Real Time-PCR HBV-DNA Analysis: Significance and First Experience in Armed Forces

Background

HBV DNA quantitation is used extensively world wide for the diagnosis and monitoring of treatment of Hepatitis B virus (HBV) infection. However, it has still to be popular in India. The aim of this study was to quantitate HBV – DNA by Real time – PCR method in Hepatitis B and in immuno-compromised patients, to compare the results with HBeAg detection and to monitor the response to therapy of chronic Hepatitis B patients to antivirals.

Methods

Ninety one serum samples of Hepatitis group of patients (all HBsAg positive), 41 samples from immuno-compromised patients (all HBsAg negative) and 49 patients of Chronic Hepatitis B group (all HBsAg positive) were the subjects of this first ever study in Armed Forces. Twenty serum samples from healthy volunteers and non-hepatitis B patients served as negative controls. The amplification detection was carried out in a Rotor-Gene 2000-sequence detector

Results

Amongst Hepatitis B group, 33% (30/91) of the samples were positive for HBV-DNA and 26% (24/91) of samples were positive for HBeAg. In the immuno-compromised group of patients 14.6% (6/11) of samples were positive for HIV-DNA and 9.7% (4/41) were positive for HBeAg. Of the Chronic Hepatitis B patients on treatment, all (100%) were positive by HBV-DNA, whereas 29/49 (59.2%) were positive by HBeAg before treatment. After treatment with antivirals, 06/49 (12.2%) were positive by both tests and 11/49 (22.5%) were positive only by HBV-DNA. 32/49 (65.3%) patients became negative serologically after therapy.

Conclusion

HBeAg status did not necessarily reflect HBV-DNA level in the serum, as 10/91 (11%) in the Hepatitis B group, 2/41 (4.9%) in the immuno compromised group and 20/49 (40.8%) patients in the Chronic Hepatitis B group were positive for HBV-DNA but negative for HBeAg. HBV-DNA was not found to be positive amongst any of the negative controls. Real time – PCR is a sensitive and reproducible assay for HBV-DNA quantitation and may be started in Armed Forces referral centers in the near future.Key Words: Real time – PCR, Chronic Hepatitis B, HBV – DNA, Antivirals     

台山市城区食品从业人员的人群HBV感染状况分析

目的 调查台山市城区食品从业人员的人群HBV感染状况与相应对策。方法 对从业人员预防性体检,抽取静脉血2ml,分离血清,采用ELISA法进行HBV标记物血清学检测。结果 2004年度体检6846人,HBsAg平均阳性率6．95％,男性高于女性,年龄分布15岁组高于30、40岁组;抗-HBsAg平均阳性率32．5％,女性高于男性,年龄分布40岁组为最低;HBeAg平均阳性率33．61％,性别年龄无差别：7种HBV标记物显示模式,俗称“大三阳”和“小三阳”占两种为主。结论 HBV感染程度接近我国流行现状．仍属中到高流行状况,应抓好防制措施,加强食品从业人员预防性体检管理,推行接种乙肝疫苗,提高人群免疫力,防止HBV的感染与传播。     

Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins

AIM: The incorporation of hepatitis B virus(HBV)preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial.Therefore our purpose was to further investigate its immunogenic domains for the epitope-based hepatitis B vaccine design. METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1(21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA. RESULTS: The results in mice showed that the immunogenic domains mainly existed in preS1(21-59) and preS1(95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1(60-70) also had some immunogenicity. More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1(34-59), were speculated to be virus-neutralizing. CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic domains in preS1(21-119) region. The preS1(34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore,the incorporation of preS1(34-59) fragments into epitope-based HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.     

HBV启动子调控的GFP在肿瘤细胞中的表达

为进一步探讨针对晚期肺癌患者治疗的新方式 ,本文根据曾有动物实验研究报告 (即ＢＣＧ辅以ＩＬ 2吸入防治实验肺癌模型 ) ,通过采用超声雾化方式 ,在临床上试行将相关免疫制剂配合化疗药物以气溶胶微粒形式吸入肺组织用以治疗肺癌 ,先后对 10例患者进行共 16例次的治疗。初步观察该方法在控制肿瘤病灶发展、减轻病痛等方面临床意义 ,现报告如下。10例患者中 ,男 7例 ,女 3例 ;年龄 6 2～ 88岁。所有患者均以临床表现 ,影像学检查 ,部分经病理证实诊断为肺癌 ,其中 ,原发性肺癌 7例 (腺癌、鳞癌各 3例 ,1例无病理 ) ;转移性肺癌 3例。ＫＰＳ…     

