British Journal of Dermatology 2001; 144: 387±392.

Isolation and polymerase chain reaction typing of Borrelia afzelii from a skin lesion in a seronegative patient with generalized ulcerating bullous lichen sclerosus et atrophicus

F.BREIER,² G.KHANAKAH,³ G.STANEK³ G.KUNZ,* E.ABERER, B.SCHMIDT AND G.TAPPEINER* Department of Dermatology, Lainz Municipal Hospital, Wolkersbergenstraûe 1, A-1130 Vienna, Austria *Department of Dermatology and ³Hygiene Institute, University of Vienna Medical Faculty, Vienna, Austria ²Department of Dermatology, University of Graz Medical Faculty, Graz, Austria Accepted for publication 24 August 2000

Summary A 64-year-old woman presented with bullous and ulcerating lichen sclerosus et atrophicus (LSA) on the neck, trunk, genital and perigenital area and the extremities. Histology of lesional skin showed the typical manifestations of LSA; in one of the biopsies were detected by silver staining. Despite treatment with four courses of ceftriaxone with or without methylprednisone for up to 20 days, progression of LSA was only stopped for a maximum of 1 year. Spirochaetes were isolated from skin cultures obtained from enlarging LSA lesions. These spirochaetes were identified as Borrelia afzelii by sodium dodecyl sulphate±polyacrylamide gel electrophoresis and polymerase chain reaction (PCR) analyses. However, serology for B. burgdorferi sensu lato was repeatedly negative. After one further 28-day course of ceftriaxone the lesions stopped expanding and sclerosis of the skin was diminished. At this time cultures for spirochaetes and PCR of lesional skin for B. afzelii DNA remained negative. These findings suggest a pathogenetic role for B. afzelii in the development of LSA and a beneficial effect of appropriate antibiotic treatment. Key words: Borrelia afzelii, Borrelia burgdorferi, isolation, lichen sclerosus et atrophicus, polymerase chain reaction, spirochaetes

Spirochaetes of the genus Borrelia were identified in the sensu lato have been demonstrated in patients with early 1980s1,2 as the pathogens causing Lyme LSA.13±19 However, the incidence and significance of borreliosis, a multisystem disease involving the skin, Borrelia in LSA is still a matter of debate, as no joints and the cardiopulmonary and central nervous trace of the organism was demonstrated in several systems. They comprise a group of organisms desig- other studies of such patients.20±24 Furthermore, a nated B. burgdorferi (Bb) sensu lato, including Bb sensu plethora of other aetiological factors of LSA have been stricto and several other closely related spirochaetes discussed: genetic, vascular, immunological, mechani- (B. afzelii, B. garinii and further strains that are mostly cal, chemical, metabolic, endocrine, psychological and not human pathogens). The strains of Bb sensu lato infectious agents have been described to be involved in are distinguished by their individual pattern of DNA the pathogenesis of LSA, usually based on scant or sequences.3,4 The chronic cutaneous disease acro- spurious evidence.11,12 We report the first patient dermatitis chronica atrophicans (ACA) is now known with genital and extragenital LSA from whose lesional also to be caused mostly by B. afzelii.5±10 skin spirochaetes were cultivated and identified by Another chronic skin disease, lichen sclerosus et polymerase chain reaction (PCR) as B. afzelii. atrophicus (LSA),11,12 was also shown to be associated 13±16 with a Bb infection by direct and indirect tech- Case report niques.17±19 During the last decade, with Bb In 1991, a 64-year-old woman presented with sclerotic Correspondence: Dr Friedrich Breier. skin lesions on the trunk and neck. The lesions con- E-mail: [email protected] sisted of polycyclic ivory-coloured patches and plaques q 2001 British Association of Dermatologists 387 388 F.BREIER et al.

Figure 1. Genital and extragenital lichen sclerosus et atrophicus with marked ulceration.

