Susceptibility Testing

All isolates were tested by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) methods, as outlined in documents M27-A3 and M27-S4 [38, 39]. The systemically active antifungal agents tested were anidulafungin, caspofungin, micafungin, fluconazole, and voriconazole. This report focuses primarily on resistance to fluconazole and the echinocandins. The results for voriconazole will only be used when discussing cross-resistance between azoles and echinocandins. The range of antifungal agent concentrations tested was 0.008–16 mg/L for the echinocandins and voriconazole and 0.12–128 mg/L for fluconazole. Minimum inhibitory concentration (MIC) results for all tested agents were determined visually after 24 hours of incubation at 35°C as the lowest concentration of agent that resulted in ≥50% inhibition of growth relative to the growth control. CLSI clinical breakpoints (CBPs) were applied for the 5 most “common” species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei), for which echinocandins, fluconazole, and voriconazole are used in treatment. Epidemiological cutoff values (ECVs) were applied when available for the other species tested [37, 38, 40]. Recognizing that CBPs for fluconazole and the echinocandins have changed over the last 20 years, we have applied only the most contemporary CBPs and ECVs to the entire Candida spp. collection [38–40]. Quality control was performed as recommended in CLSI M27-A3 using C. krusei ATCC 6258 and C. parapsilosis ATCC 22019. All results were within established ranges.