Relationship between hepatitis B virus DNA levels and liver histology in patients with chronic hepatitis B

AIM： To study the relationship between hepatitis B virus （HBV） DNA levels and liver histology in patients with chronic hepatitis B （CHB） and to determine the prevalence and characteristics of hepatitis B e antigen （HBeAg） negative patients.
METHODS： A total of 213 patients with CHB were studied, and serum HBV DNA levels were measured by the COBAS Amplicor HBV Monitor test. All patients were divided into two groups according to the HBeAg status.The correlation between serum HBV DNA levels and liver damage （liver histology and biochemistry） was explored.
RESULTS： Of the 213 patients with serum HBV DNA levels higher than 10^5 copies/mL, 178 （83.6%） were HBeAg positive, 35 （16.4%） were HBeAg negative. The serum HBV DNA levels were not correlated to the age,history of CHB, histological grade and stage of liver disease in either HBeAg negative or HBeAg positive patients. There was no correlation between serum levels of HBV DNA and alanine aminotransferanse （ALT）,aspartate aminotrans-ferase （AST） in HBeAg positive patients. In HBeAg negative patients, there was no correlation between serum levels of HBV DNA and AST,while serum DNA levels correlated with ALT （r = 0.351, P = 0.042）. The grade （G） of liver disease correlated with ALT and AST （P 〈 0.05, r = 0.205, 0.327 respectively）in HBeAg positive patients. In HBeAg negative patients,correlations were shown between ALT, AST and the G （P 〈 0.01, and r = 0.862, 0.802 respectively）. HBeAg negative patients were older （35 ± 9 years vs 30 ±9 years, P 〈 0.05 ） and had a longer history of HBV infection （8 ± 4 years vs 6 ± 4 years, P 〈 0.05） and a lower HBV DNA level than HBeAg positive patients （8.4± 1.7 Log HBV DNA vs 9.8 ± 1.3 Log HBV DNA, P 〈0.001）. There were no significant differences in sex ratio,ALT and AST levels and liver histology between the two groups.
CONCLUSION： Serum HBV DNA level is not correlated to histological grade or stage of liver disease in CHB patients with HBV DNA mor     

Failure of therapeutic vaccination using hepatitis B surface antigen vaccine in the immunotolerant phase of children with chronic hepatitis B infection

AIM: The aim of this study was to investigate the efficacy of specific hepatitis B virus (HBV) vaccination as active immunotherapy in treating chronic hepatitis B (CHB) infection during the immune-tolerant phase in children with normal aminotransferase levels and high viral load. METHODS: Fifty-one immunotolerant patients were randomly and prospectively recruited into two groups. Group 1 included 23 patients that were vaccinated with three standard injections of the GenHevac B vaccine in the deltoid or quadricep muscle, initially, and at 30 days and 60 days, for specific immunization. Group 2 contained 28 patients who did not receive any medication or vaccination and were recruited as the control group. Post-vaccination evaluation was performed at 6 months from the first injection and at the end of the 12th month by serological and virological analyses. A response criterion to therapy was defined as loss of HBV-DNA in serum and hepatitis B early antigen (HBeAg) seroconversion (loss of HBeAg, development of antibody to HBeAg (anti-HBe)). RESULTS: The mean alanine aminotransferase (ALT) value in group 1 at the beginning of the vaccination was 33.6 +/- 8.1 IU/L; this changed to 31.7 +/- 9.0 IU/L at 6 months after first injection and 29.2 +/- 7.1 IU/L at the end of 12 months (P > 0.05). In this group, mean HBV-DNA load at the starting point of the vaccination was 3,709 +/- 1,126 pg/mL; this value changed to 3,569 +/- 726 pg/mL at the sixth month and 3,295 +/- 832 pg/mL at the 12th month (P > 0.05). In group 2, the mean ALT values at the beginning of therapy, and at the 6th and 12th month were 32 +/- 8 IU/L, 31.8 +/- 8 IU/L, and 29.7 +/- 7 IU/L, respectively (P > 0.05), and the mean viral load of HBV-DNA values were 3,827 +/- 1,375 pg/mL, 3,498 +/- 886 pg/mL, and 3,059 +/- 731 pg/mL, respectively (P > 0.05). The load of HBV DNA of all patients in both groups was greater than 2,000 pg/mL. There was no statistically significant difference in the mean ALT values and mean viral load of HBV DNA (P > 0.05) between group 1 and group 2 at the end of the 6th and 12th months. Except for one each patient in each group, hepatitis B surface antigen (HBsAg) and HBeAg clearance or antibody to HBsAg (anti-HBs) and anti-HBe seroconversion were not observed during the follow-up period (P > 0.05). CONCLUSION: In this study, comparison of vaccinated and unvaccinated groups of immunotolerant children with CHB infection showed no difference in the clearance of HBV DNA and seroconversion of HBeAg to anti-HBe. Different immunization protocols should be considered for future investigations in the immunotolerant phase of children with CHB infection.     

