Publications (11)138.22 Total impact
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Article: Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia.
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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.Nature Genetics 04/2013; · 35.53 Impact Factor -
Article: Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia.
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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; · 9.68 Impact Factor -
Article: Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates.
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ABSTRACT: BACKGROUND: Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values. METHODS: Performance parameters of the W2 P. falciparum clone (considered artemisinin "sensitive") were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisininresistant area in Cambodia were compared with W2 susceptibility. RESULTS: Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy. CONCLUSION: The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.Malaria Journal 09/2012; 11(1):325. · 3.19 Impact Factor -
Article: Pharmacokinetics and Pharmacodynamics of Oral Artesunate Monotherapy in Patients with Uncomplicated Plasmodium falciparum Malaria in Western Cambodia.
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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. · 4.84 Impact Factor -
Article: Ex vivo drug sensitivity profiles of Plasmodium falciparum field isolates from Cambodia and Thailand, 2005 to 2010, determined by a histidine-rich protein-2 assay.
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ABSTRACT: In vitro drug susceptibility assay of Plasmodium falciparum field isolates processed "immediate ex vivo" (IEV), without culture adaption, and tested using histidine-rich protein-2 (HRP-2) detection as an assay, is an expedient way to track drug resistance. From 2005 to 2010, a HRP-2 in vitro assay assessed 451 P. falciparum field isolates obtained from subjects with malaria in western and northern Cambodia, and eastern Thailand, processed IEV, for 50% inhibitory concentrations (IC50) against seven anti-malarial drugs, including artesunate (AS), dihydroartemisinin (DHA), and piperaquine. In western Cambodia, from 2006 to 2010, geometric mean (GM) IC50 values for chloroquine, mefloquine, quinine, AS, DHA, and lumefantrine increased. In northern Cambodia, from 2009-2010, GM IC50 values for most drugs approximated the highest western Cambodia GM IC50 values in 2009 or 2010. Western Cambodia is associated with sustained reductions in anti-malarial drug susceptibility, including the artemisinins, with possible emergence, or spread, to northern Cambodia. This potential public health crisis supports continued in vitro drug IC50 monitoring of P. falciparum isolates at key locations in the region.Malaria Journal 06/2012; 11:198. · 3.19 Impact Factor -
Article: Artesunate dose escalation for the treatment of uncomplicated malaria in a region of reported artemisinin resistance: a randomized clinical trial.
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ABSTRACT: The emergence of artemisinin resistance has raised concerns that the most potent antimalarial drug may be under threat. The currently recommended daily dose of artesunate (AS) is 4 mg/kg, and is administered for 3 days together with a partner antimalarial drug. This study investigated the impact of different AS doses on clinical and parasitological responses in malaria patients from an area of known artemisinin resistance in western Cambodia. Adult patients with uncomplicated P. falciparum malaria were randomized into one of three 7-day AS monotherapy regimens: 2, 4 or 6 mg/kg/day (total dose 14, 28 and 42 mg/kg). Clinical, parasitological, pharmacokinetic and in vitro drug sensitivity data was collected over a 7-day inpatient period and during weekly follow-up to 42 days. 143 patients were enrolled (n = 75, 40 and 28 to receive AS 2, 4 and 6 mg/kg/day respectively). Cure rates were high in all treatment groups at 42 days despite almost half the patients remaining parasitemic on Day 3. There was no impact of increasing AS dose on median parasite clearance times, median parasite clearance rates or on the proportion of patients remaining parasitemic on Day 3. However at the lowest dose used (2 mg/kg/d) patients with parasitemia >10,000/µL had longer median (IQR) parasite clearance times than those with parasitemia <10,000/µL (63 (48-75) vs. 84 (66-96) hours, p<0.0001). 19% of patients in the high-dose arm developed neutropenia (absolute neutrophil count <1.0×10(9)/L) by Day 14 and resulted in the arm being halted early. There is no pharmacodynamic benefit of increasing the daily dose of AS (4 mg/kg) currently recommended for short-course combination treatment of uncomplicated malaria, even in regions with emerging artemisinin resistance, as long as the partner drug retains high efficacy. ClinicalTrials.gov NCT00722150.PLoS ONE 01/2011; 6(5):e19283. · 4.09 Impact Factor -
Article: Malaria and other vector-borne infection surveillance in the U.S. Department of Defense Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance program: review of 2009 accomplishments.
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ABSTRACT: Vector-borne infections (VBI) are defined as infectious diseases transmitted by the bite or mechanical transfer of arthropod vectors. They constitute a significant proportion of the global infectious disease burden. United States (U.S.) Department of Defense (DoD) personnel are especially vulnerable to VBIs due to occupational contact with arthropod vectors, immunological naiveté to previously unencountered pathogens, and limited diagnostic and treatment options available in the austere and unstable environments sometimes associated with military operations. In addition to the risk uniquely encountered by military populations, other factors have driven the worldwide emergence of VBIs. Unprecedented levels of global travel, tourism and trade, and blurred lines of demarcation between zoonotic VBI reservoirs and human populations increase vector exposure. Urban growth in previously undeveloped regions and perturbations in global weather patterns also contribute to the rise of VBIs. The Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) and its partners at DoD overseas laboratories form a network to better characterize the nature, emergence and growth of VBIs globally. In 2009 the network tested 19,730 specimens from 25 sites for Plasmodium species and malaria drug resistance phenotypes and nearly another 10,000 samples to determine the etiologies of non-Plasmodium species VBIs from regions spanning from Oceania to Africa, South America, and northeast, south and Southeast Asia. This review describes recent VBI-related epidemiological studies conducted by AFHSC-GEIS partner laboratories within the OCONUS DoD laboratory network emphasizing their impact on human populations.BMC Public Health 01/2011; 11 Suppl 2:S9. · 2.00 Impact Factor -
Article: Plasmodium falciparum gametocyte carriage is associated with subsequent Plasmodium vivax relapse after treatment.
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ABSTRACT: Mixed P. falciparum/P. vivax infections are common in southeast Asia. When patients with P. falciparum malaria are treated and followed for several weeks, a significant proportion will develop P. vivax malaria. In a combined analysis of 243 patients recruited to two malaria treatment trials in western Cambodia, 20/43 (47%) of those with P. falciparum gametocytes on admission developed P. vivax malaria by Day 28 of follow-up. The presence of Pf gametocytes on an initial blood smear was associated with a 3.5-fold greater rate of vivax parasitemia post-treatment (IRR = 3.5, 95% CI 2.0-6.0, p<0.001). The increased rate of post-treatment P. vivax infection persisted when correlates of exposure and immunity such as a history of malaria, male gender, and age were controlled for (IRR = 3.0, 95% CI 1.9-4.7, p<0.001). Polymerase chain reaction (PCR) confirmed that only a low proportion of subjects (5/55 or 9.1%) who developed vivax during follow-up had detectable Pv parasites in the peripheral blood at baseline. Molecular detection of falciparum gametocytes by reverse transcriptase PCR in a subset of patients strengthened the observed association, while PCR detection of Pv parasitemia at follow-up was similar to microscopy results. These findings suggest that the majority of vivax infections arising after treatment of falciparum malaria originate from relapsing liver-stage parasites. In settings such as western Cambodia, the presence of both sexual and asexual forms of P. falciparum on blood smear at presentation with acute falciparum malaria serves as a marker for possible occult P. vivax coinfection and subsequent relapse. These patients may benefit from empiric treatment with an 8-aminoquinolone such as primaquine.PLoS ONE 01/2011; 6(4):e18716. · 4.09 Impact Factor -
Article: Dose-dependent risk of neutropenia after 7-day courses of artesunate monotherapy in Cambodian patients with acute Plasmodium falciparum malaria.
