ArticlePDF Available

Detection of Spirochaetal DNA Simultaneously in Skin Biopsies, Peripheral Blood and Urine from Patients with Erythema Migrans



Lyme borreliosis is an emerging zoonosis transmitted by infected hard-bodied ticks. The disease is multisystemic. In the initial stage its typical manifestation is the erythema 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 involvement 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 systemically 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.
Detection of Spirochaetal DNA Simultaneously in Skin Biopsies,
Peripheral Blood and Urine from Patients with Erythema Migrans
Department of Clinical, Morphological and Technological Sciences,
Dermatology Unit and
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:
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.
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.
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 45C
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
and 1% Triton X 100 was added to
each blood sample. The samples were mixed by inversion
several times, incubated at 4C for 10 min and then
centrifuged at 6006g for 15 min at 4C. 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
45C 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 45C 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
AA-3; 80 kDa (GenBank M60802, AE001161); Primer up:
TTCTAAAGCTTCTAGC-3; Flagellin (GenBank AF244889);
Detection of spirochaetal DNA 107
Acta Derm Venereol 84
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
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
94C/1 min, annealing temperature/1 min, 72C/1 min and 65
cycles of 94C/30 sec, annealing temperature/30 sec, 72C/
30 sec. The annealing temperature for 66 kDa was 42C, for
80 kDa 50 C and for Flagellin 55C. 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 95C 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-
P]ATP (Amersham
Biosciences) for 1 h at 37C. 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
47C for 66 kDa, 42C for 80 kDa and 50C 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 10C 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.
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
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
~14.12, d.f.~2, pv0.001), whereas no differences
were detected in blood (x
~0.82, p~ 0.66) and urine
samples (x
~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.
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
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)
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).
1. Wang G, van Dam AP, Schwartz I, Dankert J. Molecular
typing of Borrelia burgdorferi sensu lato: taxonomic,
epidemiological, and clinical implications. Clin Microbiol
Rev 1999; 12: 633 653.
2. Ciceroni L, Ciarrochi S, Ciervo A, Mondarini V, Guzzo
F, Caruso G, et al. Isolation and characterization of
Borrelia burgdorferi sensu lato strains in an area of Italy
where Lyme borreliosis is endemic. J Clin Microbiol 2001;
39: 2254 2260.
3. van Dam AP, Kuiper H, Vos K, Widjojokusumo A, de
Jongh BM, Spanjaard L, et al. Different genospecies of
Borrelia burgdorferi are associated with distinct clinical
manifestations of Lyme borreliosis. Clin Infect Dis 1993;
17: 708 717.
4. Hu LT, Klempner MS. Host-pathogen interactions in the
immunopathogenesis of Lyme disease. J Clin Immunol
1997; 17: 354 365.
5. Brettschneider S, Bruckbauer H, Klugbauer N, Hofmann H.
Diagnostic value of PCR for detection of Borrelia burg-
dorferi in skin biopsy and urine samples from patients
with skin borreliosis. J Clin Microbiol 1998; 36: 2658 2665.
6. Priem S, Burmester GR, Kamradt T, Wolbart K, Rittig MG,
Krause A. Detection of Borrelia burgdorferi by poly-
merase chain reaction in synovial membrane, but not in
synovial fluid from patients with persisting Lyme arthritis
after antibiotic therapy. Ann Rheum Dis 1998; 57: 118 121.
7. Kwok S, Hiqushi R. Avoiding false positives with PCR.
Nature 1989; 339: 237 238.
8. Trevisan G, Nobile C, Bonin S, Stanta G. Detection of
Borrelia burgdorferi in skin biopsies from patients with mor-
phea by polymerase chain reaction. JEADV 1996; 6: 15 19.
9. Priem S, Rittig MG, Kamradt T, Burmester GR, Krause
A. An optimized PCR leads to rapid and highly sensitive
detection of Borrelia burgdorferi in patients with Lyme
borreliosis. J Clin Microbiol 1997; 35: 685 690.
10. Davis. In: Basic methods in molecular biology. London:
Elsevier, 1986: 44 46.
11. Lehmann U, Kreipe H. Real-time PCR analysis of DNA
and RNA extracted from formalin-fixed and paraffin-
embedded biopsies. Methods 2001; 25: 409 418.
