Anti-Taenia solium larval stage Ig G antibodies in patients with epileptic seizures

Abstract

Background:

Cysticercosis is the most common differential diagnosis for epilepsy. The present study was carried out to assess the serological response among patients with epileptic seizures visiting JIPMER Hospital Puducherry.

Materials and Methods:

A total of 934 serum samples were collected from patients with epileptic seizures. A standardized questionnaire was designed to obtain information on the demographic, socioeconomic, environmental, and behavioral characteristics related to the transmission of infection. An enzyme-linked immunosorbent assay (ELISA) was used to detect the anti-Taenia solium larval stage IgG antibodies. Samples found reactive and inconclusive by ELISA were further tested by the enzyme immunotransfer blot (EITB).

Results:

The frequency of antibodies in the serum samples of the above-mentioned population was 16.2% by EITB. Anti-Taenia solium larval stage antibodies were detected in serum samples of 163 patients, out of which 27 (16.56%) patients belonged to the 0 – 15-year age group, 82 (50.30%) patients were in the 16 – 40-year age group, and 52 (31.90%) patients were above 41 years, respectively. Although the sera from males had higher OD values than those from females, the difference was not statistically significant. Out of 163 seropositive by ELISA, 152 (93.25%) were found to be positive by EITB. Out of the 152, 61 (40.13%) were farmers and 79 (51.97%) were office or factory workers.

Conclusions:

In conclusion, the results indicate a probable endemic situation and a high prevalence of cysticercosis in patients with epileptic seizures. Living in poor sanitary conditions seems to be an important factor related to human cysticercosis in Puducherry and the neighboring districts of Tamil Nadu.

INTRODUCTION

Neurocysticercosis (NCC) is the infestation of the central nervous system (CNS) by the larval stage of pork tapeworm Taenia solium. Human beings acquire cysticercosis through fecal-oral contamination with T. solium eggs or poor hygiene practices in food handling by tapeworm carriers. The clinical presentation of NCC can be variable. Epileptic seizures are the most common presentation of NCC.[1,2] Various types of seizures have been described among patients with NCC. Recurrent seizures may occur at any stage of the disease, and may be the only symptom.[3] The seizures may be generalized or focal. NCC in humans has been reported as a major cause of epilepsy in many Latin American and African countries.[4–7]

Epilepsy due to NCC is a major problem in tropical, developing countries.[8] The incidence and prevalence of this disorder in these countries are high because of poor standards of neonatal care and high rates of infectious and parasitic diseases.[9] The prevalence of active epilepsy in India is between 2.2 and 11.93 per 1000 inhabitants.[10] The data regarding the prevalence of epilepsy due to NCC is unavailable in India.

In humans, NCC is the major cause of epileptic seizures and other neurological morbidities worldwide.[11] Given the fact that T. solium is endemic in this part of the country,[10] we studied the frequency of cysticercosis in epileptic seizure patients attending JIPMER hospital, using two serological tests ELISA and EITB for antibody detection. It has been proved that ELISA is adequate for serum screening in NCC studies.[11] The seroepidemiology of NCC in human population, in various geographical regions, has been studied using this method. The use of EITB represents a significant advance, because it allows the identification of specific antigenic proteins and eliminates false-positive results that are common when using the ELISA test.

MATERIALS AND METHODS

Serum samples

Nine hundred and thirty-four serum samples were collected from patients with epileptic seizures visiting the Departments of Medicine, Neurology, and Pediatrics, from November 2005 to March 2010. The samples were tested for the presence of antibodies to T. solium larval stage by ELISA. All the serum samples from patients with epileptic seizures were tested by ELISA. However, because of the limited availability of EITB, only those serum samples from patients which were reactive or equivocal by the ELISA were tested by EITB. The samples were analyzed in order to detect the seroprevalence of cysticercosis. Blood samples from all the subjects were obtained by venipuncture of the arm. Sera were stored at -20˚C, until the time of examination in the Parasitology laboratory. All the patients answered a questionnaire giving their demographic characteristics, hygienic habits, and sanitary conditions. Informed consent was obtained from all the adults participating in the study and from the parents or legal guardians of minors. The project was approved by the institutional review board of JIPMER..

CONTROLS

Sera collected from known positive NCC cases (confirmed by Magnetic Resonance Imaging (MRI), EITB, and ELISA) were used as positive control, as per the diagnostic criteria, for the diagnosis of NCC by Del Brutto et al.[12] For negative control, sera collected from normal healthy individuals were used. Sera collected from patients with hydatidosis (n – 20) were used to detect cross-reactivity.

