Serological follow-up after treatment of patients with erythema migrans ...

JOURNAL

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CLINICAL MICROBIOLOGY, June 1994,

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Vol. 32, No. 6

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0095-1 137/94/$04.00+0 Copyright (C 1994, American Society for Microbiology

Serological Follow-Up after Treatment of Patients with Erythema Migrans and Neuroborreliosis SUSANNE HAMMERS-BERGGREN,I* ANNE-METFE LEBECH,2 MATS KARLSSON,' BO SVENUNGSSON,3 KLAUS HANSEN,2 AND GORAN STIERNSTEDT3 Departments of Infectious Diseases, Danderyd Hospital' and Huddinge University Hospital,3 Karolinska Institut, Stockholm, Sweden, and Borrelia Laboratory, Department of Infection Immunology, Division of Biotechnology, Statens Seruminstitut, Copenhagen, Denmark2 Received 27 December 1993/Returned for modification 8 February 1994/Accepted 18 March 1994

To investigate the duration and kinetics of immunoglobulin M (IgM) and IgG antibodies against Borrelia burgdorferi in serum after treatment of Lyme borreliosis, consecutive serum samples from 30 seropositive patients with erythema migrans and 91 seropositive patients with neuroborreliosis were analyzed with a capture IgM enzyme-linked immunosorbent assay (ELISA) and an indirect IgG ELISA, both using B. burgdorferi flagella as the antigen. All the patients improved after treatment: 97 patients had a complete clinical recovery, while 24 patients had sequelae. The results showed that patients with erythema migrans and early neuroborreliosis more often initially had highly elevated IgM optical density (OD) values and low IgG OD values against B. burgdorferi, while the opposite was found in patients with late neuroborreliosis. During follow-up, the majority of patients had developed negative or significantly declining IgM ODs after 1 to 1.5 years but persistently positive IgM ODs were found up to 17 months after treatment of erythema migrans and 3 years after treatment of neuroborreliosis. IgG antibody levels declined more slowly and remained elevated to a larger extent, but more than half of the patients had developed negative IgG ODs within 5 years after therapy. However, positive IgG OD values were found after 9 to 10 years for patients treated for neuroborreliosis as well as erythema migrans. Both IgM and IgG antibodies against B. burgdorferi may persist for months to years after successful treatment of Lyme borreliosis. Consequently, a single serum sample with antibodies against B. burgdorferi must always be carefully evaluated and correlated to clinical symptoms.

Erythema migrans (EM), neuroborreliosis, arthritis, and acrodermatitis chronica atrophicans are different clinical manifestations of Lyme borreliosis, which may be diagnosed by the medical history and typical clinical findings (2, 3, 16, 18, 23, 24). However, to confirm the diagnosis, serological methods, mainly the enzyme-linked immunosorbent assay (ELISA), are available and often necessary (10, 12, 22). Serological studies have shown seropositivity rates of 25 to 50% in patients with EM and 50 to 80% in patients with neuroborreliosis (9, 10, 14, 21, 22). The diagnostic sensitivity of ELISA has been increased by the introduction of new antigens, e.g., the flagellum antigen (10, 14), and capture ELISA instead of indirect ELISA for the immunoglobulin M (IgM) analysis (12). An increase in test sensitivity is of importance for early diagnosis. Although Lyme borreliosis in all stages is a treatable disease, doubts might be raised in clinical practice whether treatment is successful. One reason for persisting symptoms after antibiotic treatment is true treatment failure, but most probably incorrect diagnosis or symptoms due to irreversible tissue damage are more common causes (1, 19). However, this points out the need for a laboratory parameter to help the clinician follow treatment efficacy. One aim of this study was to investigate whether the kinetics of the antibody response against Borrelia burgdorferi might be such a parameter for patients with Lyme borreliosis. B. burgdorferi may cause subclinical, mild clinical, or incorrectly diagnosed clinical infection, followed by a spontaneous

recovery (7, 15, 17). This may account for the high seropositivity rate among healthy people living in areas where the disease is endemic without a previous known history of Lyme borreliosis (8). If symptoms of the skin, the nervous system or joints (due to an etiological agent other than B. burgdorferi) develop in patients that are seropositive against B. burgdorferi because of previous exposure, diagnostic difficulties may appear. Earlier studies have shown the persistence of IgG against B. burgdorferi in serum for years after untreated Lyme borreliosis infection (4, 15) or the persistence of specific IgG for years in asymptomatic untreated persons in areas of endemicity (20). It has been suggested that such findings predict subsequent disease activity (4). It is therefore of interest to investigate whether the antibody response against B. burgdorferi also persists after antibiotic treatment in well-defined cases of Lyme borreliosis. To answer these questions, we analyzed consecutive serum samples from patients treated for EM or Lyme neuroborreliosis with a capture IgM ELISA and an indirect IgG ELISA, both using B. burgdorferi flagella as the antigen.

