Barbara V. Lago

Occult hepatitis B virus infection and lamivudine‐resistant mutations in isolates from renal patients undergoing hemodialysis

Background and Aims:  Patients undergoing hemodialysis are at risk of infection with both hepatitis B virus (HBV) and hepatitis C virus (HCV). Occult HBV infection is usually associated with low levels of HBV and is frequently detected in HCV‐infected patients. The aims of the present study were to compare the prevalence of occult HBV infection among anti‐HCV‐positive and anti‐HCV‐negative patients undergoing hemodialysis, and characterize the molecular patterns of HBV isolates from patients with occult infection.

Fulminant hepatitis failure in adults and children from a Public Hospital in Rio de Janeiro, Brazil.

Fulminant hepatic failure (FHF) is characterized by massive hepatocellular injury, whose physiopathology is still unclear. Hepatitis B (HBV) is probably the most common viral cause of FHF, while hepatitis A (HAV) virus seem occurs less frequently. However, the host and viral factors that determine the outcome of these infections are poorly understood. In the present study, viral load and genotyping determining regions of HAV and HBV genomes were sequenced. Eight FHF patients and one patient with severe acute hepatitis (SAH) were included. Liver and blood samples were collected during liver transplantation or necropsy procedures. HAV-RNA and HBV-DNA were extracted from serum, biopsy and paraffin liver. Nucleotide sequencing of HAV-RNA was performed from VP1/2A and HBV-DNA from PreS/S region. The amplified samples were quantified by Real-Time PCR. The cases of HAV infection were due to subgenotype IA. The cases of HBV infection were due to genotype A2 and D4. The case of HAV/HBV coinfection was infected by genotype IA and D3. Hepatitis A and B infection were associated with genotypes most prevalent in Brazil. In hepatitis A infection the mean of period evolution was 13 days. In hepatitis B, FHF patients infected by genotype D have a shorter period of evolution than FHF patients infected by genotype A (mean 15 v. 53 days). There was no association with genotype-determining region with the severity of hepatitis, however nucleotide differences and high viral load could be observed among FHF.

Hepatitis B Virus Subgenotype A1: Evolutionary Relationships between Brazilian, African and Asian Isolates

Brazil is a country of low hepatitis B virus (HBV) endemicity in which the genotype A of HBV (HBV/A) is the most prevalent. The complete nucleotide sequences of 26 HBV/A isolates, originating from eight Brazilian states, were determined. All were adw2. Twenty-three belonged to subgenotype A1 and three to A2. By phylogenetic analysis, it was shown that all the 23 HBV/A1 isolates clustered together with isolates from Bangladesh, India, Japan, Nepal, the Philippines and United Arab Emirates, but not with those of Congo, Kenya, Malawi, Rwanda, South Africa, Tanzania, Uganda and Zimbabwe. Four amino acid residues in the polymerase (His138 in the terminal protein domain, Pro18 and His90 in the spacer, and Ser109 in the reverse transcriptase), and one (Phe17) in the precore region, predominated in Latin American and Asian HBV/A1 isolates, but were rarely encountered in African isolates, with the exception of those from Somalia. Specific variations of two adjacent amino acids in the C-terminal domain of the HBx protein, namely Ala146 and Pro147, were found in all the Brazilian, but rarely in the other HBV/A1 isolates. By Bayesian analysis, the existence of an ‘Asian-American’ clade within subgenotype A1 was supported by a posterior probability value of 0.996. The close relatedness of the Brazilian, Asian and Somalian isolates suggests that the HBV/A1 strains predominant in Brazil did not originate from the five million slaves who were imported from Central and Western Africa from 1551 to 1840, but rather from the 300–400,000 captives forcibly removed from southeast Africa at the middle of the 19th century.

Phylogeography and evolutionary history of hepatitis B virus genotype F in Brazil

BackgroundHepatitis B virus (HBV) genotype F (HBV/F) is considered to be indigenous to the Americas, but its emergence and spread in the continent remain unknown. Previously, only two HBV/F complete genome sequences from Brazil were available, limiting the contribution of Brazilian isolates to the phylogenetic studies of HBV/F. The present study was carried out to assess the proportion and geographic distributions of HBV/F subgenotypes in Brazil, to determine the full-length genomic sequences of HBV/F isolates from different Brazilian geographic regions, and to investigate the detailed evolutionary history and phylogeography of HBV/F in Brazil.MethodsComplete HBV/F genomes isolated from 12 Brazilian patients, representing the HBV/F subgenotypes circulating in Brazil, were sequenced and analyzed together with sequences retrieved from GenBank, using the Bayesian coalescent and phylogeographic framework.ResultsPhylogenetic analysis using all Brazilian HBV/F S-gene sequences available in GenBank showed that HBV/F2a is found at higher frequencies countrywide and corresponds to all sequences isolated in the Brazilian Amazon Basin. In addition, the evolutionary analysis using complete genome sequences estimated an older median ancestral age for the Brazilian HBV/F2a compared to the Brazilian HBV/F1b and HBV/F4 subgenotypes, suggesting that HBV/F2a represents the original native HBV of Brazil. The phylogeographic patterns suggested a north-to-south flow of HBV/F2a from Venezuela to Brazil, whereas HBV/F1b and HBV/F4 strains appeared to have spread from Argentina to Brazil.ConclusionsThis study suggests a plausible route of introduction of HBV/F subgenotypes in Brazil and demonstrates the usefulness of recently developed computational tools for investigating the evolutionary history of HBV.

