Perelson noted that, while HCV does not cause clinically significant liver disease for decades, it is still a very dynamic process, with tremendous daily viral production, in most HCV-infected people.

Immune Responses to HCV Infection
Studies have shown that neutralizing antibodies are produced during HCV infection, but they do not appear to be protective against re-infection in humans or in chimpanzees (Ferrari 1999). The more critical determinant of the outcome of HCV infection seems to be the cell-mediated immune response, or the T-cell (CD4) response. People who are able to spontaneously clear HCV from their body have evidence of strong T-cell responses. Conversely, people who are chronically infected with HCV do not appear to either mount a strong T-cell response early after HCV infection, or maintain an initial strong T-cell response to HCV antigens (Gerlach 1999). In Gerlach's study, the 20 people (52.6%) out of 38 studied who cleared HCV infection all had a strong and sustained antiviral T-cell response to HCV, while the people who became chronically infected either showed no initial T-cell response (12, 31.6%) or did not maintain an initial strong antiviral T-cell response (six, 15.8%).

Over time, however, in most people chronically infected with HCV, T-cell responses increase but are not able to clear HCV. Ferrari and colleagues write that:

More information about the immune events taking place within the inflamed liver is required to draw a more reliable picture of HCV pathogenesis and to create the ground for more effective strategies of prevention and treatment of HCV infection. (Ferrari 1999)

Another limitation of current research is the lack of necessary cell culture systems and animal models to study the many complex factors involved in the immunologic responses to HCV infection (Cerny 1999).

Possible Strategies for a Virus to Escape Immune Elimination

1. Decrease its visibility to the immune system.
2. Decrease the effectiveness of antiviral cytokines.
3. Increase the resistance of infected cells to CTL-mediated killing.
4. Infect immunologically privileged sites.
5. Induce immunologic tolerance.
6. Immunologic evasion.

Generally neither HCV RNA levels nor alanine aminotransferase (ALT) levels (liver enzyme levels) correlate very well with the extent of liver damage seen on biopsy. (See "HCV Diagnostics" chapter.) Gretch and colleagues, however, have reported that replication of HCV in human liver tissue shows a significant correlation with the severity of HCV infection. They reported a very strong correlation with the percentage of human liver tissues cells infected with HCV in vitro, and the degree of hepatic inflammation (Gretch 1999). They suggest that the amount of damage to the liver may be a factor of the process of HCV replication, not merely the presence of HCV in the liver.

Arguments Supporting the Relevance of Immune-mediated Liver Cell Damage in HCV Infection

1. In primary HCV infection, liver cell damage correlates with the development of the host immune response--not with infection and HCV viral replication.
2. Chronic HCV replication can occur without significant liver cell damage.
3. HCV infection of liver cells does not appear to kill the infected liver cells.
4. Immunosuppression of people with HCV infection results in transient improvement in liver function tests, despite a surge in HCV RNA levels.
5. Liver cell damage is associated with an inflammatory infiltrate, and liver-infiltrating HCV immune cells associated with areas of liver damage suggest a causative role.
   (Cerny 1999)

HCV Eradication?
While HCV is an RNA virus, it is not a retrovirus; therefore, HCV does not incorporate its genetic code into the host cell's DNA. So if all infected liver cells, or hepatocytes, and any other cells in the body which are infected with HCV die–and there are no free HCV virions around to infect new cells-then the person will no longer be infected with HCV, i.e., the infection will be eradicated. Some investigators believe that most people who remain without detectable HCV RNA six months after therapy have achieved eradication (i.e., they are cured). (See "HCV Treatments" chapter.) Approximately 15% of people acutely infected with HCV are able to achieve life-long eradication of HCV from their body by their immune response. (See "HCV Natural History" chapter.) Whether people are in fact cured will require longer follow-up; however, there is an important implication to achieving eradication. People can be re-infected with HCV after they have had the initial HCV infection eradicated; there is no life-long HCV immunity (Farci 1992).

Extrahepatic Manifestations of HCV
While the primary site of clinical infection with HCV is the liver, a significant number of people develop disease symptoms at sites other than the liver, referred to as extrahepatic manifestations of chronic HCV infection. A recent report reviewed sites of HCV infection in 1,604 patients infected with HCV; it found that 74% of people had at least one extrahepatic manifestation of HCV infection (Cacoub 1999). The most common manifestations were:

• Arthralgia (joint pains)
• Paresthesia (nerve sensation abnormalities)
• Myalgias (muscle pain)
• Pruritus (itching)
• Sicca syndrome (dry eyes, skin, and mouth)

The report also noted some laboratory abnormalities such as cryoglobulinemia (increase in a certain type of "cold" antibody in the blood), antinuclear antibodies, low thyroxine (thyroid enzyme) levels, and anti-smooth-muscle antibodies. A multivariate analysis demonstrated that the following factors were associated with an increased incidence of extrahepatic manifestations:

• Age
• Female gender
• Extensive liver fibrosis (scarring)

Another study reported that 38% (122/321) of people with HCV infection had at least one extrahepatic manifestation, including joint pains (19%), skin changes (17%), dry mucous membranes (12%), and sensory nerve changes (9%). Thus, the most common symptoms involved the joints and skin. HCV/HIV coinfection was associated with an increased incidence of low platelet counts (thrombocytopenia) and autoantibodies (antibodies against body tissues) (Cacoub 2000).

All of these symptoms and laboratory abnormalities are typically seen in people with autoimmune diseases, or people with diseases that are the result of their own immune system attacking components of their own body. This observation provides additional support to the belief that much of the damage observed due to HCV infection, is actually a result of an overactive immune system, or an immune system that is mistakenly attacking components in the body, as it attempts to attack HCV.

The pathogenesis, viral dynamics, and immunologic response of HCV remains incompletely understood. Reliable and efficient cell culture systems and a small-animal model are needed to better understand these areas. It is to be hoped that further advancement in this field will lead to novel therapeutic agents to treat and cure patients with HCV.

