Real Time PCR Assay for Geomyces destructans Identification in ...
Real Time PCR Assay for Geomyces destructans Identification in ...
Real Time PCR Assay for Geomyces destructans Identification in ...
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<strong>Real</strong> <strong>Time</strong> <strong>PCR</strong> <strong>Assay</strong> <strong>for</strong><br />
<strong>Geomyces</strong> <strong>destructans</strong> <strong>Identification</strong><br />
<strong>in</strong> Fungal Cultures & Bat Tissues<br />
Sudha Chaturvedi, Ph.D.<br />
Mycology Laboratory, Wadsworth Center<br />
NYSDOH, Albany, NY
White Nose Syndrome (WNS)<br />
(<strong>Geomyces</strong> <strong>destructans</strong>)<br />
Alan Hicks
<strong>Geomyces</strong> <strong>destructans</strong><br />
4 o C 15 o C<br />
>20 o C<br />
- Psychrophilic (cold lov<strong>in</strong>g) fungus<br />
- Grows at 1-15 o C, but not above 20°C (68°F)<br />
- Thrives <strong>in</strong> darkness, cold places, & areas with high levels of humidity<br />
Bat hibernacula, along with the lowered body temperature<br />
of bats dur<strong>in</strong>g torpor make an ideal situation <strong>for</strong> the<br />
growth of <strong>Geomyces</strong> <strong>destructans</strong>
Bats Affected by Geomycosis (WNS)<br />
- Myotis lucifugus - Little brown bat<br />
- Myotis leibii - Eastern small-footed bat<br />
- Myotis septentrionalis - Northern long-eared bat<br />
- Myotis sodalis - Indiana bat<br />
- Perimyotis subflavus - Tricolored bat<br />
- Eptesicus fuscus - Big brown bat<br />
- Lasiurus borealis - Red Bat<br />
- Lasiurus c<strong>in</strong>ereus – Hoary Bat<br />
- Lasyionicterius noctivagans - Silver-haired Bat
Characteristic Pathology - Bat Sk<strong>in</strong><br />
Sebaceous gland<br />
Hair follicle
Extensive W<strong>in</strong>g Damage<br />
Cryan et al. BMC Biology 2010, 8:135
Exist<strong>in</strong>g Tests <strong>for</strong> G. <strong>destructans</strong> <strong>Identification</strong><br />
a. Histopathology<br />
b. ITS-<strong>PCR</strong><br />
c. Culture<br />
YPD Rose Bengal Agar
REAL TIME <strong>PCR</strong> ASSAY
MLST ANALYSES<br />
Maximum Parsimony Tree (alpha L-Rhamnosidase gene)<br />
The collection sites <strong>in</strong> various New York Counties are color-matched<br />
with respective <strong>Geomyces</strong> <strong>destructans</strong> isolates
<strong>Real</strong> <strong>Time</strong> <strong>PCR</strong> <strong>Assay</strong> <strong>for</strong> G. <strong>destructans</strong><br />
ALR gene<br />
Gd<br />
Gp<br />
F1 P<br />
R1<br />
Highly Sensitive with detection limit of 2 conidia/<strong>PCR</strong><br />
Quick<strong>Time</strong> and a<br />
TIFF (Uncompressed) decompressor<br />
are needed to see this picture.<br />
Quick<strong>Time</strong> and a<br />
TIFF (Uncompressed) decompressor<br />
are needed to see this picture.
