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Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata.

CHROMagar Candida is a new differential culture medium that allows selective isolation of yeasts and simultaneously identifies colonies of Candida albicans, C. tropicalis, and C. krusei. We evaluated the use of this medium with 316 yeast isolates including 247 isolated directly on CHROMagar from clinical material. Over 95% of stock and clinical isolates of C. albicans, C. tropicalis, and C. krusei were correctly identified on the basis of colony morphology and pigmentation on CHROMagar. Additionally, CHROMagar also allowed the identification of C. (Torulopsis) glabrata at a similar level of accuracy. The overall agreement between two observers in reading the CHROMagar plates was 95%. Growth of Candida sp. isolates on CHROMagar had no adverse effect on antifungal MICs or Vitek identification results. In parallel, cultures of 548 stool and rectal swab specimens set up on CHROMagar and Sabouraud glucose agar (SGA) were positive in 234 instances. CHROMagar was positive and SGA was negative for 11 specimens, and CHROMagar was negative and SGA was positive for 18 specimens. A single yeast species was isolated on both media from 162 specimens, and in 146 (90%) of these specimens the same species was detected on both CHROMagar and SGA. A total of 43 of the 234 positive cultures contained mixtures of yeast species. Twenty (47%) of these mixed cultures were detected only on CHROMagar. CHROMagar is extremely useful in making a rapid presumptive identification of common yeast species. This capability plus the ability to detect mixed cultures of Candida spp. promises to improve and streamline the work flow in the mycology and clinical microbiology laboratory.

Adult↗

Antigenic studies of Candida. II. Antigenic relation of Candida albicans group A and group B to Candida stellatoidea and Candida tropicalis.

Hasenclever, H. F. (U. S. Public Health Service, Bethesda, Md.), William O. Mitchell, and Joseph Loewe. Antigenic studies of Candida. II. Antigenic relation of Candida albicans group A and group B to Candida stellatoidea and Candida tropicalis. J. Bacteriol. 82:574-577. 1961.-Previous work in our laboratory has shown the presence of two antigenic groups within the species Candida albicans. These groups have been designated as groups A and B. Subsequent studies have been conducted to show the antigenic relationship of these groups with Candida stellatoidea and Candida tropicalis. Antisera to two strains each of C. albicans group A, C. albicans group B, C. stellatoidea, and C. tropicalis were used in this investigation. Individual suspensions from each of nine strains of C. stellatoidea and from ten strains each of the other groups and species were used to test the agglutinative properties of unadsorbed and adsorbed antisera. Separate samples of antisera to each immunizing strain were reciprocally cross adsorbed with the other immunizing strains and tested for agglutination. The results indicated that C. albicans group A and C. tropicalis were antigenically identical and C. albicans group B and C. stellatoidea were antigenically indistinguishable.

Candida↗

Cell-constituent polyamines in Candida species and new biotyping of Candida albicans, Candida tropicalis and Candida parapsilosis strains.

Intracellular amines from three Candida species were extracted and chemically derivatized by a modification of the Seiler procedure. The results of qualitative determinations of 13 amines in 20 strains each of Candida albicans, Candida tropicalis and Candida parapsilosis are presented. This report is the first to describe the detection of amines other than the classical spermine, spermidine, putrescine and cadaverine in yeasts and fungi. Characteristic profiles due to the presence or absence of particular amines in the Candida species studied are demonstrated. Although these could not be used as strict differential markers at the species level, biotyping schemes based on amines are proposed to differentiate strains of C. albicans, C. tropicalis and C. parapsilosis.

Bacterial Typing Techniques↗

Synonymy of Candida methylica with Candida boidinii and of Candida methanolophaga with Candida succiphila.

