In silico antiplasmodial effects of phytocompounds derived from Andrographis paniculata on validated drug targets of different stages of Plasmodium falciparum

Background:Andrographis paniculata has been widely reported as an herbal plant for malaria treatment.The increasing rate of resistance to recommended antimalarial drugs has justified the need for a continuous search for new and more potent drugs that target all stages of the Plasmodium falciparum life cycle from natural plant sources.This study aimed to determine the antiplasmodial effect of phytocompounds derived from A.paniculata on the stages of plasmodium falciparum.Methods:Phytocompounds from A.paniculata were identified by Gas Chromatography-Mass Spectrophotometry(GCMS)analysis.The phytocompounds were screened for their druggability using Lipinski’s rule of five and subjected to Absorption,Distribution,Metabolism,Excretion,Toxicity(ADMET)and druglikeness analysis.The phytocompounds were docked against some validated drug targets at different stages of Plasmodium falciparum(hepatic,asexual,sexual,and vector targets)using PyRx software to analyze the inhibitory potential and protein-ligand interaction.Thereafter,the stability and flexibility of the best complexes were assessed through molecular dynamics simulations at 50ns using WebGRO.Result:The 7a-Isopropenyl-4,5-dimethyloctahydroinden-4-yl exhibited a higher binding affinity and better stability throughout the simulation period with P.falciparum dihydrofolate reductase-thymidylate synthase and Plasmodium falciparum M1 alanyl aminopeptidase for asexual blood stage and gametocyte stage of Plasmodium falciparum,respectively than the existing drugs.Meanwhile,N-Ethyl-3-methoxy-4-methylphenethylamine was also found to have a higher binding affinity and more stability throughout the simulation period with P.falciparum purine nucleoside phosphorylase and Plasmodium falciparum gametocyte surface protein for Hepatic schizonts stage of Plasmodium falciparum and gametocyte transmission blocking stage,respectively,than the existing drugs.Conclusion:The 7a-Isopropenyl-4,5-dimethyloctahydroinden-4-yl and N-Ethyl-3-methoxy-4 methylphenethylamine from A.paniculata are predicted as an antimalarial drug candidate.Thus,it is recommended that in vitro and in vivo bioassays be conducted on these hit compounds to validate these predictions.

Role of toll like-receptor 2 in inflammatory activity of macrophage infected with a recombinant BCG expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum

Objective: To investigate the role of toll-like receptor 2(TLR2) in inflammatory activity of macrophage infected with the recombinant Mycobacterium bovis bacillus Calmette-Guerin(rBCG). Methods: Mouse macrophage cell line J774 A.1 was infected with Mycobacterium bovis bacillus Calmette-Guerin(BCG) and rBCG cultures for 48 h in the presence or absence of 10 μg/mL of TLR2 inhibitor. Untreated macrophages were used as a negative control while lipopolysaccharide-stimulated macrophages were used as a positive control. The ability of the macrophage to engulf the BCG and rBCG in the absence or presence of TLR2 inhibitor was assessed using a phagocytic assay, while the production of inflammatory cytokines and nitric oxide by the infected macrophages was evaluated using ELISA and Griess reagent method, while the expression of the inducible nitric oxide synthase was determined using Western blot analysis. Results: The results showed that blocking TLR2 function reduced the phagocytic activity, nitric oxide production and proinflammatory cytokine secretion such as TNF-α, IL-1β and IL-12 p40 as well as inducible nitric oxide synthase expression in the infected macrophages. These data showed the importance of TLR2 in the activation of macrophages following BCG and r BCG infections. Conclusions: Through exploring the immunological mechanism which underlies the protection conferred by the candidate vaccine, this study will improve our understanding of the vaccine candidate's mechanism to protect the host from malaria infection.

