乙型肝炎病毒前S1抗原与病毒DNA检测及相关性研究

目的了解乙型肝炎病毒(HBV)前S1(Pre-S1)抗原在乙肝患者血清中的表达及评判其与HBV复制的关系。方法采用ELISA法检测858例各型乙肝患者血清标志物,以荧光定量PCR法检测HBVDNA,并比较分析Pre-S1抗原、HBeAg与HBV复制的相互关系和临床意义。结果858例各型乙肝患者中,Pre-S1抗原阳性总检出率为30.1%(258/858),HBeAg阳性总检出率为20.7%(178/858),HBVDNA阳性总检出率为43.2%(371/858)。在258例Pre-S1抗原阳性患者中,HBV-DNA阳性检出率为94.1%(243/258),而在371例HBVDNA阳性乙肝患者中,Pre-S1抗原和HBeAg阳性率分别为65.5%(243/371)和45.6%(169/371),两者差异有显著性(P<0.01)。结论Pre-S1抗原能够比HBeAg更灵敏地反映HBV复制,临床上可作为乙肝患者体内HBV复制的重要标志,但其敏感性不如HBVDNA强。

IFN-γ基因+874位点单核苷酸多态性与乙型肝炎病毒感染关系的研究

【目的】研究干扰素(IFN-γ)基因+874位点单核苷酸多态性与乙型肝炎病毒(HBV)感染、转归的关系,探讨HBV感染的遗传易感因素。【方法】采用序列特异性引物-聚合酶链反应(PCR-SSP)技术检测231例慢性HBV感染者、165例自限性HBV感染者和135名正常对照者IFN-γ基因第一内含子+874位点T/A单核苷酸多态性。【结果】慢性HBV感染组IFN-γ基因+874位点AA基因型频率(78.8%)显著高于正常对照组(64.4%)(χ2=9.60,P=0.008),而自限性HBV感染组(62.4%)和对照组之间差异无显著性(χ2=0.16,P=0.92)。进一步比较慢性HBV感染者IFN-γ基因+874位点单核苷酸多态性与HBV-DNA复制的关系,发现IFN-γ基因+874位点基因型和等位基因频率在低水平HBV-DNA组和高水平HBV-DNA组之间的分布差异无显著性。【结论】IFN-γ基因第一内含子+874位点多态性与慢性HBV感染有关,提示该位点多态性可能在决定个体HBV感染遗传易感性方面有一定意义。

北京地区乙型肝炎病毒基因型及基因亚型的分布和临床意义

目的:调查北京地区乙型肝炎病毒(HBV)基因型及基因亚型的分布情况和临床意义。方法:应用型特异性引物聚合酶链反应法,对北京地区850份HBV感染者血清进行基因型、基因亚型检测,包括慢性乙型肝炎660例,肝硬化85例,肝癌105例。结果:B型、C型、BC混合型分布在不同类型乙肝病毒感染者中分布差异有显著性,肝硬化,肝癌患者C型所占比例明显高于慢性乙型肝炎患者。HBeAg阳性和阴性感染者中,HBV基因型分布差异无显著性。慢性肝炎、肝硬化及肝癌组中,均以C2和Ba亚型占优势。结论:在北京地区HBV感染者中,以Ba和C2亚型为主,C基因型感染者更易于发展为肝硬化、肝癌。

慢性乙型肝炎患者血清肝纤维化指标与病毒复制水平的相关性

目的:探讨HBV复制水平与血清肝纤维化指标之间的关系。方法:对156例慢性乙型肝炎患者采用荧光定量PCR及放免方法,同时对HBV-DNA和肝纤维化血清学标志透明质酸(HA)、层粘蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(Ⅳ-C)进行定量检测。结果:随慢性乙肝临床类型的加重,肝纤维化血清学标志逐渐升高(P<0.01),肝纤维化血清学标志与HBV-DNA呈正相关(P<0.05)。结论:HBV复制水平与肝纤维化之间呈正相关。

Transformation of hepatitis B serologic markers in babies born to hepatitis B surface antigen positive mothers

