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Background: Occurrence of hepatitis C virus (HCV) infection is reduced by effective risk management procedures, but patient-to-patient transmission continues to be reported in healthcare settings. Aim: To report the use of phylogenetic analysis in the clinical risk management of an HCV outbreak among 128 thalassaemia outpatients followed at a thalassaemia centre of an Italian hospital. Methods: Epidemiological investigation and root-cause analysis were performed. All patients with acute hepatitis and known chronic infection were tested for HCV RNA, HCV genotyping, and NS3, NS5A, and NS5B HCV genomic region sequencing. To identify transmission clusters, phylogenetic trees were built for each gene employing Bayesian methods. Findings: All patients with acute hepatitis were infected with HCV genotype 1b. Root-cause analysis, including a lookback procedure, excluded blood donors as the source of HCV transmission. The phylogenetic analysis, conducted on seven patients with acute infection and eight patients with chronic infection, highlighted four transmission clusters including at least one patient with chronic and one patient with acute HCV infection. All patients in the same cluster received a blood transfusion during the same day. Two patients with acute hepatitis spontaneously cleared HCV within four weeks and nine patients received ledipasvir plus sofosbuvir for six weeks, all achieving a sustained virological response. Conclusion: Combined use of root-cause analysis and molecular epidemiology was effective in ascertaining the origin of the HCV outbreak. Antiviral therapy avoided the chronic progression of the infection and further spread in care units and in the family environment.

Radionuclides for radiopharmaceuticals can be produced in cyclotrons or nuclear reactors. Each of these production modes has serious issues, such as high target costs, production of long-lived wastes and contaminants, expensive separation. For this reason, new methods are under consideration for the production of highly pure radionuclides. The ISOL (Isotope Separation On-Line) method is the major technique for the production of radioactive ion beams for nuclear physics applications. The SPES-ISOLPHARM project at INFN-LNL (Istituto Nazionale di Fisica Nucleare-Laboratori Nazionali di Legnaro) is a feasibility study for the production of medical isotopes exploiting the ISOL method. The ongoing activities concerning a recent experiment focused on 111Ag, a study performed in collaboration with Padova and Trento Universities, is presented.

Purpose: To assess the accuracy of liver stiffness quantification in patients with non-alcoholic steatohepatitis (NASH) using shear wave elastography (SWE) in comparison with transient elastography (TE). Methods and Materials: This is a prospective study performed in a single institution, including 49 patients with histological diagnosis of NASH. The stiffness of the right liver lobe was measured on the same day with two techniques: TE (FibroScan, Echosens), and SWE (RS80A ultrasound system, Samsung Medison). In the SWE evaluation, 11 patients were excluded due to more than 75% measurements fails. Receiver operating curves (ROC), areas under the ROC (AUROC) and 95% confidence intervals (CI) were calculated to assess the accuracy of TE and SWE for the diagnosis of significant fibrosis (F2-F4) and advanced fibrosis (F3-F4). Spearman’s rank coefficient (rho) was used for correlation of TE with SWE. A p

Bovine tuberculosis (bTB) is an important zoonosis, which has been re-emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis-like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU-VNTR profiles. Spoligotype SB0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU-VNTR profiles. The isolation of M. bovis in a farmer underlines the importance of M. bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection.

SummaryBackground The accuracy of available non-invasive tools for staging severe fibrosis in patients with nonalcoholic fatty liver disease (NAFLD) is still limited. Aim To assess the diagnostic performance of paired or serial combination of non-invasive tools in NAFLD patients. Methods We analysed data from 741 patients with a histological diagnosis of NAFLD. The GGT/PLT, APRI, AST/ALT, BARD, FIB-4, and NAFLD Fibrosis Score (NFS) scores were calculated according to published algorithms. Liver stiffness measurement (LSM) was performed by FibroScan. Results LSM, NFS and FIB-4 were the best non-invasive tools for staging F3-F4 fibrosis (AUC 0.863, 0.774, and 0.792, respectively), with LSM having the highest sensitivity (90%), and the highest NPV (94%), and NFS and FIB-4 the highest specificity (97% and 93%, respectively), and the highest PPV (73% and 79%, respectively). The paired combination of LSM or NFS with FIB-4 strongly reduced the likelihood of wrongly classified patients (ranging from 2.7% to 2.6%), at the price of a high uncertainty area (ranging from 54.1% to 58.2%), and of a low overall accuracy (ranging from 43% to 39.1%). The serial combination with the second test used in patients in the grey area of the first test and in those with high LSM values (>9.6 KPa) or low NFS or FIB-4 values (

The field-assisted paper spray (FAPS) - mass spectrometric method has been employed to quantify the imatinib (IMT) plasma levels in treated patients. The quantitative measurements have been performed on the collisionally generated fragment at m/z 394 of the protonated molecules of IMT and deuterated IMT (d3 -IMT), used as internal standard. The FAPS-tandem mass spectrometry (MS/MS) method exhibits some limitations, because of the high number of operative parameters that need to be carefully controlled. For this aim, papers of different geometry, thickness, and porosity were tested. To obtain a more focalized and intense electrical field, a stainless steel needle was mounted axially and placed at 4 kV voltage. The variability observed in the measurements was ascribed either to the inter-individual variability (e.g. the concomitant presence of other compounds such as proteins, lipids, drugs and/or salts in the plasma of different patients) or to the uncontrollable variables in the instrumental set-up (e.g. sample deposition, changes in paper spray conditions). Furthermore, the manual sample deposition and solvent dripping strongly affects the measure reproducibility. Despite this, it is interesting to observe that, once applied in blind on 24 real plasma samples, FAPS-MS/MS led to results analogous to those obtained by the well-consolidated liquid chromatography-MS/MS, even if the mean coefficient of variation % (CV%) values of 20.4% and 2.6% were observed for the two methods, respectively. In conclusion, despite CV values are relatively high, it is worth noting that the FAPS-MS/MS method is much more straightforward, rapid and economical than the liquid chromatography-MS/MS one, and it appears therefore very promising for applications where a high precision is not always a required task, as e.g. in some cases of therapeutic drug monitoring. Copyright © 2017 John Wiley & Sons, Ltd.

The gamma interferon (IFN-γ) assay is widely used to measure cell-mediated immune (CMI) response for the early detection of tuberculosis infection. Processing whole-blood samples for CMI-based diagnostics is time sensitive and usually must occur within 8 h of collection to ensure optimal assay performance. In this study, we developed and tested a modified protocol, in which whole-blood samples from Mycobacterium bovis -infected cattle were diluted 1:1 in RPMI medium containing 0.3% fetal bovine serum (FBS) added or not to recombinant mouse interleukin-7 (rmIL-7) or rmIL-12, alone or in combination, and stored at 4°C. At 3 and 6 days postcollection, the diluted blood samples were adjusted to 10% FBS, dispensed into culture trays, stimulated with a bovine purified protein derivative from M. bovis , and incubated at 37°C in 5% CO 2 in air. Plasma was removed and assayed for an IFN-γ response using bovine IFN-γ enzyme-linked immunosorbent assay (ELISA) (Bovigam). The results were then compared with those obtained from the conventional procedure. The IFN-γ responses of the samples stored up to 6 days postcollection in the supplemented RPMI medium were similar to those observed in the samples processed within 8 h after sampling, indicating that lymphocyte vitality and response were preserved. The addition of rmIL-7 and rmIL-12, alone or in combination, to culture medium can enhance lymphocyte survival and thus extends the time limit within which the IFN-γ assay can be applied as a diagnostic tool in bovine tuberculosis surveillance and eradication.

