# Investigation of voriconazole heteroresistance in clinical isolates of Trichosporon asahii from a multicenter study in China

## TL;DR

This study reveals that Trichosporon asahii clinical isolates show voriconazole heteroresistance, which could impact treatment effectiveness and challenge standard antifungal testing.

## Contribution

The first comprehensive characterization of voriconazole heteroresistance in Trichosporon asahii clinical isolates.

## Key findings

- Heteroresistance to voriconazole was observed in all 62 isolates, with LHV values 23 times higher than MICs.
- Most strains rapidly adapted to high voriconazole concentrations but gradually lost resistance when passaged on drug-free media.
- 90.3% of strains tolerated voriconazole at concentrations 16 times the MICs, with subpopulation frequencies between 0.002% and 0.830%.

## Abstract

Heteroresistance refers to the presence of subpopulations within seemingly homogeneous microbial cells that exhibit varying sensitivities to antifungal agents, potentially contributing to treatment failure. Our study investigated the heteroresistance levels of clinical isolates of Trichosporon asahii collected from a multicenter study in China. A total of 62 isolates from 31 research centers, representing five different genotypes, were analyzed. We assessed the minimal inhibitory concentrations (MICs) of voriconazole (VRC), the level of heteroresistance to voriconazole (LHV), the strains’ capacity for adaptation to high VRC concentrations (ADP), and the stability of the heteroresistance phenomenon. The isolates had low VRC MICs (average 0.038 µg/mL), with 95.2% (59/62) classified as wild type. Heteroresistance to VRC was observed in all isolates, with an average LHV (0.25–4 µg/mL) 23 times higher than the MICs (0.012–0.19 µg/mL). Additionally, 90.3% of strains tolerated VRC concentrations at least 16 times the MICs, with heteroresistant subpopulations appearing at frequencies ranging from 0.002% to 0.830%. Furthermore, the strains rapidly adapted to elevated drug concentrations following brief exposure to VRC, with 71.0% tolerating VRC at 8 µg/mL and an average ADP value 285 times the MICs. However, the strains gradually lost this resistance when serially passaged on drug-free SDA plates, with the time required for resistance loss varying among strains. Our study is the first to reveal the presence of heteroresistance in clinical Trichosporon asahii, adding to the body of research on fungal heteroresistance and posing challenges to the clinical interpretation of MIC values for guiding treatment.

Trichosporon asahii has become an increasingly important fungal pathogen responsible for invasive infections in clinical settings. Due to its natural resistance to flucytosine and echinocandin classes, azole drugs, especially voriconazole, are the mainstay of treatment. The phenomenon of heteroresistance may be associated with treatment failure in fungal infections; however, this phenomenon remains unclear in T. asahii. Our study provides the first comprehensive characterization of voriconazole heteroresistance in T. asahii, significantly advancing our understanding of antifungal resistance dynamics in this emerging pathogen. Our findings also challenge the reliability of conventional minimal inhibitory concentration-based therapeutic guidance for T. asahii infections.