Geographic distribution of hepatitis C virus genotypes in Brazil

Brazil is a country of continental dimension with a population of different ethnic backgrounds. Thus, a wide variation in the frequencies of hepatitis C virus (HCV) genotypes is expected to occur. To address this point, 1,688 sequential samples from chronic HCV patients were analyzed. HCV-RNA was amplified by the RT-PCR from blood samples collected from 1995 to 2000 at different laboratories located in different cities from all Brazilian States. Samples were collected in tubes containing a gel separator, centrifuged in the site of collection and sent by express mail in a refrigerated container to Laboratório Bioquímico Jardim Paulista, São Paulo, SP, Brazil. HCV- RNA was extracted from serum and submitted to RT and nested PCR using standard procedures. Nested PCR products were submitted to cycle sequencing reactions without prior purification. Sequences were analyzed for genotype determination and the following frequencies were found: 64.9% (1,095) for genotype 1, 4.6% (78) for genotype 2, 30.2% (510) for genotype 3, 0.2% (3) for genotype 4, and 0.1% (2) for genotype 5. The frequencies of HCV genotypes were statistically different among Brazilian regions (P = 0.00017). In all regions, genotype 1 was the most frequent (51.7 to 74.1%), reaching the highest value in the North; genotype 2 was more prevalent in the Center-West region (11.4%), especially in Mato Grosso State (25.8%), while genotype 3 was more common in the South (43.2%). Genotypes 4 and 5 were rarely found and only in the Southeast, in São Paulo State. The present data indicate the need for careful epidemiological surveys throughout Brazil since knowing the frequency and distribution of the genotypes would provide key information for understanding the spread of HCV.

Hepatitis C virus seroprevalence and genotypes in patients with diffuse connective tissue diseases and spondyloarthropathies

Many extrahepatic manifestations, including rheumatic diseases, have been reported to be associated with hepatitis C virus (HCV) infection. In order to investigate the prevalence of HCV infection among patients with rheumatic diseases, in the present study we interviewed 367 patients and tested their blood samples for HCV antibodies (anti-HCV) by an enzyme-linked immunosorbent assay. Anti-HCV-reactive samples were retested for confirmation by a line immunoassay and also for HCV RNA detection by the polymerase chain reaction. HCV RNA-positive samples were genotyped by INNO-LIPA. An overall HCV infection prevalence of 1.9% (7/367) was found. Of the 7 HCV-infected patients, 4 had systemic lupus erythematosus and 3 rheumatoid arthritis, resulting in positivity rates of 2.3 and 3.4%, respectively. HCV RNA genotyping revealed the presence of subtypes 1a (57.1%), 1b (28.6%) and 3a (14.3%). The clinical course was favorable for all HCV-infected patients, except one, who died due to renal insufficiency related to lupus nephritis. These results demonstrate a low HCV infection prevalence among the population studied. In the few positive cases, we observed no adverse influence of this infection on the clinical evolution of the rheumatic disease.

A prospective study of hepatitis B virus markers in patients with chronic HBV infection from Brazilian families of Western and Asian origin

The purpose of the present study was to determine the frequency of hepatitis B virus (HBV) markers in families of HBsAg-positive patients with chronic liver disease. Serum anti-HBc, HBsAg and anti-HBs were determined by enzyme immunoassay and four subpopulations were considered: genetically related (consanguineous) and non-genetically related (non-consanguineous) Asian subjects and genetically related and non-genetically related Western subjects. A total of 165 and 186 relatives of Asian and Western origin were enrolled, respectively. The occurrence of HBsAg and anti-HBs antibodies was significantly higher (P < 0.0001) in family members of Asian origin (81.8%) than in family members of Western origin (36.5%). HBsAg was also more frequent among brothers (79.6 vs 8.5%; P < 0.0001), children (37.9 vs 3.3%; P < 0.0001) and other family members (33.9 vs 16.7%; P < 0.0007) of Asian than Western origin, respectivelly. No difference between groups was found for anti-HBs, which was more frequently observed in fathers, spouses and other non-genetic relatives. HBV infection was significantly higher in children of Asian than Western mothers (P < 0.0004). In both ethnic groups, the mothers contributed more to their children's infection than the fathers (P < 0.0001). Furthermore, HBsAg was more frequent among consanguineous members and anti-HBs among non-consanguineous members. These results suggest the occurrence of vertical transmission of HBV among consanguineous members and probably horizontal sexual transmission among non-consanguineous members of a family cluster. Thus, the high occurrence of dissemination of HBV infection characterizes family members as a high-risk group that calls for immunoprophylaxis. Finally, the study showed a high familial aggregation rate for both ethnic groups, 18/19 (94.7%) and 23/26 (88.5%) of the Asian and Western origin, respectively.

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.

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.

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.

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.

