/solation of Borrelia burgdorferifrom skin 
C/inica/ st11dy 
ISOLATION OF BORRELIA BURGDORFERI FROM SKIN IN 
PATIENTS WITH ERYTHEMA MIGRANS, UNSPECIFIC 
SKIN LESIONS AND GRANULOMA ANNULARE 
F. Strle, E. Ružic, V. Preac-Mursic, J. Cimperman, B. Wilske 
ABSTRACT 
The report includes 5 patients in whom Borrelia burgdorferi was isolated from the skin. One patient had typical erythema 
migrans, another one had skin changes, which corresponded to the early. homogeneous erythema migrans, and two patients had 
non-specific skin lesions at the site of an insect bite, lasting for severa! months. Borrelia antibodies were not present in the blood 
of any of our patients. nor did any of them report a tick-bite. Also described is a case of B. burgdorferi isolated from a granuloma 
annulare. The isolated Borreliae belong to serotypes 1, 4, and 6. 
KEYWORDS: 
Lyme borreliosis, Borrelia burgdorferi, cultivationfrom skin biopsies, non-typical skin lesions, granuloma annulare 
INTRODUCTION 
In Europe and in the USA, Lyme borreliosis is the most 
widespread disease being transmitted by ticks (1,2). The 
same disease is also to be found in Slovenia (3). The term 
Lyme was taken from the county ofLyme in the USA, where 
a considerable number of patients developed this hitherto 
unknown disease over 15 years ago (4). Borreliosis was 
named after the bacterium causing the disease (5). Lyme 
borreliosis is usually transmitted by ticks. 
Like syphilis, Lyme borreliosis progresses through two 
phases, early and late infection (6,7). The early infection is 
divided into the first stage, in which a localized infection 
appears, and the second stage, characterized by a disseminated 
infection. The second stage follows within a few days or a 
few weeks from the initial stage. and is marked by intermittent 
acta dermatovenero/ogica A.P A. Vol 1, 92, No 3 
symptoms which may last for some weeks to some months. 
The late phase of the disease (persistent infection) corresponds 
to the third stage and usually begins after one year or more 
following inoculation (6, 7). The skin can be affected both in 
the early as well as in the late phase. 
In the majority of cases the disease develops about 1 O days 
(i.e. some days to some weeks) after a tick bite, when a 
redness ofthe skin appears and spreads outward. Initially the 
skin lesion is homogeneous, but later it grows paler in the 
central regi on. A typical ringlike erythemagradually enlarges, 
i.e. "migrates" outward, and is therefore named erythema 
migrans. As a general rule, Borreliae enter the skin via a bite 
of an infected tick. Changes in the skin (erythema migrans), 
following a tick bite, i;eflect a local inflammation probably 
caused by spreading of B. burgdorferi over the skin. In some 
79 
lso/ation of Borrelia burgdorferi from skin 
patients Bon-eliae enter into the blood and disseminate into 
different tissues . After a period of latency. which may last for 
some days. or. in most cases, severa! weeks or months, and 
occasionally even severa! years, the clinical evidence of 
involvement of the nervous system, heart, joints, skin and/or 
other tissues or organs may appear (7) . 
It is characteristic of Lyme bon-eliosis that its clinical 
picture is rarely complete, and sometimes any of its clinical 
stages may be completely absent (7). The pathognomonic 
clinical sign of the disease (i.e. erythema migrans, which is 
the main manifestation ofthe first stage ofLyme bon-eliosis) 
may also be either absent, or the skin lesions are atypical. In 
such cases, a bon-elial infection is usually confhmed by 
determining Bon-elia antibodies. Extreme caution, however, 
must be taken when interpreting serological tests, as negative 
results do not necessarily exclude Bon-elia infection, while 
on the other hand, positive results do not necessarily mean 
that (ali) symptoms and signs, which the patients experience, 
are a consequence of bon-elial infection. In clinically unclear 
cases, much greater significance is therefore attached to the 
isolation of B.burgdorferi than to the determination of 
antibodies. 
This report includes 5 cases of patients with different skin 
Iesions, in whom B.burgdorferi was isolated from the skin. 
Among 848 patients registered in Slovenia between 1985 and 
1988, 73 % reported a tick bite, 1 O % an insect bite. and 17 
% did not recall being bitten either by a tick or by an insect 
at the site of later skin lesi on ( 11 ). About 1 O % of the Iesions 
remained homogeneous (12). 
MATERIAL AND METHODS 
All 5 patients underwent physical examination at the 
Outpatient Clinic of the Departrnent of Infectious Diseases 
in Ljubljana. For thc present report, some patients were 
purposely selected whose clinical picture itself enabled 
recognition of the disease, while the isolation of the causing 
agent from the skin additionally confirmed an infection with 
Bon-elia. In other patients presented, the isolation of B. 
burgdorferi was essential in diagnosis of the cause of skin 
lesions. thereby enabling appropriate treatment. 
A biopsy of the skin was can-ied out for cul ture and a blood 
sample was taken for the presence of antibodies to B. 
burgdorferi in ali patients on the same day. 
The skin biopsy was performed at the periphery of the skin 
lesion after disinfection with 70 % alcohol and local 
anaesthesia with 2 % xylocaine. A piecc of tissue of about 10 
x 5 x 3 mm was removed. The obtained material was 
cultivated in MKP medi um, as previously described (8). The 
protein profile of the isolated Borrelia was determined with 
SDS-P AGE. According to the protein profile and thereactivity 
with monoclonal antibodies. the isolated Borelliae were 
categorized into serotypes, as has been reported by Wilske et 
80 
al. (9). 
The IgM and IgG antibody titers were determined with the 
IFA without absorption (10). As an antigen, Bon-elia 
burgdorferi PKo strain serotype 2. was used. Antibody titers 
of ~ 1 : 256 were considered as positive. 
CASE REPORTS 
C ase 1 : MJ, male, 49 yrs 
A 49-year-old patient was referred to our Departrnent after 
the development of dizziness, headache, nausea and generally 
feeling unwell. Ali these symptoms lasted for one day. His 
previous medica! history was uneventful. Over the last few 
years he experienced occasional pain inhis joints, particularly 
in his shoulders and elbows. In the last 6 months before 
admission he did notreceive any antibiotics. He did notrecall 
being bitten by a tick. Upon physical examination, the patient 
was afebrile. Meningeal signs were not presen t. His physical 
state did not show any abnormality except that a ringlike, 
oval-shaped erythema, measuring 30 cm in length was 
observed on the left hemithorax on his back. He was not 
aware of his skin changes. When he was asked a target 
question, he replied in the affirmative, stating that around 
July 7 he was most probably bitten on the left side ofhis back 
by an insect. Some days later, the site of the insect bite began 
to itch. A small redness appeared which after July 20 began 
to expand, but afterward he was not so aware of it anymore. 
The basic laboratory tests were within normal limits. Lyme 
serology was negative, however, B. burgdorferi, serotype 2, 
was isolated from the patient's skin. A. lumbar puncture was 
not can-ied out. The patient received 100 mg doxycycline 
twice daily over 14 consecutive days. The skin lesion 
completely disappeared 7 days after initiation of therapy. 
However, headaches, occasional dizziness and general feeling 
of being unwell persisted until the middle of December. 
Case 2: TB, male, 36 yrs 
This patient was cutting grass at the end of May and at the 
beginning of June. He was not aware ofbeing bitten by a tick 
oran insect. Around June 9th, i.e. a week prior to undergoing 
an examination at our clinic, a redness had appeared on the 
inner side of this right calf, which enlarged over the next few 
days. The last two days before the examinationhe experienced 
a buming pain in the affected area. He also had minor 
headaches, but he denied having fever or chills, or any other 
problems. 
Upon exarnination on June 16th, a homogeneous redness 
of 25 cm in lenght and around 20 cm in width was observed 
on the inner part of the patient's right calf. In the center of it 
a srna!! crust was seen. The area of redness was slightly 
warmer and its border was well-demarcated from the 
surrounding skin .. There were no other significant 
abno1malities in the patient's physical state. Serological tests 
acra dermatovenerologica AP.A. Vol 1, 92, No 3 
lsolation of Borrelia burgdorferi from skin 
failed to confirm the presence of B. burgd01i'eri antibodies, 
however, B. burgdorferi, serotype 4, was isolated from the 
material obtained at biopsy. 
