Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the TH1 and TH17 subsets of helper T cells and follicular helper T (TFH)-like cellular responses. A population of IRF4+ T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6fl/fl) in CD4+ T cells (CD4cre) resulted in reduction of TFH-like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize TFH-like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques.

Transcriptomic signatures of progression to TB disease among close contacts in Brazil.

BACKGROUND: Approximately 5% of people infected with Mycobacterium tuberculosis progress to tuberculosis (TB) disease without preventive therapy. There is a need for a prognostic test to identify those at highest risk of incident TB, so that therapy can be targeted. We evaluated host blood transcriptomic signatures for progression to TB disease. METHODS: Close contacts (≥4 hours exposure per week) of adult patients with culture-confirmed pulmonary TB were enrolled in Brazil. Investigation for incident, microbiologically-confirmed or clinically-diagnosed pulmonary or extra-pulmonary TB disease through 24 months of follow-up was symptom-triggered. Twenty previously validated blood TB transcriptomic signatures were measured at baseline by real-time quantitative PCR. Prognostic performance for incident TB was tested using receiver operating characteristic curve (ROC) analysis at 6, 9, 12, and 24 months of follow-up. RESULTS: Between June 2015 and June 2019, 1,854 close contacts were enrolled; Twenty-five progressed to incident TB, of whom 13 had microbiologically-confirmed disease. Baseline transcriptomic signature scores were measured in 1,789 close contacts. Prognostic performance for all signatures was best within 6 months of diagnosis. Seven signatures (Gliddon4, Suliman4, Roe3, Roe1, Penn-Nicholson6, Francisco2, and Rajan5) met the minimum World Health Organization target product profile (TPP) for a prognostic test through 6 months; three (Gliddon4, Rajan5, and Duffy9) through 9 months. None met the TPP threshold through 12 or more months of follow-up. CONCLUSIONS: Blood transcriptomic signatures may be useful for predicting TB risk within 9 months of measurement among TB-exposed contacts, to target preventive therapy administration.

Phospholipase C epsilon-1 (PLCƐ1) mediates macrophage activation and protection against tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infects up to a quarter of the world's population. Although immune responses can control Mtb infection, 5%-10% of infected individuals can progress to active TB disease (progressors). A myriad of host factors regulate disease progression in TB and a better understanding of immune correlates of protection and disease is pivotal for the development of new therapeutics. Comparison of human whole blood transcriptomic metadata with that of macaque TB progressors and Mtb-infected diversity outbred mice (DO) led to the identification of differentially regulated gene (DEG) signatures, associated with TB progression or control. The current study assessed the function of Phospholipase C epsilon (PLCƐ1), the top downregulated gene across species in TB progressors, using a gene-specific knockout mouse model of Mtb infection and in vitro Mtb-infected bone marrow-derived macrophages. PLCƐ1 gene expression was downregulated in TB progressors across species. PLCε1 deficiency in the mouse model resulted in increased susceptibility to Mtb infection, coincident accumulation of lung myeloid cells, and reduced ability to mount antibacterial responses. However, PLCε1 was not required for the activation and accumulation of T cells in mice. Our results suggest an important early role for PLCƐ1 in shaping innate immune response to TB and may represent a putative target for host-directed therapy.

Blood transcriptomic signatures for symptomatic TB in an African multi-cohort study.

BACKGROUND: Multiple host blood transcriptional signatures have been developed as non-sputum triage tests for tuberculosis (TB). We aimed to compare the diagnostic performance of 20 blood transcriptomic TB signatures for differentiating between symptomatic patients who have TB versus other respiratory diseases (ORD). METHODS: As part of a nested case-control study, individuals presenting with respiratory symptoms at primary health care clinics in Ethiopia, Malawi, Namibia, Uganda, South Africa, and The Gambia were enrolled. TB was diagnosed based on clinical, microbiological, and radiological findings. Transcriptomic signatures were measured in whole blood using microfluidic RT-qPCR. Diagnostic performance was benchmarked against the WHO Target Product Profile (TPP) for a non-sputum TB triage test. RESULTS: Among 541 participants, 158 had definite, microbiologically-confirmed TB, 32 had probable TB, while 389 participants had ORD. Nine signatures differentiated between ORD and TB with equivalent performance (Satproedprai7: area under the curve 0.83 [95% CI 0.79-0.87], Jacobsen3: 0.83 [0.79-0.86]; Suliman2: 0.82 [0.78-0.86]; Roe1: 0.82 [0.78-0.86]; Kaforou22: 0.82 [0.78-0.86]; Sambarey10: 0.81 [0.77-0.85]; Duffy9: 0.81 [0.76-0.86]; Gliddon3: 0.8 [0.75-0.85]; and Suliman4 0.79 [0.75-0.84]. Benchmarked against a 90% sensitivity, these signatures achieved specificities between 44% (95% CI 38-49) and 54% (49-59), not meeting the TPP criteria. Signature scores significantly varied by HIV status and country. In country-specific analyses several signatures, such as Satproedprai7 and Penn-Nicholson6, met the minimal TPP criteria for a triage test in Ethiopia, Malawi, and South Africa. CONCLUSION: No signatures met the TPP criteria in a pooled analysis of all countries, but several signatures met the minimum criteria for a non-sputum TB triage test in some countries.

Host transcriptomic signatures of tuberculosis can predict immune reconstitution inflammatory syndrome in HIV patients.

Immune reconstitution inflammatory syndrome (IRIS) can be a complication of antiretroviral therapy (ART) in patients with advanced HIV, but its pathogenesis is uncertain. In tuberculosis (TB) endemic countries, IRIS is often associated with mycobacterial infections or Bacille-Calmette-Guerin (BCG) vaccination in children. With no predictive or confirmatory tests at present, IRIS remains a diagnosis of exclusion. We tested whether RISK6 and Sweeney3, validated immune-based blood transcriptomic signatures for TB, could predict or diagnose IRIS in HIV+ children and adults. Transcripts were measured by RT-qPCR in BCG-vaccinated children and by microarray in HIV+ adults with TB including TB meningitis (TBM). Signature scores before ART initiation and up to IRIS diagnosis were compared between participants who did or did not develop IRIS. In children, RISK6 and Sweeney3 discriminated IRIS cases from non-IRIS controls before ART, and at diagnosis. In adults with TB, RISK6 discriminated IRIS cases from controls after half-week on ART and at TB-IRIS onset. In adults with TBM, only Sweeney3 discriminated IRIS cases from controls before ART, while both signatures distinguished cases from controls at TB-IRIS onset. Parsimonious whole blood transcriptomic signatures for TB showed potential to predict and diagnose IRIS in HIV+ children and adults.

Correction: Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

[This corrects the article DOI: 10.1371/journal.pmed.1002781.].

Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

BACKGROUND: A nonsputum blood test capable of predicting progression of healthy individuals to active tuberculosis (TB) before clinical symptoms manifest would allow targeted treatment to curb transmission. We aimed to develop a proteomic biomarker of risk of TB progression for ultimate translation into a point-of-care diagnostic. METHODS AND FINDINGS: Proteomic TB risk signatures were discovered in a longitudinal cohort of 6,363 Mycobacterium tuberculosis-infected, HIV-negative South African adolescents aged 12-18 years (68% female) who participated in the Adolescent Cohort Study (ACS) between July 6, 2005 and April 23, 2007, through either active (every 6 months) or passive follow-up over 2 years. Forty-six individuals developed microbiologically confirmed TB disease within 2 years of follow-up and were selected as progressors; 106 nonprogressors, who remained healthy, were matched to progressors. Over 3,000 human proteins were quantified in plasma with a highly multiplexed proteomic assay (SOMAscan). Three hundred sixty-one proteins of differential abundance between progressors and nonprogressors were identified. A 5-protein signature, TB Risk Model 5 (TRM5), was discovered in the ACS training set and verified by blind prediction in the ACS test set. Poor performance on samples 13-24 months before TB diagnosis motivated discovery of a second 3-protein signature, 3-protein pair-ratio (3PR) developed using an orthogonal strategy on the full ACS subcohort. Prognostic performance of both signatures was validated in an independent cohort of 1,948 HIV-negative household TB contacts from The Gambia (aged 15-60 years, 66% female), longitudinally followed up for 2 years between March 5, 2007 and October 21, 2010, sampled at baseline, month 6, and month 18. Amongst these contacts, 34 individuals progressed to microbiologically confirmed TB disease and were included as progressors, and 115 nonprogressors were included as controls. Prognostic performance of the TRM5 signature in the ACS training set was excellent within 6 months of TB diagnosis (area under the receiver operating characteristic curve [AUC] 0.96 [95% confidence interval, 0.93-0.99]) and 6-12 months (AUC 0.76 [0.65-0.87]) before TB diagnosis. TRM5 validated with an AUC of 0.66 (0.56-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. The 3PR signature yielded an AUC of 0.89 (0.84-0.95) within 6 months of TB diagnosis and 0.72 (0.64-0.81) 7-12 months before TB diagnosis in the entire South African discovery cohort and validated with an AUC of 0.65 (0.55-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. Signature validation may have been limited by a systematic shift in signal magnitudes generated by differences between the validation assay when compared to the discovery assay. Further validation, especially in cohorts from non-African countries, is necessary to determine how generalizable signature performance is. CONCLUSIONS: Both proteomic TB risk signatures predicted progression to incident TB within a year of diagnosis. To our knowledge, these are the first validated prognostic proteomic signatures. Neither meet the minimum criteria as defined in the WHO Target Product Profile for a progression test. More work is required to develop such a test for practical identification of individuals for investigation of incipient, subclinical, or active TB disease for appropriate treatment and care.

Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

Correction: Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

[This corrects the article DOI: 10.1371/journal.pgen.1005954.].

Inferring bona fide transfrags in RNA-Seq derived-transcriptome assemblies of non-model organisms.

BACKGROUND: De novo transcriptome assembly of short transcribed fragments (transfrags) produced from sequencing-by-synthesis technologies often results in redundant datasets with differing levels of unassembled, partially assembled or mis-assembled transcripts. Post-assembly processing intended to reduce redundancy typically involves reassembly or clustering of assembled sequences. However, these approaches are mostly based on common word heuristics and often create clusters of biologically unrelated sequences, resulting in loss of unique transfrags annotations and propagation of mis-assemblies. RESULTS: Here, we propose a structured framework that consists of a few steps in pipeline architecture for Inferring Functionally Relevant Assembly-derived Transcripts (IFRAT). IFRAT combines 1) removal of identical subsequences, 2) error tolerant CDS prediction, 3) identification of coding potential, and 4) complements BLAST with a multiple domain architecture annotation that reduces non-specific domain annotation. We demonstrate that independent of the assembler, IFRAT selects bona fide transfrags (with CDS and coding potential) from the transcriptome assembly of a model organism without relying on post-assembly clustering or reassembly. The robustness of IFRAT is inferred on RNA-Seq data of Neurospora crassa assembled using de Bruijn graph-based assemblers, in single (Trinity and Oases-25) and multiple (Oases-Merge and additive or pooled) k-mer modes. Single k-mer assemblies contained fewer transfrags compared to the multiple k-mer assemblies. However, Trinity identified a comparable number of predicted coding sequence and gene loci to Oases pooled assembly. IFRAT selects bona fide transfrags representing over 94% of cumulative BLAST-derived functional annotations of the unfiltered assemblies. Between 4-6% are lost when orphan transfrags are excluded and this represents only a tiny fraction of annotation derived from functional transference by sequence similarity. The median length of bona fide transfrags ranged from 1.5kb (Trinity) to 2kb (Oases), which is consistent with the average coding sequence length in fungi. The fraction of transfrags that could be associated with gene ontology terms ranged from 33-50%, which is also high for domain based annotation. We showed that unselected transfrags were mostly truncated and represent sequences from intronic, untranslated (5' and 3') regions and non-coding gene loci. CONCLUSIONS: IFRAT simplifies post-assembly processing providing a reference transcriptome enriched with functionally relevant assembly-derived transcripts for non-model organism.

