Evaluation of Candida ID, a new chromogenic medium for fungal isolation and preliminary identification of some yeast species

Candida ID, a new chromogenic medium, allows identification of Candida albicans (blue colonies) and preliminary identification into a group of four species (pink colonies). In comparison with Albicans ID2 and Sabouraud gentamicin chloramphenicol on 446 fungal strains, Candida ID allowed the isolation of more species than Albicans ID 2 (95.5% versus 91.2%).     

Prospective evaluation of the new chromogenic medium Candida ID, in comparison with Candiselect, for isolation of molds and isolation and presumptive identification of yeast species

We conducted a prospective evaluation of Candida ID chromogenic medium (bioMérieux, Marcy l'Etoile, France) with 786 clinical specimens in comparison with Candiselect medium (Bio-Rad, Marnes la Coquette, France). Candida ID chromogenic medium identified 97.7% of Candida albicans strains; enabled presumptive identification of C. tropicalis, C. lusitaniae, C. guillermondii, and C. kefyr and better detection of yeast combinations (11.4% more often); and was more sensitive for the isolation of filamentous fungi (17.7% more often). However, Candida ID chromogenic medium appeared to be less selective vis-à-vis bacteria, with bacterial colonies sometimes pigmented blue.     

Evaluation of CandiSelect4, a new chromogenic medium for isolation and presumptive identification of Candida species from clinical specimens

In clinical laboratories, isolation of Candida species is generally based on the culture of specimens on Sabouraud dextrose agar. This strategy does not allow species identification on primary culture and makes it difficult to detect mixed cultures. Chromogenic media contain substrates that react specifically with different Candida species, and partly overcome these difficulties.ObjectivesThe aim of this study was to compare two chromogenic media: (i) CandiSelect4 (C4), a new medium developed for direct identification of C. albicans and presumptive identification of C. krusei, C. tropicalis and C. glabrata (ii) CHROMagar Candida (CH), a medium licensed for direct identification of C. albicans, and presumptive identification of C. tropicalis and C. krusei, and employed in our laboratory since 2002.Materials and methodsAltogether, 533 clinical specimens were seeded on both media. Identification on C4 was compared to that achieved on CH, API 32 C test or an in-house C. glabrata rapid identification test.ResultsDiscrepant positive cultures occurred in 16 samples and were shared equally between C4 and CH. The ability of both media to detect associations was equivalent, with 34 associations identified with C4 and CH. No discrepancy was observed with respect to identification of C. albicans. C4 identified six of six C. krusei, but false-negative identification of C. tropicalis occurred in 2/11 cases and false-positive identification of C. glabrata occurred in 2/34 cases.ConclusionsThe overall performance of C4 and CH appeared almost identical during a two-month comparison in the clinical mycology setting.     

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.     

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%).     

Evaluation of a new chromogenic medium for isolation and identification of common urinary tract pathogens

In the study presented here, a new chromogenic medium (CPS ID 3; bioMérieux, Marcy l’Etoile, France) was compared to routine media for the isolation, identification and antimicrobial susceptibility testing of bacteria recovered from urine specimens, and a cost analysis was performed. Escherichia coli, Proteeae tribe, Pseudomonas aeruginosa, Enterococcus spp. and Streptococcus agalactiae grew on the chromogenic medium as typical differentiated colonies and were accurately identified even in mixed cultures. Although the similarity of colors produced by isolates belonging to the Klebsiella, Enterobacter, Serratia and Citrobacter (KESC) group prevents differentiation among them, members of KESC were easily identified as coliforms. No substantial difference was observed when comparing the results of antimicrobial susceptibility testing performed on colonies selected from reference media versus CPS ID 3. Use of the new medium was associated with a savings of 75% over the conventional methods and the API system. Furthermore, this medium facilitated a remarkable reduction in the laboratory workload and consequently resulted in additional time and cost savings.     

Molecular probe for identification of medically important Candida species and Torulopsis glabrata.

