What triggers seizures in neurocysticercosis? A MRI-based study in pig farming community from a district of North India

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1 Available online at Parasitology International 57 (2008) What triggers seizures in neurocysticercosis? A MRI-based study in pig farming community from a district of North India Amit Prasad a, Rakesh K. Gupta b, Sunil Pradhan c, Mukesh Tripathi d, Chandra M. Pandey e, Kashi N. Prasad a, a Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow , India b Department of Radiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India c Department of Neurology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India d Department of Anesthesia, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India e Department of Biostatistics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India Received 13 June 2007; received in revised form 25 November 2007; accepted 1 December 2007 Available online 8 December 2007 Abstract Colloidal and calcified cysts are considered responsible for seizure in neurocysticercosis (NCC); however, calcified cysts have also been reported in asymptomatic individuals. We carried out a MRI-based study in a rural pig farming community of North India to detect the various stages, locations and numbers of the cyst in asymptomatic individuals and compared them with symptomatic NCC cases to see its association with occurrence of seizures. A total of 107 asymptomatic family members of 29 symptomatic NCC confirmed cases were evaluated clinically, immunologically and by neuroimaging for NCC. MRI-based staging of the parasite was done in both groups, and compared to look for the differences, if any. Thirty-one (29.0%) asymptomatic family members of symptomatic cases were diagnosed to have NCC. There was no difference in proportion of colloidal/degenerating and calcified stages of the parasite between symptomatic and asymptomatic groups; however, significantly higher proportion of the asymptomatic populations had vesicular stage of the parasite (P = 0.029). Our findings show that a large number of individuals harboring different stages of cysticerci in their brain remain symptoms free and question the belief that the degenerating/ calcified stages of the parasite are primarily responsible for seizure occurrence in NCC Elsevier Ireland Ltd. All rights reserved. Keywords: Neurocysticercosis (NCC); Brain; Magnetic resonance imaging; Seizures; Taenia solium 1. Introduction Neurocysticercosis (NCC), caused by larval stage of the tapeworm Taenia solium, is the most common parasitic infection of the central nervous system (CNS) in humans in South Africa, Eastern Europe, South America and South East Asia; its incidence varies from per thousand individuals [1 4]. NCC is becoming a serious problem with its worldwide distribution [5]. Parenchymal NCC presents with seizures and headache in 50 80% cases [6]. Thus in endemic areas, it is considered in the differential Corresponding author. Tel.: x2265; fax: address: knprasad@sgpgi.ac.in (K.N. Prasad). diagnosis for a new-onset seizure. Besides these two common symptoms, it also produces focal neurological deficit, chronic meningitis, and hydrocephalus [7]. The symptoms and signs of NCC are non-specific and depend upon the number, topology and stage of the cyst as well as the host immune response to the parasite in the brain [8,9]. Seizures may be single, clustered or recurrent; and either focal with or without secondary generalization or may be generalized at the onset. According to present understanding, viable cysts with little or no enhancement or edema on imaging are usually not associated with symptoms [1]. Acute seizures are more frequent with the transitional form owing to the pericystic inflammatory host immune response [10]. In most patients, symptoms occur years after the initial invasion of the nervous system by the parasite, either by inflammation around the parasite, mass effect, or residual perilesional scarring [10 12]. The single /$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi: /j.parint

