Elimination of Taenia solium Transmission in Northern Peru.

BACKGROUND: Taeniasis and cysticercosis are major causes of seizures and epilepsy. Infection by the causative parasite Taenia solium requires transmission between humans and pigs. The disease is considered to be eradicable, but data on attempts at regional elimination are lacking. We conducted a three-phase control program in Tumbes, Peru, to determine whether regional elimination would be feasible. METHODS: We systematically tested and compared elimination strategies to show the feasibility of interrupting the transmission of T. solium infection in a region of highly endemic disease in Peru. In phase 1, we assessed the effectiveness and feasibility of six intervention strategies that involved screening of humans and pigs, antiparasitic treatment, prevention education, and pig replacement in 42 villages. In phase 2, we compared mass treatment with mass screening (each either with or without vaccination of pigs) in 17 villages. In phase 3, we implemented the final strategy of mass treatment of humans along with the mass treatment and vaccination of pigs in the entire rural region of Tumbes (107 villages comprising 81,170 people and 55,638 pigs). The effect of the intervention was measured after phases 2 and 3 with the use of detailed necropsy to detect pigs with live, nondegenerated cysts capable of causing new infection. The necropsy sampling was weighted in that we preferentially included more samples from seropositive pigs than from seronegative pigs. RESULTS: Only two of the strategies implemented in phase 1 resulted in limited control over the transmission of T. solium infection, which highlighted the need to intensify the subsequent strategies. After the strategies in phase 2 were implemented, no cyst that was capable of further transmission of T. solium infection was found among 658 sampled pigs. One year later, without further intervention, 7 of 310 sampled pigs had live, nondegenerated cysts, but no infected pig was found in 11 of 17 villages, including all the villages in which mass antiparasitic treatment plus vaccination was implemented. After the final strategy was implemented in phase 3, a total of 3 of 342 pigs had live, nondegenerated cysts, but no infected pig was found in 105 of 107 villages. CONCLUSIONS: We showed that the transmission of T. solium infection was interrupted on a regional scale in a highly endemic region in Peru. (Funded by the Bill and Melinda Gates Foundation and others.).

Antibody Banding Patterns of the Enzyme-Linked Immunoelectrotransfer Blot and Brain Imaging Findings in Patients With Neurocysticercosis.

Background: The enzyme-linked immunoelectrotransfer blot (EITB) assay is the reference serological test for neurocysticercosis (NCC). A positive result on EITB does not always correlate with the presence of active infections in the central nervous system (CNS), and patients with a single viable brain cyst may be EITB negative. Nonetheless, EITB antibody banding patterns appears to be related with the expression of 3 protein families of Taenia solium, and in turn with the characteristics of NCC in the CNS (type, stage, and burden of viable cysts). Methods: We evaluated EITB antibody banding patterns and brain imaging findings of 548 NCC cases. Similar banding patterns were grouped into homogeneous classes using latent class analysis. The association between classes and brain imaging findings was assessed. Results: Four classes were identified. Class 1 (patients negative or only positive to the GP50 band, related to the protein family of the same name) was associated with nonviable or single viable parenchymal cysticerci; class 2 (patients positive to bands GP42-39 and GP24, related to the T24-42 protein family, with or without anti-GP50 antibodies) was associated with intraparenchymal viable and nonviable infections; classes 3 and 4 (positive to GP50, GP42-39, and GP24 but also responding to low molecular weight bands GP21, GP18, GP14, and GP13, related to the 8 kDa protein family) were associated with extraparenchymal and intraparenchymal multiple viable cysticerci. Conclusions: EITB antibody banding patterns correlate with brain imaging findings and complement imaging information for the diagnosis of NCC and for staging NCC patients.

Seizures, cysticercosis and rural-to-urban migration: the PERU MIGRANT study.

OBJECTIVES: To examine the prevalence of seizures, epilepsy and seropositivity to cysticercosis in rural villagers (cysticercosis-endemic setting), rural-to-urban migrants into a non-endemic urban shanty town and urban inhabitants of the same non-endemic shanty town. METHODS: Three Peruvian populations (n = 985) originally recruited into a study about chronic diseases and migration were studied. These groups included rural inhabitants from an endemic region (n = 200), long-term rural-to-urban migrants (n = 589) and individuals living in the same urban setting (n = 196). Seizure disorders were detected by a survey, and a neurologist examined positive respondents. Serum samples from 981/985 individuals were processed for cysticercosis antibodies on immunoblot. RESULTS: Epilepsy prevalence (per 1000 people) was 15.3 in the urban group, 35.6 in migrants and 25 in rural inhabitants. A gradient in cysticercosis antibody seroprevalence was observed: urban 2%, migrant 13.5% and rural group 18% (P < 0.05). A similarly increasing pattern of higher seroprevalence was observed among migrants by age at migration. In rural villagers, there was strong evidence of an association between positive serology and having seizures (P = 0.011) but such an association was not observed in long-term migrants or in urban residents. In the entire study population, compared with seronegative participants, those with strong antibody reactions (≥ 4 antibody bands) were more likely to have epilepsy (P < 0.001). CONCLUSIONS: It is not only international migration that affects cysticercosis endemicity; internal migration can also affect patterns of endemicity within an endemic country. The neurological consequences of cysticercosis infection likely outlast the antibody response for years after rural-to-urban migration.

Recombinant protein- and synthetic peptide-based immunoblot test for diagnosis of neurocysticercosis.

One of the most well-characterized tests for diagnosing neurocysticercosis (NCC) is the enzyme-linked immunoelectrotransfer blot (EITB) assay developed at the CDC, which uses lentil lectin-bound glycoproteins (LLGP) extracted from Taenia solium cysticerci. Although the test is very reliable, the purification process for the LLGP antigens has been difficult to transfer to other laboratories because of the need for expensive equipment and technical expertise. To develop a simpler assay, we previously purified and cloned the diagnostic glycoproteins in the LLGP fraction. In this study, we evaluated three representative recombinant or synthetic antigens from the LLGP fraction, individually and in different combinations, using an immunoblot assay (recombinant EITB). Using a panel of 249 confirmed NCC-positive and 401 negative blood serum samples, the sensitivity of the recombinant EITB assay was determined to be 99% and the specificity was 99% for diagnosing NCC. We also tested a panel of 239 confirmed NCC-positive serum samples in Lima, Peru, and found similar results. Overall, our data show that the performance characteristics of the recombinant EITB assay are comparable to those of the LLGP-EITB assay. This new recombinant- and synthetic antigen-based assay is sustainable and can be easily transferred to other laboratories in the United States and throughout the world.

Parasite antigen in serum predicts the presence of viable brain parasites in patients with apparently calcified cysticercosis only.

BACKGROUND: Computed tomography (CT) remains the standard neuroimaging screening exam for neurocysticercosis, and residual brain calcifications are the commonest finding. Magnetic resonance imaging (MRI) is more sensitive than CT but is rarely available in endemic regions. Enzyme-linked immunoelectrotransfer blot (EITB) assay uses antibody detection for diagnosis confirmation; by contrast, enzyme-linked immunosorbent assay (ELISA) antigen detection (Ag-ELISA) detects circulating parasite antigen. This study evaluated whether these assays predict undetected viable cysts in patients with only calcified lesions on brain CT. METHODS: Serum samples from 39 patients with calcified neurocysticercosis and no viable parasites on CT were processed by Ag-ELISA and EITB. MRI was performed for each patient within 2 months of serologic testing. Conservatively high ELISA and EITB cutoffs were used to predict the finding of viable brain cysts on MRI. RESULTS: Using receiver operating characteristic-optimized cutoffs, 7 patients were Ag-ELISA positive, and 8 had strong antibody reactions on EITB. MRI showed viable brain cysts in 7 (18.0%) patients. Patients with positive Ag-ELISA were more likely to have viable cysts than Ag-ELISA negatives (6/7 vs 1/32; odds ratio, 186 [95% confidence interval, 1-34 470.0], P < .001; sensitivity 85.7%, specificity 96.9%, positive likelihood ratio of 27 to detect viable cysts). Similar but weaker associations were also found between a strong antibody reaction on EITB and undetected viable brain cysts. CONCLUSIONS: Antigen detection, and in a lesser degree strong antibody reactions, can predict viable neurocysticercosis. Serological diagnostic methods could identify viable lesions missed by CT in patients with apparently only calcified cysticercosis and could be considered for diagnosis workup and further therapy.

