2.5. Checkerboard Assay

Antifungal combination activity was assessed by a checkerboard method derived from the standardized procedure established by the CLSI for broth microdilution antifungal susceptibility testing with a few modifications [15]. In brief, the evaluation was performed in RPMI 1640 medium buffered to pH 7.0 with 0.165 mol/L 3-(N-morpholino) propanesulfonic acid buffer (MOPS). Volumes of 25 µL of each drug at a concentration of four times the targeted final concentration were dispensed in the wells of 96-well microtiter plates (Violamo, Osaka, Japan). All antifungals were dissolved in DMSO as recommended by CLSI and the final DMSO concentration did not exceed that of the standard CLSI method. The final concentrations of the antifungal agents evaluated in this study ranged from 0.015 to 8.0 µg/mL for CAS, 0.5 to 32 µg/mL for FLC, 0.125 to 8 µg/mL for ITC, 0.03 to 2 µg/mL for VRC, 0.06 to 4 µg/mL for POS, 0.03 to 2 µg/mL for AMB, and 0.0009 to 0.06 µg/mL for 5FC. All concentrations were adopted according to the MIC results and within the CLSI-recommended concentrations [15], and, except for 5FC, the CLSI-recommended concentrations were very potent and induced complete inhibition of all isolates, so lower concentrations were evaluated [7]. Yeast inocula were adjusted to a 0.5 McFarland turbidity standard, and further diluted to two times final concentration. Yeast inoculum (50 µL) was added to every well to achieve final inoculum size of 0.5 × 10³ to 2.5 × 10³ CFU/mL. The plates were incubated at 35 °C for 24 h for all isolates, with the exception of two isolates, the plates were incubated for 48 h due to the absence of growth after 24 h [16]. Antifungal combination activities were evaluated on the basis of the fractional inhibitory concentration (FIC) index, and classified as synergistic, indifferent, or antagonistic [7,11,19]. The FIC index is expressed as ΣFIC = FICA + FICB = MICA^comb/MICA^alone + MICB^comb/MICB^alone, where MICA^alone and MICB^alone are the MICs of every drug when acting alone, and MICA^comb and MICB^comb are the MICs of each antifungal in combination (in a single well). For all the antifungal combinations, the FICs were derived from IC50s [11]. The antifungal interaction was identified as synergistic if ΣFIC ≤ 0.50, indifferent if ΣFIC ranged from >0.50 to ≤4.0, and antagonistic if ΣFIC > 4.0 [7,11,19]. All antifungal combinations with synergistic action were repeated at least two independent times.