Uso de partículas magnéticas en la purificación de anticuerpos IgM contra Taenia solium / Use of magnetic particles in the purification of IgM antibodies against Taenia solium

RESUMEN Se evaluó el uso de partículas magnéticas acopladas a proteína L para la concentración y purificación de anticuerpos monoclonales inmunoglobulina M (mIgM) contra Taenia solium. Se evaluaron tres métodos de concentración y diferentes tiempos de elución y se optimizó la proporción de partículas a la proporción de mIgM. Demostramos que: 1) con el uso partículas magnéticas no se requiere de una concentración previa de mIgM, lo que disminuye la manipulación de los anticuerpos y mejora la recuperación, 2) se puede omitir el uso de un tampón de unión, ya que el pH de la mayoría de los sobrenadantes de cultivo celular son neutros, y 3) se necesitan tiempos de elución más largos (~45 minutos) para aumentar la recuperación a un nivel mayor a 80%. El estudio demuestra que el uso de partículas magnéticas acopladas a proteína L es una herramienta simple y eficiente para la concentración y purificación de mIgM.

ABSTRACT The use of L protein coupled magnetic particles for the concentration and purification of immunoglobulin M (mIgM) monoclonal antibodies against Taenia solium was evaluated. Three concentration methods and different elution times were evaluated and the ratio of particles to the ratio of mIgM was optimized. It is demonstrated that: 1) with the use of magnetic particles, a previous concentration of mIgM is not required, which reduces the manipulation of the antibodies and improves the recovery, 2) the use of a binding buffer can be omitted, since the pH of most cell culture supernatants are neutral, and 3) longer elution times (~ 45 minutes) are needed to increase recovery to a level greater than 80%. The study demonstrates that the use of L protein-coupled magnetic particles is a simple and efficient tool for mIgM concentration and purification.

Seroprevalence of human cysticercosis in Jataí, Goiás state, Brazil

ABSTRACT The Taenia solium cysticercosis affects millions of people worldwide and is considered a public health problem, especially in developing countries. The diagnosis of neurocysticercosis is complex and involves the analysis of epidemiological, clinical, neuroimaging, and immunological host data. Neurocysticercosis is endemic in Brazil, and is related to the cause of death mainly in the Southeast, South, and Central-West regions. The objective of this study was to determine the seroprevalence of cysticercosis in inhabitants of the city of Jataí, Goiás, in the Central-West region of Brazil from April to August 2012. A total of 529 serum samples were analyzed by enzyme-linked immunosorbent assay for detecting IgG antibodies against T. solium larvae, and Western blotting was used for confirming the diagnosis through the recognition of at least two specific peptides from their serum antibodies. The 351/529 (66.3%) reactive samples were analyzed by enzyme-linked immunosorbent assay and Western blotting confirmed the diagnosis in 73 samples that recognized at least two of the following peptides specific IgG antibodies for cysticercosis: 18, 24, 28-32, 39-42, 47-52, 64-68, and 70 kDa. The seroprevalence of cysticercosis was 13.8% (95% CI 5.9-21.7), demonstrating that the studied area is endemic to this disease.

Seroprevalencia y factores de riesgo de cisticercosis en dos comunidades rurales del norte del estado Anzoátegui, Venezuela / Seroprevalence and risk factors of cysticercosis in two rural communities in Anzoátegui state, Venezuela

Resumen Introducción. La cisticercosis es causada por las larvas de Taenia solium, las cuales se localizan principalmente en el sistema nervioso central y causan neurocisticercosis. En Venezuela se han hecho pocos estudios epidemiológicos de esta enfermedad. Objetivo. Determinar la seroprevalencia y los factores de riesgo de la cisticercosis en dos comunidades rurales del estado Anzoátegui, Venezuela. Materiales y métodos. Se hizo una encuesta para recolectar datos sobre los posibles factores de riesgo y los signos y síntomas de la enfermedad, y se tomaron 182 muestras de los habitantes de las comunidades de Boquerón y Punto Lindo. Se determinaron los anticuerpos IgG contra cisticercos de T. solium mediante ensayo inmunoenzimático (ELISA). Resultados. En Boquerón, se presentó una seroprevalencia de 3,3 %; debido al bajo número de muestras positivas no se pudo hacer el análisis estadístico. Sin embargo, las tres personas positivas tenían conocimiento de la enfermedad, antecedentes de tenencia de cerdos no confinados, consumo de carne de cerdo semicruda y cefalea frecuente. En Punto Lindo, la seroprevalencia fue de 28,9 %. No hubo diferencias estadísticamente significativas en cuanto al sexo y la edad, sin embargo, se encontró mayor frecuencia en menores de 20 años. Con respecto a los factores de riesgo y los signos y síntomas, se encontró asociación significativa con el consumo de carne de cerdo semicruda (odds ratio, OR=18; IC95% 5,78-55,9), cefalea frecuente (OR=3,6; IC95% 1,15-11,4), convulsiones (OR=18,9; IC95% 2,15-166,5) y problemas de visión (OR=5,7; IC95% 2,13-15,34). Conclusión. Los resultados demostraron que había poca transmisión de cisticercosis en Boquerón, pero mucha en Punto Lindo, sobre todo en niños, lo cual sugeriría que se trata de transmisión reciente.

