10 v vanha (2003) artikkeli Borrelioosin diagnostiikasta Euroopassa.http://online.liebertpub.com/doi/pdfplu ... 3322662200Diagnosis of Lyme Borreliosis in Europe
BETTINA WILSKE
ABSTRACT
In Europe, Lyme borreliosis is caused by at least three species, B. burgdorferi sensu stricto, B. afzelii and B. garinii.
Thus microbiological diagnosis in European patients must consider the heterogeneity of Lyme disease borreliae
for development of diagnostic tools such as PCR primers and diagnostic antigens. According to guidelines of the
German Society of Hygiene and Microbiology, the serological diagnosis should follow the principle of a two-step
procedure. A sensitive ELISA differentiating IgM and IgG is recommended as the first step. In case the ELISA is
reactive, it is followed by immunoblots (IgM and IgG) as the second step. The reactive diagnostic bands should
be clearly identified, which is easy if recombinant antigens are used. The sensitivity and standardization of immunoblots
has been considerably enhanced by use of recombinant antigens instead of whole cell lysates. Improved
sensitivity resulted from use of recombinant proteins that are expressed primarily in vivo (e.g., VlsE) and
combination of homologous proteins from different strains of borrelia (e.g., DbpA). It also appears promising to
use recombinant proteins (DbpA, VlsE, others) or synthetic peptides (the conserved C6 peptide derived from VlsE)
as ELISA antigens. At present, detection rates for serum antibodies are 20–50% in stage I, 70–90% in stage II, and
nearly 100% in stage III Lyme disease. The main goals for the future are to improve specificity in general and sensitivity
for diagnosis of early manifestations (stage I and II). Detection of the etiological agent by culture or PCR
should be confined to specific indications and specialised laboratories. Recommended specimens are skin biopsy
specimens, CSF and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50–70%)
and synovial tissue or fluid (50–70% with PCR). CSF yields positive results in only 10–30% of patients. Methods
that are not recommended for diagnostic purposes are antigen tests in body fluids, PCR of urine, and lymphocyte
transformation tests. Key Words: Lyme borreliosis—Borrelia burgdorferi—Diagnosis. Vector-Borne Zoonotic Dis.
3, 215–227.
INTRODUCTION
LYME BORRELIOSIS is a multisystem disease involving many organs such as the skin, the
nervous system, the joints, and the heart (Steere et al. 1989, Pfister et al. 1994). This condition is
the most frequent tick-borne disease in the northern hemisphere. Due to the diversity of
clinical symptoms, Lyme disease is often considered in a differential diagnosis. Examinations for antibodies against Borrelia burgdorferi are thus in high demand, and are among the most frequently requested serological tests
in microbiological laboratories. Microbiological diagnosis in European patients must consider the heterogeneity of Lyme disease borreliae in Europe.
HETEROGENEITY OF LYME DISEASE BORRELIAE IN EUROPE AND ITS IMPACT FOR MICROBIOLOGICAL DIAGNOSIS
In Europe, Lyme borreliosis is caused by at
least three species: B. burgdorferi sensu stricto,
Max von Pettenkofer Institute, University of Munich, National Reference Center for Borreliae, Munich, Germany.
B. afzelii, and B. garinii. In contrast, B. burgdorferi
sensu stricto is the only human-pathogenic
species in the United States (Wang et al. al
1999b). The three human-pathogenic species
comprise at least seven OspA-serotypes in Europe
(Fig. 1) (Wilske et al. 1993c). Skin isolates
primarily belong to B. afzelii (OspA-type 2), especially
those from patients with acrodermatitis
chronica athrophicans, a chronic skin disease
not present in America (Canica et al. 1993,
Ohlenbusch et al. 1996, Wilske et al. 1993c) (see
also legend of Fig. 1). Isolates from CSF and
ticks are heterogeneous with a predominance
of B. garinii (Eiffert et al. 1995, van Dam et al.
1993, Wilske et al. 1996, Wilske et al. 1993c).
Sequence analysis of polymerase chain reaction
(PCR) ospA amplicons from synovial fluid
of Lyme arthritis patients revealed heterogeneity
(Eiffert et al. 1998, Vasiliu et al. 1998),
whereas other studies found mainly B.
burgdorferi s.s. using PCR based on the 5S/23S
rRNA intergenic spacer region (Lünemann et
al. 2001) or the flagellin gene (Jaulhac et al.
1996 and 2000). The most frequent genomic
groups in Europe B. afzelii and B. garinii occur
across the continent and the islands, whereas
the third frequent group B. burgdorferi s.s. has
only rarely been isolated in Eastern Europe (for
a survey, see Hubalek et al. 1997). Strains may
be very heterogeneous even within small areas
(Eiffert et al. 1995, Gern et al. 1999, Michel
et al. 2003, Rauter et al. 2002, Rijpkema et al.
1996). On the other side a focal prevalence of
certain species or subtypes was also observed
(Michel et al. 2003, Peter et al. 1995). Mixed infections
have been repeatedly observed in ixodid
ticks (for a survey, see Hubalek et al. 1997)
and sometimes also in specimens from patients
(Demaerschalck et al. 1995, Vasiliu et al. 1998,
Wilske et al. 1996). The heterogeneity of the
causative strains (Fig. 1) is a challenge for the
microbiological diagnosis of Lyme borreliosis
in Europe and must be kept in mind for development
of diagnostic tools such as PCR
primers and diagnostic antigens. For example,
ospA PCR has been widely used. Here, it is important
to be sure that not only representatives
of the three species are detected, but also the
different ospA-types of the heterogeneous B.
garinii group (Eiffert et al. 1995). In addition,
PCR should detect B. valaisiana and the recently
detected new genotype A14S since B.
valaisiana and genotype A14S might also be
pathogenic for humans, as suggested by positive
PCR results or cultures obtained from skin
biopsy specimens in a few studies (Rijpkema
et al. 1997, Wang et al. 1999a, Wilske et al.
