Sclerosing and granulomatous lesions 
SCLEROSING AND GRANULOMATOUS 
SKIN LESIONS 
IN BORRELIA BURGDORFERI INFECTION 
F. Breier, B. Schmidt, B. Crowe, 
G. Stanek and E. Aberer 
ABSTRACT 
The pathogenetic linkage between Bo1Telia burgdo,feri and the cutaneous manifestations of Lyme Borreliosis, 
i.e. erythema migrans, borrelial lymphocytoma and acrodermatitis chronica atrophicans is well established. 
Moreover, in three other dermatoses - circumscribed scleroderma, lichen sclerosus et atrophicus and 
granuloma annulare - laboratory findings indicated that these diseases might be associated with a Bo1Telia 
burgdo,feri infection. In particular, cellular or humoral immune responses to Bo1Telia burgdo,feri, as measured 
by lymphocyte proliferation tests or by immunoblot investigations, respectively, were detected in some 
circumscribed scleroderma patients. In sporadic cases Bo1Telia burgdo,feri was even isolated from lesional skin 
of patients with circumscribed scleroderma or granuloma annulare. Recently developed molecular biological 
techniques provide tools to detect Bo1Telia burgdo,feri-DNA in severa! tissues and body fluids. Particularly the 
excretion of bo1Telia-DNA in the urine, detected by PCR, was proven to be a useful and reliable laboratory 
parameter far the clinical correlation of disease activity in erythema migrans and acrodermatitis chronica 
atrophicans. Therefare, we investigated the excretion of Bo1Telia burgdo,feri-DNA in the urine of patients with 
circumscribed scleroderma, lichen sclerosus et atrophicus and granuloma annulare by amplification of a 276 
bp flagellin gene segment using a nested PCR technique. Surprisingly, PCR was positive in 61 % of patients 
with circumscribed scleroderma, 68% of patients with lichen scleroses et atrophicus and 61 % of patients with 
granuloma annulare. These data provide evidence far the presence of a systemic Bo1Telia burgdo,feri infection 
in a subset of patients with these dermatoses and open up a field to develop new concepts in the treatment 
of these patients. 
KEY WORDS 
Bo1Telia burgdo,feri, circumscribed sclerodenna, lichen sclerosus et atrophicus, granuloma annulare, 
immunoblot-investigation, cellular immune response, polymerase chain reaction, urine 
INTRODUCTION 
The clinical spectrum of systemic and localized 
manifestations of Lyme Borreliosis (LB) increases 
steadily but has not yet been defined precisely. 
acta dermatovenerologica A.P.A. Vol 5, 96, No 3-4 
Beside the classical cutaneous manifestations of LB, 
erythema migrans (EM), borrelial lymphocytoma (BL) 
and acrodermatitis chronica atrophicans (ACA), further 
dermatoses, such as circumscribed scleroderma 
(morphea) (CS), lichen sclerosus et atrophicus (LSA) 
117 
Sclerosing and granulomatous lesions 
and granuloma annulare (GA) have been linked to 
a Borrelia burgdorferi (Bb) infection (1,2). Upon 
staining with anti-Bb polyclonal antibodies, borrelia-
Iike organisms were detected in the skin in these 
three dermatoses by immunoperoxidase methods (3,4). 
Cultivation of Bb was reported in sporadic cases of 
CS and GA (2,5). In earlier studies 30 patients with 
different types of CS were tested for serum antibodies 
against Bb determined by ELISA and Westernblot 
analysis (1,6). Bb B 31 sensu stricto was used as an 
antigen for SDS-electrophoresis. Forty-six percent of 
CS patients were IgG seropositive based on ELISA. 
W esternblot examinations confirmed the ELISA results 
in 10 out of 25 patients showing an antibody profile 
which can also be seen in the course of LB. The 
Bb seropositive patients could mostly be associated 
with a symmetric-plaque and linear-systemized type 
of morphea. In further experiments, the cellular 
immune response to Bb was investigated (7). When 
isolated peripheral blood mononuclear cells from 
CS patients were incubated with Bb, genospecies 
garinii (Bg), proliferation of these cells was induced 
in 11 (5 seropositive and 6 seronegative) out of 39 
patients. 
More recently, molecular biological techniques, 
such as PCR, made the detection of Bb-DNA in 
the skin and body fluids possible (8,9). As demon-
strated in severa! studies, Bb organisms have a high 
affinity to the urine bladder tissue and Bb-antigens 
and DNA are secreted in the urine during the 
course of LB (10-12). 
