Echinococcus multilocularis and other zoonotic helminths in red foxes (Vulpes vulpes) from a southern German hotspot for human alveolar echinococcosis.

BACKGROUND: We describe the spatial distribution of Echinococcus multilocularis in its main definitive host, the red fox, and the distribution of human cases of alveolar echinococcosis (AE) within a highly endemic focus in southern Germany (13.7-19.9/100,000 in 1992-2018). Human cases were unequally distributed within the endemicity focus. The purpose of the study was to test whether this is reflected in the small-scale distribution of E. multilocularis in foxes. METHODS: Three areas with contrasting numbers of human cases were selected within the counties of Ravensburg and Alb-Donau, Baden-Württemberg, Germany. From 2018 to 2020, a total of 240 fox carcasses were obtained from traditional hunters in these areas. Carcasses were necropsied and examined for the presence of intestinal helminths. The statistical analysis was performed with SAS version 9.4, and the geo-mapping with QGIS version 3.16.0 Hannover. RESULTS: The prevalence of E. multilocularis in foxes was 44/106 (41.5%) in area I (commune Leutkirch and environs), 30/59 (50.8%) in area II (commune Isny and environs), and 31/75 (41.3%) in area III (commune Ehingen and environs). From 1992 to 2018, a total of nine human cases of alveolar echinococcosis were recorded in area I, five cases were recorded in study area III, and no cases were recorded in area II. No statistically significant differences between the areas were observed (P > 0.05) for intestinal infections with E. multilocularis, and no apparent spatial correlation with the small-scale distribution of human cases was found. Concerning other zoonotic helminths, Toxocara spp. were equally common, with prevalence of 38.7%, 47.4% and 48.0%, respectively, while the frequency of Alaria alata varied among the study areas (0.0-9.4%), probably reflecting the specific habitat requirements for the establishment of its complex life cycle. CONCLUSIONS: Echinococcus multilocularis is highly prevalent in foxes in all the studied areas. The varying number of human AE cases within these areas should therefore be caused by factors other than the intensity of parasite transmission in foxes.

An optimized protocol for isolation of hepatic leukocytes retrieved from murine and NASH liver biopsies.

Immune dysregulation and inflammation by hepatic-resident leukocytes is considered a key step in disease progression of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis toward cirrhosis and hepatocellular carcinoma. Here, we provide a protocol for isolation and characterization of liver-resident immune cells from fine-needle biopsies obtained from a rodent model and humans. We describe steps for isolating leukocytes, cell sorting, and RNA extraction and sequencing. We then detail procedures for low-input mRNA sequencing analyses.

[Ultrasound diagnostics: Differentiated vision - upgrading - better reimbursement]. / Ultraschalldiagnostik: Differenziert sehen ­ aufwerten ­ besser vergüten.

Ultrasonography is an imaging modality of great importance in everyday clinical practice. Technical innovations continuously expand the diagnostic and therapeutic possibilities of ultrasonography requiring continuous upskilling of sonographers. Only a small number of practitioners, in hospital and practice settings in Germany, currently have the required level of skills. Therefore, these techniques are not as readily available as would be desirable. A modern high-end ultrasound scanner in the hands of a qualified sonographer is a diagnostic high-tech precision instrument, which can compete with any other imaging modality.In conclusion, basic sonography, as it is currently practised should be separated from high-end sonography, requiring extensive training and better remuneration. In this context the introduction of a new medical board speciality for ultrasonography, "Advanced Ultrasonography", with corresponding upgrading should be recommended for high-end sonography.

Detection and quantification of a focal fat deposition in a liver undergoing multiple operations for neuroendocrine tumor disease using attenuation imaging: a case report.

