Detection of B. burgdorferi by PCR in biological fluids 
DETECTION OF BORRELIA BURGDORFERI 
BY POL YMERASE CHAIN REACTION 
IN THE BIOLOGICAL FLUIDS 
E. Ružic-Sabljic 
SUMMARY 
Low bacterial loads and difficult growth in culture, slow antibody response, persistence of specific antibody 
response even after asymptomatic infections and negative results of serological tests are characteristic for B. 
burgdorferi infection. To facilitate the understanding of B. burgdorferi infection the PCR has been applied as 
a method capable of elucidating the pathogenesis of infection and as additional method of confirming 
borrelial infection. The PCR is a method for amplifying specific nucleic acid sequences by use of repeated 
cycles of DNA synthesis. 
Under appropriate conditions, the PCR can be used to identify B. burgdorferi in human tissues and 
biological fluids; less than 10 microorganisms in samples can be identified. The specificity is determined by 
the choise of an optimal DNA sequence of B. burgdorferi as a target sequence for the amplification. 
Applicability of the PCR for routine diagnostic procedure should be established by more detailed investigations. 
KEY WORDS 
Polymerase chain reaction, B. burgdorferi, blood, cerebrospinal fluid, urine 
Lyme borreliosis (LB) is characterized by a wide 
range of clinical manifestations caused by a spirochetes 
of the genus Borrelia: B burgdorferi, B. garinii and B. 
afzelii (1,2). 
In a borrelial infection, a relatively low concentration 
of spirochetes in clinical samples is characteristic 
and may result in an unsuccessful isolation procedure 
(3). B. burgdorferi can be cultivated from the skin in 
about 50 % of the cases, the recovery rate from 
acta dennatovenerologica A.P.A. Vol 3, 94, No 1/2 
cerebrospinal fluid (CSF) is 7-10 %, and only 3 % 
from blood. Because cultivation is a low-yield, tirne 
consuming and expensive procedure, it is not the 
method of choice to confirm the infection. In clinical 
practice serologic tests are usually used ( 4, 5). 
However, because of a slow antibody response early 
LB can be missed, and on the other band, because 
of persistence of specific antibody response which 
may be longstanding even after asymptomatic 
105 
Detection of B. burgdorferi by PCR in biological fluids 
infections, some other diseases may be falsely 
interpreted to be of borrelial origin. Negative results 
of serological tests do not rule out the presence of 
the causative agent nor do specific antibodies guarantee 
the presence of B. burgdorferi in the host (6). To 
facilitate the understanding of the role of B. burgdor-
feri in different stages of LB, the polymerase chain 
reaction (PCR) has been applied as a method capa-
ble of elucidating the pathogenesis of infection and 
as an additional method of confirming infection (7). 
The polymerase chain reaction (PCR) is a powerful 
technique which may detect nucleic acids of specific 
microorganism. It can detect about 10-100 bacteriae 
in a clinical sample, and with a nested PCR the 
sensitivity increases (8, 9). This "in vitro" technique 
is used to amplify specific DNA sequences of interest, 
the general sequence of the gene has to be known. 
The basic PCR cycle is as follows (7): 
l. Denaturation. Double stranded DNA is separated 
into single strands by heating to 95°C. 
2. Annealing. Two oligonucleotide primers ( about 
20 nucleotides in length) are required each flanking 
the opposite end and the opposite strand of the 
DNA template. The optimal temperature required 
for annealing ranges between 45°C and 70°C. 
3. Polymerase extension. Excess quantities of the 
four deoxynucleotide triphosphates (dATP, dTTP, 
dGTP and dCTP) and enzyme Taq polymerase are 
added to the mixture. Once the primers have annealed 
to the complementary DNA sequence, the polymerase 
will extend them from the 5' towards the 3' end. 
New DNA is synthesized complementary to the 
corresponding DNA template. 
The cycle of denaturation, annealing and chain 
extension is repeated through approximately 25 - 40 
cycles. The number of copies of amplified DNA 
increases exponentially with each cycle, leading to 
as many as 109 copies after 30 cycles. 
Low bacterial loads and difficult growth in culture 
are characteristic of B. burgdorferi infection. Successful 
detection of B. burgdorferi by the PCR is reported 
in samples with less than 10 microorganisms (7). 
This assay is capable of amplifying directly lysed B. 
burgdorferi without purifying the DNA; hence, extensive 
sample preparation is not required. Sensitivity of 
the PCR is also retained in the presence of human 
DNA, thus it should be applicable to the detection 
of B. burgdorferi in human specimens (10). 
