Probing the in-vivo reservoir of latent tuberculosis infection
Authors: Ajit Lalvani, Joe Fenn, Timesh D Pillay
Journal: The Lancet Microbe (2021)

For decades the unanswered question of where viable bacilli reside in latently infected individuals has been a central conundrum in tuberculosis. The hitherto insurmountable challenge of identifying dormant Mycobacterium tuberculosis bacilli in asymptomatic latently infected individuals is why diagnosis of latent tuberculosis infection relies on measurement of the cell-mediated immune response to M tuberculosis, by tuberculin skin test or interferon-γ release assay (IGRA).1Whitworth H Scott M Connell DW Dongés B Lalvani A IGRAs—the gateway to T cell based TB diagnosis.Methods. 2013; 61: 52-62Crossref PubMed Scopus (49) Google Scholar The first clues about M tuberculosis's anatomical niche in latent infection stretch back a century when it was shown that post-mortem cadaveric tissue homogenates, from healthy lung as well as fibrotic and granulomatous lesions, could propagate tuberculosis in challenged guinea pigs, a finding replicated in several subsequent studies.2Mayito J Andia I Belay M et al.Anatomic and cellular niches for Mycobacterium tuberculosis in latent tuberculosis infection.J Infect Dis. 2019; 219: 685-694Crossref PubMed Scopus (21) Google Scholar The past decade has seen advances in our understanding of M tuberculosis's anatomical and cellular niches in the absence of active tuberculosis. M tuberculosis DNA was identified in bone marrow mesenchymal stem cells,3Das B Kashino SS Pulu I et al.CD271(+) bone marrow mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5170ra13Crossref PubMed Scopus (129) Google Scholar and subsequently in peripheral blood pluripotent haematopoietic and mesenchymal stem cells from small numbers of treated patients with tuberculosis3Das B Kashino SS Pulu I et al.CD271(+) bone marrow mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5170ra13Crossref PubMed Scopus (129) Google Scholar as well as latently infected IGRA-positive people, but not unexposed IGRA-negative people, in a low-incidence setting.4Tornack J Reece ST Bauer WM et al.Human and mouse hematopoietic stem cells are a depot for dormant Mycobacterium tuberculosis.PLoS One. 2017; 12e0169119Crossref PubMed Scopus (33) Google Scholar Viable M tuberculosis could only be cultured from a minority of these M tuberculosis DNA-positive individuals, and M tuberculosis dormancy-associated gene-expression profiles suggested that the bacilli reside in a hypoxic yet permissive intracellular microenvironment in a state of non-replicating persistence.4Tornack J Reece ST Bauer WM et al.Human and mouse hematopoietic stem cells are a depot for dormant Mycobacterium tuberculosis.PLoS One. 2017; 12e0169119Crossref PubMed Scopus (33) Google Scholar, 5Garhyan J Bhuyan S Pulu I Kalita D Das B Bhatnagar R Preclinical and clinical evidence of Mycobacterium tuberculosis persistence in the hypoxic niche of bone marrow mesenchymal stem cells after therapy.Am J Pathol. 2015; 185: 1924-1934Summary Full Text Full Text PDF PubMed Scopus (33) Google Scholar This finding is supported by the observation that haematopoietic stem cells (HSCs) harbouring M tuberculosis from humans with latent tuberculosis infection propagated tuberculosis after adoptive transfer into mouse models.4Tornack J Reece ST Bauer WM et al.Human and mouse hematopoietic stem cells are a depot for dormant Mycobacterium tuberculosis.PLoS One. 2017; 12e0169119Crossref PubMed Scopus (33) Google Scholar Given this new insight into M tuberculosis' preferred niche during latent infection, and the fact that HSCs are able to circulate between blood, bone marrow, and tuberculous granuloma,6Raghuvanshi S Sharma P Singh S Van Kaer L Das G Mycobacterium tuberculosis evades host immunity by recruiting mesenchymal stem cells.Proc Natl Acad Sci USA. 2010; 107: 21653-21658Crossref PubMed Scopus (77) Google Scholar measurement of M tuberculosis DNA in circulating stem cells might prove useful in improving our understanding of latent infection. In an Article published in The Lancet Microbe, Mulugeta Belay and colleagues13Belay M Tulu B Younis S et al.Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study.Lancet Microbe. 2021; (published online March 31.)https://doi.org/10.1016/S2666-5247(21)00043-4Summary Full Text Full Text PDF Scopus (5) Google Scholar use digital PCR (dPCR) to identify two M tuberculosis complex DNA sequences in peripheral blood HSCs in a large cohort in a high tuberculosis incidence setting, Ethiopia. Cells bearing the CD34 cell surface glycoprotein, a marker for HSCs, were enriched from 100 mL blood drawn from each participant and dPCR applied to CD34-positive and CD34-negative cells. M tuberculosis complex DNA was detected in 156 (79%) of 197 participants and was more commonly detected in CD34-positive than in CD34-negative cells (154 [73%] of 197 vs 46 [23%] of 197; p<0·0001), indicating that the major source of M tuberculosis complex DNA in the periphery was HSCs recently egressed from the bone marrow. This cohort of asymptomatic, untreated, tuberculosis-exposed adults with normal chest radiography comprised recent household contacts of sputum smear-positive tuberculosis cases, cattle farmers with recent exposure to bovine tuberculosis, and M tuberculosis-exposed HIV-infected individuals. Given the inclusion of cattle farmers with bovine tuberculosis exposure, dPCR was specific for M tuberculosis complex, enabling detection of M bovis DNA as well as M tuberculosis DNA. The HIV-infected participants were significantly more likely to harbour dPCR-detectable bacilli (67 [89%] of 75) and to have higher insertion sequence (IS) 6110 copy number than HIV-uninfected participants (89 [73%] of 122; p=0·0065). By