Acquisition of naturally acquired antibody response to Plasmodium falciparum erythrocyte membrane protein 1-DBLα and differential regulation of IgG subclasses in severe and uncomplicated malaria

Objectives: To explore whether individuals infected with Plasmodium falciparum(P. falciparum) develop antibodies directed against Pf EMP1-DBLa, and to assess their IgG subclass distribution in severe and uncomplicated malaria.Methods: The anti-PfDBLα IgG and their IgG subclass distributions in plasma of severe(SM) and uncomplicated malaria(UCM) were assessed by enzyme-linked immunoabsorbent assay. The antibody profiles to P. falciparum blood stage antigens were evaluated. CD36 binding ability was determined by static receptor-binding assays.Rosette formation was performed by staining with acridine orange.Results: Significantly higher number of UCM(86.48%) than SM(57.78%) plasma contained total acquisition of specific IgG to P. falciparum antigens(P = 0.000). Similar manners were seen in response to P. falciparum DBLa with significant difference(UCM,59.46% vs SM, 40.00%; P = 0.014). Anti-PfDBLα-IgG1 and-IgG3 were the predominant subclasses. Similar percentage of UCM(31.82%) and SM(33.33%) plasma contained only IgG1, while 13.64% of UCM and 27.78% of SM plasma contained only IgG3. AntiPfDBLα-IgG1 coexpressed with more than one subclass was noted(UCM, 27.27%; SM,16.67%). Obviously, IgG1 coexpressed with IgG3(9.09%) was observed in only UCM plasma. IgG1 was coexpressed with IgG2 in UCM(9.09%) and SM(11.11%) plasma,while IgG1 was coexpressed with IgG4 only in UCM plasma(4.55%). IgG subclasses to P. falciparum antigens were distributed in a similar manner. Only the levels of IgG1, but not IgG3 were significantly higher in UCM than in SM.Conclusions: These data suggest that individuals infected with P. falciparum can develop the anti-Pf EMP1 antibodies with the major contribution of specific IgG subclasses. The balance and the levels of anti-PfDBLα IgG subclasses play a crucial role in antibody mediated protection against severe malaria.

Sequence Analysis and Genotypes of Glutamate Rich Protein of Plasmodium falciparum Isolates from Different Malaria Endemic Areas in China

Objective To sequence the gene encoding glutamate rich protein (GLURP) andidentify the genotypes of geographically different Plasmodium falciparum (P. f ) isolatesfrom China. Methods The gene of R2 repeat region of GLURP was amplified by nestedpolymerase chain reaction and cloned into T-vector. The nucleotide sequence of GLURPgene was determined by automatic sequencer (Dideoxy termination method) and analyzed byDNA Star software. Results At least 7 different GLURP genotypes ranging from 600 bpto 1 500 bp were found in Yunnan and Hainan provinces. R2 region of GLURP gene consistedof several repeat units. Each repeat unit was composed of 19-20 residues which were shownto be highly conserved. GLURP gene was also size polymorphic due to differences in thenumber of repeat units, whereas the repeat sequence was conserved. Sequence analysisshowed that DNA sequences and deduced amino acid sequences were highly homologousamong the geographically dispersed isolates or various isolates from the same geographicalregion. No obvious differences were found in the GLURP gene sequences amonggeographically different isolates. Conclusion GLURP gene is highly structure conservedand size polymorphic, and so is useful in searching for malaria vaccine candidate antigen anddeveloping a genotyping method for malaria research.

Haemophagocytic lymphohistiocytosis secondary to Plasmodium falciparum malaria: Case report and review of the literature

Rationale:Haemophagocytic lymphohistiocytosis is a rare complication of malaria,which is often misdiagnosed.Patient concerns:A 30-year-old male was admitted to our department for persistent fever,which began after returning from a stay in Guinea-Conakry.The laboratory investigations revealed a pancytopenia and an elevated C-reactive protein.Peripheral smear examination showed Plasmodium falciparum,therefore confirming the diagnosis of malaria.The laboratory tests showed a worsening pancytopenia.Bone marrow aspiration and biopsy revealed images of hemophagocytosis.Diagnosis:The diagnosis of haemophagocytic lymphohistiocytosis complicating malaria infection was established.Interventions:The patient was treated with artemether-lumefantrine.No immunosuppressant treatment was delivered to the patient.He received antipyretic and antimalarial treatment only.Outcomes and lessons:We report a case of haemophagocytic lymphohistiocytosis trigged by malaria infection and we review all reported cases secondary to Plasmodium falciparum malaria by searching PubMed publications till October 2019.Haemophagocytic lymphohistiocytos secondary to malaria should be suspected even in non-severe cases of malaria.

