1887

Abstract

To develop a rapid and quantitative diagnostic technique for the detection and identification of a wide range of fungi, an improved molecular method based on real-time PCR and the analysis of its products that targets the internal transcribed spacer (ITS) 2 region was established. The real-time PCR could quantitatively and specifically detect the ITS2 region from all 24 tested pathogenic fungal species at between 10 and 10 copies per test without amplification of bacterial or human DNA. The sequences of the primer-binding sites are conserved in the registered sequences of 34 other pathogenic fungal species, suggesting that the PCR would also detect these species. The hyperpolymorphic nature of the ITS2 region between fungal species in terms of length and nucleotide sequence provided valuable information for the determination of species. By labelling the 5′ end of the reverse primer with NED fluorescent dye, the fragment lengths of the real-time PCR products and their 3′-terminal fragments, derived using restriction enzyme FI digestion, were easily evaluated by capillary electrophoresis. Using this analysis, the number and species of fungi present in samples could be estimated. Moreover, sequence analysis of the real-time PCR products could accurately determine species in samples containing a single species. This diagnostic technique can estimate a wide range of fungi from various clinical samples within 1 day and accurately identify them in 2 days. Quantitative results for fungal titre in samples can also provide useful information for understanding the progression of disease and the efficacy of antifungal chemotherapy.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.006858-0
2009-08-01
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/jmm/58/8/1037.html?itemId=/content/journal/jmm/10.1099/jmm.0.006858-0&mimeType=html&fmt=ahah

