S1 De voorjaarsbijeenkomst van de Nederlandse ... - NVMM

of A. fumigatus spores in the air inhaled by these patients
is crucial for infection control. In the present study,
a new and rapid technique for the quantificatin of A.
fumigatus, based on solid phase cytometry and immunofluoresent
labelling, was developed. Air samples were
collected by impaction on a water soluble polymer that was
sub sequentely dissolved. A part of the sample was filtered
and microcolonies were allowed to form on the filter for 18
hours at 47°C. Subsequentely, labelling with a monoclonal
anti-aspergillus antibody and tyramide signal amplification
was used to detect the microcolonies with the aid of a solid
phase cytometer (ChemScan RDI). The detected spots
were microscopically validated using an epifluorescence
microscope. The specificity and sensitivity of the assay
were evaluated by testing pure cultures of 40 A. fumigatus
strains, 12 other Aspergillus species, 14 different Penicillium
species and 14 other filamentous fungi. All A. fumigatus
strains yielded labelled microcolonies, which confirmed
the sensitivity of the assay. Only Rhizopus stolonifer and
Paecilomyces varotii were labelled with the antibody and
were able to form microcolonies at 47°C. These fungi,
however, could be discriminated from A. fumigatus based
on morphology. Comparison with traditional culture-based
methods indicated that our novel approach is a rapid and
reliable alternative with a high dynamic range.
O043
Comparison of PCR-reverse line blot and real-time PCR for
the detection of dermatophytes in clinical samples
G.J. Wisselink, E. van Zanten, S. Coops,
A.M.D. Kooistra-Smid
Dept. of Research and Development, Laboratory for Infectious
Diseases, Groningen
Objectives: In a previous study PCR-Reverse Line Blot
(PCR-RLB) was compared with culture and the potassium
hydroxide test (KOH) for the detection of dermatophytes.
PCR-RLB showed to be more sensitive than culture and
KOH. Drawbacks of the PCR-RLB are the laborious nature
of the test, the difficult standardisation and the interpretation
of weak results. Therefore a multiplex real-time
PCR was developed. The aim of this study was to compare
PCR-RLB analysis with multiplex real-time PCR for the
detection of dermatophytes.
Methods: Both PCR-RLB and real-time PCR targeted the
ITS1 region located between the genes coding for 18S and
5.8S rRNA. The RLB membrane harboured 13 different
probes to identify and discriminate between 9 different
dermatophyte species. Real-time PCR consisted of two
multiplex assays. One assay targeted Trychopython rubrum,
Trychopython violaceum and Trychopython tonsurans. The
second targeted Microsporum spp., Trychopython interdigitale
group and the whole group of dermatophytes.
Ned Tijdschr Med Microbiol 2008;16:Supplement
S20
Phocine herpes virus-1 was used as internal control for the
real-time assays. Samples were processed using QIAamp ®
DNA mini kit (Qiagen, Germany) with a separate pre-lysis
step.
In total 100 clinical samples (52, 38, 10 respectively nail-,
skin- and hair samples) were analysed retrospectively by
real-time PCR and compared with PCR-RLB.
Results: Of the 100 samples, 60 were positive with the
PCR-RLB (27 T. rubrum, 14 T. interdigitale, 6 T. tonsurans, 3
T. violaceum, 1 Moraxella canis and 9 Trichophyton spp.). All
samples identified as T. rubrum, T. interdigitale, T. tonsurans
and T. violaceum by the PCR-RLB were confirmed by the
real-time PCR. The sample which tested positive for M.
canis by PCR-RLB was identified as Microsporum spp. by
real-time PCR.
The 9 samples which scored positive for Trichophyton spp.
in the PCR-RLB yielded weak results. Of these 9 samples,
real-time PCR identified 3 samples as T. interdigitale, 1 as
T. rubrum, 1 as T. tonsurans, 1 as dermatophyte positive and
3 samples remained negative.
The real-time PCR detected 8 additional samples which
scored negative with the PCR-RLB. Of these 8 samples
real-time PCR identified 4 samples as T. rubrum, 3 as T.
interdigitale and 1 as dermatophyte positive.
Conclusion: These data show that real-time PCR is a
sensitive method for detection of the most prevalent dermatophytes
in nail-, skin- and hair samples. Furthermore,
real-time PCR is more standardised and less laborious than
PCR-RLB, making it a useful tool in routine diagnostics.
O051
Cross-border dissemination of MRSA in the euregion
Meuse-Rhine
R.H. Deurenberg
Dept. of Medical Microbiology, Academic Hospital Maastricht
(azM), Maastricht
Introduction: The Euregion Meuse-Rhine (EMR) consists
of the border regions of Belgium, Germany and the
Netherlands. Cross-border patient mobility and free access
to healthcare facilities are important issues in the EMR,
but concern is rising about the possible dissemination
of methicillin-resistant Staphylococcus aureus (MRSA),
since the prevalence of MRSA is different in the three
countries (24%, 14%, and 2% in Belgium, Germany, and
the Netherlands, respectively). The aim of the study was to
investigate the dissemination of MRSA in the EMR and the
emergence and possible spread of community-associated
(CA) MRSA strains in hospitals in the EMR.
Methods: MRSA isolates (n=152; 63 from Dutch, 40 from
Belgian and 49 from German hospitals), isolated between
1999 and 2004, were characterised using pulsed-field gel
electrophoresis (PFGE), SCCmec typing and multilocus