Detection ofMycobacterium tuberculosisin human tissue via RNAin situhybridization

Abstract

ABSTRACTRationaleAccurate TB diagnosis is hampered by the variable efficacy of the widely-used Ziehl-Neelsen (ZN) staining method to identifyMycobacterium tuberculosis(Mtb) acid-fast bacilli (AFB). Here, we sought to circumvent this current limitation through direct detection ofMtbmRNA.ObjectivesTo employ RNAscope to determine the spatial distribution ofMtbmRNA 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 andMtbbiology.MethodsWe examined ante- and postmortem human TB tissue using RNAscope to detectMtbmRNA and a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B).Measurements and main resultsWe adapted RNAscope forMtband identified intact and disintegratedMtbbacilli and intra- and extracellularMtbmRNA.MtbmRNA was distributed zonally within necrotic and non-necrotic granulomas. We also foundMtbmRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchial epithelium. Intriguingly, we observed accumulation ofMtbmRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining.MtbmRNA was observed in ZN-negative antemortem lymph node biopsies.ConclusionsRNAscope has diagnostic potential and can guide therapeutic intervention as it detectsMtbmRNA and morphology in ZN-negative tissues from TB patients, andMtbmRNA in ZN-negative antemortem biopsies, respectively. Lastly, our data provide evidence that at least two phenotypically distinct populations ofMtbbacilli existin vivo.