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Serological diagnosis of Borrelia meningitis
Zbl. Bakt. H yg. A 263 , 420-424 (1986)
Serological Diagnosis of Borrelia Meningitis GO RAN STIE RNSTEDT, MA RTA G RANS T ROM, BEN GT H EDER ST EDT an d BI RGIT S K O LD E NB ER G Department s of Clinical Bacteriology and Infectious Diseases, Danderyd Hospital, and Department of Bacteriology, National Bacteriological Labora tory, Stockholm, Sweden.
Summary The antibody response against a Borrelia strain isolated from Swedish Ixodes ricinus ticks was determined by enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) of cerebrospinal fluid (CSF) and serum specimens from 45 patients with chronic meningitis. Probable Borrelia etiology could be demonstrated in 41 of 45 (91%) patients with clinical symptoms of chronic meningitis. Approxia mtely 2~ % of the patients had significantly elevated titer of antibody to the spirochete in CSF but not in serum. Patients with short duration of disease were especially prone to be antibody negative in serum but positive in CSF. Significant rise in serum antibody titers was seldom demonstrated in patients treated with antibiotics.
Introduction T ick-borne Borrelia me ningitis mig ht be a rather easy clinical diagn osis in patients wi th preceding erythema migrans and a typ ical clinical picture wi th rad icul ar pain , crania l ne uropa thy an d a lym ph ocytic meningitis . H owever, as we ha ve po inted o ut ea rlie r, Borrelia meningitis might, in accorda nce wi th anothe r spiro chet al disease, neu ro syphilis, mimic a lot of other disorders such as brain tumo ur, herniat ed disc, cer ebro va scul ar o r psych osomat ic disease (3 ). In such p at ients a sen sitive and specific serolog ica l assay is necessar y, especially as we think th at ea rly treat ment wi th intravenous peni cillin is of gr ea t imp ortan ce. At an ea rly stage o f o ur inves tiga tio ns, we noticed th at pat ients wi th Borrelia meningit is o ften had a very stro ng intra theca l anti bo dy resp onse, as measured by aga ros e electrophoresis and IgG-index (1). O ne aim of the pr esen t invest igat ion was to in vestiga te if thi s intrathecal antibody resp on se co uld be used for th e sero logica l diagnosis of Borrelia meningitis. Another ai m was to co mpa re an indi rect immunofluorscence assay (IFA) with an enzyme-linke d immunoso rb ent assay (ELISA).
Patients and Methods The serological tests were evaluated by studies on paired serum and cerebrospinal fluid (CSF) specimens from 45 patients with the clinical diagnosis of chro nic meningitis (CM), i. e. at least 2 weeks duration of disease without improvement. 43 patients has successfully been treated with high-dose intravenous penicillin G. 2 patients had improved sponta neously when Borrelia meningitis was supected. Serum and/or CSFfrom healthy individuals, patients with infectious meningitis
Sero logical Diagnosis of Borrelia Men ingitis
421
of various but proven aetiology (meningitis contro ls), pat ients with multiple sclerosis, infectious mononucleosis, and syphilis, served as control samples. The immuofluo rescence assay was performe d according to standard methods . Borrelia spirochetes isolated from Swedish Ixodes ricinus ticks were used as antigen . A conjugate detecting both IgG and IgM-antibodies, but prefe rentia lly IgG-antibodies, was used. The ELISA test has been described in detai l elsewhere (2). Briefly, the supernatant of a sonicated preparation of Borrelia spirochetes was used as antigen. The optimal coating concentrations were found to be 5 ug/ml for IgG and 10 ug/ml for IgM of prote in in phosphate-buffered saline. Separate IgG and IgM estimations of the antibody levels was perfo rmed in the ELISA test. The ELISA titer was defined as the absorbance at 405 nm (A 4os) multiplied by the serum or CSF dilution facto r. Each samp le was tested in duplicate, and the mean value was calculated. If the two values differed more than 10% from the mean, the sample was retested. If A40s was more than 1.5 or less than 0.1, the samp le was retested in a higher or lower dilution. Positive and negative contro ls were included in each test, and the time for substra te incuba tion was adjusted to these contro ls in order to eliminat e day-to-day variations.
