梅毒螺旋体特异性IgM抗体检测的临床评价

目的探讨梅毒螺旋体特异性IgM抗体检测在各个时期梅毒诊断中的意义。方法采用蛋白印迹法(WB)检测IgM、甲苯胺红不加热血清反应素试验(TRUST)、梅毒螺旋体颗粒凝集试验(TPPA)和暗视野检查梅毒螺旋体(TP)4种方法,对21例一期梅毒患者、8例二期梅毒患者、4例潜伏期梅毒患者、3例怀疑先天性梅毒婴儿、7例正规治疗18个月后梅毒患者及5例健康对照者进行检测分析。结果一期梅毒患者组IgM、TRUST、TPPA、TP阳性率分别为100.0%、42.9%、57.1%和66.7%。二期梅毒患者组、潜伏期梅毒、先天性梅毒患者组IgM、TRUST、TPPA阳性率均为100.0%。正规治疗18个月后梅毒患者组IgM、TRUST、TPPA阳性率分别为0、28.6%和100.0%。健康对照组IgM、TRUST、TPPA均为阴性。结论梅毒螺旋体特异性IgM抗体检测灵敏度高、特异性强,适用于未经治疗的各个时期的梅毒诊断。     

Evaluation of the Treponema pallidum particle agglutination technique (TP.PA) in the diagnosis of neurosyphilis

The Treponema pallidum particle agglutination technique (TP.PA) was evaluated, in comparison with the Venereal Disease Research Laboratory (VDRL) test, microhemagglutination assay for Treponema pallidum antibodies (MHA-TP), and fluorescent treponemal antibody-ABS (FTA-Abs) test for the diagnosis of neurosyphilis.We have studied 198 cerebrospinal fluid (CSF) samples from patients with syphilis, including neurosyphilis, treated syphilis, and with other neurological manifestations than neurosyphilis. All tests were nonreactive in these last group of patients. In the neurosyphilis patients, sensitivity of the TP.PA was 100%. The performance of this test in CSF from patients with primary syphilis was as good as that of the other tests. In secondary and latent syphilis, the TP.PA results (27 reactive samples/73) were similar to those of the MHA-TP (25 reactive samples/73). In the individuals treated for syphilis, the TP.PA, FTA-Abs, and MHA-TP tests were found to be reactive in eight, six, and eight samples, respectively. In conclusion, it seems that the TP.PA can be used in CSF to diagnose neurosyphilis, although as for other serological tests, interpretation of results should be done in conjunction with other neurosyphilis parameters.     

Characterization of the Western blotting IgG reactivity patterns in the clinical phases of acquired syphilis

We standardized the Western blotting (WB) method for detecting Treponema pallidum IgG (Tp-IgG) antibodies in sera samples of patients with syphilis and correlated the reactivity profile of bands with the clinical phases of the disease. The WB Tp-IgG has 100% sensitivity and 99.5% specificity. The clinical phases of the disease were associated with the reactive bands from TpN15 to TpN47. Quantitative Venereal Disease Research Laboratories was used to assist the WB Tp-IgG analysis. In primary syphilis, the reaction intensity for the antigenic band TpN47 was usually more intense when compared with other clinical phase. In secondary and sometimes in early latent syphilis, antibodies reacted with high numbers of antigenic proteins of T. pallidum. In late latent syphilis, various bands became negative, but the TpN15 and TpN47 were reactive. In tertiary syphilis, we observed reactivity with the TpN15 band and low reactivity with the TpN47. We concluded that WB Tp-IgG could be used to confirm serologic tests and characterize clinical phases of syphilis.     

