Unanticipated discovery: incidental encounter with fasciola hepatica during ERCP - a case report.

Introduction and Importance: Fasciola hepatica (FH) is a rare parasitic infection in humans. Its incidental detection during endoscopic retrograde cholangiopancreatography (ERCP) is exceptionally uncommon. This case underscores the importance of considering parasitic infections, even in low-endemicity regions, and the potential implications of dietary and environmental factors in disease transmission. Case Presentation: The authors present a case of a 31-year-old female from Dhading, Nepal, who underwent ERCP for suspected biliary stone. The patient had been experiencing recurring, nonradiating, burning epigastric pain for 5 to 7 years, which had recently intensified. Previous evaluations, including abdominal ultrasonography, CT, and MRI, revealed a dilation within the common bile duct and an obstruction in the biliary system. Clinical Discussion: During ERCP, cholangiography revealed mildly dilated extra and intrahepatic bile ducts with irregular filling defects in the common hepatic duct. Sphincterotomy was performed, followed by the extraction of multiple FH worms. A 7 Fr 7 cm double pigtail plastic stent was placed with a good flow of bile. However, the patient experienced anaphylaxis during the procedure, necessitating swift and tailored administration of appropriate medications to ensure effective management and stabilization. The patient was closely monitored in the ICU postprocedure. Conclusion: After careful monitoring and treatment, the patient fully recovered. The unexpected discovery of FH during ERCP is extremely rare. Early recognition and appropriate management of such incidental findings are crucial to ensuring optimal patient outcomes.

The Hepatic Phase of Fascioliasis Complicated with Liver Bleeding: Presentation of Three Cases.

Bleeding is a very rare complication of Fasciola species (F. hepatica or F. gigantica) infection. We present here three cases of subcapsular liver bleeding caused by the hepatic phase of Fasciola spp. infection in patients, two of whom were women, aged 22, 66, and 84 years in Diyarbakir Province, southeastern Turkey. They had symptoms of right upper quadrant pain (n = 3), nausea (n = 1), and vomiting (n = 2) for periods ranging from 6 hours to 15 days. All patients with clinical presentations ranging from moderate abdominal pain to hypovolemic shock and ischemic hepatitis were improved with supportive treatment without the need for surgery. They showed complete clinical and laboratory recovery after triclabendazole administration in their follow-up. In conclusion, Fasciola spp. infection should be considered in the etiology of bleeding from liver disease.

The effects of Fasciola hepatica recombinant proteins (peroxiredoxin and cathepsin L1) on Crohn's disease experimental model.

The immunomodulatory potential of the excretory-secretory (E/S) proteins of the helminths has been shown in previous investigations. This study evaluated the effects of the recombinants and excretory-secretory proteins of the Fasciola hepatica on induced colitis in Balb/c mice. The F. hepatica Recombinant proteins, Cathepsin L1 and Peroxiredoxin, and E/S proteins were intraperitoneally injected into the three mice groups as the case groups, while the control groups received PBS. Colitis was induced in mice by intraluminal administration of the 2, 4, 6-Trinitrobenzenesulfonic acid solution (TNBS). After 8 h, the case groups received the second dosage of the treatments, and it was repeated 24 h later. The immunological, pathological, and macroscopic changes were evaluated 3 days after colitis induction. The macroscopic evaluation revealed significantly lower inflammatory scores in the mice treated with recombinant Peroxiredoxin (rPRX) and recombinant Cathepsin L1 (rCL1). Despite the macroscopic observation, the pathological finding was insignificant between the groups. IFN-γ secretion was significantly lower in splenocytes of the groups that received rPRX, rCL1, and E/S than the controls. IL-10 showed significantly higher levels in groups treated with rPRX and rCL1 than controls, whereas the level of IL-4 was not statistically significant. Excretory-secretory proteins of the F. hepatica showed immunomodulatory potency and the main effects observed in this study were through the reduction of inflammatory cytokine and inflammation manifestation as well as induction of anti-inflammatory cytokines.

Fasciola hepatica GST mu-class suppresses the cytokine storm induced by E. coli-lipopolysaccharide, whereas it modulates the dynamic of peritoneal macrophages in a mouse model and suppresses the classical activation of macrophages.

