Building a platform to understand asymptomatic and symptomatic visceral leishmaniasis in Ethiopia

Visceral leishmaniasis (VL) is a parasitic disease caused by Leishmania (L.) donovani. It is one of the most neglected tropical diseases, affecting the poorest populations, for whom access to diagnosis and effective treatment is not readily accessible. In Ethiopia, over 3.2 million people live in areas where this disease is endemic and are therefore at risk of infection with L. donovani. While the majority of individuals infected with L. donovani show no symptoms and are therefore designated as asymptomatic; some develop a disease that, if left untreated, is almost always fatal. It is not understood why some individuals can control infection and stay asymptomatic whereas others develop progressive non-healing VL. Our aim is to identify distinct immunological and gene expression signatures that are associated with the control of parasite replication in asymptomatic individuals or uncontrolled parasite replication and dysfunctional immune responses in VL patients. To do this, we have identified a unique population of individuals in Northwest Ethiopia, in the Metema-Humera area: this is the most important endemic focus of VL in Ethiopia and accounts for 60% of the VL burden in the country. In this area, during the months of June to October, the intense seasonal agricultural activities attract >250,000 migrant workers, for weeding and harvest. The majority of these seasonal labourers come from leishmaniasis-free areas and travel for seasonal work to the Metema-Humera area where leishmaniasis is widespread. These migrant workers are at higher risk of being infected by Leishmania parasites as they live and work in areas where they are constantly being exposed to the sandflies that transmit Leishmania parasites. The environment and working conditions on these farms are extremely harsh and therefore, few studies have been made of these migrant workers, little is known about their lifestyle and their travelling and working habits. Therefore, here we propose a proof of concept study to demonstrate that we can:

- Recruit sufficient numbers of migrant workers and follow them during and after the agricultural season.
- Identify those who have been infected with L. donovani parasites by using different immunological, serological and molecular tests.
- Collect blood and store it during our visits to the farm, transport it to our laboratory, 200km away from the farm, to send to the UK to quantify genome-wide host gene expression; as well as perform basic functional assay on this farm.
- Generate preliminary epidemiological, immunological and gene expression data to identify immune signatures associated with either asymptomatic infection or VL disease.

The results of this work will serve as a foundation to plan a larger study in the Metema-Humera area to identify immunological and gene expression signatures of each stage of the development of asymptomatic infection or VL. This will be essential for the development of new strategies to prevent and treat leishmaniasis.

Visceral leishmaniasis (VL), a neglected tropical disease that causes substantial morbidity and mortality, is a growing health problem in Ethiopia. Most infections remain asymptomatic but some individuals develop symptomatic VL, which is fatal if not treated. The immunological mechanisms responsible for control of infection in asymptomatic individuals or uncontrolled parasite replication in VL patients are poorly understood. We have identified a setting in Northwest Ethiopia that will allow us to follow migrant workers when they first arrive in this endemic area, without previous exposure to Leishmania parasites; and follow them longitudinally until they are infected with Leishmania and subsequently either control the infection and stay asymptomatic, or progress to VL. Here we propose a proof of concept study to establish the potential of a farm in this area to:
1. Recruit and characterise sufficient numbers of exposed and unexposed migrant workers in the farm and follow them over 16 months.
2. Demonstrate the robustness of tests for seropositivity to Leishmania parasites in these largely asymptomatic individuals by using different diagnostic techniques.
3. Establish the best way to perform basic functional assays on-site and store blood to send to the UK for RNAseq transcriptomic analyses.
The results obtained from this study will shed new light on the demographic and epidemiological profiles of this population; the incidence of seroconversion, control of infection or development of VL; vector exposure; and will allow us to identify immune signatures associated with asymptomatic infection and VL. Taken together, these results will inform the design of a full-scale, longitudinal study to identify genes and immunological pathways that result either in the control of infection in asymptomatic individuals or in the development of VL. This understanding is an essential prerequisite to improving the prevention and treatment of this common and devastating condition.

The present project is designed as a proof of concept study to ascertain the migratory pattern of the migrant workers and the prevalence of asymptomatic individuals, as well as of those developing VL, in order to establish the potential of the Zeleke farm as a suitable site for the large-scale study. Our planned investigation will have a number of different types of impact.

Our scientific results will have direct and immediate impacts. Given the relative lack of clinical cohorts with in-depth immunological characterisation, our results will be of wide interest to basic scientists interested in immunology. In particular, researchers working on other Leishmania species will be interested in testing the generality of our findings.

In the longer term, our results will also impact more applied scientists and clinicians in both industry and academia interested in Leishmania control. The host signatures we identify should feed into the development of new-targeted immunotherapies and diagnostics that can be implemented in low- and middle-income countries. For example, diagnostic tests that could predict the likely course of a L. donovani infection would allow early treatment targeted at patients likely to progress to VL and so could have immediate impact on patient care. Following further work, there could be longer-term impacts on the treatment of leishmaniasis. Better understanding of host and parasite pathways involved in leishmaniasis pathology might also reveal novel targets for novel drugs against these diseases. These would be particularly welcome as existing drugs are either highly toxic or prohibitively expensive for many endemic regions and resistance to many anti-Leishmania compounds is an increasing problem.

In order to undertake this project we have brought together a team of specialists in immunology, epidemiology, transcriptomic and treatment of VL. This work will also impact on the careers of the staff and students involved in the project by further training and acquisition of new skills. They will be working in close association with the applicants and partners and will present their work in lab meetings and group discussions. The applicants will also interact with and train other students and visitors working on related projects in the laboratories involved. These impacts will be particularly important in Gondar, where this project should give a significant boost to the research base and contribute greatly to developing capacity for future research. We will also use the results of our research for public engagement events that will benefit the wider public by increasing awareness of leishmaniasis and neglected tropical diseases in general.