1887

Abstract

Toxoplasma reactivation is a serious complication in patients receiving allogenic stem cell transplantation. Real-time PCR assays allow a rapid diagnosis of toxoplasma infection; however, no comparative data are available on the performance of real-time PCR protocols under routine conditions. Therefore, the aim of this study was to amplify DNA from routine samples of allogenic stem cell recipients using two real-time PCR assays on a LightCycler, and using conventional nested PCR. Conventional nested PCR revealed DNA in 16 samples. Only 12 of the 16 samples yielded a positive result in both real-time PCRs. The accuracy of the conventional PCR results was demonstrated by direct sequencing. Amplification and detection of the amplicon was completed in only 1 h using the real-time PCR assays. Thus, real-time PCR substantially accelerates the detection of DNA in the majority of positive specimens; however, conventional nested PCR is required for detection of DNA in some samples.

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2004-07-01
2019-11-22
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References

  1. Bell, A. S. & Ranford-Cartwright, L. C. ( 2002;). Real-time quantitative PCR in parasitology. Trends Parasitol 18, 337–342.
    [Google Scholar]
  2. Burg, J. L., Grover, C. M., Pouletty, P. & Boothroyd, J. C. ( 1989;). Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbiol 27, 1787–1792.
    [Google Scholar]
  3. Costa, J. M., Pautas, C., Ernault, P., Foulet, F., Cordonnier, C. & Bretagne, S. ( 2000;). Real-time PCR for diagnosis and follow-up of toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes. J Clin Microbiol 38, 2929–2932.
    [Google Scholar]
  4. Homan, W. L., Vercammen, M., De Braekeleer, J. & Verschueren, H. ( 2000;). Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int J Parasitol 30, 69–75.[CrossRef]
    [Google Scholar]
  5. Mele, A., Paterson, P. J., Prentice, H. G., Leoni, P. & Kibbler, C. C. ( 2002;). Toxoplasmosis in bone marrow transplantation: a report of two cases and systematic review of the literature. Bone Marrow Transplant 29, 691–698.[CrossRef]
    [Google Scholar]
  6. Murray, L. J., Lee, R. & Martens, C. ( 1990;). In vivo cytokine gene expression in T cell subsets of the autoimmune MRL/Mp-lpr/lpr mouse. Eur J Immunol 20, 163–170.[CrossRef]
    [Google Scholar]
  7. Reischl, U., Bretagne, S., Krüger, D., Ernault, P. & Costa, J. M. ( 2003;). Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect Dis 3, 7. 7.[CrossRef]
    [Google Scholar]
  8. Roth, A., Roth, B., Hoffken, G., Steuber, S., Khalifa, K. I. & Janitschke, K. ( 1992;). Application of the polymerase chain reaction in the diagnosis of pulmonary toxoplasmosis in immunocompromised patients. Eur J Clin Microbiol Infect Dis 11, 1177–1181.[CrossRef]
    [Google Scholar]
  9. Roth, A., Mauch, H. & Göbel, U. B. ( 2001;). Quality standards for microbiological diagnostic techniques for infectious diseases. In Nucleic Acid Amplification Techniques, pp.1–28. Munich: Urban & Fischer.
  10. Teo, I. A., Choi, J. W., Morlese, J., Taylor, G. & Shaunak, S. ( 2002;). LightCycler qPCR optimisation for low copy number target DNA. J Immunol Methods 270, 119–133.[CrossRef]
    [Google Scholar]
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