Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory.

BACKGROUND: Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed. METHODS: The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis. RESULTS: Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74). CONCLUSIONS: READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs.

Exhaled Mycobacterium tuberculosis Predicts Incident Infection in Household Contacts.

BACKGROUND: Halting transmission of Mycobacterium tuberculosis (Mtb) by identifying infectious individuals early is key to eradicating tuberculosis (TB). Here we evaluate face mask sampling as a tool for stratifying the infection risk of individuals with pulmonary TB (PTB) to their household contacts. METHODS: Forty-six sputum-positive PTB patients in The Gambia (August 2016-November 2017) consented to mask sampling prior to commencing treatment. Incident Mtb infection was defined in 181 of their 217 household contacts as QuantiFERON conversion or an increase in interferon-γ of ≥1 IU/mL, 6 months after index diagnosis. Multilevel mixed-effects logistical regression analysis with cluster adjustment by household was used to identify predictors of incident infection. RESULTS: Mtb was detected in 91% of PTB mask samples with high variation in IS6110 copies (5.3 × 102 to 1.2 × 107). A high mask Mtb level (≥20 000 IS6110 copies) was observed in 45% of cases and was independently associated with increased likelihood of incident Mtb infection in contacts (adjusted odds ratio, 3.20 [95% confidence interval, 1.26-8.12]; P = .01), compared with cases having low-positive/negative mask Mtb levels. Mask Mtb level was a better predictor of incident Mtb infection than sputum bacillary load, chest radiographic characteristics, or sleeping proximity. CONCLUSIONS: Mask sampling offers a sensitive and noninvasive tool to support the stratification of individuals who are most infectious in high-TB-burden settings. Our approach can provide better insight into community transmission in complex environments.

Neutrophils Contribute to Severity of Tuberculosis Pathology and Recovery From Lung Damage Pre- and Posttreatment.

BACKGROUND: Despite microbiological cure, about 50% of tuberculosis (TB) patients have poor lung recovery. Neutrophils are associated with lung pathology; however, CD16/CD62L-defined subsets have not been studied in TB. Using flow cytometry, we monitored frequencies, phenotype, and function of neutrophils following stimulation with Mycobacterium tuberculosis (Mtb) whole cell lysate (WCL) and ESAT-6/CFP-10 fusion protein (EC) in relation to lung pathology. METHODS: Fresh blood from 42 adult, human immunodeficiency virus (HIV)-negative TB patients were analyzed pre- and post-therapy, with disease severity determined using chest radiography and bacterial load. Flow cytometry was used to monitor frequencies, phenotype, and function (generation of reactive oxygen species [ROS], together with CD11b, tumor necrosis factor, and interleukin 10 [IL-10] expression) of neutrophils following 2-hour stimulation with Mtb-specific antigens. RESULTS: Total neutrophils decreased by post-treatment compared to baseline (P = .0059); however, CD16brCD62Lbr (segmented) neutrophils increased (P = .0031) and CD16dimCD62Lbr (banded) neutrophils decreased (P = .038). Banded neutrophils were lower in patients with severe lung damage at baseline (P = .035). Following WCL stimulation, ROS from segmented neutrophils was higher in patients with low Mtb loads even after adjusting for sex (P = .038), whereas IL-10-expressing CD16dimCD62Llo cells were higher in patients with mild damage (P = .0397) at baseline. CONCLUSIONS: High ROS generation, low levels of banded neutrophils, and high levels of IL-10-expressing CD16dimCD62Llo neutrophils are associated with reduced lung pathology at diagnosis. Hence, neutrophils are potential early indicators of TB severity and promising targets for TB host-directed therapy.

Diagnostic Accuracy of the Cepheid 3-gene Host Response Fingerstick Blood Test in a Prospective, Multi-site Study: Interim Results.