Ethnobotanical database based screening and identification of potential plant species with antiplasmodial activity against chloroquine-sensitive(3D7) strain of Plasmodium falciparum

Objective: To evaluate the antiplasmodial activity of aqueous-methanolic plant extracts of nine plant species selected, based on ethnobotanical data. Methods: Based on ethnobotanical database, the selected plants were tested for their antiplasmodial activity against chloroquinesensitive(3 D7) strain of Plasmodium falciparum. Qualitative tests and high performance thin layer chromatography analysis were carried out to explore the phytocomponents present in the plant extracts. 1,1-diphenyl-2-picrylhydrazyl antioxidant activity was also determined to check the antioxidant activity of the plant extracts. Results: Moringa oleifera(IC_(50): 3.906 μg/mL),Acalypha indica(IC_(50): 3.906 μg/mL), Hyptis suaveolens(IC_(50): 3.906 μg/mL), Mangifera indica(IC_(50): 4.150 μg/mL) and Averrhoa bilimbi(IC_(50): 4.881 μg/mL) showed very good antiplasmodial activity. Conclusions: Crude extracts of Mangifera indica and Hyptis suaveolens demonstrated the most efficacious antimalarial activity. A bioassay-guided fractionation of these extracts to identify the lead compound is proved to be useful. The results validate the traditional use of the selected plants as antimalarials.

The role of heme-oxygenase-1 in pathogenesis of cerebral malaria in the co-culture model of human brain microvascular endothelial cell and ITG Plasmodium falciparum-infected red blood cells

Objective: To investigate the role of human host heme-oxygenase-1(HO-1) in pathogenesis of cerebral malaria in the in vitro model,Methods: The effect of human host HO-1 [human brain microvascular endothelial cell(HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells(i RBC) through measurement of the enzymatic products iron and bilirubin,Results: Following exposure to the HO-1 inducer Co PPIX at all concentrations,the HBMEC cells apoptosis occurred,which could be prominently observed at 15 μM of 3 h exposure,In contrast,there was no significant change in the morphology in the non-exposed i RBC at all concentrations and exposure time,This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin,of which the highest levels(106.03 and 1 753.54% of baseline level,respectively) were observed at 15 μM vs,20 μM at 3 h vs,24 h exposure,For the effect of the HO-1 inhibitor Zn PPIX,HBMEC cell morphology was mostly unchanged,but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h(37.17% of baseline level),The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed(highest effect at 10 μM and 3 h exposure),Conclusions: Results provide at least in part,insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.

Modeling and targeting an essential metabolic pathway of Plasmodium falciparum in apicoplast using Petri nets

Petri net(PN) is one of the promising computational and mathematical formalisms used to represent and study the behavior of complex metabolic networks. The various available analysis techniques of PN could be used to validate and analyze the network in different scenarios. Plasmodium falciparum is one of the threatening parasites which causes malaria, a deadly disease affecting a large number of today’s world population. The development of antimalarial drug resistance is an emerging global threat, highlighting the need to discover novel antimalarial targets. The fatty acid biosynthesis of malarial parasite is one of the essential metabolic pathways required for its growth and is present in apicoplast, a non-photosynthetic plastid. The malarial parasite obtains fatty acids by using type two fatty acid synthase(FAS II) enzyme,which is different from type one enzyme used by human host, making it an ideal drug target.This article proposes and studies the PN model of the parasite’s FAS II pathway to analyze the mechanism of potential drug targets in this pathway. The proposed PN model can serve as a base for further findings in the field of antimalarial drug targets to decrease the malaria mortality rate.