Neurocysticercosis: is serology useful in the absence of brain imaging?

Neurocysticercosis (NCC) is endemic in most parts of the world and is now recognised as an important contributor to neurological disease. Serological diagnosis of NCC improved greatly in the past two decades and contributed to demonstrating previously unsuspected regions of endemicity. Claims for an accurate serological screening tool for human cysticercosis are frequently raised. However, after symptomatic therapeutics are applied, management of NCC is driven by the characteristics of the central nervous system infection in terms of viability, number, location size and evolutionary stage of parasites, as well as by the resulting inflammation. It is unclear whether, in the absence of neuroimaging, serological confirmation of aetiology of suspected cases (neurologically symptomatic) or detection of asymptomatic cases in population screening would affect their management or prognosis.

Development and evaluation of porcine cysticercosis QuickELISA in Triturus EIA analyzer.

We evaluated three diagnostic antigens (recombinant GP50, recombinant T24H, and synthetic Ts18var1) for cysticercosis and found that all three performed well in detecting cysticercosis in humans and pigs in several assay formats. These antigens were adapted to a new antibody detection format (QuickELISA). With one single incubation step which involves all reactants except the enzyme substrate, the QuickELISA is particularly suited for automation. We formatted the QuickELISA for the Triturus EIA analyzer for testing large numbers of samples. We found that in QuickELISA formats rGP50 and rT24H have better sensitivity and specificity than sTs18var1 for detecting porcine cysticercosis.

Epilepsy and neurocysticercosis: an incidence study in a Peruvian rural population.

BACKGROUND: Epilepsy is a serious neurological disorder and neurocysticercosis (NCC), the central nervous system infection by the larvae of Taenia solium, is the main cause of acquired epilepsy in developing countries. NCC is becoming more frequent in industrialized countries due to immigration from endemic areas. Previously reported epilepsy incidences range from 30 to 50/100,000 people in industrialized countries and 90 to 122/100,000 people in developing countries. OBJECTIVES: To determine the incidence of epilepsy in a cysticercosis endemic area of Peru. METHODS: A screening survey for possible seizure cases was repeated biannually in this cohort for a period of 5 years (1999-2004) using a previously validated questionnaire. All positive respondents throughout the study were examined by a trained neurologist in the field to confirm the seizure. If confirmed, they were offered treatment, serological testing, neuroimaging (CT scans and MRI) and clinical follow-up. RESULTS: The cohort study comprised 817 individuals. The overall epilepsy incidence rate was 162.3/100,000 person-years, and for epileptic seizures, 216.6/100,000 person-years. Out of the 8 individuals who had epileptic seizures, 4 had markers for NCC (neuroimaging and/or serology). CONCLUSION: The incidence of epilepsy in this area endemic for cysticercosis is one of the highest reported worldwide.

Clustering of Necropsy-Confirmed Porcine Cysticercosis Surrounding Taenia solium Tapeworm Carriers in Peru.

