Broth microdilution assay.

Antifungals tested in the assay were amphotericin B (concentration range, 0.06 to 8 μg/ml), caspofungin (0.06 to 8 μg/ml), griseofulvin (0.06 to 8 μg/ml), terbinafine (0.125 to 16 μg/ml), fluconazole (0.06 to 128 μg/ml), ketoconazole (0.03 to 4 μg/ml), itraconazole (0.015 to 8 μg/ml), posaconazole (0.015 to 8 μg/ml), voriconazole (0.003 to 8 μg/ml) (all from Sigma-Aldrich), micafungin, and anidulafungin (all from MedChemExpress Europe, Sollentuna, Sweden). Serial 2-fold dilutions of 200× stocks of antifungals were prepared in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and stored at −80°C. Inocula (50 μl) were added to 50 μl of 2× concentrated antifungals to achieve a final cell density of 5.0 × 10³ to 5.0 × 10⁴ CFU/ml. For inoculum verification, 10 μl of a 1:10 diluted inoculum was plated onto mLN agar and incubated for 4 to 7 days at 35°C. MIC values from each well plate were deemed reliable only if counts on agar plates were within 10 to 100 CFU.

Candida parapsilosis ATCC 22019 was used as a quality control strain to assess the accuracy of our antifungal dilutions and the reproducibility of results for fluorescence and visual readings, according to CLSI (11) and EUCAST (http://www.eucast.org) guidelines.