Preventing the transmission of artemisinin resistant falciparum malaria

Malaria is a disease of devastating impact causing 584,000 deaths in 2013. Artemisinin combination therapies (ACTs) are the frontline treatment for malaria and remain safe and effective throughout most of the world. However, in recent years, resistance to artemisinin has arisen in Southeast Asia, causing slower recovery and malaria treatment failures. It is greatly feared that resistance will spread throughout the region and from there, worldwide. If this happens, in the absence of effective new drugs (that are at best several years from reaching clinical approval), malaria may become untreatable and all the gains made in recent years will be reversed.
Malaria is caused by the parasitic organism Plasmodium and transmitted through the bite of Anopheles mosquitoes. Malarial symptoms are caused by successive rounds of parasite invasion of host red blood cells (RBCs), replication, and then re-invasion of further RBCs - termed the asexual cycle. With each cycle, a small proportion of the parasites undergo an alternative developmental pathway forming the sexual stages, or male and female gametocytes. Gametocytes are responsible for onward transmission to the mosquito therefore are essential for the parasite to complete its life cycle. Very few gametocytes are ingested in a mosquito blood meal making this parasite "population bottleneck" an attractive target for therapies that prevent the spread of malaria. Only two approved antimalarial drugs are effective against this gametocyte population - primaquine (PQ) and methylene blue (MB). Given the immediate need to halt the spread of artemisinin resistance in Southeast Asia, every tool in our armoury needs to be deployed - including the aforementioned transmission-blocking drugs. To this end, it's of critical importance to identify whether artemisinin-resistant parasites will respond to PQ and MB (and any other up and coming antimalarial drugs) before embarking on widespread transmission-blocking campaigns.
Our project proposes to take already established and characterised Thai isolates of P. falciparum, some showing sensitivity and some resistance to artemisinin, and test them in an existing lab-based assay (the P. falciparum dual gamete formation assay - Pf DGFA) widely regarded as an accurate indicator of transmission-blocking. This will enable us to compare head-to-head the effectiveness of PQ and MB (and their metabolites) under standardised laboratory conditions and rank their effectiveness against artemisinin sensitive and resistant parasites. Additionally, using cutting edge genetic modification techniques (CRISPR), we will generate our own artemisinin resistant parasites by introducing successive mutations in genes known to be important for resistance. By this, we can gain an insight into how each contributes to overall resistance.
The second aspect of our project is derived from our recent understanding that the lesser population of male gametocytes are more drug-sensitive than female gametocytes. As it is so expensive and technically challenging to measure malaria transmission directly to the mosquito on a larger scale required for clinical trials, measurements of gametocyte clearance from the infected human are currently used as surrogates. These crude measurements do not measure the viability of the gametocyte, nor take into consideration differences in male and female gametocytes. We propose taking the lab-based Pf DGFA and adapting it for the field to enable an accurate assessment of transmission-blocking during clinical trials.
The benefits of our project are twofold. By understanding the effectiveness of current and future transmission-blocking drugs against artemisinin-resistant parasites we will be able to make evidence-based recommendations to the community for future trials. Then, by designing and implementing a new and more informative transmission-blocking assay, we will be giving the community the tools to accurately evaluate said transmission-blocking trials.

The potential spread of artemisinin resistant malaria throughout Southeast Asia and beyond is of grave concern to malaria elimination initiatives. To address this problem, our project aims to:
1. To understand and compare how artemisinin sensitive and resistant parasites respond to known transmission-blocking drugs
2. To develop the methodology to quantitatively assess male and female gametocyte functional viability in malarial patients
3. To adapt our Pf DGFA methodology to a form amenable for large scale implementation for transmission-blocking clinical trials
We will combine established gametocyte producing P. falciparum artemisinin-sensitive and -resistant isolates with our existing validated in vitro transmission-blocking assay (Pf Dual Gamete Formation Assay - Pf DGFA) to initially determine whether primaquine (PQ) or methylene blue (MB) (and their metabolites) show modulated transmission-blocking activity in a resistant background. As a standardised control, we will also generate a panel of lab strain parasites containing known mutations linked to artemisinin resistance by CRISPR. To extend this investigation we will also compare 34 known transmission-blocking molecules in the Pf DGFA in both sensitive and resistant backgrounds. Taking our in vitro data on PQ and MB transmission-blocking efficacy, we will repeat our investigation using ex vivo parasites taken from malarial patients under PQ/MB treatment. Finally, we will adapt the Pf DGFA into a form amenable for evaluation of transmission-blocking field trials.
Our project aims to deliver evidence-based recommendations about the use of PQ and MB for controlling the spread of artemisinin resistant parasites; a screening platform to evaluate current and future antimalarials for efficacy against artemisinin resistant parasites; and a robust tool to accurately measure transmission-blocking during field trials.

The following groups are identified as beneficiaries of our proposed project:

Malaria Policymakers:
Our project will generate the first data on how artemisinin resistant parasites respond to clinically approved transmission-blocking drugs. This information is vital for the design of future transmission-blocking campaigns in areas with artemisinin resistance to ensure that the most effective drugs are used. Consequently it will help malaria policymakers such as the World Health Organisation (WHO), Bill and Melinda Gates Foundation (BMGF) and Medicines for Malaria Venture (MMV) make informed evidence-based decisions to design malaria elimination campaigns. Communication of our data to policymakers will be an ongoing process for the duration of the project and beyond, and given the urgency to tackle artemisinin resistant malaria, we expect any significant findings to be implemented without delay.

Clinicians:
Similarly, evidence-based data on appropriate (and inappropriate) drugs to use in artemisinin-resistant areas will be highly beneficial to clinicians treating malaria to help them select the best treatment for their patients. Establishing best treatment practises for malaria in artemisinin-resistant areas will require extensive trials and so the benefits may take longer to realise. However, our data will lay the foundation for testable field trials and so help stimulate focus in this area.

Academic and Industrial Drug Discovery Groups:
Our project proposes to implement a transmission-blocking assay that can evaluate the effects of experimental antimalarials on artemisinin resistant and sensitive parasites. We anticipate that this will be in immediate high demand from both academic and industry groups to explore whether transmission-blocking compounds from their discovery programmes have the potential to act upon artemisinin resistant parasites. Given the cost of bringing a drug to the marketplace, it is of great benefit to identify early on compounds with promise (i.e. activity against resistant parasites) and therefore our assay will aid the efficiency of antimalarial drug discovery. We will utilise our links with the MMV, BMGF and GlaxoSmithKline (GSK) to accelerate adoption of the assay as a gold-standard integral part of the antimalarial test cascade.

Thai Population and Further Afield:
Living in the immediate shadow of artemisinin resistance, the Thai people are the first to feel the negative impact of artemisinin resistant malaria. As a long term beneficiary of our research, we hope that through helping to identify the most efficacious transmission-blocking therapies to use in resistant areas, we will improve the overall health of the Thai people and indeed people throughout wider Southeast Asia. With a reduction in malaria burden, we hope that our research will also benefit the Thai economy. Taking a positive view, we hope that our work will ultimately contribute to halting the spread of artemisinin resistant malaria. At worst, we hope that our work will slow the spread of artemisinin resistant malaria enough to minimise its impact until new effective replacement drugs can reach clinical approval and take over.