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## **Meet the editor**

Dr. Naglaa Allam is a professor of hepatology at the National Liver Institute (NLI) Menoufia University in Egypt. The NLI is a leading medical institution in the Middle East dedicated for the management of liver diseases as well as advanced training and research in hepatology and liver transplantation. Dr. Naglaa Allam was trained at the Northern General Hospital, UK, and

Hospital of the University of Pennsylvania in the USA. She is an editorial board member and reviewer in many medical journals and has several publications in eminent journals as well as books in the field of hepatology and liver transplantation.

## Contents

## **Preface XIII**



Chapter 9 **Response-Guided Therapy Based on the Combination of Quantitative HBsAg and HBV DNA Kinetics in Chronic Hepatitis B Patients 173** Valeriu Gheorghiță and Florin Alexandru Căruntu


#### Chapter 17 **New Strategy Treating Hepatitis B Virus (HBV) Infection: A Review of HBV Infection Biology 373** Yong-Yuan Zhang

Chapter 8 **Recent Advancement in Hepatitis B Virus, Epigenetics Alterations and Related Complications 149**

Chapter 9 **Response-Guided Therapy Based on the Combination of**

Valeriu Gheorghiță and Florin Alexandru Căruntu

**Quantitative HBsAg and HBV DNA Kinetics in Chronic Hepatitis**

Letiția Adela Maria Streba, Anca Pătrașcu, Aurelia Enescu and

Smaragdi Marinaki, Konstantinos Drouzas, Chrysanthi Skalioti and

Chapter 11 **Treatment and Prognosis of Hepatitis B Virus Concomitant with**

Thierry Burnouf, Ching-Hsuan Liu and Liang-Tzung Lin

Chapter 14 **Hepatitis C Virus Infection Treatment: Recent Advances and New Paradigms in the Treatment Strategies 285** Imran Shahid, Waleed H. AlMalki, Mohammed W. AlRabia,

Muhammad H. Hafeez and Muhammad Ahmed

Chapter 15 **Importance of MicroRNAs in Hepatitis B and C Diagnostics and**

Seyma Katrinli, H. Levent Doganay, Kamil Ozdil and Gizem Dinler-

Chih-Wen Lin, Chih-Che Lin and Sien-Sing Yang

Chapter 12 **Hepatitis B and C in Kidney Transplantation 217**

Chapter 13 **Strategies to Preclude Hepatitis C Virus Entry 251**

Mateja M. Jelen and Damjan Glavač

Chapter 16 **Can Proteomic Profiling Identify Biomarkers and/or Therapeutic Targets for Liver Fibrosis? 355**

**Section 3 Investigational Treatment Strategies 249**

Mankgopo Magdeline Kgatle

Chapter 10 **Current Management Strategies in Hepatitis B**

**During Pregnancy 189**

Costin Teodor Streba

**Alcoholism 205**

John N. Boletis

**Treatment 321**

Doganay

**B Patients 173**

**VI** Contents

## Preface

One of the greatest breakthroughs in medicine is our current capability to cure nearly everyone with chronic hepatitis C virus infection. With the emergence of direct-acting antiviral agents, hepatitis C virus infection has entered a new era. On the other hand, hepatitis B viral replication can be suppressed by potent antiviral drugs, but strategies to enhance the eradication rates of HBV infection are still needed on the horizon. Treatment of viral hepatitis is a rapidly evolving field that will continue to grow and maintain excitement over the next few years.

*"Advances in Treatment of Hepatitis C and Hepatitis B"* book is divided into two parts. The first part provides a comprehensive overview on the current state of knowledge and latest advan‐ ces in hepatitis C and hepatitis B therapeutics. The chapters include management in special conditions during pregnancy and in kidney transplantation. The second part attempts to pro‐ vide a conceptual framework of emerging and investigational treatment strategies.

This book is addressed to researchers, practicing physicians in hepatology, medical students, residents, and fellows seeking a broader understanding of updates in the treatment of viral hepatitis. The authors are bright internationally renowned experts from four continents across the globe (Africa, Asia, Europe, and the United States of America). We sincerely thank the authors for their time and expertise, and we hope the readers find their chapters useful.

I am also grateful to all my seniors and colleagues at the National Liver Institute whose zeal and dedication make the institute a vibrant, exciting place to work at.

Finally, I dedicate this book to the soul of my father, Dr. Allam, who always invested a great deal of love in supporting my work. He was excited about this book project but did not live to witness its emergence. I also acknowledge my wonderful husband, Prof. Hesham Abdel‐ dayem, for his love, encouragement, and support.

> **Naglaa Allam** National Liver Institute, Menoufia University, Egypt

**Section 1**

## **Introduction**

## **Introductory Chapter: Treatment of Viral Hepatitis - Current Challenges and Future Perspectives Introductory Chapter: Treatment of Viral Hepatitis - Current Challenges and Future Perspectives** Additional information is available at the end of the chapter**Provisional chapter Introductory Chapter: Treatment of Viral Hepatitis -**

**Current Challenges and Future Perspectives**

Naglaa Allam and Imam Waked Naglaa Allam and Imam Waked

Additional information is available at the end of the chapter Naglaa Allam and Imam Waked

http://dx.doi.org/10.5772/67420 Additional information is available at the end of the chapter

**1. Hepatitis C therapy**

Cure of hepatitis C has come true!

Clinical care for patients with hepatitis C (HCV) has advanced remarkably during the last two decades, as a result of better understanding of the pathophysiology of the disease, and because of developments in diagnostic procedures and improvements in therapy and prevention. With the introduction of genotype 1 effective directly acting antiviral agents (DAAs) in 2011, the manage‐ ment of HCV infection started to change. By 2013, second-generation pangenotypic DAAs became available, and the biggest problem was solved: interferon (IFN) became no longer necessary.

Unlike IFN regimens, which rely on upregulating the patients' own immune system, these DAAs block different stages of viral replication. There are four major groups of DAAs namely: NS5B nucleotide inhibitors, NS5B nonnucleoside inhibitors, NS5A replication complex inhibi‐ tors, and NS3/4A protease inhibitors [1]. Specific treatment regimens vary, depending on fac‐ tors such as HCV genotype, and may include multiple drugs [2]. Multiple regimens have been approved and several new regimens with high potencies, less resistance, and better safety pro‐ file are in the process of approval. Prof. Kamal elegantly describes them in detail in Chapter 2.

Sustained virological response (SVR) rates achieved in phase III clinical trials generally exceeded 90% (although real-world rates may be lower) along with reduction in treatment duration to 12 weeks or less and with fewer adverse events. An SVR is generally associated with normalization of liver enzymes and amelioration or disappearance of liver necroinflam‐ mation and fibrosis in noncirrhotic patients [3]. The use of DAAs in patients with cirrhosis, as discussed in Chapter 4, has also shown excellent results with good safety profile. SVR also improves HCV-induced portal hypertension [4]. DAAs have also begun to change the land‐ scape of management of the HCV transplant candidate on the waiting list. Prof. Al-Hamoudi provides an update on this in Chapter 5. Even patients with HCV infection and advanced kidney disease now have alternative treatment options.

and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, Thus, the era of HCV eradication and cure has begun. And in 2017, hepatologists can treat *all* patients irrespective of fibrosis score—but the job is not over yet. Real-world experience has revealed several challenges and unmet needs!

## **1.1. Challenges:**

#### **(a) Limited or absent access to therapy in the majority of infected patients**

Unfortunately, the very high cost of the new DAA drugs is creating a barrier for introduc‐ tion of treatment in most limited resource settings and may prove an obstacle on the path for elimination of HCV infection worldwide. Of course, successful treatment should prevent many late HCV complications, but even if treatment actually proves cost saving in the long run, it is too expensive and infeasible to treat all patients immediately [5].

Hence hepatitis C treatment prioritization, as the European Association for the Study of the Liver states, is necessary when resources are limited [6]. However, new data suggest that this approach may be suboptimal, if not carefully executed. Treatment prioritization is complex and may not be fair. Treating first those with the most need makes sense. But, those who need it most may also be those who benefit the least because of issues as extensive liver damage, comorbid illness, older age, or have already developed hepatocellular carcinoma? Targeting populations with high HCV prevalence like drug users, prisoners, and migrants also makes sense since they are most likely to spread infection to others. But, those most likely to transmit to others often have low disease stage. Besides they are most likely to abrogate the personal benefits of treatment by being reinfected. Treating those who have the most symptoms also makes sense, but unfortunately symptoms do not always improve with cure. And there is another major issue: who should decide whom to prioritize? [7]. Health care providers often impose socioeconomic and racial biases when prioritizing treatments [8]. Moreover, health care providers prefer not to be the barrier between patients and life‐saving therapies.

While *not* all patients require immediate treatment, an ideal strategy should treat patients before they progress toward end-stage liver disease when even highly effective treatments can confer only marginal benefit. In response, many countries have instituted coverage policies that autho‐ rize treatment only for the advanced patients, putting off therapy for less severely ill patients [5].

Aggressive action is warranted to see progress toward HCV elimination. Only if we use *efficacious therapy* in a *significant* proportion of patients will we *significantly* decrease the burden of the disease. So efforts should be made to make DAAs cost effective in all clinical scenarios and accessible to all patients. In places like Egypt and India, generic versions of the DAAs sofosbuvir and daclatasvir are being mass produced for <1% of the current US retail price and are available for a higher proportion of patients.

Another solution to reduce treatment cost is by shortening the duration of treatment without affecting efficacy. A study in China demonstrated 100% SVR with triple DAAs for only 3 weeks if the patient without cirrhosis achieved ultra-rapid virological response (HCV-RNA < 500 IU 48 hours after starting treatment) [9]. By shortening the duration of therapy from the currently recommended 12 to 3 weeks, the cost of therapy could be markedly reduced as well as the rate of adverse events. Large clinical trials should further study the application of this response‐ guided treatment approach in patients with different ethnic backgrounds and with different genotypes.

#### **(b) Emergence of drug resistance and DAA failure**

Thus, the era of HCV eradication and cure has begun. And in 2017, hepatologists can treat *all* patients irrespective of fibrosis score—but the job is not over yet. Real-world experience has

Unfortunately, the very high cost of the new DAA drugs is creating a barrier for introduc‐ tion of treatment in most limited resource settings and may prove an obstacle on the path for elimination of HCV infection worldwide. Of course, successful treatment should prevent many late HCV complications, but even if treatment actually proves cost saving in the long

Hence hepatitis C treatment prioritization, as the European Association for the Study of the Liver states, is necessary when resources are limited [6]. However, new data suggest that this approach may be suboptimal, if not carefully executed. Treatment prioritization is complex and may not be fair. Treating first those with the most need makes sense. But, those who need it most may also be those who benefit the least because of issues as extensive liver damage, comorbid illness, older age, or have already developed hepatocellular carcinoma? Targeting populations with high HCV prevalence like drug users, prisoners, and migrants also makes sense since they are most likely to spread infection to others. But, those most likely to transmit to others often have low disease stage. Besides they are most likely to abrogate the personal benefits of treatment by being reinfected. Treating those who have the most symptoms also makes sense, but unfortunately symptoms do not always improve with cure. And there is another major issue: who should decide whom to prioritize? [7]. Health care providers often impose socioeconomic and racial biases when prioritizing treatments [8]. Moreover, health

care providers prefer not to be the barrier between patients and life‐saving therapies.

While *not* all patients require immediate treatment, an ideal strategy should treat patients before they progress toward end-stage liver disease when even highly effective treatments can confer only marginal benefit. In response, many countries have instituted coverage policies that autho‐ rize treatment only for the advanced patients, putting off therapy for less severely ill patients [5]. Aggressive action is warranted to see progress toward HCV elimination. Only if we use *efficacious therapy* in a *significant* proportion of patients will we *significantly* decrease the burden of the disease. So efforts should be made to make DAAs cost effective in all clinical scenarios and accessible to all patients. In places like Egypt and India, generic versions of the DAAs sofosbuvir and daclatasvir are being mass produced for <1% of the current US retail price and

Another solution to reduce treatment cost is by shortening the duration of treatment without affecting efficacy. A study in China demonstrated 100% SVR with triple DAAs for only 3 weeks if the patient without cirrhosis achieved ultra-rapid virological response (HCV-RNA < 500 IU 48 hours after starting treatment) [9]. By shortening the duration of therapy from the currently

**(a) Limited or absent access to therapy in the majority of infected patients**

run, it is too expensive and infeasible to treat all patients immediately [5].

revealed several challenges and unmet needs!

4 Advances in Treatment of Hepatitis C and B

are available for a higher proportion of patients.

**1.1. Challenges:**

Despite improved SVR rates with DAA-based combination regimens, treatment fails to erad‐ icate HCV infection in 5–15%, dependening on the treatment regimen and treated popula‐ tion. Treatment failure is generally associated with the selection of HCV resistant-associated substitution (RAS) (or resistant-associated variant (RAV)), that is, viral molecular substitu‐ tions or variants that have reduced susceptibility to the DAA(s) administered. NS5A inhib‐ itors have a low barrier to resistance, and the variants they select confer cross‐resistance across all members of the drug class. Thus, NS5A resistance currently appears as the prin‐ cipal challenge of IFN-free, DAA-based therapy and they tend to persist for several years after treatment failure. In contrast, RASs selected by NS3/4A protease inhibitors persist for a much shorter time and are progressively replaced by wild‐type virus within a few months posttherapy. Additionally, RASs selected by the NS5B polymerase inhibitor sofosbuvir have poor viral fitness; thus, they rarely emerge in the presence of the drug and tend to rapidly disappear if selected [10]. The utility of HCV resistance testing, i.e., the determination of the sequence of the DAA target region prior to retreatment in patients who failed on any of the DAA-containing regimens is unknown. Chapter 6 summarizes the retreatment in case of drug failures.

## **(c) HCV eradication and the risk of hepatocellular carcinoma (HCC): issues with direct acting antiviral (DAA) therapy?**

Several recent publications raise concerns about unexpected high rate of HCC recurrence after undergoing direct‐acting antiviral therapy. Reig et al. showed early tumor "recurrence" in patients with HCV-related hepatocellular carcinoma (HCC) [11]. Conti et al. showed that in patients with HCV-related cirrhosis, DAA-induced resolution of HCV infection does *not* reduce recurrence of HCC, and patients previously treated for HCC have still a high risk of tumor recurrence, in the short term [12]. Kozbial et al. showed an unexpected high "occur‐ rence" in patients with advanced liver diseases after SVR [13].

In contrast, Cheung et al. found that DAA therapy in patients with decompensated cirrhosis led to sustained improvement in liver function, with no evidence of increase in HCC development in Chinese patients [14]. Also, the French ANRS study analyzed more than 6000 DAA-treated patients who underwent curative therapies for HCC and they found no increased risk of HCC [15].

Altogether these studies convey a strong message that great attention is needed to address the issue of HCC recurrence/occurrence. There is an urgent need for large prospective stud‐ ies evaluating the impact of DAA therapy on the risk of HCC in patients with HCV-related cirrhosis. For the time being, the risk of HCC development justifies HCC screening after viral clearance in patients with HCV‐related cirrhosis.

## **(d) There is no vaccine yet. Is a prophylactic vaccine still necessary?**

Obviously, therapy is not enough to surmount the burden of HCV. Is it technically possible to have vaccine? If HCV vaccines are available in the future, then vaccination program in high‐risk populations would probably have a great impact on preventing and eradicating HCV infection. An experimental protective vaccine, as shown in Chapter 14, demonstrated promise in preliminary human safety trials, and a phase II clinical trial is under way to further determine the efficacy of the vaccine.

## **1.2. Future perspectives:**

Therefore, although the DAAs have opened up new horizons for HCV cure, challenges per‐ sist in the real-world setting. It is becoming clear that developing therapeutic strategies with different modes of action would be necessary to address the various limitations of current DAAs. Third generation pangenotypic antivirals are currently in final phases of development: voxilaprevir [16], glecaprevir [17] (both NS3/NS4 protease inhibitors) and pibrentasvir (NS5A inhibitor) [17]. Antivirals with alternate mechanism of action, such as by restricting viral entry or cell-to-cell spread could help expand the scope of antiviral strategies for the management of hepatitis C. Chapters 14 and 15 describe some of the new paradigms in antiviral strategies to preclude HCV entry, such as through monoclonal antibodies and small molecules.

With these strategies, it is foreseeable, in a not too distant future, that they will help provide a better management of hepatitis C.

## **2. Hepatitis B therapy**

An overview of the six currently approved treatments is presented in Chapter 7. The advent of anti‐HBV treatment drugs has made *significant progress* in improving the health and life expectancy of patients with HBV.

But there is no cure for Hepatitis B till now!

Chronic hepatitis B remains a difficult to treat disease because at this time no treatment pro‐ vides both an optimal virological and immunological control. There is a high rate of relapse following any antiviral therapy. In addition, there are no approved therapy stopping rules, especially in HBeAg negative patients treated with nucleoside and nucleotide analogs. An early stopping rule using the combination of serum HBsAg and HBV DNA was proposed and is discussed in Chapter 9.

While there have been significant advances in the management of hepatitis B with available nucleos(t)ide analogues, there remains much work to be done to prevent HCC. Viral suppres‐ sion alone has proven not effective for the absolute prevention of HCC.

Additionally, the required long-term therapy imposes not only financial burden but also may put patients at risk for potential drug resistance and unknown toxicity. Therefore, more effec‐ tive treatment regimens aiming for HBV cure are urgently needed. New investigational thera‐ pies are in the pipeline as discussed in Chapter 17. With multiple new therapies in the pipeline, the future of treating hepatitis B is an exciting one, and there is hope that it will become a disease of the past but this will not be too soon! The new therapy will not be available soon.

Another challenge is a demand for screening pregnant females and newborns for HBV. Pregnancy screening for HBV is very defective in most countries; it is not practiced except on individual basis. Chapter 10 reviews current management strategies for hepatitis B in the pregnancy and the postpartum.

## **Conclusion**

**(d) There is no vaccine yet. Is a prophylactic vaccine still necessary?**

determine the efficacy of the vaccine.

6 Advances in Treatment of Hepatitis C and B

a better management of hepatitis C.

expectancy of patients with HBV.

But there is no cure for Hepatitis B till now!

**2. Hepatitis B therapy**

is discussed in Chapter 9.

**1.2. Future perspectives:**

Obviously, therapy is not enough to surmount the burden of HCV. Is it technically possible to have vaccine? If HCV vaccines are available in the future, then vaccination program in high‐risk populations would probably have a great impact on preventing and eradicating HCV infection. An experimental protective vaccine, as shown in Chapter 14, demonstrated promise in preliminary human safety trials, and a phase II clinical trial is under way to further

Therefore, although the DAAs have opened up new horizons for HCV cure, challenges per‐ sist in the real-world setting. It is becoming clear that developing therapeutic strategies with different modes of action would be necessary to address the various limitations of current DAAs. Third generation pangenotypic antivirals are currently in final phases of development: voxilaprevir [16], glecaprevir [17] (both NS3/NS4 protease inhibitors) and pibrentasvir (NS5A inhibitor) [17]. Antivirals with alternate mechanism of action, such as by restricting viral entry or cell-to-cell spread could help expand the scope of antiviral strategies for the management of hepatitis C. Chapters 14 and 15 describe some of the new paradigms in antiviral strategies

to preclude HCV entry, such as through monoclonal antibodies and small molecules.

With these strategies, it is foreseeable, in a not too distant future, that they will help provide

An overview of the six currently approved treatments is presented in Chapter 7. The advent of anti‐HBV treatment drugs has made *significant progress* in improving the health and life

Chronic hepatitis B remains a difficult to treat disease because at this time no treatment pro‐ vides both an optimal virological and immunological control. There is a high rate of relapse following any antiviral therapy. In addition, there are no approved therapy stopping rules, especially in HBeAg negative patients treated with nucleoside and nucleotide analogs. An early stopping rule using the combination of serum HBsAg and HBV DNA was proposed and

While there have been significant advances in the management of hepatitis B with available nucleos(t)ide analogues, there remains much work to be done to prevent HCC. Viral suppres‐

Additionally, the required long-term therapy imposes not only financial burden but also may put patients at risk for potential drug resistance and unknown toxicity. Therefore, more effec‐ tive treatment regimens aiming for HBV cure are urgently needed. New investigational thera‐ pies are in the pipeline as discussed in Chapter 17. With multiple new therapies in the pipeline,

sion alone has proven not effective for the absolute prevention of HCC.

So, in conclusion, the highly effective and well-tolerated direct-acting antiviral drugs (DAAs) for the treatment of the hepatitis C virus have revolutionized therapy for HCV. Several novel therapeutic strategies for each of HBV and HCV are under development. But until the devel‐ oping antiviral strategies are available, there is much more that can be done.

The public health burden posed by viral hepatitis should be recognized as a priority. The leading professional organizations in liver disease, the American Association for the Study of Liver Diseases (AASLD), the European Association for the Study of the Liver (EASL), and the Asian Pacific Association for the Study of the Liver (APASL) urge governments, health care organizations, and nongovernmental organizations to adopt recommendations for immuni‐ zation, screening, diagnosis and treatment and to make them available and affordable for public health programs [18].

Overall, the achievements and improvements in the field of HCV and HBV care predict that the future of HCV and HBV therapeutics is becoming brighter every day.

## **Author details**

Naglaa Allam\* and Imam Waked

\*Address all correspondence to: naglaaallam@yahoo.com

Hepatology, National Liver Institute, Menoufeya University, Egypt

## **References**


[15] ANRS collaborative study group on hepatocellular carcinoma (ANRS CO22 HEPATHER, CO12 CirVir and CO23 CUPILT cohorts). Lack of evidence of an effect of direct-acting antivirals on the recurrence of hepatocellular carcinoma: data from three ANRS cohorts. J Hepatol 2016;65(4):734–40.

[4] Mandorfer M, Kozbial K, Schwabl P, Clarissa Freissmuth C, Schwarzer R, Stern R, Chromy D Stättermayer AF, Reiberger T, Beinhardt S, Sieghart W, Trauner M, Hofer H, Ferlitsch A, Ferenci P, Markus Peck-Radosavljevic M. Sustained virologic response to interferon‐free therapies ameliorates HCV‐induced portal hypertension. J Hepatol

[5] Fox S, McCombs J. Optimizing HCV treatment – Moving beyond the cost conundrum. J

[6] World Health Organization guidelines for the screening care and treatment of chronic hepatitis C infection; 2016. Update. http://www.who.int/hiv/pub/hepatitis/hepatitis-

[7] Mehta SH, David L, Thomas DL. Doing the math on hepatitis C virus treatmentJ Hepatol

[8] Institute of Medicine Committee on U, Eliminating R, Ethnic Disparities in Health C. In: Smedley BD, Stith AY, Nelson AR, editors. Unequal treatment: confronting racial and ethnic disparities in health care. Washington (DC): National Academies Press (US); 2003.

[9] Lau G, Benhamou Y, Chen G, Li J, Shao Q, Ji D, Li F, Li B, Liu J, Hou J, Jian Sun J, Wang C, Chen J, Wu V, Wong A, Wong C, Tsang S, Wang Y, Bassit L, Tao S, Jiang Y, Hsiao H, Ke R, Perelson A, Schinazi R. Efficacy and safety of 3-week response-guided triple direct-acting antiviral therapy for chronic hepatitis Cinfection: a phase 2, open-label,

[10] Pawlotsky JM. Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-

[11] Reig M, Mariño Z, Perelló C, Iñarrairaegui M, Ribeiro A, Lens S, Díaz A, Vilana R, Darnell A, Varela M, Sangro B, Calleja JL, Forns X, Bruix J. Unexpected high rate of early tumor recurrence in patients with HCV‐related HCC undergoing interferon‐free

[12] Conti F, Buonfiglioli F, Scuteri A, Crespi C, Bolondi L, Caraceni P, Foschi FG, Lenzi M, Mazzella G, Verucchi G, Andreone P, Brillanti S. Early occurrence and recurrence of hepatocellular carcinoma in HCV‐related cirrhosis treated with direct acting antivirals.

[13] Kozbial K, Moser S, Schwarzer R, Laferl H, Al-Zoairy R, Stauber R, Stättermayer AF, Beinhardt S, Graziadei I, Freissmuth C, Maieron A, Gschwantler M, Strasser M, Peck-Radosalvjevic M, Trauner M, Hofer H, Ferenci P. Unexpected high incidence of hepato‐ cellular carcinoma in cirrhotic patients with SVR following IFN-free DAA treatment. J

[14] Cheung MC, Walker AJ, Hudson BE, Verma S, McLauchlan J, Mutimer DJ, Brown A, Gelson WT, MacDonald DC, Agarwal K, Foster GR, Irving WL; HCV Research UK. Outcomes after successful direct‐acting antiviral therapy for patients with chronic hepa‐

titis C and decompensated cirrhosis. J Hepatol 2016;65(4):741–7.

proof-of-concept study. Lancet Gastroenterol Hepatol 2016;1(2):97–104.

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therapy. J Hepatol 2016;65:719–26.

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2016;65:692–99.

8 Advances in Treatment of Hepatitis C and B

cguidelines/en/

2016;65:5–6.

Hepatol 2016;65:222–25.


**Currently Available Therapies**

#### **Chapter 2 Provisional chapter Provisional chapter**

#### **Advances in Treatment of Hepatitis C Advances in Treatment of Hepatitis C**

 Sanaa M. Kamal Sanaa M. Kamal

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66719

#### **Abstract**

Hepatitis C infection (HCV) is a major cause of chronic hepatitis and cirrhosis worldwide. Interferon-based regimen has been the sole therapy to eradicate HCV infection for decades. However, this interferon and ribavirin combination is associated with several serious adverse events and the sustained virologic response rate was suboptimal. The recent discovery of oral direct-acting antiviral agents (DAAs) heralded a revolution in the treatment of chronic HCV. This breakthrough in HCV resulted in high rates of HCV eradication with sustained virologic response rates ranging between 90 and 100% across different genotypes. New therapies were administered orally for 12 or 24 months and this resulted in better compliance and few adverse events. DAAs are categorized into four major groups namely: NS5B nucleotide inhibitors, NS5B nonnucleoside inhibitors, NS5A replication complex inhibitors, and NS3/4A protease inhibitors (PI). Several interferonfree regimens have been approved and adequately assessed and several new regimens with high potencies, less cross-resistance, and better safety profile are in the process of approval. Thus, the era of HCV eradication and cure has begun.

**Keywords:** hepatitis C, direct-acting antivirals, interferon-free regimen

## **1. Introduction**

Hepatitis C virus (HCV) is a major cause of liver cirrhosis, end-stage liver disease, and liver transplantation throughout the world [1]. Approximately 170–200 million people equating to 3% of the world's population are infected with HCV [2]. The prevalence of HCV varies in different geographic regions. The prevalence of HCV infection is greater in Africa and Asia, with infection rates exceeding 5% [3–5]. Egypt has the highest prevalence of hepatitis C in the world, with 15% of the population affected [6–8]. In the USA, nearly 2% of the population is infected [9, 10]. In Europe, the prevalence ranges from 0.1% in northern European countries and 1% in

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

countries on the Mediterranean [10, 11]. The immigration crisis may increase HCV prevalence in Europe given that immigrants originate from countries with high rates of HCV [12].

## **2. Natural history and outcome of HCV infection**

Acute HCV infection is mostly asymptomatic and rarely recognized clinically. Spontaneous viral clearance (SVC) occurs in approximately 25% of patients [13, 14]. The striking feature of HCV infection is its tendency to persist and evolve to chronic hepatitis. In some patients, chronic HCV progresses to liver cirrhosis and hepatocellular carcinoma (HCC) [14, 15].

The outcome of HCV infection depends largely on several host, viral and environmental factors. During an early stage, HCV infection triggers viral-associated molecular pattern (PAMP) receptors resulting in induction of an antiviral state through several pathways such as limiting cellular, modifying and degrading viral RNA, altering cellular vesicle trafficking and probably other not yet discovered antiviral mechanisms [15–17]. Clearance of HCV is associated with the development of robust and multispecific CD4<sup>+</sup> and CD8+ T-cell responses in blood and liver that can be maintained for years after recovery from acute disease [18–20]. In contrast, individuals who progress to chronic infection fail to mount such a response or may have inadequate production of the cytokines essential to control viral replication. Incomplete control of viral replication by CD8+ T cells in the absence of sufficient memory CD4<sup>+</sup> T cells leads to viral persistence and emergence of CTL escape mutants [21–24].

Acute resolving hepatitis has been shown to be associated with HCV homogeneity, whereas progressing hepatitis correlated with genetic diversity, presumably reflecting greater immune pressure during acute spontaneous clearance [25]. Polymorphisms of genes involved in innate immunity as well as those in genes encoding cytokines and other immunologic mediators may explain spontaneous recovery from acute HCV and influence the strength and nature of immune defense. Genes encoding the inhibitory NK cell receptor *KIR2DL3* and its human leukocyte antigen C group 1 (*HLA-C1*) ligand influenced spontaneous resolution of HCV infection suggesting that inhibitory NK cell interactions are critical for antiviral immunity [26, 27].

To date, there are no reliable methods to predict who will resolve acute HCV spontaneously and who will develop chronic HCV. Similarly, no reliable indicators exist for distinguishing chronic hepatitis C patients who may develop cirrhosis or HCC. Thus, early effective treatment of patients with HCV is necessary for prevention of progression of liver disease to cirrhosis, hepatocellular carcinoma. In the absence of a vaccine against HCV, efficient treatment is important for prevention of transmission along with adoption of infection control measures.

## **3. Evolution of HCV therapy**

The ultimate goal of hepatitis C treatment is to reduce the occurrence of end-stage liver disease and its complications including decompensated cirrhosis, liver transplantation, and HCC. Treatment success is assessed by sustained virologic response (SVR), defined undetectable HCV RNA in blood several months after completing a course of treatment [28].

countries on the Mediterranean [10, 11]. The immigration crisis may increase HCV prevalence

Acute HCV infection is mostly asymptomatic and rarely recognized clinically. Spontaneous viral clearance (SVC) occurs in approximately 25% of patients [13, 14]. The striking feature of HCV infection is its tendency to persist and evolve to chronic hepatitis. In some patients, chronic HCV progresses to liver cirrhosis and hepatocellular carcinoma (HCC) [14, 15].

The outcome of HCV infection depends largely on several host, viral and environmental factors. During an early stage, HCV infection triggers viral-associated molecular pattern (PAMP) receptors resulting in induction of an antiviral state through several pathways such as limiting cellular, modifying and degrading viral RNA, altering cellular vesicle trafficking and probably other not yet discovered antiviral mechanisms [15–17]. Clearance of HCV is associated with the

can be maintained for years after recovery from acute disease [18–20]. In contrast, individuals who progress to chronic infection fail to mount such a response or may have inadequate production of the cytokines essential to control viral replication. Incomplete control of viral repli-

Acute resolving hepatitis has been shown to be associated with HCV homogeneity, whereas progressing hepatitis correlated with genetic diversity, presumably reflecting greater immune pressure during acute spontaneous clearance [25]. Polymorphisms of genes involved in innate immunity as well as those in genes encoding cytokines and other immunologic mediators may explain spontaneous recovery from acute HCV and influence the strength and nature of immune defense. Genes encoding the inhibitory NK cell receptor *KIR2DL3* and its human leukocyte antigen C group 1 (*HLA-C1*) ligand influenced spontaneous resolution of HCV infection suggesting that inhibitory NK cell interactions are critical for antiviral immunity [26, 27]. To date, there are no reliable methods to predict who will resolve acute HCV spontaneously and who will develop chronic HCV. Similarly, no reliable indicators exist for distinguishing chronic hepatitis C patients who may develop cirrhosis or HCC. Thus, early effective treatment of patients with HCV is necessary for prevention of progression of liver disease to cirrhosis, hepatocellular carcinoma. In the absence of a vaccine against HCV, efficient treatment is important for prevention of transmission along with adoption of infection control measures.

The ultimate goal of hepatitis C treatment is to reduce the occurrence of end-stage liver disease and its complications including decompensated cirrhosis, liver transplantation, and

T cells in the absence of sufficient memory CD4<sup>+</sup>

and CD8+

T-cell responses in blood and liver that

T cells leads to viral persistence

in Europe given that immigrants originate from countries with high rates of HCV [12].

**2. Natural history and outcome of HCV infection**

development of robust and multispecific CD4<sup>+</sup>

14 Advances in Treatment of Hepatitis C and B

and emergence of CTL escape mutants [21–24].

**3. Evolution of HCV therapy**

cation by CD8+

For two decades, the standard of care (SOC) for hepatitis C infection was interferon based. IFNα has potent antiviral activity due to its ability to induce IFN-stimulated genes (ISGs) that encode proteins which inhibit various stages of viral replication [29]. Type I IFNs bind a unique ubiquitous heterodimeric receptor consisting of interferon-alpha receptor 1/2 (IFNAR1/IFNAR2), resulting in the activation of signaling pathways and induction of a large number of IFN-stimulated genes (ISGs). ISG-encoded proteins mediate the antiviral and other effects of interferons [29]. IFNAR1 and IFNAR2 are associated with the Janus-activated kinases (JAKs) tyrosine kinase 2 (TYK2) and JAK1, respectively. Binding of type I IFNs to their heterodimeric receptors leads to activation of JAKs, which results in tyrosine phosphorylation of signal transducer and activator of transcription 2 (STAT2) and STAT1; STAT1/ STAT2 migrates into the nucleus and associates with the IFN regulatory factor 9 (IRF9) to form the STAT1-STAT2-IRF9 complex. This complex then binds IFN-stimulated response elements (ISREs) inside DNA to initiate the transcription of hundreds of different ISGs [30, 31]. IFN regulatory genes (IRGs) facilitate both clearance of virus from infected cells and protection of neighboring uninfected cells from incoming viral progeny. The antiviral-associated protein kinase R (PKR) plays an important role in restricting HCV 1a replication through regulating the NF-*κ*B pathway [32, 33].

Initially, chronic hepatitis C was treated by conventional interferon (IFN) monotherapy which yielded very poor response rates. Addition of the guano sine analog, ribavirin, to conventional interferon was associated with slight improvement in sustained virologic response (SVR) although the improvement was far from satisfactory particularly in HCV genotypes 1 and 4. Pegylation of the interferon molecule resulted in modification of the pharmacokinetic profile of IFN-α-2. Both PEG-IFN-α-2a and PEG-IFN-α-2b have slower absorption, more reduced distribution and lower elimination rate than the nonpegylated IFN-α. The maintained concentrations of PEG-IFNα allowed longer periods of viral inhibition with once a week dosing. Pegylated interferon and ribavirin therapy resulted in improved sustained virologic response (SVR), defined as undetectable HCV RNA 24 weeks after completion of treatment. With pegylated interferon alpha-2 and ribavirin (RBV) combination, response rates in genotypes 2 and 3 range between 70 and 80%. However, SVR rates in chronic HCV genotypes 1 and 4 infection are suboptimal. Adverse events are common with interferon-based regimen and include fatigue, flu-like symptoms, anxiety, skin rash, and gastrointestinal symptoms such as nausea and diarrhea. Hemolytic anemia is frequent due to the use of ribavirin. Some patients treated with PEG-IFN and RBV may develop cardiac arrhythmias or severe neuropsychiatric adverse events depression and suicidal tendency. The various adverse effects, the long duration of therapy and the need to inject interferon reduce compliance and treatment adherence. These factors have driven the urgent need to develop new treatments that are safer and more effective (**Figure 1**). The discovery of direct-acting antiviral agents (DAAs) heralded the dawn of a new era of HCV cure which was a dream.

**Figure 1.** Evolution of HCV therapy.

## **4. Direct-acting antiviral agents (DAAs)**

Direct-acting antiviral agents (DAAs) represent a revolution in HCV drug discovery. DAAs were developed to improve the SVR rates, reduce adverse events, and improve adherence to therapy among HCV patients. DAAs were initially introduced as add-ons to the previous standard of care (SOC) consisting of pegylated interferon alpha plus ribavirin (PR). Recently, a breakthrough in HCV therapy has been achieved with the introduction of interferon-free all-oral DAAs, with SVR rates now in excess of 90% after 12 weeks of therapy for genotype 1 patients.

DAAs target specific steps within the HCV life cycle and disrupt viral replication in an attempt to terminate that cycle before its completion (**Figure 2**) [34]. The first step in the life cycle of the virus is cell attachment and entry of HCV RNA through hepatocyte surface receptors. The HCV RNA is then translated to one polyprotein of 3010 amino acids that is subsequently cleaved by protease. It is then processed into four structural proteins (namely Core, E1, E2, and P7) as well as the nonstructural proteins (NS2-3 and NS3-4A proteases, NS3 helicase, and NS5B RdRp). All of these enzymes are essential for the replication of the virus and are potential drug discovery targets [35–37].

## **4.1. Goals of HCV and endpoints of treatment with DAAs**

The goal of therapy is to eradicate HCV infection to prevent hepatic cirrhosis, decompensation of cirrhosis, HCC, and severe extrahepatic manifestations. The endpoint of therapy is undetectable HCV RNA in blood by a sensitive assay (with the lower limit of detection <15 IU/ml) 12 weeks (SVR-12) and/or 24 weeks (SVR-24) after the completion of treatment. Undetectable HCV core antigen (HCV c Ag) 12 or 24 weeks after the completion of therapy can be an alternative to HCVRNA testing to assess the SVR12 or the SVR24, respectively [38]. In patients with advanced fibrosis and cirrhosis, HCV eradication reduces the rate of decompensation and will reduce, albeit not abolish the risk of HCC. In these patients surveillance for HCC should be continued.

**Figure 2.** Hepatitis C life cycle and the targets of direct-acting antiviral agents.

## **4.2. Classes of DAAs**

**4. Direct-acting antiviral agents (DAAs)**

**Figure 1.** Evolution of HCV therapy.

16 Advances in Treatment of Hepatitis C and B

tial drug discovery targets [35–37].

**4.1. Goals of HCV and endpoints of treatment with DAAs**

Direct-acting antiviral agents (DAAs) represent a revolution in HCV drug discovery. DAAs were developed to improve the SVR rates, reduce adverse events, and improve adherence to therapy among HCV patients. DAAs were initially introduced as add-ons to the previous standard of care (SOC) consisting of pegylated interferon alpha plus ribavirin (PR). Recently, a breakthrough in HCV therapy has been achieved with the introduction of interferon-free all-oral DAAs, with SVR rates now in excess of 90% after 12 weeks of therapy for genotype 1 patients. DAAs target specific steps within the HCV life cycle and disrupt viral replication in an attempt to terminate that cycle before its completion (**Figure 2**) [34]. The first step in the life cycle of the virus is cell attachment and entry of HCV RNA through hepatocyte surface receptors. The HCV RNA is then translated to one polyprotein of 3010 amino acids that is subsequently cleaved by protease. It is then processed into four structural proteins (namely Core, E1, E2, and P7) as well as the nonstructural proteins (NS2-3 and NS3-4A proteases, NS3 helicase, and NS5B RdRp). All of these enzymes are essential for the replication of the virus and are poten-

The goal of therapy is to eradicate HCV infection to prevent hepatic cirrhosis, decompensation of cirrhosis, HCC, and severe extrahepatic manifestations. The endpoint of therapy is undetectable HCV RNA in blood by a sensitive assay (with the lower limit of detection <15 IU/ml) 12 weeks (SVR-12) and/or 24 weeks (SVR-24) after the completion of treatment. Undetectable HCV core antigen (HCV c Ag) 12 or 24 weeks after the completion of therapy can be an alternative to HCVRNA testing to assess the SVR12 or the SVR24, respectively [38]. In patients with advanced fibrosis and cirrhosis, HCV eradication reduces the rate of There are four classes of DAAs, which are defined by their mechanism of action and therapeutic target. DAAs include NS5B nucleotide inhibitors, NS5B nonnucleoside inhibitors, NS5A replication complex inhibitors, and NS3/4A protease inhibitors (PI) (**Figure 3**).


**Figure 3.** Resistance patterns in different direct-acting antiviral agents (DAAs).

## *4.2.1. NS3/4A protease inhibitors (PIs)*

PIs block the activity NS3/4A serine protease, an enzyme which inhibits TRIF-mediated Tolllike receptor signaling and Cardif-mediated retinoic acid–inducible gene 1 (RIG-1) signaling resulting in impaired induction of interferons and blocking viral elimination [39, 40]. PIs have been grouped according to their resistance profile into first- and second-generation agents and into separate waves according to dosing, safety, and tolerability characteristics.

## *4.2.1.1. First-generation PIs*

Telaprevir and boceprevir were the first direct-acting antivirals for treatment of HCV and represented the first generation of PIs. Telaprevir or boceprevir was used in combination with peginterferon and ribavirin for the treatment of genotype 1 [40, 41]. Although telaprevir or boceprevir regimen enhanced SVR rates; the clinical efficacy of the triple regimen was limited by narrow genotype specificity, low barrier to resistance, and drug-drug interactions. This regimen also increased adverse events such as rash and moderate to severe anemia to an extent that might require the reduction of the RBV dose. Patient adherence and tolerability to triple therapy with BOC or TPV is a challenging issue as the two DAAs should be given three times daily with food. Triple therapy was not very effective in previous PEG-IFN/ RBV dual therapy, no responders. From an economic perspective, the triple therapy dramatically increased the costs of HCV treatment which are originally prohibitive. Thus, the clinical importance of these agents diminished substantially with the discovery of subsequent-generation protease inhibitors.

## *4.2.1.2. Second-wave, first-generation protease inhibitors*

The second wave of PIs for HCV includes agents such as simeprevir, asunaprevir, danoprevir, faldaprevir, and vaniprevir [42, 43]. Simeprevir (*Olysio*) is a NS3/4A HCV PI. Simeprevir is a macrocyclic compound that noncovalently binds to and inhibits the NS3/4A HCV protease, a protein that is responsible for cleaving and processing the HCV-encoded polyprotein, a critical step in HCV viral life cycle [42, 43]. Simeprevir shows enhanced binding affinity and specificity to NS3/4A when compared with the first-generation PIs, TPV, and BOC.

The safety and efficacy of SIM/PEG-IFN/RBV combination was investigated in treatmentnaïve patients with HCV genotype 1 infection (PILLAR trial) [44]. Enrolled patients received different SIM doses administered once-daily (QD) with pegylated interferon (Peg-IFN)-α-2a and ribavirin (RBV). According to response-guided therapy (RGT) criteria, 79.2–86.1% of SMV-treated patients completed treatment by week 24; 85.2–95.6% of these subsequently achieved SVR. The safety profile of triple therapy with SIM was found to be comparable to that of PEG-IFN/RBV combination therapy [44]. In the QUEST 1 and QUEST 2 studies [45, 46], conducted on treatment-naïve genotype 1, patients were randomized to receive either triple therapy with simeprevir plus PEG-IFN and RBV using a response-guided therapy (RGT) approach or standard of care (48 weeks of PEG-IFN and RBV with placebo control). SVR12 rates were 81% in the simeprevir arm versus 50% in the control arm. The majority of simeprevir-treated patients met the RGT and received 24 weeks of treatment and 86% of these patients achieved an SVR12.

*4.2.1. NS3/4A protease inhibitors (PIs)*

18 Advances in Treatment of Hepatitis C and B

*4.2.1.1. First-generation PIs*

ation protease inhibitors.

*4.2.1.2. Second-wave, first-generation protease inhibitors*

PIs block the activity NS3/4A serine protease, an enzyme which inhibits TRIF-mediated Tolllike receptor signaling and Cardif-mediated retinoic acid–inducible gene 1 (RIG-1) signaling resulting in impaired induction of interferons and blocking viral elimination [39, 40]. PIs have been grouped according to their resistance profile into first- and second-generation agents

Telaprevir and boceprevir were the first direct-acting antivirals for treatment of HCV and represented the first generation of PIs. Telaprevir or boceprevir was used in combination with peginterferon and ribavirin for the treatment of genotype 1 [40, 41]. Although telaprevir or boceprevir regimen enhanced SVR rates; the clinical efficacy of the triple regimen was limited by narrow genotype specificity, low barrier to resistance, and drug-drug interactions. This regimen also increased adverse events such as rash and moderate to severe anemia to an extent that might require the reduction of the RBV dose. Patient adherence and tolerability to triple therapy with BOC or TPV is a challenging issue as the two DAAs should be given three times daily with food. Triple therapy was not very effective in previous PEG-IFN/ RBV dual therapy, no responders. From an economic perspective, the triple therapy dramatically increased the costs of HCV treatment which are originally prohibitive. Thus, the clinical importance of these agents diminished substantially with the discovery of subsequent-gener-

The second wave of PIs for HCV includes agents such as simeprevir, asunaprevir, danoprevir, faldaprevir, and vaniprevir [42, 43]. Simeprevir (*Olysio*) is a NS3/4A HCV PI. Simeprevir is a macrocyclic compound that noncovalently binds to and inhibits the NS3/4A HCV protease, a protein that is responsible for cleaving and processing the HCV-encoded polyprotein, a critical step in HCV viral life cycle [42, 43]. Simeprevir shows enhanced binding affinity and

The safety and efficacy of SIM/PEG-IFN/RBV combination was investigated in treatmentnaïve patients with HCV genotype 1 infection (PILLAR trial) [44]. Enrolled patients received different SIM doses administered once-daily (QD) with pegylated interferon (Peg-IFN)-α-2a and ribavirin (RBV). According to response-guided therapy (RGT) criteria, 79.2–86.1% of SMV-treated patients completed treatment by week 24; 85.2–95.6% of these subsequently achieved SVR. The safety profile of triple therapy with SIM was found to be comparable to that of PEG-IFN/RBV combination therapy [44]. In the QUEST 1 and QUEST 2 studies [45, 46], conducted on treatment-naïve genotype 1, patients were randomized to receive either triple therapy with simeprevir plus PEG-IFN and RBV using a response-guided therapy (RGT) approach or standard of care (48 weeks of PEG-IFN and RBV with placebo control). SVR12 rates were 81% in the simeprevir arm versus 50% in the control arm. The majority of

specificity to NS3/4A when compared with the first-generation PIs, TPV, and BOC.

and into separate waves according to dosing, safety, and tolerability characteristics.

Simeprevir also enhanced SVR in treatment-experienced patients. In the ASPIRE trial [47] treatment-experienced patients (with prior failure to PEG/RBV) were randomized to receive placebo plus PEG/RBV, or one of six regimens consisting of SMV plus PEG-IFN-α-2a plus ribavirin. In the SMV-treated patients, the SVR 24 rates ranged from 61 to 80% (according to the regimen used), which was significantly higher than the 23% SVR in patients treated with PEG-IFN/RBV. The safety profile observed among patients in the simeprevir arm was similar to the safety profile for patients in the placebo arm. These results were supported by those of another clinical trial conducted on treatment-experienced HCV genotype 1 patients with a history of viral relapse. The overall SVR12 in patients treated with SIM/PEG-IFN/RBV was of 79% compared to 36% for the peginterferon plus ribavirin arm. Patients with advanced fibrosis (F3-F4 by METAVIR) also had superior SVR12 rates with the addition of simeprevir (73% SVR12 compared with 24% in control arm) [48].

The efficacy of SIM/PEG-IFN/RBV in treatment-naïve and treatment-experienced patients with chronic HCV genotype 4 was evaluated in the RESTORE trial. Overall, 65.4% of the patients achieved an SRV12. The SVR12 rates varied with treatment group being 83% in treatment-naïve, 86% in treatment-experienced with prior relapse, 60% in prior partial responders, and 40% in prior null responders [49].

These trials showed that simeprevir-based/PEG-IFN/RBV triple therapy was effective, welltolerated, and safe. However, the fast-paced HCV drug discovery paved the way to new interferon-free combinations which combine efficacy, safety, and convenience. Thus, SMV was included with other DAAs such as sofosbuvir to form one of the earliest interferon-free combinations (discussed later).

*Danoprevir (DNV)* is a highly selective and potent second-wave inhibitor of HCV NS3/4A protease. Coadministration of 100 mg of ritonavir with DNV has been shown to optimize the pharmacokinetics of DNV, allowing for lower dosing and better antiviral activity The DAUPHINE trial [50] evaluated three different dosages of DNV/r: 50, 100, and 200 mg danoprevir, boosted with 100 mg ritonavir, consumed twice a day for 24 weeks. A study arm also explored danoprevir/r 100/100 mg, in a response-guided therapy (RGT) algorithm, in which patients reaching an RVR received a total of 12 weeks of treatment. Overall, the better SVR rates were achieved in higher dosage arms compared to lower dosage arms. SVR rates decreased with decreasing dosage of danoprevir/r as follows: 89.1, 78.5, and 69.1% [50]. Faldaprevir was evaluated in IFN-free regimen in combination with deleobuvir, an NS5B no nucleoside polymerase inhibitor and ribavirin, in HCV-1b patients. The combination was highly efficacious, with 95% achieving SVR12 including patients with compensated cirrhosis [51].

Taken together, the second-wave, first-generation protease inhibitors offer several benefits over the first-generation PIs, TVR, and BOC in terms of less side effect profile and more convenient dosing. However, these preparations still have low genetic barrier to resistance particularly for HCV-1a.

#### *4.2.1.3. Second-generation protease inhibitors*

Recent second-generation PIs such as the macrocyclic compound grazoprevir offer several benefits over earlier protease inhibitors, including fewer drug-drug interactions, improved dosing schedules, and less frequent and less severe side effects. Grazoprevir is distinct from earlier-generation protease inhibitors in potency against a broader array of HCV genotypes, as well as its activity against some of the major resistance-associated variants (R155K and D168Y) resulting from failure with first-generation protease inhibitors. Grazoprevir is available in combination with the NS5A inhibitor elbasvir. Elbasvir/grazoprevir (Zepatier) [52] is available as fixed dose tablets (50/100 mg) are prescribed as one tablet orally once daily, with or without food. The treatment duration and whether to take with or without ribavirin are dependent on genotypes and other patient variables [53].

The C-EDGE treatment-naïve trial assessed the safety and efficacy of the fixed-dose combination of elbasvir-grazoprevir (50/100 mg) in patients with genotype 1, 4, or 6 hepatitis C infection, with or without compensated cirrhosis. The overall SVR12 rate was 95%, with rates of 92% for genotype 1a, 99% for genotype 1b, 100% for genotype 4, and 80% for genotype 6. No statistically significant difference in SVR12 was found between cirrhotic and noncirrhotic patients [54]. The C-EDGE CO-STAR trial enrolled treatment-naïve patients who inject drugs and were infected with chronic HCV genotype 1, 4, or 6. In this difficult to treat cohort, the SVR12 were 95% [55].

Treatment-naïve patients with compensated cirrhosis and treatment-experienced patients with a prior null response to PEG plus RBV were randomized to receive elbasvir plus grazoprevir, with or without ribavirin, for 12 or 18 weeks. The SVR12 rates ranged between 90 and 97% in cirrhotics and 94% in null responder cirrhotic patients. The SVR12 was 100% for genotype 1b. No additional benefit was achieved by adding ribavirin to elbasvir plus grazoprevir in a subset of patients [56]. Treatment-experienced patients with genotype 1 HCV with previous failure of peg interferon/ribavirin (PR) and an earlier-generation protease inhibitor (BOC, TPV, or SIM) achieved SVR 24 of 96% when treated with elbasvir plus grazoprevir and RBV [57].

In the C-EDGE coinfection trial, patients with chronic hepatitis C genotype 1, 4, or 6 and HIV coinfection received elbasvir-grazoprevir once daily for 12 weeks. Patients were on antiretroviral therapy with HIV viral suppression and the median CD4 cell count was 568 cells/mm3 . The overall SVR12 rate was 96, with the breakdown by genotype SVR12 rates were 96.5, 95.5, and 96.4% for genotypes 1a, 1b, and 4, respectively. All cirrhotic patients achieved an SVR12 [58].

Thus, zepatier is active against a broad array of HCV genotypes including genotypes 1, 4, 6, as well as some of the major resistance-associated variants (R155K and D168Y) resulting from failure with first-generation protease inhibitors. Elbasvir/grazoprevir is generally well tolerated; however, the adverse effects reported include headache, nausea, fatigue, decreased appetite, anemia, pyrexia, and elevations of ALT.

#### *4.2.2. NS5B nucleoside polymerase inhibitors (NPIs)*

NS5B is an RNA-dependent RNA polymerase (RdRp) involved in posttranslational processing which is vital for HCV replication. NPIs are analogs of natural substrates that bind the active site of NS5B and terminate viral RNA chain generation. Given that the structure of NS5B is highly conserved across all HCV genotypes, NPIs are effective against all genotypes. NPIs show high antiviral activities in all genotypes and provide a high genetic barrier to resistance. Thus, NPIs are included in several efficacious all-oral combination therapies. Polymerase inhibitors are categorized according to their mode of action and specificity into NPIs and NNPIs. These two classes generally differ in specificity. Nucleoside inhibitors (NIs) bind to the catalytic site of the RNA polymerase causing chain termination. Nonnucleoside inhibitors bind to a less conserved site resulting in a conformational change that distorts the positioning of residues binding RNA resulting in inhibition of polymerization [59, 60].

## *4.2.2.1. Sofosbuvir (sovaldi)*

*4.2.1.3. Second-generation protease inhibitors*

20 Advances in Treatment of Hepatitis C and B

dependent on genotypes and other patient variables [53].

appetite, anemia, pyrexia, and elevations of ALT.

*4.2.2. NS5B nucleoside polymerase inhibitors (NPIs)*

Recent second-generation PIs such as the macrocyclic compound grazoprevir offer several benefits over earlier protease inhibitors, including fewer drug-drug interactions, improved dosing schedules, and less frequent and less severe side effects. Grazoprevir is distinct from earlier-generation protease inhibitors in potency against a broader array of HCV genotypes, as well as its activity against some of the major resistance-associated variants (R155K and D168Y) resulting from failure with first-generation protease inhibitors. Grazoprevir is available in combination with the NS5A inhibitor elbasvir. Elbasvir/grazoprevir (Zepatier) [52] is available as fixed dose tablets (50/100 mg) are prescribed as one tablet orally once daily, with or without food. The treatment duration and whether to take with or without ribavirin are

The C-EDGE treatment-naïve trial assessed the safety and efficacy of the fixed-dose combination of elbasvir-grazoprevir (50/100 mg) in patients with genotype 1, 4, or 6 hepatitis C infection, with or without compensated cirrhosis. The overall SVR12 rate was 95%, with rates of 92% for genotype 1a, 99% for genotype 1b, 100% for genotype 4, and 80% for genotype 6. No statistically significant difference in SVR12 was found between cirrhotic and noncirrhotic patients [54]. The C-EDGE CO-STAR trial enrolled treatment-naïve patients who inject drugs and were infected with chronic HCV genotype 1, 4, or 6. In this difficult to treat cohort, the SVR12 were 95% [55]. Treatment-naïve patients with compensated cirrhosis and treatment-experienced patients with a prior null response to PEG plus RBV were randomized to receive elbasvir plus grazoprevir, with or without ribavirin, for 12 or 18 weeks. The SVR12 rates ranged between 90 and 97% in cirrhotics and 94% in null responder cirrhotic patients. The SVR12 was 100% for genotype 1b. No additional benefit was achieved by adding ribavirin to elbasvir plus grazoprevir in a subset of patients [56]. Treatment-experienced patients with genotype 1 HCV with previous failure of peg interferon/ribavirin (PR) and an earlier-generation protease inhibitor (BOC, TPV, or SIM)

achieved SVR 24 of 96% when treated with elbasvir plus grazoprevir and RBV [57].

In the C-EDGE coinfection trial, patients with chronic hepatitis C genotype 1, 4, or 6 and HIV coinfection received elbasvir-grazoprevir once daily for 12 weeks. Patients were on antiretroviral therapy with HIV viral suppression and the median CD4 cell count was 568 cells/mm3

overall SVR12 rate was 96, with the breakdown by genotype SVR12 rates were 96.5, 95.5, and 96.4% for genotypes 1a, 1b, and 4, respectively. All cirrhotic patients achieved an SVR12 [58]. Thus, zepatier is active against a broad array of HCV genotypes including genotypes 1, 4, 6, as well as some of the major resistance-associated variants (R155K and D168Y) resulting from failure with first-generation protease inhibitors. Elbasvir/grazoprevir is generally well tolerated; however, the adverse effects reported include headache, nausea, fatigue, decreased

NS5B is an RNA-dependent RNA polymerase (RdRp) involved in posttranslational processing which is vital for HCV replication. NPIs are analogs of natural substrates that bind the

. The

Sofosbuvir is a nucleoside analog inhibitor of hepatitis C virus NS5B polymerase. The triphosphate form of sofosbuvir mimics the natural cellular uridine nucleotide and is incorporated by the HCV RNA polymerase into the elongating RNA primer strand, resulting in viral chain termination. Sofosbuvir is a prodrug which is rapidly converted after oral intake to GS-331007 which is taken up by hepatocytes. The cellular kinases convert GS-331007 to its pharmacologically active uridine analog 5′-triphosphate form (GS-461203) that is incorporated by the HCV RNA polymerase into the elongating RNA primer strand, resulting in chain termination. Sofosbuvir is a potent pangenotypic NS5B polymerase inhibitor with a high barrier to resistance. It is available as 400 mg tablets administered once a day with or without food. The discovery of sofosbuvir has been a breakthrough in HCV treatment. Currently, SOF represents the backbone of several interferon-free regimens for the treatment of chronic hepatitis caused by various HCV genotypes. Excretion of sofosbuvir is through the kidney (80%) [58, 59, 61].

## *4.2.3. Efficacy of sofosbuvir plus peginterferon and ribavirin*

The ATOMIC and ELECTRON (Arms 1-8 studies) [62, 63] established the effectiveness of a 12-week course of sofosbuvir plus peginterferon and ribavirin in treatment-naïve patients with HCVgenotype-1 with SVR rates ranging between 87 and 100%. In genotypes 3, the SVR 12 rates were 71% with the 16-week SOF plus RBV regimen, 84% with 24 weeks of SOV plus RBV, and 93% with 12 weeks of SOF plus RBV plus PEG-IFN. For the patients with genotype 2 infections, the SVR 12 rates were 87% with the 16-week SOF plus RBV regimen, 100% with 24 weeks of SOF plus RBV, and 94% with 12 weeks of SOF plus RBV plus PEG-IFN [64].

## *4.2.4. Efficacy of interferon-free sofosbuvir regimen*

## *4.2.4.1. Sofosbuvir (SOF) and simeprevir (SIM) combination (Olysio)*

This combination is the earliest interferon-free regimen that reached optimal results in terms of SVR. Trials showed efficacy and safety of this drug combination in treatment-naïve and treatment-experienced patients across several genotypes. The OPTIMIST-1 trial evaluated the efficacy of sofosbuvir plus simeprevir for 8 or 12 weeks in treatment naive or experienced patients with chronic HCV genotype 1 infection without cirrhosis. The sustained response rates were 97% in patients treated for 12 weeks and the SVR was 83% in patients treated for 8 weeks. These findings were further confirmed in the OPTIMIST-2 trial which demonstrated that 12-week regimen of SOF plus SIM is effective in treatment-naïve and treatment-experienced patients with cirrhosis and HCV genotype 1 infection, with the exception that patients with genotype 1a and the baseline Q80K mutation have SVR rates of only 74% [66].

A large cohort prospective study in genotype 1 patients treated with SOF plus SIM for 12–16 weeks showed that the overall SVR rate was 84%. Model-adjusted estimates demonstrated that patients with cirrhosis, prior decompensation, and previous protease inhibitor treatments were less likely to achieve an SVR. Addition of ribavirin enhances SVR rates in such patients [67].

Taken together, several clinical trials demonstrate that all-oral 12-week regimen of simeprevir plus sofosbuvir is effective and well tolerated in treatment-naïve and treatment-experienced HCV genotype 1 patients without and with cirrhosis. Ribavirin may be needed in patients with decompensation, and previous protease inhibitor treatment failure. The frequent adverse events include fatigue, headache, nausea, rashes, and insomnia. Serious adverse events and treatment discontinuation occur in only 3% of patients.

## *4.2.4.2. Sofosbuvir with the NS5A inhibitor ledipasvir (Harvoni)*

• Treatment-naïve and treatment-experienced genotype 1 patients

Gane et al. [68] evaluated an all-oral regimen comprising sofosbuvir with ledipasvir or the NS5B nonnucleoside inhibitor GS-9669 in patients with genotype 1 HCV infection. Sofosbuvir (400 mg once daily) and ledipasvir (90 mg once daily) plus ribavirin were given for 12 weeks to treatment-naïve patients and prior null responders. Sofosbuvir and GS-9669 (500 mg once daily) plus ribavirin were given for 12 weeks to treatment-naïve patients and prior null responders. Additionally, prior null responders with cirrhosis were randomly assigned to groups given a fixed-dose combination of sofosbuvir and ledipasvir, with ribavirin or without ribavirin and a group of treatment-naïve patients received sofosbuvir, ledipasvir, and ribavirin for 6 weeks. SVR12 was 100, 92, and 68% in treatment-naïve patients receiving sofosbuvir, those receiving SOV, ledipasvir, ribavirin, GS-9669, and ribavirin and patients receiving 6 weeks of sofosbuvir, ledipasvir, and ribavirin, respectively. All noncirrhotic prior null responders receiving 12 weeks of sofosbuvir along with another DAA plus RBV achieved SVR12 of 100%.

In the NIAID SYNERGY (genotype 1 study) [69], treatment-naïve patients with genotype 1 chronic HCV were randomized to receive either ledipasvir-sofosbuvir for 12 weeks; or ledipasvir-sofosbuvir (90-400 mg) plus the nonnucleoside NS5B inhibitor GS-9669 (500 mg once daily) for 6 weeks, or ledipasvir-sofosbuvir (90-400 mg) plus the NS3/4A protease inhibitor GS-9451 (80 mg once daily) for 6 weeks. Patients in the 12-week ledipasvir-sofosbuvir arm with any stage of fibrosis could be enrolled in the study. The SVR12 rates were 100, 95, and 95% in the ledipasvir-sofosbuvir arm, the ledipasvir-sofosbuvir plus GS-9669 group, and the ledipasvir-sofosbuvir plus GS-9451 group, respectively.

A trial [70] evaluated 8- and 12-week courses of the fixed-dose combination of ledipasvir (90 mg) and sofosbuvir (400 mg), with or without ribavirin in treatment-naïve and treatmentexperienced patients with chronic HCV genotype 1 infection. In all of the five study arms, SVR12 was achieved in 95–100% of patients. The regimen of ledipasvir-sofosbuvir was well tolerated; only one patient had a serious adverse event of anemia, thought to be related to ribavirin. A recent large study that enrolled 4365 genotype 1, treatment-naïve, HCV-infected patients treated with LDV/SOF±RBV demonstrated SVR rates of 91.3 and 92.0% (3191/3495) for LDV/SOF and LDV/SOF+RBV, respectively [71].

Thus, the combination of ledipasvir-sofosbuvir with or without ribavirin is highly effective in treatment-naïve and treatment-experienced patients with chronic HCV genotype 1.

• Sofosbuvir- ledipasvir in HCV nongenotype 1 patients

weeks. These findings were further confirmed in the OPTIMIST-2 trial which demonstrated that 12-week regimen of SOF plus SIM is effective in treatment-naïve and treatment-experienced patients with cirrhosis and HCV genotype 1 infection, with the exception that patients

A large cohort prospective study in genotype 1 patients treated with SOF plus SIM for 12–16 weeks showed that the overall SVR rate was 84%. Model-adjusted estimates demonstrated that patients with cirrhosis, prior decompensation, and previous protease inhibitor treatments were less likely to achieve an SVR. Addition of ribavirin enhances SVR rates in such patients [67].

Taken together, several clinical trials demonstrate that all-oral 12-week regimen of simeprevir plus sofosbuvir is effective and well tolerated in treatment-naïve and treatment-experienced HCV genotype 1 patients without and with cirrhosis. Ribavirin may be needed in patients with decompensation, and previous protease inhibitor treatment failure. The frequent adverse events include fatigue, headache, nausea, rashes, and insomnia. Serious adverse events and

Gane et al. [68] evaluated an all-oral regimen comprising sofosbuvir with ledipasvir or the NS5B nonnucleoside inhibitor GS-9669 in patients with genotype 1 HCV infection. Sofosbuvir (400 mg once daily) and ledipasvir (90 mg once daily) plus ribavirin were given for 12 weeks to treatment-naïve patients and prior null responders. Sofosbuvir and GS-9669 (500 mg once daily) plus ribavirin were given for 12 weeks to treatment-naïve patients and prior null responders. Additionally, prior null responders with cirrhosis were randomly assigned to groups given a fixed-dose combination of sofosbuvir and ledipasvir, with ribavirin or without ribavirin and a group of treatment-naïve patients received sofosbuvir, ledipasvir, and ribavirin for 6 weeks. SVR12 was 100, 92, and 68% in treatment-naïve patients receiving sofosbuvir, those receiving SOV, ledipasvir, ribavirin, GS-9669, and ribavirin and patients receiving 6 weeks of sofosbuvir, ledipasvir, and ribavirin, respectively. All noncirrhotic prior null responders receiving 12

weeks of sofosbuvir along with another DAA plus RBV achieved SVR12 of 100%.

