Screening of Strongyloides infection using an ELISA test in transplant candidates

OBJECTIVES: Hyperinfection or disseminated strongyloidiasis has been frequently reported after transplants and is related to high mortality. This study aimed to screen for strongyloidiasis using serological diagnoses in transplant candidates. METHODS: An ELISA test was performed with filariform larvae of Strongyloides venezuelensis as a source of antigen. RESULTS: In the serum from transplant candidates, anti-Strongyloides IgG antibodies were detected in 35/150 (23.3%) samples by soluble fractions in phosphate buffered saline (PBS), 31/150 (20.7%) samples by soluble fractions in Tris-HCl, 27/150 (18.0%) samples by membrane fractions in PBS and 22/150 (14.7%) samples by membrane fractions in Tris-HCl. CONCLUSIONS: The present results suggest the ELISA test, ideally using soluble fractions of filariform larvae S. venezuelensis in PBS, as an additional strategy for the diagnosis of strongyloidiasis in transplant candidates.

Producción y evaluación del antígeno recombinante TES-30 de Toxocara canis para el inmunodiagnóstico de toxocariasis / Production and evaluation of the recombinant antigen TES-30 of Toxocara canis for the immunodiagnosis of toxocariasis

Introducción. Toxocara canis es un nematodo patógeno de cánidos que accidentalmente puede ser transmitido a los humanos. A pesar de la importancia de la serología para el diagnóstico de esta zoonosis, los kits diagnósticos usan antígenos crudos de excreción-secreción, en su mayoría glucoproteínas que no son específicas de especie, por lo cual pueden presentarse reacciones cruzadas con anticuerpos generados contra otros parásitos. Objetivos. Producir el antígeno recombinante TES-30 de T. canis y evaluarlo para el inmunodiagnóstico de la toxocariasis. Materiales y métodos. Se clonó el gen que codifica TES-30 en el vector de expresión pET28a (+), usando oligonucleótidos de cadena sencilla unidos mediante reacción en cadena de la polimerasa (PCR). La proteína rTES-30 se purificó por cromotografia de afinidad (Ni 2+ ). La reacción serológica de rTES-30 se evaluó mediante immunoblot . Teniendo en cuenta que no existe una prueba de referencia , se observó el comportamiento del antigeno en comparación con la prueba de rutina para el inmunodiagnóstico de la toxocariasis, es decir, la técnica ELISA convencional con antígenos de excreción-secreción. Resultados. El rTES-30 se produjo a partir de un cultivo de Escherichia coli LB, con un rendimiento de 2,25 mg/l y 95 % de pureza. La concordancia de la reacción entre el immunoblot rTES-30 y la ELISA convencional, fue de 73 % (46/63) y de 100 % con los 21 sueros no reactivos. De los 21 sueros con diagnóstico de otras parasitosis, 19 fueron reactivos con ELISA, mientras que tan solo siete fueron positivos con el immunoblot rTES-30. La concordancia entre la ELISA y el immunoblot fue moderada (índice kappa de 0,575; IC 95% 0,41-0,74). Conclusiones. Los datos presentados respaldan la utilidad del immunoblot r TES-3 0 para la confirmación de los posibles positivos por ELISA, no solo en los estudios epidemiológicos, sino también, como candidato para el desarrollo de pruebas diagnósticas de la toxocariasis ocular en Colombia.

