Cultured Plasmodium Falciparum Research Articles

Inhibition of secondary metabolite extract of Streptomyces sp. on Plasmodium falciparum in vitro: A study of soil sediment of Papua's Hamadi mangrove forest

Background: Reports about the resistance of antimalarial drugs demand exploration and development of new drugs. Indonesia's vast maritime area and abundance of mangrove forests have the potential for the development and discovery of active anti-cancer substances, antibiotics and antiplasmodial. Studies on the potential of new drugs from this sector are still limited. Objective: The purpose of this study was to see the potential of Actinomycetes genus Streptomyces sp. as an antiplasmodial in the Hamadi Coast of Papua. Methods: Retrieval of mangrove soil sediments to isolate Streptomyces was located in the Hamadi-Jayapura mangrove forest. Selective isolation was conducted by SCA (Starch Casein Agar) selective media. Identification of Streptomyces sp was examined microscopically through observation of colony morphology and gram staining. The results of isolation of Streptomyces were then fermented on FM3 media until finally secondary metabolite extract of Streptomyces was obtained. The secondary metabolite extract of Streptomyces was tested for its inhibition on the development of Plasmodium falciparum. Result: Analysis of inhibition of the secondary metabolite extract of Streptomyces sp on the development of Plasmodium falciparum showed good results because the extract of Streptomyces sp with a concentration of 100 µg/mL could suppress average growth values of Plasmodium falciparum to only 0.66% with an average inhibitory value of 95.20%. This value followed levels of extract concentration by the lowest concentration of 0.01 µg/mL, the average value of Plasmodium falciparum growth which increased to 10.89% with an average inhibition value of 20.83%. IC50 analysis of Plasmodium falciparum in culture for 48 hours was 0.12 µg/mL, and this value was very good because a test of substance has very good inhibitory activity when the IC50 value is = 10 µg/mL. Conclusion: Streptomyces’ secondary metabolite extract from mangrove sediments showed a very good ability to inhibit the growth of Plasmodium falciparum so that it could have potential as a source of antiplasmodial.

Human microglia respond to malaria‐induced extracellular vesicles

Background and ObjectiveMicroglia are the chief immune cells of the brain and have been reported to be activated in severe malaria. Their activation may drive towards neuroinflammation in cerebral malaria. Malaria‐infected red blood cell derived‐extracellular vesicles (MiREVs) are produced during the blood stage of malaria infection. They mediate intercellular communication and immune regulation among other functions. During cerebral malaria, the breakdown of the blood‐brain barrier can promote the migration of substances such as MiREVs from the periphery into the brain targeting cells such as microglia. Microglia and extracellular vesicles interactions in different pathological conditions have been reported to induce neuroinflammation. Unlike in astrocytes, microglia‐extracellular vesicle interaction has not yet been described in malaria infection. Therefore, in this study, we aimed at investigating the uptake of malaria‐infected red blood cell derived‐extracellular vesicles (MiREVs) by human microglia cells and their cytokine response.MethodHuman blood monocyte‐derived microglia were generated from buffy coats of anonymous healthy donors using Ficoll‐Paque density gradient centrifugation. The malaria infected red blood cell derived‐EVs (MiREVs) were isolated from the Plasmodium falciparum cultures. They were purified by ultracentrifugation and labelled with PKH67 green fluorescent dye. The internalization of MiREVs by MoMi was observed after 4hours of co‐incubation on coverslips placed in a 24‐well plate at 37°C using confocal microscopy. Cytokine‐gene expression was investigated using rt‐qPCR following the stimulation of the MoMi cells with supernatants from the parasite cultures at 2,4 and 24h respectively.Results and ConclusionMiREVs were internalized by the microglia and accumulated in the perinuclear region. MiREVs‐treated cells increased gene expression of the inflammatory cytokine TNFα and reduced gene expression of the immune suppressive IL‐10. Overall, the results indicate that MiREVs may act on microglia which would contribute to enhanced inflammation in cerebral malaria.

