【作者】 陈洁；

【导师】 程浩；

【作者基本信息】 浙江大学 ， 皮肤病与性病学， 2012， 硕士

【摘要】 目的：近年来,梅毒的发病率越来越高,由于病损的多变性、临床症状的隐匿性、临床病史的不完整等因素,诊断日益困难。有时,结合血清学试验、组织病理学检查亦不能完全明确诊断。本文采用梅毒螺旋体特异性抗体的免疫组织化学法(immunohistochemistry of Treponema pallidum, TP-IHC)检测组织活检标本中的梅毒螺旋体(Treponema pallidum, TP),观察其在各期梅毒、不同皮损中的表达数量及分布情况,并与血清学试验等经典方法进行比较,旨在探讨使用免疫组化的方法检测梅毒螺旋体以协助梅毒诊断的可行性及优势,并试图阐述其应用前景。方法：共收集到浙江大学附属邵逸夫医院2004年1月-2012年5月期间,14例梅毒血清阳性患者或窗口期疑似病人的病损组织活检标本共20份,包括一期梅毒4例,标本6份；二期梅毒8例,标本10份；三期梅毒2例,标本4份,其中三份来自同一位患者,其标本组织学表现不支持梅毒。回顾患者基本信息,包括如年龄、性别、临床病损、梅毒血清学试验结果及活检组织的HE染色病理表现等。采用梅毒螺旋体为特异抗体的免疫组化法检测病损组织中的螺旋体,并记录染色阳性的螺旋体数量和分布特点。分析总结螺旋体在病损组织的表达数量与疾病分期、临床病损之间的关系,以及治疗前后螺旋体数量的变化。采用SPSS17.0, Spearman相关分析的统计学方法,检验患者RPR滴度与其组织标本内梅毒螺旋体数量之间的相关性。结果：采用梅毒螺旋体为特异抗体的免疫组化法检测病损组织中的螺旋体表达,总阳性率为80%。6份来自4例1期梅毒病人的标本中,5份TP-IHC染色呈阳性表达(阳性率83.3%),1份TP-IHC染色阴性的标本为梅毒直肠炎患者正规治疗后直肠活检标本。10份来自8例2期梅毒病人的标本中,10份TP-IHC染色均呈阳性表达(阳性率达100%)。4份来自2例3期梅毒病人的标本,1份TP-IHC染色呈阳性表达(阳性率25%),3份TP-IHC染色阴性的标本来自1例梅毒血清阳性合并HIV的患者,其病损临床及病理诊断为嗜酸细胞增多性皮病,而非梅毒相关性病损。各期梅毒病人标本阳性诊断符合率、阴性诊断符合率均为100%。染色阳性的梅毒螺旋体在组织中的分布情况：1期梅毒皮肤组织标本中梅毒螺旋体主要位于表皮下部近基底细胞层、真皮血管周围；直肠粘膜标本中主要位于近浆膜层一侧,组织内及血管周围。2期梅毒标本,皮肤组织标本中梅毒螺旋体主要位于表皮全层、附属器周围、真皮血管周围；淋巴结活检标本中梅毒螺旋体主要位于滤泡周围的炎性肉芽组织中。一份3期梅毒皮肤组织标本的血管内见螺旋体分布。染色阳性的标本内螺旋体数量与临床病损有一定相关性：硬下疳、扁平湿疣皮损活检标本及梅毒性直肠炎患者肠镜活检标本中螺旋体数量最多；鳞屑性红斑、蜊壳样斑块、三期梅毒胸壁溃疡皮肤活检标本中梅毒螺旋体表达量中等；梅毒性淋巴结炎标本中螺旋体数量最少。1例梅毒性直肠炎患者,治疗前后肠镜活检标本的免疫组化检测结果提示,治疗前螺旋体阳性,治疗后检测阴性。1例二期梅毒蜊壳样斑块患者,卞星青霉素足疗程治疗前后标本中均发现螺旋体存在,且表达量均为中等。分析梅毒患者血清RPR滴度和TP-IHC染色结果的关系发现螺旋体数量与RPR滴度间无相关性(P>0.05)。讨论与结论：采用梅毒螺旋体为特异抗体的免疫组化法检测组织中梅毒螺旋体表达有助于梅毒的诊断。本研究表明,该方法的阳性预测值、阴性预测值均达100%,由此认为,使用梅毒螺旋体为特异抗体的免疫组化法诊断梅毒病损特异性非常高,可用于协助梅毒诊断。各期梅毒患者包括窗口期疑似患者的螺旋体免疫组化法染色阳性,且早期梅毒患者皮损中螺旋体数量较多,提示该方法敏感性很高,体现了该方法在诊断早期梅毒中的优势,尤其对诊断早期血清尚未阳性的梅毒有很大帮助。一例一期梅毒直肠炎患者直肠活检标本免疫组化法检测梅毒螺旋体,治疗前阳性,治疗后阴性,提示该方法可以用于驱梅治疗的疗效评估。一例二期梅毒蜊壳样斑块患者,卞星青霉素足疗程治疗前后标本中螺旋体含量相当,表明患者对青霉素治疗反应差。因此认为,该方法可用于梅毒治疗效果的监测及对复发梅毒的判断。此外,梅毒螺旋体在皮损中的分布情况为,主要位于表皮、真皮血管周围,即其分布符合亲表皮、亲血管内皮和炎性肉芽组织三种方式。结论：1,用梅毒螺旋体为特异抗体的免疫组化法检测病损组织中梅毒螺旋体的方法敏感性很高,特异性很强,可明显提高临床梅毒诊断阳性率；2,染色阳性的梅毒螺旋体在组织中的分布主要以亲表皮、亲血管内皮细胞和炎性肉芽组织中为特点,且表达数量与RPR滴度无关；3,该方法有利于临床难确诊病例如血清尚阴性的早期疑似梅毒、RPR滴度不高的内脏梅毒、或少见部位梅毒、以及不典型病损的诊断；4,该方法有利于治疗后皮损复发的梅毒的诊断和评估；5,梅毒螺旋体为特异抗体的免疫组化法检测病损组织中梅毒螺旋体具有较好临床应用前景。更多还原