Detection of antibodies in acid eluates with the gel microcolumn assay

BACKGROUND: Gel microcolumns can be used to detect unexpected serum antibodies and to determine ABO blood group and Rh phenotype. DATs can also be performed with this system. The purpose of this study was to compare the gel microcolumn to the tube IAT using anti-IgG for the detection of antibodies eluted from RBCs. STUDY DESIGN AND METHODS: Acid eluates were prepared from 30 peripheral blood and 41 umbilical cord blood samples. Twelve of the 71 eluates made were from control samples (known DAT negative). Specificities of eluted antibodies were determined by both tube and gel assays with a three-cell screen plus A1 and B cells, as determined by blood type. RESULTS: Ten of 30 peripheral blood eluates were reactive in both assays. Eighteen were nonreactive in both assays, and two from patients with autoimmune hemolytic anemia were reactive by gel assays and nonreactive by tube assays. Thirty three of the 41 cord blood eluates were reactive in both assays. Eluates from 2 of the 35 DAT-positive samples reacted with A1 and B cells by the tube method but were nonreactive by the gel method. Of the 33 cord blood eluates that were reactive by both assays, antibody specificity differed for two samples. When tested by tube assay, these eluates reacted with both A1 and B cells, whereas the same eluates tested by gel assay showed one reacting with only A1 cells and the other with only B cells. CONCLUSIONS: Results of testing eluates in gel assays were similar to those obtained in tube assays. The gel assays may be better at detecting antibodies eluted from RBCs from patients with autoimmune hemolytic anemia, and tube assays may be better at detecting isohemagglutinins eluted from umbilical cord blood.     

先天性梅毒感染性死胎1例尸检的病理观察

目的 探讨先天性梅毒感染性胎儿的病理特点。方法 ①尸体解剖观察大体形态；②显微镜观察主要脏器组织学改变。结果 本例病理特点主要为：①胎盘苍白肿大；②多脏器纤维化伴不同程度梅毒螺旋体浸润；③梅毒感染性肝硬化、胰腺炎和关节骨软骨炎等。结论 患梅毒孕妇的梅毒螺旋体通过胎盘进入胎儿血液，并播散至肝、脾、肾上腺、心、肺等脏器中大量增殖，导致胎儿感染并胎死宫内。     

Isolation of a Treponema pallidum gene encoding immunodominant outer envelope protein P6, which reacts with sera from patients at different stages of syphilis

A phage directing the synthesis of an abundant 45-kD Treponema pallidum surface protein was isolated from an EMBL-4 bacteriophage lambda library of T. pallidum DNA. The recombinant phage was identified using an mAb that was directed toward an immunodominant, outer envelope T. pallidum protein designated P6. The recombinant P6 protein possessed the same mol mass as the native treponemal antigen detected from total T. pallidum protein preparations, confirming the cloning of the structural gene for this molecule. Furthermore, E. coli was transformed by a 4.5-kb Eco RI lambda insert fragment subcloned into the plasmid vector pUC19. These transformed cells expressed and translocated the 45-kD protein to their outer membranes. Finally, all sera from patients with different stages of syphilis (primary, secondary, and latent) contained antibody reactive to this protein.     

三种方法检测潜伏妊娠梅毒的临床评价

目的评价梅毒血清学试验检测潜伏妊娠梅毒的临床价值。方法对1236例围产保健的孕妇血清,用甲苯胺红不加热血清试验（TRUST）检测梅毒反应素,酶联免疫吸附试验（ELISA）、金免疫层析试验（GICA）和梅毒螺旋体颗粒凝集试验（TPPA）检测梅毒螺体抗体,聚合酶反应（PCR）检测梅毒螺旋体DNA。TPPA试验和PCR试验同时阳性判断为潜伏妊娠梅毒。结果 TRUST试验对潜伏妊娠梅毒的诊断灵敏度、诊断特异度、阳性预测值、阴性预测值、阳性似然比和阴性似然比分别为75.0%、97.5%、23.1%、99.7%、30.0和0.3,ELISA试验对潜伏妊娠梅毒的诊断灵敏度、诊断特异度、阳性预测值、阴性预测值、阳性似然比和阴性似然比分别为91.7%、98.8%、42.3%、99.9%、76.4和0.1,GICA试验对潜伏妊娠梅毒的诊断灵敏度、诊断特异度、阳性预测值、阴性预测值、阳性似然比和阴性似然比分别为83.3%、98.9%、43.5%、99.8%、75.7和0.2。结论临床筛查潜伏妊娠梅毒最好选择ELISA试验,同时联合TRUST、GICA检测梅毒可以提高阳性检测率,为临床预防和阻断梅毒母婴垂直传播提供更可靠的理论依据。     

Comparison of a Treponema pallidum IgM immunoblot with a 19S fluorescent treponemal antibody absorption test for the diagnosis of congenital syphilis

We compared an in-house Treponema pallidum IgM immunoblot (IB) with a 19S fluorescent treponemal antibody absorption (IgM) test during routine use for the diagnosis of congenital syphilis (CS) in a national reference laboratory in a nonendemic setting. The overall agreement between the assays was high (97%), and 19S positive samples had at least 2 reactive bands in the IB. The high agreement is mainly caused by the large number of negative results (95%). If the 19S is taken as the gold standard, the estimate sensitivity of the IB was at least 88% with a specificity of 97.2%. Analysis of the discrepancies revealed that the IB was positive with 1 or 2 specific bands in 2.8% of the cases, whereas 19S was negative, possibly indicating higher sensitivity of the IB. We conclude that the IB is a sensitive method to detect contact with T. pallidum in neonates and can replace the 19S in routine laboratory screening for CS cases.     

梅毒螺旋体酶联免疫吸附试验对梅毒筛检的可行性探讨

目的在血液筛捡中选用一种敏感性和特异性均高的检测方法对梅毒抗体进行检测,保证血液质量。方法对检测梅毒抗体的甲苯胺红不加热血清试验（TRUST）、梅毒螺旋体酶联免疫吸附试验（TP—ELISA）抗原夹心法,被动颗粒凝集试验（TPPA）3种方法进行比较。结果经过统计学处理,TP—ELISA抗原夹心法与金标准法TPPA比较,差异无统计学意义（P〉0．05）,而与TRUST比较,差异有统计学意义（P〈0．05）。结论TP—ELISA双抗原夹心法更适宜作为对血液筛检的方法。     

多种梅毒抗体检测方法的比较

目的利用梅毒酶联免疫吸附试验(TP-ELISA)、梅毒螺旋体抗体快速血浆反应素试验(RPR)、梅毒螺旋体胶凝试验(TPPA)和梅毒螺旋体抗体胶体金试纸(金标法)4种不同的方法进行梅毒抗体检测,以探讨方法学的敏感性和特异性。方法用目前常用的TP-ELISA、RPR、TPPA和金标法对67例阳性标本及80例阴性标本分别进行检测。结果 TP-ELISA、RPR、TPPA和金标法的灵敏度分别为95.5%、80.6%、98.5%、91.0%,特异性分别为91.3%、88.8%、97.5%、100.0%,阳性预期值分别为90.1%、85.7%、97.1%、100.0%,阴性预期值分别为96.1%、84.5%、98.7%、93.0%。结论 TP-ELISA是目前筛查梅毒的理想方法。RPR检测梅毒非特异性抗体用于疗效观察及过筛试验。金标法快速方便,很适合应用于急诊标本。TPPA不适合大规模的血液筛查,适用于对筛查后的阳性标本进行梅毒抗体的确证试验。     

2884例孕早期孕妇梅毒感染情况分析及处理

目的了解孕早期孕妇梅毒感染情况,采取防治策略,避免先天梅毒发生。方法对孕早期例行优生检查孕妇进行梅毒血清学检查,利用梅毒甲苯胺红试验(TRUST)检测梅毒非特异性抗体,阳性者再进行梅毒明胶颗粒试验(TPPA)检测梅毒特异性抗体,结合流行病史、临床症状及体征诊断梅毒。告知孕妇知情同意采取驱梅治疗或终止妊娠。结果 2884例孕早期孕妇实验室检查结果为:TRUST阳性18例,其中滴度1∶32有2例、1∶16有2例、1∶4有4例、1∶2有10例。18例TRUST阳性者TPPA皆阳性。诊断为一期梅毒1例,二期梅毒2例,早期隐性梅毒15例。梅毒感染率0.62%(18/2884)。18例梅毒孕妇皆接受驱梅治疗,其中3例选择终止妊娠,15例选择继续妊娠并遵医嘱定期检查。结论孕早期孕妇存在一定数量梅毒感染,应将梅毒血清学检查纳入优生检查项目,可以及早采取防治措施,避免胎传梅毒发生,保证优生优育。     

Amplification of the DNA polymerase I gene of Treponema pallidum from whole blood of persons with syphilis

Previous reports suggest that Treponema pallidum bacteremia occurs in persons with syphilis exposure (‘incubating syphilis’) and in persons with primary or secondary syphilis. During a recent syphilis outbreak, whole blood samples from 32 persons with suspected syphilis or syphilis exposure were screened using polymerase chain reaction (PCR) to amplify the DNA polymerase I gene (polA) of T. pallidum. Of the 32 samples, polA was amplified from 13 (41%). Of these 13, three were determined to have incubating syphilis; two had primary or secondary syphilis and eight had latent syphilis. This study demonstrates that spirochetemia can occur throughout the course of T. pallidum infection.     

