Publications by authors named "Dominic P Kwiatkowski"

153 Publications

An open dataset of genome variation in 7,000 worldwide samples.

Wellcome Open Res 2021 24;6:42. Epub 2021 Feb 24.

Medical Research Council Unit The Gambia, at the London School of Hygiene and Tropical Medicine, Banjul, The Gambia.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.
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http://dx.doi.org/10.12688/wellcomeopenres.16168.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008441PMC
February 2021

Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England.

Nature 2021 Mar 25. Epub 2021 Mar 25.

Department of Mathematics, Imperial College London, London, UK.

The SARS-CoV-2 lineage B.1.1.7, designated variant of concern (VOC) 202012/01 by Public Health England, was first identified in the UK in late summer to early autumn 2020. Whole-genome SARS-CoV-2 sequence data collected from community-based diagnostic testing for COVID-19 show an extremely rapid expansion of the B.1.1.7 lineage during autumn 2020, suggesting that it has a selective advantage. Here we show that changes in VOC frequency inferred from genetic data correspond closely to changes inferred by S gene target failures (SGTF) in community-based diagnostic PCR testing. Analysis of trends in SGTF and non-SGTF case numbers in local areas across England shows that B.1.1.7 has higher transmissibility than non-VOC lineages, even if it has a different latent period or generation time. The SGTF data indicate a transient shift in the age composition of reported cases, with cases of B.1.1.7 including a larger share of under 20-year-olds than non-VOC cases. We estimated time-varying reproduction numbers for B.1.1.7 and co-circulating lineages using SGTF and genomic data. The best-supported models did not indicate a substantial difference in VOC transmissibility among different age groups, but all analyses agreed that B.1.1.7 has a substantial transmission advantage over other lineages, with a 50% to 100% higher reproduction number.
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http://dx.doi.org/10.1038/s41586-021-03470-xDOI Listing
March 2021

Malaria is a cause of iron deficiency in African children.

Nat Med 2021 04 22;27(4):653-658. Epub 2021 Feb 22.

Wellcome Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

Malaria and iron deficiency (ID) are common and interrelated public health problems in African children. Observational data suggest that interrupting malaria transmission reduces the prevalence of ID. To test the hypothesis that malaria might cause ID, we used sickle cell trait (HbAS, rs334 ), a genetic variant that confers specific protection against malaria, as an instrumental variable in Mendelian randomization analyses. HbAS was associated with a 30% reduction in ID among children living in malaria-endemic countries in Africa (n = 7,453), but not among individuals living in malaria-free areas (n = 3,818). Genetically predicted malaria risk was associated with an odds ratio of 2.65 for ID per unit increase in the log incidence rate of malaria. This suggests that an intervention that halves the risk of malaria episodes would reduce the prevalence of ID in African children by 49%.
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http://dx.doi.org/10.1038/s41591-021-01238-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610676PMC
April 2021

Multi-locus genotyping reveals established endemicity of a geographically distinct Plasmodium vivax population in Mauritania, West Africa.

PLoS Negl Trop Dis 2020 12 16;14(12):e0008945. Epub 2020 Dec 16.

London School of Hygiene & Tropical Medicine, Keppel St, London, United Kingdom.

Background: Plasmodium vivax has been recently discovered as a significant cause of malaria in Mauritania, although very rare elsewhere in West Africa. It has not been known if this is a recently introduced or locally remnant parasite population, nor whether the genetic structure reflects epidemic or endemic transmission.

Methodology/principal Findings: To investigate the P. vivax population genetic structure in Mauritania and compare with populations previously analysed elsewhere, multi-locus genotyping was undertaken on 100 clinical isolates, using a genome-wide panel of 38 single nucleotide polymorphisms (SNPs), plus seven SNPs in drug resistance genes. The Mauritanian P. vivax population is shown to be genetically diverse and divergent from populations elsewhere, indicated consistently by genetic distance matrix analysis, principal components analyses, and fixation indices. Only one isolate had a genotype clearly indicating recent importation, from a southeast Asian source. There was no linkage disequilibrium in the local parasite population, and only a small number of infections appeared to be closely genetically related, indicating that there is ongoing genetic recombination consistent with endemic transmission. The P. vivax diversity in a remote mining town was similar to that in the capital Nouakchott, with no indication of local substructure or of epidemic population structure. Drug resistance alleles were virtually absent in Mauritania, in contrast with P. vivax in other areas of the world.

Conclusions/significance: The molecular epidemiology indicates that there is long-standing endemic transmission that will be very challenging to eliminate. The virtual absence of drug resistance alleles suggests that most infections have been untreated, and that this endemic infection has been more neglected in comparison to P. vivax elsewhere.
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http://dx.doi.org/10.1371/journal.pntd.0008945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773413PMC
December 2020

High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies.

Exp Biol Med (Maywood) 2021 Apr 16;246(8):916-928. Epub 2020 Dec 16.

West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, Accra, GH 0233, Ghana.

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing , , and genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including , , Influenza virus, El Tor Hemolysin, and . A large structural rearrangement and hybrid glycophorin variant, known as , which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from , other large structural variants exist, with the most common being deletion of the whole gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous DEL1 assay and the development of a novel DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.
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http://dx.doi.org/10.1177/1535370220968545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022085PMC
April 2021

Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea.

PLoS Pathog 2020 12 15;16(12):e1009133. Epub 2020 Dec 15.

