Entecavir/peginterferon alfa-2a combination in the treatment of two genotypes of chronic hepatitis B patients with lamivudine resistance

Purpose: To investigate the efficacy of entecavir plus peginterferon alfa-2a in the treatment of chronic hepatitis B (CHB) patients with different hepatitis B virus (HBV) genotypes resistant to lamivudine (LAM). Methods: 119 LAM-resistant CHB patients treated in Baoshan People's Hospital from May 2018 to May 2020 were selected. All patients received entecavir and peginterferon alfa-2a for 24 months and were scheduled for regular outpatient review and telephone follow-up. Polymerase chain reaction (PCR) was conducted to determine the HBV genotype and HBV-DNA clearance. Alanine aminotransferase (ALT) normalization rate, HBV-DNA clearance rates, and hepatitis B e-antigen (HBeAg) seroconversion rate were determined in CHB patients with different genotypes. Quality of life for all patients was assessed using SF-36 Scale. Results: Five out of 119 patients were lost during follow-up, with a follow-up rate of 95.80 %. Two HBV genotypes were identified, of which 42 (36.8 %) were type B and 72 (63.2 %) type C. At the 6th and 12th month of follow-up, the HBV-DNA clearance rate, ALT normalization rate, and HBeAg seroconversion rate were significantly higher in CHB patients with genotype B than in patients with genotype C (p < 0.05). There were no significant differences between the three rates in the two groups at 18th and 24th month of follow-up (p > 0.05). The quality of life (QOL) of the patients differed between the two groups (p < 0.05). Conclusion: Entecavir plus peginterferon alfa-2a are effective in treating LAM-resistant CHB patients with different genotypes. Genotype B CHB patients are more suitable for this combination protocol.

It has been suggested that a combination regimen of pegylated interferon α-2a and entecavir should be used in the treatment of patients with LAM-resistant chronic hepatitis B. On the one hand, it aids the inhibition of HBV self-replication, and on the other hand, compared with entecavir alone, pegylated interferon α-2a produces both immunomodulatory factors as well as antiviral proteins, which perform immunomodulatory as well as antiviral effects [7,8].Other studies have indicated that the HBV genotype of CHB patients is closely related to both HBV resistance mutations and the efficacy of antiviral therapy [9].
The combination of entecavir and peginterferon alfa-2a was used to treat LAM-resistant CHB patients, and the clinical efficacy and quality of life improvement were assessed through longterm follow-up to investigate the advantages and disadvantages of this treatment regimen in terms of virological and biological response, so as to select a more effective drug regimen for CHB patients with different HBV genotypes.

Study population and ethical consideration
One hundred and nineteen CHB patients with LAM resistance who attended the Baoshan People's Hospital from May 2018 to May 2020 were selected.The study was approved by the Ethics Committee of Baoshan People's Hospital (approval no.20180022) and followed the guidelines of the World Medical Association Declaration of Helsinki [10].

Inclusion criteria
Patients with an age range from 20 to70 years old, positive serum hepatitis B surface antigen, diagnosed as CHB patients according to the Chinese guidelines for the prevention and treatment of chronic hepatitis B (updated 2015 version) [11] and were on a combination regimen of pegylated interferon alpha-2a and entecavir applied to antiviral therapy after the development of LAM resistance were included in the study.(Patient LAM resistance criteria: elevated HBV-DNA quantification > 1 log IU/mL in more than 2 consecutive serum tests compared to the lowest level after treatment) [12].All patients signed the consent form prior to inclusion in the study.

Exclusion criteria
These included patients with renal insufficiency with creatinine clearance below 60 mL/min and patients taking other nucleoside antiviral drugs.

Patients' follow-up
All patients were followed up by regular outpatient and telephone visits at months 6, 8, 12, 18, and 24 to ensure adherence.Follow-up visits included monthly checks of HBeAg and HBV-DNA and liver function.

