Introduction
Taenia solium, also called the pork tapeworm, is a parasite belonging to the Taeniidae family, which can cause taeniasis and cysticercosis in humans. Taeniasis can be found strictly in the human host, after ingestion of raw or undercooked pork meat contaminated with the larval stage (Cysticercus cellulosae) of this tapeworm. The activity of proteolytic enzymes in the stomach liberates the larvae, which develop into adult worms in the small intestine. The body of a fully developed tapeworm can be from 2 to 7 m long, consisting of several hundred proglottids with a huge number of infective eggs (Flisser, Reference Flisser and Sun1994). As adult worms do not cause substantial damage to the intestine, they can persist there for years, usually without any symptoms (Schantz et al., Reference Schantz, Wilkins, Tsang, Scheld, Craig and Hughes1998). What causes concern regarding T. solium infection is human cysticercosis, which results either from the direct ingestion of parasitic eggs passed in human stools, or after faecal–oral transmission from food or water contaminated with eggs.
While outside of the central nervous system (CNS) tapeworm larvae cause no major symptoms, the ones in the brain (neurocysticercosis) are associated with significant morbidity and mortality (Garcia et al., Reference Garcia, Gonzalez, Evans and Gilman2003). Neurocysticercosis is considered the most common parasitic infection of the CNS and the main cause of epilepsy in developing countries (Román et al., Reference Román, Sotelo, Del Bruto, Flisser, Dumas, Wadia, Botero, Cruz, Garcia, de Bittencourt, Trelles, Arriagada, Lorenzana, Nash and Spina-França2000). Epileptic seizures are usually the only manifestation of this disease, and can be found in 50–80% of patients with parenchymal brain cysts or calcifications (Chopra et al., Reference Chopra, Kaur and Mahajan1981; Del Bruto et al., Reference Del Bruto, Santibanez, Noboa, Aguirre, Diaz and Alarcon1992; Commision on Tropical Diseases of the International League Against Epilepsy, 1994). Although cysticercosis is endemic to Asia, Africa and Latin America (Willingham & Engels, Reference Willingham and Engels2006), it has been diagnosed with increasing frequency in developed countries (Schantz et al., Reference Schantz, Wilkins, Tsang, Scheld, Craig and Hughes1998; White, Reference White2000).
According to references, 50 million people worldwide are estimated to have T. solium infection, while each year 50,000 die of cysticercosis (Eddi et al., Reference Eddi, Nari and Amanfu2003). Data on the prevalence of this infection in Europe is scarcely available and non-existent when it comes to the Croatian population. The primary goal of this research was to establish the prevalence of T. solium infection in Croatian patients with clinically manifest epilepsy.
Materials and methods
Between 2005 and 2009, a total of 770 serum samples were collected from patients aged 3–76 with a clinical diagnosis of epilepsy. All were referred to the Croatian National Institute of Public Health with a clinical diagnosis of epilepsy and no other available clinical or diagnostic data. This study was approved by the Ethical Committee of the Croatian National Institute of Public Health.
Collection and analysis of serum samples
Serum samples were obtained from patients in rural and urban areas of the Croatian mainland. Serological analysis was performed at the Parasitology Department of the Croatian National Institute of Public Health (CNIPH). For the determination of IgG-class antibodies against T. solium, a commercial qualitative enzyme immunoassay (Taenia solium IgG-ELISA, Bioactiva Diagnostica GmbH, Germany) was used, according to manufacturer's recommendations. The manufacturer also stated a diagnostic sensitivity of 93.8% and diagnostic specificity of >95%.
As cross-reactions can occur in the enzyme-linked immunosorbent assay (ELISA), positive samples were confirmed using the Western blot method. For that purpose, Cysticercosis Western Blot (WB) IgG test (LDBIO Diagnostics, Lyon, France) was used. According to the WB manufacturer's instructions, there are six bands especially chosen for their specificity for cysticercosis antibodies: 6–8 kDa, 12 kDa, 23–26 kDa, 39 kDa, 45 kDa and 50–55 kDa. The presence of a minimum of two well-defined bands of the above six on the strip is indicative of the existence of IgG-class antibodies in the tested sample. The manufacturer also stated a diagnostic sensitivity of 96.1% and diagnostic specificity of up to 100%.
Data analysis was performed using chi-square test. A P value less than 0.05 was considered to be significant.
Results
Of 770 analysed serum samples, 396 (51%) were from male and 374 (49%) from female patients. In 23 (3%) serum samples from patients with epilepsy, IgG antibodies to T. solium were detected by ELISA: 52% among male and 48% among female patients. Of the 23 positive serum samples, 11 (1.5%) were confirmed by WB. Using WB, the seroprevalence rate was established at 1.77% (7/396) for male and 1.07% (4/374) for female patients, with no statistically significant difference (P = 0.609).
The seroprevalence rate in patients residing in urban regions was 1.4% (7/484), identical to the seroprevalence rate in patients residing in rural regions (4/286).
In a group of patients under 18 years of age, a positive serum was found in only one subject, which accounts for the seroprevalence rate of 0.4% (1/249), compared to 1.92% (10/521) of positive subjects in a group of patients aged over 18. Although seropositivity differed between the two evaluated age groups, this difference was not statistically significant (P = 0.181).
