FormalPara Key Summary Points

Why carry out this study?

The number of hepatitis C patients is increasing globally, and hepatitis C disease and its complications are also a major health burden. Affordable pan-genotype treatment options remain an unmet medical need for patients with chronic hepatitis C in China.

Most of the approved direct-acting antiviral (DAA) drugs in China are specifically used for the treatment of HCV GT-1, and the accessibility of DAA drugs for GT2, GT3, and GT6 patients in China remains to be resolved.

This clinical study confirmed the efficacy and safety of alfosbuvir combined with daclatasvir for 12 weeks in adult patients with chronic hepatitis C, which may provide a new treatment option for liver cancer patients in China.

What was learned from this study?

Alfosbuvir combined with daclatasvir for 12 weeks showed good efficacy in patients with chronic hepatitis C of genotype 1, 2, 3, and 6, with a sustained virological response rate of > 95%. In particular, patients with GT-2 hepatitis C were more sensitive to the study drug, and the sustained virological response rate reached 100%.

The efficacy of the alfosbuvir combination regimen is not affected by cirrhosis status, prior interferon therapy, or baseline NS5A and/or NS5B RAS, and it has a good clinical safety profile.

Introduction

There are nearly 170 million people worldwide chronically infected with hepatitis C virus (HCV) [1]. HCV and its complications remain a significant medical burden in Asia. HCV infection has become one of the leading causes of hepatic cell carcinoma (HCC) and cirrhosis in China [2]. Since the early 1990s, China has gradually carried out regular HCV screening before blood transfusion or surgery. This means that the incidence of HCV infection in China showed a rapid decline in the past 30 years. However, considering the large population in China, hundreds of thousands of people are still expected to be infected with HCV each year [3].

In recent years, direct-acting antiviral drugs (DAAs), such as sofosbuvir and ledipasvir, have been used in patients with HCV [4, 5]. Most of the DAAs launched in China only cover a few genotypes, including the most common HCV genotype 1. In addition, many DAAs are imported, and Chinese patients face a similar insurance payment dilemma to those in the USA, resulting in an underestimation of the actual economic burden on patients [6]. Therefore, the development of pan-genotype drugs can not only meet clinical needs but also provide more treatment options for patients with HCV infection.

Alfosbuvir (formerly Holybuvir; alfosbuvir) is an HCV NS5B polymerase inhibitor independently developed by Nanjing Sanhome Pharmaceutical Co., LTD. According to the result of two unpublished preliminary clinical studies, we can see that 3 days of alfosbuvir monotherapy significantly reduced HCV RNA levels at doses of 400, 600, and 800 mg [7]. Another pan-genotypic HCV NS5A inhibitor, daclatasvir, has been approved for the treatment of chronic hepatitis C [8,9,10]. When combining alfosbuvir with daclatasvir in treatment for HCV infection, this regimen showed excellent synergistic antiviral activity without complex drug–drug interaction. Therefore, we conducted a phase 2 study to evaluate the efficacy and safety of this treatment regimen (NCT04070235). Data from the phase 2 study showed that alfosbuvir plus daclatasvir was well tolerated and highly effective for Chinese patients infected with HCV, with sustained virological response 12 weeks after the end of treatment (SVR12) of 90.2%, 95.2%, and 100% in patients receiving daclatasvir at a standard dose of 60 mg daily plus alfosbuvir at a dose of 400, 600, or 800 mg, respectively [7]. On the basis of both efficacy and safety considerations, 600 mg daily was selected as the recommended phase 3 dose for alfosbuvir.

The current study was a multicenter, open-label phase 3 study, aiming to confirm the efficacy and safety of 60 mg daclatasvir plus 600 mg alfosbuvir in treatment of Chinese patients with HCV (NCT04070235).

Methods

Study Design

This multicenter, single-arm, open-label, phase 3 study was conducted at 35 study centers in China between 7 November 2019 and 6 November 2020. Eligible patients were chronically infected with HCV genotypes 1–6 or indeterminate genotype, among which no more than 20% of the subjects had previously received an interferon-based regimen either with or without ribavirin, and no more than 20% of the subjects had compensated cirrhosis. All patients with chronic HCV infection received 600 mg alfosbuvir tablets plus 60 mg daclatasvir tablets once daily for 12 weeks. Patients were followed up at post-treatment weeks 4 and 12, and those who achieved SVR12 were followed up at post-treatment week 24.

