The expression of CTLA-4 in hepatic alveolar echinococcosis patients and blocking CTLA-4 to reverse T cell exhaustion in Echinococcus multilocularis-infected mice.

Alveolar echinococcosis (AE) is a zoonotic parasitic disease caused by the infection of Echinococcus multilocularis (E. multilocularis) larvae. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) produces inhibitory signals and induces T cell exhaustion, thereby inhibiting the parasiticidal efficacy of the liver immune system. Therefore, the purpose of this study is to explore how T-cell exhaustion contributes to AE and whether blocking CTLA-4 could reverse T cell exhaustion. Here we discovered that the expression of CTLA-4 was increased in the infiltrating margin around the lesion of the liver from AE patients by using western blot and immunohistochemistry assay. Multiple fluorescence immunohistochemistry identified that CTLA-4 and CD4/CD8 molecules were co-localized. For in vitro experiments, it was found that the sustained stimulation of E. multilocularis antigen could induce T cell exhaustion, blocking CTLA-4-reversed T cell exhaustion. For in vivo experiments, the expression of CTLA-4 was increased in the liver of E. multilocularis-infected mice, and the CTLA-4 and CD4/CD8 molecules were co-localized. Flow cytometry analysis demonstrated that the percentages of both CD4+ T cells and CD8+ T cells in the liver and peripheral blood were significantly increased and induced T exhaustion. When the mice were treated with anti-CTLA-4 antibodies, the number and weight of the lesions decreased significantly. Meanwhile, the flow cytometry results suggested that blocking CTLA-4 could effectively reverse T cell exhaustion and reactivate immune function. Our work reveals that blocking CTLA-4 could effectively reverse the T cell exhaustion caused by E. multilocularis and could be used as a novel target for the treatment of AE.

Echinococcus multilocularis specific antibody, systemic cytokine, and chemokine levels, as well as antigen-specific cellular responses in patients with progressive, stable, and cured alveolar echinococcosis: A 10-year follow-up.

BACKGROUND: The infestation with Echinococcus multilocularis larvae may persist in humans for up to decades without evident clinical symptoms. Longitudinal investigations are needed to understand the dynamic immunological processes in alveolar echinococcosis (AE) patients associated with an active and progressive, a stable or a regressive course of disease. METHODOLOGY/PRINCIPAL FINDINGS: This study evaluated the E. multilocularis specific antibody responses, systemic cytokine, and chemokine serum levels over a 10-year follow-up period, as well as cellular responsiveness in AE patients. Our results demonstrate a rapid decrease in antibodies against E. multilocularis specific antigen Em2+. Especially in cured patients, these antibodies remained negative, making them a significant predictor for cured AE. E. multilocularis specific IgG4, and indirect hemagglutination IHA decreased later in time, after around 5 years. While total IgE did not show significant dynamics over the course of disease, E. multilocularis specific IgE decreased after one to two years, and increasing levels were a significant predictor of progressive disease. There was no significant change in systemic IL-8, IL-9, CCL18 or CCL20 serum levels over time. Univariate analysis across groups indicated lower IL-8 levels in cured patients; however, this result could not be confirmed by multivariate analysis. Levels of CCL17 decreased during treatment, especially in cured patients, and thus might serve as a predictive or risk factor for progressive disease. Levels of IL-10 and CCL13 decreased during disease, especially after five and ten years of intervention. The E. multilocularis antigen (EmAg) inducible cellular productions of MCP1(CCL13), TARC(CCL17) and PARC(CCL18) were lowest in patients with cured AE and infection-free controls, while the EmAg inducible cellular production of IFN-γ increased after cure. Significant positive cytokine and chemokine correlations were observed in AE patients for IL-9, IL-10, CCL13(MCP-4), CCL17(TARC) and CCL20(LARC)(for all p<0.001). E. multilocularis specific IgG4 response correlated positively with TARC (p<0.001). Both markers enhanced over time in progressive disease and decreased after cure. The levels of IL-8, IL-10, MCP4 and LARC enhanced with AE regression. CONCLUSIONS/SIGNIFICANCE: Repeated biomarker surveys are advisable to evaluate progression or regression of disease during longitudinal follow-up and such analyses can support imaging techniques and improve staging of AE patients.

