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STUDY Possible Role of Borrelia burgdorferi Sensu Lato Infection in Lichen Sclerosus

Klaus Eisendle, MD, PhD; Tanja Grabner, MD; Heinz Kutzner, MD; Bernhard Zelger, MD, MSc

Objective: To assess the evidence for Borrelia burgdor- Results: Using focus-floating microscopy, we detected feri sensu lato infection in patients with lichen sclerosus Borrelia species in 38 of 60 cases (63%) of lichen sclero- by focus-floating microscopy. sus and in 61 of 68 (90%) of positive controls of classic borreliosis, but Borrelia species were absent in all negative Setting: Dermatology department of a university controls. Borrelia species were detected significantly more hospital. often in early inflammatory-rich (31 of 39 [80%]) than in late inflammatory-poor (7 of 21 [33.3%]) cases (P=.001). Design: Tissue sections were stained with a polyclonal Polymerase chain reaction findings were positive in 25 of B burgdorferi antibody using standard histological equip- 68 positive controls (37%) and negative in all 11 cases of ment and then scanned simultaneously in 2 planes: hori- lichen sclerosus and all 15 negative controls. zontally in a serpentinelike pattern and vertically by fo- cusingthroughthethicknessofthesection,ie,focus-floating Conclusions: Focus-floating microscopy is a reliable microscopy. Part of the material was also investigated by method to detect Borrelia species in tissue sections. The Borrelia-specific polymerase chain reaction. frequent detection of this microorganism, especially in early lichen sclerosus, points to a specific involvement Patients: The study population comprised 61 cases of lichen sclerosus and 118 controls (60 negative controls of B burgdorferi or other similar strains in the develop- and 68 positive controls). ment or as a trigger of this disease.

Main Outcome Measure: The presence of B burgdorferi sensu lato within tissue specimens. Arch Dermatol. 2008;144(5):591-598

ICHEN SCLEROSUS (LS), FRE- such as an infiltrate of lymphocytes ad- quently reported in the der- mixed with some plasma cells, an in- matologic literature as li- crease in fibrocytes and fibroblasts, and a chen sclerosus et atrophicus, diffuse dermal fibrosis to sclerosis is a chronic inflammatory (Figure 1B and C).7 These observations skinL disease of unknown etiology that leads have led several investigators to consider to substantial discomfort and morbidity. the possibility of Borrelia burgdorferi sensu It commonly affects adult woman in the lato as a common etiologic factor for both genito-anal region (Figure 1A) but also diseases. Since the first proposal of B burg- occurs elsewhere.1-4 Lichen sclerosus has dorferi as a causative agent by Aberer and Stanek8 in 1987, conflicting results have been obtained by different studies using For editorial comment serological, immunohistochemical, cul- see page 662 ture, and polymerase chain reaction (PCR) approaches. Borrelia species have frequently been detected in Europe, but not Author Affiliations: clinical and histological similarities with in cases from the United States. Studies re- Department of Dermatology morphea, and some investigators con- porting a positive association between and Venerology, Innsbruck sider this entity a superficial variant of mor- B burgdorferi infection and LS found evi- Medical University, Innsbruck, phea, an opinion supported by its fre- dence of the organism in 10% to 68% of Austria (Drs Eisendle, Grabner, quent coincidence with morphea.5,6 Lichen and Zelger); and cases; on the other hand, there are re- Dermatopathological Private sclerosus shares similarities and com- ports in which no positive cases could be 8-20 Laboratory Friedrichshafen, mon features with acrodermatitis chronica identified (Table 1). Friedrichshafen, Germany atrophicans (ACA), a chronic form of bor- One main difficulty in assessing the as- (Dr Kutzner). reliosis, particularly histological findings sociation between LS and borreliosis is the

