PCR-RFLP And Multiplex PCR for Candida Species Identification in Cancer Patients.
S. C. Gagulie 1*, P. N. Dasanayaka1, W.A.S. Wijendra2, R. Ramesh2 , A. G. G. Kaushalya2 and S. Gunasekara 3
1Department of Botany University of Sri Jayewardenepura, Sri Lanka.
2Medical Research Institute, Sri Lanka.
3Department of Microbiology and infection control, Apkesha Hospital, Maharagama, Sri Lanka.
The changing epidemiology of Candida species distribution, coupled with the escalating antifungal resistance observed in some species, necessitates the development of reliable identification methods. This study utilized Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Multiplex PCR techniques to identify Candida spp. isolated from cancer patients at Apeksha Hospital, Sri Lanka. PCR-RFLP employed ITS1 and ITS4 universal primers to amplify the ITS1-5.8S-ITS2 rDNA regions, followed by digestion with the MspI restriction enzyme. Multiplex PCR involved the use of nine specific primer pairs divided into three groups for simultaneous amplification. Both ITS1 and ITS4 primers successfully amplified the ITS region in all Candida isolates. RFLP analysis using MspI distinguished seven Candida species among the 52 isolates. The multiplex PCR procedure identified 49 isolates, while three isolates remained unidentified. Candida tropicalis exhibited the highest prevalence (38%) among the 52 isolates, followed by Candida parapsilosis (31%), Candida albicans (13%), Candida glabrata (8%), Candida krusei (4%), Candida haemulonii (4%), and Candida intermedia (2%). Multiplex PCR was unable to identify Candida intermedia and Candida haemulonii species due to the absence of species-specific primers for them. This study concludes that molecular identification techniques, offer a superior approach for identifying Candida species and recommends the use of PCR-RFLP for clinical and diagnostic purposes and multiplex PCR for research purposes.