Van alles en nog wat m.b.t. testen

[vonneke]

Correlation of tests for detection of Borrelia burgdorferi sensu lato infection in patients with diagnosed borreliosis

Ann Agric Environ Med. 2006

Chmielewska-Badora J, Cisak E, Wójcik-Fatla A, Zwoliński J, Buczek A, Dutkiewicz J.
Department of Occupational Biohazards, Institute of Agricultural Medicine, Jaczewskiego 2, 20-090 Lublin, Poland. jcb@galen.imw.lublin.pl

A group of 180 patients with diagnosed Lyme borreliosis were examined for the presence of infection with Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) by serologic tests with B. burgdorferi s.l. antigens (IgM-ELISA, IgG-ELISA, IgM-immunoblot, IgG-immunoblot) and by polymerase chain reaction (PCR, nested-PCR) for detection of B. burgdorferi s.l. DNA in peripheral blood. A total of 61.7 %, 53.9 %, 62.2 %, and 59.4 % of the examined patients' sera showed positive or borderline results in the serologic tests IgM-ELISA, IgG-ELISA, IgM-immunoblot, and IgG immunoblot, respectively. The results of the tests IgM-ELISA and IgM-immunoblot were significantly correlated (p < 0.001). A higher degree of the correlation (p < 0.000001) was found at the comparison of results obtained with IgG-ELISA and IgG-immunoblot. The correlation between the positive findings in the IgM-ELISA and detection with IgM-immunoblot the diagnostically important B. burgdorferi s.l. OspC surface protein was relatively low but statistically significant (0.01 < p < 0.05). Much higher correlation was found between the positive findings in the IgG-ELISA and detection with IgG-immunoblot other diagnostically important B. burgdorferi s.l. antigen, the VlsE protein (p < 0.000001). The presence of B. burgdorferi s.l. DNA was found by PCR in 20 out 180 examined blood samples (11.1 %). No correlation was found to exist between the PCR results and the results of any of the serologic tests for detection of anti B. burgdorferi s.l. antibodies of IgM class. PCR results correlated significantly at a relatively low level (0.01 < p < 0.05) with the results of IgG-ELISA, but not with the results of IgG-immunoblot with regard to total reactions (0.2 < p < 0.1). By contrast, a distinctly significant correlation was found between the PCR results and detection of the VlsE protein with IgG-immunoblot (0.001 < p < 0.01). In conclusion, the results of the present study suggest that antibodies of IgG class are the most reliable marker in laboratory diagnostics of Lyme borreliosis, in particular those directed against VlsE surface protein of Borrelia burgdorferi sensu lato.

PMID: 17196006

[vonneke]

Rapid Differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi Sensu Stricto by LightCycler Fluorescence Melting Curve Analysis of a PCR Product of the recA Gene

Abstract

To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recA gene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from B. burgdorferi reference strains and 16 were isolated from B. burgdorferi strains cultured from Ixodes ricinus ticks; 13 were directly isolated from nine human biopsy specimens and four I. ricinus tick midguts. The melting temperature of B. garinii was 2°C lower than that of B. burgdorferi sensu stricto and B. afzelii. Melting curve analysis offers a rapid alternative for identification and detection of B. burgdorferi sensu lato genospecies

http://www.pubmedcentral.nih.gov/articl ... rtid=87021

[vonneke]

Pediatr Infect Dis J. 2008 Jun 5. [Epub ahead of print]Links

Improved Laboratory Diagnostics of Lyme Neuroborreliosis in Children by Detection of Antibodies to New Antigens in Cerebrospinal Fluid.

Skogman BH, Croner S, Forsberg P, Ernerudh J, Lahdenne P, Sillanpää H, Seppälä I.

From the *Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University; †Center for Clinical Research Dalarna, Falun, Sweden; ‡Hospital for Children and Adolescents, Helsinki University Central Hospital; §Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki; ¶HUSLAB Laboratory Services, University Central Hospital, Helsinki, Finland; #Dr. Croner died November 29, 2007.

BACKGROUND:: Laboratory diagnostics in Lyme neuroborreliosis need improvement. We hereby investigate 4 new recombinant or peptide Borrelia antigens in cerebrospinal fluid in children with neuroborreliosis to evaluate their performance as diagnostic antigens. METHODS:: An enzyme-linked immunosorbent assay was used to detect IgG antibodies to recombinant decorin binding protein A (DbpA), BBK32, outer surface protein C (OspC), and the invariable region 6 peptide (IR6). The recombinant antigens originated from 3 pathogenic subspecies; Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto. Cerebrospinal fluid and serum from children with clinical features indicative for neuroborreliosis (n = 57) were analyzed. Classification of patients was based on clinical symptoms and laboratory findings. Controls were children with other neurologic diseases (n = 20) and adult patients with no proven infection (n = 16). RESULTS:: Sensitivity for DbpA was 82%, for BBK32 70%, for OspC 58% and for IR6 70%. Specificities were 94%, 100%, 97%, and 97%, respectively. No single antigen was superior. When new antigens were combined in a panel, sensitivity was 80% and specificity 100%. The reference flagella antigen showed a sensitivity of 60% and a specificity of 100%. Over all, the B. garinii related antigens dominated. CONCLUSIONS:: Recombinant DbpA and BBK32 as well as the peptide antigen IR6 perform well in laboratory diagnostics of neuroborreliosis in children. New antigens seem to improve diagnostic performance when compared with the routine flagella antigen. If different antigens are combined in a panel to cover the antigenic diversity, sensitivity improves further and a specificity of 100% can be achieve.

PMID: 18536620 [PubMed - as supplied by publisher]

[vonneke]

J Peripher Nerv Syst. 2008 Mar;13(1):81-91. Links

Diagnostic value of sural nerve biopsy in patients with suspected Borrelia neuropathy.

Schäfers M, Neukirchen S, Toyka KV, Sommer C.
Department of Neurology, University of Würzburg, Würzburg, Germany.

Peripheral neuropathy is a recognized but incompletely understood manifestation of borreliosis. As the pathology of this neuropathy has been described only in small case series, the value of nerve biopsy findings for the pathologic diagnosis of Borrelia-associated neuropathy is unclear. We collected and investigated 21 patients with peripheral neuropathy and with typical clinical and serologic signs of neuroborreliosis [Borrelia neuropathy (BN)]. Standard histology and immunohistochemistry were performed on sural nerve biopsies using antibodies to CD4, CD68 and membrane attack complex C5b-9, intercellular adhesion molecule (ICAM)-1, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6. Nine patients with idiopathic vasculitic neuropathy (VN) and 14 with idiopathic axonal neuropathy (AN) served as disease controls. In BN, the characteristic histology was that of an AN with transmural or perivascular lymphocytic infiltration of nerve vessels. In BN, but less in VN and AN, perineurial thickening and neovascularization were observed. For BN but not for VN, this thickening correlated with increased perineurial immunoreactivity (IR) to TNF-alpha, C5b-9, and ICAM-1. In comparison to AN, both BN and VN displayed increased perineurial T-cell infiltration and human leukocyte antigen (HLA)-DR3-IR. In the endoneurium, cytokine (IL-1beta, IL-6, TNF-alpha), HLA-DR3, and ICAM-1 expression was more pronounced in VN but not in BN. The neuropathy in patients with neuroborreliosis resembles idiopathic VN but shows some distinctive features. None of the findings of this study are disease specific but as a pattern may help support the diagnosis of inflammatory neuropathy in patients with serological evidence for Borrelia infection.