Abstract Introduction: Cysticercosis is caused by Taenia solium cysticerci, which are located mainly in the central nervous system causing neurocysticercosis. In Venezuela, few epidemiological studies on this disease have been conducted. Objective: To determine the seroprevalence and risk factors for cysticercosis in two rural communities in Anzoátegui state. Material and methods: We conducted a survey to collect data on possible risk factors and signs and symptoms of the disease, and we took 182 samples in two communities, Boquerón and Punto Lindo. Detection of IgG antibodies against T. solium cysticerci was performed by ELISA. Results: Seroprevalence in Boquerón was 3.3%; due to the low number of seropositives the statistical analysis was not possible. However, the three seropositive persons had knowledge of the disease, and a history of consumption of undercooked pork meat, and presence of headache. In Punto Lindo, seroprevalence was 28.9%. There were no significant differences by sex or age; however, we found more seropositives among individuals younger than 20 years. With regard to risk factors and signs and symptoms, significant associations were found with consumption of undercooked pork (OR=18; 95% CI: 5.78 to 55.9), headaches (OR=3.6; 95% CI: 1.15 to 11.4), seizures (OR=18.9; 95% CI: 2.15 to 166.5) and visual problems (OR=5.7; 95% CI: 2.13 to 15.34). Conclusions: The results showed low transmission of cysticercosis in Boquerón, and high in Punto Lindo, where the high prevalence in children suggests recent transmission.

Neurocysticercoiis in children presenting with febrile seizure: clinical profiile, imaging and serodiagnosis / Neurocisticercose em crianças apresentando crises afebris: perfil clínico, imagem e sorodiagnóstico

Neurocysticercosis (NCC) is one of the major causes of childhood seizures in developing countries including India and Latin America. In this study neurological pediatric cases presenting with afebrile seizures were screened for anti-Cysticercus antibodies (IgG) in their sera in order to estimate the possible burden of cysticercal etiology. The study included a total of 61 pediatric afebrile seizure subjects (aged one to 15 years old); there was a male predominance. All the sera were tested using a pre-evaluated commercially procured IgG-ELISA kit (UB-Magiwell Cysticercosis Kit ™). Anti-Cysticercus antibody in serum was positive in 23 of 61 (37.7%) cases. The majority of cases with a positive ELISA test presented with generalized seizure (52.17%), followed by complex partial seizure (26.08%), and simple partial seizure (21.73%). Headaches were the major complaint (73.91%). Other presentations were vomiting (47.82%), pallor (34.78%), altered sensorium (26.08%), and muscle weakness (13.04%). There was one hemiparesis case diagnosed to be NCC. In this study one child without any significant findings on imaging was also found to be positive by serology. There was a statistically significant association found between the cases with multiple lesions on the brain and the ELISA-positivity (p = 0.017). Overall positivity of the ELISA showed a potential cysticercal etiology. Hence, neurocysticercosis should be suspected in every child presenting with afebrile seizure especially with a radio-imaging supportive diagnosis in tropical developing countries or areas endemic for taeniasis/cysticercosis.

Neurocisticercose é uma das causas mais comuns de crises em crianças em países em desenvolvimento incluindo Índia e América Latina. Neste estudo casos neurológicos pediátricos, apresentando crises afebris foram selecionados através de anticorpos anti-Cysticercus (IgG) no seu soro para avaliar possível etiologia de Cysticercus. O estudo incluiu total de 61 casos pediátricos de indivíduos com crises afebris (idade de um a 15 anos); houve predominância de pacientes do sexo masculino. Todos os soros foram testados usando um kit comercial IgG-ELISA (UB-Magiwell Cysticercosis kit™) avaliado previamente. O anticorpo anti-Cysticercus no soro foi positivo em 23 de 61 casos (37,7%). A maioria dos casos com teste de ELISA positivo apresentava crises generalizadas (52,17%), seguida por casos de crises parciais complexas (26,08%) e crises parciais simples (21,73%). Dores de cabeça foram a queixa principal (73,91%). Outras manifestações foram vômitos (47,82%), palidez (34,78%), sensório alterado (26,08%) e fraqueza muscular (13,04%). Houve um caso de hemiparesia diagnosticado como NCC. Neste estudo uma criança sem quaisquer achados significantes às imagens apresentou sorologia positiva. Houve associação estatística significante entre os casos com múltiplas lesões no cérebro e a positividade pelo ELISA (p = 0,017). No seu conjunto a positividade pelo ELISA demonstra etiologia potencial para a cisticercose. Portanto neurocisticercose deve ser suspeitada em qualquer criança apresentado crises afebris com imagem que sugira diagnóstico em países tropicais em desenvolvimento ou em áreas endêmicas para teníase/cisticercose.