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ABSTRACT: Fears of emerging artemisinin resistance in western Cambodia have prompted a series of clinical trials investigating whether slow responses to antimalarial treatment can be overcome by increasing doses of drug. Patients with uncomplicated malaria were allocated 1 of 3 oral artesunate monotherapy regimens (2, 4, or 6 mg/kg/day for 7 days) and were observed for 42 days. A series of safety measures, including complete blood count on days 0, 3, 6, and 14, was implemented because of a lack of safety data for these experimental doses. After 3 doses, geometric mean absolute neutrophil counts were reduced in all groups, and 2 patients required artesunate to be discontinued because of neutropenia (absolute neutrophil count, <1.0 × 10(3) cells/μL). Recipients of the 6 mg/kg/day dosage had significantly lower geometric mean absolute neutrophil counts than did recipients of the 2 and 4 mg/kg/day dosages at 6 and 14 days (P < .001 for each). Overall, 5 (19%) of 26 patients who received the 6 mg/kg/day dosage became neutropenic within 14 days, triggering a cohort-halting rule and ending the trial early. Pharmacokinetic data from neutropenic patients showed wide variance, with plasma clearance occurring significantly slower in neutropenic patients than in nonneutropenic patients. Artesunate remains a crucial drug for the treatment of malaria, and determining optimal dosing regimens is vital to overcome emerging resistant parasite strains along the Thai-Cambodian border. However, future experimental dosing studies must be designed with care, because the safety of such regimens can no longer be assumed. The artemisinin derivatives remain one of the safest classes of antimalarial drugs, but this study demonstrates that the dosing limit may have been reached.Clinical Infectious Diseases 11/2010; 51(12):e105-14. · 9.15 Impact Factor -
Article: Artemisinin resistance in Cambodia: a clinical trial designed to address an emerging problem in Southeast Asia.
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ABSTRACT: Increasing rates of failure of artemisinin-based combination therapy have highlighted the possibility of emerging artemisinin resistance along the Thai-Cambodian border. We used an integrated in vivo-in vitro approach to assess the presence of artemisinin resistance in western Cambodia. This article provides additional data from a clinical trial that has been published in The New England Journal of Medicine. Ninety-four adult patients from Battambang Province, western Cambodia, who presented with uncomplicated falciparum malaria were randomized to receive high-dose artesunate therapy (4 mg/kg/day orally for 7 days) or quinine-tetracycline. Plasma concentrations of dihydroartemisinin, in vitro drug susceptibility, and molecular markers were analyzed. Cases meeting all the following criteria were classified as artemisinin resistant: failure to clear parasites within 7 days of treatment or reemergence of parasites within 28 days of follow-up; adequate plasma concentrations of dihydroartemisinin; prolonged parasite clearance; and increased in vitro drug susceptibility levels for dihydroartemisinin. Two (3.3%) of 60 artesunate-treated patients were classified as artemisinin resistant. Their parasite clearance times were prolonged (133 and 95 h, compared with a median of 52.2 h in patients who were cured). These patients had 50% inhibitory concentrations of dihydroartemisinin that were almost 10 times higher than the reference clone W2. Resistance did not appear to be mediated by the pfmdr1 copy number or selected PfATPase6 polymorphisms previously proposed to confer artemisinin resistance. Artemisinin resistance has emerged along the Thai-Cambodian border. The potentially devastating implications of spreading resistance to a drug that currently has no successor call for further studies of this emerging problem. ClinicalTrials.gov identifier NCT00479206.Clinical Infectious Diseases 10/2010; 51(11):e82-9. · 9.15 Impact Factor -
Article: Evidence of artemisinin-resistant malaria in western Cambodia.
New England Journal of Medicine 01/2009; 359(24):2619-20. · 53.30 Impact Factor
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Institutions
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2009–2011
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Armed Forces Research Institute of Medical Sciences
Bangkok, Bangkok, Thailand
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