Hepatitis C

Structure

The HCV virion is an enveloped virus that is 50 nm in diameter. The 2 envelope proteins, E1 and E2, heterodimerize and assemble into tetramers, creating a smooth outer layer, which has a “fishbone” configuration with icosahedral symmetry. The envelope proteins are anchored to a host cell–derived lipid bilayer envelope membrane that surrounds the nucleocapsid. The nucleocapsid is believed to be composed of multiple copies of the core protein and forms an internal icosahedral viral coat that encapsulates the genomic RNA. HCV circulates in various forms in the serum of an infected host, including (1) virions that are bound to VLDL and LDL and appear to represent the infectious fraction; (2) virions bound to immunoglobulin (Ig); and (3) free virions.

Genomic Organization

HCV is a single-stranded positive-sense RNA virus that belongs to the Flaviviridae family and has been classified as the sole member of the genus Hepacivirus . The genome of HCV contains approximately 9600 nucleotides with an open reading frame (ORF) that encodes one large viral polypeptide precursor of about 3000 amino acids. The HCV ORF is flanked upstream by a 5′ untranslated region (UTR) that functions as an internal ribosome entry site to direct cap-independent translation (i.e., without the addition of an extra ribonucleotide to the 5′ end of the viral messenger RNA) and downstream by a 3′ UTR that is critical for initiation of new RNA strand synthesis. The 5′ and portions of the 3′ UTR are the most conserved regions of the HCV genome.

Viral Replication and Life Cycle

Although peripheral blood mononuclear cells, B cells, T cells, and dendritic cells have been reported to support HCV replication, hepatocytes are the major site of viral replication. ^, HCV entry involves the attachment of envelope proteins E1 and E2 to cell surface molecules ( Fig. 80.1 ). The expression and function of CD81, a member of the tetraspan superfamily, is essential for HCV entry into hepatocytes. In addition, human scavenger receptor class B type 1, a selective importer of cholesteryl esters from HDL into cells, has been shown to interact with E2 and is also essential for HCV entry. Whereas CD81 and scavenger receptor class B type 1 are required early in the process of viral entry, claudin-1, a tight junction component that is highly expressed on hepatocytes, and occludin are required later in the cell entry process. ^, Heparin sulfated proteoglycans and LDL have also been shown to be involved in HCV cell entry. ^, Additional cellular factors and receptors suggested to be required for viral entry include EGF and Niemann-Pick C1-Like 1, a cholesterol uptake receptor.

Once HCV attaches to the cell, endocytosis of the bound virion is presumed to occur, as with other flaviviruses. A pH drop in the vesicle causes conformational changes in the glycoproteins that lead to fusion of the viral and cellular membranes and release of viral RNA into the cytoplasm. In the cytosol, the 5′ UTR functions as an internal ribosome entry site, which directs the RNA to its docking site on the endoplasmic reticulum and mediates cap-independent internal initiation of HCV polyprotein translation by recruiting both cellular proteins, including eukaryotic initiation factors 2 and 3, and viral proteins. The large polyprotein is co- and post-translationally processed proteolytically into at least 11 viral proteins, including both structural (nucleocapsid [C], or p21; envelope 1 [E1], or gp31; and envelope 2 [E2], or gp70) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins ( Fig. 80.2 ). The functions of these specific nonstructural proteins are described later in the chapter.

After polyprotein processing, NS4B expression causes the membrane alterations that are seen on electron microscopy known as a membranous web. The replication complex associates viral proteins, cellular components, and nascent RNA strands. HCV replication is catalyzed by the NS5B RNA-dependent RNA polymerase (RdRp). The positive-strand genomic RNA serves as a template for the synthesis of a negative-strand intermediate. The negative-strand RNA serves as a template for production of numerous strands of RNA of positive polarity that are used for polyprotein translation and synthesis of new intermediates of replication and that are packaged into new virus particles.

Finally, viral particle formation is initiated by the interaction of the core protein with genomic RNA in the endoplasmic reticulum. By analogy with pestiviruses, HCV packaging and release are likely to be inefficient because much of the virus remains in the cell. Following release, viral particles may infect adjacent hepatocytes or enter the circulation, where they are available for infection of another cell or host.

Genotypes and Quasispecies

HCV has an inherently high mutational rate that results in considerable heterogeneity throughout the genome. This high mutational rate is in part a consequence of the RdRp of HCV, which lacks 3′- to 5′-exonuclease proofreading ability that ordinarily would remove mismatched nucleotides incorporated during replication. An average of one error occurs for every 10 ⁴ to 10 ⁵ nucleotides copied. This phenomenon is favored by a high viral turnover rate; 10 ¹⁰ to 10 ¹² virions are produced per day. The estimated half-life of HCV in serum is only about 45 minutes. A substantial proportion of newly synthesized viral genomes have alterations. Because of the functional differences in HCV proteins, genetic variation in some parts of the genome confers advantages by evading or inhibiting the host immune system, whereas other mutations may be lethal to the virus if they lead to defective replication machinery. Therefore, genetic variation is distributed irregularly along the genome. Each new genetic variant is produced in a single cell and may or may not spread through the liver and into the serum. The result is not only genetic diversity in the serum, but also compartmentalization of variant virions in different parts of the liver and perhaps in extrahepatic sites.

Because of the vast genetic variation, a classification scheme was devised whereby viral sequences are given a genotype and subtype. The first division used to describe the genetic heterogeneity of HCV is the viral genotype , which refers to genetically distinct groups of HCV isolates that have arisen during the evolution of the virus. Nucleotide sequencing has shown variation of up to 34% between genotypes. The most conserved region (5′ UTR) has a maximum nucleotide sequence divergence of 9% between genotypes, whereas the highly variable regions that encode the envelope proteins (E1 and E2) exhibit a nucleotide sequence divergence of 35% to 44% between genotypes. The sequences cluster into 7 major genotypes (designated by numbers), with sequence similarities of 60% to 70%, and more than 67 subtypes (designated by a lower-case letter) within these major genotypes, with sequence similarities of 77% to 80%. In this scheme, the first variant, which was cloned by Choo and colleagues, is designated type 1a. The HCV genotype is an intrinsic characteristic of the infecting HCV strain and does not change over time; therefore, the genotype only needs to be determined once in an infected person. Mixed-genotype infections may be seen and reflect either coinfection with more than one HCV virus or methodologic problems in genotype testing. In addition, intergenotypic HCV recombinants have been described ; these are thought to arise because of recombination between different genotypes in patients with repeated exposure. The recombination events have been reported to occur in or between NS2 and NS3.

