Objective:To explore the influencing factors of BCP region A1762T/G1764A and Pre-C region G1896A mutations in patients with chronic hepatitis B virus（HBV）infection.Methods:Clinical data of 464 patients with chronic HBV infection admitted to Ningbo No.2 Hospital from October 2010 to August 2022 was retrospectively analyzed. The mutations in the BCP region A1762T/G1764A and the pre-C region G1896A were examined in all patients. Logistic regression was used to analyze the influencing factors of these mutations.Results:The mutation rate of A1762T/G1764A and G1896A was 62.1%（288/464）and 32.8%（152/464）；and the co-mutation rate of A1762T/G1764A and G1896A was 20.9%（97/464）. The mutation rate of A1762T/G1764A and the co-mutation rate of A1762T/G1764A and G1896A in patients with end-stage liver disease were higher than those in patients with chronic hepatitis B（ χ2=9.285 and 6.748，both P<0.05）. Univariate analysis showed that A1762T/G1764A mutation was associated with higher age，serum AST levels，and proportion of genotype C（ P<0.05 or <0.01）；while G1896A mutation was associated with higher age，proportion of negative HBeAg，and serum GGT levels（ P<0.05 or <0.01）. Multivariate logistic regression analysis indicated that higher age was an independent influencing factor for A1762T/G1764A mutation（ OR=1.035，95% CI 1.017-1.054， P<0.001），while HBeAg status was an independent influencing factor for G1896A mutation（ OR=0.171，95% CI 0.112-0.261， P<0.001）. Conclusion:Chronic HBV infection patients with higher age is likely to have A1762T/G1764A mutation，while those with negative HBeAg is more likely to have G1896A mutation.

Objective:To analyze the liver pathology, clinical characteristics and influence factors in patients with chronic hepatitis B virus (HBV) infection in immune tolerant phase (IT).Methods:The clinical data of 273 patients in IT phase who underwent liver biopsy from January 2015 to December 2019 were included in this study. The correlation between liver pathological changes and clinical features was analyzed.Results:There were 43 cases (15.75%) with liver histologic activity ≥ G2, 30 cases (10.99%) with liver fibrosis ≥ S2, and 55 cases (20.15%) with liver pathology ≥ G2 and/or ≥ S2. A total of 17.95% patients had liver steatosis. The majority (98.17%) of tissue samples were positive for HBsAg staining, while only 79.49% were positive for HBcAg. The characteristics of liver pathology were comparable in men from women patients. The differences of G and S were not statistically significant according to different HBsAg positivity, while those were statistically significant according to different HBcAg positivity. By univariate and multivariate analysis, the independent risk factors of pathological severity were HBcAg intensity, HBeAg level, and age. However, the differences of liver histologic activity and fibrosis were not statistically significant between those younger than 30 years old group from those older than 30 years old, neither between those younger or older than 40. Although the diagnostic value of liver inflammation and fibrosis 5 (LIF-5) was better than that of aspartate aminotransferase-to-platelet ratio index (APRI) and fibrosis 4 score (FIB-4), three diagnostic models for predicting the pathological severity were not strong enough (all area under the curves<0.8). Only the specificity of LIF-5 for predicting≥ G2, ≥ G2 and/or ≥ S2 was over 80%.Conclusions:Approximately 20% patients with chronic HBV infection in IT phase have progressive liver inflammation or fibrosis. The intensity of liver HBcAg and HBeAg level are negatively correlated with the severity of disease. The diagnostic models or most clinical indicators have low predictive effect for chronic HBV infections in IT phase.

