First Isolation of a Multi-azole-Resistant Aspergillus fumigatus cyp51A TR46/Y46F/F70L Mutant in a Patient with Fungal Keratitis.

Abstract

Introduction: Fungal keratitis (FK), a severe ocular infection caused primarily by Fusarium spp. and Aspergillus spp., remains a major cause of blindness worldwide. Current treatment relies on antifungal agents, though emerging azole resistance in Aspergillus fumigatus complicates therapeutic outcomes.

Materials and methods: We isolated a clinical FK strain and assessed its antifungal susceptibility using broth microdilution and E-test methods, followed by sequencing of the cyp51A gene's promoter region and coding sequence (CDS) to identify resistance-associated mutations.

Results: The isolate, confirmed as A. fumigatus (Aftr46-001) via ITS and β-tubulin sequencing, exhibited elevated minimum inhibitory concentrations (MICs) 16 µg/mL for itraconazole (ITC), 32 µg/mL for isavuconazole (ISA), 32 µg/mL for voriconazole (VRC), and 1 µg/mL for posaconazole (POS) by broth microdilution. E-test results revealed an ITR MIC of 12 µg/mL, ISA MIC 32 µg/mL, VRC MIC 32 µg/mL, and POS MIC of 2 µg/mL. The isolate exhibited MICs values at or above the CLSI and EUCAST epidemiological cutoff values (ECVs): 1 µg/mL for ITC, ISA, and VRC according to CLSI (with insufficient data to establish a POS ECV), and 1 µg/mL for ITC and VRC, 2 µg/mL for ISA, and 0.25 µg/mL POS per EUCAST criteria. Genotypic analysis identified a 46-nucleotide tandem repeat in the cyp51A promoter and two nonsynonymous mutations (Y46F, F70L).

Discussion: This represents the first report of a cyp51A TR46/Y46F/F70L-harboring A. fumigatus strain isolated from FK, with potential agricultural environmental origins, suggesting that this mutation may have become a potential driver of azole-resistant FK in areas with high agricultural exposure, urgently needing to be included in local treatment guidelines and active surveillance systems. The isolate's resistance to all tested azoles underscores the clinical challenge posed by this genotype. Following this combined intervention, the patient's left eye vision improved from counting fingers at 1 m upon admission to a final visual acuity of 0.3, indicating that combined surgery and local high-dose drug delivery can serve as an effective strategy for drug-resistant FK. These findings highlight the importance of monitoring azole-resistant A. fumigatus strains to guide clinical treatment. The global increase in azole drug resistance further highlights the urgency and importance of rapid and accurate pathogen identification and drug resistance testing in the current environment.