Evaluation of parameters/outcomes
Levels of HBeAg were determined using a MultiSkan enzyme marker (LABSYSTEMS Inc.) in the CHB patients.The genotype of HBV was determined through PCR using the HBV Genotyping Test Kit (Alcon Biotechnology Ltd).ABI 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific Inc.) and PCR kits (Tiangen Biochemical Technology (Beijing) Co., Ltd.) were conducted to determine serum HBV-DNA levels in CHB patients.A fluorometric quantification method was run to identify mutations in CHB patients' DNA polymerase Tyrosine-methionine-aspartateaspartate (YMDD) gene.

Liver function indicators
Colorimetric methods were used to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), and total bilirubin (TBIL) levels in CHB patients by conducting ADVIA 2400 fully automatic biochemical analyzer (Siemens Healthineers Inc.).Coagulation method was used to determine the prothrombin time (PT) of patients.The virological response and biochemical response of each patient at the 6th, 12th, 18 th , and 24th months of follow-up were determined by Eqs 1 -3.where HBV -DNA clearance (defined as HBV -DNA level < 2log IU/mL), negHBV is the number of patients with negative HBV, nALT is the number of patients with ALT normalization (defined as ALT level < 40 U/L), nHBeAg is the number of patients with HBeAg seroconversion (HBeAg is negative and anti-HBe is positive) [13], and N is the total number of patients.

Short-Form 36(SF-36) Health Survey Scale
The SF-36 Scale was performed to assess the quality of life in both groups and physical functioning (PF), social functioning (SF), role limitations due to physical health problems (RP), role limitations due to personal or emotional functioning (RE), bodily pain (BP), mental health (MH), vitality (V) and general health (GH) were included, with scores ranging from 0 to 100 [14].

Statistical analysis
Statistical Package for Social Sciences (SPSS) 19.0 was used for data analysis.All the collected data are expressed as mean ± SD.A t-test was performed for analysis of differences in the data, while chi-square test was applied for comparison between the count data.
The Kaplan-Meier method was used to compare the ALT normalization rate, cumulative HBV-DNA clearance rate, and HBeAg seroconversion rate among patients with different hepatitis B virus genotypes.
Differences were considered significant when p < 0.05.

Follow-up results and genotype distribution
A total of 2 genotypes B and 3 genotypes C were lost from the 119 CHB patients initially included, resulting in a follow-up rate of 95.80 %.Two HBV genotypes were identified in the remaining 114 patients, including 42 patients (36.8 %) with HBV genotype B and 72 patients (63.2 %) with HBV genotype C.

General information on patients with two HBV genotypes
There is no significant differences between patients with two HBV genotypes in terms of gender, age, the proportion of HBeAg positive, HBV-DNA level, YMDD mutation type, and levels of liver function (P > 0.05).As shown in Table 1.

Quality of life of patients with two HBV genotypes
After treatment with entecavir in combination with peginterferon alfa-2a, the values of PF, RP, MH, RE, and total scores were significantly higher in both groups of CHB patients than during pretreatment, while the values of SF, BP, and V were significantly lower, with significant differences between the same groups before and after treatment (p < 0.05).The QOL of CHB patients with genotype B was better than that of patients with genotype C, and the difference in QOL was significant between the two groups (p < 0.05, Table 5).