References
Cacoub P, Poynard T, Ghillani P, et al., Extrahepatic manifestations of chronic hepatitis C. Arthritis Rheum 42:2204-12, 1999.
Cacoub P, Renou C, Rosenthal E, et al. Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC. Medicine (Baltimore) 79:47-56, 2000.
Cerny A, Chisari FV. Pathogenesis of chronic hepatitis C: immunologic features of hepatic injury and viral persistence. Hepatology 30:595-601, 1999.
Chang K. The mechanisms of chronicity in hepatitis C virus infection. Gastroenterology 115: 1015-17, 1998.
Farci P, Alter HJ, Govindarajan S, et al. Lack of protective immunity against reinfection with hepatitis C virus. Science 258:135-40, 1992.
Ferrari C, Urbani S, Penna A, et al. Immunopathogenesis of hepatitis C virus infection. J Hepatol 31:31S- 38S, 1999.
Gerlach JT, Diepolder HM, Jung MC, et al. Recurrence of hepatitis C virus after loss of virus-specific CD4+ T cell response in acute hepatitis C. Gastroenterology 117:933-41, 1999.
Gretch DR, Chang M, Williams O, et al. Active replication of HCV in human liver tissue shows a significant correlation with severity of hepatitis C In vivo [abstract]. Hepatology 30:353A, 1999.
Laskus T, Radkowski M, Piasek A, et al. Hepatitis C Virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes. J Infect Dis 181:442-8, 2000.
Laskus T, Radkowski M, Wang LF, et al. Search for hepatitis C extrahepatic replication sites in patients with acquired immunodeficiency syndrome: specific detection of negative strand viral RNA in various tissues. Hepatology 28:1398-1401, 1998.
Manns MP, Rambusch EG. Autoimmunity and extrahepatic manifestations in hepatitis C virus infection. J Hepatol 31:39S-42S, 1999.
Mika BP, McCarthy ME, Layden TJ, et al. HIV/HCV co-infected patients experience HCV viral rebound after the second day of IFN treatment [abstract]. Hepatology 30:195A, 1999.
Min A, Jones J, Esposito S, et al. Early hepatitis C viral RNA decline during therapy with interferon alfa-2b and ribavirin in patients with chronic hepatitis C without sustained response to prior interferon [abstract]. Hepatology 28:A29, 1998.
Munoz S, Suvannasankha A, DiGregorio K, et al. Retreatment with ribavirin and interferon alpha 2b of relapsers and nonresponders to monotherapy [abstract]. Hepatology 28:A28, 1998.
Neumann AU, Lam NP, Dahari H, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 282:103-7, 1998.
Perelson AS. HCV dynamics: a guide to patient management [slide lecture & syllabus]. AASLD, Postgraduate Course, Dallas, 5 November 1999. Perelson AS. Viral kinetics and mathematical models. Am J Med 107:49S-52S, 1999.
Ramratnam B, Bonhoeffer S, Binley J, et al. Rapid production and clearance of HIV-1 and hepatitis C virus assessed by large volume plasma apheresis. Lancet 354:1782-85, 1999.
Seeff LB. Hepatitis C. Semin Liver Dis 15:1, 1995.
Thomas DL, Astemborski J, Vlahov D, et al. Determinants of the quantity of hepatitis C virus RNA. J of Infect Dis 181:844-51, 2000.
Vucelic B, Ostojic A, Hrstje B, et al. Viral kinetic study of induction dosing with interferon alpha and ribavirin in the treatment of chronic hepatitis C [abstract]. Hepatology 28:A32, 1998.
Walsh KM, Good T, Cameron S, et al. Viral kinetics can predict early response to alpha-interferon in chronic hepatitis C. Liver 18:191-5, 1998.
Yasui K, Okanoue T, Murakami Y, et al. Dynamics of hepatitis C viremia following interferon-alpha administration. J Infect Dis 177:1475-9, 1998.
Zeuzem S. Clinical implications of hepatitis C viral kinetics. J Hepatol 31:61S-4S, 1999.

Natural History, Clinical Manifestations, and
Prognostic Indicators of Disease Progression &
Survival of Hepatitis C Virus (HCV) Infection
by Michael Marco

The most accurate means of determining the entire spectrum of outcomes of HCV infection would be to identify a large cohort at a time of initial infection; to select a carefully matched seronegative control group who, like the seropositive group, could be maintained under active surveillance for periods of 30 years or more; and to omit therapy that might alter outcome and hence modify natural history. Obviously, these conditions cannot be met and, therefore, the desired information may never be forthcoming.

—Leonard Seeff, The Natural History of Hepatitis C—A Quandary

Introduction
Numerous HCV natural history studies have attempted to detail the course of HCV infection, yet most have been unable able to chart disease progression from initial infection to end-stage disease, and only one has more than 25 years of follow-up (Seeff 2000b). Long-term, accurate natural history data are needed to better understand the pathogenesis of the disease and to determine which patients need treatment now, which ones can wait for more effective treatment, and which ones will probably never need treatment.

Clinical Features and Outcome of Acute HCV
Acute HCV infection most often exhibits no clinical symptoms: 60% to 75% of individual are asymptomatic; 20% to 30% become jaundiced; and 10% to 20% have symptoms including fatigue, nausea, and vomiting (Dienstag 1983; Aach 1991; Koretz 1993). In those who develop jaundice, peak bilirubin levels are usually less than 12 mg/dl (mean: 8.4 mg/dl) and levels appear to resolve in less than four weeks (Esteban 1999). Fulminant hepatic failure with primary HCV infection is extremely rare.

Approximately 20% of newly individuals have symptoms that arise before the seroconversion to anti-HCV occurs (antibody-positive) (Koretz 1993). The average time from exposure to seroconversion is approximately 50 days, although it can be as long as nine months (Tremolada 1991; MJ Alter 1992).

During acute infection, a person’s aminotransferase (ALT) levels (liver enzymes) rise to 200–600 IU/I, but in 20% of cases, peak ALT levels exceed 1000 IU/l (Esteban 1999). There is often an episodic, fluctuating pattern of ALT levels during the first few months, where levels flare to a 10- to 15-fold increase. Approximately 25% of patients develop a sustained plateau pattern where ALT levels remain below 450 IU/l for many months. In others, ALT level can normalize (suggesting recovery), yet flare up again within months to years. This pattern is a tell-tale sign of chronic infection with HCV and indicates a need for ongoing ALT monitoring (MJ Alter 1992; Esteban 1999). More than 12 months after acute infection, ALT levels will continue to be elevated in about 60% of individuals (Esteban 1998).

Continued normalization of ALT levels–termed "biochemical recovery"–does not always mean a loss of anti-HCV or absence of HCV RNA (MJ Alter 1992). Likewise, continued abnormal ALT levels with anti-HCV positivity does not necessarily mean that a person is chronically infected with HCV. Fifteen to twenty-five percent of acutely HCV-infected individuals will clear their infections (Shakil1995). Complete resolution of HCV infection is defined as both the absence of HCV RNA in serum and a normalization of serum ALT level. Shakil and colleagues from the National Institutes of Health (NIH) documented the rate of chronic HCV, using PCR and bDNA assays, and histologic (liver cell) damage in 60 anti-HCV positive individuals with chronic hepatitis (Shakil 1995). Tabled below are the results of the 60 patients equally divided into three groups: group 1: normal ALT levels; group 2: elevated ALT levels less than twice the normal range; and group 3: levels more than twice the normal range.

The presence of viremia (HCV RNA in blood) in all groups did not correlate with age, route of transmission, or duration of infection. Nonetheless, those who were HCV RNA-negative were more likely to have persistently normal ALT levels (78%) compared to those who were HCV RNA-positive (20%). Likewise, most of those who were HCV RNA-negative had either normal liver histologic findings or only mild changes (Shakil 1995).