BACKGROUND: The development of a fast and accurate, non-sputum-based point-of-care triage test for tuberculosis (TB) would have a major impact on combating the TB burden worldwide. A new fingerstick blood test has been developed by Cepheid (the Xpert MTB Host Response [MTB-HR] prototype), which generates a "TB score" based on messenger RNA (mRNA) expression of 3 genes. Here we describe the first prospective findings of the MTB-HR prototype. METHODS: Fingerstick blood from adults presenting with symptoms compatible with TB in South Africa, The Gambia, Uganda, and Vietnam was analyzed using the Cepheid GeneXpert MTB-HR prototype. Accuracy of the Xpert MTB-HR cartridge was determined in relation to GeneXpert Ultra results and a composite microbiological score (GeneXpert Ultra and liquid culture) with patients classified as having TB or other respiratory diseases (ORD). RESULTS: When data from all sites (n = 75 TB, 120 ORD) were analyzed, the TB score discriminated between TB and ORD with an area under the curve (AUC) of 0.94 (95% confidence interval [CI], .91-.97), sensitivity of 87% (95% CI, 77-93%) and specificity of 94% (88-97%). When sensitivity was set at 90% for a triage test, specificity was 86% (95% CI, 75-97%). These results were not influenced by human immunodeficiency virus (HIV) status or geographical location. When evaluated against a composite microbiological score (n = 80 TB, 111 ORD), the TB score was able to discriminate between TB and ORD with an AUC of 0.88 (95% CI, .83-.94), 80% sensitivity (95% CI, 76-85%) and 94% specificity (95% CI, 91-96%). CONCLUSIONS: Our interim data indicate the Cepheid MTB-HR cartridge reaches the minimal target product profile for a point of care triage test for TB using fingerstick blood, regardless of geographic area or HIV infection status.

BCG Vaccine Protection Against Mycobacterium tuberculosis Infection by Level of Exposure in The Gambia.

In Indonesia, BCG vaccine protection against Mycobacterium tuberculosis infection decreased with increasing exposure to the pathogen. We aimed to validate these findings in Africa. Poisson regression was used to estimate BCG protection, stratified by pathogen exposure using an exposure score, against enzyme-linked immunospot assay conversion at 3 months in 220 Gambian case contacts. Although the interaction between BCG and exposure was not significant (P = .13), BCG protection was strongest in the lowest-exposure tertile (relative risk, 0.35 [95% confidence interval, .15-.82; P = .02] vs 0.50 [.30-.83; P = .008] and 0.71 (.45-1.13; P = .1] for the middle and highest-exposure tertiles, respectively. These results are consistent with those from Indonesia.

Changes in Transcript, Metabolite, and Antibody Reactivity During the Early Protective Immune Response in Humans to Mycobacterium tuberculosis Infection.

BACKGROUND: Strategies to prevent Mycobacterium tuberculosis (Mtb) infection are urgently required. In this study, we aimed to identify correlates of protection against Mtb infection. METHODS: Two groups of Mtb-exposed contacts of tuberculosis (TB) patients were recruited and classified according to their Mtb infection status using the tuberculin skin test (TST; cohort 1) or QuantiFERON (QFT; cohort 2). A negative reading at baseline with a positive reading at follow-up classified TST or QFT converters and a negative reading at both time points classified TST or QFT nonconverters. Ribonucleic acid sequencing, Mtb proteome arrays, and metabolic profiling were performed. RESULTS: Several genes were found to be differentially expressed at baseline between converters and nonconverters. Gene set enrichment analysis revealed a distinct B-cell gene signature in TST nonconverters compared to converters. When infection status was defined by QFT, enrichment of type I interferon was observed. A remarkable area under the curve (AUC) of 1.0 was observed for IgA reactivity to Rv0134 and an AUC of 0.98 for IgA reactivity to both Rv0629c and Rv2188c. IgG reactivity to Rv3223c resulted in an AUC of 0.96 and was markedly higher compared to TST nonconverters. We also identified several differences in metabolite profiles, including changes in biomarkers of inflammation, fatty acid metabolism, and bile acids. Pantothenate (vitamin B5) was significantly increased in TST nonconverters compared to converters at baseline (q = 0.0060). CONCLUSIONS: These data provide new insights into the early protective response to Mtb infection and possible avenues to interfere with Mtb infection, including vitamin B5 supplementation.Analysis of blood from highly exposed household contacts from The Gambia who never develop latent Mycobacterium tuberculosis infection shows distinct transcriptomic, antibody, and metabolomic profiles compared to those who develop latent tuberculosis infection but prior to any signs of infection.

