Skip to main content

Advertisement

SpringerLink
Log in

Evaluation of Pyrosequencing® technology for the identification of clinically relevant non-dematiaceous yeasts and related species

  • Article
  • Published:
European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  1. Walsh TJ, Groll A, Hiemenz J, Fleming R, Roilides E, Anaissie E (2004) Infections due to emerging and uncommon medically important fungal pathogens. Clin Microbiol Infect 10(Suppl 1):48–66

    Article  PubMed  Google Scholar 

  2. Vartivarian SE, Anaissie EJ, Bodey GP (1993) Emerging fungal pathogens in immunocompromised patients: classification, diagnosis, and management. Clin Infect Dis 17(Suppl 2):S487–S491

    PubMed  Google Scholar 

  3. Aperis G, Myriounis N, Spanakis EK, Mylonakis E (2006) Developments in the treatment of candidiasis: more choices and new challenges. Expert Opin Investig Drugs 15:1319–1336

    Article  PubMed  CAS  Google Scholar 

  4. Taylor LH, Latham SM, Woolhouse ME (2001) Risk factors for human disease emergence. Philos Trans R Soc Lond B Biol Sci 356:983–989

    PubMed  CAS  Google Scholar 

  5. Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20:133–163

    Article  PubMed  CAS  Google Scholar 

  6. Bhally HS, Jain S, Shields C, Halsey N, Cristofalo E, Merz WG (2006) Infection in a neonate caused by Pichia fabianii: importance of molecular identification. Med Mycol 44:185–187

    Article  PubMed  CAS  Google Scholar 

  7. Ramani R, Gromadzki S, Pincus DH, Salkin IF, Chaturvedi V (1998) Efficacy of API 20C and ID 32C systems for identification of common and rare clinical yeast isolates. J Clin Microbiol 36:3396–3398

    PubMed  CAS  Google Scholar 

  8. Coignard C, Hurst SF, Benjamin LE, Brandt ME, Warnock DW, Morrison CJ (2004) Resolution of discrepant results for Candida species identification by using DNA probes. J Clin Microbiol 42:858–861

    Article  PubMed  CAS  Google Scholar 

  9. Massonet C, Van Eldere J, Vaneechoutte M, De Baere T, Verhaegen J, Lagrou K (2004) Comparison of VITEK 2 with ITS2-fragment length polymorphism analysis for identification of yeast species. J Clin Microbiol 42:2209–2011

    Article  PubMed  CAS  Google Scholar 

  10. Chen SC, Halliday CL, Meyer W (2002) A review of nucleic acid-based diagnostic tests for systemic mycoses with an emphasis on polymerase chain reaction-based assays. Med Mycol 40:333–357

    Article  PubMed  CAS  Google Scholar 

  11. Summerbell RC, Lévesque CA, Seifert KA, Bovers M, Fell JW, Diaz MR, Boekhout T, de Hoog GS, Stalpers J, Crous PW (2005) Microcoding: the second step in DNA barcoding. Philos Trans R Soc Lond B Biol Sci 360:1897–1903

    Article  PubMed  CAS  Google Scholar 

  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 45:1152–1158

    Article  PubMed  CAS  Google Scholar 

  13. Leaw SN, Chang HC, Sun HF, Barton R, Bouchara JP, Chang TC (2006) Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. J Clin Microbiol 44:693–699

    Article  PubMed  CAS  Google Scholar 

  14. Ahmadian A, Ehn M, Hober S (2006) Pyrosequencing: history, biochemistry and future. Clin Chim Acta 363:83–94

    Article  PubMed  CAS  Google 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, California

    Google Scholar 

  16. Fakhrai-Rad H, Pourmand N, Ronaghi M (2002) Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms. Hum Mutat 19:479–485

    Article  PubMed  CAS  Google Scholar 

  17. Tavanti A, Davidson AD, Gow NAR, Maiden MCJ, Odds FC (2005) Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J Clin Microbiol 43:284–292

    Article  PubMed  CAS  Google Scholar 

  18. Gácser A, Schäfer W, Nosanchuk JS, Salomon S, Nosanchuk JD (2007) Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models. Fungal Genet Biol 44:1336–1341

    Article  PubMed  CAS  Google Scholar 

  19. Hazen KC (1995) New and emerging yeast pathogens. Clin Microbiol Rev 8:462–478

    PubMed  CAS  Google Scholar 

  20. Gharizadeh B, Norberg E, Löffler J, Jalal S, Tollemar J, Einsele H, Klingspor L, Nyrén P (2004) Identification of medically important fungi by the Pyrosequencing technology. Mycoses 47:29–33