Age and sex influence antibody profiles associated with tuberculosis progression.

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

Predictive performance of interferon-gamma release assays and the tuberculin skin test for incident tuberculosis: an individual participant data meta-analysis.

Background: Evidence on the comparative performance of purified protein derivative tuberculin skin tests (TST) and interferon-gamma release assays (IGRA) for predicting incident active tuberculosis (TB) remains conflicting. We conducted an individual participant data meta-analysis to directly compare the predictive performance for incident TB disease between TST and IGRA to inform policy. Methods: We searched Medline and Embase from 1 January 2002 to 4 September 2020, and studies that were included in previous systematic reviews. We included prospective longitudinal studies in which participants received both TST and IGRA and estimated performance as hazard ratios (HR) for the development of all diagnoses of TB in participants with dichotomised positive test results compared to negative results, using different thresholds of positivity for TST. Secondary analyses included an evaluation of the impact of background TB incidence. We also estimated the sensitivity and specificity for predicting TB. We explored heterogeneity through pre-defined sub-group analyses (e.g. country-level TB incidence). Publication bias was assessed using funnel plots and Egger's test. This review is registered with PROSPERO, CRD42020205667. Findings: We obtained data from 13 studies out of 40 that were considered eligible (N = 32,034 participants: 36% from countries with TB incidence rate ≥100 per 100,000 population). All reported data on TST and QuantiFERON Gold in-Tube (QFT-GIT). The point estimate for the TST was highest with higher cut-offs for positivity and particularly when stratified by bacillus Calmette-Guérin vaccine (BCG) status (15 mm if BCG vaccinated and 5 mm if not [TST5/15 mm]) at 2.88 (95% CI 1.69-4.90). The pooled HR for QFT-GIT was higher than for TST at 4.15 (95% CI 1.97-8.75). The difference was large in countries with TB incidence rate <100 per 100,000 population (HR 10.38, 95% CI 4.17-25.87 for QFT-GIT VS. HR 5.36, 95% CI 3.82-7.51 for TST5/15 mm) but much of this difference was driven by a single study (HR 5.13, 95% CI 3.58-7.35 for TST5/15 mm VS. 7.18, 95% CI 4.48-11.51 for QFT-GIT, when excluding the study, in which all 19 TB cases had positive QFT-GIT results). The comparative performance was similar in the higher burden countries (HR 1.61, 95% CI 1.23-2.10 for QFT-GIT VS. HR 1.72, 95% CI 0.98-3.01 for TST5/15 mm). The predictive performance of both tests was higher in countries with TB incidence rate <100 per 100,000 population. In the lower TB incidence countries, the specificity of TST (76% for TST5/15 mm) and QFT-GIT (74%) for predicting active TB approached the minimum World Health Organization target (≥75%), but the sensitivity was below the target of ≥75% (63% for TST5/15 mm and 65% for QFT-GIT). The absolute differences in positive and negative predictive values between TST15 mm and QFT-GIT were small (positive predictive values 2.74% VS. 2.46%; negative predictive values 99.42% VS. 99.52% in low-incidence countries). Egger's test did not show evidence of publication bias (0.74 for TST15 mm and p = 0.68 for QFT-GIT). Interpretation: IGRA appears to have higher predictive performance than the TST in low TB incidence countries, but the difference was driven by a single study. Any advantage in clinical performance may be small, given the numerically similar positive and negative predictive values. Both IGRA and TST had lower performance in countries with high TB incidence. Test choice should be contextual and made considering operational and likely clinical impact of test results. Funding: YH, IA, and MXR were supported by the National Institute for Health and Care Research (NIHR), United Kingdom (RP-PG-0217-20009). MQ was supported by the Medical Research Council [MC_UU_00004/07].

