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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by Wellcome ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study did not collect patient's information or require taking additional samples from patients. Informed consent is waved by the Institutional Ethical Review Committee of Hospital of Tropical Diseases in Ho Chi Minh city. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript