A real-world experience of SARS-CoV-2 infection in a tertiary referral centre of Montréal: Unexpected low prevalence and low mortality.

BACKGROUND: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in patients with chronic liver disease (CLD) and liver transplant (LT) recipients remains a concern. The aim of this study was to report the impact of coronavirus disease 2019 (COVID-19) infection among patients at the tertiary health care centre Centre hospitalier de l'Université de Montréal (CHUM) during the first wave of the SARS-CoV-2 pandemic. METHODS: This real-world, retrospective cohort included all patients admitted to our liver unit and/or seen as an outpatient with CLD with or without cirrhosis and/or LT recipients who tested positive to SARS-CoV-2 infection. Cases were considered positive as defined by the detection of SARS-CoV-2 by reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swabs. RESULTS: Between April 1 and July 31, 2020, 5,637 were admitted to our liver unit and/or seen as outpatient. Among them, 42 were positive for SARS-CoV-2. Twenty-two patients had CLD without cirrhosis while 16 patients had cirrhosis at the time of the infection (13, 2, and 1 with Child-Pugh A, B, and C scores, respectively). Four were LT recipients. Overall, 15 of 42 patients (35.7%) were hospitalized; among them, 7 of 42 (16.7%) required respiratory support and 4 of 42 (9.5%) were transferred to the intensive care unit. Only 4 of 42 (9.5%) patients died: 2 with CLD without cirrhosis and 2 with CLD with cirrhosis. Overall survival was 90.5%. CONCLUSION: This real-world study demonstrates an unexpectedly low prevalence and low mortality in the context of SARS-CoV-2 infection among patients with CLD with or without cirrhosis and LT recipients.

Gastric antral vascular ectasia is more frequent in patients with non-alcoholic steatohepatitis-induced cirrhosis.

Background: Gastric antral vascular ectasia (GAVE) is an uncommon cause of occult gastrointestinal (GI) bleeding. Based on clinical observations, we hypothesized that GAVE was more common in patients with non-alcoholic steatohepatitis (NASH) cirrhosis. Methods: We performed this retrospective study at Centre Hospitalier de l'Université de Montréal (CHUM). We included all cirrhotic patients who had undergone an esophagogastroduodenoscopy (EGD) between 2009 and 2011. GAVE was diagnosed based on a typical endoscopic appearance. NASH cirrhosis was diagnosed in patients with a metabolic syndrome after excluding other causes of liver disease. GAVE was considered symptomatic if it required treatment. Results: We included 855 cirrhotic patients in the study. The median age was 58 (range 19-88) years. The etiology of cirrhosis was as follows: NASH in 18% (n = 154), autoimmune diseases in 15.1% (n = 129), hepatitis B virus (HBV) in 6.3% (n = 54), hepatitis C virus (HCV) in 19.4% (n = 166), alcohol in 25.7% (n = 220), alcohol plus HCV in 7.8% (n = 67), cryptogenic in 2.8% (n = 24), and other etiologies in 4.8% (n = 41). GAVE was more frequently observed among patients with NASH cirrhosis than in cirrhosis of other etiologies (29.2% vs. 9.4%, respectively; p < 0.001). In multivariate analysis, NASH was strongly associated with GAVE with an odds ratio (OR) of 3.73 (95% CI 2.36 to 5.90, p < 0.001), and the association was stronger with symptomatic GAVE (OR 5.77, 95% CI 2.93 to 11.38). Conclusions: NASH cirrhosis is a major risk factor for GAVE and symptomatic GAVE.

Type 3 cytokines IL-17A and IL-22 drive TGF-ß-dependent liver fibrosis.

Inflammatory immune cells can modulate activation of hepatic stellate cells (HSCs) and progression of liver fibrosis. Type 3 inflammation characterized by production of interleukin-17A (IL-17) and IL-22 by innate and adaptive immune cells is implicated in many inflammatory conditions of the gut and can be counteracted by regulatory T cells (Tregs), but its contribution to liver fibrosis is still poorly understood. Here, we evaluated the contribution of type 3 inflammation in liver fibrosis using clinical liver biopsies, in vitro stimulation of primary HSCs, and in vivo mouse models. We report dysregulated type 3 responses in fibrotic lesions with increased IL-17+CD4+/FOXP3hiCD4+ ratio and increased IL-17 and IL-22 production in advanced liver fibrosis. Neutrophils and mast cells were the main sources of IL-17 in situ in humans. In addition, we demonstrate a new profibrotic function of IL-22 through enhancement of transforming growth factor-ß signaling in HSCs in a p38 mitogen-activated protein kinase-dependent manner. In vivo, IL-22RA1 knockout mice exhibited reduced fibrosis in response to thioacetamide and carbon tetrachloride. Blocking either IL-22 or IL-17 production using aryl hydrocarbon receptor or RAR-related orphan receptor gamma-t antagonists resulted in reduced fibrosis. Together, these data have identified a pathogenic role for type 3 immune response mediated by IL-22 in driving liver fibrosis during chronic liver injury.

