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Background: Identifying stage-specific antigens is essential for developing tuberculosis (TB) diagnostics and vaccines. In a low TB endemic country, we characterized, the Mycobacterium tuberculosis (Mtb)-specific immune response to a pool of Mtb-derived epitopes (ATB116), demonstrated as associated with TB disease., Methods: In this prospective observational cross-sectional study, we enrolled healthy donors (HD), subjects with TB disease, and TB infection (TBI) at baseline and therapy completion. T-cell response after whole blood stimulation with the peptide pools was characterized by ELISA, flow cytometry, and multiplex assay., Results: ATB116-specific IFN-γ response (by ELISA) significantly associates with Mtb regardless of infection/disease (p < 0.0001) and decreases during TB therapy (p = 0.0002). Flow cytometry confirms that ATB116-specific CD4 + T-cell response associated with Mtb regardless of infection/disease (p < 0.0001) and shows a significantly higher frequency of IFN-γ/IL-2 and central memory T-cells in TBI compared to TB (p = 0.016; p = 0.0242, respectively). CD4 + T cell-specific response decreases after TB therapy completion. The antigen-specific CD8 + T-cell response mirrors the CD4 + response. Finally, the multiplex assay analysis showed that ATB116 induces several immune factors in both TB and TBI., Conclusion: We characterized the immune response to Mtb peptide pools that is modulated by TB therapy. These results are important for our understanding of TB immunopathogenesis and vaccine design., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Baricitinib seems a promising therapy for COVID-19. To fully-investigate its effects, we in-vitro evaluated the impact of baricitinib on the SARS-CoV-2-specific-response using the whole-blood platform., Methods: We evaluated baricitinib effect on the IFN-γ-release and on a panel of soluble factors by multiplex-technology after stimulating whole-blood from 39 COVID-19 patients with SARS-CoV-2 antigens. Staphylococcal Enterotoxin B (SEB) antigen was used as a positive control., Results: In-vitro exogenous addition of baricitinib significantly decreased IFN-γ response to spike- (median: 0.21, IQR: 0.01-1; spike+baricitinib 1000 nM median: 0.05, IQR: 0-0.18; p < 0.0001) and to the remainder-antigens (median: 0.08 IQR: 0-0.55; remainder-antigens+baricitinib 1000 nM median: 0.03, IQR: 0-0.14; p = 0.0013). Moreover, baricitinib significantly decreased SEB-induced response (median: 12.52, IQR: 9.7-15.2; SEB+baricitinib 1000 nM median: 8, IQR: 1.44-12.16; p < 0.0001). Baricitinib did modulate other soluble factors besides IFN-γ, significantly decreasing the spike-specific-response mediated by IL-17, IL-1β, IL-6, TNF-α, IL-4, IL-13, IL-1ra, IL-10, GM-CSF, FGF, IP-10, MCP-1, MIP-1β (p ≤ 0.0156). The baricitinib-decreased SARS-CoV-2-specific-response was observed mainly in mild/moderate COVID-19 and in those with lymphocyte count ≥1 × 10 3 /µl., Conclusions: Exogenous addition of baricitinib decreases the in-vitro SARS-CoV-2-specific response in COVID-19 patients using a whole-blood platform. These results are the first to show the effects of this therapy on the immune-specific viral response., Competing Interests: Declaration of Competing Interest Dr Emanuele Nicastri reported consultancies from Gilead under $10,000 and outside the present work. Dr Alba Grifoni is listed as inventor on a provisional patent application on the diagnostic and therapeutic use of the MPs and peptides thereof filed on February 12, 2020. Dr Andrea Antinori reported consultancies, speaking fees and honoraria from Gilead Sciences, Janssen Cilag, Merk, VIIVHealthcare, Theratechnologies, all under $10,000 and outside the present work. Dr Alessandra Vergori received institutional grants from Gilead, travel grants and speaker's fees from Janssen and speaker's fee from MSD, all under $10,000 and outside the present work. Dr Delia Goletti reported consultancies from Quidel and Biomeriuex and speaking fees from Diasorin and Janssen, all under $10,000 and outside the present work.All the other authors have declared that no conflict of interest and/or financial disclosures exists., (Copyright © 2021. Published by Elsevier Ltd.)

