Critical Issues in HCV HCV Diagnostics: Review and Commentary By Professor Jean-Michel Pawlotsky, MD, PhD

Two categories of tests can be used in the management of patients with hepatitis C virus (HCV) infection: (1) those assessing HCV-induced liver disease, and (2) those assessing viral infection parameters.

Markers of Liver Disease
      Biological Markers of Liver Disease

Anti-tissue Antibodies
Liver Biopsy Examination
Noninvasive Markers of Fibrosis and Activity
Screening for Hepatocellular Carcinoma

Virological Markers
      Available HCV Virological Tools and Kinetics of HCV Markers

Detection of anti-HCV antibodies
HCV genotype determination
Assessment of HCV replication
HCV core antigen detection and quantification

      Practical Use and Interpretation:

Diagnois of HCV infections
Assessment of disease severity and prognosis
Treatment of chronic hepatitis C
Treatment of acute hepatitis C
Treatment of chronic hepatitis C in HIV-infected patients
Follow-up of untreated patients

Table1:   Proposed algorithm for the use of virologic tests in the treatment
               of chronic hepatitis C

A) Biological Markers of Liver Disease

Serum alanine aminotransferase activity (ALT) and aspartate aminotransferase activity (AST) are markers of liver cell damage (1) . Their elevation above the range of normal values is the most frequent feature of acute or chronic hepatitis C (1) .

However, serum aminotransferase activity elevation is not specific, because it is seen in numerous liver disorders of various etiologies. It is also poorly sensitive, since ALT and AST can remain within the normal range for long periods of time in patients with chronic HCV infection, in spite of progressive liver disease (2) .

The level of aminotransferase activity has no prognostic value, meaning that it is not related to the severity and outcome of acute or chronic liver disease. In chronic hepatitis C, ALT activity is a marker of the efficacy of antiviral treatment:

·          the biochemical response is characterized by ALT normalization during therapy;

·          the sustained biochemical response is characterized by persistently normal ALT  activity after treatment withdrawal (3) .

Bilirubin levels and alkaline phosphatase activity can be elevated in acute or chronic hepatitis C, bearing witness to associated cholestasis, an impairment of bile secretion related to liver disease. Bilirubin may also be elevated in late stage liver disease due to impaired metabolism resulting from liver cell damage.

Alkaline phosphatase has no prognostic value. Serum gamma-glutamyl transpeptidase (g-GT) activity can also be elevated in cases of cholestasis, or in the patients with chronic alcohol consumption.

B) Anti-tissue Antibodies

Various anti-tissue antibodies can be found in the serum of patients with acute or, more often, chronic hepatitis C. The most frequent are antinuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA), that can be found at low titers in up to 20% of cases in chronic HCV infection (4, 5) . Anti-liver and kidney microsomal antibodies type 1 (anti-LKM1) and anti-liver cytosol antibodies type 1 (anti-LC1) can also be observed in chronic hepatitis C (4, 5) . The presence of anti-tissue antibodies does not have any diagnostic or prognostic significance.

C) Liver Biopsy Examination

The diagnosis and the prognosis of chronic hepatitis C are currently based on histological examination of liver biopsy (6) . Several interpretation scores have been proposed, the three most widely used being the Knodell's score, the Metavir score, and the Ishak's score. There is a consensus that two parameters need to be measured in liver biopsies (6) .

(i)                   Necro-Inflammatory activity: it reflects the degree of necrosis and inflammation in the liver. Necro-inflammatory activity is the main predictor of liver disease outcome. Indeed, the patients with a high activity score are at risk of rapid fibrosis progression and cirrhosis. The degree of necro-inflammatory activity is important in assessing whether or not treatment is indicated, especially in patients without ALT elevations or with relative contra-indications to IFN-alfa-based therapy.

