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

• Published in: BMC infectious diseases Vol. 24; no. 1; p. 164
• Main Authors: Dung, Tran Thi Ngoc, Phat, Voong Vinh, Vinh, Chau, Lan, Nguyen Phu Huong, Phuong, Nguyen Luong Nha, Ngan, Le Thi Quynh, Thwaites, Guy, Thwaites, Louise, Rabaa, Maia, Nguyen, Anh T K, Duy, Pham Thanh
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 07.02.2024; BioMed Central; BMC
• Subjects: Acinetobacter baumannii; Antibiotics; Antigens; Antimicrobial agents; Antimicrobial resistance; Assaying; Bacteria; Bacteria, Pathogenic; Bacterial pneumonia; Care and treatment; Causes of; Diagnosis; Drug resistance; Drug resistance in microorganisms; E coli; Escherichia coli; Genes; Genetic aspects; Health aspects; Identification and classification; Israel; Klebsiella; Klebsiella pneumoniae; Low income groups; Lower respiratory tract infections; Melting curve; Melting curve analysis; Molecular diagnostics; Morbidity; Mortality; Multiplexing; Pathogenic microorganisms; Pathogens; Phenotypes; Pneumonia; Polymerase chain reaction; Pseudomonas aeruginosa; Real time; Real-time PCR; Respiratory tract; Respiratory tract infection; Respiratory tract infections; Sensitivity; Sputum; Staphylococcal infections; Staphylococcus aureus; Streptococcus infections; Streptococcus pneumoniae; Target detection; Vietnam; Israel; United Kingdom > UK; United States > US; Lower respiratory tract infections; Melting curve analysis; Molecular diagnostics; Antimicrobial resistance; Real-time PCR
• ISSN: 1471-2334; 1471-2334
• DOI: 10.1186/s12879-024-09028-2

Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus are major bacterial causes of lower respiratory tract infections (LRTIs) globally, leading to substantial morbidity and mortality. The rapid increase of antimicrobial resistance (AMR) in these pathogens poses significant challenges for their effective antibiotic therapy. In low-resourced settings, patients with LRTIs are prescribed antibiotics empirically while awaiting several days for culture results. Rapid pathogen and AMR gene detection could prompt optimal antibiotic use and improve outcomes. 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, limit of detection (LOD) of real-time PCR assays for pathogen detection were evaluated using DNA control mixes and spiked tracheal aspirate. The performance of RT-PCR assays was subsequently compared with the gold standard, conventional culture on 50 tracheal aspirate and sputum specimens of ICU patients. 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 of all pathogens between the two methods varied from 0.63 to 0.95. The limit of detection of target bacteria was 1600 CFU/ml. The quantitative results from the PCR assays demonstrated 100% concordance with quantitative culture of tracheal aspirates. Compared to culture, PCR assays exhibited higher sensitivity in detecting mixed infections and S. pneumoniae. There was a high level of concordance between the detection of AMR gene and AMR phenotype in single infections. Our multiplex quantitative RT-PCR assays are fast and simple, but sensitive and specific in detecting six bacterial pathogens of LRTIs and their antimicrobial resistance genes and should be further evaluated for clinical utility.