Introduction: Toxocara canis is a pathogenic nematode of canines which can be accidentally transmitted to humans. Although serology is the most important diagnostic tool for this zoonosis, diagnostic kits use crude excretion/secretion antigens, most of them being glycoproteins which are not species-specific and may cross-react with antibodies generated against other parasites. Objectives: To produce the rTES-30 recombinant antigen of Toxocara canis and evaluate it in the immunodiagnosis of toxocariasis. Materials and methods: The gene that codes for TES-30 was cloned in the expression vector pET28a (+) using single-stranded oligonucleotides united by PCR. The protein rTES-30 was purified by Ni 2+ affinity chromotography. Seroreactivity of rTES-30 was evaluated by immunoblot. Given that there is no gold standard test, the behaviour of the antigen was compared with the method that is routinely used to immunodiagnose toxocariasis, i.e., the conventional ELISA technique using excretion/secretion antigens. Results: The rTES-30 was produced from an Escherichia coli LB culture which yielded 2.25 mg/L of the antigen with a purity of 95%. The results obtained showed 73% (46/63) concordance of reactivity between the rTES-30 immunoblot and the conventional ELISA, and 100% concordance with the non-reactive sera (21). Nineteen of the 21 sera positive for other parasitoses reacted with ELISA, while only seven of these were positive with the rTES-30 immunoblot. Concordance between the ELISA and the immunoblot was moderate (kappa coefficient: 0.575; 95% CI: 0.41- 0.74). Conclusions: The data presented show the potential of the rTES-30 inmunoblot for confirmation of possible ELISA positives, not only in epidemiological studies, but also as a candidate for the development of diagnostic tests for ocular toxocariasis in Colombia.

Purificación de la fracción antigénica 27-28 KDa a partir del antígeno metabólico secretado-excretado de Fasciola hepatica / Purification of antigenic fraction 27-28 KDa from the metabolic antigen from metabolic secreted-excreted from Fasciola hepatica

En el presente estudio, las fracciones antigénicas de 27-28 KDa de Fasciola hepatica fueron purificadas por cromatografía de exclusión molecular para su aplicación en el diagnóstico de la fascioliasis humana. Se obtuvieron antígenos de excreción y secreción a partir de fasciolas adultas vivas obtenida de hígado de ovino y bovino, y cultivados en medio mínimo esencial. La reactividad y eficacia del antígeno purificado fueron evaluadas por la prueba de inmunoblot empleando cuatro sueros con fascioliasis humana; cuatro sueros con otras parasitosis, y dos sueros negativos. Se concluye que las fracciones antigénicas purificadas no presentan reacción cruzada con otras parasitosis, por inmunoblot, por lo que se considera a las proteínas purificadas como potenciales candidatas a ser utilizadas para el diagnóstico de fascioliasis humana.

Antigenic fractions of 27-28 kDa from Fasciola hepatica were purified by size-exclusion chromatography for use in the diagnosis of human fasciolosis. Excretion and secretion antigens were obtained from living adult flukes collected from sheep and cattle liver, and cultured in minimum essential medium. The reactivity of the purified antigen and efficacy were assessed by immunoblot test using four sera with human fascioliasis; four sera with other parasites, and two negative sera. We conclude that the purified antigenic fractions do not cross-react with other parasites by immunoblot. Therefore, purified proteins are considered as potential candidates to be used for the diagnosis of human fascioliasis.

Detección de coproantígenos para el diagnóstico de echinococosis canina en la zona fronteriza de La Quiaca-Villazón / Coproantigens detection for the diagnosis of canine Echinococcosis in the border area of La Quiaca-Villazón

Las técnicas de inmunodiagnóstico basadas en la identificación de antígenos parasitarios en las heces secas de los perros han sido desarrolladas para la vigilancia de la echinococosis canina. En la región fronteriza de La Quiaca-Villazón, se encontraron las condiciones ambientales que favorecerían el ciclo del parásito, dada la presencia del hospedador definitivo (perro) y de hospedadores intermediarios (ovejas y cabras). La actividad más importante de la Puna es la cría de ovinos y camélidos; la faena se realiza en el campo y a manos del dueño de los ovinos, y no se aplican medidas preventivas de sanidad. El objetivo de este trabajo fue estimar la presencia de caninos parasitados por Echinococcus granulosus en esta región. Durante el año 2006 se recolectaron 168 muestras de materia fecal. En La Quiaca se tomaron muestras de las siguientes localidades: Barrios (área semirrural), Santa Catalina, Yavi Chico, El Portillo, Pumahuasi y Cara Cara (zonas rurales) y La Quiaca (área urbana). En Villazón se seleccionó el área urbana y el área semirrural de Ojo de Agua, Lampaya y Matancillas. Las muestras se analizaron por la prueba de copro-ELISA y copro-Western blot. Las localidades de San Francisco y Barrios tuvieron una prevalencia de 14,3 % y 6,7 %, respectivamente. En Villazón se encontró un 3,4 % de prevalencia en el área urbana. En Lampaya se encontró un 30 % de prevalencia. Estos resultados sugieren la necesidad de implementar estrategias para el control de la hidatidosis, tanto a nivel urbano como rural, para evitar el aumento y la dispersión de la echinococosis en la región.

