5 Monivong Boulevard, P.O Box. 983, Phnom Penh, Cambodia [email protected]

Malaria incidence worldwide has steadily declined over the past decades. Consequently, increasingly more countries will proceed from control to elimination. The malaria distribution in low incidence settings appears patchy, and local transmission hotspots are a continuous source of infection. In this study, species-specific clusters and associated risk factors were identified based on malaria prevalence data collected in the north-east of Cambodia. In addition, Plasmodium falciparum genetic diversity, population structure and gene flows were studied. In 2012, blood samples from 5793 randomly selected individuals living in 117 villages were collected from Ratanakiri province, Cambodia. Malariometric data of each participant were simultaneously accumulated using a standard questionnaire. A two-step PCR allowed for species-specific detection of malaria parasites, and SNP genotyping of P. falciparum was performed. SaTScan was used to determine species-specific areas of elevated risk to infection, and univariate and multivariate risk analyses were carried out. PCR diagnosis found 368 positive individuals (6.4%) for malaria parasites, of which 22% contained mixed

species infections. The occurrence of these co-infections was more frequent than expected. Specific areas with elevated risk of infection were detected for all Plasmodium species. The clusters for falciparum, vivax and ovale malaria appeared in the north of the province along the main river, while the cluster for Malariae malaria was situated elsewhere. The relative risk to be a malaria parasite carrier within clusters along the river was twice that outside the area. The main risk factor associated with three out of four malaria species was overnight stay in the plot hut, a human behavior associated with indigenous farming. Haplotypes did not show clear geographical population structure, but pairwise Fst value comparison indicated higher parasite flow along the river. Spatial aggregation of malaria parasite carriers, and the identification of malaria species-specific risk factors provide key insights in malaria epidemiology in low transmission settings, which can guide targeted supplementary interventions. Consequently, future malaria programmes in the province should implement additional specific policies targeting households staying overnight at their farms outside the village, in addition to migrants and forest workers.