|Year : 2015 | Volume
| Issue : 5 | Page : 69-73
Performance of chromogenic media for Candida in rapid presumptive identification of Candida species from clinical materials
MV Pravin Charles1, Arunava Kali1, Noyal Mariya Joseph2
1 Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Puducherry, India
2 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
|Date of Submission||20-Sep-2014|
|Date of Acceptance||03-Nov-2014|
|Date of Web Publication||02-Jun-2015|
Dr. Arunava Kali
Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Puducherry - 607 402
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: In perspective of the worldwide increase in a number of immunocompromised patients, the need for identification of Candida species has become a major concern. The development of chromogenic differential media, introduced recently, facilitate rapid speciation. However, it can be employed for routine mycology workup only after an exhaustive evaluation of its benefit and cost effectiveness. This study was undertaken to evaluate the benefit and cost effectiveness of chromogenic media for speciation of Candida clinical isolates. Materials and Methods: Sputum samples of 382 patients were screened for the presence of Candida spp. by Gram stain and culture on sabouraud dextrose agar. Candida species were identified using Gram stain morphology, germ tube formation, cornmeal agar with Tween-80, sugar fermentation tests and morphology on HiCrome Candida differential agar. All the Candida isolates were inoculated on HiCrome Candida agar (HiMedia, Mumbai, India). Results: The sensitivity and specificity of HiCrome agar for identification of Candida albicans were 90% and 96.42%, respectively whereas sensitivity and specificity of carbohydrate fermentation test were 86.67% and 74.07%, respectively. Sensitivity and specificity values of HiCrome agar for detection of C. albicans, Candida parapsilosis and Candida glabrata were above 90%. Conclusions: We found HiCrome agar has high sensitivity and specificity comparable to that of the conventional method. In addition, use of this differential media could significantly cut down the turnaround time as well as cost of sample processing.
Keywords: Candida, Chromogenic differential agar, Presumptive identiﬁcation
|How to cite this article:|
Pravin Charles M V, Kali A, Joseph NM. Performance of chromogenic media for Candida in rapid presumptive identification of Candida species from clinical materials. Phcog Res 2015;7, Suppl S1:69-73
|How to cite this URL:|
Pravin Charles M V, Kali A, Joseph NM. Performance of chromogenic media for Candida in rapid presumptive identification of Candida species from clinical materials. Phcog Res [serial online] 2015 [cited 2019 May 26];7, Suppl S1:69-73. Available from: http://www.phcogres.com/text.asp?2015/7/5/69/150528
| Introduction|| |
Opportunistic fungal infections are emerging challenge in perspective of the worldwide increase in a number of immunocompromised patients.  While Candida albicans remains the leading pathogen, other Candida species have established their pathogenic role with the development of antifungal resistance.  Consequently, identification of Candida to species level has become a routine practice. The conventional speciation scheme is based on phenotypic characteristics. Microscopic morphology of yeast cells in Gram stained smear, growth on cornmeal agar, demonstration of germ tube and colony morphology on sabouraud dextrose agar (SDA) are essential components of conventional identification scheme. , Likewise, carbohydrate metabolism in terms of both carbohydrate assimilation and fermentation has been employed for Candida species identification.  Except germ tube test, which is used to provisionally identify C. albicans and Candida dubliniensis, none of these tests provides quick interpretation. Molecular methods for Candida species differentiation are considered gold standard with high sensitivity, specificity and short turnaround time.  However, these tests require proficient personnel and expensive instruments, which are often not affordable in resource poor setups. Several chromogenic differential media have been introduced recently to facilitate rapid speciation. ,, Several authors have utilized it for simultaneous isolation and identification of Candida species directly from clinical samples. , These are particularly helpful in cases where multiple yeast species are present in the sample.  However, discrepancies in interpretation may arise owing to subjective variation that is likely to compromise quality of reports as well as patient care. Therefore, it can be employed for routine mycology workup only after an exhaustive evaluation of its benefit and cost effectiveness.
| Materials and Methods|| |
This study was carried out in a tertiary care hospital in South India over a period of 9 months (from June, 2010 to March, 2011). Sputum samples of 382 in-patients and out-patients were screened for the presence of Candida spp. by Gram stain and culture on SDA. Yeast like colonies on SDA after 24 and 48 h of incubation at 37°C was subjected to Grams stain, germ tube test, urease test, fermentation test for 2% glucose, maltose, lactose and sucrose along with demonstration of chlamydospore, blastospores and pseudohyphae on cornmeal agar to differentiate Candida from other yeasts and to identify them up to species level.
