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INVESTIGATIVE REPORT
Detection of Spirochaetal DNA Simultaneously in Skin Biopsies,
Peripheral Blood and Urine from Patients with Erythema Migrans
PAOLO PAULUZZI
1
{, SERENA BONIN
1,2
*, MARIA ANGELES GONZALEZ INCHAURRAGA
1
*,
GIORGIO STANTA
2,3
and GIUSTO TREVISAN
1
1
Department of Clinical, Morphological and Technological Sciences,
2
Dermatology Unit and
3
Pathology Unit, University of Trieste, Cattinara
Hospital and ICGEB-International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
Lyme borreliosis is an emerging zoonosis transmitted by
infected hard-bodied ticks. The disea se is multisystemic.
In the initial stage its typical manifestation is the ery-
thema migrans, a cutaneous lesion that occurs in up to
90% of patients. In order to investigate the presence of
the specific agent, Borrelia burgdorferi, in the early
stages of the disease, DNA from skin biopsies, urine and
peripheral blood of 30 patients with clinically documented
erythema migrans and without apparent systemic invol ve-
ment was analysed by polymerase chain reaction.
Borrelia DNA in both blood and skin biopsies was
detected in 23 patients, while in 9 patients it was
discovered in urine and skin biopsies. These results
demonstrate that Borrelia DNA is detectable systemi-
cally also in patients with early Lyme borreliosis and
strongly suggest a possible dissemination of the causative
agents even when only a local infection is assumed. Key
words: erythema migrans; Lyme b orreliosis; paraffin-
embedded tissues; PCR analysis.
(Accepted September 8, 2003.)
Acta Derm Venereol 2004; 84: 106–110.
Serena Bonin, ICGEB-99 Padriciano, IT-34012 Trieste,
Italy. E-mail: serena@icgeb.org
Lyme borreliosis (LB) is an emerging tick-borne
spirochetosis transmitted by the bite of infected hard-
bodied ticks of the genus Ixodes. In Europe, the
principal vector of Borrelia burgdorferi is Ixodes ricinus.
LB is a multisystemic disease involving skin, joints
and the nervous system. The typical cutaneous mani-
festation of the primary stage is erythema migrans
(EM), an expanding red or bluish-red rash with central
clearing. This lesion occurs in up to 90% of patients
with objective evidence of LB (1). Discordant results
have been reported about the clinical manifestation of
LB and the different species of infecting Borrel ia.No
differences between the occurrence of EM and infection
with specific Borrelia species have been detected by
some authors (1), while others point out that strains
of B. afzelii have been found mainly in cutaneous
manifestations of the disease, such as EM, and
especially in acrodermatitis chronica atrophicans (2).
However, EM appears at the early stage of B.
burgdorferi infection, persisting from a few days to
several weeks after the bite of the infected tick, and the
lesion can be associated with systemic manifestations
in about 20% of cases (2, 3).
The progression of the disease is not fully under-
stood. The entry of the Borrelia infection is assumed to
be through the skin. However, the first stage of the
disease may lack cutaneous manifestations. There is a
local reaction at the bite site and subsequently the
blood carries the pathogens to other organs. A complex
inflammatory reaction includes plasminogen and com-
plement activation, phagocytosis of Borrelia by local
macrophages and presentation of the lysed organisms
to the immune system, which will allow a specific
immune response. At the same time, activated macro-
phages produce proinflammatory substances such as
monokines and chemokines that induce vessel dilata-
tion, increase vascular permeability, diapedesis of
granulocytes and monocytes and chemotaxis of these
cells. When the non-specific cellular and humoral
defences are unable to eliminate the spirochetes the
disease tends to progress (4).
The diagnosis of LB is primarily clinical, but
serological tests can provide useful supporting evidence.
