Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 27, Num. 1, 2009, pp. 55-58

Indian Journal of Medical Microbiology, Vol. 27, No. 1, January-March, 2009, pp. 55-58

Brief Communication

Differentiation of Candida dubliniensis on chrom agar and Pal's agar

Raut SH, Varaiya A

Department of Microbiology, S.L.Raheja Hospital, Mahim, Mumbai-400 016
Correspondence Address:Department of Microbiology, S.L.Raheja Hospital, Mahim, Mumbai-400 016, amivaraiya@yahoo.com

Date of Submission: 15-Feb-2008
Date of Acceptance: 19-May-2008

Code Number: mb09013

Abstract

Keywords: Candida dublinensis, CHROM agar, Pal′s agar

Over the past decade, there has been a significant increase in the number of reports of systemic and mucosal Candida infections with non-albicans Candida species that cause a greater proportion of nosocomial infections. Implanted devices, particularly indwelling intravascular catheters, have been a significant risk factor associated with these infections. The potential clinical importance of species-level identification has been recognized as Candida species differ in the expression of putative virulence factors and antifungal susceptibility. ^[1],[2],[3]

Candida dubliniensis was originally isolated from patients with human immunodeficiency virus (HIV) infection and recurrent oral candidiasis. ^[3] C. dubliniensis was also identified in clinical specimens from diabetics and cancer patients . ^[4] C. dubliniensis and C. albicans share many morphological and physiological characteristics, such as germ tube positivity, similar biochemical patterns and the ability to form chlamydospores in rice extract agar and cornmeal agar . ^[5],[6] This close similarity between the two species has led to the misidentification of isolates of C. dubliniensis as C. albicans .^[5],[6] The most accurate differentiation between isolates of the two species is performed in reference laboratories with the use of molecular-based techniques such as PCR or DNA fingerprinting with repetitive sequence-containing DNA probes. However, these sophisticated techniques are not readily available in routine clinical microbiology laboratories. ^[6]

CHROM agar Candida medium is widely used for the identification of Candida spp., which develop colonies with distinguishable colours. The two species could be distinguished by culture on Staib agar, Pal′s agar, modified Pal′s agar and casein agar, where C. dubliniensis produces abundant chlamydospores and rough colonies. ^[3],[7]

There is paucity of Indian data on the incidence of C. dublineinsis infections in diabetic foot infections and cancer. In view of this, the aim of the present study was to differentiate various Candida species and to find out the most prevalent species from these patients using casein agar, CHROM agar, Pal′s agar and CHROM agar supplemented with Pal′s agar.

Materials and Methods

Fifty isolates of Candida were isolated from diabetic and cancer patients over a period of 4 months - April to July 2007. With universal safety precautions, samples were collected from patients in the intensive care unit and transported and processed in the laboratory without delay. Blood cultures were processed using an automated method with Versa Trek (Trivitron, Ohio, USA). Samples were cultured on brain heart infusion blood agar and Sabourauds dextrose agar (SDA). Identification of organisms was done by the standard laboratory technique. ^[8] Further, these isolates were streaked simultaneously on Casein agar, Pal′s agar and CHROM agar Candida supplemented with Pal′s agar.

C. albicans ATCC 10231, C. krusei ATCC 14243 and C. tropicalis ATCC 66029 were used as reference strains.

The following media were used for identification and differentiation of different Candida species:

1. CHROM agar Candida ^[2],[9]

CHROM agar Candida (Hi-Media, India) contains enzymatic substrates that are linked to chromogenic substrates, which when acted upon by different enzymes produced by Candida species results in colour variations useful for the presumptive identification of the yeasts.

