ABSTRACT
The optimal strategy for chronic hepatitis B virus (HBV) infected patients with negative hepatitis B e‐antigen (HBeAg) and normal alanine aminotransferase (ALT) remains uncertain. This study aimed to evaluate the safety and efficacy of tenofovir alafenamide fumarate (TAF) treatment in this patient population. This prospective, open‐label, randomised controlled trial enrolled HBeAg‐negative patients with normal ALT and randomised them 1:1 to either the treatment group (TAF) or the control group (no treatment). The primary endpoint was the reduction in hepatitis B surface antigen (HBsAg) levels from baseline to week 48. A total of 62 patients were enrolled and followed up by 48 weeks (n = 31 per group). No serious adverse events were reported. At week 48, there was no significant difference in the change in HBsAg between the treatment and control groups [0.01 (−0.06, 0.05) vs. −0.05 (−0.12, 0.06) log10 IU/mL, p = 0.354]. However, HBV DNA levels were significantly lower in the treatment group (0 vs. 2.86 log10 IU/mL, p < 0.001). All patients achieved HBV DNA below 20 IU/mL after treatment. Additionally, chitinase‐3‐like protein 1 level was lower in the treatment group (23.8 vs. 44.8 ng/mL, p = 0.019). TAF was well‐tolerated in HBeAg‐negative patients with normal ALT and low‐level HBV DNA viremia. Treatment for 48 weeks led to a high rate of HBV DNA suppression and may potentially delay liver fibrosis progression. Accordingly, early antiviral treatment may benefit this patient population.
Trial Registration: Clinical trial number: NCT 04231565
Keywords: antiviral treatment, chronic HBV infection, efficacy, inactive, negative HBeAg
1. Introduction
Hepatitis B virus (HBV) infection remains a significant global health concern. In China, an estimated 75 million individuals are infected with HBV, with 78% of these cases being asymptomatic carriers exhibiting no signs of liver damage [1]. A study conducted in China involving 4759 chronic HBV‐infected patients revealed that inactive Hepatitis B surface antigen (HBsAg) carriers, characterised by Hepatitis B e‐antigen (HBeAg) negativity, normal alanine aminotransferase (ALT) levels and HBV DNA levels below 2000 IU/mL, comprised 36.08% of the cohort [2].
Inactive HBV infection, characterised by negative HBeAg, has traditionally been associated with a more favourable prognosis compared to chronic hepatitis B (CHB). Consequently, antiviral therapy has not been routinely recommended for this patient population. However, while the risk of hepatocellular carcinoma (HCC) is lower in inactive HBV carriers than in CHB patients, it remains elevated compared to individuals without HBV infection. A cohort study with a 13.1‐year follow‐up period demonstrated that inactive HBeAg‐negative HBV carriers with HBV DNA levels below 10,000 copies/mL and normal ALT levels exhibited a 4.6‐fold increased risk of HCC and a 2.1‐fold increased risk of liver‐related mortality compared to the general population [3].
Although the spectrum of antiviral treatment indications has broadened in recent years, only approximately 15% of hepatitis B patients in China currently receive antiviral therapy [4]. The European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Diseases (AASLD) guidelines do not recommend antiviral treatment for HBeAg‐negative chronic HBV patients with normal ALT unless they have cirrhosis, a family history of HCC, or extrahepatic manifestations [5, 6]. While the 2022 Chinese guidelines adopt a more proactive approach, a universal ‘Treat All’ strategy has not been implemented [7]. Moreover, there is a paucity of research on using nucleos(t)ide analogues (NAs) for antiviral treatment in HBeAg‐negative chronic HBV‐infected patients with normal ALT and no liver fibrosis. Consequently, this study aimed to assess the safety and efficacy of Tenofovir alafenamide fumarate (TAF) treatment in this specific patient population, providing evidence‐based support for the potential expansion of treatment indications.
2. Methods
2.1. Study Design and Participants
This was a single‐centre, prospective, open‐label, randomised, controlled study. Chronic HBV‐infected patients with negative HBeAg, HBV DNA levels between 20 and 2000 IU/mL and normal ALT levels admitted to the Third Affiliated Hospital of Sun Yat‐sen University between June 2020 and August 2023 were included. Eligible patients were randomly assigned to either a treatment group (TAF) or a control group (blank) in a 1:1 ratio. Randomisation was performed using a computer‐generated random number sequence concealed in sealed envelopes. Investigators sequentially selected envelopes and opened them to reveal the treatment allocation for each participant.
Detailed inclusion criteria were as follows: (1) HBsAg positivity for more than 6 months; (2) HBV DNA between 20 and 2000 IU/mL; (3) ALT levels ≤ 1 × upper limit of normal (ULN) (35 U/L); (4) age between 18 and 65 years; (5) no prior antiviral treatment within the past year; and (6) liver stiffness measurement (LSM) < 5.8 kPa to exclude advanced fibrosis. Exclusion criteria included: (1) use of immunosuppressants; (2) pregnancy or breastfeeding; (3) superinfection with other viral hepatitis; (4) liver cirrhosis or HCC; (5) liver diseases of other etiologies, including autoimmune liver disease, toxic or drug‐induced liver injury, alcoholic liver disease and genetic metabolic liver diseases; (6) HIV infection or other immunodeficiency disorders; (7) diabetes, renal dysfunction, autoimmune diseases, or other organ dysfunctions; (8) family history of liver cirrhosis or HCC.
