Further Evidence for Borrelia burgdoiferi Infection in Morphea and Lichen Sclerosus et Atrophicus Confirmed by DNA Amplification

Christoph Schempp, Hubertus Bocklage, Robert Lange, Hans W. Kolmel, Constantin E. Orfanos, and Harald Gollnick Department of Dermatology (CS, CE-O, HG), University Medical Center Steglitz, The Free University of Berlin; and Department of Neurology (HB, RL, HW-K), University Medical Center Charite, Humboldt University Berlin, Berlin, Germany

We present further evidence in support of the notion that either from patients with chronic eczema (three of three) or Borrelia burgdoiferi is possibly involved in the pathogenesis of psoriasis (two of two) or from normal skin (three of three). morphea and lichen sclerosus et atrophicus (LSA). Running a ~ntibodies directed against B. burgdoiferi were only detected nested polymerase chain reaction (PCR) with a primer set 111 the serum of patients with erythema chronicum migrans specific for the flagellin gene of B. burgdoiferi enabled us to (two of two) and acrodermatitis chronica atrophicans (one of demonstrate the presence of Borrelia DNA in skin biopsies of one) but were not present in cases of morphea (five of five), patients with morphea (nine of nine) or LSA (six of six). LSA (three of three), or in control subjects (three of three). lliopsy specimens obtained from patients with erythema These data suggest that B. burgdoiferi may playa role in the chronicum migrans (two patients, four of four samples) and pathogenesis of both morphea and LSA. Furthermore, we acrodermatitis chronica atrophicans (one patient, one of one conclude that PCR analysis provides an important diagnostic sample) also show~d positive P~R results: By contrast., th~re tool, even in seronegative Borrelia infections.] Inllest Dermatol was no amplification of BorrellO DNA 111 control bIOpSies 100:717-720,1993

orphea and lichen sclerosus et atrophicus (LSA) valuable tool in the detection of even small numbers of spirochetes are connective tissue disorders that share histo­ persisting in the skin [9] . pathologic features, such as cellular inflammatory . In the present study, we investigated skin biopsy samples of pa­ processes at beginning stages and sclerosis or atro­ tIents :WIth eIther morphea or LSA by PCR analysis with Borrelia phy of collagen and elastic fibers at later stages. flagellll1- gene-specific primers. American and European Borrelia MRecently, a spirochetal origin has been postulated for morphea and strains, as well as biopsies from patients with confirmed erythema [,SA on the basis of histologic, immunohistochemical, and serologic chronicum migrans (ECM) or acrodermatitis chronica atrophicans fi ndings [1 -4]; however, conflicting reports exist about a positive (ACA), were used as positive controls. Inflammatory dermatoses Borrelia bu rgdorferi serology in patients with morphea and LSA [5- and normal skin samples served as negative controls. 1]. Furthermore, because the. de~onstration and classification ~f spirochetes in tissue sections IS dIfficult, the IdentIficatIOn of artI­ MATERIALS AND METHODS flc ts cannot be completely excluded. Although some investigators Patients Tissue for PCR analysis originated either from the ar­ successfully isolated spirochetes from morphea lesions [4,8]' this chIval paraffin-embedded material of the Dermatopathological Lab­ procedure is laborious and of low yield, espe.c~ally at later ~tages of oratory ?r from paraffin-embedded biopsies prepared for routine Lyme borreliosis. Therefore, a lughly sensItive and speCIfic new dIagnostIcs that had been obtained after informed consent from method such as the polymerase chain reaction (PCR) provides a patients attending the Department of Dermatology, University Medical Center Steglitz, Berlin. The following diagnoses were es­ Manuscript received September 14, 1992; accepted for publication De­ tablished on the basis of both clinical and histopathologic evalua­ cember 31, 1992. t~on: ACA (one patient), ECM (two patients), morphea (nine pa­ Presented in part at the Annual Meeting of the European Society for tIents), LSA (SIX patients), psoriasis (two patients), and chronIC Dermatological Research, London, England, April 4- 9, 1992. eczema (three patients) (Table I) . Three patients with morphea, one Reprint requests to: Dr. Harald Gollnick, Department of Dermatology, wIth LSA, and one with ECM remembered a tick bite at the SIte of University Medical Center Steglitz, The Free University of Berlin, Hin­ the lesion (Table I). Normal skin was obtained from two healthy denburgdamm 30, D-1000 Berlin 45, Germany subjects with negative Borrelia serology and from one patient with Abbrevia tions: morphea (patient 14). In one patient with ECM (patient 3), three ACA: acrodermatitis chronica atrophicans biopsies were taken from the edge and center of the lesion and from ECM: erythema chronicum migrans EDT A: ethylenediametriaacetic acid perilesional skin. The clinical il1anifestations of morphea were of ELISA: enzyme-linked immunosorbent assay the non-disseminated plaque type and in one case (patient 18) of the LSA: lichen sclerosus et atrophicus atrophodermia Pasini - Pierini type. All but one of the patients wIth PCR: polymerase chain reaction LSA had genital LSA. In one case, the localization ofLSA was on the TE: Tris-HCI + EDTA left shoulder (patient 4). Patients with morphea or LSA were se­ Taq: thermophilus aquaticus lected for PCR analysis if there was histologic evidence of an acute

0022-202Xj93jS06.00 Copyright © 1993 by The Society for Investigative Dermatology, Inc.

