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

Malaria Journal

Methodology

Endalamaw Gadisa¹  Marga van de Vegte-Bolmer²  Kjerstin Lanke²  Wouter Graumans²  Chiara Andolina²  Robert Sauerwein²  Rianne Siebelink-Stoter²  Isaïe Reuling²  Geert-Jan van Gemert²  Karina Teelen²  Fitsum G. Tadesse³  Teun Bousema⁴  Delenasaw Yewhalaw⁵ 

[1] Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia;Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia;Institute of Biotechnology, Addis Ababa University, Addis Ababa, Ethiopia;Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, UK;Tropical and Infectious Diseases Research Center (TIDRC), Jimma University, Jimma, Ethiopia;Department of Medical Laboratory Sciences and Pathology, Faculty of Health Sciences, Jimma University, Jimma, Ethiopia;

关键词: Transmission;    Oocyst;    Sporozoite;    Anopheles;    Infectivity;    Gametocyte;   

DOI  :  10.1186/s12936-017-2011-9

 received in 2017-06-27, accepted in 2017-09-02,  发布年份 2017

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s) 2017