Please refer to usage guidelines at http://researchonline.lshtm.ac.uk/policies.html or alternatively contact researchonline@lshtm.ac.uk. Available under license: Copyright the publishersT h e ne w e ngl a nd jou r na l o f m e dic i ne n engl j med 361;5 nejm

Evolving resistance to artemisinin-based compounds threatens to derail attempts to control malaria. Resistance has been confirmed in western Cambodia, has recently emerged in western Thailand, but is absent from neighboring Laos. Artemisinin resistance results in reduced parasite clearance rates (CR) following treatment. We used a two-phase strategy to identify genome region(s) underlying this ongoing selective event. Geographical differentiation and haplotype structure at 6,969 polymorphic SNPs in 91 parasites from Cambodia, Thailand and Laos identified 33 genome regions under strong selection. We screened SNPs and microsatellites within these regions in 715 parasites from Thailand, identifying a selective sweep on chr 13 that shows strong association (P=10-6-10-12) with slow CR, illustrating the efficacy of targeted association for identifying the genetic basis of adaptive traits.

BackgroundArtemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates.ResultsIn the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype.ConclusionsThe decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.

In western Cambodia, malaria parasites clear slowly from the blood after treatment with artemisinin derivatives, but it is unclear whether this results from parasite, host, or other factors specific to this population. We measured heritability of clearance rate by evaluating patients infected with identical or nonidentical parasite genotypes, using methods analogous to human twin studies. A substantial proportion (56%-58%) of the variation in clearance rate is explained by parasite genetics. This has 2 important implications: (1) selection with artemisinin derivatives will tend to drive resistance spread and (2) because heritability is high, the genes underlying parasite clearance rate may be identified by genome-wide association.Trial Registration. ClinicalTrials.gov identifier: NCT00493363; Current Controlled Trials identifier: ISRCTN15351875.Artemisinin combination therapies (ACTs) are the mainstay of global efforts to control Plasmodium falciparum malaria [1]. Typically, a 3-day treatment course with an ACT reduces parasite densities by a factor of , and 95% of patients' malaria and there is concern that these herald the emergence of parasites resistant to artemisinin derivatives.Although slow clearance rate in western Cambodia is now undeniable, it is far from apparent whether slow clearance rate results from parasite, host, or other factors specific to this population. Parasites with slow clearance rate after ACT do not show increased resistance to artemisinin compounds with conventional in vitro testing compared with parasites from western Thailand, which show rapid clearance rate [5]. This contrasts with resistance to all other antimalarial drugs for which in vivo resistance is associated with reduced in vitro susceptibility. There are several viable alternative explanations for slow clearance rates. Host immunity and splenic function are important contributors to parasite clearance after artemisinin treatment [6]. Reduction in herd immunity, perhaps resulting from reduced transmission, could decrease parasite clearance in Cambodia. Alternatively, host factors, such as hemoglobinopathies [7] or nutritional status, could also play a role.In humans, studies of identical twins provide an effective approach in determining whether phenotypic traits have a genetic basis [8,9]. If a trait is heritable, we would expect identical twins to be more similar than fraternal twins or unrelated individuals. Such studies are most powerful when identical twins are reared in different households because this reduces common environmental influences. Studies of twins demonstrate that traits as varied as height, addictive behavior, and musical ability have a significant genetic basis. The same analytical framework can be used to analyze heritability in clonal organisms [10]. In southeast Asia, malaria parasites that are identical by descent across the genome (ie, clonally identical) are frequently found in different patients because of self-fertilization [11]. Clonally identical malaria parasites are equivalent to identical t...

Artemisinin combination therapies (ACTs) have recently been adopted as first-line therapy for Plasmodium falciparum infections in most malaria-endemic countries. In this study, we estimated the association between artesunate-mefloquine therapy failure and genetic changes in the putative transporter, pfmdr1. Blood samples were acquired from 80 patients enrolled in an 2004 in vivo efficacy study in Pailin, Cambodia, and genotyped for pfmdr1 copy number and haplotype. Having parasites with three or more copies of pfmdr1 before treatment was strongly associated with recrudescence (hazard ratio [HR] = 8.30; 95% CI: 2.60-26.43). This relationship was maintained when controlling for initial parasite density and hematocrit (HR = 7.91; 95% CI: 2.38-26.29). Artesunate-mefloquine treatment selected for increased pfmdr1 copy number, because isolates from recurrent episodes had higher copy numbers than the paired enrollment samples (Wilcoxon rank test, P = 0.040). pfmdr1 copy number should be evaluated further as a surveillance tool for artesunate-mefloquine resistance in Cambodia.

Summaryobjective To determine the efficacy of artemether-lumefantrine malaria treatment, as an alternative to artesunate + mefloquine, which is becoming ineffective in some areas of the Thai-Cambodian border. With the per-protocol analysis, the cure rate was 71.1% in study AL2002, 86.5% in study AL2003 and 92.4% in study AM2003. All the data were PCR corrected. The artemether-lumefantrine cure rate was unexpectedly low in 2002, but it increased with food supplementation in 2003. There was a significant difference (P ¼ 0.02) in lumefantrine plasma concentrations between adequate clinical and parasitological responses and treatment failure cases. In vitro susceptibility to lumefantrine was reduced for isolates sampled from patients presenting with treatment failure, but the difference was not statistically different from isolates sampled from patients who were successfully treated.conclusion Treatment failure cases of artemether-lumefantrine are most probably because of low levels of lumefantrine blood concentration. Further investigations are necessary to determine whether resistance of Plasmodium falciparum isolates to lumefantrine is present in the region.

BackgroundNew antimalarials are needed for P. vivax and P. falciparum malaria. This study compared the efficacy and safety of pyronaridine-artesunate with that of chloroquine for the treatment of uncomplicated P. vivax malaria.Methods and FindingsThis phase III randomized, double-blind, non-inferiority trial included five centers across Cambodia, Thailand, India, and Indonesia. In a double-dummy design, patients (aged >3–≤60 years) with microscopically confirmed P. vivax mono-infection were randomized (1∶1) to receive pyronaridine-artesunate (target dose 7.2∶2.4 mg/kg to 13.8∶4.6 mg/kg) or chloroquine (standard dose) once daily for three days. Each treatment group included 228 randomized patients. Outcomes for the primary endpoint, Day-14 cure rate in the per-protocol population, were 99.5%, (217/218; 95%CI 97.5, 100) with pyronaridine-artesunate and 100% (209/209; 95%CI 98.3, 100) with chloroquine. Pyronaridine was non-inferior to chloroquine: treatment difference −0.5% (95%CI −2.6, 1.4), i.e., the lower limit of the 2-sided 95%CI for the treatment difference was greater than −10%. Pyronaridine-artesunate cure rates were non-inferior to chloroquine for Days 21, 28, 35 and 42. Parasite clearance time was shorter with pyronaridine-artesunate (median 23.0 h) versus chloroquine (32.0 h; p<0.0001), as was fever clearance time (median 15.9 h and 23.8 h, respectively; p = 0.0017). Kaplan-Meier estimates of post-baseline P. falciparum infection incidence until Day 42 were 2.5% with pyronaridine-artesunate, 6.1% with chloroquine (p = 0.048, log-rank test). Post-baseline P. vivax or P. falciparum infection incidence until Day 42 was 6.8% and 12.4%, respectively (p = 0.022, log rank test). There were no deaths. Adverse events occurred in 92/228 (40.4%) patients with pyronaridine-artesunate and 72/228 (31.6%) with chloroquine. Mild and transient increases in hepatic enzymes were observed for pyronaridine-artesunate.ConclusionPyronaridine-artesunate efficacy in acute uncomplicated P. vivax malaria was at least that of chloroquine. As pyronaridine-artesunate is also efficacious against P. falciparum malaria, this combination has potential utility as a global antimalarial drug.Trial registrationClinicaltrials.gov NCT00440999

The reduced in vivo sensitivity of Plasmodium falciparum has recently been confirmed in western Cambodia. Identifying molecular markers for artemisinin resistance is essential for monitoring the spread of the resistant phenotype and identifying the mechanisms of resistance. Four candidate genes, including the P. falciparum mdr1 (pfmdr1) gene, the P. falciparum ATPase6 (pfATPase6) gene, the 6-kb mitochondrial genome, and ubp-1, encoding a deubiquitinating enzyme, of artemisinin-resistant P. falciparum strains from western Cambodia were examined and compared to those of sensitive strains from northwestern Thailand, where the artemisinins are still very effective. The artemisinin-resistant phenotype did not correlate with pfmdr1 amplification or mutations (full-length sequencing), mutations in pfATPase6 (full-length sequencing) or the 6-kb mitochondrial genome (full-length sequencing), or ubp-1 mutations at positions 739 and 770. The P. falciparum CRT K76T mutation was present in all isolates from both study sites. The pfmdr1 copy numbers in western Cambodia were significantly lower in parasite samples obtained in 2007 than in those obtained in 2005, coinciding with a local change in drug policy replacing artesunate-mefloquine with dihydroartemisinin-piperaquine. Artemisinin resistance in western Cambodia is not linked to candidate genes, as was suggested by earlier studies.Antimalarial drug resistance is the single most important threat to global malaria control. Over the past 40 years, as firstline treatments (chloroquine and sulfadoxine-pyrimethamine) failed, the malaria-attributable mortality rate rose, contributing to a resurgence of malaria in tropical countries (11). In the last decade, artemisinins, deployed as artemisinin combination therapies (ACTs), have become the cornerstone of the treatment of uncomplicated falciparum malaria (20) and, in conjunction with other control measures, have contributed to a remarkable decrease in malaria morbidity and mortality in many African and Asian countries (4). The recent confirmation of the reduced artemisinin sensitivity of Plasmodium falciparum parasites in western Cambodia has therefore alarmed the malaria community (6). A large containment effort has been launched by the World Health Organization, in collaboration with the national malaria control programs of Cambodia and neighboring Thailand. The resistant phenotype has not been well characterized and is not well reflected by the results of conventional in vitro drug susceptibility assays. No molecular marker has been identified, which impedes surveillance studies to monitor the spread of the resistant phenotype. Identification of molecular markers would give insight into the mechanisms underlying artemisinin resistance and the mechanism of antimalarial action of the artemisinins.Mutations in several candidate genes have been postulated to confer artemisinin resistance. (i) P. falciparum mdr1 (pfmdr1) encodes the P-glycoprotein homologue 1 (Pgh1), which belongs to the ATP-binding cassette transporter superfamily...