Use and interpretation of hepatitis C virus diagnostic assays

Clin Liver Dis 7 (2003) 127 – 137

Use and interpretation of hepatitis C virus diagnostic assays Jean-Michel Pawlotsky, MD, PhD Department of Virology (EA 3489), Henri Mondor Hospital, University of Paris XII, 51 avenue du Mare´chal de Lattre de Tassigny, Cre´teil 94010, France

Two types of laboratory tests are used for virologic diagnosis and monitoring of hepatitis C virus (HCV) infection: serologic assays of anti-HCV antibodies (indirect assays), and assays that detect, quantify, or characterize components of HCV particles, such as HCV RNA and core antigen (direct assays). Direct and indirect virologic assays are crucial for diagnosis, therapeutic decision-making, and monitoring the virologic response to treatment.

HCV assays Indirect assays Antibody assays Second- or third-generation enzyme immunoassays (EIAs) are generally used to detect mixtures of antibodies against various hepatitis C virus (HCV) epitopes of the core, NS3, NS4, and (third-generation assays) NS5 proteins. The viral antigens can be coated on microtiter plates, microbeads, or holders designed for ‘‘closed’’ automated devices (Table 1). Current EIAs have a specificity exceeding 99%. Given the lack of a gold standard, sensitivity is more difficult to determine. In clinical practice, EIAs are positive in more than 99% of immunocompetent patients with detectable HCV RNA [1]. However, EIAs can be negative during hemodialysis and in profoundly immunodeficient patients, despite chronic HCV replication. Although recent assays fare better [2], RNA testing has been recommended because of false negative results in this setting. Confirmatory immunoblot assays have been supplanted by anti-HCV EIAs in clinical use [3]. Blood banks still use them, however, because the positive

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Table 1 Available diagnostic EIA assays for anti-HCV antibody detection

Manufacturer

Assay

Ortho-Clinical Diagnostics, Raritan, NJ, USA

Ortho HCV 3.0 ELISA Test System with Enhanced SAVe Vitros anti-HCV 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

Abbott Diagnostic, Chicago, IL, USA

Bio-Rad, MarnesIa-Coquette, France Innogenetics, Ghent, Belgium

Holder (manual or semiautomated EIA) or device (fully automated EIA)

Epitopes

Microtiter plate

Core, NS3, NS4, NS5

Vitros ECi device Microbeads Microbeads IMx device AxSYM device Microtiter plate

Core, Core, Core, Core, Core, Core,

ACCESS device Microtiter plate

Core, NS3, NS4 Core, NS3, NS4, NS5

NS3, NS3, NS3, NS3, NS3, NS3,

NS4, NS4 NS4, NS4, NS4, NS4

NS5 NS5 NS5 NS5

Abbreviations: EIA, enzyme immunoassay; HCV, hepatitis C virus.

predictive value of EIA is far lower than in clinical use. The advent of routine molecular HCV RNA detection is likely to replace immunoblot assays in the United States and Europe [4]. HCV serotyping HCV genotype determination can be achieved by testing for type-specific antibodies with competitive EIAs. The only commercial assay available at present (MurexTM HCV Serotyping 1 –6 Assay, Murex Diagnostics, Dartford, UK) is interpretable in about 90% of cases of chronic hepatitis C in immunocompetent patients [5]. Its sensitivity is lower in hemodialysis and immunodepressed patients [6,7]. This assay can identify the type (1– 6) but not the subtype. The results are concordant with those of molecular assays in approximately 95% of cases (better with genotype 1 than other genotypes) [5,8]. When discrepancies occur, sequencing of reference genomic regions (eg, NS5B and E1) generally confirms the molecular assay result [9]. Mixed seroreactivity can occur, and this test cannot differentiate true mixed infection from cross-reactivity. Direct assays Molecular HCV genotyping methods The genotyping gold standard is direct sequencing of the NS5B or E1 region followed by sequence alignment with reference sequences and phylogenetic analysis [10]. For clinical purposes HCV can be genotyped by direct sequence analysis, reverse hybridization to genotype-specific oligonucleotide probes, or restriction fragment length polymorphism [11– 13]. There are two commercial kits

