Vikela Ekhaya: A Novel, Community-based, Tuberculosis Contact Management Program in a High Burden Setting.

BACKGROUND: The prevention of tuberculosis (TB) in child contacts of TB cases and people living with human immunodeficiency virus (HIV) is a public health priority, but global access to TB preventive therapy (TPT) remains low. In 2019, we implemented Vikela Ekhaya, a novel community-based TB contact management program in Eswatini designed to reduce barriers to accessing TPT. METHODS: Vikela Ekhaya offered differentiated TB and HIV testing for household contacts of TB cases by using mobile contact management teams to screen contacts, assess their TPT eligibility, and initiate and monitor TPT adherence in participants' homes. RESULTS: In total, 945 contacts from 244 households were screened for TB symptoms; 72 (8%) contacts reported TB symptoms, and 5 contacts (0.5%) were diagnosed with prevalent TB. A total of 322 of 330 (98%) eligible asymptomatic household contacts initiated TPT. Of 322 contacts initiating TPT, 248 children initiated 3 months of isoniazid and rifampicin and 74 children and adults living with HIV initiated 6 months of isoniazid; 298 (93%) completed TPT. In clustered logistic regression analyses, unknown HIV status (adjusted odds ratio [aOR] 5.7, P = .023), positive HIV status (aOR 21.1, P = .001), urban setting (aOR 5.6, P = .006), and low income (aOR 5.9, P = .001) predicted loss from the cascade of care among TPT-eligible contacts. CONCLUSION: Vikela Ekhaya demonstrated that community-based TB household contact management is a feasible, acceptable, and successful strategy for TB screening and TPT delivery. The results of this study support the development of novel, differentiated, community-based interventions for TB prevention and control.

Schistosomiasis Induces Persistent DNA Methylation and Tuberculosis-Specific Immune Changes.

Epigenetic mechanisms, such as DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth coinfections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)-specific and bacillus Calmette-Guérin-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared with age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA-methylated genes, with 72% hypermethylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted real-time quantitative PCR after methyl-specific endonuclease digestion confirmed DNA hypermethylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4, and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2, and IL12B (p < 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed, with helminth-infected individuals having decreased upregulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypomethylation of the IL-4 pathway and DNA hypermethylation of the Th1 pathway was confirmed by Ag-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed-rank p < 0.05). In S. haematobium-infected individuals, these DNA methylation and immune phenotypic changes persisted at least 6 mo after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth specific.

HIV Progression Perturbs the Balance of the Cell-Mediated and Anti-Inflammatory Adaptive and Innate Mycobacterial Immune Response.

INTRODUCTION: Our objective is to understand how HIV infection increases the risk of progression from latent tuberculosis (TB) to active disease. We understand now that immunity is a balance of competing immune responses by multiple cell types. Since T-lymphocyte production of interferon-gamma (IFN-γ) in response to Mycobacterium tuberculosis (Mtb) antigens fails to differentiate disease from latent infection, we applied a comprehensive profiling methodology to define immune biomarkers that reliably predict a patient's TB risk. METHODS: We established a cohort of HIV-infected adults with TB disease from Swaziland. Multiparametric flow cytometry was used to quantify the mycobacterial-specific anti-inflammatory (IL-4 and IL-10) and proinflammatory (IFN-γ) immune response. RESULTS: From 12 HIV-infected Swaziland patients with TB disease, the CD4(+), CD8(+), Double Negative, and CD56(+)CD3(-) lymphocytes increase their IL-4 : IFN-γ ratio as HIV disease worsens (Spearman r of -0.59; -0.59; -0.60; and -0.59, resp.; p < 0.05). Similarly, HIV severity is associated with an increased IL-10 : IFN-γ ratio (Spearman r of -0.76; p = 0.01). CONCLUSION: As HIV disease progresses, both the adaptive and innate branches skew away from an inflammatory and towards anti-inflammatory phenotype.

