Clinical outcomes of treatment-naïve HBeAg-negative patients with chronic hepatitis B virus infection with low serum HBsAg and undetectable HBV DNA

ABSTRACT

Serum hepatitis B surface antigen (HBsAg) level < 100 IU/ml and undetectable hepatitis B virus (HBV) DNA have been recently proposed as an alternate endpoint of “partial cure” in chronic hepatitis B (CHB). We investigated clinical outcomes of hepatitis B e antigen (HBeAg)-negative CHB patients with HBsAg <100 IU/ml and undetectable HBV DNA. Treatment-naïve HBeAg-negative CHB patients with undetectable HBV DNA and normal alanine aminotransferase were retrospectively included from three institutions. Patients were classified into the low HBsAg group (<100 IU/ml) and the high HBsAg group (≥100 IU/ml). Liver fibrosis was evaluated by noninvasive tests (NITs). A total of 1218 patients were included and the median age was 41.5 years. Patients with low HBsAg were older (45.0 vs. 40.0 years, P < 0.001) than those in the high HBsAg group, while the NIT parameters were comparable between groups. During a median follow-up of 25.7 months, patients with low HBsAg achieved a higher HBsAg clearance rate (13.0% vs. 0%, P < 0.001) and a lower rate of significant fibrosis development (2.2% vs. 7.0%, P = 0.049) compared to patients with high HBsAg. No patient developed HCC in either group. HBsAg level was negatively associated with HBsAg clearance (HR 0.213, P < 0.001) and patients with HBsAg < 100 IU/ml had a low risk of significant fibrosis development (HR 0.010, P = 0.002). The optimal cutoff value of HBsAg for predicting HBsAg clearance was 1.1 Log₁₀ IU/ml. Treatment-naïve HBeAg-negative CHB patients with HBsAg <100 IU/ml and undetectable HBV DNA had favourable outcomes with a high rate of HBsAg clearance and a low risk of fibrosis progression.

KEYWORDS:

• Chronic hepatitis B
• hepatitis B surface antigen
• liver fibrosis
• HBsAg clearance

Introduction

Chronic hepatitis B virus (HBV) infection is a global health threat resulting in approximately 800,000 deaths annually worldwide [1–3]. Patients with chronic hepatitis B (CHB) may have a high risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) [2,3]. The virological markers are commonly used to guide the management of patients with HBV infection. Hepatitis B surface antigen (HBsAg), as a hallmark of HBV infection, is confirmed to be associated with natural history, treatment response, and clinical outcomes of patients [4–6]. Serum HBsAg level has been demonstrated to be positively associated with the number of infected hepatocytes and covalently closed circular DNA (cccDNA) levels, which is associated with the development of HCC [7,8]. Suzuki et al. revealed that patients with low HBsAg levels remained at a risk of developing HBV-related HCC, especially for those with high hepatitis B core-related antigen levels [9]. However, contrary results were also reported. It is reported that patients with low HBsAg levels had better long-term outcomes and a higher likelihood of HBsAg clearance, including spontaneous and treatment-related HBsAg clearance [10,11].

Serum HBsAg loss with or without the development of hepatitis B surface antibody (HBsAb) and undetectable HBV DNA levels is regarded as a “functional cure” and an optimal treatment endpoint of CHB, which is associated with low risk of cirrhosis, HCC, and liver-related mortality [2,3]. However, only a small proportion of patients could achieve HBsAg clearance or functional cure after current therapies including pegylated interferon and nucleoside analogues [12–14]. Therefore, an alternative endpoint, “partial cure,” defined as HBeAg-negative, HBsAg <100 IU/mL, and HBV DNA < lower limit of quantitation (LLOQ) 24 weeks off-treatment has been proposed in the recently published guidance [15]. Patients who achieve partial cure have a low risk of hepatitis flare and favourable long-term outcomes according to current guidance [15]. Lee et al. reported that patients with low HBsAg levels had a lower risk of developing cirrhosis and HCC in both hepatitis B e antigen (HBeAg) positive and negative patients with CHB [16]. In addition, patients with low HBsAg levels are more likely to achieve spontaneous or treatment-induced HBsAg clearance [14,17].

However, more evidence is needed to validate that HBsAg <100 IU/ml is a clinically meaningful threshold [15]. Moreover, the clinical outcomes of treatment-naïve HBeAg-negative patients with HBsAg <100 IU/ml, undetectable HBV DNA and normal alanine aminotransferase (ALT) were not yet clear. In the present multicentre, real-world cohort study, we aimed to investigate the association of HBsAg levels and clinical outcomes in treatment-naïve HBeAg-negative patients with undetectable HBV DNA and normal ALT, with a particular focus on patients with HBsAg <100 IU/ml.

Method

Study population

Consecutive treatment-naïve HBeAg-negative patients with chronic HBV infection with undetectable HBV DNA and normal ALT were included from three medical centres between January 2016 and July 2023 in China. Patients who met any of the following exclusion criteria were excluded: (1) coexisting fatty liver disease, other viral hepatitis, immune-related liver diseases, hereditary and metabolic liver diseases; (2) coexisting HCC or other malignancy; (3) alcohol abuse; (4) detectable HBV DNA.

