Introduction and Background of the Project

Introduction

While clinical malaria cases in the Asia-Pacific and the Americas have gone down >90% in the last decade, a shift in malaria species composition has been observed, with Plasmodium vivax now being the predominant species outside Africa. This shift may relate to the unique biology of P. vivax, including the relapsing phenotype from dormant liver stages (hypnozoites). In the light of the United Nations Sustainable Development Goals (By 2030, end the epidemics of AIDS, tuberculosis, malaria...") and in the era of pursuing malaria eradication, an effective strategy to handle P. vivax malaria is indispensable. Asymptomatic hypnozoite infections form a hidden parasite reservoir in the human population that can give rise to new symptomatic and transmissible malaria weeks, months or years after primary infection, without new infection through mosquito bites. Proper diagnostic tools to identify hypnozoite-infected individuals are currently lacking, and this is mentioned as a challenge in "WHO Global Technical Strategy for Malaria 2016–2030".

Project objective

Identification of putative metabolite markers for malaria hypnozoite infection.

Project design

We plan to identify diagnostic tools for malaria hypnozoites, exploiting our unique experience in in vitro hypnozoite cultures, as well as in sensitive and quantitative metabolomics.  Specific metabolites will be identified in hypnozoite cultures, using our established P. cynomolgi hypnozoite culture technology, as well as appropriate controls. A candidate list of hypnozoite-specific metabolites will be generated, providing an in vitro Proof-of-Concept. This will be followed up by generating a detailed plan for obtaining in vivo Proof-of-Concept, initially in the P. cynomolgi-rhesus monkey model, to identify metabolite candidates for development of a rapid diagnostic test for hypnozoite infection.

How can your partnership (project) address global health challenges?

Diagnostic tools to identify hypnozoite carriers are urgently needed to further map the scale of the problem in the short-term and to allow for targeted drug treatment in the long-term. This approach of identifying and treating individuals infected with dormant malaria parasites prevents disease and further transmission of vivax malaria, while it also prevents unnecessary drug exposure of the non-infected individuals. Our unique approach would provide in vitro Proof-of-Concept for the development of such diagnostic tools and, if successful, further detailed planning for in vivo PoC and diagnostic tool development will be initiated. Once available, these important tools will significantly contribute to P. vivax malaria control and elimination.    

What sort of innovation are you bringing in your project?

We exploit our innovative malaria hypnozoite cultures to measure metabolites originating from hypnozoite infection. To obtain a broad quantitative profiling of metabolites, we will employ an innovative technology Capillary Electrophoresis Mass Spectrometry.

Role and Responsibility of Each Partner

Nagasaki University, Japan is responsible for designing and managing the metabolomic experiments. BPRC, Netherlands is responsible for all primate work as well as all hypnozoite culture work. National Institute of Technology, Kumamoto College, Japan supports the metabolome analyses. All partners will be co-responsible for data interpretation.

Final Report

1. Project objective

After a patient recovers from vivax malaria, hypnozoites remain in the liver where they can be reactivated weeks to years later to cause another round of illness and reemerged parasites can be passed along to mosquitoes. A reliable test for detecting vivax hypnozoites would enable to identify these hidden reservoirs as a target population for the treatment, however, such tests are not available. This project aims to identify metabolites outside of the liver indicating the presence of hypnozoites, which will be used to develop novel diagnostic tools for vivax hypnozoites.

2. Project design

Metabolome analyses were conducted for supernatants from in vitro culture of Plasmodium cynomolgi hypnozoites in monkey primary hepatocytes, as well as appropriate controls; non-infected hepatocytes and hepatocytes infected with a non-hypnozoite forming parasite Plasmodium knowlesi. Three biological replicates of P. cynomolgi and 1 biological replicate of P. knowlesi were prepared. A candidate list of hypnozoite-specific metabolites were generated, providing an in vitro proof-of-concept.

3. Results, lessons learned

P. cynomolgi cultures were treated with drugs between day 0-6 that would only kills developing liver stages, resulting in an enrichment in hypnozoites. Cultures without drug-treatment were also prepared. Overall ~600 and ~500 unique metabolites were identified in P. cynomolgi and P. knowlesi metabolomes, respectively. We set three ranked categories to judge if each metabolite has a potential to be hypnozoite-specific; (1st priority) detected in drug-treated cultures at day 9 (covering metabolites between day 5 and 9) or day 11 (covering metabolites between day 7 and 11) and absent in P. knowlesi, (2nd priority) metabolites that were present in both drug-treated and non-treated cultures at day 11 and absent in P. knowlesi, and (3rd priority) metabolites that were present in both drug-treated and non-treated cultures at day 9 and absent in P. knowlesi. We found 4 metabolites with known identity and 4 with unknown identity that fit to 1st priority; 3 with known identity and 2 with unknown identity that fit to 2nd priority; and 1 with known identity and 5 with unknown identity that fit to 3rd priority. Seven out of total 11 unknown metabolites were able to be further analyzed and chemical formula were predicted by high-resolution MS analysis and candidate structures were predicted based on precursor metabolite search, migration time prediction, and MS/MS analysis. It is interesting to investigate if these metabolites will be also differentially detected in vivo and serve as hypnozoite biomarkers and if they could be used as targets of diagnostic tools. If metabolites with unknown identity detected in vitro are once again identified in the serum of the monkeys and thus become focal candidates for the diagnostic tools we plan to fully identify them.