Antibodies to Measles, Mumps, and Herpes Simplex Virus in Cerebrospinal Fluid in Acute Infections and Post- Infectious Diseases of the Central Nervous System

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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, LS15 7TR

THEREhave 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, 1966). It seems likely that some of this immunoglobulin is, in fact, viral antibody. Gleiser et al. (1961), Berge et al. (1961) 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, 1966). 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.

MATERIALSAND 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 42 CSF from 41 patients originally suspected of having neurological illnesses, but subsequently diagnosed as non-neurological. The parameter range for this group was: protein 6-46 mg per 100 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 17 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, 13 with various enteroviruses and 15 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 12 cases of encephalitis. All 12 had the clinical picture of encephalitis, but only five had electro-encephalogram (EEG) studies done. Two cases of post- varicella 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 15 Apr. 1971; accepted 16 Apr. 1971. * Present address : Public Health Laboratory, Coventry and Warwickshire Hospital, Stoney Stanton Rd, Coventry CV1 4FH. Reprint requests to M. H. H. J. MED. MICROBIOL.-VOL. 5 (1972) 137 J. S. WINCHESTER AND M. H. HAMBLING

Antibody titrations. CSF and the corresponding serum specimen from any patient were always collected within 48 hr of each other. All specimens were stored at -20°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 (1962). The haemagglutination-inhibition (HI) test was based on that described by Rosen (1961). 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 1 in 8. Obviously blood-stained or xanthochromic CSF was not tested; a 1 in 5000 dilution of red cells in CSF is visually obvious (Lipton, Steigman and Dizon, 1965) 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 TABLEI Viral antibodies in cerebrospinal fluids

Number of patients Number of Number of Clinical diagnosis Number of with antibodies CSF CSF patients in the CSF examined with antibody

Control group 41 0 42 0 Bacterial meningitis 17 8 25 12 Viral meningitis 33 10 36 11 Encephalitis 12 9 18 13 Miscellaneous 6 5 6 5

Total 109 32 127 41 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 sedi- mentation recognisably different from that produced by viral antibody.

RESULTS Table I shows the results obtained with 127 CSF from 109 patients. Viral antibodies were found in 41 CSF from 32 patients (32 per cent. of CSF samples, 29 per cent. of patients). None of the 41 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 256 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 2048. Mumps CF or HI antibody was present in the serum of 16 control patients, mostly at a titre of 128 or less, although in one patient the HI antibody titre was 1024. Herpes simplex antibody was present, at a titre of 128 or less, in 11 patients. Antibody was detected in the CSF of 32 of the 68 patients with confirmed neurological disease. Forty-one of 85 specimens of CSF examined (48 per cent.) contained antibody, and among those patients with high serum-antibody levels, of 256 and greater, the frequency of antibody in the CSF was 70 per cent. Table I1 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. ANTIVIRAL ANTIBODIES IN CEREBROSPINAL FLUID 139

Seven patients in the encephalitis group had measles HI antibody in the CSF, with CSF/ serum antibody ratios ranging from 1 : 64 to 1 : 1024 (geometric mean ratio 1 : 172). Two of these patients also had measles CF antibody in the CSF with ratios of 1 : 32 and 1 : 512 (geometric mean ratio 1 : 128). 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 TABLEI1 Geometric mean CSF/serum antibody ratios in patients with uar io us neurologica1 diseases

Number of positive CSF and (in brackets) geometric mean ratio of CSF/serum antibody to

mumps virus by the herpes simplex Clinical diagnosis measles virus by the virus by the

haemagglutination- complement- haemagglutination- complement complement- inhibition test fixation test inhibition test fixation test fixation test

Bacterial meningitis 8 (1 : 62) 1 (1 : 256) 2 (1 : 12) 1 (1 : 64) 3 (1 : 101)

Viral meningitis 8 (1 : 256) 0 6 (1 : 72) 7 (1 : 58) I 0

Subacute sclerosing panencephalitis (SSPE)

Encephalitis exclud- 7 (1 : 172) 2 (1 : 128) 1(1:64) ing SSPE

Miscellaneous 4 (1 : 90) 3 (1 : 64) 0 -73 (1 : 101) 2 (1 : 90)

1 : 16 to 1 : 64 (geometric mean ratio 1 : 24) for measles HI antibody, and from 1 : 8 to 1 : 32 (geometric mean ratio 1 : 16) 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 1 : 128 to 1 : 1024 (geometric mean ratio 1 : 256). Nine patients had mumps antibody, five with HI antibody and five with CF antibody; the range of CSFlserum antibody ratios was 1 : 16 to 1 : 246 (geometric mean ratio 1 : 72) and 1 : 32 to 1 : 256 (geometric mean ratio 1 : 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 1 : 16 to 1 : 256 (geometric mean ratio 1 : 62). One patient also had measles CF antibody with a ratio of 1 : 256. Only two patients had mumps HI 140 J. S. WINCHESTER AND M. H. HAMBUNG:

