Med Microbiol Immunol (2008) 197:313–324 DOI 10.1007/s00430-007-0063-0 123 ORIGINAL INVESTIGATION A multiplex real-time PCR assay for rapid detection and diVerentiation of 25 bacterial and fungal pathogens from whole blood samples Lutz Eric Lehmann · Klaus-Peter Hunfeld · Thomas Emrich · Gerd Haberhausen · Heimo Wissing · Andreas Hoeft · Frank Stüber Received: 29 June 2007 / Published online: 16 November 2007  Springer-Verlag 2007 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. aeru- ginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identiWcation results with conventional microbiology for 1,548 clinical isolates yielded an overall speciWcity of 98.8%. The analytical speciWcity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identiWcation in clinical sepsis. Keywords Bacterial and fungal pathogens · Blood culture · ICU · Multiplex PCR assay · Sepsis · Simultaneous detection Introduction Blood culture, which currently remains the gold standard in the microbiological diagnosis of bacterial or fungal blood- stream infections (BSI), typically becomes positive 8–36 h after sampling, and therapy can then be adapted based on presumptive bacterial identiWcation suggested by Gram-stain characteristics. A more precise pathogen identiWcation and susceptibility proWle, however, is not available until up to 24–48 h [1, 2]. For the majority of patients with clinically apparent sepsis, the blood culture is negative, making the optimal antimicrobial therapy empiric [3]. Early detection and adequate treatment of causative pathogens within the Wrst 6–12 h, however, is critical for a favorable outcome in patients with BSI [4–6]. The development of rapid diagnos- tic methods for BSI has been identiWed as an important med- ical need to supplement conventional blood culture diagnostics and molecular techniques have potential to fulWll this need [1]. Nucleic acid based diagnostic systems, includ- ing polymerase chain reaction (PCR) methods as well as the application of DNA and RNA probes are well known sensi- tive techniques for a more rapid detection and the speciWc identiWcation of pathogens involved in BSI [7–15]. From a theoretical point of view, PCR-based diagnostic techniques, therefore, hold promise for sensitive and speciWc detection Lutz Eric Lehmann and Klaus-Peter Hunfeld contributed equally to this work. L. Lehmann · A. Hoeft · F. Stüber Klinik und Poliklinik für Anästhesie und operative Intensivmedizin, Universitätsklinikum Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany H. Wissing Zentrum der Anästhesiologie und Wiederbelebung, Universitätsklinikum Frankfurt am Main, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany K.-P. Hunfeld (&) Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Universitätsklinikum Frankfurt am Main, Paul-Ehrlich Str. 40, 60596 Frankfurt am Main, Germany e-mail: k.hunfeld@em.uni-frankfurt.de T. Emrich · G. Haberhausen Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany