This study included patients with CHB and compensated hepatitis B cirrhosis who visited the Gastroenterology and Hepatology Department at Tianjin Third Central Hospital from January 2018 to May 2024. The diagnostic criteria were based on the Chinese Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2022 Edition) (7).

Inclusion criteria were as follows: (1) HBsAg positivity for at least 6 months; (2) treatment with PegIFNα-2b (Pegbin^®, Xiamen Amoytop Biotech Co., Ltd.) alone or in combination with NAs for over 24 weeks; (3) achievement of HBsAg clearance during treatment; (4) regular follow-up every 3 to 6 months post-clearance.

Exclusion criteria included: (1) co-infection with other viruses (e.g., human immunodeficiency virus [HIV], hepatitis D virus [HDV], hepatitis C virus [HCV]), liver cancer, alcoholic liver disease, autoimmune hepatitis, or other liver diseases; (2) decompensated cirrhosis, including a history of complications such as gastrointestinal bleeding, ascites, or hepatic encephalopathy; (3) pregnant or breastfeeding women; (4) patients using corticosteroids, immunosuppressants, immunotherapy drugs, or those with a history of alcohol or drug abuse during the follow-up period; (5) patients who had not discontinued interferon treatment; (6) patients who did not adhere to regular follow-up. The initial dose of Peg-IFN α-2b for patients was 180 micrograms (μg), administered once weekly. During the course of treatment, dose adjustments were made according to the instructions. Dose reduction should be considered, if absolute neutrophil count (ANC) < 0.75 × 10⁹/L or PLT < 50 × 10⁹/L. Discontinuation of PegIFN was necessary if ANC < 0.50 × 10⁹/L, PLT < 25 × 10⁹/L or serious adverse events (AEs) occurred. The diagnostic criteria for MAFLD (metabolic-associated fatty liver disease) refer to the international expert consensus statement (10).

HBsAg clearance was defined as a serological conversion to negative HBsAg (HBsAg < 0.05 IU/mL) following PegIFNα-2b treatment. HBsAg sustained response was defined as sustained HBsAg loss with or without the appearance of HBsAb at the end of follow-up (EOF). HBsAg reversion was defined as the re-detection of positive HBsAg (≥ 0.05 IU/mL) during follow-up after an initial conversion to negative.

Patients were categorized into two groups based on the presence of HBsAg (≥0.05 IU/mL) during follow-up after discontinuation of PegIFNα-2b treatment: the HBsAg sustained response (HSR) group and the HBsAg reversion (HRV) group. For patients in the HRV group, the study endpoint was defined as the first detection of HBsAg reversion during follow-up, while HBsAg-negative patients continued follow-up until the conclusion of the study. Consolidation therapy (weeks) refers to the period of continued interferon treatment after HBsAg clearance; whereas, the therapy duration (weeks) represents the total time from the initiation to the cessation of interferon treatment.

Patient demographic information (including age and sex) was recorded along with baseline peripheral blood liver function tests, complete blood counts, hepatitis B serological markers including HBV DNA, quantitative HBsAg levels, the date of first HBsAg clearance, date of HBsAg reversion, types of NAs used, details of interferon treatment, and adverse reactions. After HBsAg clearance, patients were followed up approximately every 3 months, during which hepatitis B virus markers, biochemical indicators, and clinical adverse events were documented.

Quantitative HBV DNA Detection: HBV DNA was measured using the Roche COBAS AmpliPrep/COBAS TaqMan HBV Test v2.0, with a detection range of 20 to 1.70 × 10⁸ IU/mL.

Quantitative HBsAg Detection: Quantitative detection of HBsAg was performed using the Abbott ARCHITECT i4000SR chemiluminescent microparticle immunoassay (Abbott Diagnostics, Abbott Park, IL, USA), with a detection range of 0.05 to 250 IU/mL. Samples with concentrations >250 IU/mL were diluted for quantitative analysis. Levels of HBsAb and HBeAg were determined using commercial immunoassay kits.