Performance of a stool-based quantitative PCR assay for the diagnosis of tuberculosis in adolescents and adults: a multinational, prospective diagnostic accuracy study.

BACKGROUND: Despite increasing availability of rapid molecular tests for the diagnosis of tuberculosis in high-burden settings, many people with tuberculosis are undiagnosed. Reliance on sputum as the primary specimen for tuberculosis diagnostics contributes to this diagnostic gap. We evaluated the diagnostic accuracy and additive yield of a novel stool quantitative PCR (qPCR) assay for the diagnosis of tuberculosis in three countries in Africa with high tuberculosis burdens. METHODS: We undertook a prospective diagnostic accuracy study in Eswatini, Mozambique, and Tanzania from Sept 21, 2020, to Feb 2, 2023, to compare the diagnostic accuracy for tuberculosis of a novel stool qPCR test with the current diagnostic standard for Mycobacterium tuberculosis DNA detection from sputum and stool, Xpert-MTB/RIF Ultra (Xpert Ultra). Sputum, stool, and urine samples were provided by a cohort of participants, aged 10 years or older, diagnosed with tuberculosis. Participants with tuberculosis (cases) were enrolled within 72 h of treatment initiation for tuberculosis diagnosed clinically or following laboratory confirmation. Participants without tuberculosis (controls) consisted of household contacts of the cases who did not develop tuberculosis during a 6-month follow-up. The performance was compared with a robust composite microbiological reference standard (CMRS). FINDINGS: The cohort of adolescents and adults (n=408) included 268 participants with confirmed or clinical tuberculosis (cases), 147 (55%) of whom were living with HIV, and 140 participants (controls) without tuberculosis. The sensitivity of the novel stool qPCR was 93·7% (95% CI 87·4-97·4) compared with participants with detectable growth on M tuberculosis culture, and 88·1% (81·3-93·0) compared with sputum Xpert Ultra. The stool qPCR had an equivalent sensitivity as sputum Xpert Ultra (94·8%, 89·1-98·1) compared with culture. Compared with the CMRS, the sensitivity of the stool qPCR was higher than the current standard for tuberculosis diagnostics on stool, Xpert Ultra (80·4%, 73·4-86·2 vs 73·5%, 66·0-80·1; p=0·025 on paired comparison). The qPCR also identified 17-21% additional tuberculosis cases compared to sputum Xpert Ultra or sputum culture. In controls without tuberculosis, the specificity of the stool qPCR was 96·9% (92·2-99·1). INTERPRETATION: In this study, a novel qPCR for the diagnosis of tuberculosis from stool specimens had a higher accuracy in adolescents and adults than the current diagnostic PCR gold standard on stool, Xpert-MTB/RIF Ultra, and equivalent sensitivity to Xpert-MTB/RIF Ultra on sputum. FUNDING: National Institutes of Health (NIH) Allergy and Infectious Diseases, and NIH Fogarty International Center.

Community-based tuberculosis contact management: Caregiver experience and factors promoting adherence to preventive therapy.

Delivery of tuberculosis preventive therapy (TPT) for children with household exposure to tuberculosis is a globally supported intervention to reduce the impact of tuberculosis disease (TB) in vulnerable children; however, it is sub-optimally implemented in most high-burden settings. As part of a community-based household contact management program, we evaluated predictors of adherence to community based TPT in children and performed qualitative assessments of caregiver experiences. The Vikela Ekhaya (Protect the Home) project was a community-based household contact management program implemented between 2019 and 2020 in the Hhohho Region of Eswatini. At home visits, contact management teams screened children for TB, initiated TPT when indicated and performed follow-up assessments reviewing TPT adherence. TPT non-adherence was defined as either two self-reported missed doses or a pill count indicating at least two missed doses, and risk factors were evaluated using multivariate clustered Cox regression models. Semi-structured interviews were performed with caregivers to assess acceptability of home visits for TPT administration. In total, 278 children under 15 years initiated TPT and 96% completed TPT through the Vikela Ekhaya project. Risk factors for TPT non-adherence among children initiating 3HR included low family income (adjusted hazard ratio (aHR) 2.3, 95%CI 1.2-4.4), female gender of the child (aHR 2.5, 95% CI 1.4-5.0) and an urban living environment (aHR 3.1, 95%CI 1.6-6.0). Children with non-adherence at the first follow-up visit were 9.1 fold more likely not to complete therapy. Caregivers indicated an appreciation for community services, citing increased comfort, reduced cost, and support from community members. Our results are supportive of recent World Health Organization (WHO) recommendations for decentralization of TB preventive services. Here, we identify populations that may benefit from additional support to promote TPT adherence, but overall demonstrate a clear preference for and excellent outcomes with community based TPT delivery.

Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing.

BACKGROUND: Stool is an important diagnostic specimen for tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, the culture of Mycobacterium tuberculosis (M. tuberculosis) complex strains from stool perform poorly. This limits the opportunity for phenotypic drug resistance testing with this specimen. Therefore, reliable molecular methods are urgently needed for comprehensive drug resistance testing on stool specimens. METHODS: We evaluated the performance of targeted next-generation sequencing (tNGS, Deeplex® Myc-TB) for the detection of mutations associated with M. tuberculosis complex drug resistance on DNA isolated from stool specimens provided by participants from a prospective cohort of patients treated for tuberculosis in Eswatini (n = 66; 56 with and 10 participants without M. tuberculosis complex DNA detected in stool by real-time quantitative PCR), and an independent German validation cohort of participants with culture-confirmed tuberculosis (n = 21). RESULTS: The tNGS assay detected M. tuberculosis complex DNA in 38 of 56 (68%) samples; for 28 of 38 (74%) samples, a full M. tuberculosis complex drug resistance prediction report was obtained. There was a high degree of concordance with sputum phenotypic drug susceptibility results (κ = 0.82). The ability to predict resistance was concentration-dependent and successful in 7/10 (70%), 18/25 (72%), and 3/21 (14%) of samples with stool PCR concentration thresholds of > 100 femtogram per microliter (fg/µl), 1 to 100 fg/µl, and < 1 fg/µl, respectively (p = 0.0004). The German cohort confirmed these results and demonstrated a similarly high concordance between stool tNGS and sputum phenotypic drug susceptibility results (κ = 0.84). CONCLUSIONS: tNGS can identify drug resistance from stool provided by tuberculosis patients. This affords the opportunity to obtain critical diagnostic information for tuberculosis patients who struggle to provide respiratory specimens.

Increased DNA methylation, cellular senescence and premature epigenetic aging in guinea pigs and humans with tuberculosis.

BACKGROUND: Tuberculosis (TB) is the archetypical chronic infection, with patients having months of symptoms before diagnosis. In the two years after successful therapy, survivors of TB have a three-fold increased risk of death. METHODS: Guinea pigs were infected with Mycobacterium tuberculosis (Mtb) for 45 days, followed by RRBS DNA methylation analysis. In humans, network analysis of differentially expressed genes across three TB cohorts were visualized at the pathway-level. Serum levels of inflammation were measured by ELISA. Horvath (DNA methylation) and RNA-seq biological clocks were used to investigate shifts in chronological age among humans with TB. RESULTS: Guinea pigs with TB demonstrated DNA hypermethylation and showed system-level similarity to humans with TB (p-value = 0.002). The transcriptome in TB in multiple cohorts was enriched for DNA methylation and cellular senescence. Senescence associated proteins CXCL9, CXCL10, and TNF were elevated in TB patients compared to healthy controls. Humans with TB demonstrate 12.7 years (95% CI: 7.5, 21.9) and 14.38 years (95% CI: 10.23-18.53) of cellular aging as measured by epigenetic and gene expression based cellular clocks, respectively. CONCLUSIONS: In both guinea pigs and humans, TB perturbs epigenetic processes, promoting premature cellular aging and inflammation, a plausible means to explain the long-term detrimental health outcomes after TB.

CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study.

BACKGROUND: Tuberculosis remains a leading cause of global mortality, especially for adults and children living with HIV (CLHIV) underdiagnosed by sputum-based assays. Non-sputum-based assays are needed to improve tuberculosis diagnosis and tuberculosis treatment monitoring. Our aim in this study was to determine whether ultrasensitive detection of Mycobacterium tuberculosis cell-free DNA (Mtb-cfDNA) in blood can diagnose tuberculosis and evaluate tuberculosis treatment responses. METHODS: In this molecular diagnostics study we analysed archived serum from two patient populations evaluated for tuberculosis in Eswatini and Kenya to detect Mtb-cfDNA, analysing serum from all individuals who had both sufficient serum volumes and clear diagnostic results. An optimised CRISPR-mediated tuberculosis (CRISPR-TB) assay was used to detect Mtb-cfDNA in serum at enrolment from adults and children with presumptive tuberculosis and their asymptomatic household contacts, and at enrolment and during tuberculosis treatment from a cohort of symptomatic CLHIV at high risk for tuberculosis, who provided longitudinal serum at enrolment and during tuberculosis treatment. FINDINGS: CRISPR-TB identified microbiologically and clinically confirmed tuberculosis cases in the predominantly HIV-negative Eswatini adult cohort with 96% sensitivity (27 [96%] of 28, 95% CI 80-100) and 94% specificity (16 [94%] of 17, 71-100), and with 83% sensitivity (5 [83%] of 6, 36-100) and 95% specificity (21 [95%] of 22, 77-100) in the paediatric cohort, including all six cases of extrapulmonary tuberculosis. In the Kenyan CLHIV cohort, CRISPR-TB detected all (13 [100%] of 13, 75-100) confirmed tuberculosis cases and 85% (39 [85%] of 46, 71-94) of unconfirmed tuberculosis cases diagnosed by non-microbiological clinical findings. CLHIV who were CRISPR-TB positive at enrolment had a 2·4-times higher risk of mortality by 6 months after enrolment. Mtb-cfDNA signal decreased after tuberculosis treatment initiation, with near or complete Mtb-cfDNA clearance by 6 months after tuberculosis treatment initiation. INTERPRETATION: CRISPR-mediated detection of circulating Mtb-cfDNA shows promise to increase the identification of paediatric tuberculosis and HIV-associated tuberculosis, and potential for early diagnosis and rapid monitoring of tuberculosis treatment responses. FUNDING: US Department of Defense, National Institute of Child Health and Human Development, National Institute of Allergy and Infectious Diseases, University of Washington Center for AIDS Research, and the Weatherhead Presidential Endowment fund.

Child Contact Case Management-A Major Policy-Practice Gap in High-Burden Countries.

The 2021 Global Tuberculosis (TB) report shows slow progress towards closing the pediatric TB detection gap and improving the TB preventive treatment (TPT) coverage among child and adolescent contacts. This review presents the current knowledge around contact case management (CCM) in low-resource settings, with a focus on child contacts, which represents a key priority population for CCM and TPT. Compelling evidence demonstrates that CCM interventions are a key gateway for both TB case finding and identification of those in need of TPT, and their yield and effectiveness should provide a strong rationale for prioritization by national TB programs. A growing body of evidence is now showing that innovative models of care focused on community-based and patient-centered approaches to household contact investigation can help narrow down the CCM implementation gaps that we are currently facing. The availability of shorter and child-friendly TPT regimens for child contacts provide an additional important opportunity to improve TPT acceptability and adherence. Prioritization of TB CCM implementation and adequate resource mobilization by ministries of health, donors and implementing agencies is needed to timely close the gap.

DNA hypermethylation during tuberculosis dampens host immune responsiveness.

