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Journal ArticleDOI

Plasmodium falciparum clearance in clinical studies of artesunate-amodiaquine and comparator treatments in sub-Saharan Africa, 1999–2009

25 Mar 2014-Malaria Journal (BioMed Central)-Vol. 13, Iss: 1, pp 114-114

TL;DR: Within the period covered by these studies, rapid Plasmodium falciparum clearance continues to be achieved in Sub-Saharan African patients treated with ACT, and in particular with ASAQ, and the prediction formula for parasite clearance time could be a pragmatic tool for studies with binary outcomes and once-daily sampling.

AbstractArtemisinin-based combination therapy (ACT) is the recommended first-line therapy for uncomplicated Plasmodium falciparum malaria worldwide but decreased artemisinin susceptibility, phenotypically characterized as slow parasite clearance time (PCT), has now been reported in Southeast Asia. This makes it all too important to measure the dynamics of parasite clearance in African patients treated with ACT over time, to understand trends and detect changes early enough to intervene Individual patient data from 27 clinical trials of artesunate-amodiaquine (ASAQ) vs comparators conducted between 1999 and 2009 were analysed for parasite clearance on modified intent-to-treat (ITT) basis. Overall 15,017 patients treated for uncomplicated P. falciparum malaria at 44 sites in 20 sub-Saharan African countries were included in the analysis; 51% (n=7,660) vs 49% (n=7,357) were treated with ASAQ and comparator treatments, respectively. Seventy-seven per cent (77%) were children under six years of age. The proportion of the patients treated with ASAQ with persistent parasitaemia on Day 2 was 8.6%, and 1.5% on Day 3. Risk factor for not clearing parasites on Day 2 and Day 3 calculated by multivariate logistic regression with random effect on site and controlling for treatment were: high parasitaemia before treatment was (adjusted risk ratios (AOR) 2.12, 95% CI 1.91-2.35, AOR 2.43, 95% CI 1.98-3.00, respectively); non-ACT treatment (p=0.001, for all comparisons). Anaemia (p=0.001) was an additional factor for Day 2 and young age (p=0.005) for Day 3. In patients treated with ASAQ in studies who had complete parasitaemia data every 24 hours up to Day 3 and additionally Day 7, the parasite reduction ratio was 93.9% by Day 1 and 99.9% by Day 2. Using the median parasitaemia before treatment (p0=27,125 μL) and a fitted model, the predicted PCT (pPCT = 3.614*ln (p0) – 6.135, r² = 0.94) in ASAQ recipients was 31 hours. Within the period covered by these studies, rapid Plasmodium falciparum clearance continues to be achieved in Sub-Saharan African patients treated with ACT, and in particular with ASAQ. The prediction formula for parasite clearance time could be a pragmatic tool for studies with binary outcomes and once-daily sampling, both for research and monitoring purposes.

Topics: Artesunate/amodiaquine (55%)

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Journal ArticleDOI
21 Aug 2014-PLOS ONE
TL;DR: The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT, indicating that artemisinin resistance is not prevalent in Uganda.
Abstract: The emergence of resistance to artemisinin derivatives in Southeast Asia, manifested as delayed clearance of Plasmodium falciparum following treatment with artemisinins, is a major concern. Recently, the artemisinin resistance phenotype was attributed to mutations in portions of a P. falciparum gene (PF3D7_1343700) encoding kelch (K13) propeller domains, providing a molecular marker to monitor the spread of resistance. The P. falciparum cysteine protease falcipain-2 (FP2; PF3D7_1115700) has been shown to contribute to artemisinin action, as hemoglobin degradation is required for potent drug activity, and a stop mutation in the FP2 gene was identified in parasites selected for artemisinin resistance. Although delayed parasite clearance after artemisinin-based combination therapy (ACT) has not yet been noted in Uganda and ACTs remain highly efficacious, characterizing the diversity of these genes is important to assess the potential for resistance selection and to provide a baseline for future surveillance. We therefore sequenced the K13-propeller domain and FP2 gene in P. falciparum isolates from children previously treated with ACT in Uganda, including samples from 2006–7 (n = 49) and from 2010–12 (n = 175). Using 3D7 as the reference genome, we identified 5 non-synonymous polymorphisms in the K13-propeller domain (133 isolates) and 35 in FP2 (160 isolates); these did not include the polymorphisms recently associated with resistance after in vitro selection or identified in isolates from Asia. The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT. Thus, the K13-propeller and FP2 polymorphisms associated with artemisinin resistance are not prevalent in Uganda, and we did not see evidence for selection of polymorphisms in these genes.