2002). An ospA PCR for detection and differentiation
of the various European species and
OspA-types has been described by Michel
(2003).
Most of the proteins relevant for serodiagnosis
are heterogeneous. Interspecies amino
acid sequence identities are for example only
40–44% for DbpA (Osp17) and 54-68% for
OspC for representative strains of B. burgdorferi
sensu stricto, B. afzelii, and B. garinii
(strains B31, PKo, and PBi, respectively)
(Table 1). Especially DbpA has a much higher
amino acid sequence heterogeneity compared
to the DNA sequence heterogeneity indicating
immune selection. However, highly
heterogeneous proteins sometimes have conserved
immunogenic epitopes (e.g., the C6
peptide of VlsE) (Liang et al. 1999, Liang et
al. 2000).
GUIDELINES FOR THE
MICROBIOLOGICAL DIAGNOSIS
OF LYME BORRELIOSIS
The German Society of Hygiene and Microbiology
(DGHM) has recently published guidelines
for the microbiological diagnosis of Lyme
borreliosis written by an expert committee
(MiQ 12 Lyme-Borreliose) (Wilske et al. 2000).
The English version is accessible via internet
(
www.dghm.org/red/index.html?cname5MIQ). Except in cases with the pathognomic clinical
manifestation erythema migrans, the diagnosis
of Lyme borreliosis usually requires
confirmation by means of a microbiological diagnostic
assay. Antibody detection methods
mainly are used for this purpose, whereas detection
of the causative agent by culture isolation
and nucleic acid techniques is confined to
special situations, such as to clarify clinically
and serologically ambiguous findings. Application
of these methods should be reserved to
laboratories specialized in this type of examination.
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 217
FIG. 1. Heterogeneity of Lyme disease Borrelia species and OspA-serotypes in Europe. Data for skin, CSF, and ticks are based on analysis of culture isolates; data
for synovial fluid are based on analysis of ospA PCR amplicons; source of skin specimens is known in 46 patients (30 cases with erythema migrans, thereof were 1,
26, 1, and 2 cases infected with OspA-types 1, 2, 4, and 6, respectively; 16 cases with ACA, thereof were 1 and 15 cases infected with OspA-types 1 and 2, respectively).
(Modified from Figures 5 and 6 in Wilske et al., 2002, with permission of the publisher.)
SPECIMENS FOR THE
MICROBIOLOGICAL DIAGNOSIS
For culture and PCR, skin biopsy samples are
the most promising specimens. In general poor
results are obtained from body fluids with the
exception of PCR of synovial fluid. Examination
of urine (PCR, antigen detection) is not recommended
nor the examination (PCR or IFA)
of ticks removed from patients in order to decide
antibiotic prophylaxis (Brettschneider et
al. 1998, Kaiser et al. 1998, Klempner et al. 2001,
Wilske et al. 2000). Examination of ticks should
be performed only for epidemiological or other
scientific studies. For antibody determination,
serum or CSF can be investigated. CSF examination
should always be done together with
serum antibody analysis (determination of the
CSF/serum antibody index).
DIRECT DETECTION METHODS
Culture
B. burgdorferi can be cultivated in modified
Kelly’s medium (Preac-Mursic et al. 1991,
Wilske and Schriefer 2002). This, however, is a
very time-consuming method (generation time
of B. burgdorferi is about 7–20 h) characterised by
low sensitivity, especially in body fluids (Arnez
et al. 2001, Åsbrink et al. 1985, Karlsson et al.
1990, Strle et al. 1999, Zore et al. 2002) (Table 2).
Culturing may be of help in individual cases if
the clinical picture suggests Lyme borreliosis despite
a negative antibody assay (seronegative
Lyme borreliosis), for example, in atypical erythema
migrans, suspected acute neuroborreliosis
without detection of intrathecal antibodies or
in the case of suspected Lyme borreliosis in patients
with immune deficiencies.
PCR
There is no standardized method for the
preparation of specimens nor for performing
the PCR itself. For DNA amplification under
experimental conditions various target sequences
have been used by specialised laboratories,
for example, from plasmid-borne genes
such as ospA and ospB, or chromosomal genes
such as the genes for the flagellar protein or
p66 (clone 2H1), or from gene segments of
the 16S rRNA or the 5S/23S rRNA intergenic
218 WILSKE
TABLE 1. SEQUENCE IDENTITIES AMONG MAJOR IMMUNODOMINANT PROTEINS OF THE THREE GENOSPECIES
OF B. BURGDORFERI SENSU LATO (COMPARISON OF STRAINS B31, PKO AND PBI)
DNA sequences, Amino acid sequences,
Protein range (in %) range (in %)
DbpAa 51–63 40–44
OspCa 61–77 54–68
OspAa 85–86 78–81
p35a 74–85 65–80
BmpA (p39)a 91–93 89–90
p58a 90–97 90–97
Flagellin 94–95 96–97
Flagellin fragment (aa 129–251) nd 93–96
p83/100a 87–89 81–87
aSequence identities were calculated without the leader sequence of the lipoproteins.
TABLE 2. SENSITIVITY OF DIRECT PATHOGEN DETECTION METHODS IN LYME BORRELIOSIS
Specimen Sensitivity
Skin (erythema migrans, acrodermatitis) 50–70% when using culture or PCR
CSF (neuroborreliosis, stage II) 10–30% when using culture or PCR
Synovial fluida (Lyme arthritis) 50–70% when using PCR (culture is only extremely
seldom positive)
aHigher sensitivity within synovial tissue compared to synovial fluid.
spacer region (for a survey, see Schmidt et al.
1997). Borrelia PCR should allow diagnosis of
the Borrelia species, that is, the medical report
should contain information as to which of the
three species pathogenic for humans has been
found. The diagnostic sensitivity of PCR is
about the same as the sensitivity of culture.
Borreliae are detected with much more difficulty
from body fluids than from tissue specimens
by either PCR or culture (Arnez et al.
2001, Jaulhac et al. 1996, Karlsson et al. 1990).
Solely PCR of synovial fluid seems to surpass
culture significantly in sensitivity (Nocton et
al. 1994).
Sensitivity of culture and PCR
Table 2 gives a survey about sensitivity of direct
detection methods in clinical specimens
from patients with Lyme borreliosis. Culture and
PCR have the highest detection rates (50–70%) in
skin biopsies from patients with erythema migrans
or acrodermatitis chronica atrophicans
(Åsbrink et al. 1985a, van Dam et al. 1993, von
Stedingk et al. 1995, Weber et al. 1990, Zore et al.
2002). In contrast borreliae are detected by PCR
or culture in the CSF of only 10–30% of patients
with neuroborreliosis (Eiffert et al. 1995, Karlsson
et al. 1990, Wilske and Preac-Mursic 1993b).
CSF isolates are more frequently obtained from
patients with short duration of disease than from
patients with disease of long duration (Karlsson
et al. 1990). It is surprising that borreliae are detected
by PCR in 50–70% in the synovial fluids
of Lyme arthritis patients, but culture is rarely
successful (Eiffert et al. 1998, Vassiliu et al. 1998).
The best PCR results are obtained from synovial
tissue, not fluid (Jaulhac et al. 1996).
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 219
FIG. 2. Two-step approach in serodiagnosis. For criteria for positive, borderline, and negative blot, see text. (Modified
from Figure 6 in Wilske et al., 2000.)
ANTIBODY DETECTION
It is generally accepted that serological examination
should follow the principles of a two
step approach (Centers for Disease Control and
Prevention 1995, Johnson et al. 1996, Wilske et
al. 2000, Wilske and Schriefer 2002): (1) A serological
screening assay and (2) in the event of
a positive or equivocal result a confirmatory
assay. A sensitive ELISA is recommended,
which—in case it is reactive—should be confirmed
by the immunoblot (Fig. 2).
ELISA
The ELISA tests used for screening should be
at least second generation tests (Wilske et al.
2000), which have been improved with respect
to cross reactivity with other bacteria (e.g., extract
antigen with previous Reiter treponeme
adsorption) (Wilske et al. 1993a) or purified intact
flagella as antigen (Hansen et al. 1988).
Strains used as antigen source should express
OspC the immunodominant antigen of the IgM
response and DbpA an immunodominant antigen
of the IgG response (Wilske et al. 2000). Recently
specific recombinant antigens (i.e., VlsE)
or synthetic peptides (i.e., the C6 peptide derived
from VlsE) have been successfully used
in the United States (Bacon et al. 2003, Lawrenz
et al. 1999, Liang et al. 1999) and in a study with
European sera from patients with erythema migrans,
acrodermatitis, and arthritis (C6 peptide)
(Liang et al. 2000). Very recently also patients
with neuroborreliosis stage II have been
investigated with the C6 ELISA (IgG test) and
compared to the recombinant immunoblot
(Fingerle et al. 2002). Of 36 sera 31 were positive
by immunoblot and 34 by the C6-ELISA.
Two of the 31 immunoblot positive sera were
only borderline in the C6-ELISA, these sera had
antibodies against recombinant DbpA and p58
and DbpA and VlsE respectively. The C6-
ELISA appears to be sufficiently sensitive as a
screening test for IgG antibodies in patients
with neuroborreliosis if also borderline results
are included. However, VlsE has other immunodominant
epitopes besides the C6 region
that could improve diagnostic sensitivity; heterogeneity
of those immunodominant epitopes
especially must be considered in Europe (Göttner
et al. 2002). The IgM and IgG immune responses
of Lyme borreliosis patients in recombinant
immunoblots should suggest the best
combination of antigens for the development
of recombinant ELISAs.
Immunoblot
As a confirmatory assay the immunoblot
should have high specificity (at least 95%). If a
whole cell lysate is used as antigen, diagnostic
bands must be defined by monoclonal antibodies
(Fig. 3). In case of recombinant antigens,
identification of diagnostic bands is much easier.
For the whole cell lysate blot, strains expressing
immunodominant variable antigens
(OspC, DbpA5Osp17) in culture should be
used (i.e., strain PKo) (Wilske et al. 2000).
