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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., Competing Interests: TMD is an employee of and holds shares in the GSK group of companies but participated in the current work as an independent investigator. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sivakumaran, Ritz, Gjøen, Vaz, Selvam, Ottenhoff, Doherty, Jenum and Grewal.)

Pediatric tuberculosis (TB) is challenging to diagnose, confirmed by growth of Mycobacterium tuberculosis at best in 40% of cases. The WHO has assigned high priority to the development of non-sputum diagnostic tools. We therefore sought to identify transcriptional signatures in whole blood of Indian children, capable of discriminating intra-thoracic TB disease from other symptomatic illnesses. We investigated the expression of 198 genes in a training set, comprising 47 TB cases (19 definite/28 probable) and 36 asymptomatic household controls, and identified a 7- and a 10-transcript signature, both including NOD2, GBP5, IFITM1/3, KIF1B and TNIP1. The discriminatory abilities of the signatures were evaluated in a test set comprising 24 TB cases (17 definite/7 probable) and 26 symptomatic non-TB cases. In separating TB-cases from symptomatic non-TB cases, both signatures provided an AUC of 0.94 (95%CI, 0.88-1.00), a sensitivity of 91.7% (95%CI, 71.5-98.5) regardless of culture status, and 100% sensitivity for definite TB. The 7-transcript signature provided a specificity of 80.8% (95%CI, 60.0-92.7), and the 10-transcript signature a specificity of 88.5% (95%CI, 68.7-96.9%). Although warranting exploration and validation in other populations, our findings are promising and potentially relevant for future non-sputum based POC diagnostic tools for pediatric TB.