Cystic Echinococcosis is a major public health issue. Immunodiagnostic techniques based on the identification of parasite antigens in dog dry faeces have been developed as alternatives for the surveillance of canine Echinococcosis. The environmental conditions favouring the parasite cycle were met in the border of La Quiaca-Villazón, given the presence of definitive (dog) and intermediate hosts (sheep and goats). The most important activity in La Puna is sheep and goat rearing; slaughtering is carried out almost exclusively in the field by sheep's owners, and preventive health measures do not apply. The aim of this study was to determine the presence of dogs parasitized by Echinococcus granulosus in this border region. A hundred and sixty eight (168) stool specimens were collected during 2006. La Quiaca samples were taken from the following selected areas: the semi-rural area of Barrios, the rural areas of Santa Catalina, Yavi Chico, El Portillo, Pumahuasi and Cara Cara and the urban area of La Quiaca; selected urban areas in Villazón and the semi-rural area of Ojo de Agua, Lampaya and Matancillas. The samples were analyzed by copro-ELISA -Western blot test. The cities of San Francisco and Barrios had a prevalence of 14.3% and 6.7%, respectively. A prevalence of 3.4% was observed in the urban area of Villazón, which indicates that dogs become infected in the rural areas and bring the risk into the city. Lampaya showed a prevalence of 30%. These findings suggest the need to implement strategies for the control of hydatidosis, both in urban and rural areas to avoid the increase and spread of Echinococcosis in the region.

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.

Effects of Toxoplasma gondii and Toxocara canis Antigens on WEHI-164 Fibrosarcoma Growth in a Mouse Model

Cancer is the main cause of death in developed countries. However, in underdeveloped countries infections and parasitic diseases are the main causes of death. There are raising scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of Toxoplasma gondii and Toxocara canis egg antigens in comparison with Bacillus Calmette Guerin (BCG) (known to have anticancer distinctive) on WEHI-164 fibosarcoma transplanted to BALB/c mice was investigated. Groups of 6 male BALB/c mice injected with T. gondii antigen, BCG, or T. canis egg antigen as case groups and alum alone as control groups. All mice were then challenged with WEHI-164 fibrosarcoma cells. The mice were examined for growth of the solid tumor and the tumor sizes were measured every other day up to 4 wk. The mean tumor area in T. gondii, BCG, or alum alone injected mice in 4 different days of measurements was 25 mm2, 23 mm2, and 186 mm2 respectively. Also the mean tumor area in T. canis injected mice in 4 different days was 25.5 mm2 compared to the control group (alum treated) which was 155 mm2. T. gondii parasites and T. canis egg antigens induced inhibition of the tumor growth in the fibrosarcoma mouse model. We need further study to clarify the mechanisms of anti-cancer effects.

Serological reactivity of different antigenic preparations of Leishmania (Leishmania) amazonensis and the Leishmania braziliensis complex / Reatividade sorológica frente a diferentes preparações antigênicas de Leishmania (Leishmania) amazonensis e do complexo Leishmania braziliensis

Total antigen from Leishmania (Leishmania) amazonensis and isolates from the Leishmania braziliensis complex, along with their respective antigenic fractions obtained by affinity chromatography on concanavalin-A-Sepharose and jacalin-agarose columns evaluated using immunoenzymatic ELISA assay. For this, serum samples from 229 patients were used, grouped as American tegmental leishmaniasis (nº=58), visceral leishmaniasis (nº=28), Chagas disease (nº=49), malaria (nº=32), tuberculosis (nº=13) and healthy volunteers (nº=49). Samples from American tegmentary leishmaniasis showed higher reactivity with antigens isolated from the Leishmania braziliensis complex than with antigens from Leishmania amazonensis (p<0.001). ELISA assays showed a sensitivity range from 60 percent to 95 percent with antigens isolated from the Leishmania braziliensis complex. There was marked nonspecific reactivity among serum samples with the use of antigenic fractions binding with concanavalin-A and jacalin from both Leishmania complexes, in comparison with other antigens (p<0.001). The results presented in this study suggest that the use of homologous antigens increases the efficiency of anti-Leishmania immunoglobulin detection, which may be very valuable for diagnostic purposes.

