Novel N. Chegou

Host markers in QuantiFERON supernatants differentiate active TB from latent TB infection: Preliminary report

BackgroundInterferon gamma release assays, including the QuantiFERON® TB Gold In Tube (QFT) have been shown to be accurate in diagnosing Mycobacterium tuberculosis infection. These assays however, do not discriminate between latent TB infection (LTBI) and active TB disease.MethodsWe recruited twenty-three pulmonary TB patients and 34 household contacts from Cape Town, South Africa and performed the QFT test. To investigate the ability of new host markers to differentiate between LTBI and active TB, levels of 29 biomarkers in QFT supernatants were evaluated using a Luminex multiplex cytokine assay.ResultsEight out of 29 biomarkers distinguished active TB from LTBI in a pilot study. Baseline levels of epidermal growth factor (EGF) soluble CD40 ligand (sCD40L), antigen stimulated levels of EGF, and the background corrected antigen stimulated levels of EGF and macrophage inflammatory protein (MIP)-1β were the most informative single markers for differentiation between TB disease and LTBI, with AUCs of 0.88, 0.84, 0.87, 0.90 and 0.79 respectively. The combination of EGF and MIP-1β predicted 96% of active TB cases and 92% of LTBIs. Combinations between EGF, sCD40L, VEGF, TGF-α and IL-1α also showed potential to differentiate between TB infection states. EGF, VEGF, TGF-α and sCD40L levels were higher in TB patients.ConclusionThese preliminary data suggest that active TB may be accurately differentiated from LTBI utilizing adaptations of the commercial QFT test that includes measurement of EGF, sCD40L, MIP-1β, VEGF, TGF-α or IL-1α in supernatants from QFT assays. This approach holds promise for development as a rapid diagnostic test for active TB.

Hypercytokinaemia accompanies HIV-tuberculosis immune reconstitution inflammatory syndrome

Increased access to combination antiretroviral therapy in areas co-endemic for tuberculosis (TB) and HIV-1 infection is associated with an increased incidence of immune reconstitution inflammatory syndrome (TB-IRIS) whose cause is poorly understood. A case-control analysis of pro- and anti-inflammatory cytokines in TB-IRIS patients sampled at clinical presentation, and similar control patients with HIV-TB prescribed combined antiretroviral therapy who did not develop TB-IRIS. Peripheral blood mononuclear cells were cultured in the presence or absence of heat-killed Mycobacterium tuberculosis for 6 and 24 h. Stimulation with M. tuberculosis increased the abundance of many cytokine transcripts with interleukin (IL)-1&bgr;, IL-5, IL-6, IL-10, IL-13, IL-17A, interferon (IFN)-&ggr;, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF) being greater in stimulated TB-IRIS cultures. Analysis of the corresponding proteins in culture supernatants, revealed increased IL-1&bgr;, IL-2, IL-6, IL-8, IL-10, IL-12p40, IFN-&ggr;, GM-CSF and TNF in TB-IRIS cultures. In serum, higher concentrations of TNF, IL-6, and IFN-&ggr; were observed in TB-IRIS patients. Serum IL-6 and TNF decreased during prednisone therapy in TB-IRIS patients. These data suggest that cytokine release contributes to pathology in TB-IRIS. IL-6 and TNF were consistently elevated and decreased in serum during corticosteroid therapy. Specific blockade of these cytokines may be rational approach to immunomodulation in TB-IRIS.

Highly discordant T cell responses in individuals with recent exposure to household tuberculosis

Background: There are limited data comparing interferon-γ release assays (IGRAs) for the detection of Mycobacterium tuberculosis infection in highly endemic settings. Methods: A cross-sectional household contact study was conducted to measure the agreement of two IGRAs in relation to the tuberculin skin test (TST) to detect M tuberculosis infection and to assess the influence of M tuberculosis exposure and age. Results: In 82 individuals in household contact, 93% of children and 42% of adults had a high M tuberculosis contact score. The TST was positive in 78% of adults and 54% of children, the T-SPOT.TB was positive in 89% of children and 66% of adults and the QuantiFERON TB Gold (QTF) was positive in a similar proportion of adults and children (38.1% and 39.6%). In children there was poor agreement between the TST and T-SPOT.TB (κ = −0.15) and the T-SPOT.TB and the QTF (κ = −0.03), but good agreement between the TST and the QTF (κ = 0.78) using 10 mm cut-off. In adults there was fair to moderate agreement between the TST and T-SPOT.TB (κ = 0.38), the TST and QTF (κ = 0.34) and T-SPOT.TB and QTF (κ = −0.50). High levels of exposure to M tuberculosis were associated with at least a sevenfold odds of being T-SPOT.TB positive (95% CI 7.67 to 508.69) and a threefold odds of being QTF positive (95% CI 3.02 to 30.54). There was a significant difference in the magnitude of T-SPOT.TB early secretory antigenic target (ESAT)-6 and culture filtrate protein 10 kD (CFP-10) spot counts between adults and children. Conclusions: The T-SPOT.TB may be more sensitive than the TST or QTF for detecting recent M tuberculosis infection in children. Differences between assays and the predictive utility of these findings for subsequent disease development should be prospectively assessed.

