Daniele Di Paolo

HCV Genotypes Are Differently Prone to the Development of Resistance to Linear and Macrocyclic Protease Inhibitors

Background Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed whether specific HCV-genotypes are differently prone to develop resistance to linear and macrocyclic protease-inhibitors (PIs). Methods The study includes 1568 NS3-protease sequences, isolated from PI-naive patients infected with HCV-genotypes 1a (Nu200a=u200a621), 1b (Nu200a=u200a474), 2 (Nu200a=u200a72), 3 (Nu200a=u200a268), 4 (Nu200a=u200a54) 5 (Nu200a=u200a6), and 6 (Nu200a=u200a73). Genetic-barrier was calculated as the sum of nucleotide-transitions (scoreu200a=u200a1) and/or nucleotide-transversions (scoreu200a=u200a2.5) required for drug-resistance-mutations emergence. Forty-three mutations associated with PIs-resistance were analyzed (36A/M/L/G-41R-43S/V-54A/S/V-55A-Q80K/R/L/H/G-109K-138T-155K/Q/T/I/M/S/G/L-156T/V/G/S-158I-168A/H/T/V/E/I/G/N/Y-170A/T-175L). Structural analyses on NS3-protease and on putative RNA-models have been also performed. Results Overall, NS3-protease was moderately conserved, with 85/181 (47.0%) amino-acids showing <1% variability. The catalytic-triad (H57-D81-S139) and 6/13 resistance-associated positions (Q41-F43-R109-R155-A156-V158) were fully conserved (variability <1%). Structural-analysis highlighted that most of the NS3-residues involved in drug-stabilization were highly conserved, while 7 PI-resistance residues, together with selected residues located in proximity of the PI-binding pocket, were highly variable among HCV-genotypes. Four resistance-mutations (80K/G-36L-175L) were found as natural polymorphisms in selected genotypes (80K present in 41.6% HCV-1a, 100% of HCV-5 and 20.6% HCV-6; 80G present in 94.4% HCV-2; 36L present in 100% HCV-3-5 and >94% HCV-2-4; 175L present in 100% HCV-1a-3-5 and >97% HCV-2-4). Furthermore, HCV-3 specifically showed non-conservative polymorphisms (R123T-D168Q) at two drug-interacting positions. Regardless of HCV-genotype, 13 PIs resistance-mutations were associated with low genetic-barrier, requiring only 1 nucleotide-substitution (41R-43S/V-54A-55A-80R-156V/T: scoreu200a=u200a1; 54S-138T-156S/G-168E/H: scoreu200a=u200a2.5). By contrast, by using HCV-1b as reference genotype, nucleotide-heterogeneity led to a lower genetic-barrier for the development of some drug-resistance-mutations in HCV-1a (36M-155G/I/K/M/S/T-170T), HCV-2 (36M-80K-155G/I/K/S/T-170T), HCV-3 (155G/I/K/M/S/T-170T), HCV-4-6 (155I/S/L), and HCV-5 (80G-155G/I/K/M/S/T). Conclusions The high degree of HCV genetic variability makes HCV-genotypes, and even subtypes, differently prone to the development of PIs resistance-mutations. Overall, this can account for different responsiveness of HCV-genotypes to PIs, with important clinical implications in tailoring individualized and appropriate regimens.

Safety of complete and sustained prophylaxis withdrawal in patients liver-transplanted for HBV-related cirrhosis at low risk of HBV recurrence

BACKGROUND & AIMSnHBV reactivation after liver transplantation may be related to persistence of covalently closed circular (ccc) DNA. We investigated the safety of HBV prophylaxis withdrawal in selected HBV transplanted patients.nnnMETHODSnThirty patients transplanted 64-195months earlier (23 males, median age 56yrs), HBsAg-positive, HBeAg, and HBV-DNA negative at transplant (43% HCV/HDV co-infected), with undetectable intrahepatic total and ccc-DNA were enrolled. All patients underwent HBIg withdrawal and continued lamivudine with monthly HBsAg and HBV-DNA monitoring and sequential liver biopsies. Those with confirmed intrahepatic total and ccc-DNA undetectability 24weeks after stopping HBIg, also underwent lamivudine withdrawal and were followed-up without prophylaxis.nnnRESULTSnTwenty-five patients did not exhibit signs of HBV recurrence after prophylaxis withdrawal (median follow-up 28.7months, range 22-42). Five patients became HBsAg-positive: one early after HBIg withdrawal, the other four after HBIG and lamivudine withdrawal. None of these patients experienced clinically relevant events. In the first patient, HBIg were reinstituted with prompt HBsAg negativization. Of the other four, one remained HBsAg-positive with detectable HBV-DNA and mild ALT elevation and was successfully treated with tenofovir. In the remaining three, HBsAg positivity was transient and followed by anti-HBs seroconversion, thus no antiviral treatment was needed.nnnCONCLUSIONSnPatients with undetectable HBV viremia at transplant and no evidence of intrahepatic total and cccDNA may safely undergo cautious weaning of prophylaxis, showing low rate of HBV recurrence after a 2 year follow-up. Undetectability of intrahepatic ccc-DNA may help to identify patients at low-risk of recurrence, yet studies with longer follow-up are needed.

Low-dose hepatitis B immunoglobulin given "on demand" in combination with lamivudine: a highly cost-effective approach to prevent recurrent hepatitis B virus infection in the long-term follow-up after liver transplantation

Background. Cost of long-term prophylaxis with high-dose human hepatitis B immune globulin (HBIg) after liver transplantation is extremely high. The aim of the present study was to assess consumption rates of high (5,000 IU) and low (2,000 IU) doses of HBIg given intravenously “on demand,” and determine their cost-effectiveness compared with conventional fixed monthly schedules. Methods. The study included 11 male patients (mean age 53 years) who received transplants for hepatitis B virus (HBV)-related cirrhosis 29 to 96 months earlier, all receiving lamivudine (100 mg/day) prophylaxis. Each patient received three consecutive intravenous infusions of 5,000 IU HBIg, followed by three 2,000 IU infusions. HBIg consumption was assessed by serial measurement of serum hepatitis B surface antibody (HBsAb) titer at 2-week intervals. HBIg was readministered only when HBsAb titers dropped below 70 IU/L (i.e., “on demand”). Results. Mean HBsAb peak titers after high and low HBIg doses were 1,641±385 and 848±216 IU/L, respectively (P <0.0001). Mean time to reach an HBsAb titer less than 70 IU/L was 79.5±38.2 days versus 61.6±32.1 days, respectively (P =NS). Interindividual variation coefficients were 23±18% and 32±26% (5,000 IU and 2,000 IU, respectively). Using the on demand approach, maintenance of a protective anti-HBs titer required an average number of 4.0 (5,000 IU) and 5.6 (2,000 IU) HBIg administrations per year, respectively (P =NS). Conclusions. Individual HBIg consumption profiles are highly variable. A low-dose (2,000 IU) on demand HBIg administration schedule is highly cost-effective and provides more than 50% savings compared with conventional high-dose monthly schedules.

One‐year vaccination against hepatitis B virus with a MPL‐vaccine in liver transplant patients for HBV‐related cirrhosis

Conflicting results have been reported on vaccination against hepatitis B virus (HBV) as a prophylaxis against viral recurrence after liver transplantation. We investigated the efficacy of 1‐year, monthly vaccination using an adjuvant 3‐deacylated monophosphoryl‐lipid‐A (MPL) recombinant S vaccine initially administered together with hepatitis B immunoglobulins (HBIg) in 18 patients transplanted for HBV‐related cirrhosis. All received 12 vaccine doses (HBsAg, 20 mcg plus MPL, 50 mcg): the initial six doses (phase I) were administered within 7u2003days after intravenous HBIg (2000u2003IU), while the last 6 (phase II) following HBIg withdrawal. All patients received lamivudine during the study. Anti‐HBs titers were determined before each dose and then for 1u2003year after vaccination. After phase I anti‐HBs titers were greater than 100u2003IU/l in all patients and in three (16.6%) were greater than 500u2003IU/l. After phase II 10 patients (55.5%) achieved anti‐HBs titers greater than 100u2003IU/l and five (27.7%) greater than 500u2003IU/l. One year after vaccination eight patients (44.4%) maintained anti‐HBs titers greater than 100u2003IU/l, with a median titer of 234u2003IU/l (102–1205), and 2 (11.1%) greater than 500u2003IU/l. One‐year extended monthly vaccination with a MPL‐adjuvant recombinant vaccine induces a sustained protective anti‐HBs response in approximately half of transplant recipients.

