Will Resistance Associated Variants Affect the HCV Treatment Paradigm?


Specialty pharmacists play an important role in making evidence-based recommendations for the treatment of hepatitis C virus.

The arrival of direct acting antivirals (DAA) over the last several years has revolutionized the treatment of the chronic hepatitis C virus (HCV). Lengthy interferon-based treatment regimens with numerous side effects and relatively low cure rates have been abandoned in favor of simple, short, effective, all-oral regimens.

DAAs work by targeting specific nonstructural proteins on the virus to disrupt its replication and subsequent infection.1 The latest DAA, sofosbuvir/velpatasvir (Epclusa), was approved by the FDA on June 28, 2016.2

This new therapy offers patients a pangenotypic, once-daily pill that offers 99% cure rates, even in patients with cirrhosis and/or previous treatment failure, who historically have been the most difficult to treat.3 With this ever-expanding arsenal of antiviral weapons at our disposal, it can be easy to overlook the importance of properly selecting the most appropriate therapy for a patient; both in terms of drug selection as well as length of therapy.

While treatment success continues to be the new norm, recent research shows that unsuccessful treatment with DAAs may induce resistance mutations that can lower the likelihood of achieving a sustained virological response (SVR) upon retreatment.4 As specialty pharmacists, we can play an important role in making evidence-based recommendations, and help prevent resistance to these lifesaving medications by making sure they are used appropriately.

Until recently, viral resistance with HCV has historically been poorly understood. HCV replication exhibits a rapid turnover with a daily production of 1012 virions.5 HCV polymerase, a key enzyme involved in viral replication, is extremely error prone, which gives way to a virus with high genetic diversity, sometimes referred to as the HCV quasispecies.6

During the 1990s and 2000s, when interferons and ribavirin were the staples of therapy, several studies showed that patients with greater genetic heterogeneity within the quasispecies, particularly in the non-structural 5A (NS5A) region, as well as the Hypervariable Region 1 (HVR-1), prior to treatment tended to have a lower chance at achieving an SVR.7,8

However, there was little we could do with that information clinically. The exact mechanism of action of both the interferons, as well as ribavirin, was not fully understood, yet alone the affect that specific polymorphisms might have on the efficacy of their treatment in HCV.9 Resistance testing made little sense.

That changed in 2013, with the launch of the protease inhibitor, simeprevir.10 QUEST-1, a trial that compared the efficacy of simeprevir in combination with peg interferon and ribavirin, demonstrated the importance that baseline resistance testing could have. In this trial, we saw large differences in SVR rates in both genotype 1a (71% vs 46%) and 1b (90% vs 52%) compared with double therapy with just peg interferon and ribavirin.11

However, the subgroup analysis that compared these same groups of patients in the context of the resistance associated variant (RAV) mutation Q80K (a relatively common mutation found in the North American population) at baseline saw nearly identical SVR rates between the two groups (52% vs 53%).11

This provided the first context for resistance testing as part of a diagnostic workup to determine the most appropriate treatment. While the significance of this was momentarily overshadowed with the launch of sofosbuvir later that same year, resistance testing has increasingly come into focus with the recent launch of the NS5A inhibitors (ledipasvir, ombitasvir, daclatasvir, and elbasvir).

It is important to understand the potential impact of RAVs to the NS5A inhibitors as they carry with them a unique risk for viral resistance. A review of 3 different studies detected baseline NS5A RAVs in 12% to 18% of patients never previously exposed to an NS5A inhibitor.12

These baseline mutations appear to possibly lower SVR rates in genotype 1a patients, especially in those with previous treatment history and/or cirrhosis.13 Furthermore, treatment failure after exposure to NS5A therapy is almost always associated with RAVs.

In one study of ledipasvir failures, 16% of the study population started with a baseline RAV, but 99% had an NS5A RAV upon subsequent treatment failure.14 Unlike mutations to protease inhibitors that tend to be short-lived, RAVs that confer resistance to NS5A inhibitors can persist for years at a time.14

“What can be scary about ledipasvir and potentially with other agents of the NS5A class is that studies have shown after three plus years a majority of the virus is still the resistant strain,” said hepatology expert, Atif Zaman, MD of Oregon Health and Science University. “The durability and the viral fitness of these types of resistant strains seem to be more.”15

This means that patients who fail NS5A therapy may have to wait for years before they may become susceptible again to that class of antiviral agents, but that is not the only potential issue these RAVs can have. In a study of patients who previously failed therapy with 8-12 weeks of sofosbuvir/ledipasvir and who were then retreated for 24 weeks, some of the first resistant strains to sofosbuvir emerged in those who failed therapy for the second time.16

It is important to note that 60% of these patients did achieve an SVR when retreated for 24 weeks. For some patients who require urgent retreatment, it may make sense to retreat even in the presence of known RAVs, but for those who can wait it is possible retreatment may do more harm than good.