慢性乙型肝炎抗病毒疗效与外周血单个核细胞内乙型肝炎病毒DNA水平的关系

目的 探讨慢性乙型肝炎(CHB)抗病毒疗效与达到停药标准时外周血单个核细胞(PBMC)内HBV DNA水平的关系.方法 入选90例经抗病毒治疗达到停药标准的CHB患者,其中应用IFN 44例,应用核苷类药物46例.所有患者均于停药时检测PBMC内HBV DNA,比较阴性组和阳性组治疗前血清HBV DNA水平与达到停药标准时PBMC内HBV DNA的关系,观察停药时PBMC内HBV DNA水平与复发的关系.计量资料采用t检验,计数资料采用X2检验.结果 90例CHB患者停药时,PBMC内HBV DNA阴性组67例,阳性组23例.CHB患者血清HBV DNA阳转率在PBMC内HBV DNA阴性组为13.4%(9/67例),显著低于阳性组的73.9%(17/23例),差异有统计学意义(X2=30.4873,P<0.01).PBMC内HBV DNA阴性组与阳性组在肝病复发ALT升高幅度(t=0.8729,P=0.3913)、停药后复发时间(t=1.9222,P=0.0665)均差异无统计学意义,而在血清HBV DNA反弹幅度则差异有统计学意义(t=2.7493,P=0.0112).5例患者获得HBsAg血清学转换,且均未检测到PBMC内HBV DNA,随访6～12个月无一例复发.PBMC内HBV DNA阳性组治疗前血清HBV DNA水平为(7.2±1.1)lg拷贝/mL,显著高于阴性组的(5.2±2.1)lg拷贝/mL(t=4.3557,P<0.01).结论 经抗病毒治疗达到停药标准的CHB患者,其停药时的PBMC内HBV DNA水平可能是预测抗病毒疗效持久性的重要因素之一.

Abstract：

Objective To explore the relationship between the antiviral effect and peripheral blood mononuclear cell (PBMC) hepatitis B virus (HBV) DNA when the patients reach the standard of withdrawal of antiviral therapy in chronic hepatitis B (CHB).Methods Ninety CHB patients treated with interferon(n=44) or nucleot (s) ide(n=46) who reached the standard of withdrawal of antiviral therapy were recruited.HBV DNA levels in PBMCs were tested at the end of treatment,and its relationship with serum HBV DNA level before treatment in PBMC HBV DNA positive group and negative group were compared.The correlation between HBV DNA in PBMCs at the end of treatment and relapse were explored.Measurement data were analyzed by student t test and enumeration data were analyzed by X2 test.Results Among 90 patients,67(74.4%) were PBMC HBV DNA negative at the end of treatment,and 23(25.6%) were positive.The serum HBV DNA positive conversion rate in PBMC HBV DNA negative patients was 13.4%,which were significantly lower than that in positive group (73.9%) (X2=30. 4873, P<0.01 ). There were no significant differences of alanine aminotransferase (ALT) levels when hepatitis flare (t=0. 8729, P=0. 3913) and relapse time (t=1. 9222, P=0. 0665) between PBMC HBV DNA negative group and positive group after withdrawal of therapy, while the serum HBV DNA rebound was greater in positive group than that in negative group (t=2. 7493, P=0. 0112). There were five patients who achieved hepatitis B surface antigen (HBsAg) seroconversion, whose PBMC HBV DNA were all undetectable, and none relapsed during follow-up for 6-12 months. The pretreatment HBV DNA as level in PBMC HBV DNA positive was (7.2±1.1) lg copy/mL, which was much higher than that in negative group[(5.2±2.1) lg copy/mL] (t=4. 3557, P<0.01). Conclusions In patients who reach the standard of drug withdrawal,PBMC HBV DNA at the end of treatment is an important predictor for durability of antiviral therapy in CHB.     

