ANTIBODIES TO HERPES-SIMPLEX VIRUS IN THE CEREBROSPINAL FLUID OF PATIENTS WITH HER- PETIC ENCEPHALITIS

Transcription

1 ANTIBODIES TO HERPES-SIMPLEX VIRUS IN THE CEREBROSPINAL FLUID OF PATIENTS WITH HER- PETIC ENCEPHALITIS F. 0. MACCALLUM, I. J. CHINN AND J. V. T. GOSTLMG Virology Laboratory, Radclife Infirmary, Oxford and the Public Health Laboratory, St Mary s General Hospital, Portsmouth A FIRM diagnosis of herpes-simplex-virus encephalitis (HSVE) rests on demonstration of the presence of the virus in brain-biopsy specimens or ventricular fluid by isolation in tissue culture or by immunofluorescence. A rising titre of antibodies to herpes-simplex virus (HSV) in the serum suggests only that this virus is the cause of the disease. However, in 1969, MacCallum reported a rise in titre of complement-fixing (CF) and neutralising antibodies to HSV in the cerebrospinal fluid (CSF) of three patients who had survived HSVE for more than 3 weeks. It was suggested that the finding of these antibodies in the CSF might be helpful in establishing the diagnosis when virus could not be isolated from brain-biopsy material. The possible aetiological significance of their presence in the CSF of patients with dementia was also raised at that time. The present report describes the original three cases more fully and adds a further 13 cases investigated in Oxford or Portsmouth. Some of the results have already been reported briefly elsewhere (Tomlinson, 1973 ; Juel-Jensen and MacCallum, 1972; Illis and Gostling, 1972). MATERIALS AND METHODS Patients with suspected HSVE. A presumptive diagnosis of HSV infection of the central nervous system (CNS) had been made from the clinical picture. The diagnosis was confirmed in nine Oxford patients (cases 1-9) by brain biopsy (virus isolation in 7 cases, positive immunofluorescence in one case) or by virus isolation from ventricular fluid (one case). Brain biopsy was not done in the three adult Portsmouth patients (cases 13-15) and the diagnosis was based on the typical clinical picture and rising titres of serum antibody. Investigations were also performed on sera and CSFs from three children in hospital elsewhere (cases lcr12); the diagnosis was suggested by the clinical picture and confirmed in two of them (cases 10 and 11) by brain biopsy. Two or more serial samples of blood and CSF were obtained from most of the patients, but only a single, convalescent sample of CSF was available from some. In one patient (case 16) only a convalescent serum had been examined when he was ill with encephalitis in another hospital but both serum and CSF were subsequently obtained when he was admitted to an Oxford hospital for recurrence of his symptoms and signs. Control patients. It was not possible to obtain comparable numbers of CSF specimens from control patients, because, as in most diseases, repeated lumbar punctures are neither required nor desirable. However, CSFs were examined (in Oxford) from 27 patients with a variety of diseases, namely, tuberculous meningitis (including some patients given intrathecal injections of old tuberculin), suspected and proven enterovirus meningitis, various forms of Received 17 Oct. 1973; accepted 14 Nov J. MED. MICROBIOL.-VOL. 7 (1974) 325

