Differentiation of Candida dubliniensis from Candida albicans on Pal's agar

Production of a hyphal fringe around colonies grown on Pal's agar (sunflower seed agar) at 30 degrees C for 48 to 72 h provides a simple means of discriminating between isolates of C. dubliniensis and C. albicans with 100% accuracy. Of 128 C. dubliniensis isolates tested on this medium, all produced a hyphal fringe. In contrast, none of the 124 C. albicans isolates tested produced a hyphal fringe. Pal's medium has the added advantage of being prepared from inexpensive, readily available seeds.     

Rapid identification of Candida albicans by using Albicans ID and fluoroplate agar plates.

Two commercially available agar media, Albicans ID and Fluoroplate, that use a chromogenic or a fluorogenic substrate for the detection and identification of Candida albicans were evaluated. From 1,006 clinical samples containing 723 yeast strains, 352 C. albicans strains were detected with either of the two media. The sensitivity of each of the two media was 93.8% and the specificity was 98.6%, with five false-positive reactions for Candida tropicalis and no false-negative reactions.     

Tobacco agar: a new medium for chlamydosporulation in Candida albicans and Candida dubliniensis.

Chlamydospores are a distinctive morphologic feature of Candida albicans and Candida dubliniensis and aid in their identification. A new medium, tobacco agar, for chlamydosporulation in Candida is described. All the strains of C. dubliniensis and 96% of isolates of C. albicans tested produced chlamydospores after 24 h incubation on tobacco agar, whereas none of the other seven species produced chlamydospores.     

Differentiation of Candida dubliniensis from Candida albicans on staib agar and caffeic acid-ferric citrate agar

The methods currently available for the identification of the pathogenic yeast Candida dubliniensis all have disadvantages in that they are time-consuming, expensive, and/or, in some cases, unreliable. In a recent study (P. Staib and J. Morschh?user, Mycoses 42:521-524; 1999) of 14 C. dubliniensis and 11 C. albicans isolates, it was suggested that the ability of C. dubliniensis to produce rough colonies and chlamydospores (chlamydoconidia) on Staib agar (SA) provided a simple means of differentiating it from its close relative C. albicans. In the present investigation, we examined the colony morphology and chlamydospore production of 130 C. dubliniensis and 166 C. albicans isolates on SA and on the related defined medium caffeic acid-ferric citrate agar (CAF). All of the C. dubliniensis and C. albicans isolates produced chlamydospores on the control medium, i.e., rice-agar-Tween agar. However, while none of the C. albicans isolates produced chlamydospores on either SA or CAF, 85.4 and 83.8% of the C. dubliniensis isolates produced chlamydospores on SA and CAF, respectively. All of the C. albicans isolates grew as smooth, shiny colonies on SA after 48 to 72 h of incubation at 30 degrees C, while 97.7% of the C. dubliniensis isolates grew as rough colonies, many (65%) with a hyphal fringe. In contrast, 87.4% of the C. albicans and 93.8% of the C. dubliniensis isolates yielded rough colonies on CAF. Although the results of this study confirm that SA is a good medium for distinguishing between C. dubliniensis and C. albicans, we believe that discrimination between these two species is best achieved on the basis of colony morphology rather than chlamydospore production.     

Tobacco agar, a new medium for differentiating Candida dubliniensis from Candida albicans

Isolates of Candida dubliniensis may be misidentified as Candida albicans in microbiological laboratories if only the germ tube and/or the chlamydospore test is used for identification to the species level. In this study, we have evaluated the efficacy of tobacco agar for the differentiation of C. dubliniensis from C. albicans. On this medium at 28 degrees C, all 30 C. dubliniensis isolates produced yellowish-brown colonies with hyphal fringes and abundant chlamydospores, whereas 54 C. albicans isolates formed smooth, white-to-cream-colored colonies with no chlamydospore production. This medium provides a simple tool for presumptive differentiation of C. dubliniensis from C. albicans.     

Casein agar: a useful medium for differentiating Candida dubliniensis from Candida albicans

Production of chlamydospores on casein agar at 24 degrees C for 48 h provides a simple means for differentiating Candida dubliniensis from Candida albicans based on chlamydospore production. Of 109 C. dubliniensis isolates tested on this medium, 106 (97.2%) produced abundant chlamydospores and three produced few chlamydospores. In contrast, of the 120 C. albicans isolates tested, 111 (92.5%) failed to produce any chlamydospores, whereas the remaining nine isolates produced few chlamydospores. These findings indicate that abundant chlamydospore production on casein agar is a useful test for discriminating between C. dubliniensis and C. albicans.     

