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Diagnosis and treatment of herpes encephalitis
443
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Diagnosis and Treatment of Herpes Encephalitis A Multidisciplinary Approach M. RAPPEL, M. DUBOIS-DALCQ, S. S P R E C H E R , L. T H I R Y , A. L O W E N T H A L , S. PELC AND J. P. THYS Departments of Neurosurgery, Pediatric Neurology, Intensive Care Unit, Neuropathology and Electron Microscopy, Faculty of Medicine, Free University of Brussels, Pasteur Institute of Brabant, Brussels, and Department of Neurochemistry, Born-Bange Foundation, Berchem-Antwerp ( Bel,¢ium)
(Received 3 April, 1970)
INTRODUCTION Acute necrotizing encephalitis (ANE) is a well-recognized clinico-pathological entity (VAN BOGAERF et al. 1955; BRIHAYE 1959: BENNETT et al. 1962; BERGOUIGNANet al. 1968; CAMPBELL 1969; DUDGEON 1969) with a high mortality rate. However, the clinical diagnosis is only presumptive and has to be supported by laboratory investigations. There is growing evidence that most of the cases of A N E are due to herpes simplex virus (HSV) which is, like other DNA-viruses, inhibited in its growth by a thymidine analogue, the 5-iodo-2'-deoxyuridine (SKF14.287, idoxuridine, I U d R , I D U , I D U R ) as shown by HERRMANN (1961). This drug is widely used in the treatment of herpetic keratitis (KAUFMANN 1963; MAXWELL 1963). I U d R has also recently been introduced to treat herpes encephalitis, with variable results (BREEDEN et al. 1966: BUCKLEY AND MACCALLUM 1967: EVANS et al. 1967: MARSHALL 1967; BELLANTI et al. 1968; TOMLINSON AND MACCALLUM 1969). These reports raise several questions related to early diagnosis and appreciation of the efficacy of the treatment. The diagnosis is made only in the most severe cases, referred to the hospitals so that the true incidence of the disease is still unknown. A prospective multidisciplinary approach of the diagnosis should enhance further knowledge of the natural history and the aetiology of necrotizing encephalitis. This in turn will help the clinician to assess the relative usefulness of different forms of treatment. The following is a report of 5 cases of acute necrotizing encephalitis where such an approach was attempted. This work was supported by Grant No. 570 A4 from the National Multiple Sclerosis Society,U.S.A. (M. D.-D.), by a grant from the Scientific Medical Research Fund (S.S. and L.T.) and by a grant from the Belgian Ministery of National Education (A.L.). M. Dubois-Dalcq: Charge de Recherches F.N.R.S. Reprint requests to M. Rappel, Clinique Neurochirurgicale, Institut Bordet, 1, rue Hdger-Bordet, 1000 Brussels (Belgium). J. neurol. Sci., 1971, 12:443 458
0
-- (L)
. (L) .....
(R)
0
0
N.D. ANE
N.D.
N.D.
(R)
(kl
(L)
0
(R)
--
(L)
-
.
.
.
N.D.
0 N.D.
N.D.
(R)
14fll 14 very poor
?
500(56 64) poor
.
(R)
64 128 64 N.D. biopsy (7}
.
.
-
-:-
F 41 ~
(R)
(R~
200(15 i8~ good
64 256 (CSFI biopsy (12! ,.lecomr, !lSi
N.D. ANE
N.D.
:
-
0
(L)
(R)
M 13 --
~20(t5-2{}: goo;t
t6 64 N.D, biopsy i(~)and(2!~
N.D.
N.D. N.D.
N.D.
N.D.
(R)
0
?
0
F 11 12 ?
.' 3 5) ~to. ,.tled (5!
( brain
64
o
M 47
C~ase 8 Case O" Case 10 . . . . . Cas'e 11 ( B U C K L E Y (MARSHALL (BELLANT1 (W.B.) et al. 1967) 1967) et al. 1968) (TOMUNSON et al. 1969)
8 128 N.D. biopsy (56)
N.D. .
0
(R~
.
N.D.
.
N.D.
.
N.D. ANE
.
(L)
-- (L)
F 8
-Case 7 " (EVANS et al. 1967)
N.D. ANE
- (L)
(R)
'(R)
-
.
~- ( L )
-7
8 64 N.D. N.D. lobect, biopsy [7) decomp~.~'.~} decompr.¢'~!
0
0
(R) ANE
N.D.
-
-
M 34 ~
Cas~6 (BREEDEN et al. 1966)
500{~8 22) 500{9-13) 500(10 17i good no. died ( 191 good
32 128 N.D. N.D.
N.D.
N.D.
(L) N.D.
:
- (L)
-
•-
0
0
F 65 ?
R: righti L- iei:{TiN))_i not ~ione: AN E i acute necrotizing encephalitis
0 4 16 16 N.D. N.D. ]obectom3 decompress t9) (14) (27~ 500(16 24) 3 2 0 ( 1 . - - 1 ) 500(27 34~ moderate good moderate -died (63)
16 128 N.D.
0
?
N.D.
~
N.D. ANE
N.D.
-
N.D. ANE
(Rt uninterpretable
(R)
:
0
(L)
- (L)
F 45 _
case 4 ..... -C-ase 5 (J.P.) (M.D.) Nov. 1968 M a y 1969
Complement fixating antibodies given as the reciprocal of the end point dilution. ~' In parentheses: day of the d Sease (day 0 day of the acute onset).
5ibi, r-ei,iation,Ti
IDU(mgkg) > Recovery~,
Abnormal air study Optic microsc. Virus seen by EM Virus isolation (brain) Herpes antibodies fCFT)a Fluoresc. studies Surgical proced,are"
Headache Fever Epilepsy Hemiplegia or hemiparesis Meningism Confusion or psych, disturb. Coma CSF lymphocytic pleocytosis A b n o r m a l EEG Abnormal angiography Brain scan
+ (R)
M 56 ~
58
F 12
M
Age
(years)
Sex
case 3 (A.L.) Aug. 1968
Case 2 (C.D.) July 1968
Cas'e 1 (J.S.) Dec. 1967
TABLE 1
suRwY oF 11 CASESREeO~TED
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
445
PATIENTS AND TREATMENT
During a 17-month period (December 1967-May 1969) 6 cases of encephalitis were referred to the University Hospital. A provisional diagnosis of necrotizing encephalitis was made on clinical grounds in 5 of them. One patient (Case 3, A.L.) was first thought to have mumps encephalitis as he presented bilateral parotid enlargement and mumps had been diagnosed in 4 children from his village. ANE was later confirmed. Herpes virus was shown to be involved in this patient as well as in 4 other cases. The present report deals only with these 5 cases; the 6th one, where herpes virus was not demonstrated, has been described in a preliminary communication (RAPPEL AND BRIHAYE 1969). Four patients underwent temporal lobectomy and decompression ; the 5th patient received no operative therapy. The 5 patients were treated with intravenous idoxuridine. Table 1 summarizes the present 5 cases as well as the 6 other cases of herpes encephalitis treated with IUdR which have been reported in detail in the literature. Day 0 refers to the day of the acute onset of the disease. Like all the pyrimidine compounds, IUdR, in its neutral, lactame form, shows poor solubility in water. It is best dissolved in a basic solution. Therefore IUdR (500 mg/ 100 ml) was dissolved in a 5 g/100 ml glucose solution solution alkalinized by sodium bicarbonate (100 mg/100 ml). In adults the total dose of IUdR was 500 mg/kg body weight. Transient and moderate signs of hepatic toxicity and depression of bone marrow function were encountered in 4 of the patients. Cases 3 and 4 also showed a severe but transient diffuse alopecia. The 12-year-old patient (Case 2) was given 320 mg IUdR/kg body weight. No sign of toxicity was detected. Details concerning the evolution of the illness in the patients are given below.
Case I (J.S.) was not helped by the surgical procedure and became rapidly comatose and unresponsive to any stimuli. He remained pyrexial. [UdR therapy was begun 5 days after the operation. Thirty hours later, the patient responded to light stimuli and his temperature dropped. The patient awakened progressively and started speaking again. The improvement lasted 1 month, and then the patient's condition slowly deteriorated. He died on day 63 of severe bilateral bronchopneumonia. ('ase 2 (('.D.) was pyrexial, comatose and hemiplegic. Surgery immediately relieved the motor signs but the child remained confused and disorientated. IUdR treatment was started 24 hours after operation. Within 3 days, the child was greatly improved, with only a mild memory defect. [n the following months, she completely recovered her memory and her school performance returned to normal. Case 3 (A.L.) had become progressively comatose with only EEG signs of a focal lesion. There was no immediate improvement after surgery and IUdR treatment was started a few hours later. After 3 days of treatment, there was clinical improvement which continued slowly. Upon discharge, the patient was able to read and to calculate, but his mental capacity was below normal. One month after the termination of treatment, he presented a transitory thrombocytopenic purpura of the lower extremities and anti-platelet antibodies were demonstrated. Case 4 (J.P.) was pyrexial, disorientated and showed jargon aphasia on admission. Ancillary investigations revealed a left temporal mass and a rise in herpes antibodies. Within a few days, the patient became almost completely mute and then comatose. Forty-eight hours after IUdR treatment, the fever had disappeared and the patient started to speak again. Repeat arteriography and brain scan revealed disappearance of the temporal mass. The patient was discharged with. a moderate memory defect and a slight residual aphasia. The,'e has been continuous improvement with respect to each of these deficits. ('m'" 5 / M . D . ) remained deeply com~.tose after operation and IUdR treatment. She died on day 19. J. neurol. Sci., 1971, 12:443 458
446
M. RAPPELet al.
LABORATORYMETHODS Virus isolation Cerebrospinal fluid (CSF), cerebral biopsies and post-mortem brain fragments were kept at 0°C and transported to the laboratory within I-2 it after removal. Inoculation of these samples was performed intracerebrally on new-born mice, in the chorioallantoic membrane of embryonated eggs, on a human continuous cell line (KB) and on primary monkey kidney cultures. These procedures made it possible to grow poliomyelitis, Coxsackie and ECHO-viruses, as well as adenoviruses and herpes viruses. Antibody studies Several samples of sera were obtained from each patient at various stages of the disease. Antibodies were determined by a complement fixation test (CFT) using the following virus antigens: measles, mumps, Coxsackie B types 1-5, adenoviruses and herpes. In addition, neutralizing antibodies to herpes simplex hominis type 1 and 2 were assayed by a specific method (RAWLS et al. 1968). This recently-introduced method determines the rate of inactivation of each serotype by the antibodies present in the patient's sera, permitting discrimination between the antibodies to the two types of herpes virus. The results are given by the constant of inactivation, K. Protein electrophoresis CSF and serum proteins were examined by W|EME'S agar gel electrophoresis technique (1959) and the results were analyzed quantitatively according to LOWENTHAL (1964). Histology Fragments for light microscopy were immediately fixed in 10}~ formaldehyde, embedded in paraffin and stained by haematoxylin-eosin (H-E) and Nissl methods. Specimens for electron microscopy (EM) were fixed in 4.2% glutaraldehyde, postfixed in osmium, embedded in Vestopal or Epon, cut with glass on a LKB microtome and stained by uranyl acetate and by Karnovsky's lead mixture. Specimens were then studied with a Siemens Elmiskop 1.
