Incursion of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus, Brazil, 2023.

We report 4 highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b viruses in samples collected during June 2023 from Royal terns and Cabot's terns in Brazil. Phylodynamic analysis revealed viral movement from Peru to Brazil, indicating a concerning spread of this clade along the Atlantic Americas migratory bird flyway.

Neutralizing antibody response after immunization with a COVID-19 bivalent vaccine: Insights to the future.

The raising of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants led to the use of COVID-19 bivalent vaccines, which include antigens of the wild-type (WT) virus, and of the Omicron strain. In this study, we aimed to evaluate the impact of bivalent vaccination on the neutralizing antibody (NAb) response. We enrolled 93 volunteers who had received three or four doses of monovalent vaccines based on the original virus (n = 61), or a booster shot with the bivalent vaccine (n = 32). Serum samples collected from volunteers were subjected to neutralization assays using the WT SARS-CoV-2, and Omicron subvariants. In addition, immunoinformatics to quantify and localize highly conserved NAb epitopes were performed. As main result, we observed that the neutralization titers of samples from individuals vaccinated with the bivalent vaccine were higher for the original virus, in comparison to their capacity of neutralizing the Omicron variant and its subvariants. NAb that recognize epitopes mostly conserved in the WT SARS-CoV-2 were boosted, while those that recognize epitopes mostly present in the Omicron variant, and subvariants were primed. These results indicate that formulation of future vaccines shall consider to target present viruses, and not viruses that no longer circulate.

Mortality in sea lions is associated with the introduction of the H5N1 clade 2.3.4.4b virus in Brazil October 2023: whole genome sequencing and phylogenetic analysis.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 virus was detected in the South American sea lions found dead in Santa Catarina, Brazil, in October 2023. Whole genome sequencing and comparative phylogenetic analysis were conducted to investigate the origin, genetic diversity, and zoonotic potentials of the H5N1 viruses. The H5N1 viruses belonged to the genotype B3.2 of clade 2.3.4.4b H5N1 virus, which was identified in North America and disseminated to South America. They have acquired new amino acid substitutions related to mammalian host affinity. Our study provides insights into the genetic landscape of HPAI H5N1 viruses in Brazil, highlighting the continuous evolutionary processes contributing to their possible adaptation to mammalian hosts.

Effectiveness of Heterologous Coronavirus Disease 2019 (COVID-19) Vaccine Booster Dosing in Brazilian Healthcare Workers, 2021.

BACKGROUND: Little is currently known about vaccine effectiveness (VE) for either 2 doses of Oxford-AstraZeneca (ChAdOx1) viral vector vaccine or CoronaVac (Instituto Butantan) inactivated viral vaccine followed by a third dose of mRNA vaccine (Pfizer/BioNTech) among healthcare workers (HCWs). METHODS: We conducted a retrospective cohort study among HCWs (aged ≥18 years) working in a private healthcare system in Brazil from January to December 2021. VE was defined as 1 - incidence rate ratio (IRR), with IRR determined using Poisson models with the occurrence of laboratory-confirmed coronavirus disease 2019 (COVID-19) infection as the outcome, adjusting for age, sex, and job type. We compared those receiving viral vector or inactivated viral primary series (2 doses) with those who received an mRNA booster. RESULTS: A total of 11 427 HCWs met the inclusion criteria. COVID-19 was confirmed in 31.5% of HCWs receiving 2 doses of CoronaVac vaccine versus 0.9% of HCWs receiving 2 doses of CoronaVac vaccine with mRNA booster (P < .001) and 9.8% of HCWs receiving 2 doses of ChAdOx1 vaccine versus 1% among HCWs receiving 2 doses of ChAdOx1 vaccine with mRNA booster (P < .001). In the adjusted analyses, the estimated VE was 92.0% for 2 CoronaVac vaccines plus mRNA booster and 60.2% for 2 ChAdOx1 vaccines plus mRNA booster, when compared with those with no mRNA booster. Of 246 samples screened for mutations, 191 (77.6%) were Delta variants. CONCLUSIONS: While 2 doses of ChAdOx1 or CoronaVac vaccines prevent COVID-19, the addition of a Pfizer/BioNTech booster provided significantly more protection.