Detection of mixed populations of wild-type and YMDD hepatitis B variants by pyrosequencing in acutely and chronically infected patients

BackgroundLamivudine (LAM) is associated with the highest known rate of resistance mutations among nucleotide analogs used to treat chronic hepatitis B virus (HBV) infection. Despite this, LAM continues in widespread use, especially in combination therapies. The primary LAM resistance mutation (rtM204V/I) occurs in the YMDD motif of HBV polymerase. The aim of this study was to characterize Brazilian HBV isolates from acute and chronic cases by direct sequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencing method capable of detecting single-nucleotide polymorphisms. HBV DNA from serum samples of 20 individuals with acute HBV infection and 44 with chronic infection undergoing antiviral therapies containing LAM were analyzed by direct sequencing and pyrosequencing methods.ResultsPhylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D and F) for the Brazilian population in both acute and chronic infections. However, within genotype A isolates, subgenotype A2 was more frequently detected in acute cases than in chronic cases (P = 0.012). As expected, none of the individuals with acute hepatitis B had LAM-resistant isolates as a dominant virus population, whether detected by direct sequencing or pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20) had minor subpopulations (4-17%) of LAM-resistant isolates. Among chronic patients undergoing LAM treatment, YMDD mutants were frequently found as a dominant virus population. In cases where wild-type virus was the dominant population, subpopulations of YMDD variants were usually found, demonstrating the complexity of HBV quasispecies.ConclusionsYMDD variants were frequently detected as a minor population in acute HBV infection. The occurrence of pre-existing variants may lead to a high frequency of resistant mutants during antiviral therapy in the chronic phase. In chronic infection, detection of YMDD variants before virological or biochemical breakthrough might contribute to making better therapy choices and thus improving treatment outcome.

Prevalence of hepatitis B virus infection among tuberculosis patients with or without HIV in Goiânia City, Brazil

BACKGROUND Hepatitis B virus (HBV) and tuberculosis (TB) represent major public health problems. There is currently little data on HBV infection among TB patients with and without human immunodeficiency virus (HIV). OBJECTIVES To assess HBV prevalence among TB patients with and without HIV. STUDY DESIGN From April 2008 to March 2010, a cross-sectional study was conduct among TB patients attended at a reference hospital in Goiânia City, Brazil. The participants were tested for serological markers of HBV infection and HIV antibodies. HBV DNA was detected in HBsAg-positive samples, and also in HBsAg-negative/anti-HBc-positive samples to look for HBV occult infection. RESULTS Of 425 patients, 402 (94.6%) agreed to participate in the study. The overall prevalence of HBV (HBsAg and/or anti-HBc positive) and HIV infections were 25.6% (103/402) and 27.6% (111/402), respectively. A higher HBV infection rate was found among HIV-infected patients (36.9%; 41/111) compared to patients infected with TB only (20.0%; 57/285). A multivariate analysis of risk factors showed that age ≥ 50 years (p=0.03), non-injecting (p<0.01) and injecting (p<0.01) drugs use were associated with HBV infection. Among the HBsAg-positive samples (n=13), HBV DNA was detected in 10 (76.9%) samples. Of the 90 anti-HBc-positive samples, 13 were HBV DNA positive (with very low levels) resulting in an occult HBV infection rate of 14.4%. PCR-RFLP was successfully performed in 20 HBV DNA-positive samples: 15 were genotype A and 5 were genotype D. CONCLUSIONS HBV infection was common, particularly among this with HIV infection.

Prevalence and Incidence of HCV Infection among Prisoners in Central Brazil

The aim of this multicenter, cross sectional study was to assess the prevalence, incidence and associated risk factors among incarcerated populations from twelve Brazilian prisons. The total of 3,368 individuals from twelve prisons was randomly recruited between March 2013 and March 2014. Participants were interviewed, and provided blood samples which were tested for antibodies to Hepatitis C (HCV ab). One year after the first investigation, a cohort study was conducted with 1,656 inmates who participated the cross sectional study. Positive samples were tested for the presence of HCV RNA. Out of 3,368 inmates, 520 (15.4%) were females, and 2,848 (84.6%) were males. The overall prevalence of HCV was 2.4% (95% CI: 1.9 to 2.9), with 0.6% (95% CI: 0.4 to 0.8) in females, and 2.7% (95% CI: 2.1 to 3.3) in males (p<0.01). HCV RNA was detected in 51/80 (63.7%) samples. Among men prisoners, multivariate analysis of associated factors showed independent associations between HCV exposure and increasing age, inject drug use, length of incarceration, smoking hashish, sharing needle and syringe and HIV positivity. During the cohort study, 7/1,656 new cases of HCV infection were detected, and the incidence rate was 0.4/100 person-year. Once high frequency rates of specific HCV risk behaviors and new HCV infections have been identified inside prisons, effective interventions strategies such as screening, clinical evaluation and treatment to reduce the spread of HCV infection are essential.

