RESUMEN Introducción: La fasciolosis representa un grave problema para el sector pecuario y la salud humana en muchos países. En Colombia, afecta principalmente al ganado bovino y ovino, con escasos reportes en humanos. Objetivo: Estandarizar un Ensayo de Inmunoadsorción Enzimática para bovinos, ovinos y humanos como herramienta de tamizaje de fasciolosis. Metodología: Se utilizaron 50 muestras de bovinos, 50 de ovinos y 39 de humanos con diagnóstico de fasciolosis confirmado por serología y examen de materia fecal. Se utilizaron cuatro presentaciones del antígeno excretor-secretor de Fasciola hepatica. Se calcularon la sensibilidad, la especificidad y la concordancia. Resultados: La sensibilidad del ELISA en humanos, ovinos y bovinos fue de 100% y la especificidad fue de 97%, 85,2% y 96,2%, respectivamente. El coeficiente Kappa de Cohen fue superior a 0,8 en las tres especies. Conclusiones: La prueba de ELISA estandarizada para el diagnóstico de la fasciolosis humana, ovina y bovina demostró una excelente sensibilidad y buena especificidad. Se propone realizar la validación a mayor escala para su posterior uso como herramienta en el tamizaje de esta parasitosis.

ABSTRACT Introduction: Fascioliasis represents a serious problem for livestock production and human health in the world. In Colombia, it mainly affects bovine and ovine cattle, with few cases reported in humans. Objective: To standardize an Enzyme-Linked ImmunoSorbent test (ELISA) in bovine, ovine, and human individuals as a screening tool of fascioliasis. Methods: 50 bovine, 50 ovine and 39 human samples with fascioliasis confirmed by serology and stool test were used. Sensitivity, specificity and concordance were calculated. Results: Sensitivity of the ELISA in humans, bovine and ovine cattle was 100 % and specificity was 97%, 85.2% and 96.2%, respectively. Cohen´s Kappa coefficient was >0.8 in all groups. Conclusions: Standardized ELISA test for bovine, ovine and human fascioliasis exhibited an excellent sensitivity and good specificity. It is proposed to carry out a large-scale validation for its ulterior use as a screening tool for the diagnosis of this parasitic infection.

Orally transmitted Chagas disease has become a matter of concern due to outbreaks reported in four Latin American countries. Although several mechanisms for orally transmitted Chagas disease transmission have been proposed, food and beverages contaminated with whole infected triatomines or their faeces, which contain metacyclic trypomastigotes of Trypanosoma cruzi, seems to be the primary vehicle. In 2007, the first recognised outbreak of orally transmitted Chagas disease occurred in Venezuela and largest recorded outbreak at that time. Since then, 10 outbreaks (four in Caracas) with 249 cases (73.5% children) and 4% mortality have occurred. The absence of contact with the vector and of traditional cutaneous and Romana’s signs, together with a florid spectrum of clinical manifestations during the acute phase, confuse the diagnosis of orally transmitted Chagas disease with other infectious diseases. The simultaneous detection of IgG and IgM by ELISA and the search for parasites in all individuals at risk have been valuable diagnostic tools for detecting acute cases. Follow-up studies regarding the microepidemics primarily affecting children has resulted in 70% infection persistence six years after anti-parasitic treatment. Panstrongylus geniculatushas been the incriminating vector in most cases. As a food-borne disease, this entity requires epidemiological, clinical, diagnostic and therapeutic approaches that differ from those approaches used for traditional direct or cutaneous vector transmission.

Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.

