Year : 2010 | Volume
: 53 | Issue : 1 | Page : 93--95
Hichrom candida agar for identification of candida species
VP Baradkar, M Mathur, S Kumar
Department of Microbiology, Lokmanya Tilak Municipal Medical College & General Hospital, Sion, Mumbai-400 002, India
V P Baradkar
Department of Microbiology, L.T.M.M.C and L.T.M.G.H, Sion, Mumbai-400 022
Chromogenic media are frequently used in direct and rapid identification of yeasts because different Candida species produce unique colors on these media. We used 60 isolates of Candida species including 30 C. albicans, 10 C. parapsilosis, 11 C. glabrata, five C. tropicalis, and four C. dubliniensis, isolated from various clinical specimens, to evaluate the performance of HiChrome Candida agar. These strains had been identified by germ tube test, morphology on cornmeal agar, chlamydospore formation on tobacco agar and sugar assimilation tests. The sensitivity and specificity results were: C. albicans (96.55 and 96.42%); C. parapsilosis (80 and 98.03%), C. glabrata (90.90 and 88.23%), C. tropicalis (100 and 100%) and C. dubliniensis (60 and 96.55%) respectively. HiChrom Candida agaris medium has been useful and capable of presumptive, rapid identification of Candida species within 48 hours.
|How to cite this article:|
Baradkar V P, Mathur M, Kumar S. Hichrom candida agar for identification of candida species.Indian J Pathol Microbiol 2010;53:93-95
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Baradkar V P, Mathur M, Kumar S. Hichrom candida agar for identification of candida species. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Sep 20 ];53:93-95
Available from: http://www.ijpmonline.org/text.asp?2010/53/1/93/59192
Although the frequency of isolation of non-albicans candida (NAC) species is increasing gradually, C. albicans is the most common cause of candidiasis.  Other species of Candida 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.  Identification to the species level of yeasts cultured from various clinical specimen is increasingly necessary for clinical laboratories. Generally yeast identification procedures start with a germ tube test in clinical laboratories. It is a rapid method to differentiate C. albicans and C. dubliniensis from other Candida species. Although it is a rapid test it may lead to false positive and false negative results. When the yeast cannot be identified with this method, further tests such as culturing on cornmeal agar, carbohydrate fermentation and carbohydrate assimilation tests are performed. Detection of growth patterns on cornmeal agar takes 24-72 hours and sugar assimilation tests may take 72 hours to two weeks. These procedures are labor intensive and take longer to determine the diagnosis and judge the proper antifungal agent.
In order to facilitate rapid identification, several chromogenic substrate containing culture media have been developed. These special media yield microbial colonies with varying colors secondary to chromogenic substrates that react with enzymes secreted by microorganisms. ,,,
CHROMagar Candida (HiMedia, Mumbai, India) employs this methodology to differentiate several Candida yeasts by color and morphology. It identifies C. albicans by the growth of green colonies, C. tropicalis as steel blue colonies, C. glabrata as pink colored colonies, C. parapsilosis as off-white to cream colored colonies. Another potential advantage of chromogenic media is the straightforward identification of mixed yeast infections, which have a significant clinical bearing. ,,,,,,,,,,,,
Material and Methods
A total of 60 Candida strains including 30 C. albicans, 10 C. parapsilosis, 11 C. glabrata, 5 C. tropicalis, and 4 C. dubliniensis, isolated from various clinical specimens, (36 from blood cultures, 10 from sputum samples of immunocompromised patients, 10 from oral swabs and 4 from vaginal swabs) were used in this study. These strains had been identified by germ tube test, morphology on cornmeal agar, tobacco agar and carbohydrate assimilation tests. , CHROMagar Candida (HiMedia, Mumbai, India) was prepared following manufacturer's instructions. About 21.02 gram of HiChrome Candida differentiation agar base (modified) was suspended in 500 ml of distilled water. It was heated to boiling gently to dissolve the medium completely. Then it was allowed to cool to 50oC and rehydrated (one vial) contents of HiChrome Candida selective supplement was added under aseptic precautions. It was mixed well and poured into petridishes. C. albicans, C. tropicalis, C. parapsilosis, C. glabrata were identified on HiChrome agar based upon the characteristic color of the colony by subculturing from Sabouraud's dextrose agar plates and the Candida HiChrome plates were incubated at 37oC for 24- 48 hours. Sensitivity and specificity of HiChrom candida agar was calculated as below:
Of the 30 strains of C. albicans, 28 produced light green colonies [Figure 1](a) on Candida HiChrome agar while two produced dark green colonies. Of the 10 strains of C. parapsilosis, eight produced cream colored colonies ([Figure 1](c) and two produced pink colonies; of the 11 C. glabrata strains, 10 produced pink colored colonies ([Figure 1]d) and one produced a slightly creamish colony; all five C. tropicalis strains produced a bluish color [[Figure 1] (b)]. Three of the four strains of C. dubliniensis isolated from throat swabs in immunocompromised patients produced dark green colonies (as shown in [Figure 1](a)) and one showed light green colonies similar to C. albicans. However, all the strains of C. dubliniensis had produced abundant chlamydospores on tobacco agar with typical colony morphology.