Nucleocapsid single point-mutation associated with drop-out on RT-PCR assay for SARS-CoV-2 detection.

BACKGROUND: Since its beginning, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been a challenge for clinical and molecular diagnostics, because it has been caused by a novel viral agent. Whole-genome sequencing assisted in the characterization and classification of SARS-CoV-2, and it is an essential tool to genomic surveillance aiming to identify potentials hot spots that could impact on vaccine immune response and on virus diagnosis. We describe two cases of failure at the N2 target of the RT-PCR test Xpert® Xpress SARS-CoV-2. METHODS: Total nucleic acid from the Nasopharyngeal (NP) and oropharyngeal (OP) swab samples and cell supernatant isolates were obtained. RNA samples were submitted to random amplification. Raw sequencing data were subjected to sequence quality controls, removal of human contaminants by aligning against the HG19 reference genome, taxonomic identification of other pathogens and genome recovery through assembly and manual curation. RT-PCR test Xpert® Xpress SARS-CoV-2 was used for molecular diagnosis of SARS-CoV-2 infection, samples were tested in duplicates. RESULTS: We identified 27 samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing of 2 of 27 samples revealed a single common mutation in the N gene C29197T, potentially involved in the failed detection of N target. CONCLUSIONS: This study highlights the importance of genomic data to update molecular tests and vaccines.

Analysis of the complete genome of HBV genotypes F and H found in Brazil and Mexico using the next generation sequencing method.

INTRODUCTION AND OBJECTIVES: Hepatitis B Virus is classified into ten different genotypes (A- J). Genotypes F and H cluster apart from others in phylogenetic trees and is particularly frequent in the Americas. The aim of this study was to sequence complete genomes of samples of HBV genotypes F and H from Brazil and Mexico using next generation sequencing (NGS) and to study relevant characteristics for the disease associated with this virus. MATERIALS AND METHODS: Ninety plasma samples with detectable HBV DNA belonging to the F (n=59) and H (n=31) genotypes were submitted to amplification of the complete HBV genome by three different methologies. Data analysis was developed using bioinformatics tools for quality assurance and comprehensive coverage of the genome. Sequences were aligned with reference sequences for subgenotyping and detecting variants in relevant positions. A phylogenetical tree was constructed using Bayesian methods. RESULTS: HBV genome of 31 samples were amplified and 18 of them were sequenced (HBV/F=16 and HBV/H=2). One genotype F sample was co-infected with the F1b and F3 subgenotypes, while the other samples were all F2a subgenotype. Two genotype H samples clustered with other Mexican sequences. The main variants observed were found in preS and S genes (7/18) and mutations in the precore/core region (11/18). CONCLUSIONS: A NGS methodology was applied to F and H genotypes samples from Mexico and Brazil to fully characterize their sequences. This methodology will be relevant for clinical and epidemiological studies of hepatitis B in Latin America.

AIDS incidence and survival in a hospital-based cohort of HIV-positive patients from São Paulo, Brazil: The role of IFN-λ4 polymorphisms.

