Nanoparticle tracking analysis of natural hemozoin from Plasmodium parasites.

BACKGROUND: Hemozoin is a byproduct of hemoglobin digestion crucial for parasite survival. It forms crystals that can be of interest as drug targets or biomarkers of malaria infection. However, hemozoin has long been considered as an amorphous crystal of simple morphology. Studying the consequences of biomineralization of this crystal during the parasite growth may provide more comprehensive evidence of its role during malaria. OBJECTIVES: This study aimed to investigate the interest of nanoparticles tracker analysis for measuring the concentration and size of hemozoin particles produced from different parasite sources and conditions. METHODS: Hemozoin was extracted from several clones of Plasmodium falciparum both asexual and sexual parasites. Hemozoin was also extracted from blood samples of malaria patients and from saliva of asymptomatic malaria carriers. Nanoparticles tracking analysis (NTA) was performed to assess the size and concentration of hemozoin. RESULTS: NTA data showed variation in hemozoin concentration, size, and crystal clusters between parasite clones, species, and stages. Among parasite clones, hemozoin concentration ranged from 131 to 2663 particles/infected red blood cell (iRBC) and size ranged from 149.6 ± 6.3 nm to 234.8 ± 40.1 nm. The mean size was lower for Plasmodium vivax (176 ± 79.2 nm) than for Plasmodium falciparum (254.8 ± 74.0 nm). Sexual NF54 parasites showed a 7.5-fold higher concentration of hemozoin particles (28.7 particles/iRBC) compared to asexual parasites (3.8 particles/iRBC). In addition, the mean hemozoin size also increased by approximately 60 % for sexual parasites. Compared to in vitro cultures of parasites, blood samples showed low hemozoin concentrations. CONCLUSIONS: This study highlights the potential of NTA as a useful method for analyzing hemozoin, demonstrating its ability to provide detailed information on hemozoin characterization. However, further research is needed to adapt the NTA for hemozoin analysis.

Paper functionalization for detection of Plasmodium falciparum DNA using square waves voltammetry.

Malaria elimination is a major goal to be reached in the next decade. Significant progress were made in the past, and the prevalence decreased in many areas while the positive trend stalled in the last years. The exact number of asymptomatic carriers of Plasmodium parasites is unknown since this population is not detected by conventional diagnosis methods and participate in the maintenance of transmission. Molecular methods to detect low parasitemia are not available at point-of-care in low-income countries of malaria endemic areas. Adaptation of molecular methods such as loop-mediated isothermal amplification of DNA may provide effective tools but it required simplification of DNA detection. Square waves voltammetry, easily imbedded in small device such as cell phone, was largely described for DNA detection but support for reaction was an issue to address. Here we used an efficient functionalization method of paper-based material to facilitate the interactions between isothermal amplification product and methylene blue for easy-to-use DNA detection. The proof-of-concept of qualitative detection of very low parasitemia from malaria infected patients using newly chemically treated paper for square waves voltammetry was obtained with a sensitivity and specificity of 100% and a limit-of-detection of 0.1 parasite. µL-1 corresponding to a parasitemia of 0.000002%.

Pre-referral intranasal artesunate powder for cerebral malaria: a proof-of-concept study.