2.2. Procedures
Patients in the treatment group received oral TAF 25 mg daily with food. The control group received no active treatment but underwent routine follow‐up. If ALT levels exceeded 1 × ULN and HBV DNA remained positive during follow‐up, treatment with 25 mg of TAF daily was recommended, as illustrated in Figure S1. The study protocol has been previously published [8].
All subjects underwent baseline screening and were followed up with a consultation every 12 weeks post‐enrollment until the study's completion. At each follow‐up visit, patients were queried about symptoms, including fatigue, anorexia, nausea and any comorbidities. Additionally, medication adherence was confirmed. Complete blood count, biochemical parameters and serum virological markers (HBV DNA, HBsAg) were reassessed. HBV pgRNA, chitinase 3‐like protein 1 (CHI3L1), LSM and abdominal ultrasound were evaluated at baseline and every 24 weeks post‐study initiation.
2.3. Laboratory Methods
Serum HBV DNA was quantified using a qPCR internal standard method (Abbott) with a lower limit of detection of 10 IU/mL. HBsAg was measured using a quantitative detection kit (Roche) with a detection range of 0.05–52,000 IU/mL. HBeAg was detected using enzyme‐linked immunosorbent assay kits. HBV pgRNA (Supbio) was detected using a PCR fluorescent probe assay with a detection range of 15–1 × 108 copies/mL. Results above the upper limit of detection were reported as such. The CHI3L1 level (Fibro‐CHI kit, Proprium) was measured using a chemiluminescence method and liver fibrosis was considered significant when the CHI3L1 concentration was ≥ 79 ng/mL. LSM was assessed using FibroScan.
2.4. Outcomes
The primary endpoint was a significant difference in the magnitude of change in HBsAg levels between the treatment and control groups from baseline to 48 weeks. Secondary endpoints included the virological response rate (defined as HBV DNA < 20 IU/mL), changes in HBV DNA and HBV pgRNA levels from baseline and the HBsAg loss rate (defined as HBsAg < 0.05 IU/mL). Safety evaluations included the occurrence of adverse events and the incidence of dyslipidemia and ALT elevations exceeding 1 × ULN.
2.5. Statistical Analysis
Given the limited number of previous studies on TAF treatment for HBV‐infected patients with normal ALT levels, sample size estimation relied on TAF treatment data for CHB patients. According to the literature, a 24 week treatment regimen of Tenofovir disoproxil fumarate combined with pegylated interferon α resulted in a 0.91 log10 IU/mL reduction in HBsAg for patients with mild ALT elevation [9]. Another study demonstrated a 0.22 log10 IU/mL decrease in HBsAg over a median 5‐year follow‐up period in untreated HBV patients [10]. Using Power Analysis & Sample Size software, we estimated the sample sizes of the treatment and control groups to achieve 80% power (two‐sided test, α = 0.05) to detect a significant intergroup difference in HBsAg reduction from baseline. Accounting for a 20% dropout rate, 45 patients were required in each group, resulting in a total sample size of 90 patients.
According to the intention‐to‐treat principle, all patients who were randomised to treatment groups, received at least one dose of the study medication or had at least one follow‐up data point were included in the full analysis set (FAS) for efficacy and safety analyses. The primary efficacy indicators were also analysed based on the per‐protocol (PP) set, which included only those patients in the FAS who adhered strictly to the protocol.
Continuous variables were compared between the two groups using Student's t‐test for normally distributed data or the Mann–Whitney U test for non‐normally distributed data. Data were presented as mean (standard deviation) or median (interquartile range). Categorical variables were compared between the two groups using Pearson's chi‐squared test or Fisher's exact test and data are presented as n (%). Correlations were assessed using Spearman's linear regression analysis. All tests were two‐tailed, and statistical significance was defined as a p‐value less than 0.05. Statistical analyses were performed using SPSS version 23.0 software. The plot for correlations was generated using R software (v.4.2.2) package ggplot2 (v.3.4.2) through Hiplot Pro (https://hiplot.com.cn/), a comprehensive web service for biomedical data analysis and visualisation.
Patients with missing primary outcome measures (including HBsAg and HBV DNA) were excluded from the analysis. For other variables with less than 20% missing data, missing values were imputed using the average of the two nearest observed values for the same patient.
This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki (revised 1975) and the Istanbul Convention. The study protocol received approval from the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat‐sen University (approval number [2019] 02‐599‐01). All participants provided written informed consent using a form approved by the Medical Ethics Committee prior to enrollment. The study was prospectively registered on ClinicalTrials.gov (NCT 04231565).
3. Results
3.1. Patient Enrollment
From June 4, 2020 to August 5, 2023, a total of 62 eligible HBeAg‐negative patients were randomly assigned to the TAF group (n = 31) and the control group (n = 31) and had completed 48 weeks of follow‐up (FAS), as illustrated in Figure 1. One patient in the control group withdrew due to pregnancy, and 6 patients (9.7%) were lost to follow‐up, resulting in missing 48 week data. Ultimately, 55 patients were included in the PP analysis set. Patient recruitment for this study is ongoing.