IMPORTANCE: Sepsis is the consequence of a systemic bacterial infection that exacerbates the immune cell's activation via bacterial products, resulting in the augmented release of inflammatory mediators. A critical factor in the pathogenesis of sepsis is the primary component of the outer membrane of Gram-negative bacteria known as lipopolysaccharide (LPS), which is sensed by TLR4. For this reason, scientists have aimed to develop antagonists able to block TLR4 and, thereby the cytokine storm. We report here that a mixture of mu-class isoforms from the F. hepatica GST protein family administered intraperitoneally 1 h prior to a lethal LPS injection can modulate the dynamics and abundance of large peritoneal macrophages in the peritoneal cavity of septic mice while significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock. These results suggest that native F. hepatica glutathione S-transferase is a promising candidate for drug development against endotoxemia and other inflammatory diseases.

The use of cathepsin L1 (FhCL1) serological ELISA in sentinel screening for liver fluke on sheep farms.

Fasciola hepatica is a parasitic helminth (worm) that poses a significant economic threat to the ruminant livestock industry worldwide. The disease, fasciolosis, can result in a range of clinical signs including anaemia, weight loss and death, with the most severe symptoms attributed to early acute infection when the parasite is migrating through the liver. Early diagnosis and intervention are essential for the control and management of the disease to prevent productivity losses. The traditional gold standard method of diagnosis uses faecal egg counts (FEC) that is limited to detecting patent infections from 10 to 12 weeks post infection (WPI). In contrast, serological assays can detect pre-patent infections as we have shown that enzyme-linked immunosorbent assays (ELISA) using the F. hepatica cysteine peptidase cathepsin L1 (FhCL1) can detect liver fluke infections from 3 to 4 WPI. Here, we used FEC and ELISA to monitor liver fluke infections in sentinel lambs from three commercial farms in Ireland from September 2021 to March 2022. All three farms showed a significant increase in FhCL1 antibody levels and FEC over this time, with a substantial rise in positive infection detection between late November and January. However, ELISA screening detected infection at least two months prior to FEC (September). This suggests that the regular screening of sentinel lambs for F. hepatica seroconversion in a "test and treat" approach could mitigate the negative damaging impact of early fasciolosis on flock health, welfare and productivity and inform management strategies. In addition, we show that whole blood samples taken on Whatman® protein saver cards could replace conventional serum blood tubes for blood collection. Cards can be stored at room temperature for long periods of time and samples revisited at any time for re-analysis. The adoption of these cards on farm together with the FhCL1 ELISA would provide a simpler, cost-effective, and eco-friendly method for testing sentinel lambs for liver fluke disease.

How do parasitic worms prevent diabetes? An exploration of their influence on macrophage and ß-cell crosstalk.

Diabetes is the fastest growing chronic disease globally, with prevalence increasing at a faster rate than heart disease and cancer. While the disease presents clinically as chronic hyperglycaemia, two distinct subtypes have been recognised. Type 1 diabetes (T1D) is characterised as an autoimmune disease in which the insulin-producing pancreatic ß-cells are destroyed, and type 2 diabetes (T2D) arises due to metabolic insufficiency, in which inadequate amounts of insulin are produced, and/or the actions of insulin are diminished. It is now apparent that pro-inflammatory responses cause a loss of functional ß-cell mass, and this is the common underlying mechanism of both T1D and T2D. Macrophages are the central immune cells in the pathogenesis of both diseases and play a major role in the initiation and perpetuation of the proinflammatory responses that compromise ß-cell function. Furthermore, it is the crosstalk between macrophages and ß-cells that orchestrates the inflammatory response and ensuing ß-cell dysfunction/destruction. Conversely, this crosstalk can induce immune tolerance and preservation of ß-cell mass and function. Thus, specifically targeting the intercellular communication between macrophages and ß-cells offers a unique strategy to prevent/halt the islet inflammatory events underpinning T1D and T2D. Due to their potent ability to regulate mammalian immune responses, parasitic worms (helminths), and their excretory/secretory products, have been examined for their potential as therapeutic agents for both T1D and T2D. This research has yielded positive results in disease prevention, both clinically and in animal models. However, the focus of research has been on the modulation of immune cells and their effectors. This approach has ignored the direct effects of helminths and their products on ß-cells, and the modulation of signal exchange between macrophages and ß-cells. This review explores how the alterations to macrophages induced by helminths, and their products, influence the crosstalk with ß-cells to promote their function and survival. In addition, the evidence that parasite-derived products interact directly with endocrine cells to influence their communication with macrophages to prevent ß-cell death and enhance function is discussed. This new paradigm of two-way metabolic conversations between endocrine cells and macrophages opens new avenues for the treatment of immune-mediated metabolic disease.