References

  1. Chen, Y.-C., Eisner, J. D., Kattar, M. M., Rassoulian-Barrett, S. L., Lafe, K., Yarfitz, S. L., Limaye, A. P. & Cookson, B. T. ( 2000; ). Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes. J Clin Microbiol 38, 2302–2310.
    [Google Scholar]
  2. Chen, Y.-C., Eisner, J. D., Kattar, M. M., Rassoulian-Barrett, S. L., Lafe, K., Bui, U., Limaye, A. P. & Cookson, B. T. ( 2001; ). Polymorphic internal transcribed spacer region 1 DNA sequences identify medically important yeasts. J Clin Microbiol 39, 4042–4051.[CrossRef]
    [Google Scholar]
  3. De Baere, T., Claeys, G., Swinne, D., Massonet, C., Verschraegen, G., Muylaert, A. & Vaneechoutte, M. ( 2002; ). Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region. BMC Microbiol 2, 21 [CrossRef]
    [Google Scholar]
  4. Espinel-Ingroff, A., Stockman, L., Roberts, G., Pincus, D., Pollack, J. & Marler, J. ( 1998; ). Comparison of RapID yeast plus system with API 20C system for identification of common, new, and emerging yeast pathogens. J Clin Microbiol 36, 883–886.
    [Google Scholar]
  5. Esteve-Zarzoso, B., Belloch, C., Uruburu, F. & Querol, A. ( 1999; ). Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int J Syst Bacteriol 49, 329–337.[CrossRef]
    [Google Scholar]
  6. Goodrich, J. M., Reed, E. C., Mori, M., Fisher, L. D., Skerrett, S., Dandliker, P. S., Klis, B., Counts, G. W. & Meyers, J. D. ( 1991; ). Clinical features and analysis of risk factors for invasive candidal infection after marrow transplantation. J Infect Dis 164, 731–740.[CrossRef]
    [Google Scholar]
  7. Jordanides, N. E., Allan, E. K., McLintock, L. A., Copland, M., Devaney, M., Stewart, K., Parker, A. N., Johnson, P. R. E., Holyoake, T. L. & Jones, B. L. ( 2005; ). A prospective study of real-time panfungal PCR for the early diagnosis of invasive fungal infection in haemato-oncology patients. Bone Marrow Transplant 35, 389–395.[CrossRef]
    [Google Scholar]
  8. Kami, M., Fukui, T., Ogawa, S., Kazuyama, Y., Machida, U., Tanaka, Y., Kanda, Y., Kashima, T., Yamazaki, Y. & other authors ( 2001; ). Use of real-time PCR on blood samples for diagnosis of invasive aspergillosis. Clin Infect Dis 33, 1504–1512.[CrossRef]
    [Google Scholar]
  9. Kurtzman, C. P. & Robnett, C. J. ( 1997; ). Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5′ end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 35, 1216–1223.
    [Google Scholar]
  10. Leaw, S. N., Chang, H. C., Sun, H. F., Barton, R., Bouchara, J.-P. & Chang, T. C. ( 2006; ). Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. J Clin Microbiol 44, 693–699.[CrossRef]
    [Google Scholar]
  11. Löffler, J., Schumacher, U., Hebart, H., Reitze, H. & Einsele, H. ( 1996; ). Extraction of fungal DNA from cultures and blood using the QIAamp Tissue Kit. Qiagen News 4, 16–17.
    [Google Scholar]
  12. Löffler, J., Henke, N., Hebart, H., Schmidt, D., Hagmeyer, L., Schumacher, U. & Einsele, H. ( 2000; ). Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system. J Clin Microbiol 38, 586–590.
    [Google Scholar]
  13. Lott, T. J., Kuykendall, R. J. & Reiss, E. ( 1993; ). Nucleotide sequence analysis of the 5.8S rDNA and adjacent ITS2 region of Candida albicans and related species. Yeast 9, 1199–1206.[CrossRef]
    [Google Scholar]
  14. Lugert, R., Schettler, C. & Gross, U. ( 2006; ). Comparison of different protocols for DNA preparation and PCR for the detection of fungal pathogens in vitro. Mycoses 49, 298–304.[CrossRef]
    [Google Scholar]
  15. Makimura, K., Murayama, S. Y. & Yamaguchi, H. ( 1994; ). Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol 40, 358–364.[CrossRef]
    [Google Scholar]
  16. Millon, L., Piarroux, R., Drobacheff, C., Monod, M., Grenouillet, F., Bulle, B., Bole, J., Blancard, A. & Meillet, D. ( 2002; ). Evaluation of internal transcribed spacer region of ribosomal DNA sequence analysis for molecular characterization of Candida albicans and Candida dubliniensis isolates from HIV-infected patients. Med Mycol 40, 535–543.[CrossRef]
    [Google Scholar]
  17. Morrell, M., Fraser, V. J. & Kollef, M. H. ( 2005; ). Delaying the empiric treatment of Candida bloodstream infection until positive blood culture results are obtained: a potential risk factor for hospital mortality. Antimicrob Agents Chemother 49, 3640–3645.[CrossRef]
    [Google Scholar]
  18. Perfect, J. R. & Schell, W. A. ( 1996; ). The new fungal opportunists are coming. Clin Infect Dis 22, S112–S118.[CrossRef]
    [Google Scholar]
  19. Pryce, T. M., Palladino, S., Kay, I. D. & Coombs, G. W. ( 2003; ). Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis system. Med Mycol 41, 369–381.[CrossRef]
    [Google Scholar]
  20. Rakeman, J. L., Bui, U., Lafe, K., Chen, Y.-C., Honeycutt, R. J. & Cookson, B. T. ( 2005; ). Multilocus DNA sequence comparisons rapidly identify pathogenic molds. J Clin Microbiol 43, 3324–3333.[CrossRef]
    [Google Scholar]
  21. Turenne, C. Y., Sanche, S. E., Hoban, D. J., Karlowsky, J. A. & Kabani, A. M. ( 1999; ). Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system. J Clin Microbiol 37, 1846–1851.
    [Google Scholar]
  22. Velegraki, A., Kambouris, M. E., Skiniotis, G., Savala, M., Mitroussia-Ziouva, A. & Legakis, N. J. ( 1999; ). Identification of medically significant fungal genera by polymerase chain reaction followed by restriction enzyme analysis. FEMS Immunol Med Microbiol 23, 303–312.[CrossRef]
    [Google Scholar]
  23. Wald, A., Leisenring, W., Van Burik, J. A. & Bowden, R. A. ( 1997; ). Epidemiology of Aspergillus infections in a large cohort of patients undergoing bone marrow transplantation. J Infect Dis 175, 1459–1466.[CrossRef]
    [Google Scholar]
  24. Warnock, D. W. ( 2007; ). Trends in the epidemiology of invasive fungal infections. Nippon Ishinkin Gakkai Zasshi 48, 1–12.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.006858-0
Loading
/content/journal/jmm/10.1099/jmm.0.006858-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error