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Journal of Clinical Microbiology, November 2005, p. 5768-5770, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5768-5770.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Supplementation of CHROMagar Candida Medium with Pal's Medium for Rapid Identification of Candida dubliniensis

Ismail H. Sahand,1 María D. Moragues,2 Elena Eraso,1 María Villar-Vidal,1 Guillermo Quindós,1 and José Pontón1*

Departamento de Inmunología, Microbiología, y Parasitología, Facultad de Medicina y Odontología,1 Departamento de Enfermería I, Universidad del País Vasco, Apartado 699, E-48080 Bilbao, Vizcaya, Spain2

Received 21 April 2005/ Returned for modification 4 July 2005/ Accepted 15 August 2005


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ABSTRACT
 
CHROMagar Candida medium is used for the isolation and identification of Candida species, but it does not differentiate Candida albicans from Candida dubliniensis. This differentiation can be achieved by using Pal's agar, which cannot be used in primary isolation. We have combined both media to obtain a new medium that can be used for the isolation and identification of C. dubliniensis in primary cultures.


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TEXT
 
Candida dubliniensis and Candida albicans are two fungal species that show very close phenotypic characteristics that make the discrimination between them difficult. Differentiation of both species can be achieved by a limited number of both phenotypic and genotypic tests (17, 18). While molecular techniques remain the most reliable methods for identification of C. dubliniensis (17) and its described genotypes (3, 5), they are not available in many mycology laboratories. Therefore, the search for less-sophisticated, rapid, and accurate techniques for differentiating C. dubliniensis from C. albicans still continues. A number of phenotypic tests to differentiate both Candida species, including growth at 42 to 45°C (14), chla-mydospore production in different media (1, 7, 16), colony color development in differential media such as CHROMagar Candida medium (6, 17) or Candida ID medium (15), differential carbohydrate assimilation (4, 13), and an indirect immunofluorescence reaction with a specific anti-C. dubliniensis serum (2), have been described previously.

CHROMagar Candida medium is a widely used medium for the identification of Candida spp. which develop colonies with distinguishable colors. This medium allows the presumptive identification of C. albicans, Candida tropicalis, and Candida krusei (8, 12). Some authors (6, 9, 17) have observed that C. albicans and C. dubliniensis may show colonies with different shades of green; however, the color intensity is not restrictive for each individual species, and differentiation of both species based on the colony color is difficult (9, 19).

Pal's agar was originally developed for the identification of Cryptococcus neoformans (10, 11) and has been used for differentiation between C. dubliniensis and C. albicans. In this medium, C. dubliniensis forms rough colonies and chlamydo-spores, while C. albicans shows smooth colonies and does not produce chlamydospores (1). However, other species of Candida share the colony morphology characteristics of both species. In the present study, we supplemented CHROMagar Candida agar with Pal's agar to improve the differentiation of C. dubliniensis from C. albicans achieved with each culture medium individually.

Eleven reference strains were used as controls in this study. They were obtained from the National Collection of Pathogenic Fungi (NCPF), Bristol, United Kingdom, the Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands, the American Type Culture Collection (ATCC), Manassas, Va., and the Colección Española de Cultivos Tipo (CECT), Valencia, Spain, and included C. dubliniensis NCPF 3949, CBS 2747, CBS 8500, CBS 8501 and CECT 11473; C. albicans NCPF 3153; C. tropicalis NCPF 3111; Candida glabrata NCPF 3203; C. krusei ATCC 6258; Candida guilliermondii NCPF 3099; and Candida parapsilosis ATCC 22019. The remaining 131 yeast strains were clinical isolates of oral, vaginal, sputum, or blood origin previously isolated at our laboratory and included 95 C. dubliniensis strains, 29 C. albicans strains, 2 C. krusei strains, 2 C. glabrata strains, 2 C. guilliermondii strains, and 1 C. parapsilosis strain. Clinical isolates were identified by their carbohydrate assimilation patterns on ID 32C strips (bioMérieux, Marcy-l'Etoile, France), growth at 42 to 45°C, chlamydospore production in casein agar (7), and indirect immunofluorescence with a specific anti-C. dubliniensis serum (2). Genotypic identification of C. dubliniensis and C. albicans isolates was performed by PCR with specific primers. The four genotypes of C. dubliniensis were determined according to methods described previously by Brena et al. and Gee et al. (3, 5). Some C. dubliniensis and C. albicans isolates were sequenced to verify their identity. In all cases, the identity was the same as that obtained by other tests. For some experiments, 10 fresh oral swabs were directly plated onto the different culture media tested.

