Evaluation of a novel microbiological diagnostic test for latent tuberculosis infection in Ethiopia (Micro-LTBI)

Lead Research Organisation: Queen Mary, University of London
Department Name: Blizard Institute of Cell and Molecular

Abstract

Tuberculosis (TB) is a bacterial infectious disease. It causes 1.5 million deaths each year, 95% of which occur in low- and middle-income countries (LMICs). The disease particularly affects young adults, with adverse consequences for economic growth. In many LMICs, this problem is compounded by the problem of TB in cattle (bovine TB). This 'double-hit' is a particular problem in Ethiopia, which has the tenth highest burden of human TB in the world: bovine TB is widespread (prevalence 5-30%) and the country's economy is highly dependent on cattle farming. Improved TB control in humans and cattle would significantly enhance economic growth and welfare in Ethiopia and other similarly-burdened LMICs.

Most TB cases in humans arise when a dormant TB infection re-activates to cause disease. Dormant TB infection is very common in LMICs and this pool of infection will need to be treated if TB is ever going to be eliminated. Antibiotic treatment for dormant TB infection is effective, but roll-out of this treatment is limited by the lack of an accurate 'gold standard' diagnostic test for the condition. Existing tests for dormant TB infection rely on detecting an immune response to the bacteria, rather than detecting the bacteria itself. However, less than 10% of those with a positive immune test ever go on to develop active TB. This makes it difficult to target antibiotic treatment for dormant TB infection at the people who will benefit from it.

This situation could be about to change: very recently, a team of researchers in Germany (one of whom is a co-applicant on this grant) has reported that they have been able to detect TB bacteria in the blood of people with dormant TB infection. The number of bacteria is very small, and quite a large amount of blood (100 ml) is needed in order to detect them reliably. This remarkable finding has been made in just seven people living in Europe. The next step is to run the new test in a larger group of people who live in a country where TB is widespread. To take this idea forward, we have forged a new collaboration between Ethiopian and British scientists who together have the exact combination of clinical and technical skills needed to evaluate this new test.

We will carry out the following studies. First, we will recruit 200 healthy people who have recently been in contact with an infectious case of TB ('TB contacts') - 100 urban residents of Addis Ababa who have been exposed to a person with infectious lung TB living in their home, and 100 rural cattle herders who have been exposed to a cow suffering from bovine TB. We will run the new blood test in these TB contacts, and, where we find dormant TB bacteria in the blood stream, we will do genetic fingerprinting to see if the dormant bacteria found in the bloodstream are identical to those isolated from the case of infectious TB (human or bovine) to which they were exposed. If the genetic fingerprints of the TB bacteria are the same, this will suggest that detection of TB bacteria in the blood of people with dormant TB infection is very unlikely to be a 'chance finding'.

We will also recruit 40 people who are about to start antibiotic treatment for dormant TB infection, and run the new test before and after treatment. If we find that the antibiotic treatment clears dormant TB bacteria from the blood stream, this would suggest an additional potential application for the new test: monitoring response to antibiotic treatment of dormant TB infection. This is important, because existing tests cannot tell if antibiotic treatment for dormant TB infection has been effective or not.

Taken together, these studies will give us a clear idea as to whether the findings of the German team can be reproduced in a LMIC setting. If so, then we will apply for further funding to optimise the test to use smaller blood volumes, and conduct definitive studies to evaluate whether it could be integrated into existing TB control programmes in LMICs.

Technical Summary

Background

Efforts to identify and treat people with latent tuberculosis (TB) infection are impeded by the lack of a gold standard diagnostic test for this condition. Very recently, it has been reported that DNA of M. tuberculosis complex (MTBC) can be detected in CD34+ pluripotent haematopoietic stem cells isolated from peripheral blood of sensitised healthy TB contacts (n=7, Europe), using qRT-PCR.

Research Objectives

To determine whether:

a) qRT-PCR consistently detects MTBC DNA in CD34+ peripheral blood mononuclear cells (PBMC) of a larger cohort of sensitised healthy TB contacts living in Ethiopia;

b) MTBC strains isolated from human and bovine index cases of active TB in urban and rural Ethiopia are genetically identical to those detected by qRT-PCR in CD34+ PBMC of associated contacts;

c) Chemoprophylaxis renders MTBC DNA undetectable in CD34+ PBMC of Ethiopian adults who were qRT-PCR-positive at baseline.

Study design

We will conduct cross-sectional studies enrolling 200 asymptomatic contacts of infectious TB cases in humans and cattle. The novel qRT-PCR assay will be used to detect MTBC DNA in participants' PBMC. Where this is found, amplicon libraries will be generated and sequences compared to those of MTBC isolates from associated human / bovine index cases. We will also conduct a prospective study, in which results of the novel qRT-PCR assay will be compared pre- vs. post-chemoprophylaxis in 40 people with latent TB infection.

Laboratory techniques

MTBC-specific DNA in CD34+ PBMC will be quantitated by qRT-PCR. PCR-based methods will also be used to selectively amplify SNP-rich regions (amplicons) in MTBC genomic DNA detected in CD34+ PBMC of TB contacts. Amplicon sequences will be compared with those identified in MTBC isolated from index cases.

