用聚合酶链反应检测人肝细胞癌乙型肝炎病毒DNA和丙型肝炎病毒RNA

为研究乙型肝炎病毒ＤＮＡ（ＨＢＶＤＮＡ）和丙型肝炎病毒ＲＮＡ（ＨＣＶＤＮＡ）与肝细胞癌的关系，用聚合酶链反应（ＰＣＲ）和巢式ＰＣＲ（ｎｅｓｔｅｄ－ＰＣＲ）分别检测４２例肝肿瘤组织中ＨＢＶＤＮＡ和ＨＣＶＲＮＡ。结果：１例胆管细胞癌组织ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性，１例胆管囊腺瘤ＨＢＶＤＮＡ阳性。４０例肝细胞癌组织中，单纯ＨＢＶＤＮＡ阳性１９例，单纯ＨＣＶＲＮＡ阳性３例，二者均阳性１０例。ＨＢＶＤＮＡ阳性率７２．５％，ＨＣＶＲＮＡ阳性率３２．５％。ＨＢＶＤＮＡ和ＨＣＶＲＮＡ感染与肝癌组织学分型无关；且肝细胞癌中ＨＣＶ感染与ＨＢＶ未见相关。结果提示，我国ＨＢＶ感染仍是引起肝细胞癌的主要原因。但由于肝细胞癌患者中ＨＣＶ的感染率也较高，且有上升趋势，因此ＨＣＶ可能也是肝细胞癌发生的重要原因之一。     

血清学标志阴性的非甲～戊型肝炎的病原学研究

目的对血清学标志阴性的非甲～戊型肝炎进行病原学研究。方法用ＨＢＶＰＣＲ、ＨＣＶＲＴ－ＰＣＲ和ＨＥＶＲＴ－ＰＣＲ分别检测血清学标志阴性的非甲～戊型肝炎患者血清，并对其部分阳性产物进行克隆测序。结果８７例非甲～戊型肝炎血清ＨＢＶＤＮＡ均为阴性，９例（１０．３％）为ＨＣＶＲＮＡ阳性，部分经测序证实为ＨＣＶ１ｂ亚型；余７８例为ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阴性。该７８例中，１４例因无血清未作ＨＥＶＲＮＡ检测，余６４例中４９例（７６．６％）为ＨＥＶＲＮＡ阴性，１５例（２３．４％）为ＨＥＶＲＮＡ阳性。经序列分析显示，其中９例为典型的中国ＨＥＶ株基因序列，６例变异较大，与典型的中国株基因序列的同源性仅为８０％左右。４９例ＨＢＶＤＮＡ、ＨＣＶＲＮＡ和ＨＥＶＲＮＡ均阴性的血清中１６例（３２．６％）ＨＧＶＲＮＡ阳性。由此可见，该８７例中至少有９例为ＨＣＶ感染，１５例为ＨＥＶ感染，１６例为ＨＧＶ感染。结论对血清学标志阴性的非甲～戊型肝炎的病人应该用ＰＣＲ法进行病原学分型，以明确其诊断     

丙型肝炎病毒基因型与血清HCV RNA含量关系及与干扰素应答的研究

目的探讨丙型肝炎病毒（ＨＣＶ）基因型与血清ＨＣＶＲＮＡ含量的关系及其对干扰素应答的影响。方法应用定量荧光ＰＣＲ（Ａｍｐｌｉｓｅｎｓｏｒ－ＰＣＲ）技术检测了１３５例不同基因型ＨＣＶ感染的慢性丙型肝炎患者血清ＨＣＶＲＮＡ含量，另对其中７７例进行干扰素治疗并随访１２个月以上。结果ＨＣＶ－Ⅱ型感染血清ＨＣＶＲＮＡ水平（１０７．８±３．４拷贝／ｍｌ）显著高于ＨＣＶ－Ⅲ型感染（１０６．３±２．５拷贝／ｍｌ）（Ｐ＜０．０１），Ⅲ型感染的应答率（７／１３，５３．８％）显著高于Ⅱ型感染（２０／６４，３１．３％）（Ｐ＜０．０５）。应答组治疗前血清ＨＣＶＲＮＡ含量（１０６．８±２．７拷贝／ｍｌ）显著低于无应答组（１０８．３±３．２拷贝／ｍｌ）（Ｐ＜０．０１）。ＨＣＶＲＮＡ含量低于１０６．５拷贝／ｍｌ者，无论何种型别ＨＣＶ感染均应答较好，而ＨＣＶＲＮＡ高于１０８．０拷贝／ｍｌ者则应答极差。结论ＨＣＶ基因型及病毒血症水平是预测干扰素疗效的重要因素，且后者比前者意义更大。Ⅱ型感染病毒血症水平较高可能是影响其疗效的原因之一。     