Some individuals (~15%) who test HCV RNA-positive during acute or post-acute HCV infection may eventually become undetectable and, conversely, some (~19%) who initially test HCV PCR-negative may become detectable at a later date. Villano and colleagues from Johns Hopkins studied HCV RNA patterns in 43 HCV seroconverters (documented by EIA-2 and RIBA-2) who were followed for 72 months (Villano 1999). Six (14%) patients had documented viral clearance (undetectable HCV RNA, <500 copies/mL) between one and two years; one patient was initially undetectable for HCV RNA and remained negative. Two of the patients who cleared virus had ~1 million copies/mL of virus. Factors associated with viral clearance were: white race (P = 0.004); jaundice (P = 0.003); and lower peak viral titers (P = 0.003).

The presence of HCV RNA did not always precede the development of antibodies: 48% had HCV RNA at their first seroconversion visit; 33% were HCV RNA-positive a median 3.8 months before seroconversion; and 19% were HCV RNA-detectable a median 15.3 months after estimated date of seroconversion. The fact that a person who may initially test qualitatively HCV RNA-negative (no detection of HVC in serum) but later become positive suggests that "in clinical practice, virologic outcome must be determined by long-term follow-up, not a single HCV RNA level" (Villano 1999).

One significant finding in this study deserves comment. Seventy-four percent of the anti-HCV- positive patients in this study (the majority with a history of intravenous drug use (IVDU)) had been evaluated by a physician during their seroconversion intervals, yet only a few were recognized as having HCV or were screened for it. In this case, one cannot say that hepatitis was not detected because of limited access to health care. Even though HCV does not present with severe symptoms, this routine failure to diagnosis HCV in patients known to be at high risk indicates a striking lack of awareness by health care providers about HCV and its epidemiology.

Chronic Infection
Data from various studies indicate that 75-85% of individuals with acute HCV infection will become chronically infected (MJ Alter 1992; Shakil 1995; Villano 1999). The reason for such a high rate of chronicity is not completely understood. Some studies suggest that HCV persistence is related to the high mutation rate of HCV and the continual turnover of complex viral quasispecies that are able to evade the immune response of the host (Tsai 1998; Ray 1999). Farci and colleagues from the University of Cagliari and Robert Purcell's NIH laboratory recently published data which suggest that resolution of acute hepatitis is associated with relative evolutionary stasis of quasispecies, but progression to chronicity is correlated with genetic evolution of HCV (Farci 2000).

Only 60% to 70% of chronically infected individuals will have persistent or fluctuating ALT elevations; the other 30% to 40% will have normal ALT levels throughout the course of their infection. Two studies, one by Shakil and colleagues, the other by Pastore and colleagues have documented that relatively low (13-135) or normal ALT levels during acute infection were prognostic indicators predicting loss of HCV infection (Pastore 1985; Shakil 1995). In at least four other natural history studies, however, ALT levels were not found to be predictive of clearance (Mattsson 1989; Esteban 1991; MJ Alter 1992; Villano 1999). Except for the Villano study mentioned above, which noted that Whites had a higher rate of clearance than Blacks (80% of the subjects were black), and a small Swedish study (N = 37) by Mattsson that documented an increased rate of clearance among patients under 30 years old, no other major natural history studies have found any prognostic factors for disease clearance (Mattsson 1989; Villano 1999). In 2000, it is still a guessing game as to who will develop chronic infection. According to Juan Ignacio Esteban, "No clinical, biochemical or virological feature can predict the outcome of infection in a given individual" (Esteban 1999).

A liver biopsy gives the most accurate information about the degree of liver injury associated with HCV infection. There are many complex and detailed staging and scoring systems for liver biopsies (e.g., Knodell score, METAVIR fibrosis score). (For a discussion of liver biopsies and staging, see "HCV Diagnostics" chapter.)

According to Leonard Seeff, natural history studies have used one of three evaluation strategies (Seeff 1997, 2000a):

• Prospective studies starting with disease onset which have a low documentation of morbidity (symptoms, cirrhosis) and mortality (hepatic/liver failure, HCC). Follow-up tends to be short (8 to <20 years) and sample size is usually small.
• Prospective studies of patients (usually older than 45 years of age) with already established chronic liver disease who have been referred to liver disease units/specialists. These tend to have shorter follow-up and referral bias with far sicker patients.
• Retrospective/prospective (non-concurrent prospective) studies of recipients of HCV- contaminated immunoglobulin and transfused blood. These usually have an ~20 year follow-up period and contain matched non-hepatitis controls.

One of the most important prospective natural history studies, the NIH Prospective Study of HCV-Infected Donors, was recently published (HJ Alter 1997). It included 280 HCV RNA- positive blood donors followed for a median of 20 years. ALT levels were repeatedly normal in 17%; 45% had <2X the upper limit of normal; and 38% had a least one reading 5x the upper limit. Liver biopsies were performed on 81 of 280 patients, and a probable date of exposure could be ascertained in 74 of the 81. The chart below represents biopsy findings according to time from exposure:

[figure]

Only 1.3% had histologic evidence of severe hepatitis and cirrhosis following a mean interval of 18 years from time of exposure. According to Harvey Alter, "Liver-related mortality or severe morbidity is less than 10% in the first two decades after infection." (Alter 1997)

Koretz and colleagues from UCLA documented a more progressive clinical course in their study of 80 recipients of HCV transfused blood who were followed for 14 years (Koretz 1993). After 16 years of known infection, 10% had HCV-related symptoms; 18-20% had biopsy-proven cirrhosis; 1.3% developed hepatocellular carcinoma (HCC; liver cancer); and 2.5% died of liver- related complication.

Cohort studies of chronic HCV patients referred to liver-oriented tertiary-care centers suggest progressive HCV disease. Tong and colleagues followed 131 patients with HCV for approximately 22 years: mean age at transfusion was 35 years, and at time of evaluation, the mean age was 57 years (Tong 1995). After about four years of follow-up, over 67% were experiencing HCV-related symptoms (specifically fatigue); 46% had biopsy-proven cirrhosis; 10.6% had developed HCC; and 15.3 % had died of liver-related deaths. The accompanying chart documents time to hepatic event:

[figure]

Two significant retrospective natural history studies were recently published reporting the longest follow-up yet of well-characterized HCV infected cohorts (Kenny-Walsh 1999; Seeff 2000b). The first, by Kenny-Walsh and colleagues, documents the clinical course of HCV in a group of 376 HCV RNA-positive Irish women who had been infected during 1977–1978 with a batch of HCV-contaminated anti-D immune globulin (Kenny-Walsh 1999). The mean age of the women at time of infection was ~28 years, and after 17 years of living with HCV, the mean age of at screening/biopsy was 45 years. None had received any anti-HCV treatment. HCV-related symptoms, histologic grade of hepatic inflamation, and stage of fibrosis for the 363 women who underwent biopsies are documented on the next page:

Compared to other studies, this study appears to document a slower progressive course of HCV infection. After 17 years of infection, no fibrosis was documented in nearly half of the women, and cirrhosis in only 2%. Another study from Germany of 152 women infected with HCV- contaminated RH0(D) immune globulin documented a similar clinical course (Muller 1996). After 15 years, none of the women were found to have chronic active hepatitis or cirrhosis. There are many speculations as to why disease in this cohort of women was so indolent: 1) disease in women is less progressive than in men; confirmed by Poynard and colleagues (Poynard 1997); 2) patients infected at a younger age fare better than older ones; confirmed by Tong and colleagues as well as Poynard (Tong 1995; Poynard 1997); and 3) the small amount of the infecting dose of anti-D immune globulin (versus that of a blood transfusion) may have played a role (Kenny-Walsh 1999).

The 45-year HCV natural history study of 8,568 military recruits by Seeff and colleagues reports the earliest confirmed detection of HCV in the United States and has the longest follow-up of any study published to date (Seeff 2000b). Originally, 8,568 military recruits were tested for group A streptococcal infection and acute rheumatic fever between 1948 and 1954. After initial blood tests, samples were frozen and saved for 45 years. Seventeen (0.2%) individuals tested positive for HCV antibodies on EIA-3 and RIBA-3. While 17 HCV-positive patients followed in a natural history study may seem small, it's the HCV-negative control group of over 8,000 individuals (99% men) that makes this study important. Records tracking 45 years of liver abnormalities, disease, and death (using the National Death Index Plus service) as well as age, sex, race, chart review, and cause-specific mortality from death certificates were available from the Veterans Affairs' computer files.

A vast majority of the recruits were younger than 25 years of age at the original blood draw. Approximately 90%, 10%, and 0.5% were white, black and Asian, respectively. HCV infection in Blacks was significantly higher than Whites: 1.8% compared to 0.07% (RR, 25.9; CI, 8.4 to 80.0). The mean age of the surviving cohort, as of January 1997, was 64.8 years (95% were between 60 and 69 years of age). Eleven of the seventeen (65%) HCV-infected men were HCV RNA-positive; all but one had genotype 1b.

Of the seven deaths in the HCV-positive group, only one was due to liver disease. The fact that the event rate was so low in the HCV-positive group after four decades, and that there was little difference in mortality between the groups, leaves one to believe that HCV is a less progressive disease than is currently thought. According to Leonard Seeff, long-term natural history data have revealed that only 15% to 20% of HCV-infected persons will eventually develop progressive to potentially serious end-stage liver disease (namely cirrhosis) and that the remainder will die of causes other than liver disease (Seeff 2000a).

Prognostic Factors for Fibrosis Progression:
Good News for the Non-alcohol-imbibing Woman Infected with HCV Before She Was 40
A seminal natural history from France by Poynard and colleagues documents host factors, rather than virologic factors, as risks for fibrosis progression in untreated HCV-positive patients (Poynard 1997). Some 2,235 patients were selected from three well-characterized cohorts: Observatoire de l'Hépatite C (OBSVIRC); Cohorte Hépatite C Pite-Salpéteiére (DOSVIRC); and the METAVIR. All patients underwent liver biopsy, and the METAVIR¹ scoring system was used to grade the stage of fibrosis. Nine factors were assessed for effect on fibrosis progression: age at biopsy; estimated duration of infection; sex; age at infection; alcohol consumption; HCV genotype (see "HCV Virology" chapter for details); HCV viremia; method of infection; and histologic activity grade. Fibrosis progression was defined as the ratio between fibrosis stage in METAVIR units and the estimated duration of infection. (For example, for a patient with stage 2 fibrosis who had been HCV-infected for eight years, the fibrosis progression rate would be 0.25 fibrosis units per year.)

The mean and median rate of fibrosis progression per year for the 1,157 patients whose duration of infection was known was 0.252 (95% CI, 0.227-0.277) and 0.131 (95% CI, 0.125-0.143), respectively. At this rate the median time to cirrhosis was estimated as 30 years (range: 28-32 years); 33% had a median time of 20 years and 31% will never progress or will not progress for at least 50 years. Out of the nine factors assessed, three had highly significantly correlation with disease progression: gender, alcohol consumption, and age at infection. Interestingly, amount of HCV RNA and viral genotype (i.e., 1b) did not correlate significantly with disease progression. (See "HCV Treatments" chapter, where both are significant prognostic factors for success of treatment.)

Multivariate Analysis of the Three Significant Risk Factors for Fibrosis Progression in 1,038 Patients
Factor	Relative Risk	95% CI	for Fibrosis Progression in 1,038 Patients
Age at Infection (>40 years)	1.07	1.06-1.08	<0.0001
Male Sex	2.66	1.90-3.72	<0.0001
Alcohol Consumption (>50 grams/day)	1.49	1.18-3.03	<0.008
(Poynard 1997)
Association between Rate of Fibrosis Progression and Age at Infection
Age Group	Rate of Increase Fibrosis
31-40 Years vs. 21-40 Years	31%
41-50 Years vs. 31-41	45%
>50 Years vs. 41-50	67%
(Poynard 1997)

Varying prognostic factors predict a benign course of HCV for some and a severe one for others. A female infected before the age of 40 who drinks <50 grams (equals 5 glasses) of alcohol per day has an expected time to cirrhosis of 42 years compared to 15 years for a man infected after the age of 40 who drinks more than 50 grams of alcohol per day.

Selected Prognostic Factors for Disease Progression and Survival
Age (Tremolada 1992; Tong 1995; Fatovitch 1997; Poynard 1997; Neiderau 1998; Deuffic 1999); male gender (Poynard 1997; Deuffic 1999; Khan 2000); and increased alcohol intake (Donato 1997; Fattovitch 1997; Poynard 1997; Rudot-Thoraval 1997; Neiderau 1998; Wiley 1998) have been documented by many (but not all) as highly prognostic factors for HCV-related liver disease progression. However, other important prognostic factors for advanced disease progression have been less consistently documented in published natural history studies. Mode of HCV transmission: Blood transfusion vs. IVDU vs. Sporadic Cases

Two published studies have suggested that infection through blood transfusion (BT) (compared to infection via IVDU) is a highly significant prognostic factor for liver disease progression (Rudot-Thoraval 1997; Gordon 1998). Gordon and colleagues studied the clinical course of 627 chronically HCV-infected patients: 282 (45%) were BT recipients, 262 (42%) were infected via IVDU, and 83 (13%) were without known risk factors. The median estimated disease duration for all patients was 21 years (+/- 9.53 years) and the duration of follow-up ranged from 1 to 25 years. Liver histology was available on 463 patients. Cirrhosis was determined in 173/463 (37%): 118/173 (68%) were BT recipients; 40/173 (23%) were infected via IVDU (P<0.001). Below is a breakdown of those who had HCC, cirrhosis, or no cirrhosis:

Unlike the Poynard study (discussed above), in Gordon's patients, age or estimated disease duration did not predict risk of liver failure in the multivariate analysis (Poynard 1997; Gordon 1998). Rudot-Thoraval and colleagues from France also noted an increased prevalence of cirrhosis in BT patients in their survey of 6,664 chronic HCV patients (Rudot-Thoraval 1997).

Of 2,500 patients with known duration of HCV infection, the prevalence of cirrhosis for BT recipients was 22.8% compared to 5.8% for those infected via IVDU (P<0.02; OR = 0.61; 95% CI, 0.40-0.92).

"Sporadic cases" are HCV infections without identified risk factors. Fattovitch and colleagues from Italy noted that sporadic cases with compensated cirrhosis had poorer survival compared with those infected via BT or IVDU (Fattovitch 1997). Likewise, Khan and colleagues from Australia recently reported that sporadic cases were at significantly higher risk for cirrhosis, HCC, and liver transplantation or death (Khan 2000). The complete opposite was seen in a German HCV natural history study conducted by Hopf and colleagues (Hopf 1990). Finally, Poynard did not find any of these modes of transmission to be a significant prognostic factor in his study (Poynard 1997).

Abnormalities in Laboratory Values: Albumin and Bilirubin
In the 455 Australian HCV patients from the Khan study, serum albumin (a protein made by the liver that is responsible for maintaining fluid inside blood vessels) concentrations of <30 g/L at entry was associated with an 85% chance of liver-related complications at five years and a three-year mortality of 70% (Khan 2000). Gordon also noted that low serum albumin (3.2 g/L compared to 4.2 g/L) was an independent predictor of subsequent hepatic decompensation (P = 0.001; OR = 0.054; 95% CI, 0.030-0.099) (Gordon 1998). Similar findings were noted years earlier in two HCV natural history studies conducted by Fattovich and colleagues and Yano and colleagues (Yano 1996; Fattovich 1997). The deleterious effects resulting from low serum albumin levels in late-stage HCV patients prompted Hirsch and Wright to write in a Hepatology editorial:

End-stage disease from hepatitis C is one of the leading indications for liver transplantation in the United States. Currently, listing for transplantation requires significant abnormalities in at least 2 of 5 elements of the Child-Pugh [cirrhosis] classification. This may merit reassessment if the dramatic predictive value of the serum albumin alone can be validated in other large, prospective studies. (Hirsch 2000)

To a lesser extent, elevated bilirubin has been noted as a risk factor for HCV-related liver disease progression (Fattovitch 1997; Khan 2000). In their 1997 study of 384 Italians with cirrhosis, Fattovich and colleagues found that abnormally high bilirubin was a predictor of poorer survival (Fattovitch 1997). Those with bilirubin <17 mmo/L had five- and ten-year survival probabilities of 96% and 86% respectively, compared with 81% and 67% for those with bilirubin >17-<51 mmo/L (P = 0.0001).

Prolonged prothrombin time (decreased duration of blood coagulation) and decreased platelet count have been documented as significant predictors of disease progression in later-stage patients (Fatovitch 1997; Gordon 1998).

Coinfection with HAV, HBV, and HIV (See "Hepatitis & HIV Coinfection" chapter for more details)
HCV patients who acquire HAV have been found to have a substantial risk for fulminant hepatic failure² and death (Vento 1998). In an Italian HCV natural history study, 432 patients were tested thrice yearly for the development of HAV antibodies. Seventeen HCV patients (three with cirrhosis) subsequently acquired HAV infection. Ten of the patients had an uncomplicated course of HAV, but seven developed fulminant hepatic failure, and six died. There was no apparent difference in degree of baseline liver damage, yet the development of HAV posed a 41% chance of fulminant hepatitis and a 35% chance of death (Vento 1998).

Vento concludes that all HCV-infected individuals should be vaccinated for HAV, saying, "Chronic carriers of HCV who are at risk for HAV infection should be vaccinated against HAV, since superinfection with this virus may place them at risk for severe, life-threatening acute liver damage" (Vento 1998). In a subsequent Lancet editorial, Marina Berenguer and Teresa Wright agree that HAV vaccination may be warranted, but believe that Vento's results need confirmation and that a cost-benefit analysis is essential before implementing such a policy worldwide (Berenguer 1998).

HCV patients with HBV have been found to have an increased risk for cirrhosis (Roudot-Thoraval 1997; Cacciola 1999). Cacciola and colleagues from Italy tested for the presence of HBV DNA in a cohort of 200 chronically HCV-infected individuals. Sixty-six (33%) were found to have HBV sequences. Of these 66 patients, 22 (33%) had evidence of cirrhosis compared to 26 (19%) of 134 HCV-positive/HBV-negative patients (P = 0.04). This study confirmed the results of a French study of 5,786 histologically HCV-diagnosed patients (Roudot-Thoraval 1997). Cirrhosis at liver biopsy was found in 24.6% of patients positive for HBV surface antigens compared to 21.1% in those who were HBV-antigen-negative (P<0.001; OR = 1.99; 95% CI, 1.40-2.82).

Studies of people coinfected with HCV and HIV have reported that, while the progression of HIV disease is not changed by HCV, HCV infection progresses more rapidly in people with HIV. When matched for other variables, on average, people who are HIV-positive have higher levels of HCV RNA than HIV-negative people (Eyster 1993, 1994; Cribier 1995). It is important to note that a majority of natural history coinfection studies were conducted before the advent and widespread use of potent antiretroviral therapy. With control of HIV viremia and better immune status, the natural history of HCV in these patients will likely change. In immunocompetent HIV- positive individuals, HCV may replace certain opportunistic infections (i.e., pneumocystis carinii pneumonia, mycobacterium avium complex and cytomegalovirus retinitis) as a leading cause of increased morbidity and mortality.

Benhamou and colleagues from Theiry Poynard's group in France have recently reported on fibrosis progression in their well-characterized coinfected DOSVIRC cohort (Benhamou 1999a, 1999b). Low CD4 count (<200 cells/mm³), alcohol consumption of more than 50 grams/day, and age at HCV infection (>25 years old) were shown to be associated with an increased liver fibrosis progression rate (Benhamou 1999b). In a linear progression model, there was little difference in time from infection to cirrhosis between the HCV+/HIV+ patients and the matched HCV+/HIV- groups if those with coinfection who had a T-cell count of >200 and drank <50 grams/day of alcohol. The accompanying chart documents time to cirrhosis:

Virologic Determinants
Quantitative HCV RNA levels do not appear to affect the clinical course of HCV-infected individuals who are naive to treatment. Lau and colleagues found no difference in serum HCV RNA levels between patients with chronic persistent hepatitis, chronic active hepatitis, or cirrhosis (Lau 1993). These finding were confirmed in larger natural history studies (Poynard 1997; DeMoliner 1998).