“When you use Harvoni in someone with ledipasvir resistance, what you are really doing is treating them with a single agent (sofosbuvir),” Zaman said. “We know that in the HIV world when we treat with a single agent, that our chances of resistance are almost immediate. Now, in the world of hepatitis C it obviously is not almost immediate, but it is a reminder that if we keep pushing it we may see early signs. So the message is that although you get a 60% SVR, the risk you have is that in the 40% (that fail retreatment) some of those patients may start developing unique resistant strains that we have no idea what to do with.”15

This stresses the importance of properly screening for optimal therapy on initial treatment, as well as provides a strong case for resistance testing before retreatment as these mutations typically have their greatest impact on treatment success in that setting.

“Resistance testing is driving some of the theoretical directions that you can go,” Zaman elaborated. “In patients who test positive for resistance to the protease inhibitors, a NS5A regimen would make the most sense. Likewise, if a patient presents with resistance to the NS5A class, but shows susceptibility to the protease inhibitors, a simeprevir/sofosbuvir regimen would likely be a good treatment choice. If a patient failed on 12 weeks of a ledipasvir-based regimen, but does not test positive for NS5A RAVs, then you could potentially retreat them for 24 weeks, add ribavirin, and that should work because there is no resistance. In patients who are resistant to both classes, resistance testing helps us know when to potentially stop and wait for the next generation of agents.”15

AASLD guidelines currently recommend RAVs testing in limited situations at baseline for patients prescribed elbasvir/grazoprevir in genotype 1a, and in the setting of retreatment for patients who have failed a regimen of sofosbuvir/simeprevir or with an NS5A inhibitor.17

The guidelines recommend that treatment be deferred if possible, with the exception of those with cirrhosis or other factors that require retreatment urgently. They recommend testing for RAVs that confer decreased susceptibility to NS3/4A protease inhibitors, as well as the NS5A inhibitors.

The recommendations for baseline RAVs testing for elbasvir/grazoprevir are based on the observed reduction in SVR for patients in the C-EDGE and C-EDGE TE trials.18 Presence of baseline NS5A RAVs significantly reduced SVR12 in genotype 1a patients by 28%.18

In addition to these recommendations, AASLD guidelines also recommend considering RAVs testing in non-cirrhotic treatment-experienced genotype 3 patients.17 It was observed in the ALLY-3 study that the Y93H RAV can be particularly problematic, and substantially reduce the SVR rate in these patients with and without cirrhosis.21 Resistance testing can help guide providers as to how aggressively to treat this population in regards treatment length, and whether the addition of ribavirin to the regimen is necessary.

Currently, resistance testing is only available for HCV genotypes 1 and 3. These tests can help guide clinicians to the most appropriate therapy and avoid treatment with regimens that may negatively impact the efficacy. The tests that are available via LabCorp and Quest Diagnostics evaluate the following drugs and mutations:19,20


Mutation Region

Codons of Interest


1a or 1b


M28, Q30, L31, H58. Y93

Daclatasvir, Ledipasvir, Ombitasvir, Elbasvir

1a or 1b




1a or 1b








The tests do not elucidate whether therapy will fail or succeed in the presence or absence of polymorphisms, but rather predicts the probability for failure. These tests detect the most commonly reported RAVs that have been known to reduce the rates of SVR, and act as screening tools to help clinicians make informed decisions regarding whether or not to initiate therapy and what agents might be most successful.

Both tests provide an assessment on the susceptibility based on the presence of RAVs. The sequencing assays that are used analyze the regions of the virus via DNA sequencing techniques. Amino acid substitutions in the NS3/4A, NS5A, or NS5B region are identified and a viral susceptibility is determined.

The results reported via LabCorp and Quest Diagnostics are similar. Samples which test positive for the presence of RAVs which may confer a resistant profile are labeled as “resistance possible” or “probable.” Likewise, samples which are negative are labeled as “none/undetermined” or “not predicted.”

Looking on to the horizon, especially with the recent approval of sofosbuvir/velpatasvir, introduces the question of whether or not RAVs testing will continue to be appropriate and necessary as newer agents come to market with a higher barrier to resistance. The ASTRAL-1, ASTRAL-2 ASTRAL-3, and ASTRAL-4 trials collectively have shown very promising results.22 In these trials, baseline NS5A RAVs were observed in 16-70% of the treatment group, depending on which genotype was observed.