LABORATORY RESULTS
Virus isolation The isolation was attempted from temporal lobe biopsies of Cases I, 2, 3 and 5. Only the brain fragment, obtained 9 days after the onset of the disease, of Case 1 yielded a positive viral culture. All methods employed gave positive results with this fragment: a cytopathic effect in tissue culture, pocks in the chorioallantoic membrane of chick embryo and the death of baby mice. These effects were completely neutralized by a rabbit antiserum to herpes simplex, an indication that there was no other virus associated. The other biopsy samples were obtained 9, 16 and 27 days respectively after onset of the disease. In Case 1, fragments from both temporal lobes obtained at autopsy were also studied but failed to show any infectivity. Since J. neurol. Sci., 197~, 12:443 458
447
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
these fragments were larger than those of the previously-performed biopsy and were taken from several parts of the brain, the negative results suggested that the brain had been sterilized by IUdR.
Antibody studies None of the patients showed any increase in antibodies to adenovirus, Coxsackie or measles virus. An increase in antibodies to HSV was detected in Cases 1, 2 and 3 (Fig. 1) by both the CF and kinetic neutralization tests. With the latter test, there was no modification of antibody levels to herpes virus 2 (results not shown), but only to type 1 indicating that an infectious process due to this virus was developing. In Case 4, the levels of CF and neutralizing antibodies were already high at the time of collection of the first serum sample (on day 12). There was a late peak in the elevation of neutralizing antibodies, several weeks after completion of the treatment. In Case 5, the only serum sample was obtained 6 days after the beginning of the disease and no antibodies to herpes virus were present.
CF'
lIJ.S) IUdR~ , 2(C.Ol ~IUdR 16
256 128
, i
24/ / /
/I
3 AL)
~
T5 21
&(j.p}
i
e
16
8
.
,~
IU~dR ]p~
]K
1822 ] ~, " ', ~ ~~ ,
I 18
. "HI"/ i
1~
x
27 3 L
16
lO
,,,/
..8 6
2 0 Days
x ~ 5 10 f5 20 3550 5 10 15 20 25 30
/ st, 5 10 1,5 20 25
2 35
5 tO 15 20 4 5 1 6 0 ~ 0
Fig. I. 0 - - @ : titres of C F antibodies to herpes simplex virus expressed as the reciprocal of the greatest dilution showing positive results; x - - - × : neutralizing antibodies to herpes simplex virus type I, expressed as the constant of inactivation (K) of the virus.
It was considered that the presence of I U d R in blood might contribute to the activity found in the neutralizing test which may measure an inhibition &virus growth by I U d R as well as measuring the destruction of infectivity by antibodies. In order to assess whether I U d R in its active form could be detected in serum or in CSF, serial dilutions of samples from Case 1 were mixed with 100 plaque-forming units (PFU) of several viruses: pseudorabies virus (a D N A virus of the herpes group), vaccinia virus (a D N A virus of the poxvirus group) and Newcastle disease virus (a RNA virus of the myxovirus group). After 20 rain of contact, the mixtures were inoculated on primary cultures of chick embryo fibroblasts. The cytopathic effect was read every day. It was noted that the cytopathic effect of the two D N A viruses was inhibited by a final serum dilution of 1:50. This effect was observed only with the serum samples obtained 3 and 10 days after the onset of treatment, and not with the sample obtained 29 days after cessation of treatment. No inhibitory activity could be demonstrated in the CSF. No activity was noted with Newcastle disease J. ~teurol. Sci., 1971, 12 : 443-458
448
M. RAPPEL et al. TABLE
2
AGAR GEL PROTEIN ELECTROPHORESIS
All the concentrations are expressed in %. Prealbu-
Patient sample
rain
Globulins
Albumin
CSF Case 1 ( J . S . ) No. 1 No. 2
No. 3
1.1 0.4 0.5
27.8 21 2 1716
---3.9 7.8
Case 4 ( J . P . ) No. 1
4.7
31.4
4.9
Normal values
3.9
60.9
6.2 2.2 2.5
3.2
---
42
3.5 2.4
1.8 3.6
----
5.8 3.5
-
3.7
5.2
2.3
I0.0 8.3 ---
11.4 -
5.4 9.4 19.8
10.9 8.1 4,8
2 0 . 7 5.4 . . . . . 14.9 8.9 4 . 5 2 0 . 4 7.6 1 2
-----
l
6.9
.....
7.9
8.9
16.0
4.5
8.9
8.0
1.9
7.4 2.4
8.2
9.7 4.2
3.4
.........
4.4
7.7 4 . 4
1.8
Serum Case 1 (J.S.) 30.9
No. 1
Normal values
59.2
17.4 3.9
2.7
1.8
4.2
4.4
4.3
t12
.......
virus. Inhibition of the cytopathic effect with herpes virus type 1 was positive in serum samples up to a dilution of 1:20,000. Taken together these results indicate that during treatment with IUdR, the serum of the patient, but not the CSF, possessed an inhibitory activity to D N A viruses and not to R N A viruses and that this was probably due to the drug. The level of activity was low as compared to the titres of specific antibodies to herpes virus, and could not have contributed to the results presented in Fig. 1, since the method implies a 1:400 final dilution of serum at the moment when the mixture virus plus serum is inoculated on a tissue culture.
Protein electrophoresis Three samples of CSF (taken on day 28, 41 and 63) and 1 serum sample (day 63) from Case 1, and 1 CSF sample (day 167) from Case 4 were examined by agar gel electrophoresis. The results are shown in Table 2. The main characteristics of these phoretograms are: In the CSF: an enormous increase of all y-globulins with distinct peaks, not only in the fast but also in the slow and very slow fractions, the latter showing the maximal increase. There was no increase of the a-globulins, except in the last CSF sample in Case 1 where the fast a-globulins were increased. There was both a low figure for {~-globulin and an abnormal fll/z ratio in the last CSF sample of Case 1, In serum: only 1 sample was examined, e,l- and o~2-globulins were increased as well as v-globulins which upon fractionation showed the presence of slow y-globulins.
Morphological studies The results of light and electron microscopic observations are summarized in J. neurol. Sci., 1971, 1 2 : 4 4 3 - 4 5 8
---
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
449
TABLE 3 THE PATHOLOGICAL CHANGES OBSERVED IN 4 OF THE 5 CASES REPORTED
Case 1 (J.S.)
Case 2 (C.D.)
t ?
+ + : --
Case 3 (A.L.)
Case 5 (M.D.)
Light - microscopy h a e m o r r h a g i c necrosis perivascular infiltrates microglial nodules C o w d r y type A inclusions
~
ischaemic neurons other observations
? 0
Electron-mieroscopy
herpes-like particles nuclear bodies other structures
_ altered nuclei
~ + --
+ small intranuclear nucleoliform inclusions
+ r?
+ 0
not done
-+ tubules of 75 A diameter
-? --
-k-c--
Table 3. Cases 1 and 4 came to autopsy. In both cases lesions of the cortex and the white matter were seen in the right frontal and temporal lobes and to a lesser degree on the left side in Case 1. In the cortex, many neurons were destroyed and there were areas of mononuclear perivascular cuffing with extensive infiltration by macrophages and reactive astrocytes. In the white matter, there were some areas of demyelination. In both cases, Cowdry type A inclusions could not be detected after a very careful examination. Nuclear bodies (Fig. 2) were observed in the 3 cases studied by electron microscopy. These forms have already been seen in encephalitis (PERIER AND VANDERHAEGHEN 1967) and are known to be related to cellular hyperactivity (BOUTEILLE et al. 1967a). Details concerning the ultrastructural observations made in Cases 2 and 5 are given below. In both cases, there were some changes in glial nuclei suggestive of viral infection with clumping of the chromatin against an irregular nuclear membrane (Fig. 2B). In Case 2 no typical herpetic particles were found inside the nuclei, but very peculiar structures were observed in elongated necrotic cell fragments. These cell fragments were so extensively altered that it was impossible to decide whether they were of nuclear or cytoplasmic origin. At first sight these structures appeared as groups of 2-5 parallel filaments which often had small groups of lattice-like formations located nearby (Fig. 3). At a very high magnification ( x 220,000) it appeared that each filament was in fact a tubule (Fig. 3 insert). Its diameter was of 75 A and two tubules were separated from each other by 190 A. In the transverse section, the tubule was seen at each angle of the polygonal elements of the lattice-like formations. These therefore corresponded to the transverse appearance of the bundles of parallel structures. These were observed in the tissue sections of only one of the 5 blocks studied. In Case 5, typical naked herpes-like particles were detected in some sections of all J. neun,/. Sci., 1971, 12: ,1.4.3. 458
450
M. RAPPEL et al.
Fig. 2. Nuclear bodies. A : One is seen at the right of nucleus (Case 3) and corresponds to the type 2 of BOlrr~aLLE et al. (1967a). B: An unusual form seen in Case 2. There is clumping of chromatin against nuclear membrane. J. neurol. Sci., 1971, 12:44-3 458
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
451
Fig. 3. Case 2. Elongated necrotic fragment of cells contain groups of parallel filaments and a lattice-like formation. Higher magnification in the insert shows that each filament is in fact a tubule located at each angle of a polygonal element.
