High prevalence of neurocysticercosis among patients with epilepsy in a tertiary care hospital of Assam, India.

Background: Neurocysticercosis (NCC) is a parasitic disease of the central nervous system, which is caused by the metacestode of the pork tapeworm, Taenia solium. The present unicentric, hospital-based, cross-sectional study was undertaken to assess the contribution of NCC as a cause of active epilepsy among patients attending a tertiary health care center in Assam, India. Materials and
Methods: Over a period of 2 years, 152 active epilepsy patients were investigated based on clinical, epidemiological, neuroimaging (contrast-enhanced computerized tomography), and immunological techniques to establish the diagnosis of NCC. A precoded questionnaire was administered to patients and/or guardians to collect detailed medical history.
Results: Ninety-three cases (61.2%) fulfilled either definitive or probable diagnostic criteria for NCC. Anti-cysticercus immunoglobulin G antibodies were detected by ELISA and enzyme electro-immune transfer blot in 69 (45.4%) active epilepsy patients. Seroprevalence was higher in males, 46.6% (54/116); than in females, 41.7% (15/36), and increased significantly with age; peaking in the 20-39 years age group (36/76; χ2 = 5.64; P = 0.02). Among the seropositive cases, 54 (78.3%) were diagnosed with NCC. A significantly higher number of seropositive individuals were diagnosed with NCC in the 20-39 years age group as compared to the 40 years and above age group (χ2 = 6.28; P = 0.01). The association between seropositivity for NCC, and the number of lesions in the brain was statistically significant (χ2 = -8.33; P = 0.003). Conclusions: This study indicates that NCC is a major cause of active epilepsy in Assam. A high prevalence of pediatric NCC is also a major concern. Copyright:

INTRODUCTION

Epilepsy is a neurological disorder that affects >10 million patients in India.[1] It is also a major public health concern in the tea garden communities of Assam, Northeast India.[2] One of the main causes of community-acquired epilepsy in resource-poor endemic regions (Asia, Sub-Saharan Africa, and Latin America) is neurocysticercosis (NCC), which is caused by the metacestode (larva) of the pork tapeworm, Taenia solium.[34] Humans (only definitive host), suffer from taeniasis when the adult tapeworms reside in the intestine, while both humans and pigs (intermediate hosts) suffer from cysticercosis as the larvae infect various soft tissues.[56] NCC is the most severe form of cysticercosis wherein cysts develop in the central nervous system (CNS), leading to pathological symptoms, including epileptic seizures, headaches, and hydrocephalus.[4] It can be acquired under any cultural and socioeconomic conditions where there is close contact with a taeniasis carrier, but its management becomes problematic in resource-poor settings where sanitation, hygiene, and pig management practices are poor.[4]

NCC has been reported from different parts of India, but reports about its prevalence and association as a definable risk factor of epilepsy are largely lacking from Northeast India. Hence in light of the above, the current hospital-based study is aimed to determine the incidence of NCC among people with active epilepsy at a tertiary health-care facility in Assam, India.

MATERIALS AND METHODS

Ethics approval

The present study was approved by the ethics committee, and all the experiments were carried out in accordance with that approval. Written informed consent was obtained from all patients and/or their guardians (in the case of minors) before including them for this study.

Patients

This hospital-based study was conducted at Assam Medical College and Hospital in Dibrugarh-a major tertiary health care center in the study area. The sample size was calculated, assuming that the occurrence of NCC related seizures among people with epilepsy (p) would be 10% (based on data from prior clinical studies conducted in endemic regions). Other assumptions made during the sample size calculation were a 95% confidence interval (CI) with a 5% margin of error (d). This yields a sample size of 138. Assuming 10% nonresponse, we planned to enroll 152 active epilepsy patients. All patients older than 5 years, who reported seizures, were evaluated by a neurologist. Patients with a history of any other chronic disease or with a history of head injury and females with underlying pregnancy were excluded from the present study. At the end of 2 years, a total of 152 patients with active epilepsy were recruited.

