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Publication Open Access Impact of Dual Energy Window Scatter Correction on Image Quality in Tomographic Iodine 131(2018) Wirinya Saengthamchai; Supakiet Piasanthia; Kanokon Poonak; Putthiporn Charoenphun; Krisanat Chuamsaamarkkee; วิรินญา แสงธรรมชัย; ศุภเกียรติ เปียสันเทียะ; กนกอร ภู่นาค; พุทธิพรณ์ เจริญพันธุ์; กฤศณัฏฐ์ เชื่อมสามัคคี; Mahidol University. Faculty of Medicine Ramathibodi Hospital. Department of Diagnostic and Therapeutic Radiology; Navamindradhiraj University. Faculty of Medicine Vajira Hospital. Department of RadiologyBackground: The major limitation factors in single photon emission computed tomography (SPECT) detectability is the presence of scattered photons within the main photopeak. Objective: To evaluate the impact of dual-energy window (DEW) scatter... acquired 30, 40, and 90 seconds per frame for small spheres (0.52 mL, 1.15 mL, and 2.57 mL), whereas in large sphere (26.52 mL and 11.49 mL), the CNRs were varied when SC or NSC applied. Conclusions: Our results showed that the implementation of DEWPublication 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.Publication Metadata only Pooled sequencing and rare variant association tests for identifying the determinants of emerging drug resistance in malaria parasites(2015-04-01) Ian H. Cheeseman; Marina McDew-White; Aung Pyae Phyo; Kanlaya Sriprawat; Francois Nosten; Timothy J.C. Anderson; Texas Biomedical Research Institute; Mahidol University; Nuffield Department of Clinical Medicine© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. We explored the potential of pooled sequencing to swiftly and economically identify selective sweeps due to emerging artemisinin (ART) resistance in a South-East Asian malaria parasite population. ART resistance is defined by slow parasite clearance from the blood of ART-treated patients and mutations in the kelch gene (chr. 13) have been strongly implicated to play a role. We constructed triplicate pools of 70 slow-clearing (resistant) and 70 fast-clearing (sensitive) infections collected from the Thai-Myanmar border and sequenced these to high (∼150-fold) read depth. Allele frequency estimates from pools showed almost perfect correlation (Lin's concordance = 0.98) with allele frequencies at 93 single nucleotide polymorphisms measured directly from individual infections, giving us confidence in the accuracy of this approach. By mapping genome-wide divergence (FST) between pools of drug-resistant and drug-sensitive parasites, we identified two large (>150 kb) regions (on chrs. 13 and 14) and 17 smaller candidate genome regions. To identify individual genes within these genome regions, we resequenced an additional 38 parasite genomes (16 slow and 22 fast-clearing) and performed rare variant association tests. These confirmed kelch as a major molecular marker for ART resistance (P = 6.03 × 10-6). This two-tier approach is powerful because pooled sequencing rapidly narrows down genome regions of interest, while targeted rare variant association testing within these regions can pinpoint the genetic basis of resistance. We show that our approach is robust to recurrent mutation and the generation of soft selective sweeps, which are predicted to be common in pathogen populations with large effective population sizes, and may confound more traditional gene mapping approaches.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 Genetic mapping of fitness determinants across the malaria parasite Plasmodium falciparum life cycle(2019-01-01) Xue Li; Sudhir Kumar; Marina McDew-White; Meseret Haile; Ian H. Cheeseman; Scott Emrich; Katie Button-Simons; François Nosten; Stefan H.I. Kappe; Michael T. Ferdig; Tim J.C. Anderson; Ashley M. Vaughan; University of Oxford; Texas Biomedical Research Institute; University of Notre Dame; University of Tennessee, Knoxville; University of Washington, Seattle; Mahidol University; Seattle Children's Research Institute© 2019 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Determining the genetic basis of fitness is central to understanding evolution and transmission of microbial pathogens. In human malaria parasites (Plasmodium falciparum), most experimental work on fitness has focused on asexual blood stage parasites, because this stage can be easily cultured, although the transmission of malaria requires both female Anopheles mosquitoes and vertebrate hosts. We explore a powerful approach to identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-cycle stages of P. falciparum. This combines experimental genetic crosses using humanized mice, with selective whole genome amplification and pooled sequencing to determine genome-wide allele frequencies and identify genomic regions under selection across multiple lifecycle stages. We applied this approach to genetic crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, kelch13-WT) parasites, recently isolated from Southeast Asian patients. Two striking results emerge: we observed (i) a strong genome-wide skew (>80%) towards alleles from the ART-R parent in the mosquito stage, that dropped to ~50% in the blood stage as selfed ART-R parasites were selected against; and (ii) repeatable allele specific skews in blood stage parasites with particularly strong selection (selection coefficient (s) ≤ 0.18/asexual cycle) against alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 containing ARPS10. This approach robustly identifies selected loci and has strong potential for identifying parasite genes that interact with the mosquito vector or compensatory loci involved in drug resistance.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 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.
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