Analysis of HBV-RNA level in patients with Chronic hepatitis B whose HBV-DNA is negative after NAs antiviral therapy

Background: While Nucleotide Analogs (NAs) are widely used in the treatment of chronic Hepatitis B Virus (HBV) infection, it cannot clear covalently closed circular DNA from the liver, making chronic hepatitis B difficult to cure. However, NAs can effectively inhibit retroviral transcription and viral DNA synthesis, and when HBV DNA is reduced to the lower limit of detection, HBV RNA becomes a more valuable diagnostic marker. Aim: To analyze HBV RNA levels in chronic HBV patients who tested negative for HBV DNA after NAs antiviral therapy. Methods: We enrolled 60 chronic hepatitis B patients who were admitted to Tianjin Second People’s Hospital from September 2018 to March 2019 in this study. Patients were treated with NAs antiviral therapy for at least one year. HBV DNA was detected and measured using Roche reagents and fluorescence quantitative polymerase chain reaction with a detection limit of 20 IU/ ml at least two intervals of three months. These patients were also tested for HBV-RNA level, and Alamine Aminotransferase (ALT), Aspartate Transaminase (AST), γ-Glutamyl Transpeptadase (GGT), Hepatitis B Surface Antigen (HBsAg), Hepatitis B E Antigen (HBeAg), Hepatitis B Core Antibodie (HBcAb) in the meantime. Results: Patients were divided into the undetected HBV DNA and < 20 IU/ml HBV DNA groups based on HBV DNA level. The quantitative comparison between these groups showed significant differences in HBV RNA level (Z = 3.818, P < 0.05). Patients were also divided into the HBeAg-positive and HBeAg-negative groups based on HBeAg level. There were significant differences in HBV RNA level between these groups (Z= 3.363, P < 0.05). Additionally, patients were divided into the following groups based on HBsAg level: the HBsAg ≤ 500 IU/ml, 500 < HBsAg < 1500 IU/ml, and HBsAg ≥ 1500 IU/ml groups. There we no significant differences in HBV RNA level between these groups (Chi-square value = 5.891, P > 0.05). However, there were significant differences in HBV RNA level between the HBsAg ≤ 500 IU/ml and HBsAg ≥ 1500 IU/ml groups. (Z = 2.426, P < 0.05). ALT, AST, GGT, HBsAg, HBeAg, and HBcAb were included in the univariate linear regression analysis, which showed that only HBeAg level significantly correlated with HBV RNA level (P < 0.05). Conclusion: HBV RNA may still be detected in chronic HBV patients after long-term NA antiviral therapy, and HBeAg level may be associated with serum HBV RNA level.


Introduction
NAs are widely used in the treatment of chronic hepatitis B. Although active antiviral therapy can delay the progression of liver diseases and reduce the occurrence of end-stage liver diseases, few patients have achieved the clinical cure standard, which is defined as HBsAg-negative conversion. About 650,000 people still die every year due to hepatic failure, cirrhosis, and hepatocellular carcinoma caused by HBV infection [1,2]. Clinical findings have shown that, after NA treatment, while the detection of HBV DNA in chronic hepatitis B patients is lower than the detection limit, the disease may continue to progress, resulting in cirrhosis and liver cancer. However, recent studies have confirmed that serum HBV RNA is associated with the ratio of hepatic RNA to covalently closed circular DNA (cccDNA), which implies the occurrence of viral transcription. Therefore, HBV RNA can be used as a clinical marker for the transcriptional activity of cccDNA [3].
Previously, it was believed that chronic hepatitis B was difficult to cure due to the covalent closure of cyclic DNA in the nucleus of hepatocytes. Although NAs can effectively inhibit retroviral transcription and viral DNA synthesis, it cannot clear the cccDNA from the liver [4,5]. Theoretically, both serum HBV DNA and RNA can be useful markers for HBV cccDNA activity in treatment-naïve patients. However, in CHB patients under NAs therapy, HBV DNA is suppressed due to halted pgRNA reverse transcription, making RNA a more direct marker for cccDNA [6]. when NAs reduce serum HBV DNA levels to the lower limit of detection, serum HBV RNA level becomes particularly valuable for diagnosis [7]. Therefore, in this study, we analyzed the influence of HBV RNA level on chronic hepatitis B patients whose HBV DNA levels were lower than the detection limit after undergoing NAs treatment in order to guide the formulation and adjustment of clinical antiviral treatment and delay the progress of liver disease.

