Introduction
The Candida species are ubiquitous yeasts which are a part of normal flora of the alimentary tract of mammals and the mucocutaneous membranes of humans.1 It becomes an opportunistic pathogen as a result of one or more underlying pre-disposing factors.2 Until recently Candida albicans was the most common yeast isolated from infections. Epidemiologic data from the past decade reveal a paradigm shift in Candida infections with non -albicans Candida species such as C.glabrata, C.tropicalis, and C.krusei as emerging important pathogens.3, 4, 5, 6 This transition has had a significant clinical impact due to decreased susceptibility of these non -albicans yeasts to antifungal agents.5 Candida albicans and non-albicans species are closely related but differ from each other with respect to epidemiology, virulence characteristics, and fungal susceptibility. The Combined effects of increasing number of fungal infections, growing number of patients at risk, the increasing rate of fungal resistance, and the expanded antifungal armamentarium have led to an increased recognition of the need for standardized laboratory testing for antifungal drug susceptibility.7 Agar-based susceptibility testing methods have been focus of interest for many researchers, due to their simplicity, reproducibility and lack of requirements for specialized equipment.8 Various Indian workers9, 10, 11 used Mueller Hinton Agar supplemented with 2% Glucose and 0.5µg/ml Methylene Blue Dye for Antifungal Susceptibility testing by Disk Diffusion Method and reported an increased resistance of non –albicans Candida isolates, to Azole group of drugs. The present study was designed to perform antifungal susceptibility test on Candida isolates by Disk Diffusion Method and study its susceptibility pattern.
Materials and Methods
The present study was conducted in the department of Microbiology in a tertiary care hospital in Hyderabad from January 2013 to June 2014, with prior approval of the Institutional Ethics Committee. A total of 102 Candida isolates from different clinical samples were processed for species identification. Candida albicans was the major species isolated followed by C. tropicalis, C. parapsilosis, C. krusei, C. glabrata and C. dubliniensis. All the speciated Candida isolates were subjected to Antifungal susceptibility testing by disk diffusion method using Mueller-Hinton Agar + 2% Glucose and 0.5 μg/mL Methylene Blue Dye (GMB) Medium as per CLSI guidelines.12 Antifungal disc used: Amphotericin – B (100U), Clotrimazole (10µg), Fluconazole (25µg) and Voriconazole (1µg). 0.5 McFarland standards was used to standardize the inoculum density. C.albicans ATCC 90028 and C. parapsilosis ATCC 22019 were used as Controls. All the culture media, Antifungal disc, and control strains were obtained from Himedia Laboratories, Mumbai.
Results
The antifungal susceptibility pattern of candida species is shown in the following table.
Table 1
Antifungal susceptibility pattern of Candida species.
Discussion
Candida species differ in their susceptibility to antifungal agents. On comparison of susceptibility pattern of C. albicans and non-albicans Candida species it was observed that C. albicans is more susceptible to all the antifungal agents tested. In the present study all the Candida albicans showed 100% susceptibility to Amphotericin-B. Similar findings were reported by other studies.13, 14 It was observed that C. albicans showed high fluconazole susceptibility rate (95.83%) in the present study, which is consistent with other reports by Akortha et al.,15Amar etal.,16 & Pahwa et al.,17 who also observed high Fluconazole susceptibility rates of 95.7%, 92.53% and 99% respectively. 92.53% of C. albicans were susceptible to Clotrimazole in a study by Amar et al.,16 which can be compared with the present study (93.7%). Pahwa et al.,17 reported 99% of C. albicans as susceptible to Voriconazole, which can be compared with 97.91% in present study.
Among non albicans Candida spp., susceptibility to Amphotericin–B and Voriconazole varied between 83.33% to 100 %, Clotrimazole between 50% to 87.5% and Fluconazole between 25% to 100%. Non-albicans Candida showed higher resistance to all the drugs than C. albicans. Maximum resistance was shown by C. krusei (100%) and C. glabrata (75%) in the present study. Findings from the present study indicate that all the isolates of C. krusei were resistant to Fluconazole. Similar findings were shown by Hamza et al.18 Non- albicans Candida has shown an increased resistance to azoles as in other studies.19, 20 Azole resistance candidiasis appears to be on the rise. The resistance to Fluconazole is of great concern because, it is most commonly used Azole for superficial as well as deep-seated candidiasis. It was observed that out of the total 19 isolates which showed resistance to Fluconazole,14 isolates were sensitive to Voriconazole. Voriconazole seemed to be superior to Fluconazole with a better susceptibility in the Fluconazole resistant strains also. This may be due to the more effective binding of Voriconazole to Cytochrome P450 isoenzyme of Candida species.
It was observed that infections with non-albicans Candida species (NAC) have shown decreased susceptibility to azole group of drugs.
Conclusion
Findings of the antifungal susceptibility test suggest that Candida spp., differ in their susceptibility to antifungal agents. On comparison of susceptibility pattern of C. albicans and non-albicans Candida species it was observed that non-albicans Candida showed higher resistance to all the drugs than C. albicans. Resistance to azole group of drugs was more pronounced in non-albicans candida spp. Antifungal susceptibility testing of Candida isolates will be helpful in guiding physicians to select the appropriate antifungal drug so that therapeutic failures can be avoided thus decreasing patient morbidity and mortality.
Acknowledgement
I express my heartfelt gratitude to Dr. Maimoona Mustafa, Professor and HOD, Department of Microbiology, Deccan College of Medical Sciences, Hyderabad, who has always been a source of inspiration and guidance.
I am thankful to the clinical and technical staff of the hospital for extending their cooperation.
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