Identification of human and pathogen molecular variants associated to tuberculosis heterogeneity

• Author(s): Bastos, Hélder Novais
• Date: 2017
• Persistent ID: https://hdl.handle.net/1822/50226
• Origin: RepositóriUM - Universidade do Minho
• Document Type: Doctoral thesis
• Language: English
• Contributor(s): Universidade do Minho; Saraiva, Margarida; Castro, Rui Manuel Rosário Sarmento

Description

Tuberculosis (TB) imposes high human and economic tolls. One of the most striking features of TB is the variability of infection outcomes, which has been classically attributed to environmental and host determinants. More recently, studies uncovering Mycobacterium tuberculosis complex (MTBC) genomic diversity have shown the potential importance of pathogen-related factors to the disease pathogenesis. We approached this question from different angles, by combining the study of the pathogen properties, the host immune response and the clinical features of TB, within a cohort of 681 culture-confirmed pulmonary TB (PTB) cases diagnosed at the Hospital de São João, a major healthcare center in Porto, Portugal, between 2007 and 2013. We started by developing a severity assessment tool for stratifying mortality risk in PTB patients. Five risk features were selected for the prediction model: hypoxemic respiratory failure (OR 4.7, 95% CI 2.8-7.9), age ≥50 years (OR 2.9, 95% CI 1.7-4.8), bilateral lung involvement (OR 2.5, 95% CI 1.4-4.4), ≥1 significant comorbidity – HIV infection, diabetes mellitus, liver failure or cirrhosis, congestive heart failure and chronic respiratory disease – (OR 2.3, 95% CI 1.3- 3.8), and hemoglobin <12 g/dL (OR 1.8, 95% CI 1.1-3.1). A TB risk assessment tool (TReAT) was developed, stratifying patients with low (score ≤2), moderate (score 3-5) and high (score ≥6) mortality risk. The mortality associated with each group was 2.9%, 22.9% and 53.9%, respectively. The model performed equally well in the validation cohort. After focusing on the host clinical prognostic predictors, in the second part of the project we assessed the impact of M. tuberculosis diversity on the disease clinical severity. We started by developing a clinical decision tree to classify the severity of the disease and by applying it to a selected group of 133 individuals that in our cohort did not present known predictor or precipitator TB factors. We found that, for this group of patients, no association existed between the severity of disease and the phylogeny of the infecting bacteria. We also found that M. tuberculosis isolates from patients with mild disease grew significantly slower, while strains associated to moderate outcome had a longer lag phase and reached the highest plateau, after a steep exponential phase. To gain in-depth knowledge of the genetic basis for differential mycobacterial growth, we performed whole genome sequencing analysis. We detected several single nucleotide polymorphisms (SNPs) in genes that were previously associated with growth suppression and identified novel gene candidates involved in membrane transport and biosynthetic pathways. Finally, in the third part of this work, we studied the architecture of the immune response triggered by the different isolates of M. tuberculosis. Sixteen clinical isolates associated with different clinical severity of TB were selected and used to infect peripheral blood mononuclear cells (PBMCs) from nontreated/ non-recent latent TB infected (LTBI) donors or past/cured TB patients. Independently of the host genetics, we identified two distinct groups of M. tuberculosis isolates: high versus low inflammatory triggers. Furthermore, we report that PBMCs from past TB patients produced less IL-1β than those from LTBI participants in response to a variety of isolates, whereas the opposite was observed for IL-1RA. LTBI subjects elicited responses with significantly higher IL- 1β/IL-1RA ratios than those from TB patients, thus suggesting this ratio as a discriminator of risk for latent to active TB progression. Overall, we provide a new clinical prediction rule for the risk of death in TB patients and propose a new classification tree for TB severity. On the pathogen side, we unveiled the differential growth of clinical isolates associated with moderate outcomes of TB as a distinctive feature. On the host side, we suggest the ratio IL-1β/IL-1RA as a possible biomarker of disease resistance versus susceptibility to TB. Our findings present new platforms for active and latent TB management and open new avenues for basic research, to unveil host and pathogen determinants of TB outcomes.