Carmine Minichini

Occult HBV infection in HCC and cirrhotic tissue of HBsAg-negative patients: a virological and clinical study

Aim To evaluate the virological and clinical characteristics of occult HBV infection (OBI) in 68 consecutive HBsAg-negative patients with biopsy-proven cirrhosis and HCC. Methods HBV DNA was sought and sequenced in plasma, HCC tissue and non-HCC liver tissue by PCRs using primers for HBV core, surface and x regions. OBI was identified by the presence of HBV DNA in at least two different PCRs. Results OBI was detected in HCC tissue of 13 (20%) patients and in non-HCC liver tissue of 3 of these 13. OBI was detected in HCC tissue of 54.5% of 11 anti-HBs- negative/anti-HBc-positive patients, in 29.4% of 17 anti-HBs/anti-HBc-positive and in 5% of 40 anti-HBs/anti-HBc-negative (p < 0.0005). The 13 patients with OBI in HCC tissue more frequently than the 55 without showed Child-B or -C cirrhosis (53.9% vs. 5.5%, p < 0.0001) and BCLC-B or -C stages (46.1% vs. 1.8%, p < 0.0001). The pre-S1, pre-S2 and S region sequences in HCC tissue showed amino acid (AA) substitutions (F19L, P24L, S59F, T131I, Q129H) and deletions (in positions 4,8, 17 and 86) in the S region, AA substitutions (T40S, P124K, L54P, G76A, N222T and I273L) in pre-S1 region and AA substitutions in pre-S2 region (P41H and P66L). In the 3 patients showing OBI also in non-HCC liver tissue the S, pre-S1 and pre-S2 sequencing displayed patterns of mutations different. Conclusions The study showed a significant correlation between OBI and the severity of liver damage, several patterns of mutations in the S, pre-S1 and pre-S2 regions in HCC tissue, some at their first description.

Clinical impact of the hepatitis C virus mutations in the era of directly acting antivirals.

Introduced in 2013–2014, the second‐ and third‐wave directly acting antivirals (DAAs) have strongly enhanced the efficacy and tolerability of anti‐HCV treatment, with a sustained virological response (SVR) in 90–95% of cases treated. The majority of patients who did not achieve an SVR were found to be infected with HCV strains with a reduced susceptibility to these drugs. Indeed, the high error rate of the viral polymerase and a fast virion production (100‐fold higher than the human immunodeficiency virus) result in a mixture of viral genetic populations (quasi‐species) pre‐existing treatment initiation. These mutants occur frequently in the NS5A region, with a moderate frequency in the NS3/4A region and rarely in the NS5B region. Treatment‐induced resistant mutants to NS5A DAAs persist for years after treatment discontinuation, whereas those resistant to the NS3 DAAs have a shorter duration. This review focuses on the type and prevalence of viral strains with a reduced sensitivity to DAAs, their clinical impact and influence on the response to treatment and, consequently, on treatment choice for DAA‐experienced patients. J. Med. Virol. 88:1659–1671, 2016.

Hepatitis B virus infection in undocumented immigrants and refugees in Southern Italy: demographic, virological, and clinical features

BackgroundThe data on hepatitis b virus (HBV) infection in immigrants population are scanty. The porpoise of this study was to define the demographic, virological, and clinical characteristics of subjects infected with HBV chronic infection in a cohort of immigrants living in Naples, Italy.MethodsA screening for HBV infection was offered to 1,331 immigrants, of whom 1,212 (91%) (831 undocumented immigrants and 381 refugees) accepted and were screened for hepatitis B surface antigen (HBsAg) and anti-hepatitis B core antibody (HBc). Those found to be HBsAg positive were further investigated at third-level infectious disease units.ResultsOf the 1,212 immigrants screened, 116 (9.6%) were HBsAg positive, 490 (40.4%) were HBsAg negative/anti-HBc positive, and 606 (50%) were seronegative for both. Moreover, 21 (1.7%) were anti-human immunodeficiency virus positive and 45 (3.7%) were anti-hepatitis C virus positive. The logistic regression analysis showed that male sex (OR: 1.79; 95%CI: 1.28–2.51), Sub-Saharan African origin (OR: 6.18; 95%CI: 3.37–11.36), low level of schooling (OR: 0.96; 95%CI: 0.94–0.99), and minor parenteral risks for acquiring HBV infection (acupuncture, tattoo, piercing, or tribal practices, OR: 1.54; 95%CI: 1.1–2.16) were independently associated with ongoing or past HBV infection. Of the 116 HBsAg-positive immigrants, 90 (77.6%) completed their diagnostic itinerary at a third-level infectious disease unit: 29 (32.2%) were asymptomatic non-viremic HBsAg carriers, 43 (47.8%) were asymptomatic viremic carriers, 14 (15.6%) had chronic hepatitis, and four (4.4%) had liver cirrhosis, with superimposed hepatocellular carcinoma in two.ConclusionsThe data illustrate the demographic, clinical and virological characteristics of HBV infection in immigrants in Italy and indicate the need for Italian healthcare authorities to enhance their support for providing screening, HBV vaccination, treatment, and educational programs for this populations.

