This study provides information on the clinical manifestations of patients with LB serology testing in general practice in Denmark. The rates of seropositivity presented thus represent a mixture of patients with active Borrelia infection, immunological memory or non-specific cross-reactivity. In more than one third of the patients EM was suspected and these patients received antibiotic therapy also when antibody findings were negative. This is in agreement with the perception that EM with a characteristic presentation or history of an expanding rash is a distinct clinical entity and that laboratory testing is unnecessary and of limited utility[1, 11].
The average rate of IgG antibodies was low (3.4%) with a tendency to be even lower in patients suspected of Lyme arthritis (2.3%) and chronic NB (1.5%) which comes very close to the expected background seropositivity of 2%. This indicates a large proportion of false positive results and a pretest probability lower than recommended.
High IgM seropositivity rates were found in patients with suspected EM and in children (17.2 and 21.0%, respectively). A high IgG seropositivity rate (10.4%) was found in patients with suspected acrodermatitis.
Validity of the survey
The response rate for the clinical survey was 57% and there is a possibility of selection bias. However, we had access to descriptive data on all patients through the laboratory information systems of the participating departments. It was noted that compared to patients not included there were fewer patients aged 16-49 years in the study group and the rate of IgM seropositivity was slightly higher. Whether this reflected a selection bias for certain clinical manifestations is not known and overestimation of the seropositivity rate in some of the clinical subgroups is possible. From a methodological point of view, it would be desirable if the questionnaire had been completed during the first consultation with the patient, this being so before the serological results became available. Still, we believe that classification of patients was not materially influenced by the laboratory report. Altogether, there seems to have been a preference for inclusion of IgM positive patients and the seropositivity rates and OR for IgM antibody results shown in Table 1 are likely to be marginally too high.
Seropositivity rates and the clinical variables
The overall rate of seropositivity in our study compares well with another Danish study of 897 consecutive samples obtained during the winter months, December to April, where 9.9% were IgM and 2.0% were IgG positive using the same flagella based assay.
In the present study 829 (31%) of the tested patients reported a tick-bite. This is a much lower rate than seen in a Swedish survey of patients with reported LB. In this study 79% of the patients were aware of a tick bite preceding the onset of symptoms. This disparity may be explained by the difference in populations studied, as only patients with a reported diagnosis of LB were included in the Swedish study. Our study confirms that a history of a tick bite is not only a frequent cause for serological testing, but also an important motivation for the prescription of antibiotic therapy. Still, it has been shown that only about 0.5% of tick bites lead to clinical LB.
Tick bites were a fairly common complaint in our study (13.6% of patients). This was also shown in a Dutch study that reported an annual number of 4.6 patients with tick bites compared to only 0.9 patient with an EM diagnosis per doctor (average practice population 2430 people). Furthermore, reports from the USA indicate that a tick bite may lead to costly and unnecessary serologic testing or prophylactic antibiotic therapy[17, 18].
When our patients reported a tick bite it was most likely due to Ixodes ricinus and not a non specified "insect-bite" as other arthropods in Denmark do not remain attached to the skin for a longer period and has to be removed. This could explain why patients with "other insect bites" were less frequently found to be IgM and IgG antibody positive (Tables 1 and 2). More than one third (38%) of the tests were requested from patients with a rash, although this is not generally recommended due to the relatively low sensitivity of antibody testing in this group[3, 7, 19] and the clinical diagnosis is considered to be reliable [20, 21]. Treatment was given to 44.9% of patients with a rash. The rate of seropositivity for either IgG or IgM antibodies was 19.4%, indicating that the decision to treat was based on a sensible clinical evaluation.
It is recommended to perform a lumbar puncture when NB is suspected[3, 19, 22]. In our study 340 patients with suspected early NB and 130 with suspected chronic NB were not further investigated with a lumbar puncture and testing for intrathecal antibody production. As about 20% of patients with early NB may be seronegative, some cases might have been overlooked. However, we cannot preclude that some patients may have been referred to neurological evaluation, but a specialist did not deem testing for intrathecal antibody production relevant after evaluation.
Concerning possible stage 3 LB a high sensitivity of IgG antibodies is expected. Judged on the rate of IgG seropositivity in the group of patients with suspected acrodermatitis the doctors were conscious of this entity. But when using serology to support the diagnosis of Lyme arthritis the main problem was the low prevalence of this disease in the tested population. Only 2.3% of patients suspected of arthritis were found to be IgG positive in the present study (Table 1). This rate is close to the level of reactivity in the healthy adult population. Thus, in some subgroups there are few true cases of LB and the rate of seropositivity is close to the healthy background population. It must also to be expected that some patients with vague clinical symptoms are tested to rule out the diagnosis of LB rather than to confirm it. In addition, the slight or non significant variation of the monthly rates of seropositivity for both IgM and IgG support the notion, that many patients had non-typical clinical manifestations. The seasonal variation of seropositivity would have been more pronounced if more straightforward cases of EM had been included.