Ta ble 1. No. of patients with chro nic meningitis (CM) and no. of individuals in different control groups considered positive of tot al numbers tested in the different serological assays and index calculations CMpat ients
IFA Titer rise (2: 4-fold) Positive titer in serum (2: 320 ) CSF (2: 5) ELISA Titer rise (2: 2-fold)
Health y Meningitis MScontrols contro ls patients n positive/n tested ('Yo )
Syphilis pa tients
1/35 (3) 23/45 (5 1) 38/45 (84)
3/63 (6)
1/32 (3) 0/32 (0)
4/7 (57) 6/7 (86)
5/35 (14)
Positive titer in serum IgG (2: 450) serum IgM (2: 580 ) serum IgG and/or IgM CSF IgG (2: 10) CSF IgM (2: 10) CSF IgG and/or IgM
28/45 10/45 30/45 39/45 29/45 41/45
Positive index of IgG (2: 2.0) IgM (2: 1.0) IgG and/or IgM
40/45 (89) 32/45 (71 ) 41/45 (91)
(62) (22) (67) (87) (64) (91)
6/120 (5) 61120 (5) 111120 (9)
4/53 2/53 6/53 3/53 1/53 3/53
(8) (4) ( 11)
(6) (2) (6)
0/53 (0) 1/53 (2) 1/53 (2)
0/16 0/16 0/16 0/16 0/16 0/16
(0) (0) (0) (0) (0) (0)
18/25 (72) 4/25 (16) 19/25 (76) 3/12 (25) 0112 (0) 3112 (25)
0/16 (0) 0/16 (0) 0/16 (0)
Re sults
IFA. We used th e 95 percen tile tit er level o f h ealthy in d ividu al s as th e upper limit of n ormal va lues for serum titer s. In o ur test , the 95 p er centile co rresponde d to a t iter of 3 20.23 of 45 CM-patients (5 1 %) h ad titer s a bove th is lim it as compared to o nly 1 of 32 me ni ngi tis co n trol patients (T a b le 1). CSF sa m p les with detectab le anti bo dies, i. e., a
422
Goran Stiernstedt, Marta Granstrom, Bengt Hederstedt, and Birgit Skoldenberg
titer of ~ 1:5, were considered positive. Positive antibody titers in eSF were found in 38 of 45 (84% ) eM patients and in none of 32 meningitis control patients (Table 1). The results of the immunofluorescence assay are summari zed in Table 1. Both measurement of serum and eSF-titers showed a high specificity, 97 and 100 % respectively, but measurement of eSF-antibodies was consider ably more sensitive 84 %, as compared with a sensitivity of only 51 % for measurement of serum antibodies. ELISA. Also in the ELISA assay the 95% titer level of healthy individuals was used as the upper limit of normal values for serum titers . In the ELISAtest, the 95 percentile corresponded to a titer of 450 in IgG and 580 in IgM. Of 45 eM patients, 28 (62% ) were IgG positive, 10 (22% ) were IgM positive, and 8 (18%) were both IgG and IgM positive . Thus, 30 of 45 patients (67%) were positive for IgG or IgM or both (Table 1). The median titer levels were not significantl y different between healthy individuals and patients with meningitis of various etiology. Totally 4/53 (8%) meningitis controls were positive in IgG, 2/53 (4% ) in IgM and 6/53 (11 %) in either IgG or IgM, or both (Table 1). None of 16 patients with multiple sclerosis was seropositive, whereas 10 of 20 patients with mononucleosis were IgM seropositive and 2 of 20 were in both IgG and IgM positive. Among patients with other spirochetal diseases, 19 of 25 (76% ) patients with syphilis and 1 of 4 patients with leptospirosis had significantly elevated antibody titers. By measuring eSF-titers we achieved great differences in titer levels between Clvl patients and controls. A titer level of 10 was set as the upper limit of normal values for both IgG and IgM. With these limits 39 of 45 (87%) eM patients were positive in IgG, 29/45 (64%) in IgM, and 41/45 (91% ) in either IgG or IgM, or both (Table 1). Significantly elevated eSF titers (either IgG or IgM or both ) were found in 29/30 seropositive patients (data not shown ). Using the above stated limits, positive eSF titers in either IgG or IgM, or both, were found in 3/53 (6% ) meningitis controls. Since three patients were positive in IgG and one in IgM, one patient was positive in both IgG and IgM. This patient suffered from tuberculous meningitis, as did one patie nt positive in IgG only. Both patients with tuberculous meningitis were seropositive in the corresponding immunoglobulin class, and had high eSF/serum albumin ratios indicating a high degree of a damage to the blood-brain barrier. A simple measuring in eSF of serum antibodies does not consider one important fact, namely the antibody leakage over the eSF /blood barrier. We tried to compensate for the antibody-leakage by calculating a eSF-Borrelia-titer index, which is the ratio between two ratio s, namely the titer ratio and the albumin ratio ELISA eSF-titer ELISA serum-titer
eSF-albumin : serum-albumin
The calculation of a eSF-spirochete-titer index is in analogy with the calculation of an IgG-index where the IgG ratio is divided with the albumin ratio . An elevated IgGindex indicates inthrathecal antibody production of IgG-antibodies. A eSF Borrelia IgG titer index value of 2.0 was chosen as the upper limit of normal values. The corresponding value for IgM spirochete-titer -index was 1.0. Using these limits, 39/45 (87 % ) eM patients were positive in IgG, 32/45 (71% ) in IgM and 41/45 (91% ) in either IgG or IgM, or both (Table 2). Among meningitis controls, only one patient who suffered from tuberculous meningitis showed a slight increase in IgM eSF Borrelia titer index, with a value of 1.03.