In this paper we focuse on the correlation between 
different in vitro methods for laboratory diagnosis 
of Bb infection in CS, LSA, and GA. The data 
presented bere were partly published previously 
(1,7,13,14) and are reviewed in this paper. 
MATERIALS AND METHODS 
LYMPHOCYTE TRANSFORMATION TESTS AND 
IMMUNOBLOTS IN PATIENTS WITH CIRCUM-
SCRIBED SCLERODERMA 
In a first series of experiments lymphocyte trans-
formation tests (LTT) were performed in 39 patients 
with different types of CS as previously described 
(7) . In brief, 105 peripheral blood mononuclear cells 
of patients were incubated with 106 Bg organisms 
for 5 days. Eighteen hours before harvesting, 3H 
thymidine was added and its uptake by mononuclear 
cells measured in a liquid scintillation counter. The 
counts were expressed as distintegrations per minute 
118 
and a stimulation index (SI) was calculated. Control 
experiments were carried out in 6 healthy individuals, 
16 patients with non-LB-associated skin diseases and 
in patients with EM and ACA. 
lgG-immunoblots in 
patients with morphea 
'''C '''' ' '' 
.. 
~ II! 
.,. 
.., ,,,,, 
"""' .... ..... ... ...... 
"" 
"' 189 kd 
~ 
60 kd 
f lag 
39kd 
i OspC 
Fig. l. JgG-immunoblots of sera from 18 patients with 
circumscribed scleroderma. 
Right lane: Molecular weight standards; C: Control 
serum of an acrodermatitis chronica atrophicans patient. 
Arrows at the top indicate the patient's sera with 
positive lymphocyte transformation tests; arrows at the 
bottom indicate positive immunoblots in lymphocyte 
transf ormation test positive patients. Curved line s indicate 
paired sera taken from circumscribed scleroderma patients 
before and after antibiotic treatment. 
Immunoblot (surfblot, IDEA Scientific Company, 
Minneapolis, MN, U.S.A.) (IB) studies were 
performed from sera of 18 patients with CS 
(unpublished data). In 3 of these patients samples 
before and after treatment were examined. Paired 
samples were examined for LTT and 1B studies in 
14 patients. A membrane fraction of Bb, genospecies 
afzelii (Ba ), was applied on a SDS - polyacrylamid 
acta dermatovenerologica A.P.A. Vol 5, 96, No 3-4 
Sclerosing and granulomatous lesions 
electrophoresis gel. After separation, proteins were 
transferred to nitrocellulose membranes. The patients' 
sera, in a dilution of 1:250 for IgG and 1:10 for 
IgM detection, were applied to these strips and the 
bound antibodies detected by alkaline phosphatase 
conjugated goat anti-human immunoglobulin G or 
M. To determine relevant antigens, sera obtained 
from patients with the acute, disseminated and chronic 
phase of LB as well as from patients with ACA and 
healthy blood donors were also tested ( data not 
shown). Based on the statistical evaluation of the 
results obtained from patients and healthy blood 
donors seropositivity criteria of four or more reacting 
bands for IgG and three or more bands for IgM 
were established. 
PCR INVESTIGATIONS IN PATIENTS WITH CIR-
CUMSCRIBED SCLERODERMA, LICHEN SCLE-
ROSUS ET ATROPHICUS AND GRANULOMA 
ANNULARE 
In a further series of experiments the urine excretion 
of Bb-DNA was investigated in 63 patients, 31 with 
CS , 19 with LSA and 13 patients with GA 
(unpublished data). By means of a nested PCR, a 
276 bp flagellar gene sequence of Bb-DNA was 
amplified as previously described (9). In addition, 
antibodies against Bb were investigated in the sera 
of these 63 patients by ELISA (7). 
CULTURE OF BORRELIA BURGDORFERI IN 
PATIENTS WITH CIRCUMSCRIBED SCLERO-
DERMA AND LICHEN SCLEROSUS ET ATRO-
PHICUS 
Punch biopsies (4mm) were taken from the 
expanding border of CS or LSA skin lesions and 
cultured in a Barbour-Stoenner-Kelly medium (6). 
