A sero-negative arthritis is present in up to 20% of subjects with long standing HCV infection. Other common manifestations are carpal tunnel syndrome in 6%, Raynaud's phenomenon in 3.5%, and symptoms of Sjogren's syndrome in 8.7%. Up to 40% of patients with chronic hepatitis C may have low levels of cryoglobulins in serum, but only 1 percent have symptomatic cryoglobulinemia with fatigue, arthralgias, skin rash (a palpable purple rash on the arms, trunk, or legs or as a form of kidney failure called glomerulonephritis), renal disease, or neuropathy.

Centers for Disease Control and Prevention (CDC) estimate that the annual incidence of acute HCV infection in the United States decreased from an average of approximately 230,000 new cases per year in the 1980s to 38,000 cases per year in the 1990s.

Hepatitis C infection is common, affecting nearly 2 percent of the general population and a much higher percentage of people under special circumstances. Since the early 1990s, national statistics indicate that morbidity, mortality, and health care utilization associated with consequences of long-standing infection with hepatitis C are increasing in epidemic proportions. Future projection studies predict that the increase will continue in the foreseeable future.

The natural history is a product of the outcome of the acute infection as well as the outcome of the subsequent chronic hepatitis. A problematic issue is the actual timing of evolution to chronic hepatitis. Traditionally, this has been based on persistence of virus for at least 6 months. However, viremia may persist beyond this time, although it is believed that loss of virus after one year is exceptional. Prospective study has indicated that chronic hepatitis evolves in about 85 percent of acutely infected persons. On the other hand, cross-sectional studies of large, untreated anti-HCV positive cohorts, consisting mainly of young persons, many of them female, have reported absent virus in as many as 45-50 percent of instances, implying a higher rate of spontaneous recovery in some groups. Thus, spontaneous recovery from acute hepatitis C occurs in 15-45 percent of instances.

Progression to Cirrhosis
Once chronic hepatitis has developed, the question then is: What are the long-term sequelae? Numerous efforts have been made to define the frequency and rate of progression to cirrhosis and HCC. Evident in all these studies is that clinically overt liver disease is generally not seen in the first two decades following the acute infection. This does not imply that cirrhosis does not evolve during this period, but the actual timing of its onset cannot be determined without performing serial liver biopsies. Early reports, based largely on retrospective studies, indicated that, at the end of two decades of infection, about 20 percent had developed cirrhosis, although some of the studies have reported rates of almost 50 percent. The drawbacks of retrospective studies are that evaluation is limited to those who have achieved an end point and that tracing to disease onset is hindered by the paucity of symptoms at onset. Thus, ascertainment bias may exist using this approach. Later prospective studies, mainly of HCV-infected transfusion recipients, reported a lower rate of development of cirrhosis (7-16 percent), but most of these studies were too short in duration to provide an accurate assessment of the ultimate outcome. Even lower rates of cirrhosis have been reported among several groups in whom it was possible to trace back far in the past to the time of onset or near onset. Thus, among children infected through transfusion in the first years of life and traced 20 years later, and among young women infected through receipt of HCV-contaminated Rh immunoglobulin and traced over approximately the same time period, cirrhosis was noted to have occurred in about 2 percent. A similar rate was noted in a 45-year follow up of young HCV-positive military recruits who had been bled at the time of serving on a military base, the samples having been retained in a repository. The common theme of this lower rate of cirrhosis is that it was noted among persons infected at a young age.

Taking the numerous variety of studies into account, a group of Australian investigators who reviewed the world's literature for the rate of cirrhosis development at 20 years concluded that the studies could be divided into 4 broad categories: those performed in liver clinics, the mean cirrhosis rate being 22 percent (95 percent CI, 18-26 percent); post-transfusion hepatitis studies, with a mean of 24 percent (11-37 percent); studies of blood donors, with a mean of 4 percent (1-7 percent); and studies of community-based cohorts, with a mean of 7 percent (4-10 percent). They concluded that selection bias accounted for the two higher rates, and that the community-based cohort studies appeared more representative in estimating disease progression at a population level. These data provide useful figures for the frequency of progression to cirrhosis two decades after acute infection that appears to range between about 2-4 percent to 20-25 percent, depending on several factors, to be described below. However, many of those infected are young and are destined to live for several more decades. Therefore the question that must be posed is: What happens after the first two decades with regard to liver disease progression? Does fibrosis progression continue to increase at a linear rate? Does the rate level off and remain the same throughout life? Does fibrosis progression increase as age advances? Certainly, many chronically infected persons are known to live for a lifetime without succumbing to liver disease, whereas others are known to develop end-stage liver disease 30 to 60 years after acute infection. Thus, these questions can only be answered by conducting markedly extended studies, few of which have been accomplished for obvious reasons. Other approaches have been to model the expected outcome based on preconceived notions, models that may or may not turn out to be valid. Most important, is it possible to predict in the individual HCV-infected person what the outcome is likely to be? The answer is a qualified maybe, taking into account the many factors that might enhance progression.

Factors That May Determine Progression
The differing outcomes suggest that there are variables that may contribute to the rate of liver disease progression. These can be considered as being viral-related, host-related, or a consequence of external factors.

Viral-Related
Factors that might contribute include viral load, viral genotype, and quasispecies diversity. There is little evidence to indicate that viral load plays a role in disease progression; there are suggestions that progression is more likely following infection with genotypes 1a and 1b than genotype 2, although this has been disputed, most studies now reporting that there is no effect of genotype characteristics on disease outcome. While the degree of quasispecies diversity appears to play a role in evolution from acute to chronic hepatitis, there is no evidence that it enhances progression of already established chronic hepatitis.

Host-Related
One of the most important determinants is age at the time of infection, the relationship being an inverse one. What is not yet established is whether the relatively mild disease seen two decades after infection of young people will begin to accelerate with increasing age. This brings into account the fact of duration of infection, since it is rare although not unheard of, to identify end-stage liver disease in under one-and-a-half to two decades. Perhaps the flourishing of liver disease with time may be a consequence in part of age-related immune depression. Certainly, an immune suppressed state vigorously enhances disease progression as is noted among infected persons with hypogammaglobulinemia and, especially, HIV co-infection. Hepatitis B and schistosomal co-infection also increase disease progression perhaps through induced immune dysfunction as well as through direct cytotoxicity. Genetic background also may be of importance. Genes of the major histocompatability complex appear also to play a role, not so much in fibrogenesis, but in clearance of the virus. HLA class I antigens seem to be associated with viral persistence whereas class II antigens (DRB1 alleles) are identified more frequently in those who clear virus and therefore have milder disease. Inheritance of high TGF-â 1 and angiotensinogen-producing genotypes has been linked to fibrosis progression. Co-morbid conditions such as hemochromatosis and non-alcoholic steatohepatitis are also associated with advancing chronic liver disease. In addition, outcome may be influenced by gender and race. Females are reported to have a slower rate of progression, a finding that seems to be emerging also among African-Americans. Finally, the expression of the disease plays a role in outcome. HCV-infected persons with raised aminotransferase levels are far more likely to develop progressive liver disease than are those with normal serum enzymes.

External Factors
Clearly, associated chronic alcoholism is a powerful co-factor in liver disease progression. Yet to be determined is what is the least amount of alcohol and the type of drinking pattern that plays a role in advancing chronic hepatitis C. Also of note are the data suggesting that smoking may increase disease progression. Exposure to toxic products, either in the form of administered drugs that may be hepatotoxic or as environmental contaminants, may have important effects. It is noteworthy that death associated with chronic hepatitis C in the United States is more likely to be a result of end-stage liver disease rather than HCC, whereas in Japan, virtually all deaths are attributed to HCC. It has been suggested that the difference is a consequence of a longer duration of HCV infection in Japan than in the United States, a view that may or may not be valid. Another possible explanation is that toxic environmental contaminants may play a contributory role in Japan.

Indirect Tests
Anti-HCV detection. Anti-HCV is typically detected using second- or third-generation enzyme immunoassays (EIAs) that detect mixtures of antibodies directed to various HCV epitopes. The specificity of currently available EIAs for anti-HCV is higher than 99 percent. Their sensitivity is more difficult to determine in the absence of a more sensitive gold standard. EIAs for anti-HCV detect antibodies in more than 99 percent of immunocompetent patients with detectable HCV RNA. EIAs are sometimes negative despite the presence of active HCV replication in hemodialysis patients or patients with profound immunodeficiencies. Immunoblot tests have been used in the past as confirmatory assays. Given the good performance of the current anti-HCV EIAs, immunoblot tests no longer have utility in the clinical virology setting. They are still useful in the blood bank setting, where the positive predictive value of a positive EIA result is significantly lower than in the diagnostic setting.

Serological determination of HCV genotype. HCV genotype can be determined by detection of type-specific antibodies using a competitive EIA (so-called "serotyping"). This assay provides interpretable results in approximately 90 percent of immunocompetent patients with chronic hepatitis C. Its sensitivity is lower in hemodialysis or immunodepressed patients. The assay identifies the type (1 to 6) but not the subtype of HCV. Concordance with molecular assays is in the order of 95 percent. Currently, no serotyping assay is FDA-approved.

Direct Tests

Available Tests

Qualitative detection of HCV RNA. Qualitative (i.e., nonquantitative) HCV RNA detection assays are useful because they are significantly more sensitive than most available quantitative assays. The qualitative assays are based on the principle of target amplification using either polymerase chain reaction (PCR) or transcription-mediated amplification (TMA). The lower detection cutoffs of the corresponding commercial assays are 50 HCV RNA international units (IU)/ml and 10 IU/ml, respectively. Their specificity is of the order of 98-99 percent. The PCR assay is FDA-approved.

Viral level quantification. HCV RNA level can be quantified by means of target amplification techniques (PCR or TMA) or signal amplification techniques ("branched DNA" assay). The lower detection cutoffs of the current assays vary between 30 IU/ml and 615 IU/ml, and the upper limit of linear quantification between 500,000 IU/ml and 7,700,000 IU/ml. Samples with a viral level higher than the upper limit of an assay should be retested after 1/10 or 1/100 dilution. Quantification is independent of the HCV genotype. The international unit, recently defined with reference to the WHO HCV RNA standard, should be used in any HCV RNA quantitative assay in order to compare results given by different assays and to apply global recommendations. Variations of less than 0.5 logs (i.e., of less than threefold) should not be taken into account as they may relate to the intrinsic variability of the assays. No HCV RNA quantification assay is approved currently in the United States, but several are likely to be in the future.

Molecular determination of HCV genotype (genotyping). The gold standard for genotyping is direct sequencing of the NS5B or E1 regions. In clinical practice, HCV genotype can be determined by direct sequence analysis, reverse hybridization onto genotype-specific oligonucleotide probes, or restriction fragment length polymorphism analysis after PCR amplification of the 5' noncoding region. Typing errors are uncommon, but subtyping errors may occur in 10-25 percent of cases. These errors may be related to the region studied (5' noncoding) rather than the technique used. Subtyping errors have few clinical consequences because only the genotype is useful for clinical decisions. No genotyping assay is currently approved in the United States.

Detection and quantification of total HCV core antigen. Total HCV core antigen can be detected and quantified by means of EIA assay. The HCV core antigen titer (in pg/ml) correlates closely with HCV RNA level, and thus can be used as an indirect marker of viral replication. However, the current version of the assay does not detect HCV core antigen when HCV RNA is below approximately 20,000 IU/ml. This assay is not FDA-approved.

Practical Use of Virological Tests
The phrase "HCV RNA detection by means of a sensitive technique" used in this presentation refers to a technique with a lower limit of detection of 50 IU/ml or less. Furthermore, in discussing HCV RNA quantitation, it is assumed that the results are within the limits of its range of linear quantification of the assay.

Diagnosis of HCV Infection

Acute hepatitis C. During acute hepatitis of unknown origin, anti-HCV should be tested by EIA and HCV RNA by a sensitive HCV RNA technique. The presence of HCV RNA without anti-HCV is strongly indicative of acute hepatitis C, a diagnosis that can be confirmed by subsequent seroconversion. In the absence of both markers, acute hepatitis C is unlikely. In the presence of both, it is difficult to differentiate acute hepatitis C from an acute exacerbation of chronic hepatitis C or from acute hepatitis of other cause in a patient with chronic hepatitis C.

Chronic hepatitis C. In a patient with chronic liver disease, the diagnosis of chronic hepatitis C can be made based on detection of both anti-HCV and HCV RNA using a sensitive technique. The lack of anti-HCV in the presence of HCV RNA is uncommon in immunocompetent patients with chronic hepatitis C. It can occur (although rarely with the current EIAs) in hemodialysis or profoundly immunodeficient patients.

Mother-to-infant transmission. The diagnosis of HCV infection in a baby born to an HCV-infected mother should be based on the detection of HCV RNA with a sensitive technique rather than anti-HCV, because antibodies are passively transferred in utero and remain detectable for several months to more than a year after delivery regardless of whether transmission occurs. The optimal timing for HCV RNA testing for diagnosis is not known. Appropriate times are 6 to 12 months after birth.

Diagnosis of infection after an occupational exposure. HCV RNA is detectable in serum within one to two weeks after an accidental parenteral exposure. The diagnosis of acute infection should be based on detection of HCV RNA by a sensitive technique. This testing can be performed at any time after the first week after exposure, but antiviral treatment is not an emergency in this setting and can be initiated after appearance of serum aminotransferase elevations or clinical symptoms appear.

Antiviral Treatment of HCV Infection
Decision to treat. Only patients with detectable HCV RNA should be considered for treatment. HCV genotype determination should be performed before treatment as results may help in the decision to treat as well as in determining the duration of treatment. Thus, because of the high rates of response and need for 24 weeks of therapy only in patients with HCV genotypes 2 and 3, many investigators recommend therapy to all such patients provided there are no contraindications. Because response rates are only 40-45 percent and therapy must be given for 48 weeks in patients with genotype 1, the benefits of therapy must be balanced against its risks and cost. In this context, the assessment of the natural prognosis of infection by liver biopsy examination may help in making the decision to treat. In the absence of sufficient information, the same applies to genotypes 4, 5, and 6.

Virologic followup and assessment of response. Measurement of HCV RNA levels before treatment and again at 12 weeks has been proposed as an appropriate approach to monitoring patients with chronic hepatitis C who are treated with peginterferon and ribavirin. This is particularly true for patients with genotype 1. In patients infected with genotypes 2, 3, 4, 5, and 6, monitoring of HCV RNA levels may be less important, and there is little data supporting its usefulness. The basis for this will be discussed later in this conference. In all patients, however, the virological response should be assessed by testing for HCV RNA by a sensitive technique at the end of therapy. The presence of HCV RNA at the end of treatment is highly predictive of a relapse when therapy is stopped. The absence of HCV RNA at the end of 20 treatment indicates virological response and should lead to retesting for HCV RNA by a sensitive method 24 weeks later to document that the virological response is sustained.

Extrahepatic (non-liver) Manifestations of Chronic Hepatitis C
Timothy R. Morgan, MD
Chief, Hepatology, VA Medical Center, Long Beach, CA

Multiple extrahepatic (non-liver) diseases (e.g., skin, kidney, etc.) have been associated with chronic hepatitis C infection. In some extrahepatic diseases, it seems possible (or likely) that hepatitis C causes or worsens the diseases, while in other cases it is unclear what role (if any) hepatitis C plays. Interferon treatment of the hepatitis C improves some extrahepatic diseases, with relapse of the extrahepatic disease occurring if the patient relapses after the interferon is stopped. Occasionally, interferon treatment worsens the extrahepatic disease.

Essential Mixed Cryoglobulinemia (EMC)
EMC is a disease caused by the deposition of immune complexes (combinations of antibodies and hepatitis C virus) in small blood vessels (vasculitis). The typical symptoms are skin rash (palpable purpura or ulcer), joint aches, and weakness. EMC can also involve the kidneys (MPGN), nerves and brain; however, since the disease attacks blood vessels, any part of the body may be involved. There are multiple causes of EMC including HCV infection, cancer, autoimmune disease or infection with bacteria, parasites or other viruses. EMC is diagnosed by having the typical symptoms along with cyroglobulins (blood proteins [usually antibodies] that precipitate from cooled serum) in the blood. Blood complement levels (C3, C4, CH50) are usually low.

Approximately 50-80% of patients with EMC type II are infected with HCV. Furthermore, the HCV is found in the immune complex deposits in the skin and, occasionally, in the kidney, suggesting that the HCV is directly involved in causing EMC.

Cryoglobulins are found in 20-40% of European patients with hepatitis C but in fewer Americans with HCV (5-10%). In most instances, patients with cryoglobulins do not have EMC or any other problem from their cryoglobulins. At present it is not known why some patients develop cryoglobulins and others do not, nor why some patients with cryoglobulins develop EMC and others do not.

EMC is usually slowly progressive over years, although, in rare cases, it may be sudden and severe. EMC improves (in most patients) who lose HCV while on interferon treatment. EMC can return if the patient relapses after interferon is stopped.

Membranoproliferative Glomerulonephritis (MPGN)
MPGN is a disease of the glomerulus (part of the kidney) caused by the deposition of immune complexes within the glomerulus. About half the time, MPGN occurs in patients with EMC. In the other half, MPGN occurs without any other symptoms of EMC and without cryoglobulins in the blood. Patients with MPGN usually have excess protein in their urine (more than 3 grams per day) and mild renal dysfunction (average creatinine 2.0 mg/dl). Mild to moderate hypertension is also fairly common. The diagnosis of MPGN is confirmed by seeing typical changes in the glomerulus on kidney biopsy. As in EMC, cryoglobulins are frequently present in the blood and the blood level of complement is decreased.