For normally distributed continuous variables, results are expressed as mean ± standard deviation ( x ¯ ± s ) , with comparisons between groups using the t-test. Categorical data were compared using the chi-square test and trend chi-square test. For non-normally distributed continuous variables, data are presented as median (min-max) and group comparisons were conducted using the Mann-Whitney U test. A p-value of <0.05 was considered statistically significant. All statistical analyses were conducted using the R statistical software package (http://www.R-project.org, The R Foundation) and EmpoweringStats (http://www.empowerstats.com, X&Y Solutions, Inc.). Graphs were created using Origin 2022b software.

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and was approved by the Ethics Committee of Tianjin Third Central Hospital (Approval No: IRB2020-015-01). All data analyses were performed under the principle of anonymity. As a real-world cohort study, no additional specimen collection was conducted due to the study, and it did not affect patients’ treatment plans, disease progression, or prognosis; therefore, it met the criteria for a waiver of informed consent.

A total of 195 patients were enrolled in this study. Twenty patients who were still undergoing treatment and two who were lost to follow-up were excluded, resulting in a final cohort of 173 patients. The mean age of participants was 41.5 ± 9.0 years, with 117 males (67.63%), including 28 patients with compensated cirrhosis (16.19%) and 89 patients with concurrent MAFLD (51.445%). Among the 173 patients, 147 maintained HBsAg negativity throughout the follow-up period (HSR group), yielding a sustained HBsAg response rate of 84.97% (147/173). In contrast, 26 patients were found to have at least one positive HBsAg during follow-up (HRV group), resulting in an overall HBsAg reversion rate of 15.03% (26/173). The median duration of therapy for patients in the HRV group was 58.00 weeks (with a range of 42.50 to 71.25 weeks), whereas for patients in the HSR group, it was 48.00 weeks (ranging from 30.00 to 62.00 weeks). The difference between the two groups was not statistically significant. Additionally, no significant differences were observed between the two groups regarding age, types of NAs used, prior treatment history, treatment regimens, presence of MAFLD, continuation of NAs therapy after the cessation of interferon, baseline alanine aminotransferase (ALT), aspartate aminotransferase (AST), platelets (PLT), HBV DNA levels or baseline HBeAg status before interferon treatment. However, the proportion of males and baseline HBsAg levels were significantly higher in the HRV group compared to the HSR group. Additionally, there were statistically significant differences between the two groups in terms of baseline HBsAg, HBsAb level at the end of treatment and whether the consolidation therapy (weeks) ≥12 weeks (p < 0.05) ( Table 1 ).

Age, gender, types of combined NAs, prior treatment history, treatment regimen, presence of cirrhosis, presence of MAFLD, baseline liver function, HBsAg, HBV DNA, baseline HBeAg status, HBsAb levels at the end of treatment, continuation of NAs treatment after treatment completion, and consolidation therapy (weeks) ≥12 weeks, therapy duration (weeks) were used as independent variables to establish the Cox proportional risk model. The univariate analysis results indicated a significant association between HBsAg reversion and baseline HBsAg, HBsAb at the end of treatment and consolidation therapy (weeks) ≥12 weeks. Multivariate analysis results showed that a high level of HBsAb at the end of treatment was a protective factor, while baseline HBsAg was risk factor for HBsAg reversion (p < 0.05) ( Table 2 ).

From the date of initial HBsAg clearance to the conclusion of the study (August 1, 2024), the follow-up duration ranged from a minimum of 18.6 weeks to a maximum of 289.1 weeks, with a mean follow-up of 96.42 ± 54.1 weeks and a median follow-up of 89.3 weeks (range: 18.6 to 289.1 weeks). The follow-up duration from the cessation of interferon treatment to the conclusion of the study varied from 0 weeks to 277.0 weeks, with a mean of 81.64 ± 51.99 weeks and a median of 73.3 weeks (range: 0.0 to 277.0 weeks).