BACKGROUND: In patients with history of malignancy, new-onset liver lesions often present diagnostic challenges. We present the case of a patient with history of neuroendocrine tumor and new-onset echo-rich hepatic lesion, in whom attenuation imaging helped to make the diagnosis. Attenuation imaging is an ultrasound-based technique that allows for the quantification of hepatic fat content on the basis of a measurement of sound attenuation. CASE PRESENTATION: We present the case of a 62-year-old Caucasian female patient who underwent pylorus-preserving pancreaticoduodenectomy Whipple surgery in 2004 for histologically well-differentiated neuroendocrine tumor with a proliferation rate of 3% of the pancreatic head. During the course, single liver metastases were resected in 2009, 2010, and 2013. In 2019, hemihepatectomy was performed when two liver metastases recurred. The liver metastases each showed a proliferation rate of 10% with vigorous expression of chromogranin A, synaptophysin, and somatostatin. The most recent follow-up examinations showed a normal chromogranin A value and the patient reported a good general condition. However, sonography revealed a blurred, echoic lesion in the liver. On contrast-enhanced sonography, the lesion showed identical behavior to the surrounding liver parenchyma. In the asymptomatic patient, liver biopsy did not seem to be indicated at the current time. Measurement of the attenuation coefficient by attenuation imaging showed a significantly higher measurement in the area of the echo-rich lesion than in the rest of the liver. The overall findings are consistent with focal fat deposition. CONCLUSIONS: Attenuation imaging appears to be useful in the evaluation of unclear echo-rich liver lesions. In particular, primary non-malignant-appearing liver lesions that are unremarkable on abdominal contrast-enhanced ultrasound can be more accurately assessed.

[Diagnostic delaying factors in hepatic alveolar echinococcosis]. / Diagnoseverzögernde Faktoren bei hepatischer alveolärer Echinokokkose.

PURPOSE: The purpose of this study was to analyze the diagnostic workflow of patients with alveolar echinococcosis (AE) and to identify possible diagnosis-delaying factors. METHODS: The number and type of diagnostic procedures of patients diagnosed with alveolar echinococcosis were investigated. The disease history was recorded on the basis of questionnaires, the available findings, and data supplements from the hospital information system (SAP). Statistical analyses were performed using SAS version 9.4 and Microsoft Excel version 16.43. The study population of the cross-sectional study included n = 109 patients with confirmed alveolar echinococcosis. RESULTS: The definitive diagnosis of alveolar echinococcosis of the liver was made at 26.5 ± 65.0 (mean ± standard deviation) months (min - max: 0 - 344, median = 3). The majority of patients were diagnosed because of incidental imaging findings of the liver (n = 74/109 (67.9%)). A total of n = 56/74 (75.7%) of all incidental findings were diagnosed in an outpatient setting, while n = 15/74 (20.3%) of cases were diagnosed during inpatient hospitalization. On average, 1.1 ± 1.2 (0-11, median = 1) ionizing imaging modalities were used for each patient. Contrast-enhanced sonography was received by 0.3 ± 0.5 (0-2, median = 0) patients. Almost all patients (n = 104/109 (95.4%) had at least one suspected hepatic or extrahepatic malignancy at some time. Exclusion of suspected malignancy occurred at a mean of 4.1 ± 16.5 months (0 -133.8, median = 1). CONCLUSIONS: The diagnostic clarification process of AE patients is lengthy and stressful. The psychological burden of a questionable malignant diagnosis is considerable. Early use of contrast-enhanced sonography and, if necessary, puncture of unclear hepatic masses helps to shorten the difficult diagnostic process.

Differentiation of hepatic alveolar echinococcosis with a hemangioma-like pattern compared to typical liver hemangioma using contrast-enhanced ultrasound: a pilot study.

PURPOSE: Echinococcus multilocularis infects humans as a false intermediate host, primarily with intrahepatic manifestation. Incorrect diagnostic interpretation of these liver tumors, especially the hemangioma-like pattern, can lead to progressive disease. The aim of the study was to investigate the differentiation of typical hemangioma and a hemangioma-like pattern of E. multilocularis using contrast-enhanced ultrasound (CEUS). METHODS: This prospective clinical pilot study comprised patients with hemangioma (n = 14) and patients with alveolar echinococcosis (AE) and hemangioma-like pattern (n = 7). Inclusion criteria were the detection of a liver lesion according to a hemangioma-like pattern on E. multilocularis Ulm classification-ultrasound (EMUC-US) and "confirmed" or "probable" AE according to WHO case definition. The comparison group had hepatic hemangioma with typical B-scan sonographic morphology. All participants underwent conventional and contrast-enhanced ultrasonography. RESULTS: The patient group comprised five men (71.4%) and two women (28.6%) with a mean average age of 64.1 ± 11.2 years. The patient group with hemangioma comprised nine female subjects (64.3%) and five male subjects (35.7%) with a mean average age of 56.1 ± 12.0 years. Early arterial bulbous ring enhancement (p < 0.0001) and iris diaphragm phenomenon could only be visualized in the patients with hemangioma (p < 0.0001). Furthermore, the patients with hemangioma exhibited hyperenhancement in the late phase (p = 0.0003). In contrast, the patients exhibited typical early arterial rim enhancement (p < 0.0001) and, in the portal venous and late phase, complete or incomplete non-enhancement (black hole sign; p = 0.0004). CONSLUSION: The behavior of hemangioma-like AE lesions and typical liver hemangiomas is significantly different on CEUS. AE should be considered as a possible differential diagnosis, especially in high-endemic areas.

