Impact of methylene blue and atorvastatin combination therapy on the apparition of cerebral malaria in a murine model

BACKGROUND: Proveblue®, a methylene blue dye that complies with European Pharmacopoeia and contains limited organic impurities and heavy metals of recognized toxicity, showed in vitro synergy against Plasmodium falciparum when combined with atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. The objective of this study was to evaluate the in vivo efficacy of Proveblue® when combined with atorvastatin in a murine model of experimental cerebral malaria. METHODS: Forty female C57Bl6/N mice were divided into four groups (control, atorvastatin 40 mg/kg for seven days, Proveblue® 10 mg/kg for five days and atorvastatin combined with Proveblue®), infected with Plasmodium berghei ANKA parasites by intraperitoneal inoculation and observed for 45 days. RESULTS: Treatment with atorvastatin alone did not demonstrate an effect significantly different from no treatment (p = 0.0573). All the mice treated by atorvastatin alone died. Treatment with Proveblue® or a combination of Proveblue® and atorvastatin was significantly increased survival of cerebral malaria (p = 0.0011 and 0.0002, respectively). Although there was only one death in the atorvastatin and Proveblue® combination treatment group (10%) versus two deaths (22%) with Proveblue® treatment, the effect on cerebral malaria was not significant (p = 0.283). CONCLUSIONS: The present work demonstrated, for the first time, the high efficacy of Proveblue® in preventing cerebral malaria. Atorvastatin alone or in combination appears to possess limited use for preventing cerebral malaria. Combination of atorvastatin with lower doses of Proveblue® (<10 mg/kg/day) should be evaluated to show potential synergistic effects in cerebral malaria prevention

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger (Pfnhe-1) gene: the absence of association in clinical isolates from the Republic of Congo

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

Plasmodium falciparum proteome changes in response to doxycycline treatment

<p>Abstract</p> <p>Background</p> <p>The emergence of <it>Plasmodium falciparum </it>resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in <it>P. falciparum </it>have not yet been clearly defined, particularly at the protein level.</p> <p>Methods</p> <p>A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite <it>P. falciparum </it>following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and isobaric tagging reagents for relative and absolute quantification (iTRAQ).</p> <p>Results</p> <p>After doxycycline treatment, 32 and 40 <it>P. falciparum </it>proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm this hypothesis.</p> <p>Conclusions</p> <p>In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.</p

Atorvastatin treatment is effective when used in combination with mefloquine in an experimental cerebral malaria murine model

<p>Abstract</p> <p>Background</p> <p>One of the major complications of <it>Plasmodium falciparum </it>infection is cerebral malaria (CM), which causes one million deaths worldwide each year, results in long-term neurological sequelae and the treatment for which is only partially effective. Statins are recognized to have an immunomodulatory action, attenuate sepsis and have a neuroprotective effect. Atorvastatin (AVA) has shown in vitro anti-malarial activity and has improved the activity of mefloquine (MQ) and quinine.</p> <p>Methods</p> <p>The efficiency of 40 mg/kg intraperitoneal AVA, alone or in association with MQ, was assessed in an experimental <it>Plasmodium berghei </it>ANKA rodent parasite model of CM and performed according to different therapeutic schemes. The effects on experimental CM were assessed through the evaluation of brain histopathological changes and neuronal apoptosis by TUNEL staining.</p> <p>Results</p> <p>AVA alone in the therapeutic scheme show no effect on survival, but the prophylactic scheme employing AVA associated with MQ, rather than MQ alone, led to a significant delay in mouse death and had an effect on the onset of CM symptoms and on the level of parasitaemia. Histopathological findings show a correlation between brain lesions and CM onset. A neuronal anti-apoptotic effect of AVA in the AVA + MQ combination was not shown.</p> <p>Conclusions</p> <p>The combination of AVA and MQ therapy led to a significant delay in mouse mortality. There were differences in the incidence, time to cerebral malaria and the level of parasitaemia when the drug combination was administered to mice. When used in combination with MQ, AVA had a relevant effect on the in vivo growth inhibition and clinical outcome of <it>P. berghei </it>ANKA-infected mice.</p

Mosquito magnet® liberty plus trap baited with octenol confirmed best candidate for Anopheles surveillance and proved promising in predicting risk of malaria transmission in French Guiana

