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1 Hepatitis C Virus: Epidemiology, Diagnosis, and Patient Management Peter P. Chou, PhD, DABCC, FACB (Quest Diagnostics Nichols Institute, Chantilly, VA) DOI: /BNK8PH8FEPJ0VCH2 Laboratory photo courtesy of Roche Diagnostics. Used with permission. Roche labmedicine.com February 2007 Volume 38 Number 2 LABMEDICINE 85

2 Hepatitis C virus (HCV) infection is the most common chronic bloodborne infection in the United States. Globally, it is a growing and extremely challenging health care issue. Hepatitis C virus is transmitted primarily through large or repeated direct percutaneous exposures to blood containing HCV. The 2 most common exposures associated with transmission of HCV are intravenous drug use and blood transfusion performed before Laboratory tests are used for screening (liver enzyme, HCV antibody), diagnosis (HCV antibody by RIBA or HCV by PCR), disease staging (liver biopsy), prognosis (HCV genotyping and baseline HCV viral load), and patient monitoring (HCV viral load). Various treatment protocols including monotherapy and/or combination therapy with varying dosages and durations have been established. Patients under therapy need to be monitored for therapy efficacy and any side effects from the medications. Epidemiology Hepatitis C virus infection is the most common chronic bloodborne infection in United States. It is estimated that during the 1980s, an average of 230,000 new infections occurred each year. 1 The annual number of new infections has declined by more than 80% from 1989 to Data from the Third National and Nutrition Examination Survey (NHANES III), conducted during 1988 to 1994, revealed an estimated 3.9 million (1.8%) Americans had been infected with HCV and threefourth were positive for HCV RNA. This indicated that an estimated 2.7 million persons in United States were chronically infected. 3 Individuals who are chronically infected may not be aware of their infection because they are often asymptomatic. Infected persons can serve as a source of HCV transmission to others and are at risk for chronic liver disease or other HCVrelated diseases during the first 2 or 3 decades following initial infection. Furthermore, among those age 40 to 59 years and among African Americans, the prevalence of anti-hcv was even higher. Finally, the results of such serologic surveys may actually underestimate the true prevalence of HCV infections as a result of failing to include representative proportions of high-risk populations such as intravenous drug users, incarcerated individuals, and homeless persons. In the United States chronic liver disease is the tenth leading cause of death among adults. Hepatitis C virus infection accounts for approximately 40% of all chronic liver disease, results in an estimated 8,000 to 10,000 deaths annually, and is the most frequent indication for liver transplantation. 4 Projections based on the current prevalence of infection and anticipated rates of progression raise concerns over the potential impact of HCV during the next 2 decades. A computer cohort simulation of the American population for 2010 to 2020 suggests that as the duration of infection increases in the surviving cohort, the proportion with cirrhosis will increase from 16% to 32% by 2020 in an untreated population. Complications of cirrhosis also will increase dramatically during the next 20 years for hepatic decompensation, hepatocellular carcinoma, and liver-related deaths. These complications are projected to increase by 106%, 81%, and 180%, respectively. 5 On a global scale, based on current estimated that as many as 175 to 200 million persons are currently infected with HCV (Figure 1). 6 The projected morbidity, mortality, current and future health care costs associated with HCV infection are staggering. Transmission and Risk Factors Hepatitis C virus is transmitted primarily through large or repeated direct percutaneous exposures to blood containing HCV. In the United States, the relative importance of the 2 most common exposures associated with transmission of HCV [blood Figure 1_Hepatitis C; a growing and global health problem. 86 LABMEDICINE Volume 38 Number 2 February 2007 labmedicine.com

