title: Evaluation of Pyrosequencing technology for the identification of clinically relevant non-dematiaceous yeasts and related species.
abstract: Pyrosequencing was used to identify 133 isolates of clinically relevant non-dematiaceous yeasts. These included 97 ATCC strains (42 type strains), seven UAMH strains, and 29 clinical isolates. Isolates belonged to the following genera: Candida (18 species), Trichosporon (10), Cryptococcus (7), Malassezia (3), Rhodotorula (2), Geotrichum (1), Blastoschizomyces (1), and Kodamaea (1). Amplicons of a hyper-variable ITS region were obtained and analyzed using Pyrosequencing technology. The data were evaluated by a BLAST search against the GenBank database and correlated with data obtained by conventional cycle sequencing of the ITS1-5.8S-ITS2 region. Cycle sequencing identified 78.9% of the isolates to the species level. Pyrosequencing technology identified 69.1%. In 90.1% of all of the strains tested, the identification results of both sequencing methods were identical. Most Candida isolates can be identified to the species level by Pyrosequencing. Trichosporon species and some Cryptococcus species cannot be differentiated at the species level. Pyrosequencing can be used for the reliable identification of most commonly isolated non-dematiaceous yeasts, with a reduction of cost per identification compared to conventional sequencing.
authors.affiliation: Microbiology Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, U.S. Department of Health and Human Services, 10 Center Drive, Bethesda, MD 20892-1508, USA. monteroc@cc.nih.gov
authors.forename: C I
authors.initials: CI
authors.lastname: Montero
authors.forename: Y R
authors.initials: YR
authors.lastname: Shea
authors.forename: P A
authors.initials: PA
authors.lastname: Jones
authors.forename: S M
authors.initials: SM
authors.lastname: Harrington
authors.forename: N E
authors.initials: NE
authors.lastname: Tooke
authors.forename: F G
authors.initials: FG
authors.lastname: Witebsky
authors.forename: P R
authors.initials: PR
authors.lastname: Murray
mesh_terms: D002175:Candida / Q000235:genetics / Q000302:isolation & purification; D003454:Cryptococcus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000145:classification* / Q000235:genetics*; D021901:DNA, Intergenic / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D014250:Trichosporon / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D015003:Yeasts / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D004271:DNA, Fungal; D021901:DNA, Intergenic