Expression level of serum HBV RNA in HBeAg-positive chronic hepatitis B patients at different periods and its value of measurement / 临床肝胆病杂志

Objective To investigate the expression level and potential clinical value of serum HBV RNA in HBeAg-positive chronic hepatitis B (CHB) patients at different periods. Methods A total of 61 CHB patients who attended the outpatient and inpatient services of Department of Hepatology, Hangzhou Xixi Hospital, from August 2019 to December 2020 were enrolled, and according to the antiviral therapy for HBeAg-positive CHB patients, they can be divided into group A with untreated HBeAg-positive CHB (HBeAg+ and HBV DNA+) patients, group B with treatment-experienced patients before HBeAg seroconversion (HBeAg+ and HBV DNA-), and group C with treatment-experienced patients after HBeAg seroconversion (HBeAg- and HBV DNA-). Peripheral blood HBV RNA load was measured at different periods, and its correlation with HBsAg and HBV DNA was analyzed. The t -test was used for comparison of normally distributed continuous data between groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between groups; a Pearson or Spearman correlation analysis was used to describe the correlation between two variables. Results The positive rates of HBV RNA in these three groups were 100% (22/22), 88.2% (15/17), and 22.7% (6/22), respectively. In group A, HBV RNA was positively correlated with HBsAg and HBV DNA ( r =0.612 and 0.922, both P < 0.01), while in groups B and C, there was no correlation between HBV RNA and HBsAg. Group B had significantly higher levels of HBV RNA and HBsAg than group C ( Z =-4.44 and -2.41, both P < 0.05). The HBV DNA-positive group had a significantly higher level of HBV RNA than the HBV DNA-negative group ( Z =-6.16, P < 0.01). Conclusion After HBV DNA clearance achieved by antiviral therapy with nucleos(t)ide analogues in CHB patients, serum HBV RNA can still be detected in some of these patients. Since HBV RNA only comes from cccDNA in the liver, it can better reflect viral replication activity in the liver than HBV DNA and thus has a certain clinical value in the management of CHB patients.

Hepatitis B viremia in HIV-coinfected individuals under antiretroviral therapy

ABSTRACT Background: Antiretroviral therapy (ART) has decreased AIDS incidence and mortality, rendering comorbidities, such as hepatitis B more relevant for people living with human immunodeficiency virus (HIV). Since antiretroviral drugs may also inhibit hepatitis B virus (HBV) replication, analyzing the impact of ART on management of hepatitis B in this population is important. Objective: To assess HBV viremia among HIV/HBV coinfected individuals on ART and its associated factors. Method: For this cross-sectional study, HIV/HBV-coinfected individuals, aged over 18 years, who were on ART for over six months and receiving care at an outpatient clinic in São Paulo were recruited. Sociodemographic characteristics, information about viral exposure, clinical and laboratory data, including evaluation of liver fibrosis were obtained. Plasma HBV DNA was measured by polymerase chain reaction. Viral genome sequencing was conducted for genotyping and identification of drug resistance-conferring mutations if viral load exceeded 900 IU/mL. Results: Out of 2,946 patients who attended the clinic in 2015, 83 were eligible and 56 evaluated. Plasma HBV DNA was detected in 16 (28.6%) (95% CI: 18.0-41.3%), all on lamivudine and tenofovir treatment. HBV DNA detection was associated with lower education (p = 0.015), higher international normalized ratios (p = 0.045), history of an AIDS-defining illness [OR: 3.43 (95% CI: 1.10-11.50)], and HBeAg detection [OR: 6.60 (95% CI: 1.84-23.6)]. In contrast, a last CD4+ count above 500 cells/mm3 in the year prior to inclusion [OR: 0.18 (95% CI: 0.04-0.71)] and detection of anti-HBe [OR: 0.21 (95% CI: 0.04-0.99)] were negatively associated. Patients with HBV DNA above 900 IU/mL were infected with subgenotypes A1 (n = 3) and D2 (n = 1), and exhibited viral mutations associated with total resistance to lamivudine and partial resistance to entecavir. Conclusions: Despite being on ART, a significant proportion of HIV/HBV-coinfected individuals present HBV viremia. Characterization of factors that are associated with this finding may help professionals provide better management to these patients.

In vitro study on anti-HBV effects and mechanism of hypericin / 重庆医学

Objective To evaluate the anti-HBV effect of hypericin from the cellular level and to preliminarily explore its po-tential drug target point.Methods Liver cell line HepG2.2.15 cells secreting HBV particles were selected as the experimental ob-jects.Hypericin served as the HY group,lamivudine was taken as 3TC group and deionized water as the blank control group.The cells were grouped and administrated.The HBV-DNA copy level was measured at72 h after medication by Southern blot and fluo-rescent quantitative PCR;the inhibition rate of HBsAg and HBeAg was detected by using ELISA assay;the pgRNA expression level was tested by using Northern blot and fluorescent quantitative PCR;Western blot and fluorescent quantitative PCR were adopted to detect the expression of regulatory factors including HNF3β,HNF4α,PPARαand RXRα.Results Compared to the blank control group,both hypericin and lamivudine had significant inhibiting effect on HBV DNA and expression level of HBsAg and HBeAg in HepG2.2.15 cells (P <0.05).Hypericin could significantly decrease the pgRNA expression compared with the blank control group (P <0.05),while lamivudine had no obvious change (P <0.05).Moreover,hypericin exhibited significant effects on the expression of HNF3βand regulatory factor HNF4αcompared with the blank control group and 3TC group(P <0.05).Conclusion Hypericin represents a strong anti-HBV effect,moreover could increase the negative regulatory factor HNF3βn expression and decreases the positive factor HNF4αexpression,prompting that its drug target point could be pgRNA.

