Antifungal susceptibility testing of Candida parapsilosis against caspofungin and anidulafungin by the CLSI broth microdilution protocol, Etest, and a flow cytometrybased method

P. Pinto1, L. Vale-Silva2, V. Lopes1, H. Ramos1, E. Pinto2

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1Centro Hospitalar do Porto, PORTO, Portugal 2 University of Porto, PORTO, Portugal


Objectives: It is common knowledge that there has been an apparent shift in infections caused by Candida spp., with non-albicans Candida spp., like C. parapsilosis, assuming an increasing role in pathogenesis of candidemia. Candida parapsilosis have shown variable susceptibility patterns to the new antifungal agents, echinocandins. It is advisable to determine the antifungal susceptibility patterns of clinical isolates, which may assist in making appropriate decisions regarding the best therapeutic options. The objective of this study was to compare Etest and flow cytometry (FC) with the reference CLSI broth microdilution method (BMD) for antifungal susceptibility testing (AST) of C. parapsilosis to caspofungin and anidulafungin. Methods: Twenty C. parapsilosis isolates from relevant clinical samples in our Hospital were tested. The broth microdilution method was performed according to the reference CLSI protocol M27-A31. Etest MICs were determined according to the instructions from the manufacturer (AB Biodisk, Solna, Sweden). The flow cytometry method was based on a 4-h incubation of the yeast cells with two-fold dilutions of the antifungals, followed by staining with acridine orange. Quality control was performed using C. parapsilosis ATCC 22019 and C. krusei ATCC 6258. Major errors were results of susceptibility by BMD and resistance by Etest or FC. Very major errors were results of resistance by BMD and susceptibility by Etest or FC. Results: The determined caspofungin BMD MICs were 1-2 μg/mL and all strains were thus considered as susceptible. There was a 100% agreement within two log2 concentrations between the reference method and both Etest and FC and a 95% agreement within one dilution for both methods, with one major error registered with FC. Regarding anidulafungin, the BMD MICs ranged from 1 to 4 μg/mL, with three non-susceptible strains (15%). The agreements within one and two dilutions were 100% for both methods, with one very major error (5%) for each method. Conclusion: We have obtained very good correlations between MIC results for both echinocandins determined according to the reference BMD method, Etest, and a new flow cytometry-based method. The values were clustered around the 2 μg/mL breakpoint value for susceptibility, a typical finding for C. parapsilosis2, making it possible for very good MIC value agreements obtained with different methods to admit classification errors. We have found very few of those cases, however. Overall, these results appear to indicate that the three methods could be appropriate for AST of caspofungin and anidulafungin. The less time-consuming could so be employed in substitution of the reference BMD protocol. The FC method could, in addition, allow single day result availability. The authors acknowledge the Fundaçí£o para a Ciíªncia e Tecnologia (FCT) for the post-doc grant attributed to L. A. Vale-Silva (SFRH/BPD/29112/2006). References: 1. Clinical and Laboratory Standards Institute. Approved standard M27-A3, 3rd ed. 2008, Wayne, PA. 2. Van Asbeck E, Clemons KV, Martinez M, Tong AJ, Stevens DA. Diagn Microbiol Infect Dis. 2008, 62(1):106-109. show poster

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4th Trends in Medical Mycology
    • TIMM 4th (2012)