Few studies have reported the prognosis of human immunodeficiency virus (HIV)-positive patients followed for a long time in Brazil, particularly those including pre and post-HAART eras. The polymorphisms of interferon (IFN)-λ4 have been postulated as possibly associated with the pathogenesis of HIV infection. The aim of this study was to describe the incidence and mortality from a cohort of HIV-positive patients as well as whether IFN-λ4 gene polymorphisms (SNP rs8099917 and SNP rs12979860) were associated with HIV/acquired immune deficiency syndrome (AIDS) progression. We followed 402 patients for up to 30 years; 347 of them began follow-up asymptomatic, without any AIDS-defining opportunistic disease and/or a lymphocytes T CD4+ count of 350 cells/mm3 or lower. We determined the probability of the asymptomatic subjects to remain AIDS-free, and the risk of death for those entering the study already with an AIDS diagnosis, as well as for subjects developing AIDS during follow-up. We compared the prognosis of patients with two different polymorphisms for the genes encoding for IFN-λ4, variants rs8099917 and rs12979860. The follow-up time of the 347 asymptomatic-at-entry subjects was 3687 person-years. IFN-λ4 rs8099917 polymorphisms were not associated with AIDS progression, but IFN-λ4 rs12979860 wild type genotype (CC) was associated with higher mortality compared to CT and TT, with an increased probability of death from AIDS (P = .01). In conclusion, genetic variations in IFN-λ4 on rs12979860 polymorphisms in HIV-infected patients may drive mortality risk.

The role of PNPLA3 and TM6SF2 polymorphisms on liver fibrosis and metabolic abnormalities in Brazilian patients with chronic hepatitis C.

BACKGROUND: Despite the growing body of knowledge about TM6SF2 and PNPLA3 polymorphisms in non-alcoholic fatty liver disease, their influence in the spectrum of HCV liver disease is not yet fully defined. Besides that, admixed populations, such as Brazilians, were not included in most of the studies. METHODS: This cross-sectional study enrolled 365 treatment-naïve patients with HCV and 134 healthy individuals. TM6SF2 (rs58542926 c.499C > T) and PNPLA3 (rs738409 c.444C > G) polymorphisms were evaluated regarding their association with clinical and laboratory data, histological liver steatosis and fibrosis, and with components of the metabolic syndrome. RESULTS: In HCV subjects, the frequencies of TM6SF2 CC and CT + TT were 89% and 11%, while PNPLA3 frequencies of CC and CG + GG were 51.4% and 48.6%. In the univariate logistic regression analysis, the TM6SF2 CT + TT genotype in HCV was associated with significant liver fibrosis (p = 0.047; OR 1.953; 95% CI 1.009-3.788). In comparison to the CT + TT genotype, the TM6SF2 CC genotype in HCV was associated with older age (p = 0.002), higher frequency of arterial hypertension (p = 0.032), obesity (p = 0.030), metabolic syndrome (p = 0.014) and lower total cholesterol levels (p = 0.036). The PNPLA3 GG subjects had lower body mass index than CG/ CC individuals (p = 0.047). None of the polymorphisms, or their combinations, was independently associated with hepatic steatosis or fibrosis. On the other hand, older age, lower serum levels of total cholesterol, and higher serum levels of alanine aminotransferase and alkaline phosphatase were associated with liver fibrosis in the multivariate logistic regression analysis. CONCLUSION: In this evaluation of an admixed HCV population, neither TM6SF2 nor PNPLA3 polymorphisms were independently associated with hepatic steatosis or fibrosis. Other factors seem more influential than these specific polymorphisms in isolation. More studies are warranted to clarify the role of the TM6SF2 and PNPLA3 polymorphisms in Brazilians with HCV.

Sabiá Virus-Like Mammarenavirus in Patient with Fatal Hemorrhagic Fever, Brazil, 2020.

New World arenaviruses can cause chronic infection in rodents and hemorrhagic fever in humans. We identified a Sabiá virus-like mammarenavirus in a patient with fatal hemorrhagic fever from São Paulo, Brazil. The virus was detected through virome enrichment and metagenomic next-generation sequencing technology.

SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES: The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS: After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS: The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 106 TCID50/mL, titre 1.5 × 106 PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION: We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.

Validation of PNPLA3 polymorphisms as risk factor for NAFLD and liver fibrosis in an admixed population.

