July 1986, Volume 36, Issue 7

Original Article

NEUROVIROLOGICAL RESEARCH OF ENCEPHALITIDES IN AND AROUND KARACHI THE CLINICAL ASPECTS (INTERIM REPORT)

T. Takasu  ( Department of Neurology, Nihon University School of Medicine, Tokyo, Japan. )
Akhter Ahmed  ( Department of Neurology, Dow Medical College, and Civil Hospital, Karachi,Pakistan. )
R. Kono  ( Saitama Medical College, Saitama, and Special References Laboratory, Tokyo, Japan. )
K. Kondo  ( Department of Neurology, Tokyo Metropolitan Institute for Neurosciences, Tokyo, Japan. Present Address: Dept. of Public Health, Hokkaido University School of Medicine, Sapporo, Japan. )
Y. Aoyama  ( Department of Pathology, the Institute of Medical Science, Tokyo University, Tokyo, Japan. )
M. Sugamata  ( Department of Hygiene, Teikyo University School of Medicine, Tokyo, Japan. )
K. Chida  ( Department of Neurology, Nihon University School of Medicine, Tokyo, Japan. )
S. Kame  ( Department of Neurology, Dow Medical College, and Civil Hospital, Karachi,Pakistan. )

Abstract

One hundred-and-fourteen cases of encephal itides and encephalopath ies were selected from the patients admitted to the Neurology ward Civil Hospital, Karachi (CHK), during the twelve years’ period from 1971 to June 1983. There were eleven cases of subacute scierosing pan encephalities (SSPE) and seven cases of chronic progressive encephalopathies (CPE).
Forty-three cases of encephalitides and encephalopathies were collected at the CHK during the eight and a half months period (July 1983 to March 1984) on which clinical and sero­logical studies were performed.
Of these, nineteen were cases of acute encephalitides which included five cases of Japanese encephalitis and two probable and three possible cases of herpes simplex encephalitis.
In the 43, seven cases of SSPE or SSPE-like diseases were found, all of which were associated with positive antibodies against measles virus in cerebrospinal fluid (CSF).
Besides, six cases of subacute encephalitis or encephalopathy other than SSPE or SSPE or SSPE-like diseases were collected, one of which was associated with positive CSF antibodies against rubella virus, and another was a case of typhoid encephalopathy. -
Seven cases of CPE were also found in the 43, but their aetiology remains to be elucidated (JPMA 36:159, 1986).

INTRODUCTION

This Project originated from the report published in this Journal by one of the present authors in 19801 stating that on an average two to three cases of SSPE per year have been seen in the Civil Hospital, Karachi during eight to ten years prior to 1980, which would reflect an app­reciable incidence in Karachi population. The actual activity commenced in July 1983 after one year of preparatory research. It’s aim was to study the aetiology and frequency of encep­halitides in Karachi.
In view of existing complete ignorance about encephalitides in Pakistan, multidiscipli­nary approach was adopted to study not only the cases of encephalitides but to evolve information bearing on various aspects of virus and other (inflammatory) encephalitides. These aspects include mortality patterns in Karachi, and sero­epidemiological, neuro-epidemiological and entomological backgrounds.
The preliminary seroepidemiological, neuro epidemiological, and entomological aspects and backgrounds in this project are reported in this issue of the journal sepaiatély.2-5