PMID: 18346234

[vonneke]

http://jmm.sgmjournals.org/cgi/reprint/51/9/731.pdf


Recombinant OspC from Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii in the serodiagnosis of Lyme borreliosis

Genes for the outer-surface protein C (OspC) from three north European human
isolates of Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii were cloned and
sequenced. Polyhistidine-tagged recombinant OspC (rOspC) proteins were produced in
Escherichia coli and used, after biotinylation, as antigens on streptavidin-coated plates in
enzyme-linked immunosorbent assays (ELISA). In IgM ELISA, 30% (5=17) and 35%
(6=17) of patients with erythema migrans (EM) in the acute or convalescent phase,
respectively, reacted with one to three rOspCs. Of the patients, 53% (8=15) with
neuroborreliosis (NB) and 53% (8=15) with Lyme arthritis (LA) had IgM antibodies to
OspC. The immunoreactivity was stronger against rOspC from B. afzelii and B. garinii
than against rOspC from B. burgdorferi sensu stricto. In early Lyme borreliosis (LB),
rOspC and flagella performed equally well in detecting IgM antibodies. Cross-reactive
antibodies to rOspC were observed in serum samples from patients with rheumatoid
factor positivity and with syphilis or Epstein–Barr virus (EBV) infection. In IgM
ELISA, thiocyanate in the serum dilution buffer reduced EBV-associated non-specific
positive reactions. Of the patient sera examined in IgG ELISA, 30% (5=17) with EM in
the acute phase, 35% (6=17) with EM in the convalescent phase, 33% (5=15) with NB
and 60% (9=15) with LA were positive. Because of the heterogeneity of OspC, a
polyvalent antigen with several OspC variants from at least B. afzelii and B. garinii is
needed to improve the sensitivity of OspC ELISA in the serodiagnosis of LB in Europe.


The outer-surface protein C (OspC) of Borrelia
burgdorferi has been found to induce an early IgM
response [4–9]. In people vaccinated with OspA, OspC
antibody assay has been proposed for discrimination
between infection with B. burgdorferi sensu stricto and
a vaccination response [9]. In the serodiagnosis of
European LB with WCL immunoblots, specific antibody
responses to OspC of the three borrelial
genospecies as part of a combination of antigens have
been reported to increase sensitivity [10]. The best
combination of antigens for IgM WCL immunoblot
included OspC from B. afzelii and B. garinii strains.
However, false positive IgM reactions to OspC have
occurred both in ELISA [6, 9] and in immunoblot [11]
studies on patients with mononucleosis. In an IgM
ELISA, Rauer et al. [8] reported improved specificity
over WCL when recombinant OspC (rOspC) was
combined with a 14-kDa flagellin fragment. Two
peptide antigens from the conserved regions of the
OspC sequence have been evaluated in IgM ELISA, an
amino-terminal peptide at position 9–22 [12] and a
carboxy-terminal decapeptide [13]. Both peptide antigens
have shown improved discrimination between LB
patients and controls in preliminary studies

In IgG serology, a single rOspC antigen has proved to
be beneficial in some studies [4, 5, 13, 14] but has
shown low sensitivity in others [6, 9]. Recombinant
chimeric borrelial proteins, including fragments from
OspC, have also been studied as antigens in a combined
IgG-IgM ELISA [15]. Recently, a rapid immunochromatographic
assay based on these chimeric
proteins was suggested to be as sensitive as, and more
specific than, the commercial WCL ELISA [16].

A problem concerning the use of OspC as a diagnostic
antigen is the extensive structural variation of this
molecule (sequence identity ranging from 62% to 80%)
within, as well as between, subspecies of B. burgdorferi
sensu lato [17–19]. European and North American
isolates of B. burgdorferi sensu lato have been classified
into at least 16 serotypes of OspC [20, 21]: six
OspC serotypes for B. burgdorferi sensu stricto, four
for B. afzelii and six for B. garinii. In comparison, only
one OspA serotype has been reported for each
subspecies of B. burgdorferi sensu stricto and B.
afzelii, and five for B. garinii [22].


The purpose of the present study was to evaluate the
advantage of combining variant rOspC antigens from
all three pathogenic borrelial species in an ELISA
panel. This report describes the cloning and expression
of OspC proteins from three European borrelial strains
representing B. burgdorferi sensu stricto, B. afzelii and
B. garinii, and the results of ELISA assays with these
OspC recombinants as antigens. It also shows the
reduction of non-specific reactions in rOspC IgM
ELISA with sample buffer containing sodium thiocyanate
(NaSCN).


Discussion
This study showed that in serological assays, the
abilities of rOspC and flagella to detect IgM antibodies
during early LB were comparable. With both antigens,
IgM serology is complicated by false-positive reactions,
a frequent problem with IgM antibodies in general. The
results of the present study suggest that NaSCN may be
able to decrease non-specific immunoreactivity and,
thus, to improve the IgM serodiagnosis. In contrast, the
sensitivity of OspC IgG serology remained lower than
that of the conventional flagella antigen, even though
the various OspCs covered all three pathogenic species
in the antigen panel. Most probably, the results reflect
the high sequence heterogeneity of OspC.
In the early stages of LB, antibodies against borrelial
proteins are observed in only a small proportion of
patients [3]. In the present study, OspC ELISA was
compared with flagella ELISA, because the performance
of this method has been at the same level as that
of other commercially available ELISA methods [29].
In the EM serum samples, sensitivity and specificity of
rOspC seemed to be approximately equal to those of
the flagella antigen. In previous studies, IgM antibodies
to rOspC have been observed in 25–80% of patients
with EM [4–9, 14, 30] and in 48–72% of patients with
NB [4, 6]. The variability of these results may be
explained partly by the various stages of the disease
represented by the patient samples or by the origin
(subspecies) of OspC used as antigen, or both. In the
present study, IgM antibodies were most frequently
detected with rOspC from B. afzelii and B. garinii, but
only rarely with rOspC from B. burgdorferi sensu
stricto. Recently, other recombinant proteins have also
been studied as antigens in IgM serology. An earlier
study on recombinant FlaA proteins [31] showed 20–
27% or 58% sensitivity in IgM ELISA for EM or NB
samples, respectively. Rauer et al. [8] combined recombinant
OspC and recombinant 14-kDa flagellin
fragment as ELISA antigens and observed an additive
effect on the sensitivity of IgM serology.


