Sarcoidosis is a granulomatous systemic inflammatory disease predominantly affecting the lungs. It shares histopathological, clinical, and immune features with tuberculosis (TB). There are currently no diagnostic tests to formally identify sarcoidosis; instead, there is a need first to rule out the presence of other diseases, including TB. We hypothesized that Mycobacterium tuberculosis (Mtb)-specific immune signatures differ between sarcoidosis and TB. We characterized T-cell and monocyte signatures after Mtb antigen in vitro stimulation in the blood of patients with sarcoidosis compared to patients with TB disease and Mtb-sensitized and nonsensitized healthy controls using flow cytometry and transcriptomics on bulk PBMCs and sorted CD4 memory T cells. We found that sarcoidosis was associated with (1) a marked reduction in frequencies of antigen-reactive T cells in response to both Mtb peptides and Mtb lysate, (2) increased frequencies of monocytes, and (3) increased expression of monocyte-associated phagocytic genes compared to TB disease and Mtb-sensitized and nonsensitized healthy cohorts. A combination of Mtb peptide-specific T-cell and monocyte gene or flow cytometry signatures in Mtb peptide-stimulated PBMCs distinguished sarcoidosis from TB disease with high accuracy (area under the curve [AUC] = 0.91 and 0.96 for gene and flow cytometry signatures, respectively) and also distinguished sarcoidosis from Mtb-sensitized and nonsensitized healthy controls combined (AUC = 0.91 and 0.90 for gene and flow cytometry signatures, respectively). These findings highlight biological features that effectively distinguish sarcoidosis from TB and healthy populations and can be considered for the development of an optimized diagnostic method for sarcoidosis.

BACKGROUND:Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death from a single infectious agent worldwide. Exposure to Mtb results in diverse outcomes: bacterial clearance, latent infection, asymptomatic, or symptomatic TB. Current diagnostic tools cannot reliably distinguish these outcomes. Our previous studies identified Mtb proteins in extracellular vesicles (EVs) from TB patients' serum, suggesting their potential as biomarkers. Here, we aimed to discover Mtb proteins and peptides in serum EVs across TB stages, focusing on asymptomatic individuals. METHODS:Serum was obtained from healthy, HIV-negative South African adult volunteers enrolled in a TB risk study. Based on patients' outcomes, samples were classified as prevalent, incident (controls that progressed to TB), activated TB, or community controls. EVs were isolated using ExoQuick™, followed by protein digestion and mass spectrometry (MS) analysis. Data-independent acquisition (DIA) with five different data analysis strategies, and two data-dependent (DDA) methods were used to identify Mtb proteins. RESULTS:Our DIA analysis using ion-mobility and spectral libraries enriched with Mtb-MS data revealed 19 Mtb proteins. Rv2997 was significantly higher at baseline in individuals who were initially TB-negative (incident) but later became bacteriologically positive, asymptomatic-TB (activated). HspX, GroEL2, and GroES, together with a MtrB peptide were significantly different between controls and asymptomatic-TB cases. DDA approaches did not resolve additional Mtb proteins. CONCLUSIONS:Quantitative DIA analysis discovered Mtb proteins/peptides in serum-derived EVs that were differentially abundant in individuals with early, asymptomatic TB. These findings highlight their potential as biomarkers and provide insight into host-pathogen interactions during subclinical infection.

BACKGROUND:Diagnostic performance of tongue swab Mycobacterium tuberculosis PCR has been evaluated for facility-based triage of symptomatic tuberculosis (TB). It is unknown whether tongue swab performance differs for detection of asymptomatic TB in community-based screening. METHODS:Tongue swabs were collected from adult household contacts of TB patients (HHC Cohort), and symptomatic adults presenting to clinics with presumptive TB (Clinic Cohort), at eight South African sites. TB Cases were defined by positive sputum Xpert Ultra or liquid culture, performed in all participants; and matched ~1:3 (HHC Cohort) or ~1:2 (Clinic Cohort) to Controls without TB. Tongue swabs in both cohorts were tested by high-volume qPCR; and in the Clinic Cohort, also by sequence-specific magnetic capture (SSMaC) with qPCR. RESULTS:The Clinic Cohort included 217 TB Cases (100% symptomatic) and 437 Controls. The HHC Cohort included 44 TB Cases (84.1% asymptomatic) and 136 Controls. In the Clinic Cohort, sensitivity of SSMaC with qPCR was 73.2% (specificity 94.6%), but not significantly higher than high-volume qPCR (63.8%; p = 0.14) (specificity 94.4%). Sensitivity of high-volume qPCR in the Clinic Cohort (63.8%) was significantly higher than the HHC Cohort (34.1%; p = 0.0007) (specificity 91.9%). Among HHC, high-volume qPCR sensitivity was 35.1% for asymptomatic TB; 52.2% for TB with abnormal CXR; and 100% for TB with High sputum Xpert Ultra grade. CONCLUSIONS:Sensitivity of tongue swab high-volume qPCR for community-based, household screening for asymptomatic TB was low, approximately half that of facility-based triage for symptomatic TB, but increased with radiographic severity and sputum bacillary load.

Abstract Introduction Mycobacterium tuberculosis (Mtb) remains a leading cause of death worldwide. IFNγ Release Assay (IGRA) is widely used to diagnose Mtb infection by measuring the IFNγ response to Mtb antigens. However, healthy IGRA- individuals with high Mtb exposure (“resisters”) have shown evidence of infection and make Mtb-specific responses that differ from IGRA+ individuals. Human CD4 T cells are critical in controlling Mtb. IFNγ-expressing Th1 cells are largely believed to be the major protective subset. Resisters’ negative response to IGRA suggests alternative protective mechanisms of CD4 T cells. This study aims to profile the antigen-specific CD4+ T cell responses in Mtb-exposed IGRA- individuals. Methods We performed TCR sequencing on Ugandan resisters and IGRA+ individuals. TCR repertoires were analyzed with GLIPH3 algorithm we recently developed to identify resister-specific TCRs. Mtb antigenic ligands were discovered by a new T cell epitope discovery platform. Antigen-specific CD4+ T cells were then isolated using peptide-MHC multimers covering the discovered antigenic peptide, and characterized by single-cell multi-omics and Flow Cytometry. Results We identified 24 TCR specificity groups uniquely enriched in resisters. Two ligand peptides were decoded from Mtb antigens Rv2140c and ESAT6. In a parallel South African cohort, in IGRA- individuals, we detected T cell responses to ESAT6, the antigen used in IGRA test, and a robust response to Rv2140c. Notably, Rv2140c-specific CD4 T cells were predominantly follicular helper cells (Tfh), which correlated with protection from Mtb in mice, whereas IGRA+ individuals showed primarily Th1 responses. Conclusion We identified a novel Mtb antigen associated with protection, highlighting its potential as vaccine candidate. ESAT6-specific responses in IGRA- individuals confirmed underlying Mtb infection, indicating the limitations of IGRA tests. The predominance of Tfh cells reveals new protective human T cell mechanism against Mtb beyond the classic Th1 response. Funding Source Bill & Melinda Gates Foundation Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

The SHINE trial demonstrated that 4 months of treatment was non-inferior to 6 months for children with non-severe tuberculosis drug-susceptible (DS-TB) as assessed on chest radiograph. However, the optimal duration likely varies between individuals. We evaluated blood transcriptomic signatures as non-sputum biomarkers of treatment response and explored their potential to inform individualised treatment duration in children. This sub-study included children (<16 years) with non-severe TB enrolled at a trial site in Cape Town, South Africa. Participants were classified as confirmed, unconfirmed, or unlikely TB by a blinded endpoint review committee. Whole blood was collected in PAXgene tubes at enrolment (baseline), week 2, week 8, and at the End of Treatment (EoT; week 16 or 24). We measured 88 gene transcripts by microfluidic RT-qPCR, evaluated 20 published transcriptomic signatures, and used a feature selection algorithm to derive a novel treatment-monitoring signature which was evaluated in this cohort. We then evaluated this in an independent validation cohort of Indian children. Among 198 children (median 3.4 years, range 0.4-14.8), 37 (18.7%) had confirmed, 129 (65.2%) unconfirmed, and 32 (16.2%) unlikely TB. Baseline signature scores were the highest in children with confirmed TB and increased with radiographic severity. Scores declined within 2 weeks, with minimal change thereafter. Of the literature derived signatures, the Kaforou22 signature best differentiated baseline from EoT in confirmed TB (AUC 0.85, 95%CI 0.76-0.94) TB. We then discovered a novel 4-gene signature (FCGR1C, FLVCR2, LTF, and CDKN1C) that best differentiated baseline from EoT with an AUC of 0.89 (95%CI 0.81-0.97), with an AUC of 0.81 (95%CI 0.64-0.98) in an independent paediatric dataset. Blood transcriptomic signatures show promise as non-sputum tools for monitoring treatment response in children and may support biomarker-guided individualized TB treatment.

Abstract Introduction Communication between immune cells through direct contact is a critical feature of immune responses. Our previous work has highlighted that a significant fraction of immune cell doublets detected in non-imaging flow cytometry are not technical artifacts, and instead hold biological relevance. In particular, using non-imaging FACS (fluorescence-activated cell sorting), we have recently shown that circulating T cell-monocyte complexes during infection hold transcriptomic signatures of active immune interactions. However, conventional non-imaging FACS lacks the spatial resolution necessary to characterize doublets accurately. Methods In this study, we employed the recently commercialized FACSDiscoverTM S8 spectral imaging cell sorter to characterize different phenotypes of circulating T cell-monocyte complexes in a cohort of patients with tuberculosis. Using various combinations of imaging parameters, we found that T cell-monocyte doublets can be classified as either synaptic or coincidental. Synaptic doublets were defined by high spatial overlap between the two cells forming complexes, representing true biological conjugates. In contrast, coincidental doublets were non-interacting cells but in close proximity during acquisition, thus representing technical artifacts rather than biological interactions. For each patient, synaptic and coincidental T cell-monocyte doublets were sorted, resulting in physical separation of the two cells forming the doublet, and processed for single-cell droplet sequencing. Results Sorting and single-cell sequencing analysis confirmed that synaptic doublets carried unique biological gene signatures associated with high metabolic activity and immune activation, while coincidental doublets resembled singlet T cells and monocytes. Conclusion Thus, image-based cell sorting provides unprecedented granularity in the study of immune doublets and enrichment for biological doublets over technical artifacts. Funding Source The Tullie and Rickey Families SPARK Awards for Innovations in Immunology, National Institute of Allergy and Infectious Diseases, Human Immunology Project Consortium, The Conrad Prebys Foundation Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

RATIONALE:Non-sputum biomarkers to monitor tuberculosis treatment and predict poor outcomes are lacking. OBJECTIVES:To evaluate host-blood transcriptomic signatures for treatment monitoring and prognosis of death, treatment failure, and recurrence in adults with pulmonary tuberculosis. METHODS:Adults with culture-confirmed, drug-susceptible pulmonary tuberculosis were enrolled at 5 Brazilian sites. Whole-blood PAXgene samples were collected at baseline, month 2 (M2), and end of treatment (EoT). Treatment failure was defined as sputum culture positivity at month 5 or later. Participants were followed for 24 months from treatment initiation for clinical or microbiological tuberculosis recurrence. Unfavorable outcomes were matched ∼1:3 to recurrence-free cure. Twenty-two published blood transcriptomic signatures were measured by microfluidic RT-qPCR and benchmarked against the WHO Target Product Profile (TPP) criteria. MEASUREMENTS AND MAIN RESULTS:We matched 263 participants with recurrence-free cure to 33 with treatment failure, 24 who died (tuberculosis/unknown cause), and 9 with recurrence. Signature scores generally declined from baseline to EoT. Multiple signatures measured at baseline and M2 predicted recurrence (AUC range 0.71-0.91), with waning performance when measured at EoT (AUC range 0.42-0.89). Against the WHO TPP, 2/22 signatures met minimum criteria at baseline, 13/22 at M2, and none at EoT. Prediction of treatment failure was poor across timepoints (AUC < 0.70). In contrast, several signatures measured at baseline predicted death during treatment or follow-up (AUC ≥ 0.80). CONCLUSIONS:Blood transcriptomic signatures tracked treatment response and predicted recurrence and death, meeting WHO TPP benchmarks at baseline and M2. These findings support prospective, biomarker-guided trials to individualize tuberculosis therapy-shortening regimens for early responders and intensifying care for high-risk patients.

Circulating cell-cell complexes (doublets) provide critical insights into in vivo immune interactions. Here, we leverage high-dimensional imaging spectral cytometry to systematically map the landscape of immune doublets in human peripheral blood. By integrating morphometric imaging features with a 22-color spectral panel, we established a gating workflow that cleanly segregates genuine, physically interacting complexes from coincidental transits and platelet contaminants, and resolved 12 highly pure homotypic and heterotypic immune doublet configurations in peripheral blood mononuclear cells (PBMC). At steady state, each doublet population displayed unique abundance, affinity and surface phenotypic profiles relative to circulating singlets. Principal component analysis (PCA) of quantitative imaging features further revealed lineage-specific topologies distinguishing singlet cells, homotypic doublets, and heterotypic pairs. Finally, comparative cohort analysis demonstrated that while doublet frequencies, affinities and surface phenotypes remain comparable between healthy donors and patients with tuberculosis (TB) disease, imaging-derived features uncovered disease-specific alterations in doublets morphology. Specifically, high-dimensional clustering using imaging parameters showed that TB disease significantly enriches for a tightly synapsed T cell-monocyte cluster characterized by high CD3-CD14 pixel correlation. Collectively, this study establishes a robust framework for mapping circulating cell-cell networks and demonstrates the superior sensitivity of imaging features over conventional fluorescence for capturing functional immune interactions in health and disease.

Assigning antigen specificity to T cell receptor (TCR) sequences is challenging due to the TCR repertoire's diversity and the complexity of TCR-antigen recognition. We developed the peptide-driven identification of TCRs (PDI-TCR) assay that combines in vitro expansion of cells with peptide pools, bulk TCR sequencing, and statistical analysis to identify antigen-specific TCRs from human blood. A key feature of PDI-TCR is the ability to distinguish true antigen-specific TCR clonotypes from TCRs associated with unspecific bystander activation by comparing responses to nonoverlapping peptide pools. We applied PDI-TCR to tuberculosis (TB) patients, sampling blood at diagnosis and throughout treatment, and Mycobacterium tuberculosis (Mtb)-sensitized healthy individuals (IGRA+). We identified hundreds of Mtb-specific TCRs, as well as unspecific TCRs, and characterized their phenotype in each cohort by single-cell RNA sequencing ex vivo. Mtb-specific T cells were highly diverse, with short-lived effector phenotypes only present in TB at diagnosis, while memory phenotypes were maintained through treatment. In contrast, unspecific expanded T cells were more clonally restricted, had a cytotoxic phenotype, and were maintained throughout treatment. While the PDI-TCR parameters used in this study are specific to Mtb, the underlying approach is broadly applicable to the study of antigen-specific T cells and can be adapted as needed for other antigen systems. Thus, PDI-TCR is a powerful tool for identifying antigen-specific TCRs and enables direct ex vivo identification and monitoring of antigen-specific T cells.

Background:More than half of tuberculosis (TB) detected by community prevalence surveys is classified as asymptomatic. We evaluated yield of symptom and chest radiograph (CXR) screening of TB-exposed household contacts (HHC) in South Africa. Methods:Adult volunteers (≥18 years) with household exposure to pulmonary TB patients were enrolled at three sites. Systematic screening of TB symptoms (any duration), CXR (any abnormality), and sputum microscopy, Xpert Ultra, and liquid culture were performed. Serum C-reactive protein (CRP) was measured by multiplex bead array. Prevalent TB was microbiologically-confirmed (Xpert Ultra or culture). Symptomatic and asymptomatic TB were defined as prevalent TB with and without reported symptoms, respectively. Results:Between March 2021 - December 2022, 979 HHC were enrolled; 185 (18.9%) living with HIV and 187 (19.1%) with previous TB. Prevalent TB occurred in 51 (5.2%) and was asymptomatic in 42/51 (82.4%). Only 13/42 (31.0%) asymptomatic TB cases were smear-positive [8/13 (61.5%) graded scanty or 1+]. CRP did not discriminate healthy HHC from those with asymptomatic TB (AUC 0.60; 95%CI 0.47-0.73). An abnormal CXR was observed in 23/41 asymptomatic (sensitivity 56.1%, 95%CI 41.0-70.1%) versus 8/9 symptomatic (sensitivity 88.9%, 95%CI 56.5-98.0%) TB cases. Sensitivity of CXR in combination with symptom screening was 64.0% (32/50, 95%CI 50.1-75.9%) for all prevalent TB. Conclusions:More than 80% of confirmed TB cases among HHC were asymptomatic. CXR screening missed more than 40% of these asymptomatic cases. Community prevalence surveys reliant on symptom- and CXR-based approaches may significantly underestimate the prevalence of asymptomatic TB in endemic countries. Funding:Supported by RePORT South Africa through funding from the U.S. National Institutes of Health, CRDF Global, and the South African Medical Research Council. RESEARCH IN CONTEXT:Evidence before this study: World Health Organisation (WHO) guidelines for systematic tuberculosis (TB) screening recommend symptom screening and chest radiography (CXR), based on a Cochrane meta-analysis reporting 70.6% sensitivity (any TB symptom) and 94.7% sensitivity (any CXR abnormality) for bacteriologically-confirmed pulmonary TB. National TB prevalence surveys rely on a positive symptom screen or abnormal CXR to trigger diagnostic sputum testing. This approach to community screening would, by definition, miss asymptomatic TB cases without CXR evidence of disease. We reviewed the reference list of the aforementioned meta-analysis for active case-finding studies of adolescents and adults aged 15 years and older in community and contact-tracing settings. We performed forward citation-tracking and searched reference lists, including studies published in English between Jan 1, 1980, and November 1, 2024. We excluded studies that included children <15 years; or that exclusively enrolled people with additional risk factors (HIV; diabetes; latent TB infection; prior TB). We found 28 studies that performed universal sputum testing for bacteriologically-confirmed pulmonary TB and reported 51.8% (95%CI 49.9-53.7%; I 2 = 89.2%) pooled sensitivity for symptom screening (any symptom; 24 studies, 2,969 TB cases) and 62.4% (95%CI 59.3-65.3%; I 2 = 88.3%) pooled sensitivity for CXR (any abnormality; 10 studies, 1,123 TB cases). Only four studies (145 TB cases) reported accuracy of symptom screening in parallel with chest radiography (pooled sensitivity 67.3%, 95%CI 57.3-75.9%; I 2 = 87.1%), but these studies did not disaggregate symptomatic and asymptomatic disease. Added value of this study: We performed systematic screening using universal sputum microbiological testing of 978 household contacts of pulmonary TB patients in three South African communities and compared symptom (any duration) and CXR (any abnormality) screening approaches against a microbiological reference standard. We detected confirmed pulmonary TB in 5.2% of household contacts, and 82.4% of these TB cases reported no TB symptoms. Asymptomatic TB in household contacts was pauci-bacillary and associated with low serum CRP levels that were indistinguishable from healthy controls, but distinct from symptomatic TB in a comparator group of clinic attendees. Sensitivity of CXR screening for asymptomatic TB was only 56.1%; sensitivity of combined symptom and CXR screening for all TB was marginally higher at 64.0%.Implications of all the available evidence: Our findings from household contacts suggest that symptom- and CXR-based approaches are inadequate for community TB screening in South Africa and do not meet the WHO Target Product Profile for a TB screening test (minimum 90% sensitivity; 70% specificity). National TB Prevalence Surveys that omit universal sputum microbiological testing may significantly underestimate the prevalence of asymptomatic TB in high-burden countries.

RNA signatures and T cell activation responses to Mycobacterium tuberculosis (Mtb) have demonstrated good diagnostic and prognostic performance in adults. To further evaluate the implementation of these novel assays in children, we hypothesized that host genetic variation is associated with susceptibility to pediatric tuberculosis disease and blood biomarker expression. We used a case-control study with a 12–36-mo prospective observation period to examine susceptibility to tuberculosis in children aged 2 mo to 5 yr with household Mtb exposure in Worcester, South Africa. Low-pass whole-genome sequencing followed by imputation was completed for 219 cases and 207 controls. A GWAS was performed to assess for association between genetic variants and susceptibility to tuberculosis disease. Lead genetic variants were subsequently evaluated for their effect on BCG-induced innate and adaptive immune responses. Whole blood collected from an independent cohort of 189 BCG-vaccinated 10-week-old South African infants was stimulated with BCG or media and examined with flow cytometry to measure cell surface-marker and cytokine expression. We identified a genome-wide significant variant rs4600676 (p = 4.5e-08) associated with increased susceptibility to pediatric tuberculosis. rs4600676 maps to a noncoding region of DNA and is positionally nearest genes GPR45 and TGFBAP1, suggesting it may regulate cytokine signaling. We identified an additional 25 lead genetic variants across 24 risk loci mapping to 55 genes at a prespecified suggestive significance level (p < 1e-05) associated with susceptibility to pediatric tuberculosis. MAGMA gene-set analysis of these suggestive variants revealed enrichment for chemokine receptor binding (p = 6.2e-06) and IRAK4-TLR2/4 deficiency (p = 2.2e-05). Six lead variants were associated with BCG-induced immune responses (p < 0.05). Specifically, rs34925982 maps to the T cell–inhibiting gene ILDR2, is associated with decreased BCG-induced T cell responses (p = 0.007), and increased susceptibility to childhood tuberculosis (p = 4.1e-06). We identified a genome-wide significant variant associated with increased susceptibility to pediatric tuberculosis as well as an additional 26 variants suggestive of an association with tuberculosis susceptibility. These findings identify potential immunoregulatory mechanisms involved in host responses to tuberculosis that can be leveraged for therapeutic intervention. Future work will associate genetic variants with Mtb-induced RNA signatures and T cell activation responses to understand how performance of these assays may vary across populations.

Although immune responses to bacillus Calmette-Guerin (BCG)-vaccination and susceptibility to pediatric tuberculosis (TB) vary across individuals, the underlying cellular mechanism regulating this heterogeneity is poorly understood. We used a nested case-control study with a 2-yr prospective observation period to examine whether genetic variation is associated with BCG-induced innate immune responses and susceptibility to pediatric TB (N = 134 cases, 516 controls) in BCG-vaccinated infants. Whole blood collected at 10 wk of age from 189 control infants was stimulated with BCG or media and examined with flow cytometry to measure BCG-induced PDL1, CD40, and cytokine expression in myeloid (mDC) and plasmacytoid (pDC) dendritic cells, monocytes, and neutrophils. We used a cellular and clinical GWAS to assess for associations between genetic variants, BCG-induced innate immune responses, and susceptibility to TB. We identified 11 lead genetic variants at genome-wide level significance associated with BCG-induced cytokine and surface expression markers including PDL1 (5 pDCs, 3 mDCs, 1 monocytes), CD40 (1 mDCs), and IL-6 (1 monocytes). An IGLL1 variant (rs2096522) was associated with mDC CD40 expression (P = 1.6e−08) and was also discovered as a significant variant using a gene-based method. In the clinical GWAS, we identified 39 lead variants mapping to 74 genes suggestive of an association with susceptibility to pediatric TB (P < 1e−05), but no variant reached genome-wide significance. One clinical lead variant in the PDE8A region (rs1023844, P = 9.6e−07) was also an eQTL and associated with BCG-induced monocyte PDL1 expression. In summary, we identified genetic variants associated with heterogeneity in infant BCG-induced innate immune responses with potential immunoregulatory mechanisms.