Investigation of the Efficacy of Home-Based Oral Chloroquine Treatment among Under-Five Children with Plasmodium falciparum Malaria in Some Parts of Jos, Plateau State, Nigeria

Background: Nigeria is currently a malaria endemic country with an estimated 76% of her population at risk of contracting malaria [1]. According to a study in Nigeria, the first line of action mothers took when their children under 5 years have malaria showed that over 50% of them used non-prescription drugs they have at home or bought from pharmacy stores. And 60% of the most commonly used drugs for malaria treatment were chloroquine [2]. Many recent studies have demonstrated re-emergence of chloroquine-sensitive P. falciparum, suggesting a possible role in future malaria control [3]. Objective: The aim of this study was to investigate the effect of home-based oral chloroquine treatment among children under 5 years with Plasmodium falciparum malaria attending Jos University Teaching Hospital and OLA Hospital in Jos Metropolis. Method: This is a cross-sectional study of 93 malaria and non-malaria children. Malaria diagnosis was carried out using microscopical examination of Leishman’s stained thick and thin blood films, P. falciparum parasitemia was assessed by standard microscopy techniques and complete blood count was done using Beckman Coulter Analyzer. Results: The body temperature on admission was significantly lower (p ˚C ± 0.07˚C) than in the three malaria groups. The mean body temperature of chloroquine treated children with malaria was significantly lower (p Conclusion: The results obtained in this study demonstrate that there was significant positive impact of chloroquine treatment on Plasmodium falciparum parasitemia and degree of anemia in children under 5 years with Plasmodium falciparum in Jos Metropolis.

Immune evasion by Plasmodium falciparum parasites: converting a host protection mechanism for the parasite′s benefit

Immune evasion is a strategy used by pathogenic microbes to evade the host immune system in order to ensure successful propagation. Immune evasion is particularly important for the blood stages of Plasmodium falciparum, the causative agent of the deadly disease malaria tropica. Because Plasmodium blood stage parasites require human erythrocytes for replication, their ability to evade attack by the human immune system is essential for parasite survival. In order to escape immunity-induced killing, the intraerythrocytic parasites have evolved a variety of evasion mechanisms, including expansion of plasmodial surface proteins, organ-specific sequestration of the infected red blood cells and acquisition of immune-regulatory proteins by the parasite. This review aims to highlight recent advances in the molecular understanding of the immune evasion strategies by P. falciparum, including antigenic variation, surface protein polymorphisms and invasion ligand diversification. The review will further discuss new findings on the regulatory mechanisms applied by P. falciparum to avoid lysis by the human complement as well as killing by immune factors of the mosquito vector.

Molecular Investigation of Genetic Signatures of Selection in <i>Plasmodium falciparum</i>Actin-Binding Protein Coronin, Cysteine Desulfurase, and Plasmepsin 2 Gene in Mbita Field Isolates, Western Kenya

Background: Plasmodium falciparum (Pf) resistance to antimalarial drugs is a major impediment to malaria control. The Pf.Kelch 13 (PfK13) gene has been largely reported to be associated with artemisinin resistance. However, recent studies have shown artemisinin resistance without Kech13 mutations suggesting the implication of others genes in artemisinin resistance. In this current study, we focused on mutations in Pf.actin-binding protein coronin, Pf.cysteine desulfurase and Pf.plasmepsin 2 gene, three putative candidates recently were reported to be involved in artemisinin, lumefantrine and piperaquine resistance respectively. Method: Archived blood samples previously collected from asymptomatic school children from December 2016 to October 2018 were used in this study. Genomic DNA was extracted using ISOLATE II Genomic DNA kit. After PCR amplification, amplicons were purified and sequenced by capillary sequencing. Reads were analyzed for the identification of point mutations previously reported to be involved in drug selection. Results: Mutations R100K, and G50E involved in reduced artemisinin susceptibility were detected in Pfcoronin. From 2016/17 to 2018 the allele 100k increased frequency (11.2%);while 50E was only observed in 2018 time point reaching 11.1%. Lumefantrine selection marker K65, in codon (K65Q) was observed at 14.2% in Pfcysteine desulfurase, and the mutant’ allele 65Q gradually increased frequency from 28.5% in 2016/17 to 57.1% in 2018. Pf.plasmepsin 2 was the less polymorphic gene. Several other polymorphism codons and single nucleotide variants were detected. Conclusion: The findings indicate the presence of mutations associated with reduced artemisinin susceptibility and lumefantrine selection marker. Therefore, the results call for continuous monitoring of molecular makers in Mbita parasites.

