Although immune activation is associated with HIV acquisition, the nature of inflammatory profiles that increase HIV risk, which may include responses to M. tuberculosis (Mtb) infection, are not well characterized.We conducted a nested case-control study using cryopreserved samples from persons who did and did not acquire HIV during the multinational Step clinical trial of the MRKAd5 HIV-1 vaccine. PBMCs from the last HIV-negative sample from incident HIV cases and controls were stimulated with Mtb-specific antigens (ESAT-6/CFP-10) and analyzed by flow cytometry with intracellular cytokine staining and scored with COMPASS. We measured inflammatory profiles with five Correlates of TB Risk (CoR) transcriptomic signatures. Our primary analysis examined the association of latent Mtb infection (LTBI; IFNγ+CD4+ T cell frequency) or RISK6 CoR signature with HIV acquisition. Conditional logistic regression analyses, adjusted for known predictors of HIV acquisition, were employed to assess whether TB-associated immune markers were associated with HIV acquisition.Among 465 participants, LTBI prevalence (21.5% controls vs 19.1% cases, p = 0.51) and the RISK6 signature were not higher in those who acquired HIV. In exploratory analyses, Mtb antigen-specific polyfunctional CD4+ T cell COMPASS scores (aOR 0.96, 95% CI 0.77, 1.20) were not higher in those who acquired HIV. Two CoR signatures, Sweeney3 (aOR 1.38 (1.07, 1.78) per SD change) and RESPONSE5 (0.78 (0.61, 0.98)), were associated with HIV acquisition. The transcriptomic pattern used to differentiate active vs latent TB (Sweeney3) was most strongly associated with acquiring HIV.LTBI, Mtb polyfunctional antigen-specific CD4+ T cell activation, and RISK6 were not identified as risks for HIV acquisition. In exploratory transcriptomic analyses, two CoR signatures were associated with HIV risk after adjustment for known behavioral and clinical risk factors. We identified host gene expression signatures associated with HIV acquisition, but the observed effects are likely not mediated through Mtb infection.
Introduction Host blood transcriptomic biomarkers have potential as rapid point-of-care triage, diagnostic, and predictive tests for Tuberculosis disease. We aimed to summarise the performance of host blood transcriptomic signatures for diagnosis of and prediction of progression to Tuberculosis disease; and compare their performance to the recommended World Health Organisation target product profile. Methods A systematic review and meta-analysis of the performance of host blood mRNA signatures for diagnosing and predicting progression to Tuberculosis disease in HIV-negative adults and adolescents, in studies with an independent validation cohort. Medline, Scopus, Web of Science, and EBSCO libraries were searched for articles published between January 2005 and May 2019, complemented by a search of bibliographies. Study selection, data extraction and quality assessment were done independently by two reviewers. Meta-analysis was performed for signatures that were validated in ≥3 comparable cohorts, using a bivariate random effects model. Results Twenty studies evaluating 25 signatures for diagnosis of or prediction of progression to TB disease in a total of 68 cohorts were included. Eighteen studies evaluated 24 signatures for TB diagnosis and 17 signatures met at least one TPP minimum performance criterion. Three diagnostic signatures were validated in clinically relevant cohorts to differentiate TB from other diseases, with pooled sensitivity 84%, 87% and 90% and pooled specificity 79%, 88% and 74%, respectively. Four studies evaluated signatures for progression to TB disease and performance of one signature, assessed within six months of TB diagnosis, met the minimal TPP for a predictive test for progression to TB disease. Conclusion Host blood mRNA signatures hold promise as triage tests for TB. Further optimisation is needed if mRNA signatures are to be used as standalone diagnostic or predictive tests for therapeutic decision-making.
