Applied Immunohistochemistry & Molecular Morphology:
Skin Homing of Treponema pallidum in Early Syphilis: An Immunohistochemical Study
Quatresooz, Pascale MD, PhD; Piérard, Gérald E. MD, PhD
Department of Dermatopathology, University Hospital of Liège, Liège, Belgium
Supported by a grant from the “Fonds d'Investissement de la Recherche Scientifique” of the University Hospital of Liège. No other sources of funding were used to assist in the preparation of this manuscript.
The authors have no conflicts of interest that are directly relevant to the content of this review.
Reprints: Prof Gérald E. Piérard, MD, PhD, Department of Dermatopathlogy, CHU Sart Tilman, BE–4000 Liége, Belgium (e-mail: Gerald.firstname.lastname@example.org).
Received for publication January 16, 2008; accepted April 2, 2008
The incidence of syphilis is increasing in most parts of the world including some major European cities. The lesions developing during secondary syphilis may be difficult to diagnose clinically. Similarly, the histopathologic changes do not always fulfill the typical diagnostic criteria. The objective of this study was to assess the contribution of immunohistochemistry for the identification and localization of Treponema pallidum on the skin. We retrieved from our files 12 paraffin-embedded biopsies of skin lesions, which had posed diagnostic problems in the past. Only a serologic test had proven that the patients had syphilis. Controls consisted of lichenoid dermatoses unrelated to syphilis and borreliosis. Immunohistochemistry using an antispirochete (T. pallidum and Borrelia) antibody was performed retrospectively. In all samples from primary and secondary syphilis, T. pallidum was highlighted, but none of the control lesions unrelated to syphilis showed positivity. Interestingly enough, T. pallidum present in the lower mid-part of the epidermis often outnumbered that in the dermis. This difference was more striking in secondary syphilis compared with primary syphilis. Immunohistochemistry for T. pallidum considerably increased the sensitivity and the specificity of the histologic diagnosis. The strong epidermal homing of T. pallidum is highlighted in early syphilis.
Early syphilis in its primary and secondary phases is due to the intrusion and hematologic dissemination, respectively, of Treponema pallidum. Secondary syphilis usually presents with fever, malaise, lymphadenopathies, and a faint macular exanthema that progresses to a copper-red maculopapular rash. The correct diagnosis is critical to initiate therapy for preventing the evolution and sequelae of the chronic stage of the disease. However, the clinical presentation of secondary syphilis can be diverse including follicular lesions as well as eruptions resembling pityriasis lichenoides and pityriasis rosea. Owing to the variable clinical aspects, syphilis has been described as “the great imitator.” To render the situation more complex HIV infection can be associated with syphilis and can affect its presentation and progression.1–4
At the histologic examination, the diagnostic clues for syphilis are not always straightforward. For instance, plasma cells may be scanty. An appropriate sliver stain revealing the spirochetes may remain negative or doubtful.5–7 Indeed, the conventional histochemical silver stains may be difficult to interpret owing to marked background staining. Hence, there is a need for improving the diagnostic accuracy of skin biopsies.
When the clinical diagnosis of early syphilis is not established, a skin biopsy is sometimes submitted to the dermatopathologist without relevant information. We were facing some difficulties in reaching the accurate histologic diagnosis of syphilis in a number of cases because the characteristic diagnostic criteria were discrete or missing. In such a situation, immunohistochemistry had been reported to improve the histologic diagnostic accuracy of syphilis.8–12
The aim of the present study was to assess the specificity and the sensitivity of immunohistochemistry in diagnosing early syphilis. We also highlight the prominent epidermal homing of T. pallidum in early syphilis.
MATERIALS AND METHODS
A total of 12 histologic cases of primary and secondary syphilis diagnosed during the past year in patients without HIV infection were retrieved from our files. The diagnosis was confirmed serologically (Venereal Disease Research Laboratory and Treponema pallidum hemagglutination). For comparison 15 other histologic cases showing a lichenoid pattern of inflammation distinct from syphilis and borreliosis, were selected as negative controls.
Sections (5-μm thick) were cut from formalin-fixed paraffin-embedded biopsies. They were stained with silver stain using the Warthin-Starry method. Additional paraffin sections were obtained for immunohistochemical assessment using a rabbit polyclonal antibody directed to T. pallidum (1:200 Biocare Medical, Walnut Creek, CA) and the avidin-biotin peroxidase method. This antibody is highly sensitive for detecting T. pallidum using immunohistochemistry on formalin-fixed paraffin-embedded human tissues. It cross-reacts with Borrelia burgdorferi. After a 1-hour incubation time with the primary antibody, the slides were washed in Tris-buffered saline (TBS) and incubated for 30 minutes with the secondary antibody (biotinylated swine antirabbit, 1:300, Dakopatts). Slides were rinsed in TBS and covered by the EnVision (Dakopatts, Glostrup, Denmark) polymer-based revelation system. After TBS washings, Fast Red (Dakopatts) was used as chromogen substrate. The last steps consisted of counterstaining with Mayer hemalum and mounting in glycerin mounting medium (Dakopatts). Negative immunohistochemical controls were performed by omitting or substituting the primary and the secondary antibodies of the laboratory procedure.