Management of Hepatitis B Virus Infection: 2018 Guidelines from the Canadian Association for the Study of Liver Disease and Association of Medical Microbiology and Infectious Disease Canada.

Hepatitis B virus (HBV) infection is an important public health problem in Canada. In keeping with evolving evidence and understanding of HBV pathogenesis, the Canadian Association for the Study of Liver Disease periodically publishes HBV management guidelines. The goals of the 2018 guidelines are to (1) highlight the public health impact of HBV infection in Canada and the need to improve diagnosis and linkage to care, (2) recommend current best-practice guidelines for treatment of HBV, (3) summarize the key HBV laboratory diagnostic tests, and (4) review evidence on HBV management in special patient populations and include more detail on management of HBV in pediatric populations. An overview of novel HBV tests and therapies for HBV in development is provided to highlight the recent advances in HBV clinical research. The aim and scope of these guidelines are to serve as an up-to-date, comprehensive resource for Canadian health care providers in the management of HBV infection.

Novel E2 Glycoprotein Tetramer Detects Hepatitis C Virus-Specific Memory B Cells.

Acute hepatitis C virus (HCV) infection culminates in viral persistence in the majority of cases. Abs that recognize the envelope glycoproteins E1 and E2 are generated during the late stages of acute infection, yet their contribution to spontaneous viral clearance remains controversial. Investigation of the humoral responses during acute HCV infection have been limited by the inability to directly identify and characterize HCV-specific B cells. In this study we describe the development of a novel tetramer of the E2 glycoprotein ectodomain (J6, genotype 2a strain), which allowed us to visualize E2-specific B cells longitudinally in the peripheral blood of HCV-infected individuals. HCV-specific class-switched memory B cells were detected in 3 out of 7 participants during late acute infection, with a mean frequency of 0.63% for positive samples (range 0.16-0.67%) and in 7 out of 7 participants with chronic infection with a mean frequency of 0.47% (range 0.20-0.78%). In a cross-sectional study, E2 tetramer positive population was detected in 28 out of 31 chronically infected individuals. Deep sequencing of the BCR from E2-specific class-switched memory B cells sorted from two independent participants revealed a focused repertoire suggestive of clonal selection. Tetramer-specific B cells exhibited skewed CDR3 length distribution and increased mutation frequency compared with naive B cells. This BCR profile is indicative of clonal expansion and affinity maturation. E2 tetramer allows for specific and sensitive ex vivo characterization of rare HCV-specific B cells in infected individuals, and will enable researchers to gain a better understanding of humoral immunity in HCV infection.

Transarterial embolization therapies for the treatment of hepatocellular carcinoma: CEPO review and clinical recommendations.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most deadly cancers in the world and its incidence rate has consistently increased over the past 15 years in Canada. Although transarterial embolization therapies are palliative options commonly used for the treatment of HCC, their efficacy is still controversial. The objective of this guideline is to review the efficacy and safety of transarterial embolization therapies for the treatment of HCC and to develop evidence-based recommendations. METHOD: A review of the scientific literature published up to October 2013 was performed. A total of 38 studies were included. RECOMMENDATIONS: Considering the evidence available to date, the CEPO recommends the following: (i) transarterial chemoembolization therapy (TACE) be considered a standard of practice for the palliative treatment of HCC in eligible patients; (ii) drug-eluting beads (DEB)-TACE be considered an alternative and equivalent treatment to conventional TACE in terms of oncological efficacy (overall survival) and incidence of severe toxicities; (iii) the decision to treat with TACE or DEB-TACE be discussed in tumour boards; (iv) bland embolization (TAE) not be considered for the treatment of HCC; (v) radioembolization (TARE) not be considered outside of a clinical trial setting; and (vi) sorafenib combined with TACE not be considered outside of a clinical trial setting.

