HCV therapeutics: Times are changing

Infectious Disease News February 2012

by Robert T Schooley, MD

There are few areas in medicine in which the therapeutic landscape is poised to change more rapidly than in that for the therapy of hepatitis C infection. The 20 years after the introduction of interferon-alpha therapy witnessed only two advances in hepatitis C therapy.

First, ribavirin, an agent that had previously been pitched at more viruses than are included in most medical school curricula, was found to reduce the rate of relapse after a course of interferon-alpha therapy. Secondly, conjugation of interferon-alpha to polyethylene glycol was demonstrated to increase the chance of treatment and the tolerability of interferon therapy.

When these two modifications to classical interferon-alpha therapy were combined, a patient with HCV genotype-1 could look forward to a difficult year that resulted in a cure less than half of the time. In this situation, it would be expected that most patents reluctantly accepted therapy when they (and their physicians) came to the conclusion that therapy was required as a last effort to avoid end-stage liver disease or hepatocellular carcinoma.

In these circumstances, patients usually avoided therapy until their liver disease resulted in referral to a hepatologist who generally viewed their problem as a liver rather than a viral disease. We are now entering a new era in which therapeutic advances for HCV will completely change the treatment paradigm. This will be accompanied by much better outcomes for patients and will require a major expansion of the treating community in which HIV physicians will play a much greater role. These shifts will be driven by three major factors.

Success rates will increase

The addition of either of the newly approved HCV protease inhibitors — boceprevir (Victrelis, Schering) or telaprevir (Incivek, Vertex Pharmaceuticals) — to polyethylene glycol-interferon and ribavirin therapy has already increased the prospect of treatment success to about 70% in genotype-1 infection.

With the increased potency of three-agent antiviral regimens, treatment duration has decreased from 1 year to 6 months in most patients, and early assessment of the antiviral response can provide patients with prognostic information that is reliable as to whether a course of therapy is likely to be successful. Taken together, patients can expect treatment to be successful more frequently, and they will likely be inconvenienced for only half as long as before.

Furthermore, clinical trials with newer agents provide strong evidence that treatment success rates are poised to go higher and that courses of therapy will be shorter for many patients — and perhaps, most importantly, will ultimately include neither interferon nor ribavirin. These factors have already increased interest in testing for and treatment of HCV infection.

Initiation not driven by liver biopsy findings

As current therapies are limited in appeal, most patients seek therapy (and most physicians recommend therapy) only when liver disease has progressed to the point that cirrhosis is at hand or established. Although noninvasive approaches are emerging that serum markers or ultrasound are reasonably good at identifying patients with very little liver damage or substantial cirrhosis, liver biopsy is generally needed to provide sufficiently precise information in the middle spectrum of HCV-induced liver disease to make good therapeutic recommendations in most patients.

In addition, if therapy is delayed too long and decompensated cirrhosis has developed, interferon therapy can lead to liver failure and death. These factors have generally drawn hepatologists into the dialogue about whether therapy is indicated and usually keep them involved after therapy is started, if it is started later in the course of HCV-induced liver disease.

Decisions more complicated

Although art and science were involved in deciding when treatment should be recommended and when a patient should go through a course of polyethylene glycol-interferon/ribavirin therapy, the therapeutic landscape was reasonably straightforward once therapy was recommended.

Ribavirin was included in both regimens, and the two interferons differed from each other only in nuance. Thus, the treatment decision was a dichotomous one, and once therapy was started, side effects (though they were often substantial) were well delineated and predictable. In many treatment settings, a physician (usually a hepatologist) gathered the data and made recommendations about whether treatment was recommended, and care was actually delivered by physicians and nurse practitioners who were able to follow relatively simple toxicity management algorithms and manage most serious side effects with dose reduction and referrals to mental health workers for management of depression.

More than 20 directly acting anti-HCV agents are currently in various phases of development. These drugs have different molecular targets, resistance profiles, metabolic profiles, drug interactions and treatment durations — and it is clear that every regimen will require two to four of these new agents. As it becomes easier to decide who to treat, it is likely that more complex decisions will be required once therapy is started.

Decisions, not unlike those that the HIV-treating community has confronted for years, will be brought into the process, and “laminated-card driven” decision-making of the polyethylene glycol-interferon/ribavirin era will be a thing of the past. Decisions about overlapping resistance pathways and about interactions among anti-HCV agents (and between HCV agents and other drugs that patients must take) will be required. Because decisions about treatment duration and futility will be driven by close monitoring of antiviral response, multiple decisions will be required throughout the course of therapy. Although it is clear that these sorts of decisions can be made by hepatologists if they are heavily engaged in HCV therapy, it is likely that the hepatology community will see a further division into a subset of hepatologists who take care of hepatitis most of the time and a much larger subset who focus on transplantation and/or procedures.

Care models in new HCV therapy era

At present, there are approximately 4 million HCV-infected people in the United States, and it is estimated that fewer than 2,000 physicians are responsible for more than 80% of the currently administered antiviral therapy. As the number of those interested in HCV therapy increases and as the threshold to recommend therapy increasingly shifts to earlier stages of liver disease, current HCV treatment communities will become increasingly overrun. With a likely constriction in the number of hepatologists who are actively engaged in HCV therapy, it is clear that physicians from other disciplines will be drawn into HCV therapy.

Due to the complexity of HCV therapy, it will neither be good for patients nor an efficient use of resources to have thousands of generalists provide care to a few patients each. When HIV-1 emerged in the 1980s, AIDS “specialists” initially came from multiple training backgrounds, including internal medicine, infectious diseases, oncology, dermatology and family medicine.

As the field matured, most de novo training eventually became centered on infectious disease training programs. Most of the “primary” care for HIV (and many of us would also argue the best care) is now provided by multidisciplinary teams that are usually coordinated by physicians with infectious disease specialty training. These multidisciplinary teams optimally include those with complementary specialized expertise in the complications of the disease, including oncologists, hepatologists, psychiatrists, neurologists and clinical pharmacologists.

This shift occurred over time and with little explicit coordination or pre-cognition, but it has proved to be an extremely efficient model of care in which outcomes are better than in settings in which HIV care is provided by those with limited experience and expertise.

