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International audience; Background: 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 cutoff for reduced susceptibility for the two anti-malarial drugs. Methods: The distribution and range of the 50% inhibitory concentration values (IC 50) 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. Results: The PND IC 50 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 IC 50 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 IC 50 values greater than 60 nM or four-fold greater than 3D7 IC 50 are considered isolates that have reduced susceptibility to PND and those with IC 50 values greater than 135 nM or 2.3-fold greater than 3D7 IC 50 are considered isolates that have reduced susceptibility to PPQ. Conclusion: The existence of at least three phenotypes for PND and four phenotypes for PPQ was demonstrated. Based on the cutoff values, 18 isolates (5.8%) and 13 isolates (4.2%) demonstrated reduced susceptibility to PND and PPQ, respectively.

Background Dihydroartemisinin-piperaquine is a new ACT that is administered as single daily dose for three days and has been demonstrated to be tolerated and highly effective for the treatment of uncomplicated Plasmodium falciparum malaria. Piperaquine was used alone to replace chloroquine as the first-line treatment for uncomplicated malaria in China in response to increasing chloroquine resistance in the 1970s. However, the rapid emergence of piperaquine-resistant strains that resulted in the cessation of its use in China in the 1980s, suggests that there is cross-resistance between piperaquine and chloroquine. Very few data are available on cross-resistance between piperaquine and chloroquine, and the data that do exist are often contradictory. Methods In total, 280 P. falciparum isolates, collected between April 2008 and June 2012 from patients hospitalized in France with imported malaria from a malaria-endemic country, were assessed ex vivo for piperaquine and chloroquine susceptibilities by using the standard 42-hour 3H-hypoxanthine uptake inhibition method. The chloroquine resistance-associated mutation K76T in pfcrt was also investigated for the 280 isolates. Results The IC50 for piperaquine ranged from 9.8 nM to 217.3 nM (mean = 81.3 nM. The IC50 for chloroquine ranged from 5.0 nM to 1,918 nM (mean = 83.6 nM. A significant but low correlation was observed between the Log IC50 values for piperaquine and chloroquine (r = 0.145, p Pfcrt K76 wild-type group (no = 125) and 87.7 nM in the 76 T mutant group (no = 155). This difference was not significant (p = 0.875, Mann Whitney U test). Conclusions The present work demonstrates that there was no cross-resistance between piperaquine and chloroquine among 280 P. falciparum isolates and that piperaquine susceptibility is not associated with pfcrt, the gene involved in chloroquine resistance. These results confirm the efficacy of piperaquine in association with dihydroartemisinin and support its use in areas in which parasites are resistant to chloroquine.

Background Quinine is still recommended as an effective therapy for severe cases of Plasmodium falciparum malaria, but the parasite has developed resistance to the drug in some cases. Investigations into the genetic basis for quinine resistance (QNR) suggest that QNR is complex and involves several genes, with either an additive or a pairwise effect. The results obtained when assessing one of these genes, the plasmodial Na+/H+ exchanger, Pfnhe-1, were found to depend upon the geographic origin of the parasite strain. Most of the associations identified have been made in Asian strains; in contrast, in African strains, the influence of Pfnhe on QNR is not apparent. However, a recent study carried out in Kenya did show a significant association between a Pfnhe polymorphism and QNR. As genetic differences may exist across the African continent, more field data are needed to determine if this association exists in other African regions. In the present study, association between Pfnhe and QNR is investigated in a series of isolates from central Africa. Methods The sequence analysis of the polymorphisms at the Pfnhe-1 ms4760 microsatellite and the evaluation of in vitro quinine susceptibility (by isotopic assay) were conducted in 74 P. falciparum isolates from the Republic of Congo. Results Polymorphisms in the number of DNNND or NHNDNHNNDDD repeats in the Pfnhe-1 ms4760 microsatellite were not associated with quinine susceptibility. Conclusions The polymorphism in the microsatellite ms4760 in Pfnhe-1 that cannot be used to monitor quinine response in the regions of the Republic of Congo, where the isolates came from. This finding suggests that there exists a genetic background associated with geographic area for the association that will prevent the use of Pfnhe as a molecular marker for QNR. The contribution of Pfnhe to the in vitro response to quinine remains to be assessed in other regions, including in countries with different levels of drug pressure.

