影响间日疟原虫红内期短期体外培养的虫源因素研究

目的探讨影响红内期间日疟原虫短期体外培养的虫源因素。方法在间日疟流行区采集志愿者血样，以Me—Coy’s 5A为培养基，在含5％CO2的37℃培养箱中短期培养，分析间日疟原虫短期体外培养的影响因素。结果培养基可支持问日疟原虫完成1个无性周期循环。导致间日疟原虫体外短期培养失败的因素包括含虫血置于室温时间过长（〉4h）、期别单一（只见环状体）、病人服用抗疟药、服用抗生素药物、原虫密度过低。结论虫源是影响间日疟原虫短期体外培养的重要因素。     

用一种新的悬液系统体外培养伯氏疟原虫

人的恶性疟原虫体外连续培养成功后,相继有巾帽猴疟原虫、诺氏猴疟原虫和食蟹猴疟原虫培养成功的报道,但对鼠类疟原虫(伯氏疟原虫、文氏疟原虫、夏氏疟原虫和约氏疟原虫)虽作过研究,尤其是对伯氏疟原虫这种重要的实验虫种,曾用烛缸法培养,但结果并不理想,只能维持1～2个裂体增殖周期,且原虫血症迅速下降。因此作者设计一种新的悬液培养系统,结果较好,能完成多个裂殖周期,并可出现大量裂殖子和提高原虫     

关于间日疟原虫红内期的体外培养

作者报道了间日疟原虫红内期体外培养43天的结果。从曾旅居于巴西6个月的27岁年轻人采得间日疟原虫虫株,原虫密度为2.5%(大滋养体及裂殖体),用Tragcr烛缸平皿法,培养液由RPMI 1640,葡萄糖及Rh阳性的O型血清组成,不同点主要是在培养液的pH方面。在培养的头6天pH较酸,每天更换3次培养液,每2天添加红细胞1次。间日疟原虫连续培养27天,然后将其冻存于液氮中;为测定原虫活力,再将冻存的原虫进行复苏培养。到43天时中止培养再冻存于液氮。在培养过程中可见到大滋养体,未成熟裂殖体,在39天时还见到幼小滋养体及环状体。在中止培养时,原虫密度为0.25‰。     

广西凭祥一株食蟹猴疟原虫生物学特性的研究

目的 :观察 1991年 10月在中越边境广西凭祥购买的 1只野生食蟹猴 ( M4 )体内发现的疟原虫。方法 :将疟原虫血传给健康的切脾熊猴 ( M6) ,在猴体内的疟原虫配子体在 64个 /10 0 WBC时 ,给大劣按蚊叮咬感染 ,确定孢子增殖期 ,当蚊唾腺发现子孢子时 ,阳性蚊叮咬健康的熊猴 ( M5)后第 8d开始采血观察 ,确定红前期的时间。血内发现疟原虫后开始每 4 h采血 1次的定时观察 ,确定红内期裂体增殖周期及红内期各期疟原虫形态特征。结果 :该株猴疟原虫的孢子增殖期为 11d,红前期为 8d,红内期的裂体增殖周期为 4 8h。早晚期滋养体与人间日疟原虫形态相似 ,被晚期滋养体寄生的红细胞明显胀大 ,阿米巴样活动明显 ,可见薛氏小点 ,成熟的裂殖体内通常可见裂殖子 10— 15个。结论 :试验结果证实 ,该株猴疟原虫为食蟹猴疟原虫并定名为 CVH株 ( Plasmodiumcynomolgi CVH)。     

在肝肿瘤细胞中培养间日疟原虫北朝鲜株红外期

人类和猿猴类疟疾的复发目前认为源于初次红外期裂体增殖后留存在肝细胞中的休眠子。过去由于缺乏合适的红外期培养系统,复发型疟疾红外期的研究受到了限制。最近作者等(1985年)用间日疟原虫Chessen株和ONG株子孢子接种于人肝肿瘤细胞(HepG_2—A16)体外培养红外期获得成功,并发现有两种类型的红外期疟原虫。一型是快速分裂的裂殖体,于培养9天后释放出裂殖子,另一型是不分裂并在首批裂殖体自培养中消失后仍继续存留的休眠子。     

RPMI-1640培养液中加入维生素C培养食蟹猴疟原虫效果的观察

在体外连续培养红内期食蟹猴疟原虫中,为获得较高密度的虫血,我们采用周肇西等[1]方法,对RPMI-1640培养液中加与不加维生素C(VitC)的培养进行了观察。材料和方法1　虫株　为越南引进的食蟹猴疟原虫虫株,液氮保存的虫血,经室温复苏备用。2　维生素C(A.R)　每ml培养液含60μg,新鲜配制。过滤除菌。3　培养方法分为VitC培养液组和无VitC培养液组。同时进行培养。于培养前与培养72h后,分别取虫血,制成薄血膜,用吉氏染色法计数104RBC中疟原虫数。结　果VitC培养液组1次培养食蟹猴疟原虫10瓶,无VitC培养液组1次培养该虫血8瓶。培养前两组…     