Department of Tropical Medicine and Parasitology, Juntendo University Faculty of Medicine, Tokyo, Japan.

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants' genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites' drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.
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http://dx.doi.org/10.1371/journal.ppat.1009133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771869PMC
December 2020

Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites.

Commun Biol 2020 Oct 28;3(1):624. Epub 2020 Oct 28.

Department of Infection Biology, London School of Hygiene and Tropical Medicine, Keppel St, London, WC1E 7HT, UK.

Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood and difficult to measure accurately. We show intrinsic asexual blood stage multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity in patients. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous laboratory culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time in culture, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation among the isolates at each of the assayed culture timepoints robustly correlated with parasite levels seen in patients at clinical presentation, indicating innate parasite control of multiplication rate that contributes to virulence.
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http://dx.doi.org/10.1038/s42003-020-01349-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595149PMC
October 2020

Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study.

Lancet Infect Dis 2020 12 14;20(12):1470-1480. Epub 2020 Jul 14.

Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

Background: The Greater Mekong subregion is a recurrent source of antimalarial drug resistance in Plasmodium falciparum malaria. This study aimed to characterise the extent and spread of resistance across this entire region between 2007 and 2018.

Methods: P falciparum isolates from Myanmar, Thailand, Laos, and Cambodia were obtained from clinical trials and epidemiological studies done between Jan 1, 2007, and Dec 31, 2018, and were genotyped for molecular markers (pfkelch, pfcrt, pfplasmepsin2, and pfmdr1) of antimalarial drug resistance. Genetic relatedness was assessed using microsatellite and single nucleotide polymorphism typing of flanking sequences around target genes.

Findings: 10 632 isolates were genotyped. A single long pfkelch Cys580Tyr haplotype (from -50 kb to +31·5 kb) conferring artemisinin resistance (PfPailin) now dominates across the eastern Greater Mekong subregion. Piperaquine resistance associated with pfplasmepsin2 gene amplification and mutations in pfcrt downstream of the Lys76Thr chloroquine resistance locus has also developed. On the Thailand-Myanmar border a different pfkelch Cys580Tyr lineage rose to high frequencies before it was eliminated. Elsewhere in Myanmar the Cys580Tyr allele remains widespread at low allele frequencies. Meanwhile a single artemisinin-resistant pfkelch Phe446Ile haplotype has spread across Myanmar. Despite intense use of dihydroartemisinin-piperaquine in Kayin state, eastern Myanmar, both in treatment and mass drug administrations, no selection of piperaquine resistance markers was observed. pfmdr1 amplification, a marker of resistance to mefloquine, remains at low prevalence across the entire region.

Interpretation: Artemisinin resistance in P falciparum is now prevalent across the Greater Mekong subregion. In the eastern Greater Mekong subregion a multidrug resistant P falciparum lineage (PfPailin) dominates. In Myanmar a long pfkelch Phe446Ile haplotype has spread widely but, by contrast with the eastern Greater Mekong subregion, there is no indication of artemisinin combination therapy (ACT) partner drug resistance from genotyping known markers, and no evidence of spread of ACT resistant P falciparum from the east to the west. There is still a window of opportunity to prevent global spread of ACT resistance.

Funding: Thailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.
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http://dx.doi.org/10.1016/S1473-3099(20)30228-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689289PMC
December 2020

Interferon-gamma polymorphisms and risk of iron deficiency and anaemia in Gambian children.

Wellcome Open Res 2020 2;5:40. Epub 2020 Jun 2.

Kenya Medical Research Institute (KEMRI) Centre for Geographic Medicine Coast, KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya.

: Anaemia is a major public health concern especially in African children living in malaria-endemic regions. Interferon-gamma (IFN-γ) is elevated during malaria infection and is thought to influence erythropoiesis and iron status. Genetic variants in the IFN-γ gene ) are associated with increased IFN-γ production. We investigated putative functional single nucleotide polymorphisms (SNPs) and haplotypes of in relation to nutritional iron status and anaemia in Gambian children over a malaria season. We used previously available data from Gambian family trios to determine informative SNPs and then used the Agena Bioscience MassArray platform to type five SNPs from the gene in a cohort of 780 Gambian children aged 2-6 years. We also measured haemoglobin and biomarkers of iron status and inflammation at the start and end of a malaria season. We identified five haplotype-tagging SNPs ( -1616 [rs2069705], +874 [rs2430561], +2200 [rs1861493], +3234 [rs2069718] and +5612 [rs2069728]). The +2200C [rs1861493] allele was associated with reduced haemoglobin concentrations (adjusted β -0.44 [95% CI -0.75, -0.12]; Bonferroni adjusted P = 0.03) and a trend towards iron deficiency compared to wild-type at the end of the malaria season in multivariable models adjusted for potential confounders. A haplotype uniquely identified by +2200C was similarly associated with reduced haemoglobin levels and trends towards iron deficiency, anaemia and iron deficiency anaemia at the end of the malaria season in models adjusted for age, sex, village, inflammation and malaria parasitaemia. We found limited statistical evidence linking polymorphisms with a risk of developing iron deficiency and anaemia in Gambian children. More definitive studies are needed to investigate the effects of genetically influenced IFN-γ levels on the risk of iron deficiency and anaemia in children living in malaria-endemic areas.
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http://dx.doi.org/10.12688/wellcomeopenres.15750.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202087PMC
June 2020

Development of copy number assays for detection and surveillance of piperaquine resistance associated plasmepsin 2/3 copy number variation in Plasmodium falciparum.

