Clinical validation of a novel multiplex real-time PCR for diagnosing community-acquired pneumonia in the emergency department: a promising diagnostic tool for Mycoplasma pneumoniae

Community-acquired pneumonia (CAP) is a global public health burden with high morbidity, but timely diagnosis is hindered by limitations of conventional methods. We validated a novel multiplex real-time PCR (mRT-PCR) for CAP diagnosis in the emergency department, focusing on Mycoplasma pneumoniae ( M. pneumoniae ). A retrospective cohort of 371 CAP patients (2019–2021) from the Chinese PLA General Hospital was enrolled. The panel targeted 85 pathogens (40 bacteria, 18 viruses, 17 fungi, 10 other) plus 8 resistance genes, with a turnaround time (TAT) under 4 h and a cost of approximately US$100 per test. mRT-PCR showed performance comparable to mNGS for common pathogens (concordance >69% for bacteria/fungi) and high concordance with the final clinical diagnosis. Notably, it detected M. pneumoniae in 36.7% (136/371) of cases, outperforming conventional methods by identifying 204 exclusive positives (55.0% of cohort), with M. pneumoniae (40%) and viruses (30%) predominating. Polymicrobial infections occurred in 36.1% of cases; M. pneumoniae co-infections (38 cases) showed elevated neutrophils and reduced lymphocytes vs. single infections. An independent reference standard analysis excluding both index tests showed that 24.1% (210/873) of calls were independently corroborated - high for influenza, SARS-CoV-2 and M. pneumoniae , lower for S. pneumoniae , consistent with the limited sensitivity of sputum culture. Unlike conventional syndromic panels such as FilmArray, which profile fewer targets without concurrent resistance-gene detection, this mRT-PCR combines broad coverage with rapid TAT and low cost, making it promising for timely CAP diagnosis, particularly in resource-limited settings and for targeted therapy.