Juan-Juan Fu, Rui-Xia Liu, Li Li, Xiu-Cheng Pan?

1. Department of Infectious Diseases, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, China

2. Department of Infection, the Suqian People's Hospital,China

Keywords:

ABSTRACT

1. Introduction

Chronic hepatitis B (CHB) refers to chronic hepatitis with a duration of more than 6 months infected with hepatitis B virus (HBV), which may evolve into severe hepatitis, cirrhosis, liver cancer and other serious hepatitis B related diseases with the disease progression, seriously threatening people's life and health [1-3].Antihbv infection treatment is the most eあective clinical treatment for CHB, which can prevent or delay the occurrence of severe hepatitis, cirrhosis and other serious hepatitis b related diseases and improve the prognosis of patients [4-5].The current clinical anti-HBV treatment drugs mainly include interferon and nucleoside antiviral drugs, of which interferon-α (Interferon-α, IFN-α) is currently recognized at home and abroad as the most commonly used and effective drug for viral hepatitis. The polyethylene glycol longacting interferon-α (Peg IFN-α) largely compensates for the short half-life of IFN-α, which has a fixed course and does not produce virus resistance and has HBsAg and HBeAg seroconversion rates. The advantages of high response and long-lasting response are gradually being applied to the clinic [6].Hepatitis b virus covalently closed circular DNA (cccDNA) is the initial template for HBV replication, and the detection of cccDNA level in the liver nucleus is an important indicator to evaluate the eきcacy of anti-hbv and to stop anti-hbv treatment, but its detection requires liver tissue samples with certain limitations [7-8].In recent years, studies have found a correlation between serum HBV RNA quantification and HBV cccDNA in liver tissues, which can be used as an alternative marker for HBV cccDNA [9].Therefore, this study selected 34 patients with HBeAg-positive CHB who received Peg IFN-α treatment in the aきliated hospital of xuzhou medical university from February 2014 to October 2015 as the study objects, aiming to explore the changes of HBV RNA level in peripheral blood of patients during the treatment of HBeAg-positive CHB by Peg IFN-HBV and its clinical significance.

2. Materials and methods

2.1General information

34 patients with HBeAg-positive CHB who were treated with Peg IFN-α at the Affiliated Hospital of Xuzhou Medical University from February 2014 to October 2015 were selected as study subjects.There were 22 males and 12 females.The age ranged from 24 to 47, with an average age of (26.35±6.90) and of whom HBV DNA were all positive.Inclusion criteria: were in line with the guidelines for the prevention and treatment of chronic hepatitis b formulated by Chinese society of hepatology in 2019 [10]；with complete clinical data； were voluntarily receiving Peg IFN-α treatment； were informed of the content of this study and were able to cooperate with the completion of relevant indicators testing； did not receive IFN-α or NAs anti-HBV treatment within 6 months before enrollment.Exclusion criteria: people with hepatitis c (HCV), hepatitis d (HDV) or HIV；alcoholics；with severe hypertension；people with serious dysfunction of important organs；people with autoimmune diseases；Patients with tumors.This study was approved by the Medical Ethics Committee of the Affiliated Hospital of Xuzhou Medical University, and all patients were informed of the study and signed the informed consent.

2.2Method

5 mL peripheral venous blood was collected before treatment, at 12 weeks, 24 weeks and 52 weeks respectively. It was then placed in a yellow tube containing the separation glue and coagulant, centrifuged at 3500 RPM for 10 min, and the upper serum was placed in a 1.5 mL sterile EP tube, which was stored in the refrigerator at -80℃ for testing.ABI 7500 real-time quantitative PCR instrument and matching kits were used to detect serum HBV DNA levels (HBV DNA ＜5.0×102IU/mL was judged as negative, otherwise it was positive).ABI 7500 real-time quantitative PCR instrument and matching kits were used to detect serum HBV RNA levels.The serum ALT level was detected by using the Japanese OLYMPUS-5400 automatic biochemical analyzer and its supporting kit (ALT often refers to the normal range of ALT, i.e. ＜40 IU/L).HBV DNA kit was purchased from Shanghai Kehua bio-engineering Co., Ltd., and viral RNA extraction kit was purchased from tiangen biochemical technology (Beijing) Co., Ltd.