In the NIAID SYNERGY (genotype 1 study) [69], treatment-naïve patients with genotype 1 chronic HCV were randomized to receive either ledipasvir-sofosbuvir for 12 weeks; or ledipasvir-sofosbuvir (90-400 mg) plus the nonnucleoside NS5B inhibitor GS-9669 (500 mg once daily) for 6 weeks, or ledipasvir-sofosbuvir (90-400 mg) plus the NS3/4A protease inhibitor GS-9451 (80 mg once daily) for 6 weeks. Patients in the 12-week ledipasvir-sofosbuvir arm with any stage of fibrosis could be enrolled in the study. The SVR12 rates were 100, 95, and 95% in the ledipasvir-sofosbuvir arm, the ledipasvir-sofosbuvir plus GS-9669 group, and the

A trial [70] evaluated 8- and 12-week courses of the fixed-dose combination of ledipasvir (90 mg) and sofosbuvir (400 mg), with or without ribavirin in treatment-naïve and treatmentexperienced patients with chronic HCV genotype 1 infection. In all of the five study arms,

with genotype 1a and the baseline Q80K mutation have SVR rates of only 74% [66].

treatment discontinuation occur in only 3% of patients.

22 Advances in Treatment of Hepatitis C and B

ledipasvir-sofosbuvir plus GS-9451 group, respectively.

*4.2.4.2. Sofosbuvir with the NS5A inhibitor ledipasvir (Harvoni)*

• Treatment-naïve and treatment-experienced genotype 1 patients

Patients with genotype 3 and 6 achieved good SVR rates when treated with ledipasvir-sofosbuvir. The SVR 12 responses in treatment-naïve patients with genotype 3 were superior in the regimen with ribavirin (100%) than without ribavirin (64%). Among the treatment-experienced patients, 82% of treated ledipasvir-sofosbuvir plus ribavirin achieved an SVR 12 and the SVR 12 rate was 96% in the patients with genotype 6 [72].

The NIAID SYNERGY (Genotype 4) trial enrolled treatment-naïve and treatment-experienced patients with genotype 4 chronic HCV to receive ledipasvir-sofosbuvir for 12 weeks. Patients with compensated cirrhosis were allowed to enroll in the study. Overall, in the intent-to-treat SVR was 95% [73]. A recent study that enrolled treatment-naïve and -experienced patients with chronic HCV genotype 4 revealed SVR12 of 78% in patients treated with ledipasvir-sofosbuvir for 12 weeks and SVR 12 in patients treated with 24 weeks [74]. These findings suggest that further studies are still needed to optimize ledipasvir-sofosbuvir therapy in patients with different stages of chronic HCV genotype 4. To date, the efficacy and duration of DAAs in HCV genotype 4 have not been adequately studied and further trials are required to optimize therapy in this genotype.

A clinical trial assessed response to ledipasvir-sofosbuvir in 41 patients with chronic HCV genotype 5 (21 treatment-naïve and 20 treatment-experienced). The overall SVR12 was 95% in treatment-naïve and treatment-experienced patients, while the SVR12 was 97% in patients without cirrhosis and 89% in patients with cirrhosis [75].

• HCV and HIV coinfected patients

The ERADICATE trial investigated the safety and efficacy of a 12-week regimen of ledipasvirsofosbuvir in HCV treatment-naïve patients with genotype 1 chronic hepatitis C who are coinfected with HIV. Patients on antiretroviral therapy were allowed to receive tenofoviremtricitabine plus either efavirenz, raltegravir, rilpivirine, rilpivirine plus raltegravir, or efavirenz plus raltegravir. In patients taking antiretroviral therapy, SVR12 was 97% [76]. The SVR12 was 96.4% in German HIV-HCV coinfected patients [77]. A study investigated the efficacy and safety of ledipasvir/sofosbuvir plus ribavirin for 24 weeks in HCV/HIV-coinfected patients who relapsed after receiving 12 weeks of ledipasvir/sofosbuvir therapy. The SVR12 was 89% suggesting that ledipasvir/sofosbuvir can be an effective salvage therapy for patients for whom direct-acting antiviral treatment has failed [78].

Thus, ledipasvir-sofosbuvir is well tolerated and effective in patients with genotype 1 HCV and HIV coinfection. However, more studies are required to investigate the efficacy and safety of ledipasvir/sofosbuvir in treatment of HIV patients infected with various HCV genotypes. Importantly the drug-drug interactions between ledipasvir/sofosbuvir and antiretroviral therapy need extensive investigations on large cohorts.

#### -Retreatment of sofosbuvir failures

In the NIAID retreatment of sofosbuvir failures trial [79], patients with genotype 1 HCV who previously had failed a 24-week course of sofosbuvir plus ribavirin achieved SVR12 ranged between 98 and 100% when retreated with fixed-dose combination of ledipasvir-sofosbuvir for 12 weeks. Despite the small sample size in this study, the trial showed that 12-week regimen of ledipasvir-sofosbuvir without or with ribavirin was well tolerated and shows promise as a treatment option for patients with prior sofosbuvir failure.

## *4.2.4.3. Sofosbuvir/velpatasvir (Epclusa)*

Sofosbuvir-velpatasvir (sofosbuvir 400 mg plus velpatasvir 100 mg) is an oral fixed-dose combination of sofosbuvir, and the novel NS5A replication complex inhibitor, velapatasvir. Velpatasvir (formerly GS-5816) has potent *in vitro* anti-HCV activity across all genotypes at the picomolar level. The combination of sofosbuvir-velpatasvir is the first once-daily single-tablet regimen with pangenotypic activity. Sofosbuvir-velpatasvir is indicated for patients with chronic HCV genotype 1 through 6 [80]. A clinical trial [81] assessed the efficacy and safety of the combination of the nucleotide polymerase inhibitor sofosbuvir, the NS5A inhibitor velpatasvir, and the NS3/4A protease inhibitor GS-9857 in patients with hepatitis C virus (HCV) genotype 1 infection. Among treatment-naïve patients without cirrhosis, the SVR rates were 71 and 100% after 6 and 8 weeks of treatment, respectively. Among treatment-naïve patients with cirrhosis, 94% achieved SVR12 after 8 weeks therapy and 81% after 8 weeks of treatment with ribavirin. The SVR12 rates were 100% in DAA-experienced patients without cirrhosis and with cirrhosis, respectively.

Sofosbuvir-velpatasvir showed high efficacy in non-1 genotypes. ASTRAL-2 study demonstrated that the SVR12 was 99% treatment-naïve and treatment-experienced patients infected with HCV genotype 2 [82]. Among HCV with chronic HCV genotype 3 treated with sofosbuvirvelpatasvir, the ASTRAL-3 trial showed that the SVR12 rate were 93% for treatment-naïve and 89% for treatment-experienced patients [83]. SVR12 was 100% in patients with chronic HCV genotype 4 [84]. The ASTRAL-4 studies [82] demonstrated that sofosbuvir/velpatasvir plus ribavirin were effective in achieving a high SVR12 rate in patients with decompensated cirrhosis [85].

The ASTRAL-5 study investigated the safety and efficacy of 12 weeks sofosbuvir-velpatasvir in patients with HIV and HCV coinfection. Enrolled patients were infected with genotype 1, 2, 3, 4, or 6 HCV infection; 18% had compensated cirrhosis and 29% were treatment-experienced. The mean CD4 count was 583 cells/mm3 and all patients had HIV viral suppression. The antiretroviral regimens included tenofovir disoproxil fumarate (DF). The overall SVR12 rate was 95%. The presence of cirrhosis or treatment experience did not negatively influence treatment response [86].

#### *4.2.5. Nonstructural protein 5A (NS5A) inhibitors*

The NS5A protein is essential for replication and assembly of HCV. Inhibitors of NS5A block viral production at an early stage of assembly, so that no viral RNA or nucleocapsid protein is released [87]. Therefore, agents that block NS5B activity (polymerase inhibitors) inhibit the virus's RdRp [87]. Nucleoside inhibitors (NIs) bind to RdRp's active site, whereas the nonnucleoside inhibitors (NNIs) bind to the enzyme outside the active site, inducing conformational changes that downregulated RdRp's activity. As a result of mechanistic and potency differences, the NIs tend to have broad potency against multiple HCV genotypes and are less likely to select for resistant strains than are the NNIs [88]. Cyclophilin is a host protein that interacts with NS5B and appears to promote the HCV protein's ability to bind.

## *4.2.5.1. Daclatasvir (Daklinza)*

Importantly the drug-drug interactions between ledipasvir/sofosbuvir and antiretroviral ther-

In the NIAID retreatment of sofosbuvir failures trial [79], patients with genotype 1 HCV who previously had failed a 24-week course of sofosbuvir plus ribavirin achieved SVR12 ranged between 98 and 100% when retreated with fixed-dose combination of ledipasvir-sofosbuvir for 12 weeks. Despite the small sample size in this study, the trial showed that 12-week regimen of ledipasvir-sofosbuvir without or with ribavirin was well tolerated and shows promise

Sofosbuvir-velpatasvir (sofosbuvir 400 mg plus velpatasvir 100 mg) is an oral fixed-dose combination of sofosbuvir, and the novel NS5A replication complex inhibitor, velapatasvir. Velpatasvir (formerly GS-5816) has potent *in vitro* anti-HCV activity across all genotypes at the picomolar level. The combination of sofosbuvir-velpatasvir is the first once-daily single-tablet regimen with pangenotypic activity. Sofosbuvir-velpatasvir is indicated for patients with chronic HCV genotype 1 through 6 [80]. A clinical trial [81] assessed the efficacy and safety of the combination of the nucleotide polymerase inhibitor sofosbuvir, the NS5A inhibitor velpatasvir, and the NS3/4A protease inhibitor GS-9857 in patients with hepatitis C virus (HCV) genotype 1 infection. Among treatment-naïve patients without cirrhosis, the SVR rates were 71 and 100% after 6 and 8 weeks of treatment, respectively. Among treatment-naïve patients with cirrhosis, 94% achieved SVR12 after 8 weeks therapy and 81% after 8 weeks of treatment with ribavirin. The SVR12 rates were

100% in DAA-experienced patients without cirrhosis and with cirrhosis, respectively.

Sofosbuvir-velpatasvir showed high efficacy in non-1 genotypes. ASTRAL-2 study demonstrated that the SVR12 was 99% treatment-naïve and treatment-experienced patients infected with HCV genotype 2 [82]. Among HCV with chronic HCV genotype 3 treated with sofosbuvirvelpatasvir, the ASTRAL-3 trial showed that the SVR12 rate were 93% for treatment-naïve and 89% for treatment-experienced patients [83]. SVR12 was 100% in patients with chronic HCV genotype 4 [84]. The ASTRAL-4 studies [82] demonstrated that sofosbuvir/velpatasvir plus ribavirin were effective in achieving a high SVR12 rate in patients with decompensated cirrhosis [85].

The ASTRAL-5 study investigated the safety and efficacy of 12 weeks sofosbuvir-velpatasvir in patients with HIV and HCV coinfection. Enrolled patients were infected with genotype 1, 2, 3, 4, or 6 HCV infection; 18% had compensated cirrhosis and 29% were treatment-experienced. The

regimens included tenofovir disoproxil fumarate (DF). The overall SVR12 rate was 95%. The presence of cirrhosis or treatment experience did not negatively influence treatment response [86].

The NS5A protein is essential for replication and assembly of HCV. Inhibitors of NS5A block viral production at an early stage of assembly, so that no viral RNA or nucleocapsid protein is released [87]. Therefore, agents that block NS5B activity (polymerase inhibitors) inhibit the

and all patients had HIV viral suppression. The antiretroviral

apy need extensive investigations on large cohorts.

as a treatment option for patients with prior sofosbuvir failure.


24 Advances in Treatment of Hepatitis C and B

*4.2.4.3. Sofosbuvir/velpatasvir (Epclusa)*

mean CD4 count was 583 cells/mm3

*4.2.5. Nonstructural protein 5A (NS5A) inhibitors*

Daclatasvir HCV is first-in-class inhibitor of the nonstructural viral protein 5A (NS5A), a phosphoprotein that plays an important role in hepatitis C replication. The exact mechanism by which daclatasvir inhibits the NS5A replication complex is unclear, but it is believed that daclatasvir inhibits viral RNA replication and virion assembly. It may also inhibit phosphorylation of the NS4A, and therefore the formation and activation of the HCV replication complex. Based on *in vitro* data, daclatasvir has shown activity against HCV genotypes 1 through 6, with EC50 values ranging from picomolar to low nanomolar against wild-type HCV [89].

When used in combination with sofosbuvir, with or without ribavirin, daclatasvir showed high efficacy in pangenotypic all-oral regimen. According to the results of the AI444040 and ALLY-3 trials [90, 91], a 12-week regimen of daclatasvir plus sofosbuvir in patients with chronic HCV genotype 1 or 3 infection without cirrhosis resulted in high SVR12 rates, regardless of prior treatment experience. The ALLY-3 [91] trial demonstrated high SVR12 rates with a 12- or 16-week regimen of daclatasvir plus sofosbuvir and ribavirin in patients with chronic HCV genotype 3 infections and advanced fibrosis or compensated cirrhosis. A daclatasvir plus sofosbuvir-based regimen demonstrated efficacy in patients with chronic HCV genotype 1, 3, or 4 infection and advanced cirrhosis or posttransplant recurrence in the ALLY-1 trial [92], and in patients coinfected with HCV genotype 1, 3, or 4 and HIV-1 in the ALLY-2 trial [93].

Daclatasvir plus sofosbuvir with or without ribavirin was generally well tolerated. Fatigue, headache, nausea, and diarrhea were the adverse events reported in some patients treated with daclatasvir [91–93]. Daclatasvir and sofosbuvir combination can potentially cause serious bradycardia when coadministered with amiodarone. Given that daclatasvir is a substrate of CYP3A, it is contraindicated for use with drugs that are strong inducers of CYP3A, including phenytoin, carbamazepine, and rifampin [94].

Data from clinical trials showed resistance-associated substitutions in the *NS5A* gene [95]. Thus, the AASLD/IDSA HCV Guidance Panel recommends testing for these substitutions when NS5A inhibitors fail [96]. Baseline NS5A polymorphisms may also impact the emergence of NS5A resistance [96].

Taken together, daclatasvir plus sofosbuvir with or without ribavirin is an important option for use in treatment-naïve or treatment-experienced patients with chronic HCV genotype 1, 3, or 4 infections, including patients with advanced liver disease, posttransplant recurrence, and HIV-1 coinfection. Daclatasvir with sofosbuvir is a particularly useful ribavirin-free oral option for genotype 3 patients. Testing for the presence of NS5A polymorphisms is recommended at baseline for patients with HCV genotype 1a prior to initiation of treatment with in patients with genotype 1a and cirrhosis prior to sofosbuvir plus daclatasvir treatment.

## *4.2.5.2. Liedipasvir*

Ledipasvir is a potent inhibitor of HCV NS5A, a viral phosphoprotein that plays a critical role in viral replication, assembly, and secretion [97]. The results of clinical trials assessing ledipasvir combinations with SOF have been discussed previously.

Coadministration of amiodarone and ledipasvir-sofosbuvir is not recommended given that severe cases of symptomatic bradycardia have been reported. Ledipasvir-sofosbuvir has significant drug-drug interactions with P-gp inducers such as rifampin that may cause a significant reduction in levels of ledipasvir and sofosbuvir and reduced efficacy of ledipasvir-sofosbuvir [97].

#### *4.2.6. Ombitasvir-paitaprevir-ritonavir-dasabuvir (Viekira Pak)*

The four medications: ombitasvir, paritaprevir, ritonavir, and dasabuvir are combined as a fixed-dose tablet and the dasabuvir is a separate tablet. Ombitasvir is an NS5A inhibitor with potent pangenotypic picomolar antiviral activity, paritaprevir is an inhibitor of the NS3/4A serine protease, and dasabuvir is a nonnucleoside NS5B polymerase inhibitor. Ritonavir is a potent inhibitor of CYP3A4 enzymes and is used as a pharmacologic booster for paritaprevir—it significantly increases peak and trough paritaprevir plasma concentrations, as well as the area under the curve of paritaprevir [98].

PEARL III trial demonstrated the SVR12 rate of 99.5% in treatment-naïve patients with chronic HCV genotype 1b treated with ombitasvir-paritaprevir-ritonavir and dasabuvir plus ribavirin group and 99% in patients who received ombitasvir-paritaprevir-ritonavir and dasabuvir without ribavirin [99]. The TURQUOISE trial assessed the efficacy and safety of ombitasvir, paritaprevir, ritonavir, and dasabuvir plus RBV in HCV/HIV-1 coinfected patients for 12 or 24 weeks. The study enrolled HCV treatment-naïve or PEG-IFN/RBV-experienced patients, with or without Child-Pugh A cirrhosis. Patients with CD4+ count ≥200 cells/mm3 or CD4+ % ≥14%, and plasma HIV-1 RNA suppressed on a stable atazanavir- or raltegravir-inclusive antiretroviral (ART) regimen were included. Among patients treated with 3D+RBV for 12 weeks, 93.5% achieved SVR12. Among patients receiving 24 weeks of treatment, 96.9% achieved EOTR; the most common AEs were fatigue, insomnia, and nausea. Elevation in total bilirubin was the most common laboratory abnormality, occurring predominantly in patients receiving atazanavir.

This combination was effective liver transplant recipients who have recurrent hepatitis C genotype 1 infection [101]. In patients with stage 4 or 5 renal disease and patients on dialysis treated with ombitasvir-paritaprevir-ritonavir and dasabuvir., EOT response was 100% and SVR12 response was achieved in 85% of patients with genotype 1b [102].

## **5. Treatment of different HCV genotypes**

According to the 2016 HCV treatment guidelines of the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) [96] and European Association of Study of Liver Diseases (EASL) [99], chronic HCV due to any genotype can be efficiently treated using all-oral DAA interferon-free regimens.

## **5.1. HCV genotype 1 (Figure 4)**

*4.2.5.2. Liedipasvir*

26 Advances in Treatment of Hepatitis C and B

Ledipasvir is a potent inhibitor of HCV NS5A, a viral phosphoprotein that plays a critical role in viral replication, assembly, and secretion [97]. The results of clinical trials assessing

Coadministration of amiodarone and ledipasvir-sofosbuvir is not recommended given that severe cases of symptomatic bradycardia have been reported. Ledipasvir-sofosbuvir has significant drug-drug interactions with P-gp inducers such as rifampin that may cause a significant reduction in levels of ledipasvir and sofosbuvir and reduced efficacy of ledipasvir-sofosbuvir [97].

The four medications: ombitasvir, paritaprevir, ritonavir, and dasabuvir are combined as a fixed-dose tablet and the dasabuvir is a separate tablet. Ombitasvir is an NS5A inhibitor with potent pangenotypic picomolar antiviral activity, paritaprevir is an inhibitor of the NS3/4A serine protease, and dasabuvir is a nonnucleoside NS5B polymerase inhibitor. Ritonavir is a potent inhibitor of CYP3A4 enzymes and is used as a pharmacologic booster for paritaprevir—it significantly increases peak and trough paritaprevir plasma concentrations, as well as

PEARL III trial demonstrated the SVR12 rate of 99.5% in treatment-naïve patients with chronic HCV genotype 1b treated with ombitasvir-paritaprevir-ritonavir and dasabuvir plus ribavirin group and 99% in patients who received ombitasvir-paritaprevir-ritonavir and dasabuvir without ribavirin [99]. The TURQUOISE trial assessed the efficacy and safety of ombitasvir, paritaprevir, ritonavir, and dasabuvir plus RBV in HCV/HIV-1 coinfected patients for 12 or 24 weeks. The study enrolled HCV treatment-naïve or PEG-IFN/RBV-experienced patients, with or without Child-Pugh A cirrhosis. Patients with CD4+ count ≥200 cells/mm3 or CD4+ % ≥14%, and plasma HIV-1 RNA suppressed on a stable atazanavir- or raltegravir-inclusive antiretroviral (ART) regimen were included. Among patients treated with 3D+RBV for 12 weeks, 93.5% achieved SVR12. Among patients receiving 24 weeks of treatment, 96.9% achieved EOTR; the most common AEs were fatigue, insomnia, and nausea. Elevation in total bilirubin was the most common laboratory abnormality, occurring predominantly in patients receiving atazanavir.

This combination was effective liver transplant recipients who have recurrent hepatitis C genotype 1 infection [101]. In patients with stage 4 or 5 renal disease and patients on dialysis treated with ombitasvir-paritaprevir-ritonavir and dasabuvir., EOT response was 100% and

According to the 2016 HCV treatment guidelines of the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) [96] and European Association of Study of Liver Diseases (EASL) [99], chronic HCV due to any geno-

SVR12 response was achieved in 85% of patients with genotype 1b [102].

type can be efficiently treated using all-oral DAA interferon-free regimens.

**5. Treatment of different HCV genotypes**

ledipasvir combinations with SOF have been discussed previously.

*4.2.6. Ombitasvir-paitaprevir-ritonavir-dasabuvir (Viekira Pak)*

the area under the curve of paritaprevir [98].

Optimizing the regimen of therapy for chronic HCV genotype 1 depends on several factors such as whether the patient is treatment naïve or experienced and the previous therapies provided and the status of resistance in some cases. Given the high cost of IFN-free regimen and difficulties in access to this therapy in various countries, it is necessary to tailor therapy according to the patient population treated and the available therapies.

**Figure 4.** Treatment of HCV genotype 1.

#### **5.2. Ledipasvir-sofosbuvir combination**


• Treatment-experienced, DAA-naïve patients infected with genotype 1a with or without compensated cirrhosis who have NS5A RASs and resistance to ledipasvir (M28A/G/T, Q30E/G/H/K/R, L31M/V, P32L/S, H58D, and/or Y93C/H/N/S) are treated with the fixeddose combination of sofosbuvir and ledipasvir for 12 weeks with ribavirin.

## **5.3. Sofosbuvir-velpatasvir**


## **5.4. Elbasvir-grazoprevir**

Elbasvir-grazoprevir (50 mg/100 mg) therapy in chronic hepatitis C genotypes 1 is tailored according prior treatment experience and the presence of baseline polymorphisms at amino acid positions 28, 30, 31, or 93.


## *5.4.1. Sofosbuvir and daclatasvir*

In genotype 1 infections, sofosbuvir and daclatasvir are used with or without ribavirin depending on the patient population


#### *5.4.2. Ombitasvir-paritaprevir-ritonavir-dasabuvir*

In regions where this combination is available, the therapeutic strategy is recommended as follows:

• Genotype 1a, without cirrhosis: ombitasvir-paritaprevir-ritonavir and dasabuvir plus ribavirin are prescribed for 12 weeks.


Thus, Viekira Pak prescribed with ribavirin except in patients without cirrhosis

## *Genotype 2 (***Figure 5***)*

• Treatment-experienced, DAA-naïve patients infected with genotype 1a with or without compensated cirrhosis who have NS5A RASs and resistance to ledipasvir (M28A/G/T, Q30E/G/H/K/R, L31M/V, P32L/S, H58D, and/or Y93C/H/N/S) are treated with the fixed-

• Genotype 1a, regardless of the presence of cirrhosis: sofosbuvir-velpatasvir is prescribed

• Genotype 1b, regardless of the presence of cirrhosis: sofosbuvir-velpatasvir is prescribed

Elbasvir-grazoprevir (50 mg/100 mg) therapy in chronic hepatitis C genotypes 1 is tailored according prior treatment experience and the presence of baseline polymorphisms at amino

• Genotype 1a, treatment-naïve or peginterferon/ribavirin-experienced with no baseline

• Genotype 1a, treatment-naïve or peginterferon/ribavirin-experienced with baseline NS5A

• Genotype 1b, treatment-naïve or peginterferon/ribavirin-experienced: elbasvir-grazopre-

• Genotype 1a or 1b, peginterferon/ribavirin/protease inhibitor-experienced: elbasvir-grazo-

In genotype 1 infections, sofosbuvir and daclatasvir are used with or without ribavirin

• Genotype 1 with compensated cirrhosis: daclatasvir plus sofosbuvir are given for 12 weeks. • Genotype 1 with decompensated (Child-Pugh B or C) cirrhosis: daclatasvir plus sofosbuvir

In regions where this combination is available, the therapeutic strategy is recommended as

• Genotype 1a, without cirrhosis: ombitasvir-paritaprevir-ritonavir and dasabuvir plus riba-

• Genotype 1, without cirrhosis: daclatasvir plus sofosbuvir are prescribed for 12 weeks.

polymorphisms: elbasvir-grazoprevir plus ribavirin is prescribed for 16 weeks.

dose combination of sofosbuvir and ledipasvir for 12 weeks with ribavirin.

for 12 weeks without ribavirin in treatment naïve or treatment experienced.

for 12 weeks without ribavirin in treatment naïve or treatment experienced.

NS5A polymorphisms: elbasvir-grazoprevir is given for 12 weeks.

**5.3. Sofosbuvir-velpatasvir**

28 Advances in Treatment of Hepatitis C and B

**5.4. Elbasvir-grazoprevir**

acid positions 28, 30, 31, or 93.

vir is given for 12 weeks.

*5.4.1. Sofosbuvir and daclatasvir*

follows:

depending on the patient population

plus ribavirin are given for 12 weeks.

virin are prescribed for 12 weeks.

*5.4.2. Ombitasvir-paritaprevir-ritonavir-dasabuvir*

previr plus ribavirin is given for 12 weeks.

Chronic HCV genotype 2 treatment-naïve or treatment-experienced patients are treated with either with aofosbuvir/velpatasvir for 12 weeks without ribavirin or sofosbuvir and daclatasvir without ribavirin.

**Figure 5.** Treatment of HCV genotype 2.

## *HCV genotype 3 (***Figure 6***)*

Chronic HCV genotype 2 treatment-naïve patients are treated with either sofosbuvir/velpatasvir for 12 weeks without ribavirin or sofosbuvir and daclatasvir without ribavirin. Ribavirin is added for the therapy of treatment-experienced patients.

**Figure 6.** Treatment of HCV genotype 3.

## *HCV genotype 4 (***Figure 7***)*

Treatment-naïve patients with chronic hepatitis C genotype 4 can be treated by one of the following regimen according to availability:


**Figure 7.** Treatment of HCV genotype 4.

*HCV genotype 3 (***Figure 6***)*

30 Advances in Treatment of Hepatitis C and B

*HCV genotype 4 (***Figure 7***)*

**Figure 6.** Treatment of HCV genotype 3.

pensated cirrhosis.

compensated cirrhosis.

lowing regimen according to availability:

added for the therapy of treatment-experienced patients.

Chronic HCV genotype 2 treatment-naïve patients are treated with either sofosbuvir/velpatasvir for 12 weeks without ribavirin or sofosbuvir and daclatasvir without ribavirin. Ribavirin is

Treatment-naïve patients with chronic hepatitis C genotype 4 can be treated by one of the fol-




tions to the use of ribavirin or with poor tolerance to ribavirin.

## *HCV genotype 5 or 6 (***Figure 8***)*

Treatment-naïve patients with or without compensated cirrhosis patients with chronic HCV genotype 5 or 6 are treated with sofosbuvir and ledipasvir for 12 weeks without ribavirin. Treatment-experienced patients with or without compensated cirrhosis are treated with the combination of sofosbuvir and ledipasvir for 12 weeks with daily weight-based ribavirin (1000 or 1200 mg in patients <75 kg or =75 kg, respectively. Treatment-naïve and treatmentexperienced patients with or without compensated cirrhosis are treated with the fixed-dose combination of sofosbuvir and velpatasvir for 12 weeks without ribavirin, and ribavirin is added in patients with treatment-experienced patients.

**Figure 8.** Treatment of HCV genotype 5, 6. *Note*: Ledipasvir: LDV; Sofosbuvir: SOF; Ribavirin: RBV; Simeprevir: SIM, Velpatasvir: VEL; Elbasvir: EBR; Grazoprevir: GZR; Daclatasvir: DAC; Ombitasvir: OBV; Paritaprevir: PTV; Rintonavir: r.

## **6. Patients with HCV and HIV coinfection**

*HCV genotype 5 or 6 (***Figure 8***)*

32 Advances in Treatment of Hepatitis C and B

added in patients with treatment-experienced patients.

Treatment-naïve patients with or without compensated cirrhosis patients with chronic HCV genotype 5 or 6 are treated with sofosbuvir and ledipasvir for 12 weeks without ribavirin. Treatment-experienced patients with or without compensated cirrhosis are treated with the combination of sofosbuvir and ledipasvir for 12 weeks with daily weight-based ribavirin (1000 or 1200 mg in patients <75 kg or =75 kg, respectively. Treatment-naïve and treatmentexperienced patients with or without compensated cirrhosis are treated with the fixed-dose combination of sofosbuvir and velpatasvir for 12 weeks without ribavirin, and ribavirin is

**Figure 8.** Treatment of HCV genotype 5, 6. *Note*: Ledipasvir: LDV; Sofosbuvir: SOF; Ribavirin: RBV; Simeprevir: SIM, Velpatasvir: VEL; Elbasvir: EBR; Grazoprevir: GZR; Daclatasvir: DAC; Ombitasvir: OBV; Paritaprevir: PTV; Rintonavir: r.

Patients with HCV and HIV coinfection are treated according to genotype and prior treatment status as follows [96, 99, 100, 101]:

	- a. Sofosbuvir/ledipasvir for 12 weeks without ribavirin
	- b. Sofosbuvir/velpatasvir for 12 weeks without ribavirin
	- c. Ombitasvir/paritaprevir/ritonavir and dasabuvir for 12 weeks with ribavirin
	- d. Grazoprevir/elbasvir for 12 weeks without ribavirin if HCV RNA=800,000 IU/ml or 16 weeks with ribavirin if HCV RNA >800,000 IU/ml
	- e. Sofosbuvir/daclatasvir for 12 weeks without ribavirin

Genotype 1a, treatment-experienced patients may be treated with any of the following regimen:

	- a. Sofosbuvir/ledipasvir for 12 weeks without ribavirin
	- b. Sofosbuvir/velpatasvir for 12 weeks without ribavirin
	- c. Ombitasvir/paritaprevir/ritonavir and dasabuvir for 12 weeks with ribavirin
	- d. Grazoprevir/elbasvir for 12 weeks without ribavirin
	- e. Sofosbuvir/daclatasvir for 12 weeks without ribavirin
	- a. Sofosbuvir/velpatasvir for 12 weeks without ribavirin
	- b. Sofosbuvir/daclatasvir for 12 weeks without ribavirin
	- a. Sofosbuvir/ledipasvir for 12 weeks without ribavirin
	- b. Sofosbuvir/velpatasvir for 12 weeks without ribavirin
	- c. Ombitasvir/paritaprevir/ritonavir with ribavirin for 12 weeks
	- d. Grazoprevir/elbasvir for 12 weeks without ribavirin
	- e. Sofosbuvir/daclatasvir for 12 weeks without ribavirin
	- f. Sofosbuvir and simeprevir for 12 weeks without ribavirin

Genotype 4 treatment-experienced patients may be treated with any of the following regimen:


Daclatasvir, dose requirement is needed with ritonavir-boosted atazanavir and efavirenz or etravirine. Simeprevir should be used with antiretroviral drugs with which it does not have clinically significant interactions. In addition, it is recommended daily fixed doses of combined sofosbuvir (400 mg)/velpatasvir (100 mg) and of ledipasvir (90 mg)/sofosbuvir (400 mg). For combinations expected to increase tenofovir levels, baseline and ongoing assessment for tenofovir nephrotoxicity is recommended. Regarding HCV/HIV individuals, they should be treated and retreated the same as persons without HIV infection, after recognizing and managing interactions with antiretroviral medications [100, 101].

## **6.1. Treatment of patients with decompensated cirrhosis**

Patients with decompensated cirrhosis and those awaiting liver transplantation are managed according to the HCV genotype. Patients with genotypes 1 and 4 are treated with daily fixeddose combination of ledipasvir (90 mg)/sofosbuvir (400 mg) with low initial dose of ribavirin (600 mg, increased as tolerated) for 12 weeks. Another regimen is a daily fixed-dose combination of sofosbuvir (400 mg)/velpatasvir (100 mg) with weight-based ribavirin for 12 weeks. Finally, daily doses of daclatasvir (60 mg) plus sofosbuvir (400 mg) with low initial dose of ribavirin (600 mg, increased as tolerated) are given for 12 weeks. For patients who are ribavirin ineligible, the recommended regime is a daily fixed dose combination of sofosbuvir (400 mg)/velpatasvir (100 mg) for 24 weeks. Another regime is a combination of ledipasvir (90 mg)/sofosbuvir (400 mg) for 24 weeks. Patients who previously failed sofosbuvir-based treatment are given a combination of ledipasvir (90 mg)/sofosbuvir (400 mg) with low initial dose of ribavirin (600 mg, increased as tolerated) for 24 weeks [96, 99, 102**]**. Patients with HCV genotype 2 or 3 infection and decompensated cirrhosis are treated with daily fixed-dose combination sofosbuvir (400 mg)/velpatasvir (100 mg) with weight-based ribavirin for 12 weeks [96, 99, 102].

## **6.2. Patients with HCV recurrence after liver transplantation**

a. Sofosbuvir/velpatasvir for 12 weeks with ribavirin or 24 weeks without ribavirin

(5) Genotype 4 treatment-naïve patients may be treated with any of the following regimen:

Genotype 4 treatment-experienced patients may be treated with any of the following

j. Grazoprevir/elbasvir for 12 weeks without ribavirin if HCV RNA =800,000 or 16

l. Sofosbuvir and simeprevir for 12 weeks with ribavirin or 24 weeks without ribavirin

k. Sofosbuvir/daclatasvir for 12 weeks with ribavirin or 24 weeks without ribavirin

Daclatasvir, dose requirement is needed with ritonavir-boosted atazanavir and efavirenz or etravirine. Simeprevir should be used with antiretroviral drugs with which it does not have clinically significant interactions. In addition, it is recommended daily fixed doses of combined sofosbuvir (400 mg)/velpatasvir (100 mg) and of ledipasvir (90 mg)/sofosbuvir (400 mg). For combinations expected to increase tenofovir levels, baseline and ongoing assessment for tenofovir nephrotoxicity is recommended. Regarding HCV/HIV individuals, they should be treated and retreated the same as persons without HIV infection, after recognizing and

Patients with decompensated cirrhosis and those awaiting liver transplantation are managed according to the HCV genotype. Patients with genotypes 1 and 4 are treated with daily fixeddose combination of ledipasvir (90 mg)/sofosbuvir (400 mg) with low initial dose of ribavirin (600 mg, increased as tolerated) for 12 weeks. Another regimen is a daily fixed-dose combination of sofosbuvir (400 mg)/velpatasvir (100 mg) with weight-based ribavirin for 12 weeks. Finally, daily doses of daclatasvir (60 mg) plus sofosbuvir (400 mg) with low initial dose of ribavirin (600 mg, increased as tolerated) are given for 12 weeks. For patients who are ribavirin ineligible, the

g. Sofosbuvir/ledipasvir for 12 weeks with ribavirin and 24 weeks with ribavirin

b. Sofosbuvir/daclatasvir for 12 weeks with ribavirin

a. Sofosbuvir/ledipasvir for 12 weeks without ribavirin b. Sofosbuvir/velpatasvir for 12 weeks without ribavirin

d. Grazoprevir/elbasvir for 12 weeks without ribavirin e. Sofosbuvir/daclatasvir for 12 weeks without ribavirin

regimen:

34 Advances in Treatment of Hepatitis C and B

c. Ombitasvir/paritaprevir/ritonavir with ribavirin for 12 weeks

f. Sofosbuvir and simeprevir for 12 weeks without ribavirin

h. Sofosbuvir/velpatasvir for 12 weeks without ribavirin

weeks with ribavirin if HCV RNA >800,000 IU/ml

managing interactions with antiretroviral medications [100, 101].

**6.1. Treatment of patients with decompensated cirrhosis**

i. Ombitasvir/paritaprevir/ritonavir with ribavirin for 12 weeks

Patients who develop HCV after transplantation and with compensated cirrhosis are treated with daily fixed-dose combination of ledipasvir (90 mg)/sofosbuvir (400 mg) with weightbased ribavirin for 12 weeks. Treatment-naïve patients with HCV genotype 1 or 4 infection in the allograft and with compensated liver disease and who are ribavirin ineligible can be treated by a daily fixed-dose combination of ledipasvir (90 mg)/sofosbuvir (400 mg) for 24 weeks [96, 99, 103]. Patients with HCV genotype 1 infection in the allograft, including those with compensated cirrhosis can receive daily simeprevir (150 mg) plus sofosbuvir (400 mg) with or without weight-based ribavirin for 12 weeks. For those with early stage fibrosis, the recommended regimen is daily fixed-dose combination of paritaprevir (150 mg)/ritonavir (100 mg)/ombitasvir (25 mg) plus twice-daily dosed dasabuvir (250 mg) with weight-based ribavirin for 24 weeks. Treatment-naïve and -experienced patients with HCV genotype 2 infection in the allograft, including those with compensated cirrhosis, are treated with daclatasvir (60 mg) plus sofosbuvir (400 mg), with low initial dose of ribavirin (600 mg, increased as tolerated) for 12 weeks [92, 96, 99].

## **7. Patients with HCV and renal impairment**

In patients with mild to moderate renal impairment, no dosage adjustment is required when using daclatasvir (60mg), fixed-dose combination of ledipasvir (90 mg)/sofosbuvir (400 mg), fixed-dose combination of sofosbuvir (400mg)/velpatasvir (100mg), or fixed-dose combination of paritaprevir (150 mg)/ritonavir (100 mg)/ombitasvir (25 mg) with (or without for HCV genotype 4 infection) twice-daily dosed dasabuvir (250 mg), simeprevir (150 mg), or sofosbuvir (400 mg) to treat or retreat HCV infection in patients with appropriate genotypes [96, 99, 104**]**.

For patients with severe renal impairment or end stage renal disease and patients with genotype 1a, or 1b, or 4 infection and CrCl below 30 ml/min, for whom treatment has been elected before kidney transplantation, the recommended daily fixed-dose combination of elbasvir (50 mg)/grazoprevir (100mg) for 12 weeks. Genotype 1b infection patients and CrCl below 30 ml/min for whom the urgency to treat is high and treatment has been elected before kidney transplantation, daily fixed-dose combination of paritaprevir (150 mg)/ritonavir (100 mg)/ ombitasvir (25 mg) plus twice-daily dosed dasabuvir (250 mg) for 12 weeks. For patients with HCV genotype 2, 3, 5, or 6 infection and CrCl below 30 ml/min for whom the urgency to treat is high and treatment has been elected before kidney transplantation, PEG-IFN and doseadjusted ribavirin (200 mg daily) [104–107].

## **8. Retreatment of patients who failed prior therapy [57, 96, 99, 108, 109]**

Patients who failed PEG-IFN-α, ribavirin, and DAA or all DAA regimens are retreated according to the previous therapies and genotype as follows:

	- Genotypes 1, 4, 5, or 6 can be treated with sofosbuvir and ledipasvir
	- All genotypes can be treated with sofosbuvir and velpatasvir
	- Genotype 1 may be treated with ritonavir-boosted paritaprevir, ombitasvir, and dasabuvir
	- Genotype 4 is treated with ritonavir boosted, paritaprevir and ombitasvir or sofosbuvir plus simeprevir
	- Genotypes 1 or 4 are treated with grazoprevir and elbasvir for 24 weeks in F0-F2 patients with HCV RNA >800,000 IU/ml)
	- All genotypes may be treated with sofosbuvir plus daclatasvir

## **9. Treatment of HCV and HBV coinfection**

The goal of therapy in HBV and HCV coinfection is to eradicate HCV infection and inhibit HBV replication. Evaluation of liver disease progression, predominance of one virus over another, and comorbidities are essential for optimal antiviral regimens. For patients with active hepatitis C, the same regimens following the same rules as for monoinfected patients should be applied based on AASLD and EASL recommendations [96, 99]. For patients with active hepatitis B before, during or after HCV clearance or with established cirrhosis, nucleoside or nucleotide analog (NA), tenofovir or entecavir is indicated [110, 111]. Concurrent HBV nucleoside/nucleotide analog therapy is indicated either if there is a potential risk of HBV reactivation during or after HCV clearance or if HBV replication is detectable at a significant level before initiation of HCV treatment [112].

Patients should be carefully investigated for the replicative status of both HBV and HCV, and hepatitis delta virus infection prior selecting the treatment strategy. When HCV is replicating and causes liver disease, it should be treated following the same rules as applied to HCV monoinfected patients. There is a potential risk of HBV reactivation during or after HCV clearance. Prior initiating DAA-based treatment for hepatitis C, patients should be tested for HBs antigen, anti-HBc antibodies and anti-HBs antibodies. If HBs antigen is present or if HBV DNA is detectable in HBs antigen-negative, anti-HBc antibody-positive patients ("occult" hepatitis B), concurrent HBV nucleoside/nucleotide analog therapy is indicated [96, 99].

## **9.1. DAA resistance**

**8. Retreatment of patients who failed prior therapy [57, 96, 99, 108, 109]**

ing to the previous therapies and genotype as follows:

vir, with ribavirin for 12 weeks.

36 Advances in Treatment of Hepatitis C and B

dasabuvir

plus simeprevir

with HCV RNA >800,000 IU/ml)

**9. Treatment of HCV and HBV coinfection**

level before initiation of HCV treatment [112].

Patients who failed PEG-IFN-α, ribavirin, and DAA or all DAA regimens are retreated accord-



○ Genotype 1 may be treated with ritonavir-boosted paritaprevir, ombitasvir, and

○ Genotype 4 is treated with ritonavir boosted, paritaprevir and ombitasvir or sofosbuvir

○ Genotypes 1 or 4 are treated with grazoprevir and elbasvir for 24 weeks in F0-F2 patients

The goal of therapy in HBV and HCV coinfection is to eradicate HCV infection and inhibit HBV replication. Evaluation of liver disease progression, predominance of one virus over another, and comorbidities are essential for optimal antiviral regimens. For patients with active hepatitis C, the same regimens following the same rules as for monoinfected patients should be applied based on AASLD and EASL recommendations [96, 99]. For patients with active hepatitis B before, during or after HCV clearance or with established cirrhosis, nucleoside or nucleotide analog (NA), tenofovir or entecavir is indicated [110, 111]. Concurrent HBV nucleoside/nucleotide analog therapy is indicated either if there is a potential risk of HBV reactivation during or after HCV clearance or if HBV replication is detectable at a significant

Patients should be carefully investigated for the replicative status of both HBV and HCV, and hepatitis delta virus infection prior selecting the treatment strategy. When HCV is replicating and causes liver disease, it should be treated following the same rules as applied to HCV monoinfected patients. There is a potential risk of HBV reactivation during or after HCV clearance. Prior initiating DAA-based treatment for hepatitis C, patients should be tested

gylated IFN- α and ribavirin can be retreated with any of the following:

○ Genotypes 1, 4, 5, or 6 can be treated with sofosbuvir and ledipasvir

○ All genotypes can be treated with sofosbuvir and velpatasvir

○ All genotypes may be treated with sofosbuvir plus daclatasvir

Despite the great efficacy of the interferon-free DAA regimen, real-life experience revealed that approximately 5–10% of patients end up with virologic failure. Treatment failure raised the issue of resistance and occurrence of mutations. To date, the impact of such mutations on the treatment outcome is not clarified. It is not clear if the presence of mutations at baseline may independently lead to relapse [113]. HCV resistance-associated variants (RAVS) remain a challenging issue in HCV therapy. The prevalence of NS5A RAVs at baseline was shown to vary considerably across genotypes 1a, 1b, 3 and 4. Some studies showed that virologic failure tended to be more frequent when an NS5A Y93H substitution was present at baseline. Resistance-associated substitutions (RASs) have been reported both in treatment-naïve patients and following treatment with protease (NS3), phosphoprotein (NS5A) and polymerase (NS5B) inhibitors [113].

The different next-generation sequencing (NGS) technologies for (HCV) are critical for identification of both viral genotype and resistance genetic motifs in the era of DAA therapies. A study [114] compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. The study showed that the consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance [114].

In conclusion, discovery of short duration, safe and highly effective regimens has opened up new horizons for HCV cure. However, real-life experience demonstrated some challenges such as emergence of mutations and management of special patient populations. Despite the optimism for the near future and the excellent efficacy, the prohibitive cost of such regimen is a great obstacle that interferes with accessibility of patients in countries with high HCV prevalence to the new IFN-free regimens. Thus, more efforts should be made to make IFNfree cost-effective in all clinical scenarios and accessible to all patients.

## **Abbreviations**



## **Author details**

Sanaa M. Kamal

Address all correspondence to: sanaakamal@ainshamsmedicine.net

Department of Gastroenterology and Hepatology, Ain Shams Faculty of Medicine, Cairo, Egypt

## **References**

PEG IFN peginterferon

DAA direct-acting antiviral

NS5A nonstructural protein 5A

PI protease inhibitor

NNI nonnucleotide polymerase inhibitor

Nuc nucleotide polymerase inhibitor

RAV resistance-associated variant

RGT response-guided therapy

EBR-GZR elbasvir-grazoprevir

RdRp RNA-dependent RNA polymerase

EOTR ombitasvir/paritaprevir/ritonavir + dasabuvir

Address all correspondence to: sanaakamal@ainshamsmedicine.net

Department of Gastroenterology and Hepatology, Ain Shams Faculty of Medicine, Cairo,

RBV ribavirin

38 Advances in Treatment of Hepatitis C and B

GT genotype

TPV telaprevir BOC boceprevir

SIM simeprevir DNV danoprevir

SOF sofosbuvir VEL velpatasvir

GT genotype

OMV, RTV ritonavir

**Author details**

Sanaa M. Kamal

Egypt

ART antiretroviral

HCV hepatitis C virus


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**Provisional chapter**

#### **Comparative Study of IFN-Based Versus IFN-Free Regimens and Their Efficacy in Treatment of Chronic Hepatitis C Infections Comparative Study of IFN-Based Versus IFN-Free Regimens and Their Efficacy in Treatment of Chronic Provisional chapter Comparative Study of IFN-Based Versus IFN-Free Regimens and Their Efficacy in Treatment of Chronic**

Ramesh Rana, Yizhong Chang, Jing Li, Ramesh Rana, Yizhong Chang, Jing Li,

**Hepatitis C Infections**

**Hepatitis C Infections**

ShengLan Wang, Li Yang and ChangQing Yang Ramesh Rana, Yizhong Chang, Jing Li, ShengLan Wang, Li Yang and ChangQing Yang ShengLan Wang, Li Yang and ChangQing Yang

Additional information is available at the end of the chapter Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67214

#### **Abstract**

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Interferon-A2b and Ribavirin. Scand J Gastroenterol. 2009; 44: 1487–1490.

feron-Free Regimens. Gastroenterology 2016; 151(1): 70–86.

48 Advances in Treatment of Hepatitis C and B

The hepatitis C viral (HCV) infection is a global health burden, WHO estimates 130–150 million people chronically infected with hepatitis C virus worldwide. Additional 3–4 million people become newly infected annually and more than 350,000 people die each year of HCV-related liver diseases. HCV infection exhibits higher genetic diversity with regional variations in genotypic prevalence resulting big challenges on disease management. Introduction of DAAs revolutionised the new era of all oral therapy in treatment of chronic hepatitis C infection and is the regimens of choice in present days. However, IFN-based combination therapy with sofosbuvir has promising efficacy in genotypes 3, 4, 5 or 6 infections compared to genotypes 1 and 2 infections. So, these regimens could be an option in DAAs regimen failure cases. The poor availability of data on recent DAAs (IFN-free) regimens questioned on regular use and cost effectiveness is the another challenge with DAAs regimens. So phase III trials (sofosbuvir and velpatasvir) of recent DAAs with pangenotypic actions and better tolerability in HCV infected patients are the future advances in treatment of chronic hepatitis C. After all those recent combination therapies with better SVR, the combination of pegylated interferon with ribavirin is the only option available where unavailability of other regimens still exists.

**Keywords:** hepatitis C virus, HCV genotypes, pegylated interferon, direct-acting antivirals, sustained virological response

and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

## **1. Introduction**

Hepatitis C virus (HCV) is a global public health problem causing progressive liver disease. The World Health Organization (WHO) estimates 130–150 million people chronically infected worldwide, which corresponds to 2–2.5% of world's total population. Additional 3–4 million people becoming newly infected annually and more than 350,000 people die each year due to HCV-related diseases. Primary HCV infection causes acute hepatitis (AHC), asymptomatic in majorities; however, it can progress to chronicity in about 55–85% cases and spontaneous remission within 6 months without treatment in 15–45% [1–3]. Chronic hepatitis C (CHC) frequently presents with complications such as liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC). In CHC, 15–30% have risk of cirrhosis of liver within 20 years and risk of HCC in cirrhotic is approximately 2–4% per year. Decompensated cirrhosis leads to death in 50–70% of cases without liver transplantation after 5 years. Difficulties occur in determining number of new HCV infections, as most of the acute cases are not detected clinically. Less than 25% of acute cases of hepatitis C are only clinically apparent [1, 4–6].

Hepatitis C virus (HCV) is an envelope, single-stranded RNA virus of genus hepacivirus within the Flaviviridae family. HCV has seven genotypes (GT 1–7) with 67 subtypes and 20 provisional subtypes [7]. Each genotype of HCV has its own geographical variation. GT-1 is the most prevalent worldwide, one third in East Asia followed by GT-3; GT-2, 4, and 6; and GT-5 is the least prevalent [8]. The prevalence of HCV GT-1 and 3 dominate in most of the countries irrespective of economic status while HCV GT-4 and 5 are prevalent largely in countries with lower income [7, 8]. HCV subtypes 1a and 1b are the most common genotypes in the United States and also in Europe while subtype 1b is predominant in Japan [9]. HCV subtypes 2a and 2b are relatively common in North America, Europe, and Japan, subtype 2c is common in Northern Italy. Although, GT-3 has endemic strain in South Asia, 3a is especially prevalent in intravenous drug abusers in Europe and in the United States. GT-4 is prevalent in North Africa and Middle East; GT-5 seems to be confined to South Africa and GT-6 in Southeast Asia. A newly identified GT-7 isolated from a Central African (Congolese) immigrant in Canada [9]. The increased risk of HCV is highest among persons who inject drugs (PWID), global prevalence of HCV among PWID is 67%; HIV infected person, men who have sex with men (MSM); unsafe medical proceduresrecipients of infected blood products or invasive procedures in health care facilities with inadequate infection control practice. Vertical or perinatal transmission of HCV occurs in up to 4–8% of cases, and transmission risk among mothers of HIV infection is estimated 17–25% [9–11].

The HCV infection is the public health problem and global burden, and the early diagnosis and treatment are necessary. The treatment of HCV infection was begun with the approval of interferon (IFN) by the Food and Drug Administration (FDA) in 1991, followed by combined IFN with ribavirin (RBV) in 1998 and then directly acting antiviral agents (**Table 1**). Until approval of directly acting antiviral agents, combination of pegylated interferon alfa (PegIFN


PEG, pegylated interferon by injection; RBV, ribavirin (pills), HCV inhibitors are pills; SVR, sustained virological response, SVR 12, 24-viral cure.

Reference: http://hcvadvocate.org/treatment/drug-pipeline/#Quick

**1. Introduction**

50 Advances in Treatment of Hepatitis C and B

only clinically apparent [1, 4–6].

17–25% [9–11].

Hepatitis C virus (HCV) is a global public health problem causing progressive liver disease. The World Health Organization (WHO) estimates 130–150 million people chronically infected worldwide, which corresponds to 2–2.5% of world's total population. Additional 3–4 million people becoming newly infected annually and more than 350,000 people die each year due to HCV-related diseases. Primary HCV infection causes acute hepatitis (AHC), asymptomatic in majorities; however, it can progress to chronicity in about 55–85% cases and spontaneous remission within 6 months without treatment in 15–45% [1–3]. Chronic hepatitis C (CHC) frequently presents with complications such as liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC). In CHC, 15–30% have risk of cirrhosis of liver within 20 years and risk of HCC in cirrhotic is approximately 2–4% per year. Decompensated cirrhosis leads to death in 50–70% of cases without liver transplantation after 5 years. Difficulties occur in determining number of new HCV infections, as most of the acute cases are not detected clinically. Less than 25% of acute cases of hepatitis C are

Hepatitis C virus (HCV) is an envelope, single-stranded RNA virus of genus hepacivirus within the Flaviviridae family. HCV has seven genotypes (GT 1–7) with 67 subtypes and 20 provisional subtypes [7]. Each genotype of HCV has its own geographical variation. GT-1 is the most prevalent worldwide, one third in East Asia followed by GT-3; GT-2, 4, and 6; and GT-5 is the least prevalent [8]. The prevalence of HCV GT-1 and 3 dominate in most of the countries irrespective of economic status while HCV GT-4 and 5 are prevalent largely in countries with lower income [7, 8]. HCV subtypes 1a and 1b are the most common genotypes in the United States and also in Europe while subtype 1b is predominant in Japan [9]. HCV subtypes 2a and 2b are relatively common in North America, Europe, and Japan, subtype 2c is common in Northern Italy. Although, GT-3 has endemic strain in South Asia, 3a is especially prevalent in intravenous drug abusers in Europe and in the United States. GT-4 is prevalent in North Africa and Middle East; GT-5 seems to be confined to South Africa and GT-6 in Southeast Asia. A newly identified GT-7 isolated from a Central African (Congolese) immigrant in Canada [9]. The increased risk of HCV is highest among persons who inject drugs (PWID), global prevalence of HCV among PWID is 67%; HIV infected person, men who have sex with men (MSM); unsafe medical proceduresrecipients of infected blood products or invasive procedures in health care facilities with inadequate infection control practice. Vertical or perinatal transmission of HCV occurs in up to 4–8% of cases, and transmission risk among mothers of HIV infection is estimated

The HCV infection is the public health problem and global burden, and the early diagnosis and treatment are necessary. The treatment of HCV infection was begun with the approval of interferon (IFN) by the Food and Drug Administration (FDA) in 1991, followed by combined IFN with ribavirin (RBV) in 1998 and then directly acting antiviral agents (**Table 1**). Until approval of directly acting antiviral agents, combination of pegylated interferon alfa (PegIFN **Table 1.** FDA approved medications for treatment of Hepatitis C infections.

alfa) and ribavirin (RBV) was the standard treatment for all genotypic infections (**Figure 1**) [4, 12]. Over the past few years, the treatment options of HCV have exponentially grown. The development of directly acting antiviral (DAA) therapy, targeting non-structural proteins involved in replication of HCV revolutionised in the treatment of HCV infection. The combination of DAAs with or without PegIFN alfa regimens is assessed in different studies, and their efficacies in treatment of different HCV genotypes are evaluated individually. Recently, the combination of IFN-free DAAs regimens with or without ribavirin is evaluated as "All oral regimens" for treatment of HCV infection in different genotypes with better efficacy and tolerability [13]. The current treatment strategies for HCV are based on HCV genotyping; and HCV RNA load determination before, during, and after antiviral therapy; then selection of agents that are active against the isolated specific HCV genotype [4, 12, 14]. The aim of this review is to compare the efficacy of IFN-based and IFN-free regimens (DAAs combination therapy) on the basis of sustained virological response (SVR) rates in HCV genotypic infections.

**Figure 1.** Combination of PegIFN-alfa and ribavirin for the treatment of HCV infections according to genotypes [4]. HCV, hepatitis C virus; RNA, ribonucleic acid; RVR, rapid virological response; DAAs, directly acting antivirals; cEVR, complete early virological response.

## **2. Treatment**

The primary goal of HCV treatment is to cure the infection. The obtaining sustained virological response (SVR) is defined as undetectable HCV RNA in 12 weeks (SVR 12) or 24 weeks (SVR 24) after treatment completion. Cure rate, which achieves SVR, is more than 99%. SVR is generally associated with resolution of liver disease in patient without cirrhosis, but the patient with cirrhosis remains risk of life-threatening complications. However, the hepatic fibrosis may regress, and risk of complications like hepatic failure and portal hypertension is reduced. The risk of HCC and all causes of mortality are significantly reduced, nevertheless, not eliminated in cirrhotic patients who clear HCV compared to untreated patients and non-sustained virological responders [4, 15–17]. The endpoint of therapy is an SVR after therapy as assessed by sensitive molecular method with the lower limit of HCV RNA detection ≤15 International Units/ml (IU/ml) [4, 12, 14].

## **2.1. Efficacy of IFN-based versus IFN-free regimens for treatment of HCV genotype 1 infections**

their efficacies in treatment of different HCV genotypes are evaluated individually. Recently, the combination of IFN-free DAAs regimens with or without ribavirin is evaluated as "All oral regimens" for treatment of HCV infection in different genotypes with better efficacy and tolerability [13]. The current treatment strategies for HCV are based on HCV genotyping; and HCV RNA load determination before, during, and after antiviral therapy; then selection of agents that are active against the isolated specific HCV genotype [4, 12, 14]. The aim of this review is to compare the efficacy of IFN-based and IFN-free regimens (DAAs combination therapy) on the basis of sustained virological response (SVR) rates in HCV genotypic

The primary goal of HCV treatment is to cure the infection. The obtaining sustained virological response (SVR) is defined as undetectable HCV RNA in 12 weeks (SVR 12) or 24 weeks (SVR 24) after treatment completion. Cure rate, which achieves SVR, is more than 99%. SVR is generally associated with resolution of liver disease in patient without cirrhosis, but the patient with cirrhosis remains risk of life-threatening complications. However, the hepatic fibrosis may regress, and risk of complications like hepatic failure and portal hypertension is reduced. The risk of HCC and all causes of mortality are significantly reduced, nevertheless, not eliminated in cirrhotic patients who clear HCV compared to untreated patients and non-sustained

**Figure 1.** Combination of PegIFN-alfa and ribavirin for the treatment of HCV infections according to genotypes [4]. HCV, hepatitis C virus; RNA, ribonucleic acid; RVR, rapid virological response; DAAs, directly acting antivirals; cEVR,

infections.

52 Advances in Treatment of Hepatitis C and B

**2. Treatment**

complete early virological response.

HCV genotype 1 infection is the most prevalent genotype among all genotypes [8]. So, the drug trials are also largely assessed on this genotype. Previously, the combination of PegIFN alfa with ribavirin was widely used. However, after the introduction of directly acting antiviral (DAA) agents, either they were used in combination with PegIFN and ribavirin or they were used in combination with themselves as two DAAs or four DAAs regimen. The efficacy and tolerability were superior to the previously standard regimen (PegIFN alfa and ribavirin) and also duration was significantly reduced from 24–72 to 12–24 weeks. The IFN-free regimens were better preferred due to higher efficacy rate and fewer adverse effects compared to combination of PegIFN regimens. However, we cannot exclude the fact that PegIFN and ribavirin remain the ultimate option in setting where no other options are available [4, 12, 14]. The different regimens and their efficacy for treatment of genotype 1 infection are given in **Table 2**.



PegIFN alfa-pegylated interferon-alfa; RBV-ribavirin; T12PR-telaprevir, pegylated interferon-alfa and ribavirin for 12 weeks; T12PR24-telaprevir, pegylated interferon-alfa and ribavirin for 12 weeks, then pegylated interferon-alfa and ribavirin for remaining 12 weeks (total 24 weeks); T12PR48-telaprevir, pegylated interferon-alfa and ribavirin for 12 weeks then pegylated interferon alfa and ribavirin for remaining 36 weeks (total 48 weeks); NCN, non-cirrhotic naïve; C, cirrhotic; CC, compensated cirrhosis; NB, non-black patients; B, Black patients; PWID, people who inject drugs; SVR 4/8/12/24/48, sustained virological response at 4 weeks, 8 weeks, 12 weeks, 24 weeks or 48 weeks; (+) RBV, with ribavirin; (−)RBV, without ribavirin; (±) RBV, with or without ribavirin

**Table 2.** Efficacy of IFN-based vs. IFN-free regimens for treatment of HCV genotype 1 infections.

#### *2.1.1. Pegylated interferon alpha and ribavirin*

The combination of pegylated interferon alpha and ribavirin was a standard regimen previously in treatment of hepatitis C genotype 1 infection. The main drawback with this regimen was longer duration of treatment course, that is, 24–72 weeks. With this regimen, HCV genotype 1 infected patient had SVR rates of approximately 40% in North America and 50% in Western Europe [18]. The SVR rate was comparatively lower in genotype 1 than other genotypes. The previous studies showed SVR of 42–46% infected with genotype 1, treated for 24 or 48 weeks [18, 19]. The HIV co-infected patients had SVR of 40% with this regimen [20]. This regimen is contraindicated in patients with uncontrolled depression, psychosis, or epilepsy, pregnant women or couples unwilling to comply with adequate contraception, severe concurrent medical diseases and co-morbidities including retinal disease, autoimmune thyroid disease, and decompensated liver disease. In patient with hepatitis B co-infection, this regimen is used as monoinfected patients, although there is a potential risk of hepatitis B infection reactivation during HCV clearance [12].

## *2.1.2. Boceprevir in combination with pegylated interferon alfa and ribavirin*

Boceprevir is a first generation NS3/4A protease inhibitors (PIs) approved by FDA in 2011. Introduction of PIs constituted a milestone in treating CHC infection, achieved SVR rates of up to 75% in naïve and 29–88% in treatment-experienced patients with GT-1 infection [21, 22]. However, low genetic barrier to resistance is the main limitation. Introduction of newer DAAs replaced the choice of this regimen. The phase 1 and 2 double blind studies carried out for untreated HCV genotype 1 infection in non-black and black populations who were treated for 24–44 weeks showed SVR of 67–68 and 42–53%, respectively [23]. Another study of 403 patients previously treated with PegIFN alfa/RBV regimen, the triple therapy with boceprevir for 32–44 weeks showed SVR of 59–66%. Among patients with an undetectable HCV RNA level at week 8, SVR was 86 and 88% after 32 and 44 weeks of triple therapy, respectively [24]. A study done in 179 cases who inject drugs (PWID) versus non-PWID with this regimen showed SVR of 71 and 72%, respectively. Among them, 53% were advanced stage (F3–4) and 44% were on antiviral therapy [25]. The main side effect of this regimen was anaemia 21–46% for which erythropoietin has to be used or treatment had to discontinue 1–2% [24].

## *2.1.3. Telaprevir in combination with pegylated interferon alfa and ribavirin*

Telaprevir is a first generation NS3/4A protease inhibitors (PIs) approved by FDA in 2011. Telaprevir, a protease inhibitor specific to the HCV non-structural 3/4A serine protease, rapidly reduced HCV RNA levels in early studies. A study with this regimen grouped into Telaprevir/PegIFN alfa/RBV (TPR) 12 weeks; T12PR24; and T12PR48 showed sustained virological response of 35, 61 and 67%, respectively [26]. In phase 3 trial with triple therapy in previously untreated genotype 1 infected cases showed T12PR and T8PR SVR of 75 and 69%, respectively [27]. Previously in non-responders and partial responders, the SVR of 44 and 70%, respectively, was achieved [28]. A study done in 179 cases who inject drugs (PWID) versus non-PWID with this regimen showed SVR of 71 and 72%, respectively. Among them, 53% were advanced in stage F3–4 and 44% were on antiviral therapy [25]. The main adverse effect 10–21% with telaprevir was anaemia, gastrointestinal side effect, and skin rash. Rash was the most common reason for discontinuation of therapy [26, 27].