Before enrollment and initiation of any study procedures, written informed consent was obtained from all patients. The study was approved by the independent ethics committee at each participating site and was conducted in accordance with the principles of the Declaration of Helsinki, Chinese legal and regulatory requirements, and good clinical practice. This trial is registered with ClinicalTrials.gov (no. NCT04070235) and with ChinaDrugTrials.org.cn (no. CTR20182539). Written informed consent was obtained from all patients prior to enrollment and initiation of any study procedures. The study was approved by an independent ethics committee at each participating site, and we obtained ethics committee approval from all 35 research centers prior to the study commencing. It is carried out in accordance with the principles of the Declaration of Helsinki, China’s legal and regulatory requirements, and good clinical practice. The trial was conducted in mainland China, and no IRB approval was sought.

Patients

Inclusion criteria of the study were: aged ≥ 18 years; chronically infected with HCV genotype 1, 2, 3, or 6; HCV-RNA level ≥ 104 IU/mL. The enrolled patients were either treatment-naive or experienced with interferon (IFN)-based regimens (approximately 20% of them may be treatment experienced). Up to 20% of the patients might have compensated cirrhosis, which is defined as any one of the following: (i) FibroScan showing a liver stiffness value of > 12.5 kPa within ≤ 6 months of screening; (ii) liver biopsy showing cirrhosis within ≤ 12 months of screening. The exclusion criteria of the study were: received IFN-based antiviral therapy within 6 months prior to baseline; previously exposed to direct-acting antivirals (DAAs) of any source; in unstable or uncontrolled medical condition; suffering from decompensated liver disease or hepatocellular carcinoma; co-infected with hepatitis B virus (HBV) or human immunodeficiency virus (HIV). Detailed inclusion and exclusion criteria are presented in Supplementary Information 1.

Outcome Analyses

The primary efficacy endpoint was SVR12. The secondary efficacy endpoints included the proportion of patients who achieved virological response at treatment weeks 4 and 12 and at post-treatment weeks 4 and 24, the proportion of patients who experienced on-treatment virological breakthrough at treatment weeks 2, 4, 8, and 12, the proportion of patients who experienced post-treatment virological relapse at post-treatment weeks 4 and 12, and the on-treatment and post-treatment kinetics of circulating HCV RNA. Exploratory evaluation was the impact of baseline NS5A resistance-associated substitutions (RASs) and NS5B RASs on SVR12, and the occurrence of NS5A/B RASs following virological failure. The primary safety was assessed by monitoring AEs, vital signs, physical examination results, clinical laboratory results, 12-lead electrocardiography, and other safety test results.

Screening, on-treatment, and post-treatment study assessments are outlined in Supplementary Information 2. HCV RNA was measured by using a COBAS® TaqMan® HCV Test v2.0 or COBAS® AmpliPrep/COBAS® TaqMan® HCV Test v2.0 with a lower limit of quantitation (LLOQ) of 15 IU/mL. HCV RNA and genotyping assessments were conducted at Guangzhou Kingmed Center for Clinical Laboratory (Guangzhou). Blood samples to determine serum levels of HCV RNA were collected prior to initiation of alfosbuvir/daclatasvir, at treatment weeks 1, 2, 4, 8, and 12, and at post‐treatment weeks 4, 12, and 24. To evaluate drug-class resistance-associated substitutions (RASs), the HCV NS5A and NS5B coding regions were deep sequenced for all patients at baseline and for any patients with virological failure at the time of failure. Resistance testing was performed at Guangzhou Kingmed Center for Clinical Laboratory (Guangzhou). Safety assessments included monitoring of adverse events (AEs), abnormalities in clinical laboratory assessments, vital sign measurements, and physical examinations. AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 22.0.