Mycobacterium tuberculosis (M. tuberculosis) has coevolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to improve outcomes and potentially shorten existing therapy will require identification of the full complement by which host immunity is inhibited. Perturbation of host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, lymphocytic choriomeningitis virus (LCMV), and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of patients with tuberculosis (TB) and their asymptomatic household contacts and found that the patients with TB have DNA hypermethylation of the IL-2/STAT5, TNF/NF-κB, and IFN-γ signaling pathways. We performed methylation-sensitive restriction enzyme-quantitative PCR (MSRE-qPCR) and observed that multiple genes of the IL-12/IFN-γ signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1, and JAK2) were hypermethylated in patients with TB. The DNA hypermethylation of these pathways was associated with decreased immune responsiveness with decreased mitogen-induced upregulation of IFN-γ, TNF, IL-6, CXCL9, CXCL10, and IL-1ß production. The DNA hypermethylation of the IL-12/IFN-γ pathway was associated with decreased IFN-γ-induced gene expression and decreased IL-12-inducible upregulation of IFN-γ. This study demonstrates that immune cells from patients with TB are characterized by DNA hypermethylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and nonspecific immune responsiveness.

Evaluation of the QuantiFERON-Tuberculosis Gold Plus Assay in Children with Tuberculosis Disease or Following Household Exposure to Tuberculosis.

Interferon-gamma release assays are increasingly used in children to establish evidence of tuberculosis (TB) infection and to assist in the diagnosis of TB disease. The QuantiFERON-TB Gold In-Tube assay is being phased out in favor of a next-generation test, the QuantiFERON-TB Gold Plus (QFT-Plus) assay. The QFT-Plus assay is designed with two antigen tubes to differentially stimulate CD4+ and CD8+ T cells. The performance of this assay has been documented extensively in adults but has not yet been evaluated in children. Here, we compare the performance of the two assays in a cohort of 46 children exposed to TB and 12 children diagnosed with TB disease in Eswatini. The tests demonstrated excellent concordance in both TB disease (100% agreement, Cohen's kappa = 1) and TB infection (96% agreement, Cohen's kappa = 0.91). Most of the children with household exposure tested negative for TB infection by both tests, indicating the ongoing need for new tests for TB infection that can be easily implemented in TB high-burden settings at minimal cost.

Diagnostic and Treatment Monitoring Potential of A Stool-Based Quantitative Polymerase Chain Reaction Assay for Pulmonary Tuberculosis.

A quantifiable, stool-based, Mycobacterium tuberculosis (Mtb) test has potential complementary value to respiratory specimens. Limit of detection (LOD) was determined by spiking control stool. Clinical test performance was evaluated in a cohort with pulmonary tuberculosis (TB) (N = 166) and asymptomatic household TB child contacts (N = 105). Stool-quantitative polymerase chain reaction (qPCR) results were compared with sputum acid-fast bacilli (AFB) microscopy, GeneXpert MTB/RIF (Xpert MTB/RIF), and cultures. In Mtb stool-spiking studies, the LOD was 96 colony-forming units/50 mg of stool (95% confidence interval [CI]: 84.8-105.6). Among specimens collected within 72 hours of antituberculosis treatment (ATT) initiation, stool qPCR detected 22 of 23 (95%) of culture-positive cases. Among clinically diagnosed cases that were Xpert MTB/RIF and culture negative, stool qPCR detected an additional 8% (3/37). Among asymptomatic, recently TB-exposed participants, stool PCR detected Mtb in two of 105 (1.9%) patients. Two months after ATT, the Mtb quantitative burden in femtogram per microliters decreased (Wilcoxon signed-rank P < 0.001) and persistent positive stool PCR was associated with treatment failure or drug resistance (relative risk 2.8, CI: 1.2-6.5; P = 0.012). Stool-based qPCR is a promising complementary technique to sputum-based diagnosis. It detects and quantifies low levels of stool Mtb DNA, thereby supporting adjunct diagnosis and treatment monitoring in pulmonary TB.