重症乙肝患者肝、胰组织内HBsAg、HBcAg的检测及其与糖代谢异常关系的探讨

为探讨乙型肝炎病毒 (HBV)感染患者肝、胰组织中HBV检出情况及其与糖代谢异常之间的关系 ,采用免疫组化法对 2 2例重症HBV感染伴糖代谢异常患者的尸解肝、胰组织中HBsAg、HBcAg进行检测。同时用 2 2例空腹血糖正常的HBV感染者的尸解肝、胰组织作为对照。结果 :肝病伴糖代谢异常组与对照组胰腺组织内HBV检出例数有显著性差异 (P <0 .0 5)。提示 :乙型肝炎患者胰腺组织HBV感染可能是发生肝病伴糖代谢异常的原因之一。     

动态研究HBV和AFB1诱发树鼠句肝细胞癌过程中一些基因的表达

目的：探讨人乙肝病毒(HBV)和黄曲霉毒素B,(AFB1)致肝细胞癌(HCC)及协同致HCC作用的机制。方法：将树鼩分为4组：A组：感染HBV加摄入AFB,;B组：只感染HBV;C组：只摄入AFB1;D组：作空白对照。然后定期行肝活检,用免疫组化、分子生物学等技术动态观察一些癌基因(蛋白)和抑癌基因的表达。结果：①A组谷胱苷肽转移酶(GGT)阳性灶个数及面积明显多于、大于B、C组;②A组P21蛋白检出率明显高于B和C组,A组6只P21蛋白阳性的树鼩到120周时全部发生了HCC;③实验后期,A组HBxAg检出率及HBVDNA整合率明显高于B组;④在诱癌过程中,IGF-Ⅱ表达率呈波动形,带癌肝的表达率显著高于无癌肝;⑤105周时,A、B、C组P53蛋白表达率显著高于D组;在A,C组检出P53异常带;⑥第45及105周时,A组c-fos表达率分别为23．1％和20％;C组为38．5％和15．4％,B,D两组均未检出;⑦H-c-myc基因：直到120周,无癌肝组织均未见异常;在肝癌组织中,A组有4例扩增,c组无异常;@CDK4基因：只有A组在第75周有1例扩增,该树鼩在第106周时发生了HCC。在HCC中,A组有25％出现CDK。扩增,C组未见异常。结论：再次证实HBV和AFB。有协同致HCC作用;AFB、有利于HBxAg的表达和HBVDNA的整合;CDK4、Kiras和P53基因的变异参与了HCC的发生和演进;P21和c-fos蛋白的过表达与P^53蛋白的失活,促进了HCC的发生和演进;CDK4和c-myc基因的改变与HBV的感染有一定的关联。     

HBV核心蛋白结构与功能及其在基因治疗策略上的应用

核心蛋白对乙型肝炎病毒(HBV)复制过程起关键的作用。近年来对HBV核心蛋白(HBc)单体及其形成的核壳结构与功能有了深入的认识,HBc基因特定部位中插入外源基因后,其产物在细胞内仍能正确地折叠和自身装配,因而有可能在细胞内表达假HBc并掺人240个野生型HBc后,共同形成核壳结构,抑制其功能的发挥,干扰HBV复制。本文就HBc结构与功能区域的基础生物学及其在基因治疗策略上的应用进展作了综述。     

C基因区与相关调控基因变异的分子生物学

乙型肝炎病毒(HBV)C基因区的变异不仅改变了病毒本身的生物学表达,也对宿主的临床、病理生理及免疫学变化产生了影响。从分子生物学水平加深对C基因区变异的认识,有助于了解该区基因变异的意义和部分产生的原因,从而对进一步阐明变异与病毒遗传学,宿主发病等的关系有一定的意义。本文对C基因区与相关调控基因变异的分子生物学变化作了概述。     

不安全注射——扩大免疫规划面临的挑战

不安全注射正日渐成为扩大免疫规划(EPI)中不容忽视的问题.甚至在指出不安全注射有传播血源性疾病的危险之后,在发展中国家不安全注射仍被普遍实施.18个国家中有14个国家超过50%的注射是不安全的.在日本丙型肝炎的病因被认为是医源性传播.在中国只换针头的多针一管注射很普遍,一次性注射器的普及仍不能解决这一问题.提供足够的器材、培训、监督和正确的处理使用过的一次性注射器,对于保证安全注射是很关键的.