This study was approved by the Institutional Review Board, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University (Ethics number: 2008022) and registered at ClinicalTrials.gov (NCT03097952). The protocol adhered to the ethical guidelines of the Declaration of Helsinki. Due to a retrospective design, the informed consent of the patient was waived by the ethics committees.

Data collection and definition

Demographic data and clinical data of patients, including complete blood counts, liver function tests, virological markers, liver stiffness, and abdominal ultrasound results, were collected from an electronic medical record system. Serum HBsAg and HBsAb were measured by ARCHITECT assay (Abbott Gmbh, United States) in three medical centres, with positive threshold values of 0.05 IU/ml and 10 mIU/ml, respectively. Serum HBsAg <100 IU/ml and HBsAg ≥100 IU/ml were defined as low HBsAg group and high HBsAg group in the present study, respectively. Other parameters were tested by a local laboratory. The upper limit of normal (ULN) of ALT was defined as 40 U/L. Liver stiffness was measured using transient elastography technology by an image-guided detection system (FibroTouch; Wuxi Hisky Medical Technologies Co.) [18].

Liver fibrosis and cirrhosis were evaluated by aspartate aminotransferase (AST) to platelet (PLT) ratio index (APRI), fibrosis index based on 4 factors (FIB-4), transient elastography or abdominal ultrasound. The APRI and FIB-4 were calculated using the following formulas: APRI, [AST (U/L)/ULN of AST]/PLT (10⁹/L) × 100 [19]; FIB-4, [age (years) × AST (U/L)]/[PLT (10⁹/L) × (ALT [U/L])^1/2] [20]. Patients who met any of the following criteria would be considered to have significant liver fibrosis: APRI ≥1.5, FIB-4 ≥ 3.25, or liver stiffness measurement (LSM) value ≥8.0 kPa [21]. The presence of any of the following criteria would be identified as cirrhosis: APRI ≥2.0, FIB-4 ≥ 6.5, LSM value ≥11.0 kPa, or ultrasonographic findings suggestive of cirrhosis.

Follow-up and outcomes

Patients were routinely followed at six months intervals or more frequently. Liver function tests, complete blood counts, virological markers, and imaging examinations were monitored during the follow-up period. The primary outcomes were the development of significant liver fibrosis and HBsAg clearance. The definition of HBsAg clearance was HBsAg <0.05 IU/ml regardless of HBsAb status, while concurrence with HBsAg <0.05 IU/ml and HBsAb ≥10 mIU/ml was regarded as HBsAg seroconversion. Patients presenting any of the above-mentioned criteria of significant fibrosis would be regarded as significant liver fibrosis. Transitioning to reactivation status was another important outcome, and the definition of reactivation was the presence of ALT >40 U/L and HBV DNA ≥2000 IU/ml [3]. The follow-up was censored on the data of identifying HBsAg clearance, significant liver fibrosis, and reactivation status. The follow-up time was calculated from the enrolment to data of identifying outcome events or the last follow-up date for patients in the absence of outcome events. None of the patients received antiviral therapy during the follow-up period in this study.

Statistical analysis

Continuous variables were presented as medians (interquartile range [IQR]) and were compared by independent sample T test or Mann Whitney U test. Categorical data were presented as percentages and were compared by Chi-square or Fisher exact test. The propensity score matching (PSM) method was used to balance the differences in clinical features between groups. The risk factors of significant liver fibrosis and cirrhosis at baseline were identified using logistic regression analysis. Patients without significant liver fibrosis were followed longitudinally, and Cox regression analysis was used to identify the factors of significant liver fibrosis development and HBsAg clearance. Parameters with P < 0.1 in the univariate analysis were included in the multivariate analysis using an input procedure. Other potential confounders, such as age, gender, PLT, ALT, AST, gamma-glutamyl transpeptidase (GGT), and albumin (ALB) were also included in the multivariate analysis. The cumulative incidence of significant liver fibrosis and HBsAg clearance was analysed by Kaplan-Meier curves. A nomogram, for predicting significant liver fibrosis development, has been derived from the multivariable model and calculated. The predictive values of serum HBsAg levels for HBsAg clearance were evaluated using the receiver operating characteristic (ROC) curve. P < 0.05 was regarded as statistically significant. Statistical package for the social sciences software version 23.0 (IBM, Armonk, NY, USA) and R software version 4.2.0 (R Foundation, Vienna, Austria) were used for statistical analysis.

