Development and validation of multiplex real-time PCR for simultaneous detection of six bacterial pathogens causing lower respiratory tract infections and antimicrobial resistance genes

Background

Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus are the leading bacterial causes of lower respiratory tract infections (LRTIs) globally, causing significant morbidity and mortality. The rapid rise of antimicrobial resistance (AMR) in these pathogens poses significant challenges to effective antibiotic therapies. In low-resource settings, patients with LRTI are empirically prescribed antibiotics while waiting several days for culture results. Rapid pathogen and AMR gene detection may promote optimal antibiotic use and improve outcomes.

Methods

Here, we developed multiplex quantitative real-time PCR using EvaGreen dye and melting curve analysis to rapidly identify six major pathogens and fourteen AMR genes directly from respiratory samples. The reproducibility, linearity, and limit of detection (LOD) of real-time PCR assays for pathogen detection were evaluated using DNA control mixtures and spiked tracheal aspirate. The performance of RT-PCR assays was then compared with the gold standard, conventional culture, on 50 tracheal aspirate and sputum samples from intensive care patients.

Results

The sensitivity of RT-PCR assays was 100% for K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and 63.6% for S. aureus, and the specificity ranged from 87.5% to 97.6%. The kappa correlation values ​​for all pathogens between the two methods ranged from 0.63 to 0.95. The detection limit of target bacteria was 1600 CFU/ml. Quantitative results obtained from PCR assays showed 100% agreement with quantitative culture of tracheal aspirates. Compared to culture, PCR assays showed higher sensitivity in detecting mixed infections and S. pneumoniae. There was a high level of agreement between the detection of AMR gene and AMR phenotype in single infections.

Results

Our multiplex quantitative RT-PCR assays are rapid and simple, yet sensitive and specific in detecting six bacterial LRTI pathogens and their antimicrobial resistance genes, and should be further evaluated for clinical utility.