Detection of Mycobacterium tuberculosis in human tissue via RNA in situ hybridization

Rationale Accurate TB diagnosis is hampered by the variable efficacy of the widely-used Ziehl-Neelsen (ZN) staining method to identify Mycobacterium tuberculosis ( Mtb ) acid-fast bacilli (AFB). Here, we sought to circumvent this current limitation through direct detection of Mtb mRNA. Objectives To employ RNAscope to determine the spatial distribution of Mtb 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 Mtb biology. Methods We examined ante- and postmortem human TB tissue using RNAscope to detect Mtb 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 Mtb and identified intact and disintegrated Mtb bacilli and intra- and extracellular Mtb mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchial epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. Conclusions RNAscope has diagnostic potential and can guide therapeutic intervention as it detects Mtb mRNA and morphology in ZN-negative tissues from TB patients, and Mtb mRNA in ZN-negative antemortem biopsies, respectively. Lastly, our data provide evidence that at least two phenotypically distinct populations of Mtb bacilli exist in vivo . ### Competing Interest Statement The authors have declared no competing interest.