Objective: HIV-infection increases the risk to progress to active-tuberculosis (TB). Detection of latent TB infection (LTBI) is needed to eventually propose preventive-therapy and reduce TB reservoir. QuantiFERON-TB Plus (QFT-Plus)-test identifies LTBI. Currently, only two studies on QFT-Plus accuracy in HIV-infected-population are available in high TB-endemic-countries. Therefore we aimed to evaluate the effect of HIV-infection on QFT-Plus accuracy to detect LTBI in a low TB-endemic-country., Methods: We enrolled 465 participants, among the 167 HIV-infected-persons: 32 with active-TB (HIV-TB), 45 remote-LTBI (HIV-LTBI) and 90 at low M. tuberculosis (Mtb)-infection risk. Among the 298 HIV-uninfected-persons: 170 with active-TB, 76 recent-LTBI, 34 remote-LTBI and 18 with low Mtb-infection risk., Results: QFT-Plus sensitivity was similar in TB regardless of HIV-status. CD4-count did not influence the distribution of IFN-γ values in HIV-TB and HIV-LTBI. Moreover HIV-LTBI and HIV-uninfected remote LTBI had a similar proportion of results in the uncertain range (IFNγ ≥0.2 ≤ 0.7 IU/ml) differently from those LTBI-persons reporting recent-exposure (p = 0.016). Cytometry results demonstrated that CD8-response was similar in HIV-infected- and -uninfected-persons whereas CD4-response was impaired in HIV-infected-persons (p = 0.011)., Conclusions: HIV-infection does not affect QFT-Plus response in active-TB, whereas the time of exposure influences the proportion of uncertain-results in LTBI., Competing Interests: Declaration of Competing Interest DG received consultant fees for public speaking in international meetings by Qiagen and Diasorin., (Copyright © 2020 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Estimated 2017 tuberculosis (TB) incidence is 10 million and mainly depends on the reservoir of individuals with latent TB infection (LTBI). Quantiferon Ⓡ -TB Gold in-Tube (QFT-GIT) is one of the tests used for LTBI detection. Since 2015 a new version, Quantiferon Ⓡ -TB Gold Plus (QFT-Plus) is available., Objectives: To perform a systematic review and meta-analysis to assess the diagnostic accuracy for TB of QFT-Plus compared to QFT-GIT., Methods: PubMed and Scopus were used to detect records related to predefined strings from 2015 to 2018. Full text articles dealing with the sensitivity and/or specificity of the QFT-Plus vs. QFT-GIT for active-TB and LTBI detection were analyzed. Scientific quality and risk of bias were assessed using QADAS-2., Results: We selected 15 articles. Studies were mainly observational and cross-sectional, performed in 8 countries. Sample size differed in the TB group (27 to 164) compared to LTBI group (29 to 1031). Pooled sensitivity of QFT-Plus for active-TB was 0.94 (0.91 and 0.95 for TB1 and TB2, respectively), whereas pooled specificity for healthy status was 0.96. Pooled sensitivity and specificity for LTBI was 0.91 and 0.95, respectively., Conclusions: We show that QFT-Plus is more sensitive compared to QFT-GIT for detecting M. tuberculosis infection, mainly due to TB2 responses., (Copyright © 2019. Published by Elsevier Ltd.)

Objectives: Screening for latent tuberculosis infection (LTBI) diagnosis is mandatory in patients with immune-mediated inflammatory diseases (IMID) requiring biologics. QuantiFERON-TB-Plus (QFT-P), an LTBI diagnostic test, measures IFN-γ after M. tuberculosis-stimulation in TB1 and TB2 tubes in which a "CD4" or a "CD4 and CD8" response is respectively elicited. Aim of this study is to compare the response to QFT-P of IMID-LTBI patients candidates to a new biological therapy vs LTBI-subjects without IMID., Methods: We prospectively enrolled 167 subjects: 61 IMID-LTBI and 106 NON-IMID-LTBI., Results: All subjects were mitogen-responders. IFN-γ production was significantly lower in IMID-LTBI-patients compared to NON-IMID-LTBI-subjects. We observed discordant TB1 and TB2 results in 6.5% of IMID-LTBI-patients and in 8% of NON-IMID-LTBI-subjects. Applying a logistic regression analysis, we found that IMID-LTBI patients had a higher probability (TB1 stimulation OR 3.32; TB2 stimulation OR 4.33) to have IFNγ results ≤0.7 IU/mL compared to NON-IMID-LTBI-subjects. Interestingly, IMID-treatment did not interfere with the distribution of IFNγ-values., Conclusions: These results indicate that IMID-LTBI-patients have a low IFN-γ response to QFT-P, a high proportion of results ranging in the grey zone and a distribution of IFNγ-values independent from the IMID-treatment. These results are important for the management of LTBI screening in IMID patients., (Copyright © 2019. Published by Elsevier Ltd.)