Results

Baseline features of the study population

A total of 4144 untreated HBeAg-negative patients with normal ALT were screened. Of these, 2926 patients, who met exclusion criteria, were excluded, including fatty liver disease (n = 1050), HCC (n = 186), alcohol abuse (n = 164), chronic HCV infection (n = 7), and detectable HBV DNA (n = 1519). Eventually, a total of 1218 patients were included, including 501 patients with low HBsAg (HBsAg <100 IU/ml) and 717 patients with high HBsAg (HBsAg ≥100 IU/ml). Figure S1 shows the process of patient selection. Overall, the median age was 41.5 (IQR 32.0, 53.0) years and 51.3% of patients were male. The median levels of PLT, ALT, AST, and HBsAg were 192.0 (IQR 154.8, 229.0) × 10⁹/L, 19.6 (IQR 14.6, 26.0) U/L, 21.0 (IQR 18.0, 24.5) U/L, and 2.3 (IQR 1.2, 3.0) log₁₀ IU/ml, respectively. In liver fibrosis parameters, the median values of APRI, FIB-4, and LSM were 0.28 (IQR 0.21, 0.37) and 1.02 (IQR 0.68, 1.62), and 5.8 (IQR 4.6, 7.0) kPa, respectively. There were 8.9% and 4.4% of patients identified with significant liver fibrosis and cirrhosis, respectively (Table 1).

Table 1. Comparison of clinical features of CHB patients with low and high HBsAg groups.

Variables	All patients (n = 1218)	Low HBsAg group (n = 501)	High HBsAg group (n = 717)	P-value
Age (yr)	41.5 (32.0, 53.0)	45.0 (33.0, 55.0)	40.0 (32.0, 52.0)	<0.001
Male (%)	625 (51.3)	285 (56.9)	340 (47.4)	0.001
PLT (×10^9/L)	192.0 (154.8, 229.0)	191.0 (153.0, 226.5)	193.0 (157.0, 231.5)	0.659
ALT (U/L)	19.6 (14.6, 26.0)	19.4 (14.3, 26.0)	19.7 (14.8, 26.0)	0.550
AST (U/L)	21.0 (18.0, 24.5)	21.1 (18.1, 25.0)	20.7 (18.0, 24.3)	0.283
GGT (U/L)	17.4 (13.4, 24.8)	18.3 (13.6, 27.4)	17.0 (13.4, 23.5)	0.007
TBIL (μmol/L)	12.2 (9.3, 16.5)	12.5 (9.6, 16.8)	11.8 (9.2, 16.3)	0.086
ALB (g/L)	45.0 (43.2, 46.5)	44.9 (43.2, 46.4)	45.1 (43.2, 46.6)	0.212
HBsAg (log10 IU/ml)	2.3 (1.2, 3.0)	1.0 (0.2, 1.5)	2.9 (2.5, 3.3)	<0.001
APRI	0.28 (0.21, 0.37)	0.28 (0.21, 0.37)	0.27 (0.21, 0.37)	0.610
Significant fibrosis (≥1.5)	19 (1.6)	8 (1.6)	11 (1.5)	0.726
Cirrhosis (≥2.0)	10 (0.8)	5 (1.0)	5 (0.7)	0.567
FIB-4	1.02 (0.68, 1.62)	1.12 (0.71, 1.73)	0.97 (0.67, 1.52)	0.004
Significant fibrosis (≥3.25)	80 (6.6)	34 (6.8)	46 (6.4)	0.797
Cirrhosis (≥6.5)	26 (2.1)	11 (2.2)	15 (2.1)	0.902
Cirrhosis by the US				0.392
Yes	23 (5.0)	7 (3.9)	16 (5.7)	0.392
Available data	460	179	281
Liver stiffness (kPa)	5.8 (4.6, 7.0)	5.9 (4.6, 7.1)	5.8 (4.5, 6.9)	0.394
Significant fibrosis (≥8.0)	21 (14.9)	11 (18.3)	10 (12.3)	0.323
Cirrhosis (≥11.0)	9 (6.4)	5 (8.3)	4 (4.9)	0.415
Available data	141	60	81
Significant fibrosis* (%)	108 (8.9)	50 (10.0)	58 (8.1)	0.253
Cirrhosis* (%)	53 (4.4)	24 (4.8)	29 (4.0)	0.530

* Patients met any of the criteria by non-invasive tests.

ALB: albumin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: gamma-glutamyl transpeptidase; HBsAg: hepatitis B surface antigen; PLT: platelet; TBIL: total bilirubin; US: ultrasound.

In comparison, patients in the low HBsAg group were older (45.0 vs. 40.0 years, P < 0.001), and had a higher proportion of male gender (56.9% vs. 47.4%, P = 0.001) and higher levels of GGT (18.3 vs. 17.0 U/L, P = 0.007) than those in the high HBsAg group. However, other clinical parameters, including PLT, ALT, AST, ALB, APRI, FIB-4, and LSM values, were comparable between the two groups. In addition, the proportion of patients with HBsAg <100 IU/ml gradually increased with age. Male patients had a higher proportion of HBsAg <100 IU/ml (Figure 1).

Figure 1. The distribution of patients with HBsAg <100 IU/ml in different ages (A) and sex (B).

To adjust for confounding factors, PSM was conducted to match the age and sex, which yielded 482 matched pairs. After PSM, the values of APRI, FIB-4 and LSM, and proportions of significant liver fibrosis and cirrhosis remain not significantly different between these two groups (Table S1).

Factors associated with significant liver fibrosis and cirrhosis at baseline

Logistic regression analysis was conducted to identify the factors associated with significant liver fibrosis and cirrhosis at baseline (Table S2). Before PSM, the multivariate analysis revealed that age ≥40 years (odds ratio [OR] 3.500, 95% confidence interval [CI] 1.666, 7.353, P = 0.001), PLT counts (OR 0.975, 95% CI 0.969, 0.980, P < 0.001), AST (OR 1.088, 95% CI 1.052, 1.125, P < 0.001), and ALB levels (OR 0.905, 95% CI 0.844, 0.971, P = 0.006) were independent factors of significant liver fibrosis. Similar parameters were identified to be associated with cirrhosis. However, the HBsAg levels were not associated with both significant liver fibrosis and cirrhosis before and after PSM (Table S3).

Comparison of baseline features of patients in the low HBsAg group and the high HBsAg group who had available follow-up data

A total of 309 patients without significant liver fibrosis at baseline who had available follow-up data were included for analysis, including 138 patients in the low HBsAg group and 171 patients in the high HBsAg group. The low HBsAg group were older (45.0 vs. 38.0 years, P = 0.001) and presented higher values of AST (21.0 vs. 19.9 U/L, P = 0.015), APRI (0.28 vs. 0.26, P = 0.041), and FIB-4 (1.09 vs. 0.91, P = 0.003) compared with those in the high HBsAg group. The median follow-up time was comparable between these two groups (25.7 vs. 25.3 months, P = 0.632). At the end of follow-up, only 3 (2.2%) patients developed significant liver fibrosis in the low HBsAg group, while the higher incidence was observed in the high HBsAg group (7.0%, P = 0.049). The low HBsAg group also had a lower incidence of HBV DNA >2000 IU/ml than the high HBsAg group (2.9% vs. 9.9%, P = 0.014). Of note, eighteen (13.0%) patients in the low HBsAg group achieved spontaneous HBsAg clearance, while none (0%) of patients experienced HBsAg loss in the high HBsAg group (P < 0.001). Three (2.2%) patients achieved HBsAg seroconversion in the low HBsAg group (Table 2). In addition, we also compared the baseline serum HBsAg levels between patients with and without HBsAg clearance, significant liver fibrosis, and HBV DNA >2000 IU/ml, respectively. The results revealed that serum HBsAg levels in patients with HBsAg clearance were significantly lower than in patients without HBsAg clearance, while patients who developed significant liver fibrosis and HBV DNA >2000 IU/ml had higher serum HBsAg levels at baseline (Figure S2).

Table 2. Comparison of clinical features of CHB patients with low and high HBsAg groups who had follow-up data.

Variables	All patients (n = 309)	Low HBsAg group (n = 138)	High HBsAg group (n = 171)	P–value
Age (yr)	41.0 (32.0, 50.0)	45.0 (33.8, 53.2)	38.0 (32.0, 47.0)	0.001
Male (%)	156 (50.5)	69 (50.0)	87 (50.9)	0.878
PLT (×10^9/L)	194.0 (162.0, 226.5)	190.0 (157.8, 222.5)	199.0 (163.0, 230.0)	0.373
ALT (U/L)	19.2 (14.8, 24.3)	19.5 (15.3, 24.8)	19.0 (14.4, 24.0)	0.378
AST (U/L)	20.2 (18.0, 24.0)	21.0 (18.4, 25.2)	19.9 (17.6, 23.0)	0.015
GGT (U/L)	17.1 (13.5, 22.2)	17.7 (13.5, 25.2)	16.7 (13.6, 21.0)	0.187
TBIL (μmol/L)	11.7 (9.3, 15.6)	12.1 (9.4, 16.1)	11.4 (9.2, 15.5)	0.371
ALB (g/L)	45.3 (43.9, 47.0)	45.3 (44.0, 46.8)	45.3 (43.8, 47.2)	0.686
HBsAg (log10 IU/ml)	2.3 (1.2, 2.9)	1.1 (0.2, 1.6)	2.8 (2.5, 3.2)	<0.001
NIT parameters at baseline
APRI	0.26 (0.21, 0.34)	0.28 (0.22, 0.37)	0.26 (0.20, 0.33)	0.041
FIB-4	0.98 (0.70, 1.44)	1.09 (0.75, 1.76)	0.91 (0.67, 1.26)	0.003
Liver stiffness (kPa)	5.8 (4.7, 6.3)	5.4 (4.4, 6.1)	6.0 (4.7, 6.7)	0.193
NITs parameters at the end of follow-up
APRI	0.26 (0.20, 0.33)	0.26 (0.21, 0.32)	0.26 (0.20, 0.35)	0.767
Significant fibrosis (≥1.5) (%)	1 (0.3)	0	1 (0.6)	0.368
Cirrhosis (≥2.0) (%)	1 (0.3)	0	1 (0.6)	0.368
FIB-4	1.07 (0.70, 1.63)	1.12 (0.81, 1.75)	0.99 (0.67, 1.54)	0.070
Significant fibrosis (≥3.25) (%)	8 (2.6)	1 (0.7)	7 (4.1)	0.064
Cirrhosis (≥6.5) (%)	1 (0.3)	0	1 (0.6)	0.368
Cirrhosis by the US				0.922
Yes	2 (1.1)	1 (1.2)	1 (1.1)
Available data	174	81	93
Liver stiffness (kPa)	5.5 (4.3, 6.5)	5.5 (4.5, 6.9)	5.7 (4.3, 6.5)	0.961
Significant fibrosis (≥8.0) (%)	4 (2.4)	1 (2.7)	3 (1.8)	0.369
Cirrhosis (≥11.0) (%)	1 (0.6)	1 (2.7)	0	0.284
Available data	166	37	129
Significant fibrosis (%)	15 (4.9)	3 (2.2)	12 (7.0)	0.049
Cirrhosis (%)	5 (1.6)	2 (1.4)	3 (1.8)	0.833
HBsAg clearance (%)	18 (5.8)	18 (13.0)	0	<0.001
HBsAg seroconversion (%)	3 (1.0)	3 (2.2)	0	0.053
HBV DNA >2000 IU/ml	21 (6.8)	4 (2.9)	17 (9.9)	0.014
Follow-up time (months)	25.7 (16.0, 41.0)	25.7 (17.0, 40.9)	25.3 (15.2, 41.1)	0.632

