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International audience; In our ongoing search for bioactive compounds, a class of novel spiro-β-lactam isatin hybrids has been synthesized through a [2 + 2] cycloaddition reaction from 1-allyl-3-(arylimino)indolin-2-one, ketenes and various aryloxy acetic acids. The formation of all cycloadducts was confirmed by FTIR, 1H NMR, 13C NMR, and mass spectroscopy as well as elemental analyses. The new β-lactams were subsequently evaluated for their biological activities demonstrating moderate to good activities against P. falciparum K1 strain. Among them, 4b and 4e lead to the best results with IC50 of 5.04 and 7.18 µM, respectively. The molecular docking simulation of 4b with P. falciparum dihydrofolate reductase enzyme (PfDHFR) binding site presented several important intermolecular interactions. All the synthesized β-lactams were also evaluated for their antimicrobial activities against both Gram-positive (S. aureus ATCC 25923) and Gram-negative bacteria (E. coli ATCC 28922, P. aeruginosa ATCC 27853) but unfortunately MICs up to 200 µg/mL were encountered in all cases.

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

The level of human group IIA secreted phospholipase A2 (hGIIA sPLA2) is increased in the plasma of malaria patients, but its role is unknown. In parasite culture with normal plasma, hGIIA is inactive against Plasmodium falciparum, contrasting with hGIIF, hGV, and hGX sPLA2s, which readily hydrolyze plasma lipoproteins, release nonesterified fatty acids (NEFAs), and inhibit parasite growth. Here, we revisited the anti-Plasmodium activity of hGIIA under conditions closer to those of malaria physiopathology where lipoproteins are oxidized. In parasite culture containing oxidized lipoproteins, hGIIA sPLA2 was inhibitory, with a 50% inhibitory concentration value of 150.0 ± 40.8 nM, in accordance with its capacity to release NEFAs from oxidized particles. With oxidized lipoproteins, hGIIF, hGV, and hGX sPLA2s were also more potent, by 4.6-, 2.1-, and 1.9-fold, respectively. Using specific immunoassays, we found that hGIIA sPLA2 is increased in plasma from 41 patients with malaria over levels for healthy donors (median [interquartile range], 1.6 [0.7 to 3.4] nM versus 0.0 [0.0 to 0.1] nM, respectively; P < 0.0001). Other sPLA2s were not detected. Malaria plasma, but not normal plasma, contains oxidized lipoproteins and was inhibitory to P. falciparum when spiked with hGIIA sPLA2 Injection of recombinant hGIIA into mice infected with P. chabaudi reduced the peak of parasitemia, and this was effective only when the level of plasma peroxidation was increased during infection. In conclusion, we propose that malaria-induced oxidation of lipoproteins converts these into a preferential substrate for hGIIA sPLA2, promoting its parasite-killing effect. This mechanism may contribute to host defense against P. falciparum in malaria where high levels of hGIIA are observed.

Fifteen novel β-lactams bearing N-ethyl tert-butyl carbamate group 5a-o and fifteen N-(2- aminoethyl) β-lactams 6a-o were synthesized by [2+2] ketene-imine cycloaddition reaction (Staudinger). The cycloaddition reaction was found to be totally diastereoselective leading exclusively to theformation of cis-β-lactam derivatives. These newly synthesized β-lactams were evaluated for their antimalarial activity against p. falciparum K14 resistant strain and showed good to excellent EC50 values. Of the thirty β-lactams tested, 5 h, 6a and 6c showed IC50 < 20 µM while 5b, 5c, 5e, 5f, 5g, 5i, 5j, 6d, 6g and 6h exhibited IC50

