SPA-ELISA检测弓形虫感染鼠尿中的循环抗原

[目的 ]探索弓形虫感染早期诊断的简便方法。 [方法 ]用SPA ELISA检测轻度、中度和重度感染弓形虫鼠尿中弓形虫循环抗原 (TCA)。 [结果 ]轻度、中度和重度感染弓形虫 3组鼠尿中均检出TCA ,上述 3组分别于弓形虫感染后d6 、d3和d4显示阳性斑点。 [结论 ]SPA ELISA能检出弓形虫感染鼠尿中的弓形虫循环抗原 ,可用于弓形虫感染的早期诊断。     

直接凝集试验（DAT）检测弓形虫IgG抗体的研究

本文报道了国内首次建立的检测弓形虫IgG抗体的微孔板法直接凝集试验(DAT)。采用的抗原系经胰蛋白酶处理和福尔马林固定后的弓形虫速殖子全虫抗原,被检血清用α-巯基乙醇排除IgM引起的非特异性反应。对包括实验感染动物在内的不同来源的动物血清和人血清进行了检测。结果表明:该试验具有较高的敏感性、特异性和重现性,并能测出感染较早期的IgG抗体,在4～6℃低温下保存达6～9个月的抗原仍保持其原有的抗原性。     

Western blot analysis of the antibody response of patients with AIDS and toxoplasma encephalitis: antigenic diversity among Toxoplasma strains

We used immunoblotting to ascertain if toxoplasma encephalitis in disease caused by the human immunodeficiency virus (HIV) could be diagnosed by the appearance of characteristic antibodies recognizing specific Toxoplasma antigens. The profile of antibodies to Toxoplasma was examined in human serum and cerebrospinal fluid from patients with chronic and acute toxoplasmosis with or without HIV infection. Many Toxoplasma antigens were recognized by all sera; the majority were presumably surface proteins, as determined by 125I labeling. All sera recognized antigens at 38, 35, 28, and 26 kilodaltons. No specific antibody or pattern of antibodies distinguished between groups of patients. A 120-kilodalton antigen recognized by sera from Atlanta was not, however, seen in most sera from New York. Study of the recognition of the antigens of different strains of Toxoplasma gondii (RH, C56, T100) by the same human sera demonstrated strain-specific antigenic differences. These strain variations may account for the antibody diversity among the patients studied.     

我国部分地区弓形虫病流行病学监测

本文报道采用间接血凝试验对我国１６个省的部分地区进行了人畜弓形虫病流行病学调查．调查人群血清６３０９５份，阳性３６８３份，平均阳性率为５．８４％；检查畜禽３２１６５头，阳性５２７０头，平均阳性率为１６．３８％。这次调查共分离病原体３７株，其中人体３株，猪体２０株，兔体９株，鼠类５株；还从人群感染的地区、年龄、性别、职业等方面进行了分析，初步摸清了我国弓形虫病感染的基本情况．     

急性弓形虫病兔循环抗原动态的实验观察

用Dot-ELISA观察人工感染2株不同剂量的弓形虫家兔血清循环抗原(CAg)动态。结果感染RH株弓形虫速殖子10~4/只和10~2/只的两组家兔,CAg出现和全部阳转时间均在感染后3d。但RH株的感染量影响宿主血清CAg动态。感染PP株10~6/只和10~4/只的两组家兔,血清CAg变化无明显差异。RH株CAg水平变化急骤,而PP株变化较平缓,表明宿主血清CAg动态与弓形虫虫株毒力密切相关。     