γδ T Cells Kill Plasmodium falciparum in a Granzyme- and Granulysin-Dependent Mechanism during the Late Blood Stage

Plasmodium spp., the causative agent of malaria, have a complex life cycle. The exponential growth of the parasites during the blood stage is responsible for almost all malaria-associated morbidity and mortality. Therefore, tight immune control of the intraerythrocytic replication of the parasite is essential to prevent clinical malaria. Despite evidence that the particular lymphocyte subset of γδ T cells contributes to protective immunity during the blood stage in naive hosts, their precise inhibitory mechanisms remain unclear. Using human PBMCs, we confirmed in this study that γδ T cells specifically and massively expanded upon activation with Plasmodium falciparum culture supernatant. We also demonstrate that these activated cells gain cytolytic potential by upregulating cytotoxic effector proteins and IFN-γ. The killer cells bound to infected RBCs and killed intracellular P. falciparum via the transfer of the granzymes, which was mediated by granulysin in a stage-specific manner. Several vital plasmodial proteins were efficiently destroyed by granzyme B, suggesting proteolytic degradation of these proteins as essential in the lymphocyte-mediated death pathway. Overall, these data establish a granzyme- and granulysin-mediated innate immune mechanism exerted by γδ T cells to kill late-stage blood-residing P. falciparum.

Evaluating Antiplasmodial and Antimalarial Activities of Soybean (Glycine max) Seed Extracts on P. falciparum Parasite Cultures and P. berghei-Infected Mice.

Background Plasmodium parasite resistance to artemisinin-based combination therapies (ACTs) calls for development of new, affordable, safe, and effective antimalarial drugs. Studies conducted previously on soybean extracts have established that they possess antimicrobial, anti-inflammatory, anticancerous, and antioxidant properties. The activity of such extracts on Plasmodium parasite resistance to artemisinin-based combination therapies (ACTs) calls for development of new, affordable, safe, and effective antimalarial drugs. Studies conducted previously on soybean extracts have established that they possess antimicrobial, anti-inflammatory, anticancerous, and antioxidant properties. The activity of such extracts on Objectives The aim of this study was to determine the antiplasmodial activity of soybean extracts using Plasmodium falciparum cultures, followed by an in vivo evaluation of safety and antimalarial activity of the extracts in Plasmodium berghei ANKA strain-infected mice. Method Aqueous, methanol, and peptide extracts of soybean seeds were prepared. An in vitro evaluation of the extracts for antiplasmodial activity was carried out using two P. falciparum strains: D6, a chloroquine-sensitive Sierra Leone 1 strain and W2, a chloroquine-resistant Indochina 1 strain. Following the in vitro evaluation of the extracts for antiplasmodial activity was carried out using two in vivo evaluation of safety and antimalarial activity of the extracts in P. berghei ANKA strain. The two extracts were tested for their therapeutic potential (curative test). The peptide extract was further assessed to determine whether it could prevent the establishment of a P. berghei ANKA strain. The two extracts were tested for their therapeutic potential (curative test). The peptide extract was further assessed to determine whether it could prevent the establishment of a P. berghei ANKA strain. The two extracts were tested for their therapeutic potential (curative test). The peptide extract was further assessed to determine whether it could prevent the establishment of a Results Peptide and methanol extracts showed good activity with IC50 of 19.97 ± 2.57 μg/ml and 10.14 ± 9.04 μg/ml and 10.14 ± 9.04 μg/ml and 10.14 ± 9.04 μg/ml and 10.14 ± 9.04 P < 0.001) in suppression with lower doses. Conclusion The results show the presence of antimalarial properties in soybean extracts with higher curative activity when compared to the prophylactic activity. However, more research needs to be conducted on this plant to possibly establish lead compounds.