* Patients met any of the criteria by non-invasive tests.

ALB: albumin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: gamma-glutamyl transpeptidase; HBsAg: hepatitis B surface antigen; NIT: non-invasive test; PLT: platelet; TBIL: total bilirubin; US: ultrasound.

Factors associated with the development of significant liver fibrosis during follow-up

In the univariate analysis, age, sex, PLT, AST, GGT, and HBsAg were associated with the development of significant liver fibrosis. Serum HBsAg levels areclosely associated with the development of significant liver fibrosis (Table 3). In the multivariate analysis, compared to patients with serum HBsAg ≥100 IU/ml, HBsAg <100 IU/ml (HR 0.010, 95% CI 0.001, 0.177, P = 0.002) were negatively associated with the development of significant liver fibrosis after adjusting for other confounders. A nomogram based on serum HBsAg levels was established to predict significant liver fibrosis development, which showed excellent performance for predicting significant liver fibrosis (Figure S3, S4). Furthermore, the Kaplan – Meier survival analysis suggested that patients with high serum HBsAg levels had a significant higher cumulative incidence of significant liver fibrosis than those of the low HBsAg group (P = 0.020) (Figure 2(A)). Given the number of cirrhosis events was less than 10, the Cox regression analysis of cirrhosis development was not performed to avoid bias.

Figure 2. Cumulative incidence of significant liver fibrosis (A), HBsAg clearance (B), and HBV DNA >2000 IU/ml (C) in different HBsAg levels.

Table 3. Cox regression analysis of factors associated with the development of significant fibrosis.

Variables	Univariate		Multivariate-1		Multivariate-2
	HR (95% CI)	P value	HR (95% CI)	P value	HR (95% CI)	P value
Age (yr)
<40	Reference		Reference		Reference
≥40	6.264 (1.400, 28.021)	0.016	8.301 (1.400, 49.238)	0.020	6.444 (1.073, 38.706)	0.042
Sex
Female	Reference		Reference		Reference
Male	3.629 (1.012, 13.016)	0.048	4.413 (0.900, 21.645)	0.067	4.853 (0.982, 23.985)	0.053
PLT (×10^9/L)	0.981 (0.969, 0.993)	0.002	0.985 (0.973, 0.997)	0.014	0.984 (0.972, 0.997)	0.012
ALT (U/L)	1.049 (0.983, 1.121)	0.151	1.053 (0.935, 1.186)	0.391	1.053 (0.940, 1.180)	0.369
AST (U/L)	1.112 (0.996, 1.242)	0.059	1.108 (0.965, 1.274)	0.146	1.154 (1.000, 1.330)	0.049
GGT (U/L)	1.031 (1.004, 1.059)	0.024	1.027 (0.988, 1.067)	0.184	1.046 (1.003, 1.091)	0.034
ALB (g/L)	0.889 (0.730, 1.082)	0.239	0.866 (0.670, 1.119)	0.270	0.827 (0.636, 1.074)	0.154
HBsAg (Log10 IU/ml)	1.920 (1.044, 3.531)	0.036	5.149 (1.869, 14.184)	0.002
HBsAg range (IU/ml)
≥100	Reference				Reference
<100	0.202 (0.045, 0.903)	0.036			0.010 (0.001, 0.177)	0.002

ALB: albumin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CI: confidence interval; GGT: gamma-glutamyl transpeptidase; HBsAg: hepatitis B surface antigen; HR: hazard ratio; PLT: platelet.

Spontaneous HBsAg clearance/seroconversion during follow-up

We further analysed the predictors of HBsAg clearance using Cox regression analysis (Table 4). In the univariate analysis, serum HBsAg levels and AST levels were associated with the development of HBsAg clearance. The HBsAg level (HR 0.213, 95% CI 0.125, 0.364, P < 0.001) remains negatively associated with HBsAg clearance in multivariate analysis. The cumulative HBsAg clearance rate was higher in the low HBsAg group compared to the high HBsAg group (P < 0.001) (Figure 2(B)). The clinical features of 18 patients who achieved HBsAg clearance are shown in Table S4. Overall, more than half of patients were younger than 50 years and male. A majority of patients (88.9%) had very low HBsAg levels (<10 IU/ml), and none of the patients had significant liver fibrosis and cirrhosis at baseline and the last follow-up. However, only three patients achieved HBsAg seroconversion in the present study and all these patients were in the low HBsAg group. Due to the limited number of patients with HBsAg seroconversion, we did not analyse the factors associated with HBsAg seroconversion. In addition, we also analysed the optimal threshold of HBsAg level for predicting HBsAg clearance. The results suggested that the optimal cut-off value of HBsAg was 1.1 Log₁₀ IU/ml for predicting HBsAg clearance, with an area under the receiver operating characteristic curve, sensitivity, and specificity of 0.934, 83.2% and 94.4%, respectively.

Table 4. Cox regression analysis of factors associated with the development of HBsAg clearance.

Variables	Univariate		Multivariate
	HR (95% CI)	P value	HR (95% CI)	P value
Age (yr)
<40	Reference		Reference
≥40	2.063 (0.773, 5.501)	0.148	2.397 (0.696, 8.253)	0.166
Sex
Female	Reference		Reference
Male	1.927 (0.723, 5.141)	0.190	3.163 (0.836, 11.965)	0.090
PLT (×10^9/L)	0.996 (0.986, 1.005)	0.379	1.002 (0.990, 1.013)	0.798
ALT (U/L)	1.026 (0.965, 1.092)	0.406	0.969 (0.870, 1.079)	0.566
AST (U/L)	1.092 (0.988, 1.207)	0.083	0.994 (0.883, 1.119)	0.918
GGT (U/L)	1.000 (0.960, 1.041)	0.984	0.974 (0.927, 1.024)	0.307
ALB (g/L)	0.948 (0.787, 1.142)	0.577	0.953 (0.825, 1.102)	0.519
HBsAg (Log10 IU/ml)	0.212 (0.124, 0.362)	<0.001	0.213 (0.125, 0.364)	<0.001
HBsAg range (IU/ml)*
≥100	-	-	-	-
<100	-	-	-	-

* All patients who achieved HBsAg clearance had baseline HBsAg <100 IU/ml and no patients with baseline HBsAg ≥ 100 IU/ml achieved HBsAg clearance. Thus, cox regression analysis was not performed to avoid bias.

ALB: albumin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CI: confidence interval; GGT: gamma-glutamyl transpeptidase; HBsAg: hepatitis B surface antigen; HR: hazard ratio; PLT: platelet.

Development of HBV DNA >2000 IU/ml, elevated ALT, and HCC during follow-up

A total of 21 patients developed HBV DNA >2000 IU/ml regardless of ALT levels during follow-up, including 4 patients in the low HBsAg group and 17 patients in the high HBsAg group. Multivariate analysis revealed that serum HBsAg levels were a risk factor for HBV DNA >2000 IU/ml. For patients with different serum HBsAg levels, serum HBsAg levels ≥100 IU/ml (HR 4.587, 95% CI 1.430, 14.712, P = 0.010) had a significantly higher risk of HBV DNA >2000 IU/ml (Table S5). Patients in the high HBsAg group had a higher cumulative incidence of HBV DNA >2000 IU/ml compared to the low HBsAg group (P = 0.016) (Figure 2(C)). Of note, only two patients in the high HBsAg group transitioned to reactivation during thefollow-up period, characterized by ALT >40 U/L and HBV DNA ≥2000 IU/ml, while no patient was observed in the low HBsAg group. In addition, we also analysed the cumulative incidence of elevated ALT and AST, respectively. However, the cumulative rate of elevated ALT and AST were comparable between the high HBsAg group and the low HBsAg group (Figure S5). In the present study, no patient developed HCC during follow-up.

Discussion

In this multicenter real-world study, we demonstrated that the serum HBsAg levels were associated with clinical outcomes in treatment-naïve HBeAg-negative patients with undetectable HBV DNA and normal ALT. The prevalence of HBsAg <100 IU/ml was 41.1% in these patients. Patients with low HBsAg (<100 IU/ml) had a very low risk of liver fibrosis development and were more likely to achieve HBsAg clearance.