套式PCR和原位杂交技术检测肝病患者单个核细胞？…

目的 了解慢性乙型肝炎（中度）和原发性肝癌（ＨＢｓＡｇ阳性）患者ＰＢＭＣ内ＴＴＶ ＤＮＡ存在情况。方法 采用套式ＰＣＲ以及原位杂交技术检测外周血单个核细胞（ＰＢＭＣ）内ＴＴＶ ＤＮＡ。结果 套式ＰＣＲＳＷＩＭ２６例慢性乙型肝炎（中度）患者ＰＢＭＣ内ＴＴＶ ＤＮＡ阳性７例。阳性率２６．９％,非常显著高于健康对照（Ｘ＾１２＝１４．３,Ｐ〈０．００１）;２１例原发性肝癌（ＨＢｓＡｇ阳性）虱ＰＢＭＣ内ＴＴ     

乙型肝炎病毒C基因启动子及前C变异的动态研究

目的明确乙型肝炎病毒（ＨＢＶ）Ｃ基因启动子（ＢＣＰ）与前Ｃ变异株在慢性乙型肝炎患者中的动态消长,及与ＨＢｅＡｇ血清学状态的关系。方法对３２例慢性乙型肝炎患者的１０５份系列血清进行错配聚合酶链反应（ＰＣＲ）－限制性片段长度多态性分析,并对其中１５例患者系列血清标本的ＰＣＲ产物进行直接测序。结果ＢＣＰ和Ａ８３变异株感染率均明显增高（６２．５％＞４６．９％;３１．３％＞１２．５％）;ＢＰＣ变异株更多地（１４／１６）表现为优势积累,且先于ＨＢｅＡｇ血清阴转;ＢＣＰ野株／变异株比例变化影响ＨＢｅＡｇ血清学的稳定性。结论两种类型的变异株在炎症活动中均具有选择优势,最终造成ＨＢｅＡｇ（－）的ＨＢＶ感染;可能与ＨＢＶ感染之持续有关。     

广西不同人群庚型肝炎病毒感染状况的调查

目的 了解庚型肝炎病毒（ＨＧＶ）感染在广西柳州地区不同人群中的感染状况，并比较静脉毒瘾者与健康体检者ＨＧＶ，乙型肝炎病毒（ＨＢＶ）、丙型肝炎病毒（ＨＣＶ）与ＨＧＶ共同感染的特点，方法 应用酶联免疫法（ＥＬＡ）检测抗－ＨＧＶ，抗－ＨＣＶ，ＨＢＶＭ（ＨＢｓＡｇ，ＨＢｓＡｂ，ＨＢｅＡｇ，ＨＢｅＡｂ，ＨＢｃＡｂ），并对抗－ＨＧＶ阳性者随机抽取２０例（１９％）进一步用逆转录套式ＰＣＲ（ＰＴ－ｎＰＣＲ）法检测     

丙型肝炎病毒在人T淋巴细胞中的体外感染

用丙型肝炎病毒（ＨＣＶ）ＲＮＡ阳性的血清感染人类Ｔ淋巴细胞（Ｍｏｌｔ－４），１２小时后继续孵化１～２８天，将感染后的产物用聚合酶链反应（ＰＣＲ）测定，于第１、２、３天和第８天发现ＨＣＶ－ＲＮＡ的正链，第２和第８天发现ＨＣＶ－ＲＮＡ的负链。     

庚型肝炎病毒感染对慢性丙型肝炎的临床与病理学影响

目的 探讨庚型肝炎病毒（ＨＧＶ）感染对慢性丙型肝炎（ＣＨ－Ｃ）的临床与病理学影响。方法 应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）法对５３例经肝穿活检确诊的ＣＨ－Ｃ患者血清标本进行了ＨＣＶ－ＲＮＡ检测,并将合并与未合并ＨＧＶ感染者进行了临床与病理学对比。结果 ＨＣＶ－ＲＮＡ阳性１５例（２８．３０％）。合并与未合并ＨＧＶ感染者在临床表现、肝功能等生化指标水平、ＨＣＶ－ＲＮＡ阳性率及肝脏病理损害等方面差     