AIM:To better understand the clinical significance of hepatitis B seroiogic markers in babies born to hepatitis B surface antigen (HBsAg) positive mothers, the incidence of maternal seroiogic markers of hepatitis B via placenta and its transformation in these babies were investigated. METHODS: Mothers with positive HBsAg were selected in the third trimester of pregnancy. Their babies received immunoprophylaxis with hepatitis B immunoglobulin and hepatitis B vaccine after birth, and were consecutively followed up for hepatitis B seroiogic markers and HBV DNA at birth, mo 1, 4, 7, 12, and 24. RESULTS: Forty-two babies entered the study, including 16 born to hepatitis B e antigen (HBeAg)-positive HBsAg carrier mothers and 26 to HBeAg-negative HBsAg carrier mothers. Apart from four babies born to HBeAg-positive carrier mothers and demonstrated persistent positive HBeAg eventually became HBV carriers, all other babies developed anti-HBs before 12 mo of age. Among the other 12 babies born to HBeAg-positive carrier mothers, HBeAg was detected in 7 at birth, in 4 at mo 1, and in none of them thereafter. No antibody response to the transplacental HBeAg was detected. Among the babies born to HBeAg-negative carrier mothers, anti-HBe was detected 100% at birth and mo 1, in 88.5% at mo 4, in 46.2% at mo 7, in 4.2% at mo 12 and none in mo 24. Among all the immunoprophylaxis-protected babies born to either HBeAg-positive or HBeAg-negative carrier mothers, anti-HBc was detected in 100% at birth, mo 1 and mo 4, in 78.9% at mo 7, in 36.1% at mo 12 and in none at mo 24. CONCLUSION: HBeAg can pass through human placenta from mother to fetus and become undetectable before 4 mo of age, but no antibodies response to the transplacental HBeAg can be detected till mo 24 in the immunoprophylaxis-protected babies. The sole existence of anti-HBe before 1 year of age or anti-HBc before 2 years of age in babies born to HBsAg carrier mothers may simply represent the transplacental maternal antibodies, instead of indicators of HBV infection status.

Intrahepatic HBV DNA as a predictor of antivirus treatment efficacy in HBeAg-positive chronic hepatitis B patients

AIM： To evaluate the effect of antiviral agents on intrahepatic HBV DNA in HBeAg-positive chronic hepatitis B patients. METHODS： Seventy-one patients received treatment with lamivudine, interferon alpha （IFN-α2b） or sequential therapy with lamivudine-IFN-α2b for 48 wk. All subjects were followed up for 24 wk. Serum and intrahepatic HBV DNA were measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP. RESULTS： At the end of treatment, the intrahepatic HBV DNA level in 71 patients decreased from a mean of （6.1 ± 1.0） Iog10 to （4.9± 1.4） Iog10. Further, a larger decrease was seen in the intrahepatic HBV DNA level in patients with HBeAg seroconversion. Intrahepatic HBV DNA level （before and after treatment） was not significantly affected by the patients＇ HBV genotype, or by the probability of virological flare after treatment. CONCLUSION： Intrahepatic HBV DNA can be effectively lowered by antiviral agents and is a significant marker for monitoring antivirus treatment. Low intrahepatic HBV DNA level may achieve better efficacy of antivirus treatment.

Preliminary report of hepatitis B virus genotype prevalence in Iran

AIM： To determine the prevalence of hepatitis B virus （HBV） genotypes in Iranian hepatitis B surface antigen （HBsAg） carriers, chronic hepatitis B and cirrhotic patients. METHODS： A total of 109 HBsAg-positive patients were included in this study. HBV genotypes were determined by using INNO-LiPA methodology which is based on the reverse hybridization principle. RESULTS： The distribution of patients with different stages of liver disease was as follows： 95 （86.4%） chronic hepatitis, 11 （10%） liver cirrhosis, and 3 （2.7%） inactive carrier. Of the chronic hepatitis and liver cirrhosis patients, 26.4% were HBeAg-positive while 70% were HBeAg-negative. Genotype D was the only detected type found in all patients. CONCLUSION： Classifying HBV into genotypes has to be cost-effective and clinically relevant. Our study indicates that HBV genotype D prevails in the Mediterranean area, Near and Middle East, and South Asia. Continued efforts for understanding HBV genotype through international co-operation will reveal further virological differences of the genotypes and their clinical relevance.

Prevalence of occult HBV infection in haemodialysis patients with chronic HCV

AIM： To study the prevalence and clinical effects of occult HBV infection in haemodialysis patients with chronic HCV.METHODS： Fifty chronic hemodialysis patients with negative HbsAg, and positive anti-HCV were included in the study. These patients were divided into two groups： HCV-RNA positive and HCV-RNA negative, based on the results of HCV-RNA PCR. HBV-DNA was studied using the PCR method in both groups.RESULTS： None of the 22 HCV-RNA positive patients and 28 HCV-RNA negative patients revealed HBV-DNA in serum by PCR method. The average age was 47.2±17.0 in the HCV-RNA positive group and 39.6±15.6 in the HCV-RNA negative group.CONCLUSION： The prevalence of occult HBV infection is not high in haemodialysis patients with chronic HCV in our region. This result of our study has to be evaluated in consideration of the interaction between HBsAg positivity （8%-10%） and frequency of HBV mutants in our region.

