Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR
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 classific...
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doaj-984417d7182e49a2a44fc7dcbcbc8b062020-11-24T21:47:53ZengBMCMalaria Journal1475-28752017-09-0116111210.1186/s12936-017-2011-9Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCRWouter Graumans0Fitsum G. Tadesse1Chiara Andolina2Geert-Jan van Gemert3Karina Teelen4Kjerstin Lanke5Endalamaw Gadisa6Delenasaw Yewhalaw7Marga van de Vegte-Bolmer8Rianne Siebelink-Stoter9Isaïe Reuling10Robert Sauerwein11Teun Bousema12Department of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreArmauer Hansen Research Institute (AHRI)Tropical and Infectious Diseases Research Center (TIDRC), Jimma UniversityDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreDepartment of Medical Microbiology, Radboud University Nijmegen Medical CentreAbstract 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.http://link.springer.com/article/10.1186/s12936-017-2011-9TransmissionOocystSporozoiteAnophelesInfectivityGametocyte |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
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 |
spellingShingle |
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 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 Transmission Oocyst Sporozoite Anopheles Infectivity Gametocyte |
author_facet |
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 |
author_sort |
Wouter Graumans |
title |
Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR |
title_short |
Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR |
title_full |
Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR |
title_fullStr |
Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR |
title_full_unstemmed |
Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR |
title_sort |
semi-high-throughput detection of plasmodium falciparum and plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite elisa and quantitative pcr |
publisher |
BMC |
series |
Malaria Journal |
issn |
1475-2875 |
publishDate |
2017-09-01 |
description |
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. |
topic |
Transmission Oocyst Sporozoite Anopheles Infectivity Gametocyte |
url |
http://link.springer.com/article/10.1186/s12936-017-2011-9 |
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