RESULTS 
CORRELATION OF POSITIVE LYMPHOCYTE 
TRANSFORMATION TESTS AND IMMUNO-
BLOTS IN PATIENTS WITH CIRCUMSCRIBED 
SCLERODERMA 
Peripheral blood mononuclear cells from 11 out 
of 39 patients (28%) showed an elevated SI when 
exposed to Bb antigens as compared to samples of 
healthy control persons. 4-20 bands were detected 
in blots of CS patients, whereas IBs presented more 
than 20 bands in ACA. IBs were positive in 13 out 
of 18 CS serum samples (72% ), in 3 patients for 
both IgM and IgG, in 9 patients for IgG and in 1 
patient for IgM antibodies only. In 3 patients 2 
samples were examined before and after treatment 
with almost identical bands. Seven out of 14 patients 
(50%) where LTTs and IBs were performed 
simultaneously, revealed positive results in both test 
systems (Figs. 1,2). The type of CS in these patients 
belonged to a symmetric-plaque type. Moreover, 4 
patients were positive in LTTs and 3 in IBs only. 
NESTED PCR FOR DETECTION OF BORRELIA 
BURGDORFERI FLAGELLIN-GENE FRAGMENTS 
AND SEROLOGICAL FINDINGS AS DETERMI-
NED BY ELISA IN PATIENTS WITH CIRCUM-
SCRIBED SCLERODERMA, LICHEN SCLEROSUS 
ET ATROPHICUS AND GRANULOMA ANNUIARE 
Whereas in patients with EM, the early phase of 
LB, borrelia-DNA is excreted in the urine in about 
Table. Culture of Borrelia burgdorferi from skin biopsies of patients with circumscribed scleroderma and lichen 
sclerosus et atrophicus. 
Pat. Sex Diagnoš,is Clinical , Type Borrelia genospecies 
1 49 M cs1 linear-systemized B. burgdorferi sensu lato 
2 45 M cs1 linear-systemized B. afzelii 
3 55 M cs1 symmetric-plaque B. afzelii 
4 69 F LSA2 symmetric-plaque B.afzelii 
and gential area 
1 = circumscribed scleroderma 
2 = lichen sclerosus et atrophicus 
acta dermatovenerologica A.P.A. Vol 5, 96, No 3-4 119 
Sclerosing and granulomatous lesions 
90% (9), 19 out of 31 (61 % ) patients with CS were 
positive by PCR. Four of these patients were also 
seropositive, whereas 3 other patients were seropositive 
but negative in their urine PCR. In 9 patients no 
Bb-specific laboratory data could be evaluated. 
LSA patients tested PCR positive in 13/19 ( 68% ), 
7 out of these 13 patients were also seropositive, 1 
additional patient was seropositive but PCR-negative. 
In GA borrelia-DNA excretion was detectable in 
8/13 (61 % ) patients; only one of these patients was 
also seropositive. 
lgM-immunoblots in 
patients with morphea 
' 'c fffY f ff 
• 
kd 
kd 
flag 
39kd 
OspC 
Fig. 2. IgM-immunoblots of sera from 18 patients with 
circumscribed scleroderma. 
Right lane: Molecular weight standards; C: Control 
serum of an acrodermatitis chronica atrophicans patient. 
Arrows at the top indicate the patient's sera with 
positive lymphocyte transformation tests; arrows at the 
bottom indicate positive immunoblots in lymphocyte 
transformation test positive patients. Cwved lines indicate 
paired sera taken from circumscribed scleroderma patients 
before and after antibiotic treatment. 
120 
CULTURE OF BORRELIA BURGDORFERI IN 
PATIENTS WITH CIRCUMSCRIBED SCLERO-
DERMA AND LICHEN SCLEROSUS ET ATRO-
PHICUS 
As previously published, Bb was cultivated from a 
patient with a linear-systemized type of CS (6). This 
borrelia isolate was identified as Bb by immunofluo-
rescence-methods using monoclonal antibodies, but 
could not be further typed. In following culture 
trials Bb, genospecies Ba as identified by serotyping 
with monoclonal antibodies and by PCR-methods 
(7), could be isolated from two further CS and one 
LSA patient (unpublished data, see Table). 
