Juliano J.J.,University of North Carolina at Chapel Hill |
Porter K.,University of North Carolina at Chapel Hill |
Mwapasa V.,University of Malawi |
Sem R.,National Center for Parasitology |
And 6 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010
Malaria infections commonly contain multiple genetically distinct variants. Mathematical and animal models suggest that interactions among these variants have a profound impact on the emergence of drug resistance. However, methods currently used for quantifying parasite diversity in individual infections are insensitive to low-abundance variants and are not quantitative for variant population sizes. To more completely describe the in-host complexity and ecology of malaria infections, we used massively parallel pyrosequencing to characterize malaria parasite diversity in the infections of a group of patients. By individually sequencing single strands of DNA in a complex mixture, this technique can quantify uncommon variants in mixed infections. The in-host diversity revealed by this method far exceeded that described by currently recommended genotyping methods, with as many as sixfold more variants per infection. In addition, in paired pre-and posttreatment samples, we show a complex milieu of parasites, including variants likely up-selected and down-selected by drug therapy. As with all surveys of diversity, sampling limitations prevent full discovery and differences in sampling effort can confound comparisons among samples, hosts, and populations. Here, we used ecological approaches of species accumulation curves and capture-recapture to estimate the number of variants we failed to detect in the population, and show that these methods enable comparisons of diversity before and after treatment, as well as between malaria populations. The combination of ecological statistics and massively parallel pyrosequencing provides a powerful tool for studying the evolution of drug resistance and the in-host ecology of malaria infections.
Mbengue A.,University of Notre Dame |
Bhattacharjee S.,University of Notre Dame |
Pandharkar T.,University of Notre Dame |
Liu H.,University of Notre Dame |
And 19 more authors.
Nature | Year: 2015
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. © 2015 Macmillan Publishers Limited. All rights reserved.
Witkowski B.,Institute Pasteur in Cambodia |
Amaratunga C.,National Institute of Allergy and Infectious Diseases |
Khim N.,Institute Pasteur in Cambodia |
Sreng S.,National Center for Parasitology |
And 16 more authors.
The Lancet Infectious Diseases | Year: 2013
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. © 2013 Elsevier Ltd.
Lim P.,National Institute of Allergy and Infectious Diseases |
Lim P.,National Center for Parasitology |
Dek D.,National Center for Parasitology |
Try V.,National Center for Parasitology |
And 3 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2015
Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies - a molecular marker for MQ resistance - in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falciparum is regaining in vitro susceptibility to MQ. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
News Article | November 3, 2016
A frontline malaria treatment that combines fast-acting dihydroartemisinin with long-lasting piperaquine is quickly losing power in Cambodia due to the rapid spread of drug-resistant parasites. The presence of piperaquine-resistant malaria parasites in several Cambodian provinces was confirmed earlier this year by National Institutes of Health researchers and their colleagues. Now, by comparing the complete genomes of 297 parasites isolated from Cambodian malaria patients to a reference malaria parasite genome, the team has identified two genetic markers that are strongly associated with the parasites' ability to resist piperaquine. A simple test, performed after collecting blood from a finger pinprick, can show whether a malaria patient has parasites with the genetic markers. If so, dihydroartemisinin-piperaquine therapy is likely to fail, say the study authors, and an alternative drug combination (artesunate-mefloquine) should be used. Information about the distribution of these drug resistance markers is being used by officials in Cambodia and neighboring countries to map the extent and spread of piperaquine resistance and to help guide region-wide malaria treatment approaches. National Institute of Allergy and Infectious Diseases (NIAID) scientist Rick Fairhurst, M.D., Ph.D., and Roberto Amato, Ph.D., of the Wellcome Trust Sanger Institute (WTSI), Cambridge, UK, led the research team. The first marker they