建立和评价ELISA法检测血清和唾液中旋毛虫IgG抗体的研究

目的 建立检测血清和唾液中旋毛虫抗体的酶联免疫吸附试验(ELISA)方法.方法 应用方阵滴定法,筛选适宜的旋毛虫抗原(肌肉幼虫可溶性抗原、肌肉幼虫排泄分泌抗原、成虫可溶性抗原、成虫排泄分泌抗原)浓度、血清和唾液稀释度、辣根过氧化物酶标记的山羊抗兔、山羊抗人IgG抗体稀释度.共有20份旋毛虫感染兔、10份旋毛虫病患者血清和唾液用于这4种抗原的敏感性试验,20份健康兔与38份其他寄生虫感染兔和患者的血清和唾液用于这4种抗原的特异性试验.结果 这4种抗原适宜包被浓度依次为8.0 μg/ml、6.0 μg/ml、10.0 μg/ml、9.0 μg/ml.适宜的血清稀释度依次为1:100、1:200、1:50、1:200,唾液均用原液.适宜的山羊抗兔、山羊抗人IgG稀释度分别为1:2 500和l:2 000.这4种抗原检测旋毛虫感染兔血清和唾液的敏感性分别为100%和80%~100%,检测旋毛虫病患者血清和唾液的敏感性分别为100%和60%~80%;检测血清和唾液的特异性依次为81.03%、89.65%、77.59%、82.76%和93.10%、96.55%、89.65%、91.35%.结论 建立了检测兔和人血清及唾液中旋毛虫lgG抗体的ELISA方法.当采集血清标本有困难时,可将唾液替代血清用于检测旋毛虫病.     

免疫层析试纸条诊断旋毛虫病的研究

目的建立一种能诊断人体旋毛虫病及动物旋毛虫感染的快速血清学方法。方法以胶体金标SPA和旋毛虫肌幼虫ES抗原制备免疫层析试纸条(immunochromatographic strip),对旋毛虫病及其它寄生虫病患者血清、旋毛虫及其它寄生虫感染的动物血清进行检测。结果试纸条检测旋毛虫病患者与旋毛虫感染的小鼠、大鼠、兔、猪血清的阳性率分别为100%(20/20)、97.87%(92/94)、100%(5/5)、100%(5/5)及100%(25/25),15min内肉眼可观察结果;其它寄生虫病(并殖吸虫病、血吸虫病、华支睾吸虫病、囊虫病及包虫病等)患者及正常人血清、其它寄生虫感染动物及正常动物血清均为阴性。试纸条和ELISA对100条幼虫感染小鼠血清检测的阳性率分别为91.3%(21/23)和95.7%(22/23)(χ2=0.36,P>0.05),两种方法对200～500条幼虫感染小鼠血清检测的阳性率均为100%(71/71)。试纸条对乡土旋毛虫、布氏旋毛虫、伪旋毛虫及纳氏旋毛虫感染小鼠血清检测的阳性率亦均为100%。试纸条在4℃可保存13个月,检测结果在室温可保存3个月。结论该试纸条可用于人体旋毛虫病和动物旋毛虫感染的快速血清学诊断,也可用于其它种旋毛虫感染的血清流行病学调查。     

甘肃省3地区特殊人群弓形虫感染调查

目的了解甘肃省特殊人群弓形虫感染状况,为科学制定防治策略提供依据。方法随机选择兰州市、平凉市、临夏州3地区的9类特殊人群为观察组,1∶1同比选取一般人群设立对照组。采集调查对象静脉血,采用ELISA法检测弓形虫IgG抗体,对检测结果进行比较分析。结果共检测人群4040人,弓形虫IgG抗体阳性率平均为8.49%;观察组和对照组弓形虫IgG抗体阳性率分别为8.71%、8.27%;动物饲养员、屠宰工人、肉摊商贩、献血员、免疫功能低下者弓形虫IgG抗体阳性率均高于对照人群,观察组中以屠宰工人感染率最高,孕妇最低;特殊人群感染率男女无差异;感染率随调查人群文化程度增高而降低,随年龄增大而升高。结论甘肃3地区人群弓形虫感染较高,应加强动物密切接触者和育龄妇女弓形虫病防治知识的健康教育,促进自我防护,降低感染;应加强对献血人群的检测,防止健康人因受血而感染。     

弓形虫循环抗原诊断试剂的研究

目的　比较弓形虫不同免疫原所制备抗体检测CAg的效果 ,优选高效价的抗体诊断试剂。 方法　制备弓形虫的细胞质抗原 (C)、代谢抗原 (S)。用抗原C、C +S、S及活虫各免疫 2只家兔 ,收集抗血清经纯化而获得IgG抗体 (多抗 )并制备酶标记抗体 ;用上述 4种多抗与多抗 -HRP组合成 16种双抗体夹心型ELISA ,检测人血清CAg和C、S抗原 ,评价试剂的敏感性 ;抗血清与疟原虫、隐孢子虫等抗原进行交叉反应性试验 ,评价试剂的特异性。结果　各种ELISA夹心法同步检测CAg阳性样本 7例 ,阴性样本 8例 ,均未出现假阴性和假阳性反应 ,其OD均值的P/N比值依次为C +C组 33 5、S +S组 2 7 8、CS +CS组 2 1 2、P +P组 13 2 ;C和S抗原的最低检出量均为 0 5 μg/ml;与其它寄生虫未出现交叉反应。 结论　 (1) 4种抗体用于检测人血清CAg、C和S抗原以及其它抗原的结果表明 ,各种抗体试剂均具有良好的敏感性和特异性 ,其中以抗C抗体试剂最为满意。 (2 ) 16种组合形式的夹心ELISA ,检测弓形虫抗原的差异无统计学意义 ,表明弓形虫C和S抗原间存在着共同抗原成分。     