(ii)                 Fibrosis: Fibrosis assessment also has prognostic significance, because it allows to differentiate: the patients with no or mild fibrosis, who generally have early disease or are slow progressors; the patients with severe fibrosis, who have more advanced disease and are at higher risk of developing cirrhosis; the patients with cirrhosis, who are at high risk of complications and especially of developing hepatocellular carcinoma. The diagnosis of cirrhosis is particularly important when a decision-to-treat has to be made.

Although liver biopsy has been used for years as the reference assay for the assessment of liver disease in chronic hepatitis C, it still suffers from major weaknesses: minor variations are often missed by current non-continuous scoring systems; false-negative results may be found in 10%-30% of cases, mostly due to the small size of biopsy specimens and the heterogeneous distribution of fibrosis within the liver; liver biopsy itself is invasive, may have serious side effects and sometimes discourages patients to undergo evaluation for subsequent treatment.

D) Noninvasive Markers of Fibrosis and Activity

Various serological markers assessing the severity of fibrosis or the necro-inflammatory activity of liver disease have been recently proposed and are currently under clinical evaluation (7) . Scoring algorithms combining the results of several marker determinations have been derived and compared with the results of liver biopsy. The performance of these markers appears to be acceptable to discriminate between no/mild fibrosis and cirrhosis, but overlaps are still found for intermediate states. There is no doubt that noninvasive markers will ultimately replace liver biopsy when their performance is further improved and their predictive value on the natural outcome of HCV-related liver disease is better known. They already provide an interesting alternative to liver biopsy in the treatment decision process in chronically infected patients.

E. Screening for Hepatocellular Carcinoma

The patients with cirrhosis related to chronic hepatitis C are exposed to hepatocellular carcinoma occurrence. Its incidence is of the order of 1% to 4% per year and the prognosis is better if the tumor is discovered and treated early (8) . Thus, screening for hepatocellular carcinoma based on alpha-fetoprotein (AFP) levels and regular ultrasonographic evaluations are mandated in these patients. A 6-month surveillance interval is reasonable to detect tumors growing from undetectable to detectable size. A variety of radiological investigations can be used to confirm ultrasound findings in patients with cirrhosis and chronic viral hepatitis with an isolated raised AFP.

These include computerized tomography (CT), spiral CT, magnetic resonance imaging (MRI), lipiodol-CT, and hepatic angiography (8, 9) . The use of biopsy to confirm HCC remains controversial for the following reasons: it can be difficult to distinguish large cirrhotic nodules from well-differentiated HCC or low-grade dysplastic nodules from HCC in either needle or wedge biopsies; liver biopsy carries a small risk of tumor spread along the needle track; finally, fine-needle aspirates provide cells without some of the architectural abnormalities that are important in making a diagnosis.

II. Virological Markers

Virological markers can be classified into two categories: (i) indirect markers (i.e. specific antibodies), produced by immune cells in response to viral antigenic stimulation; (ii) direct markers (i.e. viral antigens and genomes), components of virions or produced during replication.

A) Available HCV Virological Tools and Kinetics of HCV Markers

1. Detection of anti-HCV antibodies

The detection of anti-HCV antibodies in plasma or serum is based on the use of enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA) which detect a mixture of antibodies directed against various viral epitopes. In these assays, serum or plasma IgG antibodies are captured onto the wells of a microtiter plate by means of recombinant HCV peptides. 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. Confirmatory assays based on immunoblot testing no longer have any diagnostic utility.

The “serologic window” between HCV infection and the detectability of specific antibodies varies from one patient to the next. On average, it is 7 to 8 weeks with current assays (10-12) . Anti-HCV antibodies are detectable in 50% to 70% of patients at the onset of initial symptoms, and later in the remaining patients (13) . In patients with spontaneously resolving infection, anti-HCV antibodies may persist throughout life, fall slightly while remaining detectable, or gradually disappear after several years (14) .

Anti-HCV antibodies always persist for life in patients who develop chronic infection, although they may become undetectable (with current assays) in hemodialysis patients or in case of profound immunodepression. Apparent seroreversions and/or seroconversions can occur in immunodepressed patients, in whom the chronic nature of the infection is confirmed by the constant presence of HCV RNA.

