Use and Interpretation of Virological Tests for Hepatitis C

Jean-Michel Pawlotsky
Four virological markers of hepatitis C virus (HCV) infection are used clinically for management of patients with hepatitis C, namely the HCV genotype, HCV RNA, HCV core antigen, and antibody to HCV (anti-HCV). The diagnosis of acute and chronic hepatitis C is based on both anti-HCV detection using enzyme immunoassays (EIA) and HCV RNA detection using a sensitive molecular biology-based technique. Other virological tools, including HCV genotype determination and HCV RNA quantication, are now used to tailor treatment to the individual patient and to determine its efcacy. This article reviews the kinetics of HCV markers during acute and chronic HCV infection, together with current assays and their practical use in the management of HCV-infected patients. (HEPATOLOGY 2002;36:
S65-S73.)

irological diagnosis and monitoring of hepatitis C virus (HCV) infection are based on 2 categories of laboratory test, namely serologic assays detecting specic antibody to HCV (anti-HCV) (indirect tests) and assays that can detect, quantify, or characterize the components of HCV viral particles, such as HCV RNA and core antigen (direct tests). Direct and indirect virological tests play a key role in the diagnosis of infection, therapeutic decision-making, and assessment of the virological response to therapy.

Virological Assay Techniques

Immunoenzymatic Techniques Immunoenzymatic techniques are widely used to detect and, sometimes, to quantify viral antigens or specic antibodies in body uids.
Abbreviations: HCV, hepatitis C virus; anti-HCV, antibody to HCV; EIA, enzyme immunoassays; ELISA, enzyme-linked immunosorbent assays; PCR, polymerase chain reaction; cDNA, complementary double-stranded DNA; TMA, transcription-mediated amplication; IU, international unit. From the Department of Virology (EA 3489), Henri Mondor Hospital, University of Paris XII, Cre teil, France. Supported in part by grants from the French Ministry for Research, the Agence Nationale de Recherches sur le SIDA et lHe patite C (ANRS), the Re seau National He patites, and the Ligue Franc aise contre le Cancer. Dr. Pawlotsky is or has been a consultant for Hoffmann-La Roche, Schering Plough Oncology, Isis Pharmaceuticals, Abbott Diagnostics, Ortho-Diagnostics, AcroMetrix, Inc, and Chiron-Blood Testing. He has received research grants from Hoffmann-La Roche and Schering Plough Oncology. Address reprint requests to: Professor Jean-Michel Pawlotsky, M.D., Ph.D., Service de Virologie, Ho pital Henri Mondor, 51 avenue du Mare chal de Lattre de Tassigny, 94010 Creteil, France. E-mail: jean-michel.pawlotsky@hmn.ap-hopparis.fr; fax: (33) 1 4981 4831. Copyright 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3605-1008$35.00/0 doi:10.1053/jhep.2002.36815