2 A. Prasad et al. / Parasitology International 57 (2008) calcified lesion is considered to be one of the causes of chronic epilepsy, as the calcified cysts with persistence of scolex may act as source of antigen that provokes inflammatory reaction [12 14]. Seizures may also be due to infarcts caused by perivascular inflammation of degenerating subarachnoid cysts in close contact with blood vessels. The clinical course of symptomatic and asymptomatic NCC is poorly understood. None of the published studies has followed the asymptomatic NCC cases for the development of seizure; most of the studies have demonstrated the association of brain imaging findings in patients with seizure [14,15]. Patients with severe refractory seizures may have only one calcified lesion; on the other hand there are patients with multiple cysts or calcifications but no seizures [16,17]. Though seizures are common events in patients with NCC in most of the studies, the exact relationship between seizure and NCC is not yet clear [18]. While working in a pig farming rural community to determine the prevalence of NCC related seizure, we screened asymptomatic family members of the individuals with NCC related seizures in order to detect asymptomatic NCC cases and the stage of the parasite. The aim of this imaging based study was to explore the association of different stages of the cysts in asymptomatic and symptomatic individuals. 2. Methods 2.1. Study population and data collection The data for this study has been taken from a rural survey to assess the magnitude of T. solium taeniasis and systemic cysticercosis of pig farming community in Mohanlalganj block, Lucknow district of Uttar Pradesh, which is the largest state of India. More than 45% populations of this block belong to pig farming community, and pig to human ratio is 1:2 in the locality. Pork and pork products are accepted as food in this community and the study area is highly endemic for T. solium taeniasis [19]. Patients with epilepsy among the pig farming community were identified during a door-to-door survey. A structured questionnaire was developed to collect information from all individuals of households having patients with epilepsy. Diagnosis of NCC in symptomatic (patients with epilepsy) and asymptomatic populations was made based on clinical, imaging (MRI), immunological (EITB) and epidemiological criteria [20,21]. Seizures in patients were classified according to the guidelines of International League Against Epilepsy [22]. A total of 60 patients with epilepsy agreed for clinical, immunological and imaging (MRI) evaluations. Definitive diagnosis of symptomatic NCC (NCC with epilepsy) was made in 29 of them. A total of 107 asymptomatic family members of these 29 NCC related seizure cases volunteered for NCC evaluations. Asymptomatic cases were defined as individuals with no evidence/history of seizure, sensory or motor neuro-focal deficit, cranial nerve involvement and psychiatric or behavioral abnormalities. We selected family members of the individuals suffering from NCC related seizures, as it was easy to convince them to volunteer for the study. Asymptomatic cases were followed up for six months for the development of any symptoms related to NCC. The Institute's ethics committee approved the work and all individuals included in the study consented for enrollment Magnetic resonance imaging (MRI) of brain With informed consent, MRI was performed in all these cases on a 1.5 Tesla scanner (Echo-speed plus, General Electric, Milwaukee, USA) using quadrature transmit receive head coil. The base line T2 [repetition time (TR)/echo time (TE)/number of excitations (NEX)=4900 ms/85 ms/2)], T1 (TR/TE/NEX=650 ms/9 ms/1) with slice thickness of 5 mm, 0.5 mm interslice gap, matrix. As a part of the routine protocol gradient echo (GRE) sequence with corrected phase was also performed to detect the calcification with TE/TR/FA=40 ms/800 ms/20 [15,23]. Postcontrast T1 imaging was also performed after injecting gadodiamide (Gd-DTPA-BMA, Omniscan, Amersham Health As, Oslo, Norway) intravenously at a dose of 0.1 mmol/kg body weight. MRI was evaluated by the radiologist who was blinded to the clinical status of the individuals. The lesions were classified into different stages (vesicular, colloidal/degenerative, calcified and more than one stage) as described earlier [23]. We also quantified the volume of edema in cases having colloidal/degenerative cyst from T2- weighted image using NIH image J software program Enzyme electroimmune transfer blot (EITB) At the time of MRI, blood was collected with consent of the individual for EITB as described earlier [24], and the sample was considered positive if one or more bands of b50 kd were detected Statistical methods Overall association between different stages was determined by Chi square test, while proportion of stages of cysts, number of cysts, location of cysts in brain and symptoms were analyzed by z-test. The difference between volumes of edema was compared by independent t-test. Analysis was done using SPSS statistical software, version 14.