Abstract
The value of detecting hepatitis B virus (HBV), pregenomic RNA (pgRNA), and hepatitis B core-related antigen (HBcrAg), both separately and jointly, in the management of HBV patients undergoing treatment with Nucleotide Analog was investigated. A total of 149 HBV patients who were being treated with Nucleotide Analog were enrolled in this study. The quantitative levels of HBV pgRNA and HBcrAg in the sera of these patients were determined, aiming to comprehend their replication levels and expression during the course of antiviral therapy. The patients were separated into 3 groups based on treatment duration: treatment time ≤ 12 months, treatment time ranging from 12 months to <60 months, and treatment time ≥ 60 months. Significantly different levels of HBcrAg and HBV pgRNA were observed among 3 groups (P < .05). In the group of patients with positive hepatitis B e antigen, both HBcrAg and pgRNA levels were higher compared to the group with negative hepatitis B e antigen, and this difference between the 2 groups was found to be statistically significant. Stratified analysis based on levels of hepatitis B surface antigen (HBsAg) revealed that the group with HBsAg levels < 100 IU/mL had lower levels of both HBcrAg and pgRNA compared to the group with HBsAg levels ≥ 100 IU/mL (P < .001). Following antiviral therapy, various degrees of transcription of covalently closed circular DNA continue to exist within the liver of HBV patients. The levels of serum HBcrAg and HBV pgRNA vary among patients with different treatment durations, indicating their efficacy in evaluating disease conditions during antiviral therapy.
Keywords: HBcrAg, HBV, HBV pgRNA, NAs
1. Introduction
Despite their utility in CHB monitoring, current hepatitis B virus (HBV) markers fail to reliably predict clinical outcomes.[1] While hepatic covalently closed circular DNA (cccDNA) remains the benchmark for tracking HBV replication, its detection and measurement still present challenges.[2,3] Serum HBV pregenomic RNA (pgRNA) has demonstrated its worth as a biomarker in various clinical scenarios.[3,4] The extended detectability of HBV pgRNA even years after commencing Nucleotide Analog (NAs) treatment[5–11] positions it as a potential surrogate for assessing cccDNA transcriptional activity in the liver. HBV core-related antigen (HBcrAg), originating from cccDNA transcription within infected hepatocyte nuclei, holds the potential to mirror cccDNA status through its serum presence and quantitative level.[3,7,12–15] This underlines the prospect of HBcrAg testing supplanting cccDNA as a pivotal factor in antiviral therapy. Building upon this foundation, we posit the hypothesis that serum HBV pgRNA and HBcrAg could serve as alternative proxies for intrahepatic cccDNA, with their quantification fluctuating throughout the antiviral therapy course in CHB patients. The combined assessment of serum HBV pgRNA and HBcrAg provides an improved evaluation of patient status, enabling more effective management of chronic HBV-infected individuals. This framework establishes the theoretical groundwork for advancing the individual or combined utilization of HBV pgRNA and HBcrAg detection in clinical CHB applications.
2. Methods
2.1. Patients
The study included CHB patients receiving oral NAs antiviral therapy as outpatients and inpatients at the Department of Infection, Affiliated Hospital of Nantong University, from November 1, 2021, to September 30, 2022. Serum specimens from these patients were stored at −80 °C. The study was ethically approved by the Ethics Committee of The Affiliated Hospital of Nantong University (2022-L001). All participants were informed about the study protocol and provided written informed consent to participate in the study.
2.2. Inclusion criterias
Participants were included based on the following criteria: ① Age between 18 and 80 years, irrespective of gender; ② positive hepatitis B surface antigen (HBsAg) for more than 6 months; ③ antiviral NAs treatment duration exceeding 3 months; ④ willingness to participate and provide informed consent.
2.3. Exclusion criterias
Patients were excluded if they met any of the following conditions: ① co-infection with hepatitis C virus, hepatitis D virus, human immunodeficiency virus, Epstein-Barr virus/human herpes virus type 4, cytomegalovirus, or herpes simplex virus; ② presence of liver diseases other than hepatitis B, such as drug-induced hepatitis, autoimmune hepatitis, alcoholic hepatitis, hepatomegaly, or other diagnosed liver conditions; ③ diagnosis of malignant tumors other than hepatitis B-related hepatocellular carcinoma (HCC); ④ use of immunosuppressants or hormones; ⑤ history of drug or alcohol abuse; ⑥ pregnancy.
2.4. Standards for clustering
Patients were separated according to: 1. treatment duration into 3 groups: treatment time ≤ 12 months, 12 months < treatment time < 60 months, and treatment time ≥ 60 months. 2. Hepatitis B e antigen (HBeAg) status, resulting in HBeAg-positive and HBeAg-negative groups. 3. Level of HBsAg expression, leading to HBsAg ≥ 100 IU/ml group and HBsAg < 100 IU/mL group.
