Laboratory detection of artemisinin-resistant Plasmodium falciparum (original) (raw)
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The Lancet Infectious Diseases, 2013
Background. Artemisinin resistance in Plasmodium falciparum has been documented in Cambodia, Thailand, Myanmar, and Vietnam. This clinical phenotype manifests as a long parasite clearance half-life during administration of artemisinin monotherapy or artemisinin-based combination therapy. The lack of in-vitro and ex-vivo correlates of artemisinin resistance makes studying this phenotype costly and logistically challenging. Methods. We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study conducted in Pursat, Cambodia, in 2010 and used novel in-vitro survival assays to explore the stage-dependent susceptibility of slow-and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the Ring-stage Survival Assay (RSA) in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia, to measure the ex-vivo responses of parasites from patients with malaria. The clinical studies are registered with ClinicalTrials.gov, numbers NCT00341003 and NCT01736319. Findings. In-vitro survival rates of culture-adapted parasites from 13 slow-and 13 fast-clearing infections differed significantly when assays were conducted on 0-3 h ring-stage parasites (RSA 0-3h , 10•9% vs. 0•23%; p=0•007). Ex-vivo survival rates significantly correlated with in-vivo parasite clearance halflives (n=30, r=0•74, 95% CI 0•50 to 0•87; p<0•0001). Interpretation. We report for the first time in-vitro and ex-vivo assays that detect artemisinin-resistant P falciparum in Cambodia. The in-vitro RSA 0-3h provides a platform for biochemical and molecular characterization of artemisinin resistance. The ex-vivo RSA can be easily implemented in countries where surveillance for artemisinin resistance is needed.
Reduced Artemisinin Susceptibility of Plasmodium falciparum Ring Stages in Western Cambodia
Antimicrobial Agents and Chemotherapy, 2013
Reduced Artemisinin Susceptibility of http://aac.asm.org/content/57/2/914 Updated information and services can be found at: These include: SUPPLEMENTAL MATERIAL Supplemental material REFERENCES http://aac.asm.org/content/57/2/914#ref-list-1 at: This article cites 26 articles, 16 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on March 14, 2013 by INSTITUT PASTEUR-Médiathèque
Laboratory detection of artemisinin resistant P. falciparum
Antimicrobial Agents and Chemotherapy, 2014
Conventional 48 hour in vitro susceptibility tests have low sensitivity in identifying artemisinin resistant P. 26 falciparum defined phenotypically by slow in vivo parasite clearance rates. We hypothesized originally that 27 this discrepancy was explained by loss of ring stage susceptibility and so developed a simple field-adapted 24 28 hour trophozoite maturation inhibition assay (TMI test) focusing on the ring stage and compared it to the 29 standard 48 hour schizont maturation inhibition (WHO test). In Pailin, Western Cambodia, where artemisinin 30 resistant P.falciparum is prevalent, the TMI test geometric mean (95% confidence interval) 50% inhibition 31 concentration [IC 50 ] for artesunate was 6.8 (5.2-8.3) ng/mL compared with 1.5 (1.2-1.8) ng/mL for the 32 standard 48 hour WHO test (P=0.001). TMI IC 50 values correlated significantly with the in vivo responses to 33 artesunate; parasite clearance time (r=0.44, P=0.001) and parasite clearance half-life (r=0.46, P=0.001) 34 whereas the standard 48 hour test values did not. On continuous culture of two resistant isolates the 35 artemisinin resistant phenotype was lost after six weeks (IC 50 values fell from 10 and 12 ng/mL to 2.7 and 3 36 ng/mL respectively). Slow parasite clearance in falciparum malaria in Western Cambodia results from 37 reduced ring stage susceptibility. 38 Abstract : 189 words 39 Keywords: P. falciparum, malaria, antimalarial drug, artemisinin resistance, in vitro-test, artesunate 40 Corporation, version 3.1, U.S.A). Correlations were assessed by the method of Spearman. Statistical 104 significance within and between groups was determined by the non-parametric Kruskal-Wallis or Mann 105 Whitney U tests. All statistical analyses were performed using the SPSS software. 106 107
Antimicrobial agents and chemotherapy, 2014
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.
Artemisinin resistance in Plasmodium falciparum malaria
2009
BACKGROUND 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. METHODS 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 pharmaco kinetics, and molecular markers of resistance. RESULTS 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. CONCLUSIONS 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.
Artemisinin Resistance inPlasmodium falciparumMalaria
The New England Journal of Medicine, 2009
BACKGROUND 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. METHODS 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 pharmaco kinetics, and molecular markers of resistance. RESULTS 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. CONCLUSIONS 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.
Emerging Infectious Diseases, 2022
A rtemisinin-based combination therapies (ACTs) have contributed greatly to the global decline of illness and death from malaria (1). However, the novel emergence of artemisinin resistance in eastern Africa has threatened the effectiveness of these breakthrough treatments (2-4). To avert potential disaster resulting from increased resistant malaria cases, the nature and extent of this resistance in Africa urgently needs to be characterized. Artemisinin resistance is conferred by some Plasmodium falciparum kelch 13 (K13) gene mutations, only a few of which are validated markers of resistance, defined by both in vitro resistance and delayed parasite clearance in treated patients. For candidate markers, only parasite clearance applies (1). In Rwanda, K13 mutations have increased over the past decade. K13 R561H, a validated marker associated with delayed parasite clearance, was recently observed in >10% of P. falciparum-positive samples (2,3,5). In neighboring Uganda, artemisinin resistance conferred by another mutation, K13 A675V, has recently been reported (4). We document in vitro artemisinin resistance in 3 P. falciparum patient isolates from Rwanda carrying K13 R561H, A675V, and C469F mutations. The Study We recruited malaria patients in Huye District, Rwanda, during September-December 2019 and documented patient characteristics and consent, ethical clearance, and K13 variants elsewhere (2). Within 6 hours of sample collection, we cryopreserved all 66 P. falciparum isolates in ethylenediaminetetraacetic acid by washing the red blood cell pellet, adding freezing solution (3% sorbitol, 28% glycerol, 0.65% NaCl), and freezing at −80°C. Eight of the 66 isolates carried nonsynonymous K13 mutations (2). We successfully thawed and culture-adapted 4 of the isolates in which we identified K13 mutations: R561H, the current prevalent mutation in Rwanda; A675V, found in 11% of P. falciparum samples in Uganda; C469F, another candidate marker; and V555A, which is of unknown significance. We conducted a 0-3-h postinvasion ring-stage susceptibility assay (RSA) with the active metabolite dihydroartemisinin (6). We exposed ring stages to a 6-h pulse of 700 nmol/L dihydroartemisinin and cultured exposed and nonexposed isolates in vitro in triplicate for 72 h. We counted parasite density per ≥10,000 red blood cells on Giemsa-stained thin blood films and calculated the means of triplicates. Dividing parasite density in dihydroartemisinin-exposed cultures by the density in nonexposed cultures provided the RSA survival rate. We considered results if 72-h growth rates exceeded 1.5× rates in the nonexposed controls and had >3 successful independent triplicate experiments per isolate. We In Vitro Confirmation of Artemisinin Resistance in Plasmodium falciparum from Patient Isolates,
Proceedings of the National Academy of Sciences of the United States of America, 2017
Artemisinin-resistant falciparum malaria, defined by a slow-clearance phenotype and the presence of kelch13 mutants, has emerged in the Greater Mekong Subregion. Naturally acquired immunity to malaria clears parasites independent of antimalarial drugs. We hypothesized that between- and within-population variations in host immunity influence parasite clearance after artemisinin treatment and the interpretation of emerging artemisinin resistance. Antibodies specific to 12 Plasmodium falciparum sporozoite and blood-stage antigens were determined in 959 patients (from 11 sites in Southeast Asia) participating in a multinational cohort study assessing parasite clearance half-life (PCt1/2) after artesunate treatment and kelch13 mutations. Linear mixed-effects modeling of pooled individual patient data assessed the association between antibody responses and PCt1/2.P. falciparum antibodies were lowest in areas where the prevalence of kelch13 mutations and slow PCt1/2 were highest [Spearman ...
Malaria Journal, 2010
Background: Artemisinin derivatives have been used for malaria treatment in Vietnam since 1989. Reported malaria cases have decreased from 1,672,000 with 4,650 deaths in 1991, to 91,635 with 43 deaths in 2006. Current national guidelines recommend artemisinin-based combination therapy (ACT), although artesunate is still available as monotherapy through the private sector. Recent reports suggest that effectiveness of ACT and artesunate monotherapy has declined in western Cambodia. This study examined Plasmodium falciparum resistance patterns over 10 years in southwest Vietnam in infected patients treated with artemisinin compounds.