Usefulness of Candida ID2 agar for the presumptive identification of Candida dubliniensis

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1 Medical Mycology November 2006, 44, Usefulness of Candida ID2 agar for the presumptive identification of Candida dubliniensis ELENA ERASO*, ISMAIL H. SAHAND*, MARÍA VILLAR-VIDAL*, CRISTINA MARCOS*, MARÍA DOLORES MORAGUES$, LUCILA MADARIAGA*, JOSÉ PONTÓN* & GUILLERMO QUINDÓS* *Laboratorio de Micología Médica, Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Universidad del País Vasco-Euskal Herriko Unibertsitatea, Bilbao, Spain, and $Departamento de Enfermería I, Universidad del País Vasco-Euskal Herriko Unibertsitatea, Bilbao, Spain CHROMagar Candida and Candida ID2 are widely used for the isolation and presumptive identification of Candida spp. based on the color of the colonies on these two media. We have studied the usefulness of these chromogenic media for differentiating Candida dubliniensis from Candida albicans isolates. One hundred isolates of C. dubliniensis and 100 C. albicans isolates were tested on Candida ID2, CHROMagar Candida (CHROMagar), and CHROMagar Candida reformulated by BBL. CHROMagar Candida and CHROMagar Candida BBL did not allow a clear differentiation of the two species based upon the shade of the green color of C. dubliniensis colonies. However, on Candida ID2, all C. dubliniensis isolates produced turquoise blue colonies whereas 91% of C. albicans colonies were cobalt blue. The sensitivity and the specificity for differentiating between C. dubliniensis fromc. albicans on Candida ID2 were 100% and 91%, respectively; whereas on CHROMagar Candida these values were 63% and 89% and on CHROMagar Candida BBL they were 18% and 98%. Candida ID2 agar provides a simple and accurate laboratory approach for the identification and differentiation of C. dubliniensis on the basis of the colony color. Keywords Candida dubliniensis, Candida albicans, chromogenic agar, candida ID2, CHROMagar candida, identification Introduction Candida spp. cause a wide range of human infections ranging from superficial mucosal diseases to lifethreatening invasive candidiasis. The most common cause of candidiasis is Candida albicans, but the reports of infections caused by non-candida C. albicans spp., such as Candida parapsilosis and Candida glabrata are increasing in the literature. Candida dubliniensis is an opportunistic yeast-like pathogen Received 30 March 2006; Accepted 30 May 2006 Correspondence: Guillermo Quindós, Laboratorio de Micología Médica, Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Universidad del País Vasco-Euskal Herriko Unibertsitatea, Apartado 699, E Bilbao, Spain. Tel: / ; Fax: / ; guillermo.quindos@ehu.es associated with colonization and infection of the oral cavity of HIV-infected patients [1 3]. In a minor frequency, C. dubliniensis has also been recovered from urinary, vaginal, respiratory, and fecal specimens from immunocompromised and immunocompetent persons [2,4]. Moreover, this species is responsible for approximately 2% of cases of candidemia in the United States [5] and Europe [6]. The vast majority of C. dubliniensis isolates are susceptible to all of the commonly used antifungal agents. However, reduced susceptibility to different antifungal agents has been observed [7 10] and azole resistance can be induced in vitro [11]. Differentiation of C. dubliniensis and C. albicans can be achieved by both phenotypic and genotypic tests. Phenotypic tools include growth at C [12], chlamydospore production in different media [13,14], colony color development on CHROMagar Candida [15], differential carbohydrate 2006 ISHAM DOI: /

2 612 Eraso et al. assimilation patterns [12] and reactivity with a specific polyclonal anti-c. dubliniensis antibody [16]. Molecular techniques remain the most reliable methods for identification and genotyping of C. dubliniensis [2,17], but they are not available in many diagnostic laboratories. Therefore, it is necessary to find simple, rapid and accurate techniques for differentiating the two species. This study was designed to test the hypothesis that C. dubliniensis and C. albicans isolates can be differentiated on the basis of colony color on chromogenic media, such as CHROMagar Candida and Candida ID2. Materials and methods Three chromogenic media were compared; Candida ID2 (biomérieux, Marcy l Étoile, France), CHROMagar Candida (CHROMagar, Paris, France), and