Sex-Differential Non-Vaccine-Specific Immunological Effects of Diphtheria-Tetanus-Pertussis and Measles Vaccination.

Vaccines can have nontargeted heterologous effects that manifest as increased protection against nonvaccine infections, as described for measles vaccine (MV), or increased susceptibility to infections and death, as described following diphtheria-tetanus-whole cell pertussis (DTP) vaccination. The mechanisms are unknown, and high-quality immunological studies are lacking. This study was designed to investigate the heterologous effects of MV and DTP in 302 Gambian infants. The results support a sex-differential immunosuppressive effect of DTP on innate proinflammatory responses and T-cell immunity. Males but not females receiving MV had enhanced proinflammatory innate responses. The results point to modified signaling via Toll-like receptor 4 (TLR4) as a possible mechanism for the effects on innate immunity. When both vaccines were administered together, purified protein derivative responses were enhanced in females but downregulated in males. Collectively, these data indicate immunological effects that could account for heterologous effects of MV and DTP, to take forward into prospective trials.

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.

BACKGROUND: User-friendly, rapid, inexpensive yet accurate TB diagnostic tools are urgently needed at points of care in resource-limited settings. We investigated host biomarkers detected in serum samples obtained from adults with signs and symptoms suggestive of TB at primary healthcare clinics in five African countries (Malawi, Namibia, South Africa, The Gambia and Uganda), for the diagnosis of TB disease. METHODS: We prospectively enrolled individuals presenting with symptoms warranting investigation for pulmonary TB, prior to assessment for TB disease. We evaluated 22 host protein biomarkers in stored serum samples using a multiplex cytokine platform. Using a pre-established diagnostic algorithm comprising of laboratory, clinical and radiological findings, participants were classified as either definite TB, probable TB, questionable TB status or non-pulmonary TB. RESULTS: Of the 716 participants enrolled, 185 were definite and 29 were probable TB cases, 6 had questionable TB disease status, whereas 487 had no evidence of TB. A seven-marker biosignature of C reactive protein, transthyretin, IFN-γ, complement factor H, apolipoprotein-A1, inducible protein 10 and serum amyloid A identified on a training sample set (n=491), diagnosed TB disease in the test set (n=210) with sensitivity of 93.8% (95% CI 84.0% to 98.0%), specificity of 73.3% (95% CI 65.2% to 80.1%), and positive and negative predictive values of 60.6% (95% CI 50.3% to 70.1%) and 96.4% (95% CI 90.5% to 98.8%), respectively, regardless of HIV infection status or study site. CONCLUSIONS: We have identified a seven-marker host serum protein biosignature for the diagnosis of TB disease irrespective of HIV infection status or ethnicity in Africa. These results hold promise for the development of a field-friendly point-of-care screening test for pulmonary TB.

C-reactive protein, Neopterin and Beta2 microglobulin levels pre and post TB treatment in The Gambia.

BACKGROUND: Tuberculosis is one of the leading causes of morbidity and mortality in developing countries. Analysis of the host immune response may help with generating point-of-care tests for personalised monitoring. Thus, the aim of this study was to assess the relationship between immune activation markers: C-reactive protein (CRP), Beta2 microglobulin (B2M) and Neopterin, disease severity prior to treatment and response to therapy in adult pulmonary TB patients. METHODS: HIV negative adult pulmonary TB index cases (n = 91) were recruited from the TB clinic at MRC, The Gambia. Plasma samples were collected at enrolment and at 2 and 6 months following TB treatment initiation. An enzyme linked immunosorbent assay (ELISA) was performed for evaluation of CRP, B2M and Neopterin levels and correlated with clinical and microbiological parameters including strain of infection. Disease severity was determined using Chest X-ray (CXR), Body Mass Index (BMI) and sputum smear grade. RESULTS: Plasma levels of all three markers were highly elevated in patients at recruitment and declined significantly during TB therapy. No correlation with disease severity was seen at recruitment. CRP showed the most significant decrease by 2 months of treatment (p < 0.0001) whereas levels of B2M and Neopterin showed little change by 2 months but a significant decrease by 6 months of treatment (p = 0.0002 and p < 0.0001 respectively). At recruitment, B2M levels were significantly higher in subjects infected with Mycobacterium africanum (Maf) compared with those infected with Mycobacterium tuberculosis sensu stricto (Mtb) (p = 0.0075). In addition, while CRP and Neopterin showed a highly significant decline post-treatment regardless of strain (p < 0.0001 for all), B2M showed differential decline depending on strain (p = 0.0153 for Mtb and p = 0.0048 for Maf) and levels were still significantly higher at 6 months in Maf compared to Mtb infected subjects (p = 0.0051). CONCLUSION: Our findings suggest that activation markers, particularly CRP, may have a role in identifying good response to TB therapy regardless of the strain of infection and could be further developed as point-of-care tests. In addition, B2M levels may allow differentiation between Mtb and Maf-infected subjects.