717 718 SCHEMPP ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table I. Characterization of Specimens Examined by PCR ELISAJ Patient Age Localization and Number' Genderb (year) Diagnosis' Lesion Type Duration IgM IgG PCR 1 F 25 ACA Left elbow 10 years + + + 2 F 66 ECM Right thigh 3 weeks + + 3a M 41 ECM' Right thigh, center of lesion 3 months + + + 3b Right thigh, edge of lesion + + + 3c Right thigh, perilesional skin + + + 4 F 71 LSA Left shoulder 2 years NO NO + 5 F 49 LSA Vulvar 4 years + 6 F 51 LSA Vulvar 20 years + 7 F 27 LSA' Vulvar 18 months + 8 M 41 LSA Preputial 5 years NO NO + 9 F 69 LSA Vulvar 1 year NO NO + 10 F 28 HEA Neck 11 F 82 PSO Back 16 years 12 M 58 PSO Abdomen 2 years NO NO 13 F 68 MOR' Abdomen, neck; multiple plaques 6 months + 14a F 16 MOF.<. Abdomen, single plaque 10 years + 14b Abdomen, healthy skin 15 M 20 MOR' Lumbar, single plaque, Wac ring 10 years + 16 F 32 MOR Back, single plaque, lilac ring 4 months NO ND + 17 F 57 MOR Trunk; multiple plaques; lilac ring 8 months NO NO + 18 F 47 MOR Disseminated, Pasini - Pierini 2 months NO NO + 19 F 57 MOR Right hip, single plaque 10 years NO NO + 20 F 29 MOR Abdomen, multiple plaques, lilac ring 20 years + 21 M 14 MOR Abdomen, single plaque 1 year + 22 F 60 HEA Back 23 M 14 ECZ Abdomen 18 months NO NO 24 M 62 ECZ Generali zed 2 months NO NO 25 M 25 ECZ Legs, forearm 6 months NO NO • Letters indicate different biopsy specimens obtained from the same patient. ! F, female; M, male. , ECM, ery thema chronicum migrans; ACA, acrodermatitis chronica atrophicans; HEA, healthy skin; LSA, lichen sc1erosus et atrophicus; MOR, morphea; PSO, psoriasis vulgaris; ECe., chronic eczema. J Flagellin ELISA (Dako, Denmark); NO, not done. , Skin lesion(s) developed at the site of a tick bite.