12. From the Centers for Disease Control and Prevention.
Recommendations for test performance and interpreta-
tion from the Second National Conference on Serologic
Diagnosis of Lyme Disease. JAMA 1995; 274: 937.
13. Nowakowski J, Schwartz I, Liveris D, Wang G, Aguero-
Rosenfeld ME, Girao G, et al. Laboratory diagnostic
techniques for patients with early Lyme disease associated
with erythema migrans: a comparison of different
techniques. Clin Infect Dis 2001; 33: 2023 2027.
14. Lebech AM, Hansen K, Brandrup F, Clemmensen O,
Halkier-Sorensen L. Diagnostic value of PCR for detec-
tion of Borrelia burgdorferi DNA in clinical specimens
from patients with erythema migrans and Lyme neuro-
borreliosis. Mol Diagn 2000; 5: 139 150.
15. Seiler KP, Weis JJ. Immunity to Lyme disease: protec-
tion, pathology and persistence. Curr Opin Immunol
1996; 8: 503 509.
16. Sigal LH. Lyme disease: a review of aspects of its
immunology and immunopathogenesis. Annu Rev Immu-
nol 1997; 15: 63 92.
17. Singh AE, Romanowski B. Syphilis: review with emphasis
on clinical, epidemiologic, and some biologic features.
Clin Microbiol Rev 1999; 12: 187 209.
18. Wicher K, Noordhoek GT, Abbruscato F, Wicher V.
Detection of Treponema pallidum in early syphilis by
DNA amplification. J Clin Microbiol 1992; 30: 497 500.
19. Burstain JM, Grimprel E, Lukehart SA, Norgard MV,
Radolf JD. Sensitive detection of Treponema pallidum by
using the polymerase chain reaction. J Clin Microbiol
1991; 29: 62 69.
110 P. Pauluzzi et al.
Acta Derm Venereol 84
... Several clinical variations have been observed, such as smaller-sized-EM of about the size of a coin, oval shaped EM with no darker outline, red-violet EM (erysipeloid), EM with vesicles which mimics herpes simplex or herpes zoster (24), painful EM (burning), itchy EM, hidden EM (scalp), and EM with atrophic evolution (25). It has been shown that in some cases of EM, Borrelia infection can already be disseminated (26). ...
... They can be located on the chromosome or on plasmid DNA. The most frequent chromosomal targets that have been reported in clinical studies are flagellin (26,164,(180)(181)(182), 16S rRNA gene (180,(183)(184)(185), the gene codifying for the 66 kDa protein (26,56,184,185), while the most used plasmid target is OspA (56,180,183,(186)(187)(188), which has been also reported to be more stable after degradation of spirochetes (178). At present the major concern in Borrelia diagnosis by PCR is the lack of standardization of the protocols and analyzed targets (167, 177,178). ...
... They can be located on the chromosome or on plasmid DNA. The most frequent chromosomal targets that have been reported in clinical studies are flagellin (26,164,(180)(181)(182), 16S rRNA gene (180,(183)(184)(185), the gene codifying for the 66 kDa protein (26,56,184,185), while the most used plasmid target is OspA (56,180,183,(186)(187)(188), which has been also reported to be more stable after degradation of spirochetes (178). At present the major concern in Borrelia diagnosis by PCR is the lack of standardization of the protocols and analyzed targets (167, 177,178). ...
Full-text available
Clinical evaluation of Lyme Borreliosis (LB) is the starting point for its diagnosis. The patient's medical history and clinical symptoms are fundamental for disease recognition. The heterogeneity in clinical manifestations of LB can be related to different causes, including the different strains of Borrelia, possible co-infection with other tick transmitted pathogens, and its interactions with the human host. This review aims at describing the heterogeneous symptoms of Lyme Borreliosis, as well as offering a practical approach for recognition of the disease, both in terms of clinical features and diagnostic/research tools.
... The most frequent target for plasmid borrelial DNA is OspA which in some cases have also been used for Borrelia genotyping [36, 39 -43]. Among chromosomal targets the most frequently assessed in PCR assays are flagellin [43 -47] and 66 kDa protein [41,44,48,49] which were historically the first targets analyzed by PCR and most recently the gene encoding the Borrelia 16S rRNA [36, 43, 48 -50]. As some plasmids may be present in more than a copy per Borrelia cell a plasmid target based PCR could be more sensitive. ...
