Background Hepatitis C virus (HCV) infection is a public health issue for which an effective universal screening method is urgently needed. An oral anti-HCV test could provide a noninvasive and rapid screening strategy for HCV infection. This study evaluated the performance of a new point-of-care oral assay developed by Well for the detection of HCV antibody. Methods Individuals from three centers with and without HCV infection were enrolled. All participants were tested for oral HCV antibody using the Well assay and for serum HCV antibody using established tests (ARCHITECT i2000 anti-HCV assay and InTec serum anti-HCV assay). For participants who obtained positive results, HCV RNA was tested for verification. Some patients underwent the OraQuick HCV test at the same time, and some self-tested with the Well assay during the same period. Results A total of 1179 participants, including 486 patients with chronic HCV infection, 108 patients with other liver diseases, and 585 individuals who underwent physical examination, were enrolled. The Well anti-HCV test had a sensitivity of 91.88% (95% confidence interval [CI]: 88.97–94.09%) and a specificity of 98.00% (96.58–98.86%) for oral HCV antibody detection. The consistency between the Well and InTec assays was 97.02% (1138/1179). The consistency between the Well and OraQuick assays was 98.50% (197/200). Furthermore, the results of self-testing were highly consistent with those of researcher-administered tests (Kappa = 0.979). In addition, the HCV RNA results also showed that HCV RNA could only be detected on 1 of the 39 false-negative samples, and for 172 positive HCV RNA results, 171 could be detected by the Well oral anti-HCV assay. Conclusions The Well oral anti-HCV test offers high sensitivity and specificity and performed comparably to both the OraQuick assay and InTec assay for HCV diagnosis. Thus, the Well test represents a new tool for universal HCV screening to identify infected patients, particularly in regions with limited medical resources.

Natural killer (NK) cells play an important role in hepatitis B virus (HBV) infection control, and are regulated by a complex network of activating and inhibitory receptors. However, NK cell activity in HBV patients remains poorly understood. The objective of this study was to investigate the phenotypic and functional characteristics of circulating NK cells in patients during different chronic hepatitis B (CHB) infection stages. We investigated NK cell phenotypes, receptor expression and function in 86 CHB patients and 20 healthy controls. NK cells were purified and NK cell subsets were characterized by flow cytometry. Cytotoxic activity (CD107a) and interferon-gamma (IFN-γ) secretion were examined, and Natural Killer p46 (NKP46) blockade and spontaneous NK cell cytolytic activity against K562, HepG2 and HepG2.215 cell lines was studied. Activating NKp46 receptor expression was higher in inactive HBsAg carriers when compared with other groups (p = 0.008). NKp46 expression negatively correlated with HBV DNA (R = -0.253, p = 0.049) and ALT (R = -0.256, p = 0.045) levels. CD107a was higher in immune-activated groups when compared with immune-tolerant groups (p = 0.039). CD107a expression was related to viral load (p = 0.02) and HBeAg status (p = 0.024). In vitro NKp46 blockade reduced NK cell cytolytic activity against HepG2 and HepG2.215 cell lines (p = 0.02; p = 0.039). Furthermore, NK cells from high viral load CHB patients displayed significantly lower specific cytolytic activity against anti-NKp46-loaded K562 targets (p = 0.0321). No significant differences were observed in IFN-γ secretion (p > 0.05). In conclusion, NKp46 expression regulates NK cell cytolytic function. NKp46 may moderate NK cell activity during HBV replication suppression and HBV-associated liver damage and may be critical for NK cell activity during CHB infection.

