J. S. WINCHESTER" AND M. H. HAMBLING Virological Department, Public Health Laboratory, Bridle Path, York Road, Leeds, LS15 7TR

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1 ANTIBODIES TO MEASLES, MUMPS, AND HERPES SIMPLEX VIRUS IN CEREBROSPINAL FLUID IN ACUTE INFECTIONS AND POST- INFECTIOUS DISEASES OF THE CENTRAL NERVOUS SYSTEM J. S. WINCHESTER" AND M. H. HAMBLING Virological Department, Public Health Laboratory, Bridle Path, York Road, Leeds, LS5 7TR THERE have been few reports of finding specific viral antibody in normal or pathological cerebrospinal fluids (CSF), although studies on the quantitative distribution of the various immunoglobulins in the CSF have been more extensive. From these it appears that definite immunoglobulin patterns are helpful in the diagnosis of certain neurological diseases (Hartley, Merrill and Claman, 966). It seems likely that some of this immunoglobulin is, in fact, viral antibody. Gleiser et al. (96), Berge et al. (96) and others have shown that the immune reaction plays an important part in the pathogenesis of viral infections of the central nervous system, and it may be that clinical disease occurs only in those individuals in whom antibody reaches the CSF at a critical stage of viral multiplication (Webb and Smith, 966). The purpose of this study was to investigate the presence of viral antibody in the CSF from acute infectious and post-infectious neurological diseases, and to provide a baseline for future antibody studies. MATERIALS AND METHODS Cerebrospinaljluids (CSF). These were divided into five groups, on the basis of clinical diagnosis and the results of biochemical, bacteriological and virological examinations of the CSF. (a) A control group of CSF from patients originally suspected of having neurological illnesses, but subsequently diagnosed as non-neurological. The parameter range for this group was: protein 6-6 mg per 00 ml, and cells 0-6 per pl. This group is not, strictly speaking, a normal control group, but it was not possible to get CSF from healthy individuals. (b) Twenty-five CSF from 7 cases of bacterial meningitis; three of these were unproven bacteriologically, but produced obviously purulent CSF. (c) Thirty-six CSF from 33 cases of viral meningitis. Twenty-eight of the cases were associated with proved virus infection, 3 with various enteroviruses and 5 with mumps virus. Five cases wefe presumed to be of viral aetiology; no agent was isolated and no serological diagnosis was made. (d) Eighteen CSF from cases of encephalitis. All had the clinical picture of encephalitis, but only five had electro-encephalogram (EEG) studies done. Two cases of postvaricella encephalitis showed a normal EEG pattern; this is not uncommon, as the varicella virus frequently involves the cerebellar lobes which may not register on the EEG. Three cases were substantiated by histological examination; a fatal case of herpes simplex encephalitis, a typical case of subacute sclerosing panencephalitis (SSPE), and a case of suspected measles encephalitis. Four cases were diagnosed from the clinical picture, together with positive virus isolation or serology; a case of SSPE, a case of Coxsackie virus type-m encephalitis, a case of post-varicella encephalitis and a case of post-measles encephalitis. (e) Six CSF from a miscellaneous group of neurological disorders, including Guillain- Barrk syndrome, viral labyrinthitis, subarachnoid haemorrhage, phenytoin encephalopathy and cerebral astrocytoma. Received 5 Apr. 97; accepted 6 Apr. 97. * Present address : Public Health Laboratory, Coventry and Warwickshire Hospital, Stoney Stanton Rd, Coventry CV FH. Reprint requests to M. H. H. J. MED. MICROBIOL.-VOL. 5 (97) 37 IP: On: Sun, 09 Sep 08 :3:9

