间日疟原虫乳酸脱氢酶编码区全长基因的克隆、序列分析及线性B细胞表位预测

目的克隆中国间日疟原虫乳酸脱氢酶(PvLDH)编码区全长基因,并对其进行生物信息学分析。方法自间日疟原虫抽提RNA,根据已知的PvLDH基因设计特异性引物,采用RT-PCR扩增PvLDH编码基因,将其克隆至pMD18-T载体,经PCR和双酶切鉴定后进行序列测定,利用生物信息学在线工具对序列进行分析并做B细胞表位预测。结果 PCR产物电泳结果显示所克隆的基因为951bp,基因测序结果与GenBank报道的基因序列有一个碱基差异,但编码氨基酸无差异。在线分析预测出12个B细胞表位,与恶性疟原虫乳酸脱氢酶预测表位对比分析,发现一个PvLDH特异性表位。结论成功克隆了我国间日疟原虫乳酸脱氢酶编码区全长基因,并预测出1个PvLDH特异性线性B细胞表位。     

间日疟原虫和恶性疟原虫乳酸脱氢酶基因的序列和重组抗原表位分析

目的对间日疟原虫和恶性疟原虫乳酸脱氢酶(LDH)基因的序列及重组抗原的表位进行比较分析。方法根据GenBank中已知的pLDH基因序列(登录号为DQ198262和DQ060151)设计特异性引物,体外扩增间日疟原虫和恶性疟原虫LDH的全长基因,对两序列进行比对分析,用SYFPEITHI软件进行表位预测分析。将Pv-LDH和Pf-LDH基因克隆入pET28a表达载体,转化至大肠埃希菌BL21(DE3)株,加入异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,Westernblotting和中和ELISA法鉴定重组蛋白。结果Pv-LDH和Pf-LDH基因的编码区全长均为951bp,编码316个氨基酸,Pf-LDH与参考序列DQ198262完全一致,而Pv-LDH与参考序列DQ060151仅在第666位有一个核苷酸的变异;两者的核苷酸和氨基酸序列的一致性分别为75.1%和90.2%。T细胞表位预测分析结果显示,能识别pLDH抗原表位的人类白细胞抗原(HLA)分子类型共28种,预测的表位数约为180个,相同或相似的表位约占总表位数的75%,Pv-LDH和Pf-LDH特异性表位分别为38个和45个。Westernblotting分析显示,Pv-LDH重组抗原可被疟疾患者血清识别,但反应强度明显低于Pv-LDH重组抗原免疫兔血清。中和ELISA试验结果显示,Pv-LDH抗原对多克隆抗体最高抑制率可达70.3%,而Pf-LDH抗原的最高抑制率仅为30.5%。结论Pv-LDH和Pf-LDH基因的核苷酸序列及其重组抗原均有差异,特异性表位诱导的抗体在整个抗体谱中相对较少。     

基于蛋白重复序列及线性B细胞表位预测筛选间日疟原虫特异性抗原肽

目的 目的 建立一种基于蛋白重复序列及线性B细胞表位预测筛选间日疟原虫 （Plasmodium vivax,P. vivax） 特异性抗 原肽的方法。方法 方法 以PlasmoDB数据为基础, 建立间日疟原虫蛋白序列数据库。编写重复序列查找软件, 逐一检索16 aa 多肽在全部蛋白序列中的重复次数, 统计出重复较高者进行连续B细胞表位预测, 优选具有间日疟原虫特异性的肽进行合 成, 偶联钥孔戚血蓝蛋白 （Keyhole limpet hemocyanin, KLH） 后免疫小鼠, 并检测抗体滴度。 结果 结果 通过软件分析间日疟原 虫全部5 432个蛋白序列中的16肽的重复次数, 利用BcePred网站预测其中重复较高的1 000个序列, 获得22个候选肽, 经 聚类分析和相似性比较, 优选到5个潜在特异性肽, 人工合成并偶联KLH后免疫小鼠诱生的抗体滴度均超过1 : 9 000。 结 结 论 论 建立了一种基于蛋白重复序列联合线性B细胞表位预测筛选间日疟原虫特异性抗原肽的方法, 得到的5个肽均能诱 导小鼠产生高滴度抗体。     

间日疟原虫海南分离株乳酸脱氢酶基因的克隆与序列分析

用PCR扩增间日疟原虫海南分离株基因组DNA中乳酸脱氢酶(LDH)全长基因,命名为PvLDH/HN(GenBank登录号为FJ527750)。生物信息学分析表明,PvLDH/HN全长951bp,编码316个氨基酸残基,与间日疟原虫SalvadorI株、Belem株LDH等分离株核苷酸序列同源性均为99.89%(950/951),氨基酸序列同源性均为100%(316/316)。拓扑结构分析显示,该蛋白具有2个α螺旋跨膜区域,可能是膜蛋白。三级结构模型显示主要抗原表位82～95aa位于蛋白表面,构成特异性底物结合环,提示该位点是可能的药物作用靶点及免疫诊断抗原表位。     

万孚疟原虫检测试剂盒检测卵形疟原虫效果评价及影响因素分析

目的 目的 评价万孚疟原虫检测试剂盒 （Pf?LDH/Pan?pLDH） 对卵形疟原虫的检测效果, 并分析原虫密度、 疟原虫乳 酸脱氢酶 （pLDH） 浓度和基因多态性等因素对检测结果的影响。方法 方法 按照万孚疟原虫检测试剂盒的操作说明书, 对经 PCR确认的100份卵形疟患者血样进行检测。采用显微镜镜检法计数患者血片的原虫密度, 以ELISA法检测血样中的 pLDH浓度, 以PCR扩增卵形疟原虫LDH （Po?LDH） 基因并测序, 并分析上述3个因素对检出率的影响。结果 结果 万孚疟原 虫检测试剂盒对上述100份卵形疟患者血样的总体检出率为70.0% （70/100）。当原虫密度 ≤ 500个/μl时, 检出率为 27.3%; 原虫密度> 500个/μl时, 检出率为75.0%～75.4%。当pLDH浓度较低时 （A值 ≤ 0.100）, 检出率为6.7%; pLDH达 到一定浓度时 （A值 > 0.100）, 检出率为95.1%～100%。Po?LDH基因序列分析结果显示所有卵形疟样本可分为2种亚 型, 分别为卵形疟原虫curtisi亚种 （P. o. curtisi） 和卵形疟原虫wallikeri亚种 （P. o. wallikeri）。2种亚型的LDH基因同源 性为97%, 共有24个单核苷酸多态性 （SNP）, 其中3个SNPs为非同义突变, 其余均为同义突变。2种亚型的LDH编码氨 基酸序列同源性为99%, 共有3个氨基酸的差异。万孚疟原虫检测试剂盒对P. o. curtisi的检出率为73.1% （38/52）, 对 P. o. wallikeri的检出率为66.7% （32/48）, 两者差异无统计学意义 （P > 0.05）。结论 结论 万孚疟原虫检测试剂盒对卵形疟原虫的 检测效果优于大部分同类产品, 其检出率与原虫密度、 pLDH浓度关系密切, 与pLDH基因多态性无关。     