The Relationship between the Plasmodium Falciparum Clearance and the Clinical Effect of the Case with Cerebral Malaria Treated with Artemisinin

Malaria is 1 mosquito-borne disease,which is most commonly caused by a parasite called Plasmodium falciparum(P.falciparum).Cerebral malaria is the most severe neurological complication presented in

Indicators of fatal outcome in severe Plasmodium falciparum malaria:a study in a tertiary-care hospital in Thailand

Objective:To illustrate the clinical features and investigate the indicators associated with a fatal outcome in adult patients with severe Plasmodium falciparum malaria admitted to the Hospital for Tropical Diseases,Bangkok,Thailand.Methods:We studied 202 adult malaria patients admitted to the Intensive Care Unit.A total of 43 clinical variables were identified by univariate and logistic regression analyses,to eliminate confounding factors.Results:Regarding the statistical methods,only 6 variables-jaundice,cerebral malaria,metabolic acidosis,body mass index,initial respiratory rate,and white blood cell count-were significant indicators of death, with adjusted odds ratios(95%CI) of 15.2(2.1-32.3).4.3(2.3-12.6),3.3(2.3-5.7),2.4(1.9-3.5),2.2 (1.5-2.6),and 1.7(1.2-3.1),respectively.Conclusions:Our study found that jaundice,cerebral malaria,metabolic acidosis,body mass index,initial respiratory rate and white blood cell count were indicators of fatal outcome in severe Plasmodium falciparum malaria.Further studies on the fatal indicators in severe malaria need to be compared with data from different geographical areas,to construct practical measures to address potentially fatal indicators in different settings.

Association of ABO blood group and Plasmodium falciparum malaria in Dore Bafeno Area,Southern Ethiopia

Objective:To assess the distribution of ABO blood group and their relationship with Plasmodium falciparum(P.falciparum) malaria among febrile outpatients who sought medical attention at Dore Bafeno Health Center,Southern Ethiopia.Methods:A total of 269 febrile outpatients who visited Dore Bafeno Health Center,Southern Ethiopia,were examined for malaria and also tested for ABO blood groups in January 2010.The blood specimens were collected by finger pricking,stained with Geimsa,and examined microscopically.Positive cases of the parasitemia were counted.CareStart^(TM) Malaria PflPv Combo was also used to test the blood specimens for malaria.ABO blood groups were determined by agglutination test using ERYCLONE antisera.Data on socio-demographic characteristics and treatment status of the participants were also collected.Chi-square and ANOVA tests were used to assess the difference between frequencies and means,respectively.Results:Out of a total of 269 participants,178(66.2%) febrile patients were found to be infected with Plasmodium parasites,among which 146(54.3%),28(10.4%),and 4(1.5%) belonged to P.falciparum,P.vivax,and mixed infections,respectively.All febrile patients were also tested for ABO blood groups and 51.3%,23.5%,21.9%and 3.3%were found to be blood types of 0,A,B and AB,respectively.Both total malaria infection and P.falciparum infection showed significant association with blood types(P<0.05).The proportion of A or B but not 0 phenotypes was higher(P<0.05) in individuals with P.falciparum as compared with non-infected individuals.The chance of having P.falciparum infection in patients with blood groups A,B and AB was 2.5,2.5 and 3.3times more than individuals showing blood 0 phenotypes,respectively.The mean P.falciparum malaria parasitemia for blood groups A,B,AB,and 0 were 3 744/μ L,1 805/ μ L,5 331/μ L,and1 515/μ L,respectively(P<0.01).Conclusions:The present findings indicate that individuals of blood groups A,B and AB are more susceptible to P.falciparum infection as compared with individuals of blood group O.Nevertheless,further in depth studies are required to clearly establish the role that ABO blood group plays in P.falciparum malaria.

In vitro antiplasmodial effect of ethanolic extracts of traditional medicinal plant Ocimum species against Plasmodium falciparum

Objective:To identify the possible antiplasmodial compounds from leaf,stem,root and flower extracts of Ocimum canum（O.canum）,Ocimum sanctum（O.sanctum） and Ocimum basilicum （O.basilicum）.Methods:The O.canum,O.sanctum and O.basilicum were collected from Ramanalhapuram District,Tamil Nadu and the extraction was carried out in ethanol.The filter sterilized extracts（100,30,23,12.5,6.23 and 3.125μg/mL） of leaf,stem,root and flower extracts of O.canum,O.sanctum and O.basilicum were tested for antiplasmodial activity against Plasmodium falciparum（P.falciparum）.The potential extracts were also tested for their phytochemical constituents.Results:The leaf extract of O.sanctum showed excellent antiplasmodial activity(IC50 3538μg/mL) followed by leaf extract of O.basilicum(IC50 4341μg/mL). The leaf extract of O.canum,root extracts of O.sanctum and O.basilicum,the stem and flower extracts of all the three tested Ocimum species showed IC50 values between 50 and 100μg/mL Statistical analysis reveals that,significant antiplasmodial activity（P【0.01） was observed between the concentrations and time of exposure.The chemical injury to erythrocytes was also carried out and it shows that,there were no morphological changes in erythrocytes by the ethanolic extract of O.canum,O.sanctum and O.basilicum.The in vitro antiplasmodial activity might be due to the presence of alkaloids,glycosides,flavonoids,phenols,saponins,triterpenoids,proteins,resins, steroids and tannins in the ethanolic extracts of tested plants.Conclusions:The ethanolic leaf extracts of O.sanctum possess lead compounds for the development of antiplasmodial drugs.

Bioinformatics analysis for structure and function of CPR of Plasmodium falciparum

Objective:To analyse the structure and function of NADPH-cytochrome p450 reductase(CYPOR or CPR) from Plasmodium falciparum(Pf),and to predict its’ drug target and vaccine target. Methods:The structure,function,drug target and vaccine target of CPR from Plasmodium falciparum were analyzed and predicted by bioinformatics methods.Results:PfCPR,which was older CPR,had close relationship with the CPR from other Plasmodium species,but it was distant from its hosts,such as Homo sapiens and Anopheles.PfCPR was located in the cellular nucleus of Plasmodium falciparum.335aa-352aa and 591aa - 608aa were inserted the interior side of the nuclear membrane,while 151aa-265aa was located in the nucleolus organizer regions.PfCPR had 40 function sites and 44 protein-protein binding sites in amino acid sequence.The teriary structure of laa-700aa was forcep-shaped with wings.15 segments of PfCPR had no homology with Homo sapien CPR and most were exposed on the surface of the protein.These segments had 25 protein-protein binding sites.While 13 other segments all possessed function sites. Conclusions:The evolution or genesis of Plasmodium falciparum is earlier than those of Homo sapiens.PfCPR is a possible resistance site of antimalarial drug and may involve immune evasion, which is associated with parasite of sporozoite in hepatocytes.PfCPR is unsuitable as vaccine target,but it has at least 13 ideal drug targets.

Chalcone analogue as potent anti-malarial compounds against Plasmodium falciparum:Synthesis,biological evaluation,and docking simulation study

Objective: To investigate in vitro antimalarial activity of chalcone derivative compounds against Plasmodum falciparum 3D7(Pf3D7) strain and in silico antimalarial activity.Methods: Synthesis of the chalcone derivatives was conducted via Claisen-Schmidt method using NaOH 60% base as catalyst. An in vitro antimalarial activity assay was carried out according to the Rieckmann method against the chloroquine-sensitive Pf3D7 strain. Molecular docking studies of the prepared compounds were performed using Discovery Studio 3.1(Accelrys, Inc., San Diego, USA) software to dihydrofolate reductases-thymidylate synthase(PfDHFR-TS) protein with Protein Data Bank ID of 1J3I.pdb(sensitive-protein) and ID: 4DP3.pdb(resistance-protein).Results: This work has successfully synthesized seven chalcone derivatives with a great antimalarial activity. It has been revealed that allyloxy, hydroxy and alkoxy functional groups could increase the antimalarial activity of the chalcone derivatives. The best antimalarial activity of the prepared compounds was possessed by 3b with an IC_(50) value of 0.59 μM and categorized as an excellent antiplasmodial. Molecular docking studies of 3b showed binding interaction with the amino acid residues such as Ala16. Ile 164. Phe58.Tyr170 of the 1J3I.pdb protein and also Ala16, Phe58, Ile 112, Met55 of the 4DP3.pdb protein.Conclusions: An in vitro antimalarial assay of the prepared chalcone derivative(3a-g)showed an excellent and good antiplasmodial activity against the chloroquine-sensitive Pf3D7 strain. In silico antimalarial studies revealed that 3a-g made binding interaction with both sensitive-protein(IJ3I.pdb) and resistance-protein(4DP3.pdb), which means that they were both active against chloroquine-sensitive and resistant plasmodium strain.

Polymorphisms of the oxidant enzymes glutathione S-transferase and glutathione reductase and their association with resistance of Plasmodium falciparum isolates to antimalarial drugs

Objective:To investigate the association between amplification of the two regulatory genes controlling glutathione(GSH) levels,glutathione reductase(PfGR) and glutathione S-transferase (PfGST) genes and sensitivity of Plasmodium falciparum(P.falciparum) isolates collected from different malaria endemic areas of Thailand to standard antimalarial drugs.Methods:A total of 70 P.falciparum isolates were collected from endemic areas of multi-drug resistance (Tak,Chantaburi and Ranong Provinces) during the year 2008-2009.The in vitro assessment of antimalarial activity of P.falciparum clones(K1- and Dd2 chloroquine resistant and 3D7- chloroquine sensitive) and isolates to chloroquine,quinine,mefloquine and arteusnate was performed based on SYBR Green modified assay.Results:68(97.14%),11(15.71%) and 28(40%) isolates respectively were classified as chloroquine-,quinine- and mefloquine-resistant isolates. With this limited number of P.falciparum isolates included in the analysis,no significant association between amplification of PfGST gene and sensitivity of the parasite to chloroquine, quinine,mefloquine and quinine was found.Based on PCR analysis,Dd2,Kl and 3D7 clones all contained only one copy of the PfGST gene.All isolates(70) also carried only one copy number of PfGST gene.There appears to be an association between amplification of PfGR gene and chloroquine resistance.The 3D7 and Dd2 clones were found to carry only one PfGR gene copy, whereas the K1 clone carried two gene copies.Conclusions:Chloroquine resistance is likely to be a consequence of multi-factors and enzymes in the GSH system may be partly involved. Larger number of parasite isolates are required to increase power of the hypothesis testing in order to confirm the involvement of both genes as well as other genes implicated in glutathione metabolism in conferring chloroquine resistance.

Schistosoma haematobium and Plasmodium falciparum coinfection with protection against Plasmodium falciparum malaria in Nigerian children

Objective:Malaria remains the single leading killer of children in sub - Sahara Africa and Schistosomiasis is considered to be second to malaria in global importance.Co - infection of malaria and urinary schistosomiasis has been reported to exacerbate disease morbidity such as anaemia.In different part of the globe,the co - infection between malaria and schistosomiasis provides some protections on the infected persons.The protective effect of this co - infection elucidated immunologically using cytokines is lacking in our locality.Methods:Urine and blood samples obtained from the 160 volunteers were subjected to standard parasitological techniques for diagnosis of urinary schistosomiasis and malaria respectively.Blood samples collected from these volunteers comprising 80 children with schistosomiasis and malaria and the 80 children who had malaria only were subjected to cytokines concentration determination using commercial standard enzyme linked immunosorbent assay kits(Abeam,UK).Results:Eighty participants with co - infection had a mean malarial parasitaemia of 662±201.1μL while the 80 participants with only P.falciparum malaria had a mean malarial parasiteamia of 5943±3270.7μL.Also the volunteers had mean haemoglobin of 11.2 g/dL for co - infected individuals and 5.7 g/dL for participants with single infection of malaria.The serum cytokine levels of the children with S. haematobium and P.falciparum and only P.falciparum infection are as follows;interleukin - 4(16.6 pg/ mL versus 5.2 pg/mL),IL - 5(501.3 pg/mL versus 357.5 pg/mL);IL -8(2 550 pg/mL versus 309 pg/mL),IL - 10(273 pg/mL versus 290 pg/mL),TNF -α(25 pg/mL versus 290 pg/mL) and IFN -γ(21.9 pg/mL versus 2.5 pg/mL).The TNF -α/IL - 10 ratio is 7 for the children with co - infection while those with only P.falciparum malaria infection had a TNF -α/IL - 10 ratio of 0.9.Conclusion:We conclude that the elevated IL - 4,IL - 5,IL - 8 and IFN -γconcentration induced by schistosomiasis altered the Th1/Th 2 profile and protected the children against the morbidity and severity of malaria attack among the children with co - infection.

Pro-and anti-inflammatory cytokines profiles among Nigerian children infected with Plasmodium falciparum malaria

Objective:To examine array of some pro- and anti-inflammatory cytokines,namely, interleukin-4(IL-4),interleukin-10(IL-10),interferon-γ(IFN-γ),interleukin-5(IL-5), interleukin-6(IL-6),interleukin-12(IL-12) and tumor necrosis factor-α(TNF-α) concentrations in some Nigerians with falciparum malaria.Methods:Sera were obtained from the blood samples of 96 Nigerian children with Plasmodium falciparum infection.The sera were subjected to cytokine evaluation using commercial standard enzyme linked immunosorbent assay kits(Abcam,UK).Results:Mean pro-inflammatory cytokines in serum of children with uncomplicated and complicated malaria were IL-5 482.2 pg/mL versus 526.7 pg/mL,IL-6 98.8 pg/mL versus 82.6 pg/mL,IL-12 24.1 pg/mL versus 15.9 pg/mL,TNF-α107 pg/mL versus 511.7 pg/mL and IFN- 7 2.1 pg/mL versus 2.5 pg/mL.The anti-inflammatory cytokines status of IL-4 were 4.7 pg/mL versus 20.3 pg/mL,and IL-10 were 216 pg/mL versus 143.8 pg/mL in uncomplicated versus complicated/severe malaria cases.Participants with uncomplicated malaria had mean parasitaemia level of 3 158.9 parasites/μL while mean parasitaemia level for participants with complicated malaria was 12 550.5 parasite/μL and this difference was statistically significant(χ~2 =5 614.6,P【0.05).The difference between mean haemoglobin level for uncomplicated malaria(9.6 g/dL) and severe malaria(3.9 g/dL) was statistically significant (χ~2 = 2.3,P【0.05).The relationship between serum level of IL-6,IL-12,IFN-γ,IL-10 and IL-4 and ages showed positive correlation at r=0.92,0.99,0.86,0.95 and 0.85,respectively;while IL-5 and TNF-αhad negative correlation at r=-0.99 and -0.99,respectively.Conclusion: IL-4,IL-5,IL-6,IL-10,IL-12,TNF-αand IFN-γare involved in the immunopathology and immunoregulation of uncomplicated and complicated malaria infections.IL-6,IL-12,IFN-γand IL-10 depressed in complicated/severe malaria may not provide any protective immunity and may be indicators of poor prognosis in Plasmodium falciparum infected Nigerian children.