Summary Background Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro ...and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an ex-vivo RSA can identify artemisinin-resistant P falciparum infections. Methods We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study done in Pursat, Cambodia, in 2010 (registered with ClinicalTrials.gov , number NCT00341003 ) and used novel in-vitro survival assays to explore the stage-dependent susceptibility of slow-clearing and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the RSA in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia ( NCT01736319 ), to measure the ex-vivo responses of parasites from patients with malaria. Continuous variables were compared with the Mann-Whitney U test. Correlations were analysed with the Spearman correlation test. Findings In-vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-clearing infections differed significantly when assays were done on 0–3 h ring-stage parasites (10·88% vs 0·23%; p=0·007). Ex-vivo survival rates significantly correlated with in-vivo parasite clearance half-lives (n=30, r =0·74, 95% CI 0·50–0·87; p<0·0001). Interpretation The in-vitro RSA of 0–3 h ring-stage parasites provides a platform for the molecular characterisation of artemisinin resistance. The ex-vivo RSA can be easily implemented where surveillance for artemisinin resistance is needed. Funding Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH.
The emergence of artemisinin resistance in Southeast Asia (SEA), where artemisinin combination therapies (ACTs) are beginning to fail, threatens global endeavors to control and eliminate Plasmodium ...falciparum malaria. Future efforts to prevent the spread of this calamity to Africa will benefit from last year's tremendous progress in understanding artemisinin resistance.
Multiple international collaborations have established that artemisinin resistance is associated with slow parasite clearance in patients, increased survival of early-ring-stage parasites in vitro, single-nucleotide polymorphisms (SNPs) in the parasite's kelch protein gene (K13), parasite 'founder' populations sharing a genetic background of four additional SNPs, parasite transcriptional profiles reflecting an 'unfolded protein response' and decelerated parasite development, and elevated parasite phosphatidylinositol-3-kinase activity. In Western Cambodia, where the K13 C580Y mutation is approaching fixation, the frontline ACT is failing to cure nearly half of patients, likely due to partner drug resistance. In Africa, where dozens of K13 mutations have been detected at low frequency, there is no evidence yet of artemisinin resistance.
In SEA, clinical and epidemiological investigations are urgently needed to stop the further spread of artemisinin resistance, monitor the efficacy of ACTs where K13 mutations are prevalent, identify currently-available drug regimens that cure ACT failures, and rapidly advance new antimalarial compounds through preclinical studies and clinical trials.
Summary Background Haemoglobinopathies can reduce the risk of malaria syndromes. We aimed to quantify the relation between different haemoglobin mutations and malaria protection to strengthen the ...foundation for translational studies of malaria pathogenesis and immunity. Methods We systematically searched the Medline and Embase databases for studies that estimated the risk of malaria in patients with and without haemoglobinopathies up to Sept 9, 2011, and identified additional studies from reference lists. We included studies that enrolled mainly children or pregnant women and had the following outcomes: Plasmodium falciparum severe malaria, uncomplicated malaria, asymptomatic parasitaemia, or pregnancy-associated malaria, and Plasmodium vivax malaria. Two reviewers identified studies independently, assessed quality of the studies, and extracted data. We produced odds ratios (ORs; 95% CIs) for case-control studies and incidence rate ratios (IRRs; 95% CIs) for prospective studies. We did the meta-analysis with a random-effects model when equivalent outcomes were reported in more than one study. Findings Of 62 identified studies, 44 reported data for haemoglobin AS, 19 for haemoglobin AC and CC, and 18 for α-thalassaemia. Meta-analysis of case-control studies showed a decreased risk of severe P falciparum malaria in individuals with haemoglobin AS (OR 0·09, 95% CI 0·06–0·12), haemoglobin CC (0·27, 0·11–0·63), haemoglobin AC (0·83, 0·67–0·96), homozygous α-thalassaemia (0·63, 0·48–0·83), and heterozygous α-thalassaemia (0·83, 0·74–0·92). In meta-analysis of prospective trials only haemoglobin AS was consistently associated with protection from uncomplicated malaria (IRR 0·69, 95% CI 0·61–0·79); no haemoglobinopathies led to consistent protection from asymptomatic parasitaemia. Few clinical studies have investigated β-thalassaemia, haemoglobin E, P vivax malaria, or pregnancy-associated malaria. Interpretation Haemoglobin AS, CC, and AC genotypes and homozygous and heterozygous α-thalassaemia provide significant protection from severe malaria syndromes, but these haemoglobinopathies differ substantially in the degree of protection provided and confer mild or no protection against uncomplicated malaria and asymptomatic parasitaemia. Through attenuation of severity of malaria, haemoglobinopathies could serve as a model for investigation of the mechanisms of malaria pathogenesis and immunity. Funding US National Institute of Allergy and Infectious Diseases.