Global geographic differences exist in the distribution of HCV genotypes, as well as in the mode of acquisition. In the USA, genotype 1a is the most prevalent, accounting for approximately 46% of HCV infections, followed by genotype 1b in 26%, genotype 2 in 11%, genotype 3 in 9%, and genotype 4, 5, 6, or mixed/other in less than 8%. Racial differences are seen in the prevalence of genotypes; approximately 90% of African Americans are infected with HCV genotype 1, whereas only 70% of whites and 71% of Hispanics are infected with genotype 1. In Europe, the most prevalent genotype is 3 (41%), followed by 1b (40%), 1a (13%), and 1c/other (18%). Genotype 4 is found mainly in Egypt, the Middle East, and Central Africa. ^, In Egypt, approximately 6.5 to 7% of the population is infected with HCV, and more than 90% have HCV genotype 4. ^, Genotype 5, although originally isolated in South Africa, is also seen in specific regions of Europe (France and Belgium) and the Middle East (Lebanon and Syria). ^, Genotype 6 is found predominantly in Asia. The distribution of genotypes is ever changing with immigration and alterations in the primary modes of viral transmission. Therefore, the frequencies of viral genotypes change over time.

In the era of IFN-based therapy, HCV genotype was an important predictor of response to treatment. Although HCV genotype as a predictor of treatment outcome is less relevant with DAA-based therapy, HCV genotype is still important, because some DAAs only have activity against specific HCV genotypes (see later). HCV genotype may also play a role in disease progression and complications of chronic HCV infection. Specifically, HCV genotype 3 has been associated with faster liver fibrosis progression, as well as with an increased risk of cirrhosis and HCC.

The second component of genetic heterogeneity is quasispecies generation. Quasispecies are closely related, yet heterogeneous, sequences of HCV RNA within a single infected person that result from mutations that occur during viral replication. The rate of nucleotide changes varies significantly among the different regions of the viral genome. The highest proportion of mutations are found in the E1 and E2 regions, particularly in HVR1. Even though this region represents only a minor part of the E2 region, it accounts for approximately 50% of the nucleotide changes and 60% of the amino acid substitutions within the envelope region.

The development of quasispecies may be one mechanism by which the virus escapes the host’s immune response and establishes persistent infection. During acute infection or during treatment, lack of diversity in the quasispecies is associated with viral clearance, and the development of numerous quasispecies is associated with viral persistence. In acute disease, patients in whom genetic variation in the HVR1 region develops after antibody seroconversion progress to chronic disease, whereas those in whom such genetic variation does not develop are more likely to achieve viral clearance. Genetic variation before seroconversion does not correlate with outcome, indicating that quasispecies formation results from antibody-mediated immune pressure. Interestingly, no intrinsically IFN-resistant variants of HCV have been defined, indicating that both viral and host factors play important roles in determining whether the virus persists or is cleared. An increased number of quasispecies has also been associated with more rapid progression to cirrhosis and the development of HCC.

Incidence and Prevalence

The worldwide prevalence of HCV infection, based on detection of HCV RNA in serum, is estimated to be 1%, with more than 71 million people infected chronically. The overall worldwide prevalence increased from 1990 to 2010. Marked geographic variation exists, with infection rates ranging from 0.1% in the Netherlands, Fiji, and Samoa, to 0.9% in the USA, 6.3% in Egypt, and 7% in Gabon. In 2002, between 3.2 and 5 million persons were infected with HCV in the USA ; however, these estimates were based on HCV seroprevalence (presence of anti-HCV antibody only). More recent studies estimate the viremic prevalence to be 2.9 million, which has been stable since 2006, although this is likely to change with the advent of highly potent IFN-free DAA therapy. ^, The highest seroprevalence in different age groups shifted from 35 to 44 years (2.5%) to 55 to 64 years in 2005 (2.7%). It had therefore been recommended that all persons born between 1945 and 1965 be tested for anti-HCV. In 2020, the Centers for Disease Control and Prevention (CDC) recommended that all adults over age 18 and all pregnant women be tested for anti-HCV unless they are in a setting in which the prevalence of HCV infection is less than 0.1% .The prevalence is higher in males (2.1%) than in females (1.1%), and in African Americans (3%) than in whites (1.5%). Other risk factors for HCV infection are injection drug use, blood transfusion before the implementation of routine blood product screening in many countries in 1992, more than 50 lifetime sexual partners, family income below the poverty level, occupational exposure, and being born in an endemic country. The prevalence of HCV infection in the USA may be underestimated because the National Health and Nutrition Examination Survey data did not evaluate persons who are homeless, incarcerated, or in the military. Trends in the epidemiology of HCV infection suggest an increase in prevalence in young drug-using persons.

Worldwide, 3 different epidemiologic patterns of HCV infection have emerged: (1) previous exposure through health care with a peak prevalence in older persons; (2) exposure through injection drug use, the major risk factor since data first became available in about 1960, with a peak prevalence among middle-aged persons; and (3) ongoing high levels of infection in areas where high rates of infection occur in all age groups.