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based on polymerase chain reaction (PCR) amplification of the 50 noncoding region. One is based on direct sequencing of PCR amplicons and database interpretation (TrugeneTM HCV 50NC Genotyping kit, Visible Genetics Inc., Toronto, Ontario, Canada). The line-probe assay (INNO-LiPA HCV II, Innogenetics, Ghent, Belgium) [11,12] is based on reverse hybridization of PCR amplicons to a nitrocellulose strip coated with genotype-specific oligonucleotide probes, followed by colorimetric revelation. Both assays can identify the six HCV types and many subtypes. Typing is reliable, but subtyping errors occur in 10% to 25% of cases, because of variability in the target 50 noncoding region. This has few consequences for clinical decision-making, which is solely type-based. HCV RNA detection tests Nonquantitative HCV RNA detection assays are significantly more sensitive than most quantitative assays. They are based on target amplification, by either PCR or transcription-mediated amplification (TMA) [14]. Two commercial kits are available (Table 2). The PCR-based assay can be fully manual or may comprise manual extraction and automated reverse transcription, amplification, and reading in the Cobas AmplicorTM device. The detection cutoff is 50 international units (IU) of HCV RNA per mL. The manual TMA assay is slightly more sensitive (10 IU/mL) [15]. Both assays are approximately 98% to 99% specific. HCV quantification Target amplification (PCR or TMA) and signal amplification (‘‘branched DNA’’) techniques can be used to determine viral copy numbers (Table 2). The World Health Organization (WHO) has established an international standard for HCV RNA units [16]. One IU represents a certain amount of HCV RNA rather than the number of viral particles [16,17]. This IU should now be universally adopted [17], because this will facilitate recommendations and guidelines for clinical practice. IU conversion factors for the units previously used in commercial HCV RNA quantitative assays have been calculated (Table 3). The detection limits of current assays vary from 30 IU/mL to 615 IU/mL (Table 2). The upper end of the linear range ranges from less than 500,000 IU/mL to 7,700,000 IU/mL (Table 2). Samples above the upper limit of a given assay must be diluted 1/10 or 1/100. These assays have a specificity of approximately 98% to 99% independent of the HCV genotype [18 –24]. Variations of less than 0.5 log (fewer than threefold) should not be taken into account, because they may be caused by intrinsic or between-patient variability [25]. HCV core antigen Total HCV core antigen can be detected and quantified with 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 marker of viral replication [26]. One picogram of total HCV core antigen per mL is equivalent to about 8000 IUs of HCV RNA in most patients [26]. The current

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Table 2 Available diagnostic HCV RNA detection and quantification assays Assay

Method

Lower limit of detection

Dynamic range of quantificationa

Roche Molecular Systems, Pleasanton, CA, USA

AmplicorTM HCV v2.0 Cobas AmplicorTM HCV v2.0 AmplicorTM HCV Monitor v2.0 Cobas Amplicor HCV MonitorTM v2.0 VersantTM HCV RNA Qualitative Assay VersantTM HCV RNA 2.0 Assay VersantTM HCV RNA 3.0 Assay LCxTM HCV RNA Quantitative assay SuperQuant

Manual qualitative RT-PCR Semiautomated qualitative RT-PCR

50 IU/mL 50 IU/mL

None None

Manual quantitative RT-PCR

600 IU/mL

600 – < 500,000 IU/mL

Semiautomated quantitative RT-PCR

600 IU/mL

600 – < 500,000 IU/mL

Manual qualitative TMA

10 IU/mL

None

Manual quantitative branched-DNA signal amplification Semiautomated quantitative branched-DNA signal amplification Semiautomated quantitative RT-PCR

