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Publication Metadata only Declining Efficacy of Artemisinin Combination Therapy Against P. Falciparum Malaria on the Thai-Myanmar Border (2003-2013): The Role of Parasite Genetic Factors(2016-09-15) Aung Pyae Phyo; Elizabeth A. Ashley; Tim J.C. Anderson; Zbynek Bozdech; Verena I. Carrara; Kanlaya Sriprawat; Shalini Nair; Marina Mc Dew White; Jerzy Dziekan; Clare Ling; Stephane Proux; Kamonchanok Konghahong; Atthanee Jeeyapant; Charles J. Woodrow; Mallika Imwong; Rose McGready; Khin Maung Lwin; Nicholas P.J. Day; Nicholas J. White; Francois Nosten; Mahidol University; University of Oxford; Texas Biomedical Research Institute; Nanyang Technological University© 2016 The Author. Published by Oxford University Press for the Infectious Diseases Society of America. Background. Deployment of mefloquine-artesunate (MAS3) on the Thailand-Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial. Methods. Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance. Results. Polymerase chain reaction (PCR)-adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8% (95% confidence interval [CI], 45.4, 68.3) compared with 97.8% (95% CI, 93.3, 99.3) in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P =. 009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%. Conclusions. The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand-Myanmar border.Publication Metadata only Fitness costs and the rapid spread of kelch13-C580Y substitutions conferring artemisinin resistance(2018-09-01) Shalini Nair; Xue Li; Grace A. Arya; Marina McDew-White; Marco Ferrari; François Nosten; Tim J.C. Anderson; Università degli Studi di Milano; University of Oxford; Texas Biomedical Research Institute; Mahidol UniversityCopyright © 2018 Nair et al. Fitness costs are key determinants of whether drug resistance alleles establish and how fast they spread within populations. More than 125 different kelch13 alleles, each containing a different amino acid substitution, have arisen in Southeast Asian malaria parasite (Plasmodium falciparum) populations under artemisinin selection over the past 15 years in a dramatic example of a soft selective event. However, just one of these alleles (C580Y) is now outcompeting other alleles in multiple different countries and is spreading toward fixation. Here we examine the fitness consequences of C580Y, relative to another less successful kelch13 mutation (R561H), to try to explain the distinctive dynamics of C580Y. We hypothesized that C580Y will show lower fitness costs than other kelch13 substitutions in the absence of artemisinin treatment. We used CRISPR/Cas9 methods to introduce single mutations (C580Y or R561H) or synonymous control edits into a wild-type parasite isolated on the Thailand-Myanmar border, conducted replicated head-to-head competition assays, and determined the outcome of competition using deep sequencing of kelch13 amplicons. Contrary to our predictions, these experiments reveal that C580Y carries higher fitness costs (s [selection coefficient] 0.15 0.008 [1 standard error {SE}]) than R561H (s 0.084 0.005). Furthermore, R561H outcompetes C580Y in direct competition (s 0.065 0.004). We conclude that fitness costs of C580Y in isolation are unlikely to explain the rapid spread of this substitution.Publication Metadata only Population parameters underlying an ongoing soft sweep in southeast asian malaria parasites(2017-01-01) Timothy J.C. Anderson; Shalini Nair; Marina McDew-White; Ian H. Cheeseman; Standwell Nkhoma; Fatma Bilgic; Rose McGready; Elizabeth Ashley; Aung Pyae Phyo; Nicholas J. White; Francois Nosten; Texas Biomedical Research Institute; Mahidol University; Nuffield Department of Clinical Medicine© 2016 The Author. Multiple kelch13 alleles conferring artemisinin resistance (ART-R) are currently spreading through Southeast Asian malaria parasite populations, providing a unique opportunity to observe an ongoing soft selective sweep, investigate why resistance alleles have evolved multiple times and determine fundamental population genetic parameters for Plasmodium. We sequenced kelch13 (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 including common mutations outside the kelch13 propeller 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. ART-R allele diversity rose until 2012 and then dropped as one allele (C580Y) spread to high frequency. The frequency with which adaptive alleles arise is determined by the rate of mutation and the population size. Two factors drive this soft sweep: (1) multiple kelch13 amino-acid mutations confer resistance providing a large mutational target-we estimate the target is 87-163bp. (2) The population mutation parameter (H = 2Nel) can be estimated from the frequency distribution of ART-R alleles and is ∼5.69, suggesting that short term effective population size is 88 thousand to 1.2 million. This is 52-705 times greater than Ne estimated from 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 and the Ne relevant for adaptation for malaria is considerably higher than previously estimated.Publication Metadata only Longitudinal genomic surveillance of Plasmodium falciparum malaria parasites reveals complex genomic architecture of emerging artemisinin resistance(2017-04-28) Gustavo C. Cerqueira; Ian H. Cheeseman; Steve F. Schaffner; Shalini Nair; Marina McDew-White; Aung Pyae Phyo; Elizabeth A. Ashley; Alexandre Melnikov; Peter Rogov; Bruce W. Birren; François Nosten; Timothy J.C. Anderson; Daniel E. Neafsey; Broad Institute; Texas