JSH Guidelines for the Management of Hepatitis C Virus Infection: A 2016 update for genotype 1 and 2.

Abstract

The Japan Society of Hepatology and Drafting Committee for Hepatitis Management Guidelines produced the first clinical practice guidelines for the management of hepatitis C virus (HCV) infection in 2012, followed by frequent updates. As English versions, we published JSH guidelines in 2013,1 and a 2014 update for genotype 1 in 2014.2 Thereafter several interferon-free regimens with direct acting antivirals (DAAs) have been launched in the clinical setting both for genotype 1 and 2 and treatment recommendations have been greatly changed with these progresses. In this year 2016, the Drafting Committee for Hepatitis Management Guidelines lauched a 2016 update for genotype 1 and 2. These JSH guidelines are intended to assist physicians and other healthcare providers to assist their decision making in the clinical process. The Committee defenitely hope these guideline help patients infected with HCV, their families and other interested individuals to overcome HCV infection and improve the outcome and quality of life with assistance of physicians and other healthcare providers. In these updated version, we focused on newly-available IFN-free DAAs and the current treatment recommendations. Please refer to the previous versions1, 2 when IFN, ribavirin, and other IFN-based DAAs (telaprevir, simeprevir) are of interst. Hepatitis C virus (HCV) was discovered by Choo et al. in the United States of America in 1989,3 and it has become clear that at least 90% of patients who were diagnosed as non-A, non-B hepatitis and at least half of all patients who were diagnosed as alcoholic liver diseases have liver damages caused by HCV. It is estimated that 170 million persons around the world and between 1.5 and 2 million persons in Japan are HCV carriers. When healthy persons are infected with HCV, around 30% have an acute course and then recover, and in around 70% the HCV infection persists and progresses to chronic hepatitis. If the infection becomes chronic, the incidence of spontaneous clearance of the virus is extremely low – 0.2% annually – and hepatic fibrosis results from inflammation caused by the infection, and the disease progresses to cirrhosis and/or hepatocellular carcinoma.4 The goal of hepatitis C therapy is to improve the long-term prognosis for chronic liver disease caused by persistent HCV infection – specifically, to prevent hepatocarcinogenesis and death associated with liver disease. To reach this goal, antiviral therapies are administered to achieve HCV clearance. Inflammation has in fact been shown to subside in patients who have achieved complete elimination of HCV RNA through interferon (IFN) therapy,5 and it has also been shown that progression to cirrhosis and hepatocarcinogenesis are prevented in such patients.6, 7 However, even in patients achieving a sustained virological response (SVR) with IFN therapy, the clearance of HCV does not by itself prevent hepatocarcinogenesis and, as described below, of patients with a mean observation period of 3.3 to 8.0 years.9-16 Furthermore, it is not at present clear whether or not the clearance of HCV through the use of IFN-free DAAs (direct-acting antivirals), which were introduced to clinical practice in 2014, will afford the same level of hepatocarcinogenesis prevention efficacy as IFN therapy. Therefore, follow-up for hepatocarcinogenesis needs to be performed to improve the long-term prognosis even after HCV clearance has been achieved through the use of IFN or DAAs. Patients who are elderly and have advanced fibrosis and are therefore at high risk for carcinogenesis need to be monitored particularly carefully. IFN therapy started to be used for hepatitis C in the clinical setting in the West in 1991 and in Japan in 1992 following confirmation by Hoofnagle et al. in 1986 that the administration of human recombinant IFN-alfa in non-A, non-B hepatitis resulted in the normalization of transaminases.17 IFN monotherapy gave way to the use of IFN in combination with ribavirin (RBV), and Peg-ylated IFN (Peg-IFN) and RBV combination therapy ended up becoming the standard antiviral therapy, and resulted in an increase in the percentage of patients achieving an SVR. However, among HCV genotype 1 and high viral load patients, who are difficult to treat, the percentage achieving SVR on this same therapy is 40% to 50%, and about half of these patients do not achieve HCV clearance. In recent years, a number of novel antiviral drugs have been developed with the aim of improving treatment efficacy or reducing the adverse reactions, and in November 2011, it became possible to use telaprevir, a first-generation protease inhibitor, in the clinical setting in genotype 1 and high viral load patients. Telaprevir + Peg-IFN + RBV combination therapy results in a higher SVR rate – 70% – in treatment-naïve patients. This therapy therefore affords better antiviral efficacy, but adverse events including the progression of severe anemia, the emergence of serious skin esions, and decreased renal function have been observed.18-22 Then, in November 2013, insurance coverage was approved for the use of simeprevir,23-25 a second-generation protease inhibitor, in genotype 1 and high viral load patients. In a Japanese clinical study of simeprevir + Peg-IFN + RBV combination therapy, the percentage of treatment-naïve patients achieving SVR rose to about 90%, and the adverse reactions were nearly identical to those seen in the placebo group.23 Then, in July 2014, IFN-free DAA combination therapy with a protease inhibitor (asunaprevir) and an NS5A inhibitor (daclatasvir) was approved, and it became possible to treat IFN-ineligible and non-responded patients, who had been difficult to treat with conventional antiviral therapies; the SVR rate in Japanese clinical studies was 80% to 90%.26 Furthermore, in a Japanese clinical study of combination therapy with an NS5B inhibitor (sofosbuvir) and an NS5A inhibitor (ledipasvir), which are second-generation IFN-free DAAs, the SVR rate was 90%, and none of the patients in the sofosbuvir plus ledipasvir combination therapy group discontinued from the study because of adverse reactions and no serious adverse reactions occurred. Furthermore, in September 2015, combination therapy with a protease inhibitor (paritaprevir), NS5A inhibitor (ombitasvir) and ritonavir, which has no antiviral effect but is added for anticipating booster effect (increasing plasma concentration and prolongation of half-life of patritaprevir) was approved, and the SVR rate in Japanese clinical trials was excellent as well, more than 95%. In genotype 2 patients, although the SVR rate on Peg-IFN + RBV combination therapy had normally been about 80%, in September 2014 is became possible to use telaprevir +Peg-IFN + RBV combination therapy in patients for whom Peg-IFN plus RBV combination therapy is not particularly effective. Then, in March 2015, the use of the IFN-free combination therapy sofosbuvir plus RBV was approved for use in genotype 2 patients as well; the SVR rate in Japanese clinical studies was 97%. Generally, the liver diseases of patients with persistent HCV infections progress gradually, accompanied by ALT increases, and the risk of carcinogenesis increases along with the progression of fibrosis.9 Conversely, carcinogenesis from a normal liver with no inflammation or fibrosis is almost never seen. Therefore, although all HCV infected patients, except for decompensated cirrhosis patients, are eligible for antiviral therapy, patients with increased ALT levels that are indicative of liver inflammation (ALT > 30 U/l) and patients with decreased platelet counts that are indicative of liver fibrosis (platelet count < 150,000/μl) are good candidates for antiviral therapy for HCV. In addition, patients with poor prognoses because of concurrent illnesses other than liver diseases should not be targeted for antiviral therapy. Although it needs to be kept in mind when determining the suitability of antiviral therapy that patients with ALT < 30 U/l and platelet count ≥ 150,000/μl are at low risk of hepatocarcinogenesis, it should also be kept in mind that the risk of carcinogenesis is not low in the elderly, even in elderly patients with ALT < 30 U/l and platelet count ≥ 150,000/μl. In addition, the patients that are believed to need rapid viral clearance are patients that are at high risk for carcinogenesis. It has become clear that in hepatitis C, the three factors “advanced age,” “advanced fibrosis,” and “male sex” are independent risk factors for hepatocarcinogenesis.6-8 Because patients with more than one of these factors are at particularly high risk for oncogenesis, early antiviral therapy induction should be considered. In analyses of hepatocarcinogenesis in hepatitis C, although the definition of who is an elderly person is not uniform, with the cutoff sometimes being 55 years, or 60 years, or 65 years, even among elderly persons the risk of carcinogenesis increases as age increases. In this guideline, elderly persons are defined as persons with “age ≥ 66 years” based on, for example, the fact that the incidence of hepatocarcinogenesis increases after age 65.10 In addition, although advanced fibrosis is defined as “hepatic fibrosis ≥ F2 or platelet count < 150,000/μl,” it is necessary to keep in mind that the risk of carcinogenesis is particularly high in those patients with “hepatic fibrosis ≥ F3 or platelet count < 120,000/μl.” In patients at high risk for carcinogenesis (patients who are both elderly and have advanced fibrosis), antiviral therapy should be initiated as soon as possible, provided the treatment is judged to be tolerable, and patients who are either elderly or have advanced fibrosis should also be started on antiviral therapy early on. However, in patients who are neither elderly nor have advanced fibrosis, who are at low risk for carcinogenesis, the suitability of antiviral therapy should be determined taking into account therapeutic efficacy and adverse reactions as well as the risk of carcinogenesis. In addition, in both groups, it is at present difficult to achieve viral clearance with antiviral therapy, and liver supporting therapies with ursodeoxycholic acid and glycyrrhizin should be administered in patients with abnormal ALT levels (< 30 U/l). Moreover, the long-term use of low doses of Peg-IFN to bring inflammation under control is another option. If such therapies fail to afford adequate efficacy, and iron overload is suspected, then switching to phlebotomy therapy or adding phlebotomy therapy as a concomitant therapy should be considered. The goal of these therapies is to maintain the ALT level at no higher than 30 U/l and to keep the ALT level as low as possible. Strict ALT control is particularly necessary in patients at high risk for carcinogenesis. Furthermore, low-dose Peg-IFN therapy should be discontinued if an improvement in ALT levels (≤40 U/l) or an improvement in AFT levels (≤10 ng/ml) is not achieved within 6 months.27, 28 SVR is defined as a negative result for HCV RNA at 24 weeks after the end of antiviral therapy. Patients who achieve SVR following IFN therapy normally remain HCV RNA negative, and the percentage of subjects achieving SVR on IFN + RBV combination therapy who remain negative has been reported to be between 99% and 100% (mean course observation period of 5.6 years; range: 1 year to 8.3 years).29, 30 In studies conducted before the year 2000, however, the percentage of patients remaining HCV RNA negative was reported to be somewhat lower: 96% to 98%.31-35 This is attributed to the fact that, in these studies, IFN monotherapy was the principal therapy administered, and to the fact that at that time HCV RNA detection sensitivity was low, and false positive results were obtained when assessing SVR. When SVR is achieved through IFN therapy, the patients remain negative for HCV RNA, and the risk of carcinogenesis developing from hepatitis C is significantly decreased.7-9, 12, 36 However, liver cancer has been reported during course observation even in patients who have achieved SVR. There are many reports in Japan of hepatocarcinogenesis following achievement of SVR,9-16 with the reported incidence of carcinogenesis ranging from 0.9% to 4.2% over a mean observation period of 3.3 years to 8.0 years. Reported risk factors for carcinogenesis include advanced age, male sex, advanced fibrosis, alcohol use, fatty liver, and insulin resistance. The time from SVR achievement to carcinogenesis is most often less than 10 years, although there have been scattered reports of carcinogenesis occurring after more than 10 years. Therefore, although no definitive conclusions have been reached about what hepatocarcinogenesis screening period is required following the achievement of SVR through IFN therapy, it appears that screening for liver cancer should be continued for 5 to 10 years after achievement of SVR. In addition, at present there is little evidence regarding whether or not a level of hepatocarcinogenesis prevention efficacy equivalent to that obtained with IFN-induced SVR is obtained when SVR is achieved with the IFN-free DAA therapies that are not being introduced to the clinical setting. Therefore, once HCV clearance is achieved through DAA therapy, patients need to be screened more closely for hepatocarcinogenesis. Careful follow-up is particularly recommended for patients at high risk of hepatocarcinogenesis (patients who are both elderly and have advanced fibrosis). The positive stranded RNA genome of the hepatitis C virus has approximately 9,600 base pairs; the non-structural region that is not incorporated into the viral particles comprises NS2 through NS5B proteins. The current targets of DAAs are the NS3/4A, NS5A, and NS5B proteins, which have, respectively, protease activity, virus genome replication complex formation, and RNA-dependent RNA polymerase activity. As of now, December 2015, five NS3/4A protease inhibitors (telaprevir, simeprevir, asunaprevir, vaniprevir and paritaprevir), two NS5A replication complex inhibitors (daclatasvir, ledipasvir and ombitasvir), and one NS5B inhibitor (sofosbuvir) have been approved and are used in daily practice (Fig. 1). Among them, combinations of NS3/4A and NS5A (daclatasvir/asunaprevir and paritaprevir/ombitasvir) and NS5A and NS5B (sofosbuvir/ledipasvir) are currently used as interferon-free DAA regimens. Daclatasvir was the first NS5A inhibitor that was developed and approved for use in the clinical setting (Fig. 1).37 The HCV nonstructural protein region NS5A is a region that codes for a phosphorylation protein comprising 447 amino acid residues. This region contains an IFN sensitivity determining region (ISDR; aa2209-2248) that correlates to the efficacy of IFN therapy and an IFN/RBV resistance-determining region (IRRDR; aa2334-2379) that correlates to the efficacy of IFN + RBV therapy. Although the functions of NS5A have not been thoroughly elucidated, NS5A is believed to play an important role in virus RNA replication; specifically, it is inferred that the NS5A protein and the core protein interact in HCV particle formation. NS5A inhibitors are low-molecular-weight inhibitors that are expected to have significant virus replication inhibition efficacy. Daclatasvir is a first-in-class, picomolar, highly selective NS5A replication complex inhibitor that exhibits effects in various genotypes. It has been reported, on the basis of investigation of its antiviral efficacy in HCV infected patients, that the HCV RNA load is markedly decreased by the oral administration of daclatasvir 10 mg and above.37 The adult dose is 60 mg as oral daclatasvir once a day. Asunaprevir, on the other hand, is similar to telaprevir and simeprevir in that it is a protease inhibitor that targets the NS3-4A region (Fig. 1).38 The HCV nonstructural protein 3-4A (NS3-4A) is a noncovalent bond complex that comprises NS3 and the cofactor thereof, NS4A. NS3 is a 70 kDa multifunctional protein with a serine protease region in the N-terminal one-third of the protein (amino acids [aa]1-180). The serine protease is a proteolytic enzyme that cleaves the NS3-5 protein. Protease inhibitors, by directly inhibiting this serine protease, inhibit the production of the virus proteins needed for virus genome replication and viral particle formation, and thus strongly inhibit virus replication. Asunaprevir, which is a second-generation protease inhibitor, possesses strong antiviral effects against genotype 1a, 1b, and 4 HCV because it possesses a mechanism of action such as that described above. The adult dose is 100 mg as oral asunaprevir twice a day. In Japan, clinical trials of daclatasvir + asunaprevir combination therapy have been conducted in IFN-ineligible or intolerant patients and in IFN-non responders, and based on the results of these studies, in July 2014 daclatasvir + asunaprevir combination therapy received health insurance coverage for use in IFN-ineligible/intolerant or non-responded patients. Another clinical trial was then conducted in treatment-naïve and relapsed patients, and based on the results of this trial the limitations on health insurance coverage for this therapy were completely lifted in March 2015, and daclatasvir +asunaprevir combination therapy is now covered by health insurance for use in all genotype 1 chronic hepatitis/compensated cirrhosis patients. This therapy is an IFN-free antiviral therapy, which makes it possible to avoid the various adverse reactions that are experienced with IFN, but which also has problematic adverse reactions of its own including elevation of liver enzymes as well as producing drug-resistance associated variants (RAVs), and which therefore needs to be administered by a physician with sufficient knowledge of and experience with the treatment of viral liver disease who has confirmed that the patient is a suitable candidate for said therapy. Similar to other DAAs, asunaprevir and daclatasvir are used together in a two-drug combination therapy because they are insufficiently effective alone. Lok et al. has reported the results of a comparison conducted in the United States in 21 genotype 1 patients who were null responders to Peg-IFN + RBV combination therapy in which 11 patients received daclatasvir + asunaprevir combination therapy (group A) and 10 patients received Peg-IFN + RBV in addition to daclatasvir and asunaprevir (group B).39 The treatment period was 24 weeks for both groups. In group A, 4 of the 11 patients achieved SVR. By genotype, the SVR rate in genotype 1a patients was 22.2% (2 of 9 patients), and all two genotype 1b patients achieved SVR. In group B, however, 9 of the 10 patients achieved SVR. These results show that daclatasvir + asunaprevir combination therapy is more effective in genotype 1b patients than in genotype 1a patients. In Japan, a phase 3 study of daclatasvir + asunaprevir combination therapy was conducted in IFN-ineligible or intolerant patients, and non-responded patients.26 Table 1 shows the backgrounds of the patients enrolled. There were 87 non-responders and 135 IFN-intolerant or ineligible patients. The median ages in these two groups were 60 years and 64 years, respectively, the sex ratios (males/females) were 39/48 and 39/97, respectively, the IL28B genotype (rs12979860) ratios (CC/CT, TT) were 16/71 and 94/41, and the median HCV RNA loads (Log IU/mL) were 6.8 and 6.6. Twenty-two patients with Child-Pugh Grade A compensated cirrhosis were included in this study. However, no studies have been performed in patients with decompensated cirrhosis. Looking at the antiviral efficacy in the study population overall, the HCV RNA virological response rate after treatment initiation was 75.2% (RVR) at week 4, 91.0% (cEVR) at week 12, and 92.3% at week 24 or at the end of treatment (EOT). The percentage of patients with HCV RNA below the limit of quantitation after EOT was 88.7% at week 4 (SVR) and 85.1% at week 12 after EOT (SVR12); the SVR24 rate in the study population overall was 84.7% (188 of 222 patients). The SVR24 rates in non-responders and IFN (+ RBV)-intolerant/ineligible patients were, respectively, 80.5% (70 of 87 patients) and 87.4% (118 of 135 patients), and 90.9% (20 of 22 patients) in the compensated cirrhosis patients (Fig. 2). Efficacy was therefore confirmed in compensated cirrhosis as well. Looking at the results of treatment by background factor, by IL28B genotype, which has a substantial effect on the therapeutic efficacy of IFN, the SVR24 rates in the TT and TG/GG groups were, respectively, 84.8% and 84.3%, so there was no difference in therapeutic efficacy between these two groups. In addition, therapeutic efficacy remained the same for the other background factors as well, such as age, sex, and HCV RNA load at initiation (Fig. 3). The number of patients who experienced relapse after EOT was 6 (7.9%) in the non-responders group and 11 (8.5%) in the IFN (+ RBV)-intolerant/ineligible group. The number of patients who experienced viral breakthrough during treatment was 10 (11.5%) in the non-responders and 4 (3.0%) in the IFN (+ RBV)-intolerant/ineligible group, and the numbers of patients in these groups who were HCV RNA positive at EOT were, respectively, 1 and 2. A phase 3 study of daclatasvir + asunaprevir combination therapy was conducted in treatment-naïve and relapsed patients.40 This study was conducted based on a protocol that called for comparing daclatasvir + asunaprevir combination therapy to telaprevir + Peg-IFN + RBV combination therapy in treatment-naïve patients, and for using only daclatasvir + asunaprevir combination therapy in relapsed patients. The backgrounds of the patients enrolled are shown in Table 2; there were 119 treatment-naïve patients and 22 relapsed patients. Because the treatment-naïve patients were studied based on a comparison versus telaprevir + Peg-IFN + RBV combination therapy, these patients were relatively young, with a median age of 57 years; none were over 70 years of age. In addition, patients with compensated cirrhosis (Fibrotest score F4) accounted for only 6 (5.0%) of the patients. The results of treatment in this study were generally good: the SVR12 rate in the treatment-naïve patients was 89.1% (compared to 62.2% in the telaprevir therapy controls) and 95.5% in the relapsed patients (Fig. 4). As in the clinical study in non-responders and IFN-ineligible/intolerant patients, no significant differences were found in the treatment results by sex, age, HCV RNA load at treatment initiation, or IL28B polymorphism. In a phase 3 study in IFN-ineligible/intolerant and non-responders, serious adverse events occurred in 13 patients (5.9%). The most common adverse events included nasopharyngitis and headache.26 The most common laboratory abnormality was elevations of AST/ALT. The study was performed using a protocol that specified that liver chemistries be performed every 2 weeks until 12 weeks after treatment initiation, and then every 4 weeks thereafter, and that treatment should be discontinued immediately if Grade 4 elavation of ALT was found. Grade 3/4 elavation of ALT or AST (Grade 3: ≥ 5 X and ≤ 10 X ULN; Grade 4: > 10 X ULN) occurred in 7.2% of patients (16 patients) and 5.4% of patients (12 patients), respectively.26 Ten patients (4.5%) discontinued study treatment. The median time to onset of ALT elevation was 10 weeks after treatment initiation, the shortest time to onset was 4 weeks, and the longest was 23 weeks; no fixed pattern was found. However, in most of the patients who experienced Grade 4 ALT elevation, the time it took from when the ALT started to increase until it reached Grade 4 was less than 28 days, with the shortest time being 5 days. All of the patients who discontinued from study treatment exhibited improvement in their ALT levels, and 8 of the 10 patients who discontinued achieved SVR. In addition, in a phase 3 study in treatment-naïve patients and relapsed patients, as well, Grade 3/4 ALT or AST elevation occurred in 15 patients (13%) and 6 patients (5%), respectively, in the treatment-naïve patients, and in 1 patient (5%) and 1 patient (5%), respectively, in relapsed patients. Although there were 5 patients (4%) in the treatment-naïve group and 1 patient (5%) in the relapsed group who discontinued from study treatment because of AST/ALT elevations, all 6 of these patients achieved SVR. In addition, although no significant differences were found in safety between compensated cirrhosis patients and other patients, decompensated cirrhosis patients were not enrolled in clinical studies, and the safety in such patients has therefore not been confirmed. Therefore, daclatasvir + asunaprevir combination therapy should not be administered to decompensated cirrhosis patients. Daclatasvir is a CYP3A4 substrate, and asunaprevir is a CYP3A and OATP1B1 and 2B1 substrate. In addition, daclatasvir has an inhibitor effect on P-glycoprotein, OATP1B1, 1B3, and BCRP, and asunaprevir has an inhibitory effect on CYP2D6, OATP1B1, 1B3, 2B1, and P-glycoprotein, and acts to induce CYP3A4. Because the concomitant use with daclatasvir and asunaprevir of the CYP3A4 inducers or inhibitors, OATP inhibitors, or CYP2D6 substrates with a narrow therapeutic range could result in decreases or increases of the blood levels of daclatasvir, asunaprevir, or the coadministered drugs, the concomitant use of these drugs is contraindicated. Drugs that when coadministered could either affect daclatasvir or asunaprevir or be affected by said coadministration should be coadministered with care.41, 43 The package insert of any drug that is going to be coadministered must be checked 3thoroughly before initiating coadministration. The following are known polymorphisms (RAVs) that significantly reduce the therapeutic efficacy of DAAs:45 for asunaprevir, which is a protease inhibitor, the NS3-4A position 168 amino acid variants D168A/E/V;43 and, for daclatasvir, the NS5A region position 31 and 91 amino acid variants L31M/V and Y93H.44 Because HCV is a virus that has tremendous diversity in its base sequences, there are patients who have these RAVs prior to DAA treatment. In a Japanese phase 3 study of daclatasvir + asunaprevir in IFN-ineligible/intolerant patients and IFN-non-responders patients, out of 214 patients in whom an exploration of HCV RAVs was performed before treatment initiation using a direct sequencing method, 30 patients (14.0%) had Y93H and 8 patients (3.7%) had L31M/V before treatment initiation. Fig. 5a and b show the treatment results by the presence or absence of NS5A RAVs before treatment. In the IFN (+ RBV)-ineligible/intolerant groups, 102 of the 107 patients who did not have Y93H RAVs before treatment achieved SVR. Therefore, when the population was limited to those patients without RAVs, the SVR rate was a good 95.3%. However, out of the 21 patients who had Y93H RAV, only 10 (47.6%) achieved SVR (Fig. 5a). In the IFN-non-responders group, the SVR rate in patients without Y93H before treatment initiation was 85.7% (66 of 77 patients), compared to a rate of 33.3% (3 of 9 patients) in patients with Y93H before treatment initiation. Of the 80 patients without L31M/V, 68 (85.0%) achieved SVR. In patients with L31M/V, of which there were admittedly few, of the 6 patients with such RAVs, only 1 (16.7%) achieved SVR (Fig. 5b). In addition, in an overseas phase 3 study (HALLMARK-DUAL), 48 patients (8%) had Y93 RAVs before treatment initiation, and the SVR rate in these patients was 38% (18 of 48), and 27 patients (5% had L31 RAVs before treatment initiation, and the SVR rate in these patients was 41% (11 of 27 patients).46 In a phase 3 study in treatment-naïve and relapsed patients, as well, the treatment results are clearly significantly worsened by the presence before treatment of NS5A RAVs. Out of 129 patients for whom NS5A RAVs were measured by a direct sequencing method, 18 (14.0%) had Y93H and 6 (4.7%) had L31I/M, and 23 (17.8%) had either one or both kinds of RAVs. Of the 106 patients who did not have any RAV, 104 (98.1%) achieved SVR12. This is in contrast to the rate of 47.8% (11 of 23 patients) in patients with either one or both kinds of RAVs (Fig. 6). Furthermore, in daclatasvir/asunaprevir combination therapy null responders, the emergence of viruses that are resistant to both drugs has been reported.47 Specifically, when RAVs were measured after breakthrough or relapse in patients who only had NS5A Y93 or L31 RAV before therapy, RAVs were found to have emerged not only in the NS5A region, but also at NS3 D168. In the overseas phase 3 HALMARK-DUAL study, L31 RAVs emerged in 63%, Y93 RAVs emerged in 58%, and NS3 D168 RAVs emerged in 92% of null responders, and NS5A and NS3 RAVs emerged in 77% of null responders.46 Of these NS5A and NS3 RAVs, the NS5A RAVs were found to persist for at least 1 year.47 In an in vitro test system, virus variants carrying multiple NS5A RAVs that had both Y93H and L31M/V were more resistant to NS5A inhibitors than viruses carrying only Y93H or L31M/V, and the emergence of a strain carrying L31V-Q54H-Y93H RAV that was a strain with a high level of resistance and high replication capability was also reported (Table 3). In patients with no history of treatment with NS5A inhibitors, it is expected that Y93H and L31M/V will be simultaneously detected in no more than 1% of patients (direct sequencing method), and that multiple NS5A RAVs will be extremely rare. However, in daclatasvir/asunaprevir combination therapy null responders, Y93H and L31M/V are detected simultaneously very frequently, and multiple NS5A RAVs thus appear to be present in a high proportion of such patients.47 Because there are currently no established therapies that are effective against such viruses carrying multiple drug resistance variants, it is of the utmost importance to prevent the emergence of such viruses. RNA-dependent RNA polymerase, which is necessary for virus replication, is enc