MATERIALS AND METHODS

1) The series of cases
All the in-patients discharge cards at the Neurology-Ward Civil Hospital, Karachi (CHK), were reviewed to pick up the cases of encepha­litides and encephalopathies. A card index .o\\n the diagnosis of each patient admitted to the ward is maintained since 1971. All the diagnoses were based solely on clinical observation. Serolo­gical studies were conducted in a limited number and autopsy was not done in any of the cases. A diagnosis of “encephalitis” was made only in the presence either of cerebrospinal fluid (CSF) pleocytosis or of fever in the course of the illness or both.
2) The new series of cases
An many cases of encephalitides and en­cephalopathies as possible were collected during the eight and a half months’ period from July 1983 to March 1984 from the Neurology, Medical and Paediatric Wards of CHK, and some from the Specialists’ Clinic, and Abbassi Shaheed Hos­pital, Karachi. Detailed clinical history and neuro­logical status were recorded in each case on a proforma. Clinical diagnoses were made after joint discussion among the authors.
3) Serological studies
One or more serum samples were taken from all the cases and CSF sample obtained mostly on the same day as the serum. All the specimens were kept at 20°C in aseptic airtight containers and transported at -20 C from Karachi to Tokyo, and stored at 80 C for less than ten months until examination. Some samples, speci­fically those transported in December 1983, were exposed to room temperature (around 10°C) for less than probably two weeks because of an accident, but there was no apparent indication of microbial proliferation in the samples examined.
The titres for antibodies against Japanese encephalities virus (JEV), West Nile virus (WNV)), Dengue-2 virus (DV), and tick-borne encephalitis virus (TBEV) were estimated by Sugamata at the Department of Hygiene, Teikyo University School of Medicine, Tokyo, Japan, with haemag­glutination-inhibition test (HI). The titres for antibodies against measles viurs (MV) and rubella virus (RV) were estimated partly by Sugamata and partly at the Special Reference Laboratory (SRL), Hachioji, Tokyo, Japan, with HI. In some of these selected samples, the titres for antibodies against MV were estimated with neutralization test (NT) by Yamanouchi and Skaguchi at Depart­ment of Veterinary Pathology, the Institute of Medical Science, Tokyo University, Tokyo, Japan. The titres for antibodies against entero­virus-70 (EV 70) were estimated at SRL with NT. The titres for antibodies against JEV, MNV, DV, TBEV, MV and RV in samples were regarded as positive if the titres were not less than 10,10,10, 10, and 8 in CSF, respectively. NT antibodies against EV7O in samples were regarded as positive if the titres were not less than 4 in serum and if they were not less than 1 in CSF. NT antibodies against MV in samples were regarded as positive if the titres were not less than 10 both in serum and in CSF.
Immunoglobulin-G (IgG) and immunoglo­bulin-M (1gM) antibodies against herpes simplex virus type 1 (HSV) were quantified separately with solidphase antigen enzyme-linked immunoa­dsorbent assay (ELISA) in 400 times diluted serum and in 40 times diluted’ CSF and with solid phase anti-immunoglobulin ELISA in 200 times diluted serum and in 100 times diluted CSF, respectively at SRL. The quantity of antibodies against HSV were expressed in grades; as for lgG antibodies a preliminary correlation study showed that +,++,+++ and ++++ are roughly equivalent to 4, 16 (4), 64 (4), and 256 (4), of comple­ment fixation titres, respectively; IgG antibodies against HSV were regarded as equivocal if the ELISA values were more than 0.15 and not more than 0.20 and as positive if they were more than 0.20 respectively. 1gM antibodies against HSV were regarded as equivocal if the ELISA values were more than 0.25 and not more than 0.35 and as positive if they were more than 0.35, respecti­vely.
IgG. concentration in CSF was determined at SRL with Laser immunoassay.

RESULTS

1) The clinical Diagnosis: One hundred and fourteen cases of encephalitis or encephalopathy collected at the Neurology Ward, CHK, during the twelve years period (1971 to 1983). The definition of “encephalitis” stated in the materials and methods. “Encephalopathies” include all the diseases of the brain itself, excluding multiple sclerosis and heredodegenerative diseases and including all cases for which we have no direct proof of infectious aetiology, but we cannot entirely rule out this possibility; meningitides were excluded.
In these 114 cases (Table 1)

seven, five eleven ad seven cases of AE, SE, SSPE or SSPE­like diseases and CPE weke found. Distribution of these cases by age and sex is shown in Table II.

One thing peculiar was that the sex ratio (M/F) of SSPE or SSPE-like diseases was 4/7 in contrast to the known male preponderance of this disease in U.S.A. ,Europe and Japan.
2) The neuro-virological diagnosis of the new series of cases. During the eight-and a half months’ period (July 1983 to March 1984) a total of 71 new cases with a tentative diagnosis of encepha­lltis or encephalopathy were collected. After discussion, 43 of them were accepted as such, 15 excluded and in the remaining 13 the decision suspended (Table III).

The distribution of the 43 cases by age and sex is shown in Table IV.


a) Acute encephalitides: Of 43 there were 19 cases of AE (Table III), thirteen of which were associated with some serological evidence for JE and /or some other arbo virus infection. Five of the latter were associated with very high titres for serum antibodies against JEV (640) together with a high titre for CSF antibodies against JEV (40) in one of them (Table V)

and a significant fall of the titres for serum antibodies against WNV and probably against JEV in the course of ifiness in another. In three of the five the aetio­logical diagnosis of JE was almost certain and in other two probable. The clinical history of the two of these five are briefly described towards the end of this section. Fourteen of the nineteen cases of AE were associated with serological evidence for HSV (Type 1) infection, in five of which the evidence was strong, four were associated with a very high ELISA value for serum antibodies (++++) and another with a significant rise of ELISA value for serum antibodies in the course of the illness (-to+++) (Table VI).

