Mycobacterium tuberculosis Latency-Associated Antigen Rv1733c SLP Improves the Accuracy of Differential Diagnosis of Active Tuberculosis and Latent Tuberculosis Infection
Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a leading global health threat, with an estimated 10 million new cases reported in 2019 alone. China, ranking third globally in TB burden, faces significant challenges in accurate diagnosis, particularly in differentiating active TB (ATB) from latent TB infection (LTBI). Traditional diagnostic methods, including smear microscopy, bacterial culture, and molecular assays like Xpert MTB/RIF, have limitations in sensitivity, timeliness, and applicability. Interferon-gamma release assays (IGRAs), which detect immune responses to MTB-specific antigens ESAT-6 and CFP-10, cannot reliably distinguish ATB from LTBI. This study addresses this diagnostic gap by evaluating the utility of MTB latency-associated antigen Rv1733c and its synthetic long peptide derivative (Rv1733c SLP) in improving diagnostic accuracy through a FluoroSpot assay measuring T-cell cytokine responses.
Study Design and Methodology
The study enrolled 93 participants: 57 ATB cases (20 pathogen-confirmed, 37 clinically diagnosed) and 36 LTBI cases. ATB patients exhibited clinical symptoms and either laboratory confirmation or positive response to anti-TB treatment. LTBI participants had no symptoms, negative imaging, and positive T-SPOT.TB results. Synthetic peptides for ESAT-6, CFP-10, Rv1733c, and Rv1733c SLP were used to stimulate peripheral blood mononuclear cells (PBMCs). Rv1733c comprised 19 overlapping 20-mer peptides, while Rv1733c SLP consisted of 19 overlapping 28-mer peptides.
The FluoroSpot assay simultaneously detected interferon-gamma (IFN-γ) and interleukin-2 (IL-2) secretion at the single-cell level. PBMCs were incubated with antigens, anti-CD28 (to enhance T-cell activation), and fluorescent antibodies. Responses were quantified as spot-forming cells (SFCs) per 2.5 × 10⁵ PBMCs. Statistical analyses included ROC curves to determine optimal diagnostic cutoffs, Mann-Whitney U tests for group comparisons, and logistic regression for combined parameter evaluation.
Key Findings
Immune Responses to ESAT-6 and CFP-10
ATB patients exhibited higher frequencies of IFN-γ-secreting T cells compared to LTBI individuals. The median frequency of single IFN-γ⁺ T cells was 55 SFCs (ATB) vs. 14 SFCs (LTBI), with a significant difference (P = 0.003). Conversely, LTBI cases showed elevated IL-2 responses: single IL-2⁺ T-cell frequencies were 7 SFCs (LTBI) vs. 3 SFCs (ATB; P = 0.004). The proportion of single IFN-γ⁺ T cells was 72.2% in ATB vs. 34.0% in LTBI (P < 0.001), highlighting IFN-γ dominance in active disease and IL-2 bias in latency.
Role of Latency-Associated Antigens
Stimulation with Rv1733c and Rv1733c SLP revealed distinct cytokine profiles. Rv1733c SLP induced stronger IL-2 responses in LTBI, with median single IL-2⁺ T-cell frequencies of 1 SFC (ATB) vs. 4 SFCs (LTBI; P < 0.001). ROC analysis identified single IL-2⁺ T-cell frequency as the best discriminator, with an AUC of 0.766 (95% CI: 0.662–0.870). A cutoff of ≥1 SFC provided 72.2% sensitivity and 73.7% specificity for distinguishing LTBI from ATB. In contrast, Rv173c alone showed lower discriminative power (AUC: 0.665), emphasizing the superiority of SLP in detecting latent infection.
Combined Diagnostic Approach
When ESAT-6/CFP-10-FluoroSpot results were combined with Rv1733c SLP data, diagnostic performance improved markedly. Using ESAT-6/CFP-10 alone, the sensitivity and specificity were 82.5% and 66.7%, respectively. Incorporating Rv1733c SLP-driven IL-2 responses elevated sensitivity to 84.2% and specificity to 83.3%, with an AUC of 0.874 (95% CI: 0.799–0.948). This combination also improved positive predictive value (88.9% vs. 79.7%) and likelihood ratios, underscoring its clinical utility.
Mechanistic Insights and Clinical Implications
The differential immune responses observed align with MTB’s pathobiology. During latency, MTB survives in hypoxic granulomas, upregulating dormancy-associated genes like those in the DosR regulon. Rv1733c, a DosR-regulated antigen, is preferentially recognized by LTBI individuals, driving IL-2 production—a cytokine critical for maintaining memory T-cell populations. In ATB, elevated bacterial load and antigen exposure skew responses toward IFN-γ, reflecting active effector responses.
Rv1733c SLP’s enhanced immunogenicity compared to Rv1733c may stem from prolonged antigen presentation and broader epitope coverage, activating more T-cell clones. This aligns with prior studies showing SLPs induce stronger CD4⁺ T-cell responses in murine models. The study’s findings suggest that incorporating latency antigens into diagnostic assays could improve LTBI detection while maintaining ATB sensitivity.
Limitations and Future Directions
While promising, the study has limitations. The case-control design risks overestimating diagnostic accuracy, and the sample size, though statistically adequate, warrants validation in larger cohorts. Prospective studies in diverse populations are needed to confirm generalizability. Additionally, Rv1733c-based vaccines, if developed, may confound diagnostic interpretations, necessitating continuous evaluation of antigen specificity.
Conclusion
This study demonstrates that combining MTB latency-associated antigen Rv1733c SLP with ESAT-6/CFP-10-FluoroSpot significantly enhances the accuracy of differentiating ATB from LTBI. By leveraging IL-2 responses specific to latent infection and IFN-γ dominance in active disease, this approach addresses a critical unmet need in TB diagnostics. Further validation and integration into clinical workflows could transform patient management in high-burden settings.
doi.org/10.1097/CM9.0000000000001858
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