基于重组优势多表位抗原的梅毒间接酶联免疫吸附试验与梅毒螺旋体血凝试验和甲苯胺红不加热血清试验检测梅毒螺旋体抗体的比较研究

目的：建立操作简便、特异性好、敏感性高的血清梅毒抗体的间接酶联免疫吸附试验（ELISA）。方法：通过计算机分析选择梅毒螺旋体优势抗原表位，构建了梅毒螺旋体多表位优势嵌合抗原(rTpN15-TpN17-TpN42-TpN47)表达载体，转化宿主菌HB101进行表达，纯化获得高纯度融合抗原。在此基础上，以纯化抗原包被酶联板，建立检测血清中梅毒抗体的间接ELISA法。利用该方法与梅毒螺旋体血凝试验（TPHA）和甲苯胺红不加热血清试验（TRUST）同时检测了126例梅毒可疑血清样本，对检测结果进行了比较研究。结果：梅毒螺旋体多表位优势嵌合抗原（rTpN15-TpN17-TpN42-TpN47)在大肠杆菌中获得了高效表达，并成功建立了检测血清中梅毒抗体的间接ELISA法及试剂。对126例梅毒可疑血清样本的检测结果，ELISA、TRUST、TPHA的检出阳性率分别为75．40％（95／126）、72．22％（91／126）、70．63％（89／126）， 其中TRUST有29例，TPHA有6例为可疑阳性。TPHA检测的6例可疑阳性中，有5例ELISA、TURST均为阳性；而TRUST测出的29例可疑样品中，仅7例ELISA、TPHA阳性。结论：多表位嵌合抗原ELISA试剂在特异性、敏感性与TPHA法相近，但明显优于TRUST法。     

梅毒螺旋体优势表位抗原嵌合表达构建双抗原夹心法的研究

目的用基因工程方法表达嵌合的梅毒螺旋体优势表位抗原,建立检测血清梅毒抗体的双抗原夹心酶联免疫方法(double antigen sandwich enzyme-linked immunosorbent assay,DAS-EIA)。方法通过计算机软件分析选择梅毒螺旋体优势抗原表位,用聚合酶链式反应(PCR)扩增优势表位基因,构建了梅毒螺旋体多优势表位嵌合抗原(rTpN15-TpN17-TpN47)表达载体,转化宿主菌BL21(DE3)进行表达,亲和层析柱法纯化获得高纯度融合抗原,并用其建立检测梅毒抗体的DAS-EIA。结果表达的优势表位嵌合抗原具有很好的抗原性。用其建立的嵌合抗原DAS-EIA检测确诊的50份阳性和30份阴性血,阳性检出率和阴性检出率都是100%。结论嵌合抗原DAS-EIA法具有比间接EIA和重组单抗原DAS-EIA更高的灵敏度和检出正确率,其检测水平已经达到国外TPHA的水平。该方法的建立为临床检测梅毒开辟了新的方法。     

螺旋体传染病

梅毒螺旋体多表位抗原的改构及在双抗原夹心检测梅毒抗体中的应用——张贺秋等(北京军事医学科学院基础医学研究所100850)《中国医学检验杂志》2004，5(6)：549-551[研制用于包被和标记重组梅毒螺旋体表位抗原，建立双抗原夹心酶联免疫检测梅毒抗体。方法：利用计算机Godlkey软件分析梅毒螺旋体基因，确立优势抗原表位(rTpN17-TpN15-TpN42-Tp-N47)，并分别克隆表达四个抗原，然后将四个抗原串联成一个嵌合表达蛋白用于包被，在此抗原的末端连接4个赖氨酸，作为辣根过氧化物酶标记用梅毒抗原，建立双抗原夹心检测梅毒抗体。     

四种梅毒螺旋体检测方法的应用分析

应用酶联免疫吸附试验(ELISA)、梅毒螺旋体血球凝集试验(TPHA)、甲苯胺红不加热血清试验(TRUST)分别检测血清中的梅毒螺旋体抗体,荧光定量PCR(FQ-PCR)法检测局部分泌物或渗出物中梅毒DNA.结果 :TRUST、TP-ELISA、TPHA、FQ-PCR法灵敏度分别为84.3%、96.1%、94.1%、97.1%,特异性分别为93.5%、98%、100%、100%.TRUST在一、三期梅毒的阳性率最低(66.7%和50%);FQ-PCR在一期梅毒阳性率最高.提示TP-ELISA可作为输血和手术前检测的一种理想筛查方法;TPHA可作为梅毒确诊方法;TRUST可应用于梅毒治疗疗效监测;早期梅毒的诊断首选FQ-PCR.     