Malar J 2020 May 13;19(1):181. Epub 2020 May 13.

Wellcome Sanger Institute, Hinxton, UK.

Background: Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the plasmepsin 2 and plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance.

Results: To accurately and quickly determine the presence of copy number variations in the plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of plasmepsin 2 in Pursat in 2013 to three copies in 2014-2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015.

Conclusions: The multiplex TaqMan assay is a robust tool for monitoring both plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring plasmepsin 2/3 amplifications. This study shows increasing levels of plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
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http://dx.doi.org/10.1186/s12936-020-03249-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218657PMC
May 2020

Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated Plasmodium falciparum malaria: a multicentre, open-label, randomised clinical trial.

Lancet 2020 04 11;395(10233):1345-1360. Epub 2020 Mar 11.

Infectious Disease Biology Unit, IGH, Rourkela Research Unit of ILS, Bhubeneswar, DBT, Rourkela, India.

Background: Artemisinin and partner-drug resistance in Plasmodium falciparum are major threats to malaria control and elimination. Triple artemisinin-based combination therapies (TACTs), which combine existing co-formulated ACTs with a second partner drug that is slowly eliminated, might provide effective treatment and delay emergence of antimalarial drug resistance.

Methods: In this multicentre, open-label, randomised trial, we recruited patients with uncomplicated P falciparum malaria at 18 hospitals and health clinics in eight countries. Eligible patients were aged 2-65 years, with acute, uncomplicated P falciparum malaria alone or mixed with non-falciparum species, and a temperature of 37·5°C or higher, or a history of fever in the past 24 h. Patients were randomly assigned (1:1) to one of two treatments using block randomisation, depending on their location: in Thailand, Cambodia, Vietnam, and Myanmar patients were assigned to either dihydroartemisinin-piperaquine or dihydroartemisinin-piperaquine plus mefloquine; at three sites in Cambodia they were assigned to either artesunate-mefloquine or dihydroartemisinin-piperaquine plus mefloquine; and in Laos, Myanmar, Bangladesh, India, and the Democratic Republic of the Congo they were assigned to either artemether-lumefantrine or artemether-lumefantrine plus amodiaquine. All drugs were administered orally and doses varied by drug combination and site. Patients were followed-up weekly for 42 days. The primary endpoint was efficacy, defined by 42-day PCR-corrected adequate clinical and parasitological response. Primary analysis was by intention to treat. A detailed assessment of safety and tolerability of the study drugs was done in all patients randomly assigned to treatment. This study is registered at ClinicalTrials.gov, NCT02453308, and is complete.

Findings: Between Aug 7, 2015, and Feb 8, 2018, 1100 patients were given either dihydroartemisinin-piperaquine (183 [17%]), dihydroartemisinin-piperaquine plus mefloquine (269 [24%]), artesunate-mefloquine (73 [7%]), artemether-lumefantrine (289 [26%]), or artemether-lumefantrine plus amodiaquine (286 [26%]). The median age was 23 years (IQR 13 to 34) and 854 (78%) of 1100 patients were male. In Cambodia, Thailand, and Vietnam the 42-day PCR-corrected efficacy after dihydroartemisinin-piperaquine plus mefloquine was 98% (149 of 152; 95% CI 94 to 100) and after dihydroartemisinin-piperaquine was 48% (67 of 141; 95% CI 39 to 56; risk difference 51%, 95% CI 42 to 59; p<0·0001). Efficacy of dihydroartemisinin-piperaquine plus mefloquine in the three sites in Myanmar was 91% (42 of 46; 95% CI 79 to 98) versus 100% (42 of 42; 95% CI 92 to 100) after dihydroartemisinin-piperaquine (risk difference 9%, 95% CI 1 to 17; p=0·12). The 42-day PCR corrected efficacy of dihydroartemisinin-piperaquine plus mefloquine (96% [68 of 71; 95% CI 88 to 99]) was non-inferior to that of artesunate-mefloquine (95% [69 of 73; 95% CI 87 to 99]) in three sites in Cambodia (risk difference 1%; 95% CI -6 to 8; p=1·00). The overall 42-day PCR-corrected efficacy of artemether-lumefantrine plus amodiaquine (98% [281 of 286; 95% CI 97 to 99]) was similar to that of artemether-lumefantrine (97% [279 of 289; 95% CI 94 to 98]; risk difference 2%, 95% CI -1 to 4; p=0·30). Both TACTs were well tolerated, although early vomiting (within 1 h) was more frequent after dihydroartemisinin-piperaquine plus mefloquine (30 [3·8%] of 794) than after dihydroartemisinin-piperaquine (eight [1·5%] of 543; p=0·012). Vomiting after artemether-lumefantrine plus amodiaquine (22 [1·3%] of 1703) and artemether-lumefantrine (11 [0·6%] of 1721) was infrequent. Adding amodiaquine to artemether-lumefantrine extended the electrocardiogram corrected QT interval (mean increase at 52 h compared with baseline of 8·8 ms [SD 18·6] vs 0·9 ms [16·1]; p<0·01) but adding mefloquine to dihydroartemisinin-piperaquine did not (mean increase of 22·1 ms [SD 19·2] for dihydroartemisinin-piperaquine vs 20·8 ms [SD 17·8] for dihydroartemisinin-piperaquine plus mefloquine; p=0·50).