In the present study, the specificity of OspC ELISA was
evaluated with control samples from patients with
syphilis, EBV infection, RF positivity or SLE, and
from healthy blood donors. EBV was especially
problematic, as the majority of samples from patients
seemed to cause non-specific reactions. Other controls
produced mainly low false-positive results. The proportion
of positive EBV patients in rOspC ELISA
decreased from 73% to 46% when NaSCN-BSA buffer
was used, and those still positive were only marginally
above the cut-off value. NaSCN did not have a
significant effect in the other control samples. Previously,
Goossens et al. [29] have suggested that, for
serodiagnosis of LB, the exclusion of false positivity by
targeted assays for EBV, syphilis infections and RF
positivity would be more successful than the two-tier
assay by successive ELISA and WB recommended by
CDC [32

A prominent feature of IgG serology was the
predominance of IgG immune reactions against OspC
from B. afzelii. The high prevalence of B. afzelii in
Scandinavia may account for this finding [23]. In
published studies, the proportion of positive samples
against single recombinant OspC has varied between
5% and 42% early in the disease [4–6, 9, 14] and
between 6% and 51% in disseminated disease [4, 6].
The findings in the present study concur with these
results, showing 6–35% positive results in the acute
phase and 18–53% in the convalescent phase of LB,
depending on the various rOspC antigens employed. It
is likely that maturation of the IgG immune response
with time would direct the specificity of the antibodies
preferentially toward epitopes of the immunising strain.
Fung et al. [4] found an IgG response in rOspC ELISA
(rOspC from B. burgdorferi sensu stricto strain 297) in
chronic LA patients but less frequently in chronic NB
patients. In keeping with these results, in the present
rOspC IgG ELISA, the patients with LA had antibodies
more frequently than the patients with EM or NB. This
may have been due to the more chronic stage of the
disease in LA than in EM or NB.

The present study used an ELISA with streptavidincoated
plates and rOspC bound to it via a biotin tag.
This procedure improved the binding of rOspC to the
ELISA plate surface (data not shown). Alternatively
Wienecke et al. [9] successfully used covalent coupling
of rOspC to plastic. The poor binding of the rOspC
constructs to plain plastic in the present study may be
associated with the low content of aromatic amino
acids in OspC. Similarly, weak binding of OspC to
nitrocellulose WCL immunoblot strips in the presence
of SDS in the transfer buffer was observed (data not
shown).
In conclusion, this study implies that, depending on the
epidemiological situation, all pathogenic borrelial
species should be covered if OspC antigens are
employed in the serology of LB. We suggest that in
the European context, on account of the heterogeneity
of OspC, a polyvalent antigen with several OspC
variants from at least B. afzelii and B. garinii is needed
to improve the serodiagnosis of LB.

[vonneke]

http://rheumatology.oxfordjournals.org/ ... 38/11/1121


Clinical evaluation of guidelines and two-test approach for Lyme disease

A. A. M. Blaauw, A. M. van Loon1, J. F. P. Schellekens2 and J. W. J. Bijlsma
Department of Rheumatology and Clinical Immunology and
1 Department of Virology, University Medical Centre, PO Box 85500, 3508 GA Utrecht and
2 National Institute of Public Health and the Environment, Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, 3720 BA Bilthoven, The Netherlands


Abstract

Objective.

The diagnosis of Lyme disease should be based on objective clinical signs and symptoms. In a clinical study, we have evaluated whether the recommended two-step approach for serodiagnosis of Lyme disease is useful in daily clinical practice and can influence clinical decision making

Methods.

The signs and symptoms of patients with ongoing musculoskeletal complaints, assumed by their referring physician or themselves to be attributable to active or chronic Lyme disease, and of patients diagnosed as having Lyme disease, were evaluated. On the basis of clinical evaluation only, patients were classified into three groups: previous Lyme disease, active Lyme disease and no Lyme disease. Antibodies to Borrelia burgdorferi were determined by means of an enzyme-linked immunosorbent assay (ELISA), followed, when positive, by immunoblotting.


Results.

One hundred and three patients (41 males and 62 females, mean age 48.7 yr) participated in the study. Of the 49 patients classified as previous Lyme disease, 25 (51%) had antibodies to B. burgdorferi. All 10 patients with active Lyme disease had positive antibodies and 12 of the 44 patients (27%) classified as no Lyme disease had positive antibodies. No statistically significant differences were found between the percentage of positive immunoblots from patients with previous Lyme disease (72%) and patients with active Lyme disease (100%). In the group of no Lyme disease, five out of 12 patients had a negative immunoblot. Concerning serological testing, immunoblotting could have added additional information. However, immunoblotting did not influence clinical decision making in this group of patients.

Conclusion.

Immunoblotting did not influence clinical decision making for the 47 patients with antibodies to B. burgdorferi in this study



Introduction

The clinical manifestations of Lyme disease have been well documented since its first description as a distinct entity in 1977 [1, 2]. In Europe, at least three distinct species of Borrelia burgdorferi sensu lato can cause the clinical syndrome of Lyme disease: B. burgdorferi sensu stricto, B. garinii and B. afzelii. Clinical manifestations correlate with the genospecies causing the infection. The clinical diagnosis of Lyme disease should be based on objective clinical signs and symptoms. Too often, the diagnosis is made in patients with non-specific and atypical features, such as headache, fatigue, arthralgia and myalgia. This has led to `overdiagnosis' of Lyme disease [3–5].

In Europe, no clinical diagnostic criteria for Lyme disease have been developed so far. Criteria developed in the USA by the Centers for Disease Control and Prevention (CDC) for surveillance purposes are often used as clinical criteria in the USA and in Europe. Since it is rather difficult to culture B. burgdorferi from specimens other than erythema migrans lesions, especially in daily clinical practice, the presence of antibodies to B. burgdorferi may confirm the clinical diagnosis. Although serological testing for Lyme disease can be performed with a high degree of sensitivity, false-negative and false-positive results continue to be an important problem [3]. Patients with Lyme disease usually remain seropositive for IgG antibodies to B. burgdorferi for years, in some cases even permanently. In these patients, a positive test can lead to `overdiagnosis' and probably `overtreatment'. False-positive results have been reported for patients with rheumatoid arthritis, systemic lupus erythematosus, infectious mononucleosis, syphilis and other spirochaetal diseases [7]. In Europe, asymptomatic seropositivity determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays (IFA) has also been demonstrated in up to 20% of the normal population as well as people at risk, such as orienteers, hunters and foresters [8–11]. As a consequence, in the absence of typical clinical signs and symptoms, diagnostic testing for antibodies to B. burgdorferi is of limited value [10


Although no consistent evidence suggests that Western blotting adds additional information to an accurate clinical history or to the results of a positive or indeterminate ELISA or indirect IFA if used in all patients, Dressler et al. and the CDC suggest that a two-step testing strategy should be used [13, 15, 16]. This two-step strategy is also adopted in Europe. Western blotting can detect and discriminate among antibodies to multiple antigens of B. burgdorferi. However, it remains unclear whether Western blotting can discriminate between true-positive test results and false-positive test results, and between active or previous infection with B. burgdorferi [17, 18]. Therefore, the clinical implications of this recommended two-step approach for the serodiagnosis of Lyme disease remain unclear and even questionable.