Antígeno total de Leishmania (Leishmania) amazonensis e isolado do complexo Leishmania brazilienis, assim como suas respectivas frações antigênicas obtidas por cromatografia de afinidade em coluna de concanavalina-A ligada a sepharose e Jacalina ligada a agarose foram avaliadas por ensaio imunoenzimático ELISA. Para tanto, foram utilizadas amostras de soros de 229 pacientes agrupadas em leishmaniose tegumentar americana (nº=58), leishmaniose visceral (nº=28), doença de Chagas (nº=49), malaria (nº=32), tuberculose (nº=13) e voluntários saudáveis (nº=49). Houve maior reatividade das amostras de leishmaniose tegumentar americana com a utilização dos antígenos obtidos do isolado do complexo Leishmania braziliensis quando comparado com antígenos de Leishmania amazonensis (p<0,001). Observou-se ainda que a sensibilidade do teste ELISA variou de 60 a 95 por cento entre os antígenos obtidos do isolado do complexo Leishmania braziliensis. Houve acentuada reatividade inespecífica das amostras de soros com a utilização das frações antigênicas ligantes de Concanavalina-A e Jacalina de ambos os complexos Leishmania em comparação aos demais antígenos (p<0,001). Os resultados apresentados no presente trabalho sugerem que a utilização de antígenos homólogos aumentam a eficiência de detecção de imunoglobulina anti-Leishmania o que pode ser de grande valia para o propósito de diagnóstico.

The ultrastructure of the gravid uterus of Brugia pahangi, another rich source of antigen of the filarial parasite.

The gravid uterus with zygotes and microfilariae in utero of Brugia pahangi, a rich source of antigen as revealed by a recent immunofluorescent technique, were studied ultrastructurally. The epithelial cells of uterus show ultrastructural features of synthetically active cells. Their secretions may provide nutrients for the egg in utero. On the basal side, the uterine epithelial cells may also secrete substances to form the basal lamina of the uterus which is rather thick and irregularly fused with the basal lamina lining the body wall where the pseudocoelomic cavity is obliterated. For the most part, the uterine basal lamina contains uniform granular material of moderate electron density. There are also elongated visceral muscle cells embeded in it, and which surround the uterus, with adjacent cells overlapping. The gravid uterus contains several stages of developing microfilariae within its lumen, the cleaving zygotes are also present at another level. The morula of zygotes are composed of several closely packed cells surrounded loosely by their own egg shell membranes. The egg shell becomes more convoluted as development proceeds. The egg shell surrounding the developing microfilariae in utero is secreted by the uterine epithelium. This structure later becomes the sheath of circulating microfilariae, and is highly antigenic as indicated by intense labeling with fluorescent antibodies.

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

Common antigenicities between Angiostrongylus cantonensis and various species of snails.

Studies on common antigenicities were carried out by using rabbit sera immunized with Angiostrongylus cantonensis adult worms or the third stage larvae and antigens of various species of snails and vice versa by the immunoblotting technique. The results obtained are summarized as follows: (1) Common antigenicities between A. cantonensis adult worms and snails susceptible to A. cantonensis were observed in a range of molecular weights of 14.3 to 200 kDa. In Puerto Rican pigmented Biomphalaria glabrata and Achatina fulca, which had high infection rates with A. cantonensis, we recognized 15 to 16 bands against the adult worm, especially the band with a molecular weight of 29 kDa, which had a more intense reaction. (2) Common antigenicities between A. cantonensis third stage larvae and snails susceptible to A. cantonensis, were observed in a range of molecular weights of 14.3 to 97.4 kDa, especially A. fulica and B. glabrata, where we detected many bands in molecular weight range of 18.4 to 43 kDa. Based on the common antigenicities between A. cantonensis and snails susceptible to A. cantonensis, it is possible that the common antigenicities are one of the factors defining the different susceptibilities of various species of snails to A. cantonensis, and more bands are seen with increasing infection rates with A. cantonensis. Of those bands, the protein with the molecular weight of 29 kDa may be the main common antigen between the A. cantonensis adult worm, the third stage larvae and the snails susceptible to A. cantonensis.