Interpretation: Dihydroartemisinin-piperaquine plus mefloquine and artemether-lumefantrine plus amodiaquine TACTs are efficacious, well tolerated, and safe treatments of uncomplicated P falciparum malaria, including in areas with artemisinin and ACT partner-drug resistance.

Funding: UK Department for International Development, Wellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, and US National Institutes of Health.
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http://dx.doi.org/10.1016/S0140-6736(20)30552-3DOI Listing
April 2020

A high throughput multi-locus insecticide resistance marker panel for tracking resistance emergence and spread in Anopheles gambiae.

Sci Rep 2019 09 16;9(1):13335. Epub 2019 Sep 16.

Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.

The spread of resistance to insecticides in disease-carrying mosquitoes poses a threat to the effectiveness of control programmes, which rely largely on insecticide-based interventions. Monitoring mosquito populations is essential, but obtaining phenotypic measurements of resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). We found resistance association in four markers, including a novel mutation in the detoxification gene Gste2 (Gste2-119V). We also identified a duplication in Gste2 combining a resistance-associated mutation with its wild-type counterpart, potentially alleviating the costs of resistance. Finally, we describe the distribution of the multiple origins of kdr resistance, finding unprecedented diversity in the DRC. This panel represents the first step towards a quantitative genotypic model of insecticide resistance that can be used to predict resistance status in An. gambiae.
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http://dx.doi.org/10.1038/s41598-019-49892-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746726PMC
September 2019

The ferroportin Q248H mutation protects from anemia, but not malaria or bacteremia.

Sci Adv 2019 09 4;5(9):eaaw0109. Epub 2019 Sep 4.

Kenya Medical Research Institute (KEMRI) Wellcome Trust Research Programme, Kilifi, Kenya.

Iron acquisition is critical for life. Ferroportin (FPN) exports iron from mature erythrocytes, and deletion of the gene results in hemolytic anemia and increased fatality in malaria-infected mice. The Q248H mutation (glutamine to histidine at position 248) renders FPN partially resistant to hepcidin-induced degradation and was associated with protection from malaria in human studies of limited size. Using data from cohorts including over 18,000 African children, we show that the Q248H mutation is associated with modest protection against anemia, hemolysis, and iron deficiency, but we found little evidence of protection against severe malaria or bacteremia. We additionally observed no excess growth in Q248H erythrocytes ex vivo, nor evidence of selection driven by malaria exposure, suggesting that the Q248H mutation does not protect from malaria and is unlikely to deprive malaria parasites of iron essential for their growth.
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http://dx.doi.org/10.1126/sciadv.aaw0109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726445PMC
September 2019

Whole-genome sequencing reveals high complexity of copy number variation at insecticide resistance loci in malaria mosquitoes.

Genome Res 2019 08 25;29(8):1250-1261. Epub 2019 Jul 25.

Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom.

Polymorphisms in genetic copy number can influence gene expression, coding sequence, and zygosity, making them powerful actors in the evolutionary process. Copy number variants (CNVs) are however understudied, being more difficult to detect than single-nucleotide polymorphisms. We take advantage of the intense selective pressures on the major malaria vector , caused by the widespread use of insecticides for malaria control, to investigate the role of CNVs in the evolution of insecticide resistance. Using the whole-genome sequencing data from 1142 samples in the 1000 genomes project, we identified 250 gene-containing CNVs, encompassing a total of 267 genes of which 28 were in gene families linked to metabolic insecticide resistance, representing significant enrichment of these families. The five major gene clusters for metabolic resistance all contained CNVs, with 44 different CNVs being found across these clusters and multiple CNVs frequently covering the same genes. These 44 CNVs are widespread (45% of individuals carry at least one of them) and have been spreading through positive selection, indicated by their high local frequencies and extended haplotype homozygosity. Our results demonstrate the importance of CNVs in the response to selection, highlighting the urgent need to identify the contribution of each CNV to insecticide resistance and to track their spread as the use of insecticides in malaria endemic countries intensifies and as the operational deployment of next-generation bed nets targeting metabolic resistance gathers pace. Our detailed descriptions of CNVs found across the species range provide the tools to do so.
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http://dx.doi.org/10.1101/gr.245795.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673711PMC
August 2019

Determinants of dihydroartemisinin-piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study.

Lancet Infect Dis 2019 09 22;19(9):952-961. Epub 2019 Jul 22.

Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom; Harvard T H Chan School of Public Health, Harvard University, Boston, MA, USA.

Background: The emergence and spread of resistance in Plasmodium falciparum malaria to artemisinin combination therapies in the Greater Mekong subregion poses a major threat to malaria control and elimination. The current study is part of a multi-country, open-label, randomised clinical trial (TRACII, 2015-18) evaluating the efficacy, safety, and tolerability of triple artemisinin combination therapies. A very high rate of treatment failure after treatment with dihydroartemisinin-piperaquine was observed in Thailand, Cambodia, and Vietnam. The immediate public health importance of our findings prompted us to report the efficacy data on dihydroartemisinin-piperaquine and its determinants ahead of the results of the overall trial, which will be published later this year.

Methods: Patients aged between 2 and 65 years presenting with uncomplicated P falciparum or mixed species malaria at seven sites in Thailand, Cambodia, and Vietnam were randomly assigned to receive dihydroartemisinin-piperaquine with or without mefloquine, as part of the TRACII trial. The primary outcome was the PCR-corrected efficacy at day 42. Next-generation sequencing was used to assess the prevalence of molecular markers associated with artemisinin resistance (kelch13 mutations, in particular Cys580Tyr) and piperaquine resistance (plasmepsin-2 and plasmepsin-3 amplifications and crt mutations). This study is registered with ClinicalTrials.gov, number NCT02453308.

