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Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infection.
Clin Microbiol Infect. 2015 May 13;
Authors: Gadsby NJ, McHugh MP, Russell CD, Mark H, Conway Morris A, Laurenson IF, Hill AT, Templeton KE
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 as 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 mRT-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 samples down to a concentration of 100 cfu/reaction (8333 cfu/ml). Furthermore, all 249 positive control isolates were correctly detected with our assay, demonstrating effectiveness on both reference strains and local clinical isolates. 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 sputa, 90% for bronchoalveolar lavage fluids 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.
PMID: 25980353 [PubMed - as supplied by publisher]