Diagnostic precision of Truenat® technique and co-relation of ALT levels with HBV-DNA viral load among HBsAg positive patients at a tertiary care hospital in Eastern Uttar Pradesh.

Background and Objectives: In India, it is estimated that there are 40 million people suffering from Hepatitis B virus (HBV). Quantification of the viral burden is an important laboratory tool in the management. However, widespread use of different HBV-DNA assays is still affected by the high cost and variable diagnostic precision. The present study was conducted to evaluate the diagnostic precision and co-relation of ALT levels with HBV-DNA by Truenat®-PCR. Materials and Methods: In this prospective cross-sectional study a total of 567 serums were collected from patients by rapid HBsAg, and processed for liver function tests (LFT). The viral HBV-DNA amplification detection was carried out through by Truenat®-PCR test. Results: Out of 567 samples, 452 samples were found to be positive by both rapid and Truenat®-PCR and 106 were negative for HBV-DNA followed by 9 invalid. High ALT level found in 73% of positive patients who had HBV-DNA level (>100000 copies/ml) which is significantly higher in 447 patients as compared to those have below ≤100000 copies/ml. Conclusion: Truenat®-PCR technique is a highly sensitive and can be performed with low resources for effective control of HBV infection. Evaluation of HBV-DNA levels and serum ALT levels showed a significant proportion of patient harbored ongoing viral replication and disease progression.

RNA Interference Therapeutics for Chronic Hepatitis B: Progress, Challenges, and Future Prospects.

Chronic hepatitis B (CHB) is a global health challenge that can result in significant liver-related morbidity and mortality. Despite a prophylactic vaccine being available, patients already living with CHB often must engage in lifelong therapy with nucleoside analogues. However, the potential of RNA interference (RNAi) therapeutics as a promising avenue for CHB treatment is being explored. RNAi, particularly using small interfering RNA (siRNA), targets viral RNA that can be used to inhibit hepatitis B virus (HBV) replication. Several candidates are currently being studied and have exhibited varying success in reducing hepatitis B surface antigen (HBsAg) levels, with some showing sustained HBsAg loss after cessation of therapy. The dynamic evolution of RNAi therapy presents a promising trajectory for the development of effective and sustained treatments for CHB. This review highlights recent findings on RNAi therapeutics, including modifications for stability, various delivery vectors, and specific candidates currently in development.

Transient Co-Expression of Bioactive Murine Interferon-Gamma and HBsAg in Tobacco and Lettuce Leaves.

Background: The synchronous expression of antigen and adjuvant proteins in plant hosts presents an intriguing potential for vaccine production and the enhancement of appropriate immune responses. In this study, we examined the expression of bioactive murine interferon-gamma (mIFN-γ) along with HBsAg in tobacco and lettuce leaves aimed to further perform the analysis of immune responses in the mouse model. Methods: Monocistronic and bicistronic cassettes, carrying genes encoding mIFN-γ and HBsAg in various orders, were constructed. These cassettes were placed under the control of the 35S CaMV promoter and included the 5' leader sequence of Tobacco Ech Virus (TEV). Through Agrobacterium infiltration, the cassettes were transferred into plant leaves. The concentration of mIFN-γ in different constructs and HBsAg was tested by ELISA. Murine IFN-γ was characterized through Western blotting, and its bioactivity was evaluated by assessing the up-regulation of MHC class II in macrophages derived from mouse bone marrow. Results: Extracts of agroinfiltrated leaves contained recombinant mIFN-γ and HBsAg proteins at about 14 unit/mg and 50 ng/mg of soluble protein, respectively. Subsequently, mIFN-γ was purified from the plant extract and its ability to up-regulate MHC class II in mouse bone marrow-derived macrophages was confirmed by immunofluorescence. Conclusion: The co-expression of recombinant HBsAg and mIFN-γ using TEV 5' leader-based cassettes in tobacco and lettuce leaves produced both proteins with active mIFN-γ in different concentrations. The attractive utility and feasibility of using plant transient co-expression systems aimed to co-delivery of vaccine antigen and appropriate cytokine to elicit immune response for different applications.

A simple-to-use score system for predicting HBsAg clearance to peginterferon alfa-2b in nucleoside analogs-experienced chronic hepatitis B patients.