2. HCV genotype determination

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 six clades or types (numbered 1 to 6), and are themselves subdivided into a large number of subclades or subtypes identified by lower-case letters (1a, 1b, 1c, etc) (15) . Phylogenetic analysis can distinguish HCV types, subtypes and isolates on the basis of average sequence divergence rates of approximately 30%, 20% and 10%, respectively (15) .

The reference method for HCV genotype determination is sequence analysis. In-house techniques have been used in many research laboratories. A standardized sequence-based assay has been developed (Trugene^TM HCV, Bayer Diagnostics, Tarrytown, New Jersey) (16, 17) . It allows to determine the nucleotide sequence of PCR amplicons and to compare it to a database including all known genotypes and subtypes. Other PCR-based genotyping techniques have been developed, such as reverse hybridization analysis after PCR using genotype-specific probes. This techniques (INNO-LiPA HCV, Innogenetics, Gent, Belgium) is available in a standardized commercial format, meaning that it can be reliably used in laboratories equipped for molecular biology (18, 19) .

The HCV genotype can also be determined by testing for type-specific antibodies with a competitive EIA (so-called “serotyping”). The available assay (Murex^TM HCV Serotyping 1-6 Assay, Murex Diagnostics, Dartford, UK) provides interpretable results in approximately 85%-90% of immunocompetent patients with chronic hepatitis C (20) . Its sensitivity is lower in hemodialysis and immunodepressed patients (21, 22) .

The assay identifies the type (1 to 6) but not the subtype. Concordance with molecular assays is of the order of 95%, and is better for genotype 1 than for other genotypes (20, 23) . In the rare cases of discrepancy, sequencing of reference genomic regions such as NS5B and E1 generally confirms the result of the molecular assay (24) . Mixed serologic reactivity is sometimes observed, and this test cannot distinguish between true mixed infection and cross-reactivity. Overall, serotyping assays provide a reliable alternative to molecular biology-based genotyping assays in the routine indication for HCV genotype determination, i.e. tailoring antiviral therapy.

3. Assessment of HCV replication

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 detectable within one to two weeks after infection. It generally increases to reach a peak, before disappearing when the infection resolves spontaneously. In contrast, in most patients progressing towards chronic infection, the fall 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 (25) . 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 lesions, except in patients with end-stage liver disease, who generally have low or even undetectable HCV RNA levels (26) . This is related to the liver lesions (hepatocyte depletion and extensive fibrosis) and not to the virus itself, as HCV recurrence after liver transplantation is generally associated with a high HCV RNA level that is facilitated by immunosuppressive treatment.

HCV RNA can be detected and/or quantified in serum or plasma by means of various categories of amplification techniques:

(i)                   Target amplification techniques. In these assays, a large number of viral genome copies are chemically synthesized in a cyclic enzymatic reaction and then detected and, eventually, quantified (27) . Two techniques are available, including "polymerase chain reaction" (PCR), in which the synthesized genome copies are double-stranded DNA molecules, and "transcription-mediated amplification" (TMA), in which the synthesized genome copies are single-stranded RNA molecules.

In current practice, HCV RNA is detected by qualitative, nonquantitative PCR or TMA assays (PCR assay: Amplicor^TM HCV v2.0 and its semi-automated version Cobas Amplicor^TM HCV v2.0, Roche Molecular Systems, Pleasanton, California; TMA assay: Versant^TM HCV RNA Qualitative Assay, Bayer Diagnostics, Tarrytown, New Jersey). The respective lower limits of detection of these assays are 50 and 10 HCV RNA international units (IU)/ml (27) . The presence of HCV RNA in qualitative assays is a marker of viral replication.

