Long-term changes in the frequency and outcome of hepatitis delta virus (HDV) infection have seldom been analysed. This retrospective, longitudinal study includes 398 consecutive hepatitis B surface antigen (HBsAg)-positive patients with anti-HDV antibodies who attended our institution between 1983 and 2008. At enrolment, 182 patients had acute and 216 chronic hepatitis. Patients were grouped into two periods. Those who attended between 1983 and 1995 and those between 1996 and 2008. The former group was significantly younger, mainly intravenous drugs users, and had a greater incidence of acute HDV and HIV and HCV coinfection. Patients with acute HBV/HDV coinfection cleared both infections in 90% of cases, while all patients with HDV superinfection evolved to chronic disease. One hundred and fifty-eight patients with chronic HDV were followed for a median period of 158months. Seventy-two per cent of the patients remained stable, 18% had hepatic decompensation, 3% developed hepatocellular carcinoma, and 8% cleared HBsAg. Liver-related death was observed in 13% of patients and mainly occurred in patients from the first period (P=0.012). These results indicate an outbreak of HDV at the end of the 1980s and the beginning of the 1990s, with a large number of acute HDV cases affecting predominately young, male intravenous drug users. Currently, patients with chronic HDV disease are older, and factors associated with worse prognosis include the presence of cirrhosis and age at the time of diagnosis.
This study contributes greatly to understanding the usefulness of genotypes of F-specific RNA bacteriophages in source tracking of faecal wastes.
The effect of natural inactivation in freshwater, chlorination, ammonia, extreme pHs, temperature, and salt content on phage inactivation was evaluated on mixtures of F-specific RNA bacteriophage isolates belonging to genotypes I, II, III, and IV. The bacteriophages studied were previously but recently isolated from natural samples, characterized as F-specific RNA bacteriophages and genotyped by plaque hybridization with genotype-specific probes. Natural inactivation in river water was modeled by in situ incubation of bacteriophages inside submerged dialysis tubes. After several days bacteriophages of genotype I showed the highest persistence, which was significantly different from that of bacteriophages of genotype II, IV, or III. The pattern of resistance of phages belonging to the various genotypes to extreme pHs, ammonia, temperature, salt concentration, and chlorination was similar. In all cases, phages of genotype I showed the highest persistence, followed by the phages of genotypes II, III, and IV. The phages of genotypes III and IV were the least resistant to all treatments, and resistance of genotypes III and IV to the treatments was similar. Bacteriophages of genotype II showed intermediate resistance to some of the treatments. The resistance of four phages of genotype I to natural inactivation and chlorination did not differ significantly. These results indicate that genotypes III and IV are much more sensitive to environmental stresses and to treatments than the other genotypes, especially than genotype I. This should be taken into consideration in future studies aimed at using genotypes of F-specific RNA bacteriophages to fingerprint the origin of fecal pollution.Fecal contamination of aquatic environments can hinder contact recreation and shellfish growing water and spoil drinking water, since it may include pathogenic microorganisms. There is a general belief that exposure to waters polluted with human feces is more hazardous than exposure to waters polluted with animal feces, although no sound epidemiological studies support this view. However, regardless of the health risks involved, management of fecal contamination of water can only improve if the sources of pollution are identified by water analysis and thus tools to distinguish between human and animal fecal sources are needed. Chemical and microbiological methods had been proposed for tracking the origin of fecal contaminants. Fecal sterols of human and animal origin have been used for this purpose (22). Microbiological indicators include the following: ratios of fecal coliforms to fecal streptococci (13); the presence of Rhodococcus coprophilus (23); the presence of some phenotypes (24) and ribotypes (25) of Bifidobacterium species; the presence of some phenotypes of Bacteroides species (21); the distribution patterns of ribotypes of Escherichia coli (7, 29); the distribution patterns of repetitive DNA sequences of E. coli (8); antibiotic resistance patterns (15); bacteriophages specific for various strains of Bacteroides fragilis (36); ph...
BackgroundSelection 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 FindingsSerum 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.ConclusionsUDPS 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 purpose of this study was to assess the use of dried blood spot (DBS) samples for hepatitis B virus (HBV) DNA quantification, HBV genotyping, and detection of G1896A precore mutants and variants in the YMDD polymerase motif. We studied DBS and serum samples from 82 patients with chronic HBV infection (23 hepatitis B e antigen [HBeAg]-positive and 39 HBeAg-negative), 20 HBeAg-inactive carriers, and 15 HBeAg-negative patients under lamivudine therapy (selected from chronic HBV patients). DBS samples consisted of approximately 20 L of blood applied to 5-mm paper disks. HBV DNA quantification and HBV precore mutant detection were done using real-time polymerase chain reaction, HBV genotyping using restriction fragment length polymorphism, and YMDD variant detection by Inno-lipa assay. DBS and serum results were compared. HBV DNA was detected in a range of 10 2 -10 8 copies/mL, with low intra-assay and inter-assay variation (<10%). Median DBS HBV DNA (copies/mL) was: 3.7 ؋ 10 6 in HBeAg-positive, 6.2 ؋ 10 5 in HBeAg-negative, and 5.5 ؋ 10 2 in inactive carriers (P < .05). HBV DNA was positive in serum (median 5 ؋ 10 3 copies/mL) but negative in DBS for five inactive carriers. The correlation coefficient between HBV DNA concentration in DBS versus serum samples was r 2 ؍ 0.96 (P < .001). The sensitivity of HBV DNA detection in DBS samples was 1 log 10 lower than in serum samples. Concordance between DBS and serum for HBV genotyping, and for precore mutant and YMDD variant detection was optimal. DBS storage for 7 days at room temperature and 21 days at ؊20°C revealed no decrease in HBV DNA levels or integrity. In conclusion, the DBS sample is useful for HBV DNA quantification, genotyping, and detection of precore mutant and YMDD variants. All four determinations can be completed with a single drop of dried blood. ( V irological diagnosis and monitoring of hepatitis B virus (HBV) infection are based on serological assays that detect specific antibodies and that detect, quantify, or characterize HBV particles at the molecular level. Several authors have suggested that the study of virological characteristics such as HBV genotypes, molecular HBV variants, and quantification of HBV-DNA levels should be included as part of the laboratory testing for routine management of chronic hepatitis B (CHB) infection. 1-3 However, implementation of this policy requires access to specific laboratory equipment that may not be readily available in some settings.Dried blood spot (DBS) samples collected on filter paper have been used for genetic screening and diagnosis of several diseases. This approach is particularly useful for programs in which samples from many areas are sent to a central laboratory. 4 Since it was first introduced for the detection of RNA human immunodeficiency virus type 1 in 1991, polymerase chain reaction (PCR)-based DBS methods have proved particularly effective for the detection of proviral HIV-1 DNA and for viral sequencing. [5][6][7] In HBV infection, DBS samples have been used mainly for testing viral an...
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