Evaluation of CLSI M44-A2 Disk Diffusion and Associated Breakpoint Testing of Caspofungin and Micafungin Using a Well-Characterized Panel of Wild-Type andfksHot Spot Mutant Candida Isolates

Abstract

ABSTRACTDisk diffusion testing has recently been standardized by the CLSI, and susceptibility breakpoints have been established for several antifungal compounds. For caspofungin, 5-μg disks are approved, and for micafungin, 10-μg disks are under evaluation. We evaluated the performances of caspofungin and micafungin disk testing using a panel ofCandidaisolates with and without knownFKSechinocandin resistance mechanisms. Disk diffusion and microdilution assays were performed strictly according to CLSI documents M44-A2 and M27-A3. Eighty-nine clinicalCandidaisolates were included:Candida albicans(20 isolates/10 mutants),C. glabrata(19 isolates/10 mutants),C. dubliniensis(2 isolates/1 mutant),C. krusei(16 isolates/3 mutants),C. parapsilosis(14 isolates/0 mutants), andC. tropicalis(18 isolates/4 mutants). Quality control strains wereC. parapsilosisATCC 22019 andC. kruseiATCC 6258. The correlations between zone diameters and MIC results were good for both compounds, with identical susceptibility classifications for 93.3% of the isolates by applying the current CLSI breakpoints. However, the numbers offkshot spot mutant isolates misclassified as being susceptible (S) (very major errors [VMEs]) were high (61% for caspofungin [S, ≥11 mm] and 93% for micafungin [S, ≥14 mm]). Changing the disk diffusion breakpoint to S at ≥22 mm significantly improved the discrimination. For caspofungin, 1 VME was detected (aC. tropicalisisolate with an F76S substitution) (3.5%), and for micafungin, 10 VMEs were detected, the majority of which were forC. glabrata(8/10). The broadest separation between zone diameter ranges for wild-type (WT) and mutant isolates was seen for caspofungin (6 to 12 mm versus −4 to 7 mm). In conclusion, caspofungin disk diffusion testing with a modified breakpoint led to excellent separation between WT and mutant isolates for allCandidaspecies.