TP-光激化学发光、TPPA、TRUST试验的临床应用评价

目的 通过对TP-光激化学发光、梅毒螺旋体明胶颗粒凝集试验（TPPA）、甲苯胺红不加热血清试验（TRUST）的临床应用进行评价,为临床医生在不同情况下选择梅毒螺旋体抗体检测方法提供依据。方法 选取2019年4月1日~7月31日我院11619例住院患者、2458例门诊患者及2983例健康体检者进行TP-光激化学发光和TRUST检测,将TP-光激化学发光检测阳性或TRUST检测阳性的标本进行TPPA法检测,比较TP-光激化学发光及TRUST检测结果,TP-光激化学发光、TRUST检测及TPPA法检测结果及检测梅毒螺旋体抗体的诊断价值,TP-光激化学发光、TRUST检测梅毒螺旋体抗体的诊断价值及TP-光激化学发光法S/CO值与TPPA结果。结果 TP-光激化学发光检测门诊阳性率为6.18%,住院阳性率为3.46%,体检阳性率为0.67%;TRUST检测门诊阳性率为3.42%,住院阳性率为1.11%,体检阳性率0.27%。对557例TP-光激化学发光阳性和222例TRUST检测阳性的标本同时进行TPPA法检测,TPPA法阳性率为79.73%,TP-光激化学发光阳性率为98.30%,TRUST阳性率为37.82%,三种方法检测结果两两比较,差异有统计学意义（P＜0.05）。TRUST检测梅毒螺旋体抗体的敏感度低于TP-光激化学发光,差异有统计学意义（P＜0.05）。以TPPA结果为标准,通过对TP-光激化学发光结果的S/CO值分段分析,结果显示S/CO值越高,与TPPA的阳性符合率越高。结论 TP-光激化学发光灵敏度高,适用于梅毒的感染筛查及辅助诊断;TPPA法检测适用于梅毒感染的确诊,能有效排除TP-光激化学发光和TRUST假阳性的问题;TRUST检测灵敏度较低,对梅毒螺旋体的筛查结果准确性不及TP-光激化学发光和TPPA法检测,不适合用于梅毒感染的筛查,可联和使用TP-光激化学发光以提高诊断梅毒的准确率。     

梅毒螺旋体特异性抗体试验方法探讨及临床应用价值评估

目的 分析化学发光微粒子免疫法(CMIA)检测梅毒螺旋体特异性抗体的性能价值,探讨其在临床应用的意义.方法 回顾性分析北京市海淀医院2019年3月至2020年7月所有采用CMIA法进行梅毒螺旋体特异性抗体检测的标本61 051例,阳性标本采用梅毒螺旋体明胶颗粒凝集试验(TPPA)进行确证,分析其相关性.结果 61 0...     

化学发光法检测新型冠状病毒特异性抗体的假阳性结果评价

目的 探讨化学发光法检测新型冠状病毒(2019 novel coronavirus)特异性抗体IgM和IgG假阳性结果的可能原因及相关性分析,为新型冠状病毒肺炎诊治提供有利依据.方法 回顾性研究2020年7月1日至2021年1月31日就诊于北京市海淀医院的患者32136例,采集血液标本用化学发光法进行特异性抗体IgM和IgG检测,阳性的患者均进行实时荧光RT-PCR新型冠状病毒核酸检测.结果 32136例样本中检出IgM阳性226例,IgG阳性452例,阳性的标本再进行核酸检测,结合诊断标准、临床表现、流行病学调查,2例为确诊阳性,其余均为假阳性.IgM抗体假阳性率为0.70％(224/32136),IgG抗体假阳性率为1.40％(450/32136),其男性女性假阳性率差异无统计学意义(P>0.05).年龄组≤20岁、21～30岁、31～40岁、41～50岁、51～60岁、>60岁组中新型冠状病毒特异性抗体IgM假阳性率均低于IgG抗体假阳性率,>60岁老年组的IgM和IgG抗体的假阳性率最高,与其他年龄组比较,差异有统计学意义(P<0.05).同时随着年龄的增加,IgG抗体假阳性率也逐渐增加.假阳性患者中肿瘤患者引起抗体假阳性率最高.结论 新冠抗体的检测虽然可以作为多次核酸检测阴性而临床有症状或影像学有特异性改变的患者的辅助诊断手段,但不适用于大规模一般人群筛查.同时还要有效识别干扰因素对新冠抗体检测结果的影响,为新型冠状病毒感染的诊断与治疗提供帮助.     

