Influence of hyaluronic acid on intra-articular friction - a biomechanical study in whole animal joints.

BACKGROUND: Cartilage is a mechanically highly stressed tissue in the human body and an important part of synovial joints. The joint cartilage is lubricated by synovial fluid with hyaluronic acid (HA) as main component. However, in joints with osteoarthritis HA has a lower concentration and molecular weight compared to healthy joints. In recent years, the intra-articular injection of therapeutic HA lubricant, has become a popular therapy. The effect of HA application on the friction of a complete joint with physiological movement needs to be further determined. METHODS: The aim of the present study was to evaluate the lubrication effect of the joint by three lubricants (NaCl, fetal calf serum (FCS) and HA) and their effect on the friction in nine complete ovine carpo-metacarpal joints. The joints were mounted on a material testing machine and a physiological movement with 10° rotation was simulated with ascending axial load (100 - 400 N). Specimens were tested native, with cartilage damage caused by drying out and relubricated. Dissipated energy (DE) as a measure of friction was recorded and compared. RESULTS: Investigating the effect of axial load, we found significant differences in DE between all axial load steps (p < .001), however, only for the defect cartilage. Furthermore, we could document an increase in DE from native (Mean: 15.0 mJ/cycle, SD: 8.98) to cartilage damage (M: 74.4 mJ/cycle, SD: 79.02) and a decrease after relubrication to 23.6 mJ/cycle (SD: 18.47). Finally, we compared the DE values for NaCl, FCS and HA. The highest values were detected for NaCl (MNorm = 16.4 mJ/cycle, SD: 19.14). HA achieved the lowest value (MNorm = 4.3 mJ/cycle, SD: 4.31), although the gap to FCS (MNorm = 5.1 mJ/cycle, SD: 7.07) was small. CONCLUSIONS: We were able to elucidate three effects in joints with cartilage damage. First, the friction in damaged joints increases significantly compared to native joints. Second, especially in damaged joints, the friction increases significantly more with increased axial load compared to native or relubricated joints. Third, lubricants can achieve an enormous decrease in friction. Comparing different lubricants, our results indicate the highest decrease in friction for HA.

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.

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.

Temporal evolution of sulfadoxine-pyrimethamine resistance genotypes and genetic diversity in response to a decade of increased interventions against Plasmodium falciparum in northern Ghana.

BACKGROUND: Anti-malarial drug resistance remains a key concern for the global fight against malaria. In Ghana sulfadoxine-pyrimethamine (SP) is used for intermittent preventive treatment of malaria in pregnancy and combined with amodiaquine for Seasonal Malaria Chemoprevention (SMC) during the high malaria season. Thus, surveillance of molecular markers of SP resistance is important to guide decision-making for these interventions in Ghana. METHODS: A total of 4469 samples from uncomplicated malaria patients collected from 2009 to 2018 was submitted to the Wellcome Trust Sanger Institute, UK for DNA sequencing using MiSeq. Genotypes were successfully translated into haplotypes in 2694 and 846 mono infections respectively for pfdhfr and pfdhps genes and the combined pfhdfr/pfdhps genes across all years. RESULTS: At the pfdhfr locus, a consistently high (> 60%) prevalence of parasites carrying triple mutants (IRNI) were detected from 2009 to 2018. Two double mutant haplotypes (NRNI and ICNI) were found, with haplotype NRNI having a much higher prevalence (average 13.8%) than ICNI (average 3.2%) across all years. Six pfdhps haplotypes were detected. Of these, prevalence of five fluctuated in a downward trend over time from 2009 to 2018, except a pfdhps double mutant (AGKAA), which increased consistently from 2.5% in 2009 to 78.2% in 2018. Across both genes, pfdhfr/pfdhps combined triple (NRNI + AAKAA) mutants were only detected in 2009, 2014, 2015 and 2018, prevalence of which fluctuated between 3.5 and 5.5%. The combined quadruple (IRNI + AAKAA) genotype increased in prevalence from 19.3% in 2009 to 87.5% in 2011 before fluctuating downwards to 19.6% in 2018 with an average prevalence of 37.4% within the nine years. Prevalence of parasites carrying the quintuple (IRNI + AGKAA or SGEAA) mutant haplotypes, which are highly refractory to SP increased over time from 14.0% in 2009 to 89.0% in 2016 before decreasing to 78.9 and 76.6% in 2017 and 2018 respectively. Though quintuple mutants are rising in prevalence in both malaria seasons, together these combined genotypes vary significantly within season but not between seasons. CONCLUSIONS: Despite high prevalence of pfdhfr triple mutants and combined pfdhfr/pfdhps quadruple and quintuple mutants in this setting SP may still be efficacious. These findings are significant as they highlight the need to continuously monitor SP resistance, particularly using deep targeted sequencing to ascertain changing resistance patterns.

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.

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.

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.

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.

Effect of graft positioning on dissipated energy in knee osteochondral autologous transplantation-A biomechanical study.

Focal cartilage defects can be treated by osteochondral autologous transplantation (OAT). High congruence of the graft with the surrounding cartilage structure is essential for a good clinical outcome, but can not always be achieved. We recently established a method to measure dissipated energy (DE) as a friction parameter in knee joints. We now investigated how autograft harvesting and implant positioning affect the DE during knee motion. Six sheep knee joints were cyclically motioned under 400 N axial load. During the cyclic motion, the flexion angle and the respective torque were recorded and the DE was calculated. Several experimental conditions were tested: first, the DE was measured after approach had been performed ("native"). Subsequently, a cylinder was removed from the medial femur condyles and a donor cylinder was inserted from an unloaded site in four different transplant positions: even, 1 mm deeper, 1 mm higher, and flush without cartilage (defect). No significant changes in friction were observed between the native knee and an even or deep OAT positioning. We, however, found a small but significant increase in DE between the "native" and "1 mm high" formations (ΔDE compared with native = 14 mJ/cycle; P = .004 after data normalization) and a large increase in defect situation (ΔDE compared with native = 119 mJ/cycle; P = .001). Considering the long-term therapeutic aim that is pursued when performing OAT, elevated graft positioning should clearly be avoided. From a biomechanical point of view, donor site morbidity after cylinder harvest can be neglected.

Influence of reduction accuracy in lateral tibial plateau fractures on intra-articular friction - a biomechanical study.

BACKGROUND: Lateral tibial split fractures (LTSF) usually require surgical therapy with screw or plate osteosynthesis. Excellent anatomical reduction of the fracture is thereby essential to avoid post-traumatic osteoarthritis. In clinical practice, a gap and step of 2 mm have been propagated as maximum tolerable limit. To date, biomechanical studies regarding tibial fractures have been limited to pressure measurement, but the relationship between dissipated energy (DE) as a friction parameter and reduction accuracy in LTSF has not been investigated. In past experiments, we developed a new method to measure DE in ovine knee joints. To determine weather non-anatomical fracture reduction with lateral gap or vertical step condition leads to relevant changes in DE in the human knee joint, we tested the applicability of the new method on human LTSFs and investigated whether the current limit of 2 mm gap and step is durable from a biomechanical point of view. METHODS: Seven right human, native knee joint specimens were cyclically moved under 400 N axial load using a robotic system. During the cyclic motion, the flexion angle and the respective torque were recorded and the DE was calculated. First, DE was measured after an anterolateral approach had been performed (condition "native"). Then a LTSF was set with a chisel, reduced anatomically, fixed with two set screws and DE was measured ("even"). DE of further reductions was then measured with gaps of 1 mm and 2 mm, and a 2 mm step down or a 2 mm step up was measured. RESULTS: We successfully established a measurement protocol for DE in human knee joints with LTSF. While gaps led to small though statistically significant increase (1 mm gap:ΔDE compared with native = 0.030 J/cycle, (+ 21%), p = 0.02; 2 mm gap:ΔDE = 0.032 J/cycle, (+ 22%), p = 0.009), this increase almost doubled when reducing in a step-down condition (ΔDE = 0.058 J/cycle, (+ 56%), p = 0.042) and even tripled in the step-up condition (ΔDE = 0.097 J/cycle, (+ 94%), p = 0.004). CONCLUSIONS: Based on our biomechanical findings, we suggest avoiding step conditions in the daily work in the operating theatre. Gap conditions can be handled a bit more generously.