Enzyme Immunoassays. In enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA), serum or plasma antibodies or viral antigens are captured on the wells of microtiter plates by using the corresponding antigen or specic (generally monoclonal) antibodies, respectively. Antigen-antibody complexes are then specifically revealed in a colorimetric enzymatic reaction. After reading in a spectrophotometer, the result is expressed as the ratio of the optical density of the test sample to that of a kit control. EIAs are easy to use, partly or fully automated, and suitable for testing large numbers of samples. Immunoblot Assays. Immunoblot tests also detect specic antibodies, but using viral antigen-coated nitrocellulose strips instead of microtiter plates. Positive reactions are characterized by the appearance of colored bands at specic positions on the strip.1 Interpretation may be visual or automated. Molecular Biology Techniques Molecular biology techniques are useful in detecting and quantifying viral genomes in body uids by means of amplication methods, such as target amplication and signal amplication.2 Viral genome sequences may also be analyzed for genotyping purposes, most frequently by means of direct sequencing or reverse hybridization.2 Target Amplication Techniques. A large number of viral genome copies (amplicons) are amplied in a cyclic enzymatic reaction. The amplicons can then be detected by various methods, and the amount of viral genomes in the clinical sample can be quantied. The polymerase chain reaction (PCR) method3 uses several temperatures and one enzyme: a thermostable DNA polymerase. An initial reverse transcription step is required in the case of
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HCV RNA in order to synthesize a complementary double-stranded DNA (cDNA) for use as template in the PCR reaction. The amplicons consist of double-stranded DNA. In transcription-mediated amplication (TMA),4 the reaction is isothermal and uses two enzymes: a reverse transcriptase and a T7 RNA polymerase. The amplicons consist of single-stranded RNA. Detection of amplicons obtained by PCR and TMA is classically based on specic hybridization to immobilized oligonucleotide probes. Amplicon-probe hybrids are revealed in an enzymatic reaction, followed by detection of a colored or luminescent signal. Quantication is based on competitive amplication of the viral template with a known amount of synthetic standard added to each reaction tube. The relative amounts of viral template and standard amplicons are measured at the end of the procedure and the results are read from a standard curve established in parallel. More recently, real-time PCR techniques have been developed. The principle is to detect amplicon synthesis and to deduce the amount of viral genomes in the starting clinical sample during rather than at the end of the PCR reaction. These methods are theoretically more sensitive than classical target amplication techniques and are not prone to carryover contamination. Their dynamic range of quantication is consistently wider, making them particularly useful for quantifying the full range of viral loads observed in untreated and treated patients with HCV infection. Signal Amplication Techniques. Viral genomes are rst hybridized to a holder, using specic capture oligonucleotide probes. Then the signal emitted by the hybrids is amplied for detection and measurement, for instance by using branched DNA amplier molecules that serve as sites for hybridization with alkaline-phosphatase-conjugated oligonucleotide probes.5 Detection is based on alkaline-phosphatase-catalyzed chemiluminescence emission from a substrate. Quantication is based on a standard curve generated simultaneously using known standards. Direct Sequencing. Sequencing is the gold standard for genomic sequence analysis. Direct sequencing of PCR amplicons is achieved with an automated DNA sequencer, giving the exact nucleotide and deduced amino acid sequences of the analyzed fragment. The generated sequences may be interpreted by direct examination of nucleotide or amino acid motifs or by phylogenetic analysis using reference sequences. Reverse Hybridization. PCR amplication of the region of interest is followed by stringent hybridization with oligonucleotide probes xed to a solid phase. The probes are designed to be complementary to the various possible

Fig. 1. Time course of HCV RNA, HCV core antigen, and anti-HCV antibodies in patients with (A) acute, spontaneously resolving hepatitis C or (B) evolution to chronic infection. White boxes: the marker is absent; black boxes: the marker is present. Alanine aminotransferase levels (ALT) are displayed in gray.

sequences. Revelation is based on a colorimetric reaction. Hybridization to a particular probe means that the analyzed fragment has the complementary sequence.

HCV Markers and Their Kinetics

Four virological markers of HCV infection can be used in management of infected patients, namely HCV genotype, HCV RNA, HCV core antigen, and anti-HCV antibodies. The kinetics of HCV markers during spontaneously resolving acute and chronic hepatitis C are shown in Figs. 1A and B, respectively. HCV Genotype. The HCV genotype is an intrinsic characteristic of the transmitted HCV strain(s) and does not change during the course of the infection. HCV genotypes form 6 clades or types (numbered 1 to 6) and are themselves subdivided into a large number of subclades or subtypes identied by lower-case letters (1a, 1b, 1c, etc).6 Phylogenetic analysis can distinguish HCV types, subtypes, and isolates on the basis of average sequence divergence rates of approximately 30%, 20%, and 10%, respectively.6 HCV RNA. The presence of HCV RNA in peripheral blood is a reliable marker of active HCV replication, which takes place principally in the liver. HCV RNA is

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Table 1. Diagnostic EIA assays for Detection of Anti-HCV

Epitopes (origin of the antigens) FDA Approval at the Date of Writing (June 2002)

Assay

Manufacturer

Technique

Ortho HCV 3.0 ELISA Test System with Enhanced SAVe Vitros anti-VHC Abbott HCV EIA 2.0 Abbott HCV EIA 3.0 IMx HCV 3.0 AxSYM HCV 3.0 Monolisa anti-HCV Plus version 2 Access HCV Ab Plus Innotest HCV Ab IV

Ortho-Clinical Diagnostics, Raritan, NJ Ortho-Clinical Diagnostics, Raritan, NJ Abbott Diagnostic, Chicago, IL Abbott Diagnostic, Chicago, IL Abbott Diagnostic, Chicago, IL Abbott Diagnostic, Chicago, IL Bio-Rad, Marnes-la-Coquette, France Bio-Rad, Marnes-la-Coquette, France Innogenetics, Ghent, Belgium