Plasmodium falciparum malaria kills over 500,000 children every year and has been a scourge of humans for millennia. Owing to the co-evolution of humans and P. falciparum parasites, the human genome ...is imprinted with polymorphisms that not only confer innate resistance to falciparum malaria, but also cause hemoglobinopathies. These genetic traits--including hemoglobin S (HbS), hemoglobin C (HbC), and α-thalassemia--are the most common monogenic human disorders and can confer remarkable degrees of protection from severe, life-threatening falciparum malaria in African children: the risk is reduced 70% by homozygous HbC and 90% by heterozygous HbS (sickle-cell trait). Importantly, this protection is principally present for severe disease and largely absent for P. falciparum infection, suggesting that these hemoglobinopathies specifically neutralize the parasite's in vivo mechanisms of pathogenesis. These hemoglobin variants thus represent a "natural experiment" to identify the cellular and molecular mechanisms by which P. falciparum produces clinical morbidity, which remain partially obscured due to the complexity of interactions between this parasite and its human host. Multiple lines of evidence support a restriction of parasite growth by various hemoglobinopathies, and recent data suggest this phenomenon may result from host microRNA interference with parasite metabolism. Multiple hemoglobinopathies mitigate the pathogenic potential of parasites by interfering with the export of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of the host red blood cell. Few studies have investigated their effects upon the activation of the innate and adaptive immune systems, although recent murine studies suggest a role for heme oxygenase-1 in protection. Ultimately, the identification of mechanisms of protection and pathogenesis can inform future therapeutics and preventive measures. Hemoglobinopathies slice the "Gordian knot" of host and parasite interactions to confer malaria protection, and offer a translational model to identify the most critical mechanisms of P. falciparum pathogenesis.
Summary Background Artemisinin resistance in Plasmodium falciparum threatens to reduce the efficacy of artemisinin combination therapies (ACTs), thus compromising global efforts to eliminate malaria. ...Recent treatment failures with dihydroartemisinin-piperaquine, the current first-line ACT in Cambodia, suggest that piperaquine resistance may be emerging in this country. We explored the relation between artemisinin resistance and dihydroartemisinin–piperaquine failures, and sought to confirm the presence of piperaquine-resistant P falciparum infections in Cambodia. Methods In this prospective cohort study, we enrolled patients aged 2–65 years with uncomplicated P falciparum malaria in three Cambodian provinces: Pursat, Preah Vihear, and Ratanakiri. Participants were given standard 3-day courses of dihydroartemisinin–piperaquine. Peripheral blood parasite densities were measured until parasites cleared and then weekly to 63 days. The primary outcome was recrudescent P falciparum parasitaemia within 63 days. We measured piperaquine plasma concentrations at baseline, 7 days, and day of recrudescence. We assessed phenotypic and genotypic markers of drug resistance in parasite isolates. The study is registered with ClinicalTrials.gov , number NCT01736319. Findings Between Sept 4, 2012, and Dec 31, 2013, we enrolled 241 participants. In Pursat, where artemisinin resistance is entrenched, 37 (46%) of 81 patients had parasite recrudescence. In Preah Vihear, where artemisinin resistance is emerging, ten (16%) of 63 patients had recrudescence and in Ratanakiri, where artemisinin resistance is rare, one (2%) of 60 patients did. Patients with recrudescent P falciparum infections were more likely to have detectable piperaquine plasma concentrations at baseline compared with non-recrudescent patients, but did not differ significantly in age, initial parasite density, or piperaquine plasma concentrations at 7 days. Recrudescent parasites had a higher prevalence of kelch13 mutations, higher piperaquine 50% inhibitory concentration (IC50 ) values, and lower mefloquine IC50 values; none had multiple pfmdr1 copies, a genetic marker of mefloquine resistance. Interpretation Dihydroartemisinin–piperaquine failures are caused by both artemisinin and piperaquine resistance, and commonly occur in places where dihydroartemisinin–piperaquine has been used in the private sector. In Cambodia, artesunate plus mefloquine may be a viable option to treat dihydroartemisinin–piperaquine failures, and a more effective first-line ACT in areas where dihydroartemisinin–piperaquine failures are common. The use of single low-dose primaquine to eliminate circulating gametocytes is needed in areas where artemisinin and ACT resistance is prevalent. Funding National Institute of Allergy and Infectious Diseases.
