Rosendo Jardi

Long-Term Immunogenicity and Efficacy of Hepatitis B Vaccine in Hemodialysis Patients

Although the efficacy of hepatitis B vaccines in patients under chronic hemodialysis treatment has been well documented, the persistence of immunity in this population remains largely unknown. In this study we have followed 60 hemodialysis patients up to 3 years after primary hepatitis B vaccination (four doses of recombinant hepatitis B vaccine; Engerix B, 20 mg/dose) to evaluate the persistence of immunity (as indicated by serum levels of antibody to hepatitis B surface antigen-anti-HBs-higher than or equal to 10 mIU/ml). Fourty-four (73%) patients developed anti-HBs levels above 10 mIU/ml after vaccination; the remaining 16 (27%) vaccinees were considered nonresponders and were given a booster dose that again failed to elicit an immunoresponse. After 3 years of follow-up, 18 out of 44 (41%) responders had no detectable anti-HBs levels in the serum (antibody loss occurring within 8 and 12 months in 3 cases, within 1 and 2 years in 13, and within 2 and 3 years in 2 other cases). When compared with the responders that lost their antibodies during the follow-up period, those who remained immunoreactive 3 years after vaccination was initiated were younger and had higher anti-HBs levels at 8 months of follow-up. Two hepatitis B virus infections were detected among nonresponders during the follow-up period. Based on these data, we conclude that patients undergoing chronic hemodialysis therapy not only have lower response rates to hepatitis B vaccination than healthy adults, but also that these are frequently transient.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative longitudinal evaluations of hepatitis delta virus RNA and hepatitis B virus DNA shows a dynamic, complex replicative profile in chronic hepatitis B and D

BACKGROUND & AIMS This study presents a real-time reverse-transcription PCR (rt-RT-PCR) assay for hepatitis delta virus (HDV) RNA quantification, designed to clarify the interplay between HDV and hepatitis B virus (HBV) in chronic infection. METHODS Serum HDV-RNA and HBV-DNA were analysed by rt-RT-PCR in a cross-sectional study of 37 untreated chronic HDV patients, 25 of whom were also longitudinally studied. RESULTS In the cross-sectional study, both viruses were active in 15 (40.5%) patients and inactive in 4 (10.8%); HDV alone was active in 12 (32.4%) and HBV in 6 (16.2%). The longitudinal study showed seven replication profiles, with considerable fluctuating activity of one or both viruses, including alternating predominance. In 20% of cases, longitudinal HBV/HDV viral loads differed from cross-sectional results, indicating a risk of misinterpreting HBV/HDV interactions when assessing a single determination. Fluctuating HBV replication only increased in the presence of fluctuating HDV activity. HBsAg levels, stable in HBV single infection, fluctuated in HDV chronic infection. The results of both the cross-sectional and longitudinal study call into question the major suppressor effect of HDV over HBV, revealing an important role of HBV. CONCLUSIONS Longitudinal evaluation of viremia shows a complex interaction between HBV and HDV and is essential to understand the pathophysiology of chronic HDV infection.

Usefulness of a national registry of alpha-1-antitrypsin deficiency. The Spanish experience☆

Severe alpha-1-antitrypsin (AAT) deficiency, phenotype Pi ZZ, is a rare condition with an estimated prevalence of 1/4500 individuals in Spain. Given this low prevalence, it seems useful to accumulate all the information derived from the care of these patients. In this context, the Spanish Registry of patients with AAT deficiency was founded in 1993; its main objectives were to establish guidelines adapted to our country for the treatment and management of AAT-deficient patients, offer expert support to physicians all over the country treating these patients, and provide technical support on the determination of Pi phenotyping and genotyping of individuals suspected of being AAT-deficient. From 1993 to January 1998 the number of enrollees increased from 48 to 223, of which 216 were Pi ZZ. Seventy-three per cent were male and only 31.5% were never smokers, mean age was 46 years (SD = 13 years) and mean FEV1 53% predicted (SD = 31%). 83% were index cases who, compared with non-index cases, were older (49 +/- 11 vs. 35 +/- 13 years, P < 0.001), more likely to have a smoking history (85% vs. 47%, P < 0.01) and displayed more severe impairment in pulmonary function (FEV1% = 40% +/- 19% vs. 96% +/- 23%, P < 0.001). Augmentation therapy was administered to 129 patients (58%). Treated patients had more severe impairment in pulmonary function than the untreated (FEV1% = 40% +/- 21% vs. 72% +/- 32%, P < 0.001) and were more likely to be index cases (81% vs. 43%, P < 0.001). Characteristics of the patients included are similar to those described for other Registries. The Registry has extended knowledge of the disease throughout the country and has established local guidelines for treatment and follow-up. It may be a valid database for future co-operation in international initiatives.

Results of a case-detection programme for α1-antitrypsin deficiency in COPD patients

α1-Antitrypsin (α1-AT) deficiency is an underdiagnosed condition in patients with chronic obstructive pulmonary disease (COPD). The present authors have conducted a nationwide case detection programme of α1-AT deficiency in unselected patients with COPD using dried blood spots. The first phase analysed samples from 971 patients by determining α1-AT concentrations and identifying the deficient Z allele by genotyping using rapid real-time PCR. The second phase analysed 1,166 samples with α1-AT concentrations and identified both the S and the Z allele, but only in samples with low α1-AT concentrations. A total of eight (0.37%) individuals with the severe deficiency PiZZ were detected. In addition, three patients were identified with the PiSZ genotype in the second phase (0.3%). The global cost of the programme was \#8364;41,512, which represents \#8364;19.42 per sample and \#8364;5,189 per PiZZ detected. A sensitivity analysis demonstrated that performing Z genotype to all samples would have resulted in increased costs of \#8364;28 per sample and \#8364;7,479.5 per PiZZ case identified. In conclusion, a case detection programme of α1-antitrypsin deficiency in patients with chronic obstructive pulmonary disease using dried blood spots is feasible and at a reasonable cost per case detected. Diagnostic yield and costs depend largely on inclusion criteria and the protocol for processing of samples.