快速诊断靶蛋白恶性疟原虫富组氨酸蛋白Ⅱ和疟原虫乳酸脱氢酶的热稳定性

在新型冠状病毒肺炎流行期间,通常需将血样进行56℃病毒灭活处理后再行病原体检测。为了解56℃灭活对血样中疟原虫抗原稳定性的影响,本研究收集2017—2019年上海市疾病预防控制中心上报的疟疾患者血样71份,其中恶性疟血样38份、三日疟血样8份、卵形疟血样11份、间日疟血样14份。应用快速诊断检测(RDT)试剂盒对56℃孵育30 min前后血样中恶性疟原虫富组氨酸蛋白Ⅱ(Pf HRPⅡ)和疟原虫乳酸脱氢酶(p LDH)的热稳定性进行测定。结果显示,热处理前T1检测线(检测目标Pf HRPⅡ)呈阳性的38份恶性疟血样,热处理后35份仍呈T1阳性(92.11%, 35/38,χ~2=3.123, P 0.05);热处理前T2检测线(检测目标pLDH)阳性的54份血样(26份恶性疟血样、 6份三日疟血样、 10份卵形疟血样和12份间日疟血样),经热处理后T2均呈阴性(阳性率为0, 0/54,χ~2=87.755, P 0.01)。表明在56℃孵育30 min条件下,Pf HRPⅡ的稳定性较好;p LDH不稳定,全部降解或失活。因此,应用RDT检测热处理后的血样,恶性疟血样的检测结果不受影响,但非恶性疟血样会被漏诊。     

间日疟原虫多表位疫苗基因的表达、纯化与初步鉴定

目的构建和筛选间日疟原虫多表位疫苗基因高拷贝Pichia pastoris菌株,研究多表位疫苗基因在酵母菌中的表达,纯化目的产物,为进一步的多表位肽免疫原性研究奠定基础。方法根据文献报道筛选间日疟原虫有效保护性抗原表位,人工合成多表位基因PvDBW,经EcoRⅠ和NotⅠ双酶切构建酵母表达载体pPIC9K-PvDBW,转化大肠埃希菌ToP10;鉴定后转化P.pastoris GS115,构建酵母表达菌株;通过G418压力筛选筛出目的基因高拷贝克隆,用甲醇诱导多表位肽基因表达,分别以RT-PCR和Western blot进行鉴定,用50%饱和硫酸铵沉淀和分子筛凝胶层析分离、纯化目的产物。结果重组质粒用EcoRⅠ和NotⅠ双酶切后可得到786 bp DNA片段;经G418压力筛选筛出两个高拷贝菌株,以α-Factor和3′AOX1为引物、重组菌基因组DNA为模板,PCR扩增出约960 bp的目的片段;SDS-PAGE显示,在GS115细胞内多表位肽呈分泌性表达,表达量为80 mg/L;分离、纯化后,纯度达90%以上;Western blot检测显示表达的多表位肽可被间日疟病人血清识别。结论间日疟原虫多期多阶段多表位疫苗基因在酵母菌中表达成功。     

不同感染来源恶性疟原虫裂殖子表面蛋白3基因多态性分析及抗原表位预测

目的分析云南省不同感染来源恶性疟原虫裂殖子表面蛋白3(PfMSP-3)基因和抗原表位多态性。方法收集2012年8月-2016年12月寄生虫病防治信息管理系统登记报告的云南省本地和输入恶性疟病例的血样和流行病学史等信息。提取血样的疟原虫DNA,半巢式PCR扩增PfMSP-3基因并测序,与Gen Bank中的恶性疟原虫Pf3D7、PfK1分离株的参比序列LN999944.1、U08851.1进行比对。用MEGA 5.04、Arlequin 3.5.2.2分析PfMSP-3基因的单倍型、期望杂合度(He)、群体间遗传分化指数(Fst)。用Network 4.6.0构建单倍型网络进化图。用Dna SP 5.10计算核苷酸的非同义(Ka)、同义置换率(Ks)。用IEDB在线预测软件预测PfMSP-3肽链T细胞、B细胞抗原表位。结果共收集190份恶性疟病例血样,其中181份扩增出长1 100 bp的PfMSP-3片段,测序成功167份(缅甸121份、非洲37份、云南本地感染9份)。序列比对结果显示,167条DNA序列存在36种单倍型,He为0.409±0.183,Ka/Ks为0.98。各单倍型沿Pf3D7型(Ⅰ)、过渡型(Ⅱ)及PfK1型(Ⅲ)等3种方向进化:H_1、H_9等7种单倍型为Pf3D7型,H_7、H_8等6种单倍型为过渡型,H_2、H_3等23种单倍型为PfK1型。Pf3D7型、过渡型和PfK1型序列的占比分别为49.7%(83/167)、12.6%(21/167)和37.7%(63/167)。PfMSP-3基因在非洲与缅甸恶性疟原虫群体间的分化程度最高,Fst=0.049;在非洲与云南本地感染恶性疟原虫群体间最小,Fst=0.032。抗原表位分析结果显示,36种单倍型的PfMSP-3肽链亲水性高于疏水性,指数分别为1.050~2.535和-2.583~-3.544。T细胞抗原表位活性为-1.1;B细胞抗原表位活性平均0.5,且表现为Pf3D7型过渡型PfK1型。结论云南省不同感染来源恶性疟原虫的PfMSP-3基因存在3类基因型,不同基因型PfMSP-3蛋白B细胞抗原表位活性的预测值较T细胞高。     

猪囊尾蚴排泄分泌抗原LRRC15蛋白真核表达及抗原表位预测

目的　对猪囊尾蚴排泄分泌抗原中差异表达蛋白富含亮氨酸重复序列结构域15（leucine⁃rich repeat containing 15, LRRC15）进行真核表达，并预测其抗原表位。方法　通过在线软件ExPASy⁃PortParam和Protean软件预测LRRC15蛋白分子质量、稳定性、氨基酸序列组成及等电点和T淋巴细胞抗原表位。采用基于PCR的精确合成（PCR⁃based accurate synthesis, PAS）技术设计全长拼接引物，合成LRRC15基因。构建重组质粒pcDNA3.4⁃LRRC15并转染至人胚胎肾细胞HEK293，表达LRRC15蛋白，并进行十二烷基磺酸钠⁃聚丙烯酰胺凝胶电泳（sodium dodecyl sulphate⁃polyacrylamide gel electrophoresis, SDS⁃PAGE）和免疫印迹试验（Western blotting）鉴定。结果　成功构建了重组质粒pcDNA3.4⁃LRRC15，表达分子质量约70 kDa的LRRC15目的蛋白。经ExPASy⁃PortParam软件预测，LRRC15属于亲水性蛋白，由644个氨基酸组成，分子质量为69.89 kDa，等电点为5.6；蛋白分子式为C3073H4942N846O953S28，不稳定系数为50.3，为一种不稳定蛋白。采用Protean软件预测发现，LRRC15蛋白位于292 ~ 295、353 ~ 361、521 ~ 526、555 ~ 564位氨基酸区段具有T细胞抗原表位优势，具有亲水性高、柔韧性好、表面可及性大、抗原性指数高的表位位于122 ~ 131、216 ~ 233、249 ~ 254、333 ~ 343、358 ~ 361、368 ~ 372、384 ~ 386、407 ~ 412、445 ~ 450、469 ~ 481、553 ~ 564、588 ~ 594、607 ~ 617、624 ~ 639位氨基酸区段。将重组质粒pcDNA3.4⁃LRRC15转染至HEK293细胞后，SDS⁃PAGE和Western blotting分析发现，在细胞分泌培养基、细胞裂解上清和沉淀中检测到LRRC15蛋白。从细胞培养基中纯化出LRRC15⁃His融合蛋白，SDS⁃PAGE显示在分子质量约为70 kDa处出现明显条带，Western blotting能够识别LRRC15重组蛋白条带。结论　成功对猪囊尾蚴排泄分泌抗原中的LRRC15蛋白进行真核表达，并对其抗原表位进行了生物信息学预测，为进一步了解该蛋白的生物学功能奠定了基础。     

间日疟原虫传播阻断疫苗候选抗原Pvs25中国分离株高度保守

目的　研究我国单纯间日疟流行区 3 1例患者 (湖北18例、浙江13例 )间日疟原虫分离株传播阻断疫苗候选抗原Pvs2 5基因多态性 ,并与 14例孟加拉间日疟原虫株进行比较分析。　方法　从干燥滤纸血膜提取疟原虫基因组DNA ,对Pvs25基因进行聚合酶链反应 (PCR)扩增、纯化和直接序列分析。　结果　与间日疟原虫标准株Sal-I相比 ,获得的 45个Pvs25全长序列中有 3处点突变 ,引起相应氨基酸的替换。并且核苷酸多态性 (π值 )检验结果显示 ,Pvs25在不同流行区或同一流行区不同分离株之间核苷酸及其相应的氨基酸序列高度保守。　结论　与红前期和红内期候选抗原相比 ,Pvs25具有有限的抗原多态性 ,提示以Pvs25为基础构建的传播阻断疫苗在我国流行区具有普遍应用的可能性     

Diagnosis of imported malaria by Plasmodium lactate dehydrogenase (pLDH) and histidine-rich protein 2 (PfHRP-2)-based immunocapture assays.

This study was conducted to evaluate the performance of two rapid non-microscopic assays: Plasmodium lactate dehydrogenase (pLDH) assay (OptiMAL) and Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) assay (ICT Malaria). The assays were used to detect malaria infection in 515 immigrants living in Kuwait. The performance of both assays was compared to that of microscopy of Giemsa-stained thick blood films and to each other. Of the 515 patients tested, 163 were positive for malaria parasites by microscopy of thick blood film. Of these, 87 were infected with Plasmodium vivax parasites, 63 with P. falciparum, 1 with Plasmodium malariae, and 12 had mixed infections of P. falciparum and P. vivax. The PfHRP-2 assay detected 53 P. falciparum infections and, as expected, failed to detect all but one case of P. vivax. Three cases of mixed infections were also not detected by this assay. The pLDH assay detected 56 P. falciparum cases and 77 P. vivax infections but failed to detect 4 cases of mixed infections. Compared to microscopy, the performance of both the assays to diagnose P. falciparum infection was comparable. The sensitivity for the PfHRP-2 assay was 82% with a specificity of 99.0% and for the pLDH assay the sensitivity was 89% with a specificity of 99.5%. The PfHRP-2 assay detected 4 false positive cases, 2 of which were also detected by the pLDH assay. These patients reported treatment with chloroquine in the last 2-5 weeks. Though the immunocapture diagnostic assays may be helpful in certain situations, microscopy of thick blood film is still the method of choice in diagnosing imported malaria.     

Malaria epidemiology in low-endemicity areas of the Atlantic Forest in the Vale do Ribeira, São Paulo, Brazil

We describe a seroepidemiological survey of malaria prevalence in two areas of low endemicity: Intervales State Park and Alto Ribeira State Tourist Park (PETAR). Both are located in the Vale do Ribeira in the state of S?o Paulo, Brazil. In this study, 318 subjects from both areas had their blood analyzed for the presence of malaria parasites by thin and thick blood smears. One hundred and sixty-three (51.2%) of the subjects were from Intervales State Park and 155 (48.7%) were from PETAR. We analyzed all the samples by indirect immunofluorescent assay (IFA) to detect antibodies against asexual forms of Plasmodium vivax and Plasmodium malariae and enzyme immunosorbent assay (ELISA) to detect the presence of antibodies against circumsporozoite proteins (CSP) from P. vivax VK210, human P. vivax-like/Plasmodium simiovale, P. vivax VK247 and Plasmodium brasilianum/P. malariae. The presence of Plasmodium species was detected by polymerase chain reaction (PCR). Eighteen of the subjects analyzed had positive IFA results for IgM against P. malariae antigens, and three others were positive for P. vivax antigens. Positivity of IgG antibodies against P. vivax detected by IFA was high in samples from both Intervales State Park and PETAR (32.0% and 49.0%, respectively), while positivity for P. malariae was lower (16.0% and 19.3% in Intervales State Park and PETAR, respectively). ELISA tests showed a higher prevalence of antibodies against P. vivax VK210 (35.0%) in samples from Intervales State Park and against human P. vivax-like (29.7%) in samples from PETAR. PCR reactions revealed the presence of parasites in several of the samples analyzed. In Intervales State Park, one subject was infected by P. malariae and two by Plasmodium falciparum, while in PETAR, one subject was positive for P. falciparum and three for both P. falciparum and P. vivax parasites. The areas where these parks are located belong to the Atlantic Forest habitat, and inhabitants frequently, see monkeys. Our data suggest that monkeys may constitute a natural reservoir for malaria in both areas.     

