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Unexpected Immunostaining of Mycobaterium leprae With a Polyclonal Antibody Against Treponema pallidum

Aparicio, M-Auxiliadora MD; Santos-Briz, Ángel MD, PhD

The American Journal of Dermatopathology: July 2012 - Volume 34 - Issue 5 - p 559–561
doi: 10.1097/DAD.0b013e3182181596
Letters to the Editor

Department of Pathology, University Hospital of Salamanca, Salamanca, Spain

The authors have no conflicts of interest to disclose.

To the Editors:

Leprosy is a granulomatous disease of the peripheral nerves and mucosa of the upper respiratory tract, and its salient clinical features are skin lesions. It caused by the bacillus, Mycobacterium leprae.1–3 The diagnosis of the disease usually relays on a combination of clinical symptoms and signs and histopathological findings of skin biopsies3,4 and can be confirmed with molecular biology tools by polymerase chain reaction amplification of the mycobacterial heat shock 65 gene (Hsp 65) from DNA extracted from lesions.5 The bacilli may be detected in tissue sections by special stains, such as the Fite–Faraco modification of the Ziehl–Neelsen staining for acid alcohol–resistant bacilli6 and the auramine-rhodamine fluorescent histochemical method.7 The bacilli may also be detected by immunocytochemistry as M. leprae possesses several antigens. Some of these antigens are species specific, whereas others are common to other Mycobacteria.8–12

Very recently, awareness has risen about the artifactual staining of bacilli of the genus Mycobacterium with antibodies against Treponema pallidum, the microorganism that causes syphilis. In a recent Letter to the Editor published in this Journal, Fernández-Flores reported that several biopsies from patients suspected to suffer mycobacterial infections contained bacilli (but not spirochetae) immunopositive for T. pallidum.13 More specifically, the application of a widely used primary polyclonal antibody against T. pallidum (Biocare Medical, Concord, California) resulted in the staining of bacilli (presumed mycobacteria) in a biopsy of the skin of a patient with and infection caused by Mycobacterium marinum, and in granulomas present in a bronchial biopsy from a patient diagnosed with tuberculosis.13

Given the possibility that commercial antisera against T. pallidum recognize antigens present in other species of mycobacteria, we set forth to analyze whether the antiserum used in most laboratories stains M. leprae bacilli. To this end, we re-assessed 3 recent cases of patients with cutaneous leprosy and stained sections of their skin biopsies with the Biocare Medical T. pallidum antibody. In the 3 cases, the diagnosis of leprosy was established clinically and histopathologically. Moreover, the presence of M. leprae was confirmed in the 3 patients by molecular biology with polymerase chain reaction. None of the patients showed clinical or analytical signs of luetic infection.

Patient number 1 was an 40-year-old Cuban male who presented an ill-defined erythematous plaque of 5 months of evolution in the anterior face of the left shoulder (Fig. 1A) and a lightly hypopigmented and hypoesthetic lesion above the right knee. Patient number 2 was a 28-years-old male from Paraguay, who visited our hospital with fever, generalized lymophadenopaty, and erythematous infiltrated cutaneus lesions involving malar zone, arms and legs, and abdominal area (Fig. 1B). Patient number 3 was a 59-year-old Spanish male who reveled a several years history of multiple and recurrent erythematous and hypoesthetic plaque located on the abdominal area (Fig. 1C).



Biopsies of 3 patients exhibited similar findings consist of lesion a noncaseating granulomatous lesion with normal orthokeratosic epidermis. The structure of the dermis was disrupted, with superficial intermediate and deep infiltrates of lymphocytes, mast cells, and macrophages, often found around blood vessels, dermal appendages, and nerves and which extended into the subcutaneous cellular tissue (Figs. 1D, E). These collections of epithelioid cells formed ill-defined granulomas without necrosis. The Fite–Faraco stain (Figs. 1G, H) and the auramine-rhodamine technique (Fig. 1F) revealed abundant acid alcohol–resistant bacilli in the dermis, both inside macrophages forming clusters (globi) and in the extracellular space. All these histological changes were consistent with “borderline leprosy” caused by M. Leprae.

We stained formalin-fixed paraffin-embedded section of the same biopsy of the lesion from the shoulder with the Biocare Medical rabbit polyclonal antiserum against T. pallidum. Immunostaining was restricted to the noncaseating granulomas. Macrophages contained a dense granular or rod-like immunostaining in their cytoplasm, similar to the globi of bacilli seen with the Fite–Faraco techniques (Fig. 2). Scarce extracellular bacilli were also observed (Figs. 2B, D, F).



Thus, in all 3 cases of leprosy, immunostaining with the Biocare Medical antiserum against T. pallidum resulted in the artifactual staining of presumed M. leprae. Therefore, M. leprae adds to the list of mycobacteria that are recognized by the T. pallidum antiserum, which, as mentioned above, stains also M. marinum and M. tuberculosis. 13

What antigen(s) of the M. leprae is (are) recognized by the T. pallidum antiserum remains unknown. It seems reasonable to assume that the antibodies bind to one or more of the antigens shared by different mycobacteria. The list of shared antigens includes 60/65-kDa and 70/70 Hsp, and other proteins, lipids, carbohydrates, and their combinations.14–20 In addition to mycobacteria, the T. pallidum antiserum has been shown to recognize antigens from other spirochetes, Escherichia coli and Helicobacter pylori.21–23 Future studies will shed light on the sources of the observed cross-reactivities, which compromises the reliability of the T. pallidum antiserum as a diagnostic tool. In the meantime, caution must be exercised when interpreting the results of infectious samples immunostained for T. pallidum, and emphasis must be placed on clinical and serological data.

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