Characterising the influence of parasite variation on visceral leishmaniasis.

Visceral leishmaniasis, also called kala azar, is a neglected infectious disease that affects the world poorest in over 98 countries, resulting in 20,000 fatalities a year. Kala azar is caused by single-celled Leishmania parasites that are transferred by sand flies and migrate to internal organs such as the spleen or bone marrow. The disease is frequently fatal if untreated. Better treatments are essential to eliminate kala azar from poor communities.

There is no vaccine to protect people in regions where sand flies carry the parasite. Drugs that are used to treat the disease can cause painful adverse effects, require hospitalisation and do not reliably kill all the parasites. Drug-resistant parasites have also evolved several times. Even when drugs do cure patients, kala azar parasites can persist in disfiguring skin lesions called post-kala azar dermal leishmaniasis (PKDL). It is important to cure PKDL, because the lesions contain live parasites, they are sources of new transmission by sand flies and may fuel new disease outbreaks. At present, we do not know why PKDL occurs, or how to stop it.

Most cases of kala azar occur in Brazil, Ethiopia, India, Kenya, Somalia, South Sudan and Sudan. Different forms of kala azar and PKDL occur between continents and within countries. For example, PKDL is common in some regions of East Africa, but never occurs in other regions. African and Indian PKDL also takes different forms. This may be due to genetic differences between the people, or because the Leishmania parasites are different, so cause different types of disease. The role of parasite variation on disease variation is not well established. It is important to understand the differences between parasites because they may require different treatments, for example it is known that some anti-Leishmania drugs are effective in India, but not effective in Brazil. Investigating parasite variation may also help us to understand how parasites evade immune systems and persist for months or years as skin lesions.

This project will study the influence of parasite genetic variation on kala azar and PKDL. It is known that East Africa contains the most genetically diverse Leishmania donovani ecotypes, so Africa is the best place to study this. We will work with a kala azar study in East Africa that is collecting detailed clinical and immune cell data from 40 patients in each of Sudan, Ethiopia, Kenya and Uganda (160 in all). Patient immune cells in blood will be taken before treatment, after treatment, and then months later when PKDL can occur. We will isolate the Leishmania parasites from each of the 160 patients when they are diagnosed and enrolled in the study. We will sequence the parasite genomes, and produce a detailed catalogue of all the genetic differences between them.

Because every species carries their evolutionary history in their genes, we can use these genomes to describe current and historic parasite migration across East Africa, showing how ecotypes are distributed. We will then use genetic methods to determine whether difference between parasites cause differences in kala azar symptoms or cause PKDL. We will be able to provide some early warning of the evolution of drug resistant parasites in Sudan, Ethiopia, Kenya or Uganda. Leishmania parasites alter host immune systems to evade detection. Because we will have detailed immune cell data from each patient, we can also determine whether some parasites are more effective at evading the immune system. Finally, we will be able to determine if specific parasite genes help them evade the immune system, cause PKDL or allow them to resist drugs.

This study will produce a better understanding of the parasites that cause kala azar and PKDL. This research may help us to design new drugs or new vaccines so we can eliminate the disease.

Visceral leishmaniasis (VL, also called kala azar) is a neglected infectious disease that affects the world poorest, resulting in 20,000 fatalities a year. VL is caused by Leishmania protozoan parasites, transmitted by sand flies. VL treatments need considerable development; no VL-vaccine is licensed and drug therapies are subject to resistance and/or require hospitalisation and do not reliably produce parasitological cure. VL can progress to persistent skin lesions (post-kala azar dermal leishmaniasis, PKDL) after successful drug treatment that are sources of transmission, likely fuel new epidemics, but is very poorly understood. VL/PKDL manifestations and drug efficacy vary between and within countries. There are indications that this variation may be due to differences between Leishmania donovani parasites, but the role of parasite variation on disease variation is not well established. This project will investigate the influence of parasite genetic variation on VL by studying L. donovani in Sudan, Ethiopia, Uganda and Kenya. We will sequence parasites genomes from 160 patients who are undergoing detailed clinical and immunological study pre- and post-drug treatment up to 12 months. We describe parasite demographics, migration, evolutionary history and patterns of molecular evolution. We will then apply genome-based restricted maximum likelihood (GREML), a sensitive quantitative genetics method, to quantify the effects of parasite variation on traits of treatment efficacy, cellular immune responses and disease manifestations such as PKDL. Genome-wide association studies (GWAS) will be used to detect parasite genes that have the strongest effects on treatment or disease. This study has potential to uncover molecular mechanisms that parasites evolve that modulate and evade immune systems, contributing to our understanding of host-parasite interactions. Study of drug treatment efficacy will detect geographically-restricted drug resistance with high sensitivity.

Visceral leishmaniasis (VL) is an infectious disease that affects the world's poorest and is not well controlled by drug treatments or vaccines. There is a clear need for development of new therapies to treat this research-neglected disease. East Africa has the highest burden of VL worldwide, but parasites from this region are very poorly studied. At present our understanding of host-parasite interactions in Africa is not sufficient to allow rational drug or vaccine design.

Our research aims to enhance quality of life for people of VL-endemic areas by contributing to the reduction of disease-causing infections and control of the parasite. At present, VL has an approximate 10% case fatality rate. Even with effective treatment, disfiguring skin lesions (PKDL) can persist, providing a reservoir for new infections and stigmatising affected people. Our research may explain why PKDL occurs and lead to cures that increase quality of life. VL treatments currently require long in-patient intravenous/intramuscular drug courses, so treatment improvements will also have long-term economic benefits to disadvantaged communities.

To ensure that our research improves the control and treatment of VL, we will promote direct communication with VL researchers and organisations that have potential to develop our findings. An important aspect of ensuring impact will be highlighting potential applications.

As fundamental research, we envisage impacts mid- or long-term. Our work will produce novel insights into host-pathogen interactions, promoting understanding of disease. As we may identify parasite genes that cause specific host immune modulations, we may provide leads to vaccine and drug development. Discovery of genetic markers for disease manifestations including PKDL may lead to predictive genetic or serological tests for PKDL risk factors.

The fundamental scientific knowledge that this project will create has potential to influence research directions. We actively promote re-use of our data and are active in publishing and international presentations. Direct influences on new researchers will be provided by teaching bioinformatics workshops to postgraduate students in West Africa (Ethiopia, Sudan), and training a PhD student in York for 11 months.

Impacts will be facilitated by the environment in the York Biomedical Research Institute (YBRI). YBRI houses a highly active environment of Leishmania researchers, with an extensive network of collaborators in VL-endemic regions of East Africa, Brazil and South Asia, including the Newton MRC/FAPESP Joint Partnership in Leishmaniasis, a UK/Brazil research network (www.york.ac.uk/biomedical-research-institute/news/jcpil/).

This New Investigator project includes VL-research collaborators from regions with the highest burdens of disease, including Sudan, Ethiopia, Kenya and Uganda. Travel funded by this award will facilitate collaboration through visits to these partners and funds to enable technical work in collaborators laboratories, enabling their research capacity. All collaborators' institutions are members of the DNDi Leishmaniasis East Africa Platform (LEAP), that aims to improve treatment and provide capacity strengthen for VL in East Africa (www.dndi.org/strengthening-capacity/leap-platform/). These connections will promote our findings regionally. The project will utilise data from the PREV_PKDL vaccine trial and phenotyping study (www.prevpkdl.eu) funded by the European and Developing Countries Clinical Trials Partnership (EDCTP). Contact with the EDCTP will help to promote wide communication of our results.