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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 (IFN\u03b3), interleukin (IL)2, and tumor necrosis factor (TNF)-secretion in response to\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">Mtb<\/jats:italic>\n                      ) antigens ESAT-6, CFP-10, and EspC. PBMCs from individuals with TBD (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 24), TBI (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 64), and IGRA-negative controls (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 26) were analyzed. The assay demonstrated high sensitivity and specificity, particularly through the detection of triple cytokine-secreting T cells (IFN\u03b3\/IL-2\/TNF), minimizing false-positive rates. Moreover, distinct cytokine secretion patterns were associated with different stages of infection. Individuals with TBD had more IFN\u03b3\/TNF-producing cells, while those with TBI had more cells producing IFN\u03b3\/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 IFN\u03b3 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-IFN\u03b3 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.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Accurate diagnosis of\n                        <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                        (\n                        <jats:italic toggle=\"yes\">Mtb<\/jats:italic>\n                        ) 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 (IFN\u03b3), interleukin 2, and tumor necrosis factor secretion in response to\n                        <jats:italic toggle=\"yes\">Mtb<\/jats:italic>\n                        -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\n                        <jats:italic toggle=\"yes\">Mtb<\/jats:italic>\n                        -specific T-cell responses may not yet produce detectable amounts of IFN\u03b3.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jcm.00894-25","type":"journal-article","created":{"date-parts":[[2025,10,29]],"date-time":"2025-10-29T13:01:34Z","timestamp":1761742894000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A multiplex Mtb-specific FluoroSpot assay measuring IFN\u03b3, IL-2, and TNF-secreting cells can improve accuracy and differentiation across the tuberculosis 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