Measurement of hepatitis B viral DNA in serum by solution hybridization and comparison with the dot-blot hybridization technique

A recently introduced method for the detection of hepatitis B viral (HBV) DNA in the serum, using solution hybridization, was assessed for its sensitivity and specificity in 242 sera from patients with chronic HBV infection, and in controls. In 87 sera the results were compared with the classical dot-blot technique. The new method gave positive results in 82% (56/68) of all dot-blot-positive sera and in 97% (35/36) of those with strong positive dot-blot reactions. All 30 hepatitis B surface antigen (HBsAg) carriers positive for hepatitis B e antigen (HBeAg) were positive in the solution hybridization assay, with a mean HBV-DNA level of 172 +/- SE 36 pg/ml. Negative results were obtained in all 20 subjects negative for all HBV markers and in all 36 HBsAg+/anti-HBe+ carriers with normal aminotransferase activity, negative for hepatitis B core antigen (HBcAg) expression in the liver. By contrast 93% (66/71) of the anti-HBe+ patients with positive HBcAg expression in the liver were positive for serum HBV-DNA in the solution hybridization assay, with a mean level of 30.5 +/- SE 6.7 pg/ml. Of 14 sera with discrepant HBV-DNA results in the two assays, 12 were solution hybridization-negative/dot-blot-positive and 2 were solution hybridization-positive/dot-blot-negative. These results indicate that the new, simple method is specific, and suitable for the determination of serum HBV-DNA in clinical practice.(ABSTRACT TRUNCATED AT 250 WORDS)     

慢性乙型肝炎重叠戊型肝炎病毒感染的研究

目的了解慢性乙型肝炎重叠戊型肝炎病毒(HEV)感染的临床特点、乙型肝炎病毒(HBV)复制指标、肝功能损伤程度及预后。方法收集慢性乙型肝炎患者和慢性乙型肝炎重叠HEV感染(重叠感染组)各115例,两组病情(轻、中、重度)和HBV DNA定量相同,对两组患者进行临床分析,慢性乙型肝炎组中74例和重叠感染组中的51例患者,在B超引导下做肝活组织检查；应用酶联免疫吸附试验和聚合酶链反应分别检测两组患者HBV标志物,HBV DNA及抗HEV lgM。结果重叠感染组重型肝炎57例,发生率49．6%,死亡29例,病死率25．2%；慢性乙型肝炎组重型肝炎5例,发生率4．4%,死亡2例,病死率1．7%,两组比较,x~2值分别为58．80和27．01,P值均＜0．01,差异有统计学意义。血清HBV DNA≥10~4患者：重叠感染组占83．7%(36/43),单纯慢性乙型肝炎组占97．1%(67/69),x~2=4．73,P＜0．05；重叠感染组总胆红素平均(495．0±217．0)μmol/L、丙氨酸氨基转移酶平均(967．0±395．0) U/L,单纯慢性乙型肝炎组总胆红素平均(216．0±195．0)μmol/L和丙氨酸氨基转移酶平均(373．0±212．0)U/L,两组比较,t值分别为10．20和14．52,P值均＜0．01,差异有统计学意义；肝组织炎症G3和G4重叠感染组33例,占64．7%,单纯慢性乙型肝炎组25例,占33．8%,x~2=12．46,P＜0．01,差异有统计学意义。结论重叠感染组肝功能损害严重,肝组织炎症程度高,HBV DNA水平低,病死率高,预后差。     

Efficacy of Pre-S-containing HBV Vaccine Combined with Lamivudine in the Treatment of Chronic HBV Infection

Treatment of chronic hepatitis B (CHB) is difficult. The response rate to interferon (IFN) as well as nucleoside analogs is not more than 30% in general. While interferon has many side effects, development of resistance in most of the nucleoside analogs precludes long-term use. Both groups of drugs are most efficacious in patients who already had or develop strong cellular immunity with treatment. A pre-S2-containing vaccine was shown to enhance cellular immunity and suppress hepatitis B virus (HBV)-DNA in subjects with chronic hepatitis B. We aimed to test the efficacy of short-term use of a nucleoside analog in combination with a pre-S2-containing vaccine in patients with CHB. In this open study, 48 consecutive patients (32 males and 16 females, mean age ± SD: 33 ± 12 years) with CHB without cirrhosis were treated with 100 mg/day lamivudine and four weekly intramuscular injections of Genhevac B 20 mcg (six doses) for 24 weeks. While 19 patients were hepatitis B e antigen (HBeAg) positive (+ve), 29 patients were Anti-HBe/HBV-DNA +ve at the outset. Response was defined as seroconversion to anti-HBe in HBeAg +ve subjects and normalization of alanine aminotransferase (ALT) with loss of HBV-DNA in anti-HBe/HBV-DNA +ve subjects. HBeAg seroconversion occurred in 5/19 subjects (26%). Eighteen of 29 anti-HBe/HBV-DNA +ves responded. In the follow-up, while relapse was not observed in any of the patients who seroconverted, 11/18 from the anti-HBe/HBV-DNA +ve group relapsed, resulting in a sustained response (SR) rate of 24% in this group. All the relapses happened in the first 48 weeks of follow-up, with no relapse thereafter. Pretreatment high serum HBV-DNA was a strong negative predictor of sustained response (SR) in HBeAg +ve group. Pretreatment serum ALT over 2 × upper limit of normal and HBV-DNA less than 200 pg/ml appeared positive predictors. None of HBeAg +ve previous interferon failures responded. Twenty-four weeks of lamivudine and hepatitis B vaccine treatment induces SR in around 1/4 of the patients with CHB. Most of the responders had high ALT and relatively low DNA.     

