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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 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.