Rapid identification of Candida dubliniensis with commercial yeast identification systems.

Candida dubliniensis is a newly described species that is closely related phylogenetically to Candida albicans and that is commonly associated with oral candidiasis in human immunodeficiency virus-positive patients. Several recent studies have attempted to elucidate phenotypic and genotypic characteristics of use in separating the two species. However, results obtained with simple phenotypic tests were too variable and tests that provided more definitive data were too complex for routine use in the clinical laboratory setting. The objective of this study was to determine if reproducible identification of C. dubliniensis could be obtained with commercial identification kits. The substrate reactivity profiles of 80 C. dubliniensis isolates were obtained by using the API 20C AUX, ID 32 C, RapID Yeast Plus, VITEK YBC, and VITEK 2 ID-YST systems. The percentages of C. dubliniensis isolates capable of assimilating or hydrolyzing each substrate were compared with the percentages from the C. albicans profiles in each kit's database, and the results were expressed as percent C. dubliniensis and percent C. albicans. Any substrate that showed >50% difference in reactivity was considered useful in differentiating the species. In addition, assimilation of methyl-alpha-D-glucoside (MDG), D-trehalose (TRE), and D-xylose (XYL) by the same isolates was investigated by the traditional procedure of Wickerham and Burton (L. J. Wickerham and K. A. Burton, J. Bacteriol. 56:363-371, 1948). At 48 h (the time recommended by the manufacturer for its new database), we found that the assimilation of four carbohydrates in the API 20C AUX system could be used to distinguish the species, i.e., glycerol (GLY; 88 and 14%), XYL (0 and 88%), MDG (0 and 85%), and TRE (15 and 97%). Similarly, results with the ID 32 C system at 48 h showed that XYL (0 and 98%), MDG (0 and 98%), lactate (LAT; 0 and 96%), and TRE (30 and 96%) could be used to separate the two species. Phosphatase (PHS; 9 and 76%) and alpha-D-glucosidase (23 and 94%) proved to be the most useful for separation of the species in the RapID Yeast Plus system. While at 24 h the profiles obtained with the VITEK YBC system showed that MDG (10 and 95%), XYL (0 and 95%), and GLY (26 and 80%) could be used to separate the two species, at 48 h only XYL (6 and 95%) could be used to separate the two species. The most useful substrates in the VITEK 2 ID-YST system were TRE (1 and 89%), MDG (1 and 99%), LAT (4 and 98%), and PHS (83 and 1%). While the latter kit was not yet commercially available at the time of the study, it would appear to be the most valuable for the identification of C. dubliniensis. Although assimilation of MDG, TRE, and XYL proved to be the most useful for species differentiation by the majority of commercial systems, the results with these carbohydrates by the Wickerham and Burton procedure were essentially the same for both species, albeit following protracted incubation. Thus, it is the rapidity of the assimilation achieved with the commercial systems that allows the differentiation of C. dubliniensis from C. albicans.     

Performance of candida ID, a new chromogenic medium for presumptive identification of Candida species, in comparison to CHROMagar Candida

Candida ID agar allows identification of Candida albicans and differentiation of other Candida species. In comparison with CHROMagar Candida, we evaluated the performance of this medium directly from 596 clinical specimens. In particular, detection of C. albicans after 24 h of incubation was easier on Candida ID (sensitivity, 96.8%) than on CHROMagar (sensitivity, 49.6%).     

Sunflower seed husk agar: a new medium for the differentiation of Candida dubliniensis from Candida albicans

A sunflower (Helianthus annuus) seed husk agar medium has been developed and evaluated for differentiation of Candida dubliniensis from Candida albicans on the basis of colony morphology and chlamydospore production. All C. dubliniensis isolates (n=40) produced rough colonies with hyphal fringes and abundant chlamydospores whereas 101 of 105 (96.2%) C. albicans isolates produced smooth colonies with no evidence of chlamydospore production. Since this medium is free from oil droplets, chlamydospores can be examined with greater clarity by Dalmau plate technique. This medium provides a simple and cost-effective tool for the presumptive differentiation of C. dubliniensis from C. albicans and is particularly suited for clinical microbiology laboratories where biochemical or molecular methods for the differentiation of these two species are not available.     

