Diagnosing Treponema pallidum in secondary syphilis by PCR and immunohistochemistry

Epidemiological aspects of syphilis in Western countries have undergone a significant change with respect to the number of cases. Detection of Treponema pallidum is difficult, and the correct diagnosis of secondary syphilis can be critical. In this study, biopsy samples from skin lesions of 12 patients with secondary syphilis were used. Diagnosis of syphilis was based on clinical presentation, dark-field microscope analysis, and serological tests. Using a polyclonal antibody directed against T. pallidum, we show the presence of T. pallidum in 90% of the samples studied with the bacteria located in the epidermis and the upper dermis. The T. pallidum 47-kDa surface protein gene could be amplified by PCR in 75% of the skin lesions. When combining both techniques, T. pallidum was detected in 92% of the samples from patients with secondary syphilis but not in the control samples. Our work suggests that both immunohistochemistry and PCR could be useful for the diagnosis of secondary syphilis and may be helpful in some rare cases when serological assays failed to detect T. pallidum antibodies.     

Molecular detection of Treponema pallidum in secondary and tertiary syphilis

Treponema pallidum can be detected by conventional techniques such as dark-field microscopy, immunofluorescence or the rabbit infectivity test, in large numbers in the skin lesions of primary and early secondary syphilis. In the skin lesions of late secondary and tertiary syphilis, conventional techniques fail to detect spirochaetes in general, perhaps due to increasing degeneration and the disappearance of treponemal spirochaetes in late syphilitic skin lesions. We used the highly sensitive technique of polymerase chain reaction (PCR) to prove the presence of Treponema pallidum -specific DNA in six lesions of late secondary syphilis and seven lesions of tertiary syphilis, including one syphilitic gumma. A Whartin-Starry stain was carried out in all 13 specimens and did not reveal any treponemal structures. Treponema pallidum-specific DNA was amplified by PCR in four of six cases of secondary syphilis and in the syphilitic gumma. These results are in favour of a direct cell-mediated immune reaction directed against treponemal antigen rather than the concept of an Id-reaction. Beside the usefulness of a PCR-based assay for understanding the aetiology of lesions of late syphilis, the assay described can be of clinical importance in various situations where traditional methods fail to detect Treponema pallidum because of lack of sensitivity.     

梅毒免疫和梅毒螺旋体的研究进展

梅毒螺旋体有着独特的基因和蛋白结构,对其基因分型有利于梅毒分子流行病学的研究.梅毒螺旋体缺乏脂多糖与外膜蛋白,但可表达多种脂蛋白,这些脂蛋白同梅毒螺旋体的组织黏附及播散有关,同时可诱导机体发生免疫应答.机体对于梅毒螺旋体的免疫应答过程十分复杂,固有免疫、体液免疫及细胞免疫均在梅毒螺旋体的感染过程中参与了应答过程.Th1型细胞免疫在梅毒的发生发展过程中起重要作用,梅毒患者在局部及系统免疫方面都存在细胞免疫的异常.     

梅毒螺旋体抗体在诊断新生儿梅毒中的应用和讨论

目的:探讨梅毒螺旋体(treponema pallidum,TP)抗体在诊断新生儿梅毒中的应用。方法:分别用甲苯胺红不加热血清试验(TRUST)、TP-ELISA和密螺旋体颗粒凝集实验(TPPA)法对84例确诊新生儿梅毒患者与100例健康新生儿血清进行检测。结果:三种检测梅毒螺旋体抗体方法的灵敏度分别为51.2%、98.8%和100%,相对而言,TRUST法不适合作为诊断新生儿梅毒的初筛试验。结论:TP-ELISA法灵敏度与特异性相对较高,操作简便易行,建议作为诊断新生儿梅毒的初筛试验;TPPA法由于操作繁琐复杂,可结合临床作为新生儿梅毒的确诊试验。     

Detection of Treponema pallidum in skin lesions of secondary syphilis and characterization of the inflammatory infiltrate

BACKGROUND: Syphilis is an ancient sexually transmitted disease. However, the pathogenesis of mucocutaneous lesions of secondary syphilis is not completely understood. METHODS: We analyzed the presence of Treponema pallidum in formalin-fixed, paraffin-embedded biopsy specimens from mucocutaneous lesions of secondary syphilis using highly sensitive nested polymerase chain reaction (PCR). The inflammatory infiltrates from the same specimens are also characterized using immunohistochemical methods. RESULTS AND CONCLUSIONS: Ten out of 24 (41.7%) specimens are T. pallidum positive using nested PCR, whereas none of them is T. pallidum positive using traditional silver staining. The presence of T. pallidum in the mucocutaneous lesions indicates that mucocutaneous lesions of secondary syphilis might be caused by direct T. pallidum invasion rather than by an allergic reaction. Furthermore, the majority of inflammatory infiltrating cells are CD45RO-positive T cells and CD68-positive macrophages, suggesting that cellular immunity plays an important role in the host reaction against T. pallidum infection in secondary syphilis.     