Prevalence of occult hepatitis b infection in iranian cancer patients before chemotherapy treatment / Prevalência de infecção oculta da hepatite B em pacientes iranianos com câncer antes do tratamento de quimioterapia

ABSTRACT Background Occult hepatitis B infection is characterized by negative hepatitis B surface antigen (HBsAg) and also detectable hepatitis B virus (HBV) -DNA, with or without hepatitis B core antibody (anti-HBc). HBV reactivation in individuals under immunosuppressive therapy is critical, occurring in occult HBV. Objective In this study, we aimed to determine the prevalence of occult HBV infection among hepatitis B surface antigen negative in cancer patients before receiving chemotherapy. Methods Sera from 204 cancer patients who were negative for HBsAg, were tested for anti-HBc antibodies. The samples that were negative for HBsAg but positive for anti-HBc also examined for HBV-DNA by polymerase chain reaction (PCR). Results Of the 204 HBsAg negative blood samples, 11 (5.4%) samples were positive for anti-HBc antibodies. HBV-DNA was detected in 9/11 (81%) of anti-HBc positive samples. Occult HBV infection in hematological cancers was more than solid cancers, 4.8% and 4.3% respectively. There was no significant difference in HBc antibody positivity based on vaccination, previous blood transfusions, history of familial hepatitis or biochemical parameters (ALT, AST, total and direct bilirubin levels) (P>0.05). Conclusion Screening of occult HBV infection by HBsAg, HBV DNA and anti HB core antibody should be suggested as a routine investigation in cancer patients before receiving chemotherapy.

RESUMO Contexto A infecção oculta da hepatite B caracteriza-se por antígeno de superfície da hepatite B (AgHBs) negativo com vírus detectável da hepatite B (HBV) -DNA, com ou sem anticorpo de núcleo da hepatite B (anti-HBc). A reativação do HBV em indivíduos sob terapia imunossupressora é crítica, originando a infecção oculta pelo VHB. Objetivo Este estudo teve como objetivo determinar a prevalência de infecção oculta pelo VHB entre em pacientes com câncer e com antígeno de superfície da hepatite B negativo antes de receber quimioterapia. Métodos Soro de 204 pacientes com câncer que foram negativos para AgHBs, foram testados para anticorpos anti-HBc. As amostras que foram negativos para AgHBs, mas positivo para anti-HBc foram também examinadas para HBV-DNA, por reação em cadeia da polimerase. Resultados Entre 204 amostras de sangue AgHBs negativas, 11 (5,4%) foram positivos para anticorpos anti-HBc. HBV-DNA foi detectado em 9/11 (81%) das amostras positivas de anti-HBc. Infecção oculta de VHB em câncer hematológico foi maior que em cânceres sólidos, 4,8% e 4,3% respectivamente. Não houve diferença significativa na positividade anti-HBc, com base na vacinação, transfusões de sangue anteriores, história de hepatite familiar ou parâmetros bioquímicos (ALT, AST, total e níveis de bilirrubina total) (P & gt; 0,05). Conclusão A triagem de infecção oculta por AgHBs, HBV-DNA e anti-anticorpo de núcleo HB deve ser sugerida como uma investigação de rotina em pacientes com câncer antes de receber a quimioterapia.

Determination of human papillomavirus in oral leukoplakia,oral lichen planus and oral squamous cell carcinoma / 北京大学学报(医学版)

Objective:To investigate the possibility for human papillomavirus (HPV)infection to be a predictable signal for the carcinogenesis of oral mucosa by comparing the prevalences of HPV in each stage of oral mucosal carcinogenesis and to compare the sensitivity differences of the two methods in de-tecting HPV infection in oral cavity.Methods:The hybrid capture (HC-Ⅱ)was used to detect infection of HPV in 255 samples taken from 1 2 cases of healthy oral mucosa,21 1 cases of patients with pathologi-cal diagnosis and 32 cases of patients with clinical diagnosis.The diagnosed cases included 8 cases of be-nign lesions of the oral mucosa,precancerous lesions [74 cases of oral leukoplakia (OLK)with hyper-plasia and 42 cases of OLK with oral epithelial dysplasia (OED)],91 cases of precancerous condition [oral lichen planus (OLP)]and 28 cases of oral squamous cell carcinoma (OSCC).And in situ hybri-dization (ISH)was used to detect infection of HPV in 33 cases of OSCC and 76 cases of OLK,including 30 cases of hyperplasia,1 5 cases of mild OED,1 5 cases of moderate OED and 1 6 cases of severe OED. Results:The prevalence of HPV in OLP samples was higher (1 2.1 2%,8/66 )than that of OLK (2.59%,3/1 1 6)(χ2 =4.666,P=0.031 )and OSCC(7.1 4%,2/28,χ2 =0.51 3,P=0.474).The prevalence of HPV in OSCC (7.1 4%,2/28)was higher than that of OLK (2.59%,3/1 1 6),and no significant difference was found.There was only one case of smoke spot and statistical analysis was not carried out.ISH was used to detect type 1 6/1 8 and type 31 /33 HPV DNA in 1 09 cases of oral mucosal lesions in paraffin sections and only one case of OSCC was HPV positive.Thirty-seven cases were detec-ted by HC-Ⅱ and ISH methods at the same time.The same negative results by the two methods were found in 94.6% samples (35/37).In the other two samples,one was OSCC with early infiltration and the other was OLK with hyperplasia,The HC-Ⅱ results were positive while the ISH results were nega-tive.The patients with OLP and HPV testing results were followed up and the average follow-up period was (36.2 ±1 0.5)months.It was found that three of them had a malignant transformation,and the ma-lignant transformation rate of HPV positive patients was 1 2.50% (1 /8),which was higher than that of HPV negative patients (3.45%,2/58),and the difference was not statistically significant,P=0.249. Conclusion:HC-Ⅱ assay was more sensitive in detecting HPV infection of oral mucosal lesions than ISH.The results of this study showed that there was insufficient evidence for taking HPV infection as a predictor of OLK carcinogenesis.Patients suffering from OLP were in a precancerous condition.The pre-valence of HPV in OLP patients of this study was higher than that in OLK and OSCC patients,suggesting that for some reason,OLP patients were susceptible to HPV.HPV testing can be considered as routine in patients with OLP,and HC-Ⅱassay was recommended.And patients with OLP and HPV positive should be followed up regularly.

