Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections,rt269L and rt269I

BACKGROUND The presence of two distinct hepatitis B virus(HBV)Pol RT polymorphisms,rt269L and rt269I,could contribute to the unique clinical or virological phenotype of HBV genotype C2.Therefore,a simple and sensitive method capable of identifying both types in chronic hepatitis B(CHB)patients infected with genotype C2 should be developed.AIM To develop a novel simple and sensitive locked nucleic acid(LNA)-real timepolymerase chain reaction(RT-PCR)method capable of identifying two rt269 types in CHB genotype C2 patients.METHODS We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types.Using synthesized DNAs of the wild type and variant forms,melting temperature analysis,detection sensitivity,and endpoint genotyping for LNA-RT-PCR were performed.The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms,and these results were compared with those obtained by a direct sequencing protocol.RESULTS The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes,two rt269L types[‘L1’(WT)and‘L2’]and one rt269I type(‘I’)in single(63 samples,72.4%)or mixed forms(24 samples,27.6%)in 87(92.6%sensitivity)of 94 samples from Korean CHB patients.When the results were compared with those obtained by the direct sequencing protocol,the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples(98.9%specificity).CONCLUSION The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms,rt269L and rt269I,in CHB patients with genotype C2 infections.This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.

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 QTAamp 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 7.5×107 and 1.67×107 copies of HBV DNA per mL in two HBV infected patients' sera, while 2.L4×105 and 3.02×105 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.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

Recent advances in molecular diagnostics of hepatitis Bvirus

Hepatitis B virus(HBV)is one of the important global health problems today.Infection with HBV can lead to a variety of clinical manifestations including severe hepatic complications like liver cirrhosis and hepatocellular carcinoma.Presently,routine HBV screening and diagnosis is primarily based on the immuno-detection of HBV surface antigen(HBsAg).However,identification of HBV DNA positive cases,who do not have detectable HBsAg has greatly encouraged the use of nucleic acid amplification based assays,that are highly sensitive,specific and are to some extent tolerant to sequence variation.In the last few years,the field of HBV molecular diagnostics has evolved rapidly with advancements in the molecular biology tools,such as polymerase chain reaction(PCR)and real-time PCR.Recently,apart of PCR based amplification methods,a number of isothermal amplification assays,such as loop mediated isothermal amplification,transcription mediated amplification,ligase chain reaction,and rolling circle amplification have been utilized for HBV diag-nosis.These assays also offer options for real time detection and integration into biosensing devices.In this manuscript,we review the molecular technologies that are presently available for HBV diagnostics,with special emphasis on isothermal amplification based technologies.We have also included the recent trends in the development of biosensors and use of next generation sequencing technologies for HBV.

Optimization of competitively differentiated poiymerase chain reaction in detection of HBV basal core promoter mutation

AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation.METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control.To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CDPCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to10 μmol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain indepe ndent of the amount of templates and the number of PCRcycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.

Hepatitis B virus genotypes in southwest Iran: Molecular, serological and clinical outcomes

AIM: To investigate the associations of hepatitis B virus (HBV) genotype with HBeAg and anti-HBe status, alanine aminotransferase (ALT) levels and HBV-DNA detection in different groups of HBV-infected patients in southwest Iran. METHODS: A total of 89 HBsAg-positive serum samples were collected from the same number of patients. All sera were then investigated to determine HBV DNA and serological markers. For all the polymerase chain reaction (PCR)-positive samples, biochemical, histopathological assays and genotyping were also performed. RESULTS: Genotype D was the only type of HBV foundin different clinical forms of acute and chronic infections. There was a high prevalence of HBeAg-negative HBV- infected patients with chronic hepatitis (52.7%). Out of 55 patients with chronic hepatitis, seven (12.7%) were diagnosed with cirrhosis. A significant association between the presence of anti-HBe antibody and an increase in ALT level, among either HBeAg-negative (P = 0.01) or HBeAg-positive (P = 0.026) patients, was demonstrated. No significant differences were observed between the clinical outcomes of HBeAg-positive and -negative individuals (P = 0.24). CONCLUSION: Genotype D has been recognized as the only type of HBV found in different clinical forms of HBV infections, including cirrhosis, among the residents of southwest Iran. Anti-HBe possibly plays a role in disease progression in some patients with chronic hepatitis, at least for a period of disease.

