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

Abstract

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.