Int. J. Med. Microbiol. 293, Suppl. 37, 174-178 (2004) © Urban& FischerVerlag http://www.urbanfischer.de/joumals/ijmrn
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Original Article Serological description of Estonian patients with Lyme disease, a comparison with control sera from endemic and non-endemic areas Kai E. Kisand a, Meeme Utt a, Kalle V. Kisand a, Tiina PrOkk b, Raivo Uibo ~ a University of Tartu, Department of Immunology, Tartu, Estonia b University of Tartu, Department of Infectious Diseases, Tartu, Estonia
Abstract Serological tests for Lyme disease are mostly not well standardized and cases of misinterpretation of test results by clinicians are rather common. The diagnostic value of serologic tests may also depend on the seroepidemiological situation of the population. The aim of the study was to compare the immunoblot pattern of Lyme borreliosis patients and control sera from endemic and non-endemic regions and to identify the most suitable interpretation criteria for our immunoblot test. Serum samples of 24 Estonian patients with Lyme disease, 12 sera from patients with tick-borne encephalitis, 40 Estonian control sera, and sera from 50 Laplanders from North Sweden where people usually never come into contact with ticks were tested for IgG antibodies to Borrelia. Sonicated lysate of Borrelia afzelii (strain ACA1) was used in immunoblot as source of antigens. In our test system the following interpretation criteria gave the specificity of 96% for Estonian population: _> 1 band from p58, p21, p17 and p14 plus _>2 bands from p83/100, p39, p34, p30 and p25; or _>4 bands from p83/100, p39, p34, p30 and p25. The comparison of Estonian controls with Laplanders showed that subclinical infections with Borrelia are rather common in Estonia. Also the rate of other infections, giving rise to cross-reactive antibodies, may be more frequent in Estonians. The frequent reactions with Borrelia antigens in a healthy population complicate the serodiagnosis of Lyme disease. Key words: Lyme disease - Borrelia - immunoblot - serology
Introduction Since the identification of Borrelia burgdorferi as the etiologic agent of Lyme borreliosis (LB) in 1983,
numerous serologic tests have been developed (Burgdorfer et al., 1983). Despite efforts to improve the sensitivity and specificity of the tests and antigen preparations, the serologic tests are still
Corresponding author: Kai E. Kisand, Department of Immunology, University of Tartu, Ravila Str. 19, Tartu 51014, Estonia, Phone: 00372-7-374 230, Fax: 00372-7-374 232, E-mail:
[email protected] 1433-1128/04/293/Suppl.37-174 $15.00/0
Lyme disease serology prone to misdiagnosis of LB (Brown et al., 1999). The major problem in diagnosis of early LB is the high percentage of seronegativity (Dattwyler et al., 1989). However, whereas erythema migrans is pathognomonic for LB, serologic tests are not of decisive importance in the diagnosis of early LB with the classical clinical picture (Steere et al., 1977). In atypical cases, when erythema migrans is missing, the diagnosis of LB is dependent on the demonstration of a specific immune response to BorreIia. Here, Western immunoblot (IB) is considered to be more specific than ELISA tests (Lange and Seyyedi, 2002). However, there is a multitude of blotting methodologies using antigens prepared from different genospecies of Borrelia burgdorferi sensu lato developed in different European countries which are difficult to standardize (Hauser et al., 1997, 1998, 1999; Engstrom et al., 1995; Nilsson and von Rosen, 1996). Big efforts have been made to find out the most suitable band combinations for all European countries (Robertson et al., 2000). Unfortunately, geographic differences of LB are so big, that no useful single European rule resulted from the study. Moreover, the published laboratory specific rules were developed regarding healthy control group from a country with low incidence of LB (Robertson et al., 2000). It has been shown that in highly endemic areas the subclinical course of infection seems to be rather c o m m o n and persons frequently exposed to vector tick bites often show strong bands in immunoblot with Borrelia antigens (Nilsson and yon Rosen 1996). Antibodies to Borrelia can persist for a long time after treatment, which makes it difficult to discriminate between active infection and past infection. Borrelia also shares many antigens with other spirochetes that are also part of the normal human microflora. Most individuals have antibodies to c o m m o n spirochetal antigens (Dattwyler et al., 1989). Misinterpretation of the serology that leads to overdiagnosis and overtreatment of chronic LB is rather common. That is why serodiagnostic criteria should be evaluated on the basis of seroepidemiological studies of a geographically relevant population. Estonia is endemic for Lyme disease. The climate and landscape with lots of brushwood is suitable for ticks (Priikk et al., 1999a) and people spend a lot of time in the countryside. The infection rate of ticks with Borrelia has been from 3.5 to 20 % in different areas (Priikk et al., 1999b). Most Borrelia infections in Estonia are caused by B. afzelii and to a lesser extent by B. garinii. In this aspect Estonia differs from North Sweden where Laplanders usually never
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come into contact with ticks and never become infected with Borrelia. The aim of the study was to compare the immunoblot pattern of LB patients and control sera from Estonians and from Laplanders and evaluate the serodiagnostic criteria on geographically relevant populations.