2. Pal′s agar ^[10],[11]

Pal′s agar was freshly prepared with unsalted sunflower seeds (including kernels and shells) and used within 5 days to ensure consistent results. First, an aqueous extract of sunflower seeds was prepared by pulverizing 50 g of seeds in a blender for 5 min and then adding the ground seeds to 1 L of distilled water, followed by boiling for 30 min. Next, the seed extract was cooled and filtered and supplemented with glucose (1 g), KH ₂ PO ₄ (1 g) and creatinine (1 g). The pH was adjusted to 5.5, the volume was readjusted to 1 L and 15 g of agar (Difco) was added before the mixture was autoclaved at 110°C for 20 min. The cooled medium (45-55°C) was poured into 90-mm-diameter petri dishes.

Khan et al . reported that the husk of sunflower seeds can be used as a substitute for whole seeds in the medium without compromising its efficacy. ^[12]

3. CHROM agar supplemented with Pal′s agar ^[10]

CHROM agar Candida medium supplemented with Pal′s medium was prepared by mixing equal volumes of prepared CHROM agar Candida medium and Pal′s agar.

4. Casein agar ^[5],[6]

Casein agar has been traditionally used to study the decomposition of casein by aerobic actinomycetes and dematiaceous fungi. Recently, casein agar has been found to be a good medium to induce the production of chlamydospores by C. dubliniensis isolates, a feature that can differentiate C. dubliniensis from C. albicans .

Preparation: 10 g of skim milk was dissolved in 90 mL of distilled water and 3 g of agar was dissolved in 97 mL of distilled water. After autoclaving both solutions separately at 121°C for 15 min they were allowed to cool to 45-50°C, after which they were mixed together. After autoclaving, 25 mL of the media was dispensed into each 90 mm-diameter petri dish.

5. Additional tests for identification of C. dubliniensis :

(i) Growth at 45^oC ^[5]

All C. dubliniensis and C. albicans isolates were isolated on sterile SDA and incubated at 45^oC for 48-72 h. Unlike C. albicans, C. dubliniensis lacks the ability to grow at 45^ξC.

(ii) Ability to reduce 2, 3, 5-triphenyltetrazolium chloride (TTC) ^[13],[14]

Tetrazolium salts possess a reducible chromogenic tetrazolium ring. These compounds are not true dyes but, in the reduced state, the five-membered ring imparts colour to the molecule. TTC is reduced by mitochondrial dehydrogenase into a water-soluble formazan product that is measured spectrophotometrically.

0.5 mL of aqueous solution (0.2%w/v) of TTC dye prepared in phosphate-buffered saline was added to the tube containing 1 mL of 0.1 optical density adjusted Candida cell culture and incubated at 37^oC for 6-48 h.

Unlike C. dubliniensis , C. albicans lacks the ability to metabolically reduce TTC.

Results

The results were reported considering CHROM agar Candida as a primary medium for differentiating between various Candida species based on their colony colour and morphology (rough/smooth). Pal′s agar was considered as a secondary medium for differentiation.

CHROM agar Candida

Based on the colony colour developed on CHROM agar Candida , 50 isolates of Candida were differentiated as: C. dubliniensis (19), C. albicans (9), C. krusei (10), C. tropicalis (9) and C. glabrata (2). One isolate showed an intermittent green-coloured colony on CHROM agar, thereby making it difficult to differentiate between C. dubliniensis and C. albicans . However, this isolate was reported as C. dubliniensis as it failed to grow on SDA and was able to reduce the TTC dye. Maximum C. dubliniensis isolates were from blood cultures followed by respiratory specimens like broncoalveolar lavage from cancer patients whereas C. dubliniensis was isolated from diabetic foot infections followed by C. krusei .

Pal′s agar

Sixteen of the 19 isolates identified as C. dubliniensis exhibited a hyphal fringe after 48 h of incubation whereas two isolates exhibited a hyphal fringe after 4 days and one isolate failed to exhibit hyphal fringe. All the nine isolates identified as C. albicans failed to exhibit the hyphal fringe. The single Candida isolate that showed an intermittent green-coloured colony on CHROM agar produced fringe on Pal′s agar after 4 days of incubation and thus was reported as C. dubliniensis . Two isolates of C. krusei also exhibited hyphal fringe.