English

The Arabs, Asians and, Traditional Health Practitioners in Mombasa county found in Kenya have been using Nigella sativa L. seeds to traditionally manage malaria associated symptoms that is, headache, fever, chills, loss of appetite among others. The present study investigated in vitro antiplasmodial, in vivo antimalarial activities and safety of different extracts of N. sativa. Five extracts obtained via aqueous extraction and sequential extraction using hexane, dichloromethane, ethyl acetate and methanol were tested against in vitro cultures of Plasmodium falciparum. The most active extracts (methanolic and ethyl acetate) were assessed for cytotoxicity and toxicity. The two active extracts were evaluated in vivo against Plasmodium berghei ANKA strain at 500, 250 and 125 mg/kg/day. On in vitro assay, methanolic and ethyl acetate extracts showed good activity with IC50 of 80.48±12.29 and 69.81±5.24 µg/ml against W2 strain and 31.93±4.31 and 53.79±6.02 µg/ml against D6 strain, respectively. The extracts exhibited weak cytotoxicity on Vero cells and high parasitemia suppression of 75.52 and 75.30% at 500 mg/kg dose of methanolic and ethyl acetate extracts respectively. Notably, there was significant decrease (p<0.001) in activity with lower doses of the extracts. The results explain the traditional use of this plant in the Middle East and Mombasa County. Key words: Nigella sativa L. seeds extracts, Plasmodium, antimalarial activity.

Human Microglia Respond to Malaria-Induced Extracellular Vesicles.

Microglia are the chief immune cells of the brain and have been reported to be activated in severe malaria. Their activation may drive towards neuroinflammation in cerebral malaria. Malaria-infected red blood cell derived-extracellular vesicles (MiREVs) are produced during the blood stage of malaria infection. They mediate intercellular communication and immune regulation, among other functions. During cerebral malaria, the breakdown of the blood–brain barrier can promote the migration of substances such as MiREVs from the periphery into the brain, targeting cells such as microglia. Microglia and extracellular vesicle interactions in different pathological conditions have been reported to induce neuroinflammation. Unlike in astrocytes, microglia–extracellular vesicle interaction has not yet been described in malaria infection. Therefore, in this study, we aimed to investigate the uptake of MiREVs by human microglia cells and their cytokine response. Human blood monocyte-derived microglia (MoMi) were generated from buffy coats of anonymous healthy donors using Ficoll-Paque density gradient centrifugation. The MiREVs were isolated from the Plasmodium falciparum cultures. They were purified by ultracentrifugation and labeled with PKH67 green fluorescent dye. The internalization of MiREVs by MoMi was observed after 4 h of co-incubation on coverslips placed in a 24-well plate at 37 °C using confocal microscopy. Cytokine-gene expression was investigated using rt-qPCR, following the stimulation of the MoMi cells with supernatants from the parasite cultures at 2, 4, and 24 h, respectively. MiREVs were internalized by the microglia and accumulated in the perinuclear region. MiREVs-treated cells increased gene expression of the inflammatory cytokine TNFα and reduced gene expression of the immune suppressive IL-10. Overall, the results indicate that MiREVs may act on microglia, which would contribute to enhanced inflammation in cerebral malaria.

In vitro and in vivo antimalarial activity of the volatile oil of Cyperus articulatus (Cyperaceae)

ABSTRACT Malaria is a disease of global tropical distribution, being endemic in more than 90 countries and responsible for about 212 million cases worldwide in 2016. To date, the strategies used to eradicate this disease have been ineffective, without specific preventive measures such as vaccines. Currently, the existing therapeutic arsenal is limited and has become ineffective against the expansion of artemisinin-resistant Plasmodium, demonstrating the need for studies that would allow the development of new compounds against this disease. In this context, we studied the volatile oil obtained from rhizomes of Cyperus articulatus (VOCA), a plant species commonly found in the Amazon region and popularly used as a therapeutic alternative for the treatment of malaria, in order to confirm its potential as an antimalarial agent by in vitro and in vivo assays. We cultured Plasmodium falciparum W2 (chloroquine-resistant) and 3D7 (chloroquine-sensitive) strains in erythrocytes and exposed them to VOCA at different concentrations in 96-well microplates. In vivo antimalarial activity was tested in BALB/c mice inoculated with approximately 106 erythrocytes infected with Plasmodium berghei. VOCA showed a high antimalarial potential against the two P. falciparum strains, with IC50 = 1.21 μg mL-1 for W2 and 2.30 μg mL-1 for 3D7. VOCA also significantly reduced the parasitemia and anemia induced by P. berghei in mice. Our results confirmed the antimalarial potential of the volatile oil of Cyperus articulatus.