The expression of cytokeratin and apoptosis-related molecules in echinococcosis related liver injury.

The study aimed to investigate the expression of cytokeratin and apoptosis-related molecules in the livers of two types of hepatic echinococcosis mice models and to preliminarily explore the relationship between the expression of cytokeratin and apoptosis in echinococcosis related liver injury. We established a mouse model infected by Echinococcus granulosus and Echinococcus multilocularis and observed the expression of cytokeratin and apoptosis related proteins in the two types of hepatic echinococcosis tissues during different stages by immunohistochemical staining. A co-culture model was established using normal hepatocytes and different concentrations of E. granulosus and E. multilocularis protoscoleces. Cell Counting Kit-8 was used to detect cell proliferation, flow cytometry was used to detect hepatocyte apoptosis, and western blot was used to quantify cytokeratin and apoptosis-related proteins, such as caspase3, caspase9, Bcl-2, and Bax. Surgical specimens were obtained from patients with hepatic echinococcosis to analyze the expressions of cytokeratin, caspase3, caspase9, Bcl-2, and Bax by western blot. The expressions of cytokeratin and caspase3 were analyzed by immunohistochemistry. The qRT-PCR method was used to determine the expression of CK8 and CK18 in the liver tissues. In vivo experiments showed that compared to that in the control group, the cytokeratin and caspase3 proteins in the liver tissues of the two types of hepatic echinococcosis were strongly expressed around the lesions of liver echinococcosis; there was a difference between cytokeratin expression of the two different echinococcosis parasites in the liver. Echinococcus granulosus and Echinococcus multilocularis in the co-culture model in vitro could promote the expression of CK, caspase3, caspase9, and Bax protein, decrease the expression of Bcl-2, promote hepatocyte apoptosis, and inhibit cell proliferation; in clinical samples, we found that compared with that in the normal tissues, the expression of cytokeratin, caspase3, caspase9, and Bax in echinococcus tissues was high, but that in Bcl-2 was low. Furthermore, the expression of CK8 and CK18 mRNA were higher in echinococcus tissues than that in the normal tissues and immunohistochemistry analysis also showed that cytokeratin and caspase3 levels were higher in echinococcus tissues than that in the normal tissues. The expression of cytokeratin and apoptosis-related molecules, reflecting liver damage, is high in the liver and is caused due to hepatic echinococcosis. This study provides the first evidence of cytokeratin could be useful for evaluating liver tissue damage caused by echinococcus infection.

Structural changes and expression of hepatic fibrosis-related proteins in coculture of Echinococcus multilocularis protoscoleces and human hepatic stellate cells.

BACKGROUND: Echinococcus multilocularis is the causative agent of human hepatic alveolar echinococcosis (AE). AE can cause damage to several organs, primarily the liver, and have severe outcomes, such as hepatic failure and encephalopathy. The main purpose of this study was to explore the interactions between hepatic stellate cells (HSCs) and E. multilocularis protoscoleces (PSCs). The results of this study provide an experimental basis for further examination of the pathogenesis of hepatic fibrosis due to AE infection. METHODS: We investigated the role of Echinococcus multilocularis (Echinococcus genus) PSCs in hepatic fibrosis by examining structural changes and measuring hepatic fibrosis-related protein levels in cocultures of PSCs and human HSCs. Structural changes were detected by transmission electron microscopy (TEM), and levels of the hepatic fibrosis-related proteins collagen I (Col-I), alpha-smooth muscle actin (α-SMA) and osteopontin (OPN) were measured by western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: Under coculture (1) both PSCs and HSCs exhibited morphological changes, as observed by TEM; (2) Col-I, α-SMA, and OPN expression levels, which were determined by western blotting and ELISA, significantly increased after 3 days of incubation. CONCLUSIONS: The results of this study provide insights into the molecular mechanisms of AE-induced hepatic fibrosis.

Impact of Albendazole on Cytokine and Chemokine Response Profiles in Echinococcus multilocularis-Inoculated Mice.