高敏PCR在HBV极低病毒载量的慢性乙型肝炎患者中检测的临床意义

目的探讨使用高敏PCR在HBV极低病毒载量(HBV DNA 10~99 IU/mL)人群检测中的意义。方法选取2019年9月—2022年2月在广州医科大学附属第五医院接受核苷(酸)类似物(NAs)治疗≥48周且经普敏HBV DNA检测(检测下限100 IU/mL)结果为低于检测下限的慢性乙型肝炎(CHB)患者,进一步行高敏HBV DNA检测(检测下限10 IU/mL),根据结果分为极低病毒载量(VLVL,HBV DNA 10~99 IU/mL)组和完全病毒学应答(CVR,HBV DNA<10 IU/mL或未检测到)组。比较两组患者的一般特征、血清病毒学指标、生化学指标、无创肝纤维化指标,评价相关血清病毒学指标对高敏HBV DNA高于检测下限的预测价值,并探讨未实现CVR的影响因素。符合正态分布的计量资料两组间比较采用成组t检验;非正态分布的计量资料两组间比较采用Mann-Whitney U检验。计数资料两组间比较采用χ^(2)检验或Fisher精确检验。采用受试者工作特征曲线(ROC曲线)评价相关血清病毒学指标对高敏HBV DNA高于检测下限的预测价值。采用二元Logistic回归分析探讨未实现CVR的影响因素。结果共纳入106例CHB患者,其中VLVL组24例、CVR组82例。VLVL组年龄小于CVR组(P=0.004),VLVL组qHBsAg水平(P=0.002)、HBeAg阳性率(P=0.002)、pgRNA阳性率(P=0.010)及ALT水平(P=0.017)高于CVR组。qHBsAg水平预测CHB患者采用高敏HBV DNA检查结果高于检测下限(>10 IU/mL)的ROC曲线下面积为0.717(P=0.002),最佳cut-off值为1214.5 IU/mL,敏感度为95.5%,特异度为53.9%。HBe Ag阳性(OR=3.654,95%CI:1.162~11.489,P=0.027)和qHBsAg(OR=2.985,95%CI:1.058~8.422,P=0.039)是未实现CVR的独立影响因素。结论CHB患者经普敏检测HBV DNA低于<100 IU/mL,但经高敏PCR检测实际仍存在VLVL。VLVL患者炎症损伤水平、pgRNA阳性率以及HBeAg阳性率均显著高于CVR者。HBeAg阳性和高qHBsAg水平是CHB患者未实现CVR的独立影响因素。临床医师不应忽视CHB患者中VLVL人群,需及时行高敏HBV DNA检测。

