Etude de l'effet de l'amodiaquine sur les globules rouges infectés par le paludisme dans les images multispectrales

Nous avons montré que l’amodiaquine se fixe préférentiellement dans les globules rouges infectés et nous avons également été capable de retrouver les concentrations du médicament dans les cellules, à partir d’images multispectrales. Une scène d'images multispectrales de frottis sanguins non marqués est obtenue à partir d’une culture de plasmodium falciparum, à laquelle nous avons ajouté différentes doses d’amodiaquine (AQ) ; Les images sont obtenues à partir d'enregistrement de treize capteurs spectraux couplés à un microscope multimodal et multispectral. Ces images sont ensuite utilisées pour étudier l’interaction du médicament avec les globules rouges en fonction des concentrations, en ayant recours à des techniques d’analyse multivariée telles que la classification hiérarchique, la méthode des k-moyennes et l’analyse en composante principale. Les résultats obtenus nous montrent que l’imagerie multispectrale est un atout majeur pour le diagnostic médical et peut donc constituer une technique de routine pour l’étude de nouvelles molécules antipaludiques, notamment issues de la pharmacopée africaine.Mots-clés: imagerie multispectrale, classification hiérarchique, analyse en composante principale, k-moyennes.Study of the Effect of Amodiaquine on Red Blood Cells Infected by Malaria in Multispectral ImagesWe have demonstrated that amodiaquine binds preferentially in the infected red blood cells and we have also been able to retrieve the drug concentrations in the cells. A scene of multispectral images of free labelled blood smears have been obtained from plasmodium falciparum culture to which we have added different quantities of amodiaquine; the images have been recorded from thirteen spectral sensors coupled with a multimodal and multispectral microscope. These images have been used to study the interaction of the drug with the red blood cells as function of drug concentration by the use of multivariate statistical analysis techniques such as hierarchical classification, k-means method and principal component analysis techniques. The results show that the multispectral imagery is a key technique in medical technologies and can therefore be used as routine method for antimalarial drug design, especially in African traditional pharmacopeia study.Keywords: multispectral imagery; hierarchical classification; principal component analysis; k-means

Synthèse et criblage antiplasmodial de quelques benzimidazolyl-chalcones

Une série d’hybrides de chalcones (6a-6g) associant le noyau benzimidazole et le groupement arylpropénone a été synthétisée par condensation entre des méthylcétones à support benzimidazole et des arylaldéhydes. L’analogue cyclohexénone (6h) a par ailleurs été préparé à l’aide de l’acétoacétate d’éthyle. Les structures chimiques des composés ont été déterminées par spectrométries RMN et de masse ESI. Leur criblage antiplasmodial vis-à-vis d’isolats de P. falciparum chloroquino-sensible et chloroquino-résistant montrent que la benzimidazolyl-chalcone est un excellent pharmacophore antiplasmodial. Les activités étaient paradoxalement toutes plus efficaces sur les isolats de P. falciparum chloroquino-résistants que chloroquinosensibles. Leur amélioration serait assujettie à l’introduction d’un chlore en C5 du benzimidazole et ne serait pas nécessairement liée, ni à la nature de l’azote pyrrolique du benzimidazole ni à la cyclisation de la propénone en cyclohexénone. La benzimidazolyl-chalcone (6e) avec une valeur de CI50 de 5,63 μM, avait le meilleur profil antiplasmodial sur l’isolat de P. falciparum chloroquino-sensible. Le composé 6f avec des valeurs de CI50 de 10,65 et 0,78 μM s’est révélé être la meilleure chalcone antiplasmodiale quel que soit le statut des isolats de P. falciparum. Ce dernier pourrait constituer une tête de série pour le développement de nouveaux agents antipaludiques.Mots clés : Benzimidazole, Chalcone, Activité antiplasmodiale, Chloroquino-résistance, Plasmodium falciparum

Targeted Next Generation Sequencing for malaria research in Africa:Current status and outlook

Targeted Next Generation Sequencing (TNGS) is an efficient and economical Next Generation Sequencing (NGS) platform and the preferred choice when specific genomic regions are of interest. So far, only institutions located in middle and high-income countries have developed and implemented the technology, however, the efficiency and cost savings, as opposed to more traditional sequencing methodologies (e.g. Sanger sequencing) make the approach potentially well suited for resource-constrained regions as well. In April 2018, scientists from the Plasmodium Diversity Network Africa (PDNA) and collaborators met during the 7th Pan African Multilateral Initiative of Malaria (MIM) conference held in Dakar, Senegal to explore the feasibility of applying TNGS to genetic studies and malaria surveillance in Africa. The group of scientists reviewed the current experience with TNGS platforms in sub-Saharan Africa (SSA) and identified potential roles the technology might play to accelerate malaria research, scientific discoveries and improved public health in SSA. Research funding, infrastructure and human resources were highlighted as challenges that will have to be mitigated to enable African scientists to drive the implementation of TNGS in SSA. Current roles of important stakeholders and strategies to strengthen existing networks to effectively harness this powerful technology for malaria research of public health importance were discussed