应用全自动微孔板法改进TRUST试验检测梅毒抗体

目的:用全自动微孔法代替传统方法检测梅毒抗体,并对改进法进行对比分析和评价.方法:用全自动酶免分析仪法与传统甲苯胺红卡片试验(TRUST)法对比检测了75例梅毒患者和1 061份献血员的血清,不符合者用螺旋体血球凝集试验(TPHA)法验证.对18 000份献血员标本用改进的方法检测,并与传统方法对比,用TPHA法验证.结果:改进方法与传统方法相比,75例梅毒患者血清均呈阳性结果,但改进方法比传统法的凝集颗粒大而均匀,更易判读.在1 061份献血员血清中,用改进的方法检出2份阳性血清,并经TPHA证实,用传统方法则判为可疑血清.用改进方法在18 000份献血员的血清中的7份标本呈阳性,TPHA方法亦为阳性,而传统方法3例呈阳性;2例可疑,2例阴性.结论:改进的全自动微孔板法比传统方法具有敏感性高、特异性好、自动化程度高、易于标准化等优点,适合献血员筛查和临床大批量梅毒患者标本的检验.     

ELISA和TRUST检测梅毒螺旋体方法比较分析

目的 比较ELISA(酶联免疫吸附试验)、TRUST(甲苯胺红不加热血清试验)法诊断梅毒螺旋体感染的方法学差异.方法 用目前国内最常用的诊断梅毒螺旋体感染的TRUST试剂及ELISA试剂检测722例标本,同时与TPPA(梅毒螺旋体明胶凝集试验)的检测结果进行比较,从而得到各试验的假阴性率和假阳性率.结果 对722份样本的检测中,ELISA和TPPA的阳性符合率为98.26％,假阴性率和假阳性率分别为0.46％和1.91％,2种方法差异无统计学意义(P ＞0.05); TRUST和TPPA的阳性符合率为84.39％,假阴性率和假阳性率分别为21.08％和5.10％;ELISA和TURST检出率差异有统计学意义(P＜0.01).结论 ELISA测定梅毒螺旋体的方法在日常大量标本检验时优于TRUST.     

ELISA法检测梅毒螺旋体的临床应用评价

长期以来临床实验室诊断梅毒螺旋体感染的血清实验主要分为两大类 ,一类为非梅毒螺旋体试验 ,代表实验有不加热血清反应素试验 (USR )、甲苯胺红不加热试验(TRUST)和快速血浆反应素试验 (RPR)等。另一类为梅毒螺旋体血清试验 ,如TPHA和TPPA等。在临床应用中前一类试验主要用于梅毒感染的初筛和疗效观察 ,后一类用于梅毒感染的确认[1] 。由于非梅毒螺旋体试验检测的抗体不是梅毒特异性抗体 ,一些其他疾病如结核、自身免疫性疾病、猩红热等也可出现阳性反应 ,加之其灵敏度和特异性也只在 70 %～ 80 % [2 ] ,故认为USR、TRUST和RPR…     

螺旋体传染病

基于重组优势多表位抗原的梅毒间接酶联免疫吸附试验与梅毒螺旋体血凝试验和甲苯胺红不加热血清试验检测梅毒螺旋体抗体的比较研究——张贺秋等(北京中国人民解放军军事医学科学院基础医学研究所100850)；《中国性病艾滋病防治&;gt;，2002，8(5)：266—268[目的：建立操     

两种检测梅毒螺旋体抗体方法的比较

近年来 ,我院以酶联免疫吸附试验 (EL ISA)法及甲苯胺红卡片试验 (TRU ST)分别检测了 34例临床梅毒患者、6例质控梅毒阳性及 6 10 9例术前验血患者血清 ,旨在比较二者梅毒螺旋体的敏感性及特异性 ,现将结果报告如下。一般资料 :检测血清共包括三种 :38份血清为我院 1998～2 0 0 3年 6 10 9例患者输血前检查梅毒螺旋体抗体的阳性标本 ,均经两种以上检测方法验证 ;34份为临床确诊梅毒患者的阳性标本 ;6份为临检部提供的质控阳性血清。方法 :TRUST采用甲苯胺红不加热血清凝集法 ;EL ISA法在包被抗原的微孔中加入 5 0 μl待测血清和 5 0…     

