Comparative analysis of immunohistochemistry, polymerase chain reaction and focus-floating microscopy for the detection of Treponema pallidum in mucocutaneous lesions of primary, secondary and tertiary syphilis

Background The incidence of syphilis is increasing in many parts of the world including a re‐emergence in Western Europe and North America. Depending on the disease stage, direct detection of Treponema pallidum in mucocutaneous lesions of syphilis may be difficult and histopathological findings are not always straightforward. Thus, the correct histological diagnosis may be challenging. Objectives Comparatively to evaluate the evidence for infection with T. pallidum by immunohistochemistry (IHC), polymerase chain reaction (PCR) and focus‐floating microscopy (FFM). Methods A series of 86 paraffin‐embedded skin biopsy samples from patients with primary, secondary or tertiary syphilis was assessed for detection of T. pallidum by IHC and FFM; 45 specimens were also investigated by a T. pallidum‐specific PCR analysis. Histopathological reaction patterns and number and distribution of treponemes were studied, and all data were re‐evaluated by clinicopathological correlation. Results Using a polyclonal antibody directed against T. pallidum, we detected the presence of T. pallidum by IHC in 42/86 (49%) samples [6/9 (67%) primary, 34/62 (55%) secondary and 2/15 (13%) tertiary syphilis]. T. pallidum‐specific DNA was detected in 31/45 (69%) specimens [4/4 (100%) primary, 26/34 (76%) secondary and 1/7 (14%) tertiary syphilis]. In comparison, FFM analysis resulted in an overall detection rate of 82/86 (95%) [9/9 (100%) primary, 60/62 (97%) secondary and 13/15 (87%) tertiary syphilis]. Significant differences were observed concerning amount and distribution of organisms (epitheliotropic vs. endotheliotropic) in correlation to the three disease stages and to histopathological reaction patterns. Conclusions FFM is a highly sensitive and specific method to detect T. pallidum in tissue from mucocutaneous syphilis lesions. Our results indicate that a combination of PCR and FFM, as the most sensitive approach, could provide an additional benefit for the histopathological diagnosis of (late) secondary and tertiary syphilis and may be helpful in cases where serological testing of T. pallidum antibodies has failed, but the clinical suspicion for syphilis remains.     

Utilidad de las nuevas pruebas treponémicas automatizadas en el diagnóstico de la sífilis precoz. A propósito de 15 casos

BackgroundSyphilis is a sexually transmitted infection caused by Treponema pallidum, subspecies pallidum. As these bacteria are difficult to culture, syphilis must be diagnosed by serologic testing. The introduction of automated treponemal tests has led to changes in the traditional diagnostic algorithm for syphilis, which began with a nontreponemal test. We present 15 cases of primary syphilis detected using these new tools and review the microbiologic techniques used for the diagnosis of early syphilis.Material and methodsWe examined all cases of syphilis diagnosed in our department between January 2013 and September 2018 and selected patients with negative nontreponemal (rapid plasma reagin [RPR]) tests.ResultsOf the 158 patients diagnosed with syphilis during the study period, 15 had a negative RPR test, and 14 of them had a positive treponemal test. Fourteen of the patients were men and ages ranged from 22 to 60 years. Polymerase chain reaction was used to detect T pallidum in the lesion exudate from 8 patients and was positive in all cases. The 15 patients were treated with a single injection of 2.4 million units of benzathine penicillin G.ConclusionChemiluminescence immunoassays and T pallidum automated enzyme-linked immunoassays are useful in the diagnosis of early syphilis, and we believe that they should be adopted as screening tools given their diagnostic sensitivity, speed, and low cost.     

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.     

A Case of Secondary Syphilis with Mucous Patches on the Hard Palate

A rare case of secondary syphilis showing mucous patches on the hard palate is reported. A 31-year-old male had two erosive patches which were slightly raised on his hard palate. A linear lesion was also present on the inside of his right alveolar process. Many red-brown macules on his abdomen and bilateral inguinal lymphadenopathy accompanied these symptoms. The serological tests for syphilis were positive: VDRL test 1:512, TPHA test 1:20,480, and IgM-FTA-ABS test 1:40. Immunohistochemical staining with rabbit monoclonal antibody to Treponema pallidum by the biotin-streptoavidin system detected treponemal organisms in the paraffin-embedded specimen from his mucous patch. A diagnosis of secondary syphilis was made, and he was given amoxicillin (AMPC) at 750 mg per day for 4 weeks. His eruptions, including the mucous patch, healed in a week.     