FIGURE 1.
Flowchart of study subjects. @According to the intention‐to‐treat (ITT) principle, sixty‐two patients were classified into full analysis set (FAS) to evaluate efficacy and safety. #fifty‐five patients were classified into per protocol analysis set (PPS) to evaluate efficacy.
3.2. Baseline Characteristics
The mean age of participants in the TAF group was higher than that in the control group (36.0 ± 6.6 vs. 32.6 ± 5.2 years, p = 0.026). No other significant differences in baseline characteristics were observed between the two groups (Table 1).
TABLE 1.
Baseline characteristics of patients (FAS).
| Overall (n = 62) | TAF (n = 31) | Control (n = 31) | p | |
|---|---|---|---|---|
| Age (years) | 34.3 ± 6.1 | 36.0 ± 6.6 | 32.6 ± 5.2 | 0.026 |
| Male, n (%) | 32 (51.6%) | 16 (51.6%) | 16 (51.6%) | 1.000 |
| WBC (109/L) | 6.0 ± 1.2 | 6.0 ± 1.2 | 5.9 ± 1.1 | 0.530 |
| HGB (g/L) | 143.4 ± 16.5 | 143.2 ± 18.6 | 143.7 ± 14.0 | 0.914 |
| PLT (109/L) | 245.3 ± 55.5 | 252.9 ± 53.8 | 236.5 ± 57.0 | 0.266 |
| ALT (U/L) | 21.2 ± 7.5 | 22.0 ± 7.3 | 20.4 ± 7.7 | 0.408 |
| AST (U/L) | 21 ± 5.5 | 22 ± 5.7 | 20 ± 5.2 | 0.158 |
| ALB (g/L) | 47.1 ± 3.3 | 47.0 ± 2.9 | 47.1 ± 3.6 | 0.709 |
| TBIL (μmol/L) | 10.3 (8.7, 12.9) | 11.2 (9.8, 12.7) | 9.7 (8.1, 13.3) | 0.234 |
| DBIL (μmol/L) | 2.3 (1.6, 3.1) | 2.4 (1.9, 3.2) | 2.2 (1.6, 3.1) | 0.588 |
| GGT (U/L) | 16 (14, 22) | 16 (14, 23.5) | 16 (13, 21) | 0.377 |
| TBA (μmol/L) | 1.7 (1.1, 3.7) | 1.7 (1, 3.2) | 1.7 (1.1, 4.2) | 1.000 |
| BUN (mmol/L) | 4.6 (4.1, 5.0) | 4.7 (4.3, 5.0) | 4.6 (4.1, 5.4) | 0.965 |
| Cr (μmol/L) | 62.6 ± 12.1 | 62.3 ± 11.2 | 62.9 ± 13.2 | 0.854 |
| eGFR (mL/min/1.73 m2) | 118.5 (112, 123.9) | 118.4 (112, 123.3) | 119.6 (112, 124.2) | 0.663 |
| P (mmol/L) | 1.1 ± 0.1 | 1.1 ± 0.1 | 1.1 ± 0.2 | 0.524 |
| CHOL (mmol/L) | 4.9 (4.5, 5.5) | 5 (4.5, 5.6) | 4.8 (4.4, 5.4) | 0.587 |
| TG (mmol/L) | 1.0 (0.8, 1.4) | 1.1 (0.8, 1.8) | 0.9 (0.8, 1.3) | 0.140 |
| LDL‐C (mmol/L) | 3 (2.6, 3.5) | 3 (2.6, 3.6) | 3 (2.7, 3.4) | 0.798 |
| HDL‐C (mmol/L) | 1.2 (1.1, 1.6) | 1.2 (1, 1.7) | 1.2 (1.1, 1.4) | 0.707 |
| AFP (ng/mL) | 2.1 (1.6, 2.9) | 2.1 (1.7, 2.9) | 2.2 (1.6, 2.9) | 0.910 |
| HBV DNA (log10, IU/mL) | 2.73 (2.36, 3.05) | 2.81 (2.38, 3.15) | 2.65 (2.23, 3.03) | 0.260 |
| HBV pgRNA (log10, copies/mL) | 0.60 (0, 1.90) | 1.34 (0, 2.45) | 0 (0, 1.51) | 0.086 |
| Negative, n (%) | 31 (50%) | 13 (41.9%) | 18 (58.1%) | 0.204 |
| HBsAg (log10, IU/mL) | 3.08 (2.59, 3.53) | 3.05 (2.59, 3.53) | 3.29 (2.58, 3.54) | 0.418 |
| CHI3L1 a (ng/mL) | 31.1 (26.5, 44.5) | 33.6 (23.3, 42.6) | 29.7 (26.6, 46.7) | 0.940 |
| LSM (KPa) | 4.9 ± 0.7 | 4.8 ± 0.8 | 5.0 ± 0.7 | 0.265 |
| NAFLD, n (%) | 6 (9.7%) | 2 (6.5%) | 4 (12.9%) | 0.668 |
Note: Data are expressed as Mean ± SD, Median (IQR), or n (%). The red highlighting indicates the items with statistically significant p‐values (p < 0.05).
20 and 22 patients underwent CHI3L1 testing in TAF and Control groups, respectively.