The immunoblot criteria recommended by
the Centers of Disease Control (CDC) for use
in the United States can not be used for Europe
(Hauser et al. 1997, Hauser et al. 1998, Robertson
et al. 2000). Dressler et al. (1994) have
shown in an immunoblot study that the immune
response of European patients is re-
220 WILSKE
FIG. 3. Standardization of the whole cell lysate immunoblot
with monoclonal antibodies (antigen, B. afzelii
strain PKo; control sera, G5IgG, M5IgM; monoclonal antibodies
(1–11). Arrows indicate closely neighbored proteins
difficult to distinguish. (Modified from Figure 3 in
Wilske et al., 2000, with permission of the publisher.)
stricted to a narrower spectrum of Borrelia proteins,
compared with that shown by American
patients. Using different serum panels (first
serum panel from Germany, second serum
panel from various European countries),
Hauser et al. demonstrated in two studies that
strain-specific interpretation rules must be defined
(Hauser et al. 1997, Hauser et al. 1998).
Figure 4 shows that immunoblot antibody
binding patterns vary considerably by strain
used as antigen. Thus different interpretation
rules are required in order to achieve equal
sensitivity and specificity when different
genospecies of Borrelia are used in preparing
the blot antigen.
Interpretation criteria for the immunoblot
recommended by the DGHM are published
in the MiQ 12 Lyme-Borreliose (Wilske et al.
2000). These are if B. afzelii strain PKo is used
as antigen source the following: The IgG blot
is positive if $2 bands of the following are present:
p83/100, p58, p43, p39, p30, OspC, p21,
Osp17, p14; the IgM blot is positive if $1 band
of the following is present: p41 (strong), p39,
OspC, DbpA (Osp17). Further interpretation
criteria (other strains, recombinant blot) are
available via internet (
www.dghm.org/red/index.
html?cname=MIQ). Borderline results are
reported if diagnostic bands are visible but the
criteria for a positive blot are not fulfilled. A
blot is negative if no diagnostic bands are visible.
Examples for IgM and IgG immunoblots are
shown in Figure 5. Patients with early manifestations
of acute neuroborreliosis have an
immune response restricted to only a few proteins.
Patients with late disease such as acrodermatitis
or arthritis have IgG antibodies to a
broad spectrum of antigens. Using recombinant
antigens for the immunoblot has several
advantages compared to the immunoblot using
whole cell lysate antigen: (a) specific antigens
can be selected (i.e., p83/100, BmpA), (b)
homologous antigens derived from different
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 221
FIG. 4. Heterogeneous IgG reactivity of sera from Lyme borreliosis patients in the immunoblot with different strains
of B. burgdorferi s.l as antigen. Strain PKa2 is B. burgdorferi s.s., strain PKo is B. afzelii, and strain PBi is B. garinii. (Modified
from Figure 2 in Hauser et al., 1997, with permission of the publisher.)
strains can be combined (i.e., DbpA (Osp17),
OspC, BmpA), (c) truncated antigens with
higher specificity can be designed (internal flagellin
fragments), and (d) antigens primarily
expressed in vivo can be used (i.e., DbpA, VlsE)
(Heikkilä et al. 2002, Schulte-Spechtel et al.
2002, Wilske et al. 1999). Commercial recombinant
antigen immunoblots are better standardised
than the conventional ones. If a broad
panel of recombinant antigens (including the
recently described VlsE) is used the recombinant
blot is at least as sensitive as the conventional
one. An in house recombinant IgG immunoblot
(Wilske et al. 1999) shown in Figure
6 could be significantly improved by addition
of recombinant VlsE and an additional DbpA
homologue (Schulte-Spechtel et al.2003). Purified
proteins and immunoblot reactivity with
sera from patients with acute neuroborreliosis
are shown in Figure 7. By addition of VlsE and
the DbpA homologue, sensitivity increased
from 52.7% to 86.1% in 36 cases of neuroborreliosis
stage II, while specificity remained unchanged.
Sensitivity was also increased compared
to the whole cell lysate immunoblot
(86.1% versus 63.8%). Thus the new recombinant
immunoblot is a considerable step towards
better standardisation and in addition is
more sensitive than the whole cell lysate blot
since homologous proteins from different
222 WILSKE
FIG. 5. Whole cell lysate immunoblot: IgM- and IgG immune response in patients with neuroborreliosis (lanes 1–7,
respectively), IgG immune response in patients with acrodermatitis (lanes 1-7). Lanes designated with 1 are IgG blots
to demonstrate a broad panel of diagnostic bands. (Modified from Figure 4 in Wilske et al., 2000, with permission of
the publisher.)
strains (especially those with low sequence
identities as DbpA, see Table 1) and in vivo expressed
proteins (as VlsE) are used as antigens.
Determination of the CSF/serum index
Methods taking into account potential dysfunction
of the blood-CSF barrier are suitable
for the detection of intrathecal antibody production
(Wilske et al. 1986, Hansen et al. 1990,
Hansen et al. 1991). Determination of the
CSF/serum index should be performed if neuroborreliosis
is considered, since a positive
CSF/serum index confirms involvement of the
CNS. It may be positive in some cases when
serum antibody tests are negative or equivocal,
especially if the patient’s illness has been of
short duration (Wilske et al. 2000). Depending
on the time elapsed since the first manifestation
of neurological symptoms, the IgG
CSF/serum index is positive for 80–90% of patients
(8–41 days after onset of the disease) up
to 100% of patients (.41 days after onset)
(Hansen et al. 1991). Detection of intrathecally
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 223
FIG. 6. Recombinant IgG immunoblot with sera from patients with neuroborreliosis stage II (old immunoblot); top
and bottoms panels are the same. (Modified from Figure 2 in Wilske et al., 1999, with permission of the publisher.)