Multiple Hepatitis B Virus (HBV) Quasispecies and Immune-Escape Mutations Are Present in HBV Surface Antigen and Reverse Transcriptase of Patients With Acute Hepatitis B

BACKGROUND This study characterizes and defines the clinical value of hepatitis B virus (HBV) quasispecies with reverse transcriptase and HBV surface antigen (HBsAg) heterogeneity in patients with acute HBV infection. METHODS Sixty-two patients with acute HBV infection (44 with genotype D infection and 18 with genotype A infection) were enrolled from 2000 to 2010. Plasma samples obtained at the time of the first examination were analyzed by ultradeep pyrosequencing. The extent of HBsAg amino acid variability was measured by Shannon entropy. RESULTS Median alanine aminotransferase and serum HBV DNA levels were 2544 U/L (interquartile range, 1938-3078 U/L) and 5.88 log10 IU/mL (interquartile range, 4.47-7.37 log10 IU/mL), respectively. Although most patients serologically resolved acute HBV infection, only 54.1% developed antibody to HBsAg (anti-HBs). A viral population with ≥1 immune-escape mutation was found in 53.2% of patients (intrapatient prevalence range, 0.16%-100%). Notably, by Shannon entropy, higher genetic variability at HBsAg amino acid positions 130, 133, and 157 significantly correlated with no production of anti-HBs in individuals infected with genotype D (P < .05). Stop codons were detected in 19.3% of patients (intrapatient prevalence range, 1.6%-47.5%) and occurred at 11 HBsAg amino acid positions, including 172 and 182, which are known to increase the oncogenic potential of HBV.Finally, ≥1 drug resistance mutation was detected in 8.1% of patients (intrapatient prevalence range, 0.11%-47.5% for primary mutations and 10.5%-99.9% for compensatory mutations). CONCLUSIONS Acute HBV infection is characterized by complex array of viral quasispecies with reduced antigenicity/immunogenicity and enhanced oncogenic potential. These viral variants may induce difficult-to-treat HBV forms; favor HBV reactivation upon iatrogenic immunosuppression, even years after infection; and potentially affect the efficacy of the current HBV vaccination strategy.

Cannabinoid Receptor 2-63 QQ Variant Is Associated with Persistently Normal Aminotransferase Serum Levels in Chronic Hepatitis C

Background and Aim To evaluate in anti-HCV-positive patients the clinical impact of the rs35761398 variant of the CNR2 gene leading to the substitution of Gln (Q) of codon 63 of the cannabinoid receptor 2 (CB2) with Arg (R). Patients and Methods 253 consecutive anti-HCV-/HCV-RNA-positive patients were enrolled, of whom 53 were HCV carriers with persistently normal ALT (PNALT group) and 200 had a history of steadily abnormal serum ALT values (abnormal ALT group). All patients were naive for antiviral therapy and were screened for the CNR2 rs35761398 polymorphism by a TaqMan assay. Results Subjects in the PNALT group, compared with those in the abnormal ALT group were older (58.5±12 vs. 50.7±12.4 years, p = 0.001), more frequently female (66% vs. 42%, p = 0.003), with lower body massindex (BMI) (24.5±3.1 vs. 26.6±4.6, p = 0.003), and more frequently with HCV genotype 2 (43.1% vs 17.7%, p = 0.0002) and CB2-63 QQ variant (34% vs. 11%, p = 0.0001). Considering all 253 patients, no difference in the demographic, biochemical, or virological data was observed between patients in the different CB2-63 variants. The logistic regression analysis identified CB2-63 QQ, HCV genotype 2, older age and lower BMI as independent predictors of PNALT (p<0.00001). Discussion The CB2-63 QQ variant in HCV patients was independently associated with the PNALT status.

Insulin resistance and steatosis in HBV-HCV co-infected patients: Role of PNPLA3 polymorphisms and impact on liver fibrosis progression.

