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Malaria cases are frequently recorded in the Ethiopian highlands even at altitudes above 2000 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 sequenced 187 Plasmodium falciparum samples from two sites in the Ethiopian highlands, Gondar (n = 159) and Ziway (n = 28), using a multiplexed droplet digital PCR (ddPCR)‐based amplicon sequencing method targeting 35 microhaplotypes and drug resistance loci. Here, we characterize the parasite population structure and genetic relatedness. We identify moderate parasite diversity (mean HE: 0.54) and low infection complexity (74.9% monoclonal). A significant percentage of infections share microhaplotypes, even across transmission seasons and sites, indicating persistent local transmission. We identify multiple clusters of clonal or near‐clonal infections, highlighting high genetic relatedness. Only 6.3% of individuals diagnosed with P. falciparum reported recent travel. Yet, in clonal or near‐clonal clusters, infections of travellers were frequently observed first in time, suggesting that parasites may have been imported and then transmitted locally. 31.1% of infections are pfhrp2‐deleted and 84.4% pfhrp3‐deleted, and 28.7% have pfhrp2/3 double deletions. Parasites with pfhrp2/3 deletions and wild‐type parasites are genetically distinct. Mutations associated with resistance to sulphadoxine–pyrimethamine or suggested to reduce sensitivity to lumefantrine are observed at near‐fixation. In conclusion, genomic data corroborate local transmission and the importance of intensified control in the Ethiopian highlands. [ABSTRACT FROM AUTHOR]

Background: Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum. Methods: Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data analysis. Results: Cpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. Conclusions: To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones. [ABSTRACT FROM AUTHOR]