Detection of Immunoglobulin M in Cerebrospinal Fluid from Syphilis Patients by Enzyme-Linked Immunosorbent Assay

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1986, p Vol. 24, No /86/ $02.00/0 Detection of Immunoglobulin M in Cerebrospinal Fluid from Syphilis Patients by Enzyme-Linked Immunosorbent Assay JUNG BOCK LEE,1t CAROL E. FARSHY,1* ELIZABETH F. HUNTER,1 EDITH A. HAMBIE,1 GARY H. WOBIG,2 AND SANDRA A. LARSEN1 Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Serology Laboratory, Mayo Clinic, Rochester, Minnesota Received 7 April 1986/Accepted 16 July 1986 Cerebrospinal fluid (CSF) samples were evaluated in an immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) for syphilis with sonic extracts of Treponema pallidum coated on polystyrene plates. The ELISA procedure was reproducible, and T. pallidum antigens were stable. A total of 15 CSF samples from patients with neurosyphilis, 18 CSF samples from patients with syphilis, 12 CSF samples from patients treated for syphilis, and 494 CSF samples from patients with neurologic or other systemic diseases were tested. The IgM ELISA gave reactive results in all of six symptomatic and congenital neurosyphilitic patients and none of nine asymptomatic neurosyphilitic patients. Of 524 CSF samples from nonneurosyphilitic individuals, 513 were nonreactive, resulting in 98% test specificity. The IgM ELISA in CSF should prove to be useful for confirmation of symptomatic neurosyphilis. During the course of early syphilis the central nervous system is frequently involved (3, 10, 18). Although changes in the cerebrospinal fluid (CSF) in early syphilis are usually transient, some untreated patients have persistently abnormal CSF, despite the resolution of the signs of early disease. In general, the risk of neurologic disease parallels the degree of CSF abnormality (19). The presence of pleocytosis, increased levels of protein (Dattner et al. [5]), and a reactive Venereal Disease Research Laboratory (VDRL) test result in CSF have been used for many years to identify active neurosyphilis. Unfortunately, these tests are of very low diagnostic value because of their low sensitivity (6, 7, 12). Others have found the CSF-fluorescent treponemal antibody (CSF-FTA) (11), CSF- FTA-absorbed (CSF-FTA-ABS) (6), and CSF microhemagglutination assay for T. pallidum antibody (MHA-TP) (16) tests to be more sensitive but less specific than the CSF- VDRL test for neurosyphilis (13). Reactive CSF tests do not necessarily indicate active disease since reactivity may be caused by transudation of serum antitreponemal immunoglobulin G (IgG) into CSF (22). Therefore, work continues toward the development of a suitable test(s) for active neurosyphilis (17, 21). Enzyme-linked immunosorbent assay (ELISA) has been used to measure antitreponemal IgG (9, 24, 25, 31) and IgM (8, 14, 23) in serum. We now report the use of ELISA to measure antitreponemal IgM in the CSF of syphilitic patients. The results of this test were compared with those obtained with the VDRL, MHA-TP, FTA, FTA-ABS, IgM FTA, and IgM FTA-ABS tests on serum and CSF. In addition, the concentration of albumin was determined in 73 pairs of serum and CSF obtained from syphilitic and nonsyphilitic patients. In this way, we were then able to compare the results of the IgM ELISA in CSF according to the integrity of the blood-csf barrier (26). * Corresponding author. t Present address: Department of Dermatology, Yonsei University College of Medicine, Seoul, South Korea. 