Establishing thresholds for azole tolerance and persistence in Aspergillus fumigatus to study their impact on voriconazole treatment in vivo

Antimicrobial tolerance and persistence are phenomena that enable pathogenic microbes to survive for extended periods in the presence of high concentrations of cidal drugs. Current evidence suggests that these phenomena may contribute to treatment failure and could even lead to the development of resistance. However, our understanding of antifungal tolerance and persistence in Aspergillus fumigatus, as well as their potential role in therapeutic failure, remains limited. In this study, we present an optimized, easy-to-perform method for detecting A. fumigatus tolerance and persistence to azole antifungals, based on a single colony-forming unit (CFU) measurement. Additionally, we developed a microscopic approach to investigate the dynamics of conidial killing in medium-throughput assays. Using these methods, we established epidemiological threshold values to classify strains as tolerant or persister and applied them to screen and categorize a collection of clinical isolates. Furthermore, we demonstrate that tolerance--but not persistence--negatively impacts the efficacy of voriconazole treatment in a Galleria mellonella infection model. Based on these findings, we propose that tolerance and persistence should be monitored in clinical isolates and potentially considered when determining therapeutic strategies.