Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence ...of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.
Artemisinin-based combination therapies (ACTs) are globally the first-line treatment for uncomplicated falciparum malaria and new compounds will not be available within the next few years. ...Artemisinin-resistant Plasmodium falciparum emerged over a decade ago in the Greater Mekong Subregion (GMS) and, compounded by ACT partner drug resistance, has caused significant ACT treatment failure. This review provides an update on the epidemiology, and mechanisms of artemisinin resistance and approaches to counter multidrug-resistant falciparum malaria.
An aggressive malaria elimination programme in the GMS has helped prevent the spread of drug resistance to neighbouring countries. However, parasites carrying artemisinin resistance-associated mutations in the P. falciparum Kelch13 gene (pfk13) have now emerged independently in multiple locations elsewhere in Asia, Africa and South America. Notably, artemisinin-resistant infections with parasites carrying the pfk13 R561H mutation have emerged and spread in Rwanda.
Enhancing the geographic coverage of surveillance for resistance will be key to ensure prompt detection of emerging resistance in order to implement effective countermeasures without delay. Treatment strategies designed to prevent the emergence and spread of multidrug resistance must be considered, including deployment of triple drug combination therapies and multiple first-line therapies.
Summary Background Evidence suggests that the PfKelch13 mutations that confer artemisinin resistance in falciparum malaria have multiple independent origins across the Greater Mekong subregion, which ...has motivated a regional malaria elimination agenda. We aimed to use molecular genotyping to assess antimalarial drug resistance selection and spread in the Greater Mekong subregion. Methods In this observational study, we tested Plasmodium falciparum isolates from Myanmar, northeastern Thailand, southern Laos, and western Cambodia for PfKelch13 mutations and for Pfplasmepsin2 gene amplification (indicating piperaquine resistance). We collected blood spots from patients with microscopy or rapid test confirmed uncomplicated falciparum malaria. We used microsatellite genotyping to assess genetic relatedness. Findings As part of studies on the epidemiology of artemisinin-resistant malaria between Jan 1, 2008, and Dec 31, 2015, we collected 434 isolates. In 2014–15, a single long PfKelch13 C580Y haplotype (−50 to +31·5 kb) lineage, which emerged in western Cambodia in 2008, was detected in 65 of 88 isolates from northeastern Thailand, 86 of 111 isolates from southern Laos, and 14 of 14 isolates from western Cambodia, signifying a hard transnational selective sweep. Pfplasmepsin2 amplification occurred only within this lineage, and by 2015 these closely related parasites were found in ten of the 14 isolates from Cambodia and 15 of 15 isolates from northeastern Thailand. C580Y mutated parasites from Myanmar had a different genetic origin. Interpretation Our results suggest that the dominant artemisinin-resistant P falciparum C580Y lineage probably arose in western Cambodia and then spread to Thailand and Laos, outcompeting other parasites and acquiring piperaquine resistance. The emergence and spread of fit artemisinin-resistant P falciparum parasite lineages, which then acquire partner drug resistance across the Greater Mekong subregion, threatens regional malaria control and elimination goals. Elimination of falciparum malaria from this region should be accelerated while available antimalarial drugs still remain effective. Funding The Wellcome Trust and the Bill and Melinda Gates Foundation.
Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens ...future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.
The ongoing novel coronavirus disease (COVID-19) pandemic is threatening the global human population, including in countries with resource-limited health facilities. Severe bilateral pneumonia is the ...main feature of severe COVID-19, and adequate ventilatory support is crucial for patient survival. Although our knowledge of the disease is still rapidly increasing, this review summarizes current guidance on the best provision of ventilatory support, with a focus on resource-limited settings. Key messages include that supplemental oxygen is a first essential step for the treatment of severe COVID-19 patients with hypoxemia and should be a primary focus in resource-limited settings where capacity for invasive ventilation is limited. Oxygen delivery can be increased by using a non-rebreathing mask and prone positioning. The presence of only hypoxemia should in general not trigger intubation because hypoxemia is often remarkably well tolerated. Patients with fatigue and at risk for exhaustion, because of respiratory distress, will require invasive ventilation. In these patients, lung protective ventilation is essential. Severe pneumonia in COVID-19 differs in some important aspects from other causes of severe pneumonia or acute respiratory distress syndrome, and limiting the positive end-expiratory pressure level on the ventilator may be important. This ventilation strategy might reduce the currently very high case fatality rate of more than 50% in invasively ventilated COVID-19 patients.
