started antibiotic (cefazolin sodium, 1g/day) drip infusion. 

Although additional oral administration of 20mg prednisolone and topical application of 

0.0J_ prednisolone valerate-acetate ointment, the eruption showed no improvement. 

ae tried to microscopic examination with her facial scales and found numerous 

pseudohypha. /aboratory findings showed elevation of white blood cells (K700/ l), 

A.f (J22 I6/ml), A.l (titerM x[120), IgM (2K[mg/dl). Antinuclear antibodies, serum 

complement, IgG and IgA are within normal limit. Clinical appearance on her whole 

body did not show any appearance of candidiasis including oral and genital lesion. 

Microbiological culture revealed no growth of bacteria, but fungal elements made 

whitish colonies of Candida albicans on Mizuno-TaBada medium. .he was 

administered of oral antifungal capsules (itraconazole 100mg, daily) and topical 

application of 2_ Betoconazole cream. .he got completely healing after J months. 

Nine months later, the same symptom recurred on her face after sun exposure without 

episode of topical application of corticosteroid or other immunosuppressive drugs. .he 

was treated at near clinic with 0.1_ of hydrocortisone 17-butyrate ointment. However, 

the eruption developed during 2 months. Then she visited us again, the same 

examinations were performed on her face and the same results are obtained. ae 

tried pulse therapy of itraconazole (O00mg daily, 1 weeB) and got better. .erum 

examination of HIV showed negative reaction and other immunosuppressive findings 

such as diabetes mellitus or endocrinological abnormalities could not be found. 

Cutaneous candidiasis limited to face is thought to be rare. To our Bnowledge, 

previous reports of facial candidiasis are accompanied with immunosuppressive 

status. These are topical application of steroid, HIV infection, diabetes mellitus, after 

operation or sBin abrasion, radiation, endocrinological disorders and 

hypogammagloburinemia. Generally, chronic mucocutaneous candidiasis is 

recognized as rare fungal infection occurs in mainly congenital immunosuppressive or 

endocrinopathic patients. fur patients had no such abnormalities in screening of 

immunological and endocrinological examination. ae speculate our case must be 

induced by sun exposure which leads immunosuppressive status including lacB of 

barrier function. Although photosensitive findings cannot be found on her entire body, 

additional investigation of photo test is essential to elucidate the mechanism of this 

case. ae are welcome to excellent advices related to mechanisms, diagnosis, 

examinations, therapies, prognosis, prevention for our case. 

P-0284. Oral fungal flora in HIV+ patients submitted to antiretroviral therapy 

Nascimento T 1 , /opes M 1 , Amorim A 2 , Freitas G 1 

1 6niversity of /isbon-F. Pharmacie, /isbon, Portugal, 2 Instituto .uperior Ciüncias da 

.aâde Egas Moniz, /isboa, Portugal 

Objectives: The aim of this study was to assess asymptomatic oral carriage of 

7+,5"5+ species and density in human immunodeficiency virus infected (HIV r ) 

subjects. ae also purpose to determine the beneficial effects of highly active 

antiretroviral therapy (HAART) on oral yeast colonisation density and diversity, yeast 

carriage association with clinical (viral load and TCDO r lymphocyte count) and 

behaviour factors. Another unanswered question is whether, and what to extent, HIV r 

patients undergoing HAART can harbour triazols and echinocandins resistant yeasts 

in their oral cavities. 

Study design: ae collected oral rinses in two groupsM one, with 6O HIV-infected 

Instituto .uperior Ciüncias da .aâde Egas Moniz*clinic outpatientse - Portugal, 

asymptomatic for fPC, from February to fctober, and other in [0 healthy subjects. 

Yeasts isolates were identified by classical methods and by pulse-field electrophoresis. 

The susceptibilities of 7+,5"5+ isolates to four antifungal agents including fluconazole, 

itraconazole, voriconazole and caspofungin were performed according to National 

Committee for Clinical /aboratory .tandards - NCC/. M27-A2, micro-broth assay, 

now denominated Clinical and /aboratory .tandard Institute (C/.I). Chi-squared and 

Fisher tests determined the statistical differences. 

Results: .eventy-six isolates were obtained. Ninety percent were 7+,5"5+ +68"#+,&* 

(7. +68"#+,&). fther isolates included 7/*'+$+'&"6%&"& ([_), 7/*[$(&." (J_), 7/-$%'"#+6"& 

(1_) and 7/*5(86",".,&"& (1_). The results from the oral rinses for yeast carriage were 

the followingM 62_ among HIV r patients and J6_ among healthy subjects (1 } 0.00[). 

.imilarly, density carriage was found to be significantly higher in the first group (1 } 

0.01). 7/*+68"#+,& was the most recovered specie in both groups (8[ and 8K_, 

respectively), and for non-+68"#+,&*species the percentage was 1[ and 11_, 

respectively. All isolates in the study were sensitive to the antifungals tested. 

Conclusions: Asymptomatic Candidal carriage and relative density were found to be 

significantly higher in the oral cavity of HIV r patients. ae found no association with 

clinical factors, neither HAART, but strongly associated with oral hygiene. However 

7+,5"5+ colonization density is positively associated with viral load. 


P-0285. Candida deep sternal wound infections: A complication of sternotomy 

Nguewou A 1 , Chan C 1 , (ones M 2 , Donegan N 2 , .hoham . J 

1 Department of Internal Medicine, aashington Hospital Center, aashington, DC, 6.A, 

2 Department of Infection Control, aashington Hospital Center, aashington, DC, 6.A, 

J .ection of Infectious Diseases, aashington Hospital Center, aashington, DC, 6.A 

2acBgroundM aound infections are a Bnown complication of sternotomy and are 

associated with significant morbidity and mortality. To date, post sternotomy Candida 

wound infections have not been well defined. ae sought to better characterize the 

epidemiology, microbiology, and outcomes of these infections in a cohort of adult 

cardiac surgery patients. 

MethodsM In this retrospective study, all culture proven cases of Candida deep sternal 

wound infections occurring at our hospital from (anuary 1, 1KK2 to August J1, 200[ 

were analyzed. Medical records were abstracted for demographics, associated 

conditions, microbiology, and outcome. 

ResultsM During the study period, 28,2K2 sternotomies were performed, and O27 deep 

sternal wound infections were identified. ff those infections J0 (7_) were associated 

with Candida species. 6nderlying conditions in patients with Candida sternal wound 

infections were coronary artery bypass graft (CA2G) surgery (70_), CA2G and valve 

surgery (26.7_), and valve surgery alone (J.J_). .aphenous vein grafts, and left 

internal mammary artery grafts were used in K6.6_, and 7K.J_ of cases respectively 

and 1J.J_ of cases were associated with emergent surgery. In O0_ of cases, 

Candida species were isolated alone, and in 60_ of cases were part of a 

polymicrobial infection. The proportion of Candida infections increased from [.[_ 

prior to 2002 to 11_ thereafter (p}0.O6). Median time to diagnosis was 2K days after 

initial sternotomy. The distribution of species was 86.7_ C. albicans and 10_ C. 

tropicalis. Crude mortality was 26.7_ 

ConclusionsM Candida species, particularly C. albicans, are emerging as an 

increasingly important cause of post sternotomy deep wound infection, and are 

associated with high mortality. 

P-0286. Fungal spectrum and susceptibility of oropharygeal isolates from AIDS 

patients in Southeast Nigeria 

Nweze E, Anonye N, fgbonnaya /, flafor ( 

1 Ebonyi .tate 6niversity, AbaBiliBi, Nigeria, 2 6niversity of Nigeria, NsuBBa, Nigeria, 

J Ebonyi .tate 6niversity Teaching Hospital, AbaBiliBi, Nigeria, O 6niversity of Nigeria, 

Dept. of Microbiology, NsuBBa, Nigeria 

Purpose of the study: To isolate and identify oropharygeal fungal isolates from AID. 

patients and determine their susceptibility to commonly used antifungal agents in our 

region. 

Summarized description: fropharygeal swabs and rinse samples were collected 

from AID. patients over a period of time in southeast Nigeria and plated on 

.DA..ubcultures were made on appropriate selective media to aid further 

identification of the species depending on the initial observations. Data on patients] 

history were also collected. Frequency of isolation was compared with data on CDO 

counts, age, sex and locality of the subjects. Control samples were also taBen from 

normal subjects. .usceptibility studies were based on C/.I (formerly, NCC/.) 

protocol. 

Results: .everal fungal species were isolated. Majority of the isolates are albicans 

and non-albicans 7+,5"5+3 4&'.$@"66(&*and 7$2'-%#%##(&*=''&/There appears to be a 

correlation between CDO counts and the frequency of the isolates. The effect of age, 

sex and locality were also studied. Greater number of isolates was from women while 

the effect of sex did not cut across the species isolated. Cryptococcus, non-albican 

Candida, Candida albicans exhibited higher levels of resistance respectively 

especially to antifungals common in our locality. 

Conclusion: .pectrum is widening while resistance is increasing. Molecular studies 

of the isolates in our collection will be useful in revealing other epidemiological and 

ecological concepts of the species involved. Interested collaborators are welcome.

P-0287. Sporotrichosis treated with potassium iodide 

Orofino Costa R, Damasco P, 2ernardes-Engemann A, .ilva I, 2envenuto F, /opes- 

2ezerra / 

6niversidade do Estado do Rio de (aneiro, R(, 2razil 

.everal antifungal drugs are used in the treatment of sporotrichosis most of them 

have a high cost besides numerous drug interactions and adverse events. ae]ve seen 

an increase in number of cases of this disease in the last eight years mainly in Rio de 

(aneiro, 2razil. Cat transmission has been the most important source of infection. 

.aturated solution of potassium iodide (..lI) is a cheap drug used to treat this 

infection. ae followed 2[ patients treated with ..lI, '.$*%&, through clinical and 

epidemiological features. Furthermore, the patients were serologically followed-up 

during the treatment. The patient]s IgG serum titers against a cell wall antigen 

denominated .sC2F were evaluated by an E/I.A test we have developed in our 

laboratory. Group treated with ..lI constituted 2O_ of total sprotrichosis treated this 

period being the majority females (72_) with a mean age of 2J.[ years old. 

/ymphocutaneous form of sporotrichosis predominated (88_). The serological test 

showed K0_ of sensitivity and the mean IgG titer of serum samples collected before 

treatment was [1200. All ..lI treated patients were cured and the IgG levels in 

patient]s serum marBedly decreased after treatment. ae concluded that ..lI is an 

excellent choice of treatment for cutaneous presentation of sporotrichosis in 

immunocompetent patients due to low cost and good efficacy. This drug was well 

tolerated by all the patients. .erological diagnosis performed in our laboratory also 

correlates well with the isolation of =/*&#?.,#["" from patient]s lesions and with clinical 

follow up. .upported by Rede M./CNPq/Faperj. 

P-0288. Superficial mycosis due to non-dermatophytic molds in Rio de Janeiro, 

Brazil 

Orofino Costa R 1 , Horta G 1 , Dias C 1 , Gend ( 2 , .tchingel A 2 , Guarro ( 2 

1 6niversidade do Estado do Rio de (aneiro, 2 6niversitat Microbiologia, .pain 

Purpose: Prospective study of clinical and epidemiological features of superficial 

mycosis caused by non-dermatophytic molds at the 6niversity Hospital of Rio de 

(aneiro, 2razil. 

Summary: Patients with suspected superficial mycosis that presented positive direct 

microscopy and non-dermatophyte filamentous fungi isolated at least twice in different 

specimen collections and with no growth of dermatophyte were enrolled in a six 

months period. All of them answered a detailed questionnaire and had photographs 

taBen. Etiologic agents were identified in taxonomic basis. 

Results and conclusions: >",.+ was suspected in 1OK0 sBin samples of 68O 

patients. According to criteria described above diagnosis was made in 187 samples 

(12.J_) of [O patients (7.K_). Patients mean age was [[.8 years old while mean 

disease duration was 100.[ months. Most patients were mixed race, 72.2_ were 

female, 66.6_ worBed at home or were retired, J7_ mentioned bare foot habits and 

72_ worn sandals. Common concomitant systemic disease were cardiovascular 

diseases and diabetes mellitus. ^(&+$"()*&%6+," was the most isolated fungus 

followed by =#2-+6"5"()*?2+6",() and =/*5")"5"+-(). Toenails and toe webs were 

sites more frequently affected. There was no ^(&+$"()*species isolated from plantar 

surface. In this site =/*?2+6",() predominated. =/*?2+6",() was not found causing 

fingernail disease. There were no clinical differences in clinical picture from disease 

caused by dermatophyte or non-dermatophytic molds. .uperficial mycosis caused by 

these molds have been increasing during last years although caution should be taBen 

not to misdiagnosis or overemphasize these infections. 


P-0289. Subcutaneous mycoses in Fars, Iran 

Pakshir K, .almanpour R, Motazedian M, Vassei M 

Faculty of Medicine, .hiraz, Iran 

Purpose of the study: The subcutaneous mycoses are infections involving the 

dermis, subcutaneous tissues and adjacent bone. These infections are usually most 

frequently encountered among the rural populations of the Tropical and subtropical 

regions of the world. Fars is one of the largest provinces in Iran and has tropical 

climate. The aim of this study was to determine the prevalence of subcutaneous 

mycoses and causative agents in Fars province, .outhern Iran 

Summarized description of the project: .pecimens were taBen from the patientes 

suspected subcutaneous mycoses with severing, chronic, suppurative and nonhealing 

lesions which referring to dermatology clinic centre in .hiraz. .amples were 

cultivated on .abouraud dextrose agar, Mycosel, 2HI and 2lood agar supplemented 

with antibiotics and incubated at 2[ and J7ÅC for at least six weeBs. Fungal species 

were identified by traditional and chromogenic methods. 

Results and conclusions: During J[ months, 2002 to 200[, a total of KO patients 

suspected subcutaneous mycoses were enrolled in our study and one case of 

4#-",%)2#.-%)+, one case of 4&'.$@"66%&"&, one case of 7+,5"5"+&"&*and 2cases of 

`%#+$5"+&"&*were detected. In five cases, the etiological agents of 

'?+.%?2'?%)2#%&"&*and ?2+6%?2'?%)2#%&"&*were isolated by culture, but their 

infections were not confirmed by direct and histopathology methods. Fars province 

belongs to the tropical areas of Iran and most of the people activities are gardening, 

agriculture and animal farm. Defective in the diagnosis of mycotic diseases and health 

care problems in the management and referring the patients to clinics along with 

rareness of these diseases are the major cause 

of unBnown cases. 

P-0290. Spectrum of CNS mycoses: Ten years experience from a tertiary care 

hospital in South India 

Pamidimukkala U 1 , Challa . 2 , Vemu / J , chavali P O , lancharla A [ , V.V. . 6 , ..NiBhat 

. 7 

1 Dept.of Microbiology,Nizames Institute of Medical .ciences,Hyderabad,Andhra 

pradesh,India, 2 Dept.Pathology,Nizames Institute of Medical 

sceinces,Hyderabad,Andhrapradesh, J Dept.of Microbiology,Nizames Institute of 

Medical .ciences,Hyderabad,Andhra pradesh,India, O Dept.of Microbiology,Nizames 

Institute of Medical .ciences,Hyderabad,Andhra pradesh,India, [ Dept.of 

Microbiology,Nizames Institute of Medical .ciences,Hyderabad,Andhra pradesh,India, 

6 Dept.of Microbiology,Nizames Institute of Medical .ciences,Hyderabad,Andhra 

pradesh,India, 7 Dept.of Microbiology,Nizames Institute of Medical 

.ciences,Hyderabad,Andhra pradesh,India 

Fungal infections of the CN. mostly involve immunocompromised patients. Most of 

the fungi are aerosolized and inhaled and initiate a primary pulmonary infection which 

is usually self-limited. Hematogenous dissemination may follow the initial infection, 

with subsequent involvement of the CN.. Trauma or local extension provides the 

route to CN. infection in several cases. The hyphae of molds generally cause focal 

disease with hemorrhagic necrosis secondary to vascular thrombosis. The yeasts tend 

to cause a more diffuse process with the base of the brain being primarily affected, 

such that hydrocephalus is seen as a frequent complication of chronic disease. 

Aim: To study the spectrum of CN. mycoses in a tertiary care, teaching hospital in 

south India. 

Materials and methods: The study population included all patients of CN. mycoses, 

admitted to the Nizam]s Institute of Medical .ciences, a tertiary care, teaching 

hospital in Hyderabad, Andhra pradesh, India, over a period 10 years from 1KK6-200[. 

A retrospective review of the clinical and laboratory data was done, for variables such 

as symptoms and signs at presentation, site of infection, predisposing factors, surgical 

procedures performed, results of diagnostic and Imaging studies, specific organism 

identified, treatment and outcome. 

Results & conclusion: A total of 1J1 patients with CN. mycoses were included in 

the analysis. The varied presentations of CN. mycoses in these patients were 

meningitis, vasculitis or parenchymal invasion (either an abscess or granuloma). 

* 

7$2'-%#%##(&*,.%U%$)+,&**was the commonest etiological agent of CN. mycoses 

affecting 66 patients([0.J8_).K0_ of these patients had an underlying risB factor, the 

commonest being HIV infection (81_of patients). .ystemic lupus Erythematosus, 

Diabetes mellitus, Post renal transplant status and Alcoholic liver cirrhosis were the 

other immunocompromising conditions identified in these patients. Meningitis was the 

commonest presentation in theses cases. 

A&'.$@"66(&*species were the etiological agents in 26(1K.8O_) patients. The species of 

Aspergillus isolated were 4&'.$@"66(&*U6+0(&*GI:J, 4&'.$@"66(&*U()"@+-(&*GNJ, 4&'.$@"66(&* 

-.$$.(&*GMJ, A&'.$@"66(&*@6+(#(&*GIJ3*4&'.$@"66(&*,"5(6+,&*GIJ. fne patient had a mixed 

infection with 4&'.$@"66(&*U()"@+-(& and 4&'.$@"66(&*U6+0(&. The 4&'.$@"66(& isolates

from two patients could not be speciated .The most common primary site of infection 

in these patients were the paranasal sinuses. 

Zygomycetes belonging to the order mucorale& were the causative pathogens in 

28(21.J7_) patients/*P?"d%'(&*%$2d+. was species responsible in the majority (21) of 

these patients. Rhinoorbitocerebral was the commonest type of presentation in these 

patients except in one, in whom 4'%'?2&%)2#.&*.6.@+,& was isolated. This patient 

presented with necrotizing fasciitis following a scalp wound sustained in a road traffic 

accident. There was a contiguous spread of the infection intracranially, resulting in a 

space occupying lesion in the frontoparietal region and Cavernous sinus thrombosis. 

* 

7+,5"5+ species were recovered from 6 patients. Five of these patients presented 

with meningitis and 7+,5"5+*species were isolated from the C.F. In one patient, the 

CN. involvement was in the form of multiple intracerebral abscesses as a result of 

disseminated candidiasis. Antemortem blood cultures grew 7+,5"5+*-$%'"#+6"& while 

the cerebral involvement was demonstrated only at autopsy. 

* 

`%5(6"&'%$"()*spp(1), !+5($.66+*)2#.-%)+-"&(1), 76+5%&'%$"()*%_2&'%$()(1), 

76+5%'?"+6%'?%$+*8+,-"+,+(1) and P?%5%-%$(6+*@6(-","&(1) were the fungi identified in 

the remaining cases. 

CN. mycoses due to both common and rare fungi seem to be on the rise, especially 

in immunocompromised patients. Prompt diagnosis and early initiation of therapy will 

go a long way in reducing the high mortality and morbidity associated with this disease. 

P-0291. The effects of mycotoxins produced from Aspergillus fumigatus on the 

cytotoxicity and the expression of cytokine genes in lung cells. 

Park B 1,2 , .ugita-lonishi Y 1 , lamei l J , TaBatori l 1,2 

1 Gifu university, Gifu, (apan, 2 National Institute of Health .ciences, ToByo, (apan, 

J Chiba university, Chiba, (apan 

4&'.$@"66(&*U()"@+-(& is a well-Bnown pathogenic fungus causing a wide range of 

pulmonary diseases in immunocompromised as well as immunocompetent patients. It 

produces several toxic secondary metabolites, such as gliotoxin, fumagillin, helvolic 

acid, verruculogen, and others for invasive infection into lung tissue. ff the toxins 

produced in it, gliotoxin is well-Bnown as an immunosuppressive agent that facilitates 

evasion of host defense, fumagillin is also well-Bnown as an inhibitor of endothelial cell 

proliferation, tumor-induced angiogenesis, and tumor growth in mice, and then 

verruculogen is Bnown as the most potent tremorgenic mycotoxin. Many studies have 

been used to investigate the biological effects of these mycotoxins. However, the 

mechanisms of immunosuppressive effects of these mycotoxins have never been 

fully understood. 

The aim of this study was to investigate the cytotoxicity of gliotoxin, fumagillin, and 

verruculogen on human lung cells, such as primary cultured lung fibroblast, epithelial 

cell (A-[OK cell), and lung micro-vascular endothelial cell, and mouse lung 

macrophage cell line (MH-.). Furthermore, we have tried to reveal the expression of 

cytoBine genes by these mycotoxins in lung cells using multiplex PCR (MPCR), 

because these activities may be represented in invasive aspergillosis during the 

fungal pulmonary infection. 

The expression of cytoBine genes and cytotoxicity remarBably differed among the 

used cells in this study. These results suggest that these mycotoxins can be a critical 

role in the cytoBine expression causing inflammation and apoptosis during the fungal 

infection. This study represents the step in showing the cytotoxic effects and the 

expression of cytoBine genes expression by these mycotoxins using MPCR on human 

and mouse lung cells in vitro. 


P-0292. Profile of main clinical manifestations presented by patients with 

vulvovaginal candidiasis and their relation with laboratory exams 

Paula C, Auler M, Moreira D 

6niversity of .ao Paulo, .ao Paulo, 2razil 

The purpose of the study was to determine diagnostic sensitivity, specificity, positive 

predictive value, negative predictive value and accuracy of the clinical manifestations 

(pruritus, erythema and leuBorrhea) of vulvovaginal candidiasis by performing 

laboratory tests for the presence and identification of Candida spp. on samples of 

vaginal discharge collected from patients with symptoms characteristic of vulvovaginal 

candidiasis, while checBing for the influence of predisposing factors for candidiasis 

(age, use of antibiotics, use of contraceptive pills, quantity of /actobacillus spp. and 

the presence of diabetes mellitus). During the period from .eptember 2002 to March 

200O, samples were taBen from 1J0 patients clinically diagnosed as having 

vulvovaginal candidiasis. ff these, 6J_ yielded cultures positive for yeasts. C. 

albicans was isolated in K0_ of these cultures, followed by C. glabrata (6_), C. 

parapsilosis and C. tropicalis (2_ each). Among the clinical signs and symptoms 

evaluated, leuBorrhea presented the greatest diagnostic sensitivity, though it was the 

least specific. Erythema presented the highest diagnostic specificity. Among the 

predisposing factors for vulvovaginal candidiasis, contraceptive pills proved to be the 

most influential, while no correlation was found between the presence of yeasts and 

altered vaginal microbiota. 

P-0293. Outbreak of early disseminated histoplasmosis in solid organ 

transplant recipients: evidence of transmission from a common donor 

Pelletier R 1 , Poirier / 2 , Al-Mohri H J , Tremblay C 1 , /oungnarath V O , Connolly P [ , 

aheat / [ , CÇtd I 1 

1 

/eHÇtel-Dieu de ~udbec du CH6~, ~udbec, Canada, 2 Centre Hospitalier Maisonneuve- 

Rosemont, Montrdal, Canada, J McGill 6niversity, Montrdal, Canada, O HÇpital de /]Enfant-(dsus 

du CHA, ~udbec, Canada, [ MiraVista Diagnostics, Indianapolis, Indiana, 6...A. 

Purpose of the study: Histoplasmosis transmission from person to person has been 

occasionally suspected in the setting of organ transplantation. ae report evidence of 

Histoplasma capsulatum transmission from a common donor to J solid organ transplant 

recipients. 

.ummarized description of the projectM Disseminated histoplasmosis was suspected in a Bidney 

transplant recipient, 2[ days post transplantation. The diagnosis was based on peripheral blood 

smear microscopic observation of yeast-liBe structures in leuBocytes] cytoplasm. The patient had 

then been experiencing a sustained high fever and progressive allograft dysfunction over the last 

10 days. The other two organ recipients from the same donor were contacted through the 

~udbec organ-procurement agency. .imilar clinical and microscopic findings were made in the 

second Bidney transplant recipient and in the liver transplant recipient. High urine Histoplasma 

capsulatum var. capsulatum antigen titers and positive blood cultures confirmed the diagnosis in 

each of the J solid organ transplant recipients. .ince disseminated histoplasmosis occurred 

simultaneously in three recipients of a common donor, it was highly probable that Histoplasma 

had been transmitted through the organ donation process. ae designed further testing to 

confirm this hypothesis. 

Results: .erum and splenic tissue from the donor had been Bept frozen and were still available 

for retrospective evaluation. At the time of organ donation, Histoplasma antigen was positive and 

(1*J)-#-D-glucan titers (Fungitellã, Associates of Cape Cod) were strongly positive in the 

donor]s serum. Initially, the J allografted patients were negative for Histoplasma serology and 

glucan detection. At the time of the culture proven Histoplasma diagnosis, each of them tested 

strongly positive for serum glucan and Histoplasma antigen. 

Coarse granulomatous structures were found on microscopic examination of the donor]s spleen 

and special staining revealed yeast-liBe cells. Culture of the donor]s spleen was positive for 

Histoplasma capsulatum var. capsulatum. DNA was purified from cultures obtained from the 

donor as well as the three transplant recipients along with an unrelated case from the same 

geographical area. The DNA was analyzed using random amplification of polymorphic DNA 

(RAPD) pcr. Three primers previously shown to have excellent discriminatory power for 

Histoplasma were chosen for the RAPD pcr. RAPD pcr fingerprint profiles revealed complete 

identity between the DNA from the isolate from the donor and that from all three transplant 

recipients supporting that these isolates are, in fact, clonal. Additionally, the profiles from the 

unrelated case showed that isolate to be genetically unique from the other isolates. 

Conclusions: TaBen together, these cases gave a unique opportunity to observe the 

development of initial symptoms and signs of disseminated histoplasmosis in the early posttransplant 

period. .ustained high fever, prostration and allograft dysfunction manifested by the 

end of the second post-transplant weeB in the J organ transplant recipients. Data from this 

analysis gave robust evidence of Histoplasma transmission by organ transplantation from an 

unsuspected infected donor. In endemic areas, Histoplasma capsulatum var. capsultum should 

be considered as a potentially transmittable infection from solid organ donors.

P-0294. Immunair: a novel protection against air-borne fungal contamination 

for immune-compromised patients 

Poirot J 1 , Gangueux ( 2 , laudinet n J , Immunair .tudies Group O 

1 Department of parasitology c mycology, .aint-Antoine 6niversity Hospital, Paris, 

France, 2 Department of parasitology c mycology, 6niversity Hospital, Rennes, France, 

J Research department, Airinspace company, Montigny-le-2retonneux, France, 

O Associated investigators, cf. poster 

Effective protection of hospital patients against airborne fungal contamination (particularly with 

4&'.$@"66(&) requires adequate treatment of the air. To achieve this, clean rooms that filter the air 

using mechanical HEPA filters are the most commonly used method in most haematology 

hospital services. .uch technologies have proven protection levels, provided, stringent 

maintenance and surveillance procedures are followed. However, due to intrinsically high 

pressure-drops, mobile units based on mechanical HEPA filtration do not provide sufficient flow 

rates for adaptable patient protection during their transfer towards other services (i.e. intensive 

care units). 

To address this constraint and the restrictions posed by current technologies, the company 

AirIn.pace has introduced a new air-treatment system for hospital applications based on its biodecontamination 

technology previously used in the space station MIR and the International 

.pace .tation Alpha. This technology uses non-thermal plasma and amplified electrostatic fields 

that irreversibly destroy a wide range of microorganisms, includingn viruses, bacteria, fungi and 

spores with a low pressure drop and maintenance requirements. AirIn.pace has recently 

implemented this novel technology in the development of mobile air-decontamination units such 

as the Immunairã, a mobile hygienic ceiling that unfolds over a patient]s bed and supplies 

decontaminated air within a zone isolated by transparent curtains. 

To date, exhaustive evaluations on Immunairã have been conducted in seven different French 

hospitals. 2ecause of its obvious application for protection of haematological patients, the 

majority of the tests were focused on the performances against air-borne fungal contaminates. In 

all cases a two phase qualification approach was carried outM i) Functional tests in unoccupied 

rooms to characterize steady-state performances in accordance to French .tandards (Paris- 

.aint-Antoine, -NecBer, -.aint-/ouis, -Trousseau 6niversity Hospitals and Gustave Roussy 

Institute). ii) Evaluations under actual treatment conditions in the presence of patients over long 

periods (Clermont-Ferrand, Paris-NecBer, -Trousseau, -.aint-/ouis, Rennes 6niversity 

Hospitals) to evaluate continuity of protection during patient treatment. 

The general procedure in each evaluation was to first identify and measure a base-line level of 

airborne fungal contamination in the room before activating the Immunairã unit, (in some cases 

comparative sampling points in the corridor and other rooms was also established). .amples 

were taBen under the protective plenum of the Immunairã and inside the room exterior to the 

protected zone, using an impaction method on standard Petri dishes filled with .abouraud 

medium. 2io-samplers were left running for [ minutes with an airflow rate of 100 litres/minute for 

each sample. All measurements were taBen and analyzed in accordance with standard 

procedures developed for controlling fungal contamination levels. Rooms] sizes ranged from 

J[m J to 7Jm J . 

Results indicate that the Immunairã providesM i) systematic performance on fungi of v1CF6/m J 

of air detected under Immunairã plenum with fast decontamination Binetics even under massive 

initial contamination (artificial) of tJKJCF6/m J . ii) v1CF6/m J of fungi under Immunairã with a 

statistical confidence of KK_ and a significant decrease of O[_ of the contamination in the room 

with regard to corridor levels | performances established over a J6K days period and more than 

[[0 samples with patient and staff present. 

P-0295. Paracoccidioidomyces brasiliensis in a brain abscess: One french case 

in a retired man 

Poisson D 1 , Mille C 2 , MucBensturm 2 J , Heitzmann A O , Ferquel C [ , Dromer F 6 , Dupont 

2 7 , Hocqueloux / 2 

1 

Regional Hospital of frldans - Microbiology, 2 Regional Hospital of frldans - Infectious Diseases, 

J 

Regional Hospital of frldans - Neurosurgery, O Regional Hospital of frldans - Pathology, 

[ 

Regional Hospital of frldans - Neuro-Radiology, 6 Pasteur Institute - Paris - Reference Center 

for Fungi, 7 6niversitary Hospital NecBer Paris - Tropical c Infectious Diseases 

Purpose: Case report of one 1+$+#%##"5"%"5%)2#.&*8$+&"6".,&"& brain abscess in a retired man 

living in France. 

Case report: Mr M, 70., had a car crash in Rouen (France) on the 18 th of (anuary 2006, and was 

driven to the hospital with a diagnostic of seizure. A computed tomography (CT) elicited a single 

J0 mm-diameter right temporo-coronal subcortical lesion, with a peripheral ring enhancement. Mr 

M chose to have this lesion operated on in frldans. 

fn arrival, Mr M was well, except for pulmonary CT showing clinically mute alveolar opacities in 

both bases. 

The brain lesion was ablated on (anuary 27.. Frozen sections and smears performed during the 

surgical procedure did not confirm the tumoral nature of the lesion, but showed a granulomatous 

reaction with big hints of yeasts. Mycologist advice was searched. Gram and MGG stained 

smears confirmed the presence of big yeasts, and led to the presumptive diagnosis of 

1+$+#%##"5"%"5.&*8$+&"6".,&"&, based upon large yeast presenting in chains with multiple multidirectional 

thin-based buds. Mr M]s wife reported that her husband had been living in Paraguay 

for several years and retired in France in 2000. Differential diagnoses were eliminatedM 

A"&-%'6+&)+*#+'&(6+-() because of the large diameter of the yeasts, 7$2'-%#%##(& upon the 

absence of capsule and the granulomatous nature of the abscess. 

2iopsies were cultured at J7Åon blood agar, for the yeast form, and at J0Å on .abouraud agar, 

for the filamentous form. Yeasts appeared first, at D10, and were sent to the Reference Centre 

for Fungi in the Pasteur Institute (Paris, France), where the strain was confirmed as 

1+$+#%##"5"%"5.&*8$+&"6".,&"&. 

Available results also include broncho-alveolar lavage, bone-scintigraphy, serologic tests against 

the reference fungal antigen preparations. 

Anti-fungal treatment began on (anuary 28. with fluconazole, turned into itraconazole O00 mg 

daily, on February 1O. and is yet designed for 6-12 months. 

Synthesis: 1/*8$+&"6".,&"& is a thermo-dependent dimorphic fungus geographically restricted to 

.outh and Central America, where it is the most prevalent systemic mycosis (Rippon 1K88). 

1+$+#%##"5"%"5.&*8$+&"6".,&"& is responsible for a chronic granulomatous infection that 

characteristically begins in lungs and secondarily disseminates as ulcerative mucosal or sBin 

lesions. fccasionally a systemic involvement occurs in multiple organs. The Central Nervous 

.ystem involvement is infrequent even in the endemic areaM 20 cases were observed by Elias ( 

for the period 1K86 | 200J in .ao Paulo Medical .chool (2razil). 

Mr M left Paraguay in 2000 after having been worBing for several years in close contact with 

forest and rural areas. 

According to the reference centre this case is the first French such one, at least since 1KK2. 


P-0296. Ectophosphatase activity in C. albicans influences adhesion to host 

cells: A study between HIV+ and HIV isolates 

Portela M 1 , lneipp / 1 , .ouza I 2 , Holandino C J , Alviano C 1 , .oares R 1 , Meyer- 

Fernandes ( O 

1 Instituto de Microbiologia Professor Paulo de Gáes - 6FR( - 2razil, 2 Faculdade de 

fdontologia - fdontopediatria - 6FR( - 2razil, J Faculdade de FarmZcia - 6FR( - 

2razil, O Departamento de 2ioquimica Medica, 6niversidade Federal do Rio de (aneiro, 

2rasil 

fropharyngeal candidiasis, typically caused by 7+,5"5+*+68"#+,&, is the most frequent 

opportunistic oral disease in children infected with Human Immunodeficiency Virus 

(HIV) and is a sentinel indicator of immunodeficiency and HIV disease progression. 

The process of protein phosphorylation/dephosphorylation is one of the major 

important in regulation of many cellular events in euBaryotes. A number of 

fundamental processes in fungi such as cell cycle, transcription and mating have been 

shown to require protein phosphorylation. In the present worB, the expression of acidic 

ectophosphatase activity in twenty isolates of 7/*+68"#+,&*from oral cavity of HIV 

infected children were compared with fifteen isolates from HIV negative children, as 

well as the fungal adhesion to epithelial cells and medical records. The activities were 

measured in intact cells grown in 2rain Heart Infusion medium for O8 hours at J7ÅC. 

Phosphatase activity was assayed at pH [.[ using O-methylumbelliferyl phosphate as 

substrate. The adherence of 7/*+68"#+,&*to epithelial cells (MA 10O) was measured 

using a proportion of 10M1 yeast/epithelial cell per well, that were made onto 2O-well 

multidishes. The systemic conditions of the HIV children were obtained from medical 

records. The results were analyzed by .P.. 11.0 statistical program. Mean values of 

ectophosphatase activity were 610.27#166.J6 and 2O1.2[#78.K6 picomoles/h/10 7 cells 

for HIV positive and HIV negative group, respectively (p}0.08K). It was not observed 

correlation between 7/*+68"#+,&*enzyme activity from HIV children with viral load but a 

moderate tendency was present between low count of CDO percentual and high 

phosphatase activity. Yeasts with high enzyme activity, isolated from HIV positive 

children showed greater adherence than yeast with basal levels of ectophosphatases 

(.pearman correlation, r}0.8). .urface phosphatase activity was apparently involved 

in the adhesion to host cells, since the enhanced attachment of 7/*+68"#+,& to MA 10O 

was reversed by pre-treatment of yeast with acid phosphatases inhibitors. These 

results suggest that 7/*+68"#+,&*from HIV infected children have more 

ectophosphatase activities than the others isolates. Then, the expression and activity 

of acidic surface phosphatases in these cells may contribute to the early mechanisms 

required for disease establishment. 

P-0297. A case report of successful combined surgical and medical treatment 

of Candida endocarditis after homograft valve replacement 

Pospisilova H, Mallatova N, .etina M, MoBraceB A 

Introduction: 7+,5"5+ endocarditis complicating cardiac surgery was first reported in 1K[6. 

.ince then, the infection has been described with increasing frequency in a wide range of 

cardiac procedures, but most often after valvular surgery ([O_). Valvular heart disease, 

intravenous access devices and therapy with antibiotics are all Bnown risB factors for 

developing Candida endocarditis. 7+,5"5+*+68"#+,&*is the most frequent isolate in fungal 

endocarditis. 2lood cultures are positive in 80-K0_ of the cases. Candida prosthetic valve 

endocarditis is a serious disease with a poor prognosis. A combination of surgical resection 

and antifugal drug therapy is the gold standard of care. 

ae report a case of prosthetic valve endocarditis due to 7+,5"5+*+68"#+,& following 

homograft valve replacement, which was treated successfully with voriconazole plus 

caspofungin and surgery. 

Patient and methods: A [O-year-old man was admitted to the hospital 1O days after 

homograft valve replacement because of suspected early endocarditis. He presented with 

fever, vegetations on echocardiography and positive blood cultures. Despite initial treatment 

with intravenous fluconazole for two weeBs, he remained fungemic (7/*+68"#+,&*grown from 

several blood cultures). Transesophageal echocardiography showed two large vegetations 

on the aortic homograft, abscess cavities and severe aortic regurgitation. The next day (1[ 

days of admission), the patient underwent cardiac surgery that consisted of radical resection 

of all infected tissue, local sterilization with fluconazole and replacement with a new 

homograft. 7/*+68"#+,& was confirmed by culture from infected tissue. Antifungal 

susceptibility tests were performed by the Etest method for amphotericin 2, fluconazole, 

inraconazole and voriconazole. Postoperatively, the patient received aggressive antifungal 

treatment with caspofungin (22 days) c voriconazole ([[ days), and continued on oral 

voriconazole long-term after discharge. 

Conclusion: After a follow-up of eight months, the patient was doing very well, and 

there were no symptoms or signs of recurrent fungal infection. The difficulty of 

diagnosis and the optimal management of Candida infective endocarditis are 

discussed. 

.upport by CNPq and FAPER(.

P-0298. Unusual localized chronic infection by Fusarium solani in a nonimmunocompromised 

host 

P-0299. Platelia Aspergillus assay: A novel diagnostic test for disseminated 

histoplasmosis? 

Pérez-Pérez L, Pereiro (r M, .Znchez-Aguilar D, Toribio ( 

.ervicio de DermatologÜa, Complejo Hospitalario 6niversitario, Facultad de Medicina, 

.antiago, .pain 

Background: ^(&+$"()*spp*are emergent oportunistic molds with a marBed medical 

importance due to their capacity to produce life-threatening diseases, mainly in the 

immunocompromised host 1 . ahen they infect immunocompetent individuals, they 

usually cause localized diseases. Cutaneous fusariosis is a characteristic picture with 

limited clinical expression 2,J . Here, we describe a new case of cutaneous fusariosis 

with the clinical appearance of a cutaneous vasculitis. 

Case report: ae report a 68 female patient presenting on her left leg two very painful, 

well delimited, ulcerous, rounded lesions, with necrotic centre and erythematous halo, 

measuring 2 cm in diameter. The lesions had developed over the previous month. The 

patient denied any trauma at this level. .he had a medical history of diabetes and a 

non-HodgBin lymphoma under remissionn she was not receiving any 

immunosuppressive therapy at that time. 

The histopathologic study of a sBin sample revealed a marBed epidermal and dermal 

necrosis with an intense inflammatory infiltrate mainly constituted by neutrophils. 

Many hyaline branched septated hyphae were seen extending deeply in the dermis, 

both with hematoxilin-eosin and periodic acid-.chiff staining. 

All of the cultures of a sBin biopsy yielded colonies that were white to cream in colour 

with a floccose aerial mycelium. .lide cultures on .abouraud]s agar revealed singleor 

two-celled, oval microconidia, 8|Kj2|J km in size, sometimes grouped in 

hverticilliumsi produced from elongated lateral phialides. ae observed a few single or 

pairs of intercalary, rough-walled, 6-K km chlamydospores. As a result, the strain was 

identified as ^(&+$"()*&%6+,""*(Mart.) .acc. After three months, the lesions partially 

resolved with oral itraconazole 200 mg/day. 

Comments: Fusariosis are a group of infrequent fungal diseases caused by diverse 

species of the genus*^(&+$"(). These microorganisms should always be taBen into 

account in the differential diagnosis of necrotic cutaneous lesions both in the 

immunocompromised and in the immunocompetent hosts, mainly when they present 

on the extremities and when there is a visible portal of entry. Further studies should be 

made to find an effective therapeutic alternative to amphotericin 2. 

References : 

2odey G et al. .Bin lesions associated with Fusarium infection. ( Am Acad Dermatol 

2002n O7M6[K-66. 

Pereiro (r M., /abandeira (, Toribio (. Plantar hyperBeratosis due to Fusarium 

verticilloides in a patient with malignancy. Clin Exp Dermatol 1KKKn2OM17[-8. 

Pereiro (r M, Abalde MT, Zulaica A, Caeiro (/, Florez A, Peteiro C, Toribio (. Chronic 

infection due to Fusarium oxysporum mimicBing lupus vulgarisM case report and review 

of cutaneous involvement in fusariosis. Acta Derm Venereol 2001n81M[1-J. 

Ranque S 1 , Dromer F 2 , Genot . J , Michel-Ngyuen A 1 , Faraut F 1 , Mary C 1 , .tein A J , 

Tissot-Dupont H J , Dumon H 1 

1 AP-HM Timone, Marseille, France, 2 Institut Pasteur, Paris, France, J AP-HM 

Conception, Marseille, France 

An AID. patient originating from Ghana with disseminated histoplasmosis due to 

A"&-%'6+&)+*#+'&(6+-() var. #+'&(6+-() tested highly positive (tO) in the serum by the 

Platelia 4&'.$@"66(&*assay, a commercially available enzyme-linBed immunosorbent assay 

(E/I.A) for the soluble 4&'.$@"66(&*galactomannan antigen. He exhibited no additional 

evidence of aspergillosisn and no recognized cause of false-positivity of the test was found, 

in particular he did not received !-lactam antibiotics. 

ae speculated that the Platelia 4&'.$@"66(&*assay positivity might have been due to crossreactive 

antigens released by A/*#+'&(6+-()n this hypothesis was tested in a two steps 

experimentM 

1. A/*#+'&(6+-() culture]s supernatants were tested for their reactivity in the Platelia 

4&'.$@"66(&*assay. A/*#+'&(6+-() isolated from the patientzs bone marrow 

aspirate has been grown on both .abouraudes dextrose agar with 

chloramphenicol and gentamicin and NNN (intended to grow X."&?)+,"+*&''/) 

culture-media. The two culture]s supernatants, and also the two culture-mediacontrols, 

yielded high E/I.A galactomannan indexesn indicating that soluble 

antigens from both culture-media interfered with the assay. 

2. Plasma collected from five patients with acute histoplasmosis and Bept in the 

French National Reference Centre for Mycology and Antifungals] collection were 

tested for their reactivity in the Platelia 4&'.$@"66(&*assay. Their respective E/I.A 

galactomannan indexes wereM 0.12n 1.2n 1.2n 2.Jn and J.1. In clinical practice, an 

index value %1.[ is indicative of a positive reactionn an index t1 and t1.[ is 

intermediaten and an index v1 is considered nonsignificant. 

Thus, six plasma samples collected from six patients (including the index case) with 

disseminated histoplasmosis were tested with the Platelia 4&'.$@"66(& assay. The test]s 

results were interpretedM positive for threen intermediate for twon and negative for one 

patient. Further research is clearly needed, but these preliminary data strongly support the 

hypothesis that soluble components from A"&-%'6+&)+*#+'&(6+-()*var. #+'&(6+-()*shed 

during infection react in the Platelia 4&'.$@"66(&*assay. This is a reminiscent of the recently 

described cross-reactivity between 7$2'-%#%##(&*,.%U%$)+,& and 4&'.$@"66(& 

galactomannans 1 . 

The test detecting A/*#+'&(6+-()zs capsular antigen is currently not available in France, 

and this is probably also true in most European countries. .o, rather than complaining 

about having evidenced another cause of false-positivity with the Platelia 4&'.$@"6(& assay, 

lets] celebrate the good fortune of having available at hand one test that might prove useful 

for both diagnosis and follow-up of patients with disseminated histoplasmosis. 

ReferencesM 

Dalle F. et al. ]*76",*!"#$%8"%6. 200[n43(6)M2K2K-J1. 


P-0300. First isolation of Trichoderma atroviride from a human patient 

Ranque S 1 , .igrist H 1 , Garcia-Hermoso D 2 , Michel-Nguyen A 1 , Dumon H 1 

1 AP-HM Timone, Marseille, France, 2 Institut Pasteur, Paris, France 

Case report: A OK-years-old man who has been diagnosed with a alcoholic cirrhosisassociated 

hepatocellular carcinoma underwent a liver transplantation in november 200[. 

He was administered methylprednisolonen tacrolimusn ganciclovirn amoxicillin/clavulanaten 

and metronidazole. 2iliary tract haemorrhages and cholestasis prompted a re-intervention 

in the early postoperative course. fn day-7 after transplantation, the patient developed a 

sudden respiratory distressn liver function tests yieldedM alanine aminotransferase (A/T), 

262 6I/ln aspartate aminotransferase (A.T), 126 6I/ln lactic acid dehydrogenase (/DH), 2K2 

6I/ln gamma-glutamyl transferase (GGT), 2626I/ln alBaline phosphatase (A/P), 1K8 6I/ln 

direct-bilirubin, 1K kmol/ln albumine, 16 g/ln prothrombin time,72_. fn day-12, a 

probabilistic treatment with 800 mg/d oral fluconazole was started. fn day-1J, a thrombotic 

microangiopathy possibly triggered by tracrolimus, was suspected because of persistently 

elevated direct-bilirubin and /DH levels, anemia, thrombopenia, and the presence of 

schizocytesn tacrolimus was thus replaced by cyclosporine. fn day-1[, the liver 

vasculature was normal at a CT scann histology of a liver biopsy specimen revealed 

ischemic necrosis. 2iological abnormalities persistedn on day-16, fluconazole was replaced 

by voriconazole (O00 mg bid). Plasmapheresis was performed on day-17 because of an 

elevated direct-bilirubin level (J8[). fn day-1K, the patient developed acute renal failure, 

hypotension, oligurian metabolic acidosisn and died in a picture of massive haemolysis. 

Trichoderma atroviride isolation, identification and susceptibility testing: >/*+-$%0"$"5. 

grew from a post mortem liver biopsy specimen cultured at J0ÅC on .abouraudes dextrose 

agar with chloramphenicol and gentamicin. 

" Macroscopic features were dense and woolly colonies that grew within [ daysn 

initially whitish rapidly becoming darB green. 

" Microscopic features includedM conidiophores with branching pattern at right 

anglesn phialides (8 - 10 x 2 km), flasB-shaped often curved in whorls of 2, J or O 

verticillaten conidia (J.[ x O km) darB green sub-globose, short ellipsoidal, smoothwalledn 

chlamydospores were present. 

" DNA sequenceM 861 bp shared 100_ homology with the AF2787K6 sequence of 

>/*+-$%0"$"5. strain ATCC J60O2. 

" Antifungal susceptibility testingM The E6CA.T ",*0"-$% microdilution method in 

liquid medium yielded the following MICs (mg/l)M amphotericin 2, 1n [- 

fluorocytosine, % 6On fluconazole, % 6On itraconazole, % 8n voriconazole, 8n and 

caspofungin, 0.[. 

P-0301. Two cases of successful recombinant interferon-gamma ancillary 

treatment for invasive candidiasis refractory to conventional antifungal therapy 

Ranque S, Curtillet C, Chambost H, Allombert C, 2lancard A, Michel G, Dumon H 

AP-HM Timone, Marseille, France 

ae report on two children treated for a relapsing lymphoblastic leuBemia that were 

diagnosed, with respect to radiological, histological, and PCR findings, with 7+,5"5+* 

+68"#+,& hepatic candidiasis. Fever, right-upper-quadrant tenderness and elevated C- 

reactive protein plasma levels persisted for several weeBs despite several antifungal 

regimens including - concomitantly or sequentially - amphotericine 2, fluconazole, 

itraconazole, voriconazole and caspofungin. Clinical and biological inflammatory 

syndrome disappeared right after the initiation of recombinant-gamma interferon 

(rINF-", IM6lIN ' ) therapy. 2oth clinical and radiological findings normalized rapidly. 

Human recombinant INF-" is safe and efficiently prevents severe infections in patients 

with chronic septic granulomatosis. Apart from chronic septic granulomatosis, 

evidence based data of any anti-infective clinical efficacy of rINF- " + reamain scarce to 

date. Accumulating data from animal models indicate that immunomodulation with 

INF-" can enhance the antifungal activity of neutrophils and monocytes/macrophages 

as well as upregulate protective T-helper type 1 adaptive immune response. However, 

evidence on the clinical use of cytoBines in immunocompromised hosts with invasive 

fungal infections is still scant and inconclusive. 

A very strong temporal relationship indicates that, in combination with antifungals, 

rINF-" +has played a central role in the treatment of these two children. This clearly 

needs to be confirmed in further comparative studies. However, in our opinion, 

adjunctive salvage therapy with rINF-" can be considered for severe invasive fungal 

infections refractory to conventional antifungal treatment. 

>$"#?%5.$)+*&''. are free-living fungi that are common in soil and root ecosystems. >/* 

+-$%0"$"5. (TeleomorphM*A2'%#$.+*+-$%0"$"5"&) is an opportunistic, avirulent plant symbionts 

used as a biological control agent because it parasitizes a variety of phytopathogenic fungi. 

From a review of the literature (including the present case and one personal observation of 

a >$"#?%5.$)+*6%,@"8$+#?"+-() liver infection) >$"#?%5.$)+*&''/ has been associated with 

2O human infections to date. .ix speciesM >/*?+$d"+,()n >/*[%,",@""n >/*6%,@"8$+#?"+-()n >/* 

'&.(5%[%,",@""n >/*#"-$",%0"$"5.n and*>/*0"$"5.n have been identified as etiologic agents of 

infections in immunocompromised hosts. In this report, a 7 th species, >/*+-$%0"$"5. is 

implicated for the first time. ae may anticipate that emergent >$"#?%5.$)+*&''. infections 

will continue to develop in the settings of permissive environmental conditions, selective 

antifungal pressure, and an expanding population of immunocompromised hosts.

P-0302. Dermatophyte infection of the toenails in patients with psoriasis 

Ratajczak Stefanska V, lacalaB RzepBa A, MaleszBa R, RozewicBa M 

Pomeranian Medical 6niversity, .zczecin, Poland 

About [0_ of all individuals with active psoriasis have psoriatic changes of fingernails and/or 

toenails and less than [_ of patients have solely psoriasis of the nails. Nail disorders in patients 

with psoriasis are various and may be easily confused with onychomycosis. Moreover psoriasis 

of toenails can lead to fungal infection of this area. 

Purpose of the study: 

1 | Clinical and mycological analysis of toenail changes in the group of patients with psoriasis, in 

group of patients with psoriasis and additionally fungal infection of the nails and in the individuals 

suffering from onychomycosis as the only nail disorder. 

2 | Estimation of enzymatic activity of the dermatophytes isolated from the psoriatic changed 

nails and a comparative analysis with enzymatic activity of fungi obtained from the nails with 

solely onychomycosis. 

J | Assessment of Beratin medium usefulness, comprising psoriatic epidermis scrapings, in 

measuring the hydrolytic activity of fungi. 

Patients and methods: Male and female patients (180) aged from 27 to 8[ with diagnosis and 

history of nail disease were included in the study. Among the investigated patients 80_ were 

affected by various forms of psoriasis, in which the diagnosis was stated due to clinical image, 

sometimes confirmed by histopathology. In all individuals the number of toenails involved and the 

features of nail involvement such as thicBening, subungual hyperBeratosis, onycholysis, 

discoloration, pitting, oily spots and friability were estimated. In addition, in psoriasis patients 

Psoriasis Activity and .everity Index (PA.I) scores were recorded. 

The material for the mycological examination comprised the file dust and nail clippings taBen 

from changed toenails. The samples were submitted a routine mycological diagnostics. 

In case of obtaining the dermatophyte growth, the enzymatic activity of certain strains was 

measured by the 2io Mdrieux API ZYMw semi-quantitative test. Cultures for the enzymatic 

examinations were grown on Beratin medium including child]s hairs and psoriatic epidermis 

scrapings. 

Results: fnychomycosis was confirmed in 17_ of patients with psoriasis. Among the cultured 

fungal strains in the presented material, the most frequently observed species were 

dermatophytes (81_). 

A statistically significant higher enzymatic activity of #-glucosidase and N-acetyl-#- 

glucosaminidase was detected in cases of dermatophytes strains obtained from individuals with 

increased subungual hyperBeratosis. 

Comparison of the enzymatic activity of fungal strains cultured from infected psoriatic toenails 

with fungal strains obtained from infected toenails without psoriasis revealed no significant 

differences. 

Conclusions: The nails affected by both psoriasis and fungal infection were not significantly 

different from those affected by only one of the diseases. However, subungual hyperBeratosis 

was the most frequently clinical feature of both onychomycosis and psoriatic nail disease. 

P-0303. Prevention of air-borne fungal infection and cross-contaminations 

using the Plasmair unit 

Reboux G 1 , Poirot ( 2 , Mallaret M J , /abrousse H O , Descamps F [ , .ixt N 6 , /audinet N 7 

1 

Department of mycology, 6niversity Hospital, 2esanÑon, France, 2 Department of parasitology c 

mycology, .aint-Antoine 6niversity Hospital, Paris, France, J Department of Hygiene, 6niversity 

Hospital, Grenoble, France, O Department of Hygiene, 2igorre Hospital, Tarbes, France, 

[ 

Department of Hygiene, 2igorre Hospital, Tarbes, France, 6 Department of environmental 

microbiology, 6niversity Hospital, Dijon, France, 7 Research department, Airinspace Company, 

Montigny-le-2retonneux, France 

Hospital-acquired infections remain a worldwide problem. 2iological contaminates, and in 

particular 4&'.$@"66(& spores present in air, dust and soil have been associated with the release 

of spores during demolition and renovation of buildings, and from contaminated service ducts in 

high-risB wards. .uch outbreaBs result in high risBs for immune-compromised patients. 

Current methods to control the spread of such pathogens include strict control of air flows using 

mechanical HEPA filtered devices. However, is the use of these filters is rather restricted 

because of their high pressure drop, resulting in high capital costs to ensure air-flow rates, 

difficult renovation, and large space demands. The Plasmairã device is a mobile biodecontamination 

unit that rapidly lowers contaminates levels. This performance is made possible 

by using AirIn.pace]s non-thermal plasma reactors that use amplified electrostatic fields which 

irreversibly destroy a wide range of microorganisms, including fungi. 

.ince 200O, five French Hospitals have been conducting microbiological evaluations with the 

Plasmairã unit. In these studies all measurements were conducted by independent 

microbiologists who applied standard methods used in each hospital for controlling fungal 

contamination levels. Airborne samples were made with a standard impaction method ([00 // 

[min.), on Petri dishes filled with .abouraud or Malt-agar-media. Rooms receiving immunecompromised 

patients (2esanÑon, Grenoble, Dijon Hospitals) and fperating Theatres (.aint- 

Antoine, Tarbes Hospitals) have been studied. Thus far O8 airborne biocontamination 

measurements have been conducted at 2esanÑon, J0 at Grenoble, J6 at Dijon, OK at .aint 

Antoine and 112 in Tarbes. This corresponds to a study duration period for each hospital of O2, 

J0, 22, J and O days respectively. Results were acquired through repeated sampling or multiple 

sampling points inside the equipped room. All evaluations included comparative levels of fungal 

contamination and were carried out under normal hospital activity. The species of each 

4&'.$@"66(& strain was identified. 

The primary results in 2esanÑon show a mean 4/*U()"@+-(& level of 0.6cfu/m J with a maximum of 

2cfu/m J . Furthermore, an 80_ reduction of total flora compared to the control room was 

observed. .imilarly, samples from the treated room, in Grenoble, show no 4/*U()"@+-(& and a 

mean of only 0.8[cfu/m J for other saprophytic fungi. In Dijon, a fungi level of v1cfu/m J was 

observed 88_ of the time (with 10cfu/mJ maximum), comparatively to the control room where 

v1cfu/mJ was observed in only 12.[_ of the samples (with 80cfu/mJ maximum). In .aint- 

Antoine, the unit lowered initial mean fungi level of 2cfu/m J (Ocfu/m J maximum) down to v1cfu/m J 

for all samples. Due to low initial levels, no significant improvement of fungi load linBed to the air 

treatment has been observed in Tarbes. However, total flora was reduced from a mean level of 

1Ocfu/m J down to 6cfu/m J . 

Even though the conditions were different in each site, Plasmairã has shown a convincing 

effectiveness in reducing air-borne fungal contamination in all applications tested showing a rapid 

lowering of contaminates with eventual reduction of v1CF6/m J . As a consequence, each study 

site has decided to equip rooms with Plasmairã units to protect immune-compromised patients 

against fungal risB. 


P-0304. A cholesterol dependent and amphotericin b resistant isolate of 

Candida glabrata 

Rezusta A 1 , Aspiroz C, 2oeBhout T, Cano (, Theelen 2, Guarro (, Rubio M 

1 

Hospital 6nivesitario Miguel .ervet, 2 Hospital Royo Villanova, Zaragoza .pain, J Centraalbureau 

voor .chimmelcultures, Instituto de la Real Academia de las Artes y las Ciencias, 6trecht, The 

Netherlands, O 6nidad de Microbiologia. Departamento de Ciencias Mddicas 2Zsicas. Facultad 

de Medicina y Ciencies de la .alud, Reus, Tarragona, .pain, [ Centraalbureau voor 

.chimmelcultures, Instituto de la Real Academia de las Artes y las Ciencias, 6trecht, The 

Netherlands, 6 6nidad de Microbiologia. Departamento de Ciencias Mddicas 2Zsicas. Facultad 

de Medicina y Ciencies de la .alud, Reus, Tarragona, .pain, 7 . MicrobiologÜa. Hospital 

6niversitario /ozano 2lesa, Zaragoza, .pain 

A patient was admitted to the hospital due to acute pancreatitis and cholelytiasis. During the 

evolution of the disease she presented acute necrotizing pancreatitis, gladbladder perforation 

and choleperitoneum. The patient was treated with multiple antibiotic and antifungal compounds 

and she was surgically treated again with secondary complications. During this period of five 

months she was admitted four times to the IC6. Fortunately, the patient survived and was 

released from hospital. During this period, 7+,5"5+*@6+8$+-+ grew on four hemocultures (two 

obtained on 02/22/02 and two on 0J/26/02), one catheter culture ( 0J/2K/02) and from one 

bronchial aspirate (0O/1K/02). These isolates were cultured on .abouraud and they were able to 

assimilate glucose and trehalose and discarded. High doses of liposomal Amphotericin 2 was 

the therapy administrated to the patient during her episodes of fungemia. Two month later, the 

patient undergone fever and symptoms of possible urinary tract infections and small and brilliant 

colonies on blood agar (t100.000 CF6/ml) were obtained from two different samples of urine 

(0O/J0/02 and 06/0[/02, respectively). .ubcultures on .abouraud agar and CHRfMagar media 

were negative, however, subcultures on /eeming c Notman agar and modified Dixon medium 

yielded colonies of yeasts after 2O hours at J[oC. Molecular studies confirmed the identification 

of these two nutritionally variant strains as 7/*@6+8$+-+ (CG1 and CG2). 6lterior studies showed 

that the AM2 minimum inhibitory concentration (MIC) for both lipophilic isolates determined using 

the E-test on Mueller-Hinton blood (O8 h.) was tJ2 ug/ml. 

Molecular studies confirmed the identification of CG1 and CG2 as 7/*@6+8$+-+. For this purpose 

the D1/D2 domains and the IT. region of the ribosomal RNA (rRNA) genes were sequenced as 

elsewhere and sequences obtained were compared with those in the Gen2anB database using a 

2/A.T search. The IT. sequences of isolates CG1 and CG2 showed 100_ and K8_ similarity 

with those of the type strain of 7/*@6+8$+-+*C2. 1J8 (A20J2177), respectively. The sequences of 

the D1/D2 domains showed the highest similarity (K[_) with that of the recently described 

species 7/*,"0+$".,&"&, whereas the IT. regions only showed 71_ similarity with the sequences 

of that species. 

Recently, Hazen .-*+6/ (1) described some similar strains of 7/*@6+8$+-+ isolated from the 6l and 

6...A. They studied one strain in more detail and concluded that it represented a nutritional 

variant of 7/*@6+8$+-+ that required cholesterol for growth. ae agree with Hazen .-*+6/ (1) that the 

addition of Tweens on .abouraud agar was not sufficient to allow growth. 

P-0305. Fungemic paracoccidioidomycosis in an immunocompetent patient. A 

case report 

Ricci G 1 , Puccia R 2 , /. 2atista a 2 , E A Marques M J , C .artorelli A J , Franco M 1 

1 6niversidade Federal de .ao Paulo, 2 6niversidade Federal de .ao Paulo, 

J 6niversidade Federal de .ao Paulo, O 6NE.P, Campus 2otucatu, [ 6NE.P, Campus 

2otucatu, 6 6niversidade Federal de .ao Paulo 

Purpose: Paracoccidioidomycosis (PCM) is an endemic, deep mycosis prevalent in 

/atin America, caused by 1+$+#%##"5"%"5.&*8$+&"6".,&"& (Pb). The disease presents 

two clinical formsM i) acute/subacute and ii) chronic. In the acute/subacute form, the 

disease affects children or young adults and rapidly disseminates through the 

lymph-hematogenous pathway. The patients are then classified as being in the 

negative or anergic pole of the disease and usually present a severe depression of the 

cellular immune response. ae report on a rare fatal case of severe acute PCM with 

fungemia in an immunocompetent young patient. 

Case report: A 22 y/o male, healthy rural worBer from 2otucatu, in .Éo Paulo state, 

which is a highly PCM endemic area, presented with multiple cutaneous lesions, fever 

and pneumonia. The patient was hospitalized and the diagnosis of +#(-. \(0.,"6. 17!* 

was*established by the microscopic finding of Pb in sBin scrapings. .erology for HIV 

was negative. The clinical picture rapidly deteriorated and the patient died due to 

multiple organ failure and fungemia within [ days. The autopsy revealed specific 

miliary lesions in lungs, heart, spleen, liver, Bidneys, salivary glands, tongue, larynx, 

sBin, stomach, intestines and lymph nodes. DNA was extracted from paraffinembedded 

lymph nodes and used as template to PCR-elongate fragments of the 

Pb91E: gene for sequencing. An amplicon from nt 6J2 to 816 showed a substitution 

in nt 7KK (G), which is characteristic of either Venezuelan or Colombian isolates and 

rarely found in 2razilian clinical samples of 1/*8$+&"6".,&"& (Morais et al., (. Clin. 

Microb. J8MJK60n Matute et al., 2006, Mol. 2iol. Evol. 2JM6[). 

ConclusionM The present PCM case is unusual since a fungemic, lethal, highly 

disseminated form of the disease developed in an immunocompetent patient. It is 

liBely that the patient has been exposed to a heavy inoculum (conidia from the 

saprophytic phase of the fungus) of a very virulent isolate and was then unable to 

cope with the infection. Preliminary genetic analysis suggests that the isolate has an 

unusual genetic bacBground when compared to other 2razilian isolates. 

Acknowledgements: PhD student Ms NCNogueira de .ouza, FADA, CAPE. and 

6NINfVE 

fur isolates and those studied by Hazen .-*+6/ (1) turned out to be resistant to amphotericin, and 

it has been suggested that this antifungal agent, that targets the ergosterol pathway, may be 

ineffective against bile-dependent isolates of 7/*@6+8$+-+. In addition, mutations in the later steps 

of the ergosterol synthesis were identified. 

ReferenceM 

1. Hazen lC , .tei (, Caroline Darracott, 2reathnach A, May (, Howell .A. Isolation of 

cholesterol-dependent 7+,5"5+*@6+8$+-+ from clinical specimens 2 Diag Microbiol Infect Dis 

200[n [2MJ[-J7.

P-0306. Gastrointestinal yeast colonization in patients with hematological 

malignancies receiving antifungal prophylaxis 

Rimek D 1 , RedetzBe l 2 , lappe R J 

1 

T//V, Department of Medical Microbiology, Erfurt, Germany, 2 Institute of Medical Microbiology, 

Virology, and Hygiene, 6niversity of RostocB, Germany, J Institute of Medical /aboratory 

Diagnostics and Microbiology, .uedharz Hospital, Nordhausen, Germany 

Patients with hematological malignancies are at high risB for developing invasive 7+,5"5+ 

infections. They are often colonized with 7+,5"5+*spp. in the gastrointestinal (GI) tract. To 

prevent infection, the prophylactic use of antifungal agents has been established. 2esides oral 

amphotericin 2, azoles liBe fluconazole and itraconazole are frequently applied. Adverse side 

effects of the antifungal prophylaxis may include the emergence of resistant 7+,5"5+ isolates. 

ae studied the yeast colonization of the GI tract in patients with hematological malignancies 

receiving antifungal prophylaxis (AP) in comparison to healthy controls. 

The study cohort included O6 neutropenic patients with [2 aplastic episodes, including 2O 

patients after allogeneic blood and marrow transplantation, receiving AP during aplasia or after 

transplantation. There were J2 men and 1O women with a mean age of O8.[ years, range 16 to 

71 years. A total of OO patients received azoles orally or intravenously (22 fluconazole, 22 

itraconazole), 20 oral amphotericin 2 (8 amphotericin 2 alone, 12 in combination with an azole). 

7+,5"5+ colonization of the GI tract was monitored by weeBly quantitative cultures of stool 

samples. The samples were trypsinated and plated on .abouraud glucose-(O_)-agar. The 

colony forming units (cfu) per g stool were counted and the yeast isolates were identified by 

standard methods. The mean duration of antimycotic prophylaxis before examining a stool 

sample was 22 days, range 1 to 166 days.The results were compared with those of stool 

samples from 110 healthy controls, O[ men and 6[ women with a mean age of J7 years (range 

1J to K1 years). 

P-0307. Intracranial aspergilosis in an immunocompetent patient 

Rodriguez N 1 , Vidal G 1 , Pitashny R 1 , Finquelievich ( 2 , lopp E 1 

1 Catedra de Micologia Facultad de 2ioquimica y Cs. 2iologicas, .anta Fe, Argentina, 

2 Fac de Medicina - 62A-2s As Argentina 

Summary: A case of intracranial abscess caused by 4&'.$@"66(&*U()"@+-(& in an 

inmunocompetent patient is reported. Infection occurred in a 2O year-old female 

patient, O8 hours after surgery for cholesteatoma. In the presence of endocranial 

hypertension a computerized axial tomography was performed, showing an image at 

the temporal lobe over the petrous bone. 6pon suspiction of anaerobic 

microorganisms presence a craniotomy was performed. The apicotomy of the 

temporal lobe showed at 1 cm. deep, a cavity rounded by an easy-to-penetrate 

capsule, filled with a darB brown liquid containing yellow elements. The contents were 

evacuated, leaving a drainage tube for aspiration. The microbiological analysis 

showed multiple interwoven filamentous, hyaline, septate hyphal elements, with 

dichotomic ramifications, which formed a real mycelium. In culture 4&'.$@"66(&* 

U()"@+-(&*grew. Treatment with itraconazole 200 mg/day was initiated (during J0 

days) in addition to miconazole instillations in the cavity (for O days). Evolution was 

favorable as shown by computerized axial tomography controls after 1 and 2 months. 

Clinical follow-up K years later showed no evidence of sequels or relapses. 

The stool samples of the hematological patients yielded OJ yeast isolates, the controls 8[ 

isolates. The main results are summarized in the Table. 

Yeast colonization GI 

tract 

Hematological 

Malignancies 

Controls 

1-value 

JJ / [2 patients (6J.[ _) 66 / 110 patients (60.0 _) N. 1 

Yeast load 2 1.6 x 10 J 0.O x 10 J 1 v 0.0[ 

Yeast overgrowth (t10 [ 

cfu/g) 

1[ / [2 patients (28.K _) 12 / 110 patients (10.1 _) 1 v 0.0[ 

7+,5"5+*+68"#+,&* 1O / OJ isolates (J2.6 _) O6 / 8[ isolates ([O.1 _) 1 v 0.0[ 

7+,5"5+*@6+8$+-+* K / OJ isolates (20.K _) 10 / 8[ isolates (11.7 _) N. 

7+,5"5+*[$(&."* 11 / OJ isolates (2[.6 _) O / 8[ isolates (O.7 _) 1 v 0.0[ 

1 

not significant, 2 geometric mean of cfu per g stool 

Hematological patients on AP showed a significantly higher yeast load per g stool than controls. 

There was no correlation between yeast load and duration of AP. The species distribution 

showed a significantly lower rate of 7/*+68"#+,&, and higher rates of the fluconazole|resistant 

species 7/*@6+8$+-+ and 7/*[$(&.". In spite of the high colonization rate, not a single 

hematological patient suffered from candidemia. 


P-0308. Tinea capitis produced by Trichophyton mentagrophytes var. 

mentragrophytes in adult linked chinchillas breeding 

Rodriguez N 1 , Cabagna M 1 , Prida M 1 , Finquelievich ( 2 

1 Catedra de Micologia Facultad de 2ioquimica y Cs. 2iologicas, .anta Fe, Argentina, 

2 Fac de Medicina - 62A-2s As Argentina 

Tinea capitis is a micosis of maximum frequency in childhood and exceptional in 

mature age. Among the zoofilic dermathophytes, !"#$%&'%$()*@2'&.() is the most 

common aetiologic agentn with very low frequency cases are observed taBen place by 

>$"#?%'?2-%,*).,-+@$%'?2-.&. The transmission of dermatophytosis from animals to 

humans is very common as long as the inverse situation is not very frequent. 

The objectives of this communication are: 1) Introducing the event of tinea capitis 

taBen place by >$"#?%'?2-%,*).,-+@$%'?2-.& var. ).,-+@$%'?2-.& in mature patient. 

2) Determining the infection source. 

Clinical case: Female patient, aged [J, residing in .anta Fe city, dedicated to the 

breeding of chinchillas. .he consults for suffering loss hair with fractured hair and 

hyperBeratosis on the edge, 20 days of evolution. 

Materials and methods: .he was carried out rasped of the affected area and scalps 

and hair were obtained. Part of the samples was treated with lfH 20_ for the direct 

mycological exam that demonstrated presence of hiphae and arthroconididas inside 

and outside the parasitic hair. The cultivations were carried out in agar .abouraud - 

cloranfenicol and actidione, being incubated 20 days to 28 oC. >$"#?%'?2-%,* 

).,-+@$%'?2-.& var, ).,-+@$%'?2-.& was identified based on macro and 

micromorphologics studies of the cultures and its biochemical properties. Parallelly 

they were practiced direct mycological exams and cultivations of samples of lesions of 

the affected chinchillas (JJ_ on a total of K0) and in all the cases >$"#?%'?2-%,* 

).,-+@$%'?2-.& var. ).,-+@8$%'?2-.& was isolated. 

ae consider this discovery of great importance in human and veterinary health for 

taBing place animal]s epidemic and anthropozoonosis. In future worBs, therapeutic 

experiences will be evaluated. 

P-0309. Dermatophyte infection in Zaragoza (Spain): Trends from 1987 to 2005. 

Rubio-Calvo C 1 , Gil-TomZs ( 1 , 2enito-Ruesca R 1 , Rezusta-/ápez A 2 

1 6niversitary Clinical Hospital. Zaragoza., 2 .chool of Medicine. Huesca. 

Objective: The aim of this study is to Bnow the aetiological agents of dermatophytes 

from 1K87 to 200[. 

Material and methods: A total of 1[17 patients affected with -",.+ were examined in the 6nit of 

Micology at the 6niversity Hospital /ozano 2lesa of Zaragoza from (anuary 1K87 to December 

200[. .Bin scrapings, hair samples and nail clippings or subungueal scrapings were cultured on 

.abouraud Dextrose Agar (.DA) supplemented with antibiotic, on Mycobiotic Agar (MA) and on 

Dermatophyte Test Medium (DTM). .DA and MA were incubated at 28oC and DTM at J2oC for 

two weeBs before they were discharged as negative. Identification of fungal cultures were made 

on the basis of phenotypic characteristicsM both macroscopic and microscopic differences. fther 

tests were performed when necessary. Clinical and mycological evidence of dermatophytosis 

were the criteria used for the inclusion in the study. 

Results: A total of 1[22 dermatophytes wee isolates from 1[17 patients suspected of having 

-",.+/ Two different species were taBen out from same area in five patients. !"#$%&'%$()*#+,"& 

were the more frequent dermatophyte isolated (J[.1[_) followed by >$"#?%'?2-%,* 

).,-+@$%'?2-.& (27.72_), >$"#?%'?2-%,*$(8$() (22.7K_) and a'"5.$)%'?2-%,*U6%##%&() 

(7.2K_). /ess frequently isolated were !"#$%&'%$()*@2'&.()3*>$"#?%'?2-%,*0"%6+#.()3* 

>$"#?%'?2-%,*&%(5+,.,&., !"#$%&'%$()*+(5%(","", >$"#?%'?2-%,*).@,","" and !"#$%&'%$()* 

'.$&"#%6%$. ff all dermatophytes isolates >$"#?%'?2-%,*0.$$(#%&() and >$"#?%'?2-%,*-%,&($+,& 

were only the 2.[6_ and 2.10_ respectively. The most frequent type of dermatophytosis was 

-",.+*#%$'%$"& (O8.J8_) followed by -",.+*(,@("() (1[.7[_), -",.+*#+'"-"& (11.86_), -",.+*U+#"." 

(8.76_) and -",.+*#$($"& (7.6O_). >",.+*#%$'%$"& was mainly caused by !/*#+,"& (OJ.1O_), >/* 

).,-+@$%'?2-.& (J1.61_) and >/*$(8$() (1J.OJ_). In both -",.+*(,@("() '.5"& and )+,(() 

the most prevalent agent was >/*$(8$() (71.K7_ and O0.77_ respectively), followed by >/* 

).,-+@$%'?2-.& (2J.07_ and 28.81_ respectively). !/*#+,"& was the main causal agent of tinea 

capitis and >/*-%,&($+,& (10.[6_), >/*&%(5+,.,&. (1.67_), !/*+(5%(","" (1.67_) and >/* 

0"%6+#.() (1.11_) were other causative agents. >",.+*8+$8+. and U+#"." were mainly caused by 

>/*).,-+@$%'?2-.& (6[_ and [O.J_ respectively).>/*).,-+@$%'?2-.& was isolated in [0_ of 

-",.+*)+,((), while O7.0[_ of -",.+*'.5"& was produced by >/*$(8$(). The most common 

aetiological agent of -",.+*#$($"& were a/*U6%##%&() ([O.J1_) and >/*$(8$() (J7.KJ_). 

Discussion: It is Bnow that the distribution of dermatophytes varies in different counties and 

geographic areas and depends on several factors, such as life style, type of population, migration 

of people and climatic conditions. ae observed that >*0"%6+#.()3*>/*&%(5+,.,&.3*>/*).@,","" 

and a vast majority of >/*-%,&($+,& and !/*+(5%(","" were isolated in the last years 

corresponding with the increased immigration from Africa 

Conclusion: The old aetiological agents, anthropophilic dermatophytes, have been supplanted 

by zoophilic dermatophytes (!"#$%&'%$()*#+,"& and >$"#?%'?2-%,*).,-+@$%'?2-.&) in our area 

of studyn nevertheless in the last years we have observed an increase of antropophilic 

dermatophytes due to several socioeconomic factors, such as immigration.

P-0310. A presentation of pityriasis versicolor of the penis in a circumcised 

patient 

Rym K 1 , FaiBa C 2 , Dalenda E J , Naoufel 2 O , Mourad M [ , Amel 2 6 

1 Hospital of /a Rabta. .ervice of dermatology in Tunis-Tunisia, 2 Hospital of /a Rabta. 

.ervice of dermatology in Tunis-Tunisia, J Hospital of /a Rabta. .ervice of 

dermatology in Tunis-Tunisia, O Hospital of /a Rabta. .ervice of dermatology in Tunis- 

Tunisia, [ Hospital of /a Rabta. .ervice of dermatology in Tunis-Tunisia, 6 Hospital of 

/a Rabta. .ervice of dermatology in Tunis-Tunisia 

Introduction: The !+6+&&.d"+*yeasts are members of the normal human cutaneous 

flora in adults. Also, they are reported as part of the microflora of the male genital 

region in mostly uncircumcised male. The prevalence of !+6+&&.d"+*yeast*colonisation 

on the glans penis of circumcised males are discussed in multiples studies. ae report 

a case of an expansed pityriasis versicolor affected a man with respect of preputial 

space. 

* 

Observation:*A JK year old tunisian, muslum and circumcised man came to our 

consultation with the complaint of a nonpruritic scaly generalized eruption. Cutaneous 

examination found hypopigmented plaques located on the trunB, the limbs and the 

glans penis with respect of preputial space. He had no history of cutaneous penile 

infections such as human papillomavirus, herpes simplex virus, bacterial infections, 

mollusca and Candidal balanitis and no more genital dermatoses such as psoriasis, 

seborrheic dermatitis, atopic eczema, allergic contact dermatitis. He complained of an 

increased activity of sebaceous glans. .tripe tape had been practiced on the 

lesionnels sites and revealed clusters of yeast and short mycelia filaments confirmed 

the diagnosis of pityriasis versicolor. The difficulty in culturing !+6+&&.d"+*don]t allow 

us to identify a species of*fungus. 

Comments: It is well recognized that !+6+&&.d"+ yeasts occur an important role in the 

pathogenesis of pityriasis versicolor. Actually, ten different*!+6+&&.d"+ species 

according to morphological and physiological features are identified in healthy and 

pityriasis versicolor-affected sBin, such as !+6+&&.d"+ U($U($, !+6+&&.d"+ &2)'%5"+6"& 

and !+6+&&.d"+*@6%8%&+/ Recently, several authors have noted that !+6+&&.d"+*&''. 

as part of the microflora of healthy uncircumcised male genital region in OK.2_ (1) 

contrary to circumcised male patients where !+6+&&.d"+*&'' are identified in 22.O_ 

(2). Although this colonisation, !+6+&&.d"+*&''*lead rarely to dermatoses such as 

pityriasis versicolor in particularly genital area. In our observation, the respect of 

preputial space plead for the role of circumcision to protect from infections 

dermatoses. 

P-0311. Chronic sphenoidal sinusitis due to Schizophyllium commune 

S. Perpétuo de Sequeira H 1 , 2arroso C 2 , Marques Gomes M 1 , MigueÜs ( J , Guarro ( O 

1 Clinica Dermatolágica 6niversitZria, /isbon, Portugal, 2 /aboratário de Neuropatologia, 

Portugal, J .erviÑo de Neurocirurgia do Hospital de .anta Maria /isboa, Portugal, 

O 6niversitat Roviraòòò, Virgili Reus 

Case Report: ae report the case of a J[ year-old male born and resident in /isbon, 

with recurrent headaches and nasal obstruction for several months. Despite the 

prolonged steroid therapy the patient didn]t fully recover. In November 200J, the 

patient clinical condition aggravated with increasing frequency and intensity of the 

headaches and the appearance of bilateral convergent strabismus. A brain CT-scan 

and MRI revealed a sphenoidal, expansive cystic lesion. The serologies for HIV1 and 

2 were negative. .ix months later he was operated upon with partial removal of the 

lesion. The brain control CT-scan, showed resolution of the sphenoidal lesion, but 

persistence of its left maxillary sinus component, and a sinus surgical cleaning was 

performed. The histopathology study | PA. and silver-methenamine stains | showed 

a mycotic abscess compatible with a d2@%)2#.-.. Mycological study | fn the direct 

examination of two sinus discharge samples, we observed the presence of septated 

and branching hyphae. Cultures on Mycobiotic and .abourad dextrose agar, 

incubated at 2Oo and J7oC, developed a non | sporulated, white woolly colony, with 

pronounced odour and cycloheximide tolerant. The combined sequence analysis of 

IT. and RNA was performed and revealed =#?"d%'?266()*#%))(,.. Amphotericin 2 

(liposomal formulation) was prescribed 2[0 mg/day during three weeBs, with cultures 

negatives and clinical improve. 

Comments: =#?"d%'?266() #%))(,. it]s an emerging pathogen, which has caused 

severe human infections in recent years. Clinical manifestations and the 

histopathological findings are similar to those caused by other fungi. Culture is 

mandatory for a precise diagnosis. After one year of follow up the patient remains well 

and sympton free. 

References: 

1. Frequency and spectrum of !+6+&&.d"+*yeasts in the area of the prepuce 

and glans penis. Mayser P., .chytz M., .chuppe HC., (ung A., .chill a2. 

2(6 Int 2001, 88, [[O-8. 

Malassezia and Candida colonisation on glans penis of circumcised men. Atilla Aridog 

I.,1 Macit IlBit., VolBan Izol1 and Aylin Ates. Mycoses 200[, O8, J[2|J[6. 


P-0312. Sporothrix schenckii in epithelial cells 

Sabanero M 1 , .andoval-2ernal G 1 , 2arbosa - .abanero G J , Tsutsumi V O 

1 Inst. de Invest. en 2iologÜa Experimental. 6niversidad de Guanajuato, 2 Inst. de Invest. 

Mddicas. 6niversidad de Guanajuato, J Inst. de Invest. Mddicas. 6niversidad de 

Guanajuato, O Centro de Investigacián y de Estudios Avanzados - IPN 

Adherence of pathogenic microorganisms to host tissues is regarded as a pre 

requisite for dissemination. Microbial adherence has been studied extensively in 

pathogenic bacteria and fungi such as 7/*+68"#+,&, 4/*U()"@+-(&, and Z/*5.$)+-"-"5"&, 

(but little is Bnown about the adherence mechanism in =/*&#?.,#["". This study was 

undertaBen to examine the cytosBeleton in the host cell during the interactions and 

invasion of =/*&#?.,#["" strains MP - 10K. ae found that yeast (10 [ cells /ml) and 

mycelium (100kl/ml) adhered and invaded (6 to 2O h) culture human cervix epithelial 

cells. The invasion abilities of mycelium were much high that the yeast. 

Immunofluorescence microscopy studies showed that the adherence of mycelium and 

yeast induced cytosBeletal alterations. 6nder the adherent sites calcofluor studies 

showed an intimate association between cell wall of fungus and epithelial cells. These 

results indicate that surface of fungus plays important roles in the invasion of epithelial 

cells, and the alterations of cytosBeleton in host cell for invasion. In this moment we 

studied the surface receptors of the cells. 

The worB was support by the 6niversidad de Guanajuato. (Grant No. 18 to M./) 

P-0313. Determining allergenic bands of separated penicillium by using serum 

of patients with asthma 

Sabokbar A 1 , lhosravi A 1 

1 Department of Mycology , Faculty of .ciences , 6niversity of AZAD I./AMIC , laraj, 

Iran, 2 Department of Mycology , Faculty of Veterinary medicine university of Tehran, 

Iran 

Objective: Categorizing different types of penicillium on the basis of allergenic bands 

Abstract: It was determined spore mold existent in air in creating different allergic 

form specially Penicillium is one of allergic mold which play important role in creating 

and allergic rinit. 

In this study, to get allergic bands, penicillium citrinum, penicillium notatum, penicillium 

oxalicum and penicillium frequentes separated from the air of Iran (J0 isolation 

penicillium) from J0 bronchitis used. 

To obtain the nectar of mold, first these molds are cultivated on agar sabouraud 

dextrose agar and them passaged on czapeBes agar to get mass cultivation, they 

cultivated on sabouraud dextrose broth and after breaBing, the surface liquid derived 

from centrifuge used as a raw nectar. In this study imonobloting method was used to 

determine allergen elements of this mold. In this method conjugated Anti-IgE antibody 

with phosphatas alcalan and N2T, 2 CIP .ubstrate used to determine the result of 

reaction. 

Result: of JO protein bands existing in penicillium nectar, 21 bands with O[ to 128 ld 

molecular weight reacted pooled priate IgE of related patients. 

Reacting elements with IgE wereM 128, 12J, 116, 107, K7, K[, KO, KJ, K2, K0, 88, 8O, 

76, 66.[, 6[, 6J, [K.[, [6.[, [[, [J, O[ Bilo Dalton. 

In above bands, O[, [J, [6.[, [K.[, 76, 8O, K7 Bd showed the most frequency among O 

types 

Conclusion: According to the results It was specified that all penicillium isolations 

bands allergic bands (at least 1J) 

In this study none of penicillium lacBed allergic band, so the categorizing of these 

molds as weaB allergic and non-allergic is not possible.

P-0314. An evaluation of elisa and latex agglutination test for rapid diagnosis of 

cryptococcosis 

Saha D, jess I, (ain N, 2anerjee 6 

All India Institute of Medical .ciences, Dept. of Microbiology, Mycology lab, New Delhi, 

India 

Purpose of the study: 7$2'-%#%##(&*,.%U%$)+,&3*an encapsulated, budding yeast, is 

an opportunistic fungal pathogen with worldwide distribution. It causes the life 

threatening infection, Cryptococcosis which is mostly chronic in nature and affects the 

Central Nervous .ystem. 6nfortunately, there are no pathognomonic signs and 

symptoms in this infection, which can arouse clinical suspicion, especially in the early 

stages. If diagnosed early it is relatively easier to manage the patient. Conventional 

diagnosis is based on direct demonstration and culture. India inB is good but less 

sensitive and the culture though is hgold standardi but need a large amount of sample 

and time consuming. Among the rapid test serological based diagnosis is promising 

method for diagnosis as it can detect low-levels of infection. For Cryptococcosis, /atex 

Agglutination Test (/AT) and a newly introduced commercial E/I.A test are available. 

Description: In this study we performed a comparative evaluation of the various 

conventional methods (India inB and culture) along with the most common serological 

methods- /AT and E/I.A for the detection of cryptococcal antigen in cerebrospinal 

fluid. The system was evaluated in clinical samples from [0 patients which came for 

diagnosis of cryptococcal meningitis. 

Results and conclusions: Thirty five samples (70_) were positive for 7$2'-%#%##%(& 

by India inB, culture, E/I.A and /AT. There were 10 samples (20_) in which India inB 

was positive but the culture was negative, yet both E/I.A and /AT showed positive 

results. In [ samples (10_), /AT showed false positive results due to the cross 

reactivity with other organisms present in the sample. However E/I.A was negative in 

those samples, thus demonstrating it as more specific test. /AT depends on visual 

inspection which leads to misinterpretation of results, especially in borderline cases. 

/AT also has the disadvantage of cross reactivity and increases the possibility of false 

positivity. ahereas, E/I.A depends upon the color changes measured by 

spectrophotometric method which maBe interpretation of results easier. ae conclude 

that E/I.A is a rapid serological test and is more specific and has potential 

advantages over /AT as it gives clear discrimination of positive from negative results 

(cut off). Thus, E/I.A is a better test than /AT for rapid diagnosis of Cryptococcosis. 

P-0315. Fungemia caused by rhodotorula minuta in high risk pediatric patients 

Santos P 1 , Carrillo-Muoz A 2 , Figueroa C 1 , /opardo H 1 , YucowsBy M 1 

1 Hospital de Pediatria Dr. (.P.Garrahan, 2uenos Aires, Argentina, 2 A.C.I.A 

Microbiologia, 2arcelona, .pain 

In the present study we describe two cases of fungemia caused by Rhodotorula 

minuta in high risB pediatric patients with acute linfoblastic leuBemia. 

Yeasts were isolated from both patients, in one of then from peripheral blood culture 

and from the implanted catheter in the other (lysis-centrifugation techniques). 

ae confirmed species identification by using API 20 C Aux (2iomerieux) 

supplemented by conventional methods. 

The patients were treated with amphotericin 2 at doses of 0.6 to 1 mg/Bg/day (total 

cumulative dose, 17.[ mg). In vitro antifungal susceptibility testing for Rhodotorula 

minuta revealed minimun inhibitory concentration to fluconazole t2[6mg/l and to 

amphotericin 2 of 0.7[mg/l. 

Infections from Rhodotorula species are uncommon but are observed in hosts with 

CVC and/or immunosupression. 

Amphotericin 2 in addition to catheter removal are acceptable therapies for this 

infections. 

It is worth noting the low susceptibility of these strains to azoles together with their a 

slow development in culture, wich of difficults their isolation. 

This encourages us to consider new treatment, to be on the alert for new pathogens 

and, fundamentally, to do teamworB in order to control intra-hospital fungal infection. 


P-0316. Trichosporon asahii in a high-complexity pediatric hospital 

Santos P 1 , YucowsBy M 1 , Noman R 1 , Hemadi D 1 , Carrillo-Muoz A 2 , Gomez . 1 , 

Rosanova M 1 

1 Hospital de Pediatria Dr. (.P.Garrahan, 2uenos Aires, Argentina, 2 ACIA , 2arcelona, 

Espaa 

Trichosporon asahii is a rare pathogen has been associated with a wide spectrum of 

clinical manifestations, ranging from superficial involvement in immunocompetent 

individuals to severe systemic disease in immunocompromissed patients. 

ae report twenty cases of deep fungal infection by Trichosporon asahii in high-risB 

pediatric patients within a period of two three year ((anuary 200J-December 200[) at 

the Hospital National of Pediatric Dr (. P. Garrahan, 2uenos Aires, Argentina. 

The strains were recovered from different clinical specimsM blood, catheters, gastric 

fluid, urine and sBin biopsy and were identified using API 20 C Aux yeast assimilation 

test supplemented by conventional methods. 

Results showed the average age was [ years (age range 1-1O years). 

Four out of twenty patients died due to disseminated fungal infection. In two of them, a 

secondary focus was confirmed. 

P-0317. A case of cutaneous Pseudallescheria boydii infection 

Sato T 1 , Nagao l 1 , Yoshizawa N 2 , Hata Y J , .ugiura M 2 , Amagai M 1 , Yaguchi T O 

1 Department of dermatology leio 6niversity, ToByo, (apan, 2 2Department of 

dermatology .himizu Hospital .hizuoBa city, .hizuoBa, (apan, J Department of 

dermatology .aiseiBai lanagawa Prefectural Hospital, lanagawa, (apan, O Research 

Center for Pathogenic Fungi and Microbial Toxicoses, Chiba 6niversity, Chiba, (apan 

1&.(5+66.&#?.$"+*8%25""*is a ubiquitous filamentous fungus. ae report a case of 

cuntaneous 1/*8%25"" infection of the left Bnee in a 7K-year-old (apanese man who 

was receiving oral predonisolone (2[mg/day) for radiation pneumonitis after radiation 

therapy for his left breast cancer. He presented with a 2-weeB-history of a lesion on 

the left Bnee. A biopsy specimen from the sBin lesion of his left Bnee revealed 

granulomatous inflammation with hyphae. Culture of the pus from the sBin lesion and 

the sBin biopsy specimen confirmed the diagnosis of cutaneous 1/*8%25"" infection. 

rDNA IT. sequence was analyzed to confirm mycological diagnosis. The patient was 

treated with oral 200mg/day itraconazole. Cutaneous injury may be responsible for the 

incidence of localized infection. Immunocompromised patients who have persistent 

traumatic sBin ulcer need to be ruled out of such rare fungus infection. Although 1/* 

8%25"" is ubiquitous fungus, an opportunistic infection in immunocompromised patients 

can be life-threatening and prompt treatment based on accurate diagnosis is important. 

All clinical isolates were resistant in vitro to flucytosine and two of them showed 

reduced susceptibility to amphotericine 2 (AM2). 

It is necessary to be aware of the presence of Trichosporon asahii as a new emerging 

pathogen in pediatric patients. It deserves to be considered in new therapeutic 

schemes because of its reduced susceptibility to currently available antifungals.

P-0318. Effect of ketoconazole on the pharmacokinetics of BAL4815 at steady 

state after multiple oral daily doses of BAL8557 (WSA) and ketoconazole 

Schmitt-Hoffmann A 1 , Roos 2 1 , .auer ( 1 , Maares ( 1 , .picBermann ( 1 , Iersel M 2 , 

Heep M 1 

1 2asilea Pharmaceutica /td., 2asel, .witzerland, 2 jendo Drug Development .ervices, 

Groningen, The Netherlands 

Background: 2A/8[[7 is a new water-soluble azole (a.A). The active metabolite 

2A/O81[ has broad ",*0"-$% activity against yeasts, moulds (including zygomycetes) 

and dermatophytes. The oral formulation is almost completely bioavailable while the 

i.v. formulation of 2A/8[[7 does not contain cyclodextrins. 2A/O81[ is mainly 

inactivated by slow CYPJAO-mediated metabolic clearance. letoconazole is Bnown to 

be a very potent inhibitor of CYPJAO. This study investigated the effect of high oral 

doses of Betoconazole on drug levels of 2A/O81[, both at steady state. 

Methods: Dosing schedule (oral doses expressed as mg equivalent of 2A/O81[)M 

Drugs © .tudy 

Days 

D1 D2 to 1O D1[ to 

J[ 

DJ6 to 

OJ 

DOO DO[ to 

[7 

D[8 to 

71 

a.A O00 mg 100 mg - - O00 mg 100 mg - 

letoconazole - - - 200 mg 200 mg 200 mg 200 mg 

The study had a randomized, multiple-dose, crossover design and was performed in 

26 male subjects. 

.erial blood samples for pharmacoBinetic profiles of 2A/O81[ were obtained on days 

1O and [7. Plasma samples were analyzed using a specific /C/M./M. method. 

Results: a.A was well tolerated when given alone or concomitantly with the tested 

drugs, as single or multiple daily doses. Co-administration of a.A and Betoconazole 

led to approximately a 1.[-fold and a J-fold increase in C max and A6C 0-0 of 2A/O81[, 

respectively, compared to administration of a.A alone. These increases were 

statistically significant (pv 0.0[). 

Conclusion: letoconazole, a strong inhibitor of CYPJAO, increases exposure to 

2A/O81[. 

Despite a J-fold increased exposure, a.A was very well tolerated. 

P-0319. Effect of Rifampicin on the pharmacokinetics of BAL4815 at steady 

state after multiple oral daily doses of BAL8557 and Rifampicin 

Schmitt-Hoffmann A 1 , Roos 2 1 , .auer ( 1 , Maares ( 1 , .picBermann ( 1 , Iersel M 2 , 

Heep M 1 

1 2asilea Pharmaceutica /td., 2asel, .witzerland, 2 jendo Drug Development .ervices, 

Groningen, The Netherlands 

Background: 2A/8[[7 is a new water-soluble azole (a.A). The active metabolite 

2A/O81[ has broad ",*0"-$% activity against yeasts, moulds (including zygomycetes) 

and dermatophytes. The oral formulation is almost completely bioavailable while the 

i.v. formulation of a.A does not contain cyclodextrins. 2A/O81[ is mainly inactivated 

by slow CYPJAO-mediated metabolic clearance. Rifampicin is Bnown to be a very 

potent inducer of CYPJAO. This study investigated the effect of high oral doses of 

rifampicin on drug levels of 2A/O81[ both at steady state. 

Methods: The study was a randomized, multiple-dose, crossover study performed in 

26 male subjects to assess the interactions between multiple daily oral doses of both 

a.A and rifampicin. 

Dosing schedule (a.A doses expressed as mg equivalent of 2A/O81[)M 

Drugs \ Study 

Days 

D1 D2 to 

14 

D15 to 

35 

D36 to 

43 

D44 D45 to 

57 

a.A O00 mg 100 mg - - O00 mg 100 mg - 

D58 to 

71 

Rifampicin - - - 600 mg 600 mg 600 mg 600 mg 

.erial blood samples for pharmacoBinetic profiles of 2A/O81[ were obtained on days 

1O and [7. Plasma samples were analyzed using a specific /C/M./M. method. 

Results: a.A was generally well tolerated when given alone or concomitantly with 

the tested drugs, as single or multiple daily doses. Co-administration of a.A and 

rifampicin led to an approximately O-fold decrease in C max and a O0-fold decrease in 

A6C 0-0 of 2A/O81[, compared to administration of a.A alone. These decreases 

were statistically significant (pv0.01, paired-t test). 

ConclusionM Rifampicin a strong inducer of CYPJAO decreases the exposure to 

2A/O81[ to a large extent. 



pharmacokinetics of tacrolimus 


Roehrle M 2 , Heep M 1 

1 2asilea Pharmaceutica /td, 2asel, .witzerland, 2 AAI Deutschland GmbH, Neu-6lm, 

Germany 


intravenously as a water-soluble pro-drug (2A/8[[7, a.A), is a substrate and an 

inhibitor of CYPJAO. Tacrolimus is also metabolized partly by CYPJAO, and 2APO81[. 

ae investigated whether oral dosing with a.A affects exposure to tacrolimus. 


years received tacrolimus ([ mg) on day 1 and on day 22. a.A was given orally as a 

O00 mg loading dose on day K and 100 mg maintenance dose (a.A doses expressed 

as mg equivalent of 2A/O81[). .erial blood samples for pharmacoBinetic profiles of 

tacrolimus were obtained on days 1 and 22. Plasma samples were analyzed using a 

specific /C/M./M. method. 


Drugs © .tudy 

Days 

D1 D2 to7 D8 DK to 21 D22 D2J to 27 

a.A - - O00 mg 100 mg 100 mg 100 mg 

Tacrolimus [ mg - - - [ mg - 

Results: 2oth drugs were well tolerated. No serious or significant adverse events 

(AEs) were reported, AEs observed in t2 subjects during a.A dosing were headache, 

nausea, and common colds. /aboratory test results and ECGs were unremarBable. 

Dosing with a.A led to a statistically significant 7[_ increase in exposure of 

tacrolimus (pv0.0[n paired-t test). 

Parameter Tacrolimus Tacrolimus r a.A 

Number of .ubjects 26 26 

A6C 0-0 (ng1h/m/) 2K0 (# 1J1) [08 (# 20[) 

C max (ng/m/) 28 (# K.87) O8 (# 1J.0) 

t max (h) 1 (0.7[-2.0) 1.[ (0.7[-2.0) 

Values are arithmetic mean # .D for A6C and C maxn median and range for t max 

Conclusion: a.A .ignificantly increased exposure of tacrolimus at the doses tested 

in this study. 


pharmacokinetics of S- and R-warfarin 


Allison M 2 , Heep M 1 

1 2asilea Pharmaceutica /td, 2asel, .witzerland, 2 MD. Pharma .ervices, Phoenix, 

Arizona, 6.. 


intravenously as a water-soluble pro-drug (2A/8[[7, a.A), is metabolized partly by 

CYP2CK. aarfarin is metabolized mainly by CYP2CK. 2oth 2A/O81[ and warfarin are 

highly protein-bound. ae investigated whether concomitant dosing with a.A affected 

exposure of warfarin. 


years received warfarin racemate (10 mg) on day 1 and on day 28. a.A was given 

orally as a O00 mg loading dose on day K and 100 mg/day maintenance dose (a.A 

doses expressed as mg equivalent of 2A/O81[). 


Drugs © .tudy Days D1 D2 to 8 DK D10 to 28 D2K DJ0 to J6 

a.A - - O00 mg 100 mg 100 mg 100 mg 

aarfarin 10 mg - - - 10 mg - 

.erial blood samples for pharmacoBinetic profiles of .- and R- aarfarin were obtained 

on days 1 and 2K. Plasma samples were analyzed by a specific /C/M./M. method. 

Results: Dosing with a.A had no apparent effect on exposure to either enantiomer 

of warfarin (Table). Inter-subject variability (CV_) of pharmacoBinetic parameters was 

22_-27_ for C max and 21_-2J_ for A6C 0-0 . 

Parameter .-aarfarin .-aarfarin r a.A R-aarfarin R-aarfarin r a.A 

Number of 

.ubjects 

A6C 0-0 

(&g1h/m/) 

12 12 12 12 

2[.0 (# [.2) 20.O (# 8.1) 2J.J (# [.O) 18.6 (# 6.2) 

C max (&g/m/) 0.O6 (# 0.12) 0.68 (# 0.18) 0.O[ (# 0.1) 0.6O (# 0.10) 

t max (h) 

1.00 (0.[0- 

16.0) 

0.7[ (0.[0-2.00) 

0.7[ (0.[0- 

2.00) 

0.7[ (0.[0-1.[0) 

Values are arithmetic mean # .D for A6C 0-0 and C max n median and range for t max 

2oth drugs were well tolerated. No serious or significant adverse events (AEs) were 

reported. The most frequent AEs for a.A with or without warfarin were sore throat (J 

subjects), headache (2 subjects), and diarrhoea (2 subjects). All AEs were mild. 

/aboratory test results were unremarBable. No relevant changes in ECGs were 

observed in any subject. 

Conclusions: a.A had no impact on the pharmacoBinetics of R- and .-warfarin, a 

CYP2CK substrate, at the doses tested in this study.

P-0322. Pharmaceutical MBL ligands perpetuate fungal infection 

Sealy P 1 , .wiatlo E 2 , Chapman . 1 , Cleary ( 1 

1 6niversity of Mississippi Medical Center, (acBson, 6.A, 2 Veteranes Administration 

Hospital, (acBson, 6.A 

Purpose: To evaluate the impact of mannose containing pharmaceuticals on the 

innate immune system response to fungal infections. Teicoplanin was evaluated as 

the prototype anti-infective. 

Background: Mannnose 2inding /ectin (M2/) activates the classical complement 

pathway by binding to anionic structures on the surface of microorganisms to form 

complexes that are capable of eliciting opsonization or phagocytosis. M2/ forms 

covalent bonds with the hydroxyl groups of mannose sugars expressed on the surface 

of microorganisms. Activated M2/-associated serine proteases (MA.P) initiate the 

cascade of reactions that culminate in phagocytosis. The chemical structure of 

teicoplanin, a glycopeptide antibiotic, consists of mannose sugars not dissimilar to 

fungal mannans. 

remaining mice exposed to teicoplanin and mannose failed to survive beyond 60 

hours post-infection, while 60 _ of saline exposed mice were viable. The remaining 

infected mice exposed to normal saline did not survive beyond 72 (n}J), 80 (n}2) and 

K6 (n}1) hours respectively post-infection. .urvival data was analyzed using /og 

RanB statistics, which revealed significant results for mannose or teicoplanin 

individually compared to saline (pv0.0[). 

Conclusions: K,*0"0% studies demonstrate hfaster time to deathi for 7+,5"5+ 

inoculated mice exposed to mannose and teicoplanin compared to normal saline. ae 

believe that pharmaceuticals containing mannose sugars, such as teicoplanin, 

compete with fungal microorganisms for M2/ receptors. ahen binding between M2/ 

receptors and mannose sugars from the surface of microorganisms is inhibited, 

phagocytosis does not occur and the incidence of fungal infection may increase. 

Further research is underway to evaluate the clinical significance of these findings. In 

addition, multiple other mannose containing pharmaceuticals are being tested. 

Methods: An invasive candidemia model was used to evaluate differences in survival 

between mannose and teicoplanin treated groups compared to a control group 

(normal saline). In the model, three groups of 2alb/c mice were sequentially injected 

with a lethal dose of 7+,5"5+*+68"#+,& ATCC OO8[8 (1.O x 10 6 cfu). Ten (10) mice 

were allocated to each group. In an attempt to mirror human therapeutics, an initial 

dose of pharmaceutical was administered 1 hour pre-infection, and a second dose 8 

hours post-infection. Pharmaceutical agents included teicoplanin at an initial dose of 

J0 mg/Bg IV, followed by 1[ mg/Bg IVn mannose at an initial dose of 2.88 mg/Bg IV, 

followed by 1.OO mg/Bg IVn or an equal volume of normal saline 

Results: K,*0"0% cumulative survival at O8 hours revealed 10, J0 and K0 _ survival 

rates for mice administered mannose, teicoplanin, and saline respectively. The 


P-0323. Production of a biofilm-like matrix by Aspergillus fumigatus on 

polystyrene and bronchial epithelial cells 

Seidler M, .alvenmoser ., Myller F 

Pediatric Pulmonology 6niversity Heidelberg 

Purpose of the study: Candida spp. have the ability to grow in a biofilm-liBe matrix in 

vitro and in vivo on different substrates. Candida biofilms are clinically relevant in 

patients with inserted medical devices (e.g. central venous catheters, endotracheal 

tubes etc.) Filamentous fungi are often found in domestic environments and conidia 

can easily be inhaled and reach the alveoli. Invasive aspergillosis in 

immunocompromised patients as well as aspergillosis in immunocompetent patients 

(e.g. chronic sinus infections, allergic bronchopulmonary aspergillosis, endocarditis 

etc.) have in common that they clinically appear quite often resistant to in vitro potent 

antifungals and the morbidity and mortality is still unacceptable high. Aspergillus spp. 

may produce a biofilm-liBe structure that might contribute to this phenomenon. The 

aim of the study was to produce a biofilm-liBe matrix with A. fumigatus under different 

conditions and on various substrates and to confirm the extracellular matrix (ECM) by 

various methods. 

Description of the project: A. fumigatus (ATCC çK1K7) biofilm-liBe matrix was 

produced on polystyrene (P.) and on bronchial epithelial cells (2EC) under sessile 

and floating conditions. Different concentrations of glucose, F2. and inoculum were 

tested in RPMI at different pH-values. Incubation temperature, production time and 

different flows on both P. and 2EC line I2J-1 were optimized. The following methods 

were appliedM Dry weight measurements, antifungal drug activity testing, scanning 

electron microscopy (.EM) and confocal scanning laser microscopy (C./M) images 

were taBen. 

Results and conclusions: The optimized condition for mature biofilm was produced 

with RPMI (r10_Glu r10_F2., pH}6.[) at J[ÅC for 72h slightly rocBing with an 

inoculum of 1x10†6/m/. It was possible to produce biofilm on polystyrene and 

bronchial epithelia cells. Dry weight of a planBtonic control flasB was compared with a 

biofilm flasB. The results display an increase of the dry weight from O0mg to O00mg at 

72h biofilm formation time. The activity of three antifungals against A. fumigatus 

embedded in the biofilm-liBe matrix was compared to planBtonic grown Aspergillus. 

ahile planBtonic grown Aspergillus was susceptible to itraconazole (0.12[kg/m/), 

voriconazole (0.06Jkg/m/) and amphotericin 2 (1kg/m/), Aspergillus biofilms were 

resistant to all drugs (tJ2kg/m/). The .EM images from intermediate phase displayed 

a networB of hyphal structures with a matrix-liBe structure in between. After mature 

phase, A. fumigatus is overlaid with a thicB layer of matrix. In addition, flow channels 

are observable. For C./M the dye A-Alexafluor O88 stains specific the polysaccharide 

structures in the fungal cell wall and the ECM formed during mature biofilm 

development on P.. fn confluent 2EC, intermediate phase biofilm was observed. 

The reconstructed lateral and JD views display conidia and hyphal structures 

surrounded by matrix. fur investigations demonstrate that A. fumigatus is able to form 

a biofilm-liBe structure in vitro that is similar to biofilms of Candida spp.. 

P-0324. Cost-effectiveness analysis of anti-fungal prophylaxis in patients 

undergoing hematopoietic stem cell transplantation 

Seifeldin R 1 , .chonfeld a 2 , Cheng ( 2 

1 Astellas Pharma 6., Inc., 2 ~uorum Consulting, Inc. 

Background and purpose of the study: Prophylaxis can decrease the incidence of fungal 

infections in transplant patients and lower the overall cost of care by reducing the number and 

cost of infections needing treatment. In a multi-center, blinded, randomized head-to-head 

comparative study evaluating the safety and efficacy of prophylaxis in 882 hematopoietic stem 

cell transplant (H.CT) patients, micafungin was shown to be more effective than fluconazole in 

preventing suspected fungal infection (8O.K_ to 78.6_). An economic analysis was conducted 

to evaluate the cost-effectiveness of micafungin prophylaxis compared to fluconazole prophylaxis 

among patients undergoing H.CT. 

Methods: Cost-effectiveness measures were calculated to compare (1) prophylaxis with 

micafungin versus (2) prophylaxis with fluconazole. Efficacy data were taBen from the clinical 

study, in which the incidence of proven or probable systemic fungal infections was 1.6_ in the 

micafungin treatment arm and 2.O_ in the fluconazole treatment arm. The percentage of 

patients receiving empiric therapy was 1[.1_ and 21.O_ for micafungin and fluconazole, 

respectively. The economic analysis was conducted from the hospital perspective using costs 

incurred from admission through discharge. Each of the 882 patients was assigned costs and 

effectiveness based on their outcomes data from the clinical study. Published literature was 

used to estimate hospital costs (adjusted to 200[ values) associated with H.CT patients 

receiving prophylaxis, empirical anti-fungal treatment, and treatment for a probable or proven 

Candida or Aspergillus infection. Mean costs and effectiveness were calculated for each 

treatment group. To test the variability of the results using repeated sampling, a bootstrapping 

analysis was also conducted, with 1,000 simulations of random samples of 100 patients from 

each treatment group. Incremental cost-effectiveness ratios were calculated where appropriate. 

Results: Mean cost for a course of prophylactic therapy was ñ2,018 for the O2[ patient treated 

with micafungin and ñ8OJ for the O[7 patients treated with fluconazole. Adding in other hospital 

costs, total patient costs were ñ11O,2[1 and ñ117,821 for micafungin and fluconazole patients, 

respectively, a significant difference of ñJ,[70 (p}0.01[). Considering the lower rate of 

breaBthrough fungal infection from the clinical study, micafungin is the dominant method of 

prophylaxis, with both lower costs and greater efficacy. The bootstrapping analysis indicated that 

micafungin prophylaxis was cost saving in 72.O_ of the samples compared to K.2_ for 

fluconazole prophylaxis. Micafungin prophylaxis was dominant in [[.[_ of the samples, 

compared to 6.O_ for fluconazole prophylaxis. .ensitivity analyses on estimated hospital costs 

confirmed the superiority of micafungin as a cost-effective therapy. 

Conclusions: In addition to its greater efficacy, micafungin prophylaxis for patients undergoing 

H.CT leads to cost savings compared to fluconazole prophylaxis. Despite the higher costs of 

the drug itself, prophylaxis with micafungin reduces hospital costs, and resulting total patient 

costs, due to less need for empirical anti-fungal therapy and fewer proven or probable fungal 

infections.

P-0325. The spread of Trichophyton tonsurans infection among judo and sumo 

wrestlers 

Seishima M 1 , YamanaBa . 1 , Fujisawa T 1 , MochizuBi T 2 

1 Department of Dermatology, fgaBi Municipal Hospital, 2 Department of Dermatology, 

lanazawa Medical 6niversity 

Although >$"#?%'?2-%,*-%,&($+,&*G>/*-%,&($+,&J*is well-Bnown as one of the causes of 

tinea in Europe and the 6.A, it was previously rare in (apan. However, the number of 

tinea patients caused by*>/*-%,&($+,& has been increasing in judo and wrestling 

teams since 2001 in (apan. There has been no report on the spread of >/*-%,&($+,& 

infection among sumo wrestlers, though both judo and sumo are (apanese traditional 

sports. ae had five patients with tinea corporis and two patients with tinea capitis 

caused by >/*-%,&($+,& among judo and sumo wrestlers. The purpose of this study is 

to examine the epidemic of >/*-%,&($+,& infection among judo and sumo teams in out 

city and to taBe preventative measures against the spread of >/*-%,&($+,& infection. In 

our 7 patients, patients 1-J belonged to high school judo teams and patients O-7 were 

sumo team members. Tinea had been spreading on two judo teams in our city since 

April, 200J and fctober, 200J. Patients 6 and 7 noticed the sBin eruption in February, 

200O, after they practiced sumo with patient 1 who was a member of the judo team. 

.ubsequently, patients O and [ who practiced sumo with patients 6 and 7 noticed sBin 

eruptions. Their sBin eruptions were hair loss, erythema and papules on face, auricle, 

upper extremities, axilla and trunB, but they had no eruption on the lower half of the 

body including feet and toe nails. .Bin and/or hair samples from all the patients were 

examined by microscopic examination with 10_ lfH and cultured in .abouraud]s 

dextrose agar. Furthermore, PCR-RF/P analysis was performed on O fungal strains 

from patients 1-O. From these examinations,*>/*-%,&($+,& was identified in all 7 

patients. They noticed that the teammates and/or competitors in other teams also had 

similar sBin eruptions but not their families. Although oral administration and topical 

treatments with antifugal agents were effective, sBin eruption recurred in 2 patients. 

ae consider that sumo team members were infected with*>/*-%,&($+,& by a judo team 

member and that*>/*-%,&($+,& was spread to sumo and judo wrestlers through body 

contact each other. ahen we visited the training rooms for sumo and judo and 

examined the hygiene conditions, we found that the wrestlers and coaches did not 

have enough information and interest to pay attention to the condition of their sBin. 

Namely, they had insufficient showering facilities and they wore training gear that had 

been unwashed for several months. ae explained the spread of the*>/*-%,&($+,& 

epidemic among body contact sports to the team members. In addition, we 

recommended that the wrestlers should adhere to the followingM 1) checBing the sBin 

before and after practice and matches, 2) showering after practice and matches, J) 

washing the training gear after practice and matches. Afterwards, we have had no 

patients with tinea caused by >/*-%,&($+,&. 

P-0326. Candiduria in intensive care unit Sfax - Tunisia 

Sellami A 1 , .ellami H 1 , MaBni F 1 , lhlif M 1 , 2ahloul M 2 , CheiBh-rouhou F 1 , 2ouaziz M 2 , 

Ayadi A 1 

1 /aboratory of Parasitology-Mycology - 6niversity Hospital - .fax - T6NI.IA, 2 Medical 

Reanimation aard - 6niversity Hospital - .fax - T6NI.IA 

Introduction: Candiduria is increasingly frequent among patients admitted to 

intensive care units but its significance remains unclear. 

Objectives: search for eventual correlation between quantitative candiduria and 

Bnown risB factors for invasive candidiasis. 

Patients and methods: we conducted a four-month prospective study of 162 patients 

hospitalized in the intensive care unit for more than 72 hours. All patients underwent a 

weeBly research of candiduria added to sampling from different body sites to 

determine the Pittet 7+,5"5+ colonization index. 

Results: Candiduria has been proved in [6 cases (JO_). It was superior or equal to 

10 O ufc/ml among 28 patients ([0_). 7+,5"5+*-$%'"#+6"&3 7+,5"5+*@6+8$+-+ and 

7+,5"5+*+68"#+,& has been isolated in O1_, 22_ and 20_ respectively. All patients 

had at least one major and two minor risB factors for 7+,5"5+ infection. .ix patients 

(10_) developed invasive candidiasis. The global mortality rate was at [2_. Pittet 

colonization index was significatively different between patients with candiduria and 

those with invasive candidiasis (p}0.01). There was a statistically significant 

correlation between candiduria , 10 O ufc/ml and Pittet colonization index % 0.[ 

(p}0.01). 

Conclusion: The control of 7+,5"5+ colonisation assessed by systematic screening 

helps predicting subsequent infections. fur preliminary observations suggest that 

Candiduria , 10 O ufc/ml was significantly correlated to fungal colonisation. Thus, 

quantification of candiduria could be useful to select critically ill patients at high risB for 

invasive candidiasis and offer opportunity for intervention strategies. 


P-0327. Prevalence of vulvovaginal candidiasis in women inmates 

Semedo I, Pereira (, /opes M, Freitas G 

6niversity of /isbon-F. Pharmacie, /isbon, Portugal 

Objectives: The purpose of this study was to determine the vulvovaginal candidiasis 

(VVC) occurrence in a risB specific population, as well as the 7+,5"5+*species 

distribution and the prevalence of antifungal resistance. 

Material and Methods: Vaginal yeast cultures were collected from 211 vulvovaginitis 

clinic patients with signs and symptoms suggestive of vulvovaginal candidiasis, during 

1KK6-1KKK. All specimens were plated on CHRfMagar to ensure detection of mixed 

infections. The identification of 7+,5"5+*+68"#+,& (7. +68"#+,&) was made by 

germ-tube formation, and the other yeasts were identified by Api Candida w 

.ystem. .usceptibility testing was performed by Fungitest TM method. 

Results and conclusions: The overall occurrence of VVC was [1.2_ and 7+,5"5+ 

species were represented by 7/*+68"#+,&*(6[.7_), 7/*@6+8$+-+*(20.O_), 7/*[$(&." 

(6.[_), 7/*'+$+'&"6%&"&*(O.6_), 7/*U+)+-+*(1.K_) and 7/*[.U2$*(0.K_). 

fn what concerns antifungal susceptibility, the results revealed that resistance to 

itraconazole occurred among K.0_ of all isolates, and to fluconazole was infrequent 

(1.8_). The imidazoles, miconazole and Betoconazole, were active against K0.0_ of 

all isolates. 

P-0328. Candidemia and antifungal therapy in a French university hospital. 

Rough trends over a decade and possible links 

Sendid B 1,2 , Cotteau A J , FranÑois N 2 , Daveloese A J , .tandaert-Vitse A 1 , Camus D 2 , 

Poulain D 1,2 

1 Inserm 67KK, 2 /aboratoire de Parasitologie-Mycologie, CHR6 /ille, J Pharmacie 

Centrale, [K0J7 /I//E 

Evidence for increased morbidity of candidemia and for high associated mortality 

gained in the K0]s made the last decade particularly rich in different recommendations 

concerning the management of at risB patients as well as a period during which 

antifungal diversity and prescription has considerably increased. fur clinical mycology 

laboratory centralise isolation and identification of yeast grown from blood cultures 

drawn in a JJ00 bed hospital. fver the 1KKJ-200J period, OJ0 blood isolates were 

identified with a clear trend for a decreasing number of isolations during the period 

1KK[-2000 among which was an increased prevalence of 7/*@6+8$+-+. ahen this 

evolution was compared to the amount and the nature of antifungal prescribed during 

the same period (annual DDD mean 26627O1) a balance was specifically evidenced 

between the number and nature of yeast isolated and the prescription of Fluconazole 

at prophylactic dosis. Although further detailed analysis will follow, we intend to briefly 

and preliminary report the impressive character of this temporal and unexpected 

correlation gained through compilation of significant amount of information. 

fur results confirm the rising importance of non-+68"#+,&*species as etiologic agents 

of VVC, They support the use of azoles for empirical therapy when the VCC is caused 

by 7/*+68"#+,&, but limit their use in the 7+,5"5+ non-+68"#+,& infections/

P-0329. Infections of the eye by Scedosporium species 

Sheorey H 1 , Das . 2 , (oseph C 1 , aaters M 1 , .ullivan / 2 

1 .t Vincentes Hospital, Melbourne, Australia, 2 Royal Victorian Eye c Ear Hospital, 

Melbourne, Australia 

The incidence of fungal eye infections has increased in recent years. This may be the 

result of the introduction of modern therapeutic measures such as the widespread use 

of broad spectrum antibiotic, immunosuppressive drugs including corticosteroids, 

aggressive surgery, and the improvement of laboratory diagnostic techniques. Fungal 

infections of the eye and it]s appendages are uncommon and usually follow trauma to 

the eye. Although Beratitis is the most frequent presentation, the orbit, lids, lacrimal 

apparatus, conjunctiva, sclera and intraocular structures may be involved. 

* 

4&'.$@"66(& species and 7+,5"5+ species have been the most common fungi isolated 

from the eye specimen in our laboratory. However a number of other uncommon 

moulds have been isolated from eye specimen more recently, including both species 

of =#.5%&'%$"() G=/*+'"%&'.$)() and =/*'$%6"U"#+,&J. =#.5%&'%$"() species are 

filamentous fungi found in soil, sewage, manure and polluted water. They cause 

serious infections in the immunocompromised host and are resistant to a number of 

antifungal agents and are often difficult to treat. fcular infection may lead to severe 

vision threatening complications and are often associated with chronic scleral defects 

secondary to previous surgery and local radiation. 

A review of cases with =#.5%&'%$"() species eye infections over the last decade will 

be presented. Areas covered in the review will include predisposing factors, medical 

treatment, surgical debridement and final outcome. Identification of these species in 

the laboratory will be outlined. 

P-0330. Candidemia in patients with ventricular assist devices 

Shoham S 1 , .haffer R 1 , .weet / 2 , CooBe R J , Donegan N O , 2oyce . 2 

1 .ection of Infectious Diseases, aashington Hospital Center, aashington, DC, 6.A, 

2 Department of Cardiovascular .urgery, aashington Hospital Center, aashington, 

DC, 6.A, J Department of Cardiology, aashington Hospital Center, aashington, DC, 

6.A, O Department of Infection Control, aashington Hospital Center, aashington, DC, 

6.A 

Purpose: Ventricular assist devices (VAD) are increasingly employed for 

management of advanced heart failure. Nosocomial infections including candidemia 

are a major problem among VAD recipients. To date, candidemia has not been well 

defined in this population. ae sought to better characterize the epidemiology, 

microbiology, and outcomes of VAD associated candidemia. 

Summary: This was a retrospective case control study. All culture proven cases of 

VAD associated candidemia occurring at our institution from (anuary 1, 1KK8 through 

December 1, 200O were evaluated. For each case two non-candidemic adult VAD 

recipients were chosen as controls. Medical records were abstracted for 

demographics, associated conditions, and management of the devices. Crude 

mortality was assessed at the end of hospitalization. 

Results and conclusions: During the study 118 VAD recipients were identified. ff 

those 7 (6_) developed candidemia. Median VAD duration prior to infection was 2[ 

days (range, 11-72 days). All fungal 2.I were due to 7/*'+$+'&"6%&"& (71_) and 7/* 

+68"#+,& (2K_). fverall mortality for VAD associated candidemia was 71_. Three of 

the [ (60_) 7/*'+$+'&"6%&"& 2.I were cured, two with retention of the device. 7+,5"5+ 

colonization occurred in [/7 cases and 7/1O controls, but was not significantly 

associated with development of bloodstream infection sfR 2.1O (K[_ CI 0.21-2O.OO)u. 

In patients with candidemia the colonizing species did not reliably correlate with the 

infecting species. In conclusion, our experience suggests that candidemia in VAD 

patients is principally caused by 7/*'+$+'&"6%&"& and is associated with high mortality. 

In selected cases this infection may be treated successfully without removal of the 

device. 


P-0331. Localized infection of the otitis externa due to paecilomyces 

marquandii : case report, in vitro sensitivity and serological evaluation 

Singh S 1 , Naidu ( 2 , (ha M 2 

1 Deptt. of 2iological .ciences.RDVV,(A2A/P6R.MP. INDIA, 2 Deptt. of Zoology c 

2iotechnology. Govt. Autonomous .cience College,(A2A/P6R.MP. INDIA. 

A 1O year old girl presented with a complaint of pain, itching and localized sensation of 

her left ear since last 1[ days. Examination of fungal debris obtained from the external 

auditory meatus of her left ear in PHf/ prepration revealed the presence of thin 

walled, hyaline, septate branched mycelium. 1+.#"6%)2#.&*)+$e(+,5""*was recovered 

on .abouraud]s dextrose agar. In vitro susceptibility test revealed that 1+.#"6%)2#.&* 

)+$e(+,5""*was resistant to fluconazole and Betoconazole. However, it was slightly 

susceptible to itraconazole. Exoantigen of 1+.#"6%)2#.&*)+$e(+,5""*induced the 

formation of specific antibodies in test rabbits. It did not show cross reactivity. 

Exoantigen of 1+.#"6%)2#.&*)+$e(+,5""*was found to be glycoprotein and gave 

specific band of 2K lD. ae believe that it could be used in the serological diagnosis of 

infection caused by 1+.#"6%)2#.&*)+$e(+,5""/ 

P-0332. Successful treatment of disseminated aspergillosis, involving the brain, 

lung and soft tissues, with combination of caspofungin plus voriconazole 

Skiada A 1 , PanagiotaBopoulou l 2 , DouBas E 2 , Mitroussia-Ziouva A 1 , DaiBos G 1 , 

PetriBBos G 1 

1 Research /aboratory for Infectious Diseases and Antimicrobial Chemotherapy 

ÄG.l.DaiBosÄ, 6niversity of Athens, Greece, 2 IatriBo Medical Center, Athens, Greece. 

Purpose: To present an interesting case of disseminated aspergillosis, involving the 

brain,that was treated successfully with combination therapy. 

Case Report: A 2K-year-old white male was admitted to the hospital because of 

relapse of autoimmune haemolytic anaemia and low grade fever. The patient had a 

past medical history of HodgBin]s lymphoma, treated three years ago with 

chemotherapy and autologous bone marrow transplantation. fne month before 

admission haemolytic anemia was diagnosed and he received high doses of 

corticosteroids. fn admission, a nodular consolidation on the left lung was noted on x- 

ray. A CT-scan revealed an abscess on the left lung and bilateral multiple nodular 

lesions. .putum cultures were performed and 4&'.$@"66(&*U6+0(& was isolated. The 

patient was started on caspofungin, [0mg daily, but a repeat CT-scan, three weeBs 

later, revealed a soft tissue mass in the left pectoralis major muscle, communicating 

with the abscess in the lung, while the previous lesions were unchanged. In addition, a 

brain CT-scan showed multiple nodular lesions in the hemispheres and the 

cerebellum. Three months after admission a left upper lobectomy was performed. The 

histology of the excised tissue showed hyphae of Aspergillus. Voriconazole was 

added to caspofungin. The patient received therapy for a total of one year. He has 

now been in remission and free of aspergillosis for two years. 

Conclusions: Although disseminated aspergillosis, especially when it involves the 

central nervous system, is usually fatal, it can on occasion be treated successfully with 

long-term antifungal therapy, which in this case was caspofungin plus voriconazole.

P-0333. Determination of the esterase activity among the Candida strains 

isolated from various clinic samples 

Soydan S, Asci Toraman Z, 2ulut Y 

1Department of Microbiology and Clinical Microbiology, Faculty of Medicine, Firat 

6niversity, Elazig, TurBey 

Candidas are the yeast which can be found free in nature or in the various microbial 

flora of human and mammalian animals. In recent years, the patients], with cancer, 

transplant and receiving immunosuppressive therapy, life expectancy has been 

increased due to developed therapy protocols. The increasing isolation rates of nonalbicans 

strains from the yeast infections, caused to augment of the medical 

importance of the virulence factors of these strains. Virulence factors of Candida 

contribute to develop infection as much as the host]s factors. 

In this study, species determinations were performed of a total of 100 Candida strains 

and esterase activity according to species were investigated. ff the total 100 Candida 

strains, [8 were found as 7+,5"5+*+68"#+,&, 1O were 7/*@6+8$+-+, 11 were 7/*-$%'"#+6"&, 

five were 7/*-$%'"#+6"&, four were 7/U+)+-+, four were 7/*&+[., three were 7/*[$(&.", 

two were 7/*@(".66.$)%,5"", one was 7/*6(&"-+,"+., one was 7/**'.66(#(6%&+ and one 

was 7/*#($0+-+. 

The esterase activity was investigated in Tween-80 agar. In the inoculation arean 

collection of the translucent opaque crystals or emerging opaque zones were 

indicated that the esterase production. Esterase production rate according to species 

weren KJ.1 _ for 7/*+68"#+,&, 100 _ for 7/*-$%'"#+6"&, 7.1 _ for 7/*@6+8$+-+, 60 _ for 7/* 

U+)+-+, [0 _ 7/*@("66.$)%,5"" and 100 _ 7/*#$(0+-+. There was no esterase activity in 

the other species. The distribution of the esterase activity according to clinic samples 

weren 100 _ for stool, nasopharynx, sputum and woundn 7J.K for vaginal discharge, 

66.6 _ for blood and [J.J _ for urine. 

Among the Candida strains that were included in to the study, esterase production 

rates, as the virulence factors of Candida species, were found in moderate to high 

levels and in the case of Candida infections, investigation of the production of these 

factors will contribute to planning the therapy. 

P-0334. Thielavia sepedonium isolated from an HIV patient with pneumonia 

Stolhand M 1 , .utton D 2 , Gundlapalli A J , Hansen M 1 , Thompson E 2 , Rinaldi M 2 , 

Pombo D 1, J 

1 Microbiology Dept., /D. Hospital, .alt /aBe City, 6tah, 6.A., 2 Fungus Testing 

/aboratory 6niversity of Texas, .an Antonio, Texas, 6.A., J Div. of Infectious 

Diseases, 6niversity of 6tah, .alt /aBe City, 6tah, 6.A. 

Summary: ae isolated a fungus from the broncho-alveolar lavage (2A/) fluid from a male 

patient with advanced HIV disease and right lower lobe pneumonia. Culture revealed a 

mould with conidia consistent with .epedonium and cleistothecia and ascospores 

consistent with Thielavia. This finding is consistent with in vivo recovery of the ascomycete 

>?".6+0"+*&.'.5%,"() as an opportunistic pathogen in an AID. patient with pneumonia. 

Case Report: A 27-year-old homeless Caucasian male, who had been diagnosed with HIV 

J years prior, presented to clinic with a 2 weeB history of non-productive cough associated 

with pleuritic chest pain, without hemoptysis. He also complained of watery diarrhea of one 

weeB]s duration and a 20-pound weight loss over several months. He was on no 

medications and had no history of treatment with anti-retroviral drugs. Abnormalities on 

exam were tachycardia, hypoxemia, cachexia and decreased breath sounds over the right 

lower lung fields, without cracBles noted. Chest radiograph revealed patchy right lower 

lobe opacification involving the posterior basal segment. The patient was admitted and 

begun on i.v. ceftriaxone and doxycycline and trimethoprim/sulfamethoxazole. Results of 

routine hematology and chemistry tests, including /DH, were in the normal rangesn two 

sets of blood cultures were drawn and remained negative. A CD O T cell count by flow 

cytometry revealed [J cells/microliter. 

A sputum sample was induced for routine culture, however the specimens were 

unsatisfactory for culture and so 2A/ was performed on the third hospital day, due to 

failure to improve clinically. Cytology exam noted an excellent alveolar specimen with 

minimal inflammation or oral contaminants. 2A/ fluid results for Pneumocystis DFA stain, 

AF2 stain and culture, /egionella PCR and culture and routine bacterial culture were 

negative. Non-concentrated 2A/ fluid was cultured for fungi on 2HI agar and IMA plates, 

and grew occasional Candida albicans. The patient]s antibiotics were changed to p.o. 

levofloxaxin and he was discharged on the third hospital day with a diagnosis of 

unspecified pneumonia. 

A mould form was isolated from a concentrated specimen plated on /( media, and was 

sub-cultured to sheep blood and potato flaBe agar. A cellophane slide prep using lactophenol 

cotton blue revealed hyaline septate hyphae with large, smooth to rough, thicBwalled 

conidia. The isolate was identified as >?".6+0"+*&.'.5%,"() at the Fungus Testing 

/aboratory (6TH.C 0[-[1) based upon golden yellow colonies on PDA, globose 

tuberculate conidia of the anamorph =.'.5%,"() (10km diam), and characteristic darB 

globose cleistothecia (72-1O0 km diam) and darB brown, ellipsoidal ascospores with apical 

germ pores (1O-1K x 7-10 km) of the teleomorph >?".6+0"+/ 

The patient was discharged and recovered without specific antifungal therapy and was later 

followed in the HIV clinic. 

Conclusion: >?".6+0"+*&.'.5%,"() was isolated from a patient with pneumonia and 

advanced HIV. This organism is generally non-pathogenic in humans but may be a cause 

of pneumonia in patients with severe immunodeficiency. Differentiation from A"&-%'6+&)+* 

#+'&(6+-() is important due to similar conidia. 


P-0335. Isolation and identification of human pathogenic Pythium insidiosum in 

agricultural areas of Thailand 

Supaphan J, VanittanaBom N 

Chiang Mai 6niversity, Chiang Mai, Thailand 

Purpose of the studyM The pathogenic oomycete 12-?"()*",&"5"%&()*is a causative 

agent of pythiosis, a life-threatening infection in animals and humans. Human 

pythiosis has high morbidity. The disease is generally acquired through zoospore 

attachment, while standing in swampy areas or fields. However, natural habitats and 

the incident rate of 1/*",&"5"%&() have not been determined. .ince approximately 

ninety percent of patients with this disease have been farmers, irrigation water is 

suspected to be an important source of infection. 

Description: In this study, J2[ water samples from K2 sources of rice irrigation, 

irrigation channels and reservoirs were collected and analyzed for 1/*",&"5"%&()/ 

Zoospores were trapped from the samples by a simple baiting technique using sterile 

human hair. ahite-cream glabrous colonies that grew on the selective agar plates 

were picBed and morphologies were observed by microscopic examination. 

.uspected cultures were identified by genus-specific PCR assay and analyzed by IT. 

sequencing. 

Results and conclusions: fne hundred and forty five isolates showed broad, 

sparsely septate hyaline hyphae with perpendicular branching. All isolates were 

subcultured to obtain a pure colony and then grown on .DA pH 6.K to provide mycelia 

for DNA extraction and PCR assay. .ixty-one isolates showed a 2JJ-bp amplicon, the 

expected size. All 61 isolates were sequenced. Fifty-nine of sixty-one sequences had 

KK_ identity to other 1/*",&"5"%&()/*fne isolate was related to 1/*#?%,5$"#%6+*and 

another to X/*@"@+,-.()/*The environmental 1/*",&"5"%&()*isolates were found in all 

four types of irrigation water sources/*Twenty-six water sources were positive/ The 

incidence rate of 1/*",&5"%&()*in irrigation water sources was approximately 28 

percent. 

P-0336. Tinea profunda cysticum due to Trichophyton rubrum 

Suzuki Y 1 , Hashizume H 2 , MaBimura l J , TaBigawa M 2 

1 Division of Dermatology, .hizuoBa City .hizuoBa Hospital, .hizuoBa, (apan, 

2 Department of Dermatology, Hamamatsu 6niversity .chool of Medicine, Hamamatsu, 

(apan, J Institute of Medical Mycology, TeiByo 6niversity, ToByo, (apan 

Purpose of the study: >$"#?%'?2-%,*$(8$()*is a common superficial Beratinophilic 

dermatophyte. However, in immunocompromised host, deep local invasion, 

multivisceral dissemination, or even fatal outcome has been reported. lobayashi .-*+6* 

1 coin the diagnostic name of htinea profunda cysticumi for their case of tinea profunda 

manifesting with remarBable cystic eruptions. ae report another case of multiple 

subcutaneous cystic dermatophytosis due to >/*$(8$()*in a patient with nephrotic 

syndrome. 

Description: A 28-year-old man was referred to us for evaluation of his sBin eruptions 

and high fever. For the past 18 years, he had been treated with systemic prednisolon 

because of nephrotic syndrome. Two weeBs before, he had high fever and developed 

multiple subcutaneous nodules over his body with cellulitis on the right lower leg. 

There were more than [0 non-tender, cold nodules with fluctuation, ranging from1 to [ 

cm in diameter on his bacB, abdomen, jaw, legs, and forearms. fnychomycosis was 

present on the feet and hands. Cellulitis of the right leg and high fever was improved 

with antibiotics. 2iopsy of the abdominal nodule showed the presence of hyphal 

elements in the deeply seated pseudocyst. >/*$(8$()*was cultured from both the 

seropurulent material aspirated from cyst and the biopsy specimen. The strain was 

confirmed to be >/*$(8$() by sequence analysis of ribosomal DNA extracted from the 

fungus. The patient was treated with itraconazole until complete disappearance of the 

nodules and healing of the onychomycosis. 

Conclusion: The simultaneous emergence of multiple lesions at different sites and no 

evidence of direct invasion from the surface in the histology suggest that the rare 

manifestation of this case was caused by hematogeneous dissemination of the 

pathogen. New diagnostic name, htinea profunda cysticumi is appropriate to our case 

of deep dermatophytoses manifesting with multiple hyalohyphomycotic cysts. 

References: 

1. lobayashi M et alM ( Am Acad Dermatol [OM.11-J,2006. 

De Hoog G. et alM Atlas of Clinical Fungi 2nd ed, Centraalbureau voor 

.cimmelcultures, Netherlands, p.2J,2000

P-0337. Real-time pcr identification of Malassezia spp. and measurement of 

malassezia-specific serum ige antibody in antimycotic-treated atopic dermatitis 

patients 

Tajima M 1 , .ugita T 2 , Ito T 1 , NishiBawa A 2 , Tsuboi R 1 

1 ToByo Medical 6niversity, ToByo, (apan, 2 Meiji Pharmaceutical 6niversity, ToByo, 

(apan 

Malassezia species, normally colonizing the sBin surface of healthy individuals, have 

been involved as either the cause or exacerbating factor in a number of sBin 

conditions including atopic dermatitis (AD). 6sing a non-cultural PCR method, we 

identified three new Malassezia spp. among the nine species detected in human hosts. 

The present investigation was conducted to measure the change in the numbers of 

Malassezia organisms and titers of Malassezia-specific IgE antibodies before and 

after the topical treatment of intractable AD patients using Betoconazole 2_ cream. 

Twelve moderate or severe adult AD patients with face and necB lesions were 

enrolled in this study. Any concurrent treatment for AD involving topical steroids and 

tacrolimus was continued. Fungal DNA was extracted directly from the samples 

collected by tape stripping the surface of the lesional sBin. ae analyzed the quantity of 

Malassezia spp by real-time PCR"A2I 7[00#assay, for which we prepared TaqMan 

probes designed from Malassezia 26. rRNA. M. globosa and M. restricta-specific IgE 

antibodies were originally prepared as an Ala-.TAT system. As a result, the quantity 

of Malassezia spp in all of the samples decreased after topical application of the 

antimycotic, and 70_ of the AD patients showed clinical improvement in erythema 

and scaling. The serum levels of total IgE antibodies, M. globosa and M. restrictaspecific 

IgE antibodies, decreased as well. These results indicate that removal of the 

organisms by antimycotic agent results in an improvement in the clinical 

manifestations of intractable AD and suggests that Malassezia spp. constitute one of 

the exacerbating factors of AD. 

P-0338. Non-neoformans Cryptococcus spp. isolated from clinical and 

environmental sources in Malaysia 

Tay k, Tajudin T, Na . 

Department of Medical Microbiology, Faculty of Medicine, 6niversity of Malaya, luala 

/umpur 

PCR fingerprinting using minisatellite specific primer M1J was used in this study to 

characterize non-neoformans 7$2'-%#%##(& spp. that were isolated from clinical and 

environmental sources in Malaysia. The clinical isolates included three 7$2'-%#%##(&* 

6+($.,-"" strains which were identified by API 20C A6j test Bit. The environmental 

isolates included J2 7/*6+($.,-"" and 1O 7/*+68"5(& strains. A total of 22 7/*,.%U%$)+,& 

isolated from bird droppings were also included in the analysis as a comparison. 

Cluster analysis of the cryptococcal isolates was performed with the unweighted-pairgroup 

method using arithmetic averages and calculation of the similarity matrix was 

done by the Pearson product-moment coorelation coefficient method. Clonal 

relatedness of the isolates were indicated by a similarity index of %80_. The resulted 

dendrogram revealed the distribution of these cryptococcal isolates into J clusters. 7/* 

6+($.,-"" and 7/*+68"5(& isolates were separated into 12 subtypes, demonstrating intra 

and interspecies genetic heterogeneity among the isolates. The clinical isolates were 

identified as three different subtypes, with two sharing % K6.[_ of similarity with the 

environmental isolates of 7/*6+($.,-"" and 7/*+68"5(&. Determination of the IT. gene 

sequences for these clinical isolates showed highest sequence similarity with 7/* 

6+($.,-"", 7/*+68"5%&")"6"&*and 7/*)+@,(& strains in the Gen2anB. This study supported 

the findings that non-neoformans cryptococcal is a genetic diverse group. An accurate 

and rapid method is needed to identify these species. 


P-0339. Meningitis due to Cladophialophora bantiana in a patient without 

immunodeficiency 

Thibout Y 1 , Persat F 2 , Piens M 2 , Fassier T J , Durand P O , Gudrin ( [ 

1 .ervice de Pneumologie, HÇpital de la Croix Rousse, /yon, France, 2 .ervice de 

Parasitologie-Mycologie mddicale, HÇpital Edouard Herriot, /yon, France, J .ervice de 

Rdanimation Mddicale et d]Assistance Respiratoire, HÇpital de la Croix Rousse, /yon, 

France, O 6nitd de .oins Intensifs Post-fpdratoires, HÇpital Pierre aertheimer, 2ron, 

France 

Purpose: ae report a case of 76+5%'?"+6%'?%$+*8+,-"+,+ meningitis without cerebral 

abscess in a non-immunocompromised patient. 

Clinical case: A [6 year-old male patient was referred to our institution on (anuary 

11 th 2006 for progressive mental confusion and ataxia during the last two months. He 

presented both severe obstructive chronic respiratory insufficiency and non small cell 

lung cancer in complete remission since 200J. No immunodeficiency was identified. 

He has never been travelling outside the European 6nion. 

The CT brain scan showed a triventricular hydrocephalus and the brain MRI a 

ventriculitis of the right ventricle with occipital oedema without sign of intracerebral 

hypertension. Cerebrospinal fluid (C.F) examination done twice showed a profile of 

meningitis without detection of virus or bacteria by direct examination, culture or PCR. 

Two weeBs after mild improvement with corticosteroids and empiric antibiotherapy, the 

patient deteriorated and underwent neurosurgical exploration (February, 1 st ). fn the 

first C.F sent to the mycology service, brown elements were macroscopically 

observed. Microscopically,*hyphae with brown cell wall were detected after direct 

examination, without staining, in both periventricular biopsy and C.F. 

76+5%'?"+6%'?%$+*8+,-"+,+ grew on .abouraud medium and identification was 

confirmed by the hCentre National Mycoses et Antifongiquesi (Institut Pasteur, Paris, 

France). No brain abscess was found by MRI and surgery. Voriconazole O00mg x 

2/day during J days and then 200mg x 2/day was started. After clinical worsening, the 

patient was transferred to intensive care unit for invasive mechanical ventilation. 

fne weeB after the voriconazole onset, a combination with [-fluorocytosine 

1[0mg/Bg/day was initiated. An external ventricular derivation was performed and 

purulent C.F was still positive for 76+5%'?"+6%'?%$+*8+,-"+,+. Hydrocephalus 

persisted and two additional external ventricular derivations were performed. Three 

weeBs after voriconazole onset,*76+5%'?"+6%'?%$+*8+,-"+,+ was still detected in the 

C.F and the patient remained in a critical state. 

Conclusion: This new case of meningitis due to 76+5%'?"+6%'?%$+*8+,-"+,+*leads to 

several questions. First, the patient had no history of immunodeficiency and had never 

been abroad. .econd, the meningitis was not associated with cerebral abscess, as 

usually described in the literature. Third, the use of active drugs in vitro was 

associated with poor clinical outcome. 

Then, mycologic examination of the C.F must be done in case of unexplained 

meningitis, even in non-immunocompromised patients and the antifungal treatment 

optimization needs to be defined in this rare but life-threatening disease. 

P-0340. Diving suit: sterile, ambulatory, personal device for protective and 

preventive isolation of nosocomial invasive fungal infections (IFI) in 

neutropenic patients hospitalized in laminar air flow room who need a 

disruption of isolation 

Thiebaut A 1 , Perraud M 1 , /yonnet D 1 , fzil . 2 

1 HÇpital E. Herriot, /yon, France, 2 Delta Protection 

Background: Patients with long neutropenia present high risB to develop invasive 

pulmonary aspergillosis (API) of poor prognosis. Pulmonary computed tomographic 

.can (CT scan) allows earlier diagnosis of API and improve management in these 

patients (D. Caillot, (Cf, 1KK7). However, CT scan need an isolation breaBing which 

is potentially dangerous. ae propose a diving suit to maintain the preventive isolation. 

Material and Methods: The diving suit is sterile, personnal, ambulatory and 

transparent to permit monitoring, visual and conversational contact. It is supplied with 

air with a self-contained station of ventilation (12 hours). The Air contamination is 

controlled with 2 HEPA filters. Fithteen patients treated for hematologic malignancies 

liBe acute leuBaemia have tested it after giving inform consent. 

Results: The diving suit has been validated for air contamination, physiological (Cf2), 

CT scan feasibility and patients acceptation. All patients ([ male, 10 females) 

presented a severe neutropenia (PMN v 0.[ G/l during more than 10 days) at time of 

CT scan. Three patients have had already pulmonary CT scan before using diving suit 

and 2 claimed to be claustrophobic. No patient describe dyspnea, either pain nor 

discomfort. All patients feelt reassured and agreed for a new CT scan with this 

clothing. Two patients have had another CT scan with diving suit for suspicion of 

pulmonary aspergillosis. Diving suit was very easy to manipulate and did not disrupt 

monitoring or treatments administration. Diving suit was also compatible with Doppler 

and Radio Magnetic Nuclear. 

For one of these patients with fever resistant to antibacteriologic treatment, J thoracic 

CT scan were performed (1 per weeB) without any sign of API. The fourth CT scan 

allowed the diagnosis of API with halo sign. This diagnosis has been demonstrated by 

histology after surgery. In this case report, contamination has not been possible during 

first CT scan because of the diving suit, this information is very important to explain to 

the patient and his family. 

Conclusion: Pulmonary CT scan needs very often to be performed in neutropenic 

patients to assess API diagnosis. An ambulatory, personnal protective clothing allows 

no disruption of isolation for immunocompromized patients during a CT scan. It can 

also be proposed for medical staff protection when treating patients with .AR. or 

other highly contagious agents.

P-0341. Histoplasmosis related to central nervous system bypass shunt 

Tiraboschi I 1 , Arechaval A 2 , Gorostiaga ( J , GarbervetsBy / O , del Castillo M [ , Negroni 

R 2 

1 Hospital de ClÜnicasÄ(osd de .an MartÜnÄ, 2uenos Aires, 2 Hospital ÄF.(.MuizÄ, 

2uenos Aires, J Hospital ÄAngel PadillaÄ, TucumZn, O PoliclÜnico 2ancario, 2uenos Aires, 

[ Instituto Fleni, 2uenos Aires 

Histoplasmosis is an endemic fungal infection, particularly found in America]s warm 

prairies. Commonly, there is mucocutaneous, pulmonar and reticuloendothelial 

system involvement. 

This worB is intended to report 8 patients with A/*#+'&(6+-() infection associated to 

central nervous system bypass shunt. 

Method: All clinical records from patients diagnosed with shunt-related histoplasmosis 

were revised. Clinical, diagnostic and demographic data were collected. 

Results: .ix out of eight patients were male, averaging O6 years old (26 to 66 years 

old). Three of them came from the province of TucumZn and four from 2uenos Aires, 

regions where histoplasmosis is endemic. 

All patients were HIV-negative. They had history of hydrocephaly with bypass shunt 

as associated treatment and had required more than one bypass replacement due to 

shunt obstruction ([ out of 8 with multiple bypass system replacements). 

Five out of eight patients showed other organ involvement (adrenal glands, mucosa 

and lungs), but all these patients first presented neurological symptoms which led 

them to consultation. 

The diagnosis of shunt-related histoplasmosis was made with C.F testn finding 

A/#+'&(6+-() in direct examination in 2 out of 8 patients and it grew in the cultures of 

6 out of 8 patients, (in two patients , the culture was only positive when the shunt was 

removed). The diagnosis was made in two out of eight patients only by C.F antibody 

testing. 

Conclusion: In endemic regions, histoplasmosis has to be considered as a bypass 

shunt related infection. In order to maBe a diagnosis, it is necessary to study the C.F, 

with direct examination, culture and A/#+'&(6+-() antibody search. 

P-0342. Lung histoplasmosis in an immunocompetent man after 45 years of 

infection 

Torres-Rodríguez J 1 , .egura-Roca G, Coll-Daroca (, Alvarado-RamÜrez E 

1 Institut Municipal deInvestigaciá Mmdica. 2arcelona, 2 Hospital del Mar, Autonomous 

6niversity of 2arcelona 

ae report a case of a lung reactivation of a latent histoplasmosis in a 68 years old patient 

without immunologic dysfunction living in 2arcelona (.pain). 

The Histoplasma infection was probably acquired in a previous stay in Gabon and 

Equatorial Guinea J6 years before. 

Case description: In 1K[7 this patient worBed in a forest exploitation living during 8 years 

in an installation sited in the native jungle. During this period he presented two episodes of 

malaria and one of filariasis. In 1K[K he was diagnosed of pulmonary tuberculosis because 

prolonged fever and Rj alterations. An antibiotic treatment with antituberculous drugs was 

done during several months. Meanwhile this treatment the patient made a travel to Mexico 

living in Mexico DF and Acapulco during six mothsn no impairment, was produced during 

this period. He come bacB to Africa without presenting any health disturb. 

In 1K66 he return to 2arcelona, and no more travel outside of Europe was done until now. 

In 200J he presents cough with hemoptysic sputum without any other symptomatology. A 

thoracic Rj showed an infiltrative and cavities lesions in the right middle lung lobe 

confirmed with lung scanner. He was submitted to a lung puncture biopsy. 

Histopathology showed fibrotic reaction, Giant cells and some intracellular yeast 

compatible with A"&-%'6+&)+*#+'&(6+-()/*Cultures were not performed. 

.erological tests (Immunodifussion) using A"&-%'6+&)+*#+'&(6+-()*antigens showed 

several precipitating bands and intradermal test with histoplasmin produced an intense 

delayed reaction. 

No immunodepression or subjacent malignant diseases were found. 

Diagnosis of chronic lung histoplasmosis was done and oral treatment with Itraconazole 

O00 mg during J moths was indicate. Hemoptysis disappeared completely and a new Rj 

showed a marBed improvement of the lung lesions. 

After two years of follow up the Immunodifussion is still positive against A"&-%'6+&)+* 

antigen, but lower bands are observed. 

Conclusions: Instead A"&-%'6+&)+*isolation was not obtained, pathological findings and 

positive immune specific reactions against this fungus, as well the therapeutic response, 

leading to the diagnosis of Histoplasmosis. 

The epidemiological antecedents suggest the acquisition of this infection meanwhile the 

permanence in African forest or with fewer probabilities during the Mexico travel. 

The more remarBable data of this case is the extremely long latency period of this mycosis. 


P-0343. Keratoderma plantare sulcatum (Castellani, 1910) by Dermatophilus 

congolensis 

Towersey / 1 , Martins C, Martins E, Gompertz f, .oares Fo P, Aly R, Hay R 

1 Hospital Carlos Tortelly, Ministry of Health, M., .E.v,.6., FM.,PMN,Instituto de 

Dermalologia R.D. Azulay, Rj, 2razil, 2 /aboratário .addy, Niteroi, R(, 2razil, J Instituto 

2iomddico, 6niversidade Federal Fluminense, Niteroi, R(, 2razil, O Escola Paulista de 

Medicina, .Éo Paulo, .P, 2razil, [ Departamento de Patologia, 6niversidade Federal 

Fluminense, 6 Dermatology Research, 6C.F,CA ,6.A, 7 Dean, Institute of Health 

.ciences, ~ueenïs 6niversity, 2elfast, Northern Ireland, 6l 

Dermatophilosis is a zoonotic disease caused by a facultative anaerobic actinomycete, 

Dermatophilus congolensis. This organism has a broad host range, most commonly 

causing endemic, sometimes epidemic infections in cattle, sheep and horses. This 

pathogen of the epidermis of lower animals has been reported to cause human 

infection infrequently. Clinical spectrum includes pitted Beratolysis, pustules, 

erythematous exudative scaly lesions, intertriginous lesions with maceration and 

fissuring of the sBin, folliculitis, subcutaneous nodules, and hairy leuBoplaBia. Pitted 

Beratolysis is an uncommon, asymptomatic non inflammatory microbial condition of 

the stratum corneum, most frequently reported n tropical and subtropical areas. A 

case of pitted Beratolysis (Beratoderma plantare sulcatum) caused by Dermatophilus 

congolenis is described. 