200,000 genome equivalentsb/mL 615 IU/mL

200,000 – 120,000,000 genome equivalentsb/mL 615 – 7,700,000 IU/mL

25 IU/mL

25 – 2,630,000 IU/mL

Semiautomated quantitative RT-PCR

30 IU/mL

30 – 1,470,000 IU/mL

Bayer Corporation, Diagnostics Division, Tarrytown, NY, USA

Abbott Diagnostic, Chicago, IL, USA National Genetics Institute, Los Angeles, CA, USA

Abbreviations: RT-PCR, reverse transcriptase-polymerase chain reaction; TMA, transcription-mediated amplification. a Quantitative assays only. b Nonstandardized HCV RNA unit used.

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Manufacturer

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Table 3 Conversion factors from nonstandardized HCV RNA quantification units used in various commercially available HCV RNA quantification assays to IUs Assay

Conversion

AmplicorTM HCV Monitor v2.0 Cobas Amplicor HCV MonitorTM v2.0 VersantTM HCV RNA 3.0 Quantitative Assay LCxTM HCV RNA Quantitative Assay SuperQuant

1 1 1 1 1

IU/mL = IU/mL = IU/mL = IU/mL = IU/mL =

0.9 2.7 5.2 3.8 3.4

copies/mL copies/mL copies/mL copies/mL copies/mL

Abbreviations: IU, international unit.

assay does not detect HCV core antigen when the HCV RNA level is under 20,000 IU/mL, restricting its clinical use [26].

Virologic assays in clinical practice Diagnosis of HCV infection Acute hepatitis C Acute hepatitis of unknown origin requires tests with an anti-HCV EIA, and with a sensitive technique for HCV RNA (‘‘sensitive’’ HCV RNA assays are assumed to have a detection limit of 50 IU/mL or less) [27]. HCV RNA positivity in an anti-HCV-negative patient strongly points to acute hepatitis C, which is confirmed by seroconversion. Subjects lacking both markers are unlikely to have acute hepatitis C, as are those with anti-HCV antibodies but no HCV RNA; most of these individuals have encountered (and cleared) HCV sometime previously. It is advisable to test such patients for HCV RNA a few weeks later, as HCV RNA can disappear transiently before chronic replication becomes detectable. Patients who have both anti-HCV antibodies and HCV RNA may have either acute hepatitis C or an acute exacerbation of chronic hepatitis C. In this setting, it is also difficult to diagnose acute hepatitis because of other causes in a patient with chronic hepatitis C. Chronic hepatitis C Chronic hepatitis C can be diagnosed confidently when a patient with chronic liver disease has both anti-HCV and HCV RNA (detected using a sensitive technique) [3]. Very few immunocompetent patients with chronic hepatitis C are anti-HCV-negative but HCV RNA-positive; by contrast, this situation is more frequent (albeit still rare with current EIAs) [2] in patients who are on hemodialysis or profoundly immunodepressed (see the article by Drs. Sulkowski and Thomas elsewhere in this issue). Detection of HCV RNA with a sensitive technique confirms chronic HCV infection in an individual who is found to be anti-HCV-positive during blood donation or screening of at-risk populations. It is difficult to distinguish patients