Some Candida species which can use methanol as a sole carbon source were studied by performing chemotaxonomic tests; we determined the ubiquinone systems of these organisms, their DNA base compositions, their electrophoretic karyotypes, and their DNA relatedness values. The type strains of Candida methanolophaga and Candida succiphila had similar DNA base compositions and exhibited 90% DNA relatedness. All Candida boidinii and Candida methylica strains had DNA G + C contents of 30.8 to 31.0 mol%, and these organisms exhibited 87 to 101% DNA relatedness to the type strain of C. boidinii. We propose that C. methanolophaga and C. methylica should be synonyms of C. succiphila and C. boidinii, respectively.

Base Composition↗

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.

BACKGROUND: Candidiasis is caused by several Candida species, of which Candida stellatoidea and C. dubliniensis are phenotypically close to C. albicans. Although current molecular biology-based techniques can distinguish between C. albicans and C. dubliniensis, a convenient tool that can distinguish C. stellatoidea from C. albicans has not yet been developed. OBJECTIVE: To develop a system that can simply, rapidly and specifically distinguish C. albicans from the related Candida species C. stellatoidea and C. dubliniensis. MATERIALS: Genomic DNAs were purified from various yeast species and amplified by primers specific for the repetitive sequence (RPS) of C. albicans. The PCR products were purified and sequenced in order to test the specificity of the PCR amplification. RESULTS: The PCR primers only amplified several products from C. albicans, C. stellatoidea and C. dubliniensis. Sequence analysis of the products revealed that C. stellatoidea and C. dubliniensis both had RPSs including alt repeats, similar to C. albicans. After the PCR amplification, each of the three Candida species showed a unique amplification profile. Furthermore, RFLP analysis of the PCR products using EcoRI and ClaI produced species-specific restriction profiles. CONCLUSIONS: This PCR-based technique targeting the alt repeats in the RPS is useful as a tool for the rapid identification and distinction of C. albicans, C. stellatoidea and C. dubliniensis.

Base Sequence↗

Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae. Inhibition with peptidomimetic inhibitors.

The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality. The secretion of aspartic proteases by Candida spp. was demonstrated to be one of the virulence determinants. Candida albicans is classified as the major human pathogen in the genus Candida. However, other species of this genus have been found to cause an increasing number of candidiases. We isolated secreted aspartic proteases (Saps) of C. albicans (Sap2p), C. tropicalis (Sapt1p), C. parapsilosis (Sapp1p), and C. lusitaniae (Saplp) from culture media. All the isolated proteases were N-terminally sequenced. Their specific proteolytic activities and sensitivity to series of peptidomimetic inhibitors modified in the type of scissile bond replacement as well as in the N- and C-termini were analyzed. The most divergent substrate specificity was observed for the Sap of C. tropicalis. The specificity of Sap of C. lusitaniae is most closely related to that of Sap of C. parapsilosis. We designed and prepared an inhibitor containing phenylstatine isoster that was equipotent towards all four proteases within the range of 10-10-10-9 M. The HIV-1 protease inhibitors ritonavir, saquinavir, indinavir, and nelfinavir were also tested for the inhibition of four Saps. Only ritonavir and saquinavir inhibited Sap2p, Sapt1p, Sapp1p, and Saplp in micromolar concentrations.

Amino Acid Sequence↗

Extracellular aspartic proteinases from Candida albicans, Candida tropicalis, and Candida parapsilosis yeasts differ substantially in their specificities.