Simultaneous Proton Transfer Reaction-Mass Spectrometry and electronic nose study of the volatile compounds released by Plasmodium falciparum infected red blood cells in vitro

The discovery that Volatile Organic Compounds (VOCs) can be biomarkers for several diseases has led to the conception of their possible application as diagnostic tools. In this study, we aimed at defining of diagnostic signatures for the presence of malaria transmissible stages in infected individuals. To do this, we compared VOCs released by asexual and sexual stage cultures of Plasmodium falciparum, the deadliest species of malaria, with those emitted by uninfected red blood cells (RBCs). VOC analysis was carried out with an innovative set-up, where each sample was simultaneously analysed by proton transfer reaction time of flight mass spectrometry (PTR-ToF-MS) and an electronic nose. PTR-Tof-MS results show that sexual stages are characterized by a larger emission of hexanal, compared with uninfected or asexual stage-infected RBCs, which makes them clearly identifiable. PTR-Tof-MS analysis also detected differences in VOC composition between asexual stages and uninfected RBCs. These results have been substantially replicated by the electronic nose analysis and may open the possibility to develop sensitive and easy-to-use devices able to detect sexual parasite stages in infected individuals. This study also demonstrates that the combination of mass spectrometry with electronic noses is a useful tool to identify markers of diseases and to support the development of optimized sensors.

An ImmunoPEGliposome for Targeted Antimalarial Combination Therapy at the Nanoscale.

Combination therapies, where two drugs acting through different mechanisms are administered simultaneously, are one of the most efficient approaches currently used to treat malaria infections. However, the different pharmacokinetic profiles often exhibited by the combined drugs tend to decrease treatment efficacy as the compounds are usually eliminated from the circulation at different rates. To circumvent this obstacle, we have engineered an immunoliposomal nanovector encapsulating hydrophilic and lipophilic compounds in its lumen and lipid bilayer, respectively. The antimalarial domiphen bromide has been encapsulated in the liposome membrane with good efficiency, although its high IC50 of ca. 1 µM for living parasites complicates its use as immunoliposomal therapy due to erythrocyte agglutination. The conjugation of antibodies against glycophorin A targeted the nanocarriers to Plasmodium-infected red blood cells and to gametocytes, the sole malaria parasite stage responsible for the transmission from the human to the mosquito vector. The antimalarials pyronaridine and atovaquone, which block the development of gametocytes, have been co-encapsulated in glycophorin A-targeted immunoliposomes. The co-immunoliposomized drugs have activities significantly higher than their free forms when tested in in vitro Plasmodium falciparum cultures: Pyronaridine and atovaquone concentrations that, when encapsulated in immunoliposomes, resulted in a 50% inhibition of parasite growth had no effect on the viability of the pathogen when used as free drugs.

Efficient synchronization of Plasmodium knowlesi in vitro cultures using guanidine hydrochloride

BackgroundLong-term in vitro culture of blood stage Plasmodium parasites invariably leads to asynchronous parasite development. The most often used technique to synchronize Plasmodium falciparum culture is sorbitol treatment, which differentially induces osmotic lysis of trophozoite- and schizont-infected red blood cells due to presence of the new permeation pathways in the membranes of these cells. However, sorbitol treatment does not work well when used to synchronize the culture-adapted Plasmodium knowlesi A1-H.1 line.MethodsA number of common solutes were tested in lieu of sorbitol for synchronization of P. knowlesi A1-H.1 ring stage.ResultsGuanidine hydrochloride was found to selectively lyse trophozoite- and schizont-infected red blood cells, yielding highly synchronous and viable rings.ConclusionsA method for synchronization of P. knowlesi in human red blood cells was developed. Requiring only common laboratory reagents, this method is simple and should be applicable to most laboratory settings.