3 transfusion and intravenous drug (IVD) use] has changed over time. Blood transfusion, which accounted for a substantial proportion of HCV infections acquired about 20 years ago, rarely accounts for recently acquired infections today. In fact, since 1994, the risk for transfusion-transmitted HCV infection has been so low that the Centers for Disease Control and Prevention (CDC) Sentinel Counties Viral Hepatitis Surveillance system has been unable to detect any reported transfusion-associated cases of acute hepatitis C infection. The risk of transfusion-associated HCV infection is small; however, it is not zero. In contrast, IVD use consistently has accounted for a substantial proportion of HCV infections and currently accounts for 60% of HCV transmission in the United States. Table 1 shows the risk factors associated with HCV transmission. There is no protective vaccine presently available for hepatitis C, largely because the virus mutates frequently, making it very difficult to develop an effective vaccine. Physical Characteristics of HCV Hepatitis C virus (Figure 2) is a member of the genus Flaviviridae. Since there is no system for growing HCV in cell culture, most of what is known about the physical characteristics of the virus is derived from cloned virus. The genetic material of HCV, or its genome, consists of a single strand of RNA approximately 9,400 nucleotides long. The HCV genome encodes 6 to 8 proteins. A diagram of the viral RNA genome is shown in Figure 3. Two non-coding regions referred to as the 5 untranslated region (UTR) and 3 UTR flank the protein-coding regions of the genome. The 5 UTR is the main target detected in the nucleic acid based assays. Serologic assays to detect HCV antibodies are directed against peptides c22-3, c33c, and c100-3, which are derived from the various viral proteins. Different strains of HCV show a considerable degree of nucleotide sequence diversity. By comparing the nucleotide sequences of different strains of HCV, the virus can be divided into 6 main groups or genotypes and more than 50 subtypes. Figure 4 illustrates phylogenetic analysis of NS-5 sequences from 76 isolates of HCV, showing 6 major HCV genotypes and some subtypes. 7 Screening and Diagnostic Tests Serologic Assays Tests to detect antibody to HCV (anti-hcv) were first licensed by the Food and Drug Administration (FDA) in Since that time, new versions of these and other FDA-approved anti-hcv tests have been used widely for clinical diagnosis and screening of asymptomatic persons. FDA-licensed or approved anti-hcv screening test kits being used in the United States comprise 3 immunoassays; 2 enzyme immunoassays (EIA) (Abbott HCV EIA 2.0, Abbott Laboratories, Abbott Park, IL, and ORTHOR HCV 3.0 ELISA, (Ortho Clinical Diagnostics, Raritan, NJ) and 1 enhanced chemiluminescence immunoassay (CIA) VITROS Anti-HCV assay, Ortho-Clinical Diagnostics, Raritan, NJ). All of these immunoassays use HCV-encoded recombinant antigens. The CDC has recommended that a person be considered to have serologic evidence of HCV infection only after an anti- HCV screening-test-positive result has been confirmed by a more specific serologic test or a nucleic acid test (NAT). 1 Table 1_Risk Factors Associated With HCV Transmission Figure 2_Hepatitis C virus. Figure 3_Hepatitis C visus genome. Figure 4_Analysis of NS5 Sequence from 76 HCV isolates. labmedicine.com February 2007 Volume 38 Number 2 LABMEDICINE 87

4 FDA-approved confirmatory tests include Chiron RIBA HCV 3.0 SIA (Chiron Corporation, Emeryville, CA), AMPLICOR HCV Test, Version 2.0 (Roche Molecular Systems, Indianapolis, IN), and COBAS AMPLICOR HCV Test (Roche Molecular Systems, Indianapolis, IN). Signal to Cut-Off (S/CO) Ratios Analysis of early versions of anti-hcv EIA results from volunteer blood donors indicated that average repeatedly reactive s/co ratios could be used to predict supplemental test-positive results. 