Neutrophil-specific transcriptomic profiling reveals a novel signature for active tuberculosis diagnosis

Tuberculosis remains a leading cause of mortality worldwide. Rapid and accurate diagnosis of active tuberculosis (ATB) is critical for its effective treatment and disease control. However, current sputum-based detection methods have limited diagnostic coverage for ATB, resulting in missed cases and treatment delay. In this study, we performed neutrophil RNA sequencing (RNA-seq) to identify ATB-specific transcriptional signatures. Through integrated analysis of differentially expressed genes (DEGs) across 173 samples (67 ATB, 42 latent tuberculosis infection [LTBI], and 64 healthy controls [HC]), followed by quantitative Real-time polymerase chain reaction (qPCR) validation using 141 samples (31 ATB, 53 LTBI, 57 HC). A novel 4-gene neutrophil-derived tuberculosis signature (neu-TB) was screened by Lasso model prediction, and further evaluation were performed in two independent cohorts (<i>n</i> = 332). The signature achieved exceptional discrimination between ATB and LTBI (area under curve [AUC] = 0.975; 95% confidence interval [CI]: 0.949-1.000; <i>P</i> < 0.0001), with 96.8% sensitivity and 92.5% specificity. When distinguishing ATB from HC, the performance remained superior (AUC = 0.980, 91.9% sensitivity, 96.5% specificity). Notably, the signature effectively differentiated ATB from other pulmonary diseases (AUC = 0.969, 95% CI: 0.943-0.995, 93.0% sensitivity, 87.9% specificity). This study represents the first application of immune cell subset-specific transcriptomic profiling to overcome the technical limitations, which are frequently confounded by heterogeneous cellular backgrounds in whole blood or peripheral blood mononuclear cell (PBMC) analyses. The development of this 4-gene neu-TB signature not only establishes a novel methodology for identifying transcriptomic biomarkers but also demonstrates promising clinical applicability for ATB diagnostics.IMPORTANCEThe most urgent need in pulmonary TB diagnosis is developing a rapid, non-sputum, biomarker-based diagnostic test. Here, we first identified a novel transcriptomic signature and addressed these challenges through two key innovations: (i) cell-type-specific resolution: by focusing on neutrophils-the most abundant immune cell in TB blood signatures and key mediators of host-pathogen interactions-we reduce biological noise and enhance biomarker precision. (ii) Rigorous multi-cohort validation: the 4-gene neutrophil-derived TB signature (neu-TB) was selected via RNA-seq of purified neutrophils (<i>n</i> = 141) and validated by qPCR in two independent cohorts (total <i>n</i> = 332), achieving 93% sensitivity/88% specificity in distinguishing active TB from latent infection and non-TB controls.