Data collection

A precoded questionnaire was administered to patients and/or guardians in the local language to collect detailed medical history, including the history of seizure as well as other known risk factors leading to epilepsy such as head trauma, alcohol consumption, and diseases like tuberculosis.

Diagnostic criteria for active epilepsy

A prevalent case of active epilepsy was defined as two or more afebrile seizures unrelated to the withdrawal of alcohol or drugs or to acute metabolic disorders within 5-year period of the interview.[7] Epileptic seizures and syndromes were grouped according to a classification suggested for developing countries, which conforms to the International League Against Epilepsy guidelines.[89]

Diagnostic criteria for neurocysticercosis

The diagnosis of NCC was based on the modified diagnostic criteria suggested by Del Brutto and guidelines specific to the Indian context by Garg.[1011] These guidelines are intended to help differentiate between cysticercus granuloma and tuberculoma as we have previously reported a high incidence of pulmonary tuberculosis in the tea garden community of Assam.[12] Based on the objective evaluation of clinical, radiological, immunological, and epidemiological data, a set of defined criteria were used to establish cases into either definitive or probable NCC.

Computed tomography scan of the brain

Contrast-enhanced computed tomography (CT) imaging of the brain was done for all cases. Neuroimaging studies were carried out to identify cystic lesions, which are pathognomonic for cysticercosis. Cysticerci in the brain parenchyma were initially classified into three groups according to definitions proposed by Carpio et al.[13] These cysticerci were then classified into four histopathological stages : Active (vascular), transitional (colloidal vascular and granular vascular), and inactive/calcified (nodular calcified); based on suggestions made by Escobar and Salgado et al.[141516] Scolex within a parenchymal cysticercus cyst was considered absolute and diagnostic of NCC. Brain imaging observations, including cystic lesion without a visible scolex, parenchymal brain calcifications, and single or multiple rings or nodular enhancing lesions; were considered highly suggestive of NCC. A diagnosis of hydrocephalus or abnormal leptomeningeal enhancement was considered compatible with NCC.[17] Solitary calcifications were considered compatible only after other forms of granulomatous diseases, mainly tuberculosis, were clinically ruled out.

Enzyme electro-immune transfer blot analysis

For the immunoblot assay, we used cystic fluid obtained from cysticerci collected from locally available infected pigs. Immunoblotting was carried out by a slight modification of the method described earlier.[18] A sample was considered positive if one or more bands <50 kDa were detected.

Seroprevalence of immunoglobulin G antibodies against Taenia solium metacestode

A total of 152 serum samples were available for the detection of specific immunoglobulin G (IgG) antibodies against T. solium metacestode. These samples were tested using an in-house ELISA method, and absorbance measurements were taken at 492 nm using a microplate reader. The sensitivity, specificity, positive, and negative predictive values of the IgG-ELISA test was determined using sera samples from confirmed NCC cases and sera from patients infected with other parasitic diseases. During our internal testing, the IgG-ELISA test had a 100% sensitivity for the diagnosis of NCC in patients with multiple (active and mixed) cysts in the brain parenchyma (unpublished data).

Data analysis

Data were compiled and subjected to analysis using IBM SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). The outcomes for continuous variables are expressed as median (interquartile range). Categorical data values are presented as numbers. For analyses, study participants of different ages were segregated by age into four groups (≤10, 11–19, 20–39, ≥40 years). The Chi-square test was performed for testing the differences between proportions across select categorical variables (https://www.medcalc.org/calc/comparison_of_proportions.php, last accessed on 22.06.2020). All statistical tests were two-tailed. A probability (P) value of < 0.05 was used as the level of significance.

RESULTS

The study made 93 (61.2%) diagnoses of NCC among the active epilepsy cases with a male, 75/116 (64.7%); to female, 18/36 (50.0%); ratio of 4.17:1 (χ2 = −2.48; P = 0.115). The age range of the male cases was 5–74 years (median age = 30 years) and that of female cases was from 8 to 65 years (median age = 27.0 years). All of these cases had parasitic lesions suggestive of NCC on CT imaging. Solitary lesions were more predominant (61/93; 65.6%) than multiple lesions (32/93; 34.4%) [Table 1] [Figure 1]. Among individuals with a solitary lesion, the active ring-enhancing lesion is the most frequent presentation (39/93).