Evaluation of adapted whole-blood interferon-γ release assays for the diagnosis of pleural tuberculosis

Background: Pleural tuberculosis (TB) remains difficult to diagnose despite numerous diagnostic tools. Recently, in vitro interferon (IFN)-γ-based assays have been introduced in the diagnosis of latent TB, but these techniques have not been established in the diagnosis of active TB disease, including pleural TB. Objectives: It was the aim of this study to assess the accuracy of the commercially available QuantiFERON® TB Gold assay and adapted variants of the assay, using pleural fluid or isolated pleural fluid cells for the diagnosis of pleural TB. Methods: We recruited 66 consecutive patients with a pleural effusion of unknown cause presenting at a tertiary academic health care centre in Cape Town, South Africa, a high prevalence area of TB. Blood and pleural fluid were collected at presentation for IFN-γ assays and the results evaluated for diagnostic accuracy. Results: The clinical diagnosis was TB in 30 (46%), malignancy in 20 (30%), parapneumonic effusion/empyema in 8 (12%) and effusion due to other causes in 8 patients (12%). Ex vivo pleural fluid IFN-γ levels accurately identified TB in all patients and were superior to the QuantiFERON In Tube assay using blood and pleural fluid (73 and 57% sensitivity, with 71 and 87% specificity, respectively) and the QuantiFERON Gold assay applied to isolated pleural fluid cells (100% sensitivity and 67% specificity). Conclusion: The ex vivo pleural fluid interferon-γ level is an accurate marker for the diagnosis of pleural TB, and the QuantiFERON TB Gold assay performed with peripheral blood or adapted for pleural fluid cells does not add diagnostic value.

Differential cytokine/chemokines and KL-6 profiles in patients with different forms of tuberculosis

Cytokines are involved in the mediation and regulation of immunity, inflammation, and haematopoiesis. Secretion patterns may reflect the pathology or etiology of different diseases. In an attempt to increase our understanding of immunopathology during different forms of tuberculosis, and identify potential biological markers that may differentiate between forms of tuberculosis, we investigated the levels of 29 cytokines and KL-6 in the plasma of HIV uninfected patients with active pulmonary tuberculosis (TB) without pleural effusions and in pleural TB with and without HIV-co-infection. Healthy individuals with latent Mycobacterium tuberculosis infection and patients with non-TB pleural effusions were used as controls, We showed that pleural TB patients had increased levels of markers associated with systemic inflammation compared to pulmonary TB (EGF, G-CSF, IL-1beta IL-6, IL-8, IL-10, MCP-1, MIP-1alpha, MIP-1beta, TNF-alpha and VEGF), whereas pulmonary TB patients without effusions had higher levels of factors involved in cell-mediated immunity (IL-12p40 and sCD40L). Plasma levels of cytokines may therefore contribute to biosignatures of diseases like TB but the data also highlight systemic differences between pulmonary TB, pleural TB and other form of pleural effusion diseases.

Potential of novel Mycobacterium tuberculosis infection phase-dependent antigens in the diagnosis of TB disease in a high burden setting

BackgroundConfirming tuberculosis (TB) disease in suspects in resource limited settings is challenging and calls for the development of more suitable diagnostic tools. Different Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens may be differentially recognized in infected and diseased individuals and therefore useful as diagnostic tools for differentiating between M.tb infection states. In this study, we assessed the diagnostic potential of 118 different M.tb infection phase-dependent antigens in TB patients and household contacts (HHCs) in a high-burden setting.MethodsAntigens were evaluated using the 7-day whole blood culture technique in 23 pulmonary TB patients and in 19 to 21 HHCs (total n = 101), who were recruited from a high-TB incidence community in Cape Town, South Africa. Interferon-gamma (IFN-γ) levels in culture supernatants were determined by ELISA.ResultsEight classical TB vaccine candidate antigens, 51 DosR regulon encoded antigens, 23 TB reactivation antigens, 5 TB resuscitation promoting factors (rpfs), 6 starvation and 24 other stress response-associated TB antigens were evaluated in the study. The most promising antigens for ascertaining active TB were the rpfs (Rv0867c, Rv2389c, Rv2450c, Rv1009 and Rv1884c), with Areas under the receiver operating characteristics curves (AUCs) between 0.72 and 0.80. A combination of M.tb specific ESAT-6/CFP-10 fusion protein, Rv2624c and Rv0867c accurately predicted 73% of the TB patients and 80% of the non-TB cases after cross validation.ConclusionsIFN-γ responses to TB rpfs show promise as TB diagnostic candidates and should be evaluated further for discrimination between M.tb infection states.