The regulatory roles of Fasciola hepatica GSTO1 protein in inflammatory cytokine expression and apoptosis in murine macrophages.

Fascioliasis, a global zoonotic parasitic disease, is mainly caused by Fasciola hepatica (F. hepatica) parasitizing in the livers of hosts, mainly humans and herbivores. Glutathione S-transferase (GST) is one of the important excretory- secretory products (ESPs) from F. hepatica, however, the regulatory roles of its Omega subtype in the immunomodulatory effects remain unknown. Here, we expressed F. hepatica recombinant GSTO1 protein (rGSTO1) in Pichia pastoris and analyzed its antioxidant properties. Then, the interaction between F. hepatica rGSTO1 and RAW264.7 macrophages and its effects on inflammatory responses and cell apoptosis were further explored. The results revealed that GSTO1 of F. hepatica owned the potent ability to resist oxidative stress. F. hepatica rGSTO1 could interact with RAW264.7 macrophages and inhibit its cell viability, furthermore, it may suppress the production of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α, but promote the expression of anti-inflammatory cytokine IL-10. In addition, F. hepatica rGSTO1 may down-regulate the ratio of Bcl-2/Bax, and increase the expression of pro-apoptotic protein caspase-3, thereby eliciting the apoptosis of macrophages. Notably, F. hepatica rGSTO1 inhibited the activation of nuclear factor-κB (NF-κB) and mitogen­activated protein kinases (MAPKs p38, ERK and JNK) pathways in LPS-activated RAW264.7 cells, exerting potent modulatory effects on macrophages. These findings suggested that F. hepatica GSTO1 can modulate the host immune response, which provided new insights into the immune evasion mechanism of F. hepatica infection in host.

Study of the cross-talk between Fasciola hepatica juveniles and the intestinal epithelial cells of the host by transcriptomics in an in vitro model.

Fasciolosis is a globally widespread trematodiasis with a major economic and veterinary impact. Therefore, this disease is responsible for millions of dollars in losses to the livestock industry, and also constitutes an emerging human health problem in endemic areas. The ubiquitous nature of Fasciola hepatica, the main causative agent, is one of the key factors for the success of fasciolosis. Accordingly, this parasite is able to subsist in a wide variety of ecosystems and hosts, thanks to the development of a plethora of strategies for adaption and immune evasion. Fasciolosis comprises a growing concern due to its high prevalence rates, together with the emergence of strains of the parasite resistant to the treatment of choice (triclabendazole). These facts highlight the importance of developing novel control measures which allow for an effective protection against the disease before F. hepatica settles in a niche inaccessible to the immune system. However, knowledge about the initial phases of the infection, including the migration mechanisms of the parasite and the early innate host response, is still scarce. Recently, our group developed an in vitro host-parasite interaction model that allowed the early events to be unveiled after the first contact between the both actors. This occurs shortly upon ingestion of F. hepatica metacercariae and the emergence of the newly excysted juveniles (FhNEJ) in the host duodenum. Here, we present a transcriptomic analysis of such model using an approach based on RNA sequencing (RNA-Seq), which reveals changes in gene expression related to proteolysis and uptake of metabolites in FhNEJ. Additionally, contact with the parasite triggered changes in host intestinal cells related to pseudogenes expression and host defence mechanisms, including immune response, among others. In sum, these results provide a better understanding of the early stages of fasciolosis at molecular level, and a pool of targets that could be used in future therapeutic strategies against the disease.

The First Case of Human Hepatic Fasciolosis Presented as Hepatic Pseudotumor Histopathologically Diagnosed in Romania-A Case Report.