3.3. Safety of Antiviral Treatment
3.3.1. Adverse Events
Only one patient in the TAF group experienced mild nausea during treatment, which resolved spontaneously. At week 12, one patient in the TAF group and one in the control group presented with elevated creatine kinase (CK) levels (4436 U/L and 1451 U/L, respectively) and muscle pain following intense physical activity within 3 days prior. The patient in the TAF group did not interrupt treatment, and symptoms resolved with 1 week of rest, with CK levels returning to below 500 U/L. No further CK elevations were observed in either patient during subsequent follow‐up. No patient discontinued treatment due to severe adverse events during the study. No cases of cirrhosis or HCC occurred. At week 48, 5 patients (17.2%) in the TAF group and 3 patients (11.5%) in the control group exhibited ultrasound findings suggestive of fatty liver (p = 0.829).
3.3.2. Biochemical Manifestations
There was no significant difference in the incidence of ALT elevations exceeding 1 × ULN within 48 weeks between the TAF group (19.4%, n = 6) and the control group (16.1%, n = 5), p = 0.74. All ALT elevations were mild (< 2 × ULN). At week 48, the TAF group exhibited slightly lower serum phosphorus levels than the control group, although both groups remained within the normal range. No significant differences were observed between the two groups in other biochemical parameters, including blood lipids (Table 2).
TABLE 2.
Biochemical parameters at week‐48.
| TAF | Control | p | 95% CI for difference | Z | |
|---|---|---|---|---|---|
| ALT (U/L) | 21.7 ± 8.9 | 23.1 ± 12.6 | 0.645 | (−7.21, 4.50) | |
| AST (U/L) | 22.5 ± 6.1 | 21.1 ± 7.0 | 0.438 | (−2.15, 4.90) | |
| ALB (g/L) | 46.1 ± 3.1 | 46.3 ± 4.2 | 0.835 | (−2.19, 1.78) | |
| TBIL (μmol/L) | 10.7 ± 4.6 | 11.3 ± 5.1 | 0.647 | (−3.2, 2.0) | |
| GGT (U/L) | 15 (13, 23.5) | 16 (13.8, 26.3) | 0.560 | −0.583 | |
| Cr (μmol/L) | 61.8 ± 12.3 | 60.6 ± 12.7 | 0.711 | (−5.52, 8.04) | |
| eGFR | 116.7 (110.1, 122.7) | 121.3 (115.4, 124.6) | 0.109 | −1.602 | |
| P (mmol/L) | 1.0 ± 0.1 | 1.1 ± 0.1 | 0.033 | (−0.15, −0.01) | |
| CHOL (mmol/L) | 5.0 (4.2, 5.6) | 5.0 (4.3, 5.4) | 0.855 | −0.182 | |
| Δ CHOL (mmol/L) | −0.24 (−0.61, 0.23) | −0.09 (−0.33, 0.26) | 0.247 | −1.159 | |
| TG (mmol/L) | 0.8 (0.7, 1.2) | 1.0 (0.8, 1.3) | 0.238 | −1.180 | |
| Δ TG (mmol/L) | −0.17 (−0.5, 0.08) | 0.03 (−0.2, 0.27) | 0.052 | −1.947 | |
| LDL‐C (mmol/L) | 3.0 (2.4, 3.8) | 3.0 (2.5, 3.4) | 0.782 | −0.276 | |
| Δ LDL‐C (mmol/L) | −0.11 (−0.59, 0.22) | −0.09 (−0.28, 0.17) | 0.430 | −0.788 | |
| HDL‐C (mmol/L) | 1.3 (1.1, 1.7) | 1.3 (1.1, 1.6) | 0.949 | −0.064 | |
| Δ HDL‐C (mmol/L) | −0.01 (−0.14, 0.08) | 0.03 (−0.05, 0.09) | 0.340 | −0.954 |
Note: Δ, changes in CHOL, TG, LDL‐C and HDL‐C from baseline to week 48. The red highlighting indicates the items with statistically significant p‐values (p < 0.05).
3.4. Treatment Efficacy
3.4.1. HBsAg
Based on the FAS, there were no significant differences in HBsAg levels between the two groups at 12, 24 and 48 weeks and no significant difference in the change in HBsAg from baseline (see Figure 2 and Table 3). At week 48, the TAF group exhibited a 0.01 log10 IU/mL increase in HBsAg from baseline (−0.06, 0.05), while the control group demonstrated a 0.05 log10 IU/mL decrease from baseline (−0.12, 0.06), p = 0.354. The results from the PP analysis were consistent with those from the FAS (see Table S1). No patient in either group achieved HBsAg loss at 48 weeks.
FIGURE 2.
Virological and serological dynamics during follow‐up in both groups. (A–C) Dynamics of HBV DNA, HBV pgRNA and HBsAg values in both groups. (D–F) Changes from baseline in HBV DNA, HBV pgRNA and HBsAg in both groups. The figures show the median and IQR.
TABLE 3.
Virological and serological parameters in both groups during follow‐up.
| TAF | Control | Z | p | |
|---|---|---|---|---|
| Week 12 | ||||
| HBV DNA (log10, IU/mL) | 0 (0, 0) | 2.54 (1.86, 3.22) | −6.501 | < 0.001 |
| Δ HBV DNA (log10, IU/mL) | −2.62 (−3.04, −2.35) | −0.18 (−0.52, 0.47) | −6.319 | < 0.001 |
| HBsAg (log10, IU/mL) | 2.94 (2.58, 3.59) | 3.26 (2.77, 3.46) | −0.808 | 0.419 |
| Δ HBsAg (log10, IU/mL) | 0.02 (−0.06, 0.08) | −0.02 (−0.06, 0.02) | −1.469 | 0.142 |
| Week 24 | ||||
| HBV DNA (log10, IU/mL) | 0 (0, 0) | 2.61 (2.25, 3.39) | −6.566 | < 0.001 |
| Δ HBV DNA (log10, IU/mL) | −2.62 (−3.11, −2.28) | −0.16 (−0.30, 0.28) | −6.521 | < 0.001 |
| HBV RNA (log10, copies/mL) | 0 (0, 2.11) | 0.68 (0, 1.94) | −0.414 | 0.679 |
| Δ HBV pgRNA (log10, copies/mL) | 0 (−1.30, 0) | 0 (0, 1.24) | −2.215 | 0.027 |
| HBsAg (log10, IU/mL) | 2.96 (2.60, 3.49) | 3.23 (2.41, 3.49) | −0.548 | 0.584 |
| Δ HBsAg (log10, IU/mL) | 0 (−0.05, 0.08) | −0.03 (−0.09, 0.03) | −1.522 | 0.128 |
| Week 48 | ||||
| HBV DNA (log10, IU/mL) | 0 (0, 0) | 2.86 (2.45, 3.32) | −6.768 | < 0.001 |
| Δ HBV DNA (log10, IU/mL) | −2.81 (−3.15, −2.37) | 0.16 (−0.09, 0.49) | −6.356 | < 0.001 |
| HBV pgRNA (log10, copies/mL) | 0 (0, 2.39) | 1.61 (0, 2.19) | −0.113 | 0.910 |
| Δ HBV pgRNA (log10, copies/mL) | 0 (−1.30, 0.50) | 0 (−0.09, 1.67) | −1.476 | 0.140 |
| HBsAg (log10, IU/mL) | 2.92 (2.66, 3.32) | 3.18 (2.37, 3.43) | −0.135 | 0.893 |
| Δ HBsAg (log10, IU/mL) | 0.01 (−0.06, 0.05) | −0.05 (−0.12, 0.06) | −0.927 | 0.354 |
Note: Δ, changes from baseline. The red highlighting indicates the items with statistically significant p‐values (p < 0.05).
3.4.2. HBV DNA
Based on the FAS analysis (see Figure 2 and Table 3), from weeks 12 to 48, HBV DNA loads were significantly lower in the TAF group compared to the control group, and the change in HBV DNA from baseline at 48 weeks was also more significant in the TAF group [−2.81 (−3.15, −2.37) vs. 0.16 (−0.09, 0.49) log10 IU/mL, p < 0.001]. The PP analysis yielded results consistent with the FAS (see Table S1). After 48 weeks of treatment, the virological response rate in the TAF group was 100%, while none of the patients in the control group achieved spontaneous HBV DNA suppression below 20 IU/mL by week 48.
3.4.3. HBV pgRNA
Analysis of the FAS showed no significant difference in HBV pgRNA levels between the two groups at 24 and 48 weeks. However, at week 24, the TAF group demonstrated a more significant reduction in HBV pgRNA from baseline compared to the control group [0 (−1.30, 0) vs. 0 (0, 1.24) log10 copies/mL, p = 0.027]. This difference was no longer evident at week 48 (p = 0.140), as shown in Figure 2 and Table 3. At week 48, the proportion of HBV pgRNA‐negative patients was 51.9% in the TAF group and 42.3% in the control group (p = 0.487). The PPS analysis showed no significant differences in HBV pgRNA levels or the change from baseline between the two groups (see Table S1).
3.5. Correlation Between Different Virological Parameters
Correlation analyses between different variables at week 48 were performed. In both the TAF and control groups, no significant correlations were observed between HBV pgRNA and levels of HBV DNA, HBsAg, or ALT. Further analysis revealed a significant positive correlation between the change in HBV pgRNA (from baseline to week 48) and the change in HBV DNA in the TAF group (r = 0.42, p = 0.03). Additionally, the change in HBV pgRNA was also significantly correlated with baseline HBV DNA and HBV pgRNA levels, with correlation coefficients of −0.51 (p = 0.006) and −0.41 (p = 0.03), respectively (Figure 3). However, in the TAF group, neither HBsAg nor Δ HBsAg at week 48 showed significant correlations with HBV DNA, HBV pgRNA, ALT at week 48, or their respective baseline levels.
FIGURE 3.
Correlation of 48‐week Δ HBV pgRNA with other variables. (A, B) Δ HBV pgRNA with Δ HBV DNA and Δ HBsAg in TAF group. (C–E) Δ HBV pgRNA with HBV DNA, HBV pgRNA and HBsAg at baseline in TAF group. (F, G) Δ HBV pgRNA with Δ HBV DNA and Δ HBsAg in Control group. (H–J) Δ HBV pgRNA with HBV DNA, HBV pgRNA and HBsAg at baseline in Control group.