It is possible that patients with clinical disease other than LB may develop cross-reacting antibodies more frequently than the healthy donor population, which was used to adjust the specificity of the IgM antibody ELISA. For all subgroups of patients in the present study the rate of IgM seropositivity was at least 5.4%. This may indicate that the finding of a solitary IgM positive test in patients with non-distinct symptoms should be interpreted with caution.
The selection of patients is assumed to be the important factor for the rates of seropositivity and the results obtained in this Danish study may not be directly generalized to other countries, where the epidemiology of LB, clinical practice, diagnostic methods, and recommendations may differ. Nevertheless, low rates of seropositivity, and thus low pretest probability, in certain patient groups may be a significant problem also in other countries. It has been recommended that the pretest probability of LB should be no less than 0.20[1, 12]. We are not aware of studies assessing whether this recommendation is fulfilled in clinical practice. As shown in this study, such a high rate of pretest probability is probably not realistic when diagnosing e.g. Lyme arthritis. Also contributing to the low rate of IgG seropositivity could be that the test has a lower sensitivity in a consecutive stream of routine patients compared to studies on selected well defined cases, as these studies may have inherent problems with representativeness.
It is generally recommended to use a "confirmatory" assay such as a Western blot (WB) in a two-tier sequence in order to improve specificity[1, 22]. However, WB is difficult to standardize[24, 25]and it has never been evaluated on consecutive clinical samples, whether this approach indeed improves the classification of patients with and without active LB. The alternative strategy is instead to use an assay with a strict cut-off allowing only 2% false positives as assessed in the local blood donor population for IgM and IgG antibody, respectively. Healthy blood donors are assumed to represent the lowest possible antibody reactivity in the population. The flagella assay is designed in accordance with this strategy and maintains an acceptable sensitivity. If using WB together with the flagella assay the cut-off would need adjustment, otherwise the sensitivity would become unacceptably low. No diagnostic laboratory in Denmark uses WB as a second line assay to modify the specificity.
To our knowledge, this study is the first allowing a crude assessment of the rate of false positive serological tests in patients who are tested but do not have active LB. We define an IgM or IgG antibody test to be falsely positive if it occurs in a patient who does not have any other indication of active LB. The maximum number of false positives in excess of the healthy donor population may be crudely estimated by subtracting 2% from rates of seropositivity in Table 1. For example in the group of patients with arthritis 3.4% are IgM positive in excess of the healthy blood donors. The low rate of IgG seropositivity shows that Lyme arthritis is indeed rare. This implies that the lowest total estimate of the specificity for the IgM antibody test could be 94.6 percent instead of the expected 98%.
False positive IgM results are documented to be common in patients with Epstein-Barr virus infection (54%) and less so in patients with cytomegalovirus infection (8%) . These rates do, however, not pertain to patients tested by general practitioners in the routine clinical setting, and it would be misleading to include them in a calculation of specificity. Moreover, these viral infections do not follow the same seasonal trend as LB where the large bulk of tests are carried out between May and October (Figure 1A). By extensive analysis of the current data we found no subgroup to fall below 5-6% in IgM seropositivity during winter months (data not shown) so the false rate could be estimated up to a maximum of 5-6%. Concerning IgG antibodies there is no indication that the specificity of IgG differs from about 98%.
An interesting finding in this study is a nearly threefold difference in the rate of IgM (10.4%) seropositivity compared to IgG (3.4%). This difference has not been found in neither blood donors or from patients with Lyme borreliosis [4, 6–8]. One hypothesis for the high frequency of IgM responses could be that many patients with short duration of clinical Lyme borreliosis are tested. Another hypothesis could be non-specific reactions in patients with other diseases. The answer is most likely a combination of both. The seasonal distribution of the frequencies of samples and IgM responses support the hypothesis that true early Borrelia infections contributes, at least partially, to the high IgM response.
The above interpretation of the rates of seropositivity above rests on the important assumptions that low rates of seropositivity indicate fewer and high rates indicate more patients with active infection due to Borrelia burgdorferi even when some of the tested patients may have non-specific antibody responses.
Still, the results obtained in this questionnaire-based study may have important implications for the guidelines provided to general practitioners for interpretation of serological tests for LB.