一步法培养恶性疟原虫及其在体外药物试验中的应用

为了克服恶性疟原虫体外标准培养法中每天更换培养液的麻烦,作者观察了连续培养3天换1次培养液时疟原虫的生长繁殖情况。所用虫种为已经在体外培养6个月的科摩罗群岛FCPS25株恶性疟原虫。开始培养时,将含虫血用培养液制成50%混悬液,原虫率分为0.25%和0.05%两组,培养皿直径皆为35mm,每个培养皿各加0.2ml含虫血混悬液,然后再分别加入含有35mMHEPES和10%人血清的RPMI 1640培养液1.5、2.25、3、3.75 ml,连续培养3天,中间不换培养液,比较二组疟原虫的繁殖情况。结果在开始培养时原虫率为0.25%组中,各培养皿     

短期体外培养间日疟原虫和卵形疟原虫用于药敏试验

Brockelman等(1989)发展了一种间日疟原虫短期体外培养系统进行药敏试验,该法基于镜下计数成熟裂殖体。作者试图用氚标记的DNA前体物作指标,对间日疟原虫和卵形疟原虫进行药敏试验,以评价此法的适用性。实验中使用了卵形疟原虫的9个分离株(P.o.-1到P.o.-9)和4株间日疟原虫的4个分离株(P.v.-1到P.v.-4),前者来自非洲移民,而间日疟原虫来自亚洲移民及     

氯喹在试管内对间日疟原虫红细胞内期无性体发育的影响

本实验的目的主要是:1)研究间日疟原虫红细胞内期无性体在试管中的发育情况;2)观察加入氯喹后对其发育有无抑制作用;3)找出可以作为鉴别原虫正常发育和药物抑制作用的定量指标的原虫形态特征或判断标准;4)得以了解应用这种方法测定间日疟原虫红细胞内期无性体对氯喹的敏感性的可能性。以13例出现间日疟无性体原虫血症者的血液作试验。在诊断并鉴定虫种后,用抗疟药治疗之前,从患者静脉采血。体外试验的方法与Riechmann等介绍的方法相似,即     

体外培养中间日疟原虫一个裂体增殖周期的观察

1976年恶性疟原虫体外连续培养成功以后,人们利用这一技术又成功地培养了巾帽猴疟原虫、食蟹猴疟原虫、豚尾猴疟原虫和诺氏猴疟原虫的红内期。但连续培养间日疟原虫的尝试始终没有成功。曾经用RPMI1640培养液蜡烛缸法培养间日疟原虫,观察     

恶性疟原虫配子体体外培养实验报告

本文报告用蜡烛缸平皿法培养恶性疟原虫配子体，对云南分离的ＦｃｃＢＨ１／ＹＮ和ＦｃｃＭＤ１两株进行培养获得成功，两株分别在４ｄ和１ｄ出现配子体，１６ｄ和１０ｄ达高峰．最高密度分别为１３５和１２３／１００００ＲＢＣ，Ｖ期配子体最高密度为２０和３０／１００００ＲＢＣ，平均８．３和４．３／１００００ＲＢＣ．雌雄配子体比例为９：１和５：１，雄配子体可见出丝１—６条，表明配子体已达到成熟阶段，为国内首次报道．     

Continuous in vitro propagation of the malaria parasite Plasmodium vivax

The difficulty in controlling Plasmodium vivax, the most common cause of human malaria, has been complicated by growing drug resistance. We have established a method to cycle parasite generations in continuous culture using human blood cells. Chesson strain parasites were passaged from owl monkey erythrocytes to human reticulocytes in McCoy’s 5A medium modified with L-glutamine with 25 mM Hepes buffer supplemented with 20% AB⁺ human serum. Reticulocytes were separated by differential centrifugation in homologous plasma from the peripheral blood of a hemochromatosis patient. Parasites were grown during each 48-hr cycle in a static candle jar environment until the beginning of schizogony, at about 36–40 hr, when reticulocytes were added and cultures transferred to a shaker for 10–12 hr. The addition of a concentration of 10% reticulocytes resulted in stabilizing parasite densities between 0.28 and 0.57 after cycle 3 and increasing the total number of parasites at least 2-fold with each generational cycle. Cultured parasites successfully infected an owl monkey. The morphology of cultured parasites was typical of P. vivax, with highly ameboid trophozoites evident; however, infected erythrocytes were enlarged and distorted on thin film preparations. The species identity of cultivated parasites was confirmed by analysis of the A and C 18S rRNA genes from genomic DNA and expression of only the A gene during erythrocytic asexual growth. The ability to culture P. vivax opens new opportunities to develop vaccines, test drugs, and clone parasites for genome sequencing.     