La infección por Echinococcus sp. es hipoendémica en Venezuela. Sólo cuatro casos de hidatidosis autóctona por E. vogeli han sido reportados, tres de ellos en la región de la Guayana venezolana. En Febrero del año 2009 se realizó el diagnóstico clínico-sero-epidemiológico de hidatidosis poliquística en una paciente femenina de la etnia Yanomami, procedente de Parima B, Alto Orinoco, en la Amazonía venezolana. Se resolvió con tratamiento médico y quirúrgico por laparoscopia y se evidenció en el quiste la presencia de ganchos rostelares compatibles con E. vogeli. En Abril del 2009 en una segunda paciente Yanomami de igual procedencia, se le diagnosticó hidatidosis por E. vogeli siendo operada exitosamente por cirugía laparoscópica asistida por robot. Dos casos humanos en una misma población y la presencia de factores de riesgo como la tenencia de perros domésticos y la comunicación por informantes indígenas del hallazgo de quistes en hígados de animales de cacería (Cuniculus paca o lapa y Dasyprocta sp. o picure), hacen pensar en transmisión activa en la cuenca del Alto Orinoco y en zonas selváticas de la Guayana venezolana. El presente, es el primer registro de casos de hidatidosis poliquística en indígenas de la etnia Yanomami.

Infection by Echinococcus sp. is hypoendemic in Venezuela. Only four cases of autochthonous E. vogeli hydatidosis have been reported, including three in the Venezuelan region of Guayana. In February 2009, based on epidemiological data, signs and symptoms and serological tests, a female patient of the Yanomami ethnic group, was diagnosed with a polycystic hydatid disease in Parima B, Alto Orinoco, in the Venezuelan Amazon. Rostellar hooks compatible with E. vogeli were found in the cyst. It was resolved with medical and surgical treatment by laparoscopy. A second Yanomami patient from the same location was diagnosed with E. vogeli hydatidosis in April 2009, being successfully operated with robot-assisted laparoscopy. Two human cases in the same population and the presence of risk factors such as domestic dog ownership and findings of cysts in livers of hunted animals (such as Cuniculus and Dasyprocta sp.) reported by indigenous informants, suggest active transmission in the Upper Orinoco basin and forested areas of the Venezuelan Guayana. These are the first reported cases of polycystic hydatid disease of the Yanomami ethnic group.

Se describe el proceso de investigación clínico-epidemiológica y de laboratorio que permitió establecer el nexo entre un caso de Enfermedad de Chagas en fase aguda aislado y una comunidad escolar urbana, así como la estrategia de diagnóstico y tratamiento de una emergencia médica para evitar mayor severidad clínica y mortalidad.

The clinical, laboratory and epidemiological investigations that established the connection between the index case of an acute episode of Chagas disease and an orally acquired outbreak in an urban school in Caracas are described. The strategy used for early diagnosis and treatment of this medical emergency prevented major clinical severity and mortality.

La Fasciolosis humana tiene baja frecuencia en Venezuela; sin embargo, el Municipio Mara es una región altamente endémica para fasciolosis bovina y no existen estudios sobre esta infección parasitaria en humanos. Con el propósito de determinar la prevalencia y factores asociados a la fasciolosis humana en el municipio Mara del estado Zulia, se recolectaron un total de 51 muestras de sangre y de heces. Los sueros se procesaron mediante la técnica de ELISA y “Western blot” (WB) con antígenos excreción-secreción de Fasciola hepatica (AFhES). A los sueros que resultaron positivos se les realizó ELISA IgG anti Toxocara spp, Toxoplasma gondii y cisticerosis. Las muestras de heces se concentraron mediante las técnicas de Ritchie y sedimentación rápida. Dos sueros (3,9%) fueron positivos a la técnica de ELISA AFhES; estos no reconocieron las moléculas específicas del WB-AFhES, detectadas por sueros de pacientes con infección absolutamente demostrada. Ambos individuos resultaron negativos a IgG anti Toxocara spp, Toxoplasma gondii y cisticerosis, así como también a parásitos intestinales. La prevalencia general de las parasitosis intestinales fue de 52,9% (27/51), siendo más frecuente los protozoarios que los helmintos. No se encontraron huevos de F. hepatica. Los dos individuos positivos a ELISA AFhES tenían en común trabajar en el corte de pasto fresco. Los resultados obtenidos sugieren que la población ha estado en contacto con F. hepatica, en ausencia de una infección activa por la falta de reconocimiento de las moléculas específicas del WB-AFhES y la ausencia de huevos en las heces. La fasciolosis humana tiene una baja frecuencia en Venezuela, razón por la cual no es sospechada su presencia, existiendo poco conocimiento de esta infección por el personal de salud y la población general.