Background The aim of this study was to evaluate the cultivation system in which the proper atmospheric conditions for growing Plasmodium falciparum parasites were maintained in a sealed bag. The Genbag® system associated with the atmospheric generators for capnophilic bacteria Genbag CO2® was used for in vitro susceptibility test of nine standard anti-malarial drugs and compared to standard incubator conditions. Methods The susceptibility of 36 pre-identified parasite strains from a wide panel of countries was assessed for nine standard anti-malarial drugs (chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone and pyrimethamine) by the standard 42-hour 3H-hypoxanthine uptake inhibition method using the Genbag CO2® system and compared to controlled incubator conditions (5% CO2 and 10% O2). Results The counts per minute values in the control wells in incubator atmospheric conditions (5% CO2 and 10% O2) were significantly higher than those of Genbag® conditions (2738 cpm vs 2282 cpm, p < 0.0001). The geometric mean IC50 estimated under the incubator atmospheric conditions was significantly lower for atovaquone (1.2 vs 2.1 nM, p = 0.0011) and higher for the quinolines: chloroquine (127 vs 94 nM, p < 0.0001), quinine (580 vs 439 nM, p < 0.0001), monodesethylamodiaquine (41.4 vs 31.8 nM, p < 0.0001), mefloquine (57.5 vs 49.7 nM, p = 0.0011) and lumefantrine (23.8 vs 21.2 nM, p = 0.0044). There was no significant difference of IC50 between the 2 conditions for dihydroartemisinin, doxycycline and pyrimethamine. To reduce this difference in term of anti-malarial susceptibility, a specific cut-off was estimated for each drug under Genbag® conditions by regression. The cut-off was estimated at 77 nM for chloroquine (vs 100 nM in 10% O2), 611 nM for quinine (vs 800 nM), 30 nM for mefloquine (vs 30 nM), 61 nM for monodesethylamodiaquine (vs 80 nM) and 1729 nM for pyrimethamine (vs 2000 nM). Conclusions The atmospheric generators for capnophilic bacteria Genbag CO2® is an appropriate technology that can be transferred to the field for epidemiological surveys of drug-resistant malaria. The present data suggest the importance of the gas mixture on in vitro microtest results for anti-malarial drugs and the importance of determining the microtest conditions before comparing and analysing the data from different laboratories and concluding on malaria resistance.

Background The aim of the present work was to assess the in vitro cross-resistance of pyronaridine with other quinoline drugs, artesunate and several other commonly used anti-malarials and to evaluate whether decreased susceptibility to pyronaridine could be associated with genetic polymorphisms in genes involved in reduced quinoline susceptibility, such as pfcrt, pfmdr1, pfmrp and pfnhe. Methods The in vitro chemosusceptibility profiles of 23 strains of Plasmodium falciparum were analysed by the standard 42-hour 3H-hypoxanthine uptake inhibition method for pyronaridine, artesunate, chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine and doxycycline. Genotypes were assessed for pfcrt, pfmdr1, pfnhe-1 and pfmrp genes. Results The IC50 values for pyronaridine ranged from 15 to 49 nM (geometric mean = 23.1 nM). A significant positive correlation was found between responses to pyronaridine and responses to artesunate (r2 = 0.20; P = 0.0317) but too low to suggest cross-resistance. No significant correlation was found between pyronaridine IC50 and responses to other anti-malarials. Significant associations were not found between pyronaridine IC50 and polymorphisms in pfcrt, pfmdr1, pfmrp or pfnhe-1. Conclusion There was an absence of cross-resistance between pyronaridine and quinolines, and the IC50 values for pyronaridine were found to be unrelated to mutations in the transport protein genes pfcrt, pfmdr1, pfmrp or pfnhe-1, known to be involved in quinoline resistance. These results confirm the interest and the efficacy of the use of a combination of pyronaridine and artesunate in areas in which parasites are resistant to quinolines.