5 blocks (Fig. 4). They were usually numerous in the nucleus, whereas in the cytoplasm they were sometimes isolated and not enveloped. The diameter of these particles varied from 85-110 m# and they sometimes possessed a well-defined hexagonal pattern. Dense core particles were very often mixed with empty ones as described by MORGANet al. (1959, 1968) and Nix et al. (1968). Most of the infected cells were very necrotic and had disrupted nuclear and cytoplasmic membranes; exact identification of the cellular type was no longer possible in this condition. DISCUSSION OF THE LABORATORY RESULTS
The true incidence of herpes encephalitis, its mortality and the usefulness of idoxuridine therapy have recently been questioned (New Engl. J. Med., Editorial 1967). LEIDER et al. (1965) and Ross AND STEVENSON(1961) have reported relatively low rates of mortality, respectively 25 and 13~. However, it is difficult to determine whether death was caused by herpes meningitis, meningo-encephalitis, encephalomyelitis or acute necrotizing encephalitis. When only ANE is considered the mortality rate is substantially higher, ranging from 50-100~. Most of the patients who survive show both physical and mental abnormalities (MILLER et al. 1966; RAWLS et al. 1966; OLSONet al. 1967). Early diagnosis of ANE is extremely important. The determination of the causal agent is based entirely upon laboratory findings. JOHNSONet al. (1968) have recently Y. neurol. Sci., 1971, 12:443-458
452
M. RAPPEL et al.
Fig. 4. Case 5. Typical naked intranuclear herpes-like particles. Some appear empty, others contain a central core.
e m p h a s i z e d the p r o b l e m of l a b o r a t o r y diagnosis of HSV infections o f the nervous system. Virus i s o l a t i o n from the brain or d e m o n s t r a t i o n o f the viral antigen by i m m u n o f l u o r e s c e n c e stud;e,~ seem to be the most convincing proofs o f the herFetic ,I. m,m'o:. Sci,, 197~. ] 2 : 4 4 3 '..58
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
453
aetiology of ANE. Electron microscopic demonstration of herpes-like particles and a 4-fold rise in complement-fixing (CF) antibodies to HSV have also been used as diagnostic tests. No correlation has ever been made of the results of these various techniques and their relative sensitivity. Negative immunofluorescence tests with successful virus isolation have been reported (MILLER AND Ross 1968) as well as positive electron microscopic studies with failure to isolate the virus (HARLAND et al. 1967). The failure to grow viruses has been explained by a low virus concentration or intense necrosis in the investigated area or by a delay greater than 15 days between the onset of the disease and the time of biopsy (DODGE AND CURE 1956; MAY et al. 1967; OLSON et al. 1967; BERGOUIGNANet al. 1968; MILLER AND ROSS 1968; NOLAN et al. 1970). However, the reasons why the herpes virus was only isolated from 1 out of the 4 patients studied in this investigation are not clear. There are no obvious differences between these cases and the 6 cases studied by other authors and presented in Table 1. The utilisation of the appearance of CF antibodies to HSV as a criterion for the diagnosis of herpes encephalitis can be criticized on the grounds that a latent infection can be reactivated as a secondary effect of another infectious disease with the resultant appearance of CF antibodies. It should be noted that herpetic encephalitis itself is not necessarily a primary infection (LEIDERet al. 1965; RAWLS et al. 1966; TOMUNSON AND M A c C A L L U M 1969). The diagnostic value of herpes-like particles discovered by electron microscopy, although denied by JOHNSON et al. (1968) is advocated by HARLAND e t a [ . (1967) and BERGOUIGNAN¢ta[. (1968). If the discovery of these particles never offers a specific diagnosis of the disease, it must be stressed that since the first observation of herpeslike particles in this disease (RYDEN et al. 1965), they have been foand in 36 out of 39 published cases of ANE (ITABASHIet al. 1966; SWANSONet al. 1966; VANDERHAEGHEN et al. 1966: BOUTEILLEet al. 1967b; CHOU AND CHERRY 1967: HADER ct al. 1967; HARLAND et al. 1967; SEITELBERGERAND SLUGA 1967 ; DELMAS-MARSALETet al. 1968 ; MORECKI AND BECKER 1968; RoY AND WOkMAN 1969; TOGA et al. 1969; HUGHES 1969; MANNWEILER AND COLMANT 1969). The particles were discovered in autopsy material in 26 cases and in brain biopsies in the 10 others. According to the strict criteria of JOHNSON et al., a herpetic aetiology was only definitely proven in our Case 1 where virus was isolated from the brain. The following points favour the herpetic aetiology in the other cases. 1. A strikingly characteristic clinico-pathological pattern, which is typical of ANE. Until now, other viruses have never been unequivocally proven to cause ANE in adults (SCHEID AND JOCHHEIM 1956; DUCKETT et al. 1963; HEATHFIELDet al. 1967). 2. The kinetic neutralizing antibodies to HSV as measured here by the very sensitive method of the constant of virus inactivation by antibody, allowing the differentiation between HSV type 1 and type 2. The neutralizing antibodies are only absent in individuals who have never been infected with HSV. Therefore, if ANE results from a primary herpetic infection, it should cause a rise of antibodies from zero. This was the case in the 2 patients (Cases 2 and 3) from whom early serum samples were obtained. In these patients it can be said that a primary herpetic infection was occurring at the very moment that encephalitis was developing. Case 4 showed high titres of J. neurol. Sci., 1971, 12:443-458
454
M. RAPPEL et al.
both types of antibodies, an indication that recent herpetic infection (recurrent or primary) had taken place, since titres as high as 1 : 32 with the CF antibody test have not been found in non-active herpetic infections in our laboratory. 3. The electron microscopic demonstration of numerous herpes-like particles m Case 5 is the only serious argument for the herpetic aetiology in this patient. In this case as well as in Case 1, light microscopic examination of cerebral tissue showed numerous intranuclear inclusions. Correlation between the electron and light microscopic findings has already been well demonstrated in 10 cases by serial semi-thin, and thin sections (ITABASHI et al. 1966; VANDERHAEGHENet al. 1966; C n o u AND CHERRY 1967; ROY AND WOLMAN 1967): it enhanced the significance of the Cowdry type A inclusions in the diagnosis of viral encephalitis. The presence of a few naked viral particles in the cytoplasm, as in Case 5, has already been mentioned (RVDEN et al. 1965; RoY AND WOLMAN 1967). This fact seems to be related to an acute cell necrosis, consisting of fragmentation of the nuclear envelope and resulting in a mingling of nuclear and cytoplasmic components, as seen after 7 days of growth of HSV in tissue culture (SnlPKEY et al. 1967). The ultrastructural tubules and the CSF electrophoretic abnormalities have never been described in the literature concerning human ANE and merit discussion. The structures observed at the electron microscopic level in Case 2 are similar to the intranuclear filaments demonstrated close to typical herpetic particles in neurons of mice, and in cell culture, infected by HSV type 2 strain (MURPHY et al. 1967: COUCH AND NAHMIAS 1969). The round unit of the lattice formation described by CoucH AND NAHMIAS (1969) in the transverse section has the same diameter as the one observed in Case 2 (120-190/~). These two groups of investigators discussed the microtubular nature of these structures and interpreted them as an abnormal assembly of viral subunits and as viral footprints. They also suggested that these structures have a proteic nature. However, in Case 2, the tubules were never observed near herpes-like particles as ill these experimental studies, and were found only in one restricted area of tissue, as already mentioned by C o u c h AND NAHMIAS 0969), The belief that these structures are somehow related to HSV type 2 is not substantiated in this case, as specific serum antibodies for HSV type 1 were found to be present. The CSF protein electrophoresis was done in only 2 patients (Cases 1 and 4) and the abnormalities seen, especially the enormous increase and fractionation of the v-globulins, have to be compared with the CSF globulin increase which occurs in other nervous system diseases. In tumours or meningitis, the increased permeability of the blood-brain barrier has never given phoretograms with such globulin fractionation. The increase and fractionation of y-globulins seen in multiple sclerosis (MS) and in subacute sclerosing panencephalitis (SSPE) differ from the fast fraction observed in the CSF of patients with acute necrotizing encephalitis. The phoretograms seen here are quantitatively comparable to those seen in some cases of neurosyphilis and trypanosomiasis. The comparison with results obtained in other types of cb_ronic or subacute encephalitis like MS, SSPE, neurosyphilis and trypanosomiasis (LoWENTHAL 1964) indicates that the CF ),-globulins are increased and fractionated in a more or less specific manner. In the only serum sample studied, increase of the s-globulins and fractionation J. neurol. Sci., 197/, 12:443458
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
455
of the 7-globulins was comparable to what was described in SSPE serum. These preliminary results show that further work with determination of immunoglobulin levels in CSF and serum and immunochemical studies of the 7-globulins could throw new light on the immunology of chronic, subacute and acute encephalitis.
DISCUSSION OF THERAPEUTIC RESULTS
There were 3 fatalities in the 11 cases treated by IUdR and analysed in Table 1. The number of cases yielding a 27.2~ mortality rate is insufficient for statistical comparison with previously-reported mortality rates in herpes encephalitis. However, it is interesting to note that in a very recent article, NOLAN et al. (1970) report full recovery in 4 cases, and 2 deaths in 6 patients where herpes encephalitis was diagnosed, and who were treated by intravenous idoxuridine. Cases 5 and 11 (Table 1) and 2 of the cases reported by NOLAN et al. (1970) were similar in that they died and did not even show transient benefit from IUdR therapy. Cases 3 and 9 showed no immediate improvement with the decompression; the condition of Case 1 was aggravated following the operation. However, the improvement in their level of consciousness began 36 to 72 h after IUdR treatment. This was also noticed in Case 4 who was not operated upon, and in Case 7 who only underwent a biopsy. The improvement was slower in Cases 6 and 10, as well as in Case 8 where the quantity of IUdR given was probably inadequate. In Cases 2, 3 and 9, where chemotherapy rapidly followed the operation, it is difficult to assess the part played by the treatment in the recovery. The fact that irt Cases 1 and 10 the brain was sterile after IUdR treatment, and that in Cases 1 and 5 the inclusions, which were so numerous in the pre-IUdR biopsy were not found at autopsy, may be related to IUdR treatment. However, these phenomena have been described in untreated cases of herpes encephalitis (YOUNG et al. 1965; MAY et al. 1967; HUGHES 1969). There is thus no absolute proof but a great deal of presumptive evidence for the effectiveness of IUdR in ANE. It must also be emphasized that TOKUMARU (1968) and TOMLINSONAND MACCALLUM(1970) presented evidences that IUdR has a a therapeutic action on herpes encephalitis induced in guinea-pigs.
CONCLUSION
This study emphasizes the need for a multidisciplinary approach and the collaboration of clinicians, morphologists, virologists and biochemists in attempting to diagnose the aetiology of an encephalitis. In the future early diagnosis should be made with new, rapid and more specific techniques such as immunofluorescence (YAMAMOTO et al. 1965) and, perhaps, negative staining on fresh tissue specimens (WATSONet al. 1963; DOANE et al. 1969). These techniques are not commonly used as they require special equipment and training, but the early diagnosis of ANE may be dependent on their utilization. Lobectomy and decompression should be performedlwhenever possible. Idoxuridine seems to be the first step in the generalization of antiviral chemotherapy: cytosinearabinoside has been recently used in a case of generalized herpes zoster infection J. neurol. Sci., 1971, 12:443458
456
M. RAPPELet al.
(MCKELVEY AND KWAAN 1969) and interferon-inducers may also be of some help. Further clinical and experimental studies will be forthcoming, and the statement by HAYSnOW (1970) that there is an urgent need for interested investigators to pool their experience in a controlled study, must be strongly emphasized.
ACKNOWLEDGEMENTS We wish to thank our colleagues for their valuable clinical and surgical contribution, Drs. O. P6rier and J. Flament for helpful criticism, all the members of the Department of Neuropathology for their collaboration, Mm. Vienne and Conreur and Mme Menu for skilled electron microscopic technical assistance and our colleagues of the Neurochemical Department of the Born-Bunge Foundation who performed the electrophoretic investigations. Our thanks are also due to Dr. Schrire of the Research Institute of Smith, Kline and French Ltd. for the kind and rapid supply of IUdR.
