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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.

Objective Artemisinin-based combination therapies have been available since 2005 in the Democratic Republic of the Congo to treat malaria and to overcome the challenge of anti-malarial drug resistance as well as to improve access to effective treatments. The private sector is the primary distribution source for anti-malarial drugs and thus, has a key position among the supply chain actors for a rational and proper use of anti-malarial drugs. We aimed to assess access to nationally recommended anti-malarial drugs in private sector pharmacies of the capital-city of Kinshasa. Method We performed a cross-sectional survey of 404 pharmacies. Results Anti-malarial drugs were stocked in all surveyed pharmacies. Non-artemisinin-based anti-malarial therapies such as quinine or sulfadoxine-pyrimethamine, were the most frequently stocked drugs (93.8% of pharmacies). Artemisinin-based combination therapies were stocked in 88% of pharmacies. Artemether-lumefantrine combinations were the most frequently dispensed drugs (93% of pharmacies), but less than 3% were quality-assured products. Other non-officially recommended artemisinin-based therapies including oral monotherapies were widely available. Conclusion Artemisinin-based combination therapies were widely available in the private pharmacies of Kinshasa. However, the private sector does not guarantee the use of nationally recommended anti-malarial drugs nor does it give priority to quality-assured anti-malarial drugs. These practices contribute to the risk of emergence and spread of resistance to anti-malarial drugs and to increasing treatment costs.

A novel series of new 3-spiro monocyclic β-lactams 3a–i have been synthesized in high chemical yields from 9H-xanthene-9-carbonyl chloride 2 as ketene source in the [2+2] cycloaddition (Staudinger reaction) with different aromatic imines. These newly synthesized β-lactams were fully characterized and evaluated for their antimalarial activities against Plasmodium falciparum K14-resistant strain with moderate to excellent EC50 values varying from 8 up to 50 µM.

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.

A new hybrid compound 5 containing a ruthenocene, a 4-aminoquinoline, and a 1,2,4-trioxane within a single molecular structure has been synthesized and evaluated for antimalarial potential. In order to ascertain the role of each component of hybrid 5, its antimalarial activity has been measured against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum in comparison with those of chloroquine, artemisinin, a ruthenocene-trioxane compound 9 not containing an aminoquinoline, the precursor trioxane ketone 4, and a trioxane dimer 10. Hybrid 5 displays high antimalarial activity, in the mid to low nanomolar IC50 range, and low cytotoxicity toward healthy mammalian cells, which translates into good selectivity indexes. The potency of 5 is consistently higher than those of chloroquine, the parent metal-free trioxane, and compound 9, the structural intermediate featuring a ruthenocene moiety bound to the trioxane but lacking the aminoquinoline. These results validate our hypothesis that it is the combination of the aminoquinoline, the ruthenocenyl moiety, and the trioxane in a single molecule that provides the enhanced antiplasmodial activity, and highlight the concept of hybrid compounds as a strategy that deserves further attention.

A new fixed-dose combination of artesunate (AS) plus amodiaquine (AQ) (TRIMALACT) was recently developed for the treatment of uncomplicated falciparum malaria. The originality of this combination lies in its galenic formulation which consists of a three-layer tablet with two layers containing each of the active ingredients, i.e. AS and AQ, and these are separated by a middle layer containing an antioxidant compound. To evaluate the efficacy and tolerability of this combination, adults with uncomplicated malaria received three administrations of two tablets (100:300 mg AS/AQ) in a 24-h interval, in Democratic Republic of Congo. Parasitemia and fever were measured and the plasma levels of parent compounds and metabolites [dihydroartemisinin (DHA) and monodesethylamodiaquine (MdAQ)] were determined by high-performance liquid chromatography. In addition, we determined the prevalence of molecular markers of resistance to chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). The AS/AQ combination TRIMALACT demonstrated a good efficacy resulting in an excellent clinical and parasitological response rate of 100% after correction for PCR results. Treatment regimen was well tolerated. The main disposition parameters to AS+AQ were: for DHA, AUC = 632 +/- 475 ng h/ml and Cmax = 432 +/- 325 ng/ml, and for MdAQ = 14268 +/- 4114 ng h/ml and Cmax = 336 +/- 225 ng/ml (mean +/- standard deviation). Parasite genotyping show high frequencies of molecular SP- and CQ-resistance markers with more 80% of the samples showing more than three mutations linked to SP resistance and 93.48% carrying parasite with the CQ-resistant haplotype. This study shows that the AS/AQ combination TRIMALACT is safe and effective in the treatment of highly drug-resistant falciparum malaria.

