The development and validation of multiplex real-time PCRs with fluorescent melting curve analysis for simultaneous detection of six bacterial pathogens of lower respiratory tract infections and antimicrobial resistance genes.

Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus are among the major bacterial causative agents of lower respiratory tract infections (LRTIs), causing substantial morbidity and mortality globally. The rapid increase of antimicrobial resistance (AMR) in these pathogens poses significant challenges for effective antibiotic therapy of LRTIs. In low-resourced settings, the diagnostics of LRTIs relies heavily on microbiological culture and patients are often treated with empirical antibiotics while awaiting several days for culture results. Rapid detection of LRTIs pathogens and AMR genes could prompt early antibiotic switching and inform antibiotic treatment duration. In this study, we developed multiplex quantitative real-time PCRs using EvaGreen dye and melting curve analysis (MCA) to rapidly identify the six major LRTIs pathogens and their AMR genes directly from the tracheal aspirate and sputum samples. The accuracy of RT-PCRs was assessed by comparing its performance against the gold standard, conventional culture method on 50 tracheal aspirate and sputum specimens. Our RT-PCR assays had 100% sensitivity for K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and 63.6% for S. aureus and the specificity ranked from 87.5% to 97.6%. The kappa correlation values of all pathogens between the two methods varied from 0.63 to 0.95. The limit of detection (LOD) of target bacteria in multiplex RT-PCRs was 1600 CFU/mL. Compared to the culture results, PCR assays exhibited higher sensitivity in detecting mixed infections and S. pneumoniae. Our findings also demonstrated a high level of concordance between the detection of AMR gene and AMR phenotype in single infections. We conclude that our multiplex quantitative RT-PCRs with fluorescence MCA is simple but sensitive and specific in detecting six major drug resistant bacterial pathogens of LRTIs and should be further evaluated for clinical utility.