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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPMENT AND APPLICATION OF MOLECULAR PHYLOGENETICS OF FUNGI TO ENHANCE FOOD SAFETY AND FOOD SECURITY

Location: Bacterial Foodborne Pathogens & Mycology Research Unit

Title: Rapid Identfication of Ascomycetous Yeasts from Clinical Specimens by a Molecular-Based Flow Cytometry Method and Comparision with Identifications from Phenotypic Assays

Authors
item Page, Brent
item Shields, Christine - JOHNS HOPKINS MED INST
item Merz, William - JOHNS HOPKINS MED INST
item Kurtzman, Cletus

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 26, 2006
Publication Date: September 1, 2006
Citation: Page, B.T., Shields, C.E., Merz, W.G., Kurtzman, C.P. 2006. Rapid identfication of ascomycetous yeasts from clinical specimens by a molecular-based flow cytometry method and comparision with identifications from phenotypic assays. Journal of Clinical Microbiology. 44(9):3167-3171.

Interpretive Summary: Previously, we reported a novel system for rapid identification of microorganisms using species-specific DNA sequences (molecular probes) attached to polystyrene microspheres that are detected and read using a laser flow cytometer. Our first application was rapid detection of clinical yeasts and was based on molecular probes developed from standard reference strains. In the present work, nearly 100 yeasts isolated from clinical specimens were identified from DNA sequencing, by our molecular probe/flow cytometry system and from standard clinical laboratory tests. The present work showed that the flow cytometry system identified all species for which there were molecular probes. The clinical strains reacted just as well with the probes as did the reference strains used for probe development. Identification time for flow cytometry was 5 hours with approximately 12 hours required for sequencing. Standard tests required 24-120 hours and frequently failed to provide an accurate identification. This study and our previous work demonstrate the value of flow cytometry-based molecular probes for rapid, accurate identification of microorganisms.

Technical Abstract: This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. LSU rRNA D1/D2 gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA tests included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activity and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA tests identified 81 of 88 strains correctly, but misidentified Candida dubliniensis (4), C. bovina (1), C. palmioleophila (1) and C. bracarensis (1). The FC method correctly identified 79 of 88 strains, did not misidentify any isolate, but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 d for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

Last Modified: 8/21/2014
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