110 citations


Cites background from "Plasmodium falciparum clearance in ..."

  • ...Second, as delayed clearance of parasites after therapy is uncommon and associated with high baseline parasitemia in Uganda, persistent parasitemia 2 days after the onset of therapy is likely not a reliable indicator of resistance [6,8]....

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Journal ArticleDOI
TL;DR: There is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation, and there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya.
Abstract: Artemether–lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995–2003) and 745 after (post-ACT; 2008–2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995–1996 to 93.2% in 2014 and 0.0% in 2002–2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya.

27 citations


Cites background from "Plasmodium falciparum clearance in ..."

  • ...However, ACTs remain highly efficacious in subSaharan Africa (SSA) with fast clearance half-lives (Ashley et al., 2014; Zwang et al., 2014)....

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Journal ArticleDOI
TL;DR: The results demonstrate the potent preclinical antileukemic activity of ARTS as well as its potential for a rapid transition to a clinical trial either alone or in combination with conventional chemotherapy or BCL-2 inhibitor, for treatment of AML.
Abstract: The artimisinins are a class of antimalarial compounds whose antiparasitic activity is mediated by induction of reactive oxygen species (ROS). Herein, we report that among the artimisinins, artesunate (ARTS), an orally bioavailable compound has the most potent antileukemic activity in AML models and primary patients' blasts. ARTS was most cytotoxic to the FLT3-ITD+ AML MV4-11 and MOLM-13 cells (IC50 values of 1.1 and 0.82μM respectively), inhibited colony formation in primary AML and MDS cells and augmented cytotoxicity of chemotherapeutics. ARTS lowered cellular BCL-2 level via ROS induction and increased the cytotoxicity of the BCL-2 inhibitor venetoclax (ABT-199). ARTS treatment led to cellular and mitochondrial ROS accumulation, double stranded DNA damage, loss of mitochondrial membrane potential and induction of the intrinsic mitochondrial apoptotic cascade in AML cell lines. The antileukemic activity of ARTS was further confirmed in MV4-11 and FLT3-ITD+ primary AML cell xenografts as well as MLL-AF9 syngeneic murine AML model where ARTS treatment resulted in significant survival prolongation of treated mice compared to control. Our results demonstrate the potent preclinical antileukemic activity of ARTS as well as its potential for a rapid transition to a clinical trial either alone or in combination with conventional chemotherapy or BCL-2 inhibitor, for treatment of AML.

18 citations


Journal ArticleDOI
TL;DR: Considerations for military involvement in regional malaria elimination efforts are reviewed, given their high mobility, frequent malaria exposure, and potential for asymptomatic carriage.
Abstract: Despite significant progress in malaria control in the Greater Mekong Subregion (GMS), malaria is still endemic, with more than 30 million people infected annually. Important gaps remain in case management, service delivery, prevention, and vector control, particularly in hard-to-reach mobile populations. Rapidly evolving drug resistance has created a new urgency to move aggressively toward elimination. However, no clear and cost-effective strategy has been identified. Although GMS militaries are under-recognized as a malaria transmission reservoir, they are an important focal point for elimination activities, given their high mobility, frequent malaria exposure, and potential for asymptomatic carriage. At the same time, military organizational capacity and proximity to other mobile populations could facilitate elimination efforts if relevant political barriers could be overcome. Here, we review considerations for military involvement in regional malaria elimination efforts.