2.5. Detection of HBV pgRNA
Approximately 2 to 5 mL venous blood was collected from study subjects into additive-free collection vessels. The blood was left at room temperature (15–30 °C) for 30 to 60 minutes, then centrifuged at 1500 to 2000 rpm for 5 minutes. The resulting separation of blood cells and serum was used for measurement. The assay used was the hepatitis B virus nucleic acid assay kit (RNA capture probe method) from Shanghai Rendu Biotechnology Co., Ltd., Shanghai. Reported HBV RNA concentration was in units of 100 copies/mL.
2.6. Detection of HBcrAg
Whole blood samples collected in serum separation tubes were incubated at room temperature for 2 hours or at 4 °C overnight. Afterward, centrifugation at 1000 × g for 20 minutes yielded the supernatant for measurement. The assay utilized was the Human hepatitis B virus core antigen associated antigen (HBcrAg) enzyme-linked immunosorbent assay kit from Shanghai Jianglai Biotechnology Co., Ltd., Shanghai.
2.7. Statistical analysis
The statistical analysis was carried out using SPSS Statistics 25.0 software. First, the Kolmogorov–Smirnov normality test was applied to the data. For data conforming to a normal distribution, descriptive statistics were presented as mean ± standard deviation. Data not adhering to a normal distribution were described using median (interquartile range) [MEDIAN (IQR)]. Notably, serum HBV DNA, HBV pgRNA, and HBcrAg mean levels were log-transformed (base 10) and expressed as median (interquartile spacing). Continuous and categorical variables across different observation groups were compared using Chi-square or Mann–Whitney U tests. To compare multiple groups, the Kruskal–Wallis test was employed. Pearson or Spearman correlation analysis was utilized to assess the correlation between 2 variables. A significance level of 0.05 was adopted, with P < .05 considered statistically significant. Graphical representations were created using GraphPad Prism 8.0 software.
3. Results
3.1. Basic data
A total of 200 CHB patients undergoing oral NAs antiviral therapy at the Affiliated Hospital of Nantong University between November 01, 2021, and September 30, 2022, were initially gathered. After excluding 51 cases due to coexisting viral infections or incomplete data, a final cohort of 149 patients was included in the study. Among these participants, 100 were male and 49 were female, with 73 patients being HBeAg(+) and 76 HBeAg(−). The distribution of patients based on treatment duration was as follows: 56 patients with treatment time ≤ 12 months, 47 patients with 12 months < treatment time < 60 months, and 46 patients with treatment time ≥ 60 months. In terms of gender distribution, there was no statistically significant variance among the 3 treatment duration groups (P > .05). The age distribution across the groups was as follows: 40.50 (33.25, 52.00) years for the treatment time ≤ 12 months group, 42.00 (32.00, 54.00) years for the 12 months < treatment time < 60 months group, and 46.50 (40.00, 55.00) years for the treatment time ≥ 60 months group. Notably, there was no significant difference in age distribution among these groups (P > .05). Further examination of AFP levels showed minimal variation between the groups: 3.20 (2.21, 4.75) ng/mL for the treatment time ≤ 12 months group, 2.80 (2.16, 3.69) ng/mL for the 12 months < treatment time < 60 months group, and 2.48 (1.82, 3.19) ng/mL for the treatment time ≥ 60 months group. The AFP level differences among these groups were not statistically significant (P > .05). However, noteworthy differences emerged when considering the levels of HBcrAg, HBV pgRNA, HBV DNA, AST, and ALT among the 3 treatment duration groups (P < .05, Table 1).
Table 1.
Basic patient information.
| Number of samples (n = 149) |
T ≤ 12 m (n = 56) |
12 < T < 60 m (n = 47) |
T ≥ 60 m (n = 46) |
P value | |
|---|---|---|---|---|---|
| Gender (male/female) | 100/49 | 34/22 | 31/16 | 35/11 | .253 |
| Age (year) | 42.00 (34.00,53.00) | 40.50 (33.25,52.00) | 42.00 (32.00,54.00) | 46.50 (40.00,55.00) | .131 |
| HBcrAg (log10 mU/mL) |
3.57 (3.10,4.16) | 4.04 (3.43,4.51) | 3.51 (3.15,3.87) | 3.31 (3.00,3.68) | <.001** |
| HBV pgRNA (log10 copies/mL) |
2.93 (2.00,5.05) | 5.01 (2.75,7.60) | 2.93 (2.08,4.56) | 2.16 (1.92,2.93) | <.001** |
| HBV DNA (log10 IU/mL) |
1.70 (1.70,2.14) | 2.25 (1.70,3.45) | 1.70 (1.70,1.70) | 1.70 (1.70,1.70) | <.001** |
| AST (U/L) | 26.00 (20.50,35.00) | 34.00 (25.50,43.00) | 24.00 (21.00,27.00) | 23.50 (19.00,30.25) | <.001** |
| ALT (U/L) | 28.00 (18.00,42.50) | 34.50 (24.00,53.25) | 27.00 (18.00,33.00) | 22.50 (17.00,39.50) | .005* |
| AFP (ng/mL) | 2.68 (1.95,3.65) | 3.20 (2.21,4.75) | 2.80 (2.16,3.69) | 2.48 (1.82,3.19) | .057 |
P < .05.