OBJECTIVE: Alveolar echinococcosis (AE) is a zoonosis caused by the larval stage of the metacestode Echinococcosis multilocularis with a tumor-like behavior in the targeted organ, especially in the liver. Surgery with albendazole is first-line modality for AE. Drug discontinuation is usually based upon the parasitic viability shown by the positron emission tomography (PET) scan. However, as a demanding and expensive method, it is not widely practiced in majority of the endemic regions. Further understanding on the cytokine and chemokine response profiles in AE patients may provide an interesting insight for potential markers in viability assessment. METHODS: Mice were inoculated with Echinococcus multilocularis intrahepatically to develop the hepatic AE murine model. Oral albendazole administration was then applied for three months after the first inoculation, and peripheral and regional immune cells including type 1 T helper cells (Th), Th2, Th17, regulatory T (Treg) cells, related cytokines, and chemokines were examined. RESULTS: The hepatic AE lesion was confirmed by ultrasound examination resulting in a successful rate of 70%. Among the 17 cytokines and chemokines detected, plasma levels of IL-23 were significantly higher in E. multilocularis-infected mice when compared to the control group; furthermore, more obvious increasing levels were found after albendazole treatment (p < 0.05). All chemokine levels other than eotaxin and MCP-3 were slightly higher in E. multilocularis-infected mice compared to the control group (p > 0.05). Eotaxin levels were significantly decreased in mice with E. multilocularis infection followed by albendazole treatment (p < 0.05). Both IL-17A and IL-23 expressions in hepatic AE lesions were significantly higher and related with disease activity. CONCLUSION: Albendazole administration influenced the balance of immune response and promotes the secretion of proinflammatory factors which is beneficial to parasite clearance. IL-23 seems to be associated with the successful albendazole treatment in mice with E. multilocularis infection; such a change could be translated into clinical application in the near future.

Response patterns in adventitial layer of Echinococcus granulosus sensu stricto cysts from naturally infected cattle and sheep.

Cystic echinococcosis is a zoonotic disease caused by the metacestode of Echinococcus granulosus sensu lato. The disease is characterized by the development of cystic structures inside viscera of the intermediate host, mainly liver and lungs. These cysts are formed by three layers: germinal, laminated, and adventitial layer, the latter being the local host immune response. Metacestodes that develop protoscoleces, the infective stage to the definitive host, are termed fertile, whereas cysts that do not produce protoscoleces are termed non-fertile. Sheep usually harbor fertile cysts while cattle usually harbor non-fertile cysts. Adventitial layers with fibrotic resolution are associated to fertile cysts, whereas a granulomatous reaction is associated with non-fertile cysts. The aim of this study was to analyze cellular distribution in the adventitial layer of fertile and non-fertile E. granulosus sensu stricto cysts found in liver and lungs of cattle and sheep. A total of 418 cysts were analyzed, 203 from cattle (8 fertile and 195 non-fertile) and 215 from sheep (64 fertile and 151 non-fertile). Fertile cysts from cattle showed mixed patterns of response, with fibrotic resolution and presence of granulomatous response in direct contact with the laminated layer, while sheep fertile cysts always displayed fibrotic resolution next to the laminated layer. Cattle non-fertile cysts display a granulomatous reaction in direct contact with the laminated layer, whereas sheep non-fertile cysts display a granulomatous reaction, but in direct contact with the fibrotic resolution. This shows that cattle and sheep cystic echinococcosis cysts have distinct local immune response patterns, which are associated to metacestode fertility.

Serology for the diagnosis of human hepatic cystic echinococcosis and its relation with cyst staging: A systematic review of the literature with meta-analysis.

