STRUCTURE

The HCV virion has been visualized by electron microscopy and is an enveloped virus 50 nm in diameter.⁸ The two envelope proteins, E1 and E2, heterodimerize and assemble into tetramers, which create a smooth outer layer. This layer 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 ribonucleic acid (RNA). HCV circulates in various forms in the serum of an infected host, including (1) virions that are bound to very-low-density and low-density lipoproteins and appear to represent the infectious fraction; (2) virions bound to immunoglobulins; and (3) free virions. In addition, viral particles that exhibit physicochemical, morphologic, and antigenic properties of nonenveloped HCV nucleocapsids have been detected in plasma.⁹

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.¹⁰ The genome of HCV contains approximately 9600 nucleotides with an open reading frame (ORF) that encodes one large viral polypeptide precursor of 3008 to 3033 amino acids. The HCV ORF is flanked upstream by a 5′ untranslated region (UTR) that functions as an internal ribosome entry site (IRES) 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.^11–13 The 5′ and portions of the 3′ UTR are the most conserved parts 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.^14,15 Understanding of the mechanisms of viral replication comes from studies in chimpanzees, extrapolation of the mechanisms used by other flaviviruses, and infection of cells in vitro with subgenomic replicons (segments of HCV RNA that are capable of amplification and synthesis of viral proteins but that do not produce mature viruses).

Early events in viral binding to the hepatocyte surface are still not completely understood (Fig. 79-1). HCV entry involves the attachment of envelope proteins E1 and E2 to cell surface molecules. The expression and function of CD81, a member of the tetraspan superfamily, are essential for HCV entry into hepatocytes.¹⁶ In addition, human scavenger receptor class B type 1 (SR-B1), a selective importer of cholesteryl esters from high-density lipoproteins (HDL) into cells, has been shown to interact with E2 and is essential for HCV entry.¹⁷ Whereas CD81 and SR-B1 are required early in the process of viral entry, claudin-1 (CLDN1), a tight junction component that is highly expressed on hepatocytes, is required later in the cell entry process.¹⁸ Heparin sulfated proteoglycans have also been shown to be essential for HCV cell entry.¹⁹ Other receptors are also likely required for viral entry.¹⁸ There is some evidence that the low-density lipoprotein (LDL) receptor is involved during endocytosis of HCV.²⁰

Figure 79-1. Putative life cycle of hepatitis C virus.

(Reproduced with permission from Pawlotsky JM, Chevaliez S, McHutchison JG. The hepatitis C virus life cycle as a target for new antiviral therapies. Gastroenterology 2007; 132:1979-98.) (See text for details and Fig. 79-2 for functions of the hepatitis C virus proteins.) NS, nonstructural; RdRp, RNA-dependent RNA polymerase.

Not only do viral receptors on hepatocytes play a role in entry of HCV into the cell, but also other cofactors affect the efficiency of viral infectivity. C-type lectins DC-SIGN (dendritic cell–specific intercellular adhesion molecule-3-grabbing nonintegrin; CD209) and L-SIGN (DC-SIGNr, liver and lymph node specific; CD209L) are expressed on dendritic cells and liver sinusoidal endothelial cells, respectively.²¹ Both receptors bind E2 on the HCV virus and facilitate infection of adjacent hepatocytes by acting as a capture and delivery mechanism. In addition, HDL increases infectivity of hepatocytes ten-fold, although the mechanism is not understood.²²

Once the HCV virus attaches to the cell, endocytosis of the bound virion is presumed to occur, as in other flaviviruses. A pH drop in the vesicle causes conformational changes in the glycoproteins that lead to fusion of the viral and cellular membranes²³ and release of viral RNA into the cytoplasm. In the cytosol, the 5′ UTR contains several highly conserved and structured domains that function as an IRES, 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 (eIF) 2 and 3, and viral proteins.^24,25

The large polyprotein generated by translation of the HCV genome 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. 79-2).^11,13 The functions of these specific proteins are described later in the chapter.