肝功能正常的慢性乙型肝炎病毒感染者血清HBeAg及HBV DNA与肝组织病理改变的关系

目的了解肝功能正常的慢性乙型肝炎病毒（HBV）感染者肝组织病理改变特征,并分析血清HBeAg及HBV DNA定量与肝组织病理改变的关系。方法选取肝功能正常的慢性HBV感染者90例,行肝穿刺病理检查,依据血清HBeAg及HBV DNA将患者分组,分别比较HBeAg阳性和阴性组及HBV DNA阳性和阴性组患者肝组织炎症分级和纤维化分期结果。结果90例患者100%存在肝组织损伤,其中肝硬化8例（8.9%）;慢性乙型肝炎轻度62例（68.9%）,中度8例（8.9%）,重度12例（13.3%）。82例病理诊断慢性乙型肝炎的患者中,炎症分级G≥2者30例（36.6%）;纤维化分期S≥2者28例（34.15%）。HBeAg阴性组病理炎症分级及纤维化分期均明显重于阳性组（P值均〈0.05）。HBV DNA阴性和阳性组肝组织炎症分级和纤维化分期差异均无统计学意义。结论肝功能正常的慢性HBV感染者,肝组织病理皆非＂正常＂;血清HBeAg、HBV DNA均不能反映肝脏损伤情况;对此类患者制定治疗方案时应考虑肝组织病理检查结果。     

Clinical outcomes of chronic hepatitis B patients with persistently detectable serum hepatitis B virus DNA during lamivudine therapy

BACKGROUND AND AIM: A small proportion of chronic hepatitis B patients have persistently detectable serum hepatitis B virus (HBV) DNA despite lamivudine therapy. The incidence and clinical outcomes of patients who persistently have detectable serum HBV-DNA during lamivudine therapy was investigated. METHOD: We enrolled 221 chronic hepatitis B patients who underwent lamivudine therapy for more than 6 months. Among them, 180 were HBeAg positive. Serum HBV-DNA, HBeAg, anti-HBe and alanine aminotransferase (ALT) levels were serially monitored. The study groups were defined, using a hybridization assay, as patients with reductions in serum HBV-DNA below the detectable level (group I) or patients with persistently detectable serum HBV-DNA (group II) during the initial 6 months of lamivudine therapy. RESULTS: The incidence of patients who had persistently detectable HBV-DNA was 7.7%. After the first year, the rates of viral breakthrough, HBeAg loss and serum ALT normalization of group I versus group II were 21% versus 63%, 38% versus 0%, and 71% versus 28%, respectively (P < 0.001). The log(10) reduction of serum HBV-DNA at 6 months was -4.58 log(10) for group I and -1.97 log(10) for group II (P < 0.001, bDNA assay). There were no pretreatment lamivudine-resistant mutants in group II. CONCLUSION: Lamivudine had little effect on serum HBV-DNA suppression, viral breakthrough suppression and rate of HBeAg loss and ALT normalization in chronic hepatitis B patients with persistently detectable serum HBV-DNA during the initial 6 months of therapy. Early termination of lamivudine therapy is advocated for these patients.     

Quantitative detection of hepatitis B virus DNA in sera from patients with acute hepatitis B

Two hundred forty-four serial serum samples from 30 adults hospitalized with benign (nonfulminant) acute hepatitis B were tested for the presence of hepatitis B virus (HBV) DNA by a quantitative solution hybridization assay using a¹²⁵I-labeled DNA probe complementary to HBV-DNA sequences. Acute hepatitis B was self-limiting in 28 and progressed to chronicity in the remaining two patients. Of the 28 patients with self-limiting hepatitis, 21 (75%) were hepatitis B e antigen (HBeAg) positive, 26 (93%) were HBV-DNA positive, and one patient (3.6%) was negative for both markers on admission to the hospital. HBV-DNA cleared after HBeAg clearance in 20 (71.4%), before HBeAg clearance in five (17.9%) and simultaneously with the loss of HBeAg in the remaining two (7.1%) of the 27 initially HBV-DNA- and/or HBeAg-positive patients. Moreover, HBV-DNA remained detectable in serum for 13.3±6.6 (range: 4–22) days after the appearance of anti-HBe in 71.4% of these patients. In contrast, HBV-DNA and HBeAg remained persistently positive in the two patients who developed chronic HBV infection. These data show that: (1) viremia frequently persists after disappearance of HBeAg and (2) appearance of anti-HBe does not indicate the cessation of HBV replication in adults with acute self-limiting hepatitis B.     