Statistical Analysis

The clinical study was designed using a single-group target value method for efficacy comparison with historical controls. Based on the results of the phase II interim analysis, it is expected that the SVR12 rate of alfosbuvir combined with daclatasvir can reach 95%, and 90% of patients can achieve optimal outcomes. After communication with the regulatory authorities, the historical control was set at 88%, and the test used a bilateral precise single-sample binomial test with a significance level of 0.05. Considering the need for safety data collection, the sample size was finally determined to be 320 cases. This 88% rate is a benchmark rate formulated according to the status of hepatitis C treatment in China. It is not simply a historical control for previous HCV treatment trials, as standard treatment varies by genotype. The rationale for adopting this design is described in the “Discussion” section.

Efficacy evaluation was based on simultaneous analysis of the full analysis set (FAS) and per protocol set (PPS). In the primary efficacy analysis of phase 3, the SVR12 rate was compared with historical data of 88%, using a two-sided exact one-sample binomial test at an α level of 0.05. For secondary efficacy endpoint, the corresponding number and proportion of subjects were calculated, and the 95% confidence intervals were calculated using the Clopper–Pearson method. Subgroup analyses of SVR12 by baseline demographics and disease characteristics were performed. Predictors of antiviral response were assessed by regression analysis. Safety analyses were conducted in all participants who received at least one dose of study medication. Safety data were summarized descriptively. AEs were coded using the Medical Dictionary for Regulatory Activities. Resistance analyses were conducted in the resistance analysis population. All statistical summaries and analyses were performed using SAS software version 9.4 or above.

Results

Patient Population

The study was conducted from November 2019 to November 2020 at 35 centers across China. Of the 421 patients screened, a total of 327 patients were enrolled in the study, and 326 patients received at least one dose of alfosbuvir plus daclatasvir. The main reason for screening failure was that patients did not meet inclusion criteria or met exclusion criteria (Fig. 1).

Fig. 1
figure 1

Trial profile

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Overall, there were similar proportions of men and women (52.8% versus 47.2%), with a median age of 50 years (range: 23, 72). The genotype of the patients was as follows: genotype 1 accounted for 51.8% of the patients, genotype 2 accounted for 29.1%, genotype 3 accounted for 6.7%, and genotype 6 accounted for 12.3%. Because of the very low prevalence of genotypes 4 and 5 in China, no genotype 4 or 5 was detected or enrolled in this study. Forty-one (12.6%) patients had compensated cirrhosis at baseline, and 23 (7.1%) had previously failed treatment with interferon-based therapy. A majority (83.1%) of patients had the IL28B CC allele, and approximately half (55.2%) of patients had baseline HCV RNA ≥ 2,000,000 IU/mL. A total of 99.4% of patients had preexisting NS5A and/or NS5B RASs at baseline. The baseline demographics and disease characteristics are presented in Table 1.

Table 1 Baseline demographics and disease characteristics
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Efficacy

Of the 326 patients who received at least one dose of the study drug, 320 (98.2%) achieved the primary endpoint, namely SVR12, which was superior to the historical SVR rate of 88% (p < 0.0001), meeting the primary efficacy endpoint of the study. All patients who had an SVR12 rate achieved SVR24, except for one patient who did not return for a visit at post-treatment week 24. Virological response rates during and after treatment with alfosbuvir plus daclatasvir are summarized in Table 2. The virological breakthrough rate at treatment weeks 2, 4, 8, and 12 was 0.6% [95% CI: 0.1–2.2], 12.9% (95% CI: 9.4–17.0), 15% (95% CI: 11.3–19.4), and 3.7% (95% CI: 1.9–6.3), respectively. The virological relapse rate at post-treatment weeks 4 and 12 was both 1.5% (95% CI: 0.5–3.5). Relative to post-treatment week 4, no new virological relapse occurred at post-treatment weeks 12 and 24. The plasma level of HCV RNA declined rapidly with the treatment. The HCV RNA level (Log10 IU/mL, mean ± SD) declined to 1.9029 ± 0.6732 after 1 week of treatment, and to 1.1461 ± 0.0000 after 12 weeks of treatment. The decrease in HCV RNA level from baseline at different test time points is shown in Fig. 2.