Serological Diagnosis of Borrelia Meningitis
423
T able 1 summa rizes th e sensitivity and specificity of th e differe nt calculations carried out by ELISA. Measurement of CSF-anti bodies was a mor e sensitive and also a mo re specific meth od th an measurement of serum an tibodies. However, th e highes t diagnostic sensitivity and specificity was achieved by calculat ion of a specific CSF Borreliatiter-index. We also evalua ted how the durat ion of disease befo re tr eatment with PcG infl uenced the antibody titer levels and found that the median titers increased in IgG and dec reased in IgM with increasi ng d uration of disease. Th e correspo nding results were achieved when we evalua ted the CSF-titers in th e sa me-way, th at is increasing median IgG-titers and decreasing median IgM-titers with increasing dur at ion of disease unt il sampling and treat ment. We also evalua ted th e influ ence of treatm ent on titer levels and found tha t th e IgG seru m tit ers increased slightly immediately post-treat ment but th en gra dually decreased. The IgM seru m titers and the CSF-titers of both IgG and IgM decreased posttreatm ertt, Another ind ication of th e same pheno mo nen was th at we ha d great difficulties in setting th e diagnosis of Borrelia meningitis by mean s of a significan t incr ease of serum titers. Onl y 3/32 pati ent s who received ant ibiotic treatm ent showed a significa nt incr ease of titer s as compared with both pati ent s not tr eat ed with antibiotics. It seems likely that ant ibiotic tr eatm ent int errupts th e an tigenic stim ulation of th e immune system resulting in decreasing tit ers.
Discussion When discuss ing th e sensitivity of th e different assays for the sero logica l diagnosis of
Borrelia meningitis, one poss ible bias is that th e sent ivitiy had to be evaluated against the clinical diagnosis of meningitis. Therefore, th e possibility of a non-spirochetal etio logy in some CM-patients can no t be excluded com pletely . Th us, the sensitivity of th e different assays may have been underes tima ted . O n the ot her hand, the selection of many patient s, who had a lo ng history of disease an d also a long time for the antibody response to develop , might have th e opposite effect, na mely th at of overes timating the sensitivity. Th e results of the present study show th at the antibo dy respo nse is rath er slow when com pa red to many other infectio us diseases. In man y patient s, 5- 10 weeks' durat ion of disease was necessary befo re sign ificantly high seru m anti bo dy titer levels were rea ched. Ano ther effect of the slow ant ibody response in seru m is th at meas ure ment of CSF antibo dies was especially imp o rtant for accurate diagnosis in pati ents w ith sho rt durat ion of disease « 5 weeks). M easurement of CSF antibo dies was necessar y for diagnosis in approx imately 25% of th e pati ents , but in pat ient s with less th an 5 weeks' duration of disease th e cor responding figure was abo ut 40%. Furthe rmore, measur ement of CSF ant ibo dies was also a more specific meth od th an measur ement of seru m anti bodies both by IFA and ELISA. Estima tion of th e degree of CSF-bloo d barri er damage by calcu lation of a CSF Borrelia titer index has some definite adva ntages . Besides being bo th the mos t sensitive and specific meth od , it also indicates a direct rela tionship to th e infection goi ng on in th e CNS, whereas positive serum an tibody tit ers migh t be due to an intercurre nt or subclinica l infection. Th e slow an tibo dy response in patient s with Borrelia infection will in th e future probably result in problems with the serological diag nosis as th e patients will be admi tted earlier than in the present stud y. Ma ny pa tients will be
424
Coran Stiernstedt, Marta Granstrom, Bengt Hederstedt, and Birgit Skoldenberg
