Comparing accuracy of lipoarabinomannan urine tests for diagnosis of pulmonary tuberculosis in children from four African countries: a cross-sectional study.

BACKGROUND: A sensitive and specific non-sputum-based test would be groundbreaking for the diagnosis of childhood tuberculosis. We assessed side by side the diagnostic accuracy of the urine-based lipoarabinomannan assays Fujifilm SILVAMP TB LAM (FujiLAM) and Alere Determine TB LAM Ag (AlereLAM) for detection of childhood tuberculosis. METHODS: In this cross-sectional study, we tested urine samples from children younger than 15 years with presumed pulmonary tuberculosis. Children were consecutively recruited from four dedicated outpatient childhood tuberculosis clinics in The Gambia, Mali, Nigeria, and Tanzania. Biobanked urine samples were thawed and tested using FujiLAM and AlereLAM assays. We measured diagnostic performance against a microbiological reference standard (confirmed tuberculosis) and a composite reference standard (confirmed and unconfirmed tuberculosis). Sensitivity and specificity were estimated with bivariate random-effects meta-analyses. FINDINGS: Between July 1, 2017, and Dec 1, 2018, we obtained and stored urine samples from 415 children. 63 (15%) children had confirmed tuberculosis, 113 (27%) had unconfirmed tuberculosis, and 239 (58%) were unlikely to have tuberculosis. 61 children were HIV-positive (prevalence 15%). Using the microbiological reference standard, the sensitivity of FujiLAM was 64·9% (95% CI 43·7-85·2; positive in 40 of 63 confirmed samples) and the sensitivity of AlereLAM was 30·7% (8·6-61·6; 19 of 63). The specificity of FujiLAM was 83·8% (95% CI 76·5-89·4; negative in 297 of 352 unconfirmed and unlikely samples) and the specificity of AlereLAM was 87·8% (79·0-93·7; 312 of 352). Against the composite reference standard, both assays had decreased sensitivity; the sensitivity of FujiLAM was 32·9% (95% CI 24·6-41·9; positive in 58 of 176 confirmed and unconfirmed samples) and the sensitivity of AlereLAM was 20·2% (12·3-29·4; 36 of 176). The specificity of FujiLAM was 83·3% (95% CI 71·8-91·7; negative in 202 of 239 unlikely samples) and the specificity of AlereLAM was 90·0% (81·6-95·6; 216 of 239). INTERPRETATION: By comparison with AlereLAM, FujiLAM showed higher sensitivity and similar specificity. FujiLAM could potentially add value to the rapid diagnosis of tuberculosis in children. FUNDING: German Federal Ministry of Education and Research, the Global Health Innovative Technology Fund, the UK Research and Innovation Global Challenges Research Fund, and the UK Medical Research Council.

A three-marker protein biosignature distinguishes tuberculosis from other respiratory diseases in Gambian children.

BACKGROUND: Our study aimed to identify a host cytokine biosignature that could distinguish childhood tuberculosis (TB) from other respiratory diseases (OD). METHODS: Cytokine responses in prospectively recruited children with symptoms suggestive of TB were measured in whole blood assay supernatants, harvested after overnight incubation, using a Luminex platform. We used logistic regression models with Least Absolute Shrinkage and Selection Operator (LASSO) penalty to identify the optimal biosignature associated with confirmed TB disease in the training set. We subsequently assessed its performance in the test set. FINDINGS: Of the 431 children included in the study, 44 had bacteriologically confirmed TB, 60 had clinically diagnosed TB while 327 had OD. All children were HIV-negative. Application of LASSO regression models to the training set (n = 260) resulted in the combination of IL-1ra, IL-7 and IP-10 from unstimulated samples as the optimally discriminant cytokine biosignature associated with bacteriologically confirmed TB. In the test set (n = 171), this biosignature distinguished children diagnosed with TB disease, irrespective of microbiological confirmation, from OD with area under the receiver operator characteristic curve (AUC) of 0•74 (95% CI: 0•67, 0•81), and demonstrated sensitivity and specificity of 72•2% (95% CI: 60•4, 82•1%) and 75•0% (95% CI: 64•9, 83•4%) respectively, with its performance independent of their age group and their age- and sex-adjusted nutritional status. INTERPRETATION: This novel biosignature of childhood TB derived from unstimulated supernatants is promising. Independent validation with further optimisation will improve its performance and translational potential. FUNDING: Steinberg Fellowship (McGill University); Grand Challenges Canada; MRC Program Grant.

