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01.12.2017 | Methodology | Ausgabe 1/2017 Open Access

Malaria Journal 1/2017

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

Zeitschrift:
Malaria Journal > Ausgabe 1/2017
Autoren:
Wouter Graumans, Fitsum G. Tadesse, Chiara Andolina, Geert-Jan van Gemert, Karina Teelen, Kjerstin Lanke, Endalamaw Gadisa, Delenasaw Yewhalaw, Marga van de Vegte-Bolmer, Rianne Siebelink-Stoter, Isaïe Reuling, Robert Sauerwein, Teun Bousema
Wichtige Hinweise

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.

Abstract

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.
Zusatzmaterial
Additional file 1: Table S1. Observations with different buffers for mosquito homogenisation. Table S2. Experimental infection data CT values by qPCR vs Optical Density in CSP-ELISA. Table S3. Overview of processing time of the four methods compared in seconds per 80 mosquitoes. Figure S1. Protocol for mixed infection detection using the same 50 µl volume. Homogenate is transferred from the prior experiments to the subsequent ones. It is important that every step is timed and thought ahead on when to start the different steps. Figure S2. Processing of mosquitoes by CS-ELISA when tested for both P. falciparum and P. vivax.
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