In this clinical study, we studied a patient population in which questions such as a true- or false-positive test result and active or previous infection are important. Clinical criteria were used as a gold standard, and ELISA and immunoblot test results were used and interpreted as in daily clinical practice. We have tried to assess whether immunoblotting should be performed in all patients with a positive ELISA and whether the suggested two-step approach can influence clinical decision making.


Patients and methods

Patients
Consecutive patients of the out-patient clinic of the Department of Rheumatology and Clinical Immunology of the University Medical Centre Utrecht, The Netherlands, were evaluated. Patients with a classic presentation of Lyme disease such as erythema migrans are, in general, diagnosed and treated by their general practitioners. Because of our special interest in clinical Lyme disease, the department serves as a secondary or tertiary referral centre for patients with presumable Lyme disease or for patients with persisting complaints thought to be chronic Lyme disease.

Patients eligible for this study were (i) patients with persisting musculoskeletal complaints assumed by their referring physicians to be attributable to active or chronic Lyme disease, (ii) patients who believed that they had active or chronic Lyme disease and (iii) patients with musculoskeletal complaints diagnosed as Lyme disease by our staff members.

All patients were seen by one of us (AAMB) who recorded a medical history and performed the physical examination. Questions were asked about past or recent tick exposure and the characteristic manifestations of Lyme disease. Clinical records and former medical correspondence were studied.

In the daily routine of our department, it is not possible to culture B. burgdorferi from clinical specimens. Genome amplification methods and urine antigen analysis are only used in experimental studies. It was therefore decided that clinical criteria were to be used as the `gold standard'. Patients were classified according to clinical criteria independent of the serological test results of (ELISA) antibodies to B. burgdorferi both at the time of (presumed) diagnosis and during the study. Because of the special referral pattern of patients seen in our department, we expected that if these patients had antibodies to B. burgdorferi, these would be IgG antibodies. IgM antibodies were not determined. If patients had (ELISA) IgG antibodies to B. burgdorferi, an IgG immunoblot was performed.

No exact figures about the incidence and prevalence of Lyme disease in The Netherlands exist. The incidence is assumed to be low and is estimated to vary from 0.01% in the general population to 2–9% in populations at risk and patients with monoarticular arthritis

Clinical criteria
On the basis of objective clinical signs and symptoms only, the patients were divided into three groups: previous Lyme disease, active Lyme disease and no Lyme disease.

The clinical criteria for Lyme disease according to literature references were defined as follows [6]: erythema migrans is defined as a red macula or papule that expanded over a period of days to weeks to form a large annular lesion, at least 5 cm in size, often with partial central clearing [1, 20, 21]. The skin lesion should have been confirmed by a physician or recognized by the patient from pictures of erythema migrans [10]. Early neurological involvement includes lymphocytic meningitis, cranial neuritis and radiculopathy, accompanied by pleocytosis of the cerebrospinal fluid [22–24]. Chronic neuroborreliosis includes encephalopathy with memory impairment, polyneuropathy with radicular pain or distal paraesthesiae and leucoencephalitis with spastic paraparesis [25]. Lyme arthritis is defined as recurrent, brief attacks of objective joint swelling in one or a few joints, especially the knees, possibly followed by chronic synovitis [26]. Lyme carditis is characterized by fluctuating degrees of atrioventricular nodal block that resolved in days to weeks, possibly associated with myocarditis [27]. Uveitis is defined as intermediate uveitis with characteristic spider web vitritis confirmed by an ophthalmologist [28]. Acrodermatitis chronica atrophicans (ACA) is the characteristic bluish-red discolouration, often with a doughy infiltration and progressing to atrophy or sclerodermic changes [21]. Borrelial lymphocytoma is the typical bluish-red tumour-like infiltrate [21]. For all patients with neurological, cardiac, eye or joint abnormalities, all other possible causes of the complaints had to be excluded. Skin lesions that developed immediately after a tick bite were not considered to be erythema migrans. Aspecific symptoms such as fatigue and arthralgia, fever, headache and paraesthesiae as a single symptom, palpitations, bundle branch block on electrocardiography, myocarditis and symmetrical polyarthritis were not accepted as clinical criteria for Lyme disease unless accompanied by objective characteristic Lyme manifestations mentioned above. A tick bite alone was also not accepted as a clinical criterion.


A clinical diagnosis of previous Lyme disease was made if at least one of the clinical criteria was present in the past and no objective manifestations of Lyme disease were present at the time of the study. Active Lyme disease was diagnosed when at least one of the clinical criteria was present at the time of the study. When no clinical criteria were present or had been present in the past, patients were assumed to have no Lyme disease.

Enzyme-linked immunosorbent assay
In daily clinical practice, our hospital uses a commercial ELISA for the detection of antibodies to B. burgdorferi (Dako A/S, Glostrup, Denmark) [29, 30]. In all cases, the IgG antibody response to B. burgdorferi was determined by this commercial ELISA. If a positive test result was obtained, the Treponema pallidum haemagglutination assay was performed to exclude false positivity due to antibodies to T. pallidum.

Patients were tested at the time they were seen in our out-patient clinic. Because of our special patient population, it is possible that patients are seen not only if they have active symptoms of Lyme disease but also years after a (presumed) diagnosis of Lyme disease. No follow-up samples were determined.

Western blot analysis
For all patients with IgG antibodies to B. burgdorferi according to the ELISA assay, IgG immunoblot analysis was performed as described in the literature [16, 30]. The IgG immunoblot was considered positive if at least five of the following eight bands were present: 17 kDa, 22 kDa, 31 kDa, 34 kDa, 39 kDa, 41 kDa, 58–74 kDa and 92 kDa, or if four bands were positive including 17 kDa or 22 kDa or 39 kDa or 92 kDa. One or more bands in the 58–74 kDa region were considered as one band [31, 32]. IgG immunoblot was considered negative if three bands were positive excluding 17 kDa, 22 kDa, 39 kDa or 92 kDa, and if less than three bands were found to be positive. IgG immunoblot was considered equivocal if four positive bands (31 kDa, 34 kDa, 41 kDa and 58–74 kDa) were found, or only three bands including 17 kDa or 22 kDa or 39 kDa or 92 kDa. Using the above criteria, the sensitivity of IgG immunoblot is 45% in early Lyme disease and 95% in disseminated or late Lyme disease; the specificity of IgG immunoblot is 98% (unpublished data, personal communication). The control population in which IgG immunoblot was tested consisted of healthy donors, pregnant women and potentially cross-reactive sera of patients with syphilis, leptospirosis or autoimmune diseases. The clinical diagnosis defined by a clinical expert was considered as the `gold standard'.