Application of results
This research could lead to the development of a more specific test for latent TB infection, with potential to revolutionise global TB control.

Planned Impact

1. Who will benefit from this research?


The following groups could potentially benefit from this research:

a) People with latent TB infection - recently or remotely acquired, living in high-, medium- or low-income settings
b) Policy-makers in the TB control community at international, national and local levels
c) The TB research community, in particular epidemiologists, clinical trialists, microbiologists and immunologists
d) Partner academic institutions
e) Study participants
f) Study staff

2. How will they benefit from this research?

If results of this study show promise, and subsequent large-scale evaluations confirm a niche for this new test in TB control programmes, the primary beneficiaries will be the wider public living in high TB transmission settings: they will benefit from the development of a more specific test for latent TB infection, that could be used to target chemoprophylaxis at specific populations who stand to benefit from it.

Policy-makers in the TB control community will also stand to benefit - the study could eventually yield a new diagnostic tool for latent TB infection that could make roll-out of chemoprophylaxis much more cost-effective. Interested parties include those working internationally (e.g. WHO Stop TB Partnership and non-governmental organisations), and nationally (e.g. Ethiopian Ministry of Health). Positive results of the study will have international relevance, as TB is a global problem. For example, results of the study could have relevance to the UK, where the vast majority of cases of active TB arise as a result of reactivation of latent TB infection: if latent infection could be diagnosed with greater specificity, and response to chemoprophylaxis could be assessed, then cost-effectiveness of efforts to identify and treat latent TB infection would be improved.

Researchers in TB epidemiology, clinical trials, microbiology and immunology could also benefit from the study. Improved knowledge about transmission pathways arising from the sequencing work proposed could inform scientific efforts to develop interventions to control TB.

Several parties directly involved in the conduct of the study will also benefit. Individual participants will benefit from screening for active TB, which they would not otherwise receive. Research staff will benefit from training in research methodology, phlebotomy and computer skills, all of which are applicable in the wider healthcare or academic sectors. Participating institutions will also benefit from new UK-Ethiopian links that could be used to conduct further collaborative projects, with potential benefit to human health in both countries.

If the new qRT-PCR test shows promise in the current study, the benefits above would be likely to be realised within the medium- to long-term. The investigation we propose is designed to rapidly inform a go/no-go decision on the utility of the novel microbiological test; definitive evaluation would require larger studies with longer follow-up, which are beyond the scope of a GCRF Foundation Award. We would seek support for such studies from MRC Response Mode Schemes if the current project yielded positive results.

Publications

10 25 50
 
Description We have discovered that tuberculosis DNA can be detected in the blood of 80% of adults studied in Ethiopia. Infection was commonest in stem cells, suggesting the bone marrow as a niche for tuberculosis bacteria in latently infected individuals. Infection was cleared by antibiotic treatment in 50% of people. Our findings have potential to form the basis for development of a new generation of microbiological biomarkers of tuberculosis, with clinical applications in diagnosis and in monitoring response to treatment.
Exploitation Route Our findings will form the basis of research by others who will want to know if they can be replicated
Sectors Healthcare

 
Description Evaluation of a novel microbiological diagnostic test for latent tuberculosis infection
Amount £50,000 (GBP)
Organisation Queen Mary University of London 
Sector Academic/University
Country United Kingdom
Start 11/2019 
End 09/2020
 
Description THRiVE-2 Clinical PhD Fellowship
Amount £23,640 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 12/2021
 
Title New microbiological biomarker of tuberculosis infection 
Description New digital PCR assay for detection of M. tuberculosis DNA in peripheral blood of latently infected individuals 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact None yet - report still in review (Lancet Microbe) 
 
Description Collaboration with Makerere University, Uganda 
Organisation Makerere University College of Health Sciences
Country Uganda 
Sector Academic/University 
PI Contribution I supervise a PhD student at Makerere (Dr Jonathan Mayito) who came to train in my lab - he worked on samples from the current project, and has set up a sister project in Uganda with independent funding from the Wellcome Trust
Collaborator Contribution Recruitment of 180 participants to a sister study; DNA extractions; PCR
Impact Publication: https://www.ncbi.nlm.nih.gov/pubmed/30376080
Start Year 2019
 
Description Collaboration with National University of Medical Sciences, Pakistan 
Organisation National University Of Medical Sciences, Pakistan
Country Pakistan 
Sector Academic/University 
PI Contribution Provision of training to post-doc from Pakistan who visited my lab; support for pilot study in Pakistan along same lines as MRC-funded study in Ethiopia
Collaborator Contribution Contribution of 9 months post-doc time to Ethiopia project; set-up of sister study in Pakistan
Impact N=41 pilot study. Manuscript in review with Lancet Microbe
Start Year 2019
 
Title New biomarker of TB infection 
Description New biomarker of latent TB infection, early development, seeking further funding from MRC / Wellcome 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Early clinical assessment
Year Development Stage Completed 2020
Development Status Actively seeking support
Impact Not yet at implementation stage