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 challenge to reliably detect B burgdorferi in tissue speci- reliosis. Therefore, negative culture findings may be at- mens. These conflicting results at least in part reflect the tributable to the fastidiousness of the organism in the difficulties of the various techniques used to document culture.26,27 The initial enthusiasm with molecular tech- the participation of Borrelia species in the disease pro- niques gave way to a more realistic evaluation of these cess. Therefore, serological techniques are unsatisfying, methods as it became clear that sensitivity varies (30%- with false-negative (20%-80%) and false-positive (20%- 90%) according to Borrelia strains, the material (fresh, 50%) results in classic manifestations of borreliosis, such frozen, or paraffin material), and the primers ap- as erythema migrans (EM), borrelial lymphocytoma (BL), plied.26,28-33 In summary, all current detection methods and ACA. A negative serological test result does not ex- seem to bear an inadequate sensitivity for the detection clude previous infection with B burgdorferi, and a posi- of Borrelia species, so even the classic cutaneous Bor- tive result may represent an endemic background.21-23 His- relia infections remain a diagnosis based on circumstan- tological, histochemical, and immunohistochemical tial evidence combining clinicopathologic and labora- detection of microorganisms has turned out to be diffi- tory information and response to therapy. cult, frequently unreliable, and almost always ex- We recently developed a highly sensitive immuno- tremely time consuming.23-26 Cultures with specific me- histochemical procedure that proved to be more sensi- dia can detect Borrelia species in all clinical forms, but tive than PCR in the detection of B burgdorferi sensu lato these techniques are not generally available and are un- in classic cutaneous borreliosis (98% vs 45%) and nearly reliable, with less than 50% sensitivity for classic bor- equally specific (99% vs 100%).34 We named this procedure focus-floating microscopy (FFM). In cases in which abundant B burgdorferi were detected by FFM were usu- A B ally positive by PCR and when fewer organisms were detected by FFM, specimens were more likely to be negative by PCR. Using this new technique, we tried to assess the evidence for infection with B burgdorferi sensu lato in patients with LS.

METHODS C PATIENTS

We searched the files of the Dermatohistopathological Labo- ratory in Innsbruck, Austria, from the years 1989 through 2006, and retrieved 61 cases of LS. Diagnoses were well established by exact correlation with clinicopathological diagnosis for LS, including photographic documentation in many instances. Figure 1. Clinical photograph of lichen sclerosus (LS) in the female genital area (A); histopathological analysis of LS at different magnifications (original Serological test results were present only for a minority of pa- magnification ϫ100 [B] and ϫ200 [C]) showing infiltrate of lymphocytes tients and were not usable because of the a priori high en- admixed with some plasma cells, an increase of fibrocytes, and a diffuse demic background of positive Borrelia serological status in our dermal sclerosis (hematoxylin-eosin). geographic area.21,22

Table 1. Results of Studies Investigating Borrelia burgdorferi in Patients With Lichen Sclerosusa

Source Country Method Results, No. Pos/Total No. Aberer et al,8 1987 Austria Immunoperoxidase 6/13 Aberer et al,9 1988 Austria Immunoperoxidase 1/2 Ross et al,10 1990 Puerto Rico Silver stain 10/21 Schempp et al,11 1993 Germany PCR (flagellin) 6/6 Ranki et al,12 1994 Finland PCR (OspA) 0/1 Dillon et al,13 1995 United States PCR (flagellin) 0/10 De Vito et al,14 1996 United States PCR (clone 2H1) 0/7 Fujiwara et al,15 1997 Japan PCR (16S RNA) 2/3 Germany PCR (16S RNA) 1/10 United States PCR (16S RNA) 0/21 Colome´-Grimmer et al,16 1997 United States PCR (flagellin) 0/10 Alonso-Llamazares et al,17 1997 Spain PCR (OspA) 0/8 Culture 0/1 Aberer et al,18 1999 Austria PCR (flagellin) 13/19 O¨ zkan et al,19 2000 Turkey PCR (flagellin) 6/12 Breier et al,20 2001 Austria Culture 1/1 Present study, 2008 Austria PCR (23S RNA) 0/11 FFM 38/60

Abbreviations: FFM, focus-floating microscopy; PCR, polymerase chain reaction; Pos, positive. a For the studies involving PCR, the amplified gene is shown in parenthesis.

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Figure 2. High-power magnifications in a case of lichen sclerosus revealing the various aspects of microorganisms at different section planes using focus-floating microscopy. Note the milky cloud, best seen in panel A (asterisk). Scanning through different levels of the tissue section shows a single microorganism in the first plane (A), multiple undulated and crossed forms in the second plane (B), plump to delicate, undulated, comma- and dot-shaped forms in the third plane (C), and slightly granular “vanishing” to delicate forms in the last plane (D). (Immunohistochemical analysis for Borrelia burgdorferi, Acris BP1002 [polyclonal rabbit antibody], no counterstain, original magnification ϫ1000.)

Sixty cases, mainly inflammatory skin lesions, served as nega- nohistochemical analysis in our laboratory with a biotinylated tive controls, and 68 cases of PCR-controlled, clinically and his- second antibody and a streptavidin-biotin horseradish peroxi- tologically characteristic Borrelia infections (15 cases of EM, dase complex as a third layer. As a final reaction product, we used 23 cases of BL, and 34 cases of ACA) served as positive con- 3-amino-9-ethylcarbazole, whose bright red color proved supe- trols. Because our archival paraffin material had been fixed in rior to the brown color of diaminobenzidine. The counterstain inadequately buffered formalin until 2004, we could perform was omitted to enable easier recognition of Borrelia species. We Borrelia-specific PCR in only 11 LS cases. In 10 of these cases, first examined all immunohistochemical stains for the presence there was enough paraffin material left to further complete FFM. of Borrelia species independently (K.E., T.G., and B.Z.), including LS cases and positive and negative controls. Absence of coun- IMMUNOHISTOCHEMICAL ANALYSIS terstains guaranteed that these sections were evaluated in a blinded fashion, with the investigator unaware of the pathological diagnosis. There was excellent interobserver reliability among the dif- Serial sections from paraffin-embedded, formalin-fixed tissue ferent investigators. In rare occasions of divergent evaluation, blocks were obtained for hematoxylin-eosin staining and immu- subtle presence of Borrelia species had been overseen by one or nohistochemical analysis as previously described.34 Briefly, we used the other investigator. After evaluation for Borrelia species by a polyclonal rabbit antibody (Acris BP1002, derived from immu- FFM, we correlated the results with the hematoxylin-eosin stains. nization with whole-cell B burgdorferi preparations [strain B31, In the case of Borrelia species detection, serial sections allowed ATCC 35210; American Type Culture Collection, Manassas, Vir- for the exact localization of microorganisms in relation to the ginia] reacting with 83-kD and 41-kD flagellin, 32-kD OspB, and disease process. 31-kD OspA antigens and their fragments in Western blots, with cross-reaction to Treponema pallidum, Borrelia hermsii, and Bor- relia parkeri) at a dilution of 1:2000, with an autoclave antigen FOCUS-FLOATING MICROSCOPY retrieval time of 30 minutes in a sodium citrate buffer (pH 6.0- 6.1) and an incubation time of 30 minutes at 37°C. Focus-floating microscopy (Figure 2) is a modified immu- Further steps followed the Ventana-KIT (Ventana Medical Sys- nohistochemical technique, which combines several strate- tems, Munich, Germany) method as routinely used for immu- gies to detect minuscule organisms in tissue sections.34 Focus-

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 floating microscopy scans through the sections in 2 planes: as inflammatory-rich when lymphocytes and plasma cells horizontally in a serpentinelike pattern, as in routine cyto- were clearly seen at scanning magnification, while inflam- logic examination, and, simultaneously, vertically by focusing matory-poor cases showed no or only scattered lympho- through the thickness of the cut (usually 3-4 µm) at an origi- ϫ ϫ cytes. Of 61 cases, 8 showed an overlap with morphea. nal magnification of 200 to 400. This holoscopic ap- Histological examination of the hematoxylin-eosin– proach allows for the detection of B burgdorferi (diameter of 0.2 µm, compared with 2.0 µm for collagen bundles), which stained sections revealed characteristic findings of LS. The pass through the section at various angles and accordingly may epidermis was usually thickened, with prominent com- appear as undulated, comma- to dot-shaped forms. In addi- pact hyperkeratosis and hypergranulosis. Acanthosis and tion, the omission of counterstaining as well as bright illumi- a moderate interface process with variable vacuoliza- nation of the scanning field proves to be helpful because the tion of basal keratocytes was frequently seen. The main bright red color of the 3-amino-9-ethylcarbazole–stained mi- pathological feature involved a markedly thickened pap- croorganisms best contrasts with the faint yellow color of un- illary dermis. In early stages, a prominent infiltrate of lym- stained collagen bundles as well as other tissue structures. phocytes and occasionally plasma cells was present, usually perivascular but sometimes lichenoid around the POLYMERASE CHAIN REACTION superficial postcapillary venules, while only scattered lymphocytes were seen around the capillaries of the papil- For molecular identification of B burgdorferi, DNA was prepared lary dermis or close to or within the grenz zone. At this from paraffin-embedded tissue. After deparaffinization with xy- stage, the papillary dermis occasionally revealed edema, lene and ethanol and digestion with 0.6-mg proteinase K for 16 yet more regularly, early fibrosis with an increase of fi- hours, the remaining DNA was purified by adsorption chroma- brocytes and fibroblasts. In later stages, the number of tography (QIAamp DNA Mini Kit; QIAGEN GmbH, Hilden, Ger- fibrocytes and fibroblasts further increased, as did fibro- many), and the concentration of the sample was adjusted to 10 mg/L. Nested PCR was performed in volumes of 25 µL with 50-ng sis and sclerosis (homogenized bundles of collagen with- DNA, 100 pmol of each primer, 10mM Tris hydrochloride (pH out interposed fibrocytes), whereas the inflammatory in- 9.0), 50mM potassium chloride, 1.5mM magnesium chloride, filtrate decreased. Very late stages finally revealed a broad, 200mM of each deoxyribonucleotide triphosphate, and 1.5 U of homogenized eosinophilic band of collagen, ie, sclero- Taq polymerase. The samples were subjected to the following con- sis without significant inflammatory infiltrate. In a small ditions: in a PTC 200 thermocycler (MJ Research Inc, Water- number of cases, the fibrosing dermatitis also involved town, Massachusetts), the first PCR was performed for 30 sec- the reticular dermis, thereby simulating features char- onds at 94°C, 30 seconds at 53°C, and 30 seconds at 72°C for 40 acteristic of morphea at its various stages. cycles; and the second PCR was performed for 30 seconds at 94°C, All forms of B burgdorferi, as described in detail by 30 seconds at 58°C, and 30 seconds at 72°C for 45 cycles. For Aberer et al,26 were seen: mostly single and paired spiro- amplification, the following primers specific for the B burgdorferi 23S ribosomal RNA gene35 were used: for the first PCR, Bor-1: chetes and, rarely, clusters and colonies. Forms varied from 5Ј-AGAAGTGCTGGAGTCGA-3Ј and Bor-2: 5Ј-TAGTGCTC- very long and undulated to comma- and dot-shaped forms, TACCTCTAT-TAA-3Ј; and for the second PCR, Bor-3: 5Ј- with their appearance ranging from delicate to plump or GCGAAAGCGAGTCTTAAAAGG-3Ј and Bor-4: 5Ј- granular (Figure 2). Generally, we found lower numbers ACTAAAATAAGGCTGAACTTAAAT-3Ј. After separation on a of Borrelia species in our LS series than in the classic forms 2% agarose gel (50 mA for 30 minutes) and staining with ethidium of Borrelia infections such as EM, BL, and ACA. The num- bromide, the PCR product of 219 base pairs was visualized un- ber of spirochetes within sections varied between a single der UV light (302 nm). spirochete (Figure 3) to multiple microorganisms in 1 high-power field (original magnification ϫ400; Figure 4). STATISTICAL ANALYSIS Detection of Borrelia species followed characteristic rules: the spirochetes were seen outside, close to, or at the pe- Data were statistically analyzed using SPSS statistical software riphery of the inflammatory process. Within the inflam- (SPSS for Windows, version 12.0; SPSS Inc, Chicago, Illinois). matory and early fibrotic center, degenerative products of Statistical comparisons were performed using the 2-tailed Pear- B burgdorferi such as swollen, granular, or clumped ma- ␹2 Ͻ son test or the Fisher exact test when appropriate. P .05 terial could be found. A faint red, diffuse staining of this was considered statistically significant. area (Figure 4) proved to be a good clue for the detection of degenerative Borrelia species within the fibrotic and/or RESULTS inflammatory center and what seemed to be vital forms around or close to the periphery of inflammation. Spiro- The median age of the patients was 58 years (mean age, chetes or their degenerative products were frequently lo- 55.1 years; minimum age, 5 years; and maximum age, cated along or in between collagen bundles (collagenot- 86 years), and 22 patients (36%) were male and 39 (64%) ropism) and were partially to completely hidden if not were female. Biopsy specimens were mainly obtained from visualized in the correct section plane. the genito-anal area (n=28 [46%]) and trunk (n=17 Table 2 gives the results of FFM, partially controlled [28%]); 7 (12%) were from the lower extremities, and 5 by PCR. Of 60 LS cases, 38 were positive by FFM (63%). (8%) were from the upper extremities, and in 4 cases (7%) The presence of Borrelia species was similar in pure LS cases we could not ascertain the localization. and in those associated with morphea (33 of 52 [64%] vs According to the presence and number of inflamma- 5 of 8 [63%]). Borrelia burgdorferi sensu lato was detected tory cells, we additionally divided our cases into inflam- significantly more often in early inflammatory-rich (31 of matory-rich (“early”; n=40 [66%]) and inflammatory- 39 [80%]) than in late inflammatory-poor (7 of 21 [33%]) poor (“late”; n=21 [34%]) forms. Cases were estimated cases (P=.001). Similarly, an early inflammatory-rich case

