<b>OBJECTIVEb>To establish a simple and accurate method for rapid detection of lamivudine related mutations in hepatitis B virus (HBV) polymerase gene.

<b>METHODSb>HBV polymerase gene fragments of covering B and C active region were amplified by nested polymerase chain reaction (nPCR) or nested mismatched PCR. The PCR products were digested with Nde I or Nia III and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were distinguished. Using this method, thirty patients with chronic hepatitis B and treated with lamivudine for at least one year were analysed for the lamivudine related mutations in polymerase gene. Sixteen cases without lamivudine therapy were used as controls. Some of the patients were also analysed by clone sequencing.

<b>RESULTSb>The nested mismatched PCR-RFLP method was simple, accurate and rapid. The whole experiments could be finished in eleven hours. The least titers of HBV DNA which could be detected was 10.3 copies/ml. The wild or mutant strains judged by RFLP were identified by clone sequencing. Mutation in the tyrosine methionine aspartic aspartic acid (YMDD) motif of HBV polymerase gene was found in eight patients and mutations of YMDD motif associated with L526M were found in another three patients. However, there were no such mutations in the control cases.

<b>CONCLUSIONb>The nested PCR-RFLP is considered as a simple and accurate method for rapid detection of lamivudine related mutations in HBV polymerase gene. It is suitable for larger number of sample detection.

Antiviral Agents ; pharmacology ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; genetics ; Hepatitis B virus ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

In order to investigate the mutation of HBV polymerase gene reverse transcription conserved region (P region) in chronic hepatitis B (CHB) patients, 212 CHB patients who took antiretroviral treatment with nucleotide analogues were chosen. The drug resistance mutations of HBV P region and HBV genotype were detected by Pyrosequencing. Sequence analysis showed that the drug resistance sites of HBV P region located at sites 173; 180; 181; 184; 204; 236 and 250. The main site of HBV P region drug resistance was 204 and 180, accounting for 35.8% and 23.5%, respectively. There were significant differences in the mutation rate of site 180 among different age groups. There were also significant differences in the mutation rate of site 204 among younger than 30 age group, 41 to 50 age group and 51 to 60 age group. (P < 0.05, P < 0.01). The mutation rate of site 180 combined with site 204 was 66.6%. The mutation rate of site 181 combined with site 236 was 23.3%. The age of C genotype infected patients was significantly older than B genotype infected patients (P < 0.01). M204V/I mutation mostly existed in the form of joint L180M mutation, the mutation rate was age-related. The detection of HBV genotypes and drug resistance sites of HBV P region have important clinical implications for the treatment and prognosis of patients with CHB.
Adult ; Aged ; Antiviral Agents ; pharmacology ; China ; Drug Resistance, Viral ; Female ; Gene Products, pol ; genetics ; Genotype ; Hepatitis B virus ; classification ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Young Adult

<b>BACKGROUNDb>To investigate the mutation of HBV polymerase gene in chronic hepatitis B patients treated with lamivudine.

<b>METHODSb>The restriction-fragment-length-polymorphism (RFLP) assay for HBV DNA sequence determination at the codon 528 and 552 in the HBV polymerase gene associated with lamivudine resistance in vitro. HBV DNA samples extracted from sera of 240 patients were subjected to PCR amplification with primer pairs F2/R2 (552), F3/R2 (528). Each PCR product was digested with Nde I or Nla III.

<b>RESULTSb>Serum HBV DNA mutation was found in 51/240 patients (38/51M552V, 26/38L528M, 13/51M552I) after therapy for 52 weeks. DNA sequence analysis was performed on samples of 3 patients, and the results were consistent with those of RFLP assay.

<b>CONCLUSIONb>The RFLP assay was able to detect the mutation of HBV DNA at codon 552 and 528 which are the principal site of HBV DNA resistant to lamivudine. The specific PCR method for HBV DNA mutation is rapid, simple and specific.