用蛋白印迹技术初步评价重组梅毒螺旋体抗原的反应性

目的：初步分析重组梅毒螺旋体抗原对梅毒螺旋体抗体的反应性。方法：用蛋白印迹技术检测与麦芽糖结合蛋白（MBP）融合的2种重组梅毒螺旋体蛋白（rTP47和rTP17)与二期梅毒患者血清的反应性。结果：30份二期梅毒患者血清均与MBP－rTP47和MBP－rTP17反应,反应强度与RPR试验测定的血清滴度无绝对一致性,但MBP－rTP17与二期梅毒患者血清的反应强于MBP－rTP47,30份正常人血清均不与MBP－rTP47和MBP－rTP17反应。结论：MBP－rTP47和MBP－rTP17对二期梅毒患者血清的反应性较强。     

4种以重组梅毒螺旋体抗原为基础的梅毒快速诊断试验的临床评价

目的：评估4种以重组梅毒螺旋体抗原为基础的梅毒快速诊断试剂盒的敏感性和特异性,及其在临床应用中的可操作性。方法：选择860例性传播疾病（STD）门诊患者,签署知情同意书并采集其静脉血。分别取全血和血清进行梅毒螺旋体血凝试验（TPHA）及4种试剂盒的检测。检测方法分别按照说明书进行。结果：以TPHA为标准,结果分别为：①Abbott Determine Syphilis TP test：血清敏感性为100％,特异性为98、9％;全血敏感性为81．9％,特异性为99．4％。②SDBIOLINE Syphilis 3．0 test：血清敏感性为95．5％,特异性为97．9％;全血敏感性为87．6％,特异性为99．4％。③VISITECT-SYPHILIS test：血清敏感性为94．0％,特异性为98．1％;全血敏感性为73．5％,特异性为99．7％。④Syphicheck-WB test：血清敏感性为67．4％,特异性为98．8％：全血敏感性为64．0％,特异性为99．7％。结论：4种试剂盒均具有优良的临床操作性能,适用于STD门诊的梅毒快速检测。而且这4种快速诊断试剂盒在血清中的检测比全血具有更高的敏感性。     

Treponema pallidum specific IgM haemagglutination test for serodiagnosis of syphilis

The Treponema pallidum specific IgM haemagglutination (TP-IgM-HA) test uses erythrocytes sensitised with antiserum to human IgM to separate IgM from IgG in serum. Specific antitreponemal IgM captured in this way is detected by adding a second reagent comprising erythrocytes sensitised with T pallidum antigen. Eighty two serum samples from 82 patients with untreated syphilis, 521 samples from 73 patients with treated syphilis, and 1872 samples from people who did not have syphilis were examined by the 19S(IgM)-TPHA (T pallidum haemagglutination), IgM-FTA-ABS (fluorescent treponemal antibody absorbed), TP-IgM-ELISA (enzyme linked immunosorbent assay), and TP-IgM-HA tests for the presence of 19S(IgM) antibodies specific to treponemes. The sensitivity of the TP-IgM-HA test was 97.6% and the specificity was 99.7%. We also traced IgM specific to treponemes in untreated patients with primary syphilis by four different tests. The TP-IgM-HA test results clearly reflected the effect of the treatment.     

Multiple primary syphilis on the lip,nipple–areola and penis: An immunohistochemical examination of Treponema pallidum localization using an anti‐T. pallidum antibody

Primary syphilis caused by Treponema pallidum usually develops after sexual contact as an initial solitary sclerosis or hard chancre in the genital region. We describe a case of primary syphilis at three sites in genital and extragenital regions of a man who had sex with men. A 29‐year‐old man visited our hospital for skin lesions on his lower lip, nipple–areola and penis. A positive syphilis serological test for rapid plasma reagin had a titer of 1:16; the patient also tested positive for specific antibodies against T. pallidum, with a cut‐off index of 39.0. Histopathological examination of a nipple–areola biopsy specimen revealed a thickened epidermis and dense infiltration of inflammatory cells extending from the upper dermal layers to the deep dermis. The inflammatory cells were composed of abundant lymphocytes, plasma cells, histiocytes and neutrophils. Immunohistochemical staining for T. pallidum using an anti‐T. pallidum antibody showed numerous spirochetes in the lower portion of the epidermis, scattered inside inflammatory cell infiltrate and perivascular sites throughout the dermis. Based on these findings, the patient was diagnosed with primary syphilis. Treatment with oral amoxicillin hydrate was started. Five days after starting treatment, a diffuse maculopapular rash (syphilitic roseola) occurred on his trunk and extremities. Perivascular cuffing due to T. pallidum was present throughout the dermis in the biopsy specimen of a localized lesion of primary syphilis. Moreover, syphilitic roseola, which indicates generalized dissemination of T. pallidum, developed during the course of treatment for primary syphilis. Therefore, we considered perivascular cuffing to be indicative of the dissemination phase.