INTRODUCTION AND AIM: Studies have shown that two polymorphisms were associated with steatosis and progression of non-alcoholic fatty liver disease (NAFLD) in different populations: the Patatin-like Phospholipase Domain Containing 3 (PNPLA3) and Transmembrane 6 Superfamily Member 2 (TM6SF2). However, the frequency and significance of these polymorphisms in an admixed population, i.e., Brazilian, is unknown. Therefore, we aimed to evaluate them in healthy subjects in comparison to patients with NAFLD. MATERIAL AND METHODS: This was a multicenter cross-sectional study in 248 patients with biopsy-proven NAFLD and in 134 healthy controls from two tertiary centers in Brazil. PNPLA3 (rs738409 c.444C>G) and TM6SF2 (rs58542926 c.449C>T) polymorphisms were evaluated. RESULTS: In controls, the frequencies of PNPLA3 CC and CG+GG were 49.25% and 50.74%, respectively; in NAFLD patients, this was 31.05% and 68.88% (p=0.0044, 95% CI 1.037-2.977). PNPLA3 GG subjects had an increased risk (3.29-fold) of having NAFLD when compared to CC subjects (p=0.0044, 95% CI 1.504-7.225). In patients with nonalcoholic steatohepatitis (NASH), PNPLA3 GG compared to CC was associated with higher AST levels [38.4±25.3 versus 36.7±40.1IU/L, p=0.0395)] and with the presence of liver fibrosis (≥F2 fibrosis, p=0.0272). TM6SF2 polymorphisms were not in Hardy-Weinberg equilibrium in our NAFLD group precluding further analysis. CONCLUSION: We demonstrated for the first time that PNPLA3 CG+GG increase the risk of NAFLD among Brazilian subjects. Moreover, PNPLA3 GG was associated with liver enzyme elevation and fibrosis in NASH patients.

Sofosbuvir inhibits yellow fever virus in vitro and in patients with acute liver failure.

INTRODUCTION AND OBJECTIVES: Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). MATERIALS AND METHODS: Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. RESULTS: Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. CONCLUSIONS: Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment.

Lipase and factor V (but not viral load) are prognostic factors for the evolution of severe yellow fever cases.

BACKGROUND: Despite a highly efficacious vaccine, yellow fever (YF) is still a major threat in developing countries and a cause of outbreaks. In 2018, the Brazilian state of São Paulo witnessed a new YF outbreak in areas where the virus has not been detected before. OBJECTIVE: The aim is to describe the clinical and laboratorial characteristics of severe cases of YF, evaluate viral to determine markers associated with fatal outcome. METHODS: Acute severe YF cases (n = 62) were admitted to the Intensive Care Unit of a reference hospital and submitted to routine laboratorial evaluation on admission. YFV-RNA was detected in serum and urine by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and then sequenced. Patients were classified in two groups: survival or death. FINDINGS: In the univariate analysis the following variables were associated with outcome: alanin aminotransferase (ALT), aspartat aminotransferase (AST), AST/ALT ratio, total bilirubin (TB), chronic kidney disease epidemiology collaboration (CKD-EPI), ammonia, lipase, factor V, international normalised ratio (INR), lactate and bicarbonate. Logistic regression model showed two independent variables associated with death: lipase [odds ratio (OR) 1.018, 95% confidence interval (CI) 1.007 to 1.030, p = 0.002], and factor V (OR -0.955, 95% CI 0.929 to 0.982, p = 0.001). The estimated lipase and factor V cut-off values that maximised sensitivity and specificity for death prediction were 147.5 U/L [area under the curve (AUC) = 0.879], and 56.5% (AUC = 0.913). MAIN CONCLUSIONS: YF acute severe cases show a generalised involvement of different organs (liver, spleen, heart, kidneys, intestines and pancreas), and different parameters were related to outcome. Factor V and lipase are independent variables associated with death, reinforcing the importance of hemorrhagic events due to fulminant liver failure and pointing to pancreatitis as a relevant event in the outcome of the disease.