CHROM agar supplemented with Pal′s agar

Out of the 20 isolates of C. dubliniensis that developed fringe on Pal′s agar, 16 isolates revealed fringe on CHROM agar supplemented with Pal′s agar [Figure - 1] whereas none of the C. albicans developed fringe on the same media. Two C. krusei isolates that revealed fringe on Pal′s agar also developed fringe on this medium.

Casein agar

All the 20 C. dubliniensis isolates revealed abundant chlamydospores.

Addition test for identification

(i) Growth at 45^oC

All C. albicans isolates were found to grow at 45^oC whereas all C. dubliniensis isolates failed to do so.

(ii) TTC reduction test

All C. dubliniensis isolates were able to reduce the TTC dye whereas all the nine C. albicans isolates failed to do so.

Discussion

C. dubliniensis has been identified in clinical specimens recovered from diabetic and cancer patients with and without symptoms of oral candidiasis . Similarity in morphological and physiological characteristics between C. dubliniensis and C. albicans leads to misidentification of C. dubliniensis as C. albicans . As both these species differ in their antifungal susceptibility, it is essential to identify and differentiate them.

In our study, C. dubliniensis was found to be the most prevalent species (38%). When subcultured on Pal′s agar, 18 of these 19 isolates exhibited hyphal fringe surrounding the colonies. In a study by Mosaid et al. , all the C. dublinensis exhibited hyphal fringe on Pal′s agar. ^[11] Sahand et al. reported that 75% of the C. dublinensis isolates exhibited hyphal fringe. ^[10]

We observed that all the C. dubliniensis isolates, including the single-fringe-negative one, developed rough colonies on Pal′s agar whereas all the C. albicans isolates developed smooth colonies. Sahand et al. in their study reported that 96% of the C. dubliniensis isolates developed rough colonies and all C. albicans isolates developed smooth colony on Pal′s after a 48 h incubation at 30^oC. ^[10] However, in the study conducted by Mosaid et al. , all C. albicans as well as C. dubliniensis grew as smooth colonies on Pal′s agar. ^[11] Hence, Pal′s agar used alone is not a good differential medium.

Among the C. krusei (10) isolates that were identified in our study, two exhibited hyphal fringe on Pal′s agar. Mosaid et al. also reported formation of rough colony with a fringe by C. krusei isolates on Pal′s agar. ^[11]

Among the 20 fringe-positive C. dubliniensis isolated on Pal′s agar, only 16 revealed fringe on CHROM agar supplemented with Pal′s agar whereas none of the C. albicans isolates developed fringe on both media, which correlates well with the study conducted by Sahand et al . ^[11]

Although chlamydospore formation by C. dubliniensis was observed on both the media, there was a reduction in the pseudohyphae production on CHROM agar supplemented with Pal′s agar. CHROM agar helps in preliminary identification of Candida species whereas Pal′s agar, due to the presence of sunflower seed as its major ingredient, facilitates fringe production by C. dubliniensis . Ability to produce hyphal fringe on Pal′s agar is attributed to pseudohyphae production by C. dubliniensis . ^[10],[12] The volume of Pal′s agar when used in combination being 50% of the total volume, could be a probable reason for the reduction of pseudohyphae in this medium.

Unlike C. albicans isolates, all C. dubliniensis isolates failed to grow at 45^oC and were able to reduce TTC, which correlates well with the study conducted by Mosca et al. and Giammanco et al . ^[5],[13] Thus, these additional identification tests were time efficient, inexpensive and easy-to-use methods for differentiation of C. dubliniensis and C. albicans isolates.

Thus, in our study, C. dubliniensis was the most prevalent Candida species in both diabetics and cancer patients. CHROM agar Candida medium and Pal′s agar when used singly is time consuming whereas CHROM agar when supplemented with Pal′s agar makes rapid differentiation of both species and can be easily incorporated in routine microbiology laboratories.

