Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in the Democratic Republic of the Congo: emergence of highly resistant pfdhfr/pfdhps alleles.

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.

Polymorphism of Plasmodium falciparum Na(+)/H(+) exchanger is indicative of a low in vitro quinine susceptibility in isolates from Viet Nam.

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 IC(50) of 682 nM versus median IC(50) of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC(50) of 704 nM versus median IC(50) 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 IC(50) 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.

Diastereoselective Synthesis of Potent Antimalarial Cis-ß-lactam Agents.

Fifteen novel ß-lactams bearing N-ethyl tert-butyl carbamate group 5a-o and fifteen N-(2- aminoethyl) ß-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-ß-lactam derivatives. These newly synthesized ß-lactams were evaluated for their antimalarial activity against p. falciparum K14 resistant strain and showed good to excellent EC50 values. Of the thirty ß-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 <50 . Compounds 5c, 5h, and 5q-t were examined for their anticancer properties against K562 Leukemia cell line and 5s showed the best activity. Compounds 3a-j, 5a-o, 6a-o, were tested against S. aureus , E. coli, C. albicans and showed no activity below 125 µg/mL.

Synthesis of New ß-Lactams Bearing the Biologically Important Morpholine Ring and POM Analyses of Their Antimicrobial and Antimalarial Activities.

Some new ß-lactams bearing biologically important morpholine ring have been synthesized by acylation of amino ß-lactams in the presence of morpholine-4-carbonyl chloride. These novel ß-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.

Artesunate-erythropoietin combination for murine cerebral malaria treatment.

Cerebral malaria is the most severe and rapidly fatal complication of Plasmodium falciparum infection. Despite appropriate anti-malarial treatment using quinine or artemisinin derivatives, 10-20% of mortality still occurs during the acute phase. To improve cerebral malaria outcome, adjunctive therapies are clearly needed. Most experiments in this area have been dedicated to immuno-modulation with various successes. Since erythropoietin has been shown to be highly effective in human ischemic stroke and in murine cerebral malaria, we addressed the issue of cerebral malaria outcome improvement by erythropoietin-artesunate drug combination. Compared to the previous study using erythropoietin high doses at the early beginning of the disease, erythropoietin treatment was decreased by six-fold and delayed to the pre-mortem phase. We studied effects on survival and on clinical recovery of the drug combination given from day 6 to day 8 post-infection to CBA/J mice infected by Plasmodium berghei ANKA. We showed that the artesunate-erythropoietin drug combination led to clinical recovery 24 h earlier for surviving mice, and to increase in the global survival rate compared to artesunate monotherapy (p<0.01). Since erythropoietin has no effect on parasite clearance, it could be stated that this drug combination is efficient and that erythropoietin could be a lead for the implementation of a new adjunctive therapy during the acute phase of cerebral malaria.

Synthesis and biological evaluation of some novel diastereoselective benzothiazole ß-lactam conjugates.

Highly diastereoselective synthesis of some novel benzothiazole-substituted ß-lactam hybrids was achieved starting from (benzo[d]thiazol-2-yl)phenol as an available precursor. This is the first time (benzo[d]thiazol-2-yl)phenoxyacetic acid has been used as ketene source in synthesizing monocyclic 2-azetidinones. These compounds were evaluated for their antimicrobial activities against a large panel of Gram-positive and Gram-negative bacterial strains and moderate activities were encountered. Antimalarial data revealed that adding methoxyphenyl or ethoxyphenyl group on the ß-lactam ring makes compounds that are more potent. Moreover, hemolytic activity and mammalian cell toxicity survey of the compounds showed their potential as a medicine.

In vitro antimalarial activity of flavonoid derivatives dehydrosilybin and 8-(1;1)-DMA-kaempferide.

Multidrug-resistant Plasmodium falciparum strains are an increasing problem in endemic areas and are partly responsible for the worsening malaria situation around the world. New cheap and effective compounds active in combination with available drug in the field are urgently needed. The aim of this work was to explore the potential antiplasmodial effect of flavonoid derivatives on parasites growth in vitro. In vitro antiplasmodial activity of dehydrosilybin and 8-(1;1)-DMA-kaempferide has been evaluated by real time PCR for five P. falciparum strains. Both revealed significative antimalarial activity against the different strains. Since this drug family has been largely used and well-tolerated in humans, flavonoid derivatives could be in the near future associated with already available drugs in order to delay the spread of P. falciparum resistance.

Synthesis of New 4-Aminoquinolines and Evaluation of Their In Vitro Activity against Chloroquine-Sensitive and Chloroquine-Resistant Plasmodium falciparum.

The efficacy of chloroquine, once the drug of choice in the fight against Plasmodium falciparum, 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-aminoquinolines 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 IC50), markedly superior to chloroquine and comparable to amodiaquine, against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, 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.

Diastereoselective synthesis of potent antimalarial cis-ß-lactam agents through a [2 + 2] cycloaddition of chiral imines with a chiral ketene.

The effect of double asymmetric induction for the synthesis of new cis-ß-lactams by [2 + 2] cycloaddition reactions of chiral imines with a chiral ketene was investigated. The cycloaddition reaction was found to be totally diastereoselective leading exclusively to the formation of the cis-ß-lactam derivatives. The newly synthesized cycloadducts were evaluated for their antimalarial activities against Plasmodium falciparum K14 resistant strain with moderate to excellent IC50 values varying from 8 to 50 µM. Of the fifteen ß-lactams tested, four showed IC50 ≤ 11 µM.

Features of apoptosis in Plasmodium falciparum erythrocytic stage through a putative role of PfMCA1 metacaspase-like protein.

The ability to undergo apoptosis, previously thought to be exclusive to multicellular organisms, has been demonstrated in unicellular parasites. On the basis of an observation that Plasmodium "crisis forms" were seen in vitro after cultivation in media containing an antimalarial drug, we attempted to determine whether Plasmodium falciparum has the ability to undergo apoptosis. By use of either the apoptosis-inducer etoposide or the antimalarial chloroquine, apoptosis in Plasmodium asexual stages was evident by the observation of DNA fragmentation and disruption of transmembrane mitochondrial potential. Next, we sought to determine whether Plasmodium produces specific cysteine proteases that can induce apoptosis. We hypothesized that the 2 metacaspase-like proteins present in the Plasmodium genome contained features typical of downstream execution steps and upstream signaling pathways such caspase activation and domain recruitment. We report that one of the metacaspase genes, PF13_0289, in addition to a universally conserved catalytic cysteine and histidine dyad required for catalysis activity, contains a putative caspase recruitment domain in the N-terminal amino acid sequence. This putative P. falciparum metacaspase protein has been designated PfMCA1. Our findings offer important insights into parasite survival strategies that could open new ways for therapeutic alternatives to drug resistance.

Comparison of a SYBR green I-based assay with a histidine-rich protein II enzyme-linked immunosorbent assay for in vitro antimalarial drug efficacy testing and application to clinical isolates.

In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC50s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC50 values when phenol red was included in the medium. The IC50s and the IC90s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC50 and IC90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.

Recombinant human erythropoietin prevents the death of mice during cerebral malaria.

Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.