DISCUSSION 
The etiology of certain dermatoses affecting the 
connective tissue, such as CS, LSA and GA, is still 
unknown. CS, which can be induced by drugs, 
mechanical and immunological factors, involves the 
dermis, subcutaneous fat and fascia leading to fibrosis 
of collagenous tissue (15). LSA is also a disease of 
unknown origin. It can be observed in genital and 
extragenital skin (16). The coexistence of CS and 
LSA in several patients favours the assumption that 
both conditions may be caused or triggered by 
analogous agents. Many reports have focused on 
their possible relationship to a Bb infection, however, 
contradicting data was published (1,17). Based on 
serological and molecular biological studies, suspected 
borrelia-induced CS and LSA have only been reported 
in European countries (18,19,20) . The association 
with a Bb infection was more likely to be observed 
in the symmetric-plaque and linear-systemized type 
of morphea as evaluated by IB and LIT investigations 
(1,7). In recent studies patients with atrophodermia 
Pasini-Pierini, the atrophic variant of CS, were mostly 
seronegative but showed positive LIT and positive 
Bb-DNA in their urine in single cases (7, unpublished 
data) . 
When comparing laboratory data of patients with 
different types of CS showing humoral or cellular 
immunity to Bb, 7 out of 14 patients (50%) were 
positive in both tests. Whereas LTTs are not 
performed routinely, IB-investigations are well esta-
blished for the diagnosis of LB. Similar to serological 
testing, however, the standardization of methods as 
well as the standardization of interpreting results 
have not yet been achieved. The main reason for 
this fact is the heterogeneity of borrelia strains and 
the variety of antigen expression which is elicited in 
the different manifestations of LB (21). Currently, 
acta dermatovenerologica A.P.A. Vol 5, 96, No 3-4 
Sclerosing and granulomatous lesions 
the Centers for Disease Control and Prevention are 
developing criteria for interpretation of Western 
blot results (22). 
Isolation of Bb from affected tissue is the most 
characteristic evidence and proof of infection. Three 
cases have been published so far where Bb was 
cultivated from a skin biopsy of CS. According to 
the clinical aspect, one patient was suffering from a 
linear-systemized type of CS (6), the two other 
reported patients from a large, partly symmetric-
plaque type of CS, one of which with coexisting 
LSA (5). It is interesting to note that in our 
studies, a further culture-positive patient showed a 
linear-systemized type of CS, one patient a symmetric-
plaque type on the trunk and an additional patient 
symmetric patches of LSA on the trunk. These 
findings, together with the high excretion rate of 
Bb-DNA in the urine, and the improvement of CS 
and LSA in some patients due to betalactam antibotics, 
underline the pathogenetic role of Bb in a subset of 
patients. 
GA was said to be caused by mechanical, auto-
immune and metabolic factors (23-25). In addition, 
infectious agents, such as viruses, were reported as 
pathogens. The development of GA was also observed 
after insect bites (23). In the literature Bb was 
isolated from a GA skin lesion of a patient with a 
history of a tick bite, arthralgias, fatigue, headache 
and seroconversion during the course of the disease 
(2). The isolated strain was of the Osp A serotype 
2 (Ba) (26). Intravenous treatment with ceftriaxone 
resulted in a remission of the clinical symtoms and 
also of GA 5 months after treatment. In our study 
a high percentage of patients with GA (61 % ) showed 
Bb-DNA excretion in the urine. An association with 
arthralgias, and a tick or insect bite was also reported 
by PCR-positive patients. 
In summary, we have shown that various laboratory 
data point to a Bb infection in a considerable 
number of patients with CS, LSA and GA In the 
future, more sensitive laboratory studies are needed 
to confirm these findings and to explore the under-
standing of pathogenetic linkages between Bb and 
sclerosing and granulomatous dermatoses. 
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AUTHORS' ADDRESSES 
122 
Friedrich Breier, MD, Department of Dermatology, Division of General Dermatology, University of 
Vienna Medical School, Wahringer Giirtel 19-20, A-1090 Vienna, Austria 
Bruno Schmidt, MD, Ludwig Boltzmann Institute for Dermato-Venerological Serodiagnosis, 
Lainz Municipal Hospital, Wolkersbergenstrasse 1, A-1130 Vienna, Austria 
Brian Crowe, PhD, Immuno AG, UferstraBe 15, A-2304 Orth, Austria 
Gerold Stanek, professor, MD, Hygiene Institute, University of Vienna Medical School, 
Kinderspitalgasse 15, A-1090 Vienna, Austria 
Elisabeth Aberer, ass.-professor, MD, Department of Dermatology and Venerology, 
University of Graz, Auenbruggerplatz 8, A-8036 Graz, Austria 
acta dennatovenerologica A.P.A. Vol 5, 96, No 3-4