References

1.	Baillie G, Douglas LJ. Iron limited biofilms of Candida albicans and their susceptibility to Amphotericin B. Antimicrob Agents Chemother 1998;42:2146-9.   Back to cited text no. 1
2.	Murray MP, Zinchuk R, Larone DH. CHROMagar Candida as the sole primary medium for isolation of yeasts and as a source medium for the rapid-assimilation of trehalose test. J Clin Microbiol 2005;43:1210-2.   Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.	Marot-Leblond A, Beucher B, David S, Nail-Billaud S, Robert R. Development and evaluation of a Rapid Latex Agglutination test using a monoclonal antibody to identify Candida dubliniensis colonies. J Clin Microbiol 2006;44:138-42.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.	Palacheck I, Strahilevi J, Sullivan D, Donnelly S, Salkin IF, Coleman DC. Recovery of Candida dubliniensis from non human immunodeficiency virus infected patient in Israel. J Clin Microbiol 2000;38:170-4.  Back to cited text no. 4
5.	Mosca C, Moragues MD, Llovo J, Al Mosaid A, Coleman DC, Ponton J. Casein agar: A useful medium for differentiating Candida dubliniensis from Candida albicans . J Clin Microbiol 2003;41:1259-62.  Back to cited text no. 5
6.	Alves SH, de Loreto ES, Linares CE, Silverira CP, Scheid LA, Pereira DI, et al . Comparison among Tomato juice agar with other three media for differentiation of C.dubliniensis from C.albicans . Rev Inst Med trop S Paulo 2006;48:119-21.   Back to cited text no. 6
7.	Staib P, Morschhδuser J. Chlamydospore formation on Staib agar as a species-specific characteristic of Candida dubliniensis . Mycoses 1999;42:521-4.  Back to cited text no. 7
8.	Forbes BA, Sham DF, Weissfeld AS. Laboratory methods in basic mycology, Chapter 65. In: Bailey and Scott's diagnostic microbiology. 10 th ed. St. Louis: The CV Mosby Company; 1998. p. 947-9.   Back to cited text no. 8
9.	Horvath LL, Hospenthal DR, Murray CK, Dooley DP. Direct isolation of Candida spp. from blood cultures on the Chromogenic medium CHROMagar Candida . J Clin Microbiol 2003;41:2629-32.  Back to cited text no. 9
10.	Sahand IH, Moragues MD, Eraso E, Villar-Vidal M, Quindós G, Pontón J. Supplementation of CHROMagar Candida medium with Pal's medium for rapid identification of Candida dubliniensis . J Clin Microbiol 2005;43:5768-70.   Back to cited text no. 10
11.	Al Mosaid A, Sullivan DJ, Coleman DC. Differentiation of Candida dubliniensis from Candida albicans on Pal's agar. J Clin Microbiol 2003;41:4787-9.   Back to cited text no. 11  [PUBMED]  [FULLTEXT]
12.	Khan ZU, Ahmad S, Mokaddas E, Al-Sweih N, Chandy R. Sunflower seed husk agar: A new medium for the differentiation of Candida dubliniensis from Candida albicans . Indian J Med Microbiol 2005;23:182-5.  Back to cited text no. 12  [PUBMED]
13.	Giammanco G, Pizzo' G, Pecorella S, Distefano S, Pecoraro V, Milici M. Identification of Candida dubliniensis among oral yeast isolates from an Italian population of human immunodeficiency virus-infected (HIV+) subjects. Oral Microbiol Immunol 2002;17:89-94.  Back to cited text no. 13
14.	Betts RP, Bankes P, Banks JG. Rapid Enumeration of viable microorganisms by staining and direct microscopy. Lett Appl Microbiol 1989;9:199-202.  Back to cited text no. 14

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