3.3. Antifungal Susceptibility Testing

In vitro antifungal susceptibility tests using minimum inhibitory concentrations (MICs) were assayed for the identified Candida species of C. albicans (n = 67), C. glabrata (n = 30), C. parapsilosis (n = 20), C. krusei (n = 12), C. tropicalis (n = 11), C. dubliniensis (n = 6), and C. africana (n = 4) according to the recommendations in the clinical and laboratory standards institute (CLSI) M27-A3 and M27-S4 documents (12, 13). Amphotericin B (Sigma, St. Louis, MO, USA), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Janssen research foundation, Beerse, Belgium), voriconazole (Pfizer), posaconazole (Schering-Plough, Kenilworth, USA), and caspofungin (Merck, Whitehouse Station, NJ, USA) were obtained from their respective manufacturers as reagent-grade powders for preparation of the CLSI microdilution trays.

The antifungal agents were diluted in the standard RPMI-1640 medium (Sigma chemical Co., USA) buffered to pH 7.0 with 0.165 M-morpholinepropanesulfonic acid (MOPS) (Sigma), with L-glutamine without bicarbonate, to yield two times their concentrations. They were dispensed into 96-well microdilution trays at a final concentration of 0.016 - 16 µg/mL for amphotericin B, itraconazole, voriconazole, and posaconazole, 0.063 - 64 µg/mL for fluconazole, and 0.008 - 8 µg/mL for caspofungin. The plates were stored at -70°C until they were used. Briefly, all isolates were grown on potato dextrose agar (PDA, Difco, Leeuwarden, the Netherlands) plates at 35°C for up to three days, with the inoculum suspensions being prepared by lightly scraping the surface of mature colonies with a loop and the resulting material being suspended in sterile saline solution.

The homogeneous conidial suspensions were then transferred to sterile tubes and the supernatants were adjusted spectrophotometrically at a wavelength of 530 nm, to an optical density (OD) that ranged from 0.12 to 0.11 (2.5 – 5 × 10⁶ CFU/mL). Microdilution plates were incubated at 35°C and examined visually after 24 and 48 hours to determine the MIC values. The MIC endpoints were determined with the aid of a reading mirror and were defined as the lowest concentration of drug that prevents any recognizable growth (i.e., exerts 100% inhibition for amphotericin B) or significant (50%) growth diminution levels (for all other agents) compared with the growth of a drug-free control. The C. parapsilosis (ATCC 22019) and C. krusei (ATCC 6258) strains were chosen as quality controls, and analysis of these strains was performed with every new batch of MIC plates.