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Figure 3. Four different cases of late lichen sclerosus, in which single spirochetes were found using focus-floating microscopy. The photographs show 4 different morphological appearances of Borrelia species: elbow form (A), bacilli form (B), undulated form (C), and bacilli and dot-shaped (D). (Immunohistochemical analysis for Borrelia burgdorferi, Acris BP1002 [polyclonal rabbit antibody], no counterstain, original magnification ϫ1000.)

of LS usually revealed more microorganisms than a late in- was subsequently further supported, at least in part, by flammatory-poor case. Statistically, all specimens of LS re- several other studies (Table 1).8-20 A bacterial cause was vealed significantly less spirochetes than cases of classic bor- further suggested because several cases of LS responded reliosis (ie, EM, BL, and ACA) (89.7% vs 63.3%; P=.001). well to therapy with antibiotics, such as dirithromycin, This significance was lost when compared with early in- penicillin, and ceftriaxone.20,36,37 flammatory-rich forms of LS (80% vs 90%; P=.16). Focus- In the present study, we detected B burgdorferi sensu floating microscopy was much more sensitive to detect Bor- lato in more than 60% of all LS cases, with a signifi- relia species in controls compared with PCR (90% vs 37%; cantly higher percentage (P=.001) in early (80%) than PϽ.001). Yet, none of 11 LS specimens was positive by PCR, in late (33%) LS, while it made no difference whether LS while 6 of 10 of these PCR-negative cases were positive by was associated with morphea. This might reflect inten- FFM, with 1 case not having material left to perform FFM tional or coincidental antibiotic exposure in longer- after PCR. term cases and/or the natural course of disease, with re- The B burgdorferi antibody showed no cross-reactions pression of the microorganism by the immune system. with other tissue structures. All 60 controls from well- The negative detection rate of Borrelia DNA by PCR in defined mainly inflammatory disorders other than bor- our study, with no positive case in 11 tested, indicates reliosis remained negative. In our experience, silver tech- the problematic role of this technique to reliably detect niques over the last decades never proved to be successful Borrelia species in tissue specimens. for the reliable detection of microorganisms in routine labo- The low number of microorganisms beyond the de- ratory procedure and thus were not performed in this study. tection threshold38 could be one explanation for the in- consistent results in PCR studies (Table 1). Other ex- COMMENT planations include old stage of disease, wrong biopsy site (eg, from negative fibrosclerotic parts), or wrong fixa- The involvement of B burgdorferi as a causative agent for tion of tissue specimens leading to DNA cross-linking (eg, LS was first proposed by Aberer and Stanek8 in 1987 and with inadequately buffered formalin). Furthermore, ex-