Dysregulation of Apoptosis Is a Risk Factor for Tuberculosis Disease Progression.

BACKGROUND: A major barrier to effective tuberculosis control is our limited understanding of risk factors for tuberculosis disease progression. This study examined the role of apoptosis in immunity to tuberculosis. METHODS: Cell subsets from tuberculosis cases and tuberculin skin test-positive (TST(+)) and TST-negative (TST(-)) household contacts (HHCs) were analyzed for expression of annexin-V and propidium iodide by flow cytometry. RNA microarrays were used to determine differences in apoptotic gene expression levels and multiplex ligation-dependent probe amplification was used to analyze gene expression in HHCs who progressed to active tuberculosis. RESULTS: T cells from TST(+)HHC exhibited higher levels of apoptosis than tuberculosis cases; however, tuberculosis cases had a higher proportion of late apoptotic cells within the CD3(+)PD-1(+) subset. Tuberculosis cases had reduced levels of antiapoptotic genes compared to HHCs with a significant reduction in BCL2 associated with disease progression at least 1 year prior to progression. CONCLUSIONS: While T cells are clearly able to mount a robust immune response to Mycobacterium tuberculosis, there are increased levels of apoptosis seen in effector T cells from tuberculosis patients. Dysregulation of several apoptotic genes suggest that apoptosis is a major functional pathway that could be targeted for future host-directed therapeutics.

Complex anemia in tuberculosis: the need to consider causes and timing when designing interventions.

BACKGROUND: Anemia is common in tuberculosis, and multiple etiologies necessitate targeted interventions. The proportion of iron-responsive anemia due to iron deficiency compared with iron-unresponsive anemia due to impaired iron absorption/redistribution from tuberculosis-associated immune activation or inflammation is unknown. This impedes selection of safe and effective treatment and appropriate intervention timing. METHODS: Baseline hemoglobin, ferritin, hepcidin, soluble transferrin receptor (sTfR), and transferrin were measured in 45 patients with confirmed pulmonary tuberculosis (cases), 47 tuberculin skin test (TST)-positive controls, and 39 TST-negative controls in The Gambia. Tuberculosis cases were additionally followed 2 and 6 months after tuberculosis treatment initiation. Mutually exclusive anemia categories based on iron biomarker concentrations were iron deficiency anemia (IDA), anemia of inflammation (AI), and multifactorial anemia (IDA+AI). RESULTS: Anemia was more frequent in tuberculosis cases (67%) than in TST-positive (36%) or TST-negative (21%) controls. AI was the predominant anemia at tuberculosis diagnosis, declining from 36% to 8% after 6 months of treatment; however, a corresponding reduction was not evident for anemia with iron-responsive components (IDA, IDA+AI). Iron biomarkers discriminated between active tuberculosis and TST-positive or TST-negative controls, as well as between active untreated and treated tuberculosis. This was most noticeable for hepcidin, which decreased from a median of 84.0 ng/mL at diagnosis to 9.7 ng/mL after 2 months (P < .001). CONCLUSIONS: Tuberculosis chemotherapy is associated with significant reductions in AI, but IDA and IDA+AI remain unresolved. Iron-based interventions are needed for IDA and IDA+AI, and monitoring of iron biomarkers reveals a window for intervention opening as early as 2 months into tuberculosis treatment.