Discussion
Epilepsy is one of the most common and challenging neurological disorders, while neurocysticercosis caused by T. solium infection of the CNS currently represents the leading cause of secondary (acquired) epilepsy in adults in Central and South America, East and South Asia, and sub-Saharan Africa (Scott et al., Reference Scott, Lhatoo and Sander2001; Raccurt et al., Reference Raccurt, Agnamey, Boncy, Henrys and Totet2009). As a result of increased migration from these endemic regions, neurocysticercosis and subsequent epilepsy are considered one of the growing public health problems in developed countries, too (Schantz et al., Reference Schantz, Wilkins, Tsang, Scheld, Craig and Hughes1998; White, Reference White2000; Carpio, Reference Carpio2002).
The overall seroprevalence rate of T. solium infection, as reported here, was 3% using ELISA and 1.5% when confirmed with WB. Although the WB assay is generally recommended for immunodiagnosis of neurocysticercosis, ELISA is more commonly used in epidemiological studies as it is technically simpler and cheaper (Carpio, Reference Carpio2002). Gekeler et al. (Reference Gekeler, Eichenlaub, Mendoza, Sotelo, Hoelscher and Löscher2002) concluded that using ELISA as a screening method and immunoblotting as a confirmatory test considerably improves the diagnosis of this disease. In order to determine the value of immunodiagnosis for neurocysticercosis, they tested 222 serum and cerebrospinal fluid samples from patients with neurocysticercosis and healthy subjects. The sensitivities of ELISA and immunoblot test in neurocysticercosis patients were almost identical (80% and 81.7%, respectively). The overall specificity of ELISA was only 75.3% due to frequent false-positive results, but the specificity of the immunoblot test was clearly higher (99.4%). Even though WB is confirmed as a superior method by a majority of studies, some authors suggest considering ELISA as the better method for the serological confirmation of neurocysticercosis in children, as it ensures higher specificity and diagnostic efficacy than the dot-blot assay in the examined population (Mandal et al., Reference Mandal, Singhi, Khandelwal and Malla2006).
Seroprevalence of this infection in the group of patients aged over 18 years was not significantly higher in comparison with the group of patients aged less than 18 (where only one case was serologically confirmed). Furthermore, no statistically significant difference was found in the infection rate in terms of gender or residence. All patients from whom sera were collected and tested were residents of Croatia at the time. It must be noted that the presence of cysticercal antibodies only suggests that the patients have acquired the infection, not necessarily in Croatia.
Several problems need to be addressed. The tests that have been used are mainly geared to detect infection with the larval stage of cysticercosis, so their validity with the adult stage is less well known. Furthermore, a positive serological test for cysticercosis may only indicate that the patient has had contact with T. solium antigens in various forms. Cysticercosis can also be situated in another organ (such as skeletal muscle), or may already be resolved with circulating antibodies (Serrano Ocana et al., Reference Serrano Ocana, Ortiz Sablon, Ochoa Tamayo, Almaguer Arena, Serrano Ocana and Govender2009). In patients with neurocysticercosis, clinical presentation and the results of neuroimaging procedures vary extensively and often do not facilitate a definite diagnosis (Gekeler et al., Reference Gekeler, Eichenlaub, Mendoza, Sotelo, Hoelscher and Löscher2002).
Studies based on hospital series have reported that 50% of the 100 patients studied in Mexico have neurocysticercosis as the main cause of late-onset epilepsy (Medina et al., Reference Medina, Rosas, Rubio-Donnadieu and Sotelo1990). In a large series studied in Colombia and Peru, where the enzyme-linked immuno-electrotransfer blot (EITB) method was used, cysticercosis was proven as the cause of epilepsy in 14% and 12% of the cases, respectively (Garcia et al., Reference Garcia, Gilman, Herrera, Diaz, Miranda, Gilman, Martinez, Alvarado, Tsang and Pilcher1993; Palacio et al., Reference Palacio, Jiménez, Garcia, Jiménez, Sánchez, Noh, Ahn, Mora, Giraldo and Tsang1998). It must be noted that there are very few studies on neurocysticercosis in Europe. Seizures were the most common inaugural sign in 29 patients with neurocysticercosis diagnosed in south-eastern France between 1988 and 1999 (Rousseau et al., Reference Rousseau, Guillotel and Delmont1999). A recent study in an Indian community suggested that the region-specific prevalence rates of epilepsy in India are partly dependent on the prevalence of neurocysticercosis in the given community (Goel et al., Reference Goel, Dhanai, Agarwal, Mehlotra and Saxena2011).
This study provides important and previously unknown data on the prevalence of T. solium infection in Croatian patients with epilepsy. Although infection with this tapeworm is rare in Croatia, this study will, hopefully, increase awareness of the importance of preventive measures and benefits of accurate and timely diagnosis among physicians and public health workers. Intervention measures for infection control are crucial, namely sanitation improvement, control of domestic pig-breeding, detailed meat inspection, detection and treatment of tapeworm carriers, hand washing and health education.