Cisteínoproteasas Catepsinas L de Taenia solium: Rol biológico en la infección y potencial uso para el inmunodiagnóstico de la neurocisticercosis / Cathepsin L Cysteine Protease from Taenia solium: Its biological role in the infection and potential use for the immunodiagnosis of neurocysticercosis

Taenia solium es un helminto aplanado responsable de la teniosis y de la cisticercosis humana, siendo esta última producida por el consumo de huevos infectivos. Los cisticercos pueden desarrollarse en diferentes tejidos del hombre, frecuentemente en el sistema nervioso central causando la neurocisticercosis (NCC). Para el diagnóstico de la NCC se requiere de una adecuada interpretación de datos clínicos, resultados de neuroimagen y pruebas serológicas. Sin embargo, las pruebas serológicas podrían mejorarse con el desarrollo de antígenos candidatos capaces de incrementar su sensibilidad y especificidad. En los últimos años se han descrito una serie de proteínas de superficie y de secreción de T. solium esenciales para la interacción parásito-hospedero. Una de estas familias son las cisteínoproteasas catepsinas L, las cuales cumplen un rol preponderante para el desarrollo y supervivencia del parásito, participando en la invasión tisular, la evasión de la respuesta inmune, el desenquistamiento y enquistamiento del cisticerco. Son consideradas como antígenos potenciales para el inmunodiagnóstico de la neurocisticercosis.

Taenia solium is a plane helminth responsible for taeniasis and human cysticercosis, the latter being the result of the consumption of infective eggs. Cysticerci can develop in different human tissues, often in the central nervous system, causing neurocysticercosis (NCC). For the diagnosis of NCC, an adequate interpretation of clinical data, neuroimaging results and serological tests are required. However, serological tests could be improved by developing candidate antigens able to increase their sensibility and specificity. In the last years, a series of surface and secretory proteins of T. solium essential for the parasite-host interaction have been described. One of these families is cathepsin L cysteine proteases, which have a predominant role in the development and survival of the parasite. They take part in the tissue invasion, immune response evasion, excystation and encystment of cysticercus. They are considered potential antigens for the immunodiagnosis of neurocysticercosis.

Antiparasitic antibodies occur with similar frequency in patients with clinically established multiple sclerosis with or without oligoclonal bands in the cerebrospinal fluid / Anticorpos antiparasitarios ocorrem com frequencia semelhante em pacientes com esclerose multipla quando ha e quando nao ha bandas oligoclonais no liquido cefalorraquidiano

The "hygiene hypothesis" postulates an inverse relationship between the prevalence of parasitic infections and the frequency of multiple sclerosis (MS). Objective: It was to study whether antibodies against parasites could be demonstrated more frequently in blood serum from MS patients with oligoclonal bands (OCB) than from MS patients without OCB. Methods: We studied serum samples from 164 patients who had previously been analyzed to investigate OCB. Parasitic antibodies were studied through unidimensional electrophoresis of proteins on polyacrylamide gel against Taenia antigens, searching for antiparasitic specific low molecular weight antibodies and also for antiparasitic nonspecific high molecular weight antibodies. Results: Two of the 103 patients with no evidence of OCB had antibodies of low molecular weight and 59 of them had antibodies of high molecular weight. Of the 61 patients with evidence of OCB, one showed antibodies of low molecular weight and 16 showed antibodies of high molecular weight. Conclusion: Antiparasitic antibodies are detected with similar frequency in MS patients with OCB and in MS patients without OCB.

A "hipótese da higiene" postula haver relação inversamente proporcional entre a prevalência de infecções por parasitas e a frequência da esclerose múltipla (EM). Objetivo: Foi verificar se em pacientes com EM aparecem anticorpos antiparasitários mais frequentemente no grupo com bandas oligoclonais (BOC) do que no grupo sem BOC. Métodos: Foram estudadas amostras de sangue de 164 pacientes previamente analisadas para investigar BOC. Foi feita eletroforese unidimensional de proteínas em gel de poliacrilamida contra antígenos de Taenia para pesquisa de anticorpos específicos de baixo peso molecular e também de anticorpos inespecíficos de alto peso molecular. Resultados: Dois dos 103 pacientes em que não havia BOC apresentaram anticorpos de baixo peso molecular, e 59 apresentaram anticorpos de alto peso molecular. Dos 61 pacientes em que não havia BOC, um apresentou anticorpos de baixo peso molecular e 16, anticorpos de alto peso molecular. Conclusão: Anticorpos antiparasitários foram detectados com frequência semelhante em doentes com EM independentemente da presença ou não de BOC.

Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

Evaluation of cysticercus-specific IgG (total and subclasses) and IgE antibody responses in cerebrospinal fluid samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies / Avaliação das respostas de anticorpos anti-cisticercos IgG (total e subclasses) e IgE em amostras de líquido cefalorraquidiano de pacientes com neurocisticercose apresentando produção intratecal de anticorpos específicos IgG

In the present study, an enzyme-linked immunosorbent assay (ELISA) standardized with vesicular fluid of Taenia solium cysticerci was used to screen for IgG (total and subclasses) and IgE antibodies in cerebrospinal fluid (CSF) samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies and patients with other neurological disorders. The following results were obtained: IgG-ELISA: 100% sensitivity (median of the ELISA absorbances (MEA)=1.17) and 100% specificity; IgG1-ELISA: 72.7% sensitivity (MEA=0.49) and 100% specificity; IgG2-ELISA: 81.8% sensitivity (MEA=0.46) and 100% specificity; IgG3-ELISA: 63.6% sensitivity (MEA=0.12) and 100% specificity; IgG4-ELISA: 90.9% sensitivity (MEA=0.85) and 100% specificity; IgE-ELISA 93.8% sensitivity (MEA=0.60) and 100% specificity. There were no significant differences between the sensitivities and specificities in the detection of IgG-ELISA and IgE-ELISA, although in CSF samples from patients with neurocysticercosis the MEA of the IgG-ELISA was significantly higher than that of the IgE-ELISA. The sensitivity and MEA values of the IgG4-ELISA were higher than the corresponding values for the other IgG subclasses. Future studies should address the contribution of IgG4 and IgE antibodies to the physiopathology of neurocysticercosis.

No presente estudo, uma reação imunoenzimática (ELISA) padronizada com o fluido vesicular de cisticercos de Taenia solium foi utilizada para avaliar as respostas de anticorpos anti-cisticercos IgG (total e subclasses) e IgE em amostras de líquido cefalorraquidiano (LCR) de pacientes com neurocisticercose apresentando produção intratecal de anticorpos específicos IgG e pacientes com outras desordens neurológicas. Os seguintes resultados foram obtidos: ELISA-IgG: 100% de sensibilidade (mediana das absorbâncias das reações ELISA (MAE)=1,17) e especificidade 100%; ELISA-IgG1: sensibilidade 72,7% (MAE=0,49) e especificidade 100%; ELISA-IgG2: sensibilidade 81,8% (MAE=0,46) e especificidade 100%; ELISA-IgG3: sensibilidade 63,6% (MAE=0,12) e especificidade 100%; ELISA-IgG4: sensibilidade 90,9% (MAE=0,85) e especificidade 100%; ELISA-IgE: sensibilidade 93,8% (MAE=0,60) e especificidade 100%. Não foram encontradas diferenças significativas entre as sensibilidades e especificidades das reações ELISA-IgG e ELISA-IgE, embora a MAE da reação ELISA-IgG em amostras de LCR de pacientes com neurocisticercose tenha sido significativamente maior que a obtida com ELISA-IgE. Os valores de sensibilidade e MAE da reação ELISA-IgG4 foram maiores que os valores correspondentes para as outras subclasses da IgG. Estudos futuros deverão abordar a contribuição dos anticorpos IgG4 e IgE na fisiopatologia da neurocisticercose.

Genetic polymorphism in Taenia solium metacestodes from different Brazilian geographic areas

The aim of the present study is to investigate genetic polymorphisms in Taenia solium metacestodes from different Brazilian geographical areas and to relate them to antibody recognition in serum samples of neurocysticercosis (NC) patients. Metacestodes were obtained from the Distrito Federal (DF), Bahia, Minas Gerais (MG) and São Paulo (SP) regions of Brazil. Samples of human sera from 49 individuals with NC, 68 individuals with other helminthiasis and 40 healthy volunteers were analysed (157 individuals in total). Antigens were prepared and used in enzyme-linked immunosorbent assay and western blotting assays to detect specific immunoglobulin G antibodies. Genetic distances between metacestode populations were analysed using random amplified polymorphic DNA (RAPD) analysis. Our results show that there was a higher frequency of reactivity in the DF region in the sera from NC patients (p < 0.05), while discrimination between active and inactive NC was seen only in extracts from the MG and SP regions (p < 0.05). Using RAPD, the sample from the DF region presented a greater increase compared to the other regions. A relationship between genetic polymorphisms among T. solium metacestodes from different areas in Brazil and the differences in antibody detection in patients with NC were established.

Evaluation of two Taenia solium cysticercal antigenic preparations (vesicular fluid and a glycoprotein fraction with affinity for lentil lectin) for the immunodiagnosis of neurocysticercosis by enzyme-linked immunosorbent assay (ELISA) / Avaliação de duas preparações antigênicas de cisticercos de Taenia solium (líquido vesicular e uma fração glicoprotéica com afinidade para lentil lectina) para o imunodiagnóstico da neurocisticercose usando uma técnica imunoenzimática (ELISA)

OBJECTIVE: To evaluate the performance of two antigenic preparations (vesicular fluid - VF and a glycoprotein fraction, LLa-Gp fraction, purified from a whole parasite extract by lentil lectin affinity chromatography) from Taenia solium cysticerci for the immunodiagnosis of neurocysticercosis. METHOD: Fifty-six cerebrospinal fluid (CSF) samples (22 from patients with neurocysticercosis and 34 from patients with other neurological disorders) and 57 serum samples (22 from patients with neurocysticercosis, 18 from patients with other infections and 17 from presumably healthy persons) were assayed for anticysticercal IgG antibodies with an enzyme-linked immunosorbent assay (ELISA). RESULTS: The VF ELISA showed 100 percent sensitivity and specificity in CSF and serum samples, whereas the sensitivity and specificity of the LLa-Gp ELISA were, respectively, 90.9 percent and 97.1 percent, with the CSF samples and 95.5 percent and 100 percent with serum samples. There was no significant difference in the sensitivity and specificity of the two antigenic preparations used to screen CSF and serum samples. CONCLUSION: Considering the complexity and high cost of obtaining the LLa-Gp fraction, VF could be more suitable for screening specific antibodies by ELISA in CSF and serum samples from patients with neurocysticercosis.

