Evaluation of Pyrosequencing® technology for the identification of clinically relevant non-dematiaceous yeasts and related species
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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.
KeywordsClinical Isolate Invasive Fungal Infection ITS2 Region Conventional Sequencing Pyrosequencing Analysis
We thank Patricia S. Conville, Department of Laboratory Medicine, Warren Grant Magnuson Clinical Center, NIH, for her technical assistance. This research was supported by the Intramural Research Program of the NIH Clinical Center. The views expressed herein are those of the authors and should not be construed as those of the U.S. Department of Health and Human Services.
- 12.Linton CJ, Borman AM, Cheung G, Holmes AD, Szekely A, Palmer MD, Bridge PD, Campbell CK, Johnson EM (2007) Molecular identification of unusual pathogenic yeast isolates by large ribosomal subunit gene sequencing: 2 years of experience at the United Kingdom mycology reference laboratory. J Clin Microbiol 45:1152–1158PubMedCrossRefGoogle Scholar
- 15.White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, CaliforniaGoogle Scholar
- 22.Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Meis JF, Gould IM, Fu W, Colombo AL, Rodriguez-Noriega E; Global Antifungal Surveillance Study (2007) Results from the ARTEMIS DISK Global Antifungal Surveillance study, 1997 to 2005: an 8.5-year analysis of susceptibilities of Candida species and other yeast species to fluconazole and voriconazole determined by CLSI standardized disk diffusion testing. J Clin Microbiol 45:1735–1745PubMedCrossRefGoogle Scholar
- 26.Hazen KC, Howell SA (2007) Candida, Cryptococcus and other yeasts of medical importance. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA (eds) Manual of clinical microbiology, 9th edn. ASM Press, Washington, DC, pp 1762–1788Google Scholar
- 27.Bovers M, Diaz MR, Hagen F, Spanjaard L, Duim B, Visser CE, Hoogveld HL, Scharringa J, Hoepelman IM, Fell JW, Boekhout T (2007) Identification of genotypically diverse Cryptococcus neoformans and Cryptococcus gattii isolates by Luminex xMAP technology. J Clin Microbiol 45:1874–1883PubMedCrossRefGoogle Scholar
- 33.de Hoog GS, Guarro J, Gene J, Figueras MJ (2000) Atlas of clinical fungi, 2nd edn. Centraalbureau voor Schimmelcultures/Universitat Rovira i Virgili, UtrechtGoogle Scholar
- 37.Centraalbureau vor Schimmelcultures (CBS) (2007) Fungal Biodiversity Center Yeast Database. Home page at: http://www.cbs.knaw.nl/yeast/BioloMICS.aspx