梅毒螺旋体重组抗原TmpA的表达及其在梅毒诊断中的应用

目的：用基因工程方法表达梅毒螺旋体的TmpA重组抗原,建立检测梅毒螺旋体抗体酶联免疫试验（EIA）。方法：用聚合酶链反应（PCR）扩增梅毒螺旋体TmpA基因,克隆到原核表达载体pQE-30中表达,用亲和层析柱法纯化该重组抗原,并用其建立检测梅毒螺旋体抗体的EIA。结果：表达的重组抗原相对分子质量构为39000,具有良好的抗原性。用其建立的EIA检测10份阳性参比血清,均为阳性,灵敏度为100%;检测20份阴性参比血清,均为阴性,特异性为100%。检测12份I期梅毒（早期感染）和24份Ⅱ期和晚期梅毒患者血清,阳性率分别为91.67%（11/12）和100%（24/24）。结论：用该重组抗原建立的EIA可检出97.2%（35/36）以上梅毒患者,仅2.8%（1/36）漏检。为进一步提高该试剂的灵敏度,可能需增加梅毒螺旋体的其它重组抗原。     

Evaluation of the fluorescent treponemal antibody absorption test for detection of antibodies (immunoglobulins G and M) to Treponema pallidum in serologic diagnosis of syphilis

We compared the fluorescent treponemal antibody-absorption (FTA-ABS) (immunoglobulin (Ig)G + IgM) assay with the (micro-) Treponema pallidum haemagglutination assay (TPHA), the T. pallidum particle agglutination assay (TPPA), the Murex syphilis ICE (ICE) enzyme-linked immunosorbent assay (ELISA), the Diesse Enzywell TP (TP) (ELISA) using 122 serum samples and the Western blot (WB) assay using 42 serum samples whose results were inharmonious with other tests. Additionally, the Captia syphilis-M (IgM) (ELISA) were performed. All sera had already been examined by the rapid plasma reagin (RPR) card test, a non-treponemal test and the TPHA, a treponemal test using routine screening tests. Agreements of the FTA-ABS with the TPHA test, the TPPA test, the ICE test and the TP test were 97.5%, 95.9%, 98.3% and 98.3%, respectively. The results suggest that the FTA-ABS test is a useful confirmatory test, but can be inadequate as a confirmatory test for serologic diagnosis of syphilis by giving equivocal and false-negative results even rarely.     

Human immunodeficiency virus infection and syphilis in Hondurian female prostitutes

The seroprevalence of human immunodeficiency virus (HIV) infection and syphilis was investigated in 181 female prostitutes in Tegucigalpa, Honduras. One particle agglutination test and two enzyme immunoassays, as well as one immunofluorescence test were used for the screening of HIV antibodies. Confirmation of positive results by the screening tests was carried out by Western blot. The prevalence of HIV seropositivity was 4% (8 women). Specific treponemal antibodies were found in 50% (90/181) of the prostitutes as judged by Treponema pallidum haemagglutination assay (TPHA) and/or fluorescent treponemal antibody-absorption (FTA-ABSIgG) test. As estimated by the positivity of any or both non-treponemal tests (VDRL and RPR), a total of 31 (17%) out of the 181 women had active syphilis. A good correlation was found between the results obtained by TPHA and FTA-ABSIgG. IgM antibodies were found in 72% of sera positive by TPHA and/or FTA-ABSIgG. Four out of the 181 women were found to have antibodies to both HIV and Treponema pallidum.     