Whole-genome sequencing of multiple related individuals with type 2 diabetes reveals an atypical likely pathogenic mutation in the PAX6 gene.

Pathogenic variants in more than 14 genes have been implicated in monogenic diabetes; however, a significant fraction of individuals with young-onset diabetes and a strong family history of diabetes have unknown genetic etiology. To identify novel pathogenic alleles for monogenic diabetes, we performed whole-genome sequencing (WGS) on four related individuals with type 2 diabetes - including one individual diagnosed at the age of 31 years - that were negative for mutations in known monogenic diabetes genes. The individuals were ascertained from a large case-control study and had a multi-generation family history of diabetes. Identity-by-descent (IBD) analysis revealed that the four individuals represent two sib-pairs that are third-degree relatives. A novel missense mutation (p.P81S) in the PAX6 gene was one of eight rare coding variants across the genome shared IBD by all individuals and was inherited from affected mothers in both sib-pairs. The mutation affects a highly conserved amino acid located in the paired-domain of PAX6 - a hotspot for missense mutations that cause aniridia and other eye abnormalities. However, no eye-related phenotype was observed in any individual. The well-established functional role of PAX6 in glucose-induced insulin secretion and the co-segregation of diabetes in families with aniridia provide compelling support for the pathogenicity of this mutation for diabetes. The mutation could be classified as "likely pathogenic" with a posterior probability of 0.975 according to the ACMG/AMP guidelines. This is the first PAX6 missense mutation that is likely pathogenic for autosomal-dominant adult-onset diabetes without eye abnormalities.

[Spread and Development of Alveolar Echinococcosis in Germany, 1992-2018]. / Untersuchung zur Ausbreitung und Entwicklung der alveolären Echinokokkose in Deutschland, 1992­2018.

GOAL OF THE STUDY: Alveolar echinococcosis (AE) is a rare parasitosis caused by the pathogen Echinococcus multilocularis. There is an increase in the number of cases in many countries. The aim of the study was to investigate the current prevalence and the change in the geographical distribution pattern. METHODOLOGY: Data were collected retrospectively for the period 1992-2018 using the registered cases in the national disease register for AE in Germany. Statistical analysis was performed using the SAS statistical analysis system version 9.4 (SAS Institute, Cary, N.C., USA). RESULTS: The study population of n=569 patients included n=322 (56,59%) women and n=247 (43,40%) men. The mean average age of patients with alveolar echinococcosis at first presentation was 53,90±17,54 years (median: 56,00 years). The Moran's I test statistic showed a positive spatial autocorrelation for the period 1992-2018 corresponding to a heterogeneous distribution of disease cases in Germany (I=0,4165; Z=10,9591, p=0,001). An increase in age- and sex-specific prevalence could be determined for the entire study period (1992-2018). The overall prevalence in the period 1992-2018 was 0,71 cases per 100,000 population. The determination of the prevalence for the period 1992-2018 resulted in 0,31 cases for men and 0,40 cases for women per 100,000 population. In the period 1992-1996, no AE cases had been registered in 11/16 (68,8%) federal states (Berlin, Brandenburg, Bremen, Hamburg, Mecklenburg-Vorpommern, Rheinland-Pfalz, Saarland, Sachsen, Sachsen-Anhalt, Schleswig-Holstein und Thüringen). The evaluation recently shows an increased occurrence of cases in the federal states of Hessen, Rheinland-Pfalz and Nordrhein-Westfalen. CONCLUSIONS: The analysis shows a rise in prevalence and an increasing number of cases outside the classic endemic areas of Baden-Württemberg and Bavaria.