Figure 79-2. Schematic representation of the hepatitis C virus polyprotein. The structural proteins C (core), E1, and E2 (envelope proteins) are cleaved from the polyprotein by the host signal peptidase. p7, a viroporin protein, is cleaved by the endoplasmic reticulum signal peptidase and forms an ion channel that is essential for assembly and release of infectious virions. The NS2 cysteine protease autocatalytically cleaves itself from the polyprotein. The NS3 protease cleaves the remainder of the nonstructural proteins: NS3 (serine protease and RNA helicase), NS4A (NS3 protease cofactor), NS4B, NS5A (RNA binding site), and NS5B (RNA-dependent RNA polymerase).

After polyprotein processing, NS4B expression causes the membrane alterations that are seen on electron microscopy as a membranous web.^26,27 The replication complex associates viral proteins, cellular components, and nascent RNA strands and is essential for HCV replication, as demonstrated in replicon cell culture systems.²⁸ 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. 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 are packaged into new virus particles.²⁹

Finally, viral particle formation is initiated by the interaction of the core protein with genomic RNA in the endoplasmic reticulum, although the details of this process and subsequent export of mature virions from the hepatocyte are poorly understood.^30,31 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.⁵⁶ This high mutational rate is in part a consequence of the RNA-dependent RNA polymerase 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 10⁴ to 10⁵ nucleotides copied. This phenomenon is favored by a high viral turnover rate; 10¹⁰ to 10¹² virions are produced per day.⁵⁷ 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.⁵⁶ 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 6 major genotypes (designated by numbers), with sequence similarities of 60% to 70%, and more than 70 subtypes (designated by a lower case letter) within these major genotypes, with sequence similarities of 77% to 80%.⁵⁶ In this scheme, the first variant, which was cloned by Choo and colleagues, is designated type 1a.⁵⁸ 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, inter-genotypic HCV recombinants have been described⁵⁹; 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 NS3.⁶⁰

Global geographic differences exist in the distribution of HCV genotypes, as well as in the mode of acquisition. In the United States, genotype 1a is the most prevalent, accounting for approximately 57% of HCV infections, followed by genotype 1b in 17%, genotype 2 in 14%, genotype 3 in 7%, and genotype 4, 5, or 6 in less than 5%.⁶¹ 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 1.⁶² In Europe, the most prevalent genotype is 1b (47%), followed by 1a (17%), 3 (16%), and 2 (13%).⁶³ Genotype 4 is found mainly in Egypt, the Middle East, and Central Africa.⁶⁴ In Egypt, approximately 15% of the population is infected with HCV, and more than 90% have HCV genotype 4. Because of the high prevalence rate in Egypt, genotype 4 represents 20% of the world’s HCV-infected population. Genotype 5, although originally isolated in South Africa, is also seen in specific regions of France, Belgium, and Spain.⁶⁵ 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.⁶⁶

An important clinical correlation with HCV genotype is response to treatment (see later). HCV genotype does not, however, appear to influence the severity of liver disease, as defined by the stage of fibrosis, or the likelihood of progression of acute to chronic HCV infection.

The second component of genetic heterogeneity is quasispecies generation.⁵⁶ 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 is 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.⁶⁷ During acute infection or during treatment, the lack of quasispecies is associated with viral clearance, and the development of numerous quasispecies is associated with viral persistence.⁶⁸ 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.⁶⁷ Genetic variation before seroconversion does not correlate with outcome, indicating that quasispecies formation results from antibody-mediated immune pressure. Interestingly, no intrinsically interferon-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.⁶⁹