P < .001.
Further subgroup comparisons yielded the following insights: The HBcrAg level in the treatment time ≥ 60 months and 12 months < treatment time < 60 months groups was lower than in the treatment time ≤ 12 months group, with significant differences (T ≥ 60 months vs T ≤ 12 months: 3.31 vs 4.04 log10 mU/mL, P < .001; 12 < T < 60 months vs. T ≤ 12 months: 3.51 vs 4.04 log10 mU/mL, P < .05). However, no statistically significant distinction was observed between the 12 months < treatment time < 60 months and treatment time ≥ 60 months groups (P > .05). HBV pgRNA levels displayed significant differences among the 3 groups (T ≤ 12 months vs T ≥ 60 months: 5.01 vs 2.16 log10 copies/mL, P < .001; 12 < T < 60 months vs T ≥ 60 months: 2.93 vs 2.16 log10 copies/mL, P < .05; 12 < T < 60 months vs T ≤ 12 months: 2.93 vs 5.01 log10 copies/mL, P < .05). Levels of HBV DNA, AST, and ALT were lower in the treatment time ≥ 60 months and 12 months < treatment time < 60 months groups compared to the treatment time ≤ 12 months group, with significant differences (P < .05). Conversely, no significant differences were noted in HBV DNA, AST, and ALT levels between the 12 months < treatment time < 60 months and treatment time ≥ 60 months groups (P > .05).
3.2. Correlation analysis
We explored the interrelationship between HBcrAg, HBV pgRNA, HBV DNA, and baseline characteristics. A mutual correlation was observed among HBcrAg, HBV pgRNA, and HBV DNA. The highest correlation coefficient was between HBV pgRNA and HBV DNA (R = 0.741, P < .001). The next in line was the correlation coefficient between HBcrAg and HBV pgRNA (R = 0.583, P < .001), and the correlation between HBcrAg and HBV DNA exhibited the lowest value (R = 0.433, P < .001). The corresponding linear regression equations were Y = 2.090 * X − 0.6229, Y = 1.899 * X − 3.263, and Y = 0.3750 * X + 2.903. The determination coefficients (R2) were 0.5484, 0.3394, and 0.1876, respectively. The substantial correlation observed between serum HBcrAg, HBV pgRNA, and HBV DNA levels can be attributed to their common derivation from cccDNA.
3.3. HBcrAg and pgRNA levels in different HBeAg states
Subgroup analysis based on HBeAg status revealed that HBcrAg levels were significantly higher in the HBeAg(+) group (3.89 [3.43, 4.44] log10 mU/mL) compared to the HBeAg(−) group (3.29 [3.00, 3.79] log10 mU/mL), with a statistically significant difference (P < .001). Similarly, the pgRNA level in the HBeAg(+) group (5.04 [3.36, 7.20] log10 copies/mL) exceeded that in the HBeAg(−) group (2.02 [1.69, 2.65] log10 copies/mL), and this difference was also statistically significant (P < .001) (Table 2). This finding suggests heightened HBeAg replication in the HBeAg(+) group compared to the HBeAg(−) group.
Table 2.
HBcrAg and pgRNA levels in different HBeAg states.
| HBeAg(+) | HBeAg(−) | P value | |
|---|---|---|---|
| (n = 73) | (n = 76) | ||
| HBcrAg | 3.89 (3.43,4.44) | 3.29 (3.00,3.79) | <.001** |
| (log10 mU/mL) | |||
| HBV pgRNA | 5.04 (3.36,7.20) | 2.02 (1.69,2.65) | <.001** |
| (log10 copies/mL) |
* P < .05.
P < .001.
Both HBcrAg and HBV pgRNA levels exhibited a significant correlation with HBeAg status. The levels of HBcrAg and pgRNA were notably elevated in the HBeAg(+) group when contrasted with the HBeAg(−) group. Subsequent ROC curve analysis highlighted the following outcomes: The AUC of HBcrAg in predicting HBeAg(+) was 0.754 (95% CI 0.677 to 0.832, P < .001). The calculated cutoff value stood at 3.20 log10 mU/mL, with sensitivity and specificity values of 91.8% and 48.7%, respectively. In comparison, the AUC of HBV pgRNA for predicting HBeAg(+) was 0.945 (95% CI 0.911 to 0.979, P < .001), using a cutoff value of 2.99 log10 copies/mL. This yielded sensitivity and specificity values of 87.7% and 89.5%, respectively (Table 3).
