Antifungal Susceptibility of Filamentous Fungi

In vitro susceptibility testing was performed using broth microdilution for filamentous fungi according to EUCAST guidelines with concentrations ranging from 0.016 µg/mL to 8 µg/mL. The antifungals used were amphotericin B (AmB), voriconazole (VRC), itraconazole (ITC), posaconazole (POS), and anidulafungin (ANI), all purchased from Sigma-Aldrich, reconstituted in DMSO and stored frozen at −80 °C until use. The MICs were determined in flat-bottomed 96-well plates with conidial suspensions prepared in RPMI 1640 supplemented with 2% glucose, buffered with MOPS, and adjusted to a final concentration of 5 × 10⁵ CFU/mL, as previously described [39 ]. Inoculated plates were incubated for 72 h at 35 °C. MICs were spectroscopically determined at 405 nm using a spectrophotometer (NEPHELOstar, BMG Labtech) as the antifungal concentration that resulted in 90% (AmB) or 50% growth inhibition (VRC, ITC, POS, ISA).