Findings: Between Sept 28, 2015, and Jan 18, 2018, 539 patients with acute P falciparum malaria were screened for eligibility, 292 were enrolled, and 140 received dihydroartemisinin-piperaquine. The overall Kaplan-Meier estimate of PCR-corrected efficacy of dihydroartemisinin-piperaquine at day 42 was 50·0% (95% CI 41·1-58·3). PCR-corrected efficacies for individual sites were 12·7% (2·2-33·0) in northeastern Thailand, 38·2% (15·9-60·5) in western Cambodia, 73·4% (57·0-84·3) in Ratanakiri (northeastern Cambodia), and 47·1% (33·5-59·6) in Binh Phuoc (southwestern Vietnam). Treatment failure was associated independently with plasmepsin2/3 amplification status and four mutations in the crt gene (Thr93Ser, His97Tyr, Phe145Ile, and Ile218Phe). Compared with the results of our previous TRACI trial in 2011-13, the prevalence of molecular markers of artemisinin resistance (kelch13 Cys580Tyr mutations) and piperaquine resistance (plasmepsin2/3 amplifications and crt mutations) has increased substantially in the Greater Mekong subregion in the past decade.

Interpretation: Dihydroartemisinin-piperaquine is not treating malaria effectively across the eastern Greater Mekong subregion. A highly drug-resistant P falciparum co-lineage is evolving, acquiring new resistance mechanisms, and spreading. Accelerated elimination of P falciparum malaria in this region is needed urgently, to prevent further spread and avoid a potential global health emergency.

Funding: UK Department for International Development, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and National Institutes of Health.
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http://dx.doi.org/10.1016/S1473-3099(19)30391-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715822PMC
September 2019

Evolution and expansion of multidrug-resistant malaria in southeast Asia: a genomic epidemiology study.

Lancet Infect Dis 2019 09 22;19(9):943-951. Epub 2019 Jul 22.

Wellcome Sanger Institute, Hinxton, UK; MRC Centre for Genomics and Global Health, Big Data Institute, University of Oxford, Oxford, UK.

Background: A multidrug-resistant co-lineage of Plasmodium falciparum malaria, named KEL1/PLA1, spread across Cambodia in 2008-13, causing high rates of treatment failure with the frontline combination therapy dihydroartemisinin-piperaquine. Here, we report on the evolution and spread of KEL1/PLA1 in subsequent years.

Methods: For this genomic epidemiology study, we analysed whole genome sequencing data from P falciparum clinical samples collected from patients with malaria between 2007 and 2018 from Cambodia, Laos, northeastern Thailand, and Vietnam, through the MalariaGEN P falciparum Community Project. Previously unpublished samples were provided by two large-scale multisite projects: the Tracking Artemisinin Resistance Collaboration II (TRAC2) and the Genetic Reconnaissance in the Greater Mekong Subregion (GenRe-Mekong) project. By investigating genome-wide relatedness between parasites, we inferred patterns of shared ancestry in the KEL1/PLA1 population.

Findings: We analysed 1673 whole genome sequences that passed quality filters, and determined KEL1/PLA1 status in 1615. Before 2009, KEL1/PLA1 was only found in western Cambodia; by 2016-17 its prevalence had risen to higher than 50% in all of the surveyed countries except for Laos. In northeastern Thailand and Vietnam, KEL1/PLA1 exceeded 80% of the most recent P falciparum parasites. KEL1/PLA1 parasites maintained high genetic relatedness and low diversity, reflecting a recent common origin. Several subgroups of highly related parasites have recently emerged within this co-lineage, with diverse geographical distributions. The three largest of these subgroups (n=84, n=79, and n=47) mostly emerged since 2016 and were all present in Cambodia, Laos, and Vietnam. These expanding subgroups carried new mutations in the crt gene, which arose on a specific genetic background comprising multiple genomic regions. Four newly emerging crt mutations were rare in the early period and became more prevalent by 2016-17 (Thr93Ser, rising to 19·8%; His97Tyr to 11·2%; Phe145Ile to 5·5%; and Ile218Phe to 11·1%).

Interpretation: After emerging and circulating for several years within Cambodia, the P falciparum KEL1/PLA1 co-lineage diversified into multiple subgroups and acquired new genetic features, including novel crt mutations. These subgroups have rapidly spread into neighbouring countries, suggesting enhanced fitness. These findings highlight the urgent need for elimination of this increasingly drug-resistant parasite co-lineage, and the importance of genetic surveillance in accelerating malaria elimination efforts.

Funding: Wellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, and UK Department for International Development.
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http://dx.doi.org/10.1016/S1473-3099(19)30392-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715858PMC
September 2019

Genomic Analysis of Plasmodium vivax in Southern Ethiopia Reveals Selective Pressures in Multiple Parasite Mechanisms.

J Infect Dis 2019 10;220(11):1738-1749

Global and Tropical Health Division, Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.

