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Background  The malaria infection status of mosquitoes is commonly determined by microscopic detection of oocysts on the dissected mosquito midgut. This method is labour-intensive, does not allow processing of large numbers of mosquitoes and can be challenging in terms of objective classification of oocysts. Here, a semi-high-throughput bead-beating ELISA method is proposed for detection of the circumsporozoite protein (CSP) followed by confirmation by quantitative PCR (qPCR).    Methods  Cultured  Plasmodium falciparum  gametocytes were offered to  Anopheles stephensi  mosquitoes and examined by microscopy. After bead-beating, mosquito homogenate was examined by CSP-ELISA and 18S qPCR. As negative controls, mosquitoes that were offered a heat-inactivated gametocyte blood meal were used. The CSP-ELISA/qPCR methodology was applied to high and low-intensity infections of cultured  P. falciparum  gametocytes. A similar methodology optimized for  P. vivax  was used on mosquitoes that were offered blood from Ethiopian donors who were naturally infected with  P. vivax .    Results  There was considerable variation in CSP-ELISA signal and qPCR values in mosquitoes with low oocyst intensities. There was a strong agreement mosquito positivity by CSP-ELISA and by qPCR in mosquitoes that fed on cultured  P. falciparum  material (agreement 96.9%; kappa = 0.97) and naturally infected  P. vivax  parasite carriers [agreement 92.4% (kappa = 0.83)].    Conclusions  The proposed bead-beating CSP-ELISA/qPCR methodology considerably increases throughput for the detection of mosquito infection. qPCR remains necessary to confirm infections in mosquitoes with low CSP-ELISA signal. This methodology may prove particularly useful for studies where very low mosquito infection prevalence is expected and study sites where experience with oocyst detection is limited.    Electronic supplementary material  The online version of this article (doi:10.1186/s12936-017-2011-9) contains supplementary material, which is available to authorized users.

malaria journal

Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR