EVALUATION OF CANDIDA SPECIES FROM CLINICAL SPECIMENS BY USING CHROMAGAR

Preeti Sharma 1 , Sorabh Singh Sambyal 1 and Divya Shrivastava 2 . 1. Ph. D Scholar, Jaipur National University. 2. Director, Life Sciences , Jaipur National University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (2), 1750-1755 1751 Material & Methods:-Present prospective study was conducted in tertiary care hospital from January 2016 to June 2016. Various samples received in laboratory from patients of all age group and both sexes with suspected Candida Infection. Clinical details were noted in the case record form. Patients who were on antifungal treatment were excluded. The specimens for laboratory investigation were collected undertaking strict aseptic precautions. The various clinical specimens collected were blood, urine, pus etc. Two swabs/specimens were taken from each case. One of the specimens was subjected for direct examination and the other for the culture. All the above samples were subjected to various mycological tests. 1) Direct examination by KOH Mount 2) Gram stain 3) Culture on SDA (at 25°C and 37°C) 4) Germ tube test for speciation: A small portion colony of the yeast to be tested was suspended in a test tube containing 0.5 ml human serum. The test tube was incubated at 35ºC for 2-3 hours. A drop of yeast-serum suspension was placed on a microscopic slide, overlaid with a cover slip and examined microscopically for presence of germ tubes. Observation: Filamentous extension from yeast cell with no constriction was considered as germ tube.

Fig B-Germ tube formation
Growth pattern on CMA for speciation:-Isolated colonies of Candida were picked up with inoculating loop. Three parallel cuts 1 cm apart was made into the surface of Cornmeal-Tween agar, holding the inoculating loop at about a 45-degree angle. A sterile cover slip was laid on the surface of agar, covering a portion of the inoculated streaks. The inoculated plates were incubated at 25-30ºC for 24-72 hours. At the end of incubation period plates were examined microscopically (under 10x and 40x) at the edge of cover slip and the pattern of growth was observed to make a presumptive identification.

Fig C-C.krusei on CMA
Growth on CHROMagar:-Isolated species were inoculated on Candida CHROMagar to improve species identification based on coloured colony morphology. These agar plates were incubated at 37 0 C for 48 hours. The species were identified by characteristic colony colour.  C. albicans-Light green coloured colonies  C. tropicalis -Blue to metallic blue coloured colonies  C. glabrata -Cream to white smooth colonies  C. krusei-Pink colonies  C. dubliniensis -Dark green colonies    11.25% C.parapsilosis 05 6.25% Table1-Total number of Candida species isolated was 80. Out of 80 isolates, Candida krusei (35. %) was the most common species followed by C.albicans 32.5%) was most common followed by C. glabrata (15%), C.tropicalis (11.25 %.), C.parapsilosis (6.25%).   Table 3-We obtained 100% sensitivity and specificity of Candida CHROMagar for C. krusei, C. albicans, C.tropicalis but sensitivity and specificity of Candida CHROMagar for C. glabrata was 100% and 72.22% respectively.

Discussion:-
The potential clinical importance of species-level identification has been recognized as Candida species differ in the expression of virulence factors and antifungal susceptibility. Non albicans Candida are on the rise due to increasing immunocompromised states. Non albicans Candida are more resistant to fluconazole, therefore species level identification has a direct impact on choice of empirical antifungal treatment. (6) The incidence of infections caused by Candida species has increased considerably over the past three decades, mainly due to the rise of the AIDS epidemic, an increasingly aged population, higher numbers of immune-compromised patients and the more widespread use of in dwelling medical devices. Candida albicans is the main cause of candidiasis; however, nonalbicans Candida species such as C. glabrata, C. tropicalis and C.parapsilosis are now frequently identified as human pathogens. (7) Total 80 Candida species were isolated from various clinical samples. Among the various clinical isolates of Candida species we obtained C.krusei (28) as the most common isolate followed by C. albicans (26), C. glabrata (12), C. tropicalis (09), C. parapsilosis (Table1). Factors like increased use of antifungal drugs, use of broad spectrum antibiotics, long term use of catheters and increase in the number of immunocompromised patients contributes to the emergence of non-albicans Candida species. (8) For differentiation among different species of Candida conventionally germ tube test, growth pattern on cornmeal agar and sugar assimilation tests are being used which are technically difficult, time consuming and difficult to interpret which may take 72 hours to two weeks for species identification (9) , (10) Chromogenic agar is technically simple, easy to interpret and rapid method to differentiate among different Candida species. It facilitates the detection and identification of Candida species and provides result in 24-48 hours. Among the new tests, Candida CHROMagar is rapid and cost effective as compared to other expensive systems like API systems, Vitek 2 ID system and molecular methods. (11) In our study, for C. glabrata specificity of Candida CHROMagar was 72.22% as 5 species of C.glabrata were falsely identified by Candida CHROMagar as C. parapsilosis. Shettar SK et al (12) reported that on Candida CHROMagar, C.parapsilosis gave same cream colour as that of C.glabrata. This may be because of C. glabrata; C. kefyr, C. parapsilosis and C.lusitaniae appear as a variety of beige/brown/yellow colours due to the mixture of 1755 natural Pigmentation and some alkaline phosphatase activity. (13) C.glabrata and C. parapsilosis can be easily differentiated from growth pattern on Cornmeal agar as C. glabrata doesn't produce pseudo-hyphae. Thus, the combination of Cornmeal agar Hi Candida agar can be used for early identification of C. glabrata. (12)

Conclusion:-
Our study showed C. krusei as most common Non -albicans Candida species causing candidiasis, which shows the rise of Non-albicans Candida among various clinical samples. CHROMagar is a simplistic, rapid, easy and inexpensive method with good sensitivity and specificity for identification of Candida species. CHROMagar can be reliably used for identification for C. krusei, C.albicans, C. tropicalis but for early identification of C. glabrata and C. parapsilosis both the corn meal agar and CHROMagar should be used.