1. Bharat Gandhi*1, and Tony Mazzulli1,2
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 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.
Method
Results
Copan ESwab
Candida krusei at 4C & 48hr in triplicate
Aspergillus niger at 4C & 48hr in triplicate
Rhizopus species at 4C & 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 These were species with broadly spreading colonies. Only Rhizopus species produced growth that was not countable though it remained viable.
Contact
References
Acknowledgments
Bharat Gandhi
Phone:
1. Clinical and Laboratory Standards Institute Quality control of microbiological transport systems; approved Standard -second edition. CLSI document M40-A2. CLSI, Wayne, PA.
2. Clinical and Laboratory Standards Institute Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved Standard -second edition. CLSI document M38-A2. CLSI, Wayne, PA.
3. Espinal-Ingroff A, Kerkering T.M Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi. J Clin Microbiol 29:
4. Ricardo A, Rodrigues A.G. Pina-Vaz C A fast practical and reproducible procedure for the standardization of the cell density of an Aspergillus suspension. Journal of Medical Microbiology 53:
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.