2 326 F. 0. MACCALLUM, I. J. CHI" AND J. V. T. GOSTLING encephalitis, cerebro-vascular accidents, epilepsy and hysteria. One of us (J. G.) also carried out neutralising antibody tests on 96 CSF specimens provided by Dr Richard Hunter from the Friern Mental Hospital in north London. Antibody techniques. CF tests were done as described by Smith, Peutherer and Mac- Callum (1967). Neutralising antibodies were estimated by mixing dilutions of serum or CSF, or undiluted CSF in the case of the control patients, with plaque-forming doses of HSV, incubating the mixtures for 30 min. at 37 C and then inoculating each mixture, in 0.2-ml amounts, on to two drained, primary human-amnion-tissue-culture monolayers. The tissueculture tubes were then kept for 30 min. at 36 C before adding 0.8 ml of maintenance medium and re-incubating at 36 C. Control positive and negative HSV antisera were included in each test. The serum titres were read usually at 48 hours as the reciprocal of the highest dilution of serum producing 50 % reduction in the number of lesions when compared with the negative serum control tubes. Tubes without any lesions at 48 hours were incubated for a further 24 hours before taking a final reading. Tests for serum neutralising antibodies against the three types of poliovirus were done in tube cultures of primary monkey-kidney cells, with 100 TCDSO of virus. Only when a titre of 64 or greater to at least one or other of the three types was found in the serum were similar tests done for poliovirus antibody in the CSF, with serial, doubling dilutions of CSF from 1 in 2 to 1 in 32; because of low levels of poliovirus antibody in the serum or insufficient CSF, this essential comparative test was carried out in only some of the patients. RESULTS The results of examining the serum and CSF of the 16 patients with proven or presumptive HSVE for herpes CF antibody and poliovirus neutralising antibody are shown in the table; herpes neutralising antibodies were also estimated but the results are not shown because they gave practically the same pattern of results as the CF test. The Oxford patients (nos. 1-9) were seen initially at different stages of illness and illustrate a variety of herpes-antibody results in the CSF, namely, no antibody when first seen but present later, antibody present on admission with the titre rising subsequently, and antibody on admission but without any increase in titre later. Seven of them had poliovirus serum-antibody titres of 64 or greater and they were examined for poliovirus antibody in the CSF; six were without detectable antibody in the CSF and one (no. 7) had a titre of only 2. On the other hand, in all nine patients the ratio of herpes serum antibody to CSF antibody was low, ranging from 1 : 1 to 16 : 1 ; this is comparable to the serum : CSF ratio of measles antibody frequently reported in subacute sclerosing panencephalitis. Patient no. 3 illustrates the time of appearance of herpes CSF antibody in an encephalitis that may have followed reactivation of latent infection; antibody was already detectable in the serum as early as the fourth day and showed no change in titre. Only this patient and no. 9 gave a history of cold sores. The remaining patients from Portsmouth and elsewhere, both adults and children, showed the same low ratio of serum : CSF herpes antibody, with the exception of no. 12. The clinical picture in this infant was typical but brain biopsy was not done, and the antibody ratio was 50 : 1. The significance of the CSF antibody, in this case, is therefore, questionable although the poliovirus serum : CSF antibody ratio was 128 : 0. Patient no. 12, however, demonstrates the very long time during which antibody may persist in the CSF. Number 11, in whom the diagnosis was proven by both immunofluorescence and the isolation

3 7* CSF ANTIBODIES IN HERPETIC ENCEPHALITIS TABLE Herpes-simplex-virus (HS V) and poliovirus antibody responses in the serum and CSF of patients with herpetic encephalitis Patient number HSV complement- Day of fixing-antibody Ratio of HSV Ratio of poliovirus Sex (years) Age illness titre' in the serum : CSF serum : CSF, - A - -, antibody antibody serum CSF 1* F < I'i :l 130 A.C. 64 2* F 53 4 < :l A.C: > (at death) 3* M < * M < I'i 1:2 64 ;o F :l 64:l I'i I'i 64 I0 256 i0 2 I'i 128 I0 5* M :l 128 : : loi Ii' * F 51 4 <4 < I'i 8* F I'i 13 2# :l 327 (Continued on next page.)

4 328 F. 0. MACCALLUM, I. J. CHINN AND J. V. T. GOSTLING TABLE (cont.) Herpes-simplex-virus (HSV) and poliovirus antibody responses in the serum and CSF of patients with herpetic encephalitis Patient number HSV complement- Age Day of fixing-antibody Ratio of HSV Ratio of poliovirus sex (years) illness, titres in the serum : CSF serum : CSF A, anti body antibody serum CSF 9* 10* F :l : i :l fi I'i 11* M 6 7 < lo :l 12 F 2& 3 < I'i F :1 M 39 6 < I'i :l 15 I F 26 3 <10 <10 7 < I'i M w I'i =No result ; A.C. = anticomplimentary. * Codinned by virus isolation or immunofluorescence. t Relapse of illness. of virus from a biopsy specimen (Silk and Roome, 1970), shows the value of this test even when only single specimens of CSF are available. Results of tests for HSV antibody in the CSF of the 27 control patients were