Prevalence of naturally occurring amino acid substitutions associated with resistance to hepatitis C virus NS3/NS4A protease inhibitors in São Paulo state.

Hepatitis C (HCV)-infected patients are treated with direct-acting antiviral agents (DAAs) in highly effective, well-tolerated, all-oral regimens. However, naturally occurring resistance-associated amino acid substitutions (RASs) may be selected during treatment. This study aimed to screen naturally occurring RASs NS3/NS4A inhibitors (PIs). Samples were obtained from DAA naïve patients, living in São Paulo state, Brazil. Screening for RASs in the HCV NS3 region was conducted in 859 samples from HCV-infected patients, of which 425 and 434 samples were subtype 1a and 1b, respectively. HCV-RNA was extracted, amplified, and sequenced. The overall prevalence of RASs to HCV PIs was 9.4%. The following RASs were observed in HCV-1a subtype infected patients: V36L (2.6%), T54S (1.6%), V55I/A (1.2% / 8.9%, respectively), Q80K (2.1%), R155K (0.5%), and D168E (0.2%); and in HCV-1b infected patients: V36L (0.7%), T54A/S (0.2% and 0.5%, respectively), V55A (0.5%), Q80K (0.2%), D168E (1.6%), and M175L (0.5%). HCV 1a infected subjects had higher serum viral load than that seen in patients infected with HCV 1b. There was no difference between the proportions of NS3 RASs with regards to geographic distribution within the investigated areas. These findings should be supported by additional studies in Brazil to help in the formation of local clinical guidelines for managing hepatitis C.

Molecular diagnosis of Strongyloides stercoralis among transplant candidates.

Strongyloidiasis can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection, principally in immunosuppressed patients. Our study aimed to evaluate the application of conventional polymerase chain reaction (cPCR) and real-time PCR (qPCR). Polymerase chain reaction (PCR) and real-time PCR (qPCR) targeting the 18S rRNA gene for detection of Strongyloides stercoralis infection among transplant candidates were applied in stool samples obtained from 150 transplant candidates, preliminarily analyzed by parasitological methods. S. stercoralis larvae were visualized in 15/150 (10.0%) transplant candidates by parasitological methods. DNA from S. stercoralis was amplified in 26/150 (17.3%) and 49/150 (32.7%) stool samples of transplant candidates, using cPCR and qPCR, respectively. The results suggest that molecular methods, especially qPCR, should be used as an additional tool for diagnostic of S. stercoralis infection among transplant candidates.

Characterization of clinical predictors of naturally occurring NS3/NS4A protease polymorphism in genotype 1 hepatitis C virus mono and HIV co-infected patients.

Spontaneously occurring resistance may impair the success of protease inhibitors based regimens in HCV treatment. This study aimed to evaluate associations between amino acid substitutions in NS3/NS4A domain and clinical features of 247 HCV mono or HCV/HIV co-infected patients. Fourteen samples (5.7%) harbored at least one resistance-associated substitution (RAS). The following RASs were detected in NS3 region: T54S (6-2.4%), V55A (7-2.8%), and Q80R (2-0.8%). S122G occurred in 86.9% of HCV genotype 1b samples with either natural polymorphisms or RASs. Advanced liver fibrosis and HIV co-infection were not related to NS3/NS4A amino acid substitutions.

Natural occurrence of NS5B inhibitor resistance-associated variants in Brazilian patients infected with HCV or HCV and HIV.

Resistance-associated variants (RAVs) represent a challenge to the success of new HCV therapies. The aim of this study was to describe the prevalence of naturally occurring NS5B RAVs in Brazilian direct acting antivirals (DAA)-naïve patients infected with HCV genotype 1, or co-infected with HIV. Patient enrollment and sample collection were performed between 2011 and 2013. Using Sanger-based sequencing, 244 sequences were obtained. RAVs detected in HCV-1a sequences were V321A (1.6 %), M414V (1.3 %), A421V (21.4-23.7 %), A421G (1.3 %) and Y448H (1.3 %); and in HCV-1b sequences were L159F (16.1 %), C316N (7.1-16.3 %) and A421V (3.2-6.3 %). Understanding the real RAVs scenario in patients is fundamental to establishing the most effective therapeutic strategy and in minimizing the risks for their selection.

