RESUMEN
AIM: To investigate the accuracy of serum alanine aminotransferase (ALT) in diagnosing lamivudine resistance and factors that contributed to abnormal serum ALT. METHODS: This was a retrospective study of chronic hepatitis B patients on lamivudine therapy who were followed for 3-mo with liver function tests and hepatitis B virus (HBV) DNA measurement. Lamivudine resistance was defined as HBV DNA ≥ 1 log from nadir on at least 2 occasions, confirmed by genotyping. Serum ALT levels in patients with lamivudine resistance were compared to serum ALT levels in those without lamivudine resistance. RESULTS: There were 111 patients with and 117 without lamivudine resistance. The area under the receiver operating characteristic of serum ALT to diagnose lamivudine resistance was 0.645 ± 0.037. Serum ALT > 42.5 U/L gave the best diagnostic accuracy with sensitivity = 61%, specificity = 60%, positive predictive value = 60%, negative predictive value = 61%, positive likelihood ratio = 1.53 and negative likelihood ratio = 0.65 for predicting lamivudine resistance, missing 39% of resistant patients. Using other serum ALT cutoffs, diagnostic accuracy was lower. By multivariate analysis, baseline abnormal serum ALT was associated with abnormal ALT during resistance (OR = 5.98, P = 0.003), and males were associated with serum ALT flares during resistance (OR = 8.9, P = 0.016). CONCLUSION: Serum ALT is inadequate for diagnosing lamivudine resistance and has implications where viral resistance testing is suboptimal and for reimbursement of rescue therapy.
Asunto(s)
Alanina Transaminasa/sangre , Antivirales/uso terapéutico , Biomarcadores/sangre , Farmacorresistencia Viral , Hepatitis B Crónica , Lamivudine/uso terapéutico , Adulto , Antivirales/farmacología , Pueblo Asiatico , ADN Viral/sangre , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Hepatitis B Crónica/sangre , Hepatitis B Crónica/tratamiento farmacológico , Humanos , Lamivudine/farmacología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND & AIMS: Although viral quasi-species evolution may be related to pathogenesis of disease, little is known about this in hepatitis B virus (HBV); consequently, we aimed to evaluate the evolution of HBV quasi-species in patients with well-characterized clinical phenotypes of chronic hepatitis B. METHODS: Four cohorts of well-defined clinical phenotypes of chronic hepatitis B, hepatitis Be antigen (HBeAg) seroconverters (spontaneous seroconverters and interferon-induced seroconverters) and nonseroconverters (controls and interferon nonresponders) were followed during 60 months on average. Serum from 4 to 5 time points was used for nested polymerase chain reaction, cloning, and sequencing of the precore/core gene (20 clones/sample). Only patients with genotype B were used. Sequences were aligned using Clustal X, then serial-sample unweighted pair grouping method with arithmetic means phylogenetic trees were constructed using Pebble 1.0 after which maximum likelihood estimates of pairwise distances under a GTR + I + G model was assessed. Viral diversity and substitution rates were then estimated. RESULTS: Analysis of 3386 sequences showed that HBeAg seroconverters had 2.4-fold higher preseroconversion viral sequence diversity (P = .0183), and 10-fold higher substitution rate (P < .0001) than did nonseroconverters, who had persistently low viral diversity (3.6 x 10(-3) substitutions/site) and substitution rate (2.2 x 10(-5) substitutions x site(-1) x month(-1)). After seroconversion, there was a striking increase in viral diversity. Most seroconverters had viral variants that showed evidence of positive selection, which was seen mainly after seroconversion. CONCLUSIONS: The high viral diversity before a reduction in HBV DNA and before HBeAg seroconversion could either be related to occurrence of stochastic mutations that lead to a break in immune tolerance or to increased immune reactivity that drives escape mutations.
Asunto(s)
Evolución Molecular , Anticuerpos contra la Hepatitis B/inmunología , Antígenos e de la Hepatitis B/inmunología , Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/virología , Adulto , Secuencia de Aminoácidos , Antivirales/uso terapéutico , Secuencia de Bases , Estudios de Cohortes , ADN Viral/genética , Femenino , Anticuerpos contra la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/inmunología , Humanos , Interferones/uso terapéutico , Masculino , Mutación/genética , Fenotipo , Filogenia , Proteínas del Núcleo Viral/metabolismoRESUMEN
In vitro studies of HBV core promoter mutations in hepatoma cell lines suggest that some mutations in core promoter transcription factor binding sites result in reduced core promoter activity and viral replication. We sought to validate this hypothesis using clinical samples with viral load differences before and after HBeAg seroconversion. A consensus sequence for transcription factor binding sites/regulatory regions was constructed based on published studies. Serum from two time points in 33 seroconverters and 10 interferon non-responders (controls) were utilized. Genotyping, HBV DNA quantification and direct sequencing of core promoter were performed. There were 216 new mutations following HBeAg seroconversion but few in controls. Mutations or mismatches to consensus transcription factor/regulatory region sequences clustered at nucleotide positions appeared genotype-specific, non-group specific or baseline mismatches and were discounted as having significant impact on viral replication. Only a few mutations in three seroconverters (9.1%) were specific, while 39.4% had no new mutations that could be attributed to reduction in viral load following HBeAg seroconversion. In 51.5% of patients, mutations were of uncertain significance because they occurred in demonstrated non-critical clustered nucleotide positions. Core promoter mutations post-seroconversion did not correlate with in vitro induced mutations that reduced the promoter activity.
