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A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

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Abstract

Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.

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Authors and Affiliations

  1. Klinik und Poliklinik für Anästhesie und operative Intensivmedizin, Universitätsklinikum Bonn, Sigmund-Freud-Str. 25, 53105, Bonn, Germany

    Lutz Eric Lehmann, Andreas Hoeft & Frank Stüber

  2. Zentrum der Anästhesiologie und Wiederbelebung, Universitätsklinikum Frankfurt am Main, Theodor-Stern-Kai 7, 60596, Frankfurt am Main, Germany

    Heimo Wissing

  3. Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Universitätsklinikum Frankfurt am Main, Paul-Ehrlich Str. 40, 60596, Frankfurt am Main, Germany

    Klaus-Peter Hunfeld

  4. Roche Diagnostics GmbH, Nonnenwald 2, 82377, Penzberg, Germany

    Thomas Emrich & Gerd Haberhausen

Authors
  1. Lutz Eric Lehmann
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  2. Klaus-Peter Hunfeld
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  3. Thomas Emrich
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  4. Gerd Haberhausen
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  5. Heimo Wissing
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  6. Andreas Hoeft
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  7. Frank Stüber
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Corresponding author

Correspondence to Klaus-Peter Hunfeld.

Additional information

Lutz Eric Lehmann and Klaus-Peter Hunfeld contributed equally to this work.

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Lehmann, L., Hunfeld, KP., Emrich, T. et al. A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples. Med Microbiol Immunol 197, 313–324 (2008). https://doi.org/10.1007/s00430-007-0063-0

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  • DOI: https://doi.org/10.1007/s00430-007-0063-0

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