The level of human group IIA secreted phospholipase A(2) (hGIIA sPLA(2)) is increased in the plasma of malaria patients, but its role is unknown. In parasite culture with normal plasma, hGIIA is inactive against Plasmodium falciparum, contrasting with hGIIF, hGV, and hGX sPLA(2)s, which readily hydrolyze plasma lipoproteins, release nonesterified fatty acids (NEFAs), and inhibit parasite growth. Here, we revisited the anti-Plasmodium activity of hGIIA under conditions closer to those of malaria physiopathology where lipoproteins are oxidized. In parasite culture containing oxidized lipoproteins, hGIIA sPLA(2) was inhibitory, with a 50% inhibitory concentration value of 150.0 ± 40.8 nM, in accordance with its capacity to release NEFAs from oxidized particles. With oxidized lipoproteins, hGIIF, hGV, and hGX sPLA(2)s were also more potent, by 4.6-, 2.1-, and 1.9-fold, respectively. Using specific immunoassays, we found that hGIIA sPLA(2) is increased in plasma from 41 patients with malaria over levels for healthy donors (median [interquartile range], 1.6 [0.7 to 3.4] nM versus 0.0 [0.0 to 0.1] nM, respectively; P < 0.0001). Other sPLA(2)s were not detected. Malaria plasma, but not normal plasma, contains oxidized lipoproteins and was inhibitory to P. falciparum when spiked with hGIIA sPLA(2). Injection of recombinant hGIIA into mice infected with P. chabaudi reduced the peak of parasitemia, and this was effective only when the level of plasma peroxidation was increased during infection. In conclusion, we propose that malaria-induced oxidation of lipoproteins converts these into a preferential substrate for hGIIA sPLA(2), promoting its parasite-killing effect. This mechanism may contribute to host defense against P. falciparum in malaria where high levels of hGIIA are observed.

Some new β-lactams bearing biologically important morpholine ring have been synthesized by acylation of amino β-lactams in the presence of morpholine-4-carbonyl chloride. These novel β-lactams were prepared under mild reaction conditions without any solvent in short reaction times. Their biological activities have been examined against microbial agents such as Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and fungi such as Candida albicans (C. albicans) and Candida glabrata (C. glabrata). They have been also tested against Plasmodium falciparum K14 resistant strain and showed moderate to good IC50 values.

International audience; Malaria is highly endemic in Umphang Valley, a district in the western edge of Tak Province, along the boundary with Kayin State of Myanmar. Although there are high indigenous malaria cases in this area every year, nothing about malaria vectors and their transmission role have been investigated before this study. The objective of this work is to characterize the Anopheles species diversity and trophic behavior of malaria vectors in the transmission area of Umphang Valley. Females of Anopheles mosquitoes were collected every two months during a two-year period. Mosquito collections were using standard collection technique, indoor and outdoor human landing collections and outdoor cattle bait collection. Anopheles mosquitoes were identified using morphological characters and multiplex AS-PCR assay for the identification of sibling species within groups and complexes present. From a total of 16,468 Anopheles females, 2723 specimens (16.54%) were collected from humans and 13,745 specimens (83.46%) were captured from cattle. From human landing collections, 2447 specimens (89.86%) of Anopheles minimus were obtained, followed by 119 Anopheles peditaeniatus (4.37%), 62 Anopheles maculatus (2.28%), 17 Anopheles dirus (0.6%), 15 Anopheles aconitus (0.5%) and 6 Anopheles sawadwongporni (0.2%) respectively. Seven putative malaria vectors, including An. minimus, An. dirus, An. baimaii, An. sawadwongporni, An. maculatus, An. pseudowillmori and An. aconitus were documented from this study and trophic behavior of each respective species were observed. Such information is definitely crucial for defining the vector capacity of each single species and for designing appropriate vector prevention and control strategies against target vector species.

