|Year : 2012 | Volume
| Issue : 3 | Page : 101-104
Evaluation of HiCrome differential agar for speciation of candida
Shyamala K. Shettar1, Asha B. Patil2, Shobha D. Nadagir2, T. A. Shepur3, B. A. Mythri2, Santosh Gadadavar2
1 Microbiologist, District Hospital, Haveri, Karnataka, India
2 Department of Microbiology, Karnataka Institute of Medical Sciences, Hubli, Karnataka, India
3 Department of Pediatrics, Karnataka Institute of Medical Sciences, Hubli, Karnataka, India
|Date of Web Publication||21-May-2014|
Dr. Asha B. Patil
Department of Microbiology, KIMS, HUBLI-31, Karnataka
Source of Support: None, Conflict of Interest: None
Background: In recent years, non-albicans Candida (NAC) are being increasingly reported in many laboratories. There is a need to diagnose them early as some of the species are resistant to antifungals. We evaluated the utility of HiCrome agar for speciation of Candida in comparison to conventional methods.
Materials and Methods: Ninety-six isolates of Candida were speciated by morphology on corn meal agar (CMA), sugar assimilation tests (SAT), and HiCrome agar. Sensitivity, specificity, and positive predictive and negative predictive values were obtained.
Results: Sensitivity of HiCrome agar in identification of Candida albicans was 97.05%, C. tropicalis was 83.33%, C. glabrata 100%, and C. krusei 87.5%; and specificity was 98.07, 91.93, 92, and 92.30%, respectively.
Conclusion: The present Candida differential agar is useful in identifying a few of the species. With further modifications this media may replace the need for conventional methods. The rapid availability of results and identification of species in mixed cultures is significant.
Keywords: Candida, HiCrome agar, speciation
|How to cite this article:|
Shettar SK, Patil AB, Nadagir SD, Shepur TA, Mythri BA, Gadadavar S. Evaluation of HiCrome differential agar for speciation of candida. J Acad Med Sci 2012;2:101-4
|How to cite this URL:|
Shettar SK, Patil AB, Nadagir SD, Shepur TA, Mythri BA, Gadadavar S. Evaluation of HiCrome differential agar for speciation of candida. J Acad Med Sci [serial online] 2012 [cited 2019 May 21];2:101-4. Available from: http://www.e-jams.org/text.asp?2012/2/3/101/132950
| Introduction|| |
The emergence of Candida species other than Candida albicans as important agents of infection is a concern in several laboratories.  Species such as C. glabrata, C. krusei, and C. tropicalis are emerging as important opportunistic pathogens and this transition has had a significant clinical impact due to decreased susceptibility of these non-albicans yeasts to antifungal agents.  The longer turnaround time taken by conventional methods of identification makes them less popular among the clinicians as early diagnosis is essential for initiating appropriate therapy. In order to facilitate rapid identification, several chromogenic substrate containing culture media have been developed.  HiCrome agar is a differential media that allows selective isolation of yeasts and identifies colonies of C. albicans, C. glabrata, C. krusei, and C. tropicalis.
| Materials and Methods|| |
A total of 96 Candida isolates from oral cavity of neonates were included in the study. These Candida species were isolated on Sabouraud's dextrose agar. Identification of the species was done by germ tube test, morphology on corn meal agar (CMA) and sugar assimilation tests (SAT) as per standard methods [Figure 1].  HiCrome Candida differential agar M1456A was obtained commercially from Hi Media, Mumbai, India. The media was prepared as per manufacturer's instructions. HiCrome agar allows differentiation of Candida species namely C. albicans, C. krusei, C. tropicalis and C. glabrata on the basis of color and colony morphology. It identifies light green colonies as C. albicans, purple fuzzy colonies as C. krusei, steel blue to blue as C. tropicalis, and cream to white as C. glabrata [Figure 2]. As many literatures suggest dark green color for C. dubliniensis, dark green colonies of Candida are identified as C. dubliniensis. ,
|Figure 1: (a) C. albicans showing large, thick-walled, terminal chlamydospores. (b) C. dubliniensis showing thick-walled, terminal chlamydospores in small bunches and pairs. (c) C. tropicalis showing blastoconidia in small groups along the pseudohyphae. (d) C. krusei showing cross matchstick- or tree-like appearance. (e) C. kefyr showing logs in stream appearance. (f) C. guilliermondii showing clusters of yeast cells with pseudohyphae with small groups of blastoconidia. (g) C. parapsilosis showing curved pseudophyphae with few blastoconidia. (h) C. glabrata showing oval budding yeast cells without pseudophyphae|
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|Figure 2: Chrome agar plate showing different Candida species. (a) Candida albicans, (b) C. dubliniensis, (c) C. tropicalis, (d) C. glabrata, and (e) C. krusei|
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Statistical analysis was done using the Statistical Package for Social Sciences (SPSS) software.
The following values were obtained: Sensitivity, specificity, and positive predictive and negative predictive values.
| Results|| |
A total of 96 Candida were isolated from 86 neonates. Mixed yeasts were isolated in 10 cases.
The species were identified by conventional methods. Distribution of various species was as follows; C. albicans 34 (35.41%), C. tropicalis 24 (25%), C. glabrata 11 (11.45%), C. krusei 08 (8.33%), C. dubliniensis 6 (6.25%), C. guillermondii 5 (5.20%), C. kefyr 4 (4.16%), and C. parapsilosis 4 (4.16%) [Table 1].