两种梅毒血清学检测方法的联合运用分析

目的进一步提高酶联免疫吸附试验(ELISA)对梅毒的筛查率,确保梅毒血清学的检测效果。方法采用ELISA联合甲苯胺红不加热血清试验(TRUST),对2011年上海浦东新区2 686例监测人群的血清样本进行梅毒血清学筛查,联合检测的血清学阳性样本用梅毒螺旋体明胶凝集试验(TPPA)甄别其生物学假阳性;再从2 397份两种方法均为阴性的样本中,随机抽取1 000份进行TPPA检测,甄别其生物学假阴性。结果 ELISA法相对于TPPA法的敏感性为98.9%,特异性为98.4%,两法间差异无统计学意义(P>0.05);ELISA与TRUST法联合应用后的敏感性接近100%,特异性为98.3%。结论 ELISA与TRUST方法的联合应用,其敏感性接近100%,表明其可用于梅毒筛查;另一方面,两者联合运用的特异性为98.3%,具有一定的假阳性率,表明其阳性样本(两法均为阳性的样本除外)还需应用TPPA方法进行最终确认。     

Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purified recombinant Treponema pallidum antigen 4D

An enzyme-linked immunosorbent assay (ELISA) for syphilis has been developed that detects IgG antibody to purified recombinant Treponema pallidum surface antigen 4D. The 4D ELISA was capable of detecting 25 ng of 4D antigen-specific antibody. Neither 172 nonsyphilitic sera nor 20 false-positive sera in the Venereal Disease Research Laboratory test reacted in the 4D ELISA. The sensitivity of the 4D ELISA was comparable to that of the adsorbed fluorescent treponemal antibody test in primary, secondary, and latent disease. Most sera from patients with yaws or pinta were also reactive, a result indicating that a 4D antigen-like molecule also exists in the closely related pathogenic treponemes Treponema pertenue and Treponema carateum.     

Evaluation of the serodia Treponema pallidum particle agglutination, the Murex Syphilis ICE and the Enzywell TP tests for serodiagnosis of syphilis

We evaluated the Treponema pallidum haemagglutination assay (TPHA), a treponemal test, with three other treponemal tests, the Serodia T. pallidum particle agglutination assay, the Murex Syphilis ICE IgG + IgM enzyme immunoassay (EIA) and the Enzywell TP IgG + M EIA (a new rapid EIA) for use in conjunction with the rapid plasma reagin test (RPR), a non-treponemal test, for serodiagnosis of syphilis. In all, 124 serum samples were found reactive with RPR and/or TPHA after testing by the routine laboratory protocol. Twenty-three (18.5%) of them were positive only by RPR test and were evaluated as biologically false-positive, 16 were positive only by the TPHA and 84 by both the RPR and TPHA tests; one sample was non-specific (heterophile reaction) in the TPHA.Agreements of the TPHA with the Serodia TPPA, the Murex Syphilis ICE and the Enzywell TP tests were 96.7%, 100% and 99.1%, respectively.We conclude that each one of the tests, the Serodia TPPA, the Murex Syphilis ICE and the Enzywell TP, is an appropriate substitute for screening for serodiagnosis of syphilis.     

Cross-reactivity in serological tests for Lyme disease and other spirochetal infections

Serum specimens from 163 persons with Lyme disease, tick-borne or louse-borne relapsing fever, yaws, syphilis, leptospirosis, or Rocky Mountain spotted fever were analyzed to assess the specificity of indirect fluorescent antibody (IFA) tests, an enzyme-linked immunosorbent assay (ELISA), and microscopic agglutination (MA) procedures. Strong cross-reactivity occurred when sera from individuals with Lyme disease, tick-borne relapsing fever, and louse-borne relapsing fever were tested against heterologous Borrelia antigens. Antibodies to Borrelia burgdorferi bound to Treponema pallidum in immunofluorescence tests for syphilis. Sera from subjects with syphilis cross-reacted in IFA tests and the ELISA for Lyme disease. Immunoglobulin antibodies to Borrelia or Treponema spirochetes, however, did not react with serovars of Leptospira interrogans in MA or IFA tests, and the prevalence of false-positive results in the reciprocal analyses was negligible.     

Influence of human immunodeficiency virus infection on treponemal serology, in patients who have been treated for syphilis

Sera from 20 homosexual men who were infected with the human immunodeficiency virus (HIV) and had been treated previously for syphilis, were examined for cardiolipin and treponemal antibodies by the Venereal Diseases Research Laboratory (VDRL) test, and the Treponema pallidum haemagglutination assay (TPHA) and fluorescent treponemal antibody absorbed test. In only one case was probable reactivation of syphilis, as judged by rising titres in the VDRL test, noted.     