Despite this, SVR results were robust across all 6 genotypes. Of the 624 patients treated in ASTRAL-1, 618 patients achieved an SVR. Of the 6 treatment failures: 2 were lost to follow up, 2 relapsed, 1 patient withdrew consent, and there was 1 patient death that was unrelated to the medication.

This is staggering data. Furthermore, ASTRAL-3 demonstrated a 95% SVR rate genotype 3 patients, including an 89% SVR rate in treatment-experienced cirrhotic patients who have now become our most difficult patient demographic to treat.23 Although the studies did not study retreatment of patients who have failed previous NS5A therapy previously, the data seems to support the use of sofosbuvir/velpatasvir as a possible salvage therapy due to its higher barrier to resistance.

It is unclear if RAVs testing in treatment-experienced patients would be beneficial prior to treatment as the presence of NS5A RAVs may not impact the patient’s outcome. Testing for Y93H RAV in genotype 3 patients may be appropriate, as it was present in 4 of the 11 treatment failures in the ASTRAL-3 trial and is associated with a higher rate of treatment failure.24

In the pipeline, data presented at the 2016 International Liver Conference showed that the combination of sofosbuvir/velpatasvir and the pangenotypic protease inhibitor, GS-9857, produced up to 100% SVR rates, dependent on genotype in patients with previous DAA failures.25 This included patients who failed previous NS5A regimens.

The lowest SVR rate among genotypes was 97% in genotype 3 patients. This is staggering data that suggests that soon, provided that we continue to use our antivirals appropriately, that no patient will be too difficult to treat.

It is an exciting time to be a health care provider treating HCV. As specialty pharmacists, we can play an important role in making sure that we get the most out of these lifesaving agents. In addition to counseling patients on appropriate use, reviewing a patient’s medical history for drug interactions, assessing for adherence, and working with providers to advocate for the most appropriate regimen, we can use our knowledge of RAVs and resistance testing to make sure we are good stewards of these drugs to maintain their efficacy.


1. Ahmed A, Felmlee D. Mechanisms of Hepatitis C Viral Resistance to Direct Acting Antivirals. Viruses. 2015;7(12):6716-6729. doi:10.3390/v7122968.

2. FDA approves Epclusa for treatment of chronic Hepatitis C virus infection. June 2016. http://www.fda.gov/newsevents/newsroom/pressannouncements /ucm508915.htm.

3. Feld JJ, Jacobson IM, Hezode C, et al. Sofosbuvir and Velpatasvir for HCV Genotype 1, 2, 4, 5, and 6 Infection. The New England Journal of Medicine. December 2015:2599-2607. doi:10.1056/NEJMoa1512610.

4. Buti M, Riveiro-Barciela M, Esteban R. Management of direct-acting antiviral agent failures. Journal of Hepatology. 2015;63(6):1511-1522. doi:10.1016/j.jhep.2015.08.010.

5. Neumann AU, Lam NP, Dahari H, et al. Hepatitis C Viral Dynamics in Vivo and the Antiviral Efficacy of Interferon-alpha Therapy. Science. 1998;282(5386):103-107. doi:10.1126/science.282.5386.103.

6. Schneider MD, Sarrazin C. Antiviral therapy of hepatitis C in 2014: Do we need resistance testing? Antiviral Research. 2014;105:64-71. doi:10.1016/j.antiviral.2014.02.011.

7. Bukh J, Miller R, Purcell R. Genetic Heterogeneity of Hepatitis C Virus: Quasispecies and Genotypes. Semin Liver Dis Seminars in Liver Disease. 1995;15(01):41-63. doi:10.1055/s-2007-1007262.

8. Enomoto N, Sato C, Kurosaki M, et al. Hepatitis C virus after interferon treatment has the variation in the hypervariable region of envelope 2 gene. Journal of Hepatology. 1994;20(2):252-261. doi:10.1016/s0168-8278(05)80066-2.

9. Chung RT, Gale M, Polyak SJ, et al. Mechanisms of action of interferon and ribavirin in chronic hepatitis C: Summary of a workshop. Hepatology. 2008;47(1):306-320. doi:10.1002/hep.22070.

10. Medications to Treat Hepatitis C — A Timeline. Medications to Treat Hepatitis C. http://www.hepatitiscentral.com/medications-to-treat-hepatitis-c-a-timeline/. Published June 2016. Accessed July 4, 2016.

11. Jacobson IM, et al. Simeprevir PEG RBV in Treatment-Naïve Genotype 1 QUEST-1 Trial. HVC Online. http://slides.hcvonline.org/uploads/57/quest1.pdf. Published 2014.