BackgroundHepatitis B virus (HBV) and hepatitis C virus (HCV) co-infections contributes to a substantial proportion of liver disease worldwide. The aim of this study was to assess the clinical and virological features of HBV-HCV co-infection.MethodsDemographic data were collected for 3238 high-risk people from an HCV-endemic region in China. Laboratory tests included HCV antibody and HBV serological markers, liver function tests, and routine blood analysis. Anti-HCV positive samples were analyzed for HCV RNA levels and subgenotypes. HBsAg-positive samples were tested for HBV DNA.ResultsA total of 1468 patients had chronic HCV and/or HBV infections. Among them, 1200 individuals were classified as HCV mono-infected, 161 were classified as HBV mono-infected, and 107 were classified as co-infected. The HBV-HCV co-infected patients not only had a lower HBV DNA positive rate compared to HBV mono-infected patients (84.1% versus 94.4%, respectively; P<0.001). The median HCV RNA levels in HBV-HCV co-infected patients were significantly lower than those in the HCV mono-infected patients (1.18[Interquartile range (IQR) 0-5.57] versus 5.87[IQR, 3.54-6.71] Log(10) IU/mL, respectively; P<0.001). Furthermore, co-infected patients were less likely to have detectable HCV RNA levels than HCV mono-infected patients (23.4% versus 56.5%, respectively; P<0.001). Those HBV-HCV co-infected patients had significantly lower median HBV DNA levels than those mono-infected with HBV (1.97[IQR, 1.3-3.43] versus 3.06[IQR, 2-4.28] Log(10) IU/mL, respectively; P<0.001). The HBV-HCV co-infection group had higher ALT, AST, ALP, GGT, APRI and FIB-4 levels, but lower ALB and total platelet compared to the HBV mono-infection group, and similar to that of the HCV mono-infected group.ConclusionThese results suggest that co-infection with HCV and HBV inhibits the replication of both viruses. The serologic results of HBV-HCV co-infection in patients suggests more liver injury compared to HBV mono-infected patients, but is similar to HCV mono-infection.

Nucleoside/tide analogs are now widely used for treatment of chronic hepatitis B (CHB). However, available nucleoside/tide analogs for treatment of CHB usually have limited therapeutic effect and potential adverse effects on CHB patients. We evaluated the anti-HBV effects of three novel derivatives (compounds 1 , 4 , and 8 ) of adefovir on the replication of hepatitis B virus (HBV) and determined their cytotoxicity. The effects of those compounds on the replication of human HBV in the HepG2 2.2.15 cell line and duck HBV in infected ducks were characterized by measuring the extracellular and intracellular HBV DNA using the quantitative real-time PCR and Southern blot assays. Their cytotoxicities against HepG2 cells were evaluated by MTT and measuring the contents of mitochondrial DNA using the dot blot assay. We found that all of the compounds inhibited the production of HBV in a dose-dependent manner, similar to that of adefovir dipivoxil. Furthermore, treatment with any of the compounds reduced significantly the replication of DHBV in ducks, accompanied by a significant reduction of inflammation in the livers, and the compound 1 appeared to be a fast-acting and long-lasting anti-DHBV reagent. Importantly, all of the compounds showed little cytotoxicity against HepG2 cells, even at a concentration of 1 mM. Collectively, those novel derivatives of adefovir had potent anti-HBV activity with little adverse effect and may be therapeutic candidates for potential clinical studies.

目的 评价国产试剂的实时荧光定量聚合酶链反应(PCR)在检测乙型肝炎病毒(HBVDNA )中的临床应用.方法 基于实时荧光定量应用瑞士罗氏公司的cobastaqman-48 仪器和美国应用生物工程公司ABI-7300 全自动系统,用国产试剂和进口试剂分别检测乙型肝炎患者血清中HBVDNA 含量,比较国产试剂与进口试剂的偏倚及两者的相关性.结果 进口试剂与国产试剂两次结果比较有显著差异(P ＜ 0.001),国产两次结果比较无差异(P ＞ 0.05).结论 本实验的实时PCR 仪间的检测值有一定的差异,提示不同型号仪器的检测值不能相互取代,对于同一患者的HBV DNA 定量监测及对抗病毒治疗疗效评价时,必须用同一型号的检测仪器(系统).