Summary Background As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong subregion, emerging resistance to partner drugs in artemisinin combination ...therapies seriously threatens global efforts to treat and eliminate this disease. Molecular markers that predict failure of artemisinin combination therapy are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in southeast Asia and beyond. Methods We did a genome-wide association study of 297 P falciparum isolates from Cambodia to investigate the relationship of 11 630 exonic single-nucleotide polymorphisms (SNPs) and 43 copy number variations (CNVs) with in-vitro piperaquine 50% inhibitory concentrations (IC50 s), and tested whether these genetic variants are markers of treatment failure with dihydroartemisinin–piperaquine. We then did a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin–piperaquine treatment. Findings Piperaquine IC50 s increased significantly from 2011 to 2013 in three Cambodian provinces (2011 vs 2013 median IC50 s: 20·0 nmol/L IQR 13·7–29·0 vs 39·2 nmol/L 32·8–48·1 for Ratanakiri, 19·3 nmol/L 15·1–26·2 vs 66·2 nmol/L 49·9–83·0 for Preah Vihear, and 19·6 nmol/L 11·9–33·9 vs 81·1 nmol/L 61·3–113·1 for Pursat; all p≤10−3 ; Kruskal-Wallis test). Genome-wide analysis of SNPs identified a chromosome 13 region that associates with raised piperaquine IC50 s. A non-synonymous SNP (encoding a Glu415Gly substitution) in this region, within a gene encoding an exonuclease, associates with parasite recrudescence following dihydroartemisinin–piperaquine treatment. Genome-wide analysis of CNVs revealed that a single copy of the mdr1 gene on chromosome 5 and a novel amplification of the plasmepsin 2 and plasmepsin 3 genes on chromosome 14 also associate with raised piperaquine IC50 s. After adjusting for covariates, both exo-E415G and plasmepsin 2–3 markers significantly associate (p=3·0 × 10−8 and p=1·7 × 10−7 , respectively) with decreased treatment efficacy (survival rates 0·38 95% CI 0·25–0·51 and 0·41 0·28–0·53, respectively). Interpretation The exo-E415G SNP and plasmepsin 2–3 amplification are markers of piperaquine resistance and dihydroartemisinin–piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since plasmepsins are involved in the parasite’s haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, plasmepsin 2–3 amplification probably mediates piperaquine resistance. Funding Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and UK Department for International Development.
Antimalarial resistance is rapidly spreading across parts of southeast Asia where dihydroartemisinin–piperaquine is used as first-line treatment for Plasmodium falciparum malaria. The first published ...reports about resistance to antimalarial drugs came from western Cambodia in 2013. Here, we analyse genetic changes in the P falciparum population of western Cambodia in the 6 years before those reports.
We analysed genome sequence data on 1492 P falciparum samples from 11 locations across southeast Asia, including 464 samples collected in western Cambodia between 2007 and 2013. Different epidemiological origins of resistance were identified by haplotypic analysis of the kelch13 artemisinin resistance locus and the plasmepsin 2–3 piperaquine resistance locus.