Hepatitis B and D viruses replication interference: Influence of hepatitis B genotype

AIM: To study the hepatitis B virus(HBV) and hepatitis D virus(HDV) replication interferences in patients with chronic hepatitis delta infected with different HBV genotypes.METHODS: We conducted a transversal study including 68 chronic hepatitis delta(CHD)(37 HIVpositive) patients and a control group of 49 chronic hepatitis B(CHB)(22 HIV-positive) patients. In addition, a dynamic follow-up was performed in 16 CHD patients. In all the samples, the surface antigen of hepatitis B(HBs Ag) serum titers were analyzed with the Monolisa HBs Ag Ultra system(Bio-Rad), using as quantification standard a serial dilution curve of an international HBs Ag standard. Serum HBV-DNA titers were analyzed using the Roche Cobas Taq Man(Roche, Barcelona, Spain), and the serum HDV-RNA using an in-house real-time q RT-PCR method, with Taq Man probes. HBV genotype was determined with the line immunoassay Li PA HBV genotyping system(Innogenetics, Ghent, Belgium). In those patients negative for Li PA assay, a nested PCR method of complete HBs Ag coding region, followed by sequence analysis was applied.RESULTS: No differences in the HBV-DNA levels were found in CHB patients infected with different HBV genotypes. However, in CHD patients the HBV-DNA levels were lower in those infected with HBV-A than in those with HBV-D, both in HIV negative [median(IQR): 1.25(1.00-1.35) vs 2.95(2.07-3.93) log10(copies/m L), P = 0.013] and HIV positive patients [2.63(1.24-2.69) vs 7.25(4.61-7.55) log10(copies/m L), P 0.001]. This was confirmed in the dynamic study of the HBV/HDV patients. These differences induce an under-estimation of HBV-A incidence in patients with CHD analyzed with Li PA assay. Finally, the HBs Ag titers reflected no significant differences in CHD patients infected with HBV-A or D.CONCLUSION: Viral replication interference between HBV and HDV is HBV-genotype dependent, and more evident in patients infected with HBV-genotype A, than with HBV-D or E.     

乙型肝炎病毒共价闭合环状DNA套式-实时荧光定量聚合酶链反应的建立

目的 建立检测HBV共价闭合环状DNA(cccDNA)的套式一实时荧光定量PCR法.方法 根据HBV cccDNA与松环DNA(rcDNA)结构上的差异,设计2对跨缺口的特异引物及1条位于负链缺口下游的特异TaqMan荧光探针.根据Plasmid-SafeTM ATP-Dependent Dnasc(PSAD)对rcDNA与cccDNA作用的不同,对模板DNA进行酶切纯化,降解reDNA,再进行套式PCR扩增,先用外引物和模板进行第一轮常规PCR,再用内引物、荧光探针和第一轮PCR产物进行实时荧光定量PCR,根据阳性参照标准品,得出待检标本定量值.结果 检测阳性参照标准品.得出该方法灵敏度可达2 lg拷贝/mL.用上述方法检测34份乙型肝炎患者血清HBV DNA阳性标本,25份血清HBVcccDNA阳性,28份外周血单个核细胞HBV cccDNA阳性.27份健康对照者血清HBV DNA阴性标本,6份HBV cccDNA阳性.对5份HBV cccDNA阳性标本扩增产物进行克隆测序,无碱基缺失、突变.与HBV不同基因型序列(A～G)比较,同源性为90.6%～99.1%,其中,与B、C基因型同源性为95.3%～99.1%,验证了方法的特异度.结论 套式-实时定量PCR法可检测乙型肝炎患者血清、PBMC中的HBV CCCDNA,且具有敏感、特异性.     