Human hepatic fasciolosis has been reported in 81 countries, some of which are endemic areas. In Europe, case reports from humans were published in Portugal, Spain, France, and Italy. Regarding Romania, we do not have any data on the prevalence of this parasitosis, with the exception of two cases of twins that were born in Romania and diagnosed in the last 37 years in Italy after joining their mother that lived there. We report the case of a patient diagnosed in Romania with chronic fasciolosis, presented as a hepatic pseudotumor that was diagnosed during the histopathological examination of the hepatic lesion. The patient received oral treatment with triclabendazole, two doses of 10 milligrams (mg) per kilogram (kg) of body weight, given 12 h apart, with no side effects during or after the treatment. The evolution of the patient was favorable. In conclusion, even in areas free of human fasciolosis, the presence of an anemic syndrome especially in children, abdominal pain in the upper quadrants, associated or not with other digestive manifestations, even more so associated with eosinophilia in the acute phase, should be carefully evaluated for ruling out a parasitosis such as fasciolosis even in countries where this diagnosis seems unlikely.

Fascioliasis associated with chronic cholecystitis in a woman from Sistan and Baluchestan province, a non-endemic region in Southeastern Iran.

BACKGROUND: Fascioliasis, caused by Fasciola hepatica, is a neglected zoonotic food-borne trematodiasis. The Caspian littoral in northern Iran is endemic for the disease, and human fascioliasis is well-known in that region. In the present study, we report the diagnosis, identification, and clinical management of a human case of fascioliasis associated with common bile duct (CBD) obstruction from a non-endemic remote area in southeastern Iran. CASE PRESENTATION: A 42-year-old female was admitted to Afzalipour Medical Center hepatobiliary surgery ward in Kerman with abdominal pain for the past three months. Dilated biliary tract and an ill-defined mass in CBD were reported in abdominal ultrasonography and magnetic resonance cholangiopancreatography, respectively. During distal CBD operation, nine leaf-like motile flatworms were isolated. A morphological study confirmed all the isolates as Fasciola, and further molecular investigations, identified the flukes as F. hepatica using both pepck multiplex PCR and cox1 sequencing. CONCLUSION: Molecular and morphological findings of the study indicated the presence of human fascioliasis in the southeastern province of Sistan and Baluchestan in Iran. Fascioliasis is among the etiologies of chronic cholecystitis, and physicians should consider chronic cholecystitis associated with fascioliasis in the differential diagnosis. In the present report, endoscopic ultrasound was usefully applied for the accurate diagnosis of biliary fasciolosis.

Extracellular vesicles from the trematodes Fasciola hepatica and Dicrocoelium dendriticum trigger different responses in human THP-1 macrophages.

Extracellular vesicles (EVs) released by the helminths Dicrocoelium dendriticum and Fasciola hepatica are important modulators of the host immune response, contributing to the establishment of the infection. Monocytes and, in particular, macrophages are major regulators of the inflammatory response and are likely responsible for the phagocytosis of most of the parasite EVs. In this study, we isolated EVs from F. hepatica (FhEVs) and D. dendriticum (DdEVs) by size exclusion chromatography (SEC) and characterized them by nanoparticle tracking analysis, transmission electron microscopy and LC-MS/MS, and analyzed the cohort of proteins. The treatment of monocytes/macrophages with FhEVs, DdEVs or EV-depleted fractions from SEC, demonstrated species-specific effects of the EVs. In particular, FhEVs reduce the migratory capacity of monocytes and the analysis of the cytokine profile showed that they induce a mixed M1/M2 response, exerting anti-inflammatory properties in Lipopolysaccharide-activated macrophages. In contrast, DdEVs do not affect monocyte migration and seem to have pro-inflammatory properties. These results correlate with the differences in the life cycle of both parasites, suggesting different host immune responses. Only F. hepatica migrates to the bile duct through the liver parenchyma, driving the host immune response to heal deep erosions. Furthermore, the proteomic analysis of the macrophages upon FhEV treatment identified several proteins that might be involved in FhEV-macrophage interactions.