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Antígenos del Núcleo de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B/virología , Mutación , Regiones Promotoras Genéticas , Carga Viral , Adulto , Sitios de Unión , Secuencia de Consenso , Análisis Mutacional de ADN , ADN Viral/química , ADN Viral/genética , Genotipo , Hepatitis B/inmunología , Antígenos e de la Hepatitis B/sangre , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/aislamiento & purificación , Virus de la Hepatitis B/fisiología , Humanos , Análisis de Secuencia de ADN , Replicación Viral/genéticaRESUMEN
AIM: We set to determine factors that determine clinical severity after the development of resistance. METHODS: Thirty-five Asian patients with genotypic lamivudine resistance were analyzed in three groups: 13/35 (37%) were non-cirrhotics with normal pre-treatment ALT (Group IA), 12/35 (34%) were non-cirrhotics with elevated pre-treatment ALT (Group IB), and 10/35 (29%) were cirrhotics (Group II). Patients were followed for a median of 98 wk (range 26-220) after the emergence of genotypic resistance. RESULTS: Group IA patients tended to retain normal ALT. Group IB patients showed initial improvement of ALT with lamivudine but 9/12 patients (75%) developed abnormal ALT subsequently. On follow-up however, this persisted in only 33%. Group II patients also showed improvement while on treatment, but they deteriorated with the emergence of resistance with 30% death from decompensated liver disease. Pretreatment ALT levels and CPT score (in the cirrhotic group) were predictive of clinical resistance and correlated with peak ALT levels and CPT score. CONCLUSION: The phenotype of lamivudine-resistant HBV correlated with the pretreatment phenotype. The clinical course was generally benign in non-cirrhotics. However, cirrhotics had a high risk of progression and death (30%) with the development of lamivudine resistance.
Asunto(s)
Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Lamivudine/administración & dosificación , Cirrosis Hepática/tratamiento farmacológico , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Adulto , Anciano , Farmacorresistencia Viral , Femenino , Genotipo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/mortalidad , Humanos , Cirrosis Hepática/mortalidad , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: p53 mutations are an early event in the multistep progression of gastric carcinoma. These mutations are often present in dysplastic and intestinal metaplastic gastric epithelium. However, the presence of immunohistochemically detectable p53 protein and p53 mutations in nondysplastic/nonmetaplastic gastric mucosa is more controversial. Recent reports have suggested that immunohistochemically detectable p53 protein may be present in the gastric epithelium of Helicobacter pylori gastritis. Furthermore, because cagA-positive H. pylori is associated with greater mucosal injury but decreased apoptosis, it would be interesting to determine if this phenotype is associated with greater immunostaining of p53, as the wild-type p53 gene helps to initiate apoptosis. METHODS: One hundred thirty-five patients with H. pylori-associated gastritis were immunohistochemically stained for p53 and quantified for the extent and intensity of the staining using a semiquantitative method (0, nil staining; 6, extensive and strong staining). The cagA status of the organism was determined by Western blot. RESULTS: Thirty-one patients (23%) showed strong p53 staining (> or = 4 of 6) in inflamed but otherwise normal gastric epithelium. In the 123 cagA-positive H. pylori gastritis patients, the average p53 staining score was 2.5 of 6. This is significantly higher than the corresponding score of 1.7 of 6 observed in the 12 patients with cagA-negative H. pylori gastritis (P = 0.04). CONCLUSIONS: Our results indicate that p53 protein is immunohistochemically detectable even before gastric metaplastic/dysplastic change occurs. The results also suggest that cagA-positive H. pylori might be associated with greater p53 immunohistochemical staining. This would indicate that p53 immunohistochemical staining does not reliably differentiate between gastric dysplasia and reactive inflammatory atypia. If the p53 protein detected is a consequence of mutation, this would help to explain why cagA-positive H. pylori gastritis is associated with decreased apoptosis.
Asunto(s)
Proteínas Bacterianas/análisis , Mucosa Gástrica/patología , Gastritis Atrófica/patología , Infecciones por Helicobacter/patología , Proteína p53 Supresora de Tumor/análisis , Enfermedad Aguda , Adolescente , Adulto , Anciano , Antígenos Bacterianos/análisis , Niño , Preescolar , Enfermedad Crónica , Femenino , Mucosa Gástrica/química , Gastritis Atrófica/metabolismo , Gastritis Atrófica/microbiología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/crecimiento & desarrollo , Helicobacter pylori/inmunología , Humanos , Inmunohistoquímica , Intestinos/química , Intestinos/patología , Masculino , Metaplasia , Persona de Mediana EdadRESUMEN
Chronic hepatitis B virus (HBV) infection is a major problem in Asia. Current therapies for chronic hepatitis B have limited efficacy. The successful use of ribozymes for intracellular inhibition of HBV gene expression was recently reported. As an alternative to ribozymes, the use of DNA-containing, phosphorothioate-modified, minimized hammerhead ribozymes (minizymes) to inhibit hepatitis B surface antigen (HBsAg) expression and viral replication was investigated. Such molecules can be synthesized and supplied exogenously. Two conserved sites within the HBsAg open reading frame (ORF) were targeted. PLC/PRF5 cells or 2.2.15 cells were treated with minizymes or antisense oligomers to assess the effects on cell viability, HBsAg expression, and viral DNA production. Treatment with the minizyme, MZPS1, resulted in >80% inhibition of HBsAg expression in PLC/PRF5 cells. MZPS1 had more inhibitory effect than the antisense oligonucletoide target at the same region, whereas the control minizyme had little effect. Another gene-specific minizyme, MZPS2, did not show any effect. Treated cells remained fully viable. Treatment of 2.2.15 cells with MZPS1 also led to decreased HBsAg expression. In addition, a 2.3-fold decrease in viral production was observed. Our data showed that minizymes can inhibit HBV gene expression and may potentially be useful for clinical therapy against chronic HBV infection.