Case-finding strategies for tuberculosis diagnosis rely on symptom screening, which is associated with poor sensitivity,1van't Hoog AH Langendam M Mitchell E et al.A systematic review of the sensitivity and specificity of symptom- and chest-radiography screening for active pulmonary tuberculosis in HIV-negative persons and persons with unknown HIV status. WHO, 2013https://www.who.int/tb/tbscreening/enDate accessed: January 25, 2020Google Scholar resultant delays in diagnosis, increased patient morbidity, and ongoing transmission. There is need for a more sensitive, non-sputum-based triage test to exclude tuberculosis at the primary care level, and for mass screening in high-burden settings.2Nathavitharana RR Yoon C Macpherson P et al.Guidance for studies evaluating the accuracy of tuberculosis triage tests.J Infect Dis. 2019; 220: S116-S125Crossref PubMed Scopus (20) Google Scholar Several blood transcriptional diagnostic signatures have been described;3Warsinske H Vashisht R Khatri P Host-response-based gene signatures for tuberculosis diagnosis: a systematic comparison of 16 signatures.PLoS Med. 2019; 16e1002786Crossref PubMed Scopus (81) Google Scholar however, these were invariably discovered and validated in carefully selected case-control cohorts, inflating diagnostic accuracy. A crucial step in the development pathway is assessment of prospective diagnostic accuracy in real-world health-care settings. In The Lancet Respiratory Medicine, Carolin Turner and colleagues4Turner CT Gupta RK Tsaliki E et al.Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.Lancet Respir Med. 2020; (published online March 13.)https://doi.org/10.1016/S2213-2600(19)30469-2Summary Full Text Full Text PDF PubMed Scopus (58) Google Scholar present the first prospective, systematic head-to-head comparison of the diagnostic accuracy of 27 blood transcriptional signatures. These were identified from their previous systematic review. From 181 symptomatic adults who presented to a primary health-care clinic in South Africa, Turner and colleagues obtained blood for RNA sequencing and sputum for culture and molecular testing using Xpert MTB/RIF (Xpert) and Xpert MTB/RIF Ultra (Ultra), and compared the diagnostic accuracy of the candidate signatures with that of culture or Xpert for active tuberculosis. Notably, no signatures met the WHO target product profile5WHOHigh-priority target product profiles for new tuberculosis diagnostics: report of a consensus meeting. World Health Organization, Geneva2014https://www.who.int/tb/publications/tpp_report/enDate accessed: January 25, 2020Google Scholar benchmark criteria for a non-sputum confirmatory tuberculosis diagnostic test (minimum 65% sensitivity, 98% specificity) or optimum criteria for a tuberculosis triage test (95% sensitivity, 80% specificity). The four best-performing signatures had similar diagnostic accuracy, independent of HIV status, and met or approached the minimum WHO criteria for a tuberculosis triage test. These results suggest that transcriptional signature diagnostic performance might have peaked and it is unlikely that new transcriptional signatures discovered in existing case-control cohorts will have better diagnostic accuracy than existing signatures. It might also prove challenging to reproduce the results achieved using batch-corrected, high-throughput methods, such as RNA sequencing, when signatures are translated to point-of-care RNA quantitation technologies in a real-world application. Tuberculosis is a spectrum that spans quiescent, latent infection through to subclinical and active symptomatic disease.6Drain PK Bajema KL Dowdy D et al.Incipient and subclinical tuberculosis: a clinical review of early stages and progression of infection.Clin Microbiol Rev. 2018; 31: e00021-e00118Crossref PubMed Scopus (221) Google Scholar The apparent ceiling and poor performance of some signatures in field settings, as tested by Turner and colleagues,4Turner CT Gupta RK Tsaliki E et al.Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.Lancet Respir Med. 2020; (published online March 13.)https://doi.org/10.1016/S2213-2600(19)30469-2Summary Full Text Full Text PDF PubMed Scopus (58) Google Scholar might be partly accounted for by discovery methods that have relied on feature selection and model construction to achieve binary differentiation between homogenous groups that represent the extremes of the spectrum. Given the difficulty in diagnosing subclinical (asymptomatic, sputum culture-positive) and paucibacillary (sputum smear-negative, culture-positive) tuberculosis, a latent class modelling approach could be considered in future tuberculosis discovery and validation studies, to account for uncertainty in disease classification.7Drain PK Gardiner J Hannah H et al.Guidance for studies evaluating the accuracy of biomarker-based nonsputum tests to diagnose tuberculosis.J Infect Dis. 2019; 220: S108-S115Crossref PubMed Scopus (26) Google Scholar Another consideration is that transcriptional