Fasciola hepatica en preescolares de un centro poblado en una región altoandina del Perú

Introducción: La infección por Fasciola hepatica es una enfermedad zoonótica de distribución mundial, desatendida y subdiagnosticada. Objetivo: Determinar la frecuencia de Fasciola hepatica en una población preescolar en Tartar Chico, distrito de Baños del Inca, en la región Cajamarca. Métodos: Estudio transversal en 48 niños de una institución educativa inicial. Los padres entregaron 3 muestras de heces para el estudio parasitológico seriado y completaron una encuesta epidemiológica. La identificación de F. hepatica y otros parásitos se realizó con las pruebas de sedimentación rápida de Lumbreras, examen directo y Kato-Katz. Para describir usamos frecuencias y porcentajes, para el análisis bivariado aplicamos Chi-cuadrado o prueba exacta de Fisher. Resultados: La frecuencia de Fasciola hepatica fue 4,17%. Además, estimamos una proporción de 8,33% para Ascaris lumbricoides, 4,17% de Diphyllobothrium pacificum y 2,08% de uncinarias; así como parásitos contaminantes Entamoeba coli, Blastocystis hominis. Conclusión: Encontramos una frecuencia de 4,17% de fascioliasis entre preescolares de una comunidad altoandina del Perú.

Introduction: Fasciola hepatica infection is a globally distributed, neglected and underdiagnosed zoonotic disease. Objectives: To determine the frequency of Fasciola hepatica infection among a preschool population in Tartar Chico, Baños del Inca, Cajamarca. Methods: Cross-sectional study in 48 children of an initial educational institution. Parents delivered 3 stool samples for the serial parasitological study and completed an epidemiological survey. The identification of F. hepatica and other parasites was carried out with the Lumbreras rapid sedimentation tests, direct examination, and Kato-Katz. For descriptive analysis, frequency and percentages were used, for the bivariate analysis, Chi-square or Fisher's exact test was used. Results: The frequency of F. hepatica was 4,17%. In addition, a proportion of 8,33% of Ascaris lumbricoides, 4,17% of Diphyllobothrium pacificum and 2,08% of hookworms; as well as contaminating parasites Entamoeba coli, Blastocystis hominis. Conclusions: A frequency of 4,17% of fascioliasis was found among preschoolers from a high Andean community in Peru.

The helminth derived peptide FhHDM-1 redirects macrophage metabolism towards glutaminolysis to regulate the pro-inflammatory response.

We have previously identified an immune modulating peptide, termed FhHDM-1, within the secretions of the liver fluke, Fasciola hepatica, which is sufficiently potent to prevent the progression of type 1 diabetes and multiple sclerosis in murine models of disease. Here, we have determined that the FhHDM-1 peptide regulates inflammation by reprogramming macrophage metabolism. Specifically, FhHDM-1 switched macrophage metabolism to a dependence on oxidative phosphorylation fuelled by fatty acids and supported by the induction of glutaminolysis. The catabolism of glutamine also resulted in an accumulation of alpha ketoglutarate (α-KG). These changes in metabolic activity were associated with a concomitant reduction in glycolytic flux, and the subsequent decrease in TNF and IL-6 production at the protein level. Interestingly, FhHDM-1 treated macrophages did not express the characteristic genes of an M2 phenotype, thereby indicating the specific regulation of inflammation, as opposed to the induction of an anti-inflammatory phenotype per se. Use of an inactive derivative of FhHDM-1, which did not modulate macrophage responses, revealed that the regulation of immune responses was dependent on the ability of FhHDM-1 to modulate lysosomal pH. These results identify a novel functional association between the lysosome and mitochondrial metabolism in macrophages, and further highlight the significant therapeutic potential of FhHDM-1 to prevent inflammation.

Challenges in Diagnosis and Treatment of Fasciola hepatica Infection.

A 57-year-old female patient presented with fever, nausea, vomiting, loss of appetite, and weight loss within the last two months. Ceftriaxone and metronidazole therapy was started upon discovery of a liver abscess but provided no benefit. Following the of abscess biopsy, the patient developed fever, itching, anemia, acute renal failure, hyperbilirubinemia, and eosinophilia that required intensive care unit (ICU) admission. The Fasciola hepatica antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Triclabendazole was started, after which the symptoms and magnetic resonance imaging (MRI) findings regressed. Even without eosinophilia, F. hepatica should be considered in cases with a liver abscess that does not respond to antibiotics.