16 citations


Journal ArticleDOI
TL;DR: An analysis of P. falciparum genetic diversity, focusing on antimalarial resistance-associated molecular markers in two socioeconomically different villages in mainland Equatorial Guinea, suggests that closer monitoring should be maintained to prevent the possible spread of artemisinin resistance in Africa.
Abstract: Efforts to control malaria may affect malaria parasite genetic variability and drug resistance, the latter of which is associated with genetic events that promote mechanisms to escape drug action. The worldwide spread of drug resistance has been a major obstacle to controlling Plasmodium falciparum malaria, and thus the study of the origin and spread of associated mutations may provide some insights into the prevention of its emergence. This study reports an analysis of P. falciparum genetic diversity, focusing on antimalarial resistance-associated molecular markers in two socioeconomically different villages in mainland Equatorial Guinea. The present study took place 8 years after a previous one, allowing the analysis of results before and after the introduction of an artemisinin-based combination therapy (ACT), i.e., artesunate plus amodiaquine. Genetic diversity was assessed by analysis of the Pfmsp2 gene and neutral microsatellite loci. Pfdhps and Pfdhfr alleles associated with sulfadoxine-pyrimethamine (SP) resistance and flanking microsatellite loci were investigated, and the prevalences of drug resistance-associated point mutations of the Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes were estimated. Further, to monitor the use of ACT, we provide the baseline prevalences of K13 propeller mutations and Pfmdr1 copy numbers. After 8 years, noticeable differences occurred in the distribution of genotypes conferring resistance to chloroquine and SP, and the spread of mutated genotypes differed according to the setting. Regarding artemisinin resistance, although mutations reported as being linked to artemisinin resistance were not present at the time, several single nucleotide polymorphisms (SNPs) were observed in the K13 gene, suggesting that closer monitoring should be maintained to prevent the possible spread of artemisinin resistance in Africa.

14 citations


Cites background from "Plasmodium falciparum clearance in ..."

  • ...falciparum resistance to artemisinin derivatives in Southeast Asia (where it was introduced for the first time in the mid-1990s), but so far it has not been detected in Africa (13)....

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References
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Journal ArticleDOI
Abstract: 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.)

2,750 citations


Journal ArticleDOI
07 Jul 2001-BMJ
TL;DR: The concept of study quality and the methods used to assess quality are discussed and the methodology for both the assessment of quality and its incorporation into systematic reviews and meta-analysis is discussed.
Abstract: This is the first in a series of four articles The quality of controlled trials is of obvious relevance to systematic reviews. If the “raw material” is flawed then the conclusions of systematic reviews cannot be trusted. Many reviewers formally assess the quality of primary trials by following the recommendations of the Cochrane Collaboration and other experts. 1 2 However, the methodology for both the assessment of quality and its incorporation into systematic reviews and meta-analysis are a matter of ongoing debate.3-5 In this article we discuss the concept of study quality and the methods used to assess quality. #### Components of internal and external validity of controlled clinical trials Internal validity —extent to which systematic error (bias) is minimised in clinical trials Quality is a multidimensional concept, which could relate to the design, conduct, and analysis of a trial, its clinical relevance, or quality of reporting.6 The validity of the findings generated by a study clearly is an important dimension of quality. In the 1950s the social scientist Campbell proposed a useful distinction between internal and external validity (see box below). 7 8 Internal validity implies that the differences observed between groups of patients allocated to different …

2,642 citations


Journal ArticleDOI
02 Jan 2014-Nature
TL;DR: 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.
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.

1,398 citations


Journal ArticleDOI
TL;DR: The in-vitro RSA of 0-3 h ring-stage parasites provides a platform for the molecular characterisation of artemisinin resistance and the ex-vivo RSA can be easily implemented where surveillance for artemis inin resistance is needed.
Abstract: Summary 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 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.

416 citations


"Plasmodium falciparum clearance in ..." refers background in this paper

  • ...Genomic studies are underway, which have generated so far two sets of putative markers of artemisinin resistance - K13-propeller polymorphism [5], and a SNP in the gene encoding a DNA repair protein RAD5 on chromosome 13 (and possibly also on chromosome 10) [6]....

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Journal ArticleDOI
TL;DR: The combination of artesunate and amodiaquine improved treatment efficacy in Gabon and Kenya, and was equivalent in Sénégal, and is a potential combination for use in Africa.
Abstract: Summary Background Increasing drug resistance limits the choice of efficacious chemotherapy against Plasmodium falciparum malaria in Africa. Amodiaquine still retains efficacy against P falciparum in many African countries. We assessed the safety, treatment efficacy, and effect on gametocyte carriage of adding artesunate to amodiaquine in three randomised trials in Kenya, Senegal, and Gabon. Methods We enrolled 941 children (400 in Kenya, 321 in Senegal, and 220 in Gabon) who were 10 years or older and who had uncomplicated P falciparum malaria. Patients were randomly assigned amodiaquine (10 mg/kg per day for 3 days) plus artesunate (4 mg/kg per day for 3 days) or amodiaquine (as above) and placebo (for 3 days). The primary endpoints were parasitological cure rates at days 14 and 28. Analysis was by intention to treat and by an evaluability method. Findings Both regimens were well tolerated. Six patients in the amodiaquine-artesunate group and five in the amodiaquine group developed early, drug-induced vomiting, necessitating alternative treatment. By intention-to-treat analysis, the day-14 cure rates for amodiaquine-artesunate versus amodiaquine were: 175/192 (91%) versus 140/188 (74%) in Kenya (Δ=16·7% [95% Cl 9·3–24·1], p Interpretation The combination of artesunate and amodiaquine improved treatment efficacy in Gabon and Kenya, and was equivalent in Senegal. Amodiaquine-artesunate is a potential combination for use in Africa. Further investigations to assess the potential effect on the evolution of drug resistance, disease transmission, and safety of amodiaquine-artesunate are warranted.

303 citations


"Plasmodium falciparum clearance in ..." refers background or methods in this paper

  • ...Predicted time of parasite clearance The number of patients included in RCT at 17 sites with complete parasitaemia record (every 24 hours from Day 0 to Day 3 plus Day 7) was 4,848 of whom 2,355 were treated with ASAQ and 2,493 with a comparator drug....

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  • ...Consecutive parasite slide results (Day 0, Day 1, Day 2, Day 3, Day 7) were available at 18 sites from five randomized controlled trials (RCT) [17-19,26,27]....

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  • ...Using multivariate logistic regression with random effect on site and controlling for treatment, the risk factors for a patient to remain positive on Day 2 were higher parasitaemia before treatment (AOR 2.12, 95% CI 1.91-2.35, p = 0.001) and anaemia (AOR 1.22, 95% CI 1.07-1.38, p = 0.001); no significant difference in the risk of being parasitaemic on Day 2 was detected in RCT comparing ASAQ to other ACT: AL (p = 0.245), DP (p = 0.762), AS + SP (p = 0.291), whereas patients treated with non-ACT were at higher risk: AQ+ SP (AOR 14.53, 95% CI 11.36-18.59, p = 0.001), CQ + SP (AOR 20.10, 95% CI 15.07-26.82, p = 0.001), or AQ (AOR 21.63, 95% CI 12.73-36.75, p = 0.001)....

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  • ...Out of the 27 studies included (conducted between 1999 and 2009), three were multi-country studies [17-19], two studies compared ASAQ fixed and loose combination [20-22] and three studies were non-comparative (in Sierra Leone [23] and Senegal [24,25])....

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