Early detection of viral resistance by determination of hepatitis B virus polymerase mutations in patients treated by lamivudine for chronic hepatitis B

We have analyzed the molecular dynamics of emergence of drug-resistant strains in patients receiving lamivudine therapy for chronic hepatitis B. Twenty consecutive patients with lamivudine resistance were studied (13 hepatitis B e antigen [HBeAg]-positive patients and 7 HBe antibody [anti-HBe]-positive patients). Determination of viral genotype, precore mutants, and polymerase gene mutants (L528M, M552V, M552I) was performed using the research version of Lipa-HBV. Quantitative analysis of HBV DNA was performed using both branched DNA (bDNA) and polymerase chain reaction (PCR) assays. Polymerase mutants (genotypic resistance) were found in 16 of 20 patients. Genotypic resistance was detected earlier than the phenotypic resistance (P =.004). Quantitative PCR allowed detection of viral DNA throughout the entire study period in 16 of 20 patients. Analysis of pretreatment variables showed that high alanine transaminase (ALT) levels (>3 x the upper limit of normal [ULN]) was associated with a more rapid selection of drug-resistant mutants (P =.027) and a high hepatitis B virus (HBV) DNA level (>1,497 Meq/mL, bDNA) with a more rapid occurrence of phenotypic resistance (P =.04). At the time of viral breakthrough, the mean serum HBV-DNA values were not different from the pretreatment values (P =.37). ALT levels were higher in anti-HBe-positive patients compared with pretreatment values and to HBeAg-positive patients (P =.01). In 8 patients, antiviral therapy was modified after viral breakthrough, with the introduction of famciclovir and/or interferon alfa. Viral DNA became undetectable by bDNA in 3 patients who received interferon. Our results suggest that genotypic assays for polymerase mutant detection and quantitative determination of viremia with highly sensitive assay are warranted for an optimal monitoring of antiviral therapy of chronic hepatitis B.     

Quantitative detection of hepatitis B virus DNA in serum by a new rapid real-time fluorescence PCR assay

A sensitive and accurate HBV DNA quantification assay is essential for monitoring hepatitis B virus (HBV) replication. This study evaluated a real‐time PCR method performed in the LightCycler^TM analyser for quantitative HBV DNA assay. HBV DNA results with this method were compared with those obtained using a branched‐chain DNA (bDNA) solution hybridization assay. Real‐time PCR was performed using two adjacent fluorescently labelled probes and primers corresponding to the HBV core gene. The same standard employed in the bDNA assay was used for calibration. Serum samples came from 193 HBV surface antigen (HBsAg)‐positive patients (34 HBV e antigen (HBeAg)‐positive and 93 with antibody to HBeAg (anti‐HBe)), and 66 asymptomatic HBV carriers. In addition, we analysed serum samples from 8 anti‐HBe‐positive patients who had been receiving lamivudine treatment for more than three years. A linear standard curve was seen in the range from 10³ to 10⁸ copies/mL. In the reproducibility analysis, intra‐assay coefficient of variation (CVs) at two known HBV DNA concentrations were 4% and 2% and interassay CVs were 6% and 4%. The median of serum HBV DNA by real‐time PCR was 9.2 × 10⁸ copies/mL in HBeAg‐positive patients with persistently elevated alanine aminotransferase (ALT) levels, 1.3 × 10⁷ copies/mL in anti‐HBe‐positive cases with persistently elevated ALT levels, 3.7 × 10⁴ copies/mL in anti‐HBe‐positive patients with fluctuating ALT levels and 10⁴ copies/mL in asymptomatic HBV carriers. The differences in HBV DNA levels among the various groups studied were statistically significant (P < 0.05). The cut‐off between chronic hepatitis patients and asymptomatic carriers was found to be at a serum HBV DNA concentration of 5 × 10⁴ copies/mL. Of the 109 serum samples with a viral load < 7.5 × 10⁵ (negative by bDNA assay) 44 (40%) were positive by real‐time PCR: 24 (56%) chronic hepatitis and 20 (33%) asymptomatic carriers. There was a positive association between HBV DNA levels determined by real‐time PCR and ALT levels (P < 0.05), which was not observed with the bDNA assay for HBV DNA quantification. At 12 months of lamivudine treatment, 6 patients (75%) showed HBV DNA levels < 5 × 10⁴ copies/mL (range < 10³–2 × 10³), significantly lower than at baseline. At 36 months, 2 of 8 (25%) showed HBV DNA levels persistently lower than 5 × 10⁴ copies/mL (1.7 × 10³, 6 × 10³). The LightCycler quantitative real‐time PCR is a practical, sensitive, reproducible single‐tube assay with a wide dynamic range of detection. The assay is automatic except for DNA extraction and the running time is only 70 min. The LightCycler real‐time PCR is useful for identifying different states of HBV infection and for evaluating the efficacy of viral therapy.     

Quantitative hepatitis B surface antigen and hepatitis B e antigen titers in prediction of treatment response to entecavir

Quantitative hepatitis B surface antigen (qHBsAg) and quantitative hepatitis B e antigen (qHBeAg) titers are emerging as useful tools for measuring viral loads and for predicting the virological response (VR) and serological response (SR) to pegylated interferon therapy. However, the clinical utility of these assays in patients taking entecavir (ETV) is largely unknown. Treatment-naive patients with chronic hepatitis B (CHB) who were taking ETV for 2 years were enrolled. The qHBsAg and qHBeAg levels were serially measured with the Architect assay. From 95 patients, 60.0% of whom were hepatitis B e antigen-positive [HBeAg(+)], 475 samples were analyzed. The median baseline log hepatitis B virus (HBV) DNA, log qHBsAg, and log qHBeAg values were 6.73 copies/mL (4.04-9.11 copies/mL), 3.58 IU/mL (1.17-5.10 IU/mL), and 1.71 Paul Ehrlich (PE) IU/mL (-0.64 to 2.63 PE IU/mL), respectively. For the prediction of VR (HBV DNA < 60 copies/mL at 24 months) in HBeAg(+) patients, baseline alanine aminotransferase (P = 0.013), HBV DNA (P = 0.040), and qHBsAg levels (P = 0.033) were significant. For the prediction of VR, the area under the curve for the baseline log qHBsAg level was 0.823 (P < 0.001); a cutoff level of 3.98 IU/mL (9550 IU/mL on a nonlogarithmic scale) yielded the highest predictive value with a sensitivity of 86.8% and a specificity of 78.9%. As for SR (HBeAg loss at 24 months), the reduction of qHBeAg was significantly greater in the SR(+) group versus the SR(-) group. The sensitivity and specificity were 75.0% and 89.8%, respectively, with a decline of 1.00 PE IU/mL at 6 months. With ETV therapy, the correlation between HBV DNA and qHBsAg peaked at 6 months in HBeAg(+) patients. CONCLUSION: Both qHBsAg and qHBeAg decreased significantly with ETV therapy. The baseline qHBsAg levels and the on-treatment decline of qHBeAg in HBeAg(+) patients were proven to be highly useful in predicting VR and SR, respectively. The determination of qHBsAg and qHBeAg can help us to select the appropriate strategy for the management of patients with CHB. However, the dynamic interplay between qHBsAg, qHBeAg, and HBV DNA during antiviral therapy remains to be elucidated.     

End-of-treatment virologic response does not predict relapse after lamivudine treatment for chronic hepatitis B

AIM: Attaining hepatitis B e antigen (HBeAg) seroconversion during lamivudine treatment is associated with fewer relapses in HBeAg-positive patients. In HBeAg-negative patients, predictors for post-treatment relapse remain largely unknown. We therefore studied whether end-of-treatment virologic response correlated with relapse after lamivudine treatment. METHODS: We prospectively analyzed 12 HBeAg-negative patients and 14 HBeAg-positive patients with chronic hepatitis B, who received at least 9 mo of lamivudine treatment and were followed up for 12 mo post-treatment. Relapse of hepatitis B activity was defined by an elevation of serum ALT level above twice the upper limit of normal as well as reappearance of serum HBV DNA by the branched DNA assay or HBeAg during the follow-up period. The serum viral loads during and at the end of treatment were further determined by a quantitative real-time polymerase chain reaction assay. RESULTS: Relapse occurred in 6 (50.0%) HBeAg-negative patients within 12 mo post-treatment. Two relapsers had end-of-treatment serum viral load <1 000 copies/mL, the proportion was not significantly different from that in the 6 non-relapsers (33.3% vs 16.7%; P = 1.00). Hepatitis B virus (HBV) DNA levels did not correlate with post-treatment relapse in HBeAg-positive patients either. However, genotype C patients tended to have a lower relapse rate than genotype B patients (14.3% vs 57.9%, P = 0.08). CONCLUSION: Our results suggest that end-of-treatment virologic response cannot predict post-treatment relapse in patients with HBeAg-negative or -positive chronic hepatitis B. The impact of HBV genotype on the response to lamivudine treatment awaits further studies.     