We identified more than 30 independent origins of artemisinin resistance, of which the KEL1 lineage accounted for 140 (91%) of 154 parasites resistant to dihydroartemisinin–piperaquine. In 2008, KEL1 combined with PLA1, the major lineage associated with piperaquine resistance. By 2013, the KEL1/PLA1 co-lineage had reached a frequency of 63% (24/38) in western Cambodia and had spread to northern Cambodia.
The KEL1/PLA1 co-lineage emerged in the same year that dihydroartemisinin–piperaquine became the first-line antimalarial drug in western Cambodia and spread rapidly thereafter, displacing other artemisinin-resistant parasite lineages. These findings have important implications for management of the global health risk associated with the current outbreak of multidrug-resistant malaria in southeast Asia.
Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, UK Department for International Development, and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases.
The widely used antimalarial combination therapy dihydroartemisinin + piperaquine (DHA + PPQ) has failed in Cambodia. Here, we perform a genomic analysis that reveals a rapid increase in the ...prevalence of novel mutations in the Plasmodium falciparum chloroquine resistance transporter PfCRT following DHA + PPQ implementation. These mutations occur in parasites harboring the K13 C580Y artemisinin resistance marker. By introducing PfCRT mutations into sensitive Dd2 parasites or removing them from resistant Cambodian isolates, we show that the H97Y, F145I, M343L, or G353V mutations each confer resistance to PPQ, albeit with fitness costs for all but M343L. These mutations sensitize Dd2 parasites to chloroquine, amodiaquine, and quinine. In Dd2 parasites, multicopy plasmepsin 2, a candidate molecular marker, is not necessary for PPQ resistance. Distended digestive vacuoles were observed in pfcrt-edited Dd2 parasites but not in Cambodian isolates. Our findings provide compelling evidence that emerging mutations in PfCRT can serve as a molecular marker and mediator of PPQ resistance.
For more than five decades, Southeast Asia (SEA) has been fertile ground for the emergence of drug-resistant Plasmodium falciparum malaria. After generating parasites resistant to chloroquine, ...sulfadoxine, pyrimethamine, quinine, and mefloquine, this region has now spawned parasites resistant to artemisinins, the world's most potent antimalarial drugs. In areas where artemisinin resistance is prevalent, artemisinin combination therapies (ACTs)-the first-line treatments for malaria-are failing fast. This worrisome development threatens to make malaria practically untreatable in SEA, and threatens to compromise global endeavors to eliminate this disease. A recent series of clinical, in vitro, genomics, and transcriptomics studies in SEA have defined in vivo and in vitro phenotypes of artemisinin resistance, identified its causal genetic determinant, explored its molecular mechanism, and assessed its clinical impact. Specifically, these studies have established that artemisinin resistance manifests as slow parasite clearance in patients and increased survival of early-ring-stage parasites in vitro; is caused by single nucleotide polymorphisms in the parasite's K13 gene, is associated with an upregulated "unfolded protein response" pathway that may antagonize the pro-oxidant activity of artemisinins, and selects for partner drug resistance that rapidly leads to ACT failures. In SEA, clinical studies are urgently needed to monitor ACT efficacy where K13 mutations are prevalent, test whether new combinations of currently available drugs cure ACT failures, and advance new antimalarial compounds through preclinical pipelines and into clinical trials. Intensifying these efforts should help to forestall the spread of artemisinin and partner drug resistance from SEA to sub-Saharan Africa, where the world's malaria transmission, morbidity, and mortality rates are highest.
The emergence and spread of artemisinin resistance, driven by mutations in Plasmodium falciparum K13, has compromised antimalarial efficacy and threatens the global malaria elimination campaign. By ...applying systems-based quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we provide evidence that K13 mutations alter multiple aspects of the parasite's intra-erythrocytic developmental program. These changes impact cell-cycle periodicity, the unfolded protein response, protein degradation, vesicular trafficking, and mitochondrial metabolism. K13-mediated artemisinin resistance in the Cambodian Cam3.II line was reversed by atovaquone, a mitochondrial electron transport chain inhibitor. These results suggest that mitochondrial processes including damage sensing and anti-oxidant properties might augment the ability of mutant K13 to protect P. falciparum against artemisinin action by helping these parasites undergo temporary quiescence and accelerated growth recovery post drug elimination.
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