Subacute sclerosing panencephalitis

Journal of the neurological Sciences, 1974, 21 : 381-390 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

381

Subacute Sclerosing Panencephalitis E l e v a t e d E p s t e i n - B a r r Virus A n t i b o d y Titers a n d F a i l u r e o f Amantadine Therapy

J. JONCAS, G. GEOFFROY, B. McLAUGHLIN, G. ALBERT, N. LAPOINTE, P. DAVID, R. LAFONTAINE AND M. GRANGER-JULIEN Institute of Microbiology and Hygiene of Montreal, the University of Montreal and Ste Justine Hospital, Montreal (Canada) (Received 5 September, 1973)

INTRODUCTION

Subacute sclerosing panencephalitis (SSPE) is a disease of children presenting usually with progressive intellectual deterioration followed by extrapyramidal motor disturbances and myoclonic jerks. The electroencephalogram (EEG) is characterized by paroxysmal slow wave dysrhythmia. Spinal fluid (CSF) and serum contain measles antibodies in high titer and usually increased amounts ofimmunoglobulins (Connolly, Haire and Hadden 1971). Measles virus has been isolated by co-cultivation of intact brain cells in several cases of the disease (Chen Tsu Teh, Watanabe, Zeman and Mealey 1969; Horta-Barbosa, Fuccillo, London, Jabbour, Zeman and Sever 1969; Payne, Baublis and Itabashi 1969). Attempts to reproduce the disease in experimental animals including primates have met with little success (Albrecht, Shabo, Burns and Tauraso 1972; Lehrich, Katz, Rorke, Barbanti-Brodano and Koprowski 1970; Wear and Rapp 1971). The present report summarizes clinical and laboratory data and discusses a few unusual findings in 12 cases of SSPE seen in 2 years at the Ste Justine Hospital in Montreal. MATERIAL AND METHODS

Clinical cases. The 12 patients included in this survey were admitted to Ste Justine Hospital from December 1970 to December 1972. The diagnostic criteria for inclusion in the group were: (1) clinical stages (Freeman 1969), (2) characteristic EEG displaying paroxysmal slow dysrhythmia, (3) first zone colloidal gold curve or elevated CSF gammaglobulins, (4) elevated measles antibody titer in the serum and in the CSF and in certain cases, histological features of the disease in brain biopsy or autopsy material. "Staging" was determined by one of the neurologists (G.G., P.D. or R.L.). -This work was supported by a Defense Research Board Grant 1880-08 (9365-15) of Canada and a Federal Provincial Public Health Research Grant 604-7-783, Quebec, Canada.

TABLE I CLINICAL AND LABORATORYI)AIA IN 12 CASES O}' SSPE

Case

I.S.M."

Adm. date

Dec. 1970

Sex

Age ( yrs )

F

6

Mea~les disease at

Lice measles vaccine

1 yr

Prodromat period heft)re diagnosis

Initial vvmptoms

5mo

ataxia

Stage and final outcome

Amantadine trial

IB liB apparent arrest

2. J.B.

April 1971 July 1971

F

March 23. 1970

5

2mo

1.5 m o

2 yrs

3.G.V.

May 1971

M

5

4. L.P.

Nov. 1971

M

7

5. S . D #

Jan. 1972

F

8

1 yr

6.S.B#

Sept.

M

13

2 yrs

2 weeks

Feb. 1971 March 1971

F

6

9 mo

4 mo

M

10

3 yrs

5 mo

9. A.V.

June 1971

M

8

2 yrs

I mo

10. M.B.

Sept. 1971 April 1972

F

8

3,5 yrs

mo

II.M.Mc

Nov. 1971

F

12.J.S.

Dec. 1971

F

Oct. 28, 1970

8. D.D.

apraxia and ataxia

3 mo

learning difficulties in school

4 mo

learning difficulties in school, behavior problem

1972

7.T.T.

ataxia

past history: epilepsy, mental retardation, dyskinesia. Present illness: spastic left hemiparesis apraxia, learning difficulties in school ataxia, poor attention span

IB J, IIA lllC death : 24.8.71 IIA ,, IIC IA ,, lIB June '72 IIA 1 llC death : 29.3.72

IIB i, II1C death : 23.10.72 lib IB

ataxia

IC

behavior problem

IA

1 IliA Jan. "72

8

2.5 yrs

1 yr

1.5 m o

learning difficulties in school, apraxia, agraphia, ataxia

IA ,L IIIA June '72

3 mo

learning difficulties in school

IliA ,L IIlC death : 12.1.72

a m a n t a d i n e 50 m g t.i.d. 8.4.71 24.8.71

amantadine 50 m g t.i.d. 26.5.71 13.7.71

amantadine 70 mg t.i.d. 9.10.72 23.10.72

EEG

coll. OoM

CSF protein~% of 7-globulin

paroxysmal and diffuse slow dysrhythmia not entirely characteristic, right frontal epileptic fOCUS

+

50-60 rag/100 ml ND

characteristic 4 days after admission

+

57 mg/100 ml ND

characteristic

+

30-64 rag/100 ml 53~o

characteristic

+

62 mg/100 ml 10~o

characteristic

+

58-70 mg/100 ml 10-21~o

characteristic

+

109 mg/100 ml ND

characteristic

ND

characteristic

+

59 mg/100 ml ND 39-59 rag/100 ml ND

characteristic

-

characteristic 1 month after admission

+

characteristic 16 days after admission

paroxysmal and diffuse slow dysrhythmia not entirely characteristic

+

ND

N ND 46-77 rag/100 m| ND

62-118 mg/100 ml 29- 44~o

64 mg/100 ml 21 ~oo

serum

Viral antibodies

R: 14,12.70:128 (NT949) 13. 1.71:1024 15.12.71: 64 H: 16.12.72: 32 CMV: 16.12.72: <4 VZ: 16.12.72: <4 EBV-IF: 16.12.72: 160 R: 6. 4.71: 512 9. 7.71:1024 H: 6. 4.71: 32

R: 19. 5.71:1024

R: R: H, CMV, VZ: EBV-IF: R: R: M: A: H, CMV: VZ: EBV-IF: R: H: VZ: CMV: EBV-IF: R: R: H: CMV: VZ: EBV-IF: R: H: M: R:

M: H, CMV, VZ: EBV-IF: R:

M: H: CMV: VZ: EBV-IF: R:

H: CMV: VZ: EBV-IF :

18. 8.72:4096 11. 9.72:2048 12. 7.72: <4 12. 7.72: 80 24. 1.72: 256 28. 1.72: 512 24. 1.72: 32 24. 1.72: 4 28. 1.72: <4 28. 1.72: 8 28. 1.72: 320 17.10.72:1024 17.10.72: 32 17.10.72: <4 17.10.72: 8 17.10.72: 5 26. 2.71:1024 18. 3.71:2048 17. 3.71:2048 6. 6.72: 512 17. 2.72: <4 17. 2.72: <4 17. 2.72: <4 17. 2.72: 40 23. 6.71:2048 23. 6.71: 16 23. 6.71: 4 24. 9.71: 512 17. 8.72: 512 26.10.72:1024 20.11.71: 16 12. 7.72: <4 12. 7.72: 5 15.11.71: 512 18. 8.72:2048 26.10.72:4096 20.11.73: 512 13. 1.72: 16 18. 8.72: 16 18. 8.72: <4 12. 7.72: 8 12. 7.72: 80 12.12.71: 256 22.12.72:1024 30.12.72: 512 30.12.72: 16 22.12.72: <4 22.12.72: 4 22.12.72: 80

CSF

16 (NT33) 32 8 <2

CSF/serum antibody ratio 1:8 1:32 1:8

<2 64 64 ND

1:8 1:16

32

1:32

32 32

1:28 1:64

<2 4

1:64

<2 <2

<2 32

1:32

ND

32

1:16

16 <2 <2 32 32 32 <2

1:128

1:16 1:16 1:32

<2 32 32 128 32 <2

1:16 1:64 1:32 1:16

<2 16 32 16 <2

1:16 1:32 1:32

<2

" Virus detection or isolation : see text. Amantadine, 8 mg/kg/day; ND, not done; EM, electron microscopy ; R, Rubeola complement fixation (CF); H, herpes simple," (CF); CMV, Cytomegalovirus (CF); VZ, Varicella-zoster (CF) M, mumps (viral antigen) (CF); A, adenovirus (CF); EBV-IF, EpsteinBarr virus indirect immunofluorescence; NT, neutralizing ~.ntibodies.

