Tuberculosis Infection - Prevalence and Host-based Biomarkers

Introduction
Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), remains a major global health problem, with an estimated 10.6 million new TB cases per year by 2023. The World Health Organization (WHO) aims to eliminate TB by 2035, which includes the management of TB infection (TBI) in low-incidence rate (IR) countries, such as Denmark. TBI is a persistent immune response to stimulation by Mtb antigens (e.g. measured by Interferon Gamma Release Assays (IGRA)) in the absence of clinical evidence of TB disease. There is still no specific standard for diagnosing TBI. In order to manage and reduce the TBI reservoir, it is necessary to determine the national prevalence of TBI and identify specific populations of interest for targeted interventions to guide a Danish TB elimination programme.

TB is a variable disease that primarily manifests in the lungs as pulmonary TB, but TB can infect all other organs (extrapulmonary TB). Diagnosing TB can be difficult due to overlapping symptoms with other diseases, low bacillary load, and the need for invasive sampling for microbiological diagnosis. The WHO desire non-sputumbased biomarker tests for TB with high specificity (target 98%) and a sensitivity of at least 65% in all groups, including extrapulmonary TB. To date, no single biomarker has been able to differentiate between TB, TBI and conditions mimicking TB (CMTB).

Micro-RNAs (miRNAs) are small (18-to 25-nucleotides), single-stranded, noncoding RNA molecules that regulate gene expression by causing translation blockage or mRNA cleavage. MiRNAs exert their functions intracellularly but are also present in plasma. The host response to Mtb infection is intricate and not yet fully comprehended, but various cytokines are involved in controlling TBI. MiRNAs and combined cytokines may act as regulators of TBI and TB by controlling the immune system and serve as markers of TB progression and regression.

Aim of thesis
The aims were to: Paper I) Estimate the period prevalence of TBI and positive IGRA in Denmark in 2014 based on TBI screening data from patients with inflammatory bowel disease (IBD) or inflammatory rheumatic disease (IRD) tested by IGRA in 2010-2018, Paper II) To determine whether T-cell and innate immunity derived cytokines and established biomarkers can discriminate 1) TB from CMTB, 2) TB from TBI, 3) TB from healthy controls (HC)), Paper III) To explore the change in 754 miRNAs between TBI and TB and validate the results. 

Methods
The thesis is based on a cross-sectional registry study, a prospective, observational, diagnostic accuracy study and a prospective, observational explorative cohort study.

Paper I. Based on nationwide Danish registries, we included all patients with IBD or IRD who had an IGRA test performed between 2010 and 2018. We estimated the prevalence of TBI and positive IGRA with 95% confidence intervals (CI) in adults aged 15-64 years after sample weighting, adjusting for distortions in the sample from the background population of sex, age groups and TB IR in the country of birth.

Paper II. We used haemoglobin, total white blood cell count, neutrophils, monocytes, C-reactive protein and ten Meso Scale Discovery analysed cytokines (interleukin (IL)-1β, IL-2, 1L-4, IL-6, Il-8, IL-10, IL-12p70, IL-13, interferon (IFN)-ɣ and tumour necrosis factor (TNF)-α) in whole blood stimulated by lipopolysaccharides (LPS), zymosan (ZYM), anti-cluster of differentiation 3/CD28 (CD3) and unstimulated (Null) TruCulture tubes to invent three index tests to differentiate TB from CMTB and TBI and TBI from HC. We applied recursive feature elimination using a random forest model to identify the most potent biomarkers.

Paper III. We analysed 754 miRNAs using the TaqMan™ Advanced miRNA Human A and B cards after exogenous and endogenous normalisation in persons with TB and TBI. We applied recursive feature elimination using a random forest model to identify the most potent miRNAs to discriminate TB from TBI. We determined these miRNAs and by Mann-Whitney-U test significant miRNAs (total 14 miRNAs) in a validation group from the same population.

Results
Paper I. In 13,574 patients with IBD or IRD, 12,892 IGRA tests (95.0%) were negative, 461 (3.4%) were positive and 221 (1.6%) were indeterminate, resulting in a weighted prevalence of TBI of 3.2% (95%CI: 2.9-3.5) and weighted positive IGRA prevalence of 3.8% (95%CI: 3.5-4.2) among adults aged 15-64 years in the background population of Denmark. The unweighted prevalence of TBI increased strongly with age and among persons born in countries with a TB IR above 10/100.000.

Paper II. In 52 persons with CMTB (n=9), TB (n=23), TBI (n=10) and HC (n=10), we found that the combination of LPS-IFN-ɣ, ZYM-IFN-ɣ, ZYM-TNF-α, ZYM-IL-1β, LPS-IL-4, ZYM-IL-10 and neutrophil count could differentiate TB from CMTB with a sensitivity of 47.8% (95%CI: 26.8 - 67.7) and specificity of 100.0 % (66.4-100.0). The combination of Null- IFN-ɣ, Null-IL-8, CD3-IL-6, CD3-IL-8, CD3-IL-13 and ZYM IL-1b could discriminate TB from TBI with a sensitivity of 73.9% (56.5-91.3) and a specificity of 100% (69.2-100.0). Cytokines and established biomarkers were unable to differentiate TBI from HC with ≥ 98% specificity.

Paper III. In 36 persons (discovery group) with TB (n=18) and TBI (n=18), we detected 495 miRNA and found the circulating miRNAs hsa-miR-148a-3p, hsa-miR204-5p and hsa-miR-584-5p the most important biomarkers to differentiate TB from TBI. Further, 11 miRNAs were differentially expressed. In the validation group of 59 individuals with TB (n=37) and TBI (n=22), many of the miRNAs showed the same pattern but the fold changes of the 14 miRNAs were minor.

Conclusion
The estimated TBI in Denmark is low. Our data suggest that a TB elimination programme should focus on persons born in high TB endemic countries and age above 44 years. Selected combinations of cytokines may serve as blood-based add-on tests to detect TB in low-endemic settings, although these results require validation. We investigated circulating miRNAs to differentiate TB from TBI but we were unable to validate the findings from the discovery group.