8 Subsequent studies and additional data from other populations generated by CDC confirmed that a specific s/co ratio could be identified that would predict a true antibody positive result greater than or equal to 95% of the time, regardless of the anti-hcv prevalence or characteristics of the population being tested. For EIA, screening-test-positive average s/co ratio greater than or equal to 3.8 was highly predictive of RIBA positivity (greater than and/or equal to 95%). However, the range of screening-test-positive s/co ratio obtained by CIA (VITROS) was greater than that obtained by EIA. Thus, s/co ratio of greater than and/or equal to 8.0 predicts RIBA positivity in 95% to 98% of the screen positive samples. Figure 5 illustrates the laboratory test algorithm and result reporting. Figure 5_Laboratory testing algorithm and result reporting. Nucleic Acid Tests FDA-approved diagnostic NATs for qualitative detection of HCV RNA includes AMPLICOR HCV Test, COBAS AMPLICOR HCV Test (Roche Molecular Systems, Indianapolis, IN), and VERSANT HCV RNA Qualitative Assay by TMA (transcription-mediated amplification, Bayer Corporation, Tarrytown, NY). Quantitative detection (viral load) of HCV RNA includes Roche COBAS and TaqMan, RT-PCR, analyte specific reagent), Bayer Corporation (Tarrytown, NY) branched chain DNA (bdna, FDA approved for viral load) and other laboratory developed assays. Genotyping can be performed using a simple technique such as the Line Probe Assay (LiPA) (Bayer Corporation, Tarrytown, NY), which uses immobilized HCV RNA sequences that are allowed to hybridize to RNA in the sample, with visual detection of hybrids by an enzyme reaction. The pattern of bands allows determination of genotype in most cases. Final Report and Interpretation of Test Results For those screen-test-positive samples that undergo reflex supplemental testing, the screening test anti-hcv results should not be reported before the results from the additional testing are available. If necessary, an interim or preliminary report can be issued indicating that the result is pending for confirmation. This procedure should be followed even if the laboratory sends the sample to a reference laboratory for such testing. After the results are received from the reference laboratory, the final results can be reported on the basis of the testing performed by both laboratories. 8 The report results should be accompanied by interpretive comments as determined by each laboratory (Table 2). 7 The content of these comments will vary on the basis of type of supplemental testing option selected by the laboratory. These comments are critical if screen-test-positive results are reported as anti-hcv positive on the basis of high s/co ratios, because the health care professional or other person interpreting the results needs to understand the limitations of the testing option used. Predictors of Treatment Outcome Baseline Viral Load Baseline viral load is a well-recognized predictor of treatment outcome. A viral load of< 2 million copies/ml (approximately 800,000 IU/mL) has been associated with a more favorable response to therapy. 9 In addition, quantitative assessment of serum HCV RNA levels allows the physicians to identify those patients with low levels of HCV RNA who may have a greater chance of achieving a durable response to treatment. Viral load testing is an important component of pretreatment patient assessment because knowledge of the magnitude of HCV infection allows the treating physician to provide patient guidance and prognosis when discussing the risks and benefits of therapy. Genotyping Various studies have suggested that baseline viral load might not be the only factor influencing the response to interferon treatment. In fact, a number of studies have shown that different HCV genotypes differ significantly in their response to this medication. 9,10 The reason for the differences in interaction sensitivity are unclear, but different subtypes of HCV may have different replication efficiencies, which give rise to different serum levels of virus and different responses to interferon. Alternatively, it has been suggested that there may be a genetic basis for viral resistance to interferon. In general, patients infected with genotypes 1, 4, and 5 tend to show less response to interferon therapy than those infected with genotypes 2 and 3. Liver Biopsy The liver biopsy results of numerous prospective and retrospective studies indicate that HCV progression to cirrhosis is <7% in infected patients in the absence of interface hepatitis (troxis necrosis). If fibrosis is absent, the long-term risk of developing cirrhosis is 20% to 30%. If fibrosis, inflammation, or necrosis is observed, then the chance of progression to cirrhosis is 70%. The prevention of progressive fibrosis and frank cirrhosis is probably the most important therapeutic end point LABMEDICINE Volume 38 Number 2 February 2007 labmedicine.com

