A Subgroup of Latently Mycobacterium tuberculosis Infected Individuals Is Characterized by Consistently Elevated IgA Responses to Several Mycobacterial Antigens.

Elevated antibody responses to Mycobacterium tuberculosis antigens in individuals with latent infection (LTBI) have previously been linked to an increased risk for progression to active disease. Studies in the field focussed mainly on IgG antibodies. In the present study, IgA and/or IgG responses to the mycobacterial protein antigens AlaDH, NarL, 19 kDa, PstS3, and MPT83 were determined in a blinded fashion in sera from 53 LTBI controls, 14 healthy controls, and 42 active TB subjects. Among controls, we found that elevated IgA levels against all investigated antigens were not randomly distributed but concentrated on a subgroup of <30%-with particular high levels in a small subgroup of ~5% comprising one progressor to active TB. Based on a specificity of 100%, anti-NarL IgA antibodies achieved with 78.6% sensitivity the highest accuracy for the detection of active TB compared to healthy controls. In conclusion, the consistently elevated IgA levels in a subgroup of controls suggest higher mycobacterial load, a risk factor for progression to active TB, and together with high IgG levels may have prognostic potential and should be investigated in future large scale studies. The novel antigen NarL may also be promising for the antibody-based diagnosis of active TB cases.

Identification of a major locus, TNF1, that controls BCG-triggered tumor necrosis factor production by leukocytes in an area hyperendemic for tuberculosis.

BACKGROUND: Tumor necrosis factor (TNF) is a key immune regulator of tuberculosis resistance, as exemplified by the highly increased risk of tuberculosis disease among individuals receiving TNF-blocker therapy. METHODS: We determined the extent of TNF production after stimulation with BCG or BCG plus interferon gamma (IFN-γ) using a whole blood assay in 392 children belonging to 135 nuclear families from an area hyperendemic for tuberculosis in South Africa. We conducted classical univariate and bivariate genome-wide linkage analysis of TNF production using the data from both stimulation protocols by means of an extension of the maximum-likelihood-binomial method for quantitative trait loci to multivariate analysis. RESULTS: Stimulation of whole blood by either BCG or BCG plus IFN-γ resulted in a range of TNF release across subjects. Extent of TNF production following both stimulation protocols was highly correlated (r = 0.81). We failed to identify genetic linkage of TNF release when considering each stimulus separately. However, using a multivariate approach, we detected a major pleiotropic locus (P < 10(-5)) on chromosome region 11p15, termed TNF locus 1 (TNF1), that controlled TNF production after stimulation by both BCG alone and BCG plus IFN-γ. CONCLUSIONS: The TNF1 locus was mapped in the vicinity of the TST1 locus, previously identified in the same family sample, that controls tuberculin skin test (TST) negativity per se, that is, T-cell-independent resistance to Mycobacterium tuberculosis infection. This suggested that there is a connection between TST negativity per se and TNF production.

MxDC and MxLIVE: software for data acquisition, information management and remote access to macromolecular crystallography beamlines.

An integrated computer software system for on-site and remote collection of macromolecular crystallography (MX) data at the Canadian Light Source (CLS) is described. The system consists of an integrated graphical user interface for data collection and beamline control [MX Data Collector (MxDC)] which provides experiment-focused control of beamline devices, and a laboratory information management system [MX Laboratory Information Virtual Environment (MxLIVE)] for managing sample and experiment information through a web browser. The system allows remote planning and transmission of sample and experiment parameters to the beamline through MxLIVE, on-site or remote data collection through MxDC guided by information from MxLIVE, and remote monitoring and download of experimental results through MxLIVE. The system is deployed and in use on both MX beamlines at the CLS which constitute the Canadian Macromolecular Crystallography Facility.

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

BACKGROUND: Confirming 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. METHODS: Antigens 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. RESULTS: Eight 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. CONCLUSIONS: IFN-γ responses to TB rpfs show promise as TB diagnostic candidates and should be evaluated further for discrimination between M.tb infection states.

Steps Toward Engagement Integrity: Learning From Participatory Visual Methods in Marginalized South African Communities.

