Dynamics of Hepatitis D (delta) virus genotype 3 in the Amazon region of South America

Hepatitis delta virus (HDV) is widely distributed and associated with fulminant hepatitis epidemics in areas with high prevalence of HBV. Several studies performed in the 1980s showed data on HDV infection in South America, but there are no studies on the viral dynamics of this virus. The aim of this study was to conduct an evolutionary analysis of hepatitis delta genotype 3 (HDV/3) prevalent in South America: estimate its nucleotide substitution rate, determine the time of most recent ancestor (TMRCA) and characterize the epidemic history and evolutionary dynamics. Furthermore, we characterized the presence of HBV/HDV infection in seven samples collected from patients who died due to fulminant hepatitis from Amazon region in Colombia and included them in the evolutionary analysis. This is the first study reporting HBV and HDV sequences from the Amazon region of Colombia. Of the seven Colombian patients, five were positive for HBV-DNA and HDV-RNA. Of them, two samples were successfully sequenced for HBV (subgenotypes F3 and Fib) and the five samples HDV positive were classified as HDV/3. By using all HDV/3 available reference sequences with sampling dates (n = 36), we estimated the HDV/3 substitution rate in 1.07 x 10(-3) substitutions per site per year (s/s/y), which resulted in a time to the most recent common ancestor (TMRCA) of 85 years. Also, it was determined that HDV/3 spread exponentially from early 1950s to the 1970s in South America. This work discusses for the first time the viral dynamics for the HDV/3 circulating in South America. We suggest that the measures implemented to control HBV transmission resulted in the control of HDV/3 spreading in South America, especially after the important raise in this infection associated with a huge mortality during the 1950s up to the 1970s. The differences found among HDV/3 and the other HDV genotypes concerning its diversity raises the hypothesis of a different origin and/or a different transmission route. (C) 2011 Elsevier B.V. All rights reserved.

Hepatitis D and B virus genotypes in chronically infected patients from the Eastern Amazon Basin

Hepatitis D virus (HDV) is a defective hepatotropic virus whose infectivity is dependent on hepatitis B virus (HBV). HDV super- or co-infiection leads to an increased risk of fulminant hepatitis or progression to severe chronic liver disease in HBV infected patients. The Brazilian Amazon Basin has been reported to be endemic for HBV and HDV, especially in the Western Amazon Basin. In this region, HDV infection is frequently associated with acute fulminant hepatitis with characteristic histologic features. HDV is classified into seven major clades (HDV-1 to HDV-7) and HBV is subdivided into eight genotypes (A-H). HDV and HBV genotypes have been shown to have a distinct geographic distribution. The aim of this study was to determine the HBV and HDV genotypes harbored by chronically infected patients from the Eastern Amazon Basin, Brazil. We studied 17 serum samples from HBV and HDV chronically infected patients admitted to a large public hospital (Santa Casa de Misericordia) at Belem, state of Para, Brazil, between 1994 and 2002. HDV-3 and HBV genotype A (subtype adw2) have been identified in all cases, in contrast to previous studies from other regions of the Amazon, where HBV genotype F has been found co-infecting patients that harbored HDV-3. The HDV-3/HBV-A co-infection suggests that there is not a specific interaction between HBV and HDV genotypes, and co-infection might merely reflect the most frequent genotypes found in a particular geographic area. The analysis of the carboxy-terminal region of the large hepatitis D antigen (L-HDAg), which interacts with the hepatitis B surface antigen (HBsAg) and is essential for HDV assembly, showed some diversity between the different isolates from the Eastern Amazon. This diversity is not observed among HDV-3 sequences from other South American regions. (C) 2008 Elsevier B.V. All rights reserved.

Population-based multicentric survey of hepatitis B infection and risk factor differences among three regions in Brazil

This multicentric population-based study in Brazil is the first national effort to estimate the prevalence of hepatitis B (HBV) and risk factors in the capital cities of the Northeast, Central-West, and Federal Districts (2004-2005). Random multistage cluster sampling was used to select persons 13-69 years of age. Markers for HBV were tested by enzyme-linked immunosorbent assay. The HBV genotypes were determined by sequencing hepatitis B surface antigen (HBsAg). Multivariate analyses and simple catalytic model were performed. Overall, 7,881 persons were included; < 70 per cent were not vaccinated. Positivity for HBsAg was less than 1 per cent among non-vaccinated persons and genotypes A, D, and F co-circulated. The incidence of infection increased with age with similar force of infection in all regions. Males and persons having initiated sexual activity were associated with HBV infection in the two settings; healthcare jobs and prior hospitalization were risk factors in the Federal District. Our survey classified these regions as areas with HBV endemicity and highlighted the risk factors differences among the settings

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

High Frequency of Lamivudine Resistance Mutations in Brazilian Patients Co-Infected With HIV and Hepatitis B

This study analyzed the genotype distribution and frequency of lamivudine (LAM) and tenofovir (TDF) resistance mutations in a group of patients co-infected with HIV and hepatitis B virus (HBV). A cross-sectional study of 847 patients with HIV was conducted. Patients provided blood samples for HBsAg detection. The load of HBV was determined using an ""in-house"" real-time polymerase chain reaction. HBV genotypes/subgenotypes, antiviral resistance, basal core promoter (BCP), and precore mutations were detected by DNA sequencing. Twenty-eight patients with co-infection were identified. The distribution of HBV genotypes among these patients was A (n = 9; 50%), D (n = 4; 22.2%), G (n = 3; 16.7%), and F (n = 2; 11.1%). Eighteen patients were treated with LAM and six patients were treated with LAM plus TDF. The length of exposure to LAM and TDF varied from 4 to 216 months. LAM resistance substitutions (rtL180M + rtM204V) were detected in 10 (50%) of the 20 patients with viremia. This pattern and an accompanying rtV173L mutation was found in four patients. Three patients with the triple polymerase substitution pattern (rtV173L+ rtL180M + rtM204V) had associated changes in the envelope gene (sE164D + sl195M). Mutations in the BCP region (A1762T, G1764A) and in the precore region (G1896A, G1899A) were also found. No putative TDF resistance substitution was detected. The data suggest that prolonged LAM use is associated with the emergence of particular changes in the HBV genome, including substitutions that may elicit a vaccine escape phenotype. No putative TDF resistance change was detected after prolonged use of TDF. J. Med. Virol. 82:1481-1488, 2010. (C) 2010 Wiley-Liss, Inc.

Hepatitis B virus and hepatitis delta virus genotypes in outbreaks of fulminant hepatitis (Labrea black fever) in the western Brazilian Amazon region

The genotypes of hepatitis B (HBV) and delta (HDV) viruses circulating among fulminant hepatitis cases from the western Amazon Basin of Brazil were characterized in this study. HBV and HDV isolates were obtained from liver samples from 14 patients who developed fulminant hepatitis and died during 1978-1989. HBV DNA and HDV RNA were detected in all samples. Phylogenetic analyses of HDV sequences showed that they all clustered with previously characterized sequences of HDV genotype 3 (HDV-3). HBV genotypes F, A and D were found in 50.0, 28.6 and 21.4% of cases, respectively. These results confirm the predominance of HDV-3 in South America and its association with the severe form of hepatitis, and the finding of the co-infection of HDV-3 with different genotypes of HBV suggests that the association between HDV-3 and HBV-F is not necessarily causally related to a more severe clinical course of infection.

A phylogenetic study of hepatitis B virus in chronically infected Brazilian patients of Western and Asian descent

Hepatitis B virus (HBV) causes one of the most important chronic viral infections worldwide. HBV is classified into eight genotypes whose epidemiology varies geographically. In Brazil, genotypes A, D, and F are more frequent, while in East Asia, genotypes B and C predominate. Several studies showed that immigrants retain the HBV infection pattern of their ancestral country. To identify HBV genotypes infecting chronic carriers in Brazilian families of Western and Asian descent by Hepatitis B surface antigen gene sequencing and analyze the route of viral transmission by phylogenetic analysis of viral sequences. Eighty-seven people chronically infected with HBV were separated into two groups: Western descent (27) and Asian descent (60). Surface and pre-core/core genes were amplified from serum HBV-DNA and sequences were subjected to phylogenetic analysis. HBV genotype A was found in 74% of Western subjects, while genotype C was found in 94% of Asian patients. Thirty-eight percent of Western families were infected with HBV with similar pre-core/core sequences, while only 25% of Asian families showed similarity in these sequences. Phylogenetical analysis of pre-core/core HBV gene suggested intra-familial transmission of HBV in 38% of Western families and 25% of Asian families. Analysis of HBsAg gene sequences helped to define the HBV genotype but did not allow inferring route of transmission as its sequences showed a smaller phylogenetic signal than pre-core/core sequences. Chronic HBV carriers of Asian descent born in or living in Brazil were infected with the same HBV genotype predominant in their ancestral country.

Follow-up of Pregnant Women With Autoimmune Hepatitis The Disease Behavior Along With Maternal and Fetal Outcomes

Goals: To assess maternal and fetal outcomes and clinical management of pregnancy in patients with autoimmune hepatitis (AIH). Background: There is a paucity of information about maternal and fetal outcomes, and AIH activity during pregnancy and in the postpartum period. There is no consensus about the administration of azathioprine during pregnancy and breastfeeding. Study: Retrospective analysis of 54 pregnancies (3 still in progress) in 39 AIH patients. Results: The median age at conception was 24 years, and 68.4% of women had liver cirrhosis. Before conception and in early pregnancy, azathioprine and prednisone were administered in 48.1%, but treatment regimen vas usually changed further to 20 mg/d prednisone and 20.4%, were off treatment. There were 36 livebirths, and fetal loss rates were 29.4% (13 miscarriages, 1 stillbirth, and 1 ectopic pregnancy). Preterm birth rate was 11.8%. In 2 cases, there was acute fetal distress; and in 2 others congenital malformations (3.9%). The rate of serious maternal complication was 7.8%, with no deaths. There were no flares in 41.2% pregnancies, but aminotransferase elevations occurred in 54.9%, 31.4% of which were true AIH relapses, only registered in the postpartum period. Conclusions: Despite the high fetal miscarriage rate, pregnancy in AIH was safe. Patients needed careful monitoring, especially in the postpartum period because of relapses. There was no evidence of a cause and effect relationship among azathioprine administration and premature births and congenital abnormalities, but more studies are necessary. Higher doses of prednisone may be an alternative option for those who prefer azathioprine withdrawal during pregnancy.

Hepatitis Delta virus genotype 8 infection in Northeast Brazil: Inheritance from African slaves?

Hepatitis Delta virus (HDV) is endemic worldwide, but its prevalence varies in different geographical areas. While in the Brazilian Amazon, HDV is known to be endemic and to represent a significant public health problem, few studies have assessed its prevalence in other regions in the country. This study evaluated the seroprevalence of HDV among HBsAg chronic carriers from Maranhao state, a region located in the Northeast of Brazil. Among 133 patients, 5 had anti-HD, of whom 3 had HDV RNA. HDV genotypes were characterized by Bayesian phylogenetic analysis of nucleotide sequences from the HDAg coding region. HDV-3 was identified in one patient who lives in Maranhao, but was born in Amazonas state (Western Amazon basin). Phylogenetic analysis shows that this HDV-3 sequence grouped with other HDV-3 sequences isolated in this state, which suggests that the patient probably contracted HDV infection there. Surprisingly, the other two patients were infected with HDV-8, an African genotype. These patients were born and have always lived in Urbano Santos, a rural county of Maranhao state, moreover they had never been to Africa and denied any contact with people from that continent. This is the first description of the HDV-8 in non-native African populations. This genotype may have been introduced to Brazil through the slaves brought to the country from the West Africa regions during the 16-18th centuries. Our results indicate that the need of clinical and epidemiological studies to investigate the presence of this infection in other areas in Brazil. (C) 2011 Elsevier B.V. All rights reserved.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.

