The minimum inhibitory concentration (MIC) values of EVL alone or in combination with ITC, VRC, POS, or AMB against various dematiaceous fungi were assessed as per the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) M38-A2. The working concentrations for the tested drugs were 0.06-4 μg/mL for ITC, VRC, POS, and AMB, and 0.25-16 μg/mL for EVL. Briefly, 50 μl of serially diluted EVL solutions were added to horizontal rows of a 96-well plate containing the conidia suspension prepared above (100 μl/well), followed by the addition of 50 μl of serially diluted ITC, VRC, POS, or AMB in the vertical columns of this plate. Plates were then incubated for 4 days at 28°C, with MIC values then being established based on the minimum drug concentration necessary to suppress 100% of fungal growth relative to control conditions.
Interactions between EVL and specific antifungal drugs were assessed based on the fractional inhibitory concentration index (FICI) as follows: FICI = (MICAc/MICAa) + (MICBc/MICBa), where MICAc and MICBc respectively correspond to test drug combinations, and MICAa and MICBa correspond to the MIC values for drugs A and B when used as single-agent treatments. A FICI ≤ 0.5 was indicative of synergism, while 0.5 < FICI < 4 indicated no interaction or indifference, and FICI ≥ 4 indicated antagonism. All experiments were independently repeated in triplicate.
Interactions between EVL and specific antifungal drugs were assessed based on the fractional inhibitory concentration index (FICI) as follows: FICI = (MICAc/MICAa) + (MICBc/MICBa), where MICAc and MICBc respectively correspond to test drug combinations, and MICAa and MICBa correspond to the MIC values for drugs A and B when used as single-agent treatments. A FICI ≤ 0.5 was indicative of synergism, while 0.5 < FICI < 4 indicated no interaction or indifference, and FICI ≥ 4 indicated antagonism. All experiments were independently repeated in triplicate.
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