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Brucellosis remains a common human zoonotic disease, especially in developing countries. Neurological involvement of the central nervous system (CNS) has been detected in 3 to 5% of the patients with brucellosis, in both the presence and absence of systemic illness (10, 13). Meningitis is the most frequently encountered clinical condition in patients with neurobrucellosis, and it occurs after direct invasion of the CNS by Brucella (7, 10, 13). Acute Brucella meningitis is usually characterized by sudden onset of fever, headache, and nuchal rigidity. Psychiatric and motor sensory disorders are also common. However, since similar symptoms may be present in some patients who have systemic brucellosis without neurological involvement and in patients who have infectious meningitis due to other microorganisms, the diagnosis of neurobrucellosis requires direct or indirect evidence of Brucella in the cerebrospinal fluid (CSF) (7, 10). Because of the low frequency of Brucella isolation from CSF (less than 20% of patients), the diagnosis of neurobrucellosis usually depends on the detection of specific antibodies in that fluid (7, 10, 13). Oligoclonal immunoglobulin G (IgG), indicative of intrathecal antibody production, is frequently detected in CSF but is not exclusively diagnostic of neurobrucellosis (7, 11). The finding of brucella-specific antibodies in the CSF is highly indicative of CNS infection; however, since these antibodies are sometimes present at low levels, agglutination tests commonly employed in the diagnosis of neurobrucellosis can give false-negative results (2, 10, 13). In contrast, an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to heat-killed Brucella antigens showed high sensitivity in the diagnosis of neurobrucellosis (1, 2). These antigens, however, are likely to contain significant amounts of lipopolysaccharide (LPS) and, as indicated by the authors, cross-reactions with other gram-negative bacteria may occur. We have previously shown that the detection of serum antibodies to cytoplasmic proteins (CP [formerly called LPS-free CYT]) of Brucella spp. is useful for the specific diagnosis of human and animal brucellosis (4, 5, 9). Here we present results which indicate that the detection of antibodies to CP in CSF makes possible the differentiation between patients who have neurobrucellosis and those who have systemic brucellosis and neurological manifestations without actual CNS infection.

Two ELISA systems were used. CSF and serum antibodies against Brucella CP antigens were detected by an indirect ELISA as described previously (9). The CP antigen is an LPS-depleted cytoplasmic fraction of Brucella abortus S19, obtained by immunosorption with an anti-LPS monoclonal antibody. Maxisorp polystyrene plates (Nunc, Roskilde, Denmark) were sensitized with 0.5 µg of CP diluted in phosphate-buffered saline (PBS) per well. Plates were blocked with 200 µl of PBS containing 1% skim milk per well. After a wash, human CSF or sera were dispensed in serial dilutions (starting at 1:100) in a solution of PBS, 0.3% skim milk, and 0.05% Tween 20. Specific antibodies were detected with polyclonal anti-human IgG- or anti-human IgM-horseradish peroxidase conjugates (diluted 1:2,000 and 1:1,000, respectively; DAKO, Carpinteria, Calif.) The reaction was developed by adding ortho-phenylenediamine (2 µg µl¹, in 0.1 M citrate-phosphate buffer containing 0.03% H₂O₂) and was stopped with 4 N H₂SO₄.

CSF and serum antibodies against LPS were detected by capture ELISA with the anti-LPS monoclonal antibody BC68 as described previously (9). Purified BC68 was adsorbed onto the well surfaces of the Maxisorp polystyrene plates, and after blocking, a cytoplasmic fraction of Brucella (9) was added to a final concentration of 5 µg of LPS per well. The testing of the samples, addition of the conjugates, and development of the reaction were performed as described above.

To establish the cutoff value of the assays, 30 serum samples from healthy subjects and 20 CSF samples from noninfected controls (mostly Alzheimer's disease patients) were assayed at a 1:100 dilution (anti-CP antibodies) or 1:200 dilution (anti-LPS antibodies) under the conditions described above. The cutoff value of each ELISA system was calculated as the mean specific optical density (OD) plus 3 standard deviations. The titer was calculated as the reciprocal of the last serum or CSF dilution giving an OD higher than the cutoff. For the assays of CSF, the cutoff values were 0.020 for anti-LPS IgM, 0.136 for anti-LPS IgG, 0.028 for anti-CP IgM, and 0.109 for anti-CP IgG.

