CLSI M38-A2 broth microdilution.

Amphotericin B, itraconazole, posaconazole, and voriconazole powders were purchased from the Sigma-Aldrich Company (St. Louis, MO, USA) and used to prepare microdilution plates. Drug dilutions were prepared following the 2-fold drug dilution scheme described in document M38-A2 to yield serial 2-fold drug dilutions (from 0.03 to 16 μg/ml), which were twice as concentrated as the final concentrations (6). The microdilution plates were stored at −70°C until use.

Aspergillus conidial inoculum suspensions were prepared from well-sporulated cultures (typically 3 days old) grown on potato dextrose agar and adjusted spectrophotometrically to a turbidity that ranged from 0.4 to 0.7 McFarland standards at 530 nm. For the M38-A2 method, the suspension was then diluted with RPMI 1640 broth to twice the density needed for the final inoculum density (0.4 × 10⁴ to 5 × 10⁴ CFU/ml), as demonstrated by quantitative colony counts.

For testing, each microdilution well containing 100 μl of a solution with the 2× drug concentration was inoculated with 100 μl of the 2× inoculum suspension. The final volume and inoculum density in each well were 200 μl and 0.4 × 10⁴ to 5 × 10⁴ CFU/ml, respectively, and the final drug concentrations were from 0.015 to 8 μg/ml. Growth (drug-free) and fungus-free controls were included. The microdilution plates were incubated at 35°C and examined for the MICs after 48 h. The MIC was the lowest drug concentration that prevented any discernible growth (100% inhibition).