P425 Approach to identify and isolate C. dubliniensis in laboratory and comparison of various methods for same
Authors: Jan Abiroo 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Introduction
C. dubliniensis, a newly recognized fungal pathogen, is clinically significant as it develops a stable fluconazole-resistant phenotype on exposure to this antifungal in vitro. C. dubliniensis is overlooked and misidentified as it shares many phenotypic traits with C. albicans. Molecular methods developed to distinguish the two species are not readily applicable. So, an easy-to-perform and an accurate phenotypic test would be a valuable tool.

Aim
This study first of its kind from Kashmir Valley (conducted in SKIMS-1200 bedded tertiary care hospital) evaluated seven phenotypic tests with PCR-RFLP as a gold standard. The best methodology for routine identification of this emerging pathogen was developed.

Materials
A total of 206 Candida spp. were tested which included 186 in-house C. albicans strains and 20 C. dubliniensis strains. The in-house 186 C. albicans strains were isolated from cancer patients. A total of 500 patients with various malignancies were screened, patients on antifungal drugs and screened once were excluded. All strains were sensitive to both fluconazole and voriconazole. Among 22°C dubliniensis strains, 14 were provided by Dr. Ziauddin Khan (Professor and Chairman, Department of Microbiology, Kuwait University) and 6 by VPCI institute Delhi.
Reference strains included in the study:
C. albicans 90028; (National culture collection of pathogenic fungi (NCCPF), Department of Medical Microbiology PGIMER Chandigarh)
C. dubliniensis (CD36) and (CBS 7987); provided by Dr. Ziauddin Khan

Methods
PCR-RFLP using BlnI (AvrII) enzyme was done to confirm the identification of all isolates (Fig. 1). Seven phenotypic tests that were evaluated include Growth on hypertonic Sabouraud Dextrose Agar (SDA), Colony color on Crome Candida Differential Agar, Growth at 45°C, Assimilation of xylose (XYL), Colony color and Chlamydospore formation on Tobacco Agar, Germ tube formation at 39°C and Fluorescence on methyl blue SDA. The variety of tests applied makes our study one of the most detailed studies for the identification of C. dubliniensis till date.

Results
No C. dubliniensis was found among the isolates recovered from cancer patients by PCR-RFLP.
Phenotypic tests that showed the best results were
Germ tube formation at 39°C was most rapid. Phenotypic methods proved to be very cost-effective than PCR-RFLP. Based on our results of growth on Hypertonic SDA and XAM and also knowing the fact that 5% of C. dubliniensis assimilate xylose and give false results of xylose assimilation, we developed a novel medium ‘Hypertonic xylose agar medium’ (HXAM). HXAM proved to be 100% accurate, simple, easy to perform, and cost-effective.

Conclusion
For routine identification of C. dubliniensis we recommend colony color on Crome Agar and growth on HXAM as they are simple, reliable, inexpensive, reproducible, and readily applicable methods. For retrospective evaluation of stored cultures, we recommend HXAM.
Based on our observations, we propose a scheme for the identification of C. dubliniensis (Fig. 2).