Table 3.
ROC curves of HBcrAg compared to HBV pgRNA predicting HBeAg(+).
| AUC | Standard error | P value | 95%CI | ||
|---|---|---|---|---|---|
| Lower limit | Upper limit | ||||
| HBcrAg | 0.754 | 0.04 | <.001 | 0.677 | 0.832 |
| HBV pgRNA | 0.945 | 0.017 | <.001 | 0.911 | 0.979 |
| HBcrAg + HBV pgRNA | 0.949 | 0.018 | <.001 | 0.914 | 0.984 |
3.4. HBcrAg, pgRNA levels corresponding to different HBsAg levels
A stratified analysis was performed based on HBsAg levels, revealing the following findings: In the HBsAg < 100 IU/mL group, HBcrAg levels were significantly lower, measuring 2.88 (2.84, 2.92) log10 mU/mL, in contrast to the HBsAg ≥ 100 IU/mL group with levels of 3.68 (3.26, 4.20) log10 mU/mL. This disparity between the 2 groups was statistically significant (P < .001). Moreover, the pgRNA levels exhibited a considerable discrepancy. The HBsAg < 100 IU/mL group demonstrated pgRNA levels of 1.69 (1.69, 2.47) log10 copies/mL, whereas the HBsAg ≥ 100 IU/mL group displayed levels of 3.04 (2.16, 5.18) log10 copies/mL. This difference was also statistically significant (P < .001) (Table 4).
Table 4.
HBcrAg and pgRNA levels corresponding to different HBsAg levels.
| HBsAg < 100 | HBsAg ≥ 100 | P value | |
|---|---|---|---|
| (n = 13) | (n = 136) | ||
| HBcrAg | 2.88 (2.84,2.92) | 3.68 (3.26,4.20) | <.001 |
| (log10 mU/mL) | |||
| HBV pgRNA | 1.69 (1.69,2.47) | 3.04 (2.16,5.18) | <.001 |
| (log10 copies/mL) |
3.5. Comparison of HBeAg-negative patients with different HBsAg levels
Within this study, a total of 76 HBeAg(−) patients were included. Stratified analysis was carried out based on HBsAg levels, yielding the subsequent outcomes: in the HBsAg < 100 IU/mL subgroup, the HBcrAg and HBV pgRNA levels were 2.89 (2.85, 2.92) log10 mU/mL and 1.69 (1.69, 1.84) log10 copies/mL, respectively. These levels were notably lower than those found in the HBsAg ≥ 100 IU/mL subgroup, which exhibited levels of 3.41 (3.07, 3.84) log10 mU/mL for HBcrAg and 2.18 (2.00, 2.67) log10 copies/mL for HBV pgRNA. Furthermore, a statistically significant distinction was evident between the 2 subgroups (P < .05) (Table 5).
Table 5.
Comparison of HBeAg-negative patients with different HBsAg levels.
| HBsAg < 100 (n = 9) |
HBsAg ≥ 100 (n = 67) |
P value | |
|---|---|---|---|
| Age (years) | 55.00 (41.50,64.00) | 44.00 (37.00,53.00) | .018 |
| HBcrAg (log10 mU/mL) |
2.89 (2.85,2.92) | 3.41 (3.07,3.84) | <.05 |
| HBV pgRNA (log10 copies/mL) |
1.69 (1.69,1.84) | 2.18 (2.00,2.67) | <.05 |
The HBcrAg and pgRNA levels demonstrated a positive correlation with HBsAg levels (R = 0.449, P < .001; R = 0.238, P < .05, respectively). Additionally, in HBeAg-negative patients with HBsAg < 100 IU/mL, both HBcrAg and pgRNA levels were lower compared to the HBsAg ≥ 100 IU/mL group. The ROC curve analysis yielded the following results: HBcrAg exhibited a robust prediction for HBsAg < 100 IU/mL, with an AUC of 0.953 (95% CI 0.901 to 1.000, P < .001) and a cutoff value of 2.94 log10 mU/mL. The sensitivity and specificity stood at 92.5% and 88.9%, respectively. In contrast, HBV pgRNA’s predictive capacity for HBsAg < 100 IU/mL was slightly lower than that of HBcrAg, with an AUC difference of 0.831 (95% CI 0.720 to 0.942, P < .05) and a cutoff value of 1.85 log10 copies/mL. Sensitivity and specificity values were 79.1% and 77.8%, respectively (Table 6).
Table 6.