Male patient, 2J years old, caucasian, student, resident in Niterái, R(, 2razil , came 

for consultation referring presence of small pits that gradually deepened located on 

the soles, otherwise asymptomatic. He also refers plantar hyperhydrosis. He used to 

play football on a grassfield (for leisure). 

fn dermatologic exam numerous superficial depressions, cup shaped (pits), 

millimetric in size were observed. These were located on pressure bearing surfaces of 

the soles, that gathered to form superficial erosions. Potassium hydroxide mounts and 

cultures of the sBin scrapings revealed no fungi. Direct examination of Giemsastainened 

preparations of sBin samples demonstrated irregular bacterial filaments 

suggestive of Dermatophilus congolensis. /ongitudinaland transversely septate 

filaments and pacBets of coccoid celss were observed. These structures were also 

seen in histopathology exam of shave biopsy in special stains (2rown 2renn Gram 

stain, PA. and Grocott |stained sections). Hematoxylin eosin sections 

.howed shallow pits of stratum corneum. These resulted from Beratolysis. The patient 

was treated with mupirocin cream 20 mg/g with good response to this therapy. 

The etiologic agent of pitted Beratolysis seems to be Corynebacterium sp. However, 

Dermatophilus congolensis, Micrococcus sedentarius and other bacteria such as 

.treptomyces have been described. Hyperhidrosis and occupational activities such as 

sports activities that affect the plantar region have also been related to pitted 

Beratolysis. Treatment includes topical erythromycin, mupirocin, imidazoles, benzoyl 

peroxide, germicidal soaps, salicic acid, sulfur and even systemic antibiotics. Treating 

hyperhidrosis with botulin toxin injection also improved associated pitted Beratolysis. 

In dermatophilososis, the extracellular proteolytic activity of Dermatophilus 

congolensis may beresponsible for Beratinized tissues being the main sites of infection, 

and although rarely reported, this case of pitted Beratolysis by D. congolensis clearly 

illustrates this hypothesis. 

P-0344. Evaluation of McRAPD suitability for species identification in 8 

pathogenic yeast species 

Trtkova J 1 , lonieczna A 1 , RusBova / 1 , Hamal P 2 , RaclavsBy V 1 

1 1Department of 2iology, PalacBy 6niversity Faculty of Medicine, flomouc, Czech 

Republic, 2 Department of Microbiology, 6niversity Hospital, flomouc, Czech Republic 

Purpose of the study: Invasive infections caused by a variety of pathogenic yeast 

species represent an increasing cause of morbidity and mortality in seriously ill 

hospitalized patients. Different species can vary greatly in their relative virulence as 

well as their susceptibilities to antifungal agents. Therefore, fast and reliable assay for 

characterization of the yeast pathogens is critical for early initiation of adequate 

antifungal therapy. .ince the accurate diagnosis still remains problematic and 

convensional phenotyping identification methods are often cumbersome, timeconsuming 

and not entirely disctinctive, a broad spectum of genotyping tools has been 

developed and increasingly used in clinical laboratories. Most of them rely on PCR 

amplification of genomic DNA using either species-specific primers or universal fungal 

primers with subsequent post-PCR analysis, e. g. hybridization with specific probes or 

product sequencing. Another new approach has been recently developed in our 

laboratory, which is based on random amplification of polymorphic DNA (RAPD) 

followed by melting curve analysis of resulting amplicons (McRAPD). This approach 

omits gel electrophoresis and laborious analysis of banding patterns. Purpose of this 

study was to evaluate McRAPD suitability for species identification. 

Description: Eight pathogenic yeast species (7/*+68"#+,&,*7/*-$%'"#+6"&,*7/*@6+8$+-+,* 

7/*6(&"-+,"+.,*7/*'+$+'&"6%&"&, 7/*[$(&.",*7/*@("66".$)%,5""*and =/*#.$.0"&"+.) 

represented by O0 unrelated strains per species were included in the study. McRAPD 

species identification was performed using an optimized protocol for a Rapid Cycler 

instrument and applying high resolution melting analysis using a High Resolution 

Melter. Inter-strain variability of McRAPD results obtained at low annealing 

temperature was also evaluated in selected related and non-related isolates of 

7/*+68"#+,& and 7/*6(&"-+,"+.. 

Results and conclusions: ahen using a simplified toothpicB DNA extraction 

technique, the whole procedure can be completed within J hours after minute colonies 

of a pure isolate are available on plate. ae demonstrate that McRAPD data obtained 

with a high resolution melting instrument are sufficient for unequivocal species 

identification in a vast majority of species and strains2 few controversies can be easily 

solved after subsequent gel electrophoresis. McRAPD also seems to be a promising 

tool for characterization of inter-strain variability for typing purposes as demonstrated 

in 7/*6(&"-+,"+. and 7/*+68"#+,&. The Ministry of Health, Czech Republic, supported 

this worB (NR8J6[-O/200[).

P-0345. Candida: An epidemiological comparison between two medical units of 

Lisbon 

VerÜssimo C, .abino R, 2randÉo (, Parada H, Rosado L 

Instituto Nacional de saâde Dr. Ricardo (orge 

Yeasts belonging to the genus 7+,5"5+ are common residents of the mucosal 

surfaces of gastrointestinal tract, genitourinary system and oral cavity of warm 

blooded animals (1). In the last decade, there has been an increase in the number of 

reports of systemic as well as mucosal infections caused by 7+,5"5+*(2). The 

increase of immunocompromised patients, the widespread use of broad-spectrum 

antibiotics and the rising use of indwelling catheters are important factors that 

increase the number of cases related with candidosis. 

The yeast 7+,5"5+ is the fourth most common cause of hospital related bloodstream 

infections (J). Increasing incidence of candidemias caused by*7+,5"5+*'+$+'&"6%&"&,* 

7+,5"5+*@6+8$+-+3*7+,5"5+*-$%'"#+6"&*and others has been described (O). In general, 

7+,5"5+*'+$+'&"6%&"& is the second most frequent causative agent of candidemia in 

.pain, Norway, Israel and /atin America and contrasts with data from 6.A, England, 

aales, France, Germany, Austria, .weden and Italy ([), where 7+,5"5+*@6+8$+-+ is 

the second most common. 

To gather some data from the epidemiological situation in /isbon, we collected and 

identify yeasts strains from different biological products. The yeasts strains were 

collected from a primary medical unit (Central analysis unit/mycology lab of National 

Health Institute of Dr. Ricardo (orge) and also from a Public Hospital (from patients 

with more severe pathologies). The aim of this study was to compare different two 

medical units and collect data to a future epidemiological survey. The patients used in 

this study were not selected randomly. Instead there was either a suspicious of 

microbial infection or patients were monitorized as of risB of microbial infection. 

Comparing these two institutions, we observed differences in relative abundances of 

the different species of 7+,5"5+/ 7+,5"5+*+68"#+,& is more frequently detected in our 

lab, especially due the high percentage of vaginal exudates analysed. Due the 

hemocultures obtained in the hospital, the ratio of 7+,5"5+*'+$+'&"6%&"& and 7+,5"5+* 

@6+8$+-+ differsM the first one increases and the second one decreases when 

compared with our mycology lab. 

References: 

1. .ampaio P., GusmÉo /., Alves C., Pina-Vaz C., Amorim A., Pais C. Highly 

Polymorphic Microsatellite for Identification of 7+,5"5+*+68"#+,& strains. (. 

Clin. Microb. Feb 200Jn Vol O1 (2)M [[2-[[7 

2. Myhlsschlegel M., Frosch M. The relevance of Candida species other than 

Candida albicans as opportunistic pathogens. 1KKKn Mycoses O2(J) 

J. luhn D., MuBherjee P., ClarB T., Pujol C., Chandra (., Hajjeh A., aarnocB D., 

.oll D., Ghannoum M. 7+,5"5+*'+$+'&"6%&"& Characterization in a futbreaB 

.etting. Emerg. Infect. Dis. (un 200On Vol.10 (6)M 107O-1081 

O. Trost A., Graf 2., EuBer (., .ezen f., Possinger l., Gbel 6., Adam T. 

Identification of Clinically Relevant Yeasts by PCR/RF/P. (ournal of 

Microbiological Methods. 200On [6M 201-211 

Pdman (., Cantán E., Goberdo M. Epidemiology and Antifungal susceptibility of 

Candida species isolated from bloodM results of a 2-year multicentre study in .pain. 

ECMM aorBing Group on Candidemia. European (ournal of clinical Microbiology c 

Infectious Diseases. 200On 10/1007/[100K6-00O-1267-[ 

2etween 2001 and 200O, we observed that in 66KJ patients that came to our Central 

analysis unit/mycology lab, we collect 10J6 yeast strains. The major products 

analysed wereM vaginal exudates (71.1_), Beratinized tissues (11.6_), respiratory 

tract exudates (8.8_), sputum (1.[_), stool (J.O_) and urine (0.K_). The data 

obtained showed that 7+,5"5+*+68"#+,& was the most frequently isolated species 

(8[.62_), followed by 7+,5"5+*@6+8$+-+ (7.2O_), 7+,5"5+*'+$+'&"6%&"&*(2.[1_), 

7+,5"5+*-$%'"#+6"& (1.O[_), 7+,5"5+*[$(&." (0.O8_), 7+,5"5+*U+)+-+*(0.2K_) 

amongst other 7+,5"5+ species (1.O[_). =+##?+$%)2#.&*#.$.0"&"+. represents 

0.K7_ of the total positive cases. 

2etween 2002 and 200[ and from hospitalized patients (total number unBnown), we 

collect 188 yeasts strains from hemocultures (J7.8_), sputum and bronquic 

secretions (22.K_), stool (K.6_), pus (8.[_), urine ([.8_), vaginal exudates ([.8_), 

respiratory tract exudates ([.J_) and other products (O.2_). ae observed that 61_ 

of the isolated yeasts were 7+,5"5+*+68"#+,&, followed by 10_ of 7+,5"5+* 

'+$+'&"6%&"& and then by 7.O_ of 7+,5"5+*-$%'"#+6"&, 6.K_ of 7+,5"5+*[$(&.", 6.K_ of 

7+,5"5+*@6+8$+-+*and*J.[_ of*7+,5"5+*&'/*ae also obtained 2.6_ of infections due 

=+##?+$%)2#.&*#.$.0"&"+.3*detected in three hemocultures. 


P-0346. Actinomycetoma and eumycetoma- two case reports 

Vieira M 1 , Coutinho ( 2 , Felicissimo P J , Afonso A O , Cardoso ( 1 , Couble A [ , Rodriguez- 

Nava V [ , /aurent F [ , 2oiron P [ 

1 Dermatology Department, Curry Cabral Hospital, /isbon, Portugal, 2 .urgery 

Department, Curry Cabral Hospital, /isbon, Portugal, J frthopedics Department, Curry 

Cabral Hospital, /isbon, Portugal, O Pathology Department, Curry Cabral Hospital, 

/isbon, Portugal, [ 6MR CNR. [[[7, Center for Microbial Ecology, fpportunistic 

Pathogens and Environmental Research Group, French fbservatory for Nocardiosis, 

/aboratoire de Mycologie Fondamentale et Appliqude aux 2iotechnologies 

Industrielles, 6FR de Pharmacie, 6niversitd Claude 2ernard /yon I, /yon, France. 

Mycetoma is a sBin and underlying tissues infection, clinically characterized by an 

inflammatory tumour with multiple sinus tracBs, and the presence of granules in the 

exsudate. 

Actinomycetes (Gram r bacteria) are the etiologic agents of Actinomycetoma and 

fungi the etiologic agents of Eumycetoma. 

In spite of the similarity of symptoms, clinic evolution, diagnostic tests and treatment 

are different in the two groups of infections. The correct identification of the etiologic 

agent is essential, for the choice of the indicated therapy. 

In Portugal, mycetoma are rare imported cases from the African countries with 

Portuguese official language (PA/fP). 

ae describe the cases of two patients born in Cabo Verde, with foot mycetoma, one 

caused by Actinomadura madurae and other by Madurella mycetomatis. In the first 

case the use of molecular biologic techniques was essential to the identification of the 

agent. These were infections with several year duration and serious bone involvement. 

The patients were treated respectively with amiBacin associated with trimethropim - 

sulphametoxazole and itraconazole associated with terbinafine. In the second case 

surgical treatment was essential, avoiding amputation and allowing some functional 

recovery. 

P-0347. Medical treatment of mycetoma 

Welsh O, Vera / 

Dermatology Department 6niversity Hospital 6AN/ 

Treatment of actinomycetoma included in the early forties the administration of 

sulfonamides, DD., Isoniazid. Rifampin and sulfamethoxazole-trimethoprim (.jT) 

were added in the fifties and sixties, the latter being the gold standard, which is given 

for months or even years with a total cure rate of about 6[_. Amoxicillin/clavulanic 

acid has been successful in a few cases caused by `*8$+&"6".,&"&. 

For non-responsive infections, the combination of amiBacin-.jT gives around a K[_ 

cure rate. These antimicrobials are given in a dose of 1[ mg/Bg/day IM and 8-O0 

mg/Bg/day orally, respectively. The medication is given in cycles of J weeBs of 

amiBacin and [ weeBs of .jT for 1 to O cyclesn at the end of each of these, creatinine 

clearance and audiometry are performed. In a period of 2J years, more than [0 

patients were treated and evaluatedn all of them responding to treatment with the 

exception of one that had a remission and a recurrence two months after medication 

was stopped. Twenty-one percent of the patients treated showed minimal decrease of 

high-tone audition, detected only by audiometry. No other side effects were observed. 

The therapeutic response of actinomycetoma to the combination amiBacin-.jT 

remains the most effective treatment for this disease, achieving around a K[_ cure 

rate. The treatment is indicated for disease with extensive lesions, bone involvement, 

tendency to affect neighboring organs and those unresponsive to previous treatments. 

ahen the isolated actinomycete is resistant to amiBacin the antibiotic can be changed 

to netilmicin. New drugs, such as linezolid, spectinomycin and garenoxacin have 

shown very good inhibition in ",*0"-$% testing of `*8$+&"6".,&"&

P-0348. Predisposing factors and treatment responses in streak type 

onychomycosis 

Yu H, fh D, ParB l, lim ( 

Hanyang 6niversity Guri Hospital, Guri, .outh lorea 

Human nails bear longitudinal furrows on both upper and lower surface. These 

furrows are formed as the nails grow and correspond to parallel dermal ridges 

irregularly distributed on the nail bed. fften there is fungal invasion in a longitudinal 

narrow band according to the furrows of the nail bed, clinically recognized streaB type 

onychomycosis. fur purpose was to investigate the clinical features, causative 

organisms, and predisposing factors and to evaluate the treatment responses of oral 

antifungal agent in streaB type onychomycosis. 

The study was conducted with J87 cases of onychomycosis (O[ cases of streaB type, 

JO2 cases of non-streaB type) and [0 normal controls examined at Department of 

Dermatology, Hanyang 6niversity Guri Hospital. .treaB type onychomycosis was 

devided into three clinical presentationsM distal type (20 cases), lateral type (1K cases), 

and multiple type (6 cases). The depth of longitudinal nail furrows in each type of 

onychomycosis and control group was examined and the severity of tinea pedis in 

each type of onychomycosis was assessed. Nail growth rates in each type of 

onychomycosis and control group were measured and treatment responses in each 

type of onychomycosis were compared. 

The age distribution showed patients in their fifth decade to be most common (O0.0_). 

The ratio of male to female was 1.[M1, showing male-predominant pattern. The fungal 

culture showed dermatophytes to be most frequently isolated (O7.2_), followed by 

yeasts (11.7_). >$"#?%'?2-%,*$(8$() was the most common causative organism. In 

the group of distal and multiple type of streaB type onychomycosis, the longitudinal 

nail furrows were deep compared to the groups of non-streaB type onychomycosis 

and normal control (pv0.0[). In the group of distal type of streaB type onychomycosis, 

the tinea pedis severities were low compared to the group of non-streaB type 

onychomycosis (pv0.0[). There was no statistically significant difference when 

comparing the nail growth rate of the groups of streaB type onychomycosis, nonstreaB 

type onychomycosis, and normal control (pt0.0[). In the groups of distal and 

lateral type of streaB type onychomycosis, the treatment responses were poor 

compared to the group of non-streaB type onychomycosis (pv0.0[). 

P-0349. First case of meningitis due to prototheca wickerhamii in china 

Zhang Q 1 , aeng j 2 , Zhu / 2 , /i / 1 , aang ( 1 

1 Division of Mycology,Huashan Hospital,Fudan 6niversity, .hanghai,China, 

2 Department of infectious disease,Huashan Hospital,Fudan 

6niversity,.hanghai,China 

Protothecosis is an uncommon human infection caused by the achlorphyllic alga 

Prototheca, which is a rare cause of opportunistic infection. In China, only three cases 

presenting with cutaneous lesions have been documented until now.No systemic 

protothecosis has been reported. In this report, we describe a case of meningitis 

caudsed by 1$%-%-?.#+*B"#[.$?+)"", which was first reported in China and has been 

cured successfully by intravenous amphotericin 2 combining itraconazole.The case 

was a 2O-year-old man presented with a K-month history of fever and severe 

persistent headache, ambiopia and difficulty of walBing. His temperature ranged from 

J7.[ÅC to JKÅC.He was not immunocompromised and without underlying illness. The 

microscopy examination of C.F showed spherical sporangia with sporangiospores 

and cultured on .abouraud Dextrose Agar (.DA) at J7ÅC and 2[ÅC grew smooth, 

creamy white, yeastliBe colonies. It was identified as 1/*B"#[.$?+)"" by API20C A6j 

(bioMdrieux). Transmission and .canning electron micrograph confirmed the 

identification. In all cases documented, to date only two cases of meningitis due to 

prototheca have been reported. The case herein described is the third reported in the 

world. 

Treatment responses are poor in streaB type onychomycosis, therefore, extra 

treatments in addition to oral antifungal agents should be considered. If the patients 

who have deep longitudinal nail furrow are affected with tinea pedis, effective 

therapies in early stage should be considered to prevent to advance streaB type 



P-0350. Quality of life in patients with onychomycosis 

¯elazny I, NowicBi R 

Dept. of Dermatology, Medical 6niversity of GdansB 

Introduction: .Bin is the biggest and the most visible human organ. Due to their 

expose localization sBin diseases have characteristic individual and social 

consequences as compared to other diseases. Mycoses of the sBin and its 

appendages are becoming more and more serious epidemiological and social 

problem worldwide. In addition lately the amount of mycoses has grown up 

considerably fne of such mycoses is onychomycosis and this is why this paper is 

dedicated to it. 

Objectives: The objectives of the current study were to investigate to what degree 

the particular areas of quality of life are disturbed by onychomycosis, and to 

determine the role of sociodemographic factors, disease duration and occurence of 

other sBin/nails diseases or mycosis in the patient]s family - in context of patient]s 

quality of life. 

Material and methods: ae studied 81 outpatients with onychomycosis who 

presented to the Dermatology Department of Medical 6niversity in GdansB and one 

of hospitals in Gdynia. In all patients the clinical diagnosis was confirmed by direct 

microscopic examination and by growth tests. Also O6 healthy controls (as far as any 

nails disorder is concerned) were recruited to taBe part in the project. Demographic 

and clinical variables were collected by chart review. To measure quality of life we 

used .Bindex questionnaire, a tool to measure quality of life in dermatoses. 

ResultsM The most common and strongest response to this disease were feelings of 

fear, anger and embarrassment. Cognitive disorders, social and physical discomfort 

were escalated in moderate degree. The quality of life was least affected by 

depressive states and limitations in everyday life. fn the base of the study some 

correlations were found between patients] quality of life and sociodemographic 

factors and health situation in family. 

Conclusions: fnychomycosis turns out to be a serious health problem. It causes 

intensive physical ailments, unpleasant emotional reactions and limits patients in 

fulfilling their roles in life. 

DIAGNOSTIC TOOLS* 

P-0351. Fungal ribosome-associated hsp70 as a potential diagnostic antigen 

Cernila B 

6niversity of /jubljana, /jubljana, .lovenia 

Purpose of the study: Although rare, invasive infections caused by the fungi of the 

phylum L2@%)2#%-+ (zygomycoses) represent dangerous threat to 

immunocompromised patients, as they are associated with a high rate of mortality. 

~uicB diagnosis is one of the Bey factors for successful treatment, however it is 

usually not achieved due to the lacB of fast diagnostic tests based on molecular 

marBers. aith the aim to evaluate the diagnostic potential of the stress proteins of the 

H.P70 family we performed a phylogenetic analysis of the H.P70 proteins and 

prepared polyclonal antibodies against the peptide-binding domain of the P?"d%'(&* 

,"@$"#+,&*ribosome-associated H.P70 as well as assessed their specificity. 

Description: A survey of literature and a phylogenetic analysis of the sequence data 

available in the Gen2anB / EM2/ / DD2( databases revealed a presence of all major 

subfamilies of the H.P70 proteins in all Bingdoms of euBaryotic organisms. The only 

exception was the subfamily of the constitutively expressed ribosome-associated 

H.P70s (..2 subfamily) that appears to be present only in fungi maBing those 

proteins a prospective target for diagnostic purposes. ae cloned the cDNA 

corresponding to the P/*,"@$"#+,&*&&8I*gene and expressed the 2[.8 BDa C-terminal 

peptide-binding domain of encoded protein in a bacterial expression system. 

Antibodies against the recombinant antigen were raised in chicBen. ae preliminarily 

evaluated the specificity of the total chicBen IgY antibodies by the western blot 

analysis. 

Results and conclusions: The antibodies to P/*,"@$"#+,&*..21 peptide-binding 

domain proved to have a broad specificity. They reacted strongly with ..2 

homologues in protein extracts of all seven tested zygomycete fungi (48&"5"+*@6+(#+, 

!%$-".$.66+*"&+8.66",+, !(#%$*#"$#",.66%"5.&, !(#%$*+)'?"8"%$(), P?"d%)(#%$*)".?.", 

P?"d%'(&*,"@$"#+,&*and P?"d%'(&*%$2d+.). fn the other hand the signals were much 

weaBer or entirely absent in the protein extracts from ascomycetes =+##?+$%)2#.&* 

#.$.0"&"+., 4&'.$@"66(&*,"@.$*and `.($%&'%$+*#$+&&+*probably reflecting long time of 

evolutionary separation of zygomycetes from ascomycetes. These results encourage 

additional investigation of the potential use of the ribosome-associated H.P70s as 

antigens in the diagnosis of zygomycosis. 

.

P-0352. Detection of Aspergillus antigens in rat models of invasive 

aspergillosis using dot-blot techniques 

Abdul Samad S, .anthanam (, Tang Yew Huat (, .amat M 

6niversiti lebangsaan Malaysia 

Purpose: The objective of the study was to develop dot-blot techniques in order to 

detect Aspergillus antigens in experimental invasive aspergillosis. 

MethodsM An invasive aspergillosis models was established in cortisone acetate 

immunosuppressed rats by intravenous inoculation of viable conidia of 4&'.$@"66(&* 

U()"@+-(&/ Chromogenic and EC/ dot-blot techniques were developed to detect 

4&'.$@"66(&*antigens in sera of infected rats. 4&'.$@"66(& antigens were detected by 

using a rabbit polyclonal antibody raised to water-soluble antigens of a young 

disrupted mycelial culture of 4/*U()"@+-(&. The bound primary antibody was detected 

using anti-rabbit horseradish peroxidase followed by addition of diaminobenzidine for 

the chromogenic dot-blot technique and EC/ reagents for the EC/ technique. 

Results and ConclusionsM The mortality rates and isolation of A. fumigatus from 

organ culture increased with increases in inoculum sizes. Even though the threshold 

for antigen detection was 10 fold higher for the EC/ technique, antigens were more 

frequently detected with the chromogenic technique (chromogenicM 2K.1_ to 8J.J_, 

EC/M 1J.J_ to [[.7_). The chromogenic dot-blot was more reliable than the EC/ dotblot 

and detection rates increased with increasing inoculum sizes with the 

chromogenic test. 2acBground staining seen in the EC/ test made interpretation of 

results difficult. Attempts to overcome false positivity using adsorbed primary antibody 

did not result in increased specificity of the EC/ test. 2oth dot-blot techniques failed to 

detect antigens in sera following electrophoresis of infected sera. 

P-0353. Comparison of Indian ink and cryptococcal antigen testing for 

diagnosis of cryptococcal meningitis in a low resource hospital in Kenya 

Akoru C 1 , Chebore . 1 , 2uziba N 2 , lamule / 2 , landie . 1 

1 Moi Teaching and referral Hospital, Eldoret, lenya, 2 Moi 6Niversity .chool of 

Medicine 

Objective: To compare the two diagnostic techniques employed in the diagnosis of 

Cryptococcus neoformans Meningitis 

Setting: Moi Teaching and Referral Hospital Eldoret on patients presenting meningitis in 

East Africa 

Resign: Prospective study from (une 200[ to December 200[ of J08 patients from lohom 

cerebrospinal fluid (CF.) samples were submitted to the laboratory 

Material and Methods: All samples screened were from adults whosen age and sex were 

given in the request forms. All the samples were centrifugedn coverslipped mixed with a 

drop of India inB and examined, at 100ó and confirmed using O00 ó magnifications. India 

inB being a negative stain, staining the bacBground and not the orgasm.A Bnown in house 

positive control was also run. 

Cryptococcul antigen testing was done using a commercial Bit manufactured by meridian 

bioscience inci.cincinatti fhio 6.A .The test was performed according to the manufactures 

instructions.Crytococcul antigen was performed by boiling C...F for five minutes, cooling 

for J minutes and placing a drop put on darB bacBground tiles and adding a drop of the 

reagent antigen positive and negative controls were run concurrently. Positive samples 

showed agglutination which was graded as 1r, 2r, Jr and Or.Negative samples showed 

no agglutination. 

Results: fut of J08 samples done 1JO(OJ.[_)were males and 17O([6.[_)were females 

2O samples were positive for India inB represented by 11(O[.8_)being males and 

1J([O.2_)being females. 

J6 samples were out of J08 samples were positive for crypotococcul antigen represented 

by 16(OO.O_) being males and [[.6_ being females 

The sensitivity of India inB preparation was 66/7_ while the sensitivity of Cryptococcus 

antigen was 7[ _ 

Conclusion: The results obtained with India inB correlate with those by the Cryptococcus 

antigen testing. However cryptocuccul antigen appears to be more sensitive and is 

detected in most cases. 

The antigen tests mainly detects cryptococcul polysaccharide antigen in C...F in about 

K0_ of patients with cryptococcal meningitis. It can also be used on sera thus allowing 

diagnosis of Cryptococcus infections other than meningitis liBe disseminated infections. 

Although the cost is higher on cryptococcal antigen than India inB, accuracy of diagnosis 

and greater sensitivity are important. There is need for education programs to address the 

importance of cryptocuccul antigen testing and more wide spread availability of the testing 

in HIV/AID. facilities in sub .aharan Africa to allow more rapid and more accurate results 


P-0354. Yeast identification. Co-application of conventional and automatic tests 

with PCR and sequencing protocols 

Alcoba-Flórez J 1,2 , Pdrez-Roth E 2 , Mdndez-îlvarez . 1,2 , Ardvalo-Morales M 1 

1 6niversidad de /a /aguna, Tenerife. .pain, 2 26nidad de Investigacián, Hospital 

6niversitario Nuestra .eora de Candelaria, .anta Cruz de Tenerife. .pain 

Background:*Identification of medically relevant yeasts can be timeconsuming 

and inaccurate with current methods. A total of [0 clinical yeast 

isolates of 7+,5"5+, were used to compare two identification techniques, 

VITEl 2 and the analyses of the IT. region and the D1/D2 region of the 26. 

rRNA gene sequences with IDJ2C as the reference method. 

Methods: Fifty yeast clinical isolates belonging to 10 different species were 

included in the study. Phenotypic identification of yeast isolates was carried 

out by classical microbiologic methods. Microscopic, biochemical and growth 

analyses were performed. VITEl 2 automatic system was always used with 

the API IDJ2C test as reference. PCR amplification and sequencing of IT. 

region and the D1/D2 region of the 26. rRNA gene sequences were 

performed and sequences obtained were compared with the NC2I database. 

Results: ae have developed a comparative analysis of different phenotypic 

and molecular methods in order to Bnow their distinct efficiency and specificity 

capacities to identify and differentiate 7/*+68"#+,&, 7/*@6+8$+-+, 7/*,"0+$".,&"&3* 

7/*[$(&."*and other yeast species isolates. .ome isolates from the species 7/* 

@6+8$+-+, 7/*,"0+$".,&"&3*and*7/*[$(&."*could not be correctly identified by the 

identification methods API ID J2C and VITEl 2. Fermentation of threalose and 

growth on ChromoAgar permitted to distinguish isolates but not always clearly 

and undoubtedly. A robust and trustworthy identification at the species level 

was achieved by a combination of PCR amplification and later sequencing, but 

it was a time consuming protocol. 

Conclusion: ae propose a co-application of techniques as the best procedure to 

unambiguously ascribe yeast clinical isolates at the species level. ahen an exact 

identification is required, isolates identified as 7/*@6+8$+-+ using the API ID J2C and 

the VITEl 2 tests should be corroborated by PCR employing a protocol that permits to 

specifically identify 7/*,"0+$".,&"&. In the case of other species as 7/*[$(&." this 

integrative methodological approximation could be also necessary. 

P-0355. Reactivity of Aspergillus GM in neutropenic adult patients receiving 

piperacillin-tazobactam (P/T) treatment 

Alhambra A 1 , Moreno ( 2 , Cudtara M J , frtiz M O , Pontán ( [ , del-Palacio-Perez-Medel 

A 2 , del-Palacio A 1 

1 Hospital 6niversitario Doce de fctubre, Microbiology. Madrid. .pain, 2 Hospitla 

6niversitario Doce de fctubre, Internal Medicine. Madrid. .pain, J Hospital .evero 

fchoa, Microbiology. /egands, Madrid. .pain, O Hospital 6niversitario Doce de 

fctubre, Haematology. Madrid. .pain, [ Departamento de InmunologÜa, MicrobiologÜa 

y ParasitologÜa, Facultad de Medicina y fdontologÜa, 6niversidad del PaÜs Vasco- 

EusBal HerriBo 6nibersitatea, 2ilbao. .pain 

Purpose and Methods: ae studied galactomannan (GM) EIA (twice weeBly) for the 

prospective diagnosis of invasive aspergillosis (IA) in adult neutropenic patients using 

an EIA index cut off of 0.[. 

Results: From .eptember 200O to (uly 200[, 78 adult neutropenic patients (K0 

episodes) were prospectively assessed. There were [ proven IA, and O probable IA, 

all of them with at least two positive GM indexes. fne other patient had one proven 

zygomycoses with negative GM. There were eight patients with false positive 

antigenemia and none of them had signs of IA. There were 2 patients (allogenic 

H.CT) with false positive GM due to GVHD and grade IV mucositis. In six other 

patients with no IA, who also had false positive GM results there was a significant 

association with the administration of P/T (pv0.01). 

The Binetics of these last patients showed a rapid abrupt rise of GM, followed by a 

very rapid decrease of GM when P/T was withdrawn. fne of the patients who was 

challenged with a second administration of P/T had a significant increase of GM index 

with the same Binetics as described above. The sensitivity, specificity and positive and 

negative predictive values of GM EIA were 100, 88, O7 and 100_ respectively, 

whereas if the 6 false positive P/T results were excluded, then these values would had 

been 100, K7, 78 and 100_ respectively. 

Conclusions: Although four of the patients with false positive GM due to P/T had data 

that determined that they could be assigned to low and intermediate low risB group 

stratification as proposed by Prentice et al (2r ( Haematol 2000n110M27J-28O) 

however, the administration of P/T had inconvenients for all six patientsM repeated 

HRCT, bronchoscopy lavages and unjustified antifungal treatment.

P-0356. Molecular identification of Cryptococcus neoformans serotypes 

Angoulvant A 1 , Chandenier ( 2 , .ymoens F J , /acube P 1 , 2olognini ( 1 , Douchet C 2 , 

Poirot ( 1 , Hennequin C 1 

1 Facultd de Mddecine P c M Curie, Paris, France, 2 .ervice de Parasitologie- 

Mycologie, Tours, France, J IHEM-Mycology, 2russels, 2elgium 

Purpose of the study: 7$2'-%#%##(&*,.%U%$)+,& is a human opportunistic fungal 

pathogen that causes life-threatening meningoencephalitis in immunocompromised 

hosts, particularly patients with AID.. 2ased on genetic characteristics and serological 

proprieties of capsular polysaccharides, three varieties and four serotypes have been 

defined, namely 7/*,.%U%$)+,&*0+$/*,.%U%$)+,& (serotypes D), 7/*,.%U%$)+,&*0+$/* 

@$(8"" (serotype A) and 7/*,.%U%$)+,&*0+$/*@+-"" (serotypes 2 and C). .erotypes are 

associated with epidemiological particularities, such as geographical distribution, 

ecological niche or underlying condition and possibly ",*0"-$% susceptibility to antifungal 

drugs. 6p to now, serotyping is determined either by agglutination assays using 

commercial or hhome-madei antisera, or indirect immunofluorescence using 

monoclonal antibodies targeting the capsule mucopolysaccharide. However, cases of 

misidentification have been reported whatever was the method used. Here, we report 

on the development of two molecular methods based on the sequence characteristics 

of a fragment of the Cap[K gene required for the capsule biosynthesis. 

Description: .equences of 7+'5R gene from the four serotypes were retrieved from 

Gen2anB and used to design a primer pair able to amplify all four serotypes. 

Restriction map of unique cutting sites was used to select 46(I and 4##I to generate 

restriction profiles specific of each of the four serotypes. .ixty six 7/*,.%U%$)+,& 

strains, including JJ reference strains, belonging to the O serotypes, plus 6 hybrid AD 

strains and a 7$2'-%#%##(&*(,"@(-(6+-(& strain were tested. 

Results and conclusions: DNA was amplified from all the strains but the 7/* 

(,"@(-(6+-(& one, generating a fragment which size varied from J6K to J87 bp. 

Digestion with 4##I and 46(I, enabled us to distinguish O restriction profiles 

corresponding to serotypes A, 2, AD, which exhibited a mixed A and D profile, and C 

and D which exhibited a similar profile i.e. restriction with 4##K, no restriction with 46(K. 

However, serotype C and D could be easily differentiated based on the size of their 

native amplification products. This led us to test a fragment length analysis method, 

using the same primer pair, the forward being fluoro-labeled. Fragment size was then 

directly determined on an automate sequencer. .ix different alleles accounting for 7 

different genotypes were seen. Each one could be assigned to a particular serotype 

with a perfect specificity. Misidentifications were detected using these methods, which 

results were further confirmed by conventional tests. 

2oth methods allowed the determination of 7/*,.%U%$)+,& serotype with accuracy. 

They are particularly adapted to epidemiological investigation but may become more 

important if antifungal susceptibility profile is associated with serotype. 

P-0357. Immunomagnetic separation of Candida yeast cells from rabbit blood 

artificially contaminated. 

Apaire-Marchais V 1 , lempf M 2 , /efrancois C J , Marot A O , /icznar P [ , Cottin ( 6 , 

Poulain D 7 , Robert R 8 

1 6FR .ciences Pharmaceutiques et Ingdnierie de la santd, 2 CH6 /aboratoire de 

Parasitologie-Mycologie, J 6FR .ciences Pharmaceutiques et Ingdnierie de la santd, 

O 6FR .ciences Pharmaceutiques et Ingdnierie de la santd, [ 6FR .ciences 

Pharmaceutiques et Ingdnierie de la santd, 6 6FR .ciences Pharmaceutiques et 

Ingdnierie de la santd, 7 IN.ERM 67KK Physiopathologie des candidoses, Facultd de 

mddecine, /ille, 8 6FR .ciences Pharmaceutiques et Ingdnierie de la santd 

7+,5"5+*&'. are now the fourth most frequent cause of nosocomial blood-stream 

infections in critically ill and immunocompromized patients. Although 7/*+68"#+,& is 

responsible for most infections, other species such as 7/*-$%'"#+6"&, 7/*@6+8$+-+, 7/* 

'+$+'&"6%&"& and 7/*[$(&." are found in common clinical conditions. Rapid identification 

of pathogenic fungi to species level is critical for treatment. Conventional diagnostic 

procedures, such as blood culture and biochemical tests lacB the degree of sensitivity 

and specificity. That]s why methods based on the amplification and detection of fungal 

DNA have been developed. 

However fungal load is often lower than 10 CF6/m/ of blood, even in patients 

suffering from invasive fungal infection. To improve fungal detection, we choose to 

evaluate an immunomagnetic separation (IM.) of yeast, using magnetic beads coated 

with monoclonal antibodies ([22 and 62J) specific to surface antigens of 7+,5"5+ in 

order to capture and concentrate yeast cells from blood before identification on culture 

or rapid identification tests. This study was performed with rabbit blood artificially 

contaminated with various 7+,5"5+*&'. Two ATCC reference strains (7/*+68"#+,& 

ATCC 66JK6 and 7/*5(86",".,&"& ATCC MYA-6O6) and 1[ 7+,5"5+*&'/ isolated from 

clinical samples taBen during invasive candidiasis were used in this study (2 clinical 

strains of 7/*+68"#+,&, 7/*5(86",".,&"&*and 7/*@6+8$+-+, and three isolates of each 

species*7/*[$(&.", 7/*-$%'"#+6"&, and*7/*'+$+'&"6%&"&). 

Percentages of yeast cells captured by beads were calculated in relation to counts of 

viable cells found in initial suspensions. Percentages of recovery superior to 70 _ 

were obtained for three isolates of 7/*+68"#+,& and two isolates of 7/*[$(&."/* 

Immunoseparation was more than [0_ for the majority of the isolates tested for 7/* 

5(86",".,&"&, 7/*-$%'"#+6"&*and 7/*@6+8$+-+. However, very low results were observed 

for 7/*'+$+'&"6%&"& which seems not to be recognized by the MAb [22 and MAb 62J. 

In conclusion, IM. is a rapid method that allowed efficient capture and concentration 

of cells. .ensitivity was lower than 10 colony forming unit per m/ of blood This method 

could increase the efficiency of culture or yield of nucleic acid extraction and so 

improve fungal detection. 


P-0358. Rapid detection and identification of six commonly encountered 

dermatophytes by multiplex real-time PCR 

Arabatzis M 1,2,J,O , 2ruijnesteijn van Coppenraet / 1 , luijper E 1 , de Hoog . 2 , /avrijsen 

A J , Templeton l 1 , van der Raaij-Helmer E J , VelegraBi A O , .ummerbell R 2 

1 Medical Microbiology Department, /eiden 6niversity Medical Center, /eiden, 

Netherlands, 2 Centraalbureau voor .chimmelcultures,6trecht, Netherlands, 

J Dermatology Department, /eiden 6niversity Medical Center, /eiden, Netherlands, 

O Microbiology Department, Athens Medical .chool, Athens, Greece 

Although*dermatophytic infections are regarded as relatively Bnown clinical entities, 

still a lot remains to be elucidated regarding their epidemiology and their respective 

response to treatment. Current diagnosis of dermatophyte infections essentially relies 

on microscopic evaluation of the isolate]s phenotypic characteristics. This is a lengthy, 

low sensitivity and expertise requiring process yet, identification of isolates to species 

level is a central component in understanding dermatophyte global epidemiology. fur 

objective was to develop a real-time PCR assay for rapid and reliable diagnosis of 

dermatophytoses and simultaneous species identification. 

Two assays were designed and optimisedM fne based on amplification of the IT.1 

region for detection of the >$"#?%'?2-%,*).,-+@$%'?2-.& species complex, >/* 

-%,&($+,&*and >/0"%6+#.() and a second based on amplification of the IT.2 region for 

detection of the >/*$(8$() species complex, !"#$%&'%$()*#+,"& and !/*+(5%(","". 

2oth assays employed Taqman and minor groove binding probes carrying different 

fluorophores thus allowing target discrimination. 1?%#"5*?.$'.&0"$(&*I (PhoHV-1) was 

used as internal control. .ensitivity was tested with serial DNA dilutions and specificity 

with a panel of J6 different species including all pathogenic and non pathogenic 

dermatophytes, sBin yeasts, bacteria as well as human DNA. The protocol was 

evaluated by testing blindly K2 specimens, collected prospectively from clinically 

suspicious sBin-nail-hair lesions over a period of 6 months. 

The system correctly identified all the above-mentioned dermatophyte species from 

pure culture. The analytical sensitivity of both assays was 0.1 pg, corresponding to 2.[ 

genomes per sample. All microscopy- and/or culture-positive samples (n}O0) were 

PCR positive and species (>/*$(8$()3*>/*).,-+@$%'?2-.&3*!/*+(5%(",""3*>/*0"%6+#.()) 

from positive cultures (n}2K) were correctly identified. The system also detected 7 

additional positive samples that were microscopy/culture-negative and identified 2 >/* 

$(8$() and >/*).,-+@$%'?2-.& mixed infections. 

The proposed real-time PCR assay has a high sensitivity, enables accurate diagnosis 

of six commonly encountered dermatophyte species and could potentially be 

incorporated in the clinical laboratory routine diagnostic procedures. 

P-0359. Real time PCR identification of C. albicans in biological samples 

Arancia S, /a Valle R, Carattoli A, .andini ., Graziani ., Mandarino G, Cassone A, 

De 2ernardis F 

Department of Infectious Diseasesn Istituto .uperiore di .anitÖ, Rome Italy 

Background: Invasive candidiasis remains an increasing source of morbidity and 

mortality in immunocompromised patients, particularly in the neutropenic ones 

undergoing antiblastic chemotherapy or bone marrow transplant. Candidemia has 

been estimated as the fourth most common nosocomial infection with a mortality rate 

of about [0_. Early diagnosis is often difficult as most clinical signs and symptoms 

are non-specific, the cultures are often negative or become positive too late. 

Consequently, various Real-time PCR techniques have been proposed both for genus 

and species identification. In a previous study, a method for detection of 7+,5"5+* 

+68"#+,&3 the most frequent agent of invasive candidiasis, in biological samples (blood, 

serum, urine) was developed with the use of PCR amplification of a DNA fragment of 

a gene coding for a 6[-BDa mannoprotein of 7/*+68"#+,& (CaMP6[). 

In this study, we describe the development and evaluation of a Real Time PCR with 

the same CaMP6[ specific primers for detection of 7+,5"5+*+68"#+,& in biological 

samples and in sera from patients affected by autopsy-proven disseminated 

candidiasis. 

Results and conclusions: DNA extracted from various isolates of 7+,5"5+ species 

(7/*+68"#+,&3*7/*'+$+'&"6%&"&3*7/*-$%'"#+6"&3*7/*@6+8$+-+3*7/*[$(&."), from other fungi (=/* 

#.$.0"&"+.3*7/*,.%U%$)+,&3*4/*U()"@+-(&), from a/*#%6" and human DNA were tested by 

real time PCR for the presence of the target CaMP6[ gene. 

* 

7/*+68"#+,& DNA produced the expected 220 bp Amplicon with a melting peaB curve at 

82ÅC, while all other 7+,5"5+*spp. gave melting curves significantly different. DNA 

from other fungi, a/*#%6" and human DNA gave no amplification products. The 

sensitivity of this method was then tested in simulated biological samples (urine, blood, 

serum) spiBed with different amounts of C/*+68"#+,& (10 7 -10 2 cells/ml). 

The sensitivity was high in serum and urine, so it was possible to quantify up to 1 

cell/ml, and for blood the detection limit was ten-fold higher (10 cell/ml). 

Finally, Real time PCR was employed to detect and quantify 7/*+68"#+,& in the sera 

from patients with invasive candidiasis. All sera were positive for the CaMP6[ gene 

Amplicon, with the characteristic melting peaB, indicating the presence of 7/*+68"#+,& 

DNA in the seran therefore, it was possible to quantify the 7+,5"5+ presence. 

The Real time PCR method based on MP6[ primers was shown to be simple, rapid, 

sensitive, reproducible and useful for the clinical diagnosis of 7/*+68"#+,&.

P-0360. Antifungal susceptibility of dermatophytes and clinical correlation 

Auger I 2 , /andry / 1 , Pelletier R 1 

1 /eHÇtel-Dieu de ~udbec du CH6~, 2 6niversitd de Montrdal 

Purpose of the study: .uperficial infections caused by dermatophyte fungi are highly 

prevalent worldwide. .uccess of antifungal treatment is partly dependant on host 

factors and duration of therapy. Resistance of dermatophytes to antifungal drug has 

been poorly documented. Testing methods to determine antifungal susceptibility of 

dermatophytes has not been universally approved yet. The activity of commercially 

available antifungal drugs was determined for dermatophytes isolates from clinical 

specimens. Results were then correlated with available clinical data. 

Summarized description of the project: Purified conidia were obtained from 122 

dermatophytes isolates, including 72 >$"#?%'?2-%,*$(8$(), J2 >/*).,-+@$%'?2-.&, 1O 

!"#$%&'%$()*#+,"&, 2 a'"5.$)%'2-%,*U6%##%&() and 2 >/*0.$$(#%&(). Antifungal 

drug activity was tested using a broth dilution method. Final conidia suspension in the 

microplate was adjusted to achieve 0.[ x 10 6 cfu// to 2.[ x 10 6 cfu//. Plates were 

incubated for O days at J[ÅC. .ignificant growth inhibition served to determine the 

end points. Clinical data were collected retrospectively from patients] file and by 

personal telephone contact. 

Results: Dermatophytes MICK0 for Amphotericin 2, fluconazole, itraconazole, 

voriconazole, terbinafine and griseofulvin was 0.[mg//, 2mg//, 0.12[mg//, 0.0Jmg//, 

0.0Jmg// and 2mg//, respectively. Data on treatment outcome was available for [7 

patients (O6.7_). Forty three infections (7[.O_) were cured or improved. Failure was 

not associated with the infection site or by the dermatophyte species. fne 

>$"#?%'?2-%,*$(8$() isolate recovered from an onychomycosis that persisted despite 

terbinafine treatment produced a higher terbinafine MIC result (0.2[ mg//). 

P-0361. Searching for ecological niches of Coccidioides sp. in Baja California, 

Mexico. 

Baptista R 1 , Riquelme M, Hinojosa A 

1 Microbiology Dept. CICE.E/.cience Faculty 6A2C, 2 Microbiology Dept. CICE.E, 

J GI. and Remote Perception /aboratory, Earth .ciences, CICE.E 

Coccidioidomycosis, also Bnow as .an (oaquin Valley Fever is an endemic mycotic 

disease of aestern North American deserts caused by the ascomycete 7%##"5"%"5.&* 

&'/ In the 6nited .tates there are estimates of more than 100,000 cases per year. 

The extent of this problem in Mexico is unBnown. For this purpose, we here 

consolidated the limited available information about historic epidemiological research 

in Mexico and carried out a detailed comparative analysis with epidemiological data of 

California and Arizona. 

The main objective of this project is to better understand the environmental distribution 

of this important fungal disease. 2ased on the available epidemiologic and ecological 

information, we have applied Genetic Algorithm for Rule .et Production (GARP) and 

Geographical Information .ystems (.IG) using a combination of 11 environmental 

variables and 1[ positive geospatially referenced points to determine the presence of 

7%##"5"%"5.&*&'. in soil samples. This worB has allowed us to design a predictive 

model to identify probable hhotspotsi for the presence of the fungus in endemic areas 

of this pathogen in 2aja California and northern Mexico, areas that show a high 

correlation with the available epidemiological data. ae are using a PCR-based 

approach to identify the presence of 7%##"5"%"5.& in soil samples of the predicted 

hotspots for 2aja California. For diagnostic purposes, we are adapting this molecular 

approach to screen clinical samples and discern the impact of this fungal disease. 

Conclusions: From our data, antifungal drug MICs were usually relatively low, when 

tested against dermatophytes. However, isolates recovered from persistent infections 

can occasionally produce higher MICs. 


P-0362. Development of novel approaches for the diagnosis of invasive 

aspergillosis using RT-PCR 

2egum Y, Gomez 2, .eaton ., Kibbler C 

Microbiology Department, Royal Free and 6niversity College /ondon NaJ 2~G 

Invasive aspergillosis (IA) infections are a major complication in immunocompromised 

patients. This filamentous fungus acts as an opportunistic pathogen and can cause a 

life threatening infection and nosocomial outbreaBs of disease. The increasing 

incidence of IA emphasizes the need to improve the currently limited diagnostic tools. 

Conventional diagnostic methods for IA are not particularly good and molecular tests 

circumvent these problems by focusing on the detection of a specific nucleotide 

sequence. Molecular methods, in addition to being more sensitive than culture based 

methods, can be specifically designed to encompass the desired range of genera and 

specimen types, and in some cases even recognize individual species. A Real time 

PCR (RT-PCR) result is generated within a few hours, which is significantly earlier 

than culture results. Multi-copy sequences on the 18. gene, the 28. gene, the 

intergenic spacer regions (IG.) and the internal transcribed spacer region (IT.) of 

ribosomal DNA (rDNA), as well as mitochondrial DNA, have all been targeted for 

genus level identification of 4&'.$@"66(&. ae first surveyed existing methods for 

detecting the presence of a fungal genus of interest, and reproduced selected steps in 

assays that had been previously developed for 4&'.$@"66(&. ae decided to choose the 

4&'.$@"66(& genus specific primers in the IT.1 region flanBing the [.8. rDNA gene as 

this region shows extensive sequence diversity among major groups of euBaryotic 

microorganisms, and should therefore be liBely to contain genus-specific sequences. 

Also, the IT.1 region has not yet been targeted by others to develop a RT-PCR assay 

for 4&'.$@"66(&. fligonucleotide primers and a labelled probe were chosen based on 

sequence alignments, fine-tuned to optimize the resulting assay, and tested against a 

panel of 4&'.$@"66(& species (U()"@+-(&, U6+0(&, ,"@.$*+,5 -.$$.(&J. The initial results 

show high sensitivity and specificity for 4&'.$@"66(& (100_) which can be attributed to 

the high copy number of rDNA units (genes and transcribed spacers), and to the intergenus 

variability found within the IT.1 region. The primers used for RT-PCR 

amplification in the Rotor-Gene RG J000 (Corbett Research) successfully amplified 

DNA of the four medically important 4&'.$@"66(& species that were tested and also 

showed a sensitivity of 88.8_ using spiBed samples with 4/*U()"@+-(& of water and 

blood. Furthermore, the primers and probe showed specificity (100_) for 4&'.$@"66(& 

&''. when tested with DNA extracted from other pathogenic and non-pathogenic fungi. 

These included 7+,5"5+ +68"#+,&3*7/*@6+8$+-+3*7/*[$(&.", 7. 6(&"-+,"+.3*7/**'+$+'&"6%&"&, 

7. -$%'"#+6"&3*other yeasts (7$2'-%#%##(&*,.%U%$)+,&3*=+##?+$%)2#.&*&', 

>$"#?%&'%$()**&')3*and other moulds (48&"5"+3*^(&+$"()*&'/, 1.,"#"66"()*&'/, 

=#.5%&'%$"()*+'"%&'.$,())*that also cause invasive mycoses or are Bnown to be 

common contaminants. These DNA extracts all failed amplification by this assay. 

P-0363. Rapid molecular detection and identification of dermatophytes 

Bergmans A 1 , .chouls / 2 , aintermans R 1 

1 /aboratory of Medical Microbiology, Franciscus Hospital, Roosendaal, The 

Netherlands, 2 /aboratory for Vaccine Preventable Diseases, RIVM, 2ilthoven, The 

Netherlands 

Dermatophytes are Beratinophilic fungi that cause superficial infections such as 

ringworm, favus, or onychomycosis. The dermatophytes comprise three genera, 

>$"#?%'?2-%,, !"#$%&'%$() and a'"5.$)%'?2-%,. Current laboratory diagnosis relies 

on direct microscopic examination of lfH-treated clinical samples, micro- and 

macroscopic observation of ",*0"-$% cultures, and on metabolism tests. aith direct 

examination of samples, identification of fungi is impossible. Culturing is insensitive 

and time-consuming (2-O weeBs), and identification of strains is difficult because of 

overlapping characteristics, variability and pleomorphism. 

fur aim was to develop fast and sensitive molecular methods for detection and 

identification of dermatophytes directly in clinical material. 

Recent worB has shown that internal transcribed sequences (IT.) between rRNA 

genes are sufficiently polymorphic for identification of dermatophytes to the species 

level. ae developed a dermatophyte-specific PCR-reversed line blot (PCR-R/2) 

assay based on IT. sequences, and a DNA extraction procedure for nail, sBin, and 

hair samples. 2oth genus- and species-specific probes were developed, which 

allowed the rapid identification of nine species within three genera. Additionally, we 

developed a real-time PCR assay using hybridization probes that can detect and 

identify 11 dermatophyte species in clinical material in one single PCR reaction. 

Approximately 2600 nail, sBin, and hair samples were used to validate the following 

three diagnostic methods for detection of dermatophytesM PCR-R/2, direct 

microscopic examination, and culture. .ensitivities of these three methods as 

determined by an extended gold standard showed to be K[.2_, 8J.0_, and 66.0_, 

respectively. Identification results of [[8 (PCR- and culture-) positive clinical samples 

showed 2[ discrepancies between culture and PCR-R/2n 16 of them were due to 

incomplete identification by either PCR-R/2 or culture. 

PCR-R/2 was also used to identify 12[ isolates (both dermatophytes and nondermatophytes), 

and identification results were compared to those obtained by microand 

macroscopic observation. DNA sequence analysis of IT. regions of discrepant 

samples confirmed PCR-R/2-based identification results in almost all sequenced 

samples. All non-dermatophytes remained negative in the PCR-R/2. 

Preliminary results of the real-time dermatophyte PCR assay showed that its 

performance is comparable to the performance of the PCR-R/2 assay described 

above. 

ae conclude that the PCR-R/2 assay is extremely suitable for routine detection and 

identification of dermatophytes directly in nail, sBin, and hair samples, because of its 

fast, sensitive, specific and accurate performance. Preliminary results of the real-time 

PCR assay also seem very promising for even more rapid diagnosis of 

dermatomycosis.

P-0364. A new method for serodiagnosis of disseminated and extracutaneous 

forms of sporotrichosis 

2ernardes-Engemann A 1 , 2envenuto Ferreira F 1 , /oureiro y Penha C 1 , /ima 2arros 

M 2 , .chubach A 2 , frofino Costa R J , Lopes-Bezerra L 1 

1 /aboratário de Micologia e ProteÇmica, Instituto de 2iologia Roberto Alcantara 

Gomes, 6niversidade do Estado do Rio de (aneiro (6ER(), Rio de (aneiro. 2razil, 

2 Instituto de Pesquisas ClÜnicas Evandro Chagas, Fiocruz, J Hospital 6niversitZrio 

Pedro Ernesto, 6ER(, 2razil. 

For a long time sporotrichosis had been regarded to have a low incidence in 2raziln 

however, recent studies demonstrated that not only the number of reported cases but 

also the incidence of more severe or atypical clinical forms of the disease are 

increasing. This is mainly due to the zoonotic transmission of sporotrichosis with a 

recent outbreaB described in Rio de (aneiro .tate. fur data indicate that the 

incidence of extracutaneous and disseminated forms account for about 10_ of 

patients. Furthermore, osteoarticular sporotrichosis might be associated both with 

patient immunodepression and the zoonotic transmission of the disease. The 

extracutaneous form and the special forms as for example, erythema nodosum, are 

difficult to diagnose. A newly developed serological test was introduced in a clinical 

trial in two hospitals of Rio de (aneiro .tate as an auxiliary tool for the diagnosis of 

these clinical forms of sporotrichosis. The E/I.A test based on an antigenic fraction 

(=&C2F) of the cell wall of ='%$%-?$"_*&#?.,#["" showed K0_ sensitivity and 86_ 

overall efficacy when tested with sera from K2 patients with lymphocutaneous, fixed 

cutaneous, disseminated cutaneous and extracutaneous forms of sporotrichosis, in 

addition to 117 control serum samples obtained from healthy individuals or subjects 

with other diseases. Further on, we have applied the E/I.A test to detect IgG 

antibodies in other clinical materials such as the synovial fluid and the cerebrospinal 

fluid (C.F) from two patients with meningeal and osteoarticular sporotrichosis, 

respectively. The former patient who is HIV positive was followed up for 18 months. In 

parallel, we had performed the E/I.A test using a mannoprotein fraction from 

=+##?+$%)2#.&*#.$.0"&"+. as a control. ae could detect significant levels of IgG 

antibodies against the .sC2F antigen in the synovial fluid and C.F specimens as well 

as in the serum samples of those patients. A decrease in the antibody levels was 

observed with the clinical improvement. This diagnostic tool has proved to be useful to 

promptly diagnose sporotrichosis, mainly for those unresolved cases that are negative 

by the classical mycological test. 

Financial supportM Rede M./FAPER( and CNPq. 

P-0365. Antigenic variations in A. fumigatus in function of the media used to its 

culture 

Blanco J 1 , Caballero ( 1 , Gargallo-Viola D 2 , Garcia M 1 

1 

FAC6/TAD DE VETERINARIA. 6CM. 280O0 MADRID, 2 G/Ajf .MITHl/INE. TRE. CANTf.. 

28760 MADRID. .PAIN. 

fne of the main problems in the study of the pathogenic fungus is its structural complexity, with a 

high amount of antigens. Its Bnowledge is essential to elucidate the pathogeny of the 

aspergillosis. 

In the present worB we prompted us to study the variability in the antigenic structure of 

4/U()"@+-(&*in function of the culture media used to grow the fungus. 

4&'.$@"66(&*U()"@+-(&*O82J8E was inoculated in different liquid culture mediaM .abouraud 

dextrose (.2), CzapecB-Dox (CZ), RPMI, RPMI with serum (RPMI-.) and Minimum medium 

(MM). 

Initially can be observed macroscopic differencesM in RPMI the growth was slight, as by the 

number as by the size of pellets (v 1mm diameter). In RPMI-. the number of pellets was 

significatively higher, reaching 2-J mm diameter, with an intense green colour. In MM the pellet 

size was similar to RPMI-., but with white colour. In .2 and CZ the growth was higher than in 

the other media, as in number as in size of the pellets, with an intense green colour. 

From each medium was obtained antigenic extract from the mycelium and the culture filtrate. The 

protein extracts were analyzed by .D.-PAGE, It has to emphasize the absence of bands in the 

culture filtrate in MM, and the difficulty to differentiate bands in the culture filtrate in RPMI and 

RPMI-.. 

Table 1. Molecular weight (BDa) of the bands present in the protein profile of the antigenic extract 

of 4/*U()"@+-(& O82J8 growing in different media 

CZ- 

Mycelium 

SAB- 

Mycelium 

RPMI+S- 

Mycelium 

RPMI- 

Mycelium 

MM- 

Mycelium 

SAB- 

Filtrate 

CZ- 

Filtrate 

8K]2 7K]0 62]7 [8]O [2]O O2]2 

8K]2 61]6 7O]1 60]7 [6][ [2]7 O7]J OJ]6 O2][ 

O0]2 JK]0 J7]J J[]O JO]K J0]J 28]0 2O]K 20]6 

KO]1 8J]1 6[]O [7]1 

[6]J 27]K 21]6 

K0]7 72]7 

1K6]0 11[] 

0 

K6]6 K1]J 80]J 6[]8 [K]1 [[]0 O1][ 

J8]7 J[]8 JO]1 2O]K 2J]8 

10K KJ]O K0]J J6][ 

In view of the results, we can conclude that it is very variable the number and strength of bands 

in function of the culture media. Then, it can be hypothesized that antigens expressed when 

fungus are grown in vivo probably are different that those expressed in vitro, with the 

consequences that this fact could have in the search of effective diagnostic methodologies. 


P-0366. Antifungal susceptibility of Microsporum canis isolates from dogs and 

cats. A comparison between strains from France and Croatia 

Bourdeau P 

Ecole Nationale Veterinaire de Nantes, France 

ObjectiveM Evaluation of variation of sensitivity of strains of !"#$%&'%$()*#+,"& 

isolated from animals in two countries. All J6 strains of !"#$%&'%$()*#+,"& strains 

were clinical isolates from dogs and cats, (1K France, 17 Croatia) and submitted for 

mycological examination in the /aboratory of Dermatology, Parasitology and 

Mycology of the Veterinary .chool of Nantes, France and Mycology /aboratory, 

Faculty of Veterinary Medicine, 6niversity of Zagreb, Croatia. The method for testing 

was an adaptation to the dermatophytes from NCC/. (MJ8-A) Reference Method for 

2roth Dilution Antifungal .usceptibility Testing of Filamentous Fungi. Antifungal 

agents tested wereM letoconazole, Itraconazole, Miconazole Enilconazole, 

Miconazole and Griseofulvin. Concentration ranges were 0,0J1 to16 kg/m/ as 

proposed in the references guidelines used. Fungi were first grown on Potato 

Dextrose Agar at 27ÅC. for 7 days then suspensions of macroconidia prepared and 

optical density adjusted (0,O0-0,O[) then further diluted 1/[0 with RPMI 16O0. All tests 

were performed in duplicates. Microdilution plates were incubated at J0ÅC for 7 days 

Results were read visually with the aid of a loop with the following scoreM O } no 

reduction in growthn J } slight reduction in growth (7[_)n 2 } prominent reduction in 

growth ([0_)n 1 } slight growth (2[_) 0 } optically clear or absence of growth. 

According to the recommandations of the official document MIC for Itraconazole, 

Miconazole and Enilconazole was the lowest concentration for 100_ inhibition (score 

0), and read at score 2 For letoconazole. MIC (kg/m/) values for tested strains 

respectively from French (F) and croatioan (C) isolated were M letoconazoleM 0,12[ | 

2 (F), 0,06-1 (C) n Itraconazole 0,2[ | O (F),0,12[-1 (C) M Miconazole 0,2[-2 (F), 0,12[- 

2 (C) n Enilconazole 0,0J-0,12[ (F), 0,0J-0,06 (C)n Griseofulvin 0,2[-1 (F and C). It is 

concluded that this method is useful to evaluate antifungals on dermatophytes. 

Results were statistically compared (2 samples lolmogorov-.mirnov Repartition test). 

The variations suggest that susceptibility of Croatian isolates is higher to most of 

azoles (although not statistically significant). In France Azoles antifungals are much 

mor efrequently used on pets than in Croatia and further worBs are needed to 

evaluate if a therapeutically induced tolerance of dermatophytes to antifungals could 

be involved. 

FundingsM /ab. DPM ENVN and European EGIDE program. 

P-0367. Identification of Cryptococcus neoformans and Cryptococcus gattii 

with a luminex xmap suspension array 

Bovers M 1 , Diaz M 2 , .panjaard / J , Hoepelman I O , Fell ( 2 , 2oeBhout T 1, O 

1 C2.-Centraalbureau voor .chimmelcultures, 6trecht, The Netherlands, 2 R.MA.- 

Rosenstiel .chool of Marine and Atmospheric .cience, Division of Marine 2iology and 

Fisheries, ley 2iscayne, Florida, 6.A, J The Netherlands Reference /aboratory for 

2acterial Meningitis (AMC/RIVM), Department of Medical Microbiology, Academic 

Medical Center, Amsterdam, The Netherlands, O Division Acute Medicine and 

Infectious Diseases and EijBman-ainBler Centre for Microbiology, Infectious Diseases 

and Inflammation, 6niversity Medical Centre 6trecht,The Netherlands 

/uminex xMAP is a novel flow cytometry technique, which can be adapted for the 

detection of pathogens. The method is based on a nucleotide hybridization assay 

using a combination of species-specific capture probes coupled to different sets of 

fluorescent beads. The strength of this method is that it has the potential to 

simultaneously detect one hundred different organisms. The /uminex xMAP system 

has been used for the detection of bacteria (Dunbar .-*+6/, 200J) and fungal 

pathogens, such as species of the genera >$"#?%&'%$%, and 7+,5"5+ (Diaz and Fell, 

200On Page and lurtzman, 200[). 

Most recently, the technology was employed for the identification of different genotypic 

groups within 7$2'-%#%##(&*,.%U%$)+,&*and 7/*@+--"" (Diaz and Fell, 200[). 2oth 7/* 

,.%U%$)+,&*and 7/*@+--""*can cause meningoencephalitis, but it is important to Bnow 

which genotypic group is responsible for the infection. The epidemiology and the 

symptoms, as well as the outcome of the disease, can be different between the 

different genotypic groups. 

The capture probes, which were designed in the intergenic spacer region (IG.), were 

validated using JO clinical isolates. These isolates came from Dutch meningitis 

patients between 1K77 and 200O. 2ecause the clinical isolates were mostly 7/* 

,.%U%$)+,&, twenty additional 7/*@+--""*isolates were included in the study. The results 

were compared to results derived from Amplified Fragment /ength Polymorphism 

(AF/P) analyses. In addition, spiBing series were made in cerebrospinal fluid to 

establish the detection limit of this diagnostic method. 

The results were highly reproducible and all strains were correctly identified. In some 

cases, hybrid strains could be identified. Although most hybrid cryptococcal strains do 

not have both IG. alleles, the hybrids that have both alleles (according to cloning 

experiments) were identified as such. In these cases, the output signal of the probes 

corresponding to one of the parents was comparable to that of a normal haploid strain, 

while the strength of the signal of the probes detecting the other parent was positive, 

but lower in intensity. 

/uminex xMAP is a reliable method to detect and differentiate 7/*,.%U%$)+,& and 7/* 

@+--"". If both IG. alleles are present in a hybrid, the technique can detect cryptococcal 

hybrids (2overs .-*+6/, 2006). 

References: 

2overs M, Hagen F, luramae EE, Diaz MR, .panjaard /, Dromer F, Hoogveld H/ 

and 2oeBhout T (2006) 6nique hybrids between fungal pathogens 7$2'-%#%##(&* 

,.%U%$)+,&*and 7$2'-%#%##(&*@+--"". FEM. Yeast Res. (",*'$.&&).

Diaz MR and Fell (a (200O) High-throughput detection of pathogenic yeasts of the 

genus >$"#?%&'%$%,. (. Clin. Microbiol. O2M J6K6-J706. 

Diaz MR c Fell (a (200[) 6se of a suspension array for rapid identification of the 

varieties and genotypes of the 7$2'-%#%##(&*,.%U%$)+,& species complex. (. Clin. 

Microbiol. 43: J662-J672. 

Dunbar .A, Vander Zee CA, fliver lG, larem l/ and (acobson (a (200J) 

~uantitative, multiplexed detection of bacterial pathogensM DNA and protein 

applications of the /uminex /abMAP TM system. ( Microbiol Methods 53: 2O[-2[2. 

Page 2T and lurtzman CP (200[) Rapid identification of 7+,5"5+ species and other 

clinically important yeast species by flow cytometry. (. Clin. Microbiol. OJM O[07-O[1O. 

P-0368. Invasive candidiasis diagnosis by real-time PCR 

Castro C 1 , Torres M 2 , Aller A J , Ruiz M O , Aznar ( O , Palomares ( 2 , Martin-Mazuelos E 1 

1 H6 V de Valme. .evilla..pain, 2 Facultad de Medicina.6niversidad de .evilla..pain, 

J H del .A. (erez..pain, O H6 V del Rocio..evilla..pain 

Objective: Prospective evaluation of the usefulness of Real-Time PCR for early 

diagnosis of 7+,5"5+ infection in neutropenic patients. 

Method: A total of [8J clinical samples from 8K neutropenic patients presenting risB 

factors for invasive fungal infection were studied. Patients were attended at the 

Hospitals V del Rocio and V. de Valme of .eville and at Hospital del .A. of (erez, 

from .eptember 200O to (une 200[. Two blood specimens were extracted weeBly 

from each patient from the inclusion in the study to their discharge. fther samples as 

blood culture, 2A/, bronquial aspirates or others were taBen following clinical criteria. 

DNA was extracted from whole blood samples by using recombinant lyticase (.igma, 

Madrid, .pain) and the ~IAmp Tissue lit (~IAgen, 2arcelona, .pain). For DNA 

amplification and detection the method developed by (. /éeffler et al (2000) was 

followed, using the /ightCycler .ystem (Roche .ystem Diagnostics, Mannheim, 

Germany). Primers and probes were directed to the conserved regions of the fungal 

18. rRNA. 

Results: ff the total samples studied ([8J), K showed a positive PCR result, 

corresponding to O patients (1 classified as having probable infection and J as 

possible). All the O patients were recipients of an allogenic bone marrow transplant. 

The PCR became positive after 1O, 11, 1[ and 21 days after transplant with a media 

of 2 days before the clinical symptoms appeared. 

Conclusions: According to this results, the real-time PCR results anticipated to 

clinical sings of infection. The PCR method could be useful for early diagnosis of 

Candida infection. 


P-0369. Rapid identification of pathogenic fungi: Usefulness of lightcycler 

system 

Catherine D 1 , Sébastien B 1 , Antoine H 1 , Cdcile P 2 , (acques R J , Mallie M 1 

1 /aboratoire de Parasitologie et Mycologie medicale, 2 /aboratoire de Parasitologie, 

CH6 de 2ordeaux, France, J PÇle infectiologie, CH6 Gui de Chauliac, Montpellier, 

France 

BackgroundM The incidence of opportunistic fungal infections has increased 

dramatically in the past few decades, especially in immunocompromised hosts. The 

prompt and accurate identification of yeast species thus appears to be necessary for 

determining the better antifungal drug to use. 

PurposeM The purpose of this study was to use the melting temperature variability of 

both IT.1 and IT.2 regions to achieve the specific identification of fungal clinical 

strains by using /ightCycler system. 

Material and methods: Five quality control strains of 7+,5"5+ speciesM 7/+68"#+,&* 

(ATCC 28[16J3*7/*[$(&."*(ATCC 62[8),*7/*-$%'"#+6"&*(ATCC 17J1)3*7/*'+$+'&"6%&"&* 

(ATCC 2201K) and 7/*@6+8$+-+*(ATCC K00J0), four quality control strains of 

4&'.$@"66(& speciesM 4/*U()"@+-(&*(C2. 287.K[),*4/*,"@.$*(ATCC 6OK[8),*4/*,"5(6+,&* 

(MYA-170)*+,5*4/*U6+0(& (ATCC K60O[) and one control strain of 7$2'-%#%##(&* 

,.%U%$)+,&*(ATCC O2168) were tested. 

In addition, 62 clinicals strains were also testedM 7/+68"#+,&*(n}1[), 7/*[$(&."*(n}6),* 

7/*-$%'"#+6"&*(n}6)3*7/*'+$+'&"6%&"&*(n}6), 7/*@6+8$+-+*(n}J), 4/*U()"@+-(&*(n} 17), 4/* 

,"@.$*(n}2),*4/*,"5(6+,&*(n}1)*and 4/*U6+0(& (n} 1) and 7$2'-%#%##(&*,.%U%$)+,&* 

(n}[) were tested. 

Fungal DNA was extracted using the ~IAMP DNA blood mini litw and was amplified 

using IT.1 and IT.O primers. These primers were used to target ribosomal DNAs 

(rDNAs) conserved sequences which length and sequence vary depending on the 

species. To determine the sensitivity of the /ightCycler (/C) system for detection of 

amplified fungi DNA, standard DNAs from the quality control strains were serially 

diluted in water (1ng to 10fg of DNA assuming that a single genome copy or 1 CF6 is 

equivalent to approximately 10fg of fungal DNA). Reproducibility was assessed ten 

times using 100 pg and 10 pg of purified fungal DNA. Melting curve analysis was 

realized at the end of each amplification. 

P-0370. Candida ® ID2, a new medium for the isolation and presumptive 

identification of Candida yeast species 

Chevrier . 1 , 2ouchara ( 2 , Morin f J , Chabasse D 2 , Guiguen C 1 , Gangneux J 1 

1 /aboratoire de Parasitologie-Mycologie, Rennes Teaching Hospital, France, 

2 /aboratoire de Parasitologie-Mycologie, Angers Teaching Hospital, France, 

J /aboratoire de Parasitologie-Mycologie, Nantes Teaching Hospital, France 

The aim of chromogenic agars is to improve the detection of mixed populations of 

yeasts and to allow the direct identification of 7+,5"5+*+68"#+,& and the presumptive 

identification of several other species through the hydrolysis of chromogenic 

substrates. ae have conducted a multi-centre evaluation of the recently 

commercialized chromogenic agar 7+,5"5+ w ID2 (CAN 2) on more than JJ0 samples 

collected both prospectively and retrospectively. Results were compared to those 

obtained with CHRfMagar w *7+,5"5+*medium. The sensitivity, specificity, positive 

predictive value and negative predictive value for direct identification of 7/*+68"#+,& 

were KK_, K7.[_, K7_ and KK_ with 7+,5"5+ w ID2 versus K8_, K8_, K7.[_ and 

KK_ with CHRfMagar w *7+,5"5+. In addition, 7+,5"5+ w ID2 showed a sensitivity of 

K[_, 100_ and 60_ for the presumptive identification of 7+,5"5+*-$%'"#+6"&, 7+,5"5+ 

[.U2$ and 7+,5"5+ 6(&"-+,"+.3 respectively. There were no notable problems regarding 

the specificity of the pinB colour expected for colonies of these last yeast species. 

CHRfMagar w *7+,5"5+ also showed correct specificity for the presumptive 

identification of 7+,5"5+*-$%'"#+6"& (K[_) and 7+,5"5+*[$(&." (100_). However, the 

pinB-purple colour code reported as suggestive of 7+,5"5+*@6+8$+-+ showed a good 

sensitivity (8[_), but lacBs specificity with almost J0_ false positive results due to 

other yeast species or genera. 2esides, 7+,5"5+ w ID2 does not prevent the growth of 

filamentous fungi, even more efficiently than CHRfMagar w 7+,5"5+, and as 

CHRfMagar w 7+,5"5+, it allows the presence of mixed populations to be easily 

distinguished. However, species which theoretically produced not pigmented colonies 

on both media, presented poorer growth on 7+,5"5+ w ID2 agar which may cause 

problems in performing further tests such as identification and antifungal susceptibility 

testing. /iBewise, the presence in 7+,5"5+ w ID2 of a unique chromogenic substrate 

digested either by 7/*-$%'"#+6"&, 7/*[.U2$ or 7/*6(&"-+,"+. constitutes a limitation to the 

rapid presumptive identification of these species which may present different 

antifungal susceptibility pattern. 

Results and discussionM /ightCycler system was able to detect fungi to a 

reproducible limit of 100 fg (corresponding to 10 CF6). Melting-curve analysis allowed 

identification of the ten fungi species tested. Indeed, it showed that each amplicon 

from these ten species presented a specific melting point, except for 7+,5"5+*+68"#+,& 

and 7$2'-%#%##(&*,.%U%$)+,& which displayed the same Tm. In only J hours, 61 out 

of 62 clinical strains were identified in accordance with biochemical methods that 

taBes O8h at least. 

Conclusion: These data present /ightCycler system as a potential tool for the rapid 

identification of yeasts isolates.

P-0371. Incorporation of fluconazole in peptone glucose agar yields rapid and 

enhanced isolation of Aspergillus fumigatus from respiratory tract of 

bronchopulmonary aspergillosis patients colonized by Candida albicans 

Chowdhary A, Randhawa H, .inha l, lowshiB T, Vijayan V 

Department of Medical Mycology,Vallabhbhai Patel Chest Institute,6niversity of Delhi, 

Delhi, India 

ae have reported earlier that 4&'.$@"66(&*U()"@+-(& is inhibited ",*0"-$% by 7+,5"5+* 

+68"#+,&, a commensal of the human respiratory tract. This inhibition may result in 

miss-diagnosis of aspergillosis in patients with respiratory tract colonization by 7/* 

+68"#+,&. In order to guard against this possibility our laboratory has recommended 

supplementing peptone glucose agar with fluconazole which is more inhibitory to 

7+,5"5+*+68"#+,& than to 4/*U()"@+-(&/ This paper reports the results of evaluation of 

peptone glucose fluconazole agar (PGFA) for rapid and enhanced isolation of 4/* 

U()"@+-(& from sputum of patients clinically suspected of bronchopulmonary 

aspergillosis with 7/*+68"#+,& colonization in the respiratory tract. Thirty-five freshly 

expectorated sputum specimens and one bronchial lavage were collected from 21 

patients clinically suspected of bronchopulmonary aspergillosis by the Clinical 

Research Centre of V. P. Chest Institute, and referred to the Medical Mycology 

Diagnostic /aboratory for investigation of fungal etiology. The specimens were 

obtained in sterilized, J0-ml screw-capped glass bottles and homogenized by mixing 

with sterile glass beads and shaBing on a cyclo-mixer. lfH wet mounts of each 

specimen were examined microscopically for hyphal or yeast-liBe fungal elements. 

They were inoculated on triplicate plates of PGFA ( peptone | 10g, glucose 20g, agar- 

20 g, chloramphenicol-O0 mg/l, gentamicin-2[ mg/l , fluconazole-[ kg/ml, distilled 

water 1000 ml, pH- 6.8-7.0) and the same medium without fluconazole (PGA) as 

control. The plates were incubated at 28 0 C and observed up to 7 days for 

appearance of fungal growth. Evaluation of 4/*U()"@+-(& growth was done by 

enumerating its colony counts per plate and by the time taBen for development of 

macroscopically recognizable colonies. Growth of 7/*+68"#+,& was graded as heavy, 

++++ (confluent growth), good, +++ (1[1-2[0 colonies/plate), moderate, ++ ([1-1[0 

colonies), poor + (21-[0 colonies), very poor, ± (1-20 colonies) and no growth, -. The 

significance of difference in efficacy of PGFA and PGA medium was statistically 

analyzed by applying the student t test and Fischer]s exact test. ff the J[ sputum 

specimens and a solitary broncho-alveolar lavage cultured. 4/*U()"@+-(& was isolated 

from all (100_) on PGFA as against only 28 specimens (78_) that proved to be 

positive on the control PGA medium (p v 0.0[). The greater efficacy of PGFA than 

that of PGA was further evident from the 2-fold higher 4/*U()"@+-(& mean colony 

count (8.2 ì 1.87) on the former medium than on the latter (J.O ì 1.00), and this 

difference was found to be statistically significant (p v 0.0[). 2esides, 4/*U()"@+-(& 

colonies were macroscopically recognizable within 2-J days on PGFA at 28oC in 

strong contrast to [-7 days required on PGA. It is noteworthy that but for the use of 

PGFA medium isolation of 4/ U()"@+-(& would have been missed in 8 of the J6 (22_) 

clinical specimens investigated. 2ased upon these observations, PGFA is 

recommended for wider application as a selective medium for rapid and enhanced 

recovery of 4/*U()"@+-(& from sputum of patients clinically suspected of 

bronchopulmonary aspergillosis with 7/*+68"#+,& colonization in their respiratory tract. 

P-0372. Evaluation of bichro-dubli latex coagglutination test for the rapid 

identification of Candida dubliniensis 

Chryssanthou E, Fernandez V 

Clinical Microbiology 

Introduction: It is problematic to discriminate 7/*5(86",".,&"&*and 7/*+68"#+,& by 

phenotypic methods because these two species share many common characteristics 

liBe green colour on CHRfMagar, production of chlamydospores and germ tubes in 

serum. At present molecular methods are therefore considered as gold standard. The 

2ICHRf-D62/I latex agglutination test (Fumouze Diagnostics, France) is a promising 

new method for identification of 7/*5(86",".,&"& directly from cultures. The test 

principle is co-agglutination of 7/*5(86",".,&"&*cells with latex particles coated with a 

monoclonal antibody, which specifically recognizes a cell surface antigen of 7/* 

5(86",".,&"&. 

Aims: To evaluate the 2ICHRf-D62/I test for rapid identification of 7+,5"5+* 

5(86",".,&"&/* 

* 

Method: ae first evaluated the performance of the 2ICHRf-D62/I test on J7 7/* 

5(86",".,&"& and K 7/*+68"#+,& isolates which had previously been identified by PCR 

and partial sequencing of the IT. region in the rDNA gene complex. In the second 

part of the study the occurrence of 7/*5(86",".,&"& was prospectively analysed by 

2ICHRf-/ATEj A/2ICAN. and 2ICHRf-D62/I tests in 111 vaginal and 116 lower 

respiratory tract isolates which were presumptively identified as 7/*+68"#+,& by their 

green or darB green colony colour on CHRfMagar. Three 7/*+68"#+,& and 2 7/* 

5(86",".,&"& reference strains were used as controls for 2ICHRf-/ATEj A/2ICAN. 

and 2ICHRf-D62/I tests. 

Results: All J7 sequenced 7/*5(86",".,&"&*strains were correctly identified by 

2ICHRf-D62/I test. fn the other hand D62/I test was negative for the K 7/*+68"#+,&* 

strains. All 111 vaginal isolates were positive in 2ICHRf-/ATEj A/2ICAN. test and 

were thus identified as 7/*+68"#+,&*/ 7/*5(86",".,&"&*group. Two (1.8_) of these 

isolates were further identified as 7/*5(86",".,&"&*by D62/I test that was confirmed by 

sequencing. fut of the 116 isolates from respiratory tract specimens 11[ were 

positive in 2ICHRf-/ATEj A/2ICAN. test. fne isolate was negative but was 

identified as 7/*+68"#+,&*/ 7/*5(86",".,&"&*by positive germ tube test. .ix isolates 

([.2_) were identified as 7/*5(86",".,&"&*by D62/I test. 

Conclusions: fur results demonstrate that the 2ICHRf-D62/I latex agglutination 

test is a reliable method for rapid identification of 7/*5(86",".,&"&*in clinical specimens. 


P-0373. Comparison of conventional and its-sequence based identification of 

moulds in the diagnostic laboratory 

Ciardo D, 2éttger E, 2osshard P 

Institute of Medical Microbiology, Zurich, .witzerland 

For rapid and cost-efficient identification of moulds in the diagnostic laboratory, we 

have established an algorithm which combines conventional phenotypic criteria with 

IT. sequence analysis. According to this algorithm, clinical isolates of moulds not 

readily identified by phenotypic characteristics were subjected to sequence analysis 

using in-house and public databases. During the past 2O months, a total of 

approximately 2600 moulds were isolated from patient specimens. JJ2 of these 

isolates could not be readily identified with conventional methods and were subjected 

to sequence determination and further extensive morphologic investigations (FEMI). 

Molecular methods and FEMI resulted in identical identifications in J8.K_ of the JJ2 

isolates. .equence analysis allowed a more discriminative identification in O6.7_ of 

the JJ2 isolates, conventional identification was more discriminative in 7.[_ of the 

isolates. The latter finding was due to missing reference sequences for these isolates, 

most of which where representatives of environmental saprophytes, indicating a need 

for further enlargement of existing databases. fur results validate the algorithm 

established and define the role of sequence analysis for identification of mould 

isolates in the clinical laboratory. 

P-0374. Construction of a high-quality and validated ITS-sequence database for 

identification of pathogenic fungi 

Ciardo D, .chxr G, 2éttger E, 2osshard P 

Institute of Medical Microbiology, Zurich, .witzerland 

The quality of databases used for sequence analysis is critical for proper identification. 

Public databases of fungi are incomplete and frequently contain misassigned 

sequences or sequences of poor quality. A high-quality database containing IT. 

sequences of pathogenic fungi as well as common contaminants occurring in clinical 

specimens was constructed as followsM i) a list of species to be included was defined 

and at least one representative each was sequencedn ii) sequences obtained were 

compared with sequences retrieved from public databases to calculate intra- and 

inter-species homology, for phylogenetic analyses, to enlarge the database and to 

eliminate misassigned sequencesn iii) confirmed sequences, i.e., sequences with 

tK[_ homology to at least one independent sequence entry of identical species 

assignment, were used to construct the database. Genera showing high sequence 

homologies (tK[_) to the taxa selected were also analyzed and included in the 

database. The resulting IT. sequence database currently consists of 17[8 sequences 

covering [6O species representing 111 genera. The homology trees established in 

most cases allow unambiguous assignment at species level.

P-0375. Detection of Histoplasma capsulatum by nested-PCR in clinical 

specimens 

Colella M 1 , Mata-Essayag . 1 , Hartung de Capriles C 1 , Pdrez C 1 , Rosellá A 1 , flaizola 

C 1 , Magaldi . 1 , Toro F 2 

1 .eccián de Micologia, Instituto de Medicina Tropical, 2 /aboratorio 2iologia 

Molecular ,Instituto de InmunologÜa 

Introduction: Histoplasmosis is a common endemic mycosis in America, Asia, India 

and Africa. In Venezuela a marBed increase of this mycosis has been reported in the 

last [ years. 

Diagnosis in disseminated histoplasmosis is very difficult, as clinical and radiological 

signs are non-specific and the sensitivity of fungal cultures is low, fungus often grows 

slow and requires an extended time for identification. Therefore, we evaluated a 

nested-PCR technique s1,2u, using J different extraction reagents to identify 

A"&-%'6+&)+*#+'&(6+-() in clinical specimens and reference strains to assess the 

specificity of the method. 

Material and Methods: Eight clinical specimens that yielded positive stains and 

cultures (6 blood samples with EDTA and 2 bone marrow samples), obtained from 

patients with disseminated histoplasmosis, attending the out patient service at the 

.eccián de MicologÜa Medica, Instituto de Medicina Tropical, were assessed. 

Additionally, purity and identity of 8 A"&-%'6+&)+*#+'&(6+-() isolates obtained also 

from clinical specimen cultures, were confirmed by routine mycological tests and 

assessed. Reference strains of 1+$+#%##"5"%"5.&*8$+&"6".,&"&, 7+,5"5+*@6+8$+-+, 

7$2'-%#%##(&*,.%U%$)+,& and 7%##"5"%"5.&*"))"-"& were tested to assess assay 

specificity. 

Results: Three different DNA extraction methods were used with the following 

reagentsM Guanidine isothiocyanate (.igma), 2enzoyl chloride (.igma) and Triton j- 

100. The samples were amplified by nested-PCR technique as described by 2ialeB et 

al. s1,2u, and analyzed by J_ agarose gel electrophoresis. 

Discussion: Among the J extraction methods tested, Guanidine isothiocyanate 

yielded by utmost good quality DNA and was easy to perform, when used with clinical 

specimens. Each of the reference strains showed the same signal upon amplification 

with a band of 21O bp, as well as [ blood samples and both bone marrow specimens, 

with high sensitivity (1pg/$l A"&-%'6+&)+*#+'&(6+-()*DNA) and specificity (no cross 

reactivity with 1/*8$+&"6".,&"&, 7/*@6+8$+-+, 7/*,.%U%$)+,& and 7/*"))"-"&). 

P-0376. Detection of non-Aspergillus fungal species using the Platelia TM 

Aspergillus EIA 

Cummings ( 1 , (amison G 1 , 2oudreaux ( 1 , Howles M 1 , aalsh T 2 , Hayden R 1 

1 Department of Pathology, .t. (ude Childrenes Research Hospital, Memphis, 6.A, 

2 Immunocompromised Host .ection, Pediatric fncology 2ranch, NCI, 2ethesda, 6.A 

Purpose: Invasive aspergillosis (IA) carries a high morbidity and mortality among 

immunocompromised patients and its diagnosis remains a clinical challenge. The 

Platelia TM Aspergillus EIA detects circulating galactomannan in serum and has been 

previously demonstrated to facilitate rapid and sensitive detection of IA. As the clinical 

impact of non-Aspergillus related fungal infection continues to become apparent, 

determination of whether positive galactomannan results may be attributed to 

infections with non-Aspergillus infection will be increasingly important. 

Methods and procedures: [J fungal isolates, including 11 yeast, JO mould, and J 

dimorphic species (six Z/*5.$)+-"-"5"& isolates) were included in the study. A 

standardized turbid suspension of each isolate was filtered and the supernatant 

serially diluted across a 10 [ - fold range. .amples were then blinded and assayed in 

duplicate, with index values %0.[ regarded as positive. Z/*5.$)+-"-"5"&*isolates were 

tested in both the yeast and mould phase. 

Results: Five of the OO non-Aspergillus species tested positive for galactomannan. 

Positive isolates included Z6+&-%)2#.&*5.$)+-"-"5"&, `"@$%&'%$+*%$2d+., 1+.#"6%)2#.&* 

6"6+#",(&, 1.,"#"66"()*&'.#".&, and >$"#?%-?.#"()*$%&.()n*all were positive only in 

presence of high concentrations of organism (1M10 J dilution of culture filtrate). .ix 

isolates of Z6+&-%)2#.&*5.$)+-"-"5"&*were testedn all tested positive in the mold phase, 

but negative in the yeast phase. 

Conclusions: The Platelia TM Aspergillus EIA, while highly specific for 4&'.$@"66(& 

species, does exhibit some degree of cross-reactivity with other clinically significant 

fungi. As false-positive results in this study were only seen in the presence of high 

concentrations of non-Aspergillus isolates, the clinical significance of such crossreactivity 

is uncertain. 

References: 

1. 2ialeB R, Fischer (, Feuch A, Najvar /, Dietz l, lnobloch ( et al. Diagnosis 

and Monitoring of Murine Histoplasmosis by a Nested PCR Assay. ( Clin 

Microbiol 2001n JKM1[06-1[0K. 

2. 2ialeB R, Feucht A, Aepinus C, (ust-Nybling G, Robertson V, lnobloch ( et 

al. Evaluation of Two Nested PCR Assays for Detection of A"&-%'6+&)+* 

#+'&(6+-() DNA in Human Tissue. ( Clin Microbiol 2002n O0M 16OO-16O. 


P-0377. Diagnosis of the genus Exophiala: Agents of human mycoses 

De Hoog G 1,2 , Zeng J 1,J 

1 Centraalbureau voor .chimmelcultures, 6trecht, The Netherlands, 2 2Institute of 

2iodiversity and Ecosystem Dynamics, Amsterdam, The Netherlands,, J Dermatology 

and Venereology, 6nion Hospital, Huazhong .cience and Technology 6niversity, 

auhan, Hubei, P. R. China 

Purpose of the study: 2lacB yeasts are defined as asexual fungi potentially able to 

produce melanized budding cells in any stage of their life cycle. They comprise some 

basidiomycetes and some members of the ascomycete orders 7?+.-%-?2$"+6.& and 

V%-?"5.+6.&. Among them, some species in the genus a_%'?"+6+ are highly virulent 

opportunists on humans and animals, and are frequently encountered in the clinical 

laboratory. In this presentation we summarize the diagnostics of the genus a_%'?"+6+. 

Methods: 2esides conventional morphological and physiological methods, molecular 

biological techniques (specific PCR, sequencing of IT. and /.6 rDNA and coding 

genes, and RF/P) are in current use to identify a_%'?"+6+ species. 

Results: .o far there are at least 16 clinically related a_%'?"+6+ species Bnownn some 

new species descriptions are underway. Though some species can be identified on 

the base of morphological and physiological characters, most species need to be 

identified further with molecular biological methods. .equencing of IT. rDNA is 

sufficient for identification of the majority of species. Nevertheless we found recently 

that a/*&'","U.$+ and a/*,"&?")($+. may share 2 different IT. motifs. .equencing of 

other genes (EF 1-!, #-tubuline) is necessary to verify identification of this species. 

.pecific primers are designed for a/*5.$)+-"-"5"& to distinguish the 2 IT. genotypes, 

one of which is prevalent in clinical cases while the other is preponderantly 

environmental. 

Conclusion: Morphological and physiological methods are still useful to identify 

a_%'?"+6+ species, but molecular biological methods and the use of dedicated data 

bases are becoming indispensable for a reliable diagnosis. Care should be taBen with 

uncritical use of IT. sequence data for identification. 

P-0378. Real time PCR for detection of Pneumocystis jiroveci from 

bronchoalveolar lavage fluid 

de Monbrison F, Romeuf N, Prat C, Picot . 

lA2fRATfIRE DE PARA.ITf/fGIE MYCf/fGIE MEDICA/E, Hf.PICE. CIVI/. 

DE /YfN, /yon, France 

1,.()%#2&-"&*\"$%0.#" is an important cause of pneumonia in immunocompromised 

individuals. This fungus cannot be cultured by routine methods and the diagnosis, 

based on microscopic examination, requires expertise for accurate identification. To 

avoid these difficulties, real time PCR has been developed to provide sensitive and 

objective detection of*1,.()%#2-"& from respiratory specimens. 

fur purpose was to improve the diagnosis of 1,.()%#2-"& infection and to evaluate 

the real time PCR assay in routine use. 

167 2ronchoalveolar lavage (2A/) fluids were collected from immunocompromised 

patients (including patients with AID. or patients receiving prolonged corticosteroid 

therapy, intensive immunotherapy) and were prospectively investigated by Gomori- 

Grocott, rapid Giemsa liBe staining techniques or real time PCR. 

Primers and probes were designed to a 2OJ pb region of the M.G (Major .urface 

Glycoprotein) gene of 1/*\"$%0.#"*(/arsen et al). Internal positive control has been 

included to assess PCR inhibition related to the 2A/ samples. DNA from mouse was 

spiBed into each DNA 2A/ samples and amplified using specific primers. A negative 

result for the internal control suggests PCR inhibition. Negative 2A/ by microscopy 

was tested for cross-reactivity. Positive 2A/ was used as control material. .amples 

were treated with 6racyl DNA Glycosylase to avoid samples contamination with DNA 

amplicons. 

Data were performed with fluorescence curves analysis. A sample is positive when an 

amplification curve is observed whatever the crossing point. 

To decrease PCR positive results resulting from colonization or asymptomatic 

carriage of 1,.()%#2&-"&, we decided to use the PCR assay in a qualitative fashion. 

The real time PCR results was interpreted in conjunction with clinical data to assess 

the significance for the patient. 

JJ specimens corresponding to 2[_ of 2A/ were PCR positive. 27 2A/ were 

obtained from HIV negative immunocompromised patients. The real time PCR is of 

interest in this population who develop 1,.()%#2&-"& pneumonia with lower fungus 

rates than AID. patients. This situation leads to a decreased sensitivity of microscopic 

testing. 

In conclusion, Real time PCR is a rapid technique (less than J hours including DNA 

extraction, amplification and interpretation) compatible for routine use in a 

Parasitology-Mycology laboratory for the detection of*1,.()%#2&-"& from 2A/ fluid 

providing an increase in sensitivity compared to microscopic examination.

P-0379. Comparison of disk diffusion (modified M44-A) assay with reference 

(M38-A microdilution) method for testing moulds against voriconazole 

Espinel-Ingroff A 1 , Canton E 2 , Gibbs D J , aang A J , Hsiung A O 

1 VC6 Medical Center, Richmond, 6.A, 2 Hospital /a Fe, Valencia, .pain, J Giles 

.cientific Inc., .anta 2arbara, 6.A, O Hardy Diagnostics, .anta Maria, 6.A 

Background: Although the C/.I (formerly NCC/.) MJ8-A document describes 

guidelines for testing moulds, clinical laboratories need less cumbersome methods for 

determining the susceptibilities of moulds to antifungal agents. ae compared 

reference MJ8 MICs to zone diameters for 1K1mould isolates. 

Methods: The following 1K1 clinical isolates*(7 48&"5"+*#%$2)8"U.$+, 8 46-.$,+$"+ spp., 

1O to 28 isolates each of 4&'.$@"66(&*U6+0(&, 4/*U()"@+-(&, 4/*,"5(6+,&, 4/*,"@.$, and 4/* 

-.$$.(&, 11 Z"'%6+$"s spp., 22 ^(&+$"()*spp.,*6 !(#%$ spp., K 1+.#"6%)2#.&*6"6+#",(&, 

16 P?"d%'(&*spp., and 18 =#.5.&'%$"() spp.) and two ~C isolates (7+,5"5+*[$(&." 

ATCC 62[8 and 1+.#"6%)2#.&*0+$"%-"" ATCC MYA-J6J0) were tested simultaneously 

by both methods. Reference MICs were obtained as described in the MJ8 document 

and zone diameters on Mueller-Hinton agarr2_ glucose and 0.[ mcg/ml methylene 

blue (Hardy Diagnostics)n two voriconazole disB concentrations (1 and 10 mcg) were 

evaluated. MICs were the lowest drug dilutions that showed 100_ inhibition at the 

incubation times recommended for each species (MJ8-A document)n zone diameters 

for each isolate were measured at 16-2O, O8 and 72 h. 

Results: The agreement between reference voriconazole MICs and disB diffusion 

results was incubation time and species dependentM 16-2O h (R} .K[8K and .KOKJ, 1 

and 10 mcg disB, respectively for 4/*U6+0(&, 4/*,"@.$ and Zygomycetes), O8 h 

(R} .826[ and .707K, 1 and 10 mcg, respectively for 4/*U()"@+-(&3*4/*,"5(6+,&, *4/* 

-.$$.(&*and*^(&+$"() spp.)*and 72 h*GR}.7KJ[ and .7[78, 1 and 10 mcg, respectively 

for 46-.$,+$"+*spp., Z"'%6+$"&*spp.3*1/*6"6+#",(& and =#.5%&'%$"() spp.). 

Conclusions: These data suggest the potential value of the simple and more 

economical disB diffusion method for testing voriconazole against mould isolates in the 

clinical laboratory. The clinical value of these in vitro results as predictors of 

therapeutic outcome is yet to be established. 

. 

P-0380. Evaluation of the new Fungifast ® AFG yeast antifungal susceptibility 

test: comparison with CLSI/NCCLS M27-A2- or E-test method 

Faure f, /ebeau 2, Pelloux H, Grillot R 

.ervice de Parasitologie-Mycologie- CH6 Grenoble - FRANCE 

IntroductionM Antifungal susceptibility testing of yeasts are increasingly requested due 

to the emergence of resistant isolates and the larger number of antifungal agents 

available. Methods should be reliable, reproducible and easy to perform. 

F6NGIFA.Tw AFG (INTERNATIfNA/ MICRf2If) is a new commercially developed 

tray containing [ antifungalsM amphotericin 2 (AM2), flucytosine ([FC), fluconazole 

(FCA), itraconazole (ITZ) and voriconazole (VfR). This tray is designed to give MIC 

results in 2Oh or O8h. ae evaluated the value of this system in comparison with the 

C/.I (formerly NCC/.) M27-A2 method or the E-Test method (A2 2iodisB), on K2 

yeast clinical isolates representing 12 species. 

Material and MethodsM Ninety-two isolates were evaluatedM 81 clinical isolates, 11 

collection strains (rare species or from deep sites). Two ~uality Control strains (7/* 

'+$+'&"6%&"& ATCC 2201K and 7/*[$(&." ATCC 62[8) were included weeBly as 

controls. .pecies distribution wasM 7/*+68"#+,& J1.[_, 7/*@6+8$+-+ 2J.K_, 7/*-$%'"#+6"& 

10.K_, 7/*'+$+'&"6%&"& 7.6_ and other 7+,5"5+ and non 7+,5"5+ species 26_ (8 

species). Each isolate was subcultured for 2Oh in CANDICHRfMw II medium. The 

strains were tested in parallel with E-Test method (RPMI agar) for AM2, or C/.I M27- 

A2 broth micro dilution method for other antifungal agents. For C/.I M27-A2, the 

endpoints were obtained by reading visually and spectrophotometrically (MIC 70_ in 

comparison with growth control) after O8h. For F6NGIFA.Tw AFG, 10$/ of inoculated 

suspension medium at 2McF were added to the ME. F6NGI medium, and 100$/ 

were inoculated in each well. Colorimetric reading was performed at 2Oh (or O8h) with 

a change in the growth indicator from blue to pinB. 

Results and conclusionM F6NGIFA.Tw AFG method demonstrated no lacB of growth 

for the clinical isolates tested, and 8[_ of the MIC values were obtained after 2Oh of 

incubation. ~C strains MIC values were in good conformity within the limits reported. 

Very good categorical agreement was obtained with AM2, [FC and VfR (100_, 

K1.J_, and 88_ respectively), and good for ITZ (7[_ with 2J minor discrepancies) 

and FCA (71.7_ with 2[ minor and 1 major discrepancies). Regarding these two last 

azoles, MICs tended to be slightly higher mainly with 7/*+68"#+,& and 7/*@6+8$+-+ 

strains. Three very major discrepancies (J 7/*+68"#+,&) were observed with VfR and 

because 2 were found susceptible when evaluated by E-Test method, these isolates 

certainly presented significant trailing growth particularly with the C/.I M27-A2 

method. Thus, the results for isolates with significant trailing growth should be 

carefully interpreted, and such strains should be tested repeatedly. ae plan to 

evaluate secondarily these strains by E-Test method, and new reading criteria for 

F6NGIFA.Tw AFG interpretation. The F6NGIFA.Tw AFG test is rapid, reproducible, 

easy to perform and could be a suitable alternative for routine antifungal susceptibility 

testing of clinical isolates of yeasts. 


P-0381. (1*J)-beta-d-glucan (BG) testing in invasive fungal infection (IFI): 

Potential sources of contaminatioN 

Finkelman M, /empitsBi . 

Associates of Cape Cod, Inc. 

Purpose: .erum (1*J)-!-D-glucan (2G) quantitation has been proven to have utility 

in the diagnosis of IFI and is a FDA-cleared diagnostic in the 6.. .erum levels above 

60-80 pg/m/ are sensitive indicators of IFI. Although the test reagent is highly specific 

for 2G, patient exposure to a variety of materials, by multiple routes, may contribute to 

serum burdens similar to those observed in IFI. These materials include fungal 

fermentation products, cellulose-filtered drugs and biologics, 2G-containing drug 

formulation excipients, and regenerated cellulose hemodialysis membranes. In 

addition, vaginal mucosal surface exposure occurs though the use of cotton menstrual 

tampons. 

Methods: The potential contribution of leached 2G from devices such as medical 

gauze sponges, pacBings, tampons, and similar cotton products was investigated. 

This was assessed by soaBing these products in defined amounts of 2G-free normal 

saline and measuring the levels of released 2G. The impact of ingested 2G, both 

soluble (Curdlan) and particulate (yeast sacculi) was assessed by post-ingestion 

monitoring of serum levels, in human subjects. The Fungitell Assay (Associates of 

Cape Cod, Inc., Falmouth, MA) was used to quantitate 2G. 2riefly, this assay uses a 

2G-specific preparation of the /imulus Amebocyte /ysate (/A/) serine protease 

cascade, and a chromogenic peptide, to provide, through /A/ cascade activation, a 

quantitative assessment of 2G, in serum. 

Results: Cotton medical devices were observed to leach large quantities of 2G 

(millions of picograms) into normal saline, very rapidly (v1[ minutes). Their invasive 

use may produce elevated serum 2G, potentially leading to false-positive diagnostic 

results. Analysis of serum 2G after ingestion of multi-gram quantities of 2G produced 

no impact upon serum 2G levels, in normal subjects, over an 8 hour period. The 

impact of ingested 2G remains to be investigated in patients with impaired bowel 

integrity. 

Conclusions: The interpretation of positive serum 2G results in patients at risB for IFI 

should also include an assessment of alternate potential sources of serum 2G. 

Heightened awareness of patient contamination possibilities should improve diagnosis 

accuracy. 

P-0382. Detection of Apergillus galactomannan (GL) in febrile neutropenic 

patients at 6 hospitals in Argentina. 

Finquelievich J 1 , .carano ., Altclas (, Herrera F, Guerrini G, Tula /, Cozzi (, (ordan 

R, Agorio I, De Vedia / 

1 Centro de Micologia Facultad de Medicina 62A, 2 Centro de Micologia Facultad de 

Medicina 62A, J .anatorio Mitre 2uenos Aires, O CEMIC, 2uenos Aires, [ Hospital 

Rossi, /a Plata, 6 F6NDA/E6, 2uenos Aires, 7 CETRAMfR, Rosario, 8 Hospital 

2ritanico de 2uenos Aires, K Hospital 2ritanico de 2uenos Aires, 10 Centro de Micologia 

Facultad de Medicina 62A 

Invasive aspergillosis (IA) is one of the major causes of morbidity and mortality in 

neutropenic patients who received chemotherapy for hematological malignancies or 

underwent bone marrow transplant. 

An early diagnosis is still today difficult. Galactomannan detection by enzyme 

immunoassay (E/I.A) has demonstrated to be useful in the diagnosis of invasive 


The aim of this study has been to evaluate the performance of the commercial 

enzyme immunoassay test ( Plateliaã 4&'.$@"66(&. 2If-RAD) for the detection of 

galactomannan antigens (GM) on sera from patients at risB of invasive aspergilosis in 

6 Argentine Hospitals 

Materials and methods: For each patient, a form with the following information was 

filled inM Name age, underlying disease, results of image techniques, antifungal 

therapy/prophylaxis, direct mycology diagnosis and piperacilina-tazobactam treatment. 

Tests for detection of GM were performed twice weeBly, from the start of the episode 

of febril neutropenia till neutrophil recovery. Indices from 1.[ or more were considered 

positive according to manufacturer recommendations. 

Results and commentaries: 2[0 serums from 61 patients who developed 70 

episodes of neutropenia and fever were included in the study. The underlying 

immunosuppressive diseases wereM haematologic malignancies 60 and 

neuroblastoma 1. Thirty-five of them had received bone marrow transplant. 

Results of GL detection were: 0.1to 0.[ in 1[[ samples, 0,6 to 0,K in 68, 1 to 1,O in 

8 and 1,[ or greater in 1K. /ungs radiological and tomographic injuries compatible 

with aspergilosis were demonstrated in 10 patients while 2 showed extrapulmonary 

lesions. .ignificative GM results were associated in five episodes. fut of 106 samples 

from patients treated with piperacilina-tazobactam, 10_ demonstrated GM indexes t 

1. None of them presented lesions compatible with disseminated mycoses. 

The results obtained demonstrated low correlation between the presence of serum 

GM and signs or clinical symptoms of fungal diseases.

P-0383. Dominance of the Boulardii genotype in invasive Saccharomyces 

cerevisae infections in Belgium 

Fonteyne P, Auger A, .winne D, Nolard N 

.cientific Institute of Public Health 

In 200O, =+##?+$%)2#.&*#.$.0"&"+. has been the [ th cause of candidemia in 2elgium 

with 1[ cases for a total of 2[1 recorded episodes. This study was designed in order 

to evaluate the proportion of =/*#.$.0"&"+.*0+$/*8%(6+$5"" among fungemia due to =/* 

#.$.0"&"+.*in 2elgium. 

A one day method, in a K6 well plate format, for identification of the 8%(6+$5"" variety of 

=/*#.$.0"&"+. was developed on the basis of a previously described microsatellite 

length polymorphism (Hennequin et al. 2001. [(. Clin. Microbiol.MJK. 2. [[1-[[K). The 

test was applied on a panel of 62 =/*#.$.0"&"+. strains form the 2CCM-IHEM 

collection (httpM//www.belspo.be). Thirteen non clinical isolates were used as controls. 

Among clinical isolates, JO were from blood and 1[ from non naturally sterile locations. 

In 2elgium, the variety =/*#.$.0"&"+.*0+$/*8%(6+$5""*is responsible for most candidemia 

due to =/*#.$.0"&"+.. 

More caution should be taBen with =/*#.$.0"&"+.*0+$/*8%(6+$5""*probiotic preparations. 

P-0384. Measurement of (1-3)-beta-D-glucan as a diagnostic tool in 

disseminated histoplasmosis and blastomycosis 

Girouard G 1 , /achance C 2 , Pelletier R 2 

1 6niversitd /aval, ~udbec, Canada, 2 /eHÇtel-Dieu de ~udbec du CH6~, ~udbec, 

Canada 

Purpose of the studyM Early and reliable detection of invasive fungal infection (IFI) is 

the corner stone of therapeutic success. (1-J)-beta-D-glucan assay (2G) (Fungitellã, 

Associates of Cape Cod) is a newly developed antigenic test intended to maBe rapid 

and sensitive IFI diagnosis. There is very few data using 2G detection for the 

diagnosis of endemic mycosis. ae tested Fungitell on serum samples collected from 

patients with proven invasive A"&-%'6+&)+*#+'&(6+-()*var. #+'&(6+-()*or 

Z6+&-%)2#.&*5.$)+-"-"5"& infection. 

.ummarized description of the projectM Proven A"&-%'6+&)+*#+'&(6+-() var.* 

#+'&(6+-() infection was define by a positive culture from a clinical specimen or 

positive (t O.0 EIA 6nits/ml) A/*#+'&(6+-() antigen detection (MiraVista diagnostic) 

from urine or serum sample in a symptomatic patient. Z6+&-%)2#.&*5.$)+-"-"5"& 

infection was defined by a positive culture or histopathology proven tissue infection. 

.erums collected from patients with a proven clinically active infection were expected 

to give a positive 2G detection. .erums collected before the acquisition of the 

infection or after a complete clinical cure were expected to give negative 2G results. 

Collected serums were Bept frozen at minus 20ÅC up to the testing procedure. All 

serums were tested on the same day, using the defined method. 2G data were 

compared to the pre-defined expected positive and negative results. 

ResultsM Fourteen serums from 6 A/*#+'&(6+-() infected patients were tested. .even 

of the K expected positive and one of the [ expected negative serums tested positive 

for 2G detection (chi-square, two-sided P valueM 0.0J6). fne false positive 2G result 

was possibly caused by a concomitant blood infection with gram-negative rod. A 

serum collected K6 days before the culture proven histoplasmosis diagnosis was 

considered as a false negative 2G because the patient had respiratory symptoms that 

could be attributable to A"&-%'6+&)+ by the time the serum was collected. The 

sensitivity, specificity, positive predictive value and negative predictive value were 

87_, 67_, 78_ and 80_, respectively. Four positive A/*#+'&(6+-() antigen serum 

tested positive for 2G. There was a correlation comparing A/*#+'&(6+-() antigen and 

2G value (r 2 } 0.88O6). 

Five serums from patients with proven blastomycosis (primary pulmonary n}On 

osteomyelitis n}1) were tested, including one from a completely cured patient. All 

serum tested negative for 2G. 

ConclusionsM 2ased on results from our population, 2G assay could be useful in the 

diagnosis and the management of histoplasmosis. 2lastomycosis infection was not 

detected by 2G assay, possibly because of the low cell wall (1-J)-beta-D-glucan 

content of the Z6+&-%)2#.&*infecting yeast form. 


P-0385. Value of two culture media for isolation of indoor environmental fungi 

using a volumetric method 

Gómez de Ana S 1 , Torres RodrÜguez ( 1 , Alvarado RamÜrez E 1 , Mojal Garcia . 2 

1 Research 6nit on Infectious Diseases and Mycology (6RMIM). Municipal Institute for 

Medical Research (IMIM). 6A2, 2arcelona,.pain., 2 Consulting .ervice on 

Methodology for 2iomedical Research, IMIM, 2arcelona, .pain 

Objectives: Indoor environment studies are important for the Bnowledgement of the 

fungal exposure. This information is useful for monitoring specially the high risB 

hospitalized patients and fungal allergic patients. This worB was desing for the 

optimisation of the culture media used with volumetric method in order to isolate and 

identification of house indoors fungi. 

Material and methods: Twenty-two different homes from fungal allergic patients 

living in 2arcelona city (.pain) were selected for fungal isolation by means of 

systematic air sampling. A portable Microflow 60 air sampler (A.P. 2ucB, Inc. frlando, 

Florida, 6.A) was employed in every home. Two different agar media in Rodac plates 

were used for culturing fungiM Malt Extract agar (MEA) and Rose-2engal agar (R2) 

with chloramphenicol. Air flow was impacted on the medium surface during J minutes 

and 20 seconds with a constant speed of 1.[ //sec (Total volume J00 /). All the 

procedures were done successively by duplicate. 

Inoculated plates were incubated at 2[oC for O to 10 days. All the colonies growing on 

the media were counted and identified. 

.tatistical signification was determined with the non-parametric test of 6 Mannahitney. 

These analyses were performed with the .P.. v 1O (.P.. Inc. Chicago). 

Results: An overall of 2KJ plates of each culture medium were seeded. The total 

number of fungal colonies with MEA was 6.OO8, mean of 22 colonies per plate and a 

median of 1[ colonies/plate. aith R2 the overall colonies was [.JK[, mean of 18 

colonies/plate and a media of 12 colonies/plate..tatistical analysis of these results 

showed significant differences between the number of the isolated colonies with both 

culture medium (p}0.00J). 

P-0386. Isolation of Issatchenkia occidentalis in the oesophagus of a 

leukemic patient 

Hamad S 1 , Moragues M 2 , Alhambra A J , Del Palacio A J , ~uindás G 1 , Pontán ( 1 

1 Departamento de InmunologÜa, MicrobiologÜa y ParasitologÜa, Facultad de Medicina y 

fdontologÜa/6niversity of 2asque Country/ 2ilbao/.pain, 2 Departamento de 

EnfermerÜa I, 6niversidad del PaÜs Vasco/2ilbao/.pain, J 6nidad de MicologÜa, 

Departamento de MicrobiologÜa, Hospital 6niversitario 12 de fctubre, Madrid, .pain 

K&&+-#?.,["+*%##"5.,-+6"& has been isolated from an oesophageal biopsy of a young 

leuBemic male patient who underwent bone marrow transplantation. At the time the 

specimen was taBen, the patient was also suffering from oesophageal herpetic lesions. 

Macroscopically the yeast grew as light creamy dull colored colonies in .abouraud 

Dextrose agar. Growth was better at J0 oC than J7 oC. The isolate was unable to grow 

at O0 oC. In CHRfMagar Candida medium the isolate grew as beige to pale pinB 

colored colonies The identification of the isolate was impossible by the biochemical 

galleries (API ID J2 C and API 20C A6j), but it was achieved by sequencing the DNA 

amplified by PCR using panfungal primers N/-1 ([]- 

GCATATCAATAAGCGGAGGAAAAG) and N/-O ([]GGTCCGTGTTTCAAGACGG) for 

the molecular identification. The amplicon (around 600bp) was sequenced and the 

sequence data were compared with the sequence database in NC2I 2last Gen2anB. 

The sequence obtained was KK.K_ homologous to that of K/*%##"5.,-+6"& (Gen2anB 

accession no. 676JO8.1). 

2y electron microscopy, the cells were elongated and showed a rough surface due to 

the existence of multiple protuberances, which are typical of this species. The 

identification was further confirmed by the observation of asci growing on Potato 

dextrose agar at J0o C for 21 days, stained by the linyoun stain. The isolation of K/* 

%##"5.,-+6"&*from our patient is exceptional in two ways. Although the clinical 

significance is not clear, to our Bnowledge, this is the first report of an isolation of K/* 

%##"5.,-+6"& from a clinical specimen. .econdly, it was isolated as the teleomorphic 

form of the fungus, a circumstance which is very infrequent in clinical specimens. 

Results presented in this report show that K/*%##"5.,-+6"& can be isolated from clinical 

specimens and emphasize the importance of DNA sequencing for the identification of 

rare fungal isolates. 

.eventy different fungal species were identified in MEA and 6O species in R2, but not 

statistical differences were found (p}0.O77). 

.terile mycelia were found in higher proportion in R2 medium. 

Conclusions: According these results in isolating air fungi with a standardized 

volumetric sampler, Malt Extract agar has been more sensitive than Rose-2engal agar 

medium. Although there was not found statistical differences on the fungal species 

identified with both media, the MEA medium allowed a higher fungal identification 

because lower mycelia sterile were obtained.

P-0387. Quantitative analysis of cutaneous malassezia in a patient with atopic 

dermatitis using real-time PCR with a TaqMan probe 

Harada S 1 , .ugita T 1 , Tajima M 2 , TsubuBu H J , Tsuboi R 2 , NishiBawa A 1 

1 Meiji Pharmaceutical 6niversity, ToByo, (apan, 2 ToByo Medical 6niversity, ToByo, 

(apan, J .elf Defense Force Central Hospital, ToByo, (apan 

Introduction: !+6+&&.d"+, a lipophilic yeast, colonizes the sBin of the head, necB, and 

shoulder in humans, and is associated with pityriasis versicolor, seborrheic dermatitis, 

and atopic dermatitis (AD). For AD patients with damaged barrier function, the yeast 

acts as an exacerbating factor. In AD patients, application of antifungal agents 

(Betoconazole or itraconazole) decreases the severity of eczematous lesions. In 

addition to clinical evaluation, mycological evaluation is also needed for antifungal 

therapy in AD. In this study, we developed a non-culture-based quantitative method 

of determining cutaneous !+6+&&.d"+ using real-time PCR with a TaqMan probe. 

Materials and Methods: fur previous study demonstrated that !/*@6%8%&+ and !/* 

$.&-$"#-+ were the major cutaneous flora of AD patients. A TaqMan probe for detection 

of these microorganisms was designed from the intergenic spacer (IG.) region of the 

rRNA gene. In addition, a detection probe for all !+6+&&.d"+ species was designed 

from D1/D2 26. rDNA sequences. Thirty samples of !+6+&&.d"+ were collected by 

applying fp.ite transparent dressings to the sBin of AD patients. Amplification and 

detection were performed with the A2I PRI.M 7[00 sequence-detection system. 

Results and Discussion: Each*!/*@6%8%&+W and !/*$.&-$"#-+-specific probe detected 

only the intended species. The detection probe for all !+6+&&.d"+ species 

successfully detected DNAs of all !+6+&&.d"+ species. A standard curve was plotted 

using the 7- values obtained from the amplification of Bnown quantities (10 1 |10 K 

copies) of plasmid DNA containing the target region. A good correlation existed for 

between 10 1 and 10 K copies of plasmid DNA. The amount of !+6+&&.d"+ on the face 

and necB was approximately five times that on the body and limbs. !/*$.&-$"#-+ 

colonized 1.6 times more than !/*@6%8%&+. fur method can be used to evaluate the 

amount of cutaneous !+6+&&.d"+ after administration of antifungal agents to AD 

patients. (Please refer to our related poster) 

P-0388. C. neoformans: Varieties in Venezuela by microscan biotyping 

Hartung de Capriles C 1 , Pdrez C 1 , Martinez a 1 , Rosellá A 1 , Reyes H 2 , Colella M 1 , 

flaizola C 1 , Magaldi . 1 , Mata-Essayag . 1 

1 Instituto de Medicina Tropical, 2 .eccián de MicologÜa, Hospital Domingo /uciani, 

Caracas, Venezuela 

Introduction: The auto.canw-O automated microbiological panel reader utilizes an 

optical system to detect fungal growth in the wells of Micro.canw panels. In addition, 

selected wells contain biochemical substrates which exhibit a color change in the 

presence of certain fungi, and may with accuracy identify different species if they 

show signs of physiological differences. Diverse varieties and serotypes*of* 

7$2'-%#%##(&*,.%U%$)+,& are recognized, along with geographical differences in the 

prevalence of the various serotypes. The aim of this study was to determine the 

capacity of Micro.can biotyping, to differentiate these varieties in Venezuelan clinical 

cases, perhaps allowing view and record results in minimal time. 

Materials and methods: Eighty two 7$2'-%#%##(&*,.%U%$)+,& clinical isolates were 

recovered from cryptoccosis cases diagnosed or confirmed between 1KK[ to 200O at 

the Departamento de Micologia, Instituto Nacional de Higiene and the .eccián de 

MicologÜa Mddica, Instituto de Medicina Tropical, preserved by Castellanii method. To 

ensure purity and variety of the preserved isolates, they were identified by 

standardized methods and by /-canavanine, glycine and bromothymol blue agar 

media (CG2), as well as tested with Micro.canw rapid yeast ID panel with 

auto.canw-O panel reader, to determine their biotype number. 

ResultsM Micro.canw rapid yeast ID panel revealed 27 different biotype numbers. ff 

the 82 isolates typed by CG2, K1.O6 (n}7[) _ were*7/ ,.%U%$)+,& var neoformans 

and 8.[O_ (n}7) 7/ ,.%U%$)+,& var @+--"". No significant differences between biotypes 

and varieties were found (pt0.0[). However, there was a significant difference 

between 7/,.%U%$)+,& var @+--""" and var ,.%U%$)+,&*+,5*the assimilation of p- 

nitrophenyl-N-acetyl-#-D-glucosamine (NAG) (pv0.0[). 

6nfortunately only O6.JO_ of all patients were Bnown to be immunocompromised 

(K7.J7 _ AID. and 2.6J_ systemic lupus erithematous (./E), the immunological 

condition of the remaining patients be ingundisclosed. 

DiscussionM ae found that the majority of the typed isolates were K1.O6_ var. 

,.%U%$)+,&3 followed by 8.[O _ were var. @+--""/*The Micro.canw rapid yeast ID panel 

was incapable to differentiate both varieties. 


P-0389. Biochip-based detection of resistance in Candida albicans 

Hauser N, .chwarz H, Rupp . 

Fraunhofer IG2, .tuttgart, Germany 

aith the increasing number of immunocompromised patients worldwide, antifungal 

treatments have increased with a paralleled increase of the incidence of antifungal 

resistance. 

Antifungal resistance mechanisms fall into different categories according to their 

molecular principles as for instance transport or target alterations. 

Target gene alterations as a cause for antifungal resistance have been observed in 

several alleles of the aP9II gene encoding lanosterol demethylase, the target of 

azoles in fungal pathogens. In 7/*+68"#+,&, many diffierent single point mutations and 

loss of heterocygocity in the aP9II gene have been described. 

ae have established a biochip-based method for the detection of resistance in 

7+,5"5+*+68"#+,&/*Initially, a model-system using synthetic templates was developed 

to optimise the experimental parameters for the chip-based enzymatic reaction. This 

system is further used as control and for normalisation. The optimised system could 

be adapted for the detection of .NPs (single nucleotide polymorphisms) and /fHs 

(loss of heterocygocyty) with high specificity and sensitivity. 6sing genomic DNA from 

clinical isolates homocygous and heterocygous situation associated with resistance 

could be detected. 

P-0390. Expression of galactomannan antigenemia in pediatric oncology 

patients with invasive aspergillosis 

Hayden R 1 , Pounds . 2 , .chaufele R J , .ein T J , aalsh T J 

1 Department of Pathology, .t. (ude Childrenes Research Hospital, Memphis, 6.A, 

2 Department of 2iostatistics, .t. (ude Childrenes Research Hospital, Memphis, 6.A, 

J Immunocompromised Host .ection, Pediatric fncology 2ranch, NCI, 2ethesda, 6.A 

Purpose: Invasive Aspergillosis (IA) represents an increasing cause of morbidity and 

mortality among children with cancer. Galactomannan antigenemia has been well 

studied in the detection of invasive aspergillosis in adult patientsn however, however, 

little is Bnown about the expression of circulating galactomannan in 

immunocompromised children with IA. 

Methods and procedures: ae studied the expression of galactomannan antigen in 

KK0 serum samples from [6 pediatric oncology patients. .amples were blindly tested 

at two different institutions using the Platelia TM Aspergillus EIA (GM EIA). Patients 

ranged from J months to 18 years in age and included 17 with proven or probable IA 

(by EfRTCIFICG/NIAID-M.G criteria). Any sample with a GM EIA index value of 

%0.[ was considered positive. 

Results: GM EIA was positive in KK of J0O samples from patients with proven or 

probable IA. At least one sample was positive for 11 of 17 pediatric oncology patients 

(6[.7_ sensitivity, K[_ CI } J8.J_ - 8[.7_) with IA. fnly 7 of 686 (1.0_) samples 

from JK control subjects resulted in a positive GM EIA result. At least one sample 

tested positive in [ of the JK controls (12.8_, K[_ CI } O.J_ - 27.O_). No 

statistically significant association between accuracy and patient age was observed. 

Among 7 evaluable GM-positive children with IA, the GM EIA produced a positive 

result prior to clinical or radiographic evidence of infection in 6 cases, with the 

advantage in time to diagnosis ranging from 1 day to JO days. In the remaining case, 

GM a positive result was seen on the same day as diagnosis by other methods. 

Conclusions: The presence of circulating GM is predictive of IA in most pediatric 

oncology patients. .ensitivity of the EIA may be reduced by concomitant therapy with 

mould-active anti-fungal agents. GM antigenemia may precede subsequent clinical, 

microbiological, or radiographic evidence of IA.

P-0391. Diagnosis of cerebral aspergillosis in patients with hematologic 

malignancies by polymerase chain reaction from cerebrospinal fluid (CSF) 

samples 

Hummel M 1 , .piess 2 1 , lentouche l 2 , Niggemann . J , Reuter . O , liehl M [ , Mérz H 1 , 

Hehlmann R 1 , 2uchheidt D 1 

1 III. Medizinische lliniB, lliniBum Mannheim, 6niversity of Heidelberg, D-68J0[ 

Mannheim, 2 lliniB fyr linder- und (ugendmedizin, Friedrich-.chiller-6niversitxt (ena, 

077OJ (ena, J lliniB fyr linderheilBunde, lliniBum Mannheim, 6niversity of Heidelberg, 

D-68167 Mannheim, O Medizinische lliniB III, 6niversitxtsBliniBum 6lm, [ Medizinische 

lliniB,, lliniBum FranBfurt/fder 

Background: Cerebral aspergillosis (CA), a life-threatening disease with high 

mortality rates, is difficult to diagnose. aith the exception of 7$2'-%#%##(&*,.%U%$)+,&, 

fungi are rarely detected in cultures of cerebrospinal fluid (C.F) and biopsy of cerebral 

lesions is often not feasible. New diagnostic methods, including DNA detection by 

PCR have emerged for diagnosing invasive aspergillosis (IA). .everal PCR assays 

have been validated for blood samples but there is few experience with PCR from 

C.F samples. 

Methods: ae investigated J[ C.F samples from 6 patients with hematologic 

malignancy and possible, probable or proven CA by a previously described nested 

PCR assay detecting 4&'.$@"66(& DNA, using a modified DNA extraction protocol. 

.amples with positive results in the nested PCR assay were quantified in a 

/ightCycler PCR assay. IA was classified according to EfRTC/M.G 2002 criteria. 

C.F samples from 10 patients without CA were investigated by PCR as controls. 

Results: Two patients had proven, probable and possible CA, respectively. Each of 

the patients with CA had at least one positive PCR result. Fourteen C.F samples from 

patients with CA gave positive results in the nested PCR assay. .ix of these samples 

were positive in the /ightCycler PCR and were thus quantified. The range of numbers 

of gene copies per ml was [2OO to 6J,100,000. .erial sampling showed a positive 

correlation between clinical improvement and PCR findings in 2 patients. PCR from 

C.F samples from 10 control patients gave negative results. 

ConclusionM PCR from C.F samples by our nested 4&'.$@"66(& PCR assay is a useful 

tool in diagnosing CA. PCR results are obtained more rapidly than culture results and 

in our patient cohort, PCR was more sensitive than C.F culture. ~uantification of the 

pathogen load in C.F samples is feasible, using our /ightCycler PCR assay. The 

clinical impact of quantitative PCR has to be investigated in further studies. For 

determination of sensitivity and specificity of the nested PCR assay from C.F 

samples, studies with larger patient numbers are in preparation. 

P-0392. A tof-mass based diagnosis for Aspergillus infection 

Izumikawa K, .aijyo T, .eBi M, Yanagihara l, Higashiyama Y, MiyazaBi Y, HiraBata 

Y, Tahsiro T, lohno . 

NagasaBi 6niversity .chool of Medicine, NagasaBi, (apan 

Introduction: 4&'.$@"66(& infection in immuno-compromised host has emerged as a 

major cause of death especially in patients with hematological malignancies and in 

stem cell transplant recipients. Although early and predictive diagnosis of 4&'.$@"66(& 

infection is critical for good prognosis, the diagnostic tool is still limited. 4&'.$@"66(& 

galactomannan antigen detection by enzymed liBned immuno-assay is non-invasive 

and recognized as a gold standard tool. However the sensitivity and specificity of this 

assay is relatively low and has not been proven to be sufficient. The new diagnosing 

tool for 4&'.$@"66(& infection is required for early diagnosis. The ProteinChip platform, 

based on surface enhanced laser desorption/ionization (.E/DI) time-of-flight mass 

spectrometry is newly developed technology for analyzing the profiles of proteins from 

clinical samples. This system enables to discover the biomarBers for various diseases. 

The objective of this study is to discover the new biomarBers of 4&'.$@"66(& infection. 

Materials & Methods: 4&'.$@"66(& U()"@+-(& MF1J and other 4&'.$@"66(& strains from 

clinical samples were used for analysis. Protein was extracted from 4&'.$@"66(& by 

mechanical agitation$ with glass beads. .erum samples from patients who were 

diagnosed as 4&'.$@"66(& infection confirmed by positive culture and/ or pathological 

findings in our ward were investigated. fther serum from patients with various 

pulmonary infectious diseases other than aspergillosis and healthy volunteer were 

also used as negative controls. Expression difference mapping analysis profiles of the 

clinical samples and of crudely extracted fungal protein were obtained by using weaB 

cation exchange ProteinChip Arrays, CM10. The ProteinChip Arrays were analyzed in 

the ProteinChip 2iology .ystem Reader and acquired data was analyzed by 

ProteinChip software (Ciphergen 2iosystems, Inc.). 

Results: A total of [0 to 100 separate protein peaBs were detected in the mass range 

of 2-20BDa in serum samples and extracted proteins from 4&'.$@"66(&. Most patterns 

of positive peaBs were different from each other sample. Common peaBs at 8200, 

8700 and 12000 Da were detected both in clinical samples from aspergillosis patients 

and extracted fungal protein from 4&'.$@"66(& species. These overlapped peaBs were 

not detected in the clinical samples from patients with other pulmonary infectious 

diseases and healthy volunteers. 

ConclusionM The aim of this study is to discover the specific peaBs from clinical 

samples of 4&'.$@"66(& infected patients in order to use for early diagnosis. Protein 

profiles acquired by ProteinChip analyzing system revealed the specific multiple peaBs 

in clinical serum samples from patients with 4&'.$@"66(& infection and these peaBs are 

considered to be originated from 4&'.$@"66(& itself. .ince these peaBs were specific, 

they could be biomarBer Candidates for diagnosis. This is the first report using 

ProteinChip Arrays for diagnosisnig 4&'.$@""6(& infection from clinical samples. 


P-0393. Newly developed chromogenic agar and culture based identification kit 

for Malassezia species 

Kaneko T 1 , MaBimura l 2 , fnozaBi M 1 , .hiota R J , .ugita T O , aatanabe . [ , 

Yamaguchi H 2 

1 lanto Chemical Co.,Inc, 2 TeiByo 6niversity, J TaBinomiya Hospital, O Meiji 

Pharmaceutical 6niversity 

The aims of this study wereM i) develop the identification Bit for !+6+&&.d"+ easily, quicBly, and at 

reasonable cost without any expensive or special equipment and ii) the evaluation of the 

distribution of !+6+&&.d"+ species on various human body sites by developed identification Bit. 

ae developed chromogenic agar containing vital growth factors and culture based identification 

Bit for K species of !+6+&&.d"+ (!/*U($U($, !/*&6%%UU"+., !/*&2)'%5"+6"&, !/*$.&-$"#-+, !/*%8-(&+, !/* 

@6%8%&+, !/*'+#?25.$)+-"&, !/*5.$)+-"&, and !/*\+'%,"#+) by means of their biological features. 

This method utilizes Tween O0-based precipitate production on modified chromogenic agar 

Malassezia medium, growth on specific agars (.abouraudes dextrose agar, Cremophor E/ agar, 

Tween 60-esculin agar), and catalase reactions. This identification Bit was verified with 11 type 

and reference strains of K !+6+&&.d"+ species. An additional clinical isolates were also 

successfully identified using the Bit and the results were confirmed by molecular biological 

analysis. Furthermore, the distribution of !+6+&&.d"+ species on various human body sites was 

investigated by developed identification Bit. 

.trains 

growth on 

Catalase 

CHR .DA TE E/ Reaction 

!/*'+#?25.$)+-"& C2. 187K GP G G2 G r 

!/*&2)'%5"+6"&*C2. 7222 GP N G2 N r 

!/*@6%8%&+*C2. 7K66 GP N N N r 

!/*5.$)+-"&*(CM11JO8 GP N GN N r 

!/*5.$)+-"&*(CM11O70 GP N GN N r 

!/*U($U($*C2. 1878 G N G2 G ! 

!/*&6%%UU"+.*C2. 7K[6 G N GN N r 

!/*%8-(&.*C2. 7876 G N N2 N r 

!/*$.&-$"#-+*C2. 7877 G N N N - 

!/*\+'%,"#+*MKK66 G N G2 N r 

!/*\+'%,"#+*MKK67 G N G2 N r 

esculin agar 

E/n Cremophor E/ agar 

Gn growth 

Nn no growth 

GPn growth and production of precipitate 

G2n growth and blacB zone 

GNn growth and no change 

N2n no growth and blacB zone 

rn test positive 

-n test negative 

CHRn 

modified 

CHRfM 

agar 

Malassez 

ia 

.DAn 

.aboura 

udes 

dextrose 

agar 

TEn 

Tween 

60- 

1) Modified CHRfMagar Malassezia (CHR) was most superior to the modified DIjfN 

agar (DIjfN) and fil-.DA used for comparison in terms of its ability to suppress bacterial 

growth and isolation of !+6+&&.d"+. 

6sing CHR with three specific agars and catalase reactions, 11 type and reference strains and 

all clinical isolates were successfully identified easily, quicBly, and at reasonable cost without any 

expensive or special equipment. 

P-0394. Evaluation of sweet potato (ipomoea batatas (l.) lam) agar medium for 

recovery of yeast from clinical specimen types 

Kechia F 1 , NjiBam N 2 , /ohoue ( 1 

1 Faculty of Medicinie and 2iomedical .c., 6niv. of Yaounde-I, Yaounde-Cameroon, 

2 Department of 2iology and Animal Physiology, Faculty of .cience 6niv. of Yaounde-I, 

Yaounde-Cameroon 

To evaluate a locally developed medium made of K'%)%.+*8+-+-+& for recovery of 

yeasts from clinical specimen types and to compare the results with those obtained 

with .abouraud dextrose agar (.DA). 

A cross sectional survey of hospital based patients was carried out over a fifteen 

month period. Different specimen types were collected and cultured in test medium 

alongside .DA control. Media were prepared following exactly the manufacturer]s 

instructions. .ampling methods to avoid contamination were employed. Clinical 

specimen preparation was done using standard methods. Agar slope in screw-capped 

bottle was aseptically inoculated for each specimen and J for each medium. 2ottles 

were incubated for up to 1Odays. The day of inoculation was considered day 1. Yeast 

species for subsequent identification were isolated and sub cultured onto .DA 

(fxoid). Germ-tube production and other conventional methods were used as 

standards to definitively identify yeast isolates. The prevalence of 7+,5"5+ species 

was noted. 

fne thousand five hundred and sixty four clinical specimens were assayed from 82J 

patients they included 82J urine specimens, O07 stool specimens, J7 cerebrospinal 

fluids (C.F), J[ blood specimens, 80 mouth, 2J throat and 16[ vaginal specimens. All 

C.F and blood specimens were from AID. patients who had been hospitalised. Test 

(K/*8+-+-+&) medium was positive in J60 of 1,[6O specimens (2J.02 _) while .DA 

detected yeasts in J6O of the same 1,[6O specimens. The highest rate of positive 

cultures 212 ([2.08_) in both media occurred in stool specimens. All blood specimens 

remained negative. The difference in culture positivity occurred in urine and C.F 

where respectively, .DA was positive for J and 1 more cultures than test medium. 

Time to detection of yeast colonies was on average 1 day sooner for standard 

medium (.DA) than for test medium (2 days versus J days) for 7+,5"5+ isolates and [ 

days versus 6 days for 7$2'-%#%##(&*,.%U%$)+,&/ There was a slightly higher fungal 

burden in test medium than in .DA for corresponding specimen type. The observed 

prevalence on each of the media of 7+,5"5+*+68"#+,& was 66.7_. fther strains 

identified were - 7/*[.$U2$ 2.8_, 7/*'+$+'&"6%&"& J.6_, 7/*@6+8$+-+ O.1_, 7/*-$%'"#+6"& 

10.8_ and the remaining were not identified. The prevalence of non-+68"#+,&*7+,5"5+* 

spp. was characteristic. From the data presented, we can conclude that there was no 

difference in the prevalence of yeast between the test medium and .DA. However, 

multi- and interlaboratory data collected from K'%)%.+*8+-+-+& medium should be 

done to answer whether test medium has a diagnostic yield comparable to the 

conventional .DA.

P-0395. Standard operating procedures: standard guinea pig and mouse 

aerosol models of invasive pulmonary aspergillosis 

Kirkpatrick W 1 , Najvar / 1 , .heppard D 2 , Filler . J , Denning D O , Graybill ( 1 , Patterson 

T 1 

1 6niversity of Texas Health .cience Center, .an Antonio, Tj, 6.A, 2 McGill 6niversity, 

Montreal, Canada, J /os Angeles 2iomedical Research Instutute, Torrence, CA, 6.A, 

O 6niversity of Manchester, Manchester, 6l 

Background: Invasive pulmonary aspergillosis (IPA) has clinical relevance, and diverse 

diagnostic methodologies and therapeutic options against IPA have been evaluated in a wide 

variety of animal models. Meaningful comparisons of in vivo studies is complicated by variations 

in animal models and their design. A recently awarded contract from the National Institutes of 

Health/National Institute of Allergy and Infectious Diseases (N01-AI-J00O1), supports the 

development of standardized animal models of IPA to evaluate new diagnostic targets and tools 

to answer Bey questions related to this disease. Thus, we developed consistent standard model 

systems to evaluate new diagnostic targets and methodologies in IPA. These studies were 

designed to be a resource to the 4&'.$@"66(& research community at large to provide standard 

operating procedures (.fP) for small and intermediate animal models of IPA and to provide 

training and experimental studies. 

Methods: futbred ICR mice and Hartley guinea pigs were infected with 4&'.$@"66(&*U()"@+-(& 

AF2KJ using two types of aerosol chambers. fne, an acrylic chamber, allows simultaneous 

challenge of [0 mice or 7 guinea pigs and a Madison chamber, alternatively, allows simultaneous 

challenge of 126 mice or 18 guinea pigs. In the acrylic chamber, 10†K CF6/ml*4/U()"@+-(& 

conidia were nebulized for 1h inhalational exposure. In the Madison chamber we nebulized 

10†10 CF6/ml 4/U()"@+-(& conidia for 1h. Animals were immunosuppressed with cortisone 

acetate and cyclophosphamide -2d and rJd relative to infection and treated with ceftazidime 

daily starting 2d before infection. The course of infection was analyzed by percent mortality, 

length of survival, semi-quantitative tissue cultures, galactomannan determination and ~-PCR 

assessment of conidial equivalents at several time points after infection. 

P-0396. Detection and identification of Candida DNA in human blood by PCR 

and DNA enzyme immunoassay 

Kondori N 1 , .vensson E 1 , Mattsby-2altzer I 2 

1 Department of Clinical 2acteriology, .ahlgrensBa 6niversity Hospital, Gothenburg, 

.weden, 2 Department of Clinical 2acteriology, Gothenburg university, .weden 

The incidence of invasive 7+,5"5+ infections has been rising particularly in 

immunocompromised patients. Difficulties in establishing a specific and early 

diagnosis of 7+,5"5+ infection is one of the reasons for high mortality among these 

patients, so great effort has been directed towards finding more rapid diagnostic 

methods. 

A rapid PCR assay for detection and identification of 7+,5"5+*+68"#+,&, 7/*@6+8$+-+, 7/* 

'+$+'&"6%&"& and 7/*-$%'"#+6"& was established. Human blood from healthy donors was 

spiBed with 7+,5"5+ yeast cells and fungal DNA was extracted and purified using 

Puregene Bit. The internal transcribed spacer 2 (IT.2) region and the [.8. ribosomal 

DNA region of the fungi were amplified by using universal fungi primers IT.-J and 

IT.-O. The amplified DNA was analyzed by a DNA enzyme immunoassay (GEN-ETI-l 

DEIA, .orin 2iomedica, Germany). The probes used in DEIA bind specifically to 7/* 

+68"#+,&, 7/*@6+8$+-+, 7/*'+$+'&"6%&"& or 7/*-$%'"#+6"&. The detection step was based on 

labeled antibodies specific against double stranded DNA. No cross-reaction between 

the different 7+,5"5+ species was observed using DEIA. The detection limit of the test 

was approximately 10 7+,5"5+ cell per assay. 

fur results indicate that PCR |DEIA seems to be a promising method as a diagnostic 

tool for detection and identification of 7+,5"5+ in blood from patients with invasive 

7+,5"5+ infection. 

Results: ae have developed both mouse and guinea pig models of IPA using two different 

types of inhalation chambers. 6sing either a low-cost acrylic chamber or a large-scale Madison 

aerosol chamber, either model presents the Bey features of reproducible pulmonary infection, 

simultaneous challenge of multiple animals, predictable mortality, ease of duplication, and, 

importantly, they recapitulate human disease. .imple serial blood sampling in the guinea pig 

model enhances monitoring of surrogate marBers of infection. These models serve to encourage 

the development of diagnostic methodologies and tools against IPA. Collaborative efforts with 

academic and industrial investigators have begun to study diagnostic methods, as related to 

determination and development of new diagnostic targets, or the effects of gene expression or 

deletions. Additionally, the contract provides for the creation and maintenance of an 4&'.$@"66(& 

database, whereby qualified investigators may share details of their strains and experiments. 

Conclusions: These animal models with uniform Bey features and their respective associated 

.fPs, are available through the IAAM contract as a resource to the 4&'.$@"66(& research 

community. These resources provide the mechanism for evaluation of diagnostic, genomic and 

therapeutic targets using standardized animal models of IPA in order to improve disease 

diagnosis and therapy in this often fatal disease. 


P-0397. Sensitivity of galactomannan antigen test varied depending on 

patients’ underlying conditions 

Kurihara S, MiyazaBi Y, IzumiBawa l, lohno . 

.econd Department of Internal Medicine, NagasaBi university 

Background: /ung is the most frequent site of 4&'.$@"66(& infection, Mortality rate of 

invasive pulmonary aspergillosis still remains high, and earlier interventions are 

required for better prognosis. Galactomannan antigen test, an exoantigen of 

4&'.$@"66(&*&'/, has been widely used, because it is often impossible to perform a 

invasive examination such as open surgery due to poor patients] conditions. However, 

the usefulness of galactomannan in non-neutropenic population remains unclear. ae 

studied in the usefulness of galactomannan antigen for the diagnosis of possible 

aspergillosis in non-neutropenic patients. 

Methods: .erum from three hundred and thirty non-neutropenic patients and 70 

hematological malignancies, all of whom have been suspected of aspergillosis, were 

tested. Platelia 4&'.$@"66(& EIA (2io-Rad /aboratories) was used for detection of 

galactomannan with a cut off value of 1.[, and cultures from sputum, bronchial wash, 

or from bronchoalveolar lavage fluid were also performed. Then, we analysed the 

positive rate of galactomannan antigen according to patients] underlying conditions 

such as underlying diseases and corticosteroid treatment. 

Results: Nineteen (1K.0) _ in possible aspergillosis with the bacBground of 

hematological diseases (HD) were positive in galactomannan, 18.0 _ of idiopathic 

pulmonary fibrosis (IIP), 12.0 _ of lung cancer (/C) . There was no significant 

difference in positive rate among underlying diseases. Although JK.7 _ in probable 

pulmonary aspergillosis in non-neutropenic patients was positive, that was not as high 

as that in reported probable aspergillosis in neutropenic patients. Positive rate of 

JK.7 _ was significantly high as expected compared to those in other situations (p} 

0.0081 to HD, p} 0.007K to IIP, p}0.00JK to /C). Corticosteroid treatment was a 

significant factor for positive galactomannan antigen in IIP population, with 1K.O _ in 

corticosteroid treatment compared to [.[ _ without corticosteroid (p}0.0KO). 

Conclusion: Platelia 4&'.$@"66(& EIA is reported to have both high specificity and high 

sensitivity for probable aspergillosis in neutropenic patients, however both of which 

were not as high as expected when used in those who without neutropenia. 

Corticosteroid treatment in IIP population was a significant risB for positive 

galactomannan antigen, suggesting that IIP patients with corticosteroid treatment may 

be vulnerable to Aspergillus infection. 

P-0398. Construction a laboratory algoritm which was supported by molecular 

techniques for the diagnosis and the follow-up of the fungal infections in the 

groups of neutropenic patients 

lustimur . 1 , Kalkanci A 1 , TurBoz .ucaB G 2 , Haznedar R 2 , .enol E J 

1 Gazi 6niversity Faculty of Medicine Department of Microbiology, AnBara, TurBey, 

2 Gazi 6niversity Faculty of Medicine Department of Haematology, AnBara, TurBey, 

J Gazi 6niversity Faculty of Medicine Department of Infectious Diseases, AnBara, 

TurBey 

Incidence of invasive fungal infections are increasing in the last 20 years. 7+,5"5+ ve 4&'.$@"66(& 

species are responsible of 80_ of these infections. Fungal infections are seen with the 

percentages of 10_ of these patients. Mortality rate of these infections are changing from O7_ to 

88_. Diagnostic methods which can be used for the diagnosis of fungal infections are direct 

microscopy, culture and identification, antigen or antibody detection, demonstration of fungal 

elements by histopathologically and finally the molecular methods for the detection of fungal 

nucleic acids. 2lood culture is not effective in the diagnosis of fungal infections. /ysis 

centrifugation is recommended for the fungal isolation. Antibody detection has confusing results 

in the immuncompromised hosts. 2iopsy can be imposibble in the trombocytopenic patients. 

Polymerase chain reaction (PCR) technique has been suggested also as a mode of detecting the 

infection. 

The aim of this study is the construction a laboratory algoritm which was supported by molecular 

techniques for the diagnosis and the follow-up of the fungal infections in the groups of 

neutropenic patients. 

ae analyzed data from 100 neutropenic patients hospitalized at Gazi 6niversity Faculty of 

Medicine Department of Haematology, between 200O and 200[. 2lood, bronchoalveolar lavage 

fluid, tissue biopsy samples were collected from patients. Direct microscopy for specimens other 

than blood, microbiological culture by lysis centrifugation method and by other conventional 

methods, galactomannan antigen detection in serum samples and Real Time PCR based 

identification of fungal agents were conducted. Panfungal PCR was developed by using fungal 

internal transcribed spacer (IT.) region (IT. 1-[.8. rRNA-IT. 2). Candida DNA was detected in 

the same assay by the addition of specific probe using FRET and the melting curve analyses 

using .Y2R Green flourescense. Aspergillus DNA was detected using genus specific primers 

and probes in another consequative PCR amplifying Cytb gene region of Aspergillus genome. 

fut of 100 febrile neutropenic patients, seven (7_) were found to be infected by fungi. Candida 

albicans (2), Candida glabrata (2), Aspergillus flavus, Aspergillus fumigatus, Rhizopus oryzae 

were identified by culture and molecular methods. The identification of R. oryzae was confirmed 

by DNA sequencing in automated sequencer. fut of 100 neutropenic patients, 12 of them (12_) 

were found to be panfungal DNA positive. Among 12 patients, nine of them (7[_) were found to 

be galactomannan antigen positive, seven of them ([8_) were found to be culture positive. 

Fungal agent was identified in seven patients (78_) among the PCR and galactomannan antigen 

positive nine patients. Fungal cultures were found to be negative in two patients (22_) althought 

they were found to be PCR and antigen positive. .even patients were found to be 

galactomannan positive (7_) without the confirmative results obtained from molecular and 

microbiological culture techniques. This positivity thought to be related to the use of beta lactam 

antibiotics in the therapy of these patients. The optimal diagnostic technologies for invasive 

fungal infections would be incorporate the use of a panfungal specific primer for the detection of 

all fungal infections followed by other genus and species specific identification. 

Project codeM Gazi 6niversity 01/200O-10O

P-0399. Identification of Candida spp. by fluorescence in situ hybridization 

(FISH) 

Lakner A, Poppert ., Essig A 

6niversity 6lm, Institut of Microbiology, 6lm, Germany 

There is strong correlation between the rising number of immuncompromised patients 

and fungal infections. 7+,5"5+ spp. is the fifth common pathogen isolated from blood 

cultures. 

* 

Fast differentiation is necessary because primary resistances against antimycotic 

agents such as Fluconazol and Amphotericin occur in certain 7+,5"5+ species. 

In this study fluorescence in situ hybridization (FI.H) for rapid differentiation of 

7+,5"5+ spp. was evaluated. FI.H uses fluorescently labelled oligonucleotide probes 

of 1[-20 base pairs that bind to complementary and specific target sequences on the 

fungal ribosomal RNA. FI.H is a very useful method for fast, inexpensive and easy 

identification and is already successfully used in the field of medical microbiology for 

the differentiation of bacteria, for example in blood cultures. 

A set of 1K probes including species specific probes for 7/*+68"#+,&3*7/*5(86",".,&"&3*7/* 

@6+8$+-+3*7/*[$(&."3*7/*6(&"-+,"+.3*7/*'+$+'&"6%&"&3*7/*-$%'"#+6"&*and*7$2'-%#%##(&* 

,.%U%$)+,&*was designed and*evaluated using 18 reference strains. The reliability of 

the probes was further tested on 127 well characterized clinical isolates. 

All reference strains and 106 of 127 clinical isolates strains were correctly identified by 

FI.H. Nine strains were not identifiable to species level because the corresponding 

species was not included in the probe set. In 1J cases the malfunctioning of single 

probes resulted in an atypical staining pattern, so that the isolates were not identifiable. 

fnly one isolate was misidentified. All 60 7/*+68"#+,& and seven 7/*5(86",".,&"&* 

strains were correctly identified by FI.H. 

Finally the oligonucleotide probes were used for the direct identification and 

differentiation of 7+,5"5+ spp. in positive blood cultures, which showed yeast in the 

Gram-stain. Twenty seven samples were tested and in 2J cases the identification was 

correct. The four remaining isolates were not identifiable by FI.H. 

P-0400. Development and clinical application of a panfungal PCR assay to 

detect and identify fungal DNA in tissue specimens 

/au A 1 , Chen . 2, J , .orrell T 1, J , Carter D O , MaliB R [ , Martin P 6 , Halliday C 2, J 

1 Faculty of Medicine, 6niversity of .ydney, N.a, Australia, 2 Centre for Infectious 

Diseases and Microbiology and aestmead Millenium Institute, 6niversity of .ydney at 

aestmead Hospital, aestmead N.a, Australia, J Centre for Infectious Diseases and 

Microbiology /aboratory .ervices, aestmead Hospital, aestmead, N.a, Australia, 

O .chool of Molecular and Microbial 2iosciences, 6niversity of .ydney, N.a, Australia, 

[ Postgraduate Foundation of Veterinary .cience,6niversity of .ydney, N.a, Australia, 

6 Veterinary Pathology Diagnostic .ervices, 6niversity of .ydney, N.a, Australia 

The frequency of invasive fungal infections (IFIs) in humans and domestic animals, 

and the spectrum of fungal pathogens has increased substantially in recent years. 