In our hospital, the clinician receives the results of immunoblotting interpreted by the laboratory as either positive, negative or equivocal. The clinician is unaware of which bands of the immunoblot are positive or negative. No follow-up samples were determined


Statistics
For categorical data, Fisher's exact test was used to test for differences between groups. A P value of <0.05 was considered statistically significant


Results

In a 4 yr period (April 1994–April 1998), 105 patients who met either one of the three eligibility criteria were seen in our department. In two patients, the ELISA for antibodies to B. burgdorferi was not performed at study entry. Of the remaining 103 patients, 41 (40%) were male and 62 (60%) were female. The mean age of the patients was 48.7 yr (range 6–82 yr).

The 103 patients were grouped according to the clinical criteria: 49 patients were classified as previous Lyme disease (48%), 10 patients as active Lyme disease (10%) and 44 as no Lyme disease (42%) (Table 1). Forty-seven of the 103 patients (46%) had IgG antibodies to B. burgdorferi according to the ELISA at study entry: 25 patients with previous Lyme disease (51%), all 10 patients with active Lyme disease (100%) and 12 patients (27%) with no Lyme disease (Table 1). Results are summarized in Table


Patients classified as previous Lyme disease
No differences for gender or mean age were found for the 49 patients classified as previous Lyme disease who did or did not have antibodies to B. burgdorferi.
Of the 25 patients with antibodies to B. burgdorferi, 15 had had early symptoms of Lyme disease defined as signs or symptoms within <1 yr after possible exposure. Fourteen of these patients had erythema migrans and one patient had neuroborreliosis. Ten patients had late symptoms defined as signs and symptoms >1 yr after possible exposure. Two of these 10 patients had ACA, one had uveitis and seven had mono- or oligoarthritis of the knees (six patients) and elbow (one patient).

Of the 24 patients without antibodies to B. burgdorferi, 19 had erythema migrans, one had erythema migrans and neuroborreliosis, and three had neuroborreliosis. One patient had late symptoms: arthritis of the knee.

Patients with antibodies to B. burgdorferi were seen on average 3.9 yr after the diagnosis of Lyme disease was made elsewhere, patients without antibodies on average after 4.6 yr. At study entry, none of these 49 patients had objective signs of Lyme disease. In 51% of these patients, antibodies to B. burgdorferi could be detected after all these years.

Patients with active Lyme disease
Ten patients were classified as having active Lyme disease based on clinical symptoms: one patient had neuroborreliosis with radiculopathy and lymphocytic meningitis, three patients had ACA, six patients had episodes of recurrent arthritis of the knee, one of them after erythema migrans. All patients received appropriate antibiotic treatment. All patients with active Lyme disease had IgG antibodies to B. burgdorferi. All patients contracted Lyme disease in Europe, except for the patient with neuroborreliosis who developed signs and symptoms after a trip to the east coast of the USA


Patients with no Lyme disease
According to our clinical criteria, 44 patients did not have objective signs of Lyme disease at study entry. Based on clinical evaluation and chart and correspondence review, they did not have objective signs or symptoms either, at the time a presumable diagnosis of Lyme disease was made elsewhere. Thirty-two patients did not have IgG antibodies to B. burgdorferi at the time of study entry. However, 12 of these 44 patients (27%) did have IgG antibodies to B. burgdorferi

Immunoblot
An IgG immunoblot was performed in all 47 patients with IgG antibodies to B. burgdorferi. Of the 25 patients classified as previous Lyme disease, 18 had a positive immunoblot (72%) and seven had a negative immunoblot. The seven patients with a negative immunoblot did have objective signs and symptoms of Lyme disease in the past. ELISA test results of these patients cannot be regarded as false positive in these seven patients. Because of the time interval between the diagnosis of Lyme disease and the time of the study, a waning humoral immunity with a `rest-response' to the 41 kDa protein is possible. All 10 patients with active Lyme disease had a positive immunoblot (100%). No statistically significant difference was found between the percentage of positive immunoblots from patients with previous Lyme disease and those with active Lyme disease. Based on clinical criteria, however, it was possible to differentiate between previous and active Lyme disease. Therefore, the use of the immunoblot assay in these two patient groups did not add any additional information to the results of the ELISA, and did not influence clinical decision making.

In the group of no Lyme disease, seven of 12 patients (58%) had a positive immunoblot and five patients (42%) had a negative immunoblot. Concerning serological testing, immunoblotting could have added additional information for five patients in this group of no Lyme disease: ELISA results were probably false positive. However, based on the clinical criteria (independent of any serological results), these patients did not have signs of Lyme disease. Therefore, immunoblotting did not influence clinical decision making for this group of patients


Seven patients classified as no Lyme disease had positive ELISA IgG antibodies to B. burgdorferi and a positive IgG immunoblot. Clinical characteristics of these patients are discussed briefly. A 37-yr-old male had systemic sclerosis and arthralgia. Because of antibodies to B. burgdorferi, he was treated with several courses of ceftriaxone i.v. without any clinical effect. A 40-yr-old male had symmetrical, rheumatoid factor-negative polyarthritis. Treatment with doxycycline and ceftriaxone had no effect. He was started on sulphasalazine as a second-line anti-rheumatic drug with excellent response. A 72-yr-old male had symmetrical, rheumatoid factor-positive, erosive polyarthritis classified as typical rheumatoid arthritis. A 56-yr-old male had eczema and arthralgia. He never had objective signs of arthritis. A 17-yr-old male was referred because of a tick bite in the past and antibodies to B. burgdorferi. He did not have any complaints or objective signs or symptoms. A 52-yr-old female had arthralgia without objective arthritis. A 23-yr-old female had arthralgia and painful knees without signs of arthritis, classified as chondromalacia patellae.

The positive results of ELISA and immunoblot in these seven patients have to be considered as true, but asymptomatic, positive. On clinical criteria, we do not feel that these patients have or had Lyme disease



Discussion

In this study, we evaluated recommendations for the use of a two-test approach for Lyme disease in a clinical situation. We especially assessed whether clinical decision making was influenced by the suggested two-test approach: a positive or undetermined ELISA should be followed by Western blot. The results indicate that immunoblotting does not reliably discriminate between previous infection and active infection with B. burgdorferi. Clinical decision making was not influenced by the results.

