The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Hepatitis B reactivation characterized by HBsAg negativity and anti-HbsAg antibodies persistence in haematopoietic stem cell transplanted patient after lamivudine withdrawal.

BACKGROUND: HBV reactivation is associated with high mortality rates in hematopoietic stem cell transplantation (HSCT) and prophylactic lamivudine (LMV) treatment is suggested to prevent this phenomenon. However, the duration of LMV treatment in HSCT patients is not fully defined and the time of immune recovery is considered the best parameter for a drug to be safely interrupted. In patients undergoing allogeneic HSCT, the time of immune recovery is not easy to define and may take years after transplantation and prolonged LMV treatments, which can lead to drug-resistant viral strains. CASE PRESENTATION: An anti-HBc-positive hematological patient who was undergoing prolonged immunosuppression and who experienced HBV reactivation 3 months after the suspension of a prolonged LMV prophylaxis is described. HBV-DNA matching an atypical serological profile characterized by HbsAg negativity and anti-HBs positivity was detected in the patient. The genotypic analysis of the HBV strain identified T127P, F170FL and S204R mutations of HbsAg, which can hinder HBsAg recognition in a diagnostic assay. CONCLUSIONS: HBV reactivation in the HSCT host can be sustained by HBsAg viral variants with characteristics of altered immunogenicity that cannot be detected by usual laboratory tests. This clinical case description suggests the importance of screening for serum HBV-DNA levels in the diagnosis of HBV reactivation and monitoring HBV-DNA after prophylaxis suspension, particularly in HSCT subjects who have undergone prolonged periods of LMV treatment.

Persistent risk of HBV reactivation despite extensive lamivudine prophylaxis in haematopoietic stem cell transplant recipients who are anti-HBc-positive or HBV-negative recipients with an anti-HBc-positive donor.

The overall rate of hepatitis B virus (HBV) reactivation was evaluated in a population of 373 haematological stem cell transplant (HSCT) patients treated with lamivudine (LMV) if they were anti-HBc-positive/HBV-DNA-negative recipients or if they were HBV-negative recipients with an anti-HBc-positive donor. The incidence of HBV reactivation was calculated in two groups of autologous (auto) or allogeneic (allo) HSCT patients who were stratified according to their HBV serostatus. The former group included 57 cases: 10 auto-HSCT and 27 allo-HSCT anti-HBc-positive recipients, two auto-HSCT and three allo-HSCT inactive carriers, and 15 allo-HSCT recipients with an anti-HBc-positive donor. Forty-seven (82.4%) patients in this group received LMV prophylaxis (the median (interquartile range, IQR) of LMV treatment was 30 (20-38) months). The second group consisted of 320 anti-HBc-negative auto-HSCT and allo-HSCT recipients with anti-HBc-negative donors. None of these patients received any prophylaxis. Two patients in the first group and two in the second group experienced reactivation of HBV infection, with an incidence of 3.5% (95% CI 0.4-12.1%) and 0.6% (95% CI 0.1-2.2%), respectively. Only one out of four reactivated patients was LMV-treated. The cumulative probability of HBV reactivation at 6 years from HSCT was 15.8% (95% CI 15.2-16.4%). Three of four viral isolates obtained from the HBV-reactivated patients harboured mutations in the immune-active HBsAg-region. In a HSCT population carefully evaluated for HBV prophylaxis, a risk of HBV reactivation persisted in the group of patients who were not LMV-treated. Only one LMV-treated patient experienced reactivation of HBV with a resistant HBV isolate.

A recent epidemiological cluster of acute hepatitis B genotype F1b infection in a restricted geographical area of Italy.

In this study, by phylogenetic analysis, we identified an epidemiological cluster involving eight individuals diagnosed with acute hepatitis B virus (HBV) infection related to unprotected sexual intercourse in a restricted area of central Italy (time period: 2011-2014). Notably, these patients (six of eight Italians) were infected by subgenotype F1b, which is not commonly found in western countries. Ultra-deep pyrosequencing confirmed a superimposable composition of HBV quasi-species in these patients. Despite the availability of effective vaccination, this study highlights the importance of not underestimating the risk of HBV infection, of continuing to set up surveillance programmes for HBV infection, and of investigating the pathogenetic potential of these atypical genotypes.

Snapshot on drug-resistance rate and profiles in patients with chronic hepatitis B receiving nucleos(t)ide analogues in clinical practice.