Objectives: Interferon-γ release assays (IGRA) are designed for diagnosis of tuberculosis (TB) infection, and do not discriminate latent TB infection (LTBI) from active TB. Heparin-binding hemagglutinin antigen (HBHA) emerged as a promising antigen for TB diagnosis when used in IGRA format. Aim of this study was to prospectively evaluate the performance of an HBHA-based IGRA to support TB diagnosis and TB therapy monitoring in children with TB infection or active TB disease., Methods: Following clinical, microbiological and radiological assessment, children (0-14 years old) were tested by the QuantiFERON TB-Gold In tube (QFT) assay and an aliquot of whole-blood was stimulated with HBHA and IFNγ evaluated only in QFT-positive subjects., Results: Among the 550 children tested, 486 (88.4%) scored negative and 64 (11.6%) positive. None of the QFT-negative had active TB. Among the QFT-positive, 45 were with LTBI and 19 active TB. HBHA-IGRA scored positive in 41/45 children (91.1%) with LTBI and in 6/19 active TB children (31.6%) at diagnosis (p = 0.001); remarkably, 5 of these 6 children with active TB scoring HBHA-positive were asymptomatic. Moreover, following TB-specific therapy, most of the non-HBHA-responding children, gained an HBHA-positive response., Conclusions: HBHA-based IGRA is a useful support in TB diagnosis and TB-therapy monitoring in children., (Copyright © 2018 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Introduction: QuantiFERON ® -TB Gold Plus (QFT-Plus) is the new generation of QuantiFERON-TB Gold In-Tube test to identify latent tuberculosis infection (LTBI). QFT-Plus includes TB1 and TB2 tubes which contain selected Mycobacterium tuberculosis (Mtb) peptides designed to stimulate both CD4 and CD8 T-cells. Aim of this study is the flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within QFT-Plus., Methods: We enrolled 27 active tuberculosis (TB) patients and 30 LTBI individuals. Following stimulation with TB1 and TB2, antigen-specific T-cells were characterized by flow cytometry. Data were also correlated with the grade of TB severity., Results: TB1 mainly elicited a CD4 T-cell response while TB2 induced both CD4 and CD8 responses. Moreover, the TB2-specific CD4 response was detected for both active TB and LTBI patients, whereas the TB2-specific CD8 response was primarily associated with active TB (p = 0.01)., Conclusions: To our knowledge, we report the first characterization of the CD4 and CD8 T-cell response to QFT-Plus. CD8 T-cell response is mainly due to TB2 stimulation which is largely associated to active TB. These results provide a better knowledge on the use of this assay., (Copyright © 2016 The British Infection Association. All rights reserved.)