(Fig. 1). She lived in a wooded area near the city of Vienna, which is endemic for Lyme borreliosis and tick- borne encephalitis; she enjoyed outdoor activities. Lesions had been progressing slowly since 1990, and she had noticed neither tick bites nor skin lesions indicative of erythema chronicum migrans. Histo- pathology of a skin biopsy taken from the right thigh was performed in April 1991 according to standard procedures, using haematoxylin and eosin (Fig. 2a) or Bosma±Steiner silver stain (Fig. 2b).25 It showed , an oedematous papillary , homo- genized collagen bundles in the reticular dermis and a perivascular and interstitial infiltrate of and plasma cells. Spirochaetes were apparent in the dermis next to collagen bundles in Bosma±Steiner- stained sections (Fig. 2b).25 At this time, culture of the skin biopsy specimen was positive. After a course of ceftriaxone 2 g twice daily for 20 days the induration of the lesions diminished and the hue faded. After treat- ment, culture and histology were negative for spiro- chaetes. The lesions stopped progressing for 6 months. Thereafter, the patient developed further lesions on the neck and trunk and new lesions on the . A further course of intravenous ceftriaxone 2 g twice daily and oral 6-methylprednisolone (initial dosage 40 mg daily) was given for 3 weeks. The lesions again improved markedly and remained unchanged for 1 year. In Figure 2. (a) Dermatopathology of lichen sclerosus et atrophicus. 1992, the patient developed vulval as well as Note the prominent homogenization of the papillary dermis and the bullous and erosive LSA lesions in the axillae and inflammatory infiltrate consisting of lymphocytes and plasma cells (haematoxylin and eosin; original magnification 80). (b) Human groins. Â The skin lesions again were histopathologically typi- skin infected with Borrelia burgdorferi sensu lato. A is shown in the extracellular matrix close to collagen bundles (arrow) cal for lichen sclerosus but also showed pronounced (modified Steiner's silver stain; original magnification 280). dermal sclerosis extending to the deep dermal tissue. A Â further course of ceftriaxone 2 g twice daily for 10 days histologically proven adenocarcinoma of the left ovary, led to a slight improvement of the skin; the perilesional a breast cancer and cholecystolithiasis; these condi- erythema faded, but this might not have been related to tions were successfully treated and the patient remains the antibiotic treatment. Routine check-up disclosed a free of malignant disease.

q 2001 British Association of Dermatologists, British Journal of Dermatology, 144, 387±392 BORRELIA IN ULCERATING BULLOUS LSA 389

One year later she developed further bullous and 4 weeks and in the fifth subculture 1 week later. The erosive lesions of LSA on the neck, trunk, vulva and organisms were identified by the methods described additionally on the legs. Ceftriaxone 2 g daily was below. administered for 10 days and oral 6-methylpredniso- lone 40 mg daily for 1 week. During treatment the Strain identification: Polymerase chain reaction lesions stopped expanding and the ulcerations healed. The skin lesions then remained unchanged for a In order to identify the species of the cultured organ- further year. isms, they were harvested by centrifugation and pre- In 1994, skin contractures developed in the left pared for analysis by PCR. Briefly, washed pellets were axilla and led to impaired mobility of the humero- boiled for 10 min and reacted with several sets of scapular joint; these were treated by excision and PCR primers in order to recognize target DNA of the grafting. After an additional course of ceftriaxone 2 g bacterial chromosome. The primers selected were those daily for 28 days the disease again went into remission described by Marconi and Garon.29 First, strains were and has remained stable to date. A skin biopsy from probed with the LD primer set (ATGCACACTTGGTGT- a residual lesion taken in January 1999 showed TAACTA and GACTTATCACCGGCAGTCTTA) which increased cellularity of the connective tissue as gives a PCR product of 357 bp with all compared with the previous biopsies and a normal borrelia. Then, for species differentiation, we used the distribution of inflammatory cells in the dermis; PCR primer set BB (GGGATGTAGCAATACATTC and ATA- and skin culture for Bb sensu lato have remained TAGTTTCCAACATAGG) to identify Bb sensu stricto, the negative. primer set BG (GGGATGTAGCAATACATCT and ATA- TAGTTTCCAACATAGT) to identify B. garinii, and the primer set BA (GCATGCAAGTCAAACGGA and ATA- Serological studies TAGTTTCCAACATAGC) to identify B. afzelii. The BB Serological tests for Bb were performed every and BG primer sets result in a PCR product of 574 bp, year from 1991 to 1994 by enzyme-linked immuno- and the BA primer set yields a 591-bp product. The sorbent assay (ELISA), using the supernatant of a Borrelia strain isolated from the skin of our patient was mixture of sonicated B. afzelii and B. garinii as antigen. identified as B. afzelii. Negative reactions were obtained Peroxidase-conjugated goat antihuman IgG with B. burgdorferi sensu stricto and B. garinii primers. (Nordic Immunological Laboratories, Stockholm, Fig. 3 shows PCR results of the LSA skin isolate (lane 3) Sweden) was used as reactant.26 The results were as compared with B. afzelii H2. analysed with a 400 AT photometer (SLT Laboratory Instruments, Salzburg, Austria) at a wavelength of Detection of Borrelia burgdorferi DNA in the urine 492 nm. The results were considered positive when a serum sample at a dilution of 1 : 1000 yielded an Urine samples30 were subjected to PCR amplification absorbency of . 0´250. Blood samples were drawn for the Bb flagellin gene,30 and for the genotype-specific from our patient and were negative at the baseline and detection of Bb sensu stricto, B. garinii and B. afzelii. at all further investigations, indicating that the con- Extraction of DNA and nested PCR were performed centration of antibodies to Bb sensu lato was as low as in according to a method recently described.30 PCR of healthy individuals. The results obtained by ELISA were urine samples was negative twice in 1994. confirmed by immunoblotting. Discussion Borrelia culture European31 and North American32 clinicians have The procedure for Borrelia cultures has recently been thought for quite some time that treatment with described in detail.27,28 In short, skin biopsy specimens b-lactam antibiotics can lead to the remission of active were taken from the periphery of clinically active disease and improvement of sclerosis in some patients lesions after surface disinfection and cultured for with LSA. This is the first report of the isolation of 4 weeks at 34 8C in Barbour±Stoenner±Kelly (BSK) II B. afzelii from skin lesions of a patient with LSA. The medium. The growth of spirochaetes was assessed by aetiological link is supported by the fact that our dark field microscopy ( 200) every 4 days. Spiro- patient's disease finally responded to treatment with  chaetes were observed in the original BSK medium after b-lactam antibiotics. A remission, now lasting for more q 2001 British Association of Dermatologists, British Journal of Dermatology, 144, 387±392 390 F.BREIER et al.