Objective : To learn the characteristics of second-line drug resistance and related gene mutations of multidrug-resistant Mycobacterium tuberculosis ( MDR-TB ) Beijing genotype strains. Methods: The MDR-TB isolates in Hwa Mei Hospital from 2017 to 2019 were enrolled and detected using RD105 deletion-targeted multiplex polymerase chain reaction (PCR). The proportion method for drug susceptibility test was used to detect the drug-resistant profiles against kanamycin, amikacin, capreomycin, ofloxacin and levofloxacin. The gene sequencing of rrs, tlyA, eis, gidB, gyrA and gyrB was conducted by PCR compared with H37RV strain. The differences in the rates of drug resistance and mutation between Beijing and non-Beijing genotype strains were examined to understand the characteristics of Beijing genotype strains. Results: There were 106 Beijing genotype and 27 non-Beijing genotype strains in 133 MDR-TB isolates. The drug resistance rates of kanamycin, amikacin, capreomycin, ofloxacin and levofloxacin in Beijing genotype strains were 9.43%, 7.55%, 3.77%, 32.08% and 32.08%, respectively. The rates of quasi-extensive and extensive drug resistance in Beijing genotype strains were 30.19% and 7.55%. The gene mutation rates of rrs, tlyA, eis, gidB, gyrA and gyrB in Beijing genotype strains were 7.55%, 7.55%, 1.89%, 2.83%, 36.79% and 2.83%, respectively. There were no significantly differences between Beijing and Non-Beijing genotype strains in the factors above ( P>0.05 ). The gene rrs, tlyA, eis, gidB, gyrA and gyrB had 2, 1, 2, 2, 5 and 3 mutation types, respectively, with single base substitution as the main type. Conclusion Beijing genotype strains are dominant in MDR-TB, with high resistance to fluoroquinolones and mainly gyrA gene mutation.

Objective:To investigate the expression of alpha fetoprotein (AFP)-L3 in chronic liver disease and its diagnostic value for hepatocellular carcinoma.Methods:From October 2013 to March 2019, 341 patients with liver diseases in Huamei Hospital, University of Chinese Academy of Sciences were selected, including 88 cases of chronic hepatitis, 97 cases of cirrhosis, 145 cases of hepatocellular carcinoma (60 cases of initial onset, 39 cases of recurrence, 23 cases treated by transcatheter arterial chemoembolization, 23 cases treated by surgery and radiofrequency therapy) and 11 cases with acute-on-chronic liver failure. The difference of AFP and AFP-L3 (%) levels between different groups was compared. The diagnostic efficacy of AFP-L3 (%) for hepatocellular carcinoma was analyzed by using receiver operating curve (ROC).Results:There were significant differences in serum AFP-L3 (%) and AFP between patients with hepatitis, cirrhosis, acute-on-chronic liver failure and hepatocellular carcinoma (initial onset) (Hc=28.384, 9.913, P=0.001, 0.019). Post hoc multiple comparisons showed that the serum AFP-L3 (%) levels of patients with hepatocellular carcinoma (initial onset) were higher than those of patients with hepatitis and cirrhosis (all P<0.05). The level of serum AFP in patients with hepatocellular carcinoma (initial onset) was higher than that of patients with cirrhosis ( P<0.05), but there was no significant difference between patients with hepatocellular carcinoma (initial onset) and patients with acute-on-chronic liver failure for AFP-L3 (%) and AFP (all P>0.05). The levels of AFP-L3 (%) and AFP in patients with recurrence of hepatocellular carcinoma were significantly higher than those in patients undergoing hepatocellular carcinoma surgery and radiofrequency therapy ( P<0.05). Tumor size and TNM stage affected serum AFP level (all P<0.05), but etiology, tumor size and number, tumor thrombus, CTP score and TNM stage had little relationship with serum AFP-L3 (%) (all P>0.05). The diagnostic value of serum AFP-L3 (%) was better than that of AFP ( Z=2.637, P=0.008); the best cut-off value of AFP-L3 in the diagnosis of hepatocellular carcinoma was 6.10%, and the specificity and sensitivity were 76.63% and 61.29%, respectively. Conclusions:The diagnostic value of serum alpha-fetoprotein heterogeneity in hepatocellular carcinoma is better than that of AFP, which is less affected by pathological factors. In order to improve the diagnostic efficiency, we can establish reliable cut-off value by validating large samples in the laboratory.