Association between proton pump inhibitor use and spontaneous bacterial peritonitis in cirrhotic patients with ascites.

BACKGROUND: There are data suggesting a link between proton pump inhibitor (PPI) use and the development of spontaneous bacterial peritonitis (SBP) in cirrhotic patients with ascites; however, these data are controversial. OBJECTIVE: To assess whether the use of PPIs in cirrhotic patients with ascites is associated with an increased risk for SBP. METHODS: A retrospective case-control study (June 2004 to June 2010) was conducted at the Centre Hospitalier de l'Université de Montréal in Montreal, Quebec. Fifty-one cirrhotic patients admitted with paracentesis-proven SBP (≥250 neutrophils/mm3), occurring within seven days of hospital admission, met the inclusion criteria. These patients were matched 1:2 (for age, Child-Pugh class and year of admission) with 102 comparable cirrhotic patients with ascites who were admitted for conditions other than SBP. RESULTS: Patients with SBP had a significantly higher rate of pre-hospital PPI use (60.8%) compared with cirrhotic patients without SBP (42.2%; P=0.03). On multivariate analysis, PPI use was the only factor independently associated with SBP (OR 2.09 [95% CI 1.04 to 4.23]; P=0.04). Thirty-five (35%) patients in both groups had no documented indication for PPI use in their charts. Forty-five percent of the remaining cirrhotic patients with SBP had an inappropriate indication, as defined in the protocol, for PPI use compared with 25% of controls. CONCLUSIONS: Cirrhotic patients with SBP were twice as likely to have taken PPIs than patients without SBP. These findings reinforce the association between PPI use and SBP observed in other studies. A high percentage of cirrhotic patients were taking a PPI without any documented indication.

Organochlorine pesticides in green mussel, Perna viridis, from the Cienfuegos Bay, Cuba.

The green mussel, Perna viridis, was used to measure bioaccumulated levels of organochlorine pesticides in the marine environment of Cuba. Samples were collected in the Cienfuegos Bay between January and December 2010. The organochlorine pesticides (i.e. DDT, Dieldrin, Chlordane, Endosulfan, HCB, Aldrin, Heptachlor and Lindane) were quantified by gas chromatography. The sum of all organochlorine pesticides in P. viridis was 6.31 ng g(-1). The concentration ranged from 3.53 to 4.42 ng g(-1) dry weight (dw) for DDTs (i.e. sum of pp' DDT, pp' DDD, op' DDE and pp' DDE); 1.7-1.9 ng g(-1) dw for Dieldrin; 0.17-0.20 ng g(-1) dw for Chlordanes; 0.14-0.16 ng g(-1) dw for Endosulfan; 0.11-0.17 ng g(-1) dw for HCB; 0.07-0.11 ng g(-1) dw for Aldrin; 0.046-0.054 ng g(-1) dw for Heptachlor and 0.035-0.039 ng g(-1) dw for Lindane. These levels can be considered as low when compared to reported values from similar studies conducted elsewhere in the world. The concentrations of all organochlorines residues detected in this study fell below the EU Maximum Residue Limits.

Management and treatment of hepatitis B virus in patients with HIV infection: A practical guide for health care professionals.

The management and treatment of HIV and hepatitis B virus (HBV)-coinfected patients present specific challenges for clinicians. The morbidity and mortality related to these concomitant infections are growing concerns, while the use of antiviral drugs effective against both viruses complicates therapeutic decision making. The present document provides guidelines for physicians regarding care and treatment of patients coinfected with HIV and HBV. Primary prevention of HBV in HIV-positive patients is achieved through appropriate vaccination schedules. Follow-up before treatment of HBV may include liver biopsy, screening for hepatocellular carcinoma and testing for esophageal varicies in cases of cirrhosis. In HBV-infected patients requiring treatment, recommendations regarding initiation, duration and choice of first-line drugs are made. Finally, in the case of resistance, appropriate alternative therapies are necessary.

Distribution of petroleum hydrocarbons and organochlorinated contaminants in marine biota and coastal sediments from the ROPME Sea Area during 2005.