Rather than to stumble into an analogous model for HCV therapy during the next decade, it is attractive to think that the HIV experience could provide yet another window on the best approach to HCV care. Optimal HCV care will require interactive multidisciplinary teams with specialized expertise. Developing centers of excellence in HCV care should be a priority for the infectious disease community that should occur in anticipation of, rather than in response to, radically changing treatment paradigms.

These centers of excellence should also include hepatologists interested in viral hepatitis in their direction and operation and will need to include translational virologists, liver transplant surgeons, psychiatrists, radiologists with expertise in noninvasive imaging of the liver and clinical pharmacologists. Based on our experience in the medical and operational aspects of HIV infection, the infectious disease community must play a major role in the initial development of these centers, and most of the leaders in this exciting area of medicine should emerge from our training programs.

We’ve applied a lot of what we learned about RNA viruses from antiretroviral drug development to HCV drug discovery. Although it has been a bit slower, some of the lessons of the benefits of closely aligning clinical and translational science in drug development have been introduced into HCV drug development. There is no reason not to apply what we learned about optimal HIV care models to the very similar multidisciplinary landscape of HCV care.

Tremendous benefits will accrue to patients, and what could be more appealing to those of us in infectious diseases than a viral disease that we can actually eradicate — all that remains is to develop a billing code for “virectomy.”

Robert T. Schooley, MD, professor and head in the division of infectious diseases, and vice-chair of the department of medicine at the University of California, San Diego. Disclosure: Dr. Schooley is a member of scientific advisory boards for Gilead Sciences, GlobeImmune, Inhibitex, Johnson & Johnson, Monogram Biosciences, and Santaris. He has consulted for Merck. Research support is provided by Boehringer Ingelheim and Bristol-Myers Squibb.

Source

Is HCV Infection a Neurologic Disorder?

Download the PDF here

Gastroenterology March 2012
CYRILLE FERAY
Service d'HEpatogastroentErologie
Hotel-Dieu Hospital
Nantes, France

Hepatitis: Brain endothelial cells support HCV entry and replication - (01/27/12)

HCV SVR Improves Quality of Life & Brain Function - (01/27/12)

from Jules of NATAP: this topic of does HCV have a neurological effect & cause neurological symptoms has been controversial with many clinicians over the years doubting that this is true and with conflicting research, for example in HIV researchers have reported from studies not finding HCV caused cognitive impairment in HIV+ coinfected individuals. In the recent year or so there have been a number of research papers just like this finding that HCV enters the brain & can cause damage. There was research years ago from Pegasys studies showing improvement in neurological symptoms in patients achieving SVR. It is clear to me this is true. After achieving an SVR about 10 yrs ago I realized how much fatigue & cognitive impairment I had previously but before achieving the SVR I had no idea how much fatigue & cognitive impairment I had & I certainly did not appreciate it could be associated with HCV. After finishing HCV therapy & achieving an SVR the I started to feel improved energy & improved cognitive abilities. And over the next several years I experienced ongoing continuing further improvements. So there is no doubt that HCV can be associated neurological impairment and that it can improve. Now I had cirrhosis before achieving SVR so having a more severe stage of HCV disease may be more associated with experiencing cognitive impairment but I think earlier HCV disease can also be associated with fatigue, depression and cognitive impairment.

Hepatitis C is known to induce chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The absence of symptoms is common even when the disease has reached the stage of cirrhosis or hepatocellular carcinoma. For this reason, infection with hepatitis C virus (HCV) is frequently called a "silent killer." Fatigue is the most frequent complaint in infected subjects. HCV infection has also been associated with cognitive dysfunction and depression, which are not correlated with the severity of liver disease and cannot be explained by hepatic encephalopathy or drug abuse.

Numerous extrahepatic disorders have been attributed to or are more frequent during HCV infection. The most common mechanism underlying these disorders is autoimmunity. Thyroiditis, arthropathies, lymphocytic sialadenitis with or without sicca syndrome, and diabetes have long been known to be more frequent with HCV.1, 2 The second most frequent disorder associated with HCV infection is type II or III cryoglobulinemia. Cryoglobulins contain immune complexes made of HCV virions and anti-HCV antibodies and are highly prevalent in infected subjects. This can lead to symptomatic vasculitis with purpura, arthralgias, and asthenia, as well as peripheral nervous system and kidney involvement. Cryoglobulins are considered to be the result of B-cell proliferation owing to chronic antigenic stimulation, and B-cell lymphomas have been reported to be slightly more common in this population.3 A direct role of HCV is suggested by the fact that antiviral therapies have an antitumoral effect.4 The third group of extrahepatic symptoms associated with HCV infection could be a direct consequence of HCV infection of components of the central nervous system. Indeed, HCV RNA has been detected at autopsy in brain tissues.5, 6, 7, 8 Recently, an elegant study using microdissection in brain tissues from HCV-positive patients obtained at autopsy combined with measurement of cytokine mRNA in cells that were positive for the HCV nonstructural 3 protein suggested that brain macrophages/microglia cells were activated in HCV-infected patients.

The purpose of the paper by Fletcher et al,9 published in this issue of Gastroenterology, was to explore the original hypothesis that HCV could infect and alter the function of blood-brain barrier (BBB) endothelial cells. The authors first demonstrated that all of the known viral receptor molecules (CD81, claudin-1, occludin, LDLR, and scavenger receptor-B1) are expressed at the surface of BBB endothelial cells. It is important to note that scavenger receptor-BI expression was restricted to the microvascular endothelium, whereas other receptors were expressed by astrocytes. In a second step, the authors convincingly showed that HCV replicates in 2 distinct cell lines derived from these BBB endothelial cells. Hepatitis C is known to infect nonhepatocyte cells. There are numerous reports in the literature describing the presence of HCV RNA in immune cells10 as well as in the brain.11 In immune cells (mainly B-cells)12 and in the central nervous system,13, 14 the detection of viral sequences different from those found in the blood or the liver illustrates the compartmentalization of viral quasispecies and supports the idea that replication takes place in these extrahepatic sites. Indeed, in the absence of local replication, the presence of HCV RNA would be because of the adsorption or internalization of circulating viral particles produced in the liver; thus, no phylogenetic differences would exist with majority variants present in the serum (unless selection of liver-generated viral variants takes place at the cell receptor level). In almost all studies, the level of extrahepatic replication of HCV has been reported to be low, so that the contribution of these sites to circulating virions is limited. The HCV genome is positively stranded, meaning that it is directly translated into the viral polyprotein in the cell cytoplasm. The detection of negatively stranded HCV RNA in tissues or cells demonstrates that viral proteins have been translated and the HCV replication complex is functional, at least for the synthesis of negatively stranded HCV RNA. This intermediate of replication has frequently been detected in extrahepatic sites. There are, however, numerous technical issues that challenge the specificity of such detection. The detection of viral proteins in tissues or cells is also difficult because of their low level of expression. However, their presence has been convincingly reported recently in lymph nodes from the liver pedicle15 and in the brain.16