Report of human fascioliasis under different climatic conditions in Brazil / Registro de fasciolose humana em diferentes condições climáticas no Brasil / Registro de fascioliasis humana bajo diferentes condiciones climáticas en Brasil

Introduction: Human fascioliasis is a plant-borne and water-borne infection caused by the trematodes Fasciola hepatica and Fasciola gigantica. It is one of the main neglected tropical diseases, and infections in humans occur via the ingestion of contaminated water and food. This study reviews all the recorded cases of human fascioliasis in Brazil under different climatic conditions in the national territory. Methodology:A survey of human fascioliasis cases in Brazil was carried out using the Google Scholar, Lilacs and PubMed databases. The climatic variables such as temperature, precipitation, moisture and altitude were obtained from the database of the Instituto Nacional de Meteorologia (INMET). Results: Between the years 1958 and 2022, sixty-six cases of human fascioliasis were recorded in places with temperature levels between 22 °C to 33 °C, humidity 78% to 86%, precipitation 90 mm to 167 mm, and at an altitude of 16 to 935 meters above sea level. Conclusion: The parasite's ability to adapt to different climatic conditions is observed in Brazil and the number of cases of human fascioliasis in the national territory may be higher due to underreporting related to the difficulty in diagnosing the infection.

Introdução: A fasciolíase humana é uma infecção de origem vegetal e hídrica, causada pelos trematódeos Fasciola hepatica e Fasciola gigantica. É uma das principais doenças tropicais negligenciadas, e as infecções em humanos ocorrem através da ingestão de água e alimentos contaminados. Este estudo revisa todos os casos registrados de fasciolíase humana no Brasil sob diferentes condições climáticas no território nacional. Metodologia: Um levantamento dos casos de fasciolíase humana no Brasil foi realizado nas bases de dados Google Scholar, Lilacs e PubMed. As variáveis climáticas como temperatura, precipitação, umidade e altitude foram obtidas do banco de dados do Instituto Nacional de Meteorologia (INMET). Resultados: Entre os anos de 1958 e 2022, sessenta e seis casos de fasciolíase humana foram registrados em locais com níveis de temperatura entre 22 °C a 33 °C, umidade de 78% a 86%, precipitação de 90 mm a 167 mm e altitude de 16 a 935 metros acima do nível do mar. Conclusão: A capacidade de adaptação do parasito a diferentes condições climáticas é observada no Brasil e o número de casos de fasciolíase humana no território nacional pode ser maior devido à subnotificação relacionada à dificuldade de diagnóstico da infecção.

Introducción: La fascioliasis humana es una infección de origen vegetal y acuático, causada por los trematodos Fasciola hepatica y Fasciola gigantica. Es una de las principales enfermedades tropicales desatendidas, y las infecciones en humanos ocurren a través de la ingestión de agua y alimentos contaminados. Este estudio revisa todos los casos registrados de fascioliasis humana en Brasil bajo diferentes condiciones climáticas en el territorio nacional. Metodología: Se realizó una encuesta de casos de fascioliasis humana en Brasil utilizando las bases de datos Google Scholar, Lilacs y PubMed. Las variables climáticas como temperatura, precipitación, humedad y altitud se obtuvieron de la base de datos del Instituto Nacional de Meteorología (INMET). Resultados: Entre los años 1958 y 2022 se registraron sesenta y seis casos de fascioliasis humana en lugares con temperatura entre 22 °C a 33 °C, humedad entre 78% y 86%, precipitación entre 90 mm y 167 mm y una altitud de 16 a 935 metros sobre el nivel del mar. Conclusión: La capacidad de adaptación del parásito a diferentes condiciones climáticas se observa en Brasil y el número de casos de fascioliasis humana en el territorio nacional puede ser mayor debido al subregistro relacionado con la dificultad en el diagnóstico de la infección.

Characterization of glutamate carboxypeptidase 2 orthologs in trematodes.

BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.

Hepatic mass caused by Fasciola hepatica: A case report.

