Efficacy and safety of entecavir for chronic HBV in HIV/HBV coinfected patients receiving lamivudine as part of antiretroviral therapy

Background: Prolonged use of lamivudine in patients coinfected with HIV and hepatitis B virus (HBV) leads to an increasing risk of lamivudine resistance in both diseases. We investigated the addition of entecavir, a potent inhibitor of HBV polymerase, to lamivudine-containing highly active antiretroviral therapy (HAART) in patients who experienced rebound in HBV viremia while maintaining Suppression of plasma HIV RNA less than 400 copies/ml. Methods: Sixty-eight patients were randomized to entecavir 1 mg (n = 51) or placebo (n = 17) once daily for 24 weeks; 65 patients continued the study with entecavir for an additional 24 weeks. Lamivudine-containing HAART was continued throughout. Results: At week 24, the mean HBV DNA in entecavir-treated patients was 5.52 log(10) - copies/ml versus 9.27 log(10) copies/ml for placebo, and at week 48, it was 4.79log(10) copies/ml versus 5.63log(10) copies/ml, respectively. The mean HBV DNA change from baseline for entecavir was -3.65 log(10) copies/ml (versus + 0.11 for placebo, P < 0.0001) and alanine aminotransferase normalization in 34%. of patients (versus 8% for placebo, P=0.08)At 48 weeks, mean change in HBV DNA reached -4.20log(10) copies/ml inpatients who received entecavir for the entire 48 weeks. The frequency of adverse events with entecavir and placebo was comparable. Through 48 weeks, no clinically relevant changes in HIV viremia or CD4 cell Counts were identified. Conclusion: In this study, entecavir was associated with rapid, clinically significant reductions in HBV DNA, with maintenance of HIV viremia suppression, in HIV/HBV coinfected patients with HBV viremia while on lamivudine treatment. (C) 2008 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

249 TP53 mutation has high prevalence and is correlated with larger and poorly differentiated HCC in Brazilian patients

Background: Ser-249 TP53 mutation (249(Ser)) is a molecular evidence for aflatoxin-related carcinogenesis in Hepatocellular Carcinoma (HCC) and it is frequent in some African and Asian regions, but it is unusual in Western countries. HBV has been claimed to add a synergic effect on genesis of this particular mutation with aflatoxin. The aim of this study was to investigate the frequency of 249Ser mutation in HCC from patients in Brazil. Methods: We studied 74 HCC formalin fixed paraffin blocks samples of patients whom underwent surgical resection in Brazil. 249Ser mutation was analyzed by RFLP and DNA sequencing. HBV DNA presence was determined by Real-Time PCR. Results: 249Ser mutation was found in 21/74 (28%) samples while HBV DNA was detected in 13/74 (16%). 249Ser mutation was detected in 21/74 samples by RFLP assay, of which 14 were confirmed by 249Ser mutant-specific PCR, and 12 by nucleic acid sequencing. All HCC cases with p53-249ser mutation displayed also wild-type p53 sequences. Poorly differentiated HCC was more likely to have 249Ser mutation (OR = 2.415, 95% CI = 1.001 - 5.824, p = 0.05). The mean size of 249Ser HCC tumor was 9.4 cm versus 5.5 cm on wild type HCC (p = 0.012). HBV DNA detection was not related to 249Ser mutation. Conclusion: Our results indicate that 249Ser mutation is a HCC important factor of carcinogenesis in Brazil and it is associated to large and poorly differentiated tumors.