International audience; Aims: Majority of deaths in children aged under 5 years are due to Plasmodium falciparum malaria. Malaria deaths in children decreased but malaria remains a major killer of children, taking the life of a child every 2 minutes. This study aims to investigate the increasing of the in vitro antiplasmodial activities by mutagenesis techniques using gamma-rays (Cs-137) or sodium azide (NaN3) as utagens. It will allow the importance of mutagenesis use as tools for improvement of secondary metabolites against malaria parasites using chemical or physical mutagens.Study Design: Laboratory experiment tests : identification of plant material, immersion of seeds in SA (sodium azide) solutions or irradiation by Gamma-rays (Cs-137) of P. odontadenius seeds for improvement of secondary metabolites against malaria parasites, in vitro culture of seeds followed by the in situ culturing of plantlets for obtaining material of study, phytochemical screening of Phyllanthus odontadenius aerial parts to determine the change of compounds in comparison to controls, in vitro antiplasmodial tests for the determination of SA concentrations or those of gammarays doses which killing 50% of malaria parasite populations (IC50). Place and Duration of Study: Department of Biotechnology and Molecular Biology, Department of Biochemistry: General Atomic Energy Commission, Regional Nuclear Studies Center of Kinshasa, P.O. Box. 868 Kinshasa XI, Democratic Republic of the Congo (DRC). National Institute of Biomedical Research (NIBR) at Kinshasa/Gombe (DRC). Laboratory of UMR-MD3, Aix-Marseille University, Faculty of Pharmacy/Marseille, France. The experiments were conducted from Junuary - December 2010; August and December 2011 and during May and September 2012 until July 2014.Methodology: Aerial parts of plants M1 and M2 from Gamma-rays irradiation of P. odontadenius seeds or from immersion of P. odontadenius seeds in sodium azide solutions used as biological material for the in vitro antiplasmodial analysis. The in vitro antiplasmodial activities assays on clinical isolates of P. falciparum and on chloroquine-resistante P. falciparum strain K1 was determined using microscopic method, the isotopic micro-test method and using HRP2-based ELISA assay.Results: Gamma-rays (Cs-137) increased (multiplied) the in vitro antiplasmodial activities from 2.48 up to 7.6 in comparison to control. Thus, the in vitro antiplasmodial activities were improved or exceeded from 147.57% up to 660% than those of control plant. SA had increased (multiplied) the in vitro antiplasmodial activities from 1.24 up to 10.15 comparing to the control plants. The antiplasmodial activities in vitro were exceeded compared to the control plants from 24.43% up to 915%. Conclusion: Treatment of P. odontadenius seeds by Gamma-rays or by SA give plants high in vitro antiplasmodial activities. Values of in vitro antiplasmodial activities varried from 1.24 (147.57%) to 10.15 (915%). 125, 150 and 225 Gy of Gamma-rays (Cs-137) for physical mutagenesis and 10.15 and 17.5 M of SA solutions for the chemical mutagenesis could be used for improving in vitro antiplasmodial activities against P. falciparum (clinical isolates of P. falciparum or chloroquine-resistant P. falciparum strain K1). Thus, plants extracts from treated seeds have justified the usefulness of mutagens in plant breeding particularly in the increasing production of secondary metabolite against malaria parasite.

Despite significant progress in prevention and therapy, malaria is still one of the world's leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL(-1). This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL(-1) and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103%, with coefficients of variation of

Background In 2005, the Democratic Republic of the Congo (DRC) switched to artesunate/amodiaquine as the first-line antimalarial in response to increasing sulfadoxine/pyrimethamine resistance and adopted intermittent preventive treatment using sulfadoxine/pyrimethamine in pregnancy. Objectives To determine the prevalence of molecular markers of sulfadoxine/pyrimethamine resistance in southwestern DRC 10 years after the new policy was instituted. Methods From March 2014 to December 2015, blood samples were collected from symptomatic patients presenting to outpatient centres in urban and rural areas. A total of 2030 confirmed Plasmodium falciparum isolates were genotyped at codons associated with sulfadoxine/pyrimethamine resistance. Results The prevalence of pfdhfr-N51I, C59R and S108N and pfdhps-A437G mutations was consistently high; the prevalence of the pfdhps-K540E mutation was low but increased since its first report in 2008 in the same region, reaching 17.6% by 2015. The pfdhps-A581G mutation increased from ∼4.5% in 2014 to ∼14.0% in 2015 at urban sites while in rural areas it remained low (∼4.0%). The mutations pfdhfr-I164L and pfdhps-A613S were detected for the first time in DRC. Also, 11 (0.8%) isolates revealed the presence of the newly described pfdhps-I431V mutation. Combining pfdhfr and pfdhps alleles, quintuple and sextuple mutations were observed, with the emergence of septuple (IRNI/IAGEGA)- and octuple (IRNI/VAGKGS)-mutant genotypes. Conclusions Intermittent preventive treatment using sulfadoxine/pyrimethamine during pregnancy remains warranted in southwestern DRC. However, the expansion of pfdhps-K540E mutation and emergence of mutants that cause higher levels of sulfadoxine/pyrimethamine resistance is concerning and may present a challenge for future preventive interventions in the country.