AIM To evaluate steatosis, insulin resistance (IR) and patatin-like phospholipase domain-containing 3 (PNPLA3) and their relation to disease progression in hepatitis B and C viruses (HCV-HBV) co-infected patients. METHODS Three hundred and thirty patients with biopsy proven chronic hepatitis were enrolled: 66 had HBV-HCV, 66 HBV and 198 HCV infection. Prevalence of steatosis, IR and PNPLA3 polymorphisms and their relation to anthropometric, biochemical, virological and histological parameters were evaluated. RESULTS Prevalence of steatosis in group HBV-HCV was similar to that in HCV (47.0% vs 49.5%, respectively); group HBV showed the lowest steatosis (33.3%). Group HBV-HCV had a lesser degree of steatosis than HCV (P = 0.016), lower HCV RNA levels (P = 0.025) and lower prevalence and degree of IR (P = 0.01). PNPLA3 polymorphisms were associated with steatosis. Group HBV-HCV showed higher levels of liver fibrosis than group HCV (P = 0.001), but similar to that observed in HBV group. In HBV-HCV group, liver fibrosis was not associated with steatosis, IR or PNPLA3. HBV infection was the independent predictor of advanced liver fibrosis. CONCLUSION HBV-HCV co-infected patients have lower degree of hepatic steatosis, IR and HCV RNA than HCV mono-infected; co-infected patients showed a more rapid liver fibrosis progression that seems to be due to the double infection and/or HBV dominance.

Clinical Findings of HCV Chronic Infection in Undocumented Immigrants and Low-Income Refugees in Three Areas of Southern Italy

INTRODUCTION AND AIM In recent decades, Italy has become a land of immigration from countries suffering a socio-economic crisis. The aim of this study was to perform an organized screening to identify and offer care to immigrants with HCV infection. MATERIAL AND METHODS The screening, performed from 2012 to 2015, involved 1,727 immigrants in the Campania and Apulia regions in southern Italy. RESULTS Screening was accepted by 1,727 (85%) out of 2,032 immigrants interviewed; 70 (4.1%) of the 1,727 were anti-HCV-positive, all unaware of their serological condition, 31 (44.3%) of whom were HCV-RNA-positive and 39 negative. The 31 HCV-RNA-positive immigrants were further investigated at a third-level clinic of infectious diseases. The HCV viral load was 2.6 x 107 ± 7.7 x107 IU/mL, and 35.5% showed HCV-genotype 1a or 1b, 23.8% genotype 2 and 22.6% genotype 3. Two immigrants had liver cirrhosis and, in accordance with the Italian Healthcare Authority guidelines, received an interferon-free regimen and achieved a sustained virological response (SVR); 18 had chronic hepatitis, 6 of whom with a high risk of progression and received interferonbased therapy, with SVR in 4, whereas 12 at low risk were put on a waiting list for future interferon-free treatment, once licensed. The remaining 11 HCV-RNA-positive immigrants were considered HCV inactive chronic carriers and were included in a long-term observational program. CONCLUSION The screening program can be considered successful since it was accepted by 85% of the subjects interviewed and identified 70 anti-HCV-positive immigrants, all unaware of their clinical and virological condition.INTRODUCTION AND AIM In recent decades, Italy has become a land of immigration from countries suffering a socio-economic crisis. The aim of this study was to perform an organized screening to identify and offer care to immigrants with HCV infection. MATERIAL AND METHODS The screening, performed from 2012 to 2015, involved 1,727 immigrants in the Campania and Apulia regions in southern Italy. RESULTS Screening was accepted by 1,727 (85%) out of 2,032 immigrants interviewed; 70 (4.1%) of the 1,727 were anti-HCV-positive, all unaware of their serological condition, 31 (44.3%) of whom were HCV-RNA-positive and 39 negative. The 31 HCV-RNA-positive immigrants were further investigated at a third-level clinic of infectious diseases. The HCV viral load was 2.6 × 107 ± 7.7 ×107 IU/mL, and 35.5% showed HCV-genotype 1a or 1b, 23.8% genotype 2 and 22.6% genotype 3. Two immigrants had liver cirrhosis and, in accordance with the Italian Healthcare Authority guidelines, received an interferon-free regimen and achieved a sustained virological response (SVR); 18 had chronic hepatitis, 6 of whom with a high risk of progression and received interferon-based therapy, with SVR in 4, whereas 12 at low risk were put on a waiting list for future interferon-free treatment, once licensed. The remaining 11 HCV-RNA-positive immigrants were considered HCV inactive chronic carriers and were included in a long-term observational program. CONCLUSION The screening program can be considered successful since it was accepted by 85% of the subjects interviewed and identified 70 anti-HCV-positive immigrants, all unaware of their clinical and virological condition.

