Tuberculosis is the world's deadliest infectious disease. Asymptomatic tuberculosis (aTB) comprises approximately 50% of prevalent TB cases and is challenging to diagnose due to clinical and molecular heterogeneity. A systems-level approach is required to untangle this variability and the underlying disease biology. In a community-wide TB screening cohort, we investigated whether we could identify sub-phenotypes of aTB based on integrated clinical, microbiological, radiological, and inflammatory (CMRI) features, and whether these phenotypes are associated with distinct biomolecular profiles.

We applied Factor Analysis of Mixed Data and k-means clustering to stratify aTB individuals, validating cluster stability through silhouette analysis and bootstrapped resampling. Differential expression analysis of a 46-analyte Luminex panel identified proteomic markers characteristic of each cluster. We identified three aTB subtypes stratified by disease severity: Cluster A comprised younger, female-predominant participants with higher BMI and minimal inflammation, lung pathology, and bacterial burden. Cluster B represented an intermediate phenotype with significant lung pathology without cavitation. Cluster C was skewed towards older males with lower BMI, elevated inflammation, higher bacterial burden, and cavitary disease. Proteomic profiling reinforced disease heterogeneity, revealing molecular differences between clusters, including high expression of immune regulators PD-L1 and MIP-3β in Cluster C. Cluster B demonstrated elevated cytokine levels compared to Cluster A, consistent with its intermediate phenotypic severity. These findings reveal aTB endotypes with differential proteomic expression, possibly indicating distinct pathways contributing to disease heterogeneity. Combined with multi-omics analyses, this stratification framework enables mechanistic understanding of aTB and facilitates development of targeted diagnostics and therapeutics.

Co-authors: Lerato Mtshali, Stephen Olivier, Helgaard Claassen, Thando Zulu, Njabulo Myeza, Mareca Sithole, Willem Hanekom, Alison Grant, Alasdair Leslie, Sarah Fortune, Andrew Fiore-Gartland, Emily Wong