Whispers in the wind: Face mask sampling for Mycobacterium tuberculosis detection in children with pulmonary tuberculosis.

BACKGROUND: Recently, face mask sampling (FMS) confirmed detection of Mycobacterium tuberculosis DNA from exhaled breath in adults with TB. To date, no study has evaluated the use of FMS to detect pulmonary Tuberculosis (TB) in children. We developed a method for FMS of M. tuberculosis-specific DNA in children and performed a clinical exploration to assess feasibility in children. METHODS: Face masks were spiked, analysed on GeneXpert-Ultra, qPCR, and tNGS. Children with pulmonary TB were asked to wear three modified FFP2 masks for 30 minutes as part of an exploratory clinical study. RESULTS: Experiments with H37Ra M. tuberculosis strain showed a limit of 95% detection of 3.75 CFU (4.85-3.11; 95%CI) on GeneXpert-Ultra. Ten children with pulmonary TB participated in the clinical study. M. tuberculosis-specific DNA was detected on none of the face masks. CONCLUSIONS: Paediatric FMS has a low limit of detection for M. tuberculosis-specific DNA in vitro. However, M. tuberculosis DNA was not detected in any of thirty masks worn by children with pulmonary TB. This suggests that FMS in this form may not be more effective for detecting M. tuberculosis in children with TB than existing methods.

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.

Gene expression signatures identify biologically and clinically distinct tuberculosis endotypes.

BACKGROUND: In vitro, animal model and clinical evidence suggests that tuberculosis is not a monomorphic disease, and that host response to tuberculosis is protean with multiple distinct molecular pathways and pathologies (endotypes). We applied unbiased clustering to identify separate tuberculosis endotypes with classifiable gene expression patterns and clinical outcomes. METHODS: A cohort comprised of microarray gene expression data from microbiologically confirmed tuberculosis patients was used to identify putative endotypes. One microarray cohort with longitudinal clinical outcomes was reserved for validation, as were two RNA-sequencing (seq) cohorts. Finally, a separate cohort of tuberculosis patients with functional immune responses was evaluated to clarify stimulated from unstimulated immune responses. RESULTS: A discovery cohort, including 435 tuberculosis patients and 533 asymptomatic controls, identified two tuberculosis endotypes. Endotype A is characterised by increased expression of genes related to inflammation and immunity and decreased metabolism and proliferation; in contrast, endotype B has increased activity of metabolism and proliferation pathways. An independent RNA-seq validation cohort, including 118 tuberculosis patients and 179 controls, validated the discovery results. Gene expression signatures for treatment failure were elevated in endotype A in the discovery cohort, and a separate validation cohort confirmed that endotype A patients had slower time to culture conversion, and a reduced cure rate. These observations suggest that endotypes reflect functional immunity, supported by the observation that tuberculosis patients with a hyperinflammatory endotype have less responsive cytokine production upon stimulation. CONCLUSION: These findings provide evidence that metabolic and immune profiling could inform optimisation of endotype-specific host-directed therapies for tuberculosis.

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.