TABLEI11 Titres of viral antibodies present in CSF and serum and CSFlserum antibody ratios in selected patients

Antibody titre in CSF/ serum Antibody antibody Clinical diagnosis CSF serum ratio Case I 1 (a)* Measles HI 4 128 1 : 32 Streptococcal meningitis (b)* Herpes simplex CF 4 64 1 : 16 2 Measles HI 8 128 1 : 16 Pneumococcal meningitis 3 Measles HI 256 1 : 128 Meningococcal meningitis Measles CF 256 1 : 256 Herpes simplex CF 256 1 : 256 4 Measles HI 512 1 : 128 Bacterial meningitis Mumps CF 512 1 : 64 Herpes simplex CF 256 1 : 256 5 Measles HI 128 1 : 64 Mumps meningitis Mumps CF 256 1 : 64 6 Measles HI 2 1024 1 : 512 Mumps meningitis Mumps CF 4 512 1 : 128 Measles HI 512 1 : 128 Mumps meningitis Mumps HI 512 1 : 128 8 Measles HI 512 1 : 512 Mumps meningitis Mumps HI 32 1 :32 9 Measles HI 2 1024 1 : 512 Mumps meningo- Mumps CF 4 256 1 : 32 encephalitis 10 Measles HI <1 256 >1 : 256 Mumps meningitis Mumps CF 1 64 1:64 11 Measles HI 2 128 1:64 Herpes encephalitis Measles CF 2 64 1 : 32 12 (Measles HI 2 512 1 : 256 Measles encephalitis 13 Measles HI 8 512 1:64 SSPE 14 (a) Measles HI 32 1024 1 : 32 Measles CF 32 1024 1 : 32 (b) Measles HI 32 512 1 : 16 Measles CF 128 1024 1:8 (c) Measles HI 128 2048 1 : 16 Measles CF 32 1024 1 : 32 (d) Measles HI 64 1024 1 : 16 Measles CF 128 1024 1 :8 15 Measles HI 4 512 1 : 128 Subarachnoid haemorrhage Mumps CF 1 256 1 : 256 16 Measles HI 2 256 1 : 128 Guillain-Barrk syndrome Measles CF 4 128 1 : 32 Mumps CF 1 32 1 : 32

~ 17 Measles HI 16 256 1 : 16 Guillain-Barr6 syndrome Measles CF 2 64 1 : 32 Herpes simplex CF 2 64 1 : 32

~ ~~ ~ 18 Mumps CF 2 256 1 : 128 Astrocytoma Herpes simplex CF 1 256 1 : 256 * (a), (b), etc., refer to different samples of CSF from the same patient. ANTIVIRAL ANTIBODIES IN CEREBROSPINAL FLUID 141

antibody in the CSF, the CSF/serum antibody ratios being 1 : 8 and 1 : 16, whilst a single patient with mumps CF antibody had a ratio of 1 : 64 for his CSF specimen. Herpes simplex antibody was found in two patients, both with CSF/serum antibody ratios of 1 : 256. 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 1 : 16 to 1 : 256 (geometric mean ratio 1 : 90), and three had measles CF antibody, with ratios of 1 : 32 to 1 : 256 (geometric mean ratio 1 : 64). Three cases had mumps CF antibody and two had herpes simplex antibody, the CSF/serum antibody ratios in both groups ranging from 1 : 32 to 1 : 256 (geometric mean ratio 1 : 64 and 1 : 101 respectively). Table I11 shows the CSF and serum antibody titres and the CSF/serum antibody ratios in 18 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 127 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 10); theyshowed markedly different CSFlserum antibody ratios for mumps and measles antibodies.

DISCUSSION Normal levels of immunoglobulin in the CSF range from 15 to 40 pg per ml, whereas normal plasma levels of IgG are 12,000-18,000 pg per ml (Schultze and Heremans, 1966). The concentration ratio for immunoglobulin in the CSF and serum would therefore be expected to lie between 1 : 300 and 1 : 1200. 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 256, 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-positiveCSF in the bacterial meningitis group (48 per cent.) than in the viral meningitis group (31 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, 72 per cent. of their CSF had antibody. The difference in antibody frequency between the encephalitis and the viral meningitis cases was significant (x2 = 4.54, 0.05 >P> 0.02), but that between the encephalitis and the bacterial meningitis cases was not (~2= 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 (1966) 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. How- ever, 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. (1967) were the 142 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 (1969) 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, 1963; Adams, 1967). 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 (1965) described a mean CSF/serum antibody ratio of 1 : 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 1 : 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 41 control patients without neurological diseases. No antibody was detected in CSF from control patients, but 48 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.

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