INCIDENCE AND PREVALENCE

The worldwide seroprevalence of HCV infection, based on detection of antibody to HCV (anti-HCV), is estimated to be 3%, with more than 170 million people infected chronically. Marked geographic variation exists, with infection rates ranging from 1.3% to 1.6% in the United States to 15% in Egypt.^1,64 Currently, between 3.2 and 5 million persons are infected with HCV in the United States.¹ The prevalence rate is higher in persons 40 to 49 years old than in older or younger persons, in males (2.1%) than in females (1.1%), and in African Americans (3%) than in whites (1.5%).¹ Other risk factors for HCV infection and the associated frequencies of infection are injection drug use (57.5%), blood transfusion before 1992 (5.8%), greater than 50 lifetime sexual partners (12%), and family income below the poverty level (3.2%). The current prevalence of HCV infection in the United States may be underestimated because the National Health and Nutrition Examination Survey (NHANES) data did not evaluate persons who are homeless, incarcerated, or in the military. Among incarcerated persons, 12% to 35% are positive for HCV RNA serum,⁷⁰ whereas those in military service have a seroprevalence rate for anti-HCV of 0.5%.⁷¹

Worldwide, three different epidemiologic patterns of HCV infection have emerged: (1) previous exposure through health care with a peak prevalence in older persons; (2) exposure through intravenous 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.⁶³

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.⁷² 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 genotypes 5 and 6 are 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.⁷³ 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 United States, Australia, and other developed countries, peak prevalence is in persons ages 40 to 49 years, and analysis of risk factors suggests that most HCV transmission occurred between the mid-1980s and the mid-1990s, through intravenous 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.⁶⁴ 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 United States, the incidence of acute hepatitis C is falling. The peak incidence was estimated to be 180,000 cases per year in the mid-1980s, but the rate declined to approximately 19,000 new cases by 2006.⁷⁴ 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.⁷⁵ 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.⁶⁶

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 United States. Currently, new HCV infections in the United States 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 “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.^97,98 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.⁹⁹ Survival in such patients is quite 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 are not readily available and most studies in man 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 as well as 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 (PAMPs) recognized by the cell.^100–102 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 tumor necrosis factor (TNF)-β and interferon-α, cytokines that are critical for dendritic cell maturation and subsequent induction of adaptive immunity.⁹⁴ After this, 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.⁹³ 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.^103,104 PAMPs activate several cellular processes including the JAK-STAT pathway and Toll-like receptor-3 (TLR-3), activation of both of which ultimately results in production of cellular interferons and interferon-regulated factors that convey antiviral properties to the cell.⁹³ NS3/4 protease degrades TRIF, an essential intermediate in this pathway, and cleaves interferon promoter stimulator-1 (IPS-1), an intermediate in the signaling cascade, to activate interferon when retinoic inducible gene-1 (RIG-1) binds viral intermediates.^47,105,106 In addition, HCV core protein promotes STAT-1 degradation, inhibits STAT-1 phosphorylation, promotes suppressor of cytokine signaling (SOCS) induction (an inhibitor of JAK-STAT signaling), and impairs interferon-stimulated gene factor-3 (ISGF3), a heterotrimer of STAT-1, STAT-2, and interferon-β promoter stimulator (IRF-9) from binding to the promoter regions of interferon-stimulated response elements (ISRE), thereby inhibiting transcription of interferon-response genes. Even when interferon-response genes are activated, NS5A and E2 both can disrupt protein kinase R (PKR) function to suppress translation, thereby allowing viral replication to continue.⁹³ In addition, NS5A inhibits 2′-5′-oligoadenylate synthetase (OAS). 2′-5′-OAS is expressed in response to HCV infection and leads to HCV RNA degradation.⁹³ Taken together, HCV is able to disorient 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.^107–109 Dendritic cells from HCV-infected patients are more likely to produce IL-10, a cytokine that inhibits antigen-specific T-cell responses. In addition, CD4⁺ T cells primed from dendritic cells of HCV-infected patients are more likely to produce IL-10. Even if an adequate T-cell response is created, HCV-infected patients have a large number of regulatory T cells in their portal tracts¹¹⁰; 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.^95,111 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.^95,111

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 in low levels and do not appear to correlate with the stage of infection or immune reactivity. Furthermore, administration of high-titer HCV-enriched or HCV-specific immunoglobulin has little effect on viral levels or persistence in humans.¹¹²

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.