DISCUSSION
With the development and improvement of pharmaceutical therapy options for CHB, the process of CHB can be slowed down by blocking HBV replication [15].However, drug resistance in some CHB patients is still difficult to overcome.Lamivudine is the first nucleotide-based, anti-HBV drug approved for marketing in China, LAM significantly reduces HBV-DNA quantification in CHB patients, enhances the seroconversion rate of HBeAg, delays liver tissues necrosis and reverses liver tissue fibrosis [16].However, LAM resistance is easy to develop in CHB patients, in the first 5 years when LAM was selected as the main drug for CHB treatment, the incidence of drug resistance increased year by year.Mutations in the DNA polymerase YMDD gene have been reported to be associated with the development of LAM resistance.
[5].Among the 114 LAM-resistant CHB patients, the proportion of YMDD gene mutations was high (98.2%), and there was 1 patient with no YMDD gene mutation in both genotype B and genotype C patients.
Peginterferon α-2a is an anti-HBV immunomodulator, but it is poorly tolerated only by injection and is expensive, all of these limits its widespread use [17].Entecavir, as one of the current anti-HBV therapeutic drugs, significantly inhibits HBV replication, reduces the occurrence of liver inflammation, decreases the activity of stellate cells and the formation of collagen fibers Post-treatment 1.33±0.54*▲ 37.83±4.44*▲ 20.07±1.79109.16±5.66*▲ *P < 0.05, Compared with the same group before treatment; ▲P < 0.05, there were significant differences between the two genotypes of CHB patients after treatment in liver tissue, and slows down the process of liver fibrosis [18].Therefore, compared with other antiviral drugs, entecavir has a more rapid viral suppressive effect and a higher safety profile, especially in patients with liver failure, and it effectively prolongs the survival of patients.Peginterferon alfa-2a and entecavir are both CHB therapeutic drugs, but their mechanisms of action are different.Therefore, the combination of them could theoretically be more effective in inhibiting HBV replication and reducing both drug resistance and liver damage [8].
Currently, at least 10 HBV genotypes (A~X) have been identified worldwide, with a more distinct geographical distribution, and with type B (southern region) and type C (northern region) predominant in China.A study by Zhang et al [6] demonstrated that there was no significant difference in the rate of drug-resistant mutations among the three HBV genotypes, B, C, and D.Moreover, the development of drug resistance was accompanied by mutations in drug-resistant genes, and such changes may increase the chances of CHB transforming into cirrhosis or liver cancer.Another study revealed that there was no significant difference between patients with hepatitis B and type C in terms of ALT recurrence rate, as well as HBeAg seroconversion rate under the same treatment regimen.There was also no significant difference in the HBV-DNA clearance rate between patients with type B and type C, except after 6 months of follow-up [19].The genotype analysis of the patients showed that the genotype of the 114 hepatitis B virus patients was predominantly genotype C, accounting for 63.2 % of the total patients.The results of the treatment regimen with peginterferon alfa-2a in combination with entecavir in patients with both hepatitis B virus genotypes have not been conclusive.However, in this study, it was found that at months 6 and 12, the HBV-DNA clearance rate, ALT regression rates, and HBeAg seroconversion rate were significantly better in patients with genotype B CHB than in patients with genotype C CHB.This is a significant difference from previous studies.In addition, the QOL of all patients was also included in the followed up, and results demonstrated that the QOL of patients treated with peginterferon alfa-2a in combination with entecavir improved significantly, with patients with genotype B CHB showing significantly better improvement than those with genotype C CHB.This further suggests that genotype B is more sensitive to the above combination regimen and has a better viral and biochemical response than genotype C.However, the mechanism is not fully yet understood, and further experiments are needed to verify this.Possible reasons for this are that genotype C has a higher frequency of mutations in the underlying core promoter A1762T/G1764A than genotype B, in patients with chronic hepatitis B. Patients with genotype C has a higher lifetime risk of developing cirrhosis and HCC than genotype B.

Limitations of the study
Although the follow-up period of this study was long, the sample size was not large enough, and the conclusions drawn may not be generalizable.

CONCLUSION
The combination of entecavir and peginterferon alfa-2a is effective in treating CHB patients with LAM-resistant.The viral and biochemical responses and the QOL scores of patients with genotype B are better than those of CHB patients with genotype C.These findings need to be validated via further experiments in vivo and in vitro.

DECLARATIONS
Chen collected and analyzed data, while Xiaoyun Peng wrote the manuscript which was approved by all authors.

Open Access
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the

Table 1 :
General information on patients with two HBV genotypes

Table 2 :
HBV-DNA clearance rates in patients with two HBV genotypes

Table 3 :
ALT normalization rate in patients with two HBV genotypes

Table 4 :
HBeAg seroconversion rates in patients with two HBV genotypes