Multinormal in vitro distribution of Plasmodium falciparum susceptibility to piperaquine and pyronaridine
© Pascual et al.; licensee BioMed Central. 2015
Received: 7 October 2014
Accepted: 26 January 2015
Published: 5 February 2015
In 2002, the World Health Organization recommended that artemisinin-based combination therapy (ACT) be used to treat uncomplicated malaria. Dihydroartemisinin-piperaquine and artesunate-pyronaridine are two of these new combinations. The aim of the present work was to assess the distribution of the in vitro values of pyronaridine (PND) and piperaquine (PPQ) and to define a cut-off for reduced susceptibility for the two anti-malarial drugs.
The distribution and range of the 50% inhibitory concentration values (IC50) of PND and PPQ were determined for 313 isolates obtained between 2008 and 2012 from patients hospitalized in France for imported malaria. The statistical Bayesian analysis was designed to answer the specific question of whether Plasmodium falciparum has different phenotypes of susceptibility to PND and PPQ.
The PND IC50 values ranged from 0.6 to 84.6 nM, with a geometric mean of 21.1 ± 16.0 nM (standard deviation). These values were classified into three components. The PPQ IC50 values ranged from 9.8 to 217.3 nM, and the geometric mean was 58.0 ± 34.5 nM. All 313 PPQ values were classified into four components. Isolates with IC50 values greater than 60 nM or four-fold greater than 3D7 IC50 are considered isolates that have reduced susceptibility to PND and those with IC50 values greater than 135 nM or 2.3-fold greater than 3D7 IC50 are considered isolates that have reduced susceptibility to PPQ.
The existence of at least three phenotypes for PND and four phenotypes for PPQ was demonstrated. Based on the cut-off values, 18 isolates (5.8%) and 13 isolates (4.2%) demonstrated reduced susceptibility to PND and PPQ, respectively.
Over the past 20 years, many strains of Plasmodium falciparum have become resistant to chloroquine and other anti-malarial drugs. In 2002, the World Health Organization (WHO) recommended that artemisinin-based combination therapy (ACT) be used to treat all cases of uncomplicated malaria. The following combinations have been evaluated: artesunate-sulphadoxine-pyrimethamine, artesunate-amodiaquine, artemether-lumefantrine, artesunate-mefloquine, artesunate-chlorproguanil-dapsone and, more recently, artesunate-pyronaridine and dihydroartemisinin-piperaquine. Most of these combinations are available as fixed-dose co-formulations that are convenient, facilitate improved adherence and help prevent misuse.
Dihydroartemisinin-piperaquine (DP) (Artekin®, Duo-Cotecxin®, Eurartesim®) is a new ACT that is administered as single daily dose for three days. It has been demonstrated to be well tolerated and highly effective for the treatment of uncomplicated malaria in Asia [1,2] and for the treatment of uncomplicated P. falciparum malaria in Africa [3-6]. DP may also have a better post-treatment prophylactic effect than does artemether-lumefantrine [7-9] and artesunate-amodiaquine . Since 2012, DP has been available for the treatment of uncomplicated falciparum malaria in France. DP has also been demonstrated to be effective for the treatment of Plasmodium vivax malaria . However, the emergence of P. falciparum resistance to DP, manifested as delayed parasite clearance following the treatment, has developed in Cambodia and Vietnam [12-14].
The piperaquine (PPQ) susceptibility of P. falciparum isolates has been assessed in studies with isolates from Africa (geometric mean IC50 = 81.3 nM and 66.8 nM) [15,16], Cameroon (39 nM) , Kenya (from 41.9 to 50 nM) [18-20], Niger (24.2 nM) , Ghana (28.3 nM) , Uganda (6.1 nM) , the China-Myanmar border (28.4 nM) , the Thai-Burmese border (49 nM) , Cambodia (22 nM) , Indonesia (21.8 nM) , and Papua New Guinea .
The pyronaridine-artesunate combination (Pyramax®) is one of the latest artemisinin-based combinations and is currently under development by the not-for-profit organization Medicines for Malaria Venture (Geneva, Switzerland) and the pharmaceutical company Shin Poong Pharmaceuticals (Seoul, Republic of Korea) for the treatment of uncomplicated P. falciparum malaria and for the blood stages of P. vivax malaria. Pyramax® has recently completed phase III trials in humans. A five-day regimen of pyronaridine (PND) alone (total dose = 1,800 mg) produced a better cure rate than did artesunate, artemether or mefloquine used alone in the same conditions in Thailand . The efficacy of PND-artesunate was not inferior to that of artemether-lumefantrine in the treatment of uncomplicated falciparum malaria in Africa and Southeast Asia [30,31]. PND-artesunate had a better efficacy than did mefloquine-artesunate in Cambodia .
The PND in vitro susceptibility was previously assessed in P. falciparum strains (1.9 to 47.8 nM and 15 to 49 nM, respectively) [33,34] and in isolates from Africa (geometric mean = 19.9 nM) , Cameroon (3.58 nM) , Gabon (3.0 nM and 1.87 nM) [36,37], Kenya (13.5 nM) , Niger (9.8 nM) , Senegal (3.8 nM and 4.52 nM) [38,39], Indonesia (1.92 nM) , and in isolates from patients in Thailand that were cured with PND (15.7 nM) or that recrudesced after PND treatment (23.0 nM) . In addition, PND is effective in vitro against P. vivax isolates (2.58 nM) .
