Journal Article

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

Nguyen, Anh T. K., Duy, Pham Thanh, Rabaa, Maia, Thwaites, Guy, Vinh, Chau, Thwaites, Louise, Dung, Tran Thi Ngoc, Ngan, Le Thi Quynh, Lan, Nguyen Phu Huong, Phat, Voong Vinh, Phuong, Nguyen Luong Nha

2024

Overview

Background 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. 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, 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. 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 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. Conclusions 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Acinetobacter baumannii / Anti-Bacterial Agents - pharmacology / Anti-Bacterial Agents - therapeutic use / Antibiotics / Antigens / Antimicrobial agents / Antimicrobial resistance / Assaying / Bacteria / Bacteria - genetics / Bacteria, Pathogenic / Bacterial pneumonia / Care and treatment / Causes of / Diagnosis / Drug resistance / Drug resistance in microorganisms / Drug Resistance, Bacterial / E coli / Escherichia coli / Escherichia coli - genetics / Genes / Genetic aspects / Health aspects / Humans / Identification and classification / Infectious Diseases / Internal Medicine / Israel / Klebsiella / Klebsiella pneumoniae / Klebsiella pneumoniae - genetics / Low income groups / Lower respiratory tract infections / Medical Microbiology / Medicine / Medicine & Public Health / Melting curve / Melting curve analysis / Molecular diagnostics / Morbidity / Mortality / Multiplex Polymerase Chain Reaction - methods / Multiplexing / Parasitology / Pathogenic microorganisms / Pathogens / Phenotypes / Pneumonia / Polymerase chain reaction / Pseudomonas aeruginosa / Real time / Real-time PCR / Real-Time Polymerase Chain Reaction - methods / Reproducibility of Results / Respiratory tract / Respiratory tract infection / Respiratory tract infections / Respiratory Tract Infections - diagnosis / Respiratory Tract Infections - microbiology / Sensitivity / Sputum / Staphylococcal infections / Staphylococcus aureus / Staphylococcus aureus - genetics / Streptococcus infections / Streptococcus pneumoniae / Streptococcus pneumoniae - genetics / Target detection / Tropical Medicine