The pork tapeworm, Taenia solium, is among the leading causes of preventable epilepsy in the world and is common in rural areas of developing countries where sanitation is limited and pigs have access to human feces. Prior studies in rural villages of Peru have observed clusters of T. solium cysticercosis among pigs that live near human tapeworm carriers. Such spatial analyses, however, have been limited by incomplete participation and substandard diagnostic tests. In this study, we evaluated the association between necropsy-confirmed cysticercosis in pigs and their distance to T. solium tapeworm carriers in six villages in northern Peru. A total of six (1.4%) tapeworm carriers were detected using copro-antigen enzyme-linked immunosorbent assay and seven of 10 (70%) pigs belonging to the tapeworm carriers were found with viable cyst infection on necropsy. This was significantly greater than the prevalence of viable cyst infection among pigs living < 500 m (11%) and > 500 m (0.5%) from a tapeworm carrier (P < 0.001 for distance trend). Similar statistically significant prevalence gradients were observed after adjustment for possible confounders and for other pig-level outcomes including infection with > 10 viable cysts, degenerated cyst infection, and serological outcomes. This investigation confirms that porcine cysticercosis clusters strongly around tapeworm carriers in endemic rural regions of northern Peru and supports interventions that target these hotspots.

Seroprevalence and risk factors for human herpesvirus 8 infection, rural Egypt.

To determine whether human herpesvirus 8 (HHV-8) is associated with schistosomal and hepatitis C virus infections in Egypt, we surveyed 965 rural household participants who had been tested for HHV-8 and schistosomal infection (seroprevalence 14.2% and 68.6%, respectively, among those <15 years of age, and 24.2% and 72.8%, respectively, among those > or =15 years of age). Among adults, HHV-8 seropositivity was associated with higher age, lower education, dental treatment, tattoos, > or =10 lifetime injections, and hepatitis C virus seropositivity. In adjusted analyses, HHV-8 seropositivity was associated with dental treatment among men (odds ratio [OR] 2.4, 95% confidence interval [CI] 1.1-5.2) and hepatitis C virus seropositivity among women (OR 3.3, 95% CI 1.4-7.9). HHV-8 association with antischistosomal antibodies was not significant for men (OR 2.1, 95% CI 0.3-16.4), but marginal for women (OR 1.5, 95% CI 1.0-2.5). Our findings suggest salivary and possible nosocomial HHV-8 transmission in rural Egypt.

False positive reactivity of recombinant, diagnostic, glycoproteins produced in High Five insect cells: effect of glycosylation.

Baculovirus-mediated expression of recombinant proteins for use in diagnostic assays is commonplace. We expressed a diagnostic antigen for cysticercosis, GP50, caused by the larval stage of Taenia solium, in both High Five and Sf9 insect cells. Upon evaluation of the specificity of recombinant GP50 (rGP50) in a western blot assay, we observed that 12.5% (21/168) of the serum samples from persons with a variety of parasitic infections other than cysticercosis reacted positive when rGP50 was produced in High Five cells. The same samples reacted negative when rGP50 was produced in Sf9 cells. The false positive reactivities of these other parasitic infection sera were abolished when rGP50, expressed in High Five cells, was deglycosylated. In addition, the same sera that reacted with rGP50 from High Five cells also reacted with recombinant human transferrin (rhTf) when expressed in High Five cells, but not Sf9 cells. High Five cells, but not Sf9 cells, modify many glycoproteins with a core alpha(1,3)-fucose. This same modification is found in the glycoproteins of several parasitic worms and is known to be immunogenic. Since the distribution of these worms is widespread and millions of people are infected, the use of recombinant proteins with N-linked glycosylation produced in High Five cells for diagnostic antigens is likely to result in a number of false positive reactions and a decrease in assay specificity.

A simple method for collecting measured whole blood with quantitative recovery of antibody activities for serological surveys.

Compliance and acceptance for the finger-prick method of blood collection is generally better than for venipuncture. A finger-prick method of blood collection with quantitative antibody recovery is even more important for seroepidemiological surveys. Finger-prick blood collected and dried onto filter paper has been used; but, unfortunately, this method has several disadvantages, including loss of antibody activity, possible contact contamination from blood spots on adjacent filter papers, and difficulties in extracting antibodies, justifying the search for other methods of collecting and transporting blood samples. We report on a simple method of collecting a measured amount of finger-prick blood onto a sample pad, which is immediately transferred to storage/extraction buffer. The diluted blood sample is never dried, and because of the storage buffer, can be transported and stored without refrigeration. Furthermore, the diluted blood samples can then be tested directly without further preparation. We systematically compared several storage/extraction buffers and commercially available filter papers. We showed that antibody recovery was not significantly affected by the type of filter papers used but was significantly affected by the storage/extraction buffer used. The best such buffer is StabilZyme Select.