or subacute inflammatory process. None of the patients had been reaction mixture was used for amplification with the internal specifically treated with antibiotics, such as penicillins, cephalospo­ primers 5'-AAGGAATTGGCAGTTCAATC-3' and 5'-ACAG­ rins, or tetracyclines, for suspected spirochetal infection before skin CAATAGCTTCATCTTG-3' [15]. Thirty amplification cycles biopsies were taken. were performed under the conditions described above. Identifi.ca­ tion ofpCR products was done by agarose gel electrophoresis. Posi­ Serologic Tests Serum samples (Table I) were tested for anti­ tive samples gave rise to a 290-bp flagellin subfragment (Fig 1). In a borrelial antibodies by a flagellin enzyme-linked immunosorbent previous experiment, we demonstrated the specificity of the result­ assay (ELISA) [10]. Results were confirmed by Western blot analysis ing product by restriction enzyme analysis [15]. Furthermore, we as previously described [11] . showed that the primers used in this PCR assay do not hybridize peR DNA was extracted from paraffin-embedded tissue samples with the flagellin gene of other bacteria, such as Treponema pallidwll, according to Rogers et al [12]. Briefly, tissue sections were deparaf­ Treponema denticola, Yersillia enterocolitica, and Escherichia co li. As posi­ finized with xylene and ethanol; 15 mg of dried tissue was sus­ tive controls, the B. b~lrgdorferi strains RTI (tick isolate; R. Lange, pended in 1 ml TE9 buffer {0.5 M Tris {hydroxymethylamino­ Berlin, Germany) [11] and B31 (ATCC 35210) were used. To avoid methane (Tris - HCI, pH 9.0; 0.02 M ethylenediaminetriacetic false-positive PCR results, the usual precautions [16] were taken acid (EDTA); 0.01 M NaCI) and digested with 500 ,ug/ml protein­ and in addition to the above-mentioned samples, at least two nega­ ase K (Merck, Darmstadt, Germany) at 55°C overnight. The DNA tive controls (distilled water) were processed in each experiment. In was then extracted by use of phenolchloroform and ether [1 3]. After one single experiment, 10 encoded samples (including water con­ subsequent ethanol precipitation, the DNA pellet was dissolved in trols) were subjected to DNA extraction. DNA amplification was 40,111 TEbuffer{10 mM Tris-HCI,pH 7.6; 2 mMEDTA). 5,ugof performed at least two times on each DNA sample. DNA suspension was used in a nested PCR, with two primer pairs RESULTS derived from the flagellin gene of B. burgdorferi. Amplification was performed in a DNA Thermal Cycler (Poly C hain II; Polygen, Lan­ A specific amplification product of 290 bp was detected in the pa­ gen, Germany). A reaction mixture of 50,111 contained: 50 mmol/I tients with morphea (nine of nine) and LSA (six of six), as well as in KCI; 10 mmol/I Tris-HCI, pH 8.3; 1.5 mmol/I MgCI2 ; 0.01% those withECM (two of two) and ACA (one of one) (Fig 1; Table I). gelatin; 200 mmol/I of each deoxynucleotide triphosphate In contrast, no specific amplification was seen in the control samples (Boehringer Mannheim, Mannheim, Germany). After addition of (samples 10- 12, 14b, and 22 - 25) processed in the same experi­ 1 U Taq (Thermophil us aquatic us) polymerase (Amersham, Ger­ ment (Fig 1; Table I) . Biopsy specimens taken from the edge and many), the mixture was covered with 100,111 mineral oil (Sigma center of ECM lesions and from perilesional skin (patient 3) were Chemical Co., St. Louis, MO). External primers were 5'-CTGCT­ PCR positive. Borrelia DNA was also detected in lesional skin from GGCATGGGAGTTTCT-3' and 5-TCAATTGCATACTCAG­ the patient with atrophoderma of Pasini-Pierini (patient 18). Of TACT-3' [14] . PCR conditions were as follows: denaturation at note, LSA was PCR positive not only in the patients with vulvar or 94 ° C for 30 sec, annealing at 48 ° C for 1 min, and extension at preputial LSA, but also in patient 4 with the extragenital manifesta­ 72°C for 2 min. After 40 cycles, a 5-,111 volume of the fi.rst PCR tion. Serologic tests revealed anti-borrelial antibodies only in the VOL. 100, NO.5 MAY 1993 BORRELIA BURGDORFERJ DNA IN MORPHEA AND lSA 719

1 2 3a 3b 3c 4 5 6 7 8 9 10 11 12 - +* .. bp is unlikely because there was DNA in all lanes (Fig 1), and the DNA resulting from phenol chloroform and ether extraction is very pure. PCR revealed Borrelia DNA in all patients with morphea (9/9) and LSA (6 /6) investigated. PCR was also positive in specimens from seronegative patients (Table I). These findings are in accordance with previous studies that have shown the lack of antispirochetal antibodies in certain forl11s of syphilis [28] and chronic Lyme borre­ liosis [29). Therefore, the presence of B. bllrgdorJeri infection cannot 587 be excluded by the absence of anti-borrelial serum antibodies. 434 In summary. we have shown that B. bllrgdorJeri can be present 290 even in the skin of seronegative patients with morphea and LSA. Thus, persistent spirochetes may contribute to the pathogenesis of morphea and LSA. The skin lesions of three patients with morphea and one with LSA developed at the site of a preceding tick bite (Table I). Because the time from the causative tick bite to the devel­ opment of the scl erotic lesions may be several years apart, it is not 'G 14a14b 15 16 17 18 19202122 2324 25 - +•• II surprising that the connection with a tick bite is rarely made by the patients. Also, it is poss ible that the location of the chronic lesions is distinct from the primary inoculation site: after hematogenous spread. the spirochetes m ay persist at sites of predilection with a lower blood supply and a lower temperature, such as the acral sites of the extremities (A CA), the pinna, the nipples (borreliallym.pho­ cytoma), or the outer genitalia (LSA); however, it remains unclear 587 whether intact B. burgdorferi orga nisms or persisting Borrelia compo­ 434 nents in the tissue are the causative agent of Illorphea and LSA or 290 whether Immunologic features of the infected patients play the predominant role in the pathogenesis of these diseases. W e conclude that patients with a cI inical diagnosis of morphea fi ure L Agarose gel electrophoresis of Borrelia DNA in skin samples and LSA should be further investigated for B. bllrgdoiferi by sensitive ~ controls. (Iall e 1) ACA; (Iau es 2 aud 3a-3c) ECM; (Iall es 4-9) LSA; methods such as PCR, especially in seronegative cases. In the ~~ nes 13 - 21) morphea; (Iau c 14b) normal skin from patient 14; (Idlles 11 present study, a small group of selected patients w ith morphea and \' d 12) psoriasis; (Iall es 23-25) ECM; (Inu es 10 MId 22) nom1al skin; (-) LSA with acute or subacute inflammatory skin lesions were exam­ ::lstilled water; (+*) B. bllrgdotjeri strains RT1;. (+•• ) B. burgd01"eri strain B31; (M) molecular weight marker (MW V; Boehnnger, Mannhelm, Germany); ined. Therefore, investigation of randomly selected larger groups is (bp) base paIr. necessary to provide statistical significa nce for the correlation of Borrelia infection and morphea and LSA. Further studies are re­ quired to determine whether the progression of morphea and LSA leSIOns can be halted by adequate antibiotic treatment in patients with confirmed Borrelia infection. c'ents with ECM (two of two) and ACA (one of one). All inves ti­ P:t~d patients with morphea (five patients). LSA (three patients). gnd psoriasis (one patl ent), as well as the two healthy control sub- 3 . We tJJallk Dr. M. OwsialJol/lski for helpfJ/1 com ments Dlld Dr. R. Soe!lllc"ell for jeets were seronegative. cooperalioll ill collectillg tissue specimells.