Preparation of Gnathostoma protein by ultrafiltration method using Nanosep membrane.

We report our experience with Gnathostoma protein preparation by the ultrafiltration method. Crude antigen was sonicated and ultrafiltrated using the Nanosep 100 K membrane. SDS-PAGE electrophoresis showed protein bands at 43, 41, 24, 22, 21, 19.5 kDa. Use of the ultrafiltration method can provide specific protein (24 kDa), similar to the non-ultrafiltration method, with the other 5 non-specific proteins. Using the non-ultrafiltration method, there were more (20) non-specific protein. The ultrafiltration method can be an alternative method for the preparation of protein, which can provide better results than non-ultrafiltration.

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis.

Detection of day blood filarial antigens by Og4C3 ELISA test using filter paper samples.

BACKGROUND: The launching of the global filariasis elimination programme has necessitated the use of highly sensitive and specific diagnostic tests. The Og4C3 monoclonal antibody-based ELISA test has been found to be highly specific and sensitive for the diagnosis of filariasis using night blood samples. However, it requires a serum sample which poses problems of transport and storage. Collection of blood samples on filter paper the will greatly circumvent these problems. Therefore, we evaluated the utility of the Og4C3 assay on filter paper samples collected during daytime. METHODS: Blood samples were collected from 63 microfilariae (mf) carriers during different time periods in a day on filter paper discs as well as venous blood for sera. The mf carriers and chronic (hydrocele n = 20; lymphoedema n = 120) and acute filariasis (adenolymphangitis n = 39) patients were from the endemic areas and the non-endemic normals were from Uthagamandalam district of Tamil Nadu, India. The filarial antigens in the samples were determined using the Og4C3 filarial antigen assay as per the manufacturer's instructions (JCU TrapBio, Australia). The sensitivity of the assay on sera and filter paper samples collected during night and also on filter paper samples collected during different time intervals of the day were compared with those of the membrane filtration technique, which was used as a gold standard. RESULTS: The geometric mean titre of the sera samples collected during night was 11 units/ml for non-endemic normals and 601.2 units/ml for mf carriers. The specificity of the assay on sera samples collected during night was 100% and the sensitivity 96.8% and the positive and negative values were 100% and 95.2%, respectively. The antigen positivity of the filter paper samples collected during morning hours was 93.3% while it was 76.6% and 86.7% for afternoon and evening hours. A significant association was observed between antigenaemia levels and mf density in the blood samples collected during the night. CONCLUSION: The samples collected on filter paper during the day can be used as an alternative to sera samples for detection of filarial antigens employing Og4C3 ELISA. Also, samples collected during morning hours yield a higher positivity. The assay when applied to serum samples will be useful especially when quantitative results are required.

The impact of repeated treatment with praziquantel of schistosomiasis in children under six years of age living in an endemic area for Schistosoma haematobium infection

Praziquantel was given every eight weeks for two years to children aged under six years of age, living in a Schistosoma haematobium endemic area. Infection with S. haematobium and haematuria were examined in urine and antibody profiles (IgA, IgE, IgM, IgG1, IgG2, IgG3, and IgG4) against S. haematobium adult worm and egg antigens were determined from sera collected before each treatment. Chemotherapy reduced infection prevalence and mean intensity from 51.8 percent and 110 eggs per 10 ml urine, respectively, before starting re-treatment programme to very low levels thereafter. Praziquantel is not accumulated after periodic administration in children. Immunoglobulin levels change during the course of treatment with a shift towards 'protective' mechanisms. The significant changes noted in some individuals were the drop in 'blocking' IgG2 and IgG4 whereas the 'protecting' IgA and IgG1 levels increased. The antibody profiles in the rest of the children remained generally unchanged throughout the study and no haematuria was observed after the second treatment. The removal of worms before production of large number of eggs, prevented the children from developing morbidity

ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci

In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra) and crude larvae extract (T-Tcra), and from Taenia solium extracts of scolex (S-Ts) and of larvae (T-Ts). A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0 per cent and 80.0 per cent sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5 per cent and 100.0 per cent. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.

