A.M. Freydiere, F. Parant, C. Chaux, Y. Gille .

1. 10th ECCMID, STOCKHOLM, May 28-31, 2000 P-278 CANDIDA ID, A NEW CHROMOGENIC MEDIUM COMPARED TO ALBICANSID2 • A.M. Freydiere, F. Parant, C. Chaux, Y. Gille. Laboratoire de Microbiologie, Hôpital de l’Antiquaille, Hospices Civils de Lyon, 69321 Lyon cedex 5, France. E-mail : am.freydiere@chu-lyon.fr • Anti-bacterial selectivity • On Candida ID, 5 bacterial strains: 2 Pseudomonas aeruginosa strains (isolated from 1 wound specimen and 1 stool specimen) and 3 Streptococcus spp. strains (isolated from 2 genital specimens and 1 stool specimen) grew. • On Albicans ID2, none bacterial strain grew. MATERIAL AND METHODS • Like the Germ Tube test and the other chromogenic media (9) Candida ID did not allow the differentiation of C. dubliniensis from C. albicans. • As C. dubliniensis strains are known to be more resistant to azole antifungal agents, in some clinical situations [e.g., oral specimens from human immunodeficiency virus (HIV)-infected and AIDS patients], it may be important to differentiate the two species. In that cases, Candida strains yielding blue or green colonies on chromogenic media should be further identified by means of commercial yeast identification systems. Since, recently, (i) Pincus et al. (10) comparing API 20C AUX, ID 32 C, RapID Yeast Plus, Vitek YBC and Vitek 2 ID-YST systems showed that the rapidity of the assimilation of Methyl D-glucose (MDG), Trehalose (TRE), and Xylose (XYL) achieved with the different commercial sytems allowed the differentiation of C. dubliniensis from C. albicans and (ii) Gales et al. (11) showed that lack of growth at 45°C and a negative XYL test with either API 20C AUX or Vitek yeast identification panel provide a presumptive identification of C. dubliniensis. • II) Results with 131 clinical specimens • Yeast isolation and discrimination • Of 131 clinical specimens tested a total of 65 yielded one or several yeast strains or a mixture of yeast and bacterial strains. (43 monomicrobial cultures and 22 mixed cultures). • ABSTRACT • Objectives: While Albicans ID2 (bioMérieux) detects only hexosaminidase enzymatic activity for the identification of Candida albicans (blue colonies), Candida ID agar medium (bioMérieux) detects a second enzymatic activity (patented) for the discrimination of different yeast species (pink colonies). This study comparatively accesses the performance of Candida ID and Albicans ID2 agar media for the identification of Candida albicans as well as yeast isolation and discrimination. • Method: 139 yeast stock strains belonging to 16 species and 131 miscellaneous clinical specimens, were streaked identically on both media. Growth and aspect of colonies were monitored after 24, 48, and 72 hrs of incubation at 36°C. All isolates were identified using conventional mycological methods including the Bichrolatex albicans test and the ID32C system. • Results: In term of yeasts growth, Candida ID and Albicans ID2 agar media showed similar performance. The second substrate included in the Candida ID agar did not allow the identification of any additional Candida species since 39 of 43 C. tropicalis, 5 of 5 C. kefyr, 2 of 5 C. sphaerica, 2 of 2 C. lusitaniae, 1 of 2 Saccharomyces cerevisiae and 1 of 1 C. utilis strains tested yielded pink colonies. • However 5 stock C. tropicalis strains showing positive hexosaminidase activity on Albicans ID2 after 48 hrs of incubation, yielded pink colonies on Candida ID. These 5 false-C. albicans blue colonies on Albicans ID2 were thus clearly distinguished from true C. albicans strains on Candida ID. • Moreover in 3 out of the 22 clinical samples yielding several yeast strains, an additional Candida strain (1 C. tropicalis, 1 C. kefyr and 1 C. parapsilosis strain) was detected on Candida ID due to the pink-colored colonies. • Conclusion: Compared to Albicans ID2, Candida ID improved specificity for C. albicans identification and differentiation of colonies in mixed fungal populations. Candida ID and Albicans ID2 (bioMérieux, France) are ready-to-use media which, use the hydrolyzing capacity of a hexosaminidase substrate to color Candida albicans colonies blue. In addition Candida ID contains a specific inhibitor of the C. tropicalis hexosaminidase enzyme and a second enzymatic substrate yielding rose-colored colonies (patented). 139 yeast stock strains encompassing 16 species and notably 43 C. tropicalis strains were subcultured on Sabouraud glucose agar at 30°C for 18 to 24 hrs, then streaked identically onto each of the two media. 131 clinical samples (59 stool specimens, 28 sputum specimens, 25 genital specimens, 8 urine specimens, 11 wound specimens) collected from hospitalized patients were tested. Each non-fluid specimen was suspended in 0.85% physiological saline; 0.01 ml of this suspension or fluid specimen was inoculated onto Candida ID and Albicans ID2 plates. For the two media, all the morphologically different colonies were identified using conventional mycological methods including the Bichrolatex Albicans (Fumouze Diagnostics, France) and when required, assimilation pattern with ID 32C (bioMerieux, France). Reading of the plates and interpretation of the results were conducted after incubation for 24, 48, and 72 hrs at 36°C. CONCLUSION Table 2: Fungi detected from 65 clinical specimens after incubation for 48 hrs at 36°C on Albicans ID2, and Candida ID • Compared to the previous formula, Candida ID plates demonstrated: • similar performance in term of yeasts growth and a lower anti-bacterial selectivity. • a slightly higher detection rate due to a better differentiation of colonies in mixed cultures. • an improved specificity for C. albicans identification. RESULTS AND DISCUSSION I) Results with 139 yeast stock strains Table 1 : Identity and colony color of 139 yeast strains grown 48hrs at 36°C on Albicans ID2 and on Candida ID INTRODUCTION Chromogenic media have been shown to allow an easier discrimination of Candida species colonies in mixed yeast populations than does Sabouraud agar, as well as rapid identification of Candida species directly on the primary isolation medium (1-8). These media may avoid or diminish the need for subculture and further biochemical tests, and considerably simplify the identification procedure. Furthermore, if more organisms can be identified within 24hrs rather than 48hrs, this reduction in turnaround time can be translated directly into a more rapid transition from empirical to directed antifungal therapy. Candida ID (bioMérieux, Marcy L'Etoile, France) is a new version of Albicans ID2, for the direct identification of Candida albicans. Compared to the previous formula Candida ID contains two chromogenic substrates and a specific inhibitor of the C. tropicalis hexosaminidase enzyme. Thus it is intended to improve differentiation of mixed cultures and specificity for direct identification of C. albicans while maintaining the same growth performance and the same C. albicans identification rate. The aim of this work is to compare the two formula (i) with 139 stock strains (ii) 131 miscellaneous clinical specimens evaluated in a true clinical laboratory context. REFERENCES 1. Baumgartner C, Freydière AM, Gille Y. Direct identification and recognition of yeast species from clinical material by using Albicans ID and CHROMagar Candida plates. J Clin Microbiol 1996, 34: 454-456. 2. De Champs C, Lebeau B, Grillot R, Ambroise-Thomas R. Evaluation of Albicans ID plates. J Clin Microbiol 1995, 33: 2227-2228. 3. Lipperheide V, Andraka L, Ponton J, Quindos G. 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Rev Iberoam Micol 1997; 14: 85-89. 9. Tintelnot K, Haase G, Seibold M, et al. Evaluation of phenotypic markers for selection and identification of C. dubliniensis. J Clin Microbiol 2000; 38: 1599-1608. 10. Pincus DH, Coleman DC, Pruitt WR, et al. Rapid identification of Candida dubliniensis with commercial yeast identification systems. J Clin Microbiol 1999; 37: 3533-3539. 11. Gales AC, Pfaller MA, Houston AK, et al. Identification of C. dubliniensis based on temperature and utilization of xylose and a-Methyl-D-glucoside as determined with the API 20C AUX and Vitek YBC systems. J Clin Microbiol 1999; 37: 3804-3808. On Candida ID, due to the pink-colored colonies, in 3/22 clinical specimens yielding several fungi, an additional Candida strain (1 C. tropicalis, 1 C. kefyr and 1 C. parapsilosis) was detected. • Identification of C. albicans. • Table 3:Candida albicans identification with Albicans ID2 and Candida ID after incubation for 24hrs and 48hrs at 36°C On Candida ID, the additional substrate enabled more specific identification of C. albicans since 5/43 C. tropicalis strains, showing positive hexosaminidase activity on Albicans ID2 after 48hrs of incubation, yielded pink or white colonies on Candida ID. These 5 false-C.albicans blue colonies on Albicans ID2 were thus clearly distinguished from true C. albicans on Candida ID. However, the additional substrate did not allow identification of any additional Candida species since 39 of 43 C. tropicalis, 5 of 5 C. kefyr, 2 of 5 C. sphaerica, 2 of 2 C. lusitaniae, 1 of 2 Saccharomyces cerevisiae and 1 of 1 C. utilis strains tested yielded pink colonies. C. glabrata, C. krusei, C. parapsilosis and other C. species, less commonly encountered, yielded white colonies. The sensitivity for growth and specific pigmentation was established on the total number of C. albicans strains isolated on at least one medium. None false positive C. albicans strain was found on both Albicans ID2 and Candida ID.