identified is a change of one subunit in a gene on the parasite's chromosome 13. Parasites with this genetic change are much more likely to be resistant to piperaquine than parasites without it. The marker, while associated with drug resistance, likely does not play a functional role in enabling parasites to resist piperaquine, according to the researchers. In contrast, the second resistance marker identified by the investigators may have such a role. That marker is an increased number of copies of two genes (plasmepsin II and plasmepsin III) in those parasites that resist piperaquine. Malaria parasites use plasmepsins to help them digest human blood and, although the exact mechanism of action of piperaquine is not known, it is believed that the drug targets plasmepsins. Parasites may react to piperaquine by increasing plasmepsin production, and any parasites with extra copies of the plasmepsin II and III genes may be better able to withstand piperaquine treatment. Of note, parasites with increased piperaquine resistance appear to have increased susceptibility to the malaria drug mefloquine. This observation hints at the possibility of devising malaria treatment regimens that combine three or more drugs to exploit opposing resistance-susceptibility attributes. In addition to NIAID and WTSI scientists, the team included investigators from the National Center for Parasitology, Entomology and Malaria Control in Phnom Penh, Cambodia, and from Mahidol University in Bangkok, Thailand. R Amato et al. Genetic markers associated with dihydroartemisinin-piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype-phenotype association study. Lancet Infectious Diseases DOI: 10.1016/S1473-3099(16)30409-1 (2016). Rick M. Fairhurst, M.D., Ph.D., Laboratory of Malaria and Vector Research, NIAID, is available to discuss this research. To schedule interviews, please contact the NIAID Office of Communications and Government Relations, (301) 402-1663, or firstname.lastname@example.org. NIAID conducts and supports research--at NIH, throughout the United States, and worldwide--to study the causes of infectious and immune-mediated diseases, and to develop better means of preventing, diagnosing and treating these illnesses. News releases, fact sheets and other NIAID-related materials are available on the NIAID website. About the National Institutes of Health (NIH): NIH, the nation's medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit http://www. .
Bustamante L.Y.,Wellcome Trust Sanger Institute |
Bartholdson S.J.,Wellcome Trust Sanger Institute |
Crosnier C.,Wellcome Trust Sanger Institute |
Campos M.G.,Wellcome Trust Sanger Institute |
And 5 more authors.
Vaccine | Year: 2013
The lack of an effective licensed vaccine remains one of the most significant gaps in the portfolio of tools being developed to eliminate Plasmodium falciparum malaria. Vaccines targeting erythrocyte invasion - an essential step for both parasite development and malaria pathogenesis - have faced the particular challenge of genetic diversity. Immunity-driven balancing selection pressure on parasite invasion proteins often results in the presence of multiple, antigenically distinct, variants within a population, leading to variant-specific immune responses. Such variation makes it difficult to design a vaccine that covers the full range of diversity, and could potentially facilitate the evolution of vaccine-resistant parasite strains. In this study, we investigate the effect of genetic diversity on invasion inhibition by antibodies to a high priority P. falciparum invasion candidate antigen, P. falciparum Reticulocyte Binding Protein Homologue 5 (PfRH5). Previous work has shown that virally delivered PfRH5 can induce antibodies that protect against a wide range of genetic variants. Here, we show that a full-length recombinant PfRH5 protein expressed in mammalian cells is biochemically active, as judged by saturable binding to its receptor, basigin, and is able to induce antibodies that strongly inhibit P. falciparum growth and invasion. Whole genome sequencing of 290 clinical P. falciparum isolates from across the world identifies only five non-synonymous PfRH5 SNPs that are present at frequencies of 10% or more in at least one geographical region. Antibodies raised against the 3D7 variant of PfRH5 were able to inhibit nine different P. falciparum strains, which between them included all of the five most common PfRH5 SNPs in this dataset, with no evidence for strain-specific immunity. We conclude that protein-based PfRH5 vaccines are an urgent priority for human efficacy trials. © 2012 Elsevier Ltd.
