Serological and molecular characterization of hepatitis B virus infection in chronic kidney disease patients from Rio de Janeiro, Brazil

ABSTRACT Chronic kidney disease (CKD) patients undergoing hemodialysis (HD) are more vulnerable to blood-borne viral infections due to frequent invasive procedures. Hepatitis B virus (HBV) infection in this cohort of patients has been a matter of concern worldwide. The objective of this cross-sectional study was to evaluate the frequency of serological markers for hepatitis B, and the occurrence of overt and occult HBV infection (OBI) and its molecular characterization in serum samples from 644 CKD patients in HD units located in Rio de Janeiro, Brazil, from 2013 to 2017. HBV DNA was investigated in HBsAg reactive and "anti-HBc alone" samples to determine infecting genotypes and genetic relatedness between sequences. The prevalence of serological markers HBsAg+, anti-HBc alone, anti-HBc+/anti-HBs+, anti-HBs+, anti-HBc/anti-HBs/HBsAg were 5.9%, 2.8%, 30.7%, 26.6%, 34.0%, respectively. HBV DNA was detected in 39.5% (15/38) of the HBsAg+ and in 5/18 (27.8%) of the "anti-HBc alone" individuals, indicating a high prevalence of OBI within this group. We found a higher prevalence of HBV/A1 (65%), followed by HBV/D3 (20%), and HBV/A2 (15%). Bayesian MCC tree with a highly supported clade, genetic distance comparison, and identical nucleotide sequences suggested a nosocomial spread of HBV in some units. The high prevalence of HBV infection and low number of individuals immune to infection reinforces the need for vaccination in this group. The presence of closely related strains in the same HD unit reinforces the importance of continuous improvement of safety control measures and laboratory surveillance of serological markers to prevent the risk of infection and transmission of HBV.

Hepatitis B virus genotypes prevalence in patients from hepatology services in Ceará, Brazil

Abstract INTRODUCTION: Hepatitis B virus (HBV) infection is a public health problem; therefore, we aimed to report HBV genotypes in Ceará, Brazil. METHODS: A total of 103 HBsAg-positive samples were subjected to HBV genotyping and subgenotyping. RESULTS: The following genetic compositions of samples were found: F-54% (F2-83.33%), A-40% (A1-65%), D-6%, C2-1%, E-1%, and G-1%. CONCLUSIONS: Some genotypes are only prevalent in certain parts of the world; however, the State of Ceará is a hub for migration and has one of the most important liver transplantation centers in Brazil, which can explain the prevalence of the F genotype.

Prevalence of hepatitis B and D virus infection in a district of Mato Grosso, bordering Amazonas and Rondônia states

Abstract INTRODUCTION: Brazil's western Amazon basin has the highest prevalence of hepatitis B virus (HBV) infection in the country. Coinfection with hepatitis D virus (HDV) is also endemic. To estimate the prevalence of HBV and HDV markers in a population inhabiting the northwest portion of Mato Grosso state in the western Amazon. METHODS: We performed a cross-sectional study of the seroprevalence of antibodies against HBV core antigen (anti-HBc) in the Três Fronteiras District northwest of Mato Grosso. Anti-HBc-positive subjects were tested for HBV surface antigen (HBsAg). Those positive for this marker were tested for HDV antibodies. Anti-HBc-negative participants were tested for anti-HBsAg. All tests were performed by EIA. RESULTS: A total of 623 individuals in the community were assessed; the majority (67.6%) were male, with a mean age of 30.8 ± 15.4 years. Two hundred and fourteen individuals (34.3%) were anti-HBc-positive, and 47 (7.5%) were HBsAg carriers. Only one individual was anti-HDV-positive. Among the 409 individuals without HBV infection, 18.3% were anti-HBsAg-positive. There was no association between HBV infection and known risk factors. CONCLUSIONS: The study area had intermediate-to-high endemicity for HBV infection, but a low prevalence of HDV. Our serological results suggesting low vaccination-induced protection indicate a need for reinforced immunization programs in the populations of northwest Mato Grosso.

Detection and molecular characterisation of a diagnosis escape variant associated with occult hepatitis B virus in Brazil

BACKGROUND Many studies have identified mutations in the hepatitis B surface antigen (HBsAg) as important factors limiting the ability of commercial serological assays to detect this viral antigen. However, an association between mutations in the HBsAg gene and the occurrence of occult HBV infection (OBI) in patients has not been established. OBJECTIVES To detect hepatitis B virus (HBV) DNA in patients with anti-HBc as a unique serological marker, a previously published, cost-effective TaqMan-based real-time polymerase chain reaction (PCR) test with minor groove binding probes was adapted for use in this study. The current study also aimed to investigate HBsAg mutations and genotypes of HBV in OBI at the Viral Hepatitis Ambulatory Clinic in Rio de Janeiro to determine any possible association. METHODS Intra-assay and inter-assay reproducibility were determined, and the mean coefficient of variation values obtained were 2.07 and 3.5, respectively. Probit analysis indicated that the 95% detection level was 25 IU/mL. The prevalence of OBI was investigated in 35 serum samples with an ‘anti-HBc alone’ profile from individuals who attended our clinic between 2011 and 2013. FINDINGS HBV DNA was detected in only one sample, resulting in an OBI rate of 2.9%. Nucleotide sequencing of the pre-S/S region was performed to genotype and analyse mutations within the HBsAg gene of this HBV DNA. The HBV in the OBI case was classified as sub-genotype A1, and a sequence analysis of the small S gene revealed 12 mutations in the major hydrophilic region compared to the consensus A1 sequence. Most of these mutations occurred in amino acid residues that have been reported as clinically relevant because they have been implicated in vaccine escape and/or inability to detect HBsAg by commercial serological assays. MAIN CONCLUSIONS Our study suggests the importance of specific HBsAg mutations, different from those in D, B, and C genotypes, in sub-genotype A1 HBV associated with OBI.

Hepatitis B virus genotypes in Southeast Brazil and its relationship with histological features

Data concerning the relationship between hepatitis B virus (HBV) genotypes and liver histology are scarce. The aim of this study was to compare HBV non-B and non-C genotypes according to demographic features, clinical status, HBV-DNA levels and liver histology in Rio de Janeiro. One hundred twenty one consecutive chronic HBV-infected patients were enrolled during two-year period and data were prospectively collected. Sera were tested for HBV genotyping using restriction fragment length polymorphism. Liver biopsy was obtained from patients with either increased alanine aminotransferase (ALT) or HBV-DNA levels. Genotype A was the most common, found in 82 (68%) patients, followed by F in 19 (15%), D in 17 (14%), B in one (1%) and C in two (2%). There was no association between HBV genotypes A, D and F and gender (p = 0.37), age (p = 0.78), race (p = 0.22), mode of infection (p = 0.94), HB "e" antigen status (p = 0.37) and HBV-DNA levels (p = 0.47). The ALT levels were lower in genotype D (75%) compared with A (47%) and F (55%) (p = 0.05). Liver biopsy showed lower inflammation [histological activity index (HAI) = 4] and fibrosis (F) (= 0) scores in genotype D than in genotypes A (HAI = 5, p < 0.001; F = 2, p = 0.008) or F (HAI = 5, p = 0.009; F = 2, p = 0.01). Genotype A was the most prevalent in chronic HBV-infected patients and genotype D patients presented with less intense liver disease.

Antiviral therapy against chronic hepatitis B in Brazil: high rates of lamivudine resistance mutations and correlation with HBV genotypes

The effectiveness of antiviral treatments of chronic hepatitis B has been poorly studied in Brazil. Here, hepatitis B virus (HBV) DNA positivity, drug resistance mutations and their association with HBV genotypes were evaluated in chronically HBV-infected patients under different drug regimens in Brazil. The study involved 129 patients under interferon or nucleos(t)ide analogue therapy for a median treatment time of 12 months. One hundred and five (81%) of these patients were treated with lamivudine (LAM), either in monotherapy or in combination with newer drugs, such as entecavir (ETV) or tenofovir (TDF). High (37.5-100%) rates of HBV DNA positivity were observed with all but one drug regimen (LAM + ETV). However, patients that were treated with ETV alone, TDF alone or with LAM combination therapies had a mean viral load that was 3-4 log lower than patients treated with LAM monotherapy. Of the patients treated with LAM, 47% developed resistance mutations. HBV genotypes A (59.1%), D (30.3%) and F (9.1%) were found. There was no association between the presence of LAM resistance mutations and genotypes, HBeAg status or treatment duration. Nevertheless, the rtM204V mutation was observed more frequently (12/13, 92%) in genotype A than in the others (p = 0.023). Six out of nine isolates that contained the rtM204I mutation belonged to genotype D and half of them displayed a single mutation. Genotype D isolates with the rtM204V variant preferentially displayed a triple mutation, while genotype A preferentially displayed a double mutation (p = 0.04).

Caracterização molecular de isolados do vírus da hepatite B em pacientes submetidos a tratamento antiviral e pacientes com infecção oculta / Molecular characterization of isolates of the virus of hepatitis B in patients undergoing antiviral therapy and patients with occult infection

Nos últimos anos, dois assuntos relacionados a infecção pelo vírus da hepatite B (HBV) vêm sendo foco de muitos debates e estudos por pesquisadores ao redor do mundo: o tratamento antiviral contra a hepatite B crônica e a infecção oculta pelo HBV. O estudo da ocorrência de mutações de resistência aos medicamentos antivirais mais potentes, os análogos de núcleos(t)ídeos (NAs), que representa o maior entrave para o sucesso do tratamento, passou a ter fundamental importância para o entendimento da dinâmica da terapia a longo prazo e para soluções para contornar este problema. A hepatite B oculta é um tema que preocupa pesquisadores do mundo inteiro pelo fato de algumas questões pertinentes ainda não tenham sido esclarecidas, como o impacto clínico da infecção oculta e seu papel na transmissão do vírus. A presente tese teve como objetivo realizar a caracterização molecular dos isolados do HBV em diversas populações de indivíduos com hepatite B: pacientes com infecção crônica submetidos a diferentes tratamentos antivirais (avaliando os tipos de tratamento antiviral atualmente utilizados no Brasil, a eficiência global de cada tratamento em reduzir os níveis de DNA do HBV e a freqüência de isolados com mutação de resistência aos medicamentos), pacientes com infecção aguda (observando a distribuição de genótipos e analisando a presença de subpopulações virais através do pirosequenciamento), e pacientes com infecção oculta pelo HBV (comparando a prevalência da infecção oculta pelo HBV em pacientes anti-HCV positivo e anti-HCV-negativo submetidos à hemodiálise) Como principais resultados obtidos nos quatro manuscritos produzidos, tivemos (i) a LAM como a terapia antiviral mais comum em uso no Brasil, com mais de 80 por cento dos pacientes analisados estando em tratamento com esta droga seja em monoterapia ou em terapia combinada com outra droga; (ii) o tratamento com IFN-alfa como o menos eficiente em reduzir o DNA do HBV e, em relação aos NAs, não foi encontrada uma diferença significativa entre a terapia com LAM e a terapia com os demais agentes orais em relação à eficiência em aduzir o DNA do HBV no soro dos pacientes, no entanto, as cargas virais nos pacientes tratados com LAM foram muito mais altas do que nos pacientes tratados com as drogas mais recentes; (iii) uma alta freqüência de isolados com mutações de resistência à LAM em pacientes submetidos à monoterapia (quase 50 por cento) ou à terapia combinada (78 por cento); (iv) uma alta incidência de isolados com a tripla mutação de resistência à LAM (6/27; 22 por cento) e uma diferença no perfil dos variantes YMDD entre os principais genótipos do HBV circulando no Brasil. Em conclusão, a alta freqüência de isolados com mutações de resistência à LAM pode limitar o uso de drogas como o ETV e a LdT para terapia de resgate em função da ocorrência de resistência cruzada entre essas drogas. As alterações de aminoácidos no HBsAg (sE164D mais sI195M) causadas pela tripla mutação de resistência à LAM, conhecidas por reduzir a afinidade de ligação deste antígeno com o anti-HBs, pode representar um sério problema epidemiológico caso esses isolados passem a circular no ambiente disseminados pela população em geral. Tais substituições podem ter contribuído para o caráter oculto da infecção por diminuírem a detecção do HBsAg por testes sorológicos, sugerindo ser esta a causa mais provável da ocorrência da infecção oculta nos pacientes em hemodiálise.