Tuberculosis assays: past, present and future.

Recent developments in the field of TB diagnostics, including the introduction of the Xpert MTB/RIF assay in field testing, raise the hope for faster and more accurate identification of active TB patients. However, there are still many issues that need to be addressed as no point-of-care tests are yet available. Furthermore, no tests are available which are universally applicable to all patients. Improvements in the microbiological and molecular-based approaches are promising and the diagnostic pipeline is encouraging. Host markers associated with active disease may hold promise, especially in situations where sputum diagnostics are problematic, including in children, HIV-infected individuals and in the case of extrapulmonary TB.

Utility of host markers detected in Quantiferon supernatants for the diagnosis of tuberculosis in children in a high-burden setting.

Background The diagnosis of childhood tuberculosis (TB) disease remains a challenge especially in young and HIV-infected children. Recent studies have identified potential host markers which, when measured in Quantiferon (QFT-IT) supernatants, show promise in discriminating between Mycobacterium tuberculosis (M.tb) infection states. In this study, the utility of such markers was investigated in children screened for TB in a setting with high TB incidence. Methodology and Principal Findings 76 children (29% HIV-infected) with or without active TB provided blood specimens collected directly into QFT-IT tubes. After overnight incubation, culture supernatants were harvested, aliquoted and frozen for future immunological research purposes. Subsequently, the levels of 12 host markers previously identified as potential TB diagnostic markers were evaluated in these supernatants for their ability to discriminate between M.tb infection and disease states using the Luminex platform. Of the 76 children included, 19 (25%) had culture confirmed TB disease; 26 (46%) of the 57 without TB had positive markers of M.tb infection defined by a positive QFT-IT test. The potentially most useful analytes for diagnosing TB disease included IFN-α2, IL-1Ra, sCD40L and VEGF and the most useful markers for discriminating between QFT-IT positive children as TB or latent infection included IL-1Ra, IP-10 and VEGF. When markers were used in combinations of four, 84% of all children were accurately classified into their respective groups (TB disease or no TB), after leave-one-out cross validation. Conclusions Measurement of the levels of IFN-α2, IL-1Ra, sCD40L, IP-10 and VEGF in QFT-IT supernatants may be a useful method for diagnosing TB disease and differentiating between active TB disease and M.tb infection in children. Our observations warrant further investigation in larger well-characterized clinical cohorts.

Analysis of Host Responses to Mycobacterium tuberculosis Antigens in a Multi-Site Study of Subjects with Different TB and HIV Infection States in Sub-Saharan Africa

Background Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas. Methods We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens. Results There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST- and TST+ contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST+ contacts (LTBI) compared to TB and TST- contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen. Conclusions Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.

Potential of Host Markers Produced by Infection Phase-Dependent Antigen-Stimulated Cells for the Diagnosis of Tuberculosis in a Highly Endemic Area

Background Recent interferon gamma (IFN-γ)-based studies have identified novel Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens as diagnostic candidates. In this study, the levels of 11 host markers other than IFN-γ, were evaluated in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens, for the diagnosis of TB disease. Methodology and Principal Findings Five M.tb infection phase-dependent antigens, comprising of three DosR-regulon-encoded proteins (Rv2032, Rv0081, Rv1737c), and two resucitation promoting factors (Rv0867c and Rv2389c), were evaluated in a case-control study with 15 pulmonary TB patients and 15 household contacts that were recruited from a high TB incidence setting in Cape Town, South Africa. After a 7-day whole blood culture, supernatants were harvested and the levels of the host markers evaluated using the Luminex platform. Multiple antigen-specific host markers were identified with promising diagnostic potential. Rv0081-specific levels of IL-12(p40), IP-10, IL-10 and TNF-α were the most promising diagnostic candidates, each ascertaining TB disease with an accuracy of 100%, 95% confidence interval for the area under the receiver operating characteristics plots, (1.0 to 1.0). Conclusions Multiple cytokines other than IFN-γ in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens show promise as diagnostic markers for active TB. These preliminary findings should be verified in well-designed diagnostic studies employing short-term culture assays.