Nowadays, HCV RNA quantification in target amplification techniques is based on "competitive" PCR, where the amount of PCR amplicons is compared with a standard curve established in each run by quantifying known amounts of standard sequences (27) . Various assays are commercially available: Amplicor HCV Monitor v2.0, and its semi-automated version Cobas Amplicor HCV Monitor v2.0 (Roche Molecular Systems); LCx HCV RNA Quantitative Assay (Abbott Diagnostic, Chicago, Illinois); and SuperQuant (National Genetics Institute, Los Angeles, California) (27) .

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 (27) . These methods are theoretically more sensitive than classical target amplification techniques and are not prone to carryover contamination. Their dynamic range of quantification is consistently wider, making them particularly useful for quantifying the full range of viral loads observed in untreated and treated patients with HCV infection.

Real-time PCR will probably become the standard for HCV RNA detection and quantification in the future.

(ii)           Signal amplification techniques. In the "branched DNA" assay (Versant HCV RNA 3.0 Quantitative Assay, Bayer Diagnostics), the viral genomes are hybridized onto microtiter plates by means of specific capture probes. Extension probes mediate fixation of preamplifier and amplifier (branched DNA) molecules that achieve amplification of the luminescent signal emitted by each hybridized genome molecule. Quantification is performed by comparison with a standard curve established in each run (27) .

4. HCV core antigen detection and quantification

An EIA detecting and quantifying HCV core antigen in serum or plasma after an initial decomplexation step that removes bound anti-core antibodies is now available (Track-C^TM, Ortho Clinical Diagnostics, Raritan, New Jersey). 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 (28) . One picogram of total HCV core antigen per milliliter is equivalent to about 8000 international units of HCV RNA in most patients (28) .

The current assay does not detect HCV core antigen when the HCV RNA level is under 10 000-20 000 IU/ml, restricting its clinical use. This assay might however prove useful in monitoring viral replication, especially in countries or regions where molecular biology-based techniques are not available or too expensive.

B) Practical use and interpretation

1. Diagnosis of HCV infections

(i)                   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, i.e. a technique detecting 50 HCV RNA IU/ml or less (29) . Detection of HCV RNA without anti-HCV is strongly indicative of acute hepatitis C; this will be confirmed by subsequent seroconversion. Acute hepatitis C is unlikely if both markers are absent.

Acute HCV infection is also unlikely if anti-HCV antibodies are present and HCV RNA absent; such cases generally correspond to patients whose liver disorders are due to another cause and who encountered and cleared HCV at some time in the past. 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 antibodies and HCV RNA are detected, it is difficult 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.

(ii)                 Chronic hepatitis C. Chronic hepatitis C is certain in a patient with chronic liver disease when both anti-HCV and HCV RNA are detected using a sensitive technique (lower limit of detection ≤ 50 IU/ml) (13) . Anti-HCV negativity with HCV RNA positivity is exceptional in an immunocompetent patient with chronic hepatitis C. This situation can arise (albeit rarely with current EIAs) when the patient is on hemodialysis or is profoundly immunodepressed.

When an individual is found to be anti-HCV-positive during blood donation or screening of at-risk populations, detection of HCV RNA with a sensitive technique confirms chronic HCV infection. When HCV RNA is undetectable on at least two occasions 6 months apart, it is difficult 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 true-positive result, whereas no conclusion can be drawn when the optical density ratio is low, because anti-HCV antibody titers may fall 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.

(iii)                Mother-to-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 (30-33) .

When transmission does occur, HCV RNA can be detectable 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 than in adults. The optimal timing of diagnostic HCV RNA testing after birth is not known, but 6 to 12 months is generally allowed. Chronicity is suspected if anti-HCV antibodies are still detectable at high titers after the first year of life, and this is confirmed by the detection of HCV RNA in the baby’s blood (30-33) .

(iv)                Accidental exposure. HCV RNA is detectable in serum within one to two weeks when accidental parenteral exposure results in infection. The diagnosis of acute infection should be based on HCV RNA testing with a sensitive technique. This can be done at any time starting one 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 (34) .