The early detection of resistance to PPQ and PND requires the establishment of the baseline parasite chemosusceptibility of current isolates from regions of endemicity. The aim of the present work was to determine the distribution and range of the 50% inhibitory concentrations (IC50) of PPQ and PND for 313 imported malaria isolates from Africa and to determine the cut-off values for in vitro reduced susceptibility to these two drugs.
Patients and sample collection
In total, 313 P. falciparum isolates were collected between April 2008 and August 2012 from patients hospitalized in France with imported malaria from a malaria-endemic country (Angola, Benin, Burkina Faso, Cameroon, Central African Republic, Chad, Comoros, Congo, Ivory Coast, Gabon, Gambia, Ghana, Guinea, Madagascar, Mali, Mauritania, Mozambique, Niger, Senegal, Togo, Zambia). Informed consent was not required for this study because the sampling procedures and testing are part of the French national recommendations for the care and surveillance of malaria. Venous blood samples were collected in Vacutainer® ACD tubes (Becton Dickinson, Rutherford, NJ, USA) before treatment and were transported at 4°C from French hospitals located in Aix en Provence, Bordeaux, Chambery, Frejus, Grenoble, Lyon, Marseille, Metz, Montpellier, Nice, Nimes, Pau, Toulon, Toulouse, and Valence to the Institute of Biomedical Research of the French Army (IRBA) in Marseille within 72 hours of collection. A Case Report Form was provided at the same time, either as a paper copy or electronically.
Thin blood smears were stained using a RAL® kit (Réactifs RAL, Paris, France) and were examined to determine the P. falciparum density and confirm mono-infection. Parasitized erythrocytes were washed three times with RPMI 1640 medium (Invitrogen, Paisley, UK), buffered with 25 mM HEPES and 25 mM NaHCO3. If the parasitaemia exceeded 0.5%, the infected erythrocytes were diluted to 0.5% with uninfected erythrocytes (human blood type A+) and re-suspended in RPMI 1640 medium supplemented with 10% human serum (Abcys S.A. Paris, France) for a final haematocrit of 1.5%. The susceptibility of the 313 isolates was assessed without culture adaptation.
PPQ and PND were obtained from Shin Poong Pharm Co. (Seoul, Korea). PPQ was first dissolved in methanol and then diluted in water to obtain final concentrations ranging from 0.8 to 1,000 nM. PND was dissolved and diluted in water to obtain concentrations ranging from 0.15 to 100 nM.
Batches of plates were tested and validated using the chloroquine-susceptible strain 3D7 (isolated in West Africa; obtained from MR4, VA, USA) in three to six independent experiments using the conditions described below. The two strains were synchronized twice with sorbitol before use , and clonality was verified every 15 days through PCR genotyping of the polymorphic genetic markers msp1 and msp2 and microsatellite loci [42,43]; additionally, clonality was verified each year by an independent laboratory from the Worldwide Anti-malarial Resistance Network (WWARN).
Ex vivo assay
For ex vivo isotopic microtests, 200 μl/well of the suspension of parasitized red blood cells (final parasitaemia, 0.5%; final haematocrit, 1.5%) were distributed in 96-well plates pre-dosed with anti-malarial drugs. Parasite growth was assessed by adding 1 μCi of tritiated hypoxanthine with a specific activity of 14.1 Ci/mmol (Perkin-Elmer, Courtaboeuf, France) to each well at time zero. The plates were then incubated for 42 hours in controlled atmospheric conditions that consisted of 10% O2, 5% CO2 and 85% N2 at 37°C with a humidity of 95%. Immediately after incubation, the plates were frozen and then thawed to lyse the erythrocytes. The contents of each well were collected on standard filter microplates (Unifilter GF/B; Perkin-Elmer) and washed using a cell harvester (Filter-Mate Cell Harvester; Perkin-Elmer). The filter microplates were dried, and 25 μl of scintillation cocktail (Microscint O; Perkin-Elmer) was placed in each well. Radioactivity incorporated in nucleotides by the parasites was measured with a scintillation counter (Top Count; Perkin-Elmer).
The drug concentration able to inhibit 50% of the parasite growth (IC50) was assessed by the drug concentration corresponding to 50% of the incorporation of tritiated hypoxanthine by the parasite in the drug-free control wells. The IC50 value was determined using a non-linear regression analysis of log-based dose-response curves (Riasmart, Packard, Meriden, USA).
