Detection of Mycobacterium tuberculosis DNA in CD34 + peripheral blood mononuclear cells of Ugandan adults with latent infection: a cross-sectional and nested prospective study.

Abstract

Background: Tuberculin skin test and interferon gamma release assay (IGRA) show limitations in diagnosing latent tuberculosis infection (LTBI) and poorly predict progression to active tuberculosis. This study will explore detection of Mycobacterium tuberculosis ( M.tb) DNA in CD34 + peripheral blood mononuclear cells (PBMCs) as a biomarker for LTBI and monitoring chemoprophylaxis response. Methods: In a cross-sectional study, 120 household contacts (60 HIV positive and 60 HIV negative) will be recruited. Also, 10 patients with sputum positive pulmonary tuberculosis and 10 visitors from low incidence countries with no history of TB treatment will be recruited as positive and negative controls, respectively. Participants will donate 100 ml (50 ml for TB patients) of blood to isolate PBMCs using density gradient centrifugation. Isolated PBMCs will be separated into CD34 + and CD34 - enriched cellular fractions. DNA from each fraction will be purified, quantified and subjected to droplet digital PCR targeting IS6110 (a M.tb Complex multi-copy gene) and rpoB, a single copy gene. Also, 4 ml of blood will be drawn for IGRA. In a nested prospective study, 60 HIV positive participants will be given 300 mg of Isoniazid Preventive Therapy (IPT) daily for six months, after which they will donate a second 100 ml blood sample that will be processed as described above. Data from the cross-sectional study will be analysed to determine the proportion of individuals in whom M.tb DNA is detectable in CD34 + and CD34 - fractions and number of M.tb genomes present. Data from the prospective study will be analysed to compare the proportion of individuals with detectable M.tb DNA in CD34 + and CD34 - fractions, and median M.tb genome copy number, post vs pre-IPT. Discussion: This study will determine whether detection of M.tb DNA in CD34 + PBMCs holds promise as a biomarker for LTBI and monitoring chemoprophylaxis response.