These assays were used to test seven CSF samples from five patients who had neurobrucellosis, as shown by signs and symptoms indicative of neurological involvement and a positive result for anti-Brucella antibodies in CSF by an agglutination or Coombs test (3) or the isolation of Brucella from CSF (Table 1). Four patients were from Lebanon, and according to the sources of infection (raw cheese or milk), all of these cases were presumed to have been caused by Brucella melitensis. The other patient was from Argentina and had meningitis caused by Brucella suis. In addition, the ELISAs were used to test CSF and serum samples from two patients who had brucellosis without neurological involvement. These latter patients had signs and symptoms typical of brucellosis and showed positive results in the standard tube agglutination and rose bengal tests. One of them also had a positive blood culture for B. abortus. Since both patients had severe headaches, CSF samples were obtained to rule out infections with neurological involvement. However, both CSF samples had normal physicochemical parameters and no microorganism was isolated. In addition, and in order to confirm the specificity of the test, the ELISAs were used for 20 CSF samples from noninfected controls and 14 CSF samples from patients who had meningitis caused by infectious agents other than Brucella (four cases caused by Staphylococcus aureus, four by Streptococcus pneumoniae, three by Neisseria meningitidis, two by Mycobacterium tuberculosis, and one by Cryptococcus neoformans). To further characterize the antibody response to Brucella proteins, CSF samples were assayed by immunoblotting against CP. Briefly, the CP antigen was electophoresed in 15% polyacrylamide gel in the presence of sodium dodecyl sulfate and electrotransferred to nitrocellulose sheets by conventional methods. After blocking with PBS containing 3% skim milk, the sheets were cut into strips, and each strip was incubated with CSF diluted 1:20 in 1% skim milk containing 0.05% Tween 20. After a subsequent incubation with peroxidase-conjugated polyclonal antibody to human IgG (diluted 1:1,000; DAKO), the reaction was developed with 4-chloro--naphthol (3 mg/ml) and H₂O₂ (0.03%) in Tris-buffered saline.

Antibodies to Brucella antigens in CSF and serum. As shown in Fig. 1, CSF samples from noninfected controls assayed at a 1:100 dilution produced very low ODs (below 0.100) in both ELISAs. At the same dilution, in contrast, the CSF from patients with neurobrucellosis produced ODs of 0.223 to 2.068 for anti-CP IgG and 0.563 to 1.882 for anti-LPS IgG. Since the respective cutoff values were 0.109 and 0.136, these samples were all considered positive for IgG to CP and IgG to LPS. Anti-LPS IgG titers from these samples ranged from 800 to 6,400, and anti-CP IgG titers ranged from 200 to 12,800. Conversely, the CSF from the two patients who had brucellosis without neurological involvement were negative for both anti-CP IgG and anti-LPS IgG. Serum samples from these two patients, however, were positive for IgM to LPS (titers of 25,600 and 1,600), IgM to CP (200 and 100), IgG to LPS (800 in both patients), and IgG to CP (200 and 400). CSF samples from patients who had brucellosis with or without neurological involvement were negative for IgM against both CP and LPS except for patient 4, whose titers were 400 and 800, respectively. In addition, CSF samples from the 14 patients with infectious nonbrucellar meningitis were negative for IgG to Brucella CP and LPS (Fig. 1), and the same was true for IgM antibodies to both antigens (not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   IgG reactivity to Brucella CP and LPS in CSF samples from noninfected subjects and from patients with neurobrucellosis, systemic brucellosis without neurological involvement, or nonbrucellar meningitis. Anti-CP and anti-LPS reactivities were determined by indirect and antigen capture ELISAs, respectively.

View larger version (75K): [in this window] [in a new window]   FIG. 2.   IgG reactivity to Brucella CP in CSF samples from patients with neurobrucellosis as assayed by Western blotting. Proteins were electrophoresed in 15% polyacrylamide gel in the presence of sodium dodecyl sulfate, transferred to nitrocellulose, and reacted to CSF diluted 1:20. Lanes 1, 2, 3, 5, and 7, diagnostic samples from patients 1 to 5, respectively; lane 4, sample taken from patient 3 60 days later; lane 6, sample taken from patient 4 30 days later.