An in vitro system for assessing the sensitivity of Plasmodium vivax to chloroquine

Following earlier observations on short-term culture of Plasmodium vivax, an in vitro test system has been developed for assessing the parasite's sensitivity to chloroquine. Fresh isolates with predominantly young trophozoites are diluted 1:19 with a (v/v=1/1) mixture of RPMI 1640 and Waymouth medium. The blood-medium mixture (BMM) is inoculated into the predosed microtitre plates before incubation in a candle jar. Incubation for 30 or 42 h yielded the best results. Incubation for 18 or 24 h was generally insufficient for an adequate development of the parasites. The reading of the test is based on stage-specific differential counts in the Giemsa-stained pre-incubation and post-incubation thick films, the evaluation on log-probit analysis of drug-related inhibition of parasite development. The test system has been evaluated on 200 fresh P. vivax isolates in an area with satisfactory clinical-parasitological response to chloroquine. At 30 or 42 h incubation 121 isolates (61.5%) showed adequate control growth and yielded valid sensitivity tests. Complete inhibition of parasite development occurred within the concentration range of 40-1280 nM. The mean EC50 for 30 h of incubation was 50.3 nM, as compared to 49.7 nM with 42 h of incubation. The geometric mean cut-off concentration of parasite development was 488 nM with 30 h of incubation as against 470 nM with 42 h of incubation.     

Population structure and transmission dynamics of Plasmodium vivax in rural Amazonia

Understanding the genetic structure of malaria parasites is essential to predict how fast some phenotypes of interest originate and spread in populations. In the present study, we used highly polymorphic microsatellite markers to analyze 74 Plasmodium vivax isolates, which we collected in cross-sectional and longitudinal surveys performed in an area of low malaria endemicity in Brazilian Amazonia, and to explore the transmission dynamics of genetically diverse haplotypes or strains. P. vivax populations are more diverse and more frequently comprise multiple-clone infections than do sympatric Plasmodium falciparum isolates, but these features paradoxically coexist with high levels of inbreeding, leading to significant multilocus linkage disequilibrium. Moreover, the high rates of microsatellite haplotype replacement that we found during 15 months of follow-up most likely do not result from strong diversifying selection. We conclude that the small-area genetic diversity in P. vivax populations under low-level transmission is not severely constrained by the low rates of effective meiotic recombination, with clear public health implications.     

PCR-ELISA检测疟原虫DNA的研究

目的：介绍一种诊断疟疾的新方法ＰＣＲ－ＥＬＩＳＡ。方法：根据业已报道的疟原虫ＳＳＵｒ－ＲＮＡ基因保守序列，设计并合成一对通用于恶性疟原虫和间日疟原虫的引物，其中一引物的５’端加以生物素标记。经ＰＣＲ扩增后，携带有生物素的扩增产物与先期包被于ＥＬＩＳＡ板上的亲和素结合，再经与恶性疟原虫、间日疟原虫特异、荧光素标记的寡核苷酸探针分别杂交，底物显色等步骤，使ＰＣＲ产物得以半定量地检出。结果：对于恶性疟原虫和间日疟原虫，本法检出最低原虫密度阈值分别为４和１０个原虫／μｌ血（取血２０μｌ），本法检测两种疟原虫未发现交叉反应。结论：本试验具有较高的敏感度和特异性，可望用于疟疾流行病学调查     

Anti-gamete antibodies block transmission of human vivax malaria to mosquitoes

Antibodies were raised in rabbits by immunizing against fresh unfixed or cryopreserved female gametes of the human malaria pathogen Plasmodium vivax. The antibodies were shown to react with the surface of gametes by the indirect immunofluorescent test. When parasite isolates from P. vivax infected individuals were fed through a membrane to Anopheles tessellatus mosquitoes in the presence of immune rabbit sera, they completely blocked the infectivity of the parasite isolates to the vector. Immunoglobulins separated from these sera also blocked infectivity to the same extent as did the immune sera indicating that antibodies were responsible for the transmission blocking effect of the sera. This study indicated that P. vivax like other malaria parasites is highly susceptible to anti gamete transmission blocking immunity.     

