Molecular Identification of Candida dubliniensis among Candida albicans Isolated from Oral Cavity of Cancer Patients using PCR-RFLP, in a Tertiary Care Hospital in Kashmir, India

Abstract

Aims: To retrospectively evaluate 186 stock strains of C. albicans strains isolated from oral cavity of HIV negative patients with various malignancies for the presence of C. dubliniensis isolates among them by PCR-RFLP. Place and Duration of Study: Department of Microbiology, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, between October 2013 and October 2014. Methodology: This study included 186 stock strains of C. albicans tentatively identified by phenotypic methods like germ tube formation in human serum, colony color on chromogenic candida differential agar, characteristic morphology on corn meal agar and assimilation of sugars isolated from HIV negative patients with various malignancies. DNA extraction was performed by chemical method. PCR amplification of ITS1-5.8S-ITS2 rDNA region was achieved using the ITS1 and ITS4 primer pairs which amplify the ITS region of both species, providing a single PCR product of expected size (540 bp). There is no visible difference between these two species with regard to their ITS PCR products. Digestion of amplified products was performed by using restriction enzyme BlnI (AvrII) which cleaves DNA where there is a CCTAGG sequence. The products of digestion generate one band of 540 bp for C. albicans, and two bands of 200 bp and 340 bp for C. dubliniensis because BlnI has one recognition site within the ITS region of C. dubliniensis, whereas none within that of C. albicans. Results: Of the 186 isolates tested, no C. dubliniensis was found by PCR-RFLP. Conclusion: Our results of not finding C. dubliniensis in this subset of patients support the need for further investigations into the prevalence of this species among other clinical samples and other susceptible patient populations.