Objective: Patients with chronic hepatitis B (CHB) often fail to achieve clearance of the hepatitis B surface antigen (HBsAg) with peginterferon treatment. Our study aimed to develop a simple-to-use scoring system to predict the likelihood of HBsAg clearance following treatment with peginterferon alfa-2b(PEG-IFN-α2b) in patients with CHB. Methods: A total of 231 patients were enrolled and divided into HBsAg clearance (n = 37) and non-HBsAg clearance (n = 194) groups. Multifactor logistic models were constructed using univariate and multiple logistic regression analyses. The area under the receiver operating characteristic curve (AUC), calibration curve, and decision curve analysis were used to evaluate the discrimination, calibration, and clinical applicability of the predictive scoring system. Results: Four clinical variables (age, baseline HBsAg level, HBsAg level decline at week 12, and alanine aminotransferase ratio at week 12) were independently associated with HBsAg clearance after PEG-IFN-α2b treatment and, therefore, were used to develop a predictive scoring system ranging from 0 to 13. The optimal cut-off value was >4, with a sensitivity of 86.49%, specificity of 72.16%, positive predictive value of 37.2%, negative predictive value of 96.6%, and an AUC of 0.872. This model exhibited good discrimination, calibration, and clinical applicability. Among patients with scores <4, 4, or > 4 HBsAg clearance was achieved in 0.85, 14.29, and 37.21% of the patients, respectively. Conclusion: The scoring system could effectively predict the predominance of HBsAg clearance after PEG-IFN-α2b treatment in the early stage. This may be helpful when making clinical decisions for the treatment of patients with CHB.

An interesting case of isolated false-negative hepatitis B surface antigen in a blood donor.

BACKGROUND AND OBJECTIVES: An important requirement for a hepatitis B surface antigen (HBsAg) screening assay is reliable detection of HBsAg mutant forms, especially in blood donation. Here we investigate and describe the case of an isolated false-negative result of commercial serology HBsAg screening assay of a blood donor. MATERIALS AND METHODS: The current donation was routinely tested for HBsAg and hepatitis B virus (HBV) DNA in the mini-pool mode nucleic acid testing (MP-NAT of six samples), and further evaluated by individual donation ID-NAT. Finally, it was quantified and sequenced. All previous donations were found to have negative HBsAg and HBV DNA, as also the subsequent sample taken 3 months after the marked donation. RESULTS: The current donation of the 53-year-old unvaccinated female with 14 previous donations was initially HBsAg negative and HBV DNA (MP-NAT) positive. Further testing showed HBsAg positive using other HBV serological assays, antibodies to HBV core antigen immunoglobulin M positive and HBV DNA ID-NAT positive, and contained 200 IU/mL of HBV DNA. The implicated donation carried genotype D strains, subtype ayw2 (F83S, V96A, V190A, L193S, I195T, L213S, F220L). The mutations in three positions, namely amino acids T118A, P120T, and P127T, were proven subsequently. CONCLUSION: This unique mutation combination near the target epitope of one of the immunoassay monoclonals is a possible cause of the reduced analytical sensitivity of the serology assay.

Comparison of HBV DNA and RNA markers in Alaska Native people who did and did not clear hepatitis B surface antigen.

In a comparison between 50 Alaska Native persons with chronic hepatitis B who cleared HBV surface antigen (HBsAg) and 50 Alaska Native age-, sex-, and HBV genotype-matched controls, we found differences in changes in HBV DNA and HBV RNA levels over time but no difference in hepatitis B core-related antigen. These findings suggest that serial HBV DNA and HBV RNA may be associated with HBV functional cure defined by HBsAg clearance.

Safety considerations for withdrawal of nucleos(t)ide analogues in patients with chronic hepatitis B: First, do no harm.

Nucleos(t)ide analogues (NA) are widely used to treat hepatitis B virus (HBV) infection, but they cannot eradicate the virus and treatment duration can be lifelong if the endpoint is set at seroclearance of the hepatitis B surface antigen (HBsAg). As an alternative strategy, finite NA therapy without the prerequisite of HBsAg seroclearance has been proposed to allow treatment cessation in patients with sustained undetectable HBV viremia for two to three years. However, reactivation of viral replication almost always follows NA withdrawal. Whereas HBV reactivation might facilitate HBsAg seroclearance in some, it could lead to serious acute flare-ups in a certain proportion of patients. Occurrence and consequences of NA withdrawal flares are complicated with various factors involving the virus, host, and treatment. Accurate risk prediction for severe flares following NA cessation is essential to ensure patient safety. The risks of life-threatening flares in patients who discontinued NA according to the stopping rules of current guidelines or local reimbursement policies have recently been quantitatively estimated in large-scale studies, which also provided empirical evidence to help identify vulnerable patients at risk of devastating outcomes. Moreover, risk predictors were further explored and validated to hopefully aid in patient selection and management. In this narrative review with a focus on patient safety, we summarize and discuss current literature on the incidence of severe flares following NA cessation, risk stratification for candidate selection, rules of posttreatment monitoring, and indications for treatment resumption. We also share our thoughts on the limitations of existing knowledge and suggestions for future research.

