Laboratory investigation Microbiological diagnosis of Lyme borreliosis 
K E Y 
WORDS 
Lyme 
borreliosis, 
diagnosis, 
isolation, 
PCR, 
serology 
Microbwlogical diagnosis 
of Lyme borreliosis 
E. Ružic-Sabljic 
SUMMAR Y 
Lyme borreliosis is a complex multisystem disorder. Clinical manifestations are variable and differential 
diagnosis is often difficult. Besides clinical criteria (erythema migrans), the diagnosis of Lyme borreliosis 
can be ascertained by demonstration of borrelial infection using either direct (isolation, PCR) or indirect 
(serology) microbiological tests. lsolation of B. burgdorferi sensu lato from clinical material represents 
the most reliable method far confirming borrelial infection. PCR has been developed far the detection of 
B. burgdorferi sensu lato DNA in clinical specimens. In contrast to culture, PCR is a rapid method but it 
is not standardized. Serologic tests represent the most commonly used method far establishing micro-
biological diagnosis of borrelial infection. Specific lgM and lgG antibodies can be detected in blood, CSF 
and synovial fluid. The percentage of seropositivity increases with duration of infection. It is not possible 
with serologic tests to distinguish between ~cute, late, active, or treated disease. In patients with Lyme 
borreliosis a specific T-cell response to B. burgdorferi sensu lato can also be detected. 
Introduction 
Lyme borreliosis is a complex multisystem disorder 
that affects persons of all ages and both sexes. Infec-
tion can manifest with protean clinical signs; different 
organs can be affected, including the skin, nervous sys-
tem, joints, and others (1). Clinical manifestations are 
variable ancl clifferential diagnosis is often difficult. The 
best clinical marker of the disease is the initial skin le-
sion e1ythema migrans (1, 2). In some patients clinical 
manifestations are not specific but indicate a borrelial 
etiology, in others they can be entirely non-specific far 
borrelial infection. In these cases, microbiological con-
firmation of borrelial infection is essential (3). 
Besides clinical criteria (primarily the presence of 
e1ythema migrans), the cliagnosis of Lyme borreliosis 
can be ascertainecl by demonstration of borrelial infec-
tion using either clirect (isolation, PCR) or indirect (se-
rology) microbiological tests. Each inclividual method 
bas its own sensitivity ancl specificity; criteria far the 
evaluation are based on clinical parameters. In many 
cases clifferent tests are combined. 
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Microbiological diagnosis of Lyme borreliosis 
Cultivation 
Bo1-relia hui·gdor/eri sensu lato has been isolated 
from different clinical specimens: skin, CSF, bloocl, syn-
ovial fluid etc. during early as well as chronic stages of 
Lyme borreliosis (4). Specimens for isolation must be 
taken uncler aseptic conditions and before the institu-
tion of antimicrobial therapy. In clinical specimens, 
Borreliae are present in low numbers and/ or periodi-
cally (for example in blood), thus specimens for isola-
tion have to be as large as possible (like 2 ml of CSF, 10 
ml of blood etc.). Because Borreliaeare susceptible to 
the environmental conditions it is recommended to in-
oculate specimens into the medium as soon as possible 
(e .g. bed side) (3, 4) . 
Borreliae grow optimally in modified Kelly medium 
at 33°C; severa! variants of basic Kelly's medium have 
been clevelopecl (5-7). Because borrelial generation tirne 
is long and ranges from 8 to 24 h (it clepends primarily 
on borrelial adaptation to the artificial medium) it is rec-
ommended to cultivate specimens for at least nine 
weeks (2,4-7). For many physicians cultivation is not 
ve1y attractive because it is a clemanc.ling, long lasting, 
anc.l expensive procedure . However, isolation of B . 
burgdo1ferisensu lato from clinical material represents 
the most reliable methocl for confirming borre lial in-
fection , especially in patients with unspecific clinical 
manifestations. 
Although B. burgdoiferi sensu lato grows well in 
laborato1y conditions, it is not easily recovered from 
clinical specimens. The frequency of recove1y of Borre-
liae from skin biopsies ranges from up to 40% in pa-
tients with acrodermatitis chronica atrophicans to 70% 
in patients with e1ythema migrans (4). Biopsies taken 
from the expancling eclge of e1ythema migrans yielcl 
comparable culture positivity as biopsies taken from the 
centre of the skin lesion (8). The frequency of recove1y 
of Borrelia from other specimens (CSF, blood, synovial 
fluid etc.) is less then 10%. Overgrowth of competing 
bacteria (for example bacteria of normal skin flora) may 
be a serious problem in culturing Borrelia from clinical 
specimens. Additionally, contamination of sam ples dur-
ing long cultivation process also decreases the fre-
quency of recove1y (4). 
Although Borreliaeare fasticlious and requesting for 
cultivation, this methocl showed some findings impor-
tant for elucidating pathogenesis of borrelial infection: 
I.) Borreliae have been isolatecl from blood and CSF of 
patients with solita1y (ancl multiple) erythema migrans. 
These finclings con firm that Borreliae clisseminate early 
after infection (9-11). 
II .) Borreliae have been isolatecl from patients with high 
antibody titers such as patients with acrodermatitis 
chronica atrophicans (12). It seems that specific anti-
boclies as demonstrated by serological tests are not able 
Laboratory investigation 
to eradicate Borreliae from the body. Moreover, in these 
patients Borreliaewere isolated not only from skin but 
also from CSF, indicating multiorgan affection. 
III.) Borreliae have been isolated from patients that had 
been treated with antimicrobial agents appropriately 
(13) . Although Borreliae are susceptible to antibiotics, 
they have some mechanisms to survive treatment. 
IV.) Genotypic and phenotypic characterisation of iso-
lated strains show different clistribution of species regard-
ing the geographical regions (Europe, USA) as well as 
biological material (humans, ticks), and inclicate rough 
association ofborrelial species and clinical manifestation 
of Lyme borreliosis (B . a/zelii with skin clisorders, B. 
gariniiwith neurologic involvement) (10,14-16). 
Polymerase chain reaction (PCR) 
PCR has been developed for the detection of B . 
hurgdor/eri sensu lato DNA directly in body fluids and 
tissue specimens (17). The target sequence for ampli-
l'ication can be borrelial chromosomal DNA (16S rRNA 
gene, flagellin gene etc .) or plasmid DNA (e.g. gene 
for OspC, OspA etc.) (17-19). For detection with PCR, 
it is not necessary that borrelial strains are alive; it is 
sufficient that their DNA is preserved. Thus, it is rea-
sonable to perform PCR even in patients treated with 
antibiotics . 
In contrast to culture, PCR is a rapid method that 
can give results in hours. But, like the culture, PCR re-
sults also depend on the concentration of spirochetes 
in the specimen taken for analysis. Although successful 
cletection of less than 10 microorganisms in the sample 
has been reportecl, in some patients culture seems to 
be more sensitive than PCR (20-21) . Inhibitory sub-
stances in the specimens ancl sample preparation pro-
cedures may negatively influence PCR sensitivity (22). 
Specificity of PCR is cleterminecl mainly by the choice 
of specific primers ancl probes. Borrelial DNA hetero-
geneity and significant sequence differences in the tar-
get gene can cause false-negative results (23) . On the 
other hand, false-positive results are possible mainly 
because of extremely high sensitivity ofthe procedure: 
they can be a result of airborne contamination with 
borrelial DNA (primarily alreacly amplified DNA) (24). 
PCR may be helpful for the diagnosis of Lyme 
borreliosis but it remains a non-standardised method. 
PCR protocols differ regarding to the sample prepara-
tion, target DNA selection, primer selection, selection 
of amplifying methocl, ancl detection of PCR-generated 
products. Thus, PCR results should be interpreted with 
caution ancl accorcling to clinical findings and the effi-
ciency of the test. 
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Microbiological diagnosis of Lyme borreliosis 
Serology 
At present, serologic tests represent the most com-
monly used methocl for establishing microbiological 
cliagnosis of borrelial infection. Methocls inclucle en-
zyme-linkecl immunosorbent assay (ELISA), immuno-
fluorescent assay (IFA) ancl Western immunoblot (WB). 
Specific IgM ancl IgG antibodies can be cletectecl in 
bloocl , CSF ancl synovial fluid. 
Hum oral antibocly response to B. burgdo1feri sensu 
lato is complex ancl varies individually. Specific IgM 
antiboclies can be detected after 3 to 6 weeks, IgG anti-
bodies some weeks later. Low sensitivity of tests at the 
beginning of the infection is a consequence of late on-
set of antibody production. The percentage of seropo-
sitivity increases with the duration of infection. About 
one fourth of patients with early infection (like erythema 
migrans) and almost all with chronic infection (such as 
acrodermatitis chronica atrophicans) are seropositive 
(3,25). In general, some patients develop a strong, the 
others a weak immune response , while in some patients 
an immune response can not be detected. On the other 
hancl, in some healthy persons, especially from endemic 
regions, specific antibodies can also be detectecl. Ab-
sence of immune response does not mean the absence 
of borrelial infection ancl, on the contra1y, presence of 
an immune response does not inclicate active infection 
(3,4) . Serologic tests alsodo not clistinguish between 
acute, late, active, or treatecl clisease. 
Phenotypic heterogeneity of Borrelia strains has 
pronouncecl impact on the antibody formation ancl de-
EFE E C ES 
Laboratory in v estigation 
tection. An infectecl person procluces antiboclies clirectecl 
against the antigens exhibited by the infecting strain. 
Because of clifferent antigen profiles and consiclerably 
distinct antibody responses, serologic tests must be ca-
pable to cletect quite heterogeneous antibody responses 
evokecl by clifferent borrelial strains (26). Commercial 
as well as home-macle serologic tests are not stanclarclisecl. 
These tests use different Borrelia species (B . afzelii, B. 
garinii, and B. burgdo1:f"eri sensu stricto) or clifferent 
strains within the same species as test antigen. Serologic 
tests also va1y regarcling the antigen preparation: whole 
Borrelia strain, purifiecl, sonicatecl , or recombinant bo-
rrelial antigens can be used (16,27). 
False positive reactions occur particularly in patients 
with syphilis or relapsing fever, ancl in patients with auto-
immune cliseases. Possible cross-reaction with antigens 
from a broacl range of microorganisms can influence 
test results. Some of the cross-reactive antibodies can 
be reclucecl by aclsorption tests ( 4,25). 
C ell-mediated immunity 
Investigations of cell-mecliatecl immunity in patients 
with Lyme borreliosis showed specific T-cell responses 
to B. burgdorferisensu lato. Some patients demonstrate 
a significant cell-mecliatecl immune response while they 
are only borclerline or low seropositive to Borreliae. 
Although applicability of T-cell proliferative assay is 
controversial, it may be a diagnostically useful in some 
groups of patients with Lyme borreliosis (28-29). 
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146 
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Eva Ružič-Sabljič, MD, PhD, Medica! Faculty, Institute oj Microbiology and 
Immunology, Zaloška 4, 1000 Ljubljana, Slovenia 
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