Objective To investigate the effects of adefovir resistance related mutations in hepatitis B virus (HBV) reverse transcription (RT) region on the viral replication and hepatitis B surface antigen (HBsAg) secretion. Methods Twelve adefovir treated chronic hepatitis B (CHB) patients who experienced a viral breakthrough were enrolled in this study. The RT region was amplified by polymerase chain reaction (PCR) using HBV DNA extracted from sera as the template. PCR products were then sequenced and analyzed to find out mutation patterns. Full-length HBV genome was amplified from 4 representative serum samples followed by direct sequencing. The dominant strain was cloned into vector PHY106 to construct a recombinant plasmid containing the 1.1 unit of HBV genome Which was transfected into Huh7 cells. HBsAg and hepatitis B e antigen (HBeAg) expression were determined by enzyme-linked immunosorbent assay (ELISA), meanwhile intracellular HBV DNA level was determined by quantitative real-time PCR. Furthermore strain harboring rtA181T/sW172 · mutation and strain without rtA181 mutation were cotransfected into Huh7 cells. HBsAg and intracellular HBV DNA were also determined after transfection. Results Ten out the 12 patients enrolled in this study exhibited mutations conferring resistance to adefovir. The rtA181T mutation was detected in 5 cases, and the rtA181T/S+rtN236T mutation was observed in 4 cases. Different mutants showed variable HBsAg secretion competency in vitro. Despite the defect of HBsAg secretion of the rtA181T/sW172 · mutant, the replication efficiency was almost the same in different mutants. When the strains with and without rtA181 mutation were cotranfected into cells, the HBsAg level increased in accordance with the amount of stains without rtA181 mutation. However, the intracellular HBV DNA level was not changed significantly. Conclusions The rtA181 mutation is common in patients with adefovir resistance, of which the rtA181T mutation is the major pattern. In vitro analysis reveals that the rtA181T/sW172 · mutant is defective in HBsAg secretion which could be rescued by coexistence of wild-type strains. The replication efficiency in various mutants shows no obvious differences.

Objective To establish a novel and convenient method to study the phenotype of drug resistant hepatitis B virus (HBV) isolates,and to analyze the drug susceptibility by replacing the reverse transcriptase (RT) domain of wild-type HBV with that of the drug resistant HBV isolates.Methods Full length of HBV isolates was amplified and cloned from the sera of patients prior to nucleoside/nucleotide analogues (NA) treatment.Wild-type full-length HBV genomes was used to construct the recombinant expression plasmids PHY536207 (genotype B) and PHY97 (genotype C).The restriction enzyme sites were introduced in the upstream and downstream region of reverse transeription (RT) domain to construct plasmid,which were named as mPHY536207 and mPHY97,respectively.Lamivudine (LAM) resistant mutant and adefovir (ADV) resistant mutant were isolated and cloned to construct recombinant expression plasmids PHY634 and PHY6923,respectively.Subsequently,the RT domain of mPHY536207 was replaced by that of drug resistant mutant to construct the plasmids RT634 (LAM-resistant) and RT6923 (ADVresistant).The HBV constructs were transfected into Huh7 cells.The HBsAg levels in supernatant were determined by enzyme-linked immunosobent assay (ELISA),and the amount of intracellular HBV DNA was assayed by real-time polymerase chain reaction and Southern blot analysis.Results The plasmids PHY536207 and PHY97 containing genotype B and genotype C wild-type fulllength HBV genomes were constructed successfully,both of which could replicate in Huh7 cells.Intracellular HBV DNA extracted from cells in each of six-well culture plates was more than 1 × 107 copy/ mL,and the introduction of Pst Ⅰ restriction enzyme site did not affect the viral replication and HBsAg secretion.PHY634 and RT634,in which mutant RT domain was replaced into a wild type HBV expressing vector,exhibited the same HBV DNA replication under the treatment with different doses of LAM,the value of 50％ inhibitory concentration (IC50) was ＞100 μmol/L,while the IC50 of mPHY536207 was 0.18μmol/L.Moreover,wild-type isolate was sensitive to ADV (IC50 =1.2 μmol/L),while PHY6923 and RT6923 were resistant to ADV treatment (IC50 ＞100 μmol/L).Conclusion The phenotypic assay is successfully developed in this study based on replacing RT domain of wild-type HBV strains with that of clinical isolated drug resistant strain.

Elimination of HBV cccDNA from hepatocytes infected with chronic HBV virus is considered to be the key to eradicating HBV. Monitoring HBV cccDNA before, during, and after viral treatment is essential for routine treatment of patients with chronic hepatitis B. With the introduction of new anti-HBV treatment technologies and new drugs targeting HBV cccDNA, Accurate and sensitive HBV cccDNA assays are urgently needed to evaluate efficacy. In recent years, HBV cccDNA detection methods have achieved gratifying results in both traditional PCR methods and digital PCR methods popular in recent years. In this paper, the advances in HBV cccDNA quantitative detection by qPCR, Magnetic bead capture hybridization, rolling circle amplification combined with in situ PCR, digital PCR and digital PCR assay in single cells were reviewed.