Twelve out of 44 patients classified as no Lyme disease at study entry had IgG antibodies to B. burgdorferi. This percentage is in contrast with the percentage of antibodies to B. burgdorferi found in the Dutch population and patients at risk [8, 10, 11]. This high percentage is probably due to referral bias. It is likely that patients with antibodies are referred, as patients without antibodies are not. Seven of these 12 patients did have a positive immunoblot. Based on their medical history and the clinical picture, these patients had not had Lyme disease. In this group of patients classified as no Lyme disease, the use of immunoblotting could have added additional information for the five patients whose immunoblot was negative. However, based on clinical criteria, these patients did not have Lyme disease at study entry or in the past. Thus, in this group of patients with no Lyme disease, the immunoblot did not influence clinical decision making either. Our study supports and strengthens the suggestion that the diagnosis of Lyme disease should be made primarily on clinical signs and symptoms [33]. Neither the results of serological testing nor the results of immunoblotting can be interpreted adequately without knowledge of clinical manifestations. Our results confirm the findings of Gern et al. [34] who stated that immunoblotting is of little help in diagnosing Lyme disease in populations at risk as well as in endemic areas where seropositivity for B. burgdorferi is common


A few points should be stressed. First, there is no gold standard for the clinical and laboratory diagnosis of Lyme disease. No generally accepted criteria have been developed, although `practice parameters' and guidelines for the diagnosis of patients with Lyme borreliosis of the nervous system and Lyme arthritis have been proposed and developed [22, 35]. Overdiagnosis as well as underdiagnosis of erythema migrans, considered as the hallmark of the disease, have been described [36, 37]. For this particular study, we used diagnostic criteria based on the literature as well as our own experience with Lyme disease patients [2, 3, 5, 12, 13, 21, 22]. We grouped our patients on the basis of these clinical criteria independently of the serological results, in an attempt to identify the possible surplus value of serology and immunoblotting. Patients could easily be assigned to one of the three groups previous Lyme disease, active Lyme disease and no Lyme disease.

Second, our study was performed in a referral hospital with a population of patients which is not representative of the total population of patients with disease due to infection with B. burgdorferi. However, these patients are a reflection of the problems which are encountered in the daily management of patients with (presumed) Lyme disease: active infection, past infection with persistent antibodies to B. burgdorferi, false-positive or asymptomatic IgG antibodies to B. burgdorferi. We have not provided any data on sensitivity, specificity, and the positive or negative predictive values of the ELISA and immunoblotting, because these data would only be applicable to a similar population and cannot be generalized for other populations. We used a standard commercial ELISA for this study as will be used in daily clinical practice by most physicians. We are aware that more sensitive assays will be available in the future. However, the possible surplus value of all these assays should be evaluated against clinical criteria.


Third, based on clinical criteria, we classified 44 patients as no Lyme disease. We used only objective signs and symptoms in our criteria. None of these 44 patients ever had any of these symptoms. This would have made their pre-test likelihood of Lyme disease <20%. These patients should not have been tested for the presence of antibodies to B. burgdorferi in the first place because a positive test is more likely to be clinically false positive than true positive [10, 13]. We assumed that it is likely that referring physicians use a negative result of ELISA for antibodies to B. burgdorferi to rule out the presence of Lyme disease and refer patients with a positive test result to our out-patient clinic.

Fourth, we studied a relatively small patient population with either positive or negative ELISA results. Larger study populations are needed for definite conclusions and for patients with possible indeterminate ELISA results. However, it can be expected that if clinicians determine the pre-test probability of Lyme disease in the diagnostic evaluation of a patient for Lyme disease, based on findings of a thorough clinical examination and knowledge of the incidence of Lyme disease in the population represented by the patient, testing for antibodies to B. burgdorferi can be avoided in patients with non-specific symptoms [13].

In view of the results of this study, we cannot recommend that, in a clinical situation, a positive ELISA for antibodies to B. burgdorferi should always be followed by immunoblotting. Immunoblotting did not influence clinical decision making for 47 patients with positive ELISA antibodies to B. burgdorferi. Results of serological testing and immunoblot analysis should not be interpreted without sufficient knowledge of the clinical picture of the patients tested.

[vonneke]

Vraag over een stukje uit bovenstaande tekst.


Hoe is dat nu in Nederland ? Houdt men zich hier nog steeds aan :

Western blot analysis
For all patients with IgG antibodies to B. burgdorferi according to the ELISA assay, IgG immunoblot analysis was performed as described in the literature [16, 30]. The IgG immunoblot was considered positive if at least five of the following eight bands were present: 17 kDa, 22 kDa, 31 kDa, 34 kDa, 39 kDa, 41 kDa, 58–74 kDa and 92 kDa, or if four bands were positive including 17 kDa or 22 kDa or 39 kDa or 92 kDa. One or more bands in the 58–74 kDa region were considered as one band [31, 32]. IgG immunoblot was considered negative if three bands were positive excluding 17 kDa, 22 kDa, 39 kDa or 92 kDa, and if less than three bands were found to be positive. IgG immunoblot was considered equivocal if four positive bands (31 kDa, 34 kDa, 41 kDa and 58–74 kDa) were found, or only three bands including 17 kDa or 22 kDa or 39 kDa or 92 kDa. Using the above criteria, the sensitivity of IgG immunoblot is 45% in early Lyme disease and 95% in disseminated or late Lyme disease; the specificity of IgG immunoblot is 98% (unpublished data, personal communication). The control population in which IgG immunoblot was tested consisted of healthy donors, pregnant women and potentially cross-reactive sera of patients with syphilis, leptospirosis or autoimmune diseases. The clinical diagnosis defined by a clinical expert was considered as the `gold standard'.

Of gebruikt men nu de criteria voor Europa ? :

However, these criteria cannot be adopted for diagnostics in Germany or Europe because they were developed using B. burgdorferi s.s. and American sera only. Dressler et al. have shown in an immunoblot study that the immune response of European patients is obviously restricted to a narrower spectrum of Borrelia proteins [8], 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 interpretation rules must be defined strain-specific [22, 24]. Thus different interpretation rules are required in order to achieve equal sensitivity and identical specificity values, resp., when different strains are used. The criteria established by Hauser have been re-evaluated by Kaiser and Brauer [31a], who also believe that they are more suitable for application to the European situation than the American rules [13].

Therefore, the rules for the whole-cell lysate immunoblot established by Hauser et al. are cited here as reliable interpretation criteria [22, 24].

According to this study, B. afzelii strain PKo is to be preferred to PBi (B. garinii) and PKa2 (B. burgdorferi s.s.) strains, because the former, showing the same sensitivity as the latter two, permits atwo-band criterion for the IgG test (at least two bands positive of p83/100, p58, p43, p39, p30, OspC, p21, Osp17 and p14).For PBi and PKa2 however, a one-band criterion applies (B. garinii PBi: at least one band positive of p83/100, p39, p30, OspC, p21, and p17; B. burgdorferi s.s. PKa2: at least one band positive of p83/100, p58, p 56, OspC, p21, and p17a). In addition, according to the general DIN recommendations on the immunoblot (DIN 58967, Part 40), at least a two-band criterion should be employed for positive evaluation of an immunoblot. The interpretation rules for the whole-cell lysate immunoblot with strain PKo are compiled in

For the recombinant IgG-immunoblot, too, a two-band criterion is recommended (cf. Table 9). The recommendations are based on comparative studies of the recombinant immunoblot and the whole-cell lysate immunoblot by Hauser

http://nrz-borrelien.lmu.de/miq-lyme/fr ... gical.html

[vonneke]

termedia.pl/showpdf.php?article_id=11571&filename=16_Frequency.pdf&priority=1 -


(ik geloof dat de link niet werkt maar weet niet hoe ik 'm goed moet krijgen.)
Bij google is het de zesde link van bovenaf http://www.google.nl/search?hl=nl&q=MAL ... eken&meta=



