Chloroquine-resistant Plasmodium falciparum malaria is a major health problem, particularly in sub-Saharan Africa. Chloroquine resistance has been associated in vitro with point mutations in two genes, pfcrt and pfmdr 1, which encode the P. falciparum digestive-vacuole transmembrane proteins PfCRT and Pgh1, respectively.

Next-generation sequencing is used here to analyse Plasmodium falciparum genome variation directly from clinical blood samples, as well as cultured isolates, from Africa, Asia and Oceania. Resistance to the major antimalarial drug artemisinin is emerging in the Plasmodium falciparum parasite across Southeast Asia, and there is concern that the increased deployment of antimalarials in pursuit of disease eradication might simply lead to increased drug resistance. To monitor these risks it is important to survey the parasite population for genetic changes. Next-generation sequencing is used here to analyse P. falciparum genome variation directly from nearly 300 clinical blood samples, and from cultured isolates from Africa, Asia and Oceania. The authors use these data to analyse the diversity of the parasite population across different geographical locations, as well as within-host diversity at the level of the whole genome, and they show how this may be used to estimate inbreeding rates, which are important for the evolution of drug resistance. Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance1,2. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Dominic Kwiatkowski and colleagues report analysis of genetic variation in 826 Plasmodium falciparum samples collected from 10 locations in West Africa and southeast Asia. They characterize the population structure of this parasite in Cambodia and find evidence for multiple distinct subpopulations showing high levels of genetic differentiation and artemisinin resistance. We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.