[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: 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.
[Show abstract][Hide abstract] ABSTRACT: Background. The emergence of Plasmodium falciparum resistance to artemisinins on the Cambodian and Myanmar-Thai borders poses severe threats to malaria control. We investigated whether increasing or splitting the dose of the short half-life drug artesunate improves parasite clearance in falciparum malaria in the two regions.Methods. In Pailin, Western Cambodia (from 2008 to 2010) and Wang Pha, North-western Thailand (2009-2010), patients with uncomplicated falciparum malaria were randomized to oral artesunate 6 mg/kg/day as single or twice-daily dose for 7 days, or artesunate 8 mg/kg/day as single or twice daily dose for 3 days, followed by mefloquine. Parasite clearance and recrudescence up to 63-days follow-up were assessed. Trial registration: ISRCTN15351875.Results. A total of 159 patients were enrolled. Overall median (IQR) parasitaemia half-life (half-life) was 6.03 (4.89-7.28) hours in Pailin versus 3.42 (2.20-4.85) hours in Wang Pha (p=0.0001). Splitting or increasing the artesunate dose did not shorten half-life in either site. Pharmacokinetic profiles of artesunate and dihydroartemisinin were similar between sites and did not correlate with half-life. Recrudescent infections occurred in 4 out of 79 patients in Pailin and 5/80 in Wang Pha and was not different between treatment arms (p=0.68). High dose artesunate was associated with transient reticulocytopenia and in one patient with transient neutropenia (0.78 x 10(3)/μL) at day 14.Conclusions. Increasing the artesunate treatment dose up to 8 mg/kg/day or splitting the dose does not improve parasite clearance in either artemisinin resistant or more sensitive infections with P. falciparum.
[Show abstract][Hide abstract] ABSTRACT: Plasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries.
A non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days) was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia), parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope), and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels.
The median (range) parasite clearance half-life and time were 4.8 (2.1-9.7) and 60 (24-96) hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours) in approximately 1/3 of infections. Fourteen of 52 participants (26.9%) had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics.
A subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread to this location from its site of origin in western Cambodia. Resistance containment efforts are underway in Myanmar.
Australian New Zealand Clinical Trials Registry ACTRN12610000896077.
PLoS ONE 01/2013; 8(3):e57689. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.
Proceedings of the National Academy of Sciences 12/2012; · 9.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted twenty isolates from Pailin and Ratanakiri (artemisinin-resistant and -susceptible areas from western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference was observed in the classical isotopic test between the two sets of isolates. However, a 6-hour pulse exposure to 700 nM dihydroartemisinin (Ring stage Survival Assay, RSA) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stages was 17-fold higher in isolates from Pailin (median: 13.5%) compared to Ratanakiri (median: 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring-stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears associated with an altered in vitro phenotype of ring-stages from Pailin in the RSA.
Antimicrobial Agents and Chemotherapy 12/2012; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This article describes 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 on dihydroartemisinin-piperaquine (DP) for the treatment of Plasmodium falciparum, and chloroquine (CQ) and DP for the treatment of P. vivax. All studies followed the standard WHO 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 PCR-corrected treatment failure rate for DP on day 42 was 25% (95%CI: 10%-51%) in Pailin and 10.7% (95%CI: 4%-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 82.6%-95.6%; 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. While DP appears to be an appropriate new first-line treatment for P. vivax in Cambodia, alternative treatments are urgently needed for P. falciparum patients in western Cambodia.