The color and colony characters on HiCrome agar was read after 48 h. Of the 34 C. albicans, 33 produced light green colonies, one produced dark green colored colonies and falsely identified as C. dubliniensis. One C. dubliniensis produced light green colonies. Three C. tropicalis gave purple, fuzzy colonies and were identified as C. krusei. One C. krusei gave bluish purple colonies. All the C. parapsilosis produced cream colonies similar to C. glabrata.
Sensitivity, specificity, and positive predictive and negative predictive values are shown in [Table 2].
| Discussion|| |
Despite the time tested methods of identification of Candida by CMA and SAT, detection of growth patterns on CMA takes 24-72 h and sugar assimilation may take 72 h-2 weeks. Besides, these procedures are labor intensive and take longer time to determine the diagnosis. In order to facilitate rapid identification, several chromogenic substrate containing culture media have been developed.  CHROMagar shows sufficient sensitivity to grow the most important yeasts. It can serve as a primary isolation and differentiation medium for clinical specimens likely to contain yeasts and also as an adjunctive differential medium for identification of yeasts isolated on other media. A major advantage of CHROMagar is the ability to detect mixed cultures of yeasts in clinical specimens. 
Varying sensitivities and specificities have been reported by several authors for identification of C. albicans. Baradkar et al., who used HiCrome media (Himedia Mumbai, India) reported a sensitivity of 96.55% and specificity of 96.42%; Willinger and Manafi  reported 98.8 and 100%, Peng et al., 100 and 94.6%;  and Yucesoy et al., reported 99.4 and 100% of sensitivity and specificity, respectively. We too found a good sensitivity (97.05%) and specificity (98.07%). Our study agrees with that of Baradkar et al. Although HiMedia does not recommend identification of C. dubliniensis, we attempted to identify based on several studies and text books. , The authors mentioned that C. dubliniensis forms dark green colonies. In present study we observed that one C. albicans was identified as C. dubliniensis on HiCrome agar. Differentiation between light green and dark green colonies posed a problem and was found to be subjective. Other tests like germ tube test, growth at 42-45°C, and chlamydospore formation on CMA and SAT need to be done in case of ambiguity.
As regards C. tropicalis, wide ranging differences in sensitivity and specificity have been reported by various authors. Sensitivity ranged from 66.7 to 100%, specificity from 78.8 to 100%. ,, Our study showed sensitivity of 83.33% and specificity of 91.33%, which is in between the rates reported by Willinger and Manafi and Yucesoy et al., We found it difficult to objectively identify colony characters like purple fuzzy and steel blue.
C. glabrata is less sensitive than other species to fluconazole and ketoconazole and C. krusei exhibits innate resistance to fluconazole.  Rapid identification of these species is therefore important. Our results of sensitivity for C. glabrata correlates well with that of Willinger and Manafi.  Very low specificity has been reported by Baradkar et al., 88.23%. Our results for specificity were between Baradkar et al., Willinger and Manafi, , and Peng et al.
In our study C. glabrata gave cream to white colonies as mentioned in HiMedia. But it needs to be highlighted that C. parapsilosis too were of the same color which were identified as C. glabrata on HiCrome agar, however these isolates were identified as C. parapsiosis based on gold standard tests like morphology on CMA and SAT. These strains can be easily differentiated from C. glabrata which does not produce even pseudohyphae on CMA. Thus, the combination of CMA and CHROMagar can be used for early identification of C. glabrata.
Rapid identification of C. krusei with chromogenic media is important because it exhibits innate resistance to fluconazole. Study done by Yucesoy et al., revealed that, all C. krusei isolates produced rough fuzzy spreading big pink colonies on CHROMagar.  Cooke et al., reported that the usefulness of colony color in identification of C. krusei appeared to be limited as several other yeasts gave pink and purple colonies on this medium. But Yucesoy et al., opines that although there are many yeast species giving the same color, the morphology of the colonies of C. krusei is distinctly different (fuzzy, rough, large, pink). 
Yucesoy et al., reported sensitivity and specificity of 100% in case of C. krusei.  Our study shows sensitivity of 87.5% and specificity of 92.30%. It was difficult to differentiate purple colonies from purple fuzzy colonies in many cases. We found such observations subjective and confusing. Expertise in using this media may decrease the ambiguity.
Baradkar et al., concluded that the use of chromogenic medium (HiCrome Candida agar) is an easy and reliable method for the presumptive identification of most commonly isolated Candida species, especially C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis with sufficient sensitivity. With typical colors shown by Candida species, this medium may well replace cornmeal agar and conventional biochemical tests, including germ tube tests, sugar fermentation and SAT, used for direct identification of C. albicans.  However, we are not entirely in agreement with this. Although identification of C. albicans and C. glabrata on HiCrome agar was satisfactory, sensitivity for identification of C. tropicalis and C. krusei was moderate. Besides the subjective nature of identification on HiCrome agar poses a problem. For instance the light green and dark green color to identify C. albicans and C. dubliniensis was found to vary between observers.
| Conclusion|| |
HiCrome agar has several advantages like rapidity, direct identification of species; thus, making it very useful in early identification and thereby early initiation of appropriate antifungal therapy. This becomes especially important in blood stream infections which have a high mortality. However, the major issue with this media is the subjective nature of the identification. Besides, the present differential media allows the identification of very few species of Candida. This may be a limiting factor as other NAC are being increasingly reported in recent years. On the other hand the long turnaround time and expertise required for the conventional tests is a problem. Thus, a combination of tests may be more useful than any single test alone. Further modification in the agar and improvement in the observer skills may increase the utility of HiCrome agar in routine mycology laboratories and may perhaps obviate the need for conventional identification methods.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]