The new RuII chloroquine complexes [Ru(η6-arene)(CQ)Cl2] (CQ = chloroquine; arene = p-cymene 1, benzene 2), [Ru(η6-p-cymene)(CQ)(H2O)2][BF4]2 (3), [Ru(η6-p-cymene)(CQ)(en)][PF6]2 (en = ethylenediamine) (4), and [Ru(η6-p-cymene)(η6-CQDP)][BF4]2 (5, CQDP = chloroquine diphosphate) have been synthesized and characterized by use of a combination of NMR and FTIR spectroscopy with DFT calculations. Each complex is formed as a single coordination isomer: in compounds 1–4 chloroquine binds to ruthenium in the η1-N mode through the quinoline nitrogen atom whereas in complex 5 an unprecedented η6 bonding through the carbocyclic ring is observed. Compounds 1, 2, 3, and 5 are active against CQ-resistant (Dd2, K1 and W2) and CQ-sensitive (FcB1, PFB, F32 and 3D7) malaria parasites (Plasmodium falciparum); importantly, the potency of these complexes against resistant parasites is consistently higher than that of the standard drug chloroquine diphosphate. Complexes 1 and 5 also inhibit the growth of colon cancer cells, independently of the p53 status and of liposarcoma tumor cell lines with the latter showing increased sensitivity, especially to complex 1 (IC50 8 µM); this is significant because this type of tumor does not respond to currently employed chemotherapies.

Background Artesunate-amodiaquine (AS-AQ) is one of the most widely used artemisinin-based combination therapies (ACTs) to treat uncomplicated Plasmodium falciparum malaria in Africa. We investigated the impact of different dosing strategies on the efficacy of this combination for the treatment of falciparum malaria. Methods Individual patient data from AS-AQ clinical trials were pooled using the WorldWide Antimalarial Resistance Network (WWARN) standardised methodology. Risk factors for treatment failure were identified using a Cox regression model with shared frailty across study sites. Results Forty-three studies representing 9,106 treatments from 1999-2012 were included in the analysis; 4,138 (45.4%) treatments were with a fixed dose combination with an AQ target dose of 30 mg/kg (FDC), 1,293 (14.2%) with a non-fixed dose combination with an AQ target dose of 25 mg/kg (loose NFDC-25), 2,418 (26.6%) with a non-fixed dose combination with an AQ target dose of 30 mg/kg (loose NFDC-30), and the remaining 1,257 (13.8%) with a co-blistered non-fixed dose combination with an AQ target dose of 30 mg/kg (co-blistered NFDC). The median dose of AQ administered was 32.1 mg/kg [IQR: 25.9-38.2], the highest dose being administered to patients treated with co-blistered NFDC (median = 35.3 mg/kg [IQR: 30.6-43.7]) and the lowest to those treated with loose NFDC-25 (median = 25.0 mg/kg [IQR: 22.7-25.0]). Patients treated with FDC received a median dose of 32.4 mg/kg [IQR: 27-39.0]. After adjusting for reinfections, the corrected antimalarial efficacy on day 28 after treatment was similar for co-blistered NFDC (97.9% [95% confidence interval (CI): 97.0-98.8%]) and FDC (98.1% [95% CI: 97.6%-98.5%]; P = 0.799), but significantly lower for the loose NFDC-25 (93.4% [95% CI: 91.9%-94.9%]), and loose NFDC-30 (95.0% [95% CI: 94.1%-95.9%]) (P

International audience; The efficacy of chloroquine, once the drug of choice in the fight against Plasmodium falcipa-rum, is now severely limited due to widespread resistance. Amodiaquine is one of the most potent antimalarial 4-aminoquinolines known and remains effective against chloroquine-resistant parasites, but toxicity issues linked to a quinone-imine metabolite limit its clinical use. In search of new compounds able to retain the antimalarial activity of amodiaquine while circumventing quinone-imine metabolite toxicity, we have synthesized five 4-amino-quinolines that feature rings lacking hydroxyl groups in the side chain of the molecules and are thus incapable of generating toxic quinone-imines. The new compounds displayed high in vitro potency (low nanomolar IC 50), markedly superior to chloroquine and comparable to amodiaquine, against chloroquine-sensitive and chloroquine-resistant strains of P. falcipa-rum, accompanied by low toxicity to L6 rat fibroblasts and MRC5 human lung cells, and metabolic stability comparable or higher than that of amodiaquine. Computational studies indicate a unique mode of binding of compound 4 to heme through the HOMO located on a biphenyl moeity, which may partly explain the high antiplasmodial activity observed for this compound.

Artesunate (ARS) is a water-soluble artemisinin derivative that is a potential alternative to quinine for the treatment of severe childhood malaria. We studied the pharmacokinetics and bioavailability of ARS given by the intramuscular (i.m.) route in an open crossover study design. Fourteen children were randomized to receive intravenous (i.v.) ARS in a loading dose (2.4 mg/kg of body weight) followed 12 h later by an i.m. dose (1.2 mg/kg) (group I), and 14 children were randomized to receive i.m. ARS (2.4 mg/kg) followed by an i.v. dose of ARS (1.2 mg/kg) (group II). We carried out a two-compartment analysis of ARS and dihydroartemisinin (DHA; the principal antimalarial metabolite) levels in 21 children (groups I and II combined). Absorption of i.m. ARS was rapid, with the maximum concentration of DHA in serum being achieved in less than 1 h in most children (median time to the maximum concentration of drug in serum, 35.1 min; range, 10.8 to 71.9 min). The absolute bioavailability of DHA was a median of 86.4% (range, 11.4 to 462.1%), the median steady-state volume of distribution was 1.3 liters/kg (range, 0.5 to 7.9 liters/kg), and the median clearance was 0.028 liters/kg/min (range, 0.001 to 1.58 liters/kg/min). There were no major adverse events attributable to ARS. Parasite clearance kinetics were comparable between the two treatment groups. These results support the use of i.m. ARS in children with severe malaria.

Background: Human malaria is still a burden in Dak Nong and Binh Phuoc Provinces in south-central Vietnam that border Cambodia. Several Anopheles species that transmit human malarial Plasmodium may also transmit Wuchereria bancrofti, the nematode that causes Bancroftian lymphatic filariasis. The objective of this study was to investigate the role of Anopheles species in the transmission of these two pathogens in the two highly malaria endemic provinces of Vietnam. Methods: Anopheles mosquitoes were collected in Dak Nong and Binh Phuoc Provinces in November and December of 2010 and 2011. Human landing catches, paired collections on human and buffalo, and resting captures were made with mouth aspirators. Collections were also made with light traps. Morphological and PCR-based methods were used to identify the species. Real-time PCR was used to detect Plasmodium species and W. bancrofti in individual mosquitoes. Results: Twenty-four Anopheles species were identified among 797 captured mosquitoes. Anopheles dirus was found in both provinces and was the predominant species in Binh Phuoc Province; An. maculatus was the most prevalent species in Dak Nong Province. Anopheles minimus was collected only in Binh Phuoc Province. Some specimens of An. minimus and An. pampanai were misidentified based on morphology. Four specimens of An. scanloni were identified, and this is the first report of this species of the Dirus Complex in Vietnam. Two females, one An. dirus and one An. pampanai, collected in Binh Phuoc Province were infected with P. vivax, for an overall infection rate of 0.41% (2/486): 0.28% for An. dirus (1/361) and 20% for An. pampanai (1/5). No mosquitoes were found to be infected with P. falciparum, P. knowlesi or W. bancrofti in either province. Conclusion: A diversity of Anopheles species occurs in Dak Nong and Binh Phuoc Provinces of Vietnam, several of which are considered to be actual and potential vectors of malarial protozoa and microfilariae. It is highly likely that two of the species, An. dirus and An. pampanai, are active in malaria transmission based on the detection of P. vivax in females of these species. This is the first report of An. scanloni in Vietnam.

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC 50 s) were determined by a modified sigmoid E max model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC 50 s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

Taxotere (docetaxel) inhibits Plasmodium falciparum erythrocytic development in vitro at nanomolar concentrations, both in chloroquine-sensitive (F32/Tanzania) and chloroquine-resistant (FcB1/Colombia, FcR3/Gambia) strains. The dose-response assays performed on asynchronous cultures during 42 hr showed clear biphasic curves with a plateau from 50 microM to 10 nM and a single sigmoid curve with a concentration inhibiting 50% of growth (IC50) of 3-6 nM observed after a 72-hr incubation. Addition of Taxotere to different stages of FcB1 revealed two types of targets: one type on ring/trophozoite-infected erythrocytes (RBCs), at the micromolar level, and another type on schizont-infected RBCs with Taxotere at micromolar concentrations inhibited the merozoite invasion of erythrocytes and parasite growth. These Taxotere-RBC interactions were stable, at least for 1 day. Pulse experiments of 5 hr with Taxotere efficiently inhibit parasite development regardless of the period of the parasite's erythrocytic life cycle. However, different cellular effects were obtained depending upon periods of drug incubations. The inhibition of P. falciparum development by Taxotere should provide additional strategies to block parasite development.