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Figure 4. Series of magnifications (original magnification ϫ100 [A], ϫ200 [B], ϫ400 [C], and ϫ1000 [D]) from the right lower part of the biopsy specimen (arrow indicates corresponding structures) allow for the identification of 3 clusters of long and partly undulated, plump, granular, or delicate microorganisms. Note the faint red, diffuse staining of subepidermal fibrosis left of the infundibula of 2 hair follicles, while clusters of spirochetes are at the periphery of the inflammatory process in the mid dermis. (Immunohistochemical analysis for Borrelia burgdorferi, Acris BP1002 [polyclonal rabbit antibody], no counterstain.)

cept for the studies by Ranki et al12 and Dillon et al,13 other rarely occurs in the United States but is commonly seen PCR studies with negative findings are lacking appropri- in Europe where B afzelii and B garinii are more preva- ate positive controls in terms of detection of Borrelia DNA lent.42 Maybe subspecies variations dictate the clinical in tissue specimens from classic borreliosis such as EM, manifestations that follow infections, with only certain BL, and ACA. Thus, the reliability of the DNA extraction strains possessing the characteristics required to initiate method for small DNA amounts or the PCR technique used the development of LS.15 Thus, another explanation for in these studies remains somewhat debatable.14,16 the moderate results by PCR might be that these tech- There is still another explanation for negative PCR re- niques use primers highly specific for known human sults: B burgdorferi sensu lato includes B burgdorferi sensu pathogenetic strains, whereas FFM uses an immunohis- stricto, Borrelia garinii and Borrelia afzelii VS461, but tochemical approach involving a less specific poly- newer Borrelia species have been identified. The patho- clonal antibody that probably detects more different Bor- genic significance of these species, such as Borrelia val- relia species. Borrelia species have not been implicated aisiana, B hermsii, Borrelia turicatae, Borrelia duttonii, as a cause of LS in the United States, and we did not have B parkeri, and most recently Borrelia spielmanii is not yet the opportunity to examine cases from other patient popu- fully answered. While B burgdorferi sensu stricto, to the lations. Therefore, it remains to be seen if the use of this best of our knowledge, is the only cause of Lyme disease technique would reveal cases of Borrelia-associated LS in the United States, B afzelii, B garinii, and probably in the United States. B valaisiana also cause Lyme disease in Europe and Asia. In any case, detection of spirochetes in pure LS and Relapsing fever borreliosis caused by B hermsii, B turi- LS associated with morphea seems to be a common de- catae, and B duttonii and EM caused by B spielmanii have nominator, which indicates the nosologic relationship of been described.39,40 The study by van Dam et al41 sug- these skin disorders.8,15 Moreover, the infectious hypoth- gests that different B burgdorferi genotypes have differ- esis with spirochetes helps to explain the most common ent pathogenic potentials. This is well documented for stereotypical presentation of LS, namely in the genito- the classic Borrelia manifestations; for example, ACA anal area. Subclinical dissemination with the spread of