DISCUSSION REFERENCES Immunologic factors [17 - 19) as well as a genetic predisposition 120,21] and hormonal factors [22J have been discussed as being 1. Aberer E, Stanek G: Histological evidence for spirochetal origin of involved in the pathogenesIs of morphea and LSA. An altered rate of morphea and lichen sclerosus et atrophicans. Am J Dermatopathol 9:374-379, 1988 collagen synthesis has been reported 111 morphe:1 [23]. The ll1vol~ e ­ ment of intercellular matrIX molecules 111 el astIc tlssue destructlon 2. Ross SA, Sanchez )L, Taboas )0: Spirochetal forms in the dermal in LSA has also been discussed [24]. Although the coexistence of leS IOns of morphea and lochen sclerosus et atrophicus. Am] Derma­ topathol 12:357 - 362, 1990 JIlorphea and LSA is often reported [25,26)' It IS stili debatable hether they are both involved in a related pathologic process [27]. 3. Schempp C, Bocklage H, Owsianowski M, Lange R, Orfanos CE, ;ecent investigations have revealed Borrelia organisms in lesional Golll11ck H: Detection of Borrelia infection by polymerase cham reaction, immunohistochemistry and ce llular immune response in a skin from patients with morphea and LSA and thu.s support a possi­ case of seronegative morphea like skin les ion. Hautarzt 44:14 - 18, bl e etiologic relationship between these two sklll dI sorders [1,2,4.8]. 1993 In one case of seronegative morphea. we were able to demonstrate 4. Aberer E, Klade H, Stanek G, Gebhart W: Borrelia bllrgdotjeri and Borrelia antigen by immunohistochemistry and PCR analysis, as different types of morph ea. Dermacologica 182: 145 -154, 1991 well as active infection by a T -cell assay [3]. To confirm our data 5. Muhlemann MF, Wright DJM, Black C: Serology of Lyme disease. from this c ase in a larger patient group, we performed a nested PCR Lancet 1:5 53 - 554, 1986 using f1agellin gene - specific primers. 6. Hoesly ]M, Mertz LE. Winkelmanll RK: Localizcd scleroderma (mor­ Because we used nes ted oligonucleotide primers and have investi­ phea) and antibody to Borrelia burgdotjeri. J Am Acad Dcrmatol gated their specificity [15]. false-positive re s ~tlt s with these primers 17:455 - 458. 1987 ~e very unlikely. In the present study, we did not amplify all sam­ 7. Vaillant L, Goudeau A: Localized scleroderma is nor 3 Borrelia bllrgdor­ ples with an internal positive control. s uch as prin:ers . for cellular Jeri infcction in Francc (letter). Dermatology 184:286, 1992 DNA. (This is commonly practiced III the quantltatlOn of Viral 8. W eber K, Preae-Mursic V, Reimers CD: Spiroc hetes iso lated from rwo ge nome to show that the samples can be amplifi ed w.ith another set paticnts with morphea. Infection 16:25 - 26, 1988 of primers.) Therefore. our negative results theoretically could be 9. Melchers W , Mcis J, Rosa P, Claas p, Nohlmanns L, Koopman R, false-negatives because they could be not ampldiable; however, tillS Horrevorts A, Ga Jama J: Amplili cation of Borrelia burgdotje ri DNA 720 SCHEMPP ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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