The specificity of the PCR is determined by the 
choice of an optimal DNA sequence of B. burgdorferi 
as a target sequence for the amplification and by 
106 
the careful choice of a pair of primers to initiate 
synthesis on each strand of a DNA template. 
Amplification produces millions of copies of the 
target DNA sequence which can be visualized, cloned 
or sequenced. The most frequently used target DNA 
sequences of B. burgdorferi are genes encoding 16S 
rRNA (11), flagellin (9,12,13), outer surface protein 
A (OspA) (7,8) and OspB (7), but any other DNA 
sequence can also be used (10). The DNA sequence 
chosen for amplification may influence the specificity 
of the reaction. It is more relevant to amplify those 
DNA regions which are specific to the pathogen on 
the species leve!, such as OspA, OspB or OspC 
genes. By amplifying genus specific DNA regions, 
such as flagellin or 16S rRNA genes, it is possible 
to identify other Borreliae beside those associated 
with LB (11,13). Molecular biology reports a high 
degree of homology as well as the lack of sequence 
conservation among the genes, so not only the 
choice of the gene, but also the choice of the 
region of the gene for amplification can influence 
on the specificity of the PCR (9,13). Results of the 
PCR always have to be contemplated in accordance 
with the clinical signs present in a patient. Under 
appropriate conditions, the PCR can be used to 
identify B. burgdorferi in human tissues and biological 
fluids. 
l. PCR detection in blood. 
B. burgdorferi can be disseminated from a primary 
skin lesion early in the course of illness, even in 
the first few days (14). Dissemination is rarely 
manifested with clinical signs or symptoms, probably 
because of a low number of Borreliae in the blood 
(1) . This also results in an unsuccessful isolation 
procedure (3). The PCR successfully identified B. 
burgdorferi in blood and confirmed suspicion of the 
early dissemination of the pathogen in some patients 
with EM (15). If a distinction between early borrelial 
skin lesions (EM, unspecific lesions, borrelial lympho-
cytoma) with and without dissemination of the 
causative agens was possible, it would be much 
easier to advise on the appropriate antibiotic treatment. 
There is no need for PCR detection in blood in 
cases of EM; it can be more useful in cases of 
.- unspecific manifestations suspected to be of borrelial 
origin (16,17). A greater part of these patients have 
negative serological tests, as well as negative cultivation 
procedure and doubts may arise how to treat them 
properly. It is expected that in some cases the PCR 
can identify borrelial DNA in blood at tirries of 
dissemination; it could probably be more effective 
when used directly in tissue. 
acta dermatovenerologica A.P.A. Vol 3, 94, No 1/2 
Detec/ion of B. burgdorferi by PCR in biological fluids 
In patients with chronic LB, the PCR can demon-
strate the presence or the absence of the spiro-
chaetemia which would help in the elucidation of 
the pathogenesis of chronic borrelial infections or it 
may be used for monitoring the effectiveness of the 
antibiotic therapy. 
One of the properties of the B. burgdorferi is the 
adherence to a number of eucaryotic cell types (18), 
so, on the PCR detection in blood may influence 
the adherence of B. burgdorferi to the blood cells 
and platelets. The real impact of this adherence on 
the efficiency of the PCR should be investigated in 
detail, in any cases, it must be considered. 
2. PCR detection in the cerebrospinal fluid 
Neurological involvement of LB may affect both 
the peripheral and central nervous systems (CNS), 
causing a wide range of acute or chronic neurological 
manifestations. Some clinical phenomena are typical 
for LB, such as Bannwarth's syndrome, others are 
non-specific and borrelial infection has to be confirmed 
by the demonstration of a specific immune response 
or by the isolation of B. burgdorferi (1,3,4). The 
PCR in the CSF can elucidate the role of this 
bacteria in the pathogenesis of neuroborreliosis, 
confirm borrelial infection and point out interactions 
between B. burgdorferi and its host (19). 
Using the PCR, it was shown that B. burgdorferi 
can disseminate into the CNS early in the course of 
LB, at the tirne when EM is present, with minimal 
or no clinical evidence of CNS involvement. At the 
tirne of dissemination, many of the patients stili 
have no specific immune response and the CSF 
chemistry and cell counts in the CSF may be 
normal (20,21). Confirmation of the presence of 
borrelial DNA in the CSF as well as in the blood 
in some cases of eariy LB gives great value to the 
PCR. The PCR results enable the selection of an 
appropriate antibiotic therapy. 
The PCR may be of value in some cases of early 
neuroborreliosis, like in the case of acute lymphocytic 
meningitis, which is sometimes the only manifestation 
of LB. To determinate the etiology of meningitis 
after a tick bite can be a real problem for clinicians, 
especially in areas such as Slovenia which are endemic 
for severa! tick-borne diseases with neurological 
involvement (22, 23). Beside B. burgdmferi, meningitis 
can be caused by other pathogens transmitted by 
ticks, for example by Arboviruses. In patients with 
meningitis due to a tick bite, specific antibodies 
against B. burgdorferi in the serum and the CSF 
acta dennatovenerologica A.P.A. Vol 3, 94, No 1/2 
may not be present in the first days or weeks of 
the illness, and isolation of B. burgdorferi from the 
CSF is low-yield procedure. With the PCR, it could 
be possible to detect DNA of B. burgdorferi in the 
CSF and to confirm the borrelial origin o( the 
meningitis. It would be very interesting to compare 
the results of isolation, serology and PCR detection 
of B. burgdorferi and other etiological agens in CSF 
in patients with lymphocytic meningitis in a prospective 
study. 
In cases of chronic neuroborreliosis, such as borrelial 
disseminate encephalomyelitis, diagnosis could usually 
be established by the detection of specific antibodies 
in the CSF and the serum ( 4, 5). In these patients, 
the PCR is not necessary for diagnosing infection, it 
can be used to study the pathogenesis of chronic 
neuroborreliosis and eventually for monitoring the 
effectiveness of antibiotics (12, 19). In chronic 
neuroborreliosis it is not necessary that ali the CSF 
specimens are positive by the PCR. Unsuccessful 
PCR detection indicate the absence of B. burgdorferi 
in the CSF and may be the consequence of B. 
burgdorferi adherence to the cells of glial origin as 
well as the result of locally synthesized antibodies 
(24). 
3.PCR detection in synovial fluid 
Joint involvement in LB may occur early after 
infection, frequently as arthralgia, rarely as migratory 
arthritis. Early joint involvement is often accompanied 
by other more specific clinical borrelial manifestations. 
The knee seems to be most commonly involved, 
followed by other large joints. Lyme arthritis is 
characterized by brief recurrent attacks of asymmetric 
swelling and pain (1). As a rule specific borrelial 
antibodies are elevated (4, 5). As in other biological 
specimens, isolation of B. burgdorferi from synovial 
fluid is unsatisfactory and is not the method of 
choice for diagnosing borrelial infection (3, 25). 
Unsuccessful isolations from synovial fluids can be 
explained by the possible persistence of B. burgdorferi 
in the synovial membrane rather then in synovial 
fluids, and only sometimes can Borreliae be found in 
the synovial fluid. It is supposed that the PCR can 
detect such a low number of bacteria in synovial 
fluid, but for the moment there are not many 
reports (7, 26). The use of the PCR in synovial 
fluid can be compared with its use in cases of 
neuroborreliosis: it could be used for studying the 
pathogenesis of infection and eventually for monitoring 
the effectiveness of antibiotic therapy. 
107 
Detec/ion of B. burgdorferi by PCR in biological fluids 
CONCLUSIONS 
l. B. burgdorferi can be successfully detected in 
biological fluids by the PCR. On the laboratory 
leve!, this technique could be maximally optimized 
in its sensitivity and specificity by choosing an 
appropriate DNA sequence of B. burgdorferi for 
amplification, by using an appropriate set of primers 
and by improving optimal reaction conditions. 
2. The PCR can confirm early dissemination of B. 
burgdorferi from the primary lesion, suggesting the 
necessity of early antibiotic treatment. 
3. Beside serological tests and the cultivation 
procedure, the PCR in organic fluids can be used 
for confirming borrelial infection. 
4. The PCR could be especially valuable in cases 
where borrelial infection can not be confirmed by 
serological tests and the cultivation technique 
5. The applicability of the PCR can be widened to 
study the pathogenesis of the infection and eventually 
for monitoring the effectiveness of antibiotics. 
6. The real value and applicability of the PCR for 
routine diagnostic procedure should be established 
by more detailed investigations. 
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AUTHOR'S ADDRESS 
Eva Ružic Sabljic, MD, MSc, Institut of Microbiology 
Medica! Faculty, University of Ljubljana, Zaloška 4, 61000 Ljubljana, Slovenia 
acta dermatovenerologica A.P.A. Vol 3, 94, No 1/2 109