Yeung S.,London School of Hygiene and Tropical Medicine |
Patouillard E.,London School of Hygiene and Tropical Medicine |
Allen H.,Population Services International |
Socheat D.,National Center for Parasitology
Malaria Journal | Year: 2011
Whilst some populations have recently experienced dramatic declines in malaria, the majority of those most at risk of Plasmodium falciparum malaria still lack access to effective treatment with artemisinin combination therapy (ACT) and others are already facing parasites resistant to artemisinins. In this context, there is a crucial need to improve both access to and targeting of ACT through greater availability of good quality ACT and parasitological diagnosis. This is an issue of increasing urgency notably in the private commercial sector, which, in many countries, plays an important role in the provision of malaria treatment. The Affordable Medicines Facility for malaria (AMFm) is a recent initiative that aims to increase the provision of affordable ACT in public, private and NGO sectors through a manufacturer-level subsidy. However, to date, there is little documented experience in the programmatic implementation of subsidized ACT in the private sector. Cambodia is in the unique position of having more than 10 years of experience not only in implementing subsidized ACT, but also rapid diagnostic tests (RDT) as part of a nationwide social marketing programme. The programme includes behaviour change communication and the training of private providers as well as the sale and distribution of Malarine, the recommended ACT, and Malacheck, the RDT. This paper describes and evaluates this experience by drawing on the results of household and provider surveys conducted since the start of the programme. The available evidence suggests that providers' and consumers' awareness of Malarine increased rapidly, but that of Malacheck much less so. In addition, improvements in ACT and RDT availability and uptake were relatively slow, particularly in more remote areas. The lack of standardization in the survey methods and the gaps in the data highlight the importance of establishing a clear system for monitoring and evaluation for similar initiatives. Despite these limitations, a number of important lessons can still be learnt. These include the importance of a comprehensive communications strategy and of a sustained and reliable supply of products, with attention to the geographical reach of both. Other important challenges relate to the difficulty in incentivising providers and consumers not only to choose the recommended drug, but to precede this with a confirmatory blood test and ensure that providers adhere to the test results and patients to the treatment regime. In Cambodia, this is particularly complicated due to problems inherent to the drug itself and the emergence of artemisinin resistance. © 2011 Yeung et al; licensee BioMed Central Ltd.
Leang R.,National Center for Parasitology |
Barrette A.,World Health Organization |
Bouth D.M.,World Health Organization |
Menard D.,World Health Organization |
And 3 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2013
We describe here the results of antimalarial therapeutic efficacy studies conducted in Cambodia from 2008 to 2010. A total of 15 studies in four sentinel sites were conducted using dihydroartemisinin-piperaquine (DP) for the treatment of Plasmodium falciparum infection and chloroquine (CQ) and DP for the treatment of P. vivax infection. All studies were performed according to the standard World Health Organization protocol for the assessment of antimalarial treatment efficacy. Among the studies of DP for the treatment of P. falciparum, an increase in treatment failure was observed in the western provinces. In 2010, the PCRcorrected treatment failure rates for DP on day 42 were 25% (95% confidence interval [CI]=10 to 51%) in Pailin and 10.7% (95% CI4 to 23%) in Pursat, while the therapeutic efficacy of DP remained high (100%) in Ratanakiri and Preah Vihear provinces, located in northern and eastern Cambodia. For the studies of P. vivax, the day 28 uncorrected treatment failure rate among patients treated with CQ ranged from 4.4 to 17.4%; DP remained 100% effective in all sites. Further study is required to investigate suspected P. falciparum resistance to piperaquine in western Cambodia; the results of in vitro and molecular studies were not found to support the therapeutic efficacy findings. The emergence of artemisinin resistance in this region has likely put additional pressure on piperaquine. Although DP appears to be an appropriate new first-line treatment for P. vivax in Cambodia, alternative treatments are urgently needed for P. falciparum-infected patients in western Cambodia. Copyright © 2013, American Society for Microbiology. All Rights Reserved.
Yasuoka J.,University of Tokyo |
Poudel K.C.,University of Tokyo |
Poudel-Tandukar K.,National Center for Global Health and Medicine |
Nguon C.,National Center for Parasitology |
And 3 more authors.