【Abstract】 Objective:The incidence of syphilis is increasing over the world. The diagnosis of syphilis remains challenging due to its diverse clinical manifestations or incomplete medical history. Similarly, the serological tests and the histopathology changes do not always fulfill the typical diagnostic criteria. Thus, we used immunohistochemistry for direct detect of Treponema pallidum in all the samples, observed the quantity and location of Treponema pallidum in different lesions and clinical phases. The object of this study was to evaluate the contribution of immunohistochemistry for the identification and localization of Treponema pallidum.Methods:A series of20paraffin-embedded lesion biopsy samples of14patients from SRRSH during2004.4to2012.5, with primary, secondary or tertiary syphilis were collected for this study. We reviewed the clinical information of these patients, including clinical manifestations, syphilis serological tests and histopathological results. All of these samples were assessed for detection of Treponema pallidum (TP) by immunohistochemistry. The number and location of TPs for each sample was recorded. The connections between the quantity/location of TPs and the clinical stages or clinical lesions were analysis. We also oberserved changes about number of TPs before and after treatment. Spearman correlation analysis (SPSS17.0) was used to test if there was any correlation between the results of RPR and TP-IHC.Results:Using immunohistochemistry for detection of Treponema pallidums in tissue, we detected the presence of TPs in80%of all the20samples.83.3%of6samples from four patients with primary syphilis,100%of10samples from eight patients with secondary syphilis, and25%of4samples from two patients with tertiary syphilis were TPs positive staining. Of these, the positive predictive value and negative predictive value for all stage was100%.In lesions from primary syphilis, TPs present in the lower part of the epidermis or around blood vessels of dermis in skin lesions, and present around blood vessels or tissues near serosa in rectal lesions. In lesions from secondary syphilis, we saw TPs presenting in all layers of epidermis, around cutaneous appendages and around blood vessels in dermis with inflammations. In an enlarged lymph node from a secondary syphilis patient, we saw that TPs present in inflammatory granulation tissues around lymphoid follicles except in the lymphoid follicles. In a sample of ulcer of chest wall from tertiary syphilis, TPs present in the blood vessels. Besides, we found the quantity of TPs was associated with clinical lesions. There were more TPs in the samples of syphilis chancre, syphilis intestinal inflammation and condyloma latum. Fewer TPs in the samples of squamous erythema, greyish-black plaque, ulcer of chest wall from tertiary syphilis. Also, we observed that the immunohistochemistry staining was TPs negative in a sample after regular therapy, while it was TPs positive before therapy. There were no correlations between the quantity of TPs and the RPR titor.Discussion and Conclusions:Discussion:In this study, immunohistochemistry of Treponema pallidum was used for the diagnosis of syphilis. The positive predictive value and negative predictive value all reached100%, showing the high specificity of this method for diagnosis of syphilis. TPs present in all lesions from patients in all stages of syphilis including one suspected patient in’window phase’. And more TPs present in the lesions of patients in early stages.The results revealed, it’s of high sensitivity for the diagnosis of syphilis and valuable for the diagnosis of syphilis in early stages, especially for patient with negative serological tests. In specimens from syphilis proctitis, we observed that the TPs staining was negative after anti-TP therapy, while TPs staining was positive before therapy.Whats more, we observed that the quantity of TPs was almost equal in the lesions from one patient with greyish-black plaque before and after Benzathine Benzylpenicillin therapy, who is resistant for that. Thus, TP-IHC can be used for the surveillance and evaluation of therapy. TPs present in lower part of epidermis or perivasculary, as’endotheliotropic pattern’and/or’epitheliotropic pattern’.Conclusions:1, Immunohistochemistry of Treponema pallidum is of both high sensitivity and specificity for the diagnosis of syphilis, improving the positive rate of clinical diagnosis for syphilis.2, TPs present in lower part of epidermis or perivasculary, as’endotheliotropic pattern’ and’epitheliotropic pattern’or in the inflammatory granulation tissue. There are no correlations between the quantity of TPs and the result of RPR.3, TP-IHC is helpful for the diagnosis of syphilis, especially for the diagnosis of early suspected syphilis with negative serological test, systemic damage of syphilis with low RPR, syphilis in untypical locations and unusual lesions.4, TP-IHC can be use for the diagnosis of syphilis relapse and the evaluation of anti-TP therapy.5, Applying TP-IHC to detect TPs in clinical lesions is a hopeful test for the clinical practice.更多还原