FIG. 7. New recombinant antigens for an improved immunoblot:
VlsE from B. burgdorferi s.s. strain PKa2 and
DbpA from B. garinii strain PBr. (a) SDS- PAGE: A—recombinant
E. coli whole cell lysate; B—purified protein. (b)
Immunoblot with immune serum against E. coli, antigens as
in a. (c) Immunoblot with sera from three patients with acute
neuroborreliosis. (Modified from Figures 1 and 3 in Schulte-
Spechtel et al., 2003, with permission of the publisher.)
produced IgM antibodies shows a high degree
of sensitivity in neuroborreliosis with short duration
of symptoms, especially in children
(Christen et al. 1993, Hansen et al. 1991).
CSF/serum index determination is especially
important for diagnosis of chronic neuroborreliosis.
A positive IgG CSF/serum index
is essential for the diagnosis of chronic borreliosis
of the central nervous system (see
EUCALB case definitions, Stanek et al. 1996),
whereas chronic peripheral polyneuropathy is
usually negative for intrathecal antibody production
(Kristoferitsch et al. 1993).
Serological findings in various
stages of the disease
Interpretation of serological test results must
always be done in context with clinical data
(Table 3). Here case definitions are helpful
(Stanek et al. 1996, Wilske et al. 2000). In stage I
(erythema migrans), only 20–50% of the patients
are seropositive for IgM and/or IgG antibodies
(Åsbrink et al. 1985b, Hansen and Åsbrink 1989,
Weber et al. 1990). IgM antibodies usually prevail.
An exception might be the immune response
against the recently detected VlsE. In
American patients with erythema migrans IgG
responses against VlsE are observed earlier than
IgM responses (in acute erythema migrans, in
44% versus 19%, in convalescent erythema migrans
in 59% versus 43%) (Bacon et al. 2003). In
European patients with erythema migrans, an
early IgG response to VlsE was observed in
20 of 23 (87%) culture-confirmed EM cases, the
IgM response has not been investigated (Liang
et al. 2000). In stage II (acute neuroborreliosis)
seropositivity (IgM and/or IgG antibodies) increases
to 70–90% (Hansen et al. 1988, Wilske et
al. 1993a). In principle, patients with early manifestations
may be seronegative especially in case
of short duration of symptoms. Then serological
follow-up is recommended. Six weeks or more
after onset of symptoms, 100% of the patients
with stage II neuroborreliosis were seropositive
(Hansen et al.1988). In cases with late disease
(stage III, acrodermatis and arthritis), IgG antibodies
were detectable in all patients tested
(Hansen and Åsbrink 1989, Johnson et al. 1996,
Wilske et al. 1993a). A negative IgG test argues
against late Lyme borreliosis. Thus, a positive
IgM test without a positive IgG test is not diagnostic
for late disease manifestations (Wilske et
al. 2000). An exception could be the situation of
a patient who received inadequate antibiotic
therapy for early disease, but sufficient drug to
abrogate IgM to IgG class switch or very short
duration of clinical symptoms. Since serological
findings vary considerably and antibodies may
persist for long time in successfully treated individuals,
serological follow up is not suitable for
determining whether further antibiotic therapy
is warranted. The presence of specific antibodies
does not prove the presence of disease; a positive
antibody test may also be due to clinical or
subclinical infections in the past. The more nonspecific
the symptoms, the lower is the predictive
value of a positive serological test. Seropositivity
in the normal healthy population varies
with age and increased outdoor activities (e.g.,
in one study in Bavaria ,5% up to 20%) (Reimer
et al. 1999).
METHODS WHICH ARE NOT
RECOMMENDED FOR
MICROBIOLOGICAL DIAGNOSIS
Recently, various methods have been used in
commercially oriented laboratories that are not
224 WILSKE
TABLE 3. SENSITIVITY OF ANTIBODY DETECTION METHODS
IN THE DIAGNOSIS OF LYME DISEASE
Stage Sensitivity Remarks
I 20–50% Predominance of IgM
II 70–90% In cases of short disease duration
predominance of IgM, in cases of long
disease duration predominance of IgG
III Nearly 100% Usually solely IgGa
aThe presence of IgM antibodies without IgG antibodies is not diagnostic for
late disease; for possible exceptions, see text.
sufficiently evaluated for diagnostic purposes.
Among them are the antigen tests in body fluids,
PCR of urine, and lymphocyte transformation
tests. These tests are not recommended
for microbiological diagnosis. They are unreliable
and some of them are in addition very expensive,
especially if used for therapy control
(Brettschneider et al. 1998, Kalish et al. 2003,
Klempner et al. 2001).
REFERENCES
Arnez, M, Ruzic-Sabljic, E, Ahcan, J, et al. Isolation of Borrelia
burgdorferi sensu lato from blood of children with
solitary erythema migrans. Pediatr Infect Dis J 2001;
3:251–255.
Åsbrink, E, Hovmark, A. Successful cultivation of spirochetes
from skin lesions of patients with erythema
chronicum migrans Afzelius and acrodermatitis chronica
atrophicans. Acta Pathol Microbiol Immunol Scand
B 1985; 93:161–163.