One of the afore-mentioned four cases was also associa­ted with a very high ELISA value (++++) for CSF antibodies. In two of the five patients the aetiological diagnosis of herpes simplex ence­phalitis (HSE) was almost certain and in the other two it was possible. The clinical history of two cases are described below, too.
b) SSPE: Among the 43 accepted cases seven cases were clinically diagnosed as SSPE (Table VII).

All had a subacute onset and their electroence­phalograms showed periodic synchronous discharge (PSD) whether they were typical or somewhat atypical of SSPE.
History of measles was noted in three and in an­other a history of rash associated with fever. Myoclonic jerks were observed in six. One thing peculiar in these cases in Karachi was again that the sex ratio (M/F) was 2/5 and another that, in six, fever apparently preceded the onset of the neurological deterioration. The details of the clinical features of these cases will be reported separately by one of the authors (A.A.).
All these cases had a high titre for serum antibodies as well as positive CSF antibodies against MV(Table VIII)

and one also had positive CSF for antibodies against RV. Moreover, in five, an abnormally high IgG content in CSF either in absolute value or in relative value to total protein was noted (Table VIII). In one (0134) brain biopsy was performed and the formalinfixed and frozen specimen were transported to Japan for detecting MV with immunofluorescence and for isolating the virus with culture, although the results were both negative. Immunofluores­cence was negative probably because the site of biopsy was inappropriate and culture was nega­tive probably because freezing was not the optimal way for preserving the virus activity.
c) Other types of SE sub-acute Encephalitis/ encephalopathy: Beside SSPE two cases of subacute encephalitis and four cases of subacute encepha­lopathy were found (Table IX).

None of the six were associated with PDS in EEG or with myoc­ionic jerks.
Serologically two cases were associated with positive CSF antibodies against MV, one with positive CSF antibodies against RV, one with a very high titres for serum antibodies against JEV and in another with a strongly positive Widal reac­tion reac­tion in serum. Aetiological meanings of these observation are still being discussed. The clinical history of case with positive CSF antibodies against RV is described later.
d) Chronic progressive encephalopathy:
Among the 43 accepted cases, seven cases of CPE were found (Table III). In One of these seven the results of antibody estimation were negative both in serum and in CSF and in another only in Serum for all eight viruses examined (Table X).

In the remaining five, the results were positive in serum for one or more of the eight, and in all of the five they were negative in CSF except for the two cases in which antibodies against HSV were weekly positive or equivocal.
e) Others: Four cases of diseases other than those classified into a, b), c) or d) were as shown in Table XI).


Over all, the causative agent was identified in 15 out of 43 cases (JEV 3, HSV 2, 6, RV 1, typhoid) and probable in other 3 (JEV 2, MV 1). If combined together, the rate of identification was 35%.