Eighteen-month lamivudine prophylaxis on preventing occult hepatitis B virus infection reactivation in patients with haematological malignancies receiving immunosuppression therapy

This study evaluated the long‐term efficacy and safety of an 18‐month lamivudine prophylaxis in 68 HBsAg‐negative/anti–HBc‐positive patients with oncohaematological disease.

Molecular diversity in irregular or refugee immigrant patients with HBV-genotype-E infection living in the metropolitan area of Naples.

In a recent testing in the metropolitan area of Naples, Italy, on 945 irregular immigrants or refugees, 87 HBsAg chronic carriers were identified, 53 of whom were infected by HBV‐genotype E. The aim of the present study was to identify the genetic diversity of HBV‐genotype E in these 53 immigrants. The 53 immigrant patients with HBV‐genotype‐E infection were born in Africa, central or eastern Asia, eastern Europe or Latin America. These patients had been seen for a clinical consultation at one of the four first‐level units from January 2012 to 2013. The first dataset contained 53 HBV‐S gene isolates plus 128 genotype/subgenotype specific reference sequences downloaded from the National Center for Biotechnology Information. The second dataset, comprising the 53 HBV‐S gene isolates, previously classified as HBV‐genotype E, was used to perform the time‐scaled phylogeny reconstruction using a Bayesian approach. Phylogenetic analysis showed that all 53 HBV‐S isolates belonged to HBV‐genotype E. Bayes factor analysis showed that the relaxed clock exponential growth model fitted the data significantly better than the other models. The time‐scaled Bayesian phylogenetic tree of the second dataset showed that the root of the tree dated back to the year 1990 (95% HPD:1984–2000). Four statistically supported clusters were identified. Cluster A dated back to 2012 (95% HPD:1997–2012); cluster B dated back to 2008 (95% HPD:2001–2015); cluster C to 2006 (95% HPD:1999–2013); cluster D to 2004 (95% HPD:1998–2011). This study disclosed the genetic evolution and phylogenesis in a group of HBV‐genotype‐E‐infected immigrants. J. Med. Virol. 89:1015–1024, 2017.

ITPase activity modulates the severity of anaemia in HCV-related cirrhosis treated with ribavirin-containing interferon-free regimens

BACKGROUND To investigate the association between inosine triphosphatase (ITPase) activity and the degree of anaemia occurring during direct-acting antiviral (DAA)/ribavirin (RBV)-based therapy in patients with cirrhosis. METHODS In a multicentre, prospective study 227 patients with HCV-related cirrhosis treated with DAA and RBV were enrolled. All patients were screened for the rs1127354 and rs7270101 ITPA single nucleotide polymorphisms using direct sequencing. RESULTS 150 (66.1%) patients had normal (100%) ITPase activity, 48 (21.1%) had moderate (60%) activity and 29 (12.8%) minimal (≤30%) activity. The ITPase activity significantly influenced the haemoglobin concentration: at day 15 it was -1.248 (sd ±0.978) in the 150 patients with an ITPase activity of 100% and -0.616 (±0.862) in the 77 patients with an ITPase activity less than 100% (P<0.000), and at day 30 it was -1.941 ±1.218 versus -1.11 ±1.218 (P<0.000). The 63 patients with a severe (at least 3/dl) haemoglobin decline, compared to those without, more frequently had an ITPase activity of 100% (82.1% versus 62.8%; P=0.021), were older (mean age ±sd: 66.7 ±8.2 versus 61.4 ±9.7 years; P=0.004) and were treated with a higher ribavirin dose (13.7 ±2.1 versus 12.8 ±2.5 mg/kg/day; P=0.008). At multivariate logistic regression analysis, the ITPase activity of 100% (OR: 2.83; 95% CI: 1.12, 7.10), male gender (OR: 3.22; 95% CI: 1.35, 7.66), body mass index (OR: 1.17; 95% CI: 1.03, 1.34) and dose of ribavirin (OR: 1.22; 95% CI: 1.06, 1.47) were independent predictors of a severe decline in haemoglobin (P<0.0001). CONCLUSIONS This study suggests that the polymorphisms in the ITPA gene influence the severity of anaemia during the first month of a DAA/RBV-based treatment in HCV-related cirrhosis.