CLINICAL INFORMATION

Clinical information was obtained directly from the patients, patient's medical records or from information provided by the patient's physicians, on a questionnaire. Determinations of clinical evidence of cysticercosis were based on the patients’ residences, travel histories, other epidemiological information, clinical signs, and symptoms (including seizures and other CNS events), findings on physical examination, radiographic or computed tomographic scan findings or MRI scan findings.

The T. solium metacestode somatic antigen

The T. solium metacestode somatic antigen was prepared from naturally infected porcine cysts (larval cysts) following the procedure described earlier.[13] punctured whole cysts (cysts after puncturing and removing the cyst fluid) were homogenized separately in a glass tissue homogenizer with phosphate buffered saline (PBS) pH 7.2, containing phenlymethylsulfonylfluoride (PMSF) 0.1,mM. Homogenization was done under cooling conditions. The homogenized tissue suspension was then sonicated eight times, at 12 kHz, with 30 seconds cooling interval. Each cycle of sonication was for one minute. The sonication was done under cooling condition, in an ice bath. The sonicated material was centrifuged at 4°C for 30 minutes at 14,000 rpm. The supernatant was collected as the somatic antigen.

ELISA for detection of anti-cysticercus IgG antibodies in serum

ELISA was performed as per the procedure described by the standard method of Crowther,[14] with a few modifications. Briefly, 1 μg protein per 100 μL concentration of the antigen was prepared in an antigen coating buffer (PBS pH 7.2) as per the method described earlier, which was used for coating plates, ( Nunc0 -Immuno Modules, Nalge Nunc0 International, Denmark) followed by blocking the non-specific binding sites by 2% Bovine serum albumin (BSA) in PBS pH 7.2. Optimum dilution (1: 50) of the test serum sample and known T. solium larval stage antibody-positive serum samples were prepared in a sample dilution buffer, added in duplicate in the wells, and goat anti-human-IgG-HRP conjugated secondary antibody (Bangalore Genei, India) was added to each well for detection. A substrate solution prepared freshly by adding 6 mg of OPD in 10 mL of PBS pH 7.2 and 10 μL of H₂O₂ (30%) was added just before use. The reaction was stopped by adding 50 μL of 2N H₂SO₄ in each well, to avoid overreaction and development of optimum color. The absorbance was taken at 492 nm in an ELISA reader (BIORAD Laboratories, Hercules, CA, USA). Various controls were used for validity of the assay, such as, antigen blank, antibody blank, negative control serum, and known positive control serum. A serum sample that gave OD492 value of more than cut-off (mean of 50 negative serum samples ±2 SD) was considered as positive. The cut-off used in the present study was 0.157±2 SD.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was performed for analyzing the protein profile of the antigen, by characterizing and separating proteins, based on their molecular weight. Briefly, the metacestode antigen was subjected to SDS-PAGE in 10% gel for resolving the antigenic peptides using a submerged gel electrophoresis apparatus (Bangalore Genei, India) and the standard protocol. The antigen preparations were separated under non-reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels, on 10 x 8 cm (1-mm thick) mini-gels (Bangalore Genei, India). The gels were stained with coomassie brilliant blue and the molecular weight was determined using standard molecular weight markers (Bangalore Genei, India). The molecular weights of the antigenic peptides were determined by comparing them with standard molecular weight markers (New England Biolab) that ran alongside.

Enzyme immunotransfer blot

The glycoprotein antigen used in Enzyme immunotransfer blot (EITB) was prepared by using metacestodes obtained from a naturally infected pig. to analyze the antibody response. For immunoblotting analysis, the above-mentioned separated antigens were electrophoretically transferred to a 0.22-μm-pore-size nitrocellulose membrane (NCM) (Pierce, Rockford, USA) in a mini-Transblot cell (Pharmacia Biotech, USA). The free binding sites were blocked by treatment with 3% BSA in PBS for two hours and washed in PBS containing 0.1% Tween 20. Analysis was carried out with human sera diluted at 1: 100 in PBS (pH 7.2) containing 0.1% Tween 20 (PBST). Horseradish peroxidase-conjugated goat antibody to human IgG diluted 1: 1000 (Bangalore Genei), was used as the secondary antibody. Antibody reactivity was visualized with a substrate solution containing diaminobenzidine (Pierce, Perbio, USA) (6 mg/mL in PBS containing one mL of 30% hydrogen peroxide (Sigma Aldrich, USA).

STATISTICS

Data were expressed as mean ± SD and assessed by the unpaired Student's t-test.

