Development and diagnostic performance of a sensitive multiplex pneumonia pathogen identification assay for emergency department patients with pneumonia

Pneumonia imposes a significant global health burden, with high morbidity and mortality, and challenges in pathogen identification due to diverse etiologies. Conventional culture-based methods are time-consuming and insufficient for fastidious organisms, necessitating advanced diagnostic tools for rapid and accurate pathogen identification, especially in emergency department (ED) settings. To address this, we developed the pneumonia pathogen identification (PPID) assay, a multiplex diagnostic platform based on the single-stranded multiplex PCR amplicons with suspension bead array technology. The PPID assay detects 27 bacterial and viral pathogens within 6 hours. Analytical validation using nucleic acids from reference strains or synthetic oligonucleotides demonstrated high specificity and sensitivity (5–500 copies per reaction). In 135 ED cases with pneumonia, the PPID assay significantly outperformed microbiological culture methods (77.8% vs 45.2%), increasing the overall detection rate to 85.9% when combined. It effectively identified polymicrobial infections in 48.6% of positive cases, including bacterial-viral co-infections, with Klebsiella pneumoniae being the most frequently identified pathogen overall and in mixed infections. These results highlight the PPID assay’s rapid and comprehensive diagnostic capability, facilitating early and targeted treatment, particularly in acute care settings. This innovative approach addresses critical unmet clinical needs and improves patient outcomes. IMPORTANCE The pneumonia pathogen identification assay is a high-throughput multiplex molecular test that detects 27 respiratory pathogens within 6 hours for up to 96 samples. It exhibits high specificity and sensitivity and enhances detection of fastidious pathogens and co-infections, addressing critical diagnostic challenges in emergency settings.