Absence of Putative Artemisinin Resistance Mutations Among Plasmodium falciparum in Sub-Saharan Africa: A Molecular Epidemiologic Study
University of North Carolina at Chapel Hill1, Duke University2, Liverpool School of Tropical Medicine3, University of Ouagadougou4, University of London5, Kwame Nkrumah University of Science and Technology6, Medical Research Council7, Centers for Disease Control and Prevention8, Kenya Medical Research Institute9, University of Malawi10, Karolinska Institutet11, University of Health and Allied Sciences12, University of California, San Francisco13, University of Kinshasa14, University of Massachusetts Amherst15
01 Mar 2015-The Journal of Infectious Diseases (Oxford University Press)-Vol. 211, Iss: 5, pp 680-688
TL;DR: An assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies is developed and validated by evaluating mixtures of laboratory parasite strains, and used to screen P. falciparum parasites from >1100 African infections collected since 2002.
Abstract: Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasite's K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance.
Citations
More filters
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
Pasteur Institute1, University of Tübingen2, Leiden University Medical Center3, International Centre for Diarrhoeal Disease Research, Bangladesh4, University of Ibadan5, Nangarhar University6, Mahidol University7, University of Melbourne8, Papua New Guinea Institute of Medical Research9, Walter and Eliza Hall Institute of Medical Research10, Mbarara University of Science and Technology11, Médecins Sans Frontières12, World Health Organization13, Jiangsu University14, National Institute of Parasitic Diseases15, Pennsylvania State University16, Cheikh Anta Diop University17, University of Lomé18, University of Douala19, Research Institute for Tropical Medicine20, Oswaldo Cruz Foundation21, University of Gondar22, Chinese Academy of Sciences23, Henry M. Jackson Foundation for the Advancement of Military Medicine24, Institut de recherche pour le développement25, University of Antwerp26, University of Kinshasa27, Thammasat University28, University of Bamako29, Medical University of Vienna30, Medicines for Malaria Venture31, Université Félix Houphouët-Boigny32, University of London33, Eijkman Institute for Molecular Biology34, Southwest University of Visual Arts35, Columbia University36, Paris Descartes University37
TL;DR: In this article, the authors analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic and identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution.
Abstract: BACKGROUND:
Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale.
METHODS:
We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci.
RESULTS:
We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay.
CONCLUSIONS:
No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).
398 citations
TL;DR: Assessment of the spread of artemisinin-resistant P falciparum in Myanmar finds Artemisinin resistance extends across much of Myanmar, and Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of warfarin resistance to other regions is to be avoided.
Abstract: Summary Background Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of molecular surveillance in remote areas through analysis of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by determining the relative prevalence of P falciparum parasites carrying K13-propeller mutations. Methods We did this cross-sectional survey at malaria treatment centres at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between January, 2013, and September, 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estimated prevalence of mutations of the K13 propeller region across Myanmar. Findings Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations. Interpretation Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided. Funding Wellcome Trust–Mahidol University–Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.
385 citations
TL;DR: Elimination of artemisinin resistance will imply elimination of all falciparum malaria from the same areas, and novel regimens and strategies using existing antimalarial drugs will be needed until novel compounds can be deployed.
Abstract: Increasing antimalarial drug resistance once again threatens effective antimalarial drug treatment, malaria control, and elimination. Artemisinin combination therapies (ACTs) are first-line treatment for uncomplicated falciparum malaria in all endemic countries, yet partial resistance to artemisinins has emerged in the Greater Mekong Subregion. Concomitant emergence of partner drug resistance is now causing high ACT treatment failure rates in several areas. Genetic markers for artemisinin resistance and several of the partner drugs have been established, greatly facilitating surveillance. Single point mutations in the gene coding for the Kelch propeller domain of the K13 protein strongly correlate with artemisinin resistance. Novel regimens and strategies using existing antimalarial drugs will be needed until novel compounds can be deployed. Elimination of artemisinin resistance will imply elimination of all falciparum malaria from the same areas. In vivax malaria, chloroquine resistance is an increasing problem.
290 citations
Cites result from "Absence of Putative Artemisinin Res..."
...…as molecular marker of artemisinin resistance (C580Y, R539T, I543T, Y493H), confirming previous smaller-sized studies (Conrad et al. 2014; Torrentino-Madamet et al. 2014; Cooper et al. 2015; Escobar et al. 2015; Hawkes et al. 2015; Isozumi et al. 2015; Kamau et al. 2015; Taylor et al. 2015)....
[...]
