Prevalence of hepatitis C virus (HCV) infection and HCV genotypes of hemodialysis patients in Salvador, Northeastern Brazil

Hepatitis C virus (HCV) infection has been identified as the major cause of chronic liver disease among patients on chronic hemodialysis (HD), despite the important reduction in risks obtained by testing candidate blood donors for anti-HCV antibodies and the use of recombinant erythropoietin to treat anemia. A cross-sectional study was performed to estimate the prevalence of HCV infection and genotypes among HD patients in Salvador, Northeastern Brazil. Anti-HCV seroprevalence was determined by ELISA in 1243 HD patients from all ten different dialysis centers of the city. HCV infection was confirmed by RT-PCR and genotyping was performed by restriction fragment length polymorphism. Anti-HCV seroprevalence among HD patients was 10.5% (95% CI: 8.8-12.3) (Murex anti-HCV, Abbott Murex, Chicago, IL, USA). Blood samples for qualitative HCV detection and genotyping were collected from 125/130 seropositive HD patients (96.2%). HCV-RNA was detected in 92/125 (73.6%) of the anti-HCV-positive patients. HCV genotype 1 (77.9%) was the most prevalent, followed by genotype 3 (10.5%) and genotype 2 (4.6%). Mixed infections of genotypes 1 and 3 were found in 7.0% of the total number of patients. The present results indicate a significant decrease in anti-HCV prevalence from 23.8% detected in a study carried out in 1994 to 10.5% in the present study. The HCV genotype distribution was closely similar to that observed in other hemodialysis populations in Brazil, in local candidate blood donors and in other groups at risk of transfusion-transmitted infection.

Co-circulation of genotypes IA and IB of hepatitis A virus in Northeast Brazil

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21% of the samples, respectively. Nucleotide sequencing was carried out in 46% of VP1/2A and in 53% of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0%. Phylogenetic analysis of the VP1/2A sequences showed that 65% belong to sub-genotype IA and 35% to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2% identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.

Prevalence of serological markers of hepatitis B virus in pregnant women from Paraná State, Brazil

The prevalence of hepatitis B virus (HBV) in Brazil increases from South to North but moderate to elevated prevalence has been detected in the Southwest of Paraná State. The prevalence of serological markers of HBV was evaluated in 3188 pregnant women from different counties in Paraná State and relevant epidemiological features were described. The prevalence of HBV markers in pregnant women for the state as a whole was 18.5% (95% CI = 17.2-19.9), ranging from 7.2% in Curitiba to 38.5% in Francisco Beltrão. The endemicity of HBV marker prevalence in pregnant women was intermediate in Cascavel, Foz do Iguaçu, and Francisco Beltrão, and low in Curitiba, Londrina, Maringá, and Paranaguá. Multiple logistic regression showed that HBV marker prevalence increased with age, was higher among black women, among women of Italian and German descent, and among women who had family members in neighboring Rio Grande do Sul State. Univariate analysis showed that HBV marker prevalence was also higher among women with no education or only primary education, with a lower family income and whose families originated from the South Region of Brazil. Pregnant women not having positive HBV markers (anti-HBc, HBsAg or anti-HBs detected by ELISA) corresponded to 73.7% of the population studied, implying that HBV vaccination needs to be reinforced in Paraná State. The highest prevalence was found in three counties that received the largest number of families from Santa Catarina and Rio Grande do Sul, where most immigrants were of German or Italian ascendance. This finding probably indicates that immigrants that came to this area brought HBV infection to Southwestern Paraná State.

The emergence of YMDD mutants precedes biochemical flare by 19 weeks in lamivudine-treated chronic hepatitis B patients: an opportunity for therapy reevaluation

Given the loss of therapeutic efficacy associated with the development of resistance to lamivudine (LMV) and the availability of new alternative treatments for chronic hepatitis B patients, early detection of viral genotypic resistance could allow the clinician to consider therapy modification before viral breakthrough and biochemical relapse occur. To this end, 28 LMV-treated patients (44 ± 12 years; 24 men), on their first therapy schedule, were monitored monthly at four Brazilian centers for the emergence of drug resistance using the reverse hybridization-based INNO-LiPA HBV DR assay and occasionally sequencing (two cases). Positive viral responses (HBV DNA clearance) after 6, 12, and 18 months of therapy were achieved by 57, 68, and 53% of patients, while biochemical responses (serum alanine aminotransferase normalization) were observed in 82, 82, and 53% of cases. All viral breakthrough cases (N = 8) were related to the emergence of YMDD variants observed in 7, 21, and 35% of patients at 6, 12, and 18 months, respectively. The emergence of these variants was not associated with viral genotype, HBeAg expression status, or pretreatment serum alanine aminotransferase levels. The detection of resistance-associated mutations was observed before the corresponding biochemical flare (41 ± 14 and 60 ± 15 weeks) in the same individuals. Then, if highly sensitive LMV drug resistance testing is carried out at frequent and regular intervals, the relatively long period (19 ± 2 weeks) between the emergence of viral resistance and the onset of biochemical relapse can provide clinicians with ample time to re-evaluate drug therapy.