In Venezuela, human Fasciolosis shows a low frequency. However, Mara Municipality is a highly endemic region for bovine fasciolosis and there are no reports of this parasite infection in humans. To determine the prevalence and associated factors to human fasciolosis in Mara municipality - Zulia state, a total of 51 blood and stool samples were collected. Serums were tested by ELISA and Western Blot (WB) assays, with excretion-secretion antigens of Fasciola hepatica (AFhES). The serum samples that resulted positive by these assays were tested by ELISA IgG anti Toxocara sp, Toxoplasma gondii and cysticerosis. Stool samples were concentrated by the Ritchie and rapid sedimentation techniques. Two serum samples were reactive to ELISA AFhES (3.9%) and these did not recognize the specific molecules of WB-AFhES detected by serum from patients with an absolutely demonstrated infection. Both participants were not positive to IgG anti Toxocara sp, Toxoplasma gondii, cysticerosis, and stool samples of these were negative to intestinal parasites. The general prevalence of intestinal parasites was 52.9% (27/51), being protozoa more frequent than helminthes. No Fasciola eggs were found. The two positives participants had in common that both had worked as fresh pasture cutters. These results suggest that the population had been in contact with F. hepatica, with no active infection because of the lack of specific molecules recognition and the absence of eggs in stool samples. Human fasciolosis has a low frequency in Venezuela and is underestimated and underrecognized by health workers and the general population.

Se realizó un estudio clínico epidemiológico para determinar la ocurrencia de fasciolosis hepática humana y parasitosis intestinales en el caserío Mesa Arriba, municipio Carache. estado Trujillo, Venezuela, zona descrita como endémica para fasciolosis bovina, donde dos casos aislados, se han reportado en humanos. Se estudiaron 98 personas previo consentimiento informado, a quienes se les realizó una encuesta epidemiológica, examen físico, hematología, aminotransferasas, estudio coproparasitológico y serológico: ELISA con antígeno de excreción-secreción de adultos de Fasciola hepatica (AFhES) y “Western blot”(WB). Con base en los datos obtenidos, se conoció que el 88,8% de los participantes había consumido berro silvestre (Rorippa nasturtium aquaticum) y 59,2% tuvo contacto con ganado vacuno. En el estudio coproparasitológico se encontró que el 47,9% presentó algún tipo de parásito intestinal, 20,4% correspondió a helmintos, 15,3% a protozoarios y 12,2% a infecciones mixtas. No se encontraron huevos de Fasciola hepatica. El 18,4% de las personas mostró eosinofilia. Se obtuvo un suero positivo por el método de ELISA, con bandas inespecíficas al WB-AFhES. Se administró tratamiento específico para parásitos intestinales. No se demostró fasciolosis humana en este trabajo, a pesar de encontrar factores favorables a la infección, pero el mismo representó una aproximación a su estudio epidemiológico en el estado Trujillo.

A clinical-epidemiological study to determine the occurrence of human hepatic fasciolosis and intestinal parasitosis was done at the Mesa Arriba Village, Carache Municipality, Trujillo State, Venezuela, area which has been described as endemic for bovine fasciolosis and where two isolated cases have been reported in humans. The study included 98 persons who, after previously signing an informed consent form, answered an epidemiological survey and were physically examined and submitted to the following laboratory tests: complete hematology, aminotransferase, and coproparasitology and serology; ELISA using Fasciola hepatica adult excretion-secretion antigen (AFhES) and “Western Blot” (WB). Based on the results obtained, it was found that 88.8% of participants had consumed wild watercress (Rorippa nasturtium aquaticum) and 59.2% had contact with bovine cattle. The coproparasitological study showed that 47.9% had some type of intestinal parasite: 20.4% corresponded to helmints, 15.3% to protozoa, and 12.2% mixed infections. There were no Fasciola hepatica eggs; 18.4% of the individuals showed eosinophilia. The ELISA test gave one positive serum with non-specific WB-AFhES bands. Specific anti-intestinal parasite treatment was indicated. Human fasciolosis was not demonstrated in this study, in spite of finding favorable factors for this infection, but is represents and approximation to its epidemiological study at Trujillo State.