...It is thought that artemisinin resistance has not been established in Africa, supported by the additional absence of evidence of invasion by Asian K13 alleles validated as molecular marker of artemisinin resistance (C580Y, R539T, I543T, Y493H), confirming previous smaller-sized studies (Conrad et al. 2014; Torrentino-Madamet et al. 2014; Cooper et al. 2015; Escobar et al. 2015; Hawkes et al. 2015; Isozumi et al. 2015; Kamau et al. 2015; Taylor et al. 2015)....
[...]
University of Oxford1, Wellcome Trust Sanger Institute2, Mahidol University3, Medical Research Council4, National Institutes of Health5, University for Development Studies6, Pasteur Institute7, University of Buea8, University of Ghana9, National Institute for Medical Research10, University of Antwerp11, University of Melbourne12, Walter and Eliza Hall Institute of Medical Research13, University of Tübingen14, Kenya Medical Research Institute15, University of London16, New York University17, Liverpool School of Tropical Medicine18, University of Bamako19, Purdue University20
TL;DR: It is shown that African kelch13 mutations have originated locally, and that kelCh13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes.
Abstract: Malaria is an infectious disease caused by a microscopic parasite called Plasmodium, which is transferred between humans by mosquitos. One species of malaria parasite called Plasmodium falciparum can cause particularly severe and life-threatening forms of the disease. Currently, the most widely used treatment for P. falciparum infections is artemisinin combination therapy, a treatment that combines the drug artemisinin (or a closely related molecule) with another antimalarial drug. However, resistance to artemisinin has started to spread throughout Southeast Asia. Artemisinin resistance is caused by mutations in a parasite gene called kelch13, and researchers have identified over 20 different mutations in P. falciparum that confer artemisinin resistance. The diversity of mutations involved, and the fact that the same mutation can arise independently in different locations, make it difficult to track the spread of resistance using conventional molecular marker approaches. Here, Amato, Miotto et al. sequenced the entire genomes of more than 3,000 clinical samples of P. falciparum from Southeast Asia and Africa, collected as part of a global network of research groups called the MalariaGEN Plasmodium falciparum Community Project. Amato, Miotto et al. found that African parasites had independently acquired many of the same kelch13 mutations that are known to cause resistance to artemisinin in Southeast Asia. However the kelch13 mutations seen in Africa remained at low levels in the parasite population, and appeared to be under much less pressure for evolutionary selection than those found in Southeast Asia. These findings demonstrate that the emergence and spread of resistance to antimalarial drugs does not depend solely on the mutational process, but also on other factors that influence whether the mutations will spread in the population. Understanding how this is affected by different patterns of drug treatments and other environmental conditions will be important in developing more effective strategies for combating malaria.
276 citations
Cites background from "Absence of Putative Artemisinin Res..."
...Thus there is considerable uncertainty about the epidemiological significance of the growing number of non-synonymous KPBD mutations reported in Africa (Ashley et al., 2014; Hopkins Sibley, 2015; Kamau et al., 2015; Taylor et al., 2015)....
[...]
...Previous studies in Africa have not identified variants known to cause resistance in Southeast Asia (Kamau et al., 2015; Taylor et al., 2015)....
[...]
References
More filters
TL;DR: SAMtools as discussed by the authors implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.
Abstract: Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.
Availability: http://samtools.sourceforge.net
Contact: [email protected]
45,957 citations
TL;DR: An advanced version of the Molecular Evolutionary Genetics Analysis software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis, is released, which enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny.
Abstract: We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
37,956 citations
TL;DR: Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
Abstract: As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
37,898 citations
01 Jan 2013
TL;DR: The Molecular Evolutionary Genetics Analysis (MEGA) software as discussed by the authors provides facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis, including the inference of timetrees.
Abstract: We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www. megasoftware.net free of charge.
30,478 citations
TL;DR: UNLABELLED Analysis of Phylogenetics and Evolution (APE) is a package written in the R language for use in molecular evolution and phylogenetics that provides both utility functions for reading and writing data and manipulating phylogenetic trees.
Abstract: Summary: Analysis of Phylogenetics and Evolution (APE) is a package written in the R language for use in molecular evolution and phylogenetics. APE provides both utility functions for reading and writing data and manipulating phylogenetic trees, as well as several advanced methods for phylogenetic and evolutionary analysis (e.g. comparative and population genetic methods). APE takes advantage of the many R functions for statistics and graphics, and also provides a flexible framework for developing and implementing further statistical methods for the analysis of evolutionary processes.
Availability: The program is free and available from the official R package archive at http://cran.r-project.org/src/contrib/PACKAGES.html#ape. APE is licensed under the GNU General Public License.
10,818 citations