EBV DNA detection in the diagnosis of Epstein-Barr virus infection realated diseases in children / 中华检验医学杂志

Objective To investigate the clinical significance of Epstein-Barr virus EBV DNA in children with Epstein-Barr virus infection realated diseases.Methods A retrospective cohort study was performed.Totally 222 blood samples were collected from children who were diagnosed as EBV infection in Shandong Provincial Hospital from June 2012 to August 2013.Fluorescent quantitative PCR( FQ-PCR) was used to analyze the EBV DNA in peripheral blood lymphocytes.ELISA was used to analyze the four EBV serology antibodies in the serum.Two groups of tested results were compared.Heart, hepatic impairment and renal function were analyzed through detecting AST, ALT, BUN, CREA, CK, CKMB.The results were grouped by EBV DNA copy number, and then non-parametric test together with correlation analysis was performed using SPSS21.0 analytics software.Results The positive rate of EBV-CA IgM and EBV DNA was 51.35%(114/222) and 72.97% (162/222) respectively, χ2 =24.01, P1 ×106 copies/ml,Ⅱ1 ×105 -1 ×106 copies/ml, Ⅲ1 ×104 -1 ×105 copies/ml, Ⅳ5 × 103 -1 ×104 copies/ml, Ⅴ<5 ×103 copies/ml), and ALT(χ2 =10.14,P<0.05), BUN(χ2 =18.17, P<0.05), CK(χ2 =13.09,P<0.05), CKMB(χ2 =17.93,P<0.01) had a statistically significant difference between each group.Well, the log value of EBV DNA copy number had a positive correlation relationship with AST(r=0.357,P=0.001), ALT(r=0.376,P=0.001), BUN(r=0.329,P=0.000), CK(r=0.235,P=0.035).Conclusions Detection of EBV DNA can be used for the early diagnosis and assessment of process of the EBV infection related disease in children.The detection of liver, kidney function and myocardial enzymes can be used for evaluating the severity of EBV infection.

The clinical evaluation of a new diagnostic kit for hepatitis B virus YMDD mutation DNA detection / 中国医师杂志

Objective To evaluate the clinical application of a novel hepatitis B virus YMDD mutation DNA diagnostic kit (magnetic beads method kit).Methods A total of 324 HBV clinical serum samples was tested with the magnetic beads method kit and another kind of fluorescence diagnostic kit (boiling method).Accuracy, specificity, and sensitivity were compared.Results The consistency of positive detection rate of two kits was 100％ (95％ CI : 98.0％ ～ 100％), negative consistency was 97.12％ (95％ CI : 92.8％ ～99.2％) and the total consistency was 98.76％ (95％ CI : 96.9％ ～99.7％).Four cases of discrepant samples were confirmed by sequencing, and statistical analysis performed by Kappa test (Kappa =0.975) shows good consistency between the two methods.Conclusions The magnetic beads method kit has good consistency compared to the regular boiling method kit, and the polymerase chain reaction (PCR) detection system contains an internal positive control (internal control) to avoid a false negative resuit, which is more suitable for clinical diagnosis.

Application of real-time PCR in pathogenic detection of necrotizing herpes stromal keratitis / 中华实验眼科杂志