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who still harbor antibodies after spontaneously resolving HCV infection in the past from patients with false-positive reactivity when HCV RNA is undetectable on at least 2 occasions 6 months apart. A high optical density ratio in EIA favors a true-positive result, but low optical density ratios are inconclusive, as anti-HCV antibody titers can fall gradually and disappear after the virus has been spontaneously cleared. Despite lacking serologic markers of HCV infection, these individuals may demonstrate recall HCV-specific T cell responses in vitro [28]. Diagnosis after occupational exposure HCV RNA becomes detectable in serum 1 to 2 weeks following exposure. The diagnosis of acute infection should be based on HCV RNA testing with a sensitive technique, at least 1 week after exposure. There is no urgent need for antiviral treatment, which can be started when symptoms or elevated serum aminotransferase activity occur [29]. Mother-to-infant transmission Babies born to HCV-infected mothers should be tested with a sensitive HCV RNA detection method rather than an anti-HCV assay, because passively transferred antibodies remain detectable for up to 1 year after delivery [30 – 33]. When transmission does occur, HCV RNA can be detected as early as a few days after delivery, then either persist or clear spontaneously. Spontaneous clearance appears to be more frequent in babies than in adults. Diagnostic HCV RNA testing should be done approximately 6 to 12 months after birth, but there is no consensus on this point. High titers of anti-HCV antibodies after the first year of life point to chronicity, and this is confirmed by the detection of HCV RNA [30 – 33]. Course of HCV disease Current markers (including the HCV RNA level and HCV genotype) do not correlate with the severity of liver damage or histologic grade/stage, and they cannot be used to predict outcome or the risk of extrahepatic disease. Antiviral treatment The treatment decision Only patients with HCV RNA detection by a sensitive technique are candidates for pegylated interferon-alpha (IFN-a)-ribavirin combination treatment. The HCV genotype determines the indication, likelihood of response to, and the duration of treatment and dose of ribavirin (Box 1). All patients with HCV genotype 2 or 3 infection should be offered treatment in the absence of contraindications, as the chances of a sustained virologic response to 24 weeks of treatment with 800 mg of ribavirin qid are high (70% –80%) [34 –36]. By contrast, only 40% to 51% of patients with genotype 1 infection respond, and treatment 48 weeks with 1000 to 1200 mg of ribavirin qid is required to achieve maximal response rates [34 – 36]. This means that the risk-benefit ratio must be

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Box 1. Proposed algorithm for the use of virologic tests in the treatment of chronic hepatitis C with the combination of pegylated IFN-b and ribavirin Genotype 2 or 3 Offer treatment in the absence of contraindications. Treat with pegylated IFN-n and ribavirin (800 mg q.i.d.) for 24 weeks. Assess end-of-treatment and sustained virologic response with a sensitive HCV RNA assay (lower limit of detection  50 IU/ml). Genotype 1 Offer treatment to the patients with a bad prognosis (ie, necroinflammatory lesions and/or fibrosis on liver biopsy) in the absence of contraindications. Treat with pegylated IFN-n and ribavirin (1000 – 1200 mg q.i.d.). Measure viral load before treatment and at week 12: 

If viral load dropped by at least 2 log (ie, 100-fold) at week 12, continue treatment for a total of 48 weeks.  If viral load dropped by less than 2 log or did nor change at week 12, stop treatment or continue with the aim to slow the progression of liver disease in patients with severe and rapidly evolving lesions on liver biopsy. Assess end-of-treatment and sustained virologic response with a sensitive HCV RNA assay (lower limit of detection  50 IU/mL) Genotypes 4, 5 and 6 (pending further studies) Offer treatment to patients who have a bad prognosis (ie, necroinflammatory lesions or fibrosis on liver biopsy) in the absence of contraindications. Treat with pegylated IFN-n and ribavirin (1000 –1200 mg qd) for 48 weeks. Assess end-of-treatment and sustained virologic response with a sensitive HCV RNA assay (lower limit of detection  50 IU/mL). weighed on a case-by-case basis in the patients with genotype 1 infection. Liver biopsy can be helpful: patients with necroinflammatory activity or fibrosis should be treated, whereas those with ‘‘mild’’ hepatitis may not. The same rules as for genotype 1 should be applied to genotype 4, 5, and 6 infection, pending further studies. The value of the baseline HCV RNA level in the decision-making