Unlabelled: There are still no reliable tests to distinguish active tuberculosis (TB) from latent TB infection (LTBI). Assessment of CD27 modulation on CD4⁺ T-cells has been suggested as a tool to diagnose different TB stages., Objectives: To use several cytometric approaches to evaluate CD27 expression on Mycobacterium tuberculosis (Mtb)-specific CD4⁺ T-cells to differentiate TB stages., Methods: 55 HIV-uninfected subjects were enrolled: 13 active TB; 12 cured TB; 30 LTBI. Whole blood was stimulated with RD1-proteins or Cytomegalovirus-lysate (CMV). Interferon (IFN)-γ response was evaluated by cytometry. The proportion of CD27(±) within the IFN-γ⁺ CD4⁺ T-cells or RATIO of the CD27-median fluorescence intensity (MFI) of CD4⁺ T-cells over the CD27 MFI of IFN-γ⁺ CD4⁺ T-cells was evaluated., Results: The greatest diagnostic accuracy in discriminating active TB vs. LTBI or cured TB was reached by evaluating the CD27(+) CD45RA(-) cells within the IFN-γ⁺ CD4⁺ T-cell subset (76.92 sensitivity for both, and 90% and 91.67% specificity, respectively), although the use of the CD27 MFI RATIO allows for stricter data analysis, independent of the operator., Conclusions: the study of CD27 expression using different approaches, whether it involves evaluation of CD45RA expression or not, is a robust biomarker for discriminating TB stages., (Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Objectives: Human Cystic Echinococcosis (CE) is estimated in 2-3 million global cases. CE diagnosis and clinical management are based on imaging and serology, which lacks sensitivity and does not provide cyst stage information. This study aimed to evaluate tools for improving diagnosis by analysing the Interleukin (IL)-4-response to Antigen B (AgB) of Echinococcus granulosus., Methods: Whole blood (WB) and peripheral blood mononuclear cells were stimulated with AgB. IL-4 levels were measured by enzyme-linked immunosorbent assay., Results: WB 1-day stimulation resulted the best experimental condition for evaluating AgB IL-4-response. IL-4 levels were significantly higher in CE patients than healthy donors (p ≤ 0.0001). A ROC analysis showed significant area under the curve (AUC) results (AUC, 0.85; p = 0.0001) identifying an IL-4 level cut-off point ≥0.39 pg/mL which predicted CE with 71.4% sensitivity and 93.3% specificity. Moreover, we found that IL-4 levels were significantly increased in patients with active cysts compared to those with inactive cysts (p ≤ 0.0001). ROC analysis showed significant AUC results (0.94; p = 0.0001) with a cut-off point of 4.6 pg/mL which predicted active cysts with 84.6% sensitivity and 92% specificity., Conclusions: We found immunological correlates associated with CE and biological cyst activity., (Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Objectives: Polyfunctional T-cells associate with chronic viral infection control while their involvement in tuberculosis (TB) is unclear. We evaluated TB-specific polyfunctional T-cell response and memory status in antiretroviral treatment (ART)-naïve HIV-infected patients from a low TB-endemic country., Methods: We prospectively enrolled HIV-infected patients, 12 with active TB (HIV-TB) and 15 with latent tuberculosis infection (LTBI). Peripheral blood cells were stimulated with TB antigens (RD1 proteins/peptides), HIV antigens, cytomegalovirus (CMV) and staphylococcal enterotoxin B (SEB) and analyzed by cytometry., Results: The HIV-TB showed a higher frequency of polyfunctional CD4(+) T-cells in response to RD1 antigens than HIV-LTBI (p = 0.007). Among the CD8(+) T-cells, both groups showed a significantly higher frequency of RD1-specific monofunctional cells than polyfunctional cells (p = 0.03). Analyzing the cytokine profile, IFNγ(+) TNFα(+) CD4(+) T-cells associated with HIV-TB (p ≤ 0.02) whereas IL2(+) TNFα(+) associated with HIV-LTBI (p = 0.009). CD4(+) T-cell response presented an effector-memory status in HIV-TB (p = 0.007) and an effector-memory terminally-differentiated phenotype in HIV-LTBI (p = 0.03). CD8(+) T-cell response presented an effector status in HIV-LTBI (p = 0.02). No significant cytokine profile pattern associated with responses to the other stimuli tested., Conclusions: In HIV-infection, polyfunctional CD4(+) T-cell-response associates with active TB, characterized by a high proportion of IFNγ(+) TNFα(+) and an effector-memory phenotype., (Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Objectives: Independent of IL-28B polymorphisms, blood IP-10 is a promising biomarker for predicting therapy response in chronic HCV infection. Urine IP-10 has been proposed as a biomarker in tuberculosis, but to date, no urine biomarkers for HCV infection have been evaluated. In this cross-sectional study, we assessed whether IP-10 is detectable in the urine of chronically HCV-infected patients, and if so, whether urine IP-10 correlates with serum IP-10 and HCV-specific clinical parameters., Methods: IP-10 was measured by ELISA in serum and urine concomitantly taken from 38 HCV-viremic patients, 10 cured-HCV subjects and 11 healthy donors enrolled as controls., Results: The urine of HCV-viremic patients showed measurable amounts