## *2.1.4. Simeprevir in combination with pegylated interferon alfa and ribavirin*

*2.1.1. Pegylated interferon alpha and ribavirin*

(−)RBV, without ribavirin; (±) RBV, with or without ribavirin

**Treatment regimens**

Sofosbuvir+ simeprevir

Sofosbuvir + ledipasvir

Sofosbuvir+ daclatasvir

Sofosbuvir + velpatasvir

Ritonavir-boosted paritaprevir, ombitasvir, dasabuvir **±** RBV

**Naïve (SVR) Treatment-**

**SVR12:** 91% (+RBV) 95% (−RBV) **SVR12**: 88% (NC) 75% (C)

54 Advances in Treatment of Hepatitis C and B

**SVR8 (NC)**: 94% (−RBV) 93% (+RBV) 95%(+RBV\* 12wks)

**SVR12**: 99% (−RBV) 97% (+RBV) **SVR24**: 98% (−RBV) 99% (+RBV)

**NC:** 100% (±RBV) **Cirrhotic**: SVR12: 84.9% SVR24: 93.4%

**SVR12-1a (NC)**: 95–97% (+RBV) 90% (−RBV) 91% (+HIV) **SVR12-1b (NC)**: 98–100% (−RBV) 97–100% (+RBV) **SVR12 (C)**: 92% (1a) 99% (1b)

– **SVR12 (overall)**:

98% (1a) 99% (1b)

**Table 2.** Efficacy of IFN-based vs. IFN-free regimens for treatment of HCV genotype 1 infections.

**SVR12 (NC)**: 96% (1a) 97% (1b) CC: SVR12: 92% SVR24: 96%

**experienced (SVR)**

SVR12: 87% (NC) 76% (C)

**SVR12 (overall)**: 94% (−RBV) 96% (+RBV) **SVR24**: 99% (−RBV) 99% (+RBV)

**Partial responders**

– – **NC**:

– Non-responders 91%

– – –

100% (−RBV) 95% (+RBV)

– – –

NC: 100% NC: 95% NC: 95%

**Null responders Relapsers**

–

–

The combination of pegylated interferon alpha and ribavirin was a standard regimen previously in treatment of hepatitis C genotype 1 infection. The main drawback with this regimen was longer duration of treatment course, that is, 24–72 weeks. With this regimen, HCV genotype 1 infected patient had SVR rates of approximately 40% in North America and 50% in Western Europe [18]. The SVR rate was comparatively lower in genotype 1 than other genotypes. The

PegIFN alfa-pegylated interferon-alfa; RBV-ribavirin; T12PR-telaprevir, pegylated interferon-alfa and ribavirin for 12 weeks; T12PR24-telaprevir, pegylated interferon-alfa and ribavirin for 12 weeks, then pegylated interferon-alfa and ribavirin for remaining 12 weeks (total 24 weeks); T12PR48-telaprevir, pegylated interferon-alfa and ribavirin for 12 weeks then pegylated interferon alfa and ribavirin for remaining 36 weeks (total 48 weeks); NCN, non-cirrhotic naïve; C, cirrhotic; CC, compensated cirrhosis; NB, non-black patients; B, Black patients; PWID, people who inject drugs; SVR 4/8/12/24/48, sustained virological response at 4 weeks, 8 weeks, 12 weeks, 24 weeks or 48 weeks; (+) RBV, with ribavirin;

> Simeprevir (TMC435) is an oral HCV NS3/4A protease inhibitor used in combination with PegIFN alfa and ribavirin to treat HCV genotype 1 infected patients. This combination is

generally well tolerated with potent antiviral activity and pharmacokinetic profile. In ASPIRE phase IIb trial done in previously treated patients with PegIFN and ribavirin, the SVR at 24 weeks was 38–59% (1a-42 and 1b-58%) in prior null responders, 48–86% (1a-56 and 1b-88%) in prior partial responders, and 77–89% (no difference) in prior relapsed cases. There were same SVR rates in patient with or without Q80k polymorphism at baseline 60.9%. In patients with cirrhosis (METAVIR score F4), combination therapy with 150 mg of simeprevir had SVR rate at 24 weeks was 73% in prior relapsers, 82% in prior partial responders, and 31% in prior null responders [29]. Another phase 3 trial on partials and null responders showed SVR of 70 and 44%, respectively [28]. According to QUEST 1 & 2 phase 3 study, overall SVR 12 in previously untreated and treated naïve patients was 81 (209/257) and 80% (1a-71 and 1b-90%). On subtype analysis, SVR rates on with or without Q80K polymorphism at baseline in 1a were 52–75 and 80–85%, respectively, and 82% in 1b. The SVR rates were comparatively higher in F0–2 83–85% than F3–4 66–70% [30, 31]. In patients who relapsed on previous therapy, the SVR 12 was 79.2%. Among them, 92.7% were enabled to shorten therapy with PR at 24 weeks [32]. The cause of treatment failure with this regimen was viral breakthrough in 10.6–13%. The main side effects were fatigue, headache, pruritus, and influenza like illness and anaemia. Skin rash and photosensitivity were also very common with simeprevir [29–32].

## *2.1.5. Sofosbuvir in combination with pegylated interferon alfa and ribavirin*

Sofosbuvir is a nucleotide analogue HCV NS5B polymerase inhibitor with similar *in vitro* activity against pan-HCV genotypes. This therapy is used for HCV pan-genotype infections (1–6 genotypes) treatment-naïve patients with or without cirrhosis but no evidence on treatment-experienced patients. In the NEUTRINO phase III trial in treatment-naïve patients, the overall SVR rate was 89% (259/291), 92% (207/225) for subtype 1a and 82% (54/66) for subtype 1b. Cirrhotic patients had a lower SVR rate than non-cirrhotic patients (80 vs. 92%, respectively) [33]. According to two large-scale US real-life studies, the overall SVR4 rate was 85% (140/164, treatment-naïve—55% and treatment-experienced—45%). SVR4 rate was 90% (114/127) in non-cirrhotic compared to 70% (26/37) in cirrhotic patients [34]. In TRIO real-life study including treatment-naïve (58%) and treatment-experienced (42%), SVR12 was 81 (112/138) and 81% (25/31) in non-cirrhotic and cirrhotic treatment-naïve patients, respectively, and 77% (30/39) in non-cirrhotic treatment-experienced and 62% (53/85) in cirrhotic treatment-experienced patients [21].

#### *2.1.6. Sofosbuvir and simeprevir plus ribavirin*

In COSMOS study, the combination of sofosbuvir and simeprevir with or without ribavirin for 12 or 24 weeks was assessed in naïve or null responders infected with genotype 1 patient without severe fibrosis. SVR12 was achieved in 91% (98/108) with ribavirin vs. 95% (56/59) of those who did not. SVR rates were similar by treatment status, treatment-naïve 95% (38/40) vs. previous non-responders 91% (116/127) or treatment duration 94% (77/82) after 12 weeks vs. 91% (77/85) after 24 weeks. Neither ribavirin nor treatment duration had clear effect on sustained virological response in HCV-infected patients with Gln80Lys polymorphism at baseline [22]. In TRIO real-life study, SVR12 achieved in 88% (68/88) of non-cirrhotic treatment-naïve and 75% (41/55) of cirrhotic treatment-naïve patients, whereas 87 (64/74) and 76% (53/70) in noncirrhotic and cirrhotic treatment-experienced patients, respectively [21].

## *2.1.7. Sofosbuvir and ledipasvir plus ribavirin*

generally well tolerated with potent antiviral activity and pharmacokinetic profile. In ASPIRE phase IIb trial done in previously treated patients with PegIFN and ribavirin, the SVR at 24 weeks was 38–59% (1a-42 and 1b-58%) in prior null responders, 48–86% (1a-56 and 1b-88%) in prior partial responders, and 77–89% (no difference) in prior relapsed cases. There were same SVR rates in patient with or without Q80k polymorphism at baseline 60.9%. In patients with cirrhosis (METAVIR score F4), combination therapy with 150 mg of simeprevir had SVR rate at 24 weeks was 73% in prior relapsers, 82% in prior partial responders, and 31% in prior null responders [29]. Another phase 3 trial on partials and null responders showed SVR of 70 and 44%, respectively [28]. According to QUEST 1 & 2 phase 3 study, overall SVR 12 in previously untreated and treated naïve patients was 81 (209/257) and 80% (1a-71 and 1b-90%). On subtype analysis, SVR rates on with or without Q80K polymorphism at baseline in 1a were 52–75 and 80–85%, respectively, and 82% in 1b. The SVR rates were comparatively higher in F0–2 83–85% than F3–4 66–70% [30, 31]. In patients who relapsed on previous therapy, the SVR 12 was 79.2%. Among them, 92.7% were enabled to shorten therapy with PR at 24 weeks [32]. The cause of treatment failure with this regimen was viral breakthrough in 10.6–13%. The main side effects were fatigue, headache, pruritus, and influenza like illness and anaemia.

Skin rash and photosensitivity were also very common with simeprevir [29–32].

Sofosbuvir is a nucleotide analogue HCV NS5B polymerase inhibitor with similar *in vitro* activity against pan-HCV genotypes. This therapy is used for HCV pan-genotype infections (1–6 genotypes) treatment-naïve patients with or without cirrhosis but no evidence on treatment-experienced patients. In the NEUTRINO phase III trial in treatment-naïve patients, the overall SVR rate was 89% (259/291), 92% (207/225) for subtype 1a and 82% (54/66) for subtype 1b. Cirrhotic patients had a lower SVR rate than non-cirrhotic patients (80 vs. 92%, respectively) [33]. According to two large-scale US real-life studies, the overall SVR4 rate was 85% (140/164, treatment-naïve—55% and treatment-experienced—45%). SVR4 rate was 90% (114/127) in non-cirrhotic compared to 70% (26/37) in cirrhotic patients [34]. In TRIO real-life study including treatment-naïve (58%) and treatment-experienced (42%), SVR12 was 81 (112/138) and 81% (25/31) in non-cirrhotic and cirrhotic treatment-naïve patients, respectively, and 77% (30/39) in non-cirrhotic treatment-experienced and 62% (53/85) in cirrhotic

In COSMOS study, the combination of sofosbuvir and simeprevir with or without ribavirin for 12 or 24 weeks was assessed in naïve or null responders infected with genotype 1 patient without severe fibrosis. SVR12 was achieved in 91% (98/108) with ribavirin vs. 95% (56/59) of those who did not. SVR rates were similar by treatment status, treatment-naïve 95% (38/40) vs. previous non-responders 91% (116/127) or treatment duration 94% (77/82) after 12 weeks vs. 91% (77/85) after 24 weeks. Neither ribavirin nor treatment duration had clear effect on sustained virological response in HCV-infected patients with Gln80Lys polymorphism at baseline [22]. In TRIO real-life study, SVR12 achieved in 88% (68/88) of non-cirrhotic treatment-naïve and

*2.1.5. Sofosbuvir in combination with pegylated interferon alfa and ribavirin*

treatment-experienced patients [21].

56 Advances in Treatment of Hepatitis C and B

*2.1.6. Sofosbuvir and simeprevir plus ribavirin*

Three phase III trials ION-1-3 have assessed the combination of sofosbuvir with ledipasvir, an NS5A inhibitor with or without ribavirin in genotype 1 infected populations. In naïve patients, including 16% compensated cirrhotic populations in ION-1 showed SVR12 in 99 (211/214) and 97% (211/217) patients after 12 weeks combination therapy without or with RBV, respectively. The SVR12 rate was 98% (212/217) in without RBV and 99% (215/217) in with RBV after 24 weeks [35]. In ION-3, non-cirrhotic treatment-naïve patients, SVR12 was 94% (202/215) without RBV for 8 weeks, 93% (201/216) with RBV for 8 weeks, and 95% (205/216) without RBV for 12 weeks. However, relapse rates were higher in 8 weeks compared to 12 weeks therapy [36]. In ION-2, in treatment-experienced patients including 20% cirrhotic patients, overall SVR12 rates were 94 (102/109) and 96% (107/111) without or with RBV, respectively. The SVR rates were 99 (108/109) and 99% (110/111) without or with RBV after 24 weeks, respectively [37]. The different phase III studies were not powered to compare responses to regimens with or without RBV or to 12 weeks or 24 weeks of treatment [38].

## *2.1.8. Sofosbuvir and daclatasvir*

Daclatasvir is a potent, pan-genotypic NS5A inhibitor with antiviral activity against HCV genotypes 1–6 *in vitro* [39], combined with sofosbuvir for treatment of hepatitis C. In phase IIb trial in patient without cirrhosis, the 24 weeks of therapy achieved SVR rates of 100% (14/14 and 15/15) without or with ribavirin, respectively, in treatment-naïve patients, and 100% (21/21) without ribavirin and 95% (19/21) with ribavirin non-responders to combination therapy of PegIFN alfa, ribavirin, and either telaprevir or boceprevir. Whereas SVR rates were achieved in 98% (40/41) of treatment-naïve without ribavirin after 12 weeks of therapy [40]. In phase II clinical trial, the efficacy of sofosbuvir plus daclatasvir with or without ribavirin for 12 or 24 weeks has been evaluated in large real-life cohort including genotype 1 cirrhotic patients. The SVR12 rates were 84.9% after weeks and 93.4% after 24 weeks of treatment. However, majority of analyses performed on data available after 4 weeks of follow up showed SVR4 rates of 85.2% with 12 weeks and 95.1% with 24 weeks of treatment without RBV, whereas 100% with 12 weeks and 98.7% with 24 weeks treatment with RBV [41]. In cirrhosis, the addition of RBV improved SVR, SVR4 of 76.5% with 12 weeks vs. 94% with 24 weeks without RBV treatment, which rose to 100 and 98.3%, respectively, with RBV. In non-cirrhotic patients, SVR4 achieved in all regardless of use of RBV or treatment duration. Without RBV, SVR4 in treatment-naïve after 12 or 24 weeks was 87.1 vs. 88.7%; however, rates increased to 100% (for both duration) with addition of RBV. In treatment-experienced patients, SVR4 without or with RBV after 12 weeks was 82.6 vs. 100%, and after 24 weeks 96.7 vs. 98.5% [41].

## *2.1.9. Sofosbuvir and velpatasvir*

Velpatasvir is a new pangenotypic HCV NS5A inhibitor with antiviral activity against HCV replicons in genotype 1–6 infections. The combination of sofosbuvir and velpatasvir for 12 weeks has been assessed in ASTRAL phase 3 trial in previously treatment-experienced patients (PegIFN/RBV with PIs) including cirrhosis, relapsed cases, patients who had detectable HCV RNA after PegIFN and ribavirin treatment. The overall sustained virological response rate was 98% in subtype 1a and 99% in subtype 1b infected patients [42]. In phase II trial in treatment-experienced patients including 50% cirrhosis and treatment failure, the combination of sofosbuvir and velpatasvir with or without ribavirin was assessed. The SVR showed 100% in without ribavirin and 96% in with ribavirin treatment patients [43]. The overall relapse rate was very low, and this regimen was well tolerated in treatment-experienced patient including cirrhosis [42, 43].

## *2.1.10. Ritonavir boosted paritaprevir, ombitasvir, and dasabuvir*

In seven phase III trials, in non-cirrhotic treatment-naïve patients, SAPPHIRE-I trial with combination therapy with RBV for 12 weeks showed SVR of 95% (307/322) in subtype 1a and 98% (148/151) in subtype 1b infected patients [44]. In PEARL-IV trial, the combination therapy without or with RBV showed SVR of 90 (185/205) vs. 97% (197/100) in subtype 1a treatmentnaïve patients, respectively [45]. In PEARL-III trial in non-cirrhotic treatment-naïve of subtype 1b patients, SVR12 rates were 99% (207/209) without RBV vs. 99% (209/210) with RBV [45]. TURQUOISE-I study in non-cirrhotic treatment-naïve patients co-infected with HIV-1 (stable on antiviral treatment – atazanavir or raltegravir), SVR12 rates were 93% (29/31) after 12 weeks vs. 91% (29/32) after 24 weeks of treatment. The SVR12 rates based on subtypes 1a and 1b were 91 (51/56) and 100% (7/7), respectively [46]. In SAPPHIRE-II trial, non-cirrhotic treatment-experienced patients (PegIFN-alfa and RBV failures) were treated with this regimen in combination with RBC for 12 weeks. The SVR12 rates were 96% (166/173) in subtype 1a vs. 97% (119/123) in subtype 1b. The overall SVR12 rates were 95% (82/86) in prior relapsers, 100% (65/65) in partial responders, and 95% (139/146) in null responders [47]. In PEARL-II trial, SVR12 achieved in 100% (91/91) without RBV vs. 97% (85/88) with RBV in subtype 1b infected patients [48]. In compensated cirrhotic treatment-naïve and treatment-experienced patients, the SVR rates were 92% (191/208) after 12 weeks vs. 96% (165/172) after 24 weeks of treatment with RBV in TURQUISE-II trial. The SVR12 rates were 92% (239/261) in subtype 1a vs. 99% (118/119) in subtype 1b infected patients [49].

## **2.2. Efficacy of IFN-based versus IFN-free regimens for treatment of HCV genotype 2 infections**

HCV genotype 2 is the third most prevalent genotype worldwide [8]. Although PegIFN alfa with ribavirin used previously, IFN-free combination of sofosbuvir with ribavirin is the best first line treatment option in genotype 2 infection [12]. Other regimens, IFN-based or IFN-free could be an option in cases who fail with this regimen (**Table 3**). The combination of PegIFN alfa and ribavirin remains acceptable when all other options are not available [12, 14].

#### *2.2.1. Pegylated interferon alfa and ribavirin*

The initial treatment of HCV genotype 2 began with PegIFN alfa alone or combination of PegIFN alfa and ribavirin. Although sustained virological response rate was lower than recent newer regimens, this regimen remains acceptable for treatment of genotype 2 where other options are not available [12]. In randomised study, the sustained virological response rates were 62% (232/372) in 16 weeks vs. 75% (268/356) in 24 weeks treatment course. The chances of relapse rates were higher among 16 weeks than 24 weeks [50]. In phase IV single arm study, 24 weeks therapy with this regimen in previously untreated naïve patients showed end of treatment (EOT) and SVR of 100 and 93%, respectively [51]. In phase III multicenter study in prior relapsers who were retreated for 24–48 weeks showed a sustained virological response rates of 53–81% in 48 weeks retreated patients vs. 75% in 24 weeks retreated patients [52].

12 weeks has been assessed in ASTRAL phase 3 trial in previously treatment-experienced patients (PegIFN/RBV with PIs) including cirrhosis, relapsed cases, patients who had detectable HCV RNA after PegIFN and ribavirin treatment. The overall sustained virological response rate was 98% in subtype 1a and 99% in subtype 1b infected patients [42]. In phase II trial in treatment-experienced patients including 50% cirrhosis and treatment failure, the combination of sofosbuvir and velpatasvir with or without ribavirin was assessed. The SVR showed 100% in without ribavirin and 96% in with ribavirin treatment patients [43]. The overall relapse rate was very low, and this regimen was well tolerated in treatment-experienced

In seven phase III trials, in non-cirrhotic treatment-naïve patients, SAPPHIRE-I trial with combination therapy with RBV for 12 weeks showed SVR of 95% (307/322) in subtype 1a and 98% (148/151) in subtype 1b infected patients [44]. In PEARL-IV trial, the combination therapy without or with RBV showed SVR of 90 (185/205) vs. 97% (197/100) in subtype 1a treatmentnaïve patients, respectively [45]. In PEARL-III trial in non-cirrhotic treatment-naïve of subtype 1b patients, SVR12 rates were 99% (207/209) without RBV vs. 99% (209/210) with RBV [45]. TURQUOISE-I study in non-cirrhotic treatment-naïve patients co-infected with HIV-1 (stable on antiviral treatment – atazanavir or raltegravir), SVR12 rates were 93% (29/31) after 12 weeks vs. 91% (29/32) after 24 weeks of treatment. The SVR12 rates based on subtypes 1a and 1b were 91 (51/56) and 100% (7/7), respectively [46]. In SAPPHIRE-II trial, non-cirrhotic treatment-experienced patients (PegIFN-alfa and RBV failures) were treated with this regimen in combination with RBC for 12 weeks. The SVR12 rates were 96% (166/173) in subtype 1a vs. 97% (119/123) in subtype 1b. The overall SVR12 rates were 95% (82/86) in prior relapsers, 100% (65/65) in partial responders, and 95% (139/146) in null responders [47]. In PEARL-II trial, SVR12 achieved in 100% (91/91) without RBV vs. 97% (85/88) with RBV in subtype 1b infected patients [48]. In compensated cirrhotic treatment-naïve and treatment-experienced patients, the SVR rates were 92% (191/208) after 12 weeks vs. 96% (165/172) after 24 weeks of treatment with RBV in TURQUISE-II trial. The SVR12 rates were 92% (239/261) in subtype 1a

**2.2. Efficacy of IFN-based versus IFN-free regimens for treatment of HCV genotype 2** 

alfa and ribavirin remains acceptable when all other options are not available [12, 14].

HCV genotype 2 is the third most prevalent genotype worldwide [8]. Although PegIFN alfa with ribavirin used previously, IFN-free combination of sofosbuvir with ribavirin is the best first line treatment option in genotype 2 infection [12]. Other regimens, IFN-based or IFN-free could be an option in cases who fail with this regimen (**Table 3**). The combination of PegIFN

The initial treatment of HCV genotype 2 began with PegIFN alfa alone or combination of PegIFN alfa and ribavirin. Although sustained virological response rate was lower than recent

patient including cirrhosis [42, 43].

58 Advances in Treatment of Hepatitis C and B

*2.1.10. Ritonavir boosted paritaprevir, ombitasvir, and dasabuvir*

vs. 99% (118/119) in subtype 1b infected patients [49].

*2.2.1. Pegylated interferon alfa and ribavirin*

**infections**


PegIFN, pegylated interferon-alfa; RBV, ribavirin; EOT, end of treatment; SVR-12/16/24, sustained virological response at 12 weeks, 16 weeks and 24 weeks; NC, non-cirrhotic; C, cirrhotic.

**Table 3.** Efficacy of IFN-based vs. IFN-free regimens for treatment of HCV genotype 2 infections.

#### *2.2.2. Pegylated interferon alfa and ribavirin plus sofosbuvir*

In LONESTAR-2 phase IIb study, in treatment-experienced patients infected with HCV genotype 2 patients including 14 with cirrhosis received therapy for 12 weeks, the sustained virological response rates were 96% [53]. Another study showed that the relapsed cases of sofosbuvir and ribavirin regimen treated for 12 weeks were retreated with this regimen for 12 weeks, achieved SVR [54]. In phase II study in previously untreated naïve patients, the sustained virological responses in 12 or 24 weeks treatment were 92% (23/25) [55]. The main side effects with this regimen were fatigue, headache, nausea, pain, and insomnia [55].

#### *2.2.3. Sofosbuvir and ribavirin*

This IFN-free combination therapy is the best first-line treatment option in HCV genotype 2 infected patients [12]. In FISSION trial in treatment-naïve patients who were treated for 12 weeks, the SVR was 95% (69/73). The virological response rate was higher in non-cirrhotic patients, 97 vs. 83% in cirrhotic patients [33]. In POSITRON trial, who were intolerant or ineligible to IFN, treated for 12 weeks, SVR was 93% (101/109) [56]. The 12 vs. 16 weeks therapy was in FUSION trial showed SVR of 82 (32/39) vs. 89% (31/35) in non-cirrhotic and 60 (6/10) vs. 78% (7/9) in cirrhotic cases, respectively. The longer than 12 weeks therapy was beneficial in cirrhotic population [56]. In VALENCE trial, the treatment was given for 12 weeks in treatment-naïve and treatment-experienced patients with or without cirrhosis. In treatment-naïve patients, the SVR rates were 97% (29/30) in without cirrhosis and 100% (2/2) in with cirrhosis. In treatment-experienced patients, SVR rates were 91 (30/33) vs. 88% (7/8) in without cirrhosis and with cirrhosis, respectively [57]. This combination therapy was well tolerated, and no virological breakthroughs were observed in treatment adherent patients [12].

## *2.2.4. Sofosbuvir and daclatasvir*

Daclatasvir, NS5A replication complex inhibitor, is active against HCV genotype 2 *in vitro*. The combination of sofosbuvir with daclatasvir therapy was observed in phase II trial showed sustained virological response of 92% (24/26) after 12 weeks of therapy and overall 93% after 24 weeks of therapy [40]. Based on data with other, 12 weeks is probably sufficient to treat more difficult-to-cure HCV genotypes. This regimen should be kept reserved for patients who failed with other options in HCV genotype 2 infections [12, 40].

#### *2.2.5. Sofosbuvir and velpatasvir*

In 2 phase III trial (open label studies), the combination of sofosbuvir (400 mg) and velpatasvir (100 mg) was assessed in patients infected with HCV genotype 2 who previously received treatment and who did not received previous treatment including compensated cirrhosis. The sustained virological response was achieved in 99% of cases with this regimen [58]. In ASTRAL phase III double blind trial in treatment-experienced patients including cirrhosis, treatment relapsed, and detectable HCV RNA under PegIFN and ribavirin therapy, the SVR12 was 100% in genotype 2 infected patients [42]. From the data available, this regimen was well tolerated with higher SVR in treatment of genotype 2 infection. However, these data have to be compared with the blind trials or studies.

## **2.3. Efficacy of IFN-based versus IFN-free regimens for treatment of HCV genotype 3 infections**

There are four treatment options available for treatment of hepatitis C genotype 3 infection including one phase III trial drug (**Table 4**). The IFN-based combination therapy-PegIFN alfa and ribavirin regimen remains acceptable only in settings where none other options are available [12]. The triple combination of PegIFN alfa, ribavirin and sofosbuvir appears to be valuable even in, who failed on sofosbuvir and ribavirin combinations. However, it has to be done in larger population infected with HCV genotype 3 patients [54]. The IFN-free combination therapy—sofosbuvir and ribavirin—appears to be suboptimal particularly in cirrhotic HCV genotype 3 infected patients, although it is the best first line treatment option for genotype 2 infection [12]. Sofosbuvir and daclatasvir with or without ribavirin are a new attractive option for patients infected with genotype 3. Ledipasvir is considerably less potent against genotype 3 *in vitro* than daclatasvir. In clinical trials, the combination of ledipasvir with sofosbuvir or other agents is not recommended in patients infected with HCV genotype 3 [12, 14].


PegIFN, pegylated interferon-alfa; RBV, ribavirin; SVR 12/24, sustained virological response at 12 weeks and 24 weeks; EOT, end of treatment; NC, non-cirrhotic; C, cirrhotic.

**Table 4.** Efficacy of IFN-based vs. IFN-free regimens for treatment of HCV genotype 3 infections.

#### *2.3.1. Pegylated interferon alfa and ribavirin*

*2.2.3. Sofosbuvir and ribavirin*

60 Advances in Treatment of Hepatitis C and B

*2.2.4. Sofosbuvir and daclatasvir*

*2.2.5. Sofosbuvir and velpatasvir*

**infections**

This IFN-free combination therapy is the best first-line treatment option in HCV genotype 2 infected patients [12]. In FISSION trial in treatment-naïve patients who were treated for 12 weeks, the SVR was 95% (69/73). The virological response rate was higher in non-cirrhotic patients, 97 vs. 83% in cirrhotic patients [33]. In POSITRON trial, who were intolerant or ineligible to IFN, treated for 12 weeks, SVR was 93% (101/109) [56]. The 12 vs. 16 weeks therapy was in FUSION trial showed SVR of 82 (32/39) vs. 89% (31/35) in non-cirrhotic and 60 (6/10) vs. 78% (7/9) in cirrhotic cases, respectively. The longer than 12 weeks therapy was beneficial in cirrhotic population [56]. In VALENCE trial, the treatment was given for 12 weeks in treatment-naïve and treatment-experienced patients with or without cirrhosis. In treatment-naïve patients, the SVR rates were 97% (29/30) in without cirrhosis and 100% (2/2) in with cirrhosis. In treatment-experienced patients, SVR rates were 91 (30/33) vs. 88% (7/8) in without cirrhosis and with cirrhosis, respectively [57]. This combination therapy was well tolerated, and no

Daclatasvir, NS5A replication complex inhibitor, is active against HCV genotype 2 *in vitro*. The combination of sofosbuvir with daclatasvir therapy was observed in phase II trial showed sustained virological response of 92% (24/26) after 12 weeks of therapy and overall 93% after 24 weeks of therapy [40]. Based on data with other, 12 weeks is probably sufficient to treat more difficult-to-cure HCV genotypes. This regimen should be kept reserved for patients who

In 2 phase III trial (open label studies), the combination of sofosbuvir (400 mg) and velpatasvir (100 mg) was assessed in patients infected with HCV genotype 2 who previously received treatment and who did not received previous treatment including compensated cirrhosis. The sustained virological response was achieved in 99% of cases with this regimen [58]. In ASTRAL phase III double blind trial in treatment-experienced patients including cirrhosis, treatment relapsed, and detectable HCV RNA under PegIFN and ribavirin therapy, the SVR12 was 100% in genotype 2 infected patients [42]. From the data available, this regimen was well tolerated with higher SVR in treatment of genotype 2 infection. However, these data

**2.3. Efficacy of IFN-based versus IFN-free regimens for treatment of HCV genotype 3** 

There are four treatment options available for treatment of hepatitis C genotype 3 infection including one phase III trial drug (**Table 4**). The IFN-based combination therapy-PegIFN alfa and ribavirin regimen remains acceptable only in settings where none other options are available [12]. The triple combination of PegIFN alfa, ribavirin and sofosbuvir appears to be valuable even in, who failed on sofosbuvir and ribavirin combinations. However, it has to be done

virological breakthroughs were observed in treatment adherent patients [12].

failed with other options in HCV genotype 2 infections [12, 40].

have to be compared with the blind trials or studies.

This combination therapy remained acceptable for treatment of genotype 3 infections until the development of other regimens with higher sustained virological response and also in setting where other options are not available [12]. In this regimen, the treatment is given for 24 weeks in genotype 3 infected patients. In phase 4 single arm study in 182 HCV genotype 3 infected population and treatment was given with this regimen for 24 weeks; the overall sustained virological response rate was observed and also at end of treatment (EOT). It showed EOT and SVR were of 93 and 79%, respectively [51]. Baseline viremia, treatment duration >16 weeks, and steatosis were independent predictors of SVR. The relapsed rate were higher among male and older age >55 years [51].

#### *2.3.2. Pegylated interferon alfa and ribavirin plus sofosbuvir*

In LONESTAR-2 phase IIb trial, in treatment-experienced patients infected with HCV genotype 3, the sustained virological response rate was 83% (20/24) including (10/12) patients with cirrhosis [53]. However, pangenotypic activity of sofosbuvir together with higher SVR in other genotypes 89% (overall in genotype 1, 4 or 6) indicates this regimen can be safely used in patients with genotype 3 infections [53]. In phase 2 trial, in non-cirrhotic treatment-naïve patients were treated for 12 weeks, the sustained virological response was achieved in 92% (23/25) cases [55]. In another study, patients who relapsed after treatment with sofosbuvir and ribavirin regimens were retreated with this triple combination therapy for 12 weeks, and the SVR was achieved in 91% (20/22) cases [54].

#### *2.3.3. Sofosbuvir and ribavirin*

The combination of sofosbuvir with daily fixed dose ribavirin is used for treatment of genotype 3 infection for 24 weeks. In FISSION trial, in treatment-naïve patients who were treated for 12 weeks, the SVR rate was 56% (102/183). The non-cirrhotic patients had better SVR of 61 vs. 34% in cirrhotic patients [33]. In POSITRON trial, patients were also treated for 12 weeks with this regimen who were ineligible or intolerant to interferon. The SVR rate was 61% (60/98) of cases. In FUSION trial, the 12 vs. 16 weeks treatment was compared. The SVR rate was significantly higher, 62% in non-cirrhotic and 61% in cirrhotic patients with 16 weeks treatment compared to 30% in non-cirrhotic and 19% in cirrhotic patient with 12 weeks treatment [56]. In VALENCE trial, treatment was given for 24 weeks in both treatment-naïve and treatment-experienced without or with cirrhosis. In treatment- naïve, the SVR24 was 94% (86/92) in non-cirrhotic and 92% (12/13) in cirrhotic patients. Whereas, in treatment-experienced, SVR 24 was 87% (87/100) in non-cirrhotic and 60% (27/45) in cirrhotic patients [57]. So based on these studies, 24 weeks treatment is appropriate for the HCV genotype 3 infected patients. Another study, in relapsed cases with sofosbuvir and ribavirin, patients were retreated for 24 weeks, achieved SVR only of 63% (24/38) of cases, indicating the regimen is suboptimal in such patients with HCV genotype 3 infection [54].

#### *2.3.4. Sofosbuvir and daclatasvir*

In treatment of HCV genotype 3 infected patients, this regimen is given for 12 weeks in non-cirrhotic patients and 24 weeks with daily weight-based ribavirin for 24 weeks in cirrhotic patients. In phase IIb trial, after 24 weeks of combination therapy, SVR rate was 89% (16/18) in treatment-naïve without cirrhosis [40]. In ALLY-3 phase III trial, after 12 weeks of combination therapy without ribavirin, SVR12 was 97% (73/75) in non-cirrhotic and 58% (11/19) in cirrhotic treatment-naïve patients, whereas SVR12 was 94 (32/34) and 69% (9/13) in treatment-experienced patients without or with cirrhosis, respectively [59]. This regimen was well tolerated with rare adverse events, and none of them discontinued treatment [12].

#### *2.3.5. Sofosbuvir and velpatasvir*

infected population and treatment was given with this regimen for 24 weeks; the overall sustained virological response rate was observed and also at end of treatment (EOT). It showed EOT and SVR were of 93 and 79%, respectively [51]. Baseline viremia, treatment duration >16 weeks, and steatosis were independent predictors of SVR. The relapsed rate were higher

In LONESTAR-2 phase IIb trial, in treatment-experienced patients infected with HCV genotype 3, the sustained virological response rate was 83% (20/24) including (10/12) patients with cirrhosis [53]. However, pangenotypic activity of sofosbuvir together with higher SVR in other genotypes 89% (overall in genotype 1, 4 or 6) indicates this regimen can be safely used in patients with genotype 3 infections [53]. In phase 2 trial, in non-cirrhotic treatment-naïve patients were treated for 12 weeks, the sustained virological response was achieved in 92% (23/25) cases [55]. In another study, patients who relapsed after treatment with sofosbuvir and ribavirin regimens were retreated with this triple combination therapy for 12 weeks, and the

The combination of sofosbuvir with daily fixed dose ribavirin is used for treatment of genotype 3 infection for 24 weeks. In FISSION trial, in treatment-naïve patients who were treated for 12 weeks, the SVR rate was 56% (102/183). The non-cirrhotic patients had better SVR of 61 vs. 34% in cirrhotic patients [33]. In POSITRON trial, patients were also treated for 12 weeks with this regimen who were ineligible or intolerant to interferon. The SVR rate was 61% (60/98) of cases. In FUSION trial, the 12 vs. 16 weeks treatment was compared. The SVR rate was significantly higher, 62% in non-cirrhotic and 61% in cirrhotic patients with 16 weeks treatment compared to 30% in non-cirrhotic and 19% in cirrhotic patient with 12 weeks treatment [56]. In VALENCE trial, treatment was given for 24 weeks in both treatment-naïve and treatment-experienced without or with cirrhosis. In treatment- naïve, the SVR24 was 94% (86/92) in non-cirrhotic and 92% (12/13) in cirrhotic patients. Whereas, in treatment-experienced, SVR 24 was 87% (87/100) in non-cirrhotic and 60% (27/45) in cirrhotic patients [57]. So based on these studies, 24 weeks treatment is appropriate for the HCV genotype 3 infected patients. Another study, in relapsed cases with sofosbuvir and ribavirin, patients were retreated for 24 weeks, achieved SVR only of 63% (24/38) of cases, indicating the regimen is suboptimal in such patients with HCV genotype 3 infection [54].

In treatment of HCV genotype 3 infected patients, this regimen is given for 12 weeks in non-cirrhotic patients and 24 weeks with daily weight-based ribavirin for 24 weeks in cirrhotic patients. In phase IIb trial, after 24 weeks of combination therapy, SVR rate was 89% (16/18) in treatment-naïve without cirrhosis [40]. In ALLY-3 phase III trial, after 12 weeks of combination therapy without ribavirin, SVR12 was 97% (73/75) in non-cirrhotic and 58% (11/19) in cirrhotic treatment-naïve patients, whereas SVR12 was 94 (32/34) and 69% (9/13) in treatment-experienced patients without or with cirrhosis, respectively [59]. This regimen was well tolerated with rare adverse events, and none of them discontinued treatment [12].

among male and older age >55 years [51].

62 Advances in Treatment of Hepatitis C and B

SVR was achieved in 91% (20/22) cases [54].

*2.3.3. Sofosbuvir and ribavirin*

*2.3.4. Sofosbuvir and daclatasvir*

*2.3.2. Pegylated interferon alfa and ribavirin plus sofosbuvir*

In phase II trial, the combination of sofosbuvir (400 mg) and velpatasvir (100 mg) with or without daily fixed dose ribavirin for 12 weeks was assessed in treatment-experienced patients with or without cirrhosis infected with HCV genotype 3. The sustained virological response was achieved 100% with or without ribavirin in non-cirrhotic patients. However, SVR of 88% without ribavirin and 96% with ribavirin was achieved in compensated cirrhotic patients [43]. In another 2 phase III trial (open label studies), in patients infected with genotype 3 who have previously received treatment and who did not receive treatment including compensated cirrhosis, 12 weeks of this regimen without ribavirin achieved SVR of 95% [58]. Based on these studies, this regimen was well tolerated in treatment of HCV genotype 3 infections.

## **2.4. Efficacy of IFN-based versus IFN-free regimens for treatment of HCV genotype 4 infections**

There are seven treatment options available for treatment of hepatitis C genotype 4 infections, including two IFN-based regimens and four IFN-free regimens (**Table 5**). The combination of PegIFN alfa and ribavirin remains acceptable only in that case where other options are not available [12].


PegIFN, pegylated interferon-alfa; RBV, ribavirin; SVR 12/24/36/48, sustained virological response at 12 weeks, 24 weeks, 36 weeks and 48 weeks; (+)C/R, with cirrhosis and relapse; (−)RBV, with ribavirin; (+) RBV, with ribavirin.

**Table 5.** Efficacy of IFN-based vs. IFN-free regimens for treatment of HCV genotype 4 infections.

## *2.4.1. Pegylated interferon alfa and ribavirin*

The combination of PegIFN alfa and ribavirin is still the option for treatment of HCV genotype 4 when other options are not available [12]. In prospective randomise controlled trial, the combination of PegIFN alfa and ribavirin was used for 24–48 weeks. The sustained virological response rates were 29 vs. 66 vs. 69% in 24 vs. 36 vs. 48 weeks of treatment, respectively [60].

## *2.4.2. Pegylated interferon alfa and ribavirin plus simeprevir*

Simeprevir is active against HCV genotype 4 *in vitro*. So, this combination therapy can be used in genotype 4 infection. However, the duration of therapy is 24 weeks (SPR 12 + PR 12 weeks) in treatment-naïve or prior relapsers including cirrhosis and 48 weeks (SPR12 + PR 36 weeks) in prior partial or null responders including cirrhosis. In phase III study, SVR12 was achieved in 83% (29/35) in treatment-naïve patients, 86% (19/22) in prior relapsers, 60% (6/10) in prior partial responders, and 40% (16/40) in prior null responders. This regimen was effective in treatment-naïve and prior relapsers, however, suboptimal in prior partial and null responders [61].

## *2.4.3. Pegylated interferon alfa and ribavirin plus sofosbuvir*

In NEUTRINO phase III in treatment-naïve patients, this combination therapy for 12 weeks was evaluated. The SVR rate was 96% (27/28) in HCV genotype 4 infected patients [33]. Those who failed in this combination therapy did not select HCV variants resistant to sofosbuvir. No data were available in treatment-experienced or HIV-coinfected patients [12].

## *2.4.4. Sofosbuvir and ledipasvir*

Sofosbuvir in combination with ledipasvir is used in treatment-naïve and treatment-experienced patients with or without cirrhosis for 12 weeks. Addition of ribavirin to this therapy has beneficial effect in cirrhotic individuals. In SYNERGY trial, efficacy and safety of combination of sofosbuvir and ledipasvir without ribavirin are assessed in patient with genotype 4 infection. The sustained virological response was achieved in 95% (20/21) of cases [62]. The shorter 8 weeks treatment duration as in patients infected with genotype 1 infections is not clear due to lack of data in genotype 4 infected cases [12].

#### *2.4.5. Sofosbuvir and simeprevir*

The unavailability of data on treatment of HCV genotype 4 infection had questioned the use of this IFN-free regimen (sofosbuvir plus simeprevir) as an option previously, however, according to a very recently published two studies, sofosbuvir (SOF) plus simeprevir (SIM) regimen with or without ribavirin, can be a good option in treating HCV genotype 4 infected cases [12, 63, 64]. A retrospective multicentre observational study in 53 patients (naïve or experienced patients) including advanced liver fibrosis or liver cirrhosis treated with SOF and SIM with or without ribavirin showed a SVR12 of 92% (49/53). In this study, treatment failures were observed in those who didn't receive ribavirin and interferon non-responders except one naïve patient [63]. Another multicentre observational study in 583 patients infected with HCV genotype 4 showed the overall SVR rates of 95.7% (558/583) with SOF/SIM regimen. Based on fibrosis stages in naïve patients, mild fibrosis score had better SVR12 of 98.9% (94/95) in F1 and 98.1% (105/107) in F2 stage than severe fibrosis score with SVR12 of 97.7% (86/88) in F3 and 80.8% (42/52) in F4 stage. While in treatment-experienced patients with severe fibrosis score, SVR12 was 94.7% (72/76) in F3 and 88.9% (40/45) in F4 stage. In addition, patients who were previously treated with interferon had SVR of 100% (45/45) in F1 and 98.7% (74/75) in F2 mild fibrosis score [64]. Therefore, this regimen can be efficacious and well tolerated in treatment-naïve and experienced patients including severe fibrosis score or liver cirrhosis. Furthermore, the addition of ribavirin could be considered especially in treatment-experienced and advanced cirrhosis patients as recommended by recent AASL and EASL guidelines [12, 14, 63, 64].

## *2.4.6. Sofosbuvir and daclatasvir*

*2.4.1. Pegylated interferon alfa and ribavirin*

64 Advances in Treatment of Hepatitis C and B

*2.4.2. Pegylated interferon alfa and ribavirin plus simeprevir*

*2.4.3. Pegylated interferon alfa and ribavirin plus sofosbuvir*

*2.4.4. Sofosbuvir and ledipasvir*

data in genotype 4 infected cases [12].

*2.4.5. Sofosbuvir and simeprevir*

The combination of PegIFN alfa and ribavirin is still the option for treatment of HCV genotype 4 when other options are not available [12]. In prospective randomise controlled trial, the combination of PegIFN alfa and ribavirin was used for 24–48 weeks. The sustained virological response

Simeprevir is active against HCV genotype 4 *in vitro*. So, this combination therapy can be used in genotype 4 infection. However, the duration of therapy is 24 weeks (SPR 12 + PR 12 weeks) in treatment-naïve or prior relapsers including cirrhosis and 48 weeks (SPR12 + PR 36 weeks) in prior partial or null responders including cirrhosis. In phase III study, SVR12 was achieved in 83% (29/35) in treatment-naïve patients, 86% (19/22) in prior relapsers, 60% (6/10) in prior partial responders, and 40% (16/40) in prior null responders. This regimen was effective in treatment-naïve and prior relapsers, however, suboptimal in prior partial and null responders [61].

In NEUTRINO phase III in treatment-naïve patients, this combination therapy for 12 weeks was evaluated. The SVR rate was 96% (27/28) in HCV genotype 4 infected patients [33]. Those who failed in this combination therapy did not select HCV variants resistant to sofosbuvir. No

Sofosbuvir in combination with ledipasvir is used in treatment-naïve and treatment-experienced patients with or without cirrhosis for 12 weeks. Addition of ribavirin to this therapy has beneficial effect in cirrhotic individuals. In SYNERGY trial, efficacy and safety of combination of sofosbuvir and ledipasvir without ribavirin are assessed in patient with genotype 4 infection. The sustained virological response was achieved in 95% (20/21) of cases [62]. The shorter 8 weeks treatment duration as in patients infected with genotype 1 infections is not clear due to lack of

The unavailability of data on treatment of HCV genotype 4 infection had questioned the use of this IFN-free regimen (sofosbuvir plus simeprevir) as an option previously, however, according to a very recently published two studies, sofosbuvir (SOF) plus simeprevir (SIM) regimen with or without ribavirin, can be a good option in treating HCV genotype 4 infected cases [12, 63, 64]. A retrospective multicentre observational study in 53 patients (naïve or experienced patients) including advanced liver fibrosis or liver cirrhosis treated with SOF and SIM with or without ribavirin showed a SVR12 of 92% (49/53). In this study, treatment failures were observed in those who didn't receive ribavirin and interferon non-responders except one naïve patient [63]. Another multicentre observational study in 583 patients infected with HCV

data were available in treatment-experienced or HIV-coinfected patients [12].

rates were 29 vs. 66 vs. 69% in 24 vs. 36 vs. 48 weeks of treatment, respectively [60].

Daclatasvir has its antiviral activity against genotype 4 *in vitro*. The combination of sofosbuvir and daclatasvir with or without ribavirin is effective in treating patients infected with HCV genotype 4. However, there is no data available with this combination in treatment of this genotype. Nevertheless, both sofosbuvir and daclatasvir have antiviral effectiveness against genotype 4 *in vitro*. So, the results in patients infected with genotype 1 can be extrapolated [12].

#### *2.4.7. Sofosbuvir and velpatasvir*

Velpatasvir and sofosbuvir have a pangenotypic action for treatment of HCV genotype 1–6 infections. The combination of sofosbuvir and velpatasvir assessed in ASTRAL phase 3 trial in previously treatment-experienced patients (PegIFN/RBV with PIs) including cirrhosis, relapsed cases, patients who had detectable HCV RNA after PegIFN and ribavirin treatment. The overall sustained virological response rate was 100% in genotype 4 infected patients. The overall relapse rate was very low, and this regimen was well tolerated in treatment-experienced patient including cirrhosis [42].

## *2.4.8. Ritonavir-boosted paritaprevir and ombitasvir*

A fixed dose ritonavir, paritaprevir, and ombitasvir with or without ribavirin treatment for 12–24 weeks were assessed in treatment-naïve and treatment-experienced patients with or without compensated cirrhosis infected with HCV genotype 4. According to PEARL-I trial in non-cirrhotic chronic HCV genotype 4 infected patients, sustained virological response rates were 100% in treatment-naïve (42/42) and treatment-experienced patients (49/49) with ribavirin regimen, whereas 90.9% (40/44) in treatment-naïve patients without ribavirin regimen for 12 weeks [65]. In AGATE-I trial with a fixed dose ritonavir, paritaprevir, and ombitasvir plus ribavirin in chronic HCV genotype 4 infected treatment-naïve and treatment-experienced patients including compensated cirrhosis, post-treatment sustained virological response rates were 97% (57/59) in 12 weeks and 98% (60/61) in 16 weeks group [66]. In addition, AGATE-II trial in Egyptian patients, SVR12 was 94% (94/100) in patients without cirrhosis, whereas SVR12 of 97% (30/31) and SVR24 of 93% (27/29) in patients with cirrhosis [67]. Extension of this treatment regimen beyond 12 weeks (16 and 24 weeks) for HCV genotype 4 infected patients with compensated cirrhosis seemed to have no additional benefits [66, 67]. This regimen was generally well tolerated by chronic HCV genotype 4 infected patients with or without compensated cirrhosis in clinical trials, so this regimen is a valuable option, although having postmarking reports of hepatic decompensation and hepatic failure mainly in patients with advanced cirrhosis [68].

## **2.5. Efficacy of IFN-based versus IFN-free regimens for treatment of HCV genotypes 5 or 6 infections**

HCV genotype 5 is the least prevalent worldwide and then genotype 6 infection [8]. The treatment options for these genotypes are one IFN-based triple combination of PegIFN alfa, ribavirin, and sofosbuvir; and three IFN-free combination therapy: sofosbuvir and ledipasvir, sofosbuvir and daclatasvir, and sofosbuvir and velpatasvir (**Table 6**). IFN-based combination of PegIFN-alfa and ribavirin remains acceptable in setting where other treatment options are not available [12].


PegIFN, pegylated interferon alfa; RBV, ribavirin; GT-5, genotype 5; GT-6, genotype 6; SVR12, sustained virological response at 12 weeks; TN+TE, treatment-naïve and treatment-experienced.

**Table 6.** Efficacy of IFN-based vs. IFN-free regimens for treatment of HCV genotype 5 or 6 infections.

#### *2.5.1. Pegylated interferon alfa and ribavirin plus sofosbuvir*

In NEUTRINO phase III trial, this combination therapy has been evaluated in treatmentnaïve patients. There were total seven patients (one infected genotype 5 and six infected with genotype 6), all patients achieved sustained virological response [33]. However, no data have been presented with this regimen in treatment-experienced patients. So, it is not clear whether longer duration of treatment is needed [12].

#### *2.5.2. Sofosbuvir and ledipasvir*

Ledipasvir is active against both genotype 5 or 6 *in vitro*. The combination of sofosbuvir and ledipasvir is used in treatment of these genotypes. Those patients without cirrhosis, including treatment-naïve and treatment-experienced should be treated for 12 weeks without ribavirin. Addition of ribavirin is recommended in cirrhotic cases. However, 24 weeks combination of sofosbuvir and ledipasvir is recommended, when ribavirin is contraindicated or with poor tolerance [12]. In multicentre open label phase II trial, in treatment-naïve and treatment-experienced patients infected with genotype 5 including cirrhosis, the overall SVR was 95% (39/42). The SVR was 95% (20/21) in treatment-naïve and 95% (19/20) in treatmentexperienced patients. However, SVR was 97% (31/32) in non-cirrhotic vs. 89% (8/9) in cirrhotic patients [69]. In phase 2 clinical trial, in treatment-naïve and treatment-experienced patients infected with HCV genotype 6, the 12 weeks treatment with this regimen without ribavirin had a sustained virological response of 96% (24/25) [12, 70].

#### *2.5.3. Sofosbuvir and daclatasvir*

patients with compensated cirrhosis seemed to have no additional benefits [66, 67]. This regimen was generally well tolerated by chronic HCV genotype 4 infected patients with or without compensated cirrhosis in clinical trials, so this regimen is a valuable option, although having postmarking reports of hepatic decompensation and hepatic failure mainly in patients

**2.5. Efficacy of IFN-based versus IFN-free regimens for treatment of HCV genotypes 5 or 6** 

HCV genotype 5 is the least prevalent worldwide and then genotype 6 infection [8]. The treatment options for these genotypes are one IFN-based triple combination of PegIFN alfa, ribavirin, and sofosbuvir; and three IFN-free combination therapy: sofosbuvir and ledipasvir, sofosbuvir and daclatasvir, and sofosbuvir and velpatasvir (**Table 6**). IFN-based combination of PegIFN-alfa and ribavirin remains acceptable in setting where other treatment options are

**Naïve Treatment-experienced Partial responders Null** 

SVR12: 90% – – – –

– – – – –

SVR (GT-5): 95% – – –

**responders**

– – –

**Relapsers**

In NEUTRINO phase III trial, this combination therapy has been evaluated in treatmentnaïve patients. There were total seven patients (one infected genotype 5 and six infected with genotype 6), all patients achieved sustained virological response [33]. However, no data have been presented with this regimen in treatment-experienced patients. So, it is not clear whether

PegIFN, pegylated interferon alfa; RBV, ribavirin; GT-5, genotype 5; GT-6, genotype 6; SVR12, sustained virological

Ledipasvir is active against both genotype 5 or 6 *in vitro*. The combination of sofosbuvir and ledipasvir is used in treatment of these genotypes. Those patients without cirrhosis,

with advanced cirrhosis [68].

66 Advances in Treatment of Hepatitis C and B

**infections**

not available [12].

**Treatment Regimens**

PegIFN/RBV + sofosbuvir

Sofosbuvir and ledipasvir

Sofosbuvir and daclatasvir

Sofosbuvir and velpatasvir

*2.5.1. Pegylated interferon alfa and ribavirin plus sofosbuvir*

SVR12 (overall): 96% SVR12 (overall):

response at 12 weeks; TN+TE, treatment-naïve and treatment-experienced.

97% (GT-5) 100% (GT-6)

**Table 6.** Efficacy of IFN-based vs. IFN-free regimens for treatment of HCV genotype 5 or 6 infections.

longer duration of treatment is needed [12].

SVR (GT-5): 95% SVR12 (TN+TE): 96%

(overall)

*2.5.2. Sofosbuvir and ledipasvir*

Daclatasvir and sofosbuvir are active against genotype 5 or 6 *in vitro*. This regimen is given for 12 weeks with or without ribavirin in these genotypes. However, in cirrhotic patients with contraindications or intolerance to ribavirin, combination therapy can be extended to 24 weeks. There were no data available with this regimen for these rare genotypes [12].

#### *2.5.4. Sofosbuvir and velpatasvir*

The combination of sofosbuvir (400 mg) and velpatasvir (100 mg) for 12 weeks was assessed in ASTRAL phase III trial for treatment of genotypes 5 and 6. In this double blind, placebo controlled trial, patients were previously treatment-experienced (PegIFN and ribavirin or PegIFN, ribavirin and protease inhibitors), relapsed cases, and who had persistent detectable HCV RNA on PegIFN alfa and ribavirin therapy. The sustained virological response achieved in patients infected with genotypes 5 and 6 were 97 and 100%, respectively. This regimen was well tolerated, with very low failure rate in treatment of HCV genotype 5 and 6 infections [42]. In another randomised trial, the overall sustained virological response was 95% in treatment-naïve patients [71].

## **3. Discussion**

The development of DAAs was the milestone in the treatment of chronic hepatitis C, and their combination therapy became the first option for almost all genotypes. However, the IFN-based combination therapies have their own role in treatment of chronic hepatitis C infections when DAAs combination regimens are unavailable or fails [12]. In genotype 1 infections, the IFNbased combination therapy: PegIFN alfa, RBV, and simeprevir, and PegIFN, RBV, and sofosbuvir combination had higher overall SVR of 60–90% including relapsers or partial/null responders compared to other three regimens. In IFN-free regimens, all the DAAs combination regimens (2/4 DAAs ± RBV) have overall SVR of above 90% [12, 14]. The combination of sofosbuvir with ledipasvir or daclatasvir velpatasvir or 4DAAs (ritonavir, paritaprevir, ombitasvir, & dasabuvir) had superior SVR rates compared to IFN-based regimens [35, 36, 40, 42, 44]. In HCV genotype 2 infections, though PegIFN alfa with RBV and sofosbuvir has higher SVR >90%, the combination of sofosbuvir and ribavirin is the first line regimen for its treatment [33]. Although combination of sofosbuvir with daclatasvir or velpatasvir has SVR > 90%, they are reserved for treatment failed options with first line drugs [40–43]. In genotype 3 infections, the combination of sofosbuvir and ribavirin is suboptimal [54], so IFN-based PegIFN/RBV/SOF regimen or IFN-free combination of SOF and daclatasvir becomes the choice of treatment [40, 54, 59]. The phase 3 trials, sofosbuvir and velpatasvir have higher SVR, so it might be the choice of regimen in future [43, 58]. In genotype 4 infections, IFN-based PegIFN, RBV with simeprevir or sofosbuvir have SVR > 90% in treatment-naïve cases. However, SVR in partial or null responders is suboptimal [33, 61]. The IFN-free two DAAs or three DAAs with or without RBV has overall SVR > 90%, although no data available for partial or null responders or relapsers cases [42, 62, 65, 72]. In genotype 5 or 6 infections, IFN-based PegIFN/RBV/SOF has SVR of 90% in treatment-naïve [33]. However, two DAAs combinations have better SVR of > 95% [12, 43, 69, 70]. In this review, it showed that IFN-free DAAs regimens have better SVR and well tolerated compared to IFN-based regimen. The combination of DAAs with or without ribavirin has almost replaced the IFN-based combination therapy in present context. Nevertheless, we cannot exclude the fact that the combination of PegIFN alfa and ribavirin still leaves us an ultimate option in setting where all other options are not available [12, 14].

## **4. Conclusion**

The combination of IFN-free DAAs regimens has superior in their efficacy and tolerability compared to IFN-based regimens in case of treatment of chronic hepatitis C in all genotypes. However, in genotypes 3, 4, 5 or 6, the IFN-based combination of pegylated interferon alfa, ribavirin, and sofosbuvir can be an option in case of treatment failure with DAAs first line regimens. Nevertheless, this is not mentioned in the retreatment guidelines, and this is just an assumed recommendation that needs to be evaluated in trials.

## **Authors' contributions**

All the authors have equally contributed to research design, editing, and finalising of this chapter.

## **Conflict of interest statement**

The authors declared that there is no conflict of interest regarding the publication of this chapter.

## **Acknowledgements**

This study was supported by the grants from the National Natural Science Foundation of China (No. 81370559), Shanghai major joint project for important diseases (2014ZYJB0201), and Shanghai joint project with advanced technology (SHDC12014122).

## **Abbreviations**

failed options with first line drugs [40–43]. In genotype 3 infections, the combination of sofosbuvir and ribavirin is suboptimal [54], so IFN-based PegIFN/RBV/SOF regimen or IFN-free combination of SOF and daclatasvir becomes the choice of treatment [40, 54, 59]. The phase 3 trials, sofosbuvir and velpatasvir have higher SVR, so it might be the choice of regimen in future [43, 58]. In genotype 4 infections, IFN-based PegIFN, RBV with simeprevir or sofosbuvir have SVR > 90% in treatment-naïve cases. However, SVR in partial or null responders is suboptimal [33, 61]. The IFN-free two DAAs or three DAAs with or without RBV has overall SVR > 90%, although no data available for partial or null responders or relapsers cases [42, 62, 65, 72]. In genotype 5 or 6 infections, IFN-based PegIFN/RBV/SOF has SVR of 90% in treatment-naïve [33]. However, two DAAs combinations have better SVR of > 95% [12, 43, 69, 70]. In this review, it showed that IFN-free DAAs regimens have better SVR and well tolerated compared to IFN-based regimen. The combination of DAAs with or without ribavirin has almost replaced the IFN-based combination therapy in present context. Nevertheless, we cannot exclude the fact that the combination of PegIFN alfa and ribavirin still leaves us an ultimate option in setting where all other options are

The combination of IFN-free DAAs regimens has superior in their efficacy and tolerability compared to IFN-based regimens in case of treatment of chronic hepatitis C in all genotypes. However, in genotypes 3, 4, 5 or 6, the IFN-based combination of pegylated interferon alfa, ribavirin, and sofosbuvir can be an option in case of treatment failure with DAAs first line regimens. Nevertheless, this is not mentioned in the retreatment guidelines, and this is just an

All the authors have equally contributed to research design, editing, and finalising of this

The authors declared that there is no conflict of interest regarding the publication of this

This study was supported by the grants from the National Natural Science Foundation of China (No. 81370559), Shanghai major joint project for important diseases (2014ZYJB0201),

and Shanghai joint project with advanced technology (SHDC12014122).

assumed recommendation that needs to be evaluated in trials.

not available [12, 14].

68 Advances in Treatment of Hepatitis C and B

**4. Conclusion**

**Authors' contributions**

**Acknowledgements**

**Conflict of interest statement**

chapter.

chapter.


## **Author details**

Ramesh Rana, Yizhong Chang, Jing Li, ShengLan Wang, Li Yang and ChangQing Yang\*

\*Address all correspondence to: cqyang@tongji.edu.cn

Division of Gastroenterology and Hepatology, Digestive Disease Institute, Tongji Hospital, Tongji University School of Medicine, Shanghai, PR China

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#### **Management of Hepatitis C Virus Infection in Patients with Cirrhosis Management of Hepatitis C Virus Infection in Patients with Cirrhosis Management of Hepatitis C Virus Infection in Patients with Cirrhosis**

Aziza Ajlan and Hussien Elsiesy Aziza Ajlan and Hussien Elsiesy Aziza Ajlan and Hussien Elsiesy

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66096

#### **Abstract**

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76 Advances in Treatment of Hepatitis C and B

459–464. DOI: 10.1016/S1473-3099(15)00529-0

gastro.2015.07.063

In this chapter, we review the history of HCV infection in patients with liver cirrhosis. Selection of appropriate regimens for HCV-infected patients with cirrhosis, consistent with approved indications, practice guidelines, and emerging data is presented. Finally, this chapter explains individualization of therapy to maximize SVR rates in HCV-infected patients with cirrhosis and to critically appraise the role of newer agents and regimens in the management of HCV-infected patients with cirrhosis.

**Keywords:** HCV, liver cirrhosis, treatment

## **1. Introduction**

Hepatitis C virus (HCV) is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC) [1]. It remains the main indication for liver transplantation in North America and Europe [2]. The indication for liver transplantation has changed in the past two decades where NASH surpasses HBV to become the second most common cause of liver transplantation but HCV remains unchanged.

Chronic hepatitis C infection in patients with cirrhosis escalates the chances of developing severe liver-related complications, including hepatic decompensation, hepatocellular cancer and subsequently, death. It is been a matter of large debate whether to treat cirrhotic patients and what could be the potential benefit as cirrhosis is irreversible. However, multiple studies have shown that successful treatment of hepatitis C in patients with compensated cirrhosis will decrease subsequent cirrhosis-related complications.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

HCV causes increased mortality compared to any other infection; therefore, both the American Association for the Study of the Liver (AASLD) and European Association for the Study of the Liver (EASL) guidelines recommend that treatment be indicated for all HCV-infected patients. However, due to outrageously high cost of the new directly acting antivirals (DAA), treating every HCV-infected patient is not practical even in countries with strong economy.

Given the high cost of the medications for HCV, both AASLD and EASL guidelines prioritize the treatment for specific population with liver cirrhosis among the top list.

The goal of HCV treatment in patients with liver cirrhosis depends on the stage of disease. For Child's class A compensated liver cirrhosis, the goal of treatment is to prevent progression or to reverse cirrhosis [3] and to decrease the prevalence of HCC [4–6].

The goal in decompensated liver cirrhosis is to reverse decompensation, delisting from the liver transplant waiting list or preventing the disease recurrence after liver transplantation [7– 9]. More importantly, achieving sustained virological response (SVR) was associated with reduced all-cause mortality in patients with advanced fibrosis related to HCV [6].

Several studies have shown reversal of cirrhosis, delisting from liver transplant waiting liver, improvement of liver function and decrease the risk of HCC in patients who achieved SVR. Decrease in model for end-stage liver disease (MELD) due to biochemical improvement without resolution of ascites may delay the liver transplantation by lowering the patient's rank on the liver transplant waiting list.

There are also studies showing prevention of disease recurrence after liver transplantation on those who achieved SVR before liver transplantation.

We predict NASH to be the leading cause of liver transplant in the next decade, not only because of the growing obesity epidemic and increasing rate of diabetes, but because of the predicted long-term effect of HCV treatment.

The HCV treatment has evolved since the introduction of Interferon monotherapy in early 1990 until having several options of highly effective interferon-free DAA.

The first randomized multicentre trial comparing interferon alfa-2b versus no treatment in compensated HCV cirrhosis did not show benefit, whoever, it was small in number, have high drop-out rate and did not evaluate the patients who achieved sustained virological response (SVR) well but established safety [10]. In the same year, a study showed that patients with chronic hepatitis C who have an SVR to IFN therapy, there is a dramatic effect on normalization of ALT levels, improvement of histological activity and slowing of fibrosis progression [11].

From 2000 to 2011, the combination of PEG-IFN/RBV became the standard of care for HCV treatment, the overall SVR is 40–50% in genotypes 1 and 4 and 70–80% in genotypes 2 and 3; however, the SVR rate was significantly lower in patients with liver cirrhosis, about 22% for genotypes 1 and 4, and 55% for genotypes 2 and 3 [12–14].

Treating patients with decompensated HCV cirrhosis was challenging, it is associated with poor tolerance, higher side effect profile, and lower SVR rate. Everson and co-workers reported the results of a low-accelerating dose regimen of IFN or PEG-IFN with RBV in 124 patients with decompensated cirrhosis. The SVR was 24%, it was significantly lower in patients with genotype 1 (13%) than in those with non-1 genotype (50%); (*P* < 0.0001). SVR was highly predictive of maintaining viral clearance after liver transplant [8].

Forns et al. evaluated the treatment with IFN a-2b/RBV in 30 patients awaiting Orthotopic liver transplant (OLT) [9]. A virological response was observed in nine patients (30%). After LT, six of them (20%) remained negative after liver transplantation.

The study by Carrion et al. evaluated PEG-IFN/RBV therapy in 51 patients with HCV and cirrhosis awaiting LT matched with 51 untreated controls [15]. The aim of this study is to evaluate both the prevention of post-transplantation recurrent HCV and the risk of bacterial infections during therapy. Only 15 patients (29%) were HCV RNA-negative at transplantation and 10 (20%) achieved an SVR after transplantation.

There is major safety concern of PEG-IFN therapy in patients with decompensated cirrhosis. The haematological side effect includes neutropenia (50–60%), thrombocytopaenia (30–50%), and anaemia (30–60%). There is an increased risk of infection (4–13%) or hepatic decompensation during therapy (11–20%) [8, 9]. Carrion et al. reported high incidence of episodes of bacterial infection, mostly spontaneous bacterial peritonitis in treated patients (25%) compared to controls (6%) (*P* = 0.01) [15]. Variables independently associated with the occurrence of bacterial infections were antiviral treatment and a Child-Pugh score of B–C. The adverse effect of this therapy increase as the child score increase, where child C patients has very high complication rate with extremely low response. We reported the safety and efficacy of PEG-IFN and ribavirin therapy in 90 patients with liver cirrhosis, 18% required dose reduction, 33% stopped treatment because of adverse effects, 9% had deterioration of liver function, 7% died and13% of patients SVR. The rate of serious complications was 16.3% in child's class A, 48% in B, and 100% in C (*P* = 0.005). Serum albumin was a significant predictor for worsening liver function (*P* = 0.007), none of the child C patients achieved SVR [16].

