1887

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Volume 52, Issue 3

Detection of seven species using the Light-Cycler system Free

Abstract

Due to the limitations of classical methods for the detection of systemic fungal infections and the high mortality rates associated with these infections, it has become essential to develop a quick, sensitive and specific detection assay. By using the Idaho Technologies Light-Cycler system, a qualitative real-time PCR system has been developed for the detection of the leading causes of systemic infection within the genus . The sensitivity of the assay was comparable to previously described PCR methods (1–5 c.f.u. ml) and, by the use of a single probe, it was able to detect, but not differentiate between, seven species of (, , , , , and ). Single-round amplification on the Light-Cycler allowed rapid turn-around of clinical samples (within one working day) and it was shown to be more sensitive than classical procedures, exposing 39 possible systemic infections that were not detected by blood culture.

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© 2003 Society for General Microbiology
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2003-03-01
2024-04-18
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References

  1. Altschul S. F, Madden T. L, Schaffer A. A, Zhang J, Zhang Z, Miller W, Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
     [Google Scholar]
  2. Ascioglu S, Rex J. H, de Pauw B. 17 other authors 2002; Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin Infect Dis 34:7–14 [CrossRef]
     [Google Scholar]
  3. 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]
  4. Corless C. E, Guiver M, Borrow R, Edwards-Jones V, Fox A. J, Kaczmarski E. B. 2001; Simultaneous detection of Neisseria meningitidis , Haemophilus influenzae , and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 39:1553–1558
     [Google Scholar]
  5. Denning D. W. 1998; Invasive aspergillosis. Clin Infect Dis 26:781–803 [CrossRef]
     [Google Scholar]
  6. Einsele H, Hebart H, Roller G. 8 other authors 1997; Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 35:1353–1360
     [Google Scholar]
  7. Funato T, Satou N, Abukawa D, Satou J, Abe Y, Ishii K. K, Iinuma K, Kaku M, Sasaski T. 2001; Quantitative evaluation of cytomegalovirus DNA in infantile hepatitis. J Viral Hepat 8:217–222 [CrossRef]
     [Google Scholar]
  8. Gleason T. G, May A. K, Caparelli D, Farr B. M, Sawyer R. G. 1997; Emerging evidence of selection of fluconazole-tolerant fungi in surgical intensive care units. Arch Surg 132:1197–1201 [CrossRef]
     [Google Scholar]
  9. Guiver M, Levi K, Oppenheim B. A. 2001; Rapid identification of Candida species by Taqman PCR. J Clin Pathol 54:362–366 [CrossRef]
     [Google Scholar]
  10. Jaeger E. E. M, Carroll N. M, Choudhury S, Dunlop A. A, Towler H. M, Matheson M. M, Adamson P, Okhravi N, Lightman S. 2000; Rapid detection and identification of Candida , Aspergillus , and Fusarium species in ocular samples using nested PCR. J Clin Microbiol 38:2902–2908
     [Google Scholar]
  11. Kawai S, Yokosuka O, Kanda T, Imazeki F, Maru Y, Saisho H. 1999; Quantification of hepatitis C virus by TaqMan PCR: comparison with HCV Amplicor Monitor assay. J Med Virol 58:121–126 [CrossRef]
     [Google Scholar]
  12. Lehmann P. F, Lin D, Lasker B. A. 1992; Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA. J Clin Microbiol 30:3249–3254
     [Google Scholar]
  13. Loeffler J, Hebart H, Schumacher U, Reitze H, Einsele H. 1997; Comparison of different methods for extraction of DNA of fungal pathogens from cultures and blood. J Clin Microbiol 35:3311–3312
     [Google Scholar]
  14. Loeffler J, Hebart H, Sepe S, Schumacher U, Klingebiel T, Einsele H. 1998; Detection of PCR-amplified fungal DNA by using a PCR-ELISA system. Med Mycol 36:275–279 [CrossRef]
     [Google Scholar]
  15. Loeffler J, Hebart H, Bialek R, Hagmeyer L, Schmidt D, Serey F. P, Hartmann M, Eucker J, Einsele H. 1999; Contaminations occurring in fungal PCR assays. J Clin Microbiol 37:1200–1202
     [Google Scholar]
  16. Loeffler J, Hagmeyer L, Hebart H, Henke N, Schumacher U, Einsele H. 2000a; Rapid detection of point mutations by fluorescence resonance energy transfer and probe melting curves in Candida species. Clin Chem 46:631–635
     [Google Scholar]
  17. Loeffler J, Henke N, Hebart H, Schmidt D, Hagmeyer L, Schumacher U, Einsele H. 2000b; Quantification of fungal DNA by using fluorescence resonance energy transfer and the Light Cycler system. J Clin Microbiol 38:586–590
     [Google Scholar]
  18. Loeffler J, Hebart H, Cox P, Flues N, Schumacher U, Einsele H. 2001; Nucleic acid sequence-based amplification of Aspergillus RNA in blood samples. J Clin Microbiol 39:1626–1629 [CrossRef]
     [Google Scholar]
  19. Mathis A, Weber R, Kuster H, Speich R. 1997; Simplified sample processing combined with a sensitive one-tube nested PCR assay for detection of Pneumocystis carinii in respiratory specimens. J Clin Microbiol 35:1691–1695
     [Google Scholar]
  20. Metzgar D, van Belkum A, Field D, Haubrich R, Wills C. 1998; Random amplification of polymorphic DNA and microsatellite genotyping of pre- and posttreatment isolates of Candida spp. from human immunodeficiency virus-infected patients on different fluconazole regimens. J Clin Microbiol 36:2308–2313
     [Google Scholar]
  21. Morace G, Sanguinetti M, Posteraro B, Lo Cascio G, Fadda G. 1997; Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis. J Clin Microbiol 35:667–672
     [Google Scholar]
  22. Mygind T, Birkelund S, Falk E, Christiansen G. 2001; Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry. J Microbiol Methods 46:241–251
     [Google Scholar]
  23. Nishikawa A, Sugita T, Shinoda T. 1999; Rapid identification of Debaryomyces hansenii / Candida famata by polymerase chain reaction. Med Mycol 37:101–104 [CrossRef]
     [Google Scholar]
  24. Palladino S, Kay I, Fonte R, Flexman J. 2001; Use of real-time PCR and the LightCycler system for the rapid detection of Pneumocystis carinii in respiratory specimens. Diagn Microbiol Infect Dis 39:233–236 [CrossRef]
     [Google Scholar]
  25. Qi Y, Patra G, Liang X, Williams L. E, Rose S, Redkar R. J, DelVecchio V. G. 2001; Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis . Appl Environ Microbiol 67:3720–3727 [CrossRef]
     [Google Scholar]
  26. Rantakokko-Jalava K, Jalava J. 2001; Development of conventional and real-time PCR assays for detection of Legionella DNA in respiratory specimens. J Clin Microbiol 39:2904–2910 [CrossRef]
     [Google Scholar]
  27. Reiss E, Morrison C. J. 1993; Nonculture methods for diagnosis of disseminated candidiasis. Clin Microbiol Rev 6:311–323
     [Google Scholar]
  28. Rimek D, Garg A. P, Haas W. H, Kappe R. 1999; Identification of contaminating fungal DNA sequences in Zymolyase. J Clin Microbiol 37:830–831
     [Google Scholar]
  29. Ririe K. M, Rasmussen R. P, Wittwer C. T. 1997; Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160 [CrossRef]
     [Google Scholar]
  30. Schroter M, Zollner B, Schafer P, Laufs R, Feucht H. H. 2001; Quantitative detection of hepatitis C virus RNA by light cycler PCR and comparison with two different PCR assays. J Clin Microbiol 39:765–768 [CrossRef]
     [Google Scholar]
  31. Schutten M, van den Hoogen B, van der Ende M. E, Gruters R. A, Osterhaus A. D, Niesters H. G. 2000; Development of a real-time quantitative RT-PCR for the detection of HIV-2 RNA in plasma. J Virol Methods 88:81–87 [CrossRef]
     [Google Scholar]
  32. van Elden L. J, Nijhuis M, Schipper P, Schuurman R, van Loon A. M. 2001; Simultaneous detection of influenza viruses A and B using real-time quantitative PCR. J Clin Microbiol 39:196–200 [CrossRef]
     [Google Scholar]
  33. Whalley S. A, Brown D, Teo C. G, Dusheiko G. M, Saunders N. A. 2001; Monitoring the emergence of hepatitis B virus polymerase gene variants during lamivudine therapy using the LightCycler. J Clin Microbiol 39:1456–1459 [CrossRef]
     [Google Scholar]
  34. Whimbey E, Kiehn T. E, Brannon P, Blevins A, Armstrong D. 1987; Bacteremia and fungemia in patients with neoplastic disease. Am J Med 82:723–730 [CrossRef]
     [Google Scholar]
  35. White T. J, Burns T, Lee S, Taylor J. 1990; Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a Guide to Methods and Applications pp 315–322 Edited by Innis M. A., Gelfand D. H., Sninsky J. J., White T. J. San Diego: Academic Press;
     [Google Scholar]
  36. Widjojoatmodjo M. N, Borst A, Schukkink R. A. F, Box A. T, Tacken N. M, Van Gemen B, Verhoef J, Top B, Fluit A. C. 1999; Nucleic acid sequence-based analysis (NASBA) detection of medically important Candida species. J Microbiol Methods 38:81–90 [CrossRef]
     [Google Scholar]
  37. Williams D. W, Wilson M. J, Lewis M. A. O, Potts A. J. C. 1995; Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA. J Clin Microbiol 33:2476–2479
     [Google Scholar]
  38. Wright W. L, Wenzel R. P. 1997; Nosocomial Candida. Epidemiology, transmission, and prevention. Infect Dis Clin North Am 11:411–425 [CrossRef]
     [Google Scholar]
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Detection of seven Candida species using the Light-Cycler system
J. Med. Microbiol. 52, 229 (2003); https://doi.org/10.1099/jmm.0.05049-0
/content/journal/jmm/10.1099/jmm.0.05049-0
/content/journal/jmm/10.1099/jmm.0.05049-0
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