Contrast-enhanced ultrasound (CEUS) in patients with metastasis-like hepatic alveolar echinococcosis: a cohort study.

PURPOSE: Hepatic alveolar echinococcosis (HAE) of the metastasis-like pattern, according to the Echinococcus Ulm classification, is usually discovered as an incidental finding, and the diagnostic differentiation from "true metastases" is difficult. The aim of this study was to investigate whether lesions of the "metastasis-like pattern" in HAE show a typical contrast behavior that can be used for differentiation from metastasis in malignancies. METHODS: This prospective clinical study included 11 patients with histologically confirmed HAE of the metastasis-like pattern (7 female and 4 male; mean age, 57.1 years; mean disease duration, 59.5 months), who had been examined by B-scan sonography and CEUS, from the National Echinococcosis Registry Germany. RESULTS: On contrast-enhanced sonography, 11/11 reference lesions showed annular rim enhancement in the arterial and portal venous phases. Throughout the entire 4-min study period, none of the reference lesions showed central contrast enhancement-i.e., all exhibited a complete "black hole sign". A small central scar was seen in 81.8% of cases. CONCLUSION: In clinically unremarkable patients with incidentally detected metastasis-like lesions of the liver, contrast-enhanced sonographic detection of rim enhancement without central contrast uptake (black hole sign) should be considered evidence supporting a diagnosis of hepatic alveolar echinococcosis with a rare metastasis-like pattern. This can help to differentiate HAE from metastases, especially in high-endemic areas.

A Systematic Comparative Study on the Diagnostic Value of Transabdominal Ultrasound in Patients with Pancreatic Cystic Lesions.

Pancreatic cystic lesions are a frequent incidental finding in abdominal imaging. Despite its usually benign background, a small fraction exhibiting features suspicious for cancerous development demands continuous follow-up or surgical removal. Current guidelines advocate magnetic resonance imaging and endoscopic ultrasound to evaluate the risk of malignancy, whereas transabdominal ultrasound is perceived as subordinate imaging. The objective of this study was to analyze cyst detection rates of latest-generation ultrasound machines compared to magnetic resonance imaging, computed tomography, and endosonographic ultrasound and to determine inter-rater reliability. The results showed that large cysts facilitate their visualization by transabdominal ultrasound while detection rates are independent of the anatomical part of the pancreas in which they were sited. Changes in the pancreatic duct width, a connection to the pancreatic duct system, and the architectural characteristics of cysts are poorly recognized by transabdominal ultrasound compared to magnetic resonance imaging and endoscopic ultrasound. Computed tomography imaging is preferred over transabdominal ultrasound to detect calcifications and regional lymphadenopathy. Even if conducted by experienced investigators, transabdominal ultrasound examinations fail to agree with magnetic resonance imaging scans regarding cyst detection rates (κ = 0.093).

Spleen and Liver Stiffness Evaluation by ARFI Imaging: A Reliable Tool for a Short-Term Monitoring of Portal Hypertension?

Background: Assessment of hepatic venous pressure gradient (HVPG) is the most reliable, though invasive method for evaluation of portal hypertension. Non-invasive, elastography-based techniques are well established in diagnosis, but not in monitoring of portal hypertension. The aim of our prospective study was to determine the value of acoustic radiation force impulse (ARFI) elastography technique of the liver and spleen in diagnosis and monitoring of portal hypertension. Methods: We prospectively assessed portal hypertension by HVPG and corresponding elastography of the liver and spleen in 31 patients with liver cirrhosis and an indication for primary prophylaxis by non-cardio selective beta-blockers. Investigations were performed at baseline and a follow-up visit after 6-8 weeks. To address the known large variability of values for spleen elastography, well-defined corresponding areas in the spleen were used for baseline and follow-up elastography. Sensitivity, specificity, and AUC-ROC values for both spleen and liver elastography monitoring of portal hypertension were calculated. Results: Liver but not spleen elastography significantly correlated with HVPG results and was suitable for initial evaluation of portal hypertension. However, changes in HVPG results did not show any correlation with alterations of ARFI values from baseline to follow-up visits both for liver and spleen elastography. Spleen stiffness results were not homogeneous across the whole organ differing significantly between the upper, hilar, and bottom placed investigation areas. Conclusions: In this prospective study ARFI-based assessment of liver elastography showed itself suitable for initial assessment but not for monitoring of portal hypertension. Spleen elastography was not appropriate for both, evaluation and monitoring of portal hypertension. A possible explanation for this new data that are in some contrast to previously published results is the degree of portal hypertension in our study, a comparatively short follow-up period, and well-defined investigation areas for spleen elastography in repetitive ARFI investigations. This trial is registered with NCT03315767.

