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

Under our previous RFP (T2017-105) we pioneered an in vitro Proof-of-Concept (PoC) towards identifying targets for diagnostic tools for malaria hypnozoites, exploiting our unique experience in in vitro P. cynomolgi hypnozoite cultures (an accessible proxy to P. vivax with near identical biology), as well as in sensitive metabolomics. Specific metabolites have been identified and prioritized based on the unique signatures found in hypnozoite-enriched cultures. A second-phase in vivo feasibility study using the P. cynomolgi-rhesus monkey model is now warranted to determine whether the specific signatures detected in the in vitro PoC are confirmed in vivo and can thus be pursued in the subsequent development phase 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 vivo PoC for the development of such diagnostic tools and, if successful, further detailed planning for 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 P. cynomolgi-rhesus monkey model 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:

Hypnozoite infection remains without symptoms and no diagnostic tools are available to identify hypnozoite carriers. Such diagnostic tools would be invaluable to precisely map the scale of the infection problem and identify individuals that would qualify for targeted drug treatment, to eliminate the reservoir of malaria parasites. In this phase of the project, in vivo feasibility studies using the Plasmodium cynomolgi-rhesus monkey model were conducted to determine whether the specific signatures detected in the in vitro Proof-of-Concept could be confirmed in vivo and could thus be pursued in the subsequent development phase of a rapid diagnostic test for hypnozoite infection.

2. Project design:

The first experiment was designed to determine whether the signatures found in vitro could be detected in vivo. Two groups of five animals were infected with a high dose of P. cynomolgi sporozoites, and either treated with chloroquine at primary parasitemia (to be close to the field situation) or prophylactically with atovaquone to attain a cleaner hyponozoite only model. An additional group was infected with a high dose of P. knowlesi (non-hypnozoite forming) as a control. The second down titration experiment was conducted to assess the sensitivity needed for the development of a diagnostic kit.

3. Results, lessons learned:

A total of 813 metabolites (first experiment) and 604 metabolites (down titration experiment) were detected by capillary electrophoresis mass spectrometry (CE-MS) at the various timepoints under consideration. These metabolites were carefully inspected and manually compared based on their mass-to-charge ratio (m/z) and retention time (RT) values. Prioritized lists based on the likelihood that metabolites that could be hypnozoite-specific were compiled: 21 metabolites (high priority), 46 metabolites (medium-high priority), 31 metabolites (medium priority) and 47 metabolites (medium-low priority). Lower priority metabolites were also identified. We focused primarily on the 21 metabolites on the high priority list. Aside from the metabolites that have been conclusively identified, an additional effort was made to structurally characterize some additional metabolites of interest through tandem mass spectrometry (MS/MS). This step first involved an assessment of whether said metabolites were abundant enough to carry out the follow up characterization. MS/MS measurements of the analyzed 8 unknown metabolites revealed that the structural estimation can be carried out for six metabolites. Of the six metabolites, three were selected for further analyses by MS/MS and high-resolution MS. These yielded low confidence structures. The candidates identified as significant in in vitro supernatants during the in vitro PoC study were all detected (at relevant timepoints) in body fluids where their concentration is expected to be many times lower, confirming the specificity of the signature. The detection sensitivity in vivo was also confirmed by varying the dosage of sporozoites. Additional metabolites were identified in vivo, which are potentially interesting for a follow up towards the development of a diagnostic tool for hypnozoite identification, but further studies will be necessary to fully characterize them, assess their specificity and sensitivity in larger groups before proceeding to deployment. A company should be involved at this stage that is willing to embark on the preliminary phases of the development of a diagnostic tool for consultation and for tailoring the work.