of IP-10, although significantly lower than in serum (p < 0.0001). Urine IP-10 was normalized with creatinuria levels and we found that the urine IP-10/creatinuria ratio was significantly higher in HCV-viremic patients than in cured-HCV subjects (p = 0.002) and healthy donors (p = 0.008), and that it significantly correlated with transaminases (p = 0.01), although the correlation was low. Similarly, the serum IP-10 level significantly associated with HCV-viremic patients (p < 0.0001) and correlated with transaminases (p < 0.0001)., Conclusions: For the first time to our knowledge, we show that IP-10 is detected and increased in the urine of HCV-viremic patients compared to healthy donors and cured-HCV subjects., (Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Objectives: Controversial results were reported on the role of polyfunctional T-cells in tuberculosis (TB). Our aim was to simultaneously characterize the Mycobacterium tuberculosis (Mtb)-specific immune response as cytokine production and memory phenotype by flow cytometry after in vitro stimulation with region of difference 1 ("RD1") recombinant proteins (ESAT-6 and CFP-10) in patients at different TB stage in a low TB endemic country. To assess the specificity of these findings, we evaluated the response to cytomegalovirus (CMV), an unrelated antigen., Methods: We enrolled subjects with active TB, cured TB, latent TB infection (LTBI). Cytokine and phenotype profiles of T-cells from whole blood stimulated with "RD1" proteins and CMV were characterized by multi-parametric flow cytometry., Results: Bifunctional IFNγ(+) TNFα(+) CD4(+) T-cells and effector memory phenotype significantly associated with active TB compared to the LTBI group (p = 0.008, at least p ≤ 0.009 respectively) whereas "RD1"-T-cell response in cured TB and LTBI was characterized by a central memory phenotype (at least p ≤ 0.013 and p ≤ 0.004 respectively vs active TB). In contrast, response to CMV antigen was not associated with a TB-specific status., Conclusion: We identified qualitative associations between Mtb-specific T-cell and TB status in terms of functional capacity and memory status. These immune correlates may be helpful to trace natural history of TB., (Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Objectives: To evaluate the T-cell interferon (IFN)-γ response to Mycobacterium tuberculosis-specific antigens during latent tuberculosis infection (LTBI) therapy in candidates for tumor necrosis factor-α inhibitors (TNFi)., Methods: 1490 Patients were screened for LTBI. One-hundred and sixty-six of them were treated for LTBI and followed-up with QuantiFERON-TB Gold (QFT-IT) testing at baseline (T0) and therapy completion (T1); 92 subjects were also tested 3-6 months after therapy completion (T2)., Results: At T1 the QFT-IT reversion and conversion rates were 24% (27/111) and 18% (10/55), respectively. By multivariate analysis, the likelihood of reversion significantly decreased with older age (>50-60), larger TST size (>15 mm) and higher IFN-γ value at T0 (>1 IU/ml); the likelihood of conversion increased with higher IFN-γ levels at T0 (1 IU/ml) and in female patients. Quantitative data among those who scored QFT-IT-positive at T0 showed a decreasing trend of IFN-γ levels between T0 and T1 that reached statistical significance when T0 was compared to T2, and T1 to T2., Conclusions: The data confirm the difficulty of interpreting the modulation of IFN-γ levels during LTBI therapy. Currently, there is no evidence to support the use of QFT-IT in the clinical practice of monitoring LTBI treatment in candidates for TNFi., (Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Objectives: To evaluate whether in vitro response to Mycobacterium tuberculosis RD1 peptides selected by computational analysis, measured by IFN-gamma, IP-10, MCP-2 or IL-2 production, is associated with active tuberculosis (TB) in a country with a high incidence of TB., Methods: 129 individuals were prospectively enrolled, 41 with active-pulmonary TB and 88 without (household contacts and community controls). A whole blood assay based on RD1 selected peptides was performed. Soluble factors were evaluated by ELISA in plasma harvested at day1-post-culture. Enrolled individuals were also tested by QuantiFERON TB-Gold In tube (QFT-IT) and tuberculin skin tests (TST)., Results: IFN-gamma response to RD1 selected peptides was significantly higher in active TB patients than in household contacts and community controls. IP-10 and MCP-2 response did not differ between active TB patients and household contacts, although it was higher in these groups compared to community controls; conversely IL-2 response did not differ among the three groups. When IFN-gamma response to RD1 selected peptides was scored based on receiver-operator-characteristic analysis, active TB was predicted with 68% sensitivity and 86% specificity. QFT-IT and TST showed a sensitivity for active TB of 90% and 68% and a specificity of 58% and 59%, respectively., Conclusions: IFN-gamma (but not IP-10, MCP-2 and IL-2) response to RD1 selected peptides is associated with active TB with a higher specificity than QFT-IT and TST.