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Accepted for Publication: April 13, 2007. Table 2. Detection of Borrelia species by FFM and PCR a Correspondence: Klaus Eisendle, MD, PhD, Depart- in Lichen Sclerosus (LS) Cases and Controls ment of Dermatology and Venerology, Innsbruck Medi- cal University, Anichstr 35, 6020 Innsbruck, Austria Borrelia-Positive Cases ([email protected]). Variable FFM PCR Author Contributions: Dr Eisendle had full access to all Classification of LS the data in the study and takes responsibility for the in- LS, “pure” 33/52 (64) 0/5 tegrity of the data and the accuracy of the data analysis. LS with morphea 5/8 (63) 0/6 Study concept and design: Eisendle, Kutzner, and Zelger. Stage (activity) of LS Acquisition of data: Eisendle, Grabner, Kutzner, and Inflammatory rich, “early” 31/39 (80) 0/7 d Zelger. Analysis and interpretation of data: Eisendle and Inflammatory poor, “late” 7/21 (33) 0/4 Zelger. Drafting of the manuscript: Eisendle and Zelger. Total 38/60 (63) 0/11 Controls Critical revision of the manuscript for important intellec- Negativeb 0/60 0/15 tual content: Grabner, Kutzner, and Zelger. Statistical Positivec 61/68 (90)e 25/68 (36.8)f analysis: Eisendle. Administrative, technical, and material support: Kutzner and Zelger. Study supervision: Abbreviations: FFM, focus-floating microscopy; PCR, polymerase chain Zelger. reaction. a Data are given as number positive/total tested (percentage). Financial Disclosure: None reported. b Includes normal skin, melanocytic nevi, atopic and stasis dermatitis, Additional Contributions: Gabriele Palmedo, PhD, prurigo, scabies, erythema multiforme, drug eruption, pityriasis rubra pilaris, Dermatohistological Private Laboratory, Friedrichs- mycosis fungoides, urticaria, Wells syndrome, lupus erythematodes hafen, Germany, contributed molecular data. Birgit Moser, (tumidus and subacute cutaneous), lichen nitidus, infectious granulomas (lepra and leishmaniasis), foreign body reaction due to ruptured infundibular Margit Abenthung, and Nadja Greier provided excel- cyst, insect bite, acne, rosacea, folliculitis decalvans, (hypertrophic) scar, lent technical assistance. keloid, dermatofibroma, Dupuytren contracture, and ganglion. c Includes 15 cases of erythema migrans, 23 cases of borrelial lymphocytoma, 34 cases of acrodermatitis chronica atrophicans. REFERENCES d Inflammatory-rich compared with inflammatory-poor cases (P = .001). e Positive controls compared with (1) all LS cases (P = .001) and (2) inflammatory-rich cases (P = .16). 1. Meffert JJ, Davis BM, Grimwood RE. Lichen sclerosus. J Am Acad Dermatol. f Polymerase chain reaction compared with FFM (P Ͻ .001). 1995;32(3):393-416. 2. Powell JJ, Wojnarowska F. Lichen sclerosus. Lancet. 1999;353(9166):1777-1783. 3. Marini A, Blecken S, Ruzicka T, Hengge UR. Lichen sclerosus: new aspects of pathogenesis and treatment [in German]. Hautarzt. 2005;56(6):550-555. Borrelia to kidneys and urine occurs in early Borrelia in- 4. Val I, Almeida G. An overview of lichen sclerosus. Clin Obstet Gynecol. 2005;48 33,43 (4):808-817. fection. Favored by the moist and frequently trauma- 5. Shono S, Imura M, Ota M, Osaku A, Shinomiya S, Toda K. Lichen sclerosus et tized conditions of genitalia, this might allow a superfi- atrophicus, morphea, and coexistence of both diseases: histological studies using cial Borrelia infection in the perigenital region (ie, LS). lectins. Arch Dermatol. 