Differences in T-cell responses between Mycobacterium tuberculosis and Mycobacterium africanum-infected patients.

In The Gambia, Mycobacterium tuberculosis (Mtb) and Mycobacterium africanum (Maf) are major causes of tuberculosis (TB). Maf is more likely to cause TB in immune suppressed individuals, implying differences in virulence. Despite this, few studies have assessed the underlying immunity to the two pathogens in human. In this study, we analyzed T-cell responses from 19 Maf- and 29 Mtb-infected HIV-negative patients before and after TB chemotherapy following overnight stimulation of whole blood with TB-specific antigens. Before treatment, percentages of early secreted antigenic target-6(ESAT-6)/culture filtrate protein-10(CFP-10) and purified protein derivative-specific single-TNF-α-producing CD4(+) and CD8(+) T cells were significantly higher while single-IL-2-producing T cells were significantly lower in Maf- compared with Mtb-infected patients. Purified protein derivative-specific polyfunctional CD4(+) T cells frequencies were significantly higher before than after treatment, but there was no difference between the groups at both time points. Furthermore, the proportion of CD3(+) CD11b(+) T cells was similar in both groups pretreatment, but was significantly lower with higher TNF-α, IL-2, and IFN-γ production in Mtb- compared with that of Maf-infected patients posttreatment. Our data provide evidence of differences in T-cell responses to two mycobacterial strains with differing virulence, providing some insight into TB pathogenesis with different Mtb strains that could be prospectively explored as biomarkers for TB protection or susceptibility.

HBHA induces IL-10 from CD4+ T cells in patients with active tuberculosis but IFN-γ and IL-17 from individuals with Mycobacterium tuberculosis infection.

Background: To effectively control tuberculosis (TB), it is crucial to distinguish between active TB disease and latent TB infection (LTBI) to provide appropriate treatment. However, no such tests are currently available. Immune responses associated with active TB and LTBI are dynamic and exhibit distinct patterns. Comparing these differences is crucial for developing new diagnostic methods and understanding the etiology of TB. This study aimed to investigate the relationship between pro- and anti-inflammatory CD4+ cytokine production following stimulation with two types of latency-associated Mycobacterium tuberculosis (M.tb) antigens to allow differentiation between active TB and LTBI. Methods: Cryopreserved PBMCs from patients with active TB disease or LTBI were stimulated overnight with replication-related antigen [ESAT-6/CFP-10 (E/C)] or two latency-associated antigens [heparin-binding hemagglutinin (HBHA) and alpha-crystallin-like protein (Acr)]. Responses were analyzed using multiparameter flow cytometry: active TB disease (n=15), LTBI (n=15) and ELISA: active TB disease (n=26) or LTBI (n=27). Results: CD4+ central memory T cells (Tcm) specific to E/C and CD4+ effector memory T cells specific to Acr and HBHA were higher in LTBI than in TB patients. IFN-γ+Tcm and IL-17+ Tem cells was higher in the LTBI group (p= 0.012 and p=0.029 respectively), but IL-10+ Tcm was higher in the active TB group (p= 0.029) following HBHA stimulation. Additionally, following stimulation with HBHA, IL-10 production from CD4+ T cells was significantly elevated in patients with active TB compared to those with LTBI (p= 0.0038), while CD4+ T cell production of IL-17 and IFN-γ was significantly elevated in LTBI compared to active TB (p= 0.0076, p< 0.0001, respectively). HBHA also induced more CCR6+IL-17+CD4Tcells and IL-17+FoxP3+CD25+CD4Tcells in LTBI than in TB patients (P=0.026 and P=0.04, respectively). HBHA also induced higher levels of IFN-γ+IL-10+CD4+ T cells in patients with active TB (Pp=0.03) and higher levels of IFN-γ+IL-17+ CD4+ T cells in those with LTBI (p=0.04). HBHA-specific cytokine production measured using ELISA showed higher levels of IFN-γ in participants with LTBI (P=0.004) and higher levels of IL-10 in those with active TB (P=0.04). Conclusion: Stimulation with HBHA and measurement of CD4+ T cell production of IFN-γ, IL-10, and IL-17 could potentially differentiate active TB from LTBI. The characteristics of cytokine-expressing cells induced by HBHA also differed between participants with active TB and LTBI.