Antimicrobial Agents and Chemotherapy 12/2012; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: By 2009, there were worrying signs from western Cambodia that parasitological responses to artesunate-containing treatment regimens for uncomplicated Plasmodium falciparum malaria were slower than elsewhere which suggested the emergence of artemisinin resistance. Vietnam shares a long land border with Cambodia with a large number of migrants crossing it on a daily basis. Therefore, there is an urgent need to investigate whether there is any evidence of a change in the parasitological response to the artemisinin derivatives in Vietnam. METHODS: From August 2010 to May 2011, a randomized controlled clinical trial in uncomplicated falciparum malaria was conducted to compare two doses of artesunate (AS) (2mg/kg/day versus 4 mg/kg/day for three days) followed by dihydroartemisinin-piperaquine (DHA-PPQ) and a control arm of DHA-PPQ. The goal was characterization of the current efficacy of artesunate in southern Vietnam. The primary endpoint of this study was the parasite clearance half-life; secondary endpoints included the parasite reduction ratios at 24 and 48 hours and the parasite clearance time. RESULTS: 166 patients were recruited into the study. The median parasite clearance half-lives were 3.54 (AS 2mg/kg), 2.72 (AS 4mg/kg), and 2.98 hours (DHA-PPQ) (p=0.19). The median parasite-reduction ratio at 24 hours was 48 in the AS 2mg/kg group compared with 212 and 113 in the other two groups, respectively (p=0.02). The proportions of patients with a parasite clearance time of >72 hours for AS 2mg/kg, AS 4mg/kg and DHA-PPQ were 27%, 27%, and 22%, respectively. Early treatment failure occurred in two (4%) and late clinical failure occurred in one (2%) of the 55 patients in the AS 2mg/kg group, as compared with none in the other two study arms. The PCR-corrected adequate clinical and parasitological response (APCR) rates in the three groups were 94%, 100%, and 100% (p=0.04). CONCLUSIONS: This study demonstrated faster P. falciparum parasite clearance in southern Vietnam than in western Cambodia but slower clearance in comparison with historical data from Vietnam. Further studies to determine whether this represents the emergence of artemisinin resistance in this area are needed. Currently, the therapeutic response to DHA-PPQ remains satisfactory in southern Vietnam.Trial registrationNTC01165372.
[Show abstract][Hide abstract] ABSTRACT: Artemisinin-resistant malaria along the Thailand-Cambodian border is an important public health concern, yet mechanisms of drug action and their contributions to the development of resistance are poorly understood. The pharmacokinetics and pharmacodynamics of oral artesunate monotherapy were explored in a dose-ranging trial in an area of emerging artesunate resistance in western Cambodia. We enrolled 143 evaluable subjects with uncomplicated Plasmodium falciparum malaria in an open label study of directly observed artesunate monotherapy at 3 dose levels (2, 4, and 6 mg/kg of body weight/day) for 7 days at Tasanh Health Center, Tasanh, Cambodia. Clinical outcomes were similar among the 3 groups. Wide variability in artesunate and dihydroartemisinin concentrations in plasma was observed. No significant dose-effect or concentration-effect relationships between pharmacokinetic (PK) and parasite clearance parameters were observed, though baseline parasitemia was modestly correlated with increased parasite clearance times. The overall parasite clearance times were prolonged compared with the clearance times in a previous study at this site in 2006 to 2007, but this did not persist when the evaluation was limited to subjects with a comparable artesunate dose (4 mg/kg/day) and baseline parasitemia from the two studies. Reduced plasma drug levels with higher presentation parasitemias, previously hypothesized to result from partitioning into infected red blood cells, was not observed in this population with uncomplicated malaria. Neither in vitro parasite susceptibility nor plasma drug concentrations appeared to have a direct relationship with the pharmacodynamic (PD) effects of oral artesunate on malaria parasites. While direct concentration-effect relationships were not found, it remains possible that a population PK modeling approach that allows modeling of greater dose separation might discern more-subtle relationships.
Antimicrobial Agents and Chemotherapy 08/2012; 56(11):5484-93. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Artemisinin-based combination therapies are the most effective drugs to treat Plasmodium falciparum malaria. Reduced sensitivity to artemisinin monotherapy, coupled with the emergence of parasite resistance to all partner drugs, threaten to place millions of patients at risk of inadequate treatment of malaria. Recognizing the significance and immediacy of this possibility, the Fogarty International Center and the National Institute of Allergy and Infectious Diseases of the U.S. National Institutes of Health convened a conference in November 2010 to bring together the diverse array of stakeholders responding to the growing threat of artemisinin resistance, including scientists from malarious countries in peril. This conference encouraged and enabled experts to share their recent unpublished data from studies that may improve our understanding of artemisinin resistance. Conference sessions addressed research priorities to forestall artemisinin resistance and fostered collaborations between field- and laboratory-based researchers and international programs, with the aim of translating new scientific evidence into public health solutions. Inspired by this conference, this review summarizes novel findings and perspectives on artemisinin resistance, approaches for translating research data into relevant public health information, and opportunities for interdisciplinary collaboration to combat artemisinin resistance.