Effect of Anti-HBs on Mortality Among Resolved HBV Infection: a Population-Based Prospective Cohort Study.

INTRODUCTION: Surveillance programs after hepatitis B surface antigen (HBsAg) loss are not yet well established, and the role of hepatitis B surface antibodies (anti-HBs) remains controversial. We aimed to evaluate the risk factors for increased mortality and the association between anti-HBs and all-cause and cause-specific mortality in a representative US (United States) population of patients with resolved HBV (Hepatitis B virus) infections. METHODS: Data were taken from the US National Health and Nutrition Examination Survey (NHANES) 1999-2018. A total of 3455 US adults with resolved HBV infection [defined as hepatitis B surface antigen (HBsAg) negative/anti-hepatitis B core antigen (anti-HBc) positive] were enrolled in this study. The primary outcome measures were all-cause and cause-specific mortality from baseline until 31 December 2019. RESULTS: During a mean follow-up of 10.3 years, 741 deaths occurred. Age, race, marital status, smoking status, physical activity level, and presence of cirrhosis, diabetes, cardiovascular diseases, chronic obstructive pulmonary diseases, cancer, and anti-HBs were significant factors for increased mortality, and a nomogram tool was developed and validated for the risk stratification of mortality. Compared with participants who were anti-HBs positive, those who were anti-HBs negative had a 23% (hazard ratio 1.23, 95% CI 1.02-1.46) higher risk of all-cause mortality in NHANES 1999-2018. For cause-specific mortality, the fully adjusted hazard ratios of participants who were anti-HBs negative were 0.71 (95% CI 0.48-1.06) for heart disease, 1.44 (95% CI 1.01-2.05) for cancer, and 1.44 (95% CI 1.13-1.83) for other conditions, compared to those of participants who were anti-HBs positive. CONCLUSIONS: Among US adults with resolved HBV infections, anti-HBs-negative status was associated with an increased risk of death from all causes and cancer, implying that the role of anti-HBs in resolved HBV infection should not be ignored. On the public health level, more rigorous surveillance was needed for populations of individuals who were isolated anti-HBc positive.

Clinical value of nucleic acid detection for hepatitis B virus screening in hospitalized patients / 中华检验医学杂志

Objective:To explore clinical value of nucleic acid detection for hepatitis B virus (HBV) screening in hospitalized patients.Methods:This cross-sectional study collected and analyzed plasma samples from patients admitted to 10 domestic medical institutions from July 2021 to December 2021. Serological immunoassay and nucleic acid screening were used to simultaneously detect hepatitis B markers such as hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), hepatitis B e Antigen (HBeAg), hepatitis B e antibody (HBeAb), hepatitis B core antibody (HBcAb),and HBV DNA. Statistical analysis was performed on the serology, nucleic acid test results and clinical information of the patients.Results:Of the 8 655 collected samples, HBsAg was positive in 216 (2.50%) samples,HBV DNA was positive in 238 (2.75%) samples ( P>0.05); 210 (2.43%) samples were positive for both HBsAg and HBV DNA, 28 (0.32%) were HBsAg negative and HBV DNA positive, 6 cases (0.07%) were HBsAg positive and HBV DNA negative. Conclusion:These results indicate that the HBV DNA testing is equally effective as hepatitis B virus serological detection for hepatitis B virus screening in hospitalized patients.

Predictive factors for functional cure after sequential therapy with nucleos(t)ide analogues and pegylated interferon alfa-2b in treatment of chronic hepatitis B / 临床肝胆病杂志