Table 1

Computed tomography scan features of active epilepsy patients with neurocysticercosis (n=93)

Type of lesion	Stage of cyst	Number (%)
Solitary cystic lesions (n=61; 65.6%)	Active	39 (63.9%)
	Inactive/calcified	22 (36.1%)
Multiple cystic lesions (n=32; 34.4%)	Active	9 (28.1%)
	degenerative	13 (40.6%)
	Inactive/calcified	10 (31.3%)
Total		93 (100%)

CT findings classified into three groups as suggested by Carpio and Escobar. CT: Computed tomography

Figure 1

Contrast-enhanced computed tomography scan showing (a) solitary, and (b) multiple ring enhancing lesions with surrounding edema

Of the 152 active epilepsy patients tested, 61 (40.1% overall) had IgG antibodies for T. solium cysticercosis by ELISA [Table 2]. Interestingly, 15 patients who tested positive had no apparent brain lesions on CT. Further, the association between seropositivity for NCC, and the number of lesions in the brain was found to be statistically significant (χ2 = 8.33; P = 0.003). Patient samples that tested negative during IgG-ELISA were further evaluated by enzyme-linked electro-immune transfer blot (EITB) assay. Overall, anti-cysticercus IgG antibodies were detected in 69 (45.4%) active epilepsy patients; the majority of whom presented solitary active lesion (29.0%) followed by multiple degenerative lesions (18.8%), multiple active lesions (13.0%), solitary calcified lesion (11.6%), and multiple calcified lesions (5.8%).

Table 2

Results of anti-cysticercus immunoglobulin G-ELISA (n=152) in active epilepsy patients with respect to type of lesions detected in brain by computed tomography

Number of lesions in brain	n (%) of sera tested positive	n (%) of sera tested negative
No lesion (normal scan), n=59	15 (25.4)	44 (74.6)
Solitary active, n=39	17 (43.6)	22 (56.4)
Solitary calcified, n=22	6 (27.3)	16 (72.7)
Multiple active, n=9	9 (100.0)	0 (0.0)
Multiple degenerative, n=13	13 (100.0)	0 (0.0)
Multiple calcified, n=10	1 (10.0)	9 (90.0)

Seroprevalence (as determined by EITB or IgG-ELISA) was higher in males, 46.6% (54/116); than in females, 41.7% (15/36), and increased significantly with age; peaking in the 20–39 years age group (36/76; Overall trend χ2 = 5.64; P = 0.02) [Table 3]. In this study group, involuntary muscle movements were the most common clinical presentation (39.7%), followed by unconsciousness (36.0%) and headaches (35.3%). Most of these active epilepsy patients (83.8%) had a history of pork consumption, which significantly increased their risk of acquiring NCC (odds ratio [OR], 2.73; 95% CI, 1.1–6.7; P = 0.03).

Table 3

Gender and age-associated changes in neurocysticercosis seropositivity in active epilepsy patients

Age (years)	n (%) of sera tested positive
Males (n=116)
≤10	1 (20.0)
11–19	6 (33.3)
20–39	29 (49.2)
≥40	18 (52.9)
Females (n=36)
≤10	0 (0.0)
11–19	3 (42.9)
20–39	7 (41.2)
≥40	5 (50.0)

Neurocysticercosis seropositivity was determined with use of the EITB or IgG-ELISA. EITB: Immunoglobulin G Enzyme electro-immune transfer blot analysis

DISCUSSION

NCC has been identified as a major cause of epilepsy in the developing world. However, in spite of India being in the endemic belt, the proportion of epilepsy due to NCC is not well documented.