Total and covalently closed circular DNA detection in liver tissue of long-term survivors transplanted for HBV-related cirrhosis

BACKGROUNDnLife-long prophylaxis against HBV recurrence is recommended in patients transplanted for HBV-related disease. The risk of HBV reactivation is due to persistence of covalently closed circular (ccc) DNA in hepatocytes. Whether cccDNA persists in livers of long-term transplant survivors who received conventional prophylaxis is unknown.nnnAIMnTo investigate the presence of intrahepatic total and cccDNA in transplanted patients with no evidence of biochemical markers of HBV recurrence.nnnMETHODSnIntrahepatic total and cccDNA were assessed using sensitive nested and real-time PCR from 44 HBsAg-positive patients (75% male; mean age 55.2+/-8.9 years) who had undetectable serum HBV-DNA at transplant. The mean follow-up after transplant was 88.3 months (range, 18-159).nnnRESULTSnOne patient underwent HBV recurrence after transplant and was the only who tested positive for both intrahepatic total HBV-DNA and cccDNA. Of the 43 patients negative for all serological markers of HBV infection, only 2 tested positive for intrahepatic total HBV-DNA, but none for cccDNA.nnnCONCLUSIONSnMost patients with undetectable HBV-DNA at transplant, who received conventional HBV prophylaxis, have no evidence of intrahepatic total HBV-DNA and cccDNA. cccDNA should be considered a new additional diagnostic tool, also to identify patients at low risk of HBV recurrence after liver transplantation.

Protective role of tauroursodeoxycholate during harvesting and cold storage of human liver: a pilot study in transplant recipients.

Background. Ischemia-reperfusion injury is a major cause of early graft dysfunction after liver transplantation. Tauroursodeoxycholic acid (TUDCA), a natural amidated hydrophilic bile salt, protects from cholestasis and hepatocellular damage in a variety of experimental models, as well as from ischemia-reperfusion injury. We investigated in the human liver transplantation setting the effect of the addition of TUDCA at time of liver harvesting and cold storage on the intra- and postoperative enzyme release and liver histopathology at the end of cold storage, at reperfusion, and 7 days after transplantation. Methods. Eighteen patients undergoing elective liver transplantation were studied, including 6 serving as controls. In six patients, TUDCA was added to the University of Wisconsin solution used during harvesting and cold storage, to reach final concentrations of 2 mM. In three of these patients, TUDCA (3 g) was infused in the portal vein of the donor before organ explantation; in the other three cases, TUDCA was given through both routes. Results. The use of TUDCA did not cause adverse events. The release of aspartate aminotransferase in the inferior vena cava blood during liver flushing was significantly lower (P =0.05) in TUDCA-treated than in control grafts, as were cytolytic enzyme levels in peripheral blood during the first postoperative week (P <0.02). At electron microscopy, an overt endothelial damage (cytoplasmic vacuolization, cell leakage, and destruction with exposure of hepatocytes to the sinusoidal lumen) was invariably found in control grafts, both at reperfusion and at day 7 after transplant. These features were significantly ameliorated by TUDCA (P <0.001). Several ultrastructural cytoplasmic abnormalities of hepatocytes were seen. Among these, damage to mitochondria matrix and crystae was significantly reduced in TUDCA-treated versus control grafts (P <0.01). Mild to severe damage of bile canaliculi was a constant feature in control biopsies, with dilatation of canalicular lumen and loss of microvilli. Both these abnormalities were markedly ameliorated (P <0.001 by TUDCA). The best preservation was observed when TUDCA was given through both routes. Conclusions. The use of TUDCA during harvesting and cold storage of human liver is associated with significant protection from ischemia-reperfusion injury. The clinical significance of this findings must be studied.

Plasma/erythrocyte ribavirin x100 ratio as an indicator of sustained virological response in HCV genotype 1 patients with early virological response