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process is unknown. Baseline HCV RNA level is not necessary in genotype 2 or 3 infection but is useful for assessing the treatment response at week 12 in patients infected by genotype 1 (see the article by Drs. Fried and McHutchison elsewhere in this issue). Virologic follow-up and response assessment HCV RNA detection at the end of treatment is highly predictive of posttreatment relapse. It is better achieved with a highly sensitive technique [15,37]. HCV RNA nondetection at the end of treatment demonstrates a virologic response; these patients should be retested for HCV RNA 24 weeks later with a sensitive method. Negative HCV RNA detection by a sensitive method 24 weeks after treatment completion indicates a sustained virologic response. HCV RNA assay before and after 12 weeks of treatment is used to monitor genotype 1 chronic HCV treated with pegylated IFN-a and ribavirin (McHutchison et al, unpublished observation) [34]. Treatment can be continued for a total of 48 weeks when a 2-log (100-fold) drop in HCV RNA level occurs or when HCV RNA is undetectable at week 12 in patients whose baseline HCV RNA level was less than 100 times the detection limit. Otherwise the likelihood of achieving a sustained virologic response is virtually nil, and treatment can be stopped, or continued in an attempt to slow liver disease progression (without clearing the virus) (McHutchison et al, unpublished observation) [34]. Total HCV core antigen assay can serve the same purpose, provided the antigen titer is more than 200 pg/ mL (the detection cutoff is 1– 2 pg/mL) [26]. Treatment of acute HCV Standard IFN-a monotherapy of acute hepatitis C has recently given encouraging results [38], but the optimal schedule is unknown, and the use of virologic assays to define the appropriate subset of patients to treat is uncertain [29]. Whatever the type, dose, and duration of IFN-a treatment, the virologic response must be assessed at the end of treatment with a sensitive HCV RNA technique. In patients with negative HCV RNA detection at the end of treatment, the nature of the response (sustained or transient) is assessed 24 weeks later; treatment has been successful if HCV RNA is negative. Chronic HVC treatment in HIV-coinfected patients The efficacy of 24 weeks of treatment with the pegylated IFN-a-ribavirin combination in patients infected by HCV genotype 2 or 3 and by HIV is unknown, as well as the optimal ribavirin dose. The predictive value of the HCV RNA level at baseline and week 12 is also unclear in patients infected by HCV genotype 1 and HIV. The virologic response to treatment must be assessed at the end of treatment and 24 weeks later in dually infected patients, as in patients infected by HCV alone, using a sensitive HCV RNA technique (see the article by Drs. Sulkowski and Thomas elsewhere in this issue).

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Monitoring of untreated patients Repeat virologic testing has no prognostic value in untreated patients whose follow-up is based on regular liver biopsy.

Summary The HCV genotype, HCV RNA, HCV core antigen, and anti-HCV antibodies are the four biologic markers currently used in hepatitis C. Acute and chronic hepatitis C are diagnosed by anti-HCV antibody (enzyme immunoassay) and HCV RNA detection with sensitive molecular biology techniques. Other virologic tools include HCV genotype determination and HCV RNA quantification; these are used to guide the individual treatment choice, and also to monitor treatment efficacy. Overall, the management of HCV infection has been vastly improved by the use of virologic assays. These assays remain to be fully standardized and automated, however, and more clinically relevant cut-off values are required on which to base management recommendations. More sensitive and accurate HCV RNA assays will improve not only the assessment of the response to antiviral treatment, but also our understanding of antiviral resistance. These improvements, and the development of new antiviral drugs (see the article by Drs. DeFrancesco and Rice elsewhere in this issue), should help to optimize the treatment of HCV infection.

