Standardization of polymerase chain reaction for detection of fluconazole resistance targeting Y132F mutation in ERG11 gene in Candida parapsilosis.

Abstract

Background and purpose: Candida parapsilosis is the third most commonly isolated species from candidemia patients admitted to Indian intensive care units. Outbreak of infection and emergence of fluconazole resistance associated with this particular species has been increasingly documented since 2018. Worldwide data has documented that Y132F substitution in the ERG11 gene is the predominant fluconazole resistance mechanism among C parapsilosis. Hence, this study aimed to detect fluconazole resistance by targeting Y132F mutation in the ERG11 gene in C. parapsilosis, by conventional polymerase chain reaction (PCR) assay with in-house designed primers.

Materials and methods: A total of 75 Candida isolates were collected from candidemia patients (Jan-Dec 2023). All the Candida isolates were subjected to phenotypic and genotypic characterization. PCR-restriction fragment length polymorphism was performed for identification and confirmation of C. parapsilosis isolates. The antifungal susceptibility testing by broth microdilution method was performed according to the Clinical and Laboratory Standards Institute guidelines (M27-A3) for all C. parapsilosis against fluconazole, itraconazole, voriconazole, and posaconazole to determine their minimum inhibitory concentration (MIC) values. Candida parapsilosis-specific PCR assay was developed with in-house designed primers to detect Y132F mutation in the ERG11 gene.

Results: In this study, among 75 candidemia patients (Jan-Dec 2023), about 24% of the candidemia was caused by C. parapsilosis. Fluconazole resistance among C. parapsilosis was found to be 16.7% with a MIC range of 32-64 µg/ml. The PCR assay successfully identified all three fluconazole-resistant C. parapsilosis with Y132F mutation, thereby confirming the PCR results. Furthermore, validation of the presence and absence of Y132F mutation in resistant and susceptible isolates by DNA sequencing showed that the results were in concordance with our PCR assay.

Conclusion: The developed PCR assay successfully detected the Y132F mutation within 3 h. This assay can be useful for early detection of fluconazole-resistant C. parapsilosis isolates in candidemia patients, which helps the provision of early antifungal treatment for better patient management.