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Journal ArticleDOI

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

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.
Abstract: Artemisinin-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.

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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.

115 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.

36 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: This study demonstrated the absence of k13-resistant genotypes in P. falciparum isolates from Cameroon, giving a baseline for the long-term monitoring of artemisinin derivative efficacy in Africa.
Abstract: The spread of Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia is a major source of concern and the emergence of resistance in Africa would have dramatic consequences, by increasing malaria mortality and morbidity. It is therefore urgent to implement regular monitoring in sentinel sites in sub-Saharan Africa using robust and easy-to-implement tools. The prevalence of k13-propeller mutations and the phenotypic profiles are poorly known in sub-Saharan Africa. Here, the k13-propeller polymorphism was compared to both ex vivo susceptibility to DHA and early parasitological and clinical responses to artemisinin combination therapy (ACT). Plasmodium falciparum isolates were collected in 2015 in Yaounde (Cameroon) from patients treated with dihydroartemisinin-piperaquine combination. Samples were analysed for their susceptibility to artemisinin using the k13-propeller sequencing, the ex vivo ring-stage survival assay, the in vivo parasite positive rate and the clinical statute at day 2. None of the collected isolates revealed the presence of resistance mutations in the k13-propeller sequence. The median ring-stage survival rate for all the 64 interpretable isolates after a 6-hour pulse of 700 nM dihydroartemisinin was low, 0.49% (IQR: 0–1.3). Total parasite clearance was observed for 87.5% of patients and the remaining parasitaemic isolates (12.5%) showed a high reduction of parasite load, ranging from 97.5 to 99.9%. Clinical symptoms disappeared in 92.8% of cases. This study demonstrated the absence of k13-resistant genotypes in P. falciparum isolates from Cameroon. Only synonymous mutations were found with a low prevalence (4.3%). A good association between k13 genotypes and the ex vivo ring-stage survival assay or parasitological and clinical data was obtained. These results give a baseline for the long-term monitoring of artemisinin derivative efficacy in Africa.

23 citations

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.

22 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.

17 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
TL;DR: The overall median clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours in Wang Pha (P<0.001) in each of the two locations as discussed by the authors.
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.)

3,010 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 …

2,746 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.

1,639 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.

491 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: Heritable artemisinin resistance is established in a second Cambodian province and future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation.
Abstract: Summary Background Artemisinin-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. Methods Between 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 μL 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, α-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. Findings We 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). Interpretation Heritable 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. Funding Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

314 citations


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

  • ...Artemisinin tolerance/resistance manifests itself with slower parasite clearance, while ACT remain generally effective both clinically and parasitologically [1-4]....

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