BACKGROUND: Malaria still kills young children in rural endemic areas because early treatment is not available. Thus, the World Health Organization recommends the administration of artesunate suppositories as pre-referral treatment before transportation to the hospital in case of severe symptoms with an unavailable parenteral and oral treatment. However, negative cultural perception of the rectal route, and limited access to artesunate suppositories, could limit the use of artesunate suppositories. There is, therefore, a need for an alternative route for malaria pre-referral treatment. The aim of this study was to assess the potential of intranasal route for malaria pre-referral treatment. METHODS: The permeability of artesunate through human nasal mucosa was tested in vitro. The Transepithelial Electrical Resistance (TEER) of the nasal mucosa was followed during the permeation tests. Beside, regional deposition of artesunate powder was assessed with an unidose drug delivery device in each nostril of a nasal cast. Artesunate quantification was performed using Liquid Chromatography coupled to tandem Mass Spectrometry. RESULTS: The experimental model of human nasal mucosa was successfully implemented. Using this model, artesunate powder showed a much better passage rate through human nasal mucosa than solution (26.8 ± 6.6% versus 2.1 ± 0.3%). More than half (62.3%) of the artesunate dose sprayed in the nostrils of the nasal cast was recovered in the olfactory areas (44.7 ± 8.6%) and turbinates (17.6 ± 3.3%) allowing nose-to-brain and systemic drug diffusion, respectively. CONCLUSION: Artesunate powder showed a good permeation efficiency on human nasal mucosa. Moreover it can be efficiently sprayed in the nostrils using unidose device to reach the olfactory area leading to a fast nose-to-brain delivery as well as a systemic effect. Taken together, those results are part of the proof-of-concept for the use of intranasal artesunate as a malaria pre-referral treatment.

Evaluation of ferrocenyl-containing γ-hydroxy-γ-lactam-derived tetramates as potential antiplasmodials.

A series of ferrocenyl-containing γ-hydroxy-γ-lactam tetramates were prepared in 2-3 steps through ring opening-ring closure (RORC) process of γ-ylidene-tetronate derivatives in the presence of ferrocenyl alkylamines. The compounds were screened in vitro for their antiplasmodial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (W2) clones of P. falciparum, displaying activity in the range of 0.12-100 µM, with generally good resistance index. The most active ferrocene in these series exhibited IC50 equal to 0.09 µM (3D7) and 0.12 µM (W2). The low cytotoxicity of the ferrocenyl-containing γ-hydroxy-γ-lactam tetramates against Human Umbilical Vein Endothelial (HUVEC) cell line demonstrated selective antiparasitic activity. The redox properties of these ferrocene-derived tetramates were studied and physico-biochemical studies evidenced that these derivatives can exert potent antimalarial activities via a mechanism distinct from ferroquine.

Assessment of quantitative and semi-quantitative biological test methods of artesunate in vitro.

Artesunate is the current most potent antimalarial drug widely used for the treatment of malaria. Considering the emergence of artemisinin resistance, several situations may require a simple method for artesunate quantification. We thus developed a quantitative and a semi-quantitative biological method for the determination of artesunate in liquid samples. The tests are based on the measurement of samples' antimalarial activity on Plasmodium falciparum 3D7 using a modified SYBR Green I drug susceptibility test. For the quantitative test, we established a standard curve that resulted from a dose-response curve and evaluated its performances using controls samples. Whereas the linear regression analysis between artesunate concentration and antimalarial activity showed promising results (linearity range 1.5-24.6 ng/mL, r2 = 0.9373), we found that artesunate content of the controls was significantly overestimated (p = 0.0313). For the semi-quantitative test, we compared the antimalarial activities of samples collected during permeation studies of artesunate to that of a reference (artesunate IC50) by statistical analysis. We demonstrated that antimalarial activities of samples from permeation tests using a powder formulation of artesunate were greater than those of samples from tests using a solution formulation. Bioassays can be simple techniques to assess artesunate in liquid samples, particularly in resource-limited settings. Comparison with reference methods is still recommended when accurate drug quantification is required.