The statistical analysis was designed to answer the specific question of whether P. falciparum has different PPQ and PND susceptibility phenotypes. A heterogeneous population of IC50 values was observed; therefore, the data were assumed to represent a univariate Gaussian mixture with k components. Each observation was assumed to originate from one of the k components, and the label of the group from which each observation arose was unknown. The unknowns of the model were the number of components, the means, variances and weights of the different components, and the vector of allocations of the observations. The analysis was performed in two steps. First, reversible jump Monte Carlo Markov Chains (RJMCMC)  samplers were used to choose a suitable number of components k, and the present algorithm followed the recommendations of Cappé et al. . After a relevant number of components was chosen, standard Gibbs samplers were run to obtain estimates of the model parameters and to classify the observations . Because of the ‘label-switching’ problem that is due to the symmetry in the likelihood of the model parameters, the mixture components should be labelled before making an inference regarding the parameters . The classical ordering constraint, which was biologically relevant here, was used. The algorithms were run for 100,000 burn-in iterations and 20,000 post-burn-in iterations. These numbers were assumed to be sufficient to obtain reliable results. Moreover, each algorithm was run three times to verify that the results obtained in two different runs were similar and that there was no convergence problem .
Statistical analysis of the 309 pyronaridine (PND) IC 50 values by year
IC 50 number
IC 50 min
IC 50 max
Parameter estimates for the three-component mixture model for the pyronaridine (PND) distribution of the 309 Plasmodium falciparum isolates
IC 50 mean (nM)
The cut-off value for in vitro reduced susceptibility to PND was estimated using the arithmetic mean plus two SDs of the IC50s of the 309 isolates and was set at 57.7 nM. Isolates with an IC50 greater than 60 nM were considered to display reduced susceptibility to PND in vitro. Eighteen isolates (5.8%) displayed reduced susceptibility to PND in vitro.
Statistical analysis of the 313 piperaquine (PPQ) IC 50 values by year
IC 50 number
IC 50 min
IC 50 max
Parameter estimates for the four-component mixture model for the piperaquine PPQ) distribution of the 313 Plasmodium falciparum isolates
IC 50 mean (nM)
The cut-off for in vitro reduced susceptibility to PPQ was estimated using the arithmetic mean plus 2 SDs of the IC50s of the 313 isolates and was found to be 135.4 nM. Isolates with an IC50 greater than 135 nM were considered to be isolates with reduced susceptibility to PPQ in vitro. Thirteen isolates (4.2%) displayed reduced susceptibility to PPQ in vitro.
The early detection of resistance to PPQ and PND requires that the baseline parasite chemosusceptibility of current isolates from regions of endemicity be established. Maximizing the efficacy and longevity of drugs as a tool to control malaria will critically depend on pursuing intensive research into identifying in vitro markers and implementing in vitro and in vivo surveillance programmes, such as those championed by WWARN [48,49]. In this context, there is a need to identify in vitro and molecular markers that predict PPQ and PND resistance and can provide an active surveillance method to monitor temporal trends in parasite susceptibility [50,51].
A Bayesian mixture modeling approach was choose. A Bayesian approach has already been proposed for anti-malarial in vitro susceptibilities; it has been used to evaluate the distribution and the cut-off for reduced susceptibility to doxycycline [52,53]. All 309 PND values were classified into three components: component A (IC50 mean 15.1 nM), component B (IC50 mean 33.9 nM) and component C (IC50 mean 59.7 nM). The proportion of isolates in each group was 58.9% for component A, 31.1% for component B and 10.0% for component C. In the previous study on doxycycline, the distribution of the doxycycline IC50 in a triple normal distribution was independent of the African origin of the isolates (imported isolates from Africa or field isolates from Senegal, Gabon or Congo) . Only the proportion of isolates predicted to belong to each component was dependent on the origin of the isolates.
In the present study, the cut-off for in vitro reduced susceptibility to PND was estimated as the geometric mean plus 2 SDs of the IC50s of the 309 isolates (57.7 nM). Isolates with an IC50 greater than 60 nM were considered to be isolates with reduced in vitro susceptibility to PND. Eighteen isolates (5.8%) demonstrated reduced susceptibility to PND in vitro. These data are consistent with clinical observations of limited failures with PND. One study of isolates in Niger reported that 3% of the isolates displayed in vitro resistance to PND, with an estimated cut-off of 20 nM .
A cut-off for in vitro resistance is defined for a specific methodology. For example, the in vitro effects and the IC50 values for doxycycline are dependent on the time incubation conditions [14-16], on gas conditions, i.e., O2 and CO2 [54,55] and on methodology, i.e., isotopic test versus immuno-enzymatic or SYBR green test [56,57]. The incubation time is the condition that interferes significantly with the IC50 values for doxycycline or other antibiotics [58,59]. The IC50 values decrease by a factor between 10 to 100 in prolonged exposure to antibiotics. The gas conditions interfere with the IC50 values for quinolines, such as chloroquine, monodesethyaodiaquine, quinine, mefloquine, or lumefantrine [55,60]. The IC50 values for quinoline drugs are significantly lower at O2 > 15% than those at 10% O2. Dissolution methods to prepare the stock solutions of anti-malarial drugs can also interfere with IC50 values. In the present study, the dissolution of PND in water was the same condition as this used in previous work [16,36,37,61]. Using an arbitrarily fixed threshold of resistance could lead to wrong decision making at the country level. To reduce the effects of the conditions of the methodology of the in vitro test, an IC50 ratio (IC50 of clinical isolate/mean IC50 of 3D7 on the same batch of plates) can be evaluated for each isolate . Another mean is to use a cut-off ratio (resistance cut-off defined for a specific methodology/mean IC50 of 3D7 tested by the same methodology). Isolates with cut-off ratio greater than four (60/16.7), i.e., isolates with IC50 four-fold greater than 3D7 IC50, are considered to be isolates with reduced in vitro susceptibility to PND. Compared to 3D7 IC50 values, the data obtained with different methodologies can be compared.