All the Candida isolates were inoculated on HiCrome Candida agar (HiMedia, Mumbai, India), a commercially available chromogenic agar for Candida (CAC). HiCrome agar colony characteristics were interpreted as per manufacturer's specifications by two personnel to avoid subjective variation. Light green colonies were identified as C. albicans isolates, whereas C. dubliniensis, Candida tropicalis, Candida krusei were considered in case of dark green, blue to purple and purple colonies respectively. Candida parapsilosis and Candida glabrata were identified by cream to white colonies on HiCrome agar.
On cornmeal agar, pseudohyphae with plenty of spherical blastoconidia at the constriction sites and thick walled large round terminal single chlamydospores were identified as C. albicans and isolates displaying pseudohyphae with clusters of terminal chlamydospores were considered as C. dubliniensis.  C. parapsilosis strains were recognized by Christmas tree appearance due to the presence of branched pseudohyphae, which gradually become smaller distally. Isolates showing long branching abundant pseudohyphae with narrow sterile apex and ellipsoid balastoconidia were labelled as C. tropicalis, whereas, clusters of small, budding blastospores without pseudohyphae was very typical of C. glabrata. 
Candida albicans ATCC 611098, C. tropicalis ATCC 13803 and C. parapsilosis ATCC 22019 strains were used for quality control. Results of HiCrome agar and carbohydrate fermentation were compared with conventional species identification based on Grams stain, germ tube and cornmeal agar test. All findings were entered in MS Excel datasheet and on completion of the study data were statistically analyzed for sensitivity, specificity, positive and negative P value.
| Results|| |
A total of 87 strains of Candida was recovered from 382 sputum samples during the study period. C. albicans (68.96%) was most common species, followed by C. tropicalis (17.24%), C. glabrata (6.89%), C. parapsilosis (4.59%), C. krusei (1.15%) and C. dubliniensis (1.15%). The distribution of Candida species is listed in [Table 1].
In this present study, both SDA and HiCrome agar were employed as primary plating medium for sputum samples. There were no major discrepancies between SDA and HiCrome agar in growth rate or colony size for primary isolation of Candida. Both media supported growth of all 87 Candida isolates. After 18-24 h of incubation, colonies of most Candida isolates on HiCrome agar started developing characteristic colors, which became more prominent after 48 h. However, false positive and false negative results were also seen. Different colony colors displayed by Candida isolates on HiCrome agar is given in [Table 2]. Results of HiCrome agar and carbohydrate fermentation are compared with conventional identification in [Table 3] and [Table 4] respectively.
|Table 3: Performance of HiCrome agar for identification of different Candida species |
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|Table 4: Performance of carbohydrate fermentation test for identification of different Candida species |
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| Discussion|| |
Until recent years, laboratory processing and identification of non-albicans Candida (NAC) from clinical specimen was in a dilemma. In most laboratories, NAC isolates were reported based on germ tube test only, without species level identification and antifungal susceptibility test.  However, NAC has emerged as an opportunistic pathogen with potential to cause life-threatening, invasive infections that are often not amenable to anti-fungal therapy.  Not only HIV, the development of advanced therapeutic modalities for the treatment of cancers, infectious diseases and immunosuppressive drugs has caused a major shift in the epidemiology of opportunistic fungal infections.  There are frequent reports of multi-species yeast infections and azole resistance among NAC. , Although C. albicans remains sensitive to most conventional and newer anti-fungal agents, the emergence of resistance to these agents in NAC is likely to limit the treatment options. ,,, Candida species is known to colonize respiratory tract, especially in patients with pulmonary tuberculosis and in patients receiving immunosuppressive or long-standing anti-microbial therapy. , Although Candida species are frequently isolated from sputum, their significance as a possible pulmonary pathogen is often doubtful. We found that C. albicans (n = 60) is the most common isolate followed by C. tropicalis (n = 15), C. glabrata (n = 6) and C. parapsilosis (n = 4) [Table 1]. This is in accordance with other Indian studies. Latha et al. found C. albicans was the predominant (45.01%) respiratory isolates of Candida and C. tropicalis, C. glabrata, C. parapsilopsis and C. krusei constituted 19.95%, 16.54%, 13.14%, and 5.10% respectively.  In another study, C. tropicalis (9.1%), C. pseudotropicalis (6.06%), and C. krusei (6.06%) have been reported. 