contrast, the IGRA was less likely to be positive, and gave weaker responses, in the HIV-infected group (25 [33%] of 75) than in the HIV-uninfected group (73 [60%] of 122; p<0·0001), reflecting HIV-associated cellular immune suppression. This finding highlights the limitations of IGRA in immunocompromised populations, whereas the higher M tuberculosis complex quantitative score (IS6110 copy number) in HIV-infected participants is congruent with the high relative risk of progression to active tuberculosis in latent tuberculosis infection with HIV coinfection. In a nested longitudinal sub-study, both the prevalence of positive M tuberculosis complex DNA results and the intra-individual IS6110 copy numbers decreased significantly following a course of isoniazid preventative therapy. Taken together, these results suggest that M tuberculosis complex DNA in circulating HSCs reflects the presence of viable bacilli and raise the question of whether such an assay might form the basis of a test of cure for latent tuberculosis infection in the future. Surprisingly, 32 (26%) of 122 IGRA-negative HIV-negative participants had detectable circulating M tuberculosis complex DNA. The presence of M tuberculosis complex DNA in these individuals means we can no longer reliably conclude that IGRA-negative tuberculosis-exposed individuals do not have latent tuberculosis infection. It is unclear where on the spectrum of tuberculosis exposure, infection, and disease these individuals lie.7Sridhar S Pollock K Lalvani A Redefining latent tuberculosis.Future Microbiol. 2011; 6: 1021-1035Crossref PubMed Scopus (19) Google Scholar However, given their IGRA-negative status, their risk of progression to active tuberculosis is probably low. Hence, the possibility arises that M tuberculosis complex DNA-positive, IGRA-negative tuberculosis-exposed individuals represent the so-called resister phenotype—ie, people who remain IGRA-negative and tuberculin skin test-negative despite repeated tuberculosis exposure.8Stein CM Nsereko M Malone LL et al.Long-term stability of resistance to latent Mycobacterium tuberculosis infection in highly exposed tuberculosis household contacts in Kampala, Uganda.Clin Infect Dis. 2019; 68: 1705-1712Crossref PubMed Scopus (16) Google Scholar Such individuals were found to have detectable non-classical (ie, interferon-γ-negative) cellular and antibody responses to M tuberculosis antigens,9Lu LL Smith MT Yu KKQ et al.IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure.Nat Med. 2019; 25: 977-987Crossref PubMed Scopus (83) Google Scholar confirming that their immune systems have encountered (or continue to encounter in vivo) M tuberculosis, despite being IGRA-negative. The IGRA-positive, PCR-negative subgroup identified by Belay and colleagues might comprise tuberculosis-exposed individuals in whom M tuberculosis in HSCs is below the limit of detection of dPCR or absent. Absence would imply such individuals have eradicated dormant bacilli but retain T cell memory to M tuberculosis antigens conferring positive IGRA status. This would be consistent with the observation that more than 90% of IGRA-positive adults in low-incidence settings never progress to active tuberculosis, which underpins the growing view that most asymptomatic IGRA-positive people whose tuberculosis exposure was more than a few years ago, and who are conventionally deemed to have latent tuberculosis infection, are no longer infected.10Behr MA Edelstein PH Ramakrishnan L Is Mycobacterium tuberculosis infection life long?.BMJ. 2019; 367l5770Crossref PubMed Scopus (46) Google Scholar, 11Ewer K Millington KA Deeks JJ Alvarez L Bryant G Lalvani A Dynamic antigen-specific T-cell responses after point-source exposure to Mycobacterium tuberculosis.Am J Respir Crit Care Med. 2006; 174: 831-839Crossref PubMed Scopus (189) Google Scholar Ultimately, understanding the clinical implications of these intriguing phenotypes, identified by discordant IGRA and M tuberculosis complex DNA results, requires large-scale longitudinal cohort studies to quantify risk of progression to tuberculosis, similar to those that established the prognostic power of the IGRA.12Abubakar I Drobniewski F Southern J et al.Prognostic value of interferon-γ release assays and tuberculin skin test in predicting the development of active tuberculosis (UK PREDICT TB): a prospective cohort study.Lancet Infect Dis. 2018; 18: 1077-1087Summary Full Text Full Text PDF PubMed Scopus (74) Google Scholar Such studies will involve thousands of participants and take several years to complete. Meanwhile, however, the application of this new probe to investigate the biology of latent tuberculosis infection and triangulation with advanced immunological and metabolic imaging tools in well defined cohorts is likely to yield important new scientific insights in the near future. AL reports issued patents underpinning IGRA and next-generation IGRA some of which were assigned by the University of Oxford to Oxford Immunotec resulting in royalty entitlements for the University of Oxford and AL. All other authors declare no competing interests. Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational studyWe report a novel molecular microbiological biomarker of latent tuberculosis infection with properties that are distinct from those of a commercial interferon-γ release assay. Our findings implicate the bone marrow as a niche for M tuberculosis in latently infected individuals. Detection of M tuberculosis complex DNA in PBMCs has potential applications in the diagnosis of latent tuberculosis infection, in monitoring response to preventive therapy, and as an outcome measure in clinical trials of interventions to prevent or treat latent tuberculosis infection. Full-Text PDF Open Access