FasciDIG. Diagnóstico oportuno y eficaz de fasciolosis humana / FasciDIG. Timely and effective diagnosis of human fasciolosis

El diagnóstico de fasciolosis humana, enfermedad zoonótica causada por el parásito Fasciola hepatica reúne los resultados de las técnicas: concentración por sedimentación (copa-cónica), FasciDIG en heces y FasciDIG en suero, además de los criterios clínico-epidemiológicos. FasciDIG constituye un ensayo inmunoenzimático que detecta antígenos de excreción-secreción de F. hepatica a partir de muestra de suero y heces. Permite diagnosticar la infección en cada una de las formas clínicas de la enfermedad y presenta una sensibilidad diagnóstica superior a las técnicas convencionales que detectan huevos del parásito (copa-cónica), por lo que se consideró oportuno abordar algunos conceptos relacionados con esta técnica inmunodiagnóstica y analizar su aplicabilidad para el diagnóstico oportuno y eficaz de esta parasitosis(AU)

Diagnosis of human fasciolosis, a zoonotic disease caused by the parasite Fasciola hepatica, combines the results of the following techniques: conical cup, feces FasciDIG and serum FasciDIG, as well as clinical-epidemiological criteria. FasciDIG is an enzyme immunoassay that detects F. hepatica excretion / secretion antigens in serum and feces samples. It makes it possible to diagnose infection at each of the clinical stages of the disease with a higher diagnostic sensitivity than conical cup. Therefore, it was considered appropriate to address a number of concepts regarding this immunodiagnostic technique and analyze its applicability in the timely and effective diagnosis of this helminth infection(AU)

First reported case of clinical fascioliasis in Santa Catarina, Brazil

Abstract Fascioliasis is a food-borne anthropozoonotic disease caused by Fasciola hepatica that affects multiple hosts, including humans. We herein report the first case of human fascioliasis in the state of Santa Catarina, Brazil. A 57-year-old female patient complaining of abdominal pain was admitted to the hospital for a clinical investigation. The diagnosis of F. hepatica was confirmed by ultrasound and indirect enzyme-linked immunosorbent assay. Authorities of the Northern coast of Santa Catarina were notified to investigate other cases and risk factors for contamination. The disease is also prevalent in cattle, which could pose as a potential route for infection.

Anticuerpos de cadena única de alpaca para la detección de antígenos de Fasciola hepática / Single-chain antibodies from alpaca for the detection of Fasciola hepatica antigens

RESUMEN Objetivo. Producir anticuerpos recombinantes de cadena única de alpaca que se unan con alta afinidad y especificidad al antígeno excretado-secretado (ES) de Fasciola hepatica para el desarrollo de tecnologías nuevas de diagnóstico de fascioliasis humana y animal. Materiales y métodos. Se ha construido una genoteca de cADNde los dominios variables de anticuerpos de cadena única pesada, conocidos como VHH, a partir de células mononucleares de sangre periférica de una alpaca inmunizada con el antígeno ES de F. hepatica. La genoteca fue tamizada con el antígeno ES por despliegue diferencial de fagos (phage display), seleccionando diez VHH que se unen específicamente a ES. El VHH anti ES fue clonado en un vector de expresión, la proteína recombinante (VHH-ES1) de 15,3 kDa fue producida por fermentación en E. coli y purificada a homogeneidad por cromatografía de afinidad. La unión del VHH-ES1 al antígeno ES fue evaluada por ELISA usando VHH-ES1 como anticuerpo de captura, antisuero policlonal anti-ES de conejo y conjugado anti IgG de conejo con peróxidasa de rábano. Resultados. Se ha identificado y producido un VHH-ES1 recombinante que se une al antígeno ES (VHH-ES1) que correspondía a un anticuerpo de la subclase IgG2 de bisagra larga. La unión del anticuerpo VHH-ES1 al antígeno muestra linealidad respecto a la concentración de ES en el rango de 50-5000 ng/mL y el valor límite de detección del antígeno está en el rango de 30-170 ng/mL de ES (R2=0,99). Conclusión . El VHH-ES1 se une con afinidad y especificidad al antígeno ES de F. hepatica y es un anticuerpo promisorio a evaluar para el desarrollo de nuevas tecnologías de diagnóstico de fascioliasis.

ABSTRACT Objectives. To produce recombinant single-chain antibodies from alpaca that will bind to the excreted-secreted (ES) Fasciola hepatica antigen with high affinity and specificity, so as to develop new diagnostic technologies of human and animal fascioliasis. Materials and Methods. A gene bank of DNA of the variable dominions of heavy single-chain antibodies (VHH) has been created, based on mononuclear cells of peripheral blood of an alpaca immunized with the ES antigen of F. hepatica. The gene bank was screened with the ES antigen by differential phage display, selecting ten VHH that bind specifically to ES. The anti-ES VHH was cloned in an expression vector, the recombinant protein (VHH-ES1) of 15.3 kDa was produced by fermentation in E. coli and purified to homogeneity by affinity chromatography. The binding of VHH-ES1 to the ES antigen was evaluated by ELISA using VHH-ES1 as capture antibody, policlonal anti-ES serum of rabbit and conjugated rabbit anti IgG with radish peroxidase. Results. A VHH that binds to the ES antigen (VHH-ES1) has been identified through differential phage display and produced by fermentation in E. coli; this corresponds to an antibody of the long-hinge IgG2 subclass. The binding of the VHH-ES1 antibody to the antigen shows linearity with respect to the concentration of ES in the 50-5,000 ng/mL range and the limit of detection value of the antigen is in the 30-170 ng/mL range of ES (R2=0.99). Conclusions. The VHH-ES1 binds with affinity and specificity to the ES antigen of F. hepatica and is a promissory antibody to be assessed for the development of new fascioliasis diagnostic technologies.

Negative serology of Fasciola hepatica infection in patients with liver cancer in Peru: a preliminary report

Abstract INTRODUCTION: The etiology of several hepatocellular carcinoma (HCC) cases remains largely unknown. Although Fasciola hepatica has been associated with liver fibrosis in Latin America, it has not yet been associated with HCC. This study aimed to determine the existence of specific IgG antibodies against F. hepatica in the serum samples of HCC patients. METHODS In total, 13 serum samples from 13 HCC patients were screened using Fas2-ELISA. RESULTS Fas2-ELISA demonstrated negative results in all HCC patients included in this study. CONCLUSIONS The pre-existence of F. hepatica infection in HCC patients needs to be further investigated in epidemiological and experimental studies.

Validity of a commercial kit for detection of antibodies in bovine serum in an endemic area for fasciolosis / Validade de um kit comercial para detecção de anticorpos no soro bovino em área endêmica de fasciolose

Abstract Fasciolosis is caused by Fasciola hepatica that affects the bile ducts and liver parenchyma of ruminants, which can result in economic loss. This study aimed to carry out the validity of the commercial kit ELISA® indirect front of the simple fecal sedimentation test used as the standard. 143 samples were collected blood and feces of cattle from Jerome, south of the Espírito Santo. Serum samples were left at -80 °C and used to perform the ELISA kit IDEXX®. All animals to stool examinations were also positive to the ELISA (22) and negative samples to test stool (121), 52 animals reacted positively against the antibody research. The frequency of fasciolosis was 15.4% in the stool examinations and 51.8% by ELISA. The validity was calculated by sensitivity (100%), specificity (57%), positive predictive value (29%) and negative predictive value (100%), and the correlation between the tests calculated using the kappa index of 0.35. The better sensitivity of the ELISA commercial kit should not be a separately evaluated, since the cost benefit and the technical facility must be considered.

Resumo Fasciolose é causada pela Fasciola hepatica, parasito que acomete os ductos biliares e o parênquima hepático dos ruminantes e pode resultar em perdas econômicas. Objetivou-se realizar a validade do kit comercial ELISA® indireto frente ao teste de sedimentação fecal simples utilizado como padrão. Foram coletadas 143 amostras de sangue e fezes de vacas provenientes do Sul do Espírito Santo. As amostras de sangue foram centrifugadas para separação do soro. As amostras de soro foram congeladas a -80ºC e utilizadas para análise com o kit de ELISA IDEXX®. Todos os 22 animais positivos ao exame coproparasitológico também foram positivos ao ELISA e, das 121 amostras negativas ao exame de fezes, 52 reagiram positivamente frente à pesquisa de anticorpos. A frequência de fasciolose foi de 15,4% no exame coproparasitológico e 51,8% pelo ELISA. A validade foi calculada pela sensibilidade (100%), especificidade (57%), valor preditivo positivo (29%) e valor preditivo negativo (100%), sendo considerada medíocre a concordância entre os testes, calculada pelo índice kappa (0,35). A maior sensibilidade obtida para o kit comercial ELISA não deve ser avaliada isoladamente, uma vez que o custo benefício e a facilidade da técnica devem ser considerados.