References [1] Colin C, Lanoir D, Touzet S, Meyaud-Kraemer L, Bailly F, Trepo C. Sensitivity and specificity of third-generation hepatitis C virus antibody detection assays: an analysis of the literature. J Viral Hepat 2001;8:87 – 95. [2] Thio CL, Nolt KR, Astemborski J, Vlahov D, Nelson KE, Thomas DL. Screening for hepatitis C virus in human immunodeficiency virus-infected individuals. J Clin Microbiol 2000;38:575 – 7. [3] Pawlotsky JM, Lonjon I, Hezode C, Raynard B, Darthuy F, Remire J, et al. What strategy should be used for diagnosis of hepatitis C virus infection in clinical laboratories? Hepatology 1998; 27:1700 – 2. [4] Stramer SL, Caglioti S, Strong DM. NAT of the United States and Canadian blood supply. Transfusion 2000;40:1165 – 8. [5] Pawlotsky JM, Prescott L, Simmonds P, Pellet C, Laurent-Puig P, Labonne C, et al. Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay. J Clin Microbiol 1997;35:1734 – 9. [6] Kobayashi M, Chayama K, Arase Y, Tsubota A, Saitoh S, Suzuki Y, et al. Enzyme-linked immunosorbent assay to detect hepatitis C virus serological groups 1 to 6. J Gastroenterol 1999;34:505 – 9. [7] Leruez-Ville M, Nguyen QT, Cohen P, Cocco S, Nouyou M, Ferriere F, et al. Large-scale analysis of hepatitis C virus serological typing assay: effectiveness and limits. J Med Virol 1998;55:18 – 23. [8] Sandres K, Dubois M, Pasquier C, Puel J, Izopet J. Determination of HCV genotype using two antibody assays and genome typing. Eur J Clin Microbiol Infect Dis 2001;20:666 – 9. [9] Prescott LE, Berger A, Pawlotsky JM, Conjeevaram P, Pike I, Simmonds P. Sequence analysis of hepatitis C virus variants producing discrepant results with two different genotyping assays. J Med Virol 1997;53:237 – 44.

136

J.-M. Pawlotsky / Clin Liver Dis 7 (2003) 127–137

[10] Simmonds P. Viral heterogeneity of the hepatitis C virus. J Hepatol 1999;31(Suppl 1):54 – 60. [11] Stuyver L, Wyseur A, van Arnhem W, Lunel F, Laurent-Puig P, Pawlotsky JM, et al. Hepatitis C virus genotyping by means of 50-UR/core line probe assays and molecular analysis of untypeable samples. Virus Res 1995;38:137 – 57. [12] Stuyver L, Wyseur A, van Arnhem W, Hernandez F, Maertens G. Second-generation line probe assay for hepatitis C virus genotyping. J Clin Microbiol 1996;34:2259 – 66. [13] Nakao T, Enomoto N, Takada N, Takada A, Date T. Typing of hepatitis C virus genomes by restriction fragment length polymorphism. J Gen Virol 1991;72(Pt 9):2105 – 12. [14] Pawlotsky JM. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;122:1554 – 68. [15] Sarrazin C, Teuber G, Kokka R, Rabenau H, Zeuzem S. Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays. Hepatology 2000;32:818 – 23. [16] Saldanha J, Lelie N, Heath A. Establishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA, WHO Collaborative Study Group. Vox Sang 1999;76:149 – 58. [17] Pawlotsky JM, Bouvier-Alias M, Hezode C, Darthuy F, Remire J, Dhumeaux D. Standardization of hepatitis C virus RNA quantification. Hepatology 2000;32:654 – 9. [18] Pawlotsky JM, Martinot-Peignoux M, Poveda JD, Bastie A, Le Breton V, Darthuy F, et al. Quantification of hepatitis C virus RNA in serum by branched DNA-based signal amplification assays. J Virol Methods 1999;79:227 – 35. [19] Yu ML, Chuang WL, Dai CY, Chen SC, Lin ZY, Hsieh MY, et al. Clinical evaluation of the automated Cobas Amplicor HCV Monitor test version 2.0 for quantifying serum hepatitis C virus RNA and comparison to the Quantiplex HCV version 2.0 test. J Clin Microbiol 2000; 38:2933 – 9. [20] Gerken G, Rothaar T, Rumi MG, Soffredini R, Trippler M, Blunk MJ, et al. Performance of the Cobas Amplicor HCV Monitor test, version 2.0, an automated reverse transcription-PCR quantitative system for hepatitis C virus load determination. J Clin Microbiol 2000;38:2210 – 4. [21] Martinot-Peignoux M, Boyer N, Le Breton V, Le Guludec G, Castelnau C, Akremi R, et al. A new step toward standardization of serum hepatitis C virus-RNA quantification in patients with chronic hepatitis C. Hepatology 2000;31:726 – 9. [22] Lee SC, Antony A, Lee N, Leibow J, Yang JQ, Soviero S, et al. Improved version 2.0 qualitative and quantitative Amplicor reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics. J Clin Microbiol 2000;38:4171 – 9. [23] Pradat P, Chossegros P, Bailly F, Pontisso P, Saracco G, Sauleda S, et al. Comparison between three quantitative assays in patients with chronic hepatitis C and their relevance in the prediction of response to therapy. J Viral Hepat 2000;7:203 – 10. [24] Reichard O, Norkrans G, Fryden A, Braconier JH, Sonnerborg A, Weiland O. Comparison of 3 quantitative HCV RNA assays: accuracy of baseline viral load to predict treatment outcome in chronic hepatitis C. Scand J Infect Dis 1998;30:441 – 6. [25] Pawlotsky JM. Measuring hepatitis C viremia in clinical samples: can we trust the assays? Hepatology 1997;26:1 – 4. [26] Bouvier-Alias M, Patel K, Dahari H, Beaucourt S, Larderie P, Blatt L, et al. Clinical utility of total HCV core antigen quantification: a new indirect marker of HCV replication. Hepatology 2002;36:211 – 8. [27] EASL International Consensus Conference on Hepatitis. Paris, 26 – 28 February, 1999, Consensus Statement. European Association for the Study of the Liver. J Hepatol 1999;30:956 – 61. [28] Takaki A, Wiese M, Maertens G, Depla E, Seifert U, Liebetrau A, et al. Cellular immune responses persist and humoral responses decrease two decades after recovery from a singlesource outbreak of hepatitis C. Nat Med 2000;6:578 – 82. [29] Hoofnagle JH. Therapy for acute hepatitis C. N Engl J Med 2001;345:1495 – 7. [30] Wejstal R, Widell A, Mansson AS, Hermodsson S, Norkrans G. Mother-to-infant transmission of hepatitis C virus. Ann Intern Med 1992;117:887 – 90.