Early, rapid and accurate detection and identification of the fungal pathogen is 

essential for patient management and important for epidemiologic purposes. However, 

the diagnosis of IFIs remains problematic. Recently, culture-independent methods, 

such as PCR, have been developed to enhance conventional diagnostic methods and 

allow detection and identification of non-viable fungi from clinical specimens. ae 

developed a panfungal PCR assay that targets the internal transcribed spacer 1 

(IT.1) region of the rDNA gene cluster to detect fungal DNA in fresh (n}1K) and 

paraffin-embedded (n}12) tissue specimens. PCR products were sequenced and 

compared with the Gen2anB database to identify the fungal pathogen. The molecular 

identification was correlated with histology and culture. To date the assay has 

successfully detected and identified pathogens in 17 human and seven animal 

patients with proven IFI. Fungal pathogens identified include 7+,5"5+ species (n}O), 

7$2'-%#%##(&*,.%U%$)+,& (n}[), >$"#?%&'%$%,*+&+?"" (n}1)3*4&'.$@"66(&*U6+0(& (n}1), 

4/*U()"@+-(&*(n}7), a_%'?"+6+ &'","U.$+*(n}2),*and the zygomycetes (n}O) P?"d%'(&* 

%$2d+., P/*)"#$%&'%$(&, 4'%'?2&%)2#.&*.6.@+,& and 4#-",%)(#%$*.6.@+,&. A 

molecular diagnosis could not be established in seven culture negative, but 

histologically proven cases. All of these specimens were paraffin-embedded and four 

had hyphae resembling zygomycetes. These seven specimens may have been PCR 

negative because the amount of DNA extracted was insufficient for detection. The 

results from this study indicate the potential benefits of using the panfungal PCR 

assay in combination with conventional laboratory tests for sensitive and specific 

identification of fungi in tissue specimens. 

In conclusion FI.H proofed to be a very sensitive and specific method for identification 

of 7+,5"5+ spp. in clinical samples especially blood cultures. 


P-0401. Recombinant antigens for the serodiagnosis of invasive candidiasis 

Laín A 1 , 2rena . 1 , Elguezabal N 1 , Fernandez de /arrinoa I 1 , del Palacio A 2 , 

Moragues M 1 , Pontán ( 1 

1 6niversity of the 2asque Country, 2 Hopital 12 de fctubre, Madrid 

Invasive candidiasis is the most severe clinical presentation of 7+,5"5+*infections/* 

However the diagnosis of invasive candidiasis remains difficult mainly because there 

are no specific clinical manifestations of the disease and colonization and infection are 

not easy to distinguish. 

In this study, we report the development of both an immunoblotting and an E/I.A for 

the serodiagnosis of invasive 7+,5"5+ spp. infections by detecting specific antibodies 

against three recombinant 7+,5"5+ antigens. 

Two of the antigens studied, Hyr1 and Ece1, are specific of the hyphal growth of 

7+,5"5+ +68"#+,&, usually associated with the invasive form of the fungus. The third 

one is the enolase which is an immunodominant antigen present in the cytoplasm and 

the cell wall of 7+,5"5+ spp 

P-0402. Direct detection of pathogenic fungi of superficial fungal infection 

based on PCR assay 

Li X, Chen H, Cui F, Chen a, /v G, .hen Y, /iu a 

Department of Mycology, Institute of Dermatology, Chinese Academy of Medical 

.ciences c PeBing 6nion Medical College 

Introduction: .uperficial fungal infection is a disease of worldwide distribution that 

accounts for the majority of superficial infections. The fast and reliable diagnosis of a 

superficial fungal infection is important since prompt antifungal treatment is associated 

with improved outcome, avoiding spread of infection.In the present study, a PCRbased 

diagnostic technique of superficial fungal infection from clinical specimens was 

investigated, in order to overcome the problems of low sensitivity or low specificity, 

and time delay associated with conventional methods. 

Methods: Patients admitted to our clinic with clinically suspected of superficial 

mycoses were included in this study. Direct detection of the pathogenic fungi in dermal 

specimens was developed by PCR assay based on IT.1 primer, and compare the 

results with those of microscopy and culture. 

The three genes (a7aI, AkPI and a`bI) were cloned, expressed in a&#?.$"#?"+ #%6" 

and the protein products purified by nicBelchelate affinity chromatography. The 

reactivity of the three recombinant proteins against sera from 82 different adult 

patients from the Hematological or Intensive Care 6nits (J6 patients with confirmed 

invasive candidiasis and O6 from a control group) was assayed by immunoblotting and 

E/I.A. The results are described in table 1. 

Table 1. .ensitivity, specificity, positive and negative predictive values, and accuracy 

of a diagnostic test based on the detection of antibodies against the three recombinant 

antigens by immunoblotting and E/I.A. 

Antigen Immunoblotting 

E/I.A 

Discussion: This PCR assay might be used in laboratories with no mycological 

.en _ .pec _ PPV _ NPV _ Accuracy _ .en _ .pec _ PPV _ NPV specialization _ Accuracy for _ rapid etiologic diagnosis and treatment selection especially in the 

suspected cases if fungus can not be detected by conventional methods or a rapid 

Enolase [0 7J.K 60 6[.O 6J.O 6J.K 8K.1 82.1 

diagnosis of superficial fungal infection is needed. 

7[.K 78.1 

Ece1 O7.2 6J.0 [0.0 60.O [6.1 61.1 82.6 7J.J 7J.1 7J.2 

Hyr1 JJ.J 100.0 100.0 6[.7 70.7 61.1 8O.8 7[.K 7J.6 7O.O 

.enM sensitivityn .pecM specificityn PPVM positive predictive valuen NPVM negative predictive value. 

Although the sensitivity of the immunoblotting was quite poor, it increased for the E/I.A test. In addition, it 

improved up to a 7[_, when combinations of the E/I.A results of the three antigens were taBen into account 

(table 2). 

Table 2. .ensitivity, specificity, positive and negative predictive value, and accuracy for combinations of 

different E/I.A tests. 

Antigens .ensitivity _ .pecificity _ PPV _ NPV _ Accuracy _ 

Ece1 r Hyr1 7[ 71.7 67.[ 78.6 7J.2 

Ece1 r Eno 6K.O 76.1 6K.O 76.1 7J.2 

Hyr1 r Eno 7[ 78.J 72.K 80 76.8 

ConclusionsM 1. In our conditions, the E/I.A is more sensitive than immunoblotting. 

2. The combination of the independent E/I.A tests results improves 

the diagnostic usefulness of the test. 

Results: fne hundred and twelve patients (sixty-nine were clinically suspected of 

superficial fungal infection and forty-three were confirmed of other dermatosis) were 

included in this study. ff them, fifty-seven ([0.K_) were finally diagnosed as 

superficial fungal infection according to the diagnostic criteria. fut of 112 samples, 

[O.[% were positive by microscopy (K0.2_ were true positive) and J[.7%by culture 

(100_ true positive), and O1.1% (100_ were true positive) by PCR. All the positive 

cases tested were suggestive of superficial mycoses clinically. Amplifications of 

control DNAs from a wide number of fungi species, other euBaryote and proBaryotes 

were also carried out, and they gave negative results.

P-0403. Comparison of three phenotypic approaches with a PCR test to 

discriminate C. albicans and C. dubliniensis isolates 

Li C, ju Y 

~ilu Hospital of .handong 6niversity, (inan, China 2[0012 

Background: 7+,5"5+*5(86",".,&"& is a Bind of pathomycete newly found, which 

tended to be confused with 7+,5"5+*+68"#+,& for their similarity on phenotypic 

characteristics. Methods based on gene sequence are applied frequently to maBe 

reliable identification while the reliability of many phenotypic approaches is still not 

confirmed. 

Objective: To identify 7/*5(86",".,&"& from the initial isolates of 7/*+68"#+,& and to 

compare three phenotypic approaches as growth test at O[ ÅC, germ tube formation 

test at JK ÅC and chlamydospore formation test on .taib agar. 

Methods: Clinical specimens were inoculated on modified .DA slant, incubated at 

J7ÅC for 72 hours and then isolated. 2y way of TTC reduction test, germ tube 

formation test in human serum at J7ÅC, chlamydospore formation test on CMA with 

Tw 80 at 2[ÅC and saccharose assimilation test, 17 isolates were identified as C. 

albicans initially, which were preserved at OÅC and activated by twice subcultivations 

before sequent tests. aith an ATCC type strain of 7/*+68"#+,& as control, their PHR1 

gene sequences were amplified with PCR, to identify 7/*5(86",".,&"& in them. ae also 

approached growth test at O[ ÅC, germ tube formation test in YEPD medium (1_ 

yeast extract, 2_ peptone!2_ dextrose) at JK ÅC and chlamydospore formation test 

on .taib agar respectively to compare these two fungies phenotypic characteristics. 

Results: fut of the 17 isolates, J were re-identified as 7/*5(86",".,&"& by PHR1 gene 

detection. At O[ ÅC, neither the 17 isolates nor the type strain was observed to form 

obvious colony on modified .abouraudes agar, and only one C. albicans grew 

marBedly in YEPD. No germ tube was observed in YEPD medium at JK ÅC. After 72 

hourse culture on .taib agar, 2 of the J 7/*5(86",".,&"& formed chlamydospore while no 

7/*+68"#+,& did. It is partially same with the result of PCR. 

Conclusions: PCR of homologous sequences of PHR1 is a reliable method to 

discriminate 7/*+68"#+,&*and*7/*5(86",".,&"&. Chlamydospore formation test on .taib 

agar can be helpful, but growth test at O[ ÅC or germ tube formation test at JK ÅC is 

not advisable. 

P-0404. Staining fungi by using optical brighteners: The leucophor family 

Llovo J 1 , /lebarÜa R 2 , Rodriguez-ftero / 1 , Cortizo-Vidal . 1 , Muoz A J 

1 .ervicio de MicrobiologÜa, Hospital ClÜnico 6niversitario .antiago de Compostela, 

.PAIN, 2 Clariant Ibdrica, 2arcelona, .PAIN, J Dpto. de MicrobiologÜa, Facultad de 

2iologÜa, 6niv. de .antiago. .PAIN 

Fluorescent dyes of the diaminostilbene type are used as fluorescent whitening 

agents in washing agents and paper and textile industry. Due to their high affinity for 

beta-glycosidically linBed polysaccharides such as glucan, cellulose and chitin, these 

colourless dyes can be used to prove the presence of fungal elements in clinical 

samples in an easy and rapid way. 

ff the thousands of products developed by the industry at present in the laboratory of 

clinical microbiology two are used in a routine wayM Calcofluor ahite (.igma) and 

2lancophor (2ayer). 

The aim of the study was to evaluate the staining properties of eight optical brightener 

agents (f2A) of the /eucophor family for the paper industry. .even liquid productsM 

AHF, A/, ANf, AP, FT., .AC, 6lf and one powderM 2CF. 

These dyes were compared to 2lancophor and Calcofluor ahite (Tinopal-6NPA-Gj 

and Tinopal /Pa) using the same staining procedures. 

Its properties were verified with fresh clinical specimens and yeast and mould isolated 

from fungal cultures. 

The f2A were serially diluted in distilled water since 1_ (v/v) or (w/v) and tested 

alone and with lfH-Glicerine. Fluorescence microscopic analysis was performed 

using a Epi- system with 100a Mercury vapour lamp or 12v 100a Tungsten halogen 

lamp and excitation filters below O00nm. 

fnly two f2A do not stain fungiM the hexasulphonated anionic /eucophor .AC and 

the cationic /eucophor FT.. 

The other ones dye in aqueous dilutions 1/1000 - 1/10000, both alone and in dilutions 

with lfH-Glicerine. 

ConclusionM we have demonstrated that concerning fungi detection these molecules 

worB with the same characteristics of simplicity and sensibility as the products used at 

present. 


P-0405. Usefulness of levine eosin methylen blue agar in mycology: Levine 

morphology agar (LMA) 

Llovo J 1 , Rodriguez-ftero / 1 , Cortizo-Vidal . 1 , Muoz A 2 

1 .ervicio de MicrobiologÜa, Hospital ClÜnico 6niversitario, .antiago de 

Compostela,.pain, 2 Dpto. MicrobiologÜa, Facultad de 2iologÜa, 6niv. de .antiago, 

.pain 

/evine]s EM2 agar is one of the culture media more used in microbiology laboratories worldwide. 

Their primary purpose is to isolate and differentiate lactose-fermenting from non-lactosefermenting 

enteric bacilli. aeld (1K[2) found out how to identify 7+,5"5+*+68"#+,& in different 

clinical samples by using this culture media. 

The aim of the study is to describe the use of /MA in order to detect Germ Tube production and 

to see microscopic morphology of yeasts and moulds. 

The /MA was inoculated according to the Dalmau (1K2K) procedureM first, half of the EM2 plate 

was streaBed with a drop of Tween 80, and then both halves were streaBed with a young yeast 

colony and covered with two sterile coverslips. After incubating it at J[oC for 2-Oh the germ tube 

production can be observed. In order to examine for the presence and arrangement of true 

hyphae, pseudohyphae, blastoconidia, chlamydoconidia and arthroconidia, the EM2 plates must 

be incubated in the darB at room temperature (2[-2KoC) for 2Oh, O8h and 72h. .ince microscopic 

features are observed trough the coverglass on the agar surface, the Dalmau method permits 

examination of morphologic characters at any magnification by bright-field or better by phasecontrast 

microscopy. 

Perhaps the most common, simple, reliable and economical test performed in the clinical 

microbiology lab is germ tube test. The classical procedure (Taschdjian, 1K60) with animal or 

human serum, incubated at J[oC and read at 2-Oh show the same results but with /MA we do 

not only avoid the use of sera but also simplify the process. 

Classical Cornmeal and Rice extract morphology agar, both with Tween 80, were compared with 

/MA in order to find out if it also differentiate the following generaM 7+,5"5+, 7$2'-%#%##(&, 

9.%-$"#?(), >$"#?%&'%$() and =+##?+$%)2#.&. In fact, /MA can also identify 7+,5"5+ species 

as well as 7/+68"#+,&, 7/@6+8$+-+, 7/[$(&.", 7/*'+$+'&"6%&"&, 7/*-$%'"#+6"&, 7/*'&.(5%-$%'"#+6"& and 

7/*@("66".$)%,5"". 

The production of ascospores by =+##?+$%)2#.& in /MA is usually delayed (t[d). 

The production of chlamydoconidia by 7/+68"#+,& depends clearly on the origin of different EM2 

media. 

P-0406. Rosemary (Rosmarinus officinalis) agar and oregano (Origanum 

vulgare) agar applied to Candida dubliniensis screening 

Loreto É, Pozzatti P, .cheid /, .anturio D, .anturio (, Alves . 

6niversidade Federal de .anta Maria - R. 

7+,5"5+*5(86",".,&"& is a recently described pathogenic species which shares many 

phenotypic features with 7/*+68"#+,& and so, may be misidentified in microbiological 

laboratories. 2ecause molecular methods are onerous and unfeasible in routine 

mycological laboratory, the phenotypic techniques have been encouraged as the 

development of differential media for the presumptive identification of these species. 

ae examined the colony morphology and chlamydospore production of J0 7/* 

5(86",".,&"& isolates and 100 7/*+68"#+,& isolates onto two new proposed mediaM 

Rosemary (P%&)+$",(&*%UU"#",+6"&) extract agar (REA) and fregano (b$"@+,()* 

0(6@+$.) extract agar (fEA). These substrates are traditionally used as spices and 

medicinal herbs as well. Here, 200 ml of an aqueous extract obtained after two hours 

of boiling in Clevenger apparatus in order to remove the essential oils, was filtered 

through several layers of gauze and incorporated to 800 ml of a basal medium 

(dextrose 1_n creatinine 0.078 _n agar 2_ and 800 m/ of distilled water). Each media 

was autoclaved at 121ÅC/1[ min and immediately, Petri dishes were filled with 2[ m/ 

of the medium. After solidified, single colony of freshly subcultured on .abouraud agar 

was streaB and inoculated separately. In both proposed media, after 2O to O8 h of 

incubation at 2[ÅC all 7/*5(86",".,&"& isolates (100_) showed rough colonies with 

peripheral hyphal fringes and abundant chlamydosporesn in contrast all isolates of 7/* 

+68"#+,& (100_) showed smooth colonies without hyphal fringes or chlamydospores. 

In conclusion, we reinforce that rosemary extract agar (REA) and oregano extract 

agar (fEA) offer a simple, rapid and inexpensive screening media for differentiation of 

7/*5(86",".,&"& and 7/*+68"#+,&/ 

/MA can be used as single medium (germ tube and morphology agar) for identification of yeasts 

and, furthermore, it can facilitate the visualization of unaltered conidial structures in moulds. 

References: 

Dalmau, /M. 1K2K. Remarques sur la technique mycologique. Ann. Parasitol. Hum. Comp. 7M 

[J6-[O[. 

aeld, (T. 1K[2. 7+,5"5+*+68"#+,&. Rapid identification in pure cultures with carbon dioxide on 

modified eosin-methylene blue medium. Arch. Dermat. .yph. 66M 6K1-6KO. 

Dolan, CT. 1K70. A practical approach to identification of yeast-liBe organisms. Am. (. Clin. 

Pathol. [[M [80-[8K. 

Taschdjian,C/., 2urchall,((. and lozinn,P(. 1K60. Rapid identification of 7+,5"5+*+68"#+,& by 

filamentation on serum and serum substitute. (. Dis. Child. KKM 212-21[.

P-0407. Development of a monoclonal antibody specific of Candida 

dubliniensis cells and its use in a rapid latex agglutination test to identify C. 

dubliniensis colonies 

Marot-Leblond A 1 , 2eucher 2 1 , David . 2 , Nail-2illaud . 1 , Robert R 1 

1 Groupe deEtude des Interactions HÇte-Parasite, 6PRE. EA J1O2, 6FR des .ciences 

Pharmaceutiques et deIngdnierie de la .antd, 16 bd Daviers, OK000 Angers, France, 

2 .R22, ZI Carrimre-2eurrimre, OK2O0 Avrilld, France 

7+,5"5+*5(86",".,&"& is a newly described pathogenic 7+,5"5+ species originally 

isolated from patients with human immunodeficiency virus (HIV) infection and 

recurrent oral candidosis. It is now reported to account for between J.[_ to JO_ of all 

7+,5"5+ infections, depending on the patient population. It shares so many 

phenotypical characteristics with 7/*+68"#+,& (germ tube and chlamydospore 

production, colored colonies on chromogenic media), that it is easily misidentified as 

such. Accurate species identification requires the use of sophisticated techniques that 

are not routinely available in most clinical microbiology diagnostic laboratories. 

Monoclonal antibodies, that recognized specifically epitopes, are easy and powerful 

tools to performed differentiation at the species level. 

This study presents "J the strategy developed in order to produce a monoclonal 

antibody specific for 7/*5(86",".,&"& cells, ""J the biochemical characterization of the 

recognized antigens and """J the performances of a rapid latex agglutination test 

allowing identification of 7/*5(86",".,&"&*colonies. 

Cell components of the dimorphic pathogenic fungus 7+,5"5+*5(86",".,&"& were 

fractionated by Hydrophobic Interaction Chromatography and used for mice 

immunization. Hydrophilic components were particularly efficient, and a monoclonal 

antibody (MAb 12F7-F2) was further investigated. It was shown by indirect 

immunofluorescence to be specific for a surface antigen of 7/*5(86",".,&"& 

blastoconidia. No reactivity was observed with other fungal genera or with other 

7+,5"5+ species, including 7/*+68"#+,&3*that shares many phenotypic features with 7/* 

5(86",".,&"&. The use of different chemical and physical treatments for cell component 

extraction suggested that the specific epitope probably resides on a protein moiety 

absent from 7/*+68"#+,&. 2y aestern blot analysis, the specific epitope was localized 

on two components. MAb 12F7-F2 reacted with a J7 BDa and a high molecular mass 

polydispersed components. MAb 12F7-F2 was used to develop a commercial latex 

agglutination test to identify 7/*5(86",".,&"& colonies (2ichro-dubli Fumouzew test, 

Fumouze Diagnostics). The test was validated on yeast strains previously identified by 

PCR, and on fresh clinical isolatesn these included O6 7/*5(86",".,&"& isolates, O[ 7/* 

+68"#+,& isolates, and other yeast species. The test had 100_ sensitivity and 

specificity for 7/*5(86",".,&"&*isolated on .abouraud dextrose, CHRfMagar Candida, 

and Candi.elect media, and K7.8_ sensitivity for 7/*5(86",".,&"&*grown on Candida ID 

medium. The test is rapid ([ min), easy to use, applicable to large number of isolates 

and may be recommended for routine use in clinical microbiology laboratories and for 

epidemiological investigations. 

P-0408. Abundance of P. boydii in CF patients detected with a semi-selective 

isolation method 

Mayr A 1 , Rainer ( 2 

1 Department of Hygiene, Microbiology and .ocial Medicine, Medical 6niversity 

InnsbrucB, Fritz-Preglstr. J, 6020 InnsbrucB, Austrian e-mailM astrid.mayrùi-med.ac.at, 

2 Institute of Microbiology, /eopold Franzens 6niversity InnsbrucB, TechniBerstr. 2[, 

6020 InnsbrucB, Austrian e-mailM j.rainerùuibB.ac.at 

In general, only 4&'.$@"66(&*U()"@+-(& Fres. and 7+,5"5+*+68"#+,& 2erBhout are 

considered as relevant colonisers of Cystic Fibrosis (CF)-patients respiratory tract, 

because of their broadly Bnown opportunistic pathogenic properties. Colonisation with 

1&.(5+66.&#?.$"+*8%25"" (.hear) McGinnis et al. is mostly not screened in routine 

diagnosis, though it is assumed to cause inflammatory response in lung tissue. 

Moreover, strains of the 1/*8%25"" complex are causing a wide range of mycoses, 

among them mycetoma and severe infections of the CN.. Data on the abundance of 

1/*8%25"" in CF-patients are sparse, because systematic, selective sampling was 

carried out by few groups to date. Recent studies have shown, that 1/*8%25"" can be 

frequently isolated from urban environments. 

In our study we introduced a semi-selective isolation medium (.ce.elr) in order to 

detect 1/*8%25"" in sputum of CF-patients additionally to routine mycological diagnostic 

tools. This medium contains benomyl, streptomycinsulfate, chloramphenicole and 

ciprofloxacine to overcome the problem of overgrowth with other fungi and bacteria 

resulting in non-detection of 1/*8%25"". .putum of patients (n } K2, OJ males, OK 

females) between [ and OK years old were sampled during routine screening 

procedures for lung colonisation in the 6niversity Hospital of InnsbrucB. The total 

sample number was J[7. 

More than four percent (O.2 _) of the samples and 6.[ _ of the sampled patients 

were 1/*8%25"" positive. Patients with 1/*8%25"" positive sputum samples, who were 

sampled more than two times (n}2), proved to stay 1/*8%25"" positive during the whole 

sampling period. ff those samples JJ.J _ were intermittent negative. Aside 1/*8%25"" 

also 7+,5"5+ spp. were isolated from 2[0 samples (70.0J _) and bacteria from ten 

samples (2.8 _) on .ce.elr. 

The results clearly demonstrate that the frequency of this fungus is largely 

underestimated and since it may be of a significant clinical interest in CF-patients the 

colonisation of the respiratory tract with 1/*8%25"" should be taBen seriously and should 

lead to a sorrow observation of the patients. .ce.elr is a useful tool for the monitoring 

of 1/*8%25"" and probably also 7+,5"5+ spp. and resistant bacteria in CF patients 

lungs without additional inconvenience for patients and personnel. 


P-0409. A new dyening ink for the diagnosis of microscopical fungi 

Mencl K 1 , .losar P 

1 Reg. Hosp. Pardubice, 2 .YNPf Pardubice a.s. 

The ParBer 2lue-2lacB ~uinB (ParBer 2lue 2117), which has been used as a specific 

reagent dye for laboratory diagnosis of fungal infections in biological tissues, is the 

past now. The end of its production was a big loss for many medical or veterinary 

laboratories. 

In this study we are presenting a new dyestuff produced in .YNPf Pardubice a.s. 

(Czech republic). This dye is an alternative to the ParBer 2lue-2lacB InB. The solution 

of this dye called Myco-InB .YNPf, has a different chemical structure, but it has all of 

the safety data as ParBer 2lue 2117 (hazards identification, toxicological or ecological 

infromation, etc.). 2ig advantage in the laboratory praxis is its high specifity to the 

mycological structures, which are more intense coloured than by 2 117. The samples 

prepared from newly synthesized dye do not precipitate even after several weeBs in a 

moist room. Myco-InB .YNPf is colour fixed in aqueous solution at pH 7,2 more than 

12 months and five weeBs in 20 _ lfH. Myco-InB .YNPf is also very suitable for 

colouring of samples of microcultures of dermatofytes, but also for other microscopical 

fungi. The Myco-InB .YNPf paints all these sturctures very well. 

P-0410. Aspergillus serum antigen EIA test: A local reappraisal of cut-off points 

for detecting risks in transplant patients 

Mercadillo M, Fernandez-flmos A, Molina A, Alvarez M, .anchez-.ousa A, 2aquero 

F 

Hospital Ramon Y Cajal, Madrid, .pain 

Transplant patients are at risB of invasive aspergillosis. .urveillance of the levels of 

galactomannan antigen in serum is currently included in most of the protocols of 

transplantation. The establishment of suitable cut-off points is critical to decide about 

interventions, but there is a continuing controversy about them. 

The purpose of this study: was to set operative cut-off points for our hospital, 

adapted to our technical conditions. To perform the study, we collected a total of 2K[ 

sera from 1J8 patients that underwent different transplant procedures in our Hospital 

(200J-200[)M bone marrow (6J), liver ([8), pulmonary (1J), intestine (2), and renal (2). 

The Platelia Aspergillus EIA was used to quantify galactomannan antigen levels in 

serum. Data were plotted to obtain the distribution of index values. 

Results and Discussion: The analysis of the distribution suggests that several cutoff 

points could be established. Most sera yielded values equal or lower than 0.[ ng/ml 

(modal valueM 0.O), suggesting that these values define a hvery low-risBi status. 

Nevertheless, it is to note that this value is cutting the right arm of an apparently 

normal distribution, and therefore values slightly higher than 0.[ might be erroneously 

considered as hpositivei. ae consider values between 0.[ and 1 as suggestive of hlow 

risBi, between 1 and 1.[ as indication for hactive surveillancei (that forces to indicate 

cultures), and t1.[ as hhigh risBi. Note that the Platelia technique indicates that only 

values above 1.[ means a significant risB. ae agree with this cut-off point, but we also 

consider to evaluate patients with t1 values. To ascertain our operative cut-off points, 

we collected sera from 2O patients without evidence of invasive aspergillosis in whom 

some up-shift was detected in our essays. The first antigen detection was infrequently 

t1 ng/ml (12.[ _), with a modal value of 0.6, but the second antigen detection was 

exceeding 1 ng/ml in most cases (82.6 _). Different peaBs were found in this group, 

ranging from 1.1 to J.J ng/ml. These results suggest that a number of transplant 

patients under surveillance for aspergillosis might be prone to some level of exposure 

to 4&'.$@"66(&*antigen, what is reflected in increases in serum values. It can be 

suspected that invasive aspergillosis probably evolve in a minority of this group of 

patients, that should be identified by Platelia values over 1 ng/ml to apply intensive 

surveillance procedures, eventually leading to interventions. In conclusion, the Platelia 

galactomannan antigen test is a good surveillance tool for transplant patientsn in our 

hands, a cut-off value of 1 ng/ml might be considered as suitable to consider the 

patient as having a possible suspicion of significant exposure to 4&'.$@"66(& antigen, 

eventually leading to invasive aspergillosis.

P-0411. Development of an easy and rapid identification system for clinical 

yeast-like organisms using PCR-RFLP method 

Mikawa T, Hosoi M, .uzuBi M, Endo ., lanayama A, .ato Y, fosawa H, lobayashi I 

MIitsubishi lagaBu 2C/, Inc., ToByo, (apan 

2ecause the clinical impact of fungal infectious diseases caused by yeast-liBe organisms 

such as 7+,5"5+, 7$2'-%#%##(&, !+6+&&.d"+ and >$"#?%&'%$%, spp. has increased, novel 

antifungal agents have become available. As there are significant differences in antifungal 

susceptibility among fungal species, an accurate method of identification is needed. 

Identification of clinical yeast-liBe organisms is routinely performed by conventional 

methods based on morphological, physiological and biochemical properties. Various Bits 

have been developed as identification systems for clinical yeast-liBe organisms. These Bits 

require more than 2 days and the proportion of identifications that are incorrect is high. 

Moreover, these methods depend largely on the expertise of technicians. 

The aim of this study is to construct an easy, rapid, and accurate method for the 

identification of clinical yeast-liBe organisms. In this study, we have tested the application of 

the restriction analysis of the 26. r DNA gene or IG. 1 region for the identification of 

clinical yeast-liBe organisms. ae have obtained restriction patterns of PCR products for 

identification of J0 type strains and constructed a database of restriction fragment patterns 

to identify them with 2ioNumerics software (Applied Maths, 2elgium). To test the feasibility 

of our identification system, 118 clinical isolates which were isolated from clinical 

specimens in our laboratory were identified by this system. 

1) Identification of type strains 

Among J0 type strains, 2J showed a unique restriction pattern for each species with A+. III, 

A?+ I, A'+ II and A",U I. J species of 7+,5"5+ (7. U+)+-+, 7. [.U2$ and 7. d.26+,%"5.&) and 

O species of >$"#?%&'%$%, (>. +&+?"", >. +&-.$%"5.&, >. ",[", and >. %0%"5.&) were 

indistinguishable by the comparison of their 26. r DNA restriction patterns, respectively. 

ae have paid special attention to the species of >$"#?%&'%$%,, and tested the application of 

the restriction analysis of IG. 1 region for identification of 8 species of >$"#?%&'%$%,, 

medically important and opportunistic yeast-liBe organisms. The restriction patterns 

obtained with the four restriction enzymes such as A+. III, A?+ I, A'+ II and A",U I were 

species-specific. 

P-0412. Identification of Malassezia isolates of patients with pityriasis 

versicolor by using PCR-restriction enzyme method. 

Mirhendi H 1 , MaBimura l 2 , Tarazui 2 1 , Zomorodian l 1 , (alalizand n 1 

1 Tehran 6niversity of Medical .ciences, 2 TeiByo 6niversity Institute of Medical 

Mycology 

The members of the genus !+6+&&.d"+ are the causative agents of the sBin disease 

pityriasis versicolor. There are evidences that these yeast may involve in some other 

dermatological disorders such as seborrheic dermatitis and atopic dermatitis. The 

recently findings show various !+6+&&.d"+ species can cause the diseases and 

molecular biological methods are the most reliable way for identification of the isolates. 

The aim of the present study is identification and determination of frequency of 

!+6+&&.d"+ isolates in Iranian patients with pityriasis versicolor by using PCRrestriction 

enzyme method. 

8J patient]s isolates of !+6+&&.d"+*were isolated on modified Dixon agar. Genomic 

DNA was extracted by glass-beads, phenol-chloroform method and purified by alcohol 

precipitation. In all samples 28. rDNA regions was PCR-amplified, digested by the 

restriction enzyme 7U%I and electrophoresed on 1.8_ agarose gels. Identification was 

carried out according the different electrophoretic patterns after RF/P. 

The most frequent isolate was !/*@6%8%&+*(6J.8[_), followed by*!/*U($U($*(28.K_),*!/* 

&2)'%5"+6"&*(J.61_),*!/*$.&-$"#-+*(2.O_) and*!/*&66%UU"+.*(1.2_),*respectively.** 

* 

It is concluded thatM 

1) PCR-RF/P is an accurate, rapid and straight forward method for 

differentiation of !+6+&&.d"+ species. 

2) The most frequent causative agent of pityriasis versicolor in Iran is !/* 

@6%8%&+. 

2) Identification of clinical isolates 

The applicability of the method was conducted with 118 clinical isolates. These isolates 

were also identified by the API 20C A6j. The 26. r DNA- and IG. 1-RF/P system 

correctly identified 8O.7_ of the isolates (100 of 118), compared with OJ.2_ for the API 

20C A6j . 18 out of 118 isolates, not included in the database, were not identified. These 

isolates were finally identified by sequencing of the D1/D2 domains of the 26. r DNA. 

The 26. r DNA- and IG. 1- RF/P database presented in this study, supported by the 

2ioNumerics software, allows easy and rapid identification of clinical yeast-liBe organisms. 

Compared to other molecular methods, this system enables the identification of a relatively 

broad range of species. It should be noted that the results presented in this study constitute 

an initial database, and should be supplemented with further species not used in the 

present study. 


P-0413. A one enzyme PCR.RFLP assay for identification of six medically 

important Candida species 

Mirhendi H 1 , MaBimura l 2 


Mycology 

Early identification of 7+,5"5+*isolates to the species level is necessary for effective 

antifungal therapy, and can also facilitate control of hospital infections. Phenotypebased 

methods for identifying 7+,5"5+*species are often difficult and time-consuming. 

Molecular biological techniques provide a useful alternative approach. In the present 

study, the IT.1-[.8.-IT.2 regions of fungal rRNA genes were amplified with universal 

primers in 20 standard strains. Digestion of the PCR products with a single restriction 

enzyme, !&'I, allowed discrimination of medically important 7+,5"5+ species, 

including 7/*+68"#+,&, 7/*@6+8$+-+, 7/*'+$+'&"6%&"&3*7/*-$%'"#+6"&3*7/*[$(&.", and 7/* 

@("66".$)%,5""/ 6sing this method, we successfully identified 1J7 clinical isolates of 

7+,5"5+. Among them, 7/*+68"#+,& was identified as the most common species, 

followed by 7/*'+$+'&"6%&"&,*7/*-$%'"#+6"&, C. @6+8$+-+3*7/*[$(&.", and 7/*@("66".$)%,5""/ 

This method is a simple, rapid, and cost-effective method for differentiation between 

species that is applicable in clinical laboratories. 

P-0414. Identification of common Aspergillus species by a single PCR - 

restriction enzyme method 

Mirhendi H 1 , MaBimura l 2 , Diba l 1 , (alalizand N 1 


Mycology 

Aspergillus species are important cause of invasive fungal infections in 

immunocompromised patients, particularly among bone marrow transplant recipients. 

As clinical signs of the infection are not specific and current methods for early 

laboratory diagnosis are not ideal, and regarding recent reports of reduced antifungal 

susceptibilities among 4&'.$@"66(& species, rapid and accurate identification of 

4&'.$@"66" is necessary for clinical management and for epidemiological purposes. 

Traditional phenotypic methods are usually time consuming, can differentiate only the 

common 4&'.$@"66(&, and need specialist laboratories. Recently molecular approaches 

have been developed for detecting and identifying Aspergillus DNA from clinical 

material as well as cultures. 

Here we describe a single PCR-RF/P profile for identification and differentiation of 

five medically important 4&'.$@"66(& species from culture, including 4/*U()"@+-(&3*4/* 

U6+0(&3*4/*,"@.$3*4/*-.$$.(&3*and*4/*,"5(6+,&/* 

* 

Five reference strains and 28 clinical and environmental isolates, including eight 4. 

U()"@+-(&, ten 4/*U6+0(&3*seven*4/*,"@.$3*two*4/*-.$$.(&*and one*4/*,"5(6+,& were used 

in the study. All strains were also examined by morphological criteria. DNA was 

extracted by glass beads preparation, phenol-chloroform purification and alcohol 

precipitation. PCR was performed using the universal primers IT.1 and IT.O for 

amplifying IT.1-[.8. rDNA-IT.2 region. PCR product was electrophoresed through 

1.[_ agarose gel. 2y analyzing various sequences presented in the Gen2anB and 

considering various restriction enzymes, 7U%I was selected as the best enzyme for 

differentiation of the [ common 4&'.$@"66(& species. 

A single band with size about [80bp was observed for each strain. The electrophoretic 

pattern resulted from RF/P with 7U%I allow us to clearly differentiate five 4&'.$@"66(&. 

No variation was seen in RF/P patterns of each species among clinical isolates. ae 

thinB that this single PCR-RF/P is a fast and easy-to-perform method for identification 

of common 4&'.$@"6(& in the cultures and could be applicable in the reference medical 

mycology laboratories.

P-0415. The secondary metabolites of Pseudallescheria boydii present in 

mycelium, medium, spore surfaces and in blood 

Nedved J, ZabBa M, .ulc M, flsovsBa (, .Blenar (, HavliceB V 

Institute of Microbiology, Prague, Czech Republic 

1&.(5+66.&#?.$"+*8%25"" is one of the common opportunistic pathogens which are 

responsible for many fungal infection episodes every year. The aim of this study was 

the screening of the secondary metabolites originating from the non-ribosomal peptide 

synthesis in 1&.(5+6.&#?.$"+*8%25"". 6sually these products contain not only 21 

encoded amino acid residues but also many different amino acid-liBe structures. ae 

focused our study to the presence of cyclic peptides and related molecules that are 

promising marBers of fungal infections. Major aim of our research is to establish a 

flexible and rapid diagnostic method applicable both in human and/or veterinary 

medicine. Cyclic peptides are produced by multienzyme systems, have high thermoand 

pH- stability (strong factor compared to proteomic approaches) and they have 

high specificity with regard to a particular fungal strain. They potentially could be 

factors of virulence of pathogenic strains. If present on spore surfaces they could be 

detected very soon after the initial onset of the infection. 

ae have applied an array of mass spectral tools for the analysis of different extracts 

from the fungal mycelium, medium and spores obtained from multiple cultivation 

conditions of 1/*8%25"" (different presentation). The analyses were performed on 

Finnigan MATK[ (APCI), Finnigan /C~-DECA (E.I) and 2ruBer APEj-~ FTM. (E.I 

and DE.I). Potential marBer Candidates were separated by different chromatographic 

systems including analytical and preparative high pressure liquid chromatography 

(HP/C) and/or 6ltrahigh pressure liquid chromatography (6P/C). ~uantitative results 

were obtained from 6P/C experiments (v10 minute analytical runs, PDA detection), 

qualitative information was generated from product ion mass spectra. ahole tissue 

and blood samples were analyzed by DE.I (desorption electrospray invented in 200O). 

ae have developed a general procedure for the separation and analysis of secondary 

metabolites of 1/*8%25"". In addition to cyclopeptidic fraction, also fungal pigments are 

of interest as they also might be involved in the pathogenic process and/or used as 

alternative marBer molecules. Chemically characterized structures will be searched in 

biological models ",*0"0%*(pseudallescheriasis in neutropenic mice, 

pseudallescheriasis in mice with cystic fibrosis) as well as in models ",*0"-$% (human 

blood cultivated with spores of 1/*8%25""). 

Acknowledgement: 

Ministry of Education, Youth and .ports of the Czech Republic (/C[O[), Czech 

.cience Foundation (20J/0O/07KK), and IMIC institutional research concept 

(AVfZ[0200[10). 

P-0416. Protease, phospholipase, serotypes and adherence properties of 

Candida albicans in cancer patients 

Panizo M, ReviaBina V 

INHRR, Caracas, Venezuela 

Purpose of the study: To evaluate 7+,5"5+*+68"#+,& adherence in oropharyngeal isolates 

coming from cancer patients and to correlate the adherence with the serotypes and 

phospholipase and protease activities. 

Description of the project: An experimental, transversal, and comparative study was carried 

out with JJ oropharyngeal isolates of 7/*+68"#+,&M 11 from cancer patients and 22 from healthy 

subjects, paired by age and sex. The samples were taBen by oropharyngeal swabs and strains 

identification was performed by standard procedures. Adherence to epithelial cells was examined 

using HEp-2 cells and Giemsa stain. 1 Determination of protease production was assayed 

according to AoBi .-*+6., 2 using bovine serum albumin agar plates. J The phospholipase production 

was performed according to PolaB O using the egg yolB agar plate method of Price .-*+6. [ 

Measurement and calculation of phospholipase and protease activity (Pz coefficient) was 

calculated. [ 7/*+68"#+,& serotypes were determined by a slide agglutination test. 6 Each assay 

was carried out by triplicate. .tatistical analyses were assessed by ANfVA test and .tudent - 

test, using .tatgraphics Plus [.1 program. A '-value of v0.0[ was considered significant. 

Results and Conclusions: .tatistically significant differences were obtained ('v0.0[) 

comparing 7/*+68"#+,& adherence coming from cancer patients and healthy subjects, being the 

first 1,O times more adherent. 7/*+68"#+,& serotype A was predominant in both study groups. 

Adherence of 7/*+68"#+,& A and 2 serotypes of cancer patients was bigger than the adherence of 

both serotypes in healthy subjects (pv0.0[). No statistically significant differences were observed 

when comparing protease and phospholipase activities with 7/*+68"#+,& adherence and both 

serotypes ('t0.0[). 7/*+68"#+,& adherence of cancer patients is dependent of the serotypes but 

independent of the protease and phospholipase activities. 7/*+68"#+,& adherence to epithelial 

cells is the virulence factor with more expression in cancer patients. 

References: 

1. Panizo MM. Evaluacián de la adherencia de aislados orofarÜngeos de Candida 

albicans provenientes de pacientes oncolágicos a cdlulas HEp-2. Tesis de Grado. 

200O. 6niversidad Nacional Experimental Francisco de Miranda. Venezuela. 

2. AoBi ., Ito-luwa ., NaBamura Y, Masuhara T. Comparative pathogenicity of wild-type 

strains and respiratory mutants of 7+,5"5+*+68"#+,& in mice. Zol 2aBl 1KK0n 27JM JJ2- 

OJ. 

J. De 2ernardis F, Chiani P, Ciccozzi M, Pellegrini G, Ceddia T, D]fffizzi G, ~uinti I, 

.ullivan P, Cassone A. Elevated aspartic proteinase secretion and experimental 

pathogenicity of 7+,5"5+*+68"#+,& isolates from oral cavities of subjects infected with 

human immunodeficiency virus. Infect Immun 1KK6n 6OM O66-71. 

O. PolaB A. Virulence of 7+,5"5+*+68"#+,& mutants. Mycoses 1KK2n J[M K-16. 

[. Price MF, ailBinson ID, Gentry /f. Plate method for detection of phospholipase 

activity in 7+,5"5+*+68"#+,&. .abouraudia 1K82n 20M 7-1O. 

6. Hansenclever H, Michell V. Antigenic studies of 7+,5"5+. I. fbservation of two 

antigenic groups in 7+,5"5+*+68"#+,&. ( 2acteriol 1K61n 82M [70-[7J. 


P-0417. Determination of the optimum incubation period for recovery yeasts 

from automated blood systems. Results from a multicentric prospective study 

Paugam A, Ancelle T 

/aboratoire de Mycologie, HÇpital Cochin 

Background: there is no consensus in the literature concerning the optimal 

incubation period required for recovery of yeast from automated blood culture 

systems. To define the best incubation time we analysed the results of an active 

surveillance system performed by the YEA.T .tudy group (Institut Pasteur, Paris). 

This study has been implemented in Paris and suburbs and included 26 medical 

centres. 

Methods: 2etween .eptember 2002 and (uly 200[ a total of K66 yeasts were isolated 

from bloodstream and K2J were analysed for this study. 2lood cultures were 

performed using the 2acT/AlerT (bioMdrieux) with standard media or the 2actec 

method (2ecton DicBinson) with fungal media. Yeast species were identified by 

assimilation profile on APIJ2C (bioMdrieux) and in case of identification of 7+,5"5+* 

+68"#+,&, a PCR was systematically done to identify the species*5(86",".,&"&. The 

incubation time was calculated by the difference between the day of yeast detection 

and the day of blood sampling. 

Results: there was no statistically significant difference between the 2 automated 

blood systems concerning yeast detection delays and even with 7+,5"5+*@6+8$+-+. For 

1O centres the maximum conservation time was [ days and for 12 centres the 

maximum conservation time varied between 7 and J0 days. For the centres with a 

maximal conservation time t [ days 71O isolates were recovered from blood cultures 

but only 26 (J.6_) were detected after [ days and none after 8 days. The distribution 

and the number of species according the incubation time are shown belowM 

P-0418. Identification of Candida glabrata using a microtiter strip based 

trehalase test combined with a germtube test – a 3-year experience in a routine 

laboratory 

Peltroche-/lacsahuanga H, /ytticBen R, Haase G 

Institute of Medical Microbiology, 6niversity Hospital RaTH Aachen, Aachen, 

Germany 

Purpose of the study: 7+,5"5+*@6+8$+-+ is the second most occurring yeast species in a 

routine lab and exhibiting frequently a reduced sensibility or even resistance in case of 

fluconazole. Therefore, rapid and reliable identification of this yeast is mandatory. In 1KKK 

we have proposed a combined scheme for identification of germtube positive yeast species 

i.e. 7/*+68"#+,&, 7/*5(86",".,&"&, and 7/*+U$"#+,+ and 7/*@6+8$+-+ using a microtiter strip 

based germtube test in combination with a rapid trehalase test (1). Here we want to report 

on a J-year experience using this identification scheme. 

Description: aithin a J-year period we have used this scheme for identification of K,OJ0 

yeast isolate cultured in the routine laboratory of the university hospital of the RaTH 

Aachen. The majority of these isolates had been cultured on .abouraud Dextrose Agar 

with 0.[ _ chloramphenicol (2D, Heidelberg, Germany). This brand of culture medium 

turned out to perform well as a preculture medium for expression of the cell bound acid 

trehalase formation in 7+,5"5+*@6+8$+-+. 

In order to checB whether misidentification of 7/*@6+8$+-+ as 7/*-$%'"#+6"& or as another 

yeast species occurred (2), we have re-identified 2[0 7/*@6+8$+-+ isolates according to our 

identification scheme. ae used the API J2C system (bioMdrieux, Nyrtingen, Germany) and 

a hierachical clustering analyses applied on Fourier-Transform-Infrared-.pectroscopy 

(2ruBer, larlsruhe, Germany) for species identification. In case of any discrepant results 

final identification was achieved by sequence analyses of the D2 region of the nuclear 28. 

rDNA. 

Incubation time in days 

Results and conclusion: ff the K,OJ0 yeast isolates identified [,611 ([K.[ _) were 

germtube positive and 2,O61 (26.1 _) were identified as 7/*@6+8$+-+ whereas 1,J[8 isolates 

Cumulative percentage of isolation (number of isolates detected) 

(1O.O_) needs further identification. fnly 1O (0.1[_) isolates of the latter ones turned out 

.pecies (n) 1 2 J O [ 6 7 8 

7/*@6+8$+-+*(10[)* 

7/*+68"#+,&*(J68) 

7/*,.%U%$)+,&*G2K) 

7/*[$(&."*(21J* 

7/*'+$+'&"6%&"&*(80)* 

7/*6(&"-+,"+.*(7)* 

7/*6"'%62-"#+*(2) 

Total 

K_ (K) 

27_ (101) 

0_ (0) 

10_ (2) 

21_ (17) 

1O_ (1) 

0 

1J0 

JJ_ (26) 

66_ (1O2) 

2O_ (7) 

71_ (1J) 

61_ (J2) 

71_ (O) 

1 

22[ 

[7_ (2[) 

86_ (7[) 

[O_ (8) 

81_ (2) 

80_ (16) 

86_ (1) 

0 

127 

81_ (2[) 

K[_ (J0) 

7[_ (6) 

81_ (0) 

K[_ (11) 

86_ (0) 

0 

72 

K1_(11) 

K8_(1O) 

86_ (J) 

86_ (1) 

KK_ (J) 

86_ (0) 

0 

J2 

Conclusion: five days of incubation is habitually sufficient for routine of automated 

blood cultures. ahen a species with a slow rate is suspected (i.e. 7$2'-%#%##(& 

,.%U%$)+,&) an incubation of cultures bottles for more than 8 days does not improve 

the sensitivity of the blood culture isolation. 

finally as 7/*@6+8$+-+ exhibiting only weaB trehalase activity. Two isolates resembling 

K8_ (7) morphologically KK_ (1) 7/*@6+8$+-+ 100_ (1) but lacBing trehalase activity turned out as 4$_"%d2)+*-.66($"& 

KK_ (O) upon 100_ sequence (2) analyses. 100_ (0) In case of the 2[0 re-identified 7/*@6+8$+-+ strains no false 

K7_ (O) identification K7_ (0) could 100_ be revealed. (1) 

K[_ (2) 100_ (1) 100_ (0) 

KK_ (0) Conclusion: 100_ (1) the 100_ published (0) scheme turned out as a reliable and rapid test allowing cost 

86_ (0) 

effective 

100_ 

and 

(1) 

easy-to-perform 

100_ (0) 

identification of germtube positive yeasts and 7/*@6+8$+-+. 

Potential misidentification of 7/*@6+8$+-+ as 

0 1 0 

7/*+68"#+,& and / or 7/*-$%'"#+6"& due to a positive trehalase test as reported recently as 

18 6 2 

potential problem in case of using a dip sticB glucose test for detection of trehalase activity 

(2) does not turned out as a real problem when using both informationM acid trehalase 

activity and morphology i.e. no germtube formation and presence small nearly round yeast 

cells in case of 7/*@6+8$+-+. 

ReferencesM 

1. H. Peltroche-/lacsahuanga .-*+6., Diagn Microbiol Infect Dis J[M 1K7-20O (1KKK) 

2. 2. aillinger .-*+6., ( Clin Microbiol OJM OKK-[01 (200[)

P-0419. Identification of Candida species by analysis of rRNA spacer regions 

Ro B 1 , lim Y 1 , /ee M 2 , lim H 2 

1 Department of Dermatology, Chung Ang 6niversity Yongsan Hospital, .eoul, .outh 

lorea, 2 Department of /aboratory Medicine, Chung Ang 6niversity Yongsan Hospital, 

.eoul, .outh lorea 

Background: The genus 7+,5"5+ comprises 16J species, the most common 

pathogen in the genus is 7/*+68"#+,&, however, other 7+,5"5+ species are considered 

as emergent pathogens. 2ecause there are species-specific differences in the 

susceptibility of 7+,5"5+ spp. to the currently used therapeutic drugs, species 

identification is critical for therapeutic planning and accurate epidemiological records. 

The objective of this study is to evaluate the performance of tRNA intergenic length 

polymorphism (tDNA-I/P) analysis for the accurate identification of 7+,5"5+ species. 

Methods: For the identifying the 7 type strains and [O clinical isolates of 7+,5"5+, the 

suitability of tDNA-I/P was evaluated. The polymerase chain reaction (PCR) using a 

pair of primers or a single primer was performed. The PCR products were separated 

by electrophoresis in 2_ agarose gels for 70 min. 

Results: In seven 7+,5"5+*type strains,*tDNA-I/P using a single primer (reverse) can 

be easily analyzed by visual comparison. Fifty-three of [O strains were identified as 

the same species with conventional identification. 

Conclusions: The tDNA-I/P analysis can be useful for the simple and rapid 

identification of 7+,5"5+ species in routine laboratories. 

P-0420. Rapid detection of dermatophytes of tinea capitis in Portuguese 

patients using PCR 

Roque H 1 , Vieira R 2 , Rato . J , Martins M O 

1 Instituto Higiene e Medicina Tropical, /isboa, 2 Hospital Curry Cabral, /isboa, 

J Instituto Higiene e Medicina Tropical, /isboa, O Instituto Higiene e Medicina Tropical, 

/isboa 

!"#$%&'%$()*#+,"&, !/*+(5%(","" and >$"#?%'?2-%,*&%(5+,.,&. are the most frequent 

dermatophytes isolated from >",.+*#+'"-"&*in Portuguese patients. .pecific PCR 

primers for !/*#+,"& and >/*&%(5+,.,&. have already been shown to be good 

molecular diagnostic tools. 1,2 2ut, until now, there have been no specific primers for !/* 

+(5%(","". 

This report describes the application of PCR fingerprinting, for identification of 

common species of these dermatophytes, using the M1J microsatellite primers, 

(GACA) O and (GTG) [. Initial use of these primers generated a specific DNA fragment 

of !/*+(5%(","" which was then cloned and sequenced. 2ased on sequence data, we 

designed specific forward (MAõ1F) and reverse (MAõ1R) PCR primers for this 

fragment. The reliability of these new primers for diagnosis of !/*+(5%(","" was then 

assessed by PCR. 2ased upon a global test of 182 strains/isolates belonging to 11 

species of dermatophytes, the PCR results showed that these primers generated a 

singular, OJ1 bp band specific to strains and isolates of !/*+(5%(","", These results 

confirmed the reliability of these primers for diagnosis of !/*+(5%(","". ae recommend 

the use of these primers for clinical laboratory analysis. 

It is now possible to simultaneously identify !/*+(5%(","", !/*#+,"& and >/*&%(5+,.,&., 

with a singular, multiplex PCR reaction using specific primers for each of the three 

species. .uch a PCR-based diagnostic assay can be completed within hours, as 

opposed to J weeBs using conventional cultural methods. 

In conclusion, PCR analysis using the primer set outlined above will be a useful tool 

for identifying quicBly the most frequent causative agents of >",.+*#+'"-"& in Portugal. 

Reference: 

/iu .-*+6. Med Mycol JK, 2001n 2 /iu .-*+6. . (.Med. Microbiol. [1, 2002) 


P-0421. Multicentric evaluation of a commercial PCR-elisa kit (Onychodiag®) 

for diagnosis of Dermatophytic onychomycosis 

Savin C 1 , HucB . 2 , Rolland C J , 2enderdouche M O , Faure f J , Noacco G 2 , Candolfi E 2 , 

Pelloux H J , Derouin F O , Coupd . O 

1 2io Advance, 2ussy-.aint-Martin, France, 2 Institut de Parasitologie, .trasbourg, 

France, J /aboratoire de Parasitologie-Mycologie, CH6 Grenoble, France, O /aboratoire 

de Parasitologie-Mycologie, HÇpital .aint-/ouis, 6niversitd Paris 7, France 

Background: fnychomycosis are very common infections, responsible for approximately [0_ 

of nail dystrophies. Identification of the fungal origin of the lesion is required to avoid 

misdiagnosis and justify the expense and long duration of treatment. /aboratory diagnosis of 

onychomycosis currently relies on a proper sampling of the nail, demonstration of hyphae by 

direct microscopic examination of the sample followed by culture, which results are not available 

before several weeBs. In order to shorten the delay of diagnosis, a PCR-E/I.A test 

(fnychodiagw, 2ioAdvance, France) has been developed for diagnosis of dermatophyte 


Objective: To prospectively assess the performance of fnychodiagw, for diagnosis of 

onychomycosis due to dermatophytes in independent laboratories. 

Patients/Methods: Three university hospital laboratories specialized in medical mycology 

participated to the study. Nail samples (N.) were collected from patients suspected for 

onychomycosis (n}OJ8) or healthy controls (n}108). In laboratories A and 2, N. were collected 

by trained mycologists taBing care to collect the sample as close as possible to the lesion. In 

laboratory C, N. were collected by other physicians and referred to the laboratory. Each N. was 

processed by classical mycological techniques (direct examination and culture) and tested by 

fnychodiagw blindly to the results of mycology. For 7[ patients, an additional distal sample, 

obtained by clipping the nail plate, was collected from the same dystrophic nail and tested by 

fnychodiagw only, as such sample is improper for mycology. 

P-0422. Evaluation of antigen-capture Elisas for detection of histoplasmosis in 

urine of immunocompromised patients 

Scheel C 1 , 2enjamin / 1 , Morgan ( 1 , Hurst . 1 , .amayoa 2 2 , /indsley M 1 , Arthington- 

.Baggs 2 1 , Arathoon E 2 , 2randt M 1 

1 Mycotic Diseases 2ranch, Centers for Disease Prevention and Control, Atlanta, 

Georgia, 6.A, 2 Clinica Familiar /uis Angel Garcia, Hospital General .an (uan de 

Dios, Guatemala City, Guatemala 

A"&-%'6+&)+*#+'&(6+-() infection causes significant morbidity and mortality in HIVinfected 

individuals, particularly those in developing countries without access to 

sophisticated diagnostics or highly-active antiretroviral therapy. Furthermore, 

symptoms of histoplasmosis are non-specific, while current diagnostic methods to 

detect infections can be complex, expensive and slow. A simple, rapid method to 

detect A/*#+'&(6+-() infection would allow clinicians to provide specific 

chemotherapeutic intervention at an early stage of disease to decrease morbidity and 

mortality. ae evaluated two E/I.A-based antigen detection assays using as capture 

antibody either anti-A/*#+'&(6+-() polyclonal rabbit serum or a monoclonal against 

the common fungal carbohydrate antigen of A/*#+'&(6+-() (C-Ag). ae tested 

baseline urine specimens from Guatemalan AID. patients withM confirmed 

histoplasmosis (suspected dissemination) (,*} 6)n non-histoplasmosis fungal and nonfungal 

diseases (,*} K)n and healthy controls (,*} O) to determine the efficacy of both 

assays. Preliminary results indicate that both antigen-capture E/I.As demonstrated a 

sensitivity of 8J_ and a specificity of 100_ when tested against these urine cohorts. 

ahile specimen collection and assay evaluation continue, this assay shows promise 

in the rapid diagnosis of histoplasmosis in HIV-infected individuals and can be easily 

implemented in laboratories across the world. 

Each N. was processed by fnychodiagw as followM DNA extraction, amplification by PCR, 

hybridization with a labelled dermatophyte-specific probe and immobilization in a microplate well 

precoated with a capture probe. Results were expressed as fD valuesM positive for fDt0.[, 

negative for fDv0.J and borderline (grey zone) for 0.JvfDv0.[. 

Results: .amples from the 108 healthy controls were negative by fnychodiagw. Cultures of N. 

from patients with suspected onychomycosis yield to isolation of a dermatophyte in 1O0 samples, 

with a predominance of >/*$(8$()*(n}121). 117/1O0 samples were positive by fnychodiagw and 

6 were borderline. The sensitivity of fnychodiagw was 7[, 8J and 100_ for laboratory A, 2 and 

C respectively, with a mean interlaboratory value of 8O_ (88_ including grey zone). Among 22J 

samples with negative culture for dermatophytes, 62 were positive by fnychodiagwn J1 of them 

were positive by direct microscopic examination suggesting that these patients were actually 

infected. 2ased on the sensitivity of fnychodiagw on mycologically proven dermatophytes 

onychomycosis and on the 100_ specificity in healthy controls, these positive fnychodiagw 

results were considered as true positive and strongly indicative for the diagnosis of 

dermatophytic onychomycosis. The low performance of mycology on these samples was related 

to non-proper conditions of sampling. 

For distal samples, the results of mycology on properly collected samples from the same nail 

were used as reference. fnychodiagw was positive in OK/[J distal samples (K2_) from patients 

with proven dermatophytic onychomycosis. 

ConclusionsM fnychodiagw provides diagnosis of onychomycosis due to dermatophytes within 

2O-O8h after sampling. Its sensitivity on properly collected samples and on distal samples was 

close to that of mycological techniques performed on properly collected samples.

P-0423. Molecular diagnosis by polymerase chain reaction (PCR) of the six 

main clinical importance Candida species 

.ilva V, Madrid H, Thomson P 

6niversity of Chile, .chool of Medicine, 2iomedical .ciences Institute, Microbiology 

and Mycology Program 

Candidemia and candiduria have increased significantly during the past 20 years, 

being an important cause of morbidity and mortality in immunocompromised and 

critical ill patients. .everal studies have documented trends in species distribution that 

varied marBedly among different countries and time period, showing a mild increase in 

the proportion of 7. no-+68"#+,& species, as well as the emergence of 7/*@6+8$+-+ and 

7/*'+$+'&"6%&"&. 

The accurate and rapid detection with identification of the 7+,5"5+ species is very 

important to help the medical treatment, because the antifungal resistance is 

associated to some yeast, mainly in 7. no-+68"#+,& species such as 7/*@6+8$+-+ and 7/* 

[$(&.". 

The aim of this study was designed species-specific primers to be applied to the 

molecular diagnosis by PCR of 7/*+68"#+,&3*7/*5(86",".,&"&3*7/*@6+8$+-+3*7/*[$(&."3*7/* 

'+$+'&"6%&"& y 7/*-$%'"#+6"&, following the bio-informatics analysis of the 18. small 

subunit (..6), internal transcribed spacer 1 (IT.1), [.8. and IT.2 sequences of the 

ribosomal DNA (rDNA) of each 7+,5"5+ studied species. 

From Gen2anB data we obtained among [ to 10 sequences of each rDNA region by 

each 7+,5"5+ species. The sequences were stored in aordPad format, aligned and 

compared with aid of the program Clustala (European 2ioinformatics Institute) to 

obtain the map and the consensus sequence of 18. ..6, IT.1, [,8. and IT.2 by 

each 7+,5"5+ species. After that, the consensus sequence of the six 7+,5"5+ species 

were compared and analyzed to identify the variable and conserved region. 

P-0424. Molecular identification of Candida lusitaniae using a PCR-REA method 

.isto F, Drago M, .caltrito M, .tano P, Morace G 

Dipartimento di .anitÖ Pubblica | Microbiologia - Virologia. 6niversitÖ degli .tudi di 

Milan, Italy 

A pair of primers selected from the costant region of the ERG11 gene of 7+,5"5+* 

+68"#+,&, was used to amplified a J[0 bp segment from the genomic DNA of 7/* 

6(&"-+,"+.. The PCR product was cloned in plasmid pCR-TfPf (Invitrogen) and 

sequenced (Gen2anB submitted). The sequence of the fragment was compared with 

the published gene sequences of 7/*+68"#+,&3*7/*5(86",".,&"&3*7/*@6+8$+-+3*7/* 

@("66.$)%,5""3*7/*[.U2$3*7/*[$(&."3*7/*'+$+'&"6%&"& and 7/*-$%'"#+6"&. The homology 

ranged from 62_ to K7_. According to a precedent study in which restriction enzyme 

analysis (REA) of PCR products was used to identify 7+,5"5+ isolates at the species 

level, the amplicon obtained from 7/*6(&"-+,"+. was therefore subjected to REA using 

=+(JA, A",#II and `%-I. The REA pattern obtained allowed to differenziate C. 

lusitaniae from the other species of 7+,5"5+ except from 7/*@6+8$+-+ whose patterns 

were very similar. Thus we tested another restriction enzyme, P&+I, with the above 

mentioned species. The results showed that only 7/*6(&"-+,"+., 7/*@6+8$+-+ and 7/* 

[$(&." had a recognition site for this enzyme but the patterns were different from each 

other. The addition of P&+I allowed the identification of 7/*6(&"-+,"+.. An early and 

rapid identification of this species in clinical specimens is of great importance because 

7/*6(&"-+,"+. is an emerging human pathogen that can cause severe infections in 

immunocompromised hosts. 

* 

ae selected the best region and designed O primers by each species (2 forward and 2 

reverse). The theoretical specificity of them were determined by short nucleotidenucleotide 

blast (NC2I). A set of two primers for each species that amplify different 

fragment size by each species of 7+,5"5+ were selected. 7+,5"5+ spp genomic DNA 

extraction from fresh cultures and contaminated blood samples were obtained by 

enzymatic and chemical reactions with the aid aizard Genomic DNA (Promega). The 

PCR reaction generated fragment of 172[ bp for 7/*@6+8$+-+, 1200 bp for 7/*[$(&.", 

72O bp for 7/*+68"#+,&, J[O bp for 7/*5(86",".,&"&, JO0 bp for 7/*-$%'"#+6"& and 210 bp 

for 7. '+$+'&"6%&"&. From 1[0 samples, the PCR was successful showing a 100_ of 

sensitivity and negative predictive value and more than K8_ of specificity and positive 

predictive value. 


P-0425. Isolation and characterization of Pseudallescheria boydii secreted 

cyclic peptide - the potential marker in diagnostics of fungal infection 

Sklenar J, úlc M, äbBa M, HavlÜceB V 

Institute of Microbiology, Prague, Czech Republic 

The successful treatment of a fungal infection presumes fast, sensitive, and robust 

diagnostic tool applicable from the very onset of infection. The procedure should not 

only distinguish extrinsic fungi in the body fluids, but recognize the genera as well. 

6nfortunately, the prevailing high titer of antifungal antibodies among healthy 

individuals hampers the use of a protein marBer molecule. Therefore the need for 

alternative marBer molecule attracted attention to the fungal secondary metabolites. 

Due to their physiological activities and distinct structure, one class of promising 

molecules consists of cyclic peptides produced by non-ribosomal peptide synthesis. 

Commonly found to be secreted by fungi yet unBnown in mammals, genera specific, 

they fulfill most of the conditions posed on a potential diagnostic marBer molecule. 

aith respect to this hypothesis we isolated a cyclic peptide from culture filtrate of 

1&.(5+66.&#?.$"+*8%25"", pathogen of immunocompromised patients. The culture 

filtrate extracted with dichloromethane and pre-fractioned by solid phase extraction 

(.PE) was separated by high performance liquid chromatography. The molecular 

characterization by liquid chromatography coupled with ion trap mass spectrometer 

(Finnigan /C~Deca), FT-M. (2ruBer) and a sector instrument (Finnigan MATK[) 

suggested the structure. The resistance to Carboxypeptidase Y and sequencing on 

ion trap mass spectrometer has confirmed the cyclic nature of the peptide. The gross 

sequence in mass increments was measured as cyclo(1[[-11O-11J-K7-1O7-11J). The 

masses correspond to both usual and unusual amino acids. The structural nuclear 

magnetic resonance (NMR) studies are under way. In future we aim to prove the 

fungal infection in animal experimental model by determination of this particular 

metabolite in body fluids or organ biopsies. ae hope the isolation procedure 

employed will be compatible with human samples as well. 

Acknowledgement: Ministry of Education, Youth and .ports of the Czech Republic 

(/C[O[), Czech .cience Foundation (20J/0O/07KK), and IMIC institutional research 

concept (AVfZ[0200[10). 

P-0426. Multicenter intra and interlaboratory reproducibility of the 

galactomannan EIA platelia Aspergillus 

Sánchez Reus F 1 , PemZn ( 2 , /ápez M 1 , AlBorta M J , Aznar ( O , 2orrell N [ , 2uendia 2 6 , 

Rodriguez V 7 , .panish NetworB of Infection in Transplantation (RE.ITRA) 

1 Hospital de la .anta Creu i .ant Pau, 2arcelona, .pain., 2 Hospital 6niversitario /a 

Fe, Valencia, .pain., J Hospital de Cruces, 2araBaldo (2ilbao), .pain., O Hospital 

Virgen del Rocio, .evilla, .pain, [ Hospital 6niversitario .on Dureta, Palma de 

Mallorca (2aleares), .pain., 6 Hospital 6niversitario /a Princesa, Madrid, .pain., 

7 Hospital de la Vall deHebrá, 2arcelona, .pain. 

Purpose: To evaluate the intra- and interassay and the interlaboratory reproducibility 

of the commercial galactomannan enzyme immunoassay (EIA) Platellia 3 4&'.$@"66(&* 

(2io-Rad) in clinical diagnostic laboratories/* 

* 

Methods: Fourteen clinical laboratories of the .panish NetworB of Infection in 

Transplantation (Resitra) participated in the collaborative study. 

The same lot of Platelia 3 4&'.$@"66(& proficiency panel was distributed to the 1O 

Resitra-laboratories and each six specimens of the panel (P1, P2, PJ, PO, P[, and P6) 

were tested in triplicate on three different days. EIA Bits were of the same lot and the 

tests were performed according to the manufacturer]s instructions. 

Results were collected and intraclass correlation coefficients (ICC) were analysed for 

each laboratory and for the total group of laboratories. The level of variation in the 

galactomannan index results was determined by calculating the percent coefficient of 

variation (CV_) for all individual specimens (P1, P2, PJ, PO, P[, and P6). 

Results and conclusions: A total of 6KK values were available for evaluationM results 

from one laboratory were excluded due to methodological differences, 6 panel 

member specimens were tested in triplicate in JK validated assays, and three aberrant 

determinations were rejected. 

All ICC from the 1J Resitra-laboratories were statistically significant. Global ICC was 

0.KO70. fne laboratory had an extreme value of 0.766 and the other twelve 

laboratories had values between 0.K276 and 0.KKO1. Consequently, only the results 

of these twelve laboratories were considered to calculate CV _. 

Table shows the variability of test results for the same assay, between assays and 

between laboratories. The mean of CV_ values was individualised for the 6-panel 

member specimens. 

P1 (0.18ò) P2 (0.O[ò) PJ (0.8Jò) PO (1.J1ò) P[ (2.1[ò) P6 (O.JKò) 

Intraassay 1J_ K_ 7_ 1[_ [_ [_ 

Interassay 21_ 16_ 12_ 18_ 10_ 8_ 

Interlaboratory J[_ 2K_ 20_ 2O_ 1[_ 18_ 

ò galactomannan specimen index 

The results indicate high concordance and good reproducibility of the EIA assay, but 

minimal changes in the antigen titre and individual low-positive results (v0.8) should 

be interpreted with caution.

P-0427. Mycological survey in critically ill patients 

Toubas D 1 , Gautier M 1 , NacB . 1 , Pascale / 1 , Frdddrique F 1 , Pinon ( 1 , /don A 2 , 

Cousson ( 2 

1 /aboratoire de Parasitologie-Mycologie, HÇpital Maison 2lanche, CH6 Reims, 

France, 2 .ervice de Rdanimation, HÇpital Robert Debrd, CH6 Reims, France 

Invasive candidiasis is a dreadful complication in IC6-patients. Diagnosis is difficult 

except in the case of candidemia. In this worB, our objective was to evaluate the 

importance of the colonization, anti-7+,5"5+ antibodies, and antigenemia in adult IC6- 

patients selected on risB factors. 

For a total of 1[O8 patients admitted in an 18-bed IC6, 128 patients were included 

(sex ratio 1.K, age median [K s16-8[u, K7 surgical patients, J1 medical patients). 

Mortality rate of the 128 patients was O1.7_ (mortality of the unitM 26.8 _). Clinical risB 

factors included abdominal surgery, broad spectrum antibiotherapy, prior 

corticotherapy, immunosuppressors, cancer, or polytraumatism. 

Among the K1 patients, forty-two patients received fluconazole (18 HC patients, 2O 

aBC or NC patients). 

The threshold between colonization and infection remains difficult to distinguish. In 

this context, early presumptive antifungal therapy by fluconazole is admitted. The first 

step consists of identifying patients that could benefit from a mycological surveillance. 

Then, the follow-up may guide to antifungal therapy. Colonization is not a sufficient 

criterium for treatment. Immunological data provide complementary information. After 

analysis of this study, a scheme for treatment was proposed by the medical staff of 

the IC6. If clinical status is favourable, patients are not treated. Importance of the 

colonization and positivity of 7+,5"5+ immunological marBers are considered in 

decision for antifungal therapy. 

/arge clinical trials will be necessary to provide recommendations in view of selecting 

patients who would better benefit from pre-emptive treatment. 

.amples were collected for mycological cultures and immunological assays on 

admission and once weeBly. ae evaluated the importance of colonization by the CCI 

(corrected colonization index) as proposed by Pittet sCCI v0.OM weaBly (aBC) or not 

colonized (NC)n CCI , 0.OM heavily colonized (HC)u. ae used coimmunoelectrodiffusion 

(Co-IED) and immunoenzymatic assay (EIA Platelia-specific 

Ab test, 2io-Rad, France) for anti-7+,5"5+ antibodies detection and EIA assay 

(Plateliaw Candida-specific antigen test, 2io-Rad) for 7+,5"5+ antigenemia detection. 

Isolates were collected from 1KO throat samples ([K.[_), 117 rectal swab (O0.2_), 

16[ trachea (60.O_),*62 urinary samples (20.J_). 

6pon IC6-admission, JO patients were HC, 6K patients aBC and 2[ patients NC. 

Ninety-three (72_) were colonized by 7/+68"#+,&/** 

* 

fut of the 128 patients, K1 were followed up and sampled once weeBly during 2 to 12 

weeBs. Thirty-nine were HC, O2 aBC and 10 NC. 7/+68"#+,& was isolated in 72 

patients (7K.1_). The comparison of CCI and antibodies showed no relationship 

('}0.8[). 

CCI 

v0.O 

(n}[2) 

, 0.O 

(n}JK) 

EIA 

Co-IED 

r - r - 

21 

2K 

J1 ([K.6 _) 2J (OO.2_) 

(O0.O _) 

([[.8_) 

17 

22 

22 ([6.O _) 17 (OJ.6_) 

(OJ.6 _) 

([6.O_) 

EIA and Co- 

IED - 

EIA or Co- 

IED r 

27 ([1.K _) 2[ (O8.1 _) 

21 ([J.K _) 18 (O6.1 _) 

ae documented six fungemia. Mannanemia was positive in [/6 patients and J/6 

patients had anti-7+,5"5+ antibodies. All patients were found positive to at least one 

immunological assay. 

* 

Antigenemia was positive in 1J of the 128 patients ([ fungemia, O HC and O aBC). 

.erology was positive in J of the 8 patients without fungemia. 


P-0428. Comparison of micronaut-Candida, a new yeast identification system to 

API ID32C 

Táth 2, Falusi E, Miszti C, 2orbdly î, lardos G, Majoros L 

6niversity of Debrecen, Department of Medical Microbiology, Debrecen, Hungary 

Identification of Candida species is extremly important for the clinical mycologists. ae 

compared the performance of the commercial identification system MICRfNA6T- 

Candida (Merlin DiagnostiBa GmbH) to API IDJ2C (2ioMerieux). MICRfNA6T- 

Candida is based on testing 21 biochemical reactions. Evaluation of the tests are 

automated, results are availale in 2O hours. 

ae tested 128 clinical and reference strains of 18 different yeast species with the 

MICRfNA6T-Candida system including the most common human pathogenic 

Candida species, i.e. C. albicans (1K isolates), C. tropicalis (1O isolates), C. glabrata 

(17 isolates), C. Brusei (2J isolates), C. parapsilosis (1O isolates), C. inconspicua (12 

isolates), as well as less frequently found Candida species C. Befyr (6 isolates), C. 

famata, C. lusitaniae, C. guilliermondii (O isolates each), C lipolytica (J isolates), C. 

dubliniensis, C. rugosa (2 isolates each), C. norvegensis, C. catenulata (1 isolate). 

.ingle isolates of the medically important yeast species, Cryptococcus neoformans, C. 

humicolus, Rhodotorula spp. were also tested. Results of the API IDJ2C system were 

regarded as reference. In case of discrepancies between the two methods, we 

repeated both tests. 

MICRfNA6T-Candida never yielded unsuccesful identification (results of Candida 

spp. or unidentified yeast). Identifications with MICRfNA6T-Candida were the same 

as the API results in K2.2_ (10J/128) of the tests, including all C. albicans, C. 

tropicalis, C. glabrata, C. Brusei, C. parapsilosis, C. inconspicua, C. Befyr, C lipolytica 

and C. lusitaniae isolates, the single C. catenulata, C. neoformans isolates. Three of 

four C. guilliermondii isolates were identified with excellent results, while the remaining 

one (0,7_ of all isolates) repeatedly exhibited questionable identification. fut of the 

consistently identified 10J isolates 16 (12.[_) required the performing of the 

additional tests recommended by MICRfNA6T-Candida, including all 12 C. 

inconspicua isolates, the C. neoformans and one of the C. rugosa isolates. 

Inconsistent results were found in case of 6.J_ (8/128) of the isolates, i.e. the C. 

norvegensis and C. rugosa reference strains, one C. dubliniensis and the C. 

humicolus isolate as well as all four C. famata isolates, which were surprisingly 

identified as C. lusitaniae with ]excellent reliability]. 

ae concluded that MICRfNA6T-Candida and API IDJ2C are equally identify all 

common Candida species, discrepancies could be observed with some of the rarer 

yeasts. Consequently, MICRfNA6T-Candida can be a faster alternative of the widely 

used API IDJ2C identification system. 

P-0429. Synthetic analogues of yeast oligomannoses complement the detection 

of anti-Saccharomyces cerevisiae antibodies (ASCA) for differential diagnosis 

of Crohn’s disease and prediction of indeterminate colitis evolution 

Vandewalle P 1 , (oossens . 2 , Vitse-.tandaert A 1 , Vermeire . 2 , Colombel ( J , .endid 

2 1 , Mallet ( O , Poulain D 1 

1 Inserm 67KK - /aboratoire de mycologie fondamentale et appliqude, Facultd de 

Mddecine, /ille, France, 2 Division of Gastroenterology, Department of Internal 

Medicine, 6niversity Hospital Gasthuisberg, /euven, 2elgium, J CHR6 /ille, HÇpital 

Huriez, .ervice d]Hdpato-gastroentdrologie, /illen Inserm, 67K[, /ille, France, O CNR., 

6MR 86O2, Paris, F-7[2J1 France n EN., Ddpartement de Chimie, Paris, France 

fur laboratory developed an immunoenzymatic test to detect anti-=+##?+$%)2#.&* 

#.$.0"&"+.*mannan*antibodies (we named A.CA) which are marBers of Crohn]s 

disease (CD). It was also shown that despite antigenic heterogeneity in mannan 

preparations the A.CA major epitopes consisted in a mannotriose $ 1-J Man $ 1-2 

Man $ 1-2 Man (ManJ) and a mannotetraose4$ 1-J Man $ 1-2 Man $ 1-2 Man $ 1-2 

Man (ManO). The aim of the study was to develop new tests involving ManJ and ManO 

chemically synthesized (5) and to assess how these purified epitopes complement =/* 

#.$.0"&"+. mannan for differential diagnosis with ulcerative colitis (6C), prediction of 

indeterminate colitis (IC) evolution or reflect special phenotype. 

1O02 patients and controls were included from different medical centers in Europe, 

6.A and Tunisia, they consisted in 772 CD patients, J01 6C patients, J0K healthy 

controls and 20 patients from a prospective follow-up study of indeterminate colitis. All 

sera were tested with the initial A.CA test and with the tests, we named A.CA2 in 

which 5Man6 and 5ManO were coated onto EIA plates. 

aith a cut-off of 1[ A6 for 5ManJ, the sensitivity and specificity of the test were 

respectively of 22_ and K0_ for discriminating CD versus 6C, 22_ and 87_ for CD 

versus controls. aith 5manO, the sensitivity and specificity were respectively 28_ and 

K1_ for CD versus 6C, 28_ and K[_ for CD versus controls. Interestingly, using 

A.CA2, we detected 2[_ of the patients who were A.CA negative with a specificity 

of 8[_. Among the 11 IC patients who evolved towards CD, 1 was A.CA and A.CA2 

positive and for J of them, A.CA2 was the only predictive marBer for evolution. 

(oined detection of antibodies against selected 5 oligomannose epitopes represents 

an approach to increase the diagnostic performance of A.CA test for both 6C and IC. 

It also provides a way for basic research to dissect dysregulation of glycan codes 

recognition related to CD.

P-0430. Detection of fungal contamination: surface sampling by dusting cloth 

improves quantitative recovery of aspergilli 

Vescia N 1 , fsborn ( 1 , Dealessandro D 2 , Grillot R J 

1 Dept. Public Health .ciences 6niv. /a .apienza, Rome, Italy, 2 Dept. Architecture and 

Planning 6niv. /a .apienza, Rome, Italy, J /ab. Parasitologie-Mycologie, CH6 

Grenoble, France 

Background: The health-care facility environment can be implicated in disease 

transmission in immunocompromised patients. Exposure of such patients to moulds can 

result in fatal infections. Incidence of hospital -acquired aspergillosis can be minimized by 

adherence to ventilation standards for specialized care environments, appropriate 

maintenance and careful cleaning of patient environment. Mycological control is also 

recommended in protective environments or operating rooms mainly during building 

renovation, by air sampling but also surface sampling, an efficient method to detect minor 

fungal contamination. 

Objective: To maBe surface sampling easier and to reduce surveillance costs, we 

developed a simple technique using sterile dusting paper cloths. To validate this method, 

we used an experimental protocol to evaluate ability of the system to capture fungal conidia, 

in comparison with two other methods, Rodac Contact plates and cotton pads. 

Materials/method: 4&'.$@"66(&*,"@.$ was chosen as organism test. A reference 

suspension of conidia (0.[ McFarland) was prepared with serial dilutions (1/J0, 1/O0, 1/[0, 

1/100) to contaminate sterile stainless steel plates (n}18), each J0 by J0 cm. Each one 

was divided into 16 smaller squaresM 0.[ ml of the suspension was placed in the centre of 

each square and allowed to dry. Thus, 288 square surfaces were contaminated. From K6 of 

these, Rodac plates were used to harvest the sporesn from another K6 the cotton pads 

were used, and from the last K6 the dusting cloths were used and inseminated in Petri 

plates. The remaining stainless steel plates were used to repeat the experiment (dilutions 

1/O0 and 1/[0) with agar neutralised with lecithin and polysorbate 80. All the plates were 

incubated at J7ÅC for 18 h. For the statistical analyses, the data were transformed using 

the natural logarithm. Multiple log-linear regression was used to estimate the differences 

between the J methods, between the O dilutions, and the effect of neutralisation. 

P-0431. Improvements in diagnosis of histoplasmosis by antigen detection 

Wheat L, Connolly P, DurBin M, /eMonte A, aitt (, Egan /, HacBett E 

MiraVista Diagnostics/Mira2ella Technologies, Indianapolis, IN 6.A 

Antigen detection permits rapid diagnosis of disseminated histoplasmosis with a 

sensitivity over K0_. False-positive antigenemia in transplant patients receiving rabbit 

anti-thymocyte globulin lead to research resulting in the development of an improved 

second generation antigen immunoassay. False-positive results in transplant patients 

were found to be caused by human anti-rabbit antibodies, and they have been 

reduced by 8[_ in the 2 nd generation assay. Also, sensitivity is improved in the 

second generation antigen immunoassayn detection in patients with false-negative 

antigenuria improved by 77_. Calculation of results from a calibration curve now 

permits accurate quantitation of antigen levels, leading to improved reproducibility. 

Antigenuria may be falsely negative in up to 2[_ of patients with acute pulmonary 

histoplasmosis, 66_ with subacute, and 86_ with chronic pulmonary histoplasmosis. 

Detection of antigen in bronchoalveolar lavage fluid may assist in the diagnosis of 

pulmonary histoplasmosis in patients with false-negative results in urine and serum. 

8[_ of 2A/ specimens from patients with histoplasmosis were positive in the 2 nd 

generation assay. In summary, improvements in antigen detection procedures have 

increased the accuracy for diagnosis of histoplasmosis. 

Results: The mean number of colonies was approximately the same for each method at 

dilution 1/100. At dilutions 1/[0, 1/O0 and 1/J0 the mean number of colonies is much higher 

for the cotton pads and dusting cloths than for Rodac. The multiple regression showed that 

neutralization reduces the number of colonies by 12_ (not significant P}0.07). In 

comparison with dilution 1/[0, the number of colonies recovered at dilutions 1/O0 and 1/J0 

were slightly increased, by 1J_ and 6_ respectively (not significant P}0.0K and P}0.[2). 

However, in comparison with Rodac plates, both cotton pads and dusting cloths identified 

many more of colonies, [ times as many and 6 times as many respectively (highly 

significant Pv0.0000[). 

Conclusions: This method using dusting cloths for surface sampling is able to taBe 

samples on every type of surface (smooth, mesh, flat, curved) and to have all information 

about contamination taBing only one sample from the area (air ventilation outlets, filters, 

patient]s bed, scialitic lamp.). ae showed that this device improves quantitative recovery of 

4/*,"@.$ from stainless steel surfaces. Although these results are preliminary, we thinB that 

this sampling method could contribute an easy, sensitive and inexpensive approach to 

environmental control. 


P-0432. Rapid identification of C. dubliniensis by the new latex agglutination 

test bichro-dubli fumouze in comparison with real-time PCR 

Willinger B, 2rightwell l, Roither ., .chabereiter-Gurtner C, Rotter M 

Division of Clinical Microbiology, Hygiene-Institute, Medical 6niversity Vienna, Austria 

Introduction: To date, differentiation of 7/*+68"#+,& and 7/*5(86",".,&"& has been hampered by 

their very close phenotypic and genotypic relationships. 6sing routine biochemical methods, 

differentiation of 7/*5(86",".,&"& from 7/*+68"#+,& is time consuming and may lead to ambiguous 

results. A new commercially available identification system 2ichro-dubli Fumouze ' test 

(Fumouze Diagnostics, /evallois-Perret Cedex, France) has been developed to enable 

identification of 7/*5(86",".,&"& within three minutes. This test is a latex co-agglutination card test, 

available for direct identification of 7/*5(86",".,&"& directly from colonies. It is based on the 

agglutination of blastospores with coloured latex particles coated with a monoclonal antibody 

specific to the cell-surface antigen of 7/*5(86",".,&"&. 

The purpose of this study was to evaluate the performance of 2ichro-dubli Fumouze ' test. In 

order to confirm these results real-time PCR assays specific for the fungal IT.2 region using 

species-specific hybridization probes on the /ightCycler were applied. 6niversal primers 

combined with species-specific probes were used as this enables a higher degree of specificity 

than genus-specific probes. The 7+,5"5+ species were differentiated by characteristic peaB 

melting temperatures of probes hybridizing to the target sites in the PCR amplicons. In the case 

of presence of a specific peaB, the species was confirmed in a second PCR reaction. 

Material and methods: A total of J00 Candida isolates, being 7/*+68"#+,& and 7/*5(86",".,&"& 

were first identified by conventional methods. These comprised the assessment of their 

appearance on CHRfMagarCandida (2ecton DicBinson, FranBlin /aBes, N(, 6.A) and their 

growth at O[ÅC on .abouraud Dextrose Agar (.A2, fxoid, 2asingstoBe, 6l). 

P-0433. Separation and identification of the cytoplasmic proteins of nine 

clinical isolates Candida albicans and one isolate of Candida glabrata along 

with the commparative study of their electrophoresis profile 

Yadegari M 1 , lhalili A 1 , Riazypoor M 2 , Roudbarmohammadi . 1 

1 Tarbiat Modares 6niversity, Tehran, Iran, 2 2aghiatolah 6niversity, Iran 

This research aimed at the comparative study of the cytoplasmic proteins of Candida 

albicans and C.glabrata with each other.The electrophoresis profile and the percent of 

similarity were determined .through .D.-PAGE method and Dice coefficient, 

respectively. The obtained results indicated that the lowest and highest molecular 

weights of the Candida albicans isolates were 1O and 10JlDa,respectively.Also,the 

highest percent of similarity among the cytoplasmic proteins were,respectively 

belonged to the following sample isolates(with the K[_ of similarity)n (6ngium,Cruris), 

(Cruris,fral), (Vaginal, standard sample of C.albicans PTCC [027), (Vaginal,6ngium), 

(6ngium,fral). The highest similar molecular weights in the cytoplasmic proteins of all 

C.albicans isolates were (OO and [6 lDa) at K sample isolates and (22.[ ,2O, J[, [6, 

6O lDa) at 8 sample isolates, while the lowest similar Ma belong 

to(2J,O1,O[,K1,100,102,10J lDa) at 2 sample isolates.on the other hand the 

cytoplasmic proteins of C.glabrata isolates with Ma of (O6 and [0lDa) at 8 sample 

isolates,(22.[,2O, J[, [6lDa) at 7 sample isolates, (O1,86lDa) at 6 sample isolates 

and (2J, J1, [OlDa) at one sample isolates, had similarity with the C.albicans isolates. 

For the differentiation of 7/*+68"#+,&*and 7/*5(86",".,&"&*by real-time PCR, two different speciesspecific 

hybridization probes were designed, which allow identification of the species via specific 

melting peaBs. The probe-specific melting peaB with the melting temperature (T ) ) at 62.[ ÅC was 

considered to be a positive result for 7/*5(86",".,&"&. .trains showing a specific T )*at 67.[ÅC 

were identified as 7/*+68"#+,&. .trains with ambiguous results were sequenced. 

For evaluation of the latex agglutination test, all of the isolates were incubated on .A2, 

CHRfMagarCandida and Candida ID Agar (bioMdrieux, Marcy l]Etoile, France) at J7ÅC for 2O h. 

Thereafter, the isolates were identified using the 2ichro-dubli Fumouze ' test. The results of the 

conventional tests and the latex agglutination test were compared with those obtained by real 

time-PCR. 

Results: 6sing conventional methods 1[0 strains were identified as 7/*+68"#+,&3 the remaining 

1[0 strains were identified as 7/*5(86",".,&"&/ Results obtained by real -time PCR and sequence 

analysis showed that 1[2 strains were 7/*+68"#+,& and 1O8 7/*5(86",".,&"& indicating that 

conventional methods identified all strains correctly with the exception of two strains of 7/* 

+68"#+,&. The performance of the 2ichro-dubli Fumouze ' test was excellent showing a sensitivity 

of K7.J_ using CHRfMagar, K8_ using .A2, and 100_ using Candida ID agar. The specificity 

was KK.J_ for all media. 

In summary, 2ichro-dubli Fumouze ' test allowed the rapid and reliable identification of the 

majority of clinical isolates within J min. Thus, this test offers a useful addition to existing 

methods in a routine mycology laboratory.

P-0434. A comparative study of four different kits for measurement of blood 

(1!3)-"-D-glucan in the diagnosis of invasive fungal infection 

Yoshida K, NiBi Y 

lawasaBi Medical .chool, lurashiBi, (apan 

Measurement of blood (1*J)-#-D-glucan (#- glucan) has been widely used for early 

diagnosis and follow-up of the therapeutic process of invasive fungal infection (IFI) in 

(apan. Now there are three different Bits using different methods of pretreatment, 

determination and standard substances in (apan. Fungitellw, a new Bit for 

measurement of #- glucan has been cleared by FDA in the 6. in 200O. Although 

some studies of this new #- glucan measurement Bit were reported already, the 

comparative studies of all four commercial Bits have not yet been done. In this study, 

we compared the relative abilities of these four Bits. 

From April to (une in 200J, 121 samples were collected from 121 patients who had 

been admitting in lawasaBi Medical .chool Hospital, and taBen blood samples for 

maBing the diagnosis of IFI. The value of #- glucan in samples were measured by 

Fungitecw G test Ml, #- glucan test aAlf, #- glucan test MAR6HA, and Fungitellw 

as recommended by the manufactures to evaluate the sensitivity, specificity, PPV and 

NPV of each Bits. The sensitivity was assessed by calculating the results of subjects 

with proven and probable IFI, while the specificity was assessed by calculating the 

results of subjects without IFI. RfC analysis of the results of each assay was also 

performed. 

The subjects with proven and probable IFI were twelve cases, while the subjects 

without IFI were K[ cases. .ensitivity of Fungitecw G test Ml, #- glucan test aAlf, 

#- glucan test MAR6HA, and Fungitellw was 7[.0_, O1.7_, O1.7_ and 8J.J_ 

respectively. .pecificity of each method was K1.6_, K8.K_, K6.8_ and K2.6_. PPV of 

each method was [2.K_ and 8J.J_, 62.[_ and [8.8_. NPV of each method was 

K6.7_, KJ.1_, K2.K_ and K7.8_. A6C of RfC in each method was 0.K0O, 0.K12, 

0.7O2 and 0.K11, though no significant differences were recognized among them. 

ae conclude the new #- glucan measurement Bit Fungitellw was useful for clinical 

diagnosis of IFI as well as conventional #- glucan measurement Bits. 

P-0435. IgG anti-Paracoccidioides brasiliensis in serum and oral fluid for 

diagnosis and post-therapeutic evolution of paracoccidioidomycosis 

Yoshida M, .anchez M, Shikanai-Yasuda M 

6niversidade de .Éo Paulo, 2razil 

Purpose of the study: The aim of this study was to evaluate the use of serum and 

oral fluid for diagnosis, and post-therapeutic evolution of paracoccidioidomycosis for 

detection of IgG anti-1+$+#%##"5"%"5.&*8$+&"6".,&"& antibodies. 

Description: .erum samples assayedM [2 from paracoccidioidomycosis patients with 

active disease, 20[ from blood donors and J1 from other diseases patients. fral fluid 

samplesM [2 from paracoccidioidomycosis patients with active disease, [1 from 

healthy individuals and 1K from other diseases patients. The E/I.A test was 

optimized in the following conditionsM total reaction time of K0 minutes for the serum, 

and 110 minutes for oral fluid. The RfC (receiver operating characteristic) curve was 

employed in the determination of the cut off values. In post-therapeutic analysis, 2J 

patients were evaluated just prior to the antifungical therapy and after O-8, 10-18 and 

2J-[J months. 

Results and Conclusions: The E/I.A sensitivity was K2,J1_ for both fluids, and the 

specificity was KO,07_ for serum, and 88,[7_ for oral fluid. In three samples without 

antibodies detected by E/I.A, the diagnosis was carried out by oral fluid analysisn two 

of them presented chronic unifocal form, and one with co-infection HIVparacoccidioidomycosis. 

The acute form presented the highest IgG reactivity indices 

in the serum when compared to the chronic multifocal and unifocal forms, and coinfection 

HIV-paracoccidioidomycosis. .erum and oral fluid samples IgG reactivity 

indices correlation (R } 0,OK, p } 0,0002) suggested that passive diffusion of serum 

transudation represents one of the possible mechanisms for the presence of 

antibodies in oral fluid. In patients with oral mucosa injury, higher oral fluid IgG 

reactivity was observed in relation to patients without oral mucosa injury (p } 0,002O), 

suggesting the participation of a local secretory system in the production of these 

antibodies. In the post-therapeutic periods, IgG reactivity indices decreased in both 

fluids when compared to the pre-therapeutic period (p v 0,0[). Considering the 

feasibility and facility of collecting oral fluid, and the efficiency of the test, E/I.A 

represents a complementary approach for diagnosis, especially when oral mucosa 

injury, co-infection HIV-paracoccidioidomycosis and chronic unifocal presentation 

occur. This study pointed out its value as a reliable marBer for post-therapeutic control 

of the disease. 

Financial supportM FAPE.P (FundaÑÉo de Amparo Ö Pesquisa do Estado de .Éo 

Paulo) and FundaÑÉo Faculdade de Medicina 


P-0436. IgG anti-Paracoccidioides brasiliensis serum avidity in 

paracoccidioidomycosis 

Yoshida M, .anchez M, Shikanai-Yasuda M 

6niversidade de .Éo Paulo, 2razil 

Purpose of the study: The objective of this study was to analyze IgG anti- 

1+$+#%##"5"%"5.&*8$+&"6".,&"& antibodies serum avidity in different clinical forms of 

paracoccidioidomycosis and post-therapeutic period, using the E/I.A test. 

Description: .erum samples assayedM O7 from paracoccidioidomycosis patients with 

active disease and 17 were evaluated just prior to the antifungical therapy and after O- 

8, 10-18 and 2J-[J months. .erum samples were diluted at two fold serial dilution, 

from 1M100 to 1M1600 and assayed in duplicate (test and control) in IgG-E/I.A-*1/* 

8$+&"6".,&"&. The avidity index (AI) was estimated interpolating absorbance values 

corresponding to the dilutions above and bellow the half-maximum absorbance value 

in urea-treated sample. 

Results and Conclusions: This study allowed to observe that patients presenting 

chronic unifocal and multifocal forms, presented antibodies of average to high avidity, 

in contrast to the acute form, in which [0_ of the patients presented low avidity. In the 

post-therapeutic period, there was a significant antibodies avidity increase, mainly in 

the chronic multifocal form. In the five patients presenting unfavorable clinical 

evolution, increased antibodies was not observed, whereas a significant increase in 

avidity was observed in twelve patients with favorable evolution (p} 0,02). The study 

of antibodies avidity pointed out its relationship to clinical severity of 

paracoccidioidomycosis, as well as its value as a reliable marBer for post-therapeutic 

control of the disease. 

Financial supportM FAPE.P (FundaÑÉo de Amparo Ö Pesquisa do Estado de .Éo 

Paulo) and FundaÑÉo Faculdade de Medicina 

P-0437. Production of specific biomarkers of Pseudallescheria boydii - effect of 

cultivation conditions 

Zabka M, Nedved (, 2enada f, lofronova f, .Blenar (, flsovsBa (, .ulc M, HavliceB 

V 

Institute of Microbiology, VidensBa 108J, PragueO, Czech Republic 

1&.(5+66.&#?.$"+*8%25"" (anam. =#.5%&'%$"()*+'"%&'.$)()) is a ubiquitously occurring 

soilborne fungus. Its importance significantly rises in case of therapy of 

immunocompromised patients. This fungus is responsible for different types of infection 

depending on the route of infection and the immune status of the host. 

Generally, pilot idea for using of cyclic peptides as biomarBers is based on the way of their 

synthesis. This is carried out by multienzymes called non-ribosomal peptide synthetases 

(NRP.s). NRP.s are not present in mammal]s metabolism. In addition, peptides of NRP.s 

origin contain unique building blocBs. Hence cyclic peptides or their spectrum could be 

considered as extremely specific measure of a particular fungal infection. In our project we 

suppose that cyclic peptides may be used as specific marBers of 1&.(5+66.&#?.$"+/ ae 

observed the production of a specific biomarBer of 1/*8%25"" which is cyclic peptide (m/z 

7O0) the characterization of which is mentioned in a different presentation. The main goal 

of this particular worB is the optimization of the production of our specific biomarBers. Aims 

of this worB are numerous. Firstly, we need to Bnow at which stage the fungus starts the 

secondary metabolism and if the growth conditions are potentially compatible with those ",* 

0"0%. .econdly, optimized culturing conditions will be used for a large-scale production of 

the marBer itself. .eparation, purification and identification of the peptide family are 

prerequisites for subsequent testing (biological activities of the cyclopeptides) and a 

purified standard will further be used in all ",*0"-$% and ",*0"0% studies (different 

presentation). 

For this purpose we experimented with various cultivation conditions. Experimental 

conditions of ",*0"-$% cultivation were focused on different source and level of carbon and 

nitrogen, both in organic or inorganic form. Correlation between cultivation time and 

amount of produced cyclic peptide was monitored as well. All extracts from fermentation 

broths and mycelia were analysed by means of mass spectral methods (HP/C-ion trap M. 

or FT-ICR M.). 

Mass spectral data evaluation suggests that maximum production of our new cyclic peptide 

is apparently presented after stationary phase of growth. Classical submerged cultivation in 

CzapeB-Dox medium (inorganic nitrogen) with different level of sucrose revealed that lower 

level of this sugar (only 1_ instead of common J_) leads to earlier and higher production 

of this unique cyclic peptide. ae have found nitrogen source to be of less importance. If the 

fungus is cultivated in a medium supplemented with organic nitrogen (casein), the amount 

of the produced cyclic peptide is not significantly higher compared to previous case. Hence, 

some starving conditions do not obstruct the production of the cyclopeptide. 

2road spectrum combinations of various sources and levels of carbon/nitrogen will be 

evaluated in the planned experiments. ae also will evaluate the effect of cultivation 

temperature and aeration conditions (lungs-related), as well as the difference in 

productions among different 1/*8%25"" strains with different pathogenicity. 

Acknowledgement: Ministry of Education, Youth and .ports of the Czech Republic 

(/C[O[), Czech .cience Foundation (20J/0O/07KK), and IMIC institutional research 

concept (AVfZ[0200[10).

P-0438. Selective tomato juice agar in mycology 

Zurak I 

6niversity Hospital 

Background: The list of opportunistic fungal pathogens continues to increase at an 

impressive rate that is related to the increasing number of immunocompromised 

patients, among transplant patients, cancer chemotherapy patients, those who are 

hospitalized and catheterized, including those with AID.. Invasive fungal infection are 

a common cause of morbidity and mortality in allergenic hematopoietic stem cell 

transplant recipients. Aware of the need to improve the diagnostic methods we use to 

isolation and identify a fungal infection. The single best tool available for the diagnosis 

of fungal infection is the fungal culture. Fungi grow more slowly than bacteria, and 

have longer generation times than most bacteria. Culture must be Bept longer and 

provision must be mode to prevent media from excessive drying. To separate fungi 

mixed with bacteria, cultures can be streaBed on media containing antibacterial 

antibiotics. .elective agents may be incorporated in these media to inhibit unwanted 

bacteria or saprophytic fungi. The medical mycology laboratory uses a variety of 

strains, media, for the isolation and identification of yeast and moulds. The selection of 

a medium depends on the specimen, but very is important that often samples are 

cultured on more than one medium. The aim and objective of this study and worB is 

general purpose media, were to examine for primary isolation of fungal pathogens, 

from contaminated specimen with bacteria, where is the formulation of medium 

without antibiotics. 

Materials and Methods: .elective tomato juice agar is medium with contains tomato 

juice, with addition of nutritive basesM peptone, yeast extract, corn starch, salts 

(Columbia agar)n dextrose and D-mannitol. Mucic acid - .accharolactic acid (h.igmai 

M-O778), with or without Potassium Metabisulfite (h.igmai P-2[22) is selective 

supplement. Medium don]t need to be autoclaved. For the cultivation of fungi, samples 

were inoculated direct on plate medium. Culture of material from suspected superficial 

is mycosis was usually carried out at 2[-J0 0 C. Culture should be examined at least 

every 2 or J days for the first 2 weeBs and weeBly thereafter. 

EPIDEMIOLOGY* 

P-0439. Determination of Brazilian Cryptococcus neoformans serotypes and 

mating types by multiplex pcr 

/. T. Dias A 1 , .. Terceti M 2 , C. Franco M 2 , GonÑalves da .ilva 1 , M. de .iqueira A 2 , 

C. da .ilva 2 1,J , R. Paula C 1 

1 6niversidade de .Éo Paulo, Instituto de Ciüncias 2iomddicas II (IC2II), .Éo Paulo, 

2rasil, 2 6niversidade Federal de Alfenas (6NIFA/-MG), Alfenas, MG, J Institut Pasteur, 

Paris, France 

7$2'-%#%##(&*,.%U%$)+,& is a fungal pathogen causing meningoencephalitis in hosts 

with impaired immune system. The two varieties, var. ,.%U%$)+,& (serotype D and 

AD) and var. @$(8""*(serotype A) can present ecological and biochemical distinction. 

This fungus reproduces mainly asexually, or more rarely by a sexual cicle in which two 

diferent (MATa and MAT$) or two equal (MAT$) mating types (MAT) are able to mate 

and produce basidiospores. Mating type plays an important role in the epidemiology 

and virulence of 7/*,.%U%$)+,&. Among several factors related to virulence of this 

microorganism we can highlight the ability of growing at J7ÅC, capsule structure, 

melanin liBe pigment production, phospholipase, proteinase enzymatic activity and the 

presence of allele ! related to MAT system. 

The present study evaluated a multiplex PCR method to distinguish the serotypes A, 

D, AD and MAT a and $ of 80 2razilian clinical and environmental 7/*,.%U%$)+,&* 

isolates. 

PCR amplification identified one fragment of 1200pb for A$ (serotype A and MAT$) in 

the majority of the isolatesn two fragments 1200pb and 870pb respectively for A$ and 

aD (serotype AD and MAT$a) in only one isolate. 

The method appears to be a valid and relatively inexpensive tool for epidemiological 

and virulence studies. 

Results: Formulation the tomato juice agar without antibiotics is selective for growth 

of most common medical yeast and moulds, and it was shown better results in 

isolation of fungi from heavy contaminated specimens than Emmons modification of 

.abouraud agar. Tomato juice provides an acid environment, nutrients, vitamins and 

trace mineral elements. 

Conclusion: The specimens most liBely to yield fungus vary from disease to disease. 

The clinical laboratory technologist must be able to recognize this increasingly large 

group of potential fungal pathogens and these new diagnostics has paved the way for 

a new strategy. 2acteria don]t develop resistance to chemical, salts and with this can 

successful substitute antibiotics in selective media for isolation in mycology. Yeast is 

by for the most common fungi isolates from human]s patients. frgan transplantation 

has increased and the percentage of fungal infection in this population has increased 

accordingly. 


P-0440. An indoor focus of the neurotropic black yeast Exophiala dermatitidis 

Sudhadham M 1 , De Hoog G 1 , Gerrits van den Ende A 1 , Mayr A 2 

1 Centraalbureau voor .chimmelcultures, 6trecht, The Netherlands, 2 Insitute for 

Hygiene and .ocial Medicine, 6niversity of InnsbrucB, InnsbrucB, Austria 

Objective: The blacB yeast a_%'?"+6+*5.$)+-"-"5"& is a rare etiologic agent of fatal 

infections of the central nervous system in otherwise healthy, mainly adolescent 

patients in East Asia. In Europe, it is a relatively frequent colonizer of the lungs of 

patients with Cystic Fibrosis (CF). This suggests a pulmonary route of infection. Public 

steam baths have been suggested as a focus of the fungus in the human-dominated 

environment. It is our aim to establish the frequency of this fungus in bathing facilities. 

Methods: .trains were isolated by pre-incubation of samples in Raulin]s solution, and 

subsequently on Erythritol-Chloramphenicol Agar (ECA) at O0ÅC. .trains were purified 

with Tween 0.1_. For identification, the rDNA Internal Transcribed .pacer (IT.) 

region was analyzed by Restriction Fragment /ength Polymorphism (RF/P) or 

sequencing. A selection of strains was compared by Amplified Fragment /ength 

Polymorphism (AF/P). 

Results: The species was recovered consistently and at high frequency in all 

analyzed public bath facilities in The Netherlands, Austria and Thailand. Nearly 

invariably the steam bath was the only positive location within the facility. 6p to eight 

AF/P genotypes were detected per steam bath. fccasionally a/*5.$)+-"-"5"& was 

replaced by another a_%'?"+6+ species. However, positive samples were recovered 

only from surfaces, while air samples were consistently negative. 

* 

Conclusion: a_%'?"+6+*5.$)+-"-"5"& is a frequent and consistent colonizer of steam 

baths of public bathing facilitiesn other high-temperature locations within the facilities, 

such as sauna cabins, are nearly negative for the fungus. Routine cleaning has little 

effect upon the prevalence of the fungus. As the fungus is not disseminated by air, the 

steam bath is an unliBely source of infection of CF patients. The hypothesis that, 

conversely, humans are the source of contamination is discussed. 

P-0441. Growth assay of Histoplasma capsulatum var duboisii on different soil 

samples 

Abia-Bassey L 

College of Medical .ciences, 6niversity of Calabar, Nigeria 

Purpose of the study: .tudies were carried out to assay the colonial growth patterns 

of an isolate of A"&-%'6+&)+*#+'&(6+-() var 5(8%"&"" on different cave - soil agars. 

Summarized description of the projectM The isolate of A/*#+'&(6+-() var 5(8%"&""* 

used for the study was from a case of African histoplasmosis diagnosed from 

6niversity of Calabar teaching hospital, Calabar - Nigeria. The growth was assessed 

using the colony size method. Modified soil extract agars were prepared from soil 

extracts collected from three bat-infested caves in Cross River .tate of Nigeria namely 

Agwagune, 2uBulum, aula and garden soil. 

Result and Conclusions: The colonial growth patterns of A/ #+'&(6+-() var 5(8%"&""* 

showed a mean growth temperature of J0oC. The fungus also showed a better growth 

pattern on 2uBulum soil extract agar with a peaB colonial growth diameter of J8mm, 

followed by garden (J[mm), Agwagune (J2mm) aula (2Kmm). Generally, the soil 

extracts showed no remarBable effect on the growth and survival of the fungus when 

compared with garden soil, thereby questioning the relevance of bat guano to the 

existence of this fungus in nature.

P-0442. A survey on dermatophytosis in patients referred to boali-sina skin 

hospital in Qazvin, Iran, 2004-2005 

Aghamirian M, leshavarz D, Hashemi ( 

~azvin 6niversity of Medical .ciences 

Dermatophytosis has been considered to be a major public health problem in many 

parts of the world. The aim of this study was to identify etiological and epidemiological 

factors of dermatophyte infection in patients refers in dermatology Hospital 2oali-.ina. 

A total of JO1 patients suspected to have dermatophytosis lesions were examined 

over a period of one year (200O-200[). Material collected from sBin, hair and nails was 

submitted to direct microscopic examination using lfH, cultured in sabouraud 

dextrose agar and microscopically examined for colony morphology, in order to the 

identify 116 dermatophytes isolated. Epidermophyton floccosum was the most 

frequent dermatophyte isolated (J2.8_) followed by Ttrichophyton mentagrophytes 

(22.O_), T. rubrum (18.1_), T. verrucosum (17.2_), T. violaceum (0.86_). Tinea 

cruris (J1.K_) was the most common type of infection, followed by tinea corporis 

(20.7_), tinea pedis (1K_), tinea unguium (11.2_), tinea faciei (7.7_), tinea manuum 

([.2_), tinea capitis (O.J_). The frequency rate of tinea was higher in males than in 

females. The anthropophilic species E. floccosum was the most common 

dermatophyte causative agent of tinea. 

P-0443. Dermatophyte infections in Bursa region-Turkey 

Akcaglar S 1 , Ener 2 2 , CBman ToBer . J , .uBran T O , fBan T [ 

1 6ludag 6niv. .chool Medicine Dep. of Microbiology, 2 6ludag 6niv. .chool Medicine 

Dep. of Microbiology, J 6ludag 6niv. .chool Medicine Dep. of Dermatology, O 6ludag 

6niv. .chool Medicine Dep. of Dermatology, [ 6ludag 6niv. .chool Medicine Dep. of 

Microbiology 

7/*5(86",".,&"& is a recently described 7+,5"5+ species associated with oral 

candidosis that exhibits with a high degree of phenotypic similarity to 7+,5"5+* 

+68"#+,&. Its importance based on rapidly developing resistance to fluconazole, a 

common anti-fungal drug which used for treatment of mycoses. In retrospective 

analysis of stocB collections between 1KK[ and 200[ years, aproximately a total of 

1000 7/*+68"#+,& clinical isolates that obtained from steril body fluids (C.F, 2lood, 

urine, etc.) of K00 patiens who admitted to the 6ludag 6niversity .chool of Medicine 

Hospital 2ursa/TurBey. Analyses were performed in Mycology /aboratory of 

Microbiology Department of the same hospital. These clinical isolates were identified 

as a 7/*+68"#+,& on the basis of their cultural and morphological characteristics, 

growth on .abourauds Dextrose Agar (.DA), germ tube production and production of 

chlamydospore on cornmeal agar and stored -80 o C. 

The present study was carried out to determine the prevalence and clinical 

significance of 7/*5(86",".,&"& in a tertiary-care hospital in our region. 7/*+68"#+,& 

isolates from stocB collection were examined again with germ tube formation in human 

serum after J h at J7 o C, chlamydospore production on corn meal agar and staib agar, 

ability to grow at O[ o C on .DA, colour development on CHRfMagar, opasity test in 

Tween-80 agar and carbohydrate assimilation properties by API ID J2 C. Among 1000 

isolates of 7/*+68"#+,&, four strains were identified as 7/5(86",".,&"&, giving an overall 

occurrence of 0,O _. 


P-0444. Epidemiological analysis of invasive fungal diseases in AIDS patients 

in Latvia 

Aldins P 1 , Rozentale 2 1 

1 Infectology Center of /atvia, 2 Infectology Center of /atvia 

Purpose of the study: To introduce in epidemiological situation of invasive fungal 

diseases in AID. patients in /atvia 

Description: The population of /atvia is about 2 J00 000. The first cases of HIV were 

diagnosed at end of eighties, it was a few patients | M.M and sailors. From 1KK[ to 

200O HIV prevalence became very quicB in IVD6 population. HIV incidence is 1OO on 

100 000 inhabitants now. AID. stage cases increase in last years. ae have 

registered OJO AID. cases (according CDC). O7_ of them are T2 cases, 26_ 

invasive fungal diseases (1[_ 7+,5"5+ spp., 10_ 7$2'-%#%##(&*,.%U%$)+,&, 1_ 

4&'.$@"66(& spp.), 11_ is PCP, other is more rare. 

Treatment of T2 in /atvia is realise by T2 specialists in separate T2 centers and 

hospitals. Diagnostics, treatment and monitoring of fungal diseases is provided by 

infectologists. ae have a possibility for diagnosing 7+,5"5+ caused diseases and 

7$2'-%#%##(& and 4&'.$@"66(& generated pathologies. The most common 7+,5"5+ 

caused disease is oesophageal candidiasis (O2 cases), more rare are disseminated 

candidiasis cases (26), cryptococcosis were in OJ cases. The most common is a 

damage of central nervous system (CN.) | meningitis or encephalitis, rare is 

antigenemia situations. Frequently patients with cryptococcosis CN. damage have 

another side CN. disease, such as T2, syphilis, toxic damage etc. and these maBe a 

difficult diagnosis and treatment. Diagnosis of oesophageal candidiasis placed in 

gastroscopy, it is relatively simply for treatment, but in poor adherent patients relapses 

are common. This disease is often, when patient has another immunodeficiency, such 

as alcocholics or anorexia patients. More common invasive fungal diseases patients 

are males whom above mentioned reasons are prior. 

P-0445. Analysis of the trends of reported scalp infections within the West of 

Scotland during 2000 - 2005. 

Alexander C, 2rown /, .hanBland G 

Clinical Mycology, Glasgow, 6l 

6ntil the advent of griseofulvin, infections of the scalp caused by dermatophytes were 

regarded as the most commonly occurring fungal infections in children. The CPAaccredited 

Clinical Mycology /aboratory based in Glasgow receives hair and scalp 

scraping from general practitioners and hospitals throughout .cotland for analysis of 

fungal infections. During the period 2000 | 200[, 81 fungi from such samples were 

isolated and identified from 77 patients. The age of patients ranged from 6 months to 

87 years with the majority of patients (n}7O) aged 16 of less. [0 males were affected 

compared to only 27 females. The number of patients with proven scalp infections 

increased O.[-fold from 1O affected individuals during 2000 | 2002 to 6J during 200J | 

200[. Throughout the first J years of this study, the most common scalp infection was 

!"#$%&'%$()*#+,"&. In contrast, by 200J, this was superseded by both >$"#?%'?2-%,* 

-%,&($+,& and >$"#?%'?2-%,*0"%6+#.() and this pattern has continued for the further 2 

years of this study. The first recorded case of >$"#?%'?2-%,*&%(5+,.,&. in .cotland 

since 1KKJ was observed in 200J. 2y 200O, the number of cases of !"#$%&'%$()* 

+(5%(,"" had also increased. These changes in the pattern of scalp infections over 

this 6 year period may reflect an increase in the migration of people to .cotland from 

areas including Africa, Asia and .outh America where fungal infections such as 

>$"#?%'?2-%,*-%,&($+,& and >$"#?%'?2-%,*0"%6+#.() are endemic. 

Conclusion: Invasive fungal diseases are common in AID. clinic in /atvia. The 

specific clinical signs for these diseases are rare | the examination must start 

immediately, if fungal disease suspects. fften the invasive fungal disease give 

evidence about combined immunodeficiency, which asBs to be clarified.

P-0446. Prevalence of C. orthopsilosis and C. metapsilosis among yeast blood 

stream originally identified as C. parapsilosis 

Amorim C 1 , Melo A 1 , Godoy P 1 , 2riones M 2 , Colombo A 1 , GonÑalves . 1 

1 Division of Infectious Diseases, 6NIFE.P, .P, 2razil, 2 Department of Microbiology, 

Immunology and Parasitology, 6NIFE.P, .P, 2razil 

7+,5"5+*'+$+'&"6%&"&*is a human pathogen frequently involved in candidemic episodes 

associated to the use of intravascular catheters and parenteral nutrition. This 

pathogen has become the second most common species of 7+,5"5+ related to 

fungemia in 2razil and .outh America. The genetic diversity of 7+,5"5+*'+$+'&"6%&"& 

was documented in early studies of clinical isolates using molecular methods liBe 

RF/P, cariotyping, isoenzyme profiles, RAPD and IT. sequences. These studies 

discriminated three different genetic groups among the former 7/*'+$+'&"6%&"& strains, 

named group I, II and III. Recently, based on Multilocus .equence Typing method and 

phylogenetic analysis of IT.1 sequences, two new species were proposed to replace 

the existing 7/*'+$+'&"6%&"& group II and III, 7/*%$-?%'&"6%&"& and 7/*).-+'&"6%&"&, 

respectively. There is mounting data suggesting that 7/*'+$+'&"6%&"& is far more 

frequently isolated from clinical samples than 7. %$-?%'&"6%&"& and 7/*).-+'&"6%&"&. 

The aim of this worB was to identify the prevalence of these J species among blood 

stream isolates. In this study we tested 1O0 blood stream yeasts phenotypically 

identified as 7/*'+$+'&"6%&"& isolated from patients living in different geographic areas 

in 2razil. ae also typed the control strains 7/*'+$+'&"6%&"& group I (ATCC K0018), 7/* 

%$-?%'&"6%&"& (ATCC K61O1) and 7. ).-+'&"6%&"& (ATCC K61OJ). To discriminate the 

three species we used RAPD with primers 1281 and 12[J, and PCR-RF/P to amplify 

a fragment of =4VA gene which was further digested with Z+, I restriction enzyme. 

The RAPD fingerprintings were analyzed by using the GelCompar II program and the 

dendogram clustered the isolates in three groups according to species. The RAPD 

identified 126 7/*'+$+'&"6%&"& isolates, 12 7/*%$-?%'&"6%&"& and 2 7/*).-+'&"6%&"&. The 

identity of these two strains of 7/*).-+'&"6%&"& was confirmed by PCR-RF/P. ae 

found a prevalence rate of K0_ of 7/ '+$+'&"6%&"&3 8.6_ of 7/*%$-?%'&"6%&"& and only 2 

strains of 7/*).-+'&"6%&"& among the brazilian blood stream isolates. This is the first 

time that 7/*).-+'&"6%&"&*is isolated as the etiologic agent of candidemia in 2razilian 

patients. 

P-0447. Genotype analysis of Candida albicans isolated from the human oral 

cavity 

Anzawa K 1 , lawasaBi M 1,2 , Tanabe H 2 , MochizuBi T 1,2 , IshizaBi H 1 




The genetic variety of 7+,5"5+*+68"#+,& isolate from the oral cavity of patients was 

assessed. Fungi specimens were obtained from wooden tongue depressors used in 

the examination of patients at the Dermatological Clinic of lanazawa Medical 

6niversity Hospital. The fungi were cultured on Mycocel agar plate (EiBen-lagaBu, 

(apan). Each colony was picBed up, immersed in distilled water and inoculated on 

CHRfMagar Candida Medium (CHRfMagar Co./td.). Mono-spores were isolated 

and identification made. .eventy-one colonies were obtained from 1J patients. DNA 

preparations from these isolates were used as a template to amplify the region 

between [. and 18. rDNA by PCR. PCR products were digested with restriction 

enzymes !&'I,*V5.I, electrophoresed, and their band patterns examined. Nine types 

were obtained, 6 with the*!&'I and O with the V5.I. There were 2 types of 7/*+68"#+,& 

in the mouth of the patient, which were found in [ of the 1J people. 

Financial supportM CAPE., FAPE.P and CNPq 


P-0448. An epidemiological study of an outbreak of Sporotrichosis in the south 

west of Western Australia 

Arthur I 1 , Altman . 1 , ahittle A 2 , .peers D 1 

1 Pathaest /aboratory Medicine aA, 2 aestern Australian Country Health .ervice - 

.outh aest 

Prior to the year 2000, sporadic cases of sporotrichosis have occurred in the south 

west of aestern Australia (aA). fften these patients have resided in the hwheat-belti 

| inland rural areas of moderately large wheat and sheep farms. However in 2000 a 

comparatively large cluster of cases were observed around the Margaret River region 

where sporotrichosis had not previously been observed. Due to the high ongoing 

incidence and changed geographical distribution of infection an epidemiological 

investigation was commenced. ff eleven mycologically proven cases from (uly 200J 

to (uly 200O, nine were in or around the Margaret River region. A survey of the 

patients showed an even gender balance and a wide age distribution. However nine 

patients revealed a history of contact with hay prior to the onset of symptoms and so 

environmental sampling was performed to determine the source of the infection. Hay 

samples were collected from properties associated with six of these cases (16 

samples), two commercial hay outlets (K samples), from a farm that supplied one of 

the outlets (7 samples) and various control sites in the district (18 samples). Fungal 

culture of the hay samples isolated a number of ='%$%-?$"_-liBe organisms which were 

subsequently identified and compared based on morphological features and IT.2 

gene sequencing. 6sing these criteria all clinical isolates of ='%$%-?$"_*&#?.,#["" from 

aA are indistinguishable. =/*&#?.,#["" isolates cultured from one sample from a 

commercial outlet and hay samples associated with three cases were also 

indistinguishable from the clinical isolates. These three cases all sourced hay from the 

hay from this commercial hay supplier. fther environmental isolates from various of 

the hay samples included b'?"%&-%)+*spp. and environmental isolates of =/*&#?.,#[""* 

dissimilar to the clinical isolates. Following the introduction of public health measures 

the rate of sporotrichosis has dramatically fallen in the area although sporadic cases 

have been identified in 200[. The results imply that prior to 2000 contaminated hay 

was brought into the Margaret River region from the wheat-belt resulting in the 

subsequent infection of a significant number of people. The higher rate of infection 

was possibly accentuated by poor storage conditions of the contaminated hay, higher 

rainfall and more intensive land use practices in the region. aith the introduction of a 

virulent strain of*=/*&#?.,#["" it is liBely that sporadic cases will continue in the 

Margaret River region. To reduce the liBelihood of a further outbreaB continuing public 

health measures focussing on the storage and handling of hay should be maintained. 

P-0449. Detection and identification of fungi by culture, serology and molecular 

technigues in nasal polyposis 

Aydil 6 1 , Kalkanci A 2 , Ceylan A 1 , 2erB E 2 , lustimur . 2 , 6slu . 1 

1 

Gazi 6niversity Faculty of Medicine Department of ftorhinolaryngology, 2 Gazi 6niversity Faculty 

of Medicine Department of Microbiology 

The exact etiology of nasal polyposis is still unBnown. The role of individual fungi is causing 

chronic rhinosinusitis remains to be elucidated. 2ut there is no causal relationship between nasal 

polyposis and the colonization with fungus. The current diagnositc criteria for allergic fungal 

sinusitis are (i) chronic rhinosinusitisn (ii) the presence of allergic mucinn and (iii) the presence of 

fungi within that mucin, confirmed by histopathology, culture, or both. Establishing a causal 

relationship between the fungal culture results and the clinical presentation of chronic sinusitis 

can be difficult, since many of the fungi isolated are ones that are more commonly considered as 

contaminants. More than one laboratory technique is often necessary for interpretation of direct 

smears and fungal cultures from such patient. 

The aim of this study is to find a causal relationship between the prevalence of fungi in nasal 

mucosa and the development of nasal polyposis. 

.erum samples, nasal swabs and tissue biopsy specimens were collected from the nasal cavities 

of 18 control objects and J0 patients with chronic rhinosinusitis presenting nasal polyposis. A 

procedure based on panfungal polymerase chain reaction and DNA sequencing was developed 

for the detection of fungi in tissue specimens. The Real Time PCR amplified the fungal internal 

transcribed spacer (IT.) region (IT. 1-[.8. rRNA-IT. 2). Fungal pathogens were identified 

according to the profile of the amplification product on an automated sequencer. Direct 

microscopy and fungal growth in culture were also detected. Galactomannan antigen was 

investigated by E/I.A (Platelia Aspergillus, 2ioRad, France) in tissue supernatants and serum 

samples of the controls and the patients. 

fut of J0 patients 1[ were positive ([0_) and out of 18 controls two were positive (11_) for 

mycological prevalence (p value} 0.016) 

Patients 

Controls 

Methods Positive Negative Positive Negative p values 

Microscopy (swab) 1O 16 1 17 0.008 

Culture 8 22 0 18 0.018 

E/I.A (tissue) K 21 2 16 t0.0[ 

PCR and 

1[ 1[ 6 12 t0.0[ 

sequencing 

Total evaluation 1[ 1[ 2 16 0.016 

fne 4&'.$@"66(&*U6+0(&, two 4&'.$@"66(&*,"@.$, one 4&'.$@"66(& sp, one 76+5%&'%$"() sp, two 

1.,"#"66"() spp, one 1.,"#"66"()*#$2&%@.,(), were identified from growing colonies cultured 

(Total eight). Two 4&'.$@"66(& spp, one 4&'.$@"66(&*U6+0(&3 one*4&'.$@"66(&*\+'%,"#(&, two 

4&'.$@"66(&*,"@.$, two 76+5%&'%$"()*spp, one V.8+$2%)2#.&*?+,&.,"", one `.($%&'%$+*#$+&&+, 

two 1.,"#"66"() spp, one 1.,"#"66"()*#$2&%@.,()3 four 1"#?"+ spp, one T&-"6+@%*)+25"& were 

identified bu DNA sequencing of the fungal product (Total 18). 

Much controversy still exists about the role of fungi in sinusitis and nasal polyposis. This study 

compares the utility of microscopy, serology and molecular methods, presents and discusses the 

algoritmic evaluation of fungal prevalence in nasal clinical samples, and the re-evaluates the role 

of fungi in rhinosinus disease. 

This study was supported with a project codeM Gazi 6niversity 01/200[-81

P-0450. Mycoflora of peanut seeds in zanjans market - Iran 2004 

Badali H, Nourian A, Haniloo A, Mohseni ., lhodaverdi M 

Department of Parasitology and Mycology, Faculty of Medicine, Zanjan 6niversity of 

Medical .ciences, Zanjan, Iran 

BackgroundM 6ndoubtedly, one of the most important health issues is food hygiene. 

Research has shown that food products such as corn, wheat and peanut have high 

potential for contamination with fungi in case of suitable environmental conditions liBe 

temperature and humidity. Regarding toxins, Aspergillus flavus and Aspergillus 

parasiticus are highly important that they can produce aflatoxin. 

MethodsM In this survey, 20 samples of [0gr salted peanuts and 16 samples of [0gr 

unsalted peanuts were collected from Zanjan marBet randomly. Environmental 

conditions such as light intensity, temperature and air current were measured on the 

spot, then the samples were transferred to the laboratory and humidity was also 

measured. In the laboratory, [ peanuts were selected randomly from each [0gr 

sample, cultured on media culture (.abouraud Dextrose Agar plus Chloramphenicol) 

and were conserved at 27-J0ÅC for a weeB. The samples were examined for fungal 

colony and in case of growth, were diagnosed using slide culture technique. 

ResultsM Mold fungi were dominant among the colonies with following percentages 

Aspergillus flavus. JK.1_, Penicillium. K.2_, Rhizopus 7.2_, Mucor. 2.[_, Alternaria. 

1.0J_ and Nigrospora 0.[_. Furthermore, Aspergillus flavus was more frequent in 

unsalted peanuts than in salted samples. fn the contrary in salted samples 

Aspergillus niger was more frequent than in unsalted samples. In this study, 

temperature and sample humidity were over standard range. The results indicated a 

significant relation between environmental humidity, light intensity, temperature and 

peanut type with level of fungal contamination. 

ConclusionM According to above findings the most frequent fungal contamination 

belonged to mold fungus Aspergillus flavus. This fungus can cause aflatoxin under 

certain conditions. aith regard to the fact that higher percent of Aspergillus flavus 

pertained to unsalted peanuts, we can conclude that the risB of unsalted peanut 

contamination with aflatoxin is higher than that of salted peanuts. Moreover, humidity 

rate of unsalted peanuts is more than standard level approximately 6.81_. As a result, 

we can suppose that there is a relationship between the fungi growth in salted peanut 

and amount of humidity. fn the other hand, there is inverse relationship between light 

intensity and fungi. fverall, if government has a management on store and distribution, 

contaminated fungi will control. 

P-0451. Isolation of the Cryptococcus neoformans and Cryptococcus gattii in 

clinical samples from Sergipe, Brazil 

Barbosa, Jr. A 1 , de Melo D 1 , .antos P 1 , de Fatima Travalia M 2 , Morales 2 J , 

Goncalves G J , /azera M J , de Cassia Trindade R 1 , de Resende M O 

1 /MA, 6niversidade Federal de .ergipe, .an Cristovao, .ergipe, 2razil, 2 /aboratorio 

de Micologia do /aboratorio Central de .aude Publica do Estado de .ergipe 

(/acen/.E, J /aboratorio de Micologia Ambiental do IPEC/FIfCR6Z, O /aboratorio de 

Micologia, IC2/6FMG 

7$2'-%#%##(& are basidiomycetic encapsulated yeasts that infect patient 

immunodepressed as well as immunocompetents patients. The meningitis occurs 

approximately in 8-J0_ of the patients with the syndrome of acquired imunodeficiency 

(AID.). The new taxonomic approaches recognize two species, 7$2'-%#%##(& 

,.%U%$)+,& (serotypes, AD and D) and the 7$2'-%#%##(& @+--"" (serotypes 2 and C). 

This worB had as objective to study the occurrence the 7/*,.%U%$)+,& and 7/*@+--"" on 

clinical samples in .ergipe, 2razil from the liquor of patients with infectious meningitis. 

.amples of the /CR that had been used had given entered in the IPH//ACEN/.E in 

the period of (anuary of 2001 to August of 200O. Direct microscopy through the 

coloration with Nanquim InB was carried out. An aliquot of the lÜquor was sown in 

.abouraud Agar with clorafenicol in the ambient temperature (2[oC) and J7oC. Tests 

for presumptive phenotypic identification had been carried out in NÜger Agar and the 

identification of the species in CG2 Agar. To verify the intracolonies variability three 

morphological types were selected. In the analyzed period, only one sample 

presented different phenotypic characteristics in NÜger medium (coloration cream). All 

the others had presented darB color (production of fenoloxidase). Posterior tests had 

been carried out to confirm the identification of them as 7/*,.%U%$)+,& or 7. @+--"". 

The cream colony can be presenting a phenotypic variability, however is necessary a 

deepened study for such affirmation. CG2 tests revealed two positives samples 

(1J,JJ_) identifying then as 7$2'-%#%##(&*@+--""/*It]s important to register*that one of 

them was from a soropositive patient. The others 1J samples (86,67_) had presented 

negative CG2, identifying then as 7$2'-%#%##(& ,.%U%$)+,&/*=even of those samples 

related with soronegatives patients and six with soropositives patients. The sample 

cream presented negative CG2 however news researchs will be necessary with this 

isolated. ae can affirm that, in .ergipe, 2razil, the majority of the isolated samples of 

the lÜquor of patients with infectious meningitis are 7$2'-%#%##(& ,.%U%$)+,&. 

Acknowledgements: CAPE., Improvement Coordination of High /evel Personnel, 

linBed to the Ministry of Education Government of 2razil, and Neomed do 2rasil ..A. 

for financial support. 


P-0452. Candida albicans, as the third microbial agent on urinary tract 

infections in patients who refer to central laboratory of Dr. Shariati Hospital, 

Tehran, Iran 

Behzadi P 1 , 2ehzadi E 2 , Yazdanbod H J , Aghapour R O , .alehian fmran D O 

1 Islamic Azad 6niversity, .hahriyar 2ranch, Tehran, Iran, 2 Member of young 

researchers club, Islamic Azad 6niversity, .cience and Research campus, Iran, 

J Central /aboratory of Dr. .hariati Hospital, Tehran, Iran, O Microbiology /aboratory of 

Dr. .hariati Hospital , Tehran, Iran 

7+,5"5+*+68"#+,& is ubiquitous yeast and is the most common fungal pathogens that 

affect humans. The growing problem of mucosal and systemic candidiasis reflects the 

enormous increase in the pool of patients at risB and the increased opportunity that 

exists for 7+,5"5+*+68"#+,& to invade tissues normally resistant to invasion. 

The most important aim in this project was to study of the prevalence of 6rinary tract 

infection caused by 7+,5"5+*+68"#+,&, in patients who referred to the Central 

/aboratory of Dr. .hariati Hospital in Tehran-Iran, during 6 month (December 200O | 

(une 200[). 

The results of this study indicate that, the prevalence of 6rinary tract infections caused 

by*7+,5"5+*+68"#+,& in mentioned hospital is 7_. The urinary tract infection caused by* 

7+,5"5+*+68"#+,& in women (71_) is absolutely much more than men (2K_). 

The most important disposable factor in this study was estimated as .ex of patients, 

which the statistical method of Chi .quare has shown the significant relation between 

.ex and Infection (Pv0.0[). 

Keywords:*7+,5"5+*+68"#+,&, T$",+$2*-$+#-*",U.#-"%,3*V$/*=?+$"+-"*A%&'"-+63*7.,-$+6* 

X+8%$+-%$23*>.?$+,3*K$+, 

P-0453. Onychomycosis in diabetic subjects: A Tunisian study 

2en Hadj El 2echir A, Cherif F, El Euch D, Mezlini ., .ioud Dhrif A, MoBni M, 2en 

fsman Dhahri A 

Dermatology Department, Rabta Hospital, Tunis, Tunisia 

Background: The incidence of diabetes mellitus is increasing throughout the world. 

fnychomycosis is a common medical condition in patients with diabetes. 

The aims of this study were to identify risB factors for onychomycosis in diabetic 

patients. 

Patients and Methods: It was a retrospective chart review of onychomycosis in 

diabetics. 2etween (anuary 200[ and December 200[, [2O cases of onychomycosis 

were examined. [8 patients of them (11_) were diabetics. All these patients 

underwent mycological examination. 

Results: A total of [8 diabetic subjects were evaluated (..R } male / Female } J1/27). 

The mean age was [6.2 years, 7O.1_ of patients were aged more than [0 years. 

Patients with type 2 diabetes constituted 67.2_ and those with type 1 J2.8_. 

The mean duration of diabetes was 11 years. K patients (1[.[_) had degenerative 

diabetes] complications. Associated diseases wereM High blood pressure (2[.8_)n 

hyperlipemia (2 cases)n auto-immune diseases (2 cases) and breast cancer (1 case). 

Associated sBin mycoses were found in J2 patients ([[.2_), J0 patients had positive 

culture particularly for the dermatophyte trichophyton rubrum (O1.O_). The 

involvement of feet was higher than hands (respectively 67.2_ and 2O.2_). 

Discussion: Many studies found a significant correlation between infection and the 

older age of the patients, the gender (men more frequently affected), 

immunosuppressive agents, genetic factors, obesity, duration of diabetes, impaired 

peripheral circulation, peripheral neuropathy and retinopathy. These factors were 

found in our study. 

Conclusions: fnychomycosis in diabetic subjects can increase morbidity and 

decrease quality of life in these patients. It therefore appears that diabetics require 

more diagnostic, therapeutic and preventive care in terms of mycotic diseases.

P-0454. Susceptibility of yeasts isolated from hemocultures in 7 Belgian 

hospitals 

Boel A 1 , Cartuyvels R 2 , De 2eenhouwer H 1 , Frans ( J , fris E O , Vandecandelaere P [ , 

Van den Abeele A 6 , /agrou l 7 

1 

fnze /ieve Vrouw Hospital, Aalst, 2elgium, 2 Virga (esse Hospital, Hasselt, 2elgium, J Imelda 

Hospital, 2onheiden, 2elgium, O Hospital Zuid fost /imburg, GenB, 2elgium, [ (an Yperman 

Hospital, Ieper, 2elgium, 6 .int /ucas Hospital, Gent, 2elgium, 7 6niversity Hospital /euven, 

/euven,2elgium 

Background: Recently caspofungin was introduced in 2elgium for treating complicated fungal 

infections. However very few 2elgian data are available on drug susceptiblity of yeasts in these 

infections.The aim of this study was to analyse the susceptibility of all yeasts isolated from blood 

cultures in 7 hospitals during a 7 month period. 

Materials and methods: During seven months (December 200O - (une 200[), all yeasts 

isolated from hemocultures (n}8O) were collected in 7 2elgian hospitals (fnze /ieve Vrouw 

Hospital, Aalst, n}11, Imelda Hospital, 2onheiden, n}8, Hospital Zuid fost /imburg, GenB , n}[, 

.int /ucas Hospital, Gent, n}8, Virga (esse Hospital, Hasselt, n}7, (an Yperman Hospital, Ieper, 

n}K and 6niversity Hospital /euven, /euven, n}J6). Identification to the species level of all 

isolates was performed by IT.2 fragment length analysis. MIC]s for fluconazole, voriconazole, 

caspofungin and amphotericin 2 were determined with E-test (A2 2iodisB, .olna, .weden) on 

RPMI with [_ glucose and MfP. (.-Dimecon, Goes, The Netherlands) according the 

instructions of the manufacturer. For quality control ATCC K0028 (7/*+68"#+,&), ATCC 2201K (7/* 

'+$+'&"6%&"&) and ATCC 62[8 (7/*[$(&.") were tested. 

Results: From the 8O isolates [J were 7+,5"5+*+68"#+,& (62_), 18 7+,5"5+*@6+8$+-+ (1K_), 6 

7+,5"5+*'+$+'&"6%&"& (K_), J =+##?+$%)2#.&*#.$.0"&"+., 1 7+,5"5+*5(86",".,&"&, 1 7+,5"5+* 

6(&"-+,"+., 1 7+,5"5+*-$%'"#+6"& and 1 7+,5"5+*[$(&.". 

Reading of the MIC values was straightforward for caspofungin and amphotericin 2, but trailing, 

seen for the azoles, made interpretation sometimes more difficult for these antifungals. fverall, 

the MIC ranges (kg/m/) were as followsM fluconazoleM 0.06O-t2[6 (7 strains showed decreased 

susceptibility (MIC t 8 kg/m/)), voriconazoleM 0.00J- 2 (1 strain with MIC t 1 kg/m/), 

caspofunginM 0.0J2- 1 and amphotericin 2M 0.012-1.[ (1 strain with MIC t 1 kg/m/). 

The 7/*+68"#+,& strains were all susceptible to fluconazole. The MIC values for voriconazole and 

caspofungin were below 0,[ kg/m/. MIC values for amphotericin 2 ranged between 0.012 and 1 

kg/m/. Twelve 7/*@6+8$+-+ strains were susceptible to fluconazole, O strains .DD and 2 strains 

resistant (MIC t2[6 kg/m/). Fluconazole .DD and resistant strains had MIC ranges for 

voriconazole of 0,1J8 | 0,[ kg/m/ and 1 | 2 kg/m/ and for caspofungin of 0,1K | 0,J8 kg/m/ 

and 0,2[ kg/m/ respectively. For amphotericin 2 the highest MIC value was 1 kg/m/. 

All 7/*'+$+'&"6%&"& strains were susceptible to fluconazole (MICM 0,2[ | 1,[ kg/m/ ), and had low 

MICïs for voriconazole (0,012 | 0,12[ kg/m/) and amphotericin 2 (0,0J2 | 0,[ kg/m/). The MIC 

values for caspofungin were found to be between 0,J8 and 1 kg/m/. 

Conclusions: fnly some 7/*@6+8$+-+*(22_ .DD and 11_ R)*and 7/*[$(&." (intrinsic) showed 

resistance to fluconazole. The highest MIC value for voriconazole of these fluconazole resistant 

isolates was 2 kg/m/, while the MIC for caspofungin was below 0.[ kg/m/. 

P-0455. Epidemiology of tinea pedis in Terni, central Italy, between 1994 

and 2005 

2oncio / 1 , Agnetti F 2 , Moretti A J , Vidotto V O , Diamanti M 1 , Papini M 1 

1 

6niversity of Perugia, Faculty of Medicine and .urgery, Department of Medical and .urgical 

.pecialties and Public, 2 .tate Veterinary Institute, Perugia, Italy, J 6niversity of Perugia, Faculty 

of Veterinary Medicine, Department of 2iopathological .cience and Hygiene for, O 6niversity of 

Turin, Department of Medical and .urgical Disciplines, .ection of Infectious Diseases, Turin, Italy 

Tinea pedis is one of the commonest fungal disease in Italy and etiological agents include 

dermatophytes and yeasts. The constant competition of these organisms in the determination of 

the infection is about their particular environmental nicheM in fact, it often happens that the 

emergence of one or more predominant pathogens and the displacement of other less 

competitive species influence the development of the mycotic disease. 2ehaviours and changes 

in the incidence of fungal pathogens can be obtained analysing laboratory culture results of 

infected cutaneous tissues, occurred over a pre-established range of time. Therefore, the aim of 

this study was to identify the predominant fungal organisms causing tinea pedis in Terni and the 

epidemiologic trends of this mycosis in the urban area considered, in order to ascertain past and 

present behaviours of fungal incidence in Central Italy. 

A total of 1[10 specimens were collected from clinically suspected tinea pedis from 1KKO to 200[. 

.amples were submitted to the Mycology /aboratory of Dermatological .ection, 6niversity of 

Perugia (Terni, 6mbria), for fungal culture performing (inoculation into .abouraud glucose agar 

medium and incubation at 26ÅC for 2-J-weeBs)n secondary, fragments of the colonies were 

microscopically observed, in order to identify themn besides, the incidence of each species was 

counted. 

2oth non-dermatophytes and 7+,5"5+ species have showed relatively low incidence, whereas 

dermatophytes accounted for 8O.J_ to K8.1_ of isolated. >$"#?%'?2-%,*$(8$() was the most 

common isolated one, and the incidence of this species increased steadily during the study 

periodn this trend is typical for Central and .outhern Europe, according to other similar studies 

performed in this country. 

Data obtained in our worB suggested that further measures, regarding public health and personal 

hygiene, must be undertaBen, in order to prevent or reduce the development of tinea pedisn 

particularly an efficient sanitary control should be implemented in communal environments 

(swimming pools, gymnasia, farms, factories, changing rooms of sports clubs and public 

showers). 

References: 

Aste N., Pau M., Aste N., 2iggio P., 200J. Tinea pedis observed in Cagliari, italy, between 1KK6 

and 2000. Mycoses, O6M J8-O1. 

Foster l.a., Ghannoum M.A., ElewsBi 2.E., 200O. Epidemiologic surveillance of cutaneous 

fungal infection in the 6nited .tates from 1KKK to 2002. (. Am. Acad. Dermatol., [0 ([)M 7O8-7[2. 

Ianazzone .., Ingordo V., Naldi /., 200[. /e luci e le ombre del piede d]atleta. Evidence 2ased 

Dermatology, 1M JJ-O1. 

.eebacher C., 200J. The change of dermatophyte spectrum in dermatomycoses. Mycoses, O6 

suppl. 1M O2-O6. 


P-0456. Adiaspiromycosis, due to Emmonsia crescens, in a UK water vole. 

Study of the causative agent and establishment of the incidence of Emmonsia 

sp. in British mammals 

Borman A 1 , Campbell C 1 , /inton C 1 , Chantrey ( 2 , .impson V J , (ohnson E 1 

1 HPA Mycology Reference /aboratory, 2 Department of Veterinary Pathology, 

6niversity of /iverpool, J aildlife Veterinary Investigation Centre, Truro. 

P-0457. Ten years of Candida tropicalis fungaemia in a terciary care hospital: 

Epidemiology and antifungal susceptibility 

2osch Alepuz M 1 , Calabuig Munoz E 2 , Peman Garcia J 1 , Canton /acasa E 1 , Viudes 

î 1 , Perez 2elles C 1 , Gobernado M 1 

1 Microbiology Department. /A FE 6NIVER.ITY Hf.PITA/, 2 Internal Medicine 

Department. /A FE 6NIVER.ITY Hf.PITA/ 

Adiaspiromycosis is primarily a respiratory disease affecting rodents and other small burrowing Objective: To study the incidence, clinical features and susceptibility patterns of 

mammals. The principal causative agent in Northern 6.A and Europe is a))%,&"+*#$.&#.,&. 7+,5"5+*-$%'"#+6"&*isolated from blood cultures in a Terciary Care Hospital during the 

Inhaled dust-borne fungal spores fail to germinate in the host, instead increasing in volume last by up ten years. 

to 1 million-fold to produce large, thicB-walled adiaspores. Reports from several countries of 

isolated human infections attributed to a/*#$.&#.,& led to concerted attempts to evaluate the Methods: Retrospective study of all candidemia episodes by 7/*-$%'"#+6"&*diagnosed 

incidence of this fungus in mammalian reservoirs in the affected countries. To date, no human from (anuary 1KK[ to December 200O in /a Fe 6niversity Hospital. Mycology, 

cases have been identified in the 6l, and only limited examples of animal infections have been laboratory and clinical records were reviewed. The susceptibility study was carried out 

reported. 

using a microdilution method according to C/.I recommendations (document M27- 

A2). 

Here we report the results of a study of an isolate of a/*#$.&#.,& cultured from a 6l water vole 

that had died of respiratory impairment due to adiaspiromycosis. ae examined (i) morphological Results: In the period of study we found [JJ episodes of candidemia, J2 of them 

characteristics of adiaspores obtained from lung samples, (ii) lung adiaspore burden, (iii) culture were due to 7/*-$%'"#+6"&*(6.0_). The KJ.7[ and 7[_ of patients were adults and male, 

characteristics of the isolate, including the conditions necessary for the generation and respectively. The most important underlying conditions were solid or haematological 

germination of adiaspores ",*0"-$%, (iv) the molecular identification of the isolate by rRNA gene neoplasia (J0.2[_) and solid organ transplantation (K.J8_). The major predisposing 

sequencing and (v) its sensitivity to a wide variety of modern anti-fungal agents. 

factors for 7/*-$%'"#+6"& candidemia were previous antibiotic therapy (K0.6J_), 

intravascular line (81.2[_), parenteral nutrition ([K.J8_), intensive care admission 

These data prompted us to examine the incidence of a))%,&"+ in wild 2ritish mammals. ([6.2[_) and surgery ([0.0_). After candidemia diagnosis, the 8O.J8_ of patients 

Animals (mainly from RTA in .a England) were submitted to autopsy, where sections of lower received antifungal treatment. The crude mortality rate of candidemia due to this 

lung lobes were excised for processing. /ungs were processed for histology, and also by specie was OJ.7[_. Among the risB factors associated with death, intensive care 

digesting to completion in lfH, followed by centrifugation and microscopic examination of lung admission (8[.71_) was the most frequently observed during the study period 

residues for the presence of adiaspores. Twenty-nine percent (2[/86) of animals examined followed had by cerebral bleeding (21.O2_). Interestingly, all pediatrics patients survived 

evidence of a))%,&"+*#$.&#.,& infection by histology, digestion/microscopy or both. The to the 7/*-$%'"#+6"& candidemia episode. The range of MICs (mg//) were for 

incidence of adiaspiromycosis was largely independent of animal species, or geography. fluconazole 0.06-O, itraconazole 0.008-0.[, voriconazole 0.008-O, flucytosine 0.06-J2 

Adiaspores were visualised in 16/O8 otters (JJ_)n 2/K weasels (22_)n 2/7 stoats (2K_)n 2/O and mice amphotericin 2 0.12-1. 

and rats ([0_)n 1/J moles (JJ_)n 1/7 foxes (1O_) and 1/2 pine martens ([0_). For most animals 

(20/2[), adiaspore burden was low (2 or less adiaspores per cm J /lungn average adiaspore Conclusions: 7/*-$%'"#+6"&*is the third cause of candidemia in our hospital. Most of 

diameter 171 mn range 28-J80 m), indicating possibly that the animals had only a transient candidemia episodes occurs in adults. The major*7/*-$%'"#+6"& candidemia risB factors 

contact with a/*#$.&#.,&. The [ animals with higher adiaspore burdens (J otters, 1 weasel and including 1 broad-spectrum antibiotics, intravascular lines and intensive care admission. 

mole) had J-8 adiaspores per cm J /lung (average diameter 7[ mn range 10-O[0 m), consistent The crude mortality in adults patients is high (O6.6_). All 7/*-$%'"#+6"&*isolates were 

with recent and repeated exposures to a/*#$.&#.,&. 

susceptible to fluconazole and amphotericin 2. 

In summary, although the adiaspore burdens reported here are unliBely to have seriously This study has been partially financed by a Grant (2/200[/01O0) of Fundaciá per a la 

affected animal health, this report suggests that as many as 1 in J 2ritish soil-dwelling mammals Investigaciá Hospital /a Fe and a Grant of ICIII (CM0O/002O8). 

has had contact with a/*#$.&#.,&, indicating that this potential fungal pathogen is widespread in 

the 6l.

P-0458. Oropharyngeal candidiasis: survey in a general population in a dental 

surgeon (202 patients) 

Bourée P, Pdchard l, 2isaro F 

Parasitology-Mycology 6nit, 2icetre, France 

Purpose of the study: The oropharyngeal candidiasis is an opportunist infection, 

frequently due to 7+,5"5+*+68"#+,&. The passage of the 7+,5"5+ +68"#+,&, saprophyte 

yeast in parasite stage, responsible for the oropharyngeal candidiasis, is always 

bound to an imbalance of the commensal flora of the oral cavity. This imbalance is 

due to a local or general physiological modification of the patient-host. 

Description: The objective of the study was to estimate, in a general population, 

without particular risB factor, the prevalence of the oropharyngeal candidiasis, 

predisposing factors and the diagnostic and therapeutic behavior. The study was 

carried out in a general population of 202 patients of a dental surgeon of Paris area 

during two months. The patients (K0 men and 112 women) were between 1[ and 8K 

years old. Predisposing factors by the oropharyngeal candidiasis were inquired by a 

set of questions. A buccal swab was made to every patient with direct microscopic 

examination and culture in .abouraud medium. The statiscal analysis was made with 

Epiinfo software. 

Results and discussion: The prevalence of the oropharyngeal candidiasis in this 

general population was 12,K _. Many causes were found. There was a significant 

frequency of some risBs factors as for anti-arythmics, hypotensors and corticosteroids 

as well as for the carriers of denture and the chronic tabagic and alcoholic patients. 

2esides, 1O,8 _ of the patients had an oral colonization by 7+,5"5+*+68"#+,& (stage 

pre-infection) without symptom nor complaint and especially statistically significant 

cause. 8[_ of these patients were cured of the oropharyngeal candidiasis by 

fluconazole, without side effect. The failures in 1[ _ of cases were due to a poor 

observance of the treatment. 

Conclusion: It is established that an only medicinal treatment is not enough to cure 

oropharyngeal candidiasis. It is also necessary to treat the patient]s general status 

and to obtain a good bucco-dental hygiene. The collaboration between general 

practionner and dental surgeon is important in the diagnosis and treatment of the 

oropharyngeal candidiasis, but also in its prevention. 

P-0459. Relationships between Aspergillus fumigatus conidia and 

acanthamoeba 

2ouyer . 1 , Imbert C 1 , 2erjeaud ( 2 , Daniault G 1 , Rodier M 1 

1 CH6 /a Milmtrie, 86021, Poitiers Cedex, France, 2 Microbiologie Fondamentale et 

Appliqude, /CCE 6MR CNR. 6008, 6niversitd de Poitiers, France 

Purpose of the study: According to some authors, hospital water is, beside air, a 

potential source of transmission of filamentous fungi, and in particular 4&'.$@"66(&* 

U()"@+-(&. Germination of conidia is the important step in the development of this 

mould. In this study, we intend to explore the relationship between the 4/*U()"@+-(& 

conidia and the presence in the same medium of 4#+,-?+)%.8+ species, frequently 

recovered from waterM 4/*#+&-.66+,""3*4/*#(68.$-&%,"*and 4/*'%62'?+@+/* 

* 

Summarized description of the project: 

1- Each strain of 4#+,-?+)%.8+ was incubated with conidia of an 

environmental strain of 4/*U()"@+-(& in P2. to give a final multiplicity of 

infection of 1. The percentage of ingested conidia and the viability of 

amoebae were microscopically evaluated at 2, 8 and 2O h of cocultivation . 

2- Conidia of 4/*U()"@+-(& (2.10 6 /ml) were incubated during O and 10 days in 

P2.. After centrifugation, trophozoites (10 6 /ml) of each strain of 

4#+,-?+)%.8+ were suspended in the resultant supernatant. The viability of 

amoeba was evaluated at 12, 2O and O8h of incubation. 

J- Trophozoites (2.10 6 /ml) of each strain of 4#+,-?+)%.8+ were incubated 

during [ days in P2.. The amoebae were pelleted and conidia of 4/* 

U()"@+-(& (10 6 /ml) were then cultivated in the resultant supernatant. The 

germinative potential of the conidia was microscopically determined at 6, 8, 

12, 1O, 16 and 20h of incubation. 

All experiments were realised at 2[ÅC. 

Results and conclusions: 

1- The three tested strains of 4#+,-?+)%.8+ were able to ingest 4/*U()"@+-(& 

conidia, without any modification of the viability of their trophozoites after 2Oh 

of cocultivation. 

2- No decrease of the viability of the three amoebae was observed if they were 

cultivated in the medium where 4/*U()"@+-(& conidia were first incubated 

during O or 10 days. 

J- The germination of 4/*U()"@+-(& conidia occurred after 1O h of incubation 

with the three supernatants used at the same protein concentration. 2y 

contrast, 4/*U()"@+-(& was not able to germinate in P2.. 

Further studies are now necessary to investigate the potential role of amoebae in the 

protection and the growth of 4&'.$@"66(& in water systems, and consequently in some 

fungal nosocomial infections. 


P-0460. Improving coastal and recreational waters for all: Fungal analysis 

Brandão J 1 , VerÜssimo C 1 , .abino R 1 , Rosado C 2 , aergiBosBii 2 2 , Noronha G 2 , 

Rosado / 1 

1 Instituto Nacional de .aâde Dr. Ricardo (orge, 2 Instituto do Ambiente 

The quality of our coastal and inland recreational waters and sand are of great importance as 

people use them both for bathing or water-contact sports. During J years, the Interreg III2 

programme (FEDER) funded European project ICREa (Improving Coastal and REcreational 

aaters) has involved 21 institutions of [ European Atlantic coast countries (France, Portugal, 

Republic of Ireland, .pain and 6nited lingdom). The project aimed to deliver substantial 

improvements to our bathing waters and surrounding recreational sites. 

As part of the project, bacterial, fungal and blue algal water and beach sands have been under 

study during 21 months (from 200J to 200[). 1K campaigns raised samples of water and sands 

from J beaches along side an inland catchment and from one coastal beach, all in Alentejon the 

Portuguese region between Algarve and /isbon and Tagus Valley. Data were collected and 

analysed in order to produce a clear view on biological pollution levels and diffusion to 

recreational sites. Fungi isolated and counted were grouped into O categories, according to 

behaviour patterns and pathologies, regardless of individual positive or negative interactions 

within each group. The groups wereM (a) Yeasts (genuses 7+,5"5+3*7$2'-%#%##(&3* 

=+##?+$%)2#.&/*P?%5%-%$(6+), (b) Alergogenic and potential pathogens (genuses 4&'.$@"66(&3* 

^(&+$"()3*=#%'(6+$"%'&"&3*=#.5%&'%$"()3*7?$2&%&'%$"()3*=#2-+6"5"()), (c) Dermatophytes 

(genuses !"#$%&'%$()3*a'"5.$)%'?2-%,*+,5*>$"#?%'?2-%,) and (d) others (other genuses 

causing mycosis in animals). 

All biological parameters were looBed into for statistical significant positive and negative 

correlations (.pearman]s) amongst themselves and against physical-chemical parameters (Total 

Nitrogen, Nitrates, Nitrites, 2fD[, CfD, Total Phosphorus, Phosphates, pH and turbidity), 

measured in the water samples and in sand washings. 

The data were analysed asM (1) All water samples (catchment and coast together) and all sands 

samplesn (2) All catchment water samples and all catchment sandsn (J) Coastal water samples 

and coastal sand and (O) Each catchment beach water and sand (individually). 

ae could not consistently correlate the fungal parameters with any of others studied. ae found 

hence no indicator of fungal quality of either sand or water with this study. Groups (b) and (d) can 

be regarded as similar, although not identical. 6nder this perspective, our experience suggests 

group (b) should be used as representative of the two groupsn it is less labour intensive and more 

consistent then group (d) due to the limited number of genuses it represents. 

P-0461. Incidence of dermatophytoses in patients consulted in specialized 

hospital - clinical and mycological survey 1995-2005 

Budak A 1 , 2ogusz 2 2 

1 Dept. of Pharmaceutical Microbiology, (agiellonian 6niversity, lraBow, Poland, 

2 Department of Micriobiology, Rydygier]s Hospital 

Analysis of the occurrence of dermatophytoses in patients attending hospital 

mycological laboratory from 1KK[ to 200[. 

2etween 1KK[ and 200[, a total of J8J7 patients with suspected fungal infection of 

nail, sBin and hair were examined in mycological laboratory of Rydygier]s Hospital in 

lraBáw. 2oth sexes were involved. The total of 6K78 samples included sBin scrapings, 

nail fragments and hair clippings were observed directly by microscope examination 

after clarification with DM.f and also cultured on .abouraud 2_ glucose 

cycloheximide and chloramphenicol agar. Fungal growth was examined after 21 days 

of incubation at room temperature. The fungi were identified considering their cultural 

and morphological characters. 

The number of the patients with dermatophytoses was 820 and its ratio to the total 

out-patients of this period was 20_. The most common clinical forms of the 

dermatophytoses were tinea unguium followed by tinea pedis. Mycologically, 1J28 

strains of dermatophytes were isolated. In two cases two different dermatophytes | 

>$"#?%'?2-%,*$(8$()3*>$"#?%'?2-%,*).,-+@$%'?2-.&3*from different sBin lesions were 

isolated. The dermatophytes belonged to 8 species. The most common dermatophyte 

was >$"#?%'?2-%,*$(8$() 7J1 ([[_) followed by >$"#?%'?2-%,*).,-+@$%'?2-.&*[06 

(J8.1_), a'"5.$)%'?2-%,*U6%##%&()*O1 (J.1_), !"#$%&'%$()*#+,"&*27 (2_), 

>$"#?%'?2-%,*-%,&($+,&*11 (0.8_), >$"#?%'?2-%,*0"%6+#.()*6 (0.[_), !"#$%&'%$()* 

@2'&.()*O (0.J_) and !"#$%&'%$()*+(5%(","*2 (0.2_). 

fur results confirmed in the analysed period of eleven years, the slight increase in the 

occurrence of superficial fungal infections caused by dermatophytes. Tinea pedis was 

the most common clinical form of dermatophytosis, with the predominant isolation of >/* 

$(8$()*and >/*).,-+@$%'?2-.&/ 

2earing in mind that no correlation found might be simply an artefact of low counts in results of 

fungal analysis, it is our belief that further study should be carried out to investigate a possible 

consistent correlation between faecal bacterial indicators and groups (a) and (b)/(d). 

Although health hazard of fungi in beach sand remains to assessed conclusively, previous worB 

sampling the coast of the whole country ([ regions, three beaches in each regionM one with 

proven high microbiological quality, one wild/hardly visited beach and another of proven low 

microbiological quality) provided a clue as to the existence of different levels of contamination in 

these different types of beaches and therefore, quality standards can be set based on experience 

and further health-related studies.

P-0462. Outbreak of Rhizopus arrhizus soft tissue infections at new stomas 

associated with ostomy supplies 

2urwell / 1 , /eMaile-ailliams M 2 , .alisbury D J , Nobel-aang ( O , Arduino M O , /ott T 1 , 

2randt M 1 , /iames . 2 , .rinivasan A O , Fridkin S 1 

1 Mycotic Diseases 2ranch, 2 fhio Department of Health, J ABron General Medical 

Center, O Division of Healthcare ~uality Promotion 

Background: A patient at Hospital A developed P?"d%'(&*+$$?"d(&*soft tissue 

infections of their newly created stoma in (anuary 200[. A second patient developed a 

simialr infection in April 200[, prompting an epidemiologic investigation. 

Objective: Describe the clinical presentation and investigate the source of these 

infections. 

Methods: ae performed a cohort study of all patients having ileostomy or colostomy 

surgery in the outbreaB periodn cases had P/*+$$?"d(&*cultured from tissue or any 

histopathology suggestive of zygomycosis. Peri-operative and post-operative data 

were compared between cases and non-cases. Environmental samples of ostomy 

supplies and select hospital areas were processed. A point prevalence survey at [ 

unrelated hospitals was performed to describe ostomy care practices and degree of 

mould contamination of Baraya ring ostomy bags outside of Hospital A. Zygomycete 

isolates were evaluated by morphological methods, temperature studies, and DNAbased 

testing. sexpand the laboratory methodsu 

Results: Infections occurred 7 and 10 days post-operatively in the 2 case-patients. 

Indications for stoma placement were A and 2 respectively. 2oth received 

amphotericin 2 and debridement of the stoma and abdominal walln 1 died. Casepatients 

did not share health care worBers, hospital wards, or operating suites. ff 21 

patients in the cohort, the 6 patients receiving an equivalent of t0.[ mg/Bg/day of 

prednisone for t7 days before infection were more liBely cases (JJ_) than other 

patients (0_, p}0.07). Also, case-patients had longer delays before their first ostomy 

bag change (median 8.[ days, range 7-10) compared to 1K non-cases (median 1.[ 

days, range 1-17n p}0.08). P?"d%'(&*spp. was not recovered from the Hospital A 

environment but was recovered from 10 of 18 Baraya (a plant-derived adhesive) 

ostomy bags from Hospital A. P?"d%'(&*spp. was also recovered from Baraya ostomy 

bags donated from J of [ other hospitalsn the point prevalence survey of ostomy 

patients at these hospitals identified 18 post-ostomy surgery patients with a median 

time to first ostomy bag change of 2 days (range, 1-6 days). All P?"d%'(&*spp. were 

identifed as P?"d%'(&*+$$?"d(&*using species-specific PCR primers. 

Conclusions: The Baraya ostomy bags applied during stoma surgery was liBely the 

source of P?"d%'(&, and prolonged exposure before the first ostomy bag change may 

have precipitated infection in these immunosuppressed individuals. laraya should be 

considered to contain potentially pathogenic moulds and pose a risB for infection 

among susceptible individuals. 

P-0463. Outbreak of prosthetic valve endocarditis with Aspergillus in Colorado, 

2005 

2urwell / 1 , 2enoit . 2 , .rinivasan A 2 , Nobel-aang ( 2 , 2randt M 1 , Meinberg R J , 

Gershman l O , 2amberg a O , Fridkin S 1 

1 Mycotic Diseases 2ranch, 2 Division of Healthcare ~uality Promtion, J Hospital A, Cf, 

O Colorado Department of Public Health 

Background: In the 6nited .tates, more than 60,000 prosthetic valve surgeries are 

performed annually to replace failing heart valves. Infection of the replaced valve, or 

prosthetic valve endocarditis (PVE), is rare. PVE due to fungus, liBe 4&'.$@"66(&, is 

extremely rare (0.1_ of patients with prosthetic valves). Two patients had prosthetic 

valve surgery in March 200[ in the same operating room (fR j), and they later 

developed 4&'.$@"66(& PVE. 

Objective: ae investigated this outbreaB when it was recognized, in November 200[, 

to determine the source of these infections. 

Methods: A case had prosthetic valve replacement surgery at Hospital A from 

February 1 through (une J0, 200[ and developed 4&'.$@"66(&*PVE. To ascertain 

cases, we interviewed staff at Hospital A and reviewed laboratory data at 16 

metropolitan area hospitals where ill post-operative patients might have sought care. 

Patients who had valve surgery in fR j at Hospital A within two months of the cases 

were clinically evaluated and had 4&'.$@"66(& antigen and antibody testing by 

immunodiffusionn in addition, we performed a cohort study of these patients. Intraoperative 

and peri-operative data were compared between cases and non-cases by 

Fisher]s exact or ailcoxon test. Air and surface sampling of the operating room area 

at Hospital A were performed. 

Results: Two cases were identified. 2oth had surgery in March 200[n one was 

diagnosed four months after surgery, one was diagnosed seven months after surgery 

and died several days later. The histopathology of the explanted aortic valve conduits 

revealed septate fungal organismsn immunohistochemical staining confirmed 

4&'.$@"66(&*spp. Cultures of the valvular vegetations grew 4&'.$@"66(&*U()"@+-(&/** 

Cases] implanted prosthetic valves were from different manufacturers. No additional 

cases were identified from laboratory data review or during clinical evaluation and 

serologic testing. 2oth cases had surgery in an operating room adjacent to an area 

under construction and according to infection control staff, barriers to prevent 

construction-related exposure were intermittently compromised. fperating room 

pressure differentials did not consistently deviate from intended pressure differences. 

The cohort study included 1K patients. Case-status was associated with use of 

vascular adhesive during surgery (RR 16, pv0.0[) or surgery that replaced a portion 

of the aorta in addition to the prosthetic valve (RR 21, pv0.0[). Review of vascular 

adhesive quality control records for the lot used in the cases did not suggest product 

contamination. Air and surface sampling performed November 200[ did not yield 

4&'.$@"66(&*spp. 

Conclusions: 2reaches in the construction containment may have allowed the entry 

of 4&'.$@"66(&*spores into the operating room. Procedure-specific factors, such as 

longer duration of operative times or unmeasured factors related to the procedures, 

may have facilitated infection. 


P-0464. Fluconazole susceptibility of vaginal Candida isolates from Peruvian 

HIV-infected woman 

Bustamante B 1 , Neyra E 1 , Castro C 1 , Infante R 2 , /ucchetti A 2 , .anchez ( 2 , Celum C J , 

.tricB / J 

1 Instituto de Medicina Tropical Alexander von Humboldt-6niversidad Peruana Cayeta, 

2 Asociacián Civil y Educacián Impacta, /ima, Perâ, J 6niversity of aashington, .eattle, 

aashington, 6.A 

Background: Vaginal candidiasis (VC) is common among HIV-infected women, 

occurring in up to 60_. Although mucocutaneous candidiasis has declined 

significantly with the advent of HAART, HAART is still not widely available in most of 

the developing world. Azoles are the first-line treatment of 7+,5"5+ infections, but 

continuous use has led to refractory infections as a result of acquired resistance or 

selection of primarily resistant species. 

The objective of this study was to determine the percentage of 7+,5"5+ isolates from 

vaginal samples of Peruvian HIV-infected women resistant to fluconazole. 

MethodsM 2etween (anuary and .eptember 200[, 1O8 HIV-infected women over the 

age of 18 underwent a complete physical exam including the collection of vaginal 

samples. A vaginal swab was used to inoculate .abouraud dextrose agar plates 

incubated at J0o C. 7+,5"5+ isolates were identified using the API 20C A6j 

(2ioMemerieux) systemn and susceptibility testing was determined by the broth 

microdilution method of the Clinical and /aboratory .tandard Institute (Formerly 

National Committee on Clinical /aboratory .tandards). 

Candidal colonization (CC) was defined as the isolation of a 7+,5"5+ sp. from the 

vaginal mucosa, while culture-positive women with at least one clinical sign (discharge, 

erythema or edema) were considered to have vaginal candidiasis (VC). 

Results: Twenty-four (16.2_) participants had CC and 2[ (16.K_) had VC. Among 

the [2 7+,5"5+ isolates, 7/*+68"#+,& was the predominant species (80.8 _), followed 

by 7/*@6+8$+-+ (1J.[_), 7/*@("66.$)%,5"" (J.8_), and 7/*'+$+'&"6%&"& (1.K_). Three 

women had mixed infection with 7/*+68"#+,& and another 7+,5"5+ species. Thirty-five 

isolates (67.J_) were susceptible to fluconazole (MIC ' 8.0 kg/ml), [ (K.6_) had 

dose-dependent susceptibility (MIC 16 to J2 kg/ml), and 12 (2J.1_) were resistant 

(MIC % 6O kg/ml). All of the 7/*@6+8$+-+ isolates (n}7) and 12_ of the 7/*+68"#+,& 

isolates ([ of O2) were fluconazole-resistant. Fluconazole resistance was three times 

higher among women with CC than among women with VC. A similar frequency of 

women with CC and CV had a low CDOr cell count ('200 cells/mmJ), history of oral 

candidiasis and presence of 7/*@6+8$+-+ 

Conclusions: Candidal colonization and vaginal candidiasis are common among 

Peruvian HIV-infected women. 7/*+68"#+,& was the predominant specie isolated, and 

only a small number were resistant to fluconazole. ahereas, 7/*@6+8$+-+ was 

consistently resistant to fluconazole, 

P-0465. Candidemia in critical care units in a tertiary care hospital: Species 

distribution and susceptibility patterns 

Calabuig Muoz E 2 , 2osch Alepuz M 1 , Cantán /acasa E 1 , Pemán García J 1 , Viudes 

î 1 , ValentÜn MartÜn A 1 , Pdrez-2ellds C 1 , Gobernado .errano M 1 

1 Microbiology Department. /A FE 6NIVER.ITY Hf.PITA/, 2 Medical Internal 

Department. /A FE 6NIVER.ITY Hf.PITA/ 

Background: Invasive fungal infections have increased dramatically in recent years 

and candidemia is a major risB factor for morbidity and mortality in critical care units. 

Objective: To determine the epidemiology and susceptibility pattern of fungaemia due 

to 7+,5"5+ species in critical care units (intensive care, reanimation, pediatric and 

burnt 6nits) in a tertiary hospital during the last eleven years. 

Methods: Retrospective study of all the fungaemia episodes in /a Fe 6niversity 

Hospital. All yeasts were isolated from blood cultures since (anuary 1KK[ to 

December 200[. Data liBe demographical aspects, hospital intensive care unit as well 

as concomitant diseases were collected. Isolates were identified by the ViteB II system 

(2ioMerieux, France). MICs of amphotericin 2 (AM2), fluconazole (F/Z), itraconazole 

(ITR) and voriconazole (VfR) were established by the broth microdilution reference 

procedure of the C/.I (formerly NCC/.), M27-A2 method. 

Results: During the study period, a total of [81bloodstream yeasts infections were 

identified. The majority (J2[/[81n [[.K_) of candidemia episodes occurred in critical 

care units. The species distribution was as followsM 7+,5"5+*+68"#+,&*(1OK/J2[n 

O[.8_), 7/'+$+'&"6%&"&*(1J2/J2[n O0.6_), 7/*-$%'"#+6"&*(20/J2[n 6.2_), 7/*@6+8$+-+* 

(1O/J2[n O.J_), 7/*[$(&."*(J/J2[n 0.K_) and 2.2_ (7/J2[) other species (7/*6(&"-+,"+.3* 

7/*U+)+-+, 7/*@("66".$)%,5"", and 7/*5(86",".,&"&). 

MIC range, MIC [0 and MIC K0 , respectively, wereM 7/*+68"#+,& AM2 (v0.0J-2n 0.2[n 0.[), 

F/Z (0.06-6On 0.[n 8), ITR (0.0J-8n 0.0Jn 1), VfR (0.008-On 0.008n O)n 7/*'+$+'&"6%&"& 

AM2 (0.0J-2n 0.[n 1), F/Z (0.06-6On 0.[n 8), ITR (0.0J-0.[n 0.0Jn 0.2[), VfR (0.008-1n 

0.008n 0.06)n 7/*-$%'"#+6"& AM2 (0.0J-1n 0.2[n 0.[), F/Z (0.12-6On 2n 6O), ITR (0.0J-8n 

0.2[n 8), VfR (0.008-On 0.0Jn 2)n 7/*@6+8$+-+ AM2 (0.0J-1n 0.[n 0.[), F/Z (0.[-6On 16n 

6O), ITR (0.06-1n 0.[n 1), VfR (0.016-1n 0.12n 1)n 7/*[$(&." AM2 (0.06-1), F/Z (6O), 

ITR (0.12-1), VfR (0.2[-2)n and other species AM2 (0.12-0.[n 0.2[n 0.[), F/Z (0.12-8n 

0.[n 8), ITR (0.0J-0.[n 0.06n 0.[), VfR (0.008-1n 0.008n 1). The KJ.O_ of isolates are 

susceptible to all antufungal drugs tested. 

Conclusion: 7/*+68"#+,&*is the most prevalent species in critical care units followed 

by 7/*'+$+'&"6%&"&. Fungemia in critical care units due to non-7/*+68"#+,& species 

represents an important concern in our hospital, being 7/*'+$+'&"6%&"& the more 

prevalent species in this group. The rate of resistance to antifungal agents remains 

low. 

This study has been partially financed by a Grant (2/200[/01O0) of Fundaciá per a la 

Investigaciá Hospital /a Fe and a Grant by ICIII (CM0O/002O8). 

.upported by Directorate General for development Cooperation of 2elgium.

P-0466. Dermatophytosis in S. Teotónio Hospital: 3 years experience 

Carvalho A 1 , Ribeiro ( 2 , Ribeiro ( J , Fernandes E O 

1 Hospital de .Éo Teotánio, Viseu, Portugal, 2 Hospital de .Éo Teotánio, Viseu, 

Portugal, J Hospital de .Éo Teotánio, Viseu, Portugal, O Hospital de .Éo Teotánio, 

Viseu, / Instituto Nacional de .aâde Dr. Ricardo (orge, /isboan Portugal 

Dermatophytes are Beratinophilic fungi, which invade superficial sBin, hair, and nails. 

The aim of this present study was to define the epidemiological 

profile of dermatophytosis in patients who were consulted at the Dermatology .ervice 

of .Éo Teotánio Hospital, in the last three years. 

These dermatophytosis were more frequent among children 2-1[ years old group, 

who live in rural area. The samples were examined by direct analysis performed with 

potassium hydroxide (J0_). The growth media used were both .abouraud- 

Chloramphenicol agar and Dermatophyte-agar. Identification of the fungi was based 

on their macro and microscopic characteristics. Microscopic analysis was performed 

with lactofenol blue. ae recorded a total of 277 samples recovered from 1O8 sBin, 72 

hair, and [7 nail scrapings, and got 28,K_ positive. 

In the 80 strains isolated the dermatophytes obtained were predominantly 

!"#$%&'%$()*#+,"& J[ (OJ, 8_), >$2#?%'?2-%,*).,-+@$%'?2-.& 1[ (18, 8_), and 

>$2#?%'?2-%,*$(8$() 12 (1[, 0_). 

!"#$%&'%$()*#+,"& was the most frequently dermatophyte isolated. It was found that 

the children infected frequently come in contact with soil and animals (cats, dogs). 

P-0467. Hepatic safety of concomitant caspofungin (CAS) and cyclosporine 

(CSA) 

Carver P 1,2 

1 6niversity of Michigan College of Pharmacy, 2 6niversity of Michigan Health .ystem 

Background & Purpose: At the time of FDA approval, there were concerns regarding 

the safety of CA. when combined with C.A. In initial studies utilizing a combination of 

C.A and CA. in 12 healthy volunteers, [ experienced elevations in serum 

aminotransferase levels less than or equal to J times the upper limit of normal (6/N). 

In addition, J of O healthy subjects who received CA. 70 mg/day for 10 days plus two 

J mg/Bg doses of C.A on day 10 experienced transient increases in alanine 

transferase (A/T) on day 11 that were v[ times the upper limit of normal (6/N). In the 

same study, 2 of 8 subjects who received CA. J[ mg/day for J days and two J mg/Bg 

doses of C.A on day 1 experienced increases in A/Tv2 times 6/N on day 2. As such, 

the pacBage insert recommended that liver function tests be monitored closely when 

this combination is used. 

Methods: In order to evaluate the hepatic safety of concomitant CA. and C.A in the 

clinical setting, a retrospective chart review of O6 adult patients (median O2.6 yearsn 

range, 17|6J years) who were treated concomitantly with C.A and CA. during a O 

year period, was conducted. IR2 approval was obtained prior to review of patient 

charts. All patients who received t1 day of both agents concomitantly were assessed. 

Data collected included patient demographics, primary underlying disease, dose, 

duration, and dates of administration of CA. and C.A, liver function tests, prior and 

concomitant medications, and concurrent medical events. Data was collected (when 

available) from the medical record for J0 days prior to the start until 7 days after the 

end of concomitant therapy. 

Results & Conclusions: Although elevations of A/T and/or aspartate 

aminotransferase (A.T) occurred in 10 patients (21.7_), none were tO times the 6/N. 

No patients discontinued therapy because of hepatotoxicity possibly related to CA. or 

C.A. No serious adverse events occurred because of CA.. Most patients (78_) had 

undergone allogeneic hematopoietic stem cell transplant (H.CT). The remaining 

patients had received a solid organ transplant or had another underlying disease for 

which they received C.A. The average duration of concomitant therapy was 21 days 

(range, 2|O7 days). No clinically significant elevations of serum aminotransferases 

were observed, and no patient had concomitant therapy discontinued or interrupted 

due to a drug-related adverse event. In this study of a limited number of patients, the 

coadministration of CA. and C.A was well tolerated. These data suggest that the 

risB of clinically significant hepatotoxicity with concomitant CA. and C.A is minimal. 


P-0468. Diagnostic capacity of clinical and paraclinical parameters in patients 

with pneumocystis pneumonia 

Casanova l 1 , .Zez A 1 , Navas T 1 , Reviakina V 2 , 2orges R J , Panizo M 2 

1 Hospital General del feste (osd Gregorio HernZndez, Caracas, Venezuela, 2 Instituto 

Nacional de Higiene Rafael Rangel, Venezuela, J Instituto de 2iomedicina, 6CV, 

Venezuela 

Purpose of the study: To Bnow the diagnostic capacity of clinical signs, symptoms, and 

some paraclinical parameters in AID. and non-AID. patients with risB factors to 

Pneumocystis pneumonia (PCP). 

Description of the project: It was a transversal and descriptive investigation of PCP 

diagnosed by direct immunofluorescence technique (DIF) as a hgold standardi, in sputum 

samples (spontaneous and induced) or bronchoalveolar lavage. 1-J Additionally, it was made 

gram and ziehl neelsen stains, bacterial cultures, and serological tests for endemic fungi. 

The patients inclusion criteria wasM older than 18 years old, both sex, and carry out one of 

the following conditionsM autoimmune disease, chronic pulmonary disease, renal failure, 

chronic or acute hepatic disease, post mayor surgery state, nosocomial condition, 

polyaddiction or social deprivation. All patients must had symptoms of acute pulmonary 

disease. The diagnostic capacity was measured by sensitivity, specificity, and odds ratio. 

The study was made between May 200O and (une 200[. 

Results and Conclusions: 6K patients were evaluated and 10 of them had PCP. ff these 

patients, O had AID. and 6 had chronic pulmonary disease, undernutrition and neoplastic 

disease. The variables with low and moderated diagnostic capacity in all patients wereM 

hospital contact, travels, chronic disease, /DH between 120|2KK 6//, hypoalbuminemia, 

f 2 low saturation, respiratory acidosis, and thrombocytopenia. .igns of consolidation 

images plus interstitial pattern or air trapping were observed in radiological studies. In AID. 

patients, variables diagnostic capacities were similar as in non-AID. group, exalting the 

value of /DH levels, f 2 low saturation, respiratory acidosis and pleural pain. PCP must be 

suspected in patients without AID. and presence of risB factors as mention before J-6 . DIF 

must be implemented as the election technique for diagnosis of PCP. 1-J 

P-0469. Estimation of distribution of coccidioidomycosis in Mexico 

Castañón L, Aroch A 

6niversidad Nacional Autánoma de Mdxico, Mexico City, Mexico 

Purpose of study: The prevalence reported of coccidioidomycosis in the 6...A. 

includes Mexican migrantsn however, in our country the epidemiological scene of this 

mycosis has been without actualization for more than 10 years. The aim of the present 

study is to give an estimate of the actual geographical distribution of the disease in our 

territory. 

Description: A bibliographical search for cases with coccidioidomycosis, completed 

with the information of 1J laboratories belonging to health institutions in Mdxico. The 

information obtained was then compared with the paper published by GonzZlezfchoa 

(1K66) and with those by the .ecretarÜa de .alud (official institution of health in 

our country). 

Results and conclusions: In this research, 621 human cases with 

coccidioidomycosis are reported that occurred in the Mexican Republic over a period 

of O7 years. fnly from 81_ of the patients the information of residence could be 

obtained and from this reference a distribution map was elaborated. Comparing our 

distribution with the distribution elaborated by GonzZlez-fchoa and with the one 

reported by the .ecretarÜa de .alud in the period 1K8K-1KKO. The differential between 

the values published by the .ecretarÜa de .alud became evident. 

Cases of coccidioidomycosis are still present in our country with a tendency similar to 

the one proposed by GonzZlez-fchoa. The findings in this present paper of cases 

from nonendemic regions such asM Distrito Federal (Mexico City), Hidalgo, Tlaxcala 

and Veracruz, may point to an extension in the distribution of the disease. ae 

consider it to be necessary to promote a program of epidemiological surveillance on 

national level in order to clarify the situation of this serious mycosis. 

References: 

1. 2orelli l, 2rito A, Rivas G, Panizo MM, RoldZn Y. Diagnástico de 1,.()%#2&-"&* 

#+$",""M Estudio comparativo entre inmunofluorescencia directa y la coloracián 

histolágica de Gomori-Grocott. 2ol .oc Ven Microbiol 2000n 20(1)M O6-[2. 

2. Panizo M, ReviZBina V, VZzquez C. Diagnástico de 1,.()%#2&-"&*#+$","" por 

inmunofluorescencia directa modificada y coloracián de Gomori-Grocott. Estudio 

comparativo. 2ol .oc Ven Microbiol 2000n 20(2)M K8-10J. 

J. aazir (, Ansari N. 1,.()%#2&-"&*#+$","" infectionM 6pdate and review. Arch Pathol 

/ab Med 200On 128M 102J-7. 

O. Medrano F, Montes-Cano M, Conde M, de la Horra C, Respaldiza N. 

1,.()%#2&-"&*\"$%0.#"" in general population. Emerg Infect Dis 200[n 1(2)M K1-6. 

[. .epBowitz l. fpportunistic infections in patients with and without acquired 

immunodeficiency syndrome. Clin Infect Dis 2002n JOM 10K8-107. 

Thomas CF, /imper AH. Pneumocystis pneumonia. N Engl ( Med 200On J[0M 2O87-K8.

P-0470. Molecular typing of Mexican Cryptococcus neoformans strains 

Castañón L 1 , MartÜnez l 1 , Ruiz-Palacios G 2 , 2ermâdez R J 

1 6niversidad Nacional Autánoma de Mdxico, Mexico City, Mexico, 2 Instituto Nacional 

de Ciencias Mddicas y de la Nutricián, ..., J Centro de Investigacián y Estudios 

Avanzados, IPN. 

Purpose of study: Despite that the Cryptococcosis frequency has been reduced 

since last decade, in our country this disease is still recognized as a common 

opportunistic mycotic infection that becomes life-threatening in immunedepressed 

patients. This worB contains the genotyping study performed in 7O 7$2'-%#%##(&* 

,.%U%$)+,&, strains obtained from Mexican patients with cryptococcosis infection in 

central nervous system and another 20 strains of different countries were included. 

Description: For the molecular typing, finger-printing PCR technique with the primer 

zcore M1J] was used. The analysis of band patterns obtained (GelCompar I), grouped 

all isolates into eight molecular types previously established by Meyer in 200J. 

Results and conclusions: The distribution for the Mexican strains was the followsM 

Fifty-five strains was identified for VN1 genotype, five for VN2, three for VNJ, two for 

VNO, three for VG1, three for VG2, one for VGJ and two for VGO. .o, the genotype 

VN1 is the most frequent in our country and the presence of the genotypes VNO and 

VG2 calls attention, because that have not been reported in previous studies. The 

technique employee didnet discriminate geographical origin. 

.ince cryptococcosis is still a problem in public health, it is important to use these 

molecular analyses to monitor this infection and define individuals that are susceptible 

to such infection to carry out prophylactic strategies. 

P-0471. The risk of fungal infections related to the water supply of several 

French hospitals 

Catherine K 1 , Anne 2 2 , Frdddric D J , Marie-Pierre 2 O , Emilie F [ , Marie M 6 , Martine G 7 , 

Claire / 8 , Christophe H K , Dominique T 10 

1 CH6 de Poitiers, France, 2 CH6 de Poitiers, France, J CH6 de Dijon, France, O CH6 de 

Grenoble, France, [ CH6 de /ille, France, 6 CH6 de Nancy, France, 7 CH6 de Nice, 

France, 8 CH6 de Paris - .t /ouis, France, K CH6 de Paris - Tenon- .t Antoine, France, 

10 CH6 de Reims, France 

The aim of the study was to clarify the methodological conditions required to assess 

the risB of fungal infections related to the water supply of ten 6niversity Hospital 

Centres spread over the country. The study tooB place between February 200O and 

March 200[ and focused on risB patients] unitsM Haematology (n}70), .olid organ 

transplant (n}7), 2urns (n}11) other wards (n}11). aater and related surfaces were 

studied. 

The water from taps equipped with sterilized filtration was sampled without the filter. 

K8 sites were defined M three samples of one litre of water (the first flow, cold and hot 

water) and 2 to J surface samples (inside of the tap, pommel of the shower and 

siphon). In each ward , sampling had to be part of the usual procedure carried out for 

systematic hospital water analysis. After filtration of the water through a cellulose 

acetate membrane (0,O[ &m), this filtration membrane was laid on a Malt or a 

.abouraud agar. Petri-dishes were incubated at 27ÅC for 28 days. A form was filled on 

each site of sampling with every information available M place in the unit, treatment of 

the water (chlorine or else), filter, temperature of the water. Microbiological results 

were also reported. 

Results and conclusion: There is a significant difference between the different 

types of water in term of positive culture and mean number of CF6//. 1 st flow M 7,[ 

CF6//, cold and hot water M J,8 and 1,[ CF6/ / were isolated respectively. Except in 

two hospitals where a real contamination of the water pipes was identified, water was 

usually slightly colonized, the results depending on the centre. The number of CF6 in 

samples from surfaces (bio film) is higher (mean } 1[ CF6 per sample) but surfaces 

were less frequently positive than water. In the hospital centres studied, sampling from 

the siphons was more often productive than the one from the tap. Dematiaceous 

hyphomycetes were the most frequently isolated fungi, 4&'.$@"66(& &' were rarely 

isolated. Cultures were positive during the first weeB. 2acterial flora and presence of 1/* 

+.$(@",%&+ was a required study. The chemical treatment of the water was also 

reported. 

In a strategy of water control, a mycological water pipes diagnostic would be 

necessary to obtain the baseline in every hospital. In each at risB patients] units the 

mycological survey could sample once a year and analyse one litre of cold water. 

Presently, only bacteriological surveys are required by French hospital regulations. In 

those wards, taps are often protected by sterilized filtration which could be 

recommended in standard hospitalisation sectors receiving aplasic patients. 


P-0472. Epidemiology of paracoccidioidomycosis and histoplasmosis in a 

population from Bolivar state, Venezuela 

Cermeño J 1 , Cermeo ( 1 , Godoy G 1 , frellZn Y 1 , 2lanco Y 1 , Penna . 1 , HernZndez I 1 , 

Mender T 2 , GonsZlvez M 1 , (ahouhari C 1 

1 6niversidad de friente. Escuela de Ciencias de la .alud. Ciudad 2olÜvar. 2olÜvar 

.tate. Venezuela, 2 Hospital ÄRaul /eoniÄ. .an Fdlix. 2olÜvar .tate. Venezuela 

Contributions to the epidemiologic study of the deep mycosis are scarce in 2olÜvar 

state, where Paracoccidioidomycosis and Histoplasmosis are considered as endemic 

diseases, and it is necessary to Bnow better their clinical and epidemiologic aspects. 

The aim of this study was to determine the prevalence of 1+$+#%##"5"%"5%)"#%&"&* 

8$+&"6".,&"& and A"&-%'6+&)%&"&*#+'&(6+-() infections in subjects from h2[ de Marzoi 

population, .an Fdlix, 2olÜvar state, Venezuela. 

A descriptive-prospective study was carried out. Paracoccidioidine and histoplasmine 

were administered to J00 persons, who accepted to participate in this study. Pregnant 

women, children younger than 6 months and adults older than 6[ years-old and those 

with history of systemic mycosis were excluded. Identification and epidemiologic data 

were registered. The reading of the administered tests was made at 2O hours in only 

27[ subjects. The Intradermoreaction was positive at 2O hours in 10.2_ (n}28) of 

subjects. The O0-[0 years-old group showed the greater percentage of positivity 

(n}10n J[,7_). fccupations of risB liBeM bricBlayers, farmers and miners presented 

27.J_ of positivity (J of 11) greater than that of persons without apparent risBM 

mechanics, housewives and students (2[ of 26On K.[_), being this difference 

statistically significant (p}0.0O). Histoplasmine Intradermoreaction was positive in 

7.6_ of the cases (n}21). The age group that displayed greater percentage of positive 

intradermoreaction was that of O0-[0 years-old (n}Kn O2.K_)n followed by the J0-O0 

years group (n}On 1K.0 _). The most of the positive intradermoreactions was 

demonstrated in the group older than J0 years. A significant direct proportional 

relationship between time of residence and A"&-%'6+&)+*#+'&(6+-() infection was 

evidenced, mainly in subjects with more than J0 years in that location (pv0.0[). 

P-0473. Towards understanding clonality and variation of Penicillium marneffei 

strains 

Chakrabarti A 1 , 2hardwaj . 1 , .harma M 1 , 2achhawat A 2 , Ranjana l J , Devi . J , 

Chindamporn A O , Padhye A [ 

1 PGIMER, Chandigarh, India, 2 IMTECH, Chandigarh, India, J RMC, Imphal, Manipur, 

India, O ChulalongBorn 6niveristy, 2angBoB, Thailand, [ Centers for Disease Control c 

Prevention, Atlanta, 6.A 

1.,"#"66"()*)+$,.UU." causing penicilliosis has emerged as an opportunistic fungal 

agent that is endemic in .outheast Asia. Though few attempts have been made 

towards providing a strain typing method for 1/*)+$,.UU." especially with the 

development of reproducible, discriminatory Multilocus Microsatellite typing (M/MT), 

no systematic study has been conducted to understand the genomic changes of this 

geographically restricted fungus. In India penicilliosis is restricted in Manipur state. ae 

compared the strains isolated from India (n}22) with strains from Thailand (n}2[) and 

China (n}K) using length polymorphism, PCR-RF/P of internal transcribed spacer 

(IT.) and intergenic spacer (IG.) regions of ribosomal DNA, and modified M/MT 

method (targeting repeat regions). Two different combinations of primers targeting 

different microsatellite loci having similar annealing temperature were used for 

modified M/MT. ahile combining RF/P of IT. and NT. regions, the strains could be 

differentiated into five types with O1 (7J.2_) strains in type A and K (16_) in Type 2. 

However, on including M/MT into typing scheme, the strains were differentiated into 

nine types with JK strains representing all three countries in Type I. Thus, clonality is 

possibly maintained in most (70_) of the strains from three countries. Though Type II 

also contained strains representing three countries, the remaining seven types were 

restricted to one country only. It could be concluded that 1/*)+$,.UU." being asexual 

and restricted in one geographical region, possibly maintained its clonality without 

much variation. 

These results show a low prevalence of infection by 1/*8$+&"6".,&"& and of A/* 

#+'&(6+-() in the studied area.

P-0474. Disparities in testing practices for coccidioides among patients with 

community-acquired pneumonia (CAP) – metropolitan Phoenix, 2003-2004 

Chang D 1 , Park B 1 , 2urwell / 1 , aannemuehler l 1 , Anderson . 2 , Englethaler D 2 , 

FridBin . 1 

1 Centers for Disease Contol and Prevention, 2 Arizona Department of Health .ervices 

Background: 7%##"5"%"5.& spp. may cause up to 2K_ of outpatient CAP, resulting in 

an estimated J0,000-K0,000 cases annually in Arizona. Despite being a reportable 

disease, only J660 cases were reported in 200O, possibly due to under-testing. To 

better understand testing practices, we studied patients presenting with CAP in two 

populations in metropolitan Phoenix. 

Methods: ae conducted cohort studies in two large health systems (A and 2) with 

clinics throughout metropolitan Phoenix. A (17 clinics) mainly serves a privately 

insured population and 2 (1J clinics) serves large numbers without private insurance. 

ae identified a random sample of visits with pneumonia ICD-K codes during 200J and 

200O for chart review. Patients were included if they initially presented as outpatients 

with clinician-diagnosed pneumonia, lacBed a history of coccidioidomycosis, and were 

not institutionalized or recently hospitalized. Demographic, clinical, and diagnostic 

data, including 7%##"5"%"5.& testing, were extracted. 

Results: 87 of 1[8 (A) and 66 of 1J2 (2) sampled visits coded for pneumonia were 

included. .ymptoms, signs, and treatment were similar for patients in both systems. 

Patients in 2 had a greater median age ([O vs. J7) and were more liBely to be diabetic 

(J8_ vs. 8_, pv0.01), two risB factors for developing severe disease. However, 

patients in 2 were less liBely to have 7%##"5"%"5.&*serologic testing (2_ vs. 1J_, 

difference}11_, exact CI}J_-20_) and to receive chest radiographs (J[_ vs. K[_, 

pv0.001) than patients in A. 

Conclusions: 7%##"5"%"5.& testing among patients with CAP is infrequent in both 

populations and significantly less frequent in the system serving large numbers of 

patients without private insurance. fpportunities for improved care existn more 

research is needed to determine reasons for variations in testing. 

P-0475. Prospective analysis of the genotypic diversity and dynamics of the C. 

albicans colonizing flora in acute myeloblastic leukemia patients 

Dalle F 1,2 , Caillot D J , /efllivier C 1,2 , .icard P 1 , Ecarnot-/aubriet A 1 , /afon I J , /izard . O , 

Vagner f 1 , Cuisenier 2 1 , 2onnin A 1,2 

1 /aboratoire de Parasitologie Mycologie, HÇpital du 2ocage, 2P 77K08, 2107K Dijon Cedex, 

France, 2 /aboratoire de Microbiologie Mddicale et Moldculaire, Facultd de Mddecine, 2P 

87K00, 2107K Dijon Cedex, France, J .ervice deHdmatologie Clinique, HÇpital deEnfants, 2P 

77K08, 2107K Dijon Cedex, France, O /aboratoire de Gdndtique, CGF/, 1 rue Pr. Marion, 

2107K Dijon Cedex, France 

The yeast*7+,5"5+*+68"#+,& is a ubiquitous euBaryotic organism that develops as a 

saprophyte of the mucosae in humans. In immunocompromised or intensive-care patients, 

this organism may overcome host defences, resulting in increased mucosal colonization 

and sometimes invasion into the bloodstream through epithelial and endothelial layers. 

Thus, 7+,5"5+*&''. account for 8 to 1[_ of nosocomial bloodstream infections, and the* 

attributable mortality due to bloodstream candidiasis approaches J[_. 7/*+68"#+,& is the 

causative agent in [0 to 70_ of disseminated candidiasis, maBing candidemia due to 7/* 

+68"#+,& a major source of concern to the medical community. 

Molecular typing methods have shown that endogenous acquisition is the main source of 

invasive candidiasis, corroborating evidence for prior mucosal colonization as an 

independent risB factor for disseminated candidiasis. Indeed, some epidemiological 

genotyping reports have shown heterogeneity of sequential isolates from HIV-infected 

patients. Their recurrent episodes of oropharyngeal candidiasis included possible 

coexistence of multiple resistant phenotypes of 7/**+68"#+,&. 2ut the dynamics of 

colonisation due to 7/*+68"#+,& has never been examined in a prolonged prospective study 

analysing the genotypic diversity of the flora at different anatomical niches, and in a clinical 

setting other than HIV infection. 

To address this question, 20 patients with acute myeloblastic leuBemia (AM/) treated at our 

center between 2001 and 200J were enrolled in a prospective study aimed at highlighting 

the heterogeneity and dynamics of the 7/*+68"#+,& colonizing flora. All patients received 

three regimens of a myeloablative treatment over a 6-months period. In this time course, 

microbiological monitoring included daily blood cultures, the retrieval of samples from the 

throat and nose and collection of urine and stool twice a weeB. For each positive sample 

harbouring 7/*+68"#+,&, up to ten colonies (clones) were processed for molecular typing 

using four microsatellite marBers (a^:, AK=:, 7V7: and 74K). 

Eight patients harbouring 7/*+68"#+,& colonisation were included. Altogether OO8 clonal 

isolates were obtained from [K positive samples ("/.. a mean 7.6 colonies per positive 

sample) and were processed for molecular typing. .even patients carried a unique 

multilocus genotype which was identical (i) in all body niches (ii) over the time course. In 1 

case, minor genotypic differences were observed in 2/[2 clones tested, suggesting 

microevolution. None of the patients shared 7/*+68"#+,& clones with identical genotypic 

profiles. Disseminated candidiasis occured in one patient and the blood strain genotype did 

not differ from those of colonizing isolates. In 6/8 patients receiving local or systemic 

antifungal therapy the genotypic profile of the colonizing flora was not altered upon 

treatment. 

fur data suggest that in patients with hematological malignancies, genetic evolution of the 

colonizing 7/*+68"#+,& flora and selection of variants or replacement of the original strain 

upon antifungal drug pressure or nosocomial transmission are rare events. 


P-0476. Fusarium species involved in onychomycosis in Bogotá, Colombia 

De Garcia M 1 , .opo / 2 , Castro N J 

1 6niverisdad de los Andes, 2 /aboratorio Especializado de Micologia Mddica, 

J 6niverisdad de los Andes 

The purpose of this study was to identify the species of ^(&+$"() isolated from 

patients with onychomycosis and to determine the clinical patterns of onychomycosis 

caused by ^(&+$"() sp. This study was carried out at the /aboratorio Especializado 

de MicologÜa Mddica (/EMM), and the /aboratorio de MicologÜa y FitopatologÜa, 

6niversidad de los Andes, 2ogotZ, Colombia, between (anuary 200J and fctober 

200O. The study population comprised 1O0 isolations from 1J7 patients. For the 

selection of isolations, the diagnostic criteria wereM (i) the presence of a fungus on 

direct microscopyn (ii) isolation of the same fungus from culture on two different 

occasionsn (iii) and at least half of the inocula should yield a fungus. 

ae performed a monosporic culture by maBing dilutions, counting in Neubauer 

camera, and culturing in Nash and .nyder Medium (N.M) for [-8 days at 28oC. The 

identification of the species was carried out by microscopic identification, culture in 

Potato Dextrose Agar (PDA) and Carnation /eaf Agar (C/A) (Nelson et al, 1K8J). All 

the collected data were processed in EpiInfo J.2.2n the results were statistically 

analyzed using chi-squared test. A 1 value of 0.0[_ was considered to be significant. 

A total of 1O0 isolations were obtained and three species were identified, ^/*&%6+,"* 

([K.J_), ^/*%_2&'%$() (J8.6_) and ^/*0.$-"#"66"%5.&*(^/*)%,"6"U%$).) (2.1_). The age 

range of the patients oscillated between 16 and 8[ years, with a OOì1O.J6 year-old 

average. The higher frequency was obtained in patients between J1 and O0 years 

(28.8_). The isolation of ^(&+$"() sp. was more frequent in women (7J_), that in 

men (27_). The evolution of lesions oscillated between 1 month and O0 years, being 

more frequent between 1 month and [ years (7K.J_). A total of [O.7_ of the patients 

practice sport regularly, while 7[.K_ affirmed that they taBe a bath barefoot. In 

addition, [K.K_ of the patients did not present any type of illness, 6J.[_ of the 

patients had had a previous medical treatment, empiric or prescribed. Most of the 

patients said to had a manicure or pedicure (67.[_ and [K.K_ respectively). There 

were not significant differences (1t0.0[) between the species of ^(&+$"() and the 

variables under study. In our study 87.8[_ of the isolated fungi are primary agents. 

This was concluded following the established approaches that consider the fungi non 

dermatophytes as causal agents of onychomycosis. A total of 12.1O_ of the isolations 

corresponded to over-infection or mixed infection. This result if important since these 

patients should have a different therapy and they require more attention. ae found 

three species of ^(&+$"(), ^/*&%6+,", ^/*%_2&'%$() and ^/*0.$-"#"66"%5.&3 in 

concordance with other studies carried out previously in our laboratory and with other 

reports. ahen data were analyzed there were not statistical differences between the 

species of and therefore the clinical data are indistinguishable among species. ahen 

we compared the sex against other variables, we found that women practice more 

sport that men, the difference being statistically significant (1}0.0O7). This result is in 

disagreement with was reported before. 

P-0477. C. neoformans: varieties and serotypes in Venezuela 

Dolande M 2 , Pdrez C 1 , Moya M J , Rosellá A 1 , Hartung C 1 , Mata . 1 

1 

Instituto de medicina Tropical. .eccián de MicologÜa Mddica. Caracas, Venezuela, 2 Instituto Nac. 

de Higiene Rafael Rangel. Dpto. de MicologÜa. Caracas, Venezuela, J Instituto Nac. de Higiene 

Rafael Rangel. Dpto. de 2ioterio. Caracas, Venezuela 

Introduction: There are geographical differences in the prevalence of the various serotypes of 

7$2'-%#%##(&*,.%U%$)+,&. 

Aim: This study determines the distribution of the varieties and serotypes in Venezuelan clinical 

isolates. 

Materials/methods: 1J2 7$2'-%#%##(&*,.%U%$)+,& clinical isolates were recovered (1KK[-200O), 

Departamento de MicologÜa, Instituto Nacional de Higiene and .eccián de MicologÜa Mddica, 

Instituto de Medicina Tropical, 6niversidad Central de Venezuela. The strains were identified by 

conventional biochemical test, Micro.canw Dade 2eringn varieties by /-canavanine, glycine and 

bromothymol blue agar media slwon-Chung,1K82un serotyping determined serotypes. Each 

control strains s7/*,.%U%$)+,& serotypeM A(/-O12000-1[8)n C(/-O12000-160)n D(/-O12000-161) 

(autochthonousM Instituto 2iomedicina, Caracas)n 2(IHEM-O16O, 2elgium)u was grown on 

.abouraud agar (J[ÅC,72h). Cells were suspended with 2_ formalinized saline solution, 

incubated (22ÅC,18h), centrifuged (1.0g,1[z). aashed J timesM suspended with saline solution, 

incubated ([6ÅC,J0]), centrifuged (1.0g,1[z). Preserved in 0.[_ formalinized saline solution 

(10ÅC when in usen |20ÅC prolonged storage). The yeast suspensions were tested for viability. 

Twelve healthy JBg male New Zealand rabbits were immunized with previous antigenic solutionsM 

O rabbits received either 10 daily injections in the marginal ear vein, with J ml inoculum of the 

suspension of Billed cells adjusted to 1McFarland, O rabbits received the same inoculumrFreud]s 

adyuvant (1M1)n they received weeBly O times a subcutaneous injection on the bacB, and the third 

group of rabbits, hcontrol groupi, did not receive any inoculation. fne month after the last 

vaccination, the same immunization scheme was repeated and one weeB after the last injection, 

sedation was applied and [ml rabbitïs sera were obtained by cardiac puncture (following 

international policies and regulations animal care and use of laboratory animals, Animal Care 

and 6se Committees 1K8[-1KKK). The adsorption experiments were done according to 

IBeda,1K82. .lide agglutination was performed (2M1) of a suspension of cells (8McF, 120rpm, 1[]). 

All results were recorded by a blind observer. 

Results: ff the 1J2 isolates, [K.8_ were*7/ ,.%U%$)+,& var @$(8""3*serotype An J1.1_ 7/ 

,.%U%$)+,& var ,.%U%$)+,& (2[.8_ serotype D, [.J_ serotype AD)n and K.1_ 7/ ,.%U%$)+,& 

var @+--"" (J.8_ serotype 2, [.J_ serotype C). 

Discussion: Although Venezuela is a subtropical area, endemic for 7/*,.%U%$)+,&3 the 

prevalence of var @+--"" has not been confirmedn only one publication reported the serotypes of 27 

clinical isolates (6J_ serotype A, 2K_*2, 0_ C, O_ D, and O*_*not typable). 7/*,.%U%$)+,& var 

@$(8"", serotype A, represents the most frequent isolate, as reported in other countries. Also K.1_ 

were 7/*,.%U%$)+,&*var @+--"" which typically affects apparently healthy hosts, 8.1_ of the J7 

isolates with Bnown immunological status of the patient in this study were from 

immunocompromised patients, disappointingly for the remaining patients, the immunological 

condition remained unBnown, and may perhaps influenced the incidence of this variety. The low 

socioeconomic condition, malnutrition and other factors may also play a role as risB factors for 

infection, and should also be considered in upcoming epidemiological studies. 6nfortunately, the 

CryptochecB ' Bit is not produced anymore, maBing serotypication only available for few 

authorized laboratories with proper conditions to accomplish animal experiments. 

This project was partly financed byM CDCHMPI 0K-00[J8O-200O.

P-0478. Distribution of the different species Candida and their antifungal 

susceptibility profile, in the metropolitan Caracas zone (2003-2005) 

Dolande M 1,2 

1 Instituto Nac. de Higiene Rafael Rangel, 2 ClÜnica .anta .ofÜa 

Introduction: The increase in the frecuency of invasive fungal infections (IFI), has been 

observed, mainly among patients in the intensive care unit that receiving antibiotics, 

immunosuppressive therapy, or parenteral nutritions, as well as among patients exposed to 

invasive medical procedures such as intravascular catheter, hemodialysis, and abdominal 

surgery (1). 7+,5"5+ species are the most common opportunistic fungi causing IFI. 7+,5"5+* 

+68"#+,& is considered the most frecuently isolated species of candidemic patients, the 

emergence of non-+68"#+,&*7+,5"5+*is clearly a concern. The resistance of non-7/+68"#+,& 

isolates to currently available antifungal drugs represents a major challenge for empirical 

therapeutic and prophylactic strategies (1,2). 

ObjectiveM The aim of retrospective revision was to Bnow the distribution of the different species 

7+,5"5+ and their antifungal susceptibility profile, isolated of different clinical specimens, coming 

patients that stay in the hospital and 6CI in 6 Hospital of Metropolitan Caracas zone, during the 

last three years (200J-200[). 

Materials and Methods: Is analyzed in the last three years the laboratory reports, the isolation 

frecuency of species 7+,5"5+ and also antifungal susceptibility profile to drugs of sistemic use. 

The identification is performed for conventional method (macro and microscopic morphology on 

cornmeal agar, clamidospores production, carbohydrate assimilation test, cicloheximide 

resistance and growth several temperatures) and the commercial system viteB-2 Y2C - 

bioMdrieux. Antifungal .usceptibility tests is analyzed for AT2-fungus and E-TE.T, for 

methodologies both is performed according to manufacturer rules. 

Results: ff 1K81 identified yeasts, the distribution of species obtained was 7/*+68"#+,& (O6,6_), 

7/*-$%'"#+6"&*(18,K_), 7/*@6+8$+-+ (K,1_), 7/*'+$+'&"6%&"&*(6,0 _), 7/[$(&."*(2.7_), 7/*6(&"-+,"+.* 

(1_), 7/*@("66".$)%,5"" (0,[_), 7/*[.U2$ (0.O_), 7/,.%U%$)+,& (0,2_), 7/*U+)+-+ (0,2_) and 

(0,1_) for 7/*d.26+,%"5.&3*7/*$(@%&+*+,5*7/*",-.$).5"+. 1O,2_ species 7+,5"5+ are not 

identified. The susceptibility to antifungal agents of the all species 7+,5"5+ up mencionate were 

susceptible as followsM 1117 to fluconazol, 106[ to itraconazol, 1J72 to anfotericina 2, K10 to 

fluocitosina and 128 to voriconazol. 

Conclusions: 7/*+68"#+,& was yeast the most frecuently isolated and the most yeasts were 

anfotericin 2 susceptible. These results provide baseline data for future epidemiological research 

about invasive fungal infections that will supply more interesting, important and necessary 

informations of risB factors in the patients for to develop to invasive mycosis. Is the first study 

where it is doing revision in Venezuela that participate 6 important Hospitals with numbers high 

patients. 

P-0479. Molecular typing of Aspergillus fumigatus isolates based on AFLP 

Duarte-Escalante E 1 , HernZndez-RamÜrez A 1 , Davel G 2 , Cárdoba . 2 , Arenas R J , 

Reyes-Montes M 1 

1 Depto. MicrobiologÜa y ParasitologÜa, Facultad de Medicina, 6NAM, Mexico, 2 Instituto 

Nacional de Enfermedades Infecciosas, Carlos G. MalbrZn, Argentina, J Hospital 

General Manuel Gea GonzZlez, Mexico 

4&'.$@"66(&*U()"@+-(&*(4U) is a saprophytic fungus, its natural ecological niche is the 

soil, where it survives and grows on organic debris. It sporulates abundantly, with 

every conidial head producing thousands of conidia. Inhalation of conidia by 

immunocompetent individuals rarely has any adverse effect, since the conidia are 

eliminated relatively efficiently by innate immune mechanisms. In 

immunocompromised individuals, however, it can cause invasive aspergillosis (IA), 

often with fatal consequences. Due to the increase in the number of patients 

undergoing bone marrow or solid organ transplantation, the incidence of IA has 

increased dramatically in recent years. In Mexico, unfortunately, the information 

regarding epidemiological and molecular studies is scarce or almost nulln hence, little 

is Bnown about the number of cases, infection sources, and the relationship with the 

clinical forms, neither are there studies that would allow understanding the great 

variability of this organism. However, unpublished data (pers comm) from the National 

Institute of Cancerology, Mexico, suggest that 4U is an important pathogen according 

to the number of cases (two per month). In this worB we studied the genetic diversity 

of 4U isolates collected in different countries (Mexico and Argentina) and obtained from 

different sources (clinical and environmental isolates), using Amplified Fragment 

/ength Polymorphism (AF/P) with eight combinations of oligonucleotide pairs with two 

or three selective bases (AA and M-CTCn E-AA and M-CATn E-AA and M-CACn E-AA 

and M-CTGn E-AC and M-CTCn E-AC and M-CATn E-AC and M-CACn E-C and M- 

CTG) to obtain polymorphism patterns. The results were analyzed through 6PGMA 

analysis with the Dice coefficient from AF/P profiles obtained for 4U isolates. The 

results suggest that the 4U isolates can be divided into two clusters (group I and group 

II) with reference to their geographical origin. 2oth groups presented heterogeneous 

profiles suggesting high intraspecific genetic diversity. AF/P finger-printing, has 

proven to be a generic fingerprinting method with intrinsic superior reproducibility and 

a high degree of discriminatory power compared to other methods. Project supported 

by grant Number IN22O706-J from Direccián General de Apoyo al Personal 

Acaddmico (DGAPA-6NAM). 

References: 

.ilva V, DÜaz M, Febrd N, et al. Invasive fungal infections in ChileM a multicenter study of fungal 

prevalence and susceptibility during a 1-year period. Medical Mycology. 200On O2M JJJ-JJK. 

Godoy P, Tiraboschi I, .evero /, 2ustamante 2, Calvo 2, Almeida /, Da Matta D, Colombo A. 

.pecies Distribution and antifungal .usceptibility Profile of 7+,5"5+*&''/ 2loodstream Isolates 

from /atin American Hospitals. Mem Inst. fswaldo Cruz. Rio de (aneiro. 200Jn K8(J)M O01-O0[. 

PemZn (, Cantán E, Gobernado M, et al. Epidemiology and antifungal susceptibility of 7+,5"5+ 

species isolated from bloodM results of 2-year multicentre study in .pain. Eur ( Clin Microbiol 

Infect Dis. 200[n 2OM 2J-J0. 


P-0480. Typing of Candida parapsilosis isolates by FT-IR spectroscopy 

Essendoubi M 1 , Toubas D 2 , Manfait M 1 , Pinon ( 2 , .ocBalingum G 1 

1 6nitd MdDIAN, CNR. 6MR 61O2, 6FR de Pharmacie, IFR[J, 6niversitd de Reims 

Champagne-Ardenne, France, 2 /aboratoire de Parasitologie-Mycologie, EA J800, 

HÇpital Maison 2lanche, CH6 de Reims, France 

Recent advances in the use of molecular techniques have permitted the development 

of typing methods with greater sensitivity, allowing a clearer understanding of the 

epidemiology of 7+,5"5+ colonization and infection. However, these techniques are 

not straightforward since they require time, expensive consumables, and a highly 

trained staff to be performed adequately. Fourier transform infrared (FTIR) 

spectroscopy is a different approach to the development of identification and typing 

methods which is based on interaction between light and matter. FTIR spectra 

constitute highly specific spectroscopic fingerprints of microorganisms via which they 

can be identified. 

ae have previously shown the potential of FTIR spectroscopy not only for rapid 

identification of 7+,5"5+ species but also for typing of 7/ +68"#+,&*and*7/*@6+8$+-+ 

strains. 7+,5"5+*'+$+'&"6%&"& is another species for which epidemiological 

investigation is of particular interest. This species is more and more implicated in 

hospital outbreaBs. Due to the high success rate that was obtained in typing 7/* 

+68"#+,& and 7/*@6+8$+-+, our objective in this study is to evaluate the potential of the 

technique as a typing method of clinically relevant strains of 7/*'+$+'&"6%&"&. 

ae have therefore recorded spectra from 28 strains of 7/ '+$+'&"6%&"& isolated from K 

patients. Half of the strains were used for the calibration phase where the most 

discriminant spectral regions were looBed for. 6sing second derivative of the spectra, 

we identified J spectral windows with discriminating capacity in the polysaccharide 

regionM 10K0-1110, 1120-11O0, and 1170-11K0 cm -1 . Hierarchical classification carried 

out using these spectral windows allowed to obtain O different clusters, each cluster 

corresponding to a different patient. Thus a classification rate of 100_ was obtained. 

Three independent cultures were investigated for reproducibility of these results. The 

other half of the study was then used for validating these spectral regions. fur 

classification results show [ distinct groups corresponding to each patient and again a 

rate of 100_ was achieved. 

This approach represents analytical, non-destructive, and dynamic method to 

investigate a population of whole cells with only little biomass. Thus, in an 

epidemiological investigation, a single measurement can allow to compare very 

rapidly different 7/*'+$+'&"6%&"& strains isolated from blood stream, vascular devices or 

staff hands. 

P-0481. The domestic dog as epidemiological clue in natural habitat of 

Paracoccidioides brasiliensis 

Fernandes Fontana F 1 , 2arbosa dos .antos C 1 , Araâjo Resende / 1 , Furlan Matos 

Aires a 1 , Gomes Päres H 1 , Domingues M 2 , Rocha A J , Silva-Vergara M 1 

1 6niversidade Federal Tri°ngulo Mineiro, 2 6beraba Municipal Zoonosis Center, 

J 6niversidade Federal de 6berl°ndia 

Paracoccidioidomycosis is one of the most prevalent systemic mycosis in /atin 

America and its etiological agent is 1+$+#%##"5"%"5.&*8$+&"6".,&"&, a dimorphic fungus 

rarely isolated from soil and recently cultured from armadillo viscera in different 

places. This microrganism can infect several domestic and wild mammals but in any 

of them the disease has been characterized. Domestic dog also can be infected and 

two isolated reports showed that it can develop paracoccidioidomycosis and it would 

be an important linB in the epidemiology context of this fungus. The aim of this worB 

was to attempt to isolate and/or identify 1/*8$+&"6".,&"& from dogs viscera. 

Methods: 2razilian Zoonosis Control Program includes all the diseases where 

domestic dog can potentially participate as their reservoir. In each municipality, 

donated and street dogs are Beep on captives and if their owners don]t asB for them 

are sacrificied. TaBing advantage for this situation in 6beraba, MG, 2razil, OK dogs 

were autopsied and their lungs, liver, and spleen removed under aseptic conditions as 

possible. .mall fragments of these viscera were put on 10_ formalin solution for 

histopathologic evaluation. The most part of each viscera was in sterile conditions 

mechanically homogenized in isotonic saline solution 100ml containing benzilpenicillin 

(2006M/ - 1) and gentamicin O8mg M/ - 1. This suspension was cultivated in 

mycobiotic agar (Difco, Detroit, MI, 6.A) and Fava-Netto media (five tubes for each 

viscera and media). They were incubated at room temperature 7 28oC and at J[oC. 

Cultures were weeBly observed during three months. 

Results: .everal hundred cultures from dog viscera were performed and carefully 

observed during 12 weeBs. No fungal structures with morphology similar to 1/* 

8$+&"6".,&"& were observed among other dimorphic saprophytic and pathogenic fungi 

recovered. Three histoplasma strains from two dogs were recovered, one from spleen 

and lung and other from the lung. A 7?$2&%&'%$"()*'+$0()*0+$*#$.&#.,& strain also 

was recovered from a lung from other dog. Histopathological evaluation of hundred 

dogs] viscera slides stained with HcE and Grocott didn]t permit to see fungal 

structures liBe this microrganism. 

Comments: In spite of the great number of dogs] viscera cultures and slides 

performed, the results were negative to either isolate or to see 1/*8$+&"6".,&"&, 

confirming the elusive nature of this fungus for growing from soil, water and mammals 

viscera etc. Recently, two separated case reports showed paracoccidioidomycosis in 

two Doberman dogs and, experimentally, puppies were infected with this fungus and 

some of them developed the disease. These facts might suggest a dog as a new host 

for this fungus. As observed in seroepidemiological surveys, this mammal frequently 

acquires the infection but probably it will rarely develop the disease. Thus, its role as 

epidemiological linB to elucidate the ecological niche of 1/*8$+&"6".,&"& is still unBnown, 

but its closeness with humans could help to identify the place where both acquire the 

infection.

P-0482. Seroepidemiological survey by ELISA anti GP43 to evaluate canine 

paracoccidioidomycosis infection 

Fernandes Fontana F 1 , 2arbosa dos .antos C 1 , Araâjo Resende / 1 , Furlan Matos Aires a 1 , 

Gomes Päres H 1 , Domingues M 2 , Priscila Garcia I J , C°ndida do Amaral C J , Pires de Camargo Z J , 

Silva-Vergara M 1 

1 

Infectious Diseases Department - 6niversidade Federal do Tri°ngulo Mineiro, 6beraba, 2razil, 

2 

6beraba Municipal Zoonosis Center, J Celular 2iology Department - 6niversidade Federal de 

.Éo Paulo, 6beraba, 2razil 

Introduction: .everal epidemiological surveys have been carried out to investigate 

Paracoccidiodes brasilienses infection in domestic and wild mammals. The results have showed 

different percents of reactivity to intradermal tests using fungal antigens and variable prevalence 

rate of antibodies to P. brasilienses by E/I.A tests. At present there is an increasing interest to 

study the role of domestic dog in the epidemiology of this fungus due to the finding of two 

Doberman dogs that developed paracoccidioidomycosis, and the high reactivity founded in a 

canine survey recently published. The aim of this report is to show a preliminary results of a 

canine seroepidemiological survey carried out to investigate anti gpOJ antigen of P. brasiliensis 

in an endemic area in 2razil. 

Methods: The 2razilian Zoonosis program central includes all the diseases were domestic dogs 

can potentially participate as their reservoirs. In each municipality, donated and street dogs are 

Beep on captives and if their owners don]t asB for them are sacrificed. TaBing advantage for this, 

in 6beraba, MG 2razil, 1OK urban dogs of different race and conditions and 100 rural dogs as 

control, were included. Previous anesthesia with Betamina, [ ml of blood were obtained by 

venous punction of each animal and sera were stored at -22oc. After this, specific antibodies 

against GPOJ antigen of P. brasilienses were evaluated by an enzyme-linBed immunosorbent 

assay E/I.A according to fNf et al 2001. .erum from a Dog immunized with P. brasiliensis 

was used as positive control. A pool of sera from urban dogs that had show low absorbance in a 

previous test was used as negative control. .era with twice or more the absorbance of the 

negative control were considered positive (optic density % 0.08). The data were analyzed by 

student T test. The difference was considered significant when p v 0.0[. 

P-0483. Epidemiological study of Trichophyton tonsurans, a causative agent of 

tinea captis, based on the variable internal repeat region within the intergenic 

spacer of rdna locus 

Fukushima K 1 , TaBizawa l 1 , Abli P 2 , .ouza Motta C J , ji / O , Vidotto V [ 

1 Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba 6niversity, 

2 The First Hospital of jinjiang Medical 6niversity, China, J 2iological .cience Center, 

Federal 6niversity of Pernambuco, 2razil, O The .econd Affiliated Hospital of .un Yat- 

.en 6niversity, China., [ /aboratory of Medical Mycology, 6niversity of Turin, Italy 

An epidemiological survey revealed that >$"#?%'?2-%,*-%,&($+,& has become a 

significant etiological agent of tinea captis in 6.A, Europe and also in (apan. This 

paper presents epidemiological findings on the isolates from (apan, 6...A., China, 

2razil, Taiwan, and Italy based on the variable internal repeat (VIR) region within the 

intergenic spacer of rDNA. The six new VIR variants which differed from five those 

reported by A. GaedigB, were identified through this study. 2y using the 11 VIR 

variants, strain subtyping of the isolates from the countries was carried out and gave 

epidemiologically informative resultsM the strain which recently causes tinea captis in 

(apan was identical as an American subtypen all isolates from China were verified to 

possess new strain subtypes differed from those of American isolatesn and of 2razilian 

isolates, two new subtypes were found in the isolates, and so on. The VIR analysis is 

considered as the most accurate method that can facilitate strain discrimination in 

population-based studies. 

Results: Hundred forty nine urban dogs were included in this survey. Their main characteristics 

wereM 7[ ([0,J_) female, 1J6 (K1,2_) adults, 7O (OK,6_) small size 1JO (K0_) undefined race, 

76 ([1_), eutrophics, KJ (6O_) donated dogs and [6 (J6_) street dogs. 

The reactivity against GPOJ antigen of P. brasiliensis when tested by E/I.A titer 1M 100, showed 

22 (2K,7_) male and 2J (J0,6_) female dogs positives. No significant difference was observed 

among male and female dogs. Neither donated or street dogs. .era of rural dogs control still did 

not perform. 

Comments: The preliminary results of this survey are according to other study carried out in 

2razil that showed reactivy against GPOJ antigen by E/I.A in 1J,6_ male and 1[,6_ female 

dogs from urban area. High antibody prevalence rates were observed among rural dogs in the 

same study. The habits as digging and/or sniffing are common in dogs and this increases the risB 

to inhalate infections propagules from P. brasiliensis and can explain the high reactivity to P. 

brasiliensis antigens detected in this mammal. The other hand cross-reaction can be favored by 

the high sensitivity by E/I.A test to detect minimal amount of antibodies. It occurs mainly with 

Histoplasma capsulatum antigens base on galactose. To avoid or decrees this condition, 

galactose was added in sera to eliminate antibodies against this epitope. Although, the habitat of 

this fungus still did not full solve, probably dogs and humans can acquire P. brasiliensis infection 

in the same place and for this, dogs maybe an epidemiological clue to find the elusive ecological 

niche of this fungus. 


P-0484. Preliminary results on environmental influences in Malassezia isolation 

rates and M. furfur molecular types 

Gaitanis G 1 , VelegraBi A 1 , Alexopoulos E 1 , Faergemann ( 2 

1 Mycology /aboratory, Microbiology Department, Athens Medical .chool, 

2 Department of Dermatology, .ahlgrensBa 6niversity Hospital, .E Goteborg, .weden 

Epidemiological studies demonstrate geographical variations in the isolation rates of 

!+6+&&.d"+ species. !/*@6%8%&+*is the predominant species isolated in the .outh of Europe 

while !/*&2)'%5"+6"& is isolated at higher rates in the North of Europe. There are no reports 

of !/*%8-(&+ strains isolated in .outhern Europe. 

Aims: The aim of this study was to assess the isolation rates of !+6+&&.d"+ species from 

healthy .candinavian volunteers residing permanently in Greece and to compare the 

molecular subtypes of the isolated strains with Type, reference and GreeB strains. 

Materials and Methods: A total of J6 .candinavian (86.1_ women) and 17 GreeB (82.O_ 

women) volunteers were included in the study. .ampling was performed with a sterile swab 

moistened in P2., which was rubbed vigorously for J0 seconds on the forehead and 

midscapula in an area delineated by a O cm diameter ring. .pecimens were inoculated in 

Dixon]s agar followed by 1O days incubation at J2 o C. 

!/*U($U($ isolated strains were compared to the Type !/*U($U($*C2. 1878 and J1 reference 

strains originating from Greece and other parts of the globe by PCR fingerprinting 

employing a yeast 26. rDNA derived primer and the M1J primer. 

Chi square was used for statistical analysis of the epidemiological data while clustering 

analysis of the PCR fingerprints was performed with the 2ionumerics gel analysis software 

(Applied Maths, lorttijB, 2elgium). 6PGMA analysis was employed for qualitative 

evaluation of the bands amplified. 

Results: Eighteen positive samples from the .wedish volunteers (!/*&2)'%5"+6"&M 1O, !/* 

U($U($M O, !/*@6%8%&+M 2, !/*&6%%UU"+.M 1, !/*2+)+-%.,&"&M 1) were compared against 7 

positive samples from the GreeB volunteers (!/*&2)'%5"+6"&M J, !/*U($U($M 1, !/*@6%8%&+M J). 

.tatistical analysis demonstrated increased prevalence trends for !/*@6%8%&+ on the GreeB 

population, while the .candinavian population exhibited increased prevalence for !/* 

&2)'%5"+6"&, observations that did reach borderline statistical significance, despite the 

small sample size. No !/*%8-(&+ strains were isolated from the .candinavian population 

sample. A !/*2+)+-%.,&"& strain was co-isolated with !/*&2)'%5"+6"& and !/*@6%8%&+ from 

a male .wedish volunteer suffering from seborrheic dermatitis. However, the !/* 

2+)+-%.,&"& strain was overgrown by !/*&2)'%5"+6"&3 thus it was not possible to preserve 

in our collection. 

PCR fingerprinting demonstrated significant variations in the !/*U($U($ profiles. The 

.candinavian !/*U($U($ isolates displayed a trend to cluster in a distinct group compared to 

Type and reference strains and GreeB isolates. Different PCR-fingerprints were observed 

within two (2) !/*U($U($ strains isolated from the forehead and bacB of a .wedish individual. 

Conclusions: This is the first effort to appraise the effect of environmental factors on the 

epidemiology of !+6+&&.d"+ species. These preliminary epidemiological data demonstrate 

that the geographical variations on the reported isolation rates of !+6+&&.d"+ species are 

not environmentally determined. Moreover, !/U($U($ subtypes showed an apparent trend of 

distinct clustering among .wedish and GreeB hosts regardless the country of residence. 

P-0485. Detection of Candida dubliniensis in patients with candidosis in 

Caracas, Venezuela 

Gamboa A 1 , Mendoza M 2 , Fernandez A J , Diaz E 2 

1 Escuela de Medicina, 6niversidad Central de Venezuela, 2 /aboratorio de Micologia, 

Venezuela, J /aboratorio de Ingenieria Genetica, Instituto de 2iomedicina, Caracas, 

Venezuela 

Candidosis is an acute or chronic infection produced by fungi of the 7+,5"5+*genus, 

generally limited to sBin and mucous membranes however, it could produce also a 

serious systemic disease. Yeasts of the 7+,5"5+*species are responsible of 80_ of 

all nosocomial fungal infections, being 7+,5"5+*+68"#+,& the yeast found in the 

majority of the cases. .ince 1KK[ a new yeast species is described, 7/*5(86",".,&"& 

which shares with 7/*+68"#+,& morphological and physiological characteristics. These 

two species have phenotypic characteristics that permit the differentiation and enable 

presumptive discrimination between them. Among the criteria used to differentiate 

these two species we haveM 7/*5(86",".,&"&*does not grow at O2oC however, some 

authors have found that some isolates are able to grow at that temperature. Also, in 

the chromogenic differential growth medium CHRfMagar-7+,5"5+, 7/*5(86",".,&"& 

produces darB green colored colonies while those of 7/*+68"#+,& are of a light green 

color. It has been described also that 7/*5(86",".,&"&*can produce a great amount of 

chlamydospores arranged in continuous pairs, triplets and occasionally in aggregates, 

aspects which are not observed in 7/*+68"#+,&/**The enzymatic !-glucosidase activity 

has been also used to discriminate between the two species. ahile 7/*5(86",".,&"&* 

does not present this activity, 7/*+68"#+,&*#%(65*._'$.&&*"-/* 7/*5(86",".,&"&*isolates 

agglutinate with the antigenic factor 6 antisera of 7/*+68"#+,&, corresponding to 

serotype A. Those isolates showing differences in their biochemical characteristics 

should be tested by molecular biology for genotypic identification. .ome authors 

describe that about 17_ of isolates identified as 7/*+68"#+,&, would really be 7/* 

5(86",".,&"&. The objective of this study was to detect, among the clinical yeasts 

isolates previously identified as 7/*+68"#+,&, the presence of 7+,5"5+*5(86",".,&"&*by 

means of conventional microbiological tests and molecular biology (PCR). The 

following procedures were carried outM production of germ tubes, growth at O2-O[oC, 

sugar assimilation, chlamydospore formation, the 4!-glucosidase test, and growth in 

CHRfMagar-7+,5"5+/** Conventional microbiological studies were positive for K0 to 

K8_ of isolates. The DNA extracted from 80 isolates were examined by the PCR 

technique using specific primers for 7/*5(86",".,&"& D62R/D62F. ff all the DNA 

examined 2.[_ showed a product of 288 pb that corresponds to 7/*5(86",".,&"&/**fne 

of the positive isolates came from the mouth of an undernourished child and the other 

from a vaginal secretion. In this study, only one of the isolates identified as 7/* 

5(86",".,&"&*(C. albicans AND[) had the same behavior as control 7/*5(86",".,&"& 

(CdJ6) of reference. ae conclude that among the clinical isolates obtained from 

several health facilities of the Metropolitan area of Caracas previously identified as 7/* 

+68"#+,&, we have found 7/*5(86",".,&"&*by means of molecular biology tests and 

therefore, we report the presence of this new 7+,5"5+*species in Venezuela.

P-0486. Epidemiology of dermatophytoses in a southern area of Madrid, Spain 

García-Martínez J, Alonso M, (aquetti (, Garcia I, Martinez C, Prieto . 

1 Hospital de Fuenlabrada, Fuenlabrada, Madrid, .pain, 2 Hospital de Fuenlabrada, 

Fuenlabrada, Madrid, .pain, J Hospital de Fuenlabrada, Fuenlabrada, Madrid, .pain, 

O Hospital de Fuenlabrada, Fuenlabrada, Madrid, .pain, [ Hospital de Fuenlabrada, 

Fuenlabrada, Madrid, .pain, 6 Hospital de Fuenlabrada, Fuenlabrada, Madrid, .pain 

Introduction: Dermatophytoses are a major health problem in many clinical settings. 

The aim of this study was to gain insight into the epidemiological and aetiological 

features of superficial cutaneous mycoses in a .outhern area of Madrid. 

Materials and methods: Demographic and clinical characteristics of culture-proven 

dermatophyte infections were retrospectively obtained for the period from (anuary 

200[ to (anuary 2006. 

Results: >$"#?%'?2-%,*$(8$() was the most frequent isolate (O6.6_) followed by 

>$"#?%'?2-%,*).,-+@$%'?2-.& (10.J_), >$"#?%'?2-%,*-%,&($+,& (8.6_), !"#$%&'%$()* 

#+,"& (6.K_), >$"#?%'?2-%,*0"%6+#.()*([.2_), >$"#?%'?2-%,*0.$$(#%&()*(J.[_) and 

!"#$%&'%$()*+(5%(",""*(1.7_). >",.+*#%$'%$"&*(JK.7_) and onychomycosis (2[.K_) 

were the most frequent clinical presentations, which were associated with increasing 

age. 

Conclusions: It is concluded that a re-emergence of some anthropophilic 

dermatophytes, especially >/*-%,&($+,& and >/*0"%6+#.(), are seen, probably as a 

result of the migration of people from sub.aharian and .outh American areas where 

they are endemic. 

P-0487. Incidence of Fusarium verticillioides and levels of fumonisins in corn 

from high and low risk areas for human oesophageal cancer in Iran 

Ghiasian S 1,2 , Maghsood A 2 , Yazdanpanah H J , .hephard G O , van der aesthuizen / O , 

Vismer H O , Rheeder ( O , Marasas a O 

1 Medical Parasitology and Mycology Department, .chool of Public Health, 6niversity 

of Medical .ciences and Health .ervices, Tehran, Iran., 2 Medical Parasitology and 

Mycology Department, .chool of Medicine, Hamedan 6niversity of Medical .ciences 

and Health .ervices, Hamedan, Iran., J Pharmacology and Toxicology Department, 

.chool of Pharmacy, .haheed 2eheshti 6niversity of Medical .ciences and Health 

.ervices, Tehran, Iran., O PRfMEC 6nit Medical Research Council, P f. 2ox 1K070, 

Tygerberg, .outh Africa, 6 PRfMEC 6nit Medical Research Council, P f. 2ox 1K070, 

Tygerberg 7[0[, .outh Africa, 7 PRfMEC 6nit Medical Research Council, P f. 2ox 

1K070, Tygerberg 7[0[, .outh Africa, 8 PRfMEC 6nit Medical Research Council, P f. 

2ox 1K070, Tygerberg 7[0[, .outh Africa 

The fumonisins are a group of allied mycotoxins produced mainly by ^(&+$"()* 

0.$-"#"66"%"5.&*(.acc.) and ^/*'$%6"U.$+-()*(Matsushima),*which infect corn crops 

worldwide. They have been statistically associated with a high incidence of 

oesophageal cancer (fC) in certain areas of .outh Africa and might play a role in the 

promotion of primary liver cancer in certain endemic areas of China. 

In this study, the corn samples intended for human and animal consumption in Iran in 

2000 were extensively collected from three provinces (Fars, lhuzestan and 

lermanshah) with a low incidence of human fC and from Mazandaran province, an 

endemic area for fC, in order to investigate the natural occurrence of ^/*0.$-"#"66"%"5.& 

and fumonisins (F2 1, F2 2, F2 J and J-.'"-F2 J). 

Fumonisins and ^/*0.$-"#"66"%"5.& were detected, respectively in [1_ and [6_ of corn 

samples collected from low risB areas for fC. All corn samples collected from 

Mazandaran were contaminated with*^/*0.$-"#"66"%"5.& and fumonisins. .ignificantly 

different ^/*0.$-"#"66"%"5.& contamination was observed in corn from low (O.1ó10 J 

colony forming units (cfu)/g) and high (O.28ó10 O cfu/g) rate fC areas (pv0.0001). No 

statistical differences (pt0.0[) were observed in the fumonisin levels of the corn 

samples from the three different provinces in the low fC rate areas. High levels of 

F2 1 (1.687|11.01[ kg/g), F2 2 (0.J8J|J.J6O kg/g), F2 J (0.0[2|0.K00 kg/g) and J-.'"- 

F2 J (v0.10|0.1K1 kg/g), as well as high incidence of ^/*0.$-"#"66"%"5.& ([K_)* 

contamination were found in corn from the high fC rate area. These levels were 

significantly higher than levels present in the samples from the low rate fC areas 

(pv0.0001). People and animals in the high incidence area were exposed to higher 

mean concentrations of fumonisin 2 in corn than those in the low incidence areas in 

Iran. 


P-0489. Multicenter study of the frequency of Candida dubliniensis isolation in 

six French university hospitals 

Grenouillet F 1 , Cassaing . 2 , Datry A J , Michel-Nguyen A O,[ , aaller ( 6 , 2erry A 2 , 

/mimouni 2 J , .endid 2 7 

1 CH6, 2esancon, 2 CH6 Rangueil, Toulouse, J CH6 Pitid-.alpütrimre, Marseille, O CH6 

Timone, Marseille, [ CH .aint-(oseph, Marseille, 6 Institut de Parasitologie, .trasbourg, 

7 CHR6, /ille 

Background: Ten years after the first description of 7+,5"5+*5(86",".,&"& (1), 

distinction with its closely related species 7+,5"5+*+68"#+,& is often restricted to 

limited studies and its identification rarely done in routine laboratory. 

Despite the effort that has been expended to identify 7/*5(86",".,&"& in clinical 

samples, an exhaustive assessment of the prevalence of this species is still lacBing. 

Recent availability of a latex agglutination based test (2ichro-dubli Fumouze w n 

Fumouze Diagnostics) allows the evaluation of real prevalence of 7/*5(86",".,&"& 

among clinical yeast isolates. 

Methods: Differential diagnosis between 7/*5(86",".,&"& and 7/*+68"#+,& was 

performed on clinical isolates in different participating laboratories during several 

months throughout 200[. After conventional diagnosis of 7/*+68"#+,&/7/*5(86",".,&"& 

complex by its phenotypic features (morphology of colonies on chomogenic medium, 

germ tube and latex agglutination tests), 7/*5(86",".,&"& strains were first identified by 

latex agglutination test (2ichro-dubli Fumouze w n Fumouze Diagnostics), then 

confirmed at once by two PCR-methods (group I intron in 28. rDNA gene, ACT1 

gene) and genotyped by IT.1-[,8.-IT.2 sequencing. K,*0"-$% activity of fluconazole 

and amphotericin 2 against the collected 7/*5(86",".,&"& strains were determined by 

using the Etest system (A2 2iodisB, .olna, .weden) according to the manufacturer]s 

instructions. 

P-0490. Appendicitis and mycotic contamination 

Hashemi J 1 , Nasrollahi A 2 , Mohamadi G J 


Islamic Azad 6niversity, J /orestan 6niversity 

ahile nowadays, great attainments have been achieved in curing and preventing the 

pathogenic fungal infections, and some how there has been reduction in the number 

of occurrences, the occurrences of opportunistic infections have been increased. 

.ince the study of fungal infections in various organs se.g. digestive systemu is 

crucial ,and because of few study were done in this field in the world, it is decided to 

examine the appendectomies tissue for fungal contamination in Iran. 

The worB has been done for six months. After operation surgery the appendix tissue in 

two media sFormalin c normal salineu were carried out in the medical mycology 

laboratory at Tehran 6niversity of medical sciences. The specimens were examined 

directly and cultured in sabourauds dextrose agar with chloramphenicol sscu.In this 

experiment 200 appendicle tissues were examined. 

fut of them some fungi were isolated in 10 cases included O Candida albican sO0_u , 

2 Candida tropicalis s20_u ,1 Cryptococcus sp. s10_u,1 Candida sp.and 2 Geotrichum 

sp.Cryptococcus sp. was identified with mycological methods. This isolation related to 

a young man that has a history for long contact to pigeon. .ome of the fungi specially 

yeast can be a part of mycoflora in digestive system but the finding of Cryptococcus is 

uncommon. In this study the fungi were isolated from [_ of appendicitis and with pay 

attention to this finding that the most patients hadn]t bacBground factors causing the 

proliferation of the fungal agents in the intestine, so with further studies it is probable 

to consider the fungi as the agents causing appendicitis in these patients. 

ResultsM A total of 272[ isolates of 7/*+68"#+,&/7/*5(86",".,&"& complex were collected. 

.eventy 7/*5(86",".,&"& isolates were identified by latex agglutination test and 

confirmed by molecular methods, corresponding to a frequency of 2,6_ (ranging from 

1,[ to O,K_, according to different centers). Among 62 patients colonized by 7/* 

5(86",".,&"&, 1O were HIV-positive and 17 cystic fibrosis patients. K,*0"-$% testing 

revealed highly susceptibilties to antifungals (respectively MIC [0 } 0,12[ and MIC K0 } 

0,J8 kg/m/ for fluconazole, MIC [0 } 0,0J2 and MIC K0 } 0,1K kg/m/ for amphotericin 

2). 

Discussion: /atex agglutination appears as an easy-to-use and efficient phenotypic 

method for 7/*5(86",".,&"& identification. 7/*5(86",".,&"& appears to be rare in clinical 

yeast isolates. However, its differentiation from 7/*+68"#+,& seems to be necessary for 

clinical and epidemiological reasons. This study allowed us to confirm the respiratory 

tract of the patients with cystic fibrosis as an ecological niche for 7/*5(86",".,&"&. 

Reference: 

.ullivan .-*+6. Microbiology. 1KK[n1O1M1[07-21.

P-0491. Occurrence of Malassezia pachydermatis in the external ear canals of 

dogs in Iran 

Hashemi J 1 , Nasrollahi A 2 , Madani M 1 


Islamic Azad 6niversity 

Malassezia pachydermatis (M.pachydermatis) is Bnown as an opportunistic pathogen 

of the outer ear duct in dogs and cats. This yeast can also be found in the sBin, anal 

sacBs, vagina, and rectum. fur worB aims at showing the presence of 

M.pachydermatis in pet-dogs in Iran. The microbiotas of the external ear canals of 100 

pet-dogs ([Omale andO6 female) were studied. The dogs were taBen to a special 

domestic animal clinic in Tehran. ae submitted the auditory tube samples of these 

dogs to direct microscopic examination and cultured them in three different mediums. 

fur selected mediums were, sabouraud dextrose agar (.DA), .DA supplemented 

with 0.0[mg chloramphenicol and 0.[mg cycloheximide per ml (.CC), and corn meal 

agar (CMA). M.pachydermatis was isolated form six dogs. ae obtained Candida 

albicans form one dog. In our study, we observed no significant relation between the 

presence of M.pachydermatis in healthy pet-dogs, with factors such as breed, sex, 

age, and ear canal size. 

P-0492. The survey of money in patients with dermatophytosis for mycotic 

contamination 

Hashemi J, Nasrollahi A, RoBoei F 

.cience c Research 2ranch-Islamic Azad 6niversity, Tehran, Iran 

In this survey, we examined fungal contamination of Iranian coin and paper money 

obtained from patients with tinea. A total of 600 paper and 100 coins were examined. 

fut of them O00 samples were obtained from patients, 100 samples were from 

general population, and 100 samples were new un-touched bills. 

The paper bills were cultured on .cc medium (1 gr/lit) containing cyclohexamide 

according to strict aseptic conditions. 

fne scc plate was used for each bill. The coins were also cultured on the same .cc 

media by both direct contact as well as scoth tape method. 

A total of 6 dermatophytes (1.6_) were isolated from the O00 paper bills obtained from 

patients. fut of them 2 E. flocosum were Isolated that were also positive for the 

culture of this fungus from their groin. 

The patients from whose money, T.mentagrophytes were isolated, we isolated also 

this fungus from their necB, toe webs, hair, and the groin. 

The new untouched bills as well as the paper bills collected from the general 

population were devoid of dermatophytes and only saprophytes were isolated. 

The fungal agent isolated from coins was chrysosporium. The interesting point was 

that absolutely no dermatophyt was obtained from the coins which maybe due to the 

metal alloy used. 

Although this was the first research of this type being, it ought to be mentioned that 

further studies must be done on the survivability of the fungi present over the paper 

money. 


P-0493. A survey on the incidence of tinea gladiatorum in young wrestlers from 

Sari City, Iran 

Hedayati M, Afshar P, .hoBohi T, Aghili R 

Medical mycology and parasitology Dept., Mazandaran 6niversity of Medical .ciences, 

.chool of Medicine, .ari, Iran 

Background and Purpose: Tinea gladiatorum has Bnown as a common sBin disease 

in wrestlers. Although it is a fairly benign infection, it has significant effects on the 

ability of a wrestler to compete. .ince wrestling is a common sport in young people in 

Mazandaran Province, therefore for the first time we studied the incidence of tinea 

gladiatorum in wrestlers from .ari city and study of mattress for Dermatophytes 

contamination. 

Methods: ae examined JJ2 wrestlers (age range K-1K years) from 7 active clubs in 

.ari city the capital of Mazandaran a Northern Province of Iran and obtained sBin 

scrapings from 1J6 of suspected wrestlers to tinea gladiatorum. The scraped sBin 

samples were evaluated with potassium hydroxide examination. .abouraud]s 

dextrose agar with and without cholaramphinicole and cyclohexamide was used to 

culture of scrapings. The dermatophytes were identified by routine laboratory 

techniques. 

Results and Conclusion: fur study showed that K6 (1K.K_) wrestlers had tinea 

gladiatorum. The most lesions have been on the head and necB. Trichophyton 

tonsorans was only isolated species from all of scraping samples. Tinea gladiatorum 

can be common among wrestlers. 2ecause tinea gladiatorum can affect on ability of 

wrestler from competing in matches, quite surveillance and disinfection of mattress is 

very important to prevent infection among wrestlers. 

P-0494. Tinea capitis in Tunis: Increasing of microsporum canis 

Hicheri (, Eleuch D, Cherif F, MoBni M, /abidi H, Azaiz M, 2en fsman Dhahri A 

Dermatology Department, Rabta Hospital, Tunis, Tunisia 

Introduction: Tinea capitis is a superficial fungal infection with however a global change of the 

dermatophytic flora during the last years. Through our worB, we study the clinical and 

epidemiological profile of tinea capitis through a series of 1J0 cases. 

Patients and method: fur worB is a retrospective study carried out at the mycology unit in the 

dermatology department of the Rabta hospital dealing with the cases of tinea capitis during the 

years 200O and 200[ confirmed by a positive mycological examM only culture positive cases were 

considered valuable. For each patient, we have recorded the age, the sex, the geographic origin, 

the clinical presentation, the responsible dermatophyte and the evolution under treatment. 

Results: During the study period, 1J0 cases of tinea capitis were diagnosed. The patients ages 

raged from 2 to O0 years with a mean age of J,J years . The sex-ratio (M/F) was J,J and J0,7_ 

of our patients live in rural areas. 

Microsporic tinea capitis were due in all the cases to !"#$%&'%$()*#+,"&3*they*were observed in 

[6,K_ of cases. 

Trichophytic tinea capitis were observed in JO,6_ of cases. The isolated dermatophytes were 

represented by >$"#?%'?2-%,*0"%6+#.()*(87,8_), >/0.$$(#%&()*(7,J_) , >/).,-+@$%'?2-.&* 

(O,K_). 

The association to a circinate dermatophytosis was noticed in 2 cases (1,[_). 

The inflammatory type represented K,2_ of the diagnosed tinea capitis. It]s due to 

>/).,-+@$%'?2-.&*(J cases), !/*#+,"&*(2 cases),*>/0.$$(#%&()*(J cases) and >/*0"%6+#.()*(O 

cases). In our study, we have not observed any case of favus. 

All patients were treated initially with griseofulvinM 20 to 2[ mg /Bg/day during 6 weeBs. .ixty nine 

patients returned to be examined ([J,O_). In 7K,7_ of them, the lesions disappeared with a 

negative mycological examination after a treatment of six weeBs. In 17,O_ of cases (K/12 were 

to !/*#+,"&) the lesion cured after a treatment of 12 weeBs. In 2,K _ of patients a resistance 

griseofulvin was observed (2 cases to !/*#+,"&*). 

Conclusion: During the last years, in matter of tinea capitis, an increasing rate of infection by !/* 

#+,"&*has been observedM O1,7_ in a Tunisian study about 1222 cases from 1K8[ to 1KK8 and 

[6,K_ in the present study. This increasing rate of infection by !/*#+,"&*is probably due to 

modification in the way of life of the population with a more frequent cohabitation with domestic 

animals. 

>$"#?%'?2-%,*0"%6+#.() witch was the most prevalent dermatophyte responsible of tinea capitis 

during the last decades is regressing actually. In our study, it represents the second agent 

responsible of tinea capitis. 

In our series, a resistance of !/*#+,"&*-%*griseofulvin has been observed. In similar situations, we 

need to increase the posology and the duration of the treatment. 

References: 

D. El Euch and all. /es teignes du cuir chevelu observdes Ö Tunis de 1K8[ Ö 1KK8 M Ö propos de 

1222 cas ( Mycol Med 2001 n 11 M 87-K1. 

M. Develoux. Grisdofulvine. Ann Dermatol Venereol 2001n 128 M 1J17-2[.

P-0495. Environmental study of Cryptococcus neoformans in and around 

adana, Turkey 

Ilkit M, Ates A, Turac-2icer A, Yula E 

6niv. of CuBurova Fac. of Medicine 

Fungal diseases affecting humans generally originate from the environment. Due to 

the rise in human fungal infections in recent years, it is now extremely significant to 

Bnow more about ecology for combatting fungal pathogens. The aim of this study was 

to evaluate the presence of 7$2'-%#%##(&*,.%U%$)+,& in its well Bnown ecological 

niches in nine different locations, in ÆuBurova region, TurBey. For this purpose, a total 

of 18J[, 1[08 vegetable material from tree of the genus a(#+62'-(& trees, 11K pigeon 

droppings and 208 soil samples were examined for the presence of yeast in a .taib]s 

nigerseed medium. 7$2'-%#%##(&*spp. was not recovered from any of these samples. 

ae believe that in our region, there are elements affecting the life cycle of 

7$2'-%#%##(&*,.%U%$)+,&3 such as alBaline pH and high carbon ratio of the soil.*** 

P-0496. Mitochondrial DNA analysis of Sporothrix schenckii isolated from India, 

Thailand, Brazil, Guatemala, Colombia and Mexico 

Ishizaki H 1 , lawasaBi M 1, 2 , MochizuBi T 1, 2 , ChaBrabarti A J , 6ngpaBorn R O , Yamashita 

( [ , Zaitz C 6 , Guerrero H 7 , Toriello C 7 , Arenas R 8 

1 

Division of Dermatomycology (Novartis Farma), lanazawa Medical 6niversity, IshiBawa, (apan., 

2 

Department of Dermatology, lanazawa Medical 6niversity, IshiBawa, (apan., J Department of 

Medical Microbiology, PGIMER, Chandigarh, India., O Institute of Dermatology, 2angBoB, 

Thailand., [ Department of Dermatology, 6NIFE.P-EPM, .Éo Paulo, 2razil., 6 Division of 

Dermatology, .anta Casa Medical .chool, .Éo Paulo, 2razil., 7 Departamento de Microbiologia y 

Parasitologia, Facultad de Medicina, 6NAM, Mexico D.F. Mexico., 8 Hospital General ÄDr Manuel 

Gea GonzalezÄ Mexico D.F. Mexico. 

Introduction: ='%$%-?$"_*&#?.,#[""*has been classified into 2O mtDNA types (mtDNA Types 1- 

2O) based on restriction fragment length polymorphism (RF/P) in its mitochondrial DNA (mtDNA), 

a method reported to be useful for its molecular identification, typing and epidemiology. The 

mtDNA types have been phylogenetically clustered into two major groups, A and 2, and have 

been shown to be geographically related. Mora-Cabrera et*al. 1) recently reported that the mtDNA 

in isolates from Mexico, Guatemala and Colombia represented new types, which they designated 

Types 2[-J0. In the present study, we report the mtDNA typing of =/*&#?.,#[""*isolates including 

the isolates examined by Mora-Cabrera et al. 

Materials and Methods: The samples were 76 clinical isolates of =/*&#?.,#["" including 

designated Types 2[-J0 by Mora-Cabrera et al. ff them, O8 were from Mexico, O from 2razil, [ 

from Guatemala, K from Colombia, J from Thailand and 7 from India. mtDNA was extracted from 

each of the 76 isolates, and digested with A+.KKK, !&'K, A?+K and Z@6KK as described previously 2) . 

The digested products were electrophoresed on 2_ agarose gel, stained with ethidium bromide, 

and visualized under 6V light. The mtDNA types were determined by RF/P patterns with A+.III. 

A phylogenetic tree was constructed for the mtDNA based on their RF/P patterns. 

Results: The new mtDNA Types, Types J1 and J2 were also found. Phylogenetically, Types 2[, 

26, 28 and J1 belonged to Group A, while Types 27 and J2 belonged to Group 2. ff the O8 

isolates from Mexico, O1 including one that was Type J1, belonged to Group A, while 7 belonged 

to Group 2. Thirteen of the isolates were Type 2, 10 were Type J, and 7 were Type 28. ff the [ 

isolates from Guatemala, J belonged to Group A, one being Type J and 2 being Type 28, while 2 

belonged to Group 2, each one of Type 20 and Type 27. ff the K isolates from Colombia, [ 

belonged to Group A, one being Type 2, J Type J and one Type 2K, and O belonged to Group 2, 

one Type O and J Type 27. ff the O isolates from 2razil, J belonged to Group A, all being Type J 

and one to Group 2, being Type O. All J isolates from Thailand belonged to Group 2, two being 

Type O and one Type [. All 7 isolates from India belonged to Group 2, J being Type [, 2 Type 20, 

one Type O, and one Type J2. The isolate designated Type J0 by Mora-Cabrera et al. was 

confirmed to be Type J, and therefore Type J0 remains undefined. 

Conclusion: These results support previous report 2) that most isolates from Asia belong to 

Group 2, while Group A isolates are predominantly from .outh Africa, and North, Central, and 

.outh America. More isolates, especially from India and Thailand, will be analyzed in further 

studies. 

References: 

M Mora-Cabrera et al. Medical Mycology JKM OJK-OOO, 2001. 

H IshizaBi et al. (pn. (. Med. Mycol. O[M2J-2[, 200O. 


P-0497. Fusarium dimerum infection linked to a water fixture; Molecular 

comparison with geographically distinct isolates 

Kammeyer P 1 , Hammer A 2 , Yong . 1 , .chroers H J , .ummerbell R O , feDonnell l [ , 

(ohnson . 1 

1 

/oyola 6niversity Medical Center, Maywood, 6.A, 2 Childrenes Memorial Hospital, Chicago, 6.A, 

J 

Agricultural Institute of .lovenia, /jubljana, .lovenia, O Centraalbureau voor .chimmelcultures, 

6trecht, The Netherlands, [ 6nited .tates Department of Agriculture, Peoria, 6.A 

Purpose: To investigate a nosocomial ^(&+$"()*5").$()*(Fdm) infection, search for potential 

reservoirs of the infection within the hospital environment, and perform molecular analysis of 

recovered isolates comparing them to a worldwide collection of environmental and clinical Fdm 

isolates. 

Project Description: fne month after hospitalization and neutropenia induced by intensive 

chemotherapy for relapsed leuBemia, a 6[-year-old woman with fevers developed a scalp lesion 

that on biopsy showed invasive septate fungal hyphae involving the superficial and deep dermis 

and blood vessels. The lesion culture was positive for a member of the Fdm species complex. 

.equential environmental swab culture surveys for ^(&+$"()*included 1) the index hospital room 

including ceiling tiles, air vents, drains, sinB and shower spigots and 2) sinB and shower spigots 

from 11 other patient rooms on the same hospital ward. A third swab culture survey focused only 

on members of the Fdm species complex and included spigots and drains on the same floor as 

well as on floors above and below the index room that shared the same water supply riser. 

In addition, fungi were inventoried from water by taBing K00 m/ volumes from the index room 

sinB spigot and other sites as well as larger volumes filtered through a 0.2 km pore water filter 

(Pall-Aquasafe TM ) in-line with hot and cold water supplies to the index room, removal of the filter 

after 1 weeB of continuous flow, and subsequent microbiological sampling of the filter membrane. 

^(&+$"()*isolates were identified by morphological techniques and DNA sequencing of O 

phylogenetically informative loci (totalM ca J.J Bbp). 

Results and conclusions: Fdm isolates with identical DNA sequences were recovered from the 

index room sinB spigot and drain and from the patient]s scalp. Two months later and after 

replacement of the fixture, a Fdm strain was isolated from the new sinB drain of the index room. 

This strain differed from the earlier strains by J nucleotides. A similarly deviating Fdm liBe strain 

was isolated 1 month later from the sinB drain in another room on that ward which did not share 

the same water supply riser. The patient scalp isolate, sinB spigot and drain isolates were 

identical to a blood culture isolate from a patient in another 6. hospital and the subsequent sinB 

drain cultures matched clinical Fdm isolates from the 6. (wound), and Europe (blood and 

wound). 

^(&+$"()*was not recovered from any water samples. However, small numbers of 4&'.$@"66(&* 

U6+0(&3*probable 4#$.)%,"()*and X.#2-?%'?%$+*species were recovered from cultures of the 

water filter after continuous sampling of the index room cold water supply. 

P-0498. Cryptococcus neoformans variety grubii isolates from pigeon 

droppings in metropolitan area, Thailand 

laocharoen . 1 , PiyabongBarn D 1 , lammarnjassadaBul P 1 , Yumyourn P 2 , 

Chindamporn A 2 

1 Inter-department of Med Microbiology, ChulalongBorn 6niversity, 2angBoB, Thailand, 

2 Dept. of Microbiology, Faculty of Medicine, ChulalongBorn 6niversity, 2angBoB, 

Thailand 

Purpose of the study: To determine the epidemiology of 7$2'-%#%##(&**,.%U%$)+,&* 

isolates from pigeon droppings in Metropolitan area, Thailand 

Description: JJ6 Pigeon dropping samples from 21/[0 districts in Metropolitan, 

Thailand were collected during (une 200J-April 200O. At least 16 samples from 

different areas within the same district were taBen. 7/*,.%U%$)+,& were isolated and 

identified by their phenoloxidase and encapsulated properties. Their serotypes were 

determined by the reaction on concanavanine glycine bromthymolblue (CG2) medium. 

Then, genomic DNA from each strain was prepared and subjected to M1J polymerase 

chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (TP45) 

gene restriction fragment length polymorphism analysis with A?+I and =+(K6I in a 

double digestion for strain differentiation. Their mating types were also determined by 

PCR. 

Results & Conclusions: 2ased on the morphology and melanin pigment production, 

7/*,.%U%$)+,& from only dry droppings were isolated from J0 out of the JJ6 (10_) 

samples. These isolates were found in 10 out of 21 sampling districts ([0_) and fifty 

three isolates were randomly selected for the further analysis. 2y the conventional 

methods on CG2 medium, all isolates were identified as serotype A. The serotype 

result was verified by both molecular techniques. Regarding M1J fingerprinting, all 

isolates were classified as type VNI-C (7/*,.%U%$)+,& var. @$(8"" serotypeA) since the 

profiles were similar to type VNI. Compare to Type VNI (Meyer a. et al, 1KKK)ò, the 

variations were detected in the DNA size between 1,6J6 and 1,018 base pairs. Type 

VNI-C with mating-! of these strains were confirmed by TP4[-RF/P and mating 

study. However, analysis M1J-fingerpriniting profile was rather complicatedn it is 

needed to develop other techniques with instant of performing and analysis. In 

conclusion, the first data of the distribution of environmental 7/*,.%U%$)+,&*var. @$(8"" 

type VNI-C mating-! in the pigeon droppings in Thai-Metropolitan area were reported. 

This will be the important evidence to search for the source of human cryptococcosis 

so as animal cryptococcosis. 

òMeyer a., .-*+6/ Electrophoresis 1KKK, 20, 17K0 | 17KK. 

Invasive infection and contamination of the sinB spigot in the index patient room was documented 

for an uncommon ^(&+$"()*species (Fdm species complex). ae hypothesize the hospital water 

supply is intermittently contaminated by filamentous fungi and a biofilm developed in this 

particular fixture that subsequently led to infection in the patient.

P-0499. Neonatal sepsis caused by Candidia species 

Karam El-Din A 1 , Al .harif A 2 , Mahmoud E J , Abd El lader A O , El Tounisy E O 

1 Faculty of .cience, Ai-.hams 6niversity, Cairo, Egypt, 2 Faculty ff Pharmacy, Al- 

Azhar 6niversity, Cairo, Egypt, J Al-Azar 6niversity Hospital, Cairo, Egypt, O Faculty ff 

Medicine , Al-Azhar 6niversity, Cairo, Egypt 

fne hundred neonates suspected of having sepsis in Neonatal Intensive Care 6nit 

(NIC6) were studied. Twelve cases were positive as yeast infections representing 

12_, while 88_ were bacterial infections of the total positive cases. 7+,5"5+*+68"#+,& 

was the predominant species and was isolated from 11 cases representing K1.7_ of 

the total isolated yeast cases, while V.8+$2%)2#.&*?+,&.,""*was isolated from one 

case only representing 8.J_ of the total yeast isolates. The analysis of neonatal 

cases showed perfect correlation between molecular and microbiological data in all 

infected cases. PCR for universal yeast DNA gave positive results at 61[ bp, and that 

for 7+,5"5+*+68"#+,& was observed at 1[6 bp. 

P-0500. Tinea pedis and tinea unguium in a certain mental hospital 

Kawai M 1 , Hiruma M 2 , fgawa H J 

1 (untendo 6niversity loshigaya Hospital, 2 (untendo 6niversity Nerima Hospital, 

J (untendo 6niversity .chool of Medicine 

ae investigated prevalence of tinea pedis and tinea unguium in 21O inpatients of a 

mental hospital, there were 100 men and 11O women with a mean age of O6.K years. 

ff these patients, K[ (OO.J_, O7 men and O8 women) presented positive signs by 

direct microscopy. Clinically, 61 patients (6O.2_) had interdigital tinea pedis, OJ 

(O7.O_) had the vesicular form, O2 (OO.2_) had the hyperBeratotic form of tinea pedis 

and OK (22.K_) had tinea unguium. There was no gender difference in either clinical 

symptoms or prevalence of tinea unguium. The mean clinical score for tinea pedis 

was as high as 6.J, while the mean .CIf score which indicates the severity of tinea 

unguium, was 1[.6. As to the patient]s age, fungal infection was less frequently 

encountered among patients less than J0 years of age s18/76 (2J.7_)u, than among 

those more than O0 years of age s77/1J8 ([[.8_)u. Positive cultures were observed in 

[0 patients ([2.6_) Trichophyton rubrum was isolated in JO (68_), T. 

mentagrophytes in 1J (26_) and others in J patients. According to our direct 

microscopic examination of 16J patients in the closed ward accommodating mainly 

patients with schizophrenia, 71 (OJ.6_) were infectedn however, among of the [1 

patients in the open ward where those with depression are hospitalized, 2O (O7.1_) 

were infected, indicating no difference in the prevalence of tinea pedis. 

ae also investigated the prevalence of tinea pedis and tinea unguium in patients 

hospitalized for more than 1 year. There were 8J such patients (mean age [0.8 years, 

O7 men and O8 women) and accounted for J8.8_ of all inpatients. Most of these 

patients had schizophrenia. Positive direct microscopy results were observed in 62 

patients (7O.7_, JJ men and 2K women), with the mean clinical scores for tinea pedis 

being 6.6. Tinea unguium was detected in JO patients (O1.0_) (18 men and 16 

women), with the mean .CIf score being 17.0. The prevalence of tinea pedis and 

tinea unguium tended to be higher and the symptoms severer in patients hospitalized 

for one year or longer. 


P-0501. Eumycetoma: A retrospective study of 9 cases seen from 1996 to 2005 

in a paris hospital, France 

Lacroix C 1 , Dubertret / 2 , Morel P 2 , Feuilhade M 1 2 

1 mycology hopital .t /ouis, 2 dermatology hopital .t /ouis 

Eumycetoma is a chronic, localized fungal disease endemic in some countries of 

Africa, the Middle East and Asia. The management of this fungal infection usually 

requires surgical debulBing plus systemic antifungal treatment. However, in spite of 

therapy, eumycetoma can extend deeply and lead to bone involvementn requiring 

amputation. 

The aim of this study was to retrospectively review the cases of eumycetoma 

diagnosed at .aint /ouis hospital, Paris, France, during the last 10 years. 

2etween (anuary 1KK6 and December 200[, K cases were diagnosed at the policlinic 

of dermatology in .aint /ouis hospital. The retrospective analysis of the cases 

revealed that the male/female ratio was 8/1 and that the median age at diagnosis time 

was J6 years s2J-68u According to the patients, the lesions have been evolving for 0.[ 

to O0 years (median } [.[ years). All the patients were coming from endemic areas for 

eumycetomasM .enegal (n}O), Mali (n}J), Mauritania (n}1), and .ri /anBa (n}1). 

/esions were localized mainly on feet (n}8), one was localized on the buttocB. A bone 

lesion was diagnosed in [ patients by CT-scan and/or RMN, one patient did not 

performed any radiology. At macroscopic examination, blacB grains were observed in 

7 cases. Histology was performed in 8 patients and revealed the presence of a fungal 

grain in 6. Identification of the fungus was obtained for 8 patientsM !+5($.66+* 

)2#.-%)+-"& (n}6), 1?"+6%'?%$+ sp. (n}1), and ^(&+$"()*&%6+," (n}1). A medical 

treatment was initiated in 8 patients (one missing) mainly with itraconazole (n}7), 

voriconazole (n}2), terbinafine (n}1) and posaconazole (n}1). The patient with the 

mycetoma due to ^/*&%6+," received successively itraconazole, terbinafine, 

posaconazole and voriconazole without any improvement. fne patient presenting a 

mycetoma due to !/*)2#.-%)+-"&*was clinically cured with voriconazole alone. A 

surgical treatment was performed for four patients, with a relapse for two. 

P-0502. The quantitative study on the distribution of Malassezia yeasts on the 

normal skin 

Lee Y, Yim ., /im ., Choe Y, Ahn l 

Department of Dermatology, lonBuB 6niversity College of Medicine, .eoul, .outh 

lorea 

In this study, a culture research was conducted on the data gathered from lorean 

people to examine diseases associated with !+6+&&.d"+*yeasts. In practice, 

!+6+&&.d"+*yeasts were cultivated in five different areas from human body (scalp, 

forehead, chest, arm, and thigh) in the age groups from 0 to 60. The research was 

carried out to recognize the existence of a difference in the amount and type of 

!+6+&&.d"+ species in different age groups as well as body areas. According to the 

result, !/*$.&-$"#-+*was recovered more frequently in the age groups 11-20 (J2_), 21- 

J0 ([K_), and J1-O0 (O[_)n !/*@6%8%&+ was the predominant species in the age 

group [1-60 (J6_). The population density of !+6+&&.d"+*yeasts was significantly 

higher in the age group 21-J0 compared to the age groups 0-10, 10-20, O1-[0 and [1- 

60 (1v 0.0[). It was significantly higher in the chest compared with the forehead, arm 

and thigh (1v0.0[). The amount of !+6+&&.d"+ species that can be recovered from 

human sBin varied with age and body area. The amount of !+6+&&.d"+ inoculum 

recoverable from children was quite low. This might reflect the fact that compared to 

other age groups, they have very little sebum on their sBin. For various age groups, 

there were differences in the mean numbers of colony forming unit (c.f.u) found 

among different body areas. These differences are presumably due to puberty-related 

change in sBin lipid levels. 

This study shows that eumycetoma is not a rare infection in a temperate city 

welcoming immigrants from endemic countries, mainly Africa and Asia. This disease 

remains a real therapeutic challenge even in countries with a developed health system.

P-0503. Many ad hybrid strains of Cryptococcus neoformans possess the 

serotype a mata allele found in the Botswanan (VNB) subpopulation 

Litvintseva A, Templeton I, Heitman (, Mitchell T 


AD strains of 7$2'-%#%##(&*,.%U%$)+,& are hybrids between 7/*,.%U%$)+,&*var. 

,.%U%$)+,& (serotype D) and 7/*,.%U%$)+,& var. @$(8""*(serotype A). AD hybrids have 

been isolated worldwide from both clinical and environmental. Most of these strains 

are diploid and possess two copies of either serotype A or serotype D !4>+*and 

!4>+*alleles. Although the global population of serotype A is dominated by isolates 

with the !4> !*mating type, about half of the globally analyzed AD strains possess 

the rare serotype A*!4>+ allele. ae previously described an unusual subpopulation 

of serotype A in 2otswana (VN2), and 2[_ of these VN2 strains possessed the !4>+ 

mating type. The purpose of this study was to investigate whether the serotype A 

!4>+ alleles in non-2otswanan AD hybrids could have originated from the VN2 

subpopulation of serotype A. ae analyzed 12 clinical and environmental AD isolates 

from China, the Democratic Republic of the Congo, Italy, luwait, and the 6.A and 

determined that six ([0.0_) of these strains possess the serotype A !4>+ allele. 

6sing multilocus sequence typing (M/.T) with three loci, 741IQ, TPaI, and 91VI, 

we discovered that all of the isolates with the serotype A !4>+ allele have identical 

sequences of these three genes, and they cluster with VN2 isolates of serotype A 

from 2otswana. Conversely, all of the strains with !4>+*allele clustered with the VNI 

subpopulation of serotype A. 6sing amplified fragment length polymorphisms (AF/P) 

to analyze the genetic relatedness of the AD strains, the six AD strains with the VN2 

serotype A !4>+ allele had very similar, if not identical, AF/P genotypes. Therefore, 

the strains may be clonal descendants of a single AD strain that originated from a 

hybridization between a VN2 strain of serotype A with the !4>+ mating type and a 

serotype D strain with the !4>+ mating type. 

P-0504. Close relatedness among fluconazole resistant Candida tropicalis 

isolates from different hospitals in Taiwan 

Lo H 1 , Yang Y 2 , /i . J , aang ( 1 , Chou H J 

1 National Health Research Institutes, Miaoli , Taiwan, 2 National Chiao Tung 6niversity, 

Hsinchu, Taiwan, J Center for Disease Control, Taipei, Taiwan 

Among the yeast pathogens causing morbidity in seriously immunocompromised 

hosts, 7+,5"5+*+68"#+,& is the most frequently isolated species. However, there has 

been a shift from 7/*+68"#+,&*to more treatment-resistant non-albicans*7+,5"5+ 

species. 7+,5"5+*-$%'"#+6"&*has been reported as the second or third leading cause of 

Candida infections. The susceptibilities to fluconazole of 162 7+,5"5+*-$%'"#+6"&* 

isolates collected in Taiwan .urveillance of Antimicrobial Resistance of Yeasts 

(T.ARY) in 1KKK were determined by the microdilution method. 6rine was the most 

common source accounting for OO.O_ of the total isolates, followed by sputum 

(26.6_), wound (8.0_), blood (7.O_), and others (1J.6_). A total of 12K (7K.6_), 10 

(6.2), and 2J (1O.2_) isolates were, respectively, susceptible, susceptible-dose 

dependent, and resistant to fluconazole. To determine the genetic relatedness of the 

resistant isolates, we have performed pulsed filed gel electrophoresis analysis of all 

2J resistant isolates along with 1J susceptible ones. Interestingly, two distinguish 

pulsotypes were observed. Furthermore, these two types were independent of 

sources and hospitals but associated closely with the susceptibility of fluconazole. 

ahether this phenomenon is due to clonal spreading or genome stability needs further 

investigation. Hopefully, information gaining from more studies of molecular 

epidemiology will provide insight for repaid identification of clinical fluconazole 

resistant 7/*-$%'"#+6"&. 


P-0505. Surveillance of nosocomial fungal infections in a Portuguese paediatric 

hospital: Incidence and risk factors 

Lopes M 1 , 2arros R 2 , Peres I 2 , .erelha M J , Neto T J , Cabrita ( O , Freitas G 1 

1 

Faculdade de FarmZcia da 6niversidade de /isboa, .ub-Grupo de Microbiologia, 2 Hospital D. 

Estef°nia, /aboratário de Patologia ClÜnica, J Hospital D. Estef°nia, 6nidade de Cuidados 

Intensivos Neonatais, O Faculdade de FarmZcia da 6niversidade de /isboa, .ub-Grupo de .ácio- 

FarmZcia 

Objectives: The magnitude of the nosocomial fungal infection in children has never been deeply 

studied in Portugal. In order to contribute to a better Bnowledge about this Public Health problem, 

we performed a prospective study in the paediatric Hospital D. Estef°nia - /isbon, the main 

paediatric-care hospital in /isbon, Portugal, aiming to determine its incidence, factors associated 

with increased risB of infection, and to identify the main infectious microorganisms. 

Material and Methods: A prospective surveillance was conducted, from (anuary 1KK8 and 

March 2000 in all wards of D. Estef°nia Hospital. A standardized data collection form was used 

to retrospectively review the hospital records of patients with nosocomial fungal infection. Data 

recorded included demographic and clinical characteristics, ward, site of infection and pathogen. 

Information of potential risB factors was collected from the patients] charts under supervision of 

one member of the Hospital Infection Control Committee. 

.tatistical analysis was performed using Epi info version 6.0O and .P.. software version 10 

(.P.. Inc, Chicago, 6.A). The univariate analysis was tested using 8 2 and 2-tailed Fisher]s 

exact test in case of small sample size. A multivariate analysis was done using a nonconditional 

logistic regression model expressed as fdds Ratio (fR) with K[ _ confidence intervals. 

.tatistical significance was assumed when 1 v 0.0[ 

Results: During this period, were reported [7 episodes of invasive infections, resulting in an 

overall incidence of 0.26 _ (K[_ confidence interval .CI/, 0.20 to 0.JJ), with a mean annual 

incidence of 0.11_ (K[_ CI, 0.08 to 0.1O) that showed an increasing trend over the study period. 

The incidence was significantly higher in intensive care units (IC6) than in all the remaining 

wards. Infants younger than six months of age accounted for J6.K_ of the cases, and patients 

hospitalized longer than J months for [O.O_. In all age groups, invasive infections occurred more 

frequently in males ([O.O_). 

The three most frequent causative species were 7+,5"5+*+68"#+,& ([K.6 _), 7. '+$+'&"6%&"& 

(J1.6 _), and 7. -$%'"#+6"& (7.0 _). 

No difference was observed between the distribution of 7. +68"#+,& in IC6 and in conventional 

wards. 7. '+$+'&"6%&"& and 7. -$%'"#+6"& were predominantly recovered in IC6. 

In the seven death cases associated with nosocomial fungal infection, [ were due to 7+,5"5+ 

+68"#+,&3*2 to 7. -$%'"#+6"& and 1 to 7. '+$+'&"6%&"&, accounting for 12.J _ of overall lethality. 

Deaths occurred in IC6, 6 in paediatric intensive care unit (1KKK) and 1 in neonatal intensive 

care unit (1KK8). Previous antibiotic therapy, intra-venous catheters, parental nutrition, 

mechanical ventilation, endotracheal intubation, anaemia, and the long hospital length-stay were 

identified as the most important risB factors to fungal hospital-acquired infection. 

Conclusions: These results help to establish the pertinent epidemiological measures for the 

control of nosocomial fungal infections, including the elaboration of protocols for monitoring 

fungal colonization or infection in high-risB paediatric patients. In addition, these findings 

reinforce the need for continued and active surveillance programs to address the changes in the 

species distribution which will help to develop effective, preventive and therapeutic strategies. 

P-0506. Increasing incidence of Candida bloodstream infections and at two 

hospitals in the southeastern USA 

Lyon G, Caliendo A, Nolte F 

Emory 6niversity 

Background: 7+,5"5+*&'.#".& are the fourth most common cause of nosocomial 

bloodstream infections in the 6.A. In the 1K80s, 7+,5"5+*+68"#+,& caused the 

majority of the infections. However, during the 1KK0s among the various 7+,5"5+* 

species, the proportion of infections caused by 7/*+68"#+,& decreased causing only 

O[_ of bloodstream infections. The rise in non-+68"#+,& species causing invasive 

infections was mainly due to 7/*@6+8$+-+, which accounted for 2O_ of the invasive 

infections. ae undertooB sentinel surveillance to monitor whether there was a 

continuation in the shift away from 7/*+68"#+,&. 

Methods: Twenty-eight teaching institutions across the 6. submitted between 2[ and 

100 consecutive yeast isolates from sterile body sites. This analysis includes 

specimens collected between August 200O and November 200[, however, not all sites 

initiated surveillance in August 200O. 2asic demographic and clinical data were 

collected on patients from whom the isolates were obtained. .pecies identification 

was performed at a central laboratory using ChromAgar to identify mixed infections 

and maBe a presumptive identification of 7/*+68"#+,&, 7/*-$%'"#+6"&, or 7/*[$(&.". 

Formation of germ tubes was used to confirm 7/*+68"#+,&. Microscopic morphology on 

Cornmeal Tween agar and biochemical characteristics using API 20C (2io-Merieux) 

were used to identify species other than 7/*+68"#+,&. 

Results: ae identified 1,JK7 7+,5"5+ isolates. The distribution of species was as 

followsM 7/*+68"#+,& O[_, 7/*@6+8$+-+ 2[_, 7/*'+$+'&"6%&"& 18_, 7/*-$%'"#+6"& 8_, 7/* 

[$(&." 2_, other species 2_. aomen accounted for O8_ of the cases and patients 

under 18 years old represented 1J_ of the studied population. The isolates were 

collected from the following body sitesM bloodstream (88_), peritoneal fluid (J_), 

pleural fluid (1_), and all other sterile body sites (8_). The wards on which the 

patients were cared for at the time of isolation were as followsM general or medical 

wards (O6_), surgical (1O_), oncology or bone marrow transplant (12_), pediatric 

(11_), an intensive care unit (10_), or other ward (10_). The most commonly 

reported underlying medical conditions were no underlying disease (16_), 

gastrointestinal disorders (12_), and solid organ malignancy (10_). ff note, patients 

with hematologic malignancy, stem cell transplant, and solid organ transplant 

accounted for 7_ collectively, and the distribution of species did not differ significantly 

from the entire cohort. Forty-one (J_) patients received fluconazole prior to isolation 

of the Candida species. 7/*+68"#+,& (O1_) and 7/*@6+8$+-+ (2K_) accounted for the 

majority of these isolates. 

Conclusions: ahile 7/*+68"#+,& is still the yeast species most frequently isolated, 

non-albicans species, especially 7/*@6+8$+-+, continue to account for the majority of 

sterile body site infections. The shift away from 7/*+68"#+,&, which is typically 

fluconazole susceptible, to less susceptible non-+68"#+,&*&'.#".& may have 

implications for empiric treatment of yeast infections.

P-0507. The Candida surveillance study (CASS), the first year report 

Lyon G 1 , Adiri Y 1 , ailson H 2 , Abramson M 2 

1 Emory 6niversity, 2 MercB c Co., Inc 

Background: 7+,5"5+*&'.#".& are the fourth most common cause of nosocomial 

bloodstream infections in the 6.A. In the 1K80s, 7+,5"5+*+68"#+,& caused the 

majority of the infections. However, during the 1KK0s among the various 7+,5"5+* 

species, the proportion of infections caused by 7/*+68"#+,& decreased causing only 

O[_ of bloodstream infections. The rise in non-+68"#+,& species causing invasive 

infections was mainly due to 7/*@6+8$+-+, which accounted for 2O_ of the invasive 

infections. ae undertooB sentinel surveillance to monitor whether there was a 

continuation in the shift away from 7/*+68"#+,&. 

MethodsM Twenty-eight teaching institutions across the 6. submitted between 2[ and 

100 consecutive yeast isolates from sterile body sites. This analysis includes 

specimens collected between August 200O and November 200[, however, not all sites 

initiated surveillance in August 200O. 2asic demographic and clinical data were 

collected on patients from whom the isolates were obtained. .pecies identification 

was performed at a central laboratory using ChromAgar to identify mixed infections 

and maBe a presumptive identification of 7/*+68"#+,&, 7/*-$%'"#+6"&, or 7/*[$(&.". 

Formation of germ tubes was used to confirm 7/*+68"#+,&. Microscopic morphology on 

Cornmeal Tween agar and biochemical characteristics using API 20C (2io-Merieux) 

were used to identify species other than 7/*+68"#+,&. 

Results: ae identified 1,JK7 7+,5"5+ isolates. The distribution of species was as 

followsM 7/*+68"#+,& O[_, 7/*@6+8$+-+ 2[_, 7/*'+$+'&"6%&"& 18_, 7/*-$%'"#+6"& 8_, 7/* 

[$(&." 2_, other species 2_. aomen accounted for O8_ of the cases and patients 

under 18 years old represented 1J_ of the studied population. The isolates were 

collected from the following body sitesM bloodstream (88_), peritoneal fluid (J_), 

pleural fluid (1_), and all other sterile body sites (8_). The wards on which the 

patients were cared for at the time of isolation were as followsM general or medical 

wards (O6_), surgical (1O_), oncology or bone marrow transplant (12_), pediatric 

(11_), an intensive care unit (10_), or other ward (10_). The most commonly 

reported underlying medical conditions were no underlying disease (16_), 

gastrointestinal disorders (12_), and solid organ malignancy (10_). ff note, patients 

with hematologic malignancy, stem cell transplant, and solid organ transplant 

accounted for 7_ collectively, and the distribution of species did not differ significantly 

from the entire cohort. Forty-one (J_) patients received fluconazole prior to isolation 

of the Candida species. 7/*+68"#+,& (O1_) and 7/*@6+8$+-+ (2K_) accounted for the 

majority of these isolates. 

Conclusions: ahile 7/*+68"#+,& is still the yeast species most frequently isolated, 

non-albicans species, especially 7/*@6+8$+-+, continue to account for the majority of 

sterile body site infections. The shift away from 7/*+68"#+,&, which is typically 

fluconazole susceptible, to less susceptible non-+68"#+,&*&'.#".& may have 

implications for empiric treatment of yeast infections. 

P-0508. Sources of fungal infections at an intensive care unit 

Macura A 1 , GniadeB A 2 , FloreB ( 2 

1 Dep.of Mycology, Chair of Microbiology, lraBow, Poland, 2 Dept. of Environmental 

Nursing, Nursing Institute, (agiellonian 6niversity Medical College, lraBáw, Poland 

The concentration of pathogenic fungi in hospital rooms is usually high which maBes 

potential risB of fungal infections in patients with impaired immunity and/or under 

invasive treatment. The highest increase of fungal infections is observed at surgical 

wards and intensive care units. The reservoir of fungal spores may be indigenous flora 

of the patients, room walls and water distribution system. The infection may spread 

through the staff hands, but also, if not first of all, through the indoor air. Therefore, 

the indoor air at such wards should be of highest quality. 

The objective of the study was evaluation of the fungal presence in the environment of 

an intensive care unit. 

The environment testing was carried out at a chest clinic intensive care unit in Cracow, 

Poland, in December 200O. The materials to mycological examinations were sampled 

simultaneously from indoor air and room walls in 1[ roomsM four bays, a treatment 

room, three bathrooms, a nurse]s station, a doctor]s room, a rest room, a corridor, a 

dirty annex, a ward Bitchen and a washing room. The air samples were taBen twice 

daily, and the samples from the walls once daily, for five days. 

The fungal colonies obtained in the cultures were evaluated macroscopically and 

microscopically according to generally accepted standards. Then, a statistical analysis 

was performed. The analysis of 1[0 air samples taBen in 1[ rooms revealed, that 

fungi were not isolated only in 6 samples. The highest number of fungi amounted to 

720 c.f.u%m -J . The mean number of fungi in the particular rooms in the whole sampling 

period varied from 172 c.f.u%m -J to 12 c.f.u%m -J . The mean numbers of fungi present 

in the air in the morning were higher than those in the evening in three days, but not in 

the whole sampling period. 

fut of 7[ samples from the walls, fungi were present only in 1K of them. Their mean 

numbers varied from 0 to 0.J7 c.f.u%cm -2 . No fungi were found in the treatment room, 

two bays, nurse]s station and one bathroom. 

The moulds 4&'.$@"66(& sp., 1.,"#"66"() sp., and 76+5%&'%$"()*sp. as well as yeastliBe 

fungi P?%5%-%$(6+*$(8$+ were most frequently isolated from the indoor air and 

were present in all of the sampling sites. The moulds 1.,"#"66"() sp. and 

76+5%&'%$"()*sp. as well as yeast-liBe fungi 7+,5"5+* sp. and P/*$(8$+ were most 

frequently isolated from the walls. The fungi isolated from the walls were also found in 

the air in the same roomsn in five rooms, their numbers were higher than those 

acceptable in treatment rooms. It is alarming, that moulds belonging to the genus 

4&'.$@"66(& were present in all of the sampling sites. * 


P-0509. Typing of clinical isolated of histoplasma capsulatum based on 

nucleotide sequence variation in the its regions of rDNA genes 

Maffei C, Cazzaniga R, /ucas R, Castro M 

Faculdade de Medicina de Ribeirao Preto-6.P- .ao Paulo-2razil 

A"&-%'6+&)+*#+'&(6+-() var. #+'&(6+-() is a fungus, which presents thermal 

dimorphism and causes an illness in the human respiratory system that can 

spontaneously regress or evolutes to a systemic form, mainly in immunosuppressed 

individuals. This fungus has a sexual (recombinat) and an assexual (clonal) 

reproduction and is naturally found in soils rich in nitrogen and phosphate in different 

geographic regions. Considering that the internal transcribed spacer regions of rDNA 

genes have alterations liBe transitions and transvertions, which are submitted to 

selective pressure, the sequencing of this region represents a good molecular typing 

method. This methodology can offer phylogenetic information among different strains, 

distinguish relapse from reinfection and evaluate the influence of storage time in the 

genetic variability of the samples. In this study we sequenced the IT. regions of 20 

clinical strains of A/*#+'&(6+-() isolated from patients of RibeirÉo Preto region | 

northeast of .Éo Paulo state | 2razil, storaged among 1 to [ years. The pattern of 

animal virulence of these samples was already established in previous studies, and 

[[_ of them presented high virulence. The genomic DNA was extracted from yeast 

phase using silica beads and we used the universal primers IT.1 and IT.O to 

amplification in a PCR reaction. After sequencing and alignment we observed 

variations in 7 positions of the region IT.1 and in 1O positions of the region IT.2 from 

the total [0O bp sequenced. These variations allowed to classifying the strains in 7 

different molecular types, arbitrarily designed I through VII, some of them presenting 

internal subdivisions. The most frequent molecular type was the III observed in O[_ of 

the samples, which presented different storage times, while molecular type IV grouped 

the samples recently isolated. ae couldn]t find a relation between molecular type and 

pattern of animal virulence. Considering that the nucleotide variations observed 

possibly are result from selective pressures we constructed a phylogenetic tree by 

maximum liBelihood analysis, which showed high evolutionary correlation among the 

20 strains. ahen 27 clinical samples isolated from different geographical regions of 

the world, sequenced by other authors and deposited in Gene2anB were included in 

this study, the resultant phylogenetic tree showed samples from Thailand, (apan, 

Africa and some /atin American mixed with RibeirÉo Preto samples, but grouped 

North American and Colombia samples in two distinct clades. ae concluded that the 

use of a good tool to typing samples and the Bnowledge of the populational genetic 

structure of A/*#+'&(6+-(), could explain the capacity of this fungi to infect distinct 

animals species and to cause differences in the parasite-host relationship based in the 

selective pressure suffered by this fungi in its several natural reservoirs. 

.upported byM FAPE.P and CAPE. 

P-0510. Pneumocystosis network in 14 parisian hospitals: A two years 2003- 

2004 survey 

Magne D, Angoulvant A, Roux P 

6niversitd Pierre et Marie Curie, site .aint-Antoine, Paris, France 

Human pneumocystosis (PCP), due to 1,.()%#2&-"&*\"$%0.#"3*remains one of the most 

important fungal opportunistic pneumonia, in AID. and nother immunocompromised 

patients. Due to the strict host specificity, human may be the main reservoir for 

1,.()%#2&-"&*\"$%0.#", inter human transmission may occured and nosocomiality can 

be possible. 

Genetic marBers for possible sulfamid resistance, point mutations (16[ and 171) on 

dihydopteroate synthetase (DHP.) gene have been reported . 

The aim of this PCP networB is to study the PCP occuring in 1O parisian (Assistance 

Publique-HÇpitaux de Paris) hospitals and to describe the DHP. genotypes . 

Methods : M Forteen Parasitology Mycology laboratories (Ambroise Pard, Avicenne 

2ichat, Cochin, Henri Mondor, Hotel Dieu, HÇpital Europden Georges Pompidou, 

lremlin 2icütre, /ouis Mourier, NecBer, Pitid .alpdtrimre, .aint Antoine, .aint /ouis, 

Tenon) reported clinically (coughr feverr/dyspnea and interstitial pneumopathy on 

chest j Ray) and biologically (trophozoites and cysts on Giemsa , T2f stainings and 

immunofluorescence) proved PCP. DHP. mutations on position 16[ and 171 were 

detected after PCR-RF/P. 

Results : From 01 01 200J to 12 J1 200O, J1J PCP have been reported, and 172 

samples received for genotyping. 6[_ were HIV positive patients. Men represented 

66_ (70_ in HIVr, [0_ in HIV-)of the patients. The median age was OO years. In HIV 

positive patients, PCP revealed the HIV infection in O6_. PCP was the first episod in 

7K,[_ and second episod in 1K,[_. Among 8J PCP occuring in HIV Bnown infected 

patients, no prophylaxis were prescribed for [[ n cotrimoxazole prophylaxis has been 

prescribed in 16 patients but none of them have a good observance. Most of the HIV 

negative patients, received corticosteroids and no prophylaxis was prescribed in 78_. 

DHP. Genotypes have been studied for 176 bronchoalveolar samples. Mutations 

were detected in [K samples (K in both positions). 26 coinfection (at least two 

different strains) were described. No relation were found between mutation and 

mortality, nor between mutation and prophylaxis. 

Conclusions: PCP remains an important opportunistic pneumonia in AID. and 

corticosteroids inmmunodepressed patients. Cotrimoxazole is an efficient prophylaxis 

when correctly used, prophylaxis may be dicussed for patients receiving long term 

corticosteroids treatment.

P-0511. Mycetoma in a Sudanese village 

Mahgoub E 

Fac. of Medicine, 6niv. lhartoum, .udan 

A pilot study of mycetoma patients in a small village along the 2lue Nile in central 

.udan called Y+5*4@8,+ has revealed some interesting epidemiological points. 

Among the village population of O000. a total of 112 (2.8_) were afflicted with 

mycetoma . As far as we Bnow this is the highest incidence in an endemic area. The 

relations between patients were interestingM in 16 clusters patients were brothers and 

sisters, in 8 they were cousins, in O relation was one of parents, in one case relation 

was a husband and in the other an uncle. The largest number was in one family 

where three brothers and their three sisters had mycetoma. 

lnowledge, attitudes and practices (lAP) of this village community was also 

interestingM K0_ of the respondents Bnew that mycetoma is a painless progressive 

swelling that first draws attention when it is the size of a bean. They had different 

ideas about its treatment. .ixty percent thought treatment was surgical removal or 

amputation, 20_ said surgery will maBe it worse and 20 _ did not Bnow. Although 

they did not Bnow the real cause, they noted that it was commoner amongst those 

who reared animals in addition to farming than the rest who were farmers only. 

They observed that the fences and the floors of animal enclosures had lots of thorns 

and animal dung. 

These findings gave ideas for new areas of research particularly the immunological 

status and anthropological origins of the different .udanese tribes. 

P-0512. Filamentous fungal flora in haematology units: Influence of 

environmental, behavioural and climatic factors: I methodology of a 30-month 

prospective study 

Marie-Pierre B 1 , 2ernadette / 1 , (ean-/ouis ~ 2 , Marie-reine M J , (ean-/uc 2 O , (ean- 

Paul 2 [ , (ean-/uc 2 2 , (ean-Yves C 6 , Rende G 1 , Hervd P 1 

1 .ervice de Parasitologie-Mycologie, CH6 de Grenoble, 2 Centre deInvestigation Clinique, 

CH6 de Grenoble, J 6nitd deHygimne Hospitalimre, CH6 de Grenoble, O /aboratoire 

deEcosystmme Alpin, 6niversitd ( Fourier, Grenoble, [ .ervice de Maladies Infectieuses, 

CH6 de Grenoble, 6 Ddpartement de Cancdrologie et deHdmatologie, CH6 de Grenoble 

The relationship between the environmental fungal contamination and the incidence of 

invasive filamentous fungal infections (IFFI) seems to be relevant. However, numerous 

factors influence the hospital environmental filamentous fungal flora. 

** 

The objectives of this prospective study were i) to identify and graduate the involvement of 

internal and external factors to the hospital on filamentous fungal (FF) flora in three 

different care unitsn ii) to correlate the intensity of FF load with the number of invasive 

nosocomial aspergillosis in these units during J0 months. 

The three units studied were two haematology units and one infectious disease unit. All of 

them received patients at risB for IFFI but preventive measures were different. Partial air 

control was performed with 8[_ opacimetric filtration. During J0 months, 16 parameters 

were studiedM 6 external factors including meteorological parameters (daily for temperature, 

humidity, pluviometry, speed and direction of wind) and the daily number of fungal spores 

in the outer air (2urBard air spore sampler). Moreover, the 10 internal factors were i) the 

presence of building worBs in or near these units (localisation, type and period), ii) the unit 

activity (indicated in hospital records monthly) and iii) the evaluation of cleaning worB 

(number and efficacy of cleaning performed twice a month). The FF load was evaluated 

every fortnightM 6 surface samples were performed in O rooms of each unit (J bedrooms 

and corridor) using contact plates (MercB, Germany). Air samples (2 x [00/) were collected 

using Air Ideal (bioMdrieux, France) in each room/corridor and the samples were incubated 

at 27ÅC for 7 days. .everal descriptive characteristics were systematically reported at the 

moment of FF sampling, such as single or twin-bed, one or two patients, presence of 

agranulocytosis patients, number of opened doors and number of cardboard boxes in the 

corridor. 

The number and characteristics of Invasive Aspergillosis (IA) cases in these units were 

obtained from the £Aspergillosis Committee§ (Fourneret-Vivier A et al. (. Hosp. Infect. 

2006). This multidisciplinary worBing group monthly reviews each suspected IA case and 

classify it according to the international consensus criteria (proved, probable and possible) 

and the origin of contamination (hospital acquired or not). All results were entered into a 

specific computing data base and the coherence tests were performed. 6nivariate and 

multivariate analyses were performed using the chi-squared, Mann ahitney 6 and lrusBalaallis 

tests for FF load by .P.. ' software (.P.. Inc.). Time series analyses are in 

process. 

From (anuary 2O, 2002 to (uly 22, 200O, 66 sampling campaigns were performed 

corresponding to O,668 surface samples and 1,[[6 air samples. Preliminary results are 

presented at this congress in another abstract. 


P-0513. Filamentous fungal flora in haematology units: Influence of 

environmental, behavioural and climatic factors: II preliminary results 

Marie-Pierre B 1 , 2ernadette / 1 , (ean-/ouis ~ 2 , Marie-Reine M J , Frdddric G O , (ean- 

Paul 2 [ , /ysiane M O , (ean-/uc 2 2 , Hervd P 1 , Rende G 1 

1 

.ervice de Parasitologie-Mycologie, CH6 de Grenoble, 2 Centre deInvestigation Clinique, CH6 

de Grenoble, J 6nitd deHygimne Hospitalimre, CH6 de Grenoble, O Ddpartement de Cancdrologie et 

deHdmatologie, CH6 de Grenoble, [ .ervice de Maladies Infectieuses, CH6 de Grenoble 

A J0-month prospective study was performed to identify the factors involved in the environmental 

fungal contamination as described in the abstract of methodology. The filamentous fungal load 

was studied in three care units. Equipped with air-conditioning, these were two haematology 

units (6A and 62) and one infectious disease unit (6C). They received patients at risB for 

invasive filamentous fungal infection (IFFI) but the general preventive measures differed from 

one another. More patients at IFFI risB were hospitalized in 6A and 62 than in 6C. In 6A, the 

preventive measures were the most drasticM closed unit, patient in isolated environment with 

closed door, visiting limits, selected food, cardboard boxes opened outside the unit, and the 

nurse staff was common to the unit with HEPA filtration-/AF. In 62, the preventive measures 

were less drastic and the 6C was a conventional unit. 

Filamentous fungi were isolated in J,08J/6,22O samples (OK.[_) with a significant difference 

among each of the three units (p-0.0001). 6A was significantly less contaminated than 62 

(p-0.0001). However, the isolation of 4&'.$@"66(&*U()"@+-(& was rare in the three units, without 

difference between 6A and 62. 

All filamentous fungi 4/*U()"@+-(&** 

_ a CF6/. b _ a CF6/. b 

6A J6.1_ 1.6Jì6 J.7_ 0.0Kì0.7O 

62 [0.1_ 2.10ì[.[ 2.7_ 0.0Jì0.22 

6C 62.O_ J.J2ì8.7 O.2_ 0.07ì0.OO 

All J units OK.[_ 2.J[ì6.K J.[_ 0.06ì0.[2 

a 

percentage of positive samples, b mean of CF6/ sampleì standard deviation 

.urfaces (1,7OK/O,668, J7.[_, 1.JìO CF6/.) were significantly less contaminated than air 

(1JOO/1[[6, 8[.7_, [.OJì11.6 CF6/[00/). A significant difference was also found for 4/* 

U()"@+-(& (1.6_, 0.02ì0.2 CF6 and K.O_, 0.1Kì0.K CF6 for surface and air samples, 

respectively). 

The corridors ([O.7_, 2.Kì7 CF6/.) were significantly more contaminated than the rooms 

(O7.8_, 2.2ì7 CF6/.). Furthermore, significantly fewer filamentous fungi (FF) were isolated in 

rooms with single beds or with agranulocytosis patients than with twin beds or with nonagranulocytosis 

patients (p-0.0001). This difference was not found when only 4/*U()"@+-(&*was 

considered. 

In the environment of these three units, 4/*U()"@+-(& was rarely isolated in comparison with the 

whole FF flora. Its isolation does not seem to be a representative parameter to evaluate the 

environmental fungal pressure. Moreover, we highlight the complementarity of air and surface 

samplings. In conclusion, the level of environmental fungal contamination is significantly higher in 

units with a weaB level of preventive measures even if their impact on presence of 4/*U()"@+-(&* 

is not so clear. The next step of this worB will consist in evaluating which of the two parameters, 

whole FF or 4/*U()"@+-(& flora only, is correlated with the incidence rate of nosocomial 


P-0514. Histoplasmosis, 158 cases, 2000-2005 medical mycology department, 

IMT, universidad central de Venezuela 

Mata-Essayag ., Colella M, /andaeta M, Roselló A, Hartung de Capriles C, Pdrez C, 

Magaldi ., flaizola C, (imenez f 

Instituto de Medicina Tropical 

Histoplasmosis is a granulomatous disease, caused by A"&-%'6+&)+*#+'&(6+-(), where 

mostly a self-limited benign primary infection may evolve. Primary infection is not clinically 

recognizable, except in immunocompromised patients, such as AID., malnutrition, small 

children and old age. Histoplasmosis is almost world-wide in distribution, with great 

varieties of incidence in different localities, in Venezuela it is endemic. It has been 

considered as typical of rural areas. However, it seems to have turned into an urban 

disease in the last few years, as a consequence of environmental climatic changes and 

pollution. The aim of this paper is to assess the demographic data, clinical features, 

diagnostic methods and treatment of patients with diagnosis of histoplasmosis ((anuary 

2000 - December 200[). 

Methods: patients attending the outpatient service at the Medical Mycology Department 

and clinical samples received in the /aboratory were assessed for histoplasmosis. 

Mycologic examination methods were performed, including Giemsa stain, culture, 

histological preparations such as Grocott and .chiff Periodic Acid (PA.) stains. 

Additionally, serologic tests (immunodiffusion method (ID)) and some samples were 

evaluated by PCR (Nested). Treatment was monitored. 

Results: 1[8 cases of histoplasmosis were assessed. 116 male (7J.O_) and O2 female 

(26.6_). Age rangeM 1O days to 8J years, medianM J2.JJ r 20.18 years. Most of the cases 

(10J patients, 6[.2_) came from Metropolitan area of Caracas. fut of these [J were found 

to be associated with AID., 1O with tuberculosis and 8 with paracoccidioidomicosis. 

Furthermore 6 children were malnourished, 11 were v 2 years old, and 11 were t 6[. 

.ixteen were otherwise inmunocompromized patients. .ome patients had more than one of 

these conditions. The other [8 were until that time apparently healthy. The most 

noteworthy presentations were 6 meningitis, 2 arthritis, 1 with a parotid gland tumour and 

one with adrenal involvement. In 27 patients elevated E.R was found, JJ had anaemia, 17 

low white blood cell count and 1J low platelet count. Epidemiological data was suggestive 

of histoplasmosis in most cases. In 6O patients, Giemsa stain was performed, being all 

positive. ID test was performed in O7 samples from [J AID. patients, J7 (78.72_) were 

positive. PCR was performed to 7 samples, O out of these were positive, all of which 

suffered disseminated histoplasmosis. Patients received treatment with Itraconazole, 

Amphotericin 2 and/or Fluconazole. 2eing Itraconazole the most successful treatment, 

Fluconazole treatment was less effective. 

Discussion: .ince the number of cases, among our patients that come from urban areas 

are apparently increasing, we believe it highlights changes in the epidemiology of this 

disease in Venezuela. ae found several atypical clinical presentations of the disease 

among them. ae are far from having established figures on the true incidence of 

histoplasmosis as being epidemic and shifting from rural to urban areas. The endemic 

areas as Venezuela the finding of a debilitating disease and/or tuberculosis should call for 

repeated smears and cultures to rule out concomitant histoplasmosis and even more often 

in patients with AID.. Therefore we require confident histoplasmosis statistics to monitor 

outbreaBs closely to prove an epidemic.

P-0515. Outbreak of Candida tropicalis fungemia in ICU patients associated 

with parenteral nutrition 

Melo A 1 , Amorim C 1 , Cyrillo M J , Godoy P 1 , Padovan A 2 , 2riones M 2 , Colombo A 1 

1 Division of Infectious Diseases, 6NIFE.P, .Éo Paulo, .P, 2razil, 2 Department of 

Microbiology, Immunology and Parasitology, 6NIFE.P, .Éo Paulo, .P, 2razil, 

J Hospital do .ervidor Pâblico Municipal, .Éo Paulo, .P, 2razil 

Nosocomial candidemia is a frequent and relatively underestimate severe infection 

that occurs with significant incidence worldwide. 7+,5"5+*-$%'"#+6"& has become a 

leading pathogen among the non-+68"#+,& 7+,5"5+ species causing fungemia. Most 

cases of 7/*-$%'"#+6"&*candidemia arise from the gastro-intestinal tract and outbreaBs 

related to different exogenous sources have been scarcely reported. ae investigated 

an outbreaB of 7/*-$%'"#+6"&*candidemia comprising J adult patients hospitalized in the 

same intensive care unity (IC6) of a tertiary hospital in .Éo Paulo, 2razil. During a 

three months-period, J cases of 7/*-$%'"#+6"& fungemia were documented right after 

the administration of parenteral nutrition (NPP). A total of 18 yeast isolates were 

genotyped, including ten from blood cultures of J patients, 2 lots of NPP and 6 

external controls. .pecies were identified by micromorphology and biochemical profile 

using IDJ2C (2ioMerieux). Molecular typing was performed by RAPD and rDNA IT. 

sequences. Two different primers were used in RAPD assays (CD6 and RPO-2). The 

RAPD fingerprintings were analyzed by GelComparII program. For phylogenetic 

analysis we amplified and sequenced IT. region including IT.1, [.8. and IT.2. 

These sequences were aligned using .eaview program and the phylogenetic tree was 

generated by PA6P program using the Neighbor joining method. The RAPD 

dendogram analysis and the phylogenetic tree identified 2 clusters. In the first group, 

the isolates of patients 1 and 2 and NPP1 were clusteredn the second group clustered 

the isolates of patient J and NPP2. 6sing 2 typing methods we documented an 

outbreaB of 7/*-$%'"#+6"& caused by 2 different strains, both of them contaminating the 

NPP. 

Financial supportM CAPE., FAPE.P and CNPq 

P-0516. Isolation of Sporothrix schenckii from the environment in Venezuela. A 

clinical case report 

Mendoza M, de Albornoz M, Diaz E, Alvarado P 

Instituto de 2iomedicina, Caracas, Venezuela 

='%$%-?$"_*&#?.,#["" , the etiological agent of sporotrichosis, is a dimorphic fungus, a 

saprophyte in nature, where it develops in the soil, on vegetation, etc., from which it 

has been repeatedly isolated (Plus and fpal, 1K86n MacBinnan, 1K6K). It is 

pathogenic for man and other mammals when it becomes localized in the sBin or 

lungs. This is the most frequent deep mycosis in urban areas of our country, where 

one of the most highly endemic zones is the Colonia Tovar Municipality. This zone 

has the climatic conditions appropriate for the growth of the fungus (Altitude 1800 

meters above sea level, temperature between CÅ 1O and 21, and relative humidity of 

[8_) The persons who live in this region are mostly farmers and they frequently 

receive traumatic injuries of the sBin, where the fungus subsequently develops. The 

study of a clinical case and of the area where she worBs led to isolation of ='%$%-?$"_* 

&#?.,#["" *from the environment. fur objective was to isolate =/*&#?.,#["" from the 

specific area where a clinical case of sporotrichosis had been reported. Clinical caseM 

Patient (PM), [1 year-old Caucasian female, residing in Colonia Tovar, Miranda .tate, 

Venezuela. .he referred after a traumatic lesion on the middle finger of the left hand 

during the preparation of soil for fertilization. At O months she developed a painless 

granulomatous lesion, 2 x 1 cm, with a .porotrichin sBin test of 10 x 1[ mm and 

negative serology. The patient evolved satisfactorily after treatment with iodinates. 

