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 (2001-2014). 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 ({Theta}=2Ne) 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 Ne estimates based on fluctuation in allele frequencies, suggesting that 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 Ne relevant for adaptation for malaria is considerably higher than previously estimated.\n\nSignificance StatementPrevious 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 (2001-14) 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 observed: the large number of amino acid changes that result in resistance (i.e. large mutational \"target size\"), the large estimated effective population size (Ne), and the fact that we were able to document this selective event in real time, rather than retrospectively.