Dog Ownership and Risk for Alveolar Echinococcosis, Germany.

Human alveolar echinococcosis is caused by the parasite Echinococcus multilocularis, and dog ownership has been identified as a risk factor. We sought to specify the factors of dog ownership underlying this risk by conducting a case-control study among dog owners in Germany. The analysis revealed an increased odds ratio of ≈7-fold for dog owners whose dogs roam unattended in fields, 13-fold for dog owners who feed their dogs organic waste daily, 4-fold for dog owners who take their dog to a veterinarian only in case of illness, and 10-fold for dog owners who have never been informed by a veterinarian about the risk for infection. The results highlight the risk for infection associated with various factors of dog ownership and the value of veterinarians informing owners about prevention.

Lower Circulating Leptin Levels Are Related to Non-Alcoholic Fatty Liver Disease in Children With Obesity.

Background: While for individuals with obesity an association between hyperleptinemia and an increased risk of non-alcoholic fatty liver disease (NAFLD) is assumed, a leptin deficiency is also related to the development of NAFLD early in life in ob/ob mice, in patients with leptin deficiency due to biallelic likely pathogenic variants in the leptin gene, and in patients with lipodystrophy. Objectives: To investigate the association of circulating leptin levels in pre-pubertal children with obesity and steatosis hepatis. Methods: The cross-sectional study consisted data of n=97 (nmale=76) pre-pubertal children (11.8 ± 1.5 years) with obesity (BMIz: 2.4 ± 0.4). Fasting concentrations of cardiometabolic parameters were measured: insulin, c-peptide, glucose, triglyceride, cholesterol, HDL, LDL, AST, ALT, GGT, leptin. Steatosis hepatis was diagnosed by an ultrasound examination (mild, moderate or severe). Patients were categorized into two groups: low z-score of circulating leptin levels (≤25th percentile) vs. normal z-score of circulating leptin levels. Results: One-third of the children with obesity were diagnosed with steatosis hepatis (I°: 63.6%, II°/III°: 36.4%). Children with steatosis hepatis had significantly lower z-scores of circulating leptin levels compared to children with an unremarkable liver ultrasonography (-2.1 ± 0.8 vs. -0.7 ± 0.6). Z-scores of circulating leptin levels correlate negatively with degree of steatosis hepatis. Children with low z-scores of circulating leptin levels had significantly higher triglyceride, fasting insulin and c-peptide levels compared to children with normal z-scores of circulating leptin levels. Conclusion: Prepubertal children with NAFLD and obesity and partial leptin deficiency might be defined as a clinical subgroup.

Evaluation of Potential Drug Interactions with AiDKlinik® in a Random Population Sample.

Purpose: Undesirable drug interactions are frequent, they endanger the success of therapy, and they lead to adverse drug reactions. The present study aimed to evaluate statistically potentially drug interactions in a locally circumscribed, random sample population. Patients and Methods: In a random sample population of 264 patients taking medications, we performed analyses with the drug information system AiDKlinik®. Statistical analysis was performed using SAS version 9.4. Results: Statistically potentially drug interactions were recorded in 82/264 (31.1%) subjects, including 39/82 (47.56%) men, and 43/82 (52.43%) women (χ 2= 0.081; p = 0.776). The average number of potential possible interactions detected per person was 1.60 ± 1.21. The regression model with the variables age, body-mass-index and number of long-term-medications shows a significant association between the number of long-term medications taken and the number of moderately severe and severe reactions to drug interactions (F(3.239) = 28.67, p < 0.0001; (t(239) 8.28; p < 0.0001)). After backward elimination, the regression model showed a significant interaction with the number of long-term medications (t (240) = 8.73, p < 0.0001) and body-mass-index (t (240) = 2.02, p = 0.0442). In descriptive analysis, the highest percentages of potential drug interactions occurred in 42/82 (51.22%) subjects with body mass indices (BMIs) >25 kg/m2 and in 28/82 (34.15%) subjects aged 61-70 years. Conclusion: Number of long-term medications use, age, and obesity may lead to increased drug-drug interactions in a random population sample.