CHROMagar Candida reformulated by BBL (Becton Dickinson, BBL, Cockeysville, MD). One hundred isolates and reference strains of C. dubliniensis and 100 of C. albicans were tested on these chromogenic media. Isolates had been previously identified by conventional mycological methods, such as the germ tube test in serum, microscopic morphology, chlamydospore production in corn meal agar with Tween 80, and carbon source assimilation with the commercial kit ID 32C (biomérieux, Marcy l Étoile, France). Reference strains were obtained from the National Collection of Pathogenic Fungi (NCPF, Bristol, UK), 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 serotype A NCPF 3153 and C. albicans serotype B NCPF 3156, ATCC 26555, ATCC 64548, ATCC 76615, ATCC 90028, and ATCC The identification of all C. dubliniensis isolates was confirmed by polymerase chain reaction (PCR) with specific primers [18]. Prior to testing, each strain was cultured for 2448 h at 378C on Sabouraud glucose agar (Difco, St. Louis, MO) to ensure viability. Experimental plates were incubated at 378C and independently read for visual colony color by three investigators after 24 and 48 h of incubation. The ability to differentiate between C. dubliniensis and C. albicans on the basis of colony color on the three chromogenic media was compared and analyzed in terms of sensitivity [number of true positives/ (number of true positives/number of false negatives)], specificity [number of true negatives/(number of true negatives/number of false positives)], positive predictive value [number of true positives/(number of true positives/number of false positives)], negative predictive value [number of true negatives/(number of true negatives/number of false negatives)], and efficiency [(number of true positives/number of true negatives)/ (number of true positives/number of true negatives/ number of false positives/number of false negatives)]. Data were statistically analyzed by the Chi square test. Results Table 1 and Fig. 1 show the results of the colony growth of C. dubliniensis and C. albicans after 48 h of incubation at 378C on the chromogenic media. All isolates grew well on both media after 24 h of incubation, but yeast colonies were smaller on CHRO- Magar Candida. After 48 h, all C. dubliniensis isolates produced turquoise blue colonies on Candida ID2 (ten of them with very light turquoise blue colonies); whereas 91 C. albicans isolates grew as blue cobalt colonies (the other nine isolates developed turquoise blue colonies (Chi square, p B/0.001). On CHROMagar Candida, 63 C. dubliniensis and 11 C. albicans gave dark green colonies (Chi square, p B/0.001) whereas on CHROMagar Candida BBL, the colony color of 18 C. dubliniensis and two C. albicans isolates was dark green. The sensitivity and the specificity for differentiating between C. dubliniensis and C. albicans on Candida ID2 chromogenic medium were 100% and 91%, respectively; whereas on CHROMagar Candida these values were 63% and 89% and on CHROMagar Candida BBL they were 18% and 98% (Table 1). Discussion For a clinical microbiology laboratory it is essential to accurately identify Candida spp. isolated from clinical samples. Due to the phenotypic similarities between C. dubliniensis and C. albicans a correct identification can be problematic. A large number of phenotypic and genotypic tests have been developed for investigating the prevalence of C. dubliniensis in the human population [1,2]. Very few of these phenotype-based identification tests are 100% accurate. More accurate discrimination between C. albicans and C. dubliniensis has been achieved by the use of improved carbohydrate assimilation test databases [19] and the application of assays based on the colony morphology when grown on different supplemented media (e.g. Birdseed, Casein, Sunflower seed, Tobacco or Tomato agars) [14,2023]. However, these non-commercial media are not readily available in many laboratories and accurate 2006 ISHAM, Medical Mycology, 44,