Characterization of a novel Taenia solium oncosphere antigen.

Infections due to Taenia solium in humans (taeniasis/cysticercosis) remain a complex health problem, particularly in developing countries. We identified two oncosphere proteins that might protect the porcine intermediate host against cysticercosis and therefore help prevent disease in humans. One of these proteins was further identified by two-dimensional gel electrophoresis and micro-sequencing. The gene encoding this protective protein was also identified, cloned and characterized. The native 31.5 kDa protein Tso31 has four variants at the cDNA level. The longest sequence from which the others seem to derive, encodes a 253 amino acid peptide. The predicted protein has a molecular weight of 25.1 kDa, one putative N-glycosylation site, two fibronectin type III domains, and one C terminal transmembrane domain. The gene structure of the protein consists of four exons and three introns. The finding of one gene and four different cDNAs for Tso31 suggests the existence of a possible mechanism of differential splicing in this parasite. The Tso31 protein is exclusive to T. solium oncospheres with a putative protein structure of an extra-cellular receptor-like protein. The Tso31 protein was expressed as a recombinant protein fused to GST and tested in a vaccine to determine its effectiveness in protecting pigs against cysticercosis. Only two pigs out of eight vaccinated were protected and although the total median number of cyst decreased in vaccinated pigs compared to controls this decrease was not statistically significant (P = 0.09).

Swine cysticercosis hotspots surrounding Taenia solium tapeworm carriers.

We estimated the Taenia solium swine cysticercosis risk gradient surrounding tapeworm carriers in seven rural communities in Peru. At baseline, the prevalences of taeniasis by microscopy and swine cysticercosis by serology were 1.2% (11 of 898) and 30.8% (280 of 908), respectively. The four-month cumulative seroincidence was 9.8% (30 of 307). The unadjusted swine seroprevalence and seroincidence rates increased exponentially by 12.0% (95% confidence [CI] = 9.7-14.3%) and 32.8% (95% CI = 25.0-41.0%), respectively when distance to carriers decreased by half. Swine seroprevalence was 18.4% at > 500 meters from a carrier, 36.5% between 51 and 500 meters, and 68.9% within 50 meters (P < 0.001). Swine seroincidence also displayed a strong gradient near tapeworm carriers (3.8%, 12.2%, and 44.0%; P < 0.001). Within 50 meters, swine seroprevalence appeared unaffected if the owners harbored tapeworms, although pigs owned by a tapeworm carrier had a four times higher seroincidence compared with other pigs (P = 0.005). In rural areas, swine cysticercosis occurs in high-risk hotspots around carriers where control interventions could be delivered.

Strategies for the elimination of taeniasis/cysticercosis.

Advances in the field of neurocysticercosis continue to shape our understanding of the disease and our efforts to control it. Several attempts have been made to eradicate the disease with active interventions such as changing domestic pig-raising practices, mass chemotherapy of porcine cysticercosis and taeniasis, selective detection and treatment of taeniasis, and community health education. Moreover, ongoing progress in the diagnosis of taeniasis and the development of a porcine vaccine against cysticercosis in Australia, Mexico and Peru has yielded at least one effective vaccine that is currently available. Thus far, however, attempted interventions have only been successful in temporarily disrupting transmission of the disease. Controlled data on the efficacy and acceptability of the different interventions is urgently needed to provide a base-line schematic for intervention which could later be tailored to each particular endemic scenario.

Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

Porcine Cysticercosis: Possible Cross-Reactivity of Taenia hydatigena to GP50 Antigen in the Enzyme-Linked Immunoelectrotransfer Blot Assay.