A cloned DNA fragment from Candida albicans containing the gene for the protein actin was used to probe the molecular structure of the actin gene of several medically important yeasts (C. albicans, Candida stellatoidea, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida parapsilosis, Candida guilliermondii, and Torulopsis glabrata). Whole-cell DNA from each species was digested with restriction endonucleases, electrophoresed on agarose gels, and transferred to nitrocellulose. Radioactively labeled C. albicans actin gene was hybridized to the DNA fragments on the nitrocellulose. The C. albicans probe produced a strong signal with all of the Candida DNAs tested, indicating considerable conservation of this gene. In addition, the actin genes of all of the species tested were found to have no internal EcoRI or SalI restriction sites. With the exception of C. guilliermondii, all of the species tested had a single internal HindIII recognition site. However, the location of flanking restriction sites was found to be species specific. For all of the enzymes tested, the locations of the flanking restriction sites in C. albicans and C. stellatoidea were identical; all of the other strains yielded fragments clearly distinct from one another. These differences provide a molecular tool for the differentiation of medically important Candida species.     

A one-enzyme PCR-RFLP assay for identification of six medically important Candida species.

Early identification of Candida isolates to the species level is necessary for effective antifungal therapy, and can also facilitate control of hospital infections. Phenotype-based methods for identifying Candida species are often difficult and time-consuming. Molecular biological techniques provide a useful alternative approach. In the present study, the ITS1-5.8S-ITS2 regions of fungal rRNA genes were amplified with universal primers in 20 standard strains. Digestion of the PCR products with one restriction enzyme, MspI, allowed discrimination of medically important Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. guilliermondii. Using this method, we successfully identified 137 clinical isolates of Candida. Among them, C. albicans was identified as the most common species, followed by C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, and C. guilliermondii. This method is a simple, rapid, and cost-effective method for differentiation between species that is applicable in clinical laboratories.     

New aniline blue dye medium for rapid identification and isolation of Candida albicans.

Organic dyes have long been used in diagnostic microbiology to differentiate species by color reactions. We studied the ability of a new noninhibitory medium, YM agar containing 0.01% aniline blue WS dye, Colour Index 42780 (YMAB), to identify Candida albicans among 1,554 yeast specimens obtained from seven clinical laboratories. Appropriate American Type Culture Collection and other characterized strains served as controls. A total of 487 of the clinical strains were identified as C. albicans. The remainder were other Candida species and non-Candida yeasts. Clinical isolates and controls were grown on Sabouraud agar for 18 h at 30 degrees C and then transferred to YMAB. Plates were incubated for 12 to 18 h at 30 degrees C, and colonies were observed for yellow-green fluorescence under long-wave UV light (A365). All control strains of C. albicans and Candida stellatoidea fluoresced, as did 480 of the 490 isolates designated as C. albicans (which included 3 strains of C. stellatoidea). Cells of C. albicans grown on YMAB produced germ tubes in serum. Only five of the other 1,062 non-C. albicans yeasts fluoresced. The sensitivity and specificity were 98.0 and 99.5%, respectively, with a predictive value of 99.1%. A fluorescent metabolite was found in cell wall particulate fractions of C. albicans sonic extracts grown on YMAB but not in non-C. albicans yeasts. This metabolite showed the same spectral curve as those of metabolites from whole cells in a recording spectrofluorometer when it was excited at 400 nm and scanned from 420 to 550 nm. Thus, growth on YMAB generates the production of a fluorescent moiety that can be used to specifically identify C. albicans within 12 to 18 h.     

Evaluation of a new chromogenic medium,Uriselect 4, for the isolation and identification of urinary tract pathogens

AIMS: To compare the performance of a new chromogenic medium, Uriselect 4, with cystine lactose electrolyte deficient (CLED) agar and an established chromogenic agar, CPS ID 2 medium, for detection of urinary tract pathogens. METHODS: Using a semiquantitative culture method, 777 samples were inoculated on to the three test media in duplicate. All bacterial strains that yielded a potentially significant growth were observed for colony colour and identified using standard methods. RESULTS: Of the 777 samples tested, 589 urine samples yielded potentially significant growth of at least one strain. A total of 811 strains were isolated on at least one of the three media. A total of 168 urine samples yielded a mixture of at least two strains. Uriselect 4 medium showed the best sensitivity of the three media and only failed to recover 14 strains (1.7%). CPS ID 2 medium failed to recover 22 strains (2.7%). CLED medium showed the worst recovery and failed to recover 74 strains (9.1%). Both chromogenic media allowed for identification of Escherichia coli with a high degree of specificity (98% for Uriselect 4, 99.7% for CPS ID 2). Inclusion of a spot indole test increased the specificity of both chromogenic media to 100% for E coli. CONCLUSIONS: Uriselect 4 and CPS ID 2 were superior to CLED medium for the isolation of urinary tract pathogens mainly because of their ability to discriminate mixed cultures. Both chromogenic media were also useful for the preliminary identification of the most common urinary tract pathogens.     