Plasmodium falciparum isolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pf crt and Pf mdr1 genes, the Pf nhe-1 Na + /H + exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pf nhe-1 gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pf nhe-1 , Pf crt , and Pf mdr1 polymorphisms in field- and culture-adapted isolates from various countries with their in vitro susceptibility to quinine. Field isolates presented a higher diversity of the Pf nhe-1 microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pf crt and Pf mdr1 genes, as well as a higher number of DNNND repeats in the Pf nhe-1 gene, were associated with a higher 50% inhibitory concentration (IC 50 ) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pf nhe-1 gene also harbored mutated Pf crt and Pf mdr1 genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pf nhe-1 gene in the modulation of the in vitro quinine response when associated with mutated Pf crt and Pf mdr1 genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pf nhe-1 polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pf nhe-1 polymorphism can be used as a molecular marker for surveillance of quinine resistance.

Marylin Torrentino-Madamet1, Lionel Almeras2, Christelle Travaillé1, Véronique Sinou1, Matthieu Pophillat3, Maya Belghazi4, Patrick Fourquet3, Yves Jammes5, Daniel Parzy11UMR-MD3, Université de la Méditerranée, Antenne IRBA de Marseille (IMTSSA, Le Pharo), 2Unité de Recherche en Biologie et Epidémiologie Parasitaires, Antenne IRBA de Marseille (IMTSSA, Le Pharo), 3Centre d'Immunologie de Marseille Luminy, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de la Méditerranée, 4Centre d'Analyse Protéomique de Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Nord, 5UMR-MD2, Physiologie et Physiopathologie en Conditions d'Oxygénations Extrêmes, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Nord, Marseille, FranceObjectives: Although human respiratory metabolism is characterized by the mitochondrial electron transport chain, some organisms present a “branched respiratory chain.” This branched pathway includes both a classical and an alternative respiratory chain. The latter involves an alternative oxidase. Though the Plasmodium falciparum alternative oxidase is not yet identified, a specific inhibitor of this enzyme, salicylhydroxamic acid (SHAM), showed a drug effect on P. falciparum respiratory function using oxygen consumption measurements. The present study aimed to highlight the metabolic pathways that are affected in P. falciparum following SHAM exposure.Design: A proteomic approach was used to analyze the P. falciparum proteome and determine the metabolic pathways altered following SHAM treatment. To evaluate the SHAM effect on parasite growth, the phenotypic alterations of P. falciparum after SHAM or/and hyperoxia exposure were observed.Results: After SHAM exposure, 26 proteins were significantly deregulated using a fluorescent two dimensional-differential gel electrophoresis. Among these deregulated proteins, some were particularly involved in energetic metabolism. And the combinatory effect of SHAM/hyperoxia seems deleterious for the growth of P. falciparum.Conclusion: Our results indicated that SHAM appears to activate glycolysis and decrease stress defense systems. These data provide a better understanding of parasite biology.Keywords: Plasmodium falciparum, salicylhydroxamic acid, hyperoxia, glycolysis, proteomic

Background The Plasmodium falciparum NA+/H+ exchanger (pfnhe1, gene PF13_0019) has recently been proposed to influence quinine (QN) susceptibility. However, its contribution to QN resistance seems to vary geographically depending on the genetic background of the parasites. Here, the role of this gene was investigated in in vitro QN susceptibility of isolates from Viet Nam. Method Ninety-eight isolates were obtained from three different regions of the Binh Phuoc and Dak Nong bordering Cambodia provinces during 2006-2008. Among these, 79 were identified as monoclonal infection and were genotyped at the microsatellite pfnhe1 ms4760 locus and in vitro QN sensitivity data were obtained for 51 isolates. Parasite growth was assessed in the field using the HRP2 immunodetection assay. Results Significant associations were found between polymorphisms at pfnhe1 microsatellite ms4760 and susceptibility to QN. Isolates with two or more DNNND exhibited much lower susceptibility to QN than those harbouring zero or one DNNND repeats (median IC50 of 682 nM versus median IC50 of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC50 of 704 nM versus median IC50 of 375 nM; p < 0.01). These QNR associated genotype features were mainly due to the over representation of profile 7 among isolates (76.5%). The majority of parasites had pfcrt76T and wild-type pfmdr1 (> 95%) thus preventing analysis of associations with these mutations. Interestingly, area with the highest median QN IC50 showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7. Conclusions The haplotype 7 which is the typical Asian profile is likely well-adapted to high drug pressure in this area and may constitute a good genetic marker to evaluate the dissemination of QNR in this part of the world.

