A multiplex Mtb-specific FluoroSpot assay measuring IFNγ, IL-2, and TNF-secreting cells can improve accuracy and differentiation across the tuberculosis spectrum.
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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 Mycobacterium tuberculosis (Mtb) antigens ESAT-6, CFP-10, and EspC. PBMCs from individuals with TBD (n = 24), TBI (n = 64), and IGRA-negative controls (n = 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 Mycobacterium tuberculosis (Mtb) 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 Mtb-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 Mtb-specific T-cell responses may not yet produce detectable amounts of IFNγ.