## **2. New direct acting antivirals (DAAs)**

HCV causes increased mortality compared to any other infection; therefore, both the American Association for the Study of the Liver (AASLD) and European Association for the Study of the Liver (EASL) guidelines recommend that treatment be indicated for all HCV-infected patients. However, due to outrageously high cost of the new directly acting antivirals (DAA), treating

Given the high cost of the medications for HCV, both AASLD and EASL guidelines prioritize

The goal of HCV treatment in patients with liver cirrhosis depends on the stage of disease. For Child's class A compensated liver cirrhosis, the goal of treatment is to prevent progression or

The goal in decompensated liver cirrhosis is to reverse decompensation, delisting from the liver transplant waiting list or preventing the disease recurrence after liver transplantation [7– 9]. More importantly, achieving sustained virological response (SVR) was associated with

Several studies have shown reversal of cirrhosis, delisting from liver transplant waiting liver, improvement of liver function and decrease the risk of HCC in patients who achieved SVR. Decrease in model for end-stage liver disease (MELD) due to biochemical improvement without resolution of ascites may delay the liver transplantation by lowering the patient's rank

There are also studies showing prevention of disease recurrence after liver transplantation on

We predict NASH to be the leading cause of liver transplant in the next decade, not only because of the growing obesity epidemic and increasing rate of diabetes, but because of the

The HCV treatment has evolved since the introduction of Interferon monotherapy in early 1990

The first randomized multicentre trial comparing interferon alfa-2b versus no treatment in compensated HCV cirrhosis did not show benefit, whoever, it was small in number, have high drop-out rate and did not evaluate the patients who achieved sustained virological response (SVR) well but established safety [10]. In the same year, a study showed that patients with chronic hepatitis C who have an SVR to IFN therapy, there is a dramatic effect on normalization of ALT levels, improvement of histological activity and slowing of fibrosis progression [11]. From 2000 to 2011, the combination of PEG-IFN/RBV became the standard of care for HCV treatment, the overall SVR is 40–50% in genotypes 1 and 4 and 70–80% in genotypes 2 and 3; however, the SVR rate was significantly lower in patients with liver cirrhosis, about 22% for

Treating patients with decompensated HCV cirrhosis was challenging, it is associated with poor tolerance, higher side effect profile, and lower SVR rate. Everson and co-workers reported the results of a low-accelerating dose regimen of IFN or PEG-IFN with RBV in 124 patients

every HCV-infected patient is not practical even in countries with strong economy.

reduced all-cause mortality in patients with advanced fibrosis related to HCV [6].

the treatment for specific population with liver cirrhosis among the top list.

to reverse cirrhosis [3] and to decrease the prevalence of HCC [4–6].

on the liver transplant waiting list.

78 Advances in Treatment of Hepatitis C and B

those who achieved SVR before liver transplantation.

until having several options of highly effective interferon-free DAA.

genotypes 1 and 4, and 55% for genotypes 2 and 3 [12–14].

predicted long-term effect of HCV treatment.

Accordingly, the AASLD-IDSA guidelines consider any patient with chronic hepatitis C infection who is diagnosed with compensated cirrhosis highest priority for hepatitis C treatment [4].

For HCV-infected patients with decompensated cirrhosis or hepatocellular cancer, treatment of HCV may provide benefit, but the treatment plans and goals may need modifying if the patient is planning to undergo liver transplantation.

## **2.1. Patients with compensated cirrhosis**

For patients with compensated cirrhosis (Child-Turcotte-Pugh Class A), including those with hepatocellular carcinoma, the AASLD/IDSA/IAS-USA guidance [4] recommends using the same general treatment approach as used for patients without cirrhosis, with several key exceptions primarily related to duration of therapy or inclusion of ribavirin.

## *2.1.1. Genotype 1*

## *2.1.1.1. Ledipasvir/sofosbuvir*

Ledipasvir (90 mg) and sofosbuvir (400 mg) are a fixed-dose combination (Harvoni®) of two direct-acting antiviral agents that were initially studied in the ION-1 trial. The trial that included 865 treatment-naïve patients, looked at the length of treatment (12 weeks versus 24 weeks) as well as the need for RBV [5]. SVR12 rates exceeded 97%, with no added benefit observed with longer treatment duration, the addition of RBV length of treatment, nor HCV genotype 1 subtype. In the study, 16% of the included patients had cirrhosis. The presence of cirrhosis did not affect SVR12 rates compared with those without cirrhosis (97%) versus (98%) [5].

## *2.1.1.2. Paritaprevir/ritonavir/ombitasvir + dasabuvir (PrOD)*

The 3D combination was studied in the TURQUOISE-II and TURQUOISE-III trials. The trail included 261, HCV genotype 1a and CTP class A, the patients were both treatment-naïve and -experienced. The study compared 12 weeks or 24 weeks of PrOD regimen with the addition of RBV. SVR12 rates were higher in patients who received 24 weeks arm (89% vs. 95%) [6]. Factors that may have contributed to these differences could be the inclusion of patients who failed previous PEG-IFN/RBV therapy. Overall, treatment-naïve patient had slightly better response to therapy (92% vs. 95%). Interestingly, in patients with HCV genotype 1b patient, the SVR12 rates reached 98.5% in the 12-week arm [6]. Subsequently, the TURQUOISE-III trail questioned the role of RBV with the 3D regimen for 12 weeks in patients with HCV genotype 1b and compensated cirrhosis. Among the 60 patients included, more than 50% of the patients had negative predictors of response as follows: 55% treatment-experienced, 83% with IL28B non-CC genotype, 22% had platelet counts of greater than 90 × 109 L−1, and 17% had albumin levels greater than 3.5 g/dL). SVR12 rates were 100%. Hence, this regimen was approved for HCV genotype 1b for 12 weeks irrespective of previous treatment history or the presence or of cirrhosis [7].

The PrOD regimen, however, carries FDA warning [8]. In October 2015, the US FDA announced that the PrOD and PrO are contraindicated in patients with Child-Turcotte-Pugh (CTP) class B or C cirrhosis. This was based on reports by the manufacturer of accelerated liver injury in patients who were receiving PrOD or PrO. The onset of liver harm and decompensating incidents were observed mainly during the first month of therapy and mainly involved a quick rise in total and direct bilirubin, as well as a concomitant increase in liver transaminases. Timely recognition and termination of PrOD or PrO resulted in resolution of injury, death was reported in two cases with compensated cirrhosis. If the decision is made to initiate treatment with PrOD or PrO, patients should be made aware of the risks associated with such therapy in addition to adequate monitoring.

#### *2.1.1.3. Simeprevir + sofosbuvir*

same general treatment approach as used for patients without cirrhosis, with several key

Ledipasvir (90 mg) and sofosbuvir (400 mg) are a fixed-dose combination (Harvoni®) of two direct-acting antiviral agents that were initially studied in the ION-1 trial. The trial that included 865 treatment-naïve patients, looked at the length of treatment (12 weeks versus 24 weeks) as well as the need for RBV [5]. SVR12 rates exceeded 97%, with no added benefit observed with longer treatment duration, the addition of RBV length of treatment, nor HCV genotype 1 subtype. In the study, 16% of the included patients had cirrhosis. The presence of cirrhosis did not affect SVR12 rates compared with those without cirrhosis (97%) versus (98%)

The 3D combination was studied in the TURQUOISE-II and TURQUOISE-III trials. The trail included 261, HCV genotype 1a and CTP class A, the patients were both treatment-naïve and -experienced. The study compared 12 weeks or 24 weeks of PrOD regimen with the addition of RBV. SVR12 rates were higher in patients who received 24 weeks arm (89% vs. 95%) [6]. Factors that may have contributed to these differences could be the inclusion of patients who failed previous PEG-IFN/RBV therapy. Overall, treatment-naïve patient had slightly better response to therapy (92% vs. 95%). Interestingly, in patients with HCV genotype 1b patient, the SVR12 rates reached 98.5% in the 12-week arm [6]. Subsequently, the TURQUOISE-III trail questioned the role of RBV with the 3D regimen for 12 weeks in patients with HCV genotype 1b and compensated cirrhosis. Among the 60 patients included, more than 50% of the patients had negative predictors of response as follows: 55% treatment-experienced, 83% with IL28B

levels greater than 3.5 g/dL). SVR12 rates were 100%. Hence, this regimen was approved for HCV genotype 1b for 12 weeks irrespective of previous treatment history or the presence or

The PrOD regimen, however, carries FDA warning [8]. In October 2015, the US FDA announced that the PrOD and PrO are contraindicated in patients with Child-Turcotte-Pugh (CTP) class B or C cirrhosis. This was based on reports by the manufacturer of accelerated liver injury in patients who were receiving PrOD or PrO. The onset of liver harm and decompensating incidents were observed mainly during the first month of therapy and mainly involved a quick rise in total and direct bilirubin, as well as a concomitant increase in liver transaminases. Timely recognition and termination of PrOD or PrO resulted in resolution of injury, death was reported in two cases with compensated cirrhosis. If the decision is made to initiate treatment with PrOD or PrO, patients should be made aware of the risks associated with such therapy in addition

L−1, and 17% had albumin

exceptions primarily related to duration of therapy or inclusion of ribavirin.

*2.1.1.2. Paritaprevir/ritonavir/ombitasvir + dasabuvir (PrOD)*

non-CC genotype, 22% had platelet counts of greater than 90 × 109

*2.1.1. Genotype 1*

[5].

of cirrhosis [7].

to adequate monitoring.

*2.1.1.1. Ledipasvir/sofosbuvir*

80 Advances in Treatment of Hepatitis C and B

Simiprevir + sofosbuvir regimen were studied in the OPTIMIST-2 trial. The single armed, openlabel trial looked at 12 weeks of simeprevir plus sofosbuvir in 103 cirrhotic patients [9]. SVR12 rates were 88% (44/50) of treatment-naïve and 79% (42/53) of treatment-experienced patients with the total SVR12 rate was 83% (86/103). Furthermore, both genotype 1a and the presence of Q80K mutation negatively affected SVR12 (genotype 1 and 1b 84% [26/31] and 92% [35/38], respectively. And 74% [25/34] with Q80K mutation. Currently, there is no data that proves that extending treatment, with or without the addition of RBV, will increase efficacy of these two groups. Hence, until further data proves otherwise, this regimen should be avoided in patients with genotype 1a or in the case Q80K mutation is present.

#### *2.1.1.4. Daclatasvir + sofosbuvir*

Cirrhotic patients tend to take advantage from extension of therapy with daclatasvir and sofosbuvir to 24 weeks, with or without RBV [10, 11]. The data from ALLY-1 trial investigated daclatasvir and sofosbuvir with RBV dosed at 600 mg, in 60 patients with advanced cirrhosis [12]. Only 76% of patients with HCV genotype 1a (*n* = 34) and 100% of patients with HCV genotype 1b (*n* = 11) achieved an SVR at 12 weeks (SVR12). It is unclear how many treatment failures were among treatment-naïve patient was 54% or those with CTP class A cirrhosis. SVR was significantly lower in CTP class C cirrhosis (54%) when compared with CTP classes A and B 92% and 94% (see **Table 1**).


&With 88% (44/50) of treatment-naïve and 79% (42/53) of treatment-experienced patients.

^With ribavirin.

\$100% SVR12 rates achieved with extending the duration to 16 weeks.

\*Treatment naïve. SOF: sofosbuvir, SIM: simiprevir, DAC: daclatasvir LED: ledipasvir, PrOD: paritaprevir, ritonavir, ombitasvir and dasabuvir. PrO: paritaprevir, ritonavir, ombitasvir. VEL: velpatasvir, GRZ: grazoprevir, ELB: elbasvir.

**Table 1.** SVR12 rates among HCV-infected patients with compensated cirrhosis.

## *2.1.1.5. Elbasvir/grazoprevir*

For genotype 1a, recommendations for cirrhotic patients are based on 92 (22%) patients in the phase III C-EDGE trial that had Metavir F4 disease [13]. SVR 12 was 97% in the subgroup of cirrhotic patients. A similar 97% (28/29) SVR 12 rate had previously been demonstrated in genotype 1 cirrhotic treatment-naïve patients treated with 12 weeks of elbasvir/grazoprevir without ribavirin in the open-label phase II C-WORTHY trial [14]. The presence or absence of compensated cirrhosis does not appear to alter the efficacy of the elbasvir/grazoprevir regimen [13, 14].

The presence of NS5A resistance-associated variants (RAVs) at baseline was found to be associated with reduced efficacy in patients with genotype 1a, and was not apparent with genotype 1b [13]. In this phase III open-label trial of elbasvir/grazoprevir that enrolled treatment-experienced patients; among 58 genotype 1a patients who received 16 weeks of therapy with elbasvir/grazoprevir plus ribavirin, there were no virologic failures and the SVR12 rates were 100% [15–17].

## *2.1.1.6. Sofosbuvir/velpatasvir*

The use of this combination in patients with decompensated cirrhosis was investigated in the ASTRAL-4 trial. The study was multicentre, open-label patients were randomly assigned in a 1:1:1 ratio to receive a fixed-dose combination tablet containing 400 mg of sofosbuvir and 100 mg of velpatasvir, administered orally once daily for 12 weeks; sofosbuvir-velpatasvir plus ribavirin once daily for 12 weeks; or sofosbuvir-velpatasvir once daily for 24 weeks. Ribavirin was administered orally with food twice daily, with the dose determined according to body weight (1000 mg daily in patients with a body weight of greater than 75 kg and 1200 mg daily in patients with a body weight ≥75 kg). The overall SVR12 rates in the three groups were 83, 94 and 86%, respectively. The study highlights a potential role of RBV in such population [18]. Nineteen percent of the patients included in the ASTRAL-1 study had cirrhosis and observed SVR12 rates of 99% when received sofosbuvir/velpatasvir for 12 weeks [19].

## *2.1.2. Genotype 2*

Sofosbuvir (400 mg daily) was combined with weight-based RBV for treatment-naïve patients with HCV genotype 2 infection in three clinical trials, each of which enrolled patients with HCV genotype 2 or 3: FISSION, POSITRON and VALENCE with very high SVR12 rates [20– 22]. However, patients with cirrhosis have lower response rates that were seen in treatmentnaïve patients with cirrhosis compared to in those without cirrhosis [23]. One may consider extending treatment duration when cirrhosis is present despite the lack of data to support such extension, as longer treatment duration is known to improve SVR in treatment-experienced patients with cirrhosis [22, 24]. Due to the small numbers of patients with HCV genotype 2 infection and cirrhosis enrolled in the registration trials, several phase III b studies are ongoing to specifically determine the appropriate length of treatment for this subgroup of patients (see **Table 1**).

#### *2.1.3. Genotype 3*

*2.1.1.5. Elbasvir/grazoprevir*

82 Advances in Treatment of Hepatitis C and B

regimen [13, 14].

*2.1.2. Genotype 2*

**Table 1**).

SVR12 rates were 100% [15–17].

*2.1.1.6. Sofosbuvir/velpatasvir*

For genotype 1a, recommendations for cirrhotic patients are based on 92 (22%) patients in the phase III C-EDGE trial that had Metavir F4 disease [13]. SVR 12 was 97% in the subgroup of cirrhotic patients. A similar 97% (28/29) SVR 12 rate had previously been demonstrated in genotype 1 cirrhotic treatment-naïve patients treated with 12 weeks of elbasvir/grazoprevir without ribavirin in the open-label phase II C-WORTHY trial [14]. The presence or absence of compensated cirrhosis does not appear to alter the efficacy of the elbasvir/grazoprevir

The presence of NS5A resistance-associated variants (RAVs) at baseline was found to be associated with reduced efficacy in patients with genotype 1a, and was not apparent with genotype 1b [13]. In this phase III open-label trial of elbasvir/grazoprevir that enrolled treatment-experienced patients; among 58 genotype 1a patients who received 16 weeks of therapy with elbasvir/grazoprevir plus ribavirin, there were no virologic failures and the

The use of this combination in patients with decompensated cirrhosis was investigated in the ASTRAL-4 trial. The study was multicentre, open-label patients were randomly assigned in a 1:1:1 ratio to receive a fixed-dose combination tablet containing 400 mg of sofosbuvir and 100 mg of velpatasvir, administered orally once daily for 12 weeks; sofosbuvir-velpatasvir plus ribavirin once daily for 12 weeks; or sofosbuvir-velpatasvir once daily for 24 weeks. Ribavirin was administered orally with food twice daily, with the dose determined according to body weight (1000 mg daily in patients with a body weight of greater than 75 kg and 1200 mg daily in patients with a body weight ≥75 kg). The overall SVR12 rates in the three groups were 83, 94 and 86%, respectively. The study highlights a potential role of RBV in such population [18]. Nineteen percent of the patients included in the ASTRAL-1 study had cirrhosis and observed

Sofosbuvir (400 mg daily) was combined with weight-based RBV for treatment-naïve patients with HCV genotype 2 infection in three clinical trials, each of which enrolled patients with HCV genotype 2 or 3: FISSION, POSITRON and VALENCE with very high SVR12 rates [20– 22]. However, patients with cirrhosis have lower response rates that were seen in treatmentnaïve patients with cirrhosis compared to in those without cirrhosis [23]. One may consider extending treatment duration when cirrhosis is present despite the lack of data to support such extension, as longer treatment duration is known to improve SVR in treatment-experienced patients with cirrhosis [22, 24]. Due to the small numbers of patients with HCV genotype 2 infection and cirrhosis enrolled in the registration trials, several phase III b studies are ongoing to specifically determine the appropriate length of treatment for this subgroup of patients (see

SVR12 rates of 99% when received sofosbuvir/velpatasvir for 12 weeks [19].

#### *2.1.3.1. Sofosbuvir/daclatasvir*

ALLY-3 is a phase III study of the once-daily NS5A inhibitor daclatasvir plus sofosbuvir for 12 weeks; the study included 101 treatment-naïve patients and demonstrated an SVR12 rate of 90%. Cirrhotic patients (Metavir F4), 58% achieved SVR12 [25]. Hence extension of therapy may be considered in such cases. European compassionate use program has supported these recommendations in cohort studies, which reported and improvement in rates of up to 70% versus 86% when daclatasvir and sofosbuvir was used for 12 weeks and 24 weeks. RBV did not seem to have a big impact on SVR12 (85.9% without RBV compared to 81.3% with RBV). SVR12 rates were also higher in those with compensated Child-Pugh A cirrhosis (85–90% compared to 70.6% in child B/C). Previous data suggested that SVR 12 rates were higher in treatment-naïve patients (91–100%) compared to experienced (81–82%) [26].

#### *2.1.4. Genotype 4*

## *2.1.4.1. Ledipasvir/sofosbuvir*

The SYNERGY trial was an open-label study evaluating 12 weeks of ledipasvir/sofosbuvir in 21 HCV genotype 4-infected patients, Among that 60% were treatment-naïve and 43% had advanced fibrosis (Metavir stage F3 or F4) [27]. All patients achieved an SVR12. Note that the study used an assay by ROCH with lower limit of quantitation (LLOQ) of 43 IU/ml, while the AASLD guidelines recommended to use an assay with LLOQ of 25 IU/ml. However, this had no impact on SVR12 results [28].

## *2.1.4.2. Paritaprevir/ritonavir/ombitasvir (PrO)*

Pro regimen has interesting SVR12 rates according to the AGATE-I trial. The trial randomized 120 subjects with genotype 4 HCV and compensated cirrhosis to 12 weeks or 16 weeks of paritaprevir/ritonavir/ombitasvir (PrO) in addition to weight-based ribavirin. The SVR12 rates were 96% and 100% in the 12 week and 16 week arms, respectively [29]. On the other hand, the AGATE-II trial randomized 60 patients with compensated (1:1) to Pro for either 12 weeks or 24 weeks. SVR12 rates in the 12 weeks group were 97% versus 93% in the 24 week group [30].

#### *2.1.4.3. Sofosbuvir/simiprevir*

In a study by Moreno et al., the combination was studied in patients with advanced fibrosis/cirrhosis. All patients achieved end of treatment response but SVR12 data were not available [31]. In another study by Kayali et al., the combination was found to achieve SVR12 rates of 77% MELD scores remain unchanged. Interestingly, black gender and BMI were identified as independent negative predictors of response in univariate regression analysis (see **Table 1**) [32].

## **3. Patients with decompensated cirrhosis**

## **3.1. Sofosbuvir/ledipasvir**

The SOLAR-1 study was a multicentre, randomized controlled trial of 108 patients with HCV genotype 1 and 4 who had decompensated cirrhosis, of whom 59 were classified as CTP class B and 49 classified as CTP class C cirrhosis. Subjects were randomly assigned to receive daily fixed dose combination ledipasvir/sofosbuvir and RBV (initial dose of 600 mg, increased as tolerated) for 12 or 24 weeks. Extension of treatment in cirrhotic patients did not seem to affect SVR rates much. For CTP B patients, SVR rates were 87% versus 89% in subjects who received 12 versus 24 weeks, respectively. Likewise, the rates of SVR CTP class C subjects were 86 and 87%, respectively, with 12 and 24 weeks of antiviral therapy [33]. During the study, only one patient with CTP class C cirrhosis died.

The SOLAR-2 study was a multicentre randomized controlled trial of 108 subjects with decompensated cirrhosis secondary to HCV genotypes 1 and 4. Some of the patients were treatment-experienced, with CTP class B cirrhosis or CTP class C cirrhosis. The patients were randomly assigned to receive daily fixed-dose combination ledipasvir/sofosbuvir and RBV (initial dose of 600 mg, increased as tolerated) for 12 weeks or 24 weeks. Sustained virologic response (SVR) was achieved in 87% of those given the 12-week treatment course and 89% of those given the 24-week treatment course. On the 4th week of treatment, the total bilirubin and serum albumin levels improved compared with baseline in all patients. Despite the fact that some patients experienced worsening of hepatic function, baseline CTP and model for endstage liver disease (MELD) scores improved in more than 50% of the treated patients. Five patients died during the study period but none of the death occurred was attributed to the study medication. Adverse events were more common in the 24-week arm (34%) than in the 12-week arm (15%). These results indicate that a 12-week course of ledipasvir/sofosbuvir and RBV (initial dose of 600 mg, increased as tolerated) is an appropriate regimen for patients with decompensated cirrhosis who are infected with HCV genotype 1 or 4. Such therapy may lead to objective improvements in hepatic function and reduce the likelihood of recurrent HCV infection after subsequent transplantation [33].

## **3.2. Sofosbuvir/daclatasvir**

Patients with advanced cirrhosis (Child-Turcotte-Pugh [CTP] class B and C; *n* = 60) were particularly investigated in the ALLY-1 study [34]. The study found the use of daclatasvir (60 mg daily) with sofosbuvir (400 mg) and low initial dose of RBV (600 mg) for 12 weeks to treatment-naïve and -experienced patients with HCV genotype 1 infection. The overall SVR12 rate was 83% among those with advanced cirrhosis. The SVR12 rate was slightly lower in patients with genotype 1a compared with patients with genotype 1b (76 and 100%, respectively). Response rates were also affected by severity of disease among those with advanced cirrhosis (94% SVR12 rates in patients with CTP class B and 56% in patients with CTP class C). Patients with genotype 3 had also lower SVR12 rates 83%.

In another real-world study by Foster et al., involving 235 genotype 1 patients with decompensated cirrhosis, the SVR rates were comparable in the genotype 1 subjects (*n* = 235) receiving SOF/LDV/RBV or SOF/LDV (86% vs. 81%) and those receiving SOF/DCV/RBV or SOF/DCV therapy (82–60%). In this study, 91% of the patients received ribavirin with 20% requiring a RBV dose reduction and only 6% discontinued RBV. Improvement in MELD scores was observed in 42% of treated patients and worsening occurred in 11%. Moreover, 14 deaths occurred with relatively higher incidence of SAE (26%) but none were attributed to study medication.

## **3.3. Genotype 2 and 3**

**3. Patients with decompensated cirrhosis**

The SOLAR-1 study was a multicentre, randomized controlled trial of 108 patients with HCV genotype 1 and 4 who had decompensated cirrhosis, of whom 59 were classified as CTP class B and 49 classified as CTP class C cirrhosis. Subjects were randomly assigned to receive daily fixed dose combination ledipasvir/sofosbuvir and RBV (initial dose of 600 mg, increased as tolerated) for 12 or 24 weeks. Extension of treatment in cirrhotic patients did not seem to affect SVR rates much. For CTP B patients, SVR rates were 87% versus 89% in subjects who received 12 versus 24 weeks, respectively. Likewise, the rates of SVR CTP class C subjects were 86 and 87%, respectively, with 12 and 24 weeks of antiviral therapy [33]. During the study, only one

The SOLAR-2 study was a multicentre randomized controlled trial of 108 subjects with decompensated cirrhosis secondary to HCV genotypes 1 and 4. Some of the patients were treatment-experienced, with CTP class B cirrhosis or CTP class C cirrhosis. The patients were randomly assigned to receive daily fixed-dose combination ledipasvir/sofosbuvir and RBV (initial dose of 600 mg, increased as tolerated) for 12 weeks or 24 weeks. Sustained virologic response (SVR) was achieved in 87% of those given the 12-week treatment course and 89% of those given the 24-week treatment course. On the 4th week of treatment, the total bilirubin and serum albumin levels improved compared with baseline in all patients. Despite the fact that some patients experienced worsening of hepatic function, baseline CTP and model for endstage liver disease (MELD) scores improved in more than 50% of the treated patients. Five patients died during the study period but none of the death occurred was attributed to the study medication. Adverse events were more common in the 24-week arm (34%) than in the 12-week arm (15%). These results indicate that a 12-week course of ledipasvir/sofosbuvir and RBV (initial dose of 600 mg, increased as tolerated) is an appropriate regimen for patients with decompensated cirrhosis who are infected with HCV genotype 1 or 4. Such therapy may lead to objective improvements in hepatic function and reduce the likelihood of recurrent HCV

Patients with advanced cirrhosis (Child-Turcotte-Pugh [CTP] class B and C; *n* = 60) were particularly investigated in the ALLY-1 study [34]. The study found the use of daclatasvir (60 mg daily) with sofosbuvir (400 mg) and low initial dose of RBV (600 mg) for 12 weeks to treatment-naïve and -experienced patients with HCV genotype 1 infection. The overall SVR12 rate was 83% among those with advanced cirrhosis. The SVR12 rate was slightly lower in patients with genotype 1a compared with patients with genotype 1b (76 and 100%, respectively). Response rates were also affected by severity of disease among those with advanced cirrhosis (94% SVR12 rates in patients with CTP class B and 56% in patients with CTP class C).

**3.1. Sofosbuvir/ledipasvir**

84 Advances in Treatment of Hepatitis C and B

patient with CTP class C cirrhosis died.

infection after subsequent transplantation [33].

Patients with genotype 3 had also lower SVR12 rates 83%.

**3.2. Sofosbuvir/daclatasvir**

A multicentre, compassionate use study included 101 genotype 3 patients to be treated with daclatasvir (60 mg), sofosbuvir (400 mg) ± RBV for 24 weeks [35]. Of those, 81% had CTP class B cirrhosis, the MELD score was higher than15 in 16%, and 7% were post-liver transplant. The reported SVR 12 data has demonstrated an SVR of 85–100%. Two patients died while 22 patients had an SAE and therapy was discontinued in five subjects. Summary of SVR in Child B and C (**Table 2**).


\*SVR12 rate was 94% among patients with CTP class B cirrhosis but only 56% among patients with CTP class C cirrhosis.

SOF: sofosbuvir, SIM: simiprevir, DAC: daclatasvir, LED: ledipasvir, PrOD: paritaprevir, ritonavir, ombitasvir and dasabuvir. PrO: paritaprevir, ritonavir, ombitasvir. VEL: velpatasvir, GRZ: grazoprevir, ELB: elbasvir.

**Table 2.** SVR12 rates in patients with Child Pugh B and/or C cirrhosis.

## **4. Summary**

There is a remarkable advance in treatment of HCV in the recent few years allowing an excellent result in difficult to treat patients with liver cirrhosis with good safety profile.

Treating HCV in patients with liver cirrhosis is a high priority to prevent decompensation and prevent HCV recurrence after liver transplantation.

## **Author details**

Aziza Ajlan1 and Hussien Elsiesy2,3\*

\*Address all correspondence to: helsiesy@gmail.com

1 Department of Pharmacy, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia

2 Department of Liver Transplantation and Hepatobiliary Surgery, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia

3 Department of Medicine, Alfaisal, Riyadh, Saudi Arabia

## **References**


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Treating HCV in patients with liver cirrhosis is a high priority to prevent decompensation and

1 Department of Pharmacy, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi

2 Department of Liver Transplantation and Hepatobiliary Surgery, King Faisal Specialist

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## **Management of Hepaitits C Virus Genotype 4 in the Liver Transplant Setting Management of Hepaitits C Virus Genotype 4 in the Liver Transplant Setting**

Waleed K. Al-Hamoudi Waleed K. Al-Hamoudi

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65473

#### **Abstract**

End-stage liver disease secondary to hepatitis C virus (HCV) infection is the major indication for orthotopic liver transplantation (OLT) worldwide. It also has a negative impact on patient and graft survival leading to an inferior transplant outcome when compared to other liver transplant indications. The percentage of HCV patients infected with genotype 4 (G4) among recipients of OLT varies depending on geographic location. In the Middle East G4 infection is the most common genotype among transplant recipients. Direct antiviral agents (DAAs) have revolutionized the management of HCV infection in the pre- and post-transplant setting. Recent clinical trials have shown high sustained virologic response rates, shorter durations of treatment, and decreased adverse events when compared with the previous treatment of pegylated interferon (PEG-IFN)-based therapy. However, most of these studies were performed in HCV-G1 infected patients. Due to the low prevalence of HCV-G4 in Europe and the USA, this genotype has not been adequately studied in prospective trials evaluating treatment outcomes. The aim of this chapter is to summarize the natural history and treatment outcome of HCV-G4 in the liver transplant setting, with particular attention to new HCV therapies.

**Keywords:** cirrhosis, direct antiviral agents, genotype 4, hepatitis C, liver transplantation

## **1. Introduction**

Hepatitis C virus (HCV) infection is the leading indication for liver transplantation (LT) and is a major cause of liver-related mortality [1, 2]. It also has a negative impact on patient and graft survival leading to an inferior transplant outcome when compared with other indications [3, 4].

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

HCV eradication prior to LT will likely improve the outcome by eliminating the risk of post transplant recurrence. In the absence of an effective HCV vaccine to prevent infection and with therapy until very recently limited to interferon (IFN)-based regimens, most HCV-infected candidates for LT patients remained untreated.

Hepatitis C genotype 4 (HCV-G4) is the most prevalent genotype in the Middle East and Northern Africa [5–8]. The frequency of infection with HCV-G4 is also increasing in European countries, particularly among intravenous drug users [9–12]. The most common genotype in Europe and the USA is genotype 1; therefore, HCV-G4 has not been adequately studied in prospective trials evaluating treatment outcomes and remains the least studied variant.

The impact of HCV-G4 on treatment outcomes in the general nontransplant population has been evaluated [13–18]. Studies from the Middle East suggest a higher rate of spontaneous resolution after acute HCV-G4 infection [19, 20]. Other studies suggest that HCV-G4 infection is associated with significant steatosis. These observations suggest that specific features of HCV-G4 infection may contribute to the natural history and treatment outcomes of the disease [21, 22].

The percentage of HCV-G4 patients among recipients of orthotopic liver transplantation (OLT) varies depending on the geographic location. HCV-G4 represents more than 90% of indications for liver transplantation in Egypt [23]. In Saudi Arabia, hepatitis C represents ∼29% of indications for liver transplantation, ∼60% of which are secondary to HCV-G4 [24]. On the other hand, HCV-G4 is a relatively uncommon indication for liver transplantation in Europe and North America [25, 26].

Until recently, interferon-based therapy was the only treatment for HCV. However, this treatment has its own drawbacks given its prolonged therapeutic course (24–48 weeks), numerous side effects, low barrier to resistance, and reduced efficacy in prior null responders or cirrhotic patient. Direct antiviral agents (DAAs) represent a breakthrough in the management of HCV. First generation DAAs (telaprevir, boceprevir) in post-liver transplant patients resulted in sustained virological response (SVR) of up to 60% with telaprevir in HCV-G1. However, significant side effects including severe anemia, skin complications and significant drug interactions resulted in major concerns [27]. These agents are currently contraindicated and are not used anymore. Second line direct-acting antiviral DAAs have emerged with better safety and efficacy profiles, leading to dramatic changes in the practice of HCV management. Multiple clinical studies have shown superiority of sofosbuvir (SOF)-based therapy when compared with the current standard of care in both treatment naïve and treatment experienced patients and across all HCV genotypes [28–34]. Because of its favorable pharmacologic profile and its reasonable drug–drug interactions, sofosbuvir has become the cornerstone in the management of HCV infection [35]. Furthermore, data are emerging on the outcome of multiple newer agents. The, aim of this chapter is to examine the natural history and treatment outcomes of HCV-G4 following liver transplantation. This review includes all published studies and abstracts involving HCV-G4 patients.

## **2. Hepatitis C genotype influences post-liver transplantation**

HCV eradication prior to LT will likely improve the outcome by eliminating the risk of post transplant recurrence. In the absence of an effective HCV vaccine to prevent infection and with therapy until very recently limited to interferon (IFN)-based regimens, most HCV-infected

Hepatitis C genotype 4 (HCV-G4) is the most prevalent genotype in the Middle East and Northern Africa [5–8]. The frequency of infection with HCV-G4 is also increasing in European countries, particularly among intravenous drug users [9–12]. The most common genotype in Europe and the USA is genotype 1; therefore, HCV-G4 has not been adequately studied in prospective trials evaluating treatment outcomes and remains the least studied variant.

The impact of HCV-G4 on treatment outcomes in the general nontransplant population has been evaluated [13–18]. Studies from the Middle East suggest a higher rate of spontaneous resolution after acute HCV-G4 infection [19, 20]. Other studies suggest that HCV-G4 infection is associated with significant steatosis. These observations suggest that specific features of HCV-G4 infection may contribute to the natural history and treatment outcomes of the disease

The percentage of HCV-G4 patients among recipients of orthotopic liver transplantation (OLT) varies depending on the geographic location. HCV-G4 represents more than 90% of indications for liver transplantation in Egypt [23]. In Saudi Arabia, hepatitis C represents ∼29% of indications for liver transplantation, ∼60% of which are secondary to HCV-G4 [24]. On the other hand, HCV-G4 is a relatively uncommon indication for liver transplantation in Europe

Until recently, interferon-based therapy was the only treatment for HCV. However, this treatment has its own drawbacks given its prolonged therapeutic course (24–48 weeks), numerous side effects, low barrier to resistance, and reduced efficacy in prior null responders or cirrhotic patient. Direct antiviral agents (DAAs) represent a breakthrough in the management of HCV. First generation DAAs (telaprevir, boceprevir) in post-liver transplant patients resulted in sustained virological response (SVR) of up to 60% with telaprevir in HCV-G1. However, significant side effects including severe anemia, skin complications and significant drug interactions resulted in major concerns [27]. These agents are currently contraindicated and are not used anymore. Second line direct-acting antiviral DAAs have emerged with better safety and efficacy profiles, leading to dramatic changes in the practice of HCV management. Multiple clinical studies have shown superiority of sofosbuvir (SOF)-based therapy when compared with the current standard of care in both treatment naïve and treatment experienced patients and across all HCV genotypes [28–34]. Because of its favorable pharmacologic profile and its reasonable drug–drug interactions, sofosbuvir has become the cornerstone in the management of HCV infection [35]. Furthermore, data are emerging on the outcome of multiple newer agents. The, aim of this chapter is to examine the natural history and treatment outcomes of HCV-G4 following liver transplantation. This review includes all published

candidates for LT patients remained untreated.

92 Advances in Treatment of Hepatitis C and B

[21, 22].

and North America [25, 26].

studies and abstracts involving HCV-G4 patients.

Campos-Varela et al. evaluated the role of the various HCV genotypes on the progression and outcome of liver transplantation. Among 745 recipients, 81% had genotype 1 (G1), 7% had genotype 2 (G2), and 12% had genotype 3 (G3). Patients were followed for a median of 3.1 years (range 2–8 years). The risk of advanced fibrosis and graft rejection was significantly higher among those infected with G1 compared with other genotypes [36]. In another multicentre European study involving 652 liver recipients, genotype 1b, age, and absence of pretransplantation coinfection by HBV are risk factors for recurrent HCV. However, graft and patient survival was comparable to other genotypes [37]. Similarly, in another prospective study involving 60 liver transplant recipients, HCV 1b was associated with more aggressive recurrent liver disease than other genotypes [38]. Gordon et al. assessed the relationship between hepatitis C genotype on posttransplant frequency of recurrent hepatitis, histologic severity of recurrence, and progression to cirrhosis. They concluded that histologic evidence of recurrent hepatitis C is seen in 90% of liver allografts; however, genotype 1b was associated with more severe histologic disease recurrence and was more likely to progress to cirrhosis when compared to non-1b genotypes [39].

By contrast, some large studies have observed no difference in the rate or degree of hepatitis or in graft or patient survival between G1 and other genotypes [40, 41]. Therefore, the impact of various genotypes on the outcome of liver transplantation remains controversial. Due to the low prevalence of HCV G-4 in western countries, these studies neglected evaluating the impact of this particular genotype.

## **3. Natural history of HCV-G4 after liver transplantation**

Re-infection of the graft is universal after liver transplantation regardless of genotype, leading to an accelerated course of liver injury in many cases [42]. Most studies of disease recurrence worldwide have investigated HCV-G1, HCV-G2, and HCV-G3, and there are few reports on post-OLT recurrence of HCV-G4.

Zekry et al. analyzed factors that predicted outcome of HCV-liver transplant recipients in the Australian and New Zealand communities. The following variables were evaluated demographic factors, coexistent pathology at the time of transplantation, HCV genotype, and donor age. In this analysis, 182 patients were transplanted for HCV including 16 patients infected with genotype 4 and the median follow-up was 4 years. Among many factors studied in univariate and multivariate analyses, HCV-G4 was associated with an increased risk of retransplantation and death. Additionally, patients infected with HCV-G4 were more likely to progress to advanced stages of fibrosis [43]. Patients infected with G2 and G3 had better posttransplant outcomes. Whether this difference in outcomes was related to the pathogenicity of HCV-G4 or to other factors not examined in this study, including donor age, immunosuppression, and compliance with medications, is not clear (**Table 1**). Furthermore, patients infected with HCV-G4 in this study were older and more likely to have coexisting hepatocellular carcinoma. Gane et al. investigated the impact of persistent HCV infection after liver transplantation on patient and graft survival and the effects of the HCV genotype on the severity of recurrent hepatitis. A group of 149 patients with HCV infection who received liver transplants were followed for a median of 36 months; 623 patients without HCV infection who underwent liver transplantation for end-stage chronic liver disease were used as a control group. Among the patient population, 14 patients were infected with HCV-G4. Approximately 50% of these patients had progressive liver disease (moderate hepatitis or cirrhosis) during the follow-up period [44]. In the same study, patients infected with G1b had the worst outcome, whereas patients infected with G2 and G3 had less severe disease recurrence. The authors speculated that patients infected with G1b had an increased replicative potential and an increased expression of viral antigen in liver tissue. A more detailed study from the UK aimed at studying the impact of HCV-G4 on transplant outcome. The study group included 128 patients who underwent transplantation for HCV infection: 28 patients, genotype 1; 11 patients, genotype 2; 19 patients, genotype 3; and 32 patients, genotype 4 [45]. A significantly higher fibrosis progression rate was observed in HCV-G4 patients compared with non-G4 patients, although their rates of survival were similar. The 5-year cumulative rates for the development of cirrhosis or severe fibrosis were 84% in HCV-G4-infected patients and 24% in patients infected with other genotypes. The HCV-G4 groups were predominantly Egyptian patients who received organs from older donors. Furthermore, the majority of these patients were placed on an alternative waiting list to be offered organs that were suitable for transplantation but unsuitable or not needed for citizens of the UK. This policy may have led to the selection of inferior grafts for the HCV-G4 patients, who were predominantly non-UK citizens, leading to inferior results in these patients.

Factors affecting transplant outcome Viral load Genotype Coinfections Alcohol Compliance Steatosis Donor age Immunosuppression Rejection

**Table 1.** Factors affecting the outcome of HCV-related transplantation.

On the other hand, studies from the Middle East show a more favorable outcome. According to reports from Saudi Arabia and Egypt, overall graft and patient survival for HCV-G4 are comparable to rates reported in the international literature. Reports from Saudi Arabia reveal an overall 3-year graft and patient survival rates of 90 and 80%, respectively [24, 46–50]. Similarly, in Egypt, where many active living-related liver transplant programs exist and HCV-G4 represents more than 90% of cases, graft and patient survival rates are ∼86% [23].

Multiple recent studies from the Middle East evaluated the natural history of HCV-G4 following liver transplantation. Mudawi et al. conducted a study to determine the epidemiological, clinical and virological characteristics of patients with biopsy-proven recurrent HCV infection and analyzed the factors that influence recurrent disease severity. They also compared disease recurrence and outcomes between HCV-4 and other genotypes [51]. Of 116 patients who underwent OLT for hepatitis C, 46 (39.7%) patients satisfied the criteria of recurrent hepatitis C. Twenty-nine (63%) patients were infected with HCV genotype 4. Among many factors included in that analysis, the only factor predictive of an advanced histological score was the HCV RNA level at the time of biopsy. The conclusion was that HCV recurrence following OLT in HCV-4 patients is not significantly different from its recurrence for other genotypes.

carcinoma. Gane et al. investigated the impact of persistent HCV infection after liver transplantation on patient and graft survival and the effects of the HCV genotype on the severity of recurrent hepatitis. A group of 149 patients with HCV infection who received liver transplants were followed for a median of 36 months; 623 patients without HCV infection who underwent liver transplantation for end-stage chronic liver disease were used as a control group. Among the patient population, 14 patients were infected with HCV-G4. Approximately 50% of these patients had progressive liver disease (moderate hepatitis or cirrhosis) during the follow-up period [44]. In the same study, patients infected with G1b had the worst outcome, whereas patients infected with G2 and G3 had less severe disease recurrence. The authors speculated that patients infected with G1b had an increased replicative potential and an increased expression of viral antigen in liver tissue. A more detailed study from the UK aimed at studying the impact of HCV-G4 on transplant outcome. The study group included 128 patients who underwent transplantation for HCV infection: 28 patients, genotype 1; 11 patients, genotype 2; 19 patients, genotype 3; and 32 patients, genotype 4 [45]. A significantly higher fibrosis progression rate was observed in HCV-G4 patients compared with non-G4 patients, although their rates of survival were similar. The 5-year cumulative rates for the development of cirrhosis or severe fibrosis were 84% in HCV-G4-infected patients and 24% in patients infected with other genotypes. The HCV-G4 groups were predominantly Egyptian patients who received organs from older donors. Furthermore, the majority of these patients were placed on an alternative waiting list to be offered organs that were suitable for transplantation but unsuitable or not needed for citizens of the UK. This policy may have led to the selection of inferior grafts for the HCV-G4 patients, who were predominantly non-UK citizens,

leading to inferior results in these patients.

**Table 1.** Factors affecting the outcome of HCV-related transplantation.

On the other hand, studies from the Middle East show a more favorable outcome. According to reports from Saudi Arabia and Egypt, overall graft and patient survival for HCV-G4 are comparable to rates reported in the international literature. Reports from Saudi Arabia reveal an overall 3-year graft and patient survival rates of 90 and 80%, respectively [24, 46–50]. Similarly, in Egypt, where many active living-related liver transplant programs exist and HCV-

G4 represents more than 90% of cases, graft and patient survival rates are ∼86% [23].

Factors affecting transplant outcome

94 Advances in Treatment of Hepatitis C and B

Viral load Genotype Coinfections Alcohol Compliance Steatosis Donor age

Immunosuppression

Rejection

In studies published from Egypt reporting on living donor related liver (LDLT) transplantation of HCV-G4 patients, similar favorable outcomes were observed. In a recent Egyptian study 74 adult hepatitis C virus positive subjects were monitored for 36 months after living-donor liver transplant and demographic and laboratory data for the recipients and donors were evaluated. HCV clinical recurrence was observed in 31% of patients and was mostly mild; 91% of patients had fibrosis scores less than F2. And during the study period 91% of patients were alive with excellent graft function. Similar to the study from Saudi Arabia, recurrent HCV was associated with a high pre- and post-transplant viral load and the presence of antibodies to hepatitis B core antigen [52]. In another study, the outcome of LDLT was evaluated in Egyptian patients with HCV-G4-related cirrhosis. Recurrence of HCV was studied in 38 of 53 adult patients who underwent LDLT. Recipient and graft survivals were 86.6% at the end of the 16 ± 8.18 months (range, 4–35 months) follow-up period. Clinical HCV recurrence was observed in 10/38 patients (26.3%). None of the recipients developed allograft cirrhosis during the follow-up period [23]. In a recent study, Allam et al. compared the outcomes of Saudi and Egyptian patients who received liver transplantation either in China or locally in Saudi Arabia (∼30% infected with HCV-G4), respective 1- and 3-year cumulative survival rates were 81 and 59% in patients transplanted in China compared with 90 and 84% for patients transplanted locally. They attributed the poorer outcomes in patients transplanted in China to liberal selection criteria, the use of donations after cardiac death, and to the limited post-transplant care [53].

The role of HCV-G4 in the natural history of this disease requires further study. Furthermore, HCV-G4 exhibits significant genetic diversity, and there are a number of viral subtypes. The impacts of the various subtypes have been demonstrated in recent studies; for example, HCV G1 subtype 1b patients were more likely to achieve a rapid virological response (RVR) compared with subtype 1a [54]. Studies performed in Egypt, where HCV-G4 subtypes 4a and 4b predominate, have consistently indicated higher rates of virological response to therapy (69–76%) compared with Saudi Arabia, where response rates are substantially lower (44–50%) [55–57]. In a retrospective analysis of HCV-G4 patients, Roulot et al. reported better sustained virological response (SVR) in 4a subtype-compared with 4d subtype-infected individuals [58]. The majority of patients involved in these European/Australian studies are Egyptians, who are likely older, have coexisting HCC and have received marginal donor grafts. Co-morbidities, such as infection with schistosomiasis, and other nonstudied variables may also have affected outcomes in these patients, leading to an impression that HCV-G4 is an aggressive virus. However, more recent studies originating from the Middle East, where HCV-G4 predominates have revealed no significant difference in outcomes between G1 and G4.

## **4. Treatment prior to transplantation**

## **4.1. Pegylated interferon and ribavirin**

Viral eradication or suppression prior to liver transplantation reduces post-transplant recurrence rates [59]. Until recently, the only available treatment regimens were interferon-based and were therefore contraindicated in patients with advanced cirrhosis [60–62].

Everson et al. evaluated the effectiveness, tolerability, and outcome of a low accelerating dose regimen (LADR) of pegylated interferon (PEG-IFN) therapy in the treatment of patients with advanced HCV. One hundred twenty-four patients were treated with LADR. Sixty-three percent had clinical complications of cirrhosis (ascites, spontaneous bacterial peritonitis, varices, variceal hemorrhage, encephalopathy). Forty-six percent were HCV RNA-negative at end of treatment, and 24% were HCV RNA-negative at last follow-up. Twelve of 15 patients who were HCV RNA-negative before transplantation remained HCV RNA-negative 6 months or more after transplantation. They concluded that LADR may result in viral eradication, stabilize clinical course, and prevent posttransplantation recurrence [61]. In a more recent study patients with various genotypes were randomized 2:1 to treatment (*n* = 31) or untreated control (*n* = 16). Of the 30 patients who were treated, 23 underwent liver transplantation, and 22% achieved a post-transplantation virological response. Although pre-transplant treatment prevented post-transplant recurrence of HCV infection in 25% of cases, including patients infected with HCV-G4, this approach was poorly tolerated and resulted in life-threatening complications [63].

## **5. Treatment of advanced disease in the new era**

The treatment of HCV patients is rapidly evolving. New oral DAAs have emerged with better safety and efficacy profiles, leading to dramatic changes in the practice of HCV management. These choices include sofosbuvir plus weight-adjusted ribavirin (RBV), ledipasvir/sofosbuvir with or without RBV, sofosbuvir/daclatasvir with or without RBV, daclatasvir/simeprevir/ sofosbuvir, ombitasvir/paritaprevir/ritonavir with weight-adjusted RBV, elbasvir-grazoprevir with or without RBV. The choice between them depends primarily on potential for drug interactions, availability, and cost. Data on the use of these new agents in cirrhotic G4 patients awaiting liver transplantation are limited. Up-to-date studies evaluating the safety and efficacy of these agents in HCV-G4 patients are summarized below.

## **5.1. Sofosbuvir and ribavirin**

outcomes in these patients, leading to an impression that HCV-G4 is an aggressive virus. However, more recent studies originating from the Middle East, where HCV-G4 predominates

Viral eradication or suppression prior to liver transplantation reduces post-transplant recurrence rates [59]. Until recently, the only available treatment regimens were interferon-based

Everson et al. evaluated the effectiveness, tolerability, and outcome of a low accelerating dose regimen (LADR) of pegylated interferon (PEG-IFN) therapy in the treatment of patients with advanced HCV. One hundred twenty-four patients were treated with LADR. Sixty-three percent had clinical complications of cirrhosis (ascites, spontaneous bacterial peritonitis, varices, variceal hemorrhage, encephalopathy). Forty-six percent were HCV RNA-negative at end of treatment, and 24% were HCV RNA-negative at last follow-up. Twelve of 15 patients who were HCV RNA-negative before transplantation remained HCV RNA-negative 6 months or more after transplantation. They concluded that LADR may result in viral eradication, stabilize clinical course, and prevent posttransplantation recurrence [61]. In a more recent study patients with various genotypes were randomized 2:1 to treatment (*n* = 31) or untreated control (*n* = 16). Of the 30 patients who were treated, 23 underwent liver transplantation, and 22% achieved a post-transplantation virological response. Although pre-transplant treatment prevented post-transplant recurrence of HCV infection in 25% of cases, including patients infected with HCV-G4, this approach was poorly tolerated and resulted in life-threatening

The treatment of HCV patients is rapidly evolving. New oral DAAs have emerged with better safety and efficacy profiles, leading to dramatic changes in the practice of HCV management. These choices include sofosbuvir plus weight-adjusted ribavirin (RBV), ledipasvir/sofosbuvir with or without RBV, sofosbuvir/daclatasvir with or without RBV, daclatasvir/simeprevir/ sofosbuvir, ombitasvir/paritaprevir/ritonavir with weight-adjusted RBV, elbasvir-grazoprevir with or without RBV. The choice between them depends primarily on potential for drug interactions, availability, and cost. Data on the use of these new agents in cirrhotic G4 patients awaiting liver transplantation are limited. Up-to-date studies evaluating the safety and efficacy

and were therefore contraindicated in patients with advanced cirrhosis [60–62].

**5. Treatment of advanced disease in the new era**

of these agents in HCV-G4 patients are summarized below.

have revealed no significant difference in outcomes between G1 and G4.

**4. Treatment prior to transplantation**

**4.1. Pegylated interferon and ribavirin**

96 Advances in Treatment of Hepatitis C and B

complications [63].

Sofosbuvir (SOF) is a novel pangenotypic nucleotide analog inhibitor that inhibits HCV RNA replication. SOF is administered orally and inhibits the HCV NS5B polymerase. SOF exerts potent antiviral activity against all HCV genotypes [28–30, 32, 64].

In a recently published open-label study, 61 patients with HCV of any genotype awaiting liver transplantation for hepatocellular carcinoma were included. The primary end point was the proportion of patients with HCV-RNA levels <25 IU/ml at 12 weeks after transplantation among patients with this HCVRNA level at their last measurement before transplantation. Patients received up to 48 weeks of SOF/RBV before liver transplantation. Of 46 patients who were transplanted, 43 had HCV-RNA levels of <25 IU/ml at the time of transplantation. Of these 43 patients, 30 (70%) exhibited a post-transplantation virological response at 12 weeks [65]. A recently published study evaluated the efficacy and safety of SOF in combination with RBV in HCV-G4 patients in patients of Egyptian ancestry. Thirty treatment-naive and thirty previously treated patients were enrolled and treated for 12 weeks (*n* = 31) or 24 weeks (*n* = 29). Overall, 23% of patients had cirrhosis. SVR12 was achieved by 68% of patients in the 12-week group, and by 93% of patients in the 24-week group. No patient discontinued treatment due to an adverse event [66]. In another Egyptian study, 103 patients' studies were treated with a combination of SOF and weight-adjusted RBV. Seventeen percentage of the study population were cirrhotic. Patients with cirrhosis at baseline had lower rates of SVR12 (63% at 12 weeks, 78% at 24 weeks) than those without cirrhosis (80% at 12 weeks, 93% at 24 weeks). However, the treatment was safe and well tolerated, with no serious drug-related adverse events [67].

However, with the emergence of other treatment options, this combination is not considered the best treatment option.

## **5.2. Ledipasvir/sofosbuvir and ribavirin**

A recently published phase 2, open-label study (Solar-1) assessed treatment with ledipasvir (LDV), SOF, and RBV in patients infected with HCV-G1 or HCV-G4. This study included a cohort of patients with cirrhosis who had not undergone liver transplantation and another cohort of patients who had undergone liver transplantation. In the nontransplant cirrhotic group, SVR12 was achieved in 86–89% of patients. There were no differences in response rates in the 12- and 24-week groups [68]. In another study, 20 (95%) of 21 patients infected with HCV-4 completed 12 weeks of treatment and achieved SVR12 including seven patients with cirrhosis. One patient was non-adherent to study drugs and withdrew from the study but was included in the intention-to-treat analysis [69].

## **5.3. Sofosbuvir/daclatasvir/ribavirin**

The ALLY-1 study evaluated daclatasvir (DCV) + SOF + RBV in patients with advanced cirrhosis or post-transplant HCV recurrence of all genotypes, including G4. DCV is a pangenotypic NS5A inhibitor with a very low potential for drug interaction and a favorable safety profile. All patients with advanced cirrhosis were treated with a combination of DCV 60 mg + S0F 400 mg + RBV (adjusted dose) for 12 weeks. Overall, 83% of the advanced cirrhosis patients achieved SVR12. SVR12 rates were higher in patients with Child-Pugh class A or B, 93%, versus class C, 56%. The response rate of cirrhotic patients infected with HCV-G4 (4 patients) was 100%. Treatment was well tolerated, with no adverse events or drug-drug interactions [70].

## **5.4. Simeprevir/daclatasvir/sofosbuvir**

The interim results of the IMPACT study indicated favorable responses to this combination in cirrhotic patients infected with G1 and G4. Simeprevir (SIM) is a NS3/4A protease inhibitor with antiviral activity against G1, G2, G4, G5, and G6. All cirrhotic patients (100%) 28/28 achieved SVR4. The treatment was safe and well tolerated, with no major adverse effects. The study is ongoing, and final results will be reported later [71]. A recent report from Qatar has examined the efficacy and safety of Sofosbuvir/daclatasvir and Sofosbuvir/Simeprevir on 85 patients. SVR4 was achieved in 96% of the study population [72].

## **5.5. Ombitasvir, ritonavir and paritaprevir**

The combination of ombitasvir, ritonavir and paritaprevir was evaluated in a large cohort of non cirrhotic genotype-4 patients. After 12 weeks of treatment, 100% of naïve patient who had RBV containing regimen achieved SVR compared to 90.9% in the RBV free regimen. Furthermore, all treatment experienced patients achieved SVR [73]. This combination when used with RBV was also found very effective in HCV genotype-4 with compensated (child A) cirrhosis. Twelve and 16 weeks of treatment resulted in SVR12 of 97 and 100%, respectively [74]. This regimen in addition to dasabuvir was also effective in cirrhotic genotype 1b patient. SVR 12 was 100% in 60 compensated cirrhotic patients [75]. The regimen is contraindicated in Child Pugh classes B and C cirrhosis. More recently, an open-label, partly randomised trial in patients with chronic HCV genotype 4 infection was conducted in Egypt. One hundred and sixty patients were included; 100 patients were assessed as not having cirrhosis and were given 12 weeks of treatment, and 60 patients assessed as having cirrhosis were randomly assigned to the 12-week treatment group (*n* = 31) or the 24-week treatment group (*n* = 29). Ninety-four (94%) of 100 patients in the without cirrhosis group, 30 (97%) of 31 patients in the cirrhosis 12 week treatment group, and 27 (93%) of 29 patients in the cirrhosis 24-week treatment group achieved SVR12. Adverse events were predominantly mild or moderate in severity, and laboratory abnormalities were not clinically meaningful. No patients discontinued treatment because of an adverse event [76].

## **5.6. Elbasvir/grazoprevir**

In a recent study, an SVR rate of 96% was achieved in 56 treatment-naïve patients receiving 12 weeks of elbasvir-grazoprevir. In contrast, SVR rates were lower with only 12 weeks among a small number of treatment-experienced patients (78% in 9 patients) but were higher with the addition of ribavirin and treatment extension to 16 weeks (100% in 8 patients). SVR rates were similar in patients with and without cirrhosis. However, this regimen is contraindicated in Child Pugh classes B and C cirrhosis [77].

## **5.7. Sofosbuvir/velpatasvir**

S0F 400 mg + RBV (adjusted dose) for 12 weeks. Overall, 83% of the advanced cirrhosis patients achieved SVR12. SVR12 rates were higher in patients with Child-Pugh class A or B, 93%, versus class C, 56%. The response rate of cirrhotic patients infected with HCV-G4 (4 patients) was 100%. Treatment was well tolerated, with no adverse events or drug-drug interactions [70].

The interim results of the IMPACT study indicated favorable responses to this combination in cirrhotic patients infected with G1 and G4. Simeprevir (SIM) is a NS3/4A protease inhibitor with antiviral activity against G1, G2, G4, G5, and G6. All cirrhotic patients (100%) 28/28 achieved SVR4. The treatment was safe and well tolerated, with no major adverse effects. The study is ongoing, and final results will be reported later [71]. A recent report from Qatar has examined the efficacy and safety of Sofosbuvir/daclatasvir and Sofosbuvir/Simeprevir on 85

The combination of ombitasvir, ritonavir and paritaprevir was evaluated in a large cohort of non cirrhotic genotype-4 patients. After 12 weeks of treatment, 100% of naïve patient who had RBV containing regimen achieved SVR compared to 90.9% in the RBV free regimen. Furthermore, all treatment experienced patients achieved SVR [73]. This combination when used with RBV was also found very effective in HCV genotype-4 with compensated (child A) cirrhosis. Twelve and 16 weeks of treatment resulted in SVR12 of 97 and 100%, respectively [74]. This regimen in addition to dasabuvir was also effective in cirrhotic genotype 1b patient. SVR 12 was 100% in 60 compensated cirrhotic patients [75]. The regimen is contraindicated in Child Pugh classes B and C cirrhosis. More recently, an open-label, partly randomised trial in patients with chronic HCV genotype 4 infection was conducted in Egypt. One hundred and sixty patients were included; 100 patients were assessed as not having cirrhosis and were given 12 weeks of treatment, and 60 patients assessed as having cirrhosis were randomly assigned to the 12-week treatment group (*n* = 31) or the 24-week treatment group (*n* = 29). Ninety-four (94%) of 100 patients in the without cirrhosis group, 30 (97%) of 31 patients in the cirrhosis 12 week treatment group, and 27 (93%) of 29 patients in the cirrhosis 24-week treatment group achieved SVR12. Adverse events were predominantly mild or moderate in severity, and laboratory abnormalities were not clinically meaningful. No patients discontinued treatment

In a recent study, an SVR rate of 96% was achieved in 56 treatment-naïve patients receiving 12 weeks of elbasvir-grazoprevir. In contrast, SVR rates were lower with only 12 weeks among a small number of treatment-experienced patients (78% in 9 patients) but were higher with the addition of ribavirin and treatment extension to 16 weeks (100% in 8 patients). SVR rates were similar in patients with and without cirrhosis. However, this regimen is contraindicated in

patients. SVR4 was achieved in 96% of the study population [72].

**5.4. Simeprevir/daclatasvir/sofosbuvir**

98 Advances in Treatment of Hepatitis C and B

**5.5. Ombitasvir, ritonavir and paritaprevir**

because of an adverse event [76].

Child Pugh classes B and C cirrhosis [77].

**5.6. Elbasvir/grazoprevir**

Sofosbuvir and velpatasvir (NS5A inhibitor) is a pangenotypic combination that was recently evaluated in the ASTRAL-1 trial that included 624 naïve and treatment experienced patients, of whom 116 (19%) were genotype-4. Patients with compensated cirrhosis (19%) were included and all genotype-4 patients achieved SVR (100%) after 12 weeks of RBV-free treatment [78]. A phase 3 open-label study involving patients infected with HCV genotypes 1 through 6 who had decompensated cirrhosis was recently conducted. Patients were randomly assigned in a 1:1:1 ratio to receive sofosbuvir and velpatasvir once daily for 12 weeks, sofosbuvir-velpatasvir plus ribavirin for 12 weeks, or sofosbuvir-velpatasvir for 24 weeks. Overall rates of sustained virologic response were 83% among patients who received 12 weeks of sofosbuvir-velpatasvir, 94% among those who received 12 weeks of sofosbuvir-velpatasvir plus ribavirin, and 86% among those who received 24 weeks of sofosbuvir-velpatasvir [79].

## **6. Treatment after liver transplantation**

Earlier studies on preemptive treatment prior to established disease recurrence were disappointing. The conclusion of these studies was that the outcome of preemptive treatment was similar to that of controls in terms of histological recurrence, graft loss, and death [80, 81]. Treatment regimens in these studies were interferon based which resulted in poor tolerability, renal impairment, cytopenias, and drug interactions. DAAs have revolutionized the management of HCV infection in the posttransplant setting. Recent clinical trials have shown high sustained virologic response rates, shorter durations of treatment, and decreased adverse events when compared with the previous PEG-INF based therapy. However, most of these studies were performed in HCV-G1-infected patients. Data on treating HCV-G4 recurrence following liver transplantation are limited (**Table 2**).

## **6.1. Pegylated interferon and ribavirin**

Reported SVR rates for pegylated interferon combination therapy following liver transplantation are lower than those in the nontransplant population. Treatment regimens have been hindered by a high incidence of adverse effects, leading to treatment withdrawal.

Dabbous et al. evaluated 243 patients transplanted for HCV-G4-related cirrhosis. All patients had a protocol biopsy 6 months post-transplant. Patients received PEG-IFN and ribavirin in case of histological recurrence. Repeated liver biopsies were performed at 3, 6, and 12 months during treatment for the detection of immune-mediated rejection induced by interferon. Fiftysix (23%) patients had evidence of histopathological disease recurrence, and 42 patients completed the treatment. Five patients were excluded due to fibrosing cholestatic hepatitis (FCH); therefore, 37 patients were included in the study. The patients received treatment in the form of combined PEG-IFN and RBV. Erythropoietin and granulocyte colony-stimulating factor were used in 70% of patients. SVR was achieved in 29 (78%) patients. The high SVR rate in this study was attributed to several factors, including the early treatment protocol, exclusion of patients with fibrosing cholestatic hepatitis and aggressive treatment of hematological


FCH = fibrosing cholestatic hepatitis,

PEG-INF = pegylated interferon.

**Table 2.** Prospective studies that included HCV-G4 patients following liver transplantation.

complications [82]. Conversely, in the largest series reported from Europe, Ponziani et al. evaluated treatment responses in 17 Italian patients with HCV-G4 recurrence following liver transplantation. The observed overall survival after LT was 100% at 1 year and 83.3% at 5 years. Thirty-five percent of patients achieved SVR. However, this retrospective study included patients treated in the 1990s with conventional interferon; the drug tolerability, the lack of aggressive management of hematological side effects and the inclusion of patients with advanced liver disease contributed to the low response rate [83]. In a recent study from Saudi Arabia, 25 patients infected with HCV-G4 were treated with PEG-IFN alpha-2a and RBV [84]. Pretreatment liver biopsies were obtained from all patients. Biochemical and virological markers were assessed before, during, and after treatment. Five patients had advanced pretreatment liver fibrosis. Eighty-eight percent achieved an early virological response; of those, 15 (60%) and 14 (56%) patients achieved end of treatment virological response and SVR, respectively. The most common adverse effects were flu-like symptoms and cytopenia. Eighteen patients (72%) required erythropoietin alpha and/or granulocyte-colony stimulating factor as a supportive measure. One patient developed severe rejection complicated by sepsis, renal failure, and death. Other adverse effects included depression, mild rejection, impotence, itching, and vitiligo. The relatively high response rate in this study may have been due to the treatment-naïve status of the patients, the use of growth factors that allowed patients to complete their course of therapy, the low treatment-withdrawal rate, and the reduction in immunosuppressive therapy during treatment.