The Horn of Africa harbors the largest reservoir of Plasmodium vivax in the continent. Most of sub-Saharan Africa has remained relatively vivax-free due to a high prevalence of the human Duffy-negative trait, but the emergence of strains able to invade Duffy-negative reticulocytes poses a major public health threat. We undertook the first population genomic investigation of P. vivax from the region, comparing the genomes of 24 Ethiopian isolates against data from Southeast Asia to identify important local adaptions. The prevalence of the Duffy binding protein amplification in Ethiopia was 79%, potentially reflecting adaptation to Duffy negativity. There was also evidence of selection in a region upstream of the chloroquine resistance transporter, a putative chloroquine-resistance determinant. Strong signals of selection were observed in genes involved in immune evasion and regulation of gene expression, highlighting the need for a multifaceted intervention approach to combat P. vivax in the region.
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http://dx.doi.org/10.1093/infdis/jiz016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804337PMC
October 2019

The origins of malaria artemisinin resistance defined by a genetic and transcriptomic background.

Nat Commun 2018 12 4;9(1):5158. Epub 2018 Dec 4.

School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore.

The predisposition of parasites acquiring artemisinin resistance still remains unclear beyond the mutations in Pfk13 gene and modulation of the unfolded protein response pathway. To explore the chain of casualty underlying artemisinin resistance, we reanalyze 773 P. falciparum isolates from TRACI-study integrating TWAS, GWAS, and eQTL analyses. We find the majority of P. falciparum parasites are transcriptomically converged within each geographic site with two broader physiological profiles across the Greater Mekong Subregion (GMS). We report 8720 SNP-expression linkages in the eastern GMS parasites and 4537 in the western. The minimal overlap between them suggests differential gene regulatory networks facilitating parasite adaptations to their unique host environments. Finally, we identify two genetic and physiological backgrounds associating with artemisinin resistance in the GMS, together with a farnesyltransferase protein and a thioredoxin-like protein which may act as vital intermediators linking the Pfk13 C580Y mutation to the prolonged parasite clearance time.
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http://dx.doi.org/10.1038/s41467-018-07588-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279830PMC
December 2018

A forward genetic screen reveals a primary role for Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a and 2b in determining alternative erythrocyte invasion pathways.

PLoS Pathog 2018 11 29;14(11):e1007436. Epub 2018 Nov 29.

Malaria Programme, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom.

Invasion of human erythrocytes is essential for Plasmodium falciparum parasite survival and pathogenesis, and is also a complex phenotype. While some later steps in invasion appear to be invariant and essential, the earlier steps of recognition are controlled by a series of redundant, and only partially understood, receptor-ligand interactions. Reverse genetic analysis of laboratory adapted strains has identified multiple genes that when deleted can alter invasion, but how the relative contributions of each gene translate to the phenotypes of clinical isolates is far from clear. We used a forward genetic approach to identify genes responsible for variable erythrocyte invasion by phenotyping the parents and progeny of previously generated experimental genetic crosses. Linkage analysis using whole genome sequencing data revealed a single major locus was responsible for the majority of phenotypic variation in two invasion pathways. This locus contained the PfRh2a and PfRh2b genes, members of one of the major invasion ligand gene families, but not widely thought to play such a prominent role in specifying invasion phenotypes. Variation in invasion pathways was linked to significant differences in PfRh2a and PfRh2b expression between parasite lines, and their role in specifying alternative invasion was confirmed by CRISPR-Cas9-mediated genome editing. Expansion of the analysis to a large set of clinical P. falciparum isolates revealed common deletions, suggesting that variation at this locus is a major cause of invasion phenotypic variation in the endemic setting. This work has implications for blood-stage vaccine development and will help inform the design and location of future large-scale studies of invasion in clinical isolates.
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http://dx.doi.org/10.1371/journal.ppat.1007436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289454PMC
November 2018

Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development.

Sci Rep 2018 10 25;8(1):15763. Epub 2018 Oct 25.

Pathogen Molecular Biology Department, London School of Hygiene and Tropical Medicine, Keppel St, London, UK.

Parasites infect hosts in widely varying environments, encountering diverse challenges for adaptation. To identify malaria parasite genes under locally divergent selection across a large endemic region with a wide spectrum of transmission intensity, genome sequences were obtained from 284 clinical Plasmodium falciparum infections from four newly sampled locations in Senegal, The Gambia, Mali and Guinea. Combining these with previous data from seven other sites in West Africa enabled a multi-population analysis to identify discrete loci under varying local selection. A genome-wide scan showed the most exceptional geographical divergence to be at the early gametocyte gene locus gdv1 which is essential for parasite sexual development and transmission. We identified a major structural dimorphism with alternative 1.5 kb and 1.0 kb sequence deletions at different positions of the 3'-intergenic region, in tight linkage disequilibrium with the most highly differentiated single nucleotide polymorphism, one of the alleles being very frequent in Senegal and The Gambia but rare in the other locations. Long non-coding RNA transcripts were previously shown to include the entire antisense of the gdv1 coding sequence and the portion of the intergenic region with allelic deletions, suggesting adaptive regulation of parasite sexual development and transmission in response to local conditions.
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http://dx.doi.org/10.1038/s41598-018-34078-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202401PMC
October 2018

Correction to: Analysis of anti-malarial resistance markers in pfmdr1 and pfcrt across Southeast Asia in the Tracking Resistance to Artemisinin Collaboration.

Malar J 2018 09 10;17(1):325. Epub 2018 Sep 10.

Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

Following publication of the original article [1], one of the authors has highlighted an xml-related discrepancy concerning the author group titled 'Additional Tracking Resistance to Artemisinin Collaboration authors (TRAC Group Authorship)', listed under the Acknowledgements section.
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http://dx.doi.org/10.1186/s12936-018-2464-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130064PMC
September 2018

Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study.

