Validation and optimization of host immunological bio-signatures for a point-of-care test for TB disease

The development of a non-sputum-based, point-of-care diagnostic test for tuberculosis (TB) is a priority in the global effort to combat this disease, particularly in resource-constrained settings. Previous studies have identified host biomarker signatures which showed potential, but there is a need to validate and refine these for development as a test. We recruited 1,403 adults presenting with symptoms suggestive of pulmonary TB at primary healthcare clinics in six countries from West, East and Southern Africa. Of the study cohort, 326 were diagnosed with TB and 787 with other respiratory diseases, from whom we randomly selected 1005 participants. Using Luminex(R) technology, we measured the levels of 20 host biomarkers in serum samples which we used to evaluate the diagnostic accuracy of previously identified and novel bio-signatures. Our previously identified seven-marker bio-signature did not perform well (sensitivity: 89%, specificity: 60%). We also identified an optimal, two-marker bio-signature with a sensitivity of 94% and specificity of 69% in patients with no history of previous TB. This signature performed slightly better than C-reactive protein (CRP) alone. The cut-off value for a positive diagnosis differed for human immuno-deficiency virus (HIV)-positive and -negative individuals. Notably, we also found that no signature was able to diagnose TB adequately in patients with a prior history of the disease. We have identified a two-marker, pan-African bio-signature which is more robust than CRP alone and meets the World Health Organization (WHO) target product profile requirements for a triage test in both HIV-negative and HIV-positive individuals. This signature could be incorporated into a point-of-care device, greatly reducing the necessity for expensive confirmatory diagnostics and potentially reducing the number of cases currently lost to follow-up. It might also potentially be useful with individuals unable to provide sputum or with paucibacillary disease. We suggest that the performance of TB diagnostic signatures can be improved by incorporating the HIV-status of the patient. We further suggest that only patients who have never had TB be subjected to a triage test and that those with a history of previous TB be evaluated using more direct diagnostic techniques.Cancer Signaling networks and Molecular Therapeutic

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. Copyright © 2008 S. Karger AG.Articl

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. © 2011 Expert Reviews Ltd.Revie

High level of discordant IGRA results in HIV-infected adults and children

SETTING: Tygerberg district, Western Cape Province, South Africa. OBJECTIVE: To measure the agreement of two interferon-gamma release assays (IGRAs) and the tuberculin skin test (TST) for the detection of Mycobacterium tuberculosis infection in human immunodeficiency virus (HIV) infected adults and children in a setting highly endemic for tuberculosis (TB). DESIGN: Cross-sectional study. RESULTS: In HIV-infected adults (n = 20) and children (n = 23), tests yielded discordant results, with 61% of individuals testing positive with T-SPOT.TB, 41% with TST and 28% with QuantiFERON® TB Gold (QTF). In children, there was poor agreement between the TST and T-SPOT.TB (kappa [κ] = -0.02), but moderate agreement between the TST and QTF (κ = 0.44). In adults, there was moderate agreement between the TST and T-SPOT.TB (κ = 0.43), and the TST and QTF (κ = 0.46). In children and adults, there was fair agreement between the T-SPOT.TB and QTF (κ = 0.33). Twenty per cent of adults had ≥1 indeterminate IGRA results. CONCLUSIONS: There is poor to moderate agreement between the TST and IGRAs in HIV-infected adults and children. T-SPOT.TB may have improved sensitivity for detection of M. tuberculosis infection in HIV-infected individuals compared to the QTF and the TST. In HIV-infected individuals, IGRA test properties are affected by test cut-off point and nil control responses. © 2008 The Union.Articl

Host Cytokine Responses Induced after Overnight Stimulation with Novel M-tuberculosis Infection Phase-Dependent Antigens Show Promise as Diagnostic Candidates for TB Disease

Immunogenetics and cellular immunology of bacterial infectious disease

Transmission Of Tuberculosis Among illicit drug use Linkages (TOTAL): A cross-sectional observational study protocol using respondent driven sampling