EIA, microtiter plate

core, NS3, NS4, NS5 (Chiron Corporation) core, NS3, NS4, NS5 (Chiron Corporation) core, NS3, NS4 (Chiron Corporation) core, NS3, NS4, NS5 (Chiron Corporation) core, NS3, NS4, NS5 (Chiron Corporation) core, NS3, NS4, NS5 (Chiron Corporation) core, NS3, NS4 (Bio-Rad) core, NS3, NS4 (Bio-Rad) core, NS3, NS4, NS5 from genotypes 1, 2, and 3a (Innogenetics)

FDA approved

EIA, automated on Vitros ECI device ELISA, microbeads ELISA, microbeads EIA, automated on IMx device EIA, automated on AxSYM device EIA, microtiter plate EIA, automated on ACCESS device EIA, microtiter plate

FDA approved FDA approved Not approved Not approved Not approved Not approved Not approved Not approved

detectable in serum within 1 to 2 weeks after infection. It generally increases to reach a peak, before disappearing when the infection resolves spontaneously. In contrast, in most patients progressing to chronic infection, the decrease in HCV RNA gradually slows then stabilizes; occasionally, however, HCV RNA may become undetectable for a few days or weeks before reappearing and reaching a plateau. HCV RNA levels are stable over time in patients with chronic infection.7 Indeed, viral kinetics are at a steady state, i.e., peripheral release of viral particles is counterbalanced by their constant peripheral degradation, while de novo infection of hepatocytes is counterbalanced by infected hepatocytes death by apoptosis.8 During this steady state, the estimated mean half-life of HCV virions in the general circulation is of the order of 3 hours, and virus turnover (production/clearance) is of the order of 1012 virions a day.8 The HCV RNA level may increase slightly after several years of chronic infection. The HCV RNA level is not affected by the severity of liver disease, except in patients with end-stage liver disease, who generally have low or even undetectable HCV RNA levels.9,10 The decrease in viral level with end-stage liver disease is probably due to hepatocyte depletion and extensive brosis rather than to changes in the virus itself, as HCV recurrence after liver transplantation is generally associated with high HCV RNA levels, which is facilitated by immunosuppressive treatment.9,10 HCV Core Antigen. The icosahedral HCV capsid is formed by polymerization of HCV core protein, a 21-kd structural phosphoprotein composed of the rst 191 amino acids of the viral polyprotein.11 It has recently been

shown that total HCV core antigen levels correlate with HCV RNA levels.12 During the preseroconversion period, core antigen is detected on average 1 to 2 days later than HCV RNA with current assays.13,14 Thereafter, core antigen kinetics run closely parallel to HCV RNA kinetics.12 The HCV core antigen titer can thus also be used as a marker of HCV replication. Anti-HCV Antibodies. The serologic window between HCV infection and the detectability of specic antibodies varies from patient to patient. With current assays, seroconversion occurs on average at 7 to 8 weeks after onset of infection.15-17 Anti-HCV is detectable in 50% to 70% of patients at the onset of clinical symptoms and later in the remaining patients.18 In patients with spontaneously resolving infection, anti-HCV may persist throughout life, or decrease slightly while remaining detectable, or gradually disappear after several years.19 AntiHCV persists indenitely in patients who develop chronic infection, although antibodies may become undetectable (with current assays) in hemodialysis patients or in cases of profound immunodepression. Apparent seroreversions and/or seroconversions can occur in immunodepressed patients, in whom the chronic nature of the infection is conrmed by the persistence of HCV RNA.

Available HCV Tests

Indirect Tests Anti-HCV Detection. Anti-HCV is typically identied by using second- or third-generation EIAs. Table 1 shows currently available anti-HCV EIAs, some of which