The results of these studies suggest that post-transplant treatment outcomes for HCV-G4 are likely better than for G1 and less favorable than for G2 and G3. This response pattern among the different genotypes parallels the response pattern in the immunocompetent population. The availability of newer treatment options with better safety profiles is drawing attention away from PEG-IFN and RBV.

## **7. HCV treatment in the new antiviral era**

## **7.1. Telaprevir and boceprevir**

complications [82]. Conversely, in the largest series reported from Europe, Ponziani et al. evaluated treatment responses in 17 Italian patients with HCV-G4 recurrence following liver transplantation. The observed overall survival after LT was 100% at 1 year and 83.3% at 5 years. Thirty-five percent of patients achieved SVR. However, this retrospective study included patients treated in the 1990s with conventional interferon; the drug tolerability, the lack of aggressive management of hematological side effects and the inclusion of patients with advanced liver disease contributed to the low response rate [83]. In a recent study from Saudi Arabia, 25 patients infected with HCV-G4 were treated with PEG-IFN alpha-2a and RBV [84].

**Table 2.** Prospective studies that included HCV-G4 patients following liver transplantation.

**Genotypes SVR Treatment protocol**

nsated cirrhosis 85–88% in cirrhosis with mild hepatic

60–75% in cirrhosis with severe hepatic dysfunction

All 96% SOF + DCV for 24 weeks

dysfunction

tients

SOF + RBV for 24 weeks

SOF + LDV + RBV for 12–24 weeks

SOF + LDV + RBV for 12–24 weeks

92% Predominant SOF/daclatasvir ± RBV

96% SOF + daclatasvir (DAC)

Ajlan [88] 36 4 91.6% SOF + RBV + PEG − INF for 12 weeks or

Dabbous [27] 39 4 76% SOF + RBV for 24 weeks Charlton [89] 40 All (1 genotype 4) 70% SOF + RBV for 24 weeks Forns [90] 104 1, 2, 3, 4 59% SOF + RBV for 24–48 weeks Abergel [91] 44 4 93% SOF + LDV for 12 weeks

**Study Sample**

**size** 

100 Advances in Treatment of Hepatitis C and B

Charlton [68] 108 1 and 4 96–98% in compe

Manns [92] 227 1(200) and 4(27) 92.5% of genotype 4 pa-

4)

4)

Dumortier [94] 125 All (11 genotype

Coilly [95] 137 All (12 genotype

FCH)

SVR = sustained virological response,

FCH = fibrosing cholestatic hepatitis, PEG-INF = pegylated interferon.

Leroy [97] 23 (all with

SOF = sofosbuvir, RBV = ribavirin, LDV = ledipsavir, DCV = daclatasvir, SIM = simeprevir,

Following the approval of telaprevir (Incivek™) and boceprevir (Victrelis™) for G1 treatment outcomes improved [85, 86]. Treatment regimens for chronic HCV-G1 infection include a combination of either of these protease inhibitors three times daily with once-weekly subcutaneous injections of PEG-IFN and twice-daily oral RBV. These new combinations increased SVR to 80% and 63–66%, respectively, in nontransplant patients. Some studies have reported poor clinical outcomes of the use of telaprevir and PEG-IFN in patients with HCV-G4 [87]. Burton et al. conducted a retrospective cohort study of 81 patients with genotype 1 HCV treated with boceprevir (10%) or telaprevir (90%) plus PEG-IFN and RBV at six US transplant centers (53% stage 3–4/4 fibrosis, 57% treatment experienced). The intent-to-treat SVR12 rate was 63%. Adverse effects were common; 21% of patients developed anemia (hemoglobin < 8 g/dl) and 57% required blood transfusions during the first 16 weeks. Twenty-seven percent were hospitalized and 9% died; all were liver-related [88]. Although the use of these two DAAs in post–liver transplant patients resulted in SVR up to 60% with telaprevir, nonresponders were observed in the boceprevir treatment, and it was associated with severe side effects, including severe anemia that required erythropoietin, RBV dose reduction and red blood cell transfusions. Significant drug interactions also occurred with immunosuppressants, requiring average cyclosporine dose reductions of 50–84% after telaprevir initiation and 33% after boceprevir initiation. Tacrolimus doses were reduced by 95% with telaprevir [27]. These significant side effects coupled with the introduction of safer antiviral drugs have shifted HCV treatment away from these agents; in fact, these agents are contraindicated by many liver association.

#### **7.2. Sofosbuvir and ribavirin**

SOF has become a cornerstone of the management of HCV infection because of its favorable pharmacological and drug interaction profiles. However, there are very limited data on the use of SOF in patients with HCV recurrence post–liver transplant, particularly G4. Ajlan et al. conducted an open label prospective cohort study at a tertiary care hospital in Saudi Arabia. The primary endpoint was SVR12 in patients treated with sofosbuvir-based therapy in postliver transplant patients with genotype 4 HCV recurrence. Thirty-six treatment-experienced liver transplant patients with HCV recurrence received sofosbuvir and ribavirin with or without PEG-INF. The majority of patients had ≥stage 2 fibrosis. Twenty-eight patients were treated with PEG-IFN and RBV in addition to SOF for 12 weeks and the remaining were treated with SOF and RBV only for 24 weeks. By week 4, only four (11.1%) patients had detectable HCV RNA. Of the 36 patients, two (5.5%) relapsed and one died (2.75%) [89]. Another recent study evaluated the efficacy, safety, and tolerability of SOF and RBV in LDLT recipients with recurrent HCV-4. In this study Thirty-nine Egyptian LDLT recipients were treated for recurrent HCV after LDLT with SOV and RBV without PEG-IFN for 6 months. Thirty eight patients completed 24 weeks of treatment and were followed for 12 weeks after end of treatment. One patient died during the first week of treatment. SVR was achieved by 76% (29/38) of recipients. SVR was significantly higher in treatment-naïve patients and in recipients with a low stage of fibrosis. Only two patients developed severe side effects wile on treatment in the form of severe pancytopenia and acute renal failure [90]. A recent prospective multicenter study enrolled 40 patients with compensated recurrent HCV infection of any genotype after a primary or secondary liver transplantation. All patients received 24 weeks of SOF 400 mg daily and RBV. Of the 40 patients enrolled and treated, 40% had biopsy proven cirrhosis, and 88% had been previously treated with interferon. SVR12 was achieved by 28 of 40 patients. Relapse accounted for all cases of virological failure, including the only patient with HCV-G4. The most common adverse events were fatigue (30%), diarrhea (28%), and headache (25%). In addition, 20% of the subjects experienced anemia. No deaths, graft losses, or episodes of rejection occurred. No interactions with any concomitant immunosuppressive agents were reported [91]. A recent post-transplantation study was conducted in which SOF and RBV were provided on a compassionate-use basis to patients with severe recurrent HCV, including those with fibrosing cholestatic hepatitis (FCH) and decompensated liver cirrhosis with a life expectancy of <1 year. Data from the first 104 patients who completed or prematurely discontinued treatment were included. All patients received SOF and RBV for 24–48 weeks. Investigators were allowed to add PEG-IFN to the regimen at their discretion. The study population included patients infected with HCV- G4. The overall SVR rate was 59% and was higher (73%) in those with early severe recurrence. At the end of the study, 57% of patients displayed clinical improvement, 22% were unchanged, 3% had worsened clinical status, and 13% had died. Overall, 123 serious adverse events occurred in 49 patients (47%). Serious adverse events associated with hepatic decompensation were the most frequent, with 26 adverse events occurring in 19 patients (18%) [92].

## **7.3. Sofosbuvir/ledipasvir with or without ribavirin**

treatment away from these agents; in fact, these agents are contraindicated by many liver

SOF has become a cornerstone of the management of HCV infection because of its favorable pharmacological and drug interaction profiles. However, there are very limited data on the use of SOF in patients with HCV recurrence post–liver transplant, particularly G4. Ajlan et al. conducted an open label prospective cohort study at a tertiary care hospital in Saudi Arabia. The primary endpoint was SVR12 in patients treated with sofosbuvir-based therapy in postliver transplant patients with genotype 4 HCV recurrence. Thirty-six treatment-experienced liver transplant patients with HCV recurrence received sofosbuvir and ribavirin with or without PEG-INF. The majority of patients had ≥stage 2 fibrosis. Twenty-eight patients were treated with PEG-IFN and RBV in addition to SOF for 12 weeks and the remaining were treated with SOF and RBV only for 24 weeks. By week 4, only four (11.1%) patients had detectable HCV RNA. Of the 36 patients, two (5.5%) relapsed and one died (2.75%) [89]. Another recent study evaluated the efficacy, safety, and tolerability of SOF and RBV in LDLT recipients with recurrent HCV-4. In this study Thirty-nine Egyptian LDLT recipients were treated for recurrent HCV after LDLT with SOV and RBV without PEG-IFN for 6 months. Thirty eight patients completed 24 weeks of treatment and were followed for 12 weeks after end of treatment. One patient died during the first week of treatment. SVR was achieved by 76% (29/38) of recipients. SVR was significantly higher in treatment-naïve patients and in recipients with a low stage of fibrosis. Only two patients developed severe side effects wile on treatment in the form of severe pancytopenia and acute renal failure [90]. A recent prospective multicenter study enrolled 40 patients with compensated recurrent HCV infection of any genotype after a primary or secondary liver transplantation. All patients received 24 weeks of SOF 400 mg daily and RBV. Of the 40 patients enrolled and treated, 40% had biopsy proven cirrhosis, and 88% had been previously treated with interferon. SVR12 was achieved by 28 of 40 patients. Relapse accounted for all cases of virological failure, including the only patient with HCV-G4. The most common adverse events were fatigue (30%), diarrhea (28%), and headache (25%). In addition, 20% of the subjects experienced anemia. No deaths, graft losses, or episodes of rejection occurred. No interactions with any concomitant immunosuppressive agents were reported [91]. A recent post-transplantation study was conducted in which SOF and RBV were provided on a compassionate-use basis to patients with severe recurrent HCV, including those with fibrosing cholestatic hepatitis (FCH) and decompensated liver cirrhosis with a life expectancy of <1 year. Data from the first 104 patients who completed or prematurely discontinued treatment were included. All patients received SOF and RBV for 24–48 weeks. Investigators were allowed to add PEG-IFN to the regimen at their discretion. The study population included patients infected with HCV- G4. The overall SVR rate was 59% and was higher (73%) in those with early severe recurrence. At the end of the study, 57% of patients displayed clinical improvement, 22% were unchanged, 3% had worsened clinical status, and 13% had died. Overall, 123 serious adverse events occurred in 49 patients (47%). Serious adverse events associated with hepatic decompensation were the most frequent, with 26 adverse events

association.

**7.2. Sofosbuvir and ribavirin**

102 Advances in Treatment of Hepatitis C and B

occurring in 19 patients (18%) [92].

Abergel evaluated the efficacy and safety of therapy with LDV and SOF in patients with HCV genotype 4. Forty-four patients (22 treatment naïve and 22 treatment experienced) received a fixed-dose combination tablet of 90 mg LDV and 400 mg SOV orally once daily for 12 weeks. Among study participants, HCV genotype 4 subtypes were well represented (4a, *n* = 25; 4d, *n* = 10; other subtypes, *n* = 9). Ten patients (23%) had compensated cirrhosis. All 44 patients completed the full 12 weeks of treatment. The SVR12 rate was 93% and was similar in treatment-naïve (95%, 21/22) and treatment-experienced (91%, 20/22) patients. The three patients who did not achieve SVR12 had virological relapse within 4 weeks of the end of treatment; all three had a high baseline HCV RNA, a non-CC IL-28B genotype, and pretreatment NS5A resistance-associated variants. None of the patients experienced a serious adverse event [93].

Cohort B (of the previously described Solar-1 study) enrolled patients who had undergone liver transplantation and included patients with various degrees of disease severity. Patients were randomly assigned to receive a fixed-dose combination tablet containing LDV and SOF plus RBV for 12 or 24 weeks. The cohort included 108 post-transplant patients. SVR12 was achieved in 96–98% of patients without cirrhosis or with compensated cirrhosis, in 85–88% of patients with moderate hepatic impairment, in 60–75% of patients with severe hepatic impairment, and in all six patients with FCH. Response rates were also similar in the 12- and 24-week groups [68]. An open-label study at 34 sites in Europe, Canada, Australia, and New Zealand recruited two groups of patients, cohort A included patients with Child-Turcotte-Pugh class B (CTP-B) or CTP-C cirrhosis who had not undergone liver transplantation. Cohort B included post-transplantation patients who had either no cirrhosis; CTP-A, CTP-B, or CTP-C cirrhosis; or fibrosing cholestatic hepatitis. Patients in each group were randomly assigned (1:1) using a computer-generated randomisation sequence to receive 12 or 24 weeks of LDV (90 mg) and SOF (400 mg) once daily, plus ribavirin (600–1200 mg daily). Of 333 patients who received treatment, 296 had genotype 1 HCV and 37 had genotype 4 HCV. Among all patients with genotype 4 HCV, SVR12 was achieved by 14 of 18 (78%) patients (12 weeks treatment) and 16 of 17 (94%) patients (24 weeks treatment). Of the five patients who did not achieve SVR12, three—all receiving 12 weeks of treatment—had virological relapse, and two died (one post-transplantation CTP-A on 12 weeks of treatment, and one untransplanted CTP-C on 24 weeks of treatment) and were not included in the analysis. Twenty five of twenty seven HCV-G4 in cohort B of the study achieved SVR the only two relapsers were cirrhotics [94]. Despite including G1 and G4 in these studies, the number of HCV-G4 infected patients was relatively small, limiting solid conclusions on the response of HCV-G4.

The safety profile of LVD/SOF with RBV was evaluated in a pooled analysis of two large multicenter studies (Solar-1 and -2). The patients involved were either cirrhotic or post–liver transplantation patients (616 G1 and 42 G4) and were randomized to 12 or 24 weeks of treatment. Of 134 SAEs, only 20 were related to treatment. RBV-associated anemia was the most common adverse effect, representing 11/20 (55%) of reported drug-related adverse events [95].

## **7.4. Sofosbuvir/daclatasvir**

Data on the use of DCV in the post-transplant setting for HCV-G4-infected patients are limited. A prospective multicenter cohort including patients with HCV-recurrence following LT treated with second generation direct antivirals was conducted. The aim of the t study was to assess efficacy and tolerance of sofosbuvir (SOF)-based regimens for the treatment of HCV recurrence in patients with severe fibrosis after LT. A SOF-based regimen was administered to 125 patients including patients infected with HCV-G4 (11 patients). The main combination regimen was SOF/DCV (73.6%). SVR12 was 92.8% (on an intent-to-treat basis); seven cases of virological failure were observed including 1 HCV-G4 patient treated with SOF/daclatasvir (DAC) combination [96]. In another multicenter prospective study137 patients with HCV recurrence receiving SOF and DCV, were included whatever the genotype or fibrosis stage. This cohort included 12 patients infected with HCV-G4. The primary efficacy end point was a sustained virological response 12 weeks after the end of treatment. The SVR rate 12 weeks after completing treatment was 96% under the intention-to treat analysis and 99% when excluding nonvirological failures. Only two patients experienced a virological failure. The serious adverse event rate reached 17.5%. Four patients (3%) stopped their treatment prematurely because of adverse events. Anaemia was the most common adverse event, with significantly more cases in the RBV group. No clinically relevant drug–drug interactions were noted, but 52% of patients required a change to the dosage of immunosuppressive drugs [97]. Fontana et al. in a retrospective multicenter study evaluated daclatasvir (DAC)/SOF combination post liver transplantation in established HCV recurrence including HCV-4 patients. Eighty seven percent of patients achieved SVR and the treatment was well tolerated [98]. Leroy et al. analyzed data from 23 patients with FCH who participated in a prospective cohort study in France and Belgium to assess the effects of antiviral agents in patients with recurrence of HCV infection after liver transplantation. Three patients with G4 infection were included in this study (one patient was treated with SOF/RBV, and two were treated with SOF/DCV). All patients survived without re-transplantation. Rapid and dramatic improvements in clinical status were observed. The patients' median bilirubin concentration decreased from 122 μmol/L at baseline to a normal value at week 12 of treatment. Twenty-two patients (96%) had a complete clinical response at week 36, and 22 patients (96%) achieved SVR12, including all 3 patients infected with G4 [99].

## **7.5. Sofosbuvir and simeprevir**

Data on the use of SIM for HCV-G4 recurrence following liver transplantation are limited to a small number of case reports and case series. In a recent report, three patients with HCV-G4 recurrence following liver transplantation were treated with SOF and SIM for 12–24 weeks. All three had high pretreatment viral loads, and one patient had established cirrhosis. SVR12 was achieved in all three patients, with no significant adverse effects or drug interactions [100]. Obed A et al. reported a patient with a recurring HCV-G4 infection and fibrosing cholestatic hepatitis following liver retransplantation, who was successfully treated with a combination therapy of SIM and SOF without PEG-INF/RBV [101].

## **8. Timing of treatment for patients on the transplant list**

**7.4. Sofosbuvir/daclatasvir**

104 Advances in Treatment of Hepatitis C and B

with G4 [99].

**7.5. Sofosbuvir and simeprevir**

therapy of SIM and SOF without PEG-INF/RBV [101].

Data on the use of DCV in the post-transplant setting for HCV-G4-infected patients are limited. A prospective multicenter cohort including patients with HCV-recurrence following LT treated with second generation direct antivirals was conducted. The aim of the t study was to assess efficacy and tolerance of sofosbuvir (SOF)-based regimens for the treatment of HCV recurrence in patients with severe fibrosis after LT. A SOF-based regimen was administered to 125 patients including patients infected with HCV-G4 (11 patients). The main combination regimen was SOF/DCV (73.6%). SVR12 was 92.8% (on an intent-to-treat basis); seven cases of virological failure were observed including 1 HCV-G4 patient treated with SOF/daclatasvir (DAC) combination [96]. In another multicenter prospective study137 patients with HCV recurrence receiving SOF and DCV, were included whatever the genotype or fibrosis stage. This cohort included 12 patients infected with HCV-G4. The primary efficacy end point was a sustained virological response 12 weeks after the end of treatment. The SVR rate 12 weeks after completing treatment was 96% under the intention-to treat analysis and 99% when excluding nonvirological failures. Only two patients experienced a virological failure. The serious adverse event rate reached 17.5%. Four patients (3%) stopped their treatment prematurely because of adverse events. Anaemia was the most common adverse event, with significantly more cases in the RBV group. No clinically relevant drug–drug interactions were noted, but 52% of patients required a change to the dosage of immunosuppressive drugs [97]. Fontana et al. in a retrospective multicenter study evaluated daclatasvir (DAC)/SOF combination post liver transplantation in established HCV recurrence including HCV-4 patients. Eighty seven percent of patients achieved SVR and the treatment was well tolerated [98]. Leroy et al. analyzed data from 23 patients with FCH who participated in a prospective cohort study in France and Belgium to assess the effects of antiviral agents in patients with recurrence of HCV infection after liver transplantation. Three patients with G4 infection were included in this study (one patient was treated with SOF/RBV, and two were treated with SOF/DCV). All patients survived without re-transplantation. Rapid and dramatic improvements in clinical status were observed. The patients' median bilirubin concentration decreased from 122 μmol/L at baseline to a normal value at week 12 of treatment. Twenty-two patients (96%) had a complete clinical response at week 36, and 22 patients (96%) achieved SVR12, including all 3 patients infected

Data on the use of SIM for HCV-G4 recurrence following liver transplantation are limited to a small number of case reports and case series. In a recent report, three patients with HCV-G4 recurrence following liver transplantation were treated with SOF and SIM for 12–24 weeks. All three had high pretreatment viral loads, and one patient had established cirrhosis. SVR12 was achieved in all three patients, with no significant adverse effects or drug interactions [100]. Obed A et al. reported a patient with a recurring HCV-G4 infection and fibrosing cholestatic hepatitis following liver retransplantation, who was successfully treated with a combination The management of hepatitis C virus (HCV) infection in patients with decompensated cirrhosis has evolved dramatically. DAAs have shown to be safe and effective in patients with decompensated cirrhosis with high SVR rates. However it is still debatable on when to initiate treatment in patients with advanced liver disease (**Figure 1**). Many factors may contribute to and affect the approach on an individual basis; for example, it may be better to defer treatment in extremely ill patients. Belli et al. assessed the impact of DAAs on patients awaiting liver transplant. They evaluated whether patients can be first inactivated due to clinicall improvement and subsequently delisted in a real life setting. They included 103 consecutive listed patients without hepatocellular carcinoma who were treated with different DAA combinations in 11 European centers. Treated patient had a significant improvement in the median model for end-stage liver disease (MELD) and Child Pugh score. They concluded that all oral DAAs were able to reverse liver dysfunction and favoured the inactivation and delisting of about one patient out-of-three and one patient out of- five in 60 weeks, respectively. Patients with lower MELD scores had higher chances to be delisted. However, the longer term benefits of therapy need to be ascertained [102]. Similarly Afdhal et al. evaluated the outcome of treatment with SOF and RBV in compensated and decompensated cirrhotic patients. They also monitored the clinical picture and measured the hepatic venous pressure gradient before and after treatment. They observed a clinically meaningful improvement in portal hypertension in addition to improvements in liver biochemistry, Child–Pugh score and model for end-stage liver disease scores [103]. The potential benefits of treating patients on the waiting list include potential improvements in overall clinical status that may salvage these patients from liver transplan-

**Figure 1.** Post transplant natural history of HCV recurrence with potential treatment strategies.

tation; reducing post-transplant recurrence; and avoiding possible post-transplant drug–drug interactions. One concern is that treating these patients may lower their MELD scores and drive them down the transplant list, thus delaying transplantation despite persistent portal hypertensive complications. The decision to treat HCV in patients with decompensated cirrhosis should be individualized till short and long term outcome data become available.

## **Author details**

Waleed K. Al-Hamoudi

Address all correspondence to: walhamoudi@gmail.com

Department of Medicine, Gastroenterology and Hepatology Unit, College of Medicine, King Saud University, Riyadh, Saudi Arabia

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Department of Medicine, Gastroenterology and Hepatology Unit, College of Medicine, King

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#### **HCV Treatment Failure in the Era of DAAs HCV Treatment Failure in the Era of DAAs**

Mohamed Hassany and Aisha Elsharkawy Mohamed Hassany and Aisha Elsharkawy

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67149

#### **Abstract**

Hepatitis C virus (HCV) has six well‐known genotypes in worldwide and has a very high genetic diversity. Introduction of DAAs leads to improvement of treatment results with SVR rates exceeding 95%. Development of HCV treatment resistance is a problematic issue that needs sufficient solutions. Many hosts, viral, and drug factors are implemented in the process of treatment resistance. Lack of clinical trials on treatment failure leads to lag in development of certain consensuses for retreatment.

**Keywords:** HCV‐DAAs, viral resistance, treatment failure

## **1. Introduction**

Chronic hepatitis C virus (HCV) infection is a major health problem all over the world. The global prevalence of viremic HCV infection was reestimated between 64 and 103 million patients [1]. Chronic HCV patients suffered a long time from the complications of their dis‐ ease until the first discovery of interferon treatment. However, its modest response rate and the development of many adverse events were the major problem. Soon the dream seems to become true with the introduction of HCV direct acting antivirals (DAAs) in 2014. Their higher rates of response and minimally observed adverse events encourage more patients to go for treatment. In addition, patients with advanced fibrosis and cirrhosis find a new hope to stop the progression of their disease. Three classes of DAAs (protease inhibitors, NS5A inhibitors, and polymerase inhibitors) targeting three HCV enzymatic nonstructural proteins were approved for treatment in many countries [2]. Variability of treatment efficacy among patients makes it difficult to control the infection; while for some patients, weak antivirals and short‐term treatments are sufficient, others require combination therapies with several highly active antivirals for longer durations [3].

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Despite the high rates of virological cure achieved with these treatments, the infection is not eliminated from a substantial number of patients (1–15%, depending on the patient status and regimen used) [4]. Patients and researchers started to face the new problem of drug resistance. In this review, HCV treatment failure in the era of DAAs will be discussed in the context of factors affecting development of resistance, diagnosis, and management.

## **2. Treatment from interferon to DAAs**

Hepatitis C virus (HCV) has six well‐known genotypes in worldwide [5–10] with multiple subtypes (a, b, c, etc.). RNA sequence may vary by 35% between different genotypes. HCV has a very high genetic diversity and very high rate of replication (>10 trillion virions/day), and due to this replication rate, significant genetic errors occur and a continuous process of correction is already running to optimize the replication and sequencing of the virus genes; failure of error correction leads to the formation of genetic drifts [5]; these drifts are repre‐ sented either in the form of genotypes or quasispecies. **Table 1** shows the difference among genotypes, subtypes, and quasispecies.

The presence of different HCV genotypes does not exhibit a major clinical implication on the nat‐ ural history of the disease and its progression, yet it has a great influence on treatment outcome. The best results for treatment in the past era of pegylated interferon (PegIFN) and ribavirin (RBV) were achieved in genotypes 2, 3 (80–90%) with less favorable results in genotypes 1, 4 (40–50%) and intermediate results in genotypes 5, 6 (60–70%). Failure of treatment during this era had no satisfactory solutions rather than retreatment using the same regimen or changing the pegylated interferon type (between alpha 2a and alpha 2b) or even extending the treatment duration.

Introduction of the direct acting antivirals (DAAs) in the playground of HCV treatment repre‐ sents a major challenge with the rising number of approved molecules and its coming followers in the pipe of production and approval as shown in **Table 2**, although the very high response to these drugs, which sometimes exceeds 95% yet its limited failure, represents a problematic issue.

DAAs permit to treat different categories of patients who could not be treated easily in the past due to the low efficacy and safety of pegylated interferon such as those with advanced liver dis‐ ease (CHILD‐PUGH B, C), autoimmune diseases, polymedicated patients, renal impairment, postorgan transplantation, etc. Implementation of larger groups to the treatment pipe leads to expulsion of more numbers of treatment failures asking for better solutions for retreatment.


**Table 1.** Differences between genotypes, subtypes, and quasispecies.


**Table 2.** DAAa pipeline current situation (April 2016).

Despite the high rates of virological cure achieved with these treatments, the infection is not eliminated from a substantial number of patients (1–15%, depending on the patient status and regimen used) [4]. Patients and researchers started to face the new problem of drug resistance. In this review, HCV treatment failure in the era of DAAs will be discussed in the context of

Hepatitis C virus (HCV) has six well‐known genotypes in worldwide [5–10] with multiple subtypes (a, b, c, etc.). RNA sequence may vary by 35% between different genotypes. HCV has a very high genetic diversity and very high rate of replication (>10 trillion virions/day), and due to this replication rate, significant genetic errors occur and a continuous process of correction is already running to optimize the replication and sequencing of the virus genes; failure of error correction leads to the formation of genetic drifts [5]; these drifts are repre‐ sented either in the form of genotypes or quasispecies. **Table 1** shows the difference among

The presence of different HCV genotypes does not exhibit a major clinical implication on the nat‐ ural history of the disease and its progression, yet it has a great influence on treatment outcome. The best results for treatment in the past era of pegylated interferon (PegIFN) and ribavirin (RBV) were achieved in genotypes 2, 3 (80–90%) with less favorable results in genotypes 1, 4 (40–50%) and intermediate results in genotypes 5, 6 (60–70%). Failure of treatment during this era had no satisfactory solutions rather than retreatment using the same regimen or changing the pegylated interferon type (between alpha 2a and alpha 2b) or even extending the treatment duration.

Introduction of the direct acting antivirals (DAAs) in the playground of HCV treatment repre‐ sents a major challenge with the rising number of approved molecules and its coming followers in the pipe of production and approval as shown in **Table 2**, although the very high response to these drugs, which sometimes exceeds 95% yet its limited failure, represents a problematic issue. DAAs permit to treat different categories of patients who could not be treated easily in the past due to the low efficacy and safety of pegylated interferon such as those with advanced liver dis‐ ease (CHILD‐PUGH B, C), autoimmune diseases, polymedicated patients, renal impairment, postorgan transplantation, etc. Implementation of larger groups to the treatment pipe leads to expulsion of more numbers of treatment failures asking for better solutions for retreatment.

> **Genotypes, subtypes Quasispecies** Difference in RNA sequence Mutation during replication Major genetic differences Minor genetic differences Does not change Continue to evolve over time

**Table 1.** Differences between genotypes, subtypes, and quasispecies.

factors affecting development of resistance, diagnosis, and management.

**2. Treatment from interferon to DAAs**

118 Advances in Treatment of Hepatitis C and B

genotypes, subtypes, and quasispecies.

All the previously mentioned molecules have different characteristics regarding the potency, genotype coverage, and barrier to resistance. **Table 3** shows the characteristics of DAAs mol‐ ecules [6].

Different continental guidelines for HCV management describe different treatment regimens:



**Table 3.** Characteristics of DAAs molecules.

## **3. Definitions**

The terms RAVs, RASs, resistant variants, and sensitive variants were recently used in clinical practice to describe the susceptibility to an administered DAA. Using these definitions paved the way to understand more about HCV treatment failure when using DAAs. Pawlotsky has described well these terms as mentioned below [4]:

#### **3.1. Viral resistance**

Positive selection of viral variants with reduced susceptibility to an administered DAA.

#### **3.2. Resistance‐associated variant (RAV)**

It is often used to indifferently describe the amino acid substitutions that reduce the sus‐ ceptibility of a virus to a drug or drug class or, alternatively, the viral variants with reduced susceptibility that carry these substitutions.

#### **3.3. Resistance‐associated substitutions (RASs)**

The amino acid substitutions that confer resistance.

#### **3.4. Resistant variants**

The viral variants carrying these RASs and thereby have reduced susceptibility to the DAA.

#### **3.5. Sensitive variants**

Viral variants that do not contain amino acids that confer reduced susceptibility to the antiviral action of an HCV DAA (contain only the original wild‐type amino acids of the viral strains).

#### **3.6. Fitness‐associated substitution(s)**

Single amino acid changes that do not alter DAA susceptibility but increase the power of replication (fitness of the resistant variants).

Prior to therapy, multiple baseline HCV resistant‐associated variants (RAVs) are already present but usually at a very low undetectable limit. After treatment with DAAs, a sharp decline of HCV viremia occurs within the first treatment days and a competition between sensitive variants and resistant variants will determine which of the following scenarios will be encountered after stoppage of the administered drug:


## **4. Factors affecting the outcome and HCV resistance**

Failure of treatment and development of resistance are a multifactorial process depending on host‐related factors, virus‐related factors, and drug‐related factors as shown in **Figure 1**.

**Figure 1.** Factors affecting treatment outcome and development of resistance.

## **4.1. Host‐related factors**

**3. Definitions**

**Table 3.** Characteristics of DAAs molecules.

120 Advances in Treatment of Hepatitis C and B

Nucleoside/nucleotide

analogues

**3.1. Viral resistance**

**3.4. Resistant variants**

**3.5. Sensitive variants**

**3.6. Fitness‐associated substitution(s)**

replication (fitness of the resistant variants).

described well these terms as mentioned below [4]:

**3.2. Resistance‐associated variant (RAV)**

susceptibility that carry these substitutions.

**3.3. Resistance‐associated substitutions (RASs)**

The amino acid substitutions that confer resistance.

The terms RAVs, RASs, resistant variants, and sensitive variants were recently used in clinical practice to describe the susceptibility to an administered DAA. Using these definitions paved the way to understand more about HCV treatment failure when using DAAs. Pawlotsky has

**Drug group Potency Genotype coverage Resistance barrier**

+++ +++ +++

NS3‐4A protease inhibitors +++ +++ ++ NS5A inhibitors +++ +++ ++

Nonnucleoside inhibitors ++ + +

Positive selection of viral variants with reduced susceptibility to an administered DAA.

It is often used to indifferently describe the amino acid substitutions that reduce the sus‐ ceptibility of a virus to a drug or drug class or, alternatively, the viral variants with reduced

The viral variants carrying these RASs and thereby have reduced susceptibility to the DAA.

Viral variants that do not contain amino acids that confer reduced susceptibility to the antiviral action of an HCV DAA (contain only the original wild‐type amino acids of the viral strains).

Single amino acid changes that do not alter DAA susceptibility but increase the power of

Introduction of DAAs eliminates multiple host factors, which affect previous treatment with PegIFN and ribavirin, yet several host factors still persist:


## **4.2. Virus‐related factors**


[12]. In addition, the presence of NS3 protease RAS Q80K was associated with a reduced rate of SVR in patients with HCV genotype 1a infection and cirrhosis, especially if they failed to respond to previous pegylated IFN–based treatment [13, 14].

## **4.3. Drugs‐related factors**

(1) Adherence to therapy: achievement of the best drug response surely will be better in case of proper administration of the drug with its proper dose at regular times and respect of

(2) HIV, post‐organ transplantation and polymedicated patients: revision of the drug‐drug interactions map is necessary in those patients to avoid the effect of other drugs in reduc‐

(3) Treatment status: most of clinical trials on DAAs showed mild better response in treat‐ ment naïve patients than those who previously failed treatment with PegIFN/RBV. (4) Hepatic fibrosis stage: patients with advanced fibrosis stage remain the most difficult to treat group even under the umbrella of DAAs which showed a wide variable results in cirrhotics ranged between 33 and 100% [7]. Addition of ribavirin and prolonged treatment duration may offer the best chance for those patients in achieving sustained virological response.

(1) Genotype: treatment with PegIFN/RBV/Sofosbuvir represents the regimen that showed remarkable potency against all HCV genotypes. IFN‐free regimens should be selected pri‐ marily based on genotype as we have pangenotypic regimens (Sofosbuvir + Velpatasvir ± RBV, Sofosbuvir + Daclatasvir ± RBV, Paritaprevir‐ritonavir/Ombitasvir ± Dasabuvir ± RBV), regimens fit for all genotypes except genotype 3 (Sofosbuvir + Simeprevir ± RBV, Sofosbuvir + Ledipasvir ± RBV), and individualized regimens for genotypes 2‐3‐4 (Sofos‐

(2) Baseline RAVs: The presence of baseline RAVs seems to be associated with variable degrees of treatment response. Zeuzem et al. [8], observed no significant difference in response in noncirrhotic genotype 1 patients treated with Sofosbuvir and ledipasvir between those with baseline RAVs and others without RAVs in different treatment status and durations (98% in RAVs group vs. 99% in no RAVs group in naïve patients treated for 8 weeks, 99% in RAVs group vs. 99% in no RAVs group in naïve patients treated for 12 weeks, 90% in RAVs group vs. 99% in no RAVs group in experienced patients treated for 12 weeks). However, a significant difference was observed in cirrhotic patients (88% in RAVs group vs. 100% in no RAVs group in naïve patients treated for 24 weeks, 87% in RAVs group vs. 100% in no RAVs group in experienced patients treated for 24 weeks) [4]. In C‐EDGE study, Zeuzem et al. showed a great influence of baseline RAVs on treatment outcome in HCV GT1 pa‐ tients treated with grazoprevir/elbasvir combined with very low SVR (22%) in GT1a pa‐ tients with NS5A baseline RAVs > fivefolds potency loss [9]. No effect on SVR in genotype 1 HCV patients with or without cirrhosis with baseline RAVs treated with combination of ombitasvir, r‐paritaprevir, and dasabuvir, with or without ribavirin, for 12 or 24 weeks in four phase three clinical trials [11]. When Sofosbuvir/Daclatasvir combination was used, the presence of NS5A baseline RAVs is associated with reduced rates of SVR in under‐ treated (too short duration, no ribavirin) patients with cirrhosis and genotype 3 infection

food relations as recommended by the manufacturer.

ing the plasma level of anti‐HCV drugs.

**4.2. Virus‐related factors**

122 Advances in Treatment of Hepatitis C and B

buvir + RBV).



**Table 4.** Resistance and cross‐resistance in NS3‐4A protease inhibitors.


**Table 5.** Resistance and cross‐resistance in NS5A inhibitors.


**Table 6.** Resistance in NS5B inhibitors.

## **5. Diagnosis of HCV RAVs**

## **5.1. Diagnosis of resistance in clinical practice is conducted by two methods**

(1) Phenotypic analysis: used to determine the optimum plasma concentration (effective con‐ centration, EC50 EC90) of the dug sufficient to inhibit the viral replication.

RAVs are typically associated with a change in the shape of the binding or interaction site of DAAs to HCV target proteins. RAVs harbor different levels of resistance due to dif‐ ferent locations within the sites of interaction and different chemical structures of DAAs targeting the same site on the same HCV protein [3].

(2) Genotypic analysis (sequence analysis): used to detect the amino acids substitutes which cause drug resistance and treatment failure [17]. Clonal and deep sequencing technologies allow reliable detection of viral variants with a frequency down to 0.5–1% and commonly accepted level reached to 15% [18]. Generally, due to the high heterogeneity of HCV iso‐ lates and methodological restrictions all sequencing technologies may miss detection of RAVs due to nonamplification based on HCV RNA secondary structures, primer selec‐ tion, and low frequencies within HCV quasispecies [3].

Resistance testing in clinical practice is not so easy, but it is actually very difficult. Limited number of well‐equipped virological labs all over the world that can deal with these tests, experienced hands and the ability to interpret the results correctly, make testing for resistance a time and money consuming procedure and balancing the benefit versus the cost should be considered especially when dealing with large populations having different genotypes.

#### **5.2. Timing of HCV resistance testing**

**Variant Sofosbuvir Dasabuvir**

**Variant Daclatasvir Ledipasvir Ombitasvir Elbasvir Velpatasvir**

M/L/L28 R ‐ ‐ ‐ ‐ P29 S ‐ ‐ ‐ ‐ Q/R/L30 R ‐ ‐ ‐ ‐ L31 R R ‐ R ‐ P32 S ‐ ‐ ‐ ‐ H/P58 R ‐ ‐ ‐ ‐ E62 S ‐ ‐ ‐ ‐ A92 S ‐ ‐ ‐ ‐ Y93 R R R R R M28 ‐ S R R ‐ Q30 ‐ R R R ‐ H58 ‐ S S ‐ ‐

S282T R R A421V ‐ R P495S/Q/L/A/T ‐ R C316Y/N ‐ R L419S ‐ R S368T ‐ R R422K ‐ R M414T/I/V/L ‐ R M423T/I/V/L ‐ R Y448C/H ‐ R I482L/V/T ‐ R G554D/S ‐ R A486/V/I/T/M ‐ R S556G ‐ R V494A ‐ R D559G ‐ R

M/L28 ‐ ‐ R ‐

**Table 5.** Resistance and cross‐resistance in NS5A inhibitors.

124 Advances in Treatment of Hepatitis C and B

**Table 6.** Resistance in NS5B inhibitors.

Because of the above‐mentioned limitations, resistance testing is not recommended before start‐ ing therapy with DAAs for the first time; especially in areas where HCV is highly endemic. Instead, trying to give patients the best chance of cure through using multiple drugs, adding rib‐ avirin or prolongation of the treatment duration if needed may be a good decision; also testing at the time of treatment failure usually associated with high prevalence of quasispecies and RAVs.

On the other hand, testing of resistance before retreatment of patients who fail to achieve virological response with DAAs may have a benefit for the proper selection of the best DAA drug for retreatment [4].

## **6. Management of drug failure and drug resistance**

Clear evidence is still not available about the best regimens, best duration, and best time for retreatment of patients with DAAs failures, yet European association for the study of the liver (EASL) [19] and American association for the study of the liver diseases (AASLD) [20] released their interim opinions for retreatment options.

EASL guidelines recommend that Sofosbuvir should be a cornerstone in any retreatment trial due to its high barrier to resistance, addition of 1 or 2 other DAAs preferably with no cross‐ resistance with the failed drug, addition of ribavirin if tolerable and prolongation of treatment duration to 24 weeks especially in cirrhotics.

AASLD guidelines using Sofosbuvir‐based triple or quadruple DAAs with ribavirin if toler‐ able for 12–24 weeks in case of failure of Sofosbuvir‐based dual regimen, RAVs testing prior to retreatment and the final treatment options is tailored based on its results.

Review of some recent published data in **Table 7** for retreatment of clinical trials appears to be insufficient to justify a competent guidelines, more data is needed to reach to the nearest figure to ideal. From these trials, we could choose one of the following models:




**Table 7.** Review of recent data for retreatment.

## **7. Conclusion**

EASL guidelines recommend that Sofosbuvir should be a cornerstone in any retreatment trial due to its high barrier to resistance, addition of 1 or 2 other DAAs preferably with no cross‐ resistance with the failed drug, addition of ribavirin if tolerable and prolongation of treatment

AASLD guidelines using Sofosbuvir‐based triple or quadruple DAAs with ribavirin if toler‐ able for 12–24 weeks in case of failure of Sofosbuvir‐based dual regimen, RAVs testing prior

Review of some recent published data in **Table 7** for retreatment of clinical trials appears to be insufficient to justify a competent guidelines, more data is needed to reach to the nearest

(1) The patients have no RAVs, so retreatment using the same failed regimen (or adding other drugs) could be allowed but add ribavirin if needed but not previously added and choose

(2) The patient has RAVs to protease or polymerase inhibitors, which will disappear after few weeks or months, so we could choose either to wait until reset point or to use another

(3) The patient has RAVs to NS5A inhibitor drug without cross‐resistance, so the failed drug

(4) The patient has RAVs to NS5A inhibitor at certain sites leading to resistance and cross‐ resistance, so the whole NS5A members from the same wave could not be used, shifting to different wave of the family or changing the whole group to protease inhibitors will

**Description Retreatment regimen Results RAVs impact**

50/51 (98%) SVR NA

76/79(96.2%) SVR

‐100% in patients without baseline RAVs ‐91.2% with baseline

NS3 RAVs ‐75% with baseline NS5A RAVs ‐66.7% in both NS3, NS5A RAVs

Sofosbuvir + Ledipasvir + Ribavirin for 12 weeks

Grazoprevir + Elbasvir + Ribavirin for 12 weeks

to retreatment and the final treatment options is tailored based on its results.

figure to ideal. From these trials, we could choose one of the following models:

the ideal duration according to the patient status.

be the best way.

Wyles et al. [21] 51 GT1 patients with

Forns et al. [22] 79 GT1 patients with

previous treatment failure 25 patients failed PegIFN/ RBV/Sofosbuvir 20 patients failed Sofosbuvir/RBV

5 failed Sofosbuvir placebo/

previous treatment failure 66 patients failed PegIFN/ RBV/protease inhibitor 12 patients intolerable to treatment with PegIFN/ RBV/protease inhibitor

PegIFN/RBV 1 failed GS‐0938 monotherapy

family of DAAs like NS5A inhibitors plus sofosbuvir.

could not be used but other drugs from the same family could be.

duration to 24 weeks especially in cirrhotics.

126 Advances in Treatment of Hepatitis C and B

HCV elimination is a worldwide goal; curing infection with oral drugs for short duration and minimal adverse events is going on. Appearance of resistance to DAAs is disappointing to the clinicians and the researchers yet choosing the proper treatment regimen initially leading to minimizing this problem. The ideal RAVs testing and interpretation lead to the best options to justify the retreatment regimen.

## **Author details**

Mohamed Hassany<sup>1</sup> and Aisha Elsharkawy2 \*

\*Address all correspondence to: a\_m\_sharkawy@yahoo.com

1 National Hepatology & Tropical Medicine Research Institute (NHTMRI), Cairo, Egypt

2 Endemic Medicine and hepatogastroentrology, Faculty of Medicine, Cairo University, Cairo, Egypt

## **References**


[14] Lawitz E, Matusow G, DeJesus E, et al. Simeprevir plus sofosbuvir in patients with chronic hepatitis C virus genotype 1 infection and cirrhosis: a phase 3 study (OPTIMIST‐2). Hepatology. 2016 Aug;64(2):360–369.

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[1] Gower E, Estes C, Blach S, Razavi‐Shearer K, Razavi H. Global epidemiology and geno‐ type distribution of the hepatitis C virus infection. J Hepatol. 2014;61:S45–S57

[2] Bartenschlager R, Lohmann V, Penin F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol. 2013;11:482–496. [3] Sarrazin C. The importance of resistance to direct antiviral drugs in HCV infection in

[4] Pawlotsky J‐M. Hepatitis C virus resistance to direct‐acting antiviral drugs in interferon‐

[5] Farci P, Purcell RH. Clinical significance of hepatitis C virus genotypes and quasispecies.

[6] Asselah T, Marcellin P. Interferon free therapy with direct acting antivirals for HCV.

[7] Majumdar A, Kitson MT, Roberts SK. Systematic review: current concepts and chal‐ lenges for the direct‐acting antiviral era in hepatitis C cirrhosis. Aliment Pharmacol

[8] Zeuzem S, Mizokami M, Pianko S, Mangia A, Han K, Martin R.et al. Prevalence of pre‐ treatment NS5A resistance associated variants in genotype 1 patients across different regions using deep sequencing and effect on treatment outcome with LDV/SOF. AASLD.

[9] Zeuzem S, Ghalib R, Reddy KR, Pockros PJ, Ben Ari Z, Zhao Y, et al. Grazoprevir‐ elbasvir combination therapy for treatment‐naive cirrhotic and noncirrhotic patients with chronic hcv genotype 1, 4, or 6 infection: a randomized trial. Ann Intern Med.

[10] Lenz O, Verbinnen T, Fevery B, Tambuyzer L, Vijgen L, Peeters M, et al. Virology anal‐ yses of HCV isolates from genotype 1‐infected patients treated with simeprevir plus peginterferon/ribavirin in Phase IIb/III studies. J Hepatol. 2015 May;62(5):1008–1014.

[11] Krishnan P, Tripathi R, Schnell G, et al. Resistance analysis of baseline and treatment‐emer‐ gent variants in hepatitis C virus genotype 1 in the AVIATOR study with paritaprevir‐ ritonavir, ombitasvir, and dasabuvir. Antimicrob Agents Chemother. 2015;59:5445–5454.

[12] 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

[13] Kwo P, Gitlin N, Nahass N, et al. Simeprevir plus sofosbuvir (12 and 8 weeks) in HCV genotype 1‐infected patients without cirrhosis: OPTIMIST‐1, a Phase 3, randomized

clinical practice. J Hepatol. 2016 Feb;64(2):486–504.

Semin Liver Dis. 2000 Jan;20(1):103–126.

Liver Int. 2013 Mar;33(Suppl 1):93–104.

Ther. 2016 Jun;43(12):1276–1292.

2015. p. Abstract 91.

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free regimens. Gastroenterology. 2016 Jul;151(1):70–86.


#### **Treatment of Chronic Hepatitis B: An Update and Prospect for Cure Treatment of Chronic Hepatitis B: An Update and Prospect for Cure Treatment of Chronic Hepatitis B: An Update and Prospect for Cure**

Andrew Dargan and Hie-Won Hann Andrew Dargan and Hie-Won Hann Andrew Dargan and Hie-Won Hann

Additional information is available at the end of the chapter Additional information is available at the end the chapterAdditional information is available at the end of the chapter

http://dx.doi.org/10.5772/66724

#### **Abstract**

Since the discovery of the hepatitis B virus (HBV) by Blumberg et al., nearly half a century ago, the subsequent development of a vaccine, understanding of the patho‐ genesis, and the advent of antiviral drugs, the prevalence of chronic hepatitis B has decreased from approximately 5% to 3.61% of the worldwide population. Despite this improvement, approximately 248 million individuals are still infected with the virus. Effective treatment of chronic hepatitis B is extremely important as a positive correla‐ tion has been observed between baseline viral load and the risk for the development of hepatocellular carcinoma (HCC). While there have been significant advancements in the management of hepatitis B virus with available nucleos(t)ide analogues, there remains much work to be done to prevent HCC. The molecular mechanism and the subsequent carcinogenesis and progression of chronic HBV carriers to HCC remain in large part poorly understood. While current treatment with nucleos(t)ide analogues has succeeded in maintaining undetectable viral levels in patients with chronic hepatitis B, eradication of the virus has not been possible, and there remains the risk of develop‐ ment of HCC. Therefore, more effective treatment regimens aiming for HBV cure are urgently needed. With multiple new therapies in the pipeline, the future of treating hepatitis B is an exciting and developing one, and hopefully, it will soon become a disease of the past.

**Keywords:** hepatitis B, hepatocellular carcinoma, anti‐HBV drugs, nucleos(t)ide analogues, HBV cure, HBV therapy

## **1. Introduction**

In the past 50 years, since the discovery of the hepatitis B virus [1], the development of a vaccine, understanding of the pathogenesis, and the advent of antiviral drugs, the preva‐ lence of chronic hepatitis B has decreased from approximately 5% to 3.61% of the worldwide

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee chapter is distributed under Attribution License distribution, and reproduction in any medium, provided the original work is properly cited.

population [2]. Nevertheless, hepatitis B remains a common and frequently encountered con‐ dition, affecting approximately 248 million individuals in the world.

The vast majority of individuals with chronic hepatitis B are located in Africa and Eastern Asia. In the United States, over 2 million Americans are afflicted and the majority (1.5 mil‐ lion) are immigrants from foreign countries [3, 4]. Effective treatment of chronic hepatitis B remains of extremely high importance, as patients who have been found to have higher baseline viral loads have been shown to have increased rates of hepatocellular carcinoma (HCC) [5]. As the treatment landscape of hepatitis B has shifted from earlier regimens of interferon and lamivudine to newer agents, namely tenofovir and entecavir, there remains much work to be done to reduce viral loads in patients and prevent long‐term sequelae of cirrhosis and HCC. This chapter will discuss the natural history and potential carcinogenesis of hepatitis B virus, and will discuss current and possible future treatments, and the hope for an eventual cure.

## **2. Natural history of hepatitis B virus**

In contrast to many known pathogens, hepatitis B virus (HBV) is not directly cytopathic to hepatocytes. Although not completely understood, the injury to the liver cells is in part through a host immune mechanism. Replicating HBV in hepatocytes produces HBsAg par‐ ticles and virions which are taken up by the antigen presenting cells. The viral proteins are degraded to peptides, which are presented on the cell surface bound to MHC class I or II molecules. MHC class I molecules are recognized by CD8 T cells and MHC II by CD4 T cells. Virus‐specific CD8+ cytotoxic T cells, with help from CD4+ T cells, recognize viral antigens presented on MHC class I chains on infected hepatocytes. This recognition reaction can lead to either direct lysis of the infected hepatocyte or the release of interferon‐**γ** and TNF‐*a*, which can down‐regulate viral replication in surrounding hepatocytes without direct cell killing [6].

In order to further discuss advancements in the understanding and treatment of HBV, it is important first to review the natural history of the disease. The cycle of chronic HBV infection primarily consists of five phases as shown in **Figure 1** [7, 8].

In the initial infection phase, or so‐called immune tolerant phase, patients have very minimal inflammation. The hallmark of this phase is that these patients are found to be positive for HBeAg with high viral loads, typically >20,000 IU/mL (> 10**<sup>5</sup>** copies/mL) [9]. Conversely, they have normal aminotransferase (ALT) levels, and near‐normal liver parenchyma on biopsy [10]. This "immune tolerant" phase is relatively short when HBV is acquired in adulthood, but can be sustained for much longer periods of time with infections acquired at birth or in early childhood [11, 12]. The risk of progressing to the chronic carrier state is significantly higher in infections acquired at a younger age, including up to a 90% risk when infected perinatally, as compared to a less than 1% risk of progression when acquired as an adult [13, 14].

population [2]. Nevertheless, hepatitis B remains a common and frequently encountered con‐

The vast majority of individuals with chronic hepatitis B are located in Africa and Eastern Asia. In the United States, over 2 million Americans are afflicted and the majority (1.5 mil‐ lion) are immigrants from foreign countries [3, 4]. Effective treatment of chronic hepatitis B remains of extremely high importance, as patients who have been found to have higher baseline viral loads have been shown to have increased rates of hepatocellular carcinoma (HCC) [5]. As the treatment landscape of hepatitis B has shifted from earlier regimens of interferon and lamivudine to newer agents, namely tenofovir and entecavir, there remains much work to be done to reduce viral loads in patients and prevent long‐term sequelae of cirrhosis and HCC. This chapter will discuss the natural history and potential carcinogenesis of hepatitis B virus, and will discuss current and possible future treatments, and the hope

In contrast to many known pathogens, hepatitis B virus (HBV) is not directly cytopathic to hepatocytes. Although not completely understood, the injury to the liver cells is in part through a host immune mechanism. Replicating HBV in hepatocytes produces HBsAg par‐ ticles and virions which are taken up by the antigen presenting cells. The viral proteins are degraded to peptides, which are presented on the cell surface bound to MHC class I or II molecules. MHC class I molecules are recognized by CD8 T cells and MHC II by CD4 T cells. Virus‐specific CD8+ cytotoxic T cells, with help from CD4+ T cells, recognize viral antigens presented on MHC class I chains on infected hepatocytes. This recognition reaction can lead to either direct lysis of the infected hepatocyte or the release of interferon‐**γ** and TNF‐*a*, which can down‐regulate viral replication in surrounding hepatocytes without direct cell

In order to further discuss advancements in the understanding and treatment of HBV, it is important first to review the natural history of the disease. The cycle of chronic HBV infection

In the initial infection phase, or so‐called immune tolerant phase, patients have very minimal inflammation. The hallmark of this phase is that these patients are found to be positive for

have normal aminotransferase (ALT) levels, and near‐normal liver parenchyma on biopsy [10]. This "immune tolerant" phase is relatively short when HBV is acquired in adulthood, but can be sustained for much longer periods of time with infections acquired at birth or in early childhood [11, 12]. The risk of progressing to the chronic carrier state is significantly higher in infections acquired at a younger age, including up to a 90% risk when infected perinatally, as compared to a less than 1% risk of progression when acquired as an adult

copies/mL) [9]. Conversely, they

dition, affecting approximately 248 million individuals in the world.

for an eventual cure.

132 Advances in Treatment of Hepatitis C and B

killing [6].

[13, 14].

**2. Natural history of hepatitis B virus**

primarily consists of five phases as shown in **Figure 1** [7, 8].

HBeAg with high viral loads, typically >20,000 IU/mL (> 10**<sup>5</sup>**

**Figure 1.** Five phases of chronic hepatitis B \*. *\* Adapted from Tong et al. (7) and modified by Halegoua‐DeMarzio and Hann (8).*

Following the "immune tolerant" phase, patients progress into the "immune clearance" phase, which again consists of high viral loads and a persistently positive HBeAg. However, at this point patients begin experiencing increased inflammation, with elevated ALT levels, and potential hepatic decompensation [15, 16]. It is at this point when the viral DNA levels of HBV begin to decline, as does the presence of HBeAg. This is in large part due to the activated T‐cell immune response, and subsequent destruction of infected hepatocytes [6]. An outcome of the immune clearance phase is HBeAg seroconversion, which has been found to be critical in predicting progression to cirrhosis and HCC.

Following HBeAg seroconversion, patients typically enter an "inactive carrier" phase, where HBeAg becomes undetectable, and HBe antibodies (anti‐HBe) appear [17]. Typically the patient's viral load is low or undetectable and ALT returns to normal. Biopsy at this time will show minimal fibrosis and mild hepatitis. If the patient had experienced severe liver injury during the "immune clearance" phase, cirrhosis can also be present [17].

During the "reactivation phase", patients who were previously infected with HBV again have a detectable viral load, elevated ALT, and inflammation seen on biopsy [18]. In contrast to the "immune tolerant" phase, however, these patients do not have HBeAg positivity, but do have positive anti‐HBe. As a result, this phase is known as the "HBeAg negative chronic hepatitis B" phase. As with the "immune clearance" phase, these patients can exhibit marked inflammation and hepatocyte destruction, and can experience hepatic decompensation during this phase.

The final phase of HBV infection is known as the "remission" or "inactive" phase, in which HBsAg may become negative, but anti‐HBe and anti‐HBc remain positive. Transaminases are normal at this time, and HBV viral loads are very low or undetectable.

It is important to remember that once patients are infected, they remain positive for anti‐HBc IgG throughout even after they lose HBsAg and after they acquire anti‐HBs. Also, anti‐HBe may often remain detectable.

Furthermore, as part of the infection of HBV into human hepatocytes, HBV DNA converts itself into a covalently closed circular DNA, known as cccDNA, inside the hepatocyte nucleus [19]. This cccDNA serves as a template for transcription of HBV viral mRNAs, which translate and produce HBV proteins as well as provide a template for HBV DNA synthesis [20]. Thus, although a patient's viral load may be undetectable and HBsAg may become negative, the patient is not cured of HBV, as cccDNA will remain within hepatocytes.

## **3. Current treatments for chronic hepatitis B**

Anti‐HBV treatment drugs have made significant progress in improving the health and lifes‐ pan of patients with HBV. Beginning with interferon in 1991, therapies have become more targeted with lower resistance profiles and more tolerable side effects. The ultimate goal of hepatitis B treatment is to achieve remission, i.e., sustained suppression of viral replication. This, in turn, will lead to prevention of progression to cirrhosis and/or HCC. Several studies have demonstrated the reduction of HCC development with antiviral drugs [21–26].

Currently there are six approved treatments for HBV. The details of drugs and efficacy are shown in **Table 1**.

*Pegylated interferon alpha‐2a***.** The first treatment approved for HBV in 1991, pegylated interferon alpha‐2a, or peg‐IFN α‐2a, is an immunomodulator, which also displays a weak effect against the virus itself [27]. It is administered as an injection, which patients receive weekly for a total treatment of 48 weeks. It has been shown to produce HBeAg seroconver‐ sion in 27% of patients, and 25% of patients develop an undetectable HBV DNA load [28]. It has been shown to have the best response for those individuals with genotype A with either ALT > 2x ULN or low HBV DNA, and for genotypes B and C with ALT > 2x ULN and low HBV DNA [29]. Although an effective treatment in the past, peg‐IFN α‐2a is a small percent‐ age of current HBV treatments in the US [30]. Much of this is likely due to the requirement of an injection weekly, a large percentage of patients who fail to respond, and a significant side effect profile.

*Lamivudine***.** The first nucleoside analogue approved for treatment of HBV, lamivudine (LMV) is a reverse transcriptase inhibitor. Functioning as a nucleoside analogue, it inhibits DNA synthesis of HBV. The treatment is extended across 1 year, and has been associated with a seroconversion rate of 16–18% at 1 year, and increases up to nearly 50% at 4 years [31, 32]. It is the least expensive of the nucleotide/nucleoside analogues, and is safe to use in pregnancy, which is one of its most common uses in current times. LMV has also been shown to reduce the rate of development of both fibrosis and HCC [33]. The most significant evidence of the effectiveness of LMV was shown in a randomized, controlled trial by Liaw et al., comparing LMV versus placebo in patients with chronic hepatitis B and high serum levels of HBV DNA

**Table 1.** Treatment options of chronic hepatitis B\*.

It is important to remember that once patients are infected, they remain positive for anti‐HBc IgG throughout even after they lose HBsAg and after they acquire anti‐HBs. Also, anti‐HBe

Furthermore, as part of the infection of HBV into human hepatocytes, HBV DNA converts itself into a covalently closed circular DNA, known as cccDNA, inside the hepatocyte nucleus [19]. This cccDNA serves as a template for transcription of HBV viral mRNAs, which translate and produce HBV proteins as well as provide a template for HBV DNA synthesis [20]. Thus, although a patient's viral load may be undetectable and HBsAg may become negative, the

Anti‐HBV treatment drugs have made significant progress in improving the health and lifes‐ pan of patients with HBV. Beginning with interferon in 1991, therapies have become more targeted with lower resistance profiles and more tolerable side effects. The ultimate goal of hepatitis B treatment is to achieve remission, i.e., sustained suppression of viral replication. This, in turn, will lead to prevention of progression to cirrhosis and/or HCC. Several studies

Currently there are six approved treatments for HBV. The details of drugs and efficacy are

*Pegylated interferon alpha‐2a***.** The first treatment approved for HBV in 1991, pegylated interferon alpha‐2a, or peg‐IFN α‐2a, is an immunomodulator, which also displays a weak effect against the virus itself [27]. It is administered as an injection, which patients receive weekly for a total treatment of 48 weeks. It has been shown to produce HBeAg seroconver‐ sion in 27% of patients, and 25% of patients develop an undetectable HBV DNA load [28]. It has been shown to have the best response for those individuals with genotype A with either ALT > 2x ULN or low HBV DNA, and for genotypes B and C with ALT > 2x ULN and low HBV DNA [29]. Although an effective treatment in the past, peg‐IFN α‐2a is a small percent‐ age of current HBV treatments in the US [30]. Much of this is likely due to the requirement of an injection weekly, a large percentage of patients who fail to respond, and a significant side

*Lamivudine***.** The first nucleoside analogue approved for treatment of HBV, lamivudine (LMV) is a reverse transcriptase inhibitor. Functioning as a nucleoside analogue, it inhibits DNA synthesis of HBV. The treatment is extended across 1 year, and has been associated with a seroconversion rate of 16–18% at 1 year, and increases up to nearly 50% at 4 years [31, 32]. It is the least expensive of the nucleotide/nucleoside analogues, and is safe to use in pregnancy, which is one of its most common uses in current times. LMV has also been shown to reduce the rate of development of both fibrosis and HCC [33]. The most significant evidence of the effectiveness of LMV was shown in a randomized, controlled trial by Liaw et al., comparing LMV versus placebo in patients with chronic hepatitis B and high serum levels of HBV DNA

have demonstrated the reduction of HCC development with antiviral drugs [21–26].

patient is not cured of HBV, as cccDNA will remain within hepatocytes.

**3. Current treatments for chronic hepatitis B**

may often remain detectable.

134 Advances in Treatment of Hepatitis C and B

shown in **Table 1**.

effect profile.

[33]. The primary endpoint was progression of liver disease identified as either an increase in Child‐Pugh score, bleeding from esophageal varices, or development of HCC. The study was discontinued early given that it demonstrated such a clear benefit of LMV compared to pla‐ cebo [33]. Despite the success that has been shown with LMV, its use is limited, mainly due to the high incidence of resistance, especially compared with newer nucleotide/nucleoside ana‐ logues [34]. In one study, however, much lower resistance was observed if the baseline HBV DNA was < 106 copies/mL [35], and there has been an extensive review as to the discrepancies of LMV resistance among the multiple studies regarding the incidence of LMV resistance [36]. LMV also reduced vertical HBV transmission from highly viremic mothers to their newborns [37]. Currently, the use of oral antiviral agents during the first and second trimesters of preg‐ nancy is not recommended.

*Adefovir dipivoxil***.** The first nucleotide analogue, adefovir dipivoxil (ADV), was approved by the FDA for use in 2002. Similar to LMV in its mechanism of action, ADV functions as a reverse transcriptase inhibitor. As compared with LMV, however, ADV had both an increased antiviral potency, and an intrinsic stereoscopic structure that prevents emergence of viral resistance. HBe seroconversion was achieved in 12% of patients after 1 year of therapy with ADV, and a 53% rate of histological improvements in patients who were positive for HBeAg [38]. Of the patients who did seroconvert, it was found to be sustained in 91% of these patients [39]. Like LMV, however, prolonged use is associated with an increase in resistance, pro‐ gressing from 3% at 2 years to 29% at 5 years [40]. Due to this, in addition to associated renal toxicity, the use of ADV has become increasingly rare with the development of newer, more effective therapies.

*Entecavir***.** The second nucleoside analogue approved for treatment of chronic HBV, entecavir (ETV), was approved by the FDA for treatment in 2005. It has been shown to be superior at reducing HBV DNA levels, as compared with LMV [41]. In a phase 3 study comparing ETV to LMV after 1 year of treatment, patients were found to have improved virological response with HBV DNA < 400 copies/mL (67% vs 36%), improvement on histologic examination (72% vs 62%), and improvement in aminotransferases, namely ALT (78% returned to normal as compared to 70%) [41]. In longer term studies, up to 96% of patients had improvement in histologic examination, and improvement in fibrosis score after 6 years [42]. Improvements were even found in patients with cirrhosis. Entecavir also was shown to keep HBeAg‐positive patients with HBV DNA levels below 300 copies/mL in 94% of patients at 5 years [43]. It has been shown to cause viral suppression quicker than ADV, and has been shown to significantly decrease the incidence of HCC in chronic HBV patients, with a 3.7% incidence in the ETV group as compared with 13.7% in the untreated group [23]. One of the most important fea‐ tures of entecavir, and a reason why it remains one of the two recommended treatments for chronic HBV today, is that it has a high genetic barrier and a very low incidence of resistance. The cumulative incidence of resistance after 6 years has been found to be 1.2% in nucleoside‐ naïve patients [44].

