How latent is “latent” tuberculosis? The radiographic, transcriptional and immunological characterisation of subclinical tuberculosis in HIV infected adults

Abstract

Background: The central hypothesis of the thesis is that the neat division of tuberculosis (TB) into states of active disease and latent infection is an oversimplification and that the transition between latent and active TB involves passage through a subclinical phase of disease, which may be prolonged, during which pathology evolves. The primary aim of this thesis is to utilise [18F]-fluoro-2-deoxy-D-glucose positron emission tomography combined with computed tomography (FDG-PET/CT) to identify and define intra-thoracic pathology consistent with subclinical TB in a cohort of asymptomatic adults diagnosed with latent TB at high risk of developing active TB (due to HIV co-infection) and then to identify transcriptional and immunological biomarkers that distinguish those with radiographic evidence of subclinical pathology. Such biomarkers may have future translational potential as tests more predictive of active TB compared to the currently available tests (tuberculin skin testing (TST) and interferon gamma release assays (IGRA)) and may also aid our understanding of the biology of TB.

Methodology: Healthy HIV infected, ART naïve, adult outpatients living in an area with very high TB burden (Khayelitsha township, Cape Town, South Africa) were screened to identify 35 participants that were asymptomatic, with CD4 count ≥ 350/mm3, evidence of latent TB (by QuantiFERON Gold in tube (QFGIT)) and with no history of previous tuberculosis or evidence of current active TB. These participants had FDG-PET/CT performed and were then commenced on isoniazid preventive therapy (IPT) or standard TB therapy if clinically indicated and had repeat FDG-PET/CT following treatment. A number of additional groups of HIV infected and uninfected control participants with and without active TB were also recruited for blood sampling. Microarray, carried out on RNA extracted from whole blood, was used to identify differentially abundant transcripts between those with and without subclinical pathology. A 38-plex assay and ELISA covering a total of 45 analytes were then used to identify serological or QFGIT plasma biomarkers that distinguish those with and without subclinical pathology.

Main Results: Parenchymal abnormalities in the 35 participants were evaluated in detail and interpreted in relation to the historical autopsy data and 28.6% were categorised as having evidence of subclinical TB pathology. Analysis of the whole blood microarray for these 35 participants along with 15 age, sex and CD4 count matched controls with clinical active TB identified 82 transcripts that clustered 80% of those with subclinical TB with active TB. Those with more metabolically active subclinical pathology, as determined by FDG uptake, clustered more effectively with clinical active TB. This signature was confirmed as specific to TB in HIV uninfected controls. Transcripts relating to the classical complement pathway and Fcγ receptor were found to be overabundant in subclinical and active TB in relation to those with latent TB with no evidence of subclinical pathology. Neutrophil related transcripts were over abundant only in clinical active TB, particularly in those that were smear positive. Network analysis of the 82 transcript signature, informed the selection of 45 soluble protein analytes. 10 analytes showed a significant difference in concentration between the 3 groups (active, subclinical and latent TB). IL-1α with a cut-off of 16.9 pg/mL and circulating immune complex (CIC) with a cut-off of 100.9 μg Eq/mL individually classified 50% and together 70% of those with subclinical TB as active TB. In addition when assessed across 5 stages of increasing disease activity by PET findings and smear status all 10 analytes showed a significant increasing trend.

Conclusion: The utility of FDG-PET/CT a novel research tool in the study of latent TB in humans has been systematically evaluated for the first time in this thesis. It has allowed for the identification of pathology within the lungs consistent with subclinical TB not reliably identified on CXR. Microarray analysis of whole blood has contributed of our understanding of which biological process may be pertinent from the early subclinical stages of disease, suggesting that the classical complement pathway and overabundance of Fcγ receptor may be important. Furthermore, the approach has lead to the identification of transcriptional and serological biomarkers that distinguish those with subclinical pathology from those without. These biomarkers may have translational potential as more predictive diagnostic tests for active TB.