Plasmodium falciparum transmission in the highlands of Ethiopia is driven by closely related and clonal parasites

Malaria cases are frequently recorded in the Ethiopian highlands even at altitudes above 2,000 m. The epidemiology of malaria in the Ethiopian highlands, and in particular the role of importation by human migration from the highly endemic lowlands is not well understood. We characterized the parasite population structure and genetic relatedness by sequencing 159 P. falciparum samples from Gondar and an additional 28 samples from Ziway using a highly multiplexed droplet digital PCR (ddPCR)-based amplicon deep sequencing method targeting 35 microhaplotypes and drug resistance loci. Diversity was moderate (mean HE: 0.54), and infection complexity was low (74.9% single clone infections). A significant percentage of infections shared genomic haplotypes, even across transmission seasons, indicating persistent local and focal transmission. Multiple clusters of clonal or near-clonal infections were identified, highlighting the overall high genetic relatedness. Frequently, infections from travelers were the earliest observed cases, suggesting that parasites may have been imported and then transmitted locally. We observed population structure between Gondar and Ziway, although some haplotypes were shared between sites. 31.1% of infections carried pfhrp2 deletions and 84.4% pfhrp3 deletions, and 28.7% pfhrp2/pfhrp3 double deletions. Parasites with pfhrp2/3 deletions and wild-type parasites were genetically distinct. Mutations associated with resistance to sulfadoxine-pyrimethamine and lumefantrine were observed at near-fixation, but no mutations in pfk13 were found. In conclusion, genomic data corroborates local transmission and the importance of intensified control in the Ethiopian highlands.