BACKGROUND:Translation of blood RNA signatures might be accelerated by identifying biomarkers composed of the minimum number of gene transcripts. We aimed to test the hypothesis that single-gene transcripts provide similar accuracy for detection of subclinical tuberculosis to multi-gene signatures and benchmark their accuracy and clinical utility against interferon-γ release assays (IGRAs). METHODS:For this individual participant data meta-analysis, we searched PubMed from database inception to June 10, 2024, using terms for "tuberculosis", "subclinical", and "RNA" to identify studies in which participants underwent whole-blood RNA sampling with at least 12 months of follow-up for development of clinical tuberculosis. We performed a one-stage individual participant data meta-analysis to compare the accuracy of multi-gene signatures against single-gene transcripts to discriminate individuals with subclinical tuberculosis-defined as asymptomatic prevalent or incident tuberculosis (diagnosed ≥21 days from enrolment, irrespective of symptoms) over a 12-month interval-from individuals who remained disease free. We performed decision curve analysis to evaluate the net benefit of using single-gene transcripts and IGRAs, alone or in combination, to stratify preventive treatment compared with strategies of treating all or no individuals. FINDINGS:276 articles were identified in the search; of these, seven met the eligibility criteria and all had IPD available. We evaluated 80 single-genes and eight multi-gene signatures in a pooled analysis of four RNA sequencing and three quantitative PCR datasets, comprising 6544 total samples and including 283 samples from 214 individuals with subclinical tuberculosis. Distributions of transcript and signature Z scores after standardisation were similar and there was little heterogeneity between datasets. Five single-gene transcripts (BATF2, FCGR1A/B, ANKRD22, GBP2, and SERPING1) had equivalent areas under the receiver operating characteristic curves (0·75 [95% CI 0·71-0·79] to 0·77 [0·73-0·81]) to the best-performing multi-gene signature over 12 months, but none met the WHO minimum target product profile (TPP) for a tuberculosis progression test. IGRAs approximated the TPP in low-burden settings but showed much lower specificity in high-burden settings (74% [95% CI 72-76] vs 32% [30-35]). By contrast, sensitivity (67% [47-82] in high-burden settings vs 78% [67-86] in low-burden settings) and specificity (72% [70-74] vs 67% [64-69]) of the best-performing single-gene transcript was similar across settings. Decision curve analysis showed that in high-burden settings, stratifying preventive treatment using single-gene transcripts had greater net benefit than using IGRAs, which offered little net benefit over treating all individuals. In low-burden settings, IGRAs offered greater net benefit than single-gene transcripts to stratify treatment, but combining both tests provided the highest net benefit for tuberculosis programmes aiming to treat fewer than 50 people to prevent a single case. INTERPRETATION:Single-gene transcripts are equivalent to multi-gene signatures for detection of subclinical tuberculosis, with consistent performance across settings. Single-gene transcripts show potential clinical utility to stratify preventive treatment, particularly when used in combination with IGRAs in low-burden settings. FUNDING:National Institute for Health Research, Wellcome Trust.

The effects of SARS-CoV-2 vaccination in children with previous multisystem inflammatory syndrome (MIS-C) are not well understood. In this study, we aimed to assess immune responses to SARS-CoV-2 vaccination in children over the age of 12 years with previous MIS-C and compare them to healthy children. Three children with previous MIS-C and four healthy children received two doses of the BNT162b2 vaccine. Blood was collected before the first dose, one week after the first dose, one week after the second dose and three weeks after the second dose. All participants had detectable SARS-CoV-2 spike IgG before vaccination. Spike binding and neutralising antibody activity increased after the first vaccine dose with no differences between children with a history of MIS-C and healthy children. Serum inflammatory cytokines and whole blood immune gene profiles did not resemble acute MIS-C and there were no differences in these between the two groups at any timepoint. All participants gained a robust SARS-CoV-2-specific T cell response by three weeks after the second dose. A transient increase in SARS-CoV-2-specific CD4 T cells expressing TCR Vβ21.3, a non-specific T cell subset previously found to be enriched in patients with MIS-C, was demonstrated in two of the children with previous MIS-C but none of the healthy children. Together, these data demonstrate that vaccination is effective at boosting SARS-CoV-2-specific immune responses in a small group of children with previous MIS-C, and that it does not induce inflammatory cytokine or gene expression responses resembling acute MIS-C. Although larger-scale studies are needed to confirm these findings, the present evidence supports SARS-CoV-2 vaccination in children with previous MIS-C.

