Malaria is one of the most important diseases in the world. A primary objective in the fight against malaria is to achieve a long-term reduction of the transmission which would result in a reduction in morbidity and mortality. Malaria transmission depends on parasite development in the mosquito that occurs only when the human host carries gametocytes. Currently, the mechanisms of malaria transmission are studied separately in the two hosts of the cycle, requiring a great deal of resources both in field and laboratory work.
The main goal of this project is to design and realize a sensitive and robust molecular method that allows analyzing the three components of the malaria cycle (human blood, parasite and mosquito) simultaneously in the infected mosquito. In particular, our interest will focus on the quantification by Real Time PCR of the Plasmodium zygote stage (few hours after the infected blood meal), that is associated with the gametocyte load and with the overall transmission capacity. The ultimate target of the project intends therefore to obtain a simple protocol applicable in future field studies to better understand the transmission of malaria, simply capturing fed mosquitoes, therefore bypassing the considerable difficulties, both organizational and practical, found to date in field studies based on human blood sampling.
The development of this protocol will include the use of fed mosquitoes (infected and not infected with P. falciparum), collected after the blood meal (when blood gametocytes mature in gametes and develop in the motile zygote, the ookinete). Nucleic acids (genomic DNA and total RNA) will be extracted from single mosquitoes to gain information on the molecular biology of Plasmodium, mosquito and on the human genetic factors that might possibly influence the transmission. The innovativeness of the project we propose relies in the use of mosquitoes as containers of information suitable to study human host, vector and parasite interactions.
In all the studies published so far, the mechanisms of malaria transmission were studied separately in the two hosts of the cycle, requiring a great deal of resources both in field work and in the laboratory [1-7]. The critical points of such studies were based on i) the microscopic and/or molecular quantification of the gametocyte stages in the peripheral blood of the human carriers and ii) the microscopic counts of the parasite's oocysts in the midgut basal lamina of infected mosquitoes. These experimental approaches implicate a relevant bureaucratic, economic, practical and ethical commitment necessary for the collection and storage of blood samples from endemic populations. Moreover, the parasite mosquito stages could be evaluated only in insectaries where the mosquitoes, collected after the infected blood meal (naturally acquired or by membrane feeding), must be kept for about 7 days to allow the parasite's oocysts to develop.
The innovativeness of the project we propose relies in the use of mosquitoes as a container of information necessary to study human, vector and parasites molecular biology and genetics. Indeed, the proposed procedure will allow the researcher photographing the stage and the load of parasites in the mosquito by quantitative RT-PCR, thus providing important information about the infection level in the invertebrate host and the interactions between the three species involved. Another important feature that contributes to the novelty of this project will count on the correlation between Real Time PCR outcomes describing stage/load of malaria parasite and the genetic background (evidenced by PCR) of both invertebrate and vertebrate host. For the first time, a single mosquito will be analysed in such a deep molecular detail to provide comprehensive information about malaria transmission and epidemiology.
Finally, we believe that this protocol, foreseeing only mosquito collection, could be very useful in large-scale field studies on human-to-mosquito malaria transmission carried out in endemic areas.
References
1. Schneider P, Wolters L, Schoone G, et al. Real-Time Nucleic Acid Sequence-Based Amplification Is More Convenient than Real-Time PCR for Quantification of Plasmodium falciparum. Journal of Clinical Microbiology, 2005;43(1):402-405.
2. Ouédraogo AL, Bousema T, Schneider P, et al. Substantial contribution of submicroscopical Plasmodium falciparum gametocyte carriage to the infectious reservoir in an area of seasonal transmission. PLoS ONE. 2009;4:e8410.
3. Schneider P, Bousema JT, Gouagna LC, et al. Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection. Am J Trop Med Hyg,. 2007;76:470¿474.
4. Gouagna LC, Bancone G, Yao F, et al. Genetic variation in human HBB is associated with Plasmodium falciparum transmission. Nat Genet, 2010; 42:328¿31.
5. Gouagna L C, Bancone G, Yao F, et al. Impact of protective haemoglobins C and S on P. falciparum malaria transmission in endemic area. Malaria Journal, 2010; 9, O17.
6. Bousema T, Dinglasan RR, Morlais I, et al. Mosquito Feeding Assays to Determine the Infectiousness of Naturally Infected Plasmodium falciparumGametocyte Carriers. PLoS ONE, 2012: 7, e42821.
7. Lin JT, Saunders D L, & Meshnick SR. The role of submicroscopic malaria in malaria transmission: what is the evidence? Trends in Parasitology, 2014: 30, 183-190.