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

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.

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

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.

Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity.

Background: Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine. Methods: Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset. Results: Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity. Conclusions: Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.

Parasite dynamics in the peripheral blood and the placenta during pregnancy-associated malaria infection.

BACKGROUND: Malaria infections during pregnancy lead to sequestration of parasite infected red blood cells in the placenta. Placental infection can result in adverse outcomes for mothers and infants. Despite many studies, it remains unclear which peripheral blood infections during pregnancy lead to development of placental malaria. Understanding the timing of peripheral infections that lead to placental malaria and the ability of intermittent preventive treatment with sulfadoxine-pyrimethamine (SP-IPT) and artemisinin-based combination therapy to clear infections will enable the rational design of new interventions to decrease the burden of malaria in pregnancy. METHODS: Microsatellite markers were used to genotype peripheral and placental malaria infections in an observational cohort in Blantyre, Malawi. Genotypes were compared to determine the timing of infections that sequester in the placenta. The effects of SP-IPT and artemether-lumefantrine as curative treatment were also evaluated by assessing the occurrence of peripheral infections or matching genotypes between peripheral and placental parasites following treatment. RESULTS: Genotypes from 92 peripheral samples prior to delivery, 26 peripheral samples at delivery, and 29 placental samples were compared. Thirty percent of women with genotyped parasites in their placentas that had peripheral infections detected during pregnancy had matching peripheral-placental genotypes. Matching genotypes were not associated with gestational age and occurred from 13 to 39 weeks. Among women with more than one genotyped peripheral infection during pregnancy, 80 % had persistent infection with the same genotype while the remaining were new infections. Among infections treated with SP or artemether-lumefantrine, 28/84 (33 %) and 9/56 (16 %) had infection detected after treatment, respectively. Recrudescent infections were detected after both treatments and occurred up to 76 days after treatment. Women treated with SP-IPT and artemether-lumefantrine had genotypes matching treated infections detected in the placenta. CONCLUSIONS: Placental malaria can occur at any time during pregnancy. In the context of late enrollment in antenatal care, interventions that protect all women of childbearing age and throughout pregnancy are needed. Currently used medications do not always clear peripheral or placental infections. The ability of anti-malarial drugs to prevent or clear placental infections should be considered in the development of future interventions.

Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia.

The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.

A Single Mutation in K13 Predominates in Southern China and Is Associated With Delayed Clearance of Plasmodium falciparum Following Artemisinin Treatment.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and poses a threat to malaria control and elimination. Mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 have been associated with delayed parasite clearance following artemisinin treatment elsewhere in the region, but not yet in China. METHODS: Therapeutic efficacy studies of artesunate and dihydroartemisinin-piperaquine were conducted from 2009 to 2012 in the Yunnan Province of China near the border with Myanmar. K13 mutations were genotyped by capillary sequencing of DNA extracted from dried blood spots collected in these clinical trials and in routine surveillance. Associations between K13 mutations and delayed parasite clearance were tested using regression models. RESULTS: Parasite clearance half-lives were prolonged after artemisinin treatment, with 44% of infections having half-lives >5 hours (n = 109). Fourteen mutations in K13 were observed, with an overall prevalence of 47.7% (n = 329). A single mutation, F446I, predominated, with a prevalence of 36.5%. Infections with F446I were significantly associated with parasitemia on day 3 following artemisinin treatment and with longer clearance half-lives. CONCLUSIONS: Plasmodium falciparum infections in southern China displayed markedly delayed clearance following artemisinin treatment. F446I was the predominant K13 mutation and was associated with delayed parasite clearance.

Hemoglobin C Trait Provides Protection From Clinical Falciparum Malaria in Malian Children.

BACKGROUND: Hemoglobin C trait, like hemoglobin S trait, protects against severe malaria in children, but it is unclear whether hemoglobin C trait also protects against uncomplicated malaria. We hypothesized that Malian children with hemoglobin C trait would have a lower risk of clinical malaria than children with hemoglobin AA. METHODS: Three hundred children aged 0-6 years were enrolled in a cohort study of malaria incidence in Bandiagara, Mali, with continuous passive and monthly active follow-up from June 2009 to June 2010. RESULTS: Compared to hemoglobin AA children (n = 242), hemoglobin AC children (n = 39) had a longer time to first clinical malaria episode (hazard ratio [HR], 0.19; P = .001; 364 median malaria-free days vs 181 days), fewer episodes of clinical malaria, and a lower cumulative parasite burden. Similarly, hemoglobin AS children (n = 14) had a longer time to first clinical malaria episode than hemoglobin AA children (HR, 0.15; P = .015; 364 median malaria-free days vs 181 days), but experienced the most asymptomatic malaria infections of any group. CONCLUSIONS: Both hemoglobin C and S traits exerted a protective effect against clinical malaria episodes, but appeared to do so by mechanisms that differentially affect the response to infecting malaria parasites.

Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia.

BACKGROUND: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. METHODS: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. RESULTS: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas. CONCLUSIONS: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

A microarray platform and novel SNP calling algorithm to evaluate Plasmodium falciparum field samples of low DNA quantity.

BACKGROUND: Analysis of single nucleotide polymorphisms (SNPs) derived from whole-genome studies allows for rapid evaluation of genome-wide diversity, and genomic epidemiology studies of Plasmodium falciparum provide insights into parasite population structure, gene flow, drug resistance and vaccine development. In areas with adequate cold chain facilities, large volumes of leukocyte-depleted patient blood can be frozen for use in parasite genomic analyses. In more remote endemic areas smaller volumes of infected blood are taken by finger prick, and dried and stored on filter paper. These dried blood spots do not generally yield enough concentrated parasite DNA for whole-genome sequencing. RESULTS: A DNA microarray was designed for use on field samples to type a genome-wide set of SNPs which prior sequencing had shown to be variable in Africa, Southeast Asia, and Papua New Guinea. An algorithm was designed to call SNPs in samples with low parasite DNA. With this new algorithm SNP-calling accuracy of 98% was measured by hybridizing purified DNA from malaria lab strains and comparing calls with SNPs called from full genome sequences. An average accuracy of >98% was likewise obtained for DNA extracted from malaria field samples collected in studies in Southeast Asia, with an average call rate of > 82%. CONCLUSION: This new high-density microarray provided high quality SNP calls from a wide range of parasite DNA quantities, and represents a robust tool for genome-wide analysis of malaria parasites in diverse settings.

A novel clade of unique eukaryotic ribonucleotide reductase R2 subunits is exclusive to apicomplexan parasites.

Apicomplexa are protist parasites of tremendous medical and economic importance, causing millions of deaths and billions of dollars in losses each year. Apicomplexan-related diseases may be controlled via inhibition of essential enzymes. Ribonucleotide reductase (RNR) provides the only de novo means of synthesizing deoxyribonucleotides, essential precursors for DNA replication and repair. RNR has long been the target of antibacterial and antiviral therapeutics. However, targeting this ubiquitous protein in eukaryotic pathogens may be problematic unless these proteins differ significantly from that of their respective host. The typical eukaryotic RNR enzymes belong to class Ia, and the holoenzyme consists minimally of two R1 and two R2 subunits (α2ß2). We generated a comparative, annotated, structure-based, multiple-sequence alignment of R2 subunits, identified a clade of R2 subunits unique to Apicomplexa, and determined its phylogenetic position. Our analyses revealed that the apicomplexan-specific sequences share characteristics with both class I R2 and R2lox proteins. The putative radical-harboring residue, essential for the reduction reaction by class Ia R2-containing holoenzymes, was not conserved within this group. Phylogenetic analyses suggest that class Ia subunits are not monophyletic and consistently placed the apicomplexan-specific clade sister to the remaining class Ia eukaryote R2 subunits. Our research suggests that the novel apicomplexan R2 subunit may be a promising candidate for chemotherapeutic-induced inhibition as it differs greatly from known eukaryotic host RNRs and may be specifically targeted.

A comprehensive survey of polymorphisms conferring anti-malarial resistance in Plasmodium falciparum across Pakistan.

