Optimized conditions for broth microdilution susceptibility testing of Trichosporon spp. to clinical antifungals

The resistance in Trichosporon species poses a significant challenge in clinical treatment, limiting the efficacy of commonly used antifungal drugs. In this context, T. asahii poses substantial risks as an opportunistic pathogen, especially in immunocompromised patients, where the effective antifungal treatment is also challenged by the absence of standardized testing methods. In this scenario, the present study evaluated and discussed the broth microdilution susceptibility testing of Trichosporon species to fluconazole and amphotericin B. A total of 33 Trichosporon spp. strains, isolated from different sources, along with reference strains, were tested. Different adaptations of the CLSI guidelines were applied to investigate optimal conditions for minimum inhibitory concentration (MIC) determination in this genus. The results revealed that fluconazole showed less variation between the tested incubation periods, with reading at 50% inhibition, while amphotericin B demonstrated more accurate results with extended incubation (48 h) and reading at 100% inhibition. Additionally, fluconazole exhibited higher MICs when isolates were incubated at 30 °C, with a range of 1-32 µg/mL, where amphotericin B showed higher MICs at 37 °C, with a range of 0.5-4 µg/mL. This work also reveals significant variability in susceptibility results, underscoring the necessity for standardized testing protocols. Based on the results, the study recommends for the optimal susceptibility testing a 48 h incubation period at 37 °C, with reading breakpoints of 50% inhibition for fluconazole and 100% inhibition for amphotericin B. Yet, this study highlights the urgent need for standardized testing methods and better understanding of antifungal resistance in Trichosporon infections.