Comparison of superb microvascular imaging (SMI) quantified with ImageJ to quantified contrast-enhanced ultrasound (qCEUS) in liver metastases-a pilot study.

Background: The aim of the study was to compare methods for the assessment of vascularisation of liver metastases (LM) between superb microvascular imaging (SMI), contrast-enhanced ultrasound, and microvascular density (MVD). Methods: SMI results were quantified as the vascularisation quotient (VQ), based on a grey-scale analysis with ImageJ image software. Those results were compared to contrast-enhanced ultrasonography (CEUS) values, calculated with VueBox®. MVD was measured with an anti-CD34 antibody. Results: This study included 13 patients with LM. The VQ showed a strong correlation with the quantified parameters of contrast-enhanced ultrasound. The parameters of quantified contrast-enhanced ultrasound compared with quantified SMI showed the following statistical correlations: peak enhancement (PE), in arbitrary unit (a.u.) (r=0.72104, P=0.0054), PE in Decibel (dB) (r=0.65918, P=0.00141), Wash-in- Area Under the Curve (WiAUC) in a.u. (r=0.63604, P=0.00194), Wash-in Perfusion-Index (WiPI) in a.u. (r=0.73337, P=0.0043), Wash-in Perfusion-Index (WiPI) in dB (r=0.65642, P=0.0194), Wash-in-Rate (WiR) in a.u. (r=0.7304, P=0.0036) and Wash-in-Rate (WiR) in dB (r=0.82897, P=0.0005). Conclusions: Comparison of the two methods, SMI and contrast-enhanced ultrasound (CEUS), for quantitative assessment of vascularisation of LM showed good correlation. The contrast-independent Doppler technique SMI can qualitatively assess the vascularisation of LM.

Echinococcosis: a Challenge for Liver Sonography.

The sonographic findings in alveolar echinococcosis (AE) and cystic echinococcosis (CE) are complex and can pose significant differential diagnostic problems. They may present like complicated cysts or malignant tumors in the liver. We will discuss the diagnostic difficulties and pitfalls of these two diseases based on case histories.

Focal fatty sparing as an indicator of higher-grade fatty liver assessed by attenuation imaging: a prospective clinical study in NAFLD population.

BACKGROUND: As part of a prospective clinical study, the degree of hepatic fatty degeneration was quantified in a patient population with nonalcoholic fatty liver disease and sonographically diagnosed with hepatic steatosis using attenuation imaging. METHODS: A total of 113 patients with hepatic steatosis were examined, of whom 35 showed focal fatty sparing. Patients with the condition after right nephrectomy, other known liver diseases, and relevant alcohol consumption were excluded from the evaluation. B-scan sonography and sonographic quantification of steatosis content using attenuation imaging (Aplio i800 Canon Medical Systems) were performed. Attenuation imaging is a new ultrasound-based measurement technique that allows objective detection and quantification of hepatic steatosis. RESULTS: The prevalence of focal fatty sparing was 31.0% in the patient population examined. Patients with focal fatty sparing showed a statistically significantly higher attenuation coefficient in contrast to patients without focal fatty sparing (0.79 ± 0.10 vs. 0.66 ± 0.09 dB/cm/MHz, p < 0.0001). CONCLUSION: Detection of focal fatty sparing is associated with an increased attenuation coefficient and is thus an expression of higher-grade hepatic fatty degeneration. Patients with focal fatty sparing are more often male and have a higher BMI and a larger liver than patients with nonalcoholic fatty liver disease without focal fatty sparing.