Original article / research
Standardisation of a Semi-nested PCR for Early Identification of Candida auris: A Cross-sectional Study from Southern India
DC17-DC20
Correspondence
Dr. Premamalini Thayanidhi,
Associate Professor, Department of Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Porur, Chennai-600116, Tamil Nadu, India.
E-mail: drtpremamalini@gmail.com
Introduction: Candida auris has drawn more clinical attention due to its multidrug re-sistance. More than 90% of C. auris isolates are known to be resistant to all the antifungal drugs. Conventionally, the identification of C. auris has not been described precisely yet but one such ability of this yeast is that they can grow at an elevated temperature and fail to grow in the presence of cycloheximide and in automated approaches like VITEK 2, Matrix-assisted Laser Desorp-tion/ionisation Time-of-flight (MALDI-TOF), Analytical Profile Index 20C AUX yeast (APIC20C-AUX) they can be misidentified as other species, which can lead to errors in the choice of antifungals.
Aim: To standardise a simple in-house semi-nested colony Polymerase Chain Reaction (PCR) for accurate identification of C. auris.
Materials and Methods: A cross-sectional study was conducted at the Department of Microbiolo-gy at Sri Ramachandra Institute of Higher Education and Research (SRIHER), Chennai, India, from March 2017 to February 2018. All the Candida isolates obtained from blood samples over a period of one year were considered for this study. All the isolates underwent conventional phenotypic identification, such as microscopy, sub-culturing on Sabouraud Dextrose Agar (SDA) with antibiotics and chromogenic me-dium and carbohydrate fermentation. Further genotypic identification was performed for all the isolates. Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) was initially performed, and the isolates that were unidentified by this method were sent for gene sequencing. C. auris-specific primers were designed manually. A semi-nested colony PCR was performed using the self-designed primers for all the isolates.
Results: A total of 87 Candida sp. were isolated and considered for this study. Phenotyp-ic and genotypic identification was performed for the isolates. In the study, it was observed that C. auris was misidentified by commonly used phenotypic methods as Candida haemulonii and Candida parapsilosis and hence, further PCR-RFLP was performed to confirm the species. The iso-lates that still couldn’t be identified by PCR-RFLP were sent for gene sequencing. All the isolates were subjected to C. auris-specific semi-nested PCR for identification. A 22 (25.3%) out of the 87 isolates produced amplicons of size 400 bp. The rest 65 (74.7%) isolates did not produce bands. All 22 isolates that produced bands were confirmed as C. auris by gene sequencing.
Conclusion: To conclude, an in-house, semi-nested colony PCR (2-hour) method for differentiat-ing C. auris from Candida haemulonii was standardised. This method not only differentiates C. auris from Candida haemulonii but also other Candida species. This test will be useful in the early identification of C. auris in any microbiological laboratory set-up.