Immunology of a unique biological structure: the Echinococcus laminated layer

The larval stages of the cestode parasites belonging to the genus Echinococcus grow within internal organs of humans and a range of animal species. The resulting diseases, collectively termed echinococcoses, include major neglected tropical diseases of humans and livestock. Echinococcus larvae are outwardly protected by the laminated layer (LL), an acellular structure that is unique to this genus. The LL is based on a fibrillar meshwork made up of mucins, which are decorated by galactose-rich O-glycans. In addition, in the species cluster termed E. granulosus sensu lato, the LL features nano-deposits of the calcium salt of myo-inositol hexakisphosphate (Insp6). The main purpose of our article is to update the immunobiology of the LL. Major recent advances in this area are (i) the demonstration of LL "debris" at the infection site and draining lymph nodes, (ii) the characterization of the decoy activity of calcium Insp6 with respect to complement, (iii) the evidence that the LL mucin carbohydrates interact specifically with a lectin receptor expressed in Kupffer cells (Clec4F), and (iv) the characterization of what appear to be receptor-independent effects of LL particles on dendritic cells and macrophages. Much information is missing on the immunology of this intriguing structure: we discuss gaps in knowledge and propose possible avenues for research.

Prevalence of Serum IgG Antibodies to Cystic Echinococcus Antigen among Patients in an Uzbekistan Emergency Hospital

Cystic echinococcosis (CE) is one of the most widespread zoonotic helminthiases, which can last an asymptomatic infection for several years. The purpose of this study was to demonstrate serum antibody prevalence of CE among asymptomatic people in Uzbekistan using ELISA. A total of 2,547 serum samples were collected, 66 from confirmed CE patients and 2,481 of patients with other diseases than CE at a hospital in Tashkent, Uzbekistan. The serum samples were screened for CE specific IgG antibodies by ELISA using cystic fluid antigen obtained from sheep. The serum antibody positive rate was 89.4% (59/66) in CE and 3.6% (89/2,481) in other disease patients. The present ELISA recognized 89.4% sensitivity and 96.4% specificity. The ELISA absorbance of positive samples was distributed 0.271-0.971 for CE and 0.273-0.887 for other disease patients. The other disease patients with high absorbance over 0.3 were 50 (2.0%) who were presumed to be active CE patients. The patients in their 40s showed the highest positive rate of 5.2% (P=0.181), and women were 4.4% while men were 3.1% positive (P=0.136). The data confirmed that there are many asymptomatic patients of CE in Tashkent. It is indicated that CE is an endemic disease of public health importance in Uzbekistan.

Urinary antingen detection for diagnosis of hydatid disease

Hydatid antigen was demonstrated for the first time in urine of patients with hydatidosis by coagglutination test [Co-A]. Urinary antigen was detected in all Co-A positive serum corresponding samples of surgically confirmed hydatid disease. The sensitivity and specificity were 100% in urine compared with the corresponding serum samples. These results clarified that the use of Co-A test for the detection of hydatid antigen in urine is an easy, simple, rapid, noninvasive and efficient method for the diagnosis of hydatidosis

Immunological follow-up of hydatid cyst cases

Hydatid disease is caused by Echinococcus granulosus. In this study, we aimed to investigate the benefit of monitoring cases with hydatid cyst by means of immune components in patients in a long-term follow-up after surgery. Eighty-four preoperative and postoperative serum samples from 14 cases undergoing surgery for hydatid disease were evaluated in terms of immune parameters, such as total and specific IgE, IgG, IgM, IgA and complement. Total and specific IgE were determined by ELISA. Specific IgG levels were measured by indirect hemaglutination.Total IgG, IgM, IgA and complement (C3 and C4) were detected by nephelometry. Imaging studies were also carried out during the follow-up. In none of the patients hydatid cysts were detected during the follow-up. Total IgE levels in the sera of the patients decreased to normal six months after surgery. Although specific IgE against echinococcal antigens decreased one year after operation, levels were still significantly high. There were no changes in the levels of anti-Echinococcus IgG and total IgG in follow-up period. Additionally, other parameters, such as IgA, IgM, C3 and C4, were not affected

Differential serodiagnosis of cystic and alveolar echinococcosis using native and recombinant antigens in Japan.

Our group at Asahikawa Medical College has established differential serodiagnosis for zoonotic larval cestodiases such as alveolar echinococcosis (AE), cystic echinococcosis (CE) and neurocysticercosis (NCC) using purified specific antigens. In this brief review, we introduce (a) four imported CE cases in Japan, easily identified serologically, (b) most recent advances in serology for differentiation of AE and monitoring of prognosis of AE in Japan. It includes application of affinity purified Em18 and prototype of a recombinant Em18 antigen. Serology using affinity purified Em18 antigens is showing much higher sensitivity for detection of AE cases which are usually undetectable by the ongoing serology for AE authorized in Hokkaido, Japan. As serology for AE, CE or NCC is still not popular in the majority of Asian countries, we expect that this review paper stimulates researchers who are interested in serology or serodiagnosis for these larval cestodiases including AE, CE and NCC.

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates

Albendazole therapy for a recurrent orbital hydatid cyst.

Till recently, the treatment of a multiloculated hydatid cyst in the confines of the orbit was every ophthalmologist's nightmare. Over the last decade, two benzimidazole compounds, mebendazole and albendazole, have been tested clinically for use in the chemotherapy of hydatid disease.

Detection of hydatid antigen in human cyst fluid by reverse latex agglutination test

The present work studied the use of reverse latex agglutination [RLA] test in the diagnosis of CE as a test that could directly detect hydatid antigen in human cyst fluid samples. The results were reliable when compared with the direct microscopic examination of cyst fluid samples. Also, it was more reliable than indirect hemagglutination test as it showed 100% sensitivity. No false positive reaction was observed with samples from cystic tumors with a resultant specificity of 100%. Moreover, the test is easy to perform with a visually interpreted results within 2-3 minutes. The reagents are inexpensive, stable and easily available. These merits make the RLA test suitable for the diagnosis of CE, particularly in suspected cases with seronegative results or cases with sterile cysts

Identification of immunodeominant antigens by immunoelectrotranfer in hydatid fluid

A través de electroforésis en geles de poliacrilamina (SDS-PAGE) e inmunoelectrotransferencia (Western Blot), fueron identificados y caracterizados los antígenos immunodominantes del líquido hidatídico. Para ello, se estudiaron 23 pacientes con hidatidosis confirmadas, 12 con sospecha clínica de infección y serología positiva con los métodos convencionales (doble difusión con detección del arco 5 y ELISA), 28 individuos sanos y 23 con otras infecciones parasitarias y no parasitarias. Los resultados demostraron 7 bandas antigénicas localizadas entre los 8 y los 120 kDa. Dos bandas immunodominantes fueron reconocidas en los pacientes con hidatidosis comprobada y con serología positiva, sus pesos moleculares (PM) fueron de 8 y 12 kDa. Dos bandas inespecíficas fueron detectadas con los sueros de individuos sanos y con cisticercosis. Se concluye que las bandas de PM de 8 y 12 kDa son las de mayor valor diagnóstico a diferencia de las de 32 y 60 kDa que son inespecíficas

Immunodiagnosis of human hydatid disease.

A study was undertaken to evaluate the efficacy of Enzyme Linked Immunosorbent Assay using locally prepared antigens for immunodiagnosis of human hydatid disease. A total of 90 cases clinically suspected to be suffering from hydatid disease and 100 controls matched for age and sex were included in the study. Two types of ELISA were performed on detected specific antihydatid antibodies belonging to IgG/IgM/IgA classes and other type detected IgE class of antibodies. Antigen prepared from the human hydatid fluid was found to be unsuitable for diagnosis as it contained host proteins i.e. IgG. Sheep hydatid fluid obtained from the fertile hydatid cyst was used to prepare and standardize the antigen. ELISA test to detect anti hydatid antibodies belonging to either IgG, IgM and or IgA was found to be highly specific (98 per cent) in surgically confirmed hydatid disease and was negative in all the controls. The results of the study indicate that ELISA along with casoni test may provide the best results in diagnosis of hydatid disease.

Foco de hidatidosis humana en la provincia del Chaco (Argentina) / Human hydatidosis in the province of Chaco (Argentina)

En Argentina han sido descritas cinco áreas endémicas de hidatidosis, no estando la Provincia de Chaco incluida en ninguna de ellas. Habiéndose notificado un caso autóctono en esta región, se decidió realizar un estudio epidemiológico para reconocer si esta área es endémica a hidatidosis. Se estudiaron 52 personas (13 mujeres-39 varones) de entre 8 y 58 años de edad, elegidos al azar entre los residentes permanentes del pueblo de Samuhú. A todos se les confeccionó una ficha epidemiológica, y se tomó muestra de sangre. Para investigar anticuerpos anti E. granulosus se empleó la prueba de ELISA y los sueros que resultaron positivos se les practicó inmunoelectroforesis (IEF) empleando en ambos casos reactivos comerciales. Diez muestras (19,3 por ciento) resultaron positivas a ELISA y de éstas, 5 (9,6 por ciento) se confirmaron por IEF (2 mujeres y 3 varones). De acuerdo a los resultados obtenidos y a la información epidemiológica recogida, se puede concluir que en la provincia del Chaco existe una área geográfica endémica a hidatidosis

Echinococcus granulosus recombinant antigens

Echinococcus granulosus is a small parasitic platyhelminth, the larval stage of which is the causative agent of cystic hydatid disease both in man and domestic ungulates. This zoonosis constitutes major economic and public (both human and veterinary) health problems in many parts of the world, including southern Brazil, where it is hyperendemic. Serodiagnosis of cystic hydatid disease has been of great importance in clinical diagnosis, posttreatment surveillance of patients and epidemiólogical surveys, since it is the most specific of the noninvasive alternatives for detecting infections with the E. granulosus metacestode. Nevertheless, immunological tests present chronical problems associated with the quality and availability of parasite antigens. In this sense, the cloning of E. granulosus antigen-encoding genes comes forth as a valuable alternative for the production of pure and well characterized antigens. In the last few years, several research groups have cloned and characterized genes that encode E. granulosus antigens, many of which are potentially useful in the immunodiagnosis of cystic hydatid disease. Furthermore, several of these antigens represent important components of the parasite biology and may be particularly relevant to vaccination, immunotherapy, or as potential targets for chemotherapy. In this review we summarize the available data concerning the production and characterization of E. granulosus recombinant antigens. We also discuss some of the perspectives opened by the use of molecular biology techniques in the diagnosis of cystic hydatid disease as well as in the study of the biology of this parasite.

Alergia a Anisakis simplex: estudios clínicos e inmunológicos y reactividad cruzada con otros parásitos / Allergy by Anisakis simplex: clinical and immunological study and cross-reactivity with other parasites

El Anisakis simplex es un parásito perteneciente a la familia Anisakidae, de la clase nematodos, que vive en el tubo digestivo de grandes mamíferos marinos y parasita accidentalmente al hombre al ingerir pescado crudo o poco cocinado. Recientemente se ha implicado como posible factor etiológico de reacciones alérgicas inducidas tras la ingesta de pescado parasitado. En el presente estudio investigamos la posible sensibilización a antígenos de A. simplex en 11 pacientes que presentaron reacciones alérgicas tras la ingesta de pescado. También se estudió la existencia de reactividad cruzada con otros parásitos. En todos los pacientes se demostró, mediante pruebas inmunológicas "in vivo" e "in vitro", la existencia de un mecanismo de hipersensibilidad inmediata a antígenos de A. simplex. Las pruebas cutáneas con el extracto comercial, así como la determinación de IgE específica por técnica CAP y la liberación de histamina frente a este parásito fueron positivos, confirmando la sensibilización mediada por anticuerpos IgE a antígenos de Anisakis. Al estudiar la posible reactividad cruzada con otros parásitos (Ascaris lumbricoides, Echinococcus granulosus) por medio del inhibición del CAP, ésta resultó muy escasa para Ascaris lumbricoides, por lo que en estos pacientes dicha reactividad cruzada parece poco relevante. En cambio la inhibición del CAP a Eschinococcus granulosus con A. simplex llegó hasta el 30-60 por ciento, sugiriendo la existencia de cierto grado de reactividad cruzada entre ambos parásitos, aunque estén más alejados taxonómicamente. Debido a que el Anisakis simplex es un parásito ubicuo, ya que infecta a pescados y mariscos en mares de todo el mundo, sugerimos que ante la historia de manifestaciones alérgicas tras la ingesta de esta clase de alimentos, se investigue la sensibilidad a este parásito, además de los antígenos propios de los frutos de mar. Por otra parte, se debería estudiar la posible sensibilización a A. simplex en los pacientes a los cuales se les detecta IgE específica a E. granulosus, descartándose con anterioridad la enfermedad hidatídica.

Isolation & immunochemical characterization of diagnostically relevant antigens of Echinococcus granulosus.

Two hydatid specific polypeptides with molecular masses of 8 kDa and 116 kDa have been successfully isolated from E. granulosus hydatid cyst fluid using affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and western immunoblot analysis under reducing and denaturing conditions indicated the 116 kDa purified antigen to be a hetero-tetramer consisting of 45 kDa, 66 kDa, 75 kDa and 116 kDa subunits linked by disulphide bonds while the 8 kDa purified antigen was found to be a monomer polypeptide. Affinity purified 116 kDa molecule was heat-labile, sensitive to treatment with pronase, trypsin and pepsin and its immunoreactivity as assessed in enzyme linked immunosorbent assay remained unaltered on treatment with sodium metaperiodate. The affinity purified 8 kDa molecule was heat-stable, sensitive to proteolytic enzymes and also sodium metaperiodate oxidation. Lectin binding studies revealed that the 8 kDa molecule specifically bound Concanavalin A and Triticum vulgaris, and thus had varies; is directly proportional to-D-glucose and N-acetyl D-glucosamine sugar moieties. The immunoreactivity of both the antigens remained unaltered on treatment with lipase. However, biochemical estimation of total lipid content revealed the affinity purified 116 kDa antigen to contain 6.25 per cent total lipids suggesting it to be lipoproteinic in nature. The 8 kDa antigen had no detectable total lipids biochemically. All sera from patients confirmed to have hydatidosis recognised the 8 kDa and 116 kDa polypeptides. However, sera from seven subjects with other parasitic infections also recognised the 116 kDa antigen though not the 8 kDa antigen. The data suggested that the recognition of 8 kDa antigen of E. granulosus has potential for specific immunodiagnosis of hydatidosis.

Recent trends in the serodiagnosis of hydatid disease.

Hydatid disease caused by Echinococcus granulosus is a zoonotic infection of cosmopolitan distribution. As the clinical manifestations of hydatid disease in man are variable, the diagnosis of the condition presents complex problems for clinicians. Since the parasitic diagnosis of the disease is difficult, the specific diagnosis of the condition relies heavily on immunodiagnostic tests. The recent approach to the diagnosis of hydatid disease in man is primarily based on: (1) a combination of two or three more serological tests to diagnose the condition, as a single test fails to detect all the cases, (2) detection of circulating hydatid antigen (CAg) in the serum by enzyme-linked immunosorbent assay (ELISA) and other assays, as the antigen detection system is useful in monitoring post-surgical and chemotherapeutic evaluation of the cases as well as in the prognosis of the condition and (3) demonstration of E. granulosus antigen in the cystic fluid to establish the etiology of the hydatid cyst. Hydatid disease is essentially a disease of poor people residing in rural areas, hence there is need for a simple, economic diagnostic immunoassay for use at the field level or in a rural health center with inadequate facilities. Counter-current-immunoelectrophoresis (CIEF) and bacterial co-agglutination (Co-A), have been standardized and evaluated in this laboratory for the first time for detection of CAg in cases of hydatid disease at the field level and rural health center.