BACKGROUND: The diagnosis of cystic echinococcosis (CE) is primarily based on imaging, while serology should be applied when imaging is inconclusive. CE cyst stage has been reported among the most important factors influencing the outcome of serodiagnosis. We performed a systematic review and meta-analysis of the relation between cyst stage of hepatic CE and diagnostic sensitivity of serological tests, to evaluate whether their relation is a consistent finding and provide guidance for the interpretation of results of serological tests. METHODOLOGY/PRINCIPAL FINDINGS: MEDLINE, EMBASE, CENTRAL, and Lilacs databases were searched on December 1st 2019. Original studies published after 2003 (year of publication of the CE cyst classification), reporting sensitivity of serological tests applied to the diagnosis of human hepatic CE, as diagnosed and staged by imaging, were included. The quality of studies was assessed using the Newcastle-Ottawa Scale. Data from 14 studies were included in the meta-analysis. Summary estimates of sensitivities and 95% confidence intervals were obtained using random effects meta-analysis. Overall, test sensitivity was highest in the presence of CE2 and CE3 (CE3a and/or CE3b), and lowest in the presence of CE5 and CE4 cysts. ELISA, ICT and WB showed the highest sensitivities, while IHA performed worst. CONCLUSIONS/SIGNIFICANCE: The results of our study confirm the presence of a clear and consistent relation between cyst stage and serological tests results. Limitations of evidence included the heterogeneity of the antigenic preparations used, which prevented to determine whether the relation between cyst stage and sensitivity was influenced by the type of antigenic preparation, the paucity of studies testing the same panel of sera with different assays, and the lack of studies assessing the performance of the same assay in both field and hospital-based settings. Our results indicate the absolute need to consider cyst staging when evaluating serological results of patients with hepatic CE.

Prevalence of Liver Hydatidosis and Its Economic Significance in Sheep Slaughtered in a Private Abattoir in Konya

Objective: This study aimed to determine the prevalence of liver hydatidosis in sheep slaughtered in a private slaughterhouse in Konya and to estimate the economic loss incurred because of the disease. Methods: The study was conducted over a period of 12 months between 1 June 2018 and 31 May 2019. Given that the aim of this investigation was to determine the prevalence of liver hydatidosis, only the livers of 41,002 sheep were examined for hydatid cysts. Results: The liver of 810 (1.97%) sheep was found to be infected with hydatid cysts during the study period. The infection rate was determined as 5.34% in animals older than one year of age and 1.68% in animals less than one year of age. Regardless of the age group, the highest infection rate was found in autumn (3.34%), while the lowest infection rate was seen in spring (0.84%). In the sheep, the highest infection rate was in December (17.2%), and in lambs, it was in June (2.9%). On the other hand, the lowest infection rate in sheep was observed in November (1.8%), while the lowest infection rate in lambs was found in April (0.7%). The total economic loss incurred due to the annihilated livers was estimated as 36,450 TL (6.417$). Regardless of the number of cysts and degree of infection, the infected livers were completely discarded. The economic loss incurred due to the discarded livers was estimated by considering the 2019 offal prices. Conclusion: Based on the data obtained from this study, it could be concluded that hydatidosis still exists in Konya as well as throughout Turkey and that it causes serious economic loss.

Copper nanoparticles: Biosynthesis, characterization, and protoscolicidal effects alone and combined with albendazole against hydatid cyst protoscoleces.

BACKGROUND: Surgery remains the preferred treatment option for hydatid cyst (cystic echinococcosis); however, recent studies have demonstrated that the current protoscolicidal agents used during surgery are associated with some adverse side effects such as biliary fibrosis, hepatic necrosis, and cirrhosis. The present study aims to evaluate the in vitro and ex vivo anti-parasitic effects of copper nanoparticles (CuNPs) alone and combined with albendazole on hydatid cyst protoscoleces. METHODS: CuNPs was green synthesized using C. spinosa extract. Various concentrations of CuNPs (250, 500, and 750 mg/mL) alone and combined with albendazole (ALZ, 200 mg/mL) were exposed to protoscoleces collected from the liver fertile hydatid cysts of infected sheep for 5-60 min in vitro and ex vivo. Next, the eosin exclusion test was applied to determine the viability of protoscoleces. Caspase-3 like activity of CuNPs-treated protoscoleces was then evaluated using the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: Scanning electron microscopy (SEM) results showed that the particle size of CuNPs was 17 and 41 nm with the maximum peak at the wavelength of 414 nm. The maximum protoscolicidal activity of CuNPs was observed at the concentration of 750 mg/mL in vitro, so that 73.3 % of protoscoleces were killed after 60 min of exposure. Meanwhile, the mortality of protoscoleces was 100 % after 10 min of exposure to 750 mg/mL of CuNPs along with ALZ (200 mg/mL). Nevertheless, the findings proved that CuNPs even in combination with ALZ required a longer time to kill protoscoleces ex vivo. After 48 h of treating protoscoleces, CuNPs in a dose-dependent manner and at doses of 250, 500, and 750 mg/mL induced the caspase enzyme activation by 20.5 %, 32.3 %, and 36.1 %, respectively. CONCLUSION: The findings of the present investigation showed potent protoscolicidal effects of CuNPs, especially combined with albendazole, which entirely eliminated the parasite after 10-20 min of exposure. The results also showed that although the possible protoscolicidal mechanisms of CuNPs are not clearly understood, the inducing apoptosis through caspases is one of the main protoscolicidal mechanisms of CuNPs. However, supplementary studies, especially in animal models and clinical settings, are needed to approve these results.