2 J. S. WINCHESTER AND M. H. HAMBLING Antibody titrations. CSF and the corresponding serum specimen from any patient were always collected within 8 hr of each other. All specimens were stored at -0 C until tested. Paired CSF and serum specimens were examined in parallel for antibodies to the following antigens : measles virus haemagglutinin, mumps virus haemagglutinin, measles virus complement-king (CF) antigen, mumps virus CF V and S antigens, and Herpesvirus hominis CF antigen. The complement-fixation test used was a modification of that described by Bradstreet and Taylor (96). The haemagglutination-inhibition (HI) test was based on that described by Rosen (96). Both were performed by a microtitration technique. In the case of CSF, since antibody titres were expected to be low, the range of dilutions examined started with undiluted CSF. Serum dilutions started at in 8. Obviously blood-stained or xanthochromic CSF was not tested; a in 5000 dilution of red cells in CSF is visually obvious (Lipton, Steigman and Dizon, 965) and less than this degree of blood contamination has little effect upon antibody levels. No attempt was made to remove non-specific inhibitors from CSF TABLE I Viral antibodies in cerebrospinal fluids Clinical diagnosis Number of patients Number of patients with antibodies in the CSF Number of CSF examined Number of CSF with antibody Control group Bacterial meningitis Viral meningitis Encephalitis Miscellaneous Total with kaolin in the HI test as this may remove small amounts of antibody as well, probably because there is insufficient protein in CSF to saturate the kaolin (Dr J. H. Connolly, personal communication). However, HI by non-specific inhibitors gives a pattern of red-cell sedimentation recognisably different from that produced by viral antibody. RESULTS Table I shows the results obtained with 7 CSF from 09 patients. Viral antibodies were found in CSF from 3 patients (3 per cent. of CSF samples, 9 per cent. of patients). None of the control patients had detectable antibody in the CSF, although many of them possessed serum antibodies. Thus, 38 had measles HI antibody in the serum, eight with titres of or more, and nine also had measles CF antibody. Seven were currently suffering from measles without neurological involvement, and six of them had measles antibody in the serum, one with an HI titre of 08. or HI antibody was present in the serum of 6 control patients, mostly at a titre of 8 or less, although in one patient the HI antibody titre was 0. Herpes simplex antibody was present, at a titre of 8 or less, in patients. Antibody was detected in the CSF of 3 of the 68 patients with confirmed neurological disease. Forty-one of 85 specimens of CSF examined (8 per cent.) contained antibody, and among those patients with high serum-antibody levels, of and greater, the frequency of antibody in the CSF was 70 per cent. Table I shows the number of CSF with detectable antibodies to measles, mumps and herpes simplex viruses in each clinical group, and the geometric mean CSF/serum antibody ratios. The two cases of SSPE are shown separately from the other encephalitis cases in order to illustrate their strikingly high CSF/serum antibody ratios. IP: On: Sun, 09 Sep 08 :3:9

3 ANTIVIRAL ANTIBODIES IN CEREBROSPINAL FLUID 39 Seven patients in the encephalitis group had measles HI antibody in the CSF, with CSF/ serum antibody ratios ranging from : 6 to : 0 (geometric mean ratio : 7). Two of these patients also had measles CF antibody in the CSF with ratios of : 3 and : 5 (geometric mean ratio : 8). Apart from mumps HI antibody in one case, measles antibody was the only antibody detected in this group of patients. The two patients with SSPE showed remarkably low CSF/serum measles antibody ratios and the antibody titres were the highest found in any of the CSF. The mean ratios for their five CSF specimens ranged from TABLE I Geometric mean CSF/serum antibody ratios in patients with uar io us neurologica diseases Number of positive CSF and (in brackets) geometric mean ratio of CSF/serum antibody to Clinical diagnosis measles virus by the mumps virus by the herpes simplex virus by the haemagglutination- complementinhibition test fixation test haemagglutinationinhibition test complement fixation test complementfixation test Bacterial meningitis 8 ( : 6) ( : ) ( : ) ( : 6) 3 ( : 0) Viral meningitis 8 ( : ) 0 6 ( : 7) 7 ( : 58) I 0 Subacute sclerosing panencephali tis (SSPE) Encephalitis excluding SSPE Miscellaneous 7 ( : 7) ( : 90) ( : 8) (:6) 3 ( : 6) ( : 0) ( : 90) : 6 to : 6 (geometric mean ratio : ) for measles HI antibody, and from : 8 to : 3 (geometric mean ratio : 6) for CF antibody. No other antibodies were detected in the CSF from these two patients. Only ten of the 33 cases of viral meningitis possessed detectable antibody in the CSF, but the range of CSF/serum antibody ratios was similar to that found in the encephalitis patients. Eight specimens from eight patients contained measles HI antibody with CSF/serum antibody ratios from : 8 to : 0 (geometric mean ratio : ). Nine patients had mumps antibody, five with HI antibody and five with CF antibody; the range of CSFlserum antibody ratios was : 6 to : 6 (geometric mean ratio : 7) and : 3 to : (geometric mean ratio : 58) respectively. Eight of the ten viral meningitis cases were diagnosed as mumps meningitis. The CSF/serum antibody ratios were much higher in bacterial meningitis than in viral meningitis. CSF antibody was found in eight cases, but only six of them had detectable measles HI antibody; the CSF/serum antibody ratios for eight CSF specimens collected from these six patients ranged from : 6 to : (geometric mean ratio : 6). One patient also had measles CF antibody with a ratio of :. Only two patients had mumps HI IP: On: Sun, 09 Sep 08 :3:9