Population genetic structure of Schistosoma haematobium and Schistosoma haematobium x Schistosoma bovis hybrids among school-aged children in Côte d'Ivoire

While population genetics of Schistosoma haematobium have been investigated in West Africa, only scant data are available from Cote d'Ivoire. The purpose of this study was to analyze both genetic variability and genetic structure among S. haematobium populations and to quantify the frequency of S. haematobium x S. bovis hybrids in school-aged children in different parts of Cote d'Ivoire. Urine samples were subjected to a filtration method and examined microscopically for Schistosoma eggs in four sites in the western and southern parts of Cote d'Ivoire. A total of 2692 miracidia were collected individually and stored on Whatman((R)) FTA cards. Of these, 2561 miracidia were successfully genotyped for species and hybrid identification using rapid diagnostic multiplex mitochondrial cox1 PCR and PCR Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the nuclear ITS2 region. From 2164 miracidia, 1966 (90.9%) were successfully genotyped using at least 10 nuclear microsatellite loci to investigate genetic diversity and population structure. Significant differences were found between sites in all genetic diversity indices and genotypic differentiation was observed between the site in the West and the three sites in the East. Analysis at the infrapopulation level revealed clustering of parasite genotypes within individual children, particularly in Duekoue (West) and Sikensi (East). Of the six possible cox1-ITS2 genetic profiles obtained from miracidia, S. bovis cox1 x S. haematobium ITS2 (42.0%) was the most commonly observed in the populations. We identified only 15 miracidia (0.7%) with an S. bovis cox1 x S. bovis ITS2 genotype. Our study provides new insights into the population genetics of S. haematobium and S. haematobium x S. bovis hybrids in humans in Cote d'Ivoire and we advocate for researching hybrid schistosomes in animals such as rodents and cattle in Cote d'Ivoire

In silico characterisation of putative Plasmodium falciparum vaccine candidates in African malaria populations.

Genetic diversity of surface exposed and stage specific Plasmodium falciparum immunogenic proteins pose a major roadblock to developing an effective malaria vaccine with broad and long-lasting immunity. We conducted a prospective genetic analysis of candidate antigens (msp1, ama1, rh5, eba175, glurp, celtos, csp, lsa3, Pfsea, trap, conserved chrom3, hyp9, hyp10, phistb, surfin8.2, and surfin14.1) for malaria vaccine development on 2375 P. falciparum sequences from 16 African countries. We described signatures of balancing selection inferred from positive values of Tajima's D for all antigens across all populations except for glurp. This could be as a result of immune selection on these antigens as positive Tajima's D values mapped to regions with putative immune epitopes. A less diverse phistb antigen was characterised with a transmembrane domain, glycophosphatidyl anchors between the N and C- terminals, and surface epitopes that could be targets of immune recognition. This study demonstrates the value of population genetic and immunoinformatic analysis for identifying and characterising new putative vaccine candidates towards improving strain transcending immunity, and vaccine efficacy across all endemic populations

The effect of dose on the antimalarial efficacy of artemether-lumefantrine: a systematic review and pooled analysis of individual patient data

Background: Artemether-lumefantrine is the most widely used artemisinin-based combination therapy for malaria, although treatment failures occur in some regions. We investigated the effect of dosing strategy on efficacy in a pooled analysis from trials done in a wide range of malaria-endemic settings. Methods: We searched PubMed for clinical trials that enrolled and treated patients with artemether-lumefantrine and were published from 1960 to December, 2012. We merged individual patient data from these trials by use of standardised methods. The primary endpoint was the PCR-adjusted risk of Plasmodium falciparum recrudescence by day 28. Secondary endpoints consisted of the PCR-adjusted risk of P falciparum recurrence by day 42, PCR-unadjusted risk of P falciparum recurrence by day 42, early parasite clearance, and gametocyte carriage. Risk factors for PCR-adjusted recrudescence were identified using Cox's regression model with frailty shared across the study sites. Findings: We included 61 studies done between January, 1998, and December, 2012, and included 14 327 patients in our analyses. The PCR-adjusted therapeutic efficacy was 97·6% (95% CI 97·4-97·9) at day 28 and 96·0% (95·6-96·5) at day 42. After controlling for age and parasitaemia, patients prescribed a higher dose of artemether had a lower risk of having parasitaemia on day 1 (adjusted odds ratio [OR] 0·92, 95% CI 0·86-0·99 for every 1 mg/kg increase in daily artemether dose; p=0·024), but not on day 2 (p=0·69) or day 3 (0·087). In Asia, children weighing 10-15 kg who received a total lumefantrine dose less than 60 mg/kg had the lowest PCR-adjusted efficacy (91·7%, 95% CI 86·5-96·9). In Africa, the risk of treatment failure was greatest in malnourished children aged 1-3 years (PCR-adjusted efficacy 94·3%, 95% CI 92·3-96·3). A higher artemether dose was associated with a lower gametocyte presence within 14 days of treatment (adjusted OR 0·92, 95% CI 0·85-0·99; p=0·037 for every 1 mg/kg increase in total artemether dose). Interpretation: The recommended dose of artemether-lumefantrine provides reliable efficacy in most patients with uncomplicated malaria. However, therapeutic efficacy was lowest in young children from Asia and young underweight children from Africa; a higher dose regimen should be assessed in these groups. Funding: Bill and Melinda Gates Foundation