736 MATERIALS AND METHODS Sera and CSF samples. The sera and CSF used in this study were divided into two groups. Group 1 consisted of 36 pairs of serum and CSF and 458 individual CSF samples from patients with neurologic or other systemic diseases; group 2 consisted of 45 pairs of serum and CSF samples from patients with syphilis. Of these 45 pairs, 12 were from patients adequately treated for 1 year before repeated examination. The remaining 33 pairs were from patients with different categories of syphilis: 1 from a patient with primary syphilis, 2 from patients with secondary syphilis, 14 from patients with latent syphilis, 1 from a patient with late congenital syphilis, 9 from patients with asymptomatic neurosyphilis, 4 from patients with symptomatic neurosyphilis, and 2 from patients with congenital neurosyphilis. Those CSF samples from patients exhibiting elevated total protein and cell count as well as an initial reactive VDRL CSF were classified asymptomatic. Group 1 samples were obtained from the Centers for Disease Control Treponemal Serum Bank and Mayo Clinic Serology Laboratory. Group 2 samples were from Yonsei University, Seoul, South Korea. Diagnoses were based on both clinical and serologic data. All specimens were coded and tested blindly. Serologic tests. The VDRL, FTA-ABS, and MHA-TP tests were performed on all pairs of serum and CSF as received and then stored at -70 C. Tests on serum samples and the VDRL-CSF were done according to published methods (30). The MHA-TP test was performed in accordance with the directions of the manufacturer (Fujirebio Pharmaceutical Co., Ltd., Tokyo, Japan). For the CSF MHA-TP the initial dilution was 1:2 in absorbing diluent. Twofold serial dilutions were prepared on the microtiter plate. The CSF-MHA- TP test results were considered reactive at a dilution of.1:80. The IgM FTA and the IgM FTA-ABS (27) tests were performed on CSF samples from 36 of the patients with neurologic disorders. Other tests on spinal fluid were performed as previously described (30). Antigens. Treponema pallidum street strain 14 was used to prepare five antigens which were adjusted to a concentration

2 VOL. 24, 1986 DETECTION OF IgM IN CSF FROM SYPHILIS PATIENTS 737 TABLE 1. Evaluation of CSF (diluted 1:10 in PBS) from relevant individuals to determine the OD cutoff for the T. pallidum-specific IgM ELISA OD at No. of No. of nonneurosyphilitics No. of neurosyphilitics 490 nm nonsyphilitics Treated Early and latenta Asymptomaticb Symptomatic Congenitalc < aone patient with late congenital syphilis is included in this category. b Criteria for asymptomatic neurosyphilis: reactive VDRL or FTA-ABS test in CSF without clinical symptoms of neurosyphilis. c Criteria for congenital neurosyphilis: reactive VDRL or FTA-ABS test in CSF and serum with maternal syphilis and no treatment; previous history for syphilis. of 108 treponemes per ml. Inoculation and extraction of infected rabbit testicular tissue were performed according to the procedure used for preparation of the FTA-ABS test antigen (7). The tissue extract was centrifuged at 1,500 rpm for 10 min to remove testicular tissue. The supernatant containing the treponemes was centrifuged at 12,000 rpm for 30 min at 5 C, and the pellet was washed twice in phosphatebuffered saline (PBS), ph 7.2 (30). After the final centrifugation, the pellet was suspended in PBS to 108 treponemes per ml, held at 5 C for 7 days, and then homogenized by ultrasonication (25). Conjugate. Horseradish peroxidase-labeled anti-human- IgM chain-specific conjugate (DAKO Chemicals, Accurate Scientific Corp., Westbury, N.Y.) diluted in 0.05% Tween 20-PBS containing 1% normal rabbit serum was used. The optimal dilution as determined by block titration was 1:500. Substrate. A stock solution of substrate was prepared by dissolving 50 mg of ortho-phenylenediamine (Eastman Kodak Co., Rochester, N.Y.) in 5 ml of absolute methanol. The working substrate solution was prepared immediately before use by adding 1 ml of stock solution and 0.1 ml of 3% hydrogen peroxide to 99 ml of distilled water (32). ELISA procedure. The ELISA procedure was similar to the assays described for IgG antibody (8, 10, 25) with these exceptions. To each well of flat-bottomed polystyrene microelisa plates (Immulon 2; Dynatech Laboratories, Inc., Alexandria, Va.) 100 pl of T. pallidum antigen suspension diluted in 0.05 M carbonate buffer, ph 9.6, was added and allowed to dry at 37 C overnight (20). Then, 50,ul of 95% ethanol was added to each well, and plates were allowed to dry at 37 C for approximately 