384

J. JONCASet al.

Seroloyy. Serological tests included complement fixation tests by the microtiter method done on sera and CSF for measles and the following other viral antigens (mumps soluble and viral antigens, adenovirus, herpes simplex, cytomegalovirus and varicella-zoster virus). In occasional cases, hemagglutination inhibiting and neutralizing measles antibody titers were also determined and in 8 cases, Epstein-Barr fluorescent antibody titers were measured by a technique previously described (Joncas and Mitnyan 1970). Virus isolation and electron microscopy. CSF samples were inoculated into HeLa and primary African Green Monkey Kidney tissue cultures which were observed for cytopathic effect and hemadsorption. Brain biopsy in 1 case and autopsy material in 2 cases (brain, lungs, liver, spleen and kidney) were co-cultivated in mixed cultures only with Hep2 and Vero cells (Horta-Barbosa et al. 1969). Hemagglutination and hemadsorption with rhesus monkey erythrocytes on supernatant and cell monolayer respectively were also carried out (Horta-Barbosa et al. 1969). Biopsy and autopsy material was embedded, thin-sectioned and examined with a Philips 200 electron microscope (Berthiaume and Joncas 1973). In addition trypsinized cells from the original biopsy or autopsy material and weekly samples of the mixed cultures were examined with the electron microscope by a previously described negative staining technique (Joncas, Berthiaume, Williams, Beaudry and Pavilanis 1969). The cocultivated cells were frozen and thawed 3 times, centrifuged at low speed and a drop of the supernatant and resuspended pellet were both examined after negative staining with the electron microscope. Cytopathic agents were identified in neutralization and in hemagglutination inhibition tests with specific (adenovirus and measles) antisera obtained from Microbiological Associates and from Grand Island Biological Co.

Amantadine trial. Amantadine hydrochloride in a syrup form* containing 50 mg/5 ml was used in the dosage of 8 mg/kg/day divided in 3 doses. Cases J.B., G. V. and S.B. only could be included in the trial with the parents' permission. RESULTS

Clininical data

A summary of our observations in 12 cases appears in Table l.Most of the patients (9) were seen in 1971. Five patients were males, 7 were females. At the times of diagnosis their ages varied from 5 to 13 years. Less than half of them (5) were from greater Montreal. A history of measles was obtained in 10 of the 12 cases. In 7 of these 10 cases, measles occurred at the age of 2 years or younger. One of these 7 cases had measles at the age of 9 months. Two of the 12 cases received live attenuated measles vaccine less than 1 year before the onset of symptoms and they apparently never had clinical measles: a 5 year-old girl (J.B.) and a 7-year-old boy (L.P.) received the vaccine respectively 11 and 10 months before the onset of SSPE. Excluding these 2 cases the interval between clinical measles and SSPE varied from 3 to 11 years. None of the other children received measles vaccine. The prodromal period prior to diagnosis varied from 1 month to 1 year in all cases. Learning difficulties in school and ataxia were the most * Symmetrel kindly supplied by the Dupont de Nemours Company, U.S.A.

SUBACUTESCLEROSINGPANENCEPHALITIS

385

common initial symptoms. Three patients were first seen in stage IA, 3 in stage IB, 1 in stage IC, 2 in stage IIA, 2 in stage IIB and 1 in stage IIIA. Four patients died within 1~1 months of the diagnosis. The majority have shown progressive downhill courses. The first patient seen (S.M.) aged 6 years, seems to be arrested in her clinical course for nearly 2 years in stage IIB.

Laboratory data The EEG was characteristic in all cases, except possibly in case S.M. and in 1 other case in which it showed diffuse in addition to paroxysmal slow dysrhythmia and also focal epileptic activity. The colloidal gold curve done in 9 of the 12 cases was of the characteristic first-zone type. CSF protein contents ranged from 58 to 109 mg/100 ml with percentages of gammaglobulin varying from 10 to 53~o. Serum and CSF complement-fixing measles antibodies were moderately to markedly elevated in all cases and the CSF/serum measles antibody ratio varied from 1/8 to 1/128. Antiviral antibodies other than measles could not be detected in the CSF. The serum antiviral antibody titers to the other viral antigens tested were unremarkable except for the EpsteinBarr virus (EBV) antibody titers which were found to be positive in all cases tested (8 cases), and elevated to a titer of 1/80 or over in 5 cases. The geometric mean titer (gmt) of 1/48 in these 8 cases was significantly higher ( P < 0.025) than the grnt of 1/16 of the same antibodies in a comparable control group of 88 children aged 1-12 years previously reported (Joncas 1972). The prevalence of the EBV antibodies in this control group was 42~o. ( P < 0.005). CSF samples did not yield any virus. Herpesvirus particles were seen with the electron microscope in the co-culture material of the brain biopsy of Case 1 (S.M.) after 21 days of co-cultivation. It had not been seen directly in the biopsy in contrast

Fig. 1. Herpesvirusparticles found in a sample of the co-cultureof the brain cells (biopsy)of case S.M. after 21 days of co-cultivation.Bar represents 100 nm.