Case 3: MK, female, 38 yrs 
The patient had been well until the onset of the present 
illness. Toward the end of May she was bitten by an insect 
(most probably a horse - fly) in her right calf. Already some 
days la ter she noticed a redness, which gradually spread. She 
occasionally experiencedminor itching and burning sensations 
in the affected area. She did not report any other symptoms 
and she denied having been bitten by a tick. She did not 
receive any antibiotics. The examination on November 23rd 
was uneventful apart from a roundish redness on the distal 
part of her right calf, measuring 4 cm in diameter, the border 
of which was slightly pronounced. The skin in the region of 
the erythema was harder, dry, and slightly flaking. The 
results of serological tests were negative, but B. burgdorferi, 
serotype 6, was isolated from the skin. Treatment with 100 
mg doxycycline twice a day over 14 consecutive days led to 
partial improvement, but complete disappearance of the 
erythema of the skin only occurred after treatment with 
ceftriaxone. 
Case 4: PD, female, 40 yrs 
The 40-year-old patient, a mother of three childreen, was 
in good health. She was able to carry out her normal duties at 
work and at home without a difficulties. ln July 1988 she was 
bitten by an insect in her right calf. A redness appeared at the 
site of the bite, but disappeared within some days. In the 
autumn of that year she developed pain in herrib cage, which 
was associated with fatigue and gradually progressing 
myalgia, arthralgia, pain, and headaches. She grew forgetful, 
had difficulties concentrating, was also psychically changed. 
In October 1988, a small redness appeared at the site of an 
insect bite, whichdidnotenlarge significantly in the following 
few months. The patient underwent several tests, which ali 
failed to show any abnormality. She was also referred to a 
neurologist, a psychiatrist, dermatologist and orthopaedic 
surgeon, who did not discover any definite cause of her 
problems. In April 1989, when the patient came to our 
Outpatient Clinic, on the anterior part of her right calf, in its 
distal third, there was a round reddish-purple erythema, 
slightly above the leve! of the skin, measuring 1 cm in 
diameter. 
Serological tests for B. burgdorferi were negative. The 
same results were obtained by repeated tests, carried out 3 
weeks later. Due to perseverance on the part of the patient's 
physician, who treated her because she was experiencing 
severe pain, a biopsy of the skin was perf01med. From the 
obtained specimen, B. burgdorferi serotype 2 was isolated. 
Although the skin lesion disappeared after antibiotic treatment, 
only slight and temporary alleviation of the remaining 
acta dermatovenerologica A.P.A. Vol 1, 92, No 3 
symptoms occurred. The patient remained serologically 
negative, however, a positive result was obtained by atest of 
transformation of lymphocytes with B. burgdoti'eri in blood. 
Case 5: MW, female, 36 yrs 
This patient's course of disease has already been desp-ibed 
elsewhere (12), she resides in an area where Lyme borreliosis 
is endemic (13). In brief, 36-year-old patient had been in 
good health until the summer of 1989, when she received a 
tick bite and several insect bites. At the end of September 
1989, a reddish-purple papule appeared on the dorsum ofher 
right foot. The papule gradu ali y increased in size and acquired 
the form of an irregularly shaped horseshoe with a diameter 
of about4 cm. In December, a similair skin change appeared 
in the area of the right foot. Later on, pain in both knees, 
extreme fatigue and almost constant headache appeared. 
Examination of the patient in the earl y April 1990 established 
no physical abnormalities other than skin changes typical of 
granuloma annulare. Serological examinations showed 
borderline titers of antibodies to B. burgdorferi (lgM 1 : 128, 
IgG 1 : 256). Histological findings in a biopsy taken from the 
right foot were typical of granuloma annulare. Borrelia 
burgdorferi was isolated from the skin Iesion. The isolated 
strain was identified as Borrelia burgdorferi with monoclonal 
antibodies (L32 IF11 L22 IF8) by Western blot. According to 
thereactivitypattem with eightmonoclonal antibodies against 
Osp A, the isolate is Osp A serotipe 2 (9). 
The patient was administered ceftriaxone 2 g daily i.v. for 
14 days. In the weeks following the therapy the headache 
ceased and arthralgia decreased. ln late September, 5 months 
after antibiotic therapy was completed, the patient was free of 
subjective problems. The skinchanges cleared and serological 
values for B. burgdorferi were negative. Tissue removed 
from immediate vicinity of the former biopsy revealed no 
signs of granuloma annulare, and culture for Borrelia 
burgdorferi was negative (Table 1). 
DISCUSSION 
Following the description ofLyme disease in the USA, the 
discovery of its causing agent, and the introduction of 
serological tests, ithas becomeclearthat thenewly discovered 
borrelial infection occurs in many different manifestations, 
which were for many years known in Europe as separate 
nosologic entities, such as acrodermatitis chronicaatrophicans 
(14), erythema migrans (15), and Garin - Bujadoux -
Bannwarth's syndrome (16). In addition to this, serological 
tests for Borrelia were also carried out and attempts for the 
isolation of the etiological agent were done in certain other, 
relatively well-defined skin lesions. The results have shown 
borrelial etiology in the majority of patients with a solitary 
lymphocytoma (17, 18, 19) and probably also in some 
patients with sclerotic and atrophic skin lesions (19). 
81 
Isolation of Borrelia burgdorferi from skin 
Table l. Basic data on patients in whom B. burgdorferi was isolatad from affected skin. 
tick insect type of serological serotype of 
Patient bite bite skin lesion tests isolated B.b. 
erythema 
1 N.A. yes migrans negative serotype 2 
2 N.A. N.A. erythema negative sorotype 4 
3 N.A. yes erythema negative sorotype 6 
4 N.A. yes erythema negative sorotype 2 
granuloma IgM 1: 128 
5 yes 
B.b. = Borrelia burgdorferi 
N.A. = not aware 
yes 
Serological tests and the visualization of spirochetes in tissue 
slides by staining suggest that some other skin entities might 
be Borrelia - induced as well. There have been reports that in 
granuloma annulare IgG antibodies to B. burgdorferi are 
positive in 26 to 65 % of cases and that skin lesions show a 
marked improvement with the use of antibiotics effective 
against B. burgdorferi (20, 21). So fare we have been aware 
ofonly onereport (12) of successful isolation ofB. burgdorferi 
from granuloma annulare skin lesi on ( our patient number 5). 
This case support the thesis that Borrelia burgdorferi may be 
thecausative agent in some patients with granuloma annulare. 
In a typical erythema migrans, borrelial etiology is 
undisputable. There are many reports on the isolation of B. 
burgdorferi from the skin in the area of erythema migrans (22 
- 27), and a few reports on the isolation of. B. burgdorferi 
from the surrounding skin, which appeared healthy (27). B. 
burgdorferi was also isolated from skin lesi on which were not 
typical of erythema migrans nor did they belong to any well-
known skin entity (24, 28). In such cases the data about a tick 
annulare IgO 1: 256 serotype 2 
bite at the site of subsequent skin changes and the presence 
ofBorrelia antibodies are diagnostically helpful. Among our 
patients with non-specific skin changes, none reported a tick 
bite nor did anyone have specific Borrelia antibodies in 
serum, despite the fact that the skin changes had lasted for 16 
weeks, 25 weeks, and 7 months respectively (cases 2, 3, and 
4) before a blood sample and a skin biopsy being taken. 
Our results confirm that in addition to typical erythema 
migrans also atypical borrelial skin changes are possible (24, 
28), and that antibodies against B. burgdorferi in blood can be 
absent even when severa! months have already elapsed from 
the start ofthe infection (29). Atypical borrelial skin lesions 
represent a diagnostic challenge and - particularly in the 
absence of antibodies - they can also give rise to severe 
diagnostic problems. Theirrecognition is critical as itenables 
appropriate treatment. 
The isolation ofthree different serotypes ofB. burgdorferi 
from skin is indicative of a diversity of Borreliae in Slovenia, 
and confirm the findings (30) about the heterogeneity of B. 
burgdorferi in Europe. 
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AUTHORS' ADDRESSES 
Franc Sterle M.D„ Ph. D, Dpt of Infectious Diseases, University Medica] Center, Japljeva 2, 61000Ljubljana, Slovenia 
Eva Ružic M.D., mr se, Institute for Microbiology, Zaloška 4, Medica! faculty, Ljubljana University, Zaloška 4, 61000 
Ljubljana . Slovenia 
Vera Preac-Mursic Ph.D., Max von Pettenkofer Institute, Medical Faculty, Miinchen University. Pettenkoferstrasse 9a, 8000 
Miinchen, Germany 
Bettina Wilske M.D., Max von Pettenkofer Institute. Medical faculty, Mtinchen University, Pettenkoferstrasse 9a, 8000 
Miinchen, Germany. 
Jože Cimperman M. D., University Medical Center. Japljeva 2, 61000 Ljubljana. Slovenia 
acta dermatovenerologica A.P A. Vol 1, 92 , No 3 83