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are not yet Food and Drug Administration (FDA)-approved. These assays detect mixtures of antibodies directed against various HCV epitopes located in the core, NS3, NS4, and (in the case of third-generation assays) NS5 proteins (Table 1). Viral antigens can be coated onto microtiter plates, microbeads, or specic holders adapted to closed automated devices (Table 1). The specicity of current EIAs for anti-HCV is greater than 99%. Their sensitivity is more difcult to determine, given the lack of a gold standard. Clinical experience indicates that antiHCV EIAs are positive in more than 99% of immunocompetent patients with detectable HCV RNA.20 These tests may be negative, despite chronic HCV replication, in hemodialysis patients and patients with profound immunodeciencies, but latest-generation assays perform better than older tests in these situations.21 Immunoblot tests have been used in the past as conrmatory assays for anti-HCV reactivity identied by EIA. Given the good performance of current anti-HCV EIAs, however, immunoblot tests are clinically obsolete.22 These tests are still used in blood banks, where low-risk populations are tested and the positive predictive value of EIA is signicantly lower than in diagnostic use. Even their utility in blood donor screening may decrease in the future with the advent of systematic molecular testing for HCV RNA in the United States and the European Union.23 Serologic Determination of the HCV Genotype. The HCV genotype can be determined by testing for type-specic antibodies with a competitive EIA (so-called serotyping). The available assay (Murex HCV Serotyping 1-6 Assay; Murex Diagnostics, Dartford, UK) provides interpretable results in approximately 90% of immunocompetent patients with chronic hepatitis C.24 Its sensitivity is lower in hemodialysis and immunodepressed patients.25,26 The assay identies the type (1 to 6), but does not discriminate between subtypes of HCV (1a v 1b, for instance). Concordance with molecular assays is of the order of 95% and is better for genotype 1 than for other genotypes.24,27 In the rare cases of discrepancy, sequencing of reference genomic regions, such as NS5B and E1, generally conrms the result of the molecular assay.28 Mixed serologic reactivity is sometimes observed, and this test cannot distinguish between true mixed infection and cross-reactivity or recovery from one genotype infection and persistence of viremia with another. No serotyping assays have received FDA approval. Direct Tests Molecular Determination of the HCV Genotype (genotyping). The gold standard for genotyping is direct sequencing of the NS5B or E1 region, followed by se-

quence alignment with reference sequences and phylogenetic analysis.6 In clinical practice, HCV can be genotyped by direct sequence analysis, by reverse hybridization to genotype-specic oligonucleotide probes, or by restriction fragment length polymorphism analysis.29-31 Two standardized kits based on PCR amplication of the 5 noncoding region are commercially available. The Trugene HCV 5NC Genotyping kit (Visible Genetics, Toronto, Ontario, Canada)32,33 is based on direct sequencing of PCR amplicons and sequence comparison with a reference sequence database. The line-probe assay (INNO-LiPA HCV II; Innogenetics, Ghent, Belgium)29,30 is based on reverse hybridization of PCR amplicons to a nitrocellulose strip coated with genotypespecic oligonucleotide probes, followed by colorimetric revelation of the hybrids. Both assays can identify the 6 HCV types and a large number of subtypes. Typing errors are uncommon, but subtyping errors may occur in 10% to 25% of cases. These errors are related to the region studied (the 5 noncoding region) rather than to the technique used. Subtyping errors have few clinical consequences, because only the type is used for clinical decision-making. No genotyping assays have been approved in the United States at the time of the Consensus Conference (June 2002). Qualitative Detection of HCV RNA. Qualitative (i.e., non-quantitative) HCV RNA detection tests are still useful, as they are signicantly more sensitive than most available quantitative assays. Qualitative assays are based on the principle of target amplication using either PCR or TMA.2 Two commercial assays are currently available. The PCR-based assay can be fully manual (Amplicor HCV v2.0; Roche Molecular Systems, Pleasanton, CA) or comprise manual extraction and automated reverse transcription, amplication, and reading in the Cobas Amplicor device (Cobas Amplicor HCV v2.0; Roche Molecular Systems). In both cases the detection cutoff is 50 international units (IU) HCV RNA per milliliter. The TMA-based assay (Versant HCV RNA Qualitative Assay, Bayer Corp., Tarrytown, NJ) is fully manual at present and is slightly more sensitive (10 IU/mL).34 The specicity of the 2 assays is 98% to 99%. Only the PCR-based Amplicor HCV v2.0 assay has received FDA approval as of June 2002. Viral Quantication. The HCV RNA level can be quantied by means of target amplication techniques (PCR or TMA) or signal amplication techniques (branched DNA assay). Until recently, the quantitative units used in the various assays did not represent the same amount of HCV RNA in a clinical sample. The World Health Organization (WHO) has now established an international standard for universal standardization of

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Table 2. Assays for Quantication of HCV RNA in Serum

Dynamic Range of Quantication FDA Approval at the Date of Writing (June 2002)

Assay

Manufacturer

Technique

Amplicor HCV Monitor v2.0 Cobas Amplicor HCV Monitor v2.0 Versant HCV RNA 2.0 Quantitative Assay Versant HCV RNA 3.0 Quantitative Assay LCx HCV RNA Quantitative Assay SuperQuant

Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Bayer Corporation, Tarrytown, NJ Bayer Corporation, Tarrytown, NJ Abbott Diagnostic, Chicago, IL National Genetics Institute, Los Angeles, CA

Manual competitive reverse transcriptase-PCR Semi-automated competitive reverse transcriptase-PCR Manual branched DNA assay Semi-automated branched DNA assay Semi-automated competitive reverse transcriptase-PCR Semi-automated competitive reverse transcriptase-PCR

600500,000 IU/mL 600500,000 IU/mL 200,000120,000,000 genome equivalents* mL 6157,700,000 IU/mL 252,630,000 IU/mL 301,470,000 IU/mL

Not approved Not approved Not approved Not approved Not approved Not approved

*Nonstandardized HCV RNA unit in this assay.

HCV RNA quantication units.35 An IU for HCV RNA has been arbitrarily dened using this standard.35,36 It does not represent the actual number of viral particles in a sample, but rather the amount of HCV RNA. All commercial HCV RNA quantitative assays now use the IU (Table 2), which should be preferred to all other units.36 Indeed, this allows recommendations and guidelines to be established in clinical trials and applied in clinical practice using any quantitative HCV RNA assay. Table 3 shows IU conversion factors for the non-standardized units previously used in commercial HCV RNA quantitative assays. This allows the results of previous studies to be converted to IU. As shown in Table 2 and Fig. 2, the lower detection cutoffs of current assays range from 30 IU/mL to 615 IU/mL, while the upper end of the linear range ranges from less than 500,000 IU/mL to 7,700,000 IU/mL. Samples with a viral content above the upper limit of a given assay must be retested after 1/10 or 1/100 dilution for accurate quantication. The specicity of the assays is of the order of 98% to 99%, and quantication is independent of the HCV genotype.37-43 Whatever the assay, differences or variations of less than 0.5 log (i.e., less than
Table 3. Conversion Factors From the Former, Nonstandardized HCV RNA Quantication Units to IU in Various Commercially Available HCV RNA Quantication Assays
Assay Conversion Factor

3-fold) should not be taken into account, as they may be due to intrinsic or between-patient variability.44 No tests for HCV RNA quantication have yet been approved in the United States, but several are likely to be approved in the future. Detection and Quantication of Total HCV Core Antigen. Total HCV core antigen can be detected and quantied by means of an EIA assay (Ortho-Clinical Diagnostics, Raritan, NJ). The HCV core antigen titer (in pg/mL) correlates closely with the HCV RNA level and can thus be used as a surrogate marker of viral replication.12 It has been estimated that 1 pg of total HCV core antigen per milliliter is equivalent to approximately 8,000 IU HCV RNA, but there are slight between-patient differences.12 The current version of the assay does not detect HCV core antigen when the HCV RNA level is below approximately 20,000 IU/mL, limiting its use in the clinical setting.12 This assay is not FDA-approved.

Amplicor HCV Monitor v2.0 (manual procedure) Cobas Amplicor HCV Monitor v2.0 (semiautomated procedure) Versant HCV RNA 3.0 Quantitative Assay LCx HCV RNA Quantitative Assay SuperQuant

1 IU/mL 0.9 copies/mL 1 1 1 1 IU/mL IU/mL IU/mL IU/mL 2.7 5.2 3.8 3.4 copies/mL copies/mL copies/mL copies/mL

NOTE. These conversion factors can be used to transform HCV RNA levels obtained with these assays before the implementation of IU standards.

Fig. 2. Comparison of the ranges of linear quantication of HCV RNA assays. The lower and upper limits of quantication of the corresponding assays are also given in Table 2. The HCV RNA levels displayed as log10 in IU/mL. In the absence of antiviral treatment, approximately 90% of patients with chronic hepatitis C harbor serum HCV RNA levels shown in the gray area.

