Instability and Subpopulation Dynamics of Azole Trailing Phenotypes in Clinical Candida tropicalis Isolates

Azole antifungal "trailing growth" in Candida spp., a tolerance phenotype distinct from resistance, complicates interpretation of in vitro susceptibility and may contribute to persistent candidemia, especially in resource-limited settings. This study investigated the stability, characteristics, and mechanisms of azole trailing, primarily in the emerging pathogen Candida tropicalis. From 847 clinical C. tropicalis isolates (2022-2024) screened by CLSI broth microdilution (BMD), 6.85% (n = 58) exhibited trailing, most frequently with fluconazole. Disk diffusion with elevated drug concentrations revealed microcolonies within inhibition zones, confirming tolerance to supra-MIC levels. Longitudinal monitoring revealed phenotypic instability: trailing was lost in 37.5% of C. tropicalis, 50% of C. albicans, and 66.7% of C. glabrata isolates after 8-12 months of storage at - 80 °C. This loss was also induced by repetitive freeze-thaw cycles in some isolates. Trailing was pH-dependent, consistently observed only at pH 6-8. Serial induction under fluconazole pressure successfully revived the phenotype in some isolates. Population Analysis Profiling (PAP) of nine trailing C. tropicalis isolates confirmed the presence of drug-tolerant subpopulations, akin to a heteroresistance reference strain. These findings demonstrate that azole trailing in C. tropicalis is a reversible, pH-dependent tolerance phenotype mediated by a minor subpopulation, which is unstable under routine laboratory storage. The discrepancy between in vitro susceptibility results and this tolerant phenotype underscores the need for adjunctive assays, like PAP, to better characterize antifungal response and improve clinical correlation.