BACKGROUND Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand–Cambodia border. Slowly clearing in fections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the “propeller” region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.)

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. 1,2 Here we describe methods for large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short term culture. Analysis of 86,158 exonic SNPs that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.

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Anthelminthic treatment of chronically infected children results in increased atopic reactivity, which indicates that helminths directly suppress allergic reactions.

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.DOI: http://dx.doi.org/10.7554/eLife.08714.001

We have analyzed the in vitro chemosensitivity profiles of 115 Kenyan isolates for chloroquine (CQ), piperaquine, lumefantrine (LM), and dihydroartemisinin in association with polymorphisms in pfcrt at codon 76 and pfmdr1 at codon 86, as well as with variations of the copy number of pfmdr1. The median drug concentrations that inhibit 50% of parasite growth (IC 50 s) were 41 nM (interquartile range [IQR], 18 to 73 nM), 50 nM (IQR, 29 to 96 nM), 32 nM (IQR, 17 to 46 nM), and 2 nM (IQR, 1 to 3 nM) for CQ, LM, piperaquine, and dihydroartemisinin, respectively. The activity of CQ correlated inversely with that of LM (r 2 ‫؍‬ ؊0.26; P ‫؍‬ 0.02). Interestingly, parasites for which LM IC 50 s were higher were wild type for pfcrt-76 and pfmdr1-86. All isolates had one pfmdr1 copy. Thus, the decrease in LM activity is associated with the selection of wild-type pfcrt-76 and pfmdr1-86 parasites, a feature that accounts for the inverse relationship between CQ and LM. Therefore, the use of LM-artemether is likely to lead to the selection of more CQ-susceptible parasites.Chemotherapy is still the main approach for the control of malaria, and current strategies for malaria treatment rely on the use of combinations of drugs that include artemisinin compounds. Although this strategy is designed to reduce the chance of resistance emerging, there is considerable concern that this will inevitably occur.For instance, the combination of lumefantrine (LM) and artemether (ATM), known as Coartem, has become the firstline treatment for malaria in many African countries, including Kenya (19). ATM is converted in vivo to dihydroartemisinin (DHA). Emerging reports indicate that the use of LM (in Coartem) selects for parasites that show increased tolerance to Coartem, and these parasites select for a wild-type pfmdr1 genotype or show increased copy numbers of pfmdr1, a gene associated with chloroquine (CQ) and mefloquine (MFQ) resistance (7,13,15,20,36,38). Thus, there is concern that resistance to LM could emerge rapidly. On the other hand, recent reports from Southeast Asia indicate that resistance to artemisinin derivatives is increasing, threatening the concept of artemisinin-based combinations (8).Another combination, piperaquine (PQ) and DHA, known as Artekin, is undergoing clinical evaluation (17,39,42). This drug is efficacious, safe, and affordable and thus is likely to become an alternative to Coartem. PQ is a bisquinoline derivative consisting of two linked CQ molecules. Although reports indicate that PQ retains potency against CQ-resistant parasites (3), there is concern that PQ could become less susceptible against a backdrop of high CQ resistance (17,22).In this paper, we sought to analyze the in vitro activities of the antimalarials LM, DHA, and PQ in relation to polymorphisms in pfcrt at codon 76 (pfcrt-76) and in pfmdr1 at codon 86 (pfmdr1-86) and in relation to pfmdr1 copy number variations in Kenyan isolates. We used CQ as a reference drug. MATERIALS AND METHODSCQ was purchased from Sigma Chemical Co. (Poole, Dorset, United...

Malaria transmission is spatially heterogeneous. This reduces the efficacy of control strategies, but focusing control strategies on clusters or ‘hotspots’ of transmission may be highly effective. Among 1500 homesteads in coastal Kenya we calculated (a) the fraction of febrile children with positive malaria smears per homestead, and (b) the mean age of children with malaria per homestead. These two measures were inversely correlated, indicating that children in homesteads at higher transmission acquire immunity more rapidly. This inverse correlation increased gradually with increasing spatial scale of analysis, and hotspots of febrile malaria were identified at every scale. We found hotspots within hotspots, down to the level of an individual homestead. Febrile malaria hotspots were temporally unstable, but 4 km radius hotspots could be targeted for 1 month following 1 month periods of surveillance.DOI: http://dx.doi.org/10.7554/eLife.02130.001