by some investigators,14,16 but not by others.22±24 Recently, DNA of B. afzelii and B. garinii was identified in skin biopsies of LSA patients from Germany and Japan, but not from U.S. patients.14 These data corro- borate our findings and underline the possibility that LSA may be induced by B. afzelii. Besides elevated antibody titres,6,18,19 a specific cellular immune response to Bb was demonstrated in two patients from Switzerland with concomitant LSA and localized .35 LSA and circumscribed scleroderma (CS) share clinical and histopathological characteristics but are thought to represent separate entities.11,12 Further- more, coexistence of LSA and morphoea has been observed in several patients.35 Recently, a spirochaetal infection5,13,35,36 was demonstrated in a subset of patients with CS by the presence of cellular35 and/ or humoral13,35 response to Bb sensu lato,orbythe observation,35 detection14,16 or even isolation28 of Borrelia from lesional skin. However, these findings could not be confirmed by other authors.35,37 LSA has also been found in six of 50 patients with ACA7 and in two patients with suspected neuro- borreliosis.10 In another study, one patient had Borrelia-associated diffuse fasciitis with eosinophilia 38 Figure 3. Agarose gel electrophoresis of the 16S rDNA specific and morphoea in addition to LSA. In all these polymerase chain reaction product of strain lichen sclerosus et patients borreliosis was confirmed by elevated serum atrophicus. Lanes 1 and 5: 100 bp DNA standard (MBI Fermentas, antibody levels; other authors could not find a humoral Germany). Lane 2: positive control, Borrelia afzelii H2. Lane 3: strain 20,21 LSA. Lane 4: negative control. response to Bb in LSA patients. Our patient had neither IgG nor IgM serum antibodies to Bb, but even chronic Lyme borreliosis may remain seronegative.36 than 8 years, was achieved: disease progression was In addition, the negativity of PCR detection of Bb DNA stopped, ulceration healed and sclerosis partially in the urine can be explained, as a third of patients with resolved after she had received a 4-week course of LSA were shown to be negative in a previous study.30 ceftriaxone; while beneficial generally, the additional The exact causes of LSA are still not known, but administration of systemic steroid in two of the treat- various factors beyond the infectious ones are very ment courses may also have had an anti-inflammatory probably involved. Bb can induce interleukin (IL) -2- effect. The relapses she repeatedly suffered despite dependent cutaneous sclerosis;39 this cytokine was initially successful antibiotic treatment could be related shown to induce fibroblasts to liberate elastase leading to the observation that Borrelia may possibly be able to to lysis of elastic fibres in LSA lesions. Furthermore, Bb remain dormant in certain tissue compartments,33 can invade human skin fibroblasts40 and even human thus escaping bactericidal antibiotic activity. This lymphocytes, inducing cytopathic killing of mononuc- would be consistent with the fact that these relapses lear cells in vitro.41 The invasion of Bb into human skin were always able to be treated successfully with a fibroblasts may lead to an impaired metabolism of course of the same antibiotics as before; this is these connective tissue cells. There may be further corroborated by a recent report that Bb may persist immunobiological links between Bb infection and the in experimentally infected dogs despite antibiotic treat- development of subtypes of LSA: Bb was shown to ment with doxycyclin or amoxycillin.34 induce IL-1 production in LSA lesions as well as large Several investigators have found spirochaetes in histo- quantities of it in human macrophages both in vitro and logical sections of LSA skin,15 as we have in our patient. in vivo.41±43 The latter may lead to sclerosis with or Bb DNA has been detected in LSA skin biopsies by PCR without autoimmune phenomena.11,12,35