Malaria Journal | Year: 2014
Background: Malaria continues to be a major public health problem in remote forested areas in Cambodia. As a national strategy to strengthen community-based malaria control, the Cambodian government has been running the Village Malaria Worker (VMW) project since 2001. This study sought to examine the nature and quality of the VMWs' services.Methods. Data collection was carried out in February and March 2008 through interviews with one of the two VMWs who takes the lead in malaria control activities in each of the 315 VMW villages (n = 251). The questionnaire addressed 1) the sociodemographic characteristics of VMWs, 2) service quality, 3) actions for malaria prevention and vector control, and 4) knowledge of malaria epidemiology and vector ecology.Results: VMWs were effective in conducting diagnosis with Rapid Diagnostic Tests (RDTs) and prescribing anti-malarials to those who had positive RDT results, skills that they had acquired through their training programmes. However, most other services, such as active detection, explanations about compliance, and follow-up of patients, were carried out by only a small proportion of VMWs. The variety of actions that VMWs took for malaria prevention and vector control was small (average action index score 12.8/23), and their knowledge was very limited with less than 20% of the VMWs giving correct answers to six out of seven questions on malaria epidemiology and vector ecology. Knowledge of vector breeding places and malaria transmission were significant determinants of both the quality of VMWs' services and the variety of their actions for malaria prevention and vector control.Conclusions: VMWs' services focused primarily on diagnosis and treatment. Their focus needs to be broadened to cover other aspects of malaria control in order to further strengthen community-based malaria control. VMWs' actions and knowledge also need substantial improvement. Strengthening training programmes can help achieve better performance by VMWs. © 2010 Yasuoka et al; licensee BioMed Central Ltd.
Yasuoka J.,University of Tokyo |
Poudel K.C.,University of Tokyo |
Ly P.,National Center for Parasitology |
Nguon C.,National Center for Parasitology |
And 2 more authors.
Malaria Journal | Year: 2012
Background: Malaria control has been scaled up in many developing countries in their efforts to achieve the Millennium Development Goals. Cambodia recently scaled up their Village Malaria Worker (VMW) project by substantially increasing the number of VMWs and expanding the project's health services to include treatment of fever, diarrhoea, and Acute Respiratory Infections (ARI) in children under five. This study examined if the scale-up interfered with VMWs' service quality, actions, and knowledge of malaria control, and analysed VMWs' overall achievements and perceptions of the newly added health services. Methods. Structured interviews were conducted pre scale-up in February-March 2008 with 251 VMWs and post scale-up in July-August 2010 with 252 VMWs. Comparing the pre and post scale-up survey results (n = 195), changes were examined in terms of VMWs' 1) service quality, 2) malaria prevention and vector control actions, and 3) knowledge of malaria epidemiology and vector ecology. In addition, VMWs' newly added health services were descriptively analysed based on the post scale-up survey (n = 252). Results: VMWs' service quality and actions significantly improved overall during the scale-up of the VMW project (mean index score: +0.805, p < 0.001; +2.923, p < 0.001; respectively). Although most of knowledge areas also showed significant improvement (between +0.256 and +0.499, p < 0.001), less than half (10.3%-47.7%) of the VMWs correctly answered a set of questions on malaria epidemiology and vector ecology, even in the post scale-up survey. About 70% of the respondents reported that their health services to control malaria remained the same or that they were more active after the scale-up. Two-thirds (66.3%) had become more enthusiastic about serving as a VMW since the scale-up, and all but one respondent reported being willing to continue the new services. Conclusions: The Cambodian experience clearly demonstrated that a nationwide scale-up of community-based malaria control can be achieved without degrading community health workers' service quality. The government's strategy to expand VMWs' health services, while providing sufficient training to maintain the quality of their original malaria control services, could have contributed to the improvement of VMW's service quality, actions, and knowledge in spite of the rapid scale-up of the project. © 2011 Yasuoka et al; licensee BioMed Central Ltd.