Colony color, sensitivity and specificity of Candida species grown on Chromagar are shown in the [Table 1].
We obtained sensitivity and specificity of HiChrom Candida agar for C. albicans as 96.55 and 96.42% respectively. Yucesoy et al.  reported low sensitivity (78%) and specificity (76%). Their study had used small culture tubes, instead of chromogenic culture plates, to demonstrate the color produced by the organism; probably this is the reason behind the low values for C. albicans. Willinger et al. reported 98.8% sensitivity and 100% specificity for C. albicans. Peng et al. studied 281 isolates of C. species previously identified by ATB ID 32 (Bio Merieux, France). The present study showed 100% sensitivity and 94.6% specificity for C. albicans on CHROMagar. Yucesoy et al. studied 270 yeast strains first identified by germ tube test and morphological characterization on cornmeal agar, Vitek 32 and API 20C AUX (Bio Merieux, France). They found that the sensitivity and specificity of CHROMagar for C. albicans strains were 99.4 and 100% respectively. Our results are comparable with these studies, which showed sensitivity and specificity values for C. albicans on CHROMagar ranging between 97-100 and 100% respectively. ,,
The sensitivity and specificity values for C. parapsilosis reported in this study are 80 and 98.03% respectively. Peng et al.  reported 90% sensitivity and 82.3% specificity for C. parapsilosis on CHROMagar. Our values are in contrast to this study showing a low sensitivity but higher specificity. As all the strains of C. parapsilosis produced characteristic morphology on Cornmeal agar (satellite colonies from original inoculum) the combination of these two media may identify all the strains of C. parapsilosis within 48 hours as compared to 48 hours to two weeks required for sugar assimilation tests.
Rapid identification of C. glabrata has a special importance because C. glabrata is less sensitive than other species to ketoconazole and fuconazole. , A sensitivity of 90.90% and specificity of 88.23% were reported in this study. Willinger et al. reported 98% sensitivity and 95.7% specificity for C. glabrata on chromagar. Peng et al.  reported sensitivity and specificity values of chrome agar for C. glabrata as 90.2 and 95.4% respectively. Yucesoy et al.  reported 90.9% sensitivity and 100% specificity of chrome agar for C. glabrata. Pfaller et al.  and Willinger et al. concluded that chrome agar allowed identification of C. glabrata. Many other authors showed that other species such as C. kefyr, C. lusitaniae, C. guilliermondi produced similar colonies. , These strains can be easily differentiated from C. glabrata as it doesn't produce even pseudo hyphae on cornmeal agar. Thus the combination of cornmeal agar and chrome agar can be used for early identification of C. glabrata. In our study only two strains of C. parapsilosis produced similar colonies. Many authors ,,,, have solved this confusion by giving special attention to the existence of pale edges on pink colonies. We could not appreciate this property for differentiation from other species.
All our five strains of C. tropicalis produced blue color on HiChrom candida agar with 100% sensitivity and specificity. In the year 1999, Willinger et al. showed sensitivity of 66.7% and specificity of 99.8% on chrome agar. Peng et al. showed 100% sensitivity and 78.8% specificity for C. tropicalis, while Yucesoy et al. showed 97% sensitivity and 100% specificity on chrome agar. Our results are in agreement with these studies.
The present study had four isolates of C. dubliniensis differentiated from C. albicans by the growth on tobacco agar (produced abundant terminal chlamydospores in clusters along with intercalary chlamydospores), no growth at 44oC and xylose assimilation tests. When these four isolates were tested on candida HiChrome agar, three isolates gave dark green colonies (fi shown in figure) and one showed pale green colonies similar to C. albicans. Thus for C. dubliniensis, HiChrom Candida agar showed low sensitivity (60%) and specificity (96.55%). It has been suggested that dark green coloration might be taken as an indication of the presence of C. dubliniensis but could not be used as criteria for identification. Jabra et al. and Pfaller et al. suggested the same color for C. dubliniensis. As the number of isolates of C. dubliniensis studied is low, it is difficult to conclude that dark green color on chrome agar can be used as a marker of C. dubliniensis.
We studied the previously identified candida strains on chromogenic medium, but this medium also carries the potential of improving identification of yeast, especially in mixed cultures. 
The use of chromogenic medium (HiChrom 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. This medium can be used as a selective isolation medium for direct identification of clinical isolates of Candida species in less than 48 hours. With typical color shown by Candida species, this medium may well replace cornmeal agar and conventional biochemical tests, including germ tube tests, sugar fermentation and sugar assimilation tests, used for direct identification of C. albicans.
Clinical microbiologists will be able to save time and costs for the diagnosis of fungi from clinical specimens specially blood cultures in which case early identification may requires change of antifungal agents as C. glabrata and C. krusei are inherently resistant to azoles.
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