5 Table 2_Recommendations for Reporting Results of Testing for Anti-HCV by Types of Reflex Supplemental Testing Performed Physicians can now take 1 of 2 approaches: (1) treat all patients with chronic HCV without first evaluating a liver biopsy, or (2) customize treatment recommendations based on histologic findings and the probable time that the host acquired HCV. The estimated cost of a liver biopsy ($500 to $1,500) and the average cost of treatment, clinic visit, and laboratory tests is $10,000. The limitation of biopsy is that this procedure samples only a very small amount of liver tissue. Therefore, it may not represent the most severe changes in the liver. Additionally liver biopsy carries a small but real risk of significant complications such as hemorrhage, bile peritonitis, etc. Table 3 summarizes various predictive factors associated with response to HCV treatment. 12 Treatment Protocols The treatment protocols for chronic HCV infection have been alpha interferon (IFN) monotherapy (Table 4). The 3 interferons that are approved for the treatment of HCV in the United States include recombinant interferon alpha-2b (rifnα2b, INTRON A, Schering-Plough Corporation, Kenilworth, NJ), interferon alfa-2a (Roferon-A, Hoffmann-La Roche Laboratories, Basle, Switzerland) and interferon alfacon-1 INTERGEN, Amgen, Thousand Oaks, CA). A fourth interferon, interferon α-in (Wellferon, Glaxo SmithKline, Research Triangle Park, NC), is also approved to treat HCV infection in a number of countries. These 4 interferons appear to be clinically equivalent. Approximately 40% of patients treated with alpha interferon monotherapy demonstrate normalization of serum liver enzyme alanine amino transferase (ALT) levels and viral disappearance at the end of therapy; however, greater than half of these responders demonstrate Table 3_Predicting Factors Associated With Response to HCV Treatment Factor Favorable Unfavorable Genotype 2,3 1,4,5,6 Baseline Viral Load <800,000 IU/mL >800,000 IU/mL Cirrhosis Absence Bridging, cirrhosis Age <40 >40 Gender Female Male Body Mass Index <28 >28 Ethnic Origin European, Hispanic African Relative increase in ALT >3 <3 ( Upper Reference Limit) Table 4_Treatment Protocols Using Various Dosing Regimens for Chronic Hepatitis C Treatment Recommended Dose Treatment Duration Monotherapy Interferon alfa-2b 6 MIU TIW 48 weeks Interferon alfa-2a 3 MIU 72 weeks Interferon alfacon-1 9 µg TIW weeks Combination Therapy Interferon alfa-2b + 3 MIU TIW + 24 weeks - Relapse Ribavirin 1,000 1,200 mg daily* weeks Naïve PEG-IFN alfa ug or 1.5 µg/kg weekly+ 24 weeks - Types 2/3 Ribavirin 1,000 1,200 mg daily* 48 weeks Type 1 Abbreviations: MIU=million international units; TIW=3 times weekly. *Dosage based on body weight: patients weighing < than or equal to 75 kg receive 1,000 mg oral ribavirin, patients weighing >75 kg receive 1,200 mg oral ribavirin. FDA approved 800 mg daily dose of ribavirin when used with PEG-IFN alfa, especially in patients who weigh <65 kg. labmedicine.com February 2007 Volume 38 Number 2 LABMEDICINE 89

6 relapse as evidenced by the re-appearance and/or increase of HCV RNA viral load after treatment cessation. 11,13 Attempts to improve patient response to therapy have included modifying the dose or dosing regimen of alpha interferon, or combining alpha interferon with other antiviral agents (eg, ribavirin). The use of daily versus 3 times weekly (TIW) interferon has been proposed by a number of investigators, and this regimen appears to increase the initial response rate and possibly the durability of response. 