Comparison of the Efficacy of Anatomic and Non-anatomic Hepatectomy for Hepatic Alveolar Echinococcosis: Clinical Experience of 240 Cases in a Single Center.

BACKGROUND: Hepatic alveolar echinococcosis (AE) is a zoonotic parasitic disease. There are more than 16,000 new cases each year, approximately 60 million people are threatened, and the annual direct economic loss is RMB 3 billion. The prevalence of AE in some areas of the Qinghai-Tibet Plateau is as high as 6.0%. Radical resection, including anatomic and non-anatomic hepatectomy, for advanced AE can significantly prolong the survival time of patients. However, there is no literature compared the efficacy of anatomic and non-anatomic hepatectomy. Therefore, by comparing various clinical evaluation indices between anatomic and non-anatomic hepatectomy, this study explored the short-term and long-term efficacy of these two surgical methods for AE. METHODS: The clinical data of patients with AE who underwent radical hepatectomy at Qinghai Provincial People's Hospital from January 2015 to January 2021 were retrospectively analyzed. The patients were divided into two groups by surgical method, that were, non-anatomic hepatectomy group and anatomic hepatectomy group. We compared these two groups focusing on basic preoperative data, such as age, sex, lesion size, and liver function parameters; main intraoperative evaluation indices, such as operation time, intraoperative porta hepatis occlusion time, intraoperative blood loss, and blood transfusion; and postoperative recovery evaluation indicators, such as postoperative liver function, incidence of surgical complications, and AE recurrence. RESULTS: A total of 240 patients were enrolled in this study, including 123 in anatomic hepatectomy group and 117 in non-anatomic hepatectomy group. There were no significant differences (P > 0.05) between baseline characteristics. Anatomic hepatectomy group was advantageous than non-anatomic hepatectomy group regarding intraoperative blood loss (P < 0.001), blood transfusion (P < 0.001), and porta hepatis occlusion time (P < 0.001). There were statistically significant differences in postoperative liver function (aspartate aminotransferase: P < 0.001; alanine aminotransferase: P < 0.001), surgical complications (P < 0.001), and AE recurrence rate (P = 0.003). The median survival of patients in the anatomic hepatectomy group was 66 months, compared to 65 months in the non-anatomic hepatectomy group (χ2 = 4.662, P = 0.031). CONCLUSIONS: Anatomic hepatectomy was not only safe for AE but also showed better short-term and long-term superiority than non-anatomic hepatectomy.

Evaluation of the prevalence of Echinococcus multilocularis in dogs that visit off-leash dog parks in southern Ontario, Canada.