Background Herpes stromal keratitis (HSK) is a common infectious ocular surface disease,with a higher recurrent rate,especially in necrotizing HSK.The diagnosis of HSK primarily depends on signs and symptoms,and specific laboratory diagnostic is lack.Objective This study was to clarify the expression of herpes simplex virus (HSV) in the corneal epithelium scrapings and tears of necrotizing HSK patients.Methods Thirty eyes of 30 patients with necrotizing HSK were enrolled in Nanjing Hospital of Nanjing Medical Hospital from September 2012 to September 2013 under the patient's informed consent.The eyes were examined by slit lamp microscope and scored.HSK patients received local and systemic therapy for 8 weeks and then an oral maintenance dose for 6 months.Corneal epithelial scrapings and tears samples were collected for HSV DNA detection by real-time PCR before and the 1 st,2nd,4th,6th and 8th week after therapy respectively.The difference of HSV positive rate was compared between corneal epithelium scrapings and tears samples using Chi-square test.Multilevel mixed effective model was employed to evaluate HSV concentration change in the samples at various time points in the HSV-positive patients of initial visit.The correlation between HSV concentration and clinical score was analyzed by Spearman rank correlation.Results HSV-positive rate was 46.4％ (13/30) in the corneal epithelial scrapings and 13.3％(4/30) in the tear samples,showing a significant difference between them (P =0.006).HSV-positive rate was significantly lower in the corneal epithelial scrapings 1 week,2 weeks and 4 weeks after treatment than before (P =0.001,0.003,0.004),and no HSV was detected 6 weeks and 8 weeks after treatment.No significant change in HSV-positive rate in tear samples in 1 week and 2 weeks after treatment in comparison with before treatment (P =1.000,0.583),and no HSV was detected after 4 weeks following treatment.The HSV concentration was 2460 (2 165-636500)/ml in initial 13 HSV-positive eyes of corneal epithelial scrapings and 0 (0-1150)/ml in initial 13 HSV-positive eyes of tear samples.Multilevel mixed effective model determined that HSV concentration was significantly lower in corneal epithelial scraping than that in tear (P =0.005),and HSV concentration was reduced with the lapse of time (P =0.001),with the faster rate of decline in the corneal epithelial scrapings (P =0.049).A positive correlation was found between initial HSV concentration and clinical scores (rs =0.844,P =0.000).Conclusiors Real-time PCR appears to be a powerful molecular tool for the detection of HSV in the HSK,especially in corneal epithelial scrapings of lesion.The initial positive outcome of viral DNA in corneal epithelial scrapings predicts a severe clinical procedure.

Design and validation of a clinical DNA sequencing assay for drug-resistance genes in hepatitis B virus / 中华检验医学杂志

Objective Under the ISO15189 and America Association of Pathologists ( CAP ) laboratory accreditation system, to establish the performance verification standards for detecting hepatitis B virus resistance gene by sanger sequencing.Methods 25 cases of HBV drug resistance outpatients and inpatients were collected from August 2012 to December in Hepatitis Clinic of Huashan Hospital Affiliated to Fudan University.Analytical performance parameters including analytical sensitivity, precision, accuracy, analytical specificity, reference range/reportable range, etc were evaluated.Sequencing quality evaluation parameters included fluorescence signal intensity overall sequencing chromatogram, signal to noise ratio, trace scores and QV value.Results 10%-20% mutation could be detected under wild-type background. The methool had good precision and accuracy.No obvious interference and cross contamination were observed.Conclusions Performance validation of the sequencing should combine with the practical application.Especially in view of the different detection subjects, and appropriately adjusted to meet the clinical needs.Detection of hepatitis B virus resistance gene by the in the test method in this study can be used in clinical detection.

Analysis of suspicious results of serum HBV DNA detected by fluorescence quantitative PCR / 中华检验医学杂志

Objective To analyze the suspicious results of serum HBV DNA by fluorescence quantitative PCR and develop appropriate countermeasures in order to improve the quality of detection of HBV DNA.Methods Blood samples of patients from the First Affiliated Hospital of Wenzhou Medical College from 2008 to 2011 were analyzed for HBV DNA by fluorescence quantitative PCR.1969 cases of suspicious results,judged by the rule of review the results of serum HBV DNA combined with the historical results,PCR amplification curve,HBV serum markers and clinical diagnosis,were analyzed and redetected by using of two different reagents,careHBV PCR Kit and careHBV PCR Kit V2,at the same time.The consistency and inconsistency ratio of the results were evaluated.Both the reasons of inconsistent and the undetected rates of careHBV PCR Kit were analyzed.The two reasons for the inconsistent results included the reagent related factors,e.g,showing no amplification curve caused by the false negative and abnormal low efficiency of amplification curve,and the non reagent related factors such as operating pollution and other sample factors.Results There were 115 154 blood samples were detected for HBV from 2008 to 2011 and 1969 samples (1.71％) with suspicious results were redetected.The consistency and inconsistency results were 1588 (80.65％) and 381 (19.35％),respectively.Every year from 2008 to 2011,the percentage of the inconsistent results caused by the reagent related factors were 18.87％,20.23％,51.33％ and 59.57％ respectively,which showed an increasing trend,and the percentage of inconsistent results caused by the nonreagent related factors were 81.13％,79.77％,48.67％ and 40.43％ respectively,which showed a declining trend year by year.The undetected rates of careHBV PCR Kit were 2.49％,4.08％,10.09％ and 14.47％ respectively,showing an increasing trend.Conclusions The redetection for the specimens with the suspicious results by using of different reagents can avoid the blind detection of HBV DNA and reduce the experimental error.All the clinical samples for quantitative HBV DNA including the mutations of HBV gene can be measured accurately and effectively,which is helpful to hepatitis B patients for antiviral therapy.