Community engagement and involvement have been increasingly recognized as an ethical and valuable component of health science research over the past two decades. Progress has been accompanied by emerging standards that emphasize participation, two-way communication, inclusion, empowerment, and ownership. Although these are important and noble benchmarks, they can represent a challenge for research conducted in marginalized contexts. This community case study reports on the methods, outcomes, constraints and learning from an NGO-led community engagement project called Bucket Loads of Health, implemented in the Western Cape province of South Africa. The independent project team used multiple participatory visual methods to foster two-way communication between members of two disenfranchised communities, Enkanini and Delft, and a group of water microbiologists at Stellenbosch University who were conducting research in Enkanini. The project was carried out during the 2018 Western Cape water crisis, under the growing threat of "Day Zero". The resulting visual outputs illustrated the negative impacts of water shortage on health and wellbeing in these community settings and showcased scientific endeavors seeking to address them. Engagement included knowledge exchange combining body maps, role play performances and films created by the community members, with hand maps, posters and presentations produced by the scientists. Whereas these engagement tools enabled reciprocal listening between all groups, their ability to respond to the issues raised was hindered by constraints in resources and capacity beyond their control. An additional core objective of the project was to bring the impacts of water shortage in participating communities, and the work of the research team, to the attention of local government. The case study demonstrates the challenges that politically ambitious community engagement faces in being acknowledged by government representatives. We further the argument that research institutions and funders need to match professed commitments to engagement with training and resources to support researchers and community members in responding to the needs and aspirations surfaced through engagement processes. We introduce the concept of engagement integrity to capture the gap between recommended standards of community engagement and what is realistically achievable in projects that are constrained by funding, time, and political interest.

High heritability of antimycobacterial immunity in an area of hyperendemicity for tuberculosis disease.

Human antimycobacterial immunity is a critical component of tuberculosis (TB) pathogenesis that is often used to infer the presence of TB infection. We report high heritability (>50%) for in vitro secretion of tumor necrosis factor alpha and interferon gamma (IFN-gamma), and the frequency of antigen-specific IFN-gamma(+)CD4(+) and IFN-gamma(+)CD8(+) cells in the response of whole blood to mycobacterial challenge. In principal component analysis, the first 3 components explain 78% of the overall variance consistent with the effect of pleiotropic regulatory genes of human antimycobacterial immunity. These results directly demonstrate the pivotal role played by host genetics in quantitative measures of antimycobacterial immunity underlying immune diagnosis of TB infection.

Biomarker discovery in heterogeneous tissue samples -taking the in-silico deconfounding approach.

BACKGROUND: For heterogeneous tissues, such as blood, measurements of gene expression are confounded by relative proportions of cell types involved. Conclusions have to rely on estimation of gene expression signals for homogeneous cell populations, e.g. by applying micro-dissection, fluorescence activated cell sorting, or in-silico deconfounding. We studied feasibility and validity of a non-negative matrix decomposition algorithm using experimental gene expression data for blood and sorted cells from the same donor samples. Our objective was to optimize the algorithm regarding detection of differentially expressed genes and to enable its use for classification in the difficult scenario of reversely regulated genes. This would be of importance for the identification of candidate biomarkers in heterogeneous tissues. RESULTS: Experimental data and simulation studies involving noise parameters estimated from these data revealed that for valid detection of differential gene expression, quantile normalization and use of non-log data are optimal. We demonstrate the feasibility of predicting proportions of constituting cell types from gene expression data of single samples, as a prerequisite for a deconfounding-based classification approach.Classification cross-validation errors with and without using deconfounding results are reported as well as sample-size dependencies. Implementation of the algorithm, simulation and analysis scripts are available. CONCLUSIONS: The deconfounding algorithm without decorrelation using quantile normalization on non-log data is proposed for biomarkers that are difficult to detect, and for cases where confounding by varying proportions of cell types is the suspected reason. In this case, a deconfounding ranking approach can be used as a powerful alternative to, or complement of, other statistical learning approaches to define candidate biomarkers for molecular diagnosis and prediction in biomedicine, in realistically noisy conditions and with moderate sample sizes.

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

BACKGROUND: Interferon 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. METHODS: We 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. RESULTS: Eight 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)-1beta 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-1beta predicted 96% of active TB cases and 92% of LTBIs. Combinations between EGF, sCD40L, VEGF, TGF-alpha and IL-1alpha also showed potential to differentiate between TB infection states. EGF, VEGF, TGF-alpha and sCD40L levels were higher in TB patients. CONCLUSION: These 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-1beta, VEGF, TGF-alpha or IL-1alpha in supernatants from QFT assays. This approach holds promise for development as a rapid diagnostic test for active TB.

Comparison of IFN-gamma responses to mycobacterial antigens as markers of response to BCG vaccination.

An increase in interferon-gamma (IFN-gamma) production to Mycobacterium tuberculosis purified protein derivative (Mtb PPD), as measured in the cultured diluted whole blood assay, is one indicator of a protective immune response to BCG vaccine. We have explored the potential for this assay to be improved by measuring IFN-gamma responses to more defined antigens of M. tuberculosis (short-term and mid-term culture filtrates, ESAT-6, 38 kDa), Mycobacterium bovis (MPB70), M. bovis BCG (Antigen 85) and Mycobacterium leprae (35 kDa), in UK teenagers before and 1 year after BCG vaccination (or no vaccination as controls). There was a significant increase in response to the culture filtrates post-vaccination, but this was no greater than that to Mtb PPD. Many teenagers responded to the purified antigens, in particular to Antigen 85, prior to vaccination, and BCG vaccination could only augment this pre-existing response to a limited extent; prior exposure to environmental mycobacteria can thus induce cross-reactive responses to antigens which complicate interpretation of in vitro assays of vaccine response. In contrast, ESAT-6 was recognised by only one teenager prior to vaccination, and, as expected, responses were not boosted by BCG. We therefore conclude that Mtb PPD is the antigen preparation of choice for assessing the immunogenicity of BCG vaccination.

