Publications (47)424.73 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases, and measured ring-stage survival rates following drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13-49% to 0.3-2.4% following the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2-29% following the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared to reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. Copyright © 2014, American Association for the Advancement of Science.
- [Show abstract] [Hide abstract] ABSTRACT: Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain-carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. Here we analyze the in-vivo transcriptomes of 1,043 P. falciparum isolates from patients with acute malaria, and show that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin resistant parasites remain in a state of decelerated development at the young ring stage while their upregulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion. Copyright © 2014, American Association for the Advancement of Science.
- [Show abstract] [Hide abstract] ABSTRACT: 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 infections (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.).
- [Show abstract] [Hide abstract] ABSTRACT: Chloroquine (CQ) is used to treat Plasmodium vivax malaria in areas where CQ resistance has not been reported. The use of artemisinin (ART)-based combination therapies (ACTs) to treat CQ-sensitive P. vivax infections is effective and convenient but may promote the emergence and worsening of ART resistance in sympatric Plasmodium falciparum populations. Here, we show that CQ effectively treats P. vivax malaria in Pursat Province, western Cambodia, where ART-resistant P. falciparum is highly prevalent and spreading. (This study has been registered at ClinicalTrials.gov under registration no. NCT00663546.)
- [Show abstract] [Hide abstract] ABSTRACT: Reduced Plasmodium falciparum sensitivity to short-course artemisinin (ART) monotherapy manifests as a long parasite clearance half-life. We recently defined three parasite founder populations with long half-lives in Pursat, western Cambodia, where reduced ART sensitivity is prevalent. Using the ring-stage survival assay, we show that these founder populations have reduced ART sensitivity in vitro at the early ring stage of parasite development and that a genetically admixed population contains subsets of parasites with normal or reduced ART sensitivity.
- [Show abstract] [Hide abstract] ABSTRACT: The ring-stage survival assay (RSA) is a powerful tool for phenotyping artemisinin-resistant Plasmodium falciparum but requires experienced microscopists to count viable parasites among 10,000 erythrocytes in Giemsa-stained thin blood smears. Here we describe a rapid flow cytometric assay that accurately counts viable parasites among 250,000 erythrocytes in suspension. This method performs as well as light microscopy and can be used to standardize the collection of RSA data between research groups in laboratory and field settings.
- [Show abstract] [Hide abstract] ABSTRACT: Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.
- [Show abstract] [Hide abstract] ABSTRACT: 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. 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. 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). 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. Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH.
- [Show abstract] [Hide abstract] ABSTRACT: In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC50s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC50s (GMIC50s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC50s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC50 and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.
Dataset: Supplementary Material 1
- [Show abstract] [Hide abstract] ABSTRACT: We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.
Dataset: Supplementary Material 3
Dataset: Supplementary Material 2
- [Show abstract] [Hide abstract] ABSTRACT: Previous microsatellite analyses of sympatric populations of Plasmodium vivax and P. falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n = 87) and P. falciparum (n = 164) parasites from Pursat province, western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P = 0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P = 0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax.
National Institute of Allergy and Infectious Diseases
Maryland, United States
- Laboratory of Parasitic Diseases (LPD)