5 CSF ANTIBODIES IN HERPETIC ENCEPHALITIS 329 essentially negative. Of 23 patients who had serum antibody titres of , none had detectable antibody in the CSF. Two patients diagnosed as zoster encephalitis did have some herpes antibody in the CSF; one had titres of 128 and 4 in blood and CSF respectively, and varicella-zoster CF antibody titres of 128 in the serum and 32 in the CSF; the other had titres of 128 and 8 respectively, and varicella-zoster antibody titres of 2256 in the serum and 2128 in the CSF. Two further, adult control patients with suspected tuberculous meningitis were being treated with daily intrathecal injections of purified protein derivative; they both had serum-antibody titres of 512, and CSF titres of 32 and 64 respectively. They were eventually given a different diagnosis and both made a complete recovery. In neither was there any other evidence of HSV infection of the brain, and it seems likely that these were simply examples of antibody leakage from the blood into the CSF. Only one of 96 patients in the mental hospital had a substantial amount of antibody (CF antibody titre >128) in the CSF; he had a serum-antibody titre of >320 and had had a severe encephalitis 3 months earlier while at sea in a trawler. None of a small number of patients, in Oxford, with recent acute dementia or temporal-lobe epilepsy had elevated titres of HSV antibody in the CSF. DISCUSSION The finding of CF antibodies to HSV in the CSF of patients with HSVE who have survived for at least 2 weeks (MacCallum, 1969), has been confirmed by our studies of further cases. These included patients diagnosed by brain biopsy, as well as others in whom the diagnosis was suspected from the clinical picture and a rising titre of serum antibody. Fifteen of the 16 patients investigated had serum : CSF antibody ratios of 1 : 1 to 16 : 1 at different stages of their illness. This low ratio, when compared with the absence of poliovirus antibody in the CSF-or its presence only in very small amount-despite relatively high titres in the serum, argues strongly in favour of the herpes antibody being made within the CNS itself rather than simply leaking into the CSF from the blood. The clear-cut difference between the results obtained from the patients and the controls confirms that a rising titre of antibody in the CSF can be accepted as diagnostic of an antigenic stimulus within the CNS, even in the absence of a very high or rising titre of serum antibody (patient 3). From the serum antibody results alone the diagnosis of HSVE in the latter patient could only have been presumptive. The value of the test obviously depends on how early the CSF specimen is collected, but this may be difficult to determine because of the variable onset of the disease. Some patients have a history of headaches for several weeks, and at the time of admission for encephalitis the titre of antibody in the serum is high but no antibody is present in the CSF (patient 2). In other patients the encephalitis has an acute onset; a four-fold or greater rise is demonstrable in the serum-antibody titre but death occurs before antibody appears in the CSF. We have also seen a few patients in Oxford with the " typical '' clinical picture of herpes encephalitis and a rising titre of antibodies