    Article  PubMed  CAS  Google Scholar 

  21. Trama JP, Mordechai E, Adelson ME (2005) Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing. Mol Cell Probes 19:145–152

    Article  PubMed  CAS  Google 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 45:1735–1745

    Article  PubMed  CAS  Google Scholar 

  23. Rodriguez-Tudela JL, Diaz-Guerra TM, Mellado E, Cano V, Tapia C, Perkins A, Gomez-Lopez A, Rodero L, Cuenca-Estrella M (2005) Susceptibility patterns and molecular identification of Trichosporon species. Antimicrob Agents Chemother 49:4026–4034

    Article  PubMed  CAS  Google Scholar 

  24. Diaz MR, Fell JW (2005) Use of a suspension array for rapid identification of the varieties and genotypes of the Cryptococcus neoformans species complex. J Clin Microbiol 43:3662–3672

    Article  PubMed  CAS  Google Scholar 

  25. Kwon-Chung KJ, Varma A (2006) Do major species concepts support one, two or more species within Cryptococcus neoformans? FEMS Yeast Res 6:574–587

    Article  PubMed  CAS  Google 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 1762–1788

    Google 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 45:1874–1883

    Article  PubMed  CAS  Google Scholar 

  28. Fell JW, Scorzetti G (2004) Reassignment of the basidiomycetous yeasts Trichosporon pullulans to Guehomyces pullulans gen. nov., comb. nov. and Hyalodendron lignicola to Trichosporon lignicola comb. nov. Int J Syst Evol Microbiol 54:995–998

    Article  PubMed  CAS  Google Scholar 

  29. Holland SM, Shea YR, Kwon-Chung J (2004) Regarding “Trichosporon pullulans infection in 2 patients with chronic granulomatous disease.” J Allergy Clin Immunol 114:205–206

    Article  PubMed  Google Scholar 

  30. Takashima M, Sugita T, Shinoda T, Nakase T (2003) Three new combinations from the Cryptococcus laurentii complex: Cryptococcus aureus, Cryptococcus carnescens and Cryptococcus peneaus. Int J Syst Evol Microbiol 53:1187–1194

    Article  PubMed  CAS  Google Scholar 

  31. Ronaghi M (2000) Improved performance of pyrosequencing using single-stranded DNA-binding protein. Anal Biochem 286:282–288

    Article  PubMed  CAS  Google Scholar 

  32. Sullivan DJ, Moran GP, Coleman DC (2005) Candida dubliniensis: ten years on. FEMS Microbiol Lett 253:9–17

    Article  PubMed  CAS  Google 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, Utrecht

    Google Scholar 

  34. Giammanco GM, Melilli D, Pizzo G (2004) Candida pararugosa isolation from the oral cavity of an Italian denture wearer. Res Microbiol 155:571–574

    Article  PubMed  Google Scholar 

  35. Bai FY, Takashima M, Nakase T (2001) Description of Bullera kunmingensis sp. nov., and clarification of the taxonomic status of Bullera sinensis and its synonyms based on molecular phylogenetic analysis. FEMS Yeast Res 1:103–109

    Article  PubMed  CAS  Google Scholar 

  36. Guého E, Midgley G, Guillot J (1996) The genus Malassezia with description of four new species. Antonie Van Leeuwenhoek 69:337–355

    Article  PubMed  Google Scholar 

  37. Centraalbureau vor Schimmelcultures (CBS) (2007) Fungal Biodiversity Center Yeast Database. Home page at: http://www.cbs.knaw.nl/yeast/BioloMICS.aspx

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Microbiology Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, U.S. Department of Health and Human Services, 10 Center Drive, MSC 1508, Bethesda, MD, 20892-1508, USA

    C. I. Montero, Y. R. Shea, P. A. Jones, F. G. Witebsky & P. R. Murray

  2. Department of Clinical Microbiology, Albany Medical Center, 43 New Scotland Ave., Albany, NY, 12208, USA

    S. M. Harrington

  3. Biotage AB, Kungsgatan 76, Uppsala, 753 18, Sweden

    N. E. Tooke

Authors
  1. C. I. Montero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Y. R. Shea
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. A. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. M. Harrington
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N. E. Tooke
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. F. G. Witebsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. P. R. Murray
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. I. Montero.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Montero, C.I., Shea, Y.R., Jones, P.A. et al. Evaluation of Pyrosequencing® technology for the identification of clinically relevant non-dematiaceous yeasts and related species. Eur J Clin Microbiol Infect Dis 27, 821–830 (2008). https://doi.org/10.1007/s10096-008-0510-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10096-008-0510-x

Keywords

Search

Navigation