The most important information in the article by Fletcher et al9 is that human cell lines derived from BBB endothelial cells can be infected by HCV. The authors first tested HCV binding using HCV pseudoparticles (HCVpp) formed by the incorporation of the envelope glycoproteins E1 and E2 into lentiviral core particles. HCVpp closely mimic the functionality of wild-type viruses during the early steps of the viral lifecycle. HCVpp binding to endothelial cells was quantitatively similar to that observed with hepatocyte cell lines. The authors then used the JFH1 strain, the only HCV strain that effectively infects and replicates in primary human hepatocytes and in the hepatocyte cell line Huh7.5.17 Not surprisingly, HCV replicated at much lower levels in endothelial cells than in hepatocytes. Definitive confirmation of viral replication in endothelial cells was provided by using specific HCV protease inhibitors, which decreased the amount of HCV RNA in these cells. Micro-RNA (miR)-122 is hepatocyte specific and its fixation at 2 sites in the 5' untranslated region of the virus genome is required for efficient HCV replication. As expected, miR-122 was not detected in endothelial cell lines. The transfection of these cells to express functionally active miR-122 RNA duplexes failed to promote HCV replication, demonstrating that replication is miR-122 independent in endothelial cells. Indirectly, this result also suggested that liver-specific factors are required for the action of miR-122 on HCV replication in hepatocytes.

Another important group of findings in this paper was the observation of functional consequences of HCV infection of endothelial cells. Indeed, HCV increased endothelial cell permeability and this effect was reversed when replication was inhibited by antiviral molecules. Neutralization of HCV infection with pooled anti-HCV immunoglobulins also restored endothelial cell permeability, demonstrating the direct effect of HCV on this parameter. Furthermore, the authors noted that HCV-infected endothelial cell lines expressing nonstructural protein NS5A were TUNEL positive, suggesting an effect of infection on brain endothelial cell apoptosis. Altogether, these findings suggest that the infection of endothelial cells by HCV and HCV replication in these cells in vivo are highly plausible.

These findings raise the important question of the impact of HCV replication in BBB endothelial cells on neurocognitive and psychological symptoms frequently reported by subjects infected by this virus. This issue has been evaluated in case-control and in longitudinal studies assessing patients before and after a sustained virologic response to antiviral therapy. In these works, neurologic involvement was evaluated by means of neurocognitive tests combined with quality-of-life questionnaires or by measuring magnetic resonance spectroscopy (MRS) signals. Such case-control studies raise the issue of matching patients with HCV infection who know their status and have past or present addictive behaviors and HCV-negative controls. Despite these limitations, most studies reported more fatigue, more depression, and less effectiveness in HCV-infected patients than in controls. The longitudinal approach also has drawbacks. Subjects know the results of therapy and have experienced the adverse effects of antiviral drugs for many months. Although numerous studies have reported an improvement of the quality of life after achieving a sustained virologic response,18 only a few recent papers investigated changes in neurocognitive functions and MRS in relation to the response to therapy. The results of these studies are conflicting. In 1 study, the authors concluded that HCV eradication had a beneficial effect on cerebral metabolism and selective aspects of neurocognitive functions, especially in patients with mild disease.19 In contrast, another comparable study concluded that HCV had a measurable effect on brain integrity in patients screened for other medical and/or psychiatric comorbidities, but that these abnormalities did not improve after viral eradication.20 Both studies were based on a very small number of patients and the evaluation of neurocognitive functions and MRS were performed early after viral clearance. The second study20 questioned the reversibility of brain involvement once HCV infection is cured. Indeed, because brain cells have a very low rate of regeneration, the effect of HCV infection could be prolonged by months or years after the virus has been cured.

The main interest of the article by Fletcher et al9 is to show that the brain is a likely target for HCV. More important, the results with BBB endothelial cells suggest that other endothelial cells could be targeted and altered by HCV. For example, a study in an Egyptian population showed higher carotid intimal media thickness in HCV-infected than in noninfected patients.21 If this is confirmed, the possibility that HCV infection could induce brain or vascular disorders could modify the current indications for therapy, which are essentially based on the severity of liver disease or the presence of extrahepatic manifestations of immune origin, such as symptomatic cryoglobulinemia or B-cell lymphoma. Further studies are thus needed in the field of HCV neuroinvasion to better define the actual consequences of infection and their reversibility if infection is eradicated. However, it must be emphasized that the effects of HCV on neurocognitive functions, depression, or fatigue are generally mild. Many HCV-infected patients are highly successful and creative. Because stigmatization of HCV infected people exists, the notion of a brain involvement in these patients could have devastating consequences. The results of the present study should therefore be interpreted as what they are, certainly not overinterpreted as the demonstration that HCV infection leads to severe brain disease (Figure 1).

Source

TMC435-TiDP16-C212 - Trial of TMC435 in Genotype 1 Hepatitis C and Human Immunodeficiency Virus Co-Infected Patients

Provided by NATAP

This study is currently recruiting participants.