3.6. Examinations Related to Liver Fibrosis
At week 48, the mean LSM values were similar in both groups, 4.5 ± 0.7 kPa in the TAF group and 4.5 ± 0.8 kPa in the Control group (p = 0.828). However, the median CHI3L1 level in the TAF group was lower than that in the Control group, with values of 23.8 (19.0, 38.1) ng/mL and 44.8 (33.7, 53.4) ng/mL, respectively (p = 0.019).
3.7. Stratified Analysis in Patients With a Low ALT Treatment Threshold
In the TAF and control groups, 22 and 21 patients, respectively, met the low ALT treatment threshold criteria (< 30 U/L for males, < 19 U/L for females). To further evaluate the treatment effect in patients with low inflammation, for whom antiviral therapy is not recommended according to current guidelines, a stratified analysis was performed. The results demonstrated that, at 48 weeks, patients with low ALT treatment thresholds in the TAF group had significantly lower HBV DNA (0 vs. 2.68 log10 IU/mL, p < 0.001) and CHI3L1 levels (26.1 vs. 44.8 ng/mL, p = 0.037) compared to the control group, similar to the findings in previous analyses. However, no significant differences were observed between the two groups in HBV pgRNA and HBsAg levels, as detailed in Table S2.
3.8. Analysis of Factors for the HBsAg Decline in the TAF Group
In the TAF group, 13 patients exhibited a decline in HBsAg from baseline at week 48 of treatment (with a median decrease of 0.06 log10 IU/mL), while 16 patients showed an increase from baseline (with a median elevation of 0.04 log10 IU/mL). To investigate potential factors that might influence the HBsAg decline in treated patients, we compared the differences in various indicators between the two subgroups. The results revealed no significant disparities between the patients with HBsAg decline and elevation in terms of age, gender and baseline ALT, AST, ALB, TBIL, GGT, AFP, LSM, HBV DNA, HBV pgRNA, HBsAg levels, as well as ΔHBV DNA and ΔHBV pgRNA during the treatment course. Detailed results are presented in Table S3.
4. Discussion
In this randomised controlled study, the participants were HBeAg‐negative, had normal ALT levels and exhibited no clinical signs of HCC, cirrhosis, or liver fibrosis. Individuals with a family history of cirrhosis or HCC were excluded. Consequently, this population is not recommended for antiviral therapy according to the AASLD guidelines.
No patient who received TAF experienced serious adverse events within 48 weeks. While one patient exhibited a significant increase in CK during treatment, given the history of intense physical activity and the rapid recovery of CK without intervention, it is unlikely that the CK elevation was medication‐related. Post‐treatment, no significant differences were observed between the two groups in biochemical parameters such as ALT, blood lipids and creatinine. Although serum phosphorus was lower in the TAF group, it remained within the normal range. These findings suggest that TAF treatment is safe for HBeAg‐negative patients with normal ALT and no liver fibrosis.
In terms of efficacy, this study demonstrated that 48 weeks of TAF treatment did not significantly impact HBsAg clearance or reduction, consistent with the findings of pre‐marketing Phase 3 clinical trials of TAF (Study 108) [11]. The primary mechanism of action of NAs is the inhibition of viral replication, thereby reducing HBsAg production. Therefore, longer‐term follow‐up is necessary to assess the long‐term impact of TAF treatment on HBsAg. However, HBV DNA levels decreased rapidly following TAF treatment. The treatment group exhibited significantly lower HBV DNA levels compared to the control group from week 12 onwards, with a 100% virological response rate at week 48. This is superior to the efficacy for patients with active CHB. Study 108 included HBeAg‐negative CHB patients with HBV DNA > 2 × 104 IU/mL and ALT > 2 × ULN. After 48 weeks of TAF treatment, 94% of them achieved HBV DNA < 29 IU/mL [11]. This difference may be attributed to the higher baseline HBV DNA levels in the Study 108 population, but it also suggests that for chronic HBV‐infected patients with normal ALT and low viral load, antiviral treatment with NAs can lead to more effective suppression of HBV DNA. Additionally, the response of serum HBV pgRNA was evaluated. Peripheral blood pgRNA may reflect the amount of cccDNA in the liver, and for patients treated with NAs, serum pgRNA may more closely reflect the transcriptional activity of cccDNA [12, 13, 14, 15]. Our correlation analysis revealed that patients with higher baseline HBV DNA and HBV pgRNA levels, as well as greater reductions in HBV DNA following TAF treatment, also exhibited more significant reductions in pgRNA. This suggests that, with the suppression of HBV DNA by TAF, the transcriptional activity of cccDNA may decrease. Furthermore, previous studies have demonstrated that, during long‐term NAs treatment, patients with high pgRNA levels have a significantly higher risk of developing HCC compared to RNA‐negative patients [16]. In our study, although there was no significant difference in pgRNA levels between the two groups at week 48, the proportion of pgRNA‐negative patients increased from 41.9% to 51.9% in the TAF group, while in the control group, this rate decreased from 51.9% at baseline to 42.3%. Therefore, we believe that the increased rate of HBV pgRNA negativity following TAF treatment may be beneficial for long‐term patient outcomes.