Reflections on the ethics of participatory visual methods to engage communities in global health research.

There is a growing body of literature describing conceptual frameworks for working with participatory visual methods (PVM). Through a global health lens, this paper examines some key themes within these frameworks. We reflect on our experiences of working with with an array of PVM to engage community members in Vietnam, Kenya, the Philippines and South Africa in biomedical research and public health. The participants that we have engaged in these processes live in under-resourced areas with high prevalence of communicable and non-communicable diseases. Our paper describes some of the challenges that we have encountered while using PVM to foster knowledge exchange, build relationships and facilitate change among individuals and families, community members, health workers, biomedical scientists and researchers. We consider multiple ethical situations that have arisen through our work and discuss the ways in which we have navigated and negotiated them. We offer our reflections and learning from facilitating these processes and in doing so we add novel contributions to ethical framework concepts.

Developing service provision for patients in primary care.

This article outlines the government's changes to the way that primary and community health services will be commissioned and provided. It also discusses the opportunities that exist for nurses to lead and develop services for the benefit of patients in the implementation of changes.

Serologic diagnosis of tuberculosis by combining Ig classes against selected mycobacterial targets.

INTRODUCTION: Accurate, simple and cost-effective diagnostic tests are needed for diagnosis of active tuberculosis (TB). Serodiagnosis is attractive as it can be harnessed for point-of-care tests. METHODS: We evaluated, in a blinded fashion, the sensitivity and specificity of serologic immunoglobulin (Ig)G, IgA and/or IgM responses to Apa, heat shock protein (HSP) 16.3, HSP20, PE35, probable thiol peroxidase Tpx and lipoarabinomannan (LAM) in 42 HIV-negative South African pulmonary TB patients and 67 control individuals. The status of latent Mycobacterium tuberculosis infection (LTBI) among controls was defined through the TST and IFN-γ release assays (IGRAs). We evaluated 47 definite LTBI (IGRA(+)/LTBI), 8 putative LTBI (IGRA(-)/TST(+)) and 12 TB-uninfected (non-LTBI) subjects. RESULTS: In contrast to anti-PE35 IgA, anti-PE35 IgG and particularly anti-Apa IgA, performances of anti-LAM IgG and selected anti-protein antibodies were less affected by inclusion of LTBI participants into the analysis. Anti-LAM IgG showed with a sensitivity/specificity of 71.4%/86.6% (p < 0.001) the best discrimination between TB and non-TB subjects. Selected five-antibody-combinations (including anti-LAM IgG, anti-LAM IgA and anti-Tpx IgG) further improved this performance to an accuracy exceeding 86%. CONCLUSIONS: Antibody responses to some Mycobacterium tuberculosis antigens often also reflect latent infection explaining the poor performance of antibody-based tests for active TB in TB-endemic settings. Our results suggest that rather a combination of serological responses against selected protein and non-protein antigens and different Ig classes should be investigated for TB serodiagnostics.

BDNF Val66Met and DRD2 Taq1A polymorphisms interact to influence PTSD symptom severity: a preliminary investigation in a South African population.

BACKGROUND: We evaluated the role that selected variants in serotonin transporter (5-HTT), dopamine receptor 2 (DRD2) and brain-derived neurotrophic factor (BDNF) genes play in PTSD symptom severity in an at-risk population. We also investigated the interaction between the genetic variants to determine whether these variables and the interactions between the variables influenced the severity of PTSD symptoms. METHODS: PTSD symptoms were quantitatively assessed using the Davidson Trauma Scale (DTS) in 150 participants from an at-risk South African population. All participants were genotyped for the 5-HTTLPR, DRD2 Taq1A and BDNF Val66Met polymorphisms. Gene-gene interactions were investigated using various linear models. All analyses were adjusted for age, gender, major depressive disorder diagnosis, level of resilience, level of social support and alcohol dependence. RESULTS: A significant interaction effect between DRD2 Taq1A and BDNF Val66Met variants on DTS score was observed. On the background of the BDNF Val66Val genotype, DTS score increased significantly with the addition of a DRD2 Taq1A A1 allele. However, on the BDNF Met66 allele background, the addition of an A1 allele was found to reduce total DTS score. CONCLUSIONS: This study provides preliminary evidence for an epistatic interaction between BDNF Val66Met and DRD2 Taq1A polymorphisms on the severity of PTSD symptoms, where both too little and too much dopamine can result in increased PTSD symptom severity.

