The frequencies of IFNγ+IL2+TNFα+ PPD-specific CD4+CD45RO+ T-cells correlate with the magnitude of the QuantiFERON® gold in-tube response in a prospective study of healthy Indian adolescents

Background: QuantiFERON-TB Gold In-Tube (QFT) is an IFNγ-release assay used in the diagnosis of Mycobacterium tuberculosis (MTB) infection. The risk of TB progression increases with the magnitude of the MTB-specific IFNγ-response. QFT reversion, also associated with low Tuberculin Skin Test responses, may therefore represent a transient immune response with control of M. tuberculosis infection. However, studies at the single cell level have suggested that the quality (polyfunctionality) of the T-cell response is more important than the quantity of cytokines produced. Objective: To explore the quality and/or magnitude of mycobacteria-specific T-cell responses associated with QFT reversion and persistent QFT-positivity. Methods: Multi-color flowcytometry on prospectively collected peripheral blood mononuclear cells was applied to assess mycobacteria-specific T-cell responses in 42 QFT positive Indian adolescents of whom 21 became QFT negative (reverters) within one year. Ten QFT consistent negatives were also included as controls. Results: There was no difference in the qualitative PPD-specific CD4+ T-cell response between QFT consistent positives and reverters. However, compared with QFT consistent positives, reverters displayed lower absolute frequencies of polyfunctional (IFNγ+IL2+TNFα+) CD4+ T-cells at baseline, which were further reduced to the point where they were not different to QFT negative controls one year later. Moreover, absolute frequencies of these cells correlated well with the magnitude of the QFT-response. Conclusion: Whereas specific polyfunctional CD4+ T-cells have been suggested to protect against TB progression, our data do not support that higher relative or absolute frequencies of PPD-specific polyfunctional CD4+ T-cells in peripheral blood can explain the reduced risk of TB progression observed in QFT reverters. On the contrary, absolute frequencies of these cells correlated with the QFT-response, suggesting that this readout reflects antigenic load

Improving Assignments for Therapeutic and Prophylactic Treatment Within TB Households. A Potential for Immuno-Diagnosis?

Delays in diagnosis and treatment of pulmonary tuberculosis (TB) can lead to more severe disease and increased transmission. Contact investigation among household contacts (HHCs) of TB patients is crucial to ensure optimal outcomes. In the context of a prospective cohort study in Palamaner, Southern India, this study attempted to assess the potential of 27 different soluble immune markers to accurately assign HHCs for appropriate treatment. A multiplex bead assay was applied on QuantiFERON (QFT)-nil supernatants collected from 89 HHCs grouped by longitudinal QFT status; M. tuberculosis (Mtb) infected (QFT positive at baseline and follow-up, n = 30), recent QFT converters (QFT-negative at baseline, n = 27) and converted to QFT-positivity within 6 months of exposure (at follow-up, n = 24) and QFT consistent negatives (n = 32). The 29 TB index cases represented Active TB. Active TB cases and HHCs with Mtb infection produced significantly different levels of both pro-inflammatory (IFNγ, IL17, IL8, IP10, MIP-1α, MIP1β, and VEGF) and anti-inflammatory (IL9 and IL1RA) cytokines. We identified a 4-protein signature (bFGF, IFNγ, IL9, and IP10) that correctly classified HHCs with Mtb infection vs. Active TB with a specificity of 92.6%, suggesting that this 4-protein signature has the potential to assign HHCs for either full-length TB treatment or preventive TB treatment. We further identified a 4-protein signature (bFGF, GCSF, IFNγ, and IL1RA) that differentiated HHCs with Mtb infection from QFT consistent negatives with a specificity of 62.5%, but not satisfactory to safely assign HHCs to no preventive TB treatment. QFT conversion, reflecting new Mtb infection, induced an elevated median concentration in nearly two-thirds (19/27) of the analyzed soluble markers compared to the levels measured at baseline. Validation in other studies is warranted in order to establish the potential of the immune biosignatures for optimized TB case detection and assignment to therapeutic and preventive treatment of Mtb infected individuals.publishedVersio

Added value of IP-10 as a read-out of <em>Mycobacterium tuberculosis</em>:Specific immunity in young children

We have explored the added value of interferon-γ (IFNγ)–inducible protein 10 as a read-out of Mycobacterium tuberculosis–specific immunity in young Indian children, where the sensitivity of the IFNγ release assays for tuberculosis is poor. Reduced frequency of indeterminate results and an increased sensitivity for tuberculosis suggest a potential for fewer missed cases with a combined IFNγ/inducible protein 10 read-out in a 4th generation IFNγ release assays

A 2-Dose AERAS-402 Regimen Boosts CD8+ Polyfunctionality in HIV-Negative, BCG-Vaccinated Recipients

Despite the widespread use of BCG, tuberculosis (TB) remains a global threat. Existing vaccine candidates in clinical trials are designed to replace or boost BCG which does not provide satisfying long-term protection. AERAS-402 is a replication-deficient Ad35 vaccine encoding a fusion protein of the M. tuberculosis (Mtb) antigens 85A, 85B, and TB10.4. The present phase I trial assessed the safety and immunogenicity of AERAS-402 in participants living in India – a highly TB-endemic area. Healthy male participants aged 18–45 years with a negative QuantiFERON-TB Gold in-tube test (QFT) were recruited. Enrolled participants (n=12) were randomized 2:1 to receive two intramuscular injections of either AERAS-402 (3 x 1010 viral particles [vp]); (n=8) or placebo (n=4) on study days 0 and 28. Safety and immunogenicity parameters were evaluated for up to 182 days post the second injection. Immunogenicity was assessed by a flow cytometry-based intracellular cytokine staining (ICS) assay and transcriptional profiling. The latter was examined using dual-color-Reverse-Transcriptase-Multiplex-Ligation-dependent-Probe-Amplification (dc-RT MLPA) assay. AERAS-402 was well tolerated, and no vaccine-related serious adverse events were recorded. The vaccine-induced CD8+ T-cell responses were dominated by cells co-expressing IFN-γ, TNF-α, and IL-2 (“polyfunctional” cells) and were more robust than CD4+ T-cell responses. Five genes (CXCL10, GNLY, IFI35, IL1B and PTPRCv2) were differentially expressed between the AERAS-402-group and the placebo group, suggesting vaccine-induced responses. Further, compared to pre-vaccination, three genes (CLEC7A, PTPRCv1 and TAGAP) were consistently up-regulated following two doses of vaccination in the AERAS-402-group. No safety concerns were observed for AERAS-402 in healthy Indian adult males. The vaccine-induced predominantly polyfunctional CD8+ T cells in response to Ag85B, humoral immunity, and altered gene expression profiles in peripheral blood mononuclear cells (PBMCs) indicative of activation of various immunologically relevant biological pathways.publishedVersio

Host blood-based biosignatures for subclinical TB and incipient TB: A prospective study of adult TB household contacts in Southern India

A large proportion of the global tuberculosis (TB) burden is asymptomatic and not detectable by symptom-based screening, driving the TB epidemic through continued M. tuberculosis transmission. Currently, no validated tools exist to diagnose incipient and subclinical TB. Nested within a large prospective study in household contacts of pulmonary TB cases in Southern India, we assessed 35 incipient TB and 12 subclinical TB cases, along with corresponding household active TB cases (n=11), and household controls (n=39) using high throughput methods for transcriptional and protein profiling. We split the data into training and test sets and applied a support vector machine classifier followed by a Lasso regression model to identify signatures. The Lasso regression model identified an 11-gene signature (ABLIM2, C20orf197, CTC-543D15.3, CTD-2503O16.3, HLADRB3, METRNL, RAB11B-AS1, RP4-614C10.2, RNA5SP345, RSU1P1, and UACA) that distinguished subclinical TB from incipient TB with a very good discriminatory power by AUCs in both training and test sets. Further, we identified an 8-protein signature comprising b-FGF, IFNγ, IL1RA, IL7, IL12p70, IL13, PDGF-BB, and VEGF that differentiated subclinical TB from incipient TB with good and moderate discriminatory power by AUCs in the training and test sets, respectively. The identified 11-gene signature discriminated well between the distinct stages of the TB disease spectrum, with very good discriminatory power, suggesting it could be useful for predicting TB progression in household contacts. However, the high discriminatory power could partly be due to over-fitting, and validation in other studies is warranted to confirm the potential of the immune biosignatures for identifying subclinical TB.publishedVersio

Protein and transcriptional biomarker profiling may inform treatment strategies in lower respiratory tract infections by indicating bacterial–viral differentiation

Lower respiratory tract infections (LRTIs) remain a significant global cause of infectious disease-related mortality. Accurate discrimination between acute bacterial and viral LRTIs is crucial for optimal patient care, prevention of unnecessary antibiotic prescriptions, and resource allocation. Plasma samples from LRTI patients with bacterial (n = 36), viral (n = 27; excluding SARS-CoV-2), SARS-CoV-2 (n = 22), and mixed bacterial–viral (n = 38) etiology were analyzed for protein profiling. Whole-blood RNA samples from a subset of patients (bacterial, n = 8; viral, n = 8; and SARS-CoV-2, n = 8) were analyzed for transcriptional profiling. Lasso regression modeling identified a seven-protein signature (CRP, IL4, IL9, IP10, MIP1α, MIP1β, and TNFα) that discriminated between patients with bacterial (n = 36) vs viral (n = 27) infections with an area under the curve (AUC) of 0.98. When comparing patients with bacterial and mixed bacterial–viral infections (antibiotics clinically justified; n = 74) vs patients with viral and SARS-CoV-2 infections (antibiotics clinically not justified; n = 49), a 10-protein signature (CRP, bFGF, eotaxin, IFNγ, IL1β, IL7, IP10, MIP1α, MIP1β, and TNFα) with an AUC of 0.94 was identified. The transcriptional profiling analysis identified 232 differentially expressed genes distinguishing bacterial (n = 8) from viral and SARS-CoV-2 (n = 16) etiology. Protein–protein interaction enrichment analysis identified 20 genes that could be useful in the differentiation between bacterial and viral infections. Finally, we examined the performance of selected published gene signatures for bacterial–viral differentiation in our gene set, yielding promising results. Further validation of both protein and gene signatures in diverse clinical settings is warranted to establish their potential to guide the treatment of acute LRTIs.publishedVersio

Host blood RNA transcript and protein signatures for sputum-independent diagnostics of tuberculosis in adults

To achieve the ambitious targets for tuberculosis (TB) prevention, care, and control stated by the End TB Strategy, new health care strategies, diagnostic tools are warranted. Host-derived biosignatures are explored for their TB diagnostic potential in accordance with the WHO target product profiles (TPPs) for point-of-care (POC) testing. We aimed to identify sputum-independent TB diagnostic signatures in newly diagnosed adult pulmonary-TB (PTB) patients recruited in the context of a prospective household contact cohort study conducted in Andhra Pradesh, India. Whole-blood mRNA samples from 158 subjects (PTB, n = 109; age-matched household controls, n = 49) were examined by dual-color Reverse-Transcriptase Multiplex Ligation-dependent Probe-Amplification (dcRT-MLPA) for the expression of 198 pre-defined genes and a Mesoscale discovery assay for the concentration of 18 cytokines/chemokines in TB-antigen stimulated QuantiFERON supernatants. To identify signatures, we applied a two-step approach; in the first step, univariate filtering was used to identify and shortlist potentially predictive biomarkers; this step may be seen as removing redundant biomarkers. In the second step, a logistic regression approach was used such that group membership (PTB vs. household controls) became the binary response in a Lasso regression model. We identified an 11-gene signature that distinguished PTB from household controls with AUCs of ≥0.98 (95% CIs: 0.94–1.00), and a 4-protein signature (IFNγ, GMCSF, IL7 and IL15) that differentiated PTB from household controls with AUCs of ≥0.87 (95% CIs: 0.75–1.00), in our discovery cohort. Subsequently, we evaluated the performance of the 11-gene signature in two external validation data sets viz, an independent cohort at the Glenfield Hospital, University Hospitals of Leicester NHS Trust, Leicester, UK (GSE107994 data set), and the Catalysis treatment response cohort (GSE89403 data set) from South Africa. The 11-gene signature validated and distinguished PTB from healthy and asymptomatic M. tuberculosis infected household controls in the GSE107994 data set, with an AUC of 0.95 (95% CI: 0.91–0.98) and 0.94 (95% CI: 0.89–0.98). More interestingly in the GSE89403 data set, the 11-gene signature distinguished PTB from household controls and patients with other lung diseases with an AUC of 0.93 (95% CI: 0.87–0.99) and 0.73 (95% CI: 0.56–0.89). These criteria meet the WHO TTP benchmarks for a non–sputum-based triage test for TB diagnosis. We suggest that further validation is required before clinical implementation of the 11-gene signature we have identified markers will be possible.publishedVersio

Diagnostic accuracy of a host response test in suspected community-Acquired pneumonia during the COVID-19 era

Community-acquired pneumonia [CAP] is a leading cause of morbidity and mortality, often complicated by diagnostic uncertainty and antibiotic overuse. This study evaluated the MeMed BV® host-response test in adults with suspected CAP, using clinical management and molecular detection as reference standards. Among 744 patients presenting with suspected CAP at Haukeland University Hospital, Bergen, Norway (2019-2023), across three prospective studies, 453 were included in the present study. Patients were classified using: [1] clinical management [antibiotic timing/duration], and [2] molecular detection via BioFire® FilmArray Pneumonia Plus panel on lower respiratory tract samples. MeMed BV® testing was performed retrospectively on stored serum/plasma, categorising results as bacterial, viral, or equivocal. Healthy controls [n = 20] were also tested. Among 442 patients classified by clinical management, the MeMed BV® test demonstrated a positive percent agreement [PPA] of 90.0% (95% CI: 86.4-92.7) and a negative percent agreement [NPA] was 44.1% (95% CI: 34.4-54.2). In 370 patients classified by molecular testing, PPA was 88.2% (95% CI: 83.6-91.7) and NPA was 30.1% (95% CI: 20.8-41.4. Equivocal results occurred in 7.5%. The test agreed with clinical management in 96.1% of cases with no detected pathogen. None of the healthy controls had bacterial scores. Conclusion: MeMed BV® demonstrated high sensitivity in identifying bacterial infections but limited specificity in viral infections, notably SARS-CoV-2. It may aid early triage when combined with clinical and microbiological data. Trial registration: ClinicalTrials.gov</p

BLR1 and FCGR1A transcripts in peripheral blood associate with the extent of intrathoracic tuberculosis in children and predict treatment outcome

Biomarkers reflecting the extent of Mycobacterium tuberculosis-induced pathology and normalization during anti-tuberculosis treatment (ATT) would considerably facilitate trials of new treatment regimens and the identification of patients with treatment failure. Therefore, in a cohort of 99 Indian children with intrathoracic tuberculosis (TB), we performed blood transcriptome kinetic analysis during ATT to explore 1) the association between transcriptional biomarkers in whole blood (WB) and the extent of TB disease at diagnosis and treatment outcomes at 2 and 6 months, and 2) the potential of the biomarkers to predict treatment response at 2 and 6 months. We present the first data on the association between transcriptional biomarkers and the extent of TB disease as well as outcome of ATT in children: Expression of three genes down-regulated on ATT (FCGR1A, FPR1 and MMP9) exhibited a positive correlation with the extent of TB disease, whereas expression of eight up-regulated genes (BCL, BLR1, CASP8, CD3E, CD4, CD19, IL7R and TGFBR2) exhibited a negative correlation with the extent of disease. Baseline levels of these transcripts displayed an individual capacity >70% to predict the six-month treatment outcome. In particular, BLR1 and FCGR1A seem to have a potential in monitoring and perhaps tailoring future antituberculosis therapy