TpN17重组抗原的表达及在梅毒血清学诊断中的初步应用

目的用基因工程技术表达梅毒螺旋体TpN17重组抗原,并建立操作简便、特异性好、敏感性高的梅毒血清抗体间接酶联免疫吸附试验(ELISA)检测。方法采用PCR技术扩增梅毒螺旋体TpN17基因,再进行T-A克隆及测序,然后亚克隆到原核表达载体中,以亲和层析法纯化重组蛋白。将重组蛋白包被于反应板,建立检测血清中梅毒抗体的ELISA间接检测法,利用该法与梅毒螺旋体血凝试验(TPHA)、甲苯胺红不加热血清试验(TRUST)同时检测血清样品,对其结果进行比较研究。结果TpN17重组蛋白在大肠杆菌BL21中得到稳定表达,并成功建立了检测血清中梅毒抗体的ELISA间接检测法及试剂。对135例梅毒可疑患者血清进行检测,ELISA、TPHA和TRUST的检出阳性率分别为82.96%、98.52%和71.11%,而TRUST测出的8例可疑样品及31例阴性样品中,TPHA结果全部阳性、而ELISA只有2例阴性。结论TpN17重组抗原ELISA试剂在特异性和敏感性上明显优于TRUST法。     

酶联免疫吸附法检测与甲苯胺红不加热血清试验在梅毒检验中应用价值的比较

目的探讨酶联免疫吸附法(ELISA)检测与甲苯胺红不加热血清试验(TRUST)在梅毒检验中应用价值。方法回顾分析260例梅毒患者,均应用ELISA法与TRUST法检测血清梅毒螺旋体,比较梅毒螺旋体检出率,并分析其敏感性及特异性。结果 ELISA法对梅毒螺旋体检出率为95.8%(249/260),显著高于TRUST法64.6%(168/260),比较具有统计学差异(P0.05);ELISA法诊断梅毒螺旋体的特异性为35.4%(92/260),阳性预测值为59.7%(249/417),阴性预测值为89.3%(92/103),准确率为65.6%(341/520)。结论 ELISA法对梅毒螺旋体检出率较高,可作为大样本梅毒筛查检验,值得临床选择。     

经化学发光免疫分析法检测梅毒抗体在梅毒患者早期诊断中的应用研究

目的:分析经化学发光免疫分析法(Chemiluminescence immunoassay,CLIA)检测梅毒抗体在梅毒患者诊断中的应用价值.方法:收集本院2019年1月至2021年2月收治且需行早期梅毒诊断的125例患者的临床资料,以梅毒螺旋体明胶凝集试验(Treponema pallidum gelatin agglutination,TPPA)检测结果作为金标准,比较甲苯胺红不加热血清学试验(Unheated serological test of toluidine red,TRUST)、CLIA检测诊断梅毒的敏感性、特异性、准确性及CLIA检测诊断效果.结果:125例患者中,TPPA检测出18例阳性,检出率为14.40％.TRUST检测出10例,检出率为8.00％.CLIA检测出21例,检出率为16.80,明显高于TRUST检测(P<0.05).但CLIA、TPPA检测比较无差异(P>0.05).TRUST检测对早期梅毒诊断敏感性、特异性及准确性分别为61.11％、98.13％、92.80％;CLIA检测分别为100.00％、97.20％、97.60％.CLIA检测诊断早期梅毒的敏感性显著高于TRUST检测(P<0.05).CLIA检测S/CO为1～5、6～10、>10者经TPPA检测为阳性的符合率分别为80.00％、100.00％、100.00％.结论:经CLIA检测梅毒抗体在梅毒患者诊断中具有较高的敏感性、特异性,且操作简便快捷,具有较高临床应用价值.     

CMIA法在输血前感染性指标检测中的应用研究

目的对化学发光微粒子免疫测定(CMIA)技术定量检测输血前感染性指标的临床应用进行评价。方法用CMIA技术对22235例患者进行输血前乙肝表面抗原(HBsAg)、抗丙型肝炎病毒抗体(Anti-HCV)、抗梅毒螺旋体特异性抗体(Anti-TP)及抗人类免疫缺陷病毒抗体(Anti-HIV)定量检测,所有阳性标本用相应的酶联免疫吸附法(ELISA)进行定性检测,Anti-TP阳性标本用梅毒螺旋体抗体明胶颗粒凝集试验(TPPA)进行确诊,Anti-HIV阳性标本送市疾控中心进行抗体确认,并对几种方法的检测结果进行分析。结果 HBsAg、Anti-HCV、Anti-TP、Anti-HIV定量检测阳性例数分别为3028、336、684、63;定量阳性标本经ELISA检测,定性阳性例数分别为2472,269,668,58;684例Anti-TP定量阳性样本中,用TPPA确认,阳性603例;63例Anti-HIV定量阳性样本送南充市疾控中心进行确认,48例确定为阳性,5例不确定,10例为阴性。结论 CMIA法定量检测输血前感染性指标可及早发现感染"窗口期"患者,降低漏检率,有利于患者的早期诊断和治疗,有利于医护人员自我防护及减少医疗纠纷。     