3 h. Plates were stored in the presence of a desiccant (silica gel) at 5 C for at least 24 h before use. Immediately before the plates were used, they were washed three times with 0.05% Tween 20-PBS. In the first well of the plate, CSF samples were diluted 1:10 with 0.05% TABLE 2. Values of OD at 490 nm in IgM ELISA with five lots of T. pallidum with the reactive control serum from a patient with secondary syphilis OD for 4 lots OD for lot El Serum dilution A B c D Time tekr 1: :1, :3, a Two values for one lot at a 2-week interval. Tween 20-PBS containing 1% normal rabbit serum, followed by serial twofold dilutions with a final dilution of 1:1,280. Positive and negative control CSF samples were included on every plate. Plates were incubated at 4 C, followed by the addition of conjugate and substrate (10). The plates were read spectrophotometrically at 490 nm on a Dynatech ELISA reader. A well containing no serum served as a blank for the assay. Stability and reproducibility. Serum from a patient with secondary syphilis and serum from a presumed normal individual were used to assess the stability of the antigen and reproducibility of the IgM ELISA. The antigen dilution was assayed by block titration in two periodic testings. Five lots of antigen were compared. Determination of albumin concentration. Determination of albumin concentrations in serum and CSF was performed by single radial immunodiffusion by using LC-Partigen albumin kits (Calbiochem-Behring, La Jolla, Calif.). RESULTS Cutoff point for OD. Optical densities (ODs) in the IgM ELISA were recorded for CSF samples from syphilitic and nonsyphilitic individuals. An OD of -0.1 at 490 nm was considered to be reactive. Table 1 shows the ODs for 1:10 dilutions of CSF from different categories of syphilitic and nonsyphilitic patients. Of 494 CSF samples from nonsyphilitic patients, 483 or 98% were nonreactive at an OD of <0.10. Of 30 samples from patients with past history of syphilis without neurologic symptoms (nonneurosyphilitic), 100% were nonreactive at an OD of '0.10. Of 15 neurosyphilis CSF samples, the 9 from asymptomatic patients were nonreactive at an OD of <0.05, while the 6 samples from symptomatic individuals were reactive at an OD of Stability of the antigen and reproducibility of the IgM ELISA. Assays were performed by using a separate antigen lot at a 2-week interval to determine the stability of the antigen. The ODs for the positive control serum, a sample from a patient with secondary syphilis, are shown in Table 2. The ODs compare favorably, especially for the 1:1,600 dilution, indicating that the antigen is stable for at least 2 weeks. Five lots of T. pallidum antigen were tested. The ODs at a 1:1,600 dilution of the same specimen varied from to Test results for sera. There were 36 patients with neurologic disease other than syphilis and for which both sera and CSF were available for evaluation. There were 45 patients with syphilis for which both sera and CSF were available for

3 738 LEE ET AL. J. CLIN. MICROBIOL. TABLE 3. CSF reactivity in nonsyphilitic and syphilitic patients No. of nonsyphilitics No.of nonneurosyphilitics No. of neurosyphilitics Serology Untreated early Treated Asymptomatic Symptomatic Congenital n = 36 n = 458 and latent (n = 12) (n = 9) (n = 4) (n = 2) (n = 18) VDRL MHA-TP FTA-ABS FTA, undiluted 0 10 NDa ND ND ND ND IgM FTA-ABS ND ND ND ND IgM FTA, undiluted ND ND ND ND IgM ELISA a ND, Not done. this study. For the nonsyphilis samples, all 36 were nonreactive in the VDRL, MHA-TP, and FTA-ABS tests. The sera from the patients with syphilis are divided into categories of nonneurosyphilis and neurosyphilis. Those in the nonneurosyphilis category were further divided into treated and untreated groups. The neurosyphilis group was divided into asymptomatic, symptomatic, and congenital neurosyphilis groups. With the exception of two patients in the nonneurosyphilis category, all 45 patients gave reactive serologic results with all three tests. The remaining two nonneurosyphilis patients