Hepatitis B virus genotype E detected in Brazil in an African patient who is a frequent traveler

Genotype E of hepatitis B virus (HBV) has not been described in Brazil and is found mainly in Africa. Genotype A is the most prevalent in Brazil, and genotypes B, C, D, and F have already been reported. We report here an HBV genotype E-infected patient and some characterization of surface (S) protein, DNA polymerase (P) and precore/core (preC/C) coding regions based on the viral genome. The patient is a 31-year-old black man with chronic hepatitis B who was born and raised in Angola. He has been followed by a hepatologist in São Paulo, Brazil, since November 2003, and he is a frequent traveler to Latin America, Africa, and Europe. In 2003, he was diagnosed with HBV infection and started treatment with lamivudine with the later addition of adefovir dipivoxil. No known risk factor was identified. Serologically, he is HBsAg and anti-HBe positive, but HBeAg and anti-HBs negative. DNA sequence analysis of the S/P region confirmed that this patient is infected with genotype E, subtype ayw4. The preC/C region showed G1896A and G1899A mutations but no mutations in the basal core promoter. Nucleotide substitutions common in genotype E were also observed (C1772, T1858 and A1757). Although this is not an autochthonous case and there is no evidence of further spread, the description of this case in Brazil highlights the current risk of viral genotypes spreading with unprecedented speed due to constant travel around the world.

Low occurrence of occult hepatitis B virus infection and high frequency of hepatitis C virus genotype 3 in hepatocellular carcinoma in Brazil

Occult hepatitis B virus (HBV) infection has been reported among patients with hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC). Our aim was to evaluate the presence of occult HBV infection in patients with HCV-related liver cirrhosis (LC) with or without HCC in São Paulo, Brazil. Serum and liver tissue samples from 50 hepatitis B surface antigen-negative patients with HCV-related LC who underwent liver transplantation at the University of São Paulo School of Medicine Hospital from 1993 to 2004 were divided into groups with LC only (N = 33) and with LC plus HCC (N = 17). HBV DNA was assayed for serum and paraffin-embedded liver tissue (tumoral and non-tumoral) using real time PCR and only 1 case with HCC had HBV DNA-positive serum. All liver samples were negative. HCV genotype 3 was detected in 17/39 (43.7%) cases. In conclusion, using a sensitive real time PCR directed to detect HBV variants circulating in Brazil, occult hepatitis B infection was not found among HCV-positive cirrhotic patients and was rarely found among HCV-positive HCC patients. These results are probably related to the low prevalence of HBV infection in our population. Furthermore, we have also shown that HCV genotype 3 is frequently found in Brazilian cirrhotic patients, particularly when they also have HCC. More studies involving a large number of cases should be carried out to confirm these data and to further characterize Brazilian HCV genotype isolates to elucidate genetic features that might be related to its carcinogenic potential.

There is no difference in hepatic fibrosis rates of patients infected with hepatitis C virus and those co-infected with HIV

Some studies have suggested that human immunodeficiency virus (HIV) infection modifies the natural history of hepatitis C virus (HCV) infection, accelerating the progression of fibrosis and the development of cirrhosis. Our objective was to evaluate the fibrosis progression rate (FPR) in HCV/HIV-co-infected patients, and to identify factors that may influence it. HCV-mono-infected and HCV/HIV-co-infected patients with a known date of HCV infection (transfusion or injection drug use) and a liver biopsy were included. The FPR was defined as the ratio between the fibrosis stage (Metavir score) and the estimated length of infection in years and the result was reported as fibrosis units per year. The factors studied were gender, age at infection, consumption of alcohol, aminotransferase levels, histological activity grade, HCV genotype and viral load, CD4 cell count, HIV viral load, and the use of antiretroviral therapy. Sixty-five HCV-infected (group 1) and 53 HCV/HIV-co-infected (group 2) patients were evaluated over a period of 19 months. The mean FPR of groups 1 and 2 was 0.086 ± 0.074 and 0.109 ± 0.098 fibrosis units per year, respectively (P = 0.276). There was a correlation between length of HCV infection and stage of fibrosis in both groups. The age at infection, the aspartate aminotransferase level (r = 0.36) and the inflammatory activity grade were correlated with the FPR (P < 0.001). No difference in FPR was found between HCV-mono-infected and HCV/HIV-co-infected patients.

