A Comparison of PCR and ELISA Methods to Detect Different Stages of Plasmodium Vivax in Anopheles Arabiensis.

Abstract Background: In characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite’s cytochrome oxidase I (COX-I) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito. Methods: A PCR-based method targeting the Plasmodium COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection timepoints (days 0.5, 1, 6, 9, 12, 15). Head and thoraces, and abdomens for each specimen were tested separately with both methods. Agreement between methods at each infection stage was measured using Cohen’s Kappa measure of test association.Results: Infection status of mosquitoes was assessed in approximately 90 head and thoraces and 90 abdomens at each time point; in total 538 head and thoraces and 534 abdomens were tested. In mosquitoes bisected after 0.5, 1-, and 6-days post-infection, the COX-I PCR detected Plasmodium DNA in both the abdomens (88%, 78%, and 67%, respectively) and head and thoraces (69%, 60%, and 44%, respectively) whilst CSP-ELISA detect sporozoites in only 1 abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9-, 12-, and 15-days post-infection in both the head and thoraces and abdomens. There was fair agreement between both methods for time points 9 -15 days post-infection (κ = 0.312, 95% CI: 0.230 – 0.394). Conclusion: The COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the COX-I PCR is a poor candidate for identifying infectious mosquitoes.