Objective To investigate the independent predictive factors for functional cure after long-term nucleos(t)ide analogue (NUC) antiviral therapy followed by pegylated interferon α-2b therapy in chronic hepatitis B (CHB) patients. Methods A total of 162 CHB patients who were admitted to several hospitals in Qingdao, China, from 2018 to 2021 were enrolled as subjects, and all patients received pegylated interferon α-2b for at least 48 weeks after NUC therapy for one year or longer. According to whether HBsAg clearance was achieved at week 48 of pegylated interferon α-2b treatment, the patients were divided into functional cure group with 79 patients and non-cure group with 83 patients, and related clinical indices were compared between the two groups. The two-independent-samples t test and the Mann-Whitney U rank sum test were used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. The Spearman correlation analysis was performed, and the univariate and multivariate logistic regression analyses were used to investigate the independent predictive factors for functional cure. The receiver operating characteristic (ROC) curve was plotted for related variables, and the area under the ROC curve (AUC) was used to evaluate the prediction accuracy of the variables. Results Compared with the non-cure group, the functional cure group had a significantly lower HBsAg level at baseline [21.63 (3.33-157.60) IU/mL vs 794.70 (336.10-1 185.34) IU/mL, Z =-8.869, P 1000 IU/mL (0 vs 8.4%, χ 2 =5.073, P =0.024), a significantly lower level of total bilirubin at baseline [12.60 (10.12-15.93) μmol/L vs 15.50 (11.80-24.10) μmol/L, Z =-3.611, P 2×upper limit of normal (16.5% vs 4.8%, χ 2 =5.835, P =0.016). The multivariate logistic regression analysis showed that baseline HBsAg (odds ratio [ OR ]=0.996, 95% confidence interval [ CI ]: 0.995-0.997, P < 0.001), HBsAg at week 12 of pegylated interferon α-2b treatment ( OR =0.990, 95% CI : 0.986-0.994, P < 0.001), HBsAg at week 24 of pegylated interferon α-2b treatment ( OR =0.983, 95% CI : 0.975-0.991, P < 0.001), and baseline total bilirubin ( OR =0.885, 95% CI : 0.826-0.949, P =0.001) were independent predictive factors for functional cure. The ROC curve of baseline HBsAg showed an AUC of 0.904 and the optimal cut-off value of 118.24 IU/mL; the ROC curve of HBsAg at week 12 of pegylated interferon α-2b treatment showed an AUC of 0.948 and the optimal cut-off value of 73.74 IU/mL; the ROC curve of HBsAg at week 24 of pegylated interferon α-2b treatment showed an AUC of 0.975 and the optimal cut-off value of 11.01 IU/mL; the ROC curve of baseline total bilirubin showed an AUC of 0.664 and the optimal cut-off value of 19.9 μmol/L. Conclusion Baseline HBsAg, HBsAg at week 12 of pegylated interferon α-2b treatment, HBsAg at week 24 of pegylated interferon α-2b, and baseline total bilirubin are independent predictive factors for functional cure at week 48 of pegylated interferon α-2b treatment in CHB patients receiving sequential therapy with NUC and pegylated interferon α-2b.

[Analysis of the therapeutic efficacy and factors influencing sequential combination of nucleos(t)ide analogues with pegylated interferon alpha for 48~96 weeks in the treatment of patients with chronic hepatitis B].

Objective: To explore the therapeutic efficacy and factors influencing the sequential combination of nucleos(t)ide analogues (NAs) with pegylated interferon alpha (Peg-IFN-α) in the treatment of patients with chronic hepatitis B (CHB). Methods: 144 CHB cases with NAs treatment for more than 1 year, HBV DNA < 20 IU/ml, hepatitis B surface antigen (HBsAg) quantification < 3 000 IU/ml, treated with a sequential combination of Peg-IFN-α treatment for 48 to 96 weeks, and followed up were selected from the Fifth Medical Center of the PLA General Hospital between May 2018 and May 2020. Intention-to-treat analysis was used to measure the HBsAg clearance rate at 96 weeks. The Kaplan-Meier method was used to compute the cumulative HBsAg clearance rate at 96 weeks. Univariate and multivariate logistic regression were used to analyze the factors influencing HBsAg clearance at 48 weeks of sequential combination therapy. Univariate and multifactorial COX proportional hazard models were used to analyze the factors influencing HBsAg clearance following 96 weeks of prolonged PEG-IFN-α treatment. The receiver operating characteristic curve was used to assess the predictive value of factors influencing HBsAg clearance. A Mann-Whitney U test was used to compare the measurement data between groups. The count data was compared using the χ(2) test between groups. Results: 41 (28.47%) cases achieved HBsAg clearance at 48 weeks of sequential combination therapy. The HBsAg clearance rate at 96 weeks was 40.28% (58/144) by intention-to-treat analysis. The Kaplan-Meier method computed that the cumulative HBsAg clearance rate at 96 weeks was 68.90%. Multivariate logistic regression analysis showed that HBsAg quantification at baseline (OR = 0.090, 95%CI: 0.034-0.240, P < 0.001) and a 24-week drop in HBsAg level (OR = 7.788, 95%CI: 3.408-17.798, P < 0.001) were independent predictors of HBsAg clearance in CHB patients treated sequentially in combination with NAs and Peg-IFN-α for 48 weeks. Receiver operating characteristic curve analysis showed that the baseline HBsAg quantification [area under the receiver operating characteristic curve (AUC), 0.911, 95% CI: 0.852-0.952)] and 24-week drop in HBsAg level (AUC = 0.881, 95%CI: 0.814-0.930) had equally good predictive value for 48-week HBsAg clearance, but there was no statistically significant difference between the two (Z = 0.638, P = 0.523). The value of the combination of baseline HBsAg quantification and 24-week drop in HBsAg level (AUC = 0.981, 95%CI: 0.941-0.997) was superior to that of single baseline HBsAg quantification (Z = 3.017, P = 0.003) and 24-week drop in HBsAg level (Z = 3.214, P = 0.001) in predicting HBsAg clearance rate at 48 weeks. Multivariate COX proportional hazards model analysis showed that HBsAg quantification at 48 weeks (HR = 0.364, 95%CI: 0.176-0.752, P = 0.006) was an independent predictor of HBsAg clearance with a prolonged course to 96 weeks of Peg-IFN-α treatment. Conclusion: The HBsAg clearance rate can be accurately predicted with baseline HBsAg quantification combined with a 24-week drop in HBsAg level in patients with CHB who are treated with a sequential combination of NAs and Peg-IFN-α therapy for 48 weeks. Prolonging the course of Peg-IFN-α treatment can enhance the HBsAg clearance rate's capability. An independent predictor of HBsAg clearance is HBsAg quantification at 48 weeks of sequential combination therapy with a prolonged course of 96 weeks of Peg-IFN-α treatment.

Comparison of hepatitis B surface antigen, anti-hepatitis B surface, and anti-hepatitis C virus prevalence in Syrian refugee pregnant women and Turkish pregnant women

SUMMARY OBJECTIVE: In this study, we aimed to determine and compare hepatitis B surface antigen level, anti-hepatitis B surface, and anti-hepatitis C virus positivity in Turkish pregnant women and Syrian refugee pregnant women residing in Turkey. METHODS: The study was conducted on Syrian refugee pregnant women aged 15-45 years and Turkish pregnant women who applied to state hospital's gynecology and obstetrics outpatient clinics between April 30, 2012, and April 30, 2022. In our study, 136,376 pregnant women (104,629 Turkish and 31,747 Syrian) tested for hepatitis B surface antigen, 72,035 pregnant women (53,070 Turkish and 18,965 Syrian) tested for anti-hepatitis B surface, and 120,611 pregnant women (92,514 Turkish and 28,097 Syrian) tested for anti-hepatitis C virus were included. The patients were divided into six groups for hepatitis B surface antigen, anti-hepatitis B surface, and anti-hepatitis C virus results based on their age: <20 years, 20-24 years, 25-29 years, 30-34 years, 35-39 years, and >40 years. For each age group, the results of Syrian refugee pregnant women and Turkish pregnant women were compared. RESULTS: Hepatitis B surface antigen positivity and anti-hepatitis B surface positivity were significantly higher in Turkish pregnant women compared to Syrian refugee pregnant women. Anti-hepatitis C virus positivity was significantly higher in Syrian refugee pregnant women compared to Turkish pregnant women. CONCLUSION: Based on the available data, we think that hepatitis B surface antigen, anti-hepatitis B surface, and anti-hepatitis C virus tests should be done routinely for pregnant women. Raising awareness among Syrian refugees about the hepatitis B virus vaccine as well as encouraging them to be vaccinated may reduce the negative impact of migration.

Dried blood spot is the feasible matrix for detection of some but not all hepatitis B virus markers of infection.