Low sensitivity in the coprologic diagnosis of human fasciolosis has motivated the development of immunodiagnosis. The excretion-secretion antigen from Fasciola hepatica adult worms (ESAFh) with ELISA is suitable for screening, although it overestimates its prevalence. However, when tested by Western blot (WB) it does not show any optimal recognition of its components. In order to obtain a better preparation, the antigen was ultrafiltered by YM 10, 30 and 50 kDa membranes. Retentates (R) were used by ELISA and WB. A higher discrimination between positives and negatives by ELISA and a better resolution in the antigen recognition in WB was achieved with the R50 fraction. Molecules of 9, 14, 65 kDa, and region about 27 kDa were detected with high sensitivity (90 to 100% of positive sera) and specificity (none of the negative sera). Among the 29 sera with other parasitic diseases, only one with Paragonimus sp. reacted to the 65 kDa molecule. ELISA-ESAFh with all filtrated fractions was useful, facilitating the infection screening even though the best results were obtained with R50. The verification of positive cases is efficiently achieved using the R50 of the ESAFh fraction by WB.

La baja sensibilidad de la coprología en el diagnóstico de la fasciolosis humana ha motivado el desarrollo del inmunodiagnóstico. El antígeno de excreción-secreción de adultos de Fasciola hepatica (AFhES) en ELISA es adecuado para el screening, aunque sobrestima la prevalencia; cuando se utiliza en Western blot (WB) no muestra un buen reconocimiento de sus componentes. Para lograr una mejor preparación, se ultrafiltró el antígeno a través de membranas YM de 10, 30 y 50 kDa. Los retenidos (R) se usaron en ELISA y WB. La mayor discriminación entre positivos y negativos en ELISA y la mejor resolución en el reconocimiento al antígeno en WB, se logró con la fracción R50. Se destacan las moléculas de 9, 14, 65 kDa y la región alrededor de 27 kDa, detectadas con alta sensibilidad (90 al 100% de los sueros positivos) y especificidad (por ningún negativo). Al ensayar 29 sueros con otras parasitosis, sólo el de una persona con Paragonimus sp. reaccionó a la molécula de 65 kDa. ELISA-AFhES con todas las fracciones filtradas fue útil, facilitando el screening de la infección, aunque con R50 se obtuvieron los mejores resultados. La comprobación de los casos positivos se logra eficientemente utilizando la fracción R50 del AFhES en WB.

De los 9 casos de fascioliasis humana registrados en Venezuela, 4 han sido publicados en los últimos 10 años, siendo 2 de éstos procedentes de la región andina. El riesgo de endemicidad frecuente en bovinos y la condición clínica de una niña de 14 años, con malestar abdominal, vómitos, eosinofilia y antecedentes de ingestión abundante de berros, motivó realizar un estudio en su lugar de procedencia (Timotes, Edo. Mérida), para conocer la exposición de la comunidad al riesgo de infección. Se evaluaron 65 personas, a quienes se les realizó historia clínica, con consentimiento previa información, toma de muestras de sangre, exámenes de heces (n=37) y ecografía abdominal (n=33). Se practicó ELISA con extracto soluble de adultos de Fasciola hepatica (ELISA-AFhES), resultando 9 personas positivas. Se practicó "Western blot" (WB) a todos los sueros positivos en ELISA, de los cuales 5 reconocieron moléculas específicas de 9, 14, 27 y 65 kDa, lo que constituye un patrón de positividad al WB. Los cuatro sueros restantes, con débil reactividad al ELISA, no reconocieron estas moléculas. Por estudios coprológicos se encontraron Entamoeba coli, Entamoeba histolytica/dispar, Blastocystis hominis, Giardia lamblia, Endolimax nana y Ascaris lumbricoides. Tres de las 5 personas positivas por ELISA-AFhES y WB-AFhES resultaron con huevos de Fasciola hepatica en las heces. Los 5 casos positivos corresponden a niñas de una familia, todas hermanas, cuyos padres resultaron sin infección, a pesar de que todos ingieren berros y ensaladas con productos de la región. El presente estudio constituye el primer reporte de transmisión a humanos en una población de un área bien delimitada en la región andina venezolana.