Antigenic components of Gnathostoma spinigerum recognized by infected human sera by two-dimensional polyacrylamide gel electrophoresis and immunoblotting.

Antigenic components of Gnathostoma spinigerum larval extract were revealed by two-dimensional gel electrophoresis (2-DE) and immunoblot analysis using sera from patients with 6 proven cases of gnathostomiasis, 5 presumptive cases of gnathostomiasis, 3 proven cases of angiostrongyliasis, 3 proven cases of cysticercosis, and pooled sera from healthy adults. By the 2-DE, the larval extract was highly complex and consisted of more than 75 polypeptides. Immunoblotting analysis of this larval extract after reaction with each of 6 proven gnathostomiasis sera revealed various numbers of antigenic spots ranging from 30 to 70 spots at the approximate molecular masses of less than 14.4 to more than 94 kDa with isoelectric points (pI) of less than 4.65 to 9.6. Antigenic spots at the approximate molecular mass of more than 30 kDa were recognized with the proven angiostrongyliasis, proven cysticercosis and healthy control sera but these sera did not react with the spots at approximate molecular masses of 23-25 kDa with pI of 8.3-8.5. The reacted spots, which consisted of at least 1 to 2 spots, were unique for the recognition of gnathostomiasis sera. Five out of 6 (83.3%) proven and 4 out of 5 (80%) presumptive gnathostomiasis sera reacted with these specific spots.

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.

IgG- and IgG4-detected antigens of Dirofilaria immitis adult worms for bancroftian filariasis by enzyme-linked immunoelectrotransfer blot.

In Thailand, Wuchereria bancrofti filariasis has persisted along the border between Thailand and Myanmar, its dynamic distribution caused by the infected transmigrants between neighboring countries, and the availability of susceptible mosquito vectors. Dirofilaria immitis adult worm was used as a source of antigens, excretory-secretory (ES) and partial surface extracts, to detect human filariasis. ES products showed several stained bands with Coomassie brilliant blue ranging from 14.5-93 kDa and mostly being glycoproteins as shown by concurrent reaction with Concanavalin A, except those at 18, 16 and 14.5 kDa which stained only with Coomassie brilliant blue. Surface proteins of 33.5-91.5 kDa were stained with Coomassie brilliant blue and showed smear bands with Concanavalin A. By enzyme-linked immunoelectrotransfer blot, Bancroftian filariasis sera gave specific reactions with glycoprotein ES antigens at MW 20.5 kDa against anti-human IgG. A prominent band of 18 kDa appeared consistently with the IgG4-ES antigen system. Surface extracts reacting with IgG and IgG4 were considered to be unsuitable as antibodies from all cases of filariasis could not detect any bands.

Neurocysticercosis and echinococcosis in Asia: recent advances in the establishment of highly reliable differential serodiagnosis for international collaboration.

Neurocysticercosis (NCC) and echinococcosis, caused by the larval stage of taeniid cestodes, are recognized as major parasitic zoonoses threatening human life worldwide. Cystic echinococcosis (CE), caused by Echinococcus granulosus, has well been known to be more widely distributing in Europe and Asia (Eurasia) than alveolar echinococcosis (AE) caused by E. multilocularis. However, it has recently been found that AE is more widely distributing or spreading in Eurasia. Furthermore, NCC caused by Taenia solium is also spreading in Eurasia. Due to the lack of reliable methodology for diagnosing these zoonotic cestodiases worldwide, prevalence rates of these diseases are extremely underestimated. Our group has been working for the establishment of differential serodiagnosis and molecular diagnosis of AE. CE and NCC as international collaboration projects sponsored by the Ministry of Education, Japan from 1994 until 2000 at least. In this paper, we introduce (1) the most recent original work on the establishment of differential serodiagnoses of NCC, AE and CE, (2) international collaboration work on epidemiology of these diseases in several countries, and discuss (3) what we can and should do for the control of such global parasitic diseases. It is stressed that international collaboration or cooperation work on the control of parasitic diseases is only successful based on the original scientific contribution of high standard.