Given the factors that influence viral diversity (see earlier), estimating the site of origin and age of HCV by phylogenetic analysis is difficult. The best estimate is that HCV originated in western and sub-Saharan Africa. Subsequent global spread probably occurred coincident with trade and human migration. Evolution of the virus led to a geographic distribution of genotypes, so that genotypes 1, 2, and 3 are most common in North America and Europe, genotype 4 is most common in the Middle East, and genotype 6 is most common in Southeast Asia. In Japan, HCV transmission transitioned from constant to exponential growth in the 1920s, and the prevalence of HCV infection is highest in older persons. In Japan, and later in southern and eastern Europe, health care–related procedures—particularly reuse of contaminated syringes—played a major role in viral spread. In the USA, Australia, and other developed countries, peak prevalence is in persons 40 to 49 years of age, and analysis of risk factors suggests that most HCV transmission occurred between the mid-1980s and the mid-1990s, through injection drug use. In Egypt, the spread of HCV increased exponentially from the 1930s to the 1980s because of mass vaccination campaigns with reuse of medical equipment. In Egypt and other developing countries, high rates of infection are observed in all age groups, suggesting that an ongoing risk of HCV acquisition exists.

In the USA, there have been distinct changes in the epidemiology of acute HCV infection. The incidence of acute HCV peaked in 1989, gradually declining over the following 15 years, before stabilizing from 2006 until 2010. The peak incidence was estimated to be 180,000 cases per year in the mid-1980s, but the rate declined to less than 20,000 cases. Many factors have contributed to the falling incidence of acute hepatitis C. In the 1980s, when blood was purchased from donors, 2% to 10% of blood units were infected with HCV, leading to a high rate of transfusion-acquired HCV infection. The institution of volunteer blood donation, creation of recombinant clotting factors, and implementation of HCV blood testing (between 1990 and 1992) dramatically decreased transfusion-acquired HCV infection. However, since 2011, the incidence of acute hepatitis C has increased 4-fold, particularly in the 18- to 39-year-old age group (a 400% increase in 18- to 29-year-olds and 325% increase in 30- to 39-year-olds), attributable to the opioid injection drug use epidemic.

An important mechanism of transmission worldwide has been the lack of sterilization of medical instruments such as syringes. Although the incidence of HCV transmission by medical instruments has also decreased markedly, the risk has not been eliminated, even in the USA. New HCV infections in the USA and other developed countries occur primarily as a result of injection drug use.

Transmission

Modes of transmission of HCV can be divided into percutaneous (blood transfusion and needlestick inoculation) and nonpercutaneous (sexual contact and perinatal exposure). Patients are often unwilling to disclose percutaneous risk factors, and therefore apparent nonpercutaneous transmission may represent occult percutaneous exposure.

Viral Mechanisms

In chronically infected patients, the pathogenesis of liver damage is largely immune mediated. In a small subset of immunocompromised HCV-infected patients among both HIV-infected patients and organ transplant recipients, however, a syndrome termed fibrosing cholestatic hepatitis develops (see Chapter 97 ). ^, Such cases are thought to result from direct viral hepatotoxicity of infected cells, because viral levels are typically greater than 30 million copies/mL and hepatocytes contain enormous concentrations of virus and viral proteins. Survival in such patients has been poor.

The majority of patients with HCV infection have a variable immune response that, although inadequate to eradicate acute infection, appears to regulate the vigor of persistent infection and avoid the development of fibrosing cholestatic hepatitis. The immune response to HCV is incompletely understood because animal models that recapitulate human disease and immunology are not readily available, and therefore most studies in humans rely on observations in peripheral blood rather than the hepatic immune environment.

Immune-Mediated Mechanisms

HCV infection elicits an immune response in the host that involves both an initial innate response and a subsequent adaptive response. The innate response is the first line of defense against the virus and includes several arms such as natural killer (NK) cell activation and cellular antiviral mechanisms triggered by pathogen-associated molecular patterns recognized by the cell (see Chapter 2 ). These processes can lead to apoptosis of infected cells within the first few hours of infection. NK cells, as the effector cells of the innate immune system, also produce TNF-β and IFN-α, cytokines that are critical for dendritic cell maturation and subsequent induction of adaptive immunity. NK cells can also attack virus-infected cells directly, as do other immune cells by different effector molecules. Subsequently, however, the virus initiates a number of mechanisms that undermine the ability of the host to control the infection.

Virus-related disruption of the innate, and later adaptive, immune response occurs at several levels. NK cell function is slowed possibly because NK cell-mediated cytotoxicity and production of cytokines are interrupted when the HCV E2 protein binds its cellular receptor CD81. Expression of TNF-related apoptosis-inducing ligand on NK cells correlates with disease activity in both acute and chronic hepatitis C, thereby suggesting that NK cells have a direct role in the immunopathogenesis of hepatitis C. Pathogen-associated molecular patterns activate several cellular processes, including the JAK-STAT (Janus kinase‒signal transducer and activator of transcription) proteins pathway and Toll-like receptor-3, activation of both of which ultimately results in production of cellular IFNs, IFN-stimulated genes (ISGs), and IFN-regulated factors that convey antiviral properties to the cell. NS3/4 protease degrades TRIF, an essential intermediate in this pathway, and cleaves IFN promoter stimulator-1, an intermediate in the signaling cascade, to block activation of IFN when retinoic inducible gene-1 binds viral intermediates. In addition, HCV core protein promotes STAT-1 degradation, inhibits STAT-1 phosphorylation, promotes suppressor of cytokine signaling induction (an inhibitor of JAK-STAT signaling), and impairs ISG factor-3 (ISGF3), a heterotrimer of STAT-1, STAT-2, and IFN-β promoter stimulator (IRF-9) from binding to the promoter regions of IFN-stimulated response elements, thereby inhibiting transcription of IFN response genes. Even when IFN response genes are activated, NS5A and E2 both can disrupt protein kinase R function to suppress translation, thereby allowing viral replication to continue. In addition, NS5A inhibits 2′-5′-oligoadenylate synthetase, which is expressed in response to HCV infection and leads to HCV RNA degradation. Taken together, HCV is able to disrupt the innate immune response at several levels, and these strategies appear to be pivotal in establishing the chronicity of infection.

