Assessing treatment with miltefosine as an intervention strategy for visceral leishmaniasis in Brazil

Leishmaniasis is a severe disease of humans and one of the world's most neglected diseases, primarily affecting the poor in developing countries. 350 million people are at risk of contracting the disease and it has severe costs in both health and economic terms and drains resources that could be used to promote growth of developing nations. There is no effective vaccine against the disease and chemotherapy is the prime means for reducing the leishmaniasis burden. Visceral Leishmaniasis (VL) is the most severe form of leishmaniasis with 6,000 cases recorded in Brazil each year; it is caused by Leishmania infantum and transmitted by sandflies. Unfortunately the drugs available to treat VL have many limitations and new drugs, or new ways of using currently licensed drugs are desperately needed. One such drug is miltefosine, which has been used successfully to treat VL in India, but which was not so successful in a clinical trial in Brazil in 2006.

Our recent research suggests that a specific section of the L. infantum genome (termed the MSL) is lost in some Brazilian strains of the parasite making them less susceptible to the drug miltefosine. The aim of our foundation project is to determine how prevalent the MSL is in the population of parasites that are found in Brazil. To do this we will validate a genetic diagnostic test using PCR, as well as sequence the genomes of L. chagasi parasites isolated from patients in the State of Piaui in Brazil. If the MSL is found to be present often, then a new clinical trial could be run, where leishmaniasis patients with parasites containing the MSL would be treated with miltefosine, whilst those leishmaniasis patients with parasites that do not contain the MSL would be treated using current drugs. We will genetically manipulate the MSL genes in Leishmania genes to find out why the MSL confers sensitivity to miltefosine. We shall also investigate why the MSL is lost in L. infantum, by testing if a natural product found in local Brazilian trees causes selection of parasites whilst in the sandfly vector.

Overall, we expect the outcome from this study to be a greatly improved understanding of how the environment in which the parasite lives influences its susceptibility to drugs and the molecular mechanisms of drug resistance.

We have identified a genomic locus, the miltefosine sensitivity locus, MSL, which is potentially a major determinant of miltefosine efficacy for the treatment of Visceral Leishmaniasis. In this proposal we shall investigate the presence or absence of the MSL in L. infantum in Brazil, as this could inform on whether a new clinical trial with stratified patients might be viable and whether a PCR-based diagnostic test for the MSL should be developed. The molecular basis by which the MSL confers susceptibility of the parasite to miltefosine will be investigated. We will also study the environmental basis for the loss of the MSL in Brazilian L. infantum. Sandflies, the insect vector that transmits Leishmania, take food from plants. We shall test the hypothesis that Brazilian trees of the family Fabaceae fed on by the sandfly contain a natural product(s) that selects for loss of the MSL via genome rearrangement in the parasite. Identification of the plant(s) and natural product (s) involved would help define the range of MSL-containing L. infantum in Brazil, though Geographic information system (GIS) mapping of the specific natural product-containing trees.
To achieve this we shall
1. Determine the prevalence of the MSL in Leishmania infantum circulating in Brazil by PCR analysis of DNA taken from infected phlebotomine flies, VL patients and domestic dogs (zoonotic reservoir). Also, to identify plants on which infected phlebotomine flies have fed.
2. Identify the molecular mechanism by which the MSL confers susceptibility to miltefosine by CRISPR-Cas9 directed genetic modification of L. infantum.
3. Test if there is a statistical association between natural products identified in specific plants and loss of MSL across geographical regions. This will combine GIS data on plant locations with identification of miltefosine-like natural products by mass spectrometry.

The primary aim of this project is to attain new knowledge, innovation, scientific advancement and the development of a sustainable UK:Brazil network. The work aims to investigate fundamental aspects of the biology of Leishmania infantum, which causes one of the world's most prevalent neglected infectious diseases (NIDs), Visceral Leishmaniasis (VL). A deeper understanding of the interaction of the pathogen with its hosts, the human and the phlebotomine fly vector, is expected to lead to the development of new treatment regimens for VL in South America; specifically a key outcome would be the initiation of a new clinical trial for patients with severe (bleeding) VL in Brazil using miltefosine alone or in combination with other anti-leishmanial drugs. Thus the ultimate beneficiaries of the work will be those in endemic areas of the world who are infected with L. infantum. The project is likely to lead to the development of new resources and tools that can be utilized by other researchers for their own investigations into NIDs.

This project will support the achievement of Sustainable Development Goals 1 (no poverty) and 3 (good health) in Lower Middle Income Countries of South America, through research contributing to the improvement of treatments for VL. Leishmaniasis is a neglected tropical disease with substantial and complex links with poverty. The burden of leishmaniasis falls disproportionately on the poorest segments of the global population, including within Brazil, where there are 30,000 cases/year. 90% of VL cases are found in six countries, Brazil, Ethiopia, India, Somalia, South Sudan and Sudan. Diagnosis and treatment of leishmaniasis is expensive and many of the drugs used are not fit for purpose; drug resistance is an increasing problem. Public investment in treatment and control would decrease the leishmaniasis disease burden and help to alleviate poverty, yet more research is required to understand the parasite and host and environmental factors that contribute to clinical outcome of disease. This project aims to use the strengths of UK science in Leishmania genetics and genomics, natural product biochemistry and environmental monitoring to increase capacity in Brazil for fundamental research into the environmental factors that influence treatment options for leishmaniasis. UK applicants are located in two world class institutions: the Department of Biology in York (Mottram and Graham) and the Royal Botanical Gardens, Kew (Simmonds). The Brazilian applicant is located in the Federal University of the state of Piaui. Visceral leishmaniasis is endemic in the State of Piaui and surrounding areas.