BACKGROUND/AIMS:Increasing evidence suggests an association between elevated serum aminotransferase levels and metabolic disorders (metabolic syndrome, hyperlipidemia and diabetes mellitus). However, the significance of relatively low levels of aminotransferases in relation to metabolic disorders has not been fully investigated in the general population. We investigated the association between serum aminotransferase levels and metabolic disorders using data from a survey in Jilin Province, China. MATERIALS AND METHODS:In 2007, a prospective survey was conducted throughout Jilin, China, covering both urban and rural areas. A total of 3835 people, 18-79 years old, were undergoing real-time ultrasonography, blood tests, and interviews with a structured questionnaire. RESULTS:Serum aminotransferase levels within the normal range were associated with metabolic syndrome independent of age, occupation, cultural and educational level, income, body mass index, waist circumference, smoking, and alcohol intake. Compared with the lowest level (< 20 IU/L), the adjusted odds ratios for alanine aminotransferase levels of 20-29, 30-39, 40-49, and >50 IU/L were 1.92, 2.50, 2.97, and 3.52 in men, and 1.38, 1.54, 3.06, and 2.62 in women, respectively. Near-normal serum aminotransferase levels associated with hyperlipidemia, non-alcoholic fatty liver disease, and diabetes mellitus were also found in the study. CONCLUSIONS:Normal to near-normal serum aminotransferase levels are associated with metabolic disorders. Serum alanine aminotransferase levels of 21-25 IU/L for men and 17-22 IU/L for women are suggested as cut-off levels that detect metabolic disorders affecting the liver.

BACKGROUND:Chronic liver diseases are a major burden in China. Alanine aminotransferase (ALT) can be used as an indicator of hepatocyte damage. In this study, we determined the prevalence and etiologies of elevated ALT in an adult population in Jilin, China.METHODS:A total of 4072 individuals aged between 18 and 79 years were first interviewed, and then underwent ultrasonography and blood tests.RESULTS:The prevalence of elevated ALT was 17.53%. The most noticeable risk factor for ALT elevation was non-alcoholic fatty liver disease (NAFLD) (accounting for 10.79%), metabolic syndrome (16.25%), or both (20.31%). The development of NAFLD occurred mostly in female peasants and small businessmen with increased income, age, fasting plasma glucose, body mass index, triglyceridemia, and low-density lipoprotein and decreased education level, high-density lipoprotein. Elevated ALT frequently occurred in low education level, male peasants and small businessmen with increased income, body mass index and triglyceride who had NAFLD and/or metabolic syndrome. However, elevated ALT with infection of hepatitis B or C virus was not associated with metabolic disorders, but rather with gender, occupation and increased age.CONCLUSION:The results from the current study demonstrate that elevated ALT is fairly high in the Northeast population (17.53%) and that the cause of its elevation is mostly due to NAFLD and metabolic syndrome.

