Optimizing Plasmodium vivax gametocyte enrichment to enhance transmission to Anopheles mosquitoes

<ns5:p> Background <ns5:italic>Plasmodium vivax</ns5:italic> is the second leading cause of malaria worldwide. The lack of continuous <ns5:italic>in vitro</ns5:italic> culture of <ns5:italic>P. vivax</ns5:italic> hampers research efforts, including research on transmission to mosquitoes and sporozoite biology <ns5:italic>.</ns5:italic> Here, we present methodological improvements to achieve high mosquito infection rates. Methods We compared two gametocyte enrichment methods: magnetic cell sorting (MACS) and Percoll density gradient centrifugation (DGC) at 1500 <ns5:italic>g</ns5:italic> or 760 <ns5:italic>g.</ns5:italic> Blood samples before and after enrichment were offered to <ns5:italic>Anopheles stephensi</ns5:italic> mosquitoes by membrane feeding. Outcomes included the proportion of mosquitoes infected, oocyst density and sporozoite positivity. Protocols were first evaluated using <ns5:italic>in vitro</ns5:italic> cultured <ns5:italic>P. falciparum</ns5:italic> , before being further optimized using gametocyte-positive samples from eight healthy, malaria-naive adults infected with a <ns5:italic>P. vivax</ns5:italic> clone ‘PvW1’ during a controlled human malaria infection. Results DGC at a speed of 1500 <ns5:italic>g</ns5:italic> achieved the highest infection rates. Oocyst density increased approximately 86-fold (95% CI 14–525; p &lt; 0.01) and the proportion of infected mosquitoes was on average 58% higher when compared to the non-enriched control (95% CI 42.6–74.0; p &lt; 0.01). DGC at 760 <ns5:italic>g</ns5:italic> increased the oocyst density 11-fold (CI 1.8–69; p &lt; 0.01) and the proportion of infected mosquitoes by 28% (CI 12.1–43.9; p &lt; 0.01). MACS did not increase <ns5:italic>P. vivax</ns5:italic> mosquito infection compared to control. Infected mosquitoes became salivary gland positive, with the mean sporozoite load in infected mosquitoes increasing with oocyst density. Conclusions This study provides the first systematic comparison of MACS and DGC as enrichment techniques to increase <ns5:italic>P. vivax</ns5:italic> transmission and presents the DGC protocol that achieved the highest proportion of infected mosquitoes and oocyst densities. This protocol will facilitate <ns5:italic>P. vivax</ns5:italic> transmission studies and enable the generation of highly infected mosquitoes and sporozoites for downstream use. </ns5:p>