LAUSR

Mycobacteria-specific T cell activation has been shown to be a robust biomarker of recent Mycobacterium tuberculosis ( M.tb ) infection, disease progression, and tuberculosis (TB) disease. We have termed an intracellular cytokine staining-based assay that measures antigen-specific activation via HLA-DR expression on cytokine-expressing T cells “TB-TASA.” In the present study, we evaluated this promising biomarker as a treatment monitoring tool in the context of a treatment-shortening study. Using a panel of mycobacterial antigens as stimulants to detect cytokine-expressing T cells, we observed higher TB-TASA scores in TB patients compared to IFNγ release assay positive (IGRA+) controls, regardless of mycobacterial antigen specificity, consistent with previous studies. We derived a TB-TASA positivity threshold of 10% HLA-DR+ mycobacteria-specific T cells using a computational flow cytometry analysis pipeline developed with the OpenCyto R package. This 10% threshold was robust across three previously published case-control studies that used different sample types ( i.e. , whole blood or PBMC), varying duration of antigen stimulation, and different flow cytometry antibody panels, all of which achieved sensitivity and specificity greater than 90% and 70%, respectively. In a prospective randomised clinical trial assessing treatment shortening in adult TB patients with less severe disease, higher TB-TASA scores were observed at treatment completion in patients who relapsed or failed treatment during follow-up compared to those successfully treated with an ROC AUC of 0.89 (95%CI 0.69–1), supporting utility of TB-TASA as a treatment monitoring tool. Importantly, we demonstrated that TB-TASA could also be reliably measured in capillary blood collected via fingerprick from 29 IGRA+ controls and 22 TB patients, achieving an ROC AUC of 0.96 (95%CI (0.9–1) and sensitivity and specificity of 95% and 69%, respectively. This provides evidence that this TB biomarker can be measured in easily accessible samples. Together, these results support the continued development of TB-TASA as a potential tool for diagnosing TB and monitoring treatment responses.