Objective: To study the activities of anti-duck hepatitis B virus (anti-DHBV) of three novel nucleoside analogues and their effects on histomorphology of duck liver, and to explore novel potential anti-HBV agents. Methods: The anti-DHBV activities were analyzed by fluorescent quantitative PCR in experimental groups with various doses of 030703, 030605, 030705 (2, 10 and 50 mg·kg -1) and adefovir dipioxil control group (10 mg·kg -1). Compounds were taken by oral administration per day and last 30 d. The serum specimens were obtained at the time of before adminstration, 15 d and 30 d after administration and 2 weeks after withdraw. The serum DHBV DNA was detected with fluorescence quantitative PCR. At the same time, DHBV DNA positive and negative groups were set up. The duck liver specimens were obtained after experiment. The histomorphological changes of duck liver caused by three tested compounds were observed under optic microscope. Results: After administration with compound 030703, there were 3 ducks (60%) in high dose group, 2 ducks (40%) in middle dose group, 1 duck (20%) in low dose group, in which the DHBV DNA contents were decreased to 1/3 of primary contents; compared with adefovir dipioxil control group, the inhibitory effects in high dose group had significant difference (P<0.01), whereas the inhibitory effects in middle and low dose groups had no significant difference (P>0.05). After administration with compound 030605, there were 4 ducks (80%) in high dose group, 3 ducks (60%) in middle dose group, 1 duck (20%) in low dose group, in which the DHBV DNA contents were decreased to 1/3 of primary contents; compared with adefovir dipioxil control groups, the inhibitory effects in high dose group and middle group had significant difference (P<0.01), whereas the inhibitory effect in low dose group had no significant difference (P>0.05). After administration with compound 030705, there were 3 ducks (60%) in high dose group, 2 ducks (40%) in middle dose group, 0 duck (0%) in low dose group, in which the DHBV DNA contents were decreased to 1/3 of primary contents; compared with adefovir dipioxil control groups, the inhibitory effects in high dose group had significant difference (P<0.01), whereas the inhibitory effects in middle and low groups had no significant difference (P>0.05). Compared with adefovir dipioxil control group, the effects of all three test compounds (high, middle and low dose groups) on duck liver inflammation were similar to that in adefovir dipioxil control group. Conclusion: Three novel test compounds 030703, 030605 and 030705 have obvious anti-DHBV activities and might improve duck liver inflammation to some extent. It has been suggested that all of these novel compounds be potential anti-HBV agents.

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is a serious health concern in China. The goal of this cross-sectional study was to determine the prevalence of NAFLD and identify the risk factors associated with this disease in Northern China. METHODS In 2007, a total of 6063 adults from Dehui, a city in Northern China, were surveyed and demographic and social-economic characteristics, life behaviors, and medical history were recorded. Among them, 3850 subjects were randomly selected for physical examination, fasting plasma glucose (FPG) test, fasting lipid and liver function profiles, hepatitis B and C infection screening, and ultrasound examination. The frequency of NAFLD in this population was analyzed by the Chi-square test and the association of potential risk factors was analyzed by logistic regression. RESULTS The prevalence of NAFLD was 15.9% in this population and the prevalence in females was significantly higher than that in males, particularly for the elderly subgroup. Obesity, hypertension, FPG, diabetes, and metabolic syndrome (MS)-related hyperlipidemia were significantly associated with NAFLD. The data indicate that MS-related multiple risk factors synergistically increase the risk for NAFLD. CONCLUSION The prevalence of NAFLD is high in Northern China, which may be associated with the high incidence of diabetes, hypertension, and MS in this area.

Background and aim: The prevalence of the hepatitis B virus (HBV) is higher in adults than in children. We determined the seroepidemiology of HBV infection in an adult population in JiLin, China, to guide effective preventive measures.Methods: A cross-sectional serosurvey was conducted throughout JiLin, China. A total of 3833 people was selected and demographic and behavioral information gathered. Serum samples were tested for HBV markers and liver enzymes.Results: The prevalence of the hepatitis B surface antigen (HBsAg), the antibody to the hepatitis B surface antigen (anti-HBs), the hepatitis B e antigen (HBeAg), the antibody to HBeAg (anti-HBe), and the antibody to the hepatitis B core antigen (anti-HBc) were 4.38%, 35.66%, 1.38%, 6.65%, and 40.88%, respectively. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly higher among HBsAg (+) than HBsAg (-) subjects. By multivariate logistic regression analysis, independent predictors for chronic HBV infection were smoking, poor sleep quality; occupation as private small-businessmen, laborers, or peasants; male gender; family history of HBV; personal history of vaccination; and older age. Independent predictors for exposure to HBV were large family size, occupation as a private small-businessman, male gender, family history of HBV, personal history of vaccination, and older age. Independent predictors for immunity by vaccination were occupation as a private small-businessman, high income, personal history of vaccination, and young age. Independent predictors for immunity by exposure were drinking, male gender, personal history of vaccination, and older age.Conclusions: The prevalence rate of HBV infection (4.38%) was lower than the previous rate of general HBV vaccination. However, 44.59% of the population remained susceptible to HBV. The prevalence of HBV infection was high in young adults, private small-businessmen, peasants, those with a family history of HBV, and males. Therefore, immunization of the non-immune population is reasonable to reduce hepatitis B transmission between adults.