BACKGROUND:Non-sputum-based, point-of-care triage tests for pulmonary tuberculosis could enhance tuberculosis diagnostic programs. We assessed the diagnostic accuracy of 2 finger-stick blood tests: the Cepheid 3 gene host-response cartridge (Xpert-HR), which measures 3 host messenger RNA transcripts, and the 3-host protein multibiomarker test (MBT). METHODS:We performed a prospective diagnostic accuracy study of consecutive participants with symptoms compatible with pulmonary tuberculosis in The Gambia, South Africa, Uganda, and Vietnam. A composite reference standard for active pulmonary tuberculosis incorporated chest radiography, symptom resolution, and sputum microbiological test results. A training-test set approach was used to evaluate test cutoff specificities at 90% sensitivity. RESULTS:Between 1 November 2020 and 1 May 2023, we screened 1262 participants aged 12-70 years with cough lasting >2 weeks and another symptom suggestive of tuberculosis. Of those who were classifiable by reference tests, 1154 participants had evaluable Xpert-HR results and 961 had evaluable MBT results. Xpert-HR had an area under the receiver operating characteristic (AUROC) curve of 0.92 at a cutoff of -1.275 or below, with a sensitivity of 92.8%, specificity of 62.5%, positive predictive value of 47.9%, and negative predictive value of 95.9%. The MBT had an AUROC of 0.91 at a cutoff of ≥0.42, with a sensitivity of 91.4%, specificity of 73.2%, positive predictive value of 52.0%, and negative predictive value of 96.4%. CONCLUSIONS:Our results show that both Xpert-HR and the MBT are promising non-sputum-based point-of-care tests. The MBT met the World Health Organization target product profile for a triage test, which suggests it should be further developed.

BACKGROUND:In a previous phase 2 trial, bacille Calmette-Guérin (BCG) revaccination was not shown to provide protection from primary Mycobacterium tuberculosis infection but prevented sustained M. tuberculosis infection, defined by an initial conversion on a QuantiFERON-TB (QFT) test (an interferon-γ release assay) from negative to positive, followed by two additional positive QFT tests at 3 and 6 months after the initial conversion (a secondary end point). A vaccine efficacy of 45% (95% confidence interval [CI], 6 to 68) was observed. METHODS:We performed a phase 2b, double-blind, randomized, placebo-controlled trial to evaluate the efficacy of BCG revaccination, as compared with placebo, for the prevention of sustained QFT test conversion (primary end point) in QFT test-negative, human immunodeficiency virus (HIV)-negative adolescents. Adverse events were assessed in a secondary analysis, and immunogenicity was assessed in an exploratory analysis. Vaccine efficacy was evaluated in the modified intention-to-treat population, which included all the participants who had undergone randomization, received the BCG vaccine or placebo, and had a negative QFT test 10 weeks after receipt of BCG vaccine or placebo; the last criterion was added to exclude participants with M. tuberculosis infection around the time that the vaccine or placebo was administered. Hazard ratios and 95% confidence intervals were estimated from a stratified Cox proportional-hazards model. RESULTS:A total of 1836 participants underwent randomization; 918 received the BCG vaccine, and 917 received placebo. After a median 30 months of follow-up, a sustained QFT test conversion was observed in 62 of 871 participants in the BCG-vaccine group and 59 of 849 participants in the placebo group. The hazard ratio for a sustained QFT test conversion (BCG vaccine vs. placebo) was 1.04 (95% CI, 0.73 to 1.48), for a vaccine efficacy point estimate of -3.8% (95% CI, -48.3 to 27.4). Adverse events occurred more frequently in the BCG-vaccine group than in the placebo group, and most were due to injection-site reactions (pain, redness, swelling, and ulceration). BCG revaccination induced cytokine-positive type 1 helper CD4 T cells. CONCLUSIONS:BCG revaccination in QFT-test negative, HIV-negative adolescents did not provide protection from sustained M. tuberculosis infection. (Funded by the Gates Foundation; ClinicalTrials.gov number NCT04152161.).