Table 2 Virological response rates during and after treatment
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Fig. 2
figure 2

Decrease of HCV RNA relative to baseline at different test time points

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The results of the subgroup analysis of SVR12 rate showed that there were some differences in the SVR12 rate between different HCV genotype subgroups. GT-1a, GT-2a, and GT-3b groups showed the best efficacy, with an SVR12 rate of 100.0%. The regimen of alfosbuvir plus daclatasvir seemed to show a similar or more potent effect in patients with HCV genotype 3 compared with the current standard treatment. Patients with GT-1b and GT-6a also responded well to the treatment, with an SVR12 rate of 98.8% and 97.1%, respectively. The SVR12 rate was also different among subgroups of patients with different cirrhosis status. In patients without cirrhosis, the SVR12 rate of the treatment was 99.3%; compared with patients without cirrhosis, the efficacy in patients with compensatory cirrhosis was slightly worse, with an SVR12 rate of 92.7%. In addition, the SVR12 rates were similar regardless of most baseline characteristics, such as prior HCV treatment history, IL28B genotype, viral load, and presence of preexisting RASs. Subgroup analysis of baseline characteristics and virological response rates at week 12 post-treatment is presented in Table 3.

Table 3 Subgroup analysis between baseline characteristics and virological response rates
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Multivariate analysis of predictors of virological response rate (Supplementary Information 3) showed that the independent variables entered into the model in the multivariate analysis of SVR12 rate were cirrhosis status (P = 0.0055) and baseline viral load (P = 0.0679), the odds ratio OR were 0.0693 (95% CI 0.0105, 0.4557) and 0.1723 (95% CI 0.0261, 1.1380), the results suggest that cirrhosis was a risk factor for treatment failure, and subjects with cirrhosis are 0.0693 times more likely to achieve SVR12 than those without cirrhosis, while the baseline viral load has no statistically significant effect on SVR12.

Resistance-Associated Substitutions

A total of five subjects did not achieve SVR12 during the study period. The drug resistance mutations before and after treatment in five patients who failed treatment were tested. The results showed that all the subjects developed new resistance mutations in the NS5A region after treatment compared with baseline, but none of the subjects developed new resistance mutations in the NS5B region after treatment (Supplementary Information 4). All amino acid mutations at positions 28, 31, 62, and 93 in the NS5A region were common drug resistance mutation sites. Among the subjects enrolled in this trial, all subjects with baseline amino acid mutations at positions 28, 31, 62, and 93 achieved SVR, except for the five subjects who had failed treatment. We therefore speculated that the treatment failure of the subjects in this study may not be clearly correlated with NS5A mutations.

Safety

The overall incidence of treatment-emergent AEs (TEAEs) was 82.2% (268/326), comprising 232 patients with grade 1 (71.2%), 106 patients with grade 2 (32.5%), 33 patients with grade 3 (10.1%), and 3 patients with grade 4 (0.9%) (Table 4). Grade 4 AEs were increased creatine phosphokinase (0.6%) and hypertriglyceridemia (0.3%). A total of 124 patients (38.0%) experienced TEAEs related to study drug, and the most often reported (≥ 1%) were hypercholesterolemia (10.1%), hypertriglyceridemia (5.8%), hyperlipidemia (6.4%), increased blood bilirubin (2.5%), increased amylase (2.1%), increased aspartate aminotransferase (1.5%), increased total bile acid (3.7%), and hypothyroidism (1.2%). Only one patient prematurely discontinued the study treatment, owing to hepatic cancer, and no deaths were reported. Twenty-five (7.7%) patients experienced serious AEs, none of which was judged to be related to alfosbuvir plus daclatasvir treatment. Grade 3 or 4 laboratory abnormalities were observed in 14 (4.3%) patients. All of them were asymptomatic and required no specific medical intervention. No patients had grade 3 or 4 elevations in alanine aminotransferase, aspartate aminotransferase, or bilirubin. In the hepatic function test, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase decreased gradually after the subjects took the drug (Supplementary Information 5). During the follow-up period after the treatment, it remained in a decreasing state without an upward trend. The result showed a trend of normalization in hepatic function throughout the study period.