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Practical Use of Virological Tests

For the purposes of this article, a sensitive technique for HCV RNA detection is a qualitative or quantitative test with a detection limit of 50 IU/mL or less. Likewise, when discussing quantitative HCV RNA assays, it is assumed that the results are within the linear range. Diagnosis of HCV Infection Acute Hepatitis C. Patients with acute hepatitis of unknown origin should be tested for anti-HCV by means of EIA and for HCV RNA with a sensitive technique.45 Detection of HCV RNA without anti-HCV is strongly indicative of acute hepatitis C, which can be conrmed by subsequent seroconversion. Acute hepatitis C is unlikely if both markers are absent. Acute HCV infection is also unlikely if anti-HCV is present without HCV RNA; this pattern is typical of patients who have recovered from hepatitis C in the past and who have a liver disease due to another cause. These subjects should, nonetheless, be retested for HCV RNA a few weeks later, as HCV RNA may disappear transiently before chronic replication becomes detectable. Finally, when both anti-HCV and HCV RNA are detected, it is difcult to distinguish acute hepatitis C from an acute exacerbation of chronic hepatitis C, and from acute hepatitis of another cause in a patient who also has chronic hepatitis C. Chronic Hepatitis C. Chronic hepatitis C is certain in a patient with chronic liver disease when both antiHCV and HCV RNA are detected using a sensitive technique.22 Anti-HCV negativity with HCV RNA positivity is exceptional, occurring rarely in immunocompetent patients with chronic hepatitis C; this situation can arise (albeit rarely with current EIAs)21 when the patient is on hemodialysis or is profoundly immunodepressed. When an individual is found to be anti-HCV-positive at the time of a blood donation or during screening of at-risk populations, detection of HCV RNA with a sensitive technique conrms chronic HCV infection. When HCV RNA is undetectable on at least 2 occasions 6 months apart, it is difcult to distinguish patients who still harbor antibodies after spontaneously resolving HCV infection in the past from patients with false-positive reactivity. A high optical density ratio in EIA favors a truepositive result, whereas no conclusion can be drawn when the optical density ratio is low, because anti-HCV titers may decrease gradually after spontaneous clearance of the virus. However, this has no implications for the patient, who can be reassured that he/she is not infected. Maternal-Infant Transmission. The diagnosis of HCV infection in a baby born to an HCV-infected mother should be based on HCV RNA detection with a

sensitive technique rather than on anti-HCV detection, because antibodies are passively transferred in utero and remain detectable for several months to more than a year after delivery, regardless of whether viral transmission occurs.46-49 When transmission does occur, HCV RNA can be detected a few days after delivery, or later on, and then persist or be cleared spontaneously. The frequency and timing of spontaneous clearance is unknown, but this outcome appears to be more frequent in newborns than in adults. The optimal timing of diagnostic HCV RNA testing after birth is not known, but testing at 6 to 12 months is generally recommended. Chronicity should be suspected if anti-HCV is still detectable at high titer after the rst year of life; this can be conrmed by the detection of HCV RNA.46-49 Diagnosis After Occupational Exposure. HCV RNA is detectable in serum within 1 to 2 weeks when accidental parenteral exposure results in infection. The diagnosis of acute infection should be based on HCV RNA testing with a sensitive technique. Testing can be performed at any time starting a week after exposure. Antiviral treatment is not urgent in this setting and can be initiated when symptoms or an increase in serum aminotransferase activity occurs.50 Prognosis of HCV Disease Virological tests have no prognostic value. Indeed, current virological markers (including HCV RNA level and the HCV genotype) do not correlate with the severity of liver injury or brosis, and they cannot be used either to predict the natural course or outcome of the infection, or the onset of extrahepatic disease. For these reasons, repeat testing for HCV RNA level is not indicated in the routine management and monitoring of patients with hepatitis C. Antiviral Treatment of HCV Infection Decision to Treat. Only patients in whom HCV RNA is detectable with a sensitive technique should be considered for treatment with peginterferon and ribavirin combination therapy. The HCV genotype should be determined before treatment, as it determines both the indication and the duration of treatment and the dose of ribavirin. Indeed, it is reasonable to offer all patients with HCV genotype 2 or 3 infection such therapy (in the absence of contraindications), as they have an excellent chance for a sustained virological response (70% to 80%) and only require therapy for 24 weeks with ribavirin 800 mg/d.51-53 In contrast, patients with genotype 1 infection have only a 40% to 45% chance of a sustained response and require a full 48 weeks of treatment with ribavirin 1,000 to 1,200 mg/d.51-53 The likely benets of therapy in these patients must therefore be weighed based on risks