RESULTS

Enzyme linked immunosorbent assay

One hundred and sixty-three (17.45%) serum samples collected from epileptic patients were found to be reactive, by ELISA. The cut-off OD at 492 nm was 0.157±2SD. T. solium larval stage somatic antigen was used to detect IgG antibodies in the serum. The test detected antibodies in the sera from known positive controls, and did not show any antibodies in the sera of negative controls or in patients with hydatid disease. The optimal dilution of the serum for ELISA, using complete somatic homogenate for detection of IgG antibodies was standardized by checkerboard titration to be 1: 200. Out of the 20 sera collected from patients with hydatidosis, two samples cross-reacted, while all the sera from healthy controls were negative.

SDS-PAGE and enzyme immunotransfer blot

Analysis of the whole cyst antigen by SDS-PAGE revealed some 10 distinct protein bands with molecular weights ranging from 106 kDa to 10 kDa. The major antigenic peptides were found to be 100 kDa, 76 kDa, 68 Da, 40 kDa, 32 kDa, 24 kDa, and 18 kDa. An immunoblot run with the sera from patients with epileptic seizures, which were positive by ELISA demonstrated major antigen-antibody activity in the 40 kDa, 32 kDa, 24 kDa, and 18 kDa band. Of the 163 serum samples tested, 152 (93.25%) had anti-T. solium larval stage antibodies by EITB. The antibody to the T. solium larval stage somatic antigen, reacting with one or more of these peptides in the serum, was considered positive by the test.

Residential, gender, age, and occupational distribution of the patients

Out of 934 study patients, 784 (83.9%) were residents of Tamil Nadu (Villupuram, Cuddalore, and Thiruvannamalai districts, which are immediately neighboring to Puducherry) and 150 (16.1%) were residents of Puducherry. Among the 934 epileptic seizure patients 568 (60.8%) were males. Among the males, 92 out of 163 (56.44%) were positive by ELISA, while 43.56% of the females were positive at an OD of 0.157±2 SD. The sera from males had (0.188±0.060) OD values and the sera from females had OD values of 0.165±0.060; the difference was not statistically significant [P<0.05]. Out of the 163 samples tested positive by ELISA, 152 (93.25%) were found to be positive by EITB. Out of the 152, 61 (40.13%) were farmers and 79 (51.97%) were office or factory workers.

DISCUSSION

According to the International League against Epilepsy, cysticercosis is probably the single most common cause of acquired epilepsy in the developing world.[15] In the Latin American countries, a high proportion of patients with late-onset epilepsy are found to have concurrent cysticercosis.[16–20] Although the conditions in much of India appear favorable for T. solium transmission, there are very few data available on the prevalence of cysticercosis, either in the human population in general or among cases of epilepsy. The prevalence rates per 100,000 population of active epilepsy, febrile seizures (FS), cerebral palsy (CP), and tic disorders (TD) with 95% confidence intervals are, 700.87 (580.60 – 838.68), 1113.14 (960.07 – 1283.59), 282.70 (CI 208.43 – 374.82), and 35.34 (12.96 – 76.92), respectively. Active epilepsy prevalence shows a rising trend and that of other disorders a declining trend, with age.[21]

Poor hygiene and sanitary infrastructures along- with the free roaming of pigs, are the major reasons for the spread of NCC, which is a known cause of epilepsy.[22] The seroprevalences of cysticercosis recorded among epileptics in the present study are similar to those seen elsewhere,[16–20] and seem to indicate that neurocysticercosis is occupying an important role in the etiology of epilepsy in India.

In India, NCC has been reported widely from different parts of the country, such as Delhi,[23] Uttaranchal region,[24] Chandigarh,[25] and Puducherry.[26] Before the advent of the computed tomography (CT)/MRI era, the frequency of NCC as a cause of epilepsy in India was reported to vary from 2.2 to 9.6%.[27,28] After the availability of CT / MRI imaging, NCC has been found to be the cause in 9 to 18.6% of the patients with epilepsy.[29,30] The neuroimaging techniques such as CT and MRI have been used for a quicker and more accurate diagnosis of NCC. Magnetic resonance imaging is more sensitive and specific for identifying NCC; however, these methods are not yet commonly available in most hospitals. Therefore, in the JIPMER Hospital, immunological methods such as ELISA and EITB have been used for the diagnosis of NCC. ELISA and EITB are the most frequently used antibody-based tests for the detection of antibodies in the serum. The ELISA has been used widely by various authors for the detection of cysticercus antibodies in the serum, with variable sensitivities of 10.4,[31] 50,[32] 79,[33] and 93%,[34] and specificities of 70[31] and 93%,[34] for the diagnosis of NCC. Also the ELISA test has demonstrated cross reactions with other parasitic diseases such as hydatid disease, schistosomiasis, and angiostrongyliasis.[32,33] In the present study, the ELISA test showed a low sensitivity of 91%, but a high specificity of 98%.

