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-1β 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.

SETTING: Cape Town, South Africa. OBJECTIVE: To develop a standardized, reliable measure of household tuberculosis (TB) exposure that considers child-specific risk factors. DESIGN: We assessed TB exposure in 536 children. Children were considered Mycobacterium tuberculosis infected if two of three tests of infection were positive. Principal component analysis identified a discrete set of components that collectively described exposure and contributed to a composite contact score. Logistic regression assessed the odds of having M. tuberculosis infection given increasing contact score while controlling for age and past TB treatment. RESULTS: Four components described 68% of data variance: 1) maternal TB and sleep proximity, 2) index case infectivity, 3) duration of exposure, and 4) exposure to multiple index cases. Components were derived from 10 binary questions that contributed to a contact score (range 1–10, median 5, 25th–75th interquartile range [IQR] 4–7). Among children aged 3 months to 6 years with household exposure, the odds of being M. tuberculosis-infected increased by 74% (OR 1.74, 95%CI 1.42–2.12) with each 1-point increase in the contact score. CONCLUSIONS: Well-quantified TB exposure is a good surrogate measure of M. tuberculosis infection in child household contacts in a high-burden setting, and could guide targeted preventive treatment in children at highest risk of M. tuberculosis infection.

Background Every year, at least one million children become ill with tuberculosis and around 200,000 children die. Xpert MTB/RIF and Xpert Ultra are World Health Organization (WHO)‐recommended rapid molecular tests that simultaneously detect tuberculosis and rifampicin resistance in adults and children with signs and symptoms of tuberculosis, at lower health system levels. To inform updated WHO guidelines on molecular assays, we performed a systematic review on the diagnostic accuracy of these tests in children presumed to have active tuberculosis. Objectives Primary objectives • To determine the diagnostic accuracy of Xpert MTB/RIF and Xpert Ultra for (a) pulmonary tuberculosis in children presumed to have tuberculosis; (b) tuberculous meningitis in children presumed to have tuberculosis; (c) lymph node tuberculosis in children presumed to have tuberculosis; and (d) rifampicin resistance in children presumed to have tuberculosis ‐ For tuberculosis detection, index tests were used as the initial test, replacing standard practice (i.e. smear microscopy or culture) ‐ For detection of rifampicin resistance, index tests replaced culture‐based drug susceptibility testing as the initial test Secondary objectives • To compare the accuracy of Xpert MTB/RIF and Xpert Ultra for each of the four target conditions • To investigate potential sources of heterogeneity in accuracy estimates ‐ For tuberculosis detection, we considered age, disease severity, smear‐test status, HIV status, clinical setting, specimen type, high tuberculosis burden, and high tuberculosis/HIV burden ‐ For detection of rifampicin resistance, we considered multi‐drug‐resistant tuberculosis burden • To compare multiple Xpert MTB/RIF or Xpert Ultra results (repeated testing) with the initial Xpert MTB/RIF or Xpert Ultra result Search methods We searched the Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, the WHO International Clinical Trials Registry Platform, ClinicalTrials.gov, and the International Standard Randomized Controlled Trials Number (ISRCTN) Registry up to 29 April 2019, without language restrictions. Selection criteria Randomized trials, cross‐sectional trials, and cohort studies evaluating Xpert MTB/RIF or Xpert Ultra in HIV‐positive and HIV‐negative children younger than 15 years. Reference standards comprised culture or a composite reference standard for tuberculosis and drug susceptibility testing or MTBDRplus (molecular assay for detection of Mycobacterium tuberculosis and drug resistance) for rifampicin resistance. We included studies evaluating sputum, gastric aspirate, stool, nasopharyngeal or bronchial lavage specimens (pulmonary tuberculosis), cerebrospinal fluid (tuberculous meningitis), fine needle aspirates, or surgical biopsy tissue (lymph node tuberculosis). Data collection and analysis Two review authors independently extracted data and assessed study quality using the Quality Assessment of Studies of Diagnostic Accuracy ‐ Revised (QUADAS‐2). For each target condition, we used the bivariate model to estimate pooled sensitivity and specificity with 95% confidence intervals (CIs). We stratified all analyses by type of reference standard. We assessed certainty of evidence using the GRADE approach. Main results For pulmonary tuberculosis, 299 data sets (68,544 participants) were available for analysis; for tuberculous meningitis, 10 data sets (423 participants) were available; for lymph node tuberculosis, 10 data sets (318 participants) were available; and for rifampicin resistance, 14 data sets (326 participants) were available. Thirty‐nine studies (80%) took place in countries with high tuberculosis burden. Risk of bias was low except for the reference standard domain, for which risk of bias was unclear because many studies collected only one specimen for culture. Detection of pulmonary tuberculosis For sputum specimens, Xpert MTB/RIF pooled sensitivity (95% CI) and specificity (95% CI) verified by culture were 64.6% (55.3% to 72.9%) (23 studies, 493 participants; moderate‐certainty evidence) and 99.0% (98.1% to 99.5%) (23 studies, 6119 participants; moderate‐certainty evidence). For other specimen types (nasopharyngeal aspirate, 4 studies; gastric aspirate, 14 studies; stool, 11 studies), Xpert MTB/RIF pooled sensitivity ranged between 45.7% and 73.0%, and pooled specificity ranged between 98.1% and 99.6%. For sputum specimens, Xpert Ultra pooled sensitivity (95% CI) and specificity (95% CI) verified by culture were 72.8% (64.7% to 79.6%) (3 studies, 136 participants; low‐certainty evidence) and 97.5% (95.8% to 98.5%) (3 studies, 551 participants; high‐certainty evidence). For nasopharyngeal specimens, Xpert Ultra sensitivity (95% CI) and specificity (95% CI) were 45.7% (28.9% to 63.3%) and 97.5% (93.7% to 99.3%) (1 study, 195 participants). For all specimen types, Xpert MTB/RIF and Xpert Ultra sensitivity were lower against a composite reference standard than against culture. Detection of tuberculous meningitis For cerebrospinal fluid, Xpert MTB/RIF pooled sensitivity and specificity, verified by culture, were 54.0% (95% CI 27.8% to 78.2%) (6 studies, 28 participants; very low‐certainty evidence) and 93.8% (95% CI 84.5% to 97.6%) (6 studies, 213 participants; low‐certainty evidence). Detection of lymph node tuberculosis For lymph node aspirates or biopsies, Xpert MTB/RIF pooled sensitivity and specificity, verified by culture, were 90.4% (95% CI 55.7% to 98.6%) (6 studies, 68 participants; very low‐certainty evidence) and 89.8% (95% CI 71.5% to 96.8%) (6 studies, 142 participants; low‐certainty evidence). Detection of rifampicin resistance Xpert MTB/RIF pooled sensitivity and specificity were 90.0% (67.6% to 97.5%) (6 studies, 20 participants; low‐certainty evidence) and 98.3% (87.7% to 99.8%) (6 studies, 203 participants; moderate‐certainty evidence). Authors' conclusions We found Xpert MTB/RIF sensitivity to vary by specimen type, with gastric aspirate specimens having the highest sensitivity followed by sputum and stool, and nasopharyngeal specimens the lowest; specificity in all specimens was > 98%. Compared with Xpert MTB/RIF, Xpert Ultra sensitivity in sputum was higher and specificity slightly lower. Xpert MTB/RIF was accurate for detection of rifampicin resistance. Xpert MTB/RIF was sensitive for diagnosing lymph node tuberculosis. For children with presumed tuberculous meningitis, treatment decisions should be based on the entirety of clinical information and treatment should not be withheld based solely on an Xpert MTB/RIF result. The small numbers of studies and participants, particularly for Xpert Ultra, limits our confidence in the precision of these estimates.

Background WHO recommends isoniazid preventive therapy (IPT) for young children in close contact with an infectious tuberculosis (TB) case. No models have examined the cost effectiveness of this recommendation. Methods A decision analysis model was developed to estimate health and economic outcomes of five TB infection screening strategies in young household contacts. In the no-testing strategy, children received IPT based on age and reported exposure. Other strategies included testing for infection with a tuberculin skin test (TST), interferon γ release assay (IGRA) or IGRA after TST. Markov modelling included age-specific disease states and probabilities while considering risk of re-infection in a high-burden country. Results Among the 0–2-year-old cohort, the no-testing strategy was most cost effective. The discounted societal cost of care per life year saved ranged from US$237 (no-testing) to US$538 (IGRA only testing). Among the 3–5-year-old cohort, strategies employing an IGRA after a negative TST were most effective, but were associated with significant incremental cost (incremental cost-effectiveness ratio >US$233 000), depending on the rate of Mycobacterium tuberculosis infection. Conclusion Screening for M tuberculosis infection and provision of IPT in young children is a highly cost-effective intervention. Screening without testing for M tuberculosis infection is the most cost-effective strategy in 0–2-year-old children and the preferred strategy in 3–5-year-old children. Lack of testing capacity should therefore not be a barrier to IPT delivery. These findings highlight the cost effectiveness of contact tracing and IPT delivery in young children exposed to TB in high-burden countries.

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.

Background Globally, children under 15 years represent approximately 12% of new tuberculosis cases, but 16% of the estimated 1.4 million deaths. This higher share of mortality highlights the urgent need to develop strategies to improve case detection in this age group and identify children without tuberculosis disease who should be considered for tuberculosis preventive treatment. One such strategy is systematic screening for tuberculosis in high‐risk groups. Objectives To estimate the sensitivity and specificity of the presence of one or more tuberculosis symptoms, or symptom combinations; chest radiography (CXR); Xpert MTB/RIF; Xpert Ultra; and combinations of these as screening tests for detecting active pulmonary childhood tuberculosis in the following groups. – Tuberculosis contacts, including household contacts, school contacts, and other close contacts of a person with infectious tuberculosis. – Children living with HIV. – Children with pneumonia. – Other risk groups (e.g. children with a history of previous tuberculosis, malnourished children). – Children in the general population in high tuberculosis burden settings. Search methods We searched six databases, including the Cochrane Central Register of Controlled Trials, MEDLINE, and Embase, on 14 February 2020 without language restrictions and contacted researchers in the field. Selection criteria Cross‐sectional and cohort studies where at least 75% of children were aged under 15 years. Studies were eligible if conducted for screening rather than diagnosing tuberculosis. Reference standards were microbiological (MRS) and composite reference standard (CRS), which may incorporate symptoms and CXR. Data collection and analysis Two review authors independently extracted data and assessed study quality using QUADAS‐2. We consolidated symptom screens across included studies into groups that used similar combinations of symptoms as follows: one or more of cough, fever, or poor weight gain and one or more of cough, fever, or decreased playfulness. For combination of symptoms, a positive screen was the presence of one or more than one symptom. We used a bivariate model to estimate pooled sensitivity and specificity with 95% confidence intervals (CIs) and performed analyses separately by reference standard. We assessed certainty of evidence using GRADE. Main results Nineteen studies assessed the following screens: one symptom (15 studies, 10,097 participants); combinations of symptoms (12 studies, 