admitted after only 1-2 weeks duration of disease, at a time when specific antibodies could neither be detected in serum nor in CSF. We have already noted this problem during the last year in some patients with a most typical clinical picture of Borrelia meningitis including preceding erythema migrans. One of the remaining problems is how to interpret serum positive but CSF titer and CSF spirochete index negative patients. In the present study, one patient had this pattern in her antibod y response. This old woman had a skin lesion, and subsequently developed neurological symptoms. Her CSF showed both pleocytosis and raised CSF protein. During 1984, we have seen some further patients who have been seropositive but CSF titer negative. They have all had cutaneous lesions followed by neurological symptoms. One possible explanation for this antibody pattern is that these patients have their primary antibod y response in serum at the time of their cutaneous lesion. If caught early in the subsequent neurological phase of disease, antibody titers in CSF of these patients might not have reached the cut-off level. Thus, at present, it seems likely that measurement of serum antibodies and CSF antibodies are complementary when ,trying to obtain a serological diagnosis of Borrelia meningitis. References 1. Seo ldenberg, B., G. Stiernstedt, A . Garde, G. Kolmodin, A . Carlstom, C. E. No rd: Chronic
meningitis caused by a penicillin-sensitive microorganism? Lancet ii (1983): 75-78 .
2. Stiernstedt G., M. Granstrom, B. Hederstedt, B. Skc ldenbcrg: Diagnosis of spirochetal menin-
gitis byenzyme-linked immunosorbent assay and indirect immunofl uorescence assay in serum and cerebrospinal fluid.] Clin Microbiol 21 (1985): 819-825. 3. Stiernstedt G.: Tick-borne Borrelia infection in Sweden. Scand.] . Infect. Dis. supp!. no.45, 1985.
Goran Stiernstedt, Dept. of Clinical Bacteriology and Infectious Diseases, Danderyd Hospital, S-1 8288 Danderyd, Sweden
Serological Diagnosis of Borrelia Meningitis GO RAN STIE RNSTEDT, MA RTA G RANS T ROM, BEN GT H EDER ST EDT an d BI RGIT S K O LD E NB ER G Department s of Clinical Bacteriology and Infectious Diseases, Danderyd Hospital, and Department of Bacteriology, National Bacteriological Labora tory, Stockholm, Sweden.
Summary The antibody response against a Borrelia strain isolated from Swedish Ixodes ricinus ticks was determined by enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) of cerebrospinal fluid (CSF) and serum specimens from 45 patients with chronic meningitis. Probable Borrelia etiology could be demonstrated in 41 of 45 (91%) patients with clinical symptoms of chronic meningitis. Approxia mtely 2~ % of the patients had significantly elevated titer of antibody to the spirochete in CSF but not in serum. Patients with short duration of disease were especially prone to be antibody negative in serum but positive in CSF. Significant rise in serum antibody titers was seldom demonstrated in patients treated with antibiotics.
Introduction T ick-borne Borrelia me ningitis mig ht be a rather easy clinical diagn osis in patients wi th preceding erythema migrans and a typ ical clinical picture wi th rad icul ar pain , crania l ne uropa thy an d a lym ph ocytic meningitis . H owever, as we ha ve po inted o ut ea rlie r, Borrelia meningitis might, in accorda nce wi th anothe r spiro chet al disease, neu ro syphilis, mimic a lot of other disorders such as brain tumo ur, herniat ed disc, cer ebro va scul ar o r psych osomat ic disease (3 ). In such p at ients a sen sitive and specific serolog ica l assay is necessar y, especially as we think th at ea rly treat ment wi th intravenous peni cillin is of gr ea t imp ortan ce. At an ea rly stage o f o ur inves tiga tio ns, we noticed th at pat ients wi th Borrelia meningit is o ften had a very stro ng intra theca l anti bo dy resp onse, as measured by aga ros e electrophoresis and IgG-index (1). O ne aim of the pr esen t invest igat ion was to in vestiga te if thi s intrathecal antibody resp on se co uld be used for th e sero logica l diagnosis of Borrelia meningitis. Another ai m was to co mpa re an indi rect immunofluorscence assay (IFA) with an enzyme-linke d immunoso rb ent assay (ELISA).