Overt and occult hepatitis B virus infection among treatment-naïve HIV-infected patients in Brazil

Although hepatitis B virus (HBV) and human immunodeficiency virus (HIV) co‐infection is common, only few data are available on HBV among HIV patients including occult hepatitis B infection (OBI), regardless of serological markers. This study aims to determine the prevalence of OBI and overall HBV infection, associated factors, HBV genotypes, and surface (S) gene mutations in a population of treatment‐naïve HIV‐infected patients in Brazil. A cross‐sectional study was conducted in treatment‐naïve HIV‐infected patients in Central Brazil. All samples were tested for HBV serological markers and HBV DNA. Sequence analysis of the S gene and overlapping polymerase gene was preformed. Overall, 25.1% (127/505) of the patients had markers of current or previous HBV infection, which was associated with age over 40 years, history of injection drug use, and homosexual sex. The hepatitis B surface antigen (HBsAg) seroprevalence was 4.9% (25/505). HBV DNA was detected in 39 out of 505 patients: 20 of them were HBsAg‐positive and 19 were HBsAg‐negative, resulting in an OBI prevalence of 3.8%. Patients with OBI had significantly higher HCV seropositivity rate compared to HBsAg‐positive patients. Sequencing of the S gene revealed Y100C, T131N, and D144A mutations. One patient had the M204I and L180M drug‐resistance mutations (polymerase). HBV genotypes A (A1, A2), D (D2, D3), and F (F2) were identified. In conclusion, OBI represented almost half of all HBV infections with detectable HBV DNA, suggesting that hepatitis B diagnosis in HIV patients should include in addition to serological markers the detection of HBV DNA. J. Med. Virol. 88:1222–1229, 2016.

Serological and molecular evidence of hepadnavirus infection in swine.

INTRODUCTION AND OBJECTIVE Recently, investigations in a swine herd identified evidence of the existence of a novel member of the Hepadnavirus family endemic in swine. The aim of this study was to investigate the serological and molecular markers of Hepadnavirus circulation in Brazilian domestic swine and wild boar herds, and to evaluate the identity with HBV and other Hepadnaviruses reported previously. MATERIALS AND METHODS For the study, 376 swine were screened for hepatitis B virus serological markers. Analyses were performed in serum samples using commercial enzyme-linked immunosorbent assay (ELISA) kits (DiaSorin®) for anti-HBc, HBsAg and anti-HBs. Reactive and undetermined swine serum samples were selected to perform DNA viral extraction (QIAamp DNA Mini Kit, Qiagen®), partial genome amplification and genome sequencing. RESULTS From 376 swine samples analysed, 28 (7.45%) were reactive to anti-HBc, 3 (0.80%) to HBsAg and 6 (1.6%) to anti-HBs. Besides, more 17 (4.52%) swine samples analyzed were classified in the grey zone of the EIA test to anti-HBc and 2 (0.53%) to HBsAg. From 49 samples molecularly analyzed after serological trial, 4 samples showed a positive result for the qualitative PCR for Hepadnavirus. Phylogenetic reconstruction using partial genome sequencing (360 bp) of 3 samples showed similarity with HBV with 90.8-96.3% of identity. CONCLUSIONS Serological and molecular data showed evidence of the circulation of a virus similar to hepatitis B virus in swine.

Hepatitis B virus genotypes A1, A2 and E in Cape Verde: Unequal distribution through the islands and association with human flows

Hepatitis B virus (HBV) diversity has not been previously studied in Cape Verde. The archipelago was discovered in 1460 by Portuguese explorers, who brought African slaves to colonise the islands. In this study, we investigated the HBV characteristics from 183 HBsAg-positive Cape Verdean individuals. Phylogenetic analysis of the pre-S/S region and the full-length genomes revealed 54 isolates with HBV/A1 (57%), 21 with HBV/A2 (22%), 19 with HBV/E (20%), and one with HBV/D (1%). HBV genotypes and subgenotypes were unequally distributed through the islands. In São Vicente, the main northern island, most isolates (84%) belonged to the African-originated HBV/A1, with the remaining isolates belonging to HBV/A2, which is prevalent in Europe. Interestingly, the HBV/A1 isolates from São Vicente were closely related to Brazilian sequences into the Asian-American clade, which suggests the dissemination of common African ancestors through slave trade. In contrast, in Santiago and nearby southern islands, where a recent influx from different populations circulates, a higher diversity of HBV was observed: HBV/A1 (40%); HBV/E (32%); HBV/A2 (28%); and HBV/D (1%). HBV/E is a recent genotype disseminated in Africa that was absent in the era of the slave trade. African and European human flows at different times of the history may explain the HBV diversity in Cape Verde. The possible origin and specifics of each HBV genotype circulating in Cape Verde are discussed.