ROC curves of HBcrAg compared with HBV pgRNA predicting HBsAg < 100 IU/mL.
| AUC | Standard error | P value | 95%CI | ||
|---|---|---|---|---|---|
| Lower limit | Upper limit | ||||
| HBcrAg | 0.953 | 0.027 | <.05 | 0.901 | 1.000 |
| HBV pgRNA | 0.831 | 0.057 | <.05 | 0.720 | 0.942 |
| HBcrAg + HBV pgRNA | 0.972 | 0.018 | <.05 | 0.937 | 1.000 |
4. Discussion
In patients with CHB undergoing NAs treatment, serum HBV DNA levels often fall below the detectable limit. This indicates that NAs effectively hinder the viral reverse transcription process, without directly affecting the transcription of cccDNA.[16,17] Meanwhile, histological alterations in the livers of CHB patients receiving NAs correlate with serum HBV RNA levels. This implies that serum HBV RNA levels could potentially serve as an indicator of intrahepatic cccDNA transcriptional activity.[6] Recent research has unveiled that serum HBV RNA is contained within prepackaged genomic RNA (pgRNA) particles, potentially residing within virus-like entities.[11,18] The combination of serum HBV DNA levels and pgRNA levels appears to provide a more comprehensive reflection of intrahepatic cccDNA activity in primary CHB patients. In this context, HBcrAg, a composite of 3 distinct proteins—HBeAg, HBcAg, and p22cr—has emerged as a proposed serum marker for disease monitoring and prognosis in CHB.[19,20] In comparison to HBsAg, HBcrAg may represent a superior alternative marker for assessing cccDNA and its transcriptional activity in the liver. The utility of HBcrAg extends beyond disease surveillance, demonstrating promise in predicting treatment response, relapse post-NA discontinuation, as well as disease progression in cirrhosis and the emergence and recurrence of HCC. The emergence of HBcrAg and HBV pgRNA as innovative biomarkers holds profound significance in the management of HBV-infected patients, offering enhanced avenues for treatment assessment and prognosis.
Within this study, serum HBV pgRNA and HBcrAg were detected, revealing a consistent trend where prolonged treatment duration corresponded to lower levels of HBcrAg and HBV pgRNA. Even after 5 years of antiviral treatment, a portion of patients still exhibited detectable levels of HBcrAg and HBV pgRNA in their serum. Wang observations align with this, as patients treated with NAs displayed reduced HBV DNA levels, yet liver histology indicated inflammation and fibrosis.[6] Wang[20] further found that 63.64% of CHB patients with HBV DNA below the detection threshold still tested positive for serum HBV pgRNA. Our study corroborates these findings, highlighting that virologic rebound in patients with positive HBV pgRNA was 7 times more prevalent than in those with negative HBV pgRNA. Thus, serum HBV pgRNA levels prove more effective in reflecting intrahepatic viral transcriptional activity and predicting virological rebound during NAs treatment. Elevated HBV pgRNA levels were noted in patients experiencing clinical relapse after drug discontinuation, while patients without clinical relapse exhibited consistently low HBV pgRNA levels. Assessing 108 CHB patients with at least a 5-year complete response to antiviral therapy and eligibility for discontinuation, Shi-Wan Zhang[21] demonstrated that HBV pgRNA and HBcrAg levels at discontinuation were notably higher in the relapse group than the non-relapse group. In our study, HBcrAg levels were lower in the groups with treatment duration ≥ 60 months and 12 months < treatment time < 60 months compared to the group with treatment time ≤ 12 months. No differences were observed between the 12 months < treatment time < 60 months group and the treatment time ≥ 60 months group. Conversely, HBV pgRNA levels differed across all 3 groups, inversely correlated with treatment duration. These outcomes emphasize the efficacy of serum HBcrAg and HBV pgRNA in assessing CHB progression during antiviral therapy. Consequently, even in cases where CHB patients receive NAs treatment for over 5 years, maintain normal liver function, and have controlled hepatitis B virus quantification within the normal range, screening for HBcrAg and HBV pgRNA remains crucial prior to medication discontinuation. The diminished expression of these markers indicates a higher margin of safety post-discontinuation. Given that this study adopted a single-center cross-sectional design with a limited sample size, future efforts will focus on expanding the sample size to validate the scientific robustness of these findings. Subsequent work will delve into exploring the utility of HBcrAg and HBV pgRNA for monitoring secure NAs discontinuation through follow-up assessments within this population.
We also investigated the correlations among HBcrAg, HBV pgRNA, and HBV DNA in relation to baseline profiles. Notably, HBcrAg, HBV pgRNA, and HBV DNA exhibited interconnections, with the highest correlation coefficient observed between HBV pgRNA and HBV DNA (R = 0.741, P < .001), and the lowest between HBcrAg and HBV DNA (R = 0.433, P < .001). As both HBV pgRNA and HBcrAg stem from cccDNA, this underlying association aids in comprehending the relatively robust link between serum HBcrAg, HBV pgRNA, and HBV DNA levels. Notably, the stronger correlation coefficients of HBV pgRNA with both HBcrAg and HBV DNA position it as a more reliable surrogate marker for cccDNA.