14 In an early study, the use of combination therapy (interferon/ribavirin) resulted in sustained responses in 40% of treated patients. Sustained response rates ranging from 30% to 70% in various HCV populations have since been reported by other investigators. Although combination treatments are more effective than monotherapy, some patients cannot tolerate ribavirin. Contraindications to ribavirin treatment include anemia, renal insufficiency, and pregnancy or the potential to become pregnant. The attachment of polyethylene glycol to the interferon molecule (PEG-IFN) is the most recent innovation in the treatment of HCV infection. Pegylation reduces the degradation and clearance thus prolonging the half-life of interferon and permitting less frequent, weekly dosing while maintaining higher and sustained interferon levels. Therefore, the current standard of care for the treatment of naïve (previously untreated) patients with chronic hepatitis C is combination PEG-IFN α by subcutaneous injection once a week and oral ribavirin daily. Table 4 shows treatment protocols involving various dosing regimens for chronic hepatitis C. Approximately 30% of all patients infected with human immunodeficiency virus (HIV) are co-infected with HCV, presumably because of shared routes of transmission. HIV/HCV co-infection is particularly common in IVD users with HIV infection, among this group of patients up to 90% may be co-infected with HCV. Therefore, all patients with HIV infection should be screened for HCV infection. HIV/HCV co-infection often is predictive of a poor outcome (eg, end stage liver disease). A majority of studies have shown Figure 6_Testing schematic. Table 5_Methodologies, Dynamic Ranges and Low Limits of Detection of Different HCV Quantitative NATs Assay Method Lower Limit Upper Limit IU/mL IU/mL Amplicor Monitor (Roche) RT-PCR ,000 Versant (Bayer) bdna 600 8,000,000 SuperQuant (NGI/LabCorp, Real Time-PCR ,000,000 Los Angeles,CA) Heptimax (Quest Diagnostics Real Time-PCR 5 50,000,000 Nichols Institute, San Juan TMA Capistrano, CA and Chantilly, VA) HCV RNA (Focus Diagnostics, Real Time-PCR ,000,000 Cypress, CA) TaqMan (Roche) Real Time-PCR 50 50,000,000 a lower rate of antibody response to HCV in patients with HIV/HCV co-infection. Nonetheless, a similar therapeutic response has been described for patients co-infected with HIV and HCV when compared with patients infected with HCV only, provided that the CD4 cell count was >500. No response to interferon was observed when CD4 cell count was < ,15-17 Monitoring of Treatment and Patient Management Baseline and 12 week monitoring of HCV RNA levels should be performed with the same quantitative assay. An early virologic response (EVR), defined as a 2 log (100 fold) reduction in HCV RNA levels during the first 12 weeks of therapy, is a valuable clinical achievement. In the absence of an EVR, the likelihood of a sustained virologic response (SVR) is less than 3%. If the only goal of therapy is to achieve an SVR, therapy can be discontinued after 12 weeks if an EVR is not achieved. However, histologic benefit may occur even in the absence of an SVR; therefore, some clinicians treat beyond 12 weeks even in the absence of an EVR. Documentation of a virologic response at the end of therapy (end-of-treatment response) or an SVR >6 months after completing therapy, requires a more sensitive quantitative assay with a low detection limit. 18 It is imperative to select the vital load assay that offers not only the wide dynamic range but also low detection limit. Table 5 lists dynamic ranges of various quantitative HCV RNA assays. 12 Figure 6 illustrates comprehensive laboratory testing used to make initial diagnosis and/or prognosis of HCV infection. Once the therapy is initiated HCV RNA results are used to judge the efficacy of treatment and to verify SVR and possible eradication of the virus. 90 LABMEDICINE Volume 38 Number 2 February 2007 labmedicine.com

7 Side effects during therapy for chronic hepatitis C with IFN or PEG-IFN include the following: (a) flu-like systemic symptoms; (b) bone marrow suppression (primarily leukopenia and thrombocytopenia); (c) behavioral effects such as irritability, difficulty concentrating, disturbed memory, and depression, and (d) autoimmune thyroiditis. Side effects related to ribavirin include hemolytic anemia, chest congestion, pruritus, rash, gout, nausea, diarrhea, and teratogenicity. Table 6 lists recommended thresholds for drug dose reductions in patients treated with PEG-IFN and ribavirin for chronic hepatitis C. Patients who have chronic liver disease caused by viral hepatitis are more susceptible to severe disease after being infected with alternate types of viral hepatitis. It has been proposed that all HCV-infected patients should undergo vaccination for hepatitis A and B, and that all patients with chronic hepatitis B should undergo vaccination for hepatitis A. Although specific recommendations for vaccination have not been defined, some consideration should be given to vaccination against influenza, pneumococcus, and Haemophilus influenza B infection. 