Clinical predictors of pulmonary tuberculosis among South African adults with HIV.

Background: Tuberculosis (TB) clinical prediction rules rely on presence of symptoms, however many undiagnosed cases in the community are asymptomatic. This study aimed to explore the utility of clinical factors in predicting TB among people with HIV not seeking care. Methods: Baseline data were analysed from an observational cohort of ambulant adults with HIV in South Africa. Participants were tested for Mycobacterium tuberculosis (Mtb) sensitisation (interferon-γ release assay, IGRA) and microbiologically-confirmed prevalent pulmonary TB disease at baseline, and actively surveilled for incident TB through 15 months. Multivariable LASSO regression with post-selection inference was used to test associations with Mtb sensitisation and TB disease. Findings: Between March 22, 2017, and May 15, 2018, 861 participants were enrolled; Among 851 participants included in the analysis, 94·5% were asymptomatic and 45·9% sensitised to Mtb. TB prevalence was 2·0% at baseline and incidence 2·3/100 person-years through 15 months follow-up. Study site was associated with baseline Mtb sensitisation (p < 0·001), prevalent (p < 0·001), and incident TB disease (p = 0·037). Independent of site, higher CD4 counts (per 50 cells/mm3, aOR 1·48, 95%CI 1·12-1·77, p = 0·006) were associated with increased IGRA positivity, and participants without TB disease (aOR 0·80, 95%CI 0·69-0·94, p = 0·006) had reduced IGRA positivity; no variables were independently associated with prevalent TB. Mixed ancestry (aHR 1·49, 95%CI 1·30->1000, p = 0·005) and antiretroviral initiation (aHR 1·48, 95%CI 1·01-929·93, p = 0·023) were independently associated with incident TB. Models incorporating clinical features alone performed poorly in diagnosing prevalent (AUC 0·65, 95%CI 0·44-0·85) or predicting progression to incident (0·67, 0·46-0·88) TB. Interpretation: CD4 count and antiretroviral initiation, proxies for immune status and HIV stage, were associated with Mtb sensitisation and TB disease. Inadequate performance of clinical prediction models may reflect predominantly subclinical disease diagnosed in this setting and unmeasured local site factors affecting transmission and progression. Funding: The CORTIS-HR study was funded by the Bill & Melinda Gates Foundation (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 regulatory sponsor was the University of Cape Town.

Evaluation of a transcriptomic signature of tuberculosis risk in combination with an interferon gamma release assay: A diagnostic test accuracy study.