Effect of a thrombin receptor (protease-activated receptor 1, PAR-1) gene polymorphism in chronic hepatitis C liver fibrosis

Background and Aim: Tissue injury leads to activation of coagulation and generation of thrombin. Inhibition of thrombin receptor protease-activated receptor 1 (PAR-1) has been shown to reduce liver fibrosis in animals. This study aimed to evaluate the effect of PAR-1 gene polymorphism on rate of liver fibrosis (RF) in chronic hepatitis C. Methods: Polymorphisms studied: C > T transition 1426 bp upstream of translation start site (-1426C/T), 13 bp repeat of preceding -506 5`-CGGCCGCGGGAAG-3` sequence (-506I/D), and A > T transversion in intervening sequence (IVS) 14 bp upstream of exon-2 start site (IVS-14A/T). A total of 287 European and 90 Brazilian patients were studied. Results: 1426C/T polymorphism: There was a trend to higher RF in patients with the TT genotype (P = 0.06) and an association between genotype CC and slow fibrosis (P = 0.03) in Europeans. In males, RF was significantly higher in those with the TT genotype compared to CT (P = 0.003) and CC (P = 0.007). There was a significant association between TT and fast fibrosis (P = 0.04). This was confirmed in an independent cohort of Brazilians where RF was higher in TT than in CC (P = 0.03). Analysis of -506I/D showed no difference in RF and distribution of slow/fast fibrosis among different genotypes in both populations. Analysis of IVS-14A/T showed no difference between genotypes. Conclusion: In conclusion, these findings suggest that PAR-1 receptor polymorphisms influence the progression of liver fibrosis.

Liver HLA-G expression is associated with multiple clinical and histopathological forms of chronic hepatitis B virus infection

As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and as the histocompatibility leucocyte antigen (HLA)-G is well described as a tolerogenic molecule, we evaluated HLA-G expression in 74 specimens of HBV liver biopsies and in 10 specimens obtained from previously healthy cadaver liver donors. HBV specimens were reviewed and classified by the METAVIR score, and HLA-G expression was assessed by immunohistochemistry. No HLA-G expression was observed in control hepatocytes. In contrast, 57 (77%) of 74 HBV specimens showed soluble and membrane-bound HLA-G expression in hepatocytes, biliary epithelial cells or both. No associations between the intensity of HLA-G expression and patient age or gender, HBeAg status, severity of liver fibrosis, and grade of histological findings were observed. Although significance was not reached (P = 0.180), patients exhibiting HLA-G expression presented a higher median HBV DNA viral load (105 copies/mL) than those who did not express HLA-G (103.7 copies/mL). These results indicate that HLA-G is expressed in most cases of chronic HBV infection in all stages and may play a role in the persistency of HBV infection.

The H63D genetic variant of the HFE gene is independently associated with the virological response to interferon and ribavirin therapy in chronic hepatitis C

Background Conflicting results have been reported in studies evaluating the relationship between serum markers of iron overload, liver iron deposits, and HFE mutations (C282Y and H63D) in chronic hepatitis C patients, and also their impact on the response to therapy in these patients. Aim To evaluate the role of HFE mutations in the severity of liver disease and in the response to therapy in chronic hepatitis C. Methods Two hundred and sixty-four hepatitis C patients treated with standard interferon and ribavirin were divided into two groups according to type of antiviral response: sustained virological response (SVR) and nonresponse or relapse. We evaluated the relationship between HFE mutation and the type of antiviral response, clinical data, biochemical tests, liver histopathology, virological data, and HFE mutations. Results Of the 264 patients, 88 (32.1%) had SVR whereas 67.9% had nonresponse or relapse. Liver iron deposits were observed in 49.2% of the patients. The factors associated with SVR were hepatitis C virus genotype 2 or 3, transferrin saturation value of 45% or less, and detection of the H63D mutation. HFE mutation was more frequent in patients with iron deposits, but without association with serum iron biochemistry or severity of liver disease. Steatosis was more frequent in patients with liver iron deposits. Conclusion The H63D mutation was an independent factor associated with SVR in chronic hepatitis C patients, as also were hepatitis C virus genotype 2 or 3 and transferrin saturation value of 45% or less. Moreover, the H63D mutation was associated with liver iron deposits. Eur J Gastroenterol Hepatol 22: 1204-1210 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Population-Based Multicentric Survey of Hepatitis B Infection and Risk Factor Differences among Three Regions in Brazil

This multicentric population-based study in Brazil is the first national effort to estimate the prevalence of hepatitis B (HBV) and risk factors in the capital cities of the Northeast. Central-West, and Federal Districts (2004-2005). Random multistage cluster sampling was used to select persons 13-69 years of age. Markers for HBV were tested by enzyme-linked immunosorbent assay. The HBV genotypes were determined by sequencing hepatitis B surface antigen (HBsAg). Multivariate analyses and simple catalytic model were performed. Overall. 7,881 persons were inculded < 70% were not vaccinated. Positivity for HBsAg was less than 1% among non-vaccinated persons and genotypes A, D, and F co-circulated. The incidence of infection increased with age with similar force of infection in all regions. Males and persons having initiated sexual activity were associated with HBV infection in the two settings: healthcare jobs and prior hospitalization were risk factors in the Federal District. Our survey classified these regions as areas with HBV endemicity and highlighted the risk factors differences among the settings.

Vitaminas D e C para poedeiras na fase inicial de produção de ovos

Um experimento foi conduzido com o objetivo de avaliar os efeitos de duas fontes de vitamina D e três níveis de vitamina C sobre as características de desempenho, a qualidade interna e externa dos ovos, os níveis de cálcio total e iônico séricos e a resistência óssea de poedeiras. Foram utilizadas 288 galinhas da linhagem ISA Babcock B300® com 23 semanas de idade, durante um período experimental de 12 semanas. Utilizou-se o delineamento inteiramente ao acaso em arranjo fatorial 2 × 3, com os fatores: fontes de vitamina D (colecalciferol e 25-hidroxicolecalciferol - 25(OH)D3) e de vitamina C (0, 100 e 200 ppm), totalizando seis tratamentos com oito repetições de seis aves. O nível basal de colecalciferol foi de 2.756 UI/kg, correspondendo a 5,51 g do produto comercial Hy.D®/t de ração, como fonte de 25(OH)D3. Os fatores estudados não influenciaram o consumo de ração, a produção, o peso e a massa de ovos. Observou-se efeito da interação de fontes de vitamina sobre a conversão alimentar, que foi melhor quando utilizado metabólito 25(OH)D3 na ausência de vitamina C. Interações foram observadas para porcentagem de albúmen e porcentagem de gema, que aumentaram na presença de 200 ppm de vitamina C. O peso específico dos ovos, as concentrações de cálcio sérico, cinzas ósseas e a resistência à quebra não foram influenciadas pelas fontes de vitamina D e C. Houve interação para porcentagem e espessura de casca, cujos maiores valores foram obtidos com a suplementação de vitamina C na presença de 25(OH)D3. Em poedeiras na fase inicial de produção, a conversão alimentar é melhor com a utilização do 25(OH)D3 e a espessura e porcentagem de casca também melhoram com a utilização de 25(OH)D3 e a suplementação de vitamina C nas dietas (100 ou 200 ppm, respectivamente).