Noscapine shows antimalarial activity against Plasmodium falciparum 3D7,its clinical isolate Pf140/SS,and Plasmodium berghei ANKA

Objective:To evaluate the antimalarial activity of noscapine against Plasmodium falciparum 3D7 strain(Pf3D7),its clinical isolate(Pf140/SS),and Plasmodium berghei ANKA(PbA).Methods:Using ring-stage survival assay,phenotypic assessments,and SYBR-green-based fluorescence assay,the antimalarial activities of noscapine were assessed compared with dihydroartemisinin(DHA)in in vivo and in vitro studies.In addition,hemolysis and cytotoxicity tests were carried out to evaluate its safety.RT-PCR assay was also conducted to determine the effect of noscapine on papain-like cysteine protease Plasmodium falciparum falcipain-2(PfFP-2).Results:The antimalarial efficacy of noscapine against Pf3D7 and Pf140/SS was comparable to DHA,with IC50 values of(7.68±0.88)and(5.57±0.74)nM/mL,respectively,and>95%inhibition of PbA infected rats.Noscapine also showed a safe profile,as evidenced by low hemolysis and cytotoxicity even at high concentrations.Moreover,PfFP-2 expression was significantly inhibited in both noscapine-treated Pf3D7 and Pf140/SS(P<0.01).Conclusions:Noscapine has antimalarial properties comparable to standard antimalarial DHA with better safety profiles,which may be further explored as a therapeutic candidate for the treatment of malaria.

Genetic diversity of the S-type small subunit ribosomal RNA gene of Plasmodium knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia

Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.

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.

Mutations in Plasmodium knowlesi Kelch protein 13 and the dihydropteroate synthase gene in clinical samples

Objective:To determine the genetic diversity,natural selection and mutations in Plasmodium(P.)knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia.Methods:P.knowlesi full-length gene sequences Kelch 13 gene(PkK13)from 40 samples and dhps gene from 30 samples originating from Malaysian Borneo were retrieved from public databases.Genetic diversity,natural selection,and phylogenetic analysis of gene sequences were analysed using DNAsp v5.10 and MEGA v5.2.Results:Seventy-two single nucleotide polymorphic sites(SNPs)across the full-length PkK13 gene(63 synonymous substitutions and 9 non-synonymous substitutions)with nucleotide diversity ofπ~0.005 was observed.Analysis of the full-length Pkdhps gene revealed 73 SNPs andπ~0.006(44 synonymous substitutions and 29 non-synonymous substitutions).A high number of haplotypes(PkK13;H=37 and Pkdhps;H=29)with haplotype diversity of Hd~0.99 were found in both genes,indicating population expansion.Nine mutant alleles were identified in PkK13 amino acid alignment of which,7(Asp3Glu,Lys50Gln,Lys53Glu,Ser123Thr,Ser127Pro,Ser149Thr and Ala169Thr)were within the Plasmodium specific domain,2(Val372Ile and Lys424Asn)were in the BTB/POZ domain and no mutation was observed within the kelch propeller domain.The 29 non-synonymous mutations in the Pkdhps gene were novel and only presented in exon 1 and 2.Conclusions:Monitoring the mutations from clinical samples collected from all states of Malaysia along with clinical efficacy studies will be necessary to determine the drug resistance in P.knowlesi.

Interleukin-33 exerts pleiotropic immunoregulatory effects in response to Plasmodium berghei ANKA(PbA)infection in mice

Objective:To determine the involvement and the modulatory effects of IL-33 during Plasmodium berghei ANKA(PbA)infection.Methods:PbA infection in male ICR mice was utilized as a model of malaria.Systemically circulating IL-33 levels were determined in blood plasma by enzyme-linked immunosorbent assay(ELISA).After 24 hours post-inoculation of PbA,recombinant IL-33 and ST2,and antibodies against IL-33 and IgG treatments were administered daily for 3 days.Tissue expression and localization of IL-33 were assessed in organs generally affected by malaria via immunohistochemistry.Moreover,histopathological examination was performed to assess the effects of the treatments.Results:The levels of systemic IL-33 were elevated at the critical phase of PbA infection.Likewise,immunohistochemical analysis revealed a significant upregulation of IL-33 expression at the critical phase in the brain,lungs,and spleen of PbA-infected mice as compared to healthy controls.Treatment with IL-33 protected against experimental cerebral malaria development and reduced pathological features in the brain and lungs of the PbA-infected mice.Conclusions:A potential critical role and involvement of IL-33 in PbA infection may hint at the resolution of immunopathological sequelae associated with malaria.

Time to stimulate Plasmodium vivax research in India: A way forwards

India bears the largest Plasmodium (P.) vivax (Pv) malaria burden and contributes 48%of Pv cases globally[1]. The efforts of government and private bodies to control malaria have successfully reduced the number of Plasmodium falciparum (Pf) malaria cases in several countries, including India.