The lentil lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB, reported sensitivity 99% and specificity 100%) is used as a serologic marker of exposure to Taenia solium in pigs. However, only a limited number of parasites have been evaluated for cross reactivity. Pigs may host other related cestode infections, including Taenia hydatigena, which have not been formally evaluated for cross-reactions. We investigated a corral in Tumbes, Peru, a region where a cysticercosis elimination demonstration project was completed in 2012. In this corral, 14/19 (73.7%) 6-8-week-old piglets were reactive to GP50 on LLGP EITB, and all had circulating Taenia sp. antigens. From eight necropsied piglets; four were infected with T. hydatigena metacestodes whereas none had evidence of T. solium infection. Two resident dogs were subsequently confirmed to have T. hydatigena taeniasis. These results suggest GP50 cross-reactivity in T. hydatigena-infected pigs, although controlled experimental infection is needed to confirm this hypothesis.

Characterization and cloning of T24, a Taenia solium antigen diagnostic for cysticercosis.

The third and final diagnostic antigen of the lentil lectin purified glycoproteins (LLGP) extracted from the larval stage of Taenia solium has been characterized, cloned, and expressed. T24 is an integral membrane protein that belongs to the tetraspanin superfamily. It migrates at a position corresponding to 24-kDa and as a homodimer at 42-kDa. Antibodies from cysticercosis patients recognize secondary structure epitopes that are dependent upon correctly formed disulfide bonds. A portion of T24, the large, extracellular loop domain, was expressed in an immunologically reactive form in insect cells. When tested in a Western blot assay with a large battery of serum samples, this protein, T24H, has a sensitivity of 94% (101/107), for detecting cases of cysticercosis with two or more viable cysts, and a specificity of 98% (284/290). The identification and expression of T24H sets the stage for the development of an ELISA suitable for testing single samples and for large-scale serosurveys that is not dependent upon the isolation and purification of antigens from parasite materials.

Transmission dynamics of Taenia solium and potential for pig-to-pig transmission.

Taenia solium taeniasis/cysticercosis is one of few potentially eradicable infectious diseases and is the target of control programs in several countries. The larval stage of this zoonotic cestode invades the human brain and is responsible for most cases of adult-onset epilepsy in the world. Our current understanding of the life cycle implicates humans as the only definitive host and tapeworm carrier, and thus the sole source of infective eggs that are responsible for cysticercosis in both human and pigs through oral-faecal transmission. Here we review transmission dynamics of porcine cysticercosis including an alternative pig-to-pig route of transmission, previously not suspected to exist. Second-hand transmission of T. solium eggs could explain the overdispersed pattern of porcine cysticercosis, with few pigs harbouring heavy parasite burdens and many more harbouring small numbers of parasites.

High Prevalence of Asymptomatic Neurocysticercosis in an Endemic Rural Community in Peru.

BACKGROUND: Neurocysticercosis is a common helminthic infection of the central nervous system and an important cause of adult-onset epilepsy in endemic countries. However, few studies have examined associations between neurologic symptoms, serology and radiographic findings on a community-level. METHODOLOGY: We conducted a population-based study of resident's ≥2 years old in a highly endemic village in Peru (pop. 454). We applied a 14 -question neurologic screening tool and evaluated serum for antibodies against Taenia solium cysticercosis using enzyme-linked immunoelectrotransfer blot (LLGP-EITB). We invited all residents ≥18 years old to have non-contrast computerized tomography (CT) of the head. PRINCIPAL FINDINGS: Of the 385 residents who provided serum samples, 142 (36.9%) were seropositive. Of the 256 residents who underwent CT scan, 48 (18.8%) had brain calcifications consistent with NCC; 8/48 (17.0%) reported a history of headache and/or seizures. Exposure to T. solium is very common in this endemic community where 1 out of 5 residents had brain calcifications. However, the vast majority of people with calcifications were asymptomatic. CONCLUSION: This study reports a high prevalence of NCC infection in an endemic community in Peru and confirms that a large proportion of apparently asymptomatic residents have brain calcifications that could provoke seizures in the future.