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.

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.

Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Interactions between thalassemia and hemochromatosis may further increase iron overload. The ethnic background of the Brazilian population is heterogeneous and studies analyzing the simultaneous presence of HFE and thalassemia-related mutations have not been carried out. The aim of this study was to evaluate the prevalence of the H63D, S65C and C282Y mutations in the HFE gene among 102 individuals with alpha-thalassemia and 168 beta-thalassemia heterozygotes and to compare them with 173 control individuals without hemoglobinopathies. The allelic frequencies found in these three groups were 0.98, 2.38, and 0.29% for the C282Y mutation, 13.72, 13.70, and 9.54% for the H63D mutation, and 0, 0.60, and 0.87% for the S65C mutation, respectively. The chi-square test for multiple independent individuals indicated a significant difference among groups for the C282Y mutation, which was shown to be significant between the beta-thalassemia heterozygote and the control group by the Fisher exact test (P value = 0.009). The higher frequency of inheritance of the C282Y mutation in the HFE gene among beta-thalassemic patients may contribute to worsen the clinical picture of these individuals. In view of the characteristics of the Brazilian population, the present results emphasize the need to screen for HFE mutations in beta-thalassemia carriers.

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 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.

The aim of the present study was to evaluate the prevalence of HEV, TTV and GBV-C/GBV-C/HGV in patients with acute viral hepatitis A, B and non-A-C. We evaluated sera of 94 patients from a sentinel program who had acute hepatitis A (N = 40), B (N = 42) and non-A-C (N = 12); 71 blood donors served as controls. IgM and anti-HEV IgG antibodies were detected by enzyme immunoassay using commercial kits. TTV and GBV-C/HGV were detected by nested PCR; genotyping was done by sequencing and phylogenetic analysis. Anti-HEV IgG was present in 38, 10 and 17% of patients with hepatitis A, B and non-A-C. Four patients with hepatitis A and 1 with non-A-C hepatitis also had anti-HEV IgM detected in serum. TTV was detected in 21% of patients with acute hepatitis and in 31% of donors. GBV-C/HGV was detected in 9% of patients with hepatitis, and in 10% of donors. We found TTV isolates of genotypes 1, 2, 3, and 4 and GBV-C/HGV isolates of genotypes 1 and 2. Mean aminotransferase levels were lower in patients who were TTV or GBV-C/HGV positive. In conclusion, the detection of anti-HEV IgM in some acute hepatitis A cases suggests co-infection with HEV and hepatitis E could be the etiology of a few cases of sporadic non-A-C hepatitis in Salvador, Brazil. TTV genotype 1, 2, 3 and 4 isolates and GBV-C/HGV genotype 1 and 2 strains are frequent in the studied population. TTV and GBV-C/HGV infection does not appear to have a role in the etiology of acute hepatitis.

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.

Occult hepatitis B virus (HBV) infection has been reported as cases in which HBV DNA was detected despite the absence of any HBV serological markers or in cases in which anti-HBc antibody was the sole marker. The aim of the present study was to determine, using the polymerase chain reaction (PCR), whether HBV infection occurs in hepatitis C and non-A-E hepatitis patients without serological evidence of hepatitis B infection in São Paulo State. Two different populations were analyzed: 1) non-A-E hepatitis patients, including 12 patients with acute and 50 patients with chronic hepatic disorders without serological evidence of infection with known hepatitis viruses; 2) 43 patients previously diagnosed as hepatitis C with positive results for anti-HCV and HCV RNA. Among hepatitis C patients, anti-HBc was detected in 18.6% of the subjects. Three different sets of primers were employed for HBV DNA detection by nested PCR, covering different HBV genes: C, S and X. HBV-DNA was not detected in any sample, whereas the positive controls did produce signals. The lack of HBV DNA detection with these pairs of primers could be due to a very low viral load or to the presence of mutations in their annealing sites. The latter is unlikely as these primers were screened against an extensive dataset of HBV sequences. The development of more sensitive methods, such as real time PCR, to detect circular covalent closed DNA is necessary in order to evaluate this question since previous studies have shown that cryptic hepatitis B might occur.

Few data are available in the literature concerning the efficacy of standard hysteroscope disinfection procedures to prevent hepatitis B transmission. The aim of the present study was to determine the risk of hepatitis B virus (HBV) transmission during hysteroscopy among anti-HBc-seropositive women. Serum and hysteroscopic samples were collected from 62 women after diagnostic hysteroscopy. All samples were tested for serologic HBV markers. Polymerase chain reactions (PCR) were carried out to amplify regions C and S of the viral genome and only samples amplified by both pairs of primers were considered to be positive. Anti-HBc was repeatedly reactive in 48 (77%) of 62 serum samples, and HBsAg was detected in 8 (13%). At least one HBV serologic marker was found in 49 (79%) samples. Only one sample was HBsAg positive and anti-HBc negative. HBV-DNA was detected by PCR in 7 serum samples but in only 3 hysteroscopic samples obtained just after hysteroscopy. It is noteworthy that high levels of anti-HBc IgM were detected in one HBsAg-negative patient who showed an HBV-DNA-positive hysteroscopic sample. An elevated sample/cut-off ratio for anti-HBc IgM suggests recent infection and reinforces the need for testing for HBsAg and anti-HBc before hysteroscopy, since acute hepatitis B can be clinically asymptomatic. Viral DNA was not detected in any hysteroscopic samples collected after washing and disinfecting procedures with glutaraldehyde. We conclude that HBV-DNA can be found in the hysteroscope soon after hysteroscopy, but standard disinfecting procedures are effective in viral removal.

