Specific Cytokines Analysis Incorporating Latency-Associated Antigens Differentiates Mycobacterium tuberculosis Infection Status: An Exploratory Study

Introduction: Current immunologic methods cannot distinguish Mycobacterium tuberculosis (Mtb) infection statuses, especially to discriminate active tuberculosis (ATB) from latent tuberculosis infection (LTBI). This study explored the potential of latency-associated antigens (Rv1733cSLP and Rv2028c) and multifactorial cytokine detection to distinguish tuberculosis infection states. Methods: ATB patients (20), LTBI healthcare workers (25), fever patients (11), and healthy controls (10) were enrolled. Cytokine levels (IFN-γ, TNF-α, IL-2, IL-6, IP-10, IL-1Ra, CXCL-1, and MCP-1) were measured using Luminex with/without MTB-specific virulence factor and latency-associated antigens stimulation. Results: Without antigen stimulation, IL-6, IP-10, MCP-1, and IL-1Ra were higher in the ATB group than in the LTBI group (p< 0.05), but no significant differences between the ATB group and the fever group. Stimulated with the four antigens, respectively, the cytokines, including IP-10 Esat− 6 , IP-10 CFP− 10 , IFN-γ Rv1733cSLP , IFN-γ Rv2028c , IL-6 Esat− 6 , IL-6 Rv1733cSLP , IL-6 Rv2028c , IL-2 Rv1733cSLP , IL-2 Rv2028c , IL-1Ra Esat− 6 , IL-1Ra CFP− 10 , IL-1Ra Rv2028c , CXCL-1 Esat− 6 , CXCL-1 CFP− 10 , CXCL-1 Rv1733cSLP , CXCL-1 Rv2028c , MCP-1 Esat− 6 and MCP-1 CFP− 10 , demonstrated accurate discrimination between ATB and LTBI ( p < 0.05). Additive concentrations demonstrated significant secretion differences of IFN-γ, IP-10 and IL-2, primarily by virulence factors in ATB and latency-associated antigens in LTBI. Latency-associated antigens synergized with virulence factors, enhancing TH1-type cytokine diagnostic efficacy for discriminating ATB from LTBI, the AUC for TNF-α increased from 0.696 to 0.820 ( p =0.038), IFN-γ increased from 0.806 to 0.962 ( p =0.025), and IL-2 increased from 0.565 to 0.868 ( p =0.007). Model selected by forward likelihood method indicated combined detection of IFN-γ CFP− 10 , IFN-γ Rv1733cSLP , IP-10 Rv1733cSLP , and CXCL-1 Rv1733cSLP achieved ATB diagnosis (AUC=0.996) and ATB-LTBI differentiation (AUC=0.992). Combined detection of IFN-γ CFP− 10 and IFN-γ Rv1733cSLP achieved tuberculosis infection diagnosis (AUC=0.943). Conclusion: Latency-associated antigens enhance multiple cytokine discriminatory ability, particularly TH1-type cytokines, for differentiating Mtb infection statuses. Keywords: active tuberculosis, cytokine, latent tuberculosis infection, latency-associated antigen, Mycobacterium tuberculosis , virulence factor