J.-M. Pawlotsky / Clin Liver Dis 7 (2003) 127–137

137

[31] Roudot-Thoraval F, Pawlotsky JM, Thiers V, Deforges L, Girollet PP, Guillot F, et al. Lack of mother-to-infant transmission of hepatitis C virus in human immunodeficiency virus-seronegative women: a prospective study with hepatitis C virus RNA testing. Hepatology 1993;17:772 – 7. [32] Ohto H, Terazawa S, Sasaki N, Hino K, Ishiwata C, Kako M, et al. Transmission of hepatitis C virus from mothers to infants. N Engl J Med 1994;330:744 – 50. [33] Zanetti AR, Tanzi E, Newell ML. Mother-to-infant transmission of hepatitis C virus. J Hepatol 1999;31(Suppl 1):96 – 100. [34] Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347:975 – 82. [35] Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001;358:958 – 65. [36] Hadziyannis SJ, Cheinquer H, Morgan T, Diago M, Jensen DM, Sette H, et al. Peginterferon alfa-2a (40 KD) (Pegasys) in combination with ribavirin (RBV): efficacy and safety results from a phase III, randomized, double-blind, multicentre study examining effect of duration of treatment and RBV dose. J Hepatol 2002;36(Suppl. 1):3. [37] Comanor L, Anderson F, Ghany M, Perrillo R, Heathcote E, Sherlock C, et al. Transcriptionmediated amplification is more sensitive than conventional PCR-based assays for detecting residual serum HCV RNA at end of treatment. Am J Gastroenterol 2001;96:2968 – 72. [38] Jaeckel E, Cornberg M, Wedemeyer H, Santantonio T, Mayer J, Zankel M, et al. Treatment of acute hepatitis C with interferon alfa-2b. N Engl J Med 2001;345:1452 – 7.