Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing...

Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.