Background & aims: The effectiveness of direct-acting antivirals (DAAs) against hepatitis C virus (HCV) after successful treatment of early hepatocellular carcinoma (HCC) has been studied extensively. However, the benefit in terms of overall survival (OS) remains to be conclusively demonstrated. The aim of this study was to assess the impact of DAAs on OS, HCC recurrence, and hepatic decompensation. Methods: We enrolled prospectively 163 consecutive patients with HCV-related cirrhosis and at first diagnosis of early Barcelona Clinic Liver Cancer (BCLC) 0/A HCC who had achieved a complete radiologic response after curative resection or ablation, subsequently treated with DAAs. DAA-untreated patients from ITA.LI.CA. cohort (n = 328) served as controls. After propensity-score matching, outcomes of 102 DAA-treated (DAA group) and 102 DAA-untreated patients (No DAA group) were compared. Results: In DAA group, 7/102 patients (6.9%) died, HCC recurred in 28/102 patients (27.5%) and hepatic decompensation occurred in 6/102 patients (5.9%), after a mean follow-up of 21.4 months. OS was significantly higher in DAA group compared with No DAA group (hazard ratio [HR] = 0.39; 95% confidence Interval [CI] = 0.17–0.91, p = 0.03). HCC recurrence was not significantly different between DAA and No DAA groups (HR = 0.70; 95%CI = 0.44–1.13, p = 0.15). A significant reduction in the rate of hepatic decompensation was observed in DAA group compared with No DAA group (HR = 0.32; 95%CI = 0.13-0.84, p = 0.02). In DAA group, sustained virologic response was a significant predictor of overall survival (HR 0.02, 95%CI 0.00–0.19, p < 0.001), HCC recurrence (HR 0.25, 95%CI 0.11–0.57, p < 0.001) and hepatic decompensation (HR 0.12, 95%CI 0.02-0.38, p = 0.02). Conclusions: In patients with HCV-related cirrhosis and previous successful treatment of early HCC, DAAs significantly improved OS compared with No DAA treatment.

Ocular squamous cell carcinoma (OSCC) in animals is a primary tumor of epithelial cells that may occur in different ocular and periocular tissues, especially the epithelial surfaces of the conjunctiva, corneoscleral junction, nictitating membrane, cornea and eyelid skin. This report describes histological and immunohistochemical features of four different clinical cases classified as OSCC in Valle del Belice sheep. Importance of solar radiation in the etiology of OSCC is discussed, together with other potential risk factors.

Summary Background The PNPLA3/Adiponutrin rs738409 C/G single nucleotide polymorphism is associated with the severity of steatosis, steatohepatitis and fibrosis in patients with non-alcoholic fatty liver disease, as well as the severity of steatosis and fibrosis in patients with chronic hepatitis C (CHC). Aim To test in genotype 1(G1)-CHC patients, the putative association between the PNPLA3 variant and histological features of steatohepatitis, as well as their impact on the severity of fibrosis. Methods Four hundred and thirty-four consecutively biopsied Caucasian G1-CHC patients were genotyped for PNPLA3 rs738409, its effect evaluated by using an additive model. Histological features of steatohepatitis in CHC were assessed using the Bedossa classification. Hepatic expression of PNPLA3 mRNA was evaluated in 63 patients. Results The prevalence of steatohepatitis increased from 16.5% in patients with PNPLA3 CC, to 23.2% in CG and 29.2% in the GG genotype (P = 0.02). By multiple logistic regression, PNPLA3 genotype (OR 1.54, 95% CI 1.03–2.30, P = 0.03), together with age (OR 1.03, 95% CI 1.00–1.05, P = 0.02), BMI ≥ 30 (OR 2.06, 95% CI 1.04–4.10, P = 0.03) and homoeostasis model assessment (HOMA, OR 1.18, 95% CI 1.04–1.32, P = 0.006) were independently linked to steatohepatitis. When stratifying for obesity, PNPLA3 was associated with NASH in non-obese patients only (12.0% in CC vs. 18.3% in CG vs. 27.3% in GG, P = 0.01), including after correction for metabolic confounders (OR 2.06, 95% CI 1.26–3.36, P = 0.004). We showed an independent association between steatohepatitis (OR 2.05, 95% CI 1.05–4.02, P = 0.003) and severe fibrosis. Higher liver PNPLA3 mRNA was associated both with the severity of steatosis (adjusted P = 0.03) and steatohepatitis after adjusting for gender, age, BMI and HOMA (P = 0.002). Conclusion In patients with genotype 1 hepatitis C, the PNPLA3 G variant is associated with a higher risk of steatosis severity and steatohepatitis, particularly among non-obese subjects.