Pal's agar was prepared by adding 50 g unsalted powdered sunflower seeds to l liter of distilled water, boiling for 30 min, and filtering through cheesecloth. This extract was supplemented with creatinine (1 g), glucose (1 g), and KH2PO4 (1 g). After pH adjustment to 5.5, agar (15 g/liter) was added, and the mixture was autoclaved at 110°C for 20 min. The cooled medium (45 to 55°C) was poured into 90-mm-diameter petri dishes (25 ml/plate). CHROMagar (Paris, France) Candida medium was prepared according to the manufacturer's instructions as follows: 47.7 g of the powder medium was slowly dispersed in 1 liter of purified water and brought to a boil by repeated heating until complete fusion of agar grains. Medium was poured into petri dishes as described above. CHROMagar Candida medium supplemented with Pal's medium was prepared by mixing equal volumes of prepared CHROMagar Candida medium and Pal's agar. After stirring well, the medium was poured into 90-mm-diameter petri dishes. Several concentrations of each medium were tested, and the best results for discrimination of C. albicans and C. dubliniensis isolates were obtained by mixing equal volumes of both media at their normal strength. Strains were grown simultaneously on Pal's agar, CHROMagar Candida medium, and CHROMagar Candida medium supplemented with Pal's agar. Inoculum of collection strains was obtained from cells grown on Sabouraud agar for 48 h at 37°C. Plates were incubated at 30°C (Pal's agar and CHROMagar Candida medium supplemented with Pal's agar) or 37°C (CHROMagar Candida medium), and colony characteristics were recorded after 24, 48, and/or 72 h of incubation by two different observers. Chlamydospore formation was detected microscopically using lactophenol cotton blue (Merck, Darmstadt, Germany) as a stain.

Table 1 shows colony color and morphology as well as chlamydospore formation by C. albicans and C. dubliniensis isolates when isolates were grown on different media. Ninety-six out of 100 C. dubliniensis isolates formed rough colonies on Pal's agar, 75% showed hyphal fringe, and 93% produced chlamydospores. On the contrary, all C. albicans strains formed smooth colonies without hyphal fringe and failed to produce chlamydospores. All three C. krusei isolates and one C. parapsilosis isolate formed rough colonies. The remaining Candida species formed smooth colonies, and none of them produced chlamydospores. Fifty-eight out of 100 C. dubliniensis strains grew as dark green colonies on CHROMagar Candida medium, and the remaining 42% grew as lighter green colonies. Thirteen out of 30 (43.3%) C. albicans isolates formed light green colonies, and the remaining 56.6% formed green and dark green colonies. The other Candida species developed different colors. Ninety-six out of 100 C. dubliniensis isolates grew as rough colonies on CHROMagar Candida medium supplemented with Pal's agar (Fig. 1), 75 isolates formed hyphal fringe, and 93 produced chlamydospores. Ninety-six isolates grew as bluish green colonies that were easy to distinguish from C. albicans colonies. On the contrary, C. albicans isolates formed smooth light green colonies (Fig. 1) and failed to produce either hyphal fringe or chlamydospores. Other Candida species formed smooth or rough colonies but with a distinguishable color, and none of them was able to form chlamydospores in this medium.


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  		TABLE 1. Colony aspects of and chlamydospore formation by C. albicans and C. dubliniensis isolates on different culture media



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  		FIG. 1. Colonies of different species of Candida after growing for 48 h at 37°C in CHROMagar Candida medium supplemented with Pal's agar.

The colony morphology (rough/smooth) and chlamydospore production results for C. dubliniensis and C. albicans isolates were the same for Pal's agar and CHROMagar Candida medium supplemented with Pal's agar. On the contrary, colony color was not definitive in CHROMagar Candida medium, as C. albicans and C. dubliniensis isolates showed the three intensities of green. However, when this medium was supplemented with Pal's agar, C. albicans isolates appeared as light green, smooth colonies, while C. dubliniensis showed a distinguishable bluish green color which was accompanied in most cases (96%) by the production of rough colonies, making differentiation of both species much easier. Other species of Candida that were assayed in this study showed rough or smooth colonies in CHROMagar Candida medium supplemented with Pal's agar; however, they never appeared as green or bluish green colonies, and in addition, they never developed chlamydospores, making their misidentification as C. albicans or C. dubliniensis highly unlikely. Interestingly, C. tropicalis and C. krusei still retained a distinguishable blue and pink-and-rough colony aspect, respectively, that allowed their identification on CHROMagar Candida medium supplemented with Pal's agar, even when used as a primary isolation medium for the fresh oral specimens assayed.

The differentiation between C. dubliniensis and C. albicans could be achieved by the observation of their colony aspects and color in CHROMagar Candida medium supplemented with Pal's agar with 92% sensitivity and 100% specificity. The remaining C. dubliniensis strains (8%) could presumptively be identified either by the rough aspect or the bluish green color of their colonies.

The C. dubliniensis reference strains from culture collections gave the expected results. The four different genotypes of C. dubliniensis were all positive in CHROMagar Candida medium supplemented with Pal's agar, growing as typical C. dubliniensis colonies.

In conclusion, CHROMagar Candida medium supplemented with Pal's agar allows the rapid and easy differentiation between C. albicans and C. dubliniensis on the basis of their colony characteristics. When used as a primary isolation medium, CHROMagar Candida medium supplemented with Pal's agar allows the identification of C. dubliniensis even in mixed cultures containing C. albicans, C. tropicalis, C. krusei, and/or C. glabrata.


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ACKNOWLEDGMENTS
 
This investigation was supported by grants 9/UPV 0093.327-13550/2001 from the Universidad del País Vasco and Etortek IE019, subproject DIAMOLFUN, from the Departamento de Industria, Comercio y Turismo from the Basque Government.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Universidad del País Vasco, Apartado 699, E-48080 Bilbao, Vizcaya, Spain. Phone: 34 94-601-2855. Fax: 34 94-601-3400. E-mail: jose.ponton{at}ehu.es. Back


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Journal of Clinical Microbiology, November 2005, p. 5768-5770, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5768-5770.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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