Development of a high-resolution melt-based assay to rapidly detect the azole-resistant Candida auris isolates

Background and Purpose: Candida auris is a multidrug-resistant yeast that rapidly spreads, making it the leading Candidate for the next pandemic. One main leading cause of emerging resistant C. auris isolates is nonsynonymous mutations. This study aimed to detect the Y۱۳۲F mutation, one of the most important azole resistance-associated mutations in the ERG-۱۱ gene of C. auris, by developing a reliable high-resolution melt (HRM)-based method.Materials and Methods: Five C. auris isolates from Iran, plus three control isolates from other Clades were used in the study. The antifungal susceptibility testing through micro broth dilution was performed to recheck their susceptibility to three azole antifungals,including fluconazole, itraconazole, and voriconazole. Moreover, the polymerase chain reaction (PCR) sequencing of the ERG-۱۱ gene was performed. Following the bioinformatic analysis and HRM-specific primer design, an HRM-based assay was developed and evaluated to detect ERG-۱۱ mutations.Results: The minimum inhibitory concentrations of fluconazole among Iranian C. auris isolates ranged from ۸ to ۶۴ μg/mL. The PCR-sequencing of the ERG-۱۱ gene and bioinformatic analyses revealed the mutation of Y۱۳۲F, a substitution consequence of A to T on codon ۳۹۵ in one fluconazole-resistant isolate (IFRC۴۰۵۰). The developed HRM assay successfully differentiated the targeted single nucleotide polymorphism between mutant and wild types (Melting temperature [Tm]: ۸۱.۷۹ ℃ - cycle threshold [CT]: ۲۰.۰۶ for suspected isolate). For both mutant and non-mutant isolates, the mean Tm range was ۸۱.۷۹-۸۲.۳۹ °C and the mean CT value was ۲۰.۰۶-۲۲.۹۳. These results were completely in accordance with the findings of DNA sequencing.Conclusion: The fast-track HRM-based method successfully detected one of the most common mechanisms of resistance in the ERG-۱۱ gene of C. auris within ۳ h. Finally, the development of more panels of HRM assays for the detection of all azole resistance mutations in C. auris ERG-۱۱ is recommended to expand the scope of the field and facilitate the elaboration of rapid and accurate methods of antifungal resistance assessment.

Keywords:

Azole resistance , Candida auris , high-resolution melt curve analysis , mutation screening , Real-Time PCR