Search

Debaryomyces hansenii is a hemiascomycetous yeast commonly found in natural substrates and in various types of cheese. Pichia guilliermondii is widely distributed in nature and is a common constituent of the normal human microflora. Both species have been described in human infections but are extremely difficult to differentiate phenotypically. Thus, frequent errors in identification occur. The 62 clinical and environmental isolates sent between 2000 and 2007 to the French National Reference Center for Mycoses and Antifungals as D. hansenii or P. guilliermondii were analyzed by using the carbon assimilation pattern, the presence of pseudohyphae, and sequencing of the ITS and D1/D2 regions of the rRNA gene. The objective of this study was to assess using nucleotide sequences whether phenotypic identification was accurate and whether phenotypic characteristics could be used to differentiate the two species when sequencing was not available. We found that 58% of the isolates were misidentified and belong to seven different species: P. guilliermondii, P. caribbica, P. jadinii, D. hansenii, Candida palmioleophila, C. haemulonii type II, and Clavispora lusitaniae. In conclusion, D. hansenii may not be as common a human pathogen as previously thought. Sequencing of either ITS or D1/D2 regions is a good tool for differentiating the species more frequently confused with D. hansenii, keeping in mind that reliable databases should be used.

Mutations in two specific regions of the Fks1 subunit of 1,3-beta-D-glucan synthase are known to confer decreased caspofungin susceptibility on Candida spp. Clinical isolates of Candida spp. (404 Candida albicans, 62 C. tropicalis, and 21 C. krusei isolates) sent to the French National Reference Center were prospectively screened for susceptibility to caspofungin in vitro by the broth microdilution reference method of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST). Twenty-eight isolates (25 C. albicans, 2 C. tropicalis, and 1 C. krusei isolate) for which the caspofungin MIC was above the MIC that inhibited 90% of the isolates of the corresponding species (MIC(90)) were subjected to molecular analysis in order to identify mutations in the fks1 gene. Substitutions in the deduced protein sequence of Fks1 were found for 8 isolates, and 20 isolates had the wild-type sequence. Among the six C. albicans isolates harboring mutations, six patterns were observed involving amino acid changes at positions 641, 645, 649, and 1358. For C. tropicalis, one isolate showed an L644W mutation, and for one C. krusei isolate, two mutations, L658W and L701M, were found. Two media, RPMI medium and AM3, were tested for their abilities to distinguish between isolates with wild-type Fks1 and those with mutant Fks1. In RPMI medium, caspofungin MICs ranged from 0.25 to 2 microg/ml for wild-type isolates and from 1 to 8 micro for mutant isolates. A sharper difference was observed in AM3: all wild-type isolates were inhibited by 0.25 micro of caspofungin, while caspofungin MICs for all mutant isolates were >or=0.5 microg/ml. These data demonstrate that clinical isolates of C. albicans, C. tropicalis, and C. krusei with decreased susceptibility to caspofungin in vitro have diverse mutations in the fks1 gene and that AM3 is potentially a better medium than RPMI for distinguishing between mutant and wild-type isolates using the AFST-EUCAST method.

Candida tropicalis is a diploid ascomycetes yeast responsible for 4%-24% of candidemia. Resistance to flucytosine is rarely described for this species but was observed for 45 (35%) of 130 C. tropicalis isolates recovered from blood cultures in the Paris area in a 4-year survey. The aims of this study were to test the hypothesis that the flucytosine-resistant isolates could represent a subgroup and to determine the relationship between epidemiologic and genomic data. Epidemiologic data and gene sequences were analyzed, and molecular typing was performed. Our results suggest that a clone of flucytosine-resistant isolates, associated with malignancies and a lower mortality than that for other C. tropicalis isolates, is widespread in the Paris area. We propose the analysis of 2 polymorphic microsatellite markers coupled with URA3 sequencing to track the clone.

For genotyping Candida albicans isolates, two PCR-based methods have recently emerged: multilocus sequence typing (MLST), based on the sequence of selected genes, and microsatellite length polymorphism (MLP), based on the length of PCR products containing variable numbers of short DNA repeats. To compare the two methods in their abilities to differentiate and group C. albicans isolates, we selected 50 independent isolates collected at the National Reference Center for Mycoses and Antifungals. MLST typing was performed using sequencing of seven loci as described at (http://test1.mlst.net). The MLP method consisted of a single multiplex PCR testing three different loci. Dendrograms were constructed by the unweighted pair group cluster method with Euclidean metric for both methods. The correlation between the distance matrices was performed with a Mantel test tested with 1,000 random permutations. The sensitivity and specificity of the MLP typing system were determined after allocating MLST groups for the greater number of isolates of each distinct MLP group. The discriminatory power index was >0.99, and the distances between the isolates were highly correlated with both systems. The Mantel coefficient and the Pearson product-moment correlation coefficient were 35,699 and 0.32, respectively (P < or = 1.2 x 10(-6)). Using MLP, the average specificity and sensitivity of clustering compared to MLST were 83% and 73%, respectively, when the singletons were excluded. The two methods are similarly discriminatory and can be interchangeable depending on the objectives. MLP is less expensive and faster than MLST. However, MLST is currently more accurate and additional standardization is needed for MLP.

The posaconazole MIC(90) for 1,903 yeast isolates from France was 1 microg/ml (range, < or =0.015 to 8 microg/ml). Ninety percent of isolates with fluconazole MICs of > or =8 microg/ml (n = 509) and 90% of those with voriconazole MICs of > or =2 microg/ml (n = 80) were inhibited by 2 and 8 microg/ml of posaconazole, respectively.