Drug Resistance, Viral ; Gene Products, pol ; genetics ; Hepatitis B virus ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; therapeutic use ; Mutation ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Reverse Transcriptase Inhibitors ; therapeutic use

BACKGROUNDS/AIMS: Continuation of lamivudine therapy is controversial for patients with chronic hepatitis B when viral breakthrough occurs. Moreover, the effect of continuous lamivudine therapy is unknown in patients with acute exacerbation after viral breakthrough. We assessed clinical course of acute exacerbation after viral breakthrough in patients who continued and discontinued lamivudine therapy. METHODS: Medical records of 109 patients with viral breakthrough during lamivudine therapy were reviewed. Of 40 patients with acute exacerbation (ALT level > 5 x ULN), adefovir dipivoxil was unavailable in 38 patients. These 38 patients (mean age 42.6 years; male/female, 34/6) were divided into continuation (n=21) and discontinuation (n=17) groups. Clinical courses of the 2 groups were compared. RESULTS: During follow-up period (mean, 27 months; range, 6-60 months), ALT levels decreased to < 2 x ULN in 11 patients (52%) of continuation group and 9 patients (53%) of discontinuation group, varied from 2 x to 5 x ULN in 9 (43%) and 5 (29%), respectively, and increased to > 5 x ULN in 1 (5%) and 3 (18%), respectively, with no statistical significance (P=.417). CONCLUSIONS: When acute exacerbation of ALT levels occurs after viral breakthrough during lamivudine administration in patients with compensated chronic hepatitis B, continuation of lamivudine may have no advantage over discontinuation.
Phosphonic Acids/therapeutic use ; Middle Aged ; Male ; Lamivudine/*administration & dosage ; Humans ; Hepatitis B, Chronic/drug therapy/enzymology/*virology ; Hepatitis B virus/*isolation & purification ; Female ; Antiviral Agents/*administration & dosage ; Alanine Transaminase/blood ; Aged ; Adult ; Adenine/analogs & derivatives/therapeutic use ; Acute Disease

OBJECTIVE: To evaluate the therapeutic effects of entecavir (ETV) and interferon- (IFN-) treatments for 48 weeks for chronic hepatitis B (CHB) in patients with different baseline alanine aminotransferase (ALT) levels. METHODS: We retrospectively analyzed the data of 369 CHB patients receiving ETV and IFN- treatments for 48 weeks. We compared the virological response rates, HBsAg clearance, and HBsAg reduction between the patients receiving ETV and IFN- treatments with different baseline ALT levels[≤ 5×upper limits of normal (ULN) level (subgroup 1), 5-10×ULN (subgroup 2), and > 10× ULN (subgroup 3)]. RESULTS: In patients receiving ETV treatment, the virological response rate was 83.3% in subgroup 1, 91.4% in subgroup 2, and 95.5% in subgroup 3, as compared with 19.7%, 40%, and 42.9% in the 3 subgroups with IFN- treatment, respectively, showing significantly differences both among different subgroups with the same treatment and between the same subgroup with different treatments ( < 0.05). HBeAg clearance rates in the 3 subgroups were 8.3%, 16.7% and 35.5% in patients with ETV treatment and were 1.8%, 41.9%, and 38.1% in patients with IFN- treatment, respectively, showing significant differences among the 3 subgroups with the same treatment ( < 0.05); in the same subgroups with different treatments, the rates differed significantly only between subgroups 2 ( < 0.05). In ETV group, the rate of HBsAg reduction to below 200 IU/ml was 2.5% in subgroup 1 and 13.8% in subgroup 2, showing no significant difference between the two subgroups; in IFN- group, the rates were also similar between subgroups 1 and 2 (30.6% 33.3%, > 0.05); but the rates differed significantly between the same subgroups with different treatments ( < 0.05). CONCLUSIONS In all the subgroups with different baseline ALT levels, ETV treatment for 48 weeks results in significantly higher virological response rates than IFN- treatment in patients with CHB. In patients with a baseline ALT of 5-10 ×ULN, IFN- can result in a higher HBeAg clearance rate than ETV. In patients with comparable baseline ALT level, IFN- more effectively reduces HBsAg level than ETV. The patients with a relatively high baseline ALT level (> 5 × ULN) show better responses to both ETV and IFN- treatment than those with ALT level below 5×ULN. We thus recommend IFN- for patients with a baseline ALT of 5-10×ULN and ETV for patients with a baseline ALT either below 5 × ULN or beyond 10×ULN.
Alanine Transaminase ; blood ; Antiviral Agents ; therapeutic use ; DNA, Viral ; Guanine ; analogs & derivatives ; therapeutic use ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; immunology ; Hepatitis B, Chronic ; drug therapy ; enzymology ; immunology ; virology ; Humans ; Interferon-alpha ; therapeutic use ; Retrospective Studies ; Time Factors ; Treatment Outcome ; Viral Load ; drug effects

<b>OBJECTIVEb>To observe the therapeutic effects of Pegasys (pegylated interferon alpha-2a) in chronic hepatitis B (CHB) patients with low-level alanine transaminase (ALT) < 2 x upper limit of normal (ULN).

<b>METHODSb>One hundred and seven CHB patients were randomized enrolled including 52 with ALT < 2 x ULN and liver tissues inflammation activity > or = G2 as observational group and 55 with ALT > 2 x ULN as control group. All the enrolled patients received pegasys treatment for 48 weeks and the responses between two groups were compared. Measurement data were analyzed using t test and numeration data were analyzed using chi square test.

<b>RESULTSb>The reductions of HBV DNA in observational group at different time points were all less than control group (all P < 0.05). At the end of treatment, the HBV DNA negative rate, HBeAg seroconversion rate and HBsAg loss rate in the observational group were 51.9%, 48.8% and 1.9% , respectively, which were all lower than control group (67.3% , 66.7% and 7. 3% , respectivley) ( all P <0. 05). The ALT normalization rates of two groups were 75% and 76.4% (P > 0. 05).

<b>CONCLUSIONb>Pegasys is efficient for CHB patients with ALT < 2 x ULN and liver tissues inflammation activity > or = G2, while it is still inferior to those with ALT > 2 x ULN.

Adolescent ; Adult ; Alanine Transaminase ; metabolism ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Female ; Hepatitis B, Chronic ; drug therapy ; enzymology ; physiopathology ; virology ; Humans ; Interferon-alpha ; therapeutic use ; Male ; Middle Aged ; Polyethylene Glycols ; therapeutic use ; Recombinant Proteins ; therapeutic use

<b>OBJECTIVEb>To study the types and emergence time of YMDD motif mutation in hepatitis B virus (HBV) polymerase gene during lamivudine treatment.

<b>METHODSb>The serum samples were collected from 33 patients with HBV DNA rebounding and 2 non-responders after at least one year lamivudine treatment. HBV polymerase gene was amplificated by PCR, then the products were detected by restriction fragment length polymorphism (RFLP) and by direct sequence analysis.

<b>RESULTSb>The variants with YMDD mutation were 14 out of the 35 patients. Mutation patterns detected in these patients included four YIDD, six YVDD, three YI/VDD and one YI/MDD. The mean emergence time of YMDD variants was 11.07+/-3.65 months after the treatment, and the earliest one and the latest one occurred 5 months and 17 months after the treatment respectively. The emergence times of YIDD, YVDD, YI/VDD were (10.00 +/- 1.41) months, (11.67 +/- 4.41) months and (13.33 +/- 3.31) months respectively, which had no statistical significance (F = 0.543, P < 0.05). Three patients treated with lamivudine 200 mg every day after the mutation were followed up for 6 months, whose HBV variants had not vanished.

<b>CONCLUSIONSb>There are many kinds of HBV variants after lamivudine treatment, including YIDD, YVDD, YI/VDD and YI/MDD. The emergence time of variants is quite variable between different types and the mean time is (11.07 +/- 3.65) months after treatment, and there is no relationship between the type of YMDD mutation and the time of lamivudine administration.

Adolescent ; Adult ; Aged ; Amino Acid Motifs ; genetics ; Child ; Cloning, Molecular ; DNA, Viral ; genetics ; Drug Resistance, Viral ; genetics ; Female ; Gene Products, pol ; genetics ; Hepatitis B virus ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Middle Aged ; Mutation ; RNA-Directed DNA Polymerase ; genetics ; Time Factors

<b>OBJECTIVEb>To characterize genotypic resistance within HBV RT region in chronic hepatitis B (CHB) patients with nucleos(t)ide analogue (NA) treatment.

<b>METHODSb>Serum samples of 229 CHB patients with NA treatment were obtained. Full-length HBV RT sequences were amplified, sequenced and analyzed, on the following NA resistant (NAr) mutations belonging to different NAr pathways.