Background Quinine (QN) remains the first line anti-malarial drug for the treatment of complicated malaria in Europe and Africa. The emergence of QN resistance has been documented. QN resistance is not yet a significant problem, but there is an urgent need to discover partners for use in combination with QN. The aim of the study was to assess the in vitro potentiating effects of atorvastatin (AVA), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with QN against Plasmodium falciparum and to evaluate whether the effects of AVA could be associated with gene copy number or mutations in genes involved in QN resistance, such as pfcrt, pfmdr1, pfmrp and pfnhe. Methods The susceptibilities to combination of AVA with QN were assessed against 21 parasite strains using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfcrt, pfmdr1, pfmdr2, pfmrp genes. In addition, the number of DNNND, DDNHNDNHNN repeats in pfnhe-1 ms4760 and the ms4760 profile were determined for each strains of P. falciparum. Results AVA demonstrated synergistic effects in combination with QN against 21 P. falciparum strains. The QN IC50 was reduced by 5% (0% to 15%; 95%CI: 1%-8%), 10% (3% to 23%; 95%CI: 7%-14%) and 22% (14% to 40%; 95%CI: 19%-25%) in presence of AVA at concentrations of 0.1, 0.5 and 1.0 μM, respectively. These reductions were all significant (p < 0.009). The reduction in the QN IC50 in presence of AVA was not significantly correlated with the QN IC50 (r = 0.22, P = 0.3288) or the AVA IC50 (r = 0.03, P = 0.8946). The synergistic effect of AVA in combination with QN was not significantly associated with polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes that could be involved in QN resistance. The synergistic effect of AVA on QN responses was not significantly associated with pfmdr1 copy number (P = 0.0428). Conclusion The synergistic effect of AVA in combination with QN was found to be unrelated to mutations occurring in transport protein genes involved in QN drug resistance. The different mechanisms of drug uptake and/or mode of action for AVA compared to the other anti-malarial drugs, as well as the AVA-mediated synergy of the anti-malarial effect of QN, suggests that AVA will be a good candidate for combinatorial malaria treatment. All of these observations support calls for both an in vivo evaluation with pharmacokinetic component and clinical trials of AVA as an anti-malarial therapy.

Background Malaria still has significant impacts on the world; particularly in Africa, South America and Asia where spread over several millions of people and is one of the major causes of death. When chloroquine diphosphate (CQDP) lost its efficiency as a first-line anti-malarial drug, this was a major setback in the effective control of malaria. Currently, malaria is treated with a combination of two or more drugs with different modes of action to provide an adequate cure rate and delay the development of resistance. Clearly, a new effective and non-toxic anti-malarial drug is urgently needed. Methods All metal-chloroquine (CQ) and metal-CQDP complexes were synthesized under N2 using Schlenk techniques. Their interactions with haematin and the inhibition of β-haematin formation were examined, in both aqueous medium and near water/n-octanol interfaces at pH 5. The anti-malarial activities of these metal- CQ and metal-CQDP complexes were evaluated in vitro against two strains, the CQ-susceptible strain (CQS) 3D7 and the CQ-resistant strain (CQR) W2. Results The previously synthesized Au(CQ)(Cl) (1), Au(CQ)(TaTg) (2), Pt(CQDP)2Cl2 (3), Pt(CQDP)2I2 (4), Pd(CQ)2Cl2 (5) and the new one Pd(CQDP)2I2 (6) showed better anti-malarial activity than CQ, against the CQS strain; moreover, complexes 2, 3 and 4 were very active against CQR strain. These complexes (1–6) interacted with haem and inhibited β-haematin formation both in aqueous medium and near water/n-octanol interfaces at pH 5 to a greater extent than chloroquine diphosphate (CQDP) and other known metal-based anti-malarial agents. Conclusions The high anti-malarial activity displayed for these metal-CQ and metal-CQDP complexes (1–6) could be attributable to their effective interaction with haem and the inhibition of β-haematin formation in both aqueous medium and near water/n-octanol interfaces at pH 5. Electronic supplementary material The online version of this article (doi:10.1186/1475-2875-13-471) contains supplementary material, which is available to authorized users.

Background There is an urgent need for the discovery of new anti-malarial drugs and combination therapy. A combinatorial approach protects each drug from the development of resistance and reduces generally the overall transmission rate of malaria. Statins, the inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and a family of lipid-lowering drugs, have in vitro anti-malarial properties, and more specially atorvastatin. However, atorvastatin has a short elimination half-life (14 hours) and an efficient combination of anti-malarial drugs must associate a drug with a short elimination half-life and a drug with a long elimination half-life. The objective of the present work was to identify new potential partners among standard new anti-malarial drugs with long elimination half-life, such as lumefantrine, piperaquine, pyronaridine and atovaquone, to improve the in vitro activity of atorvastatin against different Plasmodium falciparum strains to treat uncomplicated malaria. Methods In vitro interaction of atorvastatin in combination with lumefantrine, piperaquine, pyronaridine and atovaquone was assessed against 13 P. falciparum strains by isotopic test. Results Atorvastatin showed additive effects with pyronaridine, piperaquine and lumefantrine. Atorvastatin increased the in vitro activity of lumefantrine and piperaquine at concentrations expected in clinical observations. The average IC50 values of lumefantrine decreased significantly from 31.9 nM to 20.5 nM (a decrease of 35.7%) in combination with 1 μM of atorvastatin. Conclusions Even though in vitro data indicate that atorvastatin improved the activity of lumefantrine and piperaquine, the same may not necessarily be true in vivo. Piperaquine, a new drug with long terminal elimination half-life, is currently a very promising anti-malarial drug.