Background: We evaluated the diagnostic and prognostic performance of a transcriptomic signature of tuberculosis (TB) risk (RISK11) and QuantiFERON-TB Gold-plus (QFTPlus) as combination biomarkers of TB risk. Methods: Healthy South Africans who were HIV-negative aged 18-60 years with baseline RISK11 and QFTPlus results were evaluated in a prospective cohort study conducted between Sept 20, 2016 and Dec 20, 2019. Prevalence and incidence-rate ratios were used to evaluate risk of TB. Positive (LR+) and negative (LR-) likelihood ratios were used to compare individual tests versus Both-Positive (RISK11+/QFTPlus+) and Either-Positive (RISK11+ or QFTPlus+) combinations. Findings: Among 2912 participants, prevalent TB in RISK11+/QFTPlus+ participants was 13·3-fold (95% CI 4·2-42·7) higher than RISK11-/QFTPlus-; 2·4-fold (95% CI 1·2-4·8) higher than RISK11+/QFTPlus-; and 4·5-fold (95% CI 2·5-8·0) higher than RISK11-/QFTPlus+ participants. Risk of incident TB in RISK11+/QFTPlus+ participants was 8·3-fold (95% CI 2·5-27·0) higher than RISK11-/QFTPlus-; 2·5-fold (95% CI 1·0-6·6) higher than RISK11+/QFTPlus-; and 2·1-fold (95% CI 1·2-3·4) higher than RISK11-/QFTPlus+ participants, respectively. Compared to QFTPlus, the Both-Positive test combination increased diagnostic LR+ from 1·3 (95% CI 1·2-1·5) to 4·7 (95% CI 3·2-7·0), and prognostic LR+ from 1·4 (95% CI 1·2-1·5) to 2·8 (95% CI 1·5-5·1), but did not improve upon RISK11 alone. Compared with RISK11, the Either-Positive test combination decreased diagnostic LR- from 0·7 (95% CI 0·6-0·9) to 0·3 (95% CI 0·2-0·6), and prognostic LR- from 0·9 (95% CI 0·8-1·0) to 0·3 (0·1-0·7), but did not improve upon QFTPlus alone. Interpretation: Combining two tests such as RISK11 and QFTPlus, with discordant individual performance characteristics does not improve overall discriminatory performance, relative to the individual tests. Funding: Bill and Melinda Gates Foundation, South African Medical Research Council.

Host-Directed Targeting of LincRNA-MIR99AHG Suppresses Intracellular Growth of Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills 1.6 million people worldwide every year, and there is an urgent need for targeting host-pathogen interactions as a strategy to reduce mycobacterial resistance to current antimicrobials. Noncoding RNAs are emerging as important regulators of numerous biological processes and avenues for exploitation in host-directed therapeutics. Although long noncoding RNAs (lncRNAs) are abundantly expressed in immune cells, their functional role in gene regulation and bacterial infections remains understudied. In this study, we identify an immunoregulatory long intergenic noncoding RNA, lincRNA-MIR99AHG, which is upregulated in mouse and human macrophages upon IL-4/IL-13 stimulation and downregulated after clinical Mtb HN878 strain infection and in peripheral blood mononuclear cells from active TB patients. To evaluate the functional role of lincRNA-MIR99AHG, we used antisense locked nucleic acid (LNA) GapmeR-mediated antisense oligonucleotide (ASO) lncRNA knockdown experiments. Knockdown of lincRNA-MIR99AHG with ASOs significantly reduced intracellular Mtb growth in mouse and human macrophages and reduced pro-inflammatory cytokine production. In addition, in vivo treatment of mice with MIR99AHG ASOs reduced the mycobacterial burden in the lung and spleen. Furthermore, in macrophages, lincRNA-MIR99AHG is translocated to the nucleus and interacts with high affinity to hnRNPA2/B1 following IL-4/IL-13 stimulation and Mtb HN878 infection. Together, these findings identify lincRNA-MIR99AHG as a positive regulator of inflammation and macrophage polarization to promote Mtb growth and a possible target for adjunctive host-directed therapy against TB.

The effect of host factors on discriminatory performance of a transcriptomic signature of tuberculosis risk.