Title: Évaluation de méthodes de tests biologiques quantitatives et semi-quantitatives de l'artésunate in vitro. Abstract: L'artésunate est le médicament antipaludique le plus puissant actuellement, largement utilisé pour le traitement du paludisme. Compte tenu de l'émergence de la résistance à l'artémisinine, plusieurs situations peuvent nécessiter une méthode simple de quantification de l'artésunate. Nous avons ainsi développé un test biologique quantitatif et un test semi-quantitatif pour le dosage de l'artésunate dans des échantillons liquides. Les méthodes sont basées sur la mesure de l'activité antipaludique des échantillons sur Plasmodium falciparum 3D7 à l'aide d'un test de sensibilité aux médicaments SYBR Green I modifié. Pour le test quantitatif, nous avons établi une courbe standard issue d'une courbe dose-réponse et évalué ses performances à l'aide d'échantillons témoins. Alors que l'analyse de régression linéaire entre la concentration d'artésunate et l'activité antipaludique a montré des résultats prometteurs (gamme de linéarité de 1,5 à 24,6 ng/mL, r2 = 0,9373), nous avons constaté que la teneur en artésunate des témoins était significativement surestimée (p = 0,0313). Pour le test semi-quantitatif, nous avons comparé les activités antipaludiques d'échantillons collectés lors des études de perméation de l'artésunate à celle d'une référence (artésunate IC50) par analyse statistique. Nous avons démontré que les activités antipaludiques des échantillons provenant de tests de perméation utilisant une formulation en poudre d'artésunate étaient supérieures à celles des échantillons provenant de tests utilisant une formulation en solution. Les dosages biologiques peuvent être des techniques simples pour évaluer l'artésunate dans des échantillons liquides, en particulier dans les milieux à ressources limitées. La comparaison avec des méthodes de référence est toujours recommandée lorsqu'une quantification précise du médicament est requise.

Systematic review of artesunate pharmacokinetics: Implication for treatment of resistant malaria.

BACKGROUND: Artesunate (ART) is an artemisinin derivative used as monotherapy for the treatment of severe malaria and in combination with a partner drug for non-severe malaria. Resistance of malaria parasites to artemisinins have emerged in Southeast Asia. Adjustment of drug regimen may be an option to prevent therapeutic failures considering the relative favourable safety profile of ART high doses. METHODS: For that purpose, a systematic review was done using PubMed, Scopus and Web of Science databases. All studies on ART and DHA pharmacokinetic post-administration of artesunate in human patients or volunteers were included. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist 2009 was used. FINDINGS: Fifty studies exploring oral, intravenous, rectal, and intramuscular route (1470 persons, volunteers and patients) were included. Correlations between artesunate doses and Cmax or AUC0-∞ of dihydroartemisinin (DHA) and DHA+ART were evaluated. This correlation was good (R2>0.9) using intravenous (IV) route. DHA and ART+DHA average concentrations (Cav) were well above estimated in vivo half-maximal effective concentration (EC50) for intravenous route, but this was not the case for oral route. INTERPRETATION: The favorable Cav/EC50 ratio for IV route provides evidence that IV ART will remain efficient even in the case of increased resistance level, whereas for the oral route, a two-fold increase in EC50 may lead to therapeutic failures, thus providing a rationale for oral dose escalation. Considering the inter-individual variability of ART pharmacokinetic, Therapeutic Drug Monitoring through antimalarial stewardship activities is needed to optimize drug exposure and avoid resistance development.

Interaction between environment, nutrient-derived metabolites and immunity: A possible role in malaria susceptibility/resistance in Fulani and Dogon of Mali.