Auffällige Zunahme des Lymphogranuloma venereum unter homosexuellen Männern in Hamburg

Classical sexually transmitted diseases, including syphilis and gonorrhea, have recently increased significantly among homosexual men in Hamburg. During the last year we also observed an increase in patients with lymphogranuloma venereum (LGV) at the ifi‐institute in Hamburg. In 2003, we identified 4 homosexual patients with LGV in different clinical stages. None of the patients has traveled outside Germany. Three of these patients were HIV‐infected. In all cases Chlamydia trachomatis was identified by SDA (strand displacement amplification) in genital swabs or lymph node aspirates. In three cases sequencing of ompA PCR products was performed and in each instance revealed the C. trachomatis serovar L2. Other important genital pathogens were excluded by specific PCR tests, bacteriological and serological tests. LGV should be included in the differential diagnosis of anogenital and oral erosions, especially in homosexual patients and HIV‐infected patients. Novel nucleic acid amplification tests can be used for the rapid and reliable diagnosis of LGV by identifying Chlamydia trachomatis.     

聚合酶链反应检测梅毒螺旋体的进展

梅毒是由苍白螺旋体感染引起的一种常见系统性性传播疾病,早诊早治梅毒有利于减少传播、减轻系统损害.直接暗视野检查苍白螺旋体或血清抗体检测是诊断梅毒的主要手段,但有局限性.聚合酶链反应通过扩增苍白螺旋体靶序列诊断梅毒,而polA、tpp47基因为聚合酶链反应法检测苍白螺旋体常用靶基因.反转录聚合酶链反应和巢式聚合酶链反应的敏感性高于其他聚合酶链反应法.聚合酶链反应法检测一期梅毒拭子中苍白螺旋体敏感性可达60％以上,而检测血液及脑脊液苍白螺旋体敏感性不超过60％(胎传梅毒可达83％),特异性93％以上.患者HIV感染与聚合酶链反应检测敏感性无关.     

Seronegative secondary syphilis in an HIV-infected patient

Recently, increased rates of syphilis among gay men have been observed in American and European cities. It is important to establish the diagnosis because syphilis facilitates HIV transmission during the primary and secondary stages when sores are open on the skin. However the diagnosis can present a dilemma as negative reactions to serological tests may be observed in AIDS patients. We report here such a case in which the diagnosis was established on dark field examination.     

Use of PCR in the diagnosis of early syphilis in the United Kingdom

OBJECTIVES: To evaluate a Treponema pallidum polymerase chain reaction (PCR) test in the laboratory diagnosis of early syphilis in the United Kingdom. Subjects and setting: Men and women attending genitourinary medicine clinics in England. METHODS: A trial PCR service was offered for the analysis of swabs of ano-genital or oral ulcers suspected to be syphilitic in origin. Clinical details, results of treponemal serology, and other relevant laboratory tests carried out by the sending laboratories were obtained retrospectively by questionnaire. RESULTS: Data from 98 patients, representing 100 episodes of ulceration, were analysed. The majority of patients (70) attended clinics in the Greater Manchester area. Eighty six patients were male and 58 were men who have sex with men (MSM), of whom 24 were HIV positive. PCR results agreed with the clinical diagnosis for 95 patients; samples from 26 patients were PCR positive and serologically diagnosed as primary (18) or secondary (8) syphilis, whereas 70 patients had PCR negative samples and were not diagnosed as having active syphilis. These data include two HIV positive patients who were PCR positive 12 and 21 days before their treponemal seroconversion. One positive PCR result was not supported by positive treponemal serology (this patient coincidentally received a 10 day course of co-amoxiclav 1 week after sampling). Three patients had negative PCR results but positive syphilis serology. The sensitivity, specificity, positive and negative predictive value for primary syphilis were 94.7%, 98.6%, 94.7%, and 98.6%, respectively, and for secondary syphilis these were 80.0%, 98.6%, 88.9%, and 97.2%, respectively. CONCLUSION: PCR is a sensitive and specific test for T pallidum, and an important adjunct to dark ground microscopy and treponemal serology in diagnosing infectious syphilis in the United Kingdom.     

Are serological tests of value in diagnosing and monitoring response to treatment of syphilis in patients infected with human immunodeficiency virus?

To assess the value of serological tests in diagnosing and monitoring the response to treatment of syphilis in patients infected with the human immunodeficiency virus (HIV), case notes of eight homosexual men with a history of treated syphilis, positive reactions to serological tests for syphilis, and documented subsequent conversion to HIV seropositivity were studied. No change was noted in serological markers of syphilis after HIV infection. The case notes of one man with primary syphilis, four men with secondary syphilis, and three men with latent syphilis, of whom all were HIV seropositive, were also studied. In seven of these patients the serological responses to infection and after treatment were consistent with the experience of syphilis in HIV seronegative patients. In one man treated for secondary syphilis, and confirmed as HIV seropositive eight months after treatment, the rapid plasma reagin (RPR) test result continued to be positive at a high titre for up to 20 months after treatment.     