3 Presumptive identification of Candida dubliniensis 613 Table 1 Potential diagnostic values for discriminating Candida dubliniensis from Candida albicans Species Candida dubliniensis (n/100) Candida albicans (n/100) Candida ID2 Turquoise blue colony Cobalt blue colony 0 91 Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Efficiency (%) CHROMagar Candida Dark green colony Other shades of green colony Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Efficiency (%) CHROMagar Candida BBL Dark green colony 18 2 Other shades of green colony Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Efficiency (%) identification may require incubation periods of between two and seven days. Recently, it has been reported that very rapid and accurate discrimination Fig. 1 Colonies of Candida albicans (a) and Candida dubliniensis (b) on Candida ID2. between C. albicans and C. dubliniensis can be achieved using a latex agglutination test [24,25]. Moreover, many studies have shown that the most definitive methods of identifying C. dubliniensis are those based on differential amplification of species-specific DNA sequences using PCR [2,10,17,18,26,27]. However, most of these tests need the isolation of the etiologic agent in pure culture which can result in a h delay in identification. CHROMagar Candida and Candida ID2 are widely used for the presumptive identification of different species of Candida based upon differential colony colors. On CHROMagar Candida some authors [15,28] have observed that C. albicans and C. dubliniensis may show colonies of different shades of green. However, we have observed using two different brands of CHROMagar Candida that the colony color intensity was not restrictive for the individual species and differentiation of C. albicans and C. dubliniensis was difficult. This inaccuracy has been described by other authors using CHROMagar Candida [29]. In contrast, in these studies, differentiation between both species was established with a high certainty on Candida ID2 what with all C. dubliniensis isolates produced turquoise blue smooth colonies, while most C. albicans 2006 ISHAM, Medical Mycology

4 614 Eraso et al. isolates (91%) produced cobalt blue smooth colonies. In our experience, the sensitivity of the Candida ID2 was 100% and the specificity 91% for discriminating between the two species. The evaluation of stock culture strains could be a limitation for our study as it has been reported that the dark green color of the colonies may be lost on CHROMagar Candida under repeated subculture or storage [11,29,30]. After inspecting our more recent records of clinical specimens simultaneously plated onto Candida ID2 and CHRO- Magar Candida, we isolated five C. dubliniensis from 317 oral and vaginal specimens (data not shown). All five isolates produced turquoise blue colonies on Candida ID2. However, on CHROMagar Candida, distinctive dark green colonies were only observed in one of five cultures which contained a mixture of other species of Candida. In the other four cases, there was no agreement among readers concerning the shade of green of the colonies. In conclusion, Candida ID2 agar allows a simple and accurate laboratory approach for the identification of C. dubliniensis on the basis of the colony color and provides a simple laboratory tool for the presumptive identification of C. dubliniensis in clinical microbiology laboratories. Acknowledgements Authors have been financed in part by the projects PI030662/2003 from the Fondo de Investigación Sanitaria del Ministerio de Sanidad de España and UPV- GIU05/05 from the Universidad del País Vasco-Euskal Herriko Unibertsitatea. We are grateful to Inmaculada González Canales, Ignacio Urrechaga and José Manuel Torres from biomérieux España, for the donation of the CAID2 agar plates used in this study. References 1 Gutiérrez J, Morales P, González M A, et al. Candida dubliniensis, a new fungal pathogen. J Basic Microbiol 2002; 42: Sullivan D J, Moran G P, Pinjon E, et al. Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans. FEMS Yeast Res 2004; 4: Binolfi A, Biasoli M S, Luque A G, et al. High prevalence of oral colonization by Candida dubliniensis in HIV-positive patients in Argentina. Med Mycol 2005; 43: Kalkanci A, Kokturk N, Senol E, et al. Could Candida dubliniensis be involved in lung fungus balls? Rev Iberoam Micol 2005; 22: Jabra-Rizk M A, Johnson J K, Forrest G, et al. Prevalence of Candida dubliniensis fungemia at a large teaching hospital. Clin Infect Dis 2005; 41: Tortorano A M, Peman J, Bernhardt H, et al. Epidemiology of candidaemia in Europe: results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study. Eur J Clin Microbiol Infect Dis 2004; 23: Pfaller M A, Messer S A, Gee S, et al. In vitro susceptibilities of Candida dubliniensis isolates tested against the new triazole and