The ability of HCV to impair the innate immune response prevents development of a vigorous adaptive immune response to the infection. NK cells do not adequately activate dendritic cells, and as a result, the priming of CD8 ⁺ and CD4 ⁺ T cells in HCV-infected patients is inadequate. Even if an adequate T-cell response is created, HCV-infected patients have a large number of regulatory T cells in their portal tracts ; intrahepatic immune regulation by these cells has not been demonstrated but is presumed.

HCV-specific T cells are enriched at the site of viral replication, with an increased number in the liver when compared with the peripheral blood. CD8 ⁺ lymphocytes predominate, suggesting that cytotoxic T lymphocytes are the main perpetrators of hepatocellular injury. The T-cell immune response in the liver may result in direct lysis of infected cells and inhibition of viral replication by secreted antiviral cytokines.

Whereas the cellular immune response plays a pivotal role in the pathogenesis of HCV infection, the importance of the humoral immune response is less clear. Antibodies to viral proteins are produced and do not appear to correlate with the stage of infection or immune reactivity. Furthermore, administration of high-titer HCV-enriched or HCV-specific Ig has little effect on viral levels or persistence in humans.

In summary, viral products play an integral role in the immune regulation that leads to chronic infection instead of viral clearance. Both the virus and the immune response probably play a role in the development of hepatocellular injury. The mechanisms by which hepatocellular injury leads to hepatic fibrosis are discussed in Chapter 74 .

Acute Hepatitis C

HCV accounts for an estimated 20% of cases of acute hepatitis. Acute hepatitis C is rarely seen in clinical practice, however, because nearly all cases are asymptomatic. Within 7 to 21 days after viral transmission, HCV RNA becomes detectable in serum. Longer incubation periods can occur, especially in cases in which only a small amount of virus has been transmitted. These data suggest that the duration of the incubation period may vary among different transmission routes. HCV RNA levels rise rapidly in serum after infection, followed by a delayed increase in serum ALT levels 4 to 12 weeks after infection, indicative of hepatic injury. Serum ALT levels frequently reach values more than 10 times the upper limit of normal, with concomitant rises in the serum bilirubin level in some inidividuals ( Fig. 80.3 ). Some patients also develop clinical symptoms 2 to 12 weeks after viral transmission, but the majority of patients remain asymptomatic during the acute phase and most infected persons do not become aware of their disease. Therefore, it is not easy to study the early phase of HCV infection. Several studies have investigated patients recruited during the acute symptomatic phase of HCV infection, and 80% of the patients have presented with diverse symptoms. Even in symptomatic patients, however, most of the clinical symptoms are nonspecific. Commonly reported symptoms include fatigue, nausea, abdominal pain, loss of appetite, mild fever, itching, and myalgia. Jaundice, which is the most specific liver-related symptom, develops in 50% to 84% of patients with clinically overt acute HCV infection. ALF caused by HCV has been reported in only single cases, in contrast to infections with other hepatotropic viruses (see Chapter 95 ). The presentation may be more apparent and the clinical course more severe when acute HCV infection occurs in patients who drink large amounts of alcohol or have coinfection with HBV or HIV.

The rate of viral persistence after acute infection ranges from 45% to more than 90%. Age and gender clearly influence the risk of chronicity, with younger and female patients having the lowest rates of chronicity. Other factors that may play a role include the source of infection and size of inoculum (chronicity may be less common in PWID than in those who acquire HCV infection by blood transfusion), immune status of the host (chronicity rates are higher in persons with immunodeficiency states such as agammaglobulinemia and HIV infection), and the patient’s race (rates of viral persistence are higher in African Americans than in whites and Hispanic Americans in the USA). Finally, the rate of spontaneous clearance is higher in symptomatic patients in whom jaundice develops during acute infection than in those who remain asymptomatic.

Single nucleotide polymorphisms (SNPs) close to the IFN lambda-3, or interleukin 28B, gene ( IFN-λ3, IL28B ) have been found to be associated with the outcome of acute hepatitis C. The IFN-λ3 gene is located on chromosome 19 and encodes IFN- λ 3. Ge and colleagues reported a significant role for a specific SNP in the IFN-λ3 gene region (rs12979860 CC) in the response to pegylated IFN-α–based therapy for chronic hepatitis C in 2009. Shortly thereafter, Thomas and colleagues identified a major contribution for the same SNP in spontaneous clearance of acute HCV infection. Subsequently, these findings were confirmed by other investigators in different cohorts. ^, An association between clearance of acute HCV infection and the IFN-λ3 genotype may differ, however, between symptomatic and asymptomatic patients, because the CC IFN-λ3 polymorphism was associated with spontaneous recovery only in nonicteric patients in a cohort of East German women exposed to HCV in a single-source outbreak in the late 1970s.

Chronic Hepatitis C

Serum ALT levels are usually elevated in patients with chronic HCV infection. Because levels commonly fluctuate, however, as many as half of patients may have a normal ALT level at any given time. The ALT level may remain normal for prolonged periods of time in about 20% of cases, although transient elevations occur even in these cases. Persistently normal ALT levels are more common in women, and such cases typically are associated with lower serum HCV RNA levels and less inflammation and fibrosis on liver biopsy specimens.

Most patients with chronic hepatitis C are asymptomatic before the onset of advanced hepatic fibrosis. Patients who have been diagnosed with chronic infection, however, often complain of nonspecific symptoms such as fatigue, vague abdominal pain, or depression, and they consistently score lower than HCV-negative persons in all aspects of health-related quality of life (HRQOL). Whether the decrease in HRQOL is related to viral factors, social factors (e.g., injection drug use), social stigmatization, or worry related to the diagnosis itself is unclear. Nevertheless, HRQOL scores improve if the patient achieves a sustained response to antiviral therapy. Less common symptoms may include arthralgias, paresthesias, myalgias, sicca syndrome, nausea, anorexia, and difficulty with concentration. The severity of these symptoms may be, but is not necessarily, related to the severity of the underlying liver disease.

