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Publication Metadata only Artemisinin-resistant Plasmodium falciparum K13 mutant alleles, Thailand-Myanmar border(2016-08-01) Mikael Boullé; Benoit Witkowski; Valentine Duru; Kanlaya Sriprawat; Shalini K. Nair; Marina McDew-White; Tim J.C. Anderson; Aung Pyae Phyo; Didier Menard; François Nosten; Mahidol University; Institut Pasteur du Cambodge; Texas Biomedical Research Institute; University of OxfordPublication 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.Item Metadata only A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media(2022-05-30) Kumar S.; Li X.; McDew-White M.; Reyes A.; Delgado E.; Sayeed A.; Haile M.T.; Abatiyow B.A.; Kennedy S.Y.; Camargo N.; Checkley L.A.; Brenneman K.V.; Button-Simons K.A.; Duraisingh M.T.; Cheeseman I.H.; Kappe S.H.I.; Nosten F.; Ferdig M.T.; Vaughan A.M.; Anderson T.J.C.; Mahidol UniversityWhat genes determine in vitro growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between NF54, clone 3D7, a lab-adapted African parasite, and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: 3D7 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes 3D7 in media containing AlbuMAX, a commercial lipid-rich bovine serum formulation. To determine the basis for this polymorphism, we conducted parasite genetic crosses using humanized mice and compared genome-wide allele frequency changes in three independent progeny populations cultured in media containing human serum or AlbuMAX. This bulk segregant analysis detected three quantitative trait loci (QTL) regions [on chromosome (chr) 2 containing aspartate transaminase AST; chr 13 containing EBA-140; and chr 14 containing cysteine protease ATG4] linked with differential growth in serum or AlbuMAX in each of the three independent progeny pools. Selection driving differential growth was strong (s = 0.10 – 0.23 per 48-hour lifecycle). We conducted validation experiments for the strongest QTL on chr 13: competition experiments between ΔEBA-140 and 3D7 wildtype parasites showed fitness reversals in the two medium types as seen in the parental parasites, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of bulk segregant analysis for dissecting fitness traits in P. falciparum genetic crosses, and (ii) reveal intimate links between red blood cell invasion and nutrient composition of growth media. Use of parasite crosses combined with bulk segregant analysis will allow systematic dissection of key nutrient acquisition/metabolism and red blood cell invasion pathways in P. falciparum.Publication Metadata only The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput(2020-01-31) Sage Z. Davis; Puspendra P. Singh; Katelyn M. Vendrely; Douglas A. Shoue; Lisa A. Checkley; Marina McDew-White; Katrina A. Button-Simons; Zione Cassady; MacKenzie A.C. Sievert; Gabriel J. Foster; François H. Nosten; Timothy J.C. Anderson; Michael T. Ferdig; University of California, Riverside; Texas Biomedical Research Institute; University of Notre Dame; Mahidol University; Nuffield Department of Clinical Medicine© 2020 The Author(s). Background: Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: It is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. Methods: Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. Results: 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of-0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. Conclusions: ERRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.Publication Metadata only Pairwise growth competitions identify relative fitness relationships among artemisinin resistant Plasmodium falciparum field isolates(2019-08-28) Abigail R. Tirrell; Katelyn M. Vendrely; Lisa A. Checkley; Sage Z. Davis; Marina McDew-White; Ian H. Cheeseman; Ashley M. Vaughan; François H. Nosten; Timothy J.C. Anderson; Michael T. Ferdig; Texas Biomedical Research Institute; University of Notre Dame; Children's Hospital and Regional Medical Center; Mahidol University; Nuffield Department of Clinical Medicine© 2019 The Author(s). Background: Competitive outcomes between co-infecting malaria parasite lines can reveal fitness disparities in blood stage growth. Blood stage fitness costs often accompany the evolution of drug resistance, with the expectation that relatively fitter parasites will be more likely to spread in populations. With the recent emergence of artemisinin resistance, it is important to understand the relative competitive fitness of the metabolically active asexual blood stage parasites. Genetically distinct drug resistant parasite clones with independently evolved sets of mutations are likely to vary in asexual proliferation rate, contributing to their chance of transmission to the mosquito vector. Methods: An optimized in vitro 96-well plate-based protocol was used to quantitatively measure-head-to-head competitive fitness during blood stage development between seven genetically distinct field isolates from a hotspot of emerging artemisinin resistance and the laboratory strain, NF54. These field isolates were isolated from patients in Southeast Asia carrying different alleles of kelch13 and included both artemisinin-sensitive and artemisinin-resistant isolates. Fluorescent labeled microsatellite markers were used to track the relative densities of each parasite throughout the co-growth period of 14-60 days. All-on-all competitions were conducted for the panel of eight parasite lines (28 pairwise competitions) to determine their quantitative competitive fitness relationships. Results: Twenty-eight pairwise competitive growth outcomes allowed for an unambiguous ranking among a set of seven genetically distinct parasite lines isolated from patients in Southeast Asia displaying a range of both kelch13 alleles and clinical clearance times and a laboratory strain, NF54. This comprehensive series of assays established the growth relationships among the eight parasite lines. Interestingly, a clinically artemisinin resistant parasite line that carries the wild-type form of kelch13 outcompeted all other parasites in this study. Furthermore, a kelch13 mutant line (E252Q) was competitively more fit without drug than lines with other resistance-associated kelch13 alleles, including the C580Y allele that has expanded to high frequencies under drug pressure in Southeast Asian resistant populations. Conclusions: This optimized competitive growth assay can be employed for assessment of relative growth as an index of fitness during the asexual blood stage growth between natural lines carrying different genetic variants associated with artemisinin resistance. Improved understanding of the fitness costs of different parasites proliferating in human blood and the role different resistance mutations play in the context of specific genetic backgrounds will contribute to an understanding of the potential for specific mutations to spread in populations, with the potential to inform targeted strategies for malaria therapy.Publication Metadata only The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice(2021-12-01) Katrina A. Button-Simons; Sudhir Kumar; Nelly Carmago; Meseret T. Haile; Catherine Jett; Lisa A. Checkley; Spencer Y. Kennedy; Richard S. Pinapati; Douglas A. Shoue; Marina McDew-White; Xue Li; François H. Nosten; Stefan H. Kappe; Timothy J.C. Anderson; Jeanne Romero-Severson; Michael T. Ferdig; Scott J. Emrich; Ashley M. Vaughan; Ian H. Cheeseman; Texas Biomedical Research Institute; University of Notre Dame; The University of Tennessee, Knoxville; Mahidol University; Nuffield Department of Medicine; Seattle Biomedical Research Institute; Nimble TherapeuticsGenetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses—(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels.Item Metadata only Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1(2023-01-01) Amambua-Ngwa A.; Button-Simons K.A.; Li X.; Kumar S.; Brenneman K.V.; Ferrari M.; Checkley L.A.; Haile M.T.; Shoue D.A.; McDew-White M.; Tindall S.M.; Reyes A.; Delgado E.; Dalhoff H.; Larbalestier J.K.; Amato R.; Pearson R.D.; Taylor A.B.; Nosten F.H.; D’Alessandro U.; Kwiatkowski D.; Cheeseman I.H.; Kappe S.H.I.; Avery S.V.; Conway D.J.; Vaughan A.M.; Ferdig M.T.; Anderson T.J.C.; Mahidol UniversityMalaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.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.