Verified February 2012 by Tibotec Pharmaceuticals, Ireland

Estimated Enrollment: 100

Drug: TMC435

150 mg capsule once daily for 12 weeks in addition to peginterferon alfa-2a and ribavirin for 24 or 48 weeks

First Received on October 18, 2011. Last Updated on February 27, 2012

Recruiting TMC435-TiDP16-C212 - Trial of TMC435 in Genotype 1 Hepatitis C and Human Immunodeficiency Virus Co-Infected Patients

This is an open-label (all people know the identity of the intervention) study of TMC435 in patients who are infected with genotype 1 hepatitis C virus and co-infected with human immunodeficiency virus (HIV). Patients in this study will also receive two other drugs for their hepatitis C infection called peginterferon alfa-2a and ribavirin. The purpose of the study is to investigate the safety, tolerability, and efficacy of TMC435 against hepatitis C virus in this patient population. For the first 12 weeks all patients will receive TMC435 plus peginterferon alfa-2a and ribavirin. For the subsequent 12 weeks, patients will take peginterferon alfa-2a and ribavirin only. After that, based on prior HCV treatment experience and prespecified on-treatment response criteria, some patients will continue to take peginterferon alfa-2a and ribavirin for total treatment duration of 48 weeks. The study doctor will inform each patient about how to take their study medication and when they should stop taking it. Patients will take their own regimen of HIV medication(s) throughout the study, according to the instructions of the study doctor. After a patient stops taking study medication, they will continue to come to the study doctor's office for study visits for up to 72 weeks after they received the first dose of treatment in the study. The maximum duration of the study is 81 weeks (including screening). Patients will be monitored for safety throughout the study. Study assessments at each study visit may include, but are not limited to: blood and urine collection for testing and physical examinations.

Importance of Pharmacokinetics and Adherence to Avoid Emergence of Resistant HCV Variants

Jean-Michel Pawlotsky, MD, PhD
Professor, Department of Virology
Henri Mondor Hospital
Université Paris Est
Créteil, France

Introduction

Hepatitis C virus (HCV) resistance to a direct-acting antiviral (DAA) agent corresponds to the selection during treatment of viral variants that bear amino acid substitutions that alter the drug target; therefore, they are less susceptible to the inhibitory activity of the drug. These drug-resistant variants preexist as minor populations within the patient’s HCV quasispecies. Drug exposure profoundly inhibits replication of the dominant “wild-type” drug-sensitive viral population, and the resistant variants gradually occupy the vacant replication space. Moreover, viruses with low-level or partial resistance that can continue to replicate in the presence of drug, often favored by suboptimal drug exposure, may accumulate further mutations, leading to stepwise decreases in drug susceptibility, albeit often at a cost of reduced replicative capacity. If insufficient antiviral activity is provided because of suboptimal dosing or adherence, inadequate virologic suppression and the selection of resistance is inevitable. Therefore, to reduce the development of resistance, it is essential to achieve optimal drug concentrations through proper dosing and maximal adherence.

Factors That Influence Viral Resistance in vivo

In vivo, viral resistance is influenced by 3 major related factors: the genetic barrier to resistance, the in vivo fitness of the resistant viral population, and drug exposure.^[1]

The genetic barrier to resistance is defined as the number of amino acid substitutions needed for a viral variant to acquire full resistance to the drug in question. If a single substitution is sufficient to confer high-level resistance to a specific drug, the drug is considered to have a low genetic barrier to resistance, whereas 3 or more substitutions are required to confer resistance to a drug with a high genetic barrier. There is a low likelihood that variants bearing a large number of resistance substitutions will preexist in a given patient and be fit enough to replicate at high levels when an antiviral drug is administered. Therefore, drugs with a high genetic barrier to resistance are less likely to be associated with clinically meaningful resistance.

The in vivo fitness of the viral variant is defined as its ability to survive and grow in the replicative environment. A selected resistant variant must have the capacity to propagate to fill in the replication space left vacant by the elimination of a susceptible wild-type virus during drug exposure. Thus, a highly resistant but poorly “fit” virus will be less clinically significant than a less resistant but “fitter” virus that can replicate efficiently in the presence of the drug. The acquisition of compensatory mutations may restore the fitness of a resistant variant and allow it to replicate efficiently in the presence of the drug, possibly allowing it to persist after drug withdrawal.

Finally, drug exposure affects the development of drug resistance. The degree of drug resistance of a variant can be measured in vitro as the fold increase in the 50% and 90% inhibitory concentrations (IC₅₀ and IC₉₀ in cell-free assays) or the 50% and 90% effective concentrations (EC₅₀ and EC₉₀ in cell-culture systems), that is, the drug concentrations that inhibit the tested enzyme function or viral replication by 50% and 90%, respectively. Drug exposure is defined as the drug concentration achieved in vivo relative to the IC₅₀, IC₉₀, EC₅₀, or EC₉₀ of resistant variants. This measurement is a key determinant of the development of resistance in vivo. Indeed, if drug levels achieved in vivo are far above these IC/EC values, then resistant variants will be effectively inhibited even if they are far less susceptible than the wild-type virus in vitro. Therefore, the pharmacokinetics of the antiviral drugs and adherence to therapy are key in preventing treatment failure due to viral resistance.

Importance of Pharmacodynamics/Adherence to Interferon-Containing DAA Regimens

The importance of achieving high blood DAA concentrations to prevent the emergence of resistance was demonstrated in the first phase Ib trial with telaprevir, an NS3/4A protease inhibitor (PI) with a low genetic barrier to resistance, administered as monotherapy for 14 days.^[2] In this trial, patients were more likely to achieve either an HCV RNA plateau or virologic breakthrough during the dosing period due to selection of telaprevir-resistant variants if they received lower telaprevir doses (450 mg every 8 hours or 1250 mg every 12 hours) than if they received the higher dose (750 mg every 8 hours).^[2] However, outgrowth of resistant viral populations was only delayed in the latter group. In vivo fitness assessments showed that less resistant, but “fitter” variants were more likely to become the dominant species than more resistant, less “fit” HCV variants.^[2] These findings led the American and European regulatory agencies to limit monotherapy studies involving DAAs with low genetic barriers to resistance to only a few days. Therefore, no other studies provided sufficiently long administration to accurately assess the effect of drug exposure on the emergence of resistance.