Circular RNA is a class of non-coding RNAs, which are covalently closed and circular at both ends, showing dissimilar characteristics from linear RNA. Several studies have shown that circular RNAs play an important role in the occurrence and development of primary hepatic cancer. By combining with the latest research progress of this field at home and abroad, we summarized the mechanism regulating the occurrence and development of liver cancer, abnormal expression, and as potential molecular markers for disease diagnosis and treatment.

Objective: To analyze the distribution of HCV genotype in eastern Zhejiang Province and its correlation with sex, age, viral load, antiviral effect and so on. Methods: A total of 501 cases of HCV infection seen in Ningbo No. 2 hospital from January 2011 to April 2018 were included. The HCV genotypes and HCV RNA were detected by gene chip method and RT-PCR respectively. The liver function and blood routine tests were performed and the APRI index was calculated. The factors affecting the SVR were analyzed for the patients who were partially treated with pegylated interferon and ribavirin (PR). Results: The HCV genotypes of 501 cases were 1b、6、2a、3a、3b、1a from the higher to lower ranks, and genotype 1b was more than 50%.The distribution of HCV genotypes in different age groups was significantly different (χ²=95.433, P<0.001). The age of patients with 1a, 1b and 2 A was significantly higher than that of 3a, 3b, and type 6 (F=11.879, P<0.001). There were significant differences in viral load, AST, PLT and APRI index among different genotypes (F=2.920, χ²=12.400, F=6.294, χ²=12.700, all P<0.013), but ALT had little difference (χ²=7.994, P>0.157); 179 patients with PR standard regimen were followed up. The result showed that there was no significant difference in the overall efficacy of different genotypes (χ²=6.598, P=0.252), but the rate of sustained virological response in type 3 A was significantly higher than that of the 1b type (χ²=4.193, P=0.041). Conclusions The HCV genotypes are mainly 1b and 6 in eastern Zhejiang Province. There were significant differences in age, viral load, AST, PLT and APRI indices among patients with different HCV genotypes, and the therapeutic effect of PR regimen was better for genotype 1b than that of 3a.

Objective To analyze the heterogeneities of hepatitis B virus ( HBV ) reverse transcriptase domain (RT) gene mutations related to nucleos (t)ide analogues (NAs) resistance.Methods Blood samples from 2765 chronic hepatitis B patients with virological breakthrough or poor drug response treated in Ningbo No .2 Hospital and Ningbo Fourth Hospital from April 2011 to March 2018 were collected . According to the medication status , it was divided into LAM monotherapy group ( n =603 ) , LdT monotherapy group (n=147), ADV monotherapy group (n=68), ETV monotherapy group (n=10) and the sequential or combined drug resistance of NAs group (n=365).The resistance mutation sites and drug resistance patterns (pathways) of each group were analyzed .The SPSS 19.0 software was used to analyze the data.Results Among 2765 serum samples, the NAs-related HBV-RT resistance mutations were detected in 1193 cases with an overall mutation rate of 43.15％.The mutation rate of LAM monoclonal resistance group was 62.62％ (603/963) with 19 mutation types, the most common single point mutation was rtM204I/V (40.30％, 243/603).The mutation rate of LdT monoclonal resistance group was 45.51％(147/323), and there were 3 mutation types, with the single point mutation rtM204I/V being the most common (59.86％, 88/147).The mutation rate of the ADV monoclonal resistance group was 17.80％(68/382), mainly rtA181T single point mutation (64.71％, 44/68).The mutation rate of the ETV monoclonal resistance group was 4.06％(10/246), and the single point mutation of rtT184A/G/S/I/L/F was the most common one (80.00％, 8/10).The mutation rate of the sequential or combination therapy group was 41.91％ (365/871), among which the mutation rate of the LAM/LdT poor response or the resistance with the sequential ADV group was 63.39％(142/224), and the most single mutation point was rtA181V/T ( 35.21％, 50/142 );the mutation rate of LAM/LdT poor response or drug-resistant with combined ADV group was 42.19％ (54/128), and the most common mutation point was rtA181V/T (46.30％, 25/54);the mutation rate of LAM/LdT with poor response or resistance after sequential ETV 1.0 mg was 44.66％(117/262), and the most common mutation point was rtL180M+M204I/V+S202G/I (31.62％, 37/117);the LAM/LdT poor response or the drug-resistant ETV combined with ADV group had a mutation rate of 7.14％(5/70), all of which were multi-site mutations;the mutation rate of poor response to ADV or resistant with sequential ETV 0.5 mg group was 28.14％(47/167), all of which were multi-site mutations.Secondary ( compensation ) sites such as rtV173L, rtL180M, and rtV214A, and single-point mutations such as rtV207I/L/G, rtS213Tand rtN238T, which were not fully defined , were detected.The resistance patterns ( pathways ) of NAs monotherapy were relatively simple , and the resistance patterns ( pathways ) of NAs experienced patients ( sequential or combined treatment group ) were complex and diverse, and multiple resistance patterns (pathways) existed, along with NAs increasing in species.Non-first-line NAs-related resistance patterns ( pathways ) showed an overall downward trend sand ETV-related drug-resistant mutation showed an overall upward trend .Conclusion The NAs-related HBV resistance mutation sites ( patterns ) are complex and diverse , especially multi-site mutations , refractory drug resistance mutations, multidrug resistance mutations and cross-resistance mutations.Therefore, the optimization of antiviral treatment strategies and drug resistance management concepts need to be continuously updated .