Background and aims: Use of direct acting antivirals (DAAs) has allowed to clear HCV in almost all patients even in the presence of advanced cirrhosis. Although it has been suggested that cirrhotic portal hypertension may regress after SVR, the ultimate effect of HCV clearance on the development and progression of oesophagogastric varices (OV) is still unexplored. We assessed in a prospective cohort of patients with cirrhosis the evolution of endoscopic features of portal hypertension induced by SVR obtained with DAAs. Method: 321 consecutive patients (mean age: 65.1 ± 10.5, males: 58%) with HCV Child-Pugh A cirrhosis treated with DAAs were enrolled between January 2015 and May 2016. All patients underwent esophagogastroscopy (EGS), liver ultrasound (US), liver stiffness measurement (LSM) by Transient Elastography and laboratory tests before the starting DAAs and after achieving SVR. LS * spleen diameter/platelet ratio (LSPS) was calculated as previously described [1]. Results: Forty-one patients were excluded from the analysis, 32(10%) since they had F2/F3 OV at baseline, and 9(2.8%) since they did not achieve SVR. Overall, 280 SVR patients were analysed. At baseline, 100 patients (35.7%) did not have OV and 180(64.3%) had small OV. None received beta-blockers. After a median time of 24.6 months EGS showed de novo development of OV in 24/100(24%) patients and progression from F1 to F2/F3 OV in 30/180 patients (16.7%), p = 0.68 by Kaplan–Meier. By Cox regression analysis, LSPS as continuum variable (HR: 1.06, CI95%: 1.01–1.11, p = 0.025) or at a cut off ≥ 3(HR: 3.16, CI95%: 1.64–6.09, p = 0.001) was associated with OV progression. Age (p = 0.15), gender (p = 0.93) and BMI (p = 0.07) did not correlate with progression of OV. Conclusion: Progression of clinically significant portal hypertension, as assessed by the evolution of oesophagogastric varices, is not uncommon among patients with HCV cirrhosis after HCV clearance. Non-invasive evaluation using combined data of LS, spleen diameter, and platelet count can assist in identifying patients in whom portal hypertension is likely to progress notwithstanding SVR.

Background and aims: Large scale, real life data on the long term course of liver disease after HCV clearance obtained with DAAs are still scanty, and the separate effects on hepatic and non-hepatic causes of death still unclear. Method: We evaluated 4147 patients (mean age: 65.7 ± 11.5 years, 57.6% males) included in the prospective RESIST-HCV cohort who started DAAs treatment in 22 centres between March 2015 and April 2017. All patients were follow after SVR to register liver-related and unrelated outcomes. The primary endpoint was the evaluation of survival since starting DAAs. Cox regression analysis was used to assess the predictors of liver-related and unrelated death. Results: Patients were observed for a median of 50 weeks (range: 1–199), 934 (22.5%) had diagnosis of chronic hepatitis (F3 in >90%), 2851 (68.7%) had Child A cirrhosis and 362 (8.7%) had Child B cirrhosis. Overall, 3766 patients (90.8%) achieved SVR while 381 patients (9.2%) were HCV-RNA positive at the last control. Fifty-five patients (1.3%) died during the observation: 25 of them died for liver related causes and 30 for unrelated causes (16: cardiovascular disease, 6: sepsis, 8: other). The lack of SVR was associated with an increased incidence of overall mortality in comparison to patients with SVR (hazard ratio [HR]; 28.9; 95% confidence interval [CI]: 16.5–50.8; p < 0.001) and death from liver-related and unrelated causes (HR: 18.5, 95%CI: 8.2–41.3; p < 0.001 and HR: 45.5; 95%CI: 19.3–107.4; p < 0.001 respectively). By multivariate Cox regression analysis lack of SVR (HR: 14.9, 95%CI: 6.3–35.1; p < 0.001) and Child B cirrhosis (HR: 29.4, 95%CI: 3.8–223.9; p < 0.001) were independently related with liver mortality. Independent predictors of liver-unrelated mortality were no SVR (HR: 41.77, 95%CI: 17.30–100.87; p < 0.001), Child B cirrhosis (HR: 3.00, 95%CI: 1.36–6.22; p = 0.006), BMI (HR: 0.89, 95%CI: 0.81–0.98, p = 0.023) and diabetes (HR: 2.38, 95%CI: 1.13–5.00, p = 0.022). Conclusion: In this real world setting using a variety of DAA regimens SVR reduced overall mortality and risk of liver-related and unrelated deaths at all stages of disease, nut mostly in Child A cirrhosis. The effect on cardiovascular deaths, which is evident also in the pre-cirrhotic stages deserves further follow up and investigation.