BACKGROUND: Few studies have been conducted in Pakistan to determine the efficacy of chloroquine and sulphadoxine-pyrimethamine (SP), which remain in use as treatment for Plasmodium vivax and in combination with artesunate to treat Plasmodium falciparum, respectively. In this study, samples from several sites across Pakistan were characterized to determine prevalence of molecular resistance markers in the P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and the origin of chloroquine-resistant P. falciparum parasites. METHODS: Microscopy-confirmed malaria parasite-positive blood samples from 801 patients across the country were collected in 2011. Of these, 171 infections were identified by polymerase chain reaction (PCR) as P. falciparum and analysed by pyrosequencing for mutations conferring chloroquine resistance (pfcrt codons 72-76), multidrug resistance (pfmdr1 N86Y, Y184F, S1034C, N1042D and D1246Y), pyrimethamine resistance (pfdhfr, C50R, N51I, C59R, S108N and I164L) and sulphadoxine resistance (pfdhps, S436A, A437G, K540E, A581G and A613T/S). pfmdr1 gene copy number variation was determined by real-time PCR, and microsatellites flanking the pfcrt locus were typed to determine the origin of the chloroquine-resistant haplotype. RESULTS: The pfcrt K76T mutation was found in all samples as part of the S72/V73/M74/N75/T76 (SVMNT) haplotype. Microsatellites flanking pfcrt showed high similarity to the signature found in India and Papua New Guinea. pfmdr1 N86Y was found in 20% of samples and all samples harboured a single copy of the pfmdr1 gene. The pfdhfr double mutation C59R + S108N was present in 87% of samples while the pfdhfr triple mutant (N51I + C59R + S108N) was not detected. Pfdhps A437G was found in 60% of samples. Pure pfdhps K540E was rare, at 4%, but mixed genotype 540 K/E was found in 77% of samples. Similarly, pure pfdhps A581G was found in 4% of the isolates while mixed 581A/G was found in 39% of samples. CONCLUSIONS: These results suggest an emerging problem with multidrug resistant P. falciparum in Pakistan. The chloroquine resistance genotype has reached complete fixation in the population, with a microsatellite pattern indicative of a selective sweep. Moreover, the prevalence of mutations in both pfdhfr and pfdhps, albeit without the presence of the pfdhfr triple mutant, indicates that continued monitoring is warranted to assess whether SP remains efficacious as a partner drug for artesunate for the treatment of P. falciparum.

Clinical manifestations of new versus recrudescent malaria infections following anti-malarial drug treatment.

BACKGROUND: Distinguishing new from recrudescent infections in post-treatment episodes of malaria is standard in anti-malarial drug efficacy trials. New infections are not considered malaria treatment failures and as a result, the prevention of subsequent episodes of malaria infection is not reported as a study outcome. However, in moderate and high transmission settings, new infections are common and the ability of a short-acting medication to cure an initial infection may be outweighed by its inability to prevent the next imminent infection. The clinical benefit of preventing new infections has never been compared to that of curing the initial infection. METHODS: Children enrolled in a sulphadoxine-pyrimethamine efficacy study in Blantyre, Malawi from 1998-2004 were prospectively evaluated. Six neutral microsatellites were used to classify new and recrudescent infections in children aged less than 10 years with recurrent malaria infections. Children from the study who did not experience recurrent parasitaemia comprised the baseline group. The odds of fever and anaemia, the rate of haemoglobin recovery and time to recurrence were compared among the groups. RESULTS: Fever and anemia were more common among children with parasitaemia compared to those who remained infection-free throughout the study period. When comparing recrudescent vs. new infections, the incidence of fever was not statistically different. However, children with recrudescent infections had a less robust haematological recovery and also experienced recurrence sooner than those whose infection was classified as new. CONCLUSIONS: The results of this study confirm the paramount importance of providing curative treatment for all malaria infections. Although new and recrudescent infections caused febrile illnesses at a similar rate, recurrence due to recrudescent infection did have a worsened haemological outcome than recurrence due to new infections. Local decision-makers should take into account the results of genotyping to distinguish new from recrudescent infections when determining treatment policy on a population level. It is appropriate to weigh recrudescent malaria more heavily than new infection in assessing treatment efficacy.

A targeted amplicon sequencing panel to simultaneously identify mosquito species and Plasmodium presence across the entire Anopheles genus.

Anopheles is a diverse genus of mosquitoes comprising over 500 described species, including all known human malaria vectors. While a limited number of key vector species have been studied in detail, the goal of malaria elimination calls for surveillance of all potential vector species. Here, we develop a multilocus amplicon sequencing approach that targets 62 highly variable loci in the Anopheles genome and two conserved loci in the Plasmodium mitochondrion, simultaneously revealing both the mosquito species and whether that mosquito carries malaria parasites. We also develop a cheap, nondestructive, and high-throughput DNA extraction workflow that provides template DNA from single mosquitoes for the multiplex PCR, which means specimens producing unexpected results can be returned to for morphological examination. Over 1000 individual mosquitoes can be sequenced in a single MiSeq run, and we demonstrate the panel's power to assign species identity using sequencing data for 40 species from Africa, Southeast Asia, and South America. We also show that the approach can be used to resolve geographic population structure within An. gambiae and An. coluzzii populations, as the population structure determined based on these 62 loci from over 1000 mosquitoes closely mirrors that revealed through whole genome sequencing. The end-to-end approach is quick, inexpensive, robust, and accurate, which makes it a promising technique for very large-scale mosquito genetic surveillance and vector control.

