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

Azole resistance in <i>Aspergillus fumigatus</i> is a major clinical concern; however, treatment failures occur without the presence of classic resistance mutations. This study provides evidence that azole persistence-the ability of susceptible cells to survive drug exposure-and stress resistance precede the <i>in vivo</i> evolution of azole resistance in <i>Aspergillus fumigatus</i>. We identified two persistence types in a global isolate collection and tracked their evolution in a patient over a 2-year period. Our findings reveal that increased voriconazole persistence and stress resistance emerged before the acquisition of the <i>cyp51A</i> resistance mutation. This adaptive phase involved significant transcriptional and metabolic reprogramming, including the upregulation of secondary metabolism. We propose that persistence is not a passive survival state, but an active evolutionary stepping stone that fosters a permissive landscape for the development of resistance. Understanding this "persistence-first" pathway is crucial for developing more effective diagnostics and therapeutic strategies to combat antifungal treatment failure.