Summary Background Artemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international effort is underway to contain artemisinin-resistant Plasmodium falciparum , but ...containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand–Myanmar (Burma) border. Methods In malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≥4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms. Findings 3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5–2·7) in 2001, to 3·7 h (3·6–3·8) in 2010, compared with a mean of 5·5 h (5·2–5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≥6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identified, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010. Interpretation Genetically determined artemisinin resistance in P falciparum emerged along the Thailand–Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2–6 years. Funding The Wellcome Trust and National Institutes of Health.
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.
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.
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.
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.
UK Department for International Development, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and National Institutes of Health.
Recent gains in the fight against malaria are threatened by the emergence and spread of artemisinin and partner drug resistance in Plasmodium falciparum in the Greater Mekong Subregion (GMS). When ...artemisinins are combined with a single partner drug, all recommended artemisinin-based combination therapies have shown reduced efficacy in some countries in the GMS at some point. Novel drugs are not available for the near future. Triple artemisinin-based combination therapies, combining artemisinins with two currently available partner drugs, will provide one of the last remaining safe and effective treatments for falciparum malaria that can be deployed rapidly in the GMS, whereas their deployment beyond the GMS could delay or prevent the global emergence and spread of resistance to currently available drugs.
Artemisinin and partner drug resistance have resulted in high failure rates of artemisinin-based combination therapies (ACTs) in the GMS. Spread or emergence of resistance beyond the GMS are threats to malaria control.Triple ACTs (TACTs), combining an artemisinin and two existing partner drugs, could be a stop-gap therapy for treating multidrug-resistant malaria until new antimalarials are available. Where resistance is not established, deployment of TACTs could delay or prevent emergence of resistance and could prolong the longevity of antimalarial compounds used in any triple-drug combination.TACTs must be safe, well-tolerated, effective, and affordable. Fixed-dose combinations of three drugs in the same tablet will likely improve adherence. Barriers that hinder deployment and adherence must be identified and addressed early in the development of TACTs.
Droplet digital polymerase chain reaction (ddPCR) is a partial PCR based on water-oil emulsion droplet technology. It is a highly sensitive method for detecting and delineating minor alleles from ...complex backgrounds and provides absolute quantification of DNA targets. The ddPCR technology has been applied for detection of many pathogens. Here the sensitive assay utilizing ddPCR for detection and quantification of Plasmodium species was investigated. The assay was developed for two levels of detection, genus specific for all Plasmodium species and for specific Plasmodium species detection. The ddPCR assay was developed based on primers and probes specific to the Plasmodium genus 18S rRNA gene. Using ddPCR for ultra-sensitive P. falciparum assessment, the lower level of detection from concentrated DNA obtained from a high volume (1 mL) blood sample was 11 parasites/mL. For species identification, in particular for samples with mixed infections, a duplex reaction was developed for detection and quantification P. falciparum/ P. vivax and P. malariae/ P. ovale. Amplification of each Plasmodium species in the duplex reaction showed equal sensitivity to singleplex single species detection. The duplex ddPCR assay had higher sensitivity to identify minor species in 32 subpatent parasitaemia samples from Cambodia, and performed better than real-time PCR. The ddPCR assay shows high sensitivity to assess very low parasitaemia of all human Plasmodium species. This provides a useful research tool for studying the role of the asymptomatic parasite reservoir for transmission in regions aiming for malaria elimination.
We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified ...at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.
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