化学发光免疫法检测梅毒特异性抗体结果分析

目的 探讨化学发光法检测梅毒螺旋体特异性抗体的应用价值.方法 应用化学发光免疫测定法检测12503例住院患者梅毒螺旋体特异性抗体,并同ELISA、TPPA、RPR方法进行比较.结果 12503例住院患者化学发光法检出梅毒螺旋体特异性抗体阳性308例,同ELISA方法比较,阳性符合率99.08％,阴性符合率99.98％,总符合率99.93％.二者检测结果高度一致.同TPPA比较,阳性符合率98.7％.结论 化学发光免疫法检测梅毒特异性抗体具有特异性好、灵敏度高、结果易判断、便于自动化等优点,适合于梅毒的筛查.     

化学发光微粒子免疫分析在梅毒螺旋体抗体筛查中的应用探讨

目的 评价化学发光微粒子免疫分析法(CMIA)用于梅毒螺旋体抗体(TP-Ab)筛查的价值,并制定合理的TP-Ab血清学筛查方案.方法 收集在我院进行TP-Ab筛查的患者标本,共计30,100例,对于CMIA为阴性者直接报告结果,CMIA阳性者采用梅毒螺旋体明胶凝集试验(TPPA)确认,并同时进行快速血浆反应素环状卡片试验(RPR),分析结果的一致性.结果 在30100例筛查标本中,CMIA阳性者402例(1.3％),样本吸光度与临界质控吸光度比值(S/CO)在1.05～40.56之间.CMIA结果＞10 S/CO的标本共178例,TPPA阳性比例为100％,RPR阳性63例;在6-10 S/CO之间的标本共计50例,RPR阳性8例,TPPA阳性比例92％;在3～6 S/CO之间的标本59例,RPR阳性者5例,TPPA阳性的比例66.1％;在1～3 S/CO之间的标本115例,RPR均为阴性,TPPA阳性的比例仅为20％.结论 CMIA筛查TP-Ab具有较高的灵敏度,但特异性相对较低,采用CMIA进行TP-Ab筛查,并根据其S/CO值确定是否需要进一步采用另外一种高特异性梅毒螺旋体抗体检测试验进行确认,是TP-Ab筛查理想的方案,确认方法可以选择TPPA等试验.确认阳性者,应报告RPR结果,辅助临床区分既往感染和现症感染.     

肺炎支原体特异性抗体检测在小儿呼吸道感染中的应用

目的对小儿呼吸道感染患者进行肺炎支原体(Mp)检测,以期协助临床早诊断、早治疗.方法对680例小儿呼吸道感染者利用颗粒凝集反应法检测抗-Mp-特异性抗体.结果680例小儿呼吸道感染者,检出Mp特异性抗体阳性132例,阳性率19.4%.＜3岁、～6岁、～14岁、＞14岁4个年龄组Mp特异性抗体阳性率分别为9.4%、23.1%、29.6%、5.7%.结论颗粒凝集反应法检测血清Mp抗体可作为早期诊断Mp感染的指标之一.     

ELISA法筛查临床样本中梅毒螺旋体抗体假阳性结果分析

目的:分析酶联免疫吸附试验(ELISA)从临床样本中检测出梅毒阳性结果中的假阳性及应对措施。方法:把5886例分为老年组和中青年组,用TP-ELISA从检出阳性91例,再用蛋白印迹法(WB)进行确认。结果:TP-ELISA法从老年组的梅毒抗体检出53例(2.64%),WB法确证45例,假阳性率15.09%;TP-ELISA法从中青年组的梅毒抗体检出38例(0.98%),WB法确证37例,假阳性率2.63%。且出假阳性时TP-ELISA法的S/CO值与真阳性时的S/CO值无显著性意义。结论:为提高老年人梅毒检测准确性,实验室使用WB法对有疑问的TP-ELISA法检测阳性样本进行确认是较好的选择。     

The Treponema pallidum haemagglutination (TPHA) test in biological false positive and leprosy sera

The Treponema pallidum haemagglutination (TPHA) test was carried out on 274 sera known to show biological false positive reactions to reagin tests for syphilis. The Treponema pallidum immunization (TPI) and fluorescent treponemal antibody absorption (FTA-ABS) tests were non-reactive on all these sera. Thirty-one or 11.3% showed reactive results in the TPHA test.Sera from 267 people who had lepromatous leprosy were also tested in the TPHA test. Fourteen sera were reactive in the TPHA, TPI, and FTA-ABS tests and were from people who had both syphilis and leprosy. Biological false positive reactions were shown by 26 of the leprosy sera, of which three or 11.5% were also reactive in the TPHA test. A further four sera in the leprosy group were reactive only in the TPHA test.The possible cause of false reactive TPHA test results is discussed.It was concluded that where reagin and TPHA tests are reactive in a person who has no history or clinical signs of syphilis, the serum should be referred for TPI and FTA-ABS testing.     