All 313 PPQ values were classified into four components: component A (IC50 mean 35.4 nM), component B (IC50 mean 76.7 nM), component C (IC50 mean 103.5 nM), and component D (IC50 mean 175.0 nM). The proportion of isolates in each group was 43.5% for component A, 46.6% for component B, 7.0% for component C, and 2.9% for component D.
In the present study, PPQ was dissolved in methanol and then diluted in water. In previous works, PPQ was dissolved in methanol and then diluted in water [13,14,24,25], in methanol and hydrochloric acid and then diluted in water [20,62], in dimethyl sulphoxide (DMSO) [27,63] or in 0.5% acid lactic in water [15,28,63-65]. These several dissolution methods to prepare the PPQ stock solutions can interfere with IC50 values. The cut-off for in vitro reduced susceptibility to PPQ was estimated to be the geometric mean plus 2 SDs of the IC50s of the 313 isolates (135.4 nM). Isolates with an IC50 greater than 135 nM or 2.3-fold greater than PPQ IC50 for 3D7 were considered to be isolates with reduced in vitro susceptibility to PPQ. Thirteen isolates (4.2%) met the criteria for reduced susceptibility to PPQ in vitro. These data are consistent with previous data from Niger but not from the China-Myanmar border. In Niger, 6% of the isolates were found to be resistant to PPQ in vitro, with an estimated cut-off of 150 nM . However, 83% of the isolates from the China-Myanmar border were resistant to PPQ in vitro (the cut-off of approximately 15 nM was estimated by a three-fold decrease in susceptibility to PPQ in comparison to the strain 3D7) .
There was a significant difference in the PPQ responses among the five years of the study (p <0.0001, Kruskal-Wallis rank sum test). However, there was no significant difference in the responses to PPQ against 3D7 (geometric mean each year from 2008 to 2012: 56.7 nM, 58.4 nM, 61.0, 57.2 nM, and 62.5 nM; p = 0.6909). This absence of significant difference in the responses of the control strains makes bias due to the methodology unlikely.
The existence of at least three phenotypes for PND and four phenotypes for PPQ was demonstrated. These phenotypes may be associated with different genotypes. Genotyping analysis would be necessary to identify the molecular basis of the susceptibility differences and to correlate the genetic profiles with the phenotypes. Previous work demonstrated that the doxycycline phenotypes predicted by the Bayesian method were associated with specific genotypes [53,63]. The priority now is to investigate polymorphisms both in the genes that are known to be involved in anti-malarial drug resistance and in new genes for each phenotype.
The PND and PPQ in vitro susceptibility values ranged into three and four components, respectively. Eighteen isolates (5.8%) and 13 isolates (4.2%) demonstrated reduced in vitro susceptibility to PND and PPQ, respectively. DP is associated with a longer prophylactic time after treatment compared to the time after artemether-lumefantrine treatment [5,6,64]. However, the gametocyte carriage and malaria transmission to mosquitoes was lower after artemether-lumefantrine treatment . Despite the recent report of frequent DP failures in Cambodia and Vietnam [12-14], DP remains an effective treatment for falciparum malaria in Africa and for P. vivax [67-69]. PND-artesunate is effective for the treatment of uncomplicated falciparum malaria in Africa and Southeast Asia [30,31]. PND-artesunate successfully treats artemisinin-resistant Plasmodium berghei parasites, while artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine and dihydroartemisinin-piperaquine are not effective . Although extended parasite clearance times were indicative of artemisinin resistance, ACT remains important for the treatment of malaria.
This study was supported by the Institut de Veille Sanitaire (grant number CNR paludisme).
French National Reference Centre for Imported Malaria Study Group
V Augis, D Basset, F Benoit-Vical, A Berry, N Bourgeois, F Conquere de Monbrison, P Delaunay, J Delmont, K Ezzedine, B Faugere, C Garabedian, E Garnotel, C Lollivier, D Malvy, P Marty, D Maubon, G Menard, P Millet, P Minodier, Montaut, A Mottard, P Munier, P Parola, R Piarroux, S Picot, T Pistone, C Pomares-Estran, J Puyhardy, D Raffenot, M-C Receveur, R Saidi, H Savini, F Simon, S Vedy.
