Abstract
BackgroundThe 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).MethodsCultured 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.ResultsThere 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)].ConclusionsThe 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.
Highlights
The malaria infection status of mosquitoes is commonly determined by microscopic detection of oocysts on the dissected mosquito midgut
The transmission of malaria from human to mosquitoes depends on the presence of circulating mature sexual stages of the parasite, gametocytes, that are taken up by female Anopheles mosquitoes along with the blood meal that contains nutrients required for egg production
Uninfected and infected but heat inactivated (2 h at 56 °C) blood was used as negative control, the latter allowing the assessment of deoxyribonucleic acid (DNA) persistence in mosquitoes [15]
Summary
The malaria infection status of mosquitoes is commonly determined by microscopic detection of oocysts on the dissected mosquito midgut. Male and female gametocytes activate and fuse to form a zygote that develops into an ookinete This motile form penetrates the mosquito midgut and differentiates into an oocyst. P. vivax ELISA OD values for different A. stephensi negative controls were low in comparison with OD values for A. arabiensis mosquitoes that were fed blood meals from naturally infected P. vivax gametocyte carriers (Fig. 5a). Both CSP-ELISA OD values and qPCR CT values showed clear positivity but considerable variation for three donors with P. vivax gametocyte densities of (gametocytes/μL) of 2654, 497, and 13,616 (Fig. 5b). When CSP-ELISA data and qPCR were compared for 198 mosquitoes that were examined as part of Day Mean
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