Patients and Methods The serological tests were evaluated by studies on paired serum and cerebrospinal fluid (CSF) specimens from 45 patients with the clinical diagnosis of chro nic meningitis (CM), i. e. at least 2 weeks duration of disease without improvement. 43 patients has successfully been treated with high-dose intravenous penicillin G. 2 patients had improved sponta neously when Borrelia meningitis was supected. Serum and/or CSFfrom healthy individuals, patients with infectious meningitis
Sero logical Diagnosis of Borrelia Men ingitis
421
of various but proven aetiology (meningitis contro ls), pat ients with multiple sclerosis, infectious mononucleosis, and syphilis, served as control samples. The immuofluo rescence assay was performe d according to standard methods . Borrelia spirochetes isolated from Swedish Ixodes ricinus ticks were used as antigen . A conjugate detecting both IgG and IgM-antibodies, but prefe rentia lly IgG-antibodies, was used. The ELISA test has been described in detai l elsewhere (2). Briefly, the supernatant of a sonicated preparation of Borrelia spirochetes was used as antigen. The optimal coating concentrations were found to be 5 ug/ml for IgG and 10 ug/ml for IgM of prote in in phosphate-buffered saline. Separate IgG and IgM estimations of the antibody levels was perfo rmed in the ELISA test. The ELISA titer was defined as the absorbance at 405 nm (A 4os) multiplied by the serum or CSF dilution facto r. Each samp le was tested in duplicate, and the mean value was calculated. If the two values differed more than 10% from the mean, the sample was retested. If A40s was more than 1.5 or less than 0.1, the samp le was retested in a higher or lower dilution. Positive and negative contro ls were included in each test, and the time for substra te incuba tion was adjusted to these contro ls in order to eliminat e day-to-day variations.
Ta ble 1. No. of patients with chro nic meningitis (CM) and no. of individuals in different control groups considered positive of tot al numbers tested in the different serological assays and index calculations CMpat ients
IFA Titer rise (2: 4-fold) Positive titer in serum (2: 320 ) CSF (2: 5) ELISA Titer rise (2: 2-fold)
Health y Meningitis MScontrols contro ls patients n positive/n tested ('Yo )
Syphilis pa tients
1/35 (3) 23/45 (5 1) 38/45 (84)
3/63 (6)
1/32 (3) 0/32 (0)
4/7 (57) 6/7 (86)
5/35 (14)
Positive titer in serum IgG (2: 450) serum IgM (2: 580 ) serum IgG and/or IgM CSF IgG (2: 10) CSF IgM (2: 10) CSF IgG and/or IgM
28/45 10/45 30/45 39/45 29/45 41/45
Positive index of IgG (2: 2.0) IgM (2: 1.0) IgG and/or IgM
40/45 (89) 32/45 (71 ) 41/45 (91)
(62) (22) (67) (87) (64) (91)
6/120 (5) 61120 (5) 111120 (9)
4/53 2/53 6/53 3/53 1/53 3/53
(8) (4) ( 11)
(6) (2) (6)
0/53 (0) 1/53 (2) 1/53 (2)
0/16 0/16 0/16 0/16 0/16 0/16
(0) (0) (0) (0) (0) (0)
18/25 (72) 4/25 (16) 19/25 (76) 3/12 (25) 0112 (0) 3112 (25)
0/16 (0) 0/16 (0) 0/16 (0)
Re sults
IFA. We used th e 95 percen tile tit er level o f h ealthy in d ividu al s as th e upper limit of n ormal va lues for serum titer s. In o ur test , the 95 p er centile co rresponde d to a t iter of 3 20.23 of 45 CM-patients (5 1 %) h ad titer s a bove th is lim it as compared to o nly 1 of 32 me ni ngi tis co n trol patients (T a b le 1). CSF sa m p les with detectab le anti bo dies, i. e., a
422
Goran Stiernstedt, Marta Granstrom, Bengt Hederstedt, and Birgit Skoldenberg
titer of ~ 1:5, were considered positive. Positive antibody titers in eSF were found in 38 of 45 (84% ) eM patients and in none of 32 meningitis control patients (Table 1). The results of the immunofluorescence assay are summari zed in Table 1. Both measurement of serum and eSF-titers showed a high specificity, 97 and 100 % respectively, but measurement of eSF-antibodies was consider ably more sensitive 84 %, as compared with a sensitivity of only 51 % for measurement of serum antibodies. ELISA. Also in the ELISA assay the 95% titer level of healthy individuals was used as the upper limit of normal values for serum titers . In the ELISAtest, the 95 percentile corresponded to a titer of 450 in IgG and 580 in IgM. Of 45 eM patients, 28 (62% ) were IgG positive, 10 (22% ) were IgM positive, and 8 (18%) were both IgG and IgM positive . Thus, 30 of 45 patients (67%) were positive for IgG or IgM or both (Table 1). The median titer levels were not significantl y different between healthy individuals and patients with meningitis of various etiology. Totally 4/53 (8%) meningitis controls were positive in IgG, 2/53 (4% ) in IgM and 6/53 (11 %) in either IgG or IgM, or both (Table 1). None of 16 patients with multiple sclerosis was seropositive, whereas 10 of 20 patients with mononucleosis were IgM seropositive and 2 of 20 were in both IgG and IgM positive. Among patients with other spirochetal diseases, 19 of 25 (76% ) patients with syphilis and 1 of 4 patients with leptospirosis had significantly elevated antibody titers. By measuring eSF-titers we achieved great differences in titer levels between Clvl patients and controls. A titer level of 10 was set as the upper limit of normal values for both IgG and IgM. With these limits 39 of 45 (87%) eM patients were positive in IgG, 29/45 (64%) in IgM, and 41/45 (91% ) in either IgG or IgM, or both (Table 1). Significantly elevated eSF titers (either IgG or IgM or both ) were found in 29/30 seropositive patients (data not shown ). Using the above stated limits, positive eSF titers in either IgG or IgM, or both, were found in 3/53 (6% ) meningitis controls. Since three patients were positive in IgG and one in IgM, one patient was positive in both IgG and IgM. This patient suffered from tuberculous meningitis, as did one patie nt positive in IgG only. Both patients with tuberculous meningitis were seropositive in the corresponding immunoglobulin class, and had high eSF/serum albumin ratios indicating a high degree of a damage to the blood-brain barrier. A simple measuring in eSF of serum antibodies does not consider one important fact, namely the antibody leakage over the eSF /blood barrier. We tried to compensate for the antibody-leakage by calculating a eSF-Borrelia-titer index, which is the ratio between two ratio s, namely the titer ratio and the albumin ratio ELISA eSF-titer ELISA serum-titer
eSF-albumin : serum-albumin
The calculation of a eSF-spirochete-titer index is in analogy with the calculation of an IgG-index where the IgG ratio is divided with the albumin ratio . An elevated IgGindex indicates inthrathecal antibody production of IgG-antibodies. A eSF Borrelia IgG titer index value of 2.0 was chosen as the upper limit of normal values. The corresponding value for IgM spirochete-titer -index was 1.0. Using these limits, 39/45 (87 % ) eM patients were positive in IgG, 32/45 (71% ) in IgM and 41/45 (91% ) in either IgG or IgM, or both (Table 2). Among meningitis controls, only one patient who suffered from tuberculous meningitis showed a slight increase in IgM eSF Borrelia titer index, with a value of 1.03.
Serological Diagnosis of Borrelia Meningitis
423
T able 1 summa rizes th e sensitivity and specificity of th e differe nt calculations carried out by ELISA. Measurement of CSF-anti bodies was a mor e sensitive and also a mo re specific meth od th an measurement of serum an tibodies. However, th e highes t diagnostic sensitivity and specificity was achieved by calculat ion of a specific CSF Borreliatiter-index. We also evalua ted how the durat ion of disease befo re tr eatment with PcG infl uenced the antibody titer levels and found that the median titers increased in IgG and dec reased in IgM with increasi ng d uration of disease. Th e correspo nding results were achieved when we evalua ted the CSF-titers in th e sa me-way, th at is increasing median IgG-titers and decreasing median IgM-titers with increasing dur at ion of disease unt il sampling and treat ment. We also evalua ted th e influ ence of treatm ent on titer levels and found tha t th e IgG seru m tit ers increased slightly immediately post-treat ment but th en gra dually decreased. The IgM seru m titers and the CSF-titers of both IgG and IgM decreased posttreatm ertt, Another ind ication of th e same pheno mo nen was th at we ha d great difficulties in setting th e diagnosis of Borrelia meningitis by mean s of a significan t incr ease of serum titers. Onl y 3/32 pati ent s who received ant ibiotic treatm ent showed a significa nt incr ease of titer s as compared with both pati ent s not tr eat ed with antibiotics. It seems likely that ant ibiotic tr eatm ent int errupts th e an tigenic stim ulation of th e immune system resulting in decreasing tit ers.