signatures measure expression of non-specific inflammation, primarily comprised of interferon-stimulated genes (ISG). Because conditions other than tuberculosis, such as viral infections, induce ISG expression, false-positive results are inevitable in some symptomatic individuals without tuberculosis. A heterogenous prospective discovery cohort that is representative of the target population, in this case symptomatic clinic attendees, might result in more tuberculosis-specific gene selection and enhance downstream performance. A multinomial or multilabel classification model, in which individuals can be assigned to one or more outcomes, is an alternative strategy.8Duffy FJ Thompson EG Scriba TJ Zak DE Multinomial modelling of TB/HIV co-infection yields a robust predictive signature and generates hypotheses about the HIV+TB+ disease state.PLoS One. 2019; 14e0219322Crossref PubMed Scopus (7) Google Scholar Finally, integration of clinical variables might also provide further incremental improvements in diagnostic accuracy. Whether new biomarker discovery efforts in large, multicentre, prospective cohorts of unselected symptomatic patients seeking health care will lead to better performance remains to be seen. A key objective of tuberculosis triage tests is to rapidly screen individuals seeking care to rule out those without disease, thereby reducing the volume of more expensive confirmatory diagnostic tests.2Nathavitharana RR Yoon C Macpherson P et al.Guidance for studies evaluating the accuracy of tuberculosis triage tests.J Infect Dis. 2019; 220: S116-S125Crossref PubMed Scopus (20) Google Scholar The price of Xpert cartridges is currently fixed at US$9·98; however, it is likely that the cost of molecular testing will soon drop. It is imperative that a tuberculosis triage test should cost less than confirmatory testing, ideally closer to $2 after scale-up, as proposed by expert consensus.5WHOHigh-priority target product profiles for new tuberculosis diagnostics: report of a consensus meeting. World Health Organization, Geneva2014https://www.who.int/tb/publications/tpp_report/enDate accessed: January 25, 2020Google Scholar Whether a point-of-care transcriptional biomarker-based test could achieve the $2 price point, and be sufficiently high-throughput (>10 tests per 6 h, <1 h per test) will need to be ascertained.5WHOHigh-priority target product profiles for new tuberculosis diagnostics: report of a consensus meeting. World Health Organization, Geneva2014https://www.who.int/tb/publications/tpp_report/enDate accessed: January 25, 2020Google Scholar Given that multiple simple, fast, and affordable non-sputum-based tuberculosis screening tools have been developed, including C-reactive protein-based screening9Yoon C Semitala FC Atuhumuza E et al.Point-of-care C-reactive protein-based tuberculosis screening for people living with HIV: a diagnostic accuracy study.Lancet Infect Dis. 2017; 17: 1285-1292Summary Full Text Full Text PDF PubMed Scopus (72) Google Scholar and computer-aided radiographic detection, the impetus to take host transcriptional biomarkers to market might diminish. Ultra, which offers improved sensitivity over the previous Xpert test10Horne DJ Kohli M Zifodya JS et al.Xpert MTB/RIF and Xpert MTB/RIF Ultra for pulmonary tuberculosis and rifampicin resistance in adults.Cochrane Database Syst Rev. 2019; 6CD009593PubMed Google Scholar and other similar tests, might also supplant the need for a triage test, especially in tuberculosis high-burden settings, such as the one studied by Turner and colleagues,4Turner CT Gupta RK Tsaliki E et al.Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.Lancet Respir Med. 2020; (published online March 13.)https://doi.org/10.1016/S2213-2600(19)30469-2Summary Full Text Full Text PDF PubMed Scopus (58) Google Scholar where almost one in three symptomatic adults had microbiologically confirmed disease. However, with greater sensitivity comes a loss in specificity,10Horne DJ Kohli M Zifodya JS et al.Xpert MTB/RIF and Xpert MTB/RIF Ultra for pulmonary tuberculosis and rifampicin resistance in adults.Cochrane Database Syst Rev. 2019; 6CD009593PubMed Google Scholar probably a consequence of detecting non-viable mycobacterial DNA, a remnant of previous tuberculosis disease, or detection of early paucibacillary disease, which might self-cure. Turner and colleagues4Turner CT Gupta RK Tsaliki E et al.Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.Lancet Respir Med. 2020; (published online March 13.)https://doi.org/10.1016/S2213-2600(19)30469-2Summary Full Text Full Text PDF PubMed Scopus (58) Google Scholar found that transcriptional signatures correctly characterised Ultra-positive, culture-negative (false-positive) sputum samples, improving test specificity. This finding raises the possibility of repurposing transcriptional or other host-response biomarkers as adjunctive tests to measure Mycobacterium tuberculosis activity in Ultra-positive individuals with previous tuberculosis disease, or with trace results, to rule-out false-positives or to identify those who do not have active disease but are at high risk of disease progression. Further large-scale, prospective validation studies, ideally using RNA quantitation technologies compatible with existing point-of-care platforms, to compare multiple transcriptional signatures in real-world settings and across multiple epidemiological and geographical locations, are therefore important to advance transcriptional signatures through the diagnostics pipeline. SCM received training in research that was supported by the Fogarty International Center of the US National Institutes of Health (number D43 TW010559). TJS is a co-inventor of patents for the Zak16, Suliman4, and Penn-Nicholson6 (RISK6) signatures that were assessed in the Article by Turner and colleagues.4Turner CT Gupta RK Tsaliki E et al.Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.Lancet Respir Med. 2020; (published online March 13.)https://doi.org/10.1016/S2213-2600(19)30469-2Summary Full Text Full Text PDF PubMed Scopus (58) Google Scholar SKM and MH declare no competing interests. Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy studySelected blood transcriptional signatures met the minimum WHO benchmarks for a tuberculosis triage test but not for a confirmatory test. Further development of the signatures is warranted to investigate their possible effects on clinical and health economic outcomes as part of a triage strategy, or when used as add-on confirmatory test in conjunction with the highly sensitive Ultra test for Mycobacterium tuberculosis DNA. Full-Text PDF Open AccessConcise whole blood transcriptional signatures for incipient tuberculosis: a systematic review and patient-level pooled meta-analysisBlood transcriptional biomarkers reflect short-term risk of tuberculosis and only exceed WHO benchmarks if applied to 3–6-month intervals. Serial testing among carefully selected target groups might be required for optimal implementation of these biomarkers. Full-Text PDF Open Access
Background Adolescents in the Western Cape Province of South Africa had high force of Mycobacterium tuberculosis (MTB) infection (14% per annum) and high TB incidence (710 per 100,000 person–years) in 2005. We describe subsequent temporal changes in adolescent TB disease notification rates for the decade 2005–2015. Method We conducted an analysis of patient–level adolescent (age 10–19 years) TB disease data, obtained from an electronic TB register in the Breede Valley sub–district, Western Cape Province, South Africa, for 2005–2015. Numerators were annual TB notifications (HIV–related and HIV–unrelated); denominators were mid–year population estimates. Period averages of TB rates were obtained using time series modeling. Temporal trends in TB rates were explored using the Mann–Kendall test. Findings The average adolescent TB disease notification rate was 477 per 100,000 for all TB patients (all–TB) and 361 per 100,000 for microbiologically–confirmed patients. The adolescent all–TB rate declined by 45% from 662 to 361 per 100,000 and the microbiologically–confirmed TB rate by 38% from 492 to 305 per 100,000 between 2005–2015, driven mainly by rapid decreases for the period 2005–2009. There was a statistically significant negative temporal trend in both all–TB (per 100,000) (declined by 48%; from 662 to 343; p = 0·028) and microbiologically confirmed TB (per 100,000) (declined by 49%; from 492 to 252; p = 0·027) for 2005–2009, which was not observed for the period 2009–2015 (rose 5%; from 343 to 361; p = 0·764 and rose 21%; from 252 to 305; p = 1·000, respectively). Interpretation We observed an encouraging fall in adolescent TB disease rates between 2005–2009 with a subsequent plateau during 2010–2015, suggesting that additional interventions are needed to sustain initial advances in TB control.
The Validation of Correlates of Risk of TB Disease in High Risk Populations (CORTIS-HR) Study, a companion study of the CORTIS-01 Trial (ClinicalTrials.gov: NCT02735590), was conducted to test the diagnostic and prognostic performance of the RISK11 biomarker for tuberculosis (TB) disease in people living with HIV (PLHIV) in an ambulant community setting. The “CORTIS-HR pubdata.csv” is a public, subject-level dataset for the CORTIS-HR study containing key variables necessary to reconstruct the study findings. A data dictionary is provided below. The “CORTIS-HR PCR data.csv” provides subject-level TaqMan qPCR probe raw CT (cycle threshold) gene expression data from the Fluidigm microfluidic 96.96 Gene Expression Integrated Fluidic Circuits (chips) with sample quality control (“SAMPLE_QC”) results. Analyses of the qPCR probe data are ongoing; the embargo on this data ends 1 July 2021 when the data will be available on ZivaHub. “CORTIS-HR Protocol Version 1.0.pdf” and “CORTIS-HR SAP Version 1.0.pdf” are the protocol and the statistical analysis plan for the study respectively and have been included for reference.
Abstract We tested performance of host-blood transcriptomic tuberculosis (TB) signatures for active case-finding. Among 20,207 HIV-uninfected and 963 HIV-infected adults screened; 2,923 and 861 were enrolled from five South African communities. Eight signatures were measured by microfluidic RT-qPCR and participants were microbiologically-investigated for pulmonary TB at baseline, and actively surveilled for incident disease through 15 months. Diagnostic AUCs for 61 HIV-uninfected (weighted-prevalence 1.1%) and 10 HIV-infected (prevalence 1.2%) prevalent TB cases for the 8 signatures were 0.63–0.79 and 0.65–0.88, respectively. Thereafter, 24 HIV-uninfected and 9 HIV-infected participants progressed to incident TB (1.1 and 1.0 per 100 person-years, respectively). Prognostic AUCs through 15-months follow-up were 0.49–0.66 and 0.54–0.81, respectively. Prognostic performance for incident TB occurring within 6-12 months in HIV-negative participants was higher for all signatures. None of the signatures met WHO Target Product Profile criteria for a triage test to diagnose asymptomatic TB; most signatures met the criteria for symptomatic TB. Prognostic accuracy of most signatures for incident TB within six months of testing met the criteria for an incipient TB test.
Abstract Background Translation of blood RNA signatures may be accelerated by identifying more parsimonious biomarkers. We tested the hypothesis that single-gene transcripts provide comparable accuracy for detection of subclinical TB to multi-gene signatures and benchmarked their clinical utility to interferon-y release assays (IGRAs). Methods We identified datasets where participants underwent RNA sampling and at least 12 months of follow-up for progression to TB. We performed a one-stage individual participant data meta-analysis to compare multi-gene signatures against single-gene transcripts to detect subclinical TB, defined as asymptomatic prevalent or incident TB (diagnosed ≥21 days from enrolment, irrespective of symptoms) over a 12-month interval. We performed decision curve analysis to evaluate the net benefit of using RNA biomarkers and IGRA, alone or in combination, compared to treating all or no individuals with preventative treatment. Results We evaluated 80 single-genes and eight multi-gene signatures in a pooled analysis of four RNAseq and three qPCR datasets, comprising 6544 total samples and including 283 samples from 214 individuals with subclinical TB. Five single-gene transcripts were equivalent to the best-performing multi-gene signature over 12 months, with areas under the receiver operating characteristic curves ranging from 0.75-0.77, but none met the WHO minimum target product profile (TPP) for a TB progression test. IGRA demonstrated much lower specificity in higher burden settings, while sensitivity and specificity of RNA biomarkers were consistent across settings. In higher burden settings, RNA biomarkers had greater net benefit than IGRA, which offered little clinical utility over treating all with preventative therapy. In low burden settings, IGRA approximated the TPP and offered greater clinical utility than RNA biomarkers, but combining both tests provided the highest net benefit for services aiming to treat <50 people to prevent a single case. Interpretation Single-gene transcripts are equivalent to multi-gene signatures for detection of subclinical TB, with consistent performance across settings. Single transcripts demonstrate potential clinical utility to stratify treatment, particularly when used in combination with IGRA in low burden settings.
Abstract Six months of chemotherapy using current agents is standard of care for pulmonary, drug-sensitive tuberculosis (TB), even though some are believed to be cured more rapidly and others require longer therapy. Understanding what factors determine the length of treatment required for durable cure in individual patients would allow individualization of treatment durations, provide better clinical tools to determine the of appropriate duration of new regimens, as well as reduce the cost of large Phase III studies to determine the optimal combinations to use in TB control programs. We conducted a randomized clinical trial in South Africa and China that recruited 704 participants with newly diagnosed, drug-sensitive pulmonary tuberculosis and stratified them based on radiographic disease characteristics as assessed by FDG PET/CT scan readers. Participants with less extensive disease (N=273) were randomly assigned to complete therapy after four months or continue receiving treatment for six months. Amongst participants who received four months of therapy, 17 of 141 (12.1%) experienced unfavorable outcomes compared to only 2 of 132 (1.5%) who completed six months of treatment (treatment success 98.4% in B, 86.7% in C (difference -11.7%, 95% CI, -18.2%, -5.3%)). In the non-randomized arm that included participants with more extensive disease, only 8 of 248 (3.2%) experienced unfavorable outcomes. Total cavity volume and total lesion glycolysis at week 16 were significantly associated with risk of unfavorable outcome in the randomized participants. Based on PET/CT scans at TB recurrence, bacteriological relapses (confirmed by whole genome sequencing) predominantly occurred in the same active cavities originally present at baseline. Automated segmentation of the serial PET/CT scans was later performed, and machine-learning was used to classify participants according to their likelihood of relapse, allowing the development of predictive models with good performance based on CT, PET, microbiological and clinical characteristics. These results open the possibility for more efficient studies of future TB treatment regimens.
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