Leticia Cancella Nabuco

Hepatitis B virus genotypes circulating in Brazil: molecular characterization of genotype F isolates

BackgroundHepatitis B virus (HBV) isolates have been classified in eight genotypes, A to H, which exhibit distinct geographical distributions. Genotypes A, D and F are predominant in Brazil, a country formed by a miscegenated population, where the proportion of individuals from Caucasian, Amerindian and African origins varies by region. Genotype F, which is the most divergent, is considered indigenous to the Americas. A systematic molecular characterization of HBV isolates from different parts of the world would be invaluable in establishing HBV evolutionary origins and dispersion patterns. A large-scale study is needed to map the region-by-region distribution of the HBV genotypes in Brazil.ResultsGenotyping by PCR-RFLP of 303 HBV isolates from HBsAg-positive blood donors showed that at least two of the three genotypes, A, D, and F, co-circulate in each of the five geographic regions of Brazil. No other genotypes were identified. Overall, genotype A was most prevalent (48.5%), and most of these isolates were classified as subgenotype A1 (138/153; 90.2%). Genotype D was the most common genotype in the South (84.2%) and Central (47.6%) regions. The prevalence of genotype F was low (13%) countrywide. Nucleotide sequencing of the S gene and a phylogenetic analysis of 32 HBV genotype F isolates showed that a great majority (28/32; 87.5%) belonged to subgenotype F2, cluster II. The deduced serotype of 31 of 32 F isolates was adw4. The remaining isolate showed a leucine-to-isoleucine substitution at position 127.ConclusionThe presence of genotypes A, D and F, and the absence of other genotypes in a large cohort of HBV infected individuals may reflect the ethnic origins of the Brazilian population. The high prevalence of isolates from subgenotype A1 (of African origin) indicates that the African influx during the colonial slavery period had a major impact on the circulation of HBV genotype A currently found in Brazil. Although most genotype F isolates belonged to cluster II, the presence of some isolates belonging to clusters I (subgroup Ib) and IV suggests the existence of two or more founder viral populations of genotype F in Brazil.

Is the Rapid Virologic Response a Positive Predictive Factor of Sustained Virologic Response in all Pretreatment Status Genotype 1 Hepatitis C Patients Treated With Peginterferon-α2b and Ribavirin?

Introduction Currently it is not yet defined if the rapid virologic response (RVR) can predict a sustained virologic response (SVR) in relapsers and nonresponders. Objective To evaluate treatment-RVR as a predictive factor of SVR in genotype 1 hepatitis C treatment naive, relapsers, and nonresponder patients treated with pegylated interferon-alpha (PEG-IFN-α2b) and ribavirin. Methods One hundred sixty-seven genotype 1 hepatitis C patients who were treated with PEG-IFN-α2b and ribavirin and had SVR assessed were included. Hepatitis C virus RNA analysis at the fourth week of treatment was performed in all patients. The exclusion criteria were hepatitis B virus and/or HIV co-infection. A comparative analysis was performed between the groups with and without RVR and a logistic regression model was applied. Results One hundred sixty-seven patients were analyzed, 103 (62%) were naives, 22 (13%) relapsers, and 42 (25%) nonresponders. The SVR rates were 44% in naives, 68% in relapsers, and 12% in nonresponders. RVR was attained in 51/167 (31%) patients and in this group the SVR was higher than in those without RVR (75% vs. 23%; P<0.001). This difference was also observed in all subgroups: naives (71% vs. 29%; P=0.001), relapsers (92% vs. 40%; P=0.02), and nonresponders (50% vs. 8%; P=0.06). A stepwise logistic regression model identified RVR and absence of cirrhosis as the factors independently associated to SVR. Conclusions RVR and absence of cirrhosis are the strongest predictive factors of SVR in HCV genotype 1 patients. Assessment of RVR is very useful in all pretreatment status patients in predicting SVR and provides information for individualizing therapy.

Identification of novel recombinants of hepatitis B virus genotypes F and G in human immunodeficiency virus-positive patients from Argentina and Brazil

Hepatitis B virus (HBV) genotype G (HBV/G) infection is almost always detected along with a co-infecting HBV strain that can supply HBeAg, typically HBV/A2. In this study we describe, in two human immunodeficiency virus (HIV)-positive patients from Argentina and Brazil, the first report of HBV/G infection in Argentina and co-circulation of HBV/G, HBV/F and G/F recombinants in the American continent. HBV isolates carrying the 36 bp insertion of HBV/G were the most prevalent in both patients, with >99 % of colonies hybridizing to a probe specific for this insertion. Phylogenetic analyses of full-length genomes and precore/core fragments revealed that F4 and F1b were the co-infecting subgenotypes in the Brazilian and Argentinian patients, respectively. Bootscanning analysis provided evidence of recombination in several clones from both patients, with recombination breakpoints located mainly at the precore/core region. These data should encourage further investigations on the clinical implications of HBV/G recombinants in HBV/HIV co-infected patients.