Haematological consequences of acute uncomplicated falciparum malaria: a WorldWide Antimalarial Resistance Network pooled analysis of individual patient data

Background: Plasmodium falciparum malaria is associated with anaemia-related morbidity, attributable to host, parasite and drug factors. We quantified the haematological response following treatment of uncomplicated P. falciparum malaria to identify the factors associated with malarial anaemia. Methods: Individual patient data from eligible antimalarial efficacy studies of uncomplicated P. falciparum malaria, available through the WorldWide Antimalarial Resistance Network data repository prior to August 2015, were pooled using standardised methodology. The haematological response over time was quantified using a multivariable linear mixed effects model with nonlinear terms for time, and the model was then used to estimate the mean haemoglobin at day of nadir and day 7. Multivariable logistic regression quantified risk factors for moderately severe anaemia (haemoglobin < 7 g/dL) at day 0, day 3 and day 7 as well as a fractional fall ≥ 25% at day 3 and day 7. Results: A total of 70,226 patients, recruited into 200 studies between 1991 and 2013, were included in the analysis: 50,859 (72.4%) enrolled in Africa, 18,451 (26.3%) in Asia and 916 (1.3%) in South America. The median haemoglobin concentration at presentation was 9.9 g/dL (range 5.0–19.7 g/dL) in Africa, 11.6 g/dL (range 5.0–20.0 g/dL) in Asia and 12.3 g/dL (range 6.9–17.9 g/dL) in South America. Moderately severe anaemia (Hb < 7g/dl) was present in 8.4% (4284/50,859) of patients from Africa, 3.3% (606/18,451) from Asia and 0.1% (1/916) from South America. The nadir haemoglobin occurred on day 2 post treatment with a mean fall from baseline of 0.57 g/dL in Africa and 1.13 g/dL in Asia. Independent risk factors for moderately severe anaemia on day 7, in both Africa and Asia, included moderately severe anaemia at baseline (adjusted odds ratio (AOR) = 16.10 and AOR = 23.00, respectively), young age (age < 1 compared to ≥ 12 years AOR = 12.81 and AOR = 6.79, respectively), high parasitaemia (AOR = 1.78 and AOR = 1.58, respectively) and delayed parasite clearance (AOR = 2.44 and AOR = 2.59, respectively). In Asia, patients treated with an artemisinin-based regimen were at significantly greater risk of moderately severe anaemia on day 7 compared to those treated with a non-artemisinin-based regimen (AOR = 2.06 [95%CI 1.39–3.05], p < 0.001). Conclusions: In patients with uncomplicated P. falciparum malaria, the nadir haemoglobin occurs 2 days after starting treatment. Although artemisinin-based treatments increase the rate of parasite clearance, in Asia they are associated with a greater risk of anaemia during recovery

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website

Polymorphism of Pfatpase6 in Côte d’Ivoire: Detection of a four new point mutations

Over the past decade, the number of malaria cases has dropped by more than half in many malaria-endemic countries. However, recent parasite resistance to artemisinin undermines that progress. Artemisinin-based combination therapy (ACTs) is recommended for the treatment of Plasmodium falciparum malaria. Among the potential genes that are associated to resistance of P. falciparum to artemisinin include PfATPase6 gene that encodes the protein SERCA: the specific target of drugs in the parasite. PfATPase6 was the subject of many studies across the world to highlight its’ involvement in the resistance of P. falciprum to artemisinin. It was found in this work that this gene has a polymorphism but its’ involvement in the resistance of the parasite has not been demonstrated. The objective of this study was to describe the basic polymorphism of clinical isolates of P. falciparum in Côte d'Ivoire during the period when the country national anti- malaria program introduced ACTs in the treatment of malaria. Thus, 82 DNA fragments from 41 clinical isolates divided into regions A and B were analyzed using automatic sequencing method. The results show more points mutation of DNA fragments of PfATP6 but the most significant are D734Y (29.2%), Q254H (9.7%), N669Y (14.6%) and S670C (12.2%). Other mutations emerged in marginal proportions. We therefore recommend strict monitoring of gene polymorphism in PfATPase6 in as much as the effectiveness of artemisinin derivatives is concerned; but the fact remains that their involvement in the resistance of P falciparum to artemisinin is still very low.Key words: Côte d’Ivoire, detection, mutations, PfATPase6, polymorphism