Study Design
We enrolled 60 chronic hepatitis B patients who were admitted to Tianjin Second People's Hospital from September 2018 to March 2019. All patients were diagnosed as having chronic hepatitis B in accordance with the Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2015 edition). All patients received NA antiviral therapy for at least one year. The detection of HBV DNA via Roche reagents was less than 20 IU/ml or undetected at least two intervals of three months. Patients were excluded if they had other hepatitis virus and cytomegalovirus infections, a history of taking medication for hepatitis, alcoholic hepatitis, autoimmune liver disease, and hereditary metabolic liver disease, cirrhosis, and hepatocellular carcinoma. This study was approved by the Medical Ethics Committee of Tianjin Second People's Hospital.

Detection Methods
HBV DNA and HBV RNA were detected simultaneously. HBV-DNA was detected by real-time fluorescent quantitative PCR.HBV DNA quantitative detection reagents were purchased from Roche Diagnostic Products (Shanghai) Co., Ltd. HBV DNA detection was carried out via fluorescence quantitative polymerase chain reaction with a detection limit of 20 IU/ml. HBV-RNA was detected by real-time fluorescent quantitative PCR. HBV RNA quantitative reagents were purchased from China Hunan Shengxiang Biological Technology Co., Ltd. HBV RNA was detected with a detection limit of 100 IU/ml. HBV serum markers (quantitative method) were detected via chemiluminescence methods, and reagents were purchased from Abbott Medical (Shanghai) Co., Ltd. Liver function was evaluated using a Hitachi 7180 automatic biochemical analyzer. Whenever HBV RNA was detected, ALT, AST, GGT, and HBV serum markers were detected simultaneously.

Statistical Methods
SPSS 23.0 was used for all statistical analyses. For measurement data, normal distributions were expressed as mean ± standard deviation and non-normal distributions were expressed as median plus quartile. The Kruskal-Wallis test was used for multigroup comparisons and the rank sum test was used for pairwise comparisons between groups. The influencing factors of HBV RNA were analyzed via single-factor linear regression. A P value of < 0.05 was considered statistically significant.

Patients' Demographic and Clinical Characteristics
A total of 60 patients were enrolled in this study, including 41 males and 19 females who were 27-69 years old (Table 1).

Differences in HBV RNA level based on HBV DNA level
Patients were divided into the following two groups based on HBV DNA level: the undetected HBV DNA and < 20 IU/ml HBV DNA groups. There were significant differences in HBV RNA level between the two groups(Z = 3.818, P < 0.05) ( Table 2).

Differences in HBV RNA level based on HBeAg level
Patients were divided into either the HBeAg-positive or HBeAg-negative groups based on their HBeAg levels. The quantitative comparison between the two groups showed significant differences in HBV RNA level (Z= 3.363, P < 0.05) ( Table 3).  Note: HBV DNA and HBV RNA levels change exponentially. We converted them into logarithms so that they could become continuous variables with normal distributions.

Differences in HBV RNA based on HBsAg level
Patients were divided into the following three groups based on HBsAg level: the HBsAg ≤ 500 IU/ml, 500 IU/ml < HBsAg <1500 IU/ml, and HBsAg ≥ 1500 IU/ml groups. The quantitative comparison between these groups showed no significant differences in HBV RNA level. (Chi-square value = 5.891, P > 0.05). There were significant differences in HBV RNA level between the HBsAg ≤ 500 IU/ml and HBsAg ≥ 1500 IU/ml groups (Z = 2.426, P < 0.05) (

Analysis of the factors that influence HBV RNA level
In the univariate linear regression analysis, ALT, AST, GGT, HBsAg, HBeAg, and HBcAb levels were independent variables and serum HBV RNA level was a dependent variable. This analysis showed that only HBeAg level significantly correlated with HBV RNA level (P < 0.05) (