386

J. JONCASet al.

to our findings (to be published) in 2 cases of herpes encephalitis. No cytopathic effect nor hemadsorption nor pathogenicity for rabbits by skin and corneal scarification and subcutaneous injection was ever observed, therefore the virus could not be identified and was eventually lost (Fig. 1). An adenovirus was detected by electron microscopy in the co-culture of the kidney cells (autopsy) of Case 6 (S.B.) with Hep2 cells after 19 days of co-cultivation (Fig. 2). The isolate did not hemagglutinate rat red cells but did hemagglutinate African Green Monkey red cells. It was therefore subsequently identified as adenovirus type 21 by hemagglutination inhibition using African Green monkey red cells and adenovirus antisera against each of Rosen's group 1 adenoviruses. Identification was also confirmed in neutralization tests using adenovirus

~ 5!

Fig. 2. Adenovirus particles observed in a sample of the co-culture of the kidney cells (autopsy) of case S.B. with Hep2 cells after 19 days of co-cultivation. Subsequently identified as adenovirus type 21. Bar represents 100 nm. Fig. 3. Numerous nucleocapsid fragments 18 nm in diameter found in a sample of the co-culture of the brain cells (autopsy) of case S.D. with Vero cells after 29 days of co-cultivation. Bar represents 100 nm.

i'iJ

SUBACUTE SCLEROSING PANENCEPHALITIS

387

antisera to type 1 through 7 and to type 21. No neutralizing antibodies could be detected in the pre-mortem serum of the patient at the 1/10 dilution. Finally, a nucleocapsid 18 nm in diameter, characteristic of the paramyxovirus group, was seen with the electron microscope by negative staining in the co-culture of the brain cells (autopsy) of Case 5 (S.D.) with Vero cells after 29 days of co-cultivation (Fig. 3). A measles virus could not be identified however, although a cytopathic effect was noted transiently. Hemagglutination and hemadsorption were negative. No virus could be detected by electron microscopy in control Hep2 and Vero cells passaged and examined in parallel with the co-cultures nor directly in biopsy or autopsy material. Amantadine trial There was no improvement noted among the patients receiving amantadine even when the drug was given as soon as the diagnosis was made in stage IA (J.B.) and maintained for 5 months until death in that case. No difference could be found when the clinical course of case J.B. and that of 2 additional cases (G.V. and S.B.) treated for 2 months and 2 weeks respectively, were compared to that of the other 9 untreated cases. No significant differences in EEG, CSF proteins, serum and CSF measles antibody titers was noted between the 2 groups. Death occurred in the amantadinetreated as well as in the untreated group. No apparent toxicity from amantadine could be elicited judging from the hematology and liver function tests done (complete blood counts and serum transaminases). Minor central nervous system signs of toxicity would have been difficult to detect.

DISCUSSION

The clinical observations made in this survey are similar to those reported in the literature (Freeman 1969 ; Haslam, McQuillen and Clark 1969 ; Jabbour, Duenas, Sever, Krebs and Horta-Barbosa 1972). In particular the mean age of onset of 7.33 years is close to the 7.2 years reported by the United States SSPE registry (Jabbour etal. 1972). Similarly, more than half of the patients (7) contracted measles under the age of 2 years. Several unusual observations deserve emphasis: the occurrence of 12 new cases in 2 years (as well as 2 additional cases since the preparation of this report) must indicate a high frequency of SSPE in the Province of Quebec with a total population of 6 million inhabitants, an incidence of 1.0/million/year, when compared to the incidence of 0.1/million/year reported in the SSPE registry and that of 0.5/million/ year in Belgium (Canal and Torck 1964). The occurrence of 2 cases within 1 year after the administration of live measles vaccine without any history of clinical measles in 2 children as young as 5 and 7 years must be noted. Younger and older sibs in these families had never had measles and in 1 of the 2 families had received measles vaccine. The mean interval between measles and SSPE being 5-7 years, the possibility of unrecognized measles under 2 years in these young children is less likely although certainly not excluded. If there is a host factor conditioning the occurrence of SSPE, it would not be entirely surprising to see SSPE follow either clinical disease or vaccination in such children. Immunologic abnormalities in cases of SSPE have been reported

