Translational tuberculosis research: immune profile as biomarker of tuberculosis infection

Mtb is able to establish a chronic asintomatically infection mainly in the lung and the balance between host immune response and the mycobacteria plays a fundamental role in the control of the Mtb replication . Although the immune response to Mtb has been deeply studied, as described in the previously chapters, the host factors that leads to the development to active TB disease are not fully understood. However, on the base of immunogical findings on Mtb infection, it is possible to speculate that the containment of the latent Mtb needs an acquired cellular response with specific characteristics of immune surveillance, differently contrasting the replicating Mtb requires effector and cytotoxic proprierty. Objective of the present study is to take a picture of the immunological status of patients with Mtb infection, characterizing their Mtb specific immune response, in order to find a correlation with the different stages of Mtb infection. In the chapter 5 it is described the use of several cytometric approaches to evaluate the surface expression of the activation marker CD27 on Mtb-specific CD4+ T-cells, as a tool to diagnose active TB and LTBI. The chapter 6 is focused on flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within the QuantiFERON®-TB Gold Plus (QFT-Plus). QFT-Plus is the new generation of QuantiFERON-TB Gold In-Tube test (QFT-GIT) to identify the latent tuberculosis infection, it includes two tubes called TB1 and TB2 tubes which contain selected Mtb peptides designed to stimulate both CD4 and CD8 T-cells. Aim of the study was to analyze if the immune response to TB1 and TB2 stimulation could or not highlight differences between different TB stages. In the chapter 7, QFT-Plus performance was compared with that one of QFT-GIT in a cross sectional study of individuals with LTBI, active TB or treated for TB in the past. In this study, we wanted also to evaluate if the different ability t

Analytical evaluation of QuantiFERON- Plus and QuantiFERON- Gold In-tube assays in subjects with or without tuberculosis

The QuantiFERON-TB Gold Plus (QFT-Plus) represents the new QuantiFERON-TB Gold In-tube (QFT-GIT) to identify latent tuberculosis infection (LTBI). The main differences is the addition of a new tube containing shorter peptides stimulating CD8 T-cells. Aim of this study is to evaluate the accuracy of QFT-Plus compared with QFT-GIT in a cross sectional study of individuals with or without tuberculosis (TB). We enrolled 179 participants: 19 healthy donors, 58 LTBI, 33 cured TB and 69 active TB. QFT-Plus and QFT-GIT were performed. The two tests showed a substantial agreement. Moreover we found a similar sensitivity in active TB and same specificity in healthy donors. A higher proportion of the LTBI subjects responded to both TB1 and TB2 compared to those with active TB (97% vs 81%). Moreover, a selective response to TB2 was associated with active TB (9%) and with a severe TB disease, suggesting that TB2 stimulation induces a CD8 T-cell response in absence of a CD4-response. In conclusion, QFT-Plus and QFT-GIT assays showed a substantial agreement and similar accuracy for active TB detection. Interestingly, a higher proportion of the LTBI subjects responded concomitantly to TB1 and TB2 compared to those with active TB, whereas a selective TB2 response associated with active TB

First characterization of the CD4 and CD8 T-cell responses to QuantiFERON-TB Plus

Summary 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

Update on the diagnosis of tuberculosis

Background Tuberculosis remains a global public health threat, and the development of rapid and precise diagnostic tools is the key to enabling the early start of treatment, monitoring response to treatment, and preventing the spread of the disease. Objective An overview of recent progress in host- and pathogen-based tuberculosis diagnostics. Sources We conducted a PubMed search of recent relevant articles and guidelines on tuberculosis screening and diagnosis. Content An overview of currently used methods and perspectives in the following areas of tuberculosis diagnostics is provided: immune-based diagnostics, X-ray, clinical symptoms and scores, cough detection, culture of Mycobacterium tuberculosis and identifying its resistance profile using phenotypic and genotypic methods, including next generation sequencing, sputum- and non-sputum-based molecular diagnosis of tuberculosis and monitoring of response to treatment. Implications A brief overview of the most relevant advances and changes in international guidelines regarding screening and diagnosing tuberculosis is provided in this review. It aims at reviewing all relevant areas of diagnostics, including both pathogen- and host-based methods.PostprintPeer reviewe

Higher Frequency of T-Cell Response to M. tuberculosis Latency Antigen Rv2628 at the Site of Active Tuberculosis Disease than in Peripheral Blood

RATIONALE: Due to the invasive nature of the procedures involved, most studies of Mycobacterium tuberculosis (Mtb)-specific immunity in humans have focused on the periphery rather than the site of active infection, the lung. Recently, antigens associated with Mtb-latency and -dormancy have been described using peripheral blood (PB) cells; however their response in the lung is unknown. The objective of this report was to evaluate, in patients prospectively enrolled with suspected active tuberculosis (TB), whether the latency antigen Rv2628 induces local-specific immune response in bronchoalveolar lavage (BAL) cells compared to PB cells. MATERIAL/METHODS: Among the 41 subjects enrolled, 20 resulted with active TB. Among the 21 without active disease, 9 were defined as subjects with latent TB-infection (LTBI) [Quantiferon TB Gold In-tube positive]. Cytokine responses to Rv2628 were evaluated by enzyme linked immunospot (ELISPOT) assay and flow cytometric (FACS) analysis. RD1-secreted antigen stimulation was used as control. RESULTS: There was a significantly higher frequency of Rv2628- and RD1-specific CD4+ T-cells in the BAL of active TB patients than in PB. However the trend of the response to Rv2628 in subjects with LTBI was higher than in active TB in both PB and BAL, although this difference was not significant. In active TB, Rv2628 and RD1 induced a cytokine-response profile mainly consisting of interferon (IFN)-γ-single-positive over double-IFN-γ/interleukin (IL)-2 T-cells in both PB and BAL. Finally, BAL-specific CD4+ T-cells were mostly effector memory (EM), while peripheral T-cell phenotypes were distributed among naïve, central memory and terminally differentiated effector memory T-cells. CONCLUSIONS: In this observational study, we show that there is a high frequency of specific T-cells for Mtb-latency and RD1-secreted antigens (mostly IFN-γ-single-positive specific T-cells with an EM phenotype) in the BAL of active TB patients. These data may be important for better understanding the pathogenesis of TB in the lung

