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Background: The identification of Mycobacterium-tuberculosis (Mtb) infected individuals remains a challenge due to an insufficient understanding of immune responses detected with the current diagnostic tests for latent tuberculosis i.e. the tuberculin skin test (TST) or IFN-γ release assays (IGRAs) and an inability to distinguish infection stages with current immunologic assays. Further classification based on markers other than IFN-γ may help to define markers of early Mtb infection., Methods: We assessed the TST status of Mtb-exposed household contacts at baseline and at 6 months. Contacts were classified into those with initial positive TST (TST+); those with baseline negative TST but TST conversion at 6 months (TST converters, TSTC) and those with persistently negative TST (PTST-). We assessed their short- and long-term immune responses to PPD and ESAT-6/CFP-10 (EC) via IFN-γ ELISPOT and a multiplex cytokine array in relation to TST status and compared them to those of TB cases to identify immune profiles associated with a spectrum of infection stages., Results: After 1 and 6 days stimulation with EC, 12 cytokines (IFN-γ, IL-2, IP-10, TNF-α, IL-13, IL-17, IL-10, GMCSF, MIP-1β, MCP-3, IL-2RA and IL-1A) were not different in TSTC compared to TST+ suggesting that robust adaptive Mtb-specific immune responses precede TST conversion. Stratifying contacts by baseline IFN-γ ELISPOT to EC in combination with TST results revealed that IP-10 and IL-17 were highest in the group of TST converters with positive baseline ELISPOT, suggesting they might be markers for recent infection., Conclusion: We describe a detailed analysis of Mtb-specific biomarker profiles in exposed household contacts in a TB endemic area that provides insights into the dynamic immune responses to Mtb infection and may help to identify biomarkers for 'at-risk' populations beyond TST and IGRA.

Background: Qualitative and quantitative changes in IGRA response offer promise as biomarkers to monitor Tuberculosis (TB) drug therapy, and for the comparison of new interventions. We studied the decay kinetics of TB-specific antigen T-cell responses measured with an in-house ELISPOT assay during the course of therapy., Methods: Newly diagnosed sputum smear positive TB cases with typical TB chest radiographs were recruited. All patients were given standard anti-TB treatment. Each subject was followed up for 6 months and treatment outcomes were documented. Blood samples were obtained for the ESAT-6 and CFP-10 (EC) ELISPOT at diagnosis, 1-, 2-, 4- and 6-months. Qualitative and quantitative reversion of the ELISPOT results were assessed with McNemar test, conditional logistic regression and mixed-effects hierarchical Poisson models., Results: A total of 116 cases were recruited and EC ELISPOT was positive for 87% (95 of 109) at recruitment. There was a significant decrease in the proportion of EC ELISPOT positive cases over the treatment period (p<0.001). Most of the reversion occurred between the start and first month of treatment and at completion at 6 months. ESAT-6 had higher median counts compared to CFP-10 at all time points. Counts for each antigen declined significantly with therapy (p<0.001). Reverters had lower median SFUs at the start of treatment compared to non-Reverters for both antigens. Apart from the higher median counts for non-Reverters, no other risk factors for non-reversion were found., Conclusions: TB treatment induces qualitative and quantitative reversion of a positive in-house IGRA in newly diagnosed cases of active TB disease. As this does not occur reliably in the majority of cured individuals, qualitative and quantitative reversion of an IGRA ELISPOT has limited clinical utility as a surrogate marker of treatment efficacy.

Most people infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) actually maintain a strong immune response and are able to control bacterial growth (deemed latently infected (LTBI)), while approximately 10% progress to disease resulting in almost 2 million deaths per year. Determining the immune 'footprint' at specific stages of infection and disease will allow for better diagnostics, treatments and ultimately development of new vaccine candidates. In this study we performed multi-factorial flow cytometry on fresh blood from 56 TB cases, 46 Tuberculin Skin Test (TST) positive (LTBI) and 39 TST negative household contacts. We found a highly significant increase in granulocytes and decrease in B cells and invariant (Valpha24+Vbeta11+) NKT cells in TB cases compared to TST+ contacts (p<0.0001, p=0.007 and p=0.01 respectively) which were restored to LTBI levels following 6 months of TB treatment. Using support vector analysis, we found a combination of granulocyte and lymphocyte and/or NKT cell proportions allowed almost 90% correct classification into M. tuberculosis infection or disease. This work has important public health benefits in regards to diagnosis and treatment of TB in sub-Saharan Africa and in furthering our understanding of the requirements for protective immunity to TB.

Interferon gamma remains a key effector molecule that is still widely used as the most informative biomarker for screening human immune responses against tuberculosis, particularly in ELISPOT assays. We investigated the participation of CD4(+) and CD8(+) T lymphocytes in the PBMC responses to Mycobacterium tuberculosis (Mtb) specific antigens in 33 TB cases and 49 contacts. Responses to ESAT-6 were higher than CFP-10. There was no significant difference in responses to both Mtb antigens between cases and contacts. PBMCs response to ESAT-6 but not CFP-10 in cases was significantly reduced by depletion of CD4(+) cells whereas CD8(+) cell depletion had no impact. In conclusion, ESAT-6 is a more recognized antigen in this population, and CD4(+) lymphocytes are the main participants in IFN-gamma response by ELISPOT. Thus, a decline of CD4(+) T lymphocytes below a critical level might affect the sensitivity of IFN-gamma release assays for detecting Mtb infection.

Background: IFN-gamma Release Assays (IGRAs) have been licensed for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Their performance may depend on assay format and may vary across populations and settings. We compared the diagnostic performance of an in-house T -cell and commercial whole blood-based IGRAs for the diagnosis of LTBI and TB disease in The Gambia., Methods: Newly diagnosed sputum smear positive cases and their household contacts were recruited. Cases and contacts were bled for IGRA and contacts had a Mantoux skin test. We assessed agreement and discordance between the tests and categorized a contact's level of M. tuberculosis exposure according to where s/he slept relative to a case: the same room, same house or a different house. We assessed the relationship between exposure and test results by multiple logistic regression., Results: In 80 newly diagnosed TB cases, the sensitivity of ELISPOT was 78.7% and for QFT-GIT was 64.0% (p = 0.047). Of 194 household contacts 57.1% and 58.8% were positive for ELISPOT and QFT-GIT respectively. The overall agreement between both IGRAs for LTBI in contacts was 71.4% and there was no significant discordance (p = 0.29). There was significant discordance between the IGRAs and TST. Neither IGRA nor TST had evidence of false positive results because of Bacille Calmette Guérin (BCG) vaccination. However, agreement between QFT-GIT and TST as well as discordance between both IGRAs and TST were associated with BCG vaccination. Both IGRAs responded to the M. tuberculosis exposure gradient and were positively associated with increasing TST induration (p = 0.003 for ELISPOT and p = 0.001 for QFT-GIT)., Conclusion: The ELISPOT test is more sensitive than the QFT-GIT for diagnosing TB disease. The two tests perform similarly in the diagnosis of LTBI in TB contacts. Significant discordance between the two IGRAs and between each and the TST remain largely unexplained.