弓形虫抗体免疫印迹试剂盒的研制

目的 研制敏感、特异的检测弓形虫IgM和IgG抗体免疫印迹试剂盒。 方法 收集人工感染RH株弓形虫速殖子昆明系小鼠的腹腔液，提取弓形虫胞质蛋白，采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离弓形虫可溶性抗原，并经电泳转印至硝酸纤维膜，以无毒灵敏的四甲基联苯胺（TMB）为底物分别检测30份弓形虫IgM和28份IgG阳性血清，40份健康人血清。通过比较抗原制备方法和使用剂量、封闭剂、洗涤和稀释剂、工作浓度、作用时间以及反应带出现率，选择最佳实验条件，以敏感性、特异性、Youden指数以及稳定性作为试剂盒评价标准。 结果 用免疫印迹试剂盒检测30份弓形虫IgM和28份IgG阳性血清，敏感性分别为90.0%（27/30）和85.7%（24/28），40份健康对照者血清的弓形虫IgM和IgG抗体均为阴性，特异性均为100%，Youden指数分别为0.9和0.86。试剂盒于4℃ 保存约6个月的检测结果一致。 结论 该免疫印迹试剂盒敏感性和特异性均较高，且操作简便、快速。     

二倍体型与三倍体型卫氏并殖吸虫虫体抗原与宿主抗原的观察

用间接免疫荧光抗体法,对鼠体内两种类型卫氏并殖吸虫虫体抗原与宿主抗原做了检测。结果显示两类型童虫与感染同源卫氏并殖吸虫小鼠血清反应后,虫体抗原存在于体表皮层、肠管上皮细胞上;两类型童虫与兔抗鼠红细胞抗体血清反应后,主要在表皮层看到宿主抗原,荧光强度比虫体抗原所示为弱;三倍体型童虫的宿主抗原荧光反应有52.6～60％呈(++),而二倍体型大部只是(+)反应;这种荧光强度的差别在非免疫状态所获的童虫切片上也被看到。     

山东省弓形虫病流行病学调查研究

应用滤纸干血滴间接血凝试验（ＩＨＡＴ）对山东省９个地区１５个县市的弓形虫感染情况的调查结果表明，人群总感染率为１．５３％。调查常见动物１１种，检出阳性动物７种，感染率分别为猫４６．０％、猪３０．３４％、羊１２．８８％、狗１２．８７％、驴５．８８％、兔４．１７％、鸡４．７６％、另外４种动物牛、马、骡和鼠未发现阳性者。     

Lymphoproliferative responses of splenocytes before and after challenge with Schistosoma haematobium in C57BL/6 mice vaccinated with human anti-Idiotypes

Anti-idiotypic vaccines (anti-Id or antibody 2; Ab2) in experimental schistosomiasis engender varying degrees of resistance to challenge infection. To further characterize the mechanisms involved in the induction of protective immunity associated with such a vaccine model, spleen cells of mice vaccinated with human Ab2 (HAb2) were investigated for their lymphoproliferative responses before and after challenge infection with normal S. haematobium cercariae. HAb2 was purified from sera of chronically infected patients using protective rabbit antibodies (RAb1) isolated from sera of rabbits multiply immunized with UV-irradiated cercariae by affinity chromatography over soluble worm antigenic preparation (SWAP). Vaccination of C57BL/6 (C57) mice with HAb2 resulted in approximately 31% and approximately 36% protection in two experiments of resistance to infection. Splenocytes were collected prior to challenge at week 6-post initial immunization and after challenge at days 6, 10, 28 and 90. Prior to challenge, in vitro splenic responses of HAb2-vaccinated animals (HAb2-group) to phytohaemagglutinin (PHA) declined while both SWAP and HAb2-driven responses increased, all compared to naive control. After challenge, PHA responses increased in the two test groups on day 6 then significantly decreased to lower levels. On the other hand, SWAP- and HAb2-driven responses of HAb2 group increased by day 6 then declined while the same responses in infected control mice increased on days 10 through 28 and decreased by day 90. Generally, proliferation obtained following in vitro stimulation with HAb2 was greater than that with SWAP in the HAb2-group after challenge. These results suggested that human anti-Id antibodies could mimic at the T cell level the properties of a protective antigenic epitopes of the irradiated-cercariae vaccine.     

Detection of Trichinella spiralis antigens in urine of men and animals

The practical inability to diagnose Trichinella spiralis antibodies in man before day 20 post infection (dpi) has stimulated interest in the development of immunodiagnostic test to detect circulating antigens. Our previous experience showed that soon after infection immune complexes as well as uncomplexed parasite antigens in sera of infected rats could be detected. To diagnose the presence of antigen in urine, double sandwich-capture ELISA was applied using a peroxidase-conjugated rabbit immunoglobulin to T. spiralis larval antigens. The plates were coated with metabolic (AES) or somatic (AS) larval antigens. Mice were infected with 500 T. spiralis larvae. The urine samples from experimentally infected mice taken from 1 to 41 dpi. and the urine samples from patients of the Clinical Hospital in Bia?ystok taken from 3 to 120 dpi were examined. Before testing, the urine samples were heated for 6 min. at 100 degrees C and centrifuged for 6 min. at 5000 g, supernatants were used in ELISA. The presence of T. spiralis antigens in mice urine samples was detected between 6-26 days post infection (dpi) using double sandwich-capture ELISA. All samples taken later were negative as samples taken from uninfected mice. 3 from 9 human urine samples taken 3-10 dpi were positive, the remaining samples taken 3-10 and 10-30 dpi showed values near to "cut-off". In both mice and human urine samples the higher level of antigens was detected in ELISA when somatic larval antigen was used. The T. spiralis antigens were present in urine of infected men and mice in the first phase of infection.     

华支睾吸虫富甘氨酸2a类抗原Cs4重组蛋白的免疫学特性与定位

目的 对华支睾吸虫富甘氨酸2a(glycine rich antigen 2a,GRA2a)类抗原Cs4重组蛋白(rCs4)进行免疫学特性分析,并对GKA2a类抗原进行组织定位.方法 使用蛋白质印迹(Western印迹)分析rCs4与华支睾吸虫病患者混合血清的免疫反应性,并分析GRA2a类抗原的分泌性.应用ELISA法检测rCs4免疫小鼠血清中抗rCs4特异性IgG水平,并动态检测华支睾吸虫感染兔0～44周的血清中抗rCs4特异性IgG水平.通过免疫荧光实验(immunofluorescence assay,IFA)对华支睾吸虫GRA2a类抗原进行组织定位.结果 纯化的rCsd可被华支睾吸虫病患者混合血清识别.rCs4单抗(mAb Cs4-31)与华支睾吸虫排泄分泌抗原和可溶性抗原在相对分子质量(Mr)26 000与55 000之间分别有13、15个反应条带,均呈阶梯状分布.ELISA检测rCs4免疫小鼠抗rCs4和ESA的IgG效价均达1:64 000,感染兔的抗rCs4特异性抗体平均水平自感染第4周开始上升,于第6周达到高峰,此后逐步递减,至第20周已呈较低水平.IFA检测发现GRA2a类抗原定位于华支睾吸虫成虫的表皮.结论 Cs4蛋白是华支睾吸虫在感染早期的分泌型蛋白,具较好的抗原性.GRA2a类抗原定位于华支睾吸虫成虫表皮.