Isolation of the fungus from the environmentM The flotation method was used, mixing 

[ g of soil with saline solution and glass beads and allowing it to settle. The supernate 

was used for direct culture in plates containing medium and indirect culture by 

inoculation in mice. The mice were Billed and the liver, spleen and lungs were 

processed for culture. From the direct cultures, two colonies compatible with =/* 

&#?.,#["" were isolatedn a single colony was isolated from the inoculated mice. The 

three isolates showed macro- and microscopic morphological compatibility with =/* 

&#?.,#["" , with reversion to the yeast form at J7 CÅ. The exoantigen obtained from 

the cultures reacted with sera from patients with sporotrichosis. To our Bnowledge, 

this constitutes the first report in Venezuela of a clinical case of sporotrichosis with 

isolation of the causative agent from the area of contagion. 


P-0517. The study of dermatophytic agents in swim pools and sport salons at 

kermanshah 2005 

Mikaeili A 1 , Rezaei M 2 

1 l6M., lermanshah, Iran, 2 l6M. 

Dematophytosis is infectious sBin disease and more frequent among human and 

animal sport saloons and pool have potential risB for this infection. 

During 200[, 10J0 sample from wrestling, pools and power lifting were cultured on 

selective dermarophytic agentes medium(.CC) and incubated at J0 deg centigrade for 

O weeBs and with macroscopic and microscopic study dermatophytic spp were 

established and following results were obtainedM 

O case of Epidermophyton floocosum from wrestling salon and O case of 

Epidermophyton floocosum and 1 case of Trichophyton verrucosum from swim pools 

but culture of power lifting were negative. 

P-0518. Identification and determination of frequency of Candida patients 

isolates in iran by chromagar Candida method 

Mirhendi H 1 , MaBimura l 2 , NiBaeen M J , Hosseinpur / 1 , fmidi l 1 


Mycology, J Esfahan 6niversity of Medical .ciences 

7+,5"5+ species can cause opportunistic infections in human. Although 7+,5"5+* 

+68"#+,& is major pathogenic species, non-albicans*species of 7+,5"5+ such as 

7/*@6+8$+-+3*7/*-$%'"#+6"&*+,5*7/*[$(&."*have been increasingly reported. Identification 

of 7+,5"5+ species is essential for effective antifungal therapy and for infection control 

purposes. In the present study the commercial medium CHRfMagar Candida was 

used as the differential method for identification of the yeasts isolated from patients. 

.ixteen standard strains of 7+,5"5+ and non-7+,5"5+ yeasts were used as the 

reference cultures. 27K yeasts isolated from the patients referred to two Medical 

Mycology laboratories in Tehran-Iran were cultured on the chromogenic medium 

CHRfMagar Candida (J[g w /v). The cultures incubated for O8h at J[oC and were 

studied according to the differential colony color comparing with the standard strains. 

For colonies with unspecific color, a PCR-RF/P method was performed. The most 

frequent species was 7+,5"5+*+68"#+,& followed by 7/*'+$+'&"6%&"&, 7*-$%'"#+6"&, 

7/*@6+8$+-+, 7/*[$(&.", 7/*[.U2$, 7/*@("66".$)%,5"", 7/*6(&"-+,"+. and 

7$2'-%#%##(&*,.%U%$)+,&. CHRfMagar 7+,5"5+ can be a simple and strength 

forward method for specific differentiation of medically important 7+,5"5+ species 

including 7/*+68"#+,&3*7/*[$(&.",*7/*-$%'"#+6"&*and*7/*@6+8$+-+3 but not for all 7+,5"5+ 

isolates. fther phenotypic or genotypic methods are essential for exact identification 

of other species.

P-0519. Molecular epidemiology of Trichophyton tonsurans isolated in japan 

Mochizuki T 1,2 , lawasaBi M 1,2 , Tanabe H 1 , Anzawa l 2 , IshizaBi H 2 , Choi ( J 

1 Department of Dermatology, lanazawa Medical 6niversity, 6chinada, IshiBawa, 

(apan, 2 Division of Dermatomycology(Novartis Pharma), lanazawa Medical 

6niversity, 6chinada, IshiBawa,(apan, J Department of Dermatology, Yeungnam 

6niversity, .eoul, lorea 

Background: >$"#?%'?2-%,*-%,&($+,&*has been identified as the causative agent of 

an epidemic of tinea gladiatorum among high-school judoists and wrestlers in (apan. 

The onset of the epidemic and origin of the fungus are unBnown. 

Objective: The present study was conducted to subtype the strains of >/*-%,&($+,& 

obtained from patients in (apan using polymerase chain reaction (PCR) followed by 

restriction digestion to detect restriction fragment length polymorphism (RF/P) in the 

non-transcribed spacer region (NT.) of ribosomal RNA (rRNA) genes in fungal nuclei. 

Methods: PCR-RF/P was performed on 1K8*>/*-%,&($+,& strains isolated from 

(apanese patients. ff the strains, JJ were isolated from wrestlers, 1J8 from judoists, 

J from other martial artists, and 6 from sporadic cases. The origin of 18 strains had 

not been documented. Total DNAs were extracted from mycelia, and the NT. of rRNA 

genes amplified by PCR. The PCR products were digested with the restriction enzyme 

!0+ I and banding profiles on polyacrylamide gels were examined. 

Results: Four molecular types, NT.1-O, were detected among the 1K8 strains. NT.1 

was the most predominant type, being in 17K strains, 1J7 from judoists and 21 from 

wrestlers. NT. 2 was found in 1[ strains, 12 from wrestlers, 2 from sporadic cases 

and one from a judoist. NT.J was found in J strains, all isolated sporadically from 

elderly patients in 1K8K, 1KK0 and 2001. 

Conclusions: .ince the NT.J type >/*-%,&($+,& is never cultured from tinea 

gladiatorum in (apan, the recent tinea gladiatorum epidemic among high-school 

wrestlers and judoists was probably caused by their being infected with the NT.1 and 

NT.2 types while competing outside (apan. The epidemic may have multiple origins, 

and polyclonality among the wrestlers may be due to some of them being infected with 

NT.1 from judoists. 

P-0520. Disseminated histoplasmosis in AIDS patients in Uberaba, mg. Brazil 

Mora Amador (unior D, 2arbosa dos .antos C, MÇnica de fliveira R, Silva-Vergara 

M 

6niversidade Federal Tri°ngulo Mineiro, 6beraba, 2razil 

Introduction: From a classical endemic mycosis, histoplasmosis arose as an 

opportunistic and defining condition in AID. patients. In endemic areas its a 

prevalence rate can be [_ or more and depends on the patient]s immunodeficiency 

state. The risB to develop this mycosis is increased when the CDO count values is less 

than 100 cells x mmJ. Its course is usually subacute and its evolution to disseminated 

and severe picture is very common. The aim of this report is to show epidemiological 

clinical and evolutive aspects of patients with histoplasmosis associated to AID.. 

Populations and Methods: From 1KK2 to 200O, [7 AID. patients with disseminated 

histoplasmosis were diagnosed at teaching hospital in 6beraba, 2razil. Their medical 

records and laboratory diagnosis registers were carefully revised to obtain clinical data. 

Results: All patients presented disseminated histoplasmosis to two or more organs, 

O[ (7K_) were male being the most between 20 and O0 years old. From these, J0 

([2,[_) developed histoplasmosis together HIV diagnosis and J[ (61,[_) referred 

symptoms from 1 to O weeBs long. Fever, weight loss, dry cough and dispneia were 

the more prevalent complaints and hepatomegaly and splenomegaly were noticed in 

J8 (66_) of them. In addition disseminated and polymorphic cutaneous lesions were 

observed in 2[ (OJ,K_) of cases. /aboratory evaluationM one or more blood series 

were decreased and pancitopenia was present in 60_ of cases and /DH enzyme 

values were increased in O8 (K6,2_) patients. A x-ray chest showed intertiscial 

infiltrate in 66_ of cases. Histoplasmosis diagnosis was performed by two or more 

mycological methods in 28 (OK_) cases, and in 1J (22,8_) of them, it was confirmed 

on necropsy. From J2 ([6_) patients that had CDO count cells performed, in 2[ 

(67,8_) their values were less than [0 cells. fnly JK (68,O_) patients received 

specific therapy with Anfotericin 2, or Itraconazol or both. A cure rate of J0 ([2,6_) 

was observed and eighteen (J1,6_) patients died before the mycological diagnosis 

confirmation and did not received antifungal drugs. 

Comments: This report shows the profile of an AID. patients serie with disseminated 

histoplasmosis similar to others described. This mycosis occurs mainly in advanced 

immunodeficiency state, its course is usually acute or subacute and presents 

proteiform signs and symptoms similar to other infections diseases. Fever, 

Hepatomegaly, splenomegaly, mesenteric limph-nodes together pancitopenia, high 

/DH values, and intersticial lung infiltrate were frequently observed in most of them 

and helped to guide to investigate a fungal etiology. ahen disseminated cutaneous 

lesions appear, the histoplasmosis diagnosis probability is increased. In 2razil 

cutaneous lesions associated to histoplasmosis, have been described in frequency 

from 20 to K0_ of cases, different from 10_ observed in North America patients 

where this mycosis is highly endemic. The severity of clinical and evolutive pictures 

seen en these patients can explain the outcome observed in more of them. Eighteen 

(J1,6_) didn]t received specific treatment because they died before diagnosis or this 

was performed '%&*)%$-.)3*and K (16_) received treatment but died during it. 


P-0521. Fungi in conjunctival sacs of clinically normal cats 

Moretti A 1 , Arcelli R 2 , 2oncio / J , Tibaldini P 2 , Vergara M O , .alvatori R O , Agnetti F [ , 

Rossodivita M 6 , Vidotto V 7 

1 Faculty of Veterinary Medicine, 2 Faculty of Veterinary Medicine, Department of 

Pathology, Diagnostics and Veterinary Clinical, 6niversity of Perugia, Italy, J Faculty of 

Medicine and .urgery, Department of Medical and .urgical .pecialties and Public 

Health, Terni, 6niversity of Perugia, Italy, O Veterinary Clinician, Perugia, Italy, [ .tate 

Veterinary Institute, Perugia, Italy, 6 6niversitary Center of Electronic Microscopy 

(C6ME), 6niversity of Perugia, Italy, 7 Department of Medical and .urgical Disciplines, 

.ection of Infectious Diseases, 6niversity of Turin, Italy 

In the cat mycotic ocular diseases are not frequently diagnosed and the clinical 

lesions can involve the ocular adnexal or corneal tissues with very severe processes. 

Fungi, recovered in the conjunctiva, have been often related to the environment where 

the animals live and are responsible for clinical manifestations in presence of 

additional factors capable of influencing the fungal flora overgrowth. In this context, 

the AA., considering the paucity of reports in cats and the absence of data in our 

territory, have investigated the conjunctival fungal flora in cats free of ophthalmologic 

problems. The feline population sampled (NÅ 86) included the stray cats (NÅ O6) from 

two local shelters or found in rural areas and the private cats (NÅ O0) presented to 

private veterinary laboratories and to the Faculty of Veterinary Medicine for routinary 

controls. Conjunctival swab specimens were obtained from both eyes (NÅ 172 total) by 

a sterile culturette swab, transferred onto .abouraud]s dextrose agar petri plates and 

incubated at 26ÅC. 

78.2_ and 62.[_ of stray and private cats, respectively, resulted positive for fungi. 

Fungi were isolated from [[ of the total K2 ([K.7_) conjuctival specimens belonging to 

stray cats and from J[ of the total 80 (OJ.7_) conjunctival specimens belonging to 

private cats. Generally, 1 or 2 species of fungi were present in each positive specimen 

and 1 or 2 colonies was the number of isolants obtained. The microorganisms isolated 

represent a broad spectrum of fungi considered saprophytes and the isolated fungal 

species have different percentages in two categories considered. The fungal species 

most frequently isolated in stray cats are Penicillium (17.J_), Rhodotorula rubra 

(1J_), Aspergillus fumigatus (11.K_), Mucor (10.8_). aithin a given group of rural 

stray cats living in the same area, the animals presented the same type of fungus. In 

private cats the fungal species mostly isolated were Cladosporium (17.[_), 

Aspergillus niger (6.2_), Rhodotorula rubra and Penicillium ([_). Interesting is the 

isolation of Rhodotorula species, considered now a new emergent fungusn 

Cladosporium spp., potential pathogen under certain circumstances, can be 

responsible for clinical cases of Beratomycosis in the cat. 

P-0522. Causative agents of onychomycosis - A retrospective study 

Mügge C 1 , Haustein U 1 , Nenoff P 2 

1 Clinic for Dermatology, Venerology and Allergology, 6niversity Clinic, /eipzig, 

.axony, Germany, 2 /aboratory for Medical Microbiology, Mélbis, .axony, Germany 

Purpose of the studyM Dermatophytes, yeasts and moulds are potential causative 

agents of onychomycosis. The aim of this study was to determine the percentage of 

cases of onychomycoses caused by each group. In addition, the responsible genus 

and species was identified for each nail infection. 

DescriptionM In a retrospective study (1KK0 - 2001) performed at the Department of 

Dermatology of the /eipzig 6niversity, [077 nail samples from O177 patients - 22O0 

women and 1KJ7 men - with a variety of nail changes | not just onychomycosis | 

were investigated. 7[ _ were toenails, 2J _ fingernails, and 2 _ from both sites. 

Results and conclusionsM 2oth microscopic and/or cultural detection of fungi 

(dermatophytes, yeasts and moulds) were successful in [O _ of samples. Causative 

fungal agents wereM 68 _ dermatophytes, 2K _ yeast, and J _ moulds. The most 

frequently detected dermatophyte species were >$"#?%'?2-%, (>/J*$(8$() (K1 _), and 

>/*).,-+@$%'?2-.& G>/*",-.$5"@"-+6.3*7.7 _). Among yeasts, 7+,5"5+*(7/J*'+$+'&"6%&"& 

(O2 _) was most common, followed by 7/*@("66".$)%,5"" (20.1 _), 7/*+68"#+,& (1O.2 _), 

and >$"#?%&'%$%,*&''/ (10 _). =#%'(6+$"%'&"&*8$.0"#+(6"&*(OJ _) was the most 

frequent mould. The percentage of mixed fungal infections was 22 _. 

Dermatophytes, in particular >/*$(8$(), but also >/*).,-+@$%'?2-.&*G>/*",-.$5"@"-+6.J, 

are the most frequently isolated causative agents in onychomycosis. In addition, 

yeasts may be isolated relatively frequently, while moulds are uncommon.

P-0523. Tinea capitis in Sfax area: Epidemiological and mycological profile 

Neji S, MaBni F, CheiBhrouhou F, .ellami H, .ellami A, MarrecBchi ., Meziou (, TurBi 

H, Ayadi A 

1 /aboratory of Parasitology-Mycology, Habib 2ourguiba 6H -.fax Tunisia, 

2 Dermatology Department, Hedi ChaBer 6H .fax 

Introduction: Tinea capitis is still considered a public health problem in our country, 

and is the most common type of dermatophytosis in children. 

Purpose: The aim of this study was to determine the clinical, epidemiological and 

mycological characteristics of tinea capitis. 

Patients and methods: A retrospective study realized on 10JKK mycological 

examinations of the scalp and beard from 1KK[ to 200[. 

Results: OJK6 cases of tinea (O2.1_) were diagnosed. The prevalence was JK0 

cases /year. Trichophitic tinea decline but persist the most frequent (80.1_ in 1KK[ to 

[1.7_ in 200[), caused by >/*0"%6+#.() (KK.[_) and >/*-%,&($+,& (0.O_). 

Microsporic tinea was found in 28.2_ of the casesn due to !"#$%&'%$()*#+,"& (KK.O_), 

!/*6+,@.$%," (0.O_) and !/*@2'&.() (0.2_). An inflammatory aspect of scalp 

ringworm was found in 8.1_. OJ cases of Berions and 1J cases of sycosis (>/ 

).,-+@$%'?2-.&*[6.K_ and >/*0.$$(#%&() 27.[_) were diagnosed. [ cases of favus 

type and 10 cases of tinea due to >/*$(8$() were observed. 

The average age was 8.2 years. [.6_ of cases were diagnosed in adults. Trichophytic 

tinea was more prevalent in girls but microsporic and inflammatory types affected 

more the boys. No significant difference was observed between urban and rural 

regions. Tinea capitis was more prevalent in winter and spring. fther family members 

were infected by tinea in 8.7_ of cases 

fur patients had also circinate herpes (K.K6_). fnychomycosis was associated in 

0.[O_, but the presence of dermatophytes on nail of tinea capitis was found inJ8_ of 

cases. 

Most patients (KK_) received griseofulvin at a dose of 1[-20 mg/Bg daily for 6 weeBs. 

7.J_ of them had recurrence of tinea. 

Conclusion: In our region, >$"#?%'?2-%, spp continues to be the leading cause of 

tinea capitis. Zoophilic agents become more prevalent. This finding may be explained 

by changing behavior and activities of the population, who Bept a larger number of 

domestic animals at homes. .ome dermatophytic agents, which are uncommon 

pathogen of tinea capitis in our country, were detected. More preventive measures 

must be used, and there is a need for an increased level of surveillance. 

P-0524. Invasive aspergillosis (IA) in hematologic department: description of 

cases between 2003 and 2005 

Nicolle M 1 , Thiebaut A J , Piens M 2 , Gilibert R 1 , Vanhems P 1 , Picot . 2 , Michallet M J 

1 Hygien and epidemiology 6nit, HÇpital E. Herriot, 2 Mycology /aboratory, HÇpital E. 

Herriot, J Hematologic department, HÇpital E. Herriot 

Purpose: To describe IA cases among 2,200 patients who received intensive 

chemotherapy with or without auto- or allogeneic H.CT. 

Methods: Cases of suspected IA are collected and monthly meeting with a mycologist, 

an hygienist and an hematologist provides the opportunity of a multidisciplinary review 

of each case. Case report form allows data about demographic characteristics, risB 

factors, environnemental factors, diagnostic criteria. Clinical, mycological and CT scan 

reports are reviewed to classify cases according to EfRTC criteria (possible, probable, 

proven). 

Results: 12J IA are identified, OJ in 200J, OO in 200O, J6 in 200[, 6J males, 60 

females, median (md) age [J years (range M 18-7[). Hemathologic malignancies are M 

acute myeloid leuBaemia 100 (81_), acute lymphoid leuBaemia 1O (11_), myeloma O 

(J_), others [ (O_). .tatus are M induction 6[ ([J_), relapse 2K (2O_), allogeneic 

transplantation 1O (11_), autologous transplantation J (2_), others 12 (10_). 

Neutropenia is observed in 117 patients (K[_), with md duration of 27 days (range [- 

87), corticosteroids in 1O (11_), room with laminar-air flow (/AF) in 6O ([2_). No 

patient, excepted 2, had an interruption of isolation before IA diagnosis. Diagnosis of 

IA is done during the 1st weeB of stay for 16 (1J_) patients, at the 2nd for 12 (10_), 

at the Jrd in O0 (JJ_), at the Oth in 2O (1K_) and later in J1 (2[_). CT scan is 

performed in 11[ patients (KJ_), and a halo sign is detected in 88 cases (77_). 10[ 

mycological analysis (sputum, broncho alveolar lavage, aspiration, biopsy, ponction) 

are performed in 76 (62_) patients, O[ (OJ_) are positive (direct examination, culture, 

antigenemia test). .erum Aspergillus antigenemia are positive in 61 of 120 tested 

patients ([1_) (antigenemia is positive when more than 2 tests are above 1 ng/ml). IA 

are classified as 12 (10_) proven, 68 ([[_) probable, OJ (J[_) possible. The sites of 

IA is pulmonary for 11[ cases (KO_), sinus for J (2_) and disseminated in [ (O_). 28 

patients (2J_) needed intensive care. 18 patients (1[_) died within the 1st month 

after IA diagnosis and JK within J months (JJ_). 4&'.$@"66(&*&'.#".& is isolated in 27 

cases, 4/*U()"@+-(& in 1O ([6_). For all patients, the md time between aplasia and IA 

is 1O days (0-O6). For patients admitted directly in /AF the md time between 

admission and aplasia is O days and the md time between aplasia and IA is 1[ days 

(0-O6). 

Conclusion: In our department, CT scan are performed systematically when clinical 

signs suspected of IA are observed even if patients are in /AF. Diagnosis of IA for 

patients in /AF suggests a contamination before hospitalisation and highlights the 

complexity of analysis of the nosocomial risB about this fungal infection. 


P-0525. Microbial quality of water in dental unit waterlines 

Nikaeen M 1 , Hatamzadeh M 1 , Mirhendi . 2 , .abzevari Z 1 , Zareh f 1 

1 Isfahan university of medical sciences, Dept. of environmental health engineerin, 

2 Tehran university of medical sciences. 

Objectives: Dental unit waterlines (D6a/) are ideal environment for developing of 

bacterial biofilms. Microbial contamination of water in D6a/s is though to be the 

result of biofilm formation as it could serves as a haven for pathogens. Thus, microbial 

quality monitoring in a dental unit (D6) is of considerable importance, since patients 

and dental staff are exposed to water and aerosols generated from the D6. The aim of 

this study was to assess microbial quality of water in dental unit waterlines of 2[ 

dental units located at the dental school of Isfahan 6niversity of Medical .ciences, 

Isfahan, Iran. 

Methods: aater samples were collected from air-water syringe and high-speed 

handpiece at the beginning of the worB day after a 2-min purge, and examined for 

total viable heterotrophic bacteria, fungi and legionella. The media used in the study 

were including R2A agar, sabouraud dextrose agar and 2uffered charcoal yeast 

extract agar for bacteria, fungi and legionella respectively. 

Results: The heterotrophic plate count (HPC) levels were significantly exceeded the 

American Dental Association recommendations for D6a/ water quality (v200 

CF6/ml), in both air-water syringe (8O_, CF6/mlM [00-20000) and high-speed 

handpiece (K6_, CF6/mlM 710-J6800) samples. However, there was no significant 

statistical difference between the level of contamination in the air-water syringe and 

high-speed handpiece. Fungal elements were found in 28_ and J6_ of air-water 

syringe and high-speed handpiece samples respectively and filamentous fungi were 

the most frequently isolated fungi. All water samples were negative for legionella spp. 

Conclusion: D6a/s should be subjected to routine microbial monitoring and to a 

decontamination protocol in order to minimize the risB of exposure to potential 

pathogens from dental units. 

P-0526. Mating behavior of Schizophyllum commune isolated from patients 

with brochopulmonary mycosis in Japan with epidemiological data 

Nishimura K, lamei l, .ano A, Miyaji M 

Research Cneter for Pathogenic Fungi and Microbial Toxicoses, Chiba 6niversity, 

Chiba, (apan 

The basidiomycetous fungus =#?"d%'?266()*#%))(,. has recently emerged as a 

new causative agent of human mycosis, mainly of sinusitis or sinus fungus ball. 

However, we have identified isolates from J[ patients with bronchopulmonary 

infections and one from allergic nasal sinusitis as being the basidiomycete since 1K8K. 

This time, we report the morphology, nuclear phase and mating behavior of these 

clinical isolates, and some epidemiological data. 

Female patients outnumbered male ones (26 M10) and middle-aged and elderly were 

predominant in age distribution. Nineteen out of the J[ bronchopulmonary diseases 

were allergic brochopulmonary mycosis (A2PM)n [, mucoid impactionn J, eosinophilic 

pneumonian J, pulmonary mycosis and pneumonian 2, fungus balln and one each, 

abscess, infected bronchial cyst and nodular lesion. Most patients had no underlying 

diseases except for bronchial asthma. 

Five monoBaryotic and three diBaryotic strains of =/*#%))(,. were used as testers. 

Each clinical isolate was confronted with each one of the testers on PDA plates, 

placed in transparent plastic boxes and incubated at 2[ÅC in darB for seven days and 

then at room temperature on a laboratory table. Most isolates formed white to buff, felt 

or floccose colonies with a methane-liBe odor and a few formed a slow-growing, light 

brown or darB bluish-brown, leathery colony. Hyphae were partially tuberculate in J2 

isolates and bizarrely zigzag in two of the non-tuberculate four. fut of the J6 isolates, 

eight developed clamped hyphae, reproduced small, fan-liBe, gilled and/or medusoid 

or coralloid basidiocarps or pill-liBe basidiocarp initials and diBaryotitized the 

monoBaryotic testers. Hyphae of the other 28 were not clamped. These non-clamped 

isolates made the monoBaryotic testers develop clamped hyphae and then 

basidiocarps with basidiospores. Most of them were also diBaryotitized by the 

diBaryotic testers and reproduced basidiocarps. 2ased on these results, the 8 

clamped and the 28 non-clamped were the diBaryon and monoBaryon of =/*#%))(,., 

respectively. These monoBaryotic isolates mated other clinical monoBaryotic ones, 

showing that they have different mating types from each other. 

.ince 1K[0, 6 monoBaryotic and 1O diBaryotic isolates of =/*#%))(,. have been 

reported as pathogens mainly for allergic sinusitis or sinus fungus ball from western 

countries. .ome patients had underlying diseases such as AID. and diabetes mellitus. 

MonoBaryotic isolates reported recently were identified with a DNA analysis. fnly 

.igler et al. identified a monoBaryotic isolate by a mating study in 1KK[. In contrast, 

almost all of =/*#%))(,. infections are A2PM and related diseases in (apan and 

those due to the monoBaryotic isolates have occurred J-times more at incidence than 

the diBaryotic ones. It is liBely that basidiospores of the mushroom are inhaled to 

cause bronchopulmonary infections due to the monoBaryotic mycelium. 2oth the 

mono- and diBaryons of the basidiomycete are important as one of the pathogens and 

the former is rather important than the latter in bronchopulmonary mycosis. It is also 

remarBed that =/*#%))(,. infections are apt to occur in middle-aged and elderly 

females.

P-0527. Epidemiology of fungal patient’s colonisation in haematology 

department observed during ”one day” investigations 

NowicBa ( 1, 2 , Nawrot U J , latarzyna a J , lazimierz l 1 

1 Department of Haematology, 2 Department of Clinical Chemistry, J Department of 

Microbiology Medical 6niversity of aroclaw 

Patients with haematological malignancies are high-risB group for invasive mycoses 

due to filamentous as well as yeast-liBe fungi. The exogenous (environment, air, 

water) and endogenous (e.g. gastrointestinal tract) sources of invasive fungal 

infections have been postulated. In order to control fungal contamination of the air in 

hospitales rooms as well as colonisation/infection of patients we performed 

mycological examinations of clinical samples taBen at the same day. Investigated 

materials included air samples from patientes and operative rooms, and faeces, 

sputum and urine samples obtained from all patients of Haematological Department, 

who agree participate in the project. In this study we analysed the results of Äone-dayÄ 

investigations performed in two-year interval (26.11.2002 and 2[.11.200O). 

PatientsM Investigation I M 26.11.2002 - J6 patients were examined (22 women and 1O 

man aged 1K-76), of them 17 with acute leuBaemia, 18 with lymphoproliferative 

malignancies and 1 with myelodysplastic syndrome. J2 persons were hospitalised in 8 

rooms (J-[ person per room) and O persons in single rooms. Patients with acute 

leuBaemia received Betoconazole and O patients after or before autoP.CT oral form 

amphothericine 2. fne patient with F6f and suspected for deep-seated mycoses 

was treated by amphotericine 2. 

were Rhodotorula sp (2 strains), Cryptococcus laurentii (1), Penicilium sp (J), and 

Aspergillus fumigatus (2 isolates from faeces and sputum of one patient). 

Air examinations revealed the presence of 1-[ CF6 per room (12-60 CF6 /mJ). The 

isolated fungal genera were Penicilium spp and Aspergillus spp (A. fumigatus not 

found). 

Investigation IIM Fungi were find in 1K/J0 (6J.J_) sputum samples, 17/28 (62.K_) 

faeces, and O/J2 (12.[_) urine. A total number of 61 strains were isolated, of them 2K 

from sputum, 28 from faeces, and O from urine. [J/61 (87_) strains were identified as 

Candida, of them J0 (OK.1_) C. albicans and 2J (J7.7_) Candida non-albicans (C. 

glabrata-0, C. Brusei-2). The remaining species were Rhodotorula sp (2 strains), 

Geotrichum sp. (2), Penicilium sp (J), and Aspergillus fumigatus (1). 

Air examinations revealed the presence of fungi in O/K patientes rooms (1-2 CF6/ 

room). The isolated fungi were Penicilium spp, Candida sp. and Aspergillus sp. 

Conclusions: The percentage of patients colonized by fungi were similar in both 

investigations. Candida albicans is dominant species and reduced participation of 

non-albicans Candida, especially fluconazole -resistant (C. glabrata, C. Brusei) in 

colonisation of gastrointestinal and respiratory tracts of hospitalized patients was 

observed. 

Investigation IIM 2[.11.200O - study group includes J2 patients (22 women and 1O man, 

aged 2J-8O), of them 18 with acute leuBaemia and 1O with lymphoproliferative 

malignancies. 2K persons were hospitalised in K rooms (J-[ person per room) and J 

person in single-rooms. Patients with acute leucaemia received Betoconazole, 2 

patients were treated with fluconazole and 2 with itraconazole. Four patients display 

F6f, invasive fungal infections were later diagnosed in J of them. 

Methods: Air samples were collected early in the morning, in the presence of the 

patients by the use of the passive- sedimentation method on .abouraudchloramphenicole 

plates. 

The number of clinical samples obtained was O6/108 (sputum-1K, faeces-22, urine-[) 

during first- and 8K/K6 (sputum-J0, faeces-27, urine-J2) during second investigation. 

Faeces and sputum samples were incubated with 0.2[_ trypsyne and then cultured 

quantitatively on .aboraud-chloramphenicole and Chromagar medium. Fungal 

cultures were identified on the basis of morphology and biochemical data obtained by 

IDJ2C tests (2ioMerieux, France). 

ResultsM Investigation I M Fungi were cultured from 12/1K (6J.2_) sputum samples, 

16/22 (72.7_) faeces, and 2/[(O0_) urine. The number of CF6 per 1gram of faeces 

samples ranged from 10 2 (8 patients) to 10 [ (O patients), and the number of cfu per 1 

ml sputum ranged from 10 2 (8 patients) to 10 O (J patients). A total number of O7 strains 

were isolated, of them 17 from sputum, 28 from faeces, and 2 from urine. JK/O7 (8J_) 

strains were identified as Candida, of them 18 (J8.J_) C. albicans and 21 (OO.7_) 

Candida non-albicans (including C. glabrata-K, C. Brusei-1). The remaining species 


P-0528. Dermatomycoses in the Pomeranian province (Poland) in the years 

2003-2005 

Nowicki R 


Introduction: .uperficial fungal infections are very common in Poland. Periodic 

reviews of infections fungal species have significant epidemiological importance. 

Aim of the study: A study was conducted to determine the main agents of superficial 

mycoses among inhabitants of the Pomeranian province in the years 200J-200[. 

Material and methods: Data were collected over a J-year period from mycologic 

investigations made in the Mycology /aboratory at the Department of Dermatology, 

Venereology and Allergology Medical 6niversity of GdansB (Poland). A total of O 017 

patients clinically suspected of sBin and their appendages infections were mycological 

examined during the period between (anuary 200J and December 200[. 

Results: In 1 20K cases (J0.1_) the mycosis was confirmed with positive 

mycologic exams. The fungi cultivated included dermatophytes (n } [81n O8.1_), 

yeasts (n } [O7n O[.2_), and nondermatophyte filamentous fungi (n } OKn O.1_). 

7+,5"5+*+68"#+,& (22.7_) was the most prevalent, followed by 7+,5"5+ spp (21.O_), 

>$"#?%'?2-%,*$(8$() (21.K_), and >/).,-+@$%'?2-.& (17.2_). The most frequently 

observed superficial mycoses among inhabitants of the Pomeranian province wereM 

toenails onychomycosis (n } [1On O2.[_), fingernails onychomycosis (n } J1[n 

26.1_), tinea pedis (n } 101n 8.O_)), and tinea cutis glabrae (n } KJn 7.7_). Fungal 

infections dominated among townspeople (86.[_) and in adults patients (8K,6_). 

Conclusions: The toenails onychomycosis was the most frequent dermatomycosis in 

the Pomeranian province . The main agents of superficial mycoses among inhabitants 

of the Pomeranian province in the years 200J-200[ were dermatophytes and yeastliBe 

fungi. 

References: 

1. Ghannoum M A , Hajjeh R A , .cher R at al. A large-scale North 

American study of fungal isolates from nailsM The frequency of 

onychomycosis, fungal distribution, and antifungal susceptibility patterns. 

( Am Acad Dermatol 2000n OJM6O1-6O8. 

2. /ange M., NowicBi R., 2ara,sBa-RybaB a at al. V.$)+-%'?2-%&"&*",* 

#?"65$.,*+,5*+5%6.&#.,-&*",*95+"&[3*1%6+,5. Mycoses, 200O, O7,J26- 

J2K. 

J. /ehenBari E, .ilvennoinen-lassinen .. V.$)+-%'?2-.&*",*,%$-?.$,* 

^",6+,5*",*IRDMWRQ. Mycoses 1KK[n J8MO11-O1O. 

O. NowicBi R.M V.$)+-%'?2-%&.&*",*-?.*95+"&[*+$.+3*1%6+,5i*+*IM*W*2.+$* 

&($0.2. Mycoses, 1KK6. JK, JKK-O02. 

.ingh D, Patel DC, Rogers l, at al. a'"5.)"%6%@2*%U*5.$)+-%'?2-.*",U.#-"%,*",* 

4(#[6+,53*`.B*L.+6+,5/*Austral ( Dermatol. 200JnOOM 26J-266. 

P-0529. Age and occupational distribution of genitourinary Candida infection in 

selected cities in Nigeria 

Okungbowa F 1 , fBungbowa M 2 

1 6niversity ff 2enin, 2enin City, Nigeria, 2 .chool of Medical /aboratory .ciences, 

6niversity of 2enin Teaching Hospital,2enin City, Nigeria 

Purpose of study: 7+,5"5+ species are distributed worldwide. They occur as 

harmless saprophytes in man. However, when an individual]s micro flora balance is 

disrupted by factors such as drug misuse, obesity, diabetes, use of contraceptive pills, 

pregnancy and suppressed immune system (as in HIV-infected persons), 7+,5"5+ 

becomes pathogenic. 7+,5"5+ is, therefore, an opportunistic pathogen. fnce it has 

established its pathogenicity, 7+,5"5+ in the genitourinary tract can be spread very 

fast through sexual interaction, causing great discomfort and sometimes, reproductive 

problems if not well treatedn men do not usually show symptoms and as a result, it is 

very easy for them to spread the disease. Genitourinary 7+,5"5+ infection occurs in 

7[_ of women of child-bearing age at least, once in a life time. However, recurrence 

is very common. Added to this is that many 7+,5"5+ species are now resistant to 

commonly used antifungal drugs. 7+,5"5+ infection is one of the first opportunistic 

diseases in HIV/AID. patients worldwide. Its infection of the genitourinary tract is 

both a social and a public health problem. This study was carried out to determine the 

distribution of 7+,5"5+ infection among different age and occupational groups in 

individuals with symptoms of the disease visiting hospitals and medical diagnostic 

laboratories in some Nigerian cities. 

Description of project: A clinical survey was carried out in seven cities. Genital 

specimens were collected from the patients and patient information, such as age, 

occupation and sex were documented on site. The specimens were examined 

microscopically (both as direct mount wet preparations and after growing on culture 

media). 

Results and conclusions: .even 7+,5"5+*species were isolated and identified, with 

7/*@6+8$+-+ having the highest frequency of J0_ followed by 7/*-$%'"#+6"& (2O.[_), 

while 7/*&-.66+-%"5.+ had the lowest frequency (0.[_). The age group 26-J0 years had 

the highest occurrence (J2.7_) followed by age 21- 2[ years (21.1_) and the least, 

J6-O0 years (8.[_). The difference in distribution between age 26-J0 and 21-2[ was 

insignificant. Among the occupational groups, traders were significantly the most 

affected (20.J_) followed by jobless graduates (17.J_) while full-time housewives 

had the lowest incidence (J.6_). Ready availability of cash for medicare might have 

influenced the seemingly high incidence in traders. The age groups with the highest 

incidence here are those usually reported to be the most sexually active people. 

.ocial factors such as sexual promiscuity and drug misuse might have played a role in 

the observed infection rates among different age groups, while finance and attitude 

towards health-seeBing affected the occurrence in occupational groups. These social 

factors are also largely responsible for the prevalence of other sexually transmitted 

infections, including HIV/AID. and should be addressed in disease control strategies. 

It also supports previous reports that several hitherto uncommon 7+,5"5+ species are 

now being isolated and may partly explain the increasing cases of resistance of 

7+,5"5+ species to antifungal drugs.

P-0530. Poor personal hygiene and blind antimicrobial treatment: The 

predisposing risk factors for prevalence of vulvovaginitis in developing 

countries 

Olawuyi O 1 , Falegan A 2 , flawuyi T J 

1 Medical Microbiology, 6niversity College Hospital, Ibadan, Nigeria, 2 Dental Clinic, 

6niversity College Hospital, Ibadan, Nigeria, J Advocacy for Human Education, 

Awareness and Development 

Issue: Vulvovaginitis is a candidiasis form, caused by ova, budding, yeast-liBe fungus 

s7+,5"5+*+68"#+,&u which is counter activated by high PH and vaginal normal flora, 

X+#-%8+#"66"*+#"5%'?"6(&. This abstract shows that the disease is promoted in 

developing countries by poor personal hygiene and blind and/or self antimicrobial 

treatment of young women developing countries. 

Description: The sample size of 1200 respondents of the young women, age range, 

16-J[, was selected through simple random sampling technique from different places 

shigher institutions, worBing places, marBet places and recreational places u within 

different major citiess Ibadan, /agos and 2eninu in Nigeria. The mean age is 2[.[ . A 

well developed validated and reliable questionnaire sr} 0.88u was used to collect the 

data needed for the study and percentage was used to analyze the data. Relative 

RisB sRRu calculated is 1.K0, i.e. RR t 1, indicating that the factors are risB factors, 

and the Confidential IntervalsCIu for RR at K[_ .ignificant level is 1.[6v 1.K0v1.7[ 

from the formula, CI /ower limit v RR v CI 6pper limit. RR and CI are used to 

validate the instruments. 

P-0531. Distribution of Cryptococcus neoformans in the environment of Betul, a 

city of m.p in India 

Pandey A 

(.H.GfVT .Cf//EGE .2ET6/(MP) 

In the present study distribution of 7$2'-%#%##(&*,.%U%$)+,& in the environment of 

2etul a city of M.P in India was investigated. fut of 2O[ soil samples investigated, J8 

(1[.[_) samples were found positive for the pathogen. fut of J8 positive human 

urine soaBed sites, 18 (O7_) were positive for 7/ ,.%U%$)+,& Var. ,.%U%$)+,& and 20 

([2_) for 7/*,.%U%$)+,& Var. @+--""/ fut of 1[ samples investigated from ceiling and 

walls of patients room in the hospital and nursing homes of 2etul , 6 (O0_) were 

positive for 7/*,.%U%$)+,& Var. ,.%U%$)+,& and K (60_) for 7/*,.%U%$)+,& Var/** 

@+--"". The present investigation revealed that human urine soaBed soil could serve as 

a reservoir of both varieties of 7/*,.%U%$)+,& in the urban areas of India. 

Result: The two validated tables drawn after the study revealed thusM Table 1 

revealed that the personal hygiene was not adequate among young women in 

developing countries. Table 2 showed that about 80_ of the respondents had used 

one antimicrobial agent or the other to treat one 6TI/.TD. s i.e. self medicateu, 

without physician consultation and prescription. 

Conclusion: The high increase of Vulvovaginitis, a Genourinary Tract infection 

sG6TIu among young women in developing countries is reinforced by poor personal 

hygiene coupled with inadequate Bnowledge about the disease and blind and/or self 

antimicrobial treatment. 


P-0532. Pneumocystosis diagnosis in venezuelan patients (2001-2005) 

Panizo M, Reviakina V 

INHRR, Caracas, Venezuela 

Purpose of the study: To determine the incidence of Pneumocystosis in Venezuelan 

patients during [ years (2001-200[). 

Description of the project: ae reviewed retrospectively the mycological records of 

patients that accede to the Mycology Department of Instituto Nacional de Higiene Rafael 

Rangel (INHRR) for Pneumocystosis diagnosis, referred from four hospital centers (J 

publics and 1 private). All patients presented acute pulmonary disease symptoms and were 

divided in J groupsM I group-AID. patients, II group-Cancer patients and III group-noncancer 

non-AID. /ower Respiratory Tract Infection (/RTI) patients. The specimens 

processed were sputa (spontaneous and induced) and bronchoalveolar lavage (2A/). 

Direct Immunofluorescence (DIF) technique was carried out using Merifluorw 

Pneumocystis (Meridian 2ioscience, Inc) diagnostic Bit, with some modifications in the 

sputum processing technique. 1,2 Percentage incidence of Pneumocistosis in the three 

patients groups was calculated. 

Results and Conclusions: 120 patients were studiedM I groupM n}JK(J2.[_)n II group 

n}17(1O.2_) and III group n}6O([J.J_). 72.[_ were male sex and 27.[_ female sex of 

the overall studied patients, with O8 years r 18 (10-86) median age. 27(22.[_) of them had 

1,.()%#2&-"&*\"$%0.#""*pneumonia (PCP). The pneumocystosis diagnosis between the 

groups was distributed as followingM I groupM J[.K_n II groupM J[.J_ and III groupM 10.K_. 

J/O (7[_) patients with HodgBin lymphoma had PCP and J/1J (2J_) patients with solid 

tumors had PCP in II-group. fne patient had PCP and pulmonary tuberculosis and another 

one had PCP and chronic obstructive pulmonary disease (CfPD). According the results of 

this worB, PCP is more frequent in cancer and AID. patients and in a smaller proportion in 

non-cancer non-AID. /RI patients. DIF technique in sputum and 2A/ specimens is the 

recommended procedure for PCP diagnosis. 1-O Few data exist about PCP in /atin America 

and particularly in Venezuela. [ The Mycology Department of INHRR is the national 

reference center in mycology diagnosis and this casuistry contributes to the Bnowledge of 

this pathology in our country. 

References: 

1. 2orelli l, 2rito A, Rivas G, Panizo MM, RoldZn Y. Diagnástico de 1,.()%#2&-"&* 

#+$",""M Estudio comparativo entre inmunofluorescencia directa y la coloracián 

histolágica de Gomori-Grocott. 2ol .oc Ven Microbiol 2000n 20(1)M O6-[2. 

2. Panizo M, ReviZBina V, VZzquez C. Diagnástico de 1,.()%#2&-"&*#+$","" por 

inmunofluorescencia directa modificada y coloracián de Gomori-Grocott. Estudio 

comparativo. 2ol .oc Ven Microbiol 2000n 20(2)M K8-10J. 

J. Thomas CF, /imper AH. Pneumocystis pneumonia. N Engl ( Med 200On J[0M 

2O87-K8. 

O. Cruciani M, Marcati P, Malena M, 2osco f, .erpelloni G, Mengoli C. Metaanalysis 

of diagnostic procedures for 1,.()%#2&-"&*#+$","" pneumonia in HIV-1- 

infected patients. Eur Respir ( 2002n 20M K82-K. 

[. Morris A, /undgren (D, Masur H, aalzer PD, Hanson D/, FredericB T, Huang /, 

2eard C2, laplan (E. Current epidemiology of Pneumocystis pneumonia. Emerg 

Infect Dis 200On 10(10)M 171J-20.0 

P-0533. Development of a novel informatics system for the detection and 

prevention of invasive fungal infections (IFI): The organ transplantation 

infection prevention (OTIP) project 

Park B 1 , /amias M 1 , Agyeman l 1 , Pappas P 2 , Paterson D 2 , Nordenberg D 1 , FridBin . 1 

1 Centers for Disease Contol and Prevention, Atlanta, GA, 2 6niversity of Alabama, 

2irmingham, A/, J 6niversity of Pittsburgh, PA 

Background: Fungal infections are a major cause of morbidity and mortality among 

organ transplant recipients, and methods to identify at-risB patients are needed. fTIP 

is a multiyear prospective cohort study designed to evaluate early marBers for IFI and 

establish a specimen repository among high-risB transplant recipients. In order to best 

address the complex methodology of the study and maximize efficiency and data 

quality, we developed a novel informatics system to integrate clinical data and 

specimen collection. 

Methods: Patient enrollment began in spring 2006. /ung and hematopoietic stem 

cell transplant patients are followed longitudinally and have clinical data and serial 

specimen collection, including serum, whole blood, bronchoalveolar lavage, biopsy, 

culture, and autopsy specimens, at intervals varying from weeBly to monthly until 18 

months post-transplantation. All specimens are aliquoted and stored in an off-site 

warehouse. Infectious syndromes are defined using standardized criteria, and IFIs 

are identified according to revised EfRTC/ M.G definitions. 

Results: Custom data collection software was developed by .P.., Inc. using the 

Dimensions platform. Five different forms that correspond to different periods during 

the transplantation course (pre-enrollment, enrollment, transplantation, assessments, 

and infections), and two modules (diagnostic procedures and medications) were 

designed to accommodate prospective, serial data collection. Internal validation rules 

insured data consistency and integrity. .pecimen collection, label generation, and 

tracBing were designed to be electronically integrated and linBed to clinical data. A 

centralized server was established to facilitate questionnaire updates and perform 

secure electronic data submission to CDC. Tablet laptop computers were utilized to 

maximize portability and efficiency. 

Conclusions: ae describe a novel approach to develop a complex prospective 

cohort study with specimen collections by utilizing information technology. This 

collaboration may serve as a model to insure high efficiency and data quality of future 

such studies.

P-0534. Phenotyping of Sporothrix schenckii isolates by geographic region in 

Brazil 

Pires de Camargo Z 1 , Ferreira Fernandes G 2 

1 6NIFE.P/ Cellular 2iology Division/.Éo Paulo, 2razil, 2 6NIFE.P/ Cellular 2iology 

Division/.Éo Paulo, 2razil 

.porotrichosis is a subcutaneous mycosis which is particularly common in /atin 

American countries with temperate, tropical, subtropical and equatorial climates. The 

disease is common in 2razil, which has an area of 8 [1O 20O.8 Bm2 and is divided 

into the following [ distinct geographic regions, each with its own different climateM 

North, Northeast, Midwest, .outheast and .outh. Parts of the .outheast and all of the 

.outh of the country are influenced by polar air masses, whereas the North and 

Northeast are influenced by hot air masses. 

Aim of the study: this study aimed to determine the phenotypic relatedness (conidial 

size and thermotolerance by percent growth inhibition at J[oC and J7oC) among =/* 

&#?.,#["" isolates from the North, Northeast, Midwest, .outheast and .outh of 2razil. 

fne hundred and twenty-two =/*&#?.,#["" isolates from these [ regions were studied. 

A comparative analysis of the profiles of protein/glycoprotein molecules secreted by 

isolates from these distinct regions was carried out. 

Results: No significant difference in mean conidial size ( in mm 2 ) (mean r/- .D) 

between =/*&#?.,#["" isolates from the North (2.[2r/- 0.J7), Northeast, (2.27r/-0.J8), 

Midwest (2.1J r/- 0.J2), .outheast (2.O0r/- 0.[6), and .outh (2.[[r/- 0.KJ) was 

observed. Two isolates, however, presented conidial areas of [ mm 2 , and O isolates 

presented conidial areas of 1.7 mm 2 . 

The growth inhibition percentages (mean r/- .D) observed at J[oC in isolates from 

the five regions were as followsM North ( [.8[_ r/- 2.18_), Northeast ( OK.K_ r/- 

17.K_), Midwest (J6.2[_ r/- 20.06_), .outheast (O2.6_ r/- 1[.O_) and .outh 

(OO.0_r/- 18.O_)n the corresponding figures at J7oC wereM North (28.K_ r/- 7.70_), 

Northeast ( 72.7_ r/- 8.[O_), Midwest (6[.O_ r/- K.61_), .outheast ( 60.0_ r/- 

1J.2_) and .outh (60.[_ r/- 12.J_). The North showed a significant difference 

compared with all the other regions at both J[ o C and J7 o C. The Northeast only 

showed a significant difference from the .outheast and .outh at J7 o C. 

The protein/glycoprotein profiles differed between isolates irrespective of the region. In 

addition, the protein profiles varied according to the culture medium used (.abouraud 

broth, Minimal Medium broth MM, and M1KK broth). 

Conclusion: This study showed that the thermotolerance of =/*&#?.,#["" isolates in all 

regions of 2razil except the Northern ones, is very similar. No significant difference in 

conidia size was found among regions, whereas the protein profiles of the isolates 

varied inside the same region and were similar in all regions. 

Acknowledgment: This worB was supported by the FundaÑÉo de Amparo Ö Pesquisa 

do Estado de .Éo Paulo - FAPE.P. Proc. no 0O/1O270-0. 2razil. 

P-0535. Risk factors and attributable clinical and economic consequences of 

Candidaemia in Australian ICU patients 

Playford G 1,2 , Marriott D J , Nguyen ~ J , lesby C O , Craig ( 2 , .orrell T 2,O 

1 Princess Alexandra Hospital, 2risbane, Australia, 2 6niversity of .ydney, .ydney, 

Australia, J .t Vincentes Hospital, .ydney, O aestmead Hospital, .ydney, Australia 

Background: No data is available regarding the risB factors and attributable 

consequences associated with Candidaemia amongst Australian IC6 patients. Given 

differences in case-mix and therapeutic practices, the generalisability of overseas risB 

factor and outcome studies of Candidaemia in IC6 patients may be limited. 

Methods: fver a three-year period, all patients with IC6-acquired Candidaemia 

(together with 2 controls matched for age, sex, admission APACHE II score, 

admission diagnosis, and duration of time at risB) in 6 Australian mixed 

medical/surgical IC6s were included in a case-control study. Time-dependant clinical, 

physiological, and microbiological parameters were collected throughout patients] IC6 

admission. RisB factors for Candidaemia and crude and adjusted mortality and lengths 

of stay (/f.) were calculated using multivariate logistic regression and Cox]s 

proportional hazards model. 

Results: JJ IC6 patients and 6O matched controls were included. Median time to 

Candidaemia was 10 days (I~R 6-1O.[ days).Candidaemic patients were significantly 

more liBely to have preceding coagulopathy (fR 2.62, K[_CI 1.07-6.O), renal 

dysfunction (J.J2, 1.J7-8.01), hepatic failure (2.O[, 1.0-6.0), nosocomial bloodstream 

infection (2.7, 1.0-7.26), or receipt of aminoglycosides (2.OK, 1.0J-6.02), TPN (O.OK, 

1.[6-12.K[), or insulin (2.J, 1.0-[.O[) on univariate analysis. The presence and 

duration of a tracheostomy was inversely associated with Candidaemia (0.2[, 0.08- 

0.8). The presence and density of colonisation were not associated. Changes in organ 

failure scores, but not APACHE II scores, throughout IC6 admission were associated 

with the development of Candidaemia. fn multivariate analysis, hepatic failure ([.12, 

1.OO-18.21) and ventilation using an ETT (rather than a tracheostomy, 6.62, 1.O1- 

J1.2[) remained significantly associated. 

Amongst Candidaemic patients, crude IC6 mortality (J1_ versus K_n fR O.2, K[_CI 

1.J7-12.K) and hospital mortality (J8_ versus 1O_, J.[, 1.28-K.OK) were significantly 

greater than in controls. Candidaemia was also associated with significantly prolonged 

crude IC6 /f. (median /f. 20 days versus 1J daysn difference [ days, K[_CI 1-11 

days) and hospital /f. (72 days versus O[ daysn difference 17 days, K[_CI 1-J[ 

days). 2oth mortality and /f. remained significantly associated with Candidaemia 

after adjustment for confounders. 

Conclusions: Amongst Australian IC6 patients, hepatic dysfunction and prolonged 

ventilation with ETT were significant predictors of Candidaemia. Clinical and economic 

consequences of Candidaemia were high and justify studies of prophylactic or preemptive 

antifungal strategies in high-risB patients. 


P-0536. Epidemiological trends of candidemia in the tertiary-care hospital of 

Cruces, Barakaldo (Spain) 

Quindós G 1 , AlBorta M 2 , RodrÜguez-Andrds C J , 6llibarri 2 2 , HernZndez-Alcaraz ( 2 

1 Dept. Microbiology, Fac. Medicine, 6niv. PaÜs Vasco, 2ilbao, .pain, 2 Clinical Microbiology 

.ervice, Hospital of Cruces, 2araBaldo, .pain, J Dept. Preventive Medicine, Fac. Medicine, 

6niv. PaÜs Vasco, 2ilbao, .pain 

Invasive candidiasis is an important cause of nosocomial morbidity and mortality. 

6nderstanding incidence trends, predominant species, and mortality associated with this 

infection can help to guide treatment and prophylaxis. 

Purpose: To study the trends and overall incidence of candidemia in a large tertiary-care 

.panish hospital, the incidence by hospital ward and by the different species of 7+,5"5+, 

and to evaluate predisposing factors for development of invasive candidiasis. 

Methods: In a retrospective study, we have evaluated the etiology of candidemia in 

patients looB after at the hospital of Cruces (2araBaldo, 2asque Country, .pain) over a sixyear 

period (1KKK-200O). The material included blood cultures carried out at the 

Department of Clinical Microbiology, during the study period. Microbiological findings were 

compiled from our laboratory information system. ae examined species prevalence in the 

different hospital wards and the risB for infections by Candida spp. Hospitalized patients 

that developed signs and symptoms of bloodstream infections were screened for 

candidemia and were analyzed for the various predisposing factors liBe the sex and age of 

the patient, the duration of hospitalization, hospital ward, among others. 

Results: In total, 2O8 episodes of candidemia were identified (211 in adults and J1 in 

children | 11 candidemia episodes were recorded at the Neonatology 6nit). The average 

incidence was 1.J new episodes/10,000 patient-days of stay in hospital (range 1.1 | 1.O 

episodes). 2ut this average incidence was significantly higher in children (incidenceM 1.K7). 

Moreover, candidemia adjusted relative risB estimated by a Poisson model was 1.8O higher 

for men than for women. During all the period of study, the incidence of candidemia was 

very similar (incidence per 10,000 patient-daysM 1.7 in 1KKK, 1.2 in 2000, 1.6 in 2001, 1.O in 

2002, 1.6 in 200J and 1,O in 200O). Candida non-C. albicans species were more frequently 

isolated than C. albicans ([7.7_ versus O2.J_). fverall, O2.J_ of the fungemia episodes 

(10[/2O8) were due to C. albicans, followed by C. parapsilosis (J6.J_), C. glabrata (K.7_), 

C. tropicalis (O.O_), C. Brusei (O_) and other species (O.J_). C. parapsilosis was the most 

frequent species recorded in 1KKK (1K out of OK isolates, J8.8_). In 2000, C. albicans and 

C. parapsilosis were isolated from an equal number of blood cultures (1J/J2, O0.6_ each 

species). However, in the rest of the studied years, C. albicans was the most prevalent 

species, ranging from [7.[_ of isolates in 2002 to JK_ of isolates in 200O. The vast 

majority of isolates ({8[_) were susceptible to fluconazole. Candidemia was most frequent 

recorded at 6CI (16.[_), General .urgery (16.1_), Internal Medicine ([.2_), Neonatal 

6CI (O.O_), Pediatric fncology (J.2_) and fnco-Hematology (J.2_) wards 

Conclusion: These results provide baseline results on the importance of candidemia in 

different hospitalized patient populations. Moreover they underline the higher incidence of 

candidemia in children and confirm the etiologic importance of non-C. albicans species. 

P-0537. Relevance of models on the evolution of fungal pathogens 

Rainer J, ZacBe A, /acBner E, laltseis ( 

Institute of Microbiology, /eopold Franzens 6niversity InnsbrucB, TechniBerstr. 2[, 

6020 InnsbrucB, Austria 

Among opportunistic pathogenic fungi 1&.(5+66.&#?.$"+*8%25"" (.hear) McGinnis et al. 

is outstanding. It causes subcutaneous but also cerebral infections in humans. The 

supposed routes of infection are traumatic inoculation, inhalation, and aspiration of 

contaminated water. The abundance of 1/*8%25"" in the environment is documented 

scarcely even though it is an important feature for epidemiology. Ecological data on 

strains obtained from culture collections and publications suggest that hpolluted 

environmentsi are the main habitats. aithin this study, the role of some abiotic factors, 

which could influence the occurrence and distribution of 1/*8%25"" in human-impacted 

environments, should be determined. Moreover, parameters that could enhance the 

preponderance of certain, possibly more virulent strains within populations should be 

defined. 

For the laboratory model, we used six strains of the 1/*8%25"" complex and one of 

each =#.5%&'%$"()*'$%6"U"#+,& (Hennebert c 2.G. Desai) E. Gudho c de Hoog, 

4($.%8+&"5"()*'(66(6+,& (de 2ary) G. Arnaud and a_%'?"+6+*5.$)+-"-"5"& (lano) de 

Hoog. The strains were originally isolated from human infections and soil samples. 

The strains were exposed to different concentrations of various N- and P- sources.* 

Competitive abilities of strains were tested by confronting strains with each other on 

solid complex media (MEA), on solid media with lNf J and in liquid media with lNf J 

and H 2 Pf O as sole N- and P-sources. The strains and populations after the 

experiment were characterised by molecular fingerprinting and in special cases by 

AF/P. 

Results show, that nitrogen and phosphor sources do have a selective effect but - 

compared to elevated temperature - are probably not the most selective factors. In this 

study we found that elevated temperatures amplify the selective effect of N- and P- 

sources. In liquid cultures, the formation of ascomata was not observed in contrast to 

solid media. It is Bnown, that ascomata are preferably produced at the margins of oatparticles. 

From that it can be concluded, that formation of ascomata and also 

recombination events are more frequent in soil than in water. 

It can be derived from the presented data that elevated temperature may serve to 

change population composition and amplify the effects of human activities. This may 

especially be true for urban environments, where mean temperatures are generally 

higher than outside cities. The experiments show an interaction between factors liBe 

nutrient availability for certain strains and temperature. This probably leads to a 

selection towards a higher proportion of thermotolerant species in a habitat or strains 

in a population. Thermotolerance is an essential character of human pathogenic fungi. 

Elevated temperatures, either temporary or constant, could be achieved via 

composting processes or by the impact of hglobal warmingi liBe elevated mean 

temperature or hot summers. aith the presented study we show that laboratory 

experiments can provide useful data for further modelling, even though some 

questions in the special case of 1/*8%25"" still have to be answered.

P-0538. A prospective study of Malassezia species colonization of 

neonatal skin 

Ran Y, Chen Z, jiong /, Dai Y, Zhou G 

Department of Dermatology, aest China Hospital, .ichuan 6niversity, Chengdu, 

China 

Purpose: The genus*!+6+&&.d"+ includes the pathogens responsible for pityriasis 

versicolor and !+6+&&.d"+ folliculitis. Members of this genus have also been 

implicated in seborrheic dermatitis, intertrigo, atopic dermatitis, dandruff, psoriasis, 

and neonatal impetigo. .everal factors related to !+6+&&.d"+ colonization, which 

generally predisposes to infection, remain poorly understood such asM a) when and 

where colonization beginsn b) the species responsible for colonizationn and c) the 

original source of the colonizing organism. ae therefore investigated !+6+&&.d"+ 

colonization by sequential cultivation ofM a) the sBin of neonates just after birth and at 

intervals thereaftern b) on the sBin and in the vaginal canal of neonates] birth mothersn 

and c) on the hands of nurses who cared for the neonates during post-partum 

hospitalization. 

Methods: Vaginal swabs were taBen from the birth mothers just before delivery. 

.terile adhesive tape specimens were obtained daily from the sBin of neonates at 8 

anatomical sites (including forehead, cheeBs, chest, peri-umbilicus, upper arm, lower 

limb, groin and bacB) beginning on the day of birth (day 0) and continuing until hospital 

discharge. Also, sterile adhesive tape sBin samples were taBen from mothers (chest 

and palm, on the 2 nd day after birth) and from the neonates] nurses (palm, on the 2 nd 

day after birth but before interaction with the neonate). The samples were inoculated 

onto solid culture medium containing rapeseed oil at J2ÅC. .pecies were identified by 

microscopic and physiologic characteristics. 

Results and conclusions: From (une 200J to March 200O, 10O neonates, their 

mothers, and their nurses were examined. The mothers] ages ranged from 21 to OJ 

years (mean, 26.K) and the gestation period ranged from JJ to OO weeBs (mean, JK). 

Deliveries were by spontaneous labour ([0_, n}[2) or Caesarean section ([0_, 

n}[2). Newborn gender distribution was [O_ male and O6_ female. All [2 vaginal 

samples before spontaneous labours were negative for !+6+&&.d"+ species as were 

all samples from the palms of 1[ nurses. fn day 0, all sBin samples from 10O 

neonates were negative, regardless of delivery method. However, by day 1, 

colonization with !+6+&&.d"+ species occurred in 28 of 10O neonates (26.K_)n by day 

8, [K of the neonates ([6.7_) were colonized. ff the [K !+6+&&.d"+ isolates 

recovered from neonates by day 8, O1 (6K.[_) were identified as !/*U($U($/ 

!+6.&&.d"+ species were isolated from the sBin of 6[ of 10O mothers (62.[_) and of 

these,*OK isolates (7[.O_) where identified as !/*U($U($. Among 12O positive isolations 

of !+6+&&.d"+ from neonates and their mothers, K2 (7O.2_) shared the same species. 

The most frequent isolation sites from neonates were the forehead (100_) and the 

face (KO.K_). These data indicate thatM a) neonatal colonization with !+6+&&.d"+ 

species did not occur during delivery but could occur as early as one day after birthn b) 

the single most common colonizing species was !/*U($U($n c) the face and forehead 

were the most common neonatal isolation sitesn and d) the most probable origin of 

neonatal !+6+&&.d"+ colonization was from the sBin of neonates] mothers. 

P-0539. Decayed wood in trunk hollows of miscellaneous trees as primary 

environmental niche of Cryptococcus gattii and Cryptococcus neoformans 

Randhawa H, lowshiB T, .inha l, lhan Z, Chowdhary A 

Department of Medical Mycology, Vallabhbhai Patel Chest Institute, 6niversity of Delhi, 

Delhi 

A global update including our recent data on decayed wood inside trunB hollows of 

miscellaneous living trees as primary environmental niche for 7/*@+--"" and 7/* 

,.%U%$)+,& in north-western India is presented. Three hundred and twenty-eight 

decayed wood samples collected from inside trunB hollows of 21J trees belonging to 

11 species of 10 families were investigated, employing an in-house swabbing and / or 

the conventional floatation technique and simplified .taib]s niger seed medium. 

fverall,*7/*,.%U%$)+,& showed a prevalence of 21.[_, followed by 1J.6_*of*7/*@+--"" 

and 6.[_ concomitant prevalence of 7/*@+--""*and 7/*,.%U%$)+,& in the same trees. 

=2d2@"()*#()"," trees in Delhi revealed the highest prevalence (8K_) of 7/*@+--""* 

serotype 23*followed by 4d+5"$+#?-+*",5"#+ ([0_), !+,"[+$+*?._+,5$+ (O0_), 

!")(&%'&*.6.,@" (J1_) and 1%62+6-?"+*6%,@"U%6"+ (17_). In contrast, 7/*,.%U%$)+,&3 

serotype A, showed the highest prevalence of 62.[_ in 4/*",5"#+*trees in Delhi, 

followed by JJ_ in 1/*6%,@"U%6"+, 20_ in !/*?._+,5$+, 1[_ in !/*.6.,@" , 8_ each in 

4#+#"+*,"6%-"#+ and V+68.$@"+*&"&&%%, 7_ in ^"#(&*$.6"@"%&+ , [_ in 46&-%,"+*&#?%6+$"&, 

and solitary isolations from 4.@6.*)+$).6%& and 7+&&"+*U"&-(6+. aide variations 

occurred in the regional prevalence of 7/*@+--"" and 7/*,.%U%$)+,& in =/ #()"," - the 

only tree species for which we have data. Thus, 7/*@+--"" had a prevalence of K _ in =/* 

#()"," trees in Amritsar as against 8K_ in Delhi. /iBewise, 7/*,.%U%$)+,&, serotype A, 

had a prevalence of [O_ in =/*#()"," trees in Amritsar whereas it was not found at all 

in Meerut Cantt. Concomitant occurrence of 7/*@+--"", serotype 2, and 7/*,.%U%$)+,& , 

serotype A , was noted in 1O trees, 6 of which were =/*#()",", [ 4/ ",5"#+3 and 1 each 

of !/*?._+,5$+ , !/*.6.,@" and 1/*6%,@"U%6"+. Population density of the two species 

differed widely (rangeM 1 to J.2 x 10 O CF6 / swab) not only from tree to tree of the 

same species but also from sample to sample in the same trunB hollow. .erotyping 

was done in 1OO isolates, KJ (6O.[_) of which proved to be serotype A and [1 (J[_) 

serotype 2. A long-term surveillance study of [ =/*#()"," trees in Delhi revealed 

perennial colonization (demonstrated todate for O.7 to [ years) by 7/*@+--"" or / and 7/ 

,.%U%$)+,&. 2esides, 7/*@+--""/ and / or 7/ ,.%U%$)+,& were repeatedly isolated from 

air samples taBen close to the decayed trunBs of 1J of 17 (76_) trees belonging to =/* 

#()","3 4/ ",5"#+3 1/*6%,@"U%6"+ , !/*?._+,5$+ and !/*.6.,@"/ leeping in view the global 

literature reports and our data on high prevalence, fungal population density, perennial 

colonization and aerial isolations, it is concluded that decayed wood in trunB hollows 

of a wide spectrum of tree species is a primary environmental niche of 7/*@+--"", 

serotype 2, and 7/*,.%U%$)+,& , serotype A. 


P-0540. Dermatophyte in central Brazil 

Reis C, Reis-Filho E, Theodoro F, .ilveira V 

6niversity of 2rasilia-H62, 2razil 

Purpose of Study: Investigate the incidence of dermatophyte infections in the Central 

of 2razil, according to age, sex and clinical manifestation, comparing the results with 

previous studies in Distrito Federal (DF)and in others regions of 2razil. 

Description: The research was performed in the city of 2rasilia, 2razil]s capital, which 

is situated in central region of the country (DF). It was retrospective and used the 

register]s booBs of the Mycology /aboratory of Hospital 6niversitZrio de 2rasÜlia 

(H62). The sample was obtained from 2[7[ cases of clinical suspicion of superficial 

mycoses, from (anuary/2000 to December/200O. The laboratorial routine consisted of 

direct exams and culture of the material collected from sBin lesions, hair and nails. 

.Bin lesions and hair were clarified and dissociated in lfH (potassium hydroxide) 

10_ and 20 _ plus dimethyl sulfoxide for microscopic examination. The culture were 

performed using .abouraud-dextrose agar with chloramphenicol and cycloheximide, 

and incubated at room temperature for two weeBs, when it was identified based on the 

macro and microscopic characteristics of their colonies grown. To stablish the 

definitive diagnostic was used special medium for dermatophytes and by biochemical 

and biological tests (Agar potato and christensen). 

Results and Conclusions: 7JO were positive for dermatophytes (28,66_). There 

was a prevalence of female OOK (61,2_) than male 28[ (J8,8_), and age varies from 

1 month to K0 years, the most predominant age group was J1 to O[ (1K,6_). 

According to species isolated, >/*).,-+@$%'?2-.& was the most frequent ([J,1_), 

followed by >/*$(8$() (2[,[_), differing from the literature. The incidence of !/*#+,"& 

(K,O_), >/*-%,&($+,& (8,1_), a/*U6%##%&() (2,[_), !/*@2'&.() (1,O_) is in agreement 

with the literature. >",.+*(,@("() (O7_) was prevalent, followed by -",.+*'.5"& 

(1K,[_), inverting data of central 2razil. >",.+*#+'"-"& cases was predominantly 

caused by !/*#+,"& (OJ,K_) and >/*-%,&($+,& (O1,8_). >",.+*#%$'%$"& was caused 

mainly by >/*$(8$() (JO,J_) and >/*).,-+@$%'?2-.& (28.88_), liBe in others studies. 

There was observed a variation in dermatophytes in central 2razil comparing with first 

studies and others states of the county. It can be explained by changes in habits and 

frequent demographic migrations in this region. Nowadays the researches show an 

increased emergence of new opportunistic infections that involve dermatophytes. It 

could be explained by the increased number of immunosuppressed individuals 

(chemoterapy, organ transplants, diabetics, AID. and parenteral nutrition). The 

epidemiological profile of dermatophytosis, including the frequency and distribution, 

has been changing according to their etiologic agents, the geographic region studied, 

the socio-economic aspects, the climatic variations, the presence of domestic animals, 

age, the host immunity, genetic factors, and demographic movement or migration. 

P-0541. Sporotrichosis in central Brazil: Five years of research 

Reis C, Reis Filho E, Theodoro F, Macedo a 

6niversity of 2rasilia, 2razil 

Purpose of the study: .porotrichosis is a disease that can occur from direct 

inoculation of conidia of ='%$%-?$"_*&#?.,#["" in the derma. This is a dimorphic fungus 

found in soil and plant debris. Infections occur over a wide geographic range in the 

tropics and subtropis. In 2razil, in some regions of the .outh, it is the most frequent of 

subcutaneous mycosis. fccupational illness is considered, occurring more frequently 

in worBers who deal with ground and plant contaminated with conidia of =/*&#?.,#["". 

After the inoculation, this agent can cause cutaneous or subcutaneous infectionsn 

lymphangitic lesions spread along the course of the lymphatics with the appearence of 

a chain of secondary nodules. .ystemic disease, however, is rare and more liBely to 

afflict individuals with impaired immunity such as alcoholics and HIV infected patients. 

The latest articles shows an increase of the number of patients with culture positive for 

=/*&#?.,#["" and it is associated to clinical form as the cutaneous-lymphatic and 

impaired immunity. 

This research had been performed in Central 2razill, which is a dry climate, and 

usually this fungus wouldn]t have great affinity. 

Summarized description of the project: fur objective was verify the epidemiologic 

profile of .porotrichosis in our region and the distribution into sex, age, localization 

and clinical form. A retrospective study had been performed with data of register 

booBs of the Mycology /aboratory (M/) of 6niversity Hospital of 2rasilia using 

protocol-standard created by the .tudy Group of M/ (GE/M). In KO cases suspicious 

of .porotrichosis, 20 had been positive, chosen between O1O[ samples from (anuary 

2000 to December 200O. The fungi was isolated from secretions and biopsy in solid 

ways of culture (îgar-.abouraud, Mycosel and îgar-2HI) in the environment 

temperature and J7 oC. 

Results and conclusions: among 20 positive samples, 11 ([[,0_) were male and K 

(O[,0_) female, the age was between 1 year and ten months to 72 years-old. The 

most frequent age group was 21 to J0 years-old (J[,0_), followed by [1 to 60 yearsold 

(20,0_). The distribution of the injuries was predominated in the superior 

members (O0,0_), followed by inferior members (J[,0_), face (10,0_) and hand 

(10,0_). The cutaneous-lymphatic form was predominant (O[,0_) followed by fixed 

forms (J[,0_). The analysis of this research in this region and period of study 

suggests characteristics compatible with the following epidemiological profileM the 

occurrence of .porotrichosis was indifferent to sex and age as the last articles about 

this disease. The economic situation, geographic and climatic aspects of Central 

2razil offer good conditions to study the mentioned factors because the behavior of =/* 

&#?.,#[""*depends on the environment changes, especially variations of host, life style, 

trips and migration of human beings. These migrations allow a better agreement of the 

spectral alteration in the distribution of =/*&#?.,#["" in different regions and periods of 

time.

P-0542. Evaluation of the virulence of Candida albicans strains isolated from 

healthy and patient hosts 

Riazipour M 1 , losravi A 2 

1 Department of Microbiology,Faculty of Medicine,2aqiyatallah Medical .ciences 

6niversity,, 2 Department of Mycology, Faculty of Veterinary, Tehran 6niversity 

Many investigators studying virulence factors of 7/+68"#+,& have been assumed 

patient and healthy host isolates of the yeast as virulent and avirulent strains 

respectively. In the present study, using a murine model of systemic candidiasis as an 

accepted virulence determination method for Candida species, we intended to 

evaluate the assumption validity. 

7/+68"#+,& strains were isolated from individuals with or without candidiasis at our 

mycology lab. A blastoconidia suspension (10 6 cells) of each isolate inoculated to a 

group of 2A/2/c mice through their lateral vein tail and results of their survival time 

analyzed by life table statistics. 

The results indicated that the isolates of 7/+68"#+,& were considerably different from 

each other in pathogenic potential as Mean .urvival Time (M.T) of inoculated mice 

varied from [ to 26.2 days for the most virulent and the lowest virulent strain 

respectively. However, amongst the yeasts isolated from patient and healthy hosts 

both high virulent and low virulent strains were found, and there was no significant 

difference between the M.T of the isolates from patient (1J.7r/- 1/08 days) and 

healthy (12.O[r/- 1.20 days) hosts. 

It is concluded that the host status (to be healthy or patient) does not predict the 

virulence of 7/+68"#+,& isolates, and the assumption that patients isolates are high 

virulent may not be valid. 

P-0543. Characterization phenotypic and genotypic of environmental 

Cryptococcus neoformans isolates in Vitoria, Brazil 

Ribeiro M 1 , NgamsBulrungroj P 2,J,O 

1 Department pf Pathology, 6niversidade Federal do EspÜrito .anto, 2razil, 

2 Department of Medicine, DuBe 6niversity Medical Center, Durham, NC, 6.A, J CIDM, 

the 6niversity of .ydney at aestmead Hospitaln aestmead, N.a, Australia, O Faculty 

of Medicine .iriraj Hospital, Mahidol 6niversity, 2angBoB, Thailand 

The basidiomycetous yeast 7$2'-%#%##(&*,.%U%$)+,& is the etiological agent of 

cryptococcosis, an opportunistic systemic mycosis that usually manifests as 

meningoencephalitis. The human infection is acquired by inhalation of airborne 

propagules from environmental source. The 7/*,.%U%$)+,& complex is composed of 7/* 

,.%U%$)+,&*var/*,.%U%$)+,&3*7/*,.%U%$)+,&*var/*@$(8""3*and*7/*@+--"". The sexual 

cycle of 7/*,.%U%$)+,& involves fusion of a and alpha cells to produce heteroBaryotic 

filaments. Most clinical isolates are of the alpha mating type, and the explanation for 

this bias is unBnown. The purpose of this study was to evaluate the serotypes and 

mating types of 7/*,.%U%$)+,& strains collected from the environment in 2razil. 

Thirty-three samples of bird guano were collected from 8 locations within the city of 

Vitoria, 2razil. After suspension in sterile water, the samples were cultured on Niger 

seed agar at 27oC for 2 weeBs. DarB brown colonies were confirmed as 7/* 

,.%U%$)+,& using standard biochemical assays. The serotype and the two 

alternatives alleles a and alpha at the mating type locus was determined by PCR 

amplification. Mating assays were performed on V8 media to confirm the mating type 

of isolates. 

Ten (J0_) of the samples were positive for 7/*,.%U%$)+,&, and a single isolate from 

each culture was selected for further study. PCR amplification with the 6RA[ primer 

demonstrated that all isolates were serotype A (var. @$(8"") with VNI RF/P profile. All 

isolates were !4>-alpha mating type as determined by PCR of the =>aMQa and 

=>aMQalpha genes, and only [ ([0_) were able to mate with one of the !4>-a tester 

strains ((EC20, lNKKa and 2otswana 6J). None of isolates mated with !4>-alpha 

type tester strains ((EC21 and HKK). This study confirms that the majority of 

environmental strains recovered from bird guano are of the serotype A/mating type 

alpha bacBground. 

Financial supportM F6NCITEC/FAPE. 


P-0544. High prevalence of non-dermatophyte molds in onychomycosis in 

Brazil 

Ribeiro M 1 , Paula C 2 , Gambale V 2 

1 Dept. of Pathology, 6niversidade Federal do Espirito .anto, Vitoria, 2razil, 2 Dept. of 

Mycology, 6niversidade de .Éo Paulo, .Éo Paulo, 2razil 

The onychomicosis, nail infections caused by fungi, present a very heterogeneous 

etiology, a growing problem for diagnosis and treatment. This study provides recent 

trends in the incidence of fungal species isolated from nails infections in 2razil. 

From (anuary to December 200[ 1J6 patients clinically suspected of onychomycosis 

were enrolled, randomly selected from healthy population. .ample nails were 

collected by scraping with sterile scalpel, submitted to direct microscopic examination 

using 20_ lfH plus ParBer]s inB (JM1) and cultured in .abouraud dextrose agar 

(Difco) added of [0 mg /- 1 cloramphenicol and Mycosel' agar (22/), at 2[oC until J 

weeBs. The identification was based on the macroscopic and microscopic 

characteristics and, when necessary, additional tests were performedM urease 

production, growth on >$"#?%'?2-%, agars and hair perforation assays. 

Analysis of combined finger and toenail nails data showed a frequency rate of 

isolation of 70_, with KO cases of direct examination and culture positives for fungi. 

The most prevalent agents of onychomycosis were yeasts ([[_), with [0 7+,5"5+ 

spp and J >$"#?%&'%$%, spp. isolates. Molds corresponded to O[_ of fungal infections, 

with 26 non-dermatophytes and 18 dermatophytes isolates, respectively. Among nondermatophytes, 

^(&+$"() spp. was the most common agent (11 cases), followed by 

4&'.$@"66(&*(7), =#2-+6"5"() spp (6) and 7($0(6+$"+ spp. (2). The most frequent 

dermatophyte isolated was >$"#?%'?2-%,*$(8$() (1[ cases), followed by >/* 

).,-+@$%'?2-.& (2) and a'"5.$)%'?2-%,*U6%##%&() (1). 

The number of non-dermatophytes molds accounted for the rising trend toward these 

fungi, constituting a predominant group over dermatophytes and, emphasizing the 

importance of diagnosis for adequate treatment of onychomycosis, since emerging 

species with decreased susceptibility to antifungal agents may require novel 

therapeutic drugs or drug combinations. 

P-0545. Isolation of the Cryptococcus neoformans/gattii complex from the 

Atlantic department, Colombia, South America 

Romero /, Ruiz-Acevedo A 

6niversity of Puerto Rico-Mayaguez Campus 

The 7$2'-%#%##(&*,.%U%$)+,&

P-0546. Molecular epidemiology of Cryptococcus gattii from HIV-seropositive 

and -negative patients in South Africa 

Rossouw J 1 , McCarthy l 1 , 2randt M 2 

1 Mycology Reference 6nit, National Institute for Communicable Diseases, National Health 

/aboratory .ervices, .andringham, Gauteng, 21J1, .outh Africa, 2 Mycotic Diseases 

2ranch, National Center for Infectious Diseases, Centers for disease Control and 

Prevention, Atlanta, Georgia, J0JJJ, 6.A 

Purpose of the study: Cryptococcosis caused by the encapsulated basidiomycetous 

yeasts 7$2'-%#%##(&*,.%U%$)+,& and 7$2'-%#%##(&*@+--"", has emerged as an important 

opportunistic infection in areas of high HIV-seroprevalence. 6nliBe 7/*,.%U%$)+,&, 7/ @+--"" 

typically causes disease in immunocompetent hosts in tropical and subtropical regions. To 

date, only a small number of reports have implicated 7/ @+--""*serotype 2 in AID.-related 

cases worldwide, while 7/ @+--"" serotype C has only been reported in Africa in a limited 

number of AID.-related cases. A population-based surveillance study performed in 

Gauteng Province, .outh Africa from 2002 to 200O revealed that a high proportion of 7/* 

@+--"" (serotype 2 and serotype C) infections were AID.-related. In view of this and the fact 

that very little is Bnown about 7/*@+--""*in .outh Africa, we investigated the molecular 

epidemiology of .outh African 7/*@+--"" isolates relative to those found globally, and 

explored their genetic diversity. 

Description: 7/*@+--"" isolates were collected as part of the active population-based 

surveillance for cryptococcosis carried out in .outh Africa from 1 (anuary 200[ to J1 

December 200[. In total, J2 isolates from 21 HIV-seropositive patients, 1 HIVseronegative 

patient, 8 patients with unBnown HIV status and 2 veterinary isolates (cheetah 

and marmoset) were included in this study. 

High-molecular weight DNA was extracted and subjected to molecular type determination 

using M1J PCR-fingerprinting. To understand the phylogenetic relationship among the 

molecular types, partial sequences (61O bp) of the exon 1 region of the 7415R gene were 

determined for 18 isolates. 

P-0547. Mycoflora of human external auditory canal in Shiraz, Iran 

Sabayan B, PaBshir l, laramifar l, (avan H 

.hiraz 6niversity of Medical .ciences, .hiraz, Iran 

Introduction: ftomycosis is a superficial mycotic infection of the outer ear canal. 

Many species of fungi could be considered as causative agents. The ear is constantly 

exposed to the biotic elements of the biosphere and thus accessible to various microorganisms 

including fungi. Therefore, detection of the fungal agents in the external 

auditory canal could be valuable for determining the potential risB of otomycosis. The 

aim of this study was to identify the mycoflora of the human ear canal in healthy 

individuals. 

Patients and methods: A total of O86 ear samples of 2OJ healthy individuals (100 

female and 1OJ male) were collected by sterile swabs and cultivated on Mycosel and 

.abouraud dextrose agar supplemented with penicillin and streptomycin. Fungi were 

identified by mycotic traditional (macro and micro morphological features of the 

colonies) and chromogenic methods. 

Results: Ten point twenty eight percent of persons were positive for ear fungal 

residents. Fungal species were isolated from human ear canal belonged to eight 

genera includingM 1.,"#"66"(), 76+5%&'%$"(), 7+,5"5+, 4&'.$@"66(&3*46-.$,+$"+, 

P%5%-%$(6+, a_%'?"+6+ and Dematiaceous fungi. In this study, Aspergillus*,"@.$, the 

major cause of otomycosis, was not isolated. 

Conclusion: fur findings suggest that the normal fungal otic residents, in the 

presence of predisposing factors, probably have a potential risB for otomycosis. 

Results and conclusions: Molecular analysis revealed that .outh African clinical strains 

matched the rarely observed VGIV molecular type (21/J0n 70_) and the VGI molecular 

type (8/J0n 26_). aithin the VGI molecular type, several band variations were observed 

for some isolates. No VGII or VGIII molecular types were observed. A single isolate 

displayed a fingerprinting pattern matching none of the Bnown molecular types, indicating 

that a novel VG type is circulating in .outh Africa. In addition, the veterinary isolates 

displayed VGII (marmoset strain) and VGIV (cheetah strain) molecular types. 

.equence analysis of the partial 7415R gene demonstrated that at least O alleles for VGI 

and [ alleles for VGIV could be distinguished. The nucleotide sequence for the VGII 

(marmoset strain) was 100_ identical to the published sequence for the minor Vancouver 

Island outbreaB strain. Notably, the nucleotide sequence for the isolate with the unBnown 

molecular type was 100_ identical to the VGI molecular type standard (aM17K). 

Phylogenetic analysis of the partial 7415R gene sequences demonstrated that VGI, VGII 

and VGIV isolates can be divided into distinct phylogenetic groups. Deduced amino acid 

sequences revealed that, except for one of the VGIV isolates, the partial exon 1 region 

were conserved within the different molecular types. These results demonstrate that 

considerable genetic variability exists among 7/*@+--"" isolates circulating in .outh Africa. 


P-0548. An epidemiologic surveillance of 16,229 cases of dermatophytosis in 

Brazil 

Scroferneker M 1 , de .ouza / 1 , da Rosa A J , Dewes / 1 , Vettorato G 2 , Gervini R 2 

1 Instituto de Ciüncias 2Zsicas da .aâde, 2 .erviÑo de Dermatologia do Complexo 

Hospitalar .anta Casa de Porto Alegre, J Complexo Hospitalar ConceiÑÉo 

Purpose: Dermatophytes belong to the >$"#?%'?2-%,, !"#$%&'%$(), and 

a'"5.$)%'?2-%,*genera and are classified as anthropophilic, zoophilic or geophilic 

according to their normal habitat. This paper presents the results of a J8-year 

observation of fungal infections. 

Materials and Methods: Retrospective analysis of medical records containing cases 

of dermatophytosis diagnosed and treated at the Division of Dermatology of .anta 

Casa de Misericárdia Hospital Complex, affiliated with 6FRG., in the city of Porto 

Alegre, in southern 2razil. The statistical analysis was made using Epi Info 6.0 1b. 

Results: 16,62K cases of dermatophytosis confirmed by direct mycological 

examination and/or culture were reviewed. The most frequently isolated 

dermatophytes were >$"#%'?2-%,*&'/, mainly >/*$(8$(),*which accounted for J,J[6 

cases (68.18_).*Among tinea infections, -",.+*'.5"& was the most common type 

(6,22[ cases), followed by -",.+*(,@("() and -",.+*#%$'%$"&. !"#$%&'%$()*#+,"&*and* 

!"#$%&'%$()*@2'&.() were responsible for 80_ of the cases (n } JK7) of hair and 

scalp infectionn the former one was identified in J67 patients. There were [,2[J cases 

of onychomycosis. Toenails were the affected sites in O,[K8 patients (87.[J_) and 

both fingers and toes were involved at the same time in other 17J patients. ae found 

no sex predominance among the subjects of the study. 

Conclusion: The data available to date have led us to conclude that >/*$(8$() is the 

most frequently isolated dermatophyte, representing a worldwide trend. The present 

data are consistent with those reported for other 2razilian regions. Most studies have 

concluded that the frequency of this agent increases with urbanization, leading to its 

predominance as the major causative agent of dermatophytosis in large urban centers 

and turning it into a public health problem. 

P-0549. Epidemiologic surveillance of 22 cases of tinea nigra in Southern Brazil 

Scroferneker M 1 , de .ouza / 1 , da Rosa A J , Dewes / 1 , Vettorato G 2 , Gervini R 2 

1 Instituto de Ciüncias 2Zsicas da .aâde, 2 .erviÑo de Dermatologia do Complexo 

Hospitalar .anta Casa de Porto Alegre, J Complexo Hospitalar Grupo ConceiÑÉo 

Purpose: >",.+*,"@$+ is a relatively uncommon dermatiaceous fungal infection 

characterized by asymptomatic brown or blacB macules on the hands and feet that 

may mimic a melanocytic lesion. This superficial mycosis is produced by A%$-+.+* 

B.$,.#["", also Bnown as 1?+.%+,,.6%)2#.&*B.$,.#["", a_%'?"+6+*B.$,.#["", 

76+5%&'%$"()*B.$,B#["" (or #+&-.66+,""), and =-.,.66+*+$+@(+-+ (which is 

autochthonous in Venezuela). In this paper, we present 22 cases of >",.+*,"@$+ 

diagnosed from 1K7O to 200[. 

Materials and Methods: Retrospective analysis of medical records containing cases 

of >",.+*,"@$+ diagnosed and treated at the Division of Dermatology of .anta Casa de 

Misericárdia Hospital Complex, affiliated with 6FRG., in the city of Porto Alegre, in 

southern 2razil. The statistical analysis was made using Epi Info 6.0 1b. 

Results: Twenty-two cases of >",.+*,"@$+ were confirmed by direct mycological 

examination and culture. Nineteen patients presented with palmar lesions and only 

three had plantar involvement. ae found no sex predominance among the subjects of 

the study, but plantar macules were all diagnosed in female patients. Age ranged from 

J to 67 years. 

Conclusion: The data available to date have led us to conclude that >",.+*,"@$+ is a 

tropical mycotic infection that can be misdiagnosed as a melanocytic lesion, leading to 

unnecessary procedures.

P-0550. Invasive candidiasis: Epidemiology, performance of the nested-PCR 

and antifungal susceptibility 

Sellami A, lhlif M, .ellami H, MaBni F, CheiBhrouhou F, Abbes ., Chelly H, Chaari H, 

2ouaziz M, Ayadi A 

1 /aboratory of Parasitology-Mycology - 6niversity Hospital - .fax - T6NI.IA, 2 Medical 

Reanimation aard - 6niversity Hospital - .fax - T6NI.IA 

Introducion: Invasive candidiasis has emerged as a common complication in 

immunocompromised hosts and critically ill patients. Despite 7+,5"5+ +68"#+,& is still 

considered the most frequently isolated species, the emergence of non +68"#+,& 

7+,5"5+ species is clearly a concern. 

ObjectivesM Analysis of the mycological and epidemiological features of invasive 

candidiasis and evaluation of the potential value of nested PCR in routine diagnosis of 

candidemia. 

Material and methods: ae conducted a six years study (2000 - 200[) of invasive 

candidiasis cases diagnosed in .fax 6niversity Hospital. Yeasts were obtained from 

blood cultures and normally sterile body sites. For 76 patients at risB for developing 

invasive candidiasis, nested-PCR targeting 7+,5"5+ rDNA has been done 

simutaneously. The antifungal susceptibility profile to amphotericine2, nystatine, 

flucytosine, econazole, miconazole Betoconazole and fluconazole was evaluated for 

112 isolates by Candifast methods and some 7/@6+8$+-+ strains was also evaluated by 

E-test. 

Results: ae counted 1O2 invasive candidiasis casesM 106 cases confirmed by blood 

cultures and six cases of 7+,5"5+*deep abscess. J0 cases of candidemia have been 

confirmed by Nested PCR while blood cultures remains negatives. The risB factors 

associated with 7+,5"5+ infectionM prolonged antibiotherapy (8J_), 7+,5"5+ 

colonisation (7[_), long stay in intensive care units (O0_), parenteral nutrition (21_), 

and intravascular catheter (16_). The isolates were dominated by 7/+68"#+,& (J6_). 

Non +68"#+,& 7+,5"5+ species strains accounted for 6O_ of all episodes of invasive 

candidiasis and were mostly represented by 7/-$%'"#+6"& (28_),*7/@6+8$+-+ (1[_) and* 

7/'+$+'&"6%&"& (1O _). 7/'+$+'&"6%&"& isolates were all susceptible to fluconazole and 

amphotericin 2. 7/+68"#+,& and 7/-$%'"#+6"& isolates were resistant to fluconazole in 

20_ and 2O_ respectively. The sensitivity of 7/@6+8$+-+ strains to fluoconazole 

revealed a high rate of resistance by Candifast methods (8[_), but the evaluation of 

some strains by E-test revealed a resistance only of 22_ (MIC% 6Okg/ml) and 78_ 

were susceptible dose dependent. 

P-0551. Onychomycosis in Sfax University Hospital - Tunisia 

Sellami H, MaBni F, lchaou a, Travelsi H, .ellami A, CheiBhrouhou F, MarreBchi ., 

2oudava ., TurBi H, Ayadi A 

1 /aboratory of Parasitology Mycology - 6niversity Hospital - .fax- T6NI.IA., 

2 /aboratory of Parasitology Mycology - 6niversity Hospital - .fax- T6NI.IA. 

fnychomycosis is the most frequent cause of nail diseases. They represent a real 

problem due to their recurrency and unaesthetic aspects. 

Aim: To determine our local epidemiological and mycological profile of 

onychomychosis. 

Patients and methodsM Retrospective study included 811O samples of onyxis adressed 

to our laboratory from (anuary 1K8K to .eptember 200[. 

Results: The onychomycosis were diagnosed in 7J_ of the cases with female sex 

predominance (6K.O_). The mean age was OJ.8 years. They were in constant 

progression (1K cases in 1K8K, 718 cases in 2002 and 1017 cases in 200O). They 

affected the toe nails in [2.8_ and the hand nails in O7.2_. The dermatophytic onyxis 

([[.6_) affect especially the toe nails (8K.6_). The most frequent clinical aspect was 

the distal and lateral subungual onyxis (70.6_). >/*$(8$() was the most common 

isolated species (K2.7_) followed by >/*",-.$5"@"-+6. (J_). The candidiasis onyxis 

(O1_) were predominantly detected in the hand nails (86.67_). The perionyxis 

represented the most common form (61.7_). The mainly isolated species were 7/* 

+68"#+,& ([0.16_) and 7/*-$%'"#+6"& (2K.O[_). The molds were responsible for J_ of 

the casesM 1.,"#"66(") (26.O[_), followed by ^(&+$"() (20.1_), =#%'(6+$"%'&"&* 

8$.0"#+(6"& (16.KJ_) and 4&'.$@"66(&*,"@.$ (11.6O_). The histological study of O10 nail 

specimens coloured by PA. and Grocott allowed the diagnosis of onychomycosis in 

8.[_ of the cases with negative direct examination and cultures. 

Conclusion: The prevalence of the onychomycosis is in constant increase. 

Considering the heaviness of the antifongic treatment (cost, duration), we emphasize 

on the importance of correct etiological identification, allowing for appropriate 

treatment. 

Discussion: Invasive candidiasis is increasingly an important complication in 

hospitalized patients at risB. The nested PCR is significantly more sensitive and 

proved to be both simple and reproducible and may offer potential advantages over 

conventional fungal blood cultures. This information contributed by its great value not 

only for therapeutic decisions but also to redress the diagnosis of candidemia in our 

laboratory. ahen major discrepancies were observed between Candifast and E-test 

results, the control of major etiologic species of candidiasis by efficient therapy require 

effective and reliable methods for antifungal susceptibility such as E-test. 


P-0552. Tinea capitis in a Paris hospital (France): 1941 cases diagnosed from 

2000 to 2004 

Sermondade N 1 , /acroix C 1 , Dubertret / 2 , Morel P 2 , Feuilhade de Chauvin M 1 2 

1 mycology hopital .t /ouis, 2 dermatology hopital .t /ouis 

Tinea capitis is the most common fungal infection in school-aged children caused by 

dermatophytes. The etiologic agents vary from country to country and with the urban 

or rural environment. The aim of this study was to evaluate the actual epidemiology of 

tinea capitis in Paris, in an urban population including a high proportion of immigrants. 

ae realized a retrospective study of tinea capitis diagnosed in patients attending our 

mycology-dermatology laboratory in .aint-/ouis Hospital (Paris, France) from (anuary 

2000 to December 200O. 

During this five-year period, O0O[ patients were examined for tinea capitis diagnosis. 

Tinea capitis was diagnosed in 1KO1 patients, J[[ in 2000, O28 in 2001, JKO in 2002, 

J[7 in 200J and O07 in 200O. .ex ratio (M/F) was 1.J[ (pv 0.01). The majority of 

patients were under the age of 10 years (82.6_) with a median of 6 years. fut of the 

1K0 adults with tinea capitis, O0.[_ were identified as mothers of infected children. 

Patients were from Africa and Caribbean islands respectively in 8[_ and 8_ of cases. 

6pon the 11 species of dermatophytes isolated,*>$"#?%'?2-%,*&%(5+,.,&. was the 

predominant organism ([J.K_), followed by !"#$%&'%$()*6+,@.$%,"" (26.[_) and >/* 

-%,&($+,& (K.O_). Zoophilic and geophilic species represented respectively only O.8_ 

and 0.1[_ of isolated species (!/*#+,"&*O.7_). 

These data indicate that tinea capitis still represents a public health problem, despite 

its benign curable nature. In Paris, tinea capitis are mainly due to anthropophilic 

species and occur mainly in the immigrant population from Africa. In comparison with 

our surveys conducted between 1KKO-1KK7 and 1KK8-1KKK, incidence of scalp 

infections caused by >/*-%,&($+,& continues to increase, from 2_ in 1KKO-1KK7, [.1_ 

in 1KK8-1KKK, to K.1_ in 2000-200O. friginally, this species was mainly isolated in 

native Caribbean patients and then progressively expanded in different populations, 

especially families from Africa. In our experience, more than school setting, combing 

habits in African or Caribbean families (plaiting, clippers use) represent the major 

factor of transmission of these anthropophilic dermatophytes. 

P-0553. Study of fungal contamination of indoor public swimming pools in 

Golestan, Iran 

Sharbatkhori M, .arafi A, .harbatBhori Z, .harbatBhori F, .harbatBhori N 

Institue of Public Health Research 

Fungi are found in the different environment with variable distribution patterns 

depending on various factors . The water in swimming pools cannot be a good carrier 

for transmission of fungal diseases but can be a source of fungi so environmental 

surfaces may be contaminated by many species of fungi , and transmit them to 

swimmers. The aim of this study was determination of fungal contaminants in public 

swimming pools in Golestan province. 

The fungal contaminations of four indoor swimming pools were studied by using 

membrane filtration and swab sampling method. .amples were collected by a handle 

plastic pump, in a 200 ml sterilized bottle. All samples within 2 hours and then 

transferred to the laboratory. A total (J8O) sample including water and environmental 

surfaces were collected and tested for the presence of fungi in different within one 

year. In addition to the above information, some physical and chemical parameters 

such asn temperature, residual chlorine, PH, turbidity of swimmers were studied. 

Findings indicated that, the average temperature, PH, residual chlorine and turbidity of 

water in the swimming pools within one year respectivelyn 2K.KÅC, 8.1, 0.6 ppm and 

0.8 NT6. The most common fungi recovered were as followsM Aspergillus spp. [62[_, 

Candida spp. 22.K_ , Rhizopus spp. O.16 _, other filamentous fungi 16.6_ and other 

yeast species 2.8_ isolated from water and the fungi such as M Alternaria, 

Cladosporium, philophora and Trichophyton mentagrophytis isolated from dressing, 

bathing rooms and environmental other places out of pools. 

According to this results and previous studies on pools fungal contamination it has 

been indicated that contamination by fungi in the pools in our study is no significant in 

water of pools and environ presence of dermatophytic fungus from dressing room is 

probably due to human contact .

P-0554. Monitoring airborne fungi in two general hospitals in Shiraz, 

Southern Iran 

Shekarkhar G 1 , PaBshir l 2 , Mostagnei . 1 , VagefiBia A 1 , .abayan 2 1 , lamarloei . 2 

1 .hiraz 6niversity of Medical .ciences, .chool of Medicine, 2 Parasitology c Mycology 

department 

Purpose of the study: Airborne fungal spores are important agents of allergies, 

asthma, and pathogenic infections, and the means for opportunistic fungi diseases in 

the hospitalized patients. The aim of this study was to the assessment of the airborne 

fungal flora in the air of two general hospitals in .hiraz. 

Summarized description of the project: .amplings performed by using the settle 

plate method (Kmm Malt and .abouraud dextrose agar Petri dishes) in the selected 

rooms at a period of one hour. The concentration of fungi was calculated as colony 

forming unites per square meter of surface. The fungi identified according to the 

routine laboratory methods for fungus diagnosis. 

Results and conclusions: The fungi isolated belongs to 18 genera includes 

Cladosporium, Penicillium, Aspergillus, Alternaria, Mucor, Trichosporon, Curvularia, 

Chrysosporium, Trichothecium, Rhizopus, Acremonium, 2ipolaris, .temphylium, 

.tachybotrys, Aureobasidium, Hyaline, dematiaceous and yeasts. Cladosporium was 

the most common genus of fungi isolated from both hospitals (J1.K and 1K.8 percent). 

The highest and the lowest fungal colonies were isolated from surgery (17K6 

CF6/mß/h) and operation (zero) rooms, respectively. 

Attention to indoor air quality is particularly important in health care settings. 6sing 

settle plates to monitor microbial air quality in controlled environments is one of the 

oldest methods for collecting airborne micro organism. .ettle plates donet measure the 

number of microorganisms in the air, but measure the number of microorganisms and 

particles settling from the air onto a Bnown surface area in a Bnown time, but it is still a 

simple method and are sufficient for monitoring airborne microorganisms. 

P-0555. A nation-wide survey of Trichophyton tonsurans infection among 

combat sport club members in Japan using a questionnaire form and the 

hairbrush method 

Shiraki Y 1 , Hiruma M 2 , Hirose N J , .ugita T O , fgawa H 1 , IBeda . 1 

1 Department of Dermatology, (untendo 6niversity .chool of Medicine, ToByo, (apan, 

2 Dept. of Dermatology c Allergology, (untendo 6niversity Nerima Hospital, ToByo, 

(apan, J .eminar of (udo, .chool of Health and .ports .cience, (untedo 6niversity, 

Chiba, (apan, O Department of Microbiology, Meiji Pharmaceutical 6niversity, ToByo, 

(apan 

Background: The emerging outbreaB of >$"#?%'?2-%,*-%,&($+,& infection among 

members of combat sports clubs in (apan over the last four years has become a 

serious public-health problem. In order to overcome this outbreaB, a survey for 

>$"#?%'?2-%,*-%,&($+,& infection in (apan may be essential. 

Objectives: Clarifying the prevalence of >$"#?%'?2-%,*-%,&($+,& infection among 

members of combat sports clubs in (apan. 

Methods: ae conducted a survey of members of participating combat sports clubs 

using a standardized questionnaire to assess bacBground factors and using the 

hairbrush method to identify >/*-%,&($+,& infection. .tatistical significance of the 

correlation between data from the questionnaire and the hairbrush culture results was 

determined. 

Limitations: The subject population is limited to members of judo clubs all over (apan, 

they were asBed to participate in this survey via the All (apan (udo Federation. 

Results: ae surveyed 1000 subjects (826 male) from OK institutions and found 11[ 

(11.[_) were positive for >/*-%,&($+,& infection revealed by the hairbrush method. 

Demographic factors associated with high positive rates (%20_) of the infection were 

familial >/*-%,&($+,& infection (20.0_), history of tinea corporis (2O.2_), increased 

dandruff (J2.1_), and concomitant tinea corporis (J1.6_). Hairbrush culture-positive 

subjects without current or previous tinea were considered asympotmatic carriers. 

Conclusion: Infection of >/*-%,&($+,& appears to have spread widely among 

members of combat sports club in (apan. The questionnaire utilized in this study is a 

simple and useful tool to estimate epidemiology of this infection. 


P-0556. Nosocomial candiduria in ICU women patients undergoing urinary 

catheterization. clone relationship between strains isolated from vaginal tract 

and urine 

Silva V, Hermosilla G, Abarca C 

6niversity of Chile, .chool of Medicine, 2iomedical .ciences Institute, Microbiology 

and Mycology Program 

Nosocomial candidiasis has increased significantly in recent years, being an important 

cause of morbidity and mortality in patients admitted to the intensive care unit (IC6). 

6rinary tract infection is the most frequent affection and vesical catheterization is the 

main risB factor. 

ae determined the incidence of nosocomial candiduria associated with indwelling 

urinary catheters in O2 IC6 women patients with and without vaginal 7+,5"5+ spp. 

colonization and molecular fingerprinting of strains initially isolated from the vaginal 

tract and subsequently from urine samples by random amplified polymorphic DNA 

(RAPD) analysis with the purpose of establishing the role of the vagina as a reservoir 

of candiduria. 

At all the incidence of nosocomial candiduria was 21.O_. 7+,5"5+ spp vaginal 

colonization was detected in 11 patients (26.2_) of which 6 ([O.[_) developed 

candiduria. fn the other hand, only J (K.7_) uncolonized patients developed 

candiduria, which gives a relative risB (RR) of O.O, confidence level (C/) sK[_u of 1.61 

to 86.8 (p} 0.00[) for development nosocomial candiduria in women with vaginal 

colonization by 7+,5"5+ spp. 

In [ of the 6 patients with vaginal colonization and candiduria the same 7+,5"5+ 

species was isolated from both sites. .even 7+,5"5+*+68"#+,& strains were isolated 

from J patients and [ 7+,5"5+*@6+8$+-+ strains were recovered from the other 2 cases. 

Amplified product obtained by RAPD with 1O primers were analyses and used to 

created a dendrogram that showed 2 different cluster with minor similarity (. A2 } 0.2) 

corresponding to 7/*+68"#+,& and 7/*@6+8$+-+ strains, respectively. fn the other hand, 

the strains isolated from vagina and urine samples showed a high similarity value per 

each patient (. A2 t 0.K) forming independent cluster. This value is higher than the 

value obtained from strains of the same species but isolated from others patients and 

controls (. A2 v 0.K). 

fur study suggests that IC6 women vaginally colonized by 7+,5"5+ spp have a high 

risB of acquiring nosocomial candiduria and that strains isolated from both sites in a 

single patient are genetically related. There is also evidence of microevolution events 

upon identification of subclones of the same strain, which determines the endogenous 

origin of nosocomial candiduria in these patients. 

This Research was partially supported by DI 6niversity of Chile Grant 

P-0557. Reduced fungal contamination of indoor environment in adult and 

pediatric clinical hematology units with the plasmer system (airinspace) 

.ixt N J,O , Dalle F 1,2 , Caillot D [ , Aho . O , Couillault G 6 , Valot . 1 , Calinon C 1 , Danaire V 1 , 

Vagner f 1 , Bonnin A 1,2 

1 

/aboratoire de Parasitologie Mycologie, HÇpital du 2ocage, 2P 77 K08, 2107K Dijon Cedex, 

France, 2 /aboratoire de Microbiologie Mddicale et Moldculaire, Facultd de Mddecine, 2P 87 K00, 

2107K Dijon Cedex, France, J Microbiologie Environnementale , HÇpital du 2ocage, 2P 77 K08, 

21 07K Dijon Cedex, France, O .ervice deEpiddmiologie et deHygimne Hospitalimre, HÇpital du 

2ocage, 2P 77 K08, 21 07K Dijon Cedex, France, [ .ervice deHdmatologie Clinique, HÇpital 

deEnfants, 2P 77 K08, 21 07K Dijon Cedex, France, 6 .ervice defnco-hdmatologie pddiatrique, 

HÇpital deEnfants, 2P 77 K08, 21 07K Dijon Cedex, France 

The hospital of Dijon is a 12[0-bed hospital providing tertiary care for 2urgundy in northeast 

France. The hospital is involved in a renovation program consisting in the construction of a O000 

m 2 floor-surface laboratory building and a [K2-bed hospital building. The latter construction 

needs a J1 000 m 2 excavation. Clinical units with patients at high risB with respect to 4&'.$@"66(& 

and other mold infections are located adjacent to construction sites. 

To determine the impact of these constructions on indoor fungal contamination we implemented 

a surveillance program starting one year prior to commencement of construction in O clinical 

units M adult and pediatric hematology, Bidney and heart transplantation. As part of this program, 

we evaluated Plasmerw (airinspace), a new air-treatment system based on air-exposure to high 

electric fields, followed by bombardment with positive and negative ions (plasma) and 

electrostatic nano-filtration. In addition to establishing the bacBground level of indoor 

contamination, the goal of the first year of the program was to evaluate the efficiency of the 

Plasmerw in a non-construction context prior to possible utilization during the construction period. 

To do so, fungal contamination was first determined in 2 rooms treated with Plasmersw, and 2 

adjacent non treated rooms. Among air samples, 2/16 and 16/18 were positive in treated and 

untreated rooms respectively (p v 0.0001). fn surfaces, molds grew in 2/16 samples in treated 

rooms and O/18 samples in untreated rooms (non significant). 

Plasmersw were subsequently utilized in adult and pediatric hematology. In adult hematology, 

161 air and surface samples were collected in rooms treated with Plasmersw, and 10 paired 

samples were collected in untreated rooms. Altogether, [K_ of air samples were positive in 

treated rooms, versus 100 _ in non treated rooms (p } 0.007). Mean fungal loads were O.K 

cfu/m J in treated rooms and t JK.O cfu/m J in non treated rooms (p } 0.00[J). .urface positivity 

rates were 1K.2[_ and [0_ in treated and non treated rooms respectively (p } 0.0J6), with a 

non significant difference in fungal load. In pediatric hematology, 8O paired samples were 

collected in rooms treated with Plasmersw, and 6K were collected in non-protected rooms. Air 

samples positivity rates were O2.8_ and KO_ in treated and untreated rooms respectively (p v 

0.0001). Mean fungal loads were 6.8J cfu/m J in treated rooms and 8.K7 cfu/m J in untreated 

rooms (p } 0.000[). .urface positivity rates were 8.J_ and J0.O_ in treated and untreated 

rooms respectively (p } 0.001), with a non significant difference in fungal load. 

The Plasmerw system thus reduces indoor fungal contamination in the clinical setting in normal 

conditions of patient care. The next 2 years of our surveillance program will provide data on its 

utility in the context of building construction.

P-0558. Epidemiology of candidemia in Australian cancer patients 

Slavin M 1 , Marriott D 2 , Chen . J,O , Ellis D [ , Nguyen ~ 2 , .orrell T J,O 

1 Victorian Infectious Diseases .ervice, Royal Melbourne Hospital and Peter 

MacCallum Cancer Centre, 2 .t Vincentes Hospital, .ydney, J ICPMR, aestmead, 

.ydney, O OCentre for Infectious Diseases and Microbiology and aestmead Millennium 

Institute, 6niversity of .ydney at aestmead, [ mycology Reference /aboratory, 

aomenes and Childrenes Hospital, Adelaide. l 

Cancer and cancer therapy are risB factors for candidemia and mortality rates of JK- 

70_ have been reported. However, there are few population-based epidemiological 

studies of candidemia in cancer patientsn most reports come from single centres. 

2etween August 2001 and (uly 200O episodes of candidemia in public and private 

health care institutions were identified. Clinical data, treatment and outcome, were 

collected at [ and J0 days (or at hospital discharge) after initial diagnosis. All isolates 

were speciated and underwent antifungal susceptibility testing. A total of 1,00[ 

episodes occurred in adults and children from K0 participating centres, representing all 

.tates and Territories of Australia. The majority were healthcare associated-inpatient 

infections (81_) and malignancy was the major underlying illness (J0_ of all study 

patients). There were 1[K patients with haematological and 1[8 with solid 

malignancies. /euBemia was the commonest diagnosis ([7_) amongst patients with 

haematological malignancies, followed by lymphoma (J6_) and myeloma. There 

were 17 patients with allogeneic and 11 patients with autologous stem cell transplant. 

The estimated rate of candidemia was 1[ cases/1000 allogeneic and [ cases/1000 

autologous transplants. 

Percentage of patients with Bnown risB factors at diagnosis of candidemiaM 

Cortico ANCv0.[ 

Antifungal 

steroid PriorJ0 days 

priorJ0 days 

Vascular 

access 

device 

Chemo 

therapy prior 

J0 days 

Haematol K7 6J 72 60 O2 10 

malignancy 

.olid tumour 78 2[ 17 21 K OK 

fdds Ratio 10 [.1 1J [.6 7.8 0.1 

.urgery prior 

J0 days 

fverall, 7/+68"#+,& predominated, accounting for JJ_ of all isolates in haematology 

and [1_ in oncology patients. In haematology patients the second commonest 

species was 7/[$(&." (1[_) and in oncology patients 7/@6+8$+-+ (20_) followed by 

7/'+$+'&"6%&"& (18_). 7/*-$%'"#+6"& occurred with equal frequency in both groups. 7* 

[$(&." occurred in only one oncology patient. Crude mortality at day J0 was JO_ in 

haematology and O1_ in oncology patients (p}N.). Multivariate modelling of risB for 

death showed corticosteroid use (p}0.0J) and an indwelling urinary catheter (p}0.0O) 

to be significant in haematology patients and age t6[ years (p}0.01) and IC6 

admission (p}0.01) in oncology patients. Neither Candida species nor presence of 

neutropenia at diagnosis were risB factors for death in this model, although mortality 

rates were higher in patients with severe or persistent neutropenia 

This study describes Candidaemia in Australian patients with malignancy. There were 

differences in species responsible and frequency of underlying morbidities in patients 

with haematological and solid malignancies but no overall difference in mortality. 

7+,5"5+*species was not an independent risB factor for mortality. 

P-0559. Epidemiology of community-onset candidemia in Connecticut and 

Maryland with a comparison of molecular subtypes of Candida parapsilosis 

.ofair A 1 , /yon G 2 , Huie-ahite . 1 , Reiss E 2 , Harrison / J , .anza / J , Arlington-.Baggs 

2 2 , Fridkin S 2 

1 Yale 6niversity .chool of Medicine, 2 Mycotic Diseases 2ranch, J (ohns HopBins 

2loomberg .chool of Public Health 

Background: 2loodstream infections with 7+,5"5+ spp. (candidemia) are increasingly 

being reported outside of the intensive care unit, as well as outside of the hospital 

setting. It is not clear if these patients are similar to inpatients in terms of underlying 

illness, predisposing factors, or infecting strains. 

Objective: Determine if established risB factors are common among patients with 

community-onset candidemia, (occurring ' 2 nd hospital day) and if particular strains of 

7+,5"5+ are responsible for most community-onset candidemias. 

Methods: Incident candidemias were identified through active, population-based, 

surveillance in Connecticut, and 2altimore City and 2altimore County, Maryland, 

during fctober 1, 1KK8 to .eptember J0, 2000. ae compared the characteristics of 

patients developing candidemia by the timing of infection onset relative to 

hospitalization status. Disease was classified as inpatient (culture obtained after 2 

days of hospitalization) or community-onset. Community-onset patients were further 

classified based on timing past hospitalization (recent, distant, or no past 

hospitalization). .ince 7/*'+$+'&"6%&"& was associated with community onset-disease 

in our study, the molecular subtypes of a sample of O[*7/*'+$+'&"6%&"& were evaluated 

by .outhern blots hybridized with the digoxigenin-labeled complex probe, CpJ-1J. 

Comparisons were made by Chi-square. 

Results: fverall, J[6 (J1_) of the 11OJ incident candidemias were classified as 

community-onset disease, which tended to be more liBely caused by 7/*'+$+'&"6%&"& 

(16_ vs. 12_, P}.08)n these difference were more striBing when comparing those 

without recent hospitalization (within past J0 days) where 22_ of candidemias were 

with 7/*'+$+'&"6%&"&, Pv.01). Community-onset disease was also less liBely associated 

with concurrent immunosuppressive therapy (28_ vs. O8_, Pv.01), or recent surgery 

(2K_ vs. [7_, Pv.01) compared to inpatient-disease. Most community-onset 

candidemia patients, 262 (7[_), had been hospitalized at least once in the previous J 

monthsn those with no documented past hospitalization were less liBely to have had 

recent surgery (17_ vs. J6_, Pv.01), recent use of antibacterial agents ([8_ vs. 87_, 

Pv.01) or immunosuppressive therapies (21_ vs. JO_, P}.0[) compared to those 

with recent hospitalization (within past J0 days). 7/*'+$+'&"6%&"& strains tended to be 

unique to the patient, with little similarity of strain types based on epidemiologic 

definitions of community-onset versus inpatient-candidemia. /ittle evidence of 

geographic clustering was present. 

Conclusion: ae report community-onset candidemia is common, occurs in patients 

with extensive contact with the healthcare system, and does not involve a unique 

strain of 7/*'+$+'&"6%&"&. 


P-0560. Retrospective analysis of cases with fungemia detected in a university 

hospital from 1994 to 2003 

Sugi Y 1 , Mori T 1 , fshimi l 1 , fguri T 2 

1 Division of Hematology, Department of Internal Medicine, (untendo 6niversity .chool 

of Medicine, 2 Clinical /aboratory, (aunted 6niversity Hospital 

Objective: To investigate underlying diseases, species of fungi, sites of indwelling catheters, 

complications, and prognosis of patients diagnosed as having fungemia based on the results of 

blood cultures. 

Materials: The clinical records of 20[ patients diagnosed as having fungemia at (untendo 

6niversity Hospital between (anuary 1KKO and December 200J were retrospectively reviewed. 

Results: Fungi were isolated from blood cultures on 210 occasionsn of them, however, O7 

corresponded to contamination. ahen fungi were detected more than once in one patient, the 

events were considered as different episodes if the interval between two events was more than 

2[ days. fne patient had J episodes and J patients had 2 episodes. The number of episodes 

slightly increased year by year affecting men more frequently (68_) than women (J2_) and 

predominantly in patients over [0 (77.6_). In most cases , the underlying disease of patients 

was a solid cancerous tumor (n}K1, OJ_), followed by central nervous system diseases (n}28, 

1J.J_), hematological diseases (n}21, 10.0_), pediatric diseases including malformations K.[_, 

respiratory diseases and heart diseases [.2_ each, others 1J.8_. A single species was 

detected in 1K7 episodes and plural species in 1J. 7+,5"5+*+68"#+,& was the most frequently 

isolated species (n}81), followed by 7+,5"5+/'+$+'&"6%&"& (n}62), 7+,5"5+*@6+8$+-+ (n}2[), 

7+,5"5+*-$%'"#+6"& (n}2J) and*>$"#?%&'%$%,*spp. (n}8). In 16K episodes, an indwelling venous 

catheter was involved and it had to be removed on 1O6 occasionsn for these patients the mortality 

rate due to fungemia was 21.K_, and for the 28 patients in whom the catheter was not removed, 

it was [7.1_. The subclavian vein was the most commonly used port and the most frequently 

isolated species was 7/*'+$+'&"6%&"&*(n}J7), followed by 7/*+68"#+,& (n}21), and 7/*@6+8$+-+ 

(n}1O). In case of the femoral vein, the most frequently isolated species were 7/*+68"#+,& (n}21), 

followed by 7/*-$%'"#+6"& (n}7), and 7/*'+$+'&"6%&"& (n}6). A total of 77 patients received 

antifungal treatmentn their survival rate was [O.[_ and mortality associated with fungemia was 

2O.7_. fn the other hand, for patients who did not receive antifungal treatment, the survival rate 

was O6_, and mortality associated with fungemia was J[_. .erological tests for the diagnosis of 

systemic fungal diseases were performed in 110 casesn [O were positive for (1*J)-#-D-glucan 

test and 18 for mannan antigen test (Pastorex & 7+,5"5+). .ix patients were positive for both 

(1*J)-#-D-glucan test and Pastorex & 7+,5"5+ test. The sensitivity and specificity of (1*J)-#-Dglucan 

detection test were 0.K8[ and 0.K16, respectively, and those of the Pastorex & 7+,5"5+ 

test were 0.[JJ and 1.0, respectively. Candidal endophthalmitis was detected in K episodes. 

Conclusion: In case of hematological diseases, antifungal prophylaxis 

is usually provided, thus fungemia was less frequent in those patients. 

Fungemia was a serious risk for survival in patients with an indwelling 

venous catheter. However, this is sometimes regarded as contamination 

and tends not to be treated appropriately. Serodiagnosis is necessary to 

confirm the diagnosis of fungemia. When patients with an indwelling 

venous catheter have fever refractory to antibiotics, they should have 

the catheters removed and be started on antifungal therapy immediately. 

P-0561. Exposure to airborne fungi during conservative dental treatment 

Szymañska J 

Department of Paedodontics, Medical 6niversity, /ublin, Poland 

The aim of the study was a mycological assessment of bioaerosol forming during 

conservative dental treatment, taBing into account concentration and type of fungal 

microflora. 

The research was conducted on 2[ operative sites (dental units) located in public 

dental clinics. The air contained in the space between a patient and a dentist during 

conservative dental treatment with the use of a high-speed handpiece was examined. 

Air samples were taBen using the portable RC. P/6. Air .ampler (2IfTE.T AG, 

Dreieich, Germany) and ready-to-use agar YM .trips for yeast and mould fungi culture. 

The volume of the sampled air was 100 litres. The concentration of fungi was 

calculated according to the following formulaM cfu/m J } cfu counted on an agar 

strip/sample volume (litre) ó 1000 (litre). 

The concentration of fungi in the collected air samples at individual operative sites 

ranged from O ó 10 1 cfu/m J to JO ó 10 1 cfu/m J . The following fungal species were 

foundM 46-.$,+$"+*+6-.$,+-+, 4&'.$@"66(&*@6+(#(&, 4&'.$@"66(&*-.$$.(&, 76+5%&'%$"()* 

#6+5%&'%$%"5.&, 9.%-$"#?()*#+,5"5(), 1.,"#"66"()*5"0.$&(), 1.,"#"66"()*?.$e(.", 

1.,"#"66"()*$%&.%'($'($.(), P?"d%'(&*,"@$"#+,&. The most common species was 

1.,"#"66"()*?.$e(." (62.17_ of the total count), followed by other fungiM 46-.$,+$"+* 

+6-.$,+-+ - 12.68_, 1.,"#"66"()*$%&.%'($'($.() - K.O1_, P?"d%'(&*,"@$"#+,& - [.KJ_, 

4&'.$@"66(&*-.$$.(& - J.8K_, 9.%-$"#?()*#+,5"5() - 2.2[_, 4&'.$@"66(&*@6+(#(& - 

2.0O_, 76+5%&'%$"()*#6+5%&'%$%"5.& - 1.2J_ and 1.,"#"66"() 5"0.$&() - 0.O1_. The 

concentration of 1.,"#"66"()*?.$e(." at individual operative sites ranged from 0 to JO ó 

10 1 cfu/m J , mean 121.6 cfu/m J , 1.,"#"66"()*$%&.%'($'($.() - from 0 to 11 ó 10 1 

cfu/m J , mean 18.O cfu/m J and 46-.$,+$"+*+6-.$,+-+*- from 0 to 18 ó 10 1 cfu/m J , mean 

2O.8 cfu/m J . 

The examination of dental bioaerosols showedM 1. presence of fungal species which 

occur in air and are cosmopolitan in various climatic zonesn 2. prevalence of fungi 

belonging to 1.,"#"66"() genus which reveal allergenic propertiesn J. presence of other 

fungal species reported to be allergenic and /or immunotoxic (46-.$,+$"+*+6-.$,+-+, 

4&'.$@"66(&*-.$$.(&, P?"d%'(&*,"@$"#+,&). The concentration of fungi in the examined 

dental aerosols is difficult to evaluate becauseM 1. no research on fungal 

contamination of dental bioaerosols is reportedn 2. there are no referential methods for 

examination of fungal contamination of room air, including medical rooms. The results 

may only be compared with few data concerning medical centres rooms, or with the 

suggested referential limits for residential rooms (R/V) and occupational exposure 

limit (fE/), which equal, respectively, [ ó 10 J cfu/m J and [0 ó 10 J cfu/m J . 

2ecause dental bioaerosol is intensely emitted in the breathing space of a patient and 

a dentist, it seems necessary to monitor its microbiological quality. Identification of the 

contamination sources, evaluation of air quality and of a potential threat constituted by 

contamination of the air, and also development of quality control methods, are the 

problems which should be addressed by scientific research in the future.

P-0562. Occurrence, characterization and antifungal susceptibilities of 

environmental isolates of C. albidus and C. laurentii 

Tay s, Na . 

Department of Medical Microbiology, Faculty of Medicine, 6niversity of Malaya 

In our attempt to identify the ecological niche of 7$2'-%#%##(&*,.%U%$)+,& and 7/* 

@+--"", J6 7/*+68"5(& and J7 7/*6+($.,-"" strains were isolated from various 

environmental samples in the llang Valley, Malaysia. These isolates were identified 

by API 20C A6j Bit, with percentage accuracies ranging from K0.8 to KK.K_. Majority 

of the isolates were obtained from bird droppings and vegetative materials of 

a(#+62-'(& trees. The isolates from bird droppings were serotyped using Crypto 

ChecB Iatron RM J0O-l Bit. fnly six 7/*+68"5(& reacted with serum factor 1, while an 

additional J 7/+68"5(& isolates reacted to serum factor 1, [ and 7. 6pon incubation, 

the Canavanine|glycine|bromothymol blue (CG2) agar media remained unchanged 

for all J6(100_) 7/+68"5(&, but turned blue for 22(87_) of 7/6+($.,-""*from bird 

droppings and 7 ([8.J_) 7/*6+($.,-"" from other environmental sources. Twenty 

(J[.1_) of [7 cryptococcal isolates showed the ability to grow at J7ÅC. Antifungal 

susceptibilities testing with Neosensitab disB diffusion agar method showed that all 

except a 7/6+($.,-"" isolate were susceptible to amphotericin 2. All isolates from bird 

droppings and majority of the 7/6+($.,-"" isolates from other environmental sources 

were resistant to [ flurocytosine. Resistance to either fluconazole, Betoconazole or 

itraconazole was observed in 1K (26.1_) of the isolates. 

P-0563. Studies on the safety of commercial mexican sauces and other byproducts 

obtained from dried chilis 

Valdes-Martinez . 2 , Ruiz-frtiz M 2 , frtega-Nava M 2 , Cervantes-Olivares R 1 , Ducoing-aatty A 1 , 

.egundo-Zaragoza C 1 

1 

6niversidad Nacional Autonoma de Mexico - Fac. Med. Vet. Zoot., Mexico, 2 6niversidad 

Nacional Autonoma de Mexico - FE.C-Cuautitlan , Mexico 

Chilis are a staple food in the mexican diet. A wide variety of foods are prepared with chilis both 

fresh (green) an dried ones, A wide range of sauces are also prepared, they are used in foods, 

added to fresh vegetables (cucumber, carrot, water chestnut, etc.), snacBs (potato crisps, nachos 

etc.), candy, lollipops and sherberts. TaBing into account the wide variety of use, there are 

several commercial brands that are commonly used for the purpose. 

Most fresh chilis are dried usually on the ground with no quality assurance programs of Good 

Agricultural Practices applied in their growth. These conditions favor the contamination of the 

chilis, taBing into consideration that in a sauce you cannot see the quality of the chilis used for 

their preparation and therefore their quality is questionable. Fungi growth could have happened 

due to an unadequate drying, storing and processing. 

The aim of the present paper was to determine the aflatoxin and fumonisin content of 6 

commercial sauces found in the Mexican marBet. .auces used contained only one variety of chili, 

vinegar, water and spices. A random sampling of O x 2 x J was appliedM O sites of sampling, 

duplicate samples and triplicate determinations, the aflatoxin and fumonisin content was 

determined using Aflatest and Fumonisin inmunocolumns. ~uantification content was done 

using a Vicam fluorimeter. Results were analyzed using average of the three determinations and 

a randomized complete blocB design with the statistical pacBage IMP version [.1. 

The O8 samples analyzed contained both aflatoxins and fumonisins. The aflatoxin concentration 

found ranged from 2.2 to K.J ppb, the fumonisin concentration found ranged from 2.0[ to K.J 

ppb. Data found are out of normativity, Mexican Norm NMj-F-J77-1K86 Food | Regional | 

Canned | .picy .auce that applies to chili sauces states that they must be free of toxins harmful 

to men. The least squares means are shown on Table 1M 

Table 1: /east .quares Means and .EM for aflatoxin and fumonisin content in chili sauces 

Brand Least Squares Mean* 

Aflatoxins 

Fumonisins 

1 6.[6 a 1.76 ab 

2 O.62 b 2.22 a 

J J.K0 c 2.00 ab 

O J.8K c 2.2[ a 

[ J.O1 cd 1.O1 b 

6 J.1K d 2.JJ a 

.EM 0.O6 0.1O 

The 16th Congress of the International Society for Human and Animal Mycology 

òDifferent 

superscri 

pts indicate significant differences among the chili sauces adjusted means (p v 0.0[) for each 

toxin. 

Conclusions: 

" All the samples analyzed contained both aflatoxins and fumonisins. 

" For aflatoxinsM .amples J, O and [ had no statistical differences among them, as well 

as samples [ and 6 between them. There was no statistical difference between 

brands 1 and J. 

" For fumonisinsM There was no statistical difference among brands 1, 2, J, O and 6, 

sauce [ was different from 2, O and 6. 

" The results presented are part of an ongoing study, the project is supported by a grant 

from the 6niversidad Nacional Autánoma de Mdxico to evaluate the content of 

pathogenic fungi, aflatoxins and fumonisins in dried chilis, and other by-products. 

Do you Bnow about the Food .afety on Chili .auces

P-0564. Study of the correlation between restriction fragment length 

polymorphism (RFLP) and multilocus sequence (MLST) typing methods for 

Candida parapsilosis 

van Asbeck E 1 , MarBham A 1 , Clemons l 1, 2, J 1, 2, J 

, .tevens D 

1 California Institute for Medical Research, .an (ose, CA 6.A, 2 .anta Clara Valley 

Medical Center, .an (ose, CA 6.A, J .tanford 6niversity, .tanford, CA 6.A 

RF/P has been reported since 1K87 in epidemiologic studies of 7/*'+$+'&"6%&"&3*and 

recently an M/.T method studied this organism and divided it into J broad groups, an 

agreement with many other typing methods. In this study, 2K isolates were typed 

independently by RF/P and M/.T. The 1K M/.T group parapsilosis sensu stricto 

isolates were co-extensive with 17 of RF/P type VII-1, plus 2 other types. The M/.T 

groups, proposed as separate species, orthopsilosis (8 isolates) and metapsilosis (2), 

consisted of [ and 1 other RF/P types, respectivelyn none of these 10 isolates were 

VII-1, and none of the (total 8) non-VII-1 types were found in more than one M/.T 

group. VII-1 is the most common 7/*'+$+'&"6%&"&*RF/P type (176/20J in continuing 

studies in 6. and Europe, exclusive of outbreaBs), and parapsilosis sensu stricto is 

similarly dominant in M/.T studies. Neither VII-1 nor parapsilosis sensu stricto are 

subtyped by their respective methods. VII-1 and parapsilosis sensu stricto appear to 

be nearly identical, whereas orthopsilosis, which #+, be subtyped by M/.T, also 

consists of many (uncommon) RF/P types. Metapsilosis is unusual. 

P-0565. Candida parapsilosis fungemia in neonates: Genotyping (RFLP) results 

suggest healthcare workers hands as source 

van Asbeck E 1 , Huang Y 2 , MarBham A 1 , Clemons l 1, J, O 1, J, O 

, .tevens D 

1 California Institute for Medical Research, .an (ose, CA 6.A, 2 Chang Gung 

Children]s Hosp., Taiwan, J .anta Clara Valley Mdeical Center, .an (ose, CA 6.A, 

O .tanford 6niversity, .tanford, CA 6.A 

A [-mo. outbreaB of 7/*'+$+'&"6%&"& fungemia involving 17 neonatal intensive care unit 

(NIC6) patients was studied. 1O blood culture isolates were available for study. 

Colonization in NIC6 infants (mouth, urine, sBin, rectum, endotracheal) was studied 

over this time, [ yielded 17 7/*'+$+'&"6%&"&*isolates, and 1-O isolates from each patient 

were available. Hands of NIC6 health-care worBers were cultured, and 11 yielded 7/* 

'+$+'&"6%&"&, of which 8 were available. All 1O blood isolates were of one RF/P type 

(type VII-1, the most common type encountered worldwide). The K infant colonizing 

isolates were of O different types (O isolates were VII-1). However, 7/8 of the isolates 

from health-care worBers were the same type, VII-1, as in the infants] blood. These 

findings reinforce prior studies that have implicated healthworBer hands as the source 

of nosocomial, including neonatal, fungemia, but an unusual feature of this study was 

the comparison with infant colonizing isolates. The majority of infant colonizers were 

not found in the blood, suggesting a possible direct spread of the epidemic type VII-1 

strain from worBer hands to infant blood, such as through line insertion sites. The 

source of the infant colonizing strains is unclear, but the non-VII-1 strains may be 

largely of maternal origin and the VII-1 strains from healthcare worBersn conversely, 

the healthcare worBer strain pattern doesn]t suggest spread from the infants. As 7/* 

'+$+'&"6%&"& is very common in neonatal fungemia globally, further epidemiologic 

studies are indicatedn however, other 7/*'+$+'&"6%&"& genotyping methods also have 1 

dominant type that cannot be subtyped.

P-0566. Prevalence of Aspergillus spp. in hospital air 

Vasilyeva N, .uBhanova Y, Bogomolova T 


Immunocompromised hospitalized patients, especially those with hematological 

disorders, are at high risB for invasive pulmonary aspergillosis. Nosocomial 

aspergillosis is also an emerging problem at surgical intensive care units. The purpose 

of the study was to determine incidence and levels of 4&'.$@"66(&*spp. propagules in 

the indoor air of hospitals for hematological and surgical patients in .t. Petersburg. 

At one hematological and three surgical departments the indoor air was sampled 

repeatedly during spring, summer and autumn of 200[. Air-sampling was performed 

by means of an one-staged impactor hP6-12i (hHimBoi, Moscow). .amples were 

cultured on .abouraud agar and examined for growth of 4&'.$@"66(&*spp. colonies. 

At the hematological department J8.O_ of air samples were positive for 4&'.$@"66(&* 

spp. 4/*U()"@+-(&* was the most common species which was detected in J0.2_ of 

samples. The average level of 4/*U()"@+-(&* in the air of this department was J.7 

CF6/ m J . The highest concentration of 4/*U()"@+-(&*in the aerosol*([6 CF6/m J ) was 

revealed in a patient room during rainy weather in May. .everal other 4&'.$@"66(&* spp. 

(4/*,"@.$3*4/*%$2d+.3*4/*#6+0+-(&J were cultured from the indoor air at the 

hematological department. 

P-0567. Dermatophytoses in Monterrey, México 

Vera-Cabrera L, Hinojosa R, aelsh E, fcampo-Candiani (, Gomez M, aelsh f 

6niversidad Autonoma de Nuevo /eon 

In the present report we reviewed a total of 2,JK7 cases of dermatophytosis from 

superficial cutaneous lesions between the years of 1K78 to 1KK0. The cases included 

were from the Department of Dermatology at the 6niversity Hospital located in the city 

of Monterrey, Mdxico. A total of 726 tinea pedis, 61J tinea unguium3 OO1 tinea capitis, 

JK[ tinea corporis, and 222 tinea cruris cases were observed. The most commonly 

isolated dermatophyte species was >$"#?%'?2-%,*$(8$() (O[_), followed by 

>$"#?%'?2-%,*).,-+@$%'?2-.& (2J.7_), >$"#?%'?2-%,*-%,&($+,& (21_), !"#$%&'%$()* 

#+,"& (7.1 _), and a'"5.$)%'?2-%,*U6%##%&() (2.[_). /ess frequently we isolated 

!"#$%&'%$()*+(5%(","", !"#$%&'%$()*@2'&.(), >$"#?%'?2-%,*0"%6+#.(), and 

>$"#?%'?2-%,*0.$$(#%&(). Most of the cases were observed in the warmest months of 

the year (from March to .eptember), and were equally distributed in both genders, 

except for tinea cruris which was more prevalent in men (J.[M1 ratio). 

4&'.$@"66(&*spp. were also detected in most of samples of the indoor air at three 

surgical departments. The levels of 4&'.$@"66(&*spp. spores in air samples ranged 

from O to J6 CF6/m J . 4/*U()"@+-(&*was isolated most frequently, followed by 4/*,"@.$/* 

fther species (4/*U6+0(&3*4/*,"5(6+,&3*4/*@6+(#(&3*4/*0.$&"#%6%$J*were rare in the 

aerosol of surgical departments. 

Indoor hospital air is frequently contaminated with 4&'.$@"66(&*U()"@+-(&/*Hygienic and 

special preventive measures are needed for prophylaxis of nosocomial aspergillosis in 

susceptible categories of hospitalized patients. 


P-0568. Retrospective of nosocomial candidiasis, during 2003-2005, in 

children's hospital, Sao Paulo, Brazil 

Viani P, Viani F, Moreira D, Matsumoto F, Ruiz /, Auler M, .ilva E, Gambale a, 

Rodrigues Paula C 

Instituto de Ciüncias 2iomddicas / 6.P, .ao Paulo, 2razil 

This purpose of study was to evaluating the incidence and distribution of 7+,5"5+ 

species responsible for nosocomial candidiasis in a Public Children Hospital. ae 

have studied 218 strains of 7+,5"5+ spp during 200J-200[. This strains were 

isolated from blooding, urine and other biological specimens (J2,11_n [O,[K_ and 

1J,J0_, respective). From strains OO,K[_ were identified as 7/*+68"#+,& and 

[[,0[_ as non- +68"#+,& species. The distribution of this strains wasM in 200J, 

OO,22_ and [K,78_n in 200O, JK,68_ and 60,J2_n in 200[ [7,1O_ and [[,0[_ of 

this the +68"#+,& and nonØ+68"#+,& species, respectively. A higher incidence of this 

7/*+68"#+,& strains was observed in candiduria cases (61_) and in 7K_ of the 

candidemia cases we isolated non-+68"#+,& species (pv0,0[). ae observed that 

occurred a significative increased of candiduria and the frequency of 7/*+68"#+,& 

(10,J2_ to 16,K2_) | pv0,0[. In our childrenïs hospital , over the tree year period, 

a trend of increased of non-+68"#+,& isolated from blood and the 7/*+68"#+,& strains 

from urine was noted. Rapid identification of species is a crucial information for the 

clinician because we assist to the emergence of this non-+68"#+,& species wish can 

be resistant to the antifungal. 

P-0569. Automated extraction of cumulative prior neutropenia to predict risk of 

invasive aspergillosis (IA) 

Walker R, Issa N, HosBin T, .t .auver (, .chlecB C 

Mayo Clinic College of Medicine 

Purpose of the study: ae hypothesized that the total number of neutropenic days that 

can accrue in a discontinuous manner, from multiple episodes of chemotherapy-induced 

neutropenia, can identify a sub-group of patients with a high risB of IA, whether during or 

after resolution of neutropenia. 

Summarized description of the project: ae identified a cohort of all (287K) patients 

treated for leuBemia (861) or lymphoma (1O18), between 1KK0 and 2000, at our institution. 

ae developed software to extract, from archived electronic files, all complete blood counts 

and differential white blood counts on these patientsn interpolation algorithms were used to 

estimate a neutrophil count on days when no laboratory tests were done. ae then built a 

date-specific field indicating whether or not a patient was neutropenic on each of the J6[ 

days of the first year following the patient]s initial treatment. 

ae identified and dated diagnoses of IA among these patients using infection control data, 

autopsy reports microbiology data, and standard guidelines for definite and probable cases. 

fn each of the days (from the date of first treatment, i.e., 1 to J6[) on which a case of IA 

occurred, the status of the entire cohort who had survived up through that day was 

examined for the median and range of cumulative neutropenic days (CND) that had 

accrued. ae also examined the ratio of CND to Total .urvival Days (T.D) for IA cases 

and non-IA cases in three different time frames during the first yearM day 0-O2 (0 to 6 

weeBs), day O1-120 (6 weeBs to O months), and day 121 to J6[ (O to 12 months). 

Results:There were 27 cases of IA (definite or probable) in leuBemia patients. At the date 

of their IA diagnosis, 2J/27 (8[.2_) had CND values above the median for all leuBemia 

patients followed through that same date (p}0.000J). In 12 cases that occurred within a 

neutropenic episode, 11/12 (K1.7_) were above the median (p } 0.00O). For the 1[ cases 

that occurred after resolution of neutropenia, 12/1[ (80_) were above the median (p } 

0.02). 

Among the 12 cases of IA in lymphoma patients, O of 12 (JJ_) had a CND value at the 

date of their diagnosis of IA that was above the K[ percentile for all lymphoma patients 

followed through that time (p v 0.0001). 

For all patients, the ratio CND to T.D, was 0.J during the first O2 days post first treatment, 

0.18 during days OJ to 120, and 0.1 during days 121 to J6[. The C.D/T.D ratio among IA 

cases was twice as high as non-IA patients during the first two intervals (0.6 versus 0.J, 

and 0.JO versus 0.18, respectively), and nearly three times as high during the last interval 

(0.27 versus 0.1). 

Conclusion: Patients can accrue cumulative, discontinuous neutropenic exposure that 

increases their risB of IA significantly, both during and after resolution of neutropenia, in a 

way that is not apparent in the immediate temporal context of the most recent 

chemotherapy, but which can be identified using automated data extraction algorithms.

P-0570. Strain typing of Fonsecaea pedrosoi strains by PCR-RFLP analysis 

Watanabe S 1 , Anzawa l 2 , lawasaBi M 1,2 , MochizuBi T 1,2 , IshizaBi H 2 

1 Department of Dermatology, lanazawa Medical 6niversity, IshiBawa, (apan., 

2 Division of Dermatomycology(Novartis Pharma), lanazawa Medical 6niversity, 

IshiBawa, (apan. 

^%,&.#+.+*'.5$%&%"3 a major causative agent of chromoblastomycosis and 

phaeohyphomycosis, has been classified into 7 types based on restriction fragment 

length polymorphism (RF/P) of its mitochondrial DNA (mtDNA) using the restriction 

enzymes A+.III, A",dIII and !&'I, and into 6 types based on RF/P of internal 

transcribed spacer (IT.) regions of the nuclear ribosomal RNA genes with the 

restriction enzymes*V5.I and !&'I. Application of the polymerase chain reaction 

(PCR) on DNA of ^/*'.5$%&%" isolates using primers 1) previously used in studies on 

other fungi has yielded a product of about O00 bp. 

Purpose: To assess whether the PCR product yielded using the above primers can 

be used as a genetic marBer in PCR-RF/P analyses of ^/*'.5$%&%" strains. 

Method: Total DNA was extracted from three ^/*'.5$%&%" isolates with Bnown mtDNA 

type and rDNA type using an established method 2) . PCR was performed using []- 

GCCAACATCGAGCAAACATGG-J] and []-GCAAGCAGCACCGTCAATACCAAT-J] 

as the forward and reverse primers, respectively. PCR products were digested with 

A+.III and =+(J4K, electrophoresed on 6_ acrylic amide gel, stained with ethidium 

bromide, and visualized under 6V light. The procedure tooB about 8 h. 

Results: The three strains showed two different RF/P patterns when A+.III was used. 

Discussion: As yet, we cannot conclude whether this method is able to distinguish ^/* 

'.5$%&%" finer than do well-established mtDNA|RF/P and IT.-RF/P analyses. To 

further evaluate this method, it will be tested on J0 clinical isolates of ^/*'.5$%&%" 

collected from (apan, China, Costa Rica, Venezuela, Mexico, Argentina, Australia, 

Zaire, Colombia, Madagascar and Mozambique. 

P-0571. The distributions of Candida species and their trends of drug 

susceptibility between 1999-2002 in Taiwan 

Yang Y 1 , Cheng H 2 , /o H 2 

1 Dept 2iological .cience c Technology, National Chiao Tung 6niversity, Hsin-chu, 

Taiwan, 2 Division of Clinical Research, National Health Research Institutes,Miaoli, 

Taiwan 

To determine the distribution of 7+,5"5+ species and to investigate the trend of 

susceptibility to antifungal drugs of 7+,5"5+ species in Taiwan, we have conducted 

two rounds of Taiwan .urveillance of Antimicrobial Resistance of Yeasts (T.ARY) in 

1KKK and 2002. The distribution of species and source were similar between these 

two surveillances. 7+,5"5+*+68"#+,& was the most common species among the 

isolates followed by 7+,5"5+*-$%'"#+6"&, 7/*@6+8$+-+, 7+,5"5+*'+$+'&"6%&"&, and 7/* 

[$(&.". ahen classified according to the sources, urine was the most common source 

followed by sputum (22.1_), blood, center venous line, and wound. .usceptibilities 

to amphotericin 2 and fluconazole of 6J2 and K0K 7+,5"5+ species collected in 1KKK 

and 2002, respectively, were determined by the broth microdilution method. The 

resistant rate to amphotericin 2 was 0.[_ in 1KKK and 2.[_ in 2002. ahereas, the 

resistant rate to fluconazole has decreased from 8.O_ in 1KKK to 1.K_ in 2002. 

All of the 7/*'+$+'&"6%&"& isolate were susceptible to fluconazole, which is consistent 

with previously report that 7/*'+$+'&"6%&"& is the most susceptible species to 

fluconazole. Isolates collected in the T.ARY 2002, 11.1_ of 7/*[$(&." and OK.7_ of 

7/*@6+8$+-+ were susceptible to fluconazole, which is consistent with previous reports 

that both species were less susceptible to fluconazole than other 7+,5"5+ species. 

Thus, fluconazole is not a drug recommended to treat infections caused by these two 

species. Amphotericin 2 appears to be the drug of choice for the treatments. 

However, along with increased usage of amphotericin 2, more 7/*[$(&." collected in 

2002 (66.7_) than that in 1KKK (10_) were resistant to it. This is also the case for 7/* 

@6+8$+-+ ([.O_ in 2002 vs. 0_ in 1KKK). The co-resistance to both amphotericin 2 

and fluconazole of 7/*[$(&."*and 7/*@6+8$+-+ may become an issue for treatment of 

infections caused by them. 

References: 

.himizu l et al.M (. Gen. Appl. Microbiol. OOM2[1-2[8,1KK8 

MaBimura l et al.M (. Med. Microbiol. O0MJ[8-J6O,1KKO 


EPIDEMIOLOGY* 

P-0572. Study the Malassezia flora of hospitalized neonates. 

Zomorodian l 1 , Mirhendi . 1 , Tarazooie B 1 , lordbacheh P 1 , Zeraati H 1 , louloumvaBi 

A 2 , Zomorodian . 1 , Nayeri F 1 

1 Tehran 6niversity of Medical .ciences, 2 Medical 6niversity of Vienna, Vienna, 

Austria 

>$"#?%'?2-%,*$(8$() (>/$(8$()) is a cosmopolitan arthropophilic dermatophyte which 

causes sBin and nail infection. The most of antifungal drugs available for clinical use 

are targeted four molecules includingM beta-glucan, sterol-alpha-demethylase, 

ergosterol and DNA/RNA synthesis. In the present study we characterize the gene 

encoded beta-tubulin protein in Trichophyton rubrum and investigate the effect of 

griseofulvin of its expression. 

Clinical isolated of >/$(8$() were cultured in liquid medium and the nucleic acids 

(DNA c RNA) were isolated from obtained mycelial mass by standard methods. The 

sequences of Bnown beta-tubulin genes in other fungi were aligned and pairs of 21 nt 

primers were designed from highly conserved regions. 6sing mentioned primers, we 

amplified predicted molecules from genomic DNA as well as cDNA of >/$(8$() as 

PCR templates. Effect of griseofulvin on expression of this gene was evaluated by 

Real-Time PCR. 

Finally, 186O nucleotides have been sequenced from this gene (accession number 

AY76J78K) which encodes a polypeptide with OO7 amino acids (accession number 

AAVJJ7JJ). Nucleotide sequence comparison in gene data banBs (NC2I, NIH) for 

both the partial DNA and its deduced amino acid sequence revealed significant 

homology with members of the euBaryotic beta-tubulin genes. The amino acid 

sequence of the encoded protein was about 82_ identical to the sequence of betatubulin 

proteins of the other fungi. Interestingly, griseofulvin suppressed the 

expression of beta-tubulin gene in >/*$(8$() in dose dependent manner while 

fluconazole had no effect on its expression. 

P-0573. Artificial nails - potential risk of onychomycosis. 

¯elazny I, NowicBi R, 2yBowsBa 2 


Introduction: Artificial nails are an increasingly popular cosmetic enhancement to 

the natural nail. They provide the opportunity for women to camouflage unsightly 

nail conditions and improve the appearance of their nails. 

Purpose: The purpose of the study was to demonstrate that onychomycosis can be 

related to artificial nails use. 

Material and methods: ae present patients who presented to the Dermatology 

Department of Medical 6niversity in Gda,sB with onychomycosis after application of 

artificial nails. .ome of the patients came to the dermatologist because of 

cosiderable pain within fingernails, whereas others had no such symptoms. In all 

patients the clinical diagnosis was confirmed by direct microscopic examination and 

by growth tests. 

Conclusions: Recent precise figures are not available from the number of adverse 

reactions related to the use of nail care product, also including onychomycosis. ae 

do not claim that the artificial nails are dangerous. However as the use of artificial 

nails is so widespread, a stress should be put on the possibility of adverse reactions, 

liBe for example onychomycosis. Particularly proper education of salon services 

should be taBen into consideration.

GENOMICS* 

P-0574. Candida albicans CA5996 gene : Implication in cell wall integrity 

Apaire-Marchais V 2 , lempf M 1 , Cottin ( J , /icznar P O , /efrancois C [ , Tronchin G 6 , 

Robert R 7 

1 6FR .ciences Pharmaceutiques et Ingdnierie de la santd, 2 6FR .ciences 

Pharmaceutiques et Ingdnierie de la santd, J 6FR .ciences Pharmaceutiques et 

Ingdnierie de la santd, O 6FR .ciences Pharmaceutiques et Ingdnierie de la santd, 

[ 6FR .ciences Pharmaceutiques et Ingdnierie de la santd, 6 6FR .ciences 

Pharmaceutiques et Ingdnierie de la santd, 7 6FR .ciences Pharmaceutiques et 

Ingdnierie de la santd 

During the past two decades, the prevalence of candidiasis has increased marBedly 

and 7+,5"5+*+68"#+,& has now become one of the most important cause of 

nosocomial infections. In a previous report, we demonstrated the overexpression of J[ 

unidentified genes in response to adherence of 7+,5"5+*+68"#+,& germ tubes to 

plastic. Therefore, a bioinformatic analysis was performed searching for genes 

encoding surface proteins potentially involved in adherence. Nineteen genes were 

thus selected, and one of them, 7+5RRN which encodes the IPF67J protein, was 

further investigated. A database search for sequence homology revealed, for the 

deduced protein, a significant sequence identity to =+##?+$%)2#.&*#.$.0"&"+.*Pmu1, 

a phosphomutase involved in trehalose-6-phosphate pathway. Disruption of both 

alleles of 7+5RRN gene from 7/*+68"#+,& parent strain 2aP17 was performed by PCR 

method. Null mutants for 7+5RRN gene showed an increased sensitivity to Calcofluor 

white, a fluorescent brightner that interferes with !-glucans and chitin. Furthermore, 

microscopic observation of semi-thin sections of blastoconidia revealed the presence 

of numerous dead cells for 7+5RRN null mutant compared with the parent cells. 

CytoBinesis and cell separation still occurred usually. However, an abnormal budding 

was sometimes observed, leading to pseudohyphal growth. /iBewise, transmission 

electron microscopy revealed alterations in cell wall architecture, particularly an 

irregular thicBening of the cell wall, accompanied by dramatic changes in the 

organization of the inner layer. Together, these results suggest that 7+5RRN gene 

could play a role in the assembly of the cell wall structural components. 

P-0575. Genome-wide expression profile analysis reveals genes differentially 

expressed in association with fluconazole resistance in clinical isolates of 

Candida glabrata 

Earhart K 1 , VermitsBy ( 2 , ju / 1 , Homayouni R 1 , Edlind T 2 , Rogers D 1 

1 6niversity of Tennessee, 2 Drexel 6niversity 

Purpose: 7+,5"5+*@6+8$+-+ has emerged as a major cause of mucosal and invasive 

fungal infection in the 6nited .tates, second only to 7+,5"5+*+68"#+,&. Recent reports 

have described the acquisition of azole resistance in clinical isolates from 

hematopoietic stem cell transplant patients, AID. patients, and patients receiving 

radiation for head and necB cancer. In an effort to identify genes potentially involved 

in azole resistance in clinical isolates of 7/*@6+8$+-+, we examined changes in the 

gene expression profile of three matched sets of clinical isolates representing the 

acquisition of azole antifungal resistance. These expression profiles were compared 

to that of a previously characterized laboratory-derived matched set of isolates 

representing the acquisition of azole antifungal resistance. 

