Detection of in human tissue via RNA hybridization.

Rationale: Accurate TB diagnosis is hampered by the variable efficacy of the widely-used Ziehl-Neelsen (ZN) staining method to identify ( ) acid-fast bacilli (AFB). Here, we sought to circumvent this current limitation through direct detection of mRNA.

Objectives: To employ RNAscope to determine the spatial distribution of mRNA within tuberculous human tissue, to appraise ZN-negative tissue from confirmed TB patients, and to provide proof-of-concept of RNAscope as a platform to inform TB diagnosis and biology.

Methods: We examined ante- and postmortem human TB tissue using RNAscope to detect mRNA and a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B).

Measurements And Main Results: We adapted RNAscope for and identified intact and disintegrated bacilli and intra- and extracellular mRNA. mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchial epithelium. Intriguingly, we observed accumulation of mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. mRNA was observed in ZN-negative antemortem lymph node biopsies.

Conclusions: RNAscope has diagnostic potential and can guide therapeutic intervention as it detects mRNA and morphology in ZN-negative tissues from TB patients, and mRNA in ZN-negative antemortem biopsies, respectively. Lastly, our data provide evidence that at least two phenotypically distinct populations of bacilli exist .