There is considerable debate as to whether certain HCV genotypes, especially 1b, put patients at increased risk for disease progression. Kabayashi and colleagues found that the deterioration of liver histology during a median 9.6 years of follow-up was more common in patients with genotype 1 (68%)—namely 1b—than in those with genotype 2 (41.7%) (P<0.01) (Kobayashi 1996). Likewise, an advanced stage of liver histology was more common in the genotype 1 patients (63%) compared to those with genotype 2 (38.9%) (P<0.05). An earlier, smaller study conducted in the United Kingdom also noted that those with genotype 1 had a far more progressive histologic disease than those with genotypes 2, 3, or 4 (Dusheiko 1994). However, difference in disease outcome according to genotype has not been verified in most treatment-naive natural history studies after 1996 (Poynard 1997; Serfaty 1997; Neiderau 1998; Khan 2000). Finally, in interviews conducted with leading hepatology researchers and clinicians, none believe that genotype independently has an effect on the natural history of hepatitis in patients naive to therapy (see "Current Opinions and Controversies" chapter).

Diagnosis of Hepatitis C Virus (HCV)¹ Infection
by Michael Marco

Introduction HCV diagnostic assays (tests) are important for four specific reasons: 1) to protect the blood supply; 2) to diagnose if an individual is acutely infected with HCV; 3) to determine if an individual is a chronic carrier; and 4) to evaluate the effect of anti-HCV therapies. There are currently three types of tests used for detection of HCV infection and quantification of virus: HCV enzyme immunoassay (EIA-2 and EIA-3); reverse immunoblot assay (RIBA-2 and RIBA- 3), and polymerase chain reaction (PCR). Nonetheless, biopsy of liver tissue is still the gold standard for assessing the seriousness of hepatic disease in an individual with chronic HCV infection.

HCV EIA, RIBA, and Qualitative HCV RNA PCR
For most people, the diagnosis of HCV exposure is confirmed by detecting the presence of HCV antibodies (anti-HCV) in serum. Sensitive enzyme immunoassay (EIA) tests are commercially available for detecting anti-HCV. Three generations of these assays have been marketed to date: EIA-1, EIA-2, and EIA-3. The HCV EIA uses recombinant viral proteins that recognize epitopes of portions of the core and other viral proteins.

EIA-1 was developed in 1989 by Kuo and colleagues (Kuo 1989). It was an important breakthrough despite suboptimal sensitivity: only about 70–80% (Gretch 1997). In 1992, the much-improved EIA-2 was released, which recognized epitopes from the core (c22), NS3, and NS4 proteins. The newer test also offered much greater sensitivity and it remains the most commonly used assay—outside blood donation centers—for detecting anti-HCV. The newest HCV antibody assay, EIA-3, modified its NS3 and NS4 regions and now recognizes additional epitopes from the NS5 protein.

* Based upon detection of HCV RNA by PCR (Terrault 1999, based on Gretch 1997)
** Positive predictive value compared with RIBA

A major problem with the HCV EIA is the low positive predictive value and high false-positivity rate in low-prevalence populations (i.e., blood donors, those without known risk factors, or those with normal ALTs). In these populations, it is advised that a reactive (positive) HCV EIA be confirmed with a reverse immunoblot assay (RIBA-2 or -3), also known as a "Western blot." The newer RIBA assays detect antibodies to each of the HCV proteins (core, NS2 through 5) in a nitrocellular strip format.

Data from two studies have demonstrated that non-reactivity to RIBA-3 correlates well with an absence of HCV viremia (Uyttendaele 1994; Zein 1997). If, however, an individual’s sample is found to be reactive or to have an indeterminate reaction to the RIBA, a qualitative² HCV RNA reverse-transcription polymerase chain reaction (PCR) assay is recommended to establish the presence or absence of HCV viremia (Terrault 1999).

In a high-risk population (those with elevated ALTs or a risk factor such as history of injection drug use, multiple sexual partners, or blood transfusion before 1992), a reactive HCV EIA-2 or -3 is often sufficient to confirm HCV infection. The next logical step would be either a qualitative HCV RNA PCR to differentiate acute versus chronic infection or a quantitative2 HCV RNA PCR (to establish baseline viral load) if the individual is considering anti-HCV treatment. PCR may be indicated for immunocompromised individuals (transplant patients, those with chronic renal failure or HIV infection). These individuals are often unable to develop an adequate antibody response, and PCR (qualitative or quantitative) may be necessary to detect HCV (Terrault 1999). Likewise, there is an increased rate of false-positive anti-HCV reactions in people with HIV infection (Zylberberg 1996). The current United States Public Health Service and Infectious Disease Society of America (USPHS/IDSA) Guidelines for the Prevention of Opportunistic Infections in Persons Infected with Human Immunodeficiency Virus recommends that positive screening antibody tests for HCV in people with HIV infection be confirmed with either the RIBA or HCV RNA PCR. Also, HIV-positive people with undetectable HCV antibodies, but evidence of unexplained chronic liver disease, should have an HCV RNA test performed (CDC 1999).

A qualitative HCV RNA PCR test is the best way to determine the presence of virus during the approximately two-month "window period" between HCV exposure and the presence of antibodies. HCV RNA is detectable by PCR within one to two weeks after exposure. Schreiber and colleagues conducted a study to accurately estimate the risk of transfusion-transmitted HIV, HTLV, HBV, and HCV from screened blood which was tested and determined antibody-negative (Schreiber 1996). Using data on 586,507 persons who had given blood more than once (a total of 2,318,356 blood donations), they calculated that the risk of a donor’s being HCV RNA PCR- positive during the initial period before antibodies became detectable was 1 in 103,000 (range: 28,000–288,000). They concluded that screening blood for HCV with PCR would reduce the window of vulnerability by an estimated 59 days, and that the relative risk could be reduced by an additional 77%.

In 1999, Roth and colleagues confirmed these results and also demonstrated that PCR was suitable for rapid blood screening, testing 3,000 samples in seven to eight hours (Roth 1999). The authors make a case for routine HCV PCR screening in the blood-bank setting on the basis of improved safety as well as improved availability, with quarantine times for fresh-frozen plasma being reduced from six months after antibody testing to three to four weeks after PCR testing.

HCV RNA PCR qualitative and quantitative assays have not yet been approved by the FDA.3 Nevertheless, they are widely used in clinical practice and in treatment studies. They have become increasingly sensitive with a lower limit of detection of 10 to 1,000 genomic equivalents/mL. The major drawbacks of HCV RNA PCR assays are the wide variability and lack of standardization. Many studies have documented great variability in the detection of positive reference samples by labs using "in-house" PCR kits and labs using commercially available assays (Damen 1996). In 1997, a World Health Organization (WHO) international standard was established for HCV RNA nucleic acid testing (NAT) assays. This standard is used primarily in Europe; the U.S. FDA is currently working on its own standard.