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.

Our study demonstrates the capacity of FR160, a catechol iron chelator, to reach and accumulate into infected Plasmodium falciparum erythrocytes and parasites (cellular accumulation ratio between 12 and 43). Steady-state FR160 accumulation is obtained after 2 hr of exposure. After 2 hr exposure, it reaches intracellular levels that are 4- to 10-fold higher in infected red blood cells than those attained in normal erythrocytes. There is quite a good correlation between the accumulation of chloroquine and FR160 in the different strains ( r =0.939) and in the ic 50 values ( r =0.719). In contrast, the accumulation of FR160 and its activity is poorly correlated ( r =0.500), suggesting that activity of FR160 may be independent of its penetration into infected erythrocytes. The mechanism of accumulation is yet unknown but based on inhibitor studies, the uptake of FR160 seems to be not associated with the calcium pump or channel, the potassium channel or the Na + /H + exchanger. Combinations of FR160 with verapamil, diltiazem, clotrimazole, amiloride, diazoxide, 4-aminopyridine, and picrotoxin should be avoided (antagonistic effects). The potent in vitro activity of FR160 on chloroquine-resistant strains or isolates, its lower toxicity against Vero cells, its mechanisms of action, its capacity to reach rapidly and accumulate into infected erythrocytes suggest that FR160 holds much promise as a new structural lead and effective antimalarial agent or at least a promising adjuvant in treatment of malaria.

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.

Vinca alkaloids, vincristine and vinblastine, produce differential effects on the cell division of Trypanosoma cruzi epimastigote forms depending on drug concentrations. These effects are related to different microtubule-based mechanisms. For 15 microM vinblastine and 50 microM vincristine, the drugs inhibit both nuclear division and cytokinesis, and affect cell shape. At 3 microM vinblastine and 10 microM vincristine, however, cytokinesis is inhibited without major effect on the progression of the cell cycle; this yields giant cells having multiple nuclei, kinetoplasts and flagella. Cultures maintained over 1 week with daily drug replacement produced cells with more than 16 nuclei and 24 kinetoplasts, indicating that an equivalent of a fifth cell cycle was initiated. The ultrastructure of the multinucleate cells showed a basic organization closely similar to that of trypanosomes. Cytokinesis inhibition by vinca alkaloids seems to result from modulations of interactions between microtubules and associated proteins, rather than from an inhibition of microtubule dynamics as is usually proposed for vinca alkaloids. Cytokinesis inhibition is reversible: after removing the drug, epimastigotes emerge from the multinucleate cells. The emerging process follows a precise axis and polarity which are determined by the position of the flagellum/kinetoplast complex. This region could play an essential role in cell morphogenesis since zoids (cells without a nucleus) are frequently observed.

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.

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.

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 microtubular stabilizing agent docetaxel (Taxotere) is known to inhibit the intraerythrocytic development of Plasmodium falciparum. To investigate the mechanism(s) of inhibition, we analyzed the structural organization of the mitotic spindle by immunofluorescence and electron microscopy. When 30 microM docetaxel was applied for five hours on ring forms, alterations in the mitotic spindles leading to abnormal nuclear divisions were observed. At the trophozoite- and schizont-stage, docetaxel pulses prevent mitosis by stabilizing microtubular structures associated with the mitotic apparatus, giving abnormal spindles. However, this inhibition did not interfere with parasite DNA synthesis indicating the absence of a checkpoint that couples exit from mitosis with proper spindle assembly as observed in higher eukaryotic cells. In parallel, intraerythrocytic concentration of docetaxel was measured in parasitized erythrocytes, after incubation of cells with 3H-docetaxel for five hours. It was found to be 14-fold increased at the ring-stage of infected erythrocytes compared to normal ones, 170-fold increased at the trophozoite-stage and 1,500-fold increased at the schizont-stage. Our data show that, even though the overall intracellular concentration of docetaxel is low in docetaxel-pulsed rings, the agent might be sufficient to disturb the spindle organization. However, the existence of targets for docetaxel other than mitotic spindle microtubules, i.e. erythrocyte membrane components could interfere with mitotic spindle formation.

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.