Prior to 2012, Echinococcus multilocularis was not known to occur in any host in Ontario, Canada. However, since that year, five cases of alveolar echinococcosis have been diagnosed in dogs that resided at the western end of Lake Ontario. In addition, E. multilocularis has been shown to be a common infection in wild canids (i.e. coyotes and foxes) across southern Ontario with a high-risk infection cluster in the area surrounding the western shores of Lake Ontario and northern shores of Lake Erie. In regions endemic for E. multilocularis, dog ownership is considered a risk factor for human alveolar echinococcosis. A study was therefore carried out to determine the prevalence of E. multilocularis intestinal infections in dogs within the high-risk infection cluster. From May to November 2018, faecal samples were collected from 477 dogs aged ≥6 months that visited 12 off-leash dog parks in the Halton, Hamilton and Niagara public health units. Faecal samples were analysed via a magnetic capture probe DNA extraction and real-time PCR method for E. multilocularis DNA. Overall, 0% (97.5% CI: 0%-0.80%) of samples tested positive. This result informs preventive recommendations for E. multilocularis infections in dogs in this region.

Immunohistological detection of small particles of Echinococcus multilocularis and Echinococcus granulosus in lymph nodes is associated with enlarged lymph nodes in alveolar and cystic echinococcosis.

BACKGROUND: Alveolar (AE) and cystic echinococcosis (CE) in humans are caused by the metacestode of the tapeworms Echinococcus multilocularis and Echinococcus granulosus sensu lato (s.l.). Immunohistochemistry with the monoclonal antibodies (mAb) Em2G11, specific for AE, and the mAb EmG3, specific for AE and CE, is an important pillar of the histological diagnosis of these two infections. Our aim was to further evaluate mAb EmG3 in a diagnostic setting and to analyze in detail the localization, distribution, and impact of small particles of Echinococcus multilocularis (spems) and small particles of Echinococcus granulosus s.l. (spegs) on lymph nodes. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the mAb EmG3 in a cohort of formalin-fixed, paraffin embedded (FFPE) specimens of AE (n = 360) and CE (n = 178). These samples originated from 156 AE-patients and 77 CE-patients. mAb EmG3 showed a specific staining of the metacestode stadium of E. multilocularis and E. granulosus s.l. and had a higher sensitivity for spems than mAb Em2G11. Furthermore, we detected spegs in the surrounding host tissue and in almost all tested lymph nodes (39/41) of infected patients. 38/47 lymph nodes of AE showed a positive reaction for spems with mAb EmG3, whereas 29/47 tested positive when stained with mAb Em2G11. Spegs were detected in the germinal centers, co-located with CD23-positive follicular dendritic cells, and were present in the sinuses. Likewise, lymph nodes with spems and spegs in AE and CE were significantly enlarged in size in comparison to the control group. CONCLUSIONS/SIGNIFICANCE: mAb EmG3 is specific for AE and CE and is a valuable tool in the histological diagnosis of echinococcosis. Based on the observed staining patterns, we hypothesize that the interaction between parasite and host is not restricted to the main lesion since spegs are detected in lymph nodes. Moreover, in AE the number of spems-affected lymph nodes is higher than previously assumed. The enlargement of lymph nodes with spems and spegs points to an immunological interaction with the small immunogenic particles (spems and spegs) of Echinococcus spp.

A Case-Study of the Molecular Diagnosis of Echinococcus multilocularis in Wild Boar with Comments on its Public Health Significance in Turkey.

Echinococcus multilocularis is a parasite species of zoonotic importance which can be fatal to humans and causes Alveolar Echinococcosis (AE). This report describes the development of a cyst from the liver of a wild boar and molecular confirmation of its identification. The cyst material was obtained from the liver of a wild boar killed by hunters. Genomic DNA was extracted from the germinal layer of the cyst material, and 875 bp mitochondrial cytochrome c oxidase subunit I (COI) gene fragment was amplified by PCR and sequenced. A BLAST search matched 100% with published Echinococcus multilocularis sequences. This study confirms the occurrence of E. multilocularis in a wild boar for the first time in Turkey.

[Anaphylaxis due to a ruptured hydatid cyst]. / Anaphylaxie bei rupturierter Echinokokkuszyste.