The role of some nutrient-derived metabolites on the innate and adaptive immune responses is now established. Global research approach investigating the interplay between environment, lifestyle and the host's immune responses is crucial in the understanding of malaria susceptibility. Advanced Glycation end products (AGE), which are food-derived metabolites result from the link between reducing sugar and amino group of proteins, lipids or nucleic acids. The level of exposure to AGEs varies depending on the type of diet. The dysfunction of the immune system induced by AGE and the cellular receptors for AGEs (RAGE) in susceptibility to bacterial infection has been described. But no study has yet explored their role in susceptibility to malaria. Therefore, we aimed to evaluate systemic AGE and RAGE gene polymorphism in two sympatric populations with previously described difference of susceptibility to malaria. We measured by ELISA the plasma levels of AGEs, and their soluble receptors (sRAGE) from 170 volunteers (68 Fulani and 102 Dogon). We also determined by real-time quantitative PCR the expression of RAGE, and the -374 T/A, -429 T/C polymorphisms and 63 bp deletion by fragment length restriction polymorphism. The prevalence rate of Plasmodium in Fulani and Dogon were respectively 42.64% and 51.30% for P. falciparum, 5.88% and 6.5% for P. malariae, 0% and 2.6% for P. ovale. The average AGE was 12.65 µg/ml, and 496.48pg/ml for sRAGE. Highest levels of sRAGE were observed in Fulani (563,07pg/ml, 95% CI [547.81-580.13] vs 465.68pg/ml, 95% CI [331.19-467.51]) for Dogon, p = 0.00001. Fulani had the lowest mean of AGE (10.21µg/ml, 95% CI [8.02-10.92]) compared to Dogon (16.88µg/ml, 95% CI [13.92-17.96]; p = 0.00001. RAGE was more expressed in Dogon than Fulani (0.08 vs 0.04), P = 0.08. The -374A polymorphism vas more frequent in Fulani (32%) compared to Dogon (20%). The chronic exposure to dietary AGE could lead to immune responses impairment and polymorphism with implications in malaria susceptibility. More studies are necessary to better investigate this hypothesis.

Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for screening patients with imported malaria in a non-endemic setting.

BACKGROUND: Sensitive and easy-to-perform methods for the diagnosis of malaria are not yet available. Improving the limit of detection and following the requirements for certification are issues to be addressed in both endemic and non-endemic settings. The aim of this study was to test whether loop-mediated isothermal amplification of DNA (LAMP) may be an alternative to microscopy or real-time PCR for the screening of imported malaria cases in non-endemic area. RESULTS: 310 blood samples associated with 829 suspected cases of imported malaria were tested during a one year period. Microscopy (thin and thick stained blood slides, reference standard) was used for the diagnosis. Real-time PCR was used as a standard of truth, and LAMP (Meridian Malaria Plus) was used as an index test in a prospective study conducted following the Standards for Reporting Diagnosis Accuracy Studies. In the 83 positive samples, species identification was P. falciparum (n = 66), P. ovale (n = 9), P. vivax (n = 3) P. malariae (n = 3) and 2 co-infections with P. falciparum + P.malariae. Using LAMP methods, 93 samples gave positive results, including 4 false-positives. Sensitivity, specificity, positive predictive value and negative predictive value for LAMP tests were 100%, 98.13%, 95.51%, and 100% compared to PCR. CONCLUSION: High negative predictive value, and limit of detection suggest that LAMP can be used for screening of imported malaria cases in non-endemic countries when expert microscopists are not immediately available. However, the rare occurrence of non-valid results and the need for species identification and quantification of positive samples preclude the use of LAMP as a single reference method.

7-Chloro-4-aminoquinoline γ-hydroxy-γ-lactam derived-tetramates as a new family of antimalarial compounds.

In this Letter we report on an efficient and short 2-3 steps synthesis of γ-hydroxy-γ-lactam derived-tetramates bearing a 7-chloro-4-aminoquinoline skeleton and their evaluation as potent antimalarials. These molecules were obtained through ring opening-ring closure (RORC) process of γ-ylidene-tetronate derivatives in the presence of 7-chloro-4-aminoquinoline-derived amines. In vitro antimalarial activity of these new γ-lactams was evaluated against Plasmodium falciparum clones of variable sensitivity (3D7 and W2) and they were found to be active in the range of 14-827nM with generally good resistance index. A preliminary SAR study is also presented to explain these results. Finally, the most active compounds did not show in vitro cytotoxicity when tested against Human Umbilical Vein Endothelial Cells (HUVEC) up to concentration of 50µM and they were stable at pH 7.4 for at least 48h.

Drying anti-malarial drugs in vitro tests to outsource SYBR green assays.