Predictors of HBeAg status and hepatitis B viraemia in HIV-infected patients with chronic hepatitis B in the HAART era in Brazil

Background: HBV-HIV co-infection is associated with an increased liver-related morbidity and mortality. However, little is known about the natural history of chronic hepatitis B in HIV-infected individuals under highly active antiretroviral therapy (HAART) receiving at least one of the two drugs that also affect HBV (TDF and LAM). Information about HBeAg status and HBV viremia in HIV/HBV co-infected patients is scarce. The objective of this study was to search for clinical and virological variables associated with HBeAg status and HBV viremia in patients of an HIV/HBV co-infected cohort. Methods: A retrospective cross-sectional study was performed, of HBsAg-positive HIV-infected patients in treatment between 1994 and 2007 in two AIDS outpatient clinics located in the Sao Paulo metropolitan area, Brazil. The baseline data were age, sex, CD4 T+ cell count, ALT level, HIV and HBV viral load, HBV genotype, and duration of antiretroviral use. The variables associated to HBeAg status and HBV viremia were assessed using logistic regression. Results: A total of 86 HBsAg patients were included in the study. Of these, 48 (56%) were using combination therapy that included lamivudine (LAM) and tenofovir (TDF), 31 (36%) were using LAM monotherapy, and 7 patients had no previous use of either one. Duration of use of TDF and LAM varied from 4 to 21 and 7 to 144 months, respectively. A total of 42 (48. 9%) patients were HBeAg positive and 44 (51. 1%) were HBeAg negative. The multivariate analysis revealed that the use of TDF for longer than 12 months was associated with undetectable HBV DNA viral load (serum HBV DNA level < 60 UI/ml) (p = 0. 047). HBeAg positivity was associated with HBV DNA > 60 UI/ml (p = 0. 001) and ALT levels above normality (p = 0. 038). Conclusion: Prolonged use of TDF containing HAART is associated with undetectable HBV DNA viral load. HBeAg positivity is associated with HBV viremia and increased ALT levels.

Hepatitis B Infection Is Associated with Asymptomatic Malaria in the Brazilian Amazon

Background: Areas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question. Methodology and Findings: An observational study of 636 individuals was performed in Rondonia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic (OR: 120.13, P < 0.0001), present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (r=-0.6; P=0.0003). Conclusion: HBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions.

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.

Patterns of viral load in chronic hepatitis B patients in Brazil and their association with ALT levels and HBeAg status

Serum hepatitis B virus (HBV) DNA [eve[ is a predictor of the development of cirrhosis and hepatocellullar carcinoma in chronic hepatitis B patients. Nevertheless, the distribution of viral load levels in chronic HBV patients in Brazil has yet to be described. This cross-sectional study included 564 participants selected in nine Brazilian cities located in four of the five regions of the country using the database of a medical diagnostics company. Admission criteria included hepatitis B surface antigen seropositivity, availability of HBV viral toad samples and age >= 18 years. Mates comprised 64.5% of the study population. Mean age was 43.7 years. Most individuals (62.1%) were seronegative for the hepatitis B e antigen (HBeAg). Median serum ALT level was 34 U/L. In 58.5% of the patients HBV-DNA levels ranged from 300 to 99,999 copies/mL; however, in 21.6% levels were undetectable. Median HBV-DNA level was 2,351 copies/mL. Over 60% of the patients who tested negative for HBeAg and in whom ALT level was less than 1.5 times the upper limit of the normal range had HBV-DNA levels > 2,000 IU/mL, which has been considered a cut-off point for indicating a liver biopsy and/or treatment. In conclusion, HBV-DNA level identified a significant proportion of Brazilian individuals with chronic hepatitis B at risk of disease progression. Furthermore, this tool. enables those individuals with high HBV-DNA levels who are susceptible to disease progression to be identified among patients with normal or stightly elevated ALT.

Entecavir Treatment for up to 5 Years in Patients with Hepatitis B e Antigen-Positive Chronic Hepatitis B

Sustained virologic suppression is a primary goal of therapy for chronic hepatitis B (CHB). In study entecavir (ETV)-022, 48 weeks of entecavir 0.5 mg was superior to lamivudine for virologic suppression for hepatitis B e antigen (HBeAg)-positive CHB. A total of 183 entecavir-treated patients from ETV-022 subsequently enrolled in study ETV-901. We present the results after up to 5 years (240 weeks) of continuous entecavir therapy. The entecavir long-term cohort consists of patients who received >= 1 year of entecavir 0.5 mg in ETV-022 and then entered ETV-901 with a treatment gap <= 35 days. In ETV-901 the entecavir dose was 1.0 mg daily. For patients with samples available at Year 5, proportions with hepatitis B virus (HBV) DNA <300 copies/mL, normal alanine aminotransferase (ALT) levels, HBeAg loss, and HBeAg seroconversion were determined. In all, 146 patients met criteria for inclusion in the entecavir long-term cohort. At Year 5, 94% (88/94) had HBV DNA <300 copies/mL and 80% (78/98) had normal ALT levels. In addition to patients who achieved serologic responses during study ETV-022, 23% (33/141) achieved HBeAg seroconversion and 1.4% (2/145) lost hepatitis B surface antigen (HBsAg) during study ETV-901. Through 5 years, entecavir resistance emerged in one patient. The safety profile of entecavir was consistent with previous reports. Conclusion: Extended therapy with entecavir through 5 years maintained or increased rates of HBV DNA suppression and ALT normalization. Additional patients also achieved HBeAg loss and seroconversion. Entecavir provides sustained viral suppression with minimal resistance during long-term treatment of HBeAg-positive CHB. (HEPATOLOGY 2010;51:422-430.)

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.

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.

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.

Hepatitis B viral markers in banked human milk before and after Holder pasteurization

Background: Blood screening for hepatitis B virus (HBV) is not universally performed for donor selection in human milk banks. Objectives: To evaluate the frequency of detection of HBV surface antigen (HBsAg) and HBV-DNA in colostrum of HBV-infected nursing mothers before and after Holder pasteurization. Study design: Forty-two concentrated breast milk samples were obtained within two postnatal weeks from 24 HBsAg-positive women (4 HBeAg-positive and 20 HBeAg-negative, anti-HBe-positive) were tested for the presence of HBsAg and HBV-DNA before and after Holder pasteurization (30 min at 62.5 degrees C). Results: Before pasteurization, HBsAg and HBV-DNA were found in 14/24 (58%), and 20/24 (75%) first milk samples, respectively, obtained by 4 days after delivery. At least one marker was detected in 20/24 (83%) milk samples. Both markers were identified in milk of HBeAg-positive mothers, and most mothers with anti-HBe in blood had at least one HBV marker. Once detected, viral markers were frequently found in milk samples subsequently obtained from the same woman. Holder pasteurization did not affect the probability of detecting HBsAg (8/18, 44%), HBV-DNA (12/18, 67%). or at least one of them (15118, 83%). Conclusions: Although the biological implications of these findings remain to be determined, considering that HBV is highly contagious and most recipients of banked human milk are preterm infants, these findings should be taken into account when donors are enlisted for human milk banks without serological screening. (C) 2009 Elsevier B.V. All rights reserved.

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.

Characterization of Hepatitis B virus (HBV) genotypes in patients from Rondonia, Brazil

Background: Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondonia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM (R) 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3. Results: The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%). Conclusions: These results for the first HBV genotypic characterization in Rondonia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.

Quality evaluation of DNA obtained from stored human saliva and its applicability to identification in Forensic Dentistry

PURPOSE: This study evaluated the quality of DNA obtained from stored human saliva and its applicability to human identification. METHODS: The saliva samples of 20 subjects, collected in the form of saliva in natura and from mouth swabs and stored at -20ºC, were analyzed. After 7 days, the DNA was extracted from the 40 saliva samples and subjected to PCR and electrophoresis. After 180 days, the technique was repeated with the 20 swab samples. RESULTS: The first-stage results indicated that DNA was successfully extracted in 97.5% of reactions, 95% of saliva in natura and 100% of swab saliva samples, with no statistically significant difference between the forms of saliva. In the second phase, the result was positive for all 20 analyzed samples (100%). Subsequently, in order to analyze the quality of the DNA obtained from human saliva, the SIX3-2 gene was tested on the 20 mouth swab samples, and the PCR products were digested using the MbO1 restriction enzyme to evaluate polymorphisms in the ADRA-2 gene, with positive results for most samples. CONCLUSION: It was concluded that the quantity and quality of DNA from saliva and the techniques employed are adequate for forensic analysis of DNA.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.