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Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.

ABSTRACT: 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.

SUBMITTER: Witkowski B

PROVIDER: S-EPMC5015432 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.

Witkowski Benoit B Amaratunga Chanaki C Khim Nimol N Sreng Sokunthea S Chim Pheaktra P Kim Saorin S Lim Pharath P Mao Sivanna S Sopha Chantha C Sam Baramey B Anderson Jennifer M JM Duong Socheat S Chuor Char Meng CM Taylor Walter R J WR Suon Seila S Mercereau-Puijalon Odile O Fairhurst Rick M RM Menard Didier D

The Lancet. Infectious diseases 20130911 12

<h4>Background</h4>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 ...[more]

PMID: 24035558

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Project description:BACKGROUND: Intensified efforts are urgently needed to contain and eliminate artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion. Médecins Sans Frontières plans to support the Ministry of Health in eliminating P. falciparum in an area with artemisinin resistance in the north-east of Cambodia. As a first step, the prevalence of Plasmodium spp. and the presence of mutations associated with artemisinin resistance were evaluated in two districts of Preah Vihear Province. METHODS: A cross-sectional population-based study using a two-stage cluster sampling was conducted in the rural districts of Chhaeb and Chey Saen, from September to October 2013. In each district, 30 clusters of 10 households were randomly selected. In total, blood samples were collected for 1,275 participants in Chhaeb and 1,224 in Chey Saen. Prevalence of Plasmodium spp. was assessed by PCR on dried blood spots. Plasmodium falciparum positive samples were screened for mutations in the K13-propeller domain gene (PF3D7_1343700). RESULT: The prevalence of Plasmodium spp. was estimated at 1.49% (95% CI 0.71-3.11%) in Chhaeb and 2.61% (95% CI 1.45-4.66%) in Chey Saen. Twenty-seven samples were positive for P. falciparum, giving a prevalence of 0.16% (95% CI 0.04-0.65) in Chhaeb and 2.04% (95% CI 1.04-3.99%) in Chey Saen. Only 4.0% of the participants testing positive presented with fever or history of fever. K13-propeller domain mutant type alleles (C580Y and Y493H) were found, only in Chey Saen district, in seven out of 11 P. falciparum positive samples with enough genetic material to allow testing. CONCLUSION: The overall prevalence of P. falciparum was low in both districts but parasites presenting mutations in the K13-propeller domain gene, strongly associated with artemisinin-resistance, are circulating in Chey Saen.The prevalence might be underestimated because of the absentees - mainly forest workers - and the workers of private companies who were not included in the study. These results confirm the need to urgently develop and implement targeted interventions to contain and eliminate P. falciparum malaria in this district before it spreads to other areas.

Project description:BackgroundArtemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia.MethodsBetween June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10,000 per ?L of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, ?-thalassaemia, and a mutation of G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum: age, sex, and place of residence. This study is registered with ClinicalTrials.gov, number NCT00341003.FindingsWe assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54-6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17).InterpretationHeritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation.FundingDivision of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Project description:Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance.In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance.We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups.P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.)

Dataset Information

Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.

Publications

Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.

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