Resistance to antiviral drugs is classically prevented by combining several drugs with potent antiviral activity and no cross-resistance. Indeed, HCV resistance to DAAs is observed significantly less frequently when one of these drugs is administered in combination with peginterferon and ribavirin.^[3,4] Therefore, the triple combination of peginterferon, ribavirin, and a PI—telaprevir or boceprevir—has become the new standard-of-care therapy for both treatment-naive and treatment-experienced patients with genotype 1 HCV infection.^[5-8] For the reasons defined above, it is crucial that optimal exposure to all 3 drugs in the regimen be achieved for these patients. Telaprevir must be taken at a dose of 750 mg every 8 hours with fatty food, whereas boceprevir must be taken at the dose of 800 mg every 8 hours with food. Patients must fully adhere to the regimen for the entire treatment period because any prolonged interruption in PI administration would inevitably result in a resurgence of wild-type viruses and the opportunity for resistant variants to acquire fitness, especially if the antiviral pressure exerted by peginterferon and ribavirin is only modest. However, if the patient does miss a dose of telaprevir or boceprevir, the package inserts provide guidance on how to manage these short interruptions. For telaprevir, the prescribing information recommends that a missed dose should be skipped if more than 4 hours have passed since the time it is usually taken; however, if it is within 4 hours of the time that it is usually taken, the missed dose should be taken immediately with high-fat food.^[5,6] The recommendation for boceprevir is similar, but the timing is different. A missed boceprevir dose should be skipped if it is fewer than 2 hours before the next dose is scheduled; if it is more than 2 hours before the next scheduled dose, the missed dose should be taken immediately with food.^[7,8]

The importance of adherence to peginterferon and ribavirin has been demonstrated in the absence of DAAs; these data showed that optimal response rates were observed in patients who achieved more than 80% of their prescribed peginterferon and ribavirin doses for more than 80% of the time.^[9] The impact of poor adherence to, or dose reductions of, peginterferon, ribavirin, or both has not been extensively studied in clinical trials with the triple combination of peginterferon, ribavirin, and telaprevir or boceprevir. Nevertheless, maintaining the dose of peginterferon is likely to be key in interferon-responsive patients treated with triple therapy because treatment failure primarily results from an inadequate response to peginterferon, leading to the uncontrolled outgrowth of resistant variants selected by the PI.^[1,10-12] By contrast, a retrospective analysis of patients completing 48 weeks of peginterferon/ribavirin therapy suggested that reducing the dose of ribavirin has a negative impact on the outcome of therapy only before HCV RNA becomes undetectable, whereas the impact is modest after HCV replication is controlled.^[13] More recent studies in patients receiving PI-based therapy have shown that modest ribavirin dose reductions do not impair the likelihood of treatment success.^[14,15] Finally, a recent study showed that the cost effectiveness of triple therapy is dependent on optimal adherence.^[16]

There are several strategies that can be used to optimize adherence rates in patients receiving DAA-based therapy, some of which are under investigation. Current strategies include patient education on the importance of adherence, interventions to reduce the adverse effects of therapy, addressing comorbidities that may affect adherence to therapy, and using a multidisciplinary team to help physicians implement all of the aforementioned strategies. Regarding patient education, a prospective, multicenter study was conducted to determine the influence of patient education on adherence to peginterferon plus ribavirin therapy in patients infected with HCV.^[17] Investigators showed that patient education can significantly influence adherence to treatment as evidenced by adherence rates to ribavirin of 56% at 6 months in 175 patients not receiving education vs 70% in 208 patients receiving education (P = .006).^[17] Therapeutic education included intervention by healthcare professionals other than the prescribing physician as well as the distribution of support documents and educational materials. Another study found that physician’s treatment experience and patient motivation were associated with improved adherence to HCV therapy, indicating that empowering patients to take charge of their own treatment can impact adherence.^[18] It should be noted, however, that no data are yet available on how these strategies may affect adherence to triple combinations with the HCV PIs.

Importance of Pharmacodynamics/Adherence to Interferon-Free DAA Regimens

Maintaining optimal adherence has been shown to be an extremely effective way of minimizing the development of resistance in the HIV field.^[19] Strategies under investigation that may help optimize adherence rates to interferon-free DAA therapy include less frequent dosing and ritonavir boosting. Many DAA agents in development have half-lives that may allow for twice- or even once-daily dosing, which is encouraging.

In HIV therapy, the use of low-dose ritonavir to improve the pharmacokinetics of HIV PIs (so-called ritonavir boosting) has become standard practice. NS3/4A PIs are cytochrome P450 3A substrates; therefore, their plasma concentrations can also be substantially improved when coadministered with low-dose ritonavir, which may allow for prolonged dosing intervals and subsequent increased adherence rates. Boosting has been studied with 3 PIs—danoprevir, narlaprevir, and ABT-450—with encouraging results that suggest such strategies may support once-daily dosing, reduce adverse events (by lowering the required dose of the HCV PI), and reduce the risk of resistance.^[20-23] Results with ABT-450 showed that higher plasma trough levels were associated with a lower likelihood of selecting resistant HCV variants over a short course of administration of 3 days.^[23] However, a number of first-generation PIs can achieve high and well-tolerated plasma concentrations with once- or twice-daily dosing without ritonavir boosting.

Interferon-free regimens will reduce toxicity and adverse effects associated with peginterferon and ribavirin therapy, possibly improving adherence rates. The results of a short-term study combining the NS3/4A PI GS-9256 and the nonnucleoside RNA-dependent RNA polymerase inhibitor tegobuvir has been disappointing because this combination of agents has a low genetic barrier to resistance and frequent early virologic breakthroughs were observed.^[24] Additional studies which have combined agents with low genetic barriers to resistance include the SOUND-C1 and SOUND-C2 studies. These trials combined the NS3/4A protease inhibitor BI 201335 and nonnucleoside polymerase inhibitor BI 207127, with or without ribavirin.^[25,26] The ZENITH study combined the NS5B polymerase inhibitor VX-222 plus telaprevir.^[27] In the SOUND-C2 study, treatment response rates, and likely the selection of resistant HCV variants, appeared to be influenced by the genetic background of the host (IL28B genotype).^[26] Regimens comprising agents with higher barriers to resistance, such as the cyclophilin inhibitor alisporivir,^[28] the combination of the nucleoside analogue inhibitor mericitabine and the PI danoprevir,^[29] and the combination of the nucleotide inhibitor PSI-7977 with ribavirin have shown more promising results.^[30] Indeed, the latter combination resulted in undetectable HCV RNA at 12 weeks post-therapy (SVR12) for 10 out of 10 genotype 2/3 HCV–infected treatment-naive patients receiving this regimen for 12 weeks.