BACKGROUNDnOn-treatment predictors during antiviral therapy of HCV are useful because they allow discontinuation of an unnecessary treatment in non-responders. Our aim was to evaluate the usefulness of plasma and erythrocyte ribavirin levels in predicting sustained virological response (SVR) in HCV genotype 1 patients undergoing antiviral treatment.nnnMETHODSnA total of 40 HCV genotype 1 patients treated with pegylated interferon-alpha2a 180 microg weekly plus ribavirin 1,000 or 1,200 mg daily (according to body weight) were included in the study. Plasma and erythrocyte ribavirin levels were evaluated in all patients at week 12 by HPLC. At week 24, ribavirin levels were reassessed in those achieving early virological response (EVR).nnnRESULTSnA total of 27 patients achieved EVR, whereas 17 achieved SVR. There was no difference among EVR and non-EVR patients in terms of plasma and erythrocyte ribavirin concentrations at week 12. At week 24, EVR patients obtaining SVR exhibited higher mean +/-sd levels of ribavirin in plasma and lower levels in erythrocytes compared with non-SVR patients (in plasma 12.8 +/-10 versus 5.8 +/-4 microM [P<0.02] and in erythrocytes 1,053 +/-504 versus 1,613 +/-589 microM [P<0.03]). When the plasma ribavirin/erythrocyte ribavirin x100 ratio was compared, the difference was enhanced (1.5 +/-1.3 versus 0.4 +/-0.3 microM; P<0.01). Receiver operating characteristic curve analysis identified a cutoff for plasma ribavirin/erythrocyte ribavirin x100 ratio in predicting SVR of 0.71 with a negative predictive value of 0.8 and a positive predictive value of 0.9, whereas those related to EVR were 1 and 0.6, respectively.nnnCONCLUSIONSnPlasma ribavirin/erythrocyte ribavirin x100 ratio at week 24 seems to be a good indicator of SVR in HCV genotype 1 patients achieving EVR.

Patterns of chronic hepatitis B in Central Italy: a cross-sectional study.

We investigated the patterns of chronic hepatitis B virus (HBV)-related disease in a large cohort of HBsAg-positive patients, in Central Italy, by collecting a screening form with demographic, clinical and laboratory data. Overall, 737 HBsAg-positive cases were included (70% male; median age 52 years): 30% were inactive HBsAg carriers, 51% had chronic hepatitis B (CHB) and 19% had HBV-related cirrhosis. Patients from non-European Union (EU) countries (n = 65) were significantly younger, had a higher prevalence of HBeAg-positive infection and hepatitis delta virus (HDV) co-infection than patients of Italian origin. Therefore, as immigration from non-EU countries continues to grow, we can expect a change in the landscape of HBV-related disease in our area.

Treatment of hepatitis C recurrence is less successful in female than in male liver transplant recipients

It has been recently suggested that the risk of graft loss after liver transplantation (LT) may increase in female HCV patients. The aim of the study was to examine gender differences in HCV therapy tolerance and outcome in LT patients treated for HCV recurrence. A retrospective study was conducted on liver recipients with HCV recurrence, who were given antiviral therapy from 2001 to 2009 in 12 transplant centers in Italy. Sustained virological response (SVR), adherence‐to‐therapy, and side effects were evaluated. A multivariate logistic regression model was used after adjusting for possible confounders. The data regarding 342 treated patients were analyzed. SVR was reported in 38.8% of patients. At baseline, male and female did not differ in HCV viral load, histology, or rate of diabetes. SVR was lower in females than in males (29.5% vs. 42.1%; Pu2003=u20030.03). Adherence‐to‐therapy was also lower in females than in males 43.4% vs. 23.8%; Pu2003=u20030.001); anemia was the main reason for lower adherence. In a multivariate analysis in patients Genotypeu20031, female gender (Pu2003<u20030.04), early virological response (Pu2003<u20030.0001), and adherence to therapy (Pu2003<u20030.0001) were independent predictors for SVR. In conclusion, female gender represents an independent negative prognostic factor for the outcome of HCV antiviral therapy after LT.

Liver transplantation for the treatment of nodular regenerative hyperplasia

BACKGROUNDnNodular regenerative hyperplasia (NRH) is the leading cause of non-cirrhotic portal hypertension in Western countries. Although some patients are successfully managed medically or with shunting procedures, others require liver transplantation. The aim of this review was to assess the overall results obtained with liver transplantation and to better define its role in this setting.nnnMETHODSnSystematic review of all published studies on liver transplantation for NRH without language restrictions, in Medline, Embase and Cochrane Library databases through March 2010.nnnRESULTSn17 studies including a total of 73 patients were identified; 47 (64.3%) were excluded due to lacking inclusion criteria or clinical data and 26 (35.7%) were analysed. Before liver transplantation, the most frequent clinical presentation was gastroesophageal bleeding (65.3%) followed by ascites (61.5%), hepatic encephalopathy (30.7%) and liver failure (11.5%). The mean follow-up reported after liver transplantation was 30.6±27.6 months and patient and graft survival rate was 78.3%. Only one case reported a NRH recurrence 7 years after liver transplantation (LT).nnnCONCLUSIONSnAlthough there are no hard data supporting the role of liver transplantation in symptomatic NRH, onset of severe portal hypertension in this setting may represent a valid indication.