... All the participants have lived in villages of the transborder rural area between Italy and Slovenia and all of them reported having been bitten by ticks during their life. All the participants in the study completed a questionnaire designed by the author of the presented study, and had both serologic tests performed using immunoenzymatic test VlsE ELISA, by which the level of Borrelia burgdorferi IgM and IgG class antibodies were determined in blood serum as well as two PCR analyses on blood extracts targeting a fragment of flagellin gene and 66 kDa protein [44]. Blood and data were collected over May 2005. ...
Full-text available
Although the etiological agent of Lyme disease has been known since 1980s, diagnosis of Lyme disease is still a controversial topic because of the wide range of clinical manifestations and the limited diagnostic tools available to assess Borrelia in humans. The most used diagnostic tool for Lyme disease is currently serology, but also Polymerase chain reaction (PCR) and other methods are often used to prove Borrelia infection in different patients’ specimens. The present article deals with most of the diagnostic tools used in clinical practice for Lyme disease detection in human samples. Direct and indirect specific methods for Borrelia infection detection will be discussed. The most recent peer reviewed publications as well as original results from our study and information provided by companies’ web sites have been analyzed to compile this review article.
... It consists of a circular erythema that develops around the site of the tick bite within 5 to 30 days from the bite, it gradually enlarges and after a few months it can reach the size of 50 cm or more. Erythema migrans is an early, but not always localized form [64]. ...
Borreliaceae is a family of the phylum Spirochaetales and includes two genera, Borrelia and Cristispira genus. Borrelia genus is divided into three groups, namely Lyme group (LG), Echidna‐Reptile group (REPG) and Relapsing Fever group (RFG). All Borrelia species have an obligate parasitic lifestyle, as they depend on their hosts for most of their nutritional needs. Borreliæ are transmitted among vertebrate hosts by arthropod vectors (ticks and lice). Transtadial transmission within their carriers occurs for the Borreliæ RF Group, while this does not (or rarely occurs) for the Borreliæ Lyme Group.
... Erythema migrans (EM) is the hallmark manifestation of Lyme borreliosis (LB) in its early stage [5], representing a local dissemination of the pathogen, at least in the majority of cases [6]. EM is pathognomonic for LB, and therefore its recognition is sufficient for the diagnosis. ...
Full-text available
Background: Erythema migrans (EM) is the hallmark manifestation of the Lyme borreliosis (LB), and therefore its presence and recognition are sufficient to make a diagnosis and to start proper antibiotic treatment to attempt to eradicate the infection. Methods: In this study we compared the clinical data of 439 patients who presented an EM either according to the diagnostic modality through physical assessment or through telemedicine. Conclusions: Our data clearly show that telemedicine for EM diagnosis is useful as it enables prompt administration of appropriate antibiotic therapy, which is critical to avoid complications, especially for neurologic and articular entities. Therefore, telemedicine is a tool that could be adopted for the diagnosis of Lyme disease both by specialized centers but also by general practitioners.
... Recently, growing evidence showed that the nucleic acid of the pathogen in many infectious diseases was present in urine (8,9). The detection of Borrelia burgdorferi DNA in urine was important in the laboratory diagnosis of Lyme borreliosis (10)(11)(12). Previously, two preliminary observations in a few samples indicated that T. pallidum DNA could be detected in urine (13,14). Urine T. pallidum DNA was present in 6% of men who have sex with men (MSM) with early syphilis (15). ...
Full-text available
ABSTRACT Treponema pallidum can invade any organ, and T. pallidum DNA can be detected in various tissues and fluids. However, the knowledge of the presence and loads of T. pallidum DNA in urine is limited. For this study, we enrolled 208 syphilis patients (34 primary syphilis, 61 secondary syphilis, 68 latent syphilis, and 45 symptomatic neurosyphilis) and collected urine and plasma samples from them. polA and Tpp47 genes were amplified in urine supernatant, urine sediment, and plasma using nested PCR and droplet digital PCR assays. The detection rates were 14.9% (31 of 208) and 24.2% (50 of 207) in urine supernatant and sediment, respectively (P = 0.017). The detection rates of T. pallidum DNA in urine sediment were 47.1, 47.5, 4.4, and 4.5% for primary, secondary, latent, and symptomatic neurosyphilis, respectively. After treatment, T. pallidum DNA in urine in 20 syphilis patients turned negative. Loads of T. pallidum DNA in urine sediment were significantly higher than those in plasma and urine supernatant (both P
... It is a circular skin redness, which appears 5-30 days after the tick bite and tends to expand, reaching a diameter of even 40-50 cm after a few months. Although EM represents the first stage or localized early stage of Lyme borreliosis, Borrelia can also be found disseminated in blood and urine [243]. When EM is present, the diagnosis is certain and antibiotic treatment should be initiated immediately. ...
