Standardization of PCR for detection of fluconazole resistance targeting Y132F mutation in ERG11 gene in Candida parapsilosis

Document Type : Original Articles

Authors

1 Department of Microbiology, Sri Ramachandra Medical College & Research Institute, SRIHER, Porur, Chennai 600116, India

2 2Department of Microbiology, Sri Ramachandra Medical College & Research Institute, SRIHER, Porur, Chennai 600116, India

10.22034/cmm.2024.345209.1517

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 document that Y132F substitution in the ERG11 gene is the predominant fluconazole resistance mechanism among C. parapsilosis. Hence this study was done 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 (RFLP) was performed for identification and confirmation of C. parapsilosis isolates. The Antifungal susceptibility testing (AFST) by broth microdilution method was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (M27-A3) for all C. parapsilosis against fluconazole, itraconazole, voriconazole and posaconazole to determine their Minimum Inhibitory Concentration (MIC) values. C. parapsilosis- specific PCR assay was developed with in-house designed primers to detect Y132F mutation in the ERG11 gene.
Results: In our 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 MIC’s ranging from 32µg/ml – 64 µg/ml. The PCR assay successfully identified all 3 fluconazole-resistant C. parapsilosis with Y132F mutation, thereby confirming the PCR results. Further, 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 hours. This assay can be useful for early detection of fluconazole-resistant C. parapsilosis isolates in candidemia patients, which in turn help in providing early antifungal treatment for better patient management. 
 
 

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Current Medical Mycology

Articles in Press, Accepted Manuscript
Available Online from 27 April 2024

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History

  • Receive Date: 04 March 2024
  • Revise Date: 13 April 2024
  • Accept Date: 27 April 2024
  • First Publish Date: 27 April 2024
  • Publish Date: 27 April 2024

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