- Smithuis F, Kyaw MK, Phe O, Aye KZ, Htet L, Barends M, et al. Efficacy and effectiveness of dihydroartemisinin-piperaquine versus artesunate-mefloquine in falciparum malaria: an open label randomized comparison. Lancet. 2006;367:2075–85.View ArticlePubMedGoogle Scholar
- Thang NX, Trung TN, Phong NC, Quang HH, Dai B, Shanks GD, et al. The efficacy and tolerability of artemisinin-piperaquine (Artequick) versus artesunate-amodiaquine (Coarsucam) for the treatment of uncomplicated Plasmodium falciparum malaria in south-central Vietnam. Malar J. 2012;11:217.View ArticleGoogle Scholar
- Sylla K, Abiola A, Tine RCK, Faye B, Sow D, Ndiaye JL, et al. Monitoring the efficacy and safety of three artemisinin based-combinations therapies in Senegal: results from two years surveillance. Malar J. 2013;13:598.Google Scholar
- Yavo W, Faye B, Kuete T, Djohan V, Oga SA, Kassi RR, et al. Multicentric assessment of the efficacy and tolerability of dihydroartemisinin-piperaquine compared to artemether-lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria in sub-Saharan Africa. Malar J. 2011;10:198.View ArticlePubMed CentralPubMedGoogle Scholar
- Yeka A, Tibenderana J, Achan J, D’Alessandro U, Talisuna AO. Efficacy of quinine, artemether-lumefantrine and dihydroartemisinin-piperaquine as rescue treatment for uncomplicated malaria in Ugandan children. PLoS One. 2013;8:53772.View ArticleGoogle Scholar
- Agarwal A, McMorrow M, Onyango P, Otieno K, Odero C, Williamson J, et al. A randomized trial of artemether-lumefantrine and dihydroartemisinin-piperaquine in the treatment of uncomplicated malaria among children in western Kenya. Malar J. 2013;12:254.View ArticlePubMed CentralPubMedGoogle Scholar
- Yeka A, Dorsey G, Kamya MR, Talisuna A, Lugemwa M, Rwakimari JB, et al. Artemether-lumefantrine versus dihydroartemisinin-piperaquine for treating uncomplicated malaria: a randomized trial to guide policy in Uganda. PLoS One. 2008;3:2390.View ArticleGoogle Scholar
- Kamya MR, Yeka A, Burkirwa H, Lugemwa M, Rwakimari JB, Staedke SG, et al. Artemether-lumefantrine versus dihydroartemisinin-piperaquine for treatment of malaria: a randomized trial. PLoS Clin Trials. 2007;2:e20.View ArticlePubMed CentralPubMedGoogle Scholar
- Zongo I, Dorsey G, Rouamba N, Dokomajilar C, Séré Y, Rosenthal PJ, et al. Randomized comparison of amodiaquine plus sulfadoxine-pyrimethamine, artemether-lumefantrine, and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria in Burkina-Faso. Clin Infect Dis. 2007;45:1453–61.View ArticlePubMedGoogle Scholar
- Hasugian AR, Purba HLE, Kenangalem E, Wuwung RM, Ebsworth EP, Maristela R, et al. Dihydroartemisinin-piperaquine versus artesunate-amodiaquine: superior efficacy and posttreatment prophylaxis against multidrug resistant Plasmodium falciparum and Plasmodium vivax malaria. Clin Infect Dis. 2008;44:1067–74.View ArticleGoogle Scholar
- Pasaribu AP, Chokejindachai W, Sirivichayakul C, Tanomsing N, Chavez I, Tjitra E, et al. A randomized comparison of Dihydroartemisinin-Piperaquine and Artesunate-Amodiaquine combined with Primaquine for radical treatment of vivax malaria in Sumatera, Indonesia. J Infect Dis. 2013;208:1906–13.View ArticlePubMed CentralPubMedGoogle Scholar
- Leang R, Barrette A, Mey Bouth D, Menard D, Abdur R, Duong S, et al. Efficacy of dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010. Antimicrob Agents Chemother. 2013;57:818–26.View ArticlePubMed CentralPubMedGoogle Scholar
- Saunders DL, Vanachayangkul P, Lon C. Dihydroartemisinin-piperaquine failure in Cambodia. N Engl J Med. 2014;371:484–5.View ArticlePubMedGoogle Scholar
- Thriemer K, Van Hong N, Rosanas-Urgell A, Ha DM, Pockele E, Guetens P, et al. Delayed parasite clearance after treatment with dihydroartemisinin-piperaquine in Plasmodium falciparum malaria patients in Central Vietnam. Antimicrob Agents Chemother. 2014;58:7049–55.View ArticlePubMedGoogle Scholar
- Pascual A, Madamet M, Bertaux L, Amalvict R, Benoit N, Travers D, et al. In vitro piperaquine susceptibility is not associated with the Plasmodium falciparum chloroquine resistance transporter gene. Malar J. 2013;12:431.View ArticlePubMed CentralPubMedGoogle Scholar
- Pascual A, Parola P, Benoit-Vical F, Simon F, Malvy D, Picot S, et al. Ex vivo activity of the ACT new components pyronaridine and piperaquine in comparison with conventional ACT drugs against isolates of Plasmodium falciparum. Malar J. 2012;11:45.View ArticlePubMed CentralPubMedGoogle Scholar