The immune mechanisms induced by the Bacillus Calmette-Guérin (BCG) vaccine, and the subset of which that mediate protection against tuberculosis (TB), remain poorly understood. This is further complicated by difficulties in verifying vaccine-induced protection in humans. Although research in animal models, namely mice and nonhuman primates (NHPs), has begun to close this knowledge gap, discrepancies in the relative importance of biological pathways across species limit the utility of animal model-derived biological insights in humans. To address these challenges, we applied a systems modeling framework, Translatable Components Regression (TransCompR), to identify human blood transcriptional variability that could predict Mycobacterium tuberculosis challenge outcomes in BCG-vaccinated NHPs. These protection-associated pathways included both innate and adaptive immune activation mechanisms, along with signaling via type I IFNs and antimycobacterial Th cytokines. We further partially validated the associations between these mechanisms and protection in humans using publicly available microarray data collected from BCG-vaccinated infants who either developed TB or remained healthy during 2 years of follow-up. Overall, our work demonstrates how species translation modeling can leverage animal studies to generate hypotheses about the mechanisms that underlie human infectious disease and vaccination outcomes, which may be difficult or impossible to ascertain using human data alone.

Mycobacterium tuberculosis (Mtb) is the cause of tuberculosis that is usually transmitted through contact with TB patients. Several studies described “resisters” who exposed to Mtb for years but consistently negative for Mtb diagnosis, which assessed via IFNγ response to Mtb antigens ESAT6 and CFP10 (QFT test) and skin test. CD4 T cells are critical in combating Mtb. Delineating the T cell receptor (TCR) repertoire, antigen specificities, and phenotypes of CD4 T cells in “resisters” can uncover features of protective T cells against Mtb and guide vaccine development. However, analyzing T cells against Mtb is hindered by the vast diversity of human TCR repertoire, HLAs, and Mtb genome, and determining TCR antigen specificities has been challenging due to a lack of appropriate methods. To overcome the challenges, we used BD Rhapsody to acquire millions of TCRs of a well-described Uganda TB household cohort. Leveraging GLIPH3, an advanced TCR analysis algorithm, we identified TCR clusters uniquely enriched in “resisters”. To determine the antigen specificities of these TCRs, we developed an epitope screening system with a novel reporter T cell, which can display TCR candidates and report the recognition of TCR and peptide-MHC. With this system, we identified two epitopes in Rv2140c and ESAT6. By visiting QFT- PBMCs of Cape Town ACS cohort, we delineated unique Tfh phenotype in Rv2140c+ T cells with TGFβ expression. In contrast, the ESAT6+ T cells exhibited typical Th1 phenotype. Microbial, Parasitic, and Fungal Immunology (MPF)

BACKGROUND:Safer and more effective tuberculosis (TB) vaccines than Bacille Calmette Guérin (BCG) are needed. We evaluated the safety, reactogenicity, and immunogenicity of three dose levels of the live-attenuated Mycobacterium tuberculosis (Mtb) vaccine, MTBVAC, compared to BCG, in South African infants. METHODS:Healthy, HIV-unexposed, BCG-naïve infants were randomised to receive a single intradermal dose of BCG (2.5 × 105 CFU, n = 24) or MTBVAC (2.5 × 104, 2.5 × 105, or 2.5 × 106 CFU, each n = 25). Safety endpoints were solicited systemic, solicited injection site, and unsolicited adverse events (AE), and serious AE (SAE). Immunogenicity was measured using interferon-γ release assay (IGRA) and whole blood intracellular cytokine staining assay. Follow-up was 12 months post-vaccination. FINDINGS:Ninety-nine infants were enrolled between 18 February 2019 and 08 March 2021. Seventy-eight infants experienced reactogenicity AE (all mild except one grade 2 erythema). Induration, swelling, and erythema were more frequent as MTBVAC dose increased. All reactogenicity events were less frequent in infants receiving MTBVAC 2.5 × 105 CFU compared with BCG. Twelve infants (three BCG and nine MTBVAC recipients) experienced 14 vaccine-unrelated SAE, including one death due to bronchopneumonia (MTBVAC recipient). Eight infants were treated for unconfirmed pulmonary TB (four BCG and four MTBVAC 2.5 × 104 CFU recipients); one BCG recipient was treated for unconfirmed TB meningitis. MTBVAC was immunogenic at all 3 doses, inducing predominantly Th1-cytokine-expressing CD4 T cells, which peaked at Day 56. The 2.5 × 105 and 2.5 × 106 CFU MTBVAC doses induced similar response magnitudes and were more immunogenic than BCG. Day 56 IGRA conversion was observed in 61 (87.4%) infants receiving any MTBVAC dose, but only 28 (42.4%) remained positive by Day 365. INTERPRETATION:MTBVAC appeared safe, well-tolerated, and immunogenic at doses between 2.5 × 104 and 2.5 × 106 CFU in South African infants. The 2.5 × 105 CFU MTBVAC dose, being less reactogenic and more immunogenic than BCG, was selected for a multi-centre, phase 3 trial. FUNDING:This trial was funded by the European and Developing Countries Clinical Trials Partnership (EDCTP).