Cross‐sectional study of Treponema pallidum PCR in diagnosis of primary and secondary syphilis

Background

Syphilis remains a major challenge and a complex diagnosis. We aim to evaluate the role of polymerase chain reaction (PCR) in Treponema pallidum (Tp) detection in various types of biological samples in the diagnosis of early syphilis.

Methods

We conducted a cross‐sectional study including all attendees of the STI clinic with clinical suspicion of early syphilis. One or more specimens for the detection of Tp by PCR testing were collected.

Results

The overall sensitivity of Tp PCR test was 82.61% (95% CI: 68.6–92.2%). Tp PCR test had sensitivity of 84.6% (95% CI: 54.6–98.1%) in primary syphilis cases and 81.8% (95% CI: 64.5–93%) in secondary syphilis cases. PCR test performance was independent of HIV status.

Conclusion

Tp PCR test is a fast and reliable method for the detection of Tp in skin lesions of early syphilis, and it is a powerful tool in clinical settings.     

Origins of syphilis and management in the immunocompetent patient: Facts and controversies

Despite the continued efficacy of penicillin since the 1940s, many aspects of the natural history, diagnosis, and management of syphilis remain controversial. A key factor among the numerous factors explaining the persistence of significant areas of controversies is the absence of a gold standard direct method for distinguishing between the different stages of syphilis and appraising treatment response. This contribution presents an overview of some of the most debated aspects of the origins, diagnosis, and management of syphilis in immunocompetent patients. The two main current hypotheses on the origins of Treponema pallidum are the “Columbian” and the “Pre-Columbian” hypotheses. Strong evidence supports that Columbus' crew brought T pallidum to Europe at the time of discovery of the New World. Because T pallidum culture and inoculation to animals are not readily available methods, the gold standard method for the diagnosis of syphilis is the direct identification of T pallidum by dark field microscopy or direct fluorescent antibody tests. These methods, however, are inapplicable in many patients, and thus the diagnosis of syphilis is usually based on the clinical and serologic picture. Serologic tests should only be considered as surrogate markers of the disease and do not provide definite distinction between syphilis stages. The optimal combination of serologic tests is still undefined. Other areas of controversy include the identification of patients who would benefit from a lumbar puncture, the diagnostic criteria of neurosyphilis, and the most relevant markers of treatment response.     

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.     

Lessons from the syphilis outbreak in homosexual men in east London

OBJECTIVES: To describe the epidemiology, presentation, and diagnosis of early syphilis in 103 homosexual men in east London. METHODS: A retrospective study using data from KC60 returns, the Health Protection Agency (HPA) enhanced surveillance forms and case notes. RESULTS: 40 cases of primary (PS), 40 of secondary (SS) and 23 of early latent syphilis were identified, 33% co-infected with HIV. 41% had concurrent sexually transmitted infections (STIs). Pain featured in 35% of PS and itch in 13% of rashes. Dark ground microscopy (DGM), performed in 44 of the symptomatic cases, was positive in 37 (84%) allowing early management. Initial syphilis serology was negative in 15/40 (37%) cases of PS. 51% and 49% opted for parenteral and oral treatment, respectively. In 53/103 (51%) cases oral sex was the only risk factor. 86% of infections were UK acquired. Only 4% of contacts were seen. CONCLUSION: This outbreak, reflecting the resurgence of syphilis across the United Kingdom, highlights several important points. Painful chancres and itchy rash are common presentations. DGM is a highly sensitive diagnostic tool. Initial negative serological screening tests are common in PS and sero-surveillance for 3 months is recommended. The high prevalence of concomitant STIs indicates ongoing unprotected sexual intercourse. Oral sex is a significant risk factor and is a distinctly "unsafe" practice. Conventional partner notification is ineffective. Other methods of screening of the at-risk homosexual population are warranted. Continued education is required to reduce STI acquisition in homosexual men.     

Clinical Expression of Secondary Syphilis in a Patient with HIV Infection

There are increasing reports of unusual clinical features and atypical courses of syphilis in patients with acquired immunodeficiency syndrome. Recently, we had the opportunity to study an HIV-positive female patient with strong manifestations of secondary syphilis. The case is discussed together with the implications of secondary syphilis in her concomitant ocular affliction. Moreover, we comment on the clinico-therapeutic controversies brought about by the association of infection with Treponema pallidum and HIV.     

Value of serological diagnosis in congenital syphilis. Report of nine cases.

The diagnosis of congenital syphilis is difficult since it depends mainly on the results of serological tests. The results of five serological tests (three specific and two non-specific) in nine neonates with congenital syphilis are compared with those obtained in three with passively acquired antibodies. It appeared that the serological diagnosis of congenital syphilis must be based on the finding of specific neonatal antibodies in cord serum, which give positive results to the fluorescent treponemal antibody absorption test for immunoglobulin M, together with high titres of total IgM and negative results to latex tests. The non-specific tests are useful for confirming the efficacy of treatment. The mean number of cases of congenital syphilis in Seville is 0.81/1000 live births.     

