Submitted to: Journal of Clinical Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/9/2005
Publication Date: 6/2/2005
Citation: Page, B.T., Kurtzman, C.P. 2005. Rapid identification of Candida and other clinically important yeast species by flow cytometry. Journal of Clinical Microbiology. 43(9):4507-4514. Interpretive Summary: The identification of yeasts, whether from clinical specimens or from agricultural and food environments, is often uncertain using standard diagnostic growth tests. To solve this problem, a diagnostic gene sequence database was developed at NCAUR that allows accurate identification of yeasts from gene sequences. To facilitate use of this database in diagnostic laboratories where gene sequencing may not be possible, a system of molecular probes was developed from the database, and these probes are attached to tiny plastic beads that can be read quickly in a device called a flow cytometer that was especially developed for this procedure by the Luminex Corp. The present work focused on identification of clinical yeasts. This research provided proof of concept and demonstrated successful identification of 30 pathogenic yeast species. Identification time was 5 hours, in contrast to standard methods that may take days or weeks, and which are often inaccurate. Although this work focused on clinical yeasts, the procedure can be readily adapted to detection of food spoilage and biocontrol species as well as other microorganisms of agricultural and food safety significance.
Technical Abstract: Two methods, both utilizing the Luminex flow cytometry technology, 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 reliably identify Candida albicans, C. krusei, C. parapsilosis, C. glabrata, and C. tropicalis as well as other clinical species. The direct hybridization assay identifies a total of 21 ascomycetous yeasts, and the allele-specific primer extension assay identifies a total of 34 ascomycetous yeasts. The probes were validated against 437 strains representing 303 species. Both methods are rapid and accurate. From culture to identification the allele-specific primer extension method takes 8 hours and the direct hybridization method takes less than 5 hours to complete.