Evidence that increased azole persistence and stress resistance precede the in vivo evolution of azole resistance in Aspergillus fumigatus

Aspergillus fumigatus is the major causative agent of human aspergillosis. Azoles are the first-line therapy, but resistance is increasing. Here, we characterized voriconazole persistence by screening a global collection of 495 clinical and environmental A. fumigatus isolates. We demonstrate two persistence phenotypes: non-growth persisters (NGPs) and slow-growth persisters (SGPs). We focused on nine sequential clinical isolates from a single patient treated with voriconazole for two years. Genome sequencing and phylogenomic analyses show these isolates form a single genetically related population exhibiting within-host diversification. While temporal isolation order does not strictly correlate with genetic relatedness, we observe that increased azole persistence and stress resistance are early adaptive steps in the in vivo evolution of azole resistance. preceding the emergence of voriconazole resistance. Our findings indicate that increased persistence and stress resistance may create a permissive adaptive landscape within the host, highlighting the complex evolutionary dynamics underlying antifungal treatment failure.