388

J. JONCAS et al.

but these are not firmly established (Gerson and Haslam 1971; Lapointe, Geoffroy and Mongeau 1972; Mizutani, Mizutani, Saito, Nihei and Izuchi 1971; Saunders, Chambers, Knowles and Caspary 1969). The disease has been reported in only 1 of 2 identical twins to date (Whitaker, Sever and Engel 1972). It has been reported in sibs (Ch'ien, Wilborn and Carey 1972). The association of SSPE or other neurologic complications with measles vaccination reported to date would amount to a low incidence of the order of that of Sabin vaccine-associated poliomyelitis (Hopkins, Dismukes, Glick and Warren 1969; Jabbour et al. 1972; Landrigan and Witte 1973). Continued surveillance in years to come may help to confirm or exclude a relationship between SSPE and vaccination and, if confirmed, determine its true incidence. No difference was observed between amantadine-treated cases of SSPE and untreated cases. Parameters followed included clinical stages, EEG, CSF proteins, CSF and serum measles antibody titers. A steadily downhill course was noted in all cases even in a patient for whom the drug was given as soon as the diagnosis was made in stage IA and maintained for 5 months until death. The beneficial effects noted by Haslam (Haslam et al. 1969) may have been due to the pharmacological effect of the drug on the central nervous system (Grelak, Clark, Stump and Vernier 1970) or may have been only fortuitous. Biopsy material available in 1 case and autopsy material available in 2 cases yielded virus in each of the 3 cases. Final identification, however, was possible in only 1 case (S.B.), an adenovirus type 21 isolated in co-cultivation of kidney cells at autopsy. A virus could not be recovered to date from the co-culture of the brain cells in that case. The significance of the isolation of an adenovirus from case S.B. is not clear especially in view of the absence of adenovirus 21 neutralizing antibodies in the serum of this patient. Adenovirus type 21 has never been isolated previously in our laboratory. Paramyxovirus nucleocapsids, most probably of measles virus origin were detected by electron microscopy in the co-cultivation of the brain cells from case S.D. with Vero cells. Isolation of fully infectious virus was never achieved but similar incomplete isolations have been reported previously (Doi, Sanpe, Nakajima, Okawa, Katoh, Itoh, Sato, Oguchi, Kumanishi and Tsubaki 1972). Finally, a herpesvirus was seen by electron microscopy in the co-culture of the brain cells of case S.M. but it was not cytolytic and not pathogenic for rabbits by various routes. It could not be identified and was eventually lost. The finding of high EBV antibody titers and low or undetectable antibody titers to other viruses of the herpes group in this case suggest that it could be EBV. The finding of EBV positivity in the 8 cases of SSPE tested and of high EBV antibody titers in most of these cases is definitely different from the lower mean titer and lower prevalence (42~) of EBV antibodies in a comparable control group of 88 children of the same age previously reported (Joncas 1972). This observation suggests to us the possibility of reactivation of a latent EB virus, possibly as a direct result of SSPE, or in relation to a basic immunologic abnormality or deficiency of intracellular resistance in such patients. The sensitive cultivation technique of live cells has already uncovered latent viruses of the herpes (Baringer and Swoveland 1973; Nielsson 1971) and adenovirus groups (Evans 1958) in man. It is not therefore too surprising that viruses of the above groups could be isolated in cases of SSPE. The clinical significance of these latent infections in terms of disease in man remains to be determined (Joncas 1972).

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389

ACKNOWLEDGEMENTS The c o l l a b o r a t i o n o f L a u r e n t Berthiaume, P h . D . , electron microscopy l a b o r a t o r y a n d Jacques R e g n a r d , M. D., diagnostic virology laboratory, Institute of Microbiology a n d H y g i e n e of M o n t r e a l is gratefully acknowledged. SUMMARY Twelve new cases o f s u b a c u t e sclerosing p a n e n c e p h a l i t i s were seen over a period o f 2 years in M o n t r e a l (and 2 a d d i t i o n a l cases since the p r e p a r a t i o n o f this report) indicating a high incidence (1.0 per m i l l i o n per year) o f this disease in the Province o f Q u e b e c with a total p o p u l a t i o n o f 6 m i l l i o n i n h a b i t a n t s . A history o f measles was o b t a i n e d in 10 cases a n d o f live a t t e n u a t e d measles v a c c i n a t i o n in the other 2 cases. Measles occurred at or u n d e r the age o f 2 years in 7 cases a n d the m e a n age of onset of SSPE was 7.3 years. A few u n u s u a l o b s e r v a t i o n s were m a d e : (1) The f i n d i n g o f EBV a n t i b o d i e s in 8 cases tested, 5 o f which h a d titers a b o v e 1/80; (2) The isolation a n d / o r electron m i c r o s c o p y detection o f a p r e s u m a b l y latent herpes a n d a d e n o v i r u s 21 following c o - c u l t i v a t i o n of biopsy or a u t o p s y m a t e r i a l ; (3) The failure o f a m a n t a d i n e in 3 cases to alter the course o f the disease.

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