People who use illicit drugs (PWUDs) have been identified as a key at-risk group for tuberculosis (TB). Examination of illicit drug use networks has potential to assess the risk of TB exposure and disease progression. Research also is needed to assess mechanisms for accelerated TB transmission in this population. This study aims to 1) assess the rate of TB exposure, risk of disease progression, and disease burden among PWUD; 2) estimate the proportion of active TB cases resulting from recent transmission within this network; and 3) evaluate whether PWUD with TB disease have physiologic characteristics associated with more efficient TB transmission. Our cross-sectional, observational study aims to assess TB transmission through illicit drug use networks, focusing on methamphetamine and Mandrax (methaqualone) use, in a high TB burden setting and identify mechanisms underlying accelerated transmission. We will recruit and enroll 750 PWUD (living with and without HIV) through respondent driven sampling in Worcester, South Africa. Drug use will be measured through self-report and biological measures, with sputum specimens collected to identify TB disease by Xpert Ultra (Cepheid) and mycobacterial culture. We will co-enroll those with microbiologic evidence of TB disease in Aim 2 for molecular and social network study. Whole genome sequencing of Mycobacteria tuberculosis (Mtb) specimens and social contact surveys will be done for those diagnosed with TB. For Aim 3, aerosolized Mtb will be compared in individuals with newly diagnosed TB who do and do not smoke illicit drug. Knowledge from this study will provide the basis for a strategy to interrupt TB transmission in PWUD and provide insight into how this fuels overall community transmission. Results have potential for informing interventions to reduce TB spread applicable to high TB and HIV burden settings

Supplementary Material for: Bifunctional T-Cell-Derived Cytokines for the Diagnosis of Tuberculosis and Treatment Monitoring

<b><i>Background:</i></b> Diagnosis and treatment monitoring of patients with tuberculosis remain challenging. <b><i>Objective:</i></b> We have evaluated whether <i>Mycobacterium</i>-specific interferon (IFN)-γ and interleukin (IL)-2 bifunctional cytokine immune response assays improve the diagnosis of and correlate to treatment response in pulmonary tuberculosis. <b><i>Methods:</i></b> Early secretory antigenic target (ESAT)6/culture filtrate protein 10 (CFP10), microsomal triglyceride transfer protein 65 (MTP65) and the purified protein derivative (PPD) tuberculin-specific immune profiles were investigated in peripheral blood mononuclear cells from 19 patients with culture-confirmed tuberculosis and 23 healthy community controls (HCCs; 82.6% with latent <i>M. tuberculosis</i> infection) using a novel fluorescence-based dual-colour enzyme-linked immunospot (EliSpot) technology (FluoroSpot). <b><i>Results:</i></b> The frequency of ESAT6/CFP10-induced IFN-γ+IL-2- producing cells was elevated (p < 0.001), whereas the percentages of specific IFN-γ-IL-2+ (p = 0.002) and IFN-γ+IL-2+ double producing cells (p = 0.037) were diminished in tuberculosis patients in comparison to HCCs. A 3-host marker model using a combination of those IFN-γ and IL-2 single-cell responses showed 93.8% sensitivity and 77.8% specificity for tuberculosis. During tuberculosis treatment, the PPD-induced immune responses shifted from an IFN-γ+IL-2- dominated profile towards a balance of IFN-γ-IL-2+ and IFN-γ+IL-2+ double producing cells (all p ≤ 0.05). <b><i>Conclusions:</i></b> The addition of antigen-specific IL-2 production to IFN-γ responses by EliSpot in IFN-γ release assays increases diagnostic sensitivity for active tuberculosis

Diagnostic performance of a seven-marker serum protein biosignature for the diagnosis of active TB disease in African primary healthcare clinic attendees with signs and symptoms suggestive of TB

Microscopic imaging and technolog