Additionally, qualitative PCR is also an important tool for defining response to anti-HCV treatment with interferon (IFN) monotherapy or in combination with ribavirin (RBV). Below is a table explaining its clinical use:

(adapted from Terrault 1999)

HCV RNA PCR Quantitative Assays
HCV RNA PCR quantitative assays are used for two reasons: 1) To determine the amount of virus in an individual who is considering therapy; and 2) To observe the rate of decline of viral load during the first few weeks of treatment as a predictor of complete response (Zeuzem 1998). Although the amount of virus does not correlate with ALT levels (Ghany 1996; Zeuzem 1996) or liver histology (Lau 1993; Poynard 1997; De Moliner 1998), baseline viral loads have been shown to be a predictor of response to therapy (Davis 1997, 1998; Poynard 1998).

There are four commercially produced quantitative assays; however, the most sensitive assay, SuperQuant HCV assay, used in the interferon and ribavirin combination therapy registrational studies, is not yet commercially available. The table below, adapted from Terrault, is an analysis of all four assays based on limit of detection, units, and cost:

The Amplicor and Quantiplex assays use different types of amplification techniques to quantify viral RNA: the Amplicor uses target amplification of primers from most conserved region of the HCV genome (5' untranslated region [5'UTR]); and the Quantiplex uses signal amplification that captures and targets probes from the 5'UTR and core regions, amplifying the HCV RNA with synthetic, branched DNA oligonucleotides. Both have been compared in numerous studies (Gretch 1994; Tong 1998; Lunel 1999; Martinot-Peignoux 2000).

Martinot-Peignoux and colleagues recently published results from a study comparing three quantitative assays: Bayer’s Quantiplex (bDNA) v2.0, Roche’s new Cobas Amplicor HCV Monitor v2.0, and NGI’s non-commercially available SuperQuant (Martinot-Peignoux 2000). Both the level and range of quantification were similar among the assays, and results correlated well among various HCV genotypes. The SuperQuant detected all 22 samples with fewer than two million copies of virus compared with 17 of 22 and 19 of 22 with the bDNA and COBAS assays, respectively (P>0.05). While the bDNA assay appears less likely to accurately detect low levels of virus, it is considered the best for obtaining high-end quantification values (i.e. >5 million copies) (Reichard 1998).

Because quantitative HCV RNA measurements are beneficial only for pretreatment evaluation and for on-treatment observation, the CDC has not recommended sequential HCV RNA monitoring for all patients (CDC1998).

HCV Genotype
Not all individuals with HCV have identical viruses. Many different genetic variations (genotypes) of the virus exist. There are at least 7 distinct genotypes and at least 30 subtypes of HCV. The majority of HCV-positive individuals in the U.S. and Europe are infected with genotype 1; genotype 1a is more common in the U.S., and 1b is more common in Europe. Below is a breakdown of genotype prevalence from two recently published studies:

Nucleotide sequencing and phylogenetic analysis of NS5B and E1 regions are considered the gold standard for determining HCV genotype, but cost prohibits these methods from being used clinically (Terrault 1999). The two commercial assays most commonly used are the INNO-LiPA HCV assay (Innogenetics, Zwijnaarde, Belgium) and the HCV serotyping assay (Abbott Laboratories).

Most natural history studies have demonstrated that HCV genotype in and of itself does not play a role in the clinical course of HCV. (For a detailed discussion, see the "Natural History of HCV" chapter.) Genotype can, however, be highly predictive of response to anti-HCV treatment. Recent clinical studies have demonstrated that sustained responses to treatment are significantly less likely for individuals with genotype 1 (Davis 1988, McHutchinson 1998; Poynard 1998). The accompanying chart details the sustained virologic response rates according to genotype for patients who received either 6 or 12 months of IFN alone or in combination with RBV in the U.S. and European IFN+RBV registrational studies:

The differences in the response rates between the genotype 1 group and the genotype 2/3 group—regardless of ribavirin coadministration—are dramatic.

Liver Biopsy
Liver biopsy is considered the gold standard for clinical assessment of individuals with chronic HCV. It is the only true way to determine the severity and activity of liver disease. Because HCV can be definitively confirmed with a qualitative HCV RNA PCR, a liver biopsy is most often reserved for those considering treatment in order to assess the grade and stage of hepatitis. A liver biopsy can also help rule out other forms of liver disease such as concurrent alcoholic liver disease, medication-induced liver injury, and iron overload.

A core liver biopsy is done in order to obtain intact tissue for a pathology reading. This procedure, with the use local anesthesia, involves the passage of a thin needle between the ribs through the skin to remove a tiny (1-inch long and 1/5-inch wide) piece of liver tissue. A liver biopsy is usually done in the hospital, and an individual may leave within three to six hours. The risk of complications from a liver biopsy—primarily bleeding at the site of puncture into the liver—is less than one percent. Approximately half of individuals have no pain from the biopsy, while others experience brief localized pain.

To avoid the risk of complications, some researchers have explored doing liver biopsies with imaging-guidance techniques such as ultrasonography (Papini 1991; Lindor 1996). Ultrasonography can aid in directing the needle away from large blood vessels, bile ducts, gallbladder, and colon, and thus potentially reduce complications. This procedure has gained some followers, yet it appears that most are skeptical about its necessity and concerned with the added cost (Smith 1999).

Recently, there has been a debate among clinicians about the need for liver biopsy in patients with HCV. Some believe that with the proper information from selected assays, they can accurately determine the histologic grade and stage of a patient’s disease. An interesting study investigating clinicians’ predictions of patients’ liver histologies by surrogate markers was presented at the 1999 Digestive Disease Week annual meeting. Romagnuolo and colleagues from Canada studied 45 patients referred to their hospital for treatment (Romagnuolo 1999). All clinicians’ predictions were within one point of the actual grade and stage. Thirty-five (66%) of the patients’ inflammatory scores and 40 (75%) of the fibrosis scores were exactly predicted, including four cases of cirrhosis. Age >40, spider nevi (abnormal blood vessels on the skin of the abdomen), organomegaly (abnormal enlargement of liver and/or spleen), white blood cell count <4,000, ALT >120, bilirubin >20, albumin <3.5, and ferritin (an iron-protein complex) >200 were predictors of more severe inflammation. The same variables (except ferritin and ALT) with the addition of platelets >150,000 and prothrombin time >1.2 were significant predictors of fibrosis.

The Knodell scoring system (used mostly in the U.S.) is an important, yet complicated, tool for documenting histologic activity. It is used to grade the level and extent of inflammation, necrosis, and fibrosis of a liver biopsy. Biopsy specimens are graded in four categories:

1. Periportal +/- bridging hepatocellular necrosis I.
2. Intralobular degeneration and hepatocellular necrosis
3. Portal inflammation
4. Fibrosis

A numeric score for each category is assigned to each liver biopsy specimen, and the combined score of the four categories form the HAI (Histology Activity Index) score for that biopsy specimen. Note: The score goes from 1 to 3, omitting 2 intentionally.

