A multiplex Mtb-specific FluoroSpot assay measuring IFNγ, TNF, and IL2-secreting cells can improve accuracy and differentiation across the tuberculosis spectrum

Tuberculosis (TB) remains a major global health challenge, with approximately 25% of the global population estimated to have been infected with the causative pathogen Mycobacterium tuberculosis (Mtb) . Current diagnostic methods based on Interferon-gamma (IFNγ) release assays (IGRA) have limitations in detecting infection and cannot distinguish those with TB infection from those with disease. In this study, we evaluated a multiplex FluoroSpot assay measuring IFNγ, TNF, and IL2-secreting cells in response to the Mtb antigens ESAT-6, CFP-10, and EspC. The assay was tested on peripheral blood mononuclear cells from individuals with TB disease (n=24), TB infection (n=63), and IGRA-negative controls (n=27). Results indicate that triple cytokine-secreting cells (IFNγ/IL2/TNF) could detect Mtb -specific immune responses with higher sensitivity and specificity compared to commercially available IGRA methods. Furthermore, we identified distinct cytokine profiles associated with different stages of TB infection and disease. The study suggests that this multiplex assay could improve current TB diagnostics while also improving our understanding of Mtb -specific T cell responses associated with recent or remote TB infection and disease. Further studies are now needed to validate these findings in larger cohorts, including individuals with immunosuppressive conditions.