Quantification and genotyping of hepatitis B virus in a single reaction based on dual molecular beacon real-time PCR / 中华检验医学杂志

Objective To develop a new method for simultaneous quantifying and genotyping of HBV in a single reaction based on dual molecular beacon real-time PCR.Methods Genotype B and C recombinant plasmids were constructed as the standards and genotype-specific primers and molecular beacons were designed for each genotype.The molecular beacons of genotype B and C were labeled with FAM and Hex respectively.In this way,a simultaneous qualification and genotyping method for HBV DNA in a single real-time PCR reaction system was developed.Firstly,10-fold gradient dilution of genotype B and C standard plasmids (103-1011 kIU/L) were utilized to evaluate the linear ranges and sensitivity of this approach.The clinical specificity was tested with twenty different serum specimens (5 cases with hepatitis C virus,5 cases with herpes simplex virus and 5 cases with human papilloma virus as well as 5 healthy volunteers) ; the reproducibility was assessed by intra-assay and inter-assay coefficient of variation (CV) of cycle threshold (Ct) value through 10 repeated detections within a batch and between batches of the B,C standard plasmids (108,106 and 104 kIU/L).Then the accuracy of qualifying and genotyping of the self-built method was evaluated by a parallel examination with 132 HBV infected patients by use of two commercial kits as the references.Finally,these HBV-positive patients were divided into 4 groups:asymptomatic carrier (n =21),chronic hepatitis (n =77),liver cirrhosis (n =25) and hepatocellular carcinoma (n =9) to investigate the relationship of genotypes,stages of disease progression and HBV DNA load.Results A simultaneous qualification and genotyping assay was successfully built and its genotyping sensitivity was 103 kIU/L and the linear range was 103-1011 kIU/L.The intra-assay CV of B genotyping was 1.51％ to 1.80％ and the interassay CV was 2.11％ to 3.03％,while the intra-assay CV of C genotyping was 1.79％ to 1.95％ and the inter-assay CV was 2.53％ to 2.91％.The results of non HBV infected cases and healthy volunteers showed negative.In the test of 132 HBV infected patients,the general coincident rate of genotyping results comparing our assay and HBV DNA genotyping kit was 90.9％ (120/132,Kappa =0.832,P ＜ 0.05).The HBV DNA quatitive results between the assay[5.07 (3.89-6.33)] and HBV DNA quatitive kit [5.19 (4.15-6.32) lg kIU/L] were well correlative (R2 =0.8477,P ＜ 0.05).69 genotype B cases,51 genotype C cases and 12 B/C mixed-genotype cases were detected by dual molecular beacon real-time PCR method and their HBV DNA load were 4.54 (3.83-6.17),5.53 (4.02-6.55),4.58 (3.68-4.98) lg kIU/L respectively.Where the patients with genotype C had higher DNA load than the patients with other two genotypes (Z =-2.195and-2.162,P ＜ 0.05).The HBV DNA load of asymptomatic group,chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group were 7.02 (6.35-7.84),4.94 (4.16-6.25),4.37(3.50-5.17) and 3.45 (3.25-4.92) lg kIU/L,respectively.Among them,the asymptomatic group was significantly higher than those of other three groups (Z =-4.244,-4.568 and-3.489,P ＜0.001) and DNA load comparing with the chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group also showed statistically different (Z =-2.894 and-2.413,P ＜ 0.05).However,compared with the liver cirrhosis group and hepatocellular carcinoma group there was no significant difference (Z =-0.995,P =0.335).Conclusion A dual molecular beacon real-time PCR assay which can simultaneously quantifying and genotyping HBV DNA with highly accuracy,sensitive and specificity is successfully developed.(Chin J Lab Med,2013,36:333-338)

Efficacy of sequential add-on pegylated interferon α-2a in combination with adefovir dipivoxil in chronic hepatitis B patients with low serum HBeAg titer / 中华传染病杂志

Objective To investigate the efficacy of sequential add-on of pegylated interferon α-2a (PEGIFN-α-2a) for 48 weeks in chronic hepatitis B (CHB) patients with low serum hepatitis B e antigen (HBeAg) titer after long term adefovir dipivoxil (ADV) monotherapy.Methods This was a randomized,open and prospective clinical trial.Patients who had been treated with ADV for 72 to 144 weeks,with undetectable serum hepatitis B virus (HBV) DNA level,low HBeAg titer (5 S/CO＜ HBeAg＜50 S/CO) and serum hepatitis B surface antigen (HBsAg) ＜5000 IU/mL were included.The patients were categorized into ADV monotherapy group and ADV plus PEGIFN-a-2a combination therapy group by random number table.Patients in ADV group continued ADV monotherapy and patients in combination therapy group added PEGIFN-α-2a to ADV for 48 weeks.After the treatment,efficacy of the two therapies were assessed by comparing the reduction of serum HBeAg reduction,HBeAg loss rate,HBeAg seroconversion rate,and reduction of intrahepatic HBV DNA and HBV covalently closed circular DNA (cccDNA).Pre-and post-treatment results were compared by paired samples t test.Comparison between groups was performed using indepedent samples t test.Comparison of rates between groups was performed using x2 test.Results The trial enrolled 55 CHB patients,and there were 27 patients in ADV monotherapy group,28 patients in combination therapy group.Baseline characteristics including age distribution,sex ratio,alanine aminotransferase (ALT),aspartate aminotransferase (AST),total bilirubin (TBil),serum HBeAg and HBsAg,hepatic HBV DNA and HBV cccDNA were all comparable (all P＞0.05).Twenty-five patients in ADV monotherapy group and 26 patients in combination therapy group completed 48 weeks treatment.HBeAg loss rates and seroconversion rates of combination therapy group were higher than those of ADV monotherapy group (x2 =5.38 and 4.69,respectively,both P＜0.05).HBeAg titers of both groups were significantly lower than those of baseline (t=8.43 and 8.50,respectively,both P＜0.05).The HBeAg titer of combination therapy group was lower than that of monotherapy group (t=5.60,P＜ 0.01).HBV DNA and HBV cccDNA in liver tissue of combination therapy group was (6.934±0.52) lg IU/mg and (5.63±0.54) lg IU/mg post-treatment,respectively,which were both lower than baseline (t＝7.12.6.67,respectively,both P＜0.01).HBV DNA in liver tissue of monotherapy group was (7.09＝0.43) lg IU/mg post-treatment,which was lower than baseline (t=2.67,P=0.02).After treatment,HBV cccDNA in liver tissue of combination therapy group was lower than that of monotherapy group (t =2.87,P=0.00).Conclusions Compared with ADV monotherapy,sequential add-on of PEGIFN-a-2a in combination with ADV can achieve higher serum HBeAg loss rate and seroconversion rate and facilitate the clearance of hepatic HBV DNA and HBV cccDNA in CHB patients with low HBeAg titer after long-term ADV monotherapy.