The composition and spatial distribution of various petroleum hydrocarbons (PHs), comprising both aliphatic and polycyclic aromatic hydrocarbons (PAHs), and selected chlorinated pesticides and PCBs were measured in biota and coastal sediments from seven countries in the Persian Gulf and the Gulf of Oman (Bahrain, Iran, Kuwait, Oman, Qatar, Saudi Arabia and the United Arab Emirates). Evidence of extensive marine contamination with respect to organochlorinated compounds and PHs was not observed. Only one site, namely the BAPCO oil refinery in Bahrain, was considered to be chronically contaminated. Comparison of the results from this survey for Σ DDTs and Σ PCBs in rock oysters from the Gulf of Oman with similar measurements made at the same locations over the past two decades indicates a temporal trend of overall decreasing Σ PCB concentrations in oysters, whereas Σ DDTs levels have little changed during that period.

PCBs in sediments and oysters of Manila Bay, the Philippines.

Polychlorinated biphenyls (PCBs) were analyzed in sediment and oyster samples from coastal sites inside Manila Bay. Concentrations for 13 individual PCB congeners and total PCBs are reported herein. Median SigmaPCB concentration in sediments was 0.46 ng g(-1) dry weight (range 0.10-1.5 ng g(-1)) and in oysters it was 15 ng g(-1) dry weight (range 7-73 ng g(-1)). The most contaminated areas of the bay were the coastal zones of Metropolitan Manila and Bulacan province. A significant correlation (p < 0.01) was found between SigmaPCB concentrations in oysters and in sediments. PCB concentrations measured in the bay sediments were below acute toxic levels to marine biota. Nevertheless, consumption of oysters by human population living around the bay might originate SigmaPCB intake rates estimated at about 2.5 times lower than the tolerance limit. Further environmental surveillance is recommended in order to prevent public health risks that may be posed by these chemicals.

Organic contaminants in the marine environment of Manila Bay, Philippines.

Organochlorine pesticides (OCs) and polychlorobiphenyls (PCBs) were determined in sediments and oysters' soft tissues (Cassostrea gigas) collected in selected sites of Manila Bay. Overall, the concentration levels were very low. In sediments, PCBs were the compounds present in higher concentrations, with Sigma13PCB congeners averaging 0.69 +/- 0.46 ng/g (dry weight), followed by SigmaDDT averaging 0.53 +/- 0.28 ng/g and Sigmachlordane with 0.26 +/- 0.28 ng/g. Concentrations measured in oysters averaged 20 +/- 17 ng/g (dry weight) for Sigma13PCB and were higher than SigmaDDT, with 9.5 +/- 2.4 ng/g, and Sigmachlordane, with 3.8 +/- 3.1 ng/g. No dissolved residues of polar compounds, such as herbicides, and organophosphorous and organochlorine pesticides were found in the bay water. In general, results showed that concentrations of organochlorine pesticide residues, such as DDT, hexachlorocyclohexane, chlordane, lindane, endosulfan, and heptachlor in sediments and oysters were low in comparison with other coastal areas in Asia, although PCB concentrations in oysters were relatively high in some zones of Manila Bay and indicative of loose control of industrial chemical waste discharges into the bay. Nevertheless, current concentrations of persistent organochlorine contaminants in sediments were under threshold effect levels (TELs) and chronic toxic effects are, thus, unlikely to generate impairment of marine biota in Manila Bay.

Ecological risk assessment of PCBs and other organic contaminant residues in Laguna de Terminos, Mexico.

Laguna de Terminos, a wide coastal lagoon system in Campeche, Mexico, was investigated for the contamination by polychlorobiphenyls (PCBs). Distribution of these industrial chemical contaminants along with pesticide residues in the lagoon, as well as their sediment-water partitioning and bioaccumulation by oysters and fish were assessed. Contaminant concentrations in the lagoon were compared with toxicity data for aquatic organisms and the ecotoxicological risks discussed. Current contaminant concentrations generally were several orders of magnitude below acute toxic levels for the most sensitive aquatic species and this seems compatible with the status of nature reserve and functions aimed at Laguna de Terminos. In particular, Penaeidae shrimp species that are the most valuable fisheries resources of Campeche with important populations in the Laguna are not impaired with the current low levels of these contaminants. Nevertheless, due to known environmental persistence, the surveillance of chlorinated contaminant levels in the lagoon ecosystems is recommended.