Of the 9 cases of human fascioliasis recorded in Venezuela, 4 have been published in the last 10 years, 2 of them originating from Andean region. The well known transmission of fascioliasis in cattle and the clinical condition of a 14-year-old girl with abdominal discomfort, vomiting, eosinophilia and previous abundant ingestion of watercress, motivated the field study in the site of occurrence (Timotes, Edo. Mérida in the Andean region) to learn the risk of community infection. Sixty-five people were evaluated clinically and serologically. Coprology was done in 37 and abdominal ultrasound in 33 persons. ELISA with adult Fasciola hepatica soluble extract (AFhES- ELISA) was performed with 9 persons being positive. Western blot (WB) was carried out on all ELISA positive sera, resulting in 5 that recognized 9, 14, 27 and 65 kDa specific molecules. The remaining four sera, with weak reactivity by ELISA, did not have this recognition pattern. By stool examination, we found Entamoeba coli, E. histolytica/dispar, Blastocystis hominis, Giardia lamblia, Endolimax nana and Ascaris lumbricoides. Three of the 5 positive people by AFhES-ELISA and AFhES-WB had F. hepatica eggs in their feces. The 5 positive cases are girls of a family, sisters and whose parents do not have the infection, although all consume watercress in salads with other vegetables from that region. The present study constitutes the first report of a well established focus of fascioliasis with human repercussion in Venezuela.

La fasciolosis humana es una entidad poco frecuente reportada previamente 8 veces en Venezuela, de personas provenientes del centro y los estados occidentales. Se presenta el caso de un paciente masculino de 25 años, natural y procedente del Estado Bolívar, donde practica equitación y supervisa tareas de corral del ganado equino y bovino. La enfermedad actual se inicia con diarreas desde los 14 años, acompañada de persistente eosinofilia. ELISA e "Immunoblot", resultaron francamente positivos al antígeno de excreción- secreción de Fasciola hepática. Ante persistente dolor epigástrico, se practicó eco-endoscopia, resultando en la visualización de dilatación quística del ducto pancreático con una estructura interna probablemente correspondiente a un parásito. El examen parasitológico del contenido duodenal y heces reportó huevos de Fasciola hepática en este último material. Se administró tratamiento con Bithionol luego de lo cual disminuyó la eosinofilia y hubo franca mejoría clínica. Es el primer caso de fasciolosis humana proveniente del sureste del país, donde la fasciolosis bovina o humana no había sido descrita y en el cual además, ocurrió una localización ectópica pancreática de difícil diagnóstico clínico.

Human fasciolosis in Venezuela is an infrequent disease, reported only 8 times previously in persons coming from the center and the western states of the country. The case of a 25 years old masculine patient from Bolivar State, where he practices equitation and supervises tasks of corral of equine and bovine cattle is reported. The disease began with diarrhea at 14 years old, accompanied by persistent eosinofilia. ELISA and "Immunoblot" were strongly positive to excretion-secretion Fasciola hepatica antigen. Given the presence of persistent epigastric pain, echo-endoscopy was practiced, resulting in the visualization of cyst expansion of the pancreatic duct with an internal structure probably corresponding to a parasite. The parasitologic examination of the duodenal content and feces reported Fasciola hepatica eggs this last material. After treatment with Bithionol, eosinofilia diminished and there was frank clinical improvement. It is the first case of human fasciolosis of the southeastern part of the country, where bovine or human fasciolosis had not been described in which additionally, an ectopic pancreatic location of difficult clinical diagnosis occurred.