Background & Aims: Studies have produced conflicting results of the incidence of hepatocellular carcinoma (HCC) in patients with hepatitis C virus–associated cirrhosis treated with direct-acting antivirals (DAAs). Data from clinics are needed to accurately assess the occurrence rate of HCC in patients with cirrhosis in the real world. Methods: We collected data from a large prospective study of 2,249 consecutive patients (mean age = 65.4 years, 56.9% male) with hepatitis C virus–associated cirrhosis (90.5% with Child-Pugh class A and 9.5% with Child-Pugh class B) treated with DAAs from March 2015 through July 2016 at 22 academic and community liver centers in Sicily, Italy. HCC occurrence was evaluated by Kaplan-Meier curves. Cox regression analysis was used to identify variables associated with HCC development. Results: A sustained virologic response (SVR) was achieved by 2,140 patients (total = 95.2%; 95.9% with Child Pugh class A and 88.3% with Child Pugh class B; P

Anecdotal reports suggest that patients with chronic hepatitis C virus (HCV) hepatitis and overt or occult hepatitis B virus (HBV) coinfection may reactivate HBV when HCV is suppressed or cleared by direct-acting antivirals (DAAs). We assessed the prevalence of overt or previous HBV coinfection and the risk of HBV reactivation in patients with HCV cirrhosis treated with DAAs. This was a retrospective cohort of 104 consecutive patients with HCV cirrhosis treated with DAAs. Serum HCV-RNA and HBV-DNA were tested at weeks 4, 8 and 12 of DAAs therapy and at week 12 of follow-up. At the start of DAAs, eight patients (7.7%) were HBsAg positive/HBeAg negative with undetectable HBV-DNA and low levels of quantitative HBsAg (four on nucleos(t)ide analogues [NUCs] and four inactive carriers), 37 patients (35.6%) had markers of previous HBV infection (25 anti-HBc positive, 12 anti-HBc/anti-HBs positive) and 59 (56.7%) had no evidence of HBV infection. Sixty-seven patients (64.4%) were HCV-RNA negative at week 4 and 98 (94.2%) achieved sustained virological response. All four HBsAg-positive patients treated with NUCs remained HBV-DNA negative, but three of four untreated patients showed an increase in HBV-DNA of 2-3 log without a biochemical flare and achieved HBV-DNA suppression when given NUCs. During or after DAAs, by conventional assay, HBV-DNA remained not detectable in all 37 anti-HBc-positive patients but in three of them (8.1%) HBV-DNA became detectable with a highly sensitive PCR. HBV reactivation is likely to occur in untreated HBV/HCV-coinfected cirrhotic patients when they undergo HCV treatment with DAAs. Pre-emptive therapy with NUCs should be considered in this setting. Anti-HBc-positive patients rarely reactivate HBV without clinical or virological outcomes.