Lack of value of specific IgA detection in the postnatal diagnosis of congenital toxoplasmosis

To improve the performance of the postnatal diagnosis of congenital toxoplasmosis, we assessed the detection of IgA antibodies to Toxoplasma gondii by ELISA, compared with that of IgM by ELISA, ISAGA, and IFAT and neosynthesized antibodies using Western blot. From 1993 to 1996, IgA antibodies were detected using the Toxo IgA test (SFRI, Société Fran?aise de Recherches et d'Investissements, Bordeaux, France), in 195 serum and cord blood samples from 63 infants born to mothers who seroconverted during pregnancy. Eighteen infants had proven congenital toxoplasmosis (confirmed by the presence of IgG after 12 months of life) and 45 had no congenital toxoplasmosis (negativity of IgG after 6-12 months of life). The sensitivity of IgA detection by ELISA on serum and cord blood samples was 38.9 and 54.5% respectively, which is low when compared with the sensitivity of IgM detection by ISAGA (66.7% on serum samples, 90.9% on cord blood), ELISA (61.1% on sera, 81.8% on cord blood) and Western blot (83.3% on sera, 72.7% on cord blood). IgA antibodies were never detected by ELISA earlier than IgM or neosynthesized Ig (antibodies synthesized by infants). Thus, the detection of IgA antibodies by Toxo IgA is not useful in improving the diagnosis of congenital toxoplasmosis.     

Amplification of the DNA polymerase I gene of Treponema pallidum from whole blood of persons with syphilis

Previous reports suggest that Treponema pallidum bacteremia occurs in persons with syphilis exposure (‘incubating syphilis’) and in persons with primary or secondary syphilis. During a recent syphilis outbreak, whole blood samples from 32 persons with suspected syphilis or syphilis exposure were screened using polymerase chain reaction (PCR) to amplify the DNA polymerase I gene (polA) of T. pallidum. Of the 32 samples, polA was amplified from 13 (41%). Of these 13, three were determined to have incubating syphilis; two had primary or secondary syphilis and eight had latent syphilis. This study demonstrates that spirochetemia can occur throughout the course of T. pallidum infection.     

Molecular characterization of receptor binding proteins and immunogens of virulent Treponema pallidum

Receptor binding proteins of Treponema pallidum were identified by incubation of [35S]methionine-labeled, soluble T. pallidum preparations with formaldehyde-fixed HEp-2 cells. Three major treponemal proteins (bands 1--3) that avidly bound to the eucaryotic cell surface were detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. Brief trypsin treatment of HEp-2 cells before formaldehyde fixation reduced the extent of the interaction of these treponemal macromolecules, which implicated receptor-mediated attachment mechanisms. The presence of unlabeled T. pallidum preparations directly competed with radiolabeled T. pallidum samples for the available HEp-2 cells, which suggested a limiting number of membrane binding sites. Samples of unlabeled avirulent Reiter treponeme did not compete. T. Pallidum immunogens were examined by radioimmunoprecipitation with human and rabbit syphilitic sera. Of interest were the similarities and extent of the humoral response represented by the detection of antigen-antibody complexes against numberous treponemal proteins, including bands 1--3. T. pallidum portein band 1 appeared to be the major antigenic stimulus. Formation of antigen-antibody complexes between 35S-labeled T. pallidum proteins and human syphilitic sera was prevented by unlabeled T. pallidum but not by T. phagedenis preparations, which demonstrated specificity of the reaction. Gel profiles of radioimmunoprecipitation assays using radiolabeled T. pallidum antigens and human syphilitic and yaws sera delineated both the similarities and differences in the humoral response to these two spirochetes. The latter suggested both overlapping and distinguishing antigenic properties between T. pallidum and T. pertenue. Detection in yaws sera of specific antibody against T. pallidum protein bands 1--3 further incriminates the role of these three treponemal proteins as virulence determinants.     

不同方法检测抗梅毒抗体阳性标本的分析

目的 对TPHA检测血清抗梅毒螺旋体抗体阳性的标本,同时用另外3种方法进行检测,以探讨阳性结果是否存在方法学导致的假阳性。方法 3957例无梅毒症状的普通病人为实验组,344例性病门诊病人为对照组。用TPHA进行抗梅毒抗体筛查,检测阳性的标本再用酶免疫法、甲苯胺红不加热血清试验（TRUST）、免疫印迹法进行检测。以免疫印迹法为标准对检测结果进行对比分析。结果 实验组中检出TPHA阳性60例,经免疫印迹法检测确认阳性57例,临界2例,1例为假阳性,酶免疫法阳性53例,TRUST阳性23例。对照组中TPHA阳性40例,免疫印迹法确认阳性40例,酶免疫法阳性40例,TRUST阳性32例。结论 TPHA、EIA测定抗梅毒抗体有较高的阳性符合率,2种方法检测抗体为阳性的患者,几乎全部存在既往感染或隐性感染。对TRUST的结果则应综合分析。     

二期梅毒患者外周血白细胞介素-2水平

  目的  观察二期梅毒患者外周血中白细胞介素-2(interleukin-2, IL-2)的水平。  方法  收集2010年9月至12月在北京协和医院皮肤科性病中心就诊的二期梅毒患者26例, 其中男20例, 女6例, 年龄16~76岁, 平均(40±13)岁, 所有病例均经快速血浆反应素环状卡片试验(rapid plasma reagin, RPR)和梅毒螺旋体明胶凝结试验确诊; 应用酶联免疫吸附试验检测26例二期梅毒患者和14名年龄匹配的健康对照者血清IL-2的水平。  结果  二期梅毒患者血清IL-2水平较正常对照者明显升高[分别为(2.510±0.529) pg/ml和(2.225±0.157) pg/ml, P=0.016];而二期梅毒患者年龄、性别、是否接受治疗、IgM是否阳性及RPR滴度对IL-2水平均无明显影响。  结论  梅毒螺旋体入侵可能引起早期梅毒患者外周血中IL-2水平升高, 增强宿主对梅毒螺旋体的免疫反应, 有利于梅毒螺旋体的清除。     

An analysis of the value of some antigen-antibody interactions used as diagnostic indicators in a treponemal Western blot (TWB) test for syphilis

Densiometric quantitation and spreadsheet normalization were used to refine the parameters defining a treponemal Western blot (TWB) test for syphilis. Initially using 84 defined reactive and 105 defined non-reactive sera, we determined that the immune response to the 17 kDa antigen was the most critical of the following three candidate test determinants: the 47 kDa, 17 kDa and 15.5 kDa bands. In a second study using 124 cases of clinically diagnosed syphilis and 354 "normal" donors, a diluted serum sample was included as a minimal reactive control for the 17-kDa immune response. Reactivity to all three test determinants was obligatory for a test result to be interpreted as positive. Of the 124 cases of syphilis, 7 were nonreactive by TWB (sensitivity = 94%); of the 354 normal donors, 7 tested reactive (specificity = 98%). Forty (11%) normal serum samples had detectable but less than minimal reactivity to the 17 kDa band. Frequencies of immune response to a larger group of 12 antigens were tallied for the 124 clinically diagnosed cases of syphilis and an equal subset (124) of the normal group. In the normal subset, 72% and 52% of the samples had detectable reactivity to the 47 and 15.5 kDa antigens, respectively, while 10%, 5% and 3% reacted with the 17, 24 and 44.5 kDa antigens, respectively. Follow-up TWB testing of the clinically diagnosed cases revealed that previously untreated patients with primary or secondary syphilis were more likely to a show decrease in TWB reactivity than patients with latent symptoms who had been treated previously. As a diagnostic indicator of syphilis, the 17-kDa antigen was found to have the best combined attributes of sensitivity and specificity. Although, the highly specific 44.5 kDal and 24 kDal bands were often redundant as diagnostic indicators they are useful for the interpretation of borderline results. In addition, absence of the highly sensitive 47 and 15.5 kDa indicators should be useful in resolving some problem diagnoses.     

Cerebrospinal fluid IgG and IgM indexes as indicators of active neurosyphilis

Serological and non-serological tests were performed in matched samples of cerebrospinal fluid and serum from 236 syphilitic patients. An increased IgG or IgM index, or both, was found about 70 times more often in symptomatic neurosyphilis than in latent syphilis without involvement of the central nervous system. An increased Ig index, together with a cell count greater than 5/microL, was only found in symptomatic neurosyphilis. Although the numbers of data are small, we conclude that the IgG and IgM indexes are valuable tests in the diagnosis of syphilitic involvement of the central nervous system.     

A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d

This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti-IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti-C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti-IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post-transplant, the flow cytometry assays for anti-IgG and/or anti-C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC-associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.