Åsbrink, E, Hovmark, A, Hederstedt, B. Serologic studies
of erythema chronicum migrans Afzelius and acrodermatitis
chronica atrophicans with indirect immunofluorescence
and enzyme-linked immunosorbent assays.
Acta Derm Venereol 1985; 65:509–514.
Bacon, RM, Biggerstaff, BJ, Schriefer, M, et al. Improved
serodiagnosis of Lyme disease by kinetic ELISAs using
recombinant VlsE1 or peptide antigens of Borrelia
burgdorferi compared with two-tiered testing. J Infect
Dis 2003; 187:1187–1199.
Brettschneider, S, Bruckbauer, H, Klugbauer, N, et al. Diagnostic
value of PCR for detection of Borrelia burgdorferi
in skin biopsy and urine samples from patients with
skin borreliosis. J Clin Microbiol 1998; 36:2658–2665.
Canica, MM, Nato, F, Du Merle, L, et al. Monoclonal antibodies
for identification of Borrelia afzelii sp. nov. associated
with late cutaneous manifestations of Lyme
borreliosis. Scand J Infect Dis 1993; 25:441–448.
Centers for Disease Control and Prevention. Recommendations
for test performance and interpretation from the
second National Conference on Serologic Diagnosis of
Lyme Disease. Morbid Mortal Wkly Rprt 1995; 44:590.
Christen, H-J, Hanefeld, F, Eiffert, H, et al. Epidemiology
and clinical manifestations of Lyme borreliosis in childhood.
A prospective multicentre study with special regard
to neuroborreliosis. Acta Paediatr 1993; 386:1–76.
Demaerschalck, I, Messaoud, AB, De Kesel, M, et al. Simultaneous
presence of different Borrelia burgdorferi
genospecies in biological fluids of Lyme disease patients.
J Clin Microbiol 1995; 33:602–608.
Dressler, F, Ackermann, R, Steere, AC. Antibody responses
to the three genomic groups of Borrelia burgdorferi
in European Lyme borreliosis. J Infect Dis 1994;
169:313–318.
Eiffert, H, Karsten, A, Thomssen, R, et al. Characterization
of Borrelia burgdorferi strains in Lyme arthritis.
Scand J Infect Dis 1998; 30:265–268.
Eiffert, H, Ohlenbusch, A, Christen, H-J, et al. Nondifferentiation
between Lyme disease spirochetes from vector
ticks and human cerebrospinal fluids. J Infect Dis
1995; 171:476–479.
Gern, L, Hu, CM, Kocianova, E, et al. Genetic diversity of
Borrelia burgdorferi sensu lato isolates obtained from
Ixodes ricinus ticks collected in Slovakia. Eur J Epidemiol
1999; 15:665–669.
Göttner, G, Schulte-Spechtel, U, Wilske, B. Antigenic and
genetic heterogeneity of the immunodominant surface
protein VlsE among European B. burgdorferi s.l. strains.
Int J Med Microbiol 2002; 292:105.
Goossens, HAT, van den Bogaard, AE, Nohlmans, MKE.
Evaluation of fifteen commercially available serological
tests for diagnosis of Lyme borreliosis. Eur J Clin
Microbiol. Infect Dis 1999; 18:551–560
Fingerle, V, Schulte-Spechtel, U, Wilske, B. Evaluation of
an ELISA based on the C6-peptide of VlsE for diagnosis
of early neuroborreliosis. Int J Med Microbiol 2002;
292 (Suppl. 34): 217.
Hansen, K, Åsbrink, E. Serodiagnosis of erythema migrans
and acrodermatitis chronica atrophicans by the
Borrelia burgdorferi flagellum enzyme-linked immunosorbent
assay. J Clin Microbiol 1989; 27:545–551.
Hansen, K, Cruz, M, Link, H. Oligoclonal Borrelia burgdorferi-
specific IgG antibodies in cerebrospinal fluid in
Lyme neuroborreliosis. J Infect Dis 1990; 161:1194–1202.
Hansen, K, Hindersson, P, Pedersen, NS. Measurement of
antibodies to the Borrelia burgdorferi flagellum improves
serodiagnosis in Lyme disease. J Clin Microbiol 1988;
26:338–346.
Hansen, K, Lebech, A-M. Lyme neuroborreliosis: a new
sensitive diagnostic assay for intrathecal sythesis of Borrelia
burgdorferi–specific immunoglobulin G, A, and M.
Ann Neurol 1991; 30:197–205.
Hauser, U, Lehnert, G, Lobentanzer, R, et al. Interpretation
criteria for standardized western blots for three european
species of Borrelia burgdorferi sensu lato. J Clin
Microbiol 1997; 35:1433–1444.
Hauser, U, Lehnert, G, Wilske, B. Diagnostic value of proteins
of three Borrelia species (Borrelia burgdorferi sensu
lato) and implications for development and use of recombinant
antigens for serodiagnosis of Lyme borreliosis
in Europe. Clin Diagn Lab Immunol 1998; 5:456–462.
Heikkilä, T, Seppälä, I, Saxen, H, et al. Species-specific
serodiagnosis of Lyme arthritis and neuroborreliosis
due to Borrelia burgdorferi sensu stricto, B. afzelii, and B.
garinii by using decorin binding protein A. J Clin Microbiol
2002; 40:453–460.
Hubalek, Z, Halouzka, J. Distribution of Borrelia burgdorferi
sensu lato genomic groups in Europe, a review. Eur
J Epidemiol 1997; 13: 951–957.