A new attempt to distinguish serologically the subspecies of Treponema pallidum causing syphilis and yaws.

In an effort to serologically differentiate syphilis from yaws, 69 monoclonal antibody species raised against Treponema pallidum subsp. pallidum were tested by immunoblotting for their reactivity with Treponema pallidum subsp. pertenue. All monoclonal antibodies reacted with antigens with the same molecular weight of both subspecies. Furthermore, no differences in reactivity between sera from yaws patients and from syphilis patients were found by Western blot (immunoblot) analysis of cell lysates of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue. We tried to exploit the only known molecular difference between the subspecies. The subunits of the 190-kilodalton multimeric proteins TpF1 and TyF1 of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue, respectively, have previously been shown to differ in one amino acid residue at position 40. In this study, no difference was found in immunoreactivity of TpF1 or TyF1 with either syphilis sera or yaws sera. Synthetic peptides based on the sequence of TpF1 and of TyF1 were used in an enzyme-linked immunosorbent assay with syphilis sera and yaws sera. Again, no difference in reactivity between the T. pallidum subsp. pallidum- and T. pallidum subsp. pertenue-derived peptides was observed.     

Serospecificity of a cloned protease-resistant Treponema pallidum--specific antigen expressed in Escherichia coli.

We evaluated the serological reactivity of a protease-resistant antigen designated 4D which was encoded by Treponema pallidum DNA and was expressed in Escherichia coli from recombinant plasmid pAW329. This 19,000-molecular-weight antigen was purified in its native, non-protease-treated form from E. coli sonic extracts by molecular sieving and ion-exchange chromatography. Antibody binding to antigen 4D was detected by a radioimmunoassay. Antigen 4D-specific antibody was detected in 95% of the sera in a Centers for Disease Control syphilis serum panel. It was also detected in 55% of 121 primary syphilis patients, whereas syphilis antibody was detected in 83% of the sera by a fluorescent treponemal antibody absorption test and in 88% of the sera by a T. pallidum microhemagglutination test. In tests of 118 normal sera, less than 3% demonstrated antibody to antigen 4D; these results are similar to microhemagglutination and fluorescent treponemal antibody absorption test results. Rabbit antisera against Treponema phagedenis, Treponema refringens, Treponema denticola, and Treponema vincentii did not react with antigen 4D.     

Double-conjugate enzyme-linked immunosorbent assay for immunoglobulins G and M against Treponema pallidum

An enzyme-linked immunosorbent assay (ELISA) for the simultaneous measurement of immunoglobulin G (IgG) and IgM was developed to detect antibodies to Treponema pallidum. Wells of polystyrene microtiter plates were coated with T. pallidum antigen, diluted patient serum was added, and IgG and IgM which bound to the T. pallidum antigen were measured by the simultaneous addition of alkaline phosphatase-labeled anti-human IgG and horseradish peroxidase-labeled anti-human IgM. Bound IgG was detected first, followed by bound IgM. After development of the procedure, 145 categorized sera were evaluated: 60 from individuals without syphilis; 62 from patients with syphilis, including 22 with primary, 20 with secondary, and 20 with latent phases of syphilis; and 23 from patients with rheumatoid arthritis. Of the 60 sera from individuals without syphilis, 100% were nonreactive for IgG antibody and 16% were reactive for IgM. Of the 23 sera from patients with rheumatoid arthritis, 3 were reactive for IgG and 3 were nonreactive for IgM. Of the 62 sera from patients with syphilis, 61 (98%) were reactive for IgG antibody with increased titers as the stage of syphilis increased, whereas IgM reactivity decreased. This enzyme-linked immunosorbent assay appears to be a simple method for the simultaneous measurement of antibodies under equal assay conditions.     