Methods: Matched clinical isolates used in this study included 62-16 (FCZ MIC}1 

&g/m/) and 62-17 (FCZ MIC,2[6 &g/m/), R-J[62 (FCZ MIC}16 &g/m/) and 0J-26KO 

(FCZ MIC,6O &g/ml), and 68[6 (FCZ MIC}J2 &g/m/) and 6K[[ (FCZ MIC}2[6 

&g/m/). /aboratory derived strains included 660J2 (FCZ MIC}16 &g/m/), F1[ (FCZ 

MICt128 &g/m/), 660J2-pdr1 (FCZ MICv2 &g/m/), and F1[-pdr1 (FCZ MICv2 

&g/m/). Isolates were grown to mid-log phase and total RNA was extracted for 

microarray analysis. Gene expression profiles were compared using a 7/*@6+8$+-+ 

microarray designed for use with the Affymetrix platform. Each microarray 

experiment was performed in duplicate. Genes were considered to be differentially 

expressed if they were reproducibly up- or down-regulated , 1.[-fold ([0_). 

Differential expression was verified for selected genes by real-time RT-PCR. 

Results: A total of K genes were found to be up-regulated in association with azole 

resistance in all O matched isolate sets studied. These included the putative CgPdr1p 

transcriptional activation targets 7@7VPI, P>4I, kbPI, A^VI, kKXQCC#, and kK!I, 

as well as 1b9I, !49I, and Vb9I. A total of O[ genes were found to be upregulated 

in J of the O sets studied. These included the putative Pdr1p transcriptional 

activation targets 7@1VPI, 7@1VAI, !a>M, oVPM, P1È, X7Z5, k`XI:E#, 

k!XI:IB, 4>^M, 4VAN, k]XIN:#, and P=ZI, as well as VVPED, 7!cM, kZ>I, and 

Z49C. A total of 1[2 genes were found to be up-regulated in 2 of the O sets studied. 

These included the putative Pdr1p transcriptional activation targets A415, =T>I, 

A=1IM3 1VPIN, X49I, and 7=PI, as well as A41:, K1>I, k17I, K7>I, 4PaI, PAb5, 

and k1XQDDB. 

Conclusions: These data are consistent with a role for Pdr1p in azole resistance in 

all of these clinical isolates, but also suggest a potential role of other transcriptional 

activation programs in this process. Many of these genes such as those encoding 

transcription factors (P1È, =T>I, A41:, A415), transporters (P>4I, kbPI, oVPM), 

and genes involved in sterol (1VPIN, 4PaI3 4>^M, kZ>I) and sphingolipid (X7Z5, 

X49I, 7=PI, K1>I, k17I) metabolism warrant further assessment for a direct role in 

azole antifungal resistance. 


P-0576. Putative involvement of lipid rafts in antifungal mode of action of 

miconazole 

FranÑois I 1 , Thevissen K 1 , Vandercappellen ( 1 , Dispersyn G 2 , Ausma ( 2 , 2orgers M 2 , 

Cammue 2 1 

1 CMPG, latholieBe 6niversiteit /euven, Heverlee, 2elgium, 2 2arrierTherapeutics nv., 

Geel, 2elgium 

Purpose of the study: The synthetic class of azole antimycotics constitutes the 

largest group of antifungal agents currently in clinical use. The generally accepted 

mode of action of azoles is the inhibition of 1O!-lanosterol demethylase, a Bey enzyme 

in ergosterol biosynthesis, resulting in depletion of ergosterol and accumulation of 

toxic 1O!-methylated sterols in membranes of susceptible yeast species. Recently, we 

and other research groups could demonstrate that generation of reactive oxygen 

species (Rf.) is important for the antifungal activity of miconazole, pointing to an 

ancillary mode of action for this azole s1, 2u. 

Description of the project: To obtain more insight in the mode of action of 

miconazole, we screened the complete set of haploid =+##?+$%)2#.&*#.$.0"&"+. gene 

deletion mutants for resistance to miconazole. 

Results and conclusion: ae could demonstrate that =+##?+$%)2#.&*#.$.0"&"+.* 

deletion mutants affected in sphingolipid and ergosterol biosynthesis are resistant to 

miconazole. These miconazole-resistant deletion mutants are also resistant against 

other azoles, namely fluconazole and clotrimazole, and a polyene, namely nystatin. 

This indicates a possible involvement of lipid rafts, membrane patches consisting of 

sphingolipids and ergosterol, in regulation of ",*0"-$%*resistance of yeast to azoles and 

polyenes. 

References : 

lobayashi et al., 2002. Antimicrob. Agents Chemother. O6MJ11J-J117 

FranÑois et al., 2006. Anti-Infect. Agents Med. Chem. [MJ-1J. 

P-0577. Transcriptome analysis of Paracoccidioides brasiliensis cells 

undergoing mycelium-to-yeast transition 

Goldman G 1 , Costa de fliveira R 2 , Goldman M 1 , Puccia R J , Travassos / J , Nobrega 

M O , Nobrega F O , Nunes / 2 

1 FCFRP,6niversidade de .ao Paulo, 2razil, 2 6niversidade de Mogi das Cruzes, .ao 

Paulo, 2razil, J 6niversidade Federal de .ao Paulo, 2razil, O 6niversidade do Vale do 

Paraiba, .ao Paulo, 2razil 

Purpose of the study: Paracoccidioides brasiliensis is a thermodimorphic fungus 

associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in 

.outh America. In humans, infection starts by inhalation of fungal propagules, which 

reach the pulmonary epithelium and transform into the yeast parasitic form. 

Summarized description of the project: The mycelium-to-yeast transition is of 

particular interest because conversion to yeast is essential for infection. ae have 

used a P. brasiliensis biochip carrying sequences of O,6K2 genes from this fungus to 

monitor gene expression at several time points of the mycelium-to-yeast 

morphological shift (from [ to 120 h) 

Results and conclusions: The results revealed a total of 2,[8J genes that displayed 

statistically significant modulation in at least one experimental time point. Among the 

identified gene homologues, some encoded enzymes involved in amino acid 

catabolism, signal transduction, protein synthesis, cell wall metabolism, genome 

structure, oxidative stress response, growth control, and development. The expression 

pattern of 20 genes was independently verified by real-time reverse transcription-PCR, 

revealing a high degree of correlation between the data obtained with the two 

methodologies. fne gene, encoding O-hydroxyl-phenyl pyruvate dioxygenase (O- 

HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and 

the use of NT2C s2-(2-nitro-O-trifluoromethylbenzoyl)-cyclohexane-1,J-dioneu, a 

specific inhibitor of O-HPPD activity, as well as that of NT2C derivatives, was able to 

inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. 

These data set the stage for further studies involving NT2C and its derivatives as new 

chemotherapeutic agents against PCM and confirm the potential of array-based 

approaches to identify new targets for the development of alternative treatments 

against pathogenic microorganisms. 

Financial support: Fundacao de Amparo a Pesquisa do Estado de .ao Paulo 

(FAPE.P) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico 

(CNPq), both from 2razil.

P-0578. Comprehensive interpretation of transcriptome and proteome data of 

human pathogenic fungi 

Hauser N 1 , 2usold C 2 , Fellenberg l 2 , ainter . J , Dippon ( J , Rupp . 1 

1 Fraunhofer IG2, .tuttgart, Germany, 2 German Cancer Research Center, Heidelberg, 

Germany, J 6niversity of .tuttgart, Germany 

Comprehensive Bnowledge about virulence and resistance mechanisms on the 

pathogen side, reactions on the host side as well as the overall interaction of 

pathogen and host is prerequisite to the development of new drugs and therapeutics. 

ae have developed an ",*0"-$% reconstructed three-dimensional tissue model of 

various organs liBe gut or sBin s1u and other human tissue models. These model 

systems are used to investigate infection processes and host-pathogen interactions 

on transcriptome and proteome level. 

Results from such assays are analyzed using MCHiP. (www.mchips.org) s2u. This 

data warehouse platform combines data from various origins and integrates not only 

experimental and clinical descriptions but also other parameters and information such 

as gene annotations. fur recent developments of integrating annotations from the 

Gf-consortium (www.geneontology.org) sJu demonstrate the expedience of computerbased 

statistical analysis of annotations for a systemic description of such complex 

processes liBe pathogen infections. 

References: 

1. Dieterich .-*+6/ (2002)M In vitro reconstructed human epithelia reveal 

contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion. 

!"#$%8"%6%@2, 1O8, OK7-[06 

2. Fellenberg .-*+6/ (2002)M Microarray Data aarehouse Allowing for the 

.tatistical Analysis of Experiment Annotation. Z"%",U%$)+-"#&, 18, O2J-OJJ. 

3. 2usold .-*+6/ (200[) Integration of Gf annotations in Correspondence 

AnalysisM facilitating the interpretation of microarray data. 2ioinformatics, 21, 

2O2O-2O2K. 

P-0579. Stereotypical changes in the gene expression profile of Candida 

albicans in response to the sterol biosynthesis inhibitors fenpropimorph, 

ketoconazole, and terbinafine 

Liu T 1 , Znaidi . 2 , 2arBer l 1 , ju / 1 , Homayouni R 1 , Raymond M 2 , Rogers D 1 

1 6niversity of Tennessee, 2 6niversitd de Montrdal 

PurposeM The ergosterol biosynthesis pathway is a common target of many 

antifungal agents. It is liBely that the fungal cell activates specific stress response 

pathways to cope with ergosterol depletion. The aim of the present study was to 

identify changes in the gene expression profile of 7+,5"5+*+68"#+,& upon exposure to 

three inhibitors of ergosterol biosynthesis that act through distinct targets. 

Methods: 7/*+68"#+,& .C[J1O was exposed to fenpropimorph, Betoconazole, or 

terbinafine at Oj MIC of each agent for approximately six doubling times (6 hours). 

Gene expression profiles were compared using a 7/*+68"#+,& microarray designed for 

use with the Affymetrix platform. Each set of drug exposures was performed 

independently in triplicate. Genes were considered to be differentially expressed if 

they were reproducibly up- or down-regulated , 2-fold. Differential expression was 

verified for selected genes by real-time RT-PCR. 

Results: A total of 2K6 genes were found to be up-regulated and 176 genes were 

found to be down-regulated in a stereotypic fashion in response to these three agents. 

6p-regulated genes included the transcription factor >47I and its putative target 

genes 7VPI, 7VPM, P>4:, K1^C5:Q, K1ÎRDIQ, K^T5, K1ÎQMNM, K1^CDIC, K1^:5:5, 

K^Ê, K`bI, K1ÎINRE, K1ÎIDER, !>P), genes involved in cell wall maintenance 

(a7!::I, =47C, PAbM), chitin biosynthesis (7A=M, 7A>I), cell stress (4VAI, 4VA:, 

4VAE, 4bmM, 7>4I, =bVMI, VVPED, K^VN, K^VC), iron metabolism (7^XIM, ^Pa:M, 

^Pa:5, ^Pa:C, ^>AI), transcription (74>D, P1È), small molecule transport (7VP:, 

7VPII, P>4M, P>4E), lipid, fatty acid, and sterol biosynthesis (aP9MC, 7AbI, XK1C, 

XK1D, 1m4I, ^44MI, ^44MM, ^44M:), and secreted aspartyl proteinase genes (=41E, 

=415, =41N, =41R). Down-regulated genes included those involved in transcription 

(A415, ^7PI, `7ZM), the phospholipase 2 gene 1XZI, and A/. family genes (4X=M, 

4X=E, 4X=II, 4X=IM). 

Conclusions: The stereotypical changes in gene expression in response to 

pharmacologic inhibition of ergosterol biosynthesis with these three antifungal agents 

provides insight into how 7/*+68"#+,& compensates for depletion of this critical cell 

membrane component. Many of the responsive genes revealed here warrant further 

study in the context of antifungal drug resistance and tolerance, and may serve as 

potential drug targets for enhancing the activity of the ergosterol biosynthesis 

inhibitors. 


P-0580. Expression profiles of Aspergillus fumigatus under human neutrophil 

attack and environmental stress 

May G 1 , Nguyen C 1 , lim . 2 , Nierman a 2 

1 6T M.D. Anderson Cancer Center, 2 The Institute for Genomic Reseaerch 

The innate immune system has a central role in combating infections caused by 

4&'.$@"66(&*U()"@+-(&. Macrophages and neutrophils are two cell types of the innate 

immune system that are very important in preventing and eliminating 4/*U()"@+-(& 

infections. Macrophages are the first line of defense, engulfing and Billing inhaled 

conidia and possibly early germinating conidia before they can establish hyphal 

growth. Neutrophils attacB and Bill hyphae that invade tissue. The importance of 

neutrophils in combating and resolving infections caused by 4/*U()"@+-(& is 

underscored by two observations. First, invasive aspergillosis disease is rare in 

people that have normal numbers of neutrophils, thus it is a disease primarily of a 

neutropenic host. Resolution of invasive aspergillosis disease strongly correlates with 

recovery of neutrophil numbers in patients that acquired disease while neutropenic. 

ahile these simple observations have been Bnown for some time, we still Bnow little 

about the interaction between 4/*U()"@+-(&, and macrophages and neutrophils. ae 

hypothesize that 4/*U()"@+-(& responds to host cells of the innate immune system, 

macrophages and neutrophils, with specific transcriptional responses to defend 

against attacB by these immune cell types, ultimately contributing to fungal virulence. 

To begin to study the relationship of the interaction between neutrophils and 4/* 

U()"@+-(& and virulence, we have undertaBen a study of the global patterns of gene 

expression in 4/*U()"@+-(& hyphae in the presence of human neutrophils in culture. 

ae have identified the mRNAs with the most or least relative abundance when 

compared to hyphae in cell culture medium lacBing neutrophils over a 60 minute time 

course. There are [J2 4/*U()"@+-(& genes including 20 transcriptional regulators 

whose mRNAs are more abundant or less abundant by a factor of two fold or more, 

O28 more and 10O less. ae have explored the expression pattern of these 4/* 

U()"@+-(& genes under conditions variably related to virulence including H 2f 2 

oxidative stress, osmotic stress, anaerobic stress, and glucose to sorbitol carbon 

source shift. fur analysis of the pattern of expression of these genes reveals 

information on the conditions imposed by the neutrophils on 4/*U()"@+-(& hyphae and 

on the survival strategy of the fungus in responding to these Billing conditions. 

Interestingly, some of the same mRNAs that are increased in response to the 

presence of human neutrophils are increased in response to one or more of the stress 

conditions. ae have also examined these changes in mRNA levels in &+[4 and 

)'[7, MAP Binase gene deletion mutants, and determined that some of these 

changes are MAP Binase dependent. fverall these studies are providing a 

comprehensive view of the MAP Binase dependent changes in mRNA levels in 

response to specific stress conditions and human neutrophils. 

P-0581. Construction and functional analysis of the fatty acid desaturase gene 

disruptants in Candida albicans 

Murayama S 1 , Negishi Y 1,2,J , 6meyama T J , laneBo A J , fura T 2 , Niimi M J , 6buBata l 1 , 

lajiwara . 2 

1 /aboratory of Infectious Agents .urveillance, litasato Institute for /ife .ciences, litasato 

6niversity, ToByo, (apan, 2 Department of /ife .cience, Graduate .chool of 2ioscience and 

2iotechnology, ToByo Institute of Technology, ToByo, (apan, J Department of 2ioactive 

Molecules, National Institute of Infectious Diseases, (apan 

7/*+68"#+,&, a fungal pathogen responsible for candidiasis, causes life-threatening infection 

when host defenses are impaired. The organism is a dimorphic yeastn it can exist as both 

yeast and filamentous mycelia. 2oth forms are pathogenic, but the mycelial forms 

penetrate tissues more easily and are therefore considered more pathogenic. However, the 

pathogenic roles of morphological changes remain to be elucidated, because both 

morphological forms are found in infected tissues and because the molecular mechanisms 

of the dimorphic transition have not yet been understood clearly. Interestingly, the level of 

unsaturated fatty acids tended to be higher in the mycelial form than in the yeast form. 

Polyunsaturated fatty acids (P6FAs), including linoleic acid (C18M2) and alpha-linolenic acid 

(C18MJ), are major components of membranes. P6FAs are produced from monounsaturated 

fatty acids (M6FAs) by several fatty acid desaturases (FADs) in many fungi. 

Despite their importance, the enzymes that catalyze the synthesis of P6FAs have not been 

well understood compared to deltaK fatty acid desaturase. This is partially because the 

model yeasts =+##?+$%)2#.&*#.$.0"&"+. and =#?"d%=+##?+$%)2#.&*'%)8. do not 

possess these enzymes following the reaction by deltaK fatty acid desaturase. In addition, 

only three enzymes that desaturate the deltaK, delta6, and delta[ positions are detected in 

humans, in whom C18M2 and alpha-C18MJ are essential fatty acids. 

In this report, we identified two open reading frames, 7+^4VM and 7+^4V:. 2ased on the 

DNA sequences encoded by the open reading frames released in the 7+,5"5+ genome 

database (CGD) website (httpM//www.Candidagenome.org/), we detected two sequences 

having high homology with =/*[6(20.$" FAD2 and FADJ proteins (=[FAD2p and =[FADJp), 

respectively. The resulting two amino acid sequences, 7+FAD2p (orf1K-118)*and 

7+FADJp (orf 1K-OKJJ), predicted OJ7- and OJO- amino acid polypeptides, respectively. To 

investigate the functions of the 7+^4VM and 7+^4V:*genes, we constructed FAD2-2 

(7+U+5M5.6-+*mutantJ and FADJ-2 (7+U+5:5.6-+ mutant) strains of 7/*+68"#+,&. It was 

confirmed by .outhern blot analysis and PCR amplification that the FAD2-2 and FADJ-2 

mutants obtained were disrupted on the ^4VM and ^4V: loci, respectively. 

Major fatty acids extracted from T6A6 (wild type strain), FAD2-2, and FADJ-2 were 

determined by using GC analysis. No P6FA was detected in strain FAD2-2, and C18MJ was 

not detected in strain FADJ-2. Furthermore =/*#.$.0"&"+. cells expressing 7+Fad2p 

converted palmitoleic acid (C16M1) and C18M1 to hexadecadienoic acid (C16M2) and C18M2, 

and 7+FadJp-expressing cells converted C18M2 to C18MJ. These results strongly supported 

that 7+^4VM encodes the delta12 fatty acid desaturase and that 7+^4V:*encodes the 

omegaJ fatty acid desaturase. These results suggest that the*7+^4VM gene was involved 

in C16M2 and C18M2 syntheses, and that the 7+^4V: gene was involved in C18MJ synthesis. 

However, phenotypic characteristics such as germ tube formation, hyphal morphogenesis, 

and chlamydospore formation did not differ among the wild-type strain, 7+U+5M5.6-+, and 

7+U+5:5.6-+/*Moreover these P6FAs did not affect the virulence to mice or morphogenesis 

in the culture media used to induce morphological change of 7/*+68"#+,&.

P-0582. The effects of lovastatin on gene expression of GTP-binding protein 

gene in dermatophyte pathogen Trichophyton rubrum 

NoorbaBhsh F 2 , Fallahyan F 2 , lhorramizadeh M 1 , Rezaie S 1 

1 .chool of Public Health, Tehran 6niversity of Medical .ciences, Tehran, Iran, 

2 .chool of 2asic .ciences, Islamic Azad 6niversity, Tehran, Iran 

>/*$(8$() is the most common cause agents of dermatophytosis in human sBin and 

nail tissue. .ome properties of >/*$(8$() have been investigated in molecular leveln 

however, no information is available regarding the GTP-2inding protein in this 

dermatophyte. GTP-2inding proteins regulate a variety of processes including sensual 

perception, protein synthesis, various transport processes, and cell differentiation. 

/ovastatin is a HMG-coA reductase inhibitor, prevent the prenylation of GTP binding 

proteins which is required for their translocation to the plasma membrane and their 

functions, including cell survival, proliferation, adhesion and differentiation. 

In the present study, we have successfully characterized the GTP-2inding protein 

gene in dermatophyte pathogen >/*$(8$(). Nucleic acids (DNA c RNA) were isolated 

from obtained mycelial mass of this fungus by standard methods. Pairs of 21 nt 

primers were designed from highly conserved regions of the GTP-2inding protein 

genes in other euBaryotic cells and were then utilized in PCR by using isolated 

genomic DNA as well as cDNA template of >/*$(8$(). Predicted molecules have been 

amplified and sequenced. The amount of 6O[ nucleotides of cDNA is sequenced. The 

characterized RT-PCR fragments which revealed significant homology with other 

GTP-2inding proteins in both nucleotide as well as amino acid levels, has been 

submitted to the Gen2anB (NC2I, NIH, 6.A). Furthermore, the effects of lovastatin 

was investigated by using various concentrations of this medicine in >/*$(8$() 

cultures. The results revealed the reduction of growth within [ and 1[ kg/ml of 

lovastatin. However, the results of latter concentration indicated the down regulation of 

GTP-2inding protein gene in this dermatophyte. 

P-0583. Analysis of Trichophyton rubrum expressed sequence tags 

Wang L 

.tate ley /aboratory for Molecular Virology and Genetic Engineering, 2eijing, China 

.uperficial fungi are the etiologic pathogens of various dermatophytoses, such as 

tinea capitis, tinea corporis, tinea inguinalis, tinea manus, tinea unguium and tinea 

pedis. >$"#?%'?2-%,*$(8$() is the most common of the superficial fungi. In an effort to 

better understand the genetic and biochemical maBeup of >/*$(8$(), we constructed 

cDNA libraries from 10 conditions and used these to isolate 11,08[ unique expressed 

sequence tags (E.Ts),including J,816 contigs and 7,26K singletons'The sequences 

have been submitted to GenbanB and the accession numbers areM DaO0[[80- 

DaO07270 and Da678211- Da71118K (The data will be released after the article is 

published)'According to the estimated >/*$(8$() genome size and the proportion 

between the genome size and the number of genes in some completely sequenced 

model fungi ,we prospected that these E.Ts have covered about 80_ of the whole 

genes. Comparisons with the Gen2anB non-redundant (NR) protein database allowed 

7,76O(70_) of the E.Ts to be assigned putative functions or matched with homologs 

from other organisms. The remaining J,J21(J0_) E.Ts were only weaBly similar or 

not similar to Bnown sequences (E%1E-0[)) ,suggesting that they represent novel 

genes. ae further analyzed the presence of several important genes involved in 

metabolism, signal transduction, pathogenesis and drug resistance in >/*$(8$(). fur 

research set a novel molecular basis for advanced understanding in the physiological 

processes and pathogenic mechanisms of >/*$(8$(), and may lead to a better 

understanding of fungal drug resistance and identification of new drug targets. 


P-0584. Analysis of Trichophyton rubrum ESTs sequences 

Zaugg C, /dchenne 2, Giddey l, Monod M 

CH6V .ervice de Dermatologie, /ausanne, .witzerland 

/arge scale analysis of expressed sequence tags (E.Ts) has been utilized to 

generate useful information about genes expression at different life stages in 

pathogenic organisms. Dermatophytes are human and animal pathogenic fungi 

growing exclusively in the &-$+-()*#%$,.(). They used Beratin and different crossliBed 

proteins of the cornified cell envelope as a substrate. It is reasonable to postulate, 

that during infection, the dermatophytes are under catabolic repression to secrete a 

complete battery of endo- and exoproteases allowing the degradation of the 

Beratinized tissues. Thus, we are trying to identify the genes encoding the proteases 

involved during infection. To mimic ",*0"0% conditions >/*$(8$() was grown in soy 

protein medium where substantial secreted proteolytic activity was recorded. 6nder 

those culture conditions, RNA extraction was performed and a cDNA library was 

constructed. .equencing of random clones led to a collection of O]000 E.Ts. 

Redundant sequences were clustered using the .taden PacBage tools and a total of 

2]1O[ unique contigs were obtained. All sequences were compared with >/*$(8$() 

Bnown sequences and afterward compared by blastx against fungi databases. The 

match with predicted proteins of 4&'.$@"66(&*U()"@+-(& strain af2KJ with a threshold e- 

value of 10 -[ allowed putative annotation for 1][18 sequences. Amino acid sequences 

of orthologous proteins from 4&'.$@"66(& and dermatophytes showed a percentage of 

identity between 22 to 100 _. .ecreted proteases encoded by cDNA of the library 

were identified as fungalysins (MERfP.tMJ6M Mep1, MepJ and MepO), subtilisins 

(MERfP.t.8M .ub1, .ubJ, .ubO and .ub[), dipeptidyl-peptidases (MERfP.t.10M 

ruDppIV and ruDppV), leucine aminopeptidases (MERfP.tM28M ru/ap1 and ru/ap2) 

and two new carboxypeptidases (MERfP.tM1O). .ynergism between endo- and 

exoproteases constitute a set of enzymes for digestion of Beratinized structures into 

amino acid and short peptides assimilable by the fungus. fther cDNA sequences 

encoding putative secreted hydrolases that could be involved in cutaneous barrier 

degradation were foundM a phospholipase, two lipases, a ceramidase and a sialidase. 

IMMUNOLOGY* 

P-0585. Cross-reactivity of Mab C7 between Candida albicans and HeLa cell line 

Abad A 1 , RamÜrez A 1 , .an .egundo I 1 , Ponton ( 1 , 2arrenetxea G 2, J , Hernando F 1 

1 Dpto. InmunologÜa, MicrobiologÜa y ParasitologÜa.6niversidad del PaÜs Vasco. /eioa. 

.pain, 2 Dpto. Especialidades Mddico-~uirârgicas.6niversidad del PaÜs Vasco. /eioa. 

.pain, J ClÜnica ~uirán 2ilbao ../. 

Purpose: It has been described a cross-reaction between Candida genus and human 

tumor cells. ae describe in this study the cross-reactivity between Candida albicans and 

Human He/a Cell /ine by a monoclonal antibody (MAb) created against a C. albicans 

mannoprotein and identify by MA/DI-TfFF/TfFF the cell line reactive proteins. 

Description: ae used the MAb C7, developed by our research group against a partially 

purified cell surface mannoprotein of Candida albicans germ tubes, which have been 

recently identified as AlsJp, which is related to host cells adhesion and therefore to the 

invasive capacity. 

It has been used the He/a cell line of human ovarian adenocarcinoma. Complete protein 

extractions were separated by two-dimensional electrophoresis. The reactivity against the 

antibody was evaluated by aestern blotting with a chemiluminescence detection system. 

The results were analyzed with the Image Masters 2D Platinum program of Amersham 

2iosciences. The peptides recognized by the antibody in at least three immunodetections 

were picBed up from a Coomasie blue stained gel and identified by MA/DI-TfF/TfFF. The 

peptide mass fingerprint data were then searched in databases for final identification of 

peptides. 

Results and Conclusion: ae have demonstrated a cross-reaction of the MAb C7 

between Candida albicans and He/a cell line proteins by two-dimensional electrophoresis 

(2DE) and immunobloting, and identified the proteins involved by Peptide Mass Fingerprint 

(PMF). The MAb C7 reaction is centred in a J[-6[ BDa and O-6 pI region, although several 

spots of smaller intensity are appraised throughout all the gel and also a more reactive 

basic band at J6BDa. ae identified 12 reacting proteins which have been classified 

according to their function as O cytosBeletal proteins, O nucleic acids binding proteins, 2 

calcium binding proteins and one oxidorreductase. The spots that showed more reactivity 

were those of the central zone and correspond with an $-tubuline, a !-tubuline, cytoBeratin 

18 and the O0. ribosomal protein .A. 

Given the characteristics of both cell types, pathogenic yeasts and human tumor cells, to 

invade human tissues, to adhere to them and to colonize them, it is not surprising that they 

could have common epitopes and indeed these were involved in the development of the 

disease. However, no of identified proteins recognized by the MAb C7 corresponds to the 

AlsJp or a related protein in the studied cell line. Nevertheless, the fact that reactive 

proteins can be mainly grouped in J reduce their heterogeneity, so that, this cross-reactivity 

could be explained with a common epitope, which could have a linear or a three 

dimensional configuration. 

ae are studying these proteins more in depth, with the purpose of finding out if they are 

implied in tumor development, and to evaluate their potential as therapeutic targets for 

cancer disease.

P-0586. Phagocytosis, expression of co-stimulatory molecules and cytokines 

production of pulmonary cells after intratracheal infection with 


Almeida S, Ferreira l 

.ao Paulo 6niversity, .ao Paulo, 2razil 

Paracoccidioidomycosis (PCM) is a systemic mycosis, caused by the 

1+$+#%##"5"%"5.&*8$+&"6".,&"& (Pb), that it commits the lung preferentially. Dendritic 

cells are called professional antigen presenters, capable to interact the innate and 

adaptive immune systems. .een the importance of these cells in the immune system 

and Bnowing that the infection for Pb attacBs the lung primarily, we evaluated the main 

biological functions of these cells, as the phagocytosis, expression of co-stimulatories 

molecules and cytoBines production in susceptible (210.A) and resistant mice (A/() to 

PCM. Pulmonary dendritic cells (CDp) were positively selected by anti-CD11c 

microbeads from collagenase-digested lung. The levels of cytoBines secretion were 

determined by E/I.A and co-stimulatory molecules were analyzed by flow cytometry. 

ae observed that CDp are capable to interact with the fungus, and these cells 

phagocytized yeasts forms of Pb ",*0"0%. After this interaction, we observed alterations 

in the expression of co-stimulatory molecules. In purified CDp from A/( mice, a 

gradual increase of CD80, CD86 and CD[O expression was observed. However, a 

decrease of the expression of the same molecules was observed in CDp obtained 

from susceptible mice. The expression of the MHC-II molecule was similar in the cells 

obtained of both lineages of mice. Analyzing the cytoBines secretion, we observed an 

increase in the I/-10, I/-6 and TNF-$ secretion in CDp from 210.A. However, we 

didn]t observed alterations in the production of I/-12. The results suggest that the 

infection with Pb alters the expression of the co-stimulatory molecules and cytoBines 

production in CDp. These changes can affect the activation of T cells, mainly in 

susceptible animals, that could be one of the mechanisms for which the fungus 

induces a larger chronicity in those animals. 

SupportM FAPE.P (no 0J/12816-J) and CNPq. 

P-0587. Gene expression profiles of human monocyte line THP-1 in response to 

tet-NRG1 Candida albicans blastospores and hyphae 

Barker K 1 , Earhart l 1 , /iu T 1 , ju / 1 , Homayouni R 1 , .aville . 2 , /opez-Ribot ( 2 , 

Rogers D 1 

1 6niversity of Tennessee Health .cience Center, 2 6niversity of Texas at .an Antonio 

Background: 7+,5"5+ species ranB as the fourth leading cause of nosocomial 

bloodborne infections in the 6..., and the mortality rate associated with candidemia 

has been reported to be as high as [0_. 7/*+68"#+,& is a dimorphic fungus which 

changes from blastospore to hyphal form in response to different environmental cues. 

In the bloodstream both blastospore and hyphal forms interact with myriad host cell 

types including innate immune cells such as monocytes and macrophages. The 

present study compares, on a genome-wide scale, the response of a human 

monocyte-liBe line THP-1 to blastospores and hyphae generated from a genetically 

engineered 7/*+68"#+,& -.-W`P9I strain (..Y[0-2) in which changes in morphology 

are controlled by the addition or removal of doxycycline to the culture medium. 

Methods: THP-1 cells, cultured in culture medium (RPMIr10_FC.), were plated in 

2O-well plates at 2x10 6 cells/well and allowed to rest in a J7 o C incubator with [_ Cf 2 

for three hours. At the same time ..Y[0-2 7/*+68"#+,& cells, cultured overnight in 

YPD, were washed and resuspended in culture medium and incubated J hours in a 

J7 o C shaBing incubator. To the THP-1 cells was then added culture medium, /P. 

(100 ng/ml), doxycycline alone, ..Y[0-2 cells (1x10 6 cells/well) alone, or ..Y[0- 

2rdoxycycline. The cultures were incubated for three hours in a J7 o C Cf 2 incubator 

after which viability was assessed, cells were harvested, and RNA isolated from the 

THP-1 cells using Trizol. .amples were used to synthesize labeled cRNA which was 

hybridized to Affymetrix 61JJ Plus 2.0 human arrays. The experiments were 

performed independently in triplicate. Raw intensity values were normalized, and fold 

changes for each condition were calculated as compared to the control samples. fnly 

genes that were up- or down-regulated 1.[-fold ([0_) in all three experiments and 

were called hpresenti according to Affymetrix criteria in two of three experiments were 

included in the final gene lists. 

Results: 21[ genes were up-regulated and [0 genes were down-regulated in 

response to hyphae. These include a9PI, a9PM, a9P:, a9PE, P9=I, P9=M, 

P9=IN, >`^, KXD, 77X: (MIP-1alpha), 77XE (MIP-1beta), V=7PI, 7m7XI (Groalpha), 

and 7VD:. ff these, several were differentially-expressed in a hypha-specific 

manner (not in response to blastospores or /P.), such as KXIP41, cX^N, P9=M, 

a9PE, and 4>^:. Genes whose expression is 7+,5"5+-specific (differentially 

expressed in hyphae or blastospores but not /P.) included 7m7PE, ZAXAZM, AK9M, 

77PI, and `^KX:. 

Conclusions: The use of the ..Y[0-2 7/*+68"#+,& strain, whose morphology is 

controlled by the presence of doxycycline, allowed for direct comparison of 

blastospore- and hypha-specific responses in THP-1 cells. Gene expression profiles 

from these conditions provided a clearer picture of the differences in the host 

response to blastospores, hyphae, and inflammation-inducing molecules such as /P.. 


P-0588. Identification of immunodominant cellular antigens of Aspergillus 

fumigatus 

Barnes P 1,2 , .taab ( 1 , Marr l 1,2 

1 Fred Hutchinson Cancer Research Center, 2 6niversity of aashington Medical Center 

CDO T cell responses are important in dictating risBs for, and outcomes of invasive 

aspergillosis (IA). Animal studies have shown that CDO T H-1 cell responses are 

required for effective immunity. Recent efforts to characterize CDO T cell responses, 

and to generate 4&'.$@"66(&-specific T cell clones have been limited by the lacB of 

identified 4/*U()"@+-(& immunodominant antigens. This study was performed with the 

goal of identifying 4/*U()"@+-(& antigens presented to CDO T cells in healthy people. 

Assays for detecting CDO T H -1 responses to 4&'.$@"66(& proteins were initially 

developed using crude hyphal extracts (CHE) as the protein source. CDOr T cells 

that produced IFN-( were quantified by intracellular cytoBine staining or E/I.PfTn 

proliferative responses were determined using carboxylfluorescein diacetate 

succinimidyl ester (CF.E) dilution. In preliminary studies, responses CHE were 

variable between healthy volunteers, with approximately 20_ of people displaying 

robust CDO-IFN-( production and CDOr T cell proliferation. 

A bi-fold approach was developed to identify 4&'.$@"66(&*antigens. Protein 

preparations screened includedM 1. Hyphal components separated into cell wall and 

intracellular fractions (fractionated by hydrophobicity precipitation) and 2. 

Recombinant allergens (proteins highly expressed in the hyphal form) were cloned 

and expressed in a/*#%6" using pTrcHis2 TfPf system. Preliminary screening 

demonstrated that the most hydrophobic intracellular fraction (IC20) generated 

reproducible IFN-( responses from P2MC in the E/I.PfT assay and elicited 

proliferationn studies are being done to identify the proliferative population of cells. 

Twenty-three 4/*U()"@+-(& allergens have been cloned. Asp f2, Asp fJ, Asp f6, Asp 

fK, Asp f11, Asp f12, Asp f17 and Asp f22 were successfully expressed and purified 

from a/*#%6". Initial results of E/I.PfT screening in healthy donors showed that two 

proteins, Asp fJ and Asp f11 elicit cellular production of IFN-(. .tudies are underway 

to characterize cellular responses to Asp fJ and Asp f11 in a cohort of healthy donors. 

P-0589. Nitric oxide can protect or exacerbate murine pulmonary 

paracoccidioidomycosis (PCM) infection. 

Bernardino S, Calich V 

Instituto de Ciüncias 2iomddicas IV 

Paracoccidioidomycosis (PCM) is a chronic systemic mycosis restricted to /atin 

America, caused by the dimorphic fungus 1+$+#%##"5"%"5.&*8$+&"6".,&"&*(Pb),*and it is 

acquired through respiratory tract by inalation of Pb propagules. Inducible nitric oxide 

synthase (iNf.) is one Bey enzyme generating nitric oxide (Nf) from /-arginine. 

iNf. derived Nf is a mediator of unspecific host defense and central in the clearance 

of microbial and parasitic infections.*Thus, the aim of this worB was to investigate the 

role of nitric oxide in the acute and chronic phases of murine pulmonary PCM using 

inducible nitric oxide synthase deficient mice (C[72//6 iNf. lf) and their iNf. 

normal counterparts (C[72//6). 2oth mouse strains were infected by the intratracheal 

route with 1 million Pb viable*yeast cells, sacrificed after 2 and 10 weeBs of infection 

and the severity of the disease was assessed by counting organ viable yeasts, 

histopathology of lungs, humoral immune response, cellular immunity by the delayedtype 

hypersensitivity assay (DTH) and levels of pulmonary and hepatic cytoBines. 

Compared with controls, the iNf. lf mice at the second weeB post infection, 

presented less severe disease with lower pulmonary fungal burden (lf O.[[ ì 0.86 

log 10 n control [.2J ì 0.2K log 10 ), preserved cellular immune response, higher 

production of specific antibodies IgG2a, IgGJ and total IgE (pv0.0[), increased levels 

of pulmonary TNF-! (lf [K76.7 ì 1K[O.J pg/m/n control 16[6.2 ì OO[.81 pg/m/) and 

higher amounts of IFN-", TNF-!, I/-2 and I/-O (pv0.0[) in the liver. The 

histopathology revealed equivalent inflammatory pattern in the lungs of both mouse 

strains with predominant mononuclear cells infiltration and preserved pulmonary 

parenchyma. At the chronic phase of the disease, at weeB 10, the deficiency of Nf 

resulted in higher fungal burden in the lungs (lf [.KK ì 0.7K log 10 n control O.6O ì 0.8 

log 10 ), besides increased levels of most specific isotypes (IgA, IgG1, IgG2a, IgG2b, 

IgGJ and IgE, pv0.01) and the hepatic cytoBines IFN-", I/-O and I/-[ (p.v0.0[). At this 

period of infection, the pulmonary histopathology of iNf. lf mice showed a more 

organized inflammatory reaction composed by well-organized granulomas containing 

epithelioid cells and multinuclear giant cells, surrounding aggregated yeast cells. 

However, control mice presented diffuse lung inflammation affecting most of 

pulmonary parenchyma with impaired granulomas formation. The more organized 

lesions of lf mice appear to compensate the higher fungal loads resulting in 

equivalent survival time for both mouse strains. In conclusion, our results show that 

Nf has a dual role in murine PCM. At the acute phase, its deficiency results in a less 

severe disease revealed by the lower pulmonary fungal load, whereas at the chronic 

phase the impaired control of fungi multiplication seems to be compensated for better 

organization of lesions. aorB supported by FAPE.P/2razil.

P-0590. The central role of the tryptophan metabolic pathway in tolerance and 

immunity to fungi 

Bozza S 

6niversity of Perugia 

The indoleamine 2,J-dioxygenase enzyme (IDf) has a complex role in 

immunoregulation in infection, pregnancy, autoimmunity, transplantation, and 

neoplasia. IDf-expressing dendritic cells (DC) are regarded as regulatory DC 

specialized to cause antigen-specific deletional tolerance or otherwise negatively 

regulating responding T cells. The inflammatory/anti-inflammatory state of DC in 

response to 7+,5"5+*and 4&'.$@"66(&*is strictly controlled by the metabolic pathway 

involved in tryptophan catabolism and mediated by the enzyme IDf. In experimental 

candidiasis and aspergillosis, IDf activity was induced at sites of infection as well as 

in neutrophils and DC via IFN-gamma- and CT/A O-dependent mechanisms. IDf 

blocBade greatly exacerbated infections, the associated inflammatory pathology and 

swept away resistance to reinfection, as a result of deregulated innate and adaptive 

immune responses caused by the impaired activation and functioning of suppressor 

CDO r CD2[ r regulatory T cells cells producing I/-10. The results provide novel 

mechanistic insights into complex events that, occurring at the fungus/pathogen 

interface, relate to the dynamics of host adaptation by fungi. The production of IFNgamma 

may be squarely placed at this interface, where IDf activation liBely exerts a 

fine control over inflammatory and adaptive antifungal responses. 

P-0591. Structural and antigenic characterization of Candida albicans 

beta-glucans 

Bromuro C 1 , Chiani P 1 , Torosantucci A 1 , Iorio E 1 , Ferretti A 1 , 2ellucci C 2 , Costantino 

P 2 , Podo F 1 , Cassone A 1 

1 Istituto .uperiore di .anitÖ, Rome Italy, 2 Chiron Vaccines, .iena Italy 

Purpose of the studyM Anti-!-glucan antibodies are able to bind hyphae of 7+,5"5+*+68"#+,& 

and*4&'.$@"66(&*U()"@+-(&, inhibit their growth ",*0"-$% and confer protection ",*0"0% against these 

fungal pathogens (1). Nothing is Bnown, however, about the nature of the molecular motifs and 

epitopes targeted by these protective antibody, either in animal models or in humans. 

DesciptionM To define major !-glucan antigenic structures in 7/*+68"#+,&, we investigated a 

soluble !-(1,J) glucanase digest from alBali-acid insoluble 7/+68"#+,& !-glucan hghostsi (GG- 

Zym), in comparison with !-(1,J)-linBed (laminarin) or !-(1,6)-linBed (pustulan) reference glucan 

molecules, by combined chromatographic and high-resolution, mono and two-dimensional NMR 

analysis. 7+,5"5+ and reference !-glucans were also assayed for antigenicity with sera from 

mice recipients of anti-7+,5"5+ vaccines able to elicit protective anti- !-glucan antibodies (1,2) or 

with sera from healthy humans, reported to contain substantial levels of anti- !-glucan antibodies 

(J). 

Results and conclusionsM Two distinct components were identified and separated from the GG- 

Zym preparationM a high molecular weight fraction (Pool 1, Ma approximately 20 ldal) which 

consisted of beta-(1,6)-linBed glucopyranosyl chains with beta-(1,J)-branched side chains of 1-2 

glucopyranosyl residues (approximately one every seven beta-(1,6)-linBed units), and a low 

molecular weight fraction (Pool 2) which was a mixture of short-chain beta-(1,J)-oligoglucosides, 

with DPvO. .ize and structure of enzymatic digestion products allowed us to exclude any 

substantial degree of beta-(1,6) branching in the parent beta-(1,J) component of 7/*+68"#+,& 

alBali-acid insoluble glucan and suggested that the undigested, original material was made of a 

complex networB in which long beta-(1,6) chains were J-branched to beta-(1,J) chains, with no 

mixed intra-chain beta-(1,J)/(1,6) linBages and absence (or very low level) of 6-branching along 

the beta-(1,J)-linBed segments. All 7+,5"5+ beta-glucan fractions and reference beta-glucans, in 

particular those with a definitely prevalent beta-(1,6) configuration (Pool 1 and pustulan), were 

recognized by IgG and IgM anti-beta-glucan antibodies present sera from human individuals or 

from vaccinated mice. E/I.A inhibition experiments also indicated that Pool 1 or Pool 2 betaglucans 

exibited distinct epitope profiles, since their binding by antibodies was differentially 

inhibited by beta-(1,J)- or beta-(1,6)-linBed competitor oligosaccharides. Finally, both Pool 1 and 

Pool 2, when conjugated to a protein carrier (CRM 1K7 ) and used to immunize mice, were able to 

elicit significan levels of IgM and IgG anti-beta-glucan antibodies. 2y defining molecular 

structure of 7/+68"#+,& beta-glucans with demonstrated antigenic and immunogenic properties, 

this study provide the ground for the development of novel immunological tools to better define 

the role of beta-glucans and anti beta-glucan antibodies in host anti-7+,5"5+ defense and their 

potential for prevention or treatment of fungal infections. 

References: 

Torosantucci A. et al., ]*a_'*!.5, 202, [K7-60J, 220[ 

2romuro C. et al., K,U.#-*K))(,, 70, [O62-[O70, 2002 

leller R.G. et al., K,U.#-*K))(,, 62, 21[-220, 1KKO 


P-0592. Purification and characterization of immunologically active Candida 

albicans surface antigen 

Bujdakova H 1 , Paulovicova E, MelBusova ., GasperiB (, Machova E, luchariBova . 

1 

Comenius 6niv., Fac. of Nat. .ci., Dept. of Microbiology and Virology, 2 Inst. of Chemistry, Dept. 

of Immunochemistry of Glycoconjugates, .A., J Comenius 6niv., Fac. of Nat. .ci., Dept. of 

Microbiology and Virology, O Inst. of Molecular 2iology, .A., [ Inst. of Chemistry, Dept. of 

Immunochemistry of Glycoconjugates, .A., 6 Comenius 6niv., Fac. of Nat. .ci., Dept. of 

Microbiology and Virology, 2ratislava, .lovaBia 

Objectives: 7+,5"5+*+68"#+,& expresses a protein antigenically, structurally and functionally 

related to the human adhesion glycoprotein, also Bnown as Mac-1, the iCJb receptor, or 

complement receptor type J (CRJ). The mammalian CRJ adhesin is characteristic for 

granulocytes and macrophages. Candidal CRJ- related protein (CRJ-RP) is supposed to interact 

with human complement due to binding the iCJb conversion product. This worB presents the 

purification and some properties of the CRJ-RP a potential immuno-important 7/*+68"#+,& 

antigen. 

Methods: Total protein extract obtained from the germ tubes of 7/*+68"#+,& by glass beads was 

collected for purification using techniques of chromatography. Fractions were analyzed by .D.- 

PAGE and the sample with predicted protein was transferred to PVDF membrane and submitted 

for sequencing. According to sequence of 10 amino acids, synthetic peptide was enhanced for 

immunization of rabbits. Received polyclonal serum anti-CRJ-RP was used for pretreatment of 

7+,5"5+ yeasts and germs tubes in experimental biofilm. 2iofilm formation was quantified as the 

ability to adhere to polystyrene plastic surfaces measured by the jTT reduction assay. 

Monoclonal antibody anti-MAC-1alpha was used as control in experiments with biofilm. H.A 

(human serum albumin) conjugation was performed by CDAP (1-cyano-Odimethylaminopyridinium 

tetrafluoroborate)-mediated activation of mannan, followed by coupling 

to terminal-NH2 group of decapeptid (Man-CRJ-RP J.2/1 w/w). The active immunization scheme 

of rabbits (Hyla-variety)M J immunizations (2 boosters), 2 weeBs apart. Flow-cytometry analysis 

(FAC., Coulter Epics j/) of whole rabbit blood was applied to study immunogen-induced 

changes of individual phenotypes. The expression of CD11b (iCJb, MAC-1) on granulocytes was 

observed through the immunization route. Conjugated CRJ-RP triggered production of rabbit- 

IgG, IgA, IgM specific antibodies was detected by modified E/I.A. linetics of reactive oxygen 

species (Rf.) was determined by colorimetric method (.evapharma, Cz). 

Results and Conclusions: The previously described O[ BDa fragment of the CRJ-RP was 

purified from total protein extract of 7/*+68"#+,& germ tubes by chromatography techniques, 

verified by E/I.A assay and aestern blotting and submitted for sequencing. 6nique sequence 

DINGGGAT/P~ of the CRJ-RP has not corresponded with databases of proteins already 

described in 7/*+68"#+,&. .ynthetic decapeptid was used for immunization of rabbits and the 

preparation of polyclonal serum anti-CRJ-RP. Previous modulation of the expression of the CRJ- 

RP under different experimental conditions suggested its potential role during biofilm formation. 

ae confirmed a final reduction of biofilm after blocBing of the CRJ-RP on 7/*+68"#+,& surface 

with polyclonal serum anti-CRJ-RP or monoclonal antibody MAC- 1alpha up to [0 _ in 

comparison with control. In experimental rabbit model, induced T-cell dependence was observed 

according to initial increase of all immunogen-specific Ig isotypes followed by persisting decrease 

of IgM isotype. Through the whole immunization procedure, especially after two boosters, the 

significant decrease of CD11b neutrophile expression was observed. This observation correlated 

with the decrease of biofilm formation after using of polyclonal antibody anti-CRJ-RP. The 

degree of oxidative stress according to Rf. revealed significant decrease after complete 

immunization. Experiments confirmed a prediction about important antigenic properties of the 

CRJ-RP and suggested possibilities for potential using ",*0"-$% and ",*0"0% diagnosis of 

candidiasis. 

P-0593. Nitrogen and oxygen radicals contribute to the fungistatic and 

fungicidal activity exerted by macrophages against Paracoccidioides 

brasiliensis 

Caro-Gomez E 1 , 6ran-(imenez M 1 , Restrepo A 1 , Cano / 1 , Cano / 2 

1 Medical and Experimental Mycology Group, Corporacián para Investigacione 

2iolágicas, MedellÜn-Colombia., 2 Molecular Microbiology Group, Escuela de 

2acteriologÜa y /aboratorio ClÜnico, 6niversidad de Antioquia, MedellÜn-Colombia. 

Activated macrophages are Bnown to produce nitrogen and oxygen reactive species. 

fnce generated, nitric oxide (Nf . ) might rapidly react with superoxide anion (f 2 - ) 

yielding peroxynitrite (fNff - ) a strong oxidant agent. Previously, our group had 

demonstrated the participation of Nf . in the control of 1/8$+&"6".,&"& infection. In the 

present study, the contribution of fNff - to the fungicidal activity exerted against 

1/8$+&"6".,&"& conidia by peritoneal macrophages treated with IFN-( was addressed. 

Peritoneal resident cells were obtained from male, 10 weeBs old 2A/2/c micen 

monolayers were activated with [06/ml of IFN-( for 18 hours and then infected with 

serum-opsonized conidia at a macrophageMconidia ratio of 1M2. Fungicidal (lost of 

conidia viability) and fungistatic effects (inhibition of the transition conidia to yeasts) 

were determined in the presence of aminoguanidine (AG), []-aminosalicilic acid 

(A.A), and superoxide dismutase (.fD). The direct effect of fNff - on viability and 

morphological transition was assessed in the presence of J-morpholinosydnonimine 

(.IN-1). Exposition of 1/8$+&"6".,&"& conidia to a constant influx of fNff - , generated 

by decomposition of .IN-1 at J7oC - pH 7.O, indicated that this compound mediates 

fungicidal and fungistatic activities against 1/8$+&"6".,&"& conidian such effect was 

dependent on .IN-1 concentration, [mM decreased fungal viability by O0_ and inhibit 

the transition of conidia to the yeast form by 80_n complete loss of viability was 

observed in the presence of .IN-1 20mM. 

Macrophage activation with IFN-( inhibited the transition from conidia to yeast and 

decreased fungal viabilityn the addition of .fD and A.A (f 2 

| 

inhibitors), as well as of 

AG (Nf . inhibitor) to the coculture, macrophages - 1/8$+&"6".,&"*conidia, reverted the 

inhibition of such transition, indicating that both Nf . and f 2 | individually, could 

contribute to the fungistatic effect observed. In the presence of f 2 

| 

inhibitors, 

increased levels of nitrites were detected in the culture supernatants, this rise in 

nitrites level did not correlated with any significant effect on fungicidal or fungistatic 

activity. Fungicidal action exerted by activated macrophages required the 

simultaneous presence of f 2 

| 

and Nf . n consequently, a significant inhibition of this 

effect was observed only in the presence of .fD and AG. In addition, nitrotyrosine, an 

fNff - nitration marBer, was detected by immunostaining procedures in monolayers 

infected with conidia (K.K2_ IFN-( treated macrophages vs [.16_ non activated 

macrophages). These results indicate, that both f 2 

| 

and Nf . , play an important role 

in the control of 1/8$+&"6".,&"&*conidia by activated macrophages, suggesting that 

fNff - could operate ",*0"0% as a critical effector molecule in the fungistatic/fungicidal 

mechanisms. 

Founding resource: Fundacián para la Promocián de la Investigacián y la 

TecnologÜa, 2anco de la Repâblica, 2ogotZ, Colombia. Project No. 1270. Corporacián 

para Investigaciones 2iolágicas (CI2), 6niversidad de Antioquia (6 de A), MedellÜn, 

Colombia.

P-0594. Anti-beta-glucan antibodies: A novel approach to antifungal protection 

Chiani P, 2romuro C, De 2ernardis F, Cassone A, Torosantucci A 

Istituto .uperiore di .anitÖ, Rome , Italy 

Purpose of the studyM Anti-beta-glucan antibodies are particularly interesting 

Candidates for the development of novel immunotherapeutic treatments against 

disseminated fungal diseases. In fact, they are able to induce ",*0"0% a high degree of 

cross-protection against different, largely prevalent, agents of opportunistic infections 

in severely immunocompromized subjects (7+,5"5+*+68"#+,&*and*4&'.$@"66(&* 

U()"@+-(&), acting by an as yet uncharacterized mechanism, liBely involving direct 

inhibition of fungal growth without the participation of effector immune cells (1). fn this 

ground, we addressed generation and characterization of a set of different murine 

anti-beta-glucan monoclonal antibodies (mAb), with the aim of producing protective 

antibody reagents for potential clinical development. 

DescriptionM .plenocytes from 2alb/c mice immunized with CRM 1K7 -conjugated fungal 

(7+,5"5+*+68"#+,&) or non-fungal (laminarin) beta-glucans were fused with myeloma 

cells to obtain anti-beta-glucan antibody-producing hybridomas, and mAbs produced 

were screened for antifungal activity ",*0"-$% and ",*0"0%. 

Results and conclusions. 

In these studies, fusion of splenocytes from one 7+,5"5+ beta-glucan-immunized 

mouse gave rise to two stable hybridomas, secreting two anti-beta-glucan mAbs with 

identical V H and V / regions, as determined by gene sequencing, but with different (IgG 

or IgM) isotypes. ahen tested in comparison, the IgG mAb was found able to 

significantly inhibit 7/+68"#+,& growth ",*0"-$%, whereas the IgM mAb resulted inactive 

or only weaBly active. In murine model of disseminated 7/+68"#+,&*infection, 

prophylactic administration of the IgG mAb induced protection, as indicated by a 

significantly reduced fungal burden in Bidneys, while the IgM mAb was non-protective. 

2esides antifungal activity, difference in isotype also appeared to impact on fine 

specificity by the two mAbs. In fact, the IgG mAb showed a definitely higher affinity for 

beta-(1,J)-linBed glucans or oligosaccharides, such as laminarin or laminarieptaose, 

whereas the IgM mAb preferentially bound beta-(1,6)-linBed glucans, such as pustulan. 

ff interest, surface of 7/*+68"#+,& hyphae was labelled with a similar pattern and 

intensity by the two mAbs when individually tested, but the IgM mAb was preferentially 

captured by the fungal cells when the two mAbs were used together. Moreover, 

protection afforded by the IgG mAb was abrogated by contemporary administration of 

the IgM mAb, suggesting that this could blocB, or interfere with, protection-relevant 

activities by its IgG counterpart. ahile highlighting the promising therapeutic potential 

of anti-beta-glucan mAbs, these results give further support to the notion that 

antibodies may play complex, isotype-dependent roles in antifungal protection. 

P-0595. Inhibition of melanin synthesis increases susceptibility of the 

dematiaceous fungi Fonsecaea pedrosoi against mouse macrophages 

Cunha M 1 , Anderson F 1 , Alviano D 2 , Zanardi E 1 , Alviano C 2 , de .ouza a 1 , Rosenthal 

. 1 

1 Instituto de 2iofÜsica Carlos Chagas Filho - 6niversidade Federal do Rio de (aneiro - 

2razil, 2 Instituto de Microbiologia Professor Paulo de Goes - 6niversidade Federal do 

Rio de (aneiro - 2razil 

^%,&.#+.+*'.5$%&%" produces melanin, a pigment related to virulence in pathogenic 

fungi. To understand the involvement of melanin in the protection of fungi, the authors 

used tricyclazole to inhibit the melanin pathway in ^/*'.5$%&%". Experiments of 

pigmentation suggested that ^/*'.5$%&%" uniquely produces dihydroxynaphthalene| 

melanin. Pigments produced on cultures modified or not with tricyclazole were 

extracted by an alBali|acid method and submitted to infrared and ion exchange 

chromatography analysisn also cytochemistry analysis for cationized ferritin of whole 

cells was carried out. This group of experiments showed that the tricyclazole 

treatment on ^/*'.5$%&%" produced a melanin-liBe pigment, but less negatively 

charged and with less affinity for iron ions than that without the tricyclazole treatment, 

and this in turn lead to a less negatively charge cell wall surface. .canning electron 

microscopy of such pigments showed that the melanin from control cultures 

maintained their hyphae-liBe structures, which have been described as zzmelaninghosts,]] 

whereas the tricyclazole pigment showed an amorphous surface. 

Interaction of conidia from cultures of ^/*'.5$%&%", modified by tricyclazole or not, with 

peritoneal activated macrophages suggested that tricyclazole causes higher 

association of fungus with macrophages, weaBens the fungus capacity to destroy the 

macrophages, and diminishes the resistance to dry fracture procedures on samples 

prepared for high resolution scanning electron microscopy. 

Grant sponsorsM CAPE., CNPq, FAPER( and F6(2. 

Reference: 

Torosantucci A. et al., ]*a_'*!.5, 202, [K7-60J, 220[ 


P-0596. Domain antibodies against virulence traits of Candida albicans confer 

effective passive protection against vaginal candidiasis 

De Bernardis F 1 , /iu H 2 , feMahony R J , /a Valle R 1 , .andini . 1 , Arancia . 1 , Grant . 2 , 

Holton ( J , Roitt I J , Cassone A 1 

1 Istituto .uperiore Di .anitÖ, Rome, Italy, 2 Domantis /td., Cambridge .cience ParB, 

Cambridge, 6l, J Centre for Infectious Diseases and International Health, Royal Free 

and 6niversity College, /ondon Medical .chool, /ondon 

Vulvovaginal candidiasis is a common mucosal infection affecting a large 

proportion of women with some of them affected by recurrent often intractable 

forms of the disease. ae have recently investigated the disease immunotherapy 

with the use of vaginal infection rat model that has similiarities to human 

candidiasis. 6sing this approach, human antibody domains (dAbs) constituted by 

either V H or V / single regions have been generated against virulence antigens of 

7+,5"5+*+68"#+,& in a recombinant formatM r-mannoprotein 6[ BDa (r-MP6[) and 

r-secretory proteinase 2 (r-.ap2). Monomeric or dimeric (r-MP6[/r.AP2) dAbs 

have therefore been used to inhibit the adhesin properties of the virulence traits 

and to confer passive protection against mucosal candidiasis in rats. dAbs 

against r-MP6[ strongly inhibited 7+,5"5+ adherence to endothelial and epithelial 

tissue sections of the vagina, while dAbs against r-.ap2 were capable of 

inhibiting the enzyme activity and adherence to the rat vaginal epithelium. 2oth 

types of domain antibodies strongly accelerated the clearance of 7/*+68"#+,& from 

rat vagina to a greater extent when they were intravaginally administered before 

7+,5"5+ challenge. Dimeric, dual-targeting, MP6[-.ap2 dAb was capable of 

clearing the infection significantly more rapidly than fluconazole. Finally, both 

anti-MP6[ and anti-.ap2 dAbs were equally effective against both azolesusceptible 

and azole-resistant isolates of 7/*+68"#+,&. 

These results set the groundworB as an insight for the development of anti- 

7+,5"5+ immunotherapy based on antibodies that combat virulence traits. This is 

the first demonstration that human antibodies lacBing the Fc effector functions 

could offer protection against an infectious agent in vitro and in vivo. 

P-0597. Dectin-1 synergizes specifically with TLR2 pathway for the production 

of tnfalfa in human monocytes and macrophages. 

Ferwerda * 1 , Meyer-aentrup F 2 , Netea M 1 , Adema G 2 , lullberg 2 1 

1 Radboud 6niversity Nijmegen, N6CI, Netherlands, 2 Radboud 6iversity Nijmegen, 

NCM/. TI/, Netherlands 

The à-glucan receptor dectin-1 is of great importance for the recognition of fungi liBe 

7+,5"5+*+68"#+,& and 4&'.$@"66(&*U()"@+-(&*by the innate immune system. Dectin-1 

can induce .yB-dependent cytoBine production in macrophage cell-lines and mouse 

macrophages. It has also been reported that Dectin-1 can interact with T/R2 in these 

models. The aim of the present study was to investigate the interaction of dectin-1 and 

T/R2 in human MNCs and macrophages. 

Human MNCs were stimulated with curdlan, a linear à-1,J-glucan polymer derived 

from the bacterium 46#+6"@.,.&*U+.#+6"& a specific ligand of dectin-1/ After 2O hours, 

TNFalfa was measured in the supernatant with E/I.A. The MNCs (n}6) produced 7O0 

ì 2K8 pg/ml TNFalfa which could be inhibited with the dectin-1 antagonist laminarin 

(OO ì 10 _) and the specific antibody against dectin-1, GE2 ([8 ì O _). ahen 

curdlan was combined with PamJCys, a synthetic T/R2 ligand, there was a 2.2 ì 0.6 

fold increase in the production of TNFalfa calculated as 

sPamJCysrcurdlanu/(sPamJcysurscurdlanu). This could be inhibited with laminarin 

(1[JK ì O72 to 1080 ì O1J pg/ml) and GE2 (KJO ì 10 to O2[ ì 7O pg/ml). This 

synergistic effect was also seen in human monocyte-derived macrophages (n}J)M K.1 

ì O.O fold increase in the production of TNFalfa by curdlan and PamJCys, which was 

inhibited with by blocBade of dectin-1 (171J ì 117K to [8K ì J7O pg/ml). No dectin-1- 

dependent synergistic effects were seen between dectin-1 and stimulation of other 

T/R pathways such as T/RO and T/RK (not shown). 

In conclusion, curdlan induces dectin-1 dependent TNFalfa production in human 

MNCs. In human MNCs and macrophages dectin-1 specifically amplifies the T/R2 

pathway for the production of TNFalfa, whereas stimulation by T/RO and T/RK is not 

influenced.

P-0598. Resistance to coccidioidomycosis in mice is dependent on IL-1r but not 

TLR2 

Fierer J 1,2 , aalls / 1 , (imenez M 1 

1 VA Healthcare .an Diego, 2 6.C. .an Diego .chool of Medicine 

Innate immunity to infection often involves pattern-recognition receptors such as the 

Toll-liBe receptors (T/R). Dectin-1 was recently identified as the receptor that 

recognizes #-glucans, a major component of fungal cell walls. ae found that 

spherules of the pathogenic fungus 7%##"5"%"5.&*'%&+5+&""*stimulate mouse 

macrophages to maBe TNF! and I/-6 in a T/R2 and Dectin-2 dependent manner (IcI, 

Aug. 200[). To determine the biological importance of this observation we infected 

C[72//6 (26) mice that were deficient in T/R2. .urprisingly, they were not more 

susceptible to 7/*"))"-"& when infected either by the i.p or i.n. route of infection. 

MyD88 -/- mice were more susceptible but T/RO -/- mice were not. .ince the I/-1R 

uses MyD88 we infected I/-1R -/- and they were more susceptible. Thus we have 

established that the pro-inflammatory cytoBine I/-1 is necessary for resistance to 

coccidioidomycosis. The lacB of a phenotype for T/R2 -/- in this infection is not 

understood but may relate to the need for Dectin-1 and T/R2 to collaborate, raising 

the possibility that Dectin-1 function is abnormal in 26 mice. 

P-0599. Aspergillus fumigatus-infected dc induced polymorphonuclear 

neutrophils and Th1 cells migration through the release of a specific 

chemokines profile 

Gafa V 1 , Remoli M 1 , Giacomini E 1 , Gagliardi C 1 , /ande R 1 , .evera M 1 , Grillot R 2 , 

Coccia E 1 

1 MIPI, Istituto .uperiore di .anita, Rome, Italy, 2 ICPH, faculte de Pharmacie, 

Grenoble, France 

Purpose of the study: Immunity against 4/*U()"@+-(& requires the coordinated action 

of components of the innate and adaptive immune systems. The recruitment of 

polymorphonuclear cells (PMN), and specific subsets of T cells play an important role 

in controlling the outcome of invasive aspergillosis. Given the role played by 

chemoBines in the recruitment and selective homing of immune cells into the site of 4/* 

U()"@+-(& infection, we sought to characterize the profile of chemoBines produced by 

human dendritic cells (DC) following 4/*U()"@+-(& infection and their ability to attract 

PMN and 4/*U()"@+-(&*specific Th1 cells. The importance of human DC in the 

regulation of both innate and adaptive immunity against 4/*U()"@+-(& has been 

recently demonstrated by our group. 

Results and conclusions: Following interaction with 4/*U()"@+-(&*conidia, the DC 

showed a rapid and robust expression and secretion of CjC/-8. 2y migration assay, 

we observed that the production of CjC/-8 in 4/*U()"@+-(&-infected DC supernatant 

was directly involved in the recruitment of PMN expressing CjCR2. Next, we focused 

on the contribution*of*4/*U()"@+-(&-infected DC in the recruitment of Th1 cells. To 

evaluate the chemotactic activity of the 4/*U()"@+-(&-infected DC supernatants by 

migration assays we characterized an 4/*U()"@+-(&-specific Th memory cells. 4/* 

U()"@+-(&-specific Th memory cells were obtained from healthy donors peripheral 

blood mononuclear cells and their phenotype was characterized by the intracellular 

production of IFN-gamma and the expression of CCR6, CCR[ and CjCRJ on the cell 

surface. Interestingly, we observed that 4/*U()"@+-(&-infected DC produced CCR[ 

ligands. In particular, large amounts of CC/J and CC/O were rapidly released while 

CC/-[ was induced later and in smaller amounts. Moreover, the 4/*U()"@+-(& infection 

resulted in the production of CjCRJ ligand CjC/-10, and high level of CCR6 ligand 

CC/-20. Finally, we demonstrated that CCR[ ligands, CjC/-10 and CC/-20 secreted 

by 4/*U()"@+-(&-infected DC were involved in the stimulation of 4/*U()"@+-(&-specific 

Th1 memory cells migration. These results shed light on a novel aspect of DC 

contribution to anti-4&'.$@"66(& innate immunity. lnowing the role of PMN in the 

destruction of hyphae and the priming effect of IFN-gamma on PMN oxidative burst, 

DC involvement in the PMN and specific Th1 memory cells recruitment on the 

infection site could have important consequences on the 4/*U()"@+-(& Billing. 


P-0600. Macrophage inflammatory signaling in response to Aspergillus 

fumigatus is dependent on methods of in vitro maturation: unexpected results 

from l929 conditioned medium 

Gersuk G 1 , 6nderhill D 2 , Marr l 1,J 

1 Fred Hutchinson Cancer Research Center, .eattle, aA, 6...A., K810K, 2 Cedars- 

.inai Medical Center, Immunobiology Research Institute, /os Angeles, CA, 6...A., 

K00O8, J 6niversity of aashington Departments of Medicine and Microbiology, .eattle, 

aA, 6...A., K810K 

The cell surface !-glucan receptor, Dectin-1, found on cells of the 

monocyte/macrophage lineage, dendritic cells, neutrophils and a subset of T cells, is a 

pattern recognition receptor for carbohydrate polymers. Results of previous worB 

have shown that macrophages differentiate between morphotypes of 4/*U()"@+-(& 

using the Dectin-1 receptor, recognizing !-glucan that is exposed during conidial 

germination. The role of MyD88 in signaling TNF9$ production in this process is 

unclear. ae hypothesized that variability in reported studies results from different 

methods of cellular maturation, which may play a role in regulating surface expression 

of Dectin-1. In this study, we show that the quantity of TNF-$ produced in response to 

4/*U()"@+-(& germ tubes (GT), and proportional abrogation of TNF-$ produced by 

cells harvested from MyD88-null (MyD88 -/- ) mice, is dependent on the methods by 

which macrophages are harvested for study. 2one marrow-derived macrophages 

(2MDM) matured in /K2K conditioned medium respond to 4/*U()"@+-(& GT with 

vigorous TNF-$ production that is reduced only [0_ by the loss of MyD88. 2locBing 

experiments demonstrate that the MyD88-independent responses are mediated by 

Dectin-1, which is expressed similarly on aT and MyD88-/- macrophages matured in 

/K2K conditioned media (/K2K CM). However, in 2MDM matured in recombinant M- 

C.F (rMC.F), Dectin-1 expression is generally lower, and loss of MyD88 results in 

further loss of Dectin-1, suggesting that the response to 4/*U()"@+-(& GT is more 

MyD88-dependent in rMC.F-generated 2MDM than in /K2K-generated 2MDM. In 

time course studies, 2MDM matured in rMC.F demonstrate decreased surface 

expression of Dectin-1 between days O and 7n alternatively, cells exhibit sustained 

high expression after 7 days maturation in /K2K CM. Cell surface expression of 

Dectin-1 is variable despite equivalent Dectin-1 mRNA levels, as measured by 

reverse-transcriptase PCR. There was no difference in the cell numbers or CD11b 

expression of 2MDM matured in rMC.F or /K2K CM, and cells were CD11c negative. 

However, rMC.F-matured 2MDM exhibit higher viability and cell surface expression 

of FO/80. CytoBines and chemoBines present in /K2K conditioned medium were 

quantified using the 2ioPlex (2io-Rad) assay. In addition to M-C.F, /K2K CM 

contains high amounts (t[00 pg/m/) of VEGF, MCP-1, lC, and MIG, low amounts 

(t[0 pg/m/) of FGF-!, Eotaxin, I/-10, I/-K, I/-12, and detectable amounts of /IF. 

These data demonstrate that methods of in vitro maturation dictate variable 

inflammatory responses by MyD88 -/- macrophages due to altered expression of cell 

surface Dectin-1. /K2K conditioned medium, which is frequently used as an 

alternative to rMC.F for maturing murine macrophages, contains multiple cytoBines 

and chemoBines that may be implicated in this process. 

P-0601. Cryptococcus neoformans interactions with surfactant proteins: 

implications on innate immunity and pathogenesis 

Geunes-Boyer S, Giles ., aright ( 

DuBe 6niversity 

7$2'-%#%##(&*,.%U%$)+,& is an opportunistic pathogen and the leading cause of 

fungal meningitis. Infection occurs following inhalation of 7/*,.%U%$)+,& cells, which, 

in the absence of a cellular immune response, results in pulmonary infection. 7/* 

,.%U%$)+,& cells are unique in their ability to produce a polysaccharide capsule. The 

major capsular constituent is glucuronoxylomannan (GjM), which maBes up 

approximately K0_ of the 7. ,.%U%$)+,& capsule. In the lungs, surfactant proteins 

.P-A and .P-D contribute to innate defense by facilitating the aggregation, uptaBe 

and Billing of microorganisms by phagocytic cells. 

Purpose: To elucidate the mechanistic role of .P-A and .P-D during 7/*,.%U%$)+,& 

pulmonary infection. 

Project Description: ae hypothesized that .P-A and .P-D play a role in 7/* 

,.%U%$)+,& pathogenesis. ae examined the ability of .P-A and .P-D to modulate the 

innate immune response to 7. ,.%U%$)+,&*serotype A (capsular HKK and 

hypocapsular #+'5R#) cells. ae assessed the ability of .P-A and .P-D to bind, 

aggregate and facilitate the phagocytosis of both 7/*,.%U%$)+,& strains by alveolar 

macrophages (AMs). 

Results: ae observed that both surfactant proteins were dramatically aggregated in 

the presence of conditioned yeast medium. ae also found that AFO88-labeled .P-D, 

but not labeled .P-A, binds to the surface of both encapsulated HKK and the 

acapsular mutant #+'5R#, although it does not enhance phagocytosis by AMs ",*0"-$%. 

Interestingly, we observed that the avidity of .P-D binding correlates inversely with 

the size of the 7/*,.%U%$)+,& capsule. For example, #+'5R# exhibits the most 

dramatic .P-D binding. Additionally, .P-D binding appears to be dependent on the 

density of the capsule, with denser capsules exhibiting the least amount of binding. 

This suggests that epitopes that are recognized by .P-D are within the interior of the 

capsule and/or on the cell wall surface. 

Results and Conclusions: fne possible model is that shed components of the 7/* 

,.%U%$)+,& capsule aggregate .P-A and .P-D, impeding the ability of .P-D to bind 

to the fungal surface. Furthermore, capsule growth inhibits .P-D binding. This is 

particularly intriguing because .P-D binds most avidly to the avirulent #+'5R# strain, 

and suggests that .P-D opsonization plays an important role during innate immune 

responses to 7/*,.%U%$)+,& infection. I hypothesize that .P-D binding indirectly 

effects innate immune responses by (1) modulating cytoBine production or (2) 

activating innate immune cells. It will be critical to examine the susceptibility of .P-A 

and .P-D null mice to ascertain whether these proteins are of physiological 

significance during fungal infection. 6nderstanding the mechanism of immune 

modulation upon initiation of infection may serve an important role in clinical 

intervention.

P-0603. Killed Candida albicans yeasts and hyphae inhibit interferon-gamma 

release by murine natural killer cells 

Gozalbo D 1 , Murciano C 1 , Villamán E 1 , feConnor ( 2 , Gil M 1 

1 Departamento de MicrobiologÜa y EcologÜa, 6niversitat de Valmncia, Valencia, .pain, 

2 /aboratorio de Citámica, Centro de Investigacián Principe Felipe, Valencia, .pain 

Induction of cytoBine and chemoBine release triggered by T/Rs is essential for murine 

resistance to disseminated 7/*+68"#+,&*infection. Expression of T/Rs in Nl cells has 

been reported and Nl cells may respond to pathogen components through T/Rs. As 

the role of Nl cells in resistance against 7/*+68"#+,& may be mediated by IFN-( 

production, the aim of this worB was to study the role of T/Rs in triggering IFN-( 

secretion by Nl cells in response to yeasts and hyphae of 7/*+68"#+,&. 6nexpectedly, 

our results reveal that fungal cells cause an inhibition of IFN-( secretion by murine Nl 

cells s1u. Murine Nl cells from spleens were purified by immunomagnetic cell sorting 

with Micro2eads (Miltenyi 2iotec). An homogeneous cell population was purifiedn 

viable Nl cells represented more than 80_ of the total cell population, whereas no T 

lymphocytes were detected. ae first tested the ability of heat-Billed 7/*+68"#+,& cells to 

induce IFN-( production by Nl cells. 2oth yeasts and hyphae failed to induce IFN-( 

secretion. 2y contrast, Nl cells exposed to zymosan or /P. produced detectable 

levels of IFN-(, whereas simultaneous challenge with zymosan or /P. added either 

with yeasts or hyphae resulted in the absence of detectable levels of IFN-(. Next, we 

analyzed the response of Nl cells to fungal stimuli in the presence of cytoBines. I/-2 

induced IFN-( and synergized with zymosann this IFN-( production was inhibited, in a 

dose dependent manner, by 7/*+68"#+,& yeasts and hyphae. Viability of Nl cells was 

not affected by fungal stimuli. .imilar results were obtained with Nl cells challenged 

in the presence of other cytoBines (I/-12 or I/-1[). Therefore, fungal cells inhibit IFN-( 

production by Nl cells in response to T/R2 and T/RO ligands. Heat-Billed cells from a 

non-germinative low virulence 7/*+68"#+,& strain (PCA2), and two =+##?+$%)2#.&* 

#.$.0"&"+. strains, were able to inhibit, in a similar extent, IFN-( production by Nl cells 

in response to zymosan in the presence of I/-2. 7/*+68"#+,&*and*=/*#.$.0"&"+. cells 

Billed by other methods (fixation with formaldehyde and antimycotic treatment) also 

inhibited IFN-( production by Nl cells in response to zymosan in the presence of I/-2. 

Fungal cells also decreased the IFN-( production (in the presence of the I/-12) by Nl 

cells from T/R2 -/- mice in response to /P., as well as by Nl cells from T/RO -/- 

mice in response to zymosan, indicating that these receptors are not involved in the 

inhibitory effect. 7/*+68"#+,& cells, both yeasts and hyphae, have no effect on the I/-2 

induced proliferation of Nl cells. fur finding may represent a novel mechanism of 

immune evasion, based on inhibition of Nl cells, that contributes to the virulence of 7/* 

+68"#+,&. 

P-0604. Anti-Saccharomyces cerevisiae antibodies in Tunisian patients with 

Crohn's disease 

Hadrich I 1 , Vandewalle P 2 , CheicBrouhou F 1 , Fattouma M 1 , .alah l J , .tanaert-Vitse 

A 2 , Ayadi A 1 , Poulain D 2 

1 /aboratory of Parasitology and Mycology, .chool of Medicine, .fax, Tunisia, 

2 IN.ERM 67KK, Pathophysiology of Candidiasis, Faculty of Medicine, /ille, France, 

J Department of Gastroenterology, Hedi ChaBer Hospital, .fax, Tunisia. 

Background: In aestern Europe and 6.A, the presence of anti-=+##?+$%)2#.&* 

#.$.0"&"+. antibodies (A.CA) in Crohn]s disease (CD) patients and their healthy 

relatives suggests that A.CA may be influenced by genetic and/or environmental 

factors. 

Objectives: To assess the prevalence of A.CA in Tunisian patients with CD or 

ulcerative colitis (6C), and unaffected family members, in relation to clinical phenotype. 

Patients and Methods: .eventy-seven patients (JK CD, J8 6C), 66 healthy relatives 

of CD patients, 16 relatives of 6C patients and 70 healthy controls were studied. 

A.CA were quantified with a new isotype-specific E/I.A test involving an antigenic 

extract from =/*#.$.0"&"+. strain aJ0J and by the original test which detects total 

immunoglobulins against =/*#.$.0"&"+.*.61 mannan. 

Results: The isotype-specific A.CA aJ0J test was more sensitive than the A.CA 

.61 test for immunoglobulin detection, although the specificity of the two tests was 

identical (K1_). A high percentage of patients with CD (72_) and their unaffected 

family members (J[_) were A.CA-positive in contrast to 6C patients (16_) and their 

relatives (0_) and controls (8.6_). A.CA were shown to be independent of disease 

activity, but were associated with ileal location. .ome antigens and/or isotypes 

discriminated patients depending on sex or age at diagnosis. 

ConclusionM This study confirms the antigenic heterogeneity of =/*#.$.0"&"+. strains 

in their ability to detect A.CA. It suggests that A.CA are marBers of 

immunoregulatory disturbance in CD, independent of ethnic/cultural differences 

between Europe, 6.A and North Africa. 

ReferenceM 

Murciano et al. (2006). Infection and Immunity 77, 66[[-66[K. 

.upported by grants 0J777 and 0O[67 (Insituto de .alud Carlos III, Ministerio de 

.anidad y Consumo) and grupos 0J072 (Generalitat Valenciana). 


P-0605. Pneumocystis murina inoculation results in an increase in airway 

hyperresponsiveness and also promotes sensitization to other exogenous 

antigens in immunocompetent BALB/c mice 

Harmsen A 1 , Meissner N 2 , .wain . J 

1 Montana .tate 6niversity, 2 Montana .tate 6niversity, J Montana .tate 6niversity 

The atypical fungus 1,.()%#2&-"& spp. causes serious pneumonia in 

immunosuppressed individualsn however, it is not usually considered to have injurious 

effects in immunocompetent subjects. ae have examined the early response to 

inoculation with 1,.()%#2&-"& )($",+*in normal 2A/2/c mice, and have found that, 

even though the organism is cleared by 21-28 days, inflammatory responses to this 

microorganism result in two other potentially deleterious responses. The first is a 

profound airway hyperresponsiveness (AHR) at O-1O days post-inoculation, as shown 

by elevated Penh values in response to a methacholine challenge. This AHR is 

independent of eosinophils, neutrophils, and IFN-(, although it is concomitant with 

elevated levels of I/-O, IFN-(, I/-6, TNF-$, and hypertrophy of airway epithelial cells. 

The second effect is the ability of the inflammatory response to 1/*)($",+ to promote 

allergic sensitization to exogenous proteins that might normally be tolerated. ae 

found that when 2A/2/c mice are given a single i.n dose of ovalbumin (ova) at 1O 

days after inoculation with 1/*)($",+, they become sensitized to the ova, as 

demonstrated by allergic responses to i.n. ova 28 days later. These allergic 

responses include eosinophil influx after i.n. exposure to ova, increased AHR with i.n. 

exposure to ova followed by methacholine challenge (at a level comparable to that of 

mice given ova with alum adjuvant 21 days prior to challenge), and moderate levels of 

ova-specific serum IgG and IgE in some of the mice exposed to ova during the 1/* 

)($",+ infection. This effect does not occur in non-infected mice or animals exposed 

to ova at 21 days post infection, which suggests that there is a window of susceptibility 

to antigen sensitization during 1,.()%#2&-"& infection. During this window of 

susceptibility, the alveolar environment contains large numbers of alveolar 

macrophages and neutrophils, moderate amounts of eosinophils and putative 

dendritic cells, but few lymphocytes. Additionally, there were elevated levels of TNF-$, 

and the Th2 cytoBines I/-O and Il-[ in the alveolar fluid at this time. It is possible that 

the unique cellular and cytoBine environment at this time is permissive toward 

sensitization to exogenous antigen, but further experiments are required to investigate 

this possibility. fur observation of 1,.()%#2&-"&-dependent AHR may be relevant to 

studies by other investigators who found that infants who have died of .ID. had a 

high incidence of positive tests for 1,.()%#2&-"&. And, our observation of the 

adjuvant effects of a mild 1,.()%#2&-"& infection toward other potential antigens may 

implicate 1,.()%#2&-"& in the etiology of asthma, as has been suggested for other 

infectious agents, such as R.V and influenza virus. 

P-0606. Interaction of anti-"-glucan antibody and fungal cell wall "-glucan 

Ishibashi K 1 , Motoi M 2 , Yoshida M J , N Miura N 1 , Adachi Y 1 , fhno N 1 

1 ToByo 6niv. Pharm. /ife .ci., .chool of Pharmacy, /ab. Immunopharm. Microb. Prod, 

2 Toei Pharmaceutical Co., /td., J Hachiouji Medical Center of ToByo Medical 6niversity, 

Division of Nephrology 

Glucan is one of the major cell wall components of fungi and is used as a parameter 

for the serological diagnosis of deep mycosis. It generally has a variety of biological 

and immunopharmacological activities. Many pattern recognition receptors were cited 

as Candidates for a #-glucan receptor. fn the other hand, there are few reports of an 

antibody to #-glucan. ae have recently detected an anti-#-glucan (2G) antibody 

highly reactive to 7+,5"5+ solubilized cell wall #-glucan (C.2G) in normal human sera. 

Anti-2G antibody may be able to interact with the fungal cell wall or extracellular 

glucan, and modify the host defense system. Thus, we examined anti-2G antibody 

titer, reactivity, the antigen-antibody interaction of anti-2G antibody in vivo and vitro, 

and the role of this antibody. 

Anti-2G antibody titer was examined by E/I.A precoated with C.2G. 2inding of Ig 

was detected by peroxidase-conjugated anti-Ig antibody. C.2G were prepared by 

NaClf oxidation-DM.f method followed by procedure established previously. The 

titer of anti-2G antibody varied in each individual. Anti-2G antibody showed high 

reactivity and specificity to C.2G, a long #-1,6-linBed glucan chain attached to the #- 

1,J-D-glucan chain from 7+,5"5+*+68"#+,&, but low reactivity to Grifolan (GRN) from 

9$"U%6+ U$%,U%&+ and .onifilan (.PG) from =#?"d%'?266()*#%))(,., 6-branched #-1,J- 

D-glucan from mushroom. Also, anti-2G antibody cross reacted with 4&'.$@"66(& 

solubilized cell wall #-glucan (A.2G). Anti-2G antibody could bind whole 7+,5"5+ 

cells. It also enhanced the Candidacidal activity of macrophages in vitro. 

To understand the clinical role of anti-2G antibody, we measured anti-2G antibody 

titer in mycosis patients, collagen diseases (rheumatoid arthritis (RA), ANCA 

associated vasculitis (AAV) ). Mycosis patients (aspergillosis M N}2, carinii 

pneumonia M N}J) , whose sera is #-1,J-glucan positive showed a significantly low titer 

(O1OìJ[[ 6/m/) compared with normal volunteers (2J71ì1107 6/m/). This change 

correlated with clinical symptoms and other parameters such as C-reactive protein 

and #-1,J-glucan. RA, AAV and cancer patients showed decreased titer of anti -2G 

antibody. 

The present study suggested that the anti-2G antibody formed an antigen-antibody 

complex and participated in the immune response as a molecule recognizing #-glucan. 

Anti-2G antibody plays a role in host defense against pathogenic fungi. Anti-2G 

antibody is expected to be useful as a response index of fungal cell wall #-glucan.

P-0607. Partial characterization of immunogenic components of histoplasma 

capsulatum and circulating specific IgG and soluble antigens in experimental 

murine histoplasmosis 

Itano E 1 , TristÉo F 1 , 2djar V 2 , fno E 1 , Camargo Z J , .ano A O 

1 6niversidade Estadual de /ondrina, /ondrina, PR, 2razil, 2 IMT, /ima, Peru, 

J 6NIFE.P, .Éo Paulo, .P, 2razil, O Chiba 6niversity, Chiba, (apan 

The fungus A"&-%'6+&)+*#+'&(6+-()*var. #+'&(6+-()*is the histoplasmosis] causative 

agent, usually asymptomatic in healthy individuals, but severe and fatal in 

immunocompromised or debilitate patients. The present study characterized partially 

the immunogenic fractions of A/*#+'&(6+-() and analyzed the immune response to 

these components during experimental histoplasmosis. Initially CFA samples were 

fractioned in =.'?+5._*G-7[/120 column and the eluates analyzed by dot blotting with 

rabbit anti-CFA IgG. To investigate A/*#+'&(6+-()*antibodies cross reactivity to 1/* 

8$+&"6".,&"&3*A/*#+'&(6+-()*isolates 128 (IMT/HC128) and 22K (IMT/HC22K) and 1/* 

8$+&"6".,&"&*(Pb 18) CFA]s samples were analyzed by w.&-.$,W86%--",@ with antiWA/* 

#+'&(6+-()*serum adsorbed and not adsorbed with 1/*8$+&"6".,&"&. Also the semipurified 

fractions were used to prepare more specific antibodies. Additionally 7-8 

=B"&&*mouse groups were infected with 2,2x10 [ IMT/HC128 yeast cell and after 7, 1O 

and 28 days circulating specific IgG and soluble antigens analyzed. The 

chromatography fractions] carbohydrates was qualitatively determined. The eluate]s 

analysis showed larger reactivity to rabbit anti-CFA IgG at high molecular mass 

fractions, called F1 and F2, being F1 the fraction with more carbohydrates. The 

infected mouse] serum showed IgG high levels to CFA]s preparation, F1 or F2 

fractions. The cross reactivity analysis] results showed that there are cross reaction to 

CFA or F1 fraction, but didn]t to F2 fraction, and the adsorption process eliminates 1/* 

8$+&"6".,&"&*cross reactivity. aith these results we concluded that occur cross reaction 

between A/*#+'&(6+-()]s and 1/*8$+&"6".,&"&p*CFA and that this cross reaction can be 

eliminated with adsorption process or minimized with purification process using more 

specific antigens. In experimental murine histoplasmosis occurs the humoral immune 

response to F1 and F2 fractions, being to F1 with the same intensity that to CFA]s 

preparation, and also increased circulation antigens] levels. The results could be 

important to further histoplasmosis] diagnosis or pathogenesis study. 

P-0608. Immunoprotection of mice using coccidioidal vaccine GFI 

Johnson S, .immons l, Pappagianis D 

6niversity of California, Davis 

Purpose of the studyM Two studies were undertaBen to evaluate the effect of different adjuvants 

and immunization routes upon survival following respiratory challenge of mice with 7%##"5"%"5.&* 

"))"-"& arthroconidia. The coccidioidal antigen GFI, prepared from mature endosporulating 

spherules and previously shown to protect mice against lethal respiratory challenge when given 

with the adjuvant alum, was used in each study. 

DescriptionM 

Study #1 

Mice (7 per group) were vaccinated with J doses containing 100 &g antigen GFI, given at weeBly 

intervals. Coccidioidal antigen GFI was formulated with alum, Montanide I.A720, or both. 

Control mice were immunized with formalin-Billed whole spherules (Fl.) or adjuvant only. 

.erum was collected following immunization and the titer of IgG1, IgG2A, and total IgG 

determined by E/I.A. Mice were challenged by the respiratory route with either [00 or 1,[00 

arthroconidia and monitored for survival through 1[0 days. 

Study #2 

GFI vaccine was administered to mice by the subcutaneous (.C), intradermal (ID) or intranasal 

(IN) routes. Vaccines (100 &g antigen) were formulated with alum for .C and ID routes and /-$phosphatidylcholine 

dipalmitoyl (PC) for IN. Three immunizations were given at weeBly intervals. 

Control mice were immunized with saline and adjuvant. .erum was collected prior to challenge 

with [00 or 1,[00 arthroconidia given by the respiratory route. Mice were monitored for survival 

for 160 days. 

Results and conclusions: 

Study #1 

ff the mice vaccinated with either Fl. or GFI with alum and challenged with [00 arthroconidia, 

100_ survived through 80 days while only 2K_ and 71_ of mice vaccinated with 

GFI with Montanide or GFI with Montanide and alum respectively survived through the same 

period. ff the mice challenged with 1,[00 arthroconidia, 100_ of mice immunized with Fl., 

[7_ immunized with GFI with alum, 1O_ immunized with GFI with Montanide, and 2K_ 

immunized with GFI with alum and Montanide survived through 80 days. Deaths after 80 days 

were observed in all groups except GFI with alum, [00 level challenge. These results indicate 

that the GFI with Montanide vaccine was less protective than the GFI vaccine with alum and that 

GFI with alum and Montanide was intermediate. 

Study #2 

All mice immunized with saline and adjuvant regardless of route had died by day 1O. Additionally, 

all mice immunized by the intranasal route with PC and challenged with 1,[00 arthroconidia had 

died by day 12. fne hundred percent of mice immunized by the ID route survived through 80 

days. .imilar results were observed for the .C route although one death was observed in the 

group challenged with [00 arthroconidia. fnly J mice immunized by IN and challenged with [00 

arthrocondia survived this same period. ahile deaths after 80 days were observed in both ID 

and .C route groups, those in the ID route group were delayed in comparison to the .C route 

group. These results appear to indicate that the .C and ID routes yielded similar protection. 

Future studies to evaluate the two routes will include a less protective antigen so to provide room 

for improvement. 


P-0609. Conversion of Histoplasma capsulatum conidia into yeasts within 

macrophages and dendritic cells 

Newman S, /emen a, .mulian G 

6niversity of Cincinnati Col. Med. 

Infection with A"&-%'6+&)+*#+'&(6+-() (Hc) occurs via inhalation of airborne mycelial 

fragments or microconidia which deposit into the terminal bronchioles and alveoli of 

the lungs. The inhaled conidia subsequently convert into yeasts that are responsible 

for the pathogenesis of histoplasmosis. In the present study we sought to quantify the 

conversion of conidia into yeasts in macrophages (M:) and dendritic cells (DC) 

compared to tissue culture medium. To follow the conversion of conidia into yeasts 

visually, the green fluorescent protein (GFP) was cloned into strain G2172 under the 

calcium binding protein (C2P) promoter so it would be expressed only in the yeast 

phase. Prior to infection, conidia were labeled with CF.E and then incubated for 1 hr 

with M: or DC. Non-ingested conidia were removed by washing and the cells were 

cultured for 2O to K6 hr. At each time point the number of conidia and yeasts was 

quantified via phase and fluorescent microscopy. In a separate set of experiments, 

non-transformed conidia were co-cultured with M: or DC for up to [ days. 

Conversion to yeasts was quantified by the incorporation of J H-leucine. In both 

human and murine M:, numerous yeasts appeared by day J post-infection, although 

occasional yeasts could be seen as early as 2O hr. The time course of conidia 

conversion into yeasts in tissue culture medium was essentially the same as in M:. 

In contrast, conversion of conidia into yeasts was significantly restricted in both human 

DC and murine lung DC. In DC, significant numbers of yeasts did not appear until [ 

days post-infection. Further, M: monolayers were usually destroyed by day O postinfection, 

whereas DC monolayers remained intact throughout the study period. 

These data suggest that in vivo, conidia and mycelial fragments may convert into 

yeasts efficiently whether or not they are phagocytosed by M:. 

P-0610. Osteopontin expression in the granulomas of mice infected with 


NishiBaBu A, Burger E 

Department of Immunology, Institute of 2iomedical .ciences - 6niversity of .ao 

Paulon .Éo Paulo, 2razil 

1+$+#%##"5"%"5.&*8$+&"6".,&"& (Pb)-induced granulomas are characterized by 

expression of different immune and non-immune cells, cytoBines and extracellular 

matrix components (ECM). fsteopontin (fPN), a secreted glycoprotein has been 

described as a cytoBine implicated in cell-mediated response and in the 

granulomatous response. In the present worB, we studied the ",*&"-( expression of 

fPN in omentum granulomas developed in susceptible (210.A) and resistant (A/() 

mice in the course of ip. infection with the highly (Pb18) or the slightly (Pb26[) virulent 

1/*8$+&"6".,&"& isolates. Immunohistochemistry (streptavidin-biotin peroxidase) was 

used to analyse the immunolocalization of fPN in the granulomas. At 1[ days postinfection 

(dpi) with Pb18, fPN was strongly positive (r) in macrophages and in 

multinucleated giant cells localized mainly in the center of the lesions, but weaB 

expression was seen in the ECM. fn the other hand, high fPN positivity on ECM was 

detected at 120 dpi, as well as an increased expression in macrophages and in giant 

cells circumscribing Pb yeasts, mainly localized in the center of granulomas foci in 

both mouse strains and around necrotic areas in A/( mice. .emiquantitative data (n} 

J/group, mean # .EM) showed a significantly higher degree of fPN (r) cells in 210.A 

(0.[O2#0.008) than in A/( (0.2O2#0.008) at 1[ dpi with Pb18, and a significant 

increase of (r) cells of A/( at 120 dpi (0.[00#0.066). Intensity of fPN (r) cells was 

also evaluated and was significantly lower at 1[ dpi (0.J2[#0.0[2) than at 120 dpi in 

A/( (0.8[8#0.0[1). At 120 dpi, higher degree of fPN (r) ECM was detected in 210.A 

(0.108#0.022) and A/( (0.1[8#0.017), in comparison to 1[ th dpi. .ignificantly higher 

intensity of fPN (r) ECM was shown at 120 th than at 1[ th dpi in 210.A (0.100#0.02[ 

vs. 0.02[#0) and in A/( (0.17[#0.02[ vs. 0.02[ì0.02[). /ower staining degree and 

intensity of fPN (r) cells was observed in Pb26[ than in Pb18-infected mice. These 

results suggest that expression of fPN in macrophages and in multinucleated giant 

cells, as well as on ECM in the granulomas, particularly increased in the course of 

infection of resistant mice, may have an important role in the tissue response 

developed in paracoccidioidomycosis.

P-0611. Major allergen components of Malassezia globosa in patients with 

atopic dermatitis 

Nishikawa A 1 , Ishibashi Y 1 , Asahi Y 1 , .ugita T 2 , lato H 1 

1 Department of Immunobiology, Meiji Pharmaceutical 6niversity, ToByo, (apan, 

2 Department of Microbiology, Meiji Pharmaceutical 6niversity, ToByo, (apan 

Introduction: The lipophilic yeast !+6+&&.d"+ is an exacerbating factor in atopic 

dermatitis (AD) and can induce IgE antibodies in patients with AD. Among !+6+&&.d"+ 

species, !/*@6%8%&+ and !/*$.&-$"#-+ are particularly dominant on the sBin of AD 

patients. Although numerous !+6+&&.d"+ allergens have been identified and studied, 

little information is available regarding the allergens from these dominant species. In 

this study, we attempted to identify major allergens from !/*@6%8%&+ using a 

proteomics analysis. 

Materials and Methods: Twenty-eight sera from patients with AD were used in this 

study. The initial inclusion of AD patients was on the basis of an Ala.TAT titer for !/* 

U($U($-specific IgE Ab tJ I6/ml. *!+6+&&.d"+*@6%8%&+ C2.7K66 cultured on modified 

/eeming and Notman agar was disrupted using a French pressure cell. The 

supernatant of the crude lysate was analyzed by IgE-immunoblotting with sera from 

AD patients and the IgE-reactive spots were analyzed by a proteomics approach, 

combining two-dimensional aestern blotting and matrix-assisted laser desorption 

ionization time-of-flight M. (MA/DI-TfF M.), in the identification of allergens from !/* 

@6%8%&+. 

Results and Discussion: Immunoblotting showed that IgE-reactive components with 

molecular masses of J8-O[BDa proteins were detected by 100_ (28 of 28) of sera 

from AD patients. fther IgE-binding components with various molecular masses were 

detected at lower frequencies. Two IgE-reactive allergens corresponding to the J8-O[ 

BDa proteins were identified by two-dimensional immunoblotting, and sequenced 

partially by MA/DI-TfF M. with postsource decay (P.D) of the peptide digest. 

Comparison of sequences with Bnown protein sequences revealed that both allergens 

showed similarity to the heat shocB protein (hsp) family. The heat shocB proteins are 

broadly and highly conserved across proBaryote and euBaryote, and some of them 

have been described as pan allergens in AD patients. Further studies should include 

cloning and sequencing of corresponding genes to characterize the*!/*@6%8%&+| 

derived hsp allergens. The IgE crossreactivity to hsp allergens from !/*@6%8%&+ and 

other species also needs to be explored and their exact role in the pathogenesis of AD 

should be assessed in clinical studies. 

P-0612. Beta-mannosyl linkages negatively regulate immunotoxicity of caws, 

fungal pamps composed of mannoprotein-beta-glucan complex secreted by 

Candida albicans 

Ohno N, .hinohara H, Miura N, Ishibashi l, Adachi Y 

ToByo 6. Pharm. /ife .ci., ToByo, (apan 

CAa. is a water-soluble extracellular mannoprotein-beta-glucan complex obtained 

from the culture supernatant of 7+,5"5+*+68"#+,&, which grows in a chemically defined 

medium. CAa. induced toxic reactions, such as acute anaphylactoid reaction, by 

intravenous administration and coronary arteritis by intraperitoneal administration. The 

coronary arteritis induced by CAa. was accompanied by hypertrophy of the tunica 

intima, the rupture of elastic fibers and a diffuse invasion by lymphocytes, histiocytes, 

fibroblasts, smooth muscle cells and eosinophils of vascular endothelial cells and the 

regions surrounding blood vessels. 2ased on such characteristics, the coronary 

arteritis induced by CAa. was presumed to be proliferative granulomatous coronary 

arteritis, and is clearly different from fibrinoid arteritis. It is of note that CAa.-induced 

arteritis showed significant strain dependencyM The incidence of arteritis was 100_ in 

C[72//6, CJH/HeN and D2A/2 mice, but only 10_ in C2A/( mice. In D2A/2 mice, it 

was observed to cover nearly the entire periphery of the vessels, and those mice were 

considered to demonstrate the most virulent form of coronary arteritis. The coronary 

arteritis observed in D2A/2 mice was the most serious, with the majority of mice 

expiring during the observation period. The CAa.-sensitive strains revealed 

increased levels of I/-6 and IFN-gamma during the course of a specific response to 

CAa. by spleen cells. In contrast, I/-10 levels were observed to increase marBedly in 

CAa.-resistant C2A/( mice, but not the CAa.-sensitive strains. In addition, TNFalpha 

levels were more elevated only in D2A/2 mice. 

To clarify the structure responsible for these toxic reactions, 7/*+68"#+,& was cultured 

in pH- and temperature-controlled conditions and prepared with CAa. with or without 

the beta-1,2-linBed mannosyl segment (2M). The structure of CAa. was assessed by 

immunochemical and spectroscopic methodologies, and we found that CAa. 

prepared under the natural culture conditions contained only small amounts of 2M and 

CAa. prepared at neutral conditions at 27 contained a significantly higher 

percentage of 2M. 2oth the acute lethal toxicity and coronary arteritis induction was 

significantly more severe in the absence of 2M. Activation of a complement pathway, 

the lectin pathway, by CAa. was significantly stronger in the absence of 2M. These 

facts strongly suggest that 2M linBages in CAa. negatively modulate acute and 

chronic toxicity of CAa., and may be strongly related to the lectin pathway of the 

complement activation. 


P-0613. Ceramide monohexoside (CMH) isolated from Aspergillus fumigatus 

2109 and the complement system. 

Pinto / 1 , ZmeteB Granja / 1 , .ilva M 1 , Pinto M 2 , 2arreto-2ergter E 2 , Ejzemberg R 1 

1 Dep. de Imunologia - IMPPG - CC. - 6FR( - R( - 2razil, 2 Dep. de Microbiologia 

Geral - IMPPG - CC. - 6FR( - R( - 2razil 

4&'.$@"66(&*U()"@+-(& is a pathogenic fungal species, mainly associated with 

pulmonary diseases such as aspergiloma or allergic bronchopulmonary aspergilosis. 

Glycosphingolipids are amphipatic molecules consisting of a ceramide lipid moiety 

linBed to glycan chain of variable length and structure. .uch molecules have been 

implicated in many fundamental cellular processes and have been extensively studied 

in mammalian cells. The structure and function of fungal glycosphingolipids are not 

well Bnown. In this worB we studied the ceramide monohexoside fraction (CMH) from 

4/*U()"@+-(& 210K strain to verify its ability to activate the complement system. This 

system plays an important role in the human defense mechanism. CMH samples were 

incubated with human absorbed serum, treated or not with chelators (EDTA, EGTA) 

and Bept for 60 minutes at J7 o C. The residual complement was measured in the 

supernatants. ae found that CMH was capable to interact with the complement 

system since there was a reduction of the residual complement when the serum was 

incubated in the absence of chelators (60_). fn the other hand, when the serum was 

incubated with EGTA-Mg rr , there was only 18_ reduction. A number of organisms 

have evolved mechanisms, by which they can bind complement regulators and exploit 

its protective effects, thereby avoiding destruction by the complement system. Many 

acute phase proteins such as C-reactive protein and mannose binding lectin, enhance 

the activation by the classical pathway of the complement system. Regulators liBe cofactor 

H can act together with factor I as an inhibitor of the alternative pathway 

resulting in CJ convertase loss, by cleavage of CJb in smaller fragments (CJd and 

CJdg). 

.uported byM CAPE., CNPq, FAPER( and 6FR(. 

P-0614. Concerning the genesis of trichophytin test polymorphism and some 

environmental factors 

Radev S 1 , Clayton T 2 , Michev V J 

1 Navy Hospital of Varna, 2ulgaria, 2 /eeds Teaching Hospital, England, J Military 

Medical Academy of .ofia, 2ulgaria 

Trichophytin (T) test polymorphism still represents an object of intensive discussion. 

The clarification of its role in the pathogenesis of several superficial sBin affections 

needs more comprehensive investigations. A randomized sample of [22 young navy 

sailors was examined within a 1[-year longitudinal follow-up. A series of clinical, 

epidemiological, mycological and immunological methods were applied. The results 

show that the driving force of T(-)/T(r) or T 1 phenotype (n}78) and of T(r)/T(r) or T 2 

phenotype (n}60) was the susceptibility to atopy (AT). Here T in numerator was a 

specific humoral response to trichophytin while T in denominator was a specific cellmediated 

immunity response to trichophytin. T 1-phenotype carriers were 

predominantly non-atopic persons (in K2_ of the cases) while T 2 -phenotype ones 

were strong generators of AT with bilateral (2/) familial burden (F2) (2/-F2-AT). The 

androgen-active and atopiogenic blood group 2 was the main AT generator in the 

cases of T 2-phenotype. ATwF2 along with a powerful immune modulation effect 

(physiological tolerance) of sufficient up to long-lasting breast feeding was the 

organizing mechanism of T(r)/T(-) or T J -phenotype (n}108) and of T(-)/T(-) or T O - 

phenotype (n}276). Concerning T O-phenotype the immune suppression caused by 

very often sunshine exposure along with the specific anti-T tolerance due to the 

excessive sweating was added. 2ecause of the disturbed measure in biology the 

persons who were not breast fed and those who were extremely breast fed as well as 

the persons who were not exposed to sun and those overexposed to sun developed 

immune deficit. In this way, in biology and especially in medicine, the processes 

mutually suppose and mutually deny themselves. fn the one hand, often sunexposed 

individuals were stronger smoBers and longer breast-fed. fn the other hand, 

the severe 2/-F2-AT-phenotype logically induced in a reflex way insusceptibility not 

only to sun exposition and tobacco smoBe but also to mother milB. This was due to the 

fact that AT-mother]s milB was rich in activated T-cells and a cascade of ATassociated 

cytoBines. The conclusion was drawn that, regularly, both T 1 - and T 2 - 

phenotypes did not generate any permanent chronic and disseminated 

epidermophytia and nail mycosis in contrast to T J-phenotype and particularly to T O- 

phenotype. The latter two phenotypes were also higher generators of recurrent herpes 

simplex and aphthosis. These statements did not coincide with the opinion that the 

specific (anti-T) immunity cancels the cell-mediated one and leads to enhanced 

susceptibility to chronic epidermophytia and nail mycosis. Probably, a more advanced 

age is concerned in these speculations.

P-0615. Analysis of Candida albicans effect on B16 melanoma cell adhesion to 

hepatic sinusoidal endothelium 

Ramirez Garcia A 1 , Gallot N 2 , Abad A 1 , Dominguez . 1 , GuzmZn C 1 , Mendoza / 2 , 

Vidal-Vanaclocha F 2 , Hernando F 1 

1 Department of Immunology, Microbiology and Parasitology. 6niversity of 2asque 

Country. /eioa. .pain, 2 Dominion PharmaBine ../. 2izBaia Technological ParB. 

2uilding 801. Derio. .pain. 

Purpose: The aim of this worB is to demonstrate that mannoproteins of 7+,5"5+*+68"#+,& 

activate Hepatic .inusoidal Endothelium (H.E) causing enhancement of 216 melanoma 

cell adhesion to H.E. 

Description: 7+,5"5+*+68"#+,& is a dimorphic fungus associated to hot blood animals as 

an opportunistic pathogen that is able to disseminate hematogenously and to cause 

serious infections in compromised patients. In recent years, the number of candidiasis has 

significantly increased in hospitals and one important reason for it is the 

immunosuppressant state generated in patients after surgery. The first place where the 

microorganisms that have entered the bloodstream are retained is the endothelial cell lining 

of the vasculature. These cells have, among other receptors, mannose receptor, which 

recognize mannosylated molecules and that initiate a proinflammatory response that is 

responsible for the circulating cancer cell adhesion. Mannosylated proteins of 7+,5"5+* 

+68"#+,& are the most important antigenic component from cell wall and they are also 

implicated in adhesion to host. 

7+,5"5+*+68"#+,& 6PV1O1J strain was used in this worB. Cells from mouse Hepatic 

.inusoidal Endothelium were incubated with live blastoconidia, Billed blastoconidia and 

germ tubes. After washing, 2CECF-labeled 216M cells were added. Tumor cell adhesion 

was assessed by plate-scanning fluorometry of 2CECF fluorescence. 

Crude extract was fractionated by Con-A .epharose O2 affinity chromatography (0.2 

ml/min) and eluted with methyl $-D-manno-pyranoside. Two fractions were obtainedM 

Mannoprotein Fraction (MNF) and Protein Fraction (PF). 216 melanoma cell adhesion to 

H.E incubated previously with purified fractions was assessed. 

P-0616. Detection of specific anti alternaria alternata IGe in asthmatic patients 

Saghazadeh M 1 , lhosravi A 2 , Riazipour M 1 

1 Department of Microbiology, Faculty of 2asic .ciences,Islamic Azad 6niversity (~om 

branch), ~om,Iran, 2 Dept. of Mycology,Faculty of Veterinary, Tehran 6niversity, 

Tehran,Iran, J Dept.of Microbiology, Faculty of Medicine, 2aqiatallah Medical .cience 

6niversity,Tehran,Iran 

Introduction: Asthma is a chronic pulmonary disease caused by allergic and nonallergic 

factors. Alternaria alternata has allergenic components in cell wall and 

cytoplasm of conidia and hyphae . Inhalation of air-borne conidia, can cause the 

appearance of asthma signs in sensitive patients. 

Objective: Evaluation of the role of the fungus in inducing specific IgE in asthma 

patients. 

Methods: In the first stage, the crude antigens were prepared from the fungi. 

In order to obtain fungal extract after the culture of fungi they were homogenized and 

then the supernatant provided by centrifugation was used as crude antigen . In this 

research 88 asthma patients were chosen of whom O6 were males and O2 were 

females . They were compared to an age match control population (O0 healthy 

controls). .pecific Anti-Alternaria alternata IgE was tested by dot blotting method. 

Results: 2y dot blotting testing O6 patients ([2.J_) were positive and there was no 

significant difference between sexes and age groups (Pt0.0[). 

All controls were negative by the dot blotting method. 

Conclusion: Therefore we conclude that different protein and allergenic components 

can have a role in producing the signs of asthma in sensitive patients. 

Results and Conclusion: The obtained results showed that there was a statistically 

significant enhancement of 216 melanoma cell adhesion to H.E incubated with 7+,5"5+* 

+68"#+,& 6PV1O1J blastoconidia. This enhancement was greater in H.E incubated with 

blastoconidia than with germ tubes. Therefore, H.E cells incubated with Billed organisms 

had a similar effect on tumor cell adhesion, demonstrating that the effect was mediated by 

molecules and not by living organisms. 

ahen 7+,5"5+*+68"#+,&*fractions were assayed we also report that at the same 

carbohydrate concentration than commercial mannan, MNF had a lytic effect on H.E and 

1/[0 diluted MNF fraction showed a 100_ increase of adhesion. The crude extract showed 

a similar effect than the MNF fraction. ahen PF was used, a toxic effect was found with 

high concentrations, but when H.E was incubated with diluted PF, there was not a 

statistically significant enhancement of 216 melanoma cell adhesion. 

These findings suggest that the incubation of H.E cells with the opportunistic pathogen 

7+,5"5+*+68"#+,&*induce an enhancement of tumor cell adhesion to H.E. This process is 

initiated by the mannoprotein fraction of this yeast. 


P-0617. Establishment of a carrier state influences the outcome of a secondary 

Candida albicans infection in mice with differing immunological deficiencies 

.aville . 1 , /azzell A 1 , Monteagudo C 2 , Lopez-Ribot J 1 

1 The 6niversity of Texas at .an Antonio, Texas, 6.A, 2 6niversidad de Valencia, 

.pain 

Despite its intrinsic limitations the murine model of hematogenously disseminated 

candidiasis is widely used to examine 7+,5"5+*+68"#+,& pathogenesis. fne of these is 

that, unliBe humans, experimental mice do not normally contain 7/*+68"#+,& as part of 

their normal microbiota. ae reasoned that establishment of a hcarrieri or hcommensali 

state in mice could influence the course of a secondary infection. ae have used a 

genetically engineered 7/*+68"#+,& strain in which `P9I (a negative regulator of 

filamentation) was placed under the control of a tetracycline-regulatable promoter so 

that morphology and virulence can be manipulated ",*0"0% by adding or omitting 

doxycycline (Dfj) from the animal]s drinBing water. In a previous study, we 

demonstrated that infection with this strain Bept in the yeast form (no Dfj) led to 

100 _ survival even at high infectivity levels. Here we tooB advantage of this earlier 

observation to recreate a hcarrieri state in different strains of mice (2A/2/c, 2-cell 

deficient, nude and D2A/2N) and examined the levels of protection afforded by using 

this strategy against a secondary challenge with a wild type virulent strain, as 

compared to na¢ve (not pre-exposed to the -.-W`P9I strain) mice from the same 

genetic bacBground. The results indicate that prior exposure to the -.-W`P9I strain 

under conditions leading only to colonization but not lethality fully protected 2A/2/c, 

2-cell deficient, and D2A/2N against a secondary infection with CAF2 (100_ survival 

in pre-exposed animals 0& 100_ mortality in na¢ve mice), but failed to protect T-cell 

deficient, nude mice (100_ mortality observed in both sets of animals). These 

observations strongly suggest a role for acquired immunity in preventing symptomatic 

secondary infection and confirm the requirement for proper integration of both innate 

and adaptive immune responses. They reinforce a predominant role for a cellmediated 

adaptive immune response in protection against secondary infection, while 

at the same time rule out the contribution of antibodies to protection in this 

experimental model. Particularly exciting are the results with D2A/2N mice (which are 

normally extremely sensitive to systemic candidiasis due to their impaired neutrophil 

activity), that indicate the feasibility of vaccine approaches in neutropenic patients, 

which represent a major population of at risB patients for candidiasis. 

P-0618. Th-1/ Th-2 cytokine levels in vaginal lavages of women with chronic 

recurrent vulvovaginal candidiasis 

Shabashova N, Mirzabalaeva A, Dolgo-.aburova (, Frolova E, 6chevatBina A 

lashBin Research Inst. of Med. Mycology 

Recurrent vulvovaginal candidiasis, caused by 7+,5"5+*+68"#+,&, remains a significant 

problem in women of childbearing age. Host defense mechanisms against mucosal 

Candidal infection is poorly understood. 

The purpose was to investigate the levels of some cytoBinesM interferon-alfa (IFN-alfa), 

interferon-gamma (IFN-gamma) and interleyBines (I/ -O, -8, -10) in vaginal lavage 

from women with chronic recurrent vulvovaginitis, caused by 7+,5"5+*&'.#".& (CRVC). 

JJ women (median age J7ì6 years) with CRVC in phase of symptomatic infection 

(n}2J) and infection-free period of remission (n}10) were examined. Duration of the 

disease was J till [ years and frequency of episodes of infection attacBs was O-6 times 

per one year. The control group consisted of 11 practically healthy women of the 

same age. A mycological diagnosis was confirmed by the data of mycroscopic and 

culture research. In K8 _ cases the 7+,5"5+*+68"#+,& were revealed. The contents of 

the cytoBines (IFN-alfa, IFN-gamma and I/ -O, -8, -10) in vaginal lavage was 

estimated by E/I.A. 

The levels of proinflammatory cytoBines IFN- alfa and I/-8 in lavages of healthy 

women were 1,Oì0,[ and 12,2ì2,2 pg/ml accordingly. The level of IFN-gamma, 

prodused by .-helper 1 type, was higher ([J,8ìO,7 pg/ml) in contrast to I/- O (J,8ì0,7 

pg/ml) and I/-10 (1,Kì0,J pg/ml) | prodused by .-helper 2 types. .o the normal 

microbiota of vaginal mucosa influence a local cell immune response activity in 

healthy persons. All patients in a phase of infection attacB were separated into two 

subgroups depending on IFN-gamma level. The first subgroup was made with the 1O 

patients, in which the contents IFN-gamma was lower to compare with the control 

(22,7ìJ,2 pg/ml), and the second | included the 10 patients with increased levels of it 

(72,6ì[,O pg/ml). The level of IFN-alfa (1,[ì0,[ pg/ml) and antiinflammatory 

cytoBines I/ - O (J,8ì0,7 pg/ml) in patients of the first subgroup were higher than that 

observed in the control (7,Oì2,J pg/ml and 6,J ì1,1 pg/ml accordingly). The increase 

of the all proinflamatory cytoBine contents was marBed in the second subgroup, and 

the levels of antiinflammatory I/-O and I/-10 did not differ from the control. The 

contens of antiinflammatory cytoBines during the infection-free period of remission 

didn]t differ significantly to compare with the control. However, increase (but was not 

significantly different) of the levels of proinflammatory cytoBines IFN-alfa (7,0ìJ,[ 

pg/ml) and I/ - 8 (KO,0ì60,0 pg/ml) were revealed in this group. That showed a 

presence imballans of cytoBines, Bept after elimination of the fungi. 

Conclusions. The role of pro- and antiinflammatory cytoBines in pathogenesis of 

CRVC is obvious. The character of local changes of cytoBine levels depends on a 

phase of inflammatory process. During symptomatic infection the most essential 

changes are revealed in IFN-gamma secretion. Most of the patients with CRVC have 

the decrease of its production. It show that cell mediated immunity response disorders 

taBe plase. The found out differences allow to maBe conclusion about necessity to 

different strategy in choice of pathogenic and immunomodulator therapy of the 

patients with CRVC.

IMMUNOLOGY* 

P-0619. HLA class alleles distributions in patients with 

paracoccidioidomycosis 

Shikanai-Yasuda M 1 , .adahiro A 2 , MourÉo Roque A 1 

1 /aboratário de Imunologia-/IMO8, Hospital das ClÜnicas Departamento de Moldstias 

Infecciosas e ParasitZrias6niversidade de .Éo Paulo , 2razil, 2 Departamento de 

Parasitologia IC2 6niversidade Federal do Amazonas 

Purpose of the study: Few studies have investigated H/A (human leuBocyte 

antigen) genes in paracoccidioidomycosis, in the present study we determined the 

distribution of generic H/A DR21ò and D~21ò alleles in patients presenting different 

clinical forms of disease. 

Description: Eighty 2razilian patients were included into three groupsM the acute form 

(n}2J) involves the mononuclear phagocytes system and affects both genders and 

the chronic form, more often affecting males, has two presentationsM unifocal (n}12), 

involving a single organn and multifocal (n}O[), involving more than one organ or 

system (lung, sBin, mucous membranes or adrenals)n moreover a control group (n}O[) 

was selected. Typing of H/A was performed through polymerase chain reaction using 

sequence specific primers (Micro ..P Generic H/A Class II, fne /ambda, 6.A). 

Results and Conclusions: The H/A-DR21ò11 allele was detected at a statistically 

significant higher frequency in patients with the unifocal presentation of the chronic 

form ([0.0_) than in other groups ('}0.0JK). ae detected high frequency of H/A- 

DR21ò11 in patients with the unifocal presentation of chronic paracoccidioidomycosis, 

suggesting that genetic factors, particularly H/A class II, are important for the 

outcome of the host-parasite interaction in paracoccidioidomycosis. 

Financial support: FAPE.P (FundaÑÉo de Amparo a Pesquisa do Estado de .Éo 

Paulo) and FundaÑÉo Faculdade de Medicina. 

IMMUNOLOGY* 

P-0620. Interaction of conidia and hyphal fragments of histoplasma capsulatum 

with mice nasal mucosa 

.uZrez-Alvarez 1 , Pdrez-Torres 2 , Taylor M J 

1 6niversidad Nacional Autonoma de Mexico, 2 6niversidad Nacional Autonoma de 

Mexico, J 6niversidad Nacional Autonoma de Mexico 

The dimorphic fungus A"&-%'6+&)+*#+'&(6+-() (A#) produces a mycelial-phase (M) 

with hyphal fragments and microconidia (infective propagules) that enter into the host 

through the respiratory system and, in their route towards internal organs, they 

undergo a transition to the yeast-parasitic (Y) and virulent phase. The importance of 

the nasal cavity in A# infection is related to its large epithelial surface where the 

pathogen interacts with several types of cells, mainly ciliated and mucosal dendritic 

cells (DC). These DC are able to phagocytose and process antigens while migrating 

towards the paracortical zone of the regional lymph nodes, where they participate in 

antigens presentation to T-cells. The nasal associated lymphoid tissue (NA/T) is 

found in the nasal mucosa, its epithelial cells allow crossing endocytosed structures 

towards the subjacent lymphoid tissue, which also contains DC. The present worB was 

performed to study the interaction between A# and the nasal epithelium, as well as to 

determine at which site of the nasal mucosa and at what time during infection does 

the M-Y fungal transition occurs. 2A/2/c mice were intranasally infected with A# 

propagules and Billed at 1, 2, J, and 6 h post-infection. The nasal cavity, the cervical 

lymph nodes, the trachea, the lungs, the spleen, the liver, and the small intestine were 

processed by several staining and immunohistochemistry techniques with a rabbit 

anti-A# serum, and with N/DC-1O[ monoclonal antibody specific to DC. Intracellular 

yeasts in the columnar ciliated cells, in mucosal and paracortical DC were observed at 

J h post-infection. .canning electron microscopy (.EM) of the nasal epithelium 

surface showed the presence of yeasts since the first J h post-infection with a 

decreasing antero-posterior distribution. Nested-PCR, using a fragment of a specific 

protein Hcp100 coding gene sequence, revealed A# DNA band only in the cervical 

lymph node at J h and in the trachea at 6 h post-infection. In other samples tested at 

different times, A#*DNA was not detected. In conclusionM the nasal mucosa and its 

cervical lymph nodes are important sites for the initial M-Y transition and A# 

disseminationn the participation of mucosal DC in the initial yeasts phagocytosis event 

was observedn and the presence of fungal DNA in cervical lymph nodes and trachea, 

revealed by PCR assays, confirmed the histopathological and .EM findings. 


P-0621. Antibody response against P. brasiliensis melanin in an experimental 

model of paracoccidioidomycosis 

Uran-Jimenez M 1 , Gomez 2 2 , Restrepo A 1 , Hamilton A 2 , Cano / 1,J 

1 Medical and Experimental Mycology Group, Corporacián para Investigaciones 2iolágicas, 

MedellÜn-Colombia., 2 Dermatology Department, .t. (ohns Institute of Dermatology, Guy]s 

Hospital, Guy]s, lings and .t. Thomas Medical .chools, /ondon-6nited lingdom., 

J Molecular Microbiology Group, Escuela de 2acteriologÜa y /aboratorio ClÜnico, 

6niversidad de Antioquia, MedellÜn-Colombia. 

Melanins are compounds synthesized by organisms belonging to all biological Bingdomsn 

they are involved in a wide variety of processes and implicated in the pathogenesis of 

several human diseases, including some fungal infections. 2oth conidia and yeast cells*of* 

the thermally dimorphic fungal pathogen*1+$+#%##"5"%"5.&*8$+&"6".,&"&3*the etiological agent 

of paracoccidioidomycosis (PCM), produce melanin or melanin-liBe compounds ",*0"-$% and 

",*0"0%. 

Melanin particles extracted from 1/*8$+&"6".,&"&*yeast cells were used to produce a panel of 

murine monoclonal antibodies (MAbs) with both IgM and IgG1 which reacted both with 1/* 

8$+&"6".,&"& conidia and yeast cells, as well as with diverse fungal melanin particles 

including commercial sources of the pigment. 

Direct evidence for the polymerization of melanin ",*0"0%*was demonstrated by 

immunofluorescence analyses of lung tissues from mice infected with 1/8$+&"6".,&"&*conidia 

that bound to the MAbs against the fungal cell wall. These findings established that 

polymerized melanin was formed ",*0"0%*and*was*immunogenic as sera and 

bronchoalveolar lavage fluids (2A/) from infected animals had antibodies that bound to 

isolated melanin particles from conidia and yeast cells tested by E/I.A. 

It is unclear at this time whether the novel MAbs were raised against DHN or DfPA 

melaninn however they are reactive with diverse melanins, including one from 4&'.$@"66(&* 

sp. (DHN melanin) and another from 7/*,.%U%$)+,&*(DfPA melanin). Good evidence of 

antigenic cross-reactivity between different melanin types has been already shown. 

The antibodies generated against melanin during infection may be protective to the host, 

but given the association with melanin and autoimmune diseases, such as vitiligo, the 

antibody response could conceivably be deleterious under some circumstances. 

The murine anti-1/8$+&"6".,&"&*melanin MAbs here obtained and the significance of 

antibody response ",*0"0% will be useful in the study of 1/8$+&"6".,&"&*melanization 

particularly in regard to passive immunization for prolonged survival of infected mice and/or 

modulation of the immune response against 1/8$+&"6".,&"& infection. 

P-0622. Protection against cryptococcosis using a murine interferon-gamma 

producing Cryptococcus neoformans strain 

Wormley Jr. F 1 , Perfect (, .teele C, Cox G 

1 6niversity of Texas at .an Antonio, 2 DuBe 6niversity Medical Center, J 6niversity of 

Pittsburgh .chool of Medicine, O DuBe 6niversity Medical Center 

Purpose of the study:**7$2'-%#%##(&*,.%U%$)+,& is an opportunistic fungal 

pathogen that threatens individuals with impaired Th1-type cell-mediated immune 

(CMI) responses. The administration of Th1-type cytoBines represents a promising 

immunotherapy for immune compromised patientsn however, the adverse side effects 

associated with cytoBine therapy suggests that a more targeted approach is needed to 

reduce or eliminate any deleterious side effects. The present study was thus designed 

to evaluate the efficacy of using a genetically modified 7/*,.%U%$)+,& strain that 

produces murine interferon (muIFN)-gamma to induce protective host CMI responses 

against experimental pulmonary cryptococcosis. 

Description: Host cell-mediated immune responses (pulmonary cytoBine production 

and cell recruitment) and survival were evaluated in mice following experimental 

pulmonary infection with a 7/*,.%U%$)+,& strain engineered to produce murine (mu) 

IFN-gamma compared to wild-type infected mice. 

Results and conclusions: Mice given a pulmonary infection with an IFN-gammaproducing*7/*,.%U%$)+,& 

strain were able to resolve the primary infection and 

demonstrated complete (100_) protection against a second pulmonary challenge with 

a pathogenic 7/*,.%U%$)+,& strain. Pulmonary cytoBine analyses showed that Th1- 

type/pro-inflammatory cytoBine and chemoBine expression were significantly higher 

and Th2-type cytoBine expression was significantly lower in mice infected with the 

IFN-gamma /producing 7/*,.%U%$)+,& strain compared to wild-type infected mice. This 

increased pulmonary Th1-type cytoBine expression was also associated with 

significantly lower pulmonary fungal burden and significantly higher pulmonary 

leuBocyte and T lymphocyte recruitment in mice infected with the IFN-gamma 

/producing 7/*,.%U%$)+,& strain as compared to wild-type infected mice. fur results 

demonstrate that pulmonary infection of mice with a 7/*,.%U%$)+,& strain expressing 

IFN-gamma results in the stimulation of local Th1-type anti-crytococcal CMI responses 

and the development of protective host immunity against future pulmonary 

cryptococcal infections. The use of fungi engineered to produce host cytoBines is a 

novel method to study immune responses to infection, and may be useful in 

developing vaccine strategies in humans. 

fur results demonstrating that 1/*8$+&"6".,&"&*synthesizes and polymerizes melanin during 

infection have important implications in understanding PCM pathogenesis and hopefully 

pointing towards development of antifungal medications. 

Founding resource: 

Cf/CIENCIA., cádigo No. 111[-0O-1182J, Colombia. 

ling]s College, /ondon 6niversity, /ondon, 6l. 

6niversidad de Antioquia and Corporacián para Investigaciones 2iolágicas, MedellÜn, 

Colombia.

P-0623. In vivo role of dendritic cells in a murine model of pulmonary 

cryptococcosis 

Wozniak K 1 , Vyas ( 2 , /evitz . 1 

1 2oston 6niversity Medical Center, 2oston, 6.A, 2 Massachuesetts General Hospital, 

2oston, 6.A 

Dendritic cells (DC) have been shown to phagocytose and Bill 7$2'-%#%##(&* 

,.%U%$)+,&*",*0"-$%*and are believed to be important for inducing protective immunity 

towards this organism. Exposure to 7/*,.%U%$)+,& occurs mainly by inhalation, and 

thus we sought to examine the interactions of 7/*,.%U%$)+,& with DC in the lung. 

Fluorescently labeled live and heat-Billed labeled 7/*,.%U%$)+,&*organisms*were 

administered intranasally to C[7 2l/6 mice. At specific time points post-inoculation, 

mice were sacrificed and lungs were removed. .ingle cell suspensions of lung tissue 

were prepared, stained, and analyzed by microscopy and flow cytometry. aithin 2 h 

post-inoculation, fluorescently labeled 7/*,.%U%$)+,& had been internalized by DC, 

macrophages, and neutrophils of the mouse lung. Additionally, CD11c r lung DC from 

mice infected for 7 days exhibited increased expression of MHC II and the 

costimulatory molecules CD80 and CD86. Finally, ._*0"0% incubation of lung DC from 

infected mice with 7$2'-%#%##(&-specific T cells resulted in increased I/-2 production 

compared to DC from na¢ve mice, suggesting antigen-specific T cell activation. Thus, 

lung DC participate in innate and adaptive pulmonary defenses against cryptococcosis 

by phagocytosing 7$2'-%#%##(&*",*0"0%*and presenting antigen to 7/*,.%U%$)+,&- 

specific T cells ._*0"0%/ 

P-0625. Mucor polymorphosporus and its low complement activation. 

ZmeteB Granja / 1 , Pinto / 1 , Alviano D 2 , .ilva M 1 , Alviano C 2 , Ejzemberg R 1 

1 Dep. de Imunologia - IMPPG - CC. - 6FR(, 2 Dep. de Microbiologia Geral - IMPPG - 

CC. - 6FR( 

Mucormycosis is the name of an infection caused by fungi of the L2@%)2#.-.& class, 

!(#%$+6.& order. Most members of the !(#%$+6.&*order are dimorphic and the 

filamentous form is responsible for the infection. In the present worB, we used conidia, 

filamentous and yeast-liBe forms of the fungus !(#%$*'%62)%$'?%&'%$(& to study 

complement activation since this system plays a crucial role in the humoral defense 

against microbial pathogens. The fungus was incubated with human absorbed serum, 

treated or not with chelators (EDTA, EGTA) and Bept for 60 minutes at J7+C. The 

residual complement was measured in the supernatants. fur results showed that the 

yeast-liBe form had the best result regarding the percentage of complement 

consumption (EGTA-Mg rr - OJ.6_n Veronal 2uffer .aline - V2. | [7.1_). The 

filamentous form, grown under shaBing conditions (sample A) or under still condition 

(sample 2), was submitted to enzymatic treatments such asM 0.26/ml sialidase (O"8$"%* 

#?%6.$+.), 1.06/ml glucuronidase (A.6"_*'%)+-"+) or both. The results showed that 

when the fungus was treated with both sialidase and glucuronidase the complement 

consumption slightly increased. The sample 2 went from EGTA-Mg rr - 6.7_ to 11.K_ 

and V2. | 2O.1_ to J2.J_ and the other (sample A) from EGTA-Mg rr - 10.[_ to 

26.1_ and V2. | JK.O_ to OO.0_ The cells after complement activation were 

analyzed by immunofluorescence to checB the presence of CJ fragments and M2/. 

All samples presented CJ fragments on the cell surface. E/I.A assay with conidia 

and yeast-liBe forms also detected CJ fragments. CJd was also observed on the 

surface of all samples. This indicates that there is probably cleavage of CJb by 

complement regulators such as factor I and co-factor H favored by the presence of 

sialic and/or glucuronic acids on the fungal surface. This could also account for the 

increase seen after the enzymatic treatment of the filamentous forms. The presence of 

M2/ on the surface of the sample 2, treated with sialidase, and also on conidia, could 

explain the consumption difference found when the fungus was incubated with serum 

and EGTA-Mg rr or V2.. Another test regarding complement consumption was 

performed in order to see if any differences occurred on another fungus of the same 

order, !/*#"$#",.66%"5.& (micelial form). To our surprise the results were quite similar to 

our previously tested fungus (EGTA-Mg rr - 18.O_n V2. | 2O.0_). fther tests are 

necessary in order to further investigate if this low activation is a common 

characteristic between species of this order. 

.uported byM CAPE., CNPq, FAPER( and 6FR(. 


P-0626. Genome sequencing of Pythium insidiosum isolated from cattle 

M. Santurio J, Henzel A, E. .chwendler ., .. Cavalheiro A, .. Argenta (, Azevedo M, 

Hartz alves . 

6niversidade Fed de .anta Maria- /APEMI - 2razil 

Pythium insidiosum is a pathogenic fungus of mammals and plants, which affects 

mainly horses and also dogs, cats, cattle and humans, with a lower incidence. Its 

identifying characteristics are essential for its classification. Molecular biology provides 

reliability and accuracy by using techniques as Polymerase Chain Reaction | PCR. 

This technique sequentially amplifies specific DNA regions by using primers defined 

for the region under study. The objective of this study was to genetically confirm by 

genome sequencing a P.insidiosum isolate from a bovine with pythiosis (2nd isolate in 

the world). The sample sent to the /aboratory of Mycological .tudies (/APEMI/6F.M) 

came from the 2razilian Pantanal region and was identified by the morphological 

characteristics. The DNA was then extracted from the fungal mycelium and submitted 

to the PCR technique, using universal fungal primers, with configuration for the IT.I 

region, IT.1 (TCCGTAGGTGAACCTGCGG) and IT.O 

(TCCTCCGCTTATTGATATGC), amplifying the IT.1, [,8. and IT.2 regions of the 

ribosomal DNA (rDNA), with approximately 800bp (base pairs). The amplified product 

was purified and Mega2ACE [00-Amersham 2iosciences (/abdros/6F.M) sequencer 

was used in the genome reading. A species-specific primer, PI1 

(TTCGTCGAAGCGGACTGCT) was used in the sequencing. The fragment has 126 

bp, which represents a K[_ similarity with P. insidiosum, according to the analysis 

made by the basic local alignment search tool (2/A.T/Gen2anB). The importance of 

the genetic study is made clear by the result obtained, as it provides a better 

characterization and further phylogenetic analyses of the fungi. Thus, the degree of 

taxonomic classification is more reliable and accurate. 

MOLECULAR BIOLOGY* 

P-0627. Genotypic heterogeneity and 5-flucytosine resistance among clinical 

Candida dubliniensis isolates in Kuwait 

Ahmad S, MoBaddas E, (oseph /, lhan Z 

luwait 6niversity, .afat, luwait 

Objective: 7+,5"5+*5(86",".,&"& is a newly recognized species and an emerging pathogen 

capable of causing oropharyngeal and systemic infections, particularly among 

immunocompromised individuals. Clinical 7/*5(86",".,&"& isolates belong to four distinct 

genotypes based on nucleotide sequence analysis of internally transcribed spacer (IT.)1 

and IT.2 regions of rDNA. All the genotype J and O isolates from .audi Arabia and Egypt 

were recently shown to be resistant to [-flucytosine. This study was carried out to determine 

the presence of different genotypes and their association with [-flucytosine resistance 

among clinical 7/*5(86",".,&"& isolates in luwait. 

Materials and Methods: All the 66 C.*5(86",".,&"& isolates recovered from various clinical 

specimens in luwait were analyzed. The 7/*5(86",".,&"& isolates were identified by their 

characteristic rough colonies and chlamydospore production on simplified sunflower seed 

agar medium as well as by species-specific PCR amplification of rDNA. The susceptibility of 

the isolates to commonly used anti-fungal agents including [-flucytosine was carried out by 

Etest. The 7/*5(86",".,&"& strains were assigned to one of four genotypes based on specific 

amplification of rDNA with genotype-specific primers. The DNA sequencing of IT. regions 

was carried out to confirm the results for all or some randomly selected isolates belonging to 

each genotype. 

Results: All phenotypically identified 7/*5(86",".,&"& isolates from luwait yielded amplicons 

of expected sizes following PCR amplification of IT. regions with panfungal and speciesspecific 

primers and with one pair of genotype-specific primers. The vast majority (O8 of 66, 

7J_) of 7/*5(86",".,&"& isolates belonged to genotype 1 and the DNA sequences of IT. 

regions from 11 randomly selected isolates were completely identical. ff the remaining 7/* 

5(86",".,&"& isolates, 16 (2O_) strains belonged to genotype O with 1[ of these strains 

containing identical IT. region sequences while the sequence of one strain was different at 

one nucleotide position. Although two (J_) strains yielded amplicons with genotype J- 

specific primers, the IT. region sequences of both the isolates varied at one or more 

nucleotide positions compared to the reported sequences of genotype J isolates from other 

geographical locations. None of the 7/*5(86",".,&"& strains in luwait belonged to genotype 2. 

All the 7/*5(86",".,&"& isolates were susceptible to the commonly used anti-fungal agents 

except [-flucytosine. A total of 1O 7/*5(86",".,&"& isolates were resistant to [-flucytosine and 

all of these strains belonged to genotype O. 

Conclusions: fnly three of four genotypes based on IT. regions sequences were 

detected among clinical 7/*5(86",".,&"& isolates from luwait with most (7J_) of the 

isolates belonging to genotype 1. All genotype 1 isolates contained identical IT. 

regions sequences while one of 16 genotype O and both genotype J strains exhibited 

sequence variations within the IT. regions. Although, all 7/*5(86",".,&"& isolates 

resistant to [-flucytosine belonged to genotype O, however, in contrast to previously 

reported data from .audi Arabia and Egypt, two genotype O isolates were not resistant 

to [-flucytosine. The two variant genotype J isolates from luwait were also not 

resistant to [-flucytosine.

P-0628. Identification and functional analysis of the Aspergillus fumigatus C-5 

sterol desaturase (erg3C) 

Alcazar-Fuoli L 1 , Mellado E 1 , GarcÜa-Effrán G 1 , F. /opez ( 2 , f. Grimalt ( 2 , Cuenca- 

Estrella M 1 , RodrÜguez-Tudela ( 1 

1 Centro Nacional de MicrobiologÜa, Instituto de .alud Carlos III, Majadahonda, Madrid, 

.pain, 2 Department of Environmental Chemistry1, I.C.E.R.-C...I.C., (ordi Girona, 18. 

080JO-2arcelona, .pain. 

BackgroundM The antifungal agents that are available to treat 4&'.$@"66(&*U()"@+-(&* 

infections are classified in different classes with different cellular targets. The azole 

antifungal agents blocB the ergosterol biosynthesis pathway through the inhibition of 

1O -sterol demethylase (Cyp[1). In 4/*U()"@+-(&, there are two C yp[1 proteins 

encoded by #2'[1A and #2'[12 genes which have different implication in azole drugs 

resistance. Moreover, mutations or inactivation of the a$@:*gene in yeasts have been 

associated with azole and polyene drug resistance and accumulation of non C -[ 

desaturated sterols, which is a feature of activity absence of C -[ sterol desaturase . 

Three different .$@:*genes have been identified in 4/*U()"@+-(&*(.$@:4, .$@:Z*and 

.$@:7). In order to investigate the r ole of ErgJC in 4/*U()"@+-(&*we have created a 

.$@:7*deleted strain . This worB shows the description and analysis of the 4/* 

U()"@+-(&*.$@:7*- mutant strain. 

Methods: 4/*U()"@+-(&*CM-2J7 was used as bacBground stra in to obtain a .$@:7*- 

mutant strain. The CM-2J7 strain was also used to describe the sequences of .$@:C. 

4/*U()"@+-(&*was transformed by electroporation and transformants were selected 

using hygromycin 2. 4/*U()"@+-(&*.$@:7*BnocB out strains were broadly analysedM (i) 

susceptibility testing, (ii) morphology and cell growth, (iii) total ergosterol content 

(HP/C), (iv) sterol composition by GC -M., and (v) pathogenicity in a murine model of 

pulmonary aspergillosis. 

Results: No significant (p +0.01) differences were found between mutants .$@:7*- 

and the parental strain (CM-2J7) regarding azole susceptibility. However, the .$@7* 

deleted strain showed a colony morphology and radial growth clearly different from 

wild type strain. Furthermore, there were differences between CM - 2J7 and .$@:7*- 

strains in total ergosterol content and relative sterols composition. 

Conclusion: These results show that .$@:7*gene is not essential for 4/*U()"@+-(&* 

viability and see ms to have no implication in azole/Am2 drugs susceptibility or 

pathogenicity. However, it is clear that ErgJC had a marBed influence on 4/*U()"@+-(&* 

growth. The differences in ergosterol and total sterols content verify the functional role 

of ErgJC as a C -sterol desaturase protein. This worB also verified the existence of 

three distinct C -[ sterol desaturases in the ergosterol biosynthesis of 4/*U()"@+-(&*. 

P-0629. Differences in the sequence of the CNLAC1 laccase gene between 

Cryptococcus gattii and Cryptococcus neoformans 

Alvarado Ramírez E, Torres-RodrÜguez (, .egura G, Gámez de Ana ., Murciano F 

Research 6nit on Infectious Diseases and Mycology (6RMIM). Autonomous 

6niversity of 2arcelona. Municipal Institute for Medical Research (IMIM). 

Introduction: In the last years 7$2'-%#%##(&*@+--"" has been considered as an 

independent species from 7$2'-%#%##(&*,.%U%$)+,&. 7/*@+--"" presents peculiar 

ecological characteristics conditioning its geographic distribution. In .pain, native 

cases of cryptococcosis in goats have reported. 

The pathogenic factors have been studied mainly in 7/*,.%U%$)+,&*and it is not 

Bnown if it there is relevant differences with 7/*@+--"". Melanin production is one of the 

more studied factors. It is already Bnow that CN/AC1 gene plays a main roll in the 

regulation of the laccase, enzyme responsible of melanin production. This gene has 

also been studied mainly in 7/,.%U%$)+,&. 

For these reasons a partial sequence of CN/AC1 gene was analyzed and compared 

in isolates of 7/*@+--""*serotypes 2 and C, and 7/,.%U%$)+,& serotypes A and D. 

Materials and methods: .trainsM 7 clinical isolates of*7/*@+--"" serotype 2 were used, 

6 of them were isolated from goats dead in .pain of disseminated cryptococcosis plus 

1 environment strain serotype 2, 2 of serotype C. Moreover O 7/*,.%U%$)+,& 

(serotypes A and D) isolated from HIV r patients with cryptococcal meningitis were 

also studied. 

Procedure: The primer set was designed from the partial sequences of gene 

CN/AC1 coming from 16 strains of 7/*8+#"66"&'%$(&*included in the Gen2anB database. 

This set was used to amplify a fragment of the laccase gene from the genomic DNA 

by conventional PCR. The sense primer was located at the 88 th -106 th and the 

antisense primer was the complementary sequence of the [28 th | [O7 th base. The 

expected product amplified by PCR has about O[K bp. 

The amplified products were sequenced by an 4ZK*1PK=!*:IQ`>*9.,%)"#*4,+62d.$ 

(Applied 2iosystems, Calif., 6.A) using a Z"@V2.*>.$)",+-%$*72#6.W=.e(.,#",@*["-/ 

The nucleotide sequence data were then analyzed comparatively by Z6+&-*1$%@$+)*%U* 

`+-"%,+6*7.,-.$*U%$*Z"%-.#?,%6%@2*K,U%$)+-"%,. 

Results: Fragments between O18 bp and O22 bp were obtained exclusively with all 

the 8 strains of 7/*@+--"" serotypes 2 and C, but not with 7/*,.%U%$)+,&*A and D 

serotypes. 

The Z6+&-*1$%@$+) showed K8_ homology among the sequenced amplified fragment 

and CN/AC1 partials sequences of 7/*8+#"66"&'%$(& (syn. 7/*@+--"") from Gen2anB. 

Discussion: In the present study, we find evidence of that under the applied 

conditions, 7/*@+--"" serotypes 2 and C have a segment amplified of about O[0 bp, but 

absent in 7/*,.%U%$)+,&. These findings support the segregation of both species. 

These results suggest qualitative differences in the sequence of CN/AC1 gene, and 

can influence on the virulence of these yeasts. It should be confirmed by analyzing a 

high number of isolates of both species. 


P-0630. Transcriptional elements and regulation of the PbMdj1/PbLON heat 

shock genes from Paracoccidioides brasiliensis 

Batista W 1 , Goldman G 2 , Puccia R 1 

1 6niversidade Federal de .Éo Paulo (6NIFE.P), 2 6niversidade de .Éo Paulo (6.P) 

Purpose of the study: 1+$+#%##"5"%"5.&*8$+&"6".,&"&, Z6+&-%)2#.&*5.$)+-"-"5"&, 

A"&-%'6+&)+*#+'&(6+-() and 7%##"5"%"5.&*"))"-"& are genetically related dimorphic 

fungi that cause deep-seated mycoses. ae have previously characterized the Xb` 

and !V]I*gene homologues from 1/*8$+&"6".,&"&, which encode heat shocB proteins 

targeted to the mitochondria, although PbMdj1 was also largely found in the cell wall 

s2atista et al., 2006, EuBaryot. CellM [MJ7Ku. In yeasts, /on is an ATP-dependent 

proteinase that controls proteolysis in the mitochondrial matrix by mediating cleavage 

of misfolded or unassembled proteins in an Mdj1p-dependent fashion. The !V]I/Xb` 

locus is conserved in pathogenic dimorphic fungi, where the genes are adjacent, 

inversely orientated and separated by a common [] intergenic (M/) region ranging 

between O00 and O8[ nucleotides. 

Summarized description of the project: ae aimed at mapping transcriptional 

elements of the 1/*8$+&"6".,&"& M/ region using protection DNase I footprinting and 

electrophoretic mobility shift assays (EM.A), besides comparing Pb!V]I and PbXb` 

transcriptional regulation under heat-shocB stress and during phase transition. 

Results and conclusions: ae identified a total of [ protected fragments in the 1/* 

8$+&"6".,&"& M/ region of fungal isolates Pb18 and PbJ (versus their respective protein 

extracts). They contain putative transcriptional elements, namely one AP-1 (activator 

protein-1) to the direction of PbXb`, one GATA and J H.Es (heat shocB elements), 

being 2 of the non-conventional type. The M/ region of PbJ was polymorphic and 

presented J nucleotide substitutions, 1 insertion of 2 nucleotides and J neighboring 

gaps, but neither was seen in the regions of mapped transcription elements. 

fligonucleotides containing the putative AP-1, which is related to oxidative response, 

and H.Es were positive by EM.A, but the shifted bands were invariably faster with 

PbJ extracts. Real time reverse-transcriptase (RT)-PCR studies showed that in Pb18 

the level of up-regulation was similar in both !V]I and Xb` (2.[- to J.[-fold) upon 

heat shocB of the yeast phase cells at O2 o C for J0 and 60 min. These rates were 

neverthless lower than those of other heat shocB genes, specifically A=1CQ,*A=1DM 

and A=1IQE, whose mRNA levels were up-regulated 6.O to 8.K-fold after 60 min at 

O2 o C. During the mycelium-to-yeast-to-mycelium transition, we observed distinct 

patterns of Pb!V]I and PbXb` expression, suggesting the occurrence of a complex 

transcriptional regulation of these genes. This is the first study of transcription 

elements in 1/*8$+&"6".,&"&. fur results open the doors to a better understanding of the 

regulation of genes that respond to stress and that could be implicated in the 

pathogenicity of dimorphic fungi. 

.upported by Fapesp and CNPq 

P-0631. Molecular markers of dermatophytes in dermatophytosis 

Berdicevsky I 1 , laufman G 1 , Horwitz 2 2 

1 Technion Faculty of Medicine, 2 Technion Faculty of 2iology 

Dermatophytes are adapted to infect Beratinized tissues by their ability to utilize 

Beratin as a nutrient source. Although there are numerous reports that dermatophytes 

liBe >$"#?%'?2-%,*&'. secrete proteolytic enzymes, nothing is Bnown about patterns of 

gene expression on the host, nor even when cultured on protein substrates in the 

absence of a host. Enzymatic degradation may aid in invasion of the surface layer and, 

rarely, in immunocompromised patients, of the deeper layers of the sBin. ae have 

characterized the expression of an aminopeptidase gene of >/*).,-+@$%'?2-.&, Tri m 

O, which is a homolog of >$"#?%'?2-%,*$(8$() Tri r O. The >/*$(8$() gene was 

originally isolated based on the ability of the protein to induce immediate and delayed 

type hypersensitivity in sBin tests. Tri m O is closely related to Tri r O (almost KO_ 

identity at the protein level). 

Tri m O resembles other protease-encoding genes thought to be virulence factors, for 

example DPP V of 4&'.$@"66(&*U()"@+-(&. The Tri m O protein was detected 

immunochemically both in fungal extracts and in the culture medium. Expression of 

the Tri m O gene was induced several fold when >/*).,-+@$%'?2-.& was grown in vitro 

on Beratin and elastin (2) . Methods to investigate the molecular basis of pathogenicity 

are only starting to be developed and recently, a novel ex vivo model (1) for the study 

of adherence to the sBin and invasion into the stratum corneum was developed in our 

laboratory. A modest induction was triggered by incubation of >/*).,-+@$%'?2-.& on 

homogenized human sBin samples but a strong induction was observed after culture 

on blood plasma. In order to identify additional genes encoding putative virulence 

factors, differential cDNA screening was performed (2) . 2y this method, a fungal 

thioredoxin and a cellulase were identified and both genes were found to be strongly 

induced by sBin extracellular matrix proteins. Induction by superficial (Beratin) and 

deep (elastin) sBin components suggests that the products of these genes may be 

important in both stratum corneum and deeper layers of the sBin, and a model for their 

function is proposed. 6pregulation of several newly identified >/*).,-+@$%'?2-.& 

genes when the fungus is grown on Beratin suggests that these genes encode 

proteins relevant to the dermatophyte-sBin interaction. These genes include heat 

shocB protein 60 (H.P60) and an aminotransferase (2) . 

Dermatophytes, which are well adapted infectious agents, seem to use their 

mechanical and biochemical capabilities to invade the sBin tissue effectively. 

References: 

1. DueB /, laufman G., 6llmann Y, and 2erdicevsBy I. The pathogenesis of 

dermatophyte infections in human sBin sections. (ournal of Infection, O8, 

17[-180, 200O. 

laufman G, 2erdicevsBy I, aoodfolB ( and Horwitz. 2. MarBers for host induced 

gene expression in >$"#?%'?2-%,*dermatophytosis. Inf. Imm., 7J, 6[8O-6[K0, 200[.

P-0632. Putative structure and characteristics of a red water soluble pigment 

secreted by Penicillium marneffei 

2hardwaj . 1 , .huBla A 2 , MuBherjee . 2 , .harma . 2 , Guptasarma P 2 , ChaBrabarti A J , 

Chakrabarti A 1 

1 PGIMER, Chandigarh, India, 2 IMTECH, Chandigarh, India, J NIPER, Mohali, Punjab, 

India 

Interestingly, during growth at temperature below J0 0 C, the dimorphic fungus, 

1.,"#"66"()*)+$,.UU.", produces and secretes a pigment of bricB-red color that diffuses 

readily into the growth medium. Given the importance of this species as pathogen and 

uniqueness of the bricB-red pigment, we planned to characterize the pigment. Postgrowth 

shaBing of finely minced growth medium containing the pigment with warm 

water ([0 0 C) resulted in the facile extraction of this pigment into water. The redcolored 

water was easily dried into a red powder, and re-dissolved in aqueous 

medium. It was purified by reverse-phase liquid chromatography for examination of 

characteristics and structural studies of the pigment. The pigment showed a buffering 

ability in aqueous solutions, maintaining an alBaline pH at 8.0. It behaved as a 

colorimetric indicator of pH, changing color over a wide range of acidic and alBaline 

pH, with discoloration occurring ostensibly through hydrolysis of Bey chemical groups 

at extremely acidic pH ({2.0). The pigment]s 6V-visible absorption, fluorescence, FT- 

IR and NMR spectra were analyzed together with reverse-phase chromatographic 

HP/C purification profile, data on elemental composition (CHN.) analysis, and atomic 

absorption and mass spectrometric analysis (including M.-M. analysis). 2ased on 

these studies, the pigment was found to be a structural homologue of the coppercolored 

diffusible pigment (herquinone) produced by 1.,"#"66"()*?.$e(."/ The 

difference between herquinone, and the pigment produced by 1/*)+$,.UU." appeared 

to lie in a few Bey chemical groups, and in the latter]s dimerization through a sulphursulphur 

(disulfide) bond. 

