Terbinafine

The clinical isolate was subjected to antifungal susceptibility testing according to CLSI guidelines (M38-A document), as previously described [3 (link), 18 (link)], and the MICs of antifungal combinations were determined according to previously described methods [3 (link), 18 (link)]. Itraconazole (Xian-Janssen Pharmaceutical Ltd. Xi’ an, China), terbinafine (Beijing Novartis Pharmaceutical Ltd. (Beijing, China)) and voriconazole (Sigma, USA) were dissolved in 100% DMSO as a stock solution (3200 μg/mL). Drugs were diluted to obtain final concentrations, with Itraconazole and voriconazole from 0.008 to 8 μg/mL and terbinafine from 0.008 to 0.5 μg/mL. Isolates were sub-cultured, and spores from colonies were collected and adjusted with saline to achieve an inoculum concentration of 10⁶ conidia/mL. Each suspension was diluted 1:50–100 with RPMI 1640 to obtain the final test inoculum (0.4–5×10⁴ conidia/mL). Conidial suspension of each of the tested strains were cultivated on RPMI 1640 medium for 7 days at 35°C. Candida parapsilosis ATCC22019 (CBS604), obtained from Centraalbureau voor Schimmelcultures (CBS, the Netherlands), was used as a quality control. The final test inoculum concentration was 0.5–2.5×10³ conidia/mL.

The broth micro-dilution method (according to CLSI M38-A2 guidelines [33 ]) was used for testing the sensitivity of the dermatophyte isolates to the most commonly used antifungal drugs. Fluconazole was obtained from Pfizer International (New York, NY, USA), itraconazole, and miconazole were obtained from the Janssen Research Foundation (Beerse, Belgium), griseofulvin was purchased from Sigma Chemical Company (St. Louis, MO, USA), and terbinafine was purchased from Novartis (Basel, Switzerland). All drugs were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich), with the exception of Fluconazole, which was dissolved in RPMI1640 medium (Sigma Co. St. Louis, USA), buffered at pH 7.0 with 165 mM of 3-(N-morpholino) propanesulfonic acid (MOPS; Sigma), and serially diluted two-fold to final concentrations of 0.125–64 μg/mL for Fluconazole and 0.03–16 μg/mL for the other antifungal agents. MIC values, MIC₅₀, and MIC₉₀ were determined.