Montserrat Cotrina

HEV identified in serum from humans with acute hepatitis and in sewage of animal origin in Spain

BACKGROUND/AIMS Hepatitis E virus (HEV) is an enterically transmitted pathogen that appears sporadically in non-endemic countries. We studied HEV as a causal agent of acute hepatitis cases in the Spanish population, and the role of pigs as an animal reservoir. METHODS The presence of HEV-RNA was analysed by nested polymerase chain reaction in 37 serum samples from patients with acute viral hepatitis, 48 porcine serum samples, 6 pig faecal samples and 12 slaughter-house sewage samples. Presence of antibodies was also tested in porcine sera. RESULTS HEV-RNA was found in 3 human serum samples from patients presenting IgG anti-HEV antibodies. Nucleotide sequence analysis identified 2 strains with 93.4% identity, phylogenetically most closely related to the Greece1 isolate, and more closely related to North American and other European strains than to those from endemic regions. HEV-RNA was also detected in slaughterhouse sewage mainly from pigs, presenting 92-94% nucleotide similarity compared to the strains detected in the human sera. Twenty-five per cent of the pigs tested presented IgG anti-HEV antibodies. CONCLUSIONS These data suggest that the HEV could be more widespread than previously thought, and present new evidence of the close relationship between HEV strains detected in pigs and those from acute hepatitis patients.

Hepatitis E virus infection in acute hepatitis in Spain.

In order to study the prevalence of hepatitis E virus (HEV) infection in developed countries, IgG and IgM anti-HEV were determined in serum samples from 382 patients with acute viral hepatitis (244 hepatitis A, 48 hepatitis B and/or D, and 90 non-A, non-B, non-C hepatitis), 76 healthy subjects, 55 hemophiliacs and 50 patients on hemodialysis. IgG anti-HEV antibodies were detected and confirmed by a synthetic peptide-based EIA in 5 (5.6%) non-A, non-B, non-C acute hepatitis, in 3 (6.5%) B and D acute hepatitis, in 10 (4%) acute A hepatitis, in 3 (5.5%) of 54 healthy adults in none of the hemophiliacs and in 3 (6%) patients on hemodialysis. IgM anti-HEV antibodies were only detected in two cases of acute hepatitis B and/or D. Analysis of serial serum samples demonstrated IgG anti-HEV seroconversion in 3 of the 18 confirmed cases; one of them was also positive for IgM anti-HEV. All 3 acute anti-HEV-positive hepatitis cases occurred in adults, were community-acquired (two of them were intravenous drug addicts) and had a self-limited course. These results demonstrate that HEV is a minor cause of acute hepatitis in Spain. A similar low rate of IgG anti-HEV antibodies was detected in patients with different diseases, suggesting that HEV has a very low epidemiological impact. An apparent association of HEV infection with hepatitis B and D suggests a possible parenteral transmission of a mainly enteral pathogen.

Hepatitis delta genotypes in chronic delta infection in the northeast of Spain (Catalonia)

Abstract Background/Aims: Based on genetic analysis of variants obtained around the world, three genotypes of the hepatitis delta virus have been defined. Hepatitis delta virus variants have been associated with different disease patterns and geographic distributions. To determine the prevalence of hepatitis delta virus genotypes in the northeast of Spain (Catalonia) and the correlation with transmission routes and clinical disease, we studied the nucleotide divergence of the consensus sequence of HDV RNA obtained from 33 patients with chronic delta hepatitis (24 were intravenous drug users and nine had no risk factors), and four patients with acute self-limited delta infection. Methods: Serum HDV RNA was amplified by the polymerase chain reaction technique and a fragment of 350 nucleotides (nt 910 to 1259) was directly sequenced. Results Genetic analysis of the nucleotide consensus sequence obtained showed a high degree of conservation among sequences (93% of mean). Comparison of these sequence with those derived from different geographic areas and pertaining to genotypes I, II and III, showed a mean sequence identity of 92% with genotype I, 73% with genotype II and 61% with genotype III. At the amino acid level (aa 115 to 214), the mean identity was 87% with genotype I, 63% with genotype II and 56% with genotype III. Conserved regions included the RNA editing domain, the carboxyl terminal 19 amino acids of the hepatitis delta antigen and the polyadenylation signal of the viral mRNA. Conclusions: Hepatitis delta virus isolates in the northeast of Spain are exclusively genotype I, independently of the transmission route and the type of infection. No hepatitis delta virus subgenotypes were found, suggesting that the origin of hepatitis delta virus infection in our geographical area is homogeneous.