Frequency and specificity of the antibodies against Borrelia burgdorferi tested by Western blot method in patients with symptoms of arthritis

Abstract

Selected, highly specific antibodies to B. burgdorferi proteins are commonly used in diagnostic
serological tests for Lyme disease. From the point of view of diagnosing Borrelia burgdorferi infection,
highly immunogenic antibodies to some proteins detected in vivo after B. burgdorferi transmission to
the host human body seem to be very significant.
Knowledge on importance of proteins that are expressed in vivo alone has accumulated recently.
The proteins are: VlsE, BBA36, BB0323, Crasp3 and pG. The main goal of this study was to evaluate
the presence of IgM and IgG antibodies against expanded panel of Borrelia burgdorferi specific anti-
gens, including antigens that are expressed in vivo alone.
The study was conducted in the group of 25 patients diagnosed with borreliosis, hospitalized in
The Department of Infectious Diseases, Medical University of Lublin. The diagnosis of borreliosis
was based on patients history, clinical symptoms and ELISA serological test results. Testing for IgG
and IgM antibodies to Borrelia burgdorferi was performed using Immunoblot (Genzyme Virotech)
which included antigens for IgM: OspC, p39, EBV-VCA-gp 125; for IgG: VlsE, p39, p83, BBA36 (iv1),
BBO323 (iv2), Crasp3 (iv3), pG (iv4).
In that group IgG antibodies against full antigens panel VlsE, p39, p83, BBA36 (iv1), BB0323
(iv2), Crasp3 (iv3), pG (iv4) were observed in 3 patients (12%). In 12 patients (48%), in addition to
IgG against VlsE, p39 and p83, antibodies against BBA36 (iv1), BB0323 (iv2), Crasp3 (iv3), pG (iv4)
were detected with various frequency. The remaining 10 patients (40%) tested IgG positively based on
presence of VlsE and/or p39, p83. In spite of long term infection, the results revealed the presence of
IgM antibodies against OspC and p39 16 tested patients (64%), against OspC 5 tested patients
(20%), against p39 1 tested patients (4%).

We conclude that:
1. IgG antibodies to VlsE (antigen from in vivo group) are the most frequently produced during bor-
relia infection.
2. Membraneous proteins: p39 and p83 present in diagnostic serologic tests are reliable markers of
B. burgdorferi infection.
3. In vivo proteins alone used in diagnostic tests do not guarantee reliable results that either would
confirm or exclude B. burgdorferi infection.

(Centr Eur J Immunol 2008; 33 (4): 220-223



Introduction

The immune response to chronic B. burgdorferi in-
fection during late stage Lyme disese is still not well un-
derstood and generates a lot of questions and concerns.
In many cases natural defense mechanisms are not able
to eliminate invading pathogen which leads to a chro-
nic, long term illness. B. burgdorferi developed mecha-
nisms that allowed them to avoid complement destruc-
tion. Complement regulator-aquiring surface protein
(the Crasp proteins) are responsible for the potential of
complement inactivation. They can bind to regulatory
proteins that activate the complement, i.e. factor H and
factor H-like protein. Group Erps proteins (OspE, OspF,
Elps, p21, ErpA, ErpP) demonstrate similar properties.
The presence of membrane proteins that inactivate the
complement cascade is one of the major factors respon-
sible for B. burgdorferi transmission to the host human
body [1-4]. During early disseminated phase of borrelio-
sis as well as in its later phase other highly immunogenic
membrane associated proteins are detected, e.g. BB0323,
BmpA (p39), p83, BBA64, BBA66. Some of them are
used to diagnose borreliosis as markers of advanced sta-
ges of the disease in laboratory diagnosis [5].
Selected, highly specific antibodies to B. burgdor-
feri proteins are commonly used in diagnostic serologi-
cal tests for Lyme disease. From the point of view of
diagnosing Borrelia burgdorferi infection, highly im-
munogenic antibodies to proteins detected in vivo after
B. burgdorferi transmission to the host human body seem
to be very significant. Knowledge on importance of pro-
teins that are expressed in vivo alone has accumulated
recently. They proteins: VlsE, BBA36, BB0323, Crasp3
and pG [6].
VlsE antigen is used in the majority of commercial
ELISA and Western blot based assays. Other antigens
are not routinely included in diagnostic panels.
The main goal of this study was to evaluate the pre-
sence of IgM and IgG antibodies against expanded panel
of Borrelia burgdorferi specific antigens, including anti-
gens that are expressed in vivo alone.




Discussion

Antigens recognized in vivo are often treated as impor-
tant IgG serologic indicators in late stage borreliosis and
they could be used to evaluate immune response as they
relate to patients clinical status.
Thus a serologic test with those antigens involved cre-
ates better potential to evaluate immune response with ac-
count for clinical status of the patient.
Antigen VlsE is considered a reliable serologic mar-
ker of B. burgdorferi infection while IgM and anti-VlsE
IgG may coexist in both early and late stage of borre-
liosis. The detection VlsE antibodies can be performed
for all Borrelia burgdorferi s.l. pathogenic strains with
10 times lower false positive rate than for other Borrelia
antigens [7, 8].


In our study IgM anti-VlsE were not detected among
the patients suffering from borreliosis, which partly may
be accounted for by a substantially long period betwe-
en tick bites and a serological diagnostic test confirming
borreliosis.
IgG anti-VlsE were detected in 22 patients (88%), and
in 3 patients (12%) results were negative. Positive results
were based on IgG antibodies against other B. burgdorferi
specific antigens e.g. p39 and p83.
During B. burgdorferi infection in addition to VlsE
antigen, other highly immunogenic proteins can be de-
tected, e.g. Crasps (Crasp3), from Erp family (pG), and
immunogenic membrane-associated proteins like BB0323
[5, 6, 9].
In the patients with history of multiple tick bites IgG
anti-VlsE (22 tested patients) and BB0323 (13 tested pa-
tients) were detected.
Although in vivo antigens are considered to indicate
late stage borreliosis, the tests conducted in persons in
whom borreliosis was suspected who had erythema mi-
grans showed the presence of IgG anti-in vivo antigen
VlsE and BB0323 [10].
Our results found that in a tested panel, IgG against
other in vivo antigens were less frequent (anti BBA36
28%, Crasp3 28%, pG 20%) however they still may
have certain diagnostic value. Despite long term infection
in those patients IgM against OspC and p39 antigens were
detected.
Seropositivity which resulted from B. burgdorferi in-
fection may exists in both IgM and IgG classes for a long
time and detection of IgM is not only indicative as rein-
fection [11].


Conclusions

1. Antibodies to VlsE (antigen from in vivo group) are the
most frequently produced antibodies IgG during borre-
lia infection.
2. Membraneous proteins: p39 and p83 present in diagno-
stic serologic tests are reliable markers of B. burgdorferi
infection.
3. In vivo proteins alone used in diagnostic tests do not
guarantee reliable results that either would confirm or
exclude B. burgdorferi infection.