Gamma Delta T Cells and Its Interactions with Extracellular Vesicles during Malaria

Background and ObjectiveExtracellular vesicles (EVs) have been described as microparticles produced by different cells in the body with the capacity of mediating cell‐cell communication. A clear mechanism of how various immune cells function during malaria infection is still undergoing investigations. With various states and roles of gamma delta T cells reported, its role in human cerebral malaria has not yet been defined as a study has suggested that it is essential in the development of murine cerebral malaria through its production of interferon gamma and tumor necrosis factor. However, there is currently no report on interactions between EVs derived from Plasmodium falciparum infected red blood cells (iRC‐EVs) and human gamma delta T cells. Our aim therefore, was to investigate the interactions between iRBC‐EVs and gamma delta T cells and the resulting immunological responses that could play a role in the pathogenesis of cerebral malaria.MethodBy using Ultracentrifugation, EVs derived from Plasmodium falciparum infected red blood cell (iRBC‐EVs) culture were isolated and purified. The iRBC‐EVs were characterized by western blot, Fast protein liquid chromatography and electron microscopy. Gamma delta T cells were generated from peripheral blood mononuclear cells(PBMCs) isolated from human blood and stimulated with Plasmodium falciparum culture supernatant and IL‐2 for 14 days. The generated gamma delta T cells were treated with the iRBC‐EVs and monitored for uptake, proliferation and activation using confocal microscopy and flow cytometry.Results and ConclusionWe found out that gamma delta T cells are capable of taking up iRBC‐EVs and were proliferating on co‐incubation with iRBC‐EVs and Plasmodium falciparum culture supernatant. In conclusion, our results show that there is an influence of iRBC‐EVs on gamma delta T cells which would form a basis for further investigations on the immunological responses mediated by these cells towards the development of cerebral malaria.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Docking predictions based Plasmodium falciparum phosphoethanolamine methyl transferase inhibitor identification and in-vitro antimalarial activity analysis

The increased multidrug resistance among antimalarial drugs produces the urgency of potent anti malarial to combat resistant malaria and the malaria burden worldwide. The protein which may prevent the growth or transmission of malaria parasite may be the great target for rational drug designing. Plasmodium falciparum phosphoethanolamine methyltransferase (Pfpmt) absent in human catalyzes triple methylation of ethanolamine into phosphocholine for phosphatidylcholine biosynthesis from serine decarboxylation phosphoethanolamine methyltransferase pathway for the membrane development at asexual as well as sexual stages of parasite. The Plasmodium requires production of membrane rapidly for growth and multiplication. Hence, the phosphoethanolamine methyltransferase of Plasmodium falciparum was selected as drug target for rational drug designing. Using Discovery studio 3.5 software the library of zinc compounds was screened against target and analyzed. The compounds with better druglike properties and docking affinity and binding interaction for target protein were procured for in vitro analysis against Plasmodium falciparum culture (IC50). Compounds ZINC02103914 and ZINC12882412 were found to have good druglike properties and affinity for Pfpmt also inhibited P. falciparum growth at very low µM IC50 concentration 3.0 µM and 2.1 µM respectively also found nontoxic in vitro against HEK-293 cells. Simulation study of best inhibitor revealed the specificity for the target protein. Hence, the compounds possessed the immense probability of being inhibitors of Pfpmt and may be optimized as antimalarial agent for combinational therapy to overcome the multidrug resistance and may also be used as template for optimization and rational drug designing.

Pre-Clinical Study of Iron Nanoparticles Fortified Artesunate for Efficient Targeting of Malarial Parasite