Hepatitis B virus subgenotype C2- and B2-associated mutation patterns may be responsible for liver cirrhosis and hepatocellular carcinoma, respectively

The objective of this study was to examine hepatitis B virus (HBV) subgenotypes and mutations in enhancer II, basal core promoter, and precore regions of HBV in relation to risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Southeast China. A case-control study was performed, including chronic hepatitis B (CHB; n=125), LC (n=120), and HCC (n=136). HBV was genotyped by multiplex polymerase chain reaction and subgenotyped by restriction fragment length polymorphism. HBV mutations were measured by DNA sequencing. HBV genotype C (68.2%) predominated and genotype B (30.2%) was the second most common. Of these, C2 (67.5%) was the most prevalent subgenotype, and B2 (30.2%) ranked second. Thirteen mutations with a frequency >5% were detected. Seven mutation patterns (C1653T, G1719T, G1730C, T1753C, A1762T, G1764A, and G1799C) were associated with C2, and four patterns (C1810T, A1846T, G1862T, and G1896A) were associated with B2. Six patterns (C1653T, G1730C, T1753C, A1762T, G1764A, and G1799C) were obviously associated with LC, and 10 patterns (C1653T, G1730C, T1753C, A1762T, G1764A, G1799C, C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with CHB. Four patterns (C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with LC. Multivariate regression analyses showed that HBV subgenotype C2 and C2-associated mutation patterns (C1653T, T1753C, A1762T, and G1764A) were independent risk factors for LC when CHB was the control, and that B2-associated mutation patterns (C1810T, A1846T, G1862T, and G1896A) were independent risk factors for HCC when LC was the control.

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Occult hepatitis B virus infection in liver transplant patients in a Brazilian referral center

Estimates of occult hepatitis B virus (HBV) infection prevalence varies among different studies depending on the prevalence of HBV infection in the study population and on the sensitivity of the assay used to detect HBV DNA. We investigated the prevalence of occult HBV infection in cirrhotic patients undergoing liver transplantation in a Brazilian referral center. Frozen liver samples from 68 adults were analyzed using a nested polymerase chain reaction assay for HBV DNA. The specificity of the amplified HBV sequences was confirmed by direct sequencing of the amplicons. The patient population comprised 49 (72.1%) males and 19 (27.9%) females with a median age of 53 years (range=18-67 years). Occult HBV infection was diagnosed in three (4.4%) patients. The etiologies of the underlying chronic liver disease in these cases were alcohol abuse, HBV infection, and cryptogenic cirrhosis. Two of the patients with cryptic HBV infection also presented hepatocellular carcinoma. Markers of previous HBV infection were available in two patients with occult HBV infection and were negative in both. In conclusion, using a sensitive nested polymerase chain reaction assay to detect HBV DNA in frozen liver tissue, we found a low prevalence of occult HBV infection in cirrhotic patients undergoing liver transplant, probably due to the low prevalence of HBV infection in our population.

Intradermal vaccination of adults with three low doses (2 µg) of recombinant hepatitis B vaccine. II. Persistence of immunity and induction of immunologic memory

Of the 110 dentists who had presented seroconversion 50 days after the intradermal application of three 2 µg doses of the Belgian recombinant vaccine against hepatitis B (HB), administered eight years before at an interval of one month between the 1st and 2nd doses and of five months between the 2nd and 3rd doses, 51 were included for the assessment of the persistence of immunity. None of the dentists had hepatitis or had received HB vaccine during this period. All subjects were submitted to serological tests for the detection of the following markers of hepatitis B virus (HBV) infection: HBsAg, anti-HBc, HBeAg, anti-HBe, and anti-HBs, with no HBsAg, anti-HBc, HBeAg or anti-HBe being detected. A microparticle enzyme immunoassay (MEIA) revealed the presence of anti-HBs at protective titers (> 10 mIU/ml) in 42 dentists (82.4%), with the anti-HBs titer being higher than 100 mIU/ml in 36 of them (70.6%) (good responders), between 10 and 100 mIU/ml in 6 (11.8%) (poor responders), and lower than 10 mIU/ml in 9 (17.6%) (non-responders). According to clinical data and serological tests, none of the dentists had presented disease or latent HBV infection during the eight years following the first vaccination. A 2 µg booster dose was administered intradermally to eight dentists with anti-HBs titers lower than 10 mIU/ml (non-responders) and to six dentists with titers ranging from 10 to 100 mIU/ml (poor responders); the determination of anti-HBs one month later demonstrated the occurrence of seroconversion in the eight non-responders and an increase in anti-HBs titer in the six poor responders. In summary, the present results demonstrated the prolonged persistence of protection against HBV infection and the development of immunologic memory provided by vaccination against HB - with intra-dermal application of three 2 µg doses of the Belgian recombinant vaccine at 0, 1, and 6 months - carried out eight years before in 51 dentists.