2. Assessment of disease severity and prognosis

Virologic tests have no prognostic value. Indeed, current virologic markers (including HCV RNA level and the HCV genotype) do not correlate with the severity of liver injury or fibrosis, and they cannot be used either to predict the natural course or outcome of the infection, or the onset of extra-hepatic disease.

3. Treatment of chronic hepatitis C

The treatment of chronic hepatitis C is nowadays based on a combination of pegylated interferon (IFN) alfa, either pegylated IFN alfa-2a or pegylated IFNalfa-2b, and ribavirin (13, 35-37) .

(i)                   Decision to treat and optimal treatment schedule. Only patients in whom HCV RNA is detectable with a sensitive technique (lower limit of detection ≤ 50 IU/ml) should be considered for treatment with the combination of pegylated IFN-alfa and ribavirin (13) . The HCV genotype should be determined before treatment, as it determines both the indication, the duration and the dose of treatment (table 1) (13) :

·          in the absence of contraindications, all patients with HCV genotype 2 or 3 infection should be offered antiviral therapy, as they have a good chance of a sustained virologic response (70 to 80%). These patients only need 24 weeks of therapy and 0.8 g of ribavirin qd (13) . Baseline HCV RNA quantification is unnecessary in these patients.

·          Patients with genotype 1 infection have only a 40 to 45% chance of responding and must receive 48 weeks of treatment and 1.0-1.4 g of ribavirin qd. The likely benefits of therapy in these patients must therefore be weighed up according to the risks, cost and patient willingness to be treated. Liver biopsy (or serological markers of fibrosis and activity) can help with the treatment decision in this setting (13) . Baseline HCV RNA quantification must be performed in patients infected by genotype 1, because it serves as a reference value to assess the virologic response at week 12 (13, 35, 38) .

·          The same indication rule applies to genotypes 4, 5 and 6, pending further studies. It is still not known whether the baseline HCV RNA level should be included in the decision-making process (13) .

(ii)                 Assessment of the virological response to therapy. The main endpoint of antiviral therapy is the sustained virological response, characterized by an undetectable HCV RNA with a sensitive technique (lower limit of detection ≤ 50 IU/ml) 24 weeks after treatment withdrawal (13) . Again, the assessment of the virological response to therapy depends on the infecting HCV genotype.

·          In patients infected by HCV genotype 2 or 3, the virological response is assessed by sensitive HCV RNA assay (lower limit of detection ≤ 50 IU/ml) at the end of therapy; the presence of HCV RNA is highly predictive of post-treatment 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 (13) .

·          In the patients infected with HCV genotype 1, HCV RNA levels must be quantified before treatment and again after 12 weeks of treatment. If HCV RNA levels did not drop by 2 logs (i.e. baseline viral load remained unchanged or was divided by less than 100) at week 12, the patient has virtually no chance of achieving a sustained virological response and treatment can be stopped (ongoing studies are assessing the effect of prolonged therapy on disease progression in these patients, who are unlikely to achieve a sustained virological response) (13, 35, 38) .

·          Treatment can be continued when there is a 2-log drop in HCV RNA level or when HCV RNA is undetectable at week 12. If HCV RNA is detectable at week 24 with a sensitive assay, again treatment should be stopped because the likelihood of achieving a sustained virologic response is virtually nil (13, 35, 38) . If HCV RNA is undetectable at week 24, treatment should be continued for a total of 48 weeks. Total HCV core antigen quantification can be used to monitor the 2-log drop at week 12, provided the baseline antigen titer is more than 200 pg/ml (28) . The virologic response should be re-assessed at the end of the 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 virologic response is characterized by negative HCV RNA detection by a sensitive method 24 weeks after treatment completion.

·          In the patients infected with HCV genotypes 4, 5 or 6, it is recommended to treat for 48 weeks and to assess the virological response at the end of treatment and 24 weeks later, pending further studies (13) .