11 Conclusions Untreated chronic HCV infection can lead to cirrhosis, hepatocelluar carcinoma, and extrahepatic disease in a large number of patients. Patients should be advised of the utility of various laboratory tests such as HCV genotyping, viral quantitation, and liver biopsy. Patients should also be informed about how test information can impact their treatment options and disease prognosis. 11 The possibility of SVR or a cure, in addition to a discussion of side effects and management, should also be reviewed with the patients. Until the time arrives in which the general population can routinely be vaccinated against HCV infection, or individuals modify their high-risk behavior, infected patients should be offered therapeutic intervention. Alpha interferon has been the mainstay therapeutic option, and novel regimens continue to be evaluated in clinical investigations. The addition of ribavirin to interferon and/or pegylated interferon therapy clearly marks a significant advance in the treatment of chronic hepatitis C. However, additional work is required to ensure that new more effective therapies continue to be developed for this emerging public health threat. LM 1. CDC. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR. 1998;47(No. RR19): Alter MJ. Epidemiology of hepatitis C. Hepatology. 1997;26:62S-65S. 3. Alter MJ, Kruszon-Moran D, Nainan OV, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through N Eng J Med. 1999;341: Table 6_Recommended Thresholds for Drug Dose Reductions in Patients Treated With PEG-IFN and Ribavirin for Chronic Hepatitis C Hematologic Threshold Dose Reduction Absolute neutrophil count (/mm 3 ) Reduce PEG-IFN dose <500 Withhold PEG-IFN Platelet count (/mm 3 ) 25,000-50,000 Reduce PEG-IFN dose <25,000 Withhold PEG-IFN Hemoglobin (g/dl) <= 10 Reduce ribavirin dose <= 8.5 Withhold ribavirin 4. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med. 2001;345: Davis GL, Albright JE, Cook SE, et al. Projecting future complications of chronic hepatitis C in the United States. Liver Transpl. 2003;9: World Health Organization. Global surveillance and control of hepatitis C. Report of a WHO consultation organized in collaboration with the Viral Hepatitis Prevention Board. Antwerp, Belgium. J Viral Hepat. 1999;6: Simmonds P, Holmes EC, Cha T-A, et al. Classification of hepatitis C virus into six major genotypes and a series of subtype by phylogenetic analysis of the NS-5 region. J General Virology. 1993;74: CDC. Guidelines for laboratory testing and result reporting of antibody to hepatitis C virus. MMWR. 2003;52(No. RR3): Poynard T, Marcellin P, Lee S, et al. Randomised trial of interferon alfa-2b and ribavirin for 48 weeks or for 24 weeks versus interferon alfa-2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet. 1998;352: Davis GL, Lau JY. Factors predictive of a beneficial response to therapy of hepatitis C. Hepatology. 1997;26:122S-127S. 11. Gish RG. Standards of treatment in chronic hepatitis C. Seminars in Liver Disease. 1999;19: Dufour DR. Hepatitis C: laboratory tests for diagnosis and monitoring of infection. Clinical Laboratory News 2002;11: Heathcote EJ, Keeffe EB, Lee SS, et al. Retreatment of chronic hepatitis C with consensus interferon. Hepatology. 1998;27: Lam NP, Neumann AU, Gretch DR, et al. Dose-dependent acute clearance of hepatitis C genotype 1 virus with interferon alpha. Hepatology. 1997;26: Chamot E, Hirschel B, Wintsch J, et al. Loss of antibodies against hepatitis C virus in HIV-seropositive intravenous drug users. AIDS. 1990;4: Boyer N, Marcellin P, Degon C, et al. Recombinant interferon-alpha for chronic hepatitis C in patients positive for antibody to human immunodeficiency virus. J infect Dis. 1992;165: Marriott E, Navas S, delromero J, et al. Treatment with recombinant alphainteferon of chronic hepatitis C in anti-hiv positive patients. J Med Virol. 1993;40: Diestag JL, McHutchison JG. American Gastroenterological Association technical review on the management of hepatitis C. Gastroenterology. 2006;130: labmedicine.com February 2007 Volume 38 Number 2 LABMEDICINE 91