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

OBJECTIVES: 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 remains limited. In this study, we aimed to develop easy-to-perform methods for detecting A. fumigatus tolerance and persistence, to establish epidemiological threshold values and to evaluate their implication in treatment failure. METHODS: We developed an optimized method to detect azole tolerance and persistence 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. We utilized the Galleria mellonella infection model to investigate the implication of these phenomena in treatment failure. RESULTS: We established epidemiological threshold values to classify strains as tolerant or persister-producing and applied them to screen and categorise a collection of clinical isolates. Furthermore, we demonstrated that tolerance-but not persistence-negatively impacts the efficacy of voriconazole treatment in Galleria mellonella. CONCLUSION: Based on these findings, we propose that tolerance should be monitored in clinical isolates and potentially considered when determining therapeutic strategies.