Objective: To investigate mental health and its associated factors in college students during COVID-19 confinement in campus, and to provide a scientific basis for mental health education. Methods: A general questionnaire, the Patient Health Questionnaire 9 (PHQ-9) and the Generalized Anxiety Disorder- 7 (GAD-7) were administered. A total of 1 816 college students under COVID-19 confinement in campus in Lanzhou City were surveyed from October 18 to November 18, 2021. Data were analyzed by using the ordinal Logistic regression method. Results: The overall incidence of depressive emotions was 38.76%, and the incidences of mild, moderate to severe depression emotions were 31.33% and 7.43%, respectively. About 16.36% of students showed anxiety, with mild, moderate to severe anxiety being 13.33% and 3.03%, respectively. Multivariate analysis showed that poverty( OR =1.29), daily schedule (basically normal OR =0.33, normal OR =0.18), adaptability of online learning (moderate adaptation OR =0.45, high level of adaptation OR =0.25), concerns about the infection of oneself and family members (some concerns OR =1.73, considerable concerns OR =2.09),male( OR =0.78), and the isolation mode( OR =1.70). The music listening (sometimes OR =0.44, often OR =0.41), daily schedule (basically normal OR =0.36, normal OR =0.19), adaptability of online learning (moderate adaptation OR =0.42, high level of adaptation OR =0.28), and concerns about the infection of oneself and family members (some concerns OR =1.87, considerable concerns OR =3.27) were primary factors associated with high level of anxiety among college students( P <0.05). Conclusion The incidence of depression and anxiety among college students increased following COVID-19 confinement and centralized isolation for medical observation in campus. Universities and relevant departments should take timely and precise measures for psychological counseling.

significantly save the time of diagnosis and facilitate the clinical application of large samples.

Objective:To investigate the related factors and prognosis of invasive Klebsiella pneumoniae liver abscess syndrome (IKLAS). Methods:The in-patients diagnosed with Klebsiella pneumoniae liver abscess in the Department of Infectious Diseases, Huashan Hospital, Fudan University from January 2015 to February 2021 were retrospectively enrolled. The patients were divided into IKLAS group and non-IKLAS group according to whether they had IKLAS or not. The clinical data between the two groups were compared, including the prevalence of diabetes mellitus, the details of liver abscess, clinical symptoms such as fever and abdominal pain, as well as laboratory tests such as glycosylated hemoglobin and hemoglobin. Statistical analysis was performed using chi-square test or independent sample t test. Multivariate logistic regression analysis was used to analyze the factors influencing the occurrence of IKLAS. Results:A total of 75 patients with Klebsiella pneumoniae liver abscess were enrolled, including 55 patients (73.33%) in the IKLAS group and 20 patients (26.67%) in the non-IKLAS group. Fifty-two point seven three percent (29/55) of the patients had diabetes mellitus and 12.73%(7/55) of the patients had abdominal pain in the IKLAS group, which were 20.00%(4/20) and 45.00%(9/20) in the non-IKLAS group, respectively, and the differences were both statistically significant ( χ2=6.38 and 7.28, respectively, both P<0.05). Most of liver abscesses were single (50/75, 66.67%), and more likely to occur in the right liver (50/75, 66.67%). The maximum diameter of liver abscess in the IKLAS group was (4.58±2.04) cm, which was smaller than that in the non-IKLAS group ((6.49±3.11) cm), and the difference was statistically significant ( t=2.82, P=0.011). Compared with those in the non-IKLAS group, patients in the IKLAS group had higher glycosylated hemoglobin (8.69%±2.64% vs 6.18%±1.31%) and hemoglobin ((112.25±22.04) g/L vs (100.05±18.59) g/L), and the differences were both statistically significant ( t=-4.25 and -2.21, respectively, both P<0.05). The proportion of patients using antibiotics combined with abscess drainage in the IKLAS group was 38.18%(21/55), and that in the non-IKLAS group was 85.00%(17/20). The difference between the two groups was statistically significant ( χ2=12.86, P<0.001). A total of 16 patients (21 eyes) were diagnosed as endogenous Klebsiella pneumoniae endophthalmitis (EKPE), and all of them were IKLAS patients, and 14 patients underwent monocular/binocular eyeball injection and/or vitrectomy and silicone oil filling. The visual acuity of 13 patients decreased significantly. Multivariate logistic regression analysis showed that complicated with diabetes mellitus was an independent risk factor for IKLAS (odds ratio ( OR)=5.02, 95% confidence interval (95% CI) 1.01 to 25.03, P=0.049). The large diameter of liver abscess was a protective factor for IKLAS ( OR=0.64, 95% CI 0.47 to 0.86, P=0.003). Conclusions:The patients with IKLAS have less abdominal pain, and most of them complicate with diabetes mellitus. Diabetes mellitus is an independent risk factor for the occurrence of IKLAS, while the large diameter of liver abscess is a protective factor. EKPE is associated with poor visual prognosis.

