Uneven Plasmodium falciparum transmission and cryptic ovale transmission from the asymptomatic reservoir in Bagamoyo, Tanzania

Background Asymptomatic malaria carriers often harbor low parasite densities missed by rapid diagnostic tests (RDTs), yet they contribute to transmission. Direct skin feeding assays (DSFs) can sensitively measure their infectiousness to mosquitoes. Methods To characterize human-to-mosquito transmission from the asymptomatic reservoir in Bagamoyo, Tanzania, DSFs were performed in persons >5 years of age positive for P. falciparum by RDT or real-time PCR. Fifty colony-reared Anopheles gambiae were fed on the posterior calves. Successful mosquito infection was defined as ≥1 oocyst-positive mosquito midgut among 25 dissected eight days post-skin feeding. Results Among 491 participants with median parasite density of 5.1 parasites/uL who underwent DSF, 22% were infectious to mosquitoes. RDT-positive participants infected roughly twice as many mosquitoes compared to RDT-negative/PCR-positive persons. However, up to 21% of infectious carriers were PCR-negative at the time of skin feeding, after screening PCR-positive a few days earlier. Overall, 9.1% (342/3,741) of mosquitoes fed on infectious carriers were parasite-positive at dissection. Half of infectious individuals infected a single mosquito, while the top 16 transmitters (3% of those undergoing DSF) cumulatively infected 57% of infected mosquitoes. RDT-positive school-age children (6-15yo), 27% of the DSF cohort, contributed to 58% of infected mosquitoes. Unexpectedly, mosquito midguts from 39 DSFs (44% of oocyst-positive feeds analyzed) tested positive for Plasmodium ovale. Conclusions Parasites circulating at the limit of PCR detection commonly infect mosquitoes. However, a small proportion of highly infectious carriers contribute disproportionately to transmission, offering potential for targeted interventions. Plasmodium ovale was frequently co-transmitted with P. falciparum to mosquitoes.