ELISA and Western blot assays are the most widely
used, but, despite continuous improvements, they still
present a low specificity (1). Cross-reactive antibodies
can produce false-positive results in patients affected
by other bacterial or viral infections, autoimmune
and rheumatic diseases. False-negative reactions are
common in the early stages of LB or in immunosup-
pressed patients. Cultivation of B. burgdorferi from
body fluids is slow and inefficient, indicating the need
for new diagnostic tools (5). The polymerase chain
reaction (PCR) is a sensitive method for the diagnosis
of microorganisms that are difficult to cultivate (5).
This analytical method allows direct detection of very
few genomes of B. burgdorferi in different clinical
specimens (6), including skin biopsies, urine, peripheral
blood, synovial fluid and cerebrospinal fluid. To
investigate B. burgdorferi spreading during the first
* These authors contributed equally to the realization of this study.
{This paper is dedicated to the memory of Paolo Pauluzzi, who
passed away in January 2002.
Acta Derm Venereol 2004; 84: 106–110
Acta Derm Venereol 84 # 2004 Taylor & Francis. ISSN 0001-5555
DOI: 10.1080/00015550310006815
stages of the disease with skin local infection, we
analysed skin biopsies, urine and peripheral blood from
30 patients with clinically documented EM.
MATERIALS AND METHODS
Patients
Thirty patients with EM (17 females and 13 males) were
examined in the Dermatology Department of the University
of Trieste, Italy from January 2000 to January 2002. Their
average age was 48.4 years, ranging from 23 to 87 years; the
median age was 48.0 years (25th to 75th percentile~30.5 – 61.5).
All patients reported a tick bite 1 to 4 weeks before
dermatological examination during recreation activities in the
country around the city of Trieste, which is an endemic region
in north-eastern Italy. All patients were clinically diagnosed as
early stage skin LB on the basis of EM by experienced
dermatologists (MGI, PP or GT). A careful history focusing
on signs and/or symptoms suggestive of systemic infection
was taken for each patient. All skin lesions lasted less than
4 weeks and none had received antibiotic therapy prior to
clinical diagnosis and at least one month before the tick bite.
All patients were treated for 14 days with 1 g of
Amoxycillin three times daily in accordance with recom-
mended guidelines.
The exclusion criteria for patients in this case study were: (i)
diagnosis of LB in the past, (ii) patients who reported other
tick bites in the past; (iii) patients who could not remember
having been bitten recently by a tick; (iv) patients with EM
who reported concomitant symptoms or clinical signs of
general infection; (v) the use of any systemic antibiotic in the
preceeding 2 months; (vi) previous diagnosis of autoimmune
or rheumatic diseases; (vii) active infections of other
pathogens; and (viii) immunosuppressed patients.
Samples
Skin biopsies. Six-millimetre skin biopsies from patients
were taken from the margin of the primary EM lesion.
Biopsy specimens were formalin-fixed and paraffin-
embedded for histological examination, and subsequently 10
histological sections were submitted for PCR analyses.
Serology. Sera were examined for B. burgdorferi IgM
and IgG antibodies by ELISA (Lyme Borreliosis ELISA
kit IgG/IgM, Dako, CA, USA) in accordance with the
manufacturer’s instructions. The ELISA analysis was
performed in each patient before and after specific therapy.
DNA preparations
DNA extractions, amplifications and post-amplification pro-
cedures were performed in accordance with the precautions
suggested by Kwok & Higuchi (7).
DNA preparation from paraffin-embedded biopsies. DNA was
extracted from 10-mm sections of paraffin-embedded blocks.
Ten sections were cut from every sample with standard
microtomes. The blade was shifted after each block to prevent
cross-contamination between samples. As previously reported
(8), paraffin was removed by two washes with xylene,
followed by two washes in ethanol, 100% and 70%. After
air-drying, the tissue pellets were digested overnight at 45‡C
with proteinase K (0.5 mg/ml) in 50 mM Tris-HCl, 1 mM
EDTA, 0.5% Tween 20. To purify DNA from proteinase K
and proteolysis residues, an extraction with phenol-Tris/
chloroform was performed. The final concentration of DNA
from the solution was made by precipitation with ethanol
using 5 ml of glycogen 1 mg/ml as precipitation carrier.