套式PCR检测HBV与HCV垂直传播的比较研究

用套式ＰＣＲ法检测了２７例ＨＢｓＡｇ阳性母亲的新生儿脐带血ＨＢＶ ＤＮＡ，阳性３例（１１．１％），同时检测了７例抗－ＨＣＶ阳性母亲的新生儿脐带血ＨＣＶ ＲＮＡ及抗－ＨＣＶ，结果ＨＣＶ ＲＮＡ无１例阳性，抗－ＨＣＶ全部阳性，因此认为婴儿体内抗－ＨＣＶ是一种由母体被动获得的抗体，不能作为ＨＣＶ感染的客观指标。     

乙肝患者血清抗－HBe效价与HBeAg阴性变异株出现频率关系的研究

利用多聚酶链反应（ＰＣＲ）结合寡核苷酸探针杂交的方法，对７７例抗－ＨＢｅ阳性的乙肝病人血清进行了乙肝病毒（ＨＢＶ）ＨＢｅＡｇ阴性变异的分析，同时研究了血清抗－ＨＢｅ滴度与ＨＢｅＡｇ阴性变异出现频率的关系。结果１４例单独野毒株感染的病人，抗－ＨＢｅ平均效价为４．７９±１４．４５；１９例野毒株与变异株混合感染的病人，抗－ＨＢｅ平均效价为１２８．８３±２３．４４；１８例单独变异株感染的病人，抗－ＨＢｅ平均效价为２８．１８±３７．１５；２６例无ＨＢＶ复制的病人，抗－ＨＢｅ平均效价为８１．２８±２２．９１。单独野毒株感染组抗－ＨＢｅ效价显著低于混合感染组（Ｐ＜０．０１）及无ＨＢＶ复制组（Ｐ＜０．０５）。结果提示：高效价的抗－ＨＢｅ可能是选择ＨＢｅＡｇ阴性变异株的重要条件。在抗－ＨＢｅ效价高而ＨＢＶＤＮＡ仍为阳性的病人中，可能存在ＨＢｅＡｇ阴性变异株感染。     

临床诊断为非甲～戊型肝炎患者的病原学研究

目的探讨临床诊断为非甲-戊型肝炎患者的病原学。方法 采用巢式PCR（nPCR)检测HBV、TTV、B19、和SENV DNA；用逆转录巢式PCR（RT-nPCR)检测HGV和HCV RNA。结果 60例临床诊断为非甲-戊型肝炎患者中，单独HBVDNA阳性30例（阳性率为50.0%)，HBV和TTVDNA阳性10例（16.7%)，HBV和B19DNA阳性6例（10.0%)，HCVRNA、HBV和SENVDNA阳性1例（1.7%)，单独HCVRNA阳性1例（1.7%)，HCVRNA和B19DNA阳性1例（1.7%),HGVRNA无一例阳性，单独B19DNA阳性2例（3.3%)。单独TTVDNA阳性1例（1.7%)，另8例（13.3%)上述病毒核酸均为阴性。HBV合并感染TTV或B19对其血清学生化指标无影响。结论HBV是临床诊断为非甲-戊型肝炎的主要病原，HGV、TTV、B19和SENV与非甲-戊型肝炎无关。     

Genotypic differences in the hepatitis B virus core promoter and precore sequences during seroconversion from HBeAg to anti-HBe

Hepatitis B virus (HBV) strains from anti-HBe positive patients often show specific mutations in the precore gene, the core promoter region, or both. The dynamics of seroconversion in relation to the appearance of these mutations has not been studied and compared between defined HBV genotypes. Samples from patients followed during seroconversion from HBeAg to anti-HBe were amplified by polymerase chain reaction (PCR), sequenced and genotyped. Among 16 sets of samples, 6 belonged to genotype A, 6 to genotype D, 2 to genotype B, 1 to genotype C, and 1 to genotype E. Whereas strains from genotypes B, C and E showed changes in the core promoter, precore codon 28 or both, genotype A and D strains displayed a different pattern. In 4 of 6 anti-HBe positive samples from genotype A, the precore had a wild-type sequence while the core promoter sequence showed a specific TGA mutation. In another genotype A strain a precore stop mutation was preceded by a mutation in codon 15, thus conserving base-pairing at the pregenomic RNA level in this region. In contrast, all genotype D strains showed wild-type sequences in both the core promoter and precore codon 28 in pre- and post-seroconversion samples. Thus, in 8 patients with a mean follow-up time of 17 months, wild-type sequences in both the core promoter and precore codon 28 were found after seroconversion to anti-HBe. This study also confirmed, for genotype D, that HBeAg seroconversion often occurs earlier than genomic conversion.     