Lancet Haematol 2018 Aug 20;5(8):e333-e345. Epub 2018 Jul 20.

KEMRI/Wellcome Trust Research Programme, Kilifi, Kenya; Department of Medicine, Imperial College, St Mary's Hospital, London, UK. Electronic address:

Background: Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms-many related to the structure or function of red blood cells-and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress.

Methods: We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria.

Findings: Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11-0·20; p=2·61 × 10), blood group O (0·74, 0·66-0·82; p=6·26 × 10), and -α-thalassaemia (0·83, 0·76-0·90; p=2·06 × 10). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63-0·92; p=0·001) and FREM3 (0·64, 0·53-0·79; p=3·18 × 10). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49-0·68; p=3·22 × 10), as was homozygosity (0·26, 0·11-0·62; p=0·002).

Interpretation: Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health.

Funding: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.
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http://dx.doi.org/10.1016/S2352-3026(18)30107-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069675PMC
August 2018

Genomic analysis of a pre-elimination Malaysian Plasmodium vivax population reveals selective pressures and changing transmission dynamics.

Nat Commun 2018 07 3;9(1):2585. Epub 2018 Jul 3.

Global and Tropical Health Division, Menzies School of Health Research and Charles Darwin University, Darwin, NT, 0811, Australia.

The incidence of Plasmodium vivax infection has declined markedly in Malaysia over the past decade despite evidence of high-grade chloroquine resistance. Here we investigate the genetic changes in a P. vivax population approaching elimination in 51 isolates from Sabah, Malaysia and compare these with data from 104 isolates from Thailand and 104 isolates from Indonesia. Sabah displays extensive population structure, mirroring that previously seen with the emergence of artemisinin-resistant P. falciparum founder populations in Cambodia. Fifty-four percent of the Sabah isolates have identical genomes, consistent with a rapid clonal expansion. Across Sabah, there is a high prevalence of loci known to be associated with antimalarial drug resistance. Measures of differentiation between the three countries reveal several gene regions under putative selection in Sabah. Our findings highlight important factors pertinent to parasite resurgence and molecular cues that can be used to monitor low-endemic populations at the end stages of P. vivax elimination.
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http://dx.doi.org/10.1038/s41467-018-04965-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030216PMC
July 2018

Two complement receptor one alleles have opposing associations with cerebral malaria and interact with αthalassaemia.

Elife 2018 04 25;7. Epub 2018 Apr 25.

Centre for Immunity, Infection and Evolution, Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.

Malaria has been a major driving force in the evolution of the human genome. In sub-Saharan African populations, two neighbouring polymorphisms in the Complement Receptor One () gene, named and , occur at high frequencies, consistent with selection by malaria. Previous studies have been inconclusive. Using a large case-control study of severe malaria in Kenyan children and statistical models adjusted for confounders, we estimate the relationship between and and malaria phenotypes, and find they have opposing associations. The polymorphism is associated with markedly reduced odds of cerebral malaria and death, while the polymorphism is associated with increased odds of cerebral malaria. We also identify an apparent interaction between and αthalassaemia, with the protective association of greatest in children with normal α-globin. The complex relationship between these three mutations may explain previous conflicting findings, highlighting the importance of considering genetic interactions in disease-association studies.
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http://dx.doi.org/10.7554/eLife.31579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953541PMC
April 2018

Origins of the current outbreak of multidrug-resistant malaria in southeast Asia: a retrospective genetic study.

Lancet Infect Dis 2018 03 2;18(3):337-345. Epub 2018 Feb 2.

Wellcome Sanger Institute, Hinxton, UK; MRC Centre for Genomics and Global Health, Big Data Institute, Oxford University, Oxford, UK. Electronic address:

Background: Antimalarial resistance is rapidly spreading across parts of southeast Asia where dihydroartemisinin-piperaquine is used as first-line treatment for Plasmodium falciparum malaria. The first published reports about resistance to antimalarial drugs came from western Cambodia in 2013. Here, we analyse genetic changes in the P falciparum population of western Cambodia in the 6 years before those reports.

Methods: We analysed genome sequence data on 1492 P falciparum samples from 11 locations across southeast Asia, including 464 samples collected in western Cambodia between 2007 and 2013. Different epidemiological origins of resistance were identified by haplotypic analysis of the kelch13 artemisinin resistance locus and the plasmepsin 2-3 piperaquine resistance locus.

Findings: We identified more than 30 independent origins of artemisinin resistance, of which the KEL1 lineage accounted for 140 (91%) of 154 parasites resistant to dihydroartemisinin-piperaquine. In 2008, KEL1 combined with PLA1, the major lineage associated with piperaquine resistance. By 2013, the KEL1/PLA1 co-lineage had reached a frequency of 63% (24/38) in western Cambodia and had spread to northern Cambodia.

Interpretation: The KEL1/PLA1 co-lineage emerged in the same year that dihydroartemisinin-piperaquine became the first-line antimalarial drug in western Cambodia and spread rapidly thereafter, displacing other artemisinin-resistant parasite lineages. These findings have important implications for management of the global health risk associated with the current outbreak of multidrug-resistant malaria in southeast Asia.

Funding: Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, UK Department for International Development, and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases.
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http://dx.doi.org/10.1016/S1473-3099(18)30068-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835763PMC
March 2018

Resistance to malaria through structural variation of red blood cell invasion receptors.

