Diagnosis of Lyme Borreliosis by
Polymerase Chain Reaction
ITUM, MD, PhD
GORAZD POJE, MD
ENKA GRAHOVAC, PhD
, MD, MS
SONJA LEVANAT, PhD
orrelia burgdorferi (B. burgdorferi), a spirochete
mostly transmitted by ticks of the Ixodidae fam-
ily, causes Lyme borreliosis (LB), a multisys-
temic disease that affects the skin, joints, central ner-
vous system, and heart.
Typical and the most
common skin manifestation of Lyme borreliosis is ery-
thema migrans (EM), whereas neurologic symptoms,
joint involvement, and chronic skin alterations can de-
velop at later stages of the disease.
In northwestern Croatia, an endemic area for Lyme
borreliosis, four genomic B. burgdorferi sensu lato
groups were identified in the Ixodes ricinus ticks: B.
afzelii, B. garinii, B. valaisiana (group VS116), and B.
burgdorferi sensu stricto.
The classification of B. burg-
dorferi sensu lato into genomic groups has clinical rele-
vance for LB. The association of B. afzelii with skin
manifestations, of B. garinii with neurologic symptoms,
and of B. burgdorferi sensu stricto with arthritis has been
The pathogenetic potential of B. val-
aisiana has not yet been identified.
In Croatia, B. burgdorferi was first isolated in 1991 at
the Department of Dermatology and Venereology,
Zagreb University Hospital Center, from the skin of an
EM patient, and was named P1 Zagreb. Electrophoretic
analysis of B. burgdorferi proteins revealed six of the
most important proteins of different molecular mass:
OspA, OspB, OspC, p41, p60, and p100, classifying the
isolate into the B. burgdorferi sensu lato group.
quence data and phylogenetic analysis confirmed that
DNA isolates from the sera of patients with EM coming
from northwestern Croatia belonged to the B. afzelii
The diagnosis of LB is generally based on clinical
picture and demonstration of specific antibodies to B.
burgdorferi by indirect serologic tests. The immunoflu-
orescence assay (IFA) measuring circulating serum an-
tibody binding to B. burgdorferi antigen is most com-
monly used. Specific IgM antibodies to B. burgdorferi
occur 2 to 4 weeks after infection (in most patients,
initial IgM response decreases after 1–2 months),
whereas the presence of specific IgG antibodies in se-
rum can be demonstrated only at 6 to 8 weeks of
infection, from whence they are detectable for months
and years thereafter.
The shortcomings of this assay
are subjective interpretation of results, false-positive
reaction in patients with treponemal infection and some
and low sensitivity in stage I
Low number of B. burgdorferi in pathologic
lesions and tissue fluids, low antigen level, and the
ability of B. burgdorferi to escape the host’s immune
response result in slow, poor, and unreliable formation
of specific antibodies.
The interpretation of serologic
test results in endemic populations with subclinical B.
burgdorferi infection may frequently prove quite diffi-
The method of target-sequence DNA amplification
by repetitive cycles of DNA synthesis, polymerase
chain reaction (PCR), has proved useful in the diagnosis
It has proved especially valuable in the di-
agnosis of early dissemination of B. burgdorferi, spiro-
chetemia, and from primary skin lesions, which is
rarely manifested by clinical signs of advanced LB. In
these cases, LB poses a diagnostic problem just because
of the low B. burgdorferi concentration in blood, which
makes isolation of B. burgdorferi unsuccessful, whereas
PCR has proved to be fast, reliable, sensitive, and spe-
cies-specific even in samples containing Ͻ10 bacteria
per milliliter of tested fluid.
PCR has also been
found useful when the clinical manifestations of LB are
not typical, eg, absence of erythema chronica migrans
(ECM), presence of isolated asymmetric joint lesions, or
neuroborreliosis in the early stage of disease.
results provided by PCR allow for and help in timely
initiation of LB treatment as well as in the follow-up of
In addition, PCR has been used in
From the Department of Dermatology and Venereology, Sestre Milosrd-
nice University Hospital; the Department of Ear, Nose, and Throat, Zagreb
University Hospital Center; the Croatian Institute of Transfusion Medicine;
the Department of Dermatology and Venereology, Zagreb University Hos-
pital Center; and the Department of Molecular Medicine, Ruder Bosˇkovic´
Institute, Zagreb, Croatia.
Address correspondence to Mirna S
itum, MD, PhD, Zagreb University
Hospital Center, Department of Dermatology and Venereology, Vinogradska
29, HR-10000, Zagreb, Croatia.
E-mail address: email@example.com
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