The American journal of tropical medicine and hygiene 08/2012; 87(2):231-41. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was conducted to correlate blood concentrations of lumefantrine with treatment outcome for patients with Plasmodium falciparum malaria when the drug was given without specific instructions for administration with food. Patients with P. falciparum malaria in the highly endemic state of Orissa, India, were enrolled during 2008 and followed-up for 28 days after admistration of artemether-lumefantrine for three days according to a World Health Organization protocol. Drug concentration in whole blood was determined by using blood spots placed on filter paper on day 7. The technology is suitable for field studies. One hundred percent of the patients had an adequate clinical and parasitological response. These results confirm the efficacy of artemether-lumefantrine in persons from poor tribal communities when given without specific instructions regarding co-administration with food, despite high inter-individual variability in blood concentrations of lumefantrine.
The American journal of tropical medicine and hygiene 03/2012; 86(3):395-7. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We conducted an open-label, randomized clinical trial to assess parasite clearance times (PCT) and the efficacy of 4 mg/kg (group 1, n = 22) and 2 mg/kg (group 2, n = 22) of oral artesunate for three days followed by artemether-lumefantrine in patients with uncomplicated Plasmodium falciparum malaria at Xepon Interdistrict Hospital, Savannakhet Province in southern Laos. Slides were read in duplicate. The overall mean (95% confidence interval; range) PCT in hours was 23.2 (21.2-25.3; 12-46) and 22.4 (20.3-24.5; 12-46) for the first and second microscopists, respectively (P = 0.57). Ten (23%) patients remained parasitemic on day 1 after treatment (4 [18%] in group 1 and 6 [27%] in group 2; P = 0.47). No patient had patent asexual parasitemia on the second and third days of treatment. The 42-day polymerase chain reaction-corrected cure rates were 100% in both treatment groups. Serious adverse events did not develop during or after treatment in any patients. In conclusion, no evidence of P. falciparum in vivo resistance to artesunate was found in southern Laos.
The American journal of tropical medicine and hygiene 03/2012; 86(3):403-8. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent reports indicate that first cases of genuine artemisinin resistance have already emerged along the Thai-Cambodian border. The main objective of this trial was to track the potential emergence of artemisinin resistance in Bangladesh, which in terms of drug resistance forms a gateway to the Indian subcontinent.
We conducted an open-label, randomized, controlled 42-day clinical trial in Southeastern Bangladesh to investigate the potential spread of clinical artemisinin resistance from Southeast Asia. A total of 126 uncomplicated falciparum malaria patients were randomized to one of 3 treatment arms (artesunate monotherapy with 2 or 4 mg/kg/day once daily or quinine plus doxycycline TID for 7 days). Only cases fulfilling a stringent set of criteria were considered as being artemisinin-resistant.
The 28-day and 42-day cure rates in the artesunate monotherapy (2 and 4 mg/kg) and quinine/doxycyline arms were 97.8% (95% confidence interval, CI: 87.8-99.8%), 100% (95% CI: 91.1-100%), and 100% (95% CI: 83.4-100%), respectively. One case of re-infection was seen in the artesunate high dose arm, and a single case of recrudescence was observed in the low dose group on day 26. No differences in median parasite and fever clearance times were found between the 2 artesunate arms (29.8 h and 17.9 h vs. 29.5 h and 19.1 h). Not a single case fulfilled our criteria of artemisinin resistance. Parasite clearance times were considerably shorter and ex vivo results indicate significantly higher susceptibility (50% inhibitory concentration for dihydroartemisinin was 1.10 nM; 95% CI: 0.95-1.28 nM) to artemisinins as compared to SE-Asia.
There is currently no indication that artemisinin resistance has reached Bangladesh. However, the fact that resistance has recently been reported from nearby Myanmar indicates an urgent need for close monitoring of artemisinin resistance in the region.
