SummaryBackgroundArtemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international effort is underway to contain artemisinin-resistant Plasmodium falciparum, but containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand–Myanmar (Burma) border.MethodsIn malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≥4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms.Findings3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5–2·7) in 2001, to 3·7 h (3·6–3·8) in 2010, compared with a mean of 5·5 h (5·2–5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≥6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identified, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010.InterpretationGenetically determined artemisinin resistance in P falciparum emerged along the Thailand–Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2–6 years.FundingThe Wellcome Trust and National Institutes of Health.
Evolving resistance to artemisinin-based compounds threatens to derail attempts to control malaria. Resistance has been confirmed in western Cambodia, has recently emerged in western Thailand, but is absent from neighboring Laos. Artemisinin resistance results in reduced parasite clearance rates (CR) following treatment. We used a two-phase strategy to identify genome region(s) underlying this ongoing selective event. Geographical differentiation and haplotype structure at 6,969 polymorphic SNPs in 91 parasites from Cambodia, Thailand and Laos identified 33 genome regions under strong selection. We screened SNPs and microsatellites within these regions in 715 parasites from Thailand, identifying a selective sweep on chr 13 that shows strong association (P=10-6-10-12) with slow CR, illustrating the efficacy of targeted association for identifying the genetic basis of adaptive traits.
Pathogen control programs provide a valuable, but rarely exploited, opportunity to directly examine the relationship between population decline and population genetics. We investigated the impact of an ~12-fold decline in transmission on the population genetics of Plasmodium falciparum infections (n = 1731) sampled from four clinics on the Thai-Burma border over 10 years, and genotyped using 96 genome-wide SNPs. The most striking associated genetic change was a reduction in the frequency of infections containing multiple parasite genotypes from 63% in 2001 to 14% in 2010 (p = 3×10−15). Two measures of the clonal composition of populations (genotypic richness and the β-parameter of the Pareto distribution) declined over time as more people were infected by parasites with identical multilocus genotypes, consistent with increased selfing and a reduction in the rate at which multilocus genotypes are broken apart by recombination. We predicted that the reduction in transmission, multiple clone carriage, and outbreeding would be mirrored by an increased influence of genetic drift. However, geographical differentiation and expected heterozygosity remained stable across the sampling period. Furthermore, Ne estimates derived from allele frequencies fluctuation between years remained high (582 to ∞) and showed no downward trend. These results demonstrate how genetic data can compliment epidemiological assessments of infectious disease control programs. The temporal changes in a single declining population parallel those seen in comparisons of parasite genetics in regions of differing endemicity, strongly supporting the notion that reduced opportunity for outbreeding is the key driver of these patterns.
With the rapidly increasing abundance and accessibility of genomic data, there is a growing interest in using population genetic approaches to characterize fine-scale dispersal of organisms, providing insight into biological processes across a broad range of fields including ecology, evolution and epidemiology. For sexually recombining haploid organisms such as the human malaria parasite P. falciparum, however, there have been no systematic assessments of the type of data and methods required to resolve fine scale connectivity. This analytical gap hinders the use of genomics for understanding local transmission patterns, a crucial goal for policy makers charged with eliminating this important human pathogen. Here we use data collected from four clinics with a catchment area spanning approximately 120 km of the Thai-Myanmar border to compare the ability of divergence (FST) and relatedness based on identity by descent (IBD) to resolve spatial connectivity between malaria parasites collected from proximal clinics. We found no relationship between inter-clinic distance and FST, likely due to sampling of highly related parasites within clinics, but a significant decline in IBD-based relatedness with increasing inter-clinic distance. This association was contingent upon the data set type and size. We estimated that approximately 147 single-infection whole genome sequenced parasite samples or 222 single-infection parasite samples genotyped at 93 single nucleotide polymorphisms (SNPs) were sufficient to recover a robust spatial trend estimate at this scale. In summary, surveillance efforts cannot rely on classical measures of genetic divergence to measure P. falciparum transmission on a local scale. Given adequate sampling, IBD-based relatedness provides a useful alternative, and robust trends can be obtained from parasite samples genotyped at approximately 100 SNPs.
Malaria infections containing multiple parasite genotypes are ubiquitous in nature, and play a central role in models of recombination, intra-host dynamics, virulence, sex ratio, immunity and drug resistance evolution in Plasmodium. While these multiple infections (MIs) are often assumed to result from superinfection (bites from multiple infected mosquitoes), we know remarkably little about their composition or generation. We isolated 336 parasite clones from eight patients from Malawi (high transmission) and six from Thailand (low transmission) by dilution cloning. These were genotyped using 384 single-nucleotide polymorphisms, revealing 22 independent haplotypes in Malawi (2–6 per MI) and 15 in Thailand (2–5 per MI). Surprisingly, all six patients from Thailand and six of eight from Malawi contained related haplotypes, and haplotypes were more similar within- than between-infections. These results argue against a simple superinfection model. Instead, the observed kinship patterns may be explained by inoculation of multiple related haploid sporozoites from single mosquito bites, by immune suppression of parasite subpopulations within infections, and serial transmission of related parasites between people. That relatedness is maintained in endemic areas in the face of repeated bites from infected mosquitoes has profound implications for understanding malaria transmission, immunity and intra-host dynamics of co-infecting parasite genotypes.
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