title: Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections.
abstract: The frequent lack of a positive and timely microbiological diagnosis in patients with lower respiratory tract infection (LRTI) is an important obstacle to antimicrobial stewardship. Patients are typically prescribed broad-spectrum empirical antibiotics while microbiology results are awaited, but, because these are often slow, negative, or inconclusive, de-escalation to narrow-spectrum agents rarely occurs in clinical practice. The aim of this study was to develop and evaluate two multiplex real-time PCR assays for the sensitive detection and accurate quantification of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. We found that all eight bacterial targets could be reliably quantified from sputum specimens down to a concentration of 100 CFUs/reaction (8333 CFUs/mL). Furthermore, all 249 positive control isolates were correctly detected with our assay, demonstrating effectiveness on both reference strains and local clinical isolates. The specificity was 98% on a panel of nearly 100 negative control isolates. Bacterial load was quantified accurately when three bacterial targets were present in mixtures of varying concentrations, mimicking likely clinical scenarios in LRTI. Concordance with culture was 100% for culture-positive sputum specimens, and 90% for bronchoalveolar lavage fluid specimens, and additional culture-negative bacterial infections were detected and quantified. In conclusion, a quantitative molecular test for eight key bacterial causes of LRTI has the potential to provide a more sensitive decision-making tool, closer to the time-point of patient admission than current standard methods. This should facilitate de-escalation from broad-spectrum to narrow-spectrum antibiotics, substantially improving patient management and supporting efforts to curtail inappropriate antibiotic use.
authors.affiliation: Medical Microbiology, Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK. Electronic address: naomi.gadsby@luht.scot.nhs.uk.
authors.forename: N J
authors.initials: NJ
authors.lastname: Gadsby
authors.affiliation: Medical Microbiology, Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK.
authors.forename: M P
authors.initials: MP
authors.lastname: McHugh
authors.affiliation: Medical Microbiology, Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK.
authors.forename: C D
authors.initials: CD
authors.lastname: Russell
authors.affiliation: Medical Microbiology, Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK.
authors.forename: H
authors.initials: H
authors.lastname: Mark
authors.affiliation: Department of Anaesthesia, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK.
authors.forename: A
authors.initials: A
authors.lastname: Conway Morris
authors.affiliation: Medical Microbiology, Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK.
authors.forename: I F
authors.initials: IF
authors.lastname: Laurenson
authors.affiliation: Respiratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK.
authors.forename: A T
authors.initials: AT
authors.lastname: Hill
authors.affiliation: Medical Microbiology, Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK.
authors.forename: K E
authors.initials: KE
authors.lastname: Templeton
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D058491:Bacterial Load; D001996:Bronchopneumonia / Q000175:diagnosis* / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial
keywords: Bacteria; PCR; molecular diagnostics; quantitative; respiratory infection