Single-Cell Measurement Revolution: A New Paradigm in Tuberculosis Diagnosis

Despite a century of advances in tuberculosis (TB) diagnostics, a deceptively simple question remains unanswered: Can we reliably detect a single viable Mycobacterium tuberculosis (Mtb) bacillus in a clinical sample? In this perspective, we examine three complementary single-cell measurement platforms that face significant challenges in addressing this seemingly simple yet exceedingly difficult question. Single-cell RNA sequencing (scRNA-seq) can resolve host immune heterogeneity by identifying novel cell subsets and diagnostic biomarkers, such as GBP5, that are otherwise obscured in bulk measurements. Direct Mtb scRNA-seq offers unprecedented insights into four critical biological mechanisms: infectivity, drug resistance, persister formation, and host-pathogen interactions; however, its practical clinical application remains limited. Complementing these genomic approaches, single-cell surface-enhanced Raman spectroscopy serves as a spectroscopic platform to capture molecular heterogeneity among individual bacilli, revealing drug-resistance signatures that are typically masked by population-averaged spectra. Furthermore, single-cell fluorescent probes, arguably the most promising approach for ultimately overcoming this challenge, act as chemical measurement platforms. They enable the direct visualization of viable Mtb by leveraging pathogen-specific metabolic, enzymatic, or transport-based fluorescence activations. We critically evaluate existing translational barriers, including inadequate sensitivity across diverse clinical specimens (e.g., sputum, swabs, body fluids, tissues, and breath aerosols) as well as high costs and incompatibility with routine clinical workflows. Looking ahead, we underscore future direction in TB diagnostic measurement science, emphasizing specimen-driven probe design and the critical challenge of distinguishing slow replication-competent bacilli from metabolically active but nonreplicating populations. Collectively, these emerging technologies chart a roadmap toward the ultimate goal of detecting a single viable Mtb bacterium in clinical samples.