Another interferon-free regimen comprising agents with low barriers to resistance—the first-generation NS3/4A PI asunaprevir and the NS5A inhibitor daclatasvir—has provided valuable information on the importance of drug exposure and how this differs depending on HCV subtype.^[31,32] Protease inhibitors have a low genetic barrier to resistance; they can select fit variants that are poorly controlled at the drug concentrations achieved by doses used in clinical trials and practice. This is also true for NS5A inhibitors in genotype 1a HCV, as suggested by in vitro by studies showing a major shift in the IC₅₀s induced by single amino acid substitutions in the NS5A sequence.^[33] As a result, virologic breakthrough due to the selection of HCV variants resistant to both drugs was observed within a few days to weeks in 6 of the 9 patients infected with genotype 1a HCV.^[31] By contrast, in genotype 1b HCV models in vitro, daclatasvir retains subnanomolar potency against all variants with single amino acid substitutions; the fold-change in IC₅₀s conferred by these substitutions in the presence of the inhibitor was substantially lower than in genotype 1a.^[33] Consequently, the drug concentrations achieved in vivo in genotype 1b HCV–infected patients were able to control NS5A variants carrying these substitutions, both in the absence or presence of associated substitutions conferring resistance to the PI. As a result, the combination of asunaprevir and daclatasvir was shown to lead to high sustained viral eradication rates (~ 90%) in patients infected with genotype 1b HCV.^[31,32] To date, the available data on interferon-free regimens are more informative regarding pharmacodynamics and exposure than adherence. Additional data are awaited.

Conclusions

Although data are scarce regarding drug exposure and adherence in the context of HCV treatment with new DAA-based therapies, preliminary data and lessons learned from the HIV field suggest that optimal drug exposure and maximal adherence will be crucial to success with DAA-based therapies. In addition, the data from interferon-free regimens emphasize the importance of drug exposure, through optimal dosing and strict adherence to the prescribed regimen, when using combinations of drugs with a low to moderate barrier to resistance. Drugs with a higher barrier to resistance, such as nucleos(t)ide analogues, cyclophilin inhibitors, or second-generation PIs may theoretically be more tolerant to weaker adherence, but data are lacking thus far. When these therapies are available in clinical practice, virologic failures may be observed more often than in strictly controlled clinical trials. Vigilance and thorough patient education will be required to ensure high cure rates.

References

1. Pawlotsky JM. Treatment failure and resistance with direct-acting antiviral drugs against hepatitis C virus. Hepatology. 2011;53:1742-1751.

2. Sarrazin C, Kieffer TL, Bartels D, et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology. 2007;132:1767-1777.

3. Hézode C, Forestier N, Dusheiko G, et al. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med. 2009;360:1839-1850.

4. McHutchison JG, Everson GT, Gordon SC, et al. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N Engl J Med. 2009;360:1827-1838.

5. Incivek [package insert]. Cambridge, MA: Vertex Pharmaceuticals; 2011.

6. Incivo [telaprevir]. European Medicines Agency. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/002313/WC500115529.pdf. Accessed February 16, 2012.

7. Victrelis [package insert]. Whitehouse Station, NJ: Merck & Company; 2011.

8. Victrelis [boceprevir]. European Medicines Agency. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/002332/WC500109786.pdf. Accessed February 16, 2012.

9. McHutchison JG, Manns M, Patel K, et al. Adherence to combination therapy enhances sustained response in genotype-1-infected patients with chronic hepatitis C. Gastroenterology. 2002;123:1061-1069.

10. Poordad F, McCone J Jr, Bacon BR, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med. 2011;364:1195-1206.

11. Bacon BR, Gordon SC, Lawitz E, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N Engl J Med. 2011;364:1207-1217.

12. Zeuzem S, Andreone P, Pol S, et al. Telaprevir for retreatment of HCV infection. N Engl J Med. 2011;364:2417-2428.

13. Reddy KR, Shiffman ML, Morgan TR, et al. Impact of ribavirin dose reductions in hepatitis C virus genotype 1 patients completing peginterferon alfa-2a/ribavirin treatment. Clin Gastroenterol Hepatol. 2007;5:124-129.

14. Sulkowski MS, Poordad F, Manns MP, et al. Peginterferon alfa-2b/ribavirin with or without boceprevir is associated with higher SVR rates: analysis of previously untreated and previous treatment-failure patients. Program and abstracts of the 46th Annual Meeting of the European Association for the Study of the Liver; March 30 - April 3, 2011; Berlin, Germany. Poster 476.

15. Poordad F, Sulkowski MS, Reddy R, et al. Anemia had no effect on efficacy outcomes in treatment-naive patients who received telaprevir-based regimens in the ADVANCE and ILLUMINATE phase 3 studies. Program and abstracts of the Digestive Disease Week; May 7-10, 2011; Chicago, Illinois. Abstract 626.

16. Shan Liu SM, Cipriano LE, Holodniy M, et al. New protease inhibitors for the treatment of chronic hepatitis C. Ann Intern Med. 2012;156:279-290.

17. Cacoub P, Ouzan D, Melin P, et al. Patient education improves adherence to peg-interferon and ribavirin in chronic genotype 2 or 3 hepatitis C virus infection: a prospective, real-life, observational study. World J Gastroenterol. 2008;14:6195-6203.

18. Tanioka D, Iwasaki Y, Araki Y, et al. Factors associated with adherence to combination therapy of interferon and ribavirin for patients with chronic hepatitis C: importance of patient's motivation and physician's treatment experience. Liver Int. 2009;29:721-729.

19. Mocroft A, Phillips AN, Soriano V, et al. Reasons for stopping antiretrovirals used in an initial highly active antiretroviral regimen: increased incidence of stopping due to toxicity or patient/physician choice in patients with hepatitis C coinfection. AIDS Res Hum Retroviruses. 2005;21:743-752.