The information transfer system of halophilic archaea.

Information transfer is fundamental to all life forms. In the third domain of life, the archaea, many of the genes functioning in these processes are similar to their eukaryotic counterparts, including DNA replication and repair, basal transcription, and translation genes, while many transcriptional regulators and the overall genome structure are more bacterial-like. Among halophilic (salt-loving) archaea, the genomes commonly include extrachromosomal elements, many of which are large megaplasmids or minichromosomes. With the sequencing of genomes representing ten different genera of halophilic archaea and the availability of genetic systems in two diverse models, Halobacterium sp. NRC-1 and Haloferax volcanii, a large number of genes have now been annotated, classified, and studied. Here, we review the comparative genomic, genetic, and biochemical work primarily aimed at the information transfer system of halophilic archaea, highlighting gene conservation and differences in the chromosomes and the large extrachromosomal elements among these organisms.

Genetic surveillance in the Greater Mekong subregion and South Asia to support malaria control and elimination.

Background: National Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures. Methods: Samples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs. Results: GenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs. Conclusions: GenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community. Funding: The GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

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

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.

Genomic Epidemiology of Antimalarial Drug Resistance in Plasmodium falciparum in Southern China.

Emerging artemisinin resistance in Southeast Asia poses a significant risk to malaria control and eradication goals, including China's plan to eliminate malaria nationwide by 2020. Plasmodium falciparum was endemic in China, especially in Southern China. Parasites from this region have shown decreased susceptibility to artemisinin and delayed parasite clearance after artemisinin treatment. Understanding the genetic basis of artemisinin resistance and identifying specific genetic loci associated with this phenotype is crucial for surveillance and containment of resistance. In this study, parasites were collected from clinical patients from Yunnan province and Hainan island. The parasites were genotyped using a P. falciparum-specific single nucleotide polymorphism (SNP) microarray. The SNP profiles examined included a total of 27 validated and candidate molecular markers of drug resistance. The structure of the parasite population was evaluated by principal component analysis by using the EIGENSOFT program, and ADMIXTURE was used to calculate maximum likelihood estimates for the substructure analysis. Parasites showed a high prevalence of resistance haplotypes of pfdhfr and pfdhps and moderate prevalence of pfcrt. There was no mutation identified on pfmdr1. Candidate SNPs on chromosomes 10, 13, and 14 that were associated with delayed parasite clearance showed a low prevalence of mutants. Parasites from Southern China were clustered and separated from those from Southeast Asia. Parasites from Yunnan province were substructured from parasites from Hainan island. This study provides evidence for a genomic population with drug resistance in Southern China and also illustrates the utility of SNP microarrays for large-scale parasite molecular epidemiology.

Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination.

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.

Genomic structure and diversity of Plasmodium falciparum in Southeast Asia reveal recent parasite migration patterns.

Estimates of Plasmodium falciparum migration may inform strategies for malaria elimination. Here we elucidate fine-scale parasite population structure and infer recent migration across Southeast Asia using identity-by-descent (IBD) approaches based on genome-wide single nucleotide polymorphisms called in 1722 samples from 54 districts. IBD estimates are consistent with isolation-by-distance. We observe greater sharing of larger IBD segments between artemisinin-resistant parasites versus sensitive parasites, which is consistent with the recent spread of drug resistance. Our IBD analyses reveal actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination, as well as asymmetrical migration identifying potential sources and sinks of migrating parasites.

Mapping imported malaria in Bangladesh using parasite genetic and human mobility data.

For countries aiming for malaria elimination, travel of infected individuals between endemic areas undermines local interventions. Quantifying parasite importation has therefore become a priority for national control programs. We analyzed epidemiological surveillance data, travel surveys, parasite genetic data, and anonymized mobile phone data to measure the spatial spread of malaria parasites in southeast Bangladesh. We developed a genetic mixing index to estimate the likelihood of samples being local or imported from parasite genetic data and inferred the direction and intensity of parasite flow between locations using an epidemiological model integrating the travel survey and mobile phone calling data. Our approach indicates that, contrary to dogma, frequent mixing occurs in low transmission regions in the southwest, and elimination will require interventions in addition to reducing imported infections from forested regions. Unlike risk maps generated from clinical case counts alone, therefore, our approach distinguishes areas of frequent importation as well as high transmission.