微小RNA-127-3p在乙型肝炎病毒相关性肝癌中的表达意义及对HBV阳性肝癌细胞增殖迁移侵袭的影响

目的:探讨微小RNA-127-3p(miR-127-3p)在乙型肝炎病毒(HBV)相关性肝癌中的表达意义及其对HBV阳性肝癌细胞增殖、迁移和侵袭的影响。方法:采用实时荧光定量聚合酶链反应(RTFQ-PCR)检测2018年12月至2020年12月在医院手术切除的64例HBV相关性肝癌患者肝癌组织和癌旁组织中miR-127-3p表达量。将对数生长期肝癌HepG2细胞分别以10、20、50 pmol的miR-127-3p mimic进行转染,同时设置对照组(以0 pmol mimic进行转染),培养48 h后采用RTFQ-PCR检测各转染组细胞中miR-127-3p表达量,MTT法检测各转染组细胞增殖情况,细胞划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力。结果:肝癌组织miR-127-3p表达量(0.26±0.03)低于癌旁组织(0.53±0.06)(P<0.05)。miR-127-3p mimic转染组(10、20、50 pmol)与对照组肝癌HepG2细胞中miR-127-3p表达量分别为(0.87±0.09)、(1.23±0.13)、(1.51±0.16)、(0.28±0.04),总体比较差异有统计学意义(P<0.05);与对照组比较,miR-127-3p mimic(10、20、50 pmol)转染组miR-127-3p表达量更高(P<0.05);miR-127-3p mimic转染组中miR-127-3p表达量随转染浓度的升高而升高(P<0.05)。miR-127-3p mimic转染组(10、20、50 pmol)与对照组肝癌HepG2细胞增殖率分别为[(51.58±5.24)%、(25.42±2.55)%、(16.23±1.65)%、(78.67±7.88)%],细胞迁移距离分别为[(28.26±2.84)mm、(23.17±2.25)mm、(18.32±1.83)mm、(33.87±3.41)mm],穿膜细胞数分别为[(122.54±12.26)个、(101.26±10.45)个、(46.15±4.78)个、(131.23±13.24)个],总体比较差异均有统计学意义(P<0.05);与对照组比较,miR-127-3p mimic(10、20、50 pmol)转染组细胞增殖率更低,细胞迁移距离更短,穿膜细胞数更少(P<0.05);miR-127-3p mimic转染组细胞增殖率随转染浓度的升高而降低,细胞迁移距离随转染浓度的升高而变短,穿膜细胞数随转染浓度的升高而减少(P<0.05)。结论:miR-127-3p在HBV相关性肝癌患者中呈低表达,其可抑制HBV阳性肝癌HepG2细胞增殖、迁移和侵袭。

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).

Liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients

BACKGROUND Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver disease, but it is not widely used in resource-limited settings. Therefore, non-invasive liquid biopsy tests are needed. AIM To assess liver injury in hepatitis B patients using quantified cell free DNA combined with other serum biomarker as a liquid biopsy-based method. METHODS A cohort of 663 subjects including 313 hepatitis B patients and 350 healthy controls were enrolled. Ultrasound-guided liver biopsies followed by histopathological assessments were performed for the 263 chronic hepatitis B patients to determine the degree of liver injury. Cell-free DNA was quantified using a novel duplex real-time polymerase chain reaction assay. RESULTS Compared with healthy controls, patients with hepatitis B virus (HBV) infection had significantly higher plasma DNA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and HBV DNA levels (P<0.01). Serum ALT, AST, bilirubin, and plasma DNA levels of patients with markedsevere inflammation were significantly higher than those with mild-moderate inflammation (P<0.01). There was a statistically significant correlation between hepatocyte inflammation severity and serum bilirubin (R^2=0.673, P<0.01) or plasma DNA (R^2=0.597, P<0.01) levels. The areas under the curves of serum ALT, bilirubin, plasma DNA, and their combination to distinguish between patients with mild–moderate and marked-severe inflammation were 0.8059, 0.7910, 0.7921, and 0.9564, respectively. CONCLUSION The combination of plasma DNA, serum ALT, and bilirubin could be a candidate liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.

Prevalence of occult hepatitis B virus infection in haemodialysis patients from central Greece

AIM:To assess the hepatitis B virus(HBV)-DNA and the prevalence of occult HBV infection in end-stage renal failure(ESRF)patients from Central Greece. METHODS:Sera from 366 ESRF patients attending five out of six dialysis units from Central Greece were investigated for HBV-DNA by real-time polymerase chain reaction.Only serum samples with repeatedly detectable HBV-DNA were considered positive.IgG antibodies to hepatitis C virus(anti-HCV)were tested by a third generation enzyme linked immunosorbent assay(ELISA),while IgG antibodies to hepatitis E virus (anti-HEV)were tested by two commercially available ELISAs.RESULTS:HBV-DNA was detected in 15/366 patient (4.1%)and HBsAg in 20/366(5.5%).The prevalenc of occult HBV infection was 0.9%(3/346 HBsAg negative patients).Occult HBV was not associate with a specific marker of HBV infection or anti-HCV o anti-HEV reactivity.There was no significant differenc in HBV-DNA titres,demographic and biochemica features,between patients with occult HBV infectio and those with HBsAg-positive chronic HBV infection. CONCLUSION:In central Greece,4%of ESRF patient had detectable HBV-DNA,though in this setting,th prevalence of occult HBV seems to be very low(0.9%).