Pesticide and PCB residues in the aquatic ecosystems of Laguna de Terminos, a protected area of the coast of Campeche, Mexico.

The coastal lagoon system of Laguna de Terminos, Campeche, Mexico, a natural reserve since 1994, was investigated for contamination by agricultural and industrial chemical residues. Water, sediment and biota samples were analyzed for a wide variety of organochlorine and organophosphorus compounds. Chlorpyrifos was detected in water in concentrations up to 72 pgL(-1) and, amongst organochlorine compounds, summation operator PCB were measured averaging 1177 pgL(-1) and summation operator DDT 279 pgL(-1). Residues of chlorinated compounds were present in sediments and in biota with summation operator DDT averaging 190 pg g(-1) and 5876 pg g(-1) in sediment and oysters, respectively. Results show that the more widespread contaminants in the Laguna were residues of chlorinated hydrocarbons, such as DDTs, PCBs, endosulfan, and lindane. Concentrations of residues were not at an alarming level and were even lower than reported for other costal lagoons of the region. Still there is a need to implement control measures on persistent and bioaccumulative compounds that may reach the aquatic system of Laguna de Terminos.

Hepatitis B virus genotype G epidemiology and co-infection with genotype A in Canada.

Hepatitis B virus (HBV) genotype G (HBV/G) is an unusual variant, and little is known about its epidemiology and natural history, particularly the requirement for a co-infecting HBV genotype and their relationship during infection. This study investigated the quasispecies nature of co-infecting genotypes in 39 samples collected over a 6 year period from 13 HBV/G-infected patients. HBV/G infections were found to occur predominantly in males (92 %) and were primarily associated with male homosexual sex (67 %). All patients were infected with HBV/G and HBV/A, or a recombinant HBV/A/G strain. Co-infecting genotypic prevalence was often observed to fluctuate over time, with periods of HBV/G monoinfection in some patients. The average sequence divergence among Canadian HBV/G strains was 1.57+/-0.62 %. Thus, all HBV/G infections in Canada occur in the context of co-infection or recombination with HBV/A, and strains display increased sequence divergence compared with all known HBV/G sequences described to date.

Early interferon therapy for hepatitis C virus infection rescues polyfunctional, long-lived CD8+ memory T cells.

The majority of acute hepatitis C virus (HCV) infections progress to chronicity and progressive liver damage. Alpha interferon (IFN-alpha) antiviral therapy achieves the highest rate of success when IFN-alpha is administered early during the acute phase, but the underlying mechanisms are unknown. We used a panel of major histocompatibility complex class I tetramers to monitor the phenotypic and functional signatures of HCV-specific T cells during acute HCV infection with different infection outcomes and during early IFN therapy. We demonstrate that spontaneous resolution correlates with the early development of polyfunctional (IFN-gamma- and IL-2-producing and CD107a(+)) virus-specific CD8(+) T cells. These polyfunctional T cells are distinguished by the expression of CD127 and Bcl-2 and represent a transitional memory T-cell subset that exhibits the phenotypic and functional signatures of both central and effector memory T cells. In contrast, HCV-specific CD8(+) T cells in acute infections evolving to chronicity expressed low levels of CD127 and Bcl-2, exhibited diminished proliferation and cytokine production, and eventually disappeared from the periphery. Early therapeutic intervention with pegylated IFN-alpha rescued polyfunctional memory T cells expressing high levels of CD127 and Bcl-2. These cells were detectable for up to 1 year following discontinuation of therapy. Our results suggest that the polyfunctionality of HCV-specific T cells can be predictive of the outcome of acute HCV infection and that early therapeutic intervention can reconstitute the pool of long-lived polyfunctional memory T cells.

Adefovir dipivoxil for wait-listed and post-liver transplantation patients with lamivudine-resistant hepatitis B: final long-term results.

