Development and evaluation of a multiplex quantitative polymerase chain reaction assay for detecting bacteria associated with lower respiratory tract infection

OBJECTIVES: This study aimed to establish a multiplex quantitative polymerase chain reaction (MQ-PCR) assay for 12 bacterial pathogens found in lower respiratory tract infection (LRTI) and to evaluate its performance in a cohort of 211 patients with LRTI. METHODS: The study was divided into two stages: a pilot study to establish the methodology and a clinical validation study to evaluate its performance. In the pilot study, we established the MQ-PCR and analyzed its performance regarding limits of detection, reproducibility, specificity, and efficiency. In the clinical validation study, we obtained 211 sputum and/or bronchoalveolar lavage fluid (BALF) samples and detected pathogens by MQ-PCR. The MQ-PCR time was 3 h from sample collection to complete pathogen detection. RESULTS: copies/ml in BALF were applied, the sensitivity, specificity, and positive and negative predictive values of the MQ-PCR were 77% (95% confidence interval [CI] 67-88%), 94% (95% CI 93-95%), 25% (95% CI 19-31%), and 99% (95% CI 99-100%), respectively. CONCLUSIONS: This study demonstrates that the new MQ-PCR assay is time-saving, more effective and sensitive, and brings us closer to mainstream adoption of quantitative molecular detection of bacteria.