International journal of antimicrobial agents · 2026
Droplet digital PCR assay for simultaneous detection of the ERG11 mutation and copy number variation associated with azole resistance in Candida tropicalis.
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Fan Xin, Wang Nan, Chen Xi, Tan Chianru, Liu Feiyi, Zhao Yiying, Guo Yong, Xiao Meng
Аннотация
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.
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.