Objective: To explore the effect of hepatitis C virus (HCV) on the expression of serine protease inhibitor Kazal1 (SPINK1) and its clinical implication. Methods: mRNA and protein expression of SPINK1 in Huh7.5.1 cells infected by HCV JFH-1 and the control cells were measured by RT-PCR and western blotting, SPINK1 levels in the cell supernatants and sera of HCV patients were measured by enzyme-linked immunosorbent assay (ELISA), the difference of SPINK1 levels between healthy controls and HCV patients was analyzed. Results: Expression of SPINK1 mRNA and protein was higher in Huh7.5.1 cells infected by HCV JFH-1 than in the control cells, serum SPINK1 levels was much higher in HCV patients than in healthy controls (P=0.016). Conclusions HCV can upregulate the expression of SPINK1.

Objective To investigate the intensity of HBsAg and HBcAg expression in liver tissue of patients with chronic hepatitis B virus (HBV) infection and its clinical significance.Methods A total of 994 HBV infected patients underwent liver biopsy and histopathological examination.The expression of HBsAg and HBcAg in liver tissue was detected by histoimmunochemistry.Patients were divided into HBeAg (+)/HBVDNA(+), HBeAg(-)/HBV DNA(+) and HBeAg(-)/HBV DNA(-) groups according to HBeAg and HBV DNA levels;patients were divided into ＜2 × normal (ULN) group, 2-＜5 × ULN groupand ≥5 × ULN group according to the alanine aminotransferase (ALT) levels.The histologic activity (A), fibrosis (F), the expression of HBsAg and HBcAg in liver tissue and their correlations with clinical features were analyzed.Logistic regression analysis was used to study the factors affecting the expression of HBsAg and HBcAg in liver tissue.Results Among 994 HBV infected patients, 941 cases (94.67％) were intrahepatic HBsAg positive and 553 cases (55.63％) were intrahepatic HBcAg positive;403 cases (40.85％) were ≥A2 in histologic activity and 371 cases (36.09％) were ≥F2 in fibrosis.The degree of A and F was the highest in HBeAg (-) / HBV DNA (+) group, followed by HBeAg (-) / HBV DNA (-) group, and was the lowest in HBeAg (+) / HBV DNA (+) group.The intensity of intrahepatic HBsAg expression was significantly different among three groups (x2 =6.299, r =-0.760, P ＜ 0.05), however, the difference was not showed in pairwise comparisons.The difference of intrahepatic HBcAg intensity among three groups was statistically significant (x2 =282.995, r =-0.645, P ＜ 0.01), the intensity was the highest in HBeAg (+) / HBV DNA (+) group and the lowest in HBeAg (-) / HBV DNA (-) group.The constituent ratio of HBeAg positive and HBV DNA level were higher and the average age was lower in intrahepatic HBsAg positive group than those in HBsAg negative group.The constituent ratio of positive HBeAg, the levels of ALT, AST, PLT and HBV DNA were higher and the average age, the average FIB-4 level were lower in intrahepatic HBcAg positive group than those in HBcAg negative group.The HBV DNA level was an independent risk factor for intrahepatic HBsAg intensity, and the HBeAg positive and HBV DNA level were independent risk factors for intrahepatic HBcAg intensity.There were