showed nonreactive results in the VDRL test only. Test results for CSF. Table 3 shows the CSF reactivity in samples from the above nonsyphilitic and syphilitic patients. Additionally, 458 individual CSF samples are included from nonsyphilitic patients from which no serum samples were available. From the 494 collective CSF samples from nonsyphilitic individuals, the only reactivity was in undiluted FTA (2%) and the IgM ELISA (2%). For the 12 CSF samples from treated syphilis patients, 3 (25%) were reactive in the MHA-TP test, 2 (17%) were reactive in the FTA-ABS test, and none reacted in the IgM ELISA. For the 18 CSF samples from untreated patients with early and latent syphilis, there was no reactivity in any of the tests. For the 9 CSF samples from asymptomatic neurosyphilis patients, 2 (22%) were reactive in VDRL, 7 (78%) were reactive in MHA-TP, 9 (100%) were reactive in FTA-ABS tests, and the IgM ELISA was nonreactive. All 4 CSF samples from symptomatic neurosyphilis patients were reactive in VDRL, MHA- TP, FTA-ABS, undiluted IgM FTA, and IgM ELISA tests. Two of these were reactive in the IgM FTA-ABS test. Both CSF specimens from congenital neurosyphilis patients were reactive in VDRL, undiluted IgM FTA, and IgM ELISA tests. One specimen was also reactive in the FTA-ABS test. Serum/CSF albumin ratio. All sera and CSF samples from patients with syphilis and 28 selected sera and CSF samples from patients with neurologic disorders other than syphilis were tested to determine the protein elevation assumed to be due to impairment of the blood-csf barrier. The criteria used for determination of the impairment were the serum/ CSF albumin ratios as described by Schliep and Folgenhauer (26): slight impairment is 70 to 130, moderate is 30 to 70, severe is 10 to 30, and breakdown is below 10. The values for all specimens from nonsyphilitic individuals were above 30 with 82% above 70 (Table 4). All values from syphilitic individuals except for those with congenital syphilis were above 70. In the nonsyphilitic group 14 had values above 130, 9 had slight breakdown, and 5 had moderate breakdown. None of these CSFs was reactive in the IgM ELISA. Thirty-five of the albumin values from syphilitic patients were >130, while eight were in the range of slight breakdown. The two congenital syphilis patients had moderate or severe breakdown. CSF samples from both of these were reactive in the IgM ELISA. Out of 43 other syphilitic CSF samples, 1 was reactive in the IgM ELISA with no impairment; 8 of the 43 had slight impairment based on albumin ratios, with 3 of the 8 CSF samples reactive in IgM ELISA. DISCUSSION Currently, the diagnosis of neurosyphilis is made on the basis of a reactive VDRL, MHA-TP, or FTA-ABS test, pleocytosis, and raised protein concentrations in the CSF, as well as clinical symptoms. The occurrence of atypical forms causing only mild symptoms (i.e., mild tinnitus, deafness, or dizziness with positive serology) as well as nonspecific laboratory findings are not always helpful in the diagnosis of neurosyphilis (13, 17, 21). Luger et al. (17) recommended diagnosis by measurement of antitreponemal IgM in CSF or determination of an index relating MHA-TP titer in CSF to a ratio of albumin concentration in serum and CSF for diagnosis. Muller and Moskophidis (21) determined a local production of antitreponemal IgG in the central nervous system by estimating the CSF/serum ratio of T. pallidum TABLE 4. Serum/CSF ratio of albumin concentration in nonsyphilitic and syphilitic patients No. of nonneurosyphilitics No. of neurosyphilitics Serum/CSF ratio No. of of albumina nonsyphiliticsb Untreated early Treated Asymptomatic Symptomatic Congenital and latent > >10 a Criteria for various blood-csf barrier impairments by serum/csf ratios of albumin. Albumin concentration was determined by the method of Schliep and Folgenhauer (26): slight impairment is 70 to 130, moderate is 30 to 70, severe is 10 to 30, and breakdown is below 10. b Patients in this category have various neurologic disorders.

