Droplet digital PCR assay for simultaneous detection of the ERG11 mutation and copy number variation associated with azole resistance in Candida tropicalis.

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 normalised 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 multicentre collection of 98 invasive C. tropicalis isolates, the ddPCR assay predicted fluconazole resistance with 91.4% sensitivity and 100% specificity compared with phenotypic antifungal susceptibility results. Furthermore, our proof-of-concept evaluation demonstrated the feasibility of direct ddPCR analysis of clinical specimens.

CONCLUSIONS

This study established a mechanism-informed ddPCR assay that enables accurate detection of mutation and CNVs of azole target, providing a practical tool to address the growing clinical and public health challenges posed by the emerging azole-resistant C. tropicalis cluster.