2.5. Clinical Antifungal Drugs Susceptibility Testing

Antifungal susceptibility testing (AFST) was performed following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution reference method 9.3.1 [41]. Antifungals used were amphotericin B (Sigma-Aldrich Química, Madrid, Spain) and the azoles itraconazole (Janssen Pharmaceutica, Madrid, Spain), voriconazole (Pfizer SA, Madrid, Spain), posaconazole (Schering-Plough Research Institute, Kenilworth, NJ, USA) and isavuconazole (BasileaPharmaceutica, Basel, Switzerland (tested from January 2017)). The final concentrations tested ranged from 0.03 to 16 mg/L for amphotericin B and 0.015 to 8 mg/L for the four azoles. A. flavus ATCC 204304 and A. fumigatus ATCC 204305 were used as quality control strains in all tests performed. Minimal inhibitory concentrations (MICs) were visually read after 24 and 48 h of incubation at 37 °C in a humid atmosphere. MICs were performed at least twice for each isolate. Clinical breakpoints for interpreting AFST results established by EUCAST [42] were used for classifying the A. fumigatus strains as susceptible or resistant.