PLoS ONE 01/2012; 7(12):e52236. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chloroquine (CQ) is still the drug of choice for the treatment of vivax malaria in Ethiopia, whereas artemether-lumefantrine (AL) is for falciparum malaria. In this setting, clinical malaria cases are treated with AL. This necessitated the need to assess the effectiveness of AL for the treatment of Plasmodium vivax with CQ as a comparator. A total of 57 (80.3%) and 75 (85.2%) cases treated with CQ or AL, respectively, completed the study in an outpatient setting. At the end of the follow-up period of 28 days, a cumulative incidence of treatment failure of 7.5% (95% confidence interval [CI] = 2.9-18.9%) for CQ and 19% (95% CI = 11-31.6%) for AL was detected. CQ resistance was confirmed in three of five CQ treatment failures cases. The effectiveness of AL seems lower than CQ; however, the findings were not conclusive, because the AL evening doses were not supervised.
The American journal of tropical medicine and hygiene 01/2011; 84(1):137-40. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study describes the results of in vitro antimalarial susceptibility assays and molecular polymorphisms of Plasmodium falciparum isolates from Cambodia. The samples were collected from patients enrolled in therapeutic efficacy studies (TES) conducted by the Cambodian National Malaria Control Program for the routine efficacy monitoring of artemisinin-based combination therapy (ACT) (artesunate-mefloquine and artemether-lumefantrine combinations). The isolates (n = 2,041) were obtained from nine sentinel sites during the years 2001 to 2007. Among these, 1,588 were examined for their in vitro susceptibilities to four antimalarials (artesunate, mefloquine, chloroquine, and quinine), and 851 isolates were genotyped for single nucleotide polymorphisms (SNPs). The geometric means of the 50% inhibitory concentrations (GMIC(50)s) of the four drugs tested were significantly higher for isolates from western Cambodia than for those from eastern Cambodia. GMIC(50)s for isolates from participants who failed artesunate-mefloquine therapy were significantly higher than those for patients who were cured (P, <0.001). In vitro correlation of artesunate with the other drugs was observed. The distributions of the SNPs differed between eastern and western Cambodia, suggesting different genetic backgrounds of the parasite populations in these two parts of the country. The GMIC(50)s of the four drugs tested increased significantly in eastern Cambodia during 2006 to 2007. These results are worrisome, because they may signal deterioration of the efficacy of artesunate-mefloquine beyond the Cambodian-Thai border.
Antimicrobial Agents and Chemotherapy 03/2010; 54(5):2135-42. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current information on efficacy of antimalarials is crucial to provide early warning of resistance. A collaborative effort between the Global Malaria Program of the World Health Organization (WHO) and the WorldWide Antimalarial Resistance Network (WWARN) has recently been launched. The effort is planned as a collaboration with the scientific malaria community to create a global, comprehensive, and inclusive network that will provide quality-assured information on antimalarial drug resistance.
Trends in Parasitology 03/2010; 26(5):221-4. · 5.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum originated from at least six foci in South America, Asia, and Oceania. Malaria parasites from these locations exhibit contrasting resistance phenotypes that are distinguished by point mutations and microsatellite polymorphisms in and near the CQR transporter gene, pfcrt, and the multidrug resistance transporter gene, pfmdr1. Amodiaquine (AQ), a 4-aminoquinoline related to CQ, is recommended and often used successfully against CQ-resistant P. falciparum in Africa, but it is largely ineffective across large regions of South America. The relationship of different pfcrt and pfmdr1 combinations to these drug-resistant phenotypes has been unclear. In two P. falciparum genetic crosses, particular pfcrt and pfmdr1 alleles from South America interact to yield greater levels of resistance to monodesethylamodiaquine (MDAQ; the active metabolite of AQ) than to CQ, whereas a pfcrt allele from Southeast Asia and Africa is linked to greater CQ than MDAQ resistance with all partner pfmdr1 alleles. These results, together with (i) available haplotype data from other parasites; (ii) evidence for an emerging focus of AQ resistance in Tanzania; and (iii) the persistence of 4-aminoquinoline-resistant parasites in South America, where CQ and AQ use is largely discontinued, suggest that different histories of drug use on the two continents have driven the selection of distinct suites of pfcrt and pfmdr1 mutations. Increasing use of AQ in Africa poses the threat of a selective sweep of highly AQ-resistant, CQ-resistant parasites with pfcrt and pfmdr1 mutations that are as advantaged and persistent as in South America.
Proceedings of the National Academy of Sciences 11/2009; 106(45):18883-9. · 9.74 Impact Factor