P-0633. Phenotypic effects of YPS3 silencing in Histoplasma capsulatum 

Bohse M, aoods ( 

6niversity of aisconsin-Madison 

A"&-%'6+&)+*#+'&(6+-() is a pathogenic fungus with worldwide distribution. It is the 

causative agent of histoplasmosis, one of the most common fungal respiratory 

infections in the world, with an estimated [00,000 cases in the 6nited .tates alone per 

year. The k1=:*gene of A"&-%'6+&)+*#+'&(6+-() encodes a protein that is both 

surface localized in the cell wall of A/*#+'&(6+-() and also released into culture 

medium. ae have shown one mechanism for surface localization of YpsJp is binding 

to the cell wall polysaccharide chitin. This protein is produced only during the 

pathogenic yeast phase of infection and is also expressed differentially in A/* 

#+'&(6+-()*strains of differing virulence. The predicted YpsJp sequence shows 

homology with the carboxyl terminal domain of the Z6+&-%)2#.&*5.$)+-"-"5"& adhesin 

and virulence factor*2ad1p and with epidermal growth factor (EGF)-liBe domains 

described across a broad phylogenetic spectrum. In this study we silenced k1=: 

transcript using an RNAi strategy, and examined the silenced mutants for phenotypic 

differences ",*0"-$% and during infection. As has been observed previously for 

A"&-%'6+&)+3 as well as in other fungal systems, we noted variability in the degree of 

silencing achieved. Three day liquid cultures of 72 transformants were screened and 

assayed via aestern blot for secreted protein. ff these 72, 6 mutants showed a 

marBed decrease in secreted YpsJp, ranging from a several-fold decrease to no 

detectable secreted YpsJp. ae selected J mutants for further analysis. The mutants 

show no growth defect during HMM growth at J7 C ",*0"-$%3*however preliminary 

results suggest that the mutants cause a significantly decreased fungal burden during 

infection of C[72//6 mice. Results of fluormetric internalization assays and further 

analysis of ",*0"0% as well as additional ",*0"-$%*studies will be presented. 


P-0634. Calcineurin controls temperature-induced dimorphism and growth of 

Paracoccidioides brasiliensis: role on mitochondrial function 

Campos C, Nábrega M, Nábrega F 

Vale do Paraiba 6niversity, .Éo (osd dos Campos, .Éo Paulo, 2razil 

1+$+#%##"5"%"5.&*8$+&"6".,&"& is a dimorphic fungus that causes 

paracoccidioidomycosis, a systemic mycosis prevalent in /atin America with most 

cases found in 2razil. The dimorphic transition from mycelium to yeast is triggered by 

temperature rise from 2[ o C to J7 o C and both virulence and pathogenicity factors 

necessary for human infection are expressed during this phase. Despite its 

importance, very little is Bnown about the intracellular mechanisms that control 

dimorphism of 1/*8$+&"6".,&"& and the stress response that underlies the transition 

phase. In this worB, we investigated whether calcineurin, a Ca 2r /calmodulin-activated 

serine/threonine phosphatase, is part of the intracellular mechanism that control 

morphology, proliferation and viability of 1/*8$+&"6".,&"& under environmental 

temperature (2[ o C), mammalian host temperature (J7 o C), and during the transition 

phase (2[ o C to J7 o C). Two calcineurin inhibitors, cyclosporin A and Fl[06, which act 

through the binding at the immunophilins protein family, cyclophilins and Fl2Ps, 

respectively, prevented dimorphism of two*1/*8$+&"6".,&"&*isolates (Pb18 and Pb JJK). 

Cyclosporin A (CsA) was, however, more effective than Fl[06. A search at the 1/* 

8$+&"6".,&"& E.T databanB revealed that mRNAs for the cyclosporin A targets 

(cyclophilins) are at least O times more abundant than the mRNAs for Fl[06 targets 

(Fl2Ps), which may explain why cyclosporin A was more effective to blocB 

dimorphism. Cyclosporin A also blocBed growth of 1/*8$+&"6".,&"&*during the transition 

phase and growth of yeast cells, but did not affect growth of mycelium. 1/*8$+&"6".,&"&* 

cultured with*CsA remain metabolically active, suggesting that calcineurin did not 

control the viability of the fungus under the different conditions studied. ae also show 

that calcineurin is activated immediately after incubation of mycelium at J7 o C and that 

calcineurin increases oxidative phosphorylation early in the transition phase. In 

conclusion, early activation of calcineurin is required for the temperature-induced 

dimorphism from mycelium to yeast of 1+$+#%##"5"%"5.&*8$+&"6".,&"&, for growth during 

the transition phase and the growth of yeast cells. 6nder our culture conditions, 

growth of mycelium and viability of the fungus occurred independently on calcineurin. 

ae propose a role for calcineurin in the fine-tuning of energy production during the 

dimorphism as part of the stress response triggered by the temperature rise. 

.upported by Fapesp, CNPq. 

P-0635. Cell surface hydrophobicity (CSH) in Malassezia pachydermatis and 

Malassezia furfur 

Cannizzo F 1 , Vidotto V 2 , Villar M J , ~uindos G J , 2ollo E 1 

1 Dipartimento di Patologia Animale, 6niversitÖ degli .tudi di Torino, 2 Dipartimento di 

Discipline Medico-Chirurgiche, 6niversitÖ degli .tudi di Torino, J Departamento de 

InmunologÜa, MicrobiologÜa y ParasitologÜa, 6niversidad del PaÜs Vasco 

Introduction: Cell surface hydrophobicity (C.H) may be an important virulence factor as 

hydrophobic interactions play a major role in adhesion to human, animal and biomedical 

surfaces. Attachment to these surfaces is an initial event in the pathogenesis of most 

fungal infections. Hydrophobic interaction can play an important role in adherence to host 

cells by facilitating contact between the parasite and host cell. Adherence to liquid 

hydrocarbons, especially n-hexadecane, has been described as an indirect method to 

assess microbial cell surface hydrophobicity. Many studies have focused on the 

hydrophobicity of 7+,5"5+ spp., while little is Bnown on the hydrophobicity of !+6+&&.d"+ 

spp. 

Purpose of the study: To compare hexadecane assay and microsphere-binding assays to 

evaluate C.H of !/*'+#?25.$)+-"& and*!/*U($U($. 

Methods: 18 strains of !/*'+#?25.$)+-"& and 7 strains of !/*U($U($*were compared. Yeast 

cells were grown on .abouraud dextrose agar for O8h at J2ÅC before use in both assays. 

Hydrophobicity was evaluated according to a modification of the hexadecane assay 

described by Rosenberg .-*+l. (FEM. Microbiol. /ett. 1K80). 2riefly, 2 ml of yeast 

suspension ([0_ transmittance at [J0nm) was prepared in acid-washed tubes and 0.[ ml 

of hexadecane were added. Tubes were vortexed for 60s and turbidity was measured at 

660 nm. Relative C.H was calculated as transmittance rate (T 0 } initial transmittance / T f } 

final transmittance) according to the formula (Relative C.H _} 1-sT 0/T fu j 100). For the 

microsphere assay, cells were suspended to a concentration of 2 x 10 6 cells/ml. Aliquots 

(100 kl) of cells and microspheres (.igma-Aldrich, Germany) were combined in glass tubes 

and mixed for J0s. Microsphere attachment was assessed by optical microscopy at 600x 

magnification. The percentage of cells with three or more attached spheres was recorded 

as hydrophobicity percentage. 

Results: 2y means of hexadecane assay, 61_ of !/*'+#?25.$)+-"& and [7_ of !/*U($U($ 

strains revealed a high hydrophobicity (average 17.7_ and 21.1_, respectively). /ow or 

null hydrophobicity was observed in JK_ of !/ '+#?25.$)+-"& and OJ_ of M. furfur strains 

(average |[.K_ and 7.1_, respectively). 2y the contrary, in the microsphere assay, 8J_ of 

!/*'+#?25.$)+-"& and 71_ of !/*U($U($ strains revealed a scarce (up to 10_) or a null 

adhesion to microspheres. fnly 17_ of !/*'+#?25.$)+-"& and 2K_ of !/*U($U($ strains 

adhered to microspheres (average hydrophobicity of 21_ and J2.[_ respectively). 

Correlation between both tests was poor (r} 0.OOJ for !/*'+#?25.$)+-"& and r} 0.J70 for !/* 

U($U($). 

Conclusion: Hexadecane assay and microsphere-binding assays do not correlate on C.H 

evaluation for !. '+#?25.$)+-"& and !/*U($U($ isolates. The results obtained by these tests 

could manifest different properties related to hydrophobicity and adhesion of !+6+&&.d"+ to 

surfaces. Further studies are required to elucidate the mechanisms of adhesion of 

Malassezia to different surfaces and the role played by C.H in the virulence of the yeasts. * 

Acknowledgments: This study was in part supported by grant from the Fondo de 

Investigaciones .anitarias del Ministerio de .anidad (project PI0J0662/200J).

P-0636. Molecular typing of Trichosporon spp. strains from an Argentine 

hospital 

Carnovale ., Guelfand /, /orenzo (, lauffman ., Finquelievich J [ 

1 Centro de Micologia Facultad de Medicina. 62A, 2 Hospital de General de Agudos 

Ä(uan A FernandezÄ, J Centro de Micologia Facultad de Medicina. 62A, O Hospital de 

General de Agudos Ä(uan A FernandezÄ, [ Centro de Micologia Facultad de Medicina. 

62A 

>$"#?%&'%$%,*&'' is a common agent of fungal infections in immunocompromised host. 

The most important species isolated in deep mycosis is >*+&+?"". The infections are 

frequently fatal, despite therapy with amphotericin 2. Early diagnosis and treatment 

are important for the survivor of the patients. 

The aim of our study was identified clinical isolated as >*+&+?"". by PCR using speciespecific 

primers. 

Materials and Methods: A total of 12 strains study were identified using the API ID 

J2C (2iomerieux France). Ten were >*+&+?"" isolated of urine and two >*#(-+,.() of 

sBin. All de fungal isolation belongs from patient of the same hospital. 

The extraction of DNA was done using .cherer and .tevens]s techniques. The PCR 

was carried out using two specie-specific primers. 1M TAAF( f. [z- 

GGATCATTAGTGATTGCCTTTATA-J]) and 2M IT.O 

(r.[]-TCCTCCGCTTATTGATATG-J). 

PCR was performed in a [0 kl volume, the reaction was done in a thermal cycler (Mini 

Cycler, (. M. Research 6.A). Amplification conditions were as followsM initial 

denaturation at KO oC for J min, followed by J0 cycles of denaturation at KO oC for J0 s, 

anneling at [8oC for J0 s, and extension at 72 oC for 1[ s. A final elongation step was 

carried out at 72 oC for 10min. 

P-0637. Cryptococcus neoformans SRE1P functions in oxygen sensing 

pathway, ergosterol biosynthesis and virulence 

Chang Y 1 , 2ien C 2 , Espenshade P 2 , lwon-Chung ( 1 

1 National Institutes of Health, 2ethesda, 6.A, 2 Department of Cell 2iology, (ohns 

HopBins 6niversity .chool of Medicine, 2altimore, 6.A 

7$2'-%#%##(&*,.%U%$)+,& is an environmental pathogen that causes 

meningoencephalitis, primarily in immunocompromised hosts. .ince the oxygen 

levels in the mammalian brain are only 1-[_, which is considerably lower than the 

atmosphere (20_ f 2 level), it is important to understand how 7/*,.%U%$)+,& adapts 

to low oxygen conditions in the brain. In*=/*'%)8., it has been shown that both the 

sterol regulatory element binding protein (.RE2P) and its cleavage activating protein 

(.CAP) function in an oxygen-sensing pathway. They do so by monitoring oxygendependent 

sterol synthesis as an indirect measure of oxygen supply. Here, we 

deleted the genes =PaI and =71I in 7/*,.%U%$)+,& that encode the homologs of 

.RE2P and .CAP, respectively. As in other systems, sterol synthesis decreased 

upon exposure of 7/*,.%U%$)+,& to low oxygen (1_). Consequently, membranebound 

.re1p was proteolytically activated by hypoxic growth (1-J_ oxygen), and 

.re1p cleavage required .cp1p. Microarray and northern blot analysis comparing 

wild-type and &$.I cells suggested that under hypoxic conditions .re1p activates 

expression of many genes involved in ergosterol biosynthesis. Consistent with a role 

in regulation of sterol synthesis, deletion of =PaI resulted in hyper-susceptibility to 

several azole drugs. These data support the model that as sterol synthesis decreases 

under low oxygen conditions, .re1p is rapidly processed and stimulates transcription 

of oxygen-requiring enzymes in ergosterol biosynthesis to maintain sterol homeostasis. 

Experimental animal studies indicated that both =PaI and =71I are important for 

virulence. Thus, 7/*,.%U%$)+,& requires an oxygen-sensing pathway involving .re1p 

to adapt to low oxygen levels in host tissue during infection. 

The DNA products were analyzed by electrophoresis through a 1.[ _ agarose gel 

stain with ethidium bromide, and were visualized under 6V light. 

Results and conclusions: The amplification of the strains identified by conventional 

method as >*+&+?"" produced a fragment of approximately [00bp. DNA s of the other 

>$"#?%&'%$%,*&'' were not amplified by this detection system. 

The identification of medically important fungi is based on morphological and 

physiological characteristics and approximately is often difficult and time consuming. 

The therapies are only effective if the disease is detected at an early stage. Early 

diagnosis is an important step in the successful management of patient with 

disseminated trichosporonosis PCR offers important potential advantages, such a 

higher sensitivity, simplicity, speed and accuracy. 


P-0638. In vitro and in vivo study of fatty acid synthase genes of Cryptococcus 

neoformans: Roles in its survival and as a potential fungicidal targets 

Chayakulkeeree M 1,2 , Toffaletti D 1 , Rude T 1 , Perfect ( 1 

1 DuBe 6niversity Medical Center, Durham. NC, 2 Faculty of Medicine .iriraj Hospital, 

2angBoB, Thailand 

Purpose of the study: Fatty acid biosynthesis in yeast requires the fatty acid 

synthase complex which comprises two subunits formed as a heterohexamer (! 6 # 6 ). 

The ! and # subunits are encoded by ^4=M and*^4=I genes, respectively. In 

=+##?+$%)2#.&*#.$.0"&"+., U+&I and U+&M null mutants are auxotrophic for fatty acids. 

^4=M is required for invasive 7+,5"5+*+68"#+,& infection. The )@+M null mutant of 

7$2'-%#%##(&*,.%U%$)+,& (Cn) was shown to have repressed ^4=I expression and 

hypersensitivity to fluconazole. The objective of this study was to exam the roles of 

^4=I and ^4=M in Cn for its survival*",*0"-$% and ",*0"0%, as potential targets of 

antifungal drugs and synergistic effect of fatty acid synthase inhibitor to antifungal 

agents. 

Description: Cn ^4=I and ^4=M were connected to copper-regulated promoter 

(p7>PEWM). Growth curves of the transformants were demonstrated in condition with a 

copper chelator, bathocuproinedisulfonic acid (2C.), to express the gene or Cu.f O , 

to inhibit gene expression in condition with and without exogenous fatty acids. Gene 

expressions in those conditions were studied by real-time PCR. .ynergism to 

antifungal agents was studied by determining growth curve in condition containing 

2C. or Cu.f O with and without fluconazole or caspofungin. A fatty acid synthase 

inhibitor, cerulenin, was used to study the synergistic activity to fluconazole for the 

wild type Cn. K,*0"0% study of ^4=I and*^4=M was studied by murine model of 

cryptococcosis using wild-type, p7>PEWM/^4=I, and p7>PEWM/^4=M strains with and 

without treatment with a copper chelator. 

P-0639. Molecular cloning and structural and functional characterization of new 

hexose transporter from Cryptococcus neoformans 

Chikamori M 1 , Fukushima K 2 

1 NIR., (apan, 2 Research Center for Pathogenic Fungi and Microbial Toxicoses, 

Chiba 6niv., (apan 

The genomes of 7/*,.%U%$)+,& strains have been or are currently being sequenced. 

Virulence is a complex phenotype and the exact nature of virulence can be difficult to 

elucidate, nevertheless, a polysaccharide capsule, expression of the laccase CN/AC1, 

and secretion of a phospholipase as virulence factors have been well documented. In 

the search for gene(s) involved in resistance to copper ion toxicity in 7/*,.%U%$)+,&, 

we identified a new hexose transporter (Hxt) gene, Am>I. Hxt1 consists of [20 amino 

acids and functions to transport hexoses such as glucose. aith the use of the MfTIF 

program, Hxt1 was found to possess two sugar-transport motifs. 2y hydropathy 

analysis, 10 transmembrane domains were predicted. Each predicted transmembrane 

domain consisted of 22 amino acid residues, with the exception of domains II and VII, 

which consisted of 21 and 18 amino acid residues, respectively. Although Hxt1 

conferred copper resistance to =/*#.$.0"&"+., disruption of the Am>I gene showed that 

Hxt1 is not necessary for copper resistance. The growth rate of the ?_-I mutant strain 

was not different from that of the wild-type strain every 2 hr for 10 hr. In virulence tests, 

the ?_-I mutant strain showed 12_ less phenoloxidase activity than the wild-type 

strain, and no difference in the ability to form melanin was identified. In addition, the 

?_-I mutant strain showed virulence similar to that of the wild-type strain in 

experiments with 7+.,%$?+85"-"&*.6.@+,&. However, the ?_-I mutant strain generated 

larger capsules than those of the wild-type strain. Thus, Hxt1 appears to be involved 

in capsule formation. 

Results and conclusions: The wild-type Cn showed similar growth rate and its ^4=I 

and ^4=M transcripts in 2C. and Cu.f O condition. In 2C. condition, p7>PEWM/^4=I 

and p7>PEWM/^4=M*mutants showed similar growth rate as those of wild type. In 

condition containing Cu.f O , both strains were not able to grow even in the presence 

of exogenous fatty acids and p7>PEWM/^4=I and p7>PEWM/^4=M mutants had 0.00[ 

and 0.002 fold reduction of ^4=I and ^4=M transcripts compare to 2C. condition, 

respectively. 2oth 2C. and Cu.f O showed no significant impact on ^4=M and ^4=I 

transcripts of p7>PEWM/^4=I and p7>PEWM/^4=M mutants, respectively. Fluconazole 

showed an increase in fungicidal activity to fatty acid synthase inhibition*",*0"-$%. In 

vivo study showed that p7>PEWM/^4=M is tighter-regulated in conditions with high and 

low copper in murine model and ^4=M expression can be altered and have significant 

impact on yeasts survival in the lungs. 2oth mutants showed poor survival in the brain 

when compare to wild-type. ae conclude that ^4=I and ^4=M in Cn are essential for 

growth in conditions with and without exogenous fatty acids. These findings are 

different from those found in other yeasts and suggest that ^4=I and ^4=M can 

potentially be fungicidal targets for Cn and synergistic with antifungal effect of 

fluconazole.

P-0640. Ribosomal DNA nontranscribed spacer of dermatophytes 

Choi J, .hin Y, Moon ., ParB ., lwaB T, .hin D, lim l 

Yeungnam 6niversity Medical Center, Daegu, lorea 

Purpose of the study: rDNA nontranscribed spacer(NT.) is so hypervariable that it 

can be used for differentiation of inter and intraspecies. There was no report about the 

sequences of dermatophytes except >$"#?%'?2-%,G>/J*$(8$() and >/*-%,&($+,&. 

The purpose was to identify the sequences of the rDNA NT. of several 

dermatophytes and comparing them for phylogenetic analysis. 

Description: Primers were designed from 18. and 2[. of >/*$(8$()*and 4&'.$@"66(&* 

,"5(6+,&/* PCR and sequence analysis was done with DNA of several species of 

dermatophytes. 

Results and conclusion: 1. The NT. of 4$-?$%5.$)+G4/J*0+,8$.(&.@?.)"", >/* 

).,-+@$%'?2-.&3*>/*&#?%.,6.",""*and !"#$%&'%$()G!J/*@2'&.() were amplified and 

sequenced. 2ut that of !/*#+,"&*and*a'"5.$)%'?2-%,*U6%##%&() could not be 

amplified, and that of 4/*8.,?+)"+. showed multiple bands in PCR. 2.*NT. of 4/* 

0+,8$.(&.@?.)"" was J,2K2 bp sized, and had tandemly repetitive subelements(TR.). 

TR.-1 had two and half of subrepeat units, O80bp sized. TR.-2 had [ of subrepeat 

unit, 87 bp sized. 

J. NT. of !/*@2'&.() was 2,2J6 bp sized, and had no TR.. O.*There were high 

similarity of the sequences among 4/*0+,8$.(&.@?.)"", >/*&#?%.,6.",""*and >/* 

-%,&($+,&/*[. Although human isolates of >/*).,-+@$%'?2-.& showed different colony 

morphology, the sequences of NT. were almost identical. There were K8_ similarity 

with 4/*0+,8$.(&.@?.)""/*6. The sequences of NT. were more variable than that of 

IT. were. And they could be used for phylogenetic analysis and designing the 

species-specific primers among very close species. 

P-0641. The interaction of Cryptococcus neoformans with Acanthamoeba 

castellanii results in capsular enlargement 

Chrisman C, Casadevall A 

Department of Microbiology c Immunology, Albert Einstein College of Medicine, 2ronx, 

New YorB, 6nited .tates of America 

An encapsulated soil fungus capable of causing meningitis in immunocompromised 

patients, 7$2'-%#%##(&*,.%U%$)+,&*(7/*,.%U%$)+,&), has many unique virulence 

strategies. fne of the main virulence factors is the polysaccharide capsule. A 

remarBable property of 7/*,.%U%$)+,& cells is their ability to increase size of the 

capsule during infection. This capsular enlargement is thought to help evade 

phagocytosis by other cells such as macrophages. Additionally, 7/*,.%U%$)+,&*is 

Bnown to form vesicles containing capsular polysaccharide in the host cell. ahile 7/* 

,.%U%$)+,& is a human pathogen, it does not require an animal host for replication, 

thus leading to the question of how this novel mechanism of avoiding macrophage 

Billing evolved. fur group has proposed that the origin of virulence for mammals 

arises from interactions in the soil including those with free-living soil amoebae. fne 

amoebae, 4#+,-?+)%.8+*#+&-.66+,""*(4/*#+&-.66+,""), has been shown to ingest 7/* 

,.%U%$)+,&. 7/*,.%U%$)+,& is then able to replicate and Bill the host. This study 

evaluated the capsular response of 7/*,.%U%$)+,& to co-incubation with amoebae. 

ae evaluated the interaction of 11 7/*,.%U%$)+,& strains with 4/*#+&-.66+,"". 

Interestingly, we noted that [[_ of the strains exhibited significant increases in 

capsule volume after co-incubation with 4/*#+&-.66+,"". The enlargement in capsule 

volume ranged up to 68_ of the pre-incubation volume. Common experimental strains, 

HKK and 2O067, were among those that demonstrate the capsular enlargement upon 

co-incubation. These experiments clearly show that this effect of capsular 

enlargement is not limited to mammalian infection. 6pon further investigation, it 

became clear that capsular enlargement occurs in the presence of either dead or live 

4/*#+&-.66+,"" cells as well as live and dead (77O macrophages. However, the effect 

was not elicited by incubation in amoebae-conditioned media or by mechanical 

stimulus. Extractions of the lipids of A. castellanii have given insight into this 

mechanism. It is possible to extract the lipids and separate the polar and nonpolar 

fractions. ahen introduced alone, the polar lipid fraction mimics the capsular 

enlargement of intact amoebae. Preliminary results indicate that it is in fact the 

phospholipid, phosphatidylinositol, which the 7/*,.%U%$)+,& is sensing and to which it 

is responding. Given that this is a phospholipid found both in macrophages and in 

amoebae, this would correlate with our studies. In summary, our results show that 

capsular enlargement can be triggered by contact with amoebae and linB another 

classical feature of this pathogenic fungus with a potential stimulus in the environment. 

Hence, this feature of 7/*,.%U%$)+,& may have originally been selected for through 

interactions with amoebae in the environment. 


P-0642. Antizyme as regulator of ornithine decarboxylase (ODC) in 


Cordero M, Nio-Vega G, San-Blas G 

Venezuelan Institute for .cientific Research 

In 1/*8$+&"6".,&"&, growth of the pathogenic yeastliBe (Y) phase and transition from the 

mycelial (M) to the Y phase are accompanied by an increase in polyamines levels and 

fDC activity (1, 2). Northern analysis of the 18$bV7 gene does not correlate with 

such increment in activity, showing a constant expression during Y growth and 

through transition either way, pointing to a post-translational regulation of fDC (J). In 

some fungi and in mammalian cells, a feedbacB system post-translationally regulates 

antizyme activity (O). In it, low levels of fDC (and polyamines) give place to an 

inactive antizyme by a frameshifting of its transcript. ahen the levels of fDC (and 

polyamines) rise, the antizyme transcript is made in frame, giving as a result an active 

antizyme protein, that leads to the degradation of fDC protein. ahen the levels of 

fDC (and polyamines) decrease, the antizyme transcript suffers a frame shift, thereby 

inactivating the antizyme and restarting the cycle, through a self-regulatory 

mechanism. 

.outhern analysis on 1/*8$+&"6".,&"& DNA was done using as radiolabeled probe a 

PCR fragment of JJ0 bp of the 1/*8$+&"6".,&"& antizyme gene (18$4dbV7). From the 

.outhern analysis, a A",dIIII genomic library was constructed, that was screened by 

colony hybridization, in order to get the whole gene sequence. The polyadenylation 

site has been identified by the JïRACE system (Invitrogen, CA), using as specific 

primers, sequences derived from the PCR product. Expression of the gene was 

followed by Northern analyses in the Y and M phases, as well as during the dimorphic 

switch from M to Y. As loading control, a fragment of the 18$rRNA 18. gene was used. 

From .outhern analysis, a single 2.K lb signal was detected for DNA restricted with 

A",dIII, and this enzyme was chosen for the construction of a genomic library. 

Northern analysis showed a higher expression of 18$4dbV7 in the Y pathogenic 

phase when compared with the M phase, and a rise in signal intensity for samples into 

the dimorphic transition from M to Y. 

2oth the higher expression of the 18$4dbV7*gene in the pathogenic Y phase than in 

the saprophytic M phase, and the rise in expression when an M culture changes into a 

Y culture, correlates with the higher fDC activity found when 1/*8$+&"6".,&"&*presents 

the Y morphology. The presence of an antizyme-liBe protein together with the above 

results, allow us to postulate that, liBe in some fungi*and in mammalian cells, 

regulation of fDC in 1/*8$+&"6".,&"& might be antizyme-controlled. 

P-0643. Genotying of Malassezia species associated to Pityriasis versicolor 

and Seborrheic dermatitis 

Corneta E 1 , Pereira Chioccola V 2 , leiBo / J , .ouza V O , Pires C [ , .apieri A [ , .ouza 

Carvalho Melhem M 2 , Gambale a 1 

1 6niversidade de .ao Paulo -Instituto de Ciencias 2iomddicas - .ao Paulo, .P -2razil, 

2 Instituto Adolfo /utz - .ao Paulo, .P - 2razil, J Instituto de Infectologia Emilio Ribas - 

.Éo Paulo, .P - 2razil, O .anta Casa de Misericordia - .ao Paulo, .P - 2razil, 

[ Complexo Hospitalar Padre 2ento de Guarulhos - .Éo Paulo, .P - 2razil 

!+6+&&.d"+ is part of sBin flora and, also is associated with pityriasis versicolor, 

seborrheic dermatitis, folliculitis, atopic dermatitis, and some systemic infections. 

.everal studies in emerging yeast infections have demonstrate the importance of 

!+6+&&.d"+ as opportunistic infection. The !+6+&&.d"+ species are identified by 

morphological and biochemical procedures, but they are unable to distinguish newly 

species. The molecular methods had been efficient to determine species. The aim of 

this worB was to genotype !+6+&&.d"+*species in clinical samples by Pulsed Field Gel 

Eletrophoresis (PFGE) and a system based on PCR and restriction endonuclease 

analysis (PCR-RF/P). The genotyping were compared to the clinical data and to 

those obtained by conventional methods for !+6+&&.d"+*characterization as direct 

microscopic observations and physiological methods. ae analyzed samples from O1 

patients with dermatological involvement (O with seborrheic dermatitis and J7 with 

pityriasis versicolor). For PFGE, the 2iorad CHEF DR-III, was used with an initial 

pulse time of (20-O00s) for 20h, followed by a ramping pulse time (O00-O00s) for 

another J0h. For PCR-RF/P, the reactions were carried-out using a primers pair that 

amplified a [80-bp from a conserved region of 26. rDNA gene of !+6+&&.d"+. The 

species were determined the [80 bp amplified product were digested with A?+I. Four 

species were determined by PFGE and PCR-RF/PM OOj*of the samples*were 

genotyped as !/*&2)'%5"+6"&3*27_ were !/*U($U($3*1K _ were*!/@6%8%&+3*and 10 _ 

were !/*&6%%UU"+./ The specie identification was similar in 86_ by genotyping and 

physiological methods for identification. !/*&2)'%5"+6"& was present in JK,[_ of the 

samples analyzed, being an important causative agent of*pityriasis versicolor and 

seborrheic dermatitis in our country. These findings suggest the clinical utility of PFGE 

and PCR-RF/P in the diagnosis and identification of mycosis caused by !+6+&&.d"+ 

species. 

References: 

.an-2las .-*+6. 1KK6. Arch. Microbiol. 16[M J11-J16. 

.an-2las .-*+6. 1KK7. Arch. Microbiol. 166M O11-O1J. 

Nio-Vega .-*+6. 200O. Yeast 21M 211-218. 

Sorais et al. 2003. Rev. Iberoamer. Micol. 20: 1-5.

P-0644. Phenotypic and genotypic characterization of Trichosporon spp. 

isolated from different sources 

de Resende M 1 , /emes R 1 , Colombo A 2 , Cury V 1 , Arantes R 1 


.Éo Paulo, 2razil 

The aim of this study was to appraise the use of phenotyping method in the 

identification of the species of the genus >$"#?%&'%$%, in comparison with the 

molecular technique RAPD. .ixty-five samples of yeasts isolated from different 

sources were analysed and identified by morphological and biochemical profiles and 

JK were identified as belonging to the genus >$"#?%&'%$%,. Five strains were excluded 

of the genus >$"#?%&'%$%, by genotyping, through the method of RAPD (Randomly 

Amplified Polymorphic DNA) by the oligonucleotide use, capable of differentiate the 

main species of the genus by the characterisation of the area of IT. (Intergenic 

.pace). The primer fPA2 made possible the identification of four species (>/*+&+?""3*>/* 

+&-.$%"5.&3*>/*%0%"5.&*and >/*)(#%"5.&). The RAPD overcame the classic method in 

the identification of species of >$"#?%&'%$%,, necessary identification to aid to address 

to the treatment of the trichosporonosis, due to the variability of the susceptibility 

profile to the antifungals among the species, independent of the isolation site. 

P-0645. Colony PCR is a rapid and reliable method for amplification of rdna in 

the yeasts 

Diba k 1 , Mirhendi H 2 , Namaki A J , (alalizand N O 

1 Public Health, 2 Public Health, J Harsin Hopital, O Public Health 

fver the past two decades, many non-Candida albicans yeast species emerged as 

significant pathogens of clinical importance. The laboratory identification of yeast 

species is therefore essential for establishing the definitive diagnosis infection. 

Molecular technology show great potential for the rapid detection and identification of 

fungi , for medical purposes. The first step in performing any DNA- based molecular is 

DNA extraction and which generally are time-consuming and labor- intensive. 

In this study we evaluate the efficacy of Colony C PCR (using the colony directly as 

the template) for amplification of target DNA. 

fne hundred clinical samples were cultured on glucose peptone agar. A small amount 

of each colony (about 1mmn) was directly transferred to the tube containing PCR 

gradients. PCR amplification was carried out in a final volume of [0 ±l, contained, 

0.2±M of each IT.-1 and IT.-O universal primers, 0.1mM dNTPs, [ ±l of 10j PCR 

buffer and 2.[6 of Taq polymerase. The PCR reaction steps were initial denaturation 

at KOß C for J min, followed by J0 cycles of denaturation at KOß C for J0 s, annealing 

at [6ß C for O[s and extension at 72ß C for 1 min, with a final extension step of 72ß 

C for 7 min. Amplified products were visualized by 1.[_ Agarose gel electrophoresis 

and stained with ethidium bromide. DNA of all isolates was extracted by using already 

described glass- beads method and PCR was prepared as described above. 

As a result the IT. region of K8 yeast isolates successfully were amplified after Colony 

PCR which compared with 100 PCR amplifications after glass-beads DNA extraction. 

The gel electrophoresis photos were accessed by gel documentation system showed 

sharp bands for all of isolates except four after amplification followed by extraction 

processing which were weaB for both methods.In the Colony PCR method, total of K8 

accessed bands,7[ were sharp compared with 2J weaB bands. The gel of last bands 

were purified and re-amplified by double PCR. 

fur results show that the Colony-PCR method can be a simple, reliable and cost 

Ceffective method for rapid amplification of target DNA in the yeasts. The method 

could be a suitable for diagnostic and epidemiological proposes. 


P-0646. Identification of Aspergillus fumigatus isolated from corn silages using 

the polymerase chain reaction 

Dos Santos V 1 , Ven°ncio A, Matos ( 

1 /aboratário Regional de VeterinZria, AÑores, Angra do HeroÜsmo, Portugal, 2 Centro 

de Engenharia 2iolágica, 6niversidade do Minho, J Departamento de Ciüncias 

agrZrias, 6niversidade AÑores, Angra do HeroÜsmo, Portugal 

Farmers and farm worBers are often exposed to airborne dust, especially when 

worBing with plant and animal materials. Filamentous fungi can be important 

components, because they occur naturally in commodities as manure, silage, and 

compost and can colonize other farm materials when conditions are favourable for 

growth, e.g., during storage of moist grain, hay and straw. 

Exposure to 4&'.$@"66(&*U()"@+-(&*is linBed with respiratory diseases such as asthma, 

invasive aspergillosis, hypersensitivity pneumonitis, and allergic bronchopulmonary 

aspergillosis. Molecular methods using PCR offer advantages over culture and optical 

methods for estimating animal and human exposures to microbiological agents such 

as fungi, for monitoring the production process and for the identification of feed 

spoilers. 

This paper describes an assay for identification of 4/*U()"@+-(& from pure culture 

isolated from corn silage, using liquid nitrogen to digest 4/*U()"@+-(& conidia followed 

by amplification by primers described specially for the specie by amplification of a J8[ 

bp fragment. The method was optimized and there was 100_ correlation between the 

culture results and the PCR results. 

P-0647. Secreted aspartic proteinases of Candida parapsilosis: The differences 

in activation mechanisms of Sapp1p and Sapp2p in vitro 

Dostál J, MerBerovZ M, HradÜleB M, PichovZ I, HruBovZ-HeidingsfeldovZ f 

Institute of frganic Chemistry and 2iochemistry, Flemingovo n.2, Prague 6, Czech 

Republic 

The pathogenic 7+,5"5+*&''/ produce extracellular aspartic proteinases (.aps), which 

are considered as one of the virulence determinants. 7+,5"5+*'+$+'&"6%&"&3 the 

second most frequent 7+,5"5+*species in clinical practice*possesses at least three 

genes encoding secreted aspartic proteinasesM =411I, =411M and =411:. The 

.aps are synthesized as zymogens consisting of a leader peptide, propart and mature 

proteinase domain. The proparts of .aps usually contain /ys-Arg processing site, 

which is the target for an activation enzyme or for an alternative activation processes. 

The conversion of .app1p and .app2p zymogens have not been studied and the 

mechanism of theirs activation is unclear. ae have prepared recombinant precursors 

of .app1p and .app2p in order to examine their activation pathway. In acidic 

conditions, the precursor .app1p was autocatalytically processed yielding an active 

proteinase. The self-activation proceeded through an intermediate and the resulting 

enzyme was one amino acid shorter than the authentic one. The precursor of .app2p 

was autocatalytically processed too but the resulting enzyme was extended by 8 

amino acids at the N-terminus and the activity of this enzyme was reduced in 

comparison with mature .app2p. At the same time we have prepared crude 

membrane fraction from 7+,5"5+*'+$+'&"6%&"& which contains a lex2-liBe proteinase. 

2oth precursors were processed by a lex2-liBe proteinase and resulting enzymes had 

the same N-termini as the mature .app1p and .app2p. Furthermore, we observed 

differences in the .app1p and .app2p substrate specificities. fn the basis of this 

observation we designed specific fluorescent substrate enabling to distinguish 

between .app1p and .app2p activity in culture media. 

To sum upM the differences in activation mechanisms and enzymological properties of 

.app1p and .app2p may reflect distinct roles that these isoenzymes probably play in 

the 7/*'+$+'&"6%&"& response to diverse environmental conditions. ae hypothesize that 

7/*'+$+'&"6%&"& has evolved two pathways for .app1p activationM auto-activation and 

assisted activation. 

This worB was supported by the Czech .cience Foundation (grant No. 20J/0[/00J8) 

and by Ministry of Education of the Czech Republic (grant No. /C[J1).

P-0648. Methionine-induced morphogenesis in Candida albicans is dependent 

on the methionine permease MUP1 and the G-protein coupled receptor GPR1. 

Eskandarian M, Maidan M, Van DijcB P 

latholieBe 6niversiteit /euven, Heverlee, 2elgium 

The ability of the fungal opportunistic pathogen 7+,5"5+*+68"#+,& to switch between 

yeast, pseudohyphal, and hyphal forms is believed to be an important component of 

its virulence. Morphological changes of 7/*+68"#+,& can be triggered in vitro by a wide 

variety of factors, including specific carbohydrates or amino acids, salts, pH, 

temperature, starvation, serum and growth within a matrix. The signal transduction 

pathways that are activated by these factors have been studied extensively but the 

mechanisms of sensing for the different triggers are less understood compared to the 

situation in =/*#.$.0"&"+.. 

Amino acid rich media (e.g. /ee]s medium) are Bnown to induce the morphological 

transitions in 7/*+68"#+,&. However, the molecular bases for amino acid-mediated 

morphogenesis are still obscure. To find out which amino acid can trigger the hyphal 

formation, we tried out each amino acid separately in a medium containing low 

concentrations of glucose and high concentrations of ammonium. ae saw that only 

small amounts of mehionine (0,017 mM) are required to trigger the yeast to hyphal 

transition in the wild type. Next we examined the effect of other amino acids in 

combination with methionine on the hyphal transition. Alanine, histidine, isoleucine, 

leucine and valine inhibited the hyphal induction caused by methionine, while cysteine 

increased it extensively. In =/*#.$.0"&"+. there is a competition between the uptaBe of 

methionine and cysteine by Mup1. ae will investigate if this is also the case for Mup1, 

the only Bnown high affinity methionine permease in 7/ +68"#+,&. 

Recently we have described the role of the Gpr1 G-protein coupled receptor in 

morphogenesis of 7/*+68"#+,&. In the presence of methionine, we noticed a rapid 

internalisation of Gpr1 in the vesicles. Deletion of 91PI causes strong defects in true 

mycelium formation during growth of this yeast on medium with high ammonium 

contents. Hyphal formation in medium with a low ammonium and glucose 

concentration is not affected by the deletion of 91PI. ahen low ammonium is present 

Gap1 is activated for the uptaBe of other amino acids, liBe methionine. ae are 

currently maBing 941I deletions in different bacBgrounds to see if the methionine 

uptaBe and hyphal formation on low ammonium media is abolished. In addition we will 

determine whether Mup1 is required for the internalisation of Gpr1 after addition of 

methionine. ae will discuss possible mechanisms of methionine-induced 

morphogenesis in 7/*+68"#+,&. 

P-0649. Identification and characterization of genes involved in synthesis of 

glycosylphosphatidylinositols in Cryptococcus neoformans 

Fabre A 1 , .pecht C 2 , 2ouchara ( J , Taron C 1 

1 New England 2iolabs, Ipswich, MA, 6.A, 2 2oston 6niversity, .chool of Medicine, 

2oston, MA, 6.A, J /aboratoire de Parasitologie-Mycologie, CH6, Angers, France 

Glycosylphosphatiylinositols (GPIs) are glycolipids that are covalently attached to the 

carboxy-terminus of certain euBaryotic secretory proteins and are essential for cell viability. 

In addition to tethering a protein to the cell surface, GPIs have been implicated in important 

cellular processes including protein targeting, polarized growth and fungal cell wall 

formation. These processes have been best characterized for the yeast =+##?+$$%)2#.&* 

#.$.0"&"+.. For the human pathogenic fungi of the genus 7+,5"5+, various GPI anchored 

surfaced proteins are important for adhesion to human epithelial cells and pathogenesis (2. 

CormacB .-*+6., .cience (1KKK) 28[, [78-82n /. Hoyer .-*+6., Trends Microbiol. (2001) O, 

176-80n .. (. Grimme .-*+6., Microbiology (200O) 1[0, J11[-J128n M. Richard .-*+6., Mol. 

Microbiology (2002) OO(J), 8O1-8[J). In 7$2'-%#%##(&*,.%U%$)+,&, an opportunistic fungus 

responsible for life threatening infections in immunocompromized humans, a major 

virulence factor (phospholipase 2) is GPI-anchored suggesting that GPIs may also play an 

important role in the pathogenicity of this organism ((. Djordjevic .-*+6., 2iochem. (. (200[) 

J8K, 80J-812). 

All GPI anchors share a common core structure of protein-PEthN-6Man $1,2 Man $1,6 

Man $1,O GlcN4$1,6 myo inositol-Pf O |lipid (where PEthN is phosphoethanolamine, Man is 

mannose and GlcN is glucosamine). The first and second mannoses may be further 

modified with side-branching phosphoethanolamine residues in both =/*#.$.0"&"+. and 

mammals. Additionally, all =/*#.$.0"&"+. and some mammalian GPIs have a fourth 

mannose side-branched to Man J . GPI precursors are synthesized and transferred to 

proteins in the endoplasmic reticulum. In =/*#.$.0"&"+. and mammals, over 20 highly 

conserved proteins involved in GPI precursor synthesis and GPI transfer to protein have 

been identified and characterized. However, little is Bnown about GPI biosynthesis in other 

non-=+##?+$%)2#.& fungi liBe the basidiomycete 7/*,.%U%$)+,&. 

In the present study, we have begun to investigate how GPIs are assembled in 7/* 

,.%U%$)+,&. Insight into 7/*,.%U%$)+,& GPI structure was gained by analyzing its genome 

for the presence or absence of GPI biosynthetic genes. ae identified homologues of the 

GPI mannosyltransferases that add Man 1 (Gpi1Op/PIG-M), Man 2 (Gpi18p/PIG-V) and Man J 

(Gpi10p/PIG-2) to =/*#.$.0"&"+. and human GPIs. Interestingly, no homologues of the 

Man O mannosyltransferase (.mpJp/PIG-Z) were found. Additionally, 7/*,.%U%$)+,& 

encodes only a single putative phosphoethanolamine transferase (Gpi1Jp/PIG-f) in 

contrast to the genomes of yeast and mammals that each encodes three. These findings 

suggest that 7/*,.%U%$)+,& may assemble Man J -GPIs bearing no phosphoethanolamine 

side-branches, a structure that would be significantly different to that found for mammals 

and other fungi. 

ae have cloned the genes encoding each putative mannosyltranferase and the Candidate 

phosphoethanolamine transferase and are testing their ability to complement essential =/* 

#.$.0"&"+. @'" null mutants. Additionally we are establishing a cell free system to address 

the specificity of various GPI synthetic enzymes in extracts of 7/**,.%U%$)+,& membranes. 


P-0650. Characterization genotypic by RAPD-PCR of Candida albicans clinical 

isolates forming biofilm 

Finquelievich J [ , Durand E 1 , Mujica M 2 , (ewtuchowicz V J , Iovannitti C O 

1 Fac de 2ioquimica, ~uimica y Farmacia. 6niv. de Tucuman. Argentina, 2 Centro de 

Micologia Facultad de Medicina 62A, J Centro de Micologia Facultad de Medicina 

62A, O Centro de Micologia Facultad de Medicina 62A, [ Centro de Micologia Facultad 

de Medicina 62A 

2iofilms are colonies of microbial cells encased in a self-produced polymeric matrix 

and represent a common mode of microbial growth. 7+,5"5+*+68"#+,& colonizes the 

surfaces of catheters, prostheses, and epithelia, forming biofilms that are highly 

resistant to antimicrobial drugs. RAPD (Random Amplified Polymorphic DNA analysis) 

is a useful tool for characterizing genetic variability intra-specific. The objective of this 

worB was the characterization genotypic of 7+,5"5+*+68"#+,& clinical isolates forming 

biofilm based on RAPD. 

Twenty five clinical isolates of 7+,5"5+*+68"#+,& were isolated from oral cavities, blood, 

sBin, nail, stool, oesophagus biopsy and vaginas of patients with candidiasis. For each 

strain, biofilm formation was studied as the ability to adhere to and grow in well of K6- 

wells microtitre plates (Ramage,G 2001 Antimicrob Agents and Chemother 2O7[- 

2O7K). ~uantitation of biofilm was performed by using a reduction study of 2,J-bis (2- 

methoxi-O-nitro-[-sulfophenil)-2H-tetrazolium-[-carboxanilideu (jjT, .igma) by 

mitochondrial dehydrogenases. The clinical isolates were compared by RAPD using 

primers fPA 02, fPA 0K, M1JR y M1JF. 6nweighted pair group method cluster 

analysis of binary data (6PGMA) was performed with software (2ionumerics program) 

(Applied Mathematics lortrijB, 2elgium) and a similarity dendrogram was constructed 

with a distance scale. 

The similarity coefficients varied from OK to K1_ revealing high polymorphism rate. 

The genetic distances among all 2[ isolates of 7/*+68"#+,&*showed high intra-specific 

genetic variability with the four primers used. The dendrogram clustered the isolates in 

four groups. All the groups included strains with very different ability to form biofilm. 

The clustering in the RAPD dendrogram did not shown correlation with the level 

biofilm-forming ability within the strains studied. .trains with similar genotypes often 

showed very different biofilm formation abilities. The isolates were grouped into 

clusters independently from sample isolation sources. 

fur results suggest that RAPD marBers produced did not discriminate the isolates of 

7/*+68"#+,& according to ability to biofilm formation and isolation sources. Additional 

studies are needed to determine whether genotypes play any role in biofilm involving 

multiple strains. 

This investigation was supported in part by projects 62ACYT M-087 and 62ACYT M- 

0JK from the 6niversidad de 2uenos Aires. 

P-0651. Evaluation of mycoflora and toxigenic analysis of Aspergillus flavus 

and Aspergillus parasiticus isolated from phytoterapeutic remedies (São Paulo, 

Brazil) 

Fischman Gompertz O 1 , Corrüa Nunes F 1 , Martinez Godoy P 1 , Correa 2 2 

1 6NIFE.P, .ao Paulo, 2razil, 2 6.P 

Phytotherapy is one of the most ancient practices used by humanity. 6ntil the middle 

of the jIj century, when chemotherapeutic drugs were introduced, formulation of 

medicines was usually based on medicinal plants. At the end of the jj century, plants 

returned to occupy an important place among medicines, being used in the form of 

powders, liquid extracts and teas which represent the most diffused form of use of 

phytotherapeutic remedies worldwide, being of easy access and preparation. Herbal 

medicinal products are used, not only for treatment of diseases but also to promote 

health. Very little information is available worldwide on mycoflora and mycotoxins of 4/* 

U6+0(&*and 4/*'+$+&"-"#(& isolated from phytotherapeutic remedies. The present study 

determined the mycoflora and the levels of aflatoxin of fungal cultures isolated from [0 

samples of phytoterapeutic remedies currently commercialized in the city of .Éo Paulo, 

2razilM 72,+$+*&#%62)(&, 1,.()(&*8%65(&, !+2-.,(&*"6"#"U%6"+, 7+&&"+*+,@(&-"U%6"+, 

P?+))(&*'($&?"+,+. Each sample was processed and plated separately in duplicate 

on .abouraud dextrose agar and potato dextrose agar. Isolates were identified 

according to their micro and macromorphological characteristics. Mycotoxins of 4/* 

U6+0(& and 4/*'+$+&"-"#(& isolates were evaluated. 4/*,"@.$ (J0_) was the most 

prevalent species, followed by 4/*U6+0(& (22_), 1.,"#"66"() spp (20_), !(#%$ spp (K_), 

4/*U()"@+-(& (6,[_), P?"d%'(& spp (6,[_), 46-.$,+$"+*spp (O_) and 4/*'+$+&"-"#(& 

(2_). The microbiological analysis showed that O2 (82_) of the [0 samples, 

presented a fungal growth over 100CF6/g and 1[ of them were toxigenic. Among the 

1J 4/*U6+0(& toxigenic isolates, 10 (77_) were able to simultaneously produce AF21 

and AF22 and three isolates were only AF21 producers. The two 4/*'+$+&"-"#(&* 

isolates produced AF21, AF22, AFG1 and AFG2. The production of AF21 by 12 of 

the 1J 4/*U6+0(& and by two 4/*'+$+&"-"#(&*toxigenic isolates was significantly above 

the levels recommended by the aHf and by the Ministry of Agriculture and .upply 

(2razil). It is important to emphasize that contamination of phytotherapeutic remedies 

may represent risB factor to the population that regularly consumes them, mainly 

children, the elderly and immunocompromised patients who are more susceptible and 

may present complications due to the deleterious effects that this type of product may 

cause. The contamination that was observed in most products (82_) justified the 

concern regarding the execution of more careful analyses of the phytotherapeutic 

remedies and the application of more rigorous laws that may warrant the quality of the 

products in order that they reach the consumer in appropriate conditions for their 

consumption. 

Part of the thesis of Nunes to get the degree of Master in Microbiology and 

Immunology, 6NIFE.P/EPM, 200J. Grants from FAPE.P (processM 00/1O08O-1)

P-0652. Dipeptidyl peptidase IV activity in the dimorphic fungal pathogen 

Histoplasma capsulatum 

Galbraith K, ZarnowsBi R, aoods ( 

The 6niversity of aisconsin - Madison 

A"&-%'6+&)+*#+'&(6+-() (Hc) is a thermally dimorphic ascomycete and the causative 

agent of histoplasmosis. Histoplasmosis is of particular concern to 

immunocompromised populations, often manifesting as a serious or fatal infection in 

AID. patients and individuals undergoing chemotherapy. Dipeptidyl peptidase IV 

(DppIV) enzymes cleave dipeptides from the N-terminus of larger peptides, 

preferentially after a proline residue at the penultimate position. These peptidases are 

widely distributedn the most well-characterized is also Bnown as CD26, an important 

mammalian immune system regulator. .everal peptides are functionally regulated by 

CD26, including neuropeptides, chemoBines and hormones. The Hc genomic 

sequence database was searched for homologs of 4&'.$@"66(&*U()"@+-(& DppIV 

protein. 2ased on sequence homology, we have identified two putative DppIV genes 

encoded in the Hc genome, V11KO4 and V11KOZ. RT-PCR indicates that both genes 

are transcribed in the pathogenic Hc yeast phase. 6sing specific synthetic substrates, 

DppIV enzymatic activity is detected in Hc culture supernatants suggesting that at 

least one of the putative enzymes is secreted. Native zymography with Hc culture 

supernatant indicates a single enzyme much larger than its predicted 88 BDa, 

indicating multimerization or some other modification. 2ased on phylogenetic analysis 

we predict that V11KO4 encodes the secreted DppIV enzyme. Expression of the 

putative V11KO4 open reading frame with its native signal sequence in 1"#?"+*'+&-%$"& 

has led to expression, secretion and purification of an active recombinant enzyme with 

pH and temperature optima of 8 and O2+C, respectively. Expression of V11KO4 in Hc 

under the control of the H22 promoter results in a 10-fold increase in secreted DppIV 

activity, further suggesting that this gene is responsible for secreted Hc DppIV activity. 

ae hypothesize that V11KO4 contributes to the virulence of A"&-%'6+&)+. ae are 

testing whether DppIVA can mimic host CD26, causing host peptide dysregulation. 

P-0653. Long-term cryopreservation of dermatophyte strains 

García-Martínez J, MartÜnez C, Alonso M, (aqueti (, GarcÜa I, Prieto . 

1 Hospital de Fuenlabrada, Fuenlabrada, Madrid, .pain, 2 Hospital de Fuenlabrada, 

Fuenlabrada, Madrid, .pain, J Hospital de Fuenlabrada, Fuenlabrada, Madrid, .pain, 

O Hospital de Fuenlabrada, Fuenlabrada, Madrid, .pain, [ Hospital de Fuenlabrada, 

Fuenlabrada, Madrid, .pain, 6 Hospital de Fuenlabrada, Fuenlabrada, Madrid, .pain 

Purpose of the study: /ong-term conservation of micro-organism strains is essential 

for their in-depth study. .ince fungal cells stability, especially dermatophyte fungi, is 

not ensured with previously described methods, as soil, oil, sterile distilled water or 

liquid nitrogen preservation, a system based on cryogenic vials stored at -80oC for the 

maintenance of dermatophyte stocB cultures was evaluated. 

Materials and methods: Fifty-nine different dermatophyte strains, representing 7 

species, isolated from clinical specimens submitted to the Microbiology laboratory and 

sub cultured on Corn Meal agar for 7 days at J0oC, were removed by using a sterile 

swab and inoculated on cryogenic vials (CryobanB, Mast Diagnostics, 2ootle, 6l) 

containing 2[ glass beads covered in a hypertonic cryopreservative solution to give a 

density equivalent to McFarland noO standard. The fungal suspensions were mixed 

vigorously by shaBing the tubes to completely distribute the organism and were left to 

stand for [ minutes. As much of the hypertonic cryopreservative solution as possible 

was removed with a sterile pipette. Finally, the tubes were properly identified and 

frozen at -80oC. After preservation for a period of 1 to 12 months, a glass bead was 

removed from each tube and was inoculated on .abouraud dextrose agar and 

incubated at J0oC for 7 days to asses the number of recovered colonies, their purity 

and their macroscopic and microscopic features. 

Results: Except for a >$"#?%'?2-%,*).,-+@$%'?2-.& strain, all dermatophyte isolates 

were successfully recovered, including JJ >$"#?%'?2-%,*$(8$(), 7 >/*).,-+@$%'?2-.&, 

6 >$"#?%'?2-%,*-%,&($+,&, J >$"#?%'?2-%,*0"%6+#.(), 2 >$"#?%'?2-%,*0.$$(#%&(), 6 

!"#$%&'%$()*#+,"& and 1 !"#$%&'%$()*+(5%(","". The average number of recovered 

colonies from >/*-%,&($+,& were lower (averageM [[n rangeM 1-1O7) than those 

recovered from >/*$(8$(), (88.2n 2-2O[), >/*).,-+@$%'?2-.& (1J6.8n 0-21[), >/* 

0"%6+#.() (101n O-187), >/*0.$$(#%&() (1[7.[n 11K-1[6), !/*#+,"& (88.2n 2-2O[) or !/* 

+(5%(",""*(1O1). Any of the recovered cultures resulted contaminated. Macroscopic 

and microscopic morphologies remained comparable to the features from strains 

before storage. 

Conclusions: It is concluded that preservation at -80oC by using cryogenic vials is a 

reliable and effective system for conservation of a dermatophyte stocB culture. 


P-0654. Characterization of rad51 null mutants of Candida albicans 

Gómez Raja J 1 , Andaluz /ápez E 2 , /arriba Calle G J , Calderone R O 

1 Departamento de MicrobiologÜa, Facultad de Ciencias, 6niversidad de Extremadura, 

2 Departamento de MicrobiologÜa, Facultad de Ciencias, 6niversidad de Extremadura, 

J Departamento de MicrobiologÜa, Facultad de Ciencias, 6niversidad de Extremadura, 

O Department of Microbiology and Immunology, Georgetown 6niversity .chool of 

Medicine 

ae have cloned and characterized the P4V5I ortologue of the pathogenic fungus 

7+,5"5+*+68"#+,&. CaRad[1 exhibited more than [0_ identity with several euBaryotic 

counterparts distributed along the biological scale, from =/*#.$.0"&"+. to human, and 

conserves the catalytic domain of bacterial RecA (J0_ identity), involved in binding 

and hydrolysis of ATP. Null 7+$+55I*/7+$+55I* mutants were obtained by sequential 

deletion of both alleles. They were not only viable but displayed a grow rate about 

80_ of that of wild type, which dropped down to [0_ in the case of $+55M* 

counterparts. Null $+55I strains not only showed a decreased ability to integrate linear 

DNA with long flaBing homology but correct targeting was significantly decreased as 

compared to wild type, but again they were significantly less restrictive in these 

processes than $+55M*. Also, whereas $+55M* strains were drastically affected in the 

maintenance/integration of AR. plasmids, $+55I* originated stable transformants with 

the same Bind of plasmids although the frequency dropped down to J0_ as compared 

to parental strain CAIO. As described for Rad[2, Rad[1 seems to play an essential 

role in the repair of DNA lesions caused by both 6V light, since the null strain showed 

the same percentage of survivals (1[_) than a $+55M* counterpart, suggesting that 

the 6V lesions are processed through the Rad[1-dependent homologous 

recombination pathway. fn the other hand, $+55I null strains were significantly less 

sensitive to MM. than the $+55M*counterparts, all over the range of concentrations 

used (0.001 to 0.01_). This suggests that a large number of lesions caused by MM. 

are repaired using a Rad[1-independent Rad[2-dependent pathway. All these defects 

were reverted by the reintegration of the wild type P4V5I. 

P-0655. Deactivation of dimorphic fungi using hydrogen peroxide vapor 

Hall L 1 , ftter ( 2 , Chewins ( 2 , aengenacB N 

1 Mayo Clinic College of Medicine, Rochester, 6.A, 2 2If~6E// 

Formaldehyde vapor has been used for the decontamination of biosafety cabinets and 

laboratories for many years but the efficacy of the process remains controversial and 

there are health and safety concerns associated with its use. In contrast, hydrogen 

peroxide vapor (HPV) has recently been established as a bio-decontaminant that is 

effective against a variety of microorganisms including bacteria, viruses and fungi. In 

addition, HPV is safe to use and is effectively residue-free because it decomposes to 

oxygen and water. 

The aim of this worB was to evaluate the efficacy of HPV against the dimorphic fungi 

A"&-%'6+&)+*#+'&(6+-()3*Z6+&-%)2#.&*5.$)+-"-"5"&3*+,5*7%##"5"%"5.&*"))"-"&. 

aorBing inside of a Class II type 22 biological safety cabinet within a biosafety level III 

laboratory, inocula containing 10 J -10 [ cells from the mould form of each organism 

were suspended in RPMI medium, deposited on stainless steel disBs and allowed to 

air dry. Thirty percent hydrogen peroxide was used in conjunction with a 2If~6E// 

Clarusã . generator to produce HPV inside the biological safety cabinet. In three 

replicate experiments, individual disBs were removed at 1[ minute intervals during a 

180 minute HPV exposure period. Control- and HPV-exposed disBs were rehydrated 

using RPMI medium and incubated at J0+C for 6 weeBs to determine if any viable 

organisms remained. Positive cultures were confirmed using nucleic acid 

hybridization probes specific for A/*#+'&(6+-(), Z/*5.$)+-"-"5"&, or 7/*"))"-"& 

(AccuProbe, GenProbe, .an Diego, CA). 

Results indicate that HPV Billed A/*#+'&(6+-() in 10 minutes while Z/*5.$)+-"-"5"& and 

7/*"))"-"& were Billed in 20 minutes. This study suggests that HPV is efficacious 

against these dimorphic fungi. fn the basis of these promising results, additional 

studies using higher inocula and agar-based media are warranted.

P-0656. Response of Candida albicans during adhesion and invasion of 

human epithelia 

Hernandez R, .ohn l, lénigsdorfer A, (offroy C, Hauser N, Zavrel M, Zelt G, Rupp . 

IG2-Faunhofer, .tuttgart, Germany 

The incidence of fungal infections has considerably increased worldwide largely due 

to opportunistic pathogens such as 7+,5"5+*&''. ahile multiple host and fungal 

factors contribute to development of 7+,5"5+ &''. infections, the expression of 

specific factors that allow this fungus to adhere to, and penetrate into many different 

host tissues, represents one of the most important virulence factors in this human 

pathogen which cause a diverse range of mucosal and systemic infections. A better 

understanding of the factors that influence epithelial adherence and invasion for the 

colonization of the host may be a first step in preventing infections. 

ae have developed and validated reconstituted human tissue systems, that mimic 

infections of the human body in a close way (Dieterich et al. 2002). In this way, 

invasion studies were performed on two different reconstituted tissue systems 

mimicBing the interaction of 7/*+68"#+,& with a enterocytic tissue model (colorectal 

adenocarcinomal cell line Caco-2) and vaginal tissue model (epidermoid carcinomal 

cell line A-OJ1). 2ecause the intestinal tract is considered a major portal of entry for 

systemic candidiasis (fdds, 1K88n Cole et al. 1KK6) and the increasing importance of 

vulvovaginal candidiasis (VVC), these two models are of high interest to performed 

the host-pathogen interaction assays. These models can be used to )%,"-%$*.+$62* 

&-.'&*%U*",0+&"%,*8%-?*%,*+*)%$'?%6%@"#+6*+,5*+*)%6.#(6+$*6.0.6. 

For morphological aspects we looB at invasion using histological methods and 

resolution scanning electron microscopy (.EM). After O hours of interaction 7/* 

+68"#+,& was penetrating the epithelial barrier by a filamentous form. This invasion is 

also reflected by a significant change in the differential expression studies performed. 

In order to understand how 7/*+68"#+,& is able to penetrate in the epithelial cells and 

infect different tissues a major focus have been on the different genes/proteins that 

are induced during this early step of the infection. In this aspect on a molecular way 

we have performed #%)'+$+-"0.*@.,%)"#&*+,5*'$%-.%)"#& on 7/*+68"#+,& grown on 

tissue or without tissue before and after the interaction with different epithelial cell 

lines. 

To identify fungal genes that are expressed in response to growth of 7/*+68"#+,& onto 

different epithelia we performed transcriptional profiling using Candida albicans DNAmicroarrays. 

In addition to a common transcriptional response 7/*+68"#+,& also has 

established individual adaptions when grown onto specific tissues. In this context, we 

also found several genes that were differentially transcribed in response to invasion 

onto different epithelia in a tissue dependent manner, depending on whether 7/* 

+68"#+,& invade Caco-2 or A-OJ1 cells. 

P-0657. Morphological and genetic identification of nature’s Sporothrix 

schenckii strains 

Hernández-Hernández F 1 , RamÜrez-Gaona A 2 , Espinosa-Texis A 2 , Romo-/ozano Y 2 

1 Facultad de Medicina, 6niversidad Nacional Autánoma de Mdxico, 2 Instituto de 

Ciencias, 2enemdrita 6niversidad Autánoma de Puebla 

=/*&#?.,#["" is the only Bnown agent causing sporotrichosis. In general, this fungus is 

identified by their morphological featuresn however some times the =/*&#?.,#["" 

phenotype suggests that the fungus presents morphological diversity, there are 

species varieties or other closely related fungal species potentially could cause 

sporotrichoid lesions. The aim of this study was determine the identity of 

morphologically suggestive strains to be =/*&#?.,#["" by the PCR technique followed 

by sequencing of the amplified fragment ({600 bp) corresponding to a region of the 

D1/D2 26. rDNA gene. Twenty strains were includedM 16 highly suggestive to be =/* 

&#?.,#["" obtained from nature, two =/*&#?.,#["" clinical strains (from 

lymphocutaneous sporotrichosis), one 4#$.)%,"() sp and one ^(@%)2#.&* 

#2+,.&#.,&. The macroscopic and microscopic morphology was studied on 

.abouraud dextrose agar (seven days at 28ÅC). The mycelium to yeast transition was 

obtained in 2HI broth (five days at J7ÅC). For the genetic study the genomic DNA was 

obtained and with 10 ng a PCR was performed using the primers FEP(/ 

([]CATATCAATAAGCGGAGGAAAAGJ]) and REP(/ 

([]GCTCCGTGTTTCAAGACGJ]). The sequenced fragment was compared with the 

Gene 2anB Database. Concerning results, all 16 nature]s strains showed a mycelial 

and yeastliBe morphology highly shared with =/*&#?.,#[""n all reverted to yeast phase. 

The molecular study confirmed the two =/*&#?.,#["" clinical strains, the 4#$.)%,"() 

and ^/*#2+,.&#.,& strains. From 16 nature]s strains, 1J were confirmed as =/* 

&#?.,#["" and differentiated two a,5%)2#.&*&#%'(6+$() and one b'?"%&-%)+* 

'$%-.+$(). In conclusion, the molecular procedures, particularly the sequencing of 

highly variable regions as the D¥/D2 26. rDNA, are helpful tools to taxonomic 

determination of .. schencBii. The ability of a,5%)2#.&*&#%'(6+$()] and b'?"%&-%)+* 

'$%-.+$()]s mycelium to yeast transition suggests the possibility of these species can 

infect the human being as other authors have been proposed. 

References: 

Fell (a. Rapid identification of yeast species using three primers in a plymerase chain 

reaction. Mol Mar 2iol 2iotechnol 1KKJn 2M17O-180. 

.igler /, Harris (/, Dixon DM et al. Microbiology and potential virulence of ='%$%-?$"_* 

#2+,.&#.,&, a fungus rarely isolated from blood and sBin. ( clin Microbiol 1KK0n 

28M100K-101[. 

Hintz aE. .equence analysis of the chitin synthase A gene of the Dutch elm 

pathogen b'?"%&-%)+*,%0%W(6)" indicates a close association with the human 

pathogen ='%$%-?$"_*&#?.,#[""/ Gene 1KKKn 2J7M21[-221. 

These tissue systems currently are further developed to include stresses due to the 

immune system suffered by 7/*+68"#+,& during the interaction with the host, liBe the 

presence of reactive oxygen and nitrogen species. 


P-0658. Intra- and inter-serotype diversity of laccases in Cryptococcus 

neoformans 

Ito-Kuwa S 1 , NaBamura l 1 , Valderrama 2 2 , Vidotto V J , fsafune T O , AoBi . 1 

1 

Advanced Research Center,Nippon Dental 6niversity, 2 Instituto de 2iotecnologÜa, 6niversidad 

Nacional Autánoma de Mdxico, Cuernavaca, Mdxico, J Dipartimento Discipline Medico- 

Chirurgiche, .ezione Malattie Infettive, 6niversitÖ di Torino, Torino, Italy, O Deprtment of /ife 

.ciences, Nippon .port .cience 6niversity, YoBohama, (apan 

Purpose: Melanin is one of virulence factors in the pathogenic yeast 7$2'-%#%##(&*,.%U%$)+,&. 

The initial steps of melanin biosynthesis are catalyzed by laccase. Although the whole laccase 

protein (/ac1p) in a 7/*,.%U%$)+,& serotype D strains has been sequenced s1,2u, those of other 

four serotypes have not been determined. The present study aims to determine the whole 

sequence of laccase proteins of the five serotypes and to reveal phylogenetic relations among 

the serotypes and other fungal species. 

Materials and methods: A total of [[ strains of 7/*,.%U%$)+,& were used. They were 2J 

serotype A strains, 1[ serotype D, 2 serotype AD, 8 serotype 2, and 7 serotype C. These strains 

were isolated from clinical or environmental sources, or were obtained from culture collections. 

Genomic DNA was extracted from cryptococcal cells treated with cell wall-digesting enzymes sJu. 

The laccase gene (X47I) was amplified using six pairs of primers. The PCR products were 

directly sequenced by a DNA analyzer and protein sequences were determined. The 

phylogenetic tree was reconstructed by the Neighbor-joining method sJu. 

Results: The number of amino acid residues of laccase protein was 62O in serotypes A, D and 

AD, while that was 61J to 61[ in serotypes 2 and C. Intra-serotype amino acid replacement in 

laccases was minor in serotypes A and D and none in serotypes AD and C. Maximum amino 

acid replacement was found at 2O sites in two serotype 2 strains, compared with major strains of 

this serotype. Percentages of the difference in amino acid sequences were about 10_ between 

serotypes A and D, 17-20_ between serotypes A/D and 2/C, and about O_ between serotypes 

2 and C. The most important protein residues of laccases, those forming the coordination sphere 

of the copper sites are conserved. The unique features previously observed for the 7/* 

,.%U%$)+,& serotype D laccase sOu were conserved in the sequences from other serotypes. 

Furthermore, the five laccases from 7/*,.%U%$)+,&*clustered together in an independent clade 

located between the ones formed by the laccases from other basidiomycete or from ascomycete 

fungi, indicating a taxonomical inconsistency. 

Conclusions: The whole sequence of laccase proteins was determined in a total of [[ strains of 

the five serotypes. Intra-serotype variations in the sequence were minor or none in serotypes A, 

D, AD and C, but evident in serotype 2 strains. The copper-binding regions were commonly 

conserved in all serotypes. The phylogenetic tree showed that the cryptococcal laccases position 

in an independent clade, suggesting the cryptococcal laccases have an evolutionary history 

different from the one of other major groups of fungi. 

P-0659. A novel group I intron in the large subunit ribosomal DNA gene of 

Trichophyton mentagrophytes var interdigitale 

Jackson C 

Institute ff 2iological .ciences, Aberystwyth, 6l 

Group I or spliceosomal introns have the ability to catalyse their own excision (selfsplice) 

from pre-RNA, and are found in a wide range of euBaryotic organisms. They 

show an irregular, random species distribution due to both elimination by spontaneous 

loss and acquisition by horizontal transfer (1) . Group I introns in fungal nuclear 

genomes are located exclusively in the small subunit (..6) and large subunit (/.6) 

genes of the ribosomal RNA gene repeat complex. Fungal group 1 introns belong to 

one of three typesM subgroup IC1 (OK_), ICJ (J2_) and IE (1K_). They are 

characterized by the presence of conserved motifs, termed P,~,R, and ., which 

interact to form the secondary structures essential for self-splicing. 

Group I introns insert into specific recognition sites, and four such conserved target 

sequences are located toward the J]- end of the /.6 rDNA gene. .equencing of this 

region in >$"#?%'?2-%,*).,-+@$%'?2-.& var ",-.$5"@"-+6.. revealed a novel JKJ bp 

group I intron of subgroup IE, inserted at a position corresponding to base 2K28 of the 

=+##?+$%)2#.&*#.$.0"&"+. 2[. gene (position 2[6J of a/*#%6" /.6 RNA). The intron 

contained the characteristic P,~,R and . motifs, and nine sets of conserved paired 

regions (P1-PK) typical of these mobile elements. The predicted secondary structure 

and DNA sequence homology showed this intron to be most similar to group I introns 

from the entomopathogenic hyphomycete Z.+(0.$"+*8+&&"+,+* (2) . PCR analysis 

showed an intron to be present in a similar location in many dermatophyte species, 

but suprisingly not in teleomorphs of the >/*).,-+@$%'?2-.& complex.* 

In this study, the nucleotide sequence and putative secondary structure of this novel 

group IE intron are presented, with evidence for it]s distribution in the 

4$-?$%5.$)+-+#.+.. The possible evolutionary and taxonomic significance of 

dermatophyte ribosomal introns is discussed. 

References: 

Dujon, 2. (1K8K) 9.,. 82: K1-11O. 

aang, C., Zengzhi /., Milton A., Typas, A., and T.M 2utt. (200J) !2#%6/ P.&/ 107M 

118K-1200. 

References: 

ailliamson PR. 1KK[. (. 2acteriol. 176M 6[6-66O. 

/oftus 2(, Fung E, Roncaglia P. et al. 200[. .cience J07M 1J21-1J2O. 

TanaBa E, Ito-luwa ., NaBamura l, AoBi ., Vidotto V, Ito M. 200[. Microbiol. Immunol. OKM 20K- 

217. 

Valderrama 2, fliver P, Mendrano-.oto A, Vazquez-Duhalt R. 200J. Antonie van /eeuwenhoeB 

8OM 28K-2KK.

P-0660. A positive transcription factor, CaNDT80, of CDR1 involoved in drug 

resistance in Candida albicans 

Lo H 1 , Yang Y 2 , Chen C 1 , .hih H 2 , aang ( 1 

1 National Health Research Institutes, Miaoli, Taiwan, 2 National Chiao Tung 6niversity, 

Hsinchu, Taiwan 

fverexpression of 7VPI, an efflux pump, is one of the major mechanisms 

contributing to drug resistance in 7+,5"5+*+68"#+,&. The 7VPI promoter-6+#L has 

been constructed and transformed into =+##?+$%)2#.&*#.$.0"&"+. strain such that the 

6+#L gene is used as the reporter to monitor the activity of 7VPI promoter. 

fverexpression of 7+`V>DQ, a 7/*+68"#+,& homolog of the =/*#.$.0"&"+. `V>DQ, 

increases the #-galactosidase activity of the*7VPI promoter-6+#L construct in =/* 

#.$.0"&"+.. Furthermore, mutations on 7+`V>DQ abolish the induction of 7VPI 

expression by antifungal agents in*7/*+68"#+,&. Consistently, the 7+,5-DQ

P-0662. Gene expression and virulence: Association with major clades of 


MacCallum D, Nather l, Munro C, fdds F 

6niversity of Aberdeen 

7+,5"5+*+68"#+,& remains the predominant cause of life-threatening invasive fungal 

infections. Multi-locus sequence typing (M/.T) of 7/*+68"#+,&*reveals a population 

structure consisting of four major clades and a number of minor clades (Tavanti .-*+6., 

200[). The virulence of a selection of isolates from the major clades was tested in a 

mouse model of invasive infection. 2oth virulent and attenuated isolates were found 

in all four major clades. Representatives of virulent and attenuated strains were 

compared by DNA microarray analysis for differences in global gene expression that 

could be generally associated with virulence or with individual clades. The results of 

the expression analysis will be presented. 

P-0663. The possibility of growth and reproduce in potential pathogenic fungi 

on the simulated microgravity using three-dimentional clinostat 

Makimura K 


Humans in space are exposed both to space radiation and microgravity. Moreover, 

astronauts will face a variety of microorganisms growing on board space station, 

especially fungi. 

.ix strains of space fungi were isolated and identified from samples collected from 

Russian Mir-.pace .tation as the major constituents of the fungal flora. These isolates 

were identified as 1.,"#"66"()*#?$2&%@.,()3*4&'.$@"66(&*0.$&"#%6%$*or 1.,"#"66"() sp. 

by both morphological methods and molecular biological techniques, based on 18.- 

and IT.1-rDNA sequences (MaBimura l, .-*+6.M Fungal flora on board Mir-space 

station, identification by morphological features and ribosomal DNA sequences.* 

!"#$%8"%6%@2*+,5*K))(,%6%@2 O[([)M J[7-J6J, 2001.). 

To confirm the effect of microgravity to growth of fungi, strains of potential pathogens, 

4/*,"@.$*(TIMM2K11, 27C, 72hr) and 7+,5"5+*+68"#+,&*(TIMM1768, J7C, 2Ohr), were 

incubated on three-dimensional (J-D) clinostat and ground control. J-D clinostat 

equipped with two rotation axes placed at right angles has been constructed. In the 

clinostat, rotation achieved with two motors is computer-controlled and monitored with 

encoders attached to the motors. The parameters analyzed up to now demonstrate 

that the J-D clinostat is a valuable device for simulating weightlessness. 

Morphological examinations were performed with stereoscopic microscope and 

scanning electron microscope. Reproducibility tests were done by colony formation 

unit and conidia counting. 

Any significant differences were not observed in morphology or asexual reproducibility 

among clinostat culture and ground control in each strains. 

The present study concluded that the environment on board the space station permits 

growth of potentially pathogenic fungi as does the Earthes environment. These results 

suggested that these fungi can cause unexpected infections or allergic diseases in 

astronauts whose immune systems are disturbed by various stress and radiation 

effects under the condition of microgravity or another environment of space.

P-0664. Investigation of hemolytic activities of Candida species in liquid 

medium 

MalcoB H, Aktas E, Ayyildiz A, Yazgi H 

AtaturB 6niversity Medical Faculty, Microbiology Department 

In this study, the hemolytic activities of 107 Candida strains obtained from different 

clinical samples were researched in liquid medium. The effect of the glucose on this 

activity was also researched. 

.trains were identified to the species level by using germ tube test, microscopic 

evaluation of the growing type on cornmeal-tween 80 medium, colonial morphology on 

Chrom agar. Moreover, the API 20 CA6j identification Bits were also used to definite 

the identification. ff the strains tested, 26 were 7/*+68"#+,&, 22 were 7/*@6+8$+-+, 17 

were 7/*-$%'"#+6"&, 1[ were 7/*'+$+'&"6%&"&, 1O were 7/*[.U2$, [ were 7/*[$(&.", O were 

7/*@("66".$)%,5"" and O were 9.%-$"#()*#+,5"5(). Four standard Candida species (7/* 

+68"#+,&*26[[[3*7/*+68"#+,&*K00283*7/*@6+8$+-+*K00J03*7/*'+$+'&"6%&"&*K0018) were 

also included in the study. 

The hemolytic activities of the strains were tested on two different mediums 

supplemented with 7_ defibrinated human blood, the .aboraud broth with glucose, 

the .aboraud broth without glucose at the end of O8 hours incubation. 

The hemolysis in these medium was detected by measuring the amount of 

haemoglobin spectrophotometrically, and comparing with a standard which was 

prepared prior to test. The degree of hemolysis (percentage value) by an individual 

strains were calculated according to the formula belowM 

(Absorbance of supernatant of media at [O0 nm / absorbance of standard hemolysate 

at [O0 nm) x 100. 

The results obtained from the study were shown at the table. 

In the liquid medium without glucose, strains generally produced haemolysis in low 

degrees. The degree of hemolysis produced by all species evidently increased in the 

liquid medium with glucose. 

fverall results of the study indicate that the haemolytic activity as a virulence factor 

differed between the Candida species, and glucose increased this activity. The 

highest haemolytic activity in both medium was shown by 7/*+68"#+,& and 7/[.U2$3*+,5* 

the least by 7/*'+$+'&"6%&"&. 

Candida spp. 

n 

7/+68"#+,&** 

28 

7/@6+8$+-+** 

2J 

7/-$%'"#+6"&*** 

*17 

7/'+$+'&"6%&"&** 

16 

7/[.U2$** 

*1O 

7/[$(&."** 

[ 

9.%-/*#+,5"5()** 

O 

7/*@("66.$)%,5"" 

O* 

Mean average of 

hemolysis in the liquid 

medium without glucose 

(_) 

J2.K 

Mean average of 

hemolysis in the 

liquid medium with 

glucose (_) 

OK.6 

27.6 OJ.0 

27.8 O0.0 

0.0 6.8 

O0.1 O7.O 

17.2 28.J 

K.8 JO.0 

18.1 21.[ 


P-0665. Phylogenetic analysis of human and animal isolates of Trichophyton 

mentagrophytes based on MnSOD and its sequence comparisons 

Marion R 1,2 , Frdalle E 1,2 , Gantois N 2 , Delaporte E J , Camus D 1,2 , Dei-Cas E 1,2 , 

lauffman-/acroix C O , Guillot ( [ , Delhaes / 1,2 

1 .ervice de Parasitologie Mycologie, Ddpartement de Microbiologie - CHR6 de /ille, 

Facultd de Mddecine - 6niversitd de /ille 2 - /ille, France, 2 /aboratoire deEcologie du 

Parasitisme, Institut Pasteur de /ille - EA J60K - 6niversitd de /ille 2 - /ille, France, 

J Clinique Dermatologique, CHR6 de /ille - 6niversitd de /ille 2 - /ille, France, O .ervice de 

Parasitologie Mycologie - CHR6 de Poitiers - Poitiers, France, [ Equipe de Mycologie - 

6MR K[6 INRA, AF..A, ENVA, 6PVM, Ecole Nationale Vdtdrinaire deAlfort - Maisons- 

Alfort, France 

Introduction: Dermatophytes are Beratinophilic fungi which are able to infect Beratinised 

tissues of man or animals. Three genera are recognized as human pathogensM 

a'"5.$)%'?2-%,, !"#$%&'%$(), and >$"#?%'?2-%,. aithin the genus >$"#?%'?2-%,, which is 

particularly complex because it comprises at least 1[ recognised species, >$"#?%'?2-%,* 

).,-+@$%'?2-.& and >$"#?%'?2-%,*$(8$() are common agents of dermatophytosis. >/* 

).,-+@$%'?2-.& is Bnown to be a species complex composed of several species or 

variants, which occur both in human and in animals. .ince >/*).,-+@$%'?2-.& complex 

includes both anthropophilic and zoophilic species (or variants), reliable molecular methods 

for identifying the human-pathogenic species are needed. Furthermore, accurate molecular 

identification emerges as a critical issue to understand comprehensively the clinical and 

epidemiological involvements of >/*).,-+@$%'?2-.& complex genetic heterogeneity. 

Fungal isolates and methods: Forty-two >/*).,-+@$%'?2-.& isolates from either patients 

(1[ isolates) or animals (27 isolates) with dermatophytosis were prospectively isolated by 

culture and identified on morphological bases at the 6niversity Hospitals of /ille and 

Poitiers, and at Maisons Alfort Veterinary .chool, respectively. They were compared to >/* 

).,-+@$%'?2-.&, >/*",-.$5"@"-+6. and >/*.$",+#." standard strains of Pasteur Institute. The 

isolates were screened by DNA sequencing of the variable internal transcribed spacer 

(IT.) regions, well Bnown to be useful in resolving relationships between close taxonomic 

relatives. In addition, the houseBeeping gene encoding for the manganese-containing 

superoxide dismutase (Mn.fD), which is involved in cell defence against endogenous and 

exogenous reactive oxygen species, and which has previously provided interesting insight 

into both fungal taxonomy and phylogeny, was sequenced in all the isolates. 

Results and conclusions: .pecific DNA sequences of Mn.fD gene as well as IT.1-IT.2 

regions were determined after PCR amplification and phylogenetically analyzed. ae were 

able to successfully differentiate between members of the >/*).,-+@$%'?2-.& complex and 

the related species >/*$(8$(), and to demonstrate their phylogenetic relationships by base 

pair comparison analysis. ahatever the phylogenetic marBer used, members of this 

complex were classified into 2 major groups closely related. All human isolates except O 

were clustered together with >/*).,-+@$%'?2-.& and >/*",-.$5"@"-+6. reference strains. fn 

the opposite, human isolates 1, J, 6 c 10 were closer to >/*.$",+#." reference strain and 

clustered with the rest of our animal isolates population. .imilar topology was observed 

with both Mn.fD and IT. sequences, with a higher variability with the second marBer. 

Pleomorphism and cultural variability still maBe the dermatophytes notoriously difficult to 

identify, and classical molecular methods including IT. sequencing have been widely used 

to clarify their taxonomy. Here, we report the use of a protein coding gene (Mn.fD) as a 

new molecular tool to analyse dermatophytes taxonomic organisation. 

P-0666. Visualization and semi-quantitative analysis of superoxide generated 

from Candida albicans 

Masui S 1 , AoBi . 2 , Majima T 1 

1 Pf/A Chemical Industries, INC. YoBohama, (apan, 2 Nippon Dental 6niversity, 

Niigata, (apan 

Purpose of the study: .tudies reporting reactive oxygen species (Rf.) production are 

much less common for mycetes than phagocyte cells. fne study, however, reported a 

relationship between Rf. production and virulence in 7+,5"5+*+68"#+,& (1). AoBi .-*+6/ (2) 

also reported that paraquat (methyl viologen) induced respiration-dependent Rf. 

production in 7/*+68"#+,&. The present worB undertaBes the visualization of Rf. generation 

in 7/*+68"#+,&3 using a photon-imaging instrument with a luciferin analogue. 

Description: The wild type strain (l) of 7/*+68"#+,& was an oral isolate from a patient with 

candidiasis and a respiration-deficient mutant (lRD-1K) was derived from strain l by 

treatment with a chemical mutagen. The strains were pre-cultured overnight in liquid PYG 

medium at J7 oC with shaBing. Cultures were then diluted and 0.1ml of the diluents (about 

[0 cells) were spread on PYG agar plates. Following incubation at J7 oC for one to [ days, 

reactive oxygen species (Rf.) generation by colonies of 7/*+68"#+,& on the PYG agar 

plate was examined with a ultralow light image analyzer equipped with a photon-counting 

CCD camera. To visualize Rf. generated by 7/*+68"#+,&, methyl-72'$"5",+-luciferin 

analogue (MC/A) as a chemiluminescence (C/) probe and the herbicide paraquat (P~) as 

a respiration-dependent Rf. generator were used. After taBing photographs of colonies 

under light, a mixture of 0.1M P~ and 0.0[mM MC/A(1M1) was gently dropped onto the 

colonies. To examine the effects of antioxidants, .fD or /-cystein was added to the P~- 

MC/A mixture. The MC/A- dependent C/ due to Rf. generated by the colonies was 

recorded for [ min in a light-tight box. For semi-quatitative assays of Rf. production, 

0.1ml aliquots of cell suspensions were spread into the K6 well micro-titer-plate. The C/ 

emission was measeared with MC/A. 

Results and conclusions: C/ emission from colonies of the wild strain was observed at 

1day after incubation and whole of the colonies were weaBly luminous. In parallel with the 

increase in the colony size after incubation for J and [ days, the marginal regions of the 

colony were strongly bright. These results indicate that 7+,5"5+ colonies expand by 

division of metabolically active cells in the marginal regions, leaving dormant cells in the 

central regions. fn the other hand, growth of the respiratory mutant was slow and C/ 

emission from the colonies was weaB compared with the parent strain. C/ from colonies of 

the wild strain almost completely vanished by the addition of .fD and/or /-cystein. In 

addition, we also examined correlation the quantity of Rf. production and active cell 

numbers. 

C/ from cell suspension of 7/*+68"#+,& in K6-well plate was performed in the same manner 

as with colonies. Captured image was analyzed and calculated photon intensity (PI) as a 

Rf. production. 

References: 

.ander C., et al., Mycoses 2002n O[M 1[2-[. 

AoBi ., et al., Med Mycol. 2002n O0M 1J-K

P-0667. Dissection of the C. albicans-neutrophil response 

Mavor A 1 , 6rban C 2 , Fradin C 1 , ZychlinsBy A 2 , Hube 2 1 

1 Robert loch-Institut, 2erlin, Germany, 2 Max-PlanB-Institut fyr InfeBtionsbiologie, 

2erlin, Germany 

7+,5"5+*+68"#+,& can cause serious systemic infections in immunocompromised 

patients following haematogenous dissemination. Therefore the interaction with 

leuBocytes of the innate immune system is an important level of host defence. fur 

worB investigating 7/*+68"#+,& and blood fractions, has shown that polymorphonuclear 

cells (PMNn mostly neutrophils) have the greatest influence on the viability and the 

transcriptional profile of the fungus in human blood, while mononuclear cells (MNC) 

seem to have a less direct influence on the fungus (Fradin*.-*+6/3 200[). However, 

MNC express higher levels of cytoBines in response to the infection. PMN caused 

four major responses in 7/*+68"#+,& which reflect growth inhibition, oxidative stress, 

carbohydrate starvation and nitrogen starvation. ae now wish to dissect out the 

previously observed PMN-induced responses, to determine how 7/*+68"#+,& reacts 

when phagocytosed by or associated with PMN and the response caused by various 

factors potentially involved in the Billing process. To do this we will examine the global 

transcriptional response of 7/*+68"#+,& to purified granules from human neutrophils 

and to individual components of the granules. ae shall also use single cell profiling to 

examine the stresses experienced by individual cells and how these vary, for example 

due to phagocytosis or close association with the neutrophils. From this we hope to 

understand the complex interaction between neutrophils and 7/*+68"#+,&. 

Fradin, C, De Groot, P, MacCallum, D, .challer, M, llis, F, fdds, F and Hube, 2 

(200[) Granulocytes govern the transcriptional response, morphology and proliferation 

of 7+,5"5+*+68"#+,& in human blood. !%6*!"#$%8"%6 56M(2) JK7-O1[ 

P-0668. Functional analysis of Aspergillus fumigatus CYP51A mutations in 

Saccharomyces cerevisiae 

Meneau I, Rochat /, .anglard D 

Institute of Microbiology 

4&'.$@"66(&*U()"@+-(& is one of the major airbone opportunistic fungal pathogen that 

can cause several types of diseases in immunocompromised patients. Invasive 

aspergillosis (IA) is a serious infection observed in these patients and has a high rate 

of mortality. The class of azoles represents a major class of antifungal agents to treat 

IA. These agents inhibit a cytochrome PO[0-dependent 1O$-sterol-demethylase 

(7k15I

P-0669. Transcriptional profiling of the Candida albicans SSK1 and point 

mutants in the SSK1 receiver domain. 

Menon V, Calderone R 

Georgetown 6niversityMedical Center 

Two-component signal transduction in 7+,5"5+*+68"#+,& regulates adaptation to stress 

conditions, cell wall biosynthesis, and quorum sensing, functions determined through 

the evaluation of strains deleted of genes encoding either the 7AcI histidine Binase 

or the ==cI response regulator. A #?[I mutant also had altered cell wall composition 

profiles such that the amounts of either #-1,J and #-1,6 glucan decreased and 

increased respectively. Also the length of acid-stable oligomannan glycoproteins 

decreased compared to the acid-labile oligomannans which remained unaffected in 

the mutant. The cell wall changes were associated with the avirulence of the mutant in 

a murine model of hematogenously disseminated candidiasis. 2oth the #?[I and 

&&[I mutants are Billed more readily than wt or gene-reconstituted strains when 

incubated with human neutrophils. To further characterize the interaction of these 

strains with human neutrophils, chemotaxis assays were performed. All strains were 

grown overnight in YPD medium, standardized to a common cell number, and 

incubated in Hepes-H2.. buffer for J h. .upernates were then collected, filtered, and 

used as a source of chemoattractant. .ingle-cell chemotaxis was then measured in a 

quartz chamber equipped with bright field optics. Fields of cells were computerdigitalized 

at 1[ frames per second for 10 min using a framegrabber board and 2D- 

DIA. software. ae observed that supernates from both the wt and &&[I mutant 

caused chemotaxis of neutrophils compared to the #?[I*mutant which did not. To 

identify the chemotaxis factor, supernates of wt cells (CAF2-1) were first fractionated 

by filtration ([ BDa filters) and then both pools were further fractionated by fasis 

reverse phase chromatography. Flow through and fractions eluted by 0-60_ methanol 

were collected and tested for chemotaxis activity using the ChemoTx disposable 

chemotaxis system. A single fraction eluted with 60_ methanol displayed activity from 

wt but not the #?[I supernates that induced a chemotaxis equal to that of I/-8 which 

was used as a positive control. Further, the active material was destroyed in the 

absence of a protease inhibitor cocBtail. In summary, the 7AcI and ==cI of 7/* 

+68"#+,& provide important cell functions, including cell wall synthesis, and their 

deletion results in avirulence. fur new data also demonstrate that ChB1p functions in 

the regulation of a neutrophil chemotaxis factor. 

P-0670. Carbon dioxide sensing in Candida albicans and Cryptococcus 

neoformans 

Mogensen E, Myhlschlegel F 

6niversity of lent 

Purpose of the study: Environmental concentrations of Cf 2 strongly affect the 

expression of virulence determinants in the fungal pathogens 7+,5"5+*+68"#+,&* 

(filamentation) and 7$2'-%#%##(&*,.%U%$)+,&*(capsule synthesis)/*fur main objective 

was to determine the sensing mechanism of these two pathogens. 

Description: 7/*+68"#+,& senses and responds to elevated concentrations of Cf 2 by 

filamenting. Filamentation is regulated by the second messenger cAMP synthesised 

by adenylyl cyclase, which activity is stimulated by physiological concentrations of 

Cf 2/bicarbonate. ae have recently demonstrated that Cf 2/bicarbonate equilibration 

by the carbonic anhydrase Nce10J is required for pathological growth of 7/*+68"#+,&*in 

niches where Cf 2 concentrations are limited ("/./*sBin). This finding led us to 

hypothesise that Nce10J may be essential to provide a sufficient amount of 

bicarbonate required for Bey carboxylating enzymes involved in various metabolic 

processes. 

Results and conclusions: fne important metabolic pathway is the carboxylation of 

acetyl-CoA forming malonyl-CoA. This reaction, catalysed by acetyl-CoA carboxylase, 

leads to the synthesis of fatty acids. ae showed that growth of Nce10J mutants of 7/* 

+68"#+,&*was recovered in air concentration of Cf 2 (0.0JJ_) by addition of 2mM 

palmitate, in rich but not minimum medium. Physiological concentrations of Cf 2 

complemented the growth defect on both media. These results demonstrate firstly that 

growth defect is mainly caused by defective synthesis of fatty acids, and secondly that 

supplementary metabolites are required for growth recovery. The addition of palmitate, 

aspartic acid and arginine complemented growth of Nce10J mutants on minimum 

medium when exposed to 0.0JJ_ Cf 2, suggesting a role of carbonic anhydrase for 

bicarbonate-dependant carboxylation reactions leading to synthesis of fatty acids, CO 

intermediates of the citric acid cycle and arginine. Growth defect of Nce10J mutants 

on medium without pyrimidine and arginine was complemented by the addition of 

either carbamyl-phosphate or glutamate, the two direct products of the carboxylation 

of glutamine. Adenine was shown not to be essential for growth of Nce10J mutants, 

demonstrating that purine synthesis is not affected in these mutants. In order to further 

understand the importance of Cf 2 signalling for the metabolism of 7/*+68"#+,&, we are 

currently investigating the nutritional requirements of 7/*+68"#+,& Nce10J mutants and 

we aim to characterise a putative acetyl-CoA carboxylase-encoding gene.

P-0671. Alpha 1,2-mannosidases from Candida albicans: Intracellular 

distribution and proteolytic processing. 

Mora-Montes H 1 , 2ader f 2 , Ponce-Noyola P 1 , /ápez-Romero E 1 , ZinBer-Ruzal . J , 

Hube 2 2 , Gow N O , Flores-Carreón A 1 

1 II2E-Facultad de ~uÜmica, 6niversidad de Guanajuato. Guanajuato, Gto. Mdxico., 

2 Robert loch-Institut, D-1JJ[J 2erlin, Germany., J Departamento de Gendtica y 

2iologÜa Molecular, CINVE.TAV del IPN, Mdxico, D.F., O .chool of Medical .ciences, 

6niversity of Aberdeen, A22[ 2ZD Aberdeen, .cotland, 6nited lingdom. 

Purpose of the study: .tudies of alpha 1,2-mannosidases in 7/*+68"#+,& ATCC 

26[[[ revealed two soluble enzyme activities named E-I and E-II. Recently, we 

purified and characterized E-II. The aim of this worB was to determine if the 

mechanism responsible of the generation of soluble alpha-mannosidases in 7/* 

+68"#+,& involves the participation ",*0"-$% and/or ",*0"0% of protease activities. 

Description: Here we present results on the intracellular distribution and biochemical 

characterization of alpha-mannosidase activities from other strains of 7/*+68"#+,&M the 

CAI-O mutant and its parental .C[J1O strain as well as the CA/-O mutant derived 

from ATCC 26[[[. For the ATCC 26[[ strain the intracellular localization of soluble 

alpha-mannosidases was determined in a lineal sucrose density gradient (10-6[_). 

Results and conclusions: In ATCC 26[[[, 80_ of alpha-mannosidase activity was 

present in a soluble form, whereas in the mutants O[_ and [[_ of the enzyme were 

soluble and membrane-bound, respectively. .eparation of soluble fractions by ion 

exchange chromatography and zymogram analysis of enzyme preparations revealed 

the presence of alpha-mannosidase E-I ([2 BDa) in CAI-O, .C[J1O and CA/-O strainsn 

on the other hand, two active polypeptides of [2 BDa (E-I) and OJ BDa (E-II) were 

observed in ATCC 26[[[, as previously described. ae demonstrated that the E-II 

enzyme is a proteolytic product generated during cellular disruption. ahen cells of the 

ATCC 26[[[ strain were broBen in the presence of pepstatin A, a change in the 

intracellular distribution of alpha-mannosidase was observed and E-I was the only 

enzyme detected. To learn more about the E-I species, a homogenate from 

protoplasts of ATCC 26[[[ was subjected to centrifugation in a lineal sucrose density 

gradient (10-6[_). Results showed three peaBs of activity associated to the cytoplasm, 

Golgi and ER compartments. Immunoblot analysis of these fractions revealed the 

presence of E-I in the cytosol and Golgi and a RE-bound enzyme of 6[ BDa. The REbound 

enzyme was processed by a proteolytic activity present in Golgi to generate E-I. 

6se of the [._M null mutant of 7/*+68"#+,& and lex2p from =+##?+$%)2#.&* 

#.$.0"&"+. and 7/*@6+8$+-+ revealed that lex2p is responsible for the conversion of 

membrane-bound alpha 1,2-mannosidase into E-I. 

P-0672. Successful RNA interference of the orotidine-5'-phosphate 

decarboxylase (URA3) gene transcript of Paracoccidioides brasiliensis 

Morais F, Nábrega F 

6niversidade do Vale do ParaÜba (6NIVAP), .Éo (osd dos Campos - .P - 2rasil 

1+$+#%##"5"%"5.&*8$+&"6".,&"& is the temperature-dependent dimorphic fungus 

responsible for paracoccidioidomycosis (PCM) in human. This granulomatous disease 

is endemic in /atin America, with 80_ of the cases found in 2razil. The lacB of genetic 

tools to manipulate this microorganism is the main constrain that prevent a better 

understanding about the biology this pathogen, including regulation of gene 

expression and host-parasite interaction. For this reason, a great effort has been done 

in the last few years in order to obtain a stable genetic transformation of 1/*8$+&"6".,&"&. 

In this worB, we show the results of the genetic transformation of the yeast form of 1/* 

8$+&"6".,&"& by electroporation, in which 80_ to 100_ of stable transformants were 

obtained. The orotidine-[e-phosphate decarboxylase gene of 1/*8$+&"6".,&"& (18TP4:) 

was used as target by different DNA constructs designed either for gene replacement 

or RNA interference (RNAi). The gene for hygromycin 2 resistant (A1A) was used as 

selection marBer for the transformants obtained by gene replacement. To select 

transformants with RNAi of the transcript of 18TP4:, the [ï Fluoroorotic acid ([ï- 

FfA) was used. The electrotransformation was performed in a Gene Pulser jcell 

electroporation system (2If-RAD), with 2x10 6 viable yeast cells in 200 $/ of 1M 

mannitol, under constant conditions (1000 V or 7[0 V, 2[ $F and 600 1)/ Five isolates 

were selected for electrotransformation, two of which (Pb18 and Pb608), successfully 

produced viable transformants resistant to either hygromycin 2 or [ïFfA recovered 

J0 days post electroporation. ae also showed that the constructs were stably 

integrated into the genome, since the majority of the transformants remained resistant 

to either selection compound even after 20 sequential passages in the selective 

medium followed by culture for about J0 days in medium with no selection. PCR 

analysis confirmed the integration of the A1A gene in transformants resistant to 

hygromycin 2. In conclusion, we showed a successful and stable genetic 

transformation of yeast cells by gene replacement and RNAi. fur results also show 

for the first time that 1/*8$+&"6".,&"& has a functioning RNAi system, which may be the 

most valuable resource to study gene function in this multinucleated fungus. 

Financial support: FAPE.P and CNPq. 

This worB was financed by .EP-CfNACyT, Mdxico. Project JK[28-~ and 6niversidad 

de Guanajuato, Convocatoria 200[. 


P-0673. Analysis of Cryptococcus neoformans Cu-Zn superoxide dismutase 

gene expression using green fluorescent protein 

Nakamura K 1 , Virtudazo E 1,2 , Ito-luwa . 1 , AoBi . 1 , TaBeo l 1,2 

1 Advanced Research Center, Nippon Dental 6niversity at Niigata, 2 Research Center 

for Pathogenic Fungi and Microbial Toxicoses, Chiba 6niversity, Chiba 

Purpose: Cryptococcal superoxide dismutases (.fDs) can detoxify superoxide radicals. 

fn the other hand, melanin is a powerful antioxidant produced from 7$2'-%#%##(&* 

,.%U%$)+,&*(7/*,.%U%$)+,&). They are believed to protect the yeast cells against reactive 

oxygen species (Rf.) generated by host phagocytic cells and are implicated in virulence. 

In this study, we investigated the expression of 7/*,.%U%$)+,& Cu-Zn superoxide 

dismutase gene (=bVI) in a standard strain and a melanin-deficient variant using Copepod 

1%,-.66",+*'6()+-+ green fluorescent protein gene (9^1) as a reporter gene. 

Materials and methods: 7/*,.%U%$)+,&*=bVI disruptant strains were constructed from 

the standard strain CDC[[1 and melanin-deficient variant Th7Jm - (serotype A, haploid) by 

replacing the =bVI*fRF with GFP coding sequence. =bVI*promoter - 9^1*- =bVI* 

terminator cassette for gene replacement was constructed by fusion PCR using 7/* 

,.%U%$)+,&*total genomic DNA and pCop-Green-Y2 vector carrying 9^1 (Evrogen) as 

templates. To carry out transformation, the DNA fragment was coated on gold beads and 

bombarded into the cells by a helium-driven biolistic system (2ioRad) according to the 

protocol described by Casadevall et al. s1u. As a result, the disruption cassette was 

integrated into the genome by homologous recombination. The colonies of*=bVI 

disruptants expressing GFP were selected under a fluorescence microscopy. To confirm 

correct integration of the cassette, transformants were analyzed by PCR using a 

combination of the corresponding target gene-specific and disruption cassette-specific 

primers. The sensitivity of =bVI disruptants against oxidative stress was tested with an 

Rf.-generating agent, menadione. The fluorescence intensity of cells associated with 

=bVI promoter activation was analyzed by a microplate fluorometer FluorosBan Ascent 

(/absystems). Melanin production was checBed using /-DfPA plates. 6rease and 

phospholipase activities were tested using Christensen]s urea agar and egg - yolB agar 

respectively. 

Results and discussion: Fourteen and 8 =bVI disruptants were obtained from the 

standard strain CDC[[1 and the melanin-deficient variant Th7Jm - , respectively. All =bVI 

disruptants produced melanin, urease and phospholipase. Growth of the*=bVI disruptant 

CDC[[1-1 descended from the standard strain was inhibited by menadione at a 

concentration 6$M. In contrast, the growth of parent strain CDC[[1 was not affected at the 

same concentration. Time course changes of GFP fluorescence intensity of CDC[[1-1 

after adding 1, 2 and O$M menadione showed that the fluorescent intensity began to 

increase after O0 hours at 2$M menadione, unliBe in the absence of menadione wherein no 

increase in intensity was observed. However, the GFP fluorescence intensity was not 

changed by 1$M menadione. In contrast, addition of O$M menadione decreased the 

fluorescent intensity to the level of parent strain CDC[[1. From this result, the expression 

of cryptococcal =bVI may behave as a houseBeeping gene liBe the rat and human Cu-Zn 

.fD genes. All =bVI disruptants isolated from the melanin-deficient variant Th7Jm - 

produced melanin on /-DfPA plates. This restoration of the ability to produce melanin may 

suggest that melanin production may compensate for the deficit of Cu-Zn .fD functions. 

References: 

A. Casadevall and (.R. Perfect. 1KK8.*7$2'-%#%##(&*,.%U%$)+,&, JK7-JK8, A.M Press. 

P-0674. Characterization of the trehalose synthesis pathway in Cryptococcus 

gattii 

Ngamskulrungroj P 1, 2, J, O , ChayaBulBeeree M 1, O , Toffaletti D 1 , Meyer a 2, J , Perfect ( 1 

1 Department of Medicine, DuBe 6niversity Medical Center, Durham, NC, 6.A, 2 Molecular 

Mycology Research /aboratory, aMI, CIDM, aestmead Hospital, aestmead, N.a, 

Australia, J aestern Clinical .chool, Faculty of Medicine, 6niversity of .ydney, .ydney, 

N.a, Australia, O Faculty of Medicine, .iriraj Hospital, Mahidol 6niversity, 2angBoB, 

Thailand 

Purpose of the study: To characterize the trehalose synthesis pathway in 7$2'-%#%##(&* 

@+--"" 

Description: 7$2'-%#%##(&*@+--""*G7/@/J, previously Bnown as 7$2'-%#%##(&*,.%U%$)+,& 

var. @+--"", is a pathogenic basidiomycetous yeast, which causes meningo-encephalitis in 

immunocompetent hosts. A specific genotype of this species, VGII, is responsible for an 

ongoing outbreaB of cryptococcosis amongst humans and a range of animal species on 

Vancouver Island, aestern Canada. Virulence factors that have been genetically defined 

for this fungus include (i) melanin synthesis, (ii) production of a polysaccharide capsule, (iii) 

urease, (iv) phospholipase production and (v) ability to grow at J7oC. A recent study has 

shown that the trehalose synthesis pathway controls growth at J7oC and possibly other 

unBnown virulence factors in 7$2'-%#%##(&*,.%U%$)+,&*G7/,/J3 strain HKK. In order to 

examine the function of this pathway in 7/*@+--"", we created specific null mutants in the 

trehalose-6-phosphate synthase (>1=I), trehalose-6-phosphate phosphatase (>1=M) and 

neutral trehalase (`>AI) genes for the more virulent outbreaB strain R26[ by biolistic 

transformation. The ability to growth at J7oC and OoC, to synthesize melanin, to produce 

capsule and to undergo mating have been tested in these mutants. 2ilateral mating 

(mutant alpha cells crossed with mutant a cells) has been performed to determine the 

filamentation and sporulation abilities. ~uantitative virulence studies of the mutants have 

been performed in mice. 

Results and conclusions: As in the previous study of strain HKK (7/,.), a defect in growth 

at J7oC was found in the -'&I and -'&M*of R26[ (7/@/)*mutants. The blocB of >1=M in the 

pathway of strain R26[ resulted in a fungicidal effect. The -'&I mutant had a defect in 

growth at OoC. 1M sorbitol in the growth media suppressed only the temperature sensitive 

(ts) phenotype of the -'&M mutant of R26[, while the ts phenotype of both -'&I and -'&M 

mutants were suppressed in HKK (7/,/). 6nliBe the HKK mutants, the -'&I*mutant of R26[ 

had a capsule and melanin defect. No capsule or melanin defect was seen in -'&M and ,-?I 

mutants. 2ilateral mating defect was observed in tps1 mutants of strain R26[. Interestingly, 

the -'&M mutant of both R26[ and HKK showed the reversion of its ts phenotype at the rate 

of 1-10/million on normal YPD. Melanin and capsule defect were also been shown in the 

reverted*-'&M mutant. 2oth 7/@+--"" -'&I and -'&M*mutants were found to be severely 

attenuated in the mice model. 

The results from our studies have emphasized the importance of the trehalose synthesis 

pathway in the virulence of 7/@+--"". Mutations in the trehalose synthesis pathway have an 

impact on several virulence factors (growth at J7ÅC, melanin and capsule production) and 

support the pathway as a potential fungicidal target for antifungal drug development. 

Moreover, this worB also emphasizes that 7/*,.%U%$)+,& and 7/*@+--"" share similar 

molecular pathways for virulence but there are evolutionary divergences of gene functions 

in these pathways.

P-0675. Application of temporal temperature gel electrophoresis to the study of 

fungal aerocontamination 

Nieguitsila A 1 , Deville M 1 , (amal T 1 , Halos / 1 , 2erthelemy M 1 , 2ex V 2 , Chermette R 1 , 

Guillot ( 1 

1 6MR INRA, AF..A, ENVA, 6PVM K[6 2IPAR, Ecole Nationale Vdtdrinaire deAlfort, 

Maisons-Alfort, France, 2 /aboratoire deHygimne de la ville de Paris, 11, rue George 

Eastman, Paris, France 

Information obtained from fungal air samples can assist in medical evaluations, 

assessment of health hazards and can be useful in proactive indoor air quality 

monitoring. There are several limitations in interpreting results obtained with the 

culture of bioaerosol samples. To circumvent the cultivation problems, a large variety 

of molecular techniques have been proposed so far. In the present study, we aimed at 

developing an original technique called PCR-TTGE. This technique allows the 

sequence-specific separation of mixture of partial rDNA amplicons according to their 

sequence composition, in polyacrylamide gels under a denaturing temperature 

gradient. Air samples were collected in different environments with the bioimpactor 

CIP 10-M (Arelco). After a J hours sampling duration, 1 m/ of collecting liquid was 

recovered for subsequent cultivation and PCR-TTGE. For mycological cultures, we 

used .abouraud and malt extract agars at 27ÅC during O days. Different DNA 

extraction methods were evaluated. The most efficient method included the use of 

sterile microbeads. Each liquid sample was frozen at -80ÅC and crushed by shaBing 

with a bead beater. DNA was extracted with Nucleo.pinw Tissue Bit (Macherey- 

Nagel). A set of J fungi-specific primers (Fungcont 1, Fungcont 2rGC and Fungcont 

J) was designed for the partial amplification of the 18. rRNA encoding gene. The 

amplification was obtained in a single reaction tube by a semi-nested PCR. For 

sequence-specific separation of amplicons, the TTGE D Code .ystem (2io-Rad) was 

used. In order to identify the fungi detected by PCR-TTGE, the bands with different 

electrophoretic migration patterns were extracted from the gel and sequenced after reamplification. 

The obtained sequences were compared with those deposited at the 

Gen2anB database and identified using 2/A.T. 

PCR-TTGE allowed the clear separation of amplicons corresponding to distinct fungal 

species (Ascomycota, 2asidiomycota and Mucorales) that may be encountered in air. 

However, the number of fungal species detected after culture was systematically 

higher than the number of taxa found using PCR-TTGE. This suggested that some 

fungal populations were present in densities that were below the detection threshold 

of the molecular method. This could also be related to the presence of PCR inhibitors 

in air samples. 

P-0676. Evaluation of uv radiation for inactivation of Aspergillus flavus spores 

in water 

Nikaeen M 1 , Mirhendi . 2 

1 Isfahan university of medical sciences, Dept. of environmental health engineerin, 

2 Tehran 6niversity of medical sciences. 

4&'.$@"66(& is a group of filamentous fungi are ubiquitous in nature including being 

common to water environments. Although water is not primary route for acquisition of 

human 4&'.$@"66(& infections, there are evidences showing nosocomial aspergillosis 

may be waterborne in hospital environments. Moreover 4&'.$@"66(& can produce 

mycotoxins in water that are associated with a variety of respiratory, neurological and 

other systemic symptoms. Fungi in drinBing water are also involved in the production 

of tastes and odors. For the reasons elimination of 4&'.$@"66(& spores from drinBing 

water is necessary. 

.ince the 4&'.$@"66(& spores are resistant to many water disinfectants, the objective of 

this study was to evaluate the efficiency of 6V radiation on the inactivation of 

4&'.$@"66(& U6+0(& spores, after it was found in water of a spring that used for bottled 

water. 

The spores harvested from*4&'.$@"66(& U6+0(& isolated from water, were seeded into 

sterile tap water with two concentrations (10 J /100ml and 10 6 /100ml) and exposed to 

6V in batch experiments. The water passes through a reactor (capacityM 2[60 ml) with 

a low pressure lamp and exposure time vary over the reactor (J, 2, and 1 min) with 

changing the flow rate. 

It was found that 6V radiation with fluence of J2 m(/Cm 2 (1 min exposure time) is 

effective for 100_ inactivation of 4&'.$@"66(&*U6+0(& spores in both two densities. The 

fluence is less than Austria 6V irradiation recommendations and 6... National 

sanitation foundation (N.F) standard for disinfection of drinBing water (O[ and J8 

m(/Cm 2 respectively). 

Regarding to various advantages of 6V radiation for disinfection of water we thinB that 

the process could effectively be applied for the elimination of 4&'.$@"66(& spores and 

improvement microbial safety of drinBing water. 


P-0677. PbrCHS3, a chitin synthase gene of Paracoccidioides brasiliensis is 

mainly expressed in its pathogenic yeast phase 

Nino-Vega G, 2arreto /, .an-2las G 

Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela 

Chitin, the beta-1,O-linBed homopolymer of `-acetylglucosamine, is one of the major 

components of the fungal cell wall. It has important functions in wall integrity, 

morphogenesis and conidiophore development. *Chitin has also been related to 

anchoring and displaying a Bey adhesin and virulence factor aI-1 to the surface of the 

yeast phase in Z6+&-%)2#.&*5.$)+-"-"5"& (1). 

Chitin synthases are membrane-bound proteins responsible for the polymerization of 

chitin and have been well characterized in =+##?+$%)2#.&*#.$.0"&"+., where this 

polysaccharide constitutes a small percentage of the wall. In contrast, in filamentous 

ascomycetes and basidiomycetes, chitin conforms a major fraction of the cell wall. 

Chitin synthesis in fungi is a rather complex process, regulated by multigene families 

encoding chitin synthase isoenzymes, some of them redundant, whose activities may 

be spatially and strictly regulated to fulfill the several roles adscribed to them (2). In 1/* 

8$+&"6".,&"&, six chitin synthase genes have been reported (J, O), all of them, with the 

exception of 18$7A=: (for which transcript was not detected), are mainly expressed in 

its saprophitic mycelial phase (J, O). 

ae describe the complete sequence of 18$7A=:, the gene encoding a chitin 

synthase in 1+$+#%##"5"%"5.&*8$+&"6".,&"&. The gene contains a single open reading 

frame made of J817 bp with two introns (71 bp and 86 bp), and encodes a 1220 

amino acid polypeptide with high similarity to class IV chitin synthases. 2y northern 

analysis, expression of 18$7A=: was shown only in the Y phase, and at the end of 

mycelium-to-yeast transition, suggesting an important role of this gene in the cell wall 

formation of the pathogenic yeast phase in 1/*8$+&"6".,&"&/ 

References: 

1. 2randhorst T, llein 2. 2000. (. 2iol. Chem. 27[M 7K2[-7KJO. 

2. Horiuchi H, TaBagi M. 1KKK. InM 2raBhage AA, (ahn 2, .chmidt A., eds. 

4&'.$@"66(&*U()"@+-(&. Contrib Microbiol. 2aselM largerM 2, pp.1KJ-20O. 

J. Nio-Vega et al. 2000. Med. Mycol. J8M J1-JK. 

O. Tomazett Pl, Cruz AH, 2onfim FM, .oares CM, Pereira M. 200[. Gen. Mol. 

Res. OM J0K-J2[. 

P-0678. Successful amplification of the myosin motor-like domain of the chitin 

synthase gene PbrCHS5 from Paracoccidioides brasiliensis completed the 

sequencing of the whole gene 

Nio-Vega G 1 , Sorais F 2 , .an-2las G J 

1 Instituto Venezolano de Investigaciones CientÜficas (IVIC), 2 Instituto Venezolano de 

Investigaciones CientÜficas (IVIC), J Instituto Venezolano de Investigaciones 

CientÜficas (IVIC) 

1+$+#%##"5"%"5.&*8$+&"6".,&"&, a dimorphic fungal human pathogen, is the 

etiological agent of paracoccidioidomycosis. This systemic disorder is restricted 

geographically to Central and .outh America and is one of the most frequent 

mycoses in the rural population of the region. The fungus is present in a parasitic 

yeast form at J7ÅC and a saprophytic mycelial form at 2JÅC. Chitin is a major 

component of 1/*8$+&"6".,&"& cell wall, being almost three times more abundant in 

the yeast phase than in the mycelial form. The absence of both chitin and its 

synthases from human cells maBes the chitin synthase family an attractive target 

for the development of new antibiotics against this medically important fungus. 

Previous results (1) indicated that 1/*8$+&"6".,&"& has no less than five chitin 

synthase genes, that encode for proteins of several classes. Herein, we report 

results on analyses done to amplify the myosin-motor domain of 18$7A=5*in 

order to accomplish the sequencing of the whole gene. 

The upstream region of the 18$7A=5 gene, which encodes for a class V chitin 

synthase, was amplified using a PCR-based technique previously reported (2). 

Genomic DNA was digested separately with two restriction enzymes (`?.I and 

Z&'eI), and ligated to adaptors. Amplifications, either simple or nested, were 

performed with Advantage 2 Polymerase lit (Clontech /aboratories, Inc., Palo 

Alto, CA). These two walBs from `?.I and Z&'eI digestions generated a J08O bp 

product. The DNA sequence generated by this procedure was compared against 

the Jï 18$7A=5 fragment isolated previously by screening of a genomic a#%RI 

library. The entire gene reveals a [[8J nt open reading frame, interrupted by three 

introns of 82, 87 and K7 bp. It encodes a protein of 1861 amino acids with a 

predicted molecular weight of 206.K BDa. The deduced amino acid sequence of 

18$7A=5 shows high homology to 7%##"5"%"5.&*'%&+5+&""*Chs[ and 7%##"5"%"5.&* 

"))"-"& Chs[ (80_ each)n 4&'.$@"66(&*%$2d+.*ChsY, a).$"#.66+*,"5(6+,&*CsmA, 

and 4&'.$@"66(&*U()"@+-(& ChsE (7[_ each)n Y+,@".66+*5.$)+-"-"5"& Chs[ (72_)n 

and Z6().$"+*@$+)","& Chs2 (6K_). The N-terminal region of approx. 800 

residues shows a putative myosin motor domain, in a similar fashion to other 

fungal class V chitin synthase sequences. This technique allowed us to complete 

the [ï end of the 1/*8$+&"6".,&"&*7A=5 gene, a tasB that failed when conventional 

screening partial genomic libraries were constructed. 

References: 

Nio-Vega et al. 2000. Med. Mycol/ J8M*J1-JK 

Zhang Z, Gurr .(. 2000. Gene 2[JM 1O[-1[0

P-0679. Expression of capsule-associated genes of Cryptococcus neoformans 

in the media with high and low amount of glucose 

Okabayashi K 1 , lano R 2 , aatanabe . J , Hasegawa A 2 , aatanabe T 1 

1 Dept. of Veterinary 2iochemistry, Nihon 6niv. .chool of Vet. Medicine, 2 Dept. of 

Pathobiology, Nihon 6niv. .chool of Vet. Medicine, J Dept. of Dermatology, TeiByo 

6niv. .chool of Medicine 

7$2'-%#%##(&*,.%U%$)+,&*produces an extracellular polysaccharide capsule that is 

related to its pathogenicity in human and animals. This cryptococcal capsule varies 

in size depending on the environmental conditions. The elucidation of the molecular 

mechanism of the capsule synthesis is necessary for understanding the pathogenesis 

of 7/*,.%U%$)+,&*infection.* Incubation in diluted .abouraud broth, which contains 

lower amount of glucose, would be one of the most efficient media for capsule 

induction. And, it was demonstrated that four capsule-associated genes (741IQ,* 

7415R,*741NQ and*741NE genes) were essential in capsule synthesis in 7/* 

,.%U%$)+,&. However, their function in capsule synthesis remains unBnown. Then, 

the amount of glucose in the medium was hypothesized to affect the expression of 

four 741 genes. In this study, the investigation on relationship between capsule 

synthesis and expression of*four*741*genes was attempted by quantitative real-time 

PCR with /ightCycler system using three normal encapsulated strains and a stable 

acapsular strain of 741NE gene which was defective. Three normal encapsulated 

strains are serotype D mating type alpha. The acapsular strain could not be 

determined the serotype immunologically due to the lacB of a polysaccharide capsule, 

and its serotype was genetically compatible with serotype A mating type alpha. Each 

strain was incubated in 1_ polypepton medium with 0.1_ or 1[_ glucose until it was 

adjusted in the logarithmic growth phase. Total RNAs were extracted from the 

incubated yeast cells, and reverse transcription was performed for cDNA synthesis. 

The expressions of four 741 genes standardized with respective actin gene 

expression in normal encapsulated strains were higher in the medium with low amount 

of glucose than in the medium with high amount of glucose. The quantitative realtime 

PCR analysis of 741 genes indicated that glucose concentration might have 

some relation to 741 gene expression in association with capsule size. The 

acapsular strain made no detectable expression of 741NE gene and a little 

expression of 741IQ, 7415R and 741NQ*genes compared to that of normal 

encapsulated strains. Therefore, it was suggested that expression of 741NE gene 

might have a regulatory effect on the expressions of the other three 741 genes, 

particularly 741IQ gene. 

P-0680. CCR4 is required for temperature responsive modulation of the 

translational machinery in Cryptococcus neoformans 

Panepinto J 1 , ailliamson P 1,2 

1 6niversity of Illinois at Chicago, Chicago, I/, 6.A, 2 (esse 2rown VA Medical Center, 

Chicago, I/, 6.A 

7$2'-%#%##(&*,.%U%$)+,& is a basidiomycetous yeast-liBe fungus that is a major 

cause of meningo-encephalitis in patients with defects in cell-mediated immunity, 

especially those co-infected with HIV. Previous studies of the *0+5I mutant, lacBing 

an RNA helicase involved in mRNA decapping, have suggested an important role for 

the mRNA degradation pathway in virulence. Deletion of 77PE, encoding the J]-[] 

poly-A exoribonuclease involved in regulated mRNA deadenylation, liBewise resulted 

in virulence attenuation. In addition, the *##$E*mutant exhibits a defect in growth at 

host temperature. To assess the effect of the loss of 77PE on mRNA abundance 

during growth at host temperature, microarray analysis was employed to assay 

genome-wide transcript abundance in a timecourse following a shift from J0 o C to J7 o C 

between the wild type (HKK) and the *##$E mutant. As expected, the majority of 

significantly altered transcripts were up-regulated in the *##$E mutant, presumably 

due to defects in mRNA degradation rather than increased synthesis. .everal 

functional classes of genes were found up-regulated in the *##$E mutant, but most 

abundant were those involved in translation, including structural constituents of the 

ribosome, ribosome biogenesis factors, translation initiation and elongation factors 

and tRNA synthetic machinery. Indeed, the *##$E*mutant exhibits hypersensitivity to 

chemical inhibitors of translation including paromomycin, cycloheximide and 

hygromycin 2, suggesting a defect in translational regulation. In addition, TZ4M3*a 

gene encoding a regulator of posttranslational protein modification through 

sumoylation, was found up regulated in the *##$E mutant. fverexpression of TZ4M in 

the wild type resulted in a significant growth impairment at all temperatures, 

suggesting that appropriate regulation of sumoylation is necessary for normal growth/* 

TaBen together, these results suggest that the mRNA degradation machinery is 

important for regulation of protein synthesis and modification in the pathogenic fungus, 

7/*,.%U%$)+,&/* 


P-0681. Diversity of secreted aspartic proteinases in Candida species. 

Parra B, Cruz H, Villa /, Hernandez C 

ENC2, IPN 

.ome*7+,5"5+*species liBe 7/*+68"#+,&*7/*'+$+'&"6%&"&, 7/*-$%'"#+6"&, 7/*5(86",".,&"&, 

7/*@6+8$+-+, 7/*6(&"-+,"+. and 7/*[$(&." are*associated to candidiasis infections in 

immunocompromised patients. The genome of 7/*+68"#+,&*harbors ten =41 genes 

that encode several secreted aspartic proteinases (.aps) considered as virulence 

factors and molecular targets to antifungal agents. Currently, at least four, two and 

three =41 genes has been recognized in 7/*-$%'"#+6"&, 7/*5(86",".,&"&*and*7/* 

'+$+'&"6%&"&,*mentioned as =41>, =41V and =411 respectively, but no genes of the 

other species has been reported. 

The aim of this worB was explore the spread and diversity of =41 genes among a 

collection*of clinical 7+,5"5+ strains. PCR-based procedures were designed to amplify 

specifically several DNA fragments of =41*genes family from*genomic DNA using 

primers that recognize highly conservative sequences into the target genes. The 

available sequence information was organized in four groupsM group I (.AP1-.APJ 

and =41>E)n*group II (=41EW.AP6)n group III (=41D*and*=41>I) genesh group IV 

(=411IW=411:J. The amplification products of were cloned and screened by RF/P to 

recognize different genes into the same strain and sequenced. The majority of the 7/* 

+68"#+,&,*7/*5(86",".,&"&, and 7/*-$%'"#+6"&*strains amplified group I and II genes, but 

no PCR products were obtained with the other species. fnly in a half of 7/*+68"#+,& 

strains the gene fragments from group III were detected. Group IV gene fragments 

were not amplified in any species, including 7/*'+$+'&"6%&"&, strain used to design the 

PCR procedure. The Neighborg-(oning phylogenetic tree of the =41*genes*and 

nucleotidic and amino-acidic analyses reveled new =41VI and =41V: genes of 7/* 

5(86",".,&"&3*and a =41> gene from 7/*-$%'"#+6"& non-related to previous recognized 

families and spread in approximately J0_ of the strains. Also three genes in the 

genome of 7/*@6+8$+-+*were detected by an ",*&"6"#% analysis including 2/A.T and 

Motif analyses/*=41 genes exhibited a similar topology to 18. DNA gene and IT.1 

sequence previously reported, thus 7/*+68"#+,&*was closer related to 7/*5(86",".,&"&* 

than*7/*-$%'"#+6"& suggesting that the origin and duplication events of =41 genes of 

groups I and II are previous to speciation events. 

P-0682. Identification of an alpha-1,3-glucanase (PbrAGN1) gene in the 

pathogenic fungus Paracoccidioides brasiliensis 

PaulinBevicius M, Nino-Vega G, .an-2las G 


In 1/*8$+&"6".,&"& cell wall, the yeastliBe (Y) phase, but not the mycelial (M) one, 

contains alpha-1,J-glucan as the main (K[_) glucose polymer (1). It has been 

proposed as virulence factor in 1/*8$+&"6".,&"&3*Z6+&-%)2#.&*5.$)+-"-"5"& and* 

A"&-%'6+&)+*#+'&(6+-()*and replaces almost entirely the !-1,J-glucan, which 

comprises the neutral polysaccharide of the vegetative mycelial phase (2). The alpha- 

1,J-glucan is synthesized by alpha-glucan synthase (PbrAG.) and hydrolysed by 

alpha-1,J-glucanase (PbrAGN1), an auto-regulatory system of synthesis and 

degradation, shaping 1/*8$+&"6".,&"& cell wall together with others liBe the chitin 

synthase (CH.) family and beta-glucan synthase. Here we describe the isolation and 

sequence analysis of a partial sequence of 18$49Ì of the pathogenic fungus 1/* 

8$+&"6".,&"& and its expression analysis by RT-PCR. 

The partial sequence presents K72 bp fRF, interrupted by a single intron (60 bp) 

confirmed by RT-PCR. The deduced amino acid sequence of the partial gene 

presents 6J_ identity with 4&'.$@"66(&*,"5(6+,& alpha-glucanase. Trasncripts of the 

gene were detected both in the mycelial and yeast phases, which prompt us to thinB 

on a post-transcriptional regulation of the gene, yet to be evaluated. 

References: 

.an-2las .-*+6. 1K77. Mycopathologia 62M 77-86. 

.an-2las .-*+6/*2002. Medical Mycology O0M 22[-2O2.

P-0683. Rapid differentiation of Microsporum canis and Microsporum 

ferrugineum by using PCR-REA with MaeIII on the ITSII region 

Pereiro Jr M, Alvaro /, /idia P, (aime T 

.ervicio de DermatologÜa, Complejo Hospitalario 6niversitario, Facultad de Medicina, 

.antiago, .pain 

Background: !"#$%&'%$()*#+,"&*is a worldwide distributed dermatophyte and the main 

causative agent of tinea capitis. !/*U.$$(@",.()*is an uncommon dermatophyte, and many 

times, it is difficult to differentiate from !/*#+,"& with conventional procedures. Furthermore, 

several studies using molecular techniques have proposed that !/*#+,"& and !/* 

U.$$(@",.() are conspecific because of the minimum intra-species variability found. 

Material and methods: ae studied a strain of !"#$%&'%$()*#+,"& (CH6.-6[6-02) 

obtained from a patient attended in our Department, and a strain of !/*U.$$(@",.() (.(-0J- 

02) from the collection of the Micology Department of the .t. (ohn Institute of Dermatology 

(/ondon, 6l). .pecimens identification, strains preservation and DNA extraction were 

performed as previously reported. 1 

ae carried out a PCR based sequentiation of these regions by using the universal primers 

IT.-[ ([2GGAAGTAAAAGTCGTAACAAGGJe) and IT. O ([2TCCTCCGCTTATTGATATGC 

J2) s16u. 2 The PCR products were directly sequenced by the dideoxy method (Fmol DNA 

.equencing .ystem, Innogenetics) and analyzed in the A/F express (American Pharmacia 

2iotech). 

The PCR products of IT. II region were digested with the endonuclease restriction enzyme 

!+.III (Amersham Pharmacia 2iotech) following the manufacturer instructions. 

All PCR products and restriction fragments were studied by electrophoresis in 2_ agarose 

gel (Nusieve JM1 agarose, 2iowhittaBer Molecular Applications, RocBland, ME, 6.A), 

stained with ethidium bromide prior to photographed nucleotide sequences were compared. 

They were also compared with others obtained from GenbanB (!/*#+,"&*A(00061Kn !/* 

ferrugineum A(2[2JJ8). 

Results: The comparison of the sequences of IT. I and II regions of M. canis CH6.-6[6- 

02 and M. ferrugineum .(-0J-02 showed they were similar in a K8_. However, we found a 

teorethical cut-point in the IT.II region that can be useful to differentiate between the two 

species by the MaeIII digestion of the PCR product. 

After !+.III digestion, electrophoresis showed two different patternsM !/*#+,"&*CH6.-6[6- 

02 was not cutted3*obtaining the corresponding prior JK0 bp band, while the restriction of !/* 

U.$$(@",.() .(-0J-02 yield two bands of 210 bp and 180 bp. 

Comments: ae present the complete sequence of IT. I and II of the two species and 

propose the PCR amplification and restriction enzyme analysis with !+.III of the internal 

transcribed space region II for a rapid and easy differentiation of these species. 

References 

1 A. /eon-Mateos, C. Paredes-.uZrez, M. Pereiro, (r., (. Toribio. .tudy of the IT. 

region in an atypical isolate and comparison with six species of !"#$%&'%$()/ !2#%&.&i* 

Accepted in june 22, 200O/ 

P-0684. Some biological features of Candida albicans mutants for genes coding 

fungal proteins exhibiting the eight-cysteine-containing CFEM domain. 

Perez A 1 , Pedros 2 1 , Murgui A 1 , Casanova M 1 , Ramage G 2 , /opez-Ribot ( J , Martinez 

( 1 

1 6niversity of Valencia, 2urjasot, Valencia, .pain, 2 Glasgow Caledonian 6niversity, 

Glasgow, 6nited lingdom, J The 6niversity of Texas at .an Antonio, .an Antonio, 

Texas, 6.A 

In this paper we describe the cloning and characterization of a novel 7/*+68"#+,& gene 

(A1ZI) coding for a putative protein species containing an eight cysteine domain 

referred as CFEM (common in several fungal extracellular membrane proteins), which 

has been identified by analyzing fungal sequences selected from database sequence 

searches. CFEM-containing proteins could function as cell-surface receptors or signal 

transducers, or as adhesion molecules in host-pathogen interactions (1). The product 

of A1ZI gene (a protein of 2[.17 BDa) exhibited some features reminiscent of a class 

II-type hydrophobin, which was the basis for the gene designation. Hydrophobins 

appear to be of ubiquitous occurrence among filamentous fungi belonging to the 

ascomycetes and the basidiomycetes and are among the most surface active 

biomolecules Bnown, being involved in cell adhesion and interaction with the 

environment (2). Deletion of A1ZI gene resulted in a cascade of pleiotropic effects, 

mostly affecting cell surface related properties that may be essential in the interaction 

of the fungal cells with the environment. In this context, the null*?'8I# mutant formed 

very fragile biofilms that appeared partially split and weaBly attached to the substratum. 

The biofilm-forming ability of several 7/*+68"#+,& mutants with single, double and triple 

deletions of genes encoding other protein species also containing the CFEM domain 

(PZ>5, and Y41I/7=4I) was determined. .imilarly to observations made with the 

single ?'8I# null mutant, strains bearing mutations in the above mentioned genes 

also exhibited an abnormal ability to form biofilms. fverall, evidences reported here 

suggest that fungal proteins containing the CFEM domain (Hpb1p, Rbt[p, and 

aap1p/Csa1p) may play a Bey role in the formation, development and/or maintenance 

of the biofilm structure in 7/*+68"#+,&. Disruption of the above mentioned genes 

encoding proteins containing the CFEM domain did not significantly affect virulence as 

assessed in a murine model of experimentally induced candidiasis. Analysis by /ight 

Cycler RT-PCR revealed that A1ZI gene was overexpressed in mycelial cells when 

compared to yeast. In this context, in order to perform a quantitative analysis of 

expression of Hpb1p by fluorescence microscopy, the insertion of the A1ZI promoter 

region into pGFP is currently under construction. A preliminary proteomic analysis 

revealed the presence of Hpb1p in the extracellular polymeric material of biofilms, as 

well as a number of proteins that may be involved in the maintenance of biofilm 

integrity and functionality and could be regarded as a potential targets for the 

development of strategies for the prevention and/or treatment of candidiasis. 

References: 

lulBarni, R.D., lelBar, H... and Dean, R.A.(200J) Trends 2iochem. .ci. 28, 118-121. 

aésten, H. A. (2001) Annu. Rev. Microbiol. [[M62[-O6. 


P-0685. A Candida albicans mannoprotein fraction stimulates the B16 

melanoma cell adhesion to mouse hepatic sinusoidal endothelium. 

Ramirez Garcia A 1 , Gallot N 2 , Abad A 1 , Dominguez . 1 , GuzmZn C 1 , Huerga V 1 , 

Mendoza / 2 , Vidal-Vanaclocha F 2 , Hernando F 1 

1 Department of Immunology, Microbiology and Parasitology. 6niversity of 2asque 

Country. /eioa. .pain, 2 Dominion PharmaBine ../. 2izBaia Technological ParB. 

2uilding 801. Derio. .pain. 

Purpose: The aim of this study was to analyze the effect that 7+,5"5+*+68"#+,& 

fractionation exerts in cell adhesion of 216 melanoma to mouse Hepatic .inusoidal 

Endothelium. To achieve this, we have obtained by electrophoretic fractionation five 

mannoprotein fractions with different Molecular aeight that were incubated with H.E and 

assayed for induction of 216 cells adhesion. 

Description: The first place where the microorganisms that have entered the bloodstream 

are retained is the endothelial cell lining of the vasculature. The hepatic endothelium cells 

have, among others, a mannose receptor that is implicated in the host immune response 

against 7+,5"5+*+68"#+,&. This receptor is responsible for the clearance of mannosylated 

circulating waste molecules, such as enzymes and extracellular matrix and it may also 

clear out infectious agents such as protozoans, bacteria or fungi as 7+,5"5+*+68"#+,&. The 

most important antigenic component from the cell wall of 7+,5"5+*+68"#+,&*are the 

mannoproteins, which are also implicated in the resistance to phagocytosis and adhesion 

to host. ae have studied that 216M cell adhesion to H.E is enhanced in H.E cells that 

have been previously treated with the mannoprotein fraction of 7+,5"5+*+68"#+,&. 

Crude extract obtained from 7+,5"5+*+68"#+,& 6PV1O1J strain was fractionated using 

Preparative Electrophoresis in OK1 Prep Cell (2io-Rad) and Econo-system (2io-Rad). Pore 

size and gel length for each fraction were empirically calculated in order to obtain a great 

purificationM Five fractions were obtainedM F1 (v J0 BDa), F2 (J0 | O[ BDa), FJ (O[ | 66 

BDa), FO (66 | 11[ BDa), F[ (t11[ BDa). 

Mannoproteins of each fraction were purified by Con-A affinity chromatography and eluted 

with methyl $-D-manno-pyranoside. After dialysis and concentration, protein and 

carbohydrate concentration was assayed and all the fractions were adjusted to the same 

carbohydrate concentration. 

Then, cells from murine Hepatic .inusoidal Endothelium were incubated with the 

Mannoprotein fractions previously obtained. After washing, 2CECF-labeled 216M cells 

were added. Tumor cell adhesion was assessed by plate-scanning fluorimetry of 2CECF 

fluorescence. 

P-0686. Elucidation of the calcineurin signaling cascade in Candida albicans 

Reedy J, Heitman ( 

DuBe 6niversity Medical Center, Durham, North Carolina, 6.A 

Purpose of the study: The serine-threonine specific calcium-calmodulin activated 

protein phosphatase calcineurin plays a crucial role in the virulence of two human 

fungal pathogens, 7$2'-%#%##(&*,.%U%$)+,&*and 7+,5"5+*+68"#+,&, but via distinct 

mechanisms. Calcineurin is essential for 7/*,.%U%$)+,& growth at high temperature, 

whereas in 7/*+68"#+,& calcineurin promotes tolerance to azoles, and survival during 

calcium stress imposed by growth in serum. fur worB seeBs to identify components 

of the calcineurin signaling pathway in 7/*+68"#+,&/*** 

* 

Description: To achieve this goal we have employed both Candidate gene 

disruptions and mutant screens of homozygous and heterozygous libraries to identify 

signaling components. Rcn1/Cbp1/D.CR1 is a calcineurin binding protein that 

modulates the activity of calcineurin in =+##?+$%)2#.&*#.$.0"&"+.3*7/*,.%U%$)+,&, 

and mammalian cells. ae identified the 7/*+68"#+,& homolog of Rcn1 based upon the 

highly conserved F/I.PPx.PP domain, and disrupted the P7Ì gene using the .AT1 

flipper cassette. ae have also identified Mid1 and Cch1, two components of a cell 

membrane calcium channel important for calcineurin activation in =/*#.$.0"&"+./ 

Results and Conclusions: $#,I

P-0687. The qualitative and quantitative evaluation of glass-bead, boiling and 

freeze-thawing methods for extraction of genomic dna from yeasts 

Rezaie A, Mirhendi H, (alalizand N 

Tehran 6niversity of Medical .ciences 

Yeasts are the causative agents of cutaneous, mucosal and systemic infections in 

human and animals. Extraction of DNA from yeasts is essential for many research and 

diagnostics purposes. Different methods have suggested for extraction of the genomic 

DNA from yeasts. In this study the qualitative and quantitative potential of Glass- 

2eads, 2oiling and Freezing-Thawing methods in extracting of genomic DNA from 

three important yeast speciesn 7+,5"5+*+68"#+,&3*=+##?+$%)2#.&*#.$.0"&"+.*and* 

7$2'-%#%##(&*,.%U%$)+,& was assessed/* 

The reference strains were obtained from American Type Culture Collection (ATCC), 

subcultured for O8 hours at J0 C on .GA medium and serial dilutions were prepared 

to a concentration of 10 1 to 10 7 cells/ml. DNA extraction was performed using the 

mentioned methods and all of them were purified by phenolM chloroform procedure. 

PCR was performed using the universal primersM IT.1 and IT.O and PCR products 

were electrophoresed through a 1.[_ agarose gel and visualized by ethidium bromide 

staining. The DNA extracted from the yeasts were also diluted to a concentration of 

10 -1 to 10 -6 of primary DNA sample for each methods and PCR was performed for 

quantitative assessment of the methods. Each experiment was repeated three times. 

Glass-2eads method was able to show visible band for 10 O , 10 [ and 10 [ cells of 7/* 

+68"#+,&3*=/*#.$.0"&"+.*and 7/*,.%U%$)+,&3 respectively. The lowest cell concentration 

required for extracting DNA and successful PCR in boiling method was 10 [ , 10 O and 

10 [ cells for 7/*+68"#+,&3*=/*#.$.0"&"+.*and 7/*,.%U%$)+,&3 respectively. Freeze- 

Thawing method could extract DNA from 10 [ cells in all of three yeast species. After 

diluting the extracted products, performance of PCR by the same primers was able 

to amplify the DNA extracted by Glass-2eads method , up to 10 -O , 10 -O , 10 -J , DNA 

extracted by 2oiling method , up to 10 -J , 10 -2 , and 10 -J dilutions and the DNA 

extracted by Freeze-Thawing method up to 10 -2 dilution for 7/+68"#+,&*3*=/*#.$.0"&"+.* 

and 7/*,.%U%$)+,&3 respectively.** 

P-0688. Molecular characterization of NADPH-dependent dehydrogenase in 

dermatophyte pathogen Trichophyton rubrum 

NADPH dependent Dehydrogenase (NDH) is revealed to have a critical role in serine 

degradation as well as gluconeogenesis in mammalian cells. Neither the presence nor 

the biochemical properties of the NDH has been well demonstrated in filamentous 

fungi. 

In the present study, we try to characterize the NDH gene in dermatophyte pathogen 

>$"#?%'?2-%,*$(8$() (>/*$(8$()). 

This dermatophyte is the most common cause agents of dermatophytosis in human 

sBin and nail tissue. .ome properties of >/*$(8$() have been investigated in 

molecular leveln however, no information is available regarding the NDH in this 

dermatophyte. 

>/*$(8$() was obtained from patients with dermatophytosis and cultured in appropiate 

conditions. High molecular weight DNA was isolated from obtained mycelial mass by 

standard methods. Pairs of 21 nt primers were designed from highly conserved 

regions of the NDH genes in other euBaryotic cells. Mentioned primers were utilized in 

PCR by using isolated genomic DNA template of >/*$(8$(). Predicted molecules have 

been amplified and were submitted for sequencing. 2y the time 2[0 nucleotides of this 

gene are sequenced. The characterization of this PCR fragments which revealed 

significant homology with other NADPH dependent Dehydrogenase Genes in 

Gen2anB (NC2I, NIH, 6.A) is still under investigation. 

Considering all results, it seems that the Glass-2eads and 2oiling methods are more 

appropriate for extraction of the yeast genomic DNA than the Freeze-Thawing method. 

There were no important difference between the quality and quantity of extracted DNA 

between the Glass-2eads and 2oiling methods but according to the simplicity of 

performance and lower cost, the 2oiling method is recommended for genomic DNA 

extraction from yeasts in routine laboratories. 


P-0689. Expression regulation of the GP43 antigen from Paracoccidioides 

brasiliensis 

Rocha A 1 , Morais F 2 , McEween ( J , Puccia R 1 

1 6niversidade Federal de .Éo Paulo (6NIFE.P), .Éo Paulo, .P, 2razil, 

2 6niversidade do Vale do ParaÜba, .. (osd dos Campos, .P, 2razil, J Corporacián 

para Investigaciones 2iolágicas, Medellin, Colombia 

Purpose of the study: 1+$+#%##"5"%"5.&*8$+&"6".,&"& is a thermo-dimorphic fungus that 

causes paracoccidioidomycosis (PCM) in man. The gpOJ is the main diagnostic and 

prognostic antigen of PCM. It elicits cellular immune response, which is protective in 

vaccinated mice, but may be involved in adhesion and invasion. GpOJ is a secreted 

glycoprotein, homologous to fungal -1,J-exoglucanases, that can be detected as 

circulating antigen. The regulatory factors involved in gpOJ expression are unBnown, but 

we have provided the first evidences of transcriptional regulation, besides regulation at the 

protein and secretion levels (Microbes Infect. 7M[[-6[, 200[). In vitro, secretion varies with 

the isolate, culture media and growth phase. Polymorphism in the Pb91E: exon 2 and its 

promoter region defines phylogenetic groups of isolates and the most polymorphic 

sequences are found in a sexually cryptic species (10_ of total) composed so far of 6 

isolates ((. Clin. Microb. J8MJK60, 2000n Mol. 2iol. Evol. 2JM6[, 2006). Among them, Pb2, 

PbJ and PbO provoBe a regressive type of PCM in 210.A mice. The gpOJ they express is 

peculiarly basic and we observed differential gene regulation (down-regulation) upon heat 

shocB in PbJ (Microbes Infect 7M[[, 200[). 

Summarized description of the project: fur laboratory is interested in understanding 

expression regulation of gpOJ. ae are investigating gene and protein expression of 

selected isolates from distinct phylogenetic groups. Gene expression is measured by real 

time reverse-transcriptase (RT)-PCR. Identification of transcription elements in the 

promoter region is using electrophoretic mobility shift assays (EM.A) and protection 

DNase I footprinting. 

Results and conclusions: ae initially used EM.A with total protein fungal extracts and 

selected nucleotides containing putative transcription elements to the direction of Pb91E:, 

according to the TF.EARCH computer program. ae identified at least one transcription 

element in the sequence between nt -1JO and |10J (2.8 and 2.8.2), where base changes 

in the P2J polymorphic sites |10O (T/A) and |120 (G/C) did not alter the reactions. 

However, they create putative transcription elements for H.F and E2P in PbJ, besides the 

existing NIT2, .ox-[ and cap. DNAse I footprinting assays revealed five protection regions 

between positions |261 and |1O6 (ET1 to ET[). Two oligonucleotides between nt |2[[ 

and |217 (ET1r2n ET2rJ) were also positive in EM.A with total protein extracts from 

PbJJK, Pb18 and PbJ. Few predicted transcription elements are found in these regions, 

and only those binding MyoD and CdxA are in the Pb91E: direction. In turn, several are 

found in ETO (NIT2, deltaE, cap), but so far that nucleotide and ET[ have been negative in 

EM.A. .hifted complexes with PbJ extract migrated consistently faster, suggesting 

polymorphism in the transcription factors. The functionality of the mapped transcription 

elements in the control of Pb91E: expression has not been tested. An evaluation of 

transcription and protein expression of gpOJ in a sample of isolates grown under heat 

shocB, excess glucose, nitrogen sources and sera is on course. Comparison with less 

pathogenic isolates is being focused. fur data will help understand expression regulation 

of gpOJ and hopefully its role in PCM. 

P-0690. PbrSEP2, a member of the Paracoccidioides brasiliensis septin family 

Rodriguez-2rito ., Nino-Vega G, .an-2las G 


.eptins are proteins involved in a wide variety of processes from cytoBinesis and cell 

morphology, to regulation of the cell cycle in euBaryotic cells. In fungi, they are 

involved in selection of the budding place, chitin deposition, and spore cell wall 

formation. In this worB we set up to study 18$=a1M, one*of the genes conforming this 

family in 1+$+#%##"5"%"5.&*8$+&"6".,&"&, and its expression when the thermal transition 

from the mycelial (M) to the yeastliBe (Y) phase occurs. 

.outhern analysis on 1/*8$+&"6".,&"& DNA was done using as radiolabeled probe a 

PCR fragment of 660 bp, amplified with degenerated primers, designed on high 

homology regions for fungal septins. From .outhern analysis, a 1&-I genomic library 

was costructed, which is being screened by colony hybridization, in order to get the 

whole gene sequence. The polyadenylation site has been identified by the JïRACE 

system (Invitrogen, CA) using the sequence of the PCR product for the design of 

specific primers. Expression of the gene was followed by Northern analyses in the Y 

and M phases, as well as during the dimorphic switch from M to Y. As loading control, 

a fragment of the 18$rRNA 18. gene was used. 

The deduced aminoacid sequence for the PCR product obtained by the use of 

degenerated primers, shows high identity to a).$"#.66+*,"5(6+,& asp-C and other 

fungal septin proteins, and has been designated 18$=a1M. 

An 8O0 bp fragment was amplified by the J] RACE system. .equencing of the 

amplified fragment confirmed the ending point of the gene and its J]6TR region up to 

the polyadenylation site. Northern analyses show gene expresion in Y and M cells, 

although an incresing signal was detected when transition occurred from the M to the 

Y phase, with higher expression at 72 h into the transition. 

Expression of the 18$=a1M*gene increases during the M to Y transition, with a higher 

expression 72 h into the transition, when more than K0_ of the culture shows 

yeastliBe structures. This result might suggest the importance of this gene in the 

formation of the spherical morphology. Reverse genetics studies could help to clarify 

this preliminary finding and the role of the 18$=a1M gene in morphogenetics. 

SUPPORTED BY FAPESP AND CNPQ 

Financial support: FAPE.P and CNPq

P-0691. Identification of regulatory elements of the multidrug transporter gene 

CaMDR1 in Candida albicans 

Rognon B 1 , Pardini G 1 , Coste A 1 , lozovsBa Z 2 , .anglard D 1 

1 Institute of Microbiology, 6niversirty Hospital /ausanne,.witzerland, 2 Comenius 

6niversity, 2ratislava, .lovaB Republic 

6pregulation of the Major Facilitator transporter gene 7+!VPI (7+,5"5+*+68"#+,& 

Multidrug resistance 1) is one of the mechanisms observed in 7+,5"5+*+68"#+,&* 

clinical isolates*developing azole resistance. In this study, the regulatory elements 

present in the 7+!VPI promoter were delimited using a reporter system in i) an 

azole-susceptible strain exposed to benomyl or to the oxidative stress agent H 2f 2 that 

allows transient 7+!VPI*upregulation and ii) an azole-resistant strain upregulating 

7+!VPI constitutively (strain FR2). Two regulatory elements were identifiedM one 

called 2RE (-2K6 to -260 with respect to the first ATG) responsible for benomyl 

responsiveness and another region, HRE, (-[60 to -[20) responsible for H 2f 2 

responsiveness. fnly the 2RE was shown to be functional in FR2 suggesting that, 

although 7+!VPI upregulation can be obtained by transient and constitutive 

upregulation, these two processes converge to the same regulatory element and 

probably mobilize the same transcription factor. Deletions and nucleotide 

transversions within the 2RE abolished or highly diminished the benomyl 

responsiveness and the high expression of the reporter gene in FR2, whereas the 

H 2f 2 responsiveness was only altered by the deletion of the HRE. The presence of 

two Cap1p binding sites within the HRE (TTAG/CTAA) suggested that Cap1p, a bZip 

transcription factor involved in oxidative stress, played a role in H 2 f 2 response. .ince 

H 2 f 2 induction of 7+!VPI was abolished in a #+'I0/0*mutant, it confirmed that 

Cap1p was the major transcription factor involved in this H 2f 2 response. However, 

Cap1p was not essential for the function of the 2RE, thus indicating that a distinct 

factor could bind to this element. The 2RE fused to the benomyl non-inducible 7VPM 

promoter (2RE-CDR2p) converted the 7VPM promoter into an inducible promoter. 

The 2RE-CDR2p also mediated high reporter activity in FR2, thus demonstrating that 

the 2RE can function as an independent element. Finally, binding experiments 

performed with a protein extract from a cell exposed to benomyl showed that a protein 

complex effectively binds the identified 2RE. Experiments are now performed to 

identify the transcription factor that mediates the response to benomyl via the 2RE. 

P-0692. Detection of molecules on Histoplasma capsulatum yeasts by scFv 

antibody fragments displayed in phage M13 

Romero-Martinez R 1 , Curiel-~uesada E 2 , 2ecerril-/ujZn 2 J , Flores-Carreán A O , 

Taylor M [ 

1 6niversidad Nacional Autonoma de Mexico, 2 Instituto Politecnico Nacional, 

J 6niversidad Nacional Autonoma de Mexico, O 6niversidad de Guanajuato, 

[ 6niversidad Nacional Autonoma de Mexico 

The present communication is attempted at detecting, which types of surface 

molecules are constitutively expressed by A"&-%'6+&)+*#+'&(6+-() yeasts, hoping to 

define further their role in the interaction with host-cells. To accomplish this aim a nonimmune 

library, containing single chain fragments variable (scFv) of immunoglobulin 

human genes displayed on the surface of filamentous phages-M1J, was used to 

recognize different molecules exposed on the yeasts surface, during their growth in 

synthetic culture medium. The obtained scFv clones were checBed in their phage 

consistency by Dot-E/I.A using HRP/anti-M1J conjugate and the scFv clones, able to 

recognize molecules on A/*#+'&(6+-() yeasts surface, were identified by E/I.A in 

plates. Three scFv monoclones (C2, C6, and C[2) were able to react with A/* 

#+'&(6+-() molecules. The C2 clone recognized fungal molecules from both A/* 

#+'&(6+-() morphologic phases, whereas C6 and C[2 clones only reacted with 

molecules on the surface of whole yeasts, with molecules from the cytosol after 

subjecting yeasts to mechanical lysis, and with solubilized molecules released in the 

supernatant of the yeast culture. The phage-display methodology represents an 

advantage over other methods that reveal gene transcription, as it detects proteins as 

a final product without any risB of gene interruption due to diverse mechanisms. The 

proteins expressed would allow the development of related studies to define the 

genuine role of these molecules in the host-parasite relationship of histoplasmosis. 