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.

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

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.

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

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.

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.

Does listening to music preoperatively reduce anxiety? An evidence-based practice process for novice researchers.

This article discusses the development of a clinical research question, the execution of a systematic literature search strategy, and the critical appraisal of a selected article. It demonstrates an evidence-based review process used by nurses to critique and evaluate the evidence used to support their work. This review was conducted by a novice researcher under the supervision of his lecturer. Learning to conduct an evidence-based practice review enables health professionals to understand how to systematically review primary research relating to clinical practice. This learning experience identified the many facets of a research study that need to be considered to ascertain the validity of the results, and their relevance and application to clinical practice.

Children with cerebral malaria or severe malarial anaemia lack immunity to distinct variant surface antigen subsets.

Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or "lacunae", in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children.

Molecular markers for artemisinin and partner drug resistance in natural Plasmodium falciparum populations following increased insecticide treated net coverage along the slope of mount Cameroon: cross-sectional study.

BACKGROUND: Drug resistance is one of the greatest challenges of malaria control programmes, with the monitoring of parasite resistance to artemisinins or to Artemisinin Combination Therapy (ACT) partner drugs critical to elimination efforts. Markers of resistance to a wide panel of antimalarials were assessed in natural parasite populations from southwestern Cameroon. METHODS: Individuals with asymptomatic parasitaemia or uncomplicated malaria were enrolled through cross-sectional surveys from May 2013 to March 2014 along the slope of mount Cameroon. Plasmodium falciparum malaria parasitaemic blood, screened by light microscopy, was depleted of leucocytes using CF11 cellulose columns and the parasite genotype ascertained by sequencing on the Illumina HiSeq platform. RESULTS: A total of 259 participants were enrolled in this study from three different altitudes. While some alleles associated with drug resistance in pfdhfr, pfmdr1 and pfcrt were highly prevalent, less than 3% of all samples carried mutations in the pfkelch13 gene, none of which were amongst those associated with slow artemisinin parasite clearance rates in Southeast Asia. The most prevalent haplotypes were triple mutants Pfdhfr I 51 R 59 N 108 I 164(99%), pfcrt- C72V73 I 74 E 75 T 76 (47.3%), and single mutants PfdhpsS436 G 437K540A581A613(69%) and Pfmdr1 N86 F 184D1246 (53.2%). CONCLUSIONS: The predominance of the Pf pfcrt CVIET and Pf dhfr IRN triple mutant parasites and absence of pfkelch13 resistance alleles suggest that the amodiaquine and pyrimethamine components of AS-AQ and SP may no longer be effective in their role while chloroquine resistance still persists in southwestern Cameroon.

Community Health Warriors: Marshallese Community Health Workers' Perceptions and Experiences with CBPR and Community Engagement.

Our manuscript highlights the viewpoints and reflections of the native Marshallese community health workers (CHWs) engaged in research with the local Marshallese community in Northwest Arkansas. In particular, this paper documents the vital role Marshallese CHWs play in the success of programs and research efforts. The negative health effects of nuclear testing in the Marshall Islands has been passed down through many generations, along with unfavorable attitudes toward the U.S. government and researchers. However, the community-based participatory research (CBPR) approach used by the University of Arkansas for Medical Sciences (UAMS) has allowed the native Marshallese CHWs to become advocates for the Marshallese community. The use of native CHWs has also leveled the power dynamics that can be a barrier to community-based research, and has strengthened trust with community stakeholders. Our paper shows how using Marshallese CHWs can produce positive health outcomes for the Marshallese community.

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.