Frequent and genetically diverse Plasmodium vivax relapses contribute to malaria transmission in Cambodia

A unique feature of Plasmodium vivax is the presence of dormant hypnozoites that cause relapses weeks after the initial infection. While relapses may account for the majority of vivax malaria cases, the determinants of relapse periodicity, the genetic relationships between parasites, and the transmission potential of relapsing parasites in endemic populations remain poorly understood. We characterized P. vivax relapses in a cohort of 59 Cambodian patients treated with artesunate and relocated to a malaria-free area to prevent reinfection. Participants were followed every two days for 90 days using qPCR and microscopy. Genomes of blood stage parasites were sequenced to determine clonality and genetic relatedness between initial and relapsing infections. Membrane-feeding assays using Anopheles dirus mosquitoes were performed to evaluate the transmission potential of relapse infections. Relapses occurred frequently, with 81% of the patients experiencing at least one relapse within 90 days, with up to four consecutive relapses per individual and a median time to first relapse of 20 days. Genome sequencing revealed that relapsing parasites in monoclonal infections could be genetically identical, siblings of, or unrelated to the parasites present at enrollment. Patients with polyclonal infections at enrollment experienced significantly more relapses than those with monoclonal infections, suggesting that infection complexity may reflect the heterogeneity of the hypnozoite reservoir. Parasite growth rates during relapses varied widely among individuals but were associated with the degree of relatedness to those present in the initial infection. Despite low parasitemia and the absence of symptoms in most cases, relapse infections were frequently infectious to mosquitoes in membrane-feeding assays. Our findings show that P. vivax relapses occur commonly and frequently, and often involve genetically distinct parasites, reflecting complex hypnozoite reservoirs. Importantly, even low-density relapse infections can transmit efficiently to mosquitoes, highlighting the critical role of hypnozoites in sustaining malaria transmission and the importance of radical cure strategies for vivax malaria elimination.