Molecular epidemiological study of occult hepatitis B virus infection in blood donors / 中华传染病杂志

ObjectiveTo evaluate the prevalence of occult hepatitis B infection in blood donors and to explain the possible molecular mechanfism of occult hepatitis B infection.Methods Enzyme linked immunosorbent assay (ELISA) method was used for detection of hepatitis B virus (HBV)markers in serum samples of 594 donors which were collected from blood bank with HBsAg negative results.Nested-polymerase chain reaction (PCR) was performed to detect serum HBV DNA.In donors with occult HBV infection,the serum HBV markers were quantitatively detected by Abbott nested-PCR kit.The PCR products of S region were sequenced and sequence alignment was performed to analyze relevant virus mutations.Eleven HBsAg positive patients were randomly recruited as positive controls and S region was amplified and sequenced.The difference of S region sequences was compared between patients with occult HBV infection and HBsAg positive HBV infection.ResultsAmong 594 HBsAg negative donors,15 were diagnosed with occult HBV infection with the incidence of 2.5 ％.No correlation was found between results of serum HBV markers and occult HBV infection.Sequencing results of HBV S region were obtained from 10 cases,which revealed mutations of HBV.The amino acid mutations in the “a” determinant cluster were found in three patients,which were I126T,T140I and T140I,respectively.On the contrast,mutation in the “a” determinant cluster of T131 N was only found in one positive control.ConclusionsThe occult HBV infection exists in blood donors with negative results for HBsAg test.Genetic mutation may play a role in the occult HBV infection.

To establish a magnetic beads based nucleic acid extraction method and apply for quantitative detection of hepatitis B virus DNA / 中华检验医学杂志

ObjectiveTo facilitate the establishment of fully automatic clinical diagnostic nucleic acid assay,a study was carried out for developing a fast and convenient method of extracting highly purified viral nucleic acid from human blood with magnetic beads.Method Five KHB (Shanghai Kehua Biological Engineering Company Limited) internal strong positive HBV serum samples were used in the process,for the method development,screening and selecting the magnetic beads based on magnetism,suspension power and extraction effects of HBV DNA,the optimization of the selected key components of extraction reagents were performed,which including the quantity of magnetic beads( 1 - 1.75 μl/ml),the buffer pH of lysis (4 -9),washing conditions with or without alcohol,elution for proliferation with or without magnetic beads,and etc.The evaluation for the extraction method was carried out including linearity,precision,sensitivity,specificity,anti-interference and resistance to contamination.The magnetic beads method was compared with domestic mature boiling method in a quantitative test with 100 HBV serum samples,the magnetic beads method was also compared with the interntional Gold standard Roche COBAS Tagman test method ( Roche method) in a quantitative test with 50 HBV serum samples and the results were statistically compared for their linear regression.ResultsOptimization experiment result showed superparamagnetic beads A was the best among the candidates ( C,B,D) in terms of their physical properties and extraction effects of DNA.The optimum condition of the assay were as follow:magnetic beads best used at 1.25 μl/ml,buffer pH 7 - 8 of lysis,ethanol was eliminated from washing buffer,proliferation can be carried out with magnetic beads.The parallel comparison experiments with five KHB internal strong positive HBV serum sample and gradient diluted serum sample between beads method and boiling method were carried out.The results showed that the linear range of beads method was 4 × 101 -4 × 108 IU/ml and that of boiling method was 4 × 102 -4 × 108 IU/ml.The sensitivity for beads method was 30 IU/ml and that of boiling method was 100 IU/ml.The precision CV of beads method was 6.0％ and boiling method was 11.9％.The false positive rate was 0 for beads method vs 3.1％ for boiling method.The anti-interference ability experiment showed that when sample contained sodium heparin concentration exceed 62.5 U/ml, there was significant drop of HBV DNA detection with boiling method and no HBV DNA was detected when sodium heparin concentration in the sample reached 1000 U/ml.However,sample sodium heparin concentration had no effect on HBV DNA detection with beads method.The comparison study between beads method and boiling method was carried out by use of 100 clinical HBV positive serum samples.The result showed that the sensitivity for beads method was 86％ vs boiling method 74％.The correlation between the two method was R2 =0.948,P ＜ 0.05.The comparison study between beads method and Roche method was carried use of 50 clinical HBV DNA positive serum samples and the result showed that sensitivity for beads method was 95％ (49/50) vs Roche method was 100％ (50/50).The correlation was R2 =0.963,P ＜ 0.05.ConclusionsThe established method for automatic nucleic acid extraction with magnetic bead displayed characteristic of high yield nucleic acid extraction,broader testing range,accurate quantification and convenient in operation.This can be one choice for nucleic acid extraction and applicable for clinical automatic diagnostic system.( Chin J Lab Med,2012,35:843 -850 )