Diagnóstico de Fasciola hepatica por colangiopancreatografía retrógrada endoscópica / Diagnosis of Fasciola hepática by endoscopic retrograde cholangiopancreatography

La fasciolasis, en su fase crónica, puede causar un íctero obstructivo biliar por lo que el diagnóstico se confunde frecuentemente con litiasis de vía biliar principal. Se reportó el caso de una paciente con sospecha de colelitiasis, a la que se le realizó colangiopancreatoiografía retrógrada endoscópica. Durante el proceder se encontró un espécimen de Fasciola hepatica. A pesar que se han reportado en la literatura varios casos de esta trematodosis diagnosticados mediante técnica imagenológica, en Cuba son escasos estos reportes.

Fasciolosis, in its chronic phase, can cause biliary obstructive jaundice so the diagnosis is frequently confused with bile duct calculi. This is case report of a patient diagnosed with cholelithiasis who underwent endoscopic retrograde cholangiopancreatography (CPRE) and during the procedure, fasciola hepatica was found. Despite the cases reported in literature, in Cuba, there are few cases that have been diagnosed trematodoses by imagenological technique.

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.

Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.

Genetically confirmed fasciola hepatogigantica N.SP.

Identification of liver fluke species cannot be achieved by clinical, pathological, coprological or immunological methods. However, the differential diagnosis between F. hepatica and F. gigantica infection is very important because of their different pathological manifestations. Moreover, in countries where the two species co-exist, morphologically intermediate forms were reported. The present study aimed to identify these forms by the use of molecular characterization of DNA sequence. Based on morphometric criteria, adults of Fasciola hepatica, F. gigantica and intermediate forms were collected from naturally infected sheep and cattle from various regions of Sohag Governorate. A simple and rapid new method [QIAamp DNA Mini Kit] was used to isolate DNA from the worms and their RELP patterns were obtained after digestion of the PCR products with AvaII restriction enzymes. The result of a regular PCR experiment for the amplification of the selected 28S rDNA fragment with the designed primer set yielded identical 618- bp-long PCR products for the three types of Fasciola where the RFLP profile obtained from F. hepatica revealed four fragments of 628, 575, 165 and 95 bp, and F. gigantica generated three fragments corresponding to 628, 358 and 300 bp fragments whereas the intermediate forms revealed four fragments of 628, 541, 358 and 300 bp, which were similar to those of F. gigantica but with a distinctive fragment of 541. These results confirmed that three species are present in our locality: F. hepatica, F. gigantica and an intermediate form which was named F. hepatogigantica n.sp. on basis of having few morphometric characters from F. hepatica [length and pattern of uterine coils] but genetically they were more related to F. gigantica

Protective Role of Purified Cysteine Proteinases against Fasciola gigantica Infection in Experimental Animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG1, and IgG2 (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-gamma, and TNF-alpha, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-beta, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.

Experimental Murine Fascioliasis Derives Early Immune Suppression with Increased Levels of TGF-beta and IL-4

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19+ B cells was observed as early as week 1 post-infection while CD4+/CD8+ T cells were down-regulated. Accumulation of Mac1+ cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-alpha mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1beta expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-beta were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-beta and IL-4 during the early stages of infection.

Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática / Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens

Objetivos. Evaluar la eficacia de la técnica de electroinmunotransferencia (EITB) o Western blot utilizando antígenos de excreción-secreción de las formas adultas de Fasciola hepatica (Fh E/S Ag) para el diagnóstico de la fasciolosis humana. Materiales y métodos. Los antígenos fueron obtenidos a las 18 horas de incubación en medio Minimum Essential Eagle y preparados a la concentración proteica de 0,15 ug/uL; los cuales, al ser enfrentados con un pool de sueros de pacientes con fasciolosis confirmada por el hallazgo de huevos del parásito en las heces, se detectaron los antígenos de 10, 12, 17, 23, 27, 30, 36, 43, 66 y 136 KDa, con los cuales se desarrolló la técnica de Western blot. La sensibilidad se evaluó empleando sueros de 67 pacientes con fasciolosis, y la especificidad con sueros de 57 pacientes con otras parasitosis y diez sueros de personas no parasitadas. Resultados. De los 67 sueros, 64 reaccionaron con la banda de 23 KDa y 61 con la banda de 17KDa. Estas dos bandas no fueron detectadas por ninguno de los sueros de pacientes con otras parasitosis, ni de personas no parasitadas, siendo por ello consideradas como específicas y diagnósticas. Conclusiones. La sensibilidad de la prueba, utilizando las bandas de 17 y 23 KDa, fue de 95,5 por ciento cuando se presenta reacción positiva en una o en las dos bandas, siendo la especificidad para estos dos antígenos de 100 por ciento con un valor predictivo positivo de 100 por ciento y un valor predictivo negativo de 95,71 por ciento.

Objectives. To evaluate the performance of the enzyme-linked immunoelectrotransfer blot assay (EITB, Western blot) using excretory/secretory antigens from adult forms of Fasciola hepatica (Fh E/S Ag) for the diagnosis of human fasciolosis. Materials and methods. Antigens were obtained after 18 hours of incubation in culture medium Minimum Essential Eagle, prepared at a protein concentration of 0.15 ug/uL and run against a pool of sera of patients with proven fasciolosis (confirmed by the finding of parasite eggs in the stool microscopy). Antigens of 10, 12, 17, 23, 27, 30, 36, 43, 66 and 136 kDa were detected and used to develop the Western blot technique. The sensitivity was evaluated using sera from 67 fasciolosis patients, and the specificity using sera from 57 patients with other parasitic diseases, and 10 from healthy individuals. Results. Out of the 67 sera, 64 reacted with the 23 kDa band and 61 with the one of 17 kDa. These two bands were not detected in sera from patients with other parasitic diseases or in those from healthy volunteers and thus could be considered specific and diagnostic. Conclusions. The sensitivity of the test, using the bands of 17 and 23 kDa, was 95.5 percent for positive reactions to at least one of these two bands, being its specificity 100 percent with a positive predictive value of 100 percent and negative predictive value of 95.71 percent.

Evaluación de una prueba de ELISA con antígeno metabólico de Fasciola hepatica para el diagnóstico de fasciolosis humana en Cajamarca, Perú / Evaluation of an ELISA test with Fasciola hepatica metabolic antigen for diagnosis of human fascioliasis in Cajamarca, Peru

Se obtuvo el antígeno metabólico (antígeno excreción - secreción) de Fasciola hepatica de ovinos infectados de Cajamarca, con una concentración proteica de 1 005 μg/μL, compuesta principalmente por proteνnas de peso molecular entre 1,2 y 170 KDa. Se detectaron bandas de 170; 150; 31; 24; 18-14 y 10 kDa. Con este antνgeno se desarrollσ una prueba de ELISA y se determinσ su punto de corte en 0,140. Se evaluσ 33 sueros de pacientes con fasciolosis confirmada por visualización de huevos en heces, 177 sueros de pacientes sin fasciolosis provenientes de áreas endémicas de Cajamarca y 88 sueros de pacientes con otras infecciones parasitarias y bacterianas. Se encontró una sensibilidad de 97,0 por ciento, especificidad de 96,6 por ciento, valor predictivo positivo de 78,1 por ciento y valor predictivo negativo de 99,6 por ciento. Se encontró reacción cruzada en 9/88 sueros evaluados. Se recomienda la implementación y uso de esta prueba para el diagnóstico de fasciolosis.

Metabolic (excretion/secretion) antigen was obtained from sheep infected with Fasciola hepatica, with a 1005 μg/μL of protein concentration, composed principally by proteins of molecular weight between 1.2 and 170 KDa. Bands of 170, 150, 31, 24, 18-14 and 10 kDa were detected. With this antigen an ELISA test was developed and the cut off was determined in 0.140. We evaluated 33 serums of patient with fascioliasis confirmed by visualization of eggs in feces, 177 serums of persons without fascioliasis from endemic rural areas of Cajamarca and 88 serums of patients with others parasitic and bacterial infections. We found a 97.0 percent of sensitivity, 96.6 specificity, 78.1 percent predictive positive value, 99.6 percent predictive negative value. In 9/88 serums was found cross reactions. We recommended the implementation and use of this test for the fascioliasis diagnosis.

Evaluation of local immune response to Fasciola hepatica experimental infection in the liver and hepatic lymph nodes of goats immunized with Sm14 vaccine antigen

Protection against Fasciola hepatica in goats immunized with a synthetic recombinant antigen from Schistosoma mansoni fatty acid-binding protein 14 (rSm14) was investigated by assessing worm burdens, serum levels of hepatic enzymes, faecal egg count and hepatic damage, which was evaluated using gross and microscopic morphometric observation. The nature of the local immune response was assessed by examining the distribution of CD2+, CD4+, CD8+ and γ´+ T lymphocytes along with IgG+, IL-4+ and IFN-γ+ cells in the liver and hepatic lymph nodes (HLN). The goats used consisted of group 1 (unimmunized and uninfected), group 2 [infected control - immunized with Quillaia A (Quil A)] and group 3 (immunized with rSm14 in Quil A and infected), each containing seven animals. Immunization with rSm14 in Quil A adjuvant induced a reduction in gross hepatic lesions of 56.6 percent (p < 0.001) and reduced hepatic and HLN infiltration of CD2+, CD4+, CD8+ and γ´+ T lymphocytes as well as IL-4+ and IFN-γ+ cells (p < 0.05). This is the first report of caprine immunization against F. hepatica using a complete rSm14 molecule derived from S. mansoni. Immunization reduced hepatic damage and local inflammatory infiltration into the liver and HLN. However, considering that Quil A is not the preferential/first choice adjuvant for Sm14 immunization, further studies will be undertaken using the monophosphoryl lipid A-based family of adjuvants during clinical trials to facilitate anti-Fasciolavaccine development.

Antígeno excreción-secreción de Fasciola hepatica: ultrafiltración y aplicación en inmunodiagnóstico / Fasciola hepatica excretion-secretion antigen: ultrafiltration and application in immunodiagnosis

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.

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.

Non-resolving liver abscess with Echinococcus cross-reactivity in a non-endemic region.

A 40-year-old woman presented with high fever with chills and rigors. Imaging studies revealed multiple liver abscesses with hepatosplenomegaly and gall-stones. Ultrasound-guided aspirate revealed pus that was negative on Gram and acid-fast staining and for amebic trophozoites. ELISA for echinococcus was strongly positive, but she did not respond to albendazole therapy. At surgery, Fasciola hepatica was detected and she responded well to bithinol postoperative.

Preliminary antigenic characterisation of an adult worm vomit preparation of Fasciola hepatica by infected human sera

Fascioliasis is an emerging/re-emerging vector-borne disease with the widest known distribution. Approximately 17 million people are infected around the world, being the Andean region the most affected area. There is an important necessity to develop sensitive and specific diagnostic tools to treat patients early and to avoid complications. In this paper we evaluated the immune response of infected humans against two antigenic preparations: the total soluble extract (FhTSE) and the adult worm vomit (FhAWV) in order to identify antigenic fractions specific for Fasciola hepatica. Both preparations were processed by SDS-PAGE and Western blot with human sera with fascioliasis (F), other parasitosis and healthy individuals. In the immunoblot of FhTSE, sera F recognised 16 bands with MW between eight and 110 kDa, from which those of 8, 9, 10, 38, 45 and 57 kDa were specific. In the preparation FhAWV, sera F recognised nine bands with MW from eight to 85 kDa, from which those of 8, 12, 15 and 24 kDa were specific. Some bands of cross-reaction were evident with sera from patients with other parasitoses, more frequent with the FhTSE. Bands within the MW mentioned, particularly that of eight kDa, have been shown to be specific by others, and deserve additional characterisation for their potential use in immunodiagnosis.