HBeAg-negative patients with normal ALT and undetectable HBV DNA were considered in the HBeAg-negative chronic infection or inactive carrier phase, and this disease phase is associated with a low risk of adverse events and favourable outcomes [3]. However, previous studies found that the potential risk of reactivation and disease progression are not negligible in this phase [22]. Chen et al. reported that the risk of HCC and liver-related death in patients with HBeAg-negative chronic infection was 4.6-fold and 2.1-fold higher than in individuals not infected with HBV [23]. Although most of the patients with HBeAg-negative chronic infection had persistent biochemical and histological remission, reactivation occurred in nearly 30% of these individuals during their lifetime, which is associated with the development of liver fibrosis, cirrhosis, and HCC [2,3]. However, the factors associated with reactivation in the HBeAg-negative chronic infection phase remain lacking. Serum HBsAg has been demonstrated to associate with covalently closed circular DNA (cccDNA) levels and served as an important marker of disease progression and prognosis in patients with CHB [24]. Serum HBsAg levels present a dynamic change during chronic HBV infection, with a high level in the HBeAg-positive phase and a low level in the HBeAg-negative phase [2,3]. Serum HBsAg levels had been reported to be associated with clinical outcomes of patients with CHB. The REVEAL-HBV study has demonstrated that low HBsAg level was associated with a lower risk for HCC development in HBeAg-negative patients with CHB [16]. Moreover, serum HBsAg level was also reported as a good predictor of long-term HBsAg loss in patients with CHB [25]. In the present study, we found that the serum HBsAg levels were negatively associated with HBsAg loss and positively associated with the development of significant liver fibrosis in treatment-naïve HBeAg-negative patients with undetectable HBV DNA and normal ALT. Patients in the HBeAg-negative chronic infection phase with HBsAg <1000 IU/ml had a lower risk of HCC development, and individuals with HBsAg <100 IU/ml were more likely to achieve HBsAg clearance [26,27]. Current guidance proposed that HBsAg <100 IU/ml is a symbol of partial cure and can be considered as an alternate treatment endpoint [15]. Our results revealed that although patients with HBsAg <100 IU/ml were older, only 2.2% of patients developed significant liver fibrosis, which is significantly lower than patients with relatively high serum HBsAg. However, the rates of cirrhosis development were not significantly different between the two groups (1.4% vs. 1.8%, P = 0.833), which may be associated with a low rate of cirrhosis development during the relatively short follow-up time and limited sample size. The impact of HBsAg levels on the development of cirrhosis and other adverse outcomes needs to be evaluated in future studies with larger sample sizes and longer follow-up periods. More importantly, nearly 13% of patients achieved spontaneous HBsAg clearance in patients with low HBsAg, while none of the patient underwent HBsAg clearance in patients with relatively high HBsAg. Similar results were also reported that low HBsAg level is a crucial predictor of HBsAg clearance and seroconversion [28,29]. These results indicated that patients with HBsAg <100 IU/ml have a favourable outcome and can be an alternate endpoint for patients with chronic HBV infection. We further explored the optimal threshold of HBsAg level for predicting HBsAg clearance in patients with CHB and 1.1 Log₁₀ IU/ml was identified as the optimal threshold for predicting HBsAg clearance in this study. However, given the number of patients who achieved HBsAg clearance is limited, the optimal threshold of HBsAg for predicting HBsAg clearance needs to be validated in future studies. Of note, although all patients included in this study were HBeAg negative and undetectable HBV DNA at baseline, 8.9% and 4.4% of patients were identified as having significant liver fibrosis and cirrhosis. We suspected that liver fibrosis and cirrhosis might be caused by previous active HBV replication in these patients.

Serum HBsAg levels are interrelated with the immune response of chronic HBV infection. A recent study verified that serum HBsAg level is a biomarker for HBV-specific T and B cell responses [30]. A significant decrease in circulating HBsAg level may indicate immune control of HBV [22]. At baseline, we found that patients with low HBsAg were older compared to patients with relatively high HBsAg. Serum HBsAg levels varied during the natural history of the disease, which is a dynamic reduction with the increase of age [4,31,32]. The potential mechanism of HBsAg levels decrease may be explained by the host immunity developing and specific immune cells acting against HBsAg, and the host enters the immune clearance status with age growth [32]. We found that the HBsAg levels were lower in male patients. Similar results were also reported in previous studies, which showed a higher proportion of male patients in CHB patients with low HBsAg levels [4,31,32]. However, the potential impact of gender on HBsAg levels needs further investigation. Although patients in the low HBsAg group had higher AST and GGT levels than the high HBsAg group, the median levels were within the normal range in both groups. Therefore, the clinical significance of the association of AST, and GGT with HBsAg remains not yet clear. In addition, APRI and FIB-4 were also higher in the low HBsAg group compared to the high HBsAg group, whereas none of the patients had significant liver fibrosis at baseline. The difference between APRI and FIB-4 may result from inconsistent age and AST between the high HBsAg group and the low HBsAg group. During the follow-up period, we also observed the transition of immune status and found only two patients transitioned to HBeAg-negative reactivation status. Both patients were observed in the high HBsAg group. Overall, the risk of reactivation is relatively low in HBeAg-negative chronic infection. The cumulative rate of elevated ALT and AST were comparable between the high HBsAg group and low HBsAg group. However, more patients in the high HBsAg group presented HBV DNA ≥2000 IU/ml at the end of follow-up, and the majority of patients had normal ALT. The status of HBeAg-negative patients with HBV DNA ≥2000 IU/ml and normal ALT was regarded as the “indeterminate phase” or “grey zone.” Recently, several studies demonstrated that patients in the indeterminate phase are still at higher risk for disease progression, including liver fibrosis, cirrhosis, and HCC development [33,34]. Huang et al. reported that the risk of HCC development in the persistently indeterminate phase was 14-fold higher than inactive phase [33]. Another study also showed that untreated HBeAg-negative indeterminate phase patients with HBV DNA ≥2000 IU/ml and normal ALT had a higher risk of adverse events than treated active phase patients [34]. Therefore, the low possibility of transitioning to reactivation status or indeterminate phase may suggest a favourable clinical outcome in patients with HBsAg <100 IU/ml.

The strength of the present study is that this is a multi-centre, large sample and real-world CHB cohort study, and consecutive patient enrolment might lead to less selection bias. However, several limitations should be acknowledged. First, this is a retrospective study and the follow-up period is relatively short. Second, the number of outcome events including HBsAg clearance and the development of liver cirrhosis and HCC was small due to the limited sample size and relatively short follow-up time. Therefore, our findings need to be validated in future studies with larger sample sizes and over longer follow-up periods. Third, significant fibrosis and cirrhosis were diagnosed by non-invasive measures. Fourth, all patients included in this study were untreated. The difference in clinical outcomes between treatment-experienced cohort and treatment-naïve cohort with HBsAg <100 IU/ml remains unclear. Fifth, the HBV genotypes were not available in the current study. Previous studies have reported that the HBV genotype was associated with reactivation in patients with HBeAg-negative chronic infection [35]. Sixth, there is a significant difference in spontaneous and treatment-induced HBsAg clearance probability between Caucasians and Asians [15]. However, all patients were Asian in the present study, which may limit the generalizability of our findings to populations with different demographic and clinical characteristics. Moreover, the individualized HBsAg thresholds for predicting clinical outcomes of CHB patients from different ethnic backgrounds require further investigation. Seventh, the laboratory examination was conducted in local hospitals and the inconsistency of testing instruments, reagents, and methods among the three hospitals might have biased the results. Eighth, only 16.7% (3/18) of patients with HBsAg clearance developed HBsAb during the follow-up, but the diversity of HBsAb development is not clear. We did not detect the HBsAg-specific T cell and HBsAg-specific B cell responses due to the retrospective nature and the lack of blood samples of these patients. Last, coinfection of hepatitis D virus (HDV) in patients with CHB is associated with more severe hepatitis and an increased risk of disease progression [36,37]. However, screening for HDV is rarely performed in reality [37–39]. Not all the participants received HDV screening and the data of HDV infection was not available, due to the retrospective nature of the study.

In conclusion, serum HBsAg levels were associated with clinical outcomes in treatment-naïve HBeAg-negative patients with undetectable HBV DNA and normal ALT. Treatment-naïve HBeAg-negative patients with normal ALT, undetectable HBV DNA, and HBsAg <100 IU/ml were more likely to achieve HBsAg clearance and had a very low risk of liver fibrosis development. However, whether the cut-off of HBsAg <100 IU/ml is valid in patients who received therapies deserves further investigation and confirmation.

Author contributions

All authors contributed to this study at different levels. Study concept and design (Rui Huang, Chao Wu, Chuanwu Zhu, Jian Wang); acquisition of data (Li Zhu, Shaoqiu Zhang, Zhiyi Zhang, Tao Fan, Ye Xiong, Fei Cao, Yifan Pan, Yuanyuan Li, Chao Jiang, Shengxia Yin, Xin Tong, Yali Xiong, Juan Xia, Yuxin Chen, Xiaomin Yan, Yong Liu, Xingxiang Liu, Jie Li, Chuanwu Zhu); statistical analysis and interpretation of data (Jian Wang, Shaoqiu Zhang); drafting of the manuscript (Jian Wang, Li Zhu, Rui Huang); critical revision of the manuscript for important intellectual content (Chao Wu, Rui Huang).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the study findings are available upon reasonable request from the corresponding authors (Rui Huang and Chao Wu).

Additional information

Funding

Dr. Rui Huang acknowledges the support from the Nanjing Medical Science and Technique Development Foundation (JQX21002 and QRX17121), Natural Science Foundation of Jiangsu Province (BK20211004) and the Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2022-LCYJ-MS-07). Dr. Jian Wang acknowledges the support from the National Natural Science Foundation of China (82300719), Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2021-LCYJ-PY-43) and Nanjing Medical Science and Technique Development Foundation (YKK21067). Dr. Li Zhu acknowledges the support from the Clinical Disease Diagnosis and Treatment Technology Foundation of Suzhou (LCZX202117).