12. Wells JT, Landaverde C, Gutierrez J, et al. Management of patients with hepatitis C virus resistance-associated variants to NS5A inhibitors: Where are we now? Clinical Liver Disease. 2015;6(4):82-85. doi:10.1002/cld.507.

13. Reddy KR, Beavers K, Gordon S, et al. P0889 : Effect of baseline factors on response to the fixed-dose combination of daclatasvir (DCV), asunaprevir (ASV) and beclabuvir (BCV) in non-cirrhotic patients with HCV genotype 1 infection. Journal of Hepatology. 2015;62:S676-S677. doi:10.1016/s0168-8278(15)31092-8.

14. Dvory-Sobol H, Wyles D, Ouyang W, et al. O059 : Long-term persistence of HCV NS5A variants after treatment with NS5A inhibitor ledipasvir. Journal of Hepatology. 2015;62. doi:10.1016/s0168-8278(15)30073-8.

15. Zaman A, Bradley R, Lam S. Resistance Associated Variance in the Approach to Treatment of Chronic Hepatitis C Virus. In Person Interview. January 2016.

16. Lawitz E, Flamm S, Yang J, et al. O005 : Retreatment of patients who failed 8 or 12 weeks of ledipasvir/sofosbuvir-based regimens with ledipasvir/sofosbuvir for 24 weeks. Journal of Hepatology. 2015;62. doi:10.1016/s0168-8278(15)30012-x.

17. AASLD-IDSA. Retreatment of persons in whom prior therapy has failed. Recommendations for testing, managing, and treating hepatitis C. http://www.hcvguidelines.org/full-report/retreatment-persons-whom-prior-therapy-has-failed. [14 July 2016]

18. Zepatier®. Whitehouse Station, NJ: Merck & Co. Inc.; 2016

19. HCV GenoSure NS3/4A Drug Resistance Assay. Monogram Biosciences . http://www.monogrambio.com/hepatitis-tests/hcv-ns3-4a/. Accessed July 4, 2016.

20. Test Center. Hepatitis C Viral RNA Genotype 1 NS5A Drug Resistance. http://www.questdiagnostics.com/testcenter/testguide.action?dc=ts_hcv_ns5a_genotype. Accessed July 4, 2016.

21. Nelson DR, Cooper JN, Lalezari JP, et al. All-oral 12-week treatment with daclatasvir plus sofosbuvir in patients with hepatitis C virus genotype 3 infection: ALLY-3 phase III study. Hepatology. 2015;61(4):1127-1135. doi:10.1002/hep.27726.

22. Hezode C, Reau N, Svarovsaia ES, et al. Resistance Analysis in 1284 Patients With Genotype 1-6 HCV Infection Treated With Sofosbuvir/Velpatasvir in the Phase 3 ASTRAL-1, ASTRAL-2, ASTRAL-3, and ASTRAL-4 Studies. European Association for the Study of the Liver Meeting: April 13-17, 2106; Barcelona, Spain.

23. Foster GR, Afdhal N, Roberts SK, et al. Sofosbuvir and Velpatasvir for HCV Genotype 2 and 3 Infection. New England Journal of Medicine N Engl J Med. 2015;373(27):2608-2617. doi:10.1056/nejmoa1512612.

24. Supplement to: Foster GR, Afdhal N, Roberts SK, et al. Sofosbuvir and velpatasvir for HCV genotype 2 and 3 infection. N Engl J Med 2015;373:2608-17. DOI: 10.1056/NEJMoa1512612

25. Highleyman L. Sofosbuvir/velpatasvir GS-9857 works well for treatment-experienced hepatitis C patients. HIV & AIDS Information http://www.aidsmap.com/sofosbuvirvelpatasvir-gs-9857-works-well-for-treatment-experienced-hepatitis-c-patients/page/3050512/. Published April 15, 2016. Accessed July 4, 2016.

About the Authors

Ryan Bradley, PharmD

Dr. Ryan Bradley received his Doctor of Pharmacy degree from Pacific University in 2010. Dr. Bradley joined Ardon Health as a clinical pharmacist in 2015, and serves as the specialty pharmacy preceptor for Moda Health’s managed care residency. Previously he worked as a clinical pharmacist with CVS Health, specializing in hepatitis C and multiple sclerosis.

Steve Lam, PharmD

Dr. Steve Lam received his Doctor of Pharmacy from Oregon State University College of Pharmacy in 2015 and completed his managed care residency at Moda Health, a regional health plan in Portland Oregon, in 2016. As a resident, Dr. Lam had the unique opportunity to complete a rotation at Ardon Health, where he was exposed to care management and clinical programs.