799例重型肝炎患者的临床病原学与实验室分析

目的探讨乙型重型肝炎患者乙型肝炎病毒(HBV)DNA定量、e抗原表达与病死率的相关性,为重型肝炎临床治疗提供参考。方法统计我院2000-2004年各型重型肝炎的发病率,进一步应用荧光定量多聚酶链反应方法检测乙型重型肝炎患者血清HBV DNA,应用微粒子方法检测乙型肝炎e抗原表达情况,并分析其与病死率及抗病毒治疗的临床疗效间的关系。结果(1)重型肝炎中乙型肝炎占83．50％,慢性重型肝炎中乙型肝炎占96．77％;(2)5年间慢性乙型重型肝炎患者HBV DNA定量大于1×10,拷贝／ml组,总病死率为53．25%,小于1×105拷贝／ml组,病死率为34．50％,差异有统计学意义(P<0．01);e抗原表达对病死率无影响;(3)2004年,慢性乙型重型肝炎患者HBV DNA定量大于1×105拷贝／ml病例加用拉米夫定抗病毒治疗,病死率由2000年的54．64%下降至2004年的30．38%,差异有统计学意义(P<0．01)。结论重型肝炎以慢性乙型重型肝炎为主,病毒载量高是高病死率关键因素之一,抗病毒治疗可以降低患者的病死率。     

Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

AIM: To examine the sensitivity and accuracy of real-time polymerase chain reaction (PCR) for the quantification of hepatitis B virus (HBV) DNA in semen. METHODS: Hepatitis B viral DNA was isolated from HBV carriers' semen and sera using phenol extraction method and QIAamp DNA blood mini kit (Qiagen, Germany). HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR (quantitative polymerase chain reaction (qPCR)). The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS: Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5x10~7 and 1.67x10~7 copies of HBV DNA per mL in two HBV infected patients' sera, while 2.14x10~5 and 3.02x10~5 copies of HBV DNA per mL in the semen. CONCLUSION: Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.     

Clinical evaluation of the branched DNA assay for hepatitis B virus DNA detection in patients with chronic hepatitis B lacking hepatitis B e antigen and treated with interferon-

The aim of this study was to evaluate the Chiron branched DNA (bDNA) assay for detection of serum hepatitis B virus (HBV) DNA in patients with chronic hepatitis B lacking hepatitis B e antigen (HBeAg) and undergoing interferon (IFN) therapy. Results obtained with the bDNA assay were compared with those obtained using the Abbott liquid hybridization (LH) assay and the polymerase chain reaction (PCR). Serial samples (274) from 34 patients were analysed. Analysis of variance results indicated that bDNA values were more significantly correlated than LH values with both PCR positive/negative results (probability of artifact (Prob>F) = 0.7 and 0.09 for LH and bDNA assays, respectively) and presence/absence of precore mutations (Prob>F = 0.21 and 0.001 for LH and bDNA assays, respectively). Both bDNA and LH results correlated highly with alanine aminotransferase (ALT) values (both had Prob>F values of 0.0) while PCR was not correlated with ALT (Prob>F = 0.05). In 26 evaluable patients, a model based on a generalized Knodell score was used to predict response to IFN therapy, as defined by normalization of ALT values during therapy. This model discriminated well between non-responders and responders. The bDNA results correlated well with the generalized Knodell score, while the LH results did not (Prob>F = 0.04 and 0.19 for the bDNA and LH assays, respectively). In conclusion, the bDNA assay appears to be useful for quantification of HBV DNA levels in HBeAg-negative chronic hepatitis as it correlates with biochemical and histological indications of disease severity as well as with response to IFN therapy.     

Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection

The goals of this retrospective study were to determine whether there is a threshold hepatitis B virus (HBV) DNA value associated with spontaneous or antiviral therapy-related hepatitis B e antigen (HBeAg) clearance. We also investigated whether there is an HBV DNA value that can be used for differentiating inactive carriers from patients with HBeAg-negative chronic hepatitis B. HBV DNA levels in sequential serum samples of 165 Chinese patients with different stages of chronic HBV infection were quantified by a polymerase chain reaction (PCR)-based assay. Our results showed that almost all of the patients (89%) who remained HBeAg-positive had HBV DNA levels that were persistently above 10(5) copies/mL. Serum HBV DNA levels decreased by a mean of 3 log(10) in patients with HBeAg loss, but 51% had levels above 10(5) copies/mL at the time HBeAg first became undetectable. Mean serum HBV DNA levels were significantly lower in HBeAg-negative patients. HBV DNA value above 10(5) copies/mL would exclude all inactive carriers, but 45% of patients with HBeAg-negative chronic hepatitis would also be excluded if testing were only performed at presentation and 30% would be excluded if testing were performed on 3 occasions. In conclusion, serum HBV DNA levels decreased significantly in patients with HBeAg loss, but there was no threshold HBV DNA level associated with HBeAg clearance. Given the fluctuating course of HBeAg-negative chronic hepatitis, it is not possible to define a single cutoff HBV DNA value for differentiating inactive carriers from patients with HBeAg-negative chronic hepatitis.     

Usefulness of dried blood samples for quantification and molecular characterization of HBV-DNA

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 microL 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 x 10(6) in HBeAg-positive, 6.2 x 10(5) in HBeAg-negative, and 5.5 x 10(2) in inactive carriers (P <.05). HBV DNA was positive in serum (median 5 x 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 degrees 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.     

Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is responsible for viral persistence in the natural course of chronic HBV infection and during prolonged antiviral therapy and serves as the template for the production of HBV pregenomic RNA (pgRNA), the primary step in HBV replication. In this study, we have developed and applied sensitive and specific quantitative real-time polymerase chain reaction (PCR) assays for the measurement of intrahepatic concentration, pgRNA production, and replicative activity of cccDNA in liver biopsy samples from 34 non-treated patients with chronic hepatitis B (CHB); 12 hepatitis B e antigen (HBeAg)(+) and 22 HBeAg(-). Median copy number for cccDNA was 1.5 per cell and for pgRNA significantly higher, 6.5 copies per cell, with a good correlation between cccDNA and pgRNA levels in all samples. In HBeAg(-) patients, median values of cccDNA and pgRNA levels were 10-fold and 200-fold lower than in HBeAg(+), respectively, reflecting the differences in viral activity and clinical characteristics of the two groups. Furthermore, the replicative activity of intrahepatic cccDNA was significantly lower in HBeAg(-) patients harboring mutant HBV strains than in HBeAg(+) patients: median 3.5 versus 101 pgRNA copies per cccDNA molecule. In conclusion, the levels of both HBV cccDNA, a marker of HBV persistence, and pgRNA, an indicator of viral replication, in the liver of chronically infected patients correlate with viral activity and the phase of HBV infection. The combined measurement of cccDNA and pgRNA levels provides valuable information on the presence and replicative activity of intrahepatic HBV cccDNA.     

