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Background  The entomological inoculation rate (EIR) is an important indicator in estimating malaria transmission and the impact of vector control. To assess the EIR, the enzyme-linked immunosorbent assay (ELISA) to detect the circumsporozoite protein (CSP) is increasingly used. However, several studies have reported false positive results in this ELISA. The false positive results could lead to an overestimation of the EIR. The aim of present study was to estimate the level of false positivity among different anopheline species in Cambodia and Vietnam and to check for the presence of other parasites that might interact with the anti-CSP monoclonal antibodies.    Methods  Mosquitoes collected in Cambodia and Vietnam were identified and tested for the presence of sporozoites in head and thorax by using CSP-ELISA. ELISA positive samples were confirmed by a  Plasmodium  specific PCR. False positive mosquitoes were checked by PCR for the presence of parasites belonging to the Haemosporidia, Trypanosomatidae, Piroplasmida, and Haemogregarines. The heat-stability and the presence of the cross-reacting antigen in the abdomen of the mosquitoes were also checked.    Results  Specimens (N = 16,160) of seven anopheline species were tested by CSP-ELISA for  Plasmodium falciparum  and  Plasmodium vivax  (Pv210 and Pv247). Two new vector species were identified for the region:  Anopheles pampanai  ( P. vivax ) and  Anopheles barbirostris  ( Plasmodium malariae ). In 88% (155/176) of the mosquitoes found positive with the  P. falciparum  CSP-ELISA, the presence of  Plasmodium  sporozoites could not be confirmed by PCR. This percentage was much lower (28% or 5/18) for  P. vivax  CSP-ELISAs. False positive CSP-ELISA results were associated with zoophilic mosquito species. None of the targeted parasites could be detected in these CSP-ELISA false positive mosquitoes. The ELISA reacting antigen of  P. falciparum  was heat-stable in CSP-ELISA true positive specimens, but not in the false positives. The heat-unstable cross-reacting antigen is mainly present in head and thorax and almost absent in the abdomens (4 out of 147) of the false positive specimens.    Conclusion  The CSP-ELISA can considerably overestimate the EIR, particularly for  P. falciparum  and for zoophilic species. The heat-unstable cross-reacting antigen in false positives remains unknown. Therefore it is highly recommended to confirm all positive CSP-ELISA results, either by re-analysing the heated ELISA lysate (100°C, 10 min), or by performing  Plasmodium  specific PCR followed if possible by sequencing of the amplicons for  Plasmodium  species determination.

malaria journal

False positive circumsporozoite protein ELISA: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination