戊型肝炎病毒IgG抗体诊断试剂盒的研制及初步应用

Objective To establish an EliSA kit for serum anti-hepatitis E virus IgG detection using recombinant antigen expressed in E. Coli. Methods Utilizing the recombinant protein ORF23 of REV expressed in E. Coli as a coating antigen, the ELISA kit for detecting serum anti-HEV IgG was established. The serum samples from 100 HEV patients and 150 negative serum samples were measured, and the detecting results was compared with those of other two HEV kits(the kits from Genelabs company and Wantai Corporation). The specificity, sensitivity and stability of the kit were evaluated. The serum samples from another 500 clinical hepatitis patients were detected to evaluate the clinical application value of this kit. Results Both the specificity and sensitivity of the kit were 100% and the consistency rates to other kits were more than 95%. The kit showed a good stability and could be stable for at least 6 months under 4 ℃. 500 samples were 145 positive in 150 hepatitis E patients, the consistency rates were 96.67% . Conclusions The ELBA kit prepared with HEV recombinant antigen has good specificity and sensitivity for detecting anti-HEV IgG in serum, and may have potential evaluation in clinical diagnosis and epidemiological investigation for hepatitis E.     

肝组织中HBV cccDNA荧光定量聚合酶链反应检测法的建立

Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.     

肝组织中HBV cccDNA荧光定量聚合酶链反应检测法的建立

Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.     

脑血管疾病患者血清神经元特异性烯醇化酶水平变化及临床意义

To explore the application of NSE in the serum in the diagnosis of the cerebrovascular disease.Methods Serum levels of NSE from 195 patients with cerebrovascular disease and normal controls were detected with ELISA. The blood samples from the patients were acquired within 3 days after the outbreak of cerebrovascular disease and taken again after 2 weeks from 148 of all cases. The result of detecting of NSE was analysed in comparison with and the level of NSE in the CSF. Result There was significance difference between the level of NSE in the serum of patient with cerebrovascular diseases and that of normal controls when the Analysis of Variance was used to analyze the value of measurement. The same outcome was also got using ANOVA to analyze the different test group with the blood samples were acquired within 3 days. ( F = 24.94, P = 0. 000). But in the other hand, there was no evidently different in the different group made up patients if LSD was put in use instead of the ANOVA.Corelation analysis of NSE concentration was done between the in the blood samples in 3 days and those 2 weeks later (r = 0.884, t = 37.904, p = 0.000), NSE concentration in the blood samples and those in the CSF(r = 0.969, t = 8. 695, p = 0. 000), and the outcome show that there was difference between them.The same comparison between NSE concentration was done between the in the blood samples in 3 days and Head CT(r=O.857,p =O.000),Nerve Function Defect(r=0.848,p=0.000).Concluslon Thelevels of NSE in sera of patients with eerebrovascular disease were positive related with the degree of Cerebral infarction or volume of Hematoma shown by Head CT. Therefore, it would contribute to diagnosis of cerebrovascular disease if the detecting of NSE was applied in clinical laboratory.     

华东和华南地区商品猪戊型肝炎病毒携带率和基因分型分析

Objective To determine the prevalence and genotype of hepatitis E virus (HEV)among commercial swine population in Eastern and Southern China. Methods Six hundred specimens of swine bile collected from 5 slaughterhouses in Eastern and Southern China from 2007 to 2009 were tested for HEV RNA using nested RT-PCR. PCR products were sequenced for phylogenetic analysis. Results Forty-seven out of the 600 samples (7.83%) were positive for HEV RNA. Based on the 150 nt fragment within HEV ORF2, data from phylogenetic analysis revealed that all the 47 HEV isolates were identified to be genotype Ⅳ, sharing 75.0%-83.4%, 75.0%-84.6%, 71.9%-80.7% and 88.1%-91.5% nucleotide identities with prototype Ⅰ,Ⅱ,Ⅲ and Ⅳ HEV strains respectively while majority of the isolates clustered within their respective isolation sites. Conclusion HEV was widespread in commercial swine population in Eastern and Southern China that raised a serious concern about the safety regarding the consumption of pork products.     

华东和华南地区商品猪戊型肝炎病毒携带率和基因分型分析

Objective To determine the prevalence and genotype of hepatitis E virus (HEV)among commercial swine population in Eastern and Southern China. Methods Six hundred specimens of swine bile collected from 5 slaughterhouses in Eastern and Southern China from 2007 to 2009 were tested for HEV RNA using nested RT-PCR. PCR products were sequenced for phylogenetic analysis. Results Forty-seven out of the 600 samples (7.83%) were positive for HEV RNA. Based on the 150 nt fragment within HEV ORF2, data from phylogenetic analysis revealed that all the 47 HEV isolates were identified to be genotype Ⅳ, sharing 75.0%-83.4%, 75.0%-84.6%, 71.9%-80.7% and 88.1%-91.5% nucleotide identities with prototype Ⅰ,Ⅱ,Ⅲ and Ⅳ HEV strains respectively while majority of the isolates clustered within their respective isolation sites. Conclusion HEV was widespread in commercial swine population in Eastern and Southern China that raised a serious concern about the safety regarding the consumption of pork products.     

组氨酸脱羧酶、肠脂肪酸结合蛋白和二胺氧化酶在肠梗阻患者肠黏膜损伤诊断中的价值

Objective To investigate the clinical values of serum histidine decarboxylase(HDC),intestinal fatty acid binding protein(I-FABP),and diamine oxidase(DAO)for diagnosing intestinal mucosal injury (IMI)in patients with intestinal obstruction.Methods The expression levels of serum HDC,I-FABP,and DAO in 28 patients with strangulated intestinal obstruction,19 patients with simple intestinal obstruction,17 patients with acute simple appendicitis,and 20 healthy control were determined by enzyme-linked immunosorbent assay (ELISA)before clinical treatment,and then the areaa under receiver operating characteristic curves(AUC)of these diagnostic indicators were compared.In addition,the incidences of systemic inflammatory response syndrome (SIRS)and infectious complications were closely observed.The difference of the expressions of HDC,I-FABP,and DAO and their relationship with SIRS and infectious complications were compared among these patients and controls.Results The expression levels of serum HDC, I-FABP, and DAO were the highest in patients with strangulated intestinal obstruction (all P < 0.001), and the expression levels of these three indicators were significantly higher in patients with simple intestinal obstruction than in those with acute simple appendicitis or healthy controls (all P<0.05).The AUC of HDC (0.913) was significantly larger than that of I-FABP (0.877, P =0.000) and DAO (0.873, P = 0.000).When the cut-off value of HDC ≥31.00 ng/ml, the sensitivity, specificity, false negative rate, and false positive rate of HDC were 74.5% , 94.6% , 25.5% , and 5.4% , respectively,which were all better than those of I-FABP and DAO.There were significant differences of the incidence of SIRS ( P = 0.046) and abdominal infection (P = 0.027) among patients with strangulated intestinal obstruction, patients with simple intestinal obstruction, and patients with acute simple appendicitis, while lung infection showed no such significant difference (P = 0.728).The expression level of serum HDC was significantly higher in patients with strangulated intestinal obstruction who were also suffered from SIRS ( P = 0.000) or abdominal infection ( P =0.002) than that of uninfected patients.Meanwhile, the expression levels of serum I-FABP and DAO were significantly higher in the SIRS patients with strangulated intestinal obstruction than that of uninfected patients ( P = 0.027, P=0.017, respectively).The expression levels of HDC, I-FABP, and DAO were significantly correlated with the incideces of SIRS and abdominal infection ( all P < 0.05 ) , among which the level of HDC and the incidence of SIRS had the highest correlation (R = 0.608, P = 0.001).Conclusion HDC can be an effective indicator for diagnosing IMI in patients with intestinal obstruction.     

自身免疫性肝炎与病毒性肝炎的临床病理特点对照研究

Objective To explore the character of clinical manifestations and liver pathological features of autoimmune hepatitis (AIH) and improve the diagnostic accuracy in early stage of AIH patients. Methods 78 patients with AIH,31 patients with acute viral hepatitis(AH) and 31 patients with chronic hepatitis B (CHB) were collected. The liver function and immunoglobulins were compared among them as well as the comparison of chronic and acute onset AIH groups. A part of patients underwent liver, biopsy and HBsAg immunohistochemistry examination. Results AIH patients were dominated by female (94.87%) over 40 years old (78.21%). The level of AST/ALT, γ-glutamyl transferase (CGT), alkaline phosphatase(ALP), IgG, IgM and γ-globulin in AIH group were significantly higher than those in CHB and AH groups. Furthermore, the level of γ-globulin in chronic AIH group was higher than that in acute onset AIH group (P< 0.05). The positive rate of smooth muscle antibody(SMA) was 6.41%, and the positive rate of anti-nuclear antibody(ANA) was 79.49% in AIH patients. There was no difference in the titer of ANA between acute and chronic AIH groups(P> 0.03). Regarding the appearance of liver pathology, it was easy to distinguish AIH from AH, but difficult to differentiate AIH from CHB. liver tissues, loose degeneration, spotty necrosis and piecemeal necrosis accompanying with bridging necrosis were very common in AM. There were great numbers of lymphocyte infiltration in portal area that was often accompanied with varying degrees of fibrosis. The degree of hepatic inflammation and fibrosis were similar in acute and chronic AIH ( P > 0.05). Ihe degree of hepatic lesions in 2 AIH patients suffered with subacute liver failure didn' t agree with the severe clinical manifestations. Conclusions AIH patients are dominated with female over 40-year-old who often accompanied with elevation of AST/ALT, GGT, ALP and y-globulin as well as the appearance autoantibodies. The appearance of liver pathology is multiplicity that piecemeal of necrosis accompanying with bridging necrosis and lymphocytes infiltration is common. The degree of hepatic lesions is similar in aeute and chronic AIH. It's important to check the liver tissue for diagnose in early stage correctly.     

