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We report here that modifications of the preanalytical steps of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification of yeasts, with regard to the original protocol provided by the manufacturers, appear to be efficient for the reliable routine identification of clinical yeast isolates in medical laboratories. Indeed, when one colony was sampled instead of five and the protein extraction protocol was modified, the performance of MALDI-TOF MS was superior to that of the API ID 32C method (discrepancies were confirmed by using molecular identification), allowing the correct identification of 94% of the 335 clinical isolates prospectively tested. We then demonstrated that the time for which the primary cultures were preincubated on CHROMagar did not impact the identification of yeasts by MALDI-TOF MS, since 95.1 and 96.2% of the 183 clinical yeast isolates prospectively tested were correctly identified after 48 and 72 h of preincubation, respectively.

Analysis of a polymorphic microsatellite locus was applied to 85 Candida albicans strains from healthy individuals. Comparison with strains from nonhealthy individuals previously analyzed in our laboratory showed an overall similarity, suggesting that all commensal strains have the ability to develop as pathogens.

We developed a simple method for the identification of Candida glabrata on the basis of the ability of this species to rapidly assimilate trehalose but not sucrose. After incubation of yeasts with Rosco diagnostic tablets containing sucrose or trehalose, identification of C. glabrata was achieved in 4 h with 100% sensitivity and specificity.

Molecular typing studies have shown that the predominant form of reproduction of Candida albicans is clonal and that, in a majority of situations, persistent or recurrent infections are due to a unique strain. Characterization of distinct subpopulations and correlation with clinical features may thus be important to understanding the pathogenesis of candidiasis. In a clonal model, a unique polymorphic marker may identify populations with different biological properties. We therefore compared 48 bloodstream isolates and 48 nonbloodstream matched strains of C. albicans at the elongation factor 3-encoding gene (CEF3) polymorphic microsatellite locus of C. albicans. Sizing of the alleles was performed by automated capillary electrophoresis. A new, 137-bp allele was characterized, and seven nondescribed combinations were observed, resulting in 15 and 11 distinct CEF3 profiles in bloodstream and control strains, respectively. Genotypes 126-135, 130-136, and 131-131 accounted for 60.4% of both bloodstream and control strains. Four bloodstream isolates but no control strains displayed the 135-135 combination. None of the other genotypes was present at an increased frequency in bloodstream isolates. Bloodstream and nonbloodstream strains of C. albicans thus have a heterogeneous structure at the CEF3 locus, with three major and multiple minor allelic combinations.

We developed a PCR-based method that can be used to identify Cryptosporidium parvum in human feces. Fecal oocysts were concentrated by centrifugation on a sodium chloride gradient and filtration on a nitrocellulose filter prior to DNA extraction and PCR amplification of a 452-bp C. parvum-specific DNA sequence with a protocol including dUTP and uracil-N-glycosylase. All samples obtained from naturally infected humans (n = 10), calves (n = 4), and goats (n = 2) were positive. A 100% detection rate was achieved with both formed and solid stools (n = 10) seeded with 1,000 C. parvum oocysts per g. Procedures based on stool concentration by a modified Ritchie method and subsequent oocyst identification by immunofluorescent labeling or acid-fast staining require concentrations of 50,000 to 500,000 oocysts per g to achieve a 100% detection rate with formed stools. The described PCR-based assay thus has a 50- to 500-fold increase in sensitivity compared to those of the methods commonly used to analyze formed feces.