Evaluation of Copan ESwab Transport System for Viability of Clinically Significant Fungi by Using CLSI M40-A2
Document Parameters
Bharat Gandhi*1, and Tony Mazzulli1,2
1 Department of Microbiology, Mt. Sinai Hospital and University Health Network, and 2 University of Toronto, Canada
Revised Abstract
Objective: The objectives of this investigation were twofold:1) to evaluate the Copan ESwab transport system by performing fungal viability studies in accordance with the CLSI M40-A2 document with minimal modifications. We selected a
representative diverse test panel from the most commonly isolated group of pathogenic fungi that would challenge the transport system and 2) in connection with such tests, to conduct a pilot study to investigate a fast and practical procedure for cell
density standardization of viable fungal conidia or sporangiospore suspensions for inoculum preparation.
Method: A broad range of clinically significant yeast and filamentous fungal isolates (n=19) that were routinely isolated from swab transport system in clinical laboratories were freshly grown on potato dextrose agar, and harvested when sporulation
was visible. Propagules were adjusted to 0.5 McFarland turbidity standard, firstly by means of a manual method, the Wickerham card, and secondly by means of a fine adjustment by the DensiCHECK Plus photometer (bioMerieux).The corresponding
absorbance and % transmission values at 530nm recorded for comparison with published values, using a WP-100DPlus spectrophotometer (Walter Products Inc.). The Copan Liquid Amies ESwab transport system (Copan Diagnostics Inc.) was
validated using the CLSI M40-A2 standard roll plate method at ~ 4ºC and room temperature (RT) ~22ºC. Two modifications were made: 1) 0.5 mL sterile saline was modified with the addition of one drop of Tween 80; and, 2) 50µL (instead of 100 µL)
and 3 plates (instead of one) were used to produce isolated colonies suitable for counting in tenfold serial dilutions of adjusted inoculum.
Results: Nineteen organisms inoculated on the Copan ESwab transport system consisting of four yeasts, three dermatophytes, two dematiaceous moulds, two Zygomycetes and eight opportunistic moulds. The isolates were tested once on one
ESwab lot number and were able to produce colony counts ≥ 5 CFU at 24 hrs and 48 hrs, as compared to 0 hr counts. For all nineteen organisms tested, 0.5 McFarland adjusted suspensions produced transmission values ~ 71 to 80% T on the
spectrophotometer, similar to values cited in the literature of ~ 68 to 82% T (3).
Conclusions: The criteria set by the new CLSI M40-A2 standard for the Roll plate method standard states that for compliance of viability, any specimen held at 4ºC and RT should yield ≥ 5 CFU after a specified holding period. Results suggest that
the Copan ESwab transport system is able to maintain and recover a broad range of fungi after 48hrs at 4ºC and RT. This supports the CLSI M40-A2 document standardized for validation. Density standardization was found to be streamlined by using
the simple photometric device DensiCHECK Plus for inoculum adjustments; it proved to be a very good alternative to more expensive and less user-friendly spectrophotometers. The majority of the test isolates produced ~30 to300 colonies in the 10 4
dilution. Two of the three Zygomycetes and one dematiaceous mould produced countable colonies at 105; these were species with broadly spreading colonies. Only Rhizopus species produced growth that was not countable, though it remained
viable.
Method
Results
Concentration: 105
Candida albicans
0
Hour
RT
723
24 Hour
4`C
RT
842
822
Candida krusei
˃500
>500
Candida guilliermondii
>500
Candida glabrata
48 Hour
4`C
RT
>500 >500
24 Hour
4`C
RT
133 283
48 Hour
4`C
RT
152 >500
>500
>500
>500
297
208
>500
265
>500
>500
>500
>500
>500
456
387
>500
307
>500
>500
>500
466
>500
320
152
314
>500
332
441
>500
388
400
>500
375
>500
355
>500
>500
>500
378
>500
333
>500
>500
450
400
399
346
>500
360
>500
400
375
431
>500
480
207
61
39
229
80
100
241
28
37
43
81
100
Fusarium sp
>500
>500
>500
>500
>500
269
Trichosporon sp
134
232
100
138
143
Aspergillus fumigatus
Absidia sp
Mucor sp
>500
62
112
>500
61
120
>500
78
100
>500
63
46
Rhizopus sp
Curvularia sp
Phialophora sp
Alternaria sp
160
74
500
>500
NC
67
500
>500
NC
80
450
>500
NC
81
470
>500
Cryptococcus neoformans
Candida krusei at 4C & RT @ 48hr in triplicate
Aspergillus niger at 4C & RT @ 48hr
in triplicate
Concentration: 103
0
Hour
RT
171
Organism
Copan ESwab
Concentration: 104
Trichophyton mentagrophytes
Trichophyton tonsurans
Trichophyton rubrum
Aspergillus niger
Acremonium sp
0
Hour
RT
23
24 Hour
4`C RT
20 31
48 Hour
4`C
RT
17 >500
34
34
295
22
>500
>500
98
67
294
75
>500
236
>500
38
25
79
31
321
309
26
32
35
112
46
306
20
41
20
92
62
>500
33
34
49
80
81
33
8
4
14
13
8
44
7
3
8
11
9
72
6
4
5
12
5
37
9
2
4
13
10
315
11
2
19
12
9
260
249
269
274
32
25
34
29
32
31
27
28
31
33
11
5
4
5
6
>500
75
41
260
12
14
197
7
25
239
7
15
264
12
20
213
7
6
15
1
3
13
2
2
19
1
3
14
1
3
13
0
0
NC
45
321
>500
25
18
106
230
NC
14
98
305
NC
15
77
300
36
13
102
187
NC
10
54
199
2
2
16
16
3
1
14
24
2
2
6
28
2
1
7
25
2
1
6
18
Rhizopus species at 4C & RT @ 24 hr in triplicate
Conclusions
Results suggest that the Copan ESwab transport system is able to maintain and recover a broad range of clinically significant fungi after 48hrs at 4ºC and RT. This supports the CLSI M40-A2 document standardized for validation. Density
standardization was found to be streamlined by using the simple photometric device DensiCHECK Plus for inoculum adjustments as a very good alternative to a more expensive and less user-friendly spectrophotometers. Majority of the test
isolates produced ~30 to300 colonies in the 104 dilution. Two of the three Zygomycetes and one dematiaceous mould produced countable colonies at 105 . These were species with broadly spreading colonies. Only Rhizopus species produced
growth that was not countable though it remained viable.
Contact
References
Bharat Gandhi
Email: [email protected]
Phone: 647-234-0597
1.
2.
3.
4.
Clinical and Laboratory Standards Institute. 2010. Quality control of microbiological transport systems; approved Standard -second edition. CLSI document M40-A2. CLSI, Wayne, PA.
Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved Standard -second edition. CLSI
document M38-A2. CLSI, Wayne, PA.
Espinal-Ingroff A, Kerkering T.M. 1991. Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi. J Clin Microbiol 29:393-394.
Ricardo A, Rodrigues A.G. Pina-Vaz C. 2004. A fast practical and reproducible procedure for the standardization of the cell density of an Aspergillus suspension. Journal of Medical
Microbiology 53: 783-786.
Acknowledgments
We wish to thank Copan Diagnostic Inc. for financially supporting this poster and for providing the Copan Eswab
transport system.
We would also wish to thank Ms. Amanda Wang for her technical assistance in this project and graphic design.