Odium species in the sample. The development of more rapid immunological and molecular approaches such as the circumsporozoite protein enzyme linked immune-sorbent assay (ELISA-CSP) [11,12] and Oltipraz site PCR-based techniques rapidly got widely adopted, [13,14]. Although ELISA-CSP seems to be relatively robust and cheap, there are potential drawbacks in using this approach. A lack of specificity has been raised as an important issue because this method does not only detect the sporozoites in the salivary glands, but can also detect CSP from other PHCCC mosquito tissues [14]. An overestimation of true salivary gland infection could also result from measuring circulating CSP as this could originate from sporozoites migrating through the mosquito [15,16]. Moreover, the ELISA-CSP technique is also subjected to an underestimation of the vector’s actual level of infection because it does not target all infecting Plasmodium spp in a given mosquito species [17]. In the context of the deployment of global effort towards malaria control and elimination, it is of primary importance to develop sensitive and reliable diagnostic techniques for detecting Plasmodium spp in both humans and mosquitoes. Recently, highthroughput assays based on real-time PCR have been developed for detecting malaria parasites in humans. These methods allow some rapid and simultaneous detection, and a quantification of several target DNAs through the use of the specific fluorophorelabeled probes [7,18,19]. The benefits of these methods come from very low contamination risks and high sensitivity that reaches 100 fold over the ELISA technique [14]. The use of more sensitive and effective diagnostic technique for the detection of parasites in the vectors can ensure better estimation of transmission intensity in different malaria settings. The aim of this study was to optimize a sensitive PCR-based method that can accurately estimate mixed infection rates of Plasmodium species in An. gambiae and An. funestus, the main malaria vectors in Africa.Materials and Methods Study AreaThe Anopheles gambiae mosquitoes tested in this study were collected by the team of the Centre de Recherches Entomologiques de Cotonou (CREC) Research under the framework of the President’s Malaria Initiative (PMI) program of 24195657 the USAID. Mosquitoes were collected in five districts (Adjarra, Adjohoun, Dangbo, Misserete, and Seme) in the Oueme department ???` ` ??(6u349711E ?2u319358N) in Southern Benin. The Anopheles funestus mosquitoes were collected in 3 villages in the district of Ouidah: Tokoli (6u26957.199N, 2u09936.699E), Lokohoue (6u24924.299N, 2u10932.199E) and Kindjitokpa ` (6u26957.199N, 2u09936.699E) where this species is known to be the main malaria vector [3]. The temperatures in these areas vary between 25uC and 30uC with an annual rainfall ranging from 900 mm to 1500 mm.Mosquito Collection and Sample ProcessingIndoor and outdoor mosquito collections were conducted in two sites per village using the human landing catch technique (HLC). Collectors were hourly rotated along collection sites and/or position (indoor/outdoor). At each position, all mosquitoes caught were kept in individual tubes and in hourly bags. The collection period took place at the night between 21:00 and 05:00 AM. Mosquitoes were also captured by using window traps placed in different houses in each village. The houses were selected according to the number of the people sleeping there. Traps were placed on the outside windows in each selected.Odium species in the sample. The development of more rapid immunological and molecular approaches such as the circumsporozoite protein enzyme linked immune-sorbent assay (ELISA-CSP) [11,12] and PCR-based techniques rapidly got widely adopted, [13,14]. Although ELISA-CSP seems to be relatively robust and cheap, there are potential drawbacks in using this approach. A lack of specificity has been raised as an important issue because this method does not only detect the sporozoites in the salivary glands, but can also detect CSP from other mosquito tissues [14]. An overestimation of true salivary gland infection could also result from measuring circulating CSP as this could originate from sporozoites migrating through the mosquito [15,16]. Moreover, the ELISA-CSP technique is also subjected to an underestimation of the vector’s actual level of infection because it does not target all infecting Plasmodium spp in a given mosquito species [17]. In the context of the deployment of global effort towards malaria control and elimination, it is of primary importance to develop sensitive and reliable diagnostic techniques for detecting Plasmodium spp in both humans and mosquitoes. Recently, highthroughput assays based on real-time PCR have been developed for detecting malaria parasites in humans. These methods allow some rapid and simultaneous detection, and a quantification of several target DNAs through the use of the specific fluorophorelabeled probes [7,18,19]. The benefits of these methods come from very low contamination risks and high sensitivity that reaches 100 fold over the ELISA technique [14]. The use of more sensitive and effective diagnostic technique for the detection of parasites in the vectors can ensure better estimation of transmission intensity in different malaria settings. The aim of this study was to optimize a sensitive PCR-based method that can accurately estimate mixed infection rates of Plasmodium species in An. gambiae and An. funestus, the main malaria vectors in Africa.Materials and Methods Study AreaThe Anopheles gambiae mosquitoes tested in this study were collected by the team of the Centre de Recherches Entomologiques de Cotonou (CREC) Research under the framework of the President’s Malaria Initiative (PMI) program of 24195657 the USAID. Mosquitoes were collected in five districts (Adjarra, Adjohoun, Dangbo, Misserete, and Seme) in the Oueme department ???` ` ??(6u349711E ?2u319358N) in Southern Benin. The Anopheles funestus mosquitoes were collected in 3 villages in the district of Ouidah: Tokoli (6u26957.199N, 2u09936.699E), Lokohoue (6u24924.299N, 2u10932.199E) and Kindjitokpa ` (6u26957.199N, 2u09936.699E) where this species is known to be the main malaria vector [3]. The temperatures in these areas vary between 25uC and 30uC with an annual rainfall ranging from 900 mm to 1500 mm.Mosquito Collection and Sample ProcessingIndoor and outdoor mosquito collections were conducted in two sites per village using the human landing catch technique (HLC). Collectors were hourly rotated along collection sites and/or position (indoor/outdoor). At each position, all mosquitoes caught were kept in individual tubes and in hourly bags. The collection period took place at the night between 21:00 and 05:00 AM. Mosquitoes were also captured by using window traps placed in different houses in each village. The houses were selected according to the number of the people sleeping there. Traps were placed on the outside windows in each selected.

Odium species in the sample. The development of more rapid immunological

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