INTRODUCTION: Fascioliasis is a zoonotic infestation which presents with a wide spectrum of clinical pictures. However, it may often be overlooked, especially in the acute phase, because of uncertain symptoms. Fasciola hepatica can have an initial presentation similar to malignant liver mass or complex hepatic cyst. Here, we report a case of a hepatic mass caused by fasciola hepatica. CASE PRESENTATION: A 48-year-old woman came with chief complaints of epigastric and right hypochondrial discomfort associated with nausea and vomiting. Ultrasonography (USG) showed a heterochronic lesion in the segment VIII of the liver with few cystic lesions. CECT abdomen and pelvic gave impression of ill-defined irregular hypodense lesions in the right lobe of the liver with progressive enhancing peripheral and central cystic areas suggestive parasitic liver infestation likely echinococcus alveolaris. Right hepatectomy was done and the patient was discharged without any complications. DISCUSSION: Fascioliasis is uncommon in developed countries but more commonly seen in developing countries. The identification of fasciola hepatica eggs in the stool is a standard method for the diagnosis of fascioliasis. Fascioliasis may cause a wide variety of clinical signs ranging from asymptomatic infection to severe liver cirrhosis. Surgery for complex hydatid cysts of the liver is potentially burdened by serious complications. Technique of choice for surgical management remains inconclusive. CONCLUSION: Fasciola hepatica infection can mimic a malignant liver mass or a complex hepatic cyst because of its uncertain presentation. The disease can be prevented with public education and environmental precautions.

Case Report: "Area of Focus" Atypical Trichinellosis and Fascioliasis Coinfection.

Parasitic co-infection is commonly observed in natural populations, yet rare in the laboratory. Multiparasitism can have negative effects on the host, ranging from the atypical manifestations to increased mortality, consequently, it may be misdiagnosed and treated with unsuitable anthelmintic medicines. Therefore, reliable diagnosis is critical for appropriate treatment of parasitic co-infection. Herein, we report a case of a 31-year-old woman with persistent eosinophilia and hypoechoic liver lesion on ultrasound. The microscopic examination of multiple stool specimens did not find any pathogens. The patient had serum specific anti-Trichinella IgG antibody by Dot enzyme-linked immunosorbent assay (Dot-ELISA). After treatment with albendazole, contrast-enhanced magnetic resonance imaging (MRI) revealed more lesions in the liver. Subsequently, liver biopsy was performed in this patient and Fasciola hepatica was identified using metagenomic next-generation sequencing (mNGS) as well as polymerase chain reaction. After treatment with triclabendazole, which is the only anthelmintic drug specifically available against this fluke, her eosinophil count returned normal, and the liver lesions were significantly regressed. This case highlights the diagnostic challenge posed by parasitic co-infection, which merits more in-depth evaluation to confirm the diagnosis.

Proteomic Analysis of Extracellular Vesicles From Fasciola hepatica Hatching Eggs and Juveniles in Culture.

The identification of extracellular vesicles (EVs) in Fasciola hepatica has provided a new way to understand parasite-host communication. Most of the studies on EVs have focused on the adult stage of F. hepatica, but recently, the presence of EVs from different developmental stages has been reported. To better understand the potential role of EVs in the biology of the parasite and in the infection process, the protein cargo of EVs from embryonated eggs and newly-excysted juvenile (NEJs) flukes cultured up to 28 days, has been analyzed. EVs were isolated by size exclusion chromatography and evaluated by nanoparticle tracking analysis and transmission electron microscopy. LC-MS/MS proteomic analysis of EVs revealed the presence of 23 different proteins from embryonated egg-derived EVs and 29 different proteins from NEJ-derived EVs. Most of the identified proteins had been previously described in EVs from F. hepatica adults, including cytoskeletal proteins, glycolytic enzymes, stress-related proteins and tetraspanins. Nevertheless, EVs from hatching eggs and NEJs exhibited qualitative differences in composition, when compared to EVs form adults, including the absence of cathepsin cysteine peptidases. The differential content of the EVs released by the different developmental stages of the parasite reflect the intense activity of NEJs at this early stage, with several proteins involved in membrane traffic and cell physiology. This new set of identified proteins could help to understand key metabolic, biochemical and molecular mechanisms mediated by EVs that take place upon egg hatching and after parasite excystment.

Molecular Characteristics and Potent Immunomodulatory Activity of Fasciola hepatica Cystatin.

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-α, and promoted the expression of transforming growth factor-ß and interleukin-10. These results showed that FhCystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection.