New tool for interfering with malaria transmission

Geoff Johnston, a doctoral candidate at Columbia's PhD in Sustainable Development, is on the team behind this recent study in PNAS.  He's promised us a non-technical summary soon.

Sophie H. Adjalleya, Geoffrey L. Johnston, Tao Li, Richard T. Eastman, Eric H. Ekland, Abraham G. Eappen, Adam Richman, B. Kim Lee Sim, Marcus C. S. Lee, Stephen L. Hoffman, and David A. Fidock

Abstract: Clinical studies and mathematical models predict that, to achieve malaria elimination, combination therapies will need to incorporate drugs that block the transmission of Plasmodium falciparum sexual stage parasites to mosquito vectors. Efforts to measure the activity of existing antimalarials on intraerythrocytic sexual stage gametocytes and identify transmission-blocking agents have, until now, been hindered by a lack of quantitative assays. Here, we report an experimental system using P. falciparum lines that stably express gametocyte-specific GFP-luciferase reporters, which enable the assessment of dose- and time-dependent drug action on gametocyte maturation and transmission. These studies reveal activity of the first-line antimalarial dihydroartemisinin and the partner drugs lumefantrine and pyronaridine against early gametocyte stages, along with moderate inhibition of mature gametocyte transmission to Anopheles mosquitoes. The other partner agents monodesethyl-amodiaquine and piperaquine showed activity only against immature gametocytes. Our data also identify methylene blue as a potent inhibitor of gametocyte development across all stages. This thiazine dye almost fully abolishes P. falciparum transmission to mosquitoes at concentrations readily achievable in humans, highlighting the potential of this chemical class to reduce the spread of malaria.

From the author summary:
The scale of the malaria epidemic remains vast, causing up to 225 million symptomatic infections and ∼780,000 deaths each year, primarily in sub-Saharan Africa. Despite this sobering backdrop, there are encouraging signs that treating infected individuals with antimalarial therapies and combating the Anopheles mosquito vector with insecticides can substantially reduce the burden of disease. First-line therapies rely on pairing potent derivatives of the Chinese plant extract artemisinin with longer-lasting partner drugs in regimens referred to as artemisinin-based combination therapies. Clinical reports and mathematical models indicate that additional reductions in disease incidence will require treatments that not only cure patients but also decrease the transmission of malarial parasites to Anopheles mosquitoes (1). Here, we have investigated the ability of various antimalarial agents to inhibit transmission. This work reveals that methylene blue (MB), the first synthetic compound ever used in clinical therapy (2), has potent transmission-blocking activity superior to current first-line therapies. 
Interruption of transmission can be achieved with drugs that inhibit the development of parasite sexual forms, termed gametocytes, within red blood cells. In the case of the most lethal human malaria pathogen, Plasmodium falciparum, these gametocytes progress through five developmental stages over 10–12 d before becoming infectious to mosquitoes (Fig. P1A). Prior studies have found that some drugs that target the disease-causing asexual blood stages also inhibit early stage gametocytes (3). However, identifying compounds that inhibit the metabolically less active mature stages has proven considerably more difficult, in part because of a lack of robust experimental tools. To address this concern, we have developed recombinant parasite lines and analytical methods that enable precise measurements of drug action against gametocytes as they mature and attain infectivity. 
To investigate the abilities of known antimalarials to affect gametocyte viability at different stages, we genetically modified P. falciparum parasite lines to express GFP-luciferase reporters from gene promoters known to be active in early, mid, or late stage gametocytes. The production of gametocytes was triggered by starvation-induced stress, and their subsequent development and gametocyte maturation were monitored by quantifying luciferase activity. Measurements of the rate of action of antimalarial compounds, tested at different doses in vitro, revealed the remarkable potency of the thiazine dye MB against all developmental stages (Fig. P1A). Subsequent experiments revealed that MB almost fully blocked transmission of P. falciparum gametocytes to Anopheles mosquitoes (Fig. P1B), reducing parasite infectivity by 78–100%. The small proportion of mosquitoes that were infected had a >98% reduction in the numbers of parasites developing in the midgut. Most of the effect of MB on parasite transmission can be attributed to its potent activity against mature stage V gametocytes. Parallel studies also observed a potent effect with dihydroartemisinin, the active metabolite of artemisinin compounds, with inhibition occurring primarily against early stage gametocytes. Comparable activity against early stages was observed with key partner drugs, including amodiaquine and lumefantrine (4). 
The experimental system that we developed for these studies will enable high-throughput screening to identify additional transmission-blocking compounds. Our study also provides experimental tools to further probe gametocyte biology, including studies on the cellular processes and molecular components that dictate the formation of gametocytes and promote transmission (5). A renewed emphasis on this phase of the malarial parasite life cycle, using reporter systems such as the one described here, promises to further aid expanding efforts to roll back malaria.

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