ISOLATION, IDENTIFICATION AND ANTIFUNGAL SUSCEPTIBILITY TESTINGOF CANDIDA ISOLATES FROM VARIOUS CLINICAL SPECIMENS AT A TERTIARY CARE HOSPITAL, WESTERN RAJASTHAN

spp is a of the of the mucous gastrointestinal Candida continues to be leading cause of morbidity and mortality in large population of immunocompromised and hospitalized patients. Invasive Candidiasis due to non-albicans candida has been on the rise in last few years. This study aims to Spectate Candida using chromogenic simple, rapid and inexpensive method for identification of such species. Characterization to species level helps to identify species which might be intrinsically resistant to commonly used antifungal agents.

Background: Candida spp is a member of the normal flora of the skin, mucous membrane and gastrointestinal tract. Candida continues to be leading cause of morbidity and mortality in large population of immunocompromised and hospitalized patients. Invasive Candidiasis due to non-albicans candida has been on the rise in last few years. This study aims to Spectate Candida using chromogenic medium.The emerging pathogens are resistant to conventional antifungal therapy. Objective: To identify the various species of candida isolated from different clinical specimens and to compare the susceptibility pattern of these isolated species towards different antifungal agents. Methods: All Candida isolates recovered from various clinical samples during the period from September 2017 and august 2018 were studied., These isolates were subjected to gram's stain, germ tube test and inoculation on commercially available CHROM agar (HiMedia India). Results: A total of 155 Candida species were isolated from the different clinical specimens of suspected candida infection cases. Most of the isolates obtained were from urine samples 93 (60%) followed by blood 26(16.77%). Non albicans Candida were isolated at a higher rate 101 (65.16%) than Candida albicans 54 (34.84%) . Among 101 non C. albicans, C. tropicalis 55 (35.48%) was the most common species followed by 19  The commonly used antifungal drugs show significant variation in the susceptibility pattern among the types of Candida species. The drug resistance scenario has been increasing during last decades due to over growing use of random antifungal agents 8 . Several previous studies reported the emergence of drug resistance Candidaspecies in global scenario 9,10 . Therefore, the change in drug susceptibility pattern of Candida species and introduction of newer antifungal agents has made the invitro susceptibility testing of antifungal agents more relevant for using specific and sensitive drugs 11 .Thus, the early identification, speciation and susceptibility testing of Candida species in clinical specimens have become increasingly important to prevent the treatment failure using appropriate antifungal agent.

Aims and Objectives:-
1. To identify the various species of candida isolated from different clinical specimens. 2. To compare the susceptibility pattern of these isolated species towards different antifungal agents.

Materials And Methods:-
We enrolled 2997 clinical specimens for this study. This study was conducted between September 2017 and august 2018. The clinical specimens wereurine, pus from ear& trachea, tracheal devices, blood, CSF, sputum, throat swab, indwelling medical devices, conjunctival swab, various body fluidsand semen. These specimens were collectedfrom OPD and IPD patients of all age and sex groupsduring the period of 1 year. All specimens were investigated for fungal culture and identification of speciesafter obtaining ethical clearance from the Institutional Ethics Committee.
All clinically suspected samples were subjected to gram staining to look for presence of Gram positive yeast like budding cells with pseudohyphae indicating presence of candida species and KOH mount. The samples were inoculated on Sabouraud's dextrose agar (SDA) with chloramphenicol & Blood agar and incubated at 37⁰C and 25⁰C 24-72 hours.Further identification and speciation of Candida on SDA were confirmed by Gram's stain, Germ tube test, 0.1% Glucose agar test,Sugar fermentation test,Sugar assimilation test, CHROM agar according to standard microbiological techniques 12 .
Germ tube test was carried out by inoculating isolated yeast cells into 0.5 ml of pooled human serum in a small tube and incubation at 37ºC for 2 hours. Germ tubes formation was observed microscopically as tubular elongation extending from the yeast cells without constriction or septa at the point of attachment to the yeast cells. 0.1% Glucose agar test were inoculated and incubated at 30°C for 2-5 days and studied microscopically for the presence of pseudohyphae, chlamydospores andblastospores. Isolated candida species were sub-cultured onchromogenic Candida medium (HICHROME Candida agar) and incubated at 37ºC for 48 hours. Presumptive species identification was done based on specific colony colors produced by the chromogenic substrates in the medium. All isolates were further identified by carbohydrate assimilation.

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All the isolates were subjected to the antifungal susceptibility test according to CLSI document M 44 -A2 by disk diffusion testing method for yeasts. Muller Hinton agar supplemented with 0.5μg/ml Methylene Blue Dye and 2% Glucose (MHMB) was used for sensitivity testing. The inoculated plates were incubated at 37 0 C for 24 hours or longer 13 .

Discussion:-
Fungal infections, particularly those attributed to Candida species, are frequent complications for hospitalized patients contributing to increased morbidity and mortality and healthcare cost.Furthermore there is increasing prevalence of infections caused by non-albicans Candida worldwide with various degree of susceptibility to routinely use antifungal agents indicating the importance of laboratory diagnoses 14 .
In this study, it was observed that candidiasis can occur at all ages and in both sexes.The highest number of isolates were obtained in the age group of0-10 years 36 (23.23%), followed by the age groups of 41-50 years 24(15.48%).
Infections were more common in the 0-10 age groups in thisstudy.This can be attributed to the various co-morbid conditions and thehealth issues pertaining to the particular age-groups, as is relevant in thisstudy. However, further studies have to be carried out to justify thesignificance of the fact.In present study, Males were more affected than females with anoverall male:female ratio of (90:65)1.38. Candida tropicalis is major isolated species. In male patient Candida tropicalis 36.67% is the major isolated species whereas in female patients Candida albicans 36.92% is the major organisms.The preponderance of male patientssuffering from candidiasis in this study correlates with Singh et al 15 .

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Overall, the non-Candida albicans species showed more resistancethan C. albicans to applied antifungal agents except amphotericin-B.These similar findings corelates with study done by Duttaet al 17 .

Conclusion:-
The present study suggests an increasing prevalence of non-Candida albicans species in the various clinical samples isolated. An emergence of resistance of these Candida species isolates to the routinely used antifungals, make them difficult to treat.
Therefore, detection of distribution of Candidathrough presumptive identification, followed by confirmation and antifungal treatment, has a efficient effect on successful treatment as it helps in optimum selection of the therapeutic agent and use of CHROMagar is a simple, rapid and inexpensive method for identification of Candida species especially in the laboratory with limited resources.