Risk Factors for False-Negative T-SPOT.TB Results in Bacteriologically Confirmed Pulmonary Tuberculosis: A Retrospective Cohort Study of 4,931 Patients.

PURPOSE: T-SPOT.TB is an interferon-γ release assay that aids in detecting Mycobacterium tuberculosis infection but cannot distinguish latent infection from active tuberculosis. False-negative results may delay diagnosis and treatment in patients with active pulmonary tuberculosis (PTB). This study investigated factors associated with false-negative T-SPOT.TB results in bacteriologically confirmed PTB. PATIENTS AND METHODS: This retrospective cohort study included 4,931 adults with bacteriologically confirmed PTB hospitalized at Fuzhou Pulmonary Hospital, Fujian Province, China, between January 2018 and September 2025. The primary outcome was a false-negative T-SPOT.TB result. Demographic characteristics, comorbidities, radiological findings, inflammatory markers, and T-lymphocyte subset counts were collected. Multivariable logistic regression was used to identify independent predictors, while restricted cubic spline (RCS) analyses explored potential nonlinear dose-response relationships. RESULTS: False-negative T-SPOT.TB results occurred in 437 patients (8.86%). Compared with patients with positive results, those with false-negative results were older (median, 61 vs 57 years), had higher neutrophil-to-lymphocyte ratios (NLR; 3.85 vs 3.25), and had lower CD4⁺ T-cell counts (540 vs 594 cells/μL) (all P<0.001). Older age (OR=1.007, 95% CI: 1.001-1.014; P=0.024), chronic obstructive pulmonary disease (OR=1.848, 95% CI: 1.273-2.682; P=0.001), and elevated NLR (OR=1.018, 95% CI: 1.002-1.035; P=0.026) were independent risk factors. Diabetes mellitus, cavitary lesions, airway lesions, and higher CD4⁺ T-cell counts were independently associated with a reduced risk of false-negative results. RCS analyses demonstrated nonlinear associations for NLR (P for nonlinearity <0.001) and CD4⁺ T-cell count (P for nonlinearity=0.002), with a marked increase in false-negative risk above an NLR of 2.96 and below a CD4⁺ T-cell count of 490.7 cells/μL. CONCLUSION: Advanced age, chronic obstructive pulmonary disease, elevated NLR, and reduced CD4⁺ T-cell counts increase the likelihood of false-negative T-SPOT.TB results. In high-risk patients with TB-compatible imaging, a negative T-SPOT.TB result should not preclude further microbiological evaluation or diagnostic investigation.