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. MRI-based studies on cerebral lesions A total of 60 patients with epilepsy consented to undergo clinical, immunological (EITB) and brain MRI studies. On MRI study, 29 (48.3%) of them had cystic lesions in brain and fulfilled the definitive/probable diagnostic criteria of NCC. Among the family members of these 29 symptomatic NCC cases, 31/107 (29.0%) showed cystic lesions in the brain on neuroimaging and fulfilled the definitive/probable diagnostic criteria of NCC. Higher proportion of patients with epilepsy had NCC than the household members (48.3% vs. 29.0%; P = 0.019). Partial seizures with secondary generalization were the most common finding (14/29; 48.3%) among NCC positive patients with epilepsy, followed by complex partial seizures 7 cases (24.1%), generalized seizure 5 cases (17.2%) and unclassified seizure 3

3 168 A. Prasad et al. / Parasitology International 57 (2008) cases (10.3%). Fig. 1a c show the distribution of symptomatic and asymptomatic NCC cases according to the number, location and staging of cystic lesions in the brain, respectively. The total number of cysts identified in symptomatic cases was 62, while in asymptomatic family members it was 47. Occurrence of single cyst was the most common finding in both symptomatic and asymptomatic groups (44.8% and 64.5%, respectively) (Fig. 1a). In symptomatic group the cysts were most commonly localized at multiple sites (44.8%) followed by frontal lobe (34.5%), while in the asymptomatic group frontal lobe (45.2%) was most commonly involved followed by multiple sites (25.8%) (Fig. 1b). Cysts at the calcified stage were most common in both symptomatic and asymptomatic groups, 44.8% and 35.5%, respectively (Fig. 1c). Significantly higher proportions of asymptomatic family members had vesicular cysts in their brain as compared to symptomatic (epileptic) patients on z-test (22.6% vs. 3.4%; P = 0.029). However, no significant association was observed between presence or absence of seizures and other stages (colloidal/degenerative, calcified and more than one stage) of the parasite (P=0.176), location of cyst in the brain (P=0.359) as well as with the number of cysts present (P=0.301) on Chi square test. In both groups, single cyst (symptomatic vs. asymptomatic, 44.8% vs. 64.5%) and calcified stage (symptomatic vs. asymptomatic, 44.8% and 35.5%) were the most common finding. The representative figures of different stages of cysts among symptomatic and asymptomatic cases are shown in Figs. 2 and 3, respectively. The most interesting observation was that 5 (16.1%) NCC positive asymptomatic family members had colloidal/degenerative parasite with rim enhancement and 3 (9.7%) with calcified lesions were having perilesional edema on post-contrast study. There was no significant difference between the volume of edema around the colloidal/degenerative cyst of symptomatic and asymptomatic cases (P = 0.068) EITB studies All the symptomatic and asymptomatic NCC cases were found to be positive on immunoblot for one or more bands. The most commonly identified bands were GP24, GP 21, GP 18 and GP 14 either in isolation or in combinations by both symptomatic as well as asymptomatic cases (Fig. 4). The band GP24 was present in all the cases included in the study. 4. Discussion Our results show significantly higher proportion of NCC in symptomatic cases than asymptomatic family members. The Fig. 1. MRI-based cyst burden and staging in symptomatic (grey bar) and asymptomatic (dotted bar) individuals from pig farming community with neurocysticercosis (NCC). (a) Distribution of cyst burden in symptomatic vs. asymptomatic NCC cases; single cyst (44.8% vs. 64.5%, P=0.126), two cysts (27.6% vs. 16.2%, P=0.282), and multiple cysts (27.6% vs. 19.4%, P=0.451). (b) Distribution of cyst according to location in symptomatic vs. asymptomatic NCC cases; frontal lobe (34.5% vs. 45.2%, P=0.398), temporal lobe (13.8% vs. 12.8%, P=0.343), parietal lobe (0 vs. 9.7%, P=0.085), occipital lobe (6.9% vs. 6.5%, P =0.944), and multiple locations (44.8% vs. 25.8%, P=0.122). (c) Distribution of MRI-based staging of cysts in symptomatic vs. asymptomatic NCC cases; 1. Vesicular (3.4% vs. 22.6%, P 0.029), 2. Colloidal/degenerating (24.1% vs. 16.1%, P=0.438), 3. Calcified/healed (44.8% vs. 35.5%, P=0.460), and 4. More than one stage (27.6% vs. 25.8%, P=0.876).