SUMMARY Five cases of acute necrotizing encephalitis treated by I U d R are presented; 4 were also treated surgically. The aetiotogy of the disease was investigated by brain biopsy (in 4 cases), electron microscopy (in 3 cases) and virological studies (in 5 cases). Evidence of herpes virus infection was based upon electron microscopy (1 case), virus isolation (1 case) and the rise of complement-fixing antibodies and kinetic neutralizing antibodies (4 cases). This latter technique is specific for herpes virus hominis type 1. In 1 case, structures suggestive of incomplete viruses were seen by electron microscopy. In 2 cases, electrophoresis of the cerebrospinal fluid showed an abnormal pattern. Two patients died. The importance of a multidisciplinary approach is stressed. The problem of early diagnosis and the possible usefulness of chemotherapy are discussed.
REFERENCES ANoy. (1967) Encephalitis caused by herpes simplex virus (Editorial), New Engl. J. Med., 277: 1315. BELLANTI,J. A., G. H. GUIN, R. M. GRASSIAND E. C. OLSON(1968) Herpes simplex encephalitis. Brain biopsy and treatment with 5-iodo-2'-deoxyuridine, J. Pediat., 72: 266. BENNETT,D. R., C. H. z~r RHEINAND T. S. ROBERTS(1962) Acute necrotizing encephalitis, Arch. Neurol. (Chic.), 6: 96. BEROOUmNAN,M., J. JtmIEN, C. VrrAL AND M. DUVERT(1968) Notre experience de l'enc6phalite aigufi n6crosante. Mise en 6vidence du virus herp6tique an microscope 61ectronique: Presse mdd., 76: 709. BOGAERT,L. VAN,J. RADERMECKERANDJ. DEVOS(1955) Sur une observation mortelle d'enc6phalite aJgu~ n6crosante, Rev. neurol., 92: 329. BotrrEILLE,M., S. R. KALIFATANDJ. DELARtrE(1967a) Ultrastructural variations of nuclear bodies in human diseases, J. Ultrastr. Res., 19: 474. BOUTEILLE,M., C. VEORENNE,A. M. DuperY-CoiNANDJ. DELARUE(1967b) Inclusions intranucl6aires paxticuli~res observ6es au microscope 61ectronique dans un cas d'enc~phalite aigu6 n6crosante, Ann. Anat. path., 12: 403. BREEDEN,C. J., T. C. HALLAnt) H. R. TYLER(1966) Herpes simplex encephalitis treated with systemic 5-iodo-2'-deoxyuridine, Ann, intern. Med., 65: 1050. J. neurol. Sci., 1971, 12:443~-58
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
457
BRIHAYE, J. (1959) 12tude des enc6phalites herp6tiques et des enc6phalites n6crosantes aiguEs (Th6se), Acta neurol, psychiat, belg., 59: 1. BUCKLEY, T. F. AND F. O. MACCALLLrM(1967) Herpes simplex virus encephalitis treated with idoxuridine, Brit. reed. J., ii: 419. CAMPBELL, A. M. G. (1969) Herpes encephalitis, Part 1 (The clinical picture), Postgrad. med. J., 45: 382. CHou, S. H. AND J. D. CHERRY (1967) Ultrastructure of Cowdry type A inclusions in human herpes simplex encephalitis, Neurology (Minneap.), 17: 575. COUCH, E. F. AND A. J. NAHM1AS (1969) Filamentous structures of type 2 herpes virus hominis infection of the chorio-allantoic membrane, J. Virol., 3 : 228. DELMAS-MARSALET, P. M., M. BERGOUIGNAN, R. CASTAIGNE, P. DU PASQUIER AND C. VITAL (1968) Fr6quence actuelle des enc6phalites herp6tiques de l'adulte: sur une s6rie personnelle de huit cas confirm6s par la microscopie 6lectronique, Bull. Acad. nat. Mdd., 152: 316. DOANE, F. W., N. ANDERSON, A. ZBITNEW AND A. S. RHOBES (1969) Application of electron microscopy to the diagnosis of virus infections, Canad. rned. Ass. J., 100: 1043. DODGE, P. R. AND C. W. CURE (1956) Acute encephalitis with intranuclear cellular inclusions: a non fatal case of probable herpetic etiology diagnosed by biopsy, New Engl. J. Med., 255: 849. Dt:CKETT, S., R. KELLY AND H. C. GRANT (1963) Un cas de mgningo-enc6phalite n6crosante avec inclusions intra-cellulaires dfi ~ une infection psittacosique possible, Rev. neurol., 108: 899. DUDGEON, J. A. (1969) Herpes encephalitis, Part 2 (Pathology of herpes encephalitis), Postgrad. reed. J., 45: 386. EVANS, A. D., O. P. GRAY, M. H. MILLER, E. R. VERRIERJONES, R. D. WEEKS AND C. E. C. WELLS (1967) Herpes simplex encephalitis treated with intravenous idoxuridine, Brit. reed. J., ii: 407. HADER, W., M. BAYATPOUR, G. DEMPSTER AND B. ROZDILSKY (1967) Herpes simplex encephalitis. Two fatal cases, Canad. med. Ass. J., 96: 1565. HANSHAW, J. B. (1970) Idoxuridine in herpes virus encephalitis (Editorial), New Enfl. J. Med., 282: 47. HARLAND, W. A., J. H. ADAMS AND D. MCSEVENEY(1967) Herpes simplex particles in acute necrotizing encephalitis, Lancet, ii: 581. HEATHFIELO, K. W. G., R. PILSWORTH, B. J. WALL AND J. A. N. CORSELLIS (1967) Coxsackie B5 infections in Essex 1965, with particular reference to the nervous system, Quart. J. ?.~led., 36: 579. HERRMANN, JR.~ E. C. (1961) Plaque inhibition test for detection of specific inhibitors of D N A containing viruses, Proc. Soc. exp. Biol. (N.Y.), 107: 142. HUGHES, J. T. (1969) Pathology of herpes simplex encephalitis. In: C. W. WHITTY, J. T. HUGHES AND F. O, MCCALLUM (Eds.), Virus Diseases and the Nervous System, Blackwell, Oxford and Edinburgh, p. 29. ITABASm, H. H., D. M. BASSAND J. R. McCULLOCH (1966) Inclusion body of acute inclusion encephalitis, Arch. Neurol. (Chic.), 14: 493. JOHNSON, R. T., L. C. OLSON AND E. L. BUESCHER (1968) Herpes simplex virus infections of the nervous system. Problems in laboratory diagnosis, Arch. Neurol. (Chic.), 18: 260. KAUFMAN, H. E. (1963) Chemotherapy of virus disease, Chemotherapia, 7: 1. EEIDER, W., R. L. MAGOFFIN, E. H. LENNETTE AND L. N. R. LEONARDS (1965) Herpes simplex virus encephalitis: its possible association with reactivated latent infection, New Engl. J. Med., 273 : 341. LOWENTHAL, A. (1964) Agar Gel Electrophoresis in Neurology, Elsevier, Amsterdam. MCKELVEY, E. M. AND H. C. KWAAN (1969) Cytosine arabinoside therapy for disseminated herpes zoster in a patient with IgG pyroglobulinemia, Blood, 34: 706. MANNWEILER, K. AND H.-J. COLMANT (1969) Ultrastructural data in cases of necrotizing encephalitis without inclusion bodies. Pathogenesis and aetiology of demyelinating diseases, Int. Arch. Allergy, 36:515. MARSHALL, W . J . S . (1967) Herpes simplex encephalitis treated with idoxuridine and external decompression, Lancet, ii: 579. MAXWELL, E. (1963) Treatment of herpes keratitis with 5-iodo-2'-deoxyuridine ([DU). A clinical evaluation of 1500 cases, Amer. J. Ophthal., 56: 57l. MAY, G., R. DAHN AND K. REUSS (1967) Beitrag zum Nachweis yon Herpesvirus bei Enzephalitis, Dtsch. reed. Wschr., 92: 793. MILLER, J. D. AND C. A. C. Ross (1968) Encephalitis: a four-year survey, Lancet, i: 1121. MILLER, J. K., F. HESSER AND V. N. TOMPKINS (1966) Herpes simplex encephalitis. Report of 20 cases, Ann. intern. Meal., 64: 92. MORECKr, R. AND N. H. BECKER (1968) Human herpes virus infection. Its fine structure identification in paraffin-embedded tissue, Arch. Path., 86: 292.
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MORGAN, C., H. M. ROSE AND B. MEDNIS (1968) Electron microscopy of herpes ~irnplex virus, Part 1 (Entry), J. Virol., 2: 507. MORGAN, C., H. M. ROSE, M. HOLDEN AND E. P. JONES (1959) Electron microscopk: observations on the development of herpes simplex virus, J. exp. Med., 110: 643. MURPHY, F. A., A. K. HARRISONAND S. G. WH1TFIELD(1967) Intranuclear formation of filaments in herpes virus hominis infection of mice, Arch. ges. Viru.s~)rsch., 21 : 463. NII, S. H., C. MORGANANt) H. M. ROSE (1968) Electron microscopy of herpes simplex virus, Part 2 (Sequence of development), J. Virol., 2: 517. NOLAN, D. C., M. M. CARRtrrHERSAND A. M. LERNER(1970) Herpes virus hominis encephalitis in Michigan. Report of thirteen cases, including six treated with idoxuridine, New Engl. J. Med., 282: 10. OLSON, L. C., E. L. BUESCHER,M. S. ARENSTEINAND P. D. PARKMAN(1967) Herpes vwus infectio~ls of the human central nervous system, New Engl. J. Med., 277: 1271. PERIER, O. AND J. J. VANDERHAEGHEN(1967) Subacute sclerosing leucoencephalitis. Electron microscopic finding in 2 cases with inclusion bodies, Acta neuropath. (Berl.), 8: 362. RAPPEL, M. AND J. BRmAVE (1969) Traltement de l'enc6phalite n6crosante herp6tique par l'idoxuridine, Rev. neurol., 121: 93. RAWLS, W. E., P. J. DYCK, D. W. KLASS, H. D. GREER AND E. C. HERRMANY(1966)Encephalitis associated with. herpes simplex virus, Ann. intern. Med., 64: 104. RAWLS, W. E., D. LAUREL;J. D. MELNICK, J. M. GLtCKSMANAND R. H. KAUFMAN('1968) A search for viruses in smegma, premalignant and early malignant cervical tissues, the isolation of herpes virus with distinct antigenic properties, Amer. J. Epidem., 87: 647. Ross, C. A. C. AND J. STEVENSON(1961) Herpes simplex meningoencephalitis, Lancet. ii: 682. RoY, S. AND L. WOLMAN(1969) Electron microscopic observation on the virus particles in herpes simplex encephalitis, J. clin. Path., 22: 51. RYDEN, F. W., H. L. MOSES, C. E. GANOTTEAND D. L. BEAVER(1965) Herpetic (inclusion-body) encephalitis, Sth. reed. J., 58: 903. SCHEID, W. AND K. A. JOCHHEtM (1956) Akute Encephalomyelitis und Virus der lymphocytfiren Choriomeningitis, Nervenarzt, 27: 385. SEITELBERGER,F. AND E. SLUGA (1967) Acute hemorragic leucoencephalitis of herpes simplex virus type, J. Neuropath. exp. Neurol., 26: 124. SHtPKEY, F., R. A. ERLANDSON,R. B. BAILEY, V. I. BABCOCK AND C. M. SOUTHAM(1967) Virus biographies, Part 2 (Growth of herpes simplex virus in tissue culture), Exp. tool. Path., 6: 39. SWANSON, J. L., J. E. CRAtGHEADAND E. S. REYNOLD(1966) Electron microscopic observation on herpes virus hominis (herpes simplex virus). Encephalitis in man, Lab. Invest., 15: 1981. TOGA, M., M. F. TRtr'IER AND H. PAVAN(t969) Enc6phalite aigu~ n6crosante. A p r o p o s de quatre observations anatomo-cliniques, Ann. Anat. path., 14: 165. TOKUMARU, T. (1968) The protective effect of different immunoglobulins against herpetic encephalitis and skin infection in guinea pigs, Arch. ges. Virusforsch., 22: 332. TOMLINSON,A. H. AND F. O. MACCALLUM(1969) Herpes simplex encephalitis: virological diagnosis. In: C. W. WrftTTY, J. T. HUGHESAND F. O. MACCALLUM(Eds.), Virus Diseases aud the Nervous System, Blackwell, Oxford and Edinburgh, p. 21. TOMLtNSON, A. F[. AND F. O. MACCALLUM(1970) The effect of idoxyuridine on herpes simplex virus encephalitis in animals and man, Proceedings of the Second Conference on Antiviral Substances, N.Y. Acad. Sci., In press. VANDERHAmrrEN,J. J., O. PERtER ANt) Y. BOSSAERT(1966) Acute necrotizing encephalitis. Observations of herpes-like particles by electron microscopy, Path. europ., 1 : 29. WATSON, A. H., W. C. RUSSELLAND P. WINDY(1963) Electron microscopy particle counts on herpes virus using the phosphotungstate negative staining technique, Virology, 19: 250. WTEME, R. J. (1959) Studies on Agar Gel Electrophoresis, Arscia Editions, Brussels. YAMAMOTO,T., S. OTAMI AND H. SHIRAKI (1965) A study of the evolution of viral inti~ction in experimental herpes simplex encephalitis and rabies by means of fluorescent antibody, Aeta nearopath. (Berl.), 5: 288. YOUNG, G. F., D. L. KNOX AND P. R. DODGE (1965) Necrotizing encephalitis and chorioretinitis in a young infant, Arch. Neurol. (Chic.), 13: 15.