Significance of laboratory findings for the diagnosis of neurosyphilis

Our objective is to assess the specificity and sensitivity, and thus elaborate the relevance, of different laboratory findings for the diagnosis of neurosyphilis. One hundred and fourteen HIV-negative pairs of serum and cerebrospinal fluid (CSF) samples were examined by the Venereal Disease Research Laboratory (VDRL) test, a fluorescent treponemal antibody-absorption (FTA-ABS) test, microhaemagglutination assay with Treponema pallidum antigen (MHA-TP) test (serum) and Treponema pallidum haemagglutination assay (TPHA) test (CSF); further, albumin, total protein, and total IgG were determined and, in the CSF, cell count was performed. The donors were 60 patients with active neurosyphilis and 54 healthy persons with a former history of syphilis and with persisting positive results in the T. pallidum haemagglutination tests (serum: MHA-TP, CSF: TPHA), who supplied specimens for control. Albumin quotient, IgG index, TPHA index, modified TPHA index, Intrathecally produced T. pallidum Antigen (ITpA) index, its 2 modifications and, in 12 samples, the adenovirus group antibody (AVGA)/TPHA index were ascertained. The specificity and sensitivity of the TPHA index were 100% and 98.3%, of the modified TPHA index 50.0% and 96.7%, of the ITpA index 42.6% and 90.0%, of the modified ITpA indices 51.8% and 68.3% (first modification) and 53.7% and 63.3% (second modification). The AVGA/TPHA index yielded a specificity of 91.7% (11/12). The CSF VDRL test was positive in 55/60 (91.7%) of samples from patients with neurosyphilis and in none of the controls (0/54). A CSF-TPHA titre greater than 1:320 was observed in 59/60 (98.3%) of the neurosyphilis specimens and in none of the controls (0/54). A TPHA index above an outcome of 70, a positive CSF-TPHA test at a titre greater than 1:320 and, with lower sensitivity, the criteria of the Centers for Disease Control (CDC) guidelines yield the most reliable results for laboratory support to a diagnosis of neurosyphilis. The modified TPHA index, the ITpA index, and its 2 modifications produce results of minor sensitivity and poor specificity. Observations on the AVGA/THPA index are too limited yet for judgement. The diagnostic significance of a CSF-TPHA titre above 320 needs further confirmation on a greater number of observations made by different laboratories.     

Biological false-positive tests comprise a high proportion of Venereal Disease Research Laboratory reactions in an analysis of 300,000 sera

This retrospective study on syphilis screening at the sexually transmitted infection (STI) unit of a University Department emphasizes the necessity of a treponemal-specific test as the appropriate screening test. The Venereal Disease Research Laboratory (VDRL) test for syphilis screening may, under certain circumstances, yield positive results in patients not infected with Treponema pallidum, a phenomenon referred to as biological false-positive (BFP) VDRL test. The aim of this study was to determine the frequency of BFP tests in a large sample of sera. In this retrospective study, we analysed the results of parallel VDRL and T. pallidum haemagglutination (TPHA) testing of a total of 514,940 blood samples obtained from patients at the Vienna General Hospital between January 1988 and November 1999. Patients' sera with incomplete data on stage and sex and duplicate sera were excluded, leaving 300,000 sera for analysis. The seroprevalence for syphilis was 1.77% (n = 5320), as determined by a positive TPHA test. It was significantly higher in male than in female patients (2.03% versus 1.58%, P<0.001). Of the patients reactive in the TPHA test, 3257 (61.2%) were negative in the VDRL. With regard to reactivity in VDRL testing, 2799 patients (0.92%) of the study population were positive, of whom 736 (26%) were biological false positive. BFP reactivity was found in 0.24% of all patients and was significantly higher in women than in men (0.27% versus 0.20%, P<0.001) and in patients over 60 years of age (0.34%) as compared with those under 60 (0.25%, P<0.001). This proportion might be even higher, as reactivity in the VDRL at 1:0 and 1:2 dilutions without a positive treponemal test was not reported. The subgroup of HIV-positive patients (n = 1415) revealed a 10-fold higher rate of BFP tests (2.1% versus 0.24), an effect being statistically significant. In a low syphilis prevalence population, BFP reactions comprise a high proportion of all VDRL reactors. Therefore, the use of the VDRL as a screening procedure is challenged.