Background: Artesunate the most potent antimalarial is widely used for the treatment of multidrug-resistant malaria. The antimalarial cytotoxicity of Artesunate has been mainly attributed to its selective, irreversible and iron-catalyzed radical mediated damage of parasite biomolecules. In the present research, iron nanoparticle fortified artesunate was tested in P. falciparum and in an experimental malaria mouse model for enhancement in the selectivity and toxicity of Artesunate towards parasite. Artesunate was fortified with nontoxic biocompatible surface modified iron nanoparticle which is specially designed and synthesized for sustained pH-dependent release of Fe2+ within the parasitic food vacuole for enhanced ROS spurt. Methods: Antimalarial efficacy of Iron nanoparticle fortified Artesunate was evaluated in Plasmodium falciparum culture grown in O +ve human blood and in Plasmodium berghei ANKA infected swiss albino mice. Infected mice were daily administered intraperitoneally with Artesunate, nanoparticle fortified Artesunate and monitored for parasitemia. Internalization of iron nanoparticles, pH dependent release of Fe2+, production of reactive oxygen species and parasite biomolecule damage by iron nanoparticle fortified Artesunate was studied using various biochemical, biophysical, ultra-structural and fluorescence microscopy. Results: The nanoparticle fortified Artesunate was highly efficient in 1/8th concentration of Artesunate IC50 and led to retarded growth of P. falciparum with significant damage to macromolecules mediated via enhanced ROS production. Similarly, preclinical In vivo studies also signified a radical reduction in parasitemia with ~8-10-fold reduced dosage of Artesunate when fortified with iron nanoparticles. Interpretation: Surface coated iron-nanoparticle fortified Artesunate can be developed into a potent therapeutic agent towards multidrug-resistant malaria in humans. Funding Statement: This study is supported by Center for Study of Complex malaria in India funded by National Institute of Health, USA, and by Department of Science and Technology (DST-INDIA) and LRE JNU. DK and NY are supported by Shiv Nadar Foundation fellowships. SA is funded by ICMR, Government of India. Declaration of Interests: The authors declare that there are no conflicts of interest. Ethics Approval Statement: Animal studies were performed in accordance with guidelines of the Institutional Animal Ethics Committee (IEAC) of Jawaharlal Nehru University, Delhi and Committee for Control and Supervision of Experiments on Animals (CPCSEA).

Quantification of Free Ferriprotoporphyrin IX Heme and Hemozoin for Artemisinin Sensitive versus Delayed Clearance Phenotype Plasmodium falciparum Malarial Parasites.

We use Plasmodium falciparum culture synchronization, optimized heme and hemozoin extraction protocols, and mass spectrometry to quantify the abundance of free ferriprotoporphyrin IX (FPIX) heme and crystallized FPIX (hemozoin; Hz) for various growth stages of intraerythrocytic P. falciparum malarial parasites. Because of altered cell cycle kinetics for delayed clearance phenotype (DCP) parasites relative to that of the control, we test whether FPIX and Hz abundances differ for DCP and control parasites.

Small-scale culture of Plasmodium falciparum using μ-Slide Angiogenesis followed by automatic infection rate counting to assess drug effects

It is very important to reduce the costs involved in malarial drug development by small-scale culture of Plasmodium falciparum, and automation of the assay system for drug efficacy against the parasites for high-throughput screening. In this study, we report that P. falciparum-infected erythrocytes can be stably cultured on μ-Slide Angiogenesis, which is used to investigate angiogenesis in tube formation assays, followed by automatic counting of the infection rate (parasitaemia). After 10 μL of parasite-infected erythrocytes were added to the inner well of μ-Slide Angiogenesis to prevent a multilayer of erythrocytes, 30 μL of silicon oil was overlaid on the culture medium to avoid evaporation of the medium, leading to stable small-scale parasite cultivation. The parasites were stained with a cell-permeant fluorescent nucleic acid stain (SYTO21) followed by cultivation. After taking bright field and fluorescent images using an inverted microscope, the infection rate could be calculated automatically by counting the number of erythrocytes and parasites using MetaMorph Offline software. The effect of anti-malarial drugs on parasite growth could be investigated on μ-Slide Angiogenesis, in which the parasite culture was added to the inner wells containing the drugs followed by their cultivation. Taken together, this method may be useful for image-based screening for anti-malarial drug candidates with automatic counting of parasite infection rates.

Determining the Mode of Action of Antimalarial Drugs Using Time-Resolved LC-MS-Based Metabolite Profiling.