J. neurol. Sci., 1971, 12:443-458
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Diagnosis and Treatment of Herpes Encephalitis A Multidisciplinary Approach M. RAPPEL, M. DUBOIS-DALCQ, S. S P R E C H E R , L. T H I R Y , A. L O W E N T H A L , S. PELC AND J. P. THYS Departments of Neurosurgery, Pediatric Neurology, Intensive Care Unit, Neuropathology and Electron Microscopy, Faculty of Medicine, Free University of Brussels, Pasteur Institute of Brabant, Brussels, and Department of Neurochemistry, Born-Bange Foundation, Berchem-Antwerp ( Bel,¢ium)
(Received 3 April, 1970)
INTRODUCTION Acute necrotizing encephalitis (ANE) is a well-recognized clinico-pathological entity (VAN BOGAERF et al. 1955; BRIHAYE 1959: BENNETT et al. 1962; BERGOUIGNANet al. 1968; CAMPBELL 1969; DUDGEON 1969) with a high mortality rate. However, the clinical diagnosis is only presumptive and has to be supported by laboratory investigations. There is growing evidence that most of the cases of A N E are due to herpes simplex virus (HSV) which is, like other DNA-viruses, inhibited in its growth by a thymidine analogue, the 5-iodo-2'-deoxyuridine (SKF14.287, idoxuridine, I U d R , I D U , I D U R ) as shown by HERRMANN (1961). This drug is widely used in the treatment of herpetic keratitis (KAUFMANN 1963; MAXWELL 1963). I U d R has also recently been introduced to treat herpes encephalitis, with variable results (BREEDEN et al. 1966: BUCKLEY AND MACCALLUM 1967: EVANS et al. 1967: MARSHALL 1967; BELLANTI et al. 1968; TOMLINSON AND MACCALLUM 1969). These reports raise several questions related to early diagnosis and appreciation of the efficacy of the treatment. The diagnosis is made only in the most severe cases, referred to the hospitals so that the true incidence of the disease is still unknown. A prospective multidisciplinary approach of the diagnosis should enhance further knowledge of the natural history and the aetiology of necrotizing encephalitis. This in turn will help the clinician to assess the relative usefulness of different forms of treatment. The following is a report of 5 cases of acute necrotizing encephalitis where such an approach was attempted. This work was supported by Grant No. 570 A4 from the National Multiple Sclerosis Society,U.S.A. (M. D.-D.), by a grant from the Scientific Medical Research Fund (S.S. and L.T.) and by a grant from the Belgian Ministery of National Education (A.L.). M. Dubois-Dalcq: Charge de Recherches F.N.R.S. Reprint requests to M. Rappel, Clinique Neurochirurgicale, Institut Bordet, 1, rue Hdger-Bordet, 1000 Brussels (Belgium). J. neurol. Sci., 1971, 12:443 458
0
-- (L)
. (L) .....
(R)
0
0
N.D. ANE
N.D.
N.D.
(R)
(kl
(L)
0
(R)
--
(L)
-
.
.
.
N.D.
0 N.D.
N.D.
(R)
14fll 14 very poor
?
500(56 64) poor
.
(R)
64 128 64 N.D. biopsy (7}
.
.
-
-:-
F 41 ~
(R)
(R~
200(15 i8~ good
64 256 (CSFI biopsy (12! ,.lecomr, !lSi
N.D. ANE
N.D.
:
-
0
(L)
(R)
M 13 --
~20(t5-2{}: goo;t
t6 64 N.D, biopsy i(~)and(2!~
N.D.
N.D. N.D.
N.D.
N.D.
(R)
0
?
0
F 11 12 ?
.' 3 5) ~to. ,.tled (5!
( brain
64
o
M 47
C~ase 8 Case O" Case 10 . . . . . Cas'e 11 ( B U C K L E Y (MARSHALL (BELLANT1 (W.B.) et al. 1967) 1967) et al. 1968) (TOMUNSON et al. 1969)
8 128 N.D. biopsy (56)
N.D. .
0
(R~
.
N.D.
.
N.D.
.
N.D. ANE
.
(L)
-- (L)
F 8
-Case 7 " (EVANS et al. 1967)
N.D. ANE
- (L)
(R)
'(R)
-
.
~- ( L )
-7
8 64 N.D. N.D. lobect, biopsy [7) decomp~.~'.~} decompr.¢'~!
0
0
(R) ANE
N.D.
-
-
M 34 ~
Cas~6 (BREEDEN et al. 1966)
500{~8 22) 500{9-13) 500(10 17i good no. died ( 191 good
32 128 N.D. N.D.
N.D.
N.D.
(L) N.D.
:
- (L)
-
•-
0
0
F 65 ?
R: righti L- iei:{TiN))_i not ~ione: AN E i acute necrotizing encephalitis
0 4 16 16 N.D. N.D. ]obectom3 decompress t9) (14) (27~ 500(16 24) 3 2 0 ( 1 . - - 1 ) 500(27 34~ moderate good moderate -died (63)
16 128 N.D.
0
?
N.D.
~
N.D. ANE
N.D.
-
N.D. ANE
(Rt uninterpretable
(R)
:
0
(L)
- (L)
F 45 _
case 4 ..... -C-ase 5 (J.P.) (M.D.) Nov. 1968 M a y 1969
Complement fixating antibodies given as the reciprocal of the end point dilution. ~' In parentheses: day of the d Sease (day 0 day of the acute onset).
5ibi, r-ei,iation,Ti
IDU(mgkg) > Recovery~,
Abnormal air study Optic microsc. Virus seen by EM Virus isolation (brain) Herpes antibodies fCFT)a Fluoresc. studies Surgical proced,are"
Headache Fever Epilepsy Hemiplegia or hemiparesis Meningism Confusion or psych, disturb. Coma CSF lymphocytic pleocytosis A b n o r m a l EEG Abnormal angiography Brain scan
+ (R)
M 56 ~
58
F 12
M
Age
(years)
Sex
case 3 (A.L.) Aug. 1968
Case 2 (C.D.) July 1968
Cas'e 1 (J.S.) Dec. 1967
TABLE 1
suRwY oF 11 CASESREeO~TED
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
445
PATIENTS AND TREATMENT
During a 17-month period (December 1967-May 1969) 6 cases of encephalitis were referred to the University Hospital. A provisional diagnosis of necrotizing encephalitis was made on clinical grounds in 5 of them. One patient (Case 3, A.L.) was first thought to have mumps encephalitis as he presented bilateral parotid enlargement and mumps had been diagnosed in 4 children from his village. ANE was later confirmed. Herpes virus was shown to be involved in this patient as well as in 4 other cases. The present report deals only with these 5 cases; the 6th one, where herpes virus was not demonstrated, has been described in a preliminary communication (RAPPEL AND BRIHAYE 1969). Four patients underwent temporal lobectomy and decompression ; the 5th patient received no operative therapy. The 5 patients were treated with intravenous idoxuridine. Table 1 summarizes the present 5 cases as well as the 6 other cases of herpes encephalitis treated with IUdR which have been reported in detail in the literature. Day 0 refers to the day of the acute onset of the disease. Like all the pyrimidine compounds, IUdR, in its neutral, lactame form, shows poor solubility in water. It is best dissolved in a basic solution. Therefore IUdR (500 mg/ 100 ml) was dissolved in a 5 g/100 ml glucose solution solution alkalinized by sodium bicarbonate (100 mg/100 ml). In adults the total dose of IUdR was 500 mg/kg body weight. Transient and moderate signs of hepatic toxicity and depression of bone marrow function were encountered in 4 of the patients. Cases 3 and 4 also showed a severe but transient diffuse alopecia. The 12-year-old patient (Case 2) was given 320 mg IUdR/kg body weight. No sign of toxicity was detected. Details concerning the evolution of the illness in the patients are given below.