With respect to increasing clinical significance of NAC, isolation of Candida from clinical specimen warrants rapid identification to species level. The concept of developing chromogenic differential media for Candida came from the observation that C. albicans produces an enzyme N-acetyl-galactosaminidase, which can break down chromogenic hexosaminidase substrates incorporated in a medium.  Although chromogenic differential media for Candida were commercially available since 1990s,  these were not included in routine mycology practice and were not considered a substitute of convention speciation procedures. It has limitations like subjective variation in interpretation, alteration of color with incubation and inability to differentiate certain Candida species. Authors have reported difficulties in differentiation of C. albicans from C. dubliniensis.  C. dubliniensis is closely related to C. albicans. Both species produce the germ tube, chlamydospores and grow on CAC as green colonies. Although C. dubliniensis colonies are dark green unlike light green colonies of C. albicans, this difference may be inadequate for primary identification and often lost after serial subculture. , Hospenthal et al. reported all C. dubliniensis strains (n = 17) in their study had colonies identical to C. albicans on CAC. In this study, we isolated one C. dubliniensis, which had typical dark green colonies. However, two C. albicans and one C. tropicalis strain also displayed similar colony morphology on HiCrome agar. Sahand et al. suggested use of CAC supplemented with Pal's medium for better species discrimination.  Likewise, Candida kefyr, C. parapsilosis and C. glabrata produce identical cream to white growth on CAC, which mandates the use of other tests for identification. Cornmeal agar has a vital role in this scenario. C. glabrata can be easily differentiated from other two species by small, budding blastospores and absence of pseudohyphae, whereas C. kefyr has elongated, cylindrical blastospores in log jam appearance and C. parapsilosis displays Christmas tree appearance due to presence of branched pseudohyphae.  Bishop et al. found that Candida rugosa, Candida firmetaria and C. inconspicua might be confused with C. krusei and apart from these three common species, C. bracarensis, C. nivariensis and C. norvegensis also produced white colonies on CAC, which required molecular methods to differentiate them.  In our study, 10 isolates displayed white to cream colonies. Among these, six isolates were C. glabrata and four C. parapsilosis. Interestingly, no false positive and false negative result seen in white colony producing isolates.
We noticed HiCrome agar had high sensitivity and specificity compared to carbohydrate fermentation for all four common Candida species. C. parapsilosis and C. glabrata, in particular, had more false negative results in fermentation tests, resulting in lower sensitivity. In case of C. albicans, the sensitivity and specificity of HiCrome agar were 90% and 96.42%, respectively whereas sensitivity and specificity of carbohydrate fermentation test were 86.67% and 74.07%, respectively. This finding is in accordance with other studies. Yucesoy et al. evaluated performance of CAC and BIGGY agar and found CAC was superior to BIGGY agar, displaying above 90% sensitivity and 100% specificity for C. albicans, C. tropicalis, C. glabrata and C. krusei isolates.  In another study, sensitivity of CAC for C. albicans, C. tropicalis and C. krusei was 99%, 98% and 100%, respectively.  CAC was found by several authors as a simple method, which offers rapid, reliable identification of commonly isolated Candida species based on colony characteristics after 48 h incubation. However, color development may be unstable and incomplete within 24 h. Willinger et al. reported Candida ID medium had much higher sensitivity (96.8%) compared to CAC (49.6%), especially for rapid identification of C. albicans after 24 h of incubation.  In comparison with CAC, sugar fermentation test has several drawbacks. Prolonged incubation and a high number of false positive results from easy contamination of sugar media are common limitations. Broths containing 2% glucose, maltose, lactose and sucrose have been used in this study. In fermentation test, C. tropicalis had the highest sensitivity (96.8%) followed by C. albicans, C. parapsilosis and C. glabrata.
Although, the cost of 1 l of CAC is almost five times more than the cost of SDA,  use of CAC for primary isolation may preclude the cost of SDA, cornmeal agar, sugar media and other reagents. If samples are inoculated on either half of CAC plate, it will be further reduced the cost of sample processing. CAC is particularly important in multi-species yeast infections, where subculture on plating medium may be required to isolate different Candida strains in pure form for identification.  In this scenario, CAC offers rapid, direct identification after 48 h incubation, without the need to subculture. The main drawback of this study was a limited number of samples. Therefore, conclusions may not be representative and may require further studies for confirmation.
| Conclusion|| |
HiCrome agar showed no major discrepancies in growth potential, colony characters and growth rates for primary isolation from sputum samples as compared to SDA. With few exceptions, the colony colors developed on HiCrome agar were stable and distinct for common Candida species. However, stable color development may take 48 h. We found results sugar fermentation tests were variable and had poor sensitivity, especially for detection of C. parapsilosis and C. glabrata. Based on these findings, we recommend routine use of HiCrome agar in mycology laboratories. Use of this differential media can significantly cut down the turnaround time as well as cost of sample processing.
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[Table 1], [Table 2], [Table 3], [Table 4]