Rare alpha-1-antitrypsin variants: are they really so rare?

Alpha-1-antitrypsin (α1-AT) deficiency is mainly evaluated in the diagnostic process of chronic obstructive pulmonary disease (COPD). Around 95% of individuals with severe α1-AT deficiency carry the PI*ZZ genotype. Little is known about the epidemiology of the remaining deficient α1-AT variants, which are called ‘rare’ due to their low prevalence. The retrospective revision of 3511 α1-AT deficiency determinations performed in Barcelona from 1998 to 2010 detected 1.6% of cases with rare α1-AT alleles, a rate similar to those reported in other European studies. Among these variants, PI*I and PI*Mmalton represented 54% of cases. Hence, the so-called ‘rare’ α1-AT alleles may not be rare as has been assumed. It would be of interest to implement simple allele-specific molecular biology methods to study the most prevalent rare variants in each region. Augmentation therapy is recommended in patients with emphysema and PI*ZZ genotype, but there is little evidence regarding the implications of rare variants on therapy.

Ultra-Deep Pyrosequencing Detects Conserved Genomic Sites and Quantifies Linkage of Drug-Resistant Amino Acid Changes in the Hepatitis B Virus Genome

Background Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region. Methodology/Principal Findings Serum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples. Conclusions UDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants.

The value of quantiative detection of HBV-DNA amplified by PCR in the study of hepatitis B infection

Abstract Background/Aims: This study aimed to evaluate the usefulness of quantifying HBV-DNA amplified by polymerase chain reaction in chronic hepatitis B infection. Methods: Serum samples were obtained from 32 asymptomatic HBV carriers and 99 chronic hepatitis B patients (62 positive for anti-HBe and 37 positive for HBeAg). In addition, serial serum samples were analyzed from 15 HBeAg positive patients undergoing antiviral therapy and 17 anti-HBe positive patients with precore mutation. HBV-DNA quantification was carried out using an enzyme immunoassay with an HBV-DNA plasma standard. Results: The digoxigenin-incorporated polymerase chain reaction method detected HBV-DNA in 34.3% of the asymptomatic HBV carriers with a median HBV-DNA concentration of about 0.18×10 5 mol/ml (range 0.08–0.4), in 87%, of the anti-HBe positive chronic hepatitis cases with a range of 0.2 to >2×10 5 mol/ml and in 100% of the HBeAg positive patients, with a value in all cases over 2×10 5 mol/ml. We observed that after treatment, HBV-DNA tested negative in only two of the eight HBeAg positive chronic hepatitis patients who seroconverted to anti-HBe, and was positive in the seven remaining, with a median HBV-DNA value of about 0.2×10 5 mol/ml (0.09–0.4). In the precore mutants HBV-DNA values ranged from 0.2 to >×10 5 mol/ml. Conclusions: Polymerase chain reaction HBV-DNA quantification is a sensitive method for managing chronic hepatitis B patients, especially those with low viremia, and may be a valuable tool for evaluating the efficacy of antiviral therapy.

Analysis of hepatitis B genotype changes in chronic hepatitis B infection: Influence of antiviral therapy.

BACKGROUND/AIMS The frequency of mixed hepatitis B virus (HBV) genotypes in chronic HBV (CHB) and genotype changes during natural disease evolution and as a result of antiviral therapy were investigated. METHODS Serum samples from 103 CHB patients were included in a cross-sectional study. Longitudinal study of HBV genotypes was performed in 22 patients, 17 of them under antiviral therapy (lamivudine and/or adefovir). HBV genotyping was done by the INNO-LiPA HBV assay. RESULTS Genotypes observed in the cross-sectional study: A 32% of cases, D 42%, C 2%, F 2%, and mixed genotypes 22% (mainly A/D, followed by A/G). Genotype G was found in 7% of patients, always combined with other genotypes. In the longitudinal study, genotype changes were observed only in treated patients (9 cases). Genotype A strains were positively selected in 6 of them, mainly as mixed A/D. In 6 patients, selection coincided with a decrease in HBV-DNA levels. CONCLUSIONS A high frequency of mixed HBV genotypes was observed in our setting. Selection of genotype A strains during treatment is likely an indication that sensitivity to therapy differs between genotypes A and D. The absence of changes in untreated patients suggests that HBV genotype is stable without external factors.

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 B virus genome variability and disease progression: the impact of pre-core mutants and HBV genotypes

The hepatitis B virus (HBV), a member of the Hepadnaviridae family, is prone to mutations due to its asymmetric replication via reverse transcription of an RNA intermediate. The estimated mutation rate of the hepadnavirus genome is 2 x 10(4) base substitutions/site/year. This mutation rate is approximately 100 times higher than that of other DNA viruses but between 100 and 1000 times lower than that of RNA viruses. Analyses of both naturally occurring viral variants and in vitro mutagenesis studies have identified some mutations that have a role in viral latency, pathogenesis of liver disease, immune escape, and resistance to antiviral therapy.