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4%) and 3 (7.6%) were found. Subtype 1a (65.7%) was the most prevalent, followed by subtypes 1b (26.7%) and 3a (7.6%). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4%) were classified as genotype 1, subtypes 1a (72.6%) and 1b (19.8%), and 8 sequences (7.6%) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2% of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1% for subtype 1a and 95.2% for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

Cost-effective analysis of different algorithms for the diagnosis of hepatitis C virus infection

We compared the cost-benefit of two algorithms, recently proposed by the Centers for Disease Control and Prevention, USA, with the conventional one, the most appropriate for the diagnosis of hepatitis C virus (HCV) infection in the Brazilian population. Serum samples were obtained from 517 ELISA-positive or -inconclusive blood donors who had returned to Fundação Pró-Sangue/Hemocentro de São Paulo to confirm previous results. Algorithm A was based on signal-to-cut-off (s/co) ratio of ELISA anti-HCV samples that show s/co ratio ³95% concordance with immunoblot (IB) positivity. For algorithm B, reflex nucleic acid amplification testing by PCR was required for ELISA-positive or -inconclusive samples and IB for PCR-negative samples. For algorithm C, all positive or inconclusive ELISA samples were submitted to IB. We observed a similar rate of positive results with the three algorithms: 287, 287, and 285 for A, B, and C, respectively, and 283 were concordant with one another. Indeterminate results from algorithms A and C were elucidated by PCR (expanded algorithm) which detected two more positive samples. The estimated cost of algorithms A and B was US$21,299.39 and US$32,397.40, respectively, which were 43.5 and 14.0% more economic than C (US$37,673.79). The cost can vary according to the technique used. We conclude that both algorithms A and B are suitable for diagnosing HCV infection in the Brazilian population. Furthermore, algorithm A is the more practical and economical one since it requires supplemental tests for only 54% of the samples. Algorithm B provides early information about the presence of viremia.

Antiphospholipid antibodies in Brazilian hepatitis C virus carriers

Hepatitis C, a worldwide viral infection, is an important health problem in Brazil. The virus causes chronic infection, provoking B lymphocyte dysfunction, as represented by cryoglobulinemia, non-organ-specific autoantibody production, and non-Hodgkin's lymphoma. The aim of this research was to screen for the presence of antiphospholipid autoantibodies in 109 Brazilian hepatitis C virus carriers without clinical history of antiphospholipid syndrome. Forty healthy individuals were used as the control group. IgA, IgG, and IgM antibodies against cardiolipin and β2-glycoprotein I were measured with an enzyme-linked immunosorbent assay, using a cut-off point of either 20 UPL or 20 SBU. While 24 (22.0%) hepatitis C carriers had moderate titers of IgM anticardiolipin antibodies (median, 22.5 MPL; 95%CI: 21.5-25.4 MPL), only three carriers (<3%) had IgG anticardiolipin antibodies (median, 23 GPL; 95%CI: 20.5-25.5 GPL). Furthermore, IgA anticardiolipin antibodies were not detected in these individuals. Male gender and IgM anticardiolipin seropositivity were associated in the hepatitis C group (P = 0.0004). IgA anti-β2-glycoprotein-I antibodies were detected in 29 of 109 (27.0%) hepatitis C carriers (median, 41 SAU; 95%CI: 52.7-103.9 SAU). Twenty patients (18.0%) had IgM anti-β2-glycoprotein I antibodies (median, 27.6 SMU; 95%CI: 23.3-70.3 SMU), while two patients had IgG antibodies against this protein (titers, 33 and 78 SGU). Antiphospholipid antibodies were detected in only one healthy individual, who was seropositive for IgM anticardiolipin. We concluded that Brazilian individuals chronically infected with hepatitis C virus present a significant production of antiphospholipid antibodies, mainly IgA anti-β2-glycoprotein I antibodies, which are not associated with clinical manifestations of antiphospholipid syndrome.