<strong>Real</strong> <strong>Time</strong> <strong>PCR</strong> <strong>Assay</strong><br />
Analytical Precision<br />
Specificity Panel<br />
Organi s m Nu m b er<br />
test e d<br />
Sou r ce Al p ha --L -<br />
R h a mnosidase -<br />
PC R<br />
Ge o m y ces d estructans 17 NY S * Positive<br />
Closely r elate d mol d sp e cies<br />
Ge om yce s pannorum 10 UAM H ** Ne g ative<br />
Ge om yce s asperculatus 1 UAM H Ne g ative<br />
Ge om yce s pulverous 1 UAM H Ne g ative<br />
G y mnostellat ospora candensis 1 UAM H Ne g ative<br />
G y m nostellat ospora frigida 1 UAM H Ne g ative<br />
G y mnostellat ospora alp<strong>in</strong>a 1 UAM H Ne g ative<br />
Pseudog y mnoascus appendiculatus 1 UAM H Ne g ative<br />
Dista n tly rela ted mold s pecies<br />
Ac re moniu m spp. 1 NY S Ne g ative<br />
Aspergillus fumigatus 1 NY S Ne g ative<br />
Aspergillus versi color 1 NY S Ne g ative<br />
Blasto m yce s de r matitidis 2 NY S Ne g ative<br />
Cladosporium sp p. 1 NY S Ne g ative<br />
Chrysosporium pseud o merdari u m 2 UAM H Ne g ative<br />
Coccidioides im mitis 1 NY S Ne g ative<br />
Coccidioides posadasii 1 NY S Ne g ative<br />
Epide r mophyton floccos u m 1 NY S Ne g ative<br />
Fusariu m oxyspor u m 1 NY S Ne g ative<br />
Fusariu m solani 1 NY S Ne g ative<br />
Geotrich u m spp. 1 NY S Ne g ative<br />
Histoplasma capsulatum 2 NY S Ne g ative<br />
Micros poru m gypse u m 1 NY S Ne g ative<br />
Muco r spp. 1 NY S Ne g ative<br />
M yxotrichu m chartatum 1 UAM H Ne g ative<br />
M yxotrichu m stipitatum 1 UAM H Ne g ative<br />
Penicilliu m sp p. 1 NY S Ne g ative<br />
Rhizopus spp. 1 NY S Ne g ative<br />
Sprorothrix schen c kii 1 NY S Ne g ative<br />
Trichophyton rubrum 1 NY S Ne g ative<br />
Trichophyton m entagrophyte s 1 NY S Ne g ative<br />
Yeas t s p ecies<br />
Candida albicans 1 NY S Ne g ative<br />
Candida glabrata 1 NY S Ne g ative<br />
Candida krusei 1 NY S Ne g ative<br />
Candida parapsilos is 1 NY S Ne g ative<br />
Candida tropicalis 1 NY S Ne g ative<br />
Cryptococcus var. grubii 1 AT CC *** Ne g ative<br />
Cryptococcus var. n eo<strong>for</strong>mans 1 AT CC Ne g ative<br />
Crypt oc o cus gattii 1 AT CC<br />
Saccharo m yces cer e visiae 1 NY S Ne g ative<br />
Bact e rial sp e cies<br />
Streptococcus pne u moniae 1 NY S Ne g ative<br />
Pseud o monas aeurog<strong>in</strong>osa 1 NY S Ne g ative<br />
Escherichia coli 1 NY S Ne g ative<br />
Bacillus megateri u m 1 NY S Ne g ative<br />
Nocardia farc<strong>in</strong>ia 1 NY S Ne g at ive
Quantification of G. <strong>destructans</strong> conidia <strong>in</strong><br />
Bat Tissues (Total 147 samples; 2009-2010)<br />
Heavily<br />
<strong>in</strong>fested<br />
Moderately<br />
<strong>in</strong>fested<br />
Scantly<br />
<strong>in</strong>fested<br />
Un<strong>in</strong>fected
<strong>Real</strong> <strong>Time</strong> <strong>PCR</strong> versus Histopathology<br />
<strong>Real</strong> time <strong>PCR</strong><br />
Histopathology<br />
Group I Group II Group III
Comparison of Diagnostic Methods<br />
1<br />
Tests<br />
Positive samples<br />
Number of cases Percentage<br />
Culture 17 12<br />
ITS-<strong>PCR</strong> 37 25<br />
Histopathology 64 44<br />
<strong>Real</strong>-time <strong>PCR</strong> 80 55<br />
<strong>Real</strong> <strong>Time</strong> <strong>PCR</strong> > Histopathology > ITS-<strong>PCR</strong> > Culture
1<br />
County-wise distribution of positive tissues<br />
by real time <strong>PCR</strong> assay<br />
No. County Year-2009 Year 2010<br />
Positive/Tested Positive/Tested<br />
1 Allegany 0/6 ND *<br />
2 Broome ND 2/2<br />
3 Cattaraugus 1/8 ND<br />
4 Chatauqua 0/4 ND<br />
5 Cayuga ND 2/2<br />
6 Cl<strong>in</strong>ton 1/2 10/10<br />
7 Delaware 0/2 0/0<br />
8 Erie 3/18 10/10<br />
9 Frankl<strong>in</strong> 0/4 ND<br />
10 Liv<strong>in</strong>gston 0/3 ND<br />
11 Madison 2/2 6/6<br />
12 Niagara 0/8 2/2<br />
13 Onondaga ND 10/10<br />
14 Ontario 0/2 0/0<br />
15 Orange ND 2/2<br />
16 Orleans 1/2 ND<br />
17 Seneca 0/2 ND<br />
18 Tioga 0/2 ND<br />
19 Tompk<strong>in</strong>s 2/6 2/2<br />
20 Ulster 0/0 14/14<br />
21 Wayne 0/4 ND<br />
22 Westchester 0/2 8/8<br />
23 Wyom<strong>in</strong>g 0/2 2/2<br />
Total 10/77 70/70
A New Unanswered Question?<br />
Why are North American bats affected<br />
by <strong>Geomyces</strong> <strong>destructans</strong> but not European bats?
Possible Answers
Thanks!
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