Antifungal Susceptibility Test

The antifungal susceptibility testing was assessed by the checkerboard broth microdilution method performed according to the Clinical and Laboratory Standards Institute (CLSI) protocol M27-A4 (Clinical and Laboratory Standards Institute (CLSI), 2017). The minimum inhibitory concentration (MIC) value was determined for fluconazole (FLC), amphotericin B (AmB), 5-fluorocytosine (5-FC), itraconazole (ITC), voriconazole (VOC), posaconazole (POS), and isavuconazole (ISA). Antifungal drugs were provided as powders with known potency from Sigma Chemical Co. (St. Louis, MO, United States). Stock solutions were prepared as follows: FLC and 5-FC were dissolved in sterile distilled water to a drug storage solution of 1,280 μg/ml, while AmB, ITC, VOC, POS, and ISA were dissolved in dimethyl sulfoxide (DMSO) (Sigma Chemical Co., United States) into a stock solution of 1,600 μg/ml and stored at −20°C until needed. The final concentration ranges were 0.125–64 μg/ml for FLC, 0.00156–8 μg/ml for AmB and 5-FC, 0.0020–1 g/ml for ITC, VOC, POS, and ISA, respectively. The yeast inocula were adjusted to a concentration of 1 × 10³–5 × 10³ cfu/ml in Roswell Park Memorial Institute (RPMI) 1640 medium as measured by a hemocytometer, and an aliquot of 0.1 ml was added to each well containing various concentrations of antifungal drugs. The 96-well plates were incubated at 37°C. The assays were read 72 h after inoculation. Candida parapsilosis ATCC 22019 was used as a quality control strain for susceptibility tests.

According to the CLSI M27-A4 protocol, the MIC of AmB was defined as the lowest concentration that produced complete growth inhibition, while the MIC for other antifungal agents were defined as the lowest concentrations at which there was 50% inhibition of growth (≥50%) compared with that of drug-free control (optical clear). The MIC₅₀ and MIC₉₀, on the other hand, are the concentrations capable of inhibiting the growth of isolates by 50 and 90%, respectively (Favalessa et al., 2014). The interpretive MIC criteria for FLC were as follows: susceptible (S), ≤8 μg/ml; susceptible-dose dependent (SDD), 16–32 μg/ml; and resistance (R), ≥64 μg/ml; for 5-FC, ≤4 μg/ml (S), 8–16 μg/ml (SDD), and ≥32 μg/ml; for ITC, ≤0.125 μg/ml (S), 0.25–0.5 μg/ml (SDD), and ≥1 μg/ml; for VOR, ≤1 μg/ml (S) based on previous studies (Pfaller et al., 2005; Dias et al., 2006; Bertout et al., 2013; Gutch et al., 2015). For Cryptococcus, interpretative criteria have not been defined for POS, ISA, and AmB; hence, data available for Candida spp. were used, as previously reported (Revankar et al., 1988; Rodríguez-Tudela et al., 1995; Pfaller et al., 1999). Based on the recommendation of previous studies, the ECVs for C. neoformans var. grubii were 8.0 μg/ml for FLC, 1.0 μg/ml for AmB, 4 μg/ml for 5FC, 0.125 μg/ml for ITC, 1.0 μg/ml for VOR, 0.5 μg/ml for POS, and 0.25 μg/ml for ISA; and that for C. gattii were 8 μg/ml for FLC, 4 μg/ml for 5FC, 0.5 μg/ml for AmB, ITC, VOR, and POS, and 0.25 μg/ml for ISA (Espinel-Ingroff, 2012; Espinel-Ingroff et al., 2012, 2015; Lockhart et al., 2012). The susceptibility of each Cryptococcus spp. isolate was determined in triplicate at different times for optimal results.