Epidemiology of precore mutants of hepatitis B in the United Kingdom

A point mutation assay was used to study the codon 28 and codon 1 precore mutant status of 310 chronic hepatitis B carriers (82 HBeAg positive and 228 HBeAg negative). Fourteen of 228 (6%) of HBeAg negative carriers had high levels of serum HBV DNA. Nine of these were explained by precore variants, three by core promoter variants, and two were not explained by recognised precore changes. Nested PCR detected serum HBV DNA in 36% (82/228) of HBeAg negative carriers and 63% (52/82) of these had precore variants. Four of 82 (4%) of the HBeAg positive carriers had precore variants, all as mixed mutant/wild type populations and evidence indicated that these carriers were seroconverting. Overall 23% (52/228) of HBeAg negative carriers had both serum HBV DNA and codon 1 or 28 precore mutations. A sexual transmission event from an HBeAg negative carrier with a relatively low serum HBV DNA level (10(4)-10(6) genome copies/ml) and only core promoter mutations was observed. Despite high rates of variant carriage in the antenatal sub-group perinatal transmission was not observed. The results of direct sequencing on 45 carriers validated the point mutation assay and also showed that codon 28 mutations were only seen in carriers with the genotype CCT at codon 15. For the Caucasian population a higher prevalence of codon 28 mutations (13/25 or 52%) than expected was seen. Liver biopsy data indicated that there was no link between the presence or absence of precore mutants and the severity of liver disease.     

Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area

The sequence variability in the pre-C region of the hepatitis B virus (HBV) genome in the serum of 42 anti-HBe antibody positive carriers with chronic hepatitis B was studied by PCR and direct sequencing to determine prevalence and type of HBV pre-C mutants in a highly endemic area. Except for one, all patients were infected with viruses containing mutations in the pre-C region which prevent precore and e-antigen (HBeAg) expression: 33 were infected predominantly or exclusively with variants containing a stop codon; two had a mixture of wild-type and a pre-C stop codon mutant virus; three had precore variants with mutations of the pre-C initiation codon and two of them an additional stop codon; four had a frameshift mutation; and one had two stop codons. One patient was infected with viruses which contained a mutation creating an amino acid exchange which should not prevent precore and HBeAg expression. These data demonstrate that in an endemic area a higher prevalence and even broader spectrum of pre-C HBV mutants are found than has been recognized previously in anti-HBe positive patients with chronic hepatitis B.     

Genetic heterogeneity in the precore region of hepatitis B virus in hepatitis B e antigen-negative chronic hepatitis B Patients: Spontaneous seroconversion and interferon-induced seroconversion

To elucidate the relationship between the clinical severity of chronic liver disease and the precore mutations in hepatitis B e antigen (HBeAg)-nega-tive hepatitis B virus (HBV) carriers, mutations were investigated in the precore region of HBV DNA in 20 chronic hepatitis B patients who sero-converted either spontaneously or after the administration of α-interferon (IFN), and 5 asymptomatic carriers. The precore mutation with a stop codon at nucleotide 1896 was found in all patients, irrespective of the histology and in all asymptomatic carriers. The second mutation at nucleotide 1899 was found in 40% of cases studied but always followed by the first mutation at nucleotide 1896. The mixed viral infection of precore mutant and wild-type HBV virus was found in 40% of seroconverted cases after IFN treatment and in sera of HBV carriers obtained within a year after the spontaneous Seroconversion. These data suggest that the precore mutants prevail over wild-type HBV in all HBeAg-negative HBV carriers within several years after the sero-conversion, but their prevalence could not confine the clinical severity of chronic liver disease. © 1995 Wiley-Liss, Inc.     

Detection of Occult Hepatitis C and Hepatitis B Virus Infections from Peripheral Blood Mononuclear Cells

Purpose: To investigate the problem of occult HCV & HBV infections in patients with persistently longstanding abnormal liver function test results of unknown etiology and to investigate occult HCV in patients with sustained virological response (SVR). Methods: The study included two groups; first group included 40 patients with persistently longstanding abnormal liver function test, in addition to 62 patients with history of hepatitis C who developed SVR. HCV RNA status was tested in serum by conventional RT-PCR and by real-time PCR in Peripheral Blood Mononuclear Cells (PBMCs). HBV DNA in PBMCs was done in first group only. Results: In first group, PCR in PBMCs was positive for HCV RNA in 4 patients with elevated liver enzymes and HBV DNA was positive in PBMCs in 3 patients. In patients with SVR, 7 patients were positive for HCV RNA in PBMCs. Conclusions: Patients with long-standing abnormal results of liver-function tests with unknown etiology may have HCV RNA or HBV DNA in their PBMCs in the absence of anti-HCV antibodies, HBV markers, serum HBV DNA and serum HCV RNA.In Patients with SVR, HCV RNA in PBMCs is recommended to detect residual infection especially in those with high serum HCV RNA levels before treatment.     