BACKGROUND:Serum biochemical liver tests (LTs) (alanine aminotransferase, aspartate aminotransferase, and gamma glutamyltransferase) and platelet counts are often used to screen for chronic liver disease. We determined the prevalence and etiologies of abnormal LTs in an adult population in Jilin, China.METHODS:A total of 3791 individuals between the ages of 18 and 79 years were interviewed and then underwent ultrasonography and blood tests.RESULTS:The prevalence of abnormal LTs was 14.77% (560 out of 3791 subjects). The risk factors for abnormal LTs were non-alcoholic fatty liver disease (NAFLD) alone, which accounted for 11.61%, metabolic syndrome alone for 25%, or both for 22.14%. Abnormal LTs were more common in male than in female subjects. The development of abnormal LTs was correlated with older age males, increased daily alcohol intake, poor quality of sleep, smoking, fasting plasma glucose, body mass index, triglyceridemia, and low-density lipoprotein. Abnormal LTs in patients with metabolic syndrome and NAFLD were associated with high fasting plasma glucose, triglycerides, body mass index, low density lipoprotein, male, young age, poor sleep quality, smoking, and alcohol intake. However, abnormal LTs in patients with hepatitis B virus were associated with gender and increased age.CONCLUSIONS:The results from the current study demonstrated that the prevalence of abnormal LTs is high in the population (14.77%). Metabolic syndrome, NAFLD, and alcohol intake appear to be potentially important causes of the observed abnormal LTs.

No specific treatment can reverse the liver injury in cirrhosis. This study aims to characterize immune status and correlations between cirrhosis induced by HBV and HCV. Phenotypes of peripheral blood lymphocyte subsets (T, NK, regulatory T cells) and Th cytokine secretion were analyzed using flow cytometry in 42 HBV-cirrhotic and 40 HCV-cirrhotic patients. Cirrhotic patients had a lower proportion of CD3 + CD8 + T cells and NK cells, while the proportion of CD3 + CD4 + T cells and Treg cells were higher than those of healthy controls. The levels of Th2 cytokine (IL-6) in cirrhotic patients were increased, while only the Th1 cytokine (IFN-γ) increased in HBV-cirrhotic patients. These findings show that there is no difference between the cirrhotic groups except in the IFN-γlevel. In cirrhosis, defects in innate, adaptive immune cells are likely regardless of which virus is involved. A cytokine imbalance may play a role in the development of posthepatitic cirrhosis.

Objective To explore the association between IL-28B genetic variation,susceptibility and spontaneous clear-ance of hepatitis C virus.Methods A total of 411 hepatitis C patients(255 male and 186 female) and 100 healthy con-trols were enrolled in the study.The rs12979860 SNP of IL-28B was identified from each cohort using pyrosequencing technology.HCV-RNA was tested in hepatitis C patients.The IL28B genetic variation was compared between hepatitis C patients and healthy controls,and chronically infected and spontaneously cleared HCV groups to assess the effect of SNP.Results The minor allele frequency(rs12979860) of Chinese population was 6%.There was no genotype and allele fre-quency(rs12979860) difference between hepatitis C patients and healthy controls(P = 0.066,P = 0.072).The level of HCV-RNA in genotype CC was significantly higher than genotype CT groups(P = 0.02).There was no significant differ-ence between genotype CC and CT among individuals who spontaneously clear HCV(P = 0.64).There was no significant difference in SNP genotype(rs12979860) between chronically infected groups and healthy controls.Conclusion The SNP genotype rs12979860 may not be the predisposing gene of hepatitis C and the CC genotype cannot predict the course of spontaneous clearance of hepatitis C.