Baseline predictors of virological response for chronic hepatitis B patients

AIM： To determine which baseline factors of chronic hepatitis B patients are predictive of virological response to Peginterferon α-2b therapy. METHODS： A total of 21 HBeAg-positive chronic hepatitis B （CriB） patients treated with Peginterferon α-2b were recruited. They were treated with Peginterferon α-2b （0.5-1.0 μg/kg per week） for 24 wk and followed up for 24 wk. Clinical and laboratory data of the patients were determined at pretreatment and at week 12, at 24 during treatment, and at week 48 during follow up. RESULTS： Ten patients achieved a virological response at the end of treatment. Their baseline serum alanine aminotransferase （ALT）, thyroid-stimulating hormone （TSH）, and total thyroxin （TT4） levels were significantly different from those who failed treatment. The positive predictive values （PPV） and negative predictive values （NPV） of ALT, TSH, and TT4 were 75% and 89 %, 75% and 89 %, and 75% and 75%, respectively. Moreover, combinations of the baseline ALT and TT4, ALT and TSH, TT4 and TSH levels had much higher PPV and NPV （86% and 88%, 89% and 100%, 83% and 100%, respectively）.CONCLUSION： Baseline serum ALT, TSH, and TT4 levels, especially in combination, have high predictive values of virological response to Peginterferon α-2b in HBeAg-positive CriB patients.

Epidemiology and prevention of hepatitis B virus in China

Hepatitis B virus （HBV） infection is prevalent in China. Approximately 600 million people have ever been infected by HBV. About 130 million are HBV chronic carders and 30 million HB patients. Among them, 50% of HBV carriers are caused by carrier mothers to born infants. Around 300 000 people died of liver disease including liver cirrhosis and primary hepatocellular carcinoma each year and 50% of them died of primary hepatocellular carcinoma. HBV infection is not only the health problem but also becoming a social problem. HBV chronic carriers and patients have endured the great pressure from disease burden and social discrimination. According to the report of the national screening program of HBV released by the ministry of health in 2008, China has taken many effective measures to control the HBV infection, including vaccine immunization program, strengthening the management of blood sources and blood productions, prevention of nosocomial HBV infection, strengthening health education on HBV infection and safe injection techniques. The implementation of HB vaccine immunization program, which China officially introduced into the national immunization program since 1992, has dramatically reduced the incidence of HBV infection among infants and children. Integrated with other interventions, the rate of HBV infection decreased gradually. According to the survey of the national screening program of HBV in 2006, compared with the incidence of HBV in 1992, the incidence rate of HBsAg positive has decreased 26.36%, the number of children who have ever been infected by HBV decreased 80 million since 1992. However some problems are still existing. The solutions of low rate of vaccination in rural areas and migration population, lacking of practical measures on management of hepatitis B patients, the occurrence of health care acquired HBV infection, and low rate of vaccination among high risk groups have also been recommended.

Long-term hepatic consequences of chemotherapy-related HBV reactivation in lymphoma patients

AIM: To investigate the long-term consequences of chemotherapy-related HBV reactivation in patients with lymphoma.METHODS: This study was based on the database of published prospective study evaluating HBV reactivation in HBV lymphoma patients during chemotherapy.Deteriorated liver reserve (DLR) was defined as development of either one of the following conditions during follow-up: (1) newly onset parenchyma liver disease, splenomegaly or ascites without evidence of lymphoma involvement; (2) decrease of the ratio (albumin/globulin ratio) to less than 0.8 or increase of the ratio of INR of prothrombin time to larger than 1.2 without evidence of malnutrition or infection. Liver cirrhosis was diagnosed by imaging studies.RESULTS: A total of 49 patients were included. The median follow-up was 6.2 years (range, 3.9-8.1 years).There were 31 patients with and 18 patients without HBV reactivation. Although there was no difference of overall survival (OS) and chemotherapy response rate between the two groups, DLR developed more frequently in patients with HBV reactivation (48.4% vs 16.7%; P= 0.0342). Among the HBV reactivators, HBV genotype C was associated with a higher risk of developing DLR (P = 0.0768) and liver cirrhosis (P = 0.003). Four of five patients with sustained high titer of HBV DNA and two of three patients with multiple HBV reactivation developed DLR. Further, patients with a sustained high titer of HBV DNA had the shortest OS among the HBV reactivators (P= 0.0000). No patients in the non-HBV reactivation group developed hepatic failure or liver cirrhosis.CONCLUSION: Chemotherapy-related HBV reactivation is associated with the long-term effect of deterioration of hepatic function.