Abstract：

Objective To study immunological characteristics of Cs4 recombinant protein(rCs4)of Clonorchis sinensis,one of the glycine rich antigen 2a(GRA2a)gene family member,and the histolocalization of GRA2a antigens. Methods The immunological reactivity of Clonorcihs sinensis Cs4 protein to pooled sera from clonorchiasis patients was investigated by using purified rCs4 in Western blotting.The secretory property of GRA2a antigens was also analyzed by Western blotting.The level of specific antibodies against rCs4 in the sera from mice immunized with rCs4,and the dynamic change of antibodies against rCs4 in the sera of C. sinensisinfected New Zealand rabbits(O-44 w post infection)were examined by ELISA.Immunofluorescence assay (IFA)was performed using the anti-rCs4 monoclonal antibody Cs4-31(mAb Cs4-31)to determine the localization of GRA2a antigens in C. sinensis adult worm.Results The purified rCs4 was recognized by pooled sera from clonorchiasis patients.13 and 15 bands appeared at the sites between M,26 000 and 55 000 after mAb Cs4-31 reacting with excretory-secretory antigen(ESA)and soluble adult worm antigen(AWA),respectively.The anti-rCs4 and anti-ESA serum antibody titers in mice immunized with rCs4 were 1:64 000.In C. sinensis infected rabbits,antibody level began to elevate at 4 w after infection,peaked at 6 w,and declined rapidly to a lower level at 20 w.IFA revealed that GRA2a antigens were localized in the tegument of C.sinensis adult worm.Conclusion The Cs4 protein was a secretory antigen appeared in the early stage of infection,and exhibited high antigenieity.GRA2a antigens were localized in tIle tegument of C. sinensis adult worm.     

Composition of circulating immune complexes in acute toxoplasmosis

The circulating immune complexes (CIC) that form in the hot just in early Toxoplasma gondii invasion can be present in the blood bed for a while. At the same time, the data on the antigenic composition of CIC in toxoplasmosis are fragmentary and rather contradictory. The investigation used enzyme immunoassay (EIA) to detect specific CIC that contain antigens to T. gondii tachyzoites in the sera of different populations and studied their antigenic composition by immunoblotting after 2-6% polyethylene glycol 6000-induced deposition. Examining 464 sera from groups of individuals with varying-stage T. gondii invasion indicated CIC in each, but showing their different frequency. CIC were virtually present in all sera from children who had been diagnosed as having congenital toxoplasmosis. In groups of seropositive pregnant women, CIC detection rates were noticeably higher in the samples showing both IgG and IgM antibodies (40.2 and 66.4%, respectively). CIC were also revealed in 9.8% of the seronegative blood donors; however, immunoblotting failed to confirm that they had no components that specifically reacted with T. gondii tachyzoite antigen antibodies. There were some differences in the composition of CIC in the serum yielding positive results in both EIA and immunoblotting. The serum CIC from pregnant women that exhibited only IgG antibodies contained mainly T. gondii antigens having molecular weights of 67 and 30 kD. The serum CIC from children with congenital toxoplasmosis and from pregnant women with serologically detected IgM antibodies to Toxoplasma antigens were found to contain 55-58-, 48-, 44-, 38-, 30-, and 26-kD components. The same molecular weight proteins were detected by electrophoretic studies of Toxoplasma excretory-secretory antigen (ESA). Comparing the findings suggests that in acute toxoplasmosis, the circulating complexes mainly contain ESA of the tachyzoites which appear in human blood just at the onset of invasion. Thus, this study demonstrates that specifically CIC are detectable in the sera of individuals infected with T. gondii and their antigenic composition varies with the stage of disease. In the authors' opinion, the detection of specific CIC and the determination of their antigenic composition may be serve an additional test in diagnosing acute toxoplasmosis.     

旋毛虫氨基肽酶的表达及其血清学诊断价值的研究

目的构建旋毛虫氨基肽酶（TsAP）基因重组质粒,表达重组TsAP蛋白,评价该蛋白的血清学诊断价值。方法通过RT-PCR扩增TsAP基因。构建重组质粒pGEX-6p-1-TsAP,测序及酶切鉴定后转化E．coliBL21,用异丙基-G-D-硫代半乳糖苷（IPTG）诱导,表达产物经GSTSefinoseResin（BBI）亲和层析纯化后应用SDS-PAGE和Westernblot进行鉴定。应用旋毛虫重组rTsAP蛋白EusA（rTsAP-ELISA）对旋毛虫感染小鼠血清进行检测,观察旋毛虫感染小鼠后不同时间的血清抗体阳性率,并与旋毛虫肌幼虫ES抗原ELISA（ES-EIJIsA）的检测结果进行比较。结果构建的重组表达载体pGEX-6p-1-TsAP能表达TsAP蛋白。SDS-PAGE结果显示,rTsAP的分子质量单位约为80ku,以IPTG诱导4h后表达量最大。rTsAP-ELISA及ES-ELIS对旋毛虫感染小鼠血清的抗体检出率均为100％（40／40）,与曼氏裂头蚴、弓形虫感染小鼠及正常小鼠血清均无交叉反应,与日本血吸虫感染小鼠血清的交叉反应率分别为93．75％（15／16）和50．00％（8／16）（P〈0．05）。rTsAP-ELISA与ES-ELISA对旋毛虫感染2周的小鼠血清抗体检出率分别为50．00％（11／22）和81.82（18／22）,差异无统计学意义（P〈0．05）,至感染后6周抗体检出率均达100％。结论重组TsAP蛋白具有良好的反应原性,但与日本血吸虫感染小鼠血清有较高的交叉反应。     

Recognition of different Toxoplasma antigens by IgM and IgG antibodies in mothers and their congenitally infected newborns

The protein-blotting technique was used to determine the antigens of Toxoplasma gondii that were recognized by IgG and IgM antibodies in sera of congenitally infected newborns and their mothers. Patterns of IgG and IgM blots with sera from newborns revealed antigen-antibody reactions (bands) that were not present in the respective blots obtained with sera from their mothers. This was true for 50% of 24 congenitally infected newborns. In contrast, such a difference was noted in only one (5%) of 21 newborns who were not congenitally infected but whose mothers had serological evidence of acute infection with T. gondii acquired during gestation. Our results suggest that the protein-blotting method or an adaptation may be valuable for study of the immune response of the mother, fetus, and newborn to various antigens of infectious organisms and for diagnosis of congenital infections in the newborn.     

云南西部人类免疫缺陷病毒与弓形虫双重感染者一氧化氮水平的研究

目的了解人类免疫缺陷病毒（human immunodeficiency virus,HIV）与弓形虫双重感染者的一氧化氮（nitric oxide,NO）水平及感染弓形虫与NO之间的关系。方法收集144例HIV阳性者血清,进行弓形虫抗体检测．于9个月后再次取血．前后两次取得的血清应用硝酸还原酶法测定其NO的水平：对HIV阳性者血清进行CD4＋细胞计数．根据CDg细胞计数水平将患者分为免疫功能正常组、免疫功能受损组和免疫功能严重受损组．检测三组患者的NO水平。用SPSS16．0软件对检测结果进行统计学分析。结果HIV与弓形虫双重者感染者的血清NO值为（82．77±5．82）μmol／L。HIV阳性无弓形虫感染者的NO值为（44．56±5．38）μmol／L,二者差异有统计学意义（P〈0．05）;第一次和第二次检测的HIV与弓形虫双重感染者NO水平分别为（81．37±8．22）μmol／L和（84．18±8．28）μmol／g．差异无统计学意义（P〉0．05）;第一次和第二次检测的HIV单一感染者NO值分别为（48．35±9．16）μmol／L和（40．78±5．67）μmol／L差异无统计学意义（P〉0．05）。免疫功能正常组NO水平与免疫功能受损组、免疫功能严重受损组进行比较,差异均具有统计学意义（P〈0．05）;免疫功能受损组NO水平高于免疫功能严重受损组,但差异无统计学意义（P〉0．05）。结论HIV与弓形虫双重感染者血清NO水平比HIV阳性单一阳性者高．随着HIV阳性者CD4＋细胞计数的下降．机体产生NO的能力也在减弱。     

Use of double McAb sandwich ELISA to detect circulating antigen of Toxoplasma in infected rabbits

This paper reports on the cumulative positive frequencies of circulating antigen (CAg) detected in the sera of rabbits infected with Toxoplasma by using double-McAb sandwich ELISA. The positive frequencies of rabbits with heavy and medium infection in the incubation period are 30.8% and 11.1%. Those with medium infection in acute, subacute and early chronic period are 86.1% and 76.7%, 43.3% and 32.0% with light infection. The positive rates of CAg in rabbits of medium and light infection rose progressively in acute period, but declined in subacute and early chronic period. Cross reaction with schistosomiasis and coccidiosis was all negative. This method of high specificity, sensitivity and duplication possesses certain value in the diagnosis of acute or active Toxoplasma infection and may be useful for the diagnosis in the early period.