Alternative biomarkers of tuberculosis infection in patients with immune-mediated inflammatory diseases

IntroductionIFN-γ release assays (IGRAs) are one of the referral tests for diagnosing tuberculosis infection (TBI). To improve IGRAs accuracy, several markers have been investigated. Patients with immune-mediated inflammatory diseases (IMID), taking biological drugs, have a higher risk to progress to TB-disease compared to the general population. In several guidelines, annual TBI screening is recommended for patients undergoing biological therapy. Aim of this study was to investigate, within the QuantiFERON-TB-Plus (QFT-Plus) platform, if beside IFN-γ, alternative biomarkers help to diagnose TBI-IMID patients.MethodsWe enrolled 146 subjects: 46 with TB disease, 20 HD, 35 with TBI and 45 with TBI and IMID. Thirteen IMID subjects with a QFT-Plus negative result were diagnosed as TBI based on radiological evidence of TBI. We evaluated the IP-10 level in response to TB1 and TB2 peptides of QFT-Plus assay and we compared these results with the standardized assay based on IFN-γ. Multiplex immune assay was performed on plasma from TB1 and TB2 tubes and results were analyzed by a gradient boosting machine (GBM) as learning technique.ResultsTBI-IMID showed a significant decreased IP-10 level in response to TB1 and TB2 stimulation compared to TBI-NO IMID (p < 0.0001 and p = 0.0002). The TBI-IMID showed a moderate agreement between the IP-10-based assay and QFT-Plus scores. In TBI-IMID, QFT-Plus showed 70% sensitivity for TBI detection whereas the IP-10-based assay reached 61%. Tests combination increased the sensitivity for TBI diagnosis up to 77%. By a GBM, we explored alternative biomarkers for diagnosing TBI in IMID population reaching 89% sensitivity. In particular, the signature based on IL-2, IP-10, and IL-9 detection was associated with TB status (infection/disease). However, by applying the cut-off identified by ROC analysis, comparing TB and TBI with the HD group, within the IMID population, we did not improve the accuracy for TBI-diagnosis. Similarly, this signature did not improve TBI diagnosis in IMID with radiological evidence of TBI but negative QFT-Plus score.DiscussionTo develop alternative strategies for TBI immune-diagnosis, future studies are needed to evaluate the memory response of TBI defined by radiological tools. These results may help in tuberculosis management of patients taking lifelong immune-suppressive drugs

Serum Biomarker Profile Including CCL1, CXCL10, VEGF, and Adenosine Deaminase Activity Distinguishes Active From Remotely Acquired Latent Tuberculosis

INTRODUCTION: There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures. MATERIALS AND METHODS: The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation. RESULTS: sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants. CONCLUSION: The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals

Impact of pe_pgrs33 gene polymorphisms on mycobacterium tuberculosis infection and pathogenesis

PE_PGRS33 is a surface-exposed protein of Mycobacterium tuberculosis (Mtb) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the pe_pgrs33 gene of Mtb clinical isolates and evaluate their impact on protein functions. We sequenced pe_pgrs33 in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the Mtb complex (MTBC). Overall, an association between pe_pgrs33 alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on pe_pgrs33 against deleterious SNPs. Among a total of 19 pe_pgrs33 alleles identified in this study, 5 were cloned and used to complement the pe_pgrs33 knock-out mutant strain of Mtb H37Rv (Mtb\uce\u9433) to assess the functional impact of the respective polymorphisms in in vitro infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of Mtb H37Rv, impairing the cell entry capacity of Mtb, but neither its intracellular replication rate nor its immunomodulatory properties. In vivo studies performed in the murine model of tuberculosis (TB) demonstrated that the Mtb\uce\u9433 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, Mtb\uce\u9433 showed an enhanced virulence during the chronic steps of infection compared to Mtb H37Rv. Similarly, the complementation of Mtb\uce\u9433 with a frameshift allele also resulted in a Mtb strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of Mtb

Tuberculosis biomarkers: from diagnosis to protection

New approaches to control tuberculosis (TB) worldwide are needed. In particular, new tools for diagnosis and new biomarkers are required to evaluate both pathogen and host key elements of the response to infection. Non-sputum based diagnostic tests, biomarkers predictive of adequate responsiveness to treatment, and biomarkers of risk of developing active TB disease are major goals. Here, we review the current state of the field. Although reports on new candidate biomarkers are numerous, validation and independent confirmation are rare. Efforts are needed to reduce the gap between the exploratory <em>up-stream</em> identification of candidate biomarkers, and the validation of biomarkers against clear clinical endpoints in different populations. This will need a major commitment from both scientists and funding bodies