Extrahepatic Manifestations

Patients with HCV infection may present with extrahepatic conditions, or these manifestations may occur in patients known to have chronic HCV infection. Classification of the extrahepatic manifestations of HCV is shown in Box 80.1 and is based on the strength of available data to prove a correlation. Types 2 and 3 cryoglobulinemia, characterized by polyclonal IgG plus monoclonal IgM and polyclonal IgG plus polyclonal IgM, respectively, can both be caused by HCV infection. Among HCV-infected patients, 19% to 50% have cryoglobulins in serum, but clinical manifestations of cryoglobulinemia are reported in only 5% to 10% of these patients and are more common in patients with cirrhosis. Symptoms and signs include fatigue, arthralgias, arthritis, purpura, Raynaud phenomenon, vasculitis, peripheral neuropathy, and nephropathy. The diagnosis is clear when a rheumatoid factor is detected, cryoglobulins are present, and complement levels are low in serum; however, the reliability of cryoglobulin measurements is dependent on proper handling and processing of the sample.

Glomerular disease generally manifests as cryoglobulinemic nephropathy, membranoproliferative glomerulonephritis, and membranous nephropathy. Cryoglobulinemic nephropathy manifests as hematuria, proteinuria, edema, and renal insufficiency of varying degrees and features of membranoproliferative glomerulonephritis on renal biopsy specimens. At diagnosis, 20% of patients with type 2 cryoglobulinemia have renal involvement, and renal involvement develops in another 35% to 60% over time. In about 15% of patients, cryoglobulinemic nephropathy progresses to end-stage kidney disease requiring dialysis.

As HCV infection drives these extrahepatic manifestations, treatment of the underlying HCV infection should be considered in patients with symptomatic cryoglobulinemia. There are limited data regarding the use of IFN-free DAA regimens to treat extrahepatic manifestations of HCV, the majority of which involved treatment with the first-generation protease inhibitors combined with PegIFN/RBV, which are no longer recommended. However, given the high efficacy of the newer IFN-free DAA regimens, consensus guidelines recommend treatment of extrahepatic manifestations with IFN-free DAA regimens as per standard HCV infection treatment guidelines. In addition to antiviral therapy for HCV infection, monoclonal antibody therapy targeting B cells (anti-CD20 therapy with rituximab) has been shown to be useful for HCV-related cryoglobulinemia, particularly in patients with severe renal disease, because rituximab reduces B-cell clones that are responsible for producing cyroglobulins. ^, Although this approach has been shown to be effective in randomized controlled studies when used alone or in combination with IFN plus RBV, rituximab is not licensed for treatment of extrahepatic manifestations of HCV. Prednisone, cyclophosphamide, other chemotherapeutic agents, and plasmapheresis have been used with variable success; however, these approaches do not treat the underlying HCV infection.

Patients with vasculitis due to HCV infection may benefit from low-dose interleukin-2 therapy. This cytokine may promote the survival of immunosuppressive regulatory T cells.

HCV infection is associated with the development of B-cell non-Hodgkin lymphoma and monoclonal gammopathy of uncertain significance. The relative risk of lymphoma is small (1.28) in the USA. The most prevalent forms of lymphoma found in patients infected with HCV are follicular lymphoma, chronic lymphocytic lymphoma, lymphoplasmacytic lymphoma, and marginal zone lymphoma. Type 2 cryoglobulinemia evolves into lymphoma over time in 8% to 10% of patients. Despite the known association of HCV infection with lymphoma, HCV RNA does not integrate into the host genome and therefore HCV cannot be considered a typical oncogenic virus. Rather, HCV shows lymphotropism and may facilitate the development and selection of abnormal B-cell clones by chronic stimulation of the immune system. In addition, genetic rearrangements in B cells, specifically the Bcl2/J _H rearrangement and the t(14;18) translocation, have been found in HCV-infected patients in some, but not all, studies.

Other extrahepatic manifestations of HCV infection include porphyria cutanea tarda, lichen planus, and sicca syndrome. In addition, insulin resistance and diabetes mellitus have been thought to be associated with HCV infection, although the association has been questioned. The SVR to antiviral therapy for HCV infection is reduced in insulin-resistant patients; however, if HCV can be eradicated, insulin resistance often improves, an observation that further supports the relationship between HCV infection and insulin resistance. Although associations between HCV infection and both thyroid cancer and idiopathic pulmonary fibrosis have been described, data about the effect of HCV eradication on disease progression are lacking. A myriad of other conditions has been observed in association with HCV infection, but a true link has not been firmly established for these disorders (see Box 80.1 ).

Although not associated with disease, seropositivity for autoantibodies (e.g., ANA with a titer greater than 1:40 in 9%, smooth muscle antibodies with a titer greater than 1:40 in 20%, anti‒liver-kidney microsomal antibodies in 6%) is found in many HCV-infected persons. Therefore, the diagnosis of an autoimmune condition in a patient with HCV infection can never be based on serology alone.

The spectrum of extrahepatic manifestations may adversely impact the overall survival of HCV-infected persons. The prospective Taiwanese population-based R.E.V.E.A.L.-HCV (Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer) study, in which almost 24,000 adults 30 to 65 years of age were followed, demonstrated that HCV-infected persons had not only increased liver-related mortality, but also higher mortality from extrahepatic diseases compared with anti-HCV–negative persons.

Indirect Assays

EIAs detect antibodies against different HCV antigens. The time course of the development of symptoms, detection of anti-HCV, and appearance of HCV RNA after acute infection is shown in Fig. 80.3 . Three generations of EIAs have been developed. The third-generation EIAs detect antibodies against HCV core, NS3, NS4, and NS5 antigens as early as 7 to 8 weeks after infection, with sensitivity and specificity rates of 99%. Despite ongoing viral replication, serologic test results can be negative in patients who are on hemodialysis or are immunocompromised. Because the performance characteristics of third-generation EIAs are so good, confirmation with a recombinant immunoblot assay is no longer required. Instead, patients who are anti-HCV positive should undergo HCV RNA testing to determine if they have active viremia or have cleared the infection.