Case I (J.S.) was not helped by the surgical procedure and became rapidly comatose and unresponsive to any stimuli. He remained pyrexial. [UdR therapy was begun 5 days after the operation. Thirty hours later, the patient responded to light stimuli and his temperature dropped. The patient awakened progressively and started speaking again. The improvement lasted 1 month, and then the patient's condition slowly deteriorated. He died on day 63 of severe bilateral bronchopneumonia. ('ase 2 (('.D.) was pyrexial, comatose and hemiplegic. Surgery immediately relieved the motor signs but the child remained confused and disorientated. IUdR treatment was started 24 hours after operation. Within 3 days, the child was greatly improved, with only a mild memory defect. [n the following months, she completely recovered her memory and her school performance returned to normal. Case 3 (A.L.) had become progressively comatose with only EEG signs of a focal lesion. There was no immediate improvement after surgery and IUdR treatment was started a few hours later. After 3 days of treatment, there was clinical improvement which continued slowly. Upon discharge, the patient was able to read and to calculate, but his mental capacity was below normal. One month after the termination of treatment, he presented a transitory thrombocytopenic purpura of the lower extremities and anti-platelet antibodies were demonstrated. Case 4 (J.P.) was pyrexial, disorientated and showed jargon aphasia on admission. Ancillary investigations revealed a left temporal mass and a rise in herpes antibodies. Within a few days, the patient became almost completely mute and then comatose. Forty-eight hours after IUdR treatment, the fever had disappeared and the patient started to speak again. Repeat arteriography and brain scan revealed disappearance of the temporal mass. The patient was discharged with. a moderate memory defect and a slight residual aphasia. The,'e has been continuous improvement with respect to each of these deficits. ('m'" 5 / M . D . ) remained deeply com~.tose after operation and IUdR treatment. She died on day 19. J. neurol. Sci., 1971, 12:443 458
446
M. RAPPELet al.
LABORATORYMETHODS Virus isolation Cerebrospinal fluid (CSF), cerebral biopsies and post-mortem brain fragments were kept at 0°C and transported to the laboratory within I-2 it after removal. Inoculation of these samples was performed intracerebrally on new-born mice, in the chorioallantoic membrane of embryonated eggs, on a human continuous cell line (KB) and on primary monkey kidney cultures. These procedures made it possible to grow poliomyelitis, Coxsackie and ECHO-viruses, as well as adenoviruses and herpes viruses. Antibody studies Several samples of sera were obtained from each patient at various stages of the disease. Antibodies were determined by a complement fixation test (CFT) using the following virus antigens: measles, mumps, Coxsackie B types 1-5, adenoviruses and herpes. In addition, neutralizing antibodies to herpes simplex hominis type 1 and 2 were assayed by a specific method (RAWLS et al. 1968). This recently-introduced method determines the rate of inactivation of each serotype by the antibodies present in the patient's sera, permitting discrimination between the antibodies to the two types of herpes virus. The results are given by the constant of inactivation, K. Protein electrophoresis CSF and serum proteins were examined by W|EME'S agar gel electrophoresis technique (1959) and the results were analyzed quantitatively according to LOWENTHAL (1964). Histology Fragments for light microscopy were immediately fixed in 10}~ formaldehyde, embedded in paraffin and stained by haematoxylin-eosin (H-E) and Nissl methods. Specimens for electron microscopy (EM) were fixed in 4.2% glutaraldehyde, postfixed in osmium, embedded in Vestopal or Epon, cut with glass on a LKB microtome and stained by uranyl acetate and by Karnovsky's lead mixture. Specimens were then studied with a Siemens Elmiskop 1.
LABORATORY RESULTS
Virus isolation The isolation was attempted from temporal lobe biopsies of Cases I, 2, 3 and 5. Only the brain fragment, obtained 9 days after the onset of the disease, of Case 1 yielded a positive viral culture. All methods employed gave positive results with this fragment: a cytopathic effect in tissue culture, pocks in the chorioallantoic membrane of chick embryo and the death of baby mice. These effects were completely neutralized by a rabbit antiserum to herpes simplex, an indication that there was no other virus associated. The other biopsy samples were obtained 9, 16 and 27 days respectively after onset of the disease. In Case 1, fragments from both temporal lobes obtained at autopsy were also studied but failed to show any infectivity. Since J. neurol. Sci., 197~, 12:443 458
447
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
these fragments were larger than those of the previously-performed biopsy and were taken from several parts of the brain, the negative results suggested that the brain had been sterilized by IUdR.
Antibody studies None of the patients showed any increase in antibodies to adenovirus, Coxsackie or measles virus. An increase in antibodies to HSV was detected in Cases 1, 2 and 3 (Fig. 1) by both the CF and kinetic neutralization tests. With the latter test, there was no modification of antibody levels to herpes virus 2 (results not shown), but only to type 1 indicating that an infectious process due to this virus was developing. In Case 4, the levels of CF and neutralizing antibodies were already high at the time of collection of the first serum sample (on day 12). There was a late peak in the elevation of neutralizing antibodies, several weeks after completion of the treatment. In Case 5, the only serum sample was obtained 6 days after the beginning of the disease and no antibodies to herpes virus were present.
CF'
lIJ.S) IUdR~ , 2(C.Ol ~IUdR 16
256 128
, i
24/ / /
/I
3 AL)
~
T5 21
&(j.p}
i
e
16
8
.
,~
IU~dR ]p~
]K
1822 ] ~, " ', ~ ~~ ,
I 18
. "HI"/ i
1~
x
27 3 L
16
lO
,,,/
..8 6
2 0 Days
x ~ 5 10 f5 20 3550 5 10 15 20 25 30
/ st, 5 10 1,5 20 25
2 35
5 tO 15 20 4 5 1 6 0 ~ 0
Fig. I. 0 - - @ : titres of C F antibodies to herpes simplex virus expressed as the reciprocal of the greatest dilution showing positive results; x - - - × : neutralizing antibodies to herpes simplex virus type I, expressed as the constant of inactivation (K) of the virus.
It was considered that the presence of I U d R in blood might contribute to the activity found in the neutralizing test which may measure an inhibition &virus growth by I U d R as well as measuring the destruction of infectivity by antibodies. In order to assess whether I U d R in its active form could be detected in serum or in CSF, serial dilutions of samples from Case 1 were mixed with 100 plaque-forming units (PFU) of several viruses: pseudorabies virus (a D N A virus of the herpes group), vaccinia virus (a D N A virus of the poxvirus group) and Newcastle disease virus (a RNA virus of the myxovirus group). After 20 rain of contact, the mixtures were inoculated on primary cultures of chick embryo fibroblasts. The cytopathic effect was read every day. It was noted that the cytopathic effect of the two D N A viruses was inhibited by a final serum dilution of 1:50. This effect was observed only with the serum samples obtained 3 and 10 days after the onset of treatment, and not with the sample obtained 29 days after cessation of treatment. No inhibitory activity could be demonstrated in the CSF. No activity was noted with Newcastle disease J. ~teurol. Sci., 1971, 12 : 443-458
448
M. RAPPEL et al. TABLE
2
AGAR GEL PROTEIN ELECTROPHORESIS
All the concentrations are expressed in %. Prealbu-
Patient sample
rain
Globulins
Albumin
CSF Case 1 ( J . S . ) No. 1 No. 2
No. 3
1.1 0.4 0.5
27.8 21 2 1716
---3.9 7.8
Case 4 ( J . P . ) No. 1
4.7
31.4
4.9
Normal values
3.9
60.9
6.2 2.2 2.5
3.2
---
42
3.5 2.4
1.8 3.6
----
5.8 3.5
-
3.7
5.2
2.3
I0.0 8.3 ---
11.4 -
5.4 9.4 19.8
10.9 8.1 4,8
2 0 . 7 5.4 . . . . . 14.9 8.9 4 . 5 2 0 . 4 7.6 1 2
-----
l
6.9
.....
7.9
8.9
16.0
4.5
8.9
8.0
1.9
7.4 2.4
8.2
9.7 4.2
3.4
.........
4.4
7.7 4 . 4
1.8
Serum Case 1 (J.S.) 30.9
No. 1
Normal values
59.2
17.4 3.9
2.7
1.8
4.2
4.4
4.3
t12
.......
virus. Inhibition of the cytopathic effect with herpes virus type 1 was positive in serum samples up to a dilution of 1:20,000. Taken together these results indicate that during treatment with IUdR, the serum of the patient, but not the CSF, possessed an inhibitory activity to D N A viruses and not to R N A viruses and that this was probably due to the drug. The level of activity was low as compared to the titres of specific antibodies to herpes virus, and could not have contributed to the results presented in Fig. 1, since the method implies a 1:400 final dilution of serum at the moment when the mixture virus plus serum is inoculated on a tissue culture.
Protein electrophoresis Three samples of CSF (taken on day 28, 41 and 63) and 1 serum sample (day 63) from Case 1, and 1 CSF sample (day 167) from Case 4 were examined by agar gel electrophoresis. The results are shown in Table 2. The main characteristics of these phoretograms are: In the CSF: an enormous increase of all y-globulins with distinct peaks, not only in the fast but also in the slow and very slow fractions, the latter showing the maximal increase. There was no increase of the a-globulins, except in the last CSF sample in Case 1 where the fast a-globulins were increased. There was both a low figure for {~-globulin and an abnormal fll/z ratio in the last CSF sample of Case 1, In serum: only 1 sample was examined, e,l- and o~2-globulins were increased as well as v-globulins which upon fractionation showed the presence of slow y-globulins.
Morphological studies The results of light and electron microscopic observations are summarized in J. neurol. Sci., 1971, 1 2 : 4 4 3 - 4 5 8
---
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
449
TABLE 3 THE PATHOLOGICAL CHANGES OBSERVED IN 4 OF THE 5 CASES REPORTED
Case 1 (J.S.)
Case 2 (C.D.)
t ?
+ + : --
Case 3 (A.L.)
Case 5 (M.D.)
Light - microscopy h a e m o r r h a g i c necrosis perivascular infiltrates microglial nodules C o w d r y type A inclusions
~
ischaemic neurons other observations
? 0
Electron-mieroscopy
herpes-like particles nuclear bodies other structures
_ altered nuclei
~ + --
+ small intranuclear nucleoliform inclusions
+ r?
+ 0
not done
-+ tubules of 75 A diameter
-? --
-k-c--
Table 3. Cases 1 and 4 came to autopsy. In both cases lesions of the cortex and the white matter were seen in the right frontal and temporal lobes and to a lesser degree on the left side in Case 1. In the cortex, many neurons were destroyed and there were areas of mononuclear perivascular cuffing with extensive infiltration by macrophages and reactive astrocytes. In the white matter, there were some areas of demyelination. In both cases, Cowdry type A inclusions could not be detected after a very careful examination. Nuclear bodies (Fig. 2) were observed in the 3 cases studied by electron microscopy. These forms have already been seen in encephalitis (PERIER AND VANDERHAEGHEN 1967) and are known to be related to cellular hyperactivity (BOUTEILLE et al. 1967a). Details concerning the ultrastructural observations made in Cases 2 and 5 are given below. In both cases, there were some changes in glial nuclei suggestive of viral infection with clumping of the chromatin against an irregular nuclear membrane (Fig. 2B). In Case 2 no typical herpetic particles were found inside the nuclei, but very peculiar structures were observed in elongated necrotic cell fragments. These cell fragments were so extensively altered that it was impossible to decide whether they were of nuclear or cytoplasmic origin. At first sight these structures appeared as groups of 2-5 parallel filaments which often had small groups of lattice-like formations located nearby (Fig. 3). At a very high magnification ( x 220,000) it appeared that each filament was in fact a tubule (Fig. 3 insert). Its diameter was of 75 A and two tubules were separated from each other by 190 A. In the transverse section, the tubule was seen at each angle of the polygonal elements of the lattice-like formations. These therefore corresponded to the transverse appearance of the bundles of parallel structures. These were observed in the tissue sections of only one of the 5 blocks studied. In Case 5, typical naked herpes-like particles were detected in some sections of all J. neun,/. Sci., 1971, 12: ,1.4.3. 458
450
M. RAPPEL et al.
Fig. 2. Nuclear bodies. A : One is seen at the right of nucleus (Case 3) and corresponds to the type 2 of BOlrr~aLLE et al. (1967a). B: An unusual form seen in Case 2. There is clumping of chromatin against nuclear membrane. J. neurol. Sci., 1971, 12:44-3 458
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
451
Fig. 3. Case 2. Elongated necrotic fragment of cells contain groups of parallel filaments and a lattice-like formation. Higher magnification in the insert shows that each filament is in fact a tubule located at each angle of a polygonal element.
