title: A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples.
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.
authors.affiliation: Klinik und Poliklinik für Anästhesie und operative Intensivmedizin, Universitätsklinikum Bonn, Sigmund-Freud-Strasse 25, Bonn, Germany.
authors.forename: Lutz Eric
authors.initials: LE
authors.lastname: Lehmann
authors.forename: Klaus-Peter
authors.initials: KP
authors.lastname: Hunfeld
authors.forename: Thomas
authors.initials: T
authors.lastname: Emrich
authors.forename: Gerd
authors.initials: G
authors.lastname: Haberhausen
authors.forename: Heimo
authors.initials: H
authors.lastname: Wissing
authors.forename: Andreas
authors.initials: A
authors.lastname: Hoeft
authors.forename: Frank
authors.initials: F
authors.lastname: Stüber
mesh_terms: D016470:Bacteremia / Q000382:microbiology*; D001419:Bacteria / Q000145:classification / Q000302:isolation & purification*; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000032:analysis; D004271:DNA, Fungal / Q000032:analysis; D016469:Fungemia / Q000382:microbiology*; D005658:Fungi / Q000145:classification / Q000302:isolation & purification*; D006801:Humans; D016533:Mycological Typing Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal