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The validation of genome-wide association signals for tuberculosis (TB) susceptibility and the development of type 2 diabetes (T2D) across diverse populations remain problematic. The ancestry-specific variants (coding and non-coding) that contribute to previously identified differentially expressed genes (DEG) in patients with TB, T2D and comorbid TB-T2D, remain unknown. Identifying ancestry-specific expression quantitative trait loci (eQTLs) can aid in distinguishing the most probable disease-causing variants for population-specific therapeutic interventions. Therefore, this study conductedcis-eQTL mapping in TB, T2D and TB-T2D patients to identify variants associated with DEG. Both genotyping (Infinium H3A array with ∼2.3 M markers) and RNA sequencing data of 96 complex multi-way admixed South Africans were used for this purpose. Importantly, both global-and local ancestry adjustment were included in statistical analysis to account for complex admixture. Unique gene-variant pairs were associated with TB-T2D on chromosome 7p22 whilst adjusting for Bantu-speaking African ancestry (PRKAR1B:rs4464850; P=7.68e-07) and Khoe-San ancestry (PRKAR1B:rs117842122; P=3.66e-07). In addition,IFITM3(a biomarker for the development of TB) was associated with three SNPs (rs11025530, rs3808990, and rs10896664) on chromosome 11p15 while adjusting for Khoe-San ancestry. Our results also indicated that the upregulation of theNLRP6inflammasome is strongly associated with people with TB-T2D while adjusting for Khoe-San ancestry. Three African-specific eGenes (NLRP6, IFITM3andPRKAR1B) would have been missed if local ancestry adjustment was not conducted. This study determined a list of ancestry-specific eQTLs in TB-T2D patients that could potentially guide the search for new therapeutic targets for TB-T2D in African populations.Author SummaryThe limitation of genome-wide association study (GWAS) is that the particular biological pathway impacted by a variant might not be evident. eQTL mapping can be conducted to determine the impact that a genetic variant might have on the expression of a specific gene in a biological pathway. In this study the use ofcis-eQTL mapping was explored to elucidate the underlying genetic variants that regulate gene expression between TB-T2D and T2D patients, and between TB patients and healthy controls with multi-way genetic admixture from South Africa. Using RNA sequencing data and newly genotyped dataset of 96 individuals (Illumina Infinium H3Africa array with ∼2.5 M markers), we were able to identify ancestry-specific eQTLs. eQTLs of indigenous Khoe-San ancestral origin were identified in genetic regions previously implicated in TB and T2D in African populations. If local ancestry was not incorporated in thecis-eQTL mapping analysis these important African-specific eQTLs would have been missed. Our results provide a list of possible ancestry-specific causal variants associated with TB-T2 comorbidity that could guide the search for new therapeutic targets for African-specific populations. Including populations with complex ancestry and admixture in genetic studies is necessary to improve the quality of genetic research in sub-Saharan African groups.

The influence of smoke-derived or air pollution-derived cytoplasmic particulate matter (PM) can be detrimental and can lead to failed lung immunity. We investigated mycobacterial uptake, intracellular replication, and soluble immune-mediator responses of human bronchoalveolar lavage cells (BALCs) loaded with/without PM, to infection with mycobacterial strains. We observed that only BALCs containing PM display an ex vivo phenotypic profile dominated by spontaneous interleukin (IL)-10 production. PM-loaded BALCs retained the ability to phagocytose both Mycobacterium bovis Bacille Calmette Guérin (BCG) and Mycobacterium tuberculosis ( M.tb) ΔleuDΔpanCD at equal efficacy as clear non-PM-loaded BALCs. However, immune responsiveness, such as the production of IL-6 ( P = 0.015) and tumor necrosis factor-α (TNF)-α ( P = 0.0172) immediately post M. bovis BCG infection, were dramatically lower in black BALCs loaded with PM versus clear non-PM-loaded BALCs. By 24 h post infection, differential immune responses to M. bovis BCG between black versus clear BALC waned, and instead, production of IL-6 ( P = 0.03) and IL-1α ( P = 0.04) by black BALCs was lower versus clear BALCs following M.tb ΔleuDΔpanCD infection. Considering that TNF-α and IL-6 are characterized as critical to host protection against mycobacteria, our findings suggest that BALCs loaded with inhaled PM, display lower levels of antimycobacterial mediators and that the response magnitude differs according to infective mycobacterial strain. Even though this did not translate into altered mycobacterial killing at early time points post infection, the long-term impact of such changes remains to be established.

Summary Background A rapid, blood-based triage test that allows targeted investigation for tuberculosis at the point of care could shorten the time to tuberculosis treatment and reduce mortality. We aimed to test the performance of a host blood transcriptomic signature (RISK11) in diagnosing tuberculosis and predicting progression to active pulmonary disease (prognosis) in people with HIV in a community setting. Methods In this prospective diagnostic and prognostic accuracy study, adults (aged 18–59 years) with HIV were recruited from five communities in South Africa. Individuals with a history of tuberculosis or household exposure to multidrug-resistant tuberculosis within the past 3 years, comorbid risk factors for tuberculosis, or any condition that would interfere with the study were excluded. RISK11 status was assessed at baseline by real-time PCR; participants and study staff were masked to the result. Participants underwent active surveillance for microbiologically confirmed tuberculosis by providing spontaneously expectorated sputum samples at baseline, if symptomatic during 15 months of follow-up, and at 15 months (the end of the study). The coprimary outcomes were the prevalence and cumulative incidence of tuberculosis disease confirmed by a positive Xpert MTB/RIF, Xpert Ultra, or Mycobacteria Growth Indicator Tube culture, or a combination of such, on at least two separate sputum samples collected within any 30-day period. Findings Between March 22, 2017, and May 15, 2018, 963 participants were assessed for eligibility and 861 were enrolled. Among 820 participants with valid RISK11 results, eight (1%) had prevalent tuberculosis at baseline: seven (2·5%; 95% CI 1·2–5·0) of 285 RISK11-positive participants and one (0·2%; 0·0–1·1) of 535 RISK11-negative participants. The relative risk (RR) of prevalent tuberculosis was 13·1 times (95% CI 2·1–81·6) greater in RISK11-positive participants than in RISK11-negative participants. RISK11 had a diagnostic area under the receiver operating characteristic curve (AUC) of 88·2% (95% CI 77·6–96·7), and a sensitivity of 87·5% (58·3–100·0) and specificity of 65·8% (62·5–69·0) at a predefined score threshold (60%). Of those with RISK11 results, eight had primary endpoint incident tuberculosis during 15 months of follow-up. Tuberculosis incidence was 2·5 per 100 person-years (95% CI 0·7–4·4) in the RISK11-positive group and 0·2 per 100 person-years (0·0–0·5) in the RISK11-negative group. The probability of primary endpoint incident tuberculosis was greater in the RISK11-positive group than in the RISK11-negative group (cumulative incidence ratio 16·0 [95% CI 2·0–129·5]). RISK11 had a prognostic AUC of 80·0% (95% CI 70·6–86·9), and a sensitivity of 88·6% (43·5–98·7) and a specificity of 68·9% (65·3–72·3) for incident tuberculosis at the 60% threshold. Interpretation RISK11 identified prevalent tuberculosis and predicted risk of progression to incident tuberculosis within 15 months in ambulant people living with HIV. RISK11's performance approached, but did not meet, WHO's target product profile benchmarks for screening and prognostic tests for tuberculosis. Funding Bill & Melinda Gates Foundation and the South African Medical Research Council.

Background Diabetes mellitus (DM) patients are three times more likely to develop tuberculosis (TB) than the general population. Active TB screening in people with DM is part of a bidirectional approach. The aim of this study was to conduct pragmatic active TB screening among DM patients in four countries to inform policy. Methods DM patients were recruited in Indonesia (n=809), Peru (n=600), Romania (n=603) and South Africa (n=51). TB cases were diagnosed using an algorithm including clinical symptoms and chest X-ray. Presumptive TB patients were examined with sputum smear and culture. Results A total of 171 (8.3%) individuals reported ever having had TB (South Africa, 26%; Indonesia, 12%; Peru, 7%; Romania, 4%), 15 of whom were already on TB treatment. Overall, 14 (0.73% [95% confidence interval 0.40 to 1.23]) TB cases were identified from screening. Poor glucose control, smoking, lower body mass index, education and socio-economic status were associated with newly diagnosed/current TB. Thirteen of the 14 TB cases diagnosed from this screening would have been found using a symptom-based approach. Conclusions These data support the World Health Organization recommendation for routine symptom-based screening for TB in known DM patients in high TB-burden countries. DM patients with any symptoms consistent with TB should be investigated and diagnostic tools should be easily accessible.

Background Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) infection and is a major public health problem. Clinical challenges include the lack of a blood-based test for active disease. Current blood-based tests, such as QuantiFERON (QFT) do not distinguish active TB disease from asymptomatic Mtb infection. Methods We hypothesized that TruCulture, an immunomonitoring method for whole-blood stimulation, could discriminate active disease from latent Mtb infection (LTBI). We stimulated whole blood from patients with active TB and compared with LTBI donors. Mtb-specific antigens and live bacillus Calmette-Guérin (BCG) were used as stimuli, with direct comparison to QFT. Protein analyses were performed using conventional and digital enzyme-linked immunosorbent assay (ELISA), as well as Luminex. Results TruCulture showed discrimination of active TB cases from LTBI (P < .0001, AUC = .81) compared with QFT (P = .45, AUC = .56), based on an interferon γ (IFNγ) readout after Mtb antigen (Ag) stimulation. This result was replicated in an independent cohort (AUC = .89). In exploratory analyses, TB stratification could be further improved by the Mtb antigen to BCG IFNγ ratio (P < .0001, AUC = .91). Finally, the combination of digital ELISA and transcriptional analysis showed that LTBI donors with high IFNγ clustered with patients with TB, suggesting the possibility to identify subclinical disease. Conclusions TruCulture offers a next-generation solution for whole-blood stimulation and immunomonitoring with the possibility to discriminate active and latent infection., We tested TruCulture, an immunomonitoring tool, to identify active disease from latent Mtb infection. TruCulture showed improved discrimination of tuberculosis cases from LTBI as compared with QuantiFERON. Tuberculosis stratification could be further improved by the Mtb Ag:BCG IFNγ ratio.

Tuberculosis 133, 102169 (2022). doi:10.1016/j.tube.2022.102169, Published by Churchill Livingstone, Edinburgh

BackgroundPeople with diabetes are more likely to develop tuberculosis (TB) and to have poor TB treatment outcomes than those without. We previously showed that blood transcriptomes in people with TB-diabetes (TB-DM) co-morbidity have excessive inflammatory and reduced interferon responses at TB diagnosis. It is unknown whether this persists through treatment, potentially underlying adverse outcomes.MethodsPulmonary TB patients were recruited in South Africa, Indonesia and Romania, and classified as having TB-DM, TB with prediabetes, TB-related hyperglycaemia or uncomplicated TB, based on glycated haemoglobin (HbA1c) concentration at TB diagnosis and after 6 months of TB treatment. Gene expression in blood samples collected at diagnosis and at regular intervals throughout treatment was measured by unbiased RNA-Seq and targeted Multiplex Ligation-dependent Probe Amplification.ResultsGene expression was modulated by TB treatment in all groups but to different extents, such that differences remained in people with TB-DM relative to TB-only throughout, including genes involved in innate responses, anti-microbial immunity and the inflammasome. People with prediabetes or with TB-related hyperglycaemia had gene expression more similar to people with TB-DM than TB-only throughout treatment. The overall pattern of change was similar across clinical groups irrespective of glycaemic index, permitting models predictive of TB treatment to be developed.ConclusionsThe exacerbated transcriptome changes seen in TB-DM take longer to resolve during TB treatment, indicating that prolonged treatment or host-directed therapy may be needed to improve TB treatment outcomes. Development of transcriptome-based biomarker signatures of TB-treatment response should include people with diabetes to be useful across populations.Key PointsHost blood transcriptomes are altered in tuberculosis, and further altered with diabetes co-morbidity. We have shown that there is similar resolution of transcriptomes through treatment, but with differing magnitude and kinetics in TB patients with or without diabetes.

Objectives We evaluated longitudinal kinetics of an 11-gene blood transcriptomic tuberculosis (TB) signature, RISK11, and effects of TB preventative therapy (TPT) and respiratory organisms on RISK1...

Tuberculous granulomas that develop in response to Mycobacterium tuberculosis (M. tuberculosis) infection are highly dynamic entities shaped by the host immune response and disease kinetics. Within this microenvironment, immune cell recruitment, polarization, and activation are driven not only by coexisting cell types and multicellular interactions but also by M. tuberculosis-mediated changes involving metabolic heterogeneity, epigenetic reprogramming, and rewiring of the transcriptional landscape of host cells. There is an increased appreciation of the in vivo complexity, versatility, and heterogeneity of the cellular compartment that constitutes the tuberculosis (TB) granuloma and the difficulty in translating findings from animal models to human disease. Here, we describe a novel biomimetic in vitro three-dimensional (3D) human lung spheroid granuloma model, resembling early “innate” and “adaptive” stages of the TB granuloma spectrum, and present results of histological architecture, host transcriptional characterization, mycobacteriological features, cytokine profiles, and spatial distribution of key immune cells. A range of manipulations of immune cell populations in these spheroid granulomas will allow the study of host/pathogen pathways involved in the outcome of infection, as well as pharmacological interventions. IMPORTANCE TB is a highly infectious disease, with granulomas as its hallmark. Granulomas play an important role in the control of M. tuberculosis infection and as such are crucial indicators for our understanding of host resistance to TB. Correlates of risk and protection to M. tuberculosis are still elusive, and the granuloma provides the perfect environment in which to study the immune response to infection and broaden our understanding thereof; however, human granulomas are difficult to obtain, and animal models are costly and do not always faithfully mimic human immunity. In fact, most TB research is conducted in vitro on immortalized or primary immune cells and cultured in two dimensions on flat, rigid plastic, which does not reflect in vivo characteristics. We have therefore conceived a 3D, human in vitro spheroid granuloma model which allows researchers to study features of granuloma-forming diseases in a 3D structural environment resembling in vivo granuloma architecture and cellular orientation.

Summary Background There is an urgent need for new tools for the diagnosis of TB. We evaluated the usefulness recently described host biomarkers in supernatants from the newest generation of the QuantiFERON test (QuantiFERON Plus) as tools for the diagnosis of active TB. Methods We recruited individuals presenting at primary health care clinics in Cape Town, South Africa with symptoms requiring investigation for TB disease, prior to the establishment of a clinical diagnosis. Participants were later classified as TB or other respiratory diseases (ORD) based on the results of clinical and laboratory tests. Using a multiplex platform, we evaluated the concentrations of 37 host biomarkers in QuantiFERON Plus supernatants from study participants as tools for the diagnosis of TB. Results Out of 120 study participants, 35(29.2%) were diagnosed with active TB, 69(57.5%) with ORD whereas 16(13.3%) were excluded. 14(11.6%) of the study participants were HIV infected. Although individual host markers showed potential as diagnostic candidates, the main finding of the study was the identification of a six-marker biosignature in unstimulated supernatants (Apo-ACIII, CXCL1, CXCL9, CCL8, CCL-1, CD56) which diagnosed TB with sensitivity and specificity of 73.9%(95% CI; 51.6–87.8) and 87.6%(95% CI; 77.2–94.5), respectively, after leave-one-out cross validation. Combinations between TB-antigen specific biomarkers also showed potential (sensitivity of 77.3% and specificity of 69.2%, respectively), with multiple biomarkers being significantly different between TB patients, Quantiferon Plus Positive and Quantiferon Plus negative individuals with ORD, regardless of HIV status. Conclusions Biomarkers detected in QuantiFERON Plus supernatants may contribute to adjunctive diagnosis of TB.

Background There is an urgent need for new, accurate, rapid, and affordable tuberculosis (TB) diagnostic tests. The aim of the present study was to use mass spectrometry to identify new preliminary candidate TB diagnostic protein biomarkers in saliva obtained from individuals with TB, and patients with other respiratory diseases (ORD). Methods Saliva samples were collected from 22 individuals who self-presented with symptoms suggestive of TB as part of a larger TB biomarker project. Purified salivary proteins were subjected to tryptic digestion peptides were analyzed using a QExactive Orbitrap Mass Spectrometer. Data are available via ProteomeXchange with identifier PXD027294. Identified proteins were subjected to gene ontology and ingenuity pathway analysis for functional enrichment analysis. Results 26 of the 652 identified proteins significantly discriminated individuals with TB from those with ORD after Benjamini Hochberg correction (5% FDR), with five of these proteins diagnosing TB with an AUC ≥ 0.80. A 5-protein biosignature comprising of P01011, Q8NCW5, P28072, A0A2Q2TTZ9, and Q99574 diagnosed TB with an AUC of 1.00 (95% CI, 1.00–1.00), sensitivity of 100% (95% CI, 76.2–100%) and specificity of 90.9% (95% CI, 58.7–99.8%) after leave-one-out cross validation. Conclusions We identified novel candidate salivary protein biomarkers and biosignatures with strong potential as TB diagnostic candidates. Our results are preliminary and require validation in larger studies.

The development of a non-sputum-based, point-of-care diagnostic test for tuberculosis (TB) is a priority in the global effort to combat this disease, particularly in resource-constrained settings. Previous studies have identified host biomarker signatures which showed potential, but there is a need to validate and refine these for development as a test. We recruited 1,403 adults presenting with symptoms suggestive of pulmonary TB at primary healthcare clinics in six countries from West, East and Southern Africa. Of the study cohort, 326 were diagnosed with TB and 787 with other respiratory diseases, from whom we randomly selected 1005 participants. Using Luminex® technology, we measured the levels of 20 host biomarkers in serum samples which we used to evaluate the diagnostic accuracy of previously identified and novel bio-signatures. Our previously identified seven-marker bio-signature did not perform well (sensitivity: 89%, specificity: 60%). We also identified an optimal, two-marker bio-signature with a sensitivity of 94% and specificity of 69% in patients with no history of previous TB. This signature performed slightly better than C-reactive protein (CRP) alone. The cut-off value for a positive diagnosis differed for human immuno-deficiency virus (HIV)-positive and -negative individuals. Notably, we also found that no signature was able to diagnose TB adequately in patients with a prior history of the disease. We have identified a two-marker, pan-African bio-signature which is more robust than CRP alone and meets the World Health Organization (WHO) target product profile requirements for a triage test in both HIV-negative and HIV-positive individuals. This signature could be incorporated into a point-of-care device, greatly reducing the necessity for expensive confirmatory diagnostics and potentially reducing the number of cases currently lost to follow-up. It might also potentially be useful with individuals unable to provide sputum or with paucibacillary disease. We suggest that the performance of TB diagnostic signatures can be improved by incorporating the HIV-status of the patient. We further suggest that only patients who have never had TB be subjected to a triage test and that those with a history of previous TB be evaluated using more direct diagnostic techniques.

Summary Background Direct bronchial spread of tuberculosis was extensively described in pre-antibiotic human pathology literature but this description has been overlooked in the post-antibiotic era, in which most pathology data come from animal models that emphasise the granuloma. Modern techniques, such as [18F]2-fluoro-2-deoxy-D-glucose (FDG) PET-CT scans, might provide further insight. Our aim was to understand normal early tuberculosis resolution patterns on pulmonary PET-CT scans in treated patients with tuberculosis who were subsequently cured. Methods In this observational analysis, we analysed data from PredictTB, an ongoing, prospective, randomised clinical trial that examined sequential baseline and week 4 FDG-PET-CT scans from participants successfully treated (sputum culture negative 18 months after enrolment) for drug-susceptible pulmonary tuberculosis in South Africa and China. Participants who were aged 18–75 years, GeneXpert MTB/RIF positive for tuberculosis and negative for rifampicin resistance, had not yet started tuberculosis treatment, had not been treated for active tuberculosis within the previous 3 years, and met basic safety laboratory criteria were included and participants with diabetes, HIV infection, or with extrapulmonary tuberculosis including pleural tuberculosis were excluded. Scans were assessed by two readers for the location of tuberculosis lesions (eg, cavities and consolidations), bronchial thickening patterns, and changes from baseline to week 4 of treatment. Findings Among the first 124 participants (enrolled from June 22, 2017, to Sept 27, 2018) who were successfully treated, 161 primarily apical cavitary lesions were identified at baseline. Bronchial thickening and inflammation linking non-cavitary consolidative lesions to cavities were observed in 121 (98%) of 124 participants' baseline PET-CT scans. After 4 weeks of treatment, 21 (17%) of 124 participants had new or expanding lesions linked to cavities via bronchial inflammation that were not present at baseline, particularly participants with two or more cavities at baseline and participants from South Africa. Interpretation In participants with pulmonary tuberculosis who were subsequently cured, the location of cavitary and non-cavitary lesions at baseline and new lesions at week 4 of treatment suggest a cavitary origin of disease and bronchial spread through the lungs. Bronchial spread from cavities might play a larger role in the spread of pulmonary tuberculosis than has been appreciated. Elucidating cavity lesion dynamics and Mycobacterium tuberculosis viability within cavities might better explain treatment outcomes and why some patients are cured and others relapse. Funding Bill & Melinda Gates Foundation, European and Developing Countries Clinical Trials Partnership, China Ministry of Science and Technology, National Natural Science Foundation of China, and National Institutes of Health. Translations For the Chinese, Afrikaans and Xhosa translations of the abstract see Supplementary Materials section.

Background Inequality is rife throughout South Africa. The first wave of COVID-19 may have affected people in lower socioeconomic groups worse than the affluent. The SARS-CoV-2 seroprevalence and the specificity of anti-SARS-CoV-2 antibody tests in South Africa is not known. Methods We tested 405 volunteers representing all socioeconomic strata from the workforce of a popular shopping and tourist complex in central Cape Town with the Abbott SARS-CoV-2 IgG assay. We assessed the association between antibody positivity and COVID-19 symptom status, medical history, and sociodemographic variables. We tested 137 serum samples from healthy controls collected in Cape Town prior to the COVID-19 pandemic, to confirm the specificity of the assay in the local population. Results Of the 405 volunteers tested one month after the first peak of the epidemic in Cape Town, 96(23.7%) were SARS-CoV-2 IgG positive. Of those who tested positive, 46(47.9%) reported no symptoms of COVID-19 in the previous 6 months. Seropositivity was significantly associated with living in informal housing, residing in a subdistrict with low income-per household, and having a low-earning occupation. The specificity of the assay was 98.54%(95%CI 94.82%-99.82%) in the pre-COVID controls. Conclusions There is a high background seroprevalence in Cape Town, particularly in people of lower socioeconomic status. Almost half of cases are asymptomatic, and therefore undiagnosed by local testing strategies. These results cannot be explained by low assay specificity.

Background: The diagnosis of TB remains one of the major challenges in the control of the disease, due to limitations in the currently available diagnostic tests. There is an urgent need for new, accurate, rapid, and affordable diagnostic tests. The aim of the present study was to use mass spectrometry to identify new candidate TB diagnostic protein biomarkers in saliva obtained from individuals with TB, and patients with other respiratory diseases (ORD).Methods: Saliva samples were collected from 22 individuals who self-presented with symptoms requiring investigation for TB as part of a larger TB biomarker project. Eleven of the participants were finally diagnosed with TB using routine clinical, radiological and laboratory tests and 11 with ORD. Salivary proteins were concentrated and purified, followed by tryptic digestion. Peptides were analyzed using a QExactive Orbitrap MS coupled to a Dionex liquid chromatography system. Raw files were processed using MaxQuant software against the database of human proteins. Identified proteins were subjected to gene ontology and ingenuity pathway analysis for functional enrichment analysis.Results: We identified 1176 protein groups across all samples, of which 46 (3.91%) were contaminants, 12 (1.02%) were reverse hits and 170 (14.46%) were single-peptide protein groups. After removal of the contaminants, reverse hits and protein groups represented by single peptides, 26 of the remaining 948 proteins significantly discriminated individuals with TB from those with ORD after Benjamini Hochberg correction, with five of these proteins diagnosing TB with AUC ³ 0.80. A 5-protein biosignature comprising of P01011, Q8NCW5, P28072, A0A2Q2TTZ9 and Q99574 diagnosed TB with an AUC of 1.00 (95% CI, 1.00-1.00), sensitivity of 100% (95% CI, 76.2-100%) and specificity of 90.9% (95% CI, 58.7-99.8%) after leave-one-out cross validation. Conclusions: We identified novel salivary protein biomarkers and biosignatures with strong potential as TB diagnostic candidates. Our results are preliminary and require validation in larger studies.

Mycobacterium tuberculosis (Mtb) is extremely recalcitrant to antimicrobial chemotherapy requiring 6 months to treat drug-sensitive tuberculosis (TB). Despite this, 4–10% of cured patients will develop recurrent disease within 12 months after completing therapy. Reasons for relapse in cured TB patients remains speculative, attributed to both pathogen and host factors. Populations of dormant bacilli are hypothesized to cause relapse in initially cured TB patients however, development of tests to convincingly demonstrate their presence at the end of anti-TB treatment has been challenging. Previous studies have indicated the utility of culture filtrate supplemented media (CFSM) to detect differentially culturable tubercle bacilli (DCTB). Here, we show that 3/22 of clinically cured patients retained DCTB in induced sputum and bronchoalveolar lavage fluid (BALF), with one DCTB positive patient relapsing within the first year of completing therapy. We also show a correlation of DCTB status with “unresolved” end of treatment FDG PET-CT imaging. Additionally, 19 end of treatment induced sputum samples from patients not undergoing bronchoscopy were assessed for DCTB, identifying a further relapse case with DCTB. We further show that induced sputum is a less reliable source for the DCTB assay at the end of treatment, limiting the utility of this assay in a clinical setting. We next investigated the host proteome at the site of disease (BALF) using multiplexed proteomic analysis and compared these to active TB cases to identify host-specific factors indicative of cure. Distinct signatures stratified active from cured TB patients into distinct groups, with a DCTB positive, subsequently relapsing, end of treatment patient showing a proteomic signature closer to active TB disease than cure. This exploratory study offers evidence of live Mtb, undetectable with conventional culture methods, at the end of clinically successful treatment and putative host protein biomarkers of active disease and cure. These findings have implications for the assessment of true sterilizing cure in TB patients and opens new avenues for targeted approaches to monitor treatment response.

Pulmonary tuberculosis (PTB) is characterized by lung granulomas, inflammation and tissue destruction. Here we used within-subject peripheral blood gene expression over time to correlate with the within-subject lung metabolic activity, as measured by positron emission tomography (PET) to identify biological processes and pathways underlying overall resolution of lung inflammation. We used next-generation RNA sequencing and [18F]FDG PET-CT data, collected at diagnosis, week 4, and week 24, from 75 successfully cured PTB patients, with the [18F]FDG activity as a surrogate for lung inflammation. Our linear mixed-effects models required that for each individual the slope of the line of [18F]FDG data in the outcome and the slope of the peripheral blood transcript expression data correlate, i.e., the slopes of the outcome and explanatory variables had to be similar. Of 10,295 genes that changed as a function of time, we identified 639 genes whose expression profiles correlated with decreasing [18F]FDG uptake levels in the lungs. Gene enrichment over-representation analysis revealed that numerous biological processes were significantly enriched in the 639 genes, including several well known in TB transcriptomics such as platelet degranulation and response to interferon gamma, thus validating our novel approach. Others not previously associated with TB pathobiology included smooth muscle contraction, a set of pathways related to mitochondrial function and cell death, as well as a set of pathways connecting transcription, translation and vesicle formation. We observed up-regulation in genes associated with B cells, and down-regulation in genes associated with platelet activation. We found 254 transcription factor binding sites to be enriched among the 639 gene promoters. In conclusion, we demonstrated that of the 10,295 gene expression changes in peripheral blood, only a subset of 639 genes correlated with inflammation in the lungs, and the enriched pathways provide a description of the biology of resolution of lung inflammation as detectable in peripheral blood. Surprisingly, resolution of PTB inflammation is positively correlated with smooth muscle contraction and, extending our previous observation on mitochondrial genes, shows the presence of mitochondrial stress. We focused on pathway analysis which can enable therapeutic target discovery and potential modulation of the host response to TB.

Summary Background Targeted preventive therapy for individuals at highest risk of incident tuberculosis might impact the epidemic by interrupting transmission. We tested performance of a transcriptomic signature of tuberculosis (RISK11) and efficacy of signature-guided preventive therapy in parallel, using a hybrid three-group study design. Methods Adult volunteers aged 18–59 years were recruited at five geographically distinct communities in South Africa. Whole blood was sampled for RISK11 by quantitative RT-PCR assay from eligible volunteers without HIV, recent previous tuberculosis (ie

Early bactericidal activity studies monitor daily sputum bacterial counts in individuals with tuberculosis (TB) for 14 days during experimental drug treatment. The rate of change in sputum bacterial load over time provides an informative, but imperfect, estimate of drug activity and is considered a critical step in development of new TB drugs. In this clinical study, 160 participants with TB received isoniazid, pyrazinamide, or rifampicin, components of first-line chemotherapy, and moxifloxacin individually and in combination. In addition to standard bacterial enumeration in sputum, participants underwent 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography and computerized tomography ([18F]FDG-PET/CT) at the beginning and end of the 14-day drug treatment. Quantitating radiological responses to drug treatment provided comparative single and combination drug activity measures across lung lesion types that correlated more closely with established clinical outcomes when combined with sputum enumeration compared to sputum enumeration alone. Rifampicin and rifampicin-containing drug combinations were most effective in reducing both lung lesion volume measured by CT imaging and lesion-associated inflammation measured by PET imaging. Moxifloxacin was not superior to rifampicin in any measure by PET/CT imaging, consistent with its performance in recent phase 3 clinical trials. PET/CT imaging revealed synergy between isoniazid and pyrazinamide and demonstrated that the activity of pyrazinamide was limited to lung lesion, showing the highest FDG uptake during the first 2 weeks of drug treatment. [18F]FDG-PET/CT imaging may be useful for measuring the activity of single drugs and drug combinations during evaluation of potential new TB drug regimens before phase 3 trials.

Tuberculosis (TB) is a global health concern. Treatment is prolonged, and patients on anti-TB therapy (ATT) often experience treatment failure for various reasons. There is an urgent need to identify signatures for early detection of failure and initiation of a treatment switch. We investigated how gene biomarkers and/or basic patient characteristics could be used to define signatures for treatment outcomes in Indian adult pulmonary-TB patients treated with standard ATT. Using blood samples at baseline, a 12-gene signature combined with information on gender, previously-diagnosed TB, severe thinness, smoking and alcohol consumption was highly predictive of treatment failure at 6 months. Likewise a 4-protein biomarker signature combined with the same patient characteristics was almost as highly predictive of treatment failure. Combining biomarkers and basic patient characteristics may be useful for predicting and hence identification of treatment failure at an early stage of TB therapy., Sivakumaran et al. show that a 12-gene signature combined with gender, previously diagnosed tuberculosis (TB), severe thinness, smoking, and alcohol consumption predict the treatment outcome at 6 months. This study suggests that the combination of biomarkers and basic patient characteristics may better predict the treatment failure at an early stage of TB therapy.

Objective To investigate the potential of host urinary biomarkers as diagnostic candidates for tuberculosis (TB). Methods Adults self-presenting with symptoms requiring further investigation for TB were enrolled in Cape Town, South Africa. Participants were later classified as having TB or other respiratory diseases (ORD) using results from TB confirmatory tests. The concentrations of 29 analytes were evaluated in urine samples from participants using the Luminex platform, and their diagnostic potential was assessed using standard statistical approaches. Results Of the 151 study participants, 34 (22.5%) were diagnosed with TB and 26 (17.2%) were HIV-positive. Seven biomarkers showed potential as TB diagnostic candidates, with accuracy improving (in HIV-positives) when stratified according to HIV status (area under the receiver operating characteristics curve; AUC ≥0.80). In HIV-positive participants, a four-marker biosignature (sIL6R, MMP-9, IL-2Ra, IFN-γ) diagnosed TB with AUC of 0.96, sensitivity of 85.7% (95% confidence interval (CI) 42.1–99.6%), and specificity of 94.7% (95% CI 74.0–99.9%). In HIV-negatives, the most promising was a two-marker biosignature (sIL6R and sIL-2Ra), which diagnosed TB with AUC of 0.76, sensitivity of 53.9% (95% CI 33.4–73.4%), and specificity of 79.6% (95% CI 70.3–87.1%). Conclusions Urinary host inflammatory biomarkers possess TB diagnostic potential but may be influenced by HIV infection. The results of this study require validation in larger studies.

BackgroundDiabetes mellitus (DM) increases active tuberculosis (TB) risk and worsens TB outcomes, jeopardizing TB control especially in TB-endemic countries with rising DM prevalence rates. We assessed DM status and clinical correlates in TB patients across settings in Indonesia, Peru, Romania, and South Africa.MethodsAge-adjusted DM prevalence was estimated using laboratory glycated hemoglobin (HbA1c) or fasting plasma glucose in TB patients. Detailed and standardized sociodemographic, anthropometric, and clinical measurements were made. Characteristics of TB patients with or without DM were compared using multilevel mixed-effect regression models with robust standard errors.ResultsOf 2185 TB patients (median age 36.6 years, 61.2% male, 3.8% human immunodeficiency virus–infected), 12.5% (267/2128) had DM, one third of whom were newly diagnosed. Age-standardized DM prevalence ranged from 10.9% (South Africa) to 19.7% (Indonesia). Median HbA1c in TB–DM patients ranged from 7.4% (Romania) to 11.3% (Indonesia). Compared to those without DM, TB–DM patients were older and had a higher body mass index (BMI) (P value < .05). Compared to those with newly diagnosed DM, TB patients with diagnosed DM had higher BMI and HbA1c, less severe TB, and more frequent comorbidities, DM complications, and hypertension (P value < .05).ConclusionsWe show that DM prevalence and clinical characteristics of TB–DM vary across settings. Diabetes is primarily known but untreated, hyperglycemia is often severe, and many patients with TB–DM have significant cardiovascular disease risk and severe TB. This underlines the need to improve strategies for better clinical management of combined TB and DM.

Background: We tested diagnostic and prognostic performance of a host blood transcriptomic signature of tuberculosis (RISK11) for screening of people living with HIV (PLHIV) in a prospective, community-based cohort. Methods: Ambulant adult volunteers living with HIV were enrolled at five South African sites. RISK11 status was assessed at baseline by real-time PCR. Participants and study staff were blinded to the result. Participants underwent active surveillance for microbiologically-confirmed tuberculosis from enrolment through 15 months. The primary outcomes were prevalence and cumulative incidence of two-sputum-sample positive tuberculosis in RISK11+ versus RISK11- participants. Findings: Among 820 participants with valid RISK11 results, eight (1%) tuberculosis cases were identified at baseline. Risk of prevalent tuberculosis was 13·1-fold (95%CI 2·1-81·6) greater in RISK11+ than RISK11- participants, with tuberculosis prevalence of 2·5% and 0·2%, respectively. RISK11 had diagnostic area under the receiver-operating-characteristic curve (AUC) 88·2%; sensitivity 87·5% and specificity 65·8% at the pre-defined threshold (60%).Thereafter, eight tuberculosis cases were identified through median 15 months follow-up. Tuberculosis incidence was 2·5 vs 0·2 per 100 person-years in RISK11+ compared to RISK11- participants with cumulative incidence ratio 16·0 (95%CI 2·0-129·5); AUC 80·0%; sensitivity 88·6% and specificity 68·9%. By comparison, QuantiFERON TB Gold-Plus (QFT) had a cumulative incidence ratio of 2·0 (95%CI 0·5-8·4); AUC 70·8%; sensitivity 62·1% and specificity 56·2%. Interpretation: RISK11 identified prevalent tuberculosis and predicted risk of progression to incident tuberculosis within 15 months in ambulant PLHIV. Performance approached the World Health Organization Target Product Profile benchmarks for screening and prognostic tests for tuberculosis. QFT performance fell short of the prognostic benchmark. Funding Statement: The study was funded by the Bill & Melinda Gates Foundation (BMGF) (OPP1151915) and by the Strategic Health Innovation Partnerships Unit of the South African Medical Research Council with funds received from the South African Department of Science and Technology. The BMGF contributed to the study design. The regulatory sponsor was the University of Cape Town. Declaration of Interests: AP-N, GW, GC, TJS, and MH report grants from the Bill & Melinda Gates Foundation, during the conduct of the study; AP-N and GW report grants from the South African Medical Research Council, during the conduct of the study; GW and TJS report grants from the South African National Research Foundation, during the conduct of the study. In addition, AP-N and TJS have patents of the RISK11 and RISK6 signatures pending; GW has a patent “TB diagnostic markers” (PCT/IB2013/054377) issued and a patent “Method for diagnosing TB” (PCT/IB2017/052142) pending. The remaining authors have no competing interests to declare. Ethics Approval Statement: The study protocol (Supplement) was approved by the Institutional Human Ethics Committees of each participating site. All participants provided written informed consent in their language of choice.

Type 2 diabetes (T2D) is a prevalent risk factor for tuberculosis (TB), but most studies on TB-T2D have focused on TB patients, been limited to one community, and shown a variable impact of T2D on TB risk or treatment outcomes. We conducted a cross-sectional assessment of sociodemographic and metabolic factors in adult TB contacts with T2D (versus no T2D), from the Texas-Mexico border to study Hispanics, and in Cape Town to study South African Coloured ethnicities. The prevalence of T2D was 30.2% in Texas-Mexico and 17.4% in South Africa, with new diagnosis in 34.4% and 43.9%, respectively. Contacts with T2D differed between ethnicities, with higher smoking, hormonal contraceptive use and cholesterol levels in South Africa, and higher obesity in Texas-Mexico (p < 0.05). PCA analysis revealed striking differences between ethnicities in the relationships between factors defining T2D and dyslipidemias. Our findings suggest that screening for new T2D in adult TB contacts is effective to identify new T2D patients at risk for TB. Furthermore, studies aimed at predicting individual TB risk in T2D patients, should take into account the heterogeneity in dyslipidemias that are likely to modify the estimates of TB risk or adverse treatment outcomes that are generally attributed to T2D alone.