Extracellular aspartic proteinases have been implicated for some time as virulence factors associated with Candida opportunistic fungal infections. Our present knowledge of the enzymatic properties of these proteinases is rather limited. Information on their substrate specificity is important for understanding their roles in invasive Candida infections. We have isolated aspartic proteinases from each of the three Candida yeasts, Candida albicans, Candida tropicalis, and Candida parapsilosis, and investigated the specificities of these proteinases using a library of synthetic substrates and testing inhibition by pepstatin A. The specificities of these aspartic proteinases are different from those of major human proteinases, including gastric pepsins, renal renin, and cathepsin D. For the peptide substrate, Lys-Pro-Ala-Leu-Phe*Phe(p-NO2)-Arg-Leu, the values of kcat/Km were 2.95 x 10(6) M-1 s-1 for cleavage by Candida albicans proteinase, 1.60 x 10(6) M-1 s-1 for cleavage by Candida tropicalis proteinase, and 0.59 x 10(6) M-1 s-1 for Candida parapsilosis proteinase. Substantial differences in specificity among the Candida yeast proteinases were identified. For example, Candida tropicalis shows large changes in the kcat/Km value depending on the acidobasic character of the residue occupying the P2 position (1.6 x 10(6) M-1 s-1 for Leu, 0.47 x 10(6) M-1 s-1 for Lys, and 0.05 x 10(6) M-1 s-1 for Asp at P2, respectively). Candida parapsilosis by comparison is tolerant of these substitutions at P2 and is highly restrictive at position P4.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

DNA relatedness, karyotyping and gene probing of Candida tropicalis, Candida albicans and its synonyms Candida stellatoidea and Candida claussenii.

Isolates of Candida albicans with varied phenotypes, including sucrose-negative variants (C. stellatoidea, serotypes A and B) and avirulent germ tube-negative forms (C. claussenii) showed significant (greater than 90%) DNA relatedness to classical C. albicans, but insignificant relatedness to C. tropicalis and sucrose-negative C. tropicalis. A transverse alternating-field gel electrophoresis procedure (TAFE) showed discrete karyotype patterns among the phenotypic variants of C. albicans including the sucrose-negative C. stellatoidea. The number of chromosome-sized DNA bands for C. tropicalis (7 bands) were within the range of bands observed for C. albicans (5 to 10 bands). The general DNA-migration pattern for C. albicans appeared distinct from that of C. tropicalis. An aspartyl proteinase (PrA) gene probe from C. albicans hybridized with chromosomal DNA from C. albicans, C. claussenii and C. stellatoidea but not with that from C. tropicalis.

Aspartic Acid Endopeptidases↗

Genetic characterization of a phospholipase C gene from Candida albicans: presence of homologous sequences in Candida species other than Candida albicans.

Phospholipase C (PLC) enzymes are essential in regulating several important cellular functions in eukaryotes, including yeasts. In this study, PCR was used to identify a gene encoding PLC activity in Candida albicans, using oligonucleotide primers complementary to sequences encoding highly conserved amino acid regions within the X domains of previously characterized eukaryotic phospholipase C genes. The nucleotide sequence of the C. albicans gene, CAPLC1 (2997 bp), was determined from a recombinant clone containing C. albicans 132A genomic DNA; it encoded a polypeptide of 1099 amino acids with a predicted molecular mass of 124.6 kDa. The deduced amino acid sequence of this polypeptide (CAPLC1) exhibited many of the features common to previously characterized PLCs, including specific X and Y catalytic domains. The CAPLC1 protein also exhibited several unique features, including a novel stretch of 18-19 amino acid residues within the X domain and an unusually long N-terminus which did not contain a recognizable EF-hand Ca(2+)-binding domain. An overall amino acid homology of more than 27% with PLCs previously characterized from Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that the CAPLC1 protein is a delta-form of phosphoinositide-specific PLC (PI-PLC). PLC activity was detected in cell-free extracts of both yeast and hyphal forms of C. albicans 132A following 7 h and 24 h growth using the PLC-specific substrate p-nitrophenylphosphorylcholine (p-NPPC). In addition, CAPLC1 mRNA was detected by reverse transcriptase PCR in both yeast and hyphal forms of C. albicans 132A at the same time intervals. Expression of CAPLC1 activity was also detected in extracts of Escherichia coli DH5 alpha harbouring plasmids which contained portions of the CAPLC1 gene lacking sequences encoding part of the N-terminus. Southern hybridization and PCR analyses revealed that all C. albicans and Candida dubliniensis isolates examined possessed sequences homologous to CAPLC1. Sequences related to CAPLC1 were detected in some but not all isolates of Candida tropicalis, Candida glabrata and Candida parapsilosis tested, but not in the isolates of Candida krusei, Candida kefyr, Candida guillermondii and Candida lusitaniae examined. This paper reports the first description of the cloning and sequencing of a PLC gene from a pathogenic yeast species.