It is not crucial for individuals with HCV to know and memorize their HAI score. It can be useful, however, for them to understand the condition of their liver and to know the degree of inflammation, the stage of fibrosis, and if cirrhosis has occurred.

Fibrosis of the liver is the presence of scarring that results from the repair of hepatic tissue damage. In the case of HCV, the scarring is initiated by HCV-infected hepatocytes and the resultant inflammation. Liver fibrosis occurs slowly, first in the outer (portal) areas of the liver and then working its way in (bridging) to the central vein area.

Extensive fibrosis and deterioration of the liver’s cellular architecture is called cirrhosis. Cirrhosis results when most normal liver cells have been replaced by scar tissue. It can greatly interfere with the liver’s ability to perform many of its usual functions, including the production of proteins and enzymes, the regulation of cholesterol and storage of energy, and the metabolism of drugs and toxins. Cirrhosis can lead to internal bleeding, kidney failure, mental confusion, fluid accumulation, infection, and coma.

Below are photographs of liver biopsies documenting various histologic stages of HCV:

HCV Disease Progression
Normal	Mild Chronic Hepatitis
Moderate Chronic Hepatitis	Cirrhosis
(Courtesy Mark Sulkowski, M.D.)

References
Alter MJ, Kruszon-Moran D, Nainan OV, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 341:556-62, 1999.
Bellentani S, Pozzato G, Saccoccio G, et al. Clinical course and risk factors of hepatitis C virus related liver disease in the general population: report from the Dionysus study. Gut 44:874-80.1999.
Centers for Disease Control and Prevention. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR 47(No. RR-19):1-39, 1998.
Centers for Disease Control and Prevention. 1999 USPHS/IDSA guidelines for the prevention of opportunistic infections in persons infected with human immunodeficiency virus: U.S. Public Health Service (USPHS) and Infectious Disease Society of America (IDSA). MMWR 48(RR-10),1999.
Damen M, Zaaijer H, Reesnik H, et al. International collaborative study on the second Eurohep HCV RNA reference panel. J Virol Meth 58:175-85, 1996.
Davis G, Lau J. Factors predictive of beneficial response to therapy of hepatitis C. Hepatology 26(3 suppl 1):122S-7S, 1997.
Davis G, Esteban-Mur R, Rustgi V, et al. Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. N Engl J Med 339:1493-9, 1998.
De Moliner L, Pontisso P, De Salvo G L, et sl. Serum and liver HCV RNA levels in patients with chronic hepatitis C: correlation with clinical and histological features. Gut 42: 856-860, 1998.
Ghany MG, Chan TM, Sanchez-Pescador R, et al. Correlation between serum HCV RNA and aminotransferase levels in patients with chronic HCV infection. Dig Dis Sci 41:2213-8, 1996.
Gretch D, Corey L, Wilson J, et al. Assessment of hepatitis C virus RNA by quantitative competitive RNA polymerase chain reaction: high-titer viremia correlates with advanced stage of disease. J Infect Dis 169:1219-25, 1994.
Gretch D. Diagnostic tests for hepatitis C. Hepatology 26:43S-7S, 1997.
Kuo G, Choo QL, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-4.
Knodell RG, Ishak KG, Black WC, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1:431-35, 1981.
Lau JYN, Davis G, Kniffen, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 341:1501-04, 1993.
Lindor KD, Jorgensen RA, Rakeela J, et al. The role of ultrasonography and automatic-needle biopsy in outpatient percutaneous liver biopsy. Hepatology 23:1079-83, 1996.
Lunel F, Cresta P, Vitour D, et al. Comparative evaluation of hepatitis C virus RNA quantification by branched DNA, NASBA, and monitor assays. Hepatology 29:528-35, 1999.
Martinot-Peignoux M, Boyer N, Le Breton V, et al. A new step towards standardization of serum hepatitis C virus-RNA quantification on patients with chronic hepatitis C. Hepatology 31:726-29, 2000.
McHutchinson J, Gordon S, Schiff E, et al. Interferon alfa-2B alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 339:1485-92, 1998.
Papini E, Pacella CM, Rossi A, et al. A randomized trial of ultrasound-guided anterior subcostal liver biopsy versus conventional Menghini technique. J Hepatol 12:291-97, 1991.
Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. Lancet 349:825-32, 1997.
Poynard T, Marcellin P, Lee S, et al. Randomized trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 352:1426-32, 1998.
Reichard O, Norkrans G, Fryden A, et al. Comparison of 3 quantitative HCV-RNA assay-accuracy of baseline viral load to predict treatment outcome in chronic hepatitis C. Scand J Infect Dis 30:441-46, 1998.
Romagnuolo J, Jewell L, Jhangri G, et al. Predicting the liver histology in chronic hepatitis C: how good is the clinician? [Abstract L283]. Digestive Disease Week, Orlando, FL, 1999.
Roth WK, Weber M, Seifried E. Feasibility and efficacy of routine PCR screening of blood donations for hepatitis C virus, hepatitis B virus, and HIV-1 in a blood-bank setting. Lancet 353:359-63, 1999.
Schreiber GB, Busch MP, Kleinman SH, et al. The risk of transfusion-transmitted viral infections. N Engl J Med 334:1685-90, 1996.
Smith CI, Reddy R. Cost-effectiveness of ultrasonography in percutaneous liver biopsy [letter]. Hepatology 29:610, 1999.
Terrault N. Viral Diagnostic Studies [slide lecture & syllabus]. AASLD, Postgraduate Course, Dallas, 11/5/99.
Tong C, Hollingsworth R, Williams H, et al. Effect of genotype on the quantification of hepatitis C virus (HCV) RNA in clinical samples using the Amplicor HCV Monitor test and the Quantiplex HCV RNA 2.0 assay (bDNA). J Med Virol 55:191-6, 1998.
Zeuzem S, Franke A, Lee J-H, et al. Phylogenetic analysis of hepatitis C virus isolates and their correlation to viremia, liver function tests, and histology. Hepatology 24:1003-9, 1996.
Zeuzem S, Lee J-H, Franke A, et al.. Quantification of the initial decline of serum hepatitis C virus RNA and response to interferon alfa. Hepatology 27: 1149-56, 1998.
Uyttendaele S, Claeys H, Mertens W, et al. Evaluation of third-generation screening and confirmatory assays for HCV antibodies. Vox Sang 66:122-9, 1994.
Zein N, Germer J, Wendt N, et al. Indeterminate results of the second generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22-3 reactivity and influence of HCV genotypes. J Clin Microbiol 35:311-2, 1997.
Zylberberg H, Pol S. Reciprocal interactions between human immunodeficiency virus and hepatitis C virus infections. Clin Infect Dis 23:1117-25, 1996.