慢性乙型肝炎患者血清前S1抗原检测的意义

目的探讨PreS1抗原检测的临床价值。方法采用酶联免疫吸附试验法检测421例慢性乙型肝炎患者血清PreS1抗原和HBV标记物;采用荧光定量PCR法检测HBVDNA。结果在421例慢性乙型肝炎患者中,HBVDNA阳性者367例,其中PreS1Ag阳性者188例(51.2%),HBeAg阳性者119例(32.4%),后两者有显著性差异(P0.01);在高HBVDNA载量(105～107copies/ml和107copies/ml)组患者中,PreS1Ag阳性率(60.2%,60.0%)显著高于HBVDNA阴性组(33.3%)和低载量(103～105copies/ml)组(41.9%,P0.01);但在421例患者中,PreS1Ag阳性率(48.9%)低于HBVDNA(87.2%,P0.01)。结论 PreS1Ag能够较HBeAg更好地反映HBV在体内的复制状态,但尚不能代替HBVDNA的检测。     

Presence of precore and core promoter mutants limits the probability of response to peginterferon in hepatitis B e antigen-positive chronic hepatitis B

Peginterferon (PEG-IFN) treatment of hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) results in HBeAg loss in 30% of patients, but clearance of hepatitis B virus (HBV) DNA and hepatitis B surface antigen (HBsAg) from serum is less often achieved. We investigated whether the presence of precore (PC) and basal core promoter (BCP) mutants before PEG-IFN treatment affects serological and virological response. A total of 214 HBeAg-positive CHB patients treated with PEG-IFN ± lamivudine for 52 weeks in a global randomized trial were classified at baseline as wildtype (WT) or non-WT (detectable mutants at PC/BCP) by line-probe assay. Response was assessed at 6 months posttreatment and through long-term follow-up (LTFU). Mutants were detected in 64% of patients, in varying frequencies across HBV genotypes A through D. Patients with WT had higher baseline HBV DNA, HBeAg, and HBsAg levels than patients with non-WT. Patients with WT were more likely to achieve HBeAg loss with HBV DNA <10,000 copies/mL (response, 34 versus 11%, P < 0.001) and HBsAg clearance (18 versus 2%, P < 0.001) at week 78 than non-WT patients. Among WT patients who achieved HBeAg clearance at week 78, 78% had undetectable HBV DNA and 61% achieved HBsAg clearance at LTFU (versus 26% and 15% in non-WT patients, P < 0.001 for both). The presence of WT virus at baseline was an independent predictor of response (odds ratio [OR] 2.90, 95% confidence interval [CI]: 1.15-7.31, P = 0.023) and HBsAg clearance (OR 5.58, 95% CI: 1.26-24.63, P = 0.013) and patients with non-A genotypes with detectable mutants had a low probability of response. CONCLUSION: The presence of only WT virus at baseline is a strong predictor of response (HBeAg loss with HBV DNA <10,000 copies/mL) to PEG-IFN for HBeAg-positive CHB. Patients with detectable PC and/or BCP mutants have a lower probability of response and are less optimal candidates for PEG-IFN therapy.     

Prolonged antiviral therapy for hepatitis B virus-infected health care workers: a feasible option to prevent work restriction without jeopardizing patient safety

To prevent transmission of hepatitis B virus (HBV) from health care workers (HCWs) to patients, highly viraemic HCWs are often advised to restrict performing exposure prone procedures (EPPs). To prevent loss of highly qualified medical personnel and simultaneously minimize transmission risk to patients, we offered highly viraemic HCWs antiviral therapy and evaluated the effects of this strategy. Eighteen chronic HBV-infected HCWs have been monitored every 3-6 months for a median period of 5.6 years (range 1.1-12.5 years). Antiviral therapy was offered if HBV DNA was above 10(5) copies/mL and EPPs were performed or active liver disease was present. Median HBV DNA levels, the percentage of days with HBV DNA above 10(3), 10(4) and 10(5) copies/mL, and reduction of HBV DNA during antiviral treatment have been analysed for hepatitis B e antigen (HBeAg)-positive and HBeAg-negative HCWs separately. Prolonged viral suppression was achieved in both HBeAg-positive, as well as HBeAg-negative HCWs. In HBeAg-negative HCWs treatment with interferon or lamivudine maintained HBV DNA levels below 10(5) copies/mL. For HBeAg-positive HCWs continuous treatment with tenofovir or entecavir was essential for reaching low viraemia persistently. In 2004, median HBV DNA levels in both HBeAg-negative and HBeAg-positive HCWs were below 10(3) copies/mL and all HCWs executed their professional work full-range. For both HBeAg-positive and HBeAg-negative HCWs, antiviral treatment is effective in persistent suppression of virus levels below 10(5) copies/mL. This observation supports antiviral therapy as a viable management option instead of work restriction, with the provision of regular expert monitoring including quantification of HBV DNA.     