Simplified sunflower (Helianthus annuus) seed agar for differentiation of Candida dubliniensis from Candida albicans

This study evaluated sunflower (Helianthus annuus) seed agar (SSA) for differentiation of Candida dubliniensis from Candida albicans on the basis of colony characteristics and chlamydospore production. Simplified SSA without creatinine and KH(2)PO(4) was also used. On both media, C. dubliniensis isolates (n = 25) developed rough colonies and formed abundant chlamydospores after incubation for 24-48 h at 28 degrees C, while C. albicans isolates (n = 53) showed smooth colonies with no evidence of chlamydospore formation. Cryptococcus neoformans isolates (n = 10) formed brown colonies on both media. Simplified SSA offers a simple and inexpensive tool for presumptive differentiation of C. dubliniensis from C. albicans in clinical microbiology laboratories.     

Evaluation of a rapid immunochromatographic assay for identification of Candida albicans and Candida dubliniensis

Candida dubliniensis was first established as a novel yeast species in 1995. It is particularly associated with recurrent episodes of oral candidosis in human immunodeficiency virus (HIV)-infected patients, but it has also been detected at other anatomical sites and at a low incidence level in non-HIV-infected patients. It shares so many phenotypic characteristics with C. albicans that it is easily misidentified as such. No rapid, simple, and commercial test that allows differentiation between C. dubliniensis and C. albicans has been developed, until now. Accurate species identification requires the use of genotype-based techniques that are not routinely available in most clinical microbiology diagnostic laboratories. The present study was designed to evaluate the efficiency of a new test (the immunochromatographic membrane [ICM] albi-dubli test; SR2B, Avrille, France) to differentiate between C. albicans and C. dubliniensis. The organisms evaluated were strains whose identities had previously been confirmed by PCR tests and freshly isolated clinical strains and included 58 C. albicans isolates, 60 C. dubliniensis isolates, and 82 isolates belonging to other species of yeast. The ICM albi-dubli test is based on the principle of immunochromatographic analysis and involves the use of two distinct monoclonal antibodies that recognize two unrelated epitopes expressed by both species or specific to only one species. The assay requires no complex instrumentation for analysis and can be recommended for routine use in clinical microbiology laboratories. Results are obtained within 2 h and 30 min and are easy to interpret. This evaluation demonstrated the good performance of this immunochromatographic test for C. albicans and C. dubliniensis isolated on Sabouraud dextrose agar, CHOROMagar Candida, and CandidaSelect, with sensitivities and specificities ranging from 93.1 to 100%. These parameters decreased, however, to 91.4% when the test was performed with yeast isolated with Candida ID.     

Molecular and phenotypic identification of the yeast pathogen Candida dubliniensis

Candida dubliniensis is an emerging yeast pathogen generally misclassified as Candida albicans by standard diagnostic procedures. This study examined the efficiency of molecular identification, based on a discriminative PCR test, in a prospective study on the prevalence of C. dubliniensis among 103 oropharyngeal isolates from HIV-infected individuals or transplant recipients, and 30 vaginal isolates. All of the isolates had been classified as C. albicans by standard laboratory procedures. The PCR was evaluated in a blinded fashion against classification achieved by sequencing rDNA. Sequencing results corresponded 100% to the results of the discriminative PCR, indicating the validity of this rapid test. Twenty-one C. dubliniensis isolates were identified, all of them from HIV-infected individuals (prevalence 30%). The internal transcribed spacer regions of the C. dubliniensis isolates were sequenced. Phenotypic features of C. dubliniensis, namely abundant chlamydospore formation, atypical color on CHROMagar, growth defect at 45 degrees C, and colony morphology on Staib agar, were evaluated in a blinded fashion with respect to their discriminative potential, facilitating the design of further epidemiological studies. Carbohydrate assimilation patterns were determined for C. dubliniensis with a novel automated system showing that, in contrast to previous reports, C. dubliniensis is able to utilize D-xylose and trehalose. In evaluating these tests we present a rational approach to identification of the new species and characterization of C. dubliniensis isolates.     

Evaluation of a method for identification of Candida dubliniensis bloodstream isolates

To evaluate methods for differentiating Candida albicans and Candida dubliniensis, 772 putative C. albicans bloodstream isolates were tested for growth at 37 and 42 degrees C. Isolates showing no growth at 42 degrees C, abundant chlamydospore production, and the sugar assimilation pattern of the type strain were confirmed by DNA-based procedures to be C. dubliniensis.     