OBJECTIVES The aim was to study the pharmacokinetic profile of artesunate and its metabolite dihydroartemisinin (DHA) in a pig model. METHODS Thirteen pigs received either intravenous (iv) or intramuscular (im) artesunate (60 mg), with the alternative preparation given 24 h later in an open crossover design. Five of them also received an additional intra-arterial (ia) artesunate dose (60 mg). The plasma concentrations of artesunate and DHA were determined by high-performance liquid chromatography with electrochemical detection. Population modelling was performed with NONMEM, using a two-compartment model. RESULTS Plasma concentration-time profiles were comparable to those observed in humans, with a rapid and biphasic decline for both artesunate and DHA. Following an iv bolus, artesunate had a median maximum plasma concentration (C(max)) of 13.8 microM [interquartile range (IQR), 10.4-22.1 microM], elimination half-life (t(1/2)) = 18 min (IQR, 16-22 min), total plasma clearance (CL) = 5.58 L/h/kg (IQR, 3.31-5.91 L/h/kg) and volume of distribution (V(d)) = 1.85 L/kg (IQR, 1.27-3.20 L/kg). The median C(max) value for DHA was 3.30 microM (IQR, 2.08-5.95 microM), t(1/2) = 26 min (IQR, 23-31 min), CL/Fm = 4.37 L/h/kg (IQR, 3.29-6.87 L/h/kg) and V(d)/Fm = 2.56 L/kg (IQR, 1.93-4.49 L/kg). Artesunate and DHA pharmacokinetic parameters were similar after ia administration. Following im dosing, median artesunate C(max) was 4.81 microM (IQR, 3.74-5.40 microM), t(1/2) = 18 min (IQR, 16-28 min), CL = 4.37 L/h/kg (IQR, 4.13-4.68 L/h/kg) and V(d) = 2.07 L/kg (IQR, 1.83-2.79 L/kg); the bioavailability was 100%. For DHA, median C(max) was 1.43 microM (IQR, 1.00-1.92 microM), t(1/2) = 27 min (IQR, 25-37 min), CL/Fm = 4.68 L/h/kg (IQR, 3.35-6.73 L/h/kg) and V(d)/Fm = 3.31 L/kg (IQR, 2.89-4.27 L/kg). CONCLUSIONS The pharmacokinetic properties of artesunate and DHA in pigs were similar to those reported in humans, suggesting that the swine model is suitable for determining the preclinical pharmacokinetics of artemisinin derivatives.