Update on occult hepatitis B virus infection

The event of mutations in the surface antigen gene of hepatitis B virus(HBV) results in undetectable hepatitis B surface antigen with positive/negative anti-hepatitis B core(anti-HBc) antibody status in serum and this phenomenon is named occult hepatitis B infection(OBI). The presence of anti-HBc antibody in serum is an important key for OBI tracking, although about 20% of OBI cases are negative for anti-HBc antibody. The diagnosis of OBI is mainly based on polymerase chain reaction(PCR) and real-time PCR assays. However, real-time PCR is a more reliable method than PCR. OBI is a great issue for the public health problem and a challenge for the clinical entity worldwide. The persistence of OBI may lead to the development of cirrhosis and hepatocellular carcinoma. With regard to OBI complications, the screening of HBV DNA by the highly sensitive molecular means should be implemented for:(1) patients with a previous history of chronic or acute HBV infection;(2) patients co-infected with hepatitis C virus/human immunodeficiency virus;(3) patients undergoing chemotherapy or anti-CD20 therapy;(4) recipients of organ transplant;(5) blood donors;(6) organ transplant donors;(7) thalassemia and hemophilia patients;(8) health care workers;(9) patients with liver related disease(cryptogenic);(10) hemodialysis patients;(11) patients undergoing lamivudine or interferon therapy; and(12) children in time of HBV vaccination especially in highly endemic areas of HBV. Active HBV vaccination should be implemented for the close relatives of patients who are negative for OBI markers. Thus, the goal of this review is to evaluate the rate of OBI with a focus on status of high risk groups in different regions of the world.

To treat or not to treat the "immunotolerant phase" of hepatitis B infection:A tunnel of controversy

Hepatitis B virus(HBV) infection is a global public health problem,with an estimated 350 million people worldwide chronically infected and approximately 500000 who die annually from HBV-related liver diseases.Management of chronic HBV is challenging and waves of guidelines emerge every year.One of the hottest topics and a matter of debate is the management of patients in their early immunotolerant phase of infection.With the lack of evidence,dealing with this particular subset of patients creates a great conflict with opposing views.In this review,the author highlights the pros and cons of these views and proposes a reasonable solution to resolve this dilemma.

Increased CD163 expression is associated with acute-on-chronic hepatitis B liver failure

AIM: To assess CD163 expression in plasma and peripheral blood and analyze its association with disease in acute-on-chronic hepatitis B liver failure (ACHBLF) patients. METHODS: A retrospective study was conducted from January 1, 2011 to January 1, 2012. Forty patients with ACHBLF (mean age 44.48 ± 12.28 years, range 18-69 years), 40 patients with chronic hepatitis B (CHB) (mean age 39.45 ± 12.22 years, range 21-57 years) and 20 ageand sex-matched healthy controls (mean age 38.35 ± 11.97 years, range 28-60 years) were included in this study. Flow cytometry was used to analyze the frequency of CD163+ peripheral blood mononuclear cells (PBMCs) and surface protein expression of CD163. Real-time transcription-polymerase chain re-action was performed to assess relative CD163 mRNA levels in PBMCs. Plasma soluble CD163 (sCD163) levels were measured by enzyme-linked immunosorbent assay. Clinical variables were also recorded. Comparisons between groups were analyzed by Kruskal-Wallis H test and Mann-Whitney U test. Statistical analyses were performed using SPSS 15.0 software and a P value < 0.05 was considered statistically significant. RESULTS: Flow cytometry showed that the population of CD163+ PBMCs was significantly greater in ACHBLF patients than in CHB patients and healthy controls (47.9645% ± 17.1542%, 32.0975% ± 11.0215% vs 17.9460% ± 6.3618%, P < 0.0001). However, there were no significant differences in mean fluorescence intensity of CD163+ PBMCs within the three groups (27.4975 ± 11.3731, 25.8140 ± 10.0649 vs 20.5050 ± 6.2437, P = 0.0514). CD163 mRNA expression in ACHBLF patients was significantly increased compared with CHB patients and healthy controls (1.41 × 10 -2 ± 2.18 × 10 -2 , 5.10 × 10 -3 ± 3.61 × 10 -3 vs 37.0 × 10 -4 ± 3.55 × 10 -4 , P = 0.02). Plasma sCD163 levels in patients with ACHBLF were significantly increased compared with CHB patients and healthy controls (4706.2175 ± 1681.1096 ng/mL, 1089.7160 ± 736.8395 ng/mL vs 435.9562 ± 440.8329 ng/mL, P < 0.0001). In ACHBLF patients, plasma sCD163 levels were significantly positively associated with model for end-stage liver disease scores (r = 0.5075, P = 0.008), hepatitis B virus-DNA (r = 0.6827, P < 0.0001), and negatively associated with prothrombin activity (r = -0.3348, P = 0.0347), but had no correlation with total bilirubin (r = 0.2551, P = 0.1122). Furthermore, sCD163 was obviously elevated in non-surviving patients compared with surviving patients with ACHBLF (5344.9080 ± 1589.5199 ng/mL vs 3641.7333 ± 1264.5228 ng/mL, P = 0.0321). CONCLUSION: CD163 and sCD163 may be related to disease severity and prognosis in ACHBLF patients.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Establishment of a molecular tool for blood meal identification in Malaysia

Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in CenBank.PCR was next performed to compile gene sequences of different species of wild rodents.The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA.A total of 100 blood samples,both from laboratory animals and wild rodents were collected und analyzed.The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.Results:Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus(rattus) tanezumi,Rattus tiomanicus,Leopoldamys sabanus, Tupaia glis,Tupaia minor,Niviventor cremoriventor,Rhinosciurus laticaudatus,Calloseiurus caniseps,Sundamys muelleri,Rattus rajah,and Maxomys whitelwadi.The BLAST results confirmed the host with exact or nearly exact matches(>89%identity).Ten new gene sequences have been deposited in CenBank database since September 2010.Conclusions:This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.

B-cell clonality in the liver of hepatitis C virus-infected patients

AIM:The association of hepatitis C virus(HCV) infection with typeⅡmixed cryoglobulinemia is well established,but the role of HCV in B-cell lymphoma remains controversial.In patients with HCV infection,B-cell clonal expansions have been detected in peripheral blood and bone marrow,and a high prevalence of B-cell non-Hodgkin's lymphomas has been documented.Liver biopsies in chronic HCV infection frequently show portal lymphoid infiltrates with features of B follicles,whose clonality has not yet been investigated.The object of this study was to determine the frequency of liver-infiltrating monoclonal B-cells in 40 patients with HCV infection.METHODS:Eight hundred and forty-eight patients were studied prospectively,including 40 HCV-positive patients and 808 patients with chronic hepatitis B virus(HBV)infection.Immunohistochemical study for B-and T-cell markers was performed on the paraffin-embedded liver tissue sections.The clonality of lymphoid B-cells was tested using a polymerase chain reaction(PCR)approach designed to identify immunoglobulin heavy chain gene(IgH) rearrangements.RESULTS:Liver-infiltrating monoclonal B-cells were detected in the liver for 4(10%)of 40 HCV-positive patients but were present in only 3(0.37%)of 808 liver biopsy specimens with chronic HBV infection.Chi-square testing showed that the monoclonal B-cells infiltration in the liver was more frequent in the HCV-infected patients(P=0.000).A clonal IgH rearrangement was detected in 5(71.4%)of 7 liver biopsy specimens with monoclonal B-cells infiltration.In 2 of 5 patients with both a clonal B-cell expansion and monoclonal B-cells infiltration in the liver,a definite B-cell malignancy was finally diagnosed.CONCLUSION:Liver-infiltrating monoclonal B-cells are detected in the liver of patients with chronic HCV and HBV infection.A high percentage of patients with monoclonal B-cells infiltration and B-cell clonality in the liver were finally diagnosed as having a definite B-cell malignancy.

Interleukin-10 promoter polymorphisms in patients with hepatitis B virus infection or hepatocellular carcinoma in Chinese Han ethnic population

BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.

Research on Hepatitis B virus Genotypes and Subgenotypes among Bai Nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B+C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B+C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B+C) (P=0.077/P=0.663). In Dali, genotypes B, B+C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B+C) in patients.

Therapeutic effectiveness of interferon alpha on hepatitis B virus DNA in peripheral blood mononuclear cells

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