慢性乙型肝炎患者重叠戊型肝炎病毒感染的临床与病理学研究

目的：观察重叠戊型肝炎病毒（HEV）感染对慢性乙型肝炎（CHB）肝脏损害及HBV复制的影响。方法：应用ELISA法对122例CHB患者血清进行了抗－HEV IgkM,IgG检测,同时应用肝穿刺活检、荧光定量PCR及免疫组化等技术对重叠与未重叠HEV感染者分别进行了ALT、总胆红素（TBil)、凝血酶原活动度（PTA）、白蛋白／球蛋白（A／G）、电泳γ球蛋白（γ－EP）水平、肝脏病理学、血清HBeAg及肝组织HBcAg阳性率、血清及肝组织中HBV DNA含量对比。具有可比性的重叠（7例）与未重叠HEV感染者（14例）1年后做第2次肝穿活检并做病理学比较;HBeAg阴性重叠HEV感染者8例做HEV感染急性期、恢复期血清HBeAg定性、HBV DNA含量对比。结果：重叠HEV感染者21例（17．2％）。重叠HEV感染者较未重叠感染者ALT、TBil增高,PTA降低（P＜0．05）,但A／G、γ－EP水平未见显著差别（P＞0．05）;血清HBeAg及肝组织HBcAg阳性率、血清及肝组织HBV DNA含量低（P＜0．05）;肝组织炎症活动度重（P＜0．05）,但纤维化程度未见明显差别（P＞0．05）。两组患者1年前肝组织炎症活动度及纤维化程度无显著差别,1年后仍无显著差别（P＞0．05）。HEV感染恢复期血清HBeAg阳性率、HBV DNA含量高于急性期（P＜0．05）。结论：重叠HEV感染可加重CHB肝组织炎症活动度;对HBV复制具有短暂抑制作用。     

Serum hepatitis B virus DNA levels differentiating inactive carriers from patients with chronic hepatitis B

OBJECTIVE: We compared serum hepatitis B virus (HBV)-DNA levels in different states of hepatitis B infection, and investigated whether there is an HBV-DNA value that can be used for differentiating inactive carriers from patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis. METHODS: A retrospective study using sera at a followed endpoint from 64 Japanese patients with chronic HBV infection seen in Kobe University Hospital between 1989 and 2002. Sera of patients were assayed using a polymerase chain reaction-based assay. RESULTS: Genotype C was dominant (95.4%). Patients with chronic hepatitis with an elevation of the serum alanine aminotransferase (ALT) level had significantly higher HBV-DNA levels than patients with persistently normal ALT. For one time observation at a followed endpoint, the mean HBV-DNA level of HBeAg-negative inactive carriers was significantly lower than that of HBeAg-negative chronic hepatitis patients (3.6+/-1.0 versus 4.8+/-1.5 log copies/ml, P<0.005). The use of a cutoff value of 4.5 or 5.0 log copies/ml misclassified 23 and 18% of HBeAg-negative inactive carriers and 50 and 55% of patients with HBeAg-negative chronic hepatitis. If testing were performed on two occasions with approximately a 4-month interval, the cutoff values of 4.5 and 5.0 log copies/ml would misclassify 20 and 10% of HBeAg-negative inactive carriers and 28.6 and 28.6% of patients with HBeAg-negative chronic hepatitis. CONCLUSIONS: The measurement of serum HBV DNA more than twice is useful for assessing chronic hepatitis B surface antigen carriers and confirms that 10 copies/ml may be an appropriate level of HBV for characterizing the inactive carrier state.     

Fas、FasL在慢性乙型肝炎患者肝细胞和外周血淋巴细胞上的表达

探讨慢性乙型肝炎患者肝细胞及外周血淋巴细胞上Fas、FasL的表达与乙型肝炎病毒（HBV）复制水平及肝组织炎症程度的关系。对30例慢性乙型肝炎患者进行肝穿刺肝组织病理检查及免疫组化SP法检测肝细胞中Fas、FasL表达强度。并采用单克隆抗体经流式细胞仪检测外周血淋巴细胞上Fas、FasL表达阳性百分率;同时,采用荧光实时标记法测定血清中病毒复制指标HBV DNA水平;采用Beckman全自动生化分析仪检测肝功能并研究其相关性。慢性乙型肝炎患者肝细胞中Fas、FasL表达强度随肝组织炎症活动度加重而增强（P均〈0．001）,与白蛋白及丙氨酸转移酶（ALT）呈负相关（P〈0．005,P〈0．001）,与球蛋白、总胆红质无明显相关性（P均〉0．05）。外周血淋巴细胞上Fas、FasL的表达阳性率与血清中肝炎病毒复制指标HBV DNA水平呈正相关;与慢性肝炎分度无明显的相关性。乙型肝炎病毒感染可诱导肝细胞及外周血淋巴细胞上Fas、FasL的表达。肝细胞Fas、FasL的表达随炎症活动度加重而表达增强,但其介导的凋亡并不引起肝细胞炎症损伤即ALT活性并不升高,相反减少;同时在某种程度上影响白蛋白的合成。提示Fas—FasL介导的肝细胞凋亡是以非细胞损伤方式参与慢性乙型肝炎的发病过程。同时,随血清HBV DNA水平增高,外周血淋巴细胞上Fas、FasL的表达亦增强,淋巴细胞的凋亡因而增多,是导致乙型肝炎慢性化的原因之一。由此可见,Fas、FasL介导的凋亡参与了慢性乙型肝炎的发病机制,且在慢性乙型肝炎的发生发展中起重要作用。     