MPGN is usually slowly progressive and can, occasionally, lead to renal failure. MPGN can improve with interferon treatment, especially in patients who respond to treatment with loss of HCV.

Vasculitis
Vasculitis is an inflammation of the blood vessels, including veins or arteries of any size. In HCV, the vasculitis is usually seen in association with cryoglobulinemia (EMC) although it can develop without cryoglobulins. Patients with vasculitis can develop injury of the nerves, intestines, skin, lungs, etc. Interferon treatment should be considered.

Polyarteritis Nodosa (PAN)
Polyarteritis nodosa is a vasculitis involving small- and medium sized arteries. Typical skin findings are bruising and raised rashes. HCV has been found in 5% - 20% of patients with PAN, although how the HCV causes the PAN is unknown. Patients with PAN should be tested for HCV using PCR.

Non-Hodgkin's Lymphoma (NHL)
Non-Hodgkin's lymphoma is a rare cancer of the lymphocytes and lymph glands. Studies in Europe and the US show a definite increase in HCV infection in patients with NHL (20-30% of NHL have HCV compared with 1-5% of the general population). The way in which HCV might cause NHL is not known. It is known that HCV can infect lymphocytes and that HCV has been detected in some of the lymph nodes of patients with NHL. Many of the NHL in patients with HCV infection are low-grade lymphomas. It is not yet known what happens when NHL patients are treated with interferon.

Lichen Planus (LP)
Lichen planus is a rare disease (less than 1% of US population) of unknown cause. Typical findings are violaceous (slightly purple), scaling, raised skin rashes found on the skin or on the mucosal surfaces (e.g., mouth). The skin rash persists for a long time. Diagnosis if made by finding the typical skin rash and a skin biopsy showing the characteristic lymphocytic infiltration of the epidermis (upper layer of the skin).

Studies in Europe report HCV in 10% to 38% of patients with LP. However, it is unknown if or how HCV might cause LP. Treatment with interferon may worsen the LP, although, in the single patient I had with LP, interferon caused the LP to disappear.

Porphyria Cutanea Tarda (PCT)
PCT is a rare skin disease that is seen in patients with abnormal pophhyrin metabolism in the liver (reduced uroporphyrinogen decarboxylase activity). Typical skin findings are photosensitivity (sensitivity to sun light), fragility, bruising and vesicle or bullae formation. After a long time, the skin can become dark and thick. Extrinsic factors, such as ethanol, estrogens, or iron overload are needed for the clinical manifestations of PCT.

Studies from southern Europe report HCV in up to 75-95% of patients with PCT; HCV is also found in PCT patients in northern Europe and the US, but less frequently. It is not known if or how HCV leads to clinical PCT. All patients with PCT should be test for HCV infection. There is little information on response to interferon treatment in PCT.

Autoimmune diseases and autoantibodies
Patients with chronic HCV infection appear to have increased frequency of autoantibodies and autoimmune diseases. Autoantibodies (antibodies directed against one=s own body) reported in HCV include rheumatoid factor (in EMC), antinuclear antibody (ANA), anticardiolipin antibody, antithryoid antibody, antineutrophil cytoplasmic antibody and anti-liver-kidney-microsomal (LKM) antibody.

Thyroid disorders are the most commonly associated autoimmune diseases in patients with chronic hepatitis C (3-5% of patients). Interferon treatment may induce development of antithyroid antibodies and thyroid disease. Usually, thyroid disease resolves when the interferon is stopped. Patients should be tested for thyroid dysfunction while on interferon treatment.

Overall, it is unclear if HCV causes autoimmune diseases. However, interferon can exacerbate autoimmune diseases and should not be used in patients with known, significant autoimmune disorders.

Other reported disease
Multiple associations have been reported in patients with chronic HCV. In some cases, there are conflicting reports or only rare reports. These diseases include pulmonary fibrosis, Mooren corneal ulcers, idiopathic thrombocytopenic purpura, vascular thrombosis and antiphospholipid antibody, rheumatoid arthritis, polymyositis and dermatomyositis.

Chronic hepatitis C can lead to disease in organs other than the liver (e.g., kidney, skin, etc.). Some of these diseases are appear to be the direct result of the hepatitis C infection (or the body's response against the virus). In other instances, it is unknown how the hepatitis C virus causes the problem. Overall, relatively few people with hepatitis C develop serious extrahepatic diseases from their hepatitis C infection. Most extrahepatic diseases improve if the patient is treated with interferon and has a good response to interferon treatment. Some diseases relapse if the patient relapses after the interferon is stopped.