Prospective multicenter study identifying prognostic biomarkers and microbial profiles in severe CAP using BALF, blood mNGS, and PBMC transcriptomics.

To identify potential biomarkers associated with the prognosis and severity of severe community-acquired pneumonia (SCAP), we conducted a multi-center prospective study from January 1, 2022, to December 31, 2023, enrolling 14 mild CAP and 75 SCAP patients in Shanghai, China. Patients underwent bronchoalveolar lavage fluid (BALF) and blood metagenomic next-generation sequencing (mNGS) as well as peripheral blood mononuclear cell (PBMC) transcriptomics. Among the 75 SCAP patients, 32 died within 30 days, with older age, a history of allergies, and comorbidities like cerebrovascular disease linked to worse outcomes. BALF mNGS showed greater microbial diversity, revealing a higher prevalence of pathogens, including Acinetobacter baumannii, Klebsiella pneumoniae, and Candida albicans, compared to mild CAP patients. RNA sequencing identified 431 differentially expressed genes in deceased SCAP patients, with significant alterations in immune pathways. Notably, microbial markers such as Pneumocystis jirovecii and viral markers like Human cytomegalovirus were associated with poor outcomes. Transcriptomic biomarkers, including otoferlin (OTOF), MS4A4A, and SIGLEC1, were identified as potential prognostic indicators for SCAP severity. GSEA and traditional GO/KEGG analyses identified key immune and metabolic pathways in SCAP (death) patients, including upregulation of complement activation, oxidative phosphorylation, nitrogen metabolism, while downregulation of adaptive immune response, hematopoietic cell lineage, and antigen processing pathways. These findings underscore the superiority of BALF mNGS over blood mNGS for pathogen detection, revealing microbial differences and transcriptomic alterations between mild and severe cases while identifying prognostic markers for SCAP treatment strategies.