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.

Heparin-binding hemagglutinin induces IFN-γ(+) IL-2(+) IL-17(+) multifunctional CD4(+) T cells during latent but not active tuberculosis disease.

The mycobacterial heparin-binding hemagglutinin (HBHA) protein induces a potent gamma interferon (IFN-γ) response in latent tuberculosis (TB) infection and is a candidate vaccine and diagnostic antigen. We have assessed HBHA-specific intracellular IFN-γ, interleukin-2 (IL-2), and IL-17 production by CD4(+) T cells in TB cases and household contacts (HHCs) as well as the level of secreted IFN-γ in whole-blood culture supernatant. HHCs were further classified as tuberculin skin test (TST) positive or negative, and the group was also divided as HIV positive or negative. Our study revealed that HBHA induces multifunctional IFN-γ-, IL-2-, and IL-17-coexpressing CD4(+) T cells in HHCs but not in active TB cases; however, IFN-γ levels in culture supernatant did not differ between participant groups. Further studies are needed to completely understand how HBHA induces immune responses in different disease groups.

Biomarkers of inflammation, immunosuppression and stress with active disease are revealed by metabolomic profiling of tuberculosis patients.

Although tuberculosis (TB) causes more deaths than any other pathogen, most infected individuals harbor the pathogen without signs of disease. We explored the metabolome of >400 small molecules in serum of uninfected individuals, latently infected healthy individuals and patients with active TB. We identified changes in amino acid, lipid and nucleotide metabolism pathways, providing evidence for anti-inflammatory metabolomic changes in TB. Metabolic profiles indicate increased activity of indoleamine 2,3 dioxygenase 1 (IDO1), decreased phospholipase activity, increased abundance of adenosine metabolism products, as well as indicators of fibrotic lesions in active disease as compared to latent infection. Consistent with our predictions, we experimentally demonstrate TB-induced IDO1 activity. Furthermore, we demonstrate a link between metabolic profiles and cytokine signaling. Finally, we show that 20 metabolites are sufficient for robust discrimination of TB patients from healthy individuals. Our results provide specific insights into the biology of TB and pave the way for the rational development of metabolic biomarkers for TB.

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.

Medroxyprogesterone acetate alters Mycobacterium bovis BCG-induced cytokine production in peripheral blood mononuclear cells of contraceptive users.

Most individuals latently infected with Mycobacterium tuberculosis (M.tb) contain the infection by a balance of effector and regulatory immune responses. This balance can be influenced by steroid hormones such as glucocorticoids. The widely used contraceptive medroxyprogesterone acetate (MPA) possesses glucocorticoid activity. We investigated the effect of this hormone on immune responses to BCG in household contacts of active TB patients. Multiplex bead array analysis revealed that MPA demonstrated both glucocorticoid and progestogenic properties at saturating and pharmacological concentrations in peripheral blood mononuclear cells (PBMCs) and suppressed antigen specific cytokine production. Furthermore we showed that PBMCs from women using MPA produced significantly lower levels of IL-1α, IL-12p40, IL-10, IL-13 and G-CSF in response to BCG which corresponded with lower numbers of circulating monocytes observed in these women. Our research study is the first to show that MPA impacts on infections outside the genital tract due to a systemic effect on immune function. Therefore MPA use could alter susceptibility to TB, TB disease severity as well as change the efficacy of new BCG-based vaccines, especially prime-boost vaccine strategies which may be administered to adult or adolescent women in the future.

Higher human CD4 T cell response to novel Mycobacterium tuberculosis latency associated antigens Rv2660 and Rv2659 in latent infection compared with tuberculosis disease.

One third of the world's population is infected with Mycobacterium tuberculosis (M.tb). A vaccine that would prevent progression to TB disease will have a dramatic impact on the global TB burden. We propose that antigens of M.tb that are preferentially expressed during latent infection will be excellent candidates for post-exposure vaccination. We therefore assessed human T cell recognition of two such antigens, Rv2660 and Rv2659. Expression of these was shown to be associated with non-replicating persistence in vitro. After six days incubation of PBMC from persons with latent tuberculosis infection (LTBI) and tuberculosis (TB) disease, Rv2660 and Rv2659 induced IFN-γ production in a greater proportion of persons with LTBI, compared with TB diseased patients. Persons with LTBI also had increased numbers of viable T cells, and greater specific CD4(+) T cell proliferation and cytokine expression capacity. Persons with LTBI preferentially recognize Rv2659 and Rv2660, compared with patients with TB disease. These results suggest promise of these antigens for incorporation into post-exposure TB vaccines.