4. Treatment of acute hepatitis C

The optimal treatment schedule remains to be established for acute hepatitis C, and no recommendations can yet be made regarding the use of virologic tests in the decision to treat (34) . Whatever the type of interferon, the dose, and the duration of therapy, the virologic 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.

5. Treatment of chronic hepatitis C in HIV-coinfected patients

It is not known whether 24 weeks of treatment is also adequate in HIV-coinfected patients who are infected by HCV genotype 2 or 3. Likewise, the predictive value of the HCV RNA level at baseline and at week 12 is unknown in HIV-coinfected patients infected by HCV genotype 1. These questions are being addressed in ongoing clinical trials, the results of which will be known soon. As in patients infected by HCV alone, the virological response to therapy must be assessed at the end of treatment and 24 weeks later in dually infected patients, by means of a sensitive HCV RNA technique.

3. Follow-up of untreated patients

Repeat virologic testing is not necessary in untreated patients, as the results have no prognostic value. Follow-up in this case is based on regular liver biopsy. The interest of noninvasive markers of fibrosis in this setting remains to be established.

Table 1- Proposed algorithm for the use of virologic tests in the treatment of chronic hepatitis C with the combination of pegylated IFN-alfa and ribavirin.

Prose content of algorithm

Genotype 2 or 3

·          offer treatment in the absence of contraindications

·          treat with pegylated IFN-alfa and ribavirin (0.8 mg qd) 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 (i.e. necroinflammatory lesions and/or fibrosis on liver biopsy) in the absence of contraindications;

·          treat with pegylated IFN-alfa and ribavirin (1.0-1.4 mg qd);

·          measure viral load before treatment and at week 12:

If viral load dropped by at least 2 log (i.e. 100-fold) at week 12, continue treatment for a total of 48 weeks (provided HCV RNA is subsequently undetectable at week 24).

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 the 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 the patients with a bad prognosis (i.e. necroinflammatory lesions and/or fibrosis on liver biopsy) in the absence of contraindications;

·          treat with pegylated IFN-alfa and ribavirin (1.0-1.4 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)

Address for correspondence:

Professor Jean-Michel PAWLOTSKY, M.D., Ph.D.
Department of Virology (EA 3489)
Hôpital Henri Mondor
51 avenue du Maréchal de Lattre de Tassigny
94010 CRETEIL, France
tel: (+33) 1.49.81.28.27
fax: (+33) 1.49.81.48.31
e-mail: jean-michel.pawlotsky@hmn.ap-hop-paris.fr

REFERENCES

1.  Hoofnagle JH. Course and outcome of hepatitis C. Hepatology 2002;36: S21-29.

2.  Seeff LB. Natural history of chronic hepatitis C. Hepatology 2002; 36:S35-46.

3.  Lindsay KL. Introduction to therapy of hepatitis C. Hepatology 2002;36: S114-120.

4.  Pawlotsky JM, Ben Yahia M, Andre C, Voisin MC, Intrator L, Roudot-Thoraval F, Deforges L, et al. Immunological disorders in C virus chronic active hepatitis: a prospective case-control study. Hepatology 1994;19: 841-848.

5. Pawlotsky JM, Roudot-Thoraval F, Simmonds P, Mellor J, Ben Yahia MB, Andre C, Voisin MC, et al. Extrahepatic immunologic manifestations in chronic hepatitis C and hepatitis C virus serotypes. Ann Intern Med 1995;122: 169-173.

6. Dienstag JL. The role of liver biopsy in chronic hepatitis C. Hepatology 2002;36: S152-160.

7. Fontana RJ, Lok AS. Noninvasive monitoring of patients with chronic hepatitis C. Hepatology 2002;36: S57-64.

8. El-Serag HB. Hepatocellular carcinoma and hepatitis C in the United States. Hepatology 2002;36: S74-83.

9. Gebo KA, Chander G, Jenckes MW, Ghanem KG, Herlong HF, Torbenson MS, El-Kamary SS, et al. Screening tests for hepatocellular carcinoma in patients with chronic hepatitis C: a systematic review. Hepatology 2002;36: S84-92.

