Diagnostic value of lesion-targeted BALF immune biomarkers combined with T-SPOT.TB for smear-negative pulmonary tuberculosis

ABSTRACT Data on immune responses at local infection sites in smear-negative pulmonary tuberculosis (SNPT) remain limited. This study aimed to characterize lesion-targeted bronchoalveolar lavage fluid (BALF) cytokine and T-cell responses, and assess their diagnostic value for SNPT in clinically suspected patients. Patients were enrolled in a single-center derivation cohort between January 2021 and December 2023. Cytokine and lymphocyte subsets were measured in both peripheral blood and lesion-targeted BALF. Peripheral blood T-SPOT.TB assay and BALF targeted next-generation sequencing (tNGS) for Mycobacterium tuberculosis (Mtb) detection were performed. An immunological prediction model based on BALF immune biomarkers and peripheral blood T-SPOT.TB results was developed in the derivation cohort, and then externally validated in a three-center external validation cohort between June 2024 and May 2025. In the derivation cohort, 177 patients were included in the final analysis, including 69 SNPT patients and 108 non-SNPT patients. Stepwise logistic regression identified the BALF concentrations of IL-1β and IL-6, the BALF CD4/CD8 T-cell ratio, and the T-SPOT.TB result as independent predictors of SNPT, forming an immunological prediction model, with an area under the curve (AUC) of 0.856. Compared with T-SPOT.TB alone, the model increased specificity from 66.7% to 85.2% at an optimal predicted probability cut-off of 0.36. In the external validation cohort, another 177 suspected cases were analyzed, including 67 SNPT patients and 110 non-SNPT patients. The model achieved an AUC of 0.801, with a sensitivity of 71.6% and a specificity of 82.7% for SNPT prediction at the optimal cut-off. Parallel testing combining BALF tNGS and the prediction model yielded a sensitivity of 94.0% and a specificity of 78.0%, with an OR of 56.4 (95% CI, 18.7–170.8), exceeding that of BALF tNGS alone (OR, 18.7; 95% CI, 8.0–43.8). The measurement of BALF IL-1β and IL-6 concentrations and BALF CD4/CD8 T-cell ratio, combined with the T-SPOT.TB result, would facilitate the diagnosis of SNPT and benefit the effective initiation of anti-Mtb treatment. IMPORTANCE Smear-negative pulmonary tuberculosis (SNPT) remains a critical diagnostic challenge. Our study identifies lesion-targeted immune biomarkers as independent predictors of SNPT and integrates these biomarkers with the peripheral interferon-γ release assay to develop an immunological prediction model. This model can improve diagnostic accuracy when used in parallel testing with the nucleic acid amplification assay. Our data indicate that the application of local immune biomarkers in the diagnosis of SNPT will benefit the effective initiation of anti-tuberculosis treatment and potentially reduce the spread of the disease.