Evaluation of Saliva Specimens as an Alternative Sampling Method to Detect Hepatitis B Surface Antigen

In this study, a modified enzyme immunoassay (EIA) was evaluated for the Hepatitis B surface antigen (HBsAg) among whole saliva and oral fluid samples. Specimens were collected from 115 individuals who gave serum and oral fluid using Salivette (Sarstedt, Nümbrecht, Germany) and whole saliva. Saliva specimens were tested following a modified ELISA, and the results were compared with paired serum specimens that were tested according to the suppliers instructions. Transport buffer for the oral fluids, sample volume for assay, incubation period of sample with conjugate as well as cut‐off values were evaluated to optimize the assay. The highest sensitivity and specificity were obtained by increasing the incubation of sample and conjugate to 16 hr and using the area under the receiver operating characteristic curve to calculate cut‐off values. HBsAg was detected in 40 oral fluids and 44 whole saliva samples out of 47 paired positive serum specimens and not detected in 64 oral fluids and 63 whole saliva samples out of 68 matched negative sera samples by the ELISA assay. There was excellent agreement between the results for the serum and saliva specimens kappa value (κ): 0.80 for oral fluid and κ: 0.87 for whole saliva and there was excellent reproducibility. Using an optimized protocol, the sensitivities of whole saliva and oral fluid were 93.6 and 85.1%, respectively, whereas specificities of whole saliva and oral fluid were 92.6 and 94.1%, respectively. Our data showed a significant promise for the use of whole saliva and oral fluid together with the modified commercial EIA for Hepatitis B virus infection surveillance. J. Clin. Lab. Anal. 25:134–141, 2011.

HBV-DNA levels in HBsAg-positive blood donors and its relationship with liver histology.

Introduction and Objectives The clinical meaning of viremia, especially at low levels, in chronic hepatitis B virus (HBV)-infected patients remains unknown. The objective of the present study was to determine serum HBV-DNA levels and its relationship with liver histology in HBsAg-positive blood donors. Methods A cross-sectional study was conducted on 78 blood donors, with alanine aminotransferase (ALT) and HBeAg evaluation and quantitative determination of HBV-DNA by polymerase chain reaction (Amplicor, HBV Monitor, Roche; lower limit of sensitivity 1,000 copies/mL). Liver biopsy was obtained from all patients with detectable viremia irrespective of ALT and HBV-DNA levels. Results Among 78 blood donors, serum HBV-DNA was detected in 47 (60%) patients; 39 (83%) were males; mean age 37.6±10.4 years; 31 (66%) were HBeAg-negative, and ALT was elevated in 26 (55%). The median of HBV-DNA levels was 24,000 copies/mL and 31 (40%) subjects had no detectable serum HBV-DNA. Although the histologic lesions were mild in the majority of patients, HBV-DNA levels were significantly higher in patients with chronic hepatitis or cirrhosis when compared with patients without histologic liver disease (25,260,000 vs. 9480 copies/mL; P<0.001). There was a significant correlation between HBV-DNA levels and necroinflammatory score (r=0.59) and fibrosis (r=0.50); however, in the subset of HBeAg-negative patients with HBV-DNA levels below 30,000 copies/mL, 25% presented histologic disease related to HBV. Conclusions Most HBsAg-positive blood donors show low viral load. There is a significant association between viral replication and liver damage; however, low HBV-DNA levels do not exclude the presence of histologic disease.

Genetic variability of hepatitis B and C viruses in Brazilian patients with and without hepatocellular carcinoma

Most cases of hepatocellular carcinoma (HCC) are due to chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection worldwide. The aim of this study was to determine the viral genotypes and frequency of 17 mutations (15 for HBV and 2 for HCV), described previously as able to influence the course of chronic liver disease, in patients with and without HCC. This transversal study included 157 Brazilian patients with chronic hepatitis B (n = 51) and C (n = 106). Of these, 12 and 40 patients had HBV‐ and HCV‐related HCC, respectively. Nucleotide sequencing of core promoter, pre‐core, and pre‐S/S regions of HBV and core region of HCV strains was performed to determine their genotypes and the frequency of the respective mutations. Among the HBV isolates, subgenotype A1 was the most prevalent in both patients with (90%) and without (61%) HCC. Fourteen out of the 15 mutations under study, as well as five different pre‐S deletions, were identified. Core promoter T1753V, A1762T, and G1764A mutations were more frequent in patients with HCC than in those without, although with no statistical difference. However, a significant correlation was observed between T1753V mutation and elevation of transaminases levels (P < 0.05). As for HCV, mutation at residue 70 in the core protein of genotype 1b strains was significantly more frequent in patients with cirrhosis (56.3%) than in those without (9.1%) (P = 0.018). The detection of some key mutations in the genomes of HBV and HCV might be helpful to predict the clinical outcome of patients with chronic liver disease. J. Med. Virol. 86:217–223, 2014.