CASE REPORTS

Case 1 (case code : 0098) : JE
This female infant, aged 11 months, had fever and cough for three days, followed by fits prior to admission on 21/8/83, when she was found to be semicomatose and febrile (104° F). Her oculomotor function was intact. The muscle tone was generally increased. The deep tendon reflexes were normoactive, but with extensor planter responses. There were jerky movements in all four limbs but no neck rigidity or Kernigs sign. The peripheral leukocyte count was 16,000 per cmm with 80% polymorphonuclear cells. The CSF was clear, containing two cells per cmm, with negative Pandy test.
On 27/8/83 the CSF was clear, containing two cells per cmm, 30 mg of protein and 66 mg of sugar per lOOmi.
On 30/8/83 (13th day of illness) the titre for serum antibodies against JEV was not less than 640, although it was less than ten in CSF, i.e. the antibodies were negative in CSF (Table V).
Although paired samples were not taken, judging from her very young age and the demons­tratibn of a high titre of antibodies against JEV during the course of her AE is strongly suggestive of Japanese encephalitis (JE).
Case 2 (cases code: 0129: JE)
The girl, aged 16 years, had had headache, vomiting and fever for three days and been dis­oriented and drowsy for two days prior to adinis­sion on 19/1/84. She suffered from measles at the age of 14 years.
On admission she was drowsy and restless. There were neck rigidity and bilateral extensor plantar responses, but no paralysis. She had one generalized seizure. The CSF contained occasio­nal cells, 30 mg of protein and 61 mg of sugar per 100 ml.
EEG taken on 24/1/84 (9th day of illness) was within normal limits. On the same day the titre for serum antibodies against JEV was not less than 640, although less than ten in CSF.
On 2/2/84 (18th day of the ifiness) the titre was again not less than 640, but on 9/2/84 it was 320. She completely recovered except mild headache (Table III).
In this case the significant fall of the titre for serum antibodies against JEV demonstrated with paired samples strongly suggests that her mild acute encephalities was due to JEV ‘in­fection.
Case 3 (case code: 0117): HSE
A female infant, aged two-and-a-half years had had fever for two days prior to admis­sion on 20/12/83 for fits, vomiting and coma EEG showed generalized delta activities, which were not associated with any PSD. On 23/12/83 count was 13,800 per cmm with 75% of polymorphonuclear cells. CSF contained ten cells per cmm, 20 mg of protein and 69 mg of sugar per lOOmi.
On 2/1/83 (16th day of the illness) she was awake, but unable to sit, suck or eat. Her limbs were spastic with extensor planter responses. On the same day IgG antibodies against HSV were ++++ both in serum and in CSF and 1gM anti­bodies against the same virus was ++ in serum and negative in CSF (Table VI). On 31/1/84 (45th day of the illness) the serum antibodies against HSV were not remarkably chaiiged. On 9/2/84 she was more conscious than before but still unable to sit.
The demonstration of a very high ELISA value for IgG antibodies against HSV not only in serum but also in CSF at due time during the course of her ifiness with a significantly high value for 1gM antibodies strongly suggests that the AE in this case would probably be due to the HSV infection which occurred for the first time in her life.
Case 4 (case code: 0127): HSE
The 21-years-old young woman had had fever for five days and been drowsy for one day prior to admission on 8/1/84, when she had fever (103 F) and headache. EEG mamly showed theta activities with a few high voltage waves mixed with occasional delta waves.
On 17/1/84 (15th day of the illness) serum antibodies against HSV were negative, but on 23/2/84 (37th day of the illness) serum anti IgG antibodies were +++. On that day she responded almost normally to verbal commands.
The SE in this case was probably caused by a reactivation of HSV infection, because the significant rise in serum IgG antibodies against HSV demonstrated in paired samples during the course of her illness without any significant rise in 1gM antibodies
Case 5 (case code: 0134): SSPE
A ten-year-old boy, who had a history of measles at the age. of 6 months, had had low-grade fever and frequent falls for ten months prior to admission. He had a grandma! seizure five months prior to admission and had had violent myoclonic jerks in bed for three months prior to admission.
On admission on 5/2/84 he was moderately demented. EEG showed high voltage sharp wave complexes, each of which was followed by delta waves; myoclonus involving neck, all four limbs, and face at each myoclonic jerk he opened his mouth, assumed opisthotonic posture, extended his arms and flexed his legs at his joints. On 4/3/84 he had eyes open and followed light. His pupil were isocoric and normoreactive to light. Snout reflex was positive, Jaw jerk was exaggerated. He responded to simple verbal commands, but did not speak. There were no meningeal signs. The muscle tone was only slightly increased. There was no forced grasping. His gripping power was good. The deep tendon reflexes were rather hypoactive in upper limbs and brisk in lower limbs. There was unsustained clonus bilaterally. The plantar responses were flexor. There was no startle reaction on sound.
Serologically the NT titre for CCE antibo­dies against MV was very high (Table VII).
The results of serological studies together with the clinical course, presence of mycolonic jerks and of the PSD in EEG fovoured the dia­gnosis of SSPE caused by MV infection, although there were some atypical features; the PSD were not composed of typical high voltage delta waves but of sharp waves and the onset of his illness was apparently preceded by low grade fever. Case 6 (case code: 0105): SE
A young man, aged 21 years, had been weaker and less active than before and lost weight for one year, had low-grade fever, sometimes with chills, and fits for one month, and been unable to walk or speak; disoriented, and incon­tinent of urine and had not eaten, drunk or,ans­wered for two weeks prior to admission on 13/11/83, when he was somnolent, apathetic, having bedsores, neck rigidity, generalized spasti­city and paresis of the four limbs. The deep tendon reflexes were present.
On 14/11/83 the peripheral leukocyte count was 11,000 with 70% neutrophils. The CSF contained two cells per cmm, 30 mg of protein and 60 mg of sugar per 100 ml. The titres for serum and CSF antibodies against RV were 64 and 32, respectively (Table VI). The antibodies against RV were 64 and 32 respectively (Table VI). The CSF taken on 2/12/83 contained 11.9mg of IgG per 100 ml; EEG taken on 13/12/83 showed some slow waves.
On 5/3/84 he was unable to converse or sit up and had spastic tetraparesis. 1984).
The SE in this case was probably due to RV infection of the central nervous system reflected by the presence of antibodies against RV in serum and CSF. This case might possibly be one of progressive rubella encephalitis.