Trf4 is a useful gene for discrimination of Candida tropicalis from other medically important Candida species.

Comparative studies of random amplified polymorphic DNA (RAPD) band patterns of Candida tropicalis with those of clinically important Candida species have shown the presence of specific RAPD bands for C. tropicalis. A band specific to C. tropicalis strains (ca. 400 bp) was extracted and sequenced. It was found to belong to a fragment of the Trf4 gene, which is essential for growth of these strains and has a characteristic sequence of C. tropicalis. A PCR primer was designed specifically for C. tropicalis which amplifies the 324 bp band. The PCR primer amplified DNA products for all C. tropicalis strains tested, but did not amplify any PCR bands from C. albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. kefer, C. krusei, C. parapsilosis, or C. zeylanoides. Usefulness of the PCR primer in differentiating from clinical isolates of other fungal species is discussed.     

Comparison of the new API Candida system to the ID 32C system for identification of clinically important yeast species.

API Candida was evaluated in comparison with the ID 32C system for the identification of 619 yeast isolates. The sensitivity of API Candida for the identification of the 15 species it claims to identify with and without additional tests was 97.4% (593 of 609) and 75.2% (458 of 609), respectively. The API Candida system is easy to use and rapid (result in 18 to 24 h).     

Rapid identification of Candida species and other clinically important yeast species by flow cytometry

Two rapid diagnostic assays, utilizing two different Luminex flow cytometry methods, were developed for identification of clinically important ascomycetous yeast species. Direct hybridization and allele-specific primer extension methods were both successful in establishing a DNA-based assay that can rapidly and accurately identify Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis as well as other clinical species. The direct hybridization assay was designed to identify a total of 19 ascomycetous yeast species, and the allele-specific primer extension assay was designed to identify a total of 34 species. Probes were validated against 438 strains representing 303 species. From culture to identification, the allele-specific primer extension method takes 8 h and the direct hybridization method takes less than 5 h to complete. These assays represent comprehensive, rapid tests that are well suited for the clinical laboratory.     

Evaluation of bichro-latex albicans, a new method for rapid identification of Candida albicans.

A method for identification of Candida albicans within 5 min was evaluated by using 4,643 yeast isolates. Six false-positive and three false-negative reactions were observed. The specificity (99.87%) and sensitivity (99.74%) obtained indicate that the Bichro-latex albicans test is a useful method for the rapid identification of C. albicans colonies.     

Comparative evaluation of the Iatron serological Candida check kit and the API 20C kit for identification of medically important Candida species.

A newly developed commercial serological test (Iatron Laboratories, Inc., Tokyo, Japan) for the rapid identification of medically important species of Candida was evaluated against the API 20C (Analytab Products, Plainview, N.Y.) and the standard Wickerham assimilation and fermentation procedures. Our results indicated that the Iatron and the API 20C methods are 95% accurate since both permitted identification of 78 of 82 Candida isolates, representing eight medically important species. None of the tests on nine Cryptococcus, six Trichosporon, three Geotrichum, three Saccharomyces, and one Rhodotorula species yielded false-positive reactions. False-positive serological tests occurred with a species of Pichia and Candida rugosa. The API 20C procedure correctly identified C. rugosa but not the Pichia sp. The Iatron method permitted reliable identification of the Candida species in 10 min to 5 h, whereas the API 20C procedure required 48 to 72 h. Neither method could properly identify sucrose-negative Candida tropicalis or Candida lusitaniae isolates. In addition, Candida albicans isolates could be serotyped by the Iatron method.     

PCR identification of four medically important Candida species by using a single primer pair.

A single pair of primers was used in a PCR assay to amplify and identify the DNAs from four medically important Candida species: C. albicans, C. parapsilosis, C. tropicalis, and C. (Torulopsis) glabrata. The report describes the first successful amplification of a chitin synthase-specific fragment from the four Candida species responsible for more than 90% of all cases of neonatal candidemia. The primer pair sequence was based on that from the C. albicans chitin synthase gene, CHS1 (J. Au-Young and P.W. Robbins, Mol. Microbiol. 4:197-207, 1990). Each of the four amplified products is a single band of a different size. The DNA sequence of each PCR product was determined, and four species-specific probes were synthesized. The DNAs from as few as 10 organisms in 100 microliters of plasma could be detected after amplification and Southern blot analysis. In a retrospective study of 27 paired blood samples from 16 patients with culture-proven candidemia, PCR analysis was successful at detecting and correctly identifying to the species level 26 of the 27 Candida isolates. The speed and accuracy of this PCR-based technology make it a very powerful tool for detecting and diagnosing candidemia. Implementation of this assay for analyzing blood samples should result in the more timely treatment of neonatal candidemia, thereby reducing morbidity and mortality.     

Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus

S. aureus ID (bioMérieux, La Balme Les Grottes, France) is a new chromogenic agar medium designed to enable the isolation of staphylococci and the specific identification of Staphylococcus aureus. S. aureus produces green colonies on this medium due to production of α-glucosidase. To evaluate this medium, a total of 350 wound swabs were cultured onto S. aureus ID, CHROMagar Staph. aureus, and conventional media routinely used in our laboratory. After 18 to 20 h of incubation, 96.8% of strains formed green colonies on S. aureus ID compared with 91.1% of strains forming mauve colonies on CHROMagar Staph. aureus. A total of 94.3% of strains were recovered within 18 to 20 h with conventional media. The sensitivity was increased after 48 h of incubation to 98.7, 96.2, and 95.6% with S. aureus ID, CHROMagar Staph. aureus, and conventional media, respectively. A total of 97.4% of green colonies on S. aureus ID were confirmed as S. aureus compared with 94.4% of mauve colonies on CHROMagar Staph. aureus. We conclude that S. aureus ID is a highly sensitive and specific medium for the isolation and identification of S. aureus from wound swabs.     

Direct isolation of Candida spp. from blood cultures on the chromogenic medium CHROMagar Candida

CHROMagar Candida is a selective and differential chromogenic medium that has been shown to be useful for identification of Candida albicans, Candida krusei, Candida tropicalis, and perhaps Candida glabrata. Colony morphology and color have been well defined when CHROMagar Candida has been used to isolate yeast directly from clinical specimens, including stool, urine, respiratory, vaginal, oropharyngeal, and esophageal sources. Direct isolation of yeast on CHROMagar Candida from blood cultures has not been evaluated. We evaluated whether the color and colony characteristics produced by Candida spp. on CHROMagar Candida were altered when yeasts were isolated directly from blood cultures. Fifty clinical isolates of Candida were inoculated into aerobic and anaerobic blood culture bottles and incubated at 35 degrees C in an automated blood culture system. When growth was detected, an aliquot was removed and plated onto CHROMagar Candida. As a control, CHROMagar Candida plates were inoculated with the same isolate of yeast grown on Sabouraud dextrose agar simultaneously. No significant difference was detected in color or colony morphology between the blood and control isolates in any of the tested organisms. All C. albicans (n = 12), C. tropicalis (n = 12), C. glabrata (n = 9), and C. krusei (n = 5) isolates exhibited the expected species-specific colony characteristics and color, whether isolated directly from blood or from control cultures. CHROMagar Candida can be reliably used for direct isolation of yeast from blood cultures. Direct isolation could allow mycology laboratories to more rapidly identify Candida spp., enable clinicians to more quickly make antifungal agent selections, and potentially decrease patient morbidity and mortality.     

Immunological relatedness among Candida albicans and other pathogenic Candida species.

Membrane-mitochondrial (butanol-hot phosphate-buffered saline) and cytosol (soluble cytoplasmic substances) extracts from seven pathogenic species of Candida were used in in vivo and in vitro immunological assays to study antigenic similarities among the strains with respect to C. albicans. Mice were sensitized with C. albicans serotype A for footpad testing or to provide cells for lymphocyte stimulation assays, and guinea pigs were immunized with whole cells or butanol-hot phosphate-buffered saline extracts of C. albicans to obtain antisera for immunodiffusion assays. When extracts from each of the seven species were used in the assays, they consistently segregated, as determined by statistical or subjective analyses, into three groups. Extracts of C. albicans serotype A or B and C. stellatoidea were the most immunologically reactive in all assays, indicating close similarities between those two species, whereas extracts of C. tropicalis and C. parapsilosis elicited only moderate responses. Extracts from C. krusei, C. guilliermondii, and C. pseudotropicalis were hypo- or nonreactive in the assays, indicating a low level of antigenic relatedness to C. albicans.