Material and methods Serum samples of 24 Estonian patients with LB (diagnosed in Tartu University Clinic from 1999-2002, mean age 55 + 12; 9 males, 15 females) were enrolled in the study. 17 patients presented with typical erythema migrans, two had early generalised form without erythema migrans, two had neuroborreliosis and three had Lyme arthritis. A separate group was comprised of 12 patients with tickborne encephalitis (TBE, mean age 45 + 20, 6 males and 6 females). For comparison, samples were obtained from 32 healthy persons and 8 patients with other neurological diseases except TBE (Estonian control sera, mean age 42 ± 13; 23 males, 17 females). Sera from 50 Laplanders from North Sweden (Lapland control sera, supplied by K. Ornstein from Lund University) were also tested for antibodies to Borrelia. Sonicated lysate of Borrelia afzelii (strain ACA1) was used in immunoblot (IB) as a source of antigens (Nilsson and yon Rosen 1996). Serum samples were tested in dilution 1:100. Bound IgG was visualised using antihuman IgG conjugated with horse radish peroxidase (HRP) at a dilution of 1 : 1000 (DAKO, Denmark) in the presence of amino carbazole and peroxide. The blots were scanned with 69-710 Imaging Densitometer (Bio-Rad) and apparent molecular masses were estimated using the Quantity One Software package (Bio-Rad). After the identification of bands with the help of positive control serum (supplied by I. Nilsson, Lund University), the blots were evaluated visually. For statistical analysis Fisher's exact test (two-sided) was used.
Results A multitude of band positions from 12 to 100 kDa was revealed. The following bands with documented importance were identified: p83/100, p75, p66, p60, p58 p41, p39, p34, p30, p25, p21, p17, p14. After comparing the IB patterns of LB group and Laplander's group we discarded bands p75, p66, p60 and p41 as unspecific. Figure 1 displays the frequency of different bands in studied groups that showed significant (p < 0.05) differences between the LB and the Laplander's group. Most specific bands for LB (as for regarding Laplander's control group) were
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70
60
50
40 %
Estonian controls Lap anders
30
20
10
p83/I00
p58
p39
p34
p30
p25
p21
p17
p14
Fig. 1. Frequency plot of immunoblot bands in different study groups. LB - Lyme borreliosis, TBE - tick-borne encephalitis.
p58, p25, p21, p17 and p14 (specificity 98 - 100 %) and p83/100 (specificity 96%). However, for Estonian controls bands p83/100 and p25 were even not significant if taken separately. In Estonian healthy controls bands p39, p34, p30 and p25 were seen more frequently (p < 0.05) than in Laplanders. Also the TBE group showed more frequent bands at p83/ 100 (p < 0.01) and p30 (p < 0.05) than the Estonian control group and p83/100 was even more common in TBE than in LB group (the difference did not reach statistical significance). For obtaining interpretation criteria, first of all we tried to adjust the published European interpretation criteria to our IB results: at least two bands of p83/ 100, p58, p43, p39, p30, p25, p21, p17, p14 (Hauser et al., 1997, 1998, 1999). From these bands we could define p83/100, p58, p39, p25, p21, p17 and p14. This rule gave us specificity of 94% regarding the Laplander's control group and 70% regarding the Estonian control group. To construct the interpretation criteria suitable for Estonian population we started with most specific bands (p58, p21, p17
and p14) and added step by step the other bands. For Estonian population we got the best results by the following rule: _>1 band of p58, p21, p17 and p14 plus > 2 bands from p83/100, p39, p34, p30 and p25; or _>4 bands from p83/100, p39, p34, p30 and p25. This gave specificity of 96% and sensitivity of 54%. If we took into consideration only the cases with anamnesis longer than I month (from the tickbite to the time when the sample was drawn) the sensitivity of the test increased to 80 %. By this rule 3 cases (25 %) diagnosed as TBE gave positive IB result for LB.
Discussion Our results confirm that, although the IB test is recommended as the confirmatory test for the diagnosis of LB, the development, validation, and interpretation of the test is complicated. The IB results depend on the strain of Borrelia burgdorferi
Lyme disease serology that has been used for antigen preparation, the IB protocol used, and the local prevalences of species and strains of Borrelia burgdorferi sensu lato. European multicenter studies have resulted in several imunoblot interpretation criteria that are useful for diagnostic laboratories as a guide for the formulation of working rules suited to their methodology and local populations (Hauser et al., 1997, 1998; Robertson et al., 2000). At least two bands of p83/100, p58, p43, p39, p30, OspC, p21, p17 and p14 have been proved to be optimal for using B. afzelii strain Pko (Hauser et al., 1997). Robertson et al. (2000) identified p83/100, p58, p41, p39, OspC and p17 as most important bands. We used antigen preparation of B. afzelii strain ACA1 in our IB. Analyzing the IB patterns we found that European recommendations were not suitable for Estonian population. Adjusting these rules would have resulted in only 70% specificity that is not suitable for the confirmatory test. Band p83/100 and p25 were not specific in the Estonian population. The Estonian control group had significantly more bands at p39, p34, p30 and p25 than the sera from North Sweden where people normally never come into contact with ticks. These data support the idea that subclinical infections with Borrelia are rather common in Estonia. It has been reported that the occurrence of serum antibodies increases with time spent in the highly endemic areas (Gustavsson et al., 1993). In our study patients with documented tick bite (as diagnosed to have TBE) had more Borrelia-specific reactions than the healthy controls. Some of the patients could have mixed infection with TBE virus and Borrelia. In Estonia the infection rate of ticks with Borrelia ranges from 3.5% to 20%, there is plenty of landscape suitable for ticks, and people are used to spend lots of time in the countryside. So contacts with ticks are rather common. The differences in IB patterns of the Estonian control group and Laplanders could also be explained partially by differences in microflora or the rate of other bacterial infections giving rise to cross-reactive antibodies. Our study stresses once more that it is important to work out population-based serodiagnostic criteria for clinically significant positive/negative discrimination of IB results. Even using stringent interpretation criteria, giving high specificity, the IB test can only support the clinical diagnosis, not confirm it. Acknowledgements. We are grateful to Dr. Ingrid Nilsson (Lund University, Sweden) for the kind donation of the Borrelia strain ACA I, for her support and advise on Western blotting. We thank Dr. K. Ornstein (Lund University) for the generous donation of the Laplanders'
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sera. This work has been supported by the Estonian Science Foundation grant No. 5273.
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