Nodular secondary syphilis with associated granulomatous inflammation: case report and literature review

A 62‐year‐old male presented with a 10‐day history of a diffuse, erythematous papular rash sparing the palms and soles. Histopathologic examination of a skin lesion showed loose non‐caseating granulomas in a lymphoplasmacytic background. Scattered spirochetes were identified by Treponema pallidum immunohistochemistry, in keeping with a diagnosis of secondary syphilis. Granulomatous inflammation in secondary syphilis is uncommon. A review of the literature reveals that the majority of prior reported cases of granulomatous secondary syphilis share similar characteristics to this case; namely, a papular or nodular clinical presentation, sparing of the palms and soles, and collections of epithelioid histiocytes with associated lymphocytes and variable numbers of plasma cells.     

Syphilis serology and HIV infection in Harare, Zimbabwe

OBJECTIVE: To determine the reliability of serological tests in detecting syphilis in a factory worker cohort and examine the impact of concurrent HIV infection on serological tests for syphilis. METHOD: Reactions to non-treponemal and treponemal antigens were tested using sera from a cohort of 3401 factory workers in Harare, Zimbabwe. The participants consented to regular testing for syphilis, by VDRL, and HIV using two ELISAs. All sera from men who were VDRL positive, and a random sample of VDRL negative sera, were tested by RPR, TPHA, and where appropriate FTA-Abs. From the results, men were defined as having no syphilis, active syphilis, incident syphilis, historic syphilis, or giving biological false positive reactions. RESULTS: 709 sera were examined from 580 men. There were 78 cases of active syphilis in the cohort, giving a prevalence of 2.3%, and the seroincidence was 0.25 per 100 person years of follow up. The prevalence of HIV in the cohort was 19.8%. There was a strong association between syphilis, whether active, incident or historic, and HIV seropositivity. With both HIV positive and negative sera the negative predictive values of VDRL and RPR were > 99.9% while the positive predictive value for VDRL (30%) was lower than for RPR (39%). Biological false positive reactions were detected in 0.5% of the cohort, with in most cases a transient rise in VDRL titres up to < 1/16. Higher false positive titres occurred in five men, each of whom was HIV positive. CONCLUSIONS: The VDRL is reliable in detecting possible cases of syphilis even in a community with a high prevalence of heterosexually transmitted HIV. There is need, however, for confirmatory tests. The prevalence of syphilis in this cohort is very low in comparison with other countries in southern Africa, but is consistent with recent data from Harare. Despite a strong association between syphilis and HIV it was clear that syphilis could not be counted as a major factor fueling the HIV epidemic in Zimbabwe.     

Secondary syphilis resembling erythema multiforme

Background Secondary syphilis, which typically begins 4‐10 weeks after initial exposure to Treponema pallidum, manifests with a range of cutaneous patterns. One unusual variation features oval, targetoid plaques that may resemble erythema multiforme (EM). Methods We describe a 23‐year‐old woman with an EM‐like eruption, a mucous patch, and a prominent alopecia somewhat moth‐eaten in appearance. She had positive rapid plasma reagin and fluorescent treponemal antibody absorption (FTA‐ABS) tests, and a skin biopsy revealed swollen endothelial cells with a superficial perivascular infiltrate containing plasma cells. Response to intramuscular penicillin therapy was consistent with the empiric diagnosis of secondary syphilis. Conclusions One should consider secondary syphilis in the differential diagnosis of an eruption resembling EM.     

False‐negative anti‐treponemal immunohistochemistry in secondary syphilis

Background While serological studies are the primary tool used to confirm the diagnosis of secondary syphilis, skin biopsies are often performed to control for potential false positives and negatives. Immunohistochemistry using anti‐T. pallidum specific antibodies has proven to have a very high sensitivity and specificity for identification of spirochetes in biopsy specimens, but can occasionally fail to identify organisms in clinically and serologically confirmed cases. Methods We report two cases of apparently negative biopsy specimens in which rare organisms were subsequently identified by detailed study of multiple additional sections. Results Our experience suggests that the sensitivity of immunohistochemistry for syphilitic spirochetes may be improved by repeat immunostaining.     

Case for diagnosis. Atypical genital lesion

We present a case of a penile lesion with a clinical appearance similar to Mondor penile disease (thrombosis of the dorsal vein of the penis) or penile sclerosing lymphangitis. Laboratory evaluation, however, showed a solid lesion, with no vascular component to Doppler ultrasonography and no treponema to immunohistochemistry. Histological and serological tests were compatible with secondary syphilis. The authors reinforce the need for the inclusion of syphilis in the differential diagnosis of penile cord injuries.