[vonneke]

Competitive inhibitian Elisa for the dection of Borrelia Burgdorferi Antigens- failure to detect antigen in the cerebrospinal fluid from patients with neuroborreliosis :

http://jmm.sgmjournals.org/cgi/reprint/50/6/577.pdf

[vonneke]

http://www.bratislmedj.sk/2001/10210-02.pdf


Significance of specific antibody determination in Lyme borreliosis diagnosis

Abstract
The diagnosis of Lyme borreliosis, except in cases characterised by pathognomonic clinical manifestation,
usually requires confirmation by means of microbiological diagnostic assay, mainly by antibody
detection methods. In our study antibodies to B. burgdorferi were tested in neurological patients
with suspected Lyme borreliosis, depending on syndrome and clinical diagnosis.
Antibodies were tested with IFT, ELISA and immunoblot.
Blood samples of patients tested with IFT and ELISA tests were positive in 88 patients. Positive indirect
immunofluorescence tests were found in 83 patients; in 5 patients the antibody level was borderline.
Of these, 40 were positive also in ELISA but a correlation between IF titers and ELISA-positivity
was not established. The immunoblot method confirmed specific antibody positivity in 36 of 88 patients
(45.45 %) who were positive (or borderline positive) in the indirect IF test, and in 28 of 40 (70
%) ELISA-positive patients. Antibody specificity was found in 8 indirect IF-positive patients who were
ELISA negative. This may be explained by the higher immunoblot sensitivity in comparison with
ELISA. The Lyme borreliosis diagnosis was clinically established in 19 patients; antibodies to B.
burgdorferi were only found in 13 patients in all three tests, and in 4 patients only in the indirect IF
test.

The results of serological tests for antibodies to B. burgdorferi should be interpreted with caution, as
the tests are not standardized and may show false positive or false negative results. A two-step serological
examination with the immunoblot test is recommended, whereby some nonspecific reactions
may be eliminated. The results of serological tests have only supportive value and cannot be deemed
conclusive when establishing an etiological diagnosis.

Discussion

Determination of the presence of antibodies to B. burgdorferi
is used to support the diagnosis of Lyme borreliosis. Serological
tests are generally unsuitable in early stages of the disease
due to their low sensitivity; they are more significant in advanced
stages, when the sensitivity and specificity of tests is higher.
The importance of serological tests in Lyme borreliosis diagnoses
is frequently overestimated and may result in incorrect diagnosis
and therapy in cases when their strong and weak points
are incorrectly balanced. Reid et al. (1998) evaluated a group of
209 patients in retrospect with LB diagnosed in 1994 and 1995,
and failed to confirm the diagnosis in up to 60 % of all cases.
After ATB therapy (usually 42 days), 55 % of these patients
showed low-intensity and 6 % more intense accessory symptoms.

ELISA is the most frequently used screening test. Testing,
mainly in the past, had been based on processing whole cells of
B. burgdorferi of various strains; the present trend is to use tests
with several subunit or recombinant antigen, as they show higher
higher
specificity (Rauer et al., 1998; Magnarelli et al., 1996).
Application of the antigen mix enables to compensate for a
possible reduction in the production of antibodies to a specific
antigen, as it takes the variability of immune response of patients
and antigens of several strains in account. It also enables
to significantly reduce the false-positivity ratio, e.g. from 23 %
to 10 % in EBV infections. Increased OD values improve discrimination
between positive and negative serums. As the result,
test specificity may reach as much as 94 % according to certain
authors (Kaiser and Rauer, 1999). Positivity found in our ELISA
tests with recombinant antigens was confirmed by detecting
specific antibodies with immunoblot in 70.0 % of all cases. Positive
immunoblot was also found in 8 patients with negative
ELISA results, confirming the higher sensitivity of this
test in
accordance with other authors (Treib et al., 1998).

The indirect IF test showed high occurrence of positivity in
our patients in comparison with ELISA; however, only 45.45 %
of the patients had identical results in both tests. This may have
been influenced by selecting patients with high proportion of
demyelinating diseases and of diseases with autoimmune components,
as these could contribute to the false positivity of the
results. On the other hand, immunoblot confirmed presence of
specific antibodies in 8 patients with positive IF antibodies and
negative ELISA, indicating that this test, while being obviously
less specific, is more sensitive than ELISA.

We used two commercial immunoblot tests, and certain patients
were examined by both tests. The weak lines obtained in
these tests are sometimes difficult to interpret (Engstrom et al.,
1995), and we tried to compensate this by applying both tests
and/or by repeating them after two months. Given both the variability
the borreliae appearing in our territory and the variable
antibody response of patients, negativity in this test cannot exclude
a B. burgdorferi infection

Contemporary serodiagnostics often feel the absence of effective
tests able to prove an active infection. Prolonged retention
of IgM antibodies and frequent appearance of cross-reactions,
mainly when looking for such antibodies reduce their diagnostic
utility in comparison with other infectious diseases. The
comparison of results obtained by different diagnostic labs shows
substantial variances (Brown et al., 1999). Our sample of neurological
patients contained none with clinically diagnosed borreliosis
and negative serological examination, although such cases
have been sufficiently documented (Oksi et al., 1995).

Lyme borreliosis was clinically diagnosed in 19 patients;
however in determining the diagnosis the fact must be taken in
account that clinical symptoms indicating the disease may have
different etiologies which could not be confirmed in the framework
of broad diagnostic procedures. Also, the evaluation of
clinical symptoms of certain persistently infected patients in a
causal connection with Lyme borreliosis is problematic. Further
laboratory tests are therefore urgently needed to support the diagnosis.
Determination of the presence of intrathecal antibodies
in the liquor is an additional important diagnostic criterion; however,
none of the direct-proof methods that show various sensitivity
(cultivation, IEM, PCR) can intercept all patients, besides
being difficult to practise (Honegr et al., 2001, Mateicka and
Kmety, 2000).

To diagnose LB in neurological patients is a difficult process,
considering the wide scale of clinical symptoms. Proof of
anti-borrelia antibodies in itself does not establish the base of a
causal relationship between the infection and its clinical manifestation.
Results of serological tests should be evaluated very
carefully, as these tests are not standardised and correlation of
results among the various commercial sets and laboratories is
weak, requiring consideration of possible false-positive or falsenegative
data. Some of the problems can be resolved by immunoblot
examination that may confirm the presence of specific
antibodies and exclude certain nonspecific ones, however when
taking the variability of borreliae circulating through Europe and
variability of the immune response of patients in account even a
negative immunoblot test cannot exclude the disease. Presently
it is recommended to use immunoblot, as this test has higher
specificity in the framework of a two-step serological examination.
Results of serological tests have only supportive value in
the diagnosis, their correct evaluation requires knowledge of their
strong and weak points, thereby emphasizing the need for close
co-operation of clinical and laboratory workers.