Amino Acid Sequence↗

Detection of serum Candida antigens by enzyme-linked immunosorbent assay and a latex agglutination test with anti-Candida albicans and anti-Candida krusei antibodies.

Serum samples from 197 patients with and without candidiasis were assayed for Candida albicans mannan and Candida krusei mannan by an enzyme-linked immunosorbent assay (ELISA) and a latex agglutination test (LA) and for D-arabinitol by the enzymatic fluorometric method. Of the 43 patients positive for C. albicans mannan (> or = 0.2 ng/ml), 34 were infected with C. albicans and 9 were infected with Candida tropicalis. C. krusei mannan (> or = 0.3 ng/ml) was detected in 10 patients infected with Candida parapsilosis, 2 patients infected with Candida guilliermondii, and 1 patient infected with C. krusei. With both anti-C. albicans antibodies and anti-C. krusei antibodies, the sensitivities of ELISA and LA for detection of invasive candidiasis (58 patients) were 74 and 38%, respectively. No false-positive reactions were observed by the ELISA or the LA. The sensitivity and specificity of the D-arabinitol/creatinine ratio (> or = 1.5 mumol/mg) to invasive candidiasis were 50 and 91%, respectively. The ELISA with antibodies against both C. albicans and C. krusei may be useful in diagnosing invasive candidiasis caused by medically important Candida strains excluding Candida glabrata.

Adult↗

Candida cleridarum, Candida tilneyi and Candida powellii, three new yeast species isolated from insects associated with flowers.

Three new asexual yeast species were isolated from various floricolous insects. Candida cleridarum sp. nov. was the dominant species in clerid beetles collected in flowers of various cacti in Arizona and Southern California. The sequence of the D1D2 domains of the large-subunit rDNA showed that it is a sister species to Candida fragi (0.9% base difference), a yeast isolated once from fermenting strawberries. Candida tilneyi sp. nov. and Candida powellii sp. nov. were recovered from bees and from nitidulid beetles in flowers of two species of morning glory (Ipomoea) in north-western Costa Rica. C. tilneyi sp. nov. is most closely related to Candida geochares, but differs in the D1D2 sequence by 4.7% base substitutions. C. powellii sp. nov. is a relative of Candida batistae and Candida floricola, showing sequence differences of 5.9 and 6.9%, respectively. In all cases, the new species are phenotypically similar to their nearest relatives, but are sufficiently different to allow conventional identification. The type strains are C. cleridarum strain UWO(PS) 99-101.1T ( = CBS 8793T), C. tilneyi strain UWO(PS) 99-325.1T ( = CBS 8794T) and C. powellii strain UWO(PS) 99-325.3T ( = CBS 8795T).

Animals↗

Candida asparagi sp. nov., Candida diospyri sp. nov. and Candida qinlingensis sp. nov., novel anamorphic, ascomycetous yeast species.

Among ascomycetous yeasts that were isolated from several nature reserve areas in China, three anamorphic strains isolated from soil (QL 5-5T) and fruit (QL 21-2T and SN 15-1T) were revealed, by conventional characterization and molecular phylogenetic analysis based on internal transcribed spacer and large subunit (26S) rRNA gene D1/D2 region sequencing, to represent three novel species in the genus Candida. Candida qinlingensis sp. nov. (type strain, QL 5-5T=AS 2.2524T=CBS 9768T) was related closely to a teleomorphic species, Williopsis pratensis. The close relatives of Candida diospyri sp. nov. (type strain, QL 21-2T=AS 2.2525T=CBS 9769T) are Candida friedrichii and Candida membranifaciens. Candida asparagi sp. nov. (type strain, SN 15-1T=AS 2.2526T=CBS 9770T) forms a clade with Candida fructus.