Full-text available
Borreliae are divided into three groups, namely the Lyme group (LG), the Echidna-Reptile group (REPG) and the Relapsing Fever group (RFG). Currently, only Borrelia of the Lyme and RF groups (not all) cause infection in humans. Borreliae of the Echidna-Reptile group represent a new monophyletic group of spirochaetes, which infect amphibians and reptiles. In addition to a general description of the phylum Spirochaetales, including a brief historical digression on spirochaetosis, in the present review Borreliae of Lyme and Echidna-Reptile groups are described, discussing the ecology with vectors and hosts as well as microbiological features and molecular characterization. Furthermore, differences between LG and RFG are discussed with respect to the clinical manifestations. In humans, LG Borreliae are organotropic and cause erythema migrans in the early phase of the disease, while RFG Borreliae give high spirochaetemia with fever, without the development of erythema migrans. With respect of LG Borreliae, recently Borrelia mayonii, with intermediate characteristics between LG and RFG, has been identified. As part of the LG, it gives erythema migrans but also high spirochaetemia with fever. Hard ticks are vectors for both LG and REPG groups, but in LG they are mostly Ixodes sp. ticks, while in REPG vectors do not belong to that genus.
... Infectious agents were searched for in the patient's serum as shown in Table 1. DNA from peripheral blood was submitted to Borrelia detection as previously reported [13]. Anti-Borrelia burgdorferi antibodies were detected by Western blot using anti-Borrelia-Euroline-RN-AT (Euroimmun, Germany). ...
Some patients with a history of Borrelia burgdorferi infection develop a chronic symptomatology characterized by cognitive deficits, fatigue, and pain, despite antibiotic treatment. The pathogenic mechanism that underlines this condition, referred to as post-treatment Lyme disease syndrome (PTLDS), is currently unknown. A debate exists about whether PTLDS is due to persistent infection or to post-infectious damages in the immune system and the nervous system. We present the case of a patient with evidence of exposure to Borrelia burgdorferi sl and a long history of debilitating fatigue, cognitive abnormalities and autonomic nervous system issues. The patient had a positive Western blot for anti-basal ganglia antibodies, and the autoantigen has been identified as γ enolase, the neuron-specific isoenzyme of the glycolytic enzyme enolase. Assuming Borrelia own surface exposed enolase as the source of this autoantibody, through a mechanism of molecular mimicry, and given the absence of sera reactivity to α enolase, a bioinformatical analysis was carried out to identify a possible cross-reactive conformational B cell epitope, shared by Borrelia enolase and γ enolase, but not by α enolase. Taken that evidence, we hypothesize that this autoantibody interferes with glycolysis in neuronal cells, as the physiological basis for chronic symptoms in at least some cases of PTLDS. Studies investigating on the anti-γ enolase and anti-Borrelia enolase antibodies in PTLDS are needed to confirm our hypotheses.
The aim of this chapter is to provide a reliable method of obtaining DNA from formalin-fixed and paraffin-embedded (FFPE) tissue specimens. The application of this method allows the extraction of DNA from specimens of both biopsy and autopsy origin. The method described here is based on proteolytic digestion, phenol purification, and alcohol precipitation, using a point-to-point protocol with notes and references to guide the researcher into the laboratory practice. The DNA extracts obtained by the application of this method are suitable for most of the PCR analyses used in molecular pathology laboratories, ranging from single to complex multiplexed PCRs or B-/T-cell clonality PCR tests, as well as for PCR sequencing.
Background: Lyme disease, an infection caused by Borrelia(B.) burgdorferi, has been reported in many countries. But in Korea, only 5 cases of serologically diagnosed lyme disease have been reported. Because several strains of B. burgdorferi were isolated from Ixodes ticks which were captured in Kangwon and Chungbuk province, there might be more cases of serologically undiagnosed lyme diseases presenting with erythema migrans. Objective: To understand the clinical patterns and laboratory findings of erythema migrans in Korea. Methods: A clinical survey was retrospectively performed on 9 patients with erythema migrans which occurred after tick bites. Results: Among 9 patients with erythema migrans, 3 patients were male and 6 patients were female. The onset age of erythema migrans ranged from 26 to 71 years old (mean, 51.3 years old). The mean duration of erythema migrans after tick bite was 26.4 days and the diameter of the lesion ranged from 6 to 34 cm (mean, 18.3 cm). All cases developed from May to September and systemic symptoms such as fatigue, fever and/or chills, myalgia, palpitation, headache, arthralgia and dyspnea were present at the time of hospital visits of 3 patients. Clinically, 3 patterns of erythema migrans were seen; typical target pattern, homogenous and erythematous plaque pattern, and linear solitary plaque pattern with central postinflammatory pigmentation. Only 2 of the 7 patients (28.6%) were seropositive for IgM and IgG antibody titers by enzyme-linked immunosorbent assay in consecutive serologic tests. PCR for Borrelia DNA in paraffin-embedded tissue showed full negativity in 6 patients with erythema migrans. Conclusion: Although lyme disease is not endemic in Korea, some patients with erythema migrans might be undiagnosed as lyme disease serologically with erythema migrans. To take into consideration false negative serologic results in early erythema migrans, early oral tetracycline therapy should be included through clinical and historical diagnosis.
Full-text available
Borrelia burgdorferi sensu lato, the spirochete that causes human Lyme borreliosis (LB), is a genetically and phenotypically divergent species. In the past several years, various molecular approaches have been developed and used to determine the phenotypic and genetic heterogeneity within the LB-related spirochetes and their potential association with distinct clinical syndromes. These methods include serotyping, multilocus enzyme electrophoresis, DNA-DNA reassociation analysis, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis, plasmid fingerprinting, randomly amplified polymorphic DNA fingerprinting analysis, species-specific PCR and PCR-based restriction fragment length polymorphism (RFLP) analysis, and sequence analysis of 16S rRNA and other conserved genes. On the basis of DNA-DNA reassociation analysis, 10 different Borrelia species have been described within the B. burgdorferi sensu lato complex: B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, Borrelia andersonii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, and Borrelia bissettii sp. nov. To date, only B. burgdorferi sensu stricto, B. garinii, and B. afzelii are well known to be responsible for causing human disease. Different Borrelia species have been associated with distinct clinical manifestations of LB. In addition, Borrelia species are differentially distributed worldwide and may be maintained through different transmission cycles in nature. In this paper, the molecular methods used for typing of B. burgdorferi sensu lato are reviewed. The current taxonomic status of B. burgdorferi sensu lato and its epidemiological and clinical implications, especially correlation between the variable clinical presentations and the infecting Borrelia species, are discussed in detail.
Full-text available
By using experimentally infected rabbits as a model for early syphilis, the applicability of in vitro DNA amplification was explored for detection of Treponema pallidum. It was determined that whole blood in heparin or EDTA (but not serum), lesion exudate, and punch biopsy as well as swabs of lesions are useful specimens for examination by the polymerase chain reaction. Swabs do not require special diluents, and the specimens, whether kept at room temperature or frozen, are well suited for use in the polymerase chain reaction.
Publisher Summary The formaldehyde/agarose gel is a simple denaturing electrophoresis system that allows good size separation and resolution of single-stranded RNA. The RNA is subsequently transferred to NC from the gel and hybridized with a radiolabeled probe of interest. The time required to carry out the procedure for electrophoretic separation of the RNA and northern blot is three days. On the first day, it takes five hours to run gel and set up the blot; on the second day, it takes three hours to bake NC filters after blotting and initiate hybridization; on the third day, it takes two hours to wash and set up autoradiography. This method requires a horizontal gel electrophoresis apparatus and fume hood. This chapter lists the reagents that are needed in this method, such as agarose, formaldehyde solution, ethidium bromide, and loading buffer
Aim We looked for the evidence of Borrelia infection in patients with morphea by serologic means and by polymerase chain reaction (PCR) analysis of skin biopsy samples. Background The possible relationship between Lyme borreliosis and morphea has been suggested by certain clinical, immunological and microbiological findings, but many authors were not be able to demonstrate Borrelia burgdorferi infection in patients with morphea and cast doubts on an etiological role for B. burgdorferi in this skin lesion. Patients and methods Ten patients with morphea, 9 females (range: 8–65 years) and one 44-year-old man were examined. Serological tests for Lyme borreliosis were performed by immunofluorescence assay and flagellin enzyme-linked immunosorbent assay. Skin biopsy specimens were taken from the periphery of morphea lesions for histological examination and PCR. Results Antibodies to B. burgdorferi were detected in 3 patients and B. burgdorferi DNA was demonstrated in 5 patients. Conclusions The amplification of DNA with PCR analysis seems to open new prospects for the detection of Borrelia genome in tissues, in the present study we were able to demonstrate the presence of B. burgdorferi DNA in patients with morphea, even in seronegative patients. These data confirm that PCR is an interesting tool in skin lesion diagnosis and support the hypothesis of an etiological association between B. burgdorferi infection and some cases of morphea.
BackgroundThe aim of the study is to evaluate the diagnostic sensitivity of a 16S ribosomal RNA—based PCR on clinical specimens from patients with erythema migrans (EM) and neuroborreliosis and to compare the sensitivities with those obtained by in vitro culture and serological testing. A semiquantitative detection system, representing the input amount of specific DNA and thus the density of spirochetes in clinical specimens, indicated the preferred clinical sample to obtain for PCR testing.Methods and ResultsSkin biopsy and urine samples from 31 patients with EM and cerebrospinal fluid (CSF) and urine samples from 30 patients with neuroborreliosis were investigated. Borrelia burgdorferi DNA was detected in 71% of the skin biopsy specimens and 13% of the urine samples from patients with EM. Forty-one percent of the patients with EM were found to have B burgdorferi—specific antibodies in serum, and B burgdorferi was cultured in 29% of the EM specimens. For patients with neuroborreliosis, the diagnostic sensitivities in CSF and urine samples were 17% and 7%, respectively. Specific intrathecal antibody production was found in 90% of the patients, and 87% showed elevated B burgdorferi antibodies in serum. In general, PCR of skin biopsy samples yielded very high amounts of amplicons versus low amounts for CSF and urine samples.ConclusionsPCR of skin biopsy specimens is currently the most sensitive and specific test for the diagnosis of patients with EM, superior to culture and serological testing. For B burgdorferi—specific CSF diagnosis in patients with neuroborreliosis, the measurement of specific intrathecal antibody synthesis is superior to PCR. However, in patients with a short duration of disease (<14 days), PCR may be a useful diagnostic supplement. PCR of urine samples cannot be recommended at the present time for routine diagnosis of patients with EM or neuroborreliosis.
We have developed a sensitive assay for Treponema pallidum subsp. pallidum (T. pallidum), the agent of veneral syphilis, based upon the polymerase chain reaction (PCR). A 658-bp portion of the gene encoding the 47-kDa membrane immunogen was amplified, and the PCR products were probed by DNA-DNA hybridization with a 496-bp fragment internal to the amplitifed DNA. The assay detected approximately 0.01 pg of purified T. pallidum DNA, and positive results were obtained routinely from suspensions of treponemes calculated to contain 10 or more organism and from some suspensions calculated to contain a single organism. Specific PCR products were obtained for the closely related agent of yaws, Treponema pallidum subsp. pertenue, but not with human DNA or DNAs from other spirochetes (including Borrelia burgdoferi), skin microorganisms, sexually transmitted disease pathogens, and central nervous system pathogens. T. pallidum DNA was detected in serum, cerebrospinal fluids, and amniotic fluids from syphilis patients but not in in nonsyphilitic controls. T. pallidum DNA was also amplified from paraffin-embedded tissue. The diagnosis of syphillis by using PCR may become a significant addition to the diagnostic armamentarium and a valuable technique for the investigation of syphilis pathogenesis.
The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment. Tidiness and adherence to a strict set of protocols can avoid disaster.