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

Lehmann, Lutz Eric; Hunfeld, Klaus-Peter; Emrich, Thomas; Haberhausen, Gerd; Wissing, Heimo; Hoeft, Andreas; Stüber, Frank (2008). A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples. Medical microbiology and immunology, 197(3), pp. 313-24. Berlin: Springer 10.1007/s00430-007-0063-0

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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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