Study on the NKG2D receptor expression on the surface of natural killer cells in the patients with hepatitis B / 中华传染病杂志

Objective To investigate difference of NKG2D receptor expression level on the surface of natural killer (NK) cells in the patients with hepatitis B and its clinical significance.Methods This was a four-arm study with different types of subjects,including patients with chronic hepatitis B (CHB,n =22),HBV carriers (HBVC,n=10),patients with acute hepatitis B (AHB,n=18) and healthy donors (HD,n=18).NKG2D protein and mRNA levels on the surface NK cells in the peripheral blood were examined by reverse transcription-polymerase chain reaction assay.The relationship between NKG 2D expression and serum hepatitis B virus (HBV) DNA level was analyzed.The data were compared by analysis of variance and linear regression.Results NKG2D mRNA expression levels in groups of HBVC, HD, AHB and CHB were 0.96±0.17, 1.03±0.12,1.53±0.30 and 1.51 ± 0.35,respectively; the differences among groups were statistically significant (q=7.586,7.485,7.920 and 7.880,respectively; all P＜0.01).NKG2D protein expression levels in groups of AHB,HD,CHB and HBVC were 0.87±0.14,0.89±0.17,0.67±0.09 and 0.59±0.13,respectively; the differences among groups were statistically significant (q=6.92,7.67,7.53and 8.16,respectively; all P＜0.01).The NKG2D mRNA expression levels on NK cells were negatively correlated with serum HBV DNA viral loads in patients with CHB,AHB or HBVC (r=-0.75,-0.66 and-0.69,respectively; all P＜0.01).The NKG2D protein levels on NK cells from patients with AHB and CHB were negatively correlated with serum HBV DNA levels (r=-0.47 and -0.45,respectively; both P＜0.05).Conclusion NKG2D mediated NK cytotoxicity may play a role in viral clearance in hepatitis B.

Quantitation of hepatitis B virus total DNA, covalently closed circular DNA and HBsAg in patients with different stages of hepatitis B virus infection / 中华传染病杂志

Objective To quantitatively analyze total hepatitis B virus (HBV) DNA (HBV tDNA),covalently closed circular DNA (cccDNA) and HBsAg in patients with chronic hepatitis B (CHB),HBV-related liver cirrhosis (LC) and hepatocellular carcinoma (HCC),and to analyze the characteristics.Methods HBV tDNA and HBV cccDNA in the serum and liver biopsy samples were measured in 21 CHB,23 LC and 25 HCC patients by real-time polymerase chain reaction (PCR) assay. HBsAg titer was measured by chemiluminescence. Normally distributed variables among multiple groups were analyzed by ANOVA and t-test.Correlation between two variables was tested using Pearson correlation analysis.Skewed distribution was tested using Rank sum test.Results In CHB,LC and HCC patients,the serum HBV tDNA levels were (5.38±2.08),(4.96± 1.65) and (4.18 ± 0.91) lg copy/mL,respectively; the intrahepatic HBV tDNA levels in three groups were (7.18±1.91),(6.51±1.87) and (5.87± 1.47) lg copy/ug,respectively; the intrahepatic HBV cccDNA levels were (3.53±2.03),(2.63±2.13) and (0.58± 1.40) lg copy/μg,respectively; the serum HBsAg levels were (3.30±0.65),(3.12±0.52) and (2.60± 1.03) lg IU/mL,respectively.In CHB patients,the serum HBV tDNA,intrahepatic HBV tDNA,HBV cccDNA and HBsAg levels were all significantly higher than those of HCC patients (t=2.446,P=0.013; t=2.562,P=0.014;t=5.799,P＜0.01 ; t=2.709,P=0.003,respectively).However,only intrahepatic HBV cccDNA and HBsAg levels were statistically different between LC and HCC patients (t=-3.894,P＜0.01;t=-2.237,P=0.023,respectively).HBV cccDNA was all negative in the serum of 69 patients.The serum HBsAg level was positively correlated with serum HBV tDNA (r=0.290,P=0.016),intrahepatic HBV tDNA (r=0.372,P =0.002) and intrahepatic HBV cccDNA (r=0.378,P=0.001).Conclusions The levels of HBV tDNA,HBV cccDNA and HBsAg decrease gradually with the disease progression.The serum HBsAg level is positively correlated with serum HBV tDNA,intrahepatic HBV tDNA and intrahepatic HBV cccDNA.