29,889 participants); CXR (10 studies, 7146 participants); and Xpert MTB/RIF (2 studies, 787 participants). Several studies assessed more than one screening test. No studies assessed Xpert Ultra. For 16 studies (84%), risk of bias for the reference standard domain was unclear owing to concern about incorporation bias. Across other quality domains, risk of bias was generally low. Symptom screen (verified by CRS) One or more of cough, fever, or poor weight gain in tuberculosis contacts (4 studies, tuberculosis prevalence 2% to 13%): pooled sensitivity was 89% (95% CI 52% to 98%; 113 participants; low‐certainty evidence) and pooled specificity was 69% (95% CI 51% to 83%; 2582 participants; low‐certainty evidence). Of 1000 children where 50 have pulmonary tuberculosis, 339 would be screen‐positive, of whom 294 (87%) would not have pulmonary tuberculosis (false positives); 661 would be screen‐negative, of whom five (1%) would have pulmonary tuberculosis (false negatives). One or more of cough, fever, or decreased playfulness in children aged under five years, inpatient or outpatient (3 studies, tuberculosis prevalence 3% to 13%): sensitivity ranged from 64% to 76% (106 participants; moderate‐certainty evidence) and specificity from 37% to 77% (2339 participants; low‐certainty evidence). Of 1000 children where 50 have pulmonary tuberculosis, 251 to 636 would be screen‐positive, of whom 219 to 598 (87% to 94%) would not have pulmonary tuberculosis; 364 to 749 would be screen‐negative, of whom 12 to 18 (2% to 3%) would have pulmonary tuberculosis. One or more of cough, fever, poor weight gain, or tuberculosis close contact (World Health Organization four‐symptom screen) in children living with HIV, outpatient (2 studies, tuberculosis prevalence 3% and 8%): pooled sensitivity was 61% (95% CI 58% to 64%; 1219 screens; moderate‐certainty evidence) and pooled specificity was 94% (95% CI 86% to 98%; 201,916 screens; low‐certainty evidence). Of 1000 symptom screens where 50 of the screens are on children with pulmonary tuberculosis, 88 would be screen‐positive, of which 57 (65%) would be on children who do not have pulmonary tuberculosis; 912 would be screen‐negative, of which 19 (2%) would be on children who have pulmonary tuberculosis. CXR (verified by CRS) CXR with any abnormality in tuberculosis contacts (8 studies, tuberculosis prevalence 2% to 25%): pooled sensitivity was 87% (95% CI 75% to 93%; 232 participants; low‐certainty evidence) and pooled specificity was 99% (95% CI 68% to 100%; 3281 participants; low‐certainty evidence). Of 1000 children, where 50 have pulmonary tuberculosis, 63 would be screen‐positive, of whom 19 (30%) would not have pulmonary tuberculosis; 937 would be screen‐negative, of whom 6 (1%) would have pulmonary tuberculosis. Xpert MTB/RIF (verified by MRS) Xpert MTB/RIF, inpatient or outpatient (2 studies, tuberculosis prevalence 1% and 4%): sensitivity was 43% and 100% (16 participants; very low‐certainty evidence) and specificity was 99% and 100% (771 participants; moderate‐certainty evidence). Of 1000 children, where 50 have pulmonary tuberculosis, 31 to 69 would be Xpert MTB/RIF‐positive, of whom 9 to 19 (28% to 29%) would not have pulmonary tuberculosis; 969 to 931 would be Xpert MTB/RIF‐negative, of whom 0 to 28 (0% to 3%) would have tuberculosis. Studies often assessed more symptoms than those included in the index test and symptom definitions varied. These differences complicated data aggregation and may have influenced accuracy estimates. Both symptoms and CXR formed part of the CRS (incorporation bias), which may have led to overestimation of sensitivity and specificity. Authors' conclusions We found that in children who are tuberculosis contacts or living with HIV, screening tests using symptoms or CXR may be useful, but our review is limited by design issues with the index test and incorporation bias in the reference standard. For Xpert MTB/RIF, we found insufficient evidence regarding screening accuracy. Prospective evaluations of screening tests for tuberculosis in children will help clarify their use. In the meantime, screening strategies need to be pragmatic to address the persistent gaps in prevention and case detection that exist in resource‐limited settings.