OBJETIVO: Avaliar o desempenho de duas preparações antigênicas (líquido vesicular - LV e uma fração glicoprotéica, fração LL a-Gp, purificada do extrato total dos parasitas por cromatografia de afinidade com lentil lectina) de cisticercos de Taenia solium para o imunodiagnóstico da neurocisticercose. MÉTODO: Cinquenta e seis amostras de líquido cefalorraquidiano (LCR) (22 de pacientes com neurocisticercose e 34 de pacientes com outras doenças neurológicas) e 57 amostras de soro (22 de pacientes com neurocisticercose, 18 de pacientes com outras infecções e 17 de pessoas presumivelmente sadias) foram analisadas quanto à presença de anticorpos IgG anti-cisticercos com uma reação imunoenzimática (ELISA). RESULTADOS: A reação ELISA LV apresentou 100 por cento de sensibilidade e especificidade em amostras de LCR e soro, enquanto a sensibilidade e a especificidade da reação ELISA LLa-Gp em amostras de LCR e soro foram de 90,9 por cento e 97,1 por cento e 95,5 por cento e 100 por cento, respectivamente. Não foram encontradas diferenças significativas na sensibilidade e especificidade das duas preparações antigênicas utilizadas, tanto para amostras de LCR como para amostras de soro. CONCLUSÃO: Considerando a complexidade e o alto custo de obtenção da fração LLa-Gp, o LV pode ser mais adequado para a pesquisa de anticorpos específicos por ELISA em amostras de LCR e soro de pacientes com neurocisticercose.

Evaluation of an enzyme immunoassay for clinical diagnosis of neurocysticercosis in symptomatic patients / Avaliação de um teste imunoenzimático para o diagnóstico clínico de neurocisticercose em pacientes sintomáticos

INTRODUCTION: Neurocysticercosis is an infection of the human central nervous system caused by the metacestode larvae of Taenia solium. Neurocysticercosis is the most common parasitic disease in developing countries. Epilepsy is the most common clinical manifestation. Difficulties in confirming the diagnosis motivated the evaluation of the enzyme-linked immunosorbent assay on cerebral spinal fluid (CSF). METHODS: Twenty-two patients with NCC and 44 control patients were studied. CSF was analyzed using a commercial ELISA kit developed for NCC. Sensitivity and specificity were measured and a multivariate logistic regression was performed. RESULTS: Sensitivity and specificity of ELISA were 31.8 percent and 100 percent, respectively, with accuracy of 77.3 percent. Only the size of the lesions proved to be important for performance of the test. CONCLUSIONS: The results showed that ELISA contributes to the diagnosis of neurocysticercosis if the result is negative or if the patient has a lesion of 2 cm or more.

INTRODUÇÃO: Neurocisticercose é a infecção do sistema nervoso central causada pela larva metacestódea da Taenia solium. Neurocisticercose é a parasitose mais comum nos países em desenvolvimento. Epilepsia é a sua manifestação clínica mais comum. A dificuldade para confirmar o diagnóstico motivou a avaliação do ensaio imunoenzimático ligado à enzima no líquido cérebro-espinhal. MÉTODOS: Vinte e dois pacientes com NCC e 44 pacientes controles foram estudados. Líquido cérebro-espinhal foi analisado por um kit ELISA comercial desenvolvido para NCC. A sensibilidade e especificidade foram medidas e uma análise multivariada de regressão logística foi realizada. RESULTADOS: A sensibilidade e a especificidade de ELISA foram, respectivamente, 31,8 por cento e 100 por cento, com acurácia de 77,3 por cento. Apenas o tamanho das lesões mostrou-se importante para o desempenho do teste. CONCLUSÕES: Este estudo concluiu que ELISA contribui para o diagnóstico de NCC, caso o teste seja negativo ou caso o paciente seja portador de lesão cuja dimensão seja igual ou maior que dois centímetros.

Hydrophobic fraction of Taenia saginata metacestodes, rather than hydrophilic fraction, contains immunodominant markers for diagnosing human neurocysticercosis / Fração hidrofóbica de metacestódeos de Taenia saginata, ao contrário da fração hidrofílica, contém marcadores imunodominantes para o diagnóstico de neurocisticercose humana

INTRODUCTION: Considering that alternative antigens for diagnosing neurocysticercosis continue to be a challenge because of the increasing difficulty in obtaining parasites from naturally infected pigs for preparation of Taenia solium homologous antigen, the aim of the present study was to evaluate the detergent (D) and aqueous (A) fractions from saline extract of Taenia saginata metacestodes for diagnosing neurocysticercosis. METHODS: Taenia saginata was obtained from naturally infected bovines in the Triângulo Mineiro region, State of Minas Gerais, Brazil. The carcasses came from cold storage units and had been slaughtered in accordance with the inspection technique recommended by the Federal Inspection Service. The D and A fractions were obtained by using Triton X-114 (TX-114). Serum samples were obtained from 40 patients with a diagnosis of neurocysticercosis, 45 with other parasitic diseases and 30 from apparently normal individuals. IgG antibody levels were evaluated using the ELISA and immunoblotting assays. RESULTS: The ELISA sensitivity and specificity were 95 percent and 73.3 percent, when using saline extract; 95 percent and 82.6 percent for the D fraction; and 65 percent and 61.3 percent for the A fraction, respectively. The immunoblotting assay confirmed the ELISA results, such that the D fraction was more efficient than the other extracts, and the 70-68kDa component was immunodominant among neurocysticercosis patients. CONCLUSIONS: These results demonstrated that the D fraction from Taenia saginata metacestodes obtained using TX-114 can be used as a heterologous antigenic fraction in the immunoblotting assay for serologically diagnosing human neurocysticercosis, given its ability to select immunodominant antigens.

INTRODUÇÃO: Considerando que antígenos alternativos para o diagnóstico da neurocisticercose (NC) continua sendo um desafio devido ao aumento da dificuldade em se obter parasitas de suínos naturalmente infectados, para a preparação do antígeno homólogo de Taenia solium, o objetivo do presente estudo foi avaliar frações detergente (D) e aquosa (A), do extrato salino de metacestódeo de Taenia saginata para diagnóstico da NC. MÉTODOS: Bovinos, naturalmente infectados com Taenia saginata, procedentes da região do Triângulo Mineiro, Estado de Minas Gerais, Brasil, foram obtidos de frigoríficos e abatidos de acordo com a técnica de inspeção recomendada pelo Serviço de Inspeção Federal. As frações D e A foram obtidas utilizando Triton X-114 (TX-114). Amostras de soro foram obtidas de 40 pacientes com diagnóstico de NC, 45 com diagnóstico de outras doenças parasitárias e 30 de indivíduos aparentemente normais. Níveis de IgG foram avaliados pelos testes ELISA e Imunoblotting. RESULTADOS: A sensibilidade e especificidade do teste ELISA foram 95 por cento e 73,3 por cento, quando utilizado o extrato salino, 95 por cento e 82,6 por cento para fração D, e 65 por cento e 61,3 por cento para a fração A, respectivamente. O ensaio Imunoblotting confirmou os resultados do teste ELISA, sendo a fração D mais eficiente que os outros extratos, observando-se que o componente 70-68kDa se comportou como imunodominante para os pacientes com NC. CONCLUSÕES: Estes resultados demonstraram que a fração D de metacestódeo de Taenia saginata obtida com TX-114 pode ser utilizada como fração antigênica heteróloga pelo Imunoblotting para o diagnóstico sorológico da NC humana, considerando sua habilidade para selecionar antígenos imunodominantes.

A serological study of cysticercosis in patients with HIV / Estudo sorológico da cisticercose em pacientes com HIV

Neurocysticercosis (NCC) has attained the importance of one of the most common cause of focal brain lesions in patients infected with HIV (human immunodeficiency virus). Adequate data regarding the rate of this co-infection is lacking. Therefore, the present study was carried out to determine the prevalence of cysticercosis among HIV patients residing in Puducherry or its neighboring districts of Tamil Nadu State, India. A total of one hundred blood samples were collected from HIV seropositive cases visiting JIPMER hospital, Puducherry, between June 2007 and May 2008. Enzyme immunotransfer blot (EITB) and enzyme linked immunosorbent assay (ELISA) were used to demonstrate anti- T. solium larval stage antibodies and Co-agglutination (Co-A) test was used to detect T. solium larval stage antigens in sera. Two HIV seropositive cases were found positive for anti-T. solium larval stage antibody by EITB and four were positive by ELISA. Only one sample was positive by both EITB and ELISA. No serum sample was found positive for T. solium larval stage antigen by Co-A test. The overall seropositivity detected by all the methods was 5 percent in this study group. The accurate clinical diagnosis of NCC in HIV is difficult due to deranged immunological parameters in the HIV infected patients. The results of this study provides important data on the prevalence of cysticercosis in HIV positive patients in Puducherry and neighboring areas which was previously unknown. This study will also increase awareness among physicians and public health agencies about T. solium cysticercosis in the selected group.