The average time to HBsAg reversion for the HRV group was 41.71 ± 27.02 weeks after the initial HBsAg clearance and 30.94 ± 25.08 weeks after discontinuation of interferon treatment, with median times of 37.7 weeks and 25.95 weeks, respectively.

Among the 26 patients with HBsAg reversion, five patients (19.23%) experienced reversion within 12 weeks after stopping PegIFNα-2b treatment. Eight patients (30.77%) had reversion between 12 to 24 weeks, and another eight patients (30.77%) between 24 to 48 weeks. Four patients (15.38%) experienced reversion between 48 to 72 weeks, while one patient (3.85%) had reversion after 72 weeks. Notably, 50% (13/26) of HBsAg reversion occurred within 24 weeks, and 80.77% occurred within 48 weeks, with the rate of HBsAg reversion gradually decreasing beyond 48 weeks ( Table 3 , Figure 1 ). We grouped patients based on whether their HBsAg levels were above 500 IU/mL and whether their HBsAb levels were above 100 IU/mL, respectively, and found no statistically significant difference in the distribution of HBsAg reversion time among the groups (p > 0.05) ( Supplementary Table S1 ).

During the entire follow-up period after discontinuation of PegIFNα-2b treatment, a high sustained response rate was observed after HBsAg clearance, which increased progressively with the duration of observation. At 48 weeks post-treatment cessation, the HSR rate reached 95.45%, and this rate stabilized at 100% at 120 weeks.

Among the patients with HBsAg reversion, the latest occurrence was noted at 107.3 weeks following the cessation of PegIFNα-2b; however, this patient had not been followed up for 79 weeks prior to reversion. All cases of HBsAg reversion occurred within 72 weeks, except for this one, and no new reversion events were observed after this period. ( Table 4 , Figure 2 ).

Thus, patients who achieve HBsAg clearance following PegIFNα-2b treatment exhibit favorable long-term persistence, with most reversions occurring within 48 weeks post-treatment, and nearly no patients experiencing reversion after 72 weeks, resulting in a 0% reversion rate after 120 weeks. Clinically, HSR at 48 weeks may serve as a critical follow-up time point following interferon therapy, with HSR at 72 weeks potentially representing an ideal follow-up interval, and HSR at 120 weeks serving as a marker for extended follow-up duration.

Among the 26 patients who experienced HBsAg reversion, the median levels at the time of reversion were as follows: ALT 40.01 U/L (range: 25.02 U/L - 58.00 U/L), AST 19.00 U/L (range: 6.00 U/L - 48.00 U/L), total bilirubin (TBIL) 13.80 μmol/L (range: 5.70 μmol/L - 22.00 μmol/L). Of the 26 patients with HBsAg reversion, only one had a concurrent positive HBV DNA result (117 IU/mL), with a negative HBeAg status, while 96.1% (25/26) maintained HBV DNA negativity. Throughout the treatment and follow-up periods, no adverse events such as liver failure, decompensated cirrhosis, or hepatocellular carcinoma occurred in any patient (detailed information is presented in Table 5 ).

At the time of HBsAg relapse, the average HBsAg level was 0.697 IU/ml, and the HBsAb level was 5.44 IU/ml. During follow-up, the highest HBsAg level observed was 141.84 IU/ml. Among the 26 patients, one lost follow-up and one did not receive treatment, leaving 24 patients who underwent therapy. Of these, 10 received NAs therapy, 3 received PegIFN monotherapy, and 11 received combination therapy with NAs and PegIFN. Eleven of them achieved functional cure, resulting in a functional cure rate of 45.83% (11/24). Furthermore, the recovery rate among patients retreated with Peg-IFN was 78.57% (11/14). The 10 patients in the NA group did not achieve a second cure (detailed information is presented in Supplementary Table S2 ).