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.

食蟹猴疟原虫 (Plasmodium cynomolgi)中一个真核翻译起始因子4A(eIF-4A)同源蛋白的cDNA克隆、表达和ATP酶活性测定(英文)

真核翻译起始因子 4A(eukaryoticinitiationfactor 4A ,eIF 4A)是DEAD盒蛋白家族的ATP依赖性的RNA解旋酶类中的一个原型成员 .它在真核细胞的蛋白质合成的起始过程中起着关键性作用 .通过PCR扩增和放射探针杂交相结合的方法筛选食蟹猴疟原虫 (Plasmodiumcynomolgi)的cDNA文库 ,克隆了一个eIF 4A同源蛋白的完整cDNA序列 ,命名为CH1F .CH1F全长 1 75 3bp ,包含一个1 1 97bp的完整阅读框 ,推测编码一个由 398个氨基酸组成的蛋白 .对CH1F的蛋白序列用BlastP进行搜索和分析 ,提示它应该是DEAD盒家族的一个eIF 4A同源蛋白 ;用DNAStar将其与许多典型的DEAD盒蛋白序列进行比对分析 ,结果显示 :比起其它的DEAD盒蛋白 ,它与eIF 4A或eIF 4A的同源蛋白具有更高的同源性和更多序列上的相似结构域 .将包含完整阅读框的片段亚克隆进表达载体pET 2 8a (+) ,在大肠杆菌DH5α中表达 ,产生的融合蛋白大小在 4 5kD左右 .对该融合蛋白进行纯化、重新折叠和初步鉴定 .ATP酶活性检测显示 ,该融合蛋白只有很低的ATP酶活性 ,而且它的ATP酶活性似乎不依赖于核酸底物 .对这一检测结果给出 3种可能的原因 .这一检测结果与根据序列分析得到的推论———CH1F蛋白可能是一个eIF

Selective Effect of Qinghaosu on Different Stages of Plasmodium falciparum in Vitro

Using highly synchronous cultures of Plasmodium falciparum in vitro,the susceptibi- lity of the different stages of the intraerythrocytic parasites to Qinghaosu (QHS) was assessed.The anti- parasitic effect of QHS was measured by comparing the changes of irradiation of^3 H-hypoxanthine in- corporated into the nucleic acids of parasites exposed to various concentrations of QHS at different stages of growth.It was found that the trophozoite stage of the parasite was the most sensitive to QHS, whereas the early ring stage was the least sensitive,and the sensitivities of the late ring and schizont stages fell between those of the early ring and trophozoite stages.The results revealed the correlation of stage-dependent effects of QHS with the blockade of the protein metabolism of the parasite.

Comparison of microscopy and PCR for the detection of human Plasmodium species and Plasmodium knowlesi in southern Myanmar

To determine the distribution of Plasmodium (P) species including Plasmodium knowlesi and to compare the specificity and sensitivity of microscopy with nested PCR in malaria diagnosis.MethodsThe study was conducted in Kawthaung, southern Myanmar. Ninety clinically suspected malaria patients were screened for malaria by Giemsa stained microscopy and confirmed by nested PCR.ResultsAmong the participants, 57 (63.3%) were positive and 33 (36.7%) were negative by microscopy. Of positive samples, 39 (68.4%) were Plasmodium falciparum, 17 (29.8%) Plasmodium vivax and 1 (1.8%) Plasmodium malariae, whereas 59-amplified by PCR were 40 (67.8%), 18 (30.5%) and 1 (1.7%) respectively. PCR amplified 2 microscopy negative samples. Two samples of P. falciparum detected by microscopy were amplified as P. vivax and vice versa. All samples were negative for Plasmodium ovale, P. knowlesi and mixed infections. Microscopy had a very good measure of agreement (κ = 0.95) compared to nested PCR. Sensitivity and specificity of microscopy for diagnosis of P. falciparum were 92.5% (95% CI: 79.6-98.4) and 96.0% (95% CI: 86.3-99.5) respectively, whereas for P. vivax were 83.3% (95% CI: 58.6-96.4) and 97.2% (95% CI: 90.3-99.7).ConclusionsP. knowlesi was not detected by both microscopy and PCR. Giemsa stained microscopy can still be applied as primary method for malaria diagnosis and is considered as gold standard. As to the lower sensitivity of microscopy for vivax malaria, those with previous history of malaria and relapse cases should be diagnosed by RDT or PCR combined with microscopy. Inaccuracy of species diagnosis highlighted the requirement of training and refresher courses for microscopists.

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.

