A multiplex Mtb-specific FluoroSpot assay measuring IFNγ, IL-2, and TNF-secreting cells can improve accuracy and differentiation across the tuberculosis spectrum

Tuberculosis (TB) remains a major global health challenge, with current diagnostic tools unable to reliably distinguish between individuals at different stages of the TB spectrum of infection (TBI) and disease (TBD). Interferon-gamma release assays (IGRAs), while commonly used, are limited by their reliance on a single cytokine and inability to differentiate disease states. In this study, we evaluated a multiplex FluoroSpot assay that simultaneously detects interferon gamma (IFNγ), interleukin (IL)2, and tumor necrosis factor (TNF)-secretion in response to <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) antigens ESAT-6, CFP-10, and EspC. PBMCs from individuals with TBD (<i>n</i> = 24), TBI (<i>n</i> = 64), and IGRA-negative controls (<i>n</i> = 26) were analyzed. The assay demonstrated high sensitivity and specificity, particularly through the detection of triple cytokine-secreting T cells (IFNγ/IL-2/TNF), minimizing false-positive rates. Moreover, distinct cytokine secretion patterns were associated with different stages of infection. Individuals with TBD had more IFNγ/TNF-producing cells, while those with TBI had more cells producing IFNγ/IL-2 or IL-2/TNF. Separating individuals with TBI based on time since likely Mtb exposure revealed that more recent infection was associated with reduced IFNγ responses and a higher proportion of IL-2/TNF-secreting cells. The use of spot volume analysis provided semi-quantitative insights into cytokine production. These findings suggest that multiplex cytokine profiling can improve TB diagnostics and offer a deeper understanding of host immune responses across the TB spectrum. Including non-IFNγ responses may be particularly valuable in identifying individuals at higher risk of disease progression, such as recently infected individuals, and in populations where current diagnostics underperform.IMPORTANCEAccurate diagnosis of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infection remains a cornerstone in tuberculosis (TB) control. Current interferon-gamma release assays (IGRAs) lack the ability to distinguish between individuals at different stages of the TB infection spectrum, limiting their utility. This study evaluates a multiplex FluoroSpot assay that simultaneously detects interferon gamma (IFNγ), interleukin 2, and tumor necrosis factor secretion in response to <i>Mtb</i>-specific antigens ESAT-6, CFP-10, and EspC. The assay demonstrated improved performance compared to standard IGRA methods, particularly through the identification of triple cytokine-secreting T cells. Importantly, it revealed distinct cytokine profiles associated with different stages of TB infection, offering potential for improved risk stratification and infection monitoring. These findings support the FluoroSpot assay as a promising tool for enhancing TB diagnostics and understanding host immune responses. Its application could be especially valuable in contact tracing, where <i>Mtb</i>-specific T-cell responses may not yet produce detectable amounts of IFNγ.