Prevalence of occult hepatitis B virus infection in kidney transplant recipients

In this cross-sectional study, 207 hepatitis B surface antigen (HBsAg)-negative kidney transplant recipients were evaluated based on demographic and epidemiological data and on the levels of serological markers of hepatitis B virus (HBV) and hepatitis C virus infection and liver enzymes. Patients with HBV or human immunodeficiency virus infection were excluded. Sera were analysed for the presence of HBV-DNA. HBV-DNA was detected in two patients (1%), indicating occult hepatitis B (OHB) infection (the HBV-DNA loads were 3.1 and 3.5 IU/mL in these patients). The results of the liver function tests were normal and no serological markers indicative of HBV infection were detected. The prevalence of OHB infection was low among kidney transplant recipients, most likely due to the low HBsAg endemicity in the general population of the study area.

Effectiveness and safety of first-generation protease inhibitors in real-world patients with hepatitis C virus genotype 1 infection in Brazil: a multicenter study

OBJECTIVE: To evaluate the effectiveness and safety of first-generation protease inhibitors for the treatment of genotype 1 hepatitis C virus-infected patients at Brazilian reference centers. METHODS: This multicenter cross-sectional study included hepatitis C virus genotype 1 monoinfected patients treated with Peg-interferon, ribavirin, and either boceprevir (n=158) or telaprevir (n=557) between July 2013 and April 2014 at 15 reference centers in Brazil. Demographic, clinical, virological, and adverse events data were collected during treatment and follow-up. RESULTS: Of the 715 patients, 59% had cirrhosis and 67.1% were treatment-experienced. Based on intention-to-treat analysis, the overall sustained viral response was 56.6%, with similar effectiveness in both groups (51.9% for boceprevir and 58% for telaprevir, p=0.190). Serious adverse events occurred in 44.2% of patients, and six deaths (0.8%) were recorded. Cirrhotic patients had lower sustained viral response rates than non-cirrhotic patients (46.9% vs. 70.6%, p<0.001) and a higher incidence of serious adverse events (50.7% vs. 34.8%, p<0.001). Multivariate analysis revealed that sustained viral response was associated with the absence of cirrhosis, viral recurrence after previous treatment, pretreatment platelet count greater than 100,000/mm3, and achievement of a rapid viral response. Female gender, age>65 years, diagnosis of cirrhosis, and abnormal hemoglobin levels/platelet counts prior to treatment were associated with serious adverse events. CONCLUSION: Although serious adverse events rates were higher in this infected population, sustained viral response rates were similar to those reported for other patient cohorts.

Usefulness of in-house real time PCR for HBV DNA quantification in serum and oral fluid samples

For quantification of hepatitis B virus DNA (HBV DNA), commercial assays are used with serum or plasma samples, but oral fluid samples could be an alternative for HBV diagnosis due to ease of collection. This study aims to develop in-house real time PCR using synthetic curve for HBV DNA quantification for serum and oral fluid samples. Samples were collected from 103 individuals (55 HBsAg reactive and HBV DNA reactive by commercial assay and 48 without HBV markers) and submitted to two in-house real time PCR assays for HBV pre-S/S region with different standard curves: qPCR plasmidial and qPCR synthetic. A total of 27 serum samples were HBV DNA positive by qPCR plasmidial and 40 with qPCR synthetic (72% and 85% of concordance, respectively). Quantitative PCR synthetic presented efficiency of 99% and sensitivity of 2log10 copies/mL. Among oral fluid samples, five and ten were detected using qPCR plasmidial and synthetic, respectively. This study demonstrated that qPCR synthetic using serum samples could be used as alternative for HBV DNA quantification due to its sensitivity. In addition, it was possible to quantify HBV DNA in oral fluid samples suggesting the potential of this specimen for molecular diagnosis of HBV.

Comparison of oral fluid collection methods for the molecular detection of hepatitis B virus

BACKGROUND This study aims to compare the efficiency of four oral fluid collection methods (Salivette, FTA Card, spitting and DNA-Sal) to detect HBV DNA by qualitative PCR. MATERIALS AND METHODS Seventy-four individuals (32 HBV reactive and 42 with no HBV markers) donated serum and oral fluid. In-house qualitative PCR to detect HBV was used for both samples and commercial quantitative PCR for serum. RESULTS HBV DNA was detected in all serum samples from HBV-infected individuals, and it was not detected in control group. HBV DNA from HBV group was detected in 17 samples collected with Salivette device, 16 samples collected by FTA Card device, 16 samples collected from spitting and 13 samples collected by DNA-Sal device. Samples that corresponded to a higher viral load in their paired serum sample could be detected using all oral fluid collection methods, but Salivette collection device yielded the largest numbers of positive samples and had a wide range of viral load that was detected. CONCLUSION It was possible to detect HBV DNA using all devices tested, but higher number of positive samples was observed when samples were collected using Salivette device, which shows high concordance to viral load observed in the paired serum samples.