ABSTRACT Tuberculosis (TB, Mtb) remains one of the leading infectious disease killer globally, despite the availability of curative treatment for most individuals. The ability to identify TB which has progressed beyond bacterial containment associated with latency but not yet reached the burden of culture-positive, symptomatic active disease could have far- reaching impact in thwarting transmission and decreasing individual morbidity and mortality. The global scale-up of ultra-sensitive Mtb DNA detection assays, such as the WHO-recommended GeneXpert MTB/RIF Ultra (Ultra), presents a critical opportunity to identify this incipient, pre-cultivatable disease stage. We hypothesize that sputum Mtb DNA-positive/culture-negative individuals are more likely to have incipient TB than Mtb DNA-negative/culture-negative (TB-negative) individuals. To test this hypothesis, we initiated a substudy to an R01-funded Ultra diagnostic accuracy study of adults in Africa with signs/symptoms of TB to evaluate the biology and natural history Ultra Mtb DNA- positive/culture-negative individuals. In Aim 1, we will characterize host clinical and demographic factors associated with sputum Mtb DNA-positive/culture-negative status and characterize these individuals on the spectrum of Mtb infection using pre-validated blood transcriptional signatures predictive of progression to active TB. In Aim 2, we will evaluate and compare microbiologic indicators of TB disease between Mtb DNA positive/culture negative and TB-negative individuals. Specifically, we propose to investigate the presence of Mtb mRNA from sputum and evaluate for differentially culturable Mtb from extended incubation and culture supplemented with growth promoting factors. Finally, we will evaluate the natural history of Mtb DNA- positive/culture-negative individuals with respect to TB-negative controls using longitudinal clinical, microbiologic, and host transcriptional data collected over a 12- month follow-up period in the parent study. Together, these proposed studies will inform the utility of ultrasensitive Mtb DNA detection tests for early TB risk stratification and characterize the natural history and biomarkers of culture-negative TB.