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 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.

qPCR in a suitcase for rapid Plasmodium falciparum and Plasmodium vivax surveillance in Ethiopia.

Many Plasmodium spp. infections, both in clinical and asymptomatic patients, are below the limit of detection of light microscopy or rapid diagnostic test (RDT). Molecular diagnosis by qPCR can be valuable for surveillance, but is often hampered by absence of laboratory capacity in endemic countries. To overcome this limitation, we optimized and tested a mobile qPCR laboratory for molecular diagnosis in Ziway, Ethiopia, where transmission intensity is low. Protocols were optimized to achieve high throughput and minimize costs and weight for easy transport. 899 samples from febrile patients and 1021 samples from asymptomatic individuals were screened by local microscopy, RDT, and qPCR within a period of six weeks. 34/52 clinical Plasmodium falciparum infections were missed by microscopy and RDT. Only 4 asymptomatic infections were detected. No hrp2 deletions were observed among 25 samples typed, but 19/24 samples carried hrp3 deletions. The majority (25/41) of Plasmodium vivax infections (1371 samples screened) were found among asymptomatic individuals. All asymptomatic P. vivax infections were negative by microscopy and RDT. In conclusion, the mobile laboratory described here can identify hidden parasite reservoirs within a short period of time, and thus inform malaria control activities.