20. Gane E, Rouzier R, Stedman C, et al. Ritonavir boosting of low dose RG7227/ITMN-191, HCV NS3/4A protease inhibitor, results in robust reduction in HCV RNA at lower exposures than provided by unboosted regimens. Program and abstracts of the 45th Annual Meeting of the European Association for the Study of the Liver; April 14-18, 2010; Vienna, Austria. Abstract 38.

21. Rouzier R, Larrey D, Gane EJ, et al. Activity of danoprevir plus low-dose ritonavir (DNV/R) in combination with peginterferon alfa-2a (40KD) plus ribavirin (PEGIFNα-2A/RBV) in previous null responders. Program and abstracts of the 46th Annual Meeting of the European Association for the Study of the Liver; March 30 - April 3, 2011; Berlin, Germany. Abstract 62.

22. de Bruijne J, Bergmann JF, Reesink HW, et al. Antiviral activity of narlaprevir combined with ritonavir and pegylated interferon in chronic hepatitis C patients. Hepatology. 2010;52:1590-1599.

23. Pilot-Matias T, Tripathi R, Dekhtyar T, et al. Genotypic and phenotypic characterization of NS3 variants selected in HCV-infected patients treated with ABT-450. Program and abstracts of the 46th Annual Meeting of the European Association for the Study of the Liver; March 30 - April 3, 2011; Berlin, Germany. Abstract 1229.

24. Zeuzem S, Buggisch P, Agarwal K, et al. The protease inhibitor GS-9256 and non-nucleoside polymerase inhibitor tegobuvir alone, with RBV or peginterferon plus RBV in hepatitis C. Hepatology. 2011;[Epub ahead of print].

25. Zeuzem S, Asselah T, Angus PW, et al. High sustained virologic response following interferon-free treatment of chronic HCV GT1 infection for 4 weeks with HCV protease inhibitor BI201335, polymerase inhibitor BI207127 and ribavirin, followed by BI201335 and pegIFN/ribavirin—the SOUND-C1 study. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract 249.

26. Zeuzem S, Soriano V. Asselah T, et al. Virologic response to an interferon-free regimen of BI 201335 and BI 207127, with and without ribavirin, in treatment-naive patients with chronic genotype-1 HCV infection: Week 12 interim analysis of the SOUND-C2 study. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract LB15.

27. Nelson DR, Gane EJ, Jacobson IM, et al. VX-222/telaprevir in combination with peginterferon-alfa and ribavirin in treatment-naive genotype 1 HCV patients treated for 12 weeks: ZENITH study, SVR12 interim analysis. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract LB14.

28. Pawlotsky JM, Flisiak R, Rasenack J, et al. Once-daily alisporivir interferon (IFN)-free regimens achieve high rates of early HCV clearance in previously untreated patients with HCV genotype (G) 2 or 3. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract LB11.

29. Gane EJ, Roberts SK, Stedman CA, et al. Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial. Lancet. 2010;376:1467-1475.

30. Gane EJ, Stedman CA, Hyland RH, et al. PSI-7977: ELECTRON. Interferon is not required for sustained virologic response in treatment-naive patients with HCV GT2 or GT3. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract 34.

31. Lok AS, Gardiner DF, Lawitz E, et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. N Engl J Med. 2012;366:216-224.

32. Chayama K, Takahashi S, Kawakami Y, et al. Dual oral combination therapy with the NS5A inhibitor daclatasvir (DCV; BMS-790052) and the NS3 protease inhibitor asunaprevir (ASV; BMS-650032) achieved 90% sustained virologic response (SVR12) in Japanese HCV genotype 1b–infected null responders. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract LB4.

33. Fridell RA, Qiu D, Wang C, Valera L, Gao M. Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob Agents Chemother. 2010;54:3641-3650.

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Does Coffee Really Protect Against Liver Fibrosis?

From Medscape Gastroenterology > Ask the Experts

William F. Balistreri, MD

Posted: 03/02/2012

Question:

I heard a report that coffee may protect against liver fibrosis in patients with fatty liver disease -- is it true?

Response from William F. Balistreri, MD
Professor of Medicine, University of Cincinnati College of Medicine; Staff Physician, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Coffee and Fatty Liver Disease

That report likely refers to a recent study in which investigators attempted to correlate coffee (caffeine) consumption with the severity of nonalcoholic fatty liver disease (NAFLD).^[1] As Malloy and colleagues point out, an association between coffee consumption and a reduced risk for liver disease progression was established years ago.^[2-5] Coffee intake has been shown to correlate with lower liver enzyme levels and a reduced risk for hospitalizations and mortality in patients with cirrhosis. A recent example: Coffee consumption was linked to lower rates of clinical and pathologic progression of liver fibrosis in patients with chronic hepatitis C infection.^[6]

Investigators at Brooke Army Medical Center in Fort Sam Houston, Texas,^[1] therefore, questioned whether coffee ingestion by patients with NAFLD would similarly slow the progression of liver disease from steatosis to nonalcoholic steatosis (NASH). In their study, more than 300 patients underwent liver ultrasound; those in whom the ultrasound was negative for fatty liver served as controls. Patients with steatosis suggested by ultrasound underwent liver biopsies to categorize the patients into 3 groups:

• Bland steatosis without evidence of NASH;
• NASH with stage 0-1 fibrosis; and
• NASH with stage 2-4 fibrosis.

A validated questionnaire was used to determine whether a relationship existed between caffeine consumption (assessing the average mgs of total caffeine/coffee per day) and the degree of steatosis/NASH. A negative relationship was found between caffeine/coffee consumption and the degree of hepatic fibrosis. A significant difference in caffeine/coffee consumption was found between patients who had bland steatosis compared with those who had NASH stage 0-1, as well as between patients who had NASH stage 0-1 compared with those who had NASH stage 2-4. Therefore, this study demonstrates a histopathologic correlation between progression of fatty liver disease and estimated coffee intake. In their cohort of patients with NASH, increased intake of coffee seemed to confer a significantly reduced risk for advanced fibrosis. What is not clear from their data is the amount of coffee or caffeine that must be ingested to lower the risk for fibrosis. Also, because the study was not prospective, the impact of the observed effects on clinical outcomes over time is unknown. Further prospective studies are required.

What Is the Mechanism?

Assuming that the beneficial effects are real, what is the mechanism? Does coffee intake have a direct effect -- attributable to caffeine or to "magical powers of the bean"? Or is the effect indirect -- related to the preferential intake of coffee as a substitute for high-calorie, high-fructose-containing beverages? The investigators point out that the effects may be "more than strictly related to caffeine’s antioxidant behaviors." They cite a study in which rats receiving a high-fat diet given decaffeinated coffee had lower levels of hepatic fat and collagen, reduced liver oxidative stress (an effect of glutathione metabolism), and less liver inflammation -- emphasizing the likely importance of coffee itself, not caffeine, in preventing the progression of NASH. Potential beneficial components include aromatic extracts isolated from coffee beans and/or an elevation of glutathione levels or other antifibrogenic agents by coffee intake.^[7-9]

The Bottom Line

What advice can we offer to patients with fatty liver disease? Despite the preliminary results about the beneficial effects of vitamin E and omega-3 fatty acids, lifestyle changes (ie, weight loss through diet and exercise) are the only strategies proven to be effective.^[10,11] Moderate consumption of coffee may be a useful, benign adjunct -- but hold the cream and sugar!

References

1. Molloy JW, Calcagno CJ, Williams CD, Jones FJ, Torres DW, Harrison SA. Association of coffee and caffeine consumption with fatty liver disease, nonalcoholic steatohepatitis, and degree of hepatic fibrosis. Hepatology. 2012;55:429-436.
2. Casiglia E, Spolaore P, Ginocchio G, Ambrosio G. Unexpected effects of coffee consumption on liver enzymes. Eur J Epidemiol. 1993;9:293-297.
3. Corrao G, Zambon A, Bagnardi V, D’Amicis A, Klastky A. Coffee, caffeine, and the risk of liver cirrhosis. Ann Epidemiol. 2001;11:458-465.
4. Ruhl E, Everhart J. Coffee and caffeine consumption reduce the risk of elevated serum alanine aminotransferase activity in the United States. Gastroenterology. 2005;128:24-32.
5. Modi A, Feldman J, Park Y, et al.. Increased coffee consumption is associated with reduced hepatic fibrosis. Hepatology. 2010;51:201-209.
6. Freedman N, Everhart J, Lindsay K, et al. Coffee intake is associated with lower rates of liver disease progression in chronic hepatitis C. Hepatology. 2009;50:1360-1369.
7. Lee K, Mitchell A, Shibamoto T. Antioxidative activities of aroma extracts isolated from natural plants. BioFactors. 2000;13:173-178.
8. Huber W, Scharf G, Rossmanith W, et al. The coffee components kahweol and cafestol inducegamma-glutamylcysteine synthetase, the rate limiting enzyme of chemoprotective glutathione synthesis, in several organs of the rat. Arch Toxicol. 2002;75:685-694.
9. Scharf G, Prustomersky, Huber W. Elevation of glutathione levels by coffee components and its potential mechanisms. Adv Exp Med Biol. 2001;500:535-539.
10. Sanyal A, Chalasani N, Kowdley K, et al. Pioglitizone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med. 2010;362:1675-1685.
11. Centis E, Marzocchi R, Di Domizio S, Ciaravella MF, Marchesini G. The effect of lifestyle changes in non-alcoholic fatty liver disease. Dig Dis. 2010;28:267-273.

Source

The HCV Rooster Has Come Home to Roost

Written by Jose M Zuniga, Other, 09:50AM Feb 29, 2012

I wrote in a previous AIDScan blog about how hepatitis C virus (HCV) infection was expected to become a much larger public health problem as "baby boomers" begin to succumb to the disease. That prediction was borne out this week as research published in the Annals of Internal Medicine announced that HCV-related deaths in the United States now exceed HIV-related deaths, with an upward trend in HCV-related mortality, much of it attributed to individuals aged 45 to 64 years.¹ Equally alarming are data from a separate study also published in the Annals of Internal Medicine revealing that the majority of HCV infections in the United States are undiagnosed.²

These two papers reinforce the need to fully implement the US Department of Health and Human Services' "Action Plan for Prevention, Care, and Treatment of Viral Hepatitis," which calls for, among other things, an increase in targeted HCV screening of individuals born between 1945 and 1965.³ As the authors of the latter paper conclude, birth-cohort screening linked to HCV treatment with pegylated-interferon (PEG-IFN) and ribavirin (RBV) - which, incidentally, has been replaced with an even more therapeutically effective standard of care (see next paragraph) - might help significantly reduce HCV-related deaths versus current risk-based screening.²

In relation to HCV treatment, a new class of protease inhibitors - direct-acting antivirals (DAAs)-prescribed in combination with PEG-IFN and RBV, continues to demonstrate impressive cure rates. The HCV pipeline seems equally robust, with several more treatment options on the horizon, including potentially IFN-sparing options that will simplify dosing and address side effect concerns.

The bottlenecks to expanding access to HCV treatment, however, include both gaps in screening and diagnosis capacity, and in the numbers of clinicians able to prescribe the new standard of care for HCV infection. This, too, requires our immediate attention as well as a common understanding across medical disciplines, professions, and specialties that the needs of HCV-positive individuals require collaboration at practitioner-, clinic-, and health system-levels.

SHAMELESS PLUG WARNING: All of the issues discussed above will be addressed at the 2nd International Conference on Viral Hepatitis, March 26-27, 2012, at the New York Academy of Medicine in New York City. Visit www.iapac.org to view the program, faculty roster, and/or to register online.

1. Ly KN, Xing J, Klevens M, et al. The increasing burden of mortality from viral hepatitis in the United States between 1999 and 2007. Ann Intern Med. 2012;156:271-278.
2. Rein DB, Smith BD, Wittenborn JS, et al. The cost-effectiveness of birth-cohort screening for hepatitis C antibody in U.S. primary care settings. Ann Intern Med. 2012;156:263-260.
3. DHHS. Combating the Silent Epidemic of Viral Hepatitis: Action Plan for the Prevention, Care, and Treatment of Viral Hepatitis. 2011, US Department of Health and Human Services, Washington, DC, USA.

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