Objective:To assess the predictors of outcomes for different subtypes of liver failure, and the effectiveness of artificial liver support systems in the treatment of liver failure.Methods:The clinical data of 112 patients with hepatitis B virus (HBV)- and non-HBV-related liver failure admitted to the intensive care unit (ICU) of the Fifth People's Hospital of Wuxi were collected from January to December 2020. The relevant etiologies of acute, subacute, acute-on-chronic, subacute-on-chronic, chronic subtype liver failure were analyzed. The efficacies of artificial liver support systems in the treatment of various subtypes of liver failure were also compared. The correlation of various indicators was analyzed by Spearman correlation analysis, the risk factors affecting the prognosis of patients with liver failure were analyzed by multivariate Logistic regression equation, and receiver operator characteristic curve (ROC curve) of subjects was plotted to evaluate the predictive value of each risk factor for the prognosis of patients with liver failure.Results:Among the 112 liver failure patients, 63 were caused by hepatitis B and 49 were caused by non-hepatitis B. The liver failure caused by hepatitis B was 6 times higher than for men than for women, which was higher than that of non-HBV liver failure group (1.33 times). Antithrombin Ⅲ (AT Ⅲ) and total bilirubin (TBil) levels of subacute liver failure were higher than those of pre-liver failure in the HBV liver failure group [AT Ⅲ: (59.33±14.57)% vs. (35.66±20.72)%, TBil (μmol/L): 399.21±112.94 vs. 206.08±126.96, both P < 0.05]. The levels of AT Ⅲ in patients with pre-liver failure and chronic liver failure in the non-HBV liver failure group were significantly higher than those with acute liver failure [(58.33±15.28%), (44.00±19.10)% vs. (31.33±7.57)%, both P < 0.05], patients with acute liver failure had significantly lower level of TBil than pre-liver failure (μmol/L: 107.83±49.73 vs. 286.20±128.92, P < 0.05), the TBil levels in patients with subacute and acute-on-chronic liver failure were also significantly higher than that in pre-liver failure group (μmol/L: 417.27±118.60, 373.00±187.00 vs. 286.20±128.92, both P < 0.05). Patients with subacute liver failure, subacute-on-chronic liver failure and chronic liver failure in the non-HBV failure group were significantly longer than those in acute liver failure (days: 36.00±8.31, 27.52±11.71, 27.72±22.71 vs. 11.00±1.41, all P < 0.05). There was no statistically significant difference in the case fatality rate of using the artificial liver support system between the HBV failure group and the non-HBV failure group (55.6% vs. 50.0%, P < 0.05), the levels of AT Ⅲ in the two groups of surviving patients were significantly higher than that of the dead [HBV liver failure group: (36.20±6.26)% vs. (27.33±8.87)%, non-HBV liver failure group: (41.06±4.16)% vs. (28.71±12.35)%, both P < 0.01]. Correlation analysis showed that there was a clear positive correlation between AT Ⅲ and TBil in the dead patients of HBV liver failure group and the survival and death patients of non-HBV liver failure group ( r values were 0.069, 0.341, 0.064, and P values were 0.723, 1.196 and 0.761, respectively); there was a significant inverse correlation between AT Ⅲ and TBil in the HBV liver failure group ( r = -0.105, P = 0.745). Multivariate Logistic regression analysis showed that AT Ⅲ was an independent risk factor affecting the prognosis of patients with non-HBV liver failure [odd ratio ( OR) = 1.023, 95% confidence interval (95% CI) was -0.001 to 0.001, P = 0.007]. TBil was an independent risk factor affecting prognosis of patients with HBV liver failure ( OR = 1.005, 95% CI was -0.002 to -7.543, P = 0.033). The analysis of ROC curve showed that AT Ⅲ had a predictive value for the prognosis of patients with non-HBV liver failure, the area under the ROC curve (AUC) = 0.747, the 95% CI was 0.592-0.902, P = 0.009. When the optimal truncation value was 39.5%, its sensitivity and specificity were 83.33% and 56.25%, respectively. Conclusions:Artificial liver support system treatment of liver failure was difficult to effectively reduce the mortality of patients with end-stage liver failure. In addition to AT Ⅲ, TBil also could be used as an indicator to assess liver compensatency and predict prognosis in liver failure patients.