no significant differences in A and F among different groups of intrahepatic HBsAg intensity (x2 =1.943 and 2.630, both P ＞ 0.05).There was significant difference in F among different groups of intrahepatic HBcAg intensity (x2 =12.352, P ＜ 0.01), but not in A.The degree of F was the highest in intrahepatic HBcAg negative group.There was significant difference in intrahepatic HBcAg intensity among different groups of ALT level (x2 =16.349, P ＜ 0.01), but not in intrahepatic HBsAg intensity.The intrahepatic HBcAg intensity in ALT ＜ 2 × ULN group was lower than that in other two groups.Conclusions Most of patients with chronic HBV infection are intrahepatic HBsAg positive and more than half of them are intrahepatic HBcAg positive.The intrahepatic HBsAg intensity is not associated with A and F, but correlates with HBV DNA level.The intrahepatic HBcAg intensity is not associated with A, but it is negatively correlated with F and positively correlated with positive HBeAg expression, HBV DNA level and ALT level.

Objective To analyze the correlation between liver pathology and clinical characteristics in a large series of patients with chronic HBV infections , so as to provide the data base for non-invasive medical diagnosis .Methods Liver pathology and clinical characteristics of 1 397 patients with chronic HBV infections were retrospectively analyzed . Ridit analysis and Spearman correlation analysis were performed to investigate the correlations of clinical characteristics with liver pathology of patients .Results In 1 397 patients, there were 604 patients (43.24%) with liver inflammation grading ≥G2 and 504 patients (36.08%) with fibrosis stage ≥S2.Inflammation grade and fibrosis stage of liver tissues were both higher in male patients than those in females (u=3.093 and 2.854, P<0.01).Inflammation grade and fibrosis stage of liver tissues in patients aged ≤30 years were lower than those in patients aged >30-40 years and >40 years (r=0.259 and 0.303, P<0.01;F=4.199 and 12.226,11.610 and 24.359, P<0.05 and <0.01).Patients with HBeAg( -) and HBV DNA≥103 copies/mL were of higher degrees in liver tissue inflammation compared with those with HBeAg ( +) and those with HBeAg ( -) but HBV DNA <103 copies/mL (F=8.788 and 5.635, all P<0.01);while the fibrosis stage in patients with HBeAg (-) and HBV DNA≥103 copies/mL was only higher than that in HBeAg ( +) patients (F=12.886, P<0.01). Liver tissue inflammation and liver fibrosis aggravated with the increase of ALT ( r=0.537 and 0.517, P<0.01).There was no significant difference in liver tissue inflammation among different age groups of patients with ALT (1-<2) ×ULN and HBV DNA≥103 copies/mL (χ2 =4.365, P >0.05),but there was significant difference in liver fibrosis in patients between aged >40 years and ≤30 years ( F=3.177,P<0.05).Conclusions Liver biopsy and antiviral therapy should be considered in chronic HBV infected patients with age of >30 years, lightly elevated ALT levels , HBeAg(-) and detectable HBV DNA levels , especially in male patients .Screening for liver fibrosis should be considered in patients with HBeAg ( -) and low HBV DNA levels .