Prevalence of naturally occurring NS5A resistance-associated substitutions in patients infected with hepatitis C virus subtype 1a, 1b, and 3a, co-infected or not with HIV in Brazil.

BACKGROUND: Non-structural 5A protein (NS5A) resistance-associated substitutions (RASs) have been identified in patients infected with hepatitis C virus (HCV), even prior to exposure to direct-acting antiviral agents (DAAs). Selection for these variants occurs rapidly during treatment and, in some cases, leads to antiviral treatment failure. DAAs are currently the standard of care for hepatitis C treatment in many parts of the world. Nevertheless, in Brazil, the prevalence of pre-existing NS5A RASs is largely unknown. In this study, we evaluated the frequency of naturally occurring NS5A RASs in Brazilian patients infected with HCV as either a monoinfection or coinfection with human immunodeficiency virus (HIV). METHODS: Direct Sanger sequencing of the NS5A region was performed in 257 DAA-naïve patients chronically infected with HCV (156 monoinfected with HCV and 101 coinfected with HIV/HCV). RESULTS: The frequencies of specific RASs in monoinfected patients were 14.6% for HCV GT-1a (M28 V and Q30H/R), 6.0% for GT-1b (L31F/V and Y93H), and 22.6% for GT-3a (A30K and Y93H). For HIV/HCV-coinfected patients, the frequencies of RAS were 3.9% for GT-1a (M28 T and Q30H/R), and 11.1% for GT-1b (Y93H); no RASs were found in GT-3a sequences. CONCLUSIONS: Substitutions that may confer resistance to NS5A inhibitors exist at baseline in Brazilian DAA-naïve patients infected with HCV GT-1a, -1b, and -3a. Standardization of RAS definitions is needed to improve resistance analyses and to facilitate comparisons of substitutions reported across studies worldwide. Therapeutic strategies should be optimized to efficiently prevent DAA treatment failure due to selection for RASs, especially in difficult-to-cure patients.

Basal core promoter and precore mutations among hepatitis B virus circulating in Brazil and its association with severe forms of hepatic diseases.

BACKGROUND: In Brazil, few studies have investigated the prevalence of infection with the precore (PC) and basal core promoter (BCP) mutants of the hepatitis B virus (HBV). OBJECTIVES: This study aimed to analyse the frequency of PC and BCP mutations among patients infected with HBV and to evaluate the association between the variants and advanced hepatic disease. METHODS: A total of 161 patients infected with HBV were studied. To identify PC and BCP mutations, a 501-bp fragment of HBV DNA was amplified and sequenced. FINDINGS: PC and BCP regions from HBV strains were successfully amplified and sequenced in 129 and 118 cases, respectively. PC and BCP mutations were detected in 61.0% and 80.6% of the cases, respectively. The A1762T/G1764A variant was identified in 36.7% of the patients with grade 1 and 2 liver fibrosis (29/79) and in 81.8% of the patients with grade 3 and 4 liver fibrosis (9/11) (p < 0.01); in 76.9% of the patients with cirrhosis (10/13) and in 38.1% of the patients without cirrhosis (40/105) (p = 0.01); and in 77.8% of the patients with hepatocellular carcinoma (HCC) (7/9) and in 39.4% of the patients without HCC (43/109) (p = 0.03). MAIN CONCLUSIONS: A high prevalence of HBV PC and BCP mutants was found. The A1762T/G1764A variant was independently associated with advanced forms of liver fibrosis, hepatic cirrhosis, and HCC.