Artemisinin and its derivatives have been used against malaria in Vietnam since 1991 (4). An increase in clinical artemisinin resistance would be disastrous for malaria treatment. All possible indicators of this potential resistance must be monitored. The sarco/endoplasmic reticulum Ca2+-ATPase ortholog of Plasmodium falciparum (PfATP6) has been suggested to be the target of artemisinins (3). Consequently, the polymorphism of PfATP6 is being monitored by several scientific research teams (2, 6, 7, 9, 10, 15) We report here the genotyping results of PfATP6 from 98 P. falciparum field isolates collected in 2006 to 2007 in South Vietnam. Parasite samples were taken from patients (28.82 ± 12.31 years old) with uncomplicated P. falciparum infections before drug treatment. They were collected in Binh Phuoc and Dak Nong provinces in South Vietnam. Patients did not follow a chemoprophylaxis before sampling. Diagnosis was carried out by microscopic examination and confirmed by real-time PCR as previously described (14). The whole PfATP6 gene was sequenced once in both directions with five primer pairs (adapted from Jambou et al. [7]) and compared to the reference sequence of the 3D7 strain (“PFA0310c” in the genome annotation). We found a total of eight mutations (Table ​(Table1):1): four nonsynonymous (I89T, N463S, N465S, and N683K), three synonymous (N460N, I898I, and C1031C), and one double deletion leading to the loss of two asparagines (Δ463-464). Five of these have not been described previously (N460N, N463S, Δ463-464, N465S, and C1031C). All of the mutations were detected on different isolates, except for I898I, which was found alone or associated with others. Like Mugittu et al. in Tanzania (10) and Zhang et al. in China (15), we did not find either the S769N mutation or the A623E E431K double mutation, associated with reduced susceptibility to artemether (7). Previously, the N683K mutation was only found in Cambodia (2), suggesting that it may be specific to P. falciparum from South-East Asia. However, we did not detect this mutation in the South-East Asiatic strains W2 and Dd2 (both from Indochina, Malaria Research and Reference Reagent Resource Center), IMT-A4 (Vietnam), and IMT-K2 (Cambodia; data not shown). Interestingly, the N460N, N463S, N465S, and N683K mutations and the Δ463-464 double deletion are in a stretch of nine asparagines located in the interspecies variable region of PfATP6, a domain specific for Plasmodium species (8). Consequently, these modifications could be adaptive changes that might alter susceptibility to artemisinins. TABLE 1. Diversity of PfATP6 in P. falciparum samples from Vietnam Cojean et al. found the S769N mutation in an isolate from Africa that was susceptible to dihydroartemisinin (1), while Noedl et al. did not find this mutation in Cambodian samples that were less susceptible to artesunate (11). Consequently, we speculated on whether the correlation between the S769N mutation and the increased artemether 50% inhibitory concentration found in six isolates from French Guyana (7) should be regarded as a local case. Like other investigators, we did not detect any polymorphism in codon 263, described as the key amino acid for the interaction between PfATP6 and artemisinins (13). Mutations observed in our sequences and in those of previous studies (2, 6, 9) could be implicated indirectly in this interaction, in the case of association with artemisinin susceptibility. Considering the development of artemisinin combined therapies and the possible implication of PfATP6 in artemisinin resistance, the molecular variability of this gene should be carefully monitored.

The stage-dependent susceptibility of Plasmodium falciparum to a short exposure to docetaxel (Taxotere) was evaluated by subjecting ring-infected, trophozoite-infected, and schizont-infected erythrocytes to a 5-h exposure to various concentrations of the drug. The schizont stage was shown to be the most sensitive stage; an inhibition of more than 60% of parasite development was observed at 10 nM. At this drug concentration, the development of the younger ring and trophozoite forms was unaffected. The in vivo antimalarial activity of docetaxel against the development in blood of old trophozoites of a species that causes malaria in rodents, Plasmodium vinckei petteri, was evaluated in IOPS-OF1 mice. Two tests were performed: the 4-day suppressive test, as described by Peters (W. Peters, p. 145-273, in Chemotherapy, and Drug Resistance in Malaria, vol. 1, 1987), and the effects of a single injection of docetaxel after inoculation of the parasites. A single injection of docetaxel at 40 mg/kg of body weight was sufficient to reduce drastically the level of parasitemia; 90% inhibition of the development of parasites in blood was observed 5 days after drug injection. This program avoided the toxicity observed when mice were treated with four injections of docetaxel. The possibility of using a single bolus of taxoids to treat malaria infections is discussed.

This meeting report presents the outcomes of a workshop held in Bangkok on December 1st 2014, where the following challenges were discussed: the threat of resistance to artemisinin and artemisinin-based combination therapy in the Greater Mekong Sub-region (GMS) and in Africa; access to treatment for most at risk and hard to reach population; insecticide resistance, residual and outdoors transmission. The role of operational research and the interactions between research institutions, National Malaria Control Programmes, Civil Society Organizations, and of financial and technical partners to address those challenges and to accelerate translation of research into policies and programmes were debated. The threat and the emergency of the artemisinin resistance spread and independent emergence in the GMS was intensely debated as it is now close to the border of India. The need for key messages, based on scientific evidence and information available and disseminated without delay, was highlighted as crucial for an effective and urgent response. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0802-4) contains supplementary material, which is available to authorized users.