4 ~ 0 J. S. WINCHESTER AND M. H. HAMBUNG: (a)* (b)* Case TABLE I Titres of viral antibodies present in CSF and serum and CSFlserum antibody ratios in selected patients I Antibody Mumps HI Mumps HI Antibody titre in CSF serum 8 6 CSF/ serum antibody ratio : 3 : 6 Clinical diagnosis ( 5 : Measles encephalitis :6 SSPE (a) 3 0 : : 3 (b) 3 5 : :8 (c) 8 08 : : 3 (d) 6 0 : :8 5 5 : 8 : Subarachnoid haemorrhage Streptococcal meningitis 8 8 : 6 Pneumococcal meningitis < : 8 : : : 8 : 6 : : 6 : 6 : 5 : 8 : 8 : 8 : 5 :3 : 5 : 3 > : :6 :6 : 3 : 8 : 3 : 3 : 6 : 3 : 3 Meningococcal meningitis Bacterial meningitis Mumps meningoencephalitis Herpes encephalitis Guillain-Barrk syndrome Guillain-Barr6 syndrome : 8 : ~ ~~ ~ Astrocytoma * (a), (b), etc., refer to different samples of CSF from the same patient. IP: On: Sun, 09 Sep 08 :3:9

5 ANTIVIRAL ANTIBODIES IN CEREBROSPINAL FLUID antibody in the CSF, the CSF/serum antibody ratios being : 8 and : 6, whilst a single patient with mumps CF antibody had a ratio of : 6 for his CSF specimen. Herpes simplex antibody was found in two patients, both with CSF/serum antibody ratios of :. Five of the six patients in the miscellaneous group had antibody in the CSF. Four had measles HI antibody, with CSF/serum antibody ratios of : 6 to : (geometric mean ratio : 90), and three had measles CF antibody, with ratios of : 3 to : (geometric mean ratio : 6). Three cases had mumps CF antibody and two had herpes simplex antibody, the CSF/serum antibody ratios in both groups ranging from : 3 to : (geometric mean ratio : 6 and : 0 respectively). Table I shows the CSF and serum antibody titres and the CSF/serum antibody ratios in 8 patients who had either high antibody titres or more than one type of antibody present in the CSF. The results confirm the findings of Lipton et al. The CSF/serum antibody ratios varied widely among our patients, but the ratios for different antibodies present in the same CSF were usually very similar. Twenty-one of our 7 CSF possessed more than one type of viral antibody, and in only five did the CSF/serum antibody ratios differ significantly. Three of thesewere from cases of mumps meningitis(cases 8,9 and 0); theyshowed markedly different CSFlserum antibody ratios for mumps and measles antibodies. DISCUSSION Normal levels of immunoglobulin in the CSF range from 5 to 0 pg per ml, whereas normal plasma levels of IgG are,000-8,000 pg per ml (Schultze and Heremans, 966). The concentration ratio for immunoglobulin in the CSF and serum would therefore be expected to lie between : 300 and : 00. If specific antibodies follow the same pattern, then antibody should be detectable in the CSF of patients with serum antibody titres of 300 and greater. Nine patients in the control group had serum antibody titres greater than, but none of them had detectable antibody in the CSF. As might be expected, antibody was present in the CSF of patients with inflammatory diseases of the central nervous system, since these are characterised by altered permeability of the blood-brain barrier. In the majority of cases the CSF antibody titres were proportional to the corresponding serum antibody titres. This reflects the relative importance of transport of serum antibody across the blood-brain barrier, as the source of the immunoglobulin in the CSF. In no case did we detect specific antibody in the CSF without the presence of the same antibody in the serum. The higher proportion of antibody-positive CSF in the bacterial meningitis group (8 per cent.) than in the viral meningitis group (3 per cent.) is probably due to the greater degree of involvement of the blood-brain barrier in the inflammatory process. It was surprising, however, to find an even greater proportion of positive CSF in the encephalitis group; even without the cases of SSPE, 7 per cent. of their CSF had antibody. The difference in antibody frequency between the encephalitis and the viral meningitis cases was significant (x =.5, 0.05 > P> 0.0), but that between the encephalitis and the bacterial meningitis cases was not ( ~ = 0.67, 0.5>P>0.3). The patients with encephalitis and viral meningitis were very similar in both age-distribution and incidence and levels of antibody in the serum, and it seems unlikely that the high frequency of antibody in the CSF of the encephalitis patients can be due only to passive transfer of serum antibody across a more extensive area of inflamed theca. The high proportion of CSF with mumps antibody in our mumps meningitis cases and the greater CSF/serum antibody ratio for mumps antibody than for measles antibody in some of these cases may be relevant to the observations of Webb and Smith (966) on local production of viral antibody in the central nervous system. It is probably unwise to draw conclusions from our few cases, especially as the majority of the CSF possessing more than one type of antibody showed similar CSF/serum antibody ratios for these antibodies. However, good evidence for local production of antibody is probably afforded by our findings in the two cases of SSPE. The geometric mean CSF/serum measles HI and CF antibody ratios in these two patientswere markedlyhigher even than in the cases of bacterialmeningitis, in which gross changes in the blood-brain barrier occur. Connolly et al. (967) were the IP: On: Sun, 09 Sep 08 :3:9