5 blocks (Fig. 4). They were usually numerous in the nucleus, whereas in the cytoplasm they were sometimes isolated and not enveloped. The diameter of these particles varied from 85-110 m# and they sometimes possessed a well-defined hexagonal pattern. Dense core particles were very often mixed with empty ones as described by MORGANet al. (1959, 1968) and Nix et al. (1968). Most of the infected cells were very necrotic and had disrupted nuclear and cytoplasmic membranes; exact identification of the cellular type was no longer possible in this condition. DISCUSSION OF THE LABORATORY RESULTS
The true incidence of herpes encephalitis, its mortality and the usefulness of idoxuridine therapy have recently been questioned (New Engl. J. Med., Editorial 1967). LEIDER et al. (1965) and Ross AND STEVENSON(1961) have reported relatively low rates of mortality, respectively 25 and 13~. However, it is difficult to determine whether death was caused by herpes meningitis, meningo-encephalitis, encephalomyelitis or acute necrotizing encephalitis. When only ANE is considered the mortality rate is substantially higher, ranging from 50-100~. Most of the patients who survive show both physical and mental abnormalities (MILLER et al. 1966; RAWLS et al. 1966; OLSONet al. 1967). Early diagnosis of ANE is extremely important. The determination of the causal agent is based entirely upon laboratory findings. JOHNSONet al. (1968) have recently Y. neurol. Sci., 1971, 12:443-458
452
M. RAPPEL et al.
Fig. 4. Case 5. Typical naked intranuclear herpes-like particles. Some appear empty, others contain a central core.
e m p h a s i z e d the p r o b l e m of l a b o r a t o r y diagnosis of HSV infections o f the nervous system. Virus i s o l a t i o n from the brain or d e m o n s t r a t i o n o f the viral antigen by i m m u n o f l u o r e s c e n c e stud;e,~ seem to be the most convincing proofs o f the herFetic ,I. m,m'o:. Sci,, 197~. ] 2 : 4 4 3 '..58
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
453
aetiology of ANE. Electron microscopic demonstration of herpes-like particles and a 4-fold rise in complement-fixing (CF) antibodies to HSV have also been used as diagnostic tests. No correlation has ever been made of the results of these various techniques and their relative sensitivity. Negative immunofluorescence tests with successful virus isolation have been reported (MILLER AND Ross 1968) as well as positive electron microscopic studies with failure to isolate the virus (HARLAND et al. 1967). The failure to grow viruses has been explained by a low virus concentration or intense necrosis in the investigated area or by a delay greater than 15 days between the onset of the disease and the time of biopsy (DODGE AND CURE 1956; MAY et al. 1967; OLSON et al. 1967; BERGOUIGNANet al. 1968; MILLER AND ROSS 1968; NOLAN et al. 1970). However, the reasons why the herpes virus was only isolated from 1 out of the 4 patients studied in this investigation are not clear. There are no obvious differences between these cases and the 6 cases studied by other authors and presented in Table 1. The utilisation of the appearance of CF antibodies to HSV as a criterion for the diagnosis of herpes encephalitis can be criticized on the grounds that a latent infection can be reactivated as a secondary effect of another infectious disease with the resultant appearance of CF antibodies. It should be noted that herpetic encephalitis itself is not necessarily a primary infection (LEIDERet al. 1965; RAWLS et al. 1966; TOMUNSON AND M A c C A L L U M 1969). The diagnostic value of herpes-like particles discovered by electron microscopy, although denied by JOHNSON et al. (1968) is advocated by HARLAND e t a [ . (1967) and BERGOUIGNAN¢ta[. (1968). If the discovery of these particles never offers a specific diagnosis of the disease, it must be stressed that since the first observation of herpeslike particles in this disease (RYDEN et al. 1965), they have been foand in 36 out of 39 published cases of ANE (ITABASHIet al. 1966; SWANSONet al. 1966; VANDERHAEGHEN et al. 1966: BOUTEILLEet al. 1967b; CHOU AND CHERRY 1967: HADER ct al. 1967; HARLAND et al. 1967; SEITELBERGERAND SLUGA 1967 ; DELMAS-MARSALETet al. 1968 ; MORECKI AND BECKER 1968; RoY AND WOkMAN 1969; TOGA et al. 1969; HUGHES 1969; MANNWEILER AND COLMANT 1969). The particles were discovered in autopsy material in 26 cases and in brain biopsies in the 10 others. According to the strict criteria of JOHNSON et al., a herpetic aetiology was only definitely proven in our Case 1 where virus was isolated from the brain. The following points favour the herpetic aetiology in the other cases. 1. A strikingly characteristic clinico-pathological pattern, which is typical of ANE. Until now, other viruses have never been unequivocally proven to cause ANE in adults (SCHEID AND JOCHHEIM 1956; DUCKETT et al. 1963; HEATHFIELDet al. 1967). 2. The kinetic neutralizing antibodies to HSV as measured here by the very sensitive method of the constant of virus inactivation by antibody, allowing the differentiation between HSV type 1 and type 2. The neutralizing antibodies are only absent in individuals who have never been infected with HSV. Therefore, if ANE results from a primary herpetic infection, it should cause a rise of antibodies from zero. This was the case in the 2 patients (Cases 2 and 3) from whom early serum samples were obtained. In these patients it can be said that a primary herpetic infection was occurring at the very moment that encephalitis was developing. Case 4 showed high titres of J. neurol. Sci., 1971, 12:443-458
454
M. RAPPEL et al.
both types of antibodies, an indication that recent herpetic infection (recurrent or primary) had taken place, since titres as high as 1 : 32 with the CF antibody test have not been found in non-active herpetic infections in our laboratory. 3. The electron microscopic demonstration of numerous herpes-like particles m Case 5 is the only serious argument for the herpetic aetiology in this patient. In this case as well as in Case 1, light microscopic examination of cerebral tissue showed numerous intranuclear inclusions. Correlation between the electron and light microscopic findings has already been well demonstrated in 10 cases by serial semi-thin, and thin sections (ITABASHI et al. 1966; VANDERHAEGHENet al. 1966; C n o u AND CHERRY 1967; ROY AND WOLMAN 1967): it enhanced the significance of the Cowdry type A inclusions in the diagnosis of viral encephalitis. The presence of a few naked viral particles in the cytoplasm, as in Case 5, has already been mentioned (RVDEN et al. 1965; RoY AND WOLMAN 1967). This fact seems to be related to an acute cell necrosis, consisting of fragmentation of the nuclear envelope and resulting in a mingling of nuclear and cytoplasmic components, as seen after 7 days of growth of HSV in tissue culture (SnlPKEY et al. 1967). The ultrastructural tubules and the CSF electrophoretic abnormalities have never been described in the literature concerning human ANE and merit discussion. The structures observed at the electron microscopic level in Case 2 are similar to the intranuclear filaments demonstrated close to typical herpetic particles in neurons of mice, and in cell culture, infected by HSV type 2 strain (MURPHY et al. 1967: COUCH AND NAHMIAS 1969). The round unit of the lattice formation described by CoucH AND NAHMIAS (1969) in the transverse section has the same diameter as the one observed in Case 2 (120-190/~). These two groups of investigators discussed the microtubular nature of these structures and interpreted them as an abnormal assembly of viral subunits and as viral footprints. They also suggested that these structures have a proteic nature. However, in Case 2, the tubules were never observed near herpes-like particles as ill these experimental studies, and were found only in one restricted area of tissue, as already mentioned by C o u c h AND NAHMIAS 0969), The belief that these structures are somehow related to HSV type 2 is not substantiated in this case, as specific serum antibodies for HSV type 1 were found to be present. The CSF protein electrophoresis was done in only 2 patients (Cases 1 and 4) and the abnormalities seen, especially the enormous increase and fractionation of the v-globulins, have to be compared with the CSF globulin increase which occurs in other nervous system diseases. In tumours or meningitis, the increased permeability of the blood-brain barrier has never given phoretograms with such globulin fractionation. The increase and fractionation of y-globulins seen in multiple sclerosis (MS) and in subacute sclerosing panencephalitis (SSPE) differ from the fast fraction observed in the CSF of patients with acute necrotizing encephalitis. The phoretograms seen here are quantitatively comparable to those seen in some cases of neurosyphilis and trypanosomiasis. The comparison with results obtained in other types of cb_ronic or subacute encephalitis like MS, SSPE, neurosyphilis and trypanosomiasis (LoWENTHAL 1964) indicates that the CF ),-globulins are increased and fractionated in a more or less specific manner. In the only serum sample studied, increase of the s-globulins and fractionation J. neurol. Sci., 197/, 12:443458
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
455
of the 7-globulins was comparable to what was described in SSPE serum. These preliminary results show that further work with determination of immunoglobulin levels in CSF and serum and immunochemical studies of the 7-globulins could throw new light on the immunology of chronic, subacute and acute encephalitis.
DISCUSSION OF THERAPEUTIC RESULTS
There were 3 fatalities in the 11 cases treated by IUdR and analysed in Table 1. The number of cases yielding a 27.2~ mortality rate is insufficient for statistical comparison with previously-reported mortality rates in herpes encephalitis. However, it is interesting to note that in a very recent article, NOLAN et al. (1970) report full recovery in 4 cases, and 2 deaths in 6 patients where herpes encephalitis was diagnosed, and who were treated by intravenous idoxuridine. Cases 5 and 11 (Table 1) and 2 of the cases reported by NOLAN et al. (1970) were similar in that they died and did not even show transient benefit from IUdR therapy. Cases 3 and 9 showed no immediate improvement with the decompression; the condition of Case 1 was aggravated following the operation. However, the improvement in their level of consciousness began 36 to 72 h after IUdR treatment. This was also noticed in Case 4 who was not operated upon, and in Case 7 who only underwent a biopsy. The improvement was slower in Cases 6 and 10, as well as in Case 8 where the quantity of IUdR given was probably inadequate. In Cases 2, 3 and 9, where chemotherapy rapidly followed the operation, it is difficult to assess the part played by the treatment in the recovery. The fact that irt Cases 1 and 10 the brain was sterile after IUdR treatment, and that in Cases 1 and 5 the inclusions, which were so numerous in the pre-IUdR biopsy were not found at autopsy, may be related to IUdR treatment. However, these phenomena have been described in untreated cases of herpes encephalitis (YOUNG et al. 1965; MAY et al. 1967; HUGHES 1969). There is thus no absolute proof but a great deal of presumptive evidence for the effectiveness of IUdR in ANE. It must also be emphasized that TOKUMARU (1968) and TOMLINSONAND MACCALLUM(1970) presented evidences that IUdR has a a therapeutic action on herpes encephalitis induced in guinea-pigs.