BACKGROUND: We aimed to understand host factors that affect discriminatory performance of a transcriptomic signature of tuberculosis risk (RISK11). METHODS: HIV-negative adults aged 18-60 years were evaluated in a prospective study of RISK11 and surveilled for tuberculosis through 15 months. Generalised linear models and receiver-operating characteristic (ROC) regression were used to estimate effect of host factors on RISK11 score (%marginal effect) and on discriminatory performance for tuberculosis disease (area under the curve, AUC), respectively. FINDINGS: Among 2923 participants including 74 prevalent and 56 incident tuberculosis cases, percentage marginal effects on RISK11 score were increased among those with prevalent tuberculosis (+18·90%, 95%CI 12·66-25·13), night sweats (+14·65%, 95%CI 5·39-23·91), incident tuberculosis (+7·29%, 95%CI 1·46-13·11), flu-like symptoms (+5·13%, 95%CI 1·58-8·68), and smoking history (+2·41%, 95%CI 0·89-3·93) than those without; and reduced in males (-6·68%, 95%CI -8·31- -5·04) and with every unit increase in BMI (-0·13%, 95%CI -0·25- -0·01). Adjustment for host factors affecting controls did not change RISK11 discriminatory performance. Cough was associated with 72·55% higher RISK11 score in prevalent tuberculosis cases. Stratification by cough improved diagnostic performance from AUC = 0·74 (95%CI 0·67-0·82) overall, to 0·97 (95%CI 0·90-1·00, p < 0·001) in cough-positive participants. Combining host factors with RISK11 improved prognostic performance, compared to RISK11 alone, (AUC = 0·76, 95%CI 0·69-0·83 versus 0·56, 95%CI 0·46-0·68, p < 0·001) over a 15-month predictive horizon. INTERPRETATION: Several host factors affected RISK11 score, but only adjustment for cough affected diagnostic performance. Combining host factors with RISK11 should be considered to improve prognostic performance. FUNDING: Bill and Melinda Gates Foundation, South African Medical Research Council.

Prospective multicentre head-to-head validation of host blood transcriptomic biomarkers for pulmonary tuberculosis by real-time PCR.

Background: Sensitive point-of-care screening tests are urgently needed to identify individuals at highest risk of tuberculosis. We prospectively tested performance of host-blood transcriptomic tuberculosis signatures. Methods: Adults without suspicion of tuberculosis were recruited from five endemic South African communities. Eight parsimonious host-blood transcriptomic tuberculosis signatures were measured by microfluidic RT-qPCR at enrolment. Upper respiratory swab specimens were tested with a multiplex bacterial-viral RT-qPCR panel in a subset of participants. Diagnostic and prognostic performance for microbiologically confirmed prevalent and incident pulmonary tuberculosis was tested in all participants at baseline and during active surveillance through 15 months follow-up, respectively. Results: Among 20,207 HIV-uninfected and 963 HIV-infected adults screened; 2923 and 861 were enroled. There were 61 HIV-uninfected (weighted prevalence 1.1%) and 10 HIV-infected (prevalence 1.2%) tuberculosis cases at baseline. Parsimonious signature diagnostic performance was superior among symptomatic (AUCs 0.85-0.98) as compared to asymptomatic (AUCs 0.61-0.78) HIV-uninfected participants. Thereafter, 24 HIV-uninfected and 9 HIV-infected participants progressed to incident tuberculosis (1.1 and 1.0 per 100 person-years, respectively). Among HIV-uninfected individuals, prognostic performance for incident tuberculosis occurring within 6-12 months was higher relative to 15 months. 1000 HIV-uninfected participants were tested for respiratory microorganisms and 413 HIV-infected for HIV plasma viral load; 7/8 signature scores were higher (p < 0.05) in participants with viral respiratory infections or detectable HIV viraemia than those without. Conclusions: Several parsimonious tuberculosis transcriptomic signatures met triage test targets among symptomatic participants, and incipient test targets within 6 months. However, the signatures were upregulated with viral infection and offered poor specificity for diagnosing sub-clinical tuberculosis.

Tuberculosis alters immune-metabolic pathways resulting in perturbed IL-1 responses.

Tuberculosis (TB) remains a major public health problem and we lack a comprehensive understanding of how Mycobacterium tuberculosis (M. tb) infection impacts host immune responses. We compared the induced immune response to TB antigen, BCG and IL-1ß stimulation between latently M. tb infected individuals (LTBI) and active TB patients. This revealed distinct responses between TB/LTBI at transcriptomic, proteomic and metabolomic levels. At baseline, we identified a novel immune-metabolic association between pregnane steroids, the PPARγ pathway and elevated plasma IL-1ra in TB. We observed dysregulated IL-1 responses after BCG stimulation in TB patients, with elevated IL-1ra responses being explained by upstream TNF differences. Additionally, distinct secretion of IL-1α/IL-1ß in LTBI/TB after BCG stimulation was associated with downstream differences in granzyme mediated cleavage. Finally, IL-1ß driven signalling was dramatically perturbed in TB disease but was completely restored after successful treatment. This study improves our knowledge of how immune responses are altered during TB disease, and may support the design of improved preventive and therapeutic tools, including host-directed strategies.