In primary and secondary syphilis, the dermo-epidermal junction was blurred by a bandlike infiltrate of lymphocytes, histiocytes, admixed with a variable amount of scattered plasma cells. A few eosinophils were occasionally present. In addition to the superficial inflammatory pattern, a deeper infiltrate of similar cell composition extended along adnexal structures. Discrete foci of granulomatous formations were also incidentally seen. Endothelial cells of the superficial vascular plexus looked plump in the majority of cases. The Warthin-Starry silver stain was interpreted as inconclusive or negative owing to the melanin background in the epidermis and the presence of thin and short reticulin fibrils in the dermis.
Immunohistochemistry demonstrated spirochetes in all cases of primary (Fig. 1) and secondary (Fig. 2) syphilis. The typical spiral and threadlike organisms were highlighted by the red chromogen distinct from the brown melanin in the melanocyte dendrites. The contrast with the clean background was striking. The section of the spiral treponemes gave a tortuous or fragmented aspect to the microorganisms (Fig. 2). Intraepidermal treponemes were seen in all cases as tangled masses in the lower part of the tissue, whereas dermal spirochetes appeared much less numerous and dispersed in secondary.
All control cases had been selected because they showed a pattern of inflammatory cells reminiscent of syphilis. The Warthin-Starry stain searching for spirochetes was inconclusive because melanocyte dendrites and short reticulin fibers shed some doubts on the interpretation of data. Immunohistochemistry for spirochetes was clearly negative. However, in 1 case a short thread of artifact deposited onto the microscopic slide was stained in red. This single structure was readily distinguished from a microorganism. Further step sections of the same case showed no immunoreactivity.
Over the years, the epidemiology of syphilis has changed considerably in Western Europe. A sharp decline in incidence was observed after the introduction of penicillin. In the mid-1990s, the low incidence was in addition attributed to a better prevention benefiting from the AIDS prevention campaigns. Furthermore, AIDS mortality may have contributed to the decline in syphilis rates.13 By contrast, the past decade has shown a reverse trend with a dramatic rise in the incidence rate of syphilis. The return of the disease has been acknowledged in many European cities.14–22
The clinical diagnosis of the cutaneous manifestations of syphilis is not always straightforward.23–25 T. pallidum cannot be cultured and therefore serology is commonly relied upon, but false-positive and false-negative data exist.26,27 Histology and histochemistry detect variable numbers of positive cases, ranging 33% to 71%.11 The organisms are typically seen at the dermo-epidermal junction, and in some instances, any organisms are disclosed in the dermis. Molecular methods including polymerase chain reaction assay have been developed more recently.28–33 Immunohistochemistry is still another method which shows higher specificity and sensitivity than the conventional silver stains.8–11 This method allows to distinguish an actual syphilis lesion infiltrated by T. pallidum from other skin disorders unrelated to syphilis, but that could develop in a patient showing a true-positive or a false-positive serology for T. pallidum. Another advantage of T. pallidum immunohistochemistry is to localize the spirochetes in the skin. However, a cross-reactivity with Borrelia spp. is possible.34
The present findings are globally in line with those previously reported with the same antibody.11 However, some aspects are at variance. On one hand, the previously reported 71% sensitivity11 was lower than our present findings that were in line with another study.10 The technical conditions are indeed important to be considered. To increase the immunohistochemical performance, we used the Fast Red chromogen for a better differentiation from the brown melanin pigment, and the EnVision system to increase the immunohistochemical sensitivity. On the other hand, the prominent epidermal homing of T. pallidum appeared striking in the present cases and in other studies.10–12 Other findings showing a more frequent dermal invasion were described.11
The presence of tangled masses of treponemes in the basal layer of the epidermis is puzzling. This aspect was previously shown11,13 and the control material excludes the possibility of a cross-reactivity with melanocyte dendrites. Such homing pattern of T. pallidum appears unique as no other microorganism exhibits such characteristics. We frame as a hypothesis that some specific adhesion molecule may participate in this process.
Two factors might have influenced the differences in information gained by the published immunohistochemical studies. The selection of cases was different and the histologic presentation of our cases seems to be less typical for syphilis. It is unknown if the prominent infiltration of the lower epidermis by the spirochetes in early syphilis and their lower numerical density in the dermis, particularly in secondary syphilis were responsible for the pattern of inflammatory cell infiltration.
In conclusion, we confirm that immunohistochemistry for detecting T. pallidum in skin biopsies is much more reliable than the classic silver stain histochemistry. A strong epidermal homing of the microorganisms was highlighted in early syphilis. Despite the high sensitivity of immunohistochemistry suggested by all reported immunohistochemical studies,8–12 it is still unknown what proportion of early syphilis escape this detection method. A clever clinico-pathologic correlation combined with serology should be performed in all cases of early syphilis.
The authors appreciate the excellent secretarial assistance of Mrs Ida Leclercq.
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syphilis; Treponema pallidum; spirochete homing
© 2009 Lippincott Williams & Wilkins, Inc.
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