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.

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.

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.

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.

Tuberculin skin test and in vitro assays provide complementary measures of antimycobacterial immunity in children and adolescents.

BACKGROUND: Although many studies have compared in vitro TB diagnostic tests with the venerable tuberculin skin test (TST), there is little understanding of the quantitative relationship between critical measures of antimycobacterial immunity used to detect TB infection. We, therefore, decided to determine the degree of redundancy between quantitative read-outs of in vivo and in vitro assays of antimycobacterial immunity. METHODS: We enrolled 475 healthy HIV-negative children and young adults living in a hyperendemic area of TB. We measured in vivo TST responses, and a 1:10 diluted 3- or 7-day whole-blood assay was used to determine the in vitro antigen-specific interferon (IFN)-gamma cytokine release. The frequency of antigen-specific IFN-gamma(+)CD4(+) and IFN-gamma(+)CD8(+) cells was tested using intracellular cytokine staining after 1 day incubation. RESULTS: In vivo TST responses segregated into two well-separated groups with either no measurable response (TST induration < 5 mm; n = 164) or a normally distributed group with TST indurations > or = 5 mm with peak at 15 mm (n = 260). In vitro assays provided a less pronounced separation of responders and nonresponders. Correlation analysis of responses among persons with TST > or = 5 mm demonstrated that extent of TST response was poorly correlated with IFN-gamma release (coefficients of correlation rho = 0.17-0.22) and frequency of IFN-gamma(+)CD4(+)/CD8(+) cells (rho = 0.05-0.17) across three stimulating antigens (Mycobacterium bovis bacillus Calmette-Guérin, purified protein derivative, early-secreted antigenic target-6). CONCLUSION: We conclude that in vivo and in vitro assays are nonredundant, complementary measures of antimycobacterial immunity. Both TST and in vitro assays provided valuable information about antimycobacterial immunity and by inference latent TB in the studied high-incidence TB settings.

Two loci control tuberculin skin test reactivity in an area hyperendemic for tuberculosis.

Approximately 20% of persons living in areas hyperendemic for tuberculosis (TB) display persistent lack of tuberculin skin test (TST) reactivity and appear to be naturally resistant to infection by Mycobacterium tuberculosis. Among those with a positive response, the intensity of TST reactivity varies greatly. The genetic basis of TST reactivity is not known. We report on a genome-wide linkage search for loci that have an impact on TST reactivity, which is defined either as zero versus nonzero (TST-BINa) or as extent of TST in millimeters (TST-quantitative trait locus [QTL]) in a panel of 128 families, including 350 siblings, from an area of South Africa hyperendemic for TB. We detected a major locus (TST1) on chromosomal region 11p14 (P = 1.4 x 10(-5)), which controls TST-BINa, with a lack of responsiveness indicating T cell-independent resistance to M. tuberculosis. We also detected a second major locus (TST2) on chromosomal region 5p15 (P < 10(-5)), which controls TST-QTL or the intensity of T cell-mediated delayed type hypersensitivity (DTH) to tuberculin. Fine mapping of this region identified SLC6A3, encoding the dopamine transporter DAT1, as a promising gene for further studies. Our results pave the way for the understanding of the molecular mechanisms involved in resistance to M. tuberculosis infection in endemic areas (TST1) and for the identification of critical regulators of T cell-dependent DTH to tuberculin (TST2).

Immunogenicity of novel DosR regulon-encoded candidate antigens of Mycobacterium tuberculosis in three high-burden populations in Africa.

Increasing knowledge about DosR regulon-encoded proteins has led us to produce novel Mycobacterium tuberculosis antigens for immunogenicity testing in human populations in three countries in Africa to which tuberculosis (TB) is endemic. A total of 131 tuberculin skin test-positive and/or ESAT-6/CFP10-positive, human immunodeficiency virus-negative adult household contacts of active pulmonary TB cases from South Africa (n = 56), The Gambia (n = 26), and Uganda (n = 49) were tested for gamma interferon responses to 7 classical and 51 DosR regulon-encoded M. tuberculosis recombinant protein antigens. ESAT-6/CFP10 fusion protein evoked responses in >75% of study participants in all three countries. Of the DosR regulon-encoded antigens tested, Rv1733c was the most commonly recognized by participants from both South Africa and Uganda and the third most commonly recognized antigen in The Gambia. The four most frequently recognized DosR regulon-encoded antigens in Uganda (Rv1733c, Rv0081, Rv1735c, and Rv1737c) included the three most immunogenic antigens in South Africa. In contrast, Rv3131 induced the highest percentage of responders in Gambian contacts (38%), compared to only 3.4% of Ugandan contacts and no South African contacts. Appreciable percentages of TB contacts with a high likelihood of latent M. tuberculosis infection responded to several novel DosR regulon-encoded M. tuberculosis proteins. In addition to significant similarities in antigen recognition profiles between the three African population groups, there were also disparities, which may stem from genetic differences between both pathogen and host populations. Our findings have implications for the selection of potential TB vaccine candidates and for determining biosignatures of latent M. tuberculosis infection, active TB disease, and protective immunity.

DNA fingerprint changes in tuberculosis: reinfection, evolution, or laboratory error?

BACKGROUND: DNA fingerprint patterns of Mycobacterium tuberculosis strains vary within individuals and between epidemiologically linked individuals because of pattern evolution, new infections, and laboratory error. We explored the importance of these factors. METHODS: Cultures from individuals in northern Malawi who had been diagnosed with tuberculosis (TB) during 1996-2001 were fingerprinted with restriction fragment-length polymorphism (RFLP). Probable laboratory error was inferred by use of dates or isolated positive cultures. Pattern evolution was explored within and between individuals, and the relative importance of relapse and reinfection was estimated in individuals with recurrent TB. RESULTS: RFLP results were available for 930 isolates from 806 individuals. The maximum laboratory-error rate was 3.9%. Pattern evolution was more common in linked individuals (17%) than on relapse (11%) or during treatment (3%). Twenty individuals had recurrent TB after completing treatment: in human immunodeficiency virus (HIV)-positive individuals, 7 of 12 recurrences were due to reinfection, compared with 0 of 8 in HIV-negative individuals (P=.01). CONCLUSIONS: The rate of DNA fingerprint-pattern evolution is not linear, and rates of change calculated from repeat cultures within individuals may not be applicable to transmission between individuals. The high proportion of recurrence due to reinfection found in HIV-positive individuals suggests that secondary prophylaxis and/or antiretroviral treatments are needed for such individuals.