*Telbivudine***.** Another nucleoside analogue similar in structure to LMV, telbivudine (TLV) was approved by the FDA for treatment of chronic HBV in 2006. In HBeAg‐positive patients, the seroconversion with TLV was found to be 22% at 1 year and 30% at 2 years [45, 46]. Viral sup‐ pression to less than 300 copies of DNA/mL was found to be 60% after 1 year of TLV therapy, and 56% after two years of therapy [45, 46]. Unfortunately, although TLV was shown to have promising effects and to have a higher barrier to resistance than LMV, resistance has been found to be as high as 21.6% in HBeAg‐positive patients, and 8.6% in HBeAg‐negative patients [47]. Because of this, TLV currently is not a recommended first‐line treatment. However, TLV is shown to be highly effective for those with low baseline HBV DNA and achieves undetect‐ able HBV DNA at week 24. Therefore, TLV is highly effective for patients with the above characteristics [48]. Furthermore, recent studies report the renoprotective effect of TLV, its role in preventing ADV‐induced nephrotoxicity, and increased GFR improvement of renal function in liver transplant patients and in patients with compensated or decompensated HBV‐related liver diseases [49–52]. The rate of vertical transmission was reduced when tel‐ bivudine was given to mothers with high viral loads during the third trimester of pregnancy [53]. Currently, the use of oral antiviral agents during the first and second trimesters of preg‐ nancy is not recommended.

*Tenofovir***.** The most recent nucleotide analogue, tenofovir disoproxil fumarate (TDF), was approved by the FDA for treatment of patients with chronic hepatitis B in 2008. Structurally similar but a more potent drug than ADV, TDF has been shown to produce more viral suppression in HBeAg‐positive patients, with 76% of patients achieving viral loads < 400 copies/mL as compared with 13% of patients treated with ADV after 48 weeks of treatment [54]. ALT normalization, histologic improvement, and HBsAg loss were all also found to be significantly increased in patients treated with TDF as compared with ADV [54]. Data have shown an excellent continued response, with a 7‐year viral suppression (HBV DNA levels < both 69 IU/mL and 29 IU/mL) of greater than 99% in both HBeAg‐negative and HBeAg‐positive patients [55]. In addition to its effectiveness, TDF has also been shown to have an extremely favorable resistance profile [56]. Due to the effectiveness and the virtual absence of resistance, TDF has been recommended as a first‐line treatment in patients with chronic hepatitis B.

Several currently used guidelines are shown in **Table 2**.

of LMV resistance among the multiple studies regarding the incidence of LMV resistance [36]. LMV also reduced vertical HBV transmission from highly viremic mothers to their newborns [37]. Currently, the use of oral antiviral agents during the first and second trimesters of preg‐

*Adefovir dipivoxil***.** The first nucleotide analogue, adefovir dipivoxil (ADV), was approved by the FDA for use in 2002. Similar to LMV in its mechanism of action, ADV functions as a reverse transcriptase inhibitor. As compared with LMV, however, ADV had both an increased antiviral potency, and an intrinsic stereoscopic structure that prevents emergence of viral resistance. HBe seroconversion was achieved in 12% of patients after 1 year of therapy with ADV, and a 53% rate of histological improvements in patients who were positive for HBeAg [38]. Of the patients who did seroconvert, it was found to be sustained in 91% of these patients [39]. Like LMV, however, prolonged use is associated with an increase in resistance, pro‐ gressing from 3% at 2 years to 29% at 5 years [40]. Due to this, in addition to associated renal toxicity, the use of ADV has become increasingly rare with the development of newer, more

*Entecavir***.** The second nucleoside analogue approved for treatment of chronic HBV, entecavir (ETV), was approved by the FDA for treatment in 2005. It has been shown to be superior at reducing HBV DNA levels, as compared with LMV [41]. In a phase 3 study comparing ETV to LMV after 1 year of treatment, patients were found to have improved virological response with HBV DNA < 400 copies/mL (67% vs 36%), improvement on histologic examination (72% vs 62%), and improvement in aminotransferases, namely ALT (78% returned to normal as compared to 70%) [41]. In longer term studies, up to 96% of patients had improvement in histologic examination, and improvement in fibrosis score after 6 years [42]. Improvements were even found in patients with cirrhosis. Entecavir also was shown to keep HBeAg‐positive patients with HBV DNA levels below 300 copies/mL in 94% of patients at 5 years [43]. It has been shown to cause viral suppression quicker than ADV, and has been shown to significantly decrease the incidence of HCC in chronic HBV patients, with a 3.7% incidence in the ETV group as compared with 13.7% in the untreated group [23]. One of the most important fea‐ tures of entecavir, and a reason why it remains one of the two recommended treatments for chronic HBV today, is that it has a high genetic barrier and a very low incidence of resistance. The cumulative incidence of resistance after 6 years has been found to be 1.2% in nucleoside‐

*Telbivudine***.** Another nucleoside analogue similar in structure to LMV, telbivudine (TLV) was approved by the FDA for treatment of chronic HBV in 2006. In HBeAg‐positive patients, the seroconversion with TLV was found to be 22% at 1 year and 30% at 2 years [45, 46]. Viral sup‐ pression to less than 300 copies of DNA/mL was found to be 60% after 1 year of TLV therapy, and 56% after two years of therapy [45, 46]. Unfortunately, although TLV was shown to have promising effects and to have a higher barrier to resistance than LMV, resistance has been found to be as high as 21.6% in HBeAg‐positive patients, and 8.6% in HBeAg‐negative patients [47]. Because of this, TLV currently is not a recommended first‐line treatment. However, TLV is shown to be highly effective for those with low baseline HBV DNA and achieves undetect‐ able HBV DNA at week 24. Therefore, TLV is highly effective for patients with the above

nancy is not recommended.

136 Advances in Treatment of Hepatitis C and B

effective therapies.

naïve patients [44].

Since the majority of chronic hepatitis B patients in the United States are immigrants form endemic countries, especially from Asia, where infection takes place commonly at birth or in early childhood, Asian‐American algorithm is frequently used for treatment for this majority of HBV patients. These guidelines are as follows [7]:


*APASL (Asian Pacific Association of the Study of the Liver (74) AASLD (American Association of the Study of Liver Diseases) (75) Asian American Algorithm (7) ULN = Upper limit of normal; NS = Not stated). \* UNL: 30 IU/mL for men, 19 IU/mL for women 2000 IU/mL = 104copies/mL 20,000 IU/mL=105copies/mL*

**Table 2.** Current treatments for hepatitis B in chronic hepatitis, as recommended by different guidelines (ref. 7, 69–72).


Before the antiviral drugs became available, 25–40% of HBV‐infected individuals used to progress from chronic hepatitis to cirrhosis and eventually to HCC as shown in **Table 3**

**Table 3.** Natural history of chronic hepatitis B infection.

**6.** *Monitoring of resistance:* Viral breakthrough with confirmation of single drug resistance

**Table 2.** Current treatments for hepatitis B in chronic hepatitis, as recommended by different guidelines (ref. 7, 69–72).

**7.** Surveillance for HCC with Alpha‐fetoprotein (AFP) and abdominal ultrasound should be performed every 6 months in HBsAg‐positive patients with chronic hepatitis, cirrhosis,

**8.** *With regard to stopping treatment,* for HBeAg (+) patients, following HBeAg seroconver‐ sion, continue consolidation for 1–2 years before stopping therapy. However, the relapse rate is high, and longer consolidation therapy may be needed. For HBeAg (‐) patients,

Before the antiviral drugs became available, 25–40% of HBV‐infected individuals used to progress from chronic hepatitis to cirrhosis and eventually to HCC as shown in **Table 3**

antiviral therapy should be indefinite therapy until HBsAg seroconversion.

requires switching to another first‐line oral antiviral agent.

and for patients with a family history of HCC.

*\*EASL (European Association for the Study for the Liver) (72),*

*APASL (Asian Pacific Association of the Study of the Liver (74) AASLD (American Association of the Study of Liver Diseases) (75)*

*US Algorithm (73)*

*Asian American Algorithm (7)*

138 Advances in Treatment of Hepatitis C and B

*2000 IU/mL = 104copies/mL 20,000 IU/mL=105copies/mL*

*ULN = Upper limit of normal; NS = Not stated). \* UNL: 30 IU/mL for men, 19 IU/mL for women*

**Figure 2.** Higher baseline viral loads are associated with increased rate of HCC. \**From Chen, et al. (5).*

In their 13‐year follow‐up study of HBV‐infected carriers, Chen et al. have noted that higher baseline viral loads were associated with an increased rate of HCC [5] (**Figure 2**).

During the last 10 years, several studies have demonstrated that antiviral treatment signifi‐ cantly reduced the incidence of HCC [21–26]. However, all these treatment modalities are to suppress HBV replication. They do not fully eradicate the virus. The inability to eradi‐ cate HBV still leaves infected individuals at the risk for HCC. Current anti‐HBV treatment can achieve "functional cure" but not "complete cure", the terminology as coined by Zeisel et al. [19]. (**Table 4**).


**Table 4.** Definitions of HBV Cure.

## **4. Hepatocarcinogenesis**

The pathogenesis for HBV‐related HCC is not fully understood, but is likely multifactorial. HBV DNA level is a known factor, and the presence of HBV DNA has been shown to have a linear relationship to the development of HCC [5]. A high viral load leads to a persistent immune response against hepatocytes, with persistent inflammation, regeneration, and fibro‐ sis. This up‐regulated state of inflammation can in turn predispose to a malignant transforma‐ tion [57]. Several studies have also suggested that the integration of HBV DNA into the host DNA can lead to chromosomal instability and eventual gene rearrangement. These rearrange‐ ments can, in effect, lead to deregulation and instability of gene expression, subsequently leading to oncogenesis [58–60].

The association with chronic HBV and HCC has been described as early as the 1970s. The land‐ mark cohort study by Beasley et al. in 1981, which studied over 22,000 men in Taiwan, showed a significant association between chronic HBV carriers and the development of HCC. In this study, the relative risk of development of HCC in men with chronic HBV was determined to be 63 times higher as compared with uninfected individuals [61]. This study also designated the HBV vaccine (plasma vaccine by Blumberg and Millman followed by the recombinant vaccine) as the first "Cancer Vaccine" by the World Health Organization. The increased risk of HCC in patients with HBV has repeatedly been confirmed with smaller, more recent studies. Although HBsAg seroconversion and viral suppression are typically associated with protec‐ tion against HCC, patients who have cleared their viral load have still been found to acquire HCC. This is likely due to the continued presence of cccDNA, in a mechanism that is not well understood. Studies have also shown that HCC development is better associated with patients who have had active HBV infection for longer time periods, including patients who were infected at younger ages. Thus, it is thought that HCC progression is likely a result of HBV replication itself and subsequent liver injuries that follow [62]. It also raises the point that in individuals infected earlier, carcinogenic processes may have already been in play prior to the halt of viral replication later in life, and the ability of HBV to integrate into the infected host's hepatocyte genome is one of the most important direct pro‐oncogenic properties.

In addition to chronic HBV carrier status, other risk factors have been identified which pre‐ dispose patients with hepatitis B to HCC. These factors include co‐infection with hepatitis C (HCV), a family history of prior HCC, concurrent alcohol use, and a predominance of genotype C [63–66]. Additionally, the presence of core promoter mutations, the most com‐ mon of which is the HBx protein, a potent activator of multiple genes, including oncogenes, has been discovered [67].

## **5. Future treatments**

**4. Hepatocarcinogenesis**

**Table 4.** Definitions of HBV Cure.

*Adapted and edited from Zeisel et al. (19)*

140 Advances in Treatment of Hepatitis C and B

*\*Ab, antibodies; cccDNA, covalently closed circular DNA*

leading to oncogenesis [58–60].

has been discovered [67].

The pathogenesis for HBV‐related HCC is not fully understood, but is likely multifactorial. HBV DNA level is a known factor, and the presence of HBV DNA has been shown to have a linear relationship to the development of HCC [5]. A high viral load leads to a persistent immune response against hepatocytes, with persistent inflammation, regeneration, and fibro‐ sis. This up‐regulated state of inflammation can in turn predispose to a malignant transforma‐ tion [57]. Several studies have also suggested that the integration of HBV DNA into the host DNA can lead to chromosomal instability and eventual gene rearrangement. These rearrange‐ ments can, in effect, lead to deregulation and instability of gene expression, subsequently

The association with chronic HBV and HCC has been described as early as the 1970s. The land‐ mark cohort study by Beasley et al. in 1981, which studied over 22,000 men in Taiwan, showed a significant association between chronic HBV carriers and the development of HCC. In this study, the relative risk of development of HCC in men with chronic HBV was determined to be 63 times higher as compared with uninfected individuals [61]. This study also designated the HBV vaccine (plasma vaccine by Blumberg and Millman followed by the recombinant vaccine) as the first "Cancer Vaccine" by the World Health Organization. The increased risk of HCC in patients with HBV has repeatedly been confirmed with smaller, more recent studies. Although HBsAg seroconversion and viral suppression are typically associated with protec‐ tion against HCC, patients who have cleared their viral load have still been found to acquire HCC. This is likely due to the continued presence of cccDNA, in a mechanism that is not well understood. Studies have also shown that HCC development is better associated with patients who have had active HBV infection for longer time periods, including patients who were infected at younger ages. Thus, it is thought that HCC progression is likely a result of HBV replication itself and subsequent liver injuries that follow [62]. It also raises the point that in individuals infected earlier, carcinogenic processes may have already been in play prior to the halt of viral replication later in life, and the ability of HBV to integrate into the infected host's

hepatocyte genome is one of the most important direct pro‐oncogenic properties.

In addition to chronic HBV carrier status, other risk factors have been identified which pre‐ dispose patients with hepatitis B to HCC. These factors include co‐infection with hepatitis C (HCV), a family history of prior HCC, concurrent alcohol use, and a predominance of genotype C [63–66]. Additionally, the presence of core promoter mutations, the most com‐ mon of which is the HBx protein, a potent activator of multiple genes, including oncogenes, Most current guidelines recommend against HBV treatment of patients in the immune toler‐ ant phase (**Table 2**). However, recent reports have indicated evidence that immune reactivity is in fact present in patients during this immune tolerant stage [68–70]. There is a growing opinion that to prevent HCC, we should consider earlier treatment of chronic hepatitis B as lucidly reasoned by Zoulim and Mason [71]. Given the emergence of HCC even in patients who had become seronegative, these guidelines should be readdressed in order to treat patients starting at a younger age, in order to prevent progression of disease and the develop‐ ment of HCC, as viral suppression alone has not proven effective for the absolute prevention of HCC. Additionally, the required long‐term therapy imposes not only financial burden but may also put patients at risk for potential drug resistance and unknown toxicity.

Along with nucleoside/nucleotide analogues, treatment may need to include targeting the cccDNA and inhibiting viral entry into the newly formed hepatocytes. This may be accom‐ plished via a T‐cell vaccine which specifically targets HBV, enhancing innate immunity with toll‐like receptor agonist. Several compounds have been identified which have the


*\*cccDNA, covalently closed circular DNA; DAA, direct‐acting antiviral; FDA, US Food and Drug Administration; HCC, hepatocellular carcinoma; HTA, host‐targeting agent; IFN, interferon; LTβR, lymphotoxin‐β receptor; NTCP, sodium taurocholate co‐transporting polypeptideAdapted and edited from Zeisel et al. (19)*

**Table 5.** Emerging drugs against HBV.

potential for eradicating the virus. The clinical trials are in progress at different phases to further investigate these compounds [19]. Among these are direct‐acting antagonists against the HBV capsid, against the HBV cccDNA, and against the HBV RNA. While the targets enhancing the innate immunity are mainly in the preclinical phase, they pose excit‐ ing possibilities for the future of HBV treatment. The potential drugs in the pipelines are shown below [19] (**Table 5**).

## **6. Conclusion**

While there have been significant advancements in the understanding and management of hepatitis B virus, there remains much to be learned. The molecular mechanism and the subsequent carcinogenesis and progression of chronic HBV carriers to HCC remain in large part poorly understood. While significant improvements in treatment of HBV continue to be made, research toward HBV complete cure and the treatment landscape now is much differ‐ ent than it was at the end of the twentieth century. The development of nucleotide and nucleo‐ side analogues, particularly entecavir and tenofovir, has significantly improved the ability of chronic HBV carriers to remain with undetectable viral levels. There remains, however, the possibility of development of HCC, in part likely in the early stages of infection, as well as the viral incorporation into hepatocyte DNA. Therefore, more effective treatment regimens need to be developed, and the prospect of treating individuals at earlier stages of HBV should be addressed. With multiple new therapies in the pipeline, the future of treating hepatitis B is an exciting and developing one, and hopefully, it will soon become a disease of the past.

## **Disclosures:**

AD, no conflict, HWH, receives research grants from Bristol Myers‐Squibb and Gilead Sciences.

## **Author details**

Andrew Dargan2 and Hie‐Won Hann1, 2\*

\*Address all correspondence to: hie‐won.hann@jefferson.edu

1 Liver Disease Prevention Center, Department of Medicine, Thomas Jefferson University Hospital, Philadelphia, PA, USA

2 Division of Gastroenterology and Hepatology, Department of Medicine, Thomas Jefferson University Hospital, Philadelphia, PA, USA

## **References**

[1] Blumberg BS, Alter HJ, Visnich S. A "new" antigen in leukemia sera. JAMA. 1965; 191:541–546.

[2] Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. 2015; 386:1546–1555.

potential for eradicating the virus. The clinical trials are in progress at different phases to further investigate these compounds [19]. Among these are direct‐acting antagonists against the HBV capsid, against the HBV cccDNA, and against the HBV RNA. While the targets enhancing the innate immunity are mainly in the preclinical phase, they pose excit‐ ing possibilities for the future of HBV treatment. The potential drugs in the pipelines are

While there have been significant advancements in the understanding and management of hepatitis B virus, there remains much to be learned. The molecular mechanism and the subsequent carcinogenesis and progression of chronic HBV carriers to HCC remain in large part poorly understood. While significant improvements in treatment of HBV continue to be made, research toward HBV complete cure and the treatment landscape now is much differ‐ ent than it was at the end of the twentieth century. The development of nucleotide and nucleo‐ side analogues, particularly entecavir and tenofovir, has significantly improved the ability of chronic HBV carriers to remain with undetectable viral levels. There remains, however, the possibility of development of HCC, in part likely in the early stages of infection, as well as the viral incorporation into hepatocyte DNA. Therefore, more effective treatment regimens need to be developed, and the prospect of treating individuals at earlier stages of HBV should be addressed. With multiple new therapies in the pipeline, the future of treating hepatitis B is an

exciting and developing one, and hopefully, it will soon become a disease of the past.

and Hie‐Won Hann1, 2\*

\*Address all correspondence to: hie‐won.hann@jefferson.edu

AD, no conflict, HWH, receives research grants from Bristol Myers‐Squibb and Gilead Sciences.

1 Liver Disease Prevention Center, Department of Medicine, Thomas Jefferson University

2 Division of Gastroenterology and Hepatology, Department of Medicine, Thomas Jefferson

[1] Blumberg BS, Alter HJ, Visnich S. A "new" antigen in leukemia sera. JAMA. 1965;

shown below [19] (**Table 5**).

142 Advances in Treatment of Hepatitis C and B

**6. Conclusion**

**Disclosures:**

**Author details**

Andrew Dargan2

**References**

191:541–546.

Hospital, Philadelphia, PA, USA

University Hospital, Philadelphia, PA, USA


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**Provisional chapter**

## **Recent Advancement in Hepatitis B Virus, Epigenetics Alterations and Related Complications Recent Advancement in Hepatitis B Virus, Epigenetics Alterations and Related Complications**

Mankgopo Magdeline Kgatle Mankgopo Magdeline Kgatle

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66879

#### **Abstract**

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148 Advances in Treatment of Hepatitis C and B

Worldwide, it is estimated that more than 400 million people are currently living with chronic hepatitis B virus (HBV) infection, contributing to more than one million deaths annually as a result of liver cirrhosis and hepatocellular carcinoma (HCC). HBV DNA integrates into the cellular DNA in liver tissue of patients with chronic HBV infection and HCC. Following HBV infection, DNA methyltransferases (DNMTs) methylate any HBV DNA integrated into the human genome. This novel epigenetic mechanism enables the suppression of HBV antigens, leading to reduced viral replication. HBV is thought to induce DNA methylation via hepatitis B x (HBx) protein, which modulates cellular signalling pathways by activating DNMT 1 and 3 to benefit the virus. Activation of DNMT 1 and 3 inappropriately methylates host cellular genes including tumour suppressor genes whose disruption causes transformation of hepatocytes and hepatic malignancy. By being localised in the cytoplasm, nucleus and mitochondria of HBV-infected hepatocytes, it appears that HBx protein manages to exploit the entire body of cellular signalling pathways for viral survival and propagation. HBx protein may achieve its transcriptional transactivation action by either interacting with key genes or altering their related cellular signalling pathways or by hijacking their binding partners and taking over their roles. Although the underlying mechanisms are still unclear, processes such as cell cycle progression, calcium homeostasis, hepatic metabolism, protein ubiquitination, RNA splicing and vitamin D receptor regulation are key mechanisms that HBx protein alters to favour viral replication and cell survival. These detrimental effects would connect HBV infection to malignant transformation by inducing uncontrolled cell growth, proliferation and disrupting apoptosis.

**Keywords:** epigenetics alterations, viral integration, hepatitis B virus, hepatocellular carcinoma, hepatitis X antigen

and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

## **1. Hepatitis B virus**

Hepatitis B virus (HBV) is one of the most prevalent infections in humans and important cause of acute and chronic hepatitis. Chronic infection is defined as the presence of hepatitis B surface antigen (HBsAg) in the blood more than 6 months following initial infection. Without treatment, chronic HBV infection may result in the development of liver cirrhosis and hepatocellular carcinoma (HCC) [1–3].

HBV was first identified in the 1960s and was the first human hepatitis virus to be well characterised at a molecular level [3, 4]. Long-term inflammatory changes due to chronic hepatitis cause hepatocyte injury and the release of reactive oxygen species (ROS) and Kupffer cells activation. These produce proinflammatory and fibrogenic cytokines resulting in the recruitment of immune cells. The Kupffer cells also activate hepatic stellate cells which produce extracellular matrix proteins and cytokines. Repeating cycles of this activation and inflammation lead to cirrhosis characterised by regenerative nodules and irreversible fibrosis [2, 3, 5].

The ability of the virus to cause liver injury is associated with genetic changes that affect both viral and host DNA leading to mutations that predispose to liver injury and possible cancer. These events link chronic HBV infection with HCC. More than 80% of HCC cases arise in chronic HBV infection, strongly suggesting that HBV is an important contributor to the development of tumour [2, 3].

Possible mechanisms by which HBV infection causes HCC have been described, and these include HBV DNA integration, epigenetic alterations (change in gene expression) and aberrant transcriptional activities of HBx protein [3, 6, 7]. Nearly 90% of HBV-related HCC cases show evidence of HBV integration into the host genome [3, 8]. This is associated with genetic changes such as genomic instability, deletions and chromosomal translocations in the host cells, which may lead to accumulation of mutations and epigenetic changes with a malignant phenotype. Several contributing environmental and viral factors such as chronic tobacco smoking, alcohol consumption, aflatoxins, HBV e antigen positive status, high viral load and HBV genotype have been identified in HBV-related HCC cases and are associated with many epigenetic changes [3, 8–10].

## **1.1. Transmission Routes of HBV**

HBV can be stable for 7 days or more on dry environmental surfaces. The two major routes of HBV transmission are horizontal and perinatal or vertical transmission. The efficient modes of transmission are blood and sexual contact with an infected person. The virus is horizontally transmissible during child to child physical contact or through contact with blood or infected toys. Horizontal transmission can also occur through body fluids such as semen and vaginal secretions. Perinatal or vertical transmission of HBV occurs through blood or secretions from an infected mother to the newborn baby during delivery. Perinatal transmission is high in mothers who are positive for hepatitis B e antigen (HBeAg) at 85–90% and lower in those who are negative for HBeAg where the rate is 5–20% [1, 3, 11–13].

## **1.2. Global epidemic of HBV infection**

**1. Hepatitis B virus**

150 Advances in Treatment of Hepatitis C and B

tocellular carcinoma (HCC) [1–3].

irreversible fibrosis [2, 3, 5].

opment of tumour [2, 3].

epigenetic changes [3, 8–10].

**1.1. Transmission Routes of HBV**

Hepatitis B virus (HBV) is one of the most prevalent infections in humans and important cause of acute and chronic hepatitis. Chronic infection is defined as the presence of hepatitis B surface antigen (HBsAg) in the blood more than 6 months following initial infection. Without treatment, chronic HBV infection may result in the development of liver cirrhosis and hepa-

HBV was first identified in the 1960s and was the first human hepatitis virus to be well characterised at a molecular level [3, 4]. Long-term inflammatory changes due to chronic hepatitis cause hepatocyte injury and the release of reactive oxygen species (ROS) and Kupffer cells activation. These produce proinflammatory and fibrogenic cytokines resulting in the recruitment of immune cells. The Kupffer cells also activate hepatic stellate cells which produce extracellular matrix proteins and cytokines. Repeating cycles of this activation and inflammation lead to cirrhosis characterised by regenerative nodules and

The ability of the virus to cause liver injury is associated with genetic changes that affect both viral and host DNA leading to mutations that predispose to liver injury and possible cancer. These events link chronic HBV infection with HCC. More than 80% of HCC cases arise in chronic HBV infection, strongly suggesting that HBV is an important contributor to the devel-

Possible mechanisms by which HBV infection causes HCC have been described, and these include HBV DNA integration, epigenetic alterations (change in gene expression) and aberrant transcriptional activities of HBx protein [3, 6, 7]. Nearly 90% of HBV-related HCC cases show evidence of HBV integration into the host genome [3, 8]. This is associated with genetic changes such as genomic instability, deletions and chromosomal translocations in the host cells, which may lead to accumulation of mutations and epigenetic changes with a malignant phenotype. Several contributing environmental and viral factors such as chronic tobacco smoking, alcohol consumption, aflatoxins, HBV e antigen positive status, high viral load and HBV genotype have been identified in HBV-related HCC cases and are associated with many

HBV can be stable for 7 days or more on dry environmental surfaces. The two major routes of HBV transmission are horizontal and perinatal or vertical transmission. The efficient modes of transmission are blood and sexual contact with an infected person. The virus is horizontally transmissible during child to child physical contact or through contact with blood or infected toys. Horizontal transmission can also occur through body fluids such as semen and vaginal secretions. Perinatal or vertical transmission of HBV occurs through blood or secretions from an infected mother to the newborn baby during delivery. Perinatal transmission is high in mothers who are positive for hepatitis B e antigen (HBeAg) at 85–90% and lower in

those who are negative for HBeAg where the rate is 5–20% [1, 3, 11–13].

Worldwide, it is estimated that more than 400 million people are currently living with chronic HBV infection, contributing to more than one million deaths annually [1]. The prevalence of HBV infection is determined by the seroprevalence of HBsAg. HBV is highly endemic in Asia and sub-Saharan Africa with HBsAg seroprevalence rates exceeding 8% (**Figure 1**) [3, 14]. In these regions, the infection is typically acquired at birth or in early childhood. Progression to chronic HBV infection is common in these regions and is associated with prevalence rates of 30% for hepatic cirrhosis and 53% for HCC [16].

**Figure 1.** Global geographical distribution of chronic hepatitis B infection (Adapted from Lavanchy D **[13]**).

Annually, approximately one million people are diagnosed with HCC worldwide, and more than half of these people die within a year of diagnosis. Studies show that the highest HCC incidence rates of 70–80% occur in South-East Asia and sub-Saharan Africa, the regions with a high prevalence of chronic HBV infection [16]. This is due to various factors that include the late presentation of patients with large tumours, failure to recognise those at risk, high prevalence of risk factors in the population, lack of medical facilities for early diagnosis and limited access to effective treatment after diagnosis [3, 16].

An intermediate HBsAg seroprevalence of 2–7% is seen in some parts of Asia, Europe, America and Russia. The prevalence of HBV infection is low in Western Europe, Australia and United States where HBsAg seroprevalence is <2% [3, 17].

## **1.3. Epidemic of HBV infection in Africa**

There are 65 million individuals infected with chronic HBV in Africa and 250,000 of these people die annually due to HBV-related diseases. The prevalence of chronic HBV infection in Africa varies by geographic region. It is high in sub-Saharan Africa, with HBsAg seroprevalence rates of more than 8%. In Kenya, Sierra Leone, Zambia, Senegal and Liberia, the prevalence of HBV infection is intermediate with HBsAg seroprevalence rates ranging from 2 to 8%. North African countries including Morocco, Egypt, Algeria and Tunisia have low prevalence rates of <2%.

In South Africa and other African countries, the prevalence of HBV infection is much higher in rural compared to urban areas [18]. Low socio-economic status, infected household contact, unsafe sexual intercourse, sharing of partially eaten sweets or chewing gum, dental work and bathing towels may be some of the contributing factors for the high prevalence of HBV infection in rural areas [3, 12, 18].

## **1.4. HBV genotypes and genomic alterations**

HBV is classified into eight genotypes (A–J) with four major serotypes (adw, adr, ayw and ayr) [3, 19, 20]. HBV genotypes are differentiated by more than 8% sequence divergence in the entire genome and more than 4% at the level of S gene. They have distinct geographical distribution as illustrated in **Table 1**. Genotype A is predominant in sub-Saharan Africa, North-West Europe and North America.


*Abbreviations*: A, adenine; CpG, cytosine-phosphate-guanine; DLEC1, deleted in lung and esophageal cancer 1; G, guanine; GSTP1, glutathione S transferase pi 1; HBV, hepatitis B virus; T, thymine.

**Table 1.** The global geographic distribution of HBV genotypes, mutations and associated CpG promoter DNA methylation.

Genotype A has four subgenotypes. Subgenotype 1A is common in South Africa, Malawi, Tanzania, Uganda, Somalia, Yemen, India, Nepal, Brazil and the Philippines [3, 20]. There are three CpG islands within HBV genotype A, which are associated with methylation of the promoter of *Deleted in Lung and Esophageal Cancer 1* (*DLEC*) gene and down-regulation of its expression in HBV-induced HCC. *DLEC* is a tumour suppressor gene and has been reported to be down-regulated in ovarian, liver, lung and EBV-related cancers [3, 21].

Genotypes B and C are more prevalent in Asia, Indonesia and Vietnam [20]. Based on the phylogenetic analysis, it was demonstrated that HBV genotype C is subdivided into 5 subgenotypes (C1–C5). Geographical clustering of these subgenotypes was clear. The subgenotype C1 was found to be prevalent in East Asia, subgenotype C2 in South-East Asia, subgenotypes C3 and C4 in Southern Pacific Ocean and subgenotype C5 in Philippines [3, 22–34]. Genotype D is commonly found in the Mediterranean region and Middle East. The hepatitis B x (HBx) protein is associated with hypermethylation and down-regulation of the *GSTP1* gene which plays an important role in the development of cancer. Genotype E is found mainly in Africa. Genotype F is found in Europe and the United States, and genotype G, in France and America. Genotype H is predominant in Central America, California and Mexico. Genotype I and J are prevalent in South-East Asia and Japan, respectively [3, 20].

HBV has a mutation rate of 10%, which is relatively high compared to other viruses. It replicates via reverse transcription of RNA intermediates that result in random mismatched base errors during genomic replication. HBV DNA polymerase lacks the ability to proofread these errors, and this predisposes HBV to mutations [3, 25]. HBV develops four major mutations which are the precore, basic core promoter, tyrosine-methionine-aspartate-aspartate (YMDD) and asparagines-to-threonine (rtN236T) mutations. The precore mutants were the first to be identified and are characterised by a nonsense G1896A mutation [3, 26]. The G1896A mutation is responsible for HBeAg negativity in chronic HBV carriers and induces the down-regulation of HLA class II molecules in hepatocytes. This mutation is common in individuals infected with HBV genotype D [3, 27]. The basic core promoter mutations include A1762T and G1764A and were identified after the precore mutations. Similar to the precore mutations, the basic core promoter mutations are found in HBeAg-negative individuals where they prevent HBeAg expression [3, 28].

#### **1.5. Prevention and treatment**

of more than 8%. In Kenya, Sierra Leone, Zambia, Senegal and Liberia, the prevalence of HBV infection is intermediate with HBsAg seroprevalence rates ranging from 2 to 8%. North African countries including Morocco, Egypt, Algeria and Tunisia have low prevalence rates of <2%.

In South Africa and other African countries, the prevalence of HBV infection is much higher in rural compared to urban areas [18]. Low socio-economic status, infected household contact, unsafe sexual intercourse, sharing of partially eaten sweets or chewing gum, dental work and bathing towels may be some of the contributing factors for the high prevalence of HBV infec-

HBV is classified into eight genotypes (A–J) with four major serotypes (adw, adr, ayw and ayr) [3, 19, 20]. HBV genotypes are differentiated by more than 8% sequence divergence in the entire genome and more than 4% at the level of S gene. They have distinct geographical distribution as illustrated in **Table 1**. Genotype A is predominant in sub-Saharan Africa,

**Genotype Geographic distribution Mutation Host CpG promoter methylation**

**B** Indonesia, China, Vietnam Unknown Unknown

**E** Africa Unknown Unknown

**G** France, America Unknown Unknown

**I** South-East Asia Unknown Unknown **J** Japan Unknown Unknown

guanine; GSTP1, glutathione S transferase pi 1; HBV, hepatitis B virus; T, thymine.

G1888A 1762T1764A G1862T Induces hypomethylation and

G1896A Induces hypermethylation and

Unknown Unknown

Unknown Unknown

Unknown Unknown

gene

down-regulation of the *DLEC1*

down-regulation of *GSTP1* gene

Genotype A has four subgenotypes. Subgenotype 1A is common in South Africa, Malawi, Tanzania, Uganda, Somalia, Yemen, India, Nepal, Brazil and the Philippines [3, 20]. There

*Abbreviations*: A, adenine; CpG, cytosine-phosphate-guanine; DLEC1, deleted in lung and esophageal cancer 1; G,

**Table 1.** The global geographic distribution of HBV genotypes, mutations and associated CpG promoter DNA

tion in rural areas [3, 12, 18].

152 Advances in Treatment of Hepatitis C and B

**1.4. HBV genotypes and genomic alterations**

North-West Europe and North America.

**A** North America, Sub-Saharan

**C** East Asia, Korea, China, Japan, Polynesia, Vietnam

**D** Mediterranean area, Middle East

**F** Central and South America, Polynesia

**H** Mediterranean area, Middle East

methylation.

Africa, North-West Europe

HBV infection can be prevented by avoiding direct contact with any HBV-contaminated fluids and materials. Immunisation with recombinant hepatitis B vaccines is recommended for all infants at birth and in individuals who are at high risk of acquiring the infection. Passive immunoprophylaxis with hepatitis B immunoglobulin derived from sera of positive HBV individuals is used to prevent mother-to-child HBV transmission at birth, after liver transplantation for HBV infection, needle-stick injuries and sexual intercourse [3, 20, 29].

Acute HBV infection does not require treatment as it usually resolves spontaneously. Two major classes of drugs available for treating chronic HBV infection include the injectable standard interferon-α and pegylated interferon-α2, and the oral nucleos(t)ide analogues. Nucleoside analogues are lamivudine, entecavir, telbivudine, whilst nucleotide analoques are adefovirdipivoxil and tenofovir. The main aims of treatment are to improve long-term survival by reducing the risk of developing cirrhosis and HCC [3, 30, 31].

Treatment with oral nucleos(t)ide analogues is associated with the development of mutations. Lamivudine induces point mutations in the YMDD motif of the HBV polymerase, and these include rtM204V and rtM204I mutations. The viral replication rate increases in the presence of lamivudine resistance, and when lamivudine treatment is stopped, the wild-type virus reestablishes itself. Lamuvidine resistance mutations are responsible for the development of resistance in entecavir that is also associated with similar mutations and more including rtI169T, rtT184G, rtS202I and rtM250V [3, 32, 33]. Telbivudine has a high antiviral potency and relatively low resistance than lamuvidine and entecavir. It is associated with mutations at rtL80I/V, rtL180M, rtA181T/V, rtM204I and rtL229W/V. Telbivudine results in myoparthy and neuropathy when used simultaneously with pegylated interferon-α2, and therefore, combination of these two agents is avoided [3, 32, 34].

Adevovir treatment causes mutations that are associated with the emergence of resistant strains such as the rtN236T mutation which is downstream to the YMDD motif [35]. The use of adevovir treatment is now rare as it is associated with severe kidney injury, which may be a consequence of mitochondrial DNA depletion and activity of multidrug resistanceassociated protein 4 [3, 36].

Despite the availability of treatment for chronic HBV infection, many patients will develop cancer, and this remains a major medical problem worldwide. This may be attributed to HCC-associated risk factors such as the HBV genotype, alanine aminotransferase (ALT), HBV load and HBV surface antigen level, which may influence the response to chronic HBV treatment. The response to interferon is significantly higher in patients infected with HBV genotype A compared to D and in patients with lower levels of HBV DNA and higher levels of ALT [3, 37, 38].

Aberrant methylation of promoter CpG islands is the primary epigenetic change seen during the course of HBV infection as it progresses to cirrhosis and HCC. Such methylation is detected at higher rates in HCC tissues compared to liver cirrhosis without cancer [10]. In a recent large cohort study report by Tseng et al., high HBV surface antigen levels are associated with a risk of developing HCC even in the presence of low HBV DNA levels. This finding may be due to a higher degree of viral HBV surface antigen integration into the host genome that would result in mutations and epigenetic alteration particularly DNA methylation, causing chronic liver damage, malignant transformation and HCC [3, 38–40].

The association of DNA methylation with chronic HBV treatment was first observed during telbivudine treatment. Telbivudine is a thymidine agent that interacts with protein kinases to form telbivudine 5′–triphosphate via phosphorylation. Telbivudine 5′–triphosphate competes with thymidine 5′–triphosphate, leading to the suppression of HBV DNA polymerase and reduced viral replication. Interestingly, telbivudine was recently reported to correct HBV-induced histone methylation in HBV-infected hepatocytes [3, 41].

## **1.6. Virological characteristics of HBV**

are adefovirdipivoxil and tenofovir. The main aims of treatment are to improve long-term

Treatment with oral nucleos(t)ide analogues is associated with the development of mutations. Lamivudine induces point mutations in the YMDD motif of the HBV polymerase, and these include rtM204V and rtM204I mutations. The viral replication rate increases in the presence of lamivudine resistance, and when lamivudine treatment is stopped, the wild-type virus reestablishes itself. Lamuvidine resistance mutations are responsible for the development of resistance in entecavir that is also associated with similar mutations and more including rtI169T, rtT184G, rtS202I and rtM250V [3, 32, 33]. Telbivudine has a high antiviral potency and relatively low resistance than lamuvidine and entecavir. It is associated with mutations at rtL80I/V, rtL180M, rtA181T/V, rtM204I and rtL229W/V. Telbivudine results in myoparthy and neuropathy when used simultaneously with pegylated interferon-α2, and therefore, combi-

Adevovir treatment causes mutations that are associated with the emergence of resistant strains such as the rtN236T mutation which is downstream to the YMDD motif [35]. The use of adevovir treatment is now rare as it is associated with severe kidney injury, which may be a consequence of mitochondrial DNA depletion and activity of multidrug resistance-

Despite the availability of treatment for chronic HBV infection, many patients will develop cancer, and this remains a major medical problem worldwide. This may be attributed to HCC-associated risk factors such as the HBV genotype, alanine aminotransferase (ALT), HBV load and HBV surface antigen level, which may influence the response to chronic HBV treatment. The response to interferon is significantly higher in patients infected with HBV genotype A compared to D and in patients with lower levels of HBV DNA and higher levels

Aberrant methylation of promoter CpG islands is the primary epigenetic change seen during the course of HBV infection as it progresses to cirrhosis and HCC. Such methylation is detected at higher rates in HCC tissues compared to liver cirrhosis without cancer [10]. In a recent large cohort study report by Tseng et al., high HBV surface antigen levels are associated with a risk of developing HCC even in the presence of low HBV DNA levels. This finding may be due to a higher degree of viral HBV surface antigen integration into the host genome that would result in mutations and epigenetic alteration particularly DNA methylation, causing chronic liver damage, malignant transformation

The association of DNA methylation with chronic HBV treatment was first observed during telbivudine treatment. Telbivudine is a thymidine agent that interacts with protein kinases to form telbivudine 5′–triphosphate via phosphorylation. Telbivudine 5′–triphosphate competes with thymidine 5′–triphosphate, leading to the suppression of HBV DNA polymerase and reduced viral replication. Interestingly, telbivudine was recently reported

to correct HBV-induced histone methylation in HBV-infected hepatocytes [3, 41].

survival by reducing the risk of developing cirrhosis and HCC [3, 30, 31].

nation of these two agents is avoided [3, 32, 34].

associated protein 4 [3, 36].

154 Advances in Treatment of Hepatitis C and B

of ALT [3, 37, 38].

and HCC [3, 38–40].

HBV virions are infectious double-shelled particles of approximately 40–42 nanometre (nm) in diameter. They consist of a nucleocapsid core of 27 nm in diameter, which forms the inner part of enveloped virions known as Dane particles. The nucleocapsid core is surrounded by an outer surface antigen coat of ~4 nm thickness. It contains HBsAg and hepatitis B core antigen (HBcAg), which are detected in the sera of HBV-infected individuals in the form of spherical and filamentous particles [1, 3, 19, 42].

**Figure 2.** The structure of the HBV genome.

HBV is classified as an *Orthohepadnavirus* which belongs to the family *Hepadnaeviridae*. Contained in this family are other viruses such as the hepatic viruses of woodchucks, ducks, herons, ground and tree squirrels. These viruses replicate via reverse transcription of RNA intermediates, the step in which the DNA is packaged into hepadnaviral infectious particles. They are classified as *Hepadnaeviridae*due to their structure and genomic organisation being similar to that of HBV. HBV genome is a small and relaxed circular molecule of 3.2 kb in size. It contains two strands of different length, a long minus strand and a short plus strand as illustrated in **Figure 2**. The minus strand is terminally redundant and contains a second copy of direct repeat 1 (DR1), ε signal and poly A tail. It serves as a template for reverse transcription of a plus strand and also as a transcript for the translation of viral proteins including polymerase, HBcAg and HBeAg. The 5′ end of a minus strand is covalently linked to the viral reverse transcriptase and polymerase through a phosphor-tyrosine bond. The plus strand overlaps part of the minus strand whilst its 5′ end bears the oligoribonucleotides [3, 42, 43].

The HBV genome contains four ORFs, which have the same orientation and partially overlap. These ORFs encode the viral envelope pre-S/S, a pre-core/core, a polymerase and X proteins. The viral envelope also encodes three surface glycoproteins, which are the large (L), middle (M) and small (S) glycoproteins (**Figure 2**). These surface glycoproteins are synthesised by the initial transcription of pre-S/S. The L surface glycoprotein is important for viral assembly and infectivity, whilst the function of M surface glycoprotein is unknown. The longest open reading frame encodes the viral polymerase which serves as a reverse transcriptase and DNA polymerase. The pre-S/S envelope open reading frame overlaps the precore/core and X open reading frames and encodes HBsAg. The precore/core open reading frame produces HBeAg and HBcAg through cleavage by cellular proteases. HBcAg is the nucleocapsid and encloses the viral DNA [3, 11, 42, 43].

HBx protein is a transactivating protein that alters the expression of some genes via DNA methylation leading to tumourigenesis. It consists of 154 amino acid residues with a molecular weight of 27 kDa and is encoded by the smallest ORF. It stimulates viral replication either by activating viral transcription or by enhancing the reverse transcription of the viral polymerase [44, 45]. In hepatoma cell lines, HBx protein enhances viral replication by interacting with DNA binding protein 1 which interferes with cell growth and viability. In mice infected with wild-type HBV, viral replication is stimulated by HBx protein, suggesting that HBx protein is required for viral replication in normal hepatocyte cells [3, 44, 46, 47].

## **1.7. Life cycle of HBV**

Due to the lack of efficient in vitro infection systems and animal models in which to study the life cycle of HBV infection, a lot of data are from the duck model infected with duck hepatitis B virus (DHBV) [3, 48]. HBV life cycle begins through the interaction of HBsAg with cellular receptor/s at the surface of hepatocytes. A number of potential cellular receptors that interact with HBsAg during HBV infection have been previously identified, but the mechanisms of action still remain controversial as none of them has been proved to be functional to HBV. These receptors include retinoid X receptor (RXR), peroxisome proliferator-activated receptor (PPAR) and farnesoid X receptor (FXR) [3, 49, 50].

Sodium taurocholate cotransporting polypeptide (NTCP) was discovered as the potential receptor for HBV infection (**Figure 2**). NTCP is abundantly expressed in the liver and is involved in the transportation and clearance of bile acids from portal blood into hepatocytes. Yan et al. [51] have shown by using near-zero-distance photo-cross-linking, tandem affinity purification and mass spectrophotometry that the pre-S/S envelope domain, a key determinant for receptor/s binding, selectively interacts with NTCP to facilitate HBV infection. Knockdown of the NTCP expression in duck primary hepatocytes infected with DHBV significantly decreased HBV infection, suggesting that NTCP is actually required for HBV infection [3, 51, 52].

as illustrated in **Figure 2**. The minus strand is terminally redundant and contains a second copy of direct repeat 1 (DR1), ε signal and poly A tail. It serves as a template for reverse transcription of a plus strand and also as a transcript for the translation of viral proteins including polymerase, HBcAg and HBeAg. The 5′ end of a minus strand is covalently linked to the viral reverse transcriptase and polymerase through a phosphor-tyrosine bond. The plus strand overlaps part of the minus strand whilst its 5′ end bears the oligoribonucleotides

The HBV genome contains four ORFs, which have the same orientation and partially overlap. These ORFs encode the viral envelope pre-S/S, a pre-core/core, a polymerase and X proteins. The viral envelope also encodes three surface glycoproteins, which are the large (L), middle (M) and small (S) glycoproteins (**Figure 2**). These surface glycoproteins are synthesised by the initial transcription of pre-S/S. The L surface glycoprotein is important for viral assembly and infectivity, whilst the function of M surface glycoprotein is unknown. The longest open reading frame encodes the viral polymerase which serves as a reverse transcriptase and DNA polymerase. The pre-S/S envelope open reading frame overlaps the precore/core and X open reading frames and encodes HBsAg. The precore/core open reading frame produces HBeAg and HBcAg through cleavage by cellular proteases. HBcAg is the nucleocapsid and encloses

HBx protein is a transactivating protein that alters the expression of some genes via DNA methylation leading to tumourigenesis. It consists of 154 amino acid residues with a molecular weight of 27 kDa and is encoded by the smallest ORF. It stimulates viral replication either by activating viral transcription or by enhancing the reverse transcription of the viral polymerase [44, 45]. In hepatoma cell lines, HBx protein enhances viral replication by interacting with DNA binding protein 1 which interferes with cell growth and viability. In mice infected with wild-type HBV, viral replication is stimulated by HBx protein, suggesting that HBx pro-

Due to the lack of efficient in vitro infection systems and animal models in which to study the life cycle of HBV infection, a lot of data are from the duck model infected with duck hepatitis B virus (DHBV) [3, 48]. HBV life cycle begins through the interaction of HBsAg with cellular receptor/s at the surface of hepatocytes. A number of potential cellular receptors that interact with HBsAg during HBV infection have been previously identified, but the mechanisms of action still remain controversial as none of them has been proved to be functional to HBV. These receptors include retinoid X receptor (RXR), peroxisome proliferator-activated receptor

Sodium taurocholate cotransporting polypeptide (NTCP) was discovered as the potential receptor for HBV infection (**Figure 2**). NTCP is abundantly expressed in the liver and is involved in the transportation and clearance of bile acids from portal blood into hepatocytes. Yan et al. [51] have shown by using near-zero-distance photo-cross-linking, tandem affinity purification and mass spectrophotometry that the pre-S/S envelope domain, a key determinant for receptor/s binding, selectively interacts with NTCP to facilitate HBV infection. Knockdown of the NTCP

tein is required for viral replication in normal hepatocyte cells [3, 44, 46, 47].

[3, 42, 43].

the viral DNA [3, 11, 42, 43].

156 Advances in Treatment of Hepatitis C and B

**1.7. Life cycle of HBV**

(PPAR) and farnesoid X receptor (FXR) [3, 49, 50].

HBV requires DNA polymerase and reverse transcriptase to replicate through RNA intermediates known as pregenomic RNA. Following the interaction of surface antigen with NTCP, the viral nucleocapsid enters the host cell's nucleus to deliver dsDNA (**Figure 3**) [3, 51, 52]. In the nucleus, the dsDNA gets repaired and converted to covalently closed circular super-coiled DNA (cccDNA) by DNA polymerase. The cccDNA molecule serves as a template for the transcription of four viral RNA transcripts 3.5, 2.4, 2.1 and 0.4 kb in size, pregenomic RNA and RNA intermediate for viral replication before moving to the cytoplasm. The mRNA transcripts are then translated to produce the envelope (pre-S/S), precore/core, viral polymerase and X proteins. The 3.5 RNA transcript is reverse-transcribed into viral dsDNA [3, 8, 11, 40, 48, 53]. Some of the resulting viral DNA and polymerase-containing capsids are enveloped via budding into the endoplasmic reticulum (ER). The rest of the viral DNA is recycled or is migrated back to the nucleus where it produces new generations of cccDNA which maintains persistent HBV infection [1, 3, 11, 36, 40].

**Figure 3.** The life cycle of HBV infection and underlying mechanisms.

## **2. Epigenetics and HBV-induced hepatocarcinogenesis**

Epigenetics involves attachment of chemical compounds and proteins on the DNA sequence leading to altered gene expression and normal function. There are two major ways through which gene transcription can be regulated through epigenetic changes. One way of regulating gene transcription is directly through DNA methylation. This involves the addition of a methyl group into DNA sequence. Methyl groups are carbon and hydrogen molecules which bind to the genome through the action of methyl cytosine-phosphate-guanine (CpG)-binding proteins (MeCPs), DNA methyltransferases (DNMTs), histone acetyltransferases (HATs) and histone deacetylases (HDACs), which inactivate gene transcription. Other transcription repressors including nuclear factor kappa B (NF-κB), c-myc/c-myn, activator protein (AP)-2, E2 promoter binding factor (E2F) and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) may also be activated by methyl groups to inhibit gene transcription [3, 53, 54].

In addition to DNA methylation, epigenetics can also be regulated by histone protein modifications. Histone protein modifications may be caused by over-expression or aberrant recruitment of HDACs that remodel the chromatin shape and structure. The two basic mechanisms responsible for chromatin remodelling are histone acetylation and deacetylation [3, 53, 55]. These mechanisms are controlled by the enzyme activity of HATs and HDACs, respectively [3, 54].

Acetylation of histone proteins is generally acknowledged as playing a key role in gene regulation. For a gene to be transcribed, it must become physically accessible to the transcriptional machinery. Acetylation by HATs substitutes the positive charges on the amino terminal tails of histone proteins with an acetyl group derived from acetyl coenzyme A, causing uncoiling of the DNA and euchromatin into an open-relaxed form of chromatin. Consequently, this makes genes accessible to several binding factors such as RNA polymerase II and transcriptional factors, allowing gene expression to occur and proteins to be made. Deacetylation of histone proteins by HDACs results in the tight coiling of the DNA and closed form of chromatin regions known as heterochromatin. This prevents the interaction between DNA and transcription factors leading to suppression of gene transcription. In some cancer cells, there is increased expression or aberrant recruitment of HDACs and decreased expression of HATs. This results in the hypoacetylation of histone proteins and therefore a condensed or closed chromatin structure [3, 54–56].

Epigenetics plays important roles in oncogenic viruses including HBV, human papillomavirus and Epstein Barr virus. In episomal HBV DNA, 3 CpG islands have been identified and described. These are island 1 located on nucleotide positions 55–286, island 2 on 1224–1667 and island 3 on 2257–2443 [57]. Methylation of CpG islands in the human genome is known to regulate gene transcription. These prompted Vivekanandan et al. [58] to hypothesise that methylation of CpG islands in HBV DNA may regulate viral gene expression. To test this hypothesis, in vitro methylation of the transfected HBV DNA was done, and this resulted in decreased expression of HBV mRNA and proteins in the cells. In addition, the effect of viral cccDNA methylation in the liver tissue of patients with chronic HBV infection was investigated and found to be associated with reduced HBV replication [58]. These findings support the work of Pollicino et al. [3, 58] who showed that HBV replication is regulated by the acetylation of HBV cccDNA bound H3 and H4 histone proteins. Although these data suggest that HBV DNA methylation is a novel mechanism that influences the regulation of viral gene expression, the mechanisms of action are still not known.

**2. Epigenetics and HBV-induced hepatocarcinogenesis**

scription [3, 53, 54].

HDACs, respectively [3, 54].

158 Advances in Treatment of Hepatitis C and B

Epigenetics involves attachment of chemical compounds and proteins on the DNA sequence leading to altered gene expression and normal function. There are two major ways through which gene transcription can be regulated through epigenetic changes. One way of regulating gene transcription is directly through DNA methylation. This involves the addition of a methyl group into DNA sequence. Methyl groups are carbon and hydrogen molecules which bind to the genome through the action of methyl cytosine-phosphate-guanine (CpG)-binding proteins (MeCPs), DNA methyltransferases (DNMTs), histone acetyltransferases (HATs) and histone deacetylases (HDACs), which inactivate gene transcription. Other transcription repressors including nuclear factor kappa B (NF-κB), c-myc/c-myn, activator protein (AP)-2, E2 promoter binding factor (E2F) and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) may also be activated by methyl groups to inhibit gene tran-

In addition to DNA methylation, epigenetics can also be regulated by histone protein modifications. Histone protein modifications may be caused by over-expression or aberrant recruitment of HDACs that remodel the chromatin shape and structure. The two basic mechanisms responsible for chromatin remodelling are histone acetylation and deacetylation [3, 53, 55]. These mechanisms are controlled by the enzyme activity of HATs and

Acetylation of histone proteins is generally acknowledged as playing a key role in gene regulation. For a gene to be transcribed, it must become physically accessible to the transcriptional machinery. Acetylation by HATs substitutes the positive charges on the amino terminal tails of histone proteins with an acetyl group derived from acetyl coenzyme A, causing uncoiling of the DNA and euchromatin into an open-relaxed form of chromatin. Consequently, this makes genes accessible to several binding factors such as RNA polymerase II and transcriptional factors, allowing gene expression to occur and proteins to be made. Deacetylation of histone proteins by HDACs results in the tight coiling of the DNA and closed form of chromatin regions known as heterochromatin. This prevents the interaction between DNA and transcription factors leading to suppression of gene transcription. In some cancer cells, there is increased expression or aberrant recruitment of HDACs and decreased expression of HATs. This results in the hypoacetylation of histone proteins and

Epigenetics plays important roles in oncogenic viruses including HBV, human papillomavirus and Epstein Barr virus. In episomal HBV DNA, 3 CpG islands have been identified and described. These are island 1 located on nucleotide positions 55–286, island 2 on 1224–1667 and island 3 on 2257–2443 [57]. Methylation of CpG islands in the human genome is known to regulate gene transcription. These prompted Vivekanandan et al. [58] to hypothesise that methylation of CpG islands in HBV DNA may regulate viral gene expression. To test this hypothesis, in vitro methylation of the transfected HBV DNA was done, and this resulted in decreased expression of HBV mRNA and proteins in the cells. In addition, the effect of viral cccDNA methylation in the liver tissue of patients with chronic HBV infection was investigated

therefore a condensed or closed chromatin structure [3, 54–56].

Previous human studies have shown that DNA viruses integrate into the host genome and that the expression levels of DNMTs increase in response to active viral replication [59]. Vivekanandan et al. [58] hypothesised that the up-regulation of DNMTs gives infected cells the ability to methylate viral DNA and therefore control viral replication. To investigate this, the expression of DNMTs was measured in cell lines exposed to HBV DNA using two experimental systems, one of temporary transfection of cells and another that mimicked natural chronic infection. High-level expressions of DNMT 1, 2 and 3 were observed in response to persistent HBV infection. This correlated with suppressed viral replication associated with methylation of HBV DNA and increased methylation of host CpG islands [3, 58].

The seminal work of Vivekanandan et al. [58] allows for the development of a model that explains the development of liver injury and HCC in chronic HBV infection (**Figure 4**). In this model, infected host cells respond to HBV infection by up-regulating the expression of DNMTs. Up-regulation of DNMTs can also result from interaction with HBx transcriptional

**Figure 4.** Model of chronic HBV infection and DNA methylation.

activator protein. Once activated, DNMTs methylate HBV DNA and switch off the expression of viral mRNA and proteins, thereby reducing viral replication. The methylation of integrated HBV DNA may be detrimental to the host genome through the inappropriate methylation of the neighbouring host genome, particularly if the promoter CpG islands regions of the gene are affected. A consequence of this effect would be the transcriptional repression of host immunoregulatory and tumour suppressor genes that prevent the development of cancer [3, 58].


Abbreviations: BIRC3, baculoviral IAP repeat containing 3; CASPR3, contactin-associated protein-like 3;CCNL, cyclin L1;CHML, choroideremia-like gene; CTGF, connective tissue growth factor; DCC, deleted in colorectal cancer; DPC4, deleted in pancreatic cancer 4; EMSL, EMSL; EMX2, empty spiracle homeobox 2; ErbB3, V-erb-b2 erythroblasticleukemia viral oncogene homolog 3; FGF4, fibroblast growth factor 4; FRA, fragile site; hTERT, human telomerase reverse transcriptase; IMP-2, insulin-like growth factor II mRNA binding protein 2; IRAK2, interleukin-1 receptor-associated kinase 2; KLF1, Krueppel-like factor 1; Mill2, major histocompatibility complex I like leukocyte 2; NCF1, neutrophil cytosolic factor 1; PDGFRβ, platelet-derived growth factor receptor beta; PI3K, phosphatidylinositol 3 kinase; PTEN, phosphatise and tension homolog; RAR, retinoic acid receptor; SERCA, sarco/endoplasmic reticulum calcium transport ATPase; TCEA, transcription elongation factor A.

**Table 2.** Examples of chromosomal fragile sites associated with HBV insertions and their roles in tumour development.

HBV integrates into the host genome and promotes viral persistence. Infected cells increase the expression of DNMTs in response to viral replication. This causes methylation of HBV cccDNA and reduces viral replication. The same methylation system methylates the adjacent host tumour suppressor and immunoregulatory genes leading to hepatocarcinogenesis.

## **2.1. Integration of HBV DNA into the human genome**

activator protein. Once activated, DNMTs methylate HBV DNA and switch off the expression of viral mRNA and proteins, thereby reducing viral replication. The methylation of integrated HBV DNA may be detrimental to the host genome through the inappropriate methylation of the neighbouring host genome, particularly if the promoter CpG islands regions of the gene are affected. A consequence of this effect would be the transcriptional repression of host immunoregulatory and tumour suppressor genes that prevent the devel-

**Chromosomal fragile sites Target gene Role in tumour development** FRA1A (1p36) TCEA; RAR; CHML Alters gene expression and promote

FRA2C (1q) EMX2-like gene Modulates β-catenin signalling

FRA3D (3q25.3) IRAK2 Promotes apoptosis and tumour

FRA7 (7p) SERCA 1; NCF1 β-Catenin activation

FRA4E (4p) Cyclin A Stimulate cell cycle and anti-apoptotic

FRA5C (5p31.1) PDGFRβ Regulates DNA synthesis and fibrotic

FRA9 (9q) KLF1; CASPR3 Promote cell growth; regulates DNA

FRA10A (10q) PTEN; PI3K Promotes metastasis; promotes cell

FRA11A (11q13) EMS1, FGF4; BIRC3 Modulates β-catenin signaling

FRA12A (12q24) ErbB3; Mill2 Promotes tumour progression

FRA18 (18q) DCC; DPC4 Regulates methyl-CpG-binding

FRA19A (19q13) Cyclin E Delays DNA synthesis and promotes

FRA20 (20P12.3) hTERT Alters gene expression and promotes

Abbreviations: BIRC3, baculoviral IAP repeat containing 3; CASPR3, contactin-associated protein-like 3;CCNL, cyclin L1;CHML, choroideremia-like gene; CTGF, connective tissue growth factor; DCC, deleted in colorectal cancer; DPC4, deleted in pancreatic cancer 4; EMSL, EMSL; EMX2, empty spiracle homeobox 2; ErbB3, V-erb-b2 erythroblasticleukemia viral oncogene homolog 3; FGF4, fibroblast growth factor 4; FRA, fragile site; hTERT, human telomerase reverse transcriptase; IMP-2, insulin-like growth factor II mRNA binding protein 2; IRAK2, interleukin-1 receptor-associated kinase 2; KLF1, Krueppel-like factor 1; Mill2, major histocompatibility complex I like leukocyte 2; NCF1, neutrophil cytosolic factor 1; PDGFRβ, platelet-derived growth factor receptor beta; PI3K, phosphatidylinositol 3 kinase; PTEN, phosphatise and tension homolog; RAR, retinoic acid receptor; SERCA, sarco/endoplasmic reticulum calcium transport

**Table 2.** Examples of chromosomal fragile sites associated with HBV insertions and their roles in tumour development.

FRA13A (13q32) CTGF; CCNL; IMP-2 Tumour suppression

ATPase; TCEA, transcription elongation factor A.

cell survival

effect

genes

progression

methylation

proteins

immortalisation

cell survival

cycle progression

pathway; alters cell fate

pathway and cell survival

opment of cancer [3, 58].

160 Advances in Treatment of Hepatitis C and B

HBV integration was first discovered in 1980 using Southern blot hybridisation. It was associated with genomic instability such as loss of heterozygocity (LOH), resulting in the rearrangements, deletions, duplications and inversions of the host and viral genomic sequences. Viral integration results in the insertion of HBV DNA sequences such as HBx gene in the host genome and enables viral persistence [3, 7, 8].

Integration of HBV in the host genome also occurs in woodchucks and other animal models. In woodchucks and California ground squirrels (Spermophilusbeecheyi), HBV genome integrates close to *ras* and *myc* family oncogenes including *c-myc, N-myc1* and *N-myc2*. Modulation of *myc* and *ras* family oncogenes through *cis*-activation enhances cell proliferation and transformation. These events occur via transactivation action of HBx protein and favour the development of cancer [3, 60, 61].

The occurrence of integrated HBV DNA at preferential sites in the human chromosomes has been identified using Alu-PCR-based technique. The preferential sites are known as chromosomal fragile sites (CFS) and are non-random [3, 8]. HBV DNA integrates into the human genome soon after the repair and conversion of HBV DNA to cccDNA [3, 57, 58, 62]. The HBV genome integrates within the coding sequence or close to an array of key regulatory cellular genes that can deregulate proto-oncogenes and tumour suppressor genes. Activation or inactivation of such genes promotes genomic chromosomal instability by altering various cellular signalling pathways, triggering genetic mutations and epigenetic alteration. Mutagenesis and epigenetic alteration result in the abnormal regulation of the targeted genes. This promotes malignant transformation by altering the control of cell growth, differentiation, proliferation and apoptosis [3, 57, 58, 63]. The integration of HBV at or within *cyclin A* and *RARβ* genes is associated with increased protein activities and hepatocellular growth in HBV-induced HCC, suggesting that HBV integration contributes to hepatocytes transformation [60]. Examples of known active CFS targeted by HBV integration are outlined in **Table 2**. The 60s ribosomal protein, *hTERT, major histocompatibility complex I like leukocyte* (*Mill*), *platelet-derived growth factor receptor* (*PDGFR*) and *calcium signalling-related* genes are also common sites or targets of HBV integration. These genes are important in cellular signalling pathways that control DNA damage, oxidation stress and cell growth, and their alteration is associated with the development and progression of cancer [3, 9, 64].

## **2.2. HBx protein and its carcinogenic effects**

HBx protein is a transcriptional transactivator that HBV uses to integrate into the host cellular DNA and is associated with malignant transformation in hepatocytes. It interacts with nuclear transcription factors such as NF-κB, AP1, CREB, TATA-binding protein (TBP), peroxisome proliferator-activated receptor γ (PPARγ) and transcription factor II H (TFIIH) [44]. Interaction of HBx protein with these transcription factors disrupts multiple cellular signalling pathways that include janus kinase 1 (JAK1)-signal transducer activator of transcription (STAT), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and p53 signalling pathways. Cellular signalling pathways are important in regulating DNA repair, cell growth, differentiation, adhesion, proliferation and apoptosis. Although the precise mechanisms of action are still being elucidated, HBx protein has also been shown to induce methylation of important tumour suppressor genes critical in HBV-induced hepatocarcinogenesis by modulating DNMTs [3, 44, 45, 47, 63, 65, 66].

The transcriptional transactivation role of HBx protein on the transforming growth factor beta 1 (TGF-β1) protein may be important in explaining liver inflammation and fibrosis. TGF-β1, encoded by *TGF-β1* gene, is a cytokine that is produced in response to liver injury by activated hepatocytes, platelets and Kupffer cells. It triggers apoptosis, cell growth and differentiation in human hepatocytes, hepatoma cell lines and transgenic mice [3, 67, 68]. It promotes the development of fibrosis and cirrhosis in chronic HBV infection and other liver-related diseases. HBx protein induces the expression of TGF-β1 through the transactivation of *TGF-β1* gene, the down-regulation of α<sup>2</sup> -macroglobulin and the induction of TGF-β1 mediator Smad4. High levels of TGF-β1 protein are observed in the sera of chronic HBV-induced HCC patients and correlate with the mutation and loss of mannose-6-phosphate/IGF-II receptor that mediates TGF-β1 signalling [3, 67, 69, 70]. In addition, HBx protein alters the signalling pathway of TGF-β1 from being tumour suppressive to oncogenic in early chronic HBV infection. This occurs via the activation of c-Jun N-terminal kinase (JNK) which shifts epithelial tumour suppressive pSmad3C signal to mesenchymal oncogenic pSmadL signal pathway [3, 70].