Serial HBV DNA levels in patients with persistently normal transaminase over 10 years following spontaneous HBeAg seroconversion

Earlier studies addressing the hepatitis B virus (HBV) DNA cut-off level for inactive chronic HBV infection largely involved patients with normal alanine aminotransferase (ALT) for only 1-2 years and based on a single time HBV DNA assay. This study was conducted to address this issue using serial HBV DNA assays in patients with persistently normal ALT (PNALT) over 10 years following spontaneous hepatitis B e antigen (HBeAg) seroconversion. Serial serum specimens (mean 9 samples per patient) of 62 patients with PNALT and no disease progression over 10 years (median 18.1 years) after spontaneous HBeAg seroconversion were assayed for HBV DNA. Excluding assays within 1 year after HBeAg seroconversion, 21% and 82.3% of the patients with PNALT had HBV DNA levels persistently lower than 4 log(10) and 5 log(10) copies/mL, respectively, and only 8% had a level ≥ 5 log(10) copies/mL in at least two assays. Of the 27 patients with PNALT defined by ALT <30 U/L for male and <19 U/L for female, only 33% had serum HBV DNA level persistently <4 log(10) copies/mL. There was no significant difference in the serial HBV DNA changes among patients with different gender, HBV genotype or age at HBeAg seroconversion. Liver biopsy in nine patients invariably showed minimal necroinflammation and one showed Ishak fibrosis score 4. These results suggest that 5 log(10) copies/mL (20,000 IU/mL) is a more appropriate cut-off HBV DNA level for inactive chronic HBV infection in the setting of PNALT.     

A treatment algorithm for the management of chronic hepatitis B virus infection in the United States.

BACKGROUND AND AIMS: Chronic hepatitis B is an important public health problem worldwide and in the United States. A treatment algorithm for chronic hepatitis B virus (HBV) infection was developed by a panel of US hepatologists based on new developments in the understanding of the virology of HBV, availability of more sensitive molecular diagnostic testing, and advantages and disadvantages of currently approved therapies. METHODS: This algorithm is based on available evidence, but where data are lacking, the panel relied on clinical experience and consensus expert opinion. RESULTS: Serum HBV DNA can be detected at levels as low as 100-1000 copies/mL by using molecular assays and should be determined to establish a baseline level before treatment, monitor response to antiviral therapy, and survey for the development of drug resistance. The primary aim of antiviral therapy is durable suppression of serum HBV DNA to the lowest level possible. The threshold level of HBV DNA for determination of candidacy for therapy is >/=10(5) copies/mL for patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B. A lower serum HBV DNA threshold is appropriate for patients with HBeAg-negative chronic hepatitis B and those with decompensated cirrhosis, and the panel recommends thresholds of 10(4) copies/mL and 10(3) copies/mL, respectively. CONCLUSIONS: Interferon alfa-2b, lamivudine, and adefovir dipivoxil are all approved as initial therapy for chronic hepatitis B and have certain advantages and disadvantages. Issues for consideration include efficacy, safety, incidence of resistance, method of administration, and cost. Studies are under way to explore the safety and efficacy of combination therapy, which may prove to be more effective than monotherapy in suppressing viral replication and may decrease or delay the incidence of drug resistance.     