DNA preparation from blood samples. Five millilitres of
fresh blood from each patient was collected in an EDTA
tube and submitted to DNA extraction. Forty-five millilitres
of lysis buffer containing 0.32 M sucrose, 10 mM Tris-HCl
pH 7.5, 5 mM MgCl
2
and 1% Triton X 100 was added to
each blood sample. The samples were mixed by inversion
several times, incubated at 4‡C for 10 min and then
centrifuged at 6006g for 15 min at 4‡C. Supernatants were
decanted, the pellets were washed in 5 ml of cold PBS and
centrifuged as reported above. The resulting pellets were
resuspended in 1 ml of lysis solution containing 10 mM
Tris-HCl pH 8, 400 mM NaCl, 2 mM EDTA, 0.7% SDS,
1 mg/ml of Proteinase K. Digestion was left to proceed at
45‡C for at least 4 h under gentle shaking. To precipitate
protein debris, one-third of total volume of NaCl saturated
solution was added to samples. Tubes were vigorously
mixed for 10 sec and then centrifuged at maximum speed
(130006g) for 5 min at room temperature. Supernatants
were collected and DNAs were precipitated with one
volume of iso-propanol at room temperature. Genomic
DNAs were picked, washed with 0.5 ml of 70% ethanol, air
dried and resuspended in 100 ml of sterile water.
DNA preparation from urine samples. For the extraction of
DNA from urine samples the alkaline lysis method (9) was
avoided and a protein digestion procedure was chosen in
order to eliminate PCR protein inhibitors. Urine samples
(20 ml each) were centrifuged at 6006g for 15 min at 7‡ C.
The sediments were washed with 5 ml of PBS and
sedimented by another centrifugation step. Urine residues
were mixed with 10 volumes of digestion solution composed
of 10 mM Tris pH 7.4, 10 mM EDTA, 150 mM NaCl,
0.4% SDS and 1 mg/ml proteinase K (10). Samples were
incubated at 45‡C overnight. To eliminate protein debris an
extraction with phenol-Tris/Chloroform was performed.
DNAs were isolated by iso-propanol precipitation.
PCR amplification
We avoided nested PCR because of the high risk of carry-over
connected with this method. In order to get a higher
sensitivity, especially in paraffin-embedded tissues where
DNA is highly degraded (11), we decreased the amplicon
size to a maximum length of 100 bases and increased the
number of PCR cycles to 70. Every PCR reaction was run in
duplicate. Primer sets were chosen in regions of the Borrelia
chromosome with low variability. For the analysis, we chose a
combination of three primer sets in order to achieve high
sensitivity (5). One set of primers targeted a sequence of a
chromosomal gene encoding for a 66 kDa protein, the second
set targeted the Borrelia flagellin gene (41 kDa protein) and
the third primers set was specific for the gene encoding the
80 kDa antigen. Primer sequences are reported below: 66 kDa
(GenBank M60802, AE001174); Primer up: 5’-TGCAAA
TGGGAACTGATT-3’; Primer dw: 5’-TGAGGGTGTTTCT
TTTTT-3’; Probe: 5’-TGGACACATCTCAAAAGCAGCG
AA-3’; 80 kDa (GenBank M60802, AE001161); Primer up:
5’-GGTAAAGCCTTGGATCTTGA-3’; Primer dw: 5’-CTTC
TTCCTTGGCTTTACTT-3’; Probe: 5’-CGAGAATTAAA
TTCTAAAGCTTCTAGC-3’; Flagellin (GenBank AF244889);
Detection of spirochaetal DNA 107
Acta Derm Venereol 84
Primer up: 5’-TTCTCTGGTGAGGGAGCTCAAAC-3’;Primer
dw: 5’-CTGTTGAGCTCCTTCCTGTTG-3’; Probe: 5’-TCA
GGCTGCACCGGTTCAAGAGGGTGTT-3’. For every B.