4 A. Prasad et al. / Parasitology International 57 (2008) Fig. 2. Axial MR images of five clinically diagnosed symptomatic NCC cases with different stages (arrow) of cyst. T2 and T1 weighted axial images (upper and lower rows, respectively) at different sections show (a) vesicular cyst with scolex in the right occipital lobe, (b) colloidal/degenerating cyst with scolex and perifocal edema in the right occipital lobe, (c) healing cyst in the right frontal lobe, (d) late healing cyst in the left frontal lobe and (e) multiple calcified lesions in both cerebral hemispheres showing bloom effect on T2 weighted images consistent with calcified lesions. distribution of colloidal/degenerating and calcified stages of the cyst in symptomatic and asymptomatic cases was almost similar; however, the vesicular stage of parasite was more frequently present in asymptomatic cases. The interesting observation of the study was that (i) 5 (16.1%) asymptomatic (seizure free) cases had colloidal cyst with contrast enhancement and (ii) 11 (35.5%) had calcified lesions (3 with perilesional edema as well). However, White and Khun (1997) have reported a clear-cut association between degenerating or inflammatory cyst and seizure [10]. In recently published guideline for diagnosis of NCC, Garcia et al. (2005) have associated cysts with edema or ring enhancement with symptoms, while viable cysts with little or no enhancement or edema are usually not associated with seizure [21]. Nash et al. (2004) has suggested that calcified cysticercosis is not clinically inactive but a cause of seizures or focal symptoms, as they found a positive correlation between endemic population with increased proportions of calcification and seizure activity [17]. In our study population the presence of large number of asymptomatic NCC individuals with similar distribution of parasite load and stages of the cyst (except vesicular stage) as in symptomatic cases raises a question as to what triggers seizures in NCC. This question gains further importance as the asymptomatic NCC group had similar proportion of degenerating NCC with edema Fig. 3. Axial MR images of five asymptomatic cases with different stages (arrow) of cyst. T2 and T1 weighted axial images (upper and lower rows, respectively) at different sections show (a) vesicular cyst with scolex in the left occipital lobe, (b) colloidal/degenerating cyst with scolex and perifocal edema in the right parietooccipital lobe, (c) cyst in the left occipital lobe, (d) cyst in the right parieto-occipital lobe and (e) calcified cyst in right occipital lobe showing bloom effect on T2 weighted images consistent with calcified lesions.

5 170 A. Prasad et al. / Parasitology International 57 (2008) Fig. 4. Enzyme electroimmune transfer blot profile of four symptomatic (lanes 1 4) and four asymptomatic (lanes 5 8) NCC cases. and contrast enhancement, a finding commonly implicated to cause acute seizure in symptomatic NCC patients. Our observations are further supported by a necropsy study on war victims and road traffic accident deaths that showed a large numbers of different stages of NCC, in otherwise asymptomatic individuals [25]. A CT based study in Salama, Honduras had revealed that the majority (84%) of individuals with active or calcified NCC compatible lesions were asymptomatic [26]. Similarly in a study from rural Ecuador, 17 (14.4%) out of 118 randomly selected healthy volunteers were having the evidence of NCC on CT neuroimaging [27]. The relative contribution of the cyst stage, parasite genotype, host immune status, genetic make up of the individuals or combination of factors for the development of seizure needs to be explored. MRI is the best neuroimaging tool available for the detection of degenerating and innocuous cysticerci [28]. However, the literature on neuroimaging describes MRI as less sensitive to CT for calcified lesions and CT is considered to be the best neuroimaging procedure for patients with suspected calcified NCC lesions [23]. In general MRI provides better image detection and definition, and its high contrast resolution allows it to recognize any form of NCC not visualized on CT. To our knowledge, this is the first study on MRIbased staging of cyst in asymptomatic subjects in community setting and comparison of the occurrence of different stages of cysts, number of cysts and their location in the brain with symptomatic NCC cases. Earlier a number of studies had reported the occurrence of NCC compatible lesions in healthy controls [26,27,29,30], but none had compared them with symptomatic populations. We followed up these patients for six months and they remained asymptomatic. It has been reported that genetic differences in cysticerci may affect their infectivity and pathogenicity, and may also contribute to the severity of the disease [31]. However, to resolve these issues, further studies are required. EITB is claimed to be the most reliable serologic test with reported sensitivity of 98% and specificity of 100% [24]; however, in a recently published report from south India, authors had found only 12 of 46 (26.1%) patients with CT diagnosis of NCC were positive by EITB [32]. In a study from Honduras, EITB offered valuable information if performed in CSF [33]. We found that all symptomatic NCC patients as well as asymptomatic cases were positive on EITB for one or more bands. This is a unique observation and the probable reason for this may be that we conducted the study in a highly endemic community with reported rate of taeniasis 18.6% and epilepsy 5.6% [19]. In an epidemiologic survey on T. solium cysticercosis, Chung et al. (2005) had reported that all individuals with high antibody titer for cyst fluid antigens had multiple bands on immunoblot [34]. Casecontrol studies in larger sample size are required to evaluate the role of EITB for diagnosis of NCC in highly endemic areas and in communities at higher risk for infection. The presence of degenerating NCC in the asymptomatic group questions the need of prophylactic treatment with antiepileptic and anti-parasitic drugs for such individuals. The option to follow-up such individuals with imaging and to treat them only if they become symptomatic needs to be explored. Our data suggests that a large number of the people harboring different stages of NCC remain asymptomatic. 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