HISTORY AND CLINICAL FINDINGS: We report the case of a 27-year-old Syrian patient who came to the emergency department with a syncopal episode. No medical history could be raised due to a language barrier and so the clinical presentation was leading. INVESTIGATIONS AND DIAGNOSIS: The patient exhibited signs of shock, accompanied by an exanthema as well as perioral hematin. In an ultrasound sonography free intraabdominal fluid and an obscure change in the upper abdomen could be visualized. An esophagogastroduodenoscopy showed evidence of an ulcer, however did not explain all symptoms. In a CT abdomen, signs of a ruptured cyst could be demonstrated. TREATMENT AND COURSE: The patient stabilized under the treatment protocol for anaphylaxis. Due to the clinical course and country of origin the patient received albendazole and a partial liver resection for a suspected echinococcus cyst. CONCLUSION: Obscure clinical symptoms alongside signs of shock, should always considered to be an allergic reaction in absence of sepsis or hemorrhage. In anaphylaxis, echinococcosis should always be included in the differential diagnosis.

Alveolar echinococcosis in a dog in the eastern United States.

An 8-y-old Labrador Retriever was presented to a small animal practice in northern Virginia with a history of recent lethargy. Physical examination findings were unremarkable. Ultrasound revealed several large hepatic masses and multiple smaller masses involving the pancreas. Cytologic findings on fine-needle aspirates of the hepatic masses included inflammation and necrosis with eosinophilic, membranous oval structures consistent with cestode infection. Histopathologic findings for biopsies of these masses included extensive necrosis, inflammation, and PAS-positive hyaline-like membranous material interpreted as metacestode cyst wall. A PCR product was generated from aspirate material using primers specific for Echinococcus multilocularis. Subsequent sequence data were 100% homologous to E. multilocularis NADH dehydrogenase subunit I gene sequences. The dog received daily oral albendazole (10 mg/kg) treatment, but its condition deteriorated, and the dog was euthanized. The dog, born in Mississippi, was brought as a puppy to Virginia with no other travel history. To our knowledge, alveolar echinococcosis has not been reported previously in a dog in the United States; E. multilocularis infection was apparently acquired in the mid-Atlantic region of the United States.

Portal hypertension as a result of the incomplete surgically treated advanced alveolar echinococcosis: a case description.

BACKGROUND: Infection of Echinococcus multilocularis causes in humans the alveolar echinococcosis. Although the infection has world-wide distribution it is rarely detected. Diagnosis of alveococcosis is difficult because of not typical clinical picture and irregular results of radiological examinations suggesting neoplasmatic process which begins in the liver tissue or in the biliary tracts. The parasitic growth is slow, so the illness is quite often established in late invasion period. Treatment of long-lasting and late diagnosed infection is difficult and requires cooperation of parasitologists together with surgeons to avoid life-threatening organ dysfunction. CASE PRESENTATION: We describe a young male patient, diagnosed, according to the radiological, immunological and histological examination results, infection of Echinococcus multilocularis, who was treated with not radical resection of pathologic mass together with persistent albendazole intake. The right hepatectomy was performed. In addition, visible cysts were removed from the left lobe of the liver in nonanatomical resection and suspicious calcified lesions in hepatoduodenal ligament were also removed. After the operation portal hypertension, with splenomegaly and symptoms of the liver cirrhosis occurred (thrombocytopenia, collateral venous circulation, first degree varices oesophagii). The portal hypertension probably could be a result of incomplete surgery due to extended parasitic infection and liver anathomical changes due to performed procedures, because the portal hypertension and it's further complications had not been observed before the operation. CONCLUSIONS: Echinococcus multilocularis should be taken under consideration in differential diagnosis of irregular lesions within the liver. Lon-lasting invasion could be responsible for the irreversible secondary liver changes such as cirrhosis and portal hypertension. The surgery treatment (treatment of choice) is difficult and it's results depends on the invasion period the patient is operated on. After the surgery the patient requires careful follow - up, to detect early complications.

Fasciola hepatica coinfection modifies the morphological and immunological features of Echinococcus granulosus cysts in cattle.