Transient emergence of hepatitis B variants in a patient with chronic hepatitis B resistant to lamivudine

BACKGROUND Lamivudine is a cytosine nucleoside analogue that inhibits hepatitis B virus replication. Resistance to lamivudine monotherapy has been reported in patients who received lamivudine to prevent recurrent hepatitis B virus infection after liver transplantation. No cases of resistance have been described in patients who did not clear HBV DNA during lamivudine therapy. METHODS We report the case of an adult patient with chronic HBeAg-positive hepatitis B who had a hepatitis flare during lamivudine therapy. The patient did not respond to lamivudine and, at 4 months of treatment, developed a significant serum alanine aminotransferase elevation. Alanine aminotransferase levels remained elevated for 4 months and returned to baseline spontaneously. Lamivudine therapy was administered for 1 year (52 weeks) and after withdrawal, alanine aminotransferase levels remained elevated. RESULTS Sequencing studies of HBV DNA at week 52 showed the emergence of a lamivudine-resistant variant associated with two point mutations in the hepatitis B virus polymerase gene: one mutation led to amino acid substitution of methionine to valine at residue 552, in the highly conserved tyrosine-methionineaspartate-aspartate motif, part of the active site of the polymerase; the second mutation consisted of a substitution of leucine to methionine at residue 528. At week 54 of follow-up, both mutations were undetectable. CONCLUSION This observation demonstrates the transient emergence of HBV variants in the course of therapy in a patient resistant to lamivudine therapy.

Quantitative hepatitis B virus DNA testing for the early prediction of the maintenance of response during lamivudine therapy in patients with chronic hepatitis B.

To determine whether a dramatic decrease in hepatitis B virus (HBV) DNA levels within the first months of lamivudine therapy can predict the emergence of YMDD variants in patients with chronic hepatitis B, quantitative testing was done every 3 months on serum samples from 35 patients who were treated with lamivudine for >1 year. The decline in HBV DNA levels from baseline to month 3 was higher in 22 responders than in 13 nonresponders (mean+/-SD, 4.16+/-1.06 vs. 2.88+/-1.77 log(10) copies; P=.002), whereas no differences were observed in patients with and without YMDD variants at 1 year of therapy. At 3 months, HBV DNA was undetectable in 77% of the responders, whereas, after 1 year, it was undetectable in 23% of nonresponders, 40% of patients with YMDD variants, and 74% of those without variants. Therefore, quantitative HBV DNA testing is very useful in deciding whether to continue therapy, because of the low likelihood of response in patients who remain HBV DNA positive at month 3 of treatment.

Chronic delta hepatitis : Is the prognosis worse when associated with hepatitis C virus and human immunodeficiency virus infections ?

Eighty‐six patients were followed for 6.5 years to study the epidemiological, virological, and histological course of chronic delta hepatitis and the relationship of this disease with HIV and HCV infection.

Prevalencia e incidencia de la infección por el virus de la hepatitis A en pacientes con hepatitis crónica B y C

Fundamento Los pacientes con hepatitis cronica tienen un mayor riesgo de desarrollar una hepatitis fulminante al sobreinfec-tarse por el virus de la hepatitis A (VHA). Pacientes y metodos Se han determinado los anticuerpos IgG anti-VHA en 353 hepatitis cronicas B o C, y los IgM en las hepatitis agudas. Resultados La prevalencia de anticuerpos IgG anti-VHA fue del 81% en la hepatitis cronica C y del 77% en la hepatitis B, rela-cionandose con la edad. No se detecto ninguna hepatitis aguda A. Conclusiones La prevalencia del VHA es elevada en las hepatitis cronicas virales mientras que la incidencia es baja. La va-cunacion antihepatitis A deberra realizarse con cribado previo.

Comparative analysis of serological markers of chronic delta infection: HDV-RNA, serum HDAg and anti-HD IgM

To study the concordance, sensitivity and specificity of HDV-RNA determination by molecular hybridization, serum HDAg by immunoblot and anti-HD IgM by commercial enzyme immunoassay as compared to intrahepatic HDAg detection by an immunoperoxidase method, a statistical analysis was applied to the results of serum sample and liver biopsy determinations in 50 patients with chronic delta hepatitis (38 positive to tissue HDAg and 12 negative). Of the 38 patients with hepatic HDAg, HDV-RNA was found in 31 (82%), serum HDAg by immunoblot in 27 (71%) and anti-HD IgM in 33 (87%). Among the 12 patients without hepatic HDAg, one was found with serum HDAg using the immunoblot technique, two (17%) had HDV-RNA, and 7 (58%) had anti-HD IgM. Serum HDAg determination by immunoblot was the most specific test, followed by HDV-RNA analysis. The least specific was the anti-HD IgM technique. The anti-HD IgM test was the most sensitive, followed by HDV-RNA and serum HDAg. The concordance with intrahepatic HDAg detection was highest for HDV-RNA determination, followed by HDAg in serum. The least degree of concordance was found with anti-HD IgM determination. These results suggest that the determination of HDV-RNA by the hybridization method can be of great value for the diagnosis and monitoring of chronic delta hepatitis.

Detection of hepatitis B precore mutants by the fluorescent linear polymerase chain reaction sequencing method

To evaluate the presence of precore mutants, serum samples from 25 patients with chronic hepatitis B, HBV-DNA positive (5 HBeAg and 20 anti-HBe positive) were studied. The complete precore-core region of HBV-DNA was directly sequenced after polymerase chain reaction amplification by a fluorescent linear polymerase chain reaction sequencing method. Precore variants were detected in one HBeAg positive and in all 20 anti-HBe positive patients: in 19 cases, G to A at position 1896, with or without the substitution G to A at position 1899, in two cases C to T substitution at position 1817 which also produces a stop codon (CAA to TAA), one accompanied by the mutation G to A at position 1896. The only mutation observed in the patient who was initially HBeAg positive patient was a G to A substitution at position 1899. Consecutive serum samples from a patient with chronic hepatitis B, initially had the simultaneous presence of wild type and variant strains. Elimination of the wild-type strain was associated with reactivation of liver disease. Analysis of the sequences obtained demonstrated the heterogeneity of the hepatitis B virus genome in the precore-core region. These results indicate that the main cause of non-expression of HBeAg in chronic hepatitis B in our country is the substitution of G to A at nucleotide 1896, alone or accompanied by other variants. Fluorescent linear polymerase chain reaction is a fast and sensitive method to study heterogeneity in the precore-core region.

Interferon vs. adenine arabinoside 5'-monophosphate in patients with anti-HBe-positive chronic hepatitis.

Anti‐HBe‐positive patients with precore mutants may have severe, progressive liver disease. Therapy with interferon has been effective, but relapses are frequent. To evaluate and compare two antiviral treatments, lymphoblastoid interferon (ly‐IFN) and adenine arabinoside 5′‐monophosphate (ARA‐AMP), 20 patients with anti‐HBe‐positive chronic hepatitis (5 cirrhosis and 15 CAH) and viral replication (HBcAg in the liver and HBV DNA in serum) were treated. Patients were randomized into two groups: 11 patients received ARA‐AMP, 5 mg/kg/day during 7 weeks, and 9 received human ly‐IFN, 5,000,000 units, three times per week, during 4 months. Baseline clinical, biochemical and histological features were not significantly different between the two groups. At the end of therapy, 8 (89%) patients in the interferon group and 5 (45%) in the ARA‐AMP group showed normal ALT levels and no HBV DNA in serum by a liquid hybridization assay (P < 0.05). At 1 year of follow‐up, a persistent response was observed in 33% of ly‐IFN patients and in 27% of ARA‐AMP patients, a transient response in 56% and 18%, and nonresponse in 11% and 55%, respectively. HBV DNA remained detectable by polymerase chain reaction (PCR) in 19 of the 20 patients. Among the responders, an improvement in histological lesion and the disappearance of intrahepatic HBcAg were observed; in the nonresponders, histological lesion remained stable or worsened. In conclusion, the efficacy of interferon and ARA‐AMP was similar in treating anti‐HBe‐positive chronic hepatitis. Although interferon treatment led to initial improvement in a larger number of patients, there was a much higher rate of relapses with this drug.