The inclusion of children and adolescents in tuberculosis diagnostic development and evaluation-a consensus statement.

The diagnosis of paediatric tuberculosis remains a challenge due to the non-specificity of symptoms and the paucibacillary nature of tuberculosis in children. However, in the development of new tuberculosis diagnostics, the unique needs of children and adolescents are rarely considered in the design process, with delays in evaluation and approval. No clear guidance is available on when and how to include children and adolescents in tuberculosis diagnostic development and evaluation. To address this gap, we conducted a Delphi consensus process with 42 stakeholders, including one qualitative and two quantitative rounds. Consensus was achieved on 20 statements, with agreement that the needs and perspectives of children, adolescents, and their caregivers should be incorporated throughout diagnostic design and evaluation. Opportunities exist for the early use of well characterised samples and prospective enrolment of children and adolescents in tuberculosis diagnostic evaluation, with consideration of the type of test, expected benefit, and potential risks. Pathogen-based tests might be initially optimised and assessed in adults and adolescents, but parallel evaluation in children is needed for host-based tests. Late-stage evaluation and implementation studies should examine combination testing and integration into clinical algorithms. The statements support collaboration between developers, researchers, regulators, and users to widen and accelerate the diagnostic pipeline for paediatric tuberculosis.

Activin A levels are raised during human tuberculosis and blockade of the activin signaling axis influences murine responses to M. tuberculosis infection.

Activin A strongly influences immune responses; yet, few studies have examined its role in infectious diseases. We measured serum activin A levels in two independent tuberculosis (TB) patient cohorts and in patients with pneumonia and sarcoidosis. Serum activin A levels were increased in TB patients compared to healthy controls, including those with positive tuberculin skin tests, and paralleled severity of disease, assessed by X-ray scores. In pneumonia patients, serum activin A levels were also raised, but in sarcoidosis patients, levels were lower. To determine whether blockade of the activin A signaling axis could play a functional role in TB, we harnessed a soluble activin type IIB receptor fused to human IgG1 Fc, ActRIIB-Fc, as a ligand trap in a murine TB model. The administration of ActRIIB-Fc to Mycobacterium tuberculosis-infected mice resulted in decreased bacterial loads and increased numbers of CD4 effector T cells and tissue-resident memory T cells in the lung. Increased frequencies of tissue-resident memory T cells corresponded with downregulated T-bet expression in lung CD4 and CD8 T cells. Altogether, the results suggest a disease-exacerbating role of ActRIIB signaling pathways. Serum activin A may be useful as a biomarker for diagnostic triage of active TB or monitoring of anti-tuberculosis therapy. IMPORTANCE: Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis, can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.

Age and sex influence antibody profiles associated with tuberculosis progression.

Antibody features vary with tuberculosis (TB) disease state. Whether clinical variables, such as age or sex, influence associations between Mycobacterium tuberculosis-specific antibody responses and disease state is not well explored. Here we profiled Mycobacterium tuberculosis-specific antibody responses in 140 TB-exposed South African individuals from the Adolescent Cohort Study. We identified distinct response features in individuals progressing to active TB from non-progressing, matched controls. A multivariate antibody score differentially associated with progression (SeroScore) identified progressors up to 2 years before TB diagnosis, earlier than that achieved with the RISK6 transcriptional signature of progression. We validated these antibody response features in the Grand Challenges 6-74 cohort. Both the SeroScore and RISK6 correlated better with risk of TB progression in adolescents compared with adults, and in males compared with females. This suggests that age and sex are important, underappreciated modifiers of antibody responses associated with TB progression.

Skewing of the CD4(+) T-cell pool toward monofunctional antigen-specific responses in patients with immune reconstitution inflammatory syndrome in The Gambia.

BACKGROUND: A common complication of starting antiretroviral therapy (ART) for human immunodeficiency virus (HIV) is the development of immune reconstitution inflammatory syndrome (IRIS) in approximately 25% of patients. Despite similarities with paradoxical reactions to tuberculosis and reversal reactions in leprosy, the exact mechanisms, and therefore potential determinants, of IRIS are still unknown. METHODS: In this longitudinal cohort study, we analyzed 20 patients who developed IRIS following initiation of ART and 16 patients who did not, matched for ART time point. Peripheral blood mononuclear cells were stimulated overnight with a positive control antigen and 2 tuberculosis-specific antigens (purified protein derivative [PPD] and ESAT-6/CFP10), followed by polychromatic flow cytometry for analysis of cytokine production from CD4(+) and CD8(+) T cells. RESULTS: Responses to PPD were significantly higher in IRIS patients compared to controls during the IRIS time point, but CD4(+) and CD8(+) T-cell responses to the positive control stimulation were significantly lower in IRIS patients at all time points. Furthermore, whereas control patients had rejuvenated polyfunctional T-cell responses by 3 months after ART, IRIS patients were strikingly monofunctional (generally interferon γ alone), even up to 6 months of ART in response to all stimulations. CONCLUSIONS: Our findings suggest that the peripheral T-cell responses to the underlying pathogen are exaggerated in IRIS patients but that the overall quality of the peripheral T-cell pool is significantly reduced compared to non-IRIS patients. Furthermore, these effects are apparent at least up to 3 months after cessation of IRIS.

Immunogenic Mycobacterium africanum strains associated with ongoing transmission in The Gambia.

In West Africa, Mycobacterium tuberculosis strains co-circulate with M. africanum, and both pathogens cause pulmonary tuberculosis in humans. Given recent findings that M. tuberculosis T-cell epitopes are hyperconserved, we hypothesized that more immunogenic strains have increased capacity to spread within the human host population. We investigated the relationship between the composition of the mycobacterial population in The Gambia, as measured by spoligotype analysis, and the immunogenicity of these strains as measured by purified protein derivative-induced interferon-γ release in ELISPOT assays of peripheral blood mononuclear cells. We found a positive correlation between strains with superior spreading capacity and their relative immunogenicity. Although our observation is true for M. tuberculosis and M. africanum strains, the association was especially pronounced in 1 M. africanum sublineage, characterized by spoligotype shared international type 181, which is responsible for 20% of all tuberculosis cases in the region and therefore poses a major public health threat in The Gambia.

Histoplasma Seropositivity in TB Patients in The Gambia: Evidence to Drive Research on a High-Priority Fungal Pathogen.

Background: Inclusion of Histoplasma in the World Health Organization's first Fungal Priority Pathogens List under "high-priority" fungal species highlights the need for robust surveillance of Histoplasma spp. in endemic and underrepresented regions. Despite increasing reports of histoplasmosis in Africa, data on the burden of this fungal disease are sparse in The Gambia. This baseline study examined the human seroprevalence of anti-Histoplasma antibody in a TB patient group in The Gambia, explored associations between seropositivity and demographic and clinical variables, and proposes future research directions. Methods: Biobanked plasma samples were selected from active TB cases with variable HIV infection status. Latex agglutination tests were performed on samples from 52 study participants to detect the presence of anti-Histoplasma antibodies. Potential risk factors for Histoplasma exposure were explored using logistic regression analysis. Results: The sample seroprevalence of anti-Histoplasma antibody was 28.8% (n = 15/52; 95% CI, 17.1%-43.1%). Multivariable logistic regression analysis identified a statistically significant association between Histoplasma seropositivity and age (odds ratio, 0.91; 95% CI, 0.84-0.98; P = .008). Conclusions: This baseline study provides evidence of Histoplasma seropositivity in TB patients in The Gambia and explores risk factors for exposure. The small sample size and use of the LAT in TB and HIV-positive patient groups are significant study limitations. Future research directions are proposed to ascertain the burden of Histoplasma in general and patient populations and explore the context-specific risk factors for exposure and infection in The Gambia.