In an investigation by Van Campenhout,[22] HBeAg status emerged as the leading independent factor associated with HBV pgRNA levels. Other pertinent studies[11–17,23–27] also highlight the potential of serum HBcrAg and HBV pgRNA levels as innovative markers for predicting serological responses in HBeAg-positive patients undergoing NAs treatment. Our study concurs, observing significantly higher HBcrAg and HBV pgRNA levels in the HBeAg-positive group compared to the HBeAg-negative group—a finding aligned with a peer-reviewed study.[28] This divergence could stem from heightened cccDNA transcriptional activity in HBeAg-positive patients. Consequently, HBV pgRNA and HBcrAg emerge as valuable predictors for HBeAg serologic conversion within NAs-treated HBeAg-positive patients. Subsequent ROC curve analysis unveiled that, with a cutoff value of 2.99 log10 copies/ml, HBV pgRNA yielded an AUC of 0.945 for HBeAg(+) prediction, boasting a sensitivity of 87.7% and specificity of 89.5%. In comparison, HBcrAg’s predictive ability was slightly lower (AUC of 0.754, cutoff value of 3.20 log10 mU/ml, sensitivity of 91.8%, specificity of 48.7%). These findings imply that both HBV pgRNA and HBcrAg can identify patients at risk of HBeAg serologic conversion, illuminating the response of HBeAg-positive patients to NAs therapy. However, resource constraints prevented the undertaking of a dynamic cohort study on HBeAg-positive patients pre- and post-serologic conversion. This remains an avenue for future exploration in our research.
The 2019 conference on HBV Treatment Endpoints by EASL-AASLD noted that when HBsAg disappears, it acts as a sign of functional cure.[29,30] Once this cure is reached, the absence of HBsAg persists, showing that HBsAg negativity leads to longer suppression of HBV DNA and reduces the risk of HCC more than patients who still show positive HBsAg.[31] Sonneveld[32] studied 1216 CHB patients on NAs treatment, who were HBeAg-negative and stopped the treatment. They found that stopping the drug at HBsAg levels below 100 IU/mL was linked to sustained HBsAg negativity. Other studies also reported similar results, suggesting that having HBsAg levels below 100 IU/mL at the end of NAs treatment is the best sign for stopping.[33,34] Our analysis, focused on HBsAg levels, demonstrated that HBcrAg and pgRNA levels were much lower in the group with HBsAg levels below 100 IU/ml, compared to the group with levels above 100 IU/mL. The analysis also revealed that HBcrAg and pgRNA levels showed a positive connection with HBsAg levels, while HBV DNA levels had no significant link. When we looked at HBeAg-negative patients, ROC curve analysis indicated that HBcrAg had a 0.953 AUC in predicting HBsAg < 100 IU/mL. With a cutoff value of 2.94 log10 mU/mL, it showed 92.5% sensitivity and 88.9% specificity. This performance was better than the predictive ability of HBV pgRNA (AUC of 0.831), which had a sensitivity of 79.1% and specificity of 77.8% at a cutoff value of 1.85 log10 copies/mL. However, combining HBcrAg and HBV pgRNA prediction for HBsAg < 100IU/mL resulted in a higher AUC of 0.972, showcasing a better prediction together. The sensitivity and specificity for this combined prediction were 92.5% and 100%, respectively, for HBV pgRNA and HBV pgRNA. Based on this, we can deduce that in patients who are HBeAg-negative, having HBcrAg levels below 3.01 log10 mU/ml and HBV pgRNA levels below 1.69 log10 copies/mL greatly increases the chances of achieving HBsAg seroclearance and functional cure after stopping the treatment.
5. Conclusions
In summary, this study underscores the significance of monitoring HBV pgRNA and HBcrAg levels in NAs-treated patients. It enhances comprehension of their clinical relevance, especially in long-term therapy, offering insights into intrahepatic cccDNA transcriptional activity. The study illustrates that combined HBV pgRNA and HBcrAg assessment during NAs treatment predicts the potential for HBeAg seroconversion, aiding clinicians in identifying candidates with higher likelihood for functional cure. Patients who are negative for HBeAg and exhibit HBcrAg levels < 3.01 log10 mU/mL and HBV pgRNA levels < 1.69 log10 copies/mL possess a higher likelihood of achieving seroclearance of hepatitis B surface antigen (HBsAg) and consequently experiencing functional cure subsequent to discontinuation of the medication. Thus furnishing a foundational rationale for the broader application of HBV pgRNA and HBcrAg in CHB clinical practice.
Authors contributions
Conceptualization: Jie Lin, Shiyao Jiang, Haifeng Zhang.