Detección de inmunoglobulinas G y E específicas para E. granulosus en hidatidosis humana / Detection of G and E specific immunoglobulins for E. granulosus in human hydatidosis

Con la finalidad de conocer la utilidad clínica de la detección de anticuerpos específicos al Echinoccus granolusos de tipo inmunoglobulinas (Ig) G y E, se estudiaron 48 pacientes portadores de hidatidosis y 110 controles. Se realizó Enzima Inmuno Ensayo (ELISA) indirecto homogéneo para Ig-G y ELISA reversa para Ig-E. Se encontró una sensibilidad de 81 por ciento para IgG y de 62,5 por ciento para IgE, con una especificidad para Ig-G de 89 por ciento y para Ig-E de 96 por ciento . Los hallazgos evidencian la utilidad clínica de la detección de ambos anticuerpos específicos para el diagnóstico de la hidatidosis humana; Ig-G como primera aproximación diagnóstica y pàra estudios seroepidemiológicos e Ig-E en primoinfección, recidiva y reinfección postcirugía

Inmunología pre y postoperatoria en hidatidosis / Pre and postoperative immunology in hydatidosis

Mostramos la sensibilidad preoperatoria para el panel DD5+IgG específica y c/u por separado, además el seguimiento postoperatorio de estos marcadores biológicos en 394 pacientes(pac) portadores de quístes hidatídicos (QH) complicados o no, únicos o múltiples, pulmonares(P) o hepáticos(H) o pulmonares hepáticos concomitantes(PH), demostrados por cirugía entre enero 1980 y junio 1991 en el Hospital Regional de Temuco, IX Región de Chile. Se les determinó DD5 según patrón del Instituto de Salud Pública de Chile y/o IgE total y/o IgG específica para QH con técnicas de ELISA directa implementadas en Laboratorio de Inmunología de nuestro hospital. RESULTADOS: sensibilidad preoperatoria por separado (Total P-H-PH): DD5 225 pac 54,6 por ciento 48,3 por ciento 69 por ciento 64,7 por ciento ; IgE total 163 pac 83,4 por ciento 79,8 por ciento 84,6 por ciento 96 por ciento ; IgG específica 60 pac 81,6 por ciento 81,5 por ciento 71,4 por ciento 100 por ciento . Panel preoperatorio 23 pac 1 marcador (+) 91,3 por ciento ; 2(+) 69,5 por ciento ; 3(+) 39,7 por ciento . Seguimiento postoperatorio: DD5 37 pac(+) al 1er año 50 por ciento y al 2§ 18,1 por ciento ; IgE total 43 pac mejor regresión lineal simple llega a normalidad en 1250 días(3 años 5 meses); IgG específica 36 pac(+) al 1er año 73,3 por ciento y al 2§ 66 por ciento . CONCLUSIONES: El DD5 siendo patognomónico tiene baja sensibilidad especialmente en el QH P(38,3 por ciento ). Las IgE total e IgG específica tienen mejor sensibilidad de modo que 1 marcador(+) en el panel sube a 91,3 por ciento . El seguimiento postop. Muestra una pronta respuesta con negativización del DD5 en 81,8 por ciento en el 1er año. Las IgE total y IgG específica son más lentas necesitando 3 años 7 meses para la normalización de la primera y 66 por ciento de los pac sigue con positividad de la segunda al 2§ año postoperatorio