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.

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.

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.