Treatment of chronic viral hepatitis with nitazoxanide and second generation thiazolides

Nitazoxanide,the first thiazolide,was originally developed for the treatment of Cryptosporidium parvum.More recently,antiviral activity of nitazoxanide against hepatitis B virus(HBV)and hepatitis C virus was recognized in in vitro systems.These basic studies led to phaseⅡclinical trials that demonstrated the safety and efficacy of nitazoxanide in combination with peginterferon,with or without ribavirin,in the treatment of chronic hepatitis C genotype 4.The sustained virologic response rate was 79%and 80%in two studies,which was higher than the response rate of 50%with the standard of care with peginterferon plus ribavirin.In very preliminary studies of patients with chronic hepatitis B,nitazoxanide suppressed serum HBV DNA and led to loss of hepatitis B e antigen in the majority of patients and hepatitis B surface antigen in approximately a quarter of patients.Randomized controlled studies of naive and nonresponder patients with chronic hepatitis C genotype 1 are underway,new second generation and controlled release thiazolides are being developed,and future studies of patients with chronic hepatitis B are planned.

Quantification of HBsAg:Basic virology for clinical practice

Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B virus infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring, and there is an opportunity to make maximal use of quantitative HBsAg to elucidate its role in clinical fields. Yet, it needs to be underscored that a further understanding of HBsAg, not only from clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. It is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. In this article, we review current concepts and issues on the quantification of HBsAg titers with respect to their biologic nature, method principles, and clinically relevant topics.

Diagnostic accuracy of serum biochemical fibrosis markers in children with chronic hepatitis B evaluated by receiver operating characteristics analysis

METHODS： We determined the serum level of apolipoprotein A-I （APO A-I）, haptoglobin （HPT） and a-2 macroglobulin （A2I） with an automatic nephelometer in 63 children （age range 4-17 years, mean 10 years） with biopsy-verified chronic HBeAg-positive hepatitis B. Fibrosis stage and inflammation grade were assessed in a blinded fashion according to Batts and Ludwig. We defined mild liver fibrosis as a score ≤2 and advanced fibrosis as a score equal to 3. ROC analysis was used to calculate the power of the assays to detect advanced liver fibrosis （AccuROC, Canada）. RESULTS： Serum concentrations of APO A-I, HPT and A2M were not significantly different in patients with chronic hepatitis B compared to controls. However, APO A-I level of 1.19 ng/L had a sensitivity of 85.7% and a specificity of 60.7% （AUC = 0.7117, P = 0.035） to predict advanced fibrosis. All other serum biochemical markers and their combination did not allow a useful prediction. None of these markers was a good predictor of histologic inflammation. CONCLUSION： Apolipoprotein A-I may be a suitable serum marker to predict advanced liver fibrosis in children with chronic hepatitis B.

YMDD variants of HBV DNA polymerase gene: Rapid detection and clinicopathoiogical analysis with long-term Iamivudine therapy after liver transplantation

AIM: To look for a rapid low-cost technique for the detection of HBV variants.METHODS: Two patients who underwent orthotopic liver transplantation (OLT) for HBV infection were treated with lamivudine (100 mg daily) and HBV infection recurred in the grafted livers. The patients were monitored intensively for liver enzymes, hepatitis B surface antigen (HBsAg) and HBV DNA in serum. Liver biopsy was performed regularly. HBV DNA in a conserved polymerase domain (the YMDD locus) was amplified from serum of each patient by PCR and sequenced. HBV genotypes were analyzed by restriction fragment length polymorphism (RFLP) of the PCR products generated from a fragment of the polymerase gene.RESULTS: YMDD wild-type HBV was detected in one patient by PCR-RFLP and DNA sequencing 19 mo after OLT, and YIDD mutant-type HBV in the other patient, 16 mo after OLT.CONCLUSION: PCR-RFLP assay is an accurate and simple method for genotyping lamivudine-resistant HBV variants.

Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma

AIM： To explore the relationship between DNA methyltransferase 1 （DNMT1） and hepatitis B virus （HBV）-related hepatocellular carcinoma （HCC） and its biological significance in primary HCC. METHODS： We carried out an immunohistochemical examination of DNMT1 in both HCC and paired nonneoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC celt line SMMC-7721. RESULTS： DNMT1 protein expression was increased in HCCs compared to histologically normal nonneoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated significantly with poor tumor differentiation （P = 0.014）. There were more cases with DNMT1 overexpression in HCC with HBV （42.85%） than in HCC without HBV （28.57%）. However, no significant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen （PCNA）, even in HCC cell lines where DNMT1 was stably decreased. CONCLUSION： The findings implied that DNMT1 plays a key role in HBV-retated hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.

乙肝患者血清HBVM模式与HBV DNA定量的关系

目的:探讨HBV感染血清标志物模式(HBV M)与HBV DNA定量表达的关系。方法:采用ELISA和荧光定量聚合酶链反应(FQ-PCR)技术分别对1463份HBV感染者血清进行HBV M和HBV DNA定量检测。结果:HBsAg、HBeAg、抗HBc均阳性模式HBV DNA检出率高,且多数病毒载量大;HBsAg、抗HBe、抗HBc均阳性及其他模式HBVDNA检出率低,且多数病毒载量小。结论:HBV M与HBV DNA定量虽强相关,但各有所长。