A sero-epidemiological study of malaria in human and monkey populations in French Guiana

This paper describes a sero-epidemiological study of malaria prevalence in French Guiana. An immunofluorescence assay and an enzyme-linked immunosorbent assay were used to detect antibodies against blood-stage antigens and synthetic peptides mimicking the repetitive epitope of the sporozoites of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae/brasilianum, in 218 human sera and 113 non-human primate sera collected in French Guiana. Almost all the monkey sera tested had antibodies against malaria blood-stages (98%) and a large majority (73%) also tested positive with the P. malariae/brasilianum circumsporozoite peptide. A number of primate samples also reacted positively with P. falciparum NANP repeats in a very specific manner, suggesting that monkeys in the rainforest are bitten by mosquitoes infected with human malaria parasites. Seroprevalences were lower in the humans tested but Indian tribes on the borders with Suriname and Brazil were clearly more exposed to malaria than other ethnic groups, with a prevalence of nearly 70% seropositivity. P. vivax infections accounted for much of the observed pattern of reactivity, but there was also a high frequency of positive reactions to the P. brasilianum/malariae peptide. Similarly, a large proportion of the sera obtained from Bush Negro populations tested positive for P. malariae/brasilianum repeats. These data add to the emerging evidence that non-human primates might constitute a natural reservoir, not only for simian, but also for human malaria, and therefore suggest that they might be responsible for the maintenance of foci of P. malariae, and possibly of other malaria species, in isolated areas of the Amazonian rainforest.     

Performance of the OptiMAL assay for detection and identification of malaria infections in asymptomatic residents of Irian Jaya, Indonesia

The OptiMAL assay, a new immunochromatographic "dipstick" test for malaria based on detection of Plasmodium lactate dehydrogenase (pLDH), is purported to detect infections of approximately 200 parasites/microL of blood and to differentiate between Plasmodium falciparum and non-P. falciparum. We evaluated OptiMAL performance by comparing the test strip interpretations of two independent readers with consensus results obtained independently by expert malaria microscopists. Unbiased measures of sensitivity were derived by applying the OptiMAL test for detection and differentiation of light, asymptomatic infections by P. falciparum and Plasmodium vivax. OptiMAL readings were separated in time to determine whether the reaction signal was stable. Microscopy identified infections in 225 of 505 individuals screened; those with P. falciparum (n = 170) averaged 354 asexual forms/microL and P. vivax/Plasmodium malariae (n = 112) averaged 216 asexual forms/microL of blood. Concordance between OptiMAL and microscopy was 81% and 78% by the two independent readings. The assay's sensitivity for detection of any malaria species was 60.4% and 70.2% respectively and specificity was 97% and 89%. Most cases identified by microscopy as P. falciparum were graded as negative or non-falciparum by both OptiMAL readers. OptiMAL false negatives as well as misidentifications were related to low parasitemias (< 500/microL). The OptiMAL assay demonstrated 88-92% sensitivity for detecting infections of 500-1,000 parasites/microL, a range covering the mean parasitemia of primary symptomatic P. falciparum infections in malaria-na?ve Indonesian transmigrants. This device was markedly less sensitive than expert microscopy for discriminating between malaria species and is presently unsuited for use as an epidemiological screening tool. The OptiMAL assay is not approved for diagnostic use but is commercially available for research purposes only.     

Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients

A TaqMan real-time PCR system was used to detect and discriminate the 4 species of human malaria parasites in clinical blood samples. A 150-base pair (bp) region of the small subunit ribosomal RNA (SSU rRNA) gene of each malaria parasite, including species-specific sequences to be detected by TaqMan probe, was used as a target for PCR analysis. The PCR method used universal primers and species-specific TaqMan probes for Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The detection threshold for the method, as determined with serial dilution of cultured P. falciparum-infected erythrocytes, was 5 parasite-infected erythrocytes per reaction. Fifty blood samples of falciparum malaria and a second set of 50 samples of vivax malaria, diagnosed by microscopic examination at the Hospital for Tropical Diseases, Mahidol University, Thailand, were analyzed by real-time PCR. In the 50 samples of microscopically-diagnosed falciparum malaria, 40 were regarded as P. falciparum single infection, 7 were P. falciparum and P. vivax mixed infections, and 3 were P. vivax single infection by real-time PCR. In the second set of 50 samples of microscopically diagnosed vivax malaria, all were considered P. vivax single infection by PCR. Neither P. ovale nor P. malariae infection was identified in the 100 blood samples. Real-time PCR analysis was shown to be more sensitive and accurate than routine diagnostic methods. Application and extension of the PCR method reported here will provide a powerful tool for further studies of malaria.     

Tumor necrosis factor-alpha in patients with malaria

Serum concentration of Tumor Necrosis Factor-alpha (TNF-alpha) was observed in 54 parasitologically confirmed cases of malaria. Of them, 15 cases were Plasmodium falciparum with cerebral involvement, three cases with mixed infections of P. falciparum and P. vivax, 32 cases of P. vivax, three cases of P. malariae and one case of P. ovale. Five out of 15 patients of P. falciparum (33.3 per cent), one out of 54 patients with mixed infection of P. falciparum and P. malariae (1.8 per cent) and the sole case of P. ovale (1.8 per cent) had fatal outcome. The serum TNF-alpha measured by avidin-biotin sandwich ELISA, was found to be significantly raised in P. falciparum and more so in fatal infections. The degree of parasitaemia, due to single or double infection, had positive effect on cytokine production. The mean TNF-alpha concentration was statistically significantly higher (p < 0.001) in P. falciparum than in P. vivax parasites infection. The mean TNF-alpha values in P. falciparum and P. vivax were 915 and 280.6 against the values in normal healthy controls of 12.9 pcg/ml respectively (p < 0.001). The study thus showed that the serum concentration of TNF-alpha correlated well with severity of malaria and these values could be used as an important prognostic marker of the disease.     

Identification of a polymorphic Plasmodium vivax microsatellite marker

Microsatellite markers derived from simple sequence repeats have been useful in studying a number of human pathogens, including the human malaria parasite Plasmodium falciparum. Genetic markers for P. vivax would likewise help elucidate the genetics and population characteristics of this other important human malaria parasite. We have identified a locus in a P. vivax telomeric clone that contains simple sequence repeats. Primers were designed to amplify this region using a two-step semi-nested polymerase chain reaction protocol. The primers did not amplify template obtained from non-infected individuals, nor DNA from primates infected with the other human malaria parasites (P. ovale, P. malariae, or P. falciparum). The marker was polymorphic in P. vivax-infected field isolates obtained from Papua New Guinea, Indonesia and Guyana. This microsatellite marker may be useful in genetic and epidemiologic studies of P. vivax malaria.     

Prospective risk of morbidity in relation to malaria infection in an area of high endemicity of multiple species of Plasmodium.

In an area of Papua New Guinea with high prevalence of Plasmodium falciparum (39.6%), Plasmodium vivax (18.3%), and Plasmodium malariae (13.8%), cross-sectional analysis found P. falciparum infection to be independent of the other species despite heterogeneities in transmission. Plasmodium vivax and P. malariae infections were negatively correlated. Plasmodium malariae infection was positively associated with homologous infection four months previously and with prior P. falciparum, but not P. vivax infection. There were no other indications that any Plasmodium species protected against heterologous infection. Prospective analysis of health-center morbidity supported the idea that P. malariae infection protects against disease, but indicated greater protection against non-malaria than P. falciparum-associated fevers. Plasmodium vivax appeared to protect against P. falciparum disease but not against other forms of morbidity. Covariate adjustment had considerable effects on estimated relationships between species, and confounding variables may account for many differences among reports of inter-species interactions in human malaria.