Johnson, BJB, Robbins, KE, Balley, RE, et al. Serodiagnosis
of Lyme disease: accuracy of a two-step approach
using a flagella-based ELISA and immunoblotting. J Infect
Dis 1996; 174:346–353.
Jaulhac, B, Chary-Valckenaere, I, Sibilia, J, et al. Detection
of Borrelia burgdorferi by DNA amplification in synovial
tissue samples from patients with Lyme arthritis.
Arthritis Rheum 1996; 39:736–745.
Jaulhac, B, Heller, R, Limbach, FX, et al. Direct molecular
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 225
typing of Borrelia burgdorferi sensu lato species in synovial
samples from patients with Lyme arthritis. J Clin
Microbiol 2000; 38:1895–1900.
Kaiser, R. Teilnehmer der Expertenkonferenz: Frühsommermeningoenzephalitis
und Lyme-Borreliose-Právention
vor und nach Zeckenstich. Dtsch med Wochenschr
1998; 123:847–853.
Karlsson, M, Hovind-Hougen, K, Svenungsson, B, et al.
Cultivation and characterization of spirochetes from
cerebrospinal fluid of patients with Lyme borreliosis J
Clin Microbiol 1990; 28:473–479.
Kalish, RS, Wood, J A, Golde, W, et al. Human T lymphocyte
response to Borrelia burgdorferi infection: no
correlation between human leukocyte function antigen
type 1 peptide response and clinical status. J Infect Dis
2003; 187:102–108.
Klempner, MS, Schmid, CH, Hu, L, et al. Intralaboratory
reliability of serologic and urine testing for Lyme disease.
Am J Med 2001; 110:217–219.
Kristoferitsch, W. Chronical peripheral neuropathy. In:
Weber, K, Burgdorfer, W, eds. Aspects of Lyme Borreliosis.
Berlin: Springer, 1993:219–227.
Lawrenz, MB, Hardham, JM, Owens, RT, et al. Human
antibody responses to VlsE antigenic variation protein
of Borrelia burgdorferi. J Clin Microbiol 1999; 37:3997–4004.
Liang, FT, Alvarez, AL, Gu, Y, et al. An immunodominant
conserved region within the variable domain of
VlsE, the variable surface antigen of Borrelia burgdorferi.
J Immunol 1999; 163:5566–5573.
Liang, FT, Aberer, E, Cinco, M, et al. Antigenic conservation
of an immunodominant invariable region of the
VlsE lipoprotein among european pathogenic genospecies
of Borrelia burgdorferi sl. J Infect Dis 2000;182:
1455–1462.
Lünemann, JD, Zarmas, S, Priem, S, et al. Rapid typing of
Borrelia burgdorferi sensu lato species in specimens from
patients with different manifestations of Lyme borreliosis.
J Clin Microbiol 2001; 39:1130–1133.
Michel, H, Wilske, B, Hettche, G, et al. An ospA-polymerase
chain reaction/restriction fragment length
polymorphism-based method for sensitive detection
and reliable differentiation of all European Borrelia
burgdorferi sensu lato species and OspA types. Med Microbiol
Immunol 2003 (in press).
Nocton, JJ, Dressler, F, Rutledge, BJ, et al. Detection of
Borrelia burgdorferi DNA by polymerase chain reaction
in synovial fluid from patients with Lyme arthritis.
N Engl J Med 1994; 330:229–234
Ohlenbusch, A, Matuschka, F-R, Richter, D, et al. Etiology
of the acrodermatitis chronica atrophicans lesion
in Lyme disease. J Infect Dis 1996; 174:421–423.
Péter, O, Bretz, AG, Bee, D. Occurence of different
genospecies of Borrelia burgdorferi sensu lato in ixodid
ticks of Valais, Switzerland. Eur J Epidemiol 1995;
11:463–467.
Pfister, H-W, Wilske, B, Weber, K. Lyme borreliosis: basic
science and clinical aspects. Lancet 1994; 343:1013–1016.
Preac-Mursic, V, Wilske, B, Reinhardt, S. Culture of Borrelia
burgdorferi on six solid media. Eur J Microbiol Infect
Dis 1991; 10:1076–1079.
Rauter, C, Oehme, R, Diterich, I, et al. Distribution of clinically
relevant Borrelia genospecies in ticks assessed by
a novel, single-run, real-time PCR. J Clin Microbiol
2002; 40:36–43.
Reimer, B, Marschang, A, Fingerle, V, et al. Epedimiology
of Lyme borreliosis in South-Eastern Bavaria (Germany).
Zent Bl Bakteriol 1999; 289:653–654.
Rijpkema SGT, Tazelaar, DJ, Molkenboer, MJCH, et al.
Detection of Borrelia afzelii, Borrelia burgdorferi sensu
stricto, Borrelia garinii and group VS116 by PCR in skin
biopsies of patients with erythema migrans and acrodermatitis
chronica atrophicans. Clin Microbiol Infect
1997; 3:109–116.
Rijpkema, S, Golubic, D, Molkenboer, M, et al. Idenfication
of four genomic groups of Borrelia burgdorferi sensu
lato in Ixodes ricinus ticks collected in a Lyme borreliosis
endemic region of northern Croatia. Exp Appl Acarol
1996; 20:23–30.