Neurocisticercose (NCC) tem alcançado a importância de uma das mais comuns causas de lesões focais no cérebro em pacientes infectados pelo HIV (vírus da imunodeficiência adquirida). Dados adequados relativos à frequencia desta co-infecção estão faltando. Portanto, o presente estudo foi realizado para determinar a prevalência da cisticercose entre pacientes com HIV residindo em Puducherry ou distritos vizinhos do Estado de Tamil Nadu, India. Um total de cem amostras foram coletadas de casos soropositivos do Hospital JIPMER, Puducherry, entre junho de 2007 e maio de 2008. "Enzyme immunotransfer blot" (EITB) e ELISA foram utilizados para demonstrar anticorpos contra a fase larval do T. solium. Testes de co-aglutinação (Co-a) foram usados para demonstrar antígenos da fase larval do T. solium no soro. Dois casos HIV soropositivos foram positivos para anticorpos contra a fase larval do T. solium por EITB e quatro foram positivos por ELISA. Somente uma amostra foi positiva por ambos EITB e ELISA. Nenhuma amostra de soro foi positiva para antígeno da fase larval do T. solium pelo teste Co-a. A soropositividade total detectada por todos os métodos foi 5 por cento neste grupo de estudo. O diagnóstico clínico exato de NCC em HIV é difícil devido aos desordenados parâmetros imunológicos nos pacientes infectados pelo HIV. Os resultados deste estudo fornecem dados importantes sobre a prevalência da cisticercose em pacientes HIV positivos em Puducherry e áreas vizinhas que eram previamente desconhecidos. Este estudo também aumentará a atenção dos médicos e agências de saúde pública sobre a cisticercose por T. solium em grupo selecionado.

Evaluation of IgG4 and total IgG antibodies against cysticerci and peptide antigens for the diagnosis of human neurocysticercosis by ELISA.

To support the clinical diagnosis of human neurocysticercosis (NCC), we evaluated two peptides, HP6-3 and Ts45W-1, as well as crude saline extract (SE) of Tenia solium cysticerci as antigens for the detection of specific IgG4 subclass and total IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). The sera of definitive diagnosed NCC patients, patients infected with other parasitoses and healthy controls were examined. The diagnostic sensitivity for IgG4 and total IgG detection of the ELISA against SE antigen was 100% and 64.3% with a high amount of cross-reactions to taeniasis saginata at 88.9% (8/9) and 100% (9/9), respectively. The SE-based IgG4-ELISA showed the highest specificity (80.9%). Both peptide-based IgG4-ELISAs provided a superior sensitivity (78.6%) to the total IgG tests whereas their specificity was 66.7% for HP6-3 and 69.8% for Ts45W-1 only. The SE-based ELISA for the detection of specific IgG4 antibody can be used for the diagnosis of neurocysticercosis as well as for serological surveys of NCC endemic areas. The peptide-based IgG4 ELISAs potentially provide a reliable and cost effective alternative method independent from live parasite supply.

Verme de elite / Worms elite

Visa, de modo lúdico, trazer esclarecimentos a respeito da verdadeira forma de contágio para neurocisticercose de forma que a população passa se prevenir de forma consciente e eficiente contra esta parasitose...

Taenia solium metacestode immunodominant peptides recognized by IgG antibodies in cerebrospinal fluid and serum paired samples from patients with active and inactive neurocysticercosis

The aim of this study was to test if serological distinction between patients with active and inactive neurocysticercosis (NCC), could be accomplished by the recognition of immunodominant peptides in total saline antigenic extract of Taenia solium metacestodes by IgG antibody in cerebrospinal fluid (CSF) and serum paired samples. CSF and serum samples of 10 each, active NCC patients, inactive NCC, and individuals with other neurological disorders, were used to recognize the antigenic peptides by western blot (WB). In the active NCC the 28-32 and 39-42 kDa peptides were more frequently detected in CSF than in sera (p < 0.05). The 47-52, 64-68, and 70 kDa antigens showed high frequencies in both samples from patients with active NCC. All the CSF samples of inactive NCC and other neurological disorder (control) patients tested negative, while serum samples from these last two groups recognized mainly the 80, 86, 95, and 98 kDa bands. This finding eliminates the use of the high molecular weigh bands (> 80 kDa) for diagnosis of NCC. The final conclusions were that the difference between active and inactive NCC may be done with the detection of peptides only in the CSF samples and that the 47-52, 64-68, and 70 kDa bands may be included as specific markers for active NCC when detected in CSF samples by WB using total saline extract of T. solium metacestode.

Comparative evaluation of different immunoassays for the detection of Taenia solium cysticercosis in swine with low parasite burden

Seven swine were experimentally infected with Taenia solium eggs and blood samples from each animal were periodically collected. At the end of the experiment (t140) the animals did not show clinical aspects of cysticercosis or parasites in tongue inspection. All animals were slaughtered and cut into thin slices in searching for cysts. The number of cysts found in each animal varied from 1 to 85. Enzyme-linked immunosorbent assay (ELISA) tests for antibody (Ab) detection and for antigen (Ag) detection were performed, which presented respectively 71 and 57 percent of positivity. By immunoblot (IB), using 18/14(T. crassiceps Ag) or lentil-lectin-purified glycoproteins from T. solium Ag (LLGP) as Ag, five (71 percent) and six (86 percent) animals were positive, respectively. The association between Ag-ELISA with any IB (18/14 or LLGP) allowed the detection of all animals at 140 days post-experimental infection (days p.e.i.). The use of IB 18/14 combined to the Ag-ELISA allowed the detection of all animals since 70 days p.e.i., and the association between IB LLGP and Ag-ELISA allowed the detection of all animals since 112 days p.e.i. While all animals could be considered healthy by conventional screening tests, the use of immunoassays for detecting Ab and Ag showed better accuracy; therefore it would be more useful than usual clinical examination for screening cysticercosis in slightly infected pigs.