The development of an improved vaccine to stimulate an effective response against Mycobacterium tuberculosis (MTB) infection and disease will be a major breakthrough in the fight against TB. A lack of tools to adequately track the progression or resolution of events in TB pathogenesis that occur at bacterial loads below the threshold for culture in human samples seriously hampers vaccine development and evaluation. In this review we discuss recent studies that use new imaging applications, modalities and analysis techniques to provide insight into the dynamic processes of MTB infection and disease that are challenging to monitor. These include early infection, the spectrum of latency and subclinical disease, the paucibacillary state induced by treatment, and events leading to recurrence, including relapse.

Most infections with Mycobacterium tuberculosis (Mtb) manifest as a clinically asymptomatic, contained state, known as latent tuberculosis infection, that affects approximately one-quarter of the global population(1). Although fewer than one in ten individuals eventually progress to active disease(2), tuberculosis is a leading cause of death from infectious disease worldwide(3). Despite intense efforts, immune factors that influence the infection outcomes remain poorly defined. Here we used integrated analyses of multiple cohorts to identify stage-specific host responses to Mtb infection. First, using high-dimensional mass cytometry analyses and functional assays of a cohort of South African adolescents, we show that latent tuberculosis is associated with enhanced cytotoxic responses, which are mostly mediated by CD16 (also known as FcγRIIIa) and natural killer cells, and continuous inflammation coupled with immune deviations in both T and B cell compartments. Next, using cell-type deconvolution of transcriptomic data from several cohorts of different ages, genetic backgrounds, geographical locations and infection stages, we show that although deviations in peripheral B and T cell compartments generally start at latency, they are heterogeneous across cohorts. However, an increase in the abundance of circulating natural killer cells in tuberculosis latency, with a corresponding decrease during active disease and a return to baseline levels upon clinical cure are features that are common to all cohorts. Furthermore, by analysing three longitudinal cohorts, we find that changes in peripheral levels of natural killer cells can inform disease progression and treatment responses, and inversely correlate with the inflammatory state of the lungs of patients with active tuberculosis. Together, our findings offer crucial insights into the underlying pathophysiology of tuberculosis latency, and identify factors that may influence infection outcomes.

For the ICU physician, the failure to consider, diagnose, and treat tuberculosis (TB) results in increased morbidity and mortality, and poses risks to both patients and health care providers. At present, the diagnosis of TB depends on the detection of either mycobacteria or mycobacterial products from clinical specimens. Given the risks posed to both the patient and health care providers by undiagnosed and/or untreated TB, the ability to diagnose TB rapidly in the ICU cannot be understated. In this regard, nucleic acid amplification tests provide relatively quick information about the presence of Mycobacterium tuberculosis (Mtb) DNA. If available, a blood-based test that would accurately identify persons with TB would be of use in the ICU. Currently available tests such as the T-Spot.TB or QuantiFERON-TB Gold In-Tube can discern infection with Mtb, but are not recommended for the ICU as they cannot rule out TB. In this review, we will discuss the increasing literature that would suggest that a blood-based diagnostic that reflects the host response to TB could be used to diagnose TB in the ICU.

Improved tuberculosis diagnostics and tools for monitoring treatment response are urgently needed. We developed a robust and simple, PCR-based host-blood transcriptomic signature, RISK6, for multiple applications: identifying individuals at risk of incident disease, as a screening test for subclinical or clinical tuberculosis, and for monitoring tuberculosis treatment. RISK6 utility was validated by blind prediction using quantitative real-time (qRT) PCR in seven independent cohorts.Prognostic performance significantly exceeded that of previous signatures discovered in the same cohort. Performance for diagnosing subclinical and clinical disease in HIV-uninfected and HIV-infected persons, assessed by area under the receiver-operating characteristic curve, exceeded 85%. As a screening test for tuberculosis, the sensitivity at 90% specificity met or approached the benchmarks set out in World Health Organization target product profiles for non-sputum-based tests. RISK6 scores correlated with lung immunopathology activity, measured by positron emission tomography, and tracked treatment response, demonstrating utility as treatment response biomarker, while predicting treatment failure prior to treatment initiation. Performance of the test in capillary blood samples collected by finger-prick was noninferior to venous blood collected in PAXgene tubes. These results support incorporation of RISK6 into rapid, capillary blood-based point-of-care PCR devices for prospective assessment in field studies.

The evaluation of new tuberculosis (TB) therapies is limited by the paucity of biomarkers to monitor treatment response. Previous work detected an uncharacterized urine metabolite with a molecular mass of 874.3547 Da that showed promise as a biomarker for successful TB treatment. Using mass spectrometry combined with enzymatic digestions, the metabolite was structurally characterized as a seryl-leucine core 1 O-glycosylated peptide (SLC1G) of human origin. Examination of SLC1G in urine revealed a significant abundance increase in individuals with active TB versus their household contacts and healthy controls. Moreover, differential decreases in SLC1G levels were observed by week one in TB patients during successful treatment versus those that failed treatment. The SLC1G levels were also associated with clinical parameters used to measure bacterial burden (GeneXpert) and inflammation (positron emission tomography-computed tomography (PET-CT)). These results demonstrate the importance of metabolite identification and provide strong evidence for applying SLC1G as a biomarker of TB treatment response.