YMDD mutations in patients with chronic hepatitis B untreated with antiviral medicines

AIM: To polymerase P region (YMDD) mutations of hepatitis B virus gene (HBV DNA) in patients with chronic hepatitis B (CHB) untreated with antiviral medicines and to explore its correlation with pre-c-zone mutations, HBV genotypes and HBV DNA level, and to observe its curative effect. METHODS: A total of 104 cases (38 cases in group of familial aggregation and 66 cases in group of non-familial aggregation) were randomly chosen from 226 patients with CHB who did not receive the treatment of lamivudine (LAM) and any other antivirus drugs within the last one year. Their serum YMDD mutations were detected by microcosmic nucleic acid and cross-nucleic acid quantitative determination, HBV genotypes by PCR-microcosmic nucleic acid crossELISA, HBV DNA quantitative determination and fluorescence ration PCR analysis, hepatitis B virus markers (HBVM) by ELISA. LAM was taken by 10 patients with YMDD mutations and its curative effect was observed. RESULTS: Twenty-eight cases (26.9%) had YMDD mutations, of them 11 cases (28.9%) were in familial aggregation group (38 cases) and 17 cases (25.8%) in nonfamilial aggregation group (66 cases) with no significant difference between the two groups. Twenty-seven point one percent (16/59) cases were positive for HBeAg YMDD mutations, and 26.7% (12/45) cases were negative for HBeAg and positive for anti-HBe. There was also no significant difference between the two groups. Different YMDD incidence rate existed in different HBV genotypes. HBV DNA level did not have a positive correlation with the incidence of YMDD mutations. LAM was effective for all patients with mutations. CONCLUSION: Wild mutant strains in HBV and their incidence rate have no significant difference between familial aggregation and non-familial aggregation. It may have no significant relationship between YMDD mutations and pre-c-zone mutations. HBV DNA level may not have a positive correlation with YMDD mutations. LAM is clinically effective for CHB patients with YMDD mutations.     

舟山群岛慢性乙型肝炎病毒感染者基因型与临床病理的关系

目的观察中国舟山群岛慢性HBV感染者的基因型与临床病理变化的关系。方法180例两代以上生活在舟山群岛的当地居民,血清HBV标志物和HBV DNA均阳性的各类慢性肝病患者,男147例,女33例,年龄(39.0±11.3)岁,HBV携带者(ASC)17例,CHB轻度57例,中度48例,重度9例,重型肝炎(SHB)6例,肝炎后肝硬化(LC)39例,HCC 4例。采用PCR结合Taqman MGB探针技术,对血清中HBV基因型进行分型检测,同时检测HBV血清标志物、HBV DNA、肝功能。对其中的129例患者做肝组织活检。结果舟山群岛慢性HBV感染者HBV基因型C型135例,占75.0%,B型40例,占22.2%;B、C混合型5例,占2.8%,未见有A、D型。随着病情加重,C型所占比例逐渐增高,在ASC、CHB、LC和SHB中C型分别占41.2%(7/17例)、75.4%(86/114例)、87.2%(34/39例)、100%(6/6例);在HCC中,B、C基因型各占2例。肝组织病理学检查中,99例C基因型患者,中、重度炎症(G3～4)有84例,占84.8%,30例B基因型患者G3～4有7例,占23.3%,z=6.47,P<0.01。C基因型在中、重度纤维化(S3～4)的分布与B基因型比较,差异有统计学意义。在中、重度肝脏病理损伤中C型病理改变与临床诊断符合率较好;而在轻度肝脏病理损伤中,B型病理改变与临床诊断符合率较好,C型符合率较差。结论中国舟山群岛HBV基因型为C、B及BC混合型,以C基因型占优势,C基因型的病理损伤程度较B型严重。     

Drug Discovery Study Aimed at a Functional Cure for HBV

Hepatitis B virus (HBV) causes acute and, most importantly, chronic hepatitis B worldwide. Antiviral treatments have been developed to reduce viral loads but few patients with chronic hepatitis B (CHB) achieve a functional cure. The development of new therapeutic agents is desirable. Recently, many novel agents have been developed, including drugs targeting HBV-DNA and HBV-RNA. This review provides an overview of the developmental status of these drugs, especially direct acting antiviral agents (DAAs). Serological biomarkers of HBV infection are essential for predicting the clinical course of CHB. It is also important to determine the amount and activity of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. Hepatitis B core-associated antigen (HBcrAg) is a new HBV marker that has an important role in reflecting cccDNA in CHB, because it is associated with hepatic cccDNA, as well as serum HBV DNA. The highly sensitive HBcrAg (iTACT-HBcrAg) assay could be a very sensitive HBV activation marker and an alternative to HBV DNA testing for monitoring reactivation. Many of the drugs currently in clinical trials have shown efficacy in reducing hepatitis B surface antigen (HBsAg) levels. Combination therapies with DAAs and boost immune response are also under development; finding the best combinations will be important for therapeutic development.     