1991;127(9):1352-1356. This could explain the frequent occurrence of the dis- 6. Peterson LS, Nelson AM, Su WP. Classification of morphea (localized scleroderma). ease in the perigenital area and why other mucous mem- Mayo Clin Proc. 1995;70(11):1068-1076. 7. Asbrink E, Brehmer-Andersson E, Hovmark A. Acrodermatitis chronica atrophi- branes such as the oral or endonasal mucosa and con- cans-a spirochetosis: clinical and histopathological picture based on 32 pa- juctiva are practically never affected. The lower level of tients: course and relationship to erythema chronicum migrans Afzelius. Am J microorganisms in late LS not only indicates that the dis- Dermatopathol. 1986;8(3):209-219. ease is the consequence of the infectious agent but also 8. Aberer E, Stanek G. Histological evidence for spirochetal origin of morphea and lichen sclerosus et atrophicans. Am J Dermatopathol. 1987;9(5):374-379. reflects the challenge for the immune system and/or a 9. Aberer E, Kollegger H, Kristoferitsch W, Stanek G. Neuroborreliosis in morphea compromised immune reaction in the patients them- and lichen sclerosus et atrophicus. J Am Acad Dermatol. 1988;19(5, pt 1): selves, in whom Borrelia antigens might trigger a subse- 820-825. quent autoimmune reaction in genetically predisposed 10. Ross SA, Sanchez JL, Taboas JO. Spirochetal forms in the dermal lesions of mor- individuals via molecular mimicry.44 Thus, thyroid au- phea and lichen sclerosus et atrophicus. Am J Dermatopathol. 1990;12(4): 357-362. toantibodies have been described in 36% of patients 11. Schempp C, Bocklage H, Lange R, Kolmel HW, Orfanos CE, Gollnick H. Further 45 with LS. Borrelia burgdorferi has been proposed as an evidence for Borrelia burgdorferi infection in morphea and lichen sclerosus et environmental trigger of autoimmune thyroiditis atrophicus confirmed by DNA amplification. J Invest Dermatol. 1993;100(5): through amino acid sequence homologies between pro- 717-720. 12. Ranki A, Aavik E, Peterson P, Schauman K, Nurmilaakso P. Successful amplifi- teins of B burgdorferi and all thyroid autoantigens (eg, cation of DNA specific for Finnish Borrelia burgdorferi isolates in erythema chroni- human thyrotropin receptor, human thyroglobulin, hu- cum migrans but not in circumscribed scleroderma lesions. J Invest Dermatol. man thyroperoxidase, and human sodium iodide sym- 1994;102(3):339-345. porter) or segments thereof.46,47 The induction of auto- 13. Dillon WI, Saed GM, Fivenson DP. Borrelia burgdorferi DNA is undetectable by immunity also might explain why not all patients polymerase chain reaction in skin lesions of morphea, scleroderma, or lichen sclerosus et atrophicus of patients from North America. J Am Acad Dermatol. benefit from antibiotic therapy and makes an early an- 1995;33(4):617-620. tibiotic treatment reasonable. 14. De Vito JR, Merogi AJ, Vo T, et al. Role of Borrelia burgdorferi in the pathogen- In conclusion, FFM is a reliable method to detect Bor- esis of morphea/scleroderma and lichen sclerosus et atrophicus: a PCR study relia species in tissue sections, and the frequent detec- of thirty-five cases. J Cutan Pathol. 1996;23(4):350-358. 15. Fujiwara H, Fujiwara K, Hashimoto K, et al. Detection of Borrelia burgdorferi DNA tion of this microorganism, especially in early LS, points (B garinii or B afzelii) in morphea and lichen sclerosus et atrophicus tissues of to a specific involvement of B burgdorferi or other simi- German and Japanese but not of US patients. Arch Dermatol. 1997;133(1): lar strains in the development or as a trigger of LS. 41-44.

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