6 330 F. 0. MAcCALLUM, I. J. CHI" AND J. V. T. GOSTLING in the blood, in whom brain biopsy was not done, and no antibodies appeared in the CSF in convalescence. The diagnostic value of antibodies in the CSF became apparent in subacute sclerosing panencephalitis (SSPE), linking measles virus (or a closely related virus) aetiologically with this condition. SSPE seems to be due to reactivation of latent measles virus. Although the immunological mechanisms involved in the reactivation are obscure, cells known to be concerned with antibody production (plasma cells) can be recognised in the brain lesions. In the present study of HSVE, antibodies developed in the CSF, both in patients with primary infections and in patients with evidence of pre-existing, latent infection (cold sores). Although the antibodies may persist in the CSF for a year or more, there is usually a fall in titre after about 6 months. Since the cases that have been available to us for post-mortem examination died either too rapidly or too late after recovery-18 months or more-it is not possible to say for certain what sort of cells are responsible for the antibody production. However, from the usual histopathological descriptions of round-cell infiltration it is likely that cells of the lymphocyte series are involved here too. We have no proof that recovery from HSVE or survival with severe damage of the brain is followed by persistence of the virus in the brain, as occurs in SSPE. Unlike what is often found in the latter disease, specific IgM antibody was not found in the CSF of those of our patients so investigated, all of whom were adults (Kurtz, 1974). On the other hand, herpes infection of other parts of the body, e.g., the skin and the eye, is often followed by local recurrences and recurrent encephalitis has been demonstrated experimentally in the rabbit brain (Good and Campbell, 1948). This problem may be resolved eventually by re-isolation of virus from the brain of a long-surviving proven case, but brain biopsy for this purpose alone is not justifiable. Lerner, Bailey and Nolan (1970) failed to detect HSV neutralising antibody in the CSF of two patients with HSVE, but subsequently reported the finding of passive haemagglutinating (HA) antibodies in such patients (Lerner et al., 1972). Unfortunately, no control tests were done for other virus antibodies in the CSF that were present to high titre in the serum. As found by Kurtz (1 974) in our patients, the HA antibodies were predominantly IgG, and, as in our patients, the titres were not correlated with the protein content of the CSF or with the lymphocyte count. The passive HA test may be more sensitive than the routine CF test that we used, and might therefore be positive earlier in the disease. However, comparison of the results of Lerner et al. (1972) with ours does not show any obvious advantage for the passive HA test. SUMMARY Complement-fixing and neutralising antibodies to herpes-simplex virus usually appear in the CSF of patients with herpetic encephalitis during the second or third week of illness and may be of diagnostic value. The ratio of serum to CSF antibody titres is, as a rule, in the range of 1 : 1 to 16 : 1. The antibodies may persist in the CSF for 6 to 18 months after recovery from the acute stage of

7 CSF ANTIBODIES IN HERPETIC ENCEPHALITIS 33 1 the disease; their detection, even in the absence of an obvious history of acute encephalitis, probably implies infection of the central nervous system within the past 18 months. However, we cannot yet be certain that absence of these antibodies from the CSF excludes the diagnosis in patients with the clinical picture of herpes-simplex-virus encephalitis and a rising titre of specific antibodies in the serum. The titres are probably not affected by the systemic use of specific anti-herpetic drugs. We wish to thank our neurological colleagues at Oxford and Portsmouth for their collaboration in collecting numerous specimens from their patients, as well as Dr J. Wilson and Professor A. Dudgeon, Hospital for Sick Children, Great Ormond Street, and Drs A. B. Donnison and E. Sayle, Ascot, for specimens from patient 10 and Mr G. P. Duffy, FRCS, for specimens from patient 11. REFERENCES GOOD, R. A. AND CAMPBELL, B The precipitation of latent herpes simplex encephalitis by anaphylactic shock. Proc. SOC. exp. Biol. Med., 68, 82. ILLIS, L. S. AND GOSTLING, J. V. T Herpes simplex encephalitis. Bristol. JUEL-JENSEN, B. E. AND MACCALLUM, F Herpes simplex varicella and zoster. London. KURTZ, J. B Specific IgG and IgM antibody responses in herpes-simplex-virus infections. J. med. Microbiol., 7, 333. LERNER, A. M., BAILEY, E. J. AND NOLAN, D. C Complement-requiring neutralizing antibodies in Herpesvirus hominis encephalitis. J. Immun., 104, 607. LERNER, A. M., LAUTER, C. B., NOLAN, D. C. AND SHIPPEY, M. J Passive haemagglutinating antibodies in cerebrospinal fluids in Herpesvirus hominis encephalitis. 1. Proc. SOC. exp. Biol. Med., 140, MACCALLUM, F Antibodies in the cerebrospinal fluid in Herpesvirus hominis encephalitis. J. Path. Bact., 98, 587. SILK, B. R. AND ROOME, A. P. C. H Herpes encephalitis treated with intravenous idoxuridine. Lancet, 1,411. SMITH, I. W., PEUTHERER, J. F. AND MACCALLUM, F The incidence of Herpesvirus hominis antibody in the population. J. Hyg., Camb., 65, 395. TOMLINSON, A. H In Acute necrotising encephalitis and herpes simplex virus infection. Post-grad. med. J., 49, 406. J.ran. MICROBIOL.-VOL. 7 (1974) Z