- Basco LK, Ringwald P. In vitro activities of piperaquine and other 4-aminoquinolines against clinical isolates of Plasmodium falciparum in Cameroon. Antimicrob Agents Chemother. 2003;47:1391–4.View ArticlePubMed CentralPubMedGoogle Scholar
- Okombo J, Abdi AI, Kiara SM, Mwai L, Pole L, Sutherland CJ, et al. Repeat polymorphisms in the low-complexity regions of Plasmodium falciparum ABC transporters and associations with in vitro antimalarial responses. Antimicrob Agents Chemother. 2013;57:6196–204.View ArticlePubMed CentralPubMedGoogle Scholar
- Okombo J, Kiara SM, Mwai L, Pole L, Ohuma E, Ochola LI, et al. Baseline in vitro activities of the antimalarials pyronaridine and methylene blue against Plasmodium falciparum isolates from Kenya. Antimicrob Agents Chemother. 2012;56:1105–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Mwai L, Kiara SM, Abdirahman A, Pole L, Rippert A, Diriye A, et al. In vitro activities of piperaquine, lumefantrine, and dihydroartemisinin in Kenyan Plasmodium falciparum isolates and polymorphisms in pfcrt and pfmdr1. Antimicrob Agents Chemother. 2009;55:5069–73.View ArticleGoogle Scholar
- Issaka M, Salissou A, Arzika I, Guillebaud J, Maazou A, Specht S, et al. Ex vivo responses of Plasmodium falciparum clinical isolates to conventional and new antimalarial drugs in Niger. Antimicrob Agents Chemother. 2013;57:3415–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Quashie NB, Duah NO, Abuaku B, Quaye L, Ayanful-Torgby R, Akwoviah GA, et al. A SYBR Green 1-based in vitro test of susceptibility of Ghanaian Plasmodium falciparum clinical isolates to a panel of anti-malarial drugs. Malar J. 2013;12:450.View ArticlePubMed CentralPubMedGoogle Scholar
- Nsobya SL, Kiggundu M, Nanyunja S, Joloba M, Greenhouse B, Rosenthal PJ. In vitro sensitivity of Plasmodium falciparum to different antimalarial drugs in Uganda. Antimicrob Agents Chemother. 2010;54:1200–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Hao M, Jia D, Li Q, He Y, Yuan L, Xu S, et al. In vitro sensitivities of Plasmodium falciparum isolates from the China-Myanmar border to piperaquine and association with polymorphisms in candidate genes. Antimicrob Agents Chemother. 2013;57:1723–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Barends M, Jaidee A, Khaohirun N, Singhasivanon P, Nosten F. In vitro activity of ferroquine (SSR 97193) against Plasmodium falciparum isolates from the Thai-Burmese border. Malar J. 2007;6:81.View ArticlePubMed CentralPubMedGoogle Scholar
- Lim P, Dek D, Try V, Eastman RT, Chy S, Sreng S, et al. Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in Western, Northern, and Eastern Cambodia, 2011-2012: Association with molecular markers. Antimicrob Agents Chemother. 2013;57:5277–83.View ArticlePubMed CentralPubMedGoogle Scholar
- Marfurt J, Chalfein F, Prayoga P, Wabiser F, Kenangalem E, Piear KA, et al. Ex vivo drug susceptibility of ferroquine against chloroquine-resistant isolates of Plasmodium falciparum and P. vivax. Antimicrob Agents Chemother. 2011;55:4461–4.View ArticlePubMed CentralPubMedGoogle Scholar
- Wong RPM, Lautu D, Tavul L, Hackett SL, Siba P, Karunajeewa HA, et al. In vitro sensitivity of Plasmodium falciparum to conventional and novel antimalarial drugs in Papua New Guinea. Trop Med Int Health. 2010;15:342–9.View ArticlePubMedGoogle Scholar
- Looareesuwan S, Kyle DE, Vivaran C, Vanijanonta S, Wilairatana P, Wernsdorfer WH. Clinical study of pyronaridine for the treatment of acute uncomplicated falciparum malaria in Thailand. Am J Trop Med Hyg. 1996;54:205–9.PubMedGoogle Scholar
- Tshefu AK, Gaye O, Kayentao K, Thompson R, Bhatt KM, Sesay SS, et al. Efficacy and safety of a fixed-dose oral combination of pyronaridine-artesunate compared with artemether-lumefantrine in children and adults with uncomplicated Plasmodium falciparum malaria: a randomised non-inferiority trial. Lancet. 2010;375:1457–67.View ArticlePubMedGoogle Scholar
- Kayentao K, Doumbo OK, Pénali LK, Offianan AT, Bhatt KM, Kimani J, et al. Pyronaridine-artesunate granules versus artemether-lumefantrine crushed tablets in children with Plasmodium falciparum malaria: a randomized controlled trial. Malar J. 2012;11:364.View ArticlePubMed CentralPubMedGoogle Scholar
- Rueangweerayu R, Phyo AP, Uthaisin C, Poravuth Y, Binh TQ, Tinto H, et al. Pyronaridine–artesunate versus mefloquine plus artesunate for malaria. N Engl J Med. 2012;366:1298–309.View ArticleGoogle Scholar
- Elueze EI, Croft SL, Warhurst DC. Activity of pyronaridine and mepacrine against twelve strains of Plasmodium falciparum in vitro. J Antimicrob Chemother. 1996;37:511–8.View ArticlePubMedGoogle Scholar