The effects of hypericin on inhibition of hepatitis B virus replication in vitro / 中华传染病杂志

Objective To explore the effects of hypericin on inhibition of hepatitis 15 virus (HBV) replication.Methods The concentration gradient hypericin was added to HepG2.2.15 cell culture system and lamivudine was used as control.Enzyme-linked immunosorbent assay (ELISA) and Southern blot were used to examine HBsAg,HBeAg and HBV DNA level in the culture supernatant,respectively.Half inhibitory concentration (IC50) and half effective concentration (EC50) of hypericin were calculated.The effects of hypericin on HBV DNA polymerase were detected by 32p marked deoxy-ribonucleoside triphosphate as the substrate. Independent sample t-test and single factor analysis of variance were used to compare the data between two groups and among multiple groups.Results The inhibition rates of hypericin on HBsAg, HBeAg were enhanced with hypericin concentration increasing and those were higher than lamivudine control group when the concentration was higher than 0.5 μmol/L (t=-0.127,P＜0.05).Southern blot confirmed that hypericin was stronger in inhibition of HBV DNA (EC50=0.2 μmol/L) than lamivudine (t=-0.058,P＜0.05).Hypericin was non-toxic on HepG2.2.15 cells in the range of test with EC50 of 0.2 μmol/L and IC50 of 200 μmol/L.Hypericin did not act on HBV DNA polymerase which was quite different from lamivudine.Conclusions Hypericin can effectively inhibit HBV replication as well as antigen synthesis and is non-toxic on HepG2.2.15 cell.The anti-HBV target of hypericin is different from nucleos(t)ide analogues.

The clinical significance of quantitative detection of HBV DNA in the chronic infected patients / 中华检验医学杂志

In recent years,antiviral therapy for chronic hepatitis B infected patients has achieved great development along with the invention of nucleos (t) ide,nucleos (t) ide analogues,interferon α and pegylated interferon α.The development of antiviral medicine also proposes new demands for the clinical diagnosis and therefore promotes the development of laboratory diagnostic techniques in the detection of chronic hepatitis B.In this review,we focused on the clinical application in the quantitive detection of HBV DNA,and its significance on clinical evaluation,treatment options,follow-up and prognosis in antiviral therapy.

Comparison of HIV-1 DNA in different parts of early antiretroviral treatment patients / 中华检验医学杂志

ObjectiveTo study HIV-1 DNA levels in different parts of HIV patients during the early stage of antiretroviral therapy.MethodsThe peripheral blood,gut associated lymphoid tissues and lymph nodes samples were collected before and 12 weeks after treatment in regular follow-up HIV-1/AIDS patients in Beijing Youan Hospital ( n =11 ).The average age was 39 years old ( 25 to 55 ).Mononuclear Cells were isolated by density gradient centrifugation and then used DNA extraction kit to extract DNA.Realtime quantitative polymerase chain reaction was used to examine HIV-1 DNA copy-number.Non-parametric test was used to analyse the differences of HIV-1 DNA copy numbers among groups.Results Before treatment,HIV-1 DNA copy-number in both gut associated lymphoid tissues ( 10 714 ± 2043 ) copies/106 cells and lymph nodes (9145 ± 1202) copies/106 cells were higher than that in the peripheral blood (66 ± 8) copies/106 cells ( U =0.00,P ＜0.05 ),There was no significant difference between lymph nodes and gut associated lymphoid tissues (U =46.00,P ＞0.05).After 12 weeks of treatment,HIV-1 DNA copy-number in both gut associated lymphoid tissues (1701 ± 790) copies/106 cells and lymph node (11 591 ± 1781 ) copies/106 cells were higher than the peripheral blood ( 18 ± 3 ) copies/106 cells ( Z =- 2.934,P ＜ 0.05 ).There was a significant reduction of DNA copy-number in gut associated lymphoid tissues and peripheral blood after treatment (Z =- 2.934,P ＜ 0.05 ).Conclusion Gut associated lymphoid tissues and lymph nodes may be important latent reservoirs for HIV-1 DNA.

Influence factors of quantitative changes of dendritic cells in neonate born to HBsAg positive mother / 中华传染病杂志

Objective To investigate the influence factors of quantitative changes of dendritic cells (DC) in neonate born to HBsAg positive mother.Methods Sixty HBsAg positive mothers and their newborns were enrolled from the Third People's Hospital of Taiyuan from July 2011 to March 2012.The serum hepatitis B virus (HBV) markers and HBV DNA in mothers and newborns before vaccination were determined by chemiluminescence immunoassay (CLIA) and fluorescence quantitative polymerase chain reaction (PCR).The circulating frequencies of DC subsets were determined in the newborns by flow cytometry (FCM).The comparison of data was done by Mann-Whitney test and t test.The correlation analysis was done by Spearman rank correlation analysis and chi square test.Results Among 60 newborns,5 were HBsAg positive and HBV DNA negative.Among 60 HBsAg positive mothers,21 were HBeAg positive and 29 were HBV DNA positive.There was no significant quantitative difference of neonatal myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) between intrauterine infection group and intrauterine non-infection group (Z=-0.535,P=0.59 and Z=-0.027,P=0.98,respectively).However,mother's HBeAg positive status was closely related with neonatal HBeAg positive status (Pearson contingency coefficient was 0.928,P＜0.01).The frequencies of mDC in newborns born to HBeAg positive mothers were significantly lower than those born to HBeAg negative mothers (0.60±0.57 vs 0.87±0.58; Z=-2.085,P＜0.05).However,there was no significant quantitative differences of mDC and pDC between newborns born to HBV DNA positive mothers and born to negative mothers (Z=-1.272,P=0.20 and Z=-0.806,P=0.42,respectively).The frequencies of pDC were significantly lower in newborns born to mothers with HBV DNA＞ 1 × 107 copy/mL compared to newborns born to HBV DNA negative mothers (0.30±0.18 vs 0.64±0.55; t=-2.996,P=0.005).Conclusions HBeAg positive status of mothers may reduce neonatal frequencies of mDC.Neonatal frequencies of pDC may be reduced when the mothers' HBV DNA loads are more than 1 × 107 copy/mL.