Why are there so many independent origins of artemisinin resistance in malaria parasites?

Abstract

SummaryMultiple alleles at the kelch13 locus conferring artemisinin resistance (ART-R) are currently spreading through malaria parasite populations in Southeast Asia, providing a unique opportunity to directly observe an ongoing soft selective sweep, to investigate why resistance alleles have evolved multiple times and to determine fundamental population genetic parameters for Plasmodium. We sequenced the kelch13 gene (n=1,876), genotyped 75 flanking SNPs, and measured clearance rate (n=3,552) in parasite infections from Western Thailand (2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014). We describe 32 independent coding mutations: these included common mutations outside the kelch13 propeller region associated with significant reductions in clearance rate. Mutations were first observed in 2003 and rose to 90% by 2014, consistent with a selection coefficient of ~0.079. There was no change in diversity in flanking markers, but resistance allele diversity rose until 2012 and then dropped as one allele (C580Y) spread to high frequency. The rapid spread of C580Y suggests that the genomic signature may be considerably harder in the near future, and that retrospective studies may underestimate the complexity of selective sweeps. The frequency with which adaptive alleles arise is determined by the rate of mutation to generate beneficial alleles and the population size. Two factors drive this soft sweep: (1) multiple amino-acid mutations in kelch13 can confer resistance providing a large mutational target -we estimate the target size is between 87 and 163bp. (2) The population mutation parameter (Θ=2N e μ) can be estimated from the frequency distribution of resistant alleles and is ~ 5.69, suggesting that short term effective population size is between 88 thousand and 1.2 million. This is 52 to 705-fold greater than N e estimates based on fluctuation in allele frequencies, suggesting that . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/056291 doi: bioRxiv preprint first posted online May. 31, 2016; we have previously underestimated the capacity for adaptive evolution in Plasmodium. Our central conclusions are that retrospective studies may underestimate the complexity of selective events, ART-R evolution is not limited by availability of mutations, and the N e relevant for adaptation for malaria is considerably higher than previously estimated.Significance Statement: Previous work has identified surprisingly few origins of resistance to antimalarial drugs such as chloroquine and pyrimethamine. This has lead to optimism about prospects for minimizing resistance evolution through combination therapy. We studied a longitudinal collection of malaria parasites from the Thai-Myanmar border to examine an ongoing selective event in which ≥32 independent alleles associated with ART-R evolved. Three factors appear to explain the large number of origins obs...

Longitudinal genomic surveillance ofPlasmodium falciparummalaria parasites reveals complex genomic architecture of emerging artemisinin resistance in western Thailand

“…information potential, combined with the falling cost of genome sequencing, makes a compelling argument for prospective longitudinal genomic surveillance in pathogen populations where the evolution of resistance to therapeutics is a risk.These results, together with the very detailed longitudinal profile of the temporal dynamics of resistance mutations within the kelch13 locus itself over the same time interval[14], speak to the complexity of drug resistance evolution when observed in real time instead of after the evolution of a fit resistance genotype. This complexity could give fair warning of the emergence of resistance if changes were observed prospectively via a genome-wide surveillance program.…”

“…Parasite clearance data were used to estimate clearance half-life as described previously [14]. Ethical approval for this work was given by the Genotype filtering -Polymorphic sites meeting any of the following criteria were removed from the analysis using VCFTools [63] and custom scripts:…”

“…First, SNPs were identified that exhibited a sample frequency history strictly similar to the C580Y kelch13 mutation ('C580Y-like'), which has prevailed as the most successful ART resistance mutation in the region [14]. To be defined as C580Y-like, SNPs were required to exhibit a NRAF of 0 during the first two sampling intervals (2001-2004 and 2008) and NRAF ≥ 5% during the third sampling interval (2011-2012).…”

“…Mutations in the BTB/POZ or propeller domain of the kelch13 gene (PF3D7_1343700) have been associated with artemisinin resistance (ART-R), as evidenced by in vitro selection [7] and transfection experiments [8], and are associated with reduced cure rates following ACT [9][10][11]. Surveys have documented the rapid increase in frequency of kelch13 mutations in SE Asia over the last ten years, driven by a combination of de novo mutation creating new resistance alleles and natural selection favoring the spread of existing alleles [5,[12][13][14]. Though kelch13 resistance mutations are becoming prevalent in SE Asia, and have been observed at low frequency in Africa, South America, and other parts of the world, to date they have not been observed to spread in any location outside of SE Asia [13,15].…”

Reply to Meshnick and Hastings et al

“…Professor Meshnick believes that attempts to contain artemisinin resistance are futile because “all genetic evidence currently suggests that the K13 mutations pop up multiple times, independently, in different locations and are not spread spatially.” However, a very limited number of K13 alleles conferring artemisinin combination therapy (ACT) resistance are now replacing the throng of K13 alleles that appeared initially [4]. A more familiar pattern is emerging in which very few lineages of presumably fitter resistant parasites are increasing rapidly in frequency, causing rising rates of treatment failure and reinforcing the urgent need for an effective emergency containment response.…”