Early Lyme disease: Humoral immune status and treatment
Corresponding author and reprint requests: Galina Zhelezova Zhelezova, Hospital of Infectious Diseases, Laboratory of Clinical Immunology, 17 Dimitar Nestorov Blvd, 1606 Sofia, Bulgaria Tel: (+359 2) 54 91 21 Fax: (+359 2) 51 09 51 E-mail number: Sumerska@Bulmail.Sprint.com
Objective: To investigate the humoral immune status and the effect of antibiotic treatment in Bulgarian patients with early Lyme disease.
Method: A total of 34 early Lyme disease patients was examined, 16 with erythema migrans and 18 with non-specific systemic symptoms. Serum samples from all patients and from 12 healthy controls were tested for total immunoglobulins (IgG, IgA and IgM), hemolytic activity of complement (CH50) and immune complexes (ICs). The patients were treated with doxycycline (100 mg orally, twice daily for 10 to 15 days, in one or two courses).
Results: The patients showed significantly increased IC levels (P<0.01 for patients with erythema migrans and P<0.001 for patients with non-specific symptoms). There were no significant changes in the levels of total hemolytic complement and total immunoglobulins (IgG, IgA and IgM). The clinical outcome was satisfactory in 21 of the 34 patients (61.8%) after treatment with doxycycline for 10 to 15 days. The rest of the patients (38.2%) failed to respond to the therapy, and continued to report various complaints, such as arthralgia, myalgia, paresthesia, headache, fatigue or recurrent rash. All of these had elevated levels of IC. After a second course of treatment with the same antibiotic regimen these patients had resolution of symptoms (12 patients) or improvement (1 patient).
Conclusions: Immunologic investigation may be useful in determining treatment strategy in Lyme disease. Elevated IC levels may indicate a need for more prolonged antibiotic therapy.
Lyme disease or Lyme borreliosis is a tick-borne chronic infectious multisystem disease, caused by the spirochete Borrelia burgdorferi . There is considerable variation in the course, which is usually divided into three stages . The initial clinical manifestations (stage 1 or early Lyme disease) include a characteristic skin rash called erythema migrans (EM) and non-specific systemic symptoms such as regional lymphadenopathy, myalgia, fever and headache. In the second stage of Lyme disease (weeks or months after the tick bite) cardiac and neurologic symptoms are encountered. In the most advanced third stage of the infection, beginning months or years after the tick bite, joint dysfunction can occur.
The pathogenesis of Lyme disease involves the ability of the spirochete to enter the circulation and to persist in the infected host for prolonged periods [1–3]. The role of the immune response in the pathogenesis of Lyme disease has not been completely elucidated. Despite the high concentration of protective antibodies and circulating T-cells, the immune response itself cannot guarantee eradication of B. burgdorferi [4,5]. Sometimes the symptoms persist in patients after antibiotic treatment . Immunologic measurements might be useful in evaluation of the treatment of Lyme disease patients.
Recently, B. burgdorferi has been found in Ixodes ricinus ticks (infection rate 41.5%) collected in Bulgaria . Lyme disease patients at different stages have been diagnosed and treated.
The present study was undertaken to investigate various aspects of the humoral immune status and the effect of antibiotic treatment in Bulgarian patients with early Lyme disease. Circulating immune complexes (ICs), hemolytic activity of complement (CH50) and total immunoglobulins (IgM, IgG and IgA) in serum were determined.
PATIENTS AND METHODS
From June 1989 to May 1991 34 patients with early Lyme borreliosis were examined and treated at the Hospital of Infectious Diseases in Sofia, Bulgaria. The patients were aged from 12 to 60 years (median 38 years); 21 were female and 13 were male. Of the 34 patients, 16 had EM, the best clinical marker for the illness. Ten of these had EM accompanied by minor constitutional symptoms, such as fever, arthralgia or headache. Eighteen of the 34 patients had early Lyme disease without EM. They revealed non-specific systemic symptoms such as fever, regional lymphadenopathy, musculoskeletal discomfort, headache or fatigue.
The diagnosis of Lyme disease was based on detection of specific antiborrelial antibody in serum as described by Noeva et al., who used two methods: indirect micro-ELISA and immunodot test . Only patients with a positive serologic test for specific antibodies were included in the analysis. In addition, 26 of the 34 patients recalled tick bite 15 to 60 days before the development of symptoms. All the patients were treated with doxycycline (doxycycline hydrochloride), 100 mg twice daily, for 10 to 15 days, in one or two courses. In order to assess the response to therapy, repeated physical examinations and clinical evaluations were made after starting the treatment, and 1 month after completion.
Serum samples were obtained before treatment, or very early in treatment. Sera from 12 healthy subjects were tested as controls. Blood was allowed to clot at room temperature for 1 h, and after centrifugation (2000 g for 10 min) the serum was separated and aliquoted. The serum samples for testing the immunoglobulins and CH50 were stored at −20°C. The serum samples for determination of ICs were tested immediately, without freezing.
ICs were measured using the polyethylene glycol method . Total IgM, IgA and IgG concentrations were determined by radial immunodiffusion . CH50 titration was performed using sheep red blood cells coated with anti-sheep red cell antibodies .
Student's t-est was used to determine the statistical significance of the established differences. In all cases, a P value of less than or equal to 0.05 was considered to indicate statistical significance.
Serum immunoglobulin levels
Elevation of IgG antibodies above normal was found in two of 16 patients with EM and in five of 18 patients with non-specific systemic symptoms. IgM antibodies were elevated in one patient with EM and in two patients with non-specific systemic symptoms. In all 34 patients the levels of total IgA were in the normal range. The mean values of total immunoglobulins (IgG, IgA and IgM) are shown in Table 1. There was no statistically significant difference between patients with Lyme disease and healthy subjects.