<b>RESULTSb>Among 229 HBV isolates, 14.41% (33/229) and 85.59% (196/229) were genotype B and C, respectively; and the patients with HBV genotype C may be more susceptible to develope resistant mutations than patients with HBV genotype B(chi2 = 2.95, P < 0.05). NAr mutations were detected in 63 CHB patients. Mutations were not found at rtI169, rtT184, rtA194 or rtS202. RtM204 mutations were detected at the highest frequency among 63 mutants (40/63, 63.49%) and found to display 11 combination mutation patterns, in which rtM204I were associated with rtL80I/V and rtL180M, and rtM204V were associated with rtL1l80M, respectively. Conclusions There are complicated mutation patterns in the HBV RT region for chronic hepatitis B (CHB) patients with nucleos(t)ide analogue (NA) treatment. RtM204V/I mutation was the highest.

Adolescent ; Adult ; Aged ; Antiviral Agents ; therapeutic use ; Female ; Hepatitis B virus ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Male ; Middle Aged ; Mutation ; drug effects ; Nucleosides ; therapeutic use ; Nucleotides ; therapeutic use ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Young Adult

<b>OBJECTIVEb>To analyze genetic mutation associated with drug resistance in the reverse transcriptase (RT) domain of HBV from 40 patients with chronic hepatitis B, and to construct mutant RT gene recombinant vectors for drug-resistant phenotypic analysis.

<b>METHODSb>HBV DNA was extracted from sera of the 40 patients receiving anti-HBV nucleot (5) ide analogue. The complete RT domain-encoding gene was amplified by nested PCR, and then cloned into pGEM-T-easy vector. Three to Five clones were randomly selected for DNA sequencing. Data were analyzed by UNASTAR software. The pTriEx-HBV (C) 1.1 expression vectors were constructed by replacing the 1250-hp Xho I/Nco I fragments containing complete RT domain from individual patients samples.

<b>RESULTSb>All samples were detected with drug-resistant mutations associated with lamivudine, adefovir, and entacavir singly or in combination. Ninety-six mutant RT genes were cloned into pGEM-T-easy vector, from which 40 major mutant RT genes were replaced into pTriEx-HBV (C) 1.1 expression vectors. The construction was confinned to be successful by verifying mutation existence using DNA sequencing, and detectable HBsAg and HBeAg in the cell supernatant after transfecting recombinant expression vectors into Huh7 cells.

<b>CONCLUSIONb>The analysis of drug-resistant mutation and the construction of mutant-recombinant expression vectors were successfully implemented using the samples frum clinical patients. The work lays a foundation for drug-resistant phenotypic analysis of HBV mutants.

Adult ; Antiviral Agents ; pharmacology ; therapeutic use ; Cell Line ; Cloning, Molecular ; Drug Resistance, Viral ; Female ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B virus ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Male ; Middle Aged ; Mutation ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Young Adult

<b>OBJECTIVEb>To investigate the drug-resistant genes at hepatitis B virus (HBV) polymerase region during entecavir (ETV) treatment.

<b>METHODSb>Serum samples from chronic hepatitis B patients with virologic breakthrough during enticavir therapy were studied. The resistant mutation patterns in the polymerase gene of hepatitis B virus were analyzed using the polymerase chain reaction (PCR)-sequencing method.

<b>RESULTSb>ETV resistance was detected from 19 out of 29 ETV-refractory patients, among whom 16 (84.2%) had a history of lamivudine-refractory. The mutation patterns were diverse, while rtL180 + rtM204 + rtT184 (58.6%, 17/29) was most common in patients with ETV genotype resistance. Four of 7 patients (7/29, 24.1%) with genotype B were detected to have ETV genotype resistance, while 15 of 22 patients (22/29, 75.9%) with genotype C were detected to have ETV genotype resistance. The rate of ETV genotype resistance was 57.1% (4/7) and 68.2% (15/22) in patients with genotype B and genotype C,while no statistical difference was found(P = 0.665).

<b>CONCLUSIONSb>ETV genotype resistance is more common in patients who have been refractory to ETV and lamivudine sequential treatment. rtM204+rtL180+rtT184 mutation is common in genotype B and C ETV resistance patients.

Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; genetics ; Female ; Guanine ; analogs & derivatives ; therapeutic use ; Hepatitis B virus ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Male ; Middle Aged ; Mutation ; Viral Proteins ; genetics ; Young Adult