替比夫定对妊娠后期乙型肝炎病毒宫内感染的阻断作用

目的 探讨妊娠后期替比夫定对HBV宫内感染的阻断作用.方法 妊娠后期慢性乙型肝炎患者61例,31例给予替比夫定600mg,口服,1次/d;30例为对照,不给予抗病毒药物.观察两组患者母体HBV DNA水平的变化情况和新生儿HBsAg的阳性率.对HBsAg阳性率的差异分析采用x2检验,对计量资料采用t检验分析.结果 替比夫定组母体HBV DNA水平较服药前明显下降(t=19.09,P<0.01),且分娩前HBV DNA水平明显低于对照组(t=23.64,P<0.01).两组新生儿7月龄时HBV感染率分别为0和13.3%(4/30),x2=4.29,P<0.05.结论 妊娠后期应用替比夫定具有良好的安全性.替比夫定可显著抑制妊娠晚期孕妇血清HBV DNA水平,降低新生儿HBV感染率,可有效阻断HBV宫内感染.     

Distribution of hepatitis B virus genotypes among patients with chronic infection.

Hepatitis B virus (HBV) can be classified into at least eight genotypes, A-H. We evaluated the distribution HBV genotypes among patients with chronic infection. METHODS: We consecutively evaluated adult patients with chronic HBV infection from Salvador, Brazil. Patients were classified according to HBV infection chronic phases based on HBV-DNA levels and presence of serum HBV markers. HBV-DNA was qualitatively and quantitatively detected in serum by polymerised chain reaction (PCR). Isolates were genotyped by comparison of amino acid mutations and phylogenetic analysis. RESULTS: One-hundred and fourteen patients were evaluated. HBV-DNA was positive in 96 samples. HBV genotype was done in 76. Mean age was 36 +/- 11.3. In 61 of 76 cases subjects were classified as inactive HBsAg carriers. Their mean HBV serum level was 1760 copies/ml and 53 of 61 were infected with HBV genotype A, seven with HBV genotype F and one with genotype B. Twelve of the 76 patients had detectable hepatitis B e-antigen (HBeAg) in serum. Ten were infected with HBV genotype A and two with genotype F; most had increased alanine aminotransferase and high HBV-DNA levels. Three patients were in the immunotolerant phase, two were infected with HBV genotype A and one with genotype F. HBV subtyping showed subtypes adw2 and adw4. CONCLUSIONS: HBV genotype A adw2 and genotype F adw4 were the most prevalent isolates found. We could not find differences in genotype distribution according to HBV clinical phases and DNA levels. We did not detect HBV genotype D in contrast to a previous study in our center with acute hepatitis B. All inactive HBsAg carriers had low HBV-DNA levels.     

定量检测慢性乙型肝炎患者肝组织乙型肝炎病毒DNA的临床价值

目的探讨慢性乙型肝炎(CHB)患者肝内乙型肝炎病毒(HBV)DNA载量与血清HBV DNA、乙型肝炎病毒e抗原(HBeAg)水平的相关性及其在抗病毒治疗中的意义.方法41例HBeAg阳性CHB患者,在干扰素α和拉米夫定联合治疗前进行肝穿刺,取肝组织分别进行HBV DNA检测及组织学检查,据肝组织HBVDNA载量小于等于或大于104fg/cm3将其分为两组,治疗前及治疗期间监测其肝功能、血清HBeAg及HBV DNA情况.结果(1)肝组织HBV DNA载量高于血清HBV DNA载量(对数值4.081±1.127与3.163±1.010,t=2.218,P＜0.05),二者高度相关(r=0.840,t=4.322,P＜0.001);肝组织HBV DNA载量与血清HBeAg亦呈正相关(r=0.459,t=3.056,P＜0.005).(2)肝组织HBV DNA载量与肝组织炎症活动度呈反向关系(x2=3.874,P＜0.05).(3)治疗期间两组患者血清HBV DNA水平均明显下降,治疗前肝组织HBV DNA水平低者效果较好;治疗1年时HBeAg、抗-HBe血清转化率以肝组织HBV DNA水平低者为高(HBeAg转阴率68.4%与36.4%,x2=4.194,P＜0.05;抗-HBe阳转率73.7%与40.9%,x2=4.447,P＜0.05).结论肝组织HBV DNA水平较血清HBV DNA、HBeAg水平更能准确反映肝组织HBV DNA复制情况,且能间接反映机体的免疫状态,可作为抗病毒治疗适应证选择及疗效预测因子.