4 VOL. 24, 1986 DETECTION OF IgM IN CSF FROM SYPHILIS PATIENTS 739 hemagglutination-igg titer per milligram of total IgG. Cerny et al. described an ELISA method to detect serum contamination of CSF by the demonstration of adenovirus antibodies (1, 2) to be used along with the sensitive treponemal detection assay. Their findings await confirmation. In our study, the CSF samples from all four symptomatic and two congenital neurosyphilitic patients were reactive in the IgM ELISA. All patients with asymptomatic neurosyphilis were nonreactive in the test. Of 494 patients with nonneurosyphilis, however, 11 showed reactivity in the IgM ELISA test. Thus, the sensitivity and specificity of the IgM ELISA test were 100 and 98%, respectively. Synthesis of albumin within the central nervous system does not occur, and it thus can be assumed that albumin present in CSF is derived from serum (4). The serum/csf albumin ratio should, therefore, be a reliable parameter for evaluating the blood-brain barrier function (15), taking into account the age-dependent variation (29). Among the patients with neurosyphilis, two of nine with asymptomatic neurosyphilis and three of four with symptomatic neurosyphilis showed slight impairments of blood-csf barrier; both patients with congenital neurosyphilis showed moderate or severe impairment according to the criteria by Schliep and Folgenhauer (26), indicating early central nervous system involvement. With CSF the IgM ELISA gave positive results in all of the symptomatic and congenital neurosyphilis and none of the asymptomatic neurosyphilis patients. Of 30 patients with early, latent, or treated syphilis, 3 showed slight impairment of the blood-csf barrier. However, the IgM ELISA gave nonreactive results in all of them. These results suggest that the reactive results of IgM ELISA in CSF of neurosyphilis patients may be due to a local production of antitreponemal antibody, a finding supported by the work of Schliep and Folgenhauer (26) and Muller and Moskophidis (21). The IgM ELISA in CSF appears useful for differentiating symptomatic neurosyphilis showing atypical or mild symptoms from asymptomatic neurosyphilis. However, the test does not fill the need of identifying the asymptomatic neurosyphilitic cases. With the current low prevalence of symptomatic neurosyphilis and the unknown prevalence of asymptomatic neurosyphilis, the significance of these tests is difficult to assess. The test needs to be further evaluated with larger numbers of CSF samples from neurosyphilitics to determine the specificity of IgM reactivity and the usefulness of serum/csf albumin ratio for evaluating the blood-brain barrier function. Some cases of Lyme disease do appear to mimic neurosyphilis with sera or CSF cross-reactivity occurring in some instances (28; E. F. Hunter, H. Russell, C. E. Farshy, J. S. Sampson, and S. A. Larsen, Sex. Transm. Dis., in press). While routine syphilis serology usually provides the specific differentiation between Lyme disease and syphilis, CSF and serum pairs with histories should be included as controls in future evaluation of the IgM ELISA. ACKNOWLEDGMENTS This work was supported by a Public Health Service International Research Fellowship. J.B.L. is a recipient of the Fogarty International Fellowship of the National Institutes of Health (fellowship 1 FO5 TWO ). We thank Stuart T. Brown for aid in continuing the work. We also thank Barbara T. Craig and Elsie E. Couch for technical assistance. LITERATURE CITED 1. Cerny, E. H., E. A. Hambie, C. E. Farshy, and S. A. Larsen Detection of serum contamination of cerebrospinal fluid by measurement of adenovirus antibodies, p In R. 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