Methods for assessing the mode of action of new antimalarial compounds identified in high throughput phenotypic screens are needed to triage and facilitate lead compound development and to anticipate potential resistance mechanisms that might emerge. Here we describe a mass spectrometry-based approach for detecting metabolic changes in asexual erythrocytic stages of Plasmodium falciparum induced by antimalarial compounds. Time-resolved or concentration-resolved measurements are used to discriminate between putative targets of the compound and nonspecific and/or downstream secondary metabolic effects. These protocols can also be coupled with 13C-stable-isotope tracing experiments under nonequilibrative (or nonstationary) conditions to measure metabolic dynamics following drug exposure. Time-resolved 13C-labeling studies greatly increase confidence in target assignment and provide a more comprehensive understanding of the metabolic perturbations induced by small molecule inhibitors. The protocol provides details on the experimental design, Plasmodium falciparum culture, sample preparation, analytical approaches, and data analysis used in either targeted (pathway focused) or untargeted (all detected metabolites) analysis of drug-induced metabolic perturbations.

Identification of (4-(9H-fluoren-9-yl) piperazin-1-yl) methanone derivatives as falcipain 2 inhibitors active against Plasmodium falciparum cultures

BackgroundFalcipain 2 (FP-2) is the hemoglobin-degrading cysteine protease of Plasmodium falciparum most extensively targeted to develop novel antimalarials. However, no commercial antimalarial drugs based on FP-2 inhibition are available yet due to the low selectivity of most FP-2 inhibitors against the human cysteine proteases. MethodsA structure-based virtual screening (SVBS) using Maybridge HitFinder™ compound database was conducted to identify potential FP-2 inhibitors. In vitro enzymatic and cell-growth inhibition assays were performed for the top-scoring compounds. Docking, molecular dynamics (MD) simulations and free energy calculations were employed to study the interaction of the best hits with FP-2 and other related enzymes. Results and conclusionsTwo hits based on 4-(9H-fluoren-9-yl) piperazin-1-yl) methanone scaffold, HTS07940 and HTS08262, were identified as inhibitors of FP-2 (half-maximal inhibitory concentration (IC50) = 64 μM and 14.7 μM, respectively) without a detectable inhibition against the human off-target cathepsin K (hCatK). HTS07940 and HTS08262 inhibited the growth of the multidrug-resistant P. falciparum strain FCR3 in culture (half-maximal inhibitory concentrations (IC50) = 2.91 μM and 34 μM, respectively) and exhibited only moderate cytotoxicity against HeLa cells (Half-maximal cytotoxic concentration (CC50) = 133 μM and 350 μM, respectively). Free energy calculations reproduced the experimental affinities of the hits for FP-2 and explained the selectivity with respect to hCatK. General significanceTo the best of our knowledge, HTS07940 stands among the most selective FP-2 inhibitors identified by SBVS reported so far, displaying moderate antiplasmodial activity and low cytotoxicity against human cells. Hence, this compound constitutes a promising lead for the design of more potent and selective FP-2 inhibitors.

In situ loop-mediated isothermal amplification (LAMP) for identification of Plasmodium species in wide-range thin blood smears

BackgroundFive species of Plasmodium are known to infect humans. For proper treatment of malaria, accurate identification of the parasite species is crucial. The current gold standard for malaria diagnosis is microscopic examination of Giemsa-stained blood smears. Since the parasite species are identified by microscopists who manually search for the parasite-infected red blood cells (RBCs), misdiagnosis due to human error tends to occur in case of low parasitaemia or mixed infection. Then, molecular methods, such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are required for conclusive identification of the parasite species. However, since molecular methods are highly sensitive, false-positive results tend to occur due to contamination (carry over) or the target gene products may be detected even after clearance of the parasites from the patient’s blood. Therefore, accurate detection of parasites themselves by microscopic examination is essential for the definitive diagnosis. Thus, the method of in situ LAMP for the parasites was developed.ResultsRed blood cell suspensions, including cultured Plasmodium falciparum, strain 3D7, infected-RBCs, were dispersed on cyclic olefin copolymer (COC) plate surfaces rendered hydrophilic by reactive ion-etching treatment using a SAMCO RIE system (hydrophilic-treated), followed by standing for 10 min to allow the RBCs to settle down on the plate surface. By rinsing the plate with RPMI 1640 medium, monolayers of RBCs formed on almost the entire plate surface. The plate was then dried with a hair drier. The RBCs were fixed with formalin, followed by permeabilization with Triton X-100. Then, amplification of the P. falciparum 18S rRNA gene by the LAMP reaction with digoxigenin (DIG)-labelled dUTP and a specific primer set was performed. Infected RBCs as fluorescence-positive cells with anti-DIG antibodies conjugated with fluorescein using fluorescent microscopy could be detected.ConclusionsThe present work shows that the potential of in situ LAMP for the identification of Plasmodium species at the single cell level on hydrophilic-treated COC palates, allowing highly sensitive and accurate malaria diagnosis. The findings will improve the efficacy of the gold standard method for malaria diagnosis.