Protection against mycobacterial infection: A case-control study of mycobacterial immune responses in pairs of Gambian children with discordant infection status despite matched TB exposure.

BACKGROUND: Children are particularly susceptible to tuberculosis. However, most children exposed to Mycobacterium tuberculosis are able to control the pathogen without evidence of infection. Correlates of human protective immunity against tuberculosis infection are lacking, and their identification would aid vaccine design. METHODS: We recruited pairs of asymptomatic children with discordant tuberculin skin test status but the same sleeping proximity to the same adult with sputum smear-positive tuberculosis in a matched case-control study in The Gambia. Participants were classified as either Highly TB-Exposed Uninfected or Highly TB-Exposed Infected children. Serial luminescence measurements using an in vitro functional auto-luminescent Bacillus Calmette-Guérin (BCG) whole blood assay quantified the dynamics of host control of mycobacterial growth. Assay supernatants were analysed with a multiplex cytokine assay to measure associated inflammatory responses. FINDINGS: 29 pairs of matched Highly TB-Exposed Uninfected and Highly TB-Exposed Infected children aged 5 to 15 years old were enroled. Samples from Highly TB-Exposed Uninfected children had higher levels of mycobacterial luminescence at 96 hours than Highly TB-Exposed Infected children. Highly TB-Exposed Uninfected children also produced less BCG-specific interferon-γ than Highly TB-Exposed Infected children at 24 hours and at 96 hours. INTERPRETATION: Highly TB-Exposed Uninfected children showed less control of mycobacterial growth compared to Highly TB-Exposed Infected children in a functional assay, whilst cytokine responses mirrored infection status. FUNDING: Clinical Research Training Fellowship funded under UK Medical Research Council/Department for International Development Concordat agreement and part of EDCTP2 programme supported by European Union (MR/K023446/1). Also MRC Program Grants (MR/K007602/1, MR/K011944/1, MC_UP_A900/1122).

No added value of interferon-γ release to a prediction model for childhood tuberculosis.

The predictive value of a combination of clinical and radiological features with interferon-γ release assay (IGRA) for diagnosis of active tuberculosis (TB) disease among TB-exposed children is unknown.150 symptomatic HIV-negative children (aged 3 months to 14 years), prospectively recruited through active contact tracing, were included. Backward stepwise logistic regression and bootstrapping techniques were used for the development and internal validation of a clinical prediction model for active TB disease. Model discrimination and incremental value of a positive IGRA test were assessed by area under the receiver operating characteristic curve (AUC).35 (23%) children were diagnosed with active TB disease and started on treatment and 115 (77%) had other respiratory tract infections. A final parsimonious clinical model, comprising age <5 years (adjusted (a)OR 4.8, 95% CI 2.0-11.5) and lymphadenopathy on clinical examination (aOR 4.9, 95% CI 1.8-13.0) discriminated active TB disease from other disease with an AUC of 0.70 (95% CI 0.61-0.80). A positive IGRA result did not improve the discriminatory ability of the clinical model (c-statistic 0.72 versus 0.70; p=0.644).A clinical algorithm, including age <5 years and lymphadenopathy classified 70% of active TB disease among symptomatic TB-exposed children. IGRA does not add any discriminatory value to this prediction model.

Analysis of host responses to Mycobacterium tuberculosis antigens in a multi-site study of subjects with different TB and HIV infection states in sub-Saharan Africa.

BACKGROUND: Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas. METHODS: We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens. RESULTS: There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST(-) and TST(+) contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST(+) contacts (LTBI) compared to TB and TST(-) contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen. CONCLUSIONS: Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.