Studies show that in HBx transgenic mice and hepatoma cell lines, HBx protein can transactivate the NF-κB, MAPK/ERK, STAT3 and PI3K/Akt cellular signalling pathways by inducing the production of ROS. Accumulation of ROS in human cancers is associated with anti-apoptotic activity, DNA damage and mutations which promote malignant transformation. HBxinduced ROS and 8-oxoguanine alter the expression of PTEN protein by oxidising cysteine residues within the promoter region encoding *PTEN* gene, which activates Akt pathway and contributes to hepatocarcinogenesis [3, 65, 70–72].

## **2.3. HBx protein and DNA methylation**

HBx protein has been labelled an epigenetic deregulating agent. It uses its oncogenic ability to induce promoter methylation of some cellular tumour suppressor genes that contribute to the development of liver cancer [3, 73]. Cancer-associated DNA methylation may be global hypomethylation (less methylation) or hypermethylation (increased methylation). Abnormal hypermethylation of various cellular genes including host tumour suppressors has been described in liver cancer, and it is associated with silencing of genes critical for preventing malignant transformation [3, 56]. Altered gene expression has been reported in HBV infection where the DNA methylation machinery is induced as a host defence mechanism to suppress viral genes [3, 53, 57, 58]. This correlates with loss of normal activity in genes important for wound healing and immune processes. Disruption of these processes will interfere with normal cell proliferation and apoptosis and potentiates the ability to metastasize in abnormal cells as seen in chronic liver disease and malignant transformation [3, 58, 63]. By modulating the transcriptional activation of DNMTs, HBx protein induces the hypermethylation of tumour suppressor gene promoters and silences their expression [3, 74–77].

oxisome proliferator-activated receptor γ (PPARγ) and transcription factor II H (TFIIH) [44]. Interaction of HBx protein with these transcription factors disrupts multiple cellular signalling pathways that include janus kinase 1 (JAK1)-signal transducer activator of transcription (STAT), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and p53 signalling pathways. Cellular signalling pathways are important in regulating DNA repair, cell growth, differentiation, adhesion, proliferation and apoptosis. Although the precise mechanisms of action are still being elucidated, HBx protein has also been shown to induce methylation of important tumour suppressor genes critical in HBV-induced hepato-

The transcriptional transactivation role of HBx protein on the transforming growth factor beta 1 (TGF-β1) protein may be important in explaining liver inflammation and fibrosis. TGF-β1, encoded by *TGF-β1* gene, is a cytokine that is produced in response to liver injury by activated hepatocytes, platelets and Kupffer cells. It triggers apoptosis, cell growth and differentiation in human hepatocytes, hepatoma cell lines and transgenic mice [3, 67, 68]. It promotes the development of fibrosis and cirrhosis in chronic HBV infection and other liver-related diseases. HBx protein induces the expression of TGF-β1 through the trans-

TGF-β1 mediator Smad4. High levels of TGF-β1 protein are observed in the sera of chronic HBV-induced HCC patients and correlate with the mutation and loss of mannose-6-phosphate/IGF-II receptor that mediates TGF-β1 signalling [3, 67, 69, 70]. In addition, HBx protein alters the signalling pathway of TGF-β1 from being tumour suppressive to oncogenic in early chronic HBV infection. This occurs via the activation of c-Jun N-terminal kinase (JNK) which shifts epithelial tumour suppressive pSmad3C signal to mesenchymal onco-

Studies show that in HBx transgenic mice and hepatoma cell lines, HBx protein can transactivate the NF-κB, MAPK/ERK, STAT3 and PI3K/Akt cellular signalling pathways by inducing the production of ROS. Accumulation of ROS in human cancers is associated with anti-apoptotic activity, DNA damage and mutations which promote malignant transformation. HBxinduced ROS and 8-oxoguanine alter the expression of PTEN protein by oxidising cysteine residues within the promoter region encoding *PTEN* gene, which activates Akt pathway and

HBx protein has been labelled an epigenetic deregulating agent. It uses its oncogenic ability to induce promoter methylation of some cellular tumour suppressor genes that contribute to the development of liver cancer [3, 73]. Cancer-associated DNA methylation may be global hypomethylation (less methylation) or hypermethylation (increased methylation). Abnormal hypermethylation of various cellular genes including host tumour suppressors has been described in liver cancer, and it is associated with silencing of genes critical for preventing malignant transformation [3, 56]. Altered gene expression has been reported in HBV infection where the DNA methylation machinery is induced as a host defence mechanism to suppress viral genes [3, 53, 57, 58]. This correlates with loss of normal activity in genes important for wound healing and immune processes. Disruption of these processes will


carcinogenesis by modulating DNMTs [3, 44, 45, 47, 63, 65, 66].

activation of *TGF-β1* gene, the down-regulation of α<sup>2</sup>

genic pSmadL signal pathway [3, 70].

162 Advances in Treatment of Hepatitis C and B

contributes to hepatocarcinogenesis [3, 65, 70–72].

**2.3. HBx protein and DNA methylation**

HBx protein induces the hypermethylation of *RARβ2* gene by up-regulating DNMT1 and 3A activities and down-regulating the expression of RARβ2 protein [3, 73, 77]. *RARβ2* binds to and inactivates the E2F1 transcription factor, which is essential for cell cycle progression [3, 64, 73, 77]. Down-regulation of RARβ2 protein expression is associated with activation of E2F1 transcription factor, which abolishes the ability of retinoic acid to regulate the expression of G1 checkpoint regulators, leading to up-regulation of p16, p21 and p27 proteins. The activation of E2F1 transcription factor is associated with uncontrolled cell proliferation which contributes to carcinogenesis [3, 77].

*Insulin-like growth factor binding 3* (*IGJBP-3*) is another potential tumour suppressor gene which is both hyper- and hypomethylated in HBV-induced HCC. Hypermethylation of *IGJBP-3* gene is mediated by DNMT 1 and 3A which are upregulated via the transcriptional activities of HBx protein, and this is associated with loss of *IGJBP-3* gene expression. In contrast, HBx protein reduces the transcriptional activities of DNMT 3B, leading to hypomethylation and up-regulation of the *IGJBP-3* gene [3, 45].

*DLEC1* is a functional tumour suppressor gene silenced by promoter methylation in lung, gastric, colon and nasopharyngeal cancers. Similar methylation has also been observed in HCC where it is associated with induction of G1 cell cycle arrest and loss of gene expression. Silencing of *DLEC 1* gene expression is mediated by both DNA hypermethylation and histone acetylation [3, 21, 78]. HBx protein encoded by HBV genotype A enhances the transcription of *DLEC 1* gene by increasing the level of histone acetylation through the activation of HATs, leading to suppression of tumour progression. Through the activation of DNMT1 expression mediated by the pRB-E2F pathway, HBx protein induces DNA hypermethylation of *DLEC1* gene and suppresses its transcriptional activities [3, 78].

Caveolin-1, encoded by c*aveolin-1* gene, is an integral membrane protein abundantly expressed in adipose, fibrous and endothelial tissue. High-level expression of caveolin-1 protein disrupts growth factor signalling pathways, which in turn alters cell growth, proliferation and differentiation. HCC cells expressing high levels of caveolin-1 are associated with uncontrolled cell growth, motility, in vivo tumour aggressiveness and metastasis. Conversely, HBx-induced methylation of *Caveolin-1* gene promoter region suppresses its transcriptional activities, and this correlates with reduced tumour aggressiveness and metastasis, indicating a role of DNA methylation in HBV-related HCC [3, 80, 81].

Hypermethylation of *p16ink4a*gene is a frequent event in several malignancies including HBVinduced HCC. HBx protein silences the expression of *p16ink4a*gene through the activation of DNA methyltransferase 1 and the cyclin D1-CDK 4/6-pRb-E2F1 pathway. Methylation of *p16ink4a*gene is associated with increased viral replication, integration and loss of protein expression [3, 80, 81].

HBx-protein-induced DNA hypermethylation has also been connected with loss of expression and normal function of *LINE-1, pRB, ASPP, E-cadherin, GSTP1* and *hTERT* tumour suppressor genes [3, 76, 78, 82, 83]. This methylation is associated with increased up-regulation of DNMTs with DNMT1 being the most active one. Aberrant methylation of these genes is associated with perturbed cellular signalling pathways such as ubiquitination, DNA repair, transcription, proliferation and apoptosis, which may lead to the development of HBV-related HCC [3, 21, 45, 78].

Genome-wide studies aided in identifying DNA methylation, histone modifications and miRNA expression profiling across the entire samples with CHB and HBV-related HCC [3, 84–86]. Preliminary data conducted by Kgatle et al. [84] demonstrate that HBV-induced methylation may affect cellular processes such as cell cycle progression, calcium homeostasis, hepatic metabolism, protein ubiquitination, RNA splicing and vitamin D receptor regulation, which are key mechanisms that HBx protein alters to favour viral replication and cell survival. Disruption in these cellular processes could cause genetic instability, hepatocyte transformation and tumour development. However, amongst most conducted genome-wide studies, there are some discrepancies and data variations due to lack of proper normal control, heterogeneity of disease, variations of samples source, use of different technologies for analysis and validation with gene expression analysis, suggesting need for further validations [3, 84].

## **3. Summary**

Substantial data show that there is an association between the methylation of CpG islands and transcriptional changes in gene promoter regions. Transcriptional alterations within gene promoter regions interfere with the normal function of a wide spectrum of cellular genes including tumour suppressor genes which are potential inducers of malignancies. Oncogenic viruses integrate themselves into the human genome and alter gene transcription through DNA methylation. During HBV infection, the expression levels of DNMTs are elevated in response to viral replication as viral genes are methylated to suppress viral replication. This may result in inappropriate random methylation of neighbouring host cellular genes, including tumour suppressor genes. This would cause malignant transformation and ultimately liver cancer. In addition, other genes affected by methylation may contribute to the development of liver inflammation, fibrosis and cirrhosis. As a multifunctional viral transactivator, the HBx protein may be the driving force behind the activation of DNMTs, causing gene promoter hypermethylation and gene silencing. The epigenetic alteration of genes may affect cellular signalling pathways and favour uncontrolled hepatocyte proliferation and HBV-induced inflammation, fibrosis and cancer.

## **Author details**

Mankgopo Magdeline Kgatle

Address all correspondence to: mankgopo.kgatle@gmail.com

Department of Medicine, Faculty of Health Sciences, University of Cape Town, Groote Schuur Hospital, Cape Town, South Africa

## **References**

genes [3, 76, 78, 82, 83]. This methylation is associated with increased up-regulation of DNMTs with DNMT1 being the most active one. Aberrant methylation of these genes is associated with perturbed cellular signalling pathways such as ubiquitination, DNA repair, transcription, proliferation and apoptosis, which may lead to the development of HBV-related HCC [3, 21, 45, 78].

Genome-wide studies aided in identifying DNA methylation, histone modifications and miRNA expression profiling across the entire samples with CHB and HBV-related HCC [3, 84–86]. Preliminary data conducted by Kgatle et al. [84] demonstrate that HBV-induced methylation may affect cellular processes such as cell cycle progression, calcium homeostasis, hepatic metabolism, protein ubiquitination, RNA splicing and vitamin D receptor regulation, which are key mechanisms that HBx protein alters to favour viral replication and cell survival. Disruption in these cellular processes could cause genetic instability, hepatocyte transformation and tumour development. However, amongst most conducted genome-wide studies, there are some discrepancies and data variations due to lack of proper normal control, heterogeneity of disease, variations of samples source, use of different technologies for analysis and validation with gene expression analysis, suggesting need

Substantial data show that there is an association between the methylation of CpG islands and transcriptional changes in gene promoter regions. Transcriptional alterations within gene promoter regions interfere with the normal function of a wide spectrum of cellular genes including tumour suppressor genes which are potential inducers of malignancies. Oncogenic viruses integrate themselves into the human genome and alter gene transcription through DNA methylation. During HBV infection, the expression levels of DNMTs are elevated in response to viral replication as viral genes are methylated to suppress viral replication. This may result in inappropriate random methylation of neighbouring host cellular genes, including tumour suppressor genes. This would cause malignant transformation and ultimately liver cancer. In addition, other genes affected by methylation may contribute to the development of liver inflammation, fibrosis and cirrhosis. As a multifunctional viral transactivator, the HBx protein may be the driving force behind the activation of DNMTs, causing gene promoter hypermethylation and gene silencing. The epigenetic alteration of genes may affect cellular signalling pathways and favour uncontrolled hepatocyte proliferation and HBV-induced inflammation,

Department of Medicine, Faculty of Health Sciences, University of Cape Town, Groote Schuur

for further validations [3, 84].

164 Advances in Treatment of Hepatitis C and B

**3. Summary**

fibrosis and cancer.

**Author details**

Mankgopo Magdeline Kgatle

Hospital, Cape Town, South Africa

Address all correspondence to: mankgopo.kgatle@gmail.com


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**Provisional chapter**

## **Response-Guided Therapy Based on the Combination of Quantitative HBsAg and HBV DNA Kinetics in Chronic Hepatitis B Patients Response-Guided Therapy Based on the Combination of Quantitative HBsAg and HBV DNA Kinetics in Chronic Hepatitis B Patients**

Valeriu Gheorghiță and Florin Alexandru Căruntu Valeriu Gheorghiță and Florin Alexandru Căruntu

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67128

#### **Abstract**

Chronic hepatitis B (CHB) remains a difficult-to-treat disease because no current treatments provide an optimal virological and immunological control, there is a high rate of relapse following any antiviral therapy, and there are no identified clinical useful treatment stopping rules, especially in hepatitis B e antigen (HBeAg)-negative patients treated with nucleoside or nucleotide analogues (NUCs). Taking into account the limited options of antiviral drugs, the response-guided therapy seems to be the best approach for optimization of treatment response. Hepatitis B surface antigen (HBsAg) can be considered a surrogate marker of HBV immune control during antiviral therapy, regardless of virological response reflected by serum HBV DNA. Thus, the decrease of HBV DNA level represents a reduction of viral replication, while serum HBsAg decline signifies a reduction of messenger RNA translation. The most important on-treatment predictors of the antiviral treatment response, especially Peg-IFN α-2a, are the quantitative HBsAg and HBV DNA evolution during therapy. A combination of no HBsAg decline and <2 log10 IU/mL decrease of HBV DNA seems to be a predictor of nonresponse in European HBeAg-negative patients with genotype D. The reduction of HBsAg levels during NUCs treatment in HBeAg-positive patients may identify cases with subsequent HBeAg or HBsAg loss.

**Keywords:** chronic hepatitis B, antiviral treatment strategy, quantitative HBsAg, algorithm of chronic hepatitis B treatment

and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

## **1. Introduction**

Worldwide, hepatitis B virus (HBV) infection has a high prevalence (350–400 million people are chronic HBV surface antigen carriers) and an increased morbidity and mortality (0.5–1 million deaths annually) [1]. To date, chronic hepatitis B (CHB) remains a difficult-to-treat disease due to the inability to achieve an optimal viral and immunological control with the available treatments, the high rate of relapse following any antiviral therapy and the absence of clinically useful predictors of sustained serological and viral responses.

Although existing potent nucleoside and nucleotide analogues (NUCs) with high genetic barriers have improved patient prognosis via suppression of viral load, there are still concerns that need to be addressed such as the need for long-term therapy, reactivation of the disease after cessation of therapy, hepatocellular carcinoma (HCC) risk persistence and the low rate of hepatitis B surface antigen (HBsAg) seroconversion.

In the last years, many published studies assessed the role of serum HBsAg quantification as predictor of treatment response in CHB patients treated mainly with pegylated interferon (Peg-IFN)-based regimens [2]. Some authors have proposed an early stopping rule using the combination between serum HBsAg and HBV DNA levels for hepatitis B e antigen (HBeAg) negative CHB patients treated with Peg-IFN α-2a [2, 3].

## **2. Natural history of chronic hepatitis B**

CHB is distinguished in five different phases according to HBeAg status, HBV DNA level, HBsAg status, alanine aminotransferase (ALT) level and histologic damages [1, 4]. Thus, the five evolutionary phases are as follow: the "immune-tolerant" phase, the "immune-active" HBeAg-positive phase, the "inactive HBV carrier" state, the "immune-escape" HBeAgnegative phase and the HBsAg-negative phase or "occult" HBV infection phase [1, 4]. In the evolution of chronic HBV infection, a patient may pass through each phase consecutively, especially after vertical transmission of the virus. Also, some phases are not identifiable in every patients, either because it may not be an obligatory step in the overall natural course of the infection or because it is of very short duration [5]. This feature seems to be dependent on age at the time of infection and the host immune reactivity against the virus.

The "immune-tolerant" phase is recognized usually in perinatally HBV-infected patients, which may last for about one to four decades in different populations and individuals [4, 5]. There is a highly replicative phase of the virus denoted by the presence of the HBeAg and high levels of HBV DNA (>2 × 10<sup>7</sup> IU/mL) in the serum despite a low inflammatory reaction reflected by normal ALT levels (<19 U/L for females and <30 U/L for males) and mild or no liver inflammation and no or slow progression of fibrosis [1, 2, 4, 5].

The "immune-active" phase, in which the immune system is trying to eliminate the virus, is defined by the HBeAg positivity in conjunction with high or fluctuating serum HBV DNA levels, persistent or intermittent elevation of ALT levels and active inflammation with accelerated progression of fibrosis compared to the previous phase [1, 2, 5]. The hallmark of transition to the inactive phase of chronic HBV infection is the HBeAg seroconversion achieved in the natural course of the disease or therapeutically induced.

The "inactive HBV carrier" state represents the most desirable phase of the disease for HBsAg-positive patients. It is characterized by absence of HBeAg, positive anti-HBe, persistently normal ALT values, low or undetectable HBV DNA (usually <2000 IU/mL) and mild or no inflammatory reaction on liver histology [1, 2, 5]. In clinical practice, one of the main issues is to distinguish between truly inactive HBV carriers and HBeAg-negative active CHB phase. It is well known that HBeAg-negative CHB patients could have intermittent normal transaminases and relatively low level of viral replication. However, these patients often have a long-term chronic HBV infection with advanced fibrosis score and a high probability of progression in the absence of treatment intervention. Considering that, we reinforced the recommendation to regularly check these patients based on individual clinical and biological characteristics.

The "immune-escape" phase may follow either by a spontaneous HBeAg seroconversion to anti-HBe (10–30%) or by reactivation of HBV replication and exacerbations of hepatitis following years of persistent inactive carrier state (10–20%) [1, 2, 5]. Moreover, this phase is defined by a fluctuating evolution of the disease activity with intermittent increase in ALT and HBV DNA serum levels [2]. Most of the patients harbor a pre-core or core promoter HBV variants which are unable to express or express low levels of HBeAg [1, 2].

The "occult" HBV infection phase follows after HBsAg disappearance and represents the persistence of minimum viral replication with detectable HBV DNA into the liver and no or low levels of HBV DNA in serum (<200 IU/mL) [1]. The clinical relevance of this phase is explained by the increasing number of patients who need immunosuppressive or cytotoxic therapy. Thus, to avoid the reactivation of the HBV replication, all guidelines recommend checking for HBsAg, immune globulins G (IgG) anti-HBc, anti-HBs, ALT and HBV DNA serum levels in conjunction with preemptive antiviral therapy depending on the blood test results and type of immunosuppressive agent [1, 2, 5, 9].

## **3. Treatment objective**

**1. Introduction**

174 Advances in Treatment of Hepatitis C and B

Worldwide, hepatitis B virus (HBV) infection has a high prevalence (350–400 million people are chronic HBV surface antigen carriers) and an increased morbidity and mortality (0.5–1 million deaths annually) [1]. To date, chronic hepatitis B (CHB) remains a difficult-to-treat disease due to the inability to achieve an optimal viral and immunological control with the available treatments, the high rate of relapse following any antiviral therapy and the absence

Although existing potent nucleoside and nucleotide analogues (NUCs) with high genetic barriers have improved patient prognosis via suppression of viral load, there are still concerns that need to be addressed such as the need for long-term therapy, reactivation of the disease after cessation of therapy, hepatocellular carcinoma (HCC) risk persistence and the low rate

In the last years, many published studies assessed the role of serum HBsAg quantification as predictor of treatment response in CHB patients treated mainly with pegylated interferon (Peg-IFN)-based regimens [2]. Some authors have proposed an early stopping rule using the combination between serum HBsAg and HBV DNA levels for hepatitis B e antigen (HBeAg)-

CHB is distinguished in five different phases according to HBeAg status, HBV DNA level, HBsAg status, alanine aminotransferase (ALT) level and histologic damages [1, 4]. Thus, the five evolutionary phases are as follow: the "immune-tolerant" phase, the "immune-active" HBeAg-positive phase, the "inactive HBV carrier" state, the "immune-escape" HBeAgnegative phase and the HBsAg-negative phase or "occult" HBV infection phase [1, 4]. In the evolution of chronic HBV infection, a patient may pass through each phase consecutively, especially after vertical transmission of the virus. Also, some phases are not identifiable in every patients, either because it may not be an obligatory step in the overall natural course of the infection or because it is of very short duration [5]. This feature seems to be dependent on

The "immune-tolerant" phase is recognized usually in perinatally HBV-infected patients, which may last for about one to four decades in different populations and individuals [4, 5]. There is a highly replicative phase of the virus denoted by the presence of the HBeAg and

reflected by normal ALT levels (<19 U/L for females and <30 U/L for males) and mild or no

The "immune-active" phase, in which the immune system is trying to eliminate the virus, is defined by the HBeAg positivity in conjunction with high or fluctuating serum HBV DNA levels, persistent or intermittent elevation of ALT levels and active inflammation with accelerated

IU/mL) in the serum despite a low inflammatory reaction

age at the time of infection and the host immune reactivity against the virus.

liver inflammation and no or slow progression of fibrosis [1, 2, 4, 5].

of clinically useful predictors of sustained serological and viral responses.

of hepatitis B surface antigen (HBsAg) seroconversion.

negative CHB patients treated with Peg-IFN α-2a [2, 3].

**2. Natural history of chronic hepatitis B**

high levels of HBV DNA (>2 × 10<sup>7</sup>

As HBV cannot be truly eliminated with available treatment due to the persistence of covalently closed circular (ccc) DNA into the nuclei of the hepatocytes, the current goal of therapy in patients with CHB is improving the quality of life and prolonging their life expectancy by preventing the progression of the disease to the cirrhosis, decompensated cirrhosis, end-stage liver disease, hepatocellular carcinoma (HCC) and deaths [1, 2]. One of the efficient strategies to reach this goal is achieving and maintaining indefinitely the complete inhibition of viral replication. HBsAg loss and anti-HBs seroconversion, events rarely achieved nowadays, represent the ultimate aim of any antiviral treatment strategy and reflect especially the immune control of the virus without need for further medication, except decompensated cirrhosis or necessity of cytotoxic/immunosuppressive prolonged treatment [4].

Virtually, all patients diagnosed with chronic HBV infection are potential candidates for antiviral therapy. However, considering that current antiviral cannot completely eradicate the virus, all international guidelines agree that treatment is not required in the immune-tolerant phase and inactive carrier state of chronic HBV infection [1, 2, 5, 6]. In addition, it has been proved that patients with CHB who persist for years in immune-tolerant phase or inactive carrier state do not register a significant disease progression and the likelihood of response, in particular HBeAg seroconversion, is very low (<5%) [6, 7]. Nevertheless, even in these populations some controversy still remains about the risk of developing HCC and the risk of virus transmission into the population, respectively. The REVEAL (Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer) study has been concluded that persistently high serum HBV DNA levels are associated with increased risk of cirrhosis, HCC and liverrelated death. On the other hand, 67% of populations in this study were older than age 39 [8]. For all these reasons some experts have proposed that immune-tolerant patients older than age of 40 should receive antiviral treatment, especially if they have elevated HBV DNA (>10<sup>6</sup> IU/mL) and significant necroinflammation or fibrosis [2, 6]. Given that HBV infection is a chronic and dynamic condition, having the possibility of crossing a stage to the other and vice versa, regular monitoring is critical in patients without indication for antiviral therapy at a single-point assessment, in order to identify the best timing for treatment intervention [6].

From the clinical perspective, usually the decision to initiate the antiviral treatment in patients diagnosed with HBV infections is made by taking into consideration several important parameters: clinical status, ALT and HBV DNA levels, HBeAg status and the severity of liver inflammation and fibrosis [1, 2, 5, 6]. Indications for treatment may also depend on age, familial history of HCC, coinfection with other viruses, immunosuppression conditions, planning to become pregnant within the next 2–3 years in female patients [1, 6]. There are some absolute indications for antiviral treatment necessity such as HBV infection–associated life-threatening liver disease: acute liver failure or severe acute hepatitis (prolonged jaundice and coagulation abnormality), decompensated cirrhosis, severe exacerbation of CHB as well as for preventing reactivation in patients receiving immunosuppressive therapy, regardless of HBV DNA and ALT levels [1, 2, 5, 6]. In patients with compensated cirrhosis, we follow the European Association for the Study of the Liver (EASL) guideline which recommends antiviral treatment when the serum HBV DNA is detectable, irrespective of ALT levels [1]. In supporting of this approach, we mention the availability of potent NUCs with high genetic barrier to resistance along with slowing the progression of the disease to decompensated cirrhosis, endstage liver disease and death. In noncirrhotic CHB patients, the treatment is generally recommended when they have a serum HBV DNA levels above 2000 IU/mL, persistently increased ALT levels above upper limit of normal (ULN) and/or histologic assessment showing moderate/severe inflammation or fibrosis [1, 6]. However, there are slight differences between the guidelines regarding the cutoff for HBV DNA and ALT values and the need for liver biopsy in order to establish the indication for antiviral treatment.

The EASL guideline recommends an HBV DNA cutoff value of 2000 IU/mL for initiating treatment, irrespective of HBeAg status [1]. The American Association for the Study of Liver Disease (AASLD) and Asian Pacific Association for the Study of the Liver (APASL) guidelines suggest an HBV DNA level of 20,000 IU/mL for HBeAg-positive patients and 2000 IU/mL for HBeAg-negative patients [2]. All international guidelines agree that, for patients who fulfill the criteria for HBV DNA, treatment should be recommended whenever the ALT levels are above 2×ULN or less than 2×ULN, even in normal range, whether are evidences for moderate/ severe inflammation or fibrosis [1, 2, 5].

Virtually, all patients diagnosed with chronic HBV infection are potential candidates for antiviral therapy. However, considering that current antiviral cannot completely eradicate the virus, all international guidelines agree that treatment is not required in the immune-tolerant phase and inactive carrier state of chronic HBV infection [1, 2, 5, 6]. In addition, it has been proved that patients with CHB who persist for years in immune-tolerant phase or inactive carrier state do not register a significant disease progression and the likelihood of response, in particular HBeAg seroconversion, is very low (<5%) [6, 7]. Nevertheless, even in these populations some controversy still remains about the risk of developing HCC and the risk of virus transmission into the population, respectively. The REVEAL (Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer) study has been concluded that persistently high serum HBV DNA levels are associated with increased risk of cirrhosis, HCC and liverrelated death. On the other hand, 67% of populations in this study were older than age 39 [8]. For all these reasons some experts have proposed that immune-tolerant patients older than age of 40 should receive antiviral treatment, especially if they have elevated HBV DNA

 IU/mL) and significant necroinflammation or fibrosis [2, 6]. Given that HBV infection is a chronic and dynamic condition, having the possibility of crossing a stage to the other and vice versa, regular monitoring is critical in patients without indication for antiviral therapy at a single-point assessment, in order to identify the best timing for treatment intervention [6].

From the clinical perspective, usually the decision to initiate the antiviral treatment in patients diagnosed with HBV infections is made by taking into consideration several important parameters: clinical status, ALT and HBV DNA levels, HBeAg status and the severity of liver inflammation and fibrosis [1, 2, 5, 6]. Indications for treatment may also depend on age, familial history of HCC, coinfection with other viruses, immunosuppression conditions, planning to become pregnant within the next 2–3 years in female patients [1, 6]. There are some absolute indications for antiviral treatment necessity such as HBV infection–associated life-threatening liver disease: acute liver failure or severe acute hepatitis (prolonged jaundice and coagulation abnormality), decompensated cirrhosis, severe exacerbation of CHB as well as for preventing reactivation in patients receiving immunosuppressive therapy, regardless of HBV DNA and ALT levels [1, 2, 5, 6]. In patients with compensated cirrhosis, we follow the European Association for the Study of the Liver (EASL) guideline which recommends antiviral treatment when the serum HBV DNA is detectable, irrespective of ALT levels [1]. In supporting of this approach, we mention the availability of potent NUCs with high genetic barrier to resistance along with slowing the progression of the disease to decompensated cirrhosis, endstage liver disease and death. In noncirrhotic CHB patients, the treatment is generally recommended when they have a serum HBV DNA levels above 2000 IU/mL, persistently increased ALT levels above upper limit of normal (ULN) and/or histologic assessment showing moderate/severe inflammation or fibrosis [1, 6]. However, there are slight differences between the guidelines regarding the cutoff for HBV DNA and ALT values and the need for liver biopsy

The EASL guideline recommends an HBV DNA cutoff value of 2000 IU/mL for initiating treatment, irrespective of HBeAg status [1]. The American Association for the Study of Liver Disease (AASLD) and Asian Pacific Association for the Study of the Liver (APASL) guidelines suggest an HBV DNA level of 20,000 IU/mL for HBeAg-positive patients and 2000 IU/mL for

in order to establish the indication for antiviral treatment.

(>10<sup>6</sup>

176 Advances in Treatment of Hepatitis C and B

## **4. Antiviral treatment strategy with available options: Peg-IFN and NUCs**

To date, there are currently available two different classes of antiviral agents for treatment of CHB patients: IFN-α (conventional or pegylated) and oral drugs (NUCs) [4]. Despite the availability of seven approved drugs, only three of them are preferred as first-line options in the international American and European guidelines, as follow: Peg-IFN α-2a, entecavir (ETV) and tenofovir (TDF). Obviously, each of these agents is selected based on patient characteristics, considering that neither IFN nor NUCs are the best treatment options in any clinical condition. Thus, baseline as well as on-treatment predictive markers are needed to identify which patients benefit most from a finite course of IFN treatment or indefinite treatment with oral NUCs.

Both IFN-alfa and NUCs have different mechanisms of action in order to achieve the predefined goals of treatment in CHB patients: ALT normalization, suppression of viral replication, HBeAg and HBsAg seroconversion [9]. In addition, the reduction of the risk of progression to cirrhosis and HCC are among the desired therapeutic objectives [9].

IFN is a pro-inflammatory cytokine with dual mechanism of action, both antiviral and immunomodulatory activities, enhancing host immunity defense against HBV, which may lead to a sustained off-therapy response known as immune control [5, 6, 9]. Although the antiviral potency of IFN (Peg-IFN, respectively) is modest, the international guidelines have positioned it in the first-line treatment option, considering the major advantages associated with usage of this drug: finite duration of therapy, immunomodulatory effect with the potential to increase the chance of HBeAg and HBsAg seroconversion as well as a long-term immune control of the disease at least in a well-selected population [1, 2, 5, 10]. In addition, the National Institute for Health and Care Excellence (NICE) guideline for CHB recommend that a 48-week course of Peg-IFN α-2a should be offered as first-line treatment in adults with HBeAg-positive andnegative CHB and compensated liver disease [10].

It has been identified pretreatment predictors of IFN/Peg-IFN α-2a response in HBeAgpositive CHB patients: young age, high serum ALT levels (>2×ULN), low viral load, HBV genotype A and B, high histologic activity index and wild-type pre-core and basal-core promoter sequence [6, 9]. In HBeAg-negative patients, there were no such well-defined baseline predictors of IFN treatment [6]. From the clinical point of view, the presence of these baseline predictors in an individual patient with CHB does not assure the response to a 48-week course of Peg-IFN treatment. For this reason, of great significance are early predictive factors, such as ALT flares and quantitative HBsAg decline at 12 and 24 weeks during treatment [11–13].

On the other hand, there are some limitations of IFN-based treatment in CHB due to the parenteral weekly administration, the broad spectrum of side effects and the restriction of administration in several circumstances of the disease according to licensed indications (e.g., decompensated cirrhosis, uncontrolled psychiatric illness, pregnancy, hematologic neoplasia with need for cytotoxic or immunosuppressive treatment) [14].

NUCs, known as drugs with direct antiviral mechanism of action, targeting the HBV polymerase, represent another major class available in the therapeutic armamentarium of CHB. These antiviral drugs have become the mainstay therapy in CHB given the oral administration, the easy management, the absence of contraindications to start treatment, the high antiviral potency and a narrow spectrum of side effects [6, 9]. Among the available NUCs, lamivudine (LAM), telbivudine (LdT) and adefovir (ADV) are no longer recommended as first-line monotherapy because of the resistance concerns, while ETV and TDF are ranked by all international guidelines in the pole position of antiviral treatment [1, 2, 5].

Mitochondrial toxicity is a potential side effect of any NUCs, but fortunately is very rare event. There are reported specific side effects for each NUC, such as myopathy and neuropathy related to LdT, lactic acidosis related with administration of ETV in patients with severely impaired liver function and renal dysfunction and bone mineral density impairment in patients treated with ADV and TDF [6].

ETV and TDF suppress viral replication in over 90% of CHB patients within a defined period of time (months to years), although undetectable HBV DNA is much faster achieved when the baseline viral load is lower [9]. Furthermore, HBeAg seroconversion rate increase over time with around 40% in Asian studies and 20% of HBeAg-positive, genotype A, patients from Europe, respectively, while HBsAg seroconversion occurs in approximately 3–10% of all CHB patients over 5 years of follow-up [6, 9]. Despite the inability of NUCs to act directly on the cccDNA, the level of the intrahepatic cccDNA seems to decrease over prolonged treatment with NUCs, as the nuclear replenishment with new chains of viral DNA is interfered by blocking the transcription of pregenomic viral RNA. It is estimated that with current NUCs treatment, the median number of years needed to clear HBsAg is 52.2 years [15]. The similar study predicted a median time for HBsAg loss of 36 years in HBeAg-positive and 39 years in HBeAg-negative HBV infection, respectively [16]. High baseline ALT level seems to be the most important pretreatment predictor of response to NUC treatment in HBeAg-positive patients [6]. In the HBeAg-negative CHB patients, there have not been defined baseline predictors of treatment with NUCs [6]. Unlike Peg-IFN treatment, it has not been demonstrated that HBV genotype could influence the NUCs treatment response [6].

The long-term completely viral suppression is associated with liver histology improvement and in some patients even with reversion of cirrhosis over a treatment period of 5 years [17, 18]. The impact of long-term treatment with NUCs on HCC risk is questionable. At least from the theoretically point of view, the inhibition of viral replication could decrease the cumulative incidence of HCC, considering that HBV DNA levels have been identified as an independent risk factor for HCC occurrence [8]. Thus, there have been published some studies which established that long-term treatment with potent NUCs have been linked to the reduction of the incidence of HCC [19, 20]. On the opposite, the risk of HCC could not be eliminated with any available treatments because of the truncated sequences of HBs genes integrated in the DNA of the infected hepatocytes, which is believed to be associated with carcinogenesis.

However, it is still unresolved issue related to the NUCs treatment in CHB, such as the safety of long-term usage of these antivirals, the extent of the optimal duration of treatment and when the treatment discontinuation is suitable [21]. Despite the highest antiviral efficacy, NUCs do not have immunomodulatory effects and induce only a transient increase of immune activity, being unlikely to provide a sustained off-treatment control of viral replication [21]. Thus, treatment with NUCs is indefinite in most cases. It has been proposed that the best and safest endpoint for NUCs discontinuation in any CHB patient is HBsAg seroconversion, defined as HBsAg loss and anti-HBs appearance at a level over 100–200 IU/mL [9]. However, around 30% of patients who achieve HBsAg loss during NUCs treatment do not develop anti-HBs even with prolongation of antiviral treatment [9]. According to the guidelines, the NUCs treatment endpoints in CHB patients are different depending on the HBeAg status. In HBeAg-positive CHB, it seems reasonable to discontinue the NUCs treatment in noncirrhotic patients who undergone HBeAg seroconversion and who have maintained the undetectable HBV DNA at least 1 year thereafter, although approximately 50% relapse [1, 9]. On the other hand, indefinite treatment with NUCs is necessary for HBeAg-negative patients and HBeAgpositive patients who do not develop anti-HBe seroconversion [1]. The same approach is recommended in patients with cirrhosis irrespective of HBeAg status or anti-HBe seroconversion on treatment [1]. In some instances, discontinuations could be attempted in HBeAg-negative patients after treatment for at least 2 years (preferably 4–5 years) with undetectable HBV DNA documented on three separate occasions, 6 months apart [5, 9]. Once it has been decided to stop the NUC-based treatment, regular monitoring of virological and biochemical parameters is mandatory, considering that there are potential life-threatening clinical consequences associated with NUCs discontinuation such as hepatitis flare and hepatic decompensation [9].

## **5. Response-guided therapy based on serum HBsAg and HBV DNA kinetics**

administration in several circumstances of the disease according to licensed indications (e.g., decompensated cirrhosis, uncontrolled psychiatric illness, pregnancy, hematologic neoplasia

NUCs, known as drugs with direct antiviral mechanism of action, targeting the HBV polymerase, represent another major class available in the therapeutic armamentarium of CHB. These antiviral drugs have become the mainstay therapy in CHB given the oral administration, the easy management, the absence of contraindications to start treatment, the high antiviral potency and a narrow spectrum of side effects [6, 9]. Among the available NUCs, lamivudine (LAM), telbivudine (LdT) and adefovir (ADV) are no longer recommended as first-line monotherapy because of the resistance concerns, while ETV and TDF are ranked by

Mitochondrial toxicity is a potential side effect of any NUCs, but fortunately is very rare event. There are reported specific side effects for each NUC, such as myopathy and neuropathy related to LdT, lactic acidosis related with administration of ETV in patients with severely impaired liver function and renal dysfunction and bone mineral density impairment

ETV and TDF suppress viral replication in over 90% of CHB patients within a defined period of time (months to years), although undetectable HBV DNA is much faster achieved when the baseline viral load is lower [9]. Furthermore, HBeAg seroconversion rate increase over time with around 40% in Asian studies and 20% of HBeAg-positive, genotype A, patients from Europe, respectively, while HBsAg seroconversion occurs in approximately 3–10% of all CHB patients over 5 years of follow-up [6, 9]. Despite the inability of NUCs to act directly on the cccDNA, the level of the intrahepatic cccDNA seems to decrease over prolonged treatment with NUCs, as the nuclear replenishment with new chains of viral DNA is interfered by blocking the transcription of pregenomic viral RNA. It is estimated that with current NUCs treatment, the median number of years needed to clear HBsAg is 52.2 years [15]. The similar study predicted a median time for HBsAg loss of 36 years in HBeAg-positive and 39 years in HBeAg-negative HBV infection, respectively [16]. High baseline ALT level seems to be the most important pretreatment predictor of response to NUC treatment in HBeAg-positive patients [6]. In the HBeAg-negative CHB patients, there have not been defined baseline predictors of treatment with NUCs [6]. Unlike Peg-IFN treatment, it has not been demonstrated that HBV genotype could influence the NUCs

The long-term completely viral suppression is associated with liver histology improvement and in some patients even with reversion of cirrhosis over a treatment period of 5 years [17, 18]. The impact of long-term treatment with NUCs on HCC risk is questionable. At least from the theoretically point of view, the inhibition of viral replication could decrease the cumulative incidence of HCC, considering that HBV DNA levels have been identified as an independent risk factor for HCC occurrence [8]. Thus, there have been published some studies which established that long-term treatment with potent NUCs have been linked to the reduction of the incidence of HCC [19, 20]. On the opposite, the risk of HCC could not be eliminated with any available treatments because of the truncated sequences of HBs genes integrated in the DNA of the infected hepatocytes, which is believed to be associated with carcinogenesis.

all international guidelines in the pole position of antiviral treatment [1, 2, 5].

with need for cytotoxic or immunosuppressive treatment) [14].

in patients treated with ADV and TDF [6].

178 Advances in Treatment of Hepatitis C and B

treatment response [6].

One of the modern and cost-efficient concepts in the management of CHB in terms of antiviral treatment is "response guided therapy" depending on the kinetics of the serum HBsAg and HBV DNA levels during treatment. In the current international guidelines, there are some validated rules that support either continuation of antiviral regimen, based on a positive prediction of response, or contrary, cessation/switching therapy to another regimen, depending on a high negative prediction of sustained response.

The clinical relevance of quantitative serum HBsAg arises from the correlation with the intrahepatic amount and transcriptional activity of cccDNA, the main replicative template of HBV [22–24]. It is assumed that quantitative HBsAg could be used as a surrogate marker for immune control of the virus, regardless of the HBV DNA response during treatment and thereafter [23]. A HBV DNA decline directly reflects a reduction of viral replication, while serum HBsAg decline signifies a reduction of transcriptional activity of intranuclear cccDNA and integrated DNA sequences [24, 25].

Several studies have been published to identify the useful surrogate markers for selecting the initial antiviral regimen, for guiding the treatment as well as for early prediction of the favorable or unfavorable outcome [4]. These markers have been stratified as pretreatment and on-treatment predictors. The majority of the studies have investigated the significance of HBsAg and HBV DNA levels as the most powerful predictive factors for antiviral treatment response. It is well known that the most important decrease of HBsAg levels occurred during the Peg-IFN treatment because of the dual mechanism of action, including the modulation of the immune activity. On the other hand, the long-term treatment with NUCs induces only a minimal reduction of serum HBsAg, especially in HBeAg-positive patients. Thus, the HBsAg quantification has various benefits in the management of CHB patients depending on the HBeAg status and the antiviral treatment.

Quantification of HBsAg levels can be used to guide the treatment with Peg-IFN α-2a. In addition, different studies proposed the role of HBsAg level as a "stopping rule" at week 12 of Peg-IFN treatment in both HBeAg-positive and HBeAg-negative patients [5].

In HBeAg-positive CHB patients with genotype A and D, an absence of any HBsAg decline at 12 weeks of Peg-IFN treatment has been associated with a negative predictive value (NPV) of 97% for sustained response [26]. Moreover, in HBeAg-positive, genotype B and C chronic HBV infections, it has been observed that a level of HBsAg over 20,000 IU/mL at 12 weeks of treatment with Peg-IFN could predict a low chance of HBeAg seroconversion [1, 5]. Thus, the European and Asian guidelines have proposed an early stopping rule in CHB, HBeAgpositive patients, who do not achieve any HBsAg decline or who have an HBsAg levels over 20,000 IU/mL after 12 weeks of Peg-IFN-based treatment [1, 5]. Also, a level of HBsAg over 20,000 IU/mL at 24 weeks could be applied as another stopping rule, irrespective of HBV genotype [27]. Overall, around 20–30% of the HBeAg-positive patients would be eligible for an early stopping of treatment with Peg-IFN, at 12/24 weeks, due to the high negative prediction of the sustained response after 48 weeks course of standard of care [9]. On the other hand, it has been proved that HBeAg seroconversion rates 6 months posttreatment were significantly higher in patients with HBsAg <1500 IU/mL at weeks 12 and 24 (56.7 and 54.4%, respectively) versus patients with HBsAg <20,000 IU/mL (16.3 and 15.4%, respectively) [28]. Another ontreatment positive predictor is based on HBV DNA decline at 12 weeks. An HBV DNA level less than 20,000 IU/mL has been associated with 50% chance of anti-HBe seroconversion [29].

In HBeAg-negative genotype D patients treated with Peg-IFN α-2a, it has been validated a stopping rule depending on a combination of HBsAg and HBV DNA assessment at 12 weeks. According to this rule, we can identify early, with a NPV of 100%, all CHB, HBeAg-negative, genotype D patients who will not achieve sustained response at 48 or 96 weeks of treatment with Peg-IFN α-2a [30]. A less than 10% decline of HBsAg levels at 12 weeks for patients with nongenotype D infections and at 24 weeks for genotype D has been shown to be associated with 16% probability of treatment response at 1 year posttherapy [31]. Similar to HBeAgpositive patients, approximately 50% of HBeAg-negative CHB patients with an HBV DNA decrease <20,000 IU/mL at 12 weeks during Peg-IFN treatment would achieve a sustained off-treatment response [1].

In 2013, we published a Romanian real-life small cohort study which included 57 patients with CHB treated 48 weeks with Peg-IFN α-2a and followed for another 24 weeks. The majority of patients had HBeAg-negative CHB (68%, *n* = 39) and genotype D (approximately 80%). During treatment, patients who achieved sustained response showed a marked decrease in serum HBsAg in comparison with non-responders (mean decrease of 1.06 ± 1.3 log10 IU/mL versus 0.04 ± 0.5 log10 IU/mL at 48 weeks, *p* = 0.005). On therapy, HBV DNA reduction >2 log10 IU/mL with any decrease of HBsAg level at week 12 had a positive predictive value (PPV) of 80% (95% CI: 51.91–95.43%) for sustained response, while HBV DNA decline <2 log10 IU/mL without any decline of HBsAg had a NPV of 85.71% (95% CI: 42.23–97.63%) for sustained response. One interesting findings of our study showed that relapsers had the same HBsAg declining profile as non-responder patients [3].

and on-treatment predictors. The majority of the studies have investigated the significance of HBsAg and HBV DNA levels as the most powerful predictive factors for antiviral treatment response. It is well known that the most important decrease of HBsAg levels occurred during the Peg-IFN treatment because of the dual mechanism of action, including the modulation of the immune activity. On the other hand, the long-term treatment with NUCs induces only a minimal reduction of serum HBsAg, especially in HBeAg-positive patients. Thus, the HBsAg quantification has various benefits in the management of CHB patients depending on the

Quantification of HBsAg levels can be used to guide the treatment with Peg-IFN α-2a. In addition, different studies proposed the role of HBsAg level as a "stopping rule" at week 12

In HBeAg-positive CHB patients with genotype A and D, an absence of any HBsAg decline at 12 weeks of Peg-IFN treatment has been associated with a negative predictive value (NPV) of 97% for sustained response [26]. Moreover, in HBeAg-positive, genotype B and C chronic HBV infections, it has been observed that a level of HBsAg over 20,000 IU/mL at 12 weeks of treatment with Peg-IFN could predict a low chance of HBeAg seroconversion [1, 5]. Thus, the European and Asian guidelines have proposed an early stopping rule in CHB, HBeAgpositive patients, who do not achieve any HBsAg decline or who have an HBsAg levels over 20,000 IU/mL after 12 weeks of Peg-IFN-based treatment [1, 5]. Also, a level of HBsAg over 20,000 IU/mL at 24 weeks could be applied as another stopping rule, irrespective of HBV genotype [27]. Overall, around 20–30% of the HBeAg-positive patients would be eligible for an early stopping of treatment with Peg-IFN, at 12/24 weeks, due to the high negative prediction of the sustained response after 48 weeks course of standard of care [9]. On the other hand, it has been proved that HBeAg seroconversion rates 6 months posttreatment were significantly higher in patients with HBsAg <1500 IU/mL at weeks 12 and 24 (56.7 and 54.4%, respectively) versus patients with HBsAg <20,000 IU/mL (16.3 and 15.4%, respectively) [28]. Another ontreatment positive predictor is based on HBV DNA decline at 12 weeks. An HBV DNA level less than 20,000 IU/mL has been associated with 50% chance of anti-HBe seroconversion [29]. In HBeAg-negative genotype D patients treated with Peg-IFN α-2a, it has been validated a stopping rule depending on a combination of HBsAg and HBV DNA assessment at 12 weeks. According to this rule, we can identify early, with a NPV of 100%, all CHB, HBeAg-negative, genotype D patients who will not achieve sustained response at 48 or 96 weeks of treatment with Peg-IFN α-2a [30]. A less than 10% decline of HBsAg levels at 12 weeks for patients with nongenotype D infections and at 24 weeks for genotype D has been shown to be associated with 16% probability of treatment response at 1 year posttherapy [31]. Similar to HBeAgpositive patients, approximately 50% of HBeAg-negative CHB patients with an HBV DNA decrease <20,000 IU/mL at 12 weeks during Peg-IFN treatment would achieve a sustained

In 2013, we published a Romanian real-life small cohort study which included 57 patients with CHB treated 48 weeks with Peg-IFN α-2a and followed for another 24 weeks. The majority of patients had HBeAg-negative CHB (68%, *n* = 39) and genotype D (approximately 80%). During treatment, patients who achieved sustained response showed a marked decrease in

of Peg-IFN treatment in both HBeAg-positive and HBeAg-negative patients [5].

HBeAg status and the antiviral treatment.

180 Advances in Treatment of Hepatitis C and B

off-treatment response [1].

Considering that the rate of virological relapse after cessation of NUCs treatment is estimated to be 50%, the decline of HBsAg may help identify patients in whom treatment can be safely stopped without a high risk of relapse. Together with serum ALT and HBV DNA assessment, HBsAg quantification has been proposed as a clinically useful tool to monitor treatment responses during NUCs treatment, especially the prediction of future HBsAg loss [4].

The magnitude of HBsAg reduction during NUCs treatment could also predict the later HBsAg loss [4]. An HBsAg decline more than 1 log10 IU/mL after 1 year of oral antiviral treatment in HBeAg-positive CHB patients have been shown to predict the HBsAg loss [32].

Lower HBsAg levels at the end of treatment were predictive for later HBsAg loss, as well as for maintenance of HBV suppression after discontinuation of long-term NUCs treatment [4].

In HBeAg-positive CHB patients, an HBsAg levels <100 IU/mL was highly predictive of sustained response at 2 years off treatment [33]. In a recent Asian study, it has been showed that post-treatment virological relapse rate was significantly higher in patients over 50 years old and in patients with an HBsAg level >2 log10 IU/mL at the ETV cessation [34]. In the same study, an HBsAg level of 2.5 log10 IU/mL at HBeAg seroconversion has been established as an optimal cutoff for prediction of post-treatment virological relapse [34]. Thus, patients aged <50 years who achieved an HBsAg level <2.5 log10 IU/mL at HBeAg seroconversion had the lowest rate of relapse, 5% respectively [34]. In HBeAg-positive CHB patients treated with ETV, a serum HBsAg level below 2.5 log10 IU/mL at HBeAg seroconversion could be a useful predictor of post-treatment virological relapse [34].

Although previous studies have shown that quantitative HBsAg levels could be a useful predictor of relapse after cessation of treatment with NUCs in HBeAg-negative patients, in other recent prospective studies, neither HBsAg level at the end of treatment nor the kinetics of HBsAg were not able to predict the off-treatment relapse [35]. However, at the end of treatment, both HBsAg ≤2 log10 IU/mL and reduction by >1 log10 IU/mL from baseline were associated with a sustained virological response, defined as HBV DNA <200 IU/mL 12 month posttreatment [36].

## **6. Other clinical benefits of serum HBsAg quantifications in management of chronic hepatitis B**

Since its discovery, besides the using of qualitative HBsAg as a diagnostic marker, there have been identified several roles of HBsAg in the management of chronic HBV infections, as follows.

## **6.1. Defining different phases of CHB**

It is well known that HBsAg levels vary during the natural history of chronic HBV infections [3]. The highest values of HBsAg are reported in immune-tolerant phase (5.0 log10 IU/mL for HBsAg) and progressively decrease in "immune-active" phase (medium level of 3.0–4.0 log10 IU/mL) [24, 37, 38]. The lowest values of HBsAg levels have been reported in the "inactive carrier state" [24, 38]. Moreover, there is a variability of the quantitative HBsAg across different viral genotypes [3]. Patients with genotype A and D have the highest mean value of serum HBsAg (4.5 log10 IU/mL) compared to genotypes B and C (4.3 log10 IU/mL and 3.8 log10 IU/mL, respectively) [32, 39].

From the clinical point of view, combining a single-point determination of HBsAg <1500 IU/ mL and HBV DNA <2000 IU/mL may identify "true inactive carriers" with a NPV of 96.7% for genotype D CHB patients [40]. This strategy could be useful especially in HBeAg-negative CHB patients with an HBV DNA level around 2000 IU/mL and normal transaminases, considering that in some patients is difficult to distinguish between active HBeAg-negative hepatitis and inactive carriers.

## **6.2. Predictor of liver fibrosis**

Both HBV DNA and HBsAg levels have a declining evolution as long as liver disease progress from the immune-tolerant status to the active hepatitis and cirrhosis in HBeAg-positive patients [41]. Although previous studies showed that HBV DNA level could predict the risk of cirrhosis and HCC, it has been proved a poor correlation between HBsAg level and HBV DNA across different phases of the chronic HBV infection [8, 41]. Given that ALT measurement is a suboptimal marker for prediction of significant liver disease, it is recommended to have, as accurate as possible, an estimation of fibrosis and inflammation based on a reliable tool in order to decide antiviral treatment indication [42]. Nowadays, liver biopsy became rarely used in evaluation of patients with chronic hepatitis viral diseases due to the risk of the procedure, inter- and intraobservers variability, costs, as well as the availability of several noninvasive tests. All international guidelines agree that any HBV carriers who fulfill the criteria of HBV DNA have indication of antiviral treatment, whether there are evidences of significant necroinflammation and/or moderate/severe fibrosis [1, 2, 5]. Transient elastography, an imaging noninvasive test for assessing liver fibrosis, has a low accuracy in distinguished between intermediate stages of fibrosis (F1–F3). Also, the results are influenced by some confounding factors such as steatosis, ALT elevation [43].

There is emerging evidence suggesting association between HBsAg level and liver fibrosis stage in HBeAg-positive CHB patients. It has been proposed different cutoff levels of HBsAg for prediction of liver fibrosis among HBeAg-positive patients. Thus, serum HBsAg over 100,000 IU/mL was 100% predictive of insignificant fibrosis in patients with ALT below 2×ULN [42]. In HBeAg-positive patients with ALT ≤2×ULN, an HBsAg level over 25,000 IU/mL has been proved to be the best independent predictor of insignificant liver fibrosis (PPV of 92.7%, odds ratio 9.042) [42]. Based on these results, it has been suggested that HBeAg-positive patients with ALT ≤2×ULN and HBsAg ≥ 25,000 IU/mL could be followed without the need of liver biopsy [42]. On the other hand, there are evidences which support that lower serum levels of HBsAg are associated with more severe liver fibrosis in HBeAg-positive CHB patients [41]. A cutoff of 4.7 log10 IU/mL predicted moderate to advanced fibrosis (F2-F4) in HBeAg-positive patients, with an accuracy of 89% and a NPV of 91% [41]. Thus, a single-point baseline assessment of HBsAg level in HBeAg-positive chronic HBV-infected patients could become an accurate surrogate marker for distinguish moderate to advanced fibrosis from no or mild fibrosis [41]. However, in HBeAg-negative patients, there were no reported significant differences in serum HBsAg levels between patients with moderate to severe fibrosis and those with no or mild fibrosis [41].

## **6.3. Predictor of HCC**

**6.1. Defining different phases of CHB**

182 Advances in Treatment of Hepatitis C and B

tively) [32, 39].

and inactive carriers.

**6.2. Predictor of liver fibrosis**

founding factors such as steatosis, ALT elevation [43].

It is well known that HBsAg levels vary during the natural history of chronic HBV infections [3]. The highest values of HBsAg are reported in immune-tolerant phase (5.0 log10 IU/mL for HBsAg) and progressively decrease in "immune-active" phase (medium level of 3.0–4.0 log10 IU/mL) [24, 37, 38]. The lowest values of HBsAg levels have been reported in the "inactive carrier state" [24, 38]. Moreover, there is a variability of the quantitative HBsAg across different viral genotypes [3]. Patients with genotype A and D have the highest mean value of serum HBsAg (4.5 log10 IU/mL) compared to genotypes B and C (4.3 log10 IU/mL and 3.8 log10 IU/mL, respec-

From the clinical point of view, combining a single-point determination of HBsAg <1500 IU/ mL and HBV DNA <2000 IU/mL may identify "true inactive carriers" with a NPV of 96.7% for genotype D CHB patients [40]. This strategy could be useful especially in HBeAg-negative CHB patients with an HBV DNA level around 2000 IU/mL and normal transaminases, considering that in some patients is difficult to distinguish between active HBeAg-negative hepatitis

Both HBV DNA and HBsAg levels have a declining evolution as long as liver disease progress from the immune-tolerant status to the active hepatitis and cirrhosis in HBeAg-positive patients [41]. Although previous studies showed that HBV DNA level could predict the risk of cirrhosis and HCC, it has been proved a poor correlation between HBsAg level and HBV DNA across different phases of the chronic HBV infection [8, 41]. Given that ALT measurement is a suboptimal marker for prediction of significant liver disease, it is recommended to have, as accurate as possible, an estimation of fibrosis and inflammation based on a reliable tool in order to decide antiviral treatment indication [42]. Nowadays, liver biopsy became rarely used in evaluation of patients with chronic hepatitis viral diseases due to the risk of the procedure, inter- and intraobservers variability, costs, as well as the availability of several noninvasive tests. All international guidelines agree that any HBV carriers who fulfill the criteria of HBV DNA have indication of antiviral treatment, whether there are evidences of significant necroinflammation and/or moderate/severe fibrosis [1, 2, 5]. Transient elastography, an imaging noninvasive test for assessing liver fibrosis, has a low accuracy in distinguished between intermediate stages of fibrosis (F1–F3). Also, the results are influenced by some con-

There is emerging evidence suggesting association between HBsAg level and liver fibrosis stage in HBeAg-positive CHB patients. It has been proposed different cutoff levels of HBsAg for prediction of liver fibrosis among HBeAg-positive patients. Thus, serum HBsAg over 100,000 IU/mL was 100% predictive of insignificant fibrosis in patients with ALT below 2×ULN [42]. In HBeAg-positive patients with ALT ≤2×ULN, an HBsAg level over 25,000 IU/mL has been proved to be the best independent predictor of insignificant liver fibrosis (PPV of 92.7%, odds ratio 9.042) [42]. Based on these results, it has been suggested that HBeAg-positive patients with ALT ≤2×ULN and HBsAg ≥ 25,000 IU/mL could be followed One of the remaining concerns in the management of CHB patients is the individual prediction of the HCC risk. There is very well known that the risk of HCC cannot be eliminated with any available therapy because of integrated sequences of viral DNA into the host genome. Even in cases of acute HBV naturally resolved infections the risk of HCC is estimated to be very low but higher compared to the general populations. The REVEAL study showed that viral replication is the major driver of disease progression and is an individual risk factor for HCC occurrence in patients with baseline HBV DNA ≥2000 IU/mL [8]. From the clinical practice point of view, it is very important to identify risk factors for HCC in an individual with CHB in order to adjust our HCC screening strategy. There are preliminary data which suggest an existing correlation between higher HBsAg level and an increased risk of HCC appearance [44]. From the clinical point of view, a particular interest would be in noncirrhotic patients with low level of HBV DNA (<2000 IU/mL) in whom the risk of HCC is difficult to be estimated. Thus, in HBeAg-negative patients with HBV DNA <2000 IU/mL an HBsAg level ≥1000 IU/mL has been has been identified as a new independent risk factor of HCC with a hazard ratio of 13.7 (95% CI: 4.8–39.3) compared to patients with HBsAg level <1000 IU/mL [44]. Moreover, HBV DNA has not been associated with HCC risk in these patients. Contrary, in HBeAg-negative patients with HBV DNA level above 2000 IU/mL, the HCC risk has not been proved to be linked to serum HBsAg levels [44]. These data support the role of HBsAg as a complementary tool by the side of HBV DNA in predicting the risk of HCC occurrence. According to the existing evidences, high risk factors for HCC related to HBV chronic infection include male gender, age over 50 years, HBV genotype B and C, pre-core and basal-core promoter HBV variants, pre-S deletion mutants, high serum of ALT, HBV DNA ≥ 2000 IU/mL and last but not least HBsAg ≥1000 IU/mL in low viremic HBeAg-negative patients [45].

## **7. Conclusions**

In summary, there have been identified several clinical benefits of using quantitative HBsAg in the management of CHB. In case of IFN-based treatment, the most important role of HBsAg measurement is attributed to the highest NPV for sustained post-treatment response. Thus, in routinely clinical practice, different early stopping rules after 12 weeks of treatment can be used, depending on the HBeAg status. In HBeAg-positive CHB patients, Peg-IFN should be stopped after 12 weeks whether HBsAg does not decline more than standard error or HBsAg level is above 20,000 IU/mL. In HBeAg-negative CHB patients, an absence of HBsAg reduction combined with a less than 2 log10 IU/mL decline of HBV DNA at week 12 of treatment should be used as another stopping rule. On the other hand, in NUCs treatment, the exact roles of the HBsAg have not been defined yet. However, one of the proposed roles of HBsAg quantification during long-term NUCs therapy is identifying those patients in whom treatment discontinuation can be safely decided. Moreover, there are robust evidences that support the role of HBsAg quantification as a useful tool for identification of true inactive HBV carriers, for distinguishing between HBeAg-positive patients with moderate to advanced fibrosis and no or mild fibrosis, as well as for predicting the risk of HCC occurrence especially in HBeAg-negative low viremic patients.

## **Author details**

Valeriu Gheorghiță¹,²\* and Florin Alexandru Căruntu¹,³

\*Address all correspondence to: gvaleriu21@yahoo.com


## **References**


[8] Chen C, Yang HI, Su J, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis b virus DNA level. JAMA. 2006;295(1):65–73.

after 12 weeks whether HBsAg does not decline more than standard error or HBsAg level is above 20,000 IU/mL. In HBeAg-negative CHB patients, an absence of HBsAg reduction combined with a less than 2 log10 IU/mL decline of HBV DNA at week 12 of treatment should be used as another stopping rule. On the other hand, in NUCs treatment, the exact roles of the HBsAg have not been defined yet. However, one of the proposed roles of HBsAg quantification during long-term NUCs therapy is identifying those patients in whom treatment discontinuation can be safely decided. Moreover, there are robust evidences that support the role of HBsAg quantification as a useful tool for identification of true inactive HBV carriers, for distinguishing between HBeAg-positive patients with moderate to advanced fibrosis and no or mild fibrosis, as well as for predicting the risk of HCC occurrence especially in HBeAg-negative low viremic patients.

**Author details**

184 Advances in Treatment of Hepatitis C and B

**References**

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1 "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania

2 "Carol Davila" Central Military Emergency University Hospital, Bucharest, Romania 3 National Institute for Infectious Diseases "Prof Dr Matei Balș," Bucharest, Romania

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186 Advances in Treatment of Hepatitis C and B


#### **Current Management Strategies in Hepatitis B During Pregnancy Current Management Strategies in Hepatitis B during Pregnancy**

Letiția Adela Maria Streba, Anca Pătrașcu, Aurelia Enescu and Costin Teodor Streba Letiția Adela Maria Streba, Anca Pătrașcu, Aurelia Enescu and Costin Teodor Streba

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66068

#### **Abstract**

Hepatitis B virus (HBV) infection remains a major health problem worldwide and a major risk factor for end-stage liver disease and hepatocellular carcinoma. Notable differences of chronic hepatitis B prevalence were observed in geographic area. In highly endemic areas, at least 50 % of HBV infections are most commonly acquired either perinatally or in early childhood, during the first 5 years of life. The prevalence of chronic HBV infection in pregnant women is expected to mirror those in the general populations of each geographic area. Chronic hepatitis B during pregnancy is associated with high risk of maternal complications and an increased risk of mother-to-child transmission (MTCT). Thus, chronic hepatitis B during pregnancy can now be considered an important contributor to new HBV infections and to the global burden of disease. As a result, HBV infection during pregnancy requires management strategies for both the mother and the fetus/neonate, including prevention/elimination of MTCT and lessening the HBV effects on maternal and fetal health. This chapter will review current management strategies for hepatitis B in the pregnancy and the postpartum period, including special considerations on the effects of pregnancy on the course of HBV infection, MTCT, and antiviral therapy during the pregnancy.

**Keywords:** hepatitis B virus, pregnancy, mother-to-child transmission, disease burden, antiviral treatment, HBV vaccination, hepatitis B immune globulin (HBIG)

## **1. Introduction**

Hepatitis B is caused by hepatitis B virus (HBV), a partially double-stranded DNA virus, member of the Hepadnaviridae family. The hepatitis B virion is a 42-nm particle composed

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of a 27-nm nucleocapsid consisting of the hepatitis B core antigen (HBcAg) surrounded by an outer lipoprotein coat envelope containing the hepatitis B surface antigen (HBsAg) [1, 2].