The EITB might be expected to be more specific than the ELISA, because the latter uses crude cyst fluid as the antigen, while the former separates major glycoprotein antigens from the extracts of cysticerci; the glycoproteins are blotted onto nitrocellulose strips and appear as separate bands. Antibodies to T. solium react with at least one of these specific bands, whereas, nonspecific or cross-reacting antibodies should not. The sensitivity and specificity of the EITB are reported to be 98 and 100%,[35] respectively, in patients with two or more cysts, but the sensitivity is lower in patients with single enhancing or calcified cysts; only 28% of the patients with single lesions have had positive EITB results.[36] Several recent studies have compared the EITB and the ELISA in the detection of antibodies against T. solium cysticerci.[35,36] In general, the results indicated that the EITB was more sensitive and specific than the ELISA.

Anti-cysticercus antibodies were demonstrated by ELISA in 5.6% of 1133 sera of blood donors in Brazil[37] and by EITB in 13% sera of 108 individuals in a village in the highlands of Peru.[38] Other similar studies have been demonstrated a seroprevalence of 10.8% in Morelos, Mexico,[39] 23% in Peruvian highlands,[38] in 5.1 cases of mentally altered individuals in Columbia, and as high as 34% in the rural community in Honduras.[40] In India, similar serological studies have shown the presence of cysticercus antibodies in 17.4 to 29.2% sera in patients of epilepsy and intracranial space occupying lesions, in Chandigarh,[41] in 18.75% of epilepsy patients and 13.33% of patients with increased intracranial tension and in 5.47% CSF of children with subacute and chronic meningitis, in Bangalore.[42] In a study conducted in Vellore, the prevalence of active epilepsy was 3.83 per 1,000 people, with the prevalence in the urban clusters more than twice that in the rural clusters (6.23 vs. 3.04 per 1,000).[43] Ahuja et al., in 1998, observed a positive reaction in serum for cysticercosis in 30% of the epileptic seizures patients.[44] In the present study, we detected antibodies to the T. solium larval stage in patients with epileptic seizures visiting JIPMER Hospital, Puducherry, South India. In this study, we observed that out of 934 serum samples collected from epileptic patients, 163 (17.45%) serum samples were positive by ELISA. Out of these 163 positive sera, 152 (16.2%) were positive by EITB. This value is slightly higher where the seroprevalence of cysticercosis has been reported as 6.1% in the normal population at Puducherry. The results of the study show that cysticercosis is a major cause of epileptic seizures in patients in South India.

The reason for a high prevalence of cysticercosis in epileptic seizure patients may be many. It may be due to the low socioeconomic status of populations, where rearing of pigs is common, and many people in the rural area consume pork, as it is relatively cheaper than mutton or chicken. The infected person may also act as a source of infection by contaminating the food and water by their act of defecation in the open field, as they live in low sanitary conditions.

Gender, age, and occupation, as well as pork consumption may be associated with teniasis and cysticercosis. A higher incidence of T. solium infections in males than females has been reported from Papua, Indonesia[45,46] and vice versa in some Central American countries.[47–49] Infection risk appears to be highly dependent on the lifestyle. However, the number of cysticercosis patients are higher in males than females in South India. This might be attributed to the fact that in South India males are more involved in the outside activities and consumption of improperly made food, than females.

NCC occurred in all age groups, but the rates of infection reached a maximum in the 16 to 40-year-age group. This was almost similar to the other reports from various parts of the world. In Inner Mongolia, this age group comprised of 82% of all the patients, relatively similar to the statistics of the whole nation, which was 87.8% from age 15 to 59.[50] The age group between 7 and 30 years were mostly infected with helminths in Philippines. The overall seroprevalence of cysticercosis in this population was 24.6%, with no significant difference based on age, sex, or other helminth coinfection status.[51]

A slightly higher rate of infection was seen in people living in the urban area than in the rural area, where rearing of the pigs is common, and inadequate sanitary infrastructure is maintained similar to an earlier study.[43] The reason for the slightly high level of infection in the urban area may be due to the consumption of improperly cooked pork or the increased use of raw vegetables as salads. However, the data indicates that the prevalence of NCC is almost equal in urban and rural areas of Puducherry and Tamil Nadu.

CONCLUSION

The present study indicates that in the Indian subcontinent, epilepsy is the most predominant symptom of NCC. The commonly used methods such as ELISA- and EITB-based immunodiagnosis are an alternative to neuroimaging methods. The results of this study indicate a rising trend of NCC and its seroprevalence in patients with epilepsy.