Table 4 Frequency and severity of adverse events
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Discussion

China has a large number of patients with HCV infection, but the era of treatment with DAAs officially arrived with the approval of daclatasvir and asunaprevir in 2017. Until now, China’s domestic DAAs were limited and mainly depended on imports, resulting in a considerable economic burden on patients. Among the HCV DAA drugs that have been marketed in China, most only target HCV GT1 and/or GT4 (e.g., sofosbuvir/ribavirin/interferon, danoprevir/ribavirin/interferon, daclatasvir/asunaprevir, ombitasvir/paritaprevir/dasabuvir, elbasvir/grazoprevir). For GT2, GT3, and GT6, which account for about 40% of chronic hepatitis C patients in China, especially for GT3, the choice of DAAs is still limited [11,12,13]. In the phase III trial design of SH229 combined with daclatasvir, the incidence of SVR12 in patients with GT3 compensatory cirrhosis at 24 weeks was only 88% according to the results of the study of sofosbuvir combined with ribavirin in Chinese patients [4]. Clinical trials with similar characteristics to this study mostly compare with historical data when analyzing the SVR12 rate, such as the 85% of the ASTRAL-1 study [14,15,16]. The historical reference value of 88% has been recognized by the National Medical Products Administration.

The efficacy evaluation results of the study showed that the overall SVR12 rate was 98.2% (95% CI 96.5, 99.5), and the efficacy was significantly better than the prespecified historical control of 88% (P < 0.0001).

At post-treatment week 4, five subjects actually had virological relapse, except for one subject who did not complete follow-up; compared with post-treatment week 4, no new virological relapse occurred in the subjects at post-treatment weeks 12 and 24. The results showed that SVR4, SVR12, and SVR24 rates had good consistency. As a whole, the study drug had better efficacy in patients with HCV genotypes 1, 2, 3, and 6, achieving an SVR of more than 95.0%; patients with HCV genotype 2 were more sensitive to the study drug, achieving a 100% SVR. In terms of genotype, the efficacy of the drug was better in patients with HCV genotypes 1a, 1b, 2a, 3b, and 6a, and the SVR12 rate was over 97.0%. In addition, there were only six subjects with genotype 6n enrolled in this study, and one of them failed treatment. Therefore, the low SVR rate among subjects with genotype 6n may be related to the small sample size, which was not statistically significant. The results of multivariate analysis on predictors of virological response rate showed that the presence of cirrhosis was a risk factor for treatment failure, and subjects with cirrhosis were 0.0693 times more likely to achieve SVR12 than those without cirrhosis. The results of drug resistance mutation analysis showed that the efficacy of alfosbuvir plus daclatasvir in Chinese adults with chronic hepatitis C was not affected by baseline NS5A and/or NS5B RASs.

Three pan-genotype hepatitis C drug combinations (sofosbuvir/velpatasvir, glecaprevir/pibrentasvir and coblopasvir hydrochloride/sofosbuvir) have been approved for sale in China, and the study results show that the efficacy in patients with GT-3 hepatitis C is moderate. For example, a phase III study of sofosbuvir/velpatasvir in a predominantly Chinese Asian population showed an overall SVR12 rate of 97% for 12 weeks of sofosbuvir/velpatasvir treatment in patients with gene type 1–6 hepatitis C without cirrhosis or with compensatory cirrhosis. The SVR12 rates of genotype 1, 2, 3a, 3b, and 6 patients were 100%, 100%, 95%, 76%, and 99%, respectively [17]. Two phase III clinical studies in an Asian population dominated by the Chinese population showed that the overall SVR12 rate of glecaprevir/pibrentasvir treatment for 8 weeks was 97.2% (96.9% in the Chinese population) in patients with genotype 1–6 hepatitis C absence of cirrhosis. The rates of SVR12 in genotype 1, 2, 3, and 6 patients were 99.4%, 97.8%, 76.9%, and 100%, respectively (100%, 98.1%, 76.9%, and 100% in Chinese population, respectively) [18]. The China phase III clinical study of the coblopasvir hydrochloride/sofosbuvir therapy regimen showed that the overall SVR12 rate of coblopasvir hydrochloride/sofosbuvir therapy for 12 weeks was 97% in patients with genotype 1–6 hepatitis C without cirrhosis or with compensatory cirrhosis. The rates of SVR12 in genotype 1, 2, 3, and 6 patients were 99%, 96%, 90%, and 98%, respectively [19]. The alfosbuvir /daclatasvir combination has shown a superior virological response rate in patients with GT3 hepatitis C, particularly GT3b hepatitis C.