OBJECTIVE: Use of dried blood spots (DBS) for detection of hepatitis B virus (HBV) markers of infection has the potential to facilitate diagnosis of HBV infection especially in resource-limited countries. The aim of this study was to evaluate the feasibility of DBS for detection of various markers of HBV infections. RESULTS: Fifty-four DBS samples were engineered from well-characterized plasma samples. All DBS samples were tested for HBsAg, total anti-HBc and HBV DNA, 20 of 54 samples were also tested for HBeAg using commercially available assays. HBsAg was detected in 24 of 25 (96%), HBV DNA in 22 of 25 (88%), total anti-HBc in all 9 (100%), and HBeAg in all 7 (100%) DBS samples. The average difference in HBV DNA levels between DBS eluates and corresponding plasma samples was 2.7 log10 IU/mL. Fifteen DBS eluates positive for HBV DNA were sequenced and all of them belonged to HBV genotype A. Thirteen samples which were negative for all HBV markers showed HBeAg false positivity. Therefore, DBS is a reliable sample matrix for detection of HBsAg, total anti-HBc and HBV DNA, but not HBeAg. Further feasibility studies of DBS for diagnostic purposes and epidemiologic studies are warranted.

Randomised controlled trial: effect of metformin add-on therapy on functional cure in entecavir-treated patients with chronic hepatitis B.

INTRODUCTION AND OBJECTIVES: Hepatitis B surface antigen (HBsAg) clearance, indicating functional cure or resolved chronic hepatitis B (CHB), remains difficult to achieve via nucleos(t)ide analogue monotherapy. We investigated whether metformin add-on therapy could help achieve this goal in entecavir-treated patients with hepatitis B e antigen (HBeAg)-negative CHB. PATIENTS AND METHODS: Patients with HBeAg-negative CHB who met eligibility criteria (entecavir treatment for > 12 months, HBsAg < 1000 IU/mL) were randomly assigned (1:1) to receive 24 weeks of either metformin (1000 mg, oral, once a day) or placebo (oral, once a day) add-on therapy. The group allocation was blinded for both patients and investigators. Efficacy and safety analyses were based on the intention-to-treat set. The primary outcome, serum HBsAg level (IU/mL) at weeks 24 and 36, was analysed using mixed models. RESULTS: Sixty eligible patients were randomly assigned to the metformin (n = 29) and placebo (n = 31) groups. There was no substantial between-group difference in the HBsAg level at week 24 (adjusted mean difference 0.05, 95% confidence interval -0.04 to 0.13, p = 0.278) or week 36 (0.06, -0.03 to 0.15, p = 0.187), and no significant effect of group-by-time interaction on the HBsAg level throughout the trial (p = 0.814). The occurrence of total adverse events between the two groups was comparable (9 [31.0%] of 29 vs. 5 [16.1%] of 31, p = 0.227) and no patient experienced serious adverse events during the study. CONCLUSION: Although it was safe, metformin add-on therapy did not accelerate HBsAg clearance in entecavir-treated patients with HBeAg-negative CHB.

CpG-Containing Oligodeoxynucleotides and Freund Adjuvant in Combination with Alum Augment the Production of Monoclonal Antibodies Against Recombinant HBsAg.

Background: Adjuvants are essential to potentiate the immune response to inoculated antigens and play a central role in vaccine development. Alum is generally used as a classic adjuvant, although it does not stimulate proper immunity, and some of the immunized subjects have low or no antibody response. Efforts have been continued to find more efficient adjuvants for better antibody responses. In the present study, the efficacy of three formulations of adjuvants, i.e. Cysteine p Guanine Oligodeoxynucleotide (CpG ODN), alum, and Freund, in the production of monoclonal anti Hepatitis B Surface Antigen (HBsAg) antibodies was investigated. Methods: To immunize mice, regular hepatitis B vaccine containing recombinant HBsAg and alum was used with CpG ODN or Freund adjuvants, and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by Enzyme-Linked Immunosorbent Assay (ELISA) using HBsAg as coating antigen followed by a limited dilution process. Results: The results showed that by using all three formulations of adjuvants, monoclonal antibody (mAb) specific to HBsAg was successfully generated. It was also found that the mice immunized with (HBsAg + Alum) + CpG had the highest concentration of antibody production in serum and hybridoma supernatants as well as positive clones. Based on these findings, the addition of CpG ODN also induced a higher antibody response compared with Complete Freund's Adjuvant (CFA). Conclusion: Results of this study showed that CpG and Freund adjuvants could be efficient partners for alum in the immunization period of the process of monoclonal antibody production.

Correlation of hepatitis B surface antigen expression with clinicopathological and biochemical parameters in liver biopsies: A comprehensive study.