Discussion
In 1996, Kock et al detected HBV RNA in the sera of chronic hepatitis B patients. The clinical value of HBV RNA was gradually discovered over the course of developing antiviral treatment for chronic hepatitis B patients.Furthermore, Wang et al reported that serum HBV RNA mainly exists in the form of pre-genomic RNA and it may be a stable serological marker that can reflect the existence of cccDNA in liver tissue [8]. After NAs treatment, serum HBV RNA level may better reflect the activity of cccDNA in the liver than serum HBV DNA. It has been reported that the only source of HBV RNA in sera is cccDNA located in the nucleus of infected hepatocytes, and its production cannot be inhibited by NAs. So, HBV RNA level can reflect the real level of existence and transcriptional activity of cccDNA in hepatocytes [9,10]. Therefore, when HBV RNA is not detected in sera, it is a likely indication of the disappearance or silence of cccDNA transcription in patients' hepatocytes.
HBV DNA level can also reflect the replication activity of cccDNA in hepatocytes to some extent. However, the lower detection limit of HBV DNA can only indicate the inhibition of retroviral processes. It does not reflect the transcriptional status of cccDNA. After retroviral processes are inhibited, cccDNA continues to produce progeny viruses in the form of HBV RNA virus-like particles. So, disease rebound and recurrence rates are higher after the withdrawal of medication under the precondition of sustained virological response [11][12][13][14]. In our study, patients were divided into the undetected HBV DNA and < 20 IU/ml HBV DNA groups based on HBV DNA level. There was a statistical difference in HBV RNA level between the two groups (Z = 3.316, Volume 15; Issue 01 Ann Case Rep, an open access journal ISSN: 2574-7754 P < 0.05). This suggests that the lower HBV DNA level, the lower the serum HBV RNA level will be after long-term NA antiviral treatment. Even when HBV DNA is below the detection limit, the replication activity of cccDNA continues, which leads to the detection of HBV RNA or at a higher level.
As a result, HBV RNA can more accurately reflect the status of cccDNA than HBV DNA, making HBV RNA level a more powerful indicator of antiviral efficacy [11,15,16]. Additionally, a good correlation between the status of HBeAg and HBV DNA quantification has been reported [17]. However, a correlation between serum HBeAg status and serum HBV RNA remains to be seen. Wang et al showed that the status of serum HBeAg potentially has an effect on the correlation between serum HBV RNA and hepatic HBV cccDNA [18]. Moreover, van Bömmel F et al showed that serum HBV RNA is an early predictor of the serological conversion of HBeAg after PEG interferon alpha-2a treatment [19]. And His another study suggests that Serum HBV RNA levels may serve as a novel tool for prediction of serological response during polymerase inhibitor treatment in HBeAg-positive patients [20]. In our study, patients were divided into the HBeAgpositive and HBeAg-negative groups based on HBeAg level.
The quantitative comparison between the two groups showed significant differences in HBV RNA level (Z = 3.363, P < 0.05), which is similar to the results reported by Jansen et al [21]. Our single-factor linear regression analysis also showed that HBeAg level was the only factor that correlated with HBV RNA level (P < 0.05). After our patients underwent long-term antiviral treatment, their HBV DNA levels were lower than the detection limit, and the HBV RNA levels in HBeAg-positive patients were higher than those in HBeAg-negative patients. Therefore, HBeAg level may be associated with HBV RNA level. Further, serum HBsAg is derived from hepatic HBV cccDNA transcription, and its level is closely related to the transcriptional activity of hepatic cccDNA. The removal of serum HBsAg can significantly reduce the risk of liver cirrhosis and hepatocellular carcinoma in chronic hepatitis B patients [22]. HBV RNA can also directly reflect the level and transcriptional status of cccDNA in the liver. This is why we studied the relationship between HBsAg and HBV RNA. Patients were divided into the three following groups based on HBsAg level: the HBsAg ≤ 500, 500 < HBsAg <1500 IU/ ml, and HBsAg ≥ 1500 IU/ml groups. The quantitative comparison between the three groups showed that differences in HBV RNA level were not statistically significant. (Chi-square value = 5.891, P > 0.05). However, there were significant differences in HBV RNA level between the HBsAg ≤ 500IU/ml and HBsAg ≥1 500 IU/ml groups (Z = 2.426, P < 0.05). There was no significant difference between the remaining two groups (P > 0.05). Our univariate linear regression also showed no correlation between HBsAg level and HBV RNA level (P > 0.05). It is suggested that the HBV RNA levels in the HBsAg < 500 IU/ml group are lower than that in HBsAg < 1500 IU/ml group. However, HBsAg level was not a factor that affected HBV RNA level in our study.
Therefore, healthcare practitioners should use the lower detection limit of serum HBV RNA level as a virological response index after reaching the lower detection limit of HBV DNA in chronic hepatitis B patients who have undergone long-term antiviral therapy. It is only when both HBV DNA and RNA are below the detection limit, thereby indicating the disappearance or silence of cccDNA transcription in the liver, can it be possible to completely prevent patients' liver disease and improve the patients' quality of life. Presently, the detection of HBV RNA is not widely used in clinical practice. This study acts as a preliminary exploration of the clinical significance of HBV RNA in chronic hepatitis B patients. Our study had some limitations. A large number of clinical studies are needed to prove the significance of serum HBV RNA as an evaluation standard in antiviral therapy for chronic hepatitis B patients.

Conclusion
HBV RNA can still be detected in the absence of HBV DNA. Ours is also the first study to report the possible relationship between HBeAg and HBV RNA. Further, we believe that HBV DNA and RNA should be used concurrently as indicators of antiviral efficacy. It is only when both HBV DNA and HBV RNA are below the detection limit, thereby indicating the disappearance or silence of cccDNA transcription in the liver, can it be possible to completely prevent patients' liver disease and improve the patients' quality of life.