Clinical and Virological Aspects of Blood Donors Infected with Hepatitis B Virus Genotypes B and C

Pathogenic and therapeutic differences among hepatitis B virus (HBV) genotypes have been documented. However, the association of virological characteristics with clinical differences among HBV genotypes remains unclear. We therefore studied the clinical and virological characteristics of Taiwanese volunteer blood donors infected with HBV genotypes B and C. HBV genotypes were determined in 300 candidate blood donors positive for HBV surface antigen (HBsAg), and sequences of the precore gene of the HBV genome were determined in 50 HBV e antigen (HBeAg)-positive and 50 HBeAg-negative blood donors. Of 300 HBsAg-positive blood donors, 10% had elevated serum aminotransferase levels and 27% were positive for HBeAg. HBV genotype distribution in 264 viremic carriers was as follows: B, 221 (83.7%); C, 39 (14.8%); F, 1 (0.4%); and mixed infection, 3 (1.1%). Blood donors with genotype C infection tended to have a higher frequency of HBeAg positivity and a higher serum HBV DNA level than those with genotype B infection. The frequency of precore stop codon mutation was significantly higher in HBeAg-negative blood donors than HBeAg-positive ones, irrespective of HBV genotypes. Meanwhile, only 5% of blood donors with genotype C infection had C-1858 strains. In conclusion, mixed infection of HBV genotypes indeed occurs, and genotype C has a higher serum HBV DNA level than genotype B. Precore stop codon mutation is common in HBeAg-negative HBV carriers, irrespective of HBV genotypes. In contrast, precore C-1858 strains are rarely identified in Taiwanese HBV genotype C.     

Enzymatic amplification and sequence analysis of precore/core DNA in HBsAg-positive patients

Serum or plasma from 69 HBsAg-positive patients was tested for the presence of precore core gene specific DNA by the polymerase chain reaction (PCR). In both healthy individuals (n = 26) and chronic carriers (n = 25), there was a strong correlation between presence of circulating anti-HBe and the absence of detectable HBV genome in serum. In 18 serum samples where HBsAg was the only detectable marker, i.e., anti-HBc-negative specimens, HBV DNA could be detected in three samples. HBV strains from 21 of the 24 PCR-positive samples were sequenced over the precore/core junction. A stop codon at the end of the precore region, described by other workers, was found in strains from two blood donors, one of whom had detectable HBeAg in serum. Conversely, HBV strains from the three anti-HBc-negative patients where DNA of the HBV precore region could be amplified and who had no detectable serum HBeAg or anti-HBe did not have this stop codon. The study indicates that further investigations are required before lack of HBeAg can be correlated with evidence of mutations in the precore region.     

广州地区HBV/HCV重叠感染患者流行及临床特征研究

目的 探讨广州地区乙型肝炎病毒/丙型肝炎病毒（HBV/HCV）重叠感染患者的流行及临床特征,为提高其诊治提供依据.方法 回顾2005年4月至2011年10月我院收治慢性HBV感染患者临床资料,分析HBV/HCV重叠感染患者流行特征,按照HBeAg状态以及HBV DNA、HCV RNA水平分组分析HBV/HCV重叠感染患者临床特征.结果 HBV/HCV重叠感染率为1.9％（128/6604）,主要见于伴静脉吸毒史的男性中年患者.HBeAg阳性与阴性重叠感染患者之间的生化指标和终末期肝病发生率均无差异.HBeAg阳性患者HBV DNA阳性率高于阴性患者（P ＜0.001）,而HCV RNA水平阳性率低于阴性患者（P =0.007）.HBV DNA阳性患者终末期肝病发生率较阴性组升高（58.1％比37.9％,P=0.027）,而HCV RNA阳性与阴性患者之间发生率相似（43.6％比49.2％,P=0.540）.结论 广州地区HBV/HCV重叠感染率较低,HBV DNA水平可能与患者的疾病进展相关.