Host blood transcriptomic biomarkers of tuberculosis disease in people living with HIV: a systematic review protocol.

INTRODUCTION: Current tuberculosis triage and predictive tools offer poor accuracy and are ineffective for detecting asymptomatic disease in people living with HIV (PLHIV). Host tuberculosis transcriptomic biomarkers hold promise for diagnosing prevalent and predicting progression to incident tuberculosis and guiding further investigation, preventive therapy and follow-up. We aim to conduct a systematic review of performance of transcriptomic signatures of tuberculosis in PLHIV. METHODS AND ANALYSIS: We will search MEDLINE (PubMed), WOS Core Collection, Biological Abstracts, and SciELO Citation Index (Web of Science), Africa-Wide Information and General Science Abstracts (EBSCOhost), Scopus, and Cochrane Central Register of Controlled Trials databases for articles published in English between 1990 and 2020. Case-control, cross-sectional, cohort and randomised controlled studies evaluating performance of diagnostic and prognostic host-response transcriptomic signatures in PLHIV of all ages and settings will be included. Eligible studies will include PLHIV in signature test or validation cohorts, and use microbiological, clinical, or composite reference standards for pulmonary or extrapulmonary tuberculosis diagnosis. Study quality will be evaluated using the 'Quality Assessment of Diagnostic Accuracy Studies-2' tool and cumulative review evidence assessed using the 'Grading of Recommendations Assessment, Development and Evaluation' approach. Study selection, quality appraisal and data extraction will be performed independently by two reviewers. Study, cohort and signature characteristics of included studies will be tabulated, and a narrative synthesis of findings presented. Primary outcomes of interest, biomarker sensitivity and specificity with estimate precision, will be summarised in forest plots. Expected heterogeneity in signature characteristics, study settings, and study designs precludes meta-analysis and pooling of results. Review reporting will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses of Diagnostic Test Accuracy Studies guidelines. ETHICS AND DISSEMINATION: Formal ethics approval is not required as primary human participant data will not be collected. Results will be disseminated through peer-reviewed publication and conference presentation. PROSPERO REGISTRATION NUMBER: CRD42021224155.

Inflammatory Determinants of Differential Tuberculosis Risk in Pre-Adolescent Children and Young Adults.

The risk of progression from Mycobacterium tuberculosis (M.tb) infection to active tuberculosis (TB) disease varies markedly with age. TB disease is significantly less likely in pre-adolescent children above 4 years of age than in very young children or post-pubescent adolescents and young adults. We hypothesized that pro-inflammatory responses to M.tb in pre-adolescent children are either less pronounced or more regulated, than in young adults. Inflammatory and antimicrobial mediators, measured by microfluidic RT-qPCR and protein bead arrays, or by analyzing published microarray data from TB patients and controls, were compared in pre-adolescent children and adults. Multivariate analysis revealed that M.tb-uninfected 8-year-old children had lower levels of myeloid-associated pro-inflammatory mediators than uninfected 18-year-old young adults. Relative to uninfected children, those with M.tb-infection had higher levels of similar myeloid inflammatory responses. These inflammatory mediators were also expressed after in vitro stimulation of whole blood from uninfected children with live M.tb. Our findings suggest that myeloid inflammation is intrinsically lower in pre-pubescent children than in young adults. The lower or more regulated pro-inflammatory responses may play a role in the lower risk of TB disease in this age group.