Table 1. Serum immunologic parameters in patients with early Lyme disease (mean value ± SD)
|EM|| || || || || |
|NSS|| || || || || |
|Controls|| || || || || |
Hemolytic activity of complement
The hemolytic activity of complement varied among the patients. Two of the 18 patients with non-specific systemic symptoms, and four of the patients with EM, had values of CH50 above the range of normal individuals. Three of the 16 patients with EM and six of the 18 patients with non-specific systemic symptoms showed lower complement hemolytic activity compared with the controls. The mean CH50 values for all Lyme disease patients are presented in Table 1. There were no statistically significant differences between the patients and the controls.
Thirteen of the 16 patients with EM and 12 of the 18 patients with non-specific systemic symptoms showed elevated levels of IC. The circulating IC levels in the patients with Lyme disease were significantly higher than in the controls (Table 1). The mean IC value in patients with EM was 137.1 U/mL as against 71.7 U/mL in controls (P<0.01). The patients with nonspecific systemic symptoms had a mean IC value of 116.4 U/mL, significantly higher than in the controls (P<0.001).
Clinical course and treatment
The response to therapy was not uniform. Treatment with doxycycline (100 mg orally, twice daily for 10 to 15 days) was effective in 21 of the 34 patients. A satisfactory clinical response, with resolution of EM rash and other clinical signs and symptoms, was achieved and the patient remained asymptomatic during the 1-month post-treatment follow-up period. The rest of the patients (13) failed to respond to the therapy. After treatment, two patients (one with EM and one with non-specific systemic symptoms) continued to complain of fatigue, headache, and myalgia; five patients with EM and three with non-specific systemic symptoms complained of mild arthralgia and myalgia; one patient with non-specific systemic symptoms developed peripheral neuropathy; and two patients with EM had a recurrent rash. All these patients required retreatment because of the persistent illness. The second course of doxycycline (200 mg daily for 10 to 15 days) was effective in these patients: 12 had resolution of symptoms and one patient with arthralgia showed improvement. All 13 patients with persisting symptoms (eight patients with EM and five patients with non-specific systemic symptoms) had elevated IC levels in the initial stage of treatment. Patients with satisfactory outcome (without persisting symptoms after antibiotic treatment) varied regarding IC levels observed in the initial stage of treatment: five of eight patients with EM had elevated IC levels, as did seven of 13 patients with non-specific systemic symptoms. There is a tendency to have higher levels of IC in patients with persisting symptoms compared with patients without persisting symptoms or relapse, especially in EM patients (162.5 ± 83.5 as against 111.7 ± 61 U/mL), but there is not a statistically significant difference.
Different immunologic abnormalities have been recorded in patients with Lyme disease, and this has suggested a possible involvement of the specific or innate host responses in the pathogenesis of the disease[1,4,12,13]. In order to find out whether some humoral immunologic parameter had a prognostic value for the treatment and the clinical outcome of the disease we measured total immunoglobulins, IC levels and the hemolytic activity of complement before or early in treatment in patients with early Lyme disease.
Because of the essential bactericidal activity of antibody and complement in the human host defense against Gram-negative bacteria, we undertook an analysis of serum IgG, IgA and IgM antibodies and the hemolytic activity of complement. It is known that anti-B. burgdorferi immunoglobulin responses develop slowly and then persist [4,5]. Some studies have found signs of polyclonal B-cell activation (elevated total serum immunoglobulins) with a correlation between total serum IgM concentration and severity of clinical signs and symptoms and outcome [1,12–14]. In our study seven patients showed elevation of IgG, and three patients showed elevation of IgM antibodies. However, these differences were not significant (Table 1).
Complement activation is not a consistent feature of clinical Lyme disease[1,14,15]. We observed a broad range of hemolytic activity of complement among the 34 patients: 55.9% had normal values, 26.5% low activity and 17.6% higher activity of complement. Generally, there was no significant difference between values of complement activity in the patients with early Lyme disease and in the controls. This variability in the studied patients could be explained by differences between B. burgdorferi strains and/or different host defense factors.
It has been established that in Lyme disease  immune complexes are formed, and their possible role in the pathogenesis of the disease has been considered. ICs were determined before or early in treatment. We have established that a significant increase in the level of the IC exists in the patients with EM (81.25%, P<0.01) and in patients with non-specific systemic symptoms (66.7%, P<0.001).
As doxycycline has been recommended as an appropriate antibiotic for the treatment of early Lyme disease [6,16], we have used it for the therapy of our patients. The initial course of treatment (200 mg daily for 10 to 15 days) was effective in 61.8% of the 34 patients. The rest of the patients (13) developed persisting symptoms such as arthralgia, myalgia and relapse of EM signs. A second course with the same drug and in the same regimen was successful. Elevation of IC levels was seen in all these patients, but there was not a statistically significant difference from the IC levels in patients without persisting symptoms. However, in unsuccessfully treated patients with EM there was more prominent elevation of IC levels in comparison with patients with EM, which benefits from one course of doxycycline (162.5 ± 83.5 U/mL as against 116.3 ± 49.3 U/mL). Therefore, an assumption can be made that EM patients with more prominent IC levels need more prolonged antibiotic treatment.
In order to clarify the precise role of IC and other factors of humoral immunity in the pathogenesis of Lyme disease further investigations are necessary.
We gratefully acknowledge the technical assistance of Mrs Andreeva, Mrs Ignatova and I. Ivanov.