拉米夫定治疗慢性乙型肝炎的疗效影响因素

目的 探讨拉米夫定治疗慢性乙型肝炎患者的疗效影响因素,并初步评价基线特征、24周早期病毒学应答及治疗方案对疗效和病毒学突破(VB)发生率的影响.方法 对接受拉米夫定治疗的233例慢性乙型肝炎患者(其中90例治疗期间加用或换用阿德福韦酯)的专科门诊病历资料进行回顾性分析,采用聚合酶链反应法、酶联免疫吸附法分别检测HBV DNA水平与HBsAg、抗-HBs,HBeAg,抗-HBe水平.用SPSS17.0统计软件通过Kaplan-Meier法描述生存时间分布,并分析基线HBV DNA水平,HBeAg状态、ALT水平和疗效的关系.计量资料用t检验分析,计数资料用x2检验.结果 HBeAg阳性与HBeAg阴性患者HBV DNA阴转率分别为63.4%和84.6%,ALT复常率分别为83.8%和81.3%,VB发生率分别为31.0%和14.3%;60.6%的HBeAg阳性患者出现HBeAg阴转,28.9%出现HBeAg/抗-HBe血清学转换.HBeAg阳性患者中,与基线ALT<2.5×正常值上限(ULN)者比较,≥2.5×ULN者HBV DNA阴转率无明显变化(P>0.05),而HBeAg阴转率(66.7%与45.0%)和HBeAg血清学转换率(33.3%与17.5%)明显升高(P值均<0.05),VB发生率则明显下降(34.3%与50.0%,P<0.05),基线HBV DNA<1×106拷贝/ml者VB发生率为23.4%,与HBV DNA≥1×106拷贝/ml者的46.3%比较,差异有统计学意义(P<0.05).HBeAg阳性患者24周有初始病毒学应答(IVR)者的HBV DNA阴转率(76.3%与45.5%)、HBeAg阴转率(72.4%与43.9%)和HBeAg血清学转换率(40.8%与12.1%)均明显高于无IVR者(P值均<0.01),VB发生率较低(28.9%与45.5%,P<0.05).出现VB后,与单一拉米夫定组比较,加药或换药组中HBeAg阳性者HBV DNA阴转率(40.6%与16.7%)、HBeAg血清学转换率(21.9%与0)较高,HBeAg阴转率(37.5%与41.7%)较低,ALT复常率无差别(均为75%);而HBeAg阴性患者的HBV DNA阴转率、ALT复常率较高.结论 拉米夫定抗病毒疗效确切,基线ALT≥2.5×ULN和(或)HBV DNA水平<1×106拷贝/ml的患者疗效较好,VB发生率较低,24周IVR对拉米夫定疗效有预测价值;出现VB后,加用或者换用阿德福韦酯比继续单用拉米夫定治疗的效果好.     

Treatment of hepatitis B with lamivudine and tenofovir in HIV/HBV-coinfected patients: factors associated with response

As therapy for human immunodeficiency virus (HIV) infection evolves, optimizing hepatitis B virus (HBV) treatment and identifying factors that impact its response in the HIV/HBV-coinfected population is critical. We identified retrospectively 45 HBV/HIV-coinfected patients with detectable HBV DNA by the Bayer VERSANT HBV 3.0 bDNA assay (limit of quantification 2000 copies/mL) at baseline and/or year 1 of therapy. Patients were divided into three groups based on the active HBV agent in their antiretroviral regimen: group 1 (n = 15) received lamivudine; group 2 (n = 10), lamivudine plus tenofovir and group 3 (n = 20), lamivudine followed by lamivudine plus tenofovir. HBV genotypes and resistance profiles were determined by the Bayer Trugene HBV 1.0 assay. More patients in group 2 achieved HBV DNA suppression below 2000 copies/mL (80%), loss of HBe antigen (HBeAg) (40%) and loss of HBeAg and gain of anti-HBe (20%) than did patients in group 1 or 3. More patients with HBV genotype A, achieved HBV DNA suppression <2000 copies/mL than did patients with non-A genotypes [74% (26/35) vs 20% (2/10)], respectively (P = 0.003). Risk for virological nonresponse was significant in those with non-A genotypes [odds ratio (OR) 11.1; 95% CI: 2.0-50], previous HIV therapy (OR 6.5; 95% CI: 1.2-35) and <90% compliance (OR 3.7; 95% CI: 0.99-14.3). Simultaneous therapy with lamivudine/tenofovir suppresses HBV DNA more effectively than lamivudine or tenofovir added to lamivudine. More patients infected with HBV genotype A responded than the non-A patients, regardless of therapeutic regimen, compliance or prior HIV therapy.     