Evaluation of Taenia solium and Taenia crassiceps cysticercal antigens for immunodiagnosis of neurocysticercosis using ELISA on cerebrospinal fluid samples

The efficacy of whole parasite and vesicular fluid antigen extracts from Taenia solium and Taenia crassiceps cysticerci for immunodiagnosis of neurocysticercosis was evaluated using ELISA on cerebrospinal fluid samples. Anticysticercal IgG antibodies were assayed in cerebrospinal fluid samples from 23 patients with neurocysticercosis and 35 patients with other neurological disorders. The ELISA reaction for the whole Taenia solium cysticercal extract showed 91.3 percent sensitivity and 94.3 percent specificity, whereas the sensitivity and specificity of the ELISA for the whole Taenia crassiceps cysticercal extract were 87 percent and 94.3 percent, respectively. The ELISA reactions for vesicular fluid from Taenia solium or Taenia crassiceps showed 91.3 percent sensitivity and 97.1 percent specificity. Considering the results obtained from the four antigen preparations, vesicular fluid from Taenia solium and Taenia crassiceps cysticerci may be useful as a source of antigens for immunological reactions that are used for detecting specific antibodies in cerebrospinal fluid samples from patients with neurocysticercosis.

A eficácia de extratos antigênicos de parasitas totais e líquido vesicular de cisticercos de Taenia solium e Taenia crassiceps para o imunodiagnóstico da neurocisticercose foi avaliada por meio de reações de ELISA em amostras de líquido cefalorraquidiano. Anticorpos IgG anti-cisticercos foram pesquisados em amostras de líquido cefalorraquidiano de 23 pacientes com neurocisticercose e 35 pacientes com outras doenças neurológicas. A reação ELISA com o extrato bruto total de cisticercos de Taenia solium apresentou 91,3 por cento de sensibilidade e 94,3 por cento de especificidade, enquanto a sensibilidade e a especificidade da reação ELISA com o extrato total de cisticercos de Taenia crassiceps foram 87 por cento e 94,3 por cento, respectivamente. As reações ELISA com o líquido vesicular de Taenia solium ou Taenia crassiceps mostraram 91,3 por cento de sensibilidade e 97,1 por cento de especificidade. Considerando os resultados obtidos com as quatro preparações antigênicas, o liquido vesicular de cisticercos de Taenia solium e Taenia crassiceps pode ser útil como fonte de antígenos em reações imunológicas usadas para detectar anticorpos específicos em amostras de líquido cefalorraquidiano de pacientes com neurocisticercose.

Immunodiagnosis of human neurocysticercosis by using semi-purified scolex antigens from Taenia solium cysticerci

Crude antigen and semi-purified proteins from scolices of Taenia solium cysticerci were evaluated for the immunodiagnosis of human neurocysticercosis neurocysticercosis. Semi-purified proteins obtained by electrophoresis on polyacrylamide gel and by electroelution were tested by means of the immunoenzymatic reaction against sera from normal individuals and from patients with neurocysticercosis or other parasitic diseases. The 100kDa protein provided 100 percent sensitivity and specificity in the immunodiagnosis. When 95 or 26kDa proteins were used, 95 and 100 percent sensitivity and specificity were obtained, respectively. The assays involving crude antigen and sera from normal individuals or from patients with neurocysticercosis, diluted to 1:256, gave excellent agreement with those in which 100, 95 or 26kDa proteins were tested against the same serum samples diluted to 1:64. (Kappa: 0.95 to 1.00). Crude scolex antigen may be useful for serological screening, while 100, 95 or 26kDa protein can be used in confirmatory tests on neurocysticercosis-positive cases.

Antígeno bruto e proteínas semipurificadas de escóleces de cisticercos de Taenia solium foram avaliados para o imunodiagnóstico da neurocisticercose humana neurocisticercose. As proteínas semipurificadas, obtidas por eletroforese em gel de poliacrilamida e eletroeluição, foram testadas na reação imunoenzimática contra soros de indivíduos normais e de pacientes com neurocisticercose ou outras parasitoses. A proteína de 100kDa proporcionou 100 por cento de sensibilidade e especificidade no imunodiagnóstico. Quando a proteína de 95 ou 26kDa foi empregada, foram obtidos 95 e 100 por cento de sensibilidade e especificidade, respectivamente. Os ensaios envolvendo antígeno bruto e soros de indivíduos normais ou de pacientes com neurocisticercose, diluídos a 1:256, tiveram ótima concordância com aqueles onde a proteína de 100, 95 ou 25kDa foi testada contra os mesmas amostras de soro diluídas a 1:64 (Kappa: 0,95 a 1,00). O antígeno bruto de escolex poderá ser empregado na triagem sorológica enquanto a proteína de 100, 95 ou 26kDa nos testes confirmatórios dos casos positivos de NC.