ABSTRACT Artemisinin-based combination therapies (ACT) have been the bedrock of malaria treatment in Africa for more than a decade, spurring remarkable gains in malaria control, even as parasites tolerant to commonly used ACT partner drugs continue to circulate. In East Africa, parasites harboring mutations associated with resistance to the partner drug lumefantrine are selected through treatment and responsible for the majority of recurrent infections that arise starting 3-4 weeks following ACT. These post-treatment parasitemias likely contribute to the onward transmission of drug-resistant parasites to mosquitoes and further spread of antimalarial resistance. Now, parasites with partial artemisinin resistance that have emerged in Rwanda are spilling over into northwest Tanzania, a high malaria burden country, where 4% of the world’s malaria deaths occur. Mitigating the spread of ACT-resistant parasites across sub-Saharan Africa will require a better understanding of the transmission reservoir (who is transmitting drug-resistant parasites to mosquitoes) and ways to intervene to interrupt this transmission. While ACT and single low dose primaquine effectively clear those presenting with gametocytes, the parasite stages essential for transmission to mosquitoes, no current strategies target gametocytes that arise in the weeks following treatment. We hypothesize that gametocytes associated with post-ACT parasitemia are transmissible to mosquitoes and contribute to the spread of antimalarial resistance, even as most go undetected. This R21 proposal will determine the risk of transmission from PCR-positive individuals one month following ACT (Aim 1) and find whether drug resistant alleles that are selected during treatment are maintained through transmission (Aim 2). To accomplish these aims, we will leverage the capacity we have built over the last 5 years to characterize the infectious reservoir in Bagamoyo, Tanzania, where we have performed >600 mosquito feeding assays in asymptomatic parasite carriers, most with subpatent infection only detectable by PCR. Our team will screen >300 persons participating in a large triple ACT trial for recurrent parasitemia during weeks 3-8 of follow-up and conduct mosquito membrane feeding assays to measure their transmissibility to Anopheles gambiae and A. funestus, the primary malaria vectors in East Africa. We will sequence transmitted parasites that survive to the salivary gland sporozoite stage within mosquitoes and compare the frequency of key drug resistance alleles in these mosquitoes to blood collected from persons pre and post-treatment. We expect that the post-treatment period, 4-8 weeks after first-line ACT therapy, is indeed a vulnerable time for gametocytemia to arise without symptoms, and lead to transmission of P. falciparum parasites bearing kelch 13 and mdr1 mutations selected through treatment. However, we also expect that triple ACT therapies that reduce the rate of post-treatment parasitemia wi...