SPECIES IDENTIFICATION OF CANDIDA ISOLATES IN VARIOUS CLINICAL SAMPLES

Rachana Mehta 1 and Anupama S. Wyawahare 2 . 1. PG Student, Department of Microbiology, MGM’s Medical College & Hospital, Aurangabad, Maharashtra, India. 2. Professor, Department of Microbiology, MGM’s Medical College & Hospital, Aurangabad, Maharashtra, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Candida species especially non albicans Candida are increasingly being isolated from clinical specimens. The aim of our study was to detect the clinical distribution of Candida species in various clinical samples in a tertiary care hospital. A total of 115 Candida species were isolated from various clinical specimens submitted to Microbiology laboratory from different units of a tertiary care centre, Aurangabad Maharashtra from January 2014 to December 2014 using conventional yeast identification method after the approval from institutional ethical committee. The percentage of isolation of non albicans Candida species was 59.1% and that of C. albicans was 40.9%. The predominant species among non albicans Candida species isolated were C. tropicalis (40%) followed by C. guilliermondii (10.43%), C. krusei (4.34%), C. glabrata (2.60%), C. kefyr and C. parapsilosis (0.87%) each. As it is possible to predict the sensitivity pattern of each Candida species with high accuracy, the accurate species identification of Candida is important.

Introduction:-
Candida species are ubiquitous fungi and are the most common fungal pathogens that affect human beings. They are true opportunistic pathogens that exploit recent technological advances to gain access to the circulation and deep tissues. 1 The genus Candida comprises about 200 species, of which close to 20 have been associated with pathology in human and animal. 2 Candida species are the component of normal flora of human beings. They are commonly found on the skin throughout gastrointestinal tract and female genital tract particularly higher in vagina during pregnancy. 3 Those that are the part of normal flora can invade tissues and cause life-threatening disease in patients whose cell mediated immunity is decreased by disease or iatrogenic intervention. 4 Candida albicans is by far, the most common in all clinical forms of Candidiasis, representing 70-80% of all yeast isolates. 5 Candida species especially non albicans Candida are increasingly being isolated from clinical specimens. Speciation of Candida isolates is conventionally done by germ tube test, morphology on cornmeal agar, sugar fermentation and sugar assimilation tests. 6 Non Candida albicans like C. tropicalis, C. krusei, C. glabrata and C. parapsilosis are less susceptible to azoles, particularly fluconazole. 3,7 Therefore, correct identification of Candida species is of great importance. It presents prognostic and therapeutical significance, allowing an early and ISSN: 2320-5407

Int. J. Adv. Res. 4(8), 1340-1348
1341 appropriate antifungal therapy. Thus, the present study was undertaken for species identification of Candida isolates in various clinical specimens using conventional method.

Materials and Method:-
Study population:-All the specimens collected from IPD and OPD, patients attending various clinical services at tertiary care hospital were used in the study. These specimens were processed as per standard protocol. 3 Processing of the specimens:-Specimens like urine, pus, vaginal discharge, blood, sputum, endotracheal tube secretion, gastric lavage and other body fluids received in the Department of Microbiology for the culture were used in the study. Brain Heart Infusion broth was used for blood culture. All the specimens were screened for growth of Candida species by standard protocol 3 . Relevant clinical data was recorded. In the present study, total 115 isolates of Candida species were obtained.

Identification of Candida species:-
Isolates were preliminary identified as Candida species by direct microscopy, colony morphology and Gram staining.
All the specimens for the fungal culture were inoculated on Sabouraud's dextrose agar with chloramphenicol and incubated at 25 0 C and 37 0 C for 48 hours. Candida species were identified by standard protocol that included germ tube formation, chlamydospore production on cornmeal agar, sugar fermentation and sugar assimilation tests.

Wet preparations :
Direct microscopy :- 8 Direct examination of specimens like urine, peritoneal fluid, ascitic fluid etc. were carried out microscopically for the presence of yeast like cells.

10% KOH : 3,9
The specimens like sputum and swab from lesion were examined in KOH wet mount for the presence of yeast and pseudohyphae. The yeast cells of Candida species were approximately 4-8 μm with budding and pseudohyphae.

Gram staining : 9-11
For the specimens like pus, sputum etc Gram's stained smear was observed for the presence of oval budding yeast like cells..

Colony morphology on Sabouraud's dextrose agar : 3,9
The sabouraud's dextrose agar consisting of chloramphenicol was used for isolation of Candida species. Cream coloured pasty colonies usually appeared after 24-48 hours incubation at 35-37 0 C. The colonies had a distinctive yeast smell. Sometimes growth was observed after an overnight incubation as seen in bacteria. The lactophenol cotton blue mounts were prepared to examine detail morphological features of fungi grown on culture medium. Gram's staining was also performed from culture isolates.

Discussion:-
Candidiasis is the commonest fungal disease in humans affecting skin, nails, mucosa and internal organs of the body. Candida species is endogenous and the disease represents opportunistic infections. 15 In our study, we found highest number of Candida isolates from urine 43(37.39%). Out of 43(37.39%) isolates, 18 Candida isolates were Candida albicans and 25 were non-albicans Candida species. Among non albicans Candida species, Candida tropicalis (21) was the most predominant species. This was in agreement with Kashid et al (2011), 15 Patel et al (2012), 16 Kumar et al (2013), 17 as they found highest number of isolates from urine and among which Candida tropicalis was the most predominant species.
In our study, 34(29.56%) Candida isolates were from sputum sample. We found highest number of Candida albicans 20 from sputum sample as compared to non albicans Candida 14. This was in agreement with Patel et al (2012) 16 who found highest number of Candida albicans from sputum sample. Candida is normal inhabitant of the mouth and can be recovered from sputum in 20% to 55% of normal subjects. The prevalence and prognosis of pulmonary candidal infection is difficult to evaluate since diagnosis were seldom confirmed. Candida isolated from sputum sample is mostly a colonizer of the respiratory tract. The role of Candida in pulmonary candidiasis and its diagnosis is still controversial. 18 In our study, 15 (13.04%) of the Candida isolates were from blood. Among them 10 were Candida guilliermondii, 2 were Candida albicans and Candida krusei each and one was Candida tropicalis. We observed that Candida guilliermondii was the most predominant isolate from blood. This was in agreement with study done by Patel et al (2012) 16  In the present study, out of 115 isolates of Candida species 89(77.39%) were from IPD and 26(22.60%) were from OPD. The continuous use of invasive monitoring and surgical technologies in the intensive care unit has increased the risk of fungal infections. 20 We observed that the frequent isolation of Candida species was in the age group above 60 years (26.08%) followed by age group 0-10 years (20.87%) which was similar with the study of Kashid et al (2011) 15 who reported highest incidence in the age group above 60 years (24.48%) followed by age group 0-10 years (20.40%). Our study was also in agreement with Bineshlal et al (2011) 7 and Yashvanth et al (2013) 21 who also reported maximum isolation of Candida species in the age group above 60 years. Candida species remain the most important cause of opportunistic infections worldwide, affecting predominantly patients over 65 years old. 22