DISCUSSION

A.A. one of the present authors, reported seven cases of SSPE in 1980, which were part of the more than 25 cases probably of SSPE seen by him in Karachi over the ten years upto the year 1980. Besides SSPE, cases of CPE were also encountered in Karachi. These observations were confirmed in this study, but, aetiological diagnosis of such cases needed the introduction of more sophisticated ancillary examination as in the present study.
Nearly half as many as the total cases of encephalitis or encephalopathy collected at the Neurology Ward, CFIK during the past twelve years were gathered in this study during the eight and a half months’ period. The number of SSPE­like diseases and CPE in the new series was quite comparable with that of the past series.
Serological studies demonstrated that all the new SSPE cases, so diagnosed solely clinically, were associated with a significantly high titre for antibodies against MV both in serum and in CSF.
It has long been debated whether JE occurs west of India. The three case (0089, 008 and 0129) of the present series were associated with a specifically high titre for antibodies against JEV in serum, being exceedingly high over other arboviruses, so that in these three there must have been an actual JEV infection, which was almost certainly the cause of the AE in these cases. HSE also occurs in Karachi. Detection of JEV and HSV is needed to confirm the diagnosis of JE and HSE in Karachi.
In this study, the identification rate of aetiology was 35%. The rate would certainly improve if paired samples could be collected in more cases.

CONCLUSIONS

1) A number of cases of encephalitides and ence­phalopathies can be encountered in Karachi, and around 44% of such cases collected at the Depart­ment of Neurology CHC, from 1971 to 1984 were AE, around 16% SSPE or SSPE-like diseases, around 14% other types of SE and around 10% so collected were CPE.
2) All the SSPE or SSPE-like diseases so diag­nosed clinically were associated with a high titre for antibodies against MV both in serum and in CSF.
3) One of the cases of subacute onset encephalitis was associated with positive CSF antibodies against RV.
4) Serological studies on CPE have not given any definite conclusion about their aetiology yet. Further studies are required for elucidating the aetiology of CPE in Karachi.
5) Five cases were found in which the clinico­serological correlation strongly suggests the occurrence of JE in Karachi. In three of them the diagnosis was almost certain. However, the final conclusion should await virological confIr­mation.
6) Two cases were almost certainly those of HSE.
7) On the whole, the aetiology was identified in 15 out of 43 cases (35%).

ACKNOWLEDGEMENTS

This study has been and is being supported by a Grant-in-Aid for Overseas Scientific Survey, the Ministry of Education, Science and Culture, the Japanese Government (in 1982, No. 57042010 in 1983, No. 58041070 and in 1984, No. 5904-43064).
The authors thank Dr. M.I. Rurney, Director, National Institute of Health, Islamabad, for his help in launching the whole project here in Karachi, Pakistan, under the approval and support of the Federal Government of Pakistan of which this clinical study is a part.
The work has been and is going on in collaboration with Akhtar Ahmed’s Staffs at the Department of Neurology under the generous approval and great help of the Principal of Dow Medical College, the Medical Superintendent and many staff members of Civil Hospital Karachi. In particular, the authors would like to thank Dr. (Mrs.) Habiba Ahmed Zaki and Miss Shagufta Sheikh for their respective medical and persistent paramedical assistance. We also thank Professor Kazuya Yamanouchi and Dr. Masohiro Sakaguchi, Department of Veterinary Pathology, the Institute of Medical Science, Tokyo University, Tokyo, Japan, for estimating antibodies against MV with NT. Prof. G. Billoo, Dr. DS. Akram of Dept. of Paediatics, Dow Medical College and Dr. Yasmm Akbani of Abbasi Shaheed Hosptial, Nazimabad Karachi, for their help and permission to examine and include their cases in this series, and Prof. K. Zaki Hasan for his suggestion and support at various stages of this study.

REFERENCES

1. Ahmad, A. Subacute sclerosing panencephalitis. report of seven cases. JPMA., 1980; 30:249.
2. Sugamata, M., Kono, R., Ahmed, A. and Takasu, 1. Seroepidemiological research on viral encephalitis in Karachi, Pakistan. Preliminary report. JPMA., 1986; 36:177.
3. Kondo, K., Ahmed, A. and Takasu, T. Epidemiology of subacute scierosing panence­phalitis (SSPE) and other encephalitides in Karachi area; a progress note. JPMA., 1986; 36: 169.
4. Nakaee, K., Kondo, K., Takasu, T., Kamei, K. and Ahmed, A. Estimated mortality rate by sex age and death causes in Karachi. JPMA., 1986; 36: 174.
5. Kamimura, K., Takasu, T., Ahmed, A. and Ahmed, A. A. survey of mosquitoes in Karachi area, Pakistan.JPMA., 1986; 36: 182.

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