BACKGROUND: Measurement of anti-malarial drug efficacy and resistance relies mainly on in vivo clinical trials, in vitro/ex vivo assays and molecular markers detection. The existing in vitro/ex vivo assays, in particular those that are using non-radioactive devices, need to be standardized and adapted to field conditions. SYBR Green assay offers a rapid and cheap alternative to other in vitro assays, but it requires tools not commonly available in field laboratories. Here is described a modified SYBR green I protocol to perform the parasite growth test with blood samples in endemic areas, followed later by the SYBR green fluorescence assay performed at a specialized laboratory level. METHODS: In vitro susceptibility of Plasmodium falciparum clones HB3, 3D7, W2 and 7G8 to chloroquine (CQ), dihydroartemisinin (DHA), pyronaridine (PYD) and piperaquine (PPQ) was tested. Fresh isolates of P. falciparum from imported malaria cases were collected for ex vivo assays. The parasite suspension was added in 96-well plates predosed with anti-malarial drugs and incubated for 72 hours at 37°C, 5% CO2. SYBR green I protocol was modified to dry the plates after freeze-thawed process to mimic storage and shipping conditions. The plates were rehydrated with 200 µl of complete RPMI medium for fluorescence assay. RESULTS: There were no significant differences in IC50 values of CQ, DHA, PYD and PPQ, determined by the modified protocol, compared to standard protocol. Longer storage did not affect the IC50 values. CONCLUSION: The SYBR green I modified protocol produced reliable results and could be a suitable method for in vitro/ex vivo assays in field.

Efficacy of intranasal administration of artesunate in experimental cerebral malaria.

BACKGROUND: Improving management of patients suffering from cerebral malaria is needed to reduce the devastating mortality and morbidity of the disease in endemic areas. Intravenous artesunate is currently the first-line treatment, but the lack of material and skills in the field make it difficult to implement in endemic areas. Intranasal route provides a very easy and direct gateway to blood and brain to deliver medications, by-passing the brain blood barrier. Therefore, it could be helpful and suitable to administer artesunate in the context of cerebral malaria, especially in young children. In this study, intranasal administration of artesunate to rescue from cerebral malaria using a murine model was tested. METHODS: CBA/J mice infected with Plasmodium berghei ANKA strain received artesunate (20 mg/kg) or a placebo solution intranasally, either on day 5, 6 or 7 post-infection, during a controlled, blinded, randomized trial. Primary endpoint was mortality on day 12 post-infection. Secondary endpoints were parasitaemia and clinical stage. Pharmacokinetics data following administration were collected in blood and brains of treated mice. Local toxicity was evaluated by histopathologic examination of brain and nasal sections in blinded manner. RESULTS: Intranasal administration of artesunate dramatically reduced the mortality rate (p < 0.001), preventing death in most cases. Parasitaemia loads decreased by 88.7% (61.8-100%) within 24 hours after administration. Symptoms of cerebral malaria were prevented or reversed. Dihydroartemisinin was detected in mice blood and brain within 15 minutes of intranasal administration. No direct nasal or brain toxicity was detected. CONCLUSION: Intranasal delivery is an efficient route to timely and efficiently administer artesunate and therefore may contribute to decreasing malaria-related mortality.

New route to the 5-((arylthio- and heteroarylthio)methylene)-3-(2,2,2-trifluoroethyl)-furan-2(5H)-ones--key intermediates in the synthesis of 4-aminoquinoline γ-lactams as potent antimalarial compounds.

In this Letter we report on a multi-step synthesis of 5-((arylthio- and heteroarylthio)-methylene)-3-(2,2,2-trifluoroethyl)furan-2(5H)-ones starting from γ-keto thiolester or γ-keto carboxylic acid. The key intermediate γ-lactones were then reacted with 4-aminoquinoline-derived amines via ring opening-ring closure (RORC) process affording the corresponding γ-hydroxy-γ-lactams in moderate to good yields. In vitro antimalarial activity of the resulting new 4-aminoquinoline γ-lactams were evaluated against Plasmodium falciparum clones of variable sensitivity (3D7 and W2) and were found to be active in the range of 89-1600 nM with good resistance index and did not show cytotoxicity in vitro when tested against human umbilical vein endothelial cells (HUVEC) up to concentration of 50 µM.