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Idiopathic epilepsy is a worldwide neurological disorder that affects people of various ages. However studies of herpesvirus infections in adult epilepsy patients are still rare. This study was performed to identify the association between epilepsy and herpesvirus IgG antibodies. A cross-sectional study was conducted with serum samples obtained from epilepsy patients (n=95) and healthy controls (n=95) upon fulfilling the inclusion and exclusion criteria. The presence of specific IgG antibodies against herpes simplex virus type 1 and type 2 (HSV-1,2), varicella-zoster virus (VZV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) were detected by enzyme linked immunosorbent assay (ELISA) test kits. Among all the herpesvirus studied, only CMV IgG level showed significant association to epilepsy (X2=186.0; p<0.001). It can be concluded that cytomegalovirus infection maybe one of the factors that contributes to epilepsy. This relationship between CMV and epilepsy should be more extensively studied to investigate its mechanism in active infections.
Key words: cytomegalovirus, epilepsy, Epstein-Barr virus, herpes simplex virus, varicella zoster virus
Epilepsy is a neurological disorder that affects people of various ages in more than 50 million individual throughout the whole world 1. The World Health Organization estimated between 4 to 10 per 1000 individuals worldwide suffer from this disease 2 with a higher prevalence reported in developing countries 3.
Epilepsy affects both men and women of all ages, races and socioeconomic levels. Majority of the cases are idiopathic while the rest may be due to certain provoked forms such as traumatic brain injury, metabolic derangements, drugs withdrawal, alcohol intake, cerebral infarction and central nervous system (CNS) infection 3.
The cause of epilepsy remains elusive. Some studies link the immune system with epilepsy 4,5 while others try to relate virus infection to the seizures 6,7. Herpesvirus as aetiological agents of epilepsy have gained much attention due to the fact that the viruses have the tendency to establish latent and lifelong infections in human 8-10. They may enter CNS via endothelial cells lining the blood vessels or may enter within lymphocytes and macrophages that they infect at the peripheral neurons 6. To the best of our knowledge, most studies had focused on neonates and children. Therefore, this study was conducted to determine the association between adult epilepsy patients and infection with herpesvirus, namely herpes simplex virus type 1 and herpes simplex virus type 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV).
2. Materials and methods
2.1 Study subjects
A cross-sectional study was carried out on 95 epilepsy patients and 95 healthy subjects which started in March 2008 till February 2009. All epilepsy patients and healthy subjects were enrolled from the Hospital Universiti Sains Malaysia (HUSM) which is located in the Kota Bharu district of the Kelantan state of Malaysia. Ninety-four percent of the Kelantanese population are Malays 11, with almost two third of the population living in the rural areas 12. This study had obtained an ethical approval from the local institution of Universiti Sains Malaysia (USM).
A simple random sampling method was used to recruit epilepsy patients from a Medical Specialist Clinic, HUSM, whereby one in every four patients attending the clinic were selected after being screened using the inclusion and exclusion criteria. The inclusion criteria include epilepsy patients aged between 18 and 50 years old that were for a routine medical check-up. The diagnosis of epilepsy and seizure type was confirmed by a qualified attending Neurologist based on electroencephalography (EEG) and magnetic resonance imaging (MRI) tests. Patients who were having fever and those who were pregnant or having history of organic brain disorders, autoimmune disease, chronic infections and underlying malignancies or other acute infections were excluded.
Healthy subjects were selected from the public mostly those who are accompanying their relatives or friends who came for the check-ups. They were recruited into the study whenever they fulfil the inclusion and exclusion requirements and agreeable to be included. The inclusion criteria include healthy person aged between 18-50 years old with no medical illness, while those who are pregnant, having underlying medical illness, febrile fit, having fever or acute infections or on hospital prescriptions were totally excluded.
2.2 Blood samples
Blood samples (3 ml) were collected into plain tubes from the peripheral vein of the study subjects. Serum was separated soon after being centrifuged at 1500 g for 5 minutes following 6 hours of incubation at room temperature (27Â°C). The collected serum was kept at -80Â°C prior to testing by ELISA assay.
2.3 ELISA Procedure
Indirect ELISA tests were carried out to quantify antibodies against herpes viral antigens following the manufacturer's protocols. All ELISA kits (PlateliaTM HSV (1+2) IgG, PlateliaTM VZV IgG, PlateliaTM CMV IgG, PlateliaTM EBV-VCA IgG) were supplied by the BIO-RAD Laboratories Company (California, USA). The test procedures for all the ELISA kits are basically similar, except for PlateliaTM EBV-VCA IgG, where there was a slight difference in terms of incubation period and washing times.
Briefly, diluted patients serum (1:101) was placed in the specified well, as well as the calibrators. The ELISA plates were then incubated for 45 min at room temperature to allow the immunoglobulins to bind to the antigen before washing for several times to eliminate any residual unreacted proteins.
Further incubation was carried out with the conjugate (human IgG monoclonal antibodies labelled with peroxidase) for another 45 min before washing. The unbound conjugate was discarded and the peroxidised substrate (20 Âµl) was added.
The developed colour was proportional to the concentration of specific antibodies present in the serum sample. The absorbance (O.D.) was detected using the ELISA Analyser and the values were calculated and compared against the qualitative cut-off provided for each commercial ELISA kits used. This was to identify whether the subjects have specific antibody against viral antigen studied.
The statistical analyses were performed using SPSS software version 12.0 (SPSS Inc., Illinois, US). Normality was determined for age and gender for both study groups. The association between herpes virus and epileptic patients were analyzed via the Pearson's Chi-Square test and Fischer's Exact test and p value < 0.05 was considered to be statistically significant.