To date, 10 HBV genotypes (A–J) have been defined based on intergroup divergence of above 8 % in the complete nucleotide sequence and over 30 subgenotypes. The genotypes show heterogeneity in their global geographic distribution and have also been associated with different clinical features and different responses to antiviral therapy [3–6].

HBV infection remains a major health problem worldwide and a major risk factor for endstage liver disease and hepatocellular carcinoma. Two billion people worldwide have been infected with HBV, and more than 240 million people have chronic hepatitis B infection defined as HBsAg positive for more than 6 months. Despite the fact that in many countries HBV infections have declined substantially because of effective prevention strategies, more than 780,000 people die every year worldwide due to HBV complications, including cirrhosis and liver cancer [2].

HBV infection is transmitted by percutaneous and mucous membrane *via* blood or infected body fluids [7]. HBV mother-to-child transmission (MTCT), defined as HBsAg positivity at 6–12 months of life in an infant born to an infected mother, has been recognized as a major mode of transmission and at the same time the most important phase for the chronic hepatitis B prevention. In Asia, up to 50 % of new cases of HBV infection are due to MTCT [8–10]. In Europe, MTCT is the most important and frequent transmission route of HVB infection, which accounted for 41.1 % of all cases, according to the results of the first enhanced surveillance data collection of HVB infections across 30 countries of the European Union and the European Economic Area [11].

Infants born to HBsAg-positive mothers who do not become infected perinatally remain at risk of HBV infection during early childhood [12]. More than one third of patients with HBV acquired the infection during the perinatal period or in early childhood, even in low-endemic areas [13]. In highly endemic areas, at least 50 % of HBV infections are most commonly acquired either perinatally or in early childhood, during the first 5 years of life [2]. Moreover, the rate of chronicity is about 90 % for perinatally acquired HVB infection or during the first year of life, 30–50 % in infected children between ages 1 and 6 years, and 5–10 % in children over the age of 6 years and in adults [2, 14].

Thus, chronic hepatitis B during pregnancy is now an important contributor to the new HBV infections and to the global burden of disease.

## **2. Epidemiologic aspects of HBV infection in pregnant women**

Notable differences of chronic hepatitis B prevalence were observed by geographic area, with the highest endemicity levels in the sub-Saharan Africa and East Asia (5–10 %) and low prevalence (<1 %) in the United States (USA), Canada, and Western Europe. High rates of prevalence have also been found in the southern regions of Eastern and Central Europe [2, 15].

According to the technical report of the European Centre for Disease Prevention and Control (ECDC), based on literature review, the prevalence of HBsAg in the general population ranged from 0.1 to more than 7 % by country. Countries in the central or southern part of the Europe (EU) have a higher prevalence of HBV infection than countries in the northern or western part of the EU. Thus, Romania, Greece, and Turkey have a high HBV prevalence (>2 %); Italy has medium HBV prevalence (>1 and ≤2 %), while Belgium, France, Spain, Germany, the Netherlands, Slovakia, Sweden, Switzerland, and the United Kingdom have a low HBV prevalence (≤1 %). Among countries with available data, Turkey has the largest number of HBV-infected individuals (national and regional estimates ranged from 2.5 to 9.0 % in adults and 1.7 to 2.7 % in children only), followed by Romania (5.6 %) [16].

The prevalence of chronic HBV infection in pregnant women is expected to mirror those in the general populations of each geographic area. Thus, in higher endemicity areas, rates are proportionately higher [9].

In the United States, a country of low endemicity, estimated chronic HBV infection prevalence in pregnant women is 0.7–0.9 % [17]. In Europe, the chronic HBV infection prevalence in pregnant women is generally higher than in the general population (0.1–4.4 %) in countries where both estimates were available (e.g., Germany, Greece, Ireland, Italy, the Netherlands, and Slovakia), according to the ECDC study. This difference in prevalence can be attributed to the fact that migrant women, whom have a relatively high HBV infection prevalence, are better represented in pregnancy studies than in general population studies. Conversely, Spain reported in Catalonia in 2004 a lower prevalence of chronic hepatitis B in pregnant women than the prevalence in the general population in the same region in 2002 (0.7 %), attributing these aspects to the higher vaccination rate [16]. In France, the prevalence of chronic HBV infection is about 1 % in pregnant women [18]. In Denmark, country where all pregnant women have been screened for HBV since November 2005, the overall prevalence of HBV infection among pregnant women has increased from 0.11 % in 1971 to 0.26 % in 2007. In the same period, the prevalence among pregnant native Danes decreased from 0.11 to 0.01 % [19].

Available data suggest a wide variation in prevalence of chronic HBV infection among pregnant women globally. However, there are insufficient epidemiological data and limitations to estimate the epidemiology of HVB infection among pregnant women globally.

## **3. Serological markers of HBV infection**

of a 27-nm nucleocapsid consisting of the hepatitis B core antigen (HBcAg) surrounded by an outer lipoprotein coat envelope containing the hepatitis B surface antigen (HBsAg) [1, 2]. To date, 10 HBV genotypes (A–J) have been defined based on intergroup divergence of above 8 % in the complete nucleotide sequence and over 30 subgenotypes. The genotypes show heterogeneity in their global geographic distribution and have also been associated with dif-

HBV infection remains a major health problem worldwide and a major risk factor for endstage liver disease and hepatocellular carcinoma. Two billion people worldwide have been infected with HBV, and more than 240 million people have chronic hepatitis B infection defined as HBsAg positive for more than 6 months. Despite the fact that in many countries HBV infections have declined substantially because of effective prevention strategies, more than 780,000 people die every year worldwide due to HBV complications, including cirrhosis

HBV infection is transmitted by percutaneous and mucous membrane *via* blood or infected body fluids [7]. HBV mother-to-child transmission (MTCT), defined as HBsAg positivity at 6–12 months of life in an infant born to an infected mother, has been recognized as a major mode of transmission and at the same time the most important phase for the chronic hepatitis B prevention. In Asia, up to 50 % of new cases of HBV infection are due to MTCT [8–10]. In Europe, MTCT is the most important and frequent transmission route of HVB infection, which accounted for 41.1 % of all cases, according to the results of the first enhanced surveillance data collection of HVB infections across 30 countries of the European Union and the

Infants born to HBsAg-positive mothers who do not become infected perinatally remain at risk of HBV infection during early childhood [12]. More than one third of patients with HBV acquired the infection during the perinatal period or in early childhood, even in low-endemic areas [13]. In highly endemic areas, at least 50 % of HBV infections are most commonly acquired either perinatally or in early childhood, during the first 5 years of life [2]. Moreover, the rate of chronicity is about 90 % for perinatally acquired HVB infection or during the first year of life, 30–50 % in infected children between ages 1 and 6 years, and 5–10 % in children

Thus, chronic hepatitis B during pregnancy is now an important contributor to the new HBV

Notable differences of chronic hepatitis B prevalence were observed by geographic area, with the highest endemicity levels in the sub-Saharan Africa and East Asia (5–10 %) and low prevalence (<1 %) in the United States (USA), Canada, and Western Europe. High rates of prevalence have also been found in the southern regions of Eastern and Central

**2. Epidemiologic aspects of HBV infection in pregnant women**

ferent clinical features and different responses to antiviral therapy [3–6].

and liver cancer [2].

190 Advances in Treatment of Hepatitis C and B

European Economic Area [11].

Europe [2, 15].

over the age of 6 years and in adults [2, 14].

infections and to the global burden of disease.

Measurement of several HBV antigens and/or antibodies plays an important role in diagnosis, assessment, and monitoring the disease progression and its response to treatment.

There are three clinical useful antigen-antibody groups used in the serological diagnosis of HVB:

**1.** Hepatitis B surface antigen and antibody: antigen (HBsAg) and antibody to HBsAg (anti-HBs)


Additionally, the presence and concentration of circulating HBV DNA can also be tested [20–22].

HBsAg is the serological hallmark of both acute and chronic forms of HBV infection and the most commonly used diagnostic and blood screening marker for HBV infection. It usually appears in serum 1–10 weeks (average, 4 weeks) after acute exposure to the virus, and its persistence for six months or more implies progression to chronic HBV infection. The presence of HBsAg indicates that the person is infected with HBV and is therefore potentially infectious. More than 95–99 % of adults with acute HBV infection will recover spontaneously, without antiviral therapy [23, 24].

In patients that recover completely from their HBV infection, HBsAg usually becomes undetectable after four to six months, and its disappearance is followed several weeks later by the appearance of anti-HBs. Therefore, there is a gap ("window period") of several weeks to months between the disappearance of HBsAg and the appearance of anti-HBs, and during this period, the detectable marker of HBV infection is anti-HBc. The persistence of anti-HBs for a lifetime provides long-term immunity against HBV. Therefore, the presence of anti-HBs in serum attests to previous HBV exposure and acquired immunity. In some patients, anti-HBs may not become detectable after disappearance of HBsAg. These patients do not appear to be susceptible to recurrent infection [20, 23, 24].

Total anti-HBc (IgM and IgG) appears before anti-HBs, and its presence in serum attests both past exposure and current HBV infection. Its presence during the "window period" makes it a reliable indicator of HBV infection, in the absence of other HBV markers [25].

IgM anti-HBc develops in acute HBV infection and may usually persist for four to six months if the infection resolves [20, 22]. Although it is considered a reliable serologic marker for acute infection, IgM anti-HBc can also become positive during a chronic hepatitis B flare in patients who have long-standing hepatitis B [26, 27].

A negative IgM anti-HBc in conjunction with a positive HBsAg likely suggests a chronic HBV infection. As a result, routine testing for IgM anti-HBc is not generally recommended to screen for acutely infected patients [28, 29].

IgG anti-HBc develops in the late acute phase of infection and generally remains detectable for lifetime [20]. IgG anti-HBc may be the only serologic marker remaining in patient serum who recover from acute HBV infection. The presence of IgG anti-HBc can indicate progression to chronic disease [22].

HBeAg is a viral soluble protein that develops in the serum of persons with acute or chronic HBV infection. HBeAg appears in serum early during acute HBV infection and usually disappears about three weeks before HBsAg disappears. Persistence of HBeAg three or more months after the onset of illness indicates a carrier state and the risk of developing chronic HVB. The HBeAg presence in the serum of HBV carriers and chronic hepatitis B patients indicates greater infectivity and a high level of viral replication [20, 30].

**2.** Core antigen and antibodies: antigen (HBcAg does not appear in the blood) and antibody to HBcAg (anti-HBc), IgM antibody subclass of anti-HBc (IgM anti-HBc), and IgG antibody

Additionally, the presence and concentration of circulating HBV DNA can also be tested

HBsAg is the serological hallmark of both acute and chronic forms of HBV infection and the most commonly used diagnostic and blood screening marker for HBV infection. It usually appears in serum 1–10 weeks (average, 4 weeks) after acute exposure to the virus, and its persistence for six months or more implies progression to chronic HBV infection. The presence of HBsAg indicates that the person is infected with HBV and is therefore potentially infectious. More than 95–99 % of adults with acute HBV infection will recover spontaneously, without

In patients that recover completely from their HBV infection, HBsAg usually becomes undetectable after four to six months, and its disappearance is followed several weeks later by the appearance of anti-HBs. Therefore, there is a gap ("window period") of several weeks to months between the disappearance of HBsAg and the appearance of anti-HBs, and during this period, the detectable marker of HBV infection is anti-HBc. The persistence of anti-HBs for a lifetime provides long-term immunity against HBV. Therefore, the presence of anti-HBs in serum attests to previous HBV exposure and acquired immunity. In some patients, anti-HBs may not become detectable after disappearance of HBsAg. These patients do not appear

Total anti-HBc (IgM and IgG) appears before anti-HBs, and its presence in serum attests both past exposure and current HBV infection. Its presence during the "window period" makes it

IgM anti-HBc develops in acute HBV infection and may usually persist for four to six months if the infection resolves [20, 22]. Although it is considered a reliable serologic marker for acute infection, IgM anti-HBc can also become positive during a chronic hepatitis B flare in patients

A negative IgM anti-HBc in conjunction with a positive HBsAg likely suggests a chronic HBV infection. As a result, routine testing for IgM anti-HBc is not generally recommended to

IgG anti-HBc develops in the late acute phase of infection and generally remains detectable for lifetime [20]. IgG anti-HBc may be the only serologic marker remaining in patient serum who recover from acute HBV infection. The presence of IgG anti-HBc can indicate progression

HBeAg is a viral soluble protein that develops in the serum of persons with acute or chronic HBV infection. HBeAg appears in serum early during acute HBV infection and usually disappears about three weeks before HBsAg disappears. Persistence of HBeAg three or more months after the onset of illness indicates a carrier state and the risk of developing chronic

a reliable indicator of HBV infection, in the absence of other HBV markers [25].

**3.** Hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe)

subclass of anti-HBc (IgG anti-HBc)

192 Advances in Treatment of Hepatitis C and B

to be susceptible to recurrent infection [20, 23, 24].

who have long-standing hepatitis B [26, 27].

screen for acutely infected patients [28, 29].

to chronic disease [22].

[20–22].

antiviral therapy [23, 24].

The small-size soluble HBeAg can cross the placental barrier from the mother to the fetus especially through villous capillary endothelial cells. The maternal HBeAg-positive serological status and high serum HBV DNA levels increase the risk of MTCT. By contrast, the absence of the HBeAg in serum is associated with lower levels of viral replication and with a significantly lower risk of intrauterine HBV transmission. The infants born to HBeAg-positive mothers have up to 90 % chance of acquiring perinatal HBV without prophylaxis [13, 14, 31, 32].

Anti-HBe appears in the resolution phase of the disease, when HBeAg disappears. Its presence correlates to a decreased infectivity. A seroconversion of HBeAg to anti-HBe marks a transition to the inactive carrier state in the majority of cases [20].

Spontaneous or treatment-induced HBeAg seroconversion is associated with lower rates of disease progression [33].

In addition to viral antigens and antibodies detected or measured, serum HBV DNA can also be measured both qualitatively and quantitatively (HBV viral load). HBV DNA is the most sensitive and specific marker of viral replication [29].

Serologic pattern of acute HBV infection is characterized by the transient presence of HBsAg (<6 months) and IgM anti-HBc. HBeAg and HBV DNA are also present during the initial phase of infection. The disappearance of HBV DNA, HBeAg to anti-HBe seroconversion, and loss of HBsAg or HBsAg to anti-HBs seroconversion designate recovery. The presence of IgG anti-HBc in the absence of HBsAg usually indicates a past HBV infection, while the presence of anti-HBs only reveals immunity to HBV infection after vaccination [20, 22, 25].

Three standard tests (HBsAg, anti-HBs, and anti-HBc) are usually indicated to determine if a person is currently infected with HBV, has recovered from HBV infection, or is susceptible to HBV infection [20].

Combinations of serologic HBV markers are used to identify different phases of HBV infection (**Table 1**).



**Table 1.** Most common serological profiles of HBV infection [20, 22, 28].

## **4. Mechanisms and predictors for MTCT of HBV infection**

Perinatal transmission of hepatitis B is highest in mothers with acute hepatitis, especially in HBe-positive mothers in the third trimester (50–80 %), lower in mothers with anti-HBe (25 %), and lowest in carriers (5 %) [34].

The World Health Organization (WHO) defines "perinatal" as the time period starting at 22 completed weeks (154 days) gestation and ending seven complete days after birth [35]. However, the perinatal period is defined in various ways, and depending on the definition, it starts at the 20th–28th week of gestation and ends 1–4 weeks after birth [36]. The term MTCT is entitled and covers the transmission of all HBV infections from mother to her child during pregnancy (intrauterine transmission), childbirth, or after birth. As a result, there are three main possible routes for MTCT of HBV infection: transplacental transmission of HBV, transmission during delivery, and postnatal transmission during child care and breastfeeding [37].

Intrauterine transmission of HBV is considered the most important cause for the failure of passive-active immunoprophylaxis in preventing MTCT, although it is presumed to cause a minority of HBV infections [38]. The main risk factors for intrauterine HBV infection are maternal serum HBeAg positivity, high HBV DNA level, history of threatened preterm labor, and HBV presence in the villous capillary endothelial cells of the placenta. One of the proposed mechanisms involved in the HBV intrauterine transmission is the transplacental leakage of HBeAg-positive maternal blood induced by uterine contractions during pregnancy and by the disruption of placental barriers. In addition, HBeAg can pass through the placenta via the "cellular route." Although the risk of fetal hepatitis B infection through amniocentesis is considered to be low, the maternal HBeAg status would be valuable in the counseling regarding risks associated with amniocentesis. Another possible route of HBV intrauterine transmission could be via germ cells, maternally or paternally dependent [14, 37, 39].

HBV transmission during delivery is recognized as the most important route of MTCT in endemic areas for HBV infection, as a result of exposure to maternal cervical secretions and maternal blood that contain HBV. There is no consensus regarding the effect of delivery mode on MTCT (vaginal delivery vs. cesarean section). While some studies suggest that cesarean section might reduce the risk of MTCT, other studies assert that the mode of delivery does not influence the rate of HBV transmission as long as all infants received both hepatitis B vaccine and hepatitis B immune globulin (HBIG) at birth [37].

There is little evidence that cesarean delivery prevents HBV transmission, and current guidelines do not recommend cesarean section to decrease the risk of MTCT. As for elective cesarean section (ECS), there are studies that show alike an absolute risk reduction of MTCT of HBV compared with immunoprophylaxis alone and studies that report no benefit to ECS. According to recent clinical guidelines of American College of Gastroenterology (ACG) concerning liver disease and pregnancy, validation studies are needed to determine the relative safety and efficacy of ECS and immunoprophylaxis versus immunoprophylaxis alone in reducing MTCT of HVB [40].

Although markers of HBV are detectable in breast milk from HBsAg-positive women, there is no evidence that breastfeeding is a risk factor for HBV infection if the infant received hepatitis B vaccine and HBIG. According to the WHO and the American Academy of Pediatrics recommendations, in infants who receive full immunoprophylaxis, breastfeeding in HBs-positive mothers is not a contraindication [9, 41, 42].

## **5. Clinical and laboratory features of HBV infection in pregnancy**

**4. Mechanisms and predictors for MTCT of HBV infection**

**Serological markers Results Interpretation**

HBsAg Positive Acute HBV infection

HBsAg Positive Chronic HBV infection

Total anti-HBc Positive Resolved infection

HBsAg Negative Interpretation of isolated detection of

Anti-HBsAg Negative Window period of acute HBV (anti-

anti-HBc

HBc-predominantly IgM) False-positive test results "Low level" chronic infection

Anti-HBsAg Positive

194 Advances in Treatment of Hepatitis C and B

Total anti-HBc Positive IgM anti-HBc Positive Anti-HBsAg Negative

Total anti-HBc Positive IgM anti-HBc Negative Anti-HBsAg Negative

**Table 1.** Most common serological profiles of HBV infection [20, 22, 28].

and lowest in carriers (5 %) [34].

Perinatal transmission of hepatitis B is highest in mothers with acute hepatitis, especially in HBe-positive mothers in the third trimester (50–80 %), lower in mothers with anti-HBe (25 %),

The World Health Organization (WHO) defines "perinatal" as the time period starting at 22 completed weeks (154 days) gestation and ending seven complete days after birth [35]. However, the perinatal period is defined in various ways, and depending on the definition, it starts at the 20th–28th week of gestation and ends 1–4 weeks after birth [36]. The term MTCT is entitled and covers the transmission of all HBV infections from mother to her child during pregnancy (intrauterine transmission), childbirth, or after birth. As a result, there are three main possible routes for MTCT of HBV infection: transplacental transmission of HBV, transmission during delivery, and postnatal transmission during child care and breastfeeding [37]. Intrauterine transmission of HBV is considered the most important cause for the failure of passive-active immunoprophylaxis in preventing MTCT, although it is presumed to cause a minority of HBV infections [38]. The main risk factors for intrauterine HBV infection are maternal serum HBeAg positivity, high HBV DNA level, history of threatened preterm labor, and HBV presence in the villous capillary endothelial cells of the placenta. One of the proposed mechanisms involved in the HBV intrauterine transmission is the transplacental leakage of

The clinical manifestations of HBV infection may be variable in both acute and chronic diseases. In acute HBV infection, clinical manifestations usually range from anicteric hepatitis to icteric hepatitis, while in the chronic phase, manifestations range from an asymptomatic carrier state to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Fulminant hepatic failure, most probably due to massive immune-mediated lysis of infected hepatocytes, is unusual but can occur in some cases. Extrahepatic manifestations may be present in both acute and chronic infections [25, 40, 42].

Testing for HBsAg should be performed in all women at the first prenatal visit, even if they have been previously vaccinated or tested, and repeated later in pregnancy if appropriate [25, 43].

The first step in assessing a woman presenting at any stage of pregnancy with acute or chronic HBV infection should be the same as with any nonpregnant patient: complete history, physical


**Table 2.** Phases of chronic hepatitis B [44–46].

exam, standard serological workup, laboratory test which should include assessment of liver disease activity and function, markers of HBV replication, and tests for coinfection with hepatitis C virus [8, 16, 40, 43, 44].

The clinical spectrum of acute HBV infection in pregnant women usually is not different from that of nonpregnant women; however, the risk of preterm delivery and low birth weight is higher than in the general population [9, 14, 42]. It seems that acute HBV infection does not increase mortality or have teratogenic effects [9].

Common symptoms of acute HBV infection in pregnant women are indistinguishable from those of nonpregnant, including upper quadrant discomfort, fatigue, nausea, vomiting, diarrhea, headaches, myalgia, anorexia, low-grade fever, and jaundice. The icteric phase of acute viral hepatitis usually begins within 10 days of the initial symptoms and disappears about 4–12 weeks afterwards. Diagnosis is based on the detection of HBsAg and the presence of IgM anti-HBc. Recovery is accompanied by HBsAg clearance with seroconversion to anti-HBs, usually within 3 months. Concentrations of alanine and aspartate aminotransferase (ALT and AST) levels usually increase, with ALT typically higher than AST. In patients who recover, normalization of serum aminotransferases usually occurs within one to four months [20, 25, 42, 45].

Acute exacerbation or flare of hepatitis in chronic HBV infections can be present during pregnancy, and it may be difficult to differentiate from acute HBV infection. HBV testing with HBsAg and IgM anti-HBc is recommended in pregnant women presenting with acute hepatitis [40].

Most chronic HBV infections are asymptomatic and pregnancy is well tolerated. Some patients may complain of fatigue, anorexia, and nonspecific malaise. Significant symptoms will develop only if the liver disease progresses. Cirrhosis, condition usually associated with amenorrhea and infertility, is relatively uncommon in the younger age group of pregnant women, and severe cases are fortunately rare [9, 42, 45]. The chronic hepatitis B is usually mild in pregnant women but may flare at the end of pregnancy or shortly after delivery [9].

The natural history of chronic HBV infection consists of several phases of variable duration, which are not necessarily sequential (**Table 2**) [44–46]. Pregnancy is a hormone-induced immune-tolerant state, and there is limited understanding of the natural history of chronic HBV infection during pregnancy [47]. Increased levels of adrenal corticosteroids and estrogen hormones during pregnancy may be responsible for an increase in HBV viral load and a decrease in ALT levels. A postpartum decline in HBV DNA level, associated with increased ALT levels and active hepatitis, requires close monitoring of the mother [9, 42].

## **6. Current management strategies for chronic hepatitis B in pregnancy**

exam, standard serological workup, laboratory test which should include assessment of liver disease activity and function, markers of HBV replication, and tests for coinfection with hepa-

**Phase ALT HBV DNA HBeAg Notes**

"Immune tolerant" Normal Elevated Positive Perinatal or early

Elevated Elevated Positive Moderate-to-severe

Normal Low or undetectable Negative Low risk for cirrhosis

Elevated Elevated Negative Spontaneously or

Moderate to elevated Negative Generally in older

childhood-acquired HBV infection Patients are highly contagious Low spontaneous HBeAg loss Minimal liver inflammation and

liver inflammation or fibrosis HBeAg to anti-HBe seroconversion possible, leading to "immune-control"

fibrosis

phase

Minimal liver necroinflammation, variable fibrosis

persons Liver necroinflammation Risk for fibrosis or cirrhosis

precipitated by immunosuppressive

therapy, transplantation, antiviral resistance, HIV infection, withdrawal of antiviral therapy Moderateto-severe liver necroinflammation and

fibrosis

The clinical spectrum of acute HBV infection in pregnant women usually is not different from that of nonpregnant women; however, the risk of preterm delivery and low birth weight is higher than in the general population [9, 14, 42]. It seems that acute HBV infection does not

Common symptoms of acute HBV infection in pregnant women are indistinguishable from those of nonpregnant, including upper quadrant discomfort, fatigue, nausea, vomiting,

titis C virus [8, 16, 40, 43, 44].

**Table 2.** Phases of chronic hepatitis B [44–46].

HBeAg-positive immune-active phase "Immune active"

196 Advances in Treatment of Hepatitis C and B

Inactive chronic hepatitis "Immune

HBeAg-negative chronic hepatitis "Immune escape mutant"

"Reactivation" or "acute-on-chronic hepatitis" or HBeAgnegative immune reactivation phase

control"

increase mortality or have teratogenic effects [9].

Elevated persistent or intermittently

> HBV infection during pregnancy requires management strategies for both mother and fetus/ neonate, including prevention/elimination of MTCT and lessening the HBV effects on maternal and fetal health [48, 49].

> Current management strategies for hepatitis B during pregnancy include antenatal maternal screening for HBV infection, initial assessment of mother with HBV infection (severity of liver disease, level of viral replication, presence of comorbidities), prophylactic HBV vaccination and HBIG administration to all infants born to HBV-infected mothers as soon as possible after birth, the use of antiviral medications for pregnant women with chronic hepatitis B, safe delivery practices, and strengthened maternal and child health services [8, 40, 45, 50].

> Few countries have national hepatitis strategies, plans, and budgets, and as a consequence, the WHO recently published a 5-year global health sector strategy on viral hepatitis. This

includes testing algorithms, strategies for hepatitis B, diagnosis and management of acute hepatitis B, as well as management of advanced liver disease [8, 45].

Antenatal screening for HBV infection in all pregnant women is a well-established, evidencebased standard of practice to prevent MTCT. Therefore, the first step is to identify all HBsAgpositive pregnant women in the first trimester by universal screening [45].

All pregnant women who are HBsAg positive should be assessed the same way as any nonpregnant individual: a complete history with special emphasis on risk factors for coinfection, physical exam and laboratory tests for assessment of liver disease activity and function, markers of HBV replication, and tests for coinfection (hepatitis C virus, hepatitis delta virus, or human immunodeficiency virus in those at risk) [24, 44, 48, 50].

Assessment of the severity of liver disease should include measurement of ALT, AST, alkaline phosphatase (ALP), gamma-glutamyl transpeptidase, total bilirubin, full blood count, serum albumin and globulins, prothrombin time, and an ultrasound examination. Assessment of the level of viral replication in chronic hepatitis B using quantification of serum HBV DNA and HBeAg and anti-HBe is an important step in determining the risk of MTCT and therefore in guiding antiviral therapy decisions and the need for surveillance [24, 44, 48, 50]. Elevated serum ALT and HBV DNA levels are strongly predictive of risk of liver complications [44].

According to the WHO Strategic Advisory Group of Experts, the currently recommended practice to reduce perinatal MTCT of HBV relies on the administration of HBV vaccine and, in some countries, concurrent administration of HBIG. The infants of all HBsAg-positive women should receive immunoprophylaxis with HBV vaccination ± HBIG. Hepatitis B vaccine and HBIG should be administered at different injection sites [45].

The timing of administration of the first dose of hepatitis B vaccine to infants in relation to birth is the most important factor in determining the efficacy of vaccination [41, 51]. As a result, the recommended timing of administration of the first dose of hepatitis B vaccine in newborns has evolved in the last decades, in order to optimize prevention of MTCT hepatitis B infections. The WHO recommends that all infants receive the hepatitis B vaccine as soon as possible after birth, within 24 h of the birth [2].

Passive immunization against hepatitis B with HBIG in conjunction with HBV vaccination may be of additional benefit for newborn whose mothers are HBsAg positive, particularly if they are also HBeAg positive [45]. According to the Centers for Disease Control, all preterm infants born to HBsAg-positive mothers and mothers with unknown HBsAg status must receive HBIG and hepatitis B vaccine within 12 h of birth [52].

Unfortunately, despite postnatal active-passive immunization of the newborns, MTCT of HBV still occurs, especially if the mother has very a high maternal concentration of HBV DNA, typically observed in HBeAg-positive women [45].

There are emerging data based on open-label nonrandomized studies which suggest that short-term maternal antiviral therapy used in pregnant women with stable liver disease during the third trimester may reduce the risk of MTCT occurring during the perinatal period, by lowering maternal viral load prior to delivery [24, 47].

Current guidelines of the American Association for the Study of Liver Diseases (AASLD), ACG, European Association for the Study of the Liver (EASL), and Asian Pacific Association for the Study of the Liver (APASL) suggest or recommend antiviral therapy to reduce the risk of perinatal transmission of hepatitis B in HBsAg-positive pregnant women with a HBV DNA above 200,000 IU/mL [24, 44, 48, 50]. As for the WHO current position, the Guidelines Development Group did not make a formal recommendation on the use of antiviral therapy to prevent MTCT, due to the fact that key trials are still ongoing and there is a lack of consensus regarding the programmatic implications of a policy of more widespread antiviral use in pregnancy [45].

includes testing algorithms, strategies for hepatitis B, diagnosis and management of acute

Antenatal screening for HBV infection in all pregnant women is a well-established, evidencebased standard of practice to prevent MTCT. Therefore, the first step is to identify all HBsAg-

All pregnant women who are HBsAg positive should be assessed the same way as any nonpregnant individual: a complete history with special emphasis on risk factors for coinfection, physical exam and laboratory tests for assessment of liver disease activity and function, markers of HBV replication, and tests for coinfection (hepatitis C virus, hepatitis delta virus, or

Assessment of the severity of liver disease should include measurement of ALT, AST, alkaline phosphatase (ALP), gamma-glutamyl transpeptidase, total bilirubin, full blood count, serum albumin and globulins, prothrombin time, and an ultrasound examination. Assessment of the level of viral replication in chronic hepatitis B using quantification of serum HBV DNA and HBeAg and anti-HBe is an important step in determining the risk of MTCT and therefore in guiding antiviral therapy decisions and the need for surveillance [24, 44, 48, 50]. Elevated serum ALT and HBV DNA levels are strongly predictive of risk of liver complications [44].

According to the WHO Strategic Advisory Group of Experts, the currently recommended practice to reduce perinatal MTCT of HBV relies on the administration of HBV vaccine and, in some countries, concurrent administration of HBIG. The infants of all HBsAg-positive women should receive immunoprophylaxis with HBV vaccination ± HBIG. Hepatitis B vaccine and

The timing of administration of the first dose of hepatitis B vaccine to infants in relation to birth is the most important factor in determining the efficacy of vaccination [41, 51]. As a result, the recommended timing of administration of the first dose of hepatitis B vaccine in newborns has evolved in the last decades, in order to optimize prevention of MTCT hepatitis B infections. The WHO recommends that all infants receive the hepatitis B vaccine as soon as

Passive immunization against hepatitis B with HBIG in conjunction with HBV vaccination may be of additional benefit for newborn whose mothers are HBsAg positive, particularly if they are also HBeAg positive [45]. According to the Centers for Disease Control, all preterm infants born to HBsAg-positive mothers and mothers with unknown HBsAg status must

Unfortunately, despite postnatal active-passive immunization of the newborns, MTCT of HBV still occurs, especially if the mother has very a high maternal concentration of HBV

There are emerging data based on open-label nonrandomized studies which suggest that short-term maternal antiviral therapy used in pregnant women with stable liver disease during the third trimester may reduce the risk of MTCT occurring during the perinatal period, by

hepatitis B, as well as management of advanced liver disease [8, 45].

198 Advances in Treatment of Hepatitis C and B

human immunodeficiency virus in those at risk) [24, 44, 48, 50].

HBIG should be administered at different injection sites [45].

receive HBIG and hepatitis B vaccine within 12 h of birth [52].

DNA, typically observed in HBeAg-positive women [45].

lowering maternal viral load prior to delivery [24, 47].

possible after birth, within 24 h of the birth [2].

positive pregnant women in the first trimester by universal screening [45].

There are only three therapeutic antiviral agents studied and used for the treatment of chronic hepatitis B in pregnant women: lamivudine, telbivudine (nucleoside analogues (NAs)), and tenofovir disoproxil fumarate (nucleotide analogue). According to the US Food and Drug Administration classification of oral antiviral, based on the risk of teratogenicity in preclinical evaluation, only two drugs from the nucleoside/nucleotide analogues (NAs) class—tenofovir and telbivudine—are classified in risk category B (no risk in animal studies, but unknown in humans), while lamivudine, entecavir, and adefovir dipivoxil are classified as category C drugs (teratogenic in animals, but unknown in humans) [24, 44]. Additionally, tenofovir received category B classification based on data collected from human exposure [53].

Lamivudine, the first and the most studied NAs in pregnant women with chronic hepatitis B, is not considered an optimal choice for prevention of MTCT due to its poor antiviral activity and low barrier to resistance. Its administration, even for short periods, is associated with the selection of resistant mutants. Lamivudine reaches higher concentrations in amniotic fluid than in serum and has been found to be excreted in breast milk [49, 54, 55].

The results of small human pregnancy trials show that telbivudine reduces MTCT in highly viremic pregnant women and its use appears to be safe in late pregnancy [47].

Tenofovir is considered a preferred choice in pregnant women with chronic hepatitis B, due to its antiviral potency, the available safety data of use during pregnancy, and its better resistance profile [44, 45].

As for other antiviral drugs, the safety of entecavir in pregnancy is not known, and interferon (IFN) therapy is contraindicated during pregnancy [44, 45].

Antiviral therapy was started at 28–32 weeks of gestation in most studies, and therefore NAs starting from 28 to 32 weeks of gestation are recommended [24, 45]. A careful examination to exclude maternal systemic disorder and fetal anomalies is required prior to the administration of NAs [44, 50]. For pregnant women with immune-active chronic hepatitis B, monitoring therapeutic response to NAs, both serological and virological, as well as for potential side effects, should be based on recommendations for nonpregnant women [24, 44, 45]. Tenofovir therapy requires monitoring serum creatinine and serum phosphate levels every three months, due to potential nephrotoxicity. The risks of maternal liver disease, fetal development, HBV MTCT, and long-term plan for treatment should be discussed with pregnant women [24, 50].

Although there are no studies on the duration of NA therapy (cessation at delivery vs. after delivery), cessation of NA therapy (at delivery or 4–12 weeks after delivery) is recommended in females without ALT flares [24, 44, 45]. According to EASL guidelines, if NA therapy is given only for prevention of MTCT, it may be discontinued within the first 3 months after delivery [50]. If the anti-HBV therapy is discontinued during pregnancy or early after delivery, women need to be closely monitored for the risk of hepatic flares, especially after delivery [44, 50].

In certain situations, such as ALT flares detected during the treatment period, continuation of antiviral treatment after delivery is needed. As a result, this raises the issue of safety of NA therapy during breastfeeding. Due to limited data on the effect of these medications on infants, the safety of NA therapy during breastfeeding is considered uncertain [24, 50].

The safety of lamivudine and tenofovir during breastfeeding in HBV infection has not been well studied. Additionally, tenofovir and lamivudine concentrations in breast milk have been reported. However, due to its poor oral bioavailability, the breastfeeding infants are exposed to only small tenofovir concentrations [50].

According to drug labels, tenofovir disoproxil fumarate and lamivudine should not be used during breastfeeding. Breastfeeding is discouraged during maternal NA treatment according to APASL current guidelines, but in the case of ALT flares, continuation of antiviral may be indicated, depending on the liver disease status of mother [24]. A recent review of available data concluded that tenofovir and lamivudine should not be contraindicated during breastfeeding. However, there are insufficient data based on long-term studies to establish the safety of infant exposure to different antiviral therapies during breastfeeding [56].

## **7. Conclusions**

Despite advancements in the prevention, diagnosis, and treatment of HBV infection, it remains a serious global health issue and one of major risk factors for end-stage liver disease and hepatocellular carcinoma. Given that chronic hepatitis B in pregnant women is an important contributor worldwide to the new HBV infections, most effective and sustainable measures are required for prevention of MTCT. Universal screening of pregnant women for HBsAg and passive and active immunoprophylaxis are important tools in MTCT of HBV. The causes of immunoprophylaxis failure in some infants are not yet not fully understood, and, therefore, studies are needed in order to clarify this issue. Longitudinal cohort studies are also required to determine the safety of infant exposure to different NA therapies during breastfeeding.

## **Author details**

Letiția Adela Maria Streba, Anca Pătrașcu, Aurelia Enescu and Costin Teodor Streba\*

\*Address all correspondence to:costinstreba@gmail.com

University of Medicine and Pharmacy of Craiova, Romania

## **References**

Although there are no studies on the duration of NA therapy (cessation at delivery vs. after delivery), cessation of NA therapy (at delivery or 4–12 weeks after delivery) is recommended in females without ALT flares [24, 44, 45]. According to EASL guidelines, if NA therapy is given only for prevention of MTCT, it may be discontinued within the first 3 months after delivery [50]. If the anti-HBV therapy is discontinued during pregnancy or early after delivery, women need to be closely monitored for the risk of hepatic flares, especially after delivery [44, 50].

In certain situations, such as ALT flares detected during the treatment period, continuation of antiviral treatment after delivery is needed. As a result, this raises the issue of safety of NA therapy during breastfeeding. Due to limited data on the effect of these medications on infants, the safety of NA therapy during breastfeeding is considered uncertain [24, 50].

The safety of lamivudine and tenofovir during breastfeeding in HBV infection has not been well studied. Additionally, tenofovir and lamivudine concentrations in breast milk have been reported. However, due to its poor oral bioavailability, the breastfeeding infants are exposed

According to drug labels, tenofovir disoproxil fumarate and lamivudine should not be used during breastfeeding. Breastfeeding is discouraged during maternal NA treatment according to APASL current guidelines, but in the case of ALT flares, continuation of antiviral may be indicated, depending on the liver disease status of mother [24]. A recent review of available data concluded that tenofovir and lamivudine should not be contraindicated during breastfeeding. However, there are insufficient data based on long-term studies to establish the

Despite advancements in the prevention, diagnosis, and treatment of HBV infection, it remains a serious global health issue and one of major risk factors for end-stage liver disease and hepatocellular carcinoma. Given that chronic hepatitis B in pregnant women is an important contributor worldwide to the new HBV infections, most effective and sustainable measures are required for prevention of MTCT. Universal screening of pregnant women for HBsAg and passive and active immunoprophylaxis are important tools in MTCT of HBV. The causes of immunoprophylaxis failure in some infants are not yet not fully understood, and, therefore, studies are needed in order to clarify this issue. Longitudinal cohort studies are also required to determine the safety of infant exposure to different NA therapies during breastfeeding.

Letiția Adela Maria Streba, Anca Pătrașcu, Aurelia Enescu and Costin Teodor Streba\*

\*Address all correspondence to:costinstreba@gmail.com

University of Medicine and Pharmacy of Craiova, Romania

safety of infant exposure to different antiviral therapies during breastfeeding [56].

to only small tenofovir concentrations [50].

200 Advances in Treatment of Hepatitis C and B

**7. Conclusions**

**Author details**


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[17] Dionne-Odom J, Tita AT, Silverman NS. #38: Hepatitis B in pregnancy screening, treatment, and prevention of vertical transmission. Am J Obstet Gynecol 2016;214(1):6–14.

[18] Fouquet A, Jambon AC, Canva V, Bocket-Mouton L, Gottrand F, Subtil D. Hepatitis B and pregnancy. Part 1. Thirteen practical issues in antenatal period. J Gynecol Obstet

[19] Hansen N, Hay G, Cowan S, Jepsen P, Bygum Krarup H, Obel N, Weis N, Brehm Christensen P. Hepatitis B prevalence in Denmark – an estimate based on nationwide registers and a national screening programme, as on 31 December 2007. Euro Surveill 2013;18(47):pii=20637. DOI: http://dx.doi.org/10.2807/1560-7917.ES2013.18.47.20637 [20] World Health Organization. Hepatitis B. 2016. Available from: http://www.who.int/csr/ disease/hepatitis/HepatitisB\_whocdscsrlyo2002\_2.pdf?ua=1 [Accessed June 17]

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[22] Centers for Disease Control and Prevention. Recommendations for Identification and Public Health Management of Persons with Chronic Hepatitis B Virus Infection. MMWR 2008;57(RR-8):3. Available at: http://www.cdc.gov/mmwr/PDF/rr/rr5708.pdf [Accessed

[23] Chen Y-P, Qiao Y-Y, Zhao X-H, Chen H-S, Wang Y, Wang Z. Rapid detection of hepatitis B virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis B virus surface antigen. Clin Vaccine

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[25] Petersen J. Hepatitis B. In Mauss S, Berg T, Rockstroh J, Sarrazin C, Wedemeyer H. Hepatology: A clinical textbook. 7th ed., Fokus Verlag, Hamburg, 2016, pp. 145–155. [26] Maruyama T, Schodel F, Iino S, Koike K, Yasuda K, Peterson D, Milich DR. Distinguishing between acute and symptomatic chronic hepatitis B virus infection. Gastroenterology

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#### **Treatment and Prognosis of Hepatitis B Virus Concomitant with Alcoholism Treatment and Prognosis of Hepatitis B Virus Concomitant with Alcoholism Treatment and Prognosis of Hepatitis B Virus Concomitant with Alcoholism**

Chih-Wen Lin, Chih-Che Lin and Sien-Sing Yang Chih-Wen Lin, Chih-Che Lin and Sien-Sing Yang Yang

Additional information is available at the end of the chapter Additional information is available at the end of the chapterAdditional information is available at the end of the chapter

Chih-Wen Lin, Chih-Che Lin and Sien-Sing

http://dx.doi.org/10.5772/66981

#### **Abstract**

[44] Terrault NA, Bzowej NH, Chang KM, Hwang JP, Jonas MM, Murad MH. AASLD guide-

[45] World Health Organization, Guidelines for the Prevention, Care and Treatment of Persons with Chronic Hepatitis B Infection, WHO, 2015, http://apps.who.int/iris/bitstr

[46] World Gastroenterology Organisation Global Guideline. Hepatitis B 2015. Available from: http://www.spg.pt/wp-content/uploads/2015/11/2015-hepatitis-b.pdf [Accessed

[47] Pan CQ, Lee HM. Antiviral therapy for chronic hepatitis B in pregnancy. Semin Liver

[48] Visvanathan K, Dusheiko G, Giles M, et al. Managing HBV in pregnancy. Prevention, prophylaxis, treatment and follow-up: position paper produced by Australian, UK and

[49] Degli Esposti S, Shah D. Hepatitis B in pregnancy: challenges and treatment. Gastroenterol

[50] European Association for the Study of the Liver. EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. J Hepatol 2012;57(1):167–185.

[51] André FE, Zuckerman AJ. Review: protective efficacy of hepatitis B vaccines in neonates.

[52] Centers for Disease Control and Prevention. Hepatitis B in the Pink Book: Course Textbook. 13th ed. 2015. Available from: http://www.cdc.gov/vaccines/pubs/pinkbook/

[53] Guclu E, Karabay O. Choice of drugs in the treatment of chronic hepatitis B in pregnancy. World J Gastroenterol 2013;19(10):1671–1672. DOI: 10.3748/wjg.v19.i10.1671. [54] Han L, Zhang H-W, Xie J-X, Zhang Q, Wang H-Y, Cao G-W. A meta-analysis of lamivudine for interruption of mother-to-child transmission of hepatitis B virus. World J

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[56] Ehrhardt S, Xie C, Guo N, Nelson K, Thio CL. Breastfeeding while taking lamivudine or tenofovir disoproxil fumarate: a review of the evidence. Clin Infect Dis 2015;60:275–278.

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downloads/hepb.pdf

Hepatitis B virus (HBV) infection is a global disease worldwide. The Asia-Pacific region has a high prevalence of viral hepatitis, and Taiwan is a region of high prevalence of chronic hepatitis B (CHB) with increasing alcoholic liver disease. We have investigated the prognosis and treatment of patients with concomitant hepatitis B virus (HBV) infection and alcoholism. The 10-year cumulative incidence of hepatocellular carcinoma (HCC) is much higher in patients with concomitant alcoholism and HBV infection than in those with alcoholism or HBV infection alone. Treatment with antiviral therapy and abstinence may be started in patients with decompensated cirrhosis and compensated cirrhosis with high HBV DNA. In pre-cirrhotic cases, treatment with antiviral therapy and abstinence may be started in patients with persistently elevated ALT levels and high HBV DNA, and significant fibrosis with minimal elevated or normal ALT levels and mild high HBV DNA. Treatment with antiviral therapy and abstinence reduces the incidence of HCC in patients with concomitant HBV infection and alcoholism. In conclusion, patients with concomitant HBV infection and alcoholism have high incidence of cirrhosis, HCC, and mortality. Treatment with antiviral therapy and abstinence may be started to reduce the incidence of cirrhosis, HCC, and mortality in these patients.

**Keywords:** chronic hepatitis B, hepatitis B virus DNA, nucleos(t)ides analogues, alcoholism, hepatocellular carcinoma, treatment, prognosis

## **1. Introduction**

Hepatitis B virus (HBV) infection is a global disease, affecting approximately 350 million people worldwide [1]. The Asia-Pacific region has a high prevalence of viral hepatitis, and Taiwan is a region with high prevalence of chronic hepatitis B (CHB) [2]. It is particularly

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

endemic in Taiwan, where the infection is usually acquired perinatally or in early childhood [2]. The morbidity and mortality associated with CHB are substantial in that 15% to approximately 40% of infected patients will develop serious sequels including persistent hepatitis, hepatic failure, liver cirrhosis and hepatocellular carcinoma (HCC) during their lifetime [2].

Alcohol-related morbidity and mortality represent a major public health issue worldwide [3, 4]. The United States National Institute on Alcohol Abuse and Alcoholism defines "heavy drinking" as consuming more than fourteen drinks per week for males and seven drinks per week for females. The risk threshold for developing alcohol-related liver disease is consuming 20–30 g of alcohol per day, and the development of cirrhosis occurs in 10–20% of those consuming more than 80 g of alcohol daily [3]. The Asia-Pacific region has a high prevalence of viral hepatitis, and Taiwan is a region of high prevalence of chronic hepatitis B (CHB) with increasing alcoholic liver disease [5–7]. The affordability of alcohol and changes in life style and drinking behavior have the causes for the increase in cases of hospitalization for alcoholic liver disease [6].

In the animal model system, mice fed with ethanol have an increased serum hepatitis B surface antigen (HBsAg) by up to seven folds accompanied by an increased in viral DNA load [8]. In addition, these ethanol-fed mice have elevated expression of HBV surface, core, and X antigens in the liver, accompanied by an increase in HBV RNA levels. Chronic ethanol consumption is found to stimulate hepatitis B virus replication and gene expression in HBV transgenic mice [8]. Our recent study also reveals that patients with concomitant alcoholism and HBV infection have high percentages of hepatitis B viral load in clinics [9]. Moreover, the lipid composition of cellular membranes in lipid rafts is altered by alcohol exposure, and alcohol exposure may thereby influences HBV infectivity [10]. Furthermore, alcohol can influence anti-HBV immunity, an effect involving the cellular membrane as well as the lipid rafts. HBV is known to interfere with the T-cell receptor (TCR) responsible for interacting and recognizing foreign antigens, thereby preventing the initiation of an immune response. This results in a defective adaptive immune response during chronic HBV infection [8, 11]. Thus, alcohol can acts synergistically with HBV to limit antiviral immunity. Since the adaptive immunity plays a key role in viral clearance, the consequences of alcohol's effects on the TCR of HBV infection are of high interest in the field of hepatology [12].

## **2. Epidemiology**

HBV infection is a serious global health problem, with 2 billion people infected worldwide and 350 million suffering from chronic HBV infection. HBV infections result in 0.5–1.2 million deaths per year caused by chronic hepatitis, cirrhosis, and HCC. HBV-related end-stage liver disease or HCC is responsible for over 0.5–1 million deaths per year and currently represents 5–10% of cases of liver transplantation. Morbidity and mortality in CHB are linked to persistence of viral replication and evolution to cirrhosis and/or HCC [1, 2].

In Taiwan, the introduction of universal vaccination of neonates in 1983–1985 has drastically decreased the prevalence of HBsAg in children below the age of 15 from 9.8 % in 1984 to 0.5 % in 2004 [13]. This is accompanied by a significant decrease in the incidence of infant fulminant hepatitis associated with chronic liver disease, mortality, and HCC [14, 15].

Alcohol is abused by more than 18 million adults in the United States. A daily consumption of alcohol exceeding 80 g for more than 10 years increases the risk for HCC by fivefold, while daily consumption of alcohol below 80 g is not significantly associated with an increased risk for HCC [3, 4]. The risk for HCC in decompensated alcoholic cirrhosis is close to 1% per year [3, 4]. Alcohol consumption is one of the top five causes of disease and disability in almost all European countries [16]. In the United States, about 50% of liver-related death is attributed by alcohol consumption, accounting for \$3 billion annually loss, and is the third leading cause of preventable deaths in the U.S. [17]. It is estimated that alcohol is responsible for 5.9% of global mortality worldwide [18] and 2.5 million deaths per annual [19, 20].

## **3. Prognosis**

endemic in Taiwan, where the infection is usually acquired perinatally or in early childhood [2]. The morbidity and mortality associated with CHB are substantial in that 15% to approximately 40% of infected patients will develop serious sequels including persistent hepatitis, hepatic failure, liver cirrhosis and hepatocellular carcinoma (HCC) during their

Alcohol-related morbidity and mortality represent a major public health issue worldwide [3, 4]. The United States National Institute on Alcohol Abuse and Alcoholism defines "heavy drinking" as consuming more than fourteen drinks per week for males and seven drinks per week for females. The risk threshold for developing alcohol-related liver disease is consuming 20–30 g of alcohol per day, and the development of cirrhosis occurs in 10–20% of those consuming more than 80 g of alcohol daily [3]. The Asia-Pacific region has a high prevalence of viral hepatitis, and Taiwan is a region of high prevalence of chronic hepatitis B (CHB) with increasing alcoholic liver disease [5–7]. The affordability of alcohol and changes in life style and drinking behavior have the causes for the increase in cases of hospitalization for alcoholic

In the animal model system, mice fed with ethanol have an increased serum hepatitis B surface antigen (HBsAg) by up to seven folds accompanied by an increased in viral DNA load [8]. In addition, these ethanol-fed mice have elevated expression of HBV surface, core, and X antigens in the liver, accompanied by an increase in HBV RNA levels. Chronic ethanol consumption is found to stimulate hepatitis B virus replication and gene expression in HBV transgenic mice [8]. Our recent study also reveals that patients with concomitant alcoholism and HBV infection have high percentages of hepatitis B viral load in clinics [9]. Moreover, the lipid composition of cellular membranes in lipid rafts is altered by alcohol exposure, and alcohol exposure may thereby influences HBV infectivity [10]. Furthermore, alcohol can influence anti-HBV immunity, an effect involving the cellular membrane as well as the lipid rafts. HBV is known to interfere with the T-cell receptor (TCR) responsible for interacting and recognizing foreign antigens, thereby preventing the initiation of an immune response. This results in a defective adaptive immune response during chronic HBV infection [8, 11]. Thus, alcohol can acts synergistically with HBV to limit antiviral immunity. Since the adaptive immunity plays a key role in viral clearance, the consequences of alcohol's effects on the TCR of HBV infection are of high interest in the field of

HBV infection is a serious global health problem, with 2 billion people infected worldwide and 350 million suffering from chronic HBV infection. HBV infections result in 0.5–1.2 million deaths per year caused by chronic hepatitis, cirrhosis, and HCC. HBV-related end-stage liver disease or HCC is responsible for over 0.5–1 million deaths per year and currently represents 5–10% of cases of liver transplantation. Morbidity and mortality in CHB are linked to persis-

tence of viral replication and evolution to cirrhosis and/or HCC [1, 2].

lifetime [2].

206 Advances in Treatment of Hepatitis C and B

liver disease [6].

hepatology [12].

**2. Epidemiology**

Based on a large nationwide Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer-Hepatitis B Virus (REVEAL-HBV) study performed in Taiwan for CHB without alcoholism, detectable serum HBV DNA at study entry is demonstrated to be a significant risk predictor of HCC in HBV patients [21–23]. Those with detectable HBsAg are at 5- to 98-fold higher risk of developing HCC [24]. The seropositivity for HBeAg is also found associated with an increase in risk for HCC [25]. Compared to those who are seronegative for HBsAg and HBeAg, the hazard ratio (HR) of developing HCC is about 10 and 60, respectively, for individuals with seropositivity for HBsAg and both HBsAg and HBeAg [25, 26]. The serum level of HBV DNA is therefore a strong risk predictor of HCC [21], and it is also an important and independent risk factor for disease progression prognosis (including cirrhosis, risk of death, metastasis, and recurrence following surgery) in chronic hepatitis B [22]. Alcohol has a synergistic effect in increasing the risk of HCC incidence in HBsAgpositive men [27].

In one of our study, 966 cirrhotic patients in Taiwan, consisting of 632 patients with HBV infection, 132 patients with HBV infection and alcoholism, and 202 patients with alcoholism, are evaluated for HCC development [6]. We show that 15.8, 28.8 and 10.4% of the patients with HBV infection alone, concomitant HBV infection and alcoholism, and alcoholism alone, respectively, are found to have newly developed HCC after a period of 10 years of follow-up. The 1-, 3-, 5-, and 10-year cumulative incidence of HCC is 1.2, 9.4, 18.4, and 39.8%, respectively, for patients with HBV infection alone; 3.1, 28.7, 36.8, and 52.8%, respectively, for patients with concomitant HBV infection and alcoholism; and 1.1, 6.1, 10.7, and 25.6%, respectively, for patients with alcoholism alone (**Figure 1**). The 10-year cumulative incidence of HCC is much higher in patients with concomitant alcoholism and HBV infection than in those with alcoholism alone or HBV infection alone (52.8% vs. 25.6% vs. 39.8%, p < 0.001). The mean

**Figure 1.** Cirrhotic patients with concomitant HBV infection and alcoholism have higher cumulative incidence of HCC than those with alcoholism alone or HVB infection alone.

follow-up period is 2.9, 5.2, and 3.9 years for patients with concomitant HBV infection and alcoholism, alcoholism alone, and HBV infection alone, respectively. The annual incidence of HCC is 9.9, 2.1, and 4.1%, respectively, for patients with concomitant HBV infection and alcoholism, alcoholism alone, and HBV infection alone. Our findings reveal that heavy alcohol consumption significantly increases the risk of developing HCC in HBV-related cirrhotic patients [6].

The baseline serum HBV DNA level, antiviral nucleos(t)ide analogues [NA(s)] therapy, serum α-fetoprotein, daily amount of alcohol intake, and years of alcohol intake are also found to be significantly associated with the incidence of HCC by univariate analyses. In multivariate logistic regression analyses, antiviral NUCs therapy (OR = 0.01) and baseline high serum HBV DNA levels (OR = 16.8) are significantly linked to a reduction in the incidence of HCC. In addition, the cumulative incidence of HCC during the follow-up period is significantly higher in patients with higher baseline serum HBV DNA levels than those with lower baseline serum HBV DNA levels. Alcoholic cirrhotic patients with higher serum HBV DNA levels have higher incidence of HCC than those with lower serum HBV DNA levels, and increasing HBV DNA levels precipitates the progression of liver cirrhosis to HCC [6].

In another case-control and hospital-based study conducted in Italy, the relative risks of HCC for HBsAg and heavy alcohol intake are 11.4 and 4.6, respectively [28]. Positive synergisms between HBsAg positivity and heavy alcohol intake are reported, suggesting a stronger additive effect of viral infections and alcohol drinking on the risk of HCC. On the basis of population attributable risks (AR), heavy alcohol intake seems to be the single most relevant cause of HCC in this area (AR: 45%) followed by HBV (AR: 22%) infection [28]. Similarly, another study by Sagnelli and colleagues has demonstrates that alcohol abuse can increase the risk of hepatitis B infection progressing to liver cirrhosis by threefold [29].

Furthermore, in another hospital-based, case-control study carried out in USA, the ORs for HCC based on multivariate analysis are 12.6, 4.5, and 4.3, respectively, for patients with HBsAg, heavy alcohol consumption (daily consumption of more than 80 mL of alcohol), and diabetes mellitus. Based on the additive model, synergistic interactions are observed between heavy alcohol consumption and diabetes mellitus (OR, 9.9) and chronic hepatitis virus infection (OR, 53.9). The significant synergy observed between heavy alcohol consumption, hepatitis virus infection, and diabetes mellitus may suggest the presence of a common pathway for hepatocarcinogenesis [30].

In another Taiwanese men prospective and community-based study carried out in the REVEAL-HBV study cohort over a period of 14 years, 20% of the patients are reported to be alcohol users [27]. Based on analyses adjusted for multivariable, alcohol abuse and extreme obesity (BMI ≥30 kg/m<sup>2</sup> ) have synergistic effects on the risk of incident HCC (HR, 3.40). Obesity and alcohol are also reported to have synergistic effects in increasing risk of incident HCC in HBsAg-positive men [27]. It is therefore concluded that lifestyle interventions might significantly reduce the incidence of HCC [27].

## **4. Treatment in patients with concomitant HBV infection and alcoholism**

follow-up period is 2.9, 5.2, and 3.9 years for patients with concomitant HBV infection and alcoholism, alcoholism alone, and HBV infection alone, respectively. The annual incidence of HCC is 9.9, 2.1, and 4.1%, respectively, for patients with concomitant HBV infection and alcoholism, alcoholism alone, and HBV infection alone. Our findings reveal that heavy alcohol consumption significantly increases the risk of developing HCC in HBV-related

**Figure 1.** Cirrhotic patients with concomitant HBV infection and alcoholism have higher cumulative incidence of HCC

The baseline serum HBV DNA level, antiviral nucleos(t)ide analogues [NA(s)] therapy, serum α-fetoprotein, daily amount of alcohol intake, and years of alcohol intake are also found to be significantly associated with the incidence of HCC by univariate analyses. In multivariate logistic regression analyses, antiviral NUCs therapy (OR = 0.01) and baseline high serum HBV DNA levels (OR = 16.8) are significantly linked to a reduction in the incidence of HCC. In addition, the cumulative incidence of HCC during the follow-up period is significantly higher in patients with higher baseline serum HBV DNA levels than those with lower baseline serum HBV DNA levels. Alcoholic cirrhotic patients with higher serum HBV DNA levels have higher incidence of HCC than those with lower serum HBV DNA levels, and increasing

HBV DNA levels precipitates the progression of liver cirrhosis to HCC [6].

cirrhotic patients [6].

than those with alcoholism alone or HVB infection alone.

208 Advances in Treatment of Hepatitis C and B

Antiviral therapies including lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, and Peg-interferon have been widely prescribed for the treatment of HBV-related liver diseases worldwide [14, 31, 32]. Several large population-based and international studies have reveal that antiviral therapy could reduce the incidence of hepatic failure, cirrhosis, HCC, and mortality in CHB patients without alcoholism [33–40].

In patients with concomitant HBV infection and alcoholism, the prescription of both antiviral therapy and abstinence is important for the treatment of disease progression. Oral NA(s) can reduce the disease progression for HBV infection-induced liver diseases. Abstinence is one of the most important therapies for patients with alcohol-induced liver diseases [41]. In addition, abstinence has been shown to improve the histological features of hepatic injury and reduce the outcome of disease progression to cirrhosis, HCC, and mortality in patients with alcoholic liver diseases [5, 6, 41–45].

The indications of treatment for patients with concomitant HBV infection and alcoholism are based on three criteria: severity of liver disease, serum HBV DNA levels, and serum ALT



**Table 1.** Treatment indications for patients with concomitant HBV infection and alcoholism.

**Alcoholic patients with HBsAg positive**

210 Advances in Treatment of Hepatitis C and B

Severe reactivation of chronic HBV

Non-cirrhotic HBeAgpositive chronic hepatitis B

Non-cirrhotic HBeAgnegative chronic hepatitis B **HBV DNA (IU/mL) ALT Treatment**

Detectable Elevated Treat with NA(s) or Peg-

>20,000 >2× ULN Observation for 3 months.

2000–20,000 Any ALT Monitor every 3 months.

<2000 <ULN Monitor every 3 months

Undetectable Any ALT Treat with abstinence

>2000 >2× ULN Observation for 3 months.

abstinence

immediately

1–2× ULN Monitor every 3 months

Persistently normal Monitor every 3 months

>ULN Monitor every 3 months

1–2× ULN Monitor every 3 months

interferon and abstinence

Treat with NA(s) or Peginterferon and abstinence

Treat with NA(s) or Peginterferon and abstinence if noninvasive tests suggest significant fibrosis

Treat with NA(s) or Peginterferon and abstinence if noninvasive tests suggest significant fibrosis

Assess fibrosis noninvasively. Monitor every 3 months Treat with NA(s) and abstinence if noninvasive tests suggest evidence of significant fibrosis.

Treat with NA(s) or Peginterferon and abstinence if noninvasive tests suggest significant fibrosis

Treat with NA(s) or Peginterferon and abstinence if noninvasive tests suggest significant fibrosis

Treat with NA(s) or Peginterferon and abstinence

Treat with NA(s) or Peginterferon and abstinence if noninvasive tests suggest significant fibrosis

Decompensated cirrhosis Detectable Any Treat with NA(s) and

Compensated cirrhosis >2000 Any Treat NA(s) and abstinence

levels [14]. The treatment in patients with concomitant HBV infection and alcoholism is summarized in **Table 1**.

## **4.1. In cirrhotic patients or patient with severe HBV reactivation with concomitant HBV infection and alcoholism**


## **4.2. In pre-cirrhotic patients with concomitant HBV infection and alcoholism**


Treatment with antiviral therapy [NA(s) or Peg-interferon] and abstinence may be started. Antiviral therapy and abstinence prevent further progression of fibrosis and other complications of liver disease.


Our previous study shows that oral antiviral therapy significantly reduces the incidence of HCC in alcoholic cirrhotic patients with concomitant HBV infection (**Figure 2**) [6]. Therefore, aggressive NA(s) therapy should be considered in patients with alcoholic cirrhosis and detectable serum HBV DNA, in order to reduce the incidence of HCC [6].

**Figure 2.** The cumulative incidence of HCC in cirrhotic patients with concomitant alcoholism and HBV infection is significantly reduced in patients receiving oral antiviral therapy.

## **5. Conclusion**

Treatment with antiviral therapy [NA(s) or Peg-interferon] and abstinence may be started. Antiviral therapy and abstinence prevent further progression of fibrosis and

**3.** Patients have persistently elevated, minimally elevated, or normal ALT levels or HBV DNA <20,000 IU/mL if HBeAg positive and <2000 IU/mL if HBeAg negative, and a noninvasive method shows the presence of a significant fibrosis. Treatment with antiviral therapy [NA(s) or Peg-interferon] and abstinence may be started. NA(s) and abstinence prevent further progression of fibrosis and other complications of liver

**4.** Patients have normal or elevated ALT levels and undetectable HBV DNA. Treatment with abstinence may be started. Abstinence prevents further progression of fibrosis and

Our previous study shows that oral antiviral therapy significantly reduces the incidence of HCC in alcoholic cirrhotic patients with concomitant HBV infection (**Figure 2**) [6]. Therefore, aggressive NA(s) therapy should be considered in patients with alcoholic cirrhosis and detect-

**Figure 2.** The cumulative incidence of HCC in cirrhotic patients with concomitant alcoholism and HBV infection is

other complications of liver disease.

212 Advances in Treatment of Hepatitis C and B

other complications of liver disease.

significantly reduced in patients receiving oral antiviral therapy.

able serum HBV DNA, in order to reduce the incidence of HCC [6].

disease.

Patients with concomitant HBV infection and alcoholism have high incidence of cirrhosis, HCC, and mortality. Treatment with antiviral therapy and abstinence may be started with the aim to reduce the incidence of cirrhosis, HCC, and mortality in patients with concomitant HBV infection and alcoholism.

## **Acknowledgements**

We thank Jen-Chien Chen, Chia-Chang Hsu, and Kah Wee Koh for collection of the data.

## **Author details**

Chih-Wen Lin1,2,3, \*, Chih-Che Lin<sup>4</sup> and Sien-Sing Yang<sup>5</sup>

\*Address all correspondence to: lincw66@gmail.com

1 School of Medicine, College of Medicine, I-Shou University, Kaohsiung, Taiwan

2 Division of Gastroenterology and Hepatology, Department of Medicine, E-DA Dachang Hospital, I-Shou University, Kaohsiung, Taiwan

3 Department of Health Examination, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan

4 Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan

5 Liver Unit, Cathay General Hospital and Fu-Jen Catholic University, Taipei, Taiwan

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