Direct Assays

Quantitative, highly sensitive, “real-time” HCV RNA tests represent the state of the art for determining HCV viremia in anti-HCV–positive persons. The lower limit of detection of most assays varies from 10 to 15 international units (IU)/mL. These assays have a linear dynamic range of 1 to 7 log ₁₀ IU/mL and are the preferred testing method in practice. Transcription-mediated amplification is also extremely sensitive, but available assays are not quantitative in the lower dynamic range of the test. The advantages of these very sensitive tests include positivity within 1 to 3 weeks after acute infection and detection of low-level residual infection during antiviral therapy.

A disadvantage of all quantitative tests is the lack of comparability among different assays. Although conversion to a standard IU/mL concentration attempted to resolve such discrepancies, results are still variable. Reported conversion factors vary from 0.9 copies/mL to 5.2 copies/mL per IU/mL. For this reason, the same laboratory and assay are recommended during antiviral treatment monitoring.

A cheaper and faster alternative to nucleic acid testing for HCV RNA to confirm HCV viremia is the HCV core antigen assay. Fully automated immunoassays have been developed that detect the HCV core antigen, and the assays have proved to be robust across HCV genotypes and in different patient populations, ^, but with major limitations in sensitivity. Therefore, the assay cannot be used to monitor response to antiviral therapy and make decisions regarding therapy. If viremia needs just to be confirmed, however, HCV core antigen testing is a reasonable alternative to HCV RNA testing.

HCV Genotype

Identifying the genotype and subtype of HCV is important because some DAA regimens are only recommended for certain HCV genotypes and subtypes. HCV genotyping can be accomplished by several methods. The most accurate approach uses PCR methodology and direct sequencing of the NS5B or E1 region; however, this approach is not practical in clinical practice. HCV genotyping can be performed by evaluating type-specific antibodies and has a 90% concordance in immunocompetent patients when results are compared with sequence analysis of the HCV genome. Testing can also be accomplished with reverse hybridization to genotype-specific probes, restriction fragment length polymorphism analysis, or PCR amplification of the 5′ noncoding region of the HCV genome. These tests have 92% to 96% concordance with the correct genotype; genotype 1 is identified with the highest accuracy. Because of mutations in the regions studied, errors in subtype identification occur in 10% to 25% of cases regardless of the technique used. A line-probe assay (INNO-LiPA) using genotype-specific probes for reverse transcription of the 5′ portion of the HCV genome is the most popular commercial assay for HCV genotyping.

Selection of Serologic and Virologic Tests

For patients at low risk for HCV infection, a negative result of an EIA for anti-HCV is sufficient to exclude HCV infection. HCV RNA testing should be performed to confirm active infection if a positive anti-HCV test is returned. In high-risk patients, such as those with an elevated serum ALT level who have a known risk factor for HCV, have experienced recent exposure, or are either immunocompromised or on dialysis, a positive anti-HCV result is sufficient to confirm HCV infection; however, HCV RNA testing should also be performed to confirm that the infection is active. If the anti-HCV result is negative, then HCV RNA testing should be performed in patients with a recent exposure in case the anti-HCV result is falsely negative because of insufficient time for anti-HCV to develop or immunocompromise in the host with failure to produce sufficient anti-HCV.

Factors Associated with Progression

Age is one of the most important risk factors for fibrosis progression in chronic HCV infection (see Table 80.2 ). A longer duration of infection has also been associated with a higher stage of liver fibrosis, but HCV infection acquired during childhood seems to follow a milder course. Overall, the development of HCV-related cirrhosis seems to be a dynamic process that accelerates exponentially with increasing age. The mechanisms by which progression of fibrosis accelerates with aging are not well defined. Changes in the regenerative capacity of the liver, alterations in the immune system, and telomere shortening may play roles. A higher risk for fibrosis progression in patients older than 40 years has been described in patients with various causes of liver disease. Some studies, but not others, have suggested that older age in general, and more specifically older age at the time of infection, is a risk factor for progression of fibrosis. Overall, cirrhosis has been predicted to develop in most patients with hepatitis C by about 65 years of age, irrespective of the age at infection.

Some studies have suggested that the mode of viral transmission may influence the degree of liver damage; however, the role of the route of transmission in fibrosis progression remains controversial. By contrast, female gender seems to be protective, and fibrosis progression is much faster in HCV-infected men, thereby suggesting that hormonal factors may be important in the regulation of liver fibrosis. Genetic factors also play a role in the development of cirrhosis. Histologic activity and the frequency of cirrhosis are lower in African Americans than in Caucasians. Several specific genes have been suggested to be involved in fibrosis progression; these include certain variants of the HLA class I and II antigens. A cirrhosis risk score based on polymorphisms in 7 genes has been proposed for patients with HCV infection ; this score was able to predict fibrosis progression in patients with initially mild chronic hepatitis C.

Elevated serum aminotransferase levels are used widely as a surrogate for ongoing intrahepatic inflammation, and elevated serum ALT levels during chronic hepatitis C are associated with an increased risk of liver fibrosis progression. Lower progression rates of fibrosis are reported in patients with normal serum ALT levels, but normal levels do not exclude the possibility of fibrosis progression.

Differences in the natural history of hepatitis C have been reported for different HCV genotypes. Several studies have described accelerated disease progression in patients infected with HCV genotype 3, which is consistent with higher reported mortality rates in patients infected with HCV genotype 3. Flares of hepatitis seem to occur more frequently in HCV genotype 2 infection and may result in a more severe course of liver disease. By contrast, viral load is not related to the degree of liver damage or fibrosis.

Hepatic steatosis is a histologic hallmark of chronic hepatitis C. Several studies have shown that steatosis is linked to the stage of liver fibrosis in patients with chronic HCV infection. Some studies suggest that HCV infection itself can trigger hepatic steatosis as well as NASH, and HCV infection may cause insulin resistance. There is also evidence for a direct association between HCV infection and hepatic steatosis. The strongest association exists between HCV genotype 3 infection and steatosis, and a direct molecular effect has been shown in a mouse model in which HCV genotype 3 is expressed.