While the selection of complex HBV drug-resistance patterns on therapeutic failure can compromise the efficacy of anti-HBV therapies, recent data show that patients failing treatment without drug-resistance have a rate of virological success close to drug-naive patients. The goal of this study is defining, in clinical practice, the burden of drug-resistance mutations in a cohort of patients treated with anti-HBV drugs. Prevalence and patterns of drug-resistance were analyzed by RT-sequencing in 204 patients infected chronically: 148 experiencing virological rebound (defined as an increase in serum HBV-DNA > 20 IU/ml after achieving virological success [HBV-DNA < 20 IU/ml]), and 56 null/partial responders (always detectable serum HBV-DNA [>20 IU/ml] within 48 weeks of therapy). The highest rate of drug-resistance was observed in patients experiencing virological rebound (prevalence, 79.1%). Conversely, almost half (46.4%) null/partial responders have no evidence of drug-resistance. The rate of drug-resistance was higher in patients treated with lamivudine (76.8% [109/142]) and telbivudine (83.3% [5/6]), followed by adefovir (62.5% [15/24]), and entecavir (52.2% [12/23]). Complex mutational patterns characterized by the co-presence of rtM204V/I-rtA181T/V (impairing the efficacy of all anti-HBV drugs) were detected in four patients (2.7%) with virological rebound. Drug-resistance is the main cause of failure to therapy in patients experiencing virological rebound, supporting the need of rapid switch to anti-HBV drugs with higher genetic barrier and potency (entecavir/tenofovir). Conversely, nearly half of null/partial responders shows no evidence of drug-resistance mutations, maintaining high chance of achieving therapeutic success with the same class of drug. In this setting, genotypic resistance may help in selecting patients still carrying wild-type viruses, that may take major benefits from antiviral treatment.

Characterization of drug-resistance mutations in HBV D-genotype chronically infected patients, naïve to antiviral drugs.

Presence of drug-resistance mutations in drug-naïve hepatitis B virus (HBV) infected patients can seriously compromise response to antiviral treatment. Therefore, our study was aimed at defining the prevalence of HBV drug-resistance in a population of 140 patients, all infected with HBV-D-genotype (the most common HBV-genotype in Eastern Europe, Mediterranean countries and Middle East) and naïve to antiviral therapy. HBV reverse-transcriptase (RT) region was sequenced and analyzed for 20 mutations, confirmed by in vitro studies as associated with resistance to nucleos(t)ide HBV-RT inhibitors (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C/G/I-rtM204V/I-rtN236T-rtM250V). Amino acid changes at other six RT positions, potentially associated with resistance, were also analyzed (rtV84M-rtV191I-rtV207L-rtV214A-rtQ215S-rtI233V). Overall, only 2/140 (1.4%) patients carried primary drug-resistance mutations [rtA181V (0.7%), and rtA194T (0.7%)], while 3/140 (2.1%) patients harbored the secondary mutations rtV173L (1.4%) and rtL180M (0.7%). Additionally, five polymorphic mutations, with a suggested role in drug resistance, were detected [rtQ215S (12.8%), rtI233V (4.3%), rtV214A (3.6%), rtV191I (0.7%), rtV207L (0.7%)]. Notably, no YMDD mutations, namely rtM204V/I, were found. Taken together, the rate of important drug resistance mutations in naïve HBV D-genotype infected patients is today very low, and suggests the potential full efficacy of new-generation antiviral drugs used in first line therapy. Whether such low rate can be extrapolated to non HBV-D subtypes, requires a detailed investigation to be performed in a different cohort of patients.

Successful switch to tenofovir after suboptimal response to entecavir in an immunocompromised patient with chronic hepatitis B and without genotypic hepatitis B virus resistance.

We report a case of an immunocompromised patient affected by chronic hepatitis B virus (HBV) with high basal HBV viremia (>8 log(10) IU/ml) who failed an entecavir regimen, despite the absence of primary or secondary drug resistance mutations. The patient achieved sustained virological success (serum HBV DNA <12 IU/ml) when tenofovir was added to the treatment. This case highlights the difficulty in choosing an optimal therapy in such specific conditions and supports the concept of tailoring therapy (including combination regimens) on the basis of the particular conditions of each individual patient.

Performance evaluation of an in-house human immunodeficiency virus type-1 protease-reverse transcriptase genotyping assay in Cameroon.

Most commercial HIV-1 genotyping assays are hampered by high cost in resource-limited settings. Moreover, their performance might be influenced over time by HIV genetic heterogeneity and evolution. An in-house genotyping protocol was developed, and its sequencing performance and reproducibility were compared to that of ViroSeq™. One hundred ninety plasma samples from HIV-1-infected subjects in Cameroon, a resource-limited setting with a high HIV genetic variability, were processed for pol gene sequencing with an in-house protocol, ViroSeq™, or both. Only non-B subtypes were found. The in-house sequencing performance was 98.7% against 92.1% with ViroSeq™. Among 36 sequence pairs obtained using both assays, the overall rate of discordant amino acid positions was negligible (0.24%). With its high sensitivity and reproducibility, as well as its affordable cost (about half of ViroSeq™: 92 euros vs. 217 euros), this in-house assay is a suitable alternative for HIV-1 genotyping in resource-limited and/or in high-genetic-diversity settings.