MATERIALS AND METHODS Patients. Patients with EM and patients with Lyme neuroborreliosis were included in this retrospective study. The patients were admitted to the Department of Infectious Diseases or Department of Dermatology, Danderyd Hospital, or the Department of Infectious Diseases, Roslagstull Hospital (present Huddinge Hospital), Stockholm, Sweden, between 1973 and 1991. Only patients with positive IgM and/or IgG antibodies against B. burgdorferi in serum before or within a

* Corresponding author. Mailing address: Department of Infectious Diseases, Danderyd Hospital, S-182 88 Danderyd, Sweden. Phone: 46 /8/ 655 50 00. Fax: 46 /8/ 755 12 37.

month after the start of antibiotic therapy were included. Serological testing of individual patients was performed until

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HAMMERS-BERGGREN ET AL.

TABLE 1. Clinical data for patients with EM Characteristic Value for group No. of patients ............................ 30) Male/female (no. of patients) ..................... 14/16 32-68 (54) Age (median) in yrs ............................ Preceding tick bite (no. of cases) ............... 2) (67%) Clinical manifcstations (no. of cases) 14 Localized EM ............................ Singlc EM + gencral symptoms'............. 11 2 Multiple EM ............................. Multiple EM + general symptoms .........3 Duration of symptoms pretrcatmcnt ..........2 days-7 mo (median 3.5 wk) Treatment 1t)-14 days (no. of patients) Oral pcV, I g q8-12h" ............................ 20) Oral doxycycline, 2t)t) mg q24h ...............7 Oral erythromycin, St()(mg ql2h ............3 Retreatment for 14 days within 3 mo with oral pcV-*oral doxycycline 2 (no. of patients) ............................. 3 mo-9 yr (mcdian 3 yr) Follow-up time ............................ Improvement (no. of patients) ....................30 Complete recovery ............................ 29 Sequclac ......... ................... I (arthralgia aftcr 1 yr) General symptoms: fever, myalgia, and headache. " q8-12h, every 8 to 12 h.

seronegativity occurred or until the end of this study. Additional serological testing of patients who had become seronegative was also performed if new symptoms compatible with Lyme borreliosis appeared. EM. A total of 30 patients with EM were included in the study. The diagnosis of EM was made on the basis of clinical examination. Clinical data from the 30 patients are presented in Table 1. Neuroborreliosis. A total of 91 patients with neuroborreliosis were included in the study. The diagnosis of neuroborreliosis was made on the basis of a typical clinical picture (n = 91), the presence of pleocytosis in cerebrospinal fluid (n = 91), and intrathecal antibody production against B. burgdorferi (n = 62). In the 29 patients without specific intrathecal antibody production at diagnosis, a majority of whom had a disease duration of less than 1 month, preceding or present EM (n = 8), positive IgM antibodies against B. burgdorferi in serum (n = 7), or both (n = 14) were required for inclusion. Clinical data from the 91 patients are presented in Table 2. ELISA. All serum samples, stored at -70°C, were analyzed with a capture IgM ELISA and an indirect IgG ELISA, both using B. burgdoiferi flagella as the antigen. The methods have earlier been described in detail (10, 12). In brief, in the capture IgM ELISA the solid phase was coated with ,u-chain-specific rabbit anti-human IgM, binding to human IgM antibodies in sera diluted 1/200. B. burgdorferi-specific IgM antibodies reacted with the test antigen consisting of a B. burgdorferi flagellum-biotin-avidin-peroxidase complex. Bound flagella were visualized by the addition of O-phenylenediamine substrate. In the indirect IgG ELISA, microdilution plates were coated with purified B. burgdorferi flagella. Serum samples diluted 1/200 were added, and specific antibodies were bound. The binding was detected by the addition of peroxidaseconjugated rabbit anti-human IgG antibodies and O-phenylenediamine substrate. In both assays the optical density (OD) was read spectrophotometrically and cutoff was defined as the 98th percentile of OD values obtained among sera from 200 healthy controls and was 0.500 in the IgM ELISA and 0.180 in the IgG ELISA (10, 12). Consecutive serum samples

J. CLIN. MICRC)BIC)L.

TABLE 2. Clinical data for patients with neuroborreliosis Characteristic Value for group No. of patients ................................ 91 Male/female (no. of patients) ............................34/57 6-88 (49) Age (median) in yrs ................................ Preceding tick bite (no. of cases) ......................1 7 (19%) Preceding EM (no. of cases) ..............................21 (23%) Preceding tick bite and EM (no. of cases) .......17 (19%) Clinical manifestations (no. of cases)

Meningitis ..........

......................

14

Meningoradiculitis, cranial neuritis ...............66 Encephalomyelitis ................................. 1.1 91 (t)t)%) Pleocytosis ............................... Leukocyte count (10"/liter) .............................6-1,4)0 (median 128) Intrathecal antibody production (no. of cases) ............................... Duration of neurological symptoms

pretreatment ................................

62 (68%)

3 days-2 yr (median I mo)

Treatment 10-14 days (no. of patients) i.v. pcG, 3 g q6-8h" (15t) mg/kg/day for

63 children) ............................... i.v. cefuroxime, 3 g q8h .................................1 i.v. doxycycline, 200 mg q24h .........................4 Oral doxycycline, 200 mg q24h ......................2) i.v. penicillin + oral doxycycline (5 days) and oral doxycycline (9 days) .....................3 Retreatment for 14 days within 8 mo (no. of patients) with: Oral doxycycline--i.v. cefuroxime .................1 i.v. pcG--i.v. cefotaxime, 3 g ql2h ................2 3 mo-10 yr (median 2.5 yr) Follow-up time ............................... Improvement (no. of patients) ...........................91 68 (75%) Complete recovery ................................ 23 (25%) Sequelac ................................ Impaired sensitivity, pain ............................6

Impaired hearing .................................1 Unilateral partial facial palsy .....................6 Bilateral partial facial palsy ........................I Monoparesis of extremity ...........................2 Paraparesis .................................3 Reduced intellectual capacity .....................3 Asthenia ................................1 " q6-8h, every 6 to 8 h.

from each patient were analyzed simultaneously on the same microdilution plate. A twofold decrease or increase of the OD in consecutive serum samples was considered significant for both the IgM capture ELISA and the indirect IgG ELISA. For patients with neuroborreliosis, intrathecal antibody production against B. burgdorferi was analyzed in paired serumcerebrospinal fluid samples drawn before or within a month of start of antibiotic treatment. The ELISA methods used were either a capture IgM and IgG ELISA, using B. burgdorferi flagella as the antigen (11), or an indirect IgM and IgG ELISA, using whole-cell sonicated B. burgdorferi spirochetes as the

antigen (22).

RESULTS EM pretreatment to 1 month after start of antibiotic treatment. Nine of the thirty patients had only positive IgM ODs, 12 patients had both positive IgM and IgG ODs, and 9 patients were only positive by IgG ELISA. For 21 of 30 patients with positive IgM ODs, the range was 0.540 to >2.500

(median, 1.160) and for 21 of 30 patients with positive IgG ODs the range varied from 0.180 to 1.090 (median, 0.340) in

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the first serum sample drawn from each patient during the period. EM follow-up. (i) IgM OD values. The kinetics of IgM OD in serum are demonstrated in Fig. IA. The range and median of the OD values at different time intervals of follow-up are shown in Table 3. In summary, 17 of 21 initially IgM-positive patients (81%) became negative by the IgM ELISA during follow-up from 3 weeks to 9 years (median, 3 years), while 4 patients (19%) still had positive ODs of IgM against B. burgdorferi 7, 12, 15, and 17 months after antibiotic therapy. (ii) IgG OD values. The kinetics of IgG OD in serum are demonstrated in Fig. lB. The range and median of the OD values are shown in Table 3. In summary, 9 of 21 initially IgG-positive patients (43%) became IgG negative during follow-up from 4 months to 9 years (median, 3 years). The remaining 12 patients (57%) still had positive IgG ODs after 3, 6, 12, 15, and 17 (n = 2) months and 2.5 (n = 2), 3 (n = 2), and 9 (n = 2) years of follow-up. Retreated EM patients. Serum samples from two patients treated by a second antibiotic regimen showed declining IgM OD levels before retreatment. Both patients had negative IgG ODs. Comparison of EM patients who recovered and those with sequelae. The only patient with persistent symptoms at follow-up (arthralgia 1 year posttreatment) became seronegative. Among the 29 patients with a complete clinical recovery, 1 patient (3%) still had a positive IgM OD after 7 months, 3 patients (10%) were still positive by both IgM and IgG ELISA after 12 to 17 months (median, 15 months), and 9 patients (31%) had positive IgG ODs at follow-up after 3 months to 9 years (median, 2.5 years). The remaining 16 patients (55%) without residual symptoms became seronegative during a follow-up time which varied from 4 months to 9 years (median, 3 years). Neuroborreliosis. To illustrate the kinetics of the IgM and IgG ODs for patients with neuroborreliosis graphically (Fig. 2, 3, and 4), the 91 patients were divided into three different groups according to disease duration (2 months [n = 26]). Neuroborreliosis pretreatment to 1 month after start of treatment. Ten patients had only ODs positive for IgM, 53 patients were positive by both IgM and IgG ELISA, and 28 patients had only ODs positive for IgG. In 63 of 91 patients with ODs positive for IgM against B. burgdorferi, the range varied from 0.500 to >2.500 (median, 1.140) and in 81 of 91 patients positive for IgG in serum, the range was 0.180 to >2.500 (median, 0.490) in the first serum sample drawn from each patient during the period. One of the 10 patients with only positive IgM was initially IgG negative but had seroconverted at 1.5 months of follow-up

SEROLOGICAL FOLLOW-UP OF LYME BORRELIOSIS

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