ACUTE AND CHRONIC HEPATITIS C

HCV accounted for an estimated 20% of cases of acute hepatitis in 2006.⁷⁴ Acute hepatitis C is rarely seen in clinical practice because nearly all cases are asymptomatic.^87,113 Jaundice probably occurs in about 10% of patients with acute HCV infection, whereas 20% to 30% of patients present with nonspecific symptoms such as fatigue, nausea, and vomiting. HCV RNA is detectable within 2 to 3 weeks of exposure, and anti-HCV seroconversion occurs between day 15 and month 3. Serum aminotransferase levels peak at about the first month after exposure (Fig. 79-3), exceed 1000 IU/L in 20% of cases, and generally follow a fluctuating pattern for the first few months. In patients in whom jaundice develops, peak serum bilirubin levels usually are less than 12 mg/dL, and jaundice typically resolves within one month. Severe impairment of liver function and liver failure are rare. 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.

Figure 79-3. Serologic pattern of acute hepatitis C virus (HCV) infection followed by recovery. Symptoms may or may not be present during acute infection. ALT, serum alanine aminotransferase level; anti-HCV, antibody to hepatitis C virus.

(Modified from the Centers for Disease Control and Prevention, www.cdc.gov/hepatitis/Resources/Professionals/Training/Serology/training.htm#one.)

The rate of viral persistence after acute infection varies, ranging 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 injection drug users 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 United States). Finally, the rate of spontaneous clearance is higher in symptomatic patients in whom jaundice develops during acute infection than in those who remain asymptomatic.^87,113,114 In patients with community-acquired hepatitis C in whom the infection resolves spontaneously, loss of HCV RNA from serum usually occurs within three to four months of the onset of clinical disease.¹¹⁴

Serum alanine aminotransferase (ALT) levels are usually elevated in patients with chronic HCV infection. Because levels commonly fluctuate, however, as many as one half of patients may have a normal ALT level at any given time.¹ The ALT level may remain normal for prolonged periods of time in about 20% of cases,¹¹⁵ although transient elevations occur even in these cases.¹¹⁶ 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.¹¹⁵

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 fatigue or depression, and they consistently score lower than HCV-negative persons in all aspects of health-related quality of life (HRQL).^117,118 Whether the decrease in HRQL is related to viral factors, social factors (e.g., intravenous drug use), social stigmatization, or worry related to the diagnosis itself is unclear. Nonetheless, HRQL scores improve if the patient achieves a sustained response to antiviral therapy (see later). 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 hepatitis C. Classification of the extrahepatic manifestations of HCV is shown in Table 79-1 and is based on the strength of available data to prove a correlation.¹¹⁹ Types 2 and 3 cryoglobulinemia, characterized by polyclonal immunoglobulin G (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’s 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.

Table 79-1 Extrahepatic Manifestations of Hepatitis C Virus Infection

Glomerular disease generally manifests as cryoglobulinemic nephropathy, membranoproliferative glomerulonephritis (MPGN), and membranous nephropathy.^119,120 Cryoglobulinemic nephropathy manifests as hematuria, proteinuria, edema, and renal insufficiency of varying degrees, and on renal biopsy specimens it has features of MPGN. 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.

Therapy should be considered in patients with symptomatic cryoglobulinemia. Cryoglobulinemia resolves in patients who achieve an SVR with pegylated interferon and ribavirin therapy (see later).¹¹⁹ Unfortunately, patients with significant renal involvement are at a disadvantage with respect to antiviral therapy because administration of ribavirin is generally contraindicated if the creatinine clearance is less than 50 mL/min. In these patients, treatment with rituximab should be considered.^121,122 A durable clinical response to four doses of rituximab has been reported, although no prospective clinical trials have been completed to date. Prednisone, cyclophosphamide, other chemotherapeutic agents as well as plasmapheresis have been used with variable success; however, these approaches do not treat the underlying HCV infection.¹¹⁹ If cryoglobulinemia and renal disease improve with such treatment, then subsequent treatment of the HCV infection with pegylated interferon-α and ribavirin should be reconsidered.