Data curation: Jie Lin, Shiyao Jiang, Xiangyu Chen.
Formal analysis: Min Zhu.
Writing – original draft: Jie Lin, Shiyao Jiang.
Abbreviations:
- cccDNA
- covalently closed circular DNA
- HBcrAg
- hepatitis B core-related antigen
- HBeAg
- hepatitis B e antigen
- HBsAg
- hepatitis B surface antigen
- HBV
- hepatitis B virus
- HCC
- hepatocellular carcinoma
- NAs
- Nucleotide Analog
- pgRNA
- pregenomic RNA
JL and SJ contributed equally to this work.
This study was supported by a grant from the Social Application Research Plans Foundation of Nantong (grant no. MSZ2022002).
The authors have no conflicts of interest to disclose.
The study was ethically approved by the Ethics Committee of The Affiliated Hospital of Nantong University (2022-L001). All participants were informed about the study protocol and provided written informed consent to participate in the study. I confirm that all methods were performed in accordance with the relevant guidelines. All procedures were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
All data generated or analyzed during this study are included in this published article [and its supplementary information files].
How to cite this article: Lin J, Jiang S, Chen X, Zhu M, Zhang H. The significance of detecting HBV pgRNA and HBcrAg in HBV patients treated with NAs. Medicine 2024;103:14(e37752).
Contributor Information
Jie Lin, Email: linjiejsnt@163.com.
Shiyao Jiang, Email: jiangshiyao0116@163.com.
Xiangyu Chen, Email: 1569884859@qq.com.
Min Zhu, Email: 1454241149@qq.com.
References
- [1].Höner Zu SC, Maasoumy B, Cornberg M. What is new on HBsAg and other diagnostic markers in HBV infection? Best Pract Res Clin Gastroenterol. 2017;31:281–9. [DOI] [PubMed] [Google Scholar]
- [2].Xia Y, Guo H. Hepatitis B virus cccDNA: formation, regulation and therapeutic potential. Antiviral Res. 2020;180:104824. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [3].Dong J, Ying J, Qiu X, et al. Advanced strategies for eliminating the cccDNA of HBV. Dig Dis Sci. 2018;63:7–15. [DOI] [PubMed] [Google Scholar]
- [4].Liu Y, Veeraraghavan V, Pinkerton M, et al. Viral biomarkers for hepatitis B virus-related hepatocellular carcinoma occurrence and recurrence. Front Microbiol. 2021;12:665201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [5].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? J Clin Microbiol. 2017;55:2972–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [6].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. J Hepatol. 2018;68:16–24. [DOI] [PubMed] [Google Scholar]
- [7].Chen E-Q, Wang M-L, Tao Y-C. Serum HB crAg is better than HBV RNA and HBsAg in reflecting intrahepatic covalently closed circular DNA. J Viral Hepat. 2019;26:586–95. [DOI] [PubMed] [Google Scholar]
- [8].Shen S, Xie Z, Cai D, et al. Biogenesis and molecular characteristics of serum hepatitis B virus RNA. PLoS Pathog. 2020;16:e1008945. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [9].Lin N, Ye A, Lin J, et al. Diagnostic value of detection of pregenomic RNA in Sera of Hepatitis B Virus-infected patients with different clinical outcomes. J Clin Microbiol. 2020;58:e01275–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [10].Mak LY, Cloherty G, Wong DK, et al. HBV RNA profiles in patients with chronic Hepatitis B under different disease phases and antiviral therapy. Hepatology (Baltimore, Md). 2021;73:2167–79. [DOI] [PubMed] [Google Scholar]
- [11].Wang J, Shen T, Huang X, et al. Serum hepatitis B virus RNA is encapsidated pregenome RNA that may be associated with persistence of viral infection and rebound. J Hepatol. 2016;65:700–10. [DOI] [PubMed] [Google Scholar]
- [12].Hong X, Luckenbaugh L, Mendenhall M, et al. Characterization of Hepatitis B Precore/Core-Related Antigens. J Virol. 2021;95:e01695–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [13].Hong X, Luckenbaugh L, Perlman D, et al. Characterization and application of precore/core-related antigens in animal models of hepatitis B virus infection. Hepatology (Baltimore, Md). 2021;74:99–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].Honda M, Shirasaki T, Terashima T, et al. Hepatitis B virus (HBV) core-related antigen during nucleos(t)ide analog therapy is related to intra-hepatic HBV replication and development of hepatocellular carcinoma. J Infect Dis. 2016;213:1096–106. [DOI] [PubMed] [Google Scholar]