肝活检对HBV携带者的诊断意义

目的探讨肝活检对HBV携带者的诊断意义。方法回顾性分析134例HBV携带者,在B超引导下行1秒钟快速肝穿刺活检术,行肝组织病理学检查。结果在134例患者中,仅2例（1.49%）肝组织病理学检查完全正常（G0S0）;在105例HBV DNA≥10^5copies/ml（或HBeAg阴性者HBV DNA≥10^4copies/ml）的HBV携带者中,肝组织炎症活动度≥G2者80例（76.19%）,即可实施抗病毒治疗;在100例HBeAg（＋）与34例HBeAg（-）感染者,肝组织炎症活动度和纤维化程度比较无明显统计学差异（P=0.308）;在不同肝组织炎症活动度和纤维化程度感染者,HBV DNA载量无明显的统计学差异（P=0.557）,HBV DNA≥10^7copies/ml与HBV DNA〈10^7copies/ml感染者比,肝组织炎症活动度和纤维化程度存在统计学差异（P=0.007,P=0.001）。结论对于慢性HBV携带者,应及时进行肝组织活检。     

替比夫定治疗HBeAg阳性慢性乙型肝炎的疗效及相关预测因子的Logistic回归分析

目的观察替比夫定（LdT）治疗HBeAg阳性慢性乙型肝炎（CHB）患者3年的疗效,应用Logistic回归探讨HBeAg血清学转换的预测因子。方法收集58例采用LdT治疗的HBeAg阳性CHB患者,分析其性别、年龄、基线ALT水平、基线HBV DNA载量、基线HBeAg和HBsAg滴度与治疗3年时ALT复常率、HBV DNA阴转率、HBeAg阴转率和HBeAg血清转换率的相关性;采用Logistic回归分析HBeAg血清转换的相关因素。结果治疗3年时ALT复常率为84.48%,HBV DNA阴转率为70.69%,HBeAg阴转率为50.00%,HBeAg血清转换率为43.10%。与ALT≤2倍正常值上限（2×ULN）相比,基线ALT〉5×ULN的患者HBeAg转换率显著增高（P〈0.05）;与HBeAg≤100（S/CO）组相比,基线HBeAg〉200 S/CO的患者HBeAg的阴转率和血清转换率均显著下降（P〈0.05）;与HBV DNA≤6 log拷贝/ml组相比,HBV DNA〉7 log拷贝/ml的患者HBV DNA转阴率、HBeAg转阴率和HBeAg转换率下降显著（P〈0.05）;患者性别、年龄及基线HBsAg滴度对以上疗效指标无影响（P〉0.05）。多因素Logistic回归分析发现仅基线HBeAg滴度低的患者更易出现HBeAg血清学转换。结论 LdT能有效恢复肝功能,抑制HBV复制和提高HBeAg血清转换;基线HBeAg滴度可预测LdT治疗HBeAg阳性CHB患者的HBeAg血清转换率。     

Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients

This study examined a signal amplification assay, the Invader assay, for the quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver biopsies and sera. DNA was extracted from liver biopsy and serum samples were collected from 16 hepatitis B e antigen (HBeAg)-positive and 36 antibody-to-HBeAg-positive (anti-HBe-positive) chronic hepatitis B patients. The amount of total HBV DNA and cccDNA was measured using the Invader assay. Anti-HBe-positive patients had lower median total intrahepatic HBV DNA (P < .001) and intrahepatic cccDNA levels (P = .001) than HBeAg-positive patients. Intrahepatic cccDNA correlated positively with the total intrahepatic HBV DNA (r = 0.950, P < .001). However, the proportion of intrahepatic HBV DNA in the form of cccDNA was inversely related to the amount of total intrahepatic HBV DNA (r = -0.822, P < .001). A small amount of cccDNA was detected in 39 of 52 (75%) serum samples. Anti-HBe-positive patients had lower median serum cccDNA levels than HBeAg-positive patients (P = .002). Serum HBV DNA correlated positively with intrahepatic total HBV DNA (r = 0.778, P < .001) and intrahepatic cccDNA (r = 0.481, P = .002). In conclusion, the Invader assay is a reliable assay for the quantitation of cccDNA. Serum and intrahepatic total HBV DNA and cccDNA levels become lower as the disease progresses from HBeAg-positive to anti-HBe-positive phase, with cccDNA becoming the predominant form of intrahepatic HBV DNA.