In the present study, a high prevalence of NCC was recorded among active epilepsy patients attending a tertiary care center in Dibrugarh, Assam. Ninety-three cases (61.2%) fulfilled either definitive or probable diagnostic criteria for NCC; which is greater than those reported by previous hospital-based studies from NCC endemic areas in South Africa (61.1%), Bhutan (25.4%), Rwanda (23.3%), Nepal (16.0%), Tanzania (2.0%–13.7%), Peru (12.0%), and rest of India including Odisha (43.7%), and Andhra Pradesh (27.5%) suggesting that NCC is a larger public health problem in this region.[192021222324252627]

Solitary lesions were the predominant finding in 65.6% of patients. This observation is in agreement with prior clinical and community studies from India, wherein NCC presents itself as solitary cysts but unlike reports from Latin America and China where most cases present more than one viable lesion.[27282930313233] In this study, all the cases presented parenchymal NCC. Parenchymal NCC is more common in the Indian subcontinent, while extraparenchymal NCC is frequent in Latin America.[33] This variability could be related to a complex range of host-parasite and environmental interactions.

Seroprevalence against T. solium was also evaluated using both ELISA and EITB. While EITB is the preferred method over ELISA, studies have described that ELISA based on cystic fluid/vesicular fluid of T. solium cysticerci is highly dependable for immunodiagnosis of NCC.[34] As determined by ELISA, 40.1% (61/152) active epilepsy patients and 49.5% (46/93) NCC patients were seropositive. The seronegativity observed during ELISA may be partly associated with the fact that the average time between acquisition of parasite and development of symptomatic infection is about 7 years, and the fact that a sizable number of calcified lesions (solitary, 22/61 and multiple, 10/32) were recorded among NCC patients, which are known to illicit minimal immune response.[2535]

In our case-series, specific serology with EITB method is shown to be better sensitive, especially in cases with “calcified” lesions. The EITB assay detected circulating anti–T. solium antibodies in eight ELISA negative, CT-positive cases (8/47; calcified 62.5% vs. active 37.5%). ELISA negative individuals, with no evidence of cysticercosis of the CNS (NCC) were not considered for EITB. These results are consistent with previous findings that the diagnostic performance of EITB (sensitivity and specificity) when performed using serum is better than that of ELISA.[2036] Furthermore, detection of antibody in the serum of patients who presented only calcified NCC lesions does not necessarily imply the existence of undetected viable brain cysts as circulating antibodies may persist over a period of several months or years after the parasites have died.[36]

Seropositivity rate was significantly higher among patients with late-onset epilepsy (after age 20) than in those with earlier onset epilepsy (χ2 = −2.31; P = 0.04). Within this cohort, seroprevalence was higher in males, 46.6% (54/116); than in females, 41.7% (15/36), and increased significantly with age; peaking in the 20–39 years age group (36/76; overall trend χ2 = −5.64; P = 0.02) [Table 3]. Although statistically insignificant, data generated during a regression analysis suggest that individuals in the 20–39 years age group were 2.7-fold more likely to suffer from epilepsy due to NCC (OR, 2.72;95% CI, 0.6–13.1). There is also a significant difference in the number of seropositive individuals diagnosed with NCC in the older age groups (χ2 = −6.28; P = 0.01)-88.9% in the 20–39 years age group versus 60.9% in the 40 years and above age group. Such age-dependent difference in the manifestation of NCC has been reported previously and draws us toward concluding that individuals aged 40 years and above may have developed immunity against T. solium NCC.[33]

Interestingly, in the pediatric age group (all patients aged 1–16 years, n = 23), 4 out of 5 (80.0%) of the seropositive cases and 8 out of 18 (44.4%) of the seronegative cases were diagnosed with NCC (12/23, 52.2%). Although the sample was relatively small and validation requires further research, these rates are greater than those reported by previous hospital-based studies from Nepal (overall frequency 43.0%), Italy (0.4%), Madagascar (17.6%), and rest of India, including Andhra Pradesh (37.7%), and Puducherry (22.8%).[3738394041] The prevalence, however, is less than that reported from Haryana (54.4%).[42] NCC is less frequently among children (52.2% vs. 62.8%), probably due to age-based differences on the mode of disease acquisition and differences in immune reactivity against the parasite (seropositivity, 21.7% vs. 49.6%).[43]

CONCLUSIONS

Our study identified NCC as a major cause of epilepsy in this region. A high prevalence of pediatric NCC is of major concern and warrants further investigation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.