间日疟原虫、恶性疟原虫和伯氏疟原虫各期的提纯和分离

用纤维素(Cellulose,CF-11)过滤去除白细胞后再用 Percoll 密度梯度离心方法提纯分离间日疟(P.v.)、恶性疟(P.f.)和伯氏疟(P.b.)3种疟原虫。经纤维素过滤后,P.v.病人血和P.b.感染鼠血中白细胞去除率分别平均达 94.7％和 75.4％,而且不影响原虫密度和各期构成比。多个 Percoll 不连续梯度离心测试3种疟原虫裂殖体、滋养体、环状体和未感染红细胞的相对平均密度(kg/L):P.v.分别为 1.059、1.072、1.096 和 1.099;P.f.分别为 1.070、1.079、1.10 和 1.10,配子体 1.085;P.b.分别为 1.059、1.062、1.103 和 1.104。并据此选择各自合适的 Percoll 梯度分离3 种疟原虫,将裂殖体、滋养体同环状体分开,获取原虫纯度(原虫数/100 个红细胞),P.v.由分离前 0.8％至分离后 93.5％,P.f.由2.3％至100％,P.b.由 30.6％至93％,分别提高 113.8、43.4和 2倍。     

Genetic complexity of Plasmodium vivax parasites in individual human infections analyzed with monoclonal antibodies against variant epitopes on a single parasite protein.

Monoclonal antibodies against variant epitopes of a highly polymorphic protein (PV200) in schizonts of Plasmodium vivax have been used to analyze the variety of genetically distinct populations of parasites present in the peripheral blood of individual P. vivax infections in Sri Lanka. In 9 out of 10 isolates of freshly drawn P. vivax infected blood from different individuals, parasites of only 1 PV200 serotype was found within each individual infection, even though parasites were serotypically distinct between individuals. In 1 isolate parasite population, 3 distinct PV200 serotypes were identified. Thus, most P. vivax infections appeared to consist of a single genetically homogeneous population of parasites within the detection limits of the technique. The prevalence of P. vivax infections in an area of malaria transmission in southern Sri Lanka and the densities of oocysts in mosquitoes fed on P. vivax infected individuals indicated that parasite populations would be transmitted many times before encountering parasites of other origins, and that individual populations would tend to reduce to genetic homogeneity during transmission. These expectations are consistent with the high proportion of genetically homogeneous P. vivax isolates observed.     

Evaluation of a rapid diagnostic test specific for Plasmodium vivax

Plasmodium vivax is the only human malaria indigenous to the Republic of Korea (ROK). A rapid and sensitive diagnostic test (RDT) that detects P. vivax is appropriate for evaluating suspected malaria patients with no travel history abroad. The RDTs, SD Malaria Antigen P.v (SD diagnostic, Kyonggi, ROK) specific for P. vivax and the well documented OptiMAL (DiaMed, Cressier, Switzerland) were compared among 282 volunteers for specificity and sensitivity of P. vivax and Plasmodium falciparum malaria infections against Giemsa-stained blood smears read by an experienced microscopist. A total of 137 volunteers were diagnosed with P. vivax, 45 cases (returned travellers from overseas) were diagnosed with P. falciparum and 100 healthy volunteers were diagnosed as negative for malaria. Correspondingly, the SD Malaria Antigen P.v test identified P. vivax infections in 128/137 malaria patients (93.4%) and 0/100 (0%) healthy volunteers. Three patients identified with P. falciparum also were interpreted as P. vivax by the SD Malaria Antigen P.v test; however, these patients were later confirmed as mixed infections of P. vivax and P. falciparum by polymerase chain reaction. OptiMAL interpreted the three mixed infections only as P. falciparum and detected 130/137 (94.9%) patients with P. vivax. The sensitivity of the SD Malaria Antigen P.v test decreased from 100% (>5000 parasite/microl) to 81.3% (1-100 parasites/microl) as parasitaemia levels declined. For the regions where P. vivax is the primary malaria parasite, the SD P. vivax-specific rapid diagnostic test may be useful for screening suspected malaria patients when sufficient material and human resources (e.g. trained microscopists) are unavailable for malaria diagnosis.     

In vitro anti-malarial drug susceptibility of temperate Plasmodium vivax from central China

In the face of recent increase of Plasmodium vivax malaria in central China, we conducted a study to evaluate in vitro susceptibility of temperate-zone P. vivax parasites to antimalarial drugs. During 2005-2006, in vitro drug susceptibility was measured for 42 clinical P. vivax isolates by using a schizont maturation inhibition technique. Geometric means of 50% inhibitory concentrations (IC(50)s) and 95% confidence intervals (CIs) were 10.87 (4.50-26.26) ng/mL for chloroquine, 4.21 (1.88-9.42-8) ng/mL for mefloquine, 11.82 (6.20-22.56) ng/mL for quinine, 0.13 (0.09-0.20) ng/mL for artesunate, 18.32 (8.08-41.50) ng/mL for pyrimethamine, and 17.73 (10.29-30.57) ng/mL for piperaquine. The IC(50) for chloroquine was lower than those obtained from isolates from Thailand and South Korea, suggesting that chloroquine remained effective against P. vivax malaria in central China. The results further indicated that temperate-zone P. vivax isolates from China were more susceptible to chloroquine, quinine, and mefloquine than isolates from Thailand.