BACKGROUND: In French Guiana, Mosquito Magnet(®) Liberty Plus trap baited with octenol (MMoct) has been proposed for sampling Anopheles darlingi after comparison with CDC light trap and Human landing catch (HLC). However, other available lures were not tested. The current study compared MMoct and MM baited with Lurex™ (MMlur) to HLC, and analysed entomological data from MMoct collection with malaria cases to facilitate malaria surveillance. METHODS: Two independent experiments were conducted during 2012 and 2013 in Saint-Georges town, French Guiana. The first experiment used Latin square design to compare MMoct and MMlur to HLC between 18:30 to 22:30 and 05:00 to 07:00. Parity rate was determined for An. darlingi from each sampling system. In the second experiment, a 24:00 hour collection was done for four consecutive days during the first week of each month and every four days for the rest of the month using MMoct. Portion of the 24 hour collection was dissected for parity rate. All anophelines were screened for Plasmodium infection by PCR. Data for number of malaria cases was analysed for association with density of An. darlingi. RESULTS: In the first experiment, 3,721 anopheline mosquitoes were collected over 21 nights. Of these, 95.7% was identified morphologically to five species and An. darlingi contributed 98.4%, mainly from HLC (75.1%, CI 95% [73.2-77.0]) than MMoct (14.1%, CI 95% [12.6-15.7]) and MMlur (10.8%, CI 95% [9.4-12.2]). Species richness was highest in HLC meanwhile species diversity index was greatest in MMoct. MMoct collected more parous An. darlingi than HLC (p < 0.0001) and MMlur (p = 0.0021). The second experiment amounted to 2035 females, 60.8% belonging to 10 species. Anopheles darlingi constituted 85.0% of the species and had parity rate of 52.3%. Specimens were uninfected with Plasmodium. Density of An. darlingi best correlated with malaria cases observed six weeks later (p = 0.0016; r = 0.4774). CONCLUSION: Though MMoct and MMlur performed well in sampling An. darlingi, MMoct captured more species and, therefore, would be useful for surveillance. Even if it collected mostly parous mosquitoes, MMoct proved useful in collecting entomological data required for predicting malaria emergence. It is a potential replacement for HLC

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay

OBJECTIVE: The main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC(50)) for chloroquine. METHODS: The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC(50 )for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake. RESULTS: From 5% O(2 )to 21% O(2 )conditions, no parasiticide effect of O(2 )concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O(2 )with an increase in the length of schizogony. The chloroquine IC(50 )at 10% O(2 )were significantly higher than those at 21% O(2), means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC(50 )> 100 nM) at 10% O(2), 17 were sensitive to chloroquine (IC(50 )< 100 nM) at 21% O(2). CONCLUSION: Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance

Early treatment failure during treatment of Plasmodium falciparum malaria with atovaquone-proguanil in the Republic of Ivory Coast

The increased spread of drug-resistant malaria highlights the need for alternative drugs for treatment and chemoprophylaxis. The combination of atovaquone‐proguanil (Malarone®) has shown high efficacy against Plasmodium falciparum with only mild side-effects. Treatment failures have been attributed to suboptimal dosages or to parasite resistance resulting from a point mutation in the cytochrome b gene. In this paper, a case of early treatment failure was reported in a patient treated with atovaquone-proguanil; this failure was not associated with a mutation in the parasite cytochrome b gene, with impaired drug bioavailability, or with re-infection

Determinants of compliance with anti-vectorial protective measures among non-immune travellers during missions to tropical Africa

International audienceThe effectiveness of anti-vectorial malaria protective measures in travellers and expatriates is hampered by incorrect compliance. The objective of the present study was to identify the determinants of compliance with anti-vectorial protective measures (AVPMs) in this population that is particularly at risk because of their lack of immunity

Quinine-Resistant Malaria in Traveler Returning from Senegal, 2007

We describe clinical and parasitologic features of in vivo and in vitro Plasmodium falciparum resistance to quinine in a nonimmune traveler who returned to France from Senegal in 2007 with severe imported malaria. Clinical quinine failure was associated with a 50% inhibitory concentration of 829 nmol/L. Increased vigilance is required during treatment follow-up

Antimalarial drug use in general populations of tropical Africa

<p>Abstract</p> <p>Background</p> <p>The burden of <it>Plasmodium falciparum </it>malaria has worsened because of the emergence of chloroquine resistance. Antimalarial drug use and drug pressure are critical factors contributing to the selection and spread of resistance. The present study explores the geographical, socio-economic and behavioural factors associated with the use of antimalarial drugs in Africa.</p> <p>Methods</p> <p>The presence of chloroquine (CQ), pyrimethamine (PYR) and other antimalarial drugs has been evaluated by immuno-capture and high-performance liquid chromatography in the urine samples of 3,052 children (2–9 y), randomly drawn in 2003 from the general populations at 30 sites in Senegal (10), Burkina-Faso (10) and Cameroon (10). Questionnaires have been administered to the parents of sampled children and to a random sample of households in each site. The presence of CQ in urine was analysed as dependent variable according to individual and site characteristics using a random – effect logistic regression model to take into account the interdependency of observations made within the same site.</p> <p>Results</p> <p>According to the sites, the prevalence rates of CQ and PYR ranged from 9% to 91% and from 0% to 21%, respectively. In multivariate analysis, the presence of CQ in urine was significantly associated with a history of fever during the three days preceding urine sampling (OR = 1.22, p = 0.043), socio-economic level of the population of the sites (OR = 2.74, p = 0.029), age (2–5 y = reference level; 6–9 y OR = 0.76, p = 0.002), prevalence of anti-circumsporozoite protein (CSP) antibodies (low prevalence: reference level; intermediate level OR = 2.47, p = 0.023), proportion of inhabitants who lived in another site one year before (OR = 2.53, p = 0.003), and duration to reach the nearest tarmacked road (duration less than one hour = reference level, duration equal to or more than one hour OR = 0.49, p = 0.019).</p> <p>Conclusion</p> <p>Antimalarial drug pressure varied considerably from one site to another. It was significantly higher in areas with intermediate malaria transmission level and in the most accessible sites. Thus, <it>P. falciparum </it>strains arriving in cross-road sites or in areas with intermediate malaria transmission are exposed to higher drug pressure, which could favour the selection and the spread of drug resistance.</p