- [15].Suzuki F, Miyakoshi H, Kobayashi M, et al. Correlation between serum hepatitis B virus core-related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients. J Med Virol. 2009;81:27–33. [DOI] [PubMed] [Google Scholar]
- [16].Sarin SK, Kumar M, Lau GK, et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update. Hepatol Int. 2016;10:1–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [17].Levrero M, Testoni B, Zoulim F. HBV cure: why, how, when? Current Opinion Virol. 2016;18:135–43. [DOI] [PubMed] [Google Scholar]
- [18].Jansen L, Kootstra NA, van Dort KA, et al. Hepatitis B virus pregenomic RNA is present in virions in plasma and is associated with a response to pegylated interferon Alfa-2a and Nucleos(t)ide Analogues. J Infect Dis. 2016;213:224–32. [DOI] [PubMed] [Google Scholar]
- [19].Mak LY, Wong DK, Cheung KS, et al. Review article: hepatitis B core-related antigen (HBcrAg): an emerging marker for chronic hepatitis B virus infection. Aliment Pharmacol Ther. 2018;47:43–54. [DOI] [PubMed] [Google Scholar]
- [20].Wang Y, Liao H, Deng Z, et al. Serum HBV RNA predicts HBeAg clearance and seroconversion in patients with chronic hepatitis B treated with nucleos(t)ide analogues. J Viral Hepat. 2022;29:420–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [21].Wang L, Cao X, Wang Z. Correlation analysis between the expression levels of HBV pgRNA and HBcrAg in circulating serum of patients with chronic hepatitis B after discontinuing nucleoside (acid) analogues and recurrence. J Clin Hepatobiliary Dis. 2023;39:56–62. [Google Scholar]
- [22].van Bömmel F, Bartens A, Mysickova A, et al. Serum hepatitis B virus RNA levels as an early predictor of hepatitis B envelope antigen seroconversion during treatment with polymerase inhibitors. Hepatology (Baltimore, Md). 2015;61:66–76. [DOI] [PubMed] [Google Scholar]
- [23].Luo H, Tan N, Kang Q, et al. Hepatitis B virus pregenomic RNA status can reveal the long-term prognoses of chronic hepatitis B patients treated with nucleos(t)ide analogues. J Viral Hepat. 2020;27:323–8. [DOI] [PubMed] [Google Scholar]
- [24].Caviglia GP, Armandi A, Rosso C, et al. Hepatitis B core-related antigen as surrogate biomarker of intrahepatic Hepatitis B Virus covalently-closed-circular DNA in patients with chronic Hepatitis B: a meta-analysis. Diagnostics (Basel). 2021;11:187. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [25].Chen EQ, Feng S, Wang ML, et al. Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B. Sci Rep. 2017;7:173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [26].Chen S, Jia J, Gao Y, et al. Clinical evaluation of hepatitis B core-related antigen in chronic hepatitis B and hepatocellular carcinoma patients. Clinica chimica acta. 2018;486:237–44. [DOI] [PubMed] [Google Scholar]
- [27].Testoni B, Lebossé F, Scholtes C, et al. Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients. J Hepatol. 2019;70:615–25. [DOI] [PubMed] [Google Scholar]
- [28].Tsuge M, Chayama K. Availability of monitoring serum HBV DNA plus RNA during nucleot(s)ide analogue therapy. J Gastroenterol. 2013;48:779–80. [DOI] [PubMed] [Google Scholar]
- [29].Tsounis EP, Tourkochristou E, Mouzaki A, et al. Toward a new era of hepatitis B virus therapeutics: the pursuit of a functional cure. World J Gastroenterol. 2021;27:2727–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [30].Cornberg M, Lok AS, Terrault NA, et al. Guidance for design and endpoints of clinical trials in chronic hepatitis B – report from the 2019 EASL-AASLD HBV treatment endpoints conference (‡). J Hepatol. 2020;72:539–57. [DOI] [PubMed] [Google Scholar]
- [31].Gehring AJ, Protzer U. Targeting innate and adaptive immune responses to cure chronic HBV Infection. Gastroenterology. 2019;156:325–37. [DOI] [PubMed] [Google Scholar]
- [32].Sonneveld MJ, Chiu SM, Park JY, et al. Probability of HBsAg loss after nucleo(s)tide analogue withdrawal depends on HBV genotype and viral antigen levels. J Hepatol. 2022;76:1042–50. [DOI] [PubMed] [Google Scholar]
- [33].Kao JH, Jeng WJ, Ning Q, et al. APASL guidance on stopping nucleos(t)ide analogues in chronic hepatitis B patients. Hepatol Int. 2021;15:833–51. [DOI] [PubMed] [Google Scholar]
- [34].Tseng TN, Hu TH, Wang JH, et al. Incidence and factors associated with HBV relapse after cessation of entecavir or tenofovir in patients with HBsAg below 100 IU/mL. Clin Gastroenterol Hepatol. 2020;18:2803–2812.e2. [DOI] [PubMed] [Google Scholar]