Candida↗

Karyotyping of Candida albicans and Candida glabrata from patients with Candida sepsis.

The aim of this study was to determine the relatedness of Candida strains from patients suffering from Candida septicaemia by typing of Candida isolates from blood cultures and different body sites by pulsed field gel electrophoresis (PFGE using a contour-clamped homogenous electric field, CHEF). We studied 17 isolates of Candida albicans and 10 isolates of Candida glabrata from six patients. Four patients suffered from a C. albicans septicaemia, one patient from a C. glabrata septicaemia, and one patient had a mixed septicaemia with C. albicans and C. glabrata. Eight isolates from blood cultures were compared with 19 isolates of other sites (stool six, urine four, genital swab four, tip of central venous catheter three, tracheal secretion one, sputum one). PFGE typing resulted in 10 different patterns, four with C. albicans and six with C. glabrata. Five of the six patients had strains of identical PFGE patterns in the blood and at other sites. Seven isolates of a 58-year-old female with a C. glabrata septicaemia fell into five different PFGE patterns. However, they showed minor differences only, which may be due to chromosomal rearrangements within a single strain. Thus it appears, that the colonizing Candida strains were identical to the circulating strains in the bloodstream in at least five of six patients.

Aged↗

Antigenic studies of Candida. III. Comparative pathogenicity of Candida albicans group A, group B, and Candida stellatoidea.

Hasenclever, H. F. (U. S. Public Health Service, Bethesda, Md.), and William O. Mitchell. Antigenic studies of Candida. III. Comparative pathogenicity of Candida albicans group A, group B, and Candida stellatoidea. J. Bacteriol. 82:578-581. 1961.-Since the antigenic differences between Candida albicans group A and C. albicans group B had been established, the problem of a difference in pathogenicity for laboratory animals was studied. Candida stellatoidea was also included in this investigation. Similar experiments with Candida tropicalis have been reported. The virulence of 24 strains of C. albicans group A, 23 strains of C. albicans group B, and 11 strains of C. stellatoidea for mice was determined. Five C. albicans group B and four C. stellatoidea strains were tested for lethality in rabbits.C. albicans group A and C. albicans group B displayed about the same degree of virulence for mice. Although C. stellatoidea in general showed less virulence for mice than C. albicans, some of its strains were more virulent than the least virulent C. albicans strains. All five C. albicans group B strains tested in rabbits demonstrated considerably more virulence for this laboratory animal than any of the four C. stellatoidea strains tested.C. albicans group B was isolated from more diverse clinical specimens than C. albicans group A, but only a small number of specimens were studied.

Animals↗

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%).

Candida↗

Candida glabrata, Candida krusei, non-albicans Candida spp., and other fungal organisms in a sixty-bed national cancer center in 1989-1993: no association with the use of fluconazole.

During the 5-year period 1989-1993, the incidence of Candida krusei, and other non-albicans Candida spp., was analyzed in a 60-bed cancer department. The frequency of C. krusei, before fluconazole was introduced into therapeutic protocols in 1990, was 16.5%, and after introduction of fluconazole into prophylaxis in acute leukemia in 1991, the incidence of C. krusei was 12.7%. After 3 years of using this drug in therapy and prophylaxis, the incidence of C. krusei in 1993 was 14.8%, what was lower than before this drug was introduced in our country. 97.6% of all isolated fungi were yeasts and only 2.4% were molds. Among yeasts, the most frequently isolated pathogen was Candida albicans with 64.3% in 1989 and 74.2% in 1993. The next was C. krusei with 21.2% in 1992 and 16.5% in 1989, but 14.8% in 1993, and Candida tropicalis and Candida glabrata with 9.03% in 1989 and 2.7% in 1993. Among the molds, Aspergillus spp. was the most frequently isolated genus. Analyzing the etiology of mycologically proven fungal infections confirmed by positive blood cultures or biopsies, C. albicans and Aspergillus spp. were the most common causative organisms.

Cancer Care Facilities↗