During the study, the incidence of TEAEs was 82.2% (268/326), and the incidence of ARs was 38.0% (124/326). Lipid metabolism abnormalities such as hypercholesterolemia (10.1%), hyperlipidemia (6.4%), and hypertriglyceridemia (5.8%) were the most common AEs of the alfosbuvir plus daclatasvir hydrochloride treatment regimen. There have been many literature reports highlighting the safety risks of DAAs, which can cause abnormal lipid metabolism abnormalities. Endo et al. found that, in patients with chronic hepatitis C (N = 276) who received sofosbuvir/ledipasvir for 12 weeks or daclatasvir/asunaprevir for 24 weeks and achieved SVR, significant increases in total cholesterol (TC), low-density cholesterol (LDL-C), and high-density cholesterol (HDL-C) levels can be observed [20]; Jain et al. found that, in patients with chronic hepatitis C (N = 50) who received sofosbuvir/daclatasvir for 12 weeks, the levels of TC and LDL-C also showed a significant increase after treatment [21]. Graf et al. reported that patients with chronic hepatitis C (N = 46) who received sofosbuvir/velpatasvir, gorcarrevir/pibrentasvir, elbasvir/grazoprevir, sofosbuvir/velpatasvir/voxilaprevir, and other DAA regimens exhibited a significant increase in the average level of TC, LDL-C, HDL-C, and liver fat content [22]. Doyle et al. reported that patients with chronic hepatitis C (N = 24) who received ombitasvir/dasabuvir/ribavirin also exhibited a significant increase in TC, LDL-C, and triglyceride (TG) levels and liver fat content [23]. The above findings suggest that DAAs exert a significant impact on the lipid metabolism of the host, irrespective of the target and structure of the DAAs. The possible mechanisms by which DAAs affect lipid metabolism are as follows: Upon entering the blood circulation, HCV binds to apolipoproteins (ApoB, ApoE, and ApoC), cholesterol lipids, phospholipids, cholesterol, triglycerides, and other lipids in the blood of the host to form lipoviral particles (LVPs), and subsequently enters liver cells via LDL receptors. This process may cause a decrease in the content of lipids in the blood, while DAAs can reverse the above process. When HCV is cleared, the lipids bound to HCV are released, thus leading to an increase in the concentration of lipids in the blood [22,23,24]. The incidence of SAEs during the study was 7.7% (25/326), none of which were related to the study drug. Most AEs were controllable and tolerable. No patients died during the study.

In the clinical study of alfosbuvir combined with daclatasvir, only historical controls were used for study design, which lacked direct comparison with the effectiveness and safety of listed DAA drugs, and the study results were one-sided. In this study, owing to the widespread spread of COVID-19 in China, it is difficult to collect clinical research data, and part of the interview results are missing. Although these data have been properly processed in the statistical process, it still has a slight impact on the overall trial.

In general, the clinical effectiveness, good tolerability, and safety characteristics of the alfosbuvir plus daclatasvir treatment regimen show a good benefit–risk ratio, supporting the use of this drug in patients with chronic hepatitis C genotypes 1, 2, 3, and 6 who are newly treated or treated with interferon. Treatment with the pan-genotypic regimen of alfosbuvir plus daclatasvir for 12 weeks was highly effective and safe in Chinese patients infected with HCV genotypes 1, 2, 3, or 6, suggesting that this regimen could be a promising option for HCV treatment in China, irrespective of genotype. The clinical benefits of this combination regimen address unmet medical needs in China, which will facilitate the WHO goal of HCV elimination.

Conclusions

Overall, the clinical efficacy, tolerability, and safety profile of alfosbuvir in combination with daclatasvir demonstrated a favorable benefit–risk ratio, supporting the use of alfosbuvir in the treatment of adults with genotype 1, 2, 3, and 6 chronic hepatitis C with initial or interferon-treated treatment. Compared with the three pan-genotype hepatitis C drug combinations approved for market in mainland China (sofosbuvir/velpatasvir, glecaprevir/pibrentasvir, and coblopasvir hydrochloride/sofosbuvir), the alfosbuvir/daclatasvir combination regimen demonstrated a superior virological response rate in patients with GT3 hepatitis C, especially GT3b hepatitis C patients. It provides a new choice for the treatment of hepatitis C patients in China.