3.1 Sociodemographic factors.
A normal distribution were obtained for both groups of epilepsy patients (n=95) and healthy controls (n=95). The sociodemographic factors that include age, gender, ethnicity and education level were calculated and compared between both study groups (Table 1). Male and female distribution for epilepsy group is also normally distributed with the ratio of male to female was 1.2:1. Seventy nine percent of the epilepsy patients came from rural areas whereas the remaining 11% were from the urban areas. Majority of the healthy controls (73%) were also from rural areas which are comparable to epilepsy patients in terms of study population. The age at first seizure onset, type of seizure, occurrence of febrile fits and family history of epilepsy disorder were obtained and analysed (Table 1). The mean age at first seizure onset is 15.58 years. All epilepsy patients were diagnosed to be suffering from idiopathic epilepsy with no identifiable causes present. 80 out of the 95 patients had temporal lobe epilepsy with or without secondary generalisation. Single half hour recording of EEG picked up temporal lobe focus in 35 patients, 14 on the right side and 21 on the left side. "PLEASE INSERT TABLE I HERE"
3.2 The association between epilepsy and previous herpes virus infection.
Among the specific IgG of the four herpesvirus reported in this study, only CMV IgG showed an association with epileptic subjects (Ð¥2 = 186.0 ; p< 0.001) (Table II). The extremely high X2 value among CMV infected epileptic patients suggests a strong association between CMV and epilepsy. The specific IgG results of the study subjects are illustrated in Figure I.
"PLEASE INSERT TABLE II AND FIGURE I HERE"
The findings from this study showed that less than 10% of the epilepsy patients had family history of their close relatives (parents or siblings) suffering from similar disorder. This result suggests that genetic inheritance is not a major cause contributing to epilepsy 13 and there is possibility that some other causative factor plays a role in its pathogenesis.
According to our study, antibody against CMV IgG infection was significantly associated with epilepsy whereas VZV IgG, EBV IgG and HSV (1+2) IgG were not. A previous study using polymerase chain reaction (PCR) on hippocampal brain tissue samples from patients with temporal lobe epilepsy (TLE) found that 25% of the study subjects were positive for CMV DNA 14. Another study using in-situ hybridization method on brain tissue biopsy of Rasmussen Syndrome patients found that 70% of their study subjects were positive for CMV DNA 15. Our present study on the detection of herpesvirus specific IgG from peripheral serum samples clearly shows that presence of CMV antibody is significantly related to all the epilepsy patients. However, since only 36.8% of our study subjects suffered from TLE we think that this could be the reason (apart from the different method used) for the difference seen when comparing the results of this study to the above mentioned studies.
Suzuki et al. (2008) on the other hand, studied epilepsy in congenital CMV and found that seven of 19 subjects with congenital CMV developed epilepsy 16. The findings were found to be congruent with our present study when looking at the relationship between CMV and epilepsy, however we could not say whether our epilepsy patients were infected by CMV during infancy (congenital) as the relevant data is not presently available.
Cytomegalovirus is ubiquitous and infections are usually acquired very early during childhood with majority of the infections asymptomatic. Four point two percent of our epilepsy patients experienced febrile fits during their childhood, but majority of study subjects had their first seizure onset at the mean age of 16 years old. Most researchers agrees on the fact that the risk to develop epilepsy following episodes of febrile fit is quite small and not significant 17, only 2-10%, depending on the type of febrile fit they encountered 18. Moreover, only about 10-15% of CMV infected infants actually present with the clinical symptoms while the rest remains asymptomatic 10,16.
A previous report 19 found that children from low socioeconomic backgrounds have a higher incidence of being infected with CMV when compared to those who lived in the urban areas; which is comparable to our study whereby majority of the patients came from rural areas.
A total of 98.9% of study subjects studied were found to be immune to EBV. This is comparable to a study conducted by Norzuriza et al. (2008) who found that 77.5% of the public in Malaysia between the age 0-20 years, and 100% of people aged above 60 years had previous infection with EBV 20. A similar study conducted in Bangladesh showed that 85% of the study group had positive EBV IgG after the age of 10 21 while a research in Papua New Guinea reported that approximately 93% people in urban areas and 98% people in rural areas were positive for EBV antibodies 22. Infections with EBV is thus very common, with the prevalence varies among different countries due to environmental, geographical, socioeconomic and hygienic conditions 23.
Eighty-seven percent of our study subjects, both control and epilepsy group were found to be immune to VZV, most probably as the result of previous chicken pox and shingles. This result is comparable to a report that stated a near 100% seropositivity to VZV in USA 24. Varicella-zoster virus infection is also as common as EBV with childhood infection the norm; chicken pox being the usual clinical manifestation even though severe infection may occurs in immunocompromised patients 10,24.
The proportion of study subjects with HSV specific antibody is relatively high in both groups, even though not significantly associated to epilepsy. The former result is comparable with a previous study that showed a HSV specific antibodies prevalence of 61.4% in the peninsular part of Malaysia 25.
As a conclusion, our findings revealed that there is a significant association between epilepsy and CMV IgG that suggests previous CMV infections may play a role in contributing to the pathogenesis of epilepsy. Therefore, CMV infections among epilepsy patients should be more extensively studied to investigate its mechanism and function in active and latent infections. Further studies with a larger cohort are necessary to confirm our findings and to also study the association of TLE with other herpesvirus such as HHV-6 and HHV-7.
The authors gratefully acknowledge the support from the Research University Grant (1001/PSKBP/8120192). Special thanks goes to the staffs of Immunology Department and Serology Laboratory of Department of Medical Microbiology and Parasitology, School of Medical Sciences USM, Specialist Clinic of HUSM and those who were involved direct or indirectly to this study.