Biomedical Research Institute; Mahidol University; Nuffield Department of Clinical Medicine© 2017 The Author(s). Background: Artemisinin-based combination therapies are the first line of treatment for Plasmodium falciparum infections worldwide, but artemisinin resistance has risen rapidly in Southeast Asia over the past decade. Mutations in the kelch13 gene have been implicated in this resistance. We used longitudinal genomic surveillance to detect signals in kelch13 and other loci that contribute to artemisinin or partner drug resistance. We retrospectively sequenced the genomes of 194 P. falciparum isolates from five sites in Northwest Thailand, over the period of a rapid increase in the emergence of artemisinin resistance (2001-2014). Results: We evaluate statistical metrics for temporal change in the frequency of individual SNPs, assuming that SNPs associated with resistance increase in frequency over this period. After Kelch13-C580Y, the strongest temporal change is seen at a SNP in phosphatidylinositol 4-kinase, which is involved in a pathway recently implicated in artemisinin resistance. Furthermore, other loci exhibit strong temporal signatures which warrant further investigation for involvement in artemisinin resistance evolution. Through genome-wide association analysis we identify a variant in a kelch domain-containing gene on chromosome 10 that may epistatically modulate artemisinin resistance. Conclusions: This analysis demonstrates the potential of a longitudinal genomic surveillance approach to detect resistance-associated gene loci to improve our mechanistic understanding of how resistance develops. Evidence for additional genomic regions outside of the kelch13 locus associated with artemisinin-resistant parasites may yield new molecular markers for resistance surveillance, which may be useful in efforts to reduce the emergence or spread of artemisinin resistance in African parasite populations.Publication Metadata only Optimal health and disease management using spatial uncertainty: A geographic characterization of emergent artemisinin-resistant Plasmodium falciparum distributions in Southeast Asia(2016-10-24) Eric P.M. Grist; Jennifer A. Flegg; Georgina Humphreys; Ignacio Suay Mas; Tim J.C. Anderson; Elizabeth A. Ashley; Nicholas P.J. Day; Mehul Dhorda; Arjen M. Dondorp; M. Abul Faiz; Peter W. Gething; Tran T. Hien; Tin M. Hlaing; Mallika Imwong; Jean Marie Kindermans; Richard J. Maude; Mayfong Mayxay; Marina McDew-White; Didier Menard; Shalini Nair; Francois Nosten; Paul N. Newton; Ric N. Price; Sasithon Pukrittayakamee; Shannon Takala-Harrison; Frank Smithuis; Nhien T. Nguyen; Kyaw M. Tun; Nicholas J. White; Benoit Witkowski; Charles J. Woodrow; Rick M. Fairhurst; Carol Hopkins Sibley; Philippe J. Guerin; WorldWide Antimalarial Resistance Network (WWARN); Nuffield Department of Clinical Medicine; Monash University; Texas Biomedical Research Institute; Mahidol University; National Institute of Allergy and Infectious Diseases; Dev Care Foundation; University of Oxford; Oxford University Clinical Research Unit; Defence Services Medical Research Centre; Medecins Sans Frontieres, Brussels; Pasteur Institute in Cambodia; Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU); University of Maryland School of Medicine; Menzies School of Health Research; Myanmar Oxford Clinical Research Unit; University of Washington, Seattle; Harvard School of Public Health© 2016 The Author(s). Background: Artemisinin-resistant Plasmodium falciparum malaria parasites are now present across much of mainland Southeast Asia, where ongoing surveys are measuring and mapping their spatial distribution. These efforts require substantial resources. Here we propose a generic 'smart surveillance' methodology to identify optimal candidate sites for future sampling and thus map the distribution of artemisinin resistance most efficiently. Methods: The approach uses the 'uncertainty' map generated iteratively by a geostatistical model to determine optimal locations for subsequent sampling. Results: The methodology is illustrated using recent data on the prevalence of the K13-propeller polymorphism (a genetic marker of artemisinin resistance) in the Greater Mekong Subregion. Conclusion: This methodology, which has broader application to geostatistical mapping in general, could improve the quality and efficiency of drug resistance mapping and thereby guide practical operations to eliminate malaria in affected areas.Publication Metadata only Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: A cross-sectional survey of the K13 molecular marker(2015-04-01) Kyaw M. Tun; Mallika Imwong; Khin M. Lwin; Aye A. Win; Tin M. Hlaing; Thaung Hlaing; Khin Lin; Myat P. Kyaw; Katherine Plewes; M. Abul Faiz; Mehul Dhorda; Phaik Yeong Cheah; Sasithon Pukrittayakamee; Elizabeth A. Ashley; Tim J.C. Anderson; Shalini Nair; Marina McDew-White; Jennifer A. Flegg; Eric P.M. Grist; Philippe Guerin; Richard J. Maude; Frank Smithuis; Arjen M. Dondorp; Nicholas P.J. Day; François Nosten; Nicholas J. White; Charles J. Woodrow; Myanmar Oxford Clinical Research Unit; Defence Services Medical Research Centre; Mahidol University; Shoklo Malaria Research Unit; Institute of Medicine (1); Ministry of Health; Department of Medical Research; WorldWide Antimalarial Resistance Network; Nuffield Department of Clinical Medicine; Dev Care Foundation; Texas Biomedical Research Institute; Monash University; Medical Action Myanmar© 2015 Tun et al. 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.