自身免疫性肝炎与病毒性肝炎的临床病理特点对照研究

Objective To explore the character of clinical manifestations and liver pathological features of autoimmune hepatitis (AIH) and improve the diagnostic accuracy in early stage of AIH patients. Methods 78 patients with AIH,31 patients with acute viral hepatitis(AH) and 31 patients with chronic hepatitis B (CHB) were collected. The liver function and immunoglobulins were compared among them as well as the comparison of chronic and acute onset AIH groups. A part of patients underwent liver, biopsy and HBsAg immunohistochemistry examination. Results AIH patients were dominated by female (94.87%) over 40 years old (78.21%). The level of AST/ALT, γ-glutamyl transferase (CGT), alkaline phosphatase(ALP), IgG, IgM and γ-globulin in AIH group were significantly higher than those in CHB and AH groups. Furthermore, the level of γ-globulin in chronic AIH group was higher than that in acute onset AIH group (P< 0.05). The positive rate of smooth muscle antibody(SMA) was 6.41%, and the positive rate of anti-nuclear antibody(ANA) was 79.49% in AIH patients. There was no difference in the titer of ANA between acute and chronic AIH groups(P> 0.03). Regarding the appearance of liver pathology, it was easy to distinguish AIH from AH, but difficult to differentiate AIH from CHB. liver tissues, loose degeneration, spotty necrosis and piecemeal necrosis accompanying with bridging necrosis were very common in AM. There were great numbers of lymphocyte infiltration in portal area that was often accompanied with varying degrees of fibrosis. The degree of hepatic inflammation and fibrosis were similar in acute and chronic AIH ( P > 0.05). Ihe degree of hepatic lesions in 2 AIH patients suffered with subacute liver failure didn' t agree with the severe clinical manifestations. Conclusions AIH patients are dominated with female over 40-year-old who often accompanied with elevation of AST/ALT, GGT, ALP and y-globulin as well as the appearance autoantibodies. The appearance of liver pathology is multiplicity that piecemeal of necrosis accompanying with bridging necrosis and lymphocytes infiltration is common. The degree of hepatic lesions is similar in aeute and chronic AIH. It's important to check the liver tissue for diagnose in early stage correctly.     

Diagnostic value of vascular endothelial growth factor combined with β-human chorionic gonadotropin for suspected ectopic pregnancy

Objective To explore the diagnostic values of vascular endothelial growth factor (VEGF) and p -human chorionic gonadotropin( β -hCG) for early ectopic pregnancy. Methods The serum levels of VEGF and β-hCG were measured in 109 patients with suspected ectopic pregnancy by ELISA and RIA respectively, receiver operating characteristic (ROC) curvers were established, the diagnostic values of VEGF and β -hCG for ectopic pregnancy were analyzed. Results One hundred and nine patients with suspected ectopic pregnancy were diagnosed in the end: 62 cases with ectopic pregnancy and 47 cases with intrauterine pregnancy. The levels of VEGF in ectopic pregnancy were significantly higher than those in intrauterine pregnancy (P ＜ 0.05), but the levels of β -hCG were significantly lower than those in intrauterine pregnancy (P ＜ 0.05). The areas under ROC curves of VEGF were 0.87, the areas under ROC curves of β -hCG were 0.71. In VEGF ＞ 140 ng/L for diagnosis of ectopic pregnancy, the sensitivity and specificity were 75.8% (47/62) and 80.9% (38/47) respectively. In β-hCG ＜ 1200 U/L for diagnosis of ectopic pregnancy, the sensitivity and specificity were 64.5%(40/62) and 61.7%(29/47) respectively. The sensitivity and specificity of combined detection of VEGF and β -hCG were 80.6%(50/62) and 57.4%( 27/47) respectively. Conclusions The diagnostic values of VEGF are better than those of β -hCG. The combined detection of VEGF and β -hCG can increase the rate of early diagnosis and shorten observation time.