HCV infection is associated with the development of B-cell non-Hodgkin’s lymphoma and monoclonal gammopathy of uncertain significance.^119,123,124 The relative risk of lymphoma is small (1.28) in the United States.¹²³ The most prevalent forms of lymphoma found in patients infected with HCV are follicular lymphoma, chronic lymphocytic lymphoma, lymphoplasmacytic lymphoma, and marginal zone lymphoma. In addition, marginal splenic lymphoma has been reported to regress after therapy for HCV infection alone. An estimated 8% to 10% of patients with type 2 cryoglobulinemia evolve into lymphoma over time. Despite the known association of HCV infection with lymphoma, HCV RNA does not integrate into the host genome and 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.^125,126 Patients with B-cell genetic alterations are less likely to respond to antiviral therapy.¹²⁷

Other extrahepatic manifestations of HCV infection include porphyria cutanea tarda, lichen planus, and sicca syndrome.¹¹⁹ In addition, insulin resistance and diabetes mellitus are associated with HCV infection. 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.¹²⁸ 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 (see Table 79-1). A myriad of other conditions have been observed in association with HCV infection, but a true link has not been firmly established for these disorders.¹¹⁹

Although not associated with disease, seropositivity for autoantibodies is found in many HCV-infected persons (e.g., antinuclear antibodies 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%).¹²⁹ In addition, anti-thyroid peroxidase is found in serum in 5% to 12% of HCV-infected patients, although associated thyroid disease is found in only 2% to 5% of patients.¹¹⁹ Therefore, the diagnosis of an autoimmune condition in a patient with HCV infection can never be based on serology alone.

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 Figure 79-3. Three generations of EIAs have been developed. The latest, 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%.¹³¹ Despite ongoing viral replication, serologic test results can be negative in patients who are on hemodialysis or are immunocompromised.⁸⁰ Because the performance characteristics of third-generation EIAs are so good, confirmation with a recombinant immunoblot assay (RIBA) 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

Two general types of direct assays exist: qualitative and quantitative HCV RNA tests.¹³⁰ Qualitative HCV RNA nucleic acid tests (NAT) only report whether HCV RNA is found in serum or not and do not quantitate the amount of HCV RNA. These tests should be used only for screening purposes now (e.g., screening of blood donated to a blood bank) and should not be used in clinical practice. As a result of NAT technology, transfusion-related HCV infection has decreased to less than one case per two million units of blood transfused.^76,77

Unlike NAT testing, quantitative HCV RNA tests are essential for monitoring the response to antiviral therapy (see later). Currently “real-time” tests, such as Taqman, are performed using polymerase chain reaction (PCR) methodology, with a lower limit of detection of 10 to 15 international units (IU)/mL.¹³² These assays have a linear dynamic range of 1 to 7 log₁₀ IU/mL and are the preferred testing method in practice. Transcription-mediated amplification (TMA) 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 one to three 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. Thus, conversion factors vary from 0.9 copies/mL to 5.2 copies/mL per IU/mL reported. For this reason, the same laboratory and assay should be used during antiviral treatment when comparisons of levels at different points in time are critical to decision making about treatment.

SELECTION OF SEROLOGIC AND VIROLOGIC TESTS

For patients at low risk for HCV infection, a negative result on an EIA for anti-HCV is sufficient to exclude HCV infection. A positive anti-HCV result is sufficient to confirm the diagnosis if the serum ALT level is elevated. If the serum ALT level is not elevated, HCV RNA should be measured. In high-risk patients, such as those with an elevated 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. If the anti-HCV result is negative, then HCV RNA testing should be done.