- Pradines B, Briolant S, Henry M, Oeuvray C, Baret E, Amalvict R, et al. Absence of association between pyronaridine in vitro responses and polymorphisms in genes involved in quinoline resistance in Plasmodium falciparum. Malar J. 2010;9:337.View ArticleGoogle Scholar
- Ringwald P, Moukoko Eboumbou CE, Bickii J, Basco LK. In vitro activities of pyronaridine, alone and in combination with other antimalarial drugs, against Plasmodium falciparum. Antimicrob Agents Chemother. 1999;43:1525–7.PubMed CentralPubMedGoogle Scholar
- Pradines B, Mabika Mamfoumbi M, Parzy D, Owono Medang M, Lebeau C, Mourou Mbina JR, et al. In vitro susceptibility of African isolates of Plasmodium falciparum from Gabon to pyronaridine. Am J Trop Med Hyg. 1999;60:105–8.PubMedGoogle Scholar
- Kurth F, Pongratz P, Bélard S, Mordmüller B, Kremsner PG, Ramharter M. In vitro activity of pyronaridine against Plasmodium falciparum and comparative evaluation of anti-malarial drug susceptibility assays. Malar J. 2009;8:79.View ArticlePubMed CentralPubMedGoogle Scholar
- Pradines B, Tall A, Parzy D, Spiegel A, Fusai T, Hienne R, et al. In vitro activity of pyronaridine and amodiaquine against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial agents. J Antimicrob Chemother. 1998;42:333–9.View ArticlePubMedGoogle Scholar
- Pradines B, Tall A, Ramiandrasoa F, Spiegel A, Sokhna C, Fusai T, et al. In vitro activity of iron-binding compounds against Senegalese isolates of Plasmodium falciparum. J Antimicrob Chemother. 2006;57:1093–9.View ArticlePubMedGoogle Scholar
- Price RN, Marfurt J, Chalfein F, Kenagalem E, Piera KA, Tjitra E, et al. In vitro activity of pyronaridine against multidrug-resistant Plasmodium falciparum and Plasmodium vivax. Antimicrob Agents Chemother. 2010;54:5146–50.View ArticlePubMed CentralPubMedGoogle Scholar
- Lambros C, Vanderberg JP. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979;65:418–20.View ArticlePubMedGoogle Scholar
- Bogreau H, Renaud F, Bouchiba H, Durand P, Assi SB, Henry MC, et al. Genetic diversity and structure of African Plasmodium falciparum populations in urban and rural areas. Am J Trop Med Hyg. 2006;74:953–9.PubMedGoogle Scholar
- Henry M, Diallo I, Bordes J, Ka S, Pradines B, Diatta B, et al. Urban malaria in Dakar, Senegal: chemosusceptibility and genetic diversity of Plasmodium falciparum isolates. Am J Trop Med Hyg. 2006;75:146–51.PubMedGoogle Scholar
- Richardson S, Green PJ. On Bayesian analysis of mixtures with an unknown number of components (with discussion). J Roy Stat Soc. 1997;B59:731–92.View ArticleGoogle Scholar
- Cappé O, Robert CP, Rydén T. Reversible jump, birth-and-death and more general continuous time Markov chain Monte Carlo samplers. J Roy Stat Soc. 2003;B65:679–700.View ArticleGoogle Scholar
- Diebolt J, Robert CP. Estimation of finite mixture distributions through Bayesian sampling. J Roy Stat Soc. 1994;56:363–75.Google Scholar
- Jasra A, Holmes CC, Stephens DA. Markov chain Monte Carlo methods and the label switching problem in Bayesian mixture modeling. Stat Sci. 2005;20:50–67.View ArticleGoogle Scholar
- Sibley CH, Barnes KI, Plowe CV. The rationale and plan for creating a world antimalarial resistance network (WARN). Malar J. 2007;6:118.View ArticlePubMed CentralPubMedGoogle Scholar
- Sibley CH, Barnes KI, Watkins WM, Plowe CV. A network to monitor antimalarial drug resistance: a plan for moving forward. Trends Parasitol. 2008;24:43–8.View ArticlePubMedGoogle Scholar
- Bacon DJ, Jambou R, Fandeur T, Le Bras J, Wongsrichanalai C, Fukuda MM, et al. World Antimalarial Resistance Network (WARN) II: in vitro antimalarial drug susceptibility. Malar J. 2007;6:120.View ArticlePubMed CentralPubMedGoogle Scholar
- Plowe CV, Rooper C, Barnwell JW, Happi CT, Joshi HH, Mbacham W, et al. World Antimalarial Resistance Network (WARN) III: molecular markers for drug resistant malaria. Malar J. 2007;6:121.View ArticlePubMed CentralPubMedGoogle Scholar
- Briolant S, Baragatti M, Parola P, Simon F, Tall A, Sokhna C, et al. Multinormal in vitro distribution model suitable for the distribution of Plasmodium falciparum chemosusceptibility to doxycycline. Antimicrob Agents Chemother. 2009;53:688–95.View ArticlePubMed CentralPubMedGoogle Scholar
- Gaillard T, Briolant S, Houzé S, Baragatti M, Wurtz N, Hubert V, et al. PftetQ and pfmdt copy numbers as predictive molecular markers of decreased ex vivo doxycycline susceptibility in imported Plasmodium falciparum malaria. Malar J. 2013;12:414.View ArticlePubMed CentralPubMedGoogle Scholar