burgdorferi gene, three oligonucleotides were synthesized, two
in DNA sense and one in antisense orientation. The first
sense and the antisense oligonucleotides were used for the
amplification reaction. The second sense oligonucleotide was
used as internal probe for the amplified product in order to
detect only specific amplicons. In this way a further test of
specificity was performed. To evaluate the sensitivity of the
primers sets in blood, urine and paraffin-embedded biopsy
tissues (PET), 0.01 pg, 1 pg, 10 pg and 100 pg of Borrelia
DNA were diluted in 1 mg of DNA derived from PET (free
from LB) and in 2 mg of DNA derived from blood and urine
derived from a disease-free donor. Every test was run in
duplicate. Positivity was detected for every sample and every
primer set. To assess the sensitivity of the different primer
sets, serial dilutions of specific Borrelia DNA were performed.
With the proposed sets of primers 0.01 – 0.02 pg of Borrelia
DNA was detected, corresponding to 5 – 10 Borrelia genomes.
The efficiency of the primers was initially evaluated using PET
from patients clinically positive for LB. Borrelia DNA was
detected in 97% of the cases. In order to assess the specificity
of the three primer sets, PET of different microbial skin
lesions were analysed. DNAs were obtained from 2 samples of
cutaneous tuberculosis and 2 scraps of primary syphilis lesion.
Specificity tests were also done using DNA of Mycobacterium
avium and Candida albicans (generously provided by the
bacteriology unit of ICGEB) and E. coli. In none of the
samples was Borrelia DNA detected utilising the proposed
analytical method.
The PCR mixture (total volume 50 ml) contained the
isolated DNA, 15 pmol of each primer, 200 mM of each
dNTP, 10 mM Tris-HCl, 50 mM KCl, 1.0 mM MgCl
2
and
1.25 U of TaqPolymerase (Amersham Biosciences, Uppsala,
Sweden). Amplifications were carried out for 70 cycles as
follows: 1 denaturation step at 94‡ C for 3 min; 5 cycles of
94‡C/1 min, annealing temperature/1 min, 72‡C/1 min and 65
cycles of 94‡C/30 sec, annealing temperature/30 sec, 72‡C/
30 sec. The annealing temperature for 66 kDa was 42‡C, for
80 kDa 50‡ C and for Flagellin 55‡C. Two micrograms of
DNA obtained for blood and urine samples was submitted to
PCR amplification and 1 mg of DNA obtained from PET. For
every PCR analysis negative controls containing DNA
obtained from healthy donors were included. In addition,
pure genomic B. burgdorferi DNA was used as positive
control. For positive controls, 5 ng of specific DNA obtained
from B. garinii, afzelii and sensu stricto were used.
Dot-blot hybridization
The amplifications were tested by dot blot with specific
internal probe hybridization. Twenty microlitres of amplified
material were denatured for 10 min at 95‡C and chilled on ice.
After this step, 30 ml of SSC (saline sodium citrate) 206 and
1 ml of dye for dot blot (0.25% bromophenol blue, 2.5% ficoll
in sterile water) were added to each sample. Specimens were
spotted on a pre-equilibrated Hybond Nz membrane
(Amersham Biosciences) using a dot blot apparatus. The
membrane was air-dried and cross-linked twice in UV-
Stratalinker (Stratagene, La Jolla, CA, USA). In order to
overcome the detection of unspecific products, the third
oligonucleotide, internal to the amplification fragment, was
used as a probe after a kinasation step. Reaction was
performed with 500 ng of oligonucleotide using 10 units of
T4 polynucleotide kinase (New England Biolabs Inc.,
Beverly, MA, USA) and 50 mCi of [c-
32
P]ATP (Amersham
Biosciences) for 1 h at 37‡C. Labelled probe was then purified
onto a G-25 Sephadex (Amersham Biosciences) minicolumn.
After pre-hybridization for 1 h the membranes were
hybridized overnight at the proper temperature in SSC 66,
0.25% milk powder. The hybridization temperatures were
47‡C for 66 kDa, 42‡C for 80 kDa and 50‡C for flagellin.