Distribution of the human leukocyte antigen class II alleles in Brazilian patients with chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a global medical problem. The current standard of treatment consists of the combination of peginterferon plus ribavirin. This regimen eradicates HCV in 55% of cases. The immune response to HCV is an important determinant of disease evolution and can be influenced by various host factors. HLA class II may play an important role in immune response against HCV. The objective of the present study was to determine the distribution of HLA class II (DRB1 and DQB1) alleles, their association with chronic HCV infection and their response to interferon therapy. One hundred and two unrelated white Brazilian patients with chronic HCV infection, 52 responders (45 males and 7 females) and 50 non-responders (43 males and 7 females) to antiviral treatment, were included in the study. Healthy Brazilian bone marrow donors of Caucasian origin from the same geographic area constituted the control group (HLA-DRB1, N = 99 and HLA-DQB1, N = 222 individuals). HLA class II genotyping was performed using a low-resolution DRB1, DQB1 sequence-specific primer amplification. There were higher frequencies of HLA-DRB1*13 (26.5 vs 14.1%) and HLA-DQB1*02 (52.9 vs 38.7%) in patients compared with controls; however, these were not significantly different after P correction (Pc = 0.39 and Pc = 0.082, respectively). There was no significant difference between the phenotypic frequencies of HLA-DRB1 (17.3 vs 14.0%) and HLA-DQB1 alleles in responder and non-responder HCV patients. The HLA-DRB1*07 allele was significantly more common in HCV patients (33.3 vs 12.1%) than in controls (Pc = 0.0039), suggesting that the HLA-DRB1*07 allele is associated with chronic HCV infection.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilson’s disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8% of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3% of the patients with fibrosis grade 1+2 (OR = 1.8; 95%CI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95%CI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients.

Clinical features of patients with type 2 diabetes mellitus and hepatitis C infection

The objective of the present cross-sectional study was to assess the prevalence and the clinical and laboratory features of hepatitis C virus (HCV)-positive patients with type 2 diabetes mellitus (DM) attending either an outpatient clinic or hemodialysis units. Serologic-HCV testing was performed in 489 type 2 DM patients (303 outpatients and 186 on dialysis). A structured assessment of clinical, laboratory and DM-related complications was performed and the patients were then compared according to HCV infection status. Mean patient age was 60 years; HCV positivity (HCV+) was observed in 39 of 303 (12.9%) outpatients and in 34 of 186 (18.7%) dialysis patients. Among HCV+ patients, 32 were men (43.8%). HCV+ patients had higher serum levels of aspartate aminotransferase (0.90 ± 0.83 vs 0.35 ± 0.13 µKat/L), alanine aminotransferase (0.88 ± 0.93 vs 0.38 ± 0.19 µKat/L), gamma-glutamyl transferase (1.57 ± 2.52 vs 0.62 ± 0.87 µKat/L; P < 0.001), and serum iron (17.65 ± 6.68 vs 14.96 ± 4.72 µM; P = 0.011), and lower leukocyte and platelet counts (P = 0.010 and P < 0.001, respectively) than HCV-negative (HCV-) patients. HCV+ dialysis patients had higher diastolic blood pressure than HCV- patients (87.5 ± 6.7 vs 81.5 ± 6.0 mmHg; P = 0.005) and a lower prevalence of diabetic retinopathy (75 vs 92.7%; P = 0.007). In conclusion, our study showed that HCV is common among subjects with type 2 DM but is not associated with a higher prevalence of chronic diabetic complications.

Role of p53 codon 72 polymorphism in chromosomal aberrations and mitotic index in patients with chronic hepatitis B

Polymorphisms of the p53 gene, which participates in DNA repair, can affect the functioning of the p53 protein. The Arg and Pro variants in p53 codon 72 were shown to have different regulation properties of p53-dependent DNA repair target genes that can affect various levels of cytogenetic aberrations in chronic hepatitis B patients. The present study aimed to examine the frequency of chromosomal aberrations and the mitotic index in patients with chronic hepatitis B and their possible association with p53 gene exon 4 codon 72 Arg72Pro (Ex4+119 G>C; rs1042522) polymorphism. Fifty-eight patients with chronic hepatitis B and 30 healthy individuals were genotyped in terms of the p53 gene codon 72 Arg72Pro polymorphism by PCR-RFLP. A 72-h cell culture was performed on the same individuals and evaluated in terms of chromosomal aberrations and mitotic index. A high frequency of chromosomal aberrations and low mitotic index were detected in the patient group compared to the control group. A higher frequency of chromosomal aberrations was detected in both the patient and the control groups with a homozygous proline genotype (13 patients, 3 control subjects) compared to patients and controls with other genotypes [Arg/Pro (38 patients, 20 control subjects) and Arg/Arg (7 patients, 7 control subjects)]. We observed an increased frequency of cytogenetic aberrations in patients with chronic hepatitis B. In addition, a higher frequency of cytogenetic aberrations was observed in p53 variants having the homozygous proline genotype compared to variants having other genotypes both in patients and healthy individuals.

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.