Fasciolíase é uma doença emergente/re-emergente transmitida por vetores com a distribuição sabidamente mais ampla. Existem aproximadamente 17 milhões de pessoas infectadas em todo mundo, sendo a região andina a área mais afetada. Há uma necessidade importante para desenvolver ferramentas diagnósticas sensíveis e específicas para tratar cedo os pacientes e para evitar complicações. Neste trabalho avaliamos a resposta imune de seres humanos infectados comparando a duas preparações antigênicas: o extrato solúvel total (FhTSE) e o vômito (FhAWV) do verme adulto a fim de identificar as frações antigênicas específicas para Fasciola hepatica. Ambas as preparações foram processadas por SDS-PAGE e Western blot com os soros humanos de portadores de fasciolíase (F), outras parasitoses e indivíduos saudáveis. No immunoblot de FhTSE, os soros F reconheceram 16 faixas com PM entre 8 e 110 kDa, das quais as de 8, 9, 10, 38, 45 e 57 kDa foram específicas. Na preparação de FhAWV, os soros F reconheceram 9 faixas com PM entre 8 e 85 kDa, das quais as de 8, 12, 15 e 24 kDa foram específicas. Algumas faixas com reação cruzada foram evidentes com os soros dos pacientes com outras parasitoses, mais freqüentes com o FhTSE. As faixas dentro do PM mencionado, particularmente aquela de 8 kDa, mostraram ser específicas por outros autores, e merecem a caracterização adicional para seu uso potencial no diagnóstico imunológico.

Excretory-secretory product of fasciola hepatica worm protects against Schistosoma mansoni infection in mice.

The objective of this study was to evaluate the protective immunity of excretory-secretory products of Fasciola hepatica (FhES) worm against S.mansoni infection in mice. Evaluation of FhES antigen was through measuring worm burden, ova count, granuloma size and frequency as well as the histopathological picture of the liver. The study was extended to determine the level of free radical scavengers; lipid peroxide, glutathione (GSH), vitamin C, vitamin E, catalase and superoxide dismutase (SOD). Liver function enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were also taken into consideration. Four groups of eight mice each were selected for this study. Group 1 served as control group. Group 2: normal healthy mice vaccinated with FhES product. Group 3: S.mansoni infected mice for 2 months and group 4: infected mice pre-vaccinated with FhES antigen. Vaccination schedule comprised of a single subcutaneous injection of FhES antigen emulsified with Freund's complete adjuvant in a dose 0.5 mg protein/mouse, followed by intraperitoneal injections of the same antigen without adjuvant in 3 doses/week for 3 successive weeks. The total antigen inoculation was 5 mg protein/mouse. The present results revealed a drastic change in all the measured parameters after S.mansoni infection and a noticeable improved level after vaccination with FhES antigen. It can be concluded that FhES antigen succeeded to protect mice against schistosomiasis by a significant reduction in worm burden, ova count, granuloma size and number, improvement in the histopathological architecture of the liver as well as amelioration in the antioxidant levels under investigation.

Fascioliasis in relatives of patients with Fasciola hepatica infection in Peru

Altas tasas de fasciolosis humana han sido descritas en varias regiones del Perú. Estudiamos 20 familias en una área endémica del Perú para determinar la proporción de infección con F. hepatica en los familiares de los sujetos diagnosticados y para identificar factores de riesgo asociados. El estudio incluyó un total de 93 sujetos, quienes contribuyeron con muestras de heces y sangre. Las edades comprendieron desde 1 a 53 años (media = 18.6; DS = 14.2). La prevalencia general de fasciolosis por exámenes de heces fue 33.3% (n = 83) y por serología, 51.9% (n = 86). La prevalencia en el grupo de edad I (< 19 años de edad) por pruebas coprológicas y serológicas fueron 61.4% y 75.9%, respectivamente; en el grupo II (> 19 años de edad) 15.4% y 37.5%. El principal factor de riesgo asociado con fasciolosis fue comer ensaladas (OR = 3.29, IC = 1.2-9.0, p = 0.02). En conclusión, la fasciolosis humana es altamente prevalente en familiares de los casos índices y el factor de riesgo más significante para adquirirla en la familia es comer ensaladas en las áreas endémicas.