After hybridization, 2 washes in 66 SSC, 0.1% SDS at
room temperature, 2 washes in 36 SSC, 0.1% SDS and 2 in
16 SSC, 0.1% SDS at 10‡C more than the hybridization
temperature were performed. Membrane positivity was
detected using a Cyclon Storage Phosphor System (Packard
Instrument, Meriden, CT, USA).
Statistical analysis
The chi-square test was performed to assess the differences in
the results obtained using the three PCR systems. Statistical
analyses were performed using the EPI6 software dedicated to
epidemiology studies.
RESULTS
We examined 30 patients with early LB with clinically
defined EM. The serological tests with the ELISA
method gave positive results for specific IgM antibodies
in 8 patients. A control serological examination carried
out 2 months after the end of therapy showed sero-
conversion with specific IgG antibodies in 3 patients
only.
For every patient, the biological specimens were
investigated in duplicate by PCR amplification. Blood,
urine and skin (PET) were analysed with the three
different PCR primer sets specific for different
sequences of the B. burgdorferi genome (B-80, 66 kDa
protein, flagellin). PCR results for each patient are
reported in Table I. The percentage of positive results
for B-80 in blood was 47%, in urine 10% and in PET
50% (Table II). For 66 kDa protein the rate of positive
results was 53% in blood, 20% in urine and only 37% in
PET. The third set, detecting the B. burgdorferi flagellin
gene, gave the best results in archive biopsies (PET)
with 86% positivity, 37% in blood and 13% in urine
(Table II). Complete concordance of the three sets was
detected in 13% of blood samples (4/30), in no urine
sample and in 6% (2/30) of PET. B. burgdorferi DNA
could be detected by at least one of the three sets in
76% (23/30) of blood samples, in 30% of urine samples
(9/30) and in 100% (30/30) of PET. No B. burgdorferi
DNA was detectable in control patients related to
dermatological diseases different from LB.
Discordant results were observed for the three sets
of primers with statistically significant differences: in
PET the flagellin primer set was the more sensitive
(x
2
~14.12, d.f.~2, pv0.001), whereas no differences
were detected in blood (x
2
~0.82, p~ 0.66) and urine
samples (x
2
~1.26, p~0.53).
The PCR analyses indicated that 7 patients were
positive in all 3 biological specimens examined (blood,
urine and PET), 16 had positive results in PET and
108 P. Pauluzzi et al.
Acta Derm Venereol 84
blood, 2 in urine and PET and 5 had positive results
only in PET. No one was completely negative.
DISCUSSION
In this study, 30 patients with early LB, characterized
by the presence of EM, were investigated. Three
biological specimens were analysed for each patient:
skin (PET), blood and urine. Patients with EM, but
without any other secondary manifestation connected
with Borrelia infection, were selected. The goal of the
study was the evaluation of the dissemination of
Borrelia at an early stage of LB infection without
clinical systemic involvement. EM is the dermatological
hallmark of early LB.
EM presents a non-specific histological pattern with
dermal lymphocytes infiltration confined mainly to the
perivascular area. ELISA is currently the method of
choice for laboratory confirmation of LB, but negative
serology does not exclude LB, especially in the early
phases of the disease. Specific IgM antibodies to
B. burgdorferi usually appear 3 to 4 weeks after onset
of the infection (12), but a prompt antibiotic treatment
of the clinical manifestation may sometimes suppress
the antibody response. Previous studies have demon-
strated that PCR amplification of sequences of the
B. burgdorferi genome is a valuable tool for supporting
the clinical diagnosis of LB (9), especially in the absence
of a serologic response in the early stage of the infection
(13). PCR can in addition detect non-viable organisms.
Thus, a positive PCR result does not establish whether
the infection is active or not. In our study, all patients
satisfied the Centres for Disease Control and Preven-
tion’s Surveillance definition of Lyme disease, and no
one was treated with antibiotics in the prior 2 months.
Therefore B. burgdorferi specific DNA detected in
patient’s specimens can be considered as confirmation
of an active infection.