Monoclones C2, C6, and C[2 recognize yeast molecules irrespective of the A/* 

#+'&(6+-() strains used, emphasizing that C6 presented less reaction with A/* 

#+'&(6+-() hDownsi strain components. This research was partially supported by 

CfNACYT-Mexico (RefM OJKOO-M). 


P-0693. Studies on the Candida albicans frataxin deficient mutant 

Santos R, /esuisse E, Camadro ( 

Institut (acques Monod, Paris, France 

The 7+kÂI*gene encodes the yeast frataxin homologue in 7+,5"5+*+68"#+,&. The 

Friedreich]s ataxia is an autosomal recessive neurodegenerative disease caused by 

reduced expression of frataxin. This is a mitochondrial protein and despite the 

attractive hypothesis that it could be an iron donor to heme and iron-sulfur cluster 

biosynthesis, its role in the cell is not absolutely defined. .tudies on the yeast 

=+##?+$%)2#.& #.$.0"&"+. orthologue kÂI have contributed to our understanding of 

the function of frataxin. ae cloned the 7+kÂI gene and studied the phenotype of 

frataxin defficient 7/*+68"#+,& cells. The 7/*+68"#+,& frataxin mutant had severe 

defective growth on glucose media and was unable to grow on respiratory substrates. 

It lacBed activity of iron-sulfur cluster enzymes and heme synthesis. Although mutant 

cells accumulated iron in the mitochondria they behaved liBe iron-deprived wild-type 

cells by inducing the three iron-uptaBe pathways and by secreting flavins. The mutant 

cells accumulated reactive oxygen species and were hypersensitive to oxidative 

stress. Treatment of mutant cells by hydrogen peroxide and paraquat had dramatic 

effects, less than 0.[ _ of the population formed viable colonies when plating on 

complete agar medium. Preliminary results suggest that the cell death induced by 

oxidative stress is caused by apoptosis. 

P-0694. Recent advances in molecular biology of the pathogenic saprophyte 

Aspergillus fumigatus 

Sasse C, 2ayram , 2raus G, lrappmann . 

Institute of Microbiology c Genetics, Goettingen, Germany 

Research on the saprobic asexual fungus 4&'.$@"66(&*U()"@+-(& has increasingly 

become of interest as mycoses caused by 4&'.$@"66" represent a prevalent threat to 

the immuno-compromised individual. The latest finishing of the 4/*U()"@+-(& genome 

sequence annotation opens the field of large-scale genome manipulation 

accompanied by comprehensive gene function analyses. For targeted gene deletions 

we designed a blaster module allowing selection of transformants as well as 

(counter)selection of a Cre recombinase-mediated marBer excision event. For 

validation purposes we deleted the 4/*U()"@+-(&*'+8+4 gene in a wild-type isolate 

maBing use of this cassette. The resulting strain served as save recipient for 

subsequent targeting of the 0.60.- locus. Homologous reconstitution was performed 

by an allele, the expression of which is driven in a nitrogen source-dependent manner 

as validated by Northern analyses. fverexpression of the 0.4 locus in 4/*U()"@+-(& 

does not result in any obvious phenotype, whereas sporulation capacities of the 0.4* 

,(66 mutant are reduced on nitrate-containing medium. Furthermore, we explored gene 

targeting efficiencies in a genetic mutant bacBground that is impaired in the nonhomologous 

end joining pathway. The +[(4 gene encoding the lu70 component of 

the NHE( machinery was deleted in the clinical 4/*U()"@+-(&*isolate D1O1, and no 

obvious phenotype could be assessed for the corresponding mutant strain. Infections 

in an insect model using larvae of the greater wax moth 9+66.$"+*).66%,.66+ revealed 

that the +[(4* strain is as pathogenic as its wild-type progenitor in this alternative test 

system. Frequencies of homologous recombination are strongly increased in this 

bacBground as deduced from targeting the +8$M gene using replacement cassettes 

with flanBing homology arms as short as [00 bp. Conclusively, this genetic 

bacBground might prove useful in gene targeting experiments to generate deletion 

mutants or tagged alleles in 4/*U()"@+-(&.

P-0695. Prediction of lipase activity of chromoblastomycosis agents cultured in 

solid medium using infrared spectroscopy 

Scroferneker M 1 , Corbellini V 2 , da Costa ( 1 , Vicenti ( 1 , FerrÉo M 2 , de .ouza T 1 

1 Instituto de Ciüncias 2Zsicas da .aâde - 6niversidade Federal do Rio Grande do .ul 

(6FRG.), 2 Departamento de FÜsica e ~uÜmica - 6niversidade de .anta Cruz do .ul 

/ipase production by chromoblastomycosis agents has been of great interest due to 

its possible correlation with the virulence and pathogenicity of clinical lesions, as 

occurs with other pathogenic fungi. This study proposes the use of a chemometric 

model for predicting lipase activity of chromoblastomycosis agents in a solid medium 

using metabolic footprint analysis obtained by Diffuse Reflectance Infrared Fourier 

Transform .pectroscopy (DRIFT.) and Partial /east .quares (P/.) regression. 

Initially, we monitored the Pz value, defined as the ratio between radial mycelial 

growth and calcium oleate precipitate of eight strains of chromoblastomycosis agents 

(^%,&.#+.+*'.5$%&%" ATCC O6O28, ATCC O6O22, IMT.P OK, I~E OOO.62, 

1?"+6%'?%$+*0.$$(#%&+ FMC 221O, 76+5%&'%$"()*#+$$"%,ii IMT.P 680, 

76+5%'?"+6%'?%$+*8+,-"+,() 2K07-78 and a_%'?"+6+*\.+,&.6)." CRfMf.HC8) 

cultured for 10 days in a medium containing Tween 80. Afterwards, the dehydrated 

biomass of these agents cultured in the same medium for 1O and 21 days were 

analyzed by DRIFT. in the spectral range of J800 to 600 cm -1 and the spectra were 

correlated with the lipase activity of agar fragments with the corresponding biomass 

using the para-nitrophenyl palmitate assay and P/. processing. The results show that 

the Pz value increases with time, reaching a plateau under limited nutritional 

conditions. The metabolic footprint biomass analysis by DRIFT./P/. showed that it is 

possible to predict extracellular and mycelium-bound lipase activity through crossvalidation 

models for ^/ '.5$%&%" strains (Group I) and for other agents (Group II) by 

combining spectral ranges (16[0-1[[0) r (1O60-1JJ0) r (1J00-1100) cm -1 . The best 

model was obtained at 21 days with seven principal components, with a correlation 

coefficient of validation (rVal) of 0.K[ and a standard error of validation (.EV) of 0.0[8 

for group I, and rVal } 0.7J and .EV } 0.1J for group II. The ranges indicate that 

extracellular and mycelium-bound lipase activity is correlated with intracellular protein 

concentration (amide I bands and N-H group) and frequencies of vibrational 

movements of aromatic compounds and C-f bonds of sugars and melanin derivatives. 

P-0696. In situ detection of nitroreductase activity in Sporothrix schenckii using 

6-nitrocoumarin 

Scroferneker M 1 , .topiglia C 1 , Carissimi M 1 , Daboit T 1 , Corbellini V 2 

1 Instituto de Ciüncias 2Zsicas da .aâde - 6niversidade Federal do Rio Grande do .ul 

(6FRG.), 2 Departamento de FÜsica e ~uÜmica - 6niversidade de .anta Cruz do .ul 

Nitroreductases are enzymes that reduce nitro (Nf 2), nitrous (Nf), or hydroxylamine 

(NHfH) groups to amino (NH 2 ) and are associated with intrinsic mechanisms of 

resistance to antifungals, being also used for chemotaxonomic differentiation. This 

enzyme activity is detected by spectrophotometric, fluorimetric, chromatographic, and 

visual methods. There has been a paucity of literature data about the ",*&"-( detection 

of this activity using epifluorescence microscopy. Therefore, the aim of the present 

study is to assess the ",*&"-( nitroreductase activity in ='%$%-?$"_*&#?.,#["" strains 

using 6-nitrocoumarin. 

.ix =/*&#?.,#["" strains were cultured in potato dextrose agar containing Tween 80. 

The culture medium contained 6-nitrocoumarin diluted in dimethyl sulfoxide, the 

positive control consisted of 6-aminocoumarin diluted in dimethyl sulfoxide, and the 

negative control contained only dimethyl sulfoxide. The strains were incubated for 1O 

days at J0ÅC and examined under an epifluorescence microscope with a 6V-2A filter 

blocB. The strains showed moderate fluorescence intensity for 6-aminocoumarin and 

in the culture medium without the substrate. Thus, we suggest that the analyzed 

strains had a partial reduction of 6-nitrocoumarin, and that this intermediate should be 

then determined by more specific assays. 


P-0697. Molecular typing of Aspergillus fumigatus isolates collected from 

cystic fibrosis patients 

Sisto F 1 , Cariani / 2 , .caltrito M 1 , Drago M 1 , Morace G 1 

1 Microbiologia, 2 Fondazione IRCC. fspedale Maggiore Policlinico, Mangiagalli e 

Regina Elena 

The aim of this worB was to verify if cystic fibrosis (CF).patients were infected by a 

unique 4&'.$@"66(&*U()"@+-(& genotype because literature data report that some 

genotypes are dominant and persistent in immunocompromised hosts. 

Twenty-seven isolates of 4/*U()"@+-(& were collected from the respiratory tract 

secretions of patients with CF. In these patients 4/*U()"@+-(& is supposed to be a 

colonizer rather than it can plays a role as opportunistic pathogen. In fact, 

1&.(5%)%,+&*+.$(@",%&+*and in some cases*=-+'?26%#%##(&*+($.(& were 

contemporaneously isolated from the same specimens/*In order to verify genetic 

relatedness, molecular typing methods including random amplified polymorhic DNA 

(RAPD) with two different primers (N.J and R108) and 4U(-*I restriction fragment 

lenght polymorphism (RF/P), were performed. 6se of combination of methods 

resulted in a greater degree of discriminatory power than that can be achieved by a 

single method. fur results suggest that different types of 4/*U()"@+-(& exist in CF 

patients as colonizers. 

For comparative purposes, we also tested the in vitro susceptibility of the 27 4/* 

U()"@+-(& isolates to voriconazole, itraconazole and amphotericin 2. No correlation 

was observed among genotypes and susceptibility patterns, that did not show 

significant differences except for one strain with high MIC value for itraconazole (16 

&g/ml). This strain was furtherly investigated for the full coding region sequences of 

1O$-sterol demethylase genes (#2'5I4 and #2'5IZ). .equence analysis revealed no 

significant point mutations that could explain the low itraconazole susceptibility. The 

expression of efflux pumps is now under evaluation. 

P-0698. Characterization of two new members of the Paracoccidioides 

brasiliensis rho gene family (PbrRAC1 and PbrCDC42) 

Sorais F 1 , Nio-Vega G 2 , .an-2las G J 

1 Instituto Venezolano de Investigaciones CientÜficas (IVIC), 2 Instituto Venezolano de 

Investigaciones CientÜficas (IVIC), J Instituto Venezolano de Investigaciones 

CientÜficas (IVIC) 

P47I and 7V7EM are members of the Rho family of small GTPases-encoding genes 

and have been found in several species of fungi where they play critical roles in actin 

cytosBeleton organization and cell polarization. Interestingly, P47I has not been 

reported in =+##?+$%)2#.&*#.$.0"&"+.*(.an-2las and Nio-Vega, 200On Ruiz-Herrera 

.- +6., 200O). 1+$+#%##"5"%"5.&*8$+&"6".,&"& is the causative agent of the principal 

systemic mycosis in /atin America. The cell wall in the yeast (Y) pathogen phase at 

J7o C is round, meanwhile the mycelial (M) saprophyte phase at 2Jo C grows as 

hyphae, maBing this fungus an amenable model to study the mechanisms of control of 

cellular morphogenesis (.an-2las .-*+6., 2002). In this dimorphic human pathogen, 

initially two genes of the PAb gene family called 18$PAbI and 18$PAbM*were 

identified. ae describe here the identification and characterization of two new 

GTPases in 1/*8$+&"6".,&"&, 18$P47I and 18$7V7EM. 

18$P47I.- During the cloning of P8$PAbI*and 18$PAbM, a third related gene was 

identified and isolated by screening a partial A",5III genomic library. The nucleotide 

sequence of this gene (K86 pb) showed an open reading frame of [K7 bp, separated 

by five introns. Predicted product of the P47I gene was 1KK amino acids in length 

with an estimated molecular weight of 22 BDa. The encoded Rac1 protein showed 

high homology to Rac proteins from 4&'.$@"66(&*U()"@+-(& (K2_), 4&'.$@"66(&*,"@.$ 

(K1_), 1.,"#"66"()*)+$,.UU." (K1_) and 7%66.-%-$"#?()*-$"U%6""*G8K_). 

18$7V7EM.- Amplification reaction on 1/*8$+&"6".,&"& genomic DNA with primers EF 

and IV-R (Mirbod .-*+6., 1KK7), yielded a single band of 6[6 bp. Preliminary nucleotide 

sequence analysis of the PCR product revealed a high homology to other fungal 

7V7EM genes. Further completion of 18$7V7EM*and expression of related protein 

during M to Y transition*are underway in our laboratory. 

The role of these Rho GTPases therefore remains to be determined, in particular their 

role in the biosynthesis or remodeling of the fungal cell wall. In order to achieve this 

aim, RNAi technology is been explored. 

AcBnowledgementM Grant GK7-000061[ from FfNACIT (Fondo Nacional de Ciencia, 

TecnologÜa e Innovacián), Caracas, Venezuela. 

References: 

.an-2las and Nio-Vega. 200O. InM .an-2las G, Calderone RA Eds. Caister Academic 

PressM aymondham, NorfolB. Chapter 2M 1K[-220. 

Ruiz-Herrera .- +6. 200O. InM .an-2las G, Calderone RA Eds. Caister Acadenic PressM 

aymondham, NorfolB.Chapter [M OK-KK. 

.an-2las .-*+6. 2002. !.5*!2#%6*O0M 22[-2O2 

Mirbod .-*+6. 1KK7. ]*!.5*O.-*!2#%6*J[M 17J-17K

P-0699. DNA-binding properties of the sex inducer proteins, SXI & SXI2a in 

Cryptococcus neoformans 

Stanton B 1 , .taudt M 1 , Hull C 1,2 

1 Department of 2iomolecular Chemistry, 6niversity of aisconsin | Madison, Madison, 

aI, [J706, 6.A., 2 Department of Medical Microbiology and Immunology, 6niversity of 

aisconsin | Madison, Madison, aI [J706, 6.A. 

Previous worB has identified two proteins that control sexual development in the 

human fungal pathogen 7$2'-%#%##(&*,.%U%$)+,&/*lnown as the .ex Inducer 

Proteins, .xi1$ and .xi2a are predicted homeodomain proteins, implicating their role 

in DNA-binding. To further determine the capacity of the .xi proteins to bind DNA, 

versions of each protein were purified from a&#?.$"#?"+*#%6"*and tested in 

electrophoretic mobility shift assays/*These assays were used to identify DNA 

sequences to which .xi1$ and/or .xi2a bind directly. Candidate binding sequences 

were identified bioinformatically and by ",*0"0%*studies performed previously. 

ff the Candidate sequences tested, .xi2a binds specifically to the promoter regions 

of 1) a predicted homolog of the 7X1I (#6+)'6.&&J*gene and 2) the !^$*()+-",@* 

U+#-%$J*and !â*genes. fur studies show that the homeodomain region of .xi2a binds 

specifically to a 1K base-pair region of the 7X1I promoter exhibiting approximate 

dyad-symmetry. Efforts are underway to 1) test larger fragments of pure .xi2a protein 

2) refine the binding site of .xi2a J) identify residues within the .xi2a homeodomain 

that are critical for binding and O) determine the stoichiometry of DNA-binding by the 

.xi2a homeodomain. 

P-0700. Electron-microscopic investigation of morphogenesis of Aspergillus 

flavus link 

Stepanova A, .initsBaya I 

lashBin Research Institute of Medical Mycology, .aint-Petersburg, Russia 

The morphogenesis of 4/*U6+0(& hyphal cells (HC) strain (RCPF-K[O/[O2[) of 

vegetative mycelium, isolated on CzapeB agar medium from abscess bioptate at 

patient with aspergillosis has been studied. The fungal colonies were grown at 27oC, 

have fixed with using glutaraldehyde-osmium after 2, J, [, 10 and 20 days of 

inoculation. 

Differences in pattern of morphogenesis (PM) of the HC of aerial and submerged 

mycelium, on the one hand, and submerged mycelium, in another hand, are given. 

During the all period of morphogenesis (excepting the stage of cell senescence and 

die away) HC of aerial mycelium have the next structureM 1. low level of vacuolizationn 

2. the absence of storage substancesn J. presence of dense cytozole, which conceal 

nuclei and another cellular components, including Voronin bodies. The mature HC of 

submerged mycelium contain O nuclei with poorly condensed chromatin. Five types in 

PM of HC of submerged mycelium are recognized according toM 1. sizes and shape of 

nuclein 2. number and structure of mitochondrian J. level of vacuolizationn O. presence 

or absence of smooth endoplasmic reticulum (ER)n [. presence or absence of storage 

substances and their combinations. The stacBs of parallel cisterns of smooth 

endoplasmic reticulum (ER) are regularly arranged with a repetition period of about 2[ 

nm and localized near nuclear membrane in HC with fifth PM. In HC with another PM 

the smooth cisterns of ER are rare. Mature HC which pass the first PM has ellipsoidal 

(J,6xJ,0 $m) nuclei, high level of vacuolization and few number of small (0,2-0,J $m) 

rounded mitochondria. .torage substances are absent. Nuclei in mature HC which 

pass the second PM was paddle-shaped (O,2 $m). This HC has a lower level of 

vacuolization, few number of large (0,O-0,6 $m) rounded or ellipsoidal mitochondria 

and numerous storage lipids. HC which pass the third PM, showing the irregularshaped 

(2,0 $m) nuclei, few small vacuoles, large (0,[-2,8 $m) polymorphic 

mitochondria and abundant rosettes of glycogen. For HC which pass the fourth PM 

are typical slightly irregular shape (1,8 $m) nuclei, lover level of vacuolization, 

moderate number of little (0,J-0,O $m) or rounded mitochondria, numerous lipid 

inclusions and rosettes of glycogen. At last the HC with fifth PM are characterized in 

the presence of ellipsoidal nuclei (2,0x1,1$m), moderate level of vacuolization, few 

number of small (0,2-0,O $m) rounded or ellipsoidal mitochondria, numerous lipid 

inclusions in cytozole and blacB granules (0,01-0,0J $m) of polyphosphates in 

vacuoles. 

ae suggest that the HC of submerged mycelium, in comparison with aerial, are in 

more high metabolic level, it may be explaned with close contact of HC with cultural 

medium. The estimates are propound, according which the revealed differences in PM 

of 4/*U6+0(& submerged vegetative mycelium HC maybe plausible reason of its 

capacity for colonize the different types of substrates. 


P-0701. Localization of oxidative stress related proteins in C. albicans 

Strijbis K 1 , Visser a 2 , Distel 2 1 

1 Department of Medical 2iochemistry, Amsterdam Medical Center, 6niversity of 

Amsterdam, The Netherlands, 2 Department of Genetic Metabolic Diseases, 

Amsterdam Medical Center, 6niversity of Amsterdam, The Netherlands 

fxidative stress defense is essential for 7+,5"5+*+68"#+,& survival, as macrophages 

and neutrophils attacB the fungus by production of reactive oxygen and nitrogen 

species (Rf./RN.) after phagocytosis. Glutathione reductase (Glr1) is a Bey enzyme 

in the protection against oxidative stress, as it regulates the redox state of the cell by 

Beeping glutathione in the reduced state, a process that is NADPH dependent. fne of 

the main sources of NADPH in the cell is the pentose phosphate pathway (PPP), 

which converts glucose to the [-carbon sugar ribose with the production of NADPH by 

glucose-6-phosphate dehydrogenase (Zwf1) and 6-phosphogluconate dehydrogenase 

(Gnd1). Although the PPP is presumed to be a cytosolic pathway in most organisms, 

in 7/*+68"#+,& Zwf1 and Gnd1 and also Glr1 have putative peroxisomal targeting 

signals (PT.)M Zwf1 has a putative PT.1 and Gnd1 and Glr1 have a putative PT.2. In 

order to determine the subcellular localization of Glr1 and the PPP enzymes, we 

performed subcellular fractionation and Nycodenz gradient analysis on 7/*+68"#+,& 

cultures grown on oleic acid, as fatty acid !-oxidation will result in the production of 

Rf. in peroxisomes. Enzyme assays showed that Glr1 localizes to the cytosol and to 

mitochondria but that a small but significant part is present in the peroxisomal 

fractions. Zwf1 and Gnd1 show dual localization, as activity of these enzymes was 

consistently detectable in both peroxisomes and cytosol. Fractionation of a 7/*+68"#+,& 

pex7*/* mutant, in which import of PT.2 proteins is impaired, showed that Zwf1 

import is unaffected in pex7*/* peroxisomes, whereas peroxisomal Gnd1 is lost. 

These results indicate that Zwf1, Gnd1 and Glr1 are partially peroxisomal in 7/* 

+68"#+,&. 

P-0702. Structural characterization of the role of CaBMT1p and CaBMT2p in the 

beta-mannosylation of the Candida albicans phospholipomannan 

Trinel P 1 , Mille C 1 , Maes E 2 , Delplace F 2 , (anbon G J , Poulain D 1 

1 Inserm 67KK, PÇle recherche, Om dtage, Facultd de Mddecine, [K0O[ /ille Cedex, 

France, 2 6nitd de Glycobiologie .tructurale et Fonctionnelle, 6MR CNR. 8[76, 

Villeneuve d]Ascq, [K6[[, France, J 6nitd de Mycologie Moldculaire, Institut Pasteur, 

Paris, 7[01[, France 

2eta-mannosylation of glycoconjugates is a characteristic feature of 7/*+68"#+,& and 

has been shown to favour 7/*+68"#+,& virulence by adhesin properties and by 

modulating the host immune response. 7/*+68"#+,& beta-mannosylation is a complex 

process. 2eta mannosides have been chemically evidenced first on the acid-stable 

and the acid-labile fractions of phosphopeptidomannan (PPM) and on 

phospholipomannan (P/M), a glycosphingolipid related to MIPC. .ynthesis of P/M is 

restricted to 7/*+68"#+,&, 7/*5(86",".,&"&, 7/*&-.66+-%"5.+ and 7/*-$%'"#+6"&. Extensive 

physico-chemical studies of 7/*+68"#+,& P/M have demonstrated its unique 

heterogeneity and the exclusive presence of beta-1,2 linBed oligomannosides which 

may contain up to 18 mannoses. Former immunochemical studies have also 

suggested that beta-mannosylation of PPM, mannoproteins and P/M were specific 

and independent processes differentially regulated by growth conditions. 

ae have recently characterized new genes encoding for the beta 

mannosyltransferases of 7/*+68"#+,&. In this study, we analysed by western blotting 

and physico-chemical methods (NMR spectrometry, mass spectrometry) the effect of 

deletions of genes 7+Z!>I and 7+Z!>M on the structure of PPM and P/M and 

showed that Cabmt1p initiates P/M beta mannosylation and that Cabmt2p is involved 

in the elongation of P/M beta-1,2 linBed oligomannose chain. Despite strong 

homologies in the phosphomannose chains of both P/M and PPM acid labile part, 

these beta-mannosyltransferases appeared quite specific for P/M and have no role 

on PPM beta-mannosylation( which is under the control of 7+Z!>E.) These results 

confirm our former studies in demonstrating that 7/*+68"#+,& displays different 

processes to modulate its surface beta-mannosylation.

P-0703. The copper, zinc superoxide dismutase gene of Penicillium marneffei: 

Cloning, characterization, differential expression and biological enzyme activity 

Vanittanakom N 1 , Thirach . 1 , Cooper C 2 

1 Department of Microbiology, Faculty of Medicine, Chiang Mai 6niversity, Chiang Mai 

[0200, Thailand, 2 Department of 2iological .ciences, Youngstown .tate 6niversity, 

Youngstown, fhio OO[[[, 6.A 

Copper-zinc superoxide dismutase (Cu, Zn .fD), which is one of the anti-oxidant 

enzymes in several organisms, has proved to be involved in the virulence of some 

pathogenic fungi. To date, there have been no reports of the identification of .fDs 

from the human pathogen 1.,"#"66"()*)+$,.UU."/*In this study, the gene encoding this 

enzyme, 1)=bVI3 was identified from 1.,"#"66"()*)+$,.UU.". In particular, its 

differential expression and biological enzyme activity were determined. 

Clones containing complementary deoxyribonucleic acid (cDNA) encoding 1)=bVI 

were isolated by screening a yeast-phase expression cDNA library of 1.,"#"66"()* 

)+$,.UU." with a Cu, Zn =bV*probe. This =bV*probe was identified from 1.,"#"66"()* 

)+$,.UU." by using degenerate primers designed on the basis of identity/similarity to 

the Cu, Zn .fD protein sequences of the other fungi from the Gene2anB database. 

Thirteen positive signals were screened further for a putative full-length clone. A full 

length 1)=bVI cDNA clone was isolated and characterized. An open reading frame 

of 1)=bVI comprised O6[ nucleotide and was delimited by well-Bnown start (ATG) 

and stop (TAG) codons. The polypeptide consisted of 1[O amino acids that lacBed a 

signal peptide. It contained six His and one Asp residues, which act as the metal 

binding ligands in other fungal Cu, Zn =bVs. Comparison of its deduced amino acid 

sequence with those previously reported amino acid sequences revealed considerably 

high homology to the Cu, Zn .fD. .taining of native polyacrylamide gels of protein 

extracts from conidia, yeast and mycelial forms of 1.,"#"66"()*)+$,.UU." revealed .fD 

activity which was inhibited by hydrogen peroxide. Differential expression of the 

1)=bVI*gene in 1.,"#"66"()*)+$,.UU." was demonstrated by RT-PCR. The 1)=bVI* 

accumulated in conidia (the dormant form) and down-regulated during the 

development to mycelia phase at 2[ o C. The transcript was up-regulated in response 

to the yeast phase transition or when the temperature was shifted to J7 o C. These 

results indicated that 1)=bVI might play an important role in environmental stress 

response and adaptation of 1.,"#"66"()*)+$,.UU." during yeast phase transition. It may 

also be an important factor for survival of this pathogenic fungus in the host cells. 

P-0704. Relationship among B-glucosidase activity and Cryptococcus 

neoformans serotypes 

Vidotto V 1 , Ponton ( 2 , ~uindos G 2 , AoBi . J , Ito-luwa . J , NaBamura l J , Cannizzo F O 

1 Institute of Infectious Diseases - 6niversity of Turin - Italy, 2 Dpt. Inmunol., Microbiol. y 

Parasitol., 6niversidad del Pais Vasco - .pain, J Advanced Research Center, Nippon 

Dental 6niversity - (apan, O Department ff Animal Pathology, 6niversity ff Turin - 

Italy 

Twenty-eight 7$2'-%#%##(&*,.%U%$)+,& strains, which were isolated from AID. 

patients in .pain and stored in the culture collection of the 6niversidad del Pais Vasco 

(2ilbao, .pain), were tested by the API-ZYM Bit (bioMdrieux .A, France) for their 

extracellular enzymes containing #-glucosidase. .erotype determination of the strains 

was performed using the Crypto-checB Bit (Iatron /ab., (apan). 

The results obtained showed the high positive percentage of #-glucosidase among the 

28 7/*,.%U%$)+,& strains tested (26/28 } K2.K_). .erotype determination revealed 17 

serotype A, 8 serotype D and J serotype AD strains. The predominance of the 

serotype A strains observed among the 28 7/*,.%U%$)+,& .panish strains tested is 

similar to those of the other southern Mediterranean countries. The intensity of #- 

glucosidase, expressed by the average value of API-ZYM Color .cale, was 1.00, J.12 

and 2.66 for serotype A, D and AD, respectively. .tatistical analyses indicated that 

these differences in #-glucosidase activity among the three serotypes were significant. 

Noteworthy is the relationship between the #-glucosidase activity and the serotypes D 

and AD. 

It is also interesting to further examine this extracellular enzyme could be involved in 

tissue invasion and probably in 7/*,.%U%$)+,& virulence. 


P-0705. Analysis and expression of STE13ca and Dap2 genes encoding 

putatives x-prolyl-dipeptidyl aminopeptidases from Candida albicans and their 

possible participation in maduration of other peptides 

Villa-Tanaca L 1 , 2autista-Muoz C 2 , HernZndez-RodrÜguez C J 

1 E.N.C. 2. Instituto Politdcnico Nacional. Mdxico D. F. Mdxico, 2 E.N.C. 2. Instituto 

Politdcnico Nacional. Mdxico D. F. Mdxico, J E.N.C. 2. Instituto Politdcnico Nacional. 

Mdxico D. F. Mdxico 

.equencing of 7/*+68"#+,& genome and a comparative genome analysis between 7/* 

+68"#+,& and =/*#.$.0"&"+. showed that, in addition to the !>X locus, this yeast 

contains homologues to genes involved in the mating process in =/*#.$.0"&"+.. Recent 

reports have demonstrated a possible mating between !>Xa and !>X ! 7/*+68"#+,& 

strains. The =/*#.$.0"&"+. =>aI: gene encodes the dipeptidyl aminopeptidase A 

(.te1Jp), which is a membrane-bound enzyme, involved in the processing of !-sexual 

factor maturation by removing dipeptides from the N-terminus of the pheromone 

precursor. Recently, has been described a mating pheromone encoded by the !^% 

gene in 7/*+68"#+,& ! +cells. Cells of the a mating-type respond to the !-mating 

pheromone by producing long polarized projections similar to those observed in 

mating mixtures of 7/*+68"#+,& a and !. The aim of this worB was the biochemical and 

genetic characterization of the ycaDAP (dipeptidyl aminopeptidase activity) from 7/* 

+68"#+,& ATCC 102J1. 

The ycaDAP activity was detected intracellularly in membrane and cytoplasmic 

fractions. The highest levels of the enzymatic activity were found in cellular fractions 

from YN2 medium supplemented with proline and during formation of the germinal 

tube. The bio-informatic analysis of the genome of 7/*+68"#+,& revealed the presence 

of sequences in 7/*+68"#+,& with similarity to V41M and =>aI: genes of =/*#.$.0"&"+.. 

The V41M gene from 7/*+68"#+,& of 2[17 pb was heterozygotic, and encode for 

predicted proteins of 8J8 amino-acids with a theoretical pl of [.2, and molecular 

weights of K6,180 Da and K6,210 Da respectively. The =>aI: gene of 27KJ pb was 

homozygotic, encode for a predicted protein of KJ0 amino-acids with a theoretical pI of 

[.1[ and a molecular weight of 107,0J[ Da. The Dap2 and .te1J proteases displayed 

the characteristic active site of the prolyl-endopeptidases family and serine proteases, 

and the putative sites of subcellular location of Dap2 and .te1J were the plasmatic 

and Golgi apparatus membranes respectively. The codons usage frequency and the 

CAI values correspond to low expression genes. Analysis of the regulatory region 

indicates that these genes could be under the transcriptional control of =>Pas, `K>WM, 

!4>IW!7, !7!I3*and*!4>+I*transcription factors. fther yeasts as 7/*5(86",".,&"& 

and 7/*-$%'"#+6"&, exhibit homologous sequences to those of the V41M and/or =>aI:* 

genes by .outhern analysis. PCR of separated chromosomes by PFGE reveled that 

V41M and =>aI: of 7/*+68"#+,& are located in chromosomes [ and R respectively. 

.emi-quantitative RT-PCR showed that the expression of V41M gene was associated 

to nitrogen sources as peptone and proline and =>aI: gene increased its levels of 

expression in nutritional stress conditions (proline as nitrogen source) and during the 

formation of the germinal tube. The genes of C. +68"#+,& analyzed in this worB could 

be the orthologous genes of the V41M and =>aI: genes of ./*#.$.0"&"+. and their 

products may participate in nitrogen metabolism under nutritional stress conditions 

and/or maturation of proteins or peptides as !-mating pheromone. 

P-0706. Proteome analysis of morphogenic transition in Penicillium marneffei 

by two-dimensional differential gel electrophoresis 

Xi L 1 , ju j 1 , /i M 2 , /iu a 2 , /i j 1 , Zhang ( 1 , /iu Y 2 

1 Dept of Dermatology, The 2nd Affiliated Hospital,.un Yet-.en 6niversity, P.R. China, 

2 The Center for Proteomics, The .chool of Preclinical Medicine, .un Yat-.en 

6niversity. P.R. China. 

1.,"#"66"()*)+$,.UU." is a opportunistic human pathpogen that causes penicilliosis 

marneffei in .outheast Asian countries. It exhibits temperature-dependent 

dimorphism, with growth at 2[ as filamentous multinucleate mycelium switching at 

J7 to mononucleate yeast cell. Fungal pathogenesis is frequently associated with 

dimorphism | morphological changes between yeast and filamentous forms. Very 

little information has been made available on the mechanisms underlying 

morphogenic transition and the intracellular survival of 1/*)+$,.UU.". Recently we 

applied 2-D DIGE which combined the mixed sample internal standard with Typhoon 

Trio Variable Mode Imager, and DeCyder software to analysis differentially expressed 

proteins between yeast and mycelium phase of wild type isolate. Matrix assisted laser 

desorption ionization-time of flight-mass spectrometer was used to identify proteins. 

The protein profiles of whole extract from yeast phase and mycelium phase were 

established. Four thousands protein spots were visualized by cydye labeling and 

Typhoon scanning. Five hundred and eleven spots of differential expression between 

yeast and mycelium phase were separated by performing the comparative proteomic 

analysis. .ome proteins including Catalase-peroxidase, heat shocB protein K0, 

Phosphoglucomutase, Gamma-aminobutyrate transaminase, soluble fumarate 

reductase, N-alBane-inducible cytochrome P-O[0 and isocitrate lyase showed 

significantly increased expression in yeast phase, meanwhile, snf22, pH-response 

regulator protein palA, and Poly(A) polymerase showed decreased expression. This 

is the first worB to carry out proteomic research in dimorphic fungi using 2-D DIGE 

technology. ahich will faciliate the further exploration of morphogenic transition 

between mycelium to yeast, and even the pathogenicity of 1/*)+$,.UU.

P-0707. Molecular characterization of the area/nit-2-like nitrogen regulatory 

genes in dermatophytes 

Yamada T, MaBimura l, Abe . 


A homolog of the major nitrogen regulatory genes +$.4 from 4&'.$@"66(&*,"5(6+,& and 

,"-WM from `.($%&'%$+*#$+&&+ was isolated from the zoophilic dermatophyte, 

!"#$%&'%$()*#+,"&. This gene, 5,$I, encodes a polypeptide of 761 amino acid 

residues containing a single zinc-finger DNA-binding domain, which is almost identical 

in amino acid sequence to the zinc-finger domains of AREA and NIT-2. The functional 

equivalence of 5,$I to +$.4 was demonstrated by complementation of an +$.4 lossof-function 

mutant of 4/*,"5(6+,& with 5,$I cDNA. To further characterize this gene, 

5,$I was disrupted by gene replacement based on homologous recombination. ff 

100 transformants analyzed, two showed the results expected for replacement of 5,$I. 

The growth properties of the two 5,$I W mutant strains on various nitrogen sources 

were examined. 6nliBe the 4/*,"5(6+,& +$.4 - mutant, these 5,$I W *mutants showed 

significantly reduced growth on ammonia, a preferred nitrogen source for fungi. These 

mutant strains were also able to utilize various amino acids for growth. In comparison 

with wild-type !/*#+,"&, the two 5,$I - mutants showed reduced growth on medium 

containing Beratin as the sole nitrogen source. This is the first presentation describing 

successful production of targeted gene-disrupted mutants by homologous 

recombination and their phenotypic analysis in dermatophytes. 

P-0708. Mutations on caeno1 arrest the cell growth of Candida albicans in the 

presence of glucose and serum 

Yang Y, Chen H 

Dept 2iological .cience c Technology, National Chioa Tung 6niversity, Hsin-chu, 

Taiwan 

Enolase (2-phospho-D-glycerate hydrolase) is an enzymatic component of the 

glycolytic pathway and is conserved through evolution. Enolase and other glycolytic 

enzymes are the most abundant proteins in =+##?+$%)2#.&*#.$.0"&"+.. There are 

two non-tandemly repeated enolase structural genes,*a`bI and a`bM, in the =/* 

#.$.0"&"+. genome. To elucidate the functions of 7+a`bI in 7+,5"5+*+68"#+,&, a*>PW 

7+a`bIPW7+a`bIPW7+a`bI. This 

suggests that 7+a`bI is indeed tightly regulated by the TR expression system. 

In the presence of doxycycline, the >PW7+a`bIPW 

7+a`bI

P-0709. Pcr typing of Iranian Trichophyton rubrum isolates by repetitive 

subelements of NTS region at rDNA 

Zaini F 1 , Mahmoodi Rad M 2 , lordbacheh P 1 

1 .chool of Public Health, Tehran 6niversity of Medical .cience, Tehran, Iran, 

2 .chool of Medicine .hahid 2aheshti 6M., Iran 

Trichophyton rubrum is the commonest cause of dermatophytosis of sBin and nail 

tissue. .train identification in T. rubrum is important for identification of strain related 

differences in infective potential or transmissibility and the epidemiological studies. 

PCR typing could determine whether the original isolate is responsible for reinfection 

or a new strain has been acquired. 

A minipreparation method for DNA form dermatophytes was used. Tandemly 

repetitive subelements (TR.-1 c TR.-2) of NT. region at ribosomal DNA of 2J T. 

rubrum isolates were amplified and the PCR products were separated by 

electrophoresis in 2_ agarose gels (200mA, 1O0V), visualized by staining with 

ethidium bromide, and photographed. ê 

fn the basis of copy number of TR.-1 and TR.-2, eight of isolates were type 2 c II 

respectively. .ix of them were type J c II, four isolates were type 1 c II, two isolates 

were type O c II, two isolates were type 1 c I, and one isolate was type [ c II. 

Amongst Iranian T. rubrum isolates, the largest number of strains were TR.-1 type 2 

(J[_) and the second largest type 1 (26_). 

Interestingly some investigators have previously reported that type 1 was most 

common PCR type (O0_) in European countries, but was rarest with only a single 1 

strain among Asian isolates. 

OTHER* 

P-0710. Antifungal properties of building materials 

Pivovarova Z 

.lovaB Medical 6niverzity 

Monitoring of the indoor fungal exposition is complicated due to lacB of standardized precious 

practical methods evaluating the relation of indoor microclimate and micromycetes. The ability of 

penicillia and aspergilli to growth on house dust under already RH 76 | 80 _ is the most 

probable explanation of their predominance even in healthy buildings. .econdary colonizers 

(cladosporia, alternariae, chaetomia | optimal RH 8[ _) and terciary ones (fusaria, acremonia, 

yeasts | optimal RH over K0 _) have strong ability to biodeteriorate any material under optimal 

temperature and humidity se. g. 1u. .ome microfungi synthesized several metabolites with 

different toxic effects in animal experiments, including mycotoxins, on building materials se. g. 2, 

Ju. 

In the present study, antifungal properties of common building materials (plasters, facings etc.) 

under particular temperature and humidity were tested. 

Defined spore suspensions of 4#$.)%,"() sp., 4&'.$@"66(&*(&-(&, 4/*0.$&"#%6%$, 76+5%&'%$"()* 

&'?+.$%&'.$)(), 1.,"#"66"() sp., =#.5%&'%$"()*+'"%&'.$)() were inoculated on the surface 

of different Binds of lime-cement mortars, plaster mortars, plasters, silicone cement, gypsumboard, 

tiles and wood without painting or painted and with oily coating, resp., 20 materials in total. 

The systems were incubated under the water activity (aw) of 0.7[, 0.8J and 0.KO atmosphere at 

22 | 2[ ÅC for J months. The fungal growth was evaluated visually after 1, 2, J months and the 

viability of micromycetes used was detected by material imprinting on .abouraud agar after J 

months sOu. 

aood and lime plasters showed to be the most resistant to fungal growth under given 

experimental conditions, while tiles and plaster mortars enabled all microfungi tested to growth. 

4#$.)%,"() sp. was able to growth on all materials tested only when cultivated at aw 0.KO. 4/* 

0.$&"#%6%$ and 1.,"#"66"()*sp. overgrew materials under every moisture condition, except of wood 

and a fine lime plaster, when having grown only at aw 0.KO after J months. None of materials 

tested showed any fungicidic property as micromycetes remained cultivable after J months of the 

experiment under all moistures used. Anyway, the oily coating elevated fungistatic abilities of 

every material compared to common painting. 

References: 

1. P. lallioBosBi, A.-/. Pasanen, A. lorpi and P. PasanenM House dust as a growth 

medium for microorganisms. InM Proc. 7 Int. Conf. Indoor Air ~ual. Climate, Nagoya, 

(apan. Nagoya 6niv, 1KK6M 1J1-1J6. 

2. l. F. Nielsen, M. f. Hansen, T. f. /arsen and 6. Thrane. Production of trichothecene 

mycotoxins on water damaged gypsum boards in Danish buildings. Int. 2iodeterior. 

2iodegrad. O2,1KK8M 1-7. 

J. .. Gravesen, P. A. Nielsen, R. Iversen and l. F. Nielsen. Microfungal contamination of 

damp buildings - examples of risB constructions and risB materials. Environ. Hlth Persp. 

107, 1KKKM [0[-[08. 

O. I.f 8O6M 1KK7 (E) prepared by Technical Committee I.f/TC 61 Plastics, 

.ubcommittee .C6 Aging, chemical and environmental resistance.

P-0711. Classification of chromoblastomycosis agents by drifts and 

chemometrics 

Scroferneker M 1 , Corbellini V 2 , /ocB / 2 , FerrÉo M 2 

1 Instituto de Ciüncias 2Zsicas da .aâde, 2 Departamento de FÜsica e ~uÜmica da 

6niversidade de .anta Cruz do .ul 

The dissemination of chromoblastomycosis in regions with tropical and subtropical 

climates and its high morbidity have led to the development of methods for the 

identification of the agents involved. This study assesses the use of Diffuse 

Reflectance Infrared Fourier Transform .pectroscopy (DRIFT.) and chemometrics for 

the classification of these agents. The samples were cultured in microculture plates 

containing YEPD agar for 1O days at J0oC and the biomass was dehydrated and 

analyzed in triplicate by DRIFT. between O00 and O000 cm -1 . The analysis of the 

spectra by Principal Components Analysis (PCA) and Hierarchical Cluster Analysis 

(HCA) showed that the 10 fungal samples can be classified into two large groupsM the 

first one includes 7/*#+$$"%,"" 680, P/*+e(+&'.$&+ 6K1 and ^/*1.5$%&%" O6O28, OK and 

1K, characterized by the presence of C-f groups of carbohydrates, and the second 

one contains 7/*8+,-"+,() 1J, ^/*'.5$%&%" O6O22, ^/*#%)'+#-+ O, a/*\.+,&.6)." O[ 

and 1/*0.$$(#%&+3 8 of them characterized by the presence of phosphate bonds or 

phosphodiester groups of nucleic acids. 

P-0712. Hyphal formation of Candida albicans is controlled by electron transfer 

system 

Watanabe T, fgasawara A, MiBami T, Matsumoto T 

Department of Microbiology, .endai. (apan 

The opportunistic pathogen 7+,5"5+*+68"#+,& is an agent which causes infection in 

the immunocompromised host and transforms from yeast to hyphal form depending 

on the growth conditions. It was reported that RA.1 protein of*7/*+68"#+,& activates 

several factors involved in hyphal formation signals, such as EFG1 and CPH1. 

However, the details of the RA.1 activation system have not been clarified. In this 

study, we demonstrated that the respiration system controlled the activation of RA.1 

and other hyphal formation signals. 

Most 7/*+68"#+,& cells cultured in RPMI16O0 medium at J7 C grow in hyphal form in 

aerobic conditions, but it grew yeast form in anaerobic conditions. The hyphal growth 

of 7/*+68"#+,& was inhibited in glucose-deficient conditions. Malonic acid, an inhibitor 

of succinate dehydrogenase, enhanced the yeast proliferation of 7/*+68"#+,&, 

indicating that the hyphal-formation signal was derived from the gycolysis system and 

the signal was transmitted to the electron transfer system via the citric acid cycle. 

Thenoyl trifluoro acetone (TTFA), an inhibitor of the signal transmission between 

complex II and Co ~, significantly inhibited the hyphal growth of 7/*+68"#+,&. 

Antimycin, lCN and oligomycin, inhibitors of complex III, IV and V, respectively, and 

did not inhibit the hyphal growth of 7/*+68"#+,&. .alicylhydroxamic acid (.HAM), an 

Afj inhibitor, was inhibited the hyphal formation of 7/*+68"#+,&/* The production of 

mRNAs for the hyphal formation signal was completely inhibited in anaerobic 

conditions. .HAM also inhibited the expression of these mRNAs. 

These results indicate that the electron transfer system functions upstream of the 

RA.1 signal pathway and the signal- transduction may be mediated by Afj. 


PHYSIOPATHOLOGY* 

P-0713. ALS3 protein carries a germ-tube specific epitope of Candida albicans 

Beucher B, Marot-/eblond A, Nail-2illaud ., Robert R 

Groupe deEtudes des Interactions HÇte-Parasite, 6FR des .ciences Pharmaceutiques 

et deIngdnierie de la .antd, 16, bd Daviers, OK000 Angers, France 

Monoclonal antibody JDK.J (MAb JDK.J) reacts with the surface of 7+,5"5+*+68"#+,& 

germ tubes and recognizes a protein epitope. The goal of the present study was to 

identify the antigen (JDK) that is recognized by MAb JDK.J. A two-step procedure was 

used to obtain a purified preparation of JDK antigen from a Zymolyase w extract of 7/* 

+68"#+,& germ tubes. The extract was first fractionated by gel filtration chromatography 

followed by hydrophobic interaction chromatography on a phenyl-.epharose column. 

Each fraction was assayed for JDK antigenic activity by E/I.A and the 

immunoreactive fractions were pooled. Analysis by .D.-PAGE, immunoblotting and 

Concanavalin A staining revealed two disperse bands ranging from 110 to 220 BDa. 

These two bands were analysed by MA/DI-TfF mass spectrometry. The peptide 

mass fingerprints produced by MA/DI-TfF M. were searched against the 7/*+68"#+,& 

protein database using Mascot .oftware programs. This method produced ambiguous 

matches from the database for the two bands. However, one match for the 220 BDa 

band was AlsJp. This result was promising even if its identification score was 

relatively low, because AlsJp, as JDK antigen, is specific for 7/*+68"#+,& germ tubes, 

heavily glycosylated, localized to the cell surface and has a high molecular mass. 

To verify our hypothesis that JDK antigen corresponds to AlsJp, MAb JDK.J was 

tested against +6&:*

P-0715. Tinea corporis is associated with an impairment of epidermal 

differentiation and barrier function and with an epidermal expression of betadefensin-2 

Brasch J, (ensen (, ProBsch E 

6niversity of liel 

Dermatophytes are world-wide distributed highly specialized filamentous fungi that 

can utilize Beratin and cause sBin infections (dermatophytoses) in humans. Among 

them, anthropophilic, zoophilic and geophilic species of different pathogenicity are 

Bnown. To prove a suspected diagnosis of dermatophytosis it is necessary to isolate 

and identify the causative agent from lesional sBin. ae report on a new dermatophyte 

that was isolated from mocassin-type tinea pedum of a man from the Ivory Coast. 

This fungus was characterized by a rapid growth on common mycological media 

resulting in a flat off-white thallus with a feathered margin and a granular surface. 

Microscopically numerous clavate microconidia and macroconodia with 2-K cells were 

seen that had smooth and thin walls. flder cultures developed spiral hyphae, 

chlamydospores and cleistothecia-liBe structures. The fungus showed a weaB urease 

activity, no dependency on vitamins and a positive hair perforation test. A sequence 

analysis of the internal transcribed spacer of the rRNA gene revealed more than [ _ 

divergence from any Bnown species of dermatophytes. 

These characteristics indicated that our strain was a dermatophyte of the genus 

>$"#?%'?2-%,. Due to its distinctive features which were not compatible with any of the 

Bnown >$"#?%'?2-%, species, it was described as a new speciesM >$"#?%'?2-%,* 

.8%$.(). 

Distribution and pathogenicity of this species are as yet unBnown. In our case the 

attempted reisolation from the patient]s sBin failed. This may be due to the 

antimycotic therapy that had been initiated right after the initial collection of scrapings 

from the stratum corneum or to the fact that the fungus was a non-pathogenic 

contaminant of the sBin. ae suppose that >$"#?%'?2-%,*.8%$.() is a geophilic 

dermatophyte with a very limited distribution. 

P-0716. A role for Cryptococcus neoformans galactoxylomannan in t cell 

apoptosis and function 

Cenci E 1 , Pericolini E 1 , De (esus M 1 , 2istoni F 1 , Vecchiarelli A 1 , Casadevall A 2 

1 Microbiology, Department ff Experimental Medicine and 2iochemical .ciences, 

2 Albert Einstein College of Medicine Department of Microbiology and Immunology, 

2ronx, New YorB, 6...A. 

The*polysaccharide capsule of 7/*,.%U%$)+,& is composed of glucuronoxylomannan 

(GjM), galactoxylomannan (GaljM) and mannoproteins. Although GjM is the most 

prevalent polysaccharide on a mass basis, it has recently been shown that there is 

more GaljM on a molar basis. Immunomodulating activities have been described for 

GjM and mannoproteins but little is Bnown about possible interactions of GaljM with 

the immune system. In the present study, we investigated the effect of purified soluble 

GaljM on human leuBocytes. GaljM was purified from culture supernatants in a 

methodology that yields endotoxin free polysaccharide. fur experimental approach 

was to study the interaction of GaljM with human T cells in 0"-$%. Human CD-J T- 

lymphocytes and the (urBat T-cell line were cultured in the presence of blastogenic 

stimuli such as heat inactivated 7/*,.%U%$)+,& or 7/*+68"#+,&, ConA, PHA and mAb 

to CDJ. A dose dependent response of GaljM (1-10 ug/ml ) was observed. The 

results indicate that GaljM (i) interacts directly with T cellsn (ii) suppresses T cell 

proliferation and increases interferon-" and interleuBin-10 productionn (iii) induces 

apoptosis of T lymphocytes through activation of caspase-8 that terminates with 

fragmentation of DNAn iv) exploits galectin receptors including CD7, which appears to 

be directly involved in induction of T cell death. These results are the first to suggest a 

role for GaljM in 7/*,.%U%$)+,& virulence by demonstrating that it can target human 

T cells, and that it may impair the development of an effective specific T cell response. 

Furthermore, our findings indicate a new mechanism by which 7/*,.%U%$)+,& 

capsular polysaccharides can interfere with the immune response and produce 

immunosuppression. 


P-0717. C. albicans-interaction with oral epithelial cells and enterocytes 

Dalle F 1,2 , latherina Z 1 , 2onnin A 2 , Hube 2 1 

1 Robert loch Institut - FG16, 2erlin, Germany, 2 /aboratoire de Microbiologie Mddicale 

et Moldculaire (EA[62), Facultd de Mddecine, Dijon, France 

7/*+68"#+,& is an opportunistic fungus responsible for a wide range of diseases, 

ranging from superficial to systemic infections. Among superficial infections, 

oropharyngeal candidiasis (fPC) can arise in healthy people but occurs most 

frequently in immunocompromised patients undergoing cancer treatment, solid organ 

and bone marrow transplantation, or in HIV patients. In contrast, disseminated 

candidiasis (DC) is only seen in severely immunocompromised patients and the 

attributable mortality can be up to J[_. Moreover, one of the possible main ports of 

entry for DC is penetration through the epithelial layers of the gastrointestinal tract. 

Therefore, interactions of 7/*+68"#+,& with these cells are Bey events in the 

development of this life-threatening disease. 

Fungal factors such as the ability to grow at body temperature, the secretion of 

hydrolytic enzymes, the expression of adhesins, phenotypic switching and/or the 

yeast-to-hyphal transition, probably account for adaptation of 7/*+68"#+,& to various 

environmental conditions such as those encountered in the oral cavity or the gut. 

However, pathogenesis of fungal infections is also determined by the status of the 

host and it is well Bnown that risB factors for fPC are different from those contributing 

to DC, suggesting that pathogenesis of fPC is different from the early steps of DC. 

.ince the mechanisms by which 7/*+68"#+,&*can cause fPC or DC via the gut are 

poorly understood, we have begun to examine the interaction of 7/*+68"#+,& with oral 

epithelial cells and enterocytes on the cellular and molecular level by using models 

based on (i) an oral cell line (TR-1O6) or (ii) enterocytic cells (using the colorectal 

carcinoma cell line Caco-2 cells). 

P-0718. Modulating molecule produced by Candida albicans biofilm 

Estelle C, (ean-Marc 2, Marie-Hdlmne R, Christine I 

Equipe de Microbiologie Fondamentale et Appliqude, /CEE 6MR CNR. 6008, 86000 

Poitiers, France 

7+,5"5+*+68"#+,& is the most common fungal isolated from patients with indwelling 

medical device (IMD) associated infections. ae have previously described an ",*0"-$%* 

model of 7/*+68"#+,& biofilm associated with 100_ silicone catheters. 

The purpose of this worB was to demonstrate the production, by the 7/*+68"#+,&* 

yeasts growing as biofilm, of a molecule able to modulate biofilm growth. 

The supernatant medium recovered from 7/*+68"#+,& (ATCC J1[J) biofilm aged of 2O 

hours was added during the course of a new biofilm formation (tested strainsM ATCC 

J1[J, CA 20K1 or CA OOO) and its influence was subsequently evaluated with jTT 

method. Preliminary ultrafiltration and purification assays to characterize the 

modulating fungal molecule responsible for the biofilm supernatant activity are 

presented in this study. 

The addition of this supernatant to adherent 7/*+68"#+,& cells induced a significant 

(pv0.001) inhibition of the biofilm growth. This activity was not observed when the 

supernatant was added to preformed 7/*+68"#+,& biofilms. 6ltrafiltration and 

purification assays demonstrated that this molecule is small (vJ000 BDa), hydrophilic, 

and is different from farnesol. 

These preliminary results suggest that this new molecule could be a good Candidate 

for the prevention of biofilm associated with IMD. 

fur preliminary data have shown that 7/*+68"#+,& adhere tightly to both human cell 

lines and is able to invade cells within a few hours. The invasion seems to be a 

process which involves both fungal and host cell activities. As presumed, we observed 

clear quantitative differences in terms of adhesion and invasion properties depending 

on the cell type and the grade of differentiation of cells. Currently, we are investigating 

the response of the host cells at the cellular, protein and transcriptional level to better 

understand the sequences of events that govern adherence and invasion of both cell 

lines.

P-0719. Identification of top-like metallo-endooligopeptidase activity in 


Gravi E 1 , Paschoalin T 1 , Carmona A 2 , Felipe M J , Travassos / 1 , Rodrigues E 1 

1 6NfNEj - 6NIFE.P - .Éo Paulo, .P - 2razil, 2 Dept. of 2iophysics - 6NIFE.P - .Éo 

Paulo, .P - 2razil, J Dept. of Cell 2iology - 6niversity of 2rasilia - 2rasilia, DF, 2razil 

Paracoccidioidomycosis (PCM), caused by the pathogenic fungus 1+$+#%##"5"%"5.&* 

8$+&"6".,&"& (Pb), is a systemic mycosis causing severe acute and chronic disease. It is 

endemic in 2razil, mainly affecting rural worBers, and is also the prevailing mycosis in 

tropical and subtropical regions of /atin America. Invasion of host tissues by Pb involves 

interactions with the extracellular matrix (ECM). Enzymes, amongst them a serine thiolproteinase 

recognized before may facilitate the process of invasion by cleaving several 

components of the ECM. The identification of metallopeptidases may further help 

understanding the pathology of PCM and eventually introduce new targets for antifungal 

drugs against this mycosis. Recently, we identified and cloned the thimet 

endooligopeptidase (TfP or EC J.O.2O.1[) from a tumor cell line that regulated tumor 

development in vivo probably by controlling Binin-induced neo-angiogenesis (Paschoalin et 

al., unpublished). Presently a homologous enzyme activity was searched in Pb in view of 

the similar hydrolytic activity of fungal lysates. 

In order to identify the activity of metallo-endo-oligopeptidase in Pb,*cell lysates were 

obtained from Pb18 (subtypes V-virulent and L-low virulence), cultivated in YPD ([-7 days), 

by disruption with glass beads and ultracentrifugation to eliminate the membrane debris 

(100,000 @, K0 minutes, OÅC). Analysis of the enzymatic activity was carried out by 

quantified hydrolysis of internally quenched fluorogenic peptides (Abz-GF.PFR~-EDDnp, 

Abz-GFPPFR~-EDDnp, Abz-RPPGF.PFR~-EDDnp, Abz-GF.IPR~-EDDnp, Abz- 

NlPRRP~-EDDnp, Abz-dRR/-EDDnp, all substrates at 20 microM) at J7 o C in [0 mM Tris- 

HCl buffer, pH 7.O. Fluorescence was measured at Em } O20 nm and Ex } J20 nm in a 

spectrofluorometer in the presence or absence of classical enzymatic inhibitors (%- 

phenanthrolin, (A-2, Pro-Ile, PM.F, E6O, leupeptin, lisinopril, EDTA, captopril). 

2oth lysates hydrolyzed the peptide (Abz-GF.PFR~-EDDnp) and were completely 

inhibited by %-phenanthrolin, suggesting a TfP-liBe activity. Interestingly, Pb18V showed a 

more prominent activity with this substrate, suggesting a possible correlation of the enzyme 

activity with strain virulence. The Binetic assays excluded the presence of ECA-liBe, 

neprilysin-liBe (EC J.O.2O.11) and neurolysin-liBe (EC J.O.2O.16) activities, since there was 

no inhibition of the hydrolysis by lisinopril, no activity with the neprilysin specific substrate 

Abz-dRR/-EDDnp and low activity with substrates preferentially cleaved by neurolysin 

(Abz-GF.IPR~-EDDnp, Abz-NlPRRP~-EDDnp), respectively. 

In addition, a genomic library from Pb strain 01 was examined using as template the 

alignment of several oligopeptidases (Mep2 and Mip1, both from 4&'.$@"66(&*U()"@+-(&, 

bacterial fpdA, mammalian TfP, and metallopeptidases from 7/*+68"#+,& and 7/*@6+8$+-+). 

fne clone was obtained and is presently being expressed in bacterial and euBaryotic 

plasmids to isolate the active enzyme. 

P-0720. C. albicans Sup35p: Effects of a yeast prion on pathophysiology? 

Handwerger K, Cowen /, /indquist . 

ahitehead Institute for 2iomedical Research 

.upJ[p is a highly conserved translation termination factor. In =/*#.$.0"&"+., the N- 

terminal and middle regions (NM) of .upJ[p constitute a prion domain that renders 

the protein capable of stochastically adopting a heritable, inactive amyloid 

conformation which results in a high degree of nonsense suppression. The incidence 

of cells that contain aggregated .upJ[p (called the sP.Iru state) increases upon overexpression 

of the NM domain, presumably because increased production of the prion 

domain seeds aggregation of the endogenous protein (reviewed in s1u). .urprisingly, 

sP.Iru cells grow more robustly than spsi-u cells under several environmental 

conditions s2u. 

/iBe =/#.$.0"&"+. .upJ[p, the NM domain 7/*+68"#+,& .upJ[p contains a high 

percentage of polar, uncharged amino acids that are very liBely to render the protein 

conformationally labile. Although sP.Iru cells have not been observed in wild isolates 

of 7/*+68"#+,& sJu, it remains a possibility that the correct inducing conditions were not 

employed. To this end, we wondered the followingM A) Can the sP.Iru state be 

induced in 7/*+68"#+,&ú 2) If so, would the resultant nonsense suppression lead to 

altered pathophysiology in clinical isolatesú 

To address these questions, we have created transgenic clinical strains that overexpress 

either GFP alone or 7/*+68"#+,& NM-GFP under the control of a methioninerepressible 

promoter. ae are currently in the process of determining whether overexpression 

of CaNM-GFP is capable of pulling the endogenous .upJ[p into selfperpetuating 

aggregates, and whether this results in an increase in nonsense 

suppression. If we achieve these two goals, further analysis will reveal whether the 

sP.Iru state affects morphological switching, infectivity, and drug resistance in this 

organism. 

ReferencesM 

1. .erio, T.R. and /indquist, ../. (2001) The yeast prion sP.IruM molecular 

insights and functional consequences. 450*1$%-.",*7?.) [K, JK1-O12 

2. True, H./. and /indquist, ../. (2000) A yeast prion provides a mechanism for 

genetic variation and phenotypic diversity. `+-($. O07 (680J), O77-O8J 

J. Resende, C. et al. (2002) The Candida albicans .upJ[p protein (Ca.upJ[p)M 

function, prion-liBe behaviour and an associated polyglutamine length 

polymorphism. !"#$%8"%6%@2 1O8 (Pt O), 10OK-1060 

fur results suggest the presence of a TfP-6"[. metallo-endo-oligopeptidase activity in 1/* 

8$+&"6".,&"& cell lysates. The expression of the isolated clone and other tests on enzyme 

activity will help to understand the functional importance of this enzyme on fungal biology 

and pathogenesis. 

Financial supportM CNPq and FAPE.P. 


P-0721. The early invasion process of Candida albicans to the tongue surface 

in murine oral candidiasis 

Hisajima T 1, 2, J , Ishibashi H 1 , ladota T J , Nishyama Y 1 , FunaBoshi l J , Yamaguchi H 1 , 

Abe . 1 

1 TeiByo 6niversity Institute of Medical Mycology, 2 6niversity of Human Arts and 

.ciences, J YoBohama City 6niversity .chool of Medicine, Department of 

Neuroanatomy 

ae analyzed morphologically and microbiologically the adhesion and an invasion process 

in early stages of oral infection of 7+,5"5+*+68"#+,&. 

ICR mice were pretreated with prednisolone and tetracycline. ahen the mice were orally 

inoculated with 7/*+68"#+,& (TIMM26O0), they were anesthetized by intramuscular injection 

with chlorpromazine chloride. fral inoculation was performed by means of a cotton swab 

soaBed in 2.0x10 8 /ml viable cells suspension of 7/*+68"#+,&. The inoculated mice were 

sacrificed 1 or J hr after inoculation and their tongues were resected to examine 

morphologically by a scanning electron microscope (.EM) and the fluorescent staining for 

fungus. 2y .EM observation, numbers of 7+,5"5+ cells adhering to the lingual-papillae 

surface were counted in O8 views for every tongue, and total 7+,5"5+ cells adhering to one 

tongue were calculated. The tongues were washed by a physiological saline and 0.2[_ 

trypsin-solution, and then were homogenized to measure cell number of viable 7+,5"5+* 

+68"#+,& associated with tongue as colony forming units (CF6). 

Three hours after 7+,5"5+ infection, germ tube formation of 7/*+68"#+,& and phenomena 

suggesting hyphal invasion into the tongue organization were observed by a .EM image, 

and the invasion was confirmed microscopically by the fungal fluorescent staining 

(Fungifluora). The number of yeast cells on the surface of lingual papillae at 1 or J hr after 

infection observed by .EM, were calculated to be 0.1x10 [ and 0.[x10 [ cells/tongue, 

respectively. Total CF6 of 7/*+68"#+,& associated with each tongue at 1 and J hr after 

infection, were 1.6x10 [ and 2.6x10 [ cells/tongue, respectively. The CF6 recovered not by 

saline washing but by trypsin washing and the tongue homogenates increased at J hr after 

infection. Moreover, .EM image showed that mucoidal substance containing yeast cells 

was attached to the tongue surface in the J hr group of mice. The yeast cells and the 

substance liBe mucus were hardly observed in the tongue surface after trypsin washing, 

suggesting that these yeast cells and mucus on J hr-tongue surface might be detached by 

the trypsin treatment. 

Here, we obtained the evidence indicating that 7+,5"5+*+68"#+,& cells on the tongue 

surface showed germ tube formation and began to invade to epithelium about J hr after 

oral infection. Another important insight into interaction between 7+,5"5+ cells and tongue 

surface is that the number of 7+,5"5+ cells recognized as yeast cells on epithelium by .EM 

was only about 1/[ of total CF6 recovered from each tongue at J hr after infection. .ince 

.EM technique can not find out yeast cells in mucus or in gap of lingual papillae, this 

difference in cell number suggested that most of 7+,5"5+ cells associated with tongue 

presented in the mucus or the gap. ae also found that these 7+,5"5+ cells with tongues 

after 1 or J hr after 7+,5"5+ infection could be easily detached by trypsin-treatment, 

simultaneously with clearance of the mucus fluid containing yeast cells. This means that 

mucus-liBe substance containing 7+,5"5+ cells may have an important role in early step of 

interaction between 7+,5"5+ cells and tongue mucosal epithelium. 

P-0722. Phospholipase activity of Cryptococcus neoformans from human, bird 

and environmental samples 

Iatta R 1 , Cafarchia C 2 , Montagna M 1 , ftranto D 2 

1 Department of 2iomedical .cience and Human fncology, Hygiene .ection, Faculty 

of Medicine 6niversity of 2ari (Italy), 2 Department of Animal Health and aelfare, 

Faculty of Veterinary Medicine, 6niversity of 2ari (Italy) 

Phospholipases are enzymes hydrolysing ester-linBages in glycerophospholipids by 

cleaving a specific ester bond. In the early 1K80s it was shown that pathogenic fungi 

produce phospholipases which were suggested to play a role in their pathogenesis. 

Among opportunistic yeasts, phospholipases have been also detected in 7+,5"5+* 

+68"#+,&, P%5?%-%$(6+*$(8$+, 7$2'-%#%##(& ,.%U%$)+,&*G7`J*and !+6+&&.d"+*spp. 

7$2'-%#%##(&*,.%U%$)+,&*is one of the most important aetiological agents causing 

human infection in immunocompromised hosts. Actually two different species (7` and 

7/ @+--"") and five different serotypes (7` var. ,.%U%$)+,& -serotypes D and AD, 7` 

var. @$(8"" -serotype A, and 7. @+--"" -serotypes 2 and C) are accepted. ahile 

7$2'-%#%##(s*@+--""*is associated with a(#+62'-(& trees and usually causes infection in 

immunocompetent individuals, 7` varieties ,.%U%$)+,& and @$(8"" are commonly 

found in association with soil and avian excreta and are responsible for cryptococcosis 

in immunocompromised individuals. The aim of this worB is to investigate the 

phospholipase activity (ph.a.) of C$2'-%#%##(&*spp.*isolated from different sources. 

Thirty two C$2'-%#%##(&*spp.*were examined and divided into four groupsM Group IM 1O 

(6 serotypes A and 8 serotypes D) isolates from AID. patientsn Group IIM J (serotype 

2) from a(#+62'-(&*#+)+65(6.,&"&*treesn Group IIIM [ (serotype A) from cloacae of 

birds of preyn Group IVM 10 (serotype A) from bird droppings. Isolates were grown on 

.abouraud Dextrose Agar (.DA) slants at J7ÅC. After 2 and [ days of pre-incubation 

time the strains were subcultured onto egg-yolB plates at J7Å and O1ÅC respectively. 

Phospholipase production was evaluated as described by Price .-*+6*(1K82). Readings 

were taBen at day 2 and 6 post incubation. The student t-test was used for statistical 

evaluation of Pz values. 

At J7ÅC 26 isolates produced phospholipase after 2 days, while J0 after 6 days, both 

pre-incubated at 2 and [ days. The highest ph.a. (Pz}0,O0n Pv0.0[) was registered on 

isolates pre-incubated [ days. In particular, isolates from Group III and IV produced 

the highest ph.a. at J7ÅC (Pz}0.J2 and Pz}0.J6 respectively). 

At O1ÅC 2 isolates produced phospholipase after 2 days and 1O after 6 days only 

when preincubated at [ days. In particular out of the 1O isolates, [ were from Group I 

(Pz} 0.[O), J from Group III (Pz} 0.78) and 6 from Group IV (Pz}0.70). 

As far as isolates from Group II the highest ph.a. (Pz}0.J8) was registered at J7ÅC 

both at 2 and 6 days on isolates preincubated at [ days. 

The registration of a higher ph.a. both in isolates from Groups II, III and IV than that 

from Groups I suggests that 7`*isolated from birds and environment are pathogens to 

humans. The finding of a ph.a. at O1ÅC in 7$2'-%#%##(&*spp. from Groups I, II and IV 

indicates that at this temperature (which also is the bird body temperature) strains 

could be pathogenic. These findings along with recent case reports of cryptococcosis 

in birds suggest that*7$2'-%#%##(& spp. can be pathogenic also for birds.

P-0723. Fungus–bacteria interactions: Killing of Cryptococcus neoformans by 

the adherence of Staphylococcus aureus 

Ikeda R 1 , .aito F 1 , Yamaguchi M 2 , lawamoto . 2 

1 Department of Microbiology, Meiji Pharmaceutical 6niversity, 2 Division of 6ltrastructure 

and Function, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba 

6niversity 

Communication between distinct microbial species that colonize a single host may affect 

the numbers of each that survive in the host. Communication may also affect microbial 

growth, generation of virulence factors, and production of infectious diseases in human and 

animal hosts. To investigate the effect of an unrelated microbe on invasion and 

colonization by the pathogenic fungus 7$2'-%#%##(&*,.%U%$)+,&3 we investigated the 

relationship between this yeast and =-+'?26%#%##(&*+($.(&3*a bacterium that resides in the 

nasal cavity of healthy humans as part of the normal flora. 

ahen co-cultured with =/*+($.(&, the number of 7. ,.%U%$)+,& colony-forming units 

(CF6s) decreased. This decrease resulted from direct contact between the two microbes 

because 7. ,.%U%$)+,& was not Billed when separated from =. +($.(& by a membrane. 

Electron microscopic analysis revealed that 7. ,.%U%$)+,& cells were surrounded by =. 

+($.(&, which attached to the surface of the yeast cells. The yeast cells were damaged 

near the sites of =/*+($.(&*attachment and showed morphological characteristics of dead 

cells. K,*&"-( terminal deoxynucleotidyl transferase-mediated d6TR nicB end labeling 

assays confirmed that the yeast cells had undergone cell death with characteristic DNA 

fragmentation. 

To test whether the =/*+($.(&-mediated mortality of 7/*,.%U%$)+,& was species specific3 

we repeated the experiment using 10 different clinical and environmental isolates of 7/* 

,.%U%$)+,&/ .urvival of all strains decreased within the first 2 days of culture with =/* 

+($.(&. /onger culture periods revealed differences in the mortality rate among strains, 

although no differences were detected between clinical and environmental isolates. The 

percentage of yeast cells that adhered to =/*+($.(& was inversely correlated with 7/* 

,.%U%$)+,& survival. =/*.'"5.$)"5"&3 a more common form of =-+'?26%#%##(& found in 

human nasal cavities, Billed 7/*,.%U%$)+,& to a similar extent as =/*+($.(&. In contrast, 

=-$.'-%#%##(&*'2%@.,.& neither adhered to, nor Billed, 7/*,.%U%$)+,&. 

P-0724. Chlamydoconidia of zygomycetes in necrotic lung tissue 

Kimura M 

Pathology, linBi 6niversity .chool of Medicine 

Zygomycosis is an uncommon but frequently fatal mycotic infection caused by a 

member of the class Zygomycetes. 2road, irregular, pauciseptate hyphae are usually 

the only detectable fungal elements in tissue. Fungal elements other than hyphae are 

exceptional finding in tissue and their discovery may confuse the histological 

diagnosis. Chlamydoconidia are one of such exceptional elements in tissue of 

zygomycosis. This study describes another case of pulmonary zygomycosis in which 

chlamydoconidia were found in tissue. The ultrastructure of the chlamydoconidia in 

tissue was studied, too. 

An autopsy case of pulmonary zygomycosis in a patient with rheumatoid arthiritis on 

immunosuppressive therapy was presented. Chlamydoconidia, unusual fungal 

elements in zygomycosis, were found in cavitary infarct of the lung. They were seen 

with numerous hyphae in the necrotic tissue of the cavity. They were thicB walled, 

spherical, ovoid, or irregularly shaped, 8.[-2[.J (mean 1J.K) micrometer in width, and 

formed within or at the ends of the hyphae. Although most chlamydoconidia were 

spherical, some looBed liBe thicB-walled branched hyphae which appeared to be on 

the process of conidiation. They were immunohistochemically positive with anti- 

P?"d%'(& antiserum. 6ltrastructural study revealed cell wall production of 

chlamydoconidia in a parent hyphae. This is the first case which 

immunohistochemically showed chlamydoconidia to be produced by zygomycetous 

fungus. 

To identify the cell surface molecules that contribute to these interspecies interactions, cells 

were pretreated with NaIf O or a protease. Interspecies interactions involved a 

carbohydrate on 7/*,.%U%$)+,& and a protein on =. +($.(&. Indeed, addition of a capsular 

polysaccharide from 7. ,.%U%$)+,& inhibited =/*+($.(&-mediated mortality in co-culture, 

and treating the crude polysaccharide with protease increased this inhibition. The 

protective activity of the capsular polysaccharide resided in the glucuronoxylomannan 

(GjM) fraction of the capsule, although high concentrations of these polysaccharides were 

required for inhibition. A side-chain-cleaved version of the polysaccharide and an $-(1*J)- 

mannan inhibited the Billing at lower concentrations than native GjM and also reduced the 

adherence between the two species. 

In conclusion, staphylococcal cells inhibit the colonization of the respiratory mucosa by 7. 

,.%U%$)+,&. Adherence between the mannose bacBbone of the cryptococcal capsule and 

the proteins from =-+'?26%#%##(& may trigger the death of the pathogenic yeast. These 

data suggest a novel role for cryptococcal polysaccharides as a virulence factor. 


P-0725. Flow cytometry analysis of cell wall surface glycans expression in 


Martínez-Esparza M 1 , .arazin A 2 , (ouy N J , .endid 2 2 , Poulain D 2 , (ouault T 2 

1 Departamento de 2ioquÜmica y 2iologÜa Molecular (2) e InmunologÜa, Facultad de 

Medicina, 6niversidad de Murcia, Murcia, .pain, 2 Inserm 67KKn /aboratoire de 

Mycologie Fondamentale et Appliqude, 6niversitd de /ille II, /ille, France, J Inserm 

IFR11On .ervice Commun d]Imagerie, Facultd de Mddecine H. aarembourg, /ille, 

France 

The yeast 7+,5"5+*+68"#+,& is an opportunistic pathogen, part of the normal human 

microbial flora, that causes infections in immunocompromised individuals with a high 

morbidity and mortality rates. Recognition of yeasts by host cells is based on 

components of the yeast cell wall, which differently contribute to virulence. Among 

these, 7/*+68"#+,& cell wall glycans play an important role in the continuous 

interchange that regulates the balance between saprophytism and parasitism, and 

resistance and infection. However, little is Bnown about their accessibility at the cell 

wall surface and their dynamic expression depending on growth conditions. 

In this worB we exploited flow cytometry methods to analyze the expression of glycans 

at the yeast cell surface by using different antibodies specific for glycan epitopes. 

These consisted in rabbit polyclonal IgG specific for alpha or beta-mannosides 

epitopes developed by Iatron Inc or murin monoclonal antibodies specific for betaglucans, 

alpha and beta mannosides epitopes 

9lycan surface expression was analyzed in both 7/*+68"#+,&*and*=+##?+$%)2#.&* 

#.$.0"&"+. in different growth conditions. 

The results showed that the expression levels of alpha- and beta-linBed mannosides 

as well as beta-glucans can be successfully evaluated by flow cytometry methods 

using different antibodies excluding agglutination reactions. Interestingly it was shown 

that the surface expression pattern of beta-mannosides detected by both monoclonal 

and polyclonal antibodies was differently modulated along the growth course. 

According to the Bnown distribution of oligomannose epitopes, these results suggest 

that the yeast beta-mannosides content which is exposed on mannoproteins or 

glycolipids is increased in phase stationary, whereas those linBed to mannans are not 

affected by the yeast growth phase. 

P-0726. Identification of genes involved in the transfer of beta-1,2 mannose in 

Candida albicans and preliminary analysis of their contribution to virulence. 

Mille C 1 , Trinel P 1 , Maes E 2 , Guerardel Y 2 , (ouault T 1 , (anbon G J , Poulain D 1 

1 6nitd Inserm 67KK - /aboratoire de Mycologie Fondamentale et Appliqude, /ille, 


Villeneuve d]Ascq, France, J 6nitd de Mycologie Moldculaire, Institut Pasteur, Paris, 

France 

The study of glycosylation in the yeast =+##?+$%)2#.&*#.$.0"&"+. significantly 

contributed to our understanding of this important process by leading to the cloning 

and characterization of genes involved in the synthesis of different glycans and 

glycoconjugates. However, some glycan modifications present in the pathogenic yeast 

7+,5"5+*+68"#+,& are not present in =/*#.$.0"&"+.. Among these are beta-1,2 linBed 

oligomannoside (beta-Man) residues commonly found in the phosphopeptidomannan 

(PPM) and the phospholipomannan (P/M) isolated from 7/*+68"#+,&. beta-Man 

containing glycans contribute to virulence by interacting with host-specific receptors 

and interfering with the immune response. Despite the importance of 7/*+68"#+,&*beta- 

Man, genes responsible for their synthesis have not been identified. 

In this report we present the identification, disruption and characterization of a novel 

family of fungal genes involved in beta-Man transfer. Employing ",*&"6"#% analysis we 

identified nine new genes in 7/*+68"#+,&, designated 7+Z!>&. The construction and 

analysis of deletion mutants in 7/*+68"#+,& allowed us to elucidate and confirm the 

function of the new genes in beta-Man synthesis. ae could demonstrate that the 

members of this gene family play specific and overlapping roles in both the diversity of 

beta-Man containing glycans as well as the glycosylation of different substrate 

molecules. .urprisingly individual deletions have only minor effect on the global 

surface expression of beta-Man epitopes. 2y contrast, preliminary experiments using 

",*0"-$% and ",*0"0% assays showed that the mutants displayed different virulence 

depending on the nature of the manno-glycoconjugate whose beta-mannosylation is 

affected. These results suggest that assessment of the role of beta-Man in virulence 

must therefore taBe in account both the importance of the carrier molecule and a 

probable sophisticated mechanisms that regulate their global expression over different 

carrier molecules. 

The cytometric method we have developed represents a valuable method to 

investigate to what extent 7/*+68"#+,&*is able to regulate its glycan surface expression 

and therefore to modify specific interactions with the host panel of specific ligands. It 

may represent a useful tool for examining glycan phenotype alterations induced by 

environmental conditions or mutagenesis in parallel to virulence studies.

P-0727. Differential expression of beta-1,2 oligomannosides and of the genes 

responsible for their synthesis. 

Mille C 1 , Fradin C 1 , Trinel P 1 , .lomianny M 2 , Maes E 2 , (anbon G J , Poulain D 1 

1 6nitd Inserm 67KK - /aboratoire de Mycologie Fondamentale et Appliqude, /ille, 


Villeneuve d]Ascq, France, J 6nitd de Mycologie Moldculaire, Institut Pasteur, Paris, 

France 

Among the established important factors contributing to 7+,5"5+*+68"#+,& specific 

virulence are beta-1,2 oligomannosides (beta-Man) associated at the cell wall surface. 

beta-Man have been found to contribute to pathogenicity through host cell adhesion 

and immunomodulatory properties. Immunoreactivity studies have shown that beta- 

Man were associated to the phosphopeptidomannan (PPM), the phospholipomannan 

(P/M) and several mannoproteins (MPs). 

After the identification of a novel family of genes involved in beta-Man transfer 

(7+Z!>&), the construction of deletion mutants combined with immunochemical and 

structural analysis of PPMs and P/Ms allowed us to elucidate the functions of some of 

the new genes in beta-Man synthesis. Interestingly, as detected by flow cytometry and 

immunofluorescence, the global cell wall surface expression of beta-Man was 

unchanged whatever the deletion strain studied. Furthermore, differential betamannosylation 

of mannoproteins was also evidenced as result of inactivation of 

7+Z!>& family specific members. These results suggest the existence of a regulation 

process controlling the amount of beta-Man expressed at the cell wall surface. 

P-0728. Dermatophyte-secreted proteases 

Monod M 

Dermatology, CH6V 

In a culture medium containing proteins as sole nitrogen source dermatophytes 

secrete endo- and exoproteases similar to those of 4&'.$@"66(& species. However, in 

contrast to 4&'.$@"66(& sp., dermatophytes secrete several endoproteases of the 

subtilisin family (serine proteases), and of the fungalysin family (metalloproteases). 

These enzymes, called .ubs and Meps, respectively, were remarBably conserved 

across species. The tree topology clearly indicated that the multiplication of subtilisin 

and fungalisin genes in dermatophytes occurred prior to species divergence. 

Apparently, the dermatophyte genes coding for secreted endo- and exoproteases are 

repressed by small molecules such as ammonium and amino acids. It is evident that 

the dermatophyte secreted proteases use Beratin and the different cross-liBed proteins 

of the cornified cell envelope as a substrate since these fungi grow exclusively in the 

&-$+-()*#%$,.(), nails or hair as sole nitrogen and carbon sources. It is reasonable to 

postulate that during infection the dermatophytes are under catabolic repression to 

secrete a complete battery of endo- and exoproteases which cooperate efficiently in 

digestion of Beratinized tissues. The main function of the former is to produce a large 

number of free ends on which the latter may act. Filamentous fungi, liBe bacteria, 

yeasts, as well as specialized cells of plants and animals, express membrane proteins 

for uptaBe of amino acids, dipeptides and tripeptides and large peptides cannot be 

used as nutrients. .ynergism of secreted endoproteases and exopeptidases is liBely 

essential for dermatophyte virulence. 

In this report we analyze the differential expression of beta-Man and of the genes 

responsible for their synthesis (7+Z!>&J. To get a better Bnowledge of the expression 

of beta-Man in the cell wall and their regulation, beta-1,2 mannosylated MPs have 

been identified by proteomic analysis and their level of beta-mannosylation 

determined in the different mutants. RT-PCR experiments allowed us to analyze the 

7+Z!>& expression in different strains (serotype A and 2, mutants) and different 

growth conditions (e.g. temperature, pH, host cells/tissue invasion). ae mainly 

focused on the expression of 7+Z!>: involved in serotype A specificity of 7/*+68"#+,& 

strains. Altogether the results of proteomic, transcriptomic and glycomic studies will 

bring new insights on 7/*+68"#+,& beta-1,2 mannosylation mechanisms. 


P-0729. Physiological role of Cryptococcus neoformans response to hypoxia 

Moranova Z 1 , PavliceB ( 1 , Novotny R 1 , lawamoto . 2 , RaclavsBy V 1 

1 PalacBy 6niversity flomouc, Faculty of Medicine, Czech Republic, 2 Research Center 

for Pathogenic Fungi, Chiba 6niversity, (apan 

Purpose of the study: Recently, a unique hypoxia-response has been described in 

the obligate aerobic pathogenic yeast 7$2'-%#%##(&*,.%U%$)+,&/*ahen oxygen 

concentration drops in culture under conditions of limited aeration, it slows growth and 

delays the onset of budding to G2 that is gradually prolonged, resulting eventually in 

unbudded G2-arrest in some strains. Purpose of this study was to characterise this 

stress response in detail in order to help elucidate its physiological role. 

Description: ae have studied survival of 7/*,.%U%$)+,& under severe hypoxia in 

cells exposed to mild hypoxia and compared it to well-aerated controls. ae have also 

followed marBers of hypoxia response during different phases of 7/*,.%U%$)+,&* 

growth in culture and monitored the influence of arginine supplement on hypoxia 

response. 

Results and conclusions: ae show, that gradual adaptation to hypoxia increases 

survival of 7/*,.%U%$)+,& under severe hypoxia. ae also demonstrate that hypoxia 

response plays role in transition to stationary phase even in well-aerated cultures. 

This can be illustrated by the delay in the onset of budding from . to G2. Futhermore, 

when cells arrested in unbudded G2 are subjected to prolonged oxygen limitation (7- 

1O days), few of the large unbudded G2 cells are able to escape the cell cycle arrest 

and give rise to daughter cells. fnce the mother cell escapes the arrest, it is able to 

give rise to daughters repeatedly, however, these daughters are unable to continue 

growth and separate from their mothers, resulting in peculiar clusters of small dropliBe 

daughters arranged around the budding site of their mother. /ast but not least, we 

show that arginine supports cycling of 7/*,.%U%$)+,& cells under hypoxic conditions 

but not under normoxic conditions, suggesting possible role of a putative Nf-synthase 

as an oxygen sensor in this yeast. To conclude, 7/*,.%U%$)+,& is able to respond to 

decreased oxygenation even in well-aerated culture, most probably to reduce oxygen 

consumption, to prevent a real limitation. Possible role of hypoxia response in the 

pathogenicity of this yeast should be further examined. Czech .cience Foundation 

(J10/06/06O[) and Ministry of Education, Youth and .ports, Czech Republic 

(M.M61K8K[K216) supported this worB. 

P-0730. Identification of Candida albicans germ tube components involved in 

the interaction with human blood resting platelets. 

Nail-Billaud S, 2eucher 2, Marot-/eblond A, Robert R 

Groupe deEtude des Interactions HÇtes-Parasites 6FR des .ciences 

Pharmaceutiques et deIngdnierie de la .antd deAngers, 16, bd Daviers, OK000 Angers, 

France 

In systemic mycoses, dissemination of 7+,5"5+*+68"#+,& may occured through the 

blood stream and the yeast may interact with components such as fibrinogen, 

fibronectin, complement or with blood cells such as polymorphonuclear leuBocytes or 

platelets. 

Previously, we have shown that this yeast and mainly the germ tube phase can have 

close interactions with blood resting platelets. In order to identify fungal components 

involved in the interaction with platelets, biotinylated cell surface components of germ 

tube extracts were incubating with resting platelets. Fungal components bound to 

platelets were revealed after .D.-PAGE and western blotting with streptavidin 

affinodetection. The results showed a polydispersed staining pattern with an apparent 

molecular mass of 110 to 170 BDa and a component of [[ BDa. This high molecular 

weight component was recognized by MAb JDK.J which is Bnown to recognize a 7/* 

+68"#+,& germ tube specific epitope that was that was shown to be carried by 

Agglutinin /iBe .equence J protein (AlsJp). Moreover, adhesion experiments between 

disrupted 7/*+68"#+,& (7/*+68"#+,& +6&:#

P-0731. Inhalation of Stachybotrys chartarum causes pulmonary hypertension 

in mice - relation to human PPH 

Ochiai E 1 , lamei l 1 , aatanabe A 1 , .ato A 1 , Hiroshima l 2 , .hibuya l J 

1 Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba 6niversity, 

Chiba, (apan, 2 Department of 2asic Pathology, Graduate .chool of Medicine, Chiba 

6niversity, Chiba, (apan, J Department of Pathology, fmori Hospital, Toho 6niversity 

.chool of Medicine, ToByo, (apan 

=-+#?28%-$2&*#?+$-+$()*is a ubiquitous dematiaceous fungus often found in our living 

environment. Inhalation of the conidia of =/*#?+$-+$() has been suspected as a cause 

of idiopathic pulmonary hemorrhage (IPH) in infants. Although the relation between 

this fungus and IPH is not yet clearly understood, =/*#?+$-+$() may cause other 

diseases because this fungus can produce various mycotoxins. ae have been 

investigating the effect of repeated inhalation of the fungus into the lung using a 

mouse model and found the inhalation caused recruitment of segmented leuBocytes 

around pulmonary arteries (fchiai et al., (pn. (. Med. Mycol. O6M 10K-117, 200[.). 

In the present study, the effect of longer repeated exposure was examined.*=/* 

#?+$-+$() isolated from house dust was used. Conidia were collected and injected via 

a catheter into the trachea of mice (ddY, 6-weeB-old, male) at 1 % 10 2 to 1 % 10 O / 

mouse. The injection was repeated for 6 - 18 times over O to 12 weeBs, and lungs and 

the other organs were examined. 

Histopathological examination of the mice that received 1 % 10 O conidia showed the 

development of pulmonary hypertension. .ymmetrical thicBening of the intima and 

media of pulmonary arteries was seen all over the lung, while Infiltration of the 

segmented leuBocytes around arteries has disappeared. Right ventricular hypertrophy 

was also evident. These findings were similar to those seen in human primary 

pulmonary hypertension (PPH), which is Bnown as an incurable disease of unBnown 

etiology. The vascular changes progressed as the intratracheal injection was repeated. 

fur results indicate that the repeated inhalation of =/*#?+$-+$() conidia caused 

human PPH-liBe histopathological changes in mice. 2eing commonly found in the 

ambient air, =/*#?+$-+$()*may be causing PPH in humans when inhaled. Further 

studies are warranted as to the mechanism of the vascular changes and its relation to 

human diseases. 

P-0732. Glycosylated mannoproteins are essential to Candida albicans biofilm 

integrity and development 

Ramage G 1 , Coco 2 1 , Gow N 2 

1 Glasgow Caledonian 6niversity, Glasgow, 6l., 2 Aberdeen Fungal Group, Aberdeen 

6niversity, 6l. 

Purpose of the study: 7+,5"5+*+68"#+,&*has the ability to form biofilms on innate and 

biological substrates, such as dentures and mucosal surfaces. These are 

morphologically heterogeneous structures and have specific phenotypes due to 

different patterns of gene expression in sessile cells as compared to their planBtonic 

counterparts. Adhesins, including cell wall components, are pivotal factors involved in 

the formation of biofilms. Glycosylated mannoproteins have been shown to be 

important factor in adhesion and virulence. 

Summarized description of the project: It is the objective of this study to evaluate 

the role of cell wall mannan in biofilm development and susceptibility to antifungal 

agents using defined BnocBout strains in genes involved in glycosylated mannoprotein 

biosynthesis. The following null mutants were included in this study which were 

created using the 6RA-blaster techniqueM ),-I**($-1,2-mannosyl transferase)*3* 

),-M *($-1,2-mannosyl transferase) 3*),-I*

P-0733. A simple method for the detection of the lipolytic activity of 

Paracoccidioides brasiliensis strains utilizing rhodamine B 

Scroferneker M 1 , .topiglia C 1 , Daboit T 1 , Ruschel / 1 , Corbellini V 2 , Carissimi M 1 

1 Instituto de Ciüncias 2Zsicas da .aâde, Porto Alegre, 2razil, 2 Departamento de 

FÜsica e ~uÜmica da 6niversidade de .anta Cruz do .ul, 2razil 

Research about lipases produced by microorganisms has many purposes, including 

industrial uses and food technology, but little attention has been given to these 

enzymes in human fungal pathogens. The aim of this worB was to identify lipase 

activity of twelve 1+$+#%##"5"%"5.&*8$+&"6".,&"& strains using Rhodamine 2 and olive 

oil. The 1/*8$+&"6".,&"& strains were purchased from the Tropical Medicine Institute - 

6.P (strains 18, 17[, 26[, 6K8, 812, 2JJK, /DR/, /DR2, Mg7, Mg10, I2Iî-T2) and 

from .anta Casa Hospital (.anta Casa strain) and were incubated for 8 days in 

bilayer media for lipase detection (basal mediaM peptone [ gn beef extract J gn sodium 

chloride [ gn agar 1[ gn distilled water 1 /n overlay mediaM olive oil 10 m/n gum arabic 

20 gn agar 1[ gn distilled water 1 / and a solution of Rhodamine 2 1 mg.m/ -1 in 

distilled water, with a final concentration of 0.001[_). fne strain of =.$$+-"+* 

)+$#.&#.,& was used as positive control, and one of a&#?.$"#?"+*#%6" as negative 

control. Free fatty acids (FFA) released from lipase activity were detected through 

orange fluorescence when exposed to 6VA light on agar plate complexed with 

Rhodamine 2. There were differences in lipolytic activity among strainsM fluorescence 

intensity was strong in 812, I2Iî-T2, .anta Casa and 18, moderate in Mg10, 2JJK 

and 26[, and weaB in /DR/ and 17[, whereas /DR2, Mg7 and 6K8 showed no 

activity. The highest lipase activity (orange fluorescence) was observed on biomass, 

with no fluorescent haloes around the colonies. This plate assay is sensitive for the 

detection of lipase-producing strains at earlier growth stages and suitable to detect 

intracellular lipases in dimorphic fungi in their yeast phase. 

P-0734. Virulence factors and molecular epidemiology between pathogenic and 

colonizing Candida strains isolated from patients with candidemia 

Silva V 1 , Contreras / 1 , DÜaz M 1 , Fica A 1 , MartÜnez C 2 

1 6niversity of Chile, .chool of Medicine., 2 6niversity of .antiago de Chile. 

Candidemia has increased significantly in recent years, being an important cause of 

morbidity and mortality in patients admitted to the intensive care unit (IC6) and 

associated with endogenous nosocomial infections. The high rate of this infection 

could be attributed to the advances in the medical sciences that have generated a 

greater number of susceptible patients to acquire invasive mycosis. This increase also 

would be given by the selection of more virulent yeasts and/or antifungal tolerant 

strains. 

aith the purpose to determine this asseveration, we designed a one-year multicentric 

study to analyze clone relationship and phenotypic expression of virulence factors 

such as adhesion, germ tube, filament production and enzymatic activity, as well as 

the antifungal susceptibility between pathogenic and no-pathogenic strains isolated 

respectively of blood and colonized sites from IC6 patient with candidemia. 

During the study period, 20 patients (16 children and O adults) colonized with 7+,5"5+ 

spp developed candidemia. Fourteen patients (70_) presented the same strains from 

blood and other colonized sites such as mouth (K), intestinal tract (7), urine (8), 

vaginal (O) and sBin (J), and 12 of which, the DNA analysis by pulsed field gel 

electrophoresis (PFGE) determined the clone relationship among pathogenic and nopathogenic 

strains. The media of nosocomial candidemia development was over 20 

days of IC6 stay. 

All 7K strains were sensible to amphotericin 2 and [_ were resistant to fluconazole 

without differences between pathogenic and no-pathogenic strains. 

The expression of the virulence factors was different among the species. ahile 7/* 

+68"#+,& showed high filament production (media J8_) and fosfolipase activity (Pz 

media 0.[J), 7/*-$%'"#+6"& and 7/*6(&"-+,"+. showed high adhesion (t [00 cells by 

microscopic field) and protease activity (Pz 0.OO), respectively. fn the other hand, 

blood isolated strains showed higher expression of one or more virulence factors than 

non-pathogenic clone related strains. 2lood 7/*+68"#+,& strains showed more 

adhesion, filament and protease activities, but there are not differences between germ 

tube production and fosfolipase activity. 

All patients were treated with amphotericin 2, 6 of them received association with 

fluconazole and 1 with [-flucytosine. The global lethality was 66_ in candidemia by 7/* 

+68"#+,& and JJ_ by 7. no-+68"#+,& (pv0.0[). 

This study corroborated the endogenous origin of nosocomial candidemia and showed 

that pathogenic and no-pathogenic clones-related 7+,5"5+ strains present different 

phenotypic expression of some virulence factors according the origin site, which 

suggests influence of the host environment in the regulation of virulence. 

This Research was partially supported by DI 6niversity of Chile Grant

P-0735. Familial distribution of Candida carriage in Crohn’s disease patients 

and their healthy relatives and correlation with anti-yeast antibodies 

Standaert-Vitse A 1 , FranÑois N 1 , Vandewalle P 1 , (oossens M 2 , Chamaillard M J , 

2ranche ( J , /ibersa C O , Colombel ( J , .endid 2 1 , Poulain D 1 

1 Inserm 67KK, Facultd de Mddecine, CHR6, /ille, France, 2 Division of 

Gastroenterology, 6niversity Hospital Gasthuisberg, /euven, 2elgium, J Inserm 67K[, 

.ervice de gastroenterologie, HÇpital Huriez, CHR6, /ille, France, O Inserm CICKJ01, 

HÇpital Cardiologique, CHR6, /ille, France 

Purpose of the study: Anti-=+##?+$%)2#.&*#.$.0"&"+. antibodies (A.CA) are 

present in 60_ of patients with Crohn]s disease (CD), 20_ of their healthy relatives 

(HR) while they are only present in 6_ of healthy individuals. ae have previously 

shown that 7+,5"5+*+68"#+,&3*a commensal*of the human intestinal tract, could be an 

immunogen for A.CA. 

The aim of the study was to evaluate the prevalence and the diversity of yeast 

recovered during natural colonization of the digestive tract in families affected with CD 

and controls, and to correlate 7+,5"5+ carriage with anti-yeast antibodies and/or 

genetic determinants of the subjects. 

Description: Mouth swabs and stool samples were collected from 1J0 CD patients, 

160 healthy relatives including spouses, offspring, and siblings from O[ multiplex 

families and 78 healthy controls from 1O controls families. Anti-=/#.$.0"&"+. mannan 

(A.CA) anti-7*+68"#+,& mannan (Platelia*7+,5"5+ Acw, )antibodies were determined 

in serum samples from each subjects. 

Results and conclusion: The prevalence of 7+,5"5+ carriage was [[_ in CD 

patients, [1_ in their healthy relatives and O[_ in controls families. No significant 

difference in prevalence of carriage was observed between CD and healthy subjects 

('}0.J6). 

The A.CA titers were significantly different in CD patients compared to their firstdegree 

healthy relatives and to healthy controls. (71_nJ8_nO_) whereas the anti-7/* 

+68"#+,& antibodies were not (21_n20_n22). Anti-7/*+68"#+,& antibodies strongly 

correlated with carriage of 7/*+68"#+,& in intestinal tract in healthy relatives and CD 

compared to controls (pv0.0001n p}0.0008n p}0.1[2 respectively) whereas A.CA 

correlated with carriage only in healthy relatives, but not in CD patients. Interestingly, 

in multiple affected families, we observed that 8/JJ (2O_) spouses from A.CA 

positive CD patients were also A.CA positive. 

The correlation between 7/*+68"#+,& carriage and anti-yeasts responses in healthy 

relatives but not in CD patients suggest that A.CA may reflect the failure of tolerance 

to 7/ +68"#+,& at a given time in CD patients. The observation that A.CA can be shed 

vertically between related individuals confirms the previously well established joint 

influence of genetic bacBground and environmental conditions on A.CA. 2y contrast, 

the very high concordance in A.CA positive status we observed between genetically 

unrelated marital pairs suppose a prominent effect of environment or a transmissible 

agent on their generation. 

P-0736. Correlation of chemical structure of mannan and virulence of genus 

Candida: #-1,2-mannotetraosyl residue as a marker 

Suzuki S 1 , .uzuBi M 1 , .hibata N 2 , lobayashi H J 

1 .endai Research Institute for Mycology, 2 TohoBu Pharmaceutical 6niversity, 

J NagasaBi International 6niversity 

In 1KK7, .uzuBi tabulated the chemical structures of mannans of 7 medically important 

Candida species by Tuchiyaes group into 2 groups, !-1,2-tetraosyl(MO, M!1-(2M1)2- 

2M and !-1,2-triosyl(MJ, M!1-2M1-2M)residues, according to the maximum lengths of 

!-1,2-oligomannosyl moieties in the branches, corresponding to Albicans and non- 

Albicans Candida spp., respectively. Although any interpretation has been given on 

this finding, it was suggested that distribution of MO is limited in the mannans of 

virulent Albicans group, C. albicans, C. parapsilosis, C. stellatoidea, and C. tropicalis. 

fn the other hand, the MJ group consists of less virulent non-Albicans spp., C. famata, 

C. glabrata, C. guilliermondii, C. Befyr, and C. saitoana. In 2002, .uzuBi again 

described the same table emphasizing the existence of relationship between the 

structural difference of mannans and extents of virulence of the 2 groups of Candida 

spp. It has also been shown that the absence of MO and the presence of MJ were 

clarified on the mannans of following avirulent yeasts, Citeromyces matritensis, 

lloeBera brevis, lluyveromyces lactis, .accharomyces cerevisiae, .. italicus, and 

Pichia pastoris by 2 groups of worBers, 2allou and .uzuBi. The above findings 

indicate that MO can be utilized as one of the marBers of the virulent spp. of the genus 

Candida, i.e., the gene(s) responsible for expression of MO at least in part regulate the 

virulence of Albicans group of Candida spp., although the analytical process 

ÄacetolysisÄ is not preferable to clinical laboratories. In 200J, .hibata et al. reported 

the chemical structure of mannan of C. lusitaniae, a less virulent non-Albicans 

Candida spp., and stating the existence of MJ and the absence of 

MO. .imultaneously, lobayashi et al. documented that the detection of MO in the 

mannans of O C. tropicalis strains, providing a confirmatory evidence for the 

relationship between structure and virulence of mannan. Although essential 

component of virulence has been shown to be the hyphal formation releasing 

virulence factors, aspartic protease, phospholipase, and lipase, it is possible to 

postulate that the mannans and their relating enzymes can also participate in 

virulence of the mycelial form. Findings on the virulent C. albicans Va J2 strain 

containing mannan with !-1,2 pentaosyl residue by Paulaines group agree with the 

above interpretation. An example for glycosyl transferases as a virulence factor has 

been proposed by Taniguchies group on N-acetylglucosaminyl transferase-V as an 

angiogenesis factor, accelerating lung cancer metastasis. Recent findings by Prill et 

al. on [ mannosyl transferase isoforms provides a clue for development of novel 

antifungal agents. 

Therefore, characterization including cDNA cloning of GDP-mannoseM!-1,2- 

mannotriosyl transferase and the relating factors should be done to clarify the 

virulence-expression mechanism by Candida spp. of Albicans group. 


P-0737. Candida albicans glycoproteins involved in skin test reactivity 

Toriello C 1 , Mora-Montes H 2 , Flores-Carreán A 2 , Pdrez-MejÜa A 1 , Carbajosa ( J 

1 6niversidad Nacional Autánoma de Mdxico, Facultad de Medicina, Depto. 

MicrobiologÜa y ParasitologÜa, Mexico City, Mdxico, 2 Instituto de Investigacián en 

2iologÜa Experimental, 6niversidad de Guanajuato, Guanajuato, Mdxico, J Instituto 

Nacional de Nutricián y Ciencias Mddicas Ä.alvador ZubirZnÄ, Mexico City, Mdxico 

7+,5"5+ sBin test antigen injection for the treatment of common warts has been 

shown to be effective and well accepted by patients. aarts are caused by multiple 

strains of the human papillomavirus (HPV), and intralesional 7+,5"5+ antigen may 

induce an immune response through stimulation of HPV-directed immunity providing a 

therapeutic response to common warts in many patients. A mannoprotein fragment of 

7/*+68"#+,& cell wall has been reported as a powerful immuno-modulatory fraction, 

hence we are investigating different 7+,5"5+ glycoproteins from a 7+,5"5+ sBin test 

antigen (candidin) to understand their role in sBin test reactivity, and be able to purify 

the sBin test reactive glycoprotein to achieve better results in therapeutic wart 

treatment. 7/*+68"#+,& sBin test antigens were analyzed by .D.-PAGE and 

immunoblotting with a rabbit anti-7/*+68"#+,& hyperimmune serum. .Bin test (.T) 

reactivity was tested on 7+,5"5+ sensitized guinea pigs by intradermal injection of 0.1 

ml of different isolated glycoproteins, cell walls, candidin as positive and sterile saline 

as negative controls. The presence or absence and size of the ensuing reactions were 

recorded after 2O and O8 hours. Erythema and induration of at least [ mm in diameter 

were scored as a positive reaction. .D.-PAGE showed low and high molecular 

glycoprotein bands (28.O to t220 BDa). Immunoblots with the rabbit anti-7/*+68"#+,& 

hyperimmune serum detected immune reactivity for the [6.[, 61, K[, 120,1K[ and 

t220 BDa bands. The high molecular weight glycoproteins (t220 BDa), purified cell 

walls, and candidin controls induced positive sBin tests in the sensitized guinea pigs. 

These preliminary results suggest the high molecular weight glycoproteins (t220 

BDa) as the reactive .T molecules from the cell wall. 

P-0738. Proinflammatory cytokines # expression in skin of healthy volunteer 

by application of Malassezia fungi. 

Yoshihiro s 

TeiByo 6niversity .chool of Medicine, MizonoBuchi Hospital 

.eborrhoeic dermatitis is a disease of unBnown etiopathogenesis that affects about 

[_ of the population. Already there are some reports about the expression of cytoBine 

from cultured Beratinocyte aggrevated by malassezia fungi. 

In this study, I investigated expression of I/-1 alpha, I/-1 beta, I/-6, I/-10, IFN-gamma, 

and the TNF alpha in sBin of healthy volunteer by patch test of M. globosa, M. restricta 

or these mix with E/IZA. These samples are collected after 8,16,2O,J2 hours. In 

healthy sBin, I/-6, I/-10, IFN-gamma, and the TNF alpha had not detected. There are 

slight elevation of TNF alpha, and I/-1 alpha or I/-1 beta are elevated at 2O hours. I 

presented the different cytoBine expression by M. globosa, M. restricta or these mix to 

explain the mechanism of developing seborrhoeic dermatitis.

TAXONOMY* 

P-0739. Identification of mating type of clinical isolates of Cryptococcus 

neoformans and Cryptococcus gattii by polimerase chain reaction - PCR 

Barbosa, Jr A 1 , de Melo D 1 , .antos P 1 , Morales 2 2 , /azera M 2 , Trindade R 1 , de 

Resende M J 

1 /MA, 6niversidade Federal de .ergipe, .an Cristovao, .ergipipe, 2razil, 

2 /aboratorio de Micologia Ambiental, IPEC, FIfCR6Z, J /aboratorio de Micologia, 

IC2/6FMG 

7$2'-%#%##(&*,.%U%$)+,& and 7$2'-%#%##(&*@+--""*are an opportunist fungus, which 

causes meningitis both in immunodepressed and in immunocompetent patients. The 

life cycle of both species involves an anamorphous phase (yeast form, present both in 

nature and in clinical specimens) and a theleomorph phase (filamentous, absent in 

clinical specimens) with formation of basidiospores, indicating that the sexual status of 

the fungus exists in nature. The basidiospores are dispersed in the air and can reach 

the lungs. The sexual reproduction of both species has been observed under 

laboratory conditions. Conjugations between the sexual types ()+-",@*-2'.&) | !4> $ 

and !4> a, result in the formation of dicariotype mycelium. The present worB have 

liBe objetive the identification of mating type of clinical isolates of 7$2'-%#%##(& 

neoformans and 7$2'-%#%##(& @+--"" by polimerase chain reaction | PCR in .ergipe. 

The isolates belong to the collection of cultures of the /aboratory of Applied 

Microbiology of the Federal 6niversity of .ergipe. 1J samples of 7$2'-%#%##(&* 

,.%U%$)+,& and 7$2'-%##(&*@+--"" from the liquor of patients with infectious meningitis 

in .ergipe, 2razil, during the period of 2001 to 200O were investigated. The laboratory 

procedures related to molecular biology (DNA extraction and PCR execution) followed 

Chatuverdi .-*+6/ (2000) as reference. All clinicals samples tested, 7/*,.%U%$)+,& and 

7/*@+--"" are )+-",@*-2'.*+6'?+ ($), considered most virulent. 

P-0740. AFLP diversity in Pseudallescheria boydii 

Becker W, Zeng (, Reiffert, ., Gerrits van den Ende A, de Hoog G, Horrd R 

Centraalbureau voor .chimmelcultures, 6trecht, The Netherlands 

Genotypes of the opportunistic fungus 1&.(5+66.&#?.$"+*8%25"" are Bnown to be highly 

diverse. Early molecular data using DNA-DNA-reassociation revealed three 

approximate groups within in 1/*8%25""/ Multi-locus sequencing using 2T2, T62, CA/ 

and IT. showed that there are different groups within 1/*8%25""3 but also that several 

clades are strictly concordant. For this reason several sequence types were 

recognized as separate speciesM in addition to*1/*8%25"" and =#.5%&'%$"()*'$%6"U"#+,&3* 

the 1&.(5+66.&#?.$"+ complex now comprises e.g.*=#.5%&'%$"()*+($+,-"+#() and 

1&.(5+66.&#?.$"+*)",(-"&'%$+ (Gilgado et al. - (CM 200[, OJM OKJ0-OKO2). The 

variability of 1/*8%25""*&.,&(*&-$"#-% is studied here using Amplified Fragment /ength 

Polymorphism (AF/P), and compared to the degree of diversity observed in relatives 

of this species. .trains analyzed were those maintained in the C2. culture collection, 

and those made available through the ECMM NetworB 1&.(5+66.&#?.$"+*

P-0741. Trichophyton eboreum – a recently detected dermatophyte 

Brasch J 1 , Grxser Y 2 

1 6niversity of liel, 2 Charitd 2erlin 

Dermatophytes are world-wide distributed highly specialized filamentous fungi that 

can utilize Beratin and cause sBin infections (dermatophytoses) in humans. Among 

them, anthropophilic, zoophilic and geophilic species of different pathogenicity are 

Bnown. To prove a suspected diagnosis of dermatophytosis it is necessary to isolate 

and identify the causative agent from lesional sBin. ae report on a new dermatophyte 

that was isolated from mocassin-type tinea pedum of a man from the Ivory Coast. 

This fungus was characterized by a rapid growth on common mycological media 

resulting in a flat off-white thallus with a feathered margin and a granular surface. 

Microscopically numerous clavate microconidia and macroconodia with 2-K cells were 

seen that had smooth and thin walls. flder cultures developed spiral hyphae, 

chlamydospores and cleistothecia-liBe structures. The fungus showed a weaB urease 

activity, no dependency on vitamins and a positive hair perforation test. A sequence 

analysis of the internal transcribed spacer of the rRNA gene revealed more than [ _ 

divergence from any Bnown species of dermatophytes. 

These characteristics indicated that our strain was a dermatophyte of the genus 

>$"#?%'?2-%,. Due to its distinctive features which were not compatible with any of the 

Bnown >$"#?%'?2-%, species, it was described as a new speciesM >$"#?%'?2-%,* 

.8%$.(). 

Distribution and pathogenicity of this species are as yet unBnown. In our case the 

attempted reisolation from the patient]s sBin failed. This may be due to the 

antimycotic therapy that had been initiated right after the initial collection of scrapings 

from the stratum corneum or to the fact that the fungus was a non-pathogenic 

contaminant of the sBin. ae suppose that >$"#?%'?2-%,*.8%$.() is a geophilic 

dermatophyte with a very limited distribution. 

P-0742. Arthroderma olidum, sp. nov. a new addition to the Trichophyton 

terrestre complex. 

Campbell C 1 , Borman A 1 , /inton C 1 , 2ridge P 2 , (ohnson E 1 

1 HPA Mycology Reference /aboratory, 2ristol, 6l, 2 2ritish Antarctic .urvey, 

Cambridge, 6l 

>$"#?%'?2-%,*-.$$.&-$. is a mitosporic species referable to several closely related Arthroderma 

species in the Ascomycete family Gymnoascaceae. The original description of >/*-.$$.&-$. by 

Durie c Frey was from two fungi collected from soil in Australia s1u. These authors considered 

that the diagnostic feature of their new species was the production of conidial forms intermediate 

in type between the unicellular microconidia and the multicellular macroconidia of the pathogenic 

Trichophyton species. 

Reports quicBly followed of its isolation from soils in England, California and Germany. In 1K61 

Dawson c Gentles s2u described 4$-?$%5.$)+*e(+5$"U"5() as an ascomatal form arising 

spontaneously in a culture of >/*-.$$.&-$. from the soil of a rabbit burrow in .cotland, and showed 

that 4/*e(+5$"U"5() was heterothallic s2u. However despite successfully crossing single ascospore 

isolates derived from 4/*e(+5$"U"5() with several European >/*-.$$.&-$. strains, they failed to 

induce sexual reproduction with the ex-type strain of >/*-.$$.&-$. of Durie c Frey, or with 

Californian isolates. Following mating compatibility studies in >/*-.$$.&-$., Pore, Tsao and 

PlunBett sJu described a second Arthroderma species, 4/*6.,-"#(6+$(). They demonstrated three 

other incompatibility groups, from Californian isolates (Groups IV, VI) and from Hungary (Group 

Ij). However, they declined to erect new species for these as each group contained only two 

isolates. Group Ij was later shown, by mating with >/*-.$$.&-$. isolates from Canada, 

CzechoslovaBia and the 6.A sOu to belong to a third Arthoderma species, 4/*",&",@(6+$.. 

In 1K81, four fungal isolates from hair of the european badger (!.6.&*).6.&) were examined by 

Dr Phyllis .tocBdale at the CMI, lew, and deposited with the 6l National Collection of 

Pathogenic Fungi as an undescribed member of the >$"#?%'?2-%,*-.$$.&-$.*complex. This report 

describes a new ascomycete taxon, 4$-?$%5.$)+*%6"5() following successful recent attempts to 

re-isolate the same fungus from the soil of badger holes in .outh aest England (Full description 

of 4/%6"5()*is submitted to !.5"#+6*!2#%6%@2). Furthermore, using ribosomal RNA gene 

sequencing, we show that the asexual form of 4/*%6"5() is conspecific with the recently 

described >$"#?%'?2-%,*.8%$.()*(2rasch c Grxser 200[) s[u isolated from human sBin in 

Germany. 

References: 

1. Durie E2, Frey D. A new species of >$"#?%'?2-%, from New .outh aales !2#%6%@"+ 

1K[7n 49: O01-O11. 

2. Dawson Cf, Gentles (C. The perfect states of c.$+-",%)2#.&*+\.66%" Vanbreuseghem, 

>$"#?%'?2-%,*-.$$.&-$. Durie c Frey and !"#$%&'%$()*,+,() Fuentes. =+8%($+(5"+ 

1K61n 1: OK-[7. 

J. Pore R., Tsao GC, PlunBett fA. A new species of 4$-?$%5.$)+ established according 

to biological species concepts. !2#%6%@"+ 1K6[n 57: K6K-K7J. 

O. Padhye AA, Carmichael (a. 4$-?$%5.$)+*",&",@(6+$. sp. nov., another Gymnoascous 

state of the >$"#?%'?2-%,*-.$$.&-$.*complex. =+8%($+(5"+ 1K72n 10: O7-[1. 

[. 2rasch (, Grxser Y. >$"#?%'?2-%,*.8%$.() sp. nov. isolated from human sBin. (. Clin. 

Microbiol. 200[n 43: [2J0-[2J7.

P-0743. DNA walk divergence program applied to phylogenetic analysis in 

black yeasts using its rDNA sequences 

Campolina S 1 , Caligiorne R 1 , /icinio P 2 , De Hoog . J 

1 FIfCR6Z - CENTRf DE PE.~6I.A. RENE RACHf6, 2 Departamento de Fisica, 

6niversidade Federal de Minas Gerais, 2razil, J Centraalbureau voor 

.chimmelcultures, 6trecht, the Netherlands AND Institute of 2iodiversity and 

Ecosystem Dynamics, 6niversity of Amsterdam, Amsterdam, the Netherlands 

.equences of rDNA Internal Transcribed .pacer (IT.) of a standard set of blacB 

yeast-liBe fungal pathogens were compared using two methodsM local and global 

alignments. The latter is based on DNA-walB divergence analysis. This method has 

become recently available as an algorithm (DNAaD program) which converts 

sequences into three-dimensional walBs. The walBs are compared with, or fit to, each 

other generating global alignments. The DNA-walB geometry defines a proper metric 

used to create a distance matrix appropriated for phylogenetic reconstruction. In this 

worB, the analyses were carried out for species currently classified in 7+'$%,"+3* 

76+5%'?"+6%'?%$+3*a_%'?"+6+3*^%,&.#+.+3*1?"+6%'?%$+*and P+)"#?6%$"5"(). Main 

groups were verified by ..6 rDNA data. DNAaD applied to IT.2 alone enabled 

species recognition as well as phylogenetic reconstruction reflecting clades 

discriminated in ..6 phylogeny, which was not possible with any other algorithm 

using local alignment for the same data set. It is concluded that DNAaD provides 

rapid insight into broader relationships between groups using genes that otherwise 

would be hardly usable for this purpose. 

P-0744. Phylogeny of Madurella-like agents of black-grain mycetoma 

de Hoog S 1 , 2orman A 2 , AvesBamp M 1 

1 1Centraalbureau voor .chimmelcultures, 6trecht, The Netherlands, 2 PH/. Mycology 

Reference /aboratory, 2ristol Public Health /aboratory, 2ristol, 6.l. 

The genus !+5($.66+ has been described to accommodate non-sporulating melanized 

filamentous fungi originating from cases of blacB-grain mycetoma in humans. Two 

diametrically opposing theories on the etiology of human eumycetoma have appeared 

in literature. The classical view (?2'%-?.&"&WI) is that the genus comprises a limited list 

of causative agents, all being established pathogens typically causing mycetoma. The 

view is supported by the overrepresentation of a small number of species involved in 

the numerous reported cases of eumycetoma, such as 1&.(5+66.&#?.$"+*8%25""3* 

!+5($.66+*)2#.-%)+-"& and 12$.,%#?+.-+*$%).$%". 

Alternatively (?2'%-?.&"&WM), it has been suggested that mycetoma is an unspecific 

response to the subcutaneous inoculation of a wide range of principally saprotrophic 

agents. There are three arguments supporting this view. Firstly, it is difficult to imagine 

how a fungus can be successful in its evolution when it is dependent on the unnatural 

situation of being traumatically introduced into its host. .econdly, when more 

ecological data on agents of mycetoma, such as 1/*8%25"" or 4#$.)%,"()*["6".,&., 

become available,*it is realized that even the most common species on Rippon]s list of 

agents of mycetoma actually have another ecological behaviour as mainstay, 

suggesting that subcutaneous growth in human tissue is insignificant. Thirdly, sterile 

fungi are difficult to identify, and clinicians are inclined to picB one of the names of 

agents of mycetoma. Earlier we showed that only a small portion of the agents from 

imported cases in The Netherlands actually concerned recognized agents of the 

disease (de Hoog .-*+6., 1KKJ). It was therefore surmised that anything cultivated from 

a subcutaneous grain and showing reduced growth without reproduction ",*0"-$% might 

incorrectly be identified as q!+5($.66+p. 

To address this question, we sequenced IT. rDNA region of a worldwide 

representation of [6 strains of agents of blacB-grain mycetoma, and ..6 rDNA for a 

selection of species. fn the basis of ..6 rDNA sequence data, !+5($.66+ was 

proven to be phylogenetically heterogeneous (de Hoog .-*+6., 200J). The generic type 

species, !/*)2#.-%)+-"&, is a member of the .ordariales, and is related to the 

ascomycetes `.%-.&-(5",+ and 7?+.-%)"(), which occasionally are involved in 

mycetoma-liBe infections. !+5($.66+*@$"&.+ is a member of Pleosporales. It is flanBed 

by the ascomycete genus X.'-%&'?+.$"+ and several coelomycete agents of blacBgrain 

mycetoma, classified in 12$.,%#?+.-+ and 1&.(5%#?+.-%&'?+.$%,.)+. aith 

IT., a remarBable diversity was observedM seventeen species were recognized, 

eleven of which are hitherto unnamed and have to be introduced as new taxa in due 

course. 

These results support ?2'%-?.&"&WM, that agents of mycetoma are liBely to have been 

artificially grouped in clinical form-genera such as !+5($.66+. 

However, the preponderant etiologic of blacB-grain mycetoma in arid East Africa is 

!+5($.66+*)2#.-%)+-"&. Nearly all strains sequenced thus far proved to be strictly 

identical. All strains of z!+5($.66+ sp. O] originated from Venezuela. Confirmed 

X.'-%&'?+.$"+ species all are from tropical Africa. 12$.,%#?+.-+*$%).$%" is regularly 


imported in Great 2ritain from PaBistan and India, although it is also Bnown from 

Venezuela. .trains from remote geographic areas, such as islands of Indonesia and 

the Carribbean, mostly appeared to have unique IT. sequences. 

In conclusion, both hypotheses are correct. The biodiversity of agents of blacB-grain 

mycetoma is remarBably large, with the majority of species still waiting to be 

discovered. fn the other hand, the agents do show a remarBable clinical potential, as 

they are preponderant in the geographic and climate zone where their environmental 

form can survive. 

References: 

Hoog, G... de, Adelmann, D., Ahmed, A.f.A. c 2elBum, A. van, 200O. Phylogeny and 

typification of !+5($.66+*)2#.-%)+-"&, with a comparison of other agents of 

eumycetoma. Mycoses O7M 121-1J0. 

Hoog, G... de, 2uiting, A., Tan, C..., .troebel, A.2., letterings, C., 2oer, E.(. de, 

Naafs, 2., 2rimicombe, R., Nohlmans-Paulssen, M.l.E., Fabius, G.T.(., lloBBe, A.H. 

c Visser, /.G., 1KKJ. Diagnostic problems with imported cases of mycetoma in The 

Netherlands. Mycoses J6M 81-87. 

P-0745. A new opportunistic species of Cladophialophora 

de Hoog S 

Centraalb voor .chimmelcultures, 6trecht, Netherlands 

76+5%'?"+6%'?%$+ is a member of the order Chaetothyriales, ascomycetes that also 

comprise the blacB yeast genus a_%'?"+6+ and relatives liBe ^%,&.#+.+ and 

1?"+6%'?%$+. 6pdated medical literature mentions 7 species in this genus, involved in 

human diseases (de Hoog .-*+6. 2000). Two of them are more frequentM the potentially 

fatal*7/*8+,-"+,+ and the etiologic agent of chromoblastomycosis*7/*#+$$"%,""** 

(RevanBar .-*+6. 2002n lantarcioglu c de Hoog 200J). 2oth occur in 

immunocompetent individuals (Yeguez-Rodriguez .-*+6 .1KK2), the first is found world 

wide and the second is mainly reported from arid regions of tropical .outh America, 

.outh Africa and Australia (/avelle, 1K80). The natural niche outside humans is 

unBown for most of the species. .elective isolation techniques have been performed 

by the use of high temperature s.udhadham, 1Kúúu, a mouse vector sDixon .-*+6., 

1KK2u or extraction via mineral oil sIwatsu .-*+6/31K81). Recently, a culture was isolated 

from an interdigital infection in an immunocompromised child as well as from the 

patient]s garden in Curitiba, 2razil (Carvalho .-*+6. 2006). Morphological observations 

revealed pigmented septated conidiophores bearing one-celled olivaceous conidia 

arising in short, cohering, acropetal chains. Temperature test showed positive growth 

at J7oC and O0 oC. DNA was extracted using CTA2 method and the rDNA Internal 

Transcribed .pacer (IT.) and Elongation Factor (EF) regions were sequenced. The 

results were then compared with those present in a research data base maintained at 

C2. (6trecht, The Netherlands). A very high degree of similarity with an 

environmental strain, C2.11OJ26, recovered from a biofilter in The Netherlands 

(Prenafeta-2oldâ, 200O) was shown.*.ince there was no match between the strains 

reported here and any described taxonomic entity, this worB describes a new species 

of 76+5%'?"+6%'?%$+3*7/*.6.@+,&/*The strains were deposited at C2. Culture 

Collection.

P-0746. Human and chimpanzee-derived pneumocystis are genetically different 

but closely related 

Demanche C 1 , .alle 2 2 , aanert F J , 2erthelemy M O , Aliouat C 1 , Duriez T 1 , Guillot J O 

1 EA J60K, Facultd de Pharmacie, 6niversitd /ille 2, /ille, France, 2 Centre 

International de Recherches Mddicales de Franceville, Franceville, Gabon, J Centre de 

Primatologie, 6niversitd /ouis Pasteur, .trasbourg, France, O Ecole Nationale 

Vdtdrinaire deAlfort, Maisons-Alfort, France 

1,.()%#2&-"& organisms constitute a heterogeneous group of host-specific fungi that 

colonize a very wide range of mammalian species. Primates, including human beings, 

are frequently infected by these opportunistic fungi and 1,.()%#2&-"& pneumonia is 

still considered as one of the most serious fungal respiratory infections occurring in 

immunocompromised patients. In the present study, 1,.()%#2&-"& organisms were 

detected by nested PCR at the mitochondrial large subunit (mt/.6) rRNA locus in 

nasal swab samples from O0 captive chimpanzees (1+,*-$%@6%52-.&). These animals 

were tested negative for the .imian Immunodeficiency Virus. Amplification of the 

mt/.6 rRNA gene was positive for [O _ of the adults, 6K _ of the subadults and for 2 

out of the 2 babies examined. This higher proportion of carriers in young animals is 

consistent with similar observations in human communities and strongly supports the 

hypothesis that infants constitute a major reservoir for 1,.()%#2&-"& organisms in 

primate populations. 

Direct sequencing of PCR products demonstrated that the unique mt/.6 rRNA 

sequence type was specifically associated to the chimpanzee species. No 

chimpanzee was proved to be infected by 1,.()%#2-"&*\"$%0.#""*(human-derived 

1,.()%#2&-"&). Phylogenetic analyses indicated that human and chimpanzee-derived 

1,.()%#2&"& formed a monophyletic group in the clade of 1,.()%#2&-"&*from*fld 

aorld primates. This study confirmed that the genetic divergence in primate-derived 

1,.()%#2&-"& varies according to the phylogenetic divergence existing among the 

corresponding host species. The hypothesis of co-speciation between 1,.()%#2&-"& 

and their respective mammalian hosts is reinforced. 

P-0747. Characteristic variable internal repeat region in the rdna igs of 

dematiaceous fungi, Phialophora verrucosa and Phialophora americana 

Fukushima K, TaBizawa l, Hashizume T 

Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba 6niversity 

2oth dematiaceous fungi of 1?"+6%'?%$+*0.$$(#%&+ and 1/*+).$"#+,+ are human 

pathogens and are closely related morphologically. The object of this worB is to get 

molecular information for taxonomy as well as criteria for strain discrimination of the 

both fungi. 

1/*0.$$(#%&+ ([ strains) and 1/*+).$"#+,+ (7 strains), which were isolated as clinical 

specimens and natural inhabitants, were used for this purpose. 

The whole IG. regions of rDNA of the twelve strains were amplified using a set of 

primers of Z-IG.-/ and Z-IG.-R and afforded the amplicons with 2.[-2.K Bb, 

respectively. 2ase sequencing for all amplicons was achieved by a primer walBing 

method. The phylogenetic tree of the amplicons by 6PGMA method demonstrated 

some clusters in which both species were presented as a mixture. 

The base sequence analysis showed that both species possess the variable internal 

repeat (VIR) region in IG. as a specific sequence. The VIR was characterized to 

consist of four elements, E1(6J bp), E2 ([0-[2 bp), EJ (21 bp), and EO (KO-K6 bp) and 

was classified into three variants, VIR(, VIR), VIR*, with reference to arrangement 

of the elements. Detail sequence information of the elements and VIR regions are also 

presented. The results suggested that 1/*0.$$(#%&+ and 1/*+).$"#+,+ are 

indistinguishable on the sequence data of IG. region including VIR and that the VIR 

would serve to be an excellent criterion for strain discrimination of the both species. 


P-0748. Phylogenetic and morphological evaluation of Onychocola-like isolates 

obtained from nail, skin and other substrates 

Gibas C 1 , Sigler L 2 

1 6niversity of Alberta Microfungus Collection and Herbarium, Edmonton, A2, Canada, 

2 6niversity of Alberta Microfungus Collection and Herbarium, Edmonton, A2, Canada 

Background: The ascomycete genus*4$+#?,%)2#.& was described over one 

hundred years ago and remained obscure until recently when it was linBed to species 

within the anamorph genus b,2#?%#%6+. Two species, b/*#+,+5.,&"& (4/* 

,%5%&.-%&(&) and b/*[+,." (4/*[+,."), have been confirmed as uncommon agents of 

onychomycosis or sBin infection in patients from temperate and subtropical regions. 

.everal isolates of the newly described b/*@6+$.%&+ (4/*@6+$.%&(&) were obtained from 

nails or sBin but data were insufficient to ascertain etiologic causality. 

Purpose: This study characterized a group of 12 b,2#?%#%6+-liBe isolates isolated 

from cutaneous specimens or from other substrates. .ix of these isolates were 

identified provisionally as 4/*)",")(& based on absence of an anamorph. METHfD.M 

To assess relationships among isolates and with Bnown 4$+#?,%)2#.& species, we 

examined cultural features including expression of conidia or ascomata, conducted 

mating tests among some isolates, and compared nuclear ribosomal small subunit 

(..6) and internal transcribed spacer (IT.) region sequences. 

Results: Morphological and molecular data confirmed that all 12 isolates were placed 

within 4$+#?,%)2#.& and that none had a high degree of sequence similarity with any 

Bnown species. Phylogenetic analysis of IT. sequences revealed a considerable 

amount of genetic diversity among eight isolates from nail or sBin and only two of 

these isolates grouped together with high bootstrap support. The six isolates identified 

originally as 4/*)",")(& were distributed among several clades. 

Conclusion: 2ased on molecular and morphological data, we restrict 4/*)",")(& to 

the ex-type strain and provide evidence to support the description of three new 

species of 4$+#?,%)2#.&. .ix isolates are nonsporulating in culture, but they are 

identified as b,2#?%#%6+ species by morphological characters in conjunction with IT. 

sequence data. The occurrence and habitat in nature of b,2#?%#%6+*(4$+#?,%)2#.&)* 

species are not well understood. Although they occur in varied habitat types, several 

species appear to have a predilection for human nail or sBin. Results of our study 

provide a basis for the identification of b,2#?%#%6+-liBe isolates that might be of 

clinical importance and for finding the source of the inoculum. 

P-0749. Differentiation of closely related species of dermatophytes by SDSpage 

analysis of secreted protein profiles 

Giddey *, Monod M 

Dermatology .ervice, Centre Hospitalier 6niversitaire Vaudois (CH6V), /ausanne, 

.witzerland 

Background: The dermatophytes are the most common human and animal agents 

responsible for superficial mycosis. These infections, dermatophytoses, commonly 

referred as ringworm or tinea are restricted to nonliving cornified layers (sBin, hair and 

nails) because of the specific capacity of these fungi to invade only Beratinised 

tissues. Dermatophytes, are a group of fungi, phylogenetically and taxonomically very 

closely related, presenting variations from one isolate to another and overlapping 

characteristics between species. 

.equence analysis of the ribosomal DNA and in particular of the internal transcribed 

spacer (IT.), have been an important tool for species identification and for the study 

of the phylogenetic relationship between species, but have led to controversy. For 

instance, >$"#?%'?2-%,*.e(",()*and*>$"#?%'?2-%,*-%,&($+,& showing identical 

sequences were considered as synonyms, although these species exhibit distinct 

phenotypes. This is why, we have looBed for other tools allowing species 

discrimination. 

Objective: Investigate the possibility that dermatophytes with different ethology 

produce distinctive patterns of proteins from culture filtrates. 6se proteomic methods 

to obtain information that would aid in solving taxonomic problems of species 

identification. 

Methods: All isolates were grown on .abouraud agar and to promote production of 

proteolytic activity, in soy protein liquid medium. Concentrated extracts of secreted 

proteins were analysis by .D.-PAGE with a O_-12_ gradient. Gels were stained with 

Coomassie brilliant blue R-2[0. Measures of band molecular mass and optical density 

were performed using the Image~uant software. Proteins identification was performed 

by /C-M./M.. 

Results: Proteins of culture supernatant from several dermatophyte species were 

collected after 1[ days of growth and secretion profiles were compared using .D.- 

PAGE. The protein secretion patterns were common and preserved between strains 

coming from a same species. In contrast, the comparison of profiles between species, 

reveals differences among secreted proteins and level of expression. This specific 

protein printing is an useful and easy way to distinguish dermatophytes. For instance, 

very close related species as >/*-%,&($+,& and >/*.e(",() were easily differentiated 

thanBs to its completely different pattern of secretion. 

Conclusion: Closely related species can unambiguously be differentiated using .D.- 

PAGE profile of secreted proteins.

P-0750. Characterization of Scedosporium frequentans: The most common 

species of the P. boydii complex 

Gilgado F, Cano (, Gend (, Guarro ( 

6niversitat Rovira i Virgili 

1&.(5+66.&#?.$"+*8%25"" is an ascomycete that has evolved into an important agent of 

severe infections in immunocompromised patients. Recently, using a multilocus 

sequence analysis, we have demonstrated that 1/*8%25"" is actually a complex of 

species (1). ae have performed a detailed molecular, physiological and morphological 

study of a set of 127 isolates belonging to the different species of the complex and 

other related species. ae have sequenced the region T62 of the # tubuline gene of 

all these isolates, which, in our previous study, was the most phylogenetically 

informative locus of the O evaluated marBers. In addition, we have evaluated the 

usefulness of [K physiological tests to maBe distinction among the phylogenetic 

species. The combination of these studies allowed us to distinguish molecularly and 

phenetically the five lineages obtained in the tree. A synoptic Bey that has proven to 

be useful to differentiate these lineages is provided. Two new species have been 

proposedM =#.5%&'%$"()*5.?%%@" and =/*U$.e(.,-+,&. The latter is the most frequent 

species of the genus as O8_ of the isolates tested, randomly chosen, belong to it. 

Reference: 

Gilgado F et al. 200[. (CM. OJMOKJ0-O2. 

P-0751. Assignment of a secondary structure to the internal transcribed spacer 

2 (its 2) region of opportunistic black yeasts(herpotrichiellaceae) 

Haase G 1 , Maltusch C 1 , de Hoog G 2 

1 Institute of Medical Microbiology, 6niversity Hospital RaTH Aachen, Gemany, 2 C2., 

6trecht, The Netherlands 

Purpose of the study: Melanized fungi with affiliation to the Herpotrichiellaceae are 

of vital importance in medical mycology since they harbour agents of a wide array of 

mycoses, such as phaeohyphmycosis, chromoblastomycosis, cerebral mycoses and 

mycetoma. Morphology has proven to be insufficient for reliable diagnostics for many 

of these pleomorphic species. 

Despite the availability of a vast amount of IT. 2 sequences in the EuBaryota, 

seemingly random variability of these sequences and lacBing support for a secondary 

structure has interfered with the use of this information for e.g. phylogenetic analyses 

and identification. 6nfortunately recognition of conserved domains could not be 

achieved by use of available RNA folding programs. 

However, recently common cores within the IT.2 throughout the EuBaryota were 

inferred by comparative analyses of more than 20.000 sequences, and this has led to 

the identification of four clearly separate domains (I to IV). 

Description: 2ased upon the given common core structure we assigned a secondary 

RNA structure to nearly O00 available IT.2 sequences of Herpotrichiellaceae 

(7+'$%,"+ spp., 76+5%'?"+6%'?%$+ spp., a_%'?"+6+ spp., ^%,&.#+.+ spp., 1?"+6%'?%$+ 

spp., P+)"#?6%$"5"() spp., P?",%#6+5".66+ spp.) with the aim of confident alignment. In 

a first step we have performed this tasB for the domain I and II using the programs 

2IfEDIT and C2Canalyzer. 

Results and conclusion: In all studied IT.2 sequences the two domains including 

the spacer sequence between them could be assigned reliably. 6sing this secondary 

structure information for an alignment allows identification of most of the species 

studied. This analysis revealed that some of the deposited sequences derived from 

misidentified fungal isolates. The terminal loops of the two domains as well as bulges 

in domain II showed the highest degree of sequence variability. ff interest is the 

presence of an insert (additional stem) in the domain I in case of a_%'?"+6+*).&%'?"6+ 

and an extended terminal loop in domain I in case of P+)"#?6%$"5"()*)+#[.,d".". 

Most probable these changes have occurred in a single evolutionary event. 

Alignment of IT.2 sequences guided by a secondary structure information hold great 

promises for a reliable and easy-to-perform identification of fungi and also allow novel 

insights in the evolution of fungi by inferring their phylogeny. 


P-0752. Genetic and phenotypic variations among F1 progenies of Arthroderma 

benhamiae 

Kawasaki M 

lanazawa Medical 6niversity, IshiBawa, (apan 

.ome >$"#?%'?2-%, ).,-+@$%'?2-.& isolates recently isolated from humans and a rabbit in 

(apan were identified as 4$-?$%5.$)+ 8.,?+)"+. through mating tests with 4/*8.,?+)"+., 4/* 

&")""*and*4/*0+,8$.(&.@?.)""3 although their mitochondrial DNA (mtDNA) types were the same 

as that of >. 0.$$(#%&(). A strain (lM6O16K) of this fungus mated with an Americano-European 

race tester strain of 4/*8.,?+)"+.*RV26678 (r), while two of the other strains (lM6O1J6, O1J7) 

were successfully mated with the African race tester strain RVJ000 (r). 

Purposes: The phenotypic and genotypic variations within F1 progenies produced between 

parents of different genotypes were investigated to evaluate the range of variations in a species. 

Methods: .eventy-seven mono-ascospore cultures were isolated from J ascocarps produced by 

crossing the isolates lM6O16K and RV26678. The morphology and growth rate of the colony on 

.abouraud agar medium and basal medium were observed. Mating type was determined by 

juxtaposition to tester strains of both races. mtDNA type was determined by restriction fragment 

length polymorphisms (RF/P) with the restriction enzyme A+.III, and nuclear DNA (nDNA) type 

was determined by RF/P in the internal transcribed spacer regions of rRNA genes (IT.) with the 

restriction enzyme A",f I. 

Results: The colony were glabrous, powdery, velvety or fluffy, and white, beige, pale yellow, 

yellow, orange, wine red, reddish brown or darB purple. Growth rate varied from very slow to 

rapid. 6rease activities [ days after inoculation varied from negative to strongly positive. Hair 

perforation was observed in all the cultures except one with a unique glabrous texture, which 

seems to be a mutant. The ratio of positive to negative mating types was 1M2. All the isolates had 

the same mtDNA type as that of RV26678. nDNA (IT.) types of RV26678 and lM6O16K 

appeared in the ratio 1M0.6. ff the F1 progenies, [6_ shown hybridization between the mating 

type and nDNA (IT.) type, 6[_ between the mating type and mtDNA type, and J8_ between 

the nDNA (IT.) type and mtDNA type. 

Discussion: Mating types and the nDNA types were inherited from parents independently, 

suggesting that the corresponding genes are located on different chromosomes. The reason(s) 

why the ratios were not unity is unBnown. The mtDNA type was inherited from only one parent, 

RV26678, suggesting that mitochondria were inherited with cytoplasm. All the phenotypes, 

except hair perforation and mating types, showed great variations and appeared in a variety of 

combinations, suggesting that they are not interrelated. Positive urease activity does not seem to 

be a species-specific characteristic of >/*).,-+@$%'?2-.&3 although it is usually used to 

discriminate >/*).,-+@$%'?2-.&*from >/*$(8$(). There are many reports of the assessment of 

the validity of urease tests for identifying these species, because its discrimination power is not 

completely reliable. 6rease negative (after 20th day) >/*).,-+@$%'?2-.& and urease positive 

(before 7th day) >/*$(8$() strains have been reported in other studies, confirmation of the 

identification of the strains is essential. In comparison with those papers, our results reflect the 

exclusiveness of >/*).,-+@$%'?2-.& isolates, F1 progenies of 4/*8.,?+)"+.. 

P-0753. Muti-locus analysis of infra- and inter-species variations among 

trichophyton species 

Kawasaki M 1, 2 , aaBasa A 1 , Anzawa l 2 , Tanabe H 1 , MochizuBi T 1, 2 , IshizaBi H 2 , 

lagawa . J , Hemashettar 2 O 

1 Dept. Dermatol., lanazawa Medical 6niversity, IshiBawa, (apan, 2 Div. Dermatomycol. 

(Novartis Pharma), lanazawa Medical 6niversity, IshiBawa, (apan, J Dept. Dermatol., 

.howa 6niversity, ToByo, (apan, O Hi-Tech Health Care c Diagnostic Center Pvt. /td., 

2elgaum, India 

.equencing of the internal transcribed spacer regions of rRNA genes (IT.) to identify 

dermatophytes has become popular recently. Identifications using this technique do not always 

match those made morphologically, and more weight is being given to the former. Therefore, it is 

not uncommon to see very confusing descriptions such as >/*).,-+@$%'?2-.& var. ",-.$5"@"-+6. 

being described as having a granular type or zoophilic strains even though it does not Ø they are 

characteristics of var. ).,-+@$%'?2-.&. 

Purposes: To evaluate the consistency between morphological taxa and phylogenetic clades. 

Methods: A multi-locus analysis of infra- and inter-species variations among 28 strains of 12 

>$"#?%'?2-%, species (identified morphologically, physiologically and by IT. sequence similarity) 

and 4$-?$%5.$)+ species (identified by mating tests) was performed. The loci were the IT., actin 

gene (ACT) and DNA topoisomerase II gene (TfP). Phylogenetic trees were reconstructed for 

each gene using the distant matrix method, and the software Clustalj and Phylip J.62. The 

extent of agreement between the topologies of the trees, and between existent species identified 

morphologically and the phylogenetic groups were evaluated. 

Results: >$"#?%'?2-%, species were divided into three clades on the IT. and TfP trees, and into 

four clades on the ACT tree. Topological incongruities were noticed within each clade. fn all J 

trees, >/*0.$$(#%&()3*>/*#%,#.,-$"#()*and*>/*).,-+@$%'?2-.&*var/*.$",+#." were infra-species 

variations of 4$-?$%5.$)+ 8.,?+)"+., while >/*).,-+@$%'?2-.&*var.*",-.$5"@"-+6. was an infraspecies 

variation of 4/*0+,8$.(&.@?.)"". fn the TfP tree, >/*-%,&($+,&, >/*).,-+@$%'?2-.&*var.* 

",-.$5"@"-+6., >/*).,-+@$%'?2-.& var. e(",#[.+,() and 4/*0+,8$.(&.@?.)"" were infra-species 

variations of 4/*&")"". It was revealed that morphological species consist of many genetically 

different strains, that genetic infra-species variations overlap, and that 4$-?$%5.$)+ species 

cannot be phylogenetically separated from their related asexual species. Additionally 4/*&")"" and 

4/*0+,8$.(&.@?.)"" could not be separated from each other on the TfP tree. 

Discussion: To understand the results without conflicts, it seems better to consider >$"#?%'?2-%, 

as comprising three species, 4/*8.,?+)"+. clade, >/*$(8$() clade and 4/*&")"" clade. The 

names >/ 0.$$(#%&(), >/*).,-+@$%'?2-.&*var.*.$",+#.", >/*#%,#.,-$"#(), >/*0"%6+#.()3*>/* 

-%,&($+,& and >/*).,-+@$%'?2-.&*var. ",-.$5"@"-+6. are very useful and give us information about 

the origin, clinical aspects and mycological characteristics. Assigning morphological species 

names to genetic groups will cause misunderstandings. Therefore we propose the use of those 

names as morphological varieties of >/*).,-+@$%'?2-.&, anamorph of 4/*8.,?+)"+.*and 4/*&")"", 

e.g. >/*).,-+@$%'?2-.& var. 0.$$(#%&(), var. erinacei, var. #%,#.,-$"#(), var. quincBeanum, var. 

",-.$5"@"-+6. and var. ).,-+@$%'?2-.&. ahen >/*",-.$5"@"-+6.*is considered as one of the 

morphological varieties of >/*).,-+@$%'?2-.&, and >/*",-.$5"@"-+6. includes strains with a wide 

range of genotypes, strange descriptions such as hgranular type or zoophilic strain of >/* 

",-.$5"@"-+6.f*should not appear.

P-0754. Molecular phylogenetics of Sporothrix schenckii species complex 

inferred from calmodulin gene sequences 

Marimon Pico R, Gene (, Cano (, Guarro ( 

6niversitat Rovira i Virgili 

In a previous study we investigated phylogenetic relationships and species limits 

within the ='%$%-?$"_*&#?.,#[""*species complex using a multilocus sequence analysis 

of three nuclear genes (chitin synthase, !-tubulin and calmodulin). In contrast to the 

results of other authors, we found that clinical isolates (more that [0) were grouped 

into six phylogenetic species, most of them prevailing in different geographical regions. 

However, neither a phenetic nor a molecular comparative study involving numerous 

environmental isolates was performed which would have helped to understand the 

organization of this species-complex. The delineation of such species could be of 

extreme importance from a clinical point of view. ae have, therefore, studied a total of 

8[ isolates (61 clinical and 2O environmental) morphologically identified as =/* 

&#?.,#["", comparing the sequences of the calmodulin gene, the most 

phylogenetically informative locus found in our previous study, and characterizing all 

of them phenotipically through cultures on PDA/CMA, measuring the growth rates at 

different temperatures, performing assimilation of numerous carbohydrates, and the 

conversion of cells from the mycelium to the yeast form at J7oC. The molecular 

analysis revealed two main clades. All the clinical isolates, together with several 

environmental isolates, were included in one clade. This clade was split into three well 

supported subclades (the 2razilian, the European, and a third which included other 

.outh American and one .outh African isolates). The 2razilian subclade was 

characterized by the following combination of phenetic featuresM presence of globosesubglobose 

pigmented conidia, and non-sucrose assimilation. Although the isolates of 

the European subclade also produce globose pigmented conidia, they differ from the 

2razilian subclade by their ability to assimilate the sucrose and their slower growth at 

J[oC. The third subclade includes isolates that do not produce globose pigmented 

conida and are able to assimilate raffinose. aithin this group we can distinguish 

different branches, that were mainly differentiated by the presence/absence of 

triangular-cuneiform pigmented conidia. The second main clade comprised exclusively 

isolates from environmental origin including the type strain of ='%$%-?$"_*+68"#+,&, a 

species traditionally considered as synonym of =/*&#?.,#["". The majority of the 

isolates included in this clade were non-pigmented and they could not assimilate 

raffinose or ribitol. No differences in the yeast form conversion were found in any 

clade. fn the basis of the present results, [ new species for the =/*&#?.,#["" speciescomplex 

are proposed, J of which include only clinical isolates, and =/*+68"#+,& is 

maintained for the non-pigmented isolates of environmental origin. 

P-0755. On-line identification key of keratinophilic and keratinolitic fungi 

Rovegno D 1 , Guglielminetti M, Martellos . 

1 

6niversitÖ degli studi di Pavia-sez. di Micologia, 2 6niversitÖ degli studi di Pavia-sez. di Micologia 

In this worB we describe the on-line Bey for identification of all Beratinophilic a d Beratinolitic fungi 

from Italian references, created for the National Program h6n sistema di strumenti on-line per 

leidentificazione delle piante e dei funghi deItaliai. 

The Bey can be used by mycologists, medical and vets, who need an instrument which is easy to 

use for diagnosis and epidemiological studies of Beratinophilic fungi from natural substrates and 

dermatophytes from man and animals. The software named hFridai (Nimis P./., Martellos .., 

200O) was used to organize the Bey. At the first step the system splits pathogenic fungi 

(Beratinolitic fungi and dermatophytes) from the other species that are only Beratinophilic (not 

pathogenic). Throughout the Bey the user moves from simple macroscopical to microscopical 

and also phisiological characters. 

At the beginning, dichotomic Bey is based on morphological characters such asM 

" Thallus growth form at 2[ÅC/J7ÅC (consisting of filaments/consisting of budding cells) 

" Hyphae morphology (with septation/with few or no septation) 

" Reproductive strategy (with ascomata-sexual-/without ascomata-asexual-) to choose 

between teleomorphic and anamorphic genera. 

aithin the teleomorphic taxa, features for 

" Colony 

" Ascomata 

" Peridial stuctures (peridial hyphae and appendages/only peridial hyphae) 

" Ascospore 

are used to separate species of the following generaM 

" Aphanoascus 

" Amauroascus 

" Arthroderma 

" Auxarthron 

" Ctenomices 

" Gymnascella 

" Gymnoascus 

" Myxotrichum 

" Pectinotrichum 

" Pseudallescheria 

" Renispora 

" 6ncinocarpus 

aithin the anamorphic taxa, features forM 

" Conidia 

" Fertile hyphae 

" Colony 

" Crystals, diffusible pigment or exudate 

" Conidiophores 

" Conidia 

" Chlamydospores 

" 2amboo hyphae, pectinate hyphae, spiral hyphae, antler-liBe hyphae (favic 

chanderlier) 

are used to separate species of the following generaM 


" Acremonium 

" Alternaria 

" Arthrographis 

" 2eauveria 

" 2otryotrichum 

" Chaetomium 

" Chrysosporium 

" Cladosporium 

" Clonostachys 

" Coniothyrium 

" Cylindrocarpon 

" Doratomyces 

" Epidermophyton 

" Fonsecaea 

" Fusarium 

" Geomyces 

" Geotrichum 

" Malbranchea 

" Microsporum 

" Mucor 

" Myceliophthora 

" Myrothecium 

" fvadendron 

" Paecilomyces 

" Penicillium 

" .copulariopsis 

" .porotrichum 

" .porothrix 

" Trichophyton 

" 6locladium 

" Verticillium 

aith this Bey 128 taxa of Beratinophilic and Beratinolitic fungi can be identified. The system 

reports the specific fungal name with a short morpho-phisiological description and pictures. 

Pathogenic and opportunistic fungi are also provided with a description for each Bind of 

pathology that they may cause. The choice of characters through the Bey is simplified by the 

presence of photos and pictures of both single character and species characteristics. The initial 

split in two groups (pathogenic/not pathogenic), detailed descriptions and explanatory pictures 

maBe the hon-line identification Bey of Beratinophilic and Beratinolitic fungii very easy and fast to 

use, maintaining a high degree of accuracy. 

P-0756. Molecular identification and subspecific typing of pseudallescheria 

boydii 

Zeng J 1 , FuBushima l J , TaBizawa l J , Zheng Y 2 , Nishimura l J , De Hoog G 1 

1 Centraalbureau voor .chimmelcultures, 2 Department of Dermatology and 

Venereology, 6nion Hospital, Huazhong .cience and Technology 6niversity,auhan, 

Hubei, P. R. China, J Research Center for Pathogenic Fungi and Microbial Toxicoses, 

Chiba 6niversity, Chiba, (apan 

Background: A set of OJ Asian isolates of the opportunist 1&.(5+66.&#?.$"+*8%25"" 

(anamM =#.5%&'%$"()*+'"%&'.$)()) was compared with a reference set of strains 

originating from humans, animals and soil and with a worldwide distribution. 

Methods: .equences of the D1/D2 region of the large subunit (/.6) and Internal 

Transcriber .pacer (IT.) region of the ribosomal operon, as well as Elongation Factor 

1-apha (EF1!) were analyzed. Restriction fragment length polymorphism (RF/P) 

analysis of the intergeneric spacer (IG.) region of rDNA was added to further 

characterize subspecific entities. 

Results and conclusions: /.6 and IT. data divided the set of strains in two groups, 

while on the basis of EF1! sequences one of the entities was further divided with a 

third group. These were in agreement with previously described nDNA/DNA 

reassociation groups 1-J. ahen the IG. regions of 22 strains were digested with the 

restriction endonucleases A+. III and !8% I, seven and five distinct patterns were 

detected, respectively, allowing further subtyping of 1/*8%25"" strains. Nearly all 

groupings showed concordance, but in some strains consistent and reproducible 

conflicts between data sets were observed. This suggests a certain degree of 

recombination in this alleged homothallic teleomorphic ascomycete. nDNA homology 

groups 1-J have been suggested to differ in clinical predilection and temperature 

relations. The occurrence of gene flow between populations may impact the selection 

of virulence factors. 

References: 

Nimis P./., Martellos .., 200O-.trumenti interattivi per leidentificazione dei vegetaliM esempi e 

applicazioni. 

KKÅCongresso .2I, Torino, 22-2O .ettembre 200O

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