Science 2017 06 18;356(6343). Epub 2017 May 18.

International Blood Group Reference Laboratory, National Health Service (NHS) Blood and Transplant, 500 North Bristol Park, Filton, Bristol BS34 7QH, UK.

The malaria parasite invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes and We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of and gain of two hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.
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http://dx.doi.org/10.1126/science.aam6393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575826PMC
June 2017

Massive introgression drives species radiation at the range limit of Anopheles gambiae.

Sci Rep 2017 04 18;7:46451. Epub 2017 Apr 18.

Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Lisbon, Portugal.

Impacts of introgressive hybridisation may range from genomic erosion and species collapse to rapid adaptation and speciation but opportunities to study these dynamics are rare. We investigated the extent, causes and consequences of a hybrid zone between Anopheles coluzzii and Anopheles gambiae in Guinea-Bissau, where high hybridisation rates appear to be stable at least since the 1990s. Anopheles gambiae was genetically partitioned into inland and coastal subpopulations, separated by a central region dominated by A. coluzzii. Surprisingly, whole genome sequencing revealed that the coastal region harbours a hybrid form characterised by an A. gambiae-like sex chromosome and massive introgression of A. coluzzii autosomal alleles. Local selection on chromosomal inversions may play a role in this process, suggesting potential for spatiotemporal stability of the coastal hybrid form and providing resilience against introgression of medically-important loci and traits, found to be more prevalent in inland A. gambiae.
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http://dx.doi.org/10.1038/srep46451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394460PMC
April 2017

Population genetic structure and adaptation of malaria parasites on the edge of endemic distribution.

Mol Ecol 2017 Jun 15;26(11):2880-2894. Epub 2017 Mar 15.

Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, Keppel St, London, WC1E 7HT, UK.

To determine whether the major human malaria parasite Plasmodium falciparum exhibits fragmented population structure or local adaptation at the northern limit of its African distribution where the dry Sahel zone meets the Sahara, samples were collected from diverse locations within Mauritania over a range of ~1000 km. Microsatellite genotypes were obtained for 203 clinical infection samples from eight locations, and Illumina paired-end sequences were obtained to yield high coverage genomewide single nucleotide polymorphism (SNP) data for 65 clinical infection samples from four locations. Most infections contained single parasite genotypes, reflecting low rates of transmission and superinfection locally, in contrast to the situation seen in population samples from countries further south. A minority of infections shared related or identical genotypes locally, indicating some repeated transmission of parasite clones without recombination. This caused some multilocus linkage disequilibrium and local divergence, but aside from the effect of repeated genotypes there was minimal differentiation between locations. Several chromosomal regions had elevated integrated haplotype scores (|iHS|) indicating recent selection, including those containing drug resistance genes. A genomewide F scan comparison with previous sequence data from an area in West Africa with higher infection endemicity indicates that regional gene flow prevents genetic isolation, but revealed allele frequency differentiation at three drug resistance loci and an erythrocyte invasion ligand gene. Contrast of extended haplotype signatures revealed none to be unique to Mauritania. Discrete foci of infection on the edge of the Sahara are genetically highly connected to the wider continental parasite population, and local elimination would be difficult to achieve without very substantial reduction in malaria throughout the region.
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http://dx.doi.org/10.1111/mec.14066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485074PMC
June 2017

THE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites.

PLoS Comput Biol 2017 01 26;13(1):e1005348. Epub 2017 Jan 26.

Department of Medicine, University of California, San Francisco, San Francisco, California, United States.

As many malaria-endemic countries move towards elimination of Plasmodium falciparum, the most virulent human malaria parasite, effective tools for monitoring malaria epidemiology are urgent priorities. P. falciparum population genetic approaches offer promising tools for understanding transmission and spread of the disease, but a high prevalence of multi-clone or polygenomic infections can render estimation of even the most basic parameters, such as allele frequencies, challenging. A previous method, COIL, was developed to estimate complexity of infection (COI) from single nucleotide polymorphism (SNP) data, but relies on monogenomic infections to estimate allele frequencies or requires external allele frequency data which may not available. Estimates limited to monogenomic infections may not be representative, however, and when the average COI is high, they can be difficult or impossible to obtain. Therefore, we developed THE REAL McCOIL, Turning HEterozygous SNP data into Robust Estimates of ALelle frequency, via Markov chain Monte Carlo, and Complexity Of Infection using Likelihood, to incorporate polygenomic samples and simultaneously estimate allele frequency and COI. This approach was tested via simulations then applied to SNP data from cross-sectional surveys performed in three Ugandan sites with varying malaria transmission. We show that THE REAL McCOIL consistently outperforms COIL on simulated data, particularly when most infections are polygenomic. Using field data we show that, unlike with COIL, we can distinguish epidemiologically relevant differences in COI between and within these sites. Surprisingly, for example, we estimated high average COI in a peri-urban subregion with lower transmission intensity, suggesting that many of these cases were imported from surrounding regions with higher transmission intensity. THE REAL McCOIL therefore provides a robust tool for understanding the molecular epidemiology of malaria across transmission settings.
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http://dx.doi.org/10.1371/journal.pcbi.1005348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300274PMC
January 2017

Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

Nature 2017 02 25;542(7639):101-104. Epub 2017 Jan 25.

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.
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http://dx.doi.org/10.1038/nature21038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326575PMC
February 2017