echinocandin antifungal agents. J Clin Microbiol 1999; 37: Quindós G, Carrillo-Muñoz A J, Arévalo M P, et al. In vitro susceptibility of Candida dubliniensis to current and new antifungal agents. Chemotherapy 2000; 46: Quindós G, Ruesga M T, Martín-Mazuelos E, et al. In vitro activity of 5-fluorocytosine against 1,021 Spanish clinical isolates of Candida and other medically important yeasts. Rev Iberoam Micol 2004; 21: Al Mosaid A, Sullivan D J, Polacheck I, et al. Novel 5-flucytosine-resistant clade of Candida dubliniensis from Saudi Arabia and Egypt identified by Cd25 fingerprinting. J Clin Microbiol 2005; 43: Moran G P, Sanglard D, Donnelly S M, et al. Identification and expression of multidrug transporters responsible for fluconazole resistance in Candida dubliniensis. Antimicrob Agents Chemother 1998; 42: Pinjon E, Sullivan D, Salkin I, Shanley D, Coleman D. Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. J Clin Microbiol 1998; 36: Staib P, Morschhauser J. Chlamydospore formation on Staib agar as a species-specific characteristic of Candida dubliniensis. Mycoses 1999; 42: Sahand I H, Moragues M D, Eraso E, et al. Supplementation of CHROMagar Candida medium with Pal s medium for rapid identification of Candida dubliniensis. J Clin Microbiol 2005; 43: Jabra-Rizk M A, Brenner T M, Romagnoli M, et al. Evaluation of a reformulated CHROMagar Candida. J Clin Microbiol 2001; 39: Bikandi J, San Millán R, Moragues M D, et al. Rapid identification of Candida dubliniensis by indirect immunofluorescence based on differential localization of antigens on C. dubliniensis blastospores and Candida albicans germ tubes. J Clin Microbiol 1998; 36: Brena S, Rubio M C, Salesa R, et al. Genotipos de Candida dubliniensis en aislamientos clínicos. Rev Iberoam Micol 2003; 21: Kanbe T, Horii T, Arishima T, Ozeki M, et al. PCR-based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes. Yeast 2002; 19: Cárdenes-Perera C D, Torres-Lana A, Alonso-Vargas R, et al. Evaluation of API ID 32C and VITEK-2 to identify Candida dubliniensis. Diagn Microbiol Infect Dis 2004; 50: Al Mosaid A, Sullivan D J, Coleman D C. Differentiation of Candida dubliniensis from Candida albicans on Pal s Agar. J Clin Microbiol 2003; 41: Mosca C O, Moragues M D, Llovo J, et al. Casein agar: a useful medium for differentiating Candida dubliniensis from Candida albicans. J Clin Microbiol 2003; 41: Kumar C P, Menon T. Tobacco agar: a new medium for chlamydosporulation in Candida albicans and Candida dubliniensis. Med Mycol 2005; 43: Alves S H, Linares C E, Loreto E S, et al. Utilization of tomato juice agar (V8 agar) in the presumptive identification of Candida dubliniensis. Rev Soc Bras Med Trop 2006; 39: [In Portuguese] ISHAM, Medical Mycology, 44,

5 Presumptive identification of Candida dubliniensis Marot-Leblond A, Beucher B, David S, et al. Development and evaluation of a rapid latex agglutination test using a monoclonal antibody to identify Candida dubliniensis colonies. J Clin Microbiol 2006; 44: Sahand I H, Moragues M D, Robert R, et al. Evaluation of Bichro-Dubli Fumouze to distinguish Candida dubliniensis from Candida albicans. Diagn Microbiol Infect Dis 2006; 55: Alonso-Vargas R, Garaizar J, Pontón J, et al. Utilidad de la amplificación aleatoria de ADN en la diferenciación de Candida albicans y Candida dubliniensis. Rev Iberoam Micol 2000; 17: Kanbe T, Kurimoto K, Hattori H, et al. Rapid identification of Candida albicans and its related species Candida stellatoidea and Candida dubliniensis by a single PCR amplification using primers specific for the repetitive sequence (RPS) of Candida albicans. J Dermatol Sci 2005; 40: Kirkpatrick W R, Revankar S G, McAtee R K, et al. Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus-infected patients in North America by primary CHROMagar Candida screening and susceptibility testing of isolates. J Clin Microbiol 1998; 36: Hospenthal D R, Beckius M L, Floyd K L, et al. Presumptive identification of Candida species other than C. albicans, C. krusei, and C. tropicalis with the chromogenic medium CHROMagar Candida. Ann Clin Microbiol Antimicrob 2006; 5: 1. Available online at (accessed on 13 January 2006). 30 Schoofs A, Odds FC, Colebunders R, et al. Isolation of Candida species on media with and without added fluconazole reveals high variability in relative growth susceptibility phenotypes. Antimicrob Agents Chemother 1997; 41: ISHAM, Medical Mycology