Treponema pallidum surface immunofluorescence assay for serologic diagnosis of syphilis

A surface immunofluorescence assay (SIFA) using live spirochetes was analyzed and compared with Western blot (WB), fluorescent treponemal antibody absorption (FTA-ABS), microhemagglutination (MHA-TP), and Treponema pallidum immobilization (TPI) assays for detecting serum antibodies to T. pallidum in patients with syphilis, in disease controls, and in healthy subjects. SIFA and WB were 99% sensitive (99 of 100 positive specimens) and specific (140 of 140 negative specimens); FTA-ABS showed a sensitivity and a specificity of 90 and 89% (90 of 100 positive and 125 of 140 negative specimens), respectively. MHA-TP showed a sensitivity of 84% (84 of 100 positive specimens) and a specificity of 98.5% (138 of 140 negative specimens). Finally, TPI had a sensitivity of 52% (52 of 100 positive specimens) and a specificity of 100% (140 of 140 negative specimens). The T. pallidum SIFA was therefore highly specific, showing no equivocal reactivities with control sera, and sensitive. The results suggest the possible use of SIFA as a confirmatory test in the serologic diagnosis of syphilis.     

Molecular analysis of immunoglobulins M and G immune response to protein antigens of Treponema pallidum in human syphilis.

Protein antigens of Treponema pallidum precipitated by immunoglobulin M (IgM) and IgG antibodies of sera from patients with untreated primary and secondary syphilis as well as treated secondary syphilis were characterized on a molecular basis. T. pallidum was labeled internally with [35S]methionine and solubilized in 0.1% sodium dodecyl sulfate-1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5% gels followed by autoradiography revealed 32 distinct proteins with molecular weights between 13,500 and 200,000. Twenty-three proteins of T. pallidum with molecular weights between 15,500 and 115,000 were identified as antigens by double antibody radioimmunoprecipitation with IgM and IgG antibodies of sera from syphilitic patients. The molecular analysis of the IgM and IgG immune response to T. pallidum in human syphilis is in accord with earlier immunological observations. Finally, utilizing syphilitic human sera, we characterized 15 protein antigens of T. pallidum that are common to Treponema phagedenis by partial absorption of IgM and IgG antibodies with an ultrasonicate of T. phagedenis.     

Decline in prevalence of HIV-1 infection and syphilis among young women attending antenatal care clinics in Addis Ababa,Ethiopia: results from sentinel surveillance, 1995-2001

From 1995 to 2001, five rounds of sentinel surveillance were carried out for young women attending antenatal care clinics at four health centers in Addis Ababa, the capital city of Ethiopia, to monitor trends in the prevalence of HIV infection and syphilis. Serum samples were tested for antibodies to HIV (enzyme-linked immunosorbent assay and Western blotting) and antibodies to Treponema pallidum (T. pallidum hemagglutination assay and rapid plasma reagin test). Prevalence ratios for an increase in one calendar year were estimated using log-binomial models. Between 1995 and 2001, the prevalence of HIV infection among young women (age range, 15-24 years) attending antenatal care clinics in inner city health centers declined from 24.2% to 15.1% (prevalence ratio for an increase in one calendar year, 0.91; 95% confidence interval, 0.87-0.95). No change was observed for older age groups or in outer city health centers. The decline in the prevalence of active syphilis (T. pallidum hemagglutination assay and rapid plasma reagin testing positive for antibodies to T. pallidum) was more pronounced among and also restricted to the young age groups (age range, 15-24 years) in the inner city (from 7.6% in 1995 to 1.3% in 2001; prevalence ratio, 0.69; 95% confidence interval, 0.59-0.80). The declining trends in the prevalence of HIV infection and syphilis among young women attending antenatal care clinics in the inner city are encouraging, but these findings require confirmation in future years and for other population groups.     

Tpr homologs in Treponema paraluiscuniculi Cuniculi A strain

Treponema paraluiscuniculi, the etiologic agent of rabbit venereal syphilis, is morphologically indistinguishable from Treponema pallidum subsp. pallidum (T. pallidum), the human syphilis treponeme, and induces similar immune responses and histopathologic changes in the infected host. Because of their high degree of relatedness, comparative studies are likely to identify genetic determinants that contribute to pathogenesis or virulence in human syphilis. The tpr (Treponema pallidum repeat) genes are believed to code for potential virulence factors. In this study, we identified 10 tpr homologs in Treponema paraluiscuniculi Cuniculi A strain and determined their sequence architecture. Half of this group of paralogous genes were predicted to be nonfunctional due to the presence of frameshifts and premature stop codons. Furthermore, the immune response against the T. paraluiscuniculi Tpr homologs in long-term-infected rabbits was studied by enzyme-linked immunosorbent assay and lymphocyte proliferation assay, showing that TprK is the only target of the antibody and T-cell responses during experimental infection and emphasizing the importance of this putative virulence factor in venereal treponematosis.