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and costs. Liver biopsy can help with the treatment decision in this setting (genotype 1): patients with marked necroinammatory activity and/or brosis should be treated, while those with so-called mild hepatitis should not. The same approach applies to patients infected with HCV genotypes 4, 5, and 6, pending further studies. The role of measurement of baseline HCV RNA levels in the decision for or against therapy is still unresolved. Baseline HCV RNA quantication is unnecessary in patients infected by genotype 2 or 3, whereas it serves as a reference value to assess the virological response at week 12 in patients infected by genotype 1. Virological Follow-Up and Response Evaluation. In patients infected by HCV genotype 2 or 3, the virological response is assessed by sensitive HCV RNA assay at the end of therapy; the presence of HCV RNA is highly predictive of posttreatment relapse. The absence of HCV RNA at the end of treatment indicates a virological response, and such patients should be retested for HCV RNA with a sensitive method 24 weeks later to show whether the response is sustained. Measurement of HCV RNA levels before treatment and again after 12 weeks of treatment is an appropriate way to monitor patients with genotype 1 chronic hepatitis C who are treated with peginterferon alfa and ribavirin.53 Treatment can be continued (for a total of 48 weeks) when there is a 2-log drop in HCV RNA level (i.e., a baseline HCV RNA level divided by 100 or more) or when HCV RNA is undetectable at week 12 (in patients whose baseline HCV RNA level was less than 100 times the lower detection cutoff of the assay). In contrast, in the absence of such a decrease, the likelihood of achieving a sustained virological response is virtually nil, and treatment can either be stopped or continued (to slow liver disease progression without clearing the virus).53 Total HCV core antigen quantication can be used for the same purpose, provided the antigen titer is more than 200 pg/mL (the detection cutoff of the assay being approximately 1 to 2 pg/mL).12 The virological response should be reassessed after 48 weeks of therapy by testing for HCV RNA with a sensitive technique. The presence of HCV RNA at the end of treatment is highly predictive of relapse when therapy is stopped, whereas a sustained virological response is characterized by negative HCV RNA detection by a sensitive method 24 weeks after treatment completion. Figure 3 shows an algorithm for the use of virological tests in the treatment of chronic hepatitis C. Treatment of Acute Hepatitis C Encouraging results were recently published with standard interferon alfa monotherapy of acute hepatitis C.54 However, the optimal schedule remains to be established,

Fig. 3. Algorithm for the use of virological tests before, during, and after treatment of chronic hepatitis C using the combination of peginterferon alfa and ribavirin. The Metavir scoring system (A0 to 3 and F0 to 4) is used for liver biopsy interpretation.55 NB: HCV core antigen quantication can be used instead of HCV RNA quantication to evaluate the response at week 12, provided that baseline antigenemia is at least 200 pg/mL.

and no recommendations can yet be made regarding the use of virological tests in the decision to treat.50 Whatever the type of interferon, the dose, and the duration of therapy, the virological response must be assessed at the end of therapy by means of a sensitive HCV RNA technique. When HCV RNA is negative at the end of treatment, the sustained or transient nature of the response is assessed 24 weeks later; negative HCV RNA detection at this time indicates that therapy has been successful. Treatment of Chronic Hepatitis C in Patients Coinfected by Human Immunodeciency Virus It is not known whether 24 weeks of treatment with the combination of peginterferon alfa and ribavirin is also adequate in human immunodeciency virus (HIV)/HCV coinfected patients with HCV genotype 2 or 3. Likewise, the predictive value of the HCV RNA level at baseline and at week 12 is unknown in coinfected patients with genotype 1. These issues are being addressed in ongoing clin-

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ical trials. As in patients infected by HCV alone, the virological response to therapy should be assessed at the end of treatment and 24 weeks later in coinfected patients by means of a sensitive HCV RNA technique. Follow-up of Untreated Patients. Repeat virological testing is not necessary in untreated patients, as the results have no prognostic value. Clinically useful information is obtained by serial testing for serum aminotransferase levels and liver biopsy.

Future Research Needs

Virological tests are invaluable in the management of HCV infection. They can be used to diagnose active infection, to guide treatment decisions, and to assess the virological response to therapy. Further work is required to fully standardize these tests, to improve automation, and to dene more clinically relevant thresholds on which to base universal recommendations for patient care. The development of increasingly sensitive and accurate assays for HCV RNA detection and quantication is necessary to improve not only the assessment of the response to antiviral therapy, but also our understanding of the mechanisms underlying antiviral resistance. Together with the development of new antiviral drugs and novel therapeutic approaches, these improvements should allow us to optimize the treatment of HCV infection and improve the global results.

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