2.3Statistical methods

All data were analyzed and processed by SPSS22.0 statistical software.The measurement data are expressed as (±s ), and the independent sample t test is conducted；The counting data were presented as n (%) for 2 test.Pearson analysis was used for correlation analysis.P＜0.05 was considered statistically significant.

3. Results

3.1 Clinical efficacy of Peg IFN- in the treatment of CHB patients

At 12, 24 and 52 weeks after treatment, ALT and AST levels were significantly lower than before treatment.While ALT levels in patients at 24 and 52 weeks after treatment were significantly lower than those at 12 weeks after treatment.Moreover, the ALT level of patients at 52 weeks of treatment was significantly lower than that at 24 weeks of treatment, and the diあerences were statistically significant (P ＜0.05), as was shown in Figure 1.What`s more,at the 12th week of the treatment, ALT recurrence rate was 41.18% (14/34), HBeAg negative conversion rate was 26.47% (9/34), and HBV DNA negative conversion rate was 38.23% (13/34).At the 24th week of the treatment, ALT recurrence rate was 55.88% (19/34), HBeAg negative conversion rate was 29.41% (10/34), and HBV DNA negative conversion rate was 50.00% (17/34).At the 52th week of the treatment, ALT recurrence rate was 76.47% (26/34), HBeAg negative conversion rate was 47.06% (16/34), and HBV DNA negative conversion rate was 61.76% (21/34), as was shown in table 1.

Figure 1 The change of serum ALT level in patients during the treatment with Peg IFN-α

Table 1 The clinical eきcacy of IFN-α in patients with CHB

3.2 Changes of serum HBV RNA levels in patients during Peg IFN-α treatment

During the treatment of Peg IFN- HBV, the serum HBV RNA level of the patients showed a general decreasing trend.The serum HBV RNA levels of patients at 12 weeks, 24 weeks and 52 weeks were significantly lower than before treatment, and the diあerences were statistically significant (P ＜0.05), as was shown in Figure 2.

Figure 2 The change of serum HBV RNA level in patients during the treatment with Peg IFN-α

3.3 Correlation between serum HBV RNA levels and ALT normalization

At 12, 24, and 52 weeks after treatment, serum HBV RNA levels in patients with recurrent ALT levels were significantly lower than those in patients without recurrent ALT levels, with statistically significant diあerences (P＜0.05), as shown in table 2.

Table 2 The correlation between serum HBV RNA level and ALT recurrence in patients

3.4 Correlation between serum HBV RNA level and HBeAg negative rotation in patients

At 12, 24, and 52 weeks after treatment, serum HBV RNA levels in patients with negative transformation of HBeAg were significantly lower than those without negative transformation, with statistically significant diあerences (P＜0.05), as was shown in table 3.

Table 3 The correlation between serum HBV RNA level and HBeAg negative rotation in patients

3.5 Correlation between serum HBV RNA level and complete response in patients

The simultaneous removal of serum HBeAg and negative conversion of HBV DNA (below the lower limit of detection) during the treatment were defined as a Complete Response (CR), otherwise a Noncomplete Response (NCR).At 12 weeks, 24 weeks and 52 weeks after treatment, serum HBV RNA levels in patients with complete response were significantly lower than those in patients with incomplete response, with statistically significant diあerences (P＜0.05), as was shown in table 4.

Table 4 The correlation between serum HBV RNA level and complete response in patients

3.6 Correlation analysis between serum HBV RNA levels and ALT, HBsAg, HBV DNA levels in patients

The correlation analysis results of Person showed that the serum HBV RNA level of patients had no significant correlation with ALT level and HBsAg level (P＞0.05), and the serum HBV RNA level of patients had a significant positive correlation with HBV DNA level (r=0.408, P=0.009, Fig.3), as was shown in table 5.