Robertson, J, Guy, E, Andrews, N, et al. European multicenter
study of immunoblotting in serodiagnosis of
Lyme borreliosis. J Clin Microbiol 2000; 38:2097–
2102.
Schmidt, BL. PCR in laboratory diagnosis of human Borrelia
burgdorferi infections. Clin Microbiol Rev 1997;
10:185–201.
Schulte-Spechtel, U. Lehnert, G, Liegl, G, et al. Significant
improvement of the recombinant Borrelia IgG immunoblot
by addition of VlsE and a DbpA homologue
derived from B. garinii for the diagnosis of early neuroborreliosis.
J Clin Microbiol 2003; 41:1299–1303.
Stanek, G, O’Connell, S, Cimmino, M, et al. European
Union concerted action on risk assessment in Lyme borreliosis:
clinical case definitions for Lyme borreliosis.
Wien Klin Wochenschr 1996; 108/23:741–747.
Steere, AC. Medical progress—Lyme disease. N Engl J
Med 1989; 321:586–596.
Strle, F, Nadelman, RB, Cimperman, J, et al. Comparison
of culture-confirmed erythema migrans caused by Borrelia
burgdorferi sensu stricto in New York State and
by Borrelia afzelii in Slovenia. Ann Intern Med 1999;
130:32–36.
Van Dam, AP, Kuiper, H, Vos, K, et al. Different
genospecies of Borrelia burgdorferi are associated with
distinct clinical manifestations of Lyme borreliosis. Clin
Infect Dis 1993; 17:708–717.
Vasiliu, V, Herzer, P, Rössler, D, et al. Heterogeneity of
Borrelia burgdorferi sensu lato demonstrated by an ospAtype-
specific PCR in synovial fluid from patients with
Lyme arthritis. Med Microbiol Immunol 1998;
187:97–102.
Von Stedingk, LV, Olsson, I, Hanson, HS, et al. Polymerase
chain reaction for detection of Borrelia burgdorferi
DNA in skin lesions of early and late Lyme borreliosis.
Eur J Clin Microbiol Infect Dis 1995; 14:1–5.
Wang, G, van Dam, AP, Dankert, J. Phenotypic and genetic
characterization of a novel Borrelia burgdorferi
sensu lato isolate from a patient with Lyme borreliosis.
J Clin Microbiol 1999; 37:3025–3028.
Wang, G, van Dam, AP, Schwartz, I, et al. Molecular typing
of Borrelia burgdorferi sensu lato taxonomic, epi-
226 WILSKE
demiological, and clinical implications. Clin Microbiol
Rev 1999; 12:633–653.
Weber, K, Preac-Mursic, V, Wilske, B, et al. A randomized
trial of ceftriaxone versus oral penicillin for treatment
of early Lyme borreliosis. Infection 1990; 18:91–96
Wilske, B. Microbiological diagnosis in Lyme borreliosis.
Int J Med Microbiol 2002; 291:33:114–119.
Wilske, B, Busch, U, Eiffert, H, et al. Diversity of OspA
and OspC among cerebrospinal fluid isolates of Borrelia
burgdorferi sensu lato from patients with neuroborreliosis
in Germany. Med Microbiol Immunol 1996;
184:195–201.
Wilske, B, Fingerle, V, Herzer, P, et al. Recombinant immunoblot
in the serodiagnosis of Lyme borreliosis,
comparison with indirect immunofluoerescence and
enzyme-linked immnosorbent assay. Med Microbiol
Immunol 1993; 182:255–270.
Wilske, B, Habermann, C, Fingerle, V, et al. An improved
recombinant IgG immunoblot for serodiagnosis of
Lyme borreliosis. Med Microbiol Immunol 1999;
188:139–144.
Wilske, B, Preac-Mursic, V. Microbiological diagnosis of
Lyme borreliosis. In: Weber, K, Burgdorfer, W, eds. Aspects
of Lyme Borreliosis. Berlin: Springer; 1993:267–300.
Wilske, B, Preac-Mursic, V, Göbel, UB, et al. An OspA
serotyping system for Borrelia burgdorferi based on reactivity
with monoclonal antibodies and OspA sequence
analysis. J Clin Microbiol 1993; 31:340–350.
Wilske, B, Schierz, G, Preac-Mursic, V, et al. Intrathecal
production of specific antibodies against Borrelia
burgdorferi in patients with lymphocytic meningoradiculitis
(Bannwarth’s syndrome). J Infect Dis 1986;
153:304–314.
Wilske, B, Schriefer, M. Borrelia. Murray, PR, Baron, EJ,
Jorgensen, JH, eds. In: Manual of Clinical Microbiology,
8th ed. Washington, DC: ASM Press; 2003:937–954.
Wilske, B, Zöller, L, Brade, V, et al. MIQ 12 Lyme-borreliose.
In: Qualitätsstandards in der mikrobiologisch infektiologischen
Diagnostik. Mauch, H, Lütticken, R, eds. Munich:
Urban & Fischer Verlag, 2000.
Zore, A, Ruzic-Sabljic, E, Maraspin, V, et al. Sensitiviy of
culture and polymerase chain reaction for the etiologic
diagnosis of erythema migrans. Wien Klin Wochenschr
2002; 114:606–609.
Address reprint requests to:
Bettina Wilske, M.D., Ph.D.
Max von Pettenkofer Institute
University of Munich
National Reference Center for Borreliae
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E-mail:
Bettina.Wilske@mvp-bak.med.unimuenchen.
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