Mild to moderately increased hepatic iron stores are associated with more advanced liver fibrosis. A consistent relationship between C282Y or H63D heterozygosity (see Chapter 75 ) and increased progression of fibrosis in patients with HCV infection has not been established, however. A reduction in hepatic iron concentrations does not reduce the risk of progression of fibrosis or improve the response to antiviral treatment.

Excessive alcohol consumption is clearly an independent major cause of cirrhosis, and chronic alcohol intake of more than 50 g/day is associated with a remarkable increase in the risk of cirrhosis in HCV-infected patients. On the other hand, coffee consumption has been reported to have a beneficial effect on overall mortality from HCV infection in population-based studies, and drinking coffee has been associated with a more favorable course of liver disease in general. Freedman and colleagues have also shown that greater coffee consumption correlates with a lower stage of liver fibrosis, reduced fibrosis progression, less steatosis and insulin resistance, and lower serum ALT levels; the best outcomes occur in persons who drink 3 or more cups per day. ^,

HCC

The incidence of HCC has been rising rapidly in the industrial countries since the 1980s (see Chapter 96 ). In the USA, the incidence of HCC is 3 times higher than in 1975, and the global HCV epidemic has contributed to the rising incidence of HCC worldwide. Overall, chronic hepatitis C is responsible for approximately 25% of cases of HCC worldwide, with particularly high prevalence rates in East Asia. Because the development of HCC in HCV-infected patients is an indolent and age-dependent process, the peak incidence of HCV-related HCC has not been reached yet in Europe and the USA, where the majority of infections occurred in the 1970s and 1980s. In some European countries such as Italy, however, the peak rate of HCC-related mortality may have been reached. In contrast to chronic hepatitis B, HCC due to HCV usually does not develop in noncirrhotic livers, although HCC may be detected in some patients in whom cirrhosis has not yet developed. Lok and colleagues reported an HCC frequency rate of 0.8% per year in noncirrhotic patients with chronic hepatitis C who had advanced liver fibrosis ; however, the risk is much higher in patients with cirrhosis, with a rate of 1.4% to 4.9% per year. The overall 5-year risk of HCC has been reported to be as high as 7% to 30% in patients with HCV-related cirrhosis. ^, The appearance of HCC is frequently the first clinical complication of HCV-related cirrhosis and often occurs before hepatic decompensation becomes evident.

Risk factors for the development of HCC in patients with chronic HCV infection are similar to those associated with the development of cirrhosis. For example, older age is related to a higher frequency of HCC, and male gender and substantial alcohol consumption are well-established risk factors. Moreover, type 2 diabetes mellitus has been identified as an important independent risk factor. ^, Coinfection with HBV increases the risk of HCC. Importantly, the various risk factors act synergistically to enhance the overall risk of HCC. Genetic factors also contribute to the development of HCC. Kumar and colleagues performed a genome-wide association study in 721 persons with HCV-related HCC and showed that a SNP (rs2596542) at the gene encoding MICA (major histocompatibility class I polypeptide-related sequence A) was strongly associated with the development of HCC in HCV-infected persons. Conversely, coffee consumption has been associated with a reduced risk of HCC.

Goals

The primary goal of therapy for HCV infection is eradication of the virus. A consequence of achieving this goal is prevention of liver-related deaths associated with the development of decompensated cirrhosis and HCC. SVR—the absence of detectable virus in blood 12 weeks after completion of therapy—is an excellent surrogate marker for the resolution of HCV infection. Late relapses are rare. Long-term follow-up studies confirm sustained responses in more than 99% of cases if the patient is HCV RNA negative in serum 12 weeks after completion of DAA therapy. SVR is also associated with a reduction in hepatic inflammation and regression of fibrosis during IFN plus RBV therapy ( Fig. 80.6 ). Moreover, an improvement in HRQOL has been documented in patients successfully treated with IFN plus RBV. Data regarding these endpoints during DAA therapy are limited owing to the more recent implementation of these regimens and thus limited long-term follow-up; however, elastography fibrosis scores improve following successful DAA therapy, although it is unclear if this represents true regression of fibrosis or resolution of hepatic inflammation. ^,

Antiviral treatment prevents the development of clinical endpoints. A significant reduction in liver-related deaths and hepatic decompensation can be observed following SVR with DAAs even in patients who already have advanced liver fibrosis. A reduction in the frequency of HCC has also been documented in patients who respond during antiviral therapy. ^, An SVR is also associated with improved overall survival in patients who had advanced fibrosis or liver cirrhosis at the time of therapy. ^, In particular, an SVR following PegIFN/RBV has been associated with a lower frequency of end-stage renal failure, cardiovascular disease, ^, and cerebrovascular disease. ^,

Indications and Contraindications

The development of highly efficacious and well-tolerated IFN-free DAA regimens, together with the clear benefit of HCV eradication on all-cause and liver-related mortality and quality of life, means that antiviral therapy should be considered in all patients with chronic hepatitis C. Furthermore, with the increasingly broad range of IFN-free and many RBV-free DAA regimens with different pharmacokinetic and drug-drug interaction (DDI) properties available, there are very few clinical scenarios for which treatment is contraindicated, with the exceptions being early acute HCV infection, decompensated cirrhosis with a high MELD score in a patient waitlisted for LT (see later and Chapter 97 ), and pregnancy.

Because RBV is a teratogen, unwillingness of the patient and his or her partner to practice adequate contraception and avoid pregnancy during treatment and for 6 months after the discontinuation of therapy is an absolute contraindication to starting or continuing a DAA regimen that includes RBV. There are insufficient data regarding the safety of the new DAAs in pregnancy, and, therefore, they are not recommended during pregnancy.

