Novel RNA biomarkers improve discrimination of children with tuberculosis disease from those with non-TB pneumonia after in vitro stimulation

The diagnosis of pediatric tuberculosis (TB) poses a challenge for clinical teams worldwide. TB-mediated changes in the expression of host genes in the peripheral blood can serve as diagnostic biomarkers and can provide better insights into the host immune mechanisms of childhood TB. Peripheral blood mononuclear cells (PBMCs) from children (n=102) with microbiologically confirmed TB disease, TB infection (TBI), pneumonia, and healthy controls (HC) were stimulated with either the Purified Protein Derivative (PPD) or the Early Secretory Antigen 6kDa-Culture Filtrate Protein 10 (ESAT6-CFP10) complex of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>). RNA was extracted and quantified using gene expression microarrays. Differential expression analysis was performed comparing microbiologically confirmed TB to the other diagnostic groups for the stimulated and unstimulated samples. Using variable selection, we identified sparse diagnostic gene signatures; one gene (<i>PID1</i>) was able to distinguish TB from pneumonia after ESAT6-CFP10 stimulation with an AUC of 100% in the test set, while a combination of two genes (<i>STAT1</i> and <i>IFI44</i>) achieved an AUC of 91.7% (CI_95% 75.0%-100%) in the test set after PPD stimulation. The number of significantly differentially expressed (SDE) genes was higher when contrasting TB to pneumonia or HC in stimulated samples, compared to unstimulated ones, leading to a larger pool of candidate diagnostic biomarkers. Our approach provides enlightened aspects of peripheral TB-specific responses and can form the basis for a point of care test meeting the World Health Organization (WHO) Target Product Profile (TPP) for pediatric TB.