BACKGROUND: Chronic viral B hepatitis (CHB) is a potentially life-threatening liver disease that may progress to liver failure and cirrhosis. Currently, although combinations of different laboratory methods are used in the follow-up and treatment of CHB, the failure of these procedures in some cases has led to the necessity of developing new approaches. In CHB, the intrahepatic expression pattern of viral antigens, including hepatitis B surface antigen (HBsAg), is related to different phases of inflammation. However, many studies have focused on the intracytoplasmic properties of HBsAg staining, and HBsAg positivity in liver tissue has not been evaluated by objective quantitative methods. AIM: To investigate the relationship of image analysis-based quantitative HBsAg expression and its staining patterns with clinicopathological factors and treatment in CHB. METHODS: A total of 140 liver biopsies from treatment-naïve cases with CHB infection were included in this study. Following diagnosis, all patients were treated with entecavir (0.5 mg) and followed up at three-month intervals. The percentage of immunohistochemical HBsAg (p-HBsAg) expression in the liver was determined in whole tissue sections of biopsies from each case by image analysis. The immunohistochemical staining pattern was also evaluated separately according to 3 different previously defined classifications. RESULTS: A positive correlation between p-HBsAg and serum levels of hepatitis B virus (HBV) DNA and HBsAg was observed (P < 0.001). The p-HBsAg value was significantly higher in younger patients than in older patients. When the groups were categorized according to the hepatitis B e antigen (HBeAg) status in HBeAg-positive cases, p-HBsAg was correlated with HBV DNA, hepatitis activity index (HAI) and fibrosis scores (P < 0.001). In this group, p-HBsAg and HBsAg expression patterns were also correlated with the viral response (VR) and the serological response (SR) (P < 0.001). Multivariate analysis revealed that p-HBsAg was an independent predictor of either VR or SR (P < 0.001). In HBeAg-negative patients, although HBsAg expression patterns were correlated with both HAI and fibrosis, no relationship was observed among p-HBsAg, clinicopathological factors and VR. CONCLUSION: In pretreatment liver biopsies, the immunohistochemical determination of HBsAg expression by quantitative methods, beyond its distribution within the cell, may be a good predictor of the treatment response, especially in HBeAg-positive cases.

RNA interference as a novel treatment strategy for chronic hepatitis B infection.

Chronic hepatitis B (CHB) is a major cause of liver-related morbidity and mortality. Functional cure of CHB, defined as sustainable hepatitis B surface antigen (HBsAg) seroclearance, is associated with improved clinical outcomes. However, functional cure is rarely attainable by current treatment modalities. RNA interference (RNAi) by small-interfering RNA (siRNA) and anti-sense oligonucleotide (ASO) has been studied as a novel treatment strategy for CHB. RNAi targets post-transcriptional messenger RNAs and pregenomic RNAs to reduce hepatitis B virus (HBV) antigen production and viral replication. By reducing viral antigens, host immune reconstitution against HBV may also be attained. Phase I/II trials on siRNAs have demonstrated them to be safe and well-tolerated. siRNA is effective when given in monthly doses with different total number of doses according to different trial design, and can lead to sustainable dose-dependent mean HBsAg reduction by 2-2.5 log. Incidences of HBsAg seroclearance after siRNA therapy have also been reported. ASOs have also been studied in early phase trials, and a phase Ib study using frequent dosing regimen within 4 weeks could achieve similar HBsAg reduction of 2 log from baseline. Given the established efficacy and safety of nucleos(t) ide analogues (NAs), future RNAi regimens will likely include NA backbone. While the current evidence on RNAi appears promising, it remains undetermined whether the potent HBsAg reduction by RNAi can result in a high rate of HBsAg seroclearance with durability. Data on RNAi from phase IIb/III trials are keenly anticipated.

Effects of hepatitis B vaccination on hepatitis B surface antigen in neonates and its change in vivo.

Background: Vaccination is effective to prevent hepatitis B virus (HBV) infection. However, there is still a risk of infection after vaccination. In clinical work, we found that newborns were positive for HBV surface antigen (HBsAg) after vaccination. Objectives: To determine the effect of hepatitis B vaccination on the detection of HBsAg trend in newborns. Methods: We collected data at birth, history of vaccination for hepatitis B, quantitative HBsAg results, and other information about newborns born in our hospital from July 2017 to July 2020. Serum samples from healthy neonates were randomly selected to be supplemented with recombinant hepatitis B vaccine on a concentration gradient, and HBsAg was measured quantitatively. Results: Data from 1417 neonates were included in the study; 306 (21.6%) were HBsAg positive within 8 d after vaccination, with levels ranging from 0.104 IU/mL to 0.339 IU/mL. The proportion of neonates with HBsAg-positive serum was significantly correlated with the level of hepatitis B surface antibodies (anti-HBs) in the serum of their mothers (P < 0.01). Experiments in vitro showed that the proportion of neonates with HBsAg-positive serum was correlated with the dose of the hepatitis B vaccine, and when the concentration of the hepatitis B vaccine reached 5 ng/mL and 10 ng/mL, the serum HBsAg levels showed a significant negative correlation with the original concentration of serum anti-HBs. Conclusions: Hepatitis B vaccination can affect the level of HBsAg detected in neonatal serum, and the effect could be mitigated by delaying the measurement. Moreover, maternal anti-HBs offset the effects of neonatal vaccination on HBsAg serum levels.

Contrast-enhanced US diagnostic algorithm of hepatocellular carcinoma in patients with occult hepatitis B.

PURPOSE: To assess the diagnostic performance of contrast-enhanced (CE) US Liver Imaging Reporting and Data System (LI-RADS) version 2017 and propose a diagnostic algorithm in diagnosing hepatocellular carcinoma (HCC) in patients with occult HBV infection (OBI). METHODS: 251 OBI patients with 251 newly diagnosed focal liver lesions were retrospectively enrolled. Each nodule was evaluated according to CEUS LI-RADS. The subgroup analyses were also performed in patients with alpha-fetoprotein (AFP) more than 20ug/L or not. Diagnostic performance of CEUS LI-RADS for diagnosing HCC was validated via sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV), respectively. RESULTS: There were 90 HCCs (90 of 251, 35.9%), of which 2 (2.0%), 53 (53.5%), and 35 (35.4%) were classified as LR-4, LR-5, and LR-M, respectively. The sensitivity, specificity, accuracy, PPV, and NPV of CEUS LR-5 for HCC diagnosis were 58.9%, 88.8%, 78.1%, 74.6%, and 79.4%, respectively. AFP increased in 50.6% (45/89) HCCs. Using a proposed diagnostic algorithm (for OBI patients with AFP more than 20 ug/L, LR-5 nodules were diagnosed as definitely HCC), the sensitivity, specificity, accuracy, PPV, and NPV were 62.2%, 71.4%, 63.5%, 93.3%, and 22.7%, respectively. Therefore, 12.2% (30 of 246) nodules could be confirmed as HCC by CEUS without biopsy. CONCLUSION: HCC diagnosis in patients with OBI is challenging. However, using LR-5 as a noninvasively diagnostic standard in OBI patients with AFP more than 20ug/L, HCC could be confirmed by CEUS without biopsy.

Detection of the Hepatitis B Surface Antigen in Patients with Occult Hepatitis B by Use of an Assay with Enhanced Sensitivity.

Patients with occult hepatitis B infection (OBI) have undetectable hepatitis B surface antigen (HBsAg) by conventional assays but detectable hepatitis B virus (HBV) DNA in blood/liver. We evaluated the key performance characteristics of a sensitive HBsAg assay (Architect HBsAg Next qualitative assay, referred to as NEXT) with respect to HBsAg detection. Assay precision, sample carryover, and seroconversion sensitivity of NEXT were evaluated. HBsAg was measured by NEXT in 1,138 individuals, including 1,038 patients who attended liver clinics in a tertiary hospital (200 HBV DNA-positive blood donors whose HBsAg was undetectable by conventional assays, 38 patients receiving immunosuppressive therapy, and 800 chronic hepatitis B patients with HBsAg seroclearance) and 100 HBsAg-negative subjects recruited from a community project. The within-run and within-laboratory coefficients of variation were <6% for the positive sample pools. In 9 seroconversion panels tested, NEXT allowed an earlier HBsAg detection than conventional assays. NEXT detected HBsAg in 10/200 (5%) HBsAg-negative blood donors, 1/20 (5%) and 0/18 HBsAg-negative patients with and without HBV reactivation, respectively, and 59/800 (7.3%) patients with HBsAg seroclearance. HBsAg was detectable by NEXT in 27.8%, 8.2%, 6.9%, 3.8%, and 1.9% samples at <3, 3 to 5, >5 to 8, >8 to 11, and >11 years after HBsAg seroclearance, respectively. Seven out of 100 HBsAg-negative community-identified subjects were tested positive by NEXT. Compared with conventional HBsAg assays, NEXT demonstrated a higher sensitivity and conferred an increment of 5 to 7% detection rate in patients with OBI, thereby helping in identifying HBV carriers and prevention of OBI-associated HBV transmission and reactivation.