B. burgdorferi DNA could be detected in 100%
Table I. Individual PRC result for Borellia DNA positivity in blood, urine and paraffin-embedded tissue (PET) of 30 patients
with erythema migrans using 3 different markers.
Pat. no.
B-80 66 kDa 41 kDa (Flagellin)
Blood Urine PET Blood Urine PET Blood Urine PET
1 22222z 2 zz
2 22222222z
3 zz2 zzz22z
4 22222zz 2 z
5 22222z 22z
6 z 2 zz 22zzz
7 z 2 zz z z22z
8 z 2 zz 22z 22
9 22zz 2 z 222
10 z 2 zz z zz z 2
11 22222222z
12 z 222 222 2z
13 222z 22z 2 z
14 z 22z 2 zz 2 z
15 222z 2 z 22z
16 2 zzz 2 zz z z
17 222z 22z 2 z
18 z 22z 222 2z
19 22z 2 z 22 2 z
20 z 2 zz 222 22
21 z 2 z 2 z 22 2 z
22 z 2 z 22222z
23 z 2 zz 222 2z
24 222222z 2 z
25 222z 222 2z
26 22z 22z 22z
27 22222222z
28 zzz2 z 2 z 2 z
29 z 2 z 22222z
30 22zz 22z 2 z
Table II. Summary of the positive results for each primer
set
1
Markers Blood Urine Skin (PET)
B-80 47% (14/30) 10% (3/30) 50% (15/30)
66 kDa 53% (16/30) 20% (6/30) 37% (11/30)
Flagellin (41 kDa) 37% (11/30) 13% (4/30) 86% (26/30)
1
Same patients and samples as in Table I.
Detection of spirochaetal DNA 109
Acta Derm Venereol 84
of PET from EM skin by application of the three
Borrelia genome sequence amplifications. Even if the
sensitivity of PCR in PET is lower than in fresh tissues
(5), our results demonstrate that with a set of primers it
is possible to use PET to detect Borrelia DNA reliably.
In order to confirm the results in every sample,
duplicate tests were performed. Positive results were
given only when verified by the second test. In our
experience, positivity was confirmed in every case, but
sometimes with different intensity.
The rate of positive results is different for distinct
primers, especially in PET where the flagellin sequence
gave the most frequent positive results. For this
sequence, 86% (26 out of 30 cases) were positive for
Borrelia, which is in agreement with the results
obtained by Lebech et al. (14), who detected Borrelia
DNA in 71% of skin biopsies but only in 13% of urine
samples from patients with EM. The reason for the
enhanced positivity of this marker in archive tissue is
not clear; a possible gene amplification has been
suggested. PET-DNA analysis has a lower efficiency
than the analysis performed in DNA obtained from
fresh tissue, but it is well known that in blood and urine
the micro-organism is more diluted (14).
The diagnostic value of urine PCR in early infection is
unclear and the previously reported results are contro-
versial (5). It is well known that the diagnostic sensitivity
in urine samples is low due to the decreased number of
targets (14). The percentages of positive results detected
in urine samples using the three different primer sets were
comparable and consistent with other reports (14). Also
in blood the rate of positive results for the different
sequence systems presents no significant differences.
B. burgdorferi spreads locally in the skin, but at the
same time probably slips in between the endothelial
cells (15, 16). The PCR simultaneously identified
Borrelia in blood and skin of 23 patients, thereby
confirming the suspicion of early dissemination of the
pathogen in a major fraction of patients with EM. In a
further 9 patients it was detected in both skin and
urine. These findings contrast with the usual clinical
division of LB into local infection (Stage I that includes
EM) and disseminated infection (Stages II and III).
According to our results the infections seem to be
disseminated already at the initial stages. This spread-
ing of the microorganism at the early phase of the
disease concurs with the pathogenesis of syphilis, where
Treponema pallidum is known to disseminate somati-
cally even at an early stage of the infection (17). Also
for this spirochete PCR analysis was reported as a
valuable tool for pathogen detection in different types of
biological samples (18, 19).
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