Aim: agrin plays a critical role in myoneural junction formation and maintenance, and also regulates neurite formation, elongation, and maintenance. agrin expression products distribute extensively in body. In this study, we designed a perfect Real-Time PCR method to quantitate rat agrin mRNA and test the specificity, sensitivity and repeatability of this method. Methods: The experiment was performed at the Third Combined Laboratory of Jilin University and the Laboratory of Department of Infectious Disease of Jilin University from April to November 2007. The perfect Real-Time PCR primers to detect rat agrin mRNA and rat ACTB mRNA were designed by Primer3 software. After the specificity of these primers was proved in Blast, the primer to construct standard preparation of rat agrin mRNA was designed by Primer5 software. Wistar rats (clean grade) were selected and mated to get neonate rat. After operation, brain tissue of 3-day neonate rat was used for mRNA harvest. The reverse transcription-polymerase chain reaction was performed to obtain 554 bp DNA fragment of rat agrin. After T7 promotor was connected to rat agrin DNA by PCR, standard preparation of rat agrin mRNA was constructed by T7 RNA Polymerase. DNA was digested and the concentration of standard preparation of rat agrin mRNA was detected by spectrophotometer. Rat agrin mRNA was diluted by multiproportion of 1 : 5 for 3 times, and standard preparation of rat agrin cDNA was obtained by reverse transcription reaction. The standard preparation of rat ACTB mRNA was constructed by reverse transcription reaction while the perfect Real-Time PCR primer was used. mRNA was diluted by multiproportion of 1: 5 for 3 times, and standard preparation of rat ACTB cDNA was obtained. Reaction conditions were confirmed and optimized using PCR proceeding. The standard preparations were sequenced to test theirs specificity. Dependablity of standard curve, specificity of dilapsus curve and repeatability of the method were detected. Results: 1The primer of Real-Time PCR was set: Rat agrin mRNA sense GAA GGC CAG GTG CGA ATC A, antisense CAC AGG TAG CAG TGG AGC CAA G; internal standard: Rat ACTB mRNA sense GGA GAT TAC TGC CCT GGC TCC TA, antisense GAC TCA TCG TAC TCC TGC TTG CTG. Optimized PCR proceed was an initial denaturation cycle of 5 seconds at 95 °C, followed by 40 cycles (5 seconds at 95 °C, 20 seconds at 60 °C and 20 seconds at 72 °C). 2The rate of dependablity about the standard curves of rat agrin mRNA and rat ACTB mRNA >0.95. 3Electrophoresis, sequencing result of PCR products and dilapsus curve of Real-Time PC verified the specificity of the method. The repeatability of the method was good, and the sensitivity met the requirements. Conclusion: The perfect Real-Time PCR method to quantitate rat agrin mRNA is tested and can be used as a standard method.

Aim: Platelet-derived growth factor (PDGF)-A plays an important role in leading to proliferative vitreous retinopathy through inducing the migration of retinal pigment epithelium. This study was designed to establish the recombinant plasmid of small hairpin interfering RNA (shRNA) against human PDGF-A and observe silence effect of the human PDGF-A expression. Methods: The experiment was conducted at the Infection Laboratory of the First Affiliated Hospital of Jilin University from April to July in 2007. Recombinant plasmid was constructed based on human PDGF-A mRNA sequence. Three pairs of oligo nucleotides were synthesized and inserted into plasmid pGPU6/GFP/Neo to generate shRNA eukaryotic expression vectors. Interfering plasmid from GAPDH were established as positive control and interfering plasmid targeting none gene served as negative control. The recombinant were verified by enzyme digestion and sequencing. Results: The recombinant plasmid sequence was the same as the sequence of shRNA transcribed sequence. So the recombinant vectors were established successfully. Conclusion: The shRNA expression vector targeting human PDGF-A can be established successfully by RNA interference technique.