Evaluation of phenotypic markers for selection and identification of Candida dubliniensis

Candida dubliniensis is often associated with C. albicans in cultures. Easy-to-perform selective isolation procedures for these closely related species do not exist. Therefore, we evaluated previously described discriminatory phenotypic markers for C. dubliniensis. A total of 150 oral rinses from human immunodeficiency virus (HIV)-infected patients were cultured on CHROMagar Candida. Dark green colonies described as being indicative of C. dubliniensis and other green colonies, 170 in total, were isolated. Chlamydospore formation, intracellular beta-D-glucosidase activity, ability to grow at 42 degrees C, carbohydrate assimilation pattern obtained by the API ID 32C, and Fourier transform infrared (FT-IR) spectroscopy were used for phenotypic characterization. Sequencing of the 5' end of the nuclear large-subunit (26S) ribosomal DNA gene was used for definitive species identification for C. dubliniensis. C. dubliniensis was found in 34% of yeast-colonized HIV-infected patients. The color of the colonies on CHROMagar Candida proved to be insufficient for selecting C. dubliniensis, since only 30 of 53 proven C. dubliniensis isolates showed a dark green color in primary cultures. The described typical chlamydospore formation can give only some indication of C. dubliniensis. The assimilation pattern proved to be insufficient to discriminate C. dubliniensis from C. albicans. All C. dubliniensis strains showed no or highly restricted growth at 42 degrees C and a lack of beta-D-glucosidase activity. Unfortunately, atypical C. albicans strains can also exhibit these phenotypic traits. FT-IR spectroscopy combined with hierarchical clustering proved to be as reliable as genotyping for discriminating the two species.     

A rapid heat-resistant technique for presumptive identification ofSalmonella on desoxycholate-citrate agar

A caprylate esterase chromogenic test, which was considered to indicate positivity in 5 minutes if a bright colour appeared on the filter paper inoculum site to which one colony had been applied, was used to test 534Salmonella, 535 other bacteria capable of growth on desoxycholatecitrate agar (DCA), and 517 non-lactose fermenting colonies from stool cultures on DCA. It was found to be 100 % sensitive forSalmonella, 99 % specific, and more accurate than either direct antiserum agglutination or a urease test in these respects. The test kit could be stored at room temperature.     

Supplementation of CHROMagar Candida medium with Pal's medium for rapid identification of Candida dubliniensis

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.     

Evaluation of three commercial agar preparations for the presumptive identification of significant urinary isolates

Three commercially available pre-poured chromogenic preparations--chromogenic urinary tract infection (UTI) medium (chromogenic UTI, Oxoid), CHROMagar Orientation (Becton Dickinson) and CPS ID2 (bioMérieux)--are evaluated in comparison to routine urine microbiology using cysteine lactose electrolyte-deficient (CLED) medium and conventional methods of identification and susceptibility testing by Vitek 1 for the majority of isolates. Most isolates were Escherichia coli, and a chromogenic medium has been shown to be a reliable, rapid and more economic medium on which to presumptively identify these organisms due to the substrates the strain utilises in the plate and the chromogen subsequently produced. However, the opacity of chromogenic UTI made the medium difficult to inoculate and read, although the colours were clear and strong. Although there was no statistical difference between CHROMagar Orientation and CPS ID2, the colours observed on the former were stronger. This meant that colony counting was possible at significant concentrations of 10(4) and 10(5) colony-forming units (cfu)/mL and it may be easier to detect mixtures that would indicate contamination. Chromogenic media are richer than CLED and a number of Lactobacillus spp. (normally regarded as normal flora) grew on this medium. These were not considered to be significant.     

Genotypic identification of Candida dubliniensis isolated from HIV patients by MLEE.

Candida dubliniensis is a novel species only recently described. This emerging pathogen shares some of the phenotypic characteristics specific to C. albicans but is genetically different. In this study we typed four strains of atypical C. albicans isolated in our laboratory and compared them to 41 strains of C. albicans and 11 strains of C. dubliniensis by several phenotypic methods and by multilocus enzyme electrophoresis. Using factorial correspondence analysis, we distinguished C. dubliniensis and the atypical C. albicans strains from all strains of C. albicans. Atypical C. albicans strains were identified as C. dubliniensis.     

CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species.

CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species. We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material. After 2 days of incubation at 37 degrees C, 285 C. albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species. A total of 54 C. tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar. C. krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C. norvegensis. Trichosporon spp. (n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation. Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint. The only exceptions were found among isolates identified as Geotrichum sp. or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar. The specificity and sensitivity of the new medium for the presumptive identification of C. albicans, C. krusei, and C. tropicalis exceeded 99% for all three species. A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C. albicans, C. tropicalis, C, krusei, and Trichosporon spp. None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs. The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants. In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances. CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.