- Divo AA, Geary TG, Jensen JB. Oxygen- and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in culture. Antimicrob Agents Chemother. 1985;27:21–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Pascual A, Basco LK, Baret E, Amalvict R, Travers D, Rogier C, et al. Use of the atmospheric generators for capnophilic bacteria Genbag-CO2 for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs. Malar J. 2011;10:8.View ArticlePubMed CentralPubMedGoogle Scholar
- Fall B, Diawara S, Sow K, Baret E, Diatta B, Fall KB, et al. Ex vivo susceptibility of Plasmodium falciparum isolates from Dakar, Senegal, to seven standard anti-malarial drugs. Malar J. 2011;10:310.View ArticlePubMed CentralPubMedGoogle Scholar
- Wein S, Mynadier M, Tran Van Ba C, Cerdan R, Peyrottes S, Fraisse L, et al. Reliability of antimalarial sensitivity tests depends on drug mechanisms of action. J Clin Microbiol. 2010;48:1651–60.View ArticlePubMed CentralPubMedGoogle Scholar
- Pradines B, Rogier C, Fusai T, Mosnier J, Daries W, Barret E, et al. In vitro activities of antibiotics against Plasmodium falciparum are inhibited by iron. Antimicrob Agents Chemother. 2001;45:1746–50.View ArticlePubMed CentralPubMedGoogle Scholar
- Sidhu ABS, Sun Q, Nkrumah LJ, Dunne MW, Sacchettini JC, Fidock DA. In vitro efficacy, resistance selection, and structural modeling studies implicate the malarial parasite apicoplast as the target of azithromycin. J Biol Chem. 2007;282:2494–504.View ArticlePubMedGoogle Scholar
- Briolant S, Parola P, Fusai T, Madamet-Torrentino M, Baret E, Mosnier J, et al. Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of standardized in vitro assay. Malar J. 2007;6:44.View ArticlePubMed CentralPubMedGoogle Scholar
- Pradines B, Tall A, Fusai T, Spiegel A, Hienne R, Rogier C, et al. In vitro activities of benflumetol against 158 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs. Antimicrob Agents Chemother. 1999;43:418–20.PubMed CentralPubMedGoogle Scholar
- Muangnoicharoen S, Johnson DJ, Looareesuwan S, Krudsood S, Ward SA. Role of known molecular markers of resistance in the antimalarial potency of piperaquine and dihydroartemisinin in vitro. Antimicrob Agents Chemother. 2009;53:1362–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Hofer S, Brun R, Maerki S, Matile H, Scheurer C, Wittlin S. In vitro assessment of the pharmacodynamics properties of DB75, piperaquine, OZ277 and OZ401 in cultures of Plasmodium falciparum. J Antimicrob Chemother. 2008;62:1061–4.View ArticlePubMedGoogle Scholar
- Tinto H, Bonkian LN, Nana LA, Yerbanga I, Lingani M, Kazienga A, et al. Ex vivo anti-malarial drugs sensitivity profile of Plasmodium falciparum field isolates from Burkina Faso five years after the national policy change. Malar J. 2014;13:207.View ArticlePubMed CentralPubMedGoogle Scholar
- Tyner SD, Lon C, Se Y, Bethell D, Socheat D, Noedl H, et al. Ex vivo drug sensitivity profiles of Plasmodium falciparum field isolates from Cambodia and Thailand, 2005 to 2010, determined by a histidine-rich protein assay. Malar J. 2012;11:198.View ArticlePubMed CentralPubMedGoogle Scholar
- Sawa P, Shekalaghe SA, Drakeley CJ, Sutherland CJ, Mweresa CK, Baidjoe AY, et al. Malaria transmission after artemether-lumefantrine and dihydroartemisinin-piperaquine: A randomized trial. J Infect Dis. 2013;207:1637–45.View ArticlePubMedGoogle Scholar
- Onyamboko MA, Fanello CI, Wongsaen K, Tarning J, Cheah PY, Tshefu KA, et al. Randomized comparison of the efficacies and tolerabilities of three artemisinin-based combination treatments for children with acute Plasmodium falciparum malaria in the Democratic Republic of the Congo. Antimicrob Agents Chemother. 2014;58:5528–36.View ArticlePubMed CentralPubMedGoogle Scholar
- Wanzira H, Kakuru A, Arinaitwe E, Bigira V, Muhindo MK, Conrad M, et al. Longitudinal outcomes in a cohort of Ugandan children randomized to artemether-lumefantrine versus dihydroartemisinin-piperaquine for the treatment of malaria. Clin Infect Dis. 2014;59:509–16.View ArticlePubMedGoogle Scholar
- Naing C, Racloz V, Whittaker MA, Aung K, Reid SA, Mak JW, et al. Efficacy and safety of dihydroartemisinin-piperaquine for treatment of Plasmodium vivax malaria in endemic countries: meta-analysis of randomized controlled studies. PLoS One. 2013;8:78819.View ArticleGoogle Scholar
- Henrich PP, O’Brien C, Sáenz FE, Cremers S, Kyle DE, Fidock DA. Evidence for pyronaridine as a highly effective partner drug for treatment of artemisinin-resistant malaria in a rodent model. Antimicrob Agents Chemother. 2014;58:183–95.View ArticlePubMed CentralPubMedGoogle Scholar
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