Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia
Medical Research Council1, Wellcome Trust Centre for Human Genetics2, Wellcome Trust Sanger Institute3, National Institutes of Health4, University of Oxford5, University for Development Studies6, Mahidol University7, University of Maryland, Baltimore8, World Health Organization9, Pasteur Institute10, University of London11, Charles Darwin University12
TL;DR: An analysis of genome variation in 825 P. falciparum samples from Asia and Africa is described that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia, and a catalog of SNPs that show high levels of differentiation in the art Artemisinin-resistant subpopulations are provided.
Abstract: 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.
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TL;DR: Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing, and the incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission.
Abstract: BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.).
1,777 citations
TL;DR: Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance.
Abstract: Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.
1,639 citations
Cites background from "Multiple populations of artemisinin..."
...…Sokunthea Sreng6, Seila Suon6, Char Meng Chuor6, Denis Mey Bout7, Sandie Ménard8{, William O. Rogers9, Blaise Genton10, Thierry Fandeur1,3, Olivo Miotto11,12,13, Pascal Ringwald14, Jacques Le Bras15, Antoine Berry8{, Jean-Christophe Barale1,2{, Rick M. Fairhurst4*, Françoise…...
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...10.1038/nature12876. pasteur-00921203 ARTICLE doi:10.1038/nature12876 A molecular marker of artemisininresistant Plasmodium falciparummalaria Frédéric Ariey1,2{, Benoit Witkowski3, Chanaki Amaratunga4, Johann Beghain1,2{, Anne-Claire Langlois1,2, Nimol Khim3, Saorin Kim3, Valentine Duru3, Christiane Bouchier5, Laurence Ma5, Pharath Lim3,4,6, Rithea Leang6, Socheat Duong6, Sokunthea Sreng6, Seila Suon6, Char Meng Chuor6, Denis Mey Bout7, Sandie Ménard8{, William O. Rogers9, Blaise Genton10, Thierry Fandeur1,3, Olivo Miotto11,12,13, Pascal Ringwald14, Jacques Le Bras15, Antoine Berry8{, Jean-Christophe Barale1,2{, Rick M. Fairhurst4*, Françoise Benoit-Vical16,17*, Odile Mercereau-Puijalon1,2* & Didier Ménard3* Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatensmalaria control and elimination activities worldwide....
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TL;DR: The data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites and imperils efforts to reduce the global malaria burden.
Abstract: The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.
570 citations
TL;DR: This Review describes the metabolic engineering and synthetic biology approaches that were used to develop this important antimalarial drug precursor and illuminates how lessons learned from this work could be applied to the production of other pharmaceutical agents.
Abstract: Recent developments in synthetic biology, combined with continued progress in systems biology and metabolic engineering, have enabled the engineering of microorganisms to produce heterologous molecules in a manner that was previously unfeasible. The successful synthesis and recent entry of semi-synthetic artemisinin into commercial production is the first demonstration of the potential of synthetic biology for the development and production of pharmaceutical agents. In this Review, we describe the metabolic engineering and synthetic biology approaches that were used to develop this important antimalarial drug precursor. This not only demonstrates the incredible potential of the available technologies but also illuminates how lessons learned from this work could be applied to the production of other pharmaceutical agents.
559 citations
TL;DR: Biochemical and cellular evidence is provided that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action and present PI3P as the key mediator of art Artemisinin resistance and the sole PfPI3k as an important target for malaria elimination.
Abstract: Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.
515 citations
References
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TL;DR: The neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods for reconstructing phylogenetic trees from evolutionary distance data.
Abstract: A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
57,055 citations
TL;DR: Burrows-Wheeler Alignment tool (BWA) is implemented, a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps.
Abstract: Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals.
Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ~10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package.
Availability: http://maq.sourceforge.net
Contact: [email protected]
43,862 citations
Additional excerpts
...The sites are (1) Banjul, The Gambia; (2) Bamako, Mali; (3) Bobo‐Dioulasso, Burkina Faso; (4) Navrongo, Ghana; (5) Mae Sot, Thailand; (6) Ratanakiri, Cambodia; (7) Binh Phuoc province, Vietnam; (8) Pailin, (9) Tasanh and (10)...
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TL;DR: The results show that ADMIXTURE's computational speed opens up the possibility of using a much larger set of markers in model-based ancestry estimation and that its estimates are suitable for use in correcting for population stratification in association studies.
Abstract: Population stratification has long been recognized as a confounding factor in genetic association studies. Estimated ancestries, derived from multi-locus genotype data, can be used to perform a statistical correction for population stratification. One popular technique for estimation of ancestry is the model-based approach embodied by the widely applied program structure. Another approach, implemented in the program EIGENSTRAT, relies on Principal Component Analysis rather than model-based estimation and does not directly deliver admixture fractions. EIGENSTRAT has gained in popularity in part owing to its remarkable speed in comparison to structure. We present a new algorithm and a program, ADMIXTURE, for model-based estimation of ancestry in unrelated individuals. ADMIXTURE adopts the likelihood model embedded in structure. However, ADMIXTURE runs considerably faster, solving problems in minutes that take structure hours. In many of our experiments, we have found that ADMIXTURE is almost as fast as EIGENSTRAT. The runtime improvements of ADMIXTURE rely on a fast block relaxation scheme using sequential quadratic programming for block updates, coupled with a novel quasi-Newton acceleration of convergence. Our algorithm also runs faster and with greater accuracy than the implementation of an Expectation-Maximization (EM) algorithm incorporated in the program FRAPPE. Our simulations show that ADMIXTURE's maximum likelihood estimates of the underlying admixture coefficients and ancestral allele frequencies are as accurate as structure's Bayesian estimates. On real-world data sets, ADMIXTURE's estimates are directly comparable to those from structure and EIGENSTRAT. Taken together, our results show that ADMIXTURE's computational speed opens up the possibility of using a much larger set of markers in model-based ancestry estimation and that its estimates are suitable for use in correcting for population stratification in association studies.
5,846 citations
David R. Bentley1, Shankar Balasubramanian2, Harold Swerdlow1, Harold Swerdlow3 +198 more•Institutions (4)
TL;DR: An approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost is reported, effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.
Abstract: DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.
3,802 citations
Additional excerpts
...The sites are (1) Banjul, The Gambia; (2) Bamako, Mali; (3) Bobo‐Dioulasso, Burkina Faso; (4) Navrongo, Ghana; (5) Mae Sot, Thailand; (6) Ratanakiri, Cambodia; (7) Binh Phuoc province, Vietnam; (8) Pailin, (9) Tasanh and (10)...
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TL;DR: In this paper, the authors derived necessary and sufficient conditions for a solution to exist in real Euclidean space for a multivariate multivariate sample of size n as points P1, P2,..., PI in a Euclidian space and discussed the interpretation of the distance A(Pi, Pj) between the ith and jth members of the sample.
Abstract: SUMMARY This paper is concerned with the representation of a multivariate sample of size n as points P1, P2, ..., PI in a Euclidean space. The interpretation of the distance A(Pi, Pj) between the ith andjth members of the sample is discussed for some commonly used types of analysis, including both Q and R techniques. When all the distances between n points are known a method is derived which finds their co-ordinates referred to principal axes. A set of necessary and sufficient conditions for a solution to exist in real Euclidean space is found. Q and R techniques are defined as being dual to one another when they both lead to a set of n points with the same inter-point distances. Pairs of dual techniques are derived. In factor analysis the distances between points whose co-ordinates are the estimated factor scores can be interpreted as D2 with a singular dispersion matrix.
3,746 citations