Virologic Response

During IFN-based therapy, the rate of HCV clearance from the circulation was an important predictor of subsequent SVR24. Predictors of an SVR included a rapid virologic response (RVR, negative HCV RNA level at treatment week 4) and a complete early virologic response (negative HCV RNA level at treatment week 12). However, with the extremely potent DAAs, the majority of patients achieve an RVR during DAA therapy. Furthermore, failure to attain an RVR does not preclude subsequent SVR12 with DAA-based therapy. ^, The AASLD/Infectious Diseases Society of America (IDSA) Guidance recommends HCV RNA testing at treatment week 4, not to determine treatment stopping rules, but to ensure treatment adherence.

Drugs

Acute Hepatitis C

Although postexposure prophylaxis against HCV is not effective, treatment of acute HCV infection is effective. However, early treatment of acute HCV was more important in the IFN era when various cohort studies demonstrated higher (>85%) response rates if acute hepatitis C was treated with IFN early after acquisition than when HCV infection was chronic and response rates were only 54% to 56% overall. Because a substantial proportion of patients may clear the acute infection spontaneously without any antiviral intervention, different strategies have been proposed to avoid unnecessary treatment, particularly with IFN. ^, The patient’s IFN-λ3 genotype can provide an indication as to whether a patient is more or less likely to recover spontaneously ^, ; however, the positive predictive value of IFN-λ3 testing is below 90%, and treatment decisions cannot be based solely on the patient’s IFN-λ3 genotype. A prospective randomized trial compared immediate treatment of acute hepatitis C with PegIFN alone and a delayed approach of starting combination therapy with PegIFN and RBV after 12 weeks in those patients who did not become HCV RNA negative spontaneously. Delayed therapy resulted in a lower SVR rate on an intention-to-treat analysis but gave similar response rates on an “adherence-to-therapy” analysis. Therefore, delayed therapy is effective, but early treatment has advantages because fewer patients are lost to follow up. Data are emerging on the efficacy of IFN-free DAA regimens for both acute HCV monoinfection and acute HCV infection in HIV-positive individuals ^, ; however, these studies are small, and consensus on the optimal treatment regimen, timing of initiation of therapy, treatment duration, and cost-effectiveness has not been established.

A reasonable approach for managing acute hepatitis C is to monitor the serum HCV RNA for at least 12 to 16 weeks before DAA treatment is considered to allow adequate time for spontaneous clearance. Treatment of acute hepatitis C in the context of HIV coinfection is similar to that for patients with HCV monoinfection. ^{, ,} Given the high efficacy and favorable safety profiles of the DAA regimens, the same regimens used for chronic HCV infection are recommended in acute HCV infection (see later). ^, IFN-based regimens are no longer recommended.

Chronic Hepatitis C

In contrast to most other chronic viral infections, cure of HCV infection is possible. HCV has an entirely cytoplasmic life cycle; therefore, suppression of viral replication in the absence of resistance can cure HCV-infected cells.

Until 2011, the standard of care for the treatment of HCV infection was the combination of a PegIFN plus RBV. However, IFN-based therapy was associated with poor overall cure rates (54% to 56%), ^{, ,} with significant toxicity leading to discontinuation of therapy in 14% of patients. In addition, many patients were either ineligible for or intolerant of IFN, thereby limiting treatment options in these patients.

The first DAAs (telaprevir, boceprevir, simeprevir, and sofosbuvir) were developed as “triple” therapy to be used in combination with PegIFN plus RBV. Although SVR rates were higher, limitations included restricted HCV genotypic activity, additional toxicity beyond PegIFN/RBV, poor response in cirrhotic patients and prior null responders, and RAS considerations, as well as a requirement for PegIFN/RBV eligibility. Subsequently, combination IFN-free DAA regimens were developed (see earlier). IFN-based regimens are no longer recommended for treatment of HCV infection.

Combinations of DAAs that target distinct and complementary steps in the HCV life cycle (see Fig. 80.1 ) have revolutionized the treatment of HCV infection, yielding SVR rates in excess of 95% with minimal toxicity. The first proof-of-concept study showing an SVR in 4 patients with chronic hepatitis C with an IFN-free DAA combination therapy was published in 2012. The compounds investigated were the HCV NS5A inhibitor daclatasvir and the HCV NS3 protease inhibitor asunaprevir. SVR rates were only 22% (2/9) in HCV genotype 1a patients; however, SVR rates were considerably higher in HCV genotype 1b patients (90% for treatment-naïve patients and 82% for patients who were prior nonresponders to or ineligible for IFN), providing proof-of-concept that IFN-free therapy can cure HCV, but that the efficacy of all-oral combination regimens may differ among HCV genotypes and subtypes. Since this study, many DAAs have undergone clinical development, 13 of which have been approved by the FDA as of early 2020 (5 protease inhibitors, 6 NS5A inhibitors, and 2 NS5B inhibitors). The oldest IFN-free DAA regimens are HCV genotype specific, whereas the more recently approved IFN-free regimens have pangenotypic coverage. IFN-free DAA regimens are discussed in the context of each HCV genotype/subtype, presence or absence of cirrhosis, and treatment history, because these factors are important considerations when choosing an IFN-free DAA regimen. Special populations, including persons with decompensated cirrhosis, HCV recurrence following LT, and renal impairment are discussed later.

The 13 DAAs approved by the FDA for the treatment of chronic hepatitis C infection include the NS3/4A protease inhibitors simeprevir, paritaprevir (ritonavir boosted), grazoprevir, glecaprevir, and voxilaprevir; the NS5A inhibitors daclatasvir, ombitasvir, ledipasvir, elbasvir, velpatasvir, and pibrentasvir; and the NS5B inhibitors sofosbuvir and dasabuvir (see Table 80.3 ). Pangenotypic drugs or FDCs include sofosbuvir, sofosbuvir/velpatasvir, sofosbuvir/velpatasvir/voxilaprevir, and glecaprevir/pibrentasvir. Genotype-specific drugs or FDCs include sofosbuvir/ledipasvir, and elbasvir/grazoprevir. RBV can be added to some IFN-free DAA regimens in more difficult-to-treat populations, such as HCV genotype 3 cirrhotic patients.