Wait-listed (n = 226) or post-liver transplantation (n = 241) chronic hepatitis B (CHB) patients with lamivudine-resistant hepatitis B virus (HBV) were treated with adefovir dipivoxil for a median of 39 and 99 weeks, respectively. Among wait-listed patients, serum HBV DNA levels became undetectable (<1,000 copies/mL) in 59% and 65% at weeks 48 and 96, respectively. After 48 weeks, alanine aminotransferase (ALT), albumin, bilirubin, and prothrombin time normalized in 77%, 76%, 60%, and 84% of wait-listed patients, respectively. Among posttransplantation patients, serum HBV DNA levels became undetectable in 40% and 65% at weeks 48 and 96, respectively. After 48 weeks, ALT, albumin, bilirubin, and prothrombin time normalized in 51%, 81%, 76%, and 56% of posttransplantation patients, respectively. Among wait-listed patients who underwent on-study liver transplantation, protection from graft reinfection over a median of 35 weeks was similar among patients who did (n = 34) or did not (n = 23) receive hepatitis B immunoglobulin (HBIg). Hepatitis B surface antigen was detected on the first measurement only in 6% and 9% of patients who did or did not receive HBIg, respectively. Serum HBV DNA was detected on consecutive visits in 6% and 0% of patients who did or did not receive HBIg, respectively. Treatment-related adverse events led to discontinuation of adefovir dipivoxil in 4% of patients. Cumulative probabilities of resistance were 0%, 2%, and 2% at weeks 48, 96, and 144, respectively. In conclusion, adefovir dipivoxil is effective and safe in wait-listed or posttransplantation CHB patients with lamivudine-resistant HBV and prevents graft reinfection with or without HBIg.

Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient.

BACKGROUND/AIMS: Complex mutants may be selected under sequential anti-VHB pressures. We analyzed the genotypic and phenotypic evolution of the viral quasi-species of a patient who developed resistance to entecavir following lamivudine breakthrough. METHODS: The polymerase gene was amplified, cloned and sequenced at different time points. Hepatoma cell lines were transfected to compare the replication capacity of HBV mutants and their drug susceptibility. RESULTS: A mixture of lamivudine-resistant HBV strains coexisted following viral breakthrough to lamivudine, all harboring the rtM204V mutation. The rtV173L+L180M+M204V dominant mutant displayed strong lamivudine-resistance and the highest replication capacity. Following the switch to entecavir, the viral load dropped but the lamivudine-resistant strains continued to be selected. Three years later, the viral load rose again, and a complex mixture of entecavir-resistant strains, all harboring the lamivudine-resistance signature rtL180M+M204V and the rtS202G mutation were observed. Although the rtL180M+S202G+M204V variant, that prevailed at the end of entecavir therapy, did not show the highest viral genome replication capacity, it conferred one of the strongest resistance levels to entecavir. CONCLUSIONS: We report the selection of complex HBV mutants that escaped lamivudine and entecavir antiviral pressures. Genotypic and phenotypic analysis provided additional information to understand the process of HBV variant selection.

Selection of a multiple drug-resistant hepatitis B virus strain in a liver-transplanted patient.

BACKGROUND & AIMS: Sequential anti-hepatitis B virus (HBV) therapy may lead to the selection of complex mutants. We analyzed the genetic and phenotypic evolution of the viral quasispecies of a patient who received successively lamivudine, add-on adefovir+lamivudine, followed by lamivudine+adefovir+hepatitis B immunoglobulins (HBIg) after orthotopic liver transplantation. METHODS: For genotypic analysis, a 1310-bp region of the polymerase gene was amplified, cloned, and sequenced. Huh-7 cells were transfected to compare the replication fitness of HBV mutants and their susceptibility to drugs. RESULTS: At baseline, all HBV genomes carried a wild-type (wt) RT gene but 22% harbored the sP120S and 55% the sC107stop mutations within the surface (S) gene associated with vaccine escape. Following viral breakthrough to lamivudine monotherapy, a complex mixture of lamivudine-resistant HBV strains prevailed. Interestingly, among these mutants emerged a population harboring only the rtL180M+A181V mutations, conferring lamivudine-resistance in vitro. After addition of adefovir to the ongoing treatment, viral load dropped, and the patient underwent an orthotopic liver transplantation and received HBIg. As viral load rose again, a single viral population was progressively selected, harboring the rtV173L+L180M+A181V+N236T and sP120S mutations. In vitro, this last mutant showed a level of replication reduced by only 30% compared to wt HBV and a strong resistance to both lamivudine (>1000-fold) and adefovir (>10-fold). It remained sensitive to tenofovir both in vitro and in vivo. CONCLUSIONS: We report the selection of a complex HBV mutant that escaped the antiviral pressure of lamivudine, adefovir, and HBIg, and provide insight on the process of selection via genotypic and phenotypic analysis.