Discussion When discuss ing th e sensitivity of th e different assays for the sero logica l diagnosis of
Borrelia meningitis, one poss ible bias is that th e sent ivitiy had to be evaluated against the clinical diagnosis of meningitis. Therefore, th e possibility of a non-spirochetal etio logy in some CM-patients can no t be excluded com pletely . Th us, the sensitivity of th e different assays may have been underes tima ted . O n the ot her hand, the selection of many patient s, who had a lo ng history of disease an d also a long time for the antibody response to develop , might have th e opposite effect, na mely th at of overes timating the sensitivity. Th e results of the present study show th at the antibo dy respo nse is rath er slow when com pa red to many other infectio us diseases. In man y patient s, 5- 10 weeks' durat ion of disease was necessary befo re sign ificantly high seru m anti bo dy titer levels were rea ched. Ano ther effect of the slow ant ibody response in seru m is th at meas ure ment of CSF antibo dies was especially imp o rtant for accurate diagnosis in pati ents w ith sho rt durat ion of disease « 5 weeks). M easurement of CSF antibo dies was necessar y for diagnosis in approx imately 25% of th e pati ents , but in pat ient s with less th an 5 weeks' duration of disease th e cor responding figure was abo ut 40%. Furthe rmore, measur ement of CSF ant ibo dies was also a more specific meth od th an measur ement of seru m anti bodies both by IFA and ELISA. Estima tion of th e degree of CSF-bloo d barri er damage by calcu lation of a CSF Borrelia titer index has some definite adva ntages . Besides being bo th the mos t sensitive and specific meth od , it also indicates a direct rela tionship to th e infection goi ng on in th e CNS, whereas positive serum an tibody tit ers migh t be due to an intercurre nt or subclinica l infection. Th e slow an tibo dy response in patient s with Borrelia infection will in th e future probably result in problems with the serological diag nosis as th e patients will be admi tted earlier than in the present stud y. Ma ny pa tients will be
424
Coran Stiernstedt, Marta Granstrom, Bengt Hederstedt, and Birgit Skoldenberg
admitted after only 1-2 weeks duration of disease, at a time when specific antibodies could neither be detected in serum nor in CSF. We have already noted this problem during the last year in some patients with a most typical clinical picture of Borrelia meningitis including preceding erythema migrans. One of the remaining problems is how to interpret serum positive but CSF titer and CSF spirochete index negative patients. In the present study, one patient had this pattern in her antibod y response. This old woman had a skin lesion, and subsequently developed neurological symptoms. Her CSF showed both pleocytosis and raised CSF protein. During 1984, we have seen some further patients who have been seropositive but CSF titer negative. They have all had cutaneous lesions followed by neurological symptoms. One possible explanation for this antibody pattern is that these patients have their primary antibod y response in serum at the time of their cutaneous lesion. If caught early in the subsequent neurological phase of disease, antibody titers in CSF of these patients might not have reached the cut-off level. Thus, at present, it seems likely that measurement of serum antibodies and CSF antibodies are complementary when ,trying to obtain a serological diagnosis of Borrelia meningitis. References 1. Seo ldenberg, B., G. Stiernstedt, A . Garde, G. Kolmodin, A . Carlstom, C. E. No rd: Chronic
meningitis caused by a penicillin-sensitive microorganism? Lancet ii (1983): 75-78 .
2. Stiernstedt G., M. Granstrom, B. Hederstedt, B. Skc ldenbcrg: Diagnosis of spirochetal menin-
gitis byenzyme-linked immunosorbent assay and indirect immunofl uorescence assay in serum and cerebrospinal fluid.] Clin Microbiol 21 (1985): 819-825. 3. Stiernstedt G.: Tick-borne Borrelia infection in Sweden. Scand.] . Infect. Dis. supp!. no.45, 1985.
Goran Stiernstedt, Dept. of Clinical Bacteriology and Infectious Diseases, Danderyd Hospital, S-1 8288 Danderyd, Sweden