Background Identification of blood biomarkers that prospectively predict progression of Mycobacterium tuberculosis infection to tuberculosis disease may lead to interventions that impact the epidemic. Methods Healthy, M. tuberculosis infected South African adolescents were followed for 2 years; blood was collected every 6 months. A prospective signature of risk was derived from whole blood RNA-Sequencing data by comparing participants who ultimately developed active tuberculosis disease (progressors) with those who remained healthy (matched controls). After adaptation to multiplex qRT-PCR, the signature was used to predict tuberculosis disease in untouched adolescent samples and in samples from independent cohorts of South African and Gambian adult progressors and controls. The latter participants were household contacts of adults with active pulmonary tuberculosis disease. Findings Of 6,363 adolescents screened, 46 progressors and 107 matched controls were identified. A 16 gene signature of risk was identified. The signature predicted tuberculosis progression with a sensitivity of 66·1% (95% confidence interval, 63·2–68·9) and a specificity of 80·6% (79·2–82·0) in the 12 months preceding tuberculosis diagnosis. The risk signature was validated in an untouched group of adolescents (p=0·018 for RNA-Seq and p=0·0095 for qRT-PCR) and in the independent South African and Gambian cohorts (p values <0·0001 by qRT-PCR) with a sensitivity of 53·7% (42·6–64·3) and a specificity of 82·8% (76·7–86) in 12 months preceding tuberculosis. Interpretation The whole blood tuberculosis risk signature prospectively identified persons at risk of developing active tuberculosis, opening the possibility for targeted intervention to prevent the disease. Funding Bill and Melinda Gates Foundation, the National Institutes of Health, Aeras, the European Union and the South African Medical Research Council (detail at end of text).

SummaryMycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

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