Growth of Plasmodium falciparum in response to a rotating magnetic field

BackgroundPlasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing because of pathogen drug resistance. Increasing knowledge of the parasite life cycle and mechanism of infection may provide new models for improved treatment paradigms. This study sought to investigate the paramagnetic nature of the parasite’s haemozoin to inhibit parasite viability.ResultsParamagnetic haemozoin crystals, a byproduct of the parasite’s haemoglobin digestion, interact with a rotating magnetic field, which prevents their complete formation, causing the accumulation of free haem, which is lethal to the parasites. Plasmodium falciparum cultures of different stages of intraerythrocytic growth (rings, trophozoites, and schizonts) were exposed to a magnetic field of 0.46 T at frequencies of 0 Hz (static), 1, 5, and 10 Hz for 48 h. The numbers of parasites were counted over the course of one intraerythrocytic life cycle via flow cytometry. At 10 Hz the schizont life stage was most affected by the rotating magnetic fields (p = 0.0075) as compared to a static magnetic field of the same strength. Parasite growth in the presence of a static magnetic field appears to aid parasite growth.ConclusionsSequestration of the toxic haem resulting from haemoglobin digestion is key for the parasites’ survival and the focus of almost all existing anti-malarial drugs. Understanding how the parasites create the haemozoin molecule and the disruption of its creation aids in the development of drugs to combat this disease.

Efficacy and safety of methylene blue in the treatment of malaria: a systematic review

BackgroundMethylene blue (MB) was the first synthetic antimalarial to be discovered and was used during the late 19th and early 20th centuries against all types of malaria. MB has been shown to be effective in inhibiting Plasmodium falciparum in culture, in the mouse model and in rhesus monkeys. MB was also shown to have a potent ex vivo activity against drug-resistant isolates of P. falciparum and P. vivax. In preclinical studies, MB acted synergistically with artemisinin derivates and demonstrated a strong effect on gametocyte reduction in P. falciparum. MB has, thus, been considered a potentially useful partner drug for artemisinin-based combination therapy (ACT), particularly when elimination is the final goal. The aim of this study was to review the scientific literature published until early 2017 to summarise existing knowledge on the efficacy and safety of MB in the treatment of malaria.MethodsThis systematic review followed PRISMA guidelines. Studies reporting on the efficacy and safety of MB were systematically searched for in relevant electronic databases according to a pre-designed search strategy. The search (without language restrictions) was limited to studies of humans published until February 2017.ResultsOut of 474 studies retrieved, a total of 22 articles reporting on 21 studies were eligible for analysis. The 21 included studies that reported data on 1504 malaria patients (2/3 were children). Older studies were case series and reports on MB monotherapy while recent studies were mainly controlled trials of combination regimens. MB was consistently shown to be highly effective in all endemic areas and demonstrated a strong effect on P. falciparum gametocyte reduction and synergy with ACT. MB treatment was associated with mild urogenital and gastrointestinal symptoms as well as blue coloration of urine. In G6PD-deficient African individuals, MB caused a slight but clinically non-significant haemoglobin reduction.ConclusionsMore studies are needed to define the effects of MB in P. falciparum malaria in areas outside Africa and against P. vivax malaria. Adding MB to ACT could be a valuable approach for the prevention of resistance development and for transmission reduction in control and elimination programs.Systematic review registrationThis study is registered at PROSPERO (registration number CRD42017062349).