6 J. S. WINCHESTER AND M. H. HAMBLJNG first to report high titres of measles antibody in the CSF of patients with SSPE, and this has since been substantiated by others. Many of the CSF were collected during the acute stage of the illnesses. If sequential CSF had been available for study antibody might have been detected in more patients, as Kaldor and Ferris (969) have demonstrated significantly higher levels of IgG and IgA in late specimens of CSF. In agreement with other workers we have shown that measles HI and CF antibodies are much more easily demonstrated in the CSF than mumps and herpes simplex virus antibodies (Sibley and Foley, 963; Adams, 967). This is probably a reflection of the persistence of relatively high titres of measles antibody in the serum of most of the population. Measles antibody may, therefore, provide a useful parameter for the measurement of CSF/serum antibody ratios. Unfortunately, no one has yet provided a baseline ratio for measles antibody in normal, healthy individuals. Clarke, Dane and Dick (965) described a mean CSF/serum antibody ratio of : 505 for poliovirus-neutralising antibodies in control patients without neurological disease. We were unable to detect measles antibody in the CSF of any of our control patients, and our findings suggest that the CSF/serum measles antibody ratio may well be considerably higher than : 505. Estimation of antibodies in the CSF is not a routine procedure. Apart from their known association with SSPE, their diagnostic significance is still uncertain. SUMMARY The titres of antibodies against three viruses, measles, mumps and herpes simplex, were determined in the cerebrospinal fluid (CSF) and in the serum of 68 patients with various forms of meningitis and encephalitis, as well as in control patients without neurological diseases. No antibody was detected in CSF from control patients, but 8 per cent. of CSF from patients with neurological involvement had detectable antibody. The ratio of CSF/serum antibody was highest in subacute sclerosing panencephalitis and bacterial meningitis. We would like to thank Dr J. Stevenson of the Infectious Diseases Unit at Seacroft Hospital for providing most of the clinical information and specimens for this study, and we are grateful to Dr C. M. Patricia Bradstreet of the Standards Laboratory, Central Public Health Laboratory, Colindale, for provision of the complement-fixing antigens. REFERENCES ADAMS, J. M Measles antibodies in patients with multiple sclerosis. Neurology, Minneap., 7, 707. BERGE, T. O., GLEISER, C. A., GOCHENOUR, W. S., JR, MEISSE, MARIE L., AND TIGERTT, W. D. 96. Studies on the virus of Venezuelan equine encephalomyelitis.. Modification by specific immune serum of response of central nervous system of mice. J. Immun., 87, 509. BRADSTREET, C. M. PATRICIA, AND TAYLOR, C. E. D. 96. Technique of complement fixation test applicable to the diagnosis of virus diseases. Mon. Bull. Minist. Hlth,,96. CLARKE, J. K., DANE, D. S., AND DICK, G. W. A Viral antibody in the cerebrospinal fluid and serum of multiple sclerosis patients. Brain, 88, 953. CONNOLLY, J. H., ALLEN, INGRID V., HURWITZ, L. J., AND MILLAR, J. H. D Measlesvirus antibody and antigen in subacute sclerosing panencephalitis. Lancet,, 5. GLEISER, C. A., GOCHENOTJR, W. S., BERGE, T. O., AND TIGERTT, W. D. 96. Studies on the virus of Venezuelan equine encephalomyelitis. I. Modification by cortisone of the response of the central nervous system of Macaca muhtta. J. Immun., 87, 50. HARTLEY, T. F., MERRILL, DEBORAH A., AND CLAMAN, H. N Quantitation of immunoglobulins in cerebrospinal fluid. Archs Neurol., Chicago, 5, 7. IP: On: Sun, 09 Sep 08 :3:9

7 ANTIVIRAL ANTIBODIES IN CEREBROSPINAL FLUID 3 KALDOR, J., AND FERRIS, A. A Immunoglobulin levels in cerebro-spinal fluids in viral and bacterial meningitis. Med. J. Austral.,, 06. LIPTON, M. M., STEIGMAN, A. J., AND DIZON, F. C Poliovirus neutralizing antibody in cerebrospinal fluid. J. Infect. Dis., 5, 356. ROSEN, L. 96. Hemagglutination and hemagglutination-inhibition with measles virus. Virology, 3, 39. SCHULTZE, H. E., AND HEREMANS, J. F Molecular biology of human proteins, Amsterdam, vol., pp. 735 and 8. STSLEY, W. A,, AND FOLEY, J. M Measles antibodies in multiple sclerosis. Trans. Amer. Neurol. ASSOC., 88, 77. WEBB, H. E., AND SMITH, C. E. G Relation of immune response to development of central nervous system lesions in virus infections of man. Br. Med. J.,, 79. IP: On: Sun, 09 Sep 08 :3:9