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We have presented strong evidence for an association sclerosus et atrophicus tissues of German and Japanese but not of of Bb infection and LSA in our patient: even though she US patients. Arch Dermatol 1997; 133:41±4. 15 Ross SA, Sanchez JL, Taboas JO. Spirochetal forms in the dermal was seronegative for Bb, spirochaetes were found in lesions of and lichen sclerosus et atrophicus. Am J histopathology slides from biopsies, and Bb was culti- Dermatopathol 1990; 12: 357±62. vated in lesional tissue and characterized by sodium 16 Schempp C, Bocklage H, Lange R et al. Further evidence for dodecyl sulfate±polyacrylamide gel electrophoresis and Borrelia burgdorferi infection in morphea and lichen sclerosus et atrophicus confirmed by DNA amplification. J Invest Dermatol PCR. This report is not meant to imply that LSA is 1993; 100: 717±20. always caused by Borrelia. It does, however, emphasize 17 Aberer E, Neumann R, Lubec G. Acrodermatitis chronica that these patients may benefit from adequate, pro- atrophicans in association with lichen sclerosus et atrophicans: longed antibiotic therapy. The rarely occurring sclerosis tubulo-interstitial nephritis and urinary excretion of spirochete- in ACA may be clinically and histopathologically like organisms. Acta Derm Venereol (Stockh) 1987; 67:62±5. 7 18 Garbe C. Borrelia infections of the skinÐprogress of knowl- indistinguishable from linear LSA. In order to eluci- edge since the discovery of Lyme disease. Hautarzt 1991; 42: date an underlying Bb infection in LSA patients, these 356±65. individuals should be evaluated using cultures from 19 Tuffanelli D. Do some patients with morphea and lichen sclerosus skin biopsies, serology and PCR studies. Thus, addi- et atrophicans have a Borrelia infection? Am J Dermatopathol 1987; 9: 371±3. tional epidemiological information will become avail- 20 Afa G, Caprilli F, Crescimbeni E et al. Anti-Borrelia burgdorferi able and diagnostic and therapeutic strategies for LSA antibodies in chronic , benign lymphadenosis will improve. cutis, scleroderma and scleroatrophic lichen. G Ital Dermatol Venereol 1990; 125: 369±73. 21 Alonso-Llamazares J, Persing DH, Anda P et al. No evidence for Borrelia burgdorferi infection in lesions of morphea and lichen References sclerosus et atrophicus in SpainÐa prospective study and literature review. Acta Derm Venereol (Stockh) 1997; 77: 1 Burgdorfer W, Barbour AG, Hayes SF et al. Lyme disease: a tick- 299±304. borne spirochetosis? Science 1982; 216: 1317±19. 22 ColomeÂ-Grimmer MI, Payne DA, Tyring SK, SaÂnchez RL. Borrelia 2 Steere AC, Grodzicki RL, Kornblatt AN et al. The spirochetal burgdorferi DNA and Borrelia hermsii DNA are not associated etiology of Lyme disease. N Engl J Med 1983; 308: 733±40. with morphea or lichen sclerosus et atrophicus in the south- 3 Postic D, Assous M, Belfaiza J et al. 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