CONCLUSION
This study emphasizes the need for a multidisciplinary approach and the collaboration of clinicians, morphologists, virologists and biochemists in attempting to diagnose the aetiology of an encephalitis. In the future early diagnosis should be made with new, rapid and more specific techniques such as immunofluorescence (YAMAMOTO et al. 1965) and, perhaps, negative staining on fresh tissue specimens (WATSONet al. 1963; DOANE et al. 1969). These techniques are not commonly used as they require special equipment and training, but the early diagnosis of ANE may be dependent on their utilization. Lobectomy and decompression should be performedlwhenever possible. Idoxuridine seems to be the first step in the generalization of antiviral chemotherapy: cytosinearabinoside has been recently used in a case of generalized herpes zoster infection J. neurol. Sci., 1971, 12:443458
456
M. RAPPELet al.
(MCKELVEY AND KWAAN 1969) and interferon-inducers may also be of some help. Further clinical and experimental studies will be forthcoming, and the statement by HAYSnOW (1970) that there is an urgent need for interested investigators to pool their experience in a controlled study, must be strongly emphasized.
ACKNOWLEDGEMENTS We wish to thank our colleagues for their valuable clinical and surgical contribution, Drs. O. P6rier and J. Flament for helpful criticism, all the members of the Department of Neuropathology for their collaboration, Mm. Vienne and Conreur and Mme Menu for skilled electron microscopic technical assistance and our colleagues of the Neurochemical Department of the Born-Bunge Foundation who performed the electrophoretic investigations. Our thanks are also due to Dr. Schrire of the Research Institute of Smith, Kline and French Ltd. for the kind and rapid supply of IUdR.
SUMMARY Five cases of acute necrotizing encephalitis treated by I U d R are presented; 4 were also treated surgically. The aetiotogy of the disease was investigated by brain biopsy (in 4 cases), electron microscopy (in 3 cases) and virological studies (in 5 cases). Evidence of herpes virus infection was based upon electron microscopy (1 case), virus isolation (1 case) and the rise of complement-fixing antibodies and kinetic neutralizing antibodies (4 cases). This latter technique is specific for herpes virus hominis type 1. In 1 case, structures suggestive of incomplete viruses were seen by electron microscopy. In 2 cases, electrophoresis of the cerebrospinal fluid showed an abnormal pattern. Two patients died. The importance of a multidisciplinary approach is stressed. The problem of early diagnosis and the possible usefulness of chemotherapy are discussed.
REFERENCES ANoy. (1967) Encephalitis caused by herpes simplex virus (Editorial), New Engl. J. Med., 277: 1315. BELLANTI,J. A., G. H. GUIN, R. M. GRASSIAND E. C. OLSON(1968) Herpes simplex encephalitis. Brain biopsy and treatment with 5-iodo-2'-deoxyuridine, J. Pediat., 72: 266. BENNETT,D. R., C. H. z~r RHEINAND T. S. ROBERTS(1962) Acute necrotizing encephalitis, Arch. Neurol. (Chic.), 6: 96. BEROOUmNAN,M., J. JtmIEN, C. VrrAL AND M. DUVERT(1968) Notre experience de l'enc6phalite aigufi n6crosante. Mise en 6vidence du virus herp6tique an microscope 61ectronique: Presse mdd., 76: 709. BOGAERT,L. VAN,J. RADERMECKERANDJ. DEVOS(1955) Sur une observation mortelle d'enc6phalite aJgu~ n6crosante, Rev. neurol., 92: 329. BotrrEILLE,M., S. R. KALIFATANDJ. DELARtrE(1967a) Ultrastructural variations of nuclear bodies in human diseases, J. Ultrastr. Res., 19: 474. BOUTEILLE,M., C. VEORENNE,A. M. DuperY-CoiNANDJ. DELARUE(1967b) Inclusions intranucl6aires paxticuli~res observ6es au microscope 61ectronique dans un cas d'enc~phalite aigu6 n6crosante, Ann. Anat. path., 12: 403. BREEDEN,C. J., T. C. HALLAnt) H. R. TYLER(1966) Herpes simplex encephalitis treated with systemic 5-iodo-2'-deoxyuridine, Ann, intern. Med., 65: 1050. J. neurol. Sci., 1971, 12:443~-58
DIAGNOSIS AND TREATMENT OF HERPES ENCEPHALITIS
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BRIHAYE, J. (1959) 12tude des enc6phalites herp6tiques et des enc6phalites n6crosantes aiguEs (Th6se), Acta neurol, psychiat, belg., 59: 1. BUCKLEY, T. F. AND F. O. MACCALLLrM(1967) Herpes simplex virus encephalitis treated with idoxuridine, Brit. reed. J., ii: 419. CAMPBELL, A. M. G. (1969) Herpes encephalitis, Part 1 (The clinical picture), Postgrad. med. J., 45: 382. CHou, S. H. AND J. D. CHERRY (1967) Ultrastructure of Cowdry type A inclusions in human herpes simplex encephalitis, Neurology (Minneap.), 17: 575. COUCH, E. F. AND A. J. NAHM1AS (1969) Filamentous structures of type 2 herpes virus hominis infection of the chorio-allantoic membrane, J. Virol., 3 : 228. DELMAS-MARSALET, P. M., M. BERGOUIGNAN, R. CASTAIGNE, P. DU PASQUIER AND C. VITAL (1968) Fr6quence actuelle des enc6phalites herp6tiques de l'adulte: sur une s6rie personnelle de huit cas confirm6s par la microscopie 6lectronique, Bull. Acad. nat. Mdd., 152: 316. DOANE, F. W., N. ANDERSON, A. ZBITNEW AND A. S. RHOBES (1969) Application of electron microscopy to the diagnosis of virus infections, Canad. rned. Ass. J., 100: 1043. DODGE, P. R. AND C. W. CURE (1956) Acute encephalitis with intranuclear cellular inclusions: a non fatal case of probable herpetic etiology diagnosed by biopsy, New Engl. J. Med., 255: 849. Dt:CKETT, S., R. KELLY AND H. C. GRANT (1963) Un cas de mgningo-enc6phalite n6crosante avec inclusions intra-cellulaires dfi ~ une infection psittacosique possible, Rev. neurol., 108: 899. DUDGEON, J. A. (1969) Herpes encephalitis, Part 2 (Pathology of herpes encephalitis), Postgrad. reed. J., 45: 386. EVANS, A. D., O. P. GRAY, M. H. MILLER, E. R. VERRIERJONES, R. D. WEEKS AND C. E. C. WELLS (1967) Herpes simplex encephalitis treated with intravenous idoxuridine, Brit. reed. J., ii: 407. HADER, W., M. BAYATPOUR, G. DEMPSTER AND B. ROZDILSKY (1967) Herpes simplex encephalitis. Two fatal cases, Canad. med. Ass. J., 96: 1565. HANSHAW, J. B. (1970) Idoxuridine in herpes virus encephalitis (Editorial), New Enfl. J. Med., 282: 47. HARLAND, W. A., J. H. ADAMS AND D. MCSEVENEY(1967) Herpes simplex particles in acute necrotizing encephalitis, Lancet, ii: 581. HEATHFIELO, K. W. G., R. PILSWORTH, B. J. WALL AND J. A. N. CORSELLIS (1967) Coxsackie B5 infections in Essex 1965, with particular reference to the nervous system, Quart. J. ?.~led., 36: 579. HERRMANN, JR.~ E. C. (1961) Plaque inhibition test for detection of specific inhibitors of D N A containing viruses, Proc. Soc. exp. Biol. (N.Y.), 107: 142. HUGHES, J. T. (1969) Pathology of herpes simplex encephalitis. In: C. W. WHITTY, J. T. HUGHES AND F. O, MCCALLUM (Eds.), Virus Diseases and the Nervous System, Blackwell, Oxford and Edinburgh, p. 29. ITABASm, H. H., D. M. BASSAND J. R. McCULLOCH (1966) Inclusion body of acute inclusion encephalitis, Arch. Neurol. (Chic.), 14: 493. JOHNSON, R. T., L. C. OLSON AND E. L. BUESCHER (1968) Herpes simplex virus infections of the nervous system. Problems in laboratory diagnosis, Arch. Neurol. (Chic.), 18: 260. KAUFMAN, H. E. (1963) Chemotherapy of virus disease, Chemotherapia, 7: 1. EEIDER, W., R. L. MAGOFFIN, E. H. LENNETTE AND L. N. R. LEONARDS (1965) Herpes simplex virus encephalitis: its possible association with reactivated latent infection, New Engl. J. Med., 273 : 341. LOWENTHAL, A. (1964) Agar Gel Electrophoresis in Neurology, Elsevier, Amsterdam. MCKELVEY, E. M. AND H. C. KWAAN (1969) Cytosine arabinoside therapy for disseminated herpes zoster in a patient with IgG pyroglobulinemia, Blood, 34: 706. MANNWEILER, K. AND H.-J. COLMANT (1969) Ultrastructural data in cases of necrotizing encephalitis without inclusion bodies. Pathogenesis and aetiology of demyelinating diseases, Int. Arch. Allergy, 36:515. MARSHALL, W . J . S . (1967) Herpes simplex encephalitis treated with idoxuridine and external decompression, Lancet, ii: 579. MAXWELL, E. (1963) Treatment of herpes keratitis with 5-iodo-2'-deoxyuridine ([DU). A clinical evaluation of 1500 cases, Amer. J. Ophthal., 56: 57l. MAY, G., R. DAHN AND K. REUSS (1967) Beitrag zum Nachweis yon Herpesvirus bei Enzephalitis, Dtsch. reed. Wschr., 92: 793. MILLER, J. D. AND C. A. C. Ross (1968) Encephalitis: a four-year survey, Lancet, i: 1121. MILLER, J. K., F. HESSER AND V. N. TOMPKINS (1966) Herpes simplex encephalitis. Report of 20 cases, Ann. intern. Meal., 64: 92. MORECKr, R. AND N. H. BECKER (1968) Human herpes virus infection. Its fine structure identification in paraffin-embedded tissue, Arch. Path., 86: 292.
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