Table 5 The correlation analysis of serum HBV RNA level with ALT, HBsAg and HBV DNA level

Figure 3. The correlation between serum HBV RNA and HBV DNA

4. Discussion

CHB is a major risk factor for the induction of severe hepatitis, cirrhosis, and even liver cancer, and anti-HBV infection treatment is still a hot topic around the world [11-13].The objective of anti-HBV treatment is to make serological transformation of HBeAg happen as soon as possible by inhibiting viral replication, and finally realize HBV-DNA negative transformation, so as to alleviate the disease [1,4-5].HBV-DNA negative conversion is a commonly used indicator to evaluate the eきcacy of anti-HBV infection treatment in the clinic. HBeAg positive indicates that the patient is in the active stage of HBV replication, which greatly increases the risk of inflammatory necrosis of liver cells and liver fibrosis, and accelerates the progress of CHB disease [11-12].Peg IFN-α, with the advantages of fixed course of treatment, no virus resistance, high HBsAg and HBeAg serum conversion rate and persistent response, is currently clinically preferred to Peg IFN-α extended treatment for patients who are young, expect to complete treatment in a short period of time and receive the first treatment [6].The results of this study showed that among the 34 HBeAg-positive CHB patients receiving Peg IFN-α anti-HBV treatment, 9 patients showed HBeAg negative conversion (negative conversion rate was 26.47%) and 13 patients showed HBV DNA negative conversion (negative conversion rate was 38.23%) at 12 weeks after treatment.At 24 weeks after treatment, HBeAg negative rotation was observed in 10 patients with a rate of 29.41%, and HBV DNA negative rotation was observed in 17 patients with a rate of 50.00%.By 52 weeks of treatment, 16 patients had negative conversion of HBeAg, with a negative conversion rate of 47.06%, and 21 patients had negative conversion of HBV DNA, with a negative conversion rate of 61.76%, suggesting that the eあect of Peg IFN-α anti-HBV treatment is ideal, consistent with expected.In addition, the results of this study confirm that with the extension of the antiviral time of Peg IFN-α, the patient's liver function index ALT has gradually improved. By 52 weeks of treatment, the ALT normalization rate has reached 76.47%, and the liver function recovery is ideal.

This study used real-time fluorescent quantitative PCR system for quantitative HBeAg positive patients with CHB serum HBV RNA detection, the results showed that Peg IFN-α treatment in patients with serum HBV RNA level in the process of a downward trend overall, treatment for 12 weeks and 24 weeks and the 52 weeks in patients with serum HBV RNA level before treatment were significantly reduced, the diあerences were statistically significant (P＜0.05), suggesting serum HBV RNA level possible for evaluating anti HBV infection treatment eきcacy of an auxiliary index,Recent clinical reports by van B?mmel F[14] and Wen Jia[15] also confirmed this point.Further analysis showed that at 12, 24, and 52 weeks of treatment, serum HBV RNA levels in patients with normal ALT levels were significantly lower than those in non-normal ALT levels, and serum HBV RNA levels in patients with negative HBeAg conversion were significantly lower than those in patients with negative ALT.And the serum HBV RNA level of patients with complete response was significantly lower than that of patients with incomplete response, while the diあerences were statistically significant (P＜0.05), suggesting that the serum HBV RNA level of patients during the treatment of Peg IFN-HBV antiviral therapy may be correlated with liver function, HBeAg, and HBV RDA status during the treatment, further suggesting that serum HBV RNA level may be used as an auxiliary indicator to evaluate the eきcacy of anti-HBV infection treatment.The results of Person correlation analysis showed that there was a significant positive correlation between serum HBV RNA level and HBV DNA level (r=0.408, P=0.009), indicating that regular detection of serum HBV RNA level in patients during the treatment of Peg IFN-HBV was helpful in evaluating the therapeutic eあect.

In summary, the serum HBV RNA levels of patients during the treatment of Peg IFN-α generally showed a downward trend, which was significantly positively correlated with HBV DNA levels. It is expected to serve as an auxiliary indicator for evaluating the eきcacy of anti-HBV infection treatment, and has certain clinical value.