Abstract

A single ddPCR assay was developed to simultaneous detect ERG11 A395T/W mutations and allele-resolved quantification of wild-type and mutant ERG11 copy numbers in Candida tropicalis . • Allele-resolved ERG11 copy number measurement enables accurate prediction of fluconazole resistance, bridging genomic resistance mechanisms and routine antifungal susceptibility testing. • ddPCR measurements show excellent quantitative agreement with whole-genome sequencing while providing a scalable quantitative tool for investigating ERG11 -mediated resistance dynamics in experimental and longitudinal studies. • Direct testing of a clinical specimen demonstrates the potential for earlier, culture-independent resistance assessment in invasive C. tropicalis infections. Background Azole resistance in Candida tropicalis has increased worldwide and is largely driven by an emerging population previously reported Cluster AZR/X. This cluster exhibits high-level azole resistance driven by ERG11 A395T/W mutations accompanied by amplification of mutant ERG11 alleles, underscoring the clinical need for a quantitative, mechanism-informed molecular assay. Methods We developed and validated a droplet digital PCR (ddPCR)-based assay enabling simultaneous detection of the ERG11 mutation and quantitative assessment of ERG11 copy number variations (CNVs). The assay employs allele-specific probes to detect wild-type and mutant ERG11 alleles, and normalized against endogenous reference genes for allele-resolved quantification. Validation of the ddPCR assay was carried out in 93 clinical isolates, and the results were compared with whole-genome sequencing data. Results The assay demonstrated excellent concordance for both ERG11 A395T/W mutation detection (κ = 1.00) and CNV estimation (R² = 0.98), and exhibited high repeatability and specificity. When further applied to a multicenter collection of 98 invasive C. tropicalis isolates, the ddPCR assay predicted fluconazole resist