The CHI3L1 gene is highly expressed in liver tissue [17]. Due to its significant role in the development and progression of liver fibrosis and cirrhosis [18], CHI3L1 is considered a valuable biomarker for fibrosis, enhancing the accuracy of liver transient elastography diagnostics [19]. One study, including CHB patients with HBV DNA < 103 copies/mL and ALT ≤ 2 × ULN, used liver histology as the gold standard and demonstrated that CHI3L1 is a strong indicator for detecting significant liver fibrosis (≥ S2), with an AUC of 0.94 [17]. Another study found that after 78 weeks of entecavir treatment in CHB patients, CHI3L1 levels significantly decreased, which paralleled improvements in liver pathology, suggesting that CHI3L1 can not only assess liver fibrosis in hepatitis B patients before treatment but also monitor changes in fibrosis during NA therapy [20]. In the present study, CHI3L1 levels were lower in the TAF group compared to the control group at week 48. Although both groups had levels below the threshold for significant liver fibrosis, CHI3L1 levels decreased from baseline in the TAF group, while they increased in the control group. This may indicate that TAF antiviral treatment could help delay the progression of liver fibrosis in inactive HBeAg‐negative HBV‐infected patients.
The primary limitation of this study is the relatively short follow‐up period. Forty‐eight weeks of antiviral treatment may be insufficient to observe a significant decline in HBsAg levels from baseline, and longer‐term treatment is necessary for further evaluation. To mitigate this limitation, we intend to extend the duration of the study to allow for a more comprehensive evaluation of the long‐term efficacy of the antiviral therapy.
5. Conclusion
This study showed that TAF was well‐tolerated in HBeAg‐negative patients with normal ALT and low‐level HBV DNA viremia, for whom antiviral treatment is not currently recommended under the AASLD and EASL guidelines. Treatment for 48 weeks led to a high rate of HBV DNA suppression and may potentially delay liver fibrosis progression. Accordingly, early antiviral treatment may benefit this patient population.
Author Contributions
Study concept and design (L.P., C.X.), acquisition of data (Q.L., Y.Z.), analysis and interpretation of data (Q.L., W.X.), drafting of the manuscript (Q.L.), critical revision of the manuscript for important intellectual content (L.P.), administrative, technical, or material support (W.X., C.X.), study supervision (L.P.) and investigation (X.L., J.L., J.L., X.Z., H.D., L.C., X.Z.). All authors have made a significant contribution to this study and have approved the final manuscript.
Ethics Statement
The study protocol was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat‐sen University (approval number [2019] 02‐599‐01) in accordance with the Helsinki Declaration (revised 1975). The written consent form was obtained prior to enrollment.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Figure S1: Study protocol.
Table S1: Virological and serological parameters at 24 and 48 weeks (PPS).
Table S2: Stratified analysis in patients with a low ALT treatment threshold.
Table S3: Factors for the changes of HBsAg in the TAF group.
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China (82070611) from Liang Peng, Basic and Applied Basic Research Foundation of Guangdong Province (2021A1515220029) from Chan Xie, Guangzhou Municipal Science and Technology Program Key Projects (2023B03J1287 and 2023B01J1007) from Liang Peng, Guangzhou Municipal Science and Technology Program (202102010204) from Chan Xie, Sun Yat‐sen University Clinical Research 5010 Program (2020007) from Liang Peng and the Five‐Year Plan of Third Affiliated Hospital of Sun Yat‐sen University (2024W106) from Liang Peng.
Luo Q., Xu W., Zhang Y., et al., “Tenofovir Alafenamide Fumarate Reduces Virological Replication in HBeAg‐Negative Patients With Normal Alanine Aminotransferase: A 48‐Week Randomised Controlled Trial,” Journal of Viral Hepatitis 33, no. 3 (2026): e70141, 10.1111/jvh.70141.
Contributor Information
Chan Xie, Email: xchan@mail.sysu.edu.cn.
Liang Peng, Email: pliang@mail.sysu.edu.cn.
Data Availability Statement
The datasets generated and analysed during the present study are available from the corresponding author upon reasonable request.
References
- 1. Zheng H., Wang Y., Wang F., et al., “New Progress in HBV Control and the Cascade of Health Care for People Living With HBV in China: Evidence From the Fourth National Serological Survey, 2020,” Lancet Regional Health—Western Pacific 51 (2024): 101193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Yao K., Liu J., Wang J., et al., “Distribution and Clinical Characteristics of Patients With Chronic Hepatitis B Virus Infection in the Greyzone,” Journal of Viral Hepatitis 28, no. 7 (2021): 1025–1033. [DOI] [PubMed] [Google Scholar]
- 3. Chen J. D., Yang H. I., Iloeje U. H., et al., “Carriers of Inactive hepatitisB Virus Are Still at Risk for Hepatocellular Carcinoma and Liverrelated Death,” Gastroenterology 138, no. 5 (2010): 1747–1754. [DOI] [PubMed] [Google Scholar]
- 4. Polaris Observatory Collaborators , “Global Prevalence, Cascade of Care, and Prophylaxis Coverage of Hepatitis B in 2022: A Modelling Study,” Lancet Gastroenterology & Hepatology 8, no. 10 (2023): 879–907. [DOI] [PubMed] [Google Scholar]
- 5. European Association for the Study of the Liver , “EASL 2017 Clinical Practice Guidelines on the Management of Hepatitis B Virus Infection,” Journal of Hepatology 67, no. 2 (2017): 370–398. [DOI] [PubMed] [Google Scholar]
- 6. Terrault N. A., Lok A. S. F., McMahon B. J., et al., “Update on Prevention, Diagnosis, and Treatment of Chronic Hepatitis B: AASLD 2018 Hepatitis B Guidance,” Hepatology 67, no. 4 (2018): 1560–1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Chinese Society of Hepatology, Chinese Medical Association and Chinese Society of Infectious Diseases, Chinese Medical Association , “Guidelines for the Prevention and Treatment of Chronic Hepatitis B (Version 2022),” Chinese Journal of Hepatology 30, no. 12 (2022): 1309–1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Lu W., Lina W., Xuejun L., et al., “Tenofovir Alafenamide Fumarate Therapy in Subjects With Positive HBV‐DNA and Normal Levels of Alanine Transaminase: A Study Protocol for a Randomised Controlled Trial,” BMJ Open 11, no. 8 (2021): e048410. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Perrillo R., Lok A. S., Leonard K., et al., “Association of Alanine Aminotransferase Flares to Hepatitis B Surface Decline During Tenofovir Alone or With Pegylated Interferon Alfa,” American Journal of Gastroenterology 118, no. 11 (2023): 2075–2079. [DOI] [PubMed] [Google Scholar]
- 10. ShihCheng Y., ShengNan L., ChuanMo L., et al., “Combining the HBsAg Decline and HBV DNA Levels Predicts Clinical Outcomes in Patients With Spontaneous HBeAg Seroconversion,” Hepatology International 7, no. 2 (2013): 489–499. [DOI] [PubMed] [Google Scholar]
- 11. Maria B., Edward G., Wai Kay S., et al., “Tenofovir Alafenamide Versus Tenofovir Disoproxil Fumarate for the Treatment of Patients With HBeAg‐Negative Chronic Hepatitis B Virus Infection: A Randomised, Double‐Blind, Phase 3, Non‐Inferiority Trial,” Lancet Gastroenterology & Hepatology 1, no. 3 (2016): 196–206. [DOI] [PubMed] [Google Scholar]
- 12. Wang J., Yu Y., Li G., et al., “Natural History of Serum HBV‐RNA in Chronic HBV Infection,” Journal of Viral Hepatitis 5, no. 9 (2018): 1038–1047. [DOI] [PubMed] [Google Scholar]
- 13. Wang X., Chi X., Wu R., et al., “Serum HBV RNA Correlated With Intrahepatic cccDNA More Strongly Than Other HBV Markers During Peg‐Interferon Treatment,” Virology Journal 18, no. 1 (2021): 4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Gao Y., Li Y., Meng Q., et al., “Serum Hepatitis B Virus DNA, RNA, and HBsAg: Which Correlated Better With Intrahepatic Covalently Closed Circular DNA Before and After Nucleos(t)Ide Analogue Treatment?,” Journal of Clinical Microbiology 55, no. 10 (2017): 2972–2982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Wang J., Yu Y., Li G., et al., “Relationship Between Serum HBV‐RNA Levels and Intrahepatic Viral as Well as Histologic Activity Markers in Entecavir‐Treated Patients,” Journal of Hepatology 68, no. 1 (2018): 16–24. [DOI] [PubMed] [Google Scholar]
- 16. Liu S., Deng R., Zhou B., et al., “Association of Serum Hepatitis B Virus RNA With Hepatocellular Carcinoma Risk in Chronic Hepatitis B Patients Under Nucleos(t)ide Analogues Therapy,” Journal of Infectious Diseases 226, no. 5 (2022): 881–890. [DOI] [PubMed] [Google Scholar]
- 17. Huang H., Wu T., Mao J., et al., “CHI3L1 Is a Liver‐Enriched, Noninvasive Biomarker That Can Be Used to Stage and Diagnose Substantial Hepatic Fibrosis,” OMICS: A Journal of Integrative Biology 19, no. 6 (2015): 339–345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Tao H., Yang J.‐J., Shi K.‐H., et al., “The Significance of YKL‐40 Protein in Liver Fibrosis,” Inflammation Research 63 (2014): 249–254. [DOI] [PubMed] [Google Scholar]
- 19. Rath T., Roderfeld M., Guler C., et al., “YKL‐40 and Transient Elastography, a Powerful Team to Assess Hepatic Fibrosis,” Scandinavian Journal of Gastroenterology 46 (2011): 1369–1380. [DOI] [PubMed] [Google Scholar]
- 20. Wang L., Liu T., Zhou J., You H., and Jia J., “Changes in Serum Chitinase 3‐Like 1 Levels Correlate With Changes in Liver Fibrosis Measured by Two Established Quantitative Methods in Chronic Hepatitis B Patients Following Antiviral Therapy,” Hepatology Research 48, no. 3 (2018): E283–E290. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1: Study protocol.
Table S1: Virological and serological parameters at 24 and 48 weeks (PPS).
Table S2: Stratified analysis in patients with a low ALT treatment threshold.
Table S3: Factors for the changes of HBsAg in the TAF group.
Data Availability Statement
The datasets generated and analysed during the present study are available from the corresponding author upon reasonable request.