Parvovirus B19 has been associated by some investigators with cases of severe hepatitis. The aim of the present study was to determine the presence of active parvovirus B19 infection among 129 Brazilian patients with non-A-E hepatitis. The patients were assayed for antibodies against parvovirus B19, IgM class, by ELISA. In IgM-positive cases, parvovirus B19 DNA was assayed by PCR in serum and liver tissue and parvovirus VP1 antigen in liver tissue was assayed by immunohistochemistry. Antibodies against parvovirus B19, IgM class, were detected in 3 (2.3%) of 129 patients with non-A-E hepatitis. Previous surgery and blood transfusions were reported by these 3 patients. One patient was a 56-year-old female with severe hepatitis, with antimitochondrial antibody seropositivity and submassive necrosis at liver biopsy, who responded to corticosteroid therapy. Strong evidence for active parvovirus B19 infection was found in this patient, with parvovirus B19 DNA being detected by PCR in liver tissue. Furthermore, parvovirus VP1 antigen was also detected in liver tissue by immunohistochemistry. The other two IgM-positive patients were chronic hepatitis cases, but active infection was not proven, since neither viral DNA nor antigen were detected in their liver tissues. This and other reports suggest a possible relation between parvovirus B19 infection and some cases of hepatitis.

O vírus da Hepatite G ou vírus GB-C é um novo vírus recentemente descoberto em casos de hepatites não A-E. Neste estudo, casos de hepatite crônica não-B, não-C foram testados com uma reação de amplificação específica para GBV-C. Este vírus foi detectado em 1 entre os 13 casos estudados. Este paciente apresentava insuficiência hepática severa, tinha habitado por vários anos na Amazônia Ocidental e nenhuma outra causa para este quadro clínico havia sido relatada. O impacto da descoberta deste novo agente está ainda sendo investigado. O estudo da prevalência deste vírus entre pacientes com hepatite crônica e entre indivíduos sadios (como, por exemplo, doadores de sangue) fornecerá subsídios para o estabelecimento de sua real patogenicidade.

Hepatitis G virus/ GB virus C is a novel flavivirus recently detected in hepatitis non A-E cases. In this study, the presence of this virus in chronic non-B, non-C hepatitis patients was evaluated using GBV-C specific PCR and this virus was detected in one out of thirteen patients. This patient has presented a severe liver failure, has lived for a long time in the Western Amazon basin and no other cause for this clinical picture was reported. The impact of the discovery of this new agent is still under evaluation throughout the world. The study of the prevalence of this virus among chronic hepatitis patients and healthy individuals (as blood donors) will furnish subside to evaluate its real pathogenicity.

A detecção do DNA do Vírus da Hepatite B pela Reação em Cadeia da Polimerase (PCR) foi comparada com os outros marcadores sorológicos virais (AgHBs, AgHBe e anti-HBe) numa série de 49 pacientes com hepatite crônica B, incluindo 12 que apresentaram clareamento espontâneo do AgHBs. Nenhum caso AgHBs negativo foi PCR positivo, mas 33/37 (89,2%) dos casos AgHBs positivos foram PCR positivos (p < 0,0001). Entre as amostras AgHBs positivas, 9 foram AgHBc positivas e anti-HBe negativas, todas elas PCR positivas. Outros 3 pacientes foram AgHBe e anti-HBe positivos, todos também PCR positivos. Um terceiro grupo incluía 21 pacientes anti-HBe positivos e anti-HBe negativos: 19 foram PCR positivos e 2 PCR negativos. Os últimos 4 casos foram AgHBe e anti-HBe negativos, sendo 2 destes PCR positivos. A detecção de casos virêmicos anti-HBe positivos sugere que as variantes preC do VHB podem também estar presentes em nosso país. Em conclusão, a "fase de integração viral" da hepatite crônica B parece ser muito menos frequente do que se imaginava previamente, quando apenas se dispunha das técnicas de detecção de AgHBe e DNA por hibridização. O novo termo "fase de baixa replicação baixa" parece ser melhor que o anterior "fase de integração viral" .

Detection of HBV-DNA by PCR was compared with other serological markers (HBsAg, HBeAg and anti-HBe) in a series of49 Chronic Hepatitis B patients, including 12 with a spontaneous clearance of HBsAg. None of these HBsAg negative cases were PCR positive, but 33/37 (89.2%) HBsAg positive cases were PCR positive (p < 0.0001). Among HBsAg positive samples, nine cases were HBeAg positive and anti-HBe negative, all of them PCR positive. Other 3 patients were HBeAg and anti-HBe positive and these cases were also found PCR positive. A third group included 21 patients anti-HBe positive and HBeAg negative: 19 of them were PCR positive and 2 were PCR negative. The last 4 cases were HBeAg and anti-HBe negative, two of them were PCR positive. The detection of anti-HBe viremic cases in the present series suggest that preC variants could occur in our country. In conclusion, the integrated phase o f chronic hepatitis B seems to be less frequent than it was assumed, when only HBeAg or dot blot hybridization techniques were used. The new term "low replication phase" might favorably replace the former "integrated phase".