Polyparasitism occurs when animals harbour multiple parasites concomitantly. It is a common occurrence but is generally understudied in wild and domestic animals. Fasciola hepatica and Echinococcus granulosus, which are helminths of ungulates, frequently coinfect cattle. The effects of this particular type of polyparasitism are not well documented. The metacestode of Echinococcus granulosus is surrounded by the adventitial layer, which constitutes the host immune response to the parasite. This layer in cattle is produced by a granulomatous reaction and is involved in echinococcal cyst (EC) fertility. Due to the systemic immune-modulating abilities of Fasciola hepatica, coinfection possibly generates a favourable environment for EC growth. A total of 203 Echinococcus granulosus sensu stricto cysts were found in 82 cattle, of which 42 ECs were found in 31 animals coinfected with Fasciola hepatica. The overall infection intensity was 3 cysts per animal. Coinfection with Fasciola hepatica decreased the mean infection intensity to 1.4 cysts per animal. Regarding EC size, coinfection resulted in smaller ECs (15.91 vs 22.09 mm), especially for infertile lung cysts. The adventitial layer of ECs in coinfected animals lacked lymphoid follicles and palisading macrophages, which are generally hallmarks of the granulomatous immune response. The ECs in coinfected animals had organized laminated layers, whereas those in animals without coinfection did not. Although coinfection was not statistically associated with EC fertility, we did not find fertile cysts in the livers of coinfected animals. We concluded that coinfection with Fasciola hepatica and Echinococcus granulosus has a detrimental effect on ECs, particularly infertile cysts.

Successful robotic radical resection of hepatic echinococcosis located in posterosuperior liver segments.

BACKGROUND: Radical resection is an important treatment method for hepatic echinococcosis. The posterosuperior segments of the liver remain the most challenging region for laparoscopic or robotic hepatectomy. AIM: To demonstrate the safety and preliminary experience of robotic radical resection of cystic and alveolar echinococcosis in posterosuperior liver segments. METHODS: A retrospective analysis was conducted on the clinical data of 5 patients with a median age of 37 years (21-56 years) with cystic and alveolar echinococcosis in difficult liver lesions admitted to two centers from September to December 2019. The surgical methods included total pericystectomy, segmental hepatectomy, or hemihepatectomy. RESULTS: Among the 5 patients, 4 presented with cystic echinococcosis and 1 presented with alveolar echinococcosis, all of whom underwent robotic radical operation successfully without conversion to laparotomy. Total caudate lobectomy was performed in 2 cases, hepatectomy of segment VII in 1 case, total pericystectomy of segment VIII in 1 case, and right hemihepatectomy in 1 case. Operation time was 225 min (175-300 min); blood loss was 100 mL (50-600 mL); and postoperative hospital stay duration was 10 d (5-19 d). The Clavien-Dindo complication grade was I in 4 cases and II in 1 case. No recurrence of echinococcosis was found in any patient at the 3 mo of follow-up. CONCLUSION: Robotic radical surgery for cystic and selected alveolar echinococcosis in posterosuperior liver segments is safe and feasible.

Molecular survey on cattle and sheep hydatidosis and first detection of Echinococcus canadensis (G6/G7) in sheep in Turkey.

Larval stage of genus Echinococcus is the causing agent for the zoonotic infection which is life threatening known as Echinococcosis. The purpose of this study was the identification, molecular analysis and characterization of Echinococcus spp. in sheep and cattle. The sampling was done from slaughterhouse of Elazig, Turkey. A total of 85 isolates (sheep, n = 19 and cattle, n = 66) have been collected after slaughtering. Following the gDNA isolation and PCR products of mt-CO1 gene (446 bp) of all the samples were sequenced. Out of 85 isolates, 84 were recognized as Echinococcus granulosus sensu stricto and one sheep isolate was found as Echinococcus canadensis (G6/G7 ) which is identified for the first time in Turkey. However, single nucleotide polymorphism has been observed not only in samples of different animals but also in samples collected from the same cattle. Six liver and three lung hydatid cysts have been detected in cattle. Although no nucleotide differences have been observed in the liver samples, there was single nucleotide polymorphism (C→T) in 40th nucleotide of two lung cysts. As a result of haplotype analysis, 16 haplotypes of E. granulosus s.s. were detected in 66 cattle isolates whereas 7 haplotypes of E. granulosus s.s. were identified in 19 sheep samples.