IL-10在Plasmodium yoelii 17XL和Plasmodium chabaudi AS疟原虫混合感染宿主病理损伤中的作用研究

为探讨IL-10在致死型约氏疟原虫(Plasmodium yoelii 17XL,P.y17XL)和夏氏疟原虫(Plasmodiumchabaudi AS,P.cAS)混合感染宿主病理损伤中的作用,用P.y17XL、P.cAS和P.y17XL+P.cAS分别感染DBA/2小鼠,计数红细胞感染率;感染后第3、5、8、10、12和19天分别尾静脉取血,肝素抗凝后短暂离心,采用高纯度DNA提取试剂盒抽提DNA,实时定量PCR检测虫负荷水平;感染后第0、1、3、5、8、10、12和15天制备脾细胞悬液,ELISA检测脾细胞培养上清中IL-10水平。实验结果发现,P.y17XL单独感染和混合感染小鼠IL-10水平在感染后第5天和第8天分别达峰值,随后开始下降至正常水平,小鼠虫血症均达中等水平,存活率100%;相比P.cAS感染小鼠IL-10在感染后第3天突然出现高水平升高并且维持时间较长;于感染后第8天达峰值,是同天P.y17XL单独感染和混合感染小鼠IL-10水平的2倍,虫血症水平较高,小鼠全部死亡。同时实时定量PCR结果发现,混合感染小鼠,于感染后3～12 d P.y17XL增殖占优势,而感染后15～19 d则P.cAS增殖处于优势状态。表明以IL-10为核心的免疫调节网络与疟疾感染过程中病理损伤密切相关。同时提示混合感染小鼠应答模式与P.y17XL感染小鼠的应答模式相同。

Antimalarial potential of kolaviron,a biflavonoid from Garcinia kola seeds,against Plasmodium berghei infection in Swiss albino mice

Objective:To investigate the antimalarial potential of kolaviron(KV),a biflavonoid fraction from Garcinia kola seeds,against Plasmodium berghei(P.berghei)infection in Swiss albino mice.Methods:The study consists of seven groups of ten mice each.Groups I,II and III were normal mice that received com oil.KV1 and chloroquine(CQ),respectively.Groups IV,V,ⅥandⅦwere infected mice that received corn oil.CQ,KYI and KV2.respectively.CQ.KY1 and KV2were given at 10-,100-and 200-mg/kg daily,respectively for three consecutive days.Results:Administration of KV1 and KV2 significantly(P<0.05)suppressed P.berghei-infection in the mice by 85%and 90%.respectively,while CQ produced 87%suppression relative to untreated infected group after the fifth day of treatment.Also,KV2 significantly(P<0.05)increased the mean survival time of the infected mice by 175%.The biflavonoid prevented a drastic reduction in HCV from day4 of treatment,indicating its efficacy in ameliorating anaemia.Significant(P<0.05)oxidative stress assessed by the elevation of serum and hepatic malondialdehydewere observed in unlrealed P.berghei-infected mice.Specifically,senum and hepatic malondialdehyde levels increased by93%and 78%,resjiectively in the unlrealed infecled mice.Furlhennore,antioxidant indices,viz;superoxide dismutase.catalase,glutathione-s-transferasc.glualhione peroxidase and reduced gluathione decreased significantly(P<0.05)in the tissues of untreated P.berghei-infected mice.KV significantly(P<0.05)ameliorated the P.berghei-induced decrease in antioxidant status of the infected mice.Conclusions:This study shows that kolaviron,especially at 200 mg/kg,has high antimalarial activities in P.berghei-infected mice,in addition to its known antioxidant properties.

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.

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.

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.

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.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

Objective:To compare the applicability of the SYBK Grcen-Ⅰ assay with the standard schizont maturalion assay,for determination of sensitivity of Plasmodium vivax(P.vivax) to chloroquine and a new antifolale WR 99210.Methods:The study was conducted at Mae Tao Clinic for migrant workers,Tak Province during April 2009 to July 2010.A total of 64 blood samples(1 mL blood collected into sodium heparinized plastic tube) were collected from patients with monoinfection with P.vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P.vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-Ⅰ assays.Results:A total of 30 out of 64 blood samples collected from patients with P.vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays.The failure rates of the schizont maturation inhibition assay(50%) and the SYBR Green-I assay(54%) were similar(P=0.51).The median IC_(10)s,IC_(50)s and IC_(90)s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P.vivax tested.Based on the cut-off of 100 nM,the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates,respectively.The strength of agreement between the two methods was very poor for both chloroquine and WR992I0.Conclusions:On the basis of this condition and its superior sensitivity,the microscopic method appears better than the SYUK Green-I Green assay for assessing in vitro sensitivity of fresh P.vivax isolates to antimalarial drugs.

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(IC_(50) 3538μg/mL) followed by leaf extract of O.basilicum(IC_(50) 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 IC_(50) 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.

Comparative evaluation of microscopy,OptiMAL® and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner,India

Objective:To evaluate microscopy,OptiMAL? and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL? and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL? was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL?.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL?,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.