10. Farci P, Alter HJ, Wong D, Miller RH, Shih JW, Jett B, Purcell RH. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991;325: 98-104.

11. Hino K, Sainokami S, Shimoda K, Niwa H, Iino S. Clinical course of acute hepatitis C and changes in HCV markers. Dig Dis Sci 1994;39: 19-27.

12. Puoti M, Zonaro A, Ravaggi A, Marin MG, Castelnuovo F, Cariani E. Hepatitis C virus RNA and antibody response in the clinical course of acute hepatitis C virus infection. Hepatology 1992;16: 877-881.

13. National Institutes of Health Consensus Development Conference Statement: Management of hepatitis C: 2002--June 10-12, 2002. Hepatology 2002; 36: S3-20.

14. Lefrere JJ, Guiramand S, Lefrere F, Mariotti M, Aumont P, Lerable J, Petit JC, et al. Full or partial seroreversion in patients infected by hepatitis C virus. J Infect Dis 1997;175: 316-322.

15. Simmonds P. Viral heterogeneity of the hepatitis C virus. J Hepatol 1999;31 (Suppl 1):54-60.

16. Ansaldi F, Torre F, Bruzzone BM, Picciotto A, Crovari P, Icardi G. Evaluation of a new hepatitis C virus sequencing assay as a routine method for genotyping. J Med Virol 2001;63: 17-21.

17. Ross RS, Viazov SO, Holtzer CD, Beyou A, Monnet A, Mazure C, Roggendorf M. Genotyping of hepatitis C virus isolates using CLIP sequencing. J Clin Microbiol 2000;38: 3581-3584.

18. Stuyver L, Rossau R, Wyseur A, Duhamel M, Vanderborght B, Van Heuverswyn H, Maertens G. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. J Gen Virol 1993;74: 1093-1102.

19. 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: 2259-2266.

20. Pawlotsky JM, Prescott L, Simmonds P, Pellet C, Laurent-Puig P, Labonne C, Darthuy F, et al. Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay. J Clin Microbiol 1997;35:1734-1739.

21. Kobayashi M, Chayama K, Arase Y, Tsubota A, Saitoh S, Suzuki Y, Ikeda K, et al. Enzyme-linked immunosorbent assay to detect hepatitis C virus serological groups 1 to 6. J Gastroenterol 1999;34: 505-509.

22. Leruez-Ville M, Nguyen QT, Cohen P, Cocco S, Nouyou M, Ferriere F, Deny P. Large-scale analysis of hepatitis C virus serological typing assay: effectiveness and limits. J Med Virol 1998;55: 18-23.

23. 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: 666-669.

24. 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: 237-244.

25. Nguyen TT, Sedghi-Vaziri A, Wilkes LB, Mondala T, Pockros PJ, Lindsay KL, McHutchison JG. Fluctuations in viral load (HCV RNA) are relatively insignificant in untreated patients with chronic HCV infection. J Viral Hepat 1996;3: 75-78.

26. Duvoux C, Pawlotsky JM, Bastie A, Cherqui D, Soussy CJ, Dhumeaux D. Low HCV replication levels in end-stage hepatitis C virus-related liver disease. J Hepatol 1999;31: 593-597.

27. Pawlotsky JM. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;122: 1554-1568.

28. Bouvier-Alias M, Patel K, Dahari H, Beaucourt S, Larderie P, Blatt L, Hezode C, et al. Clinical utility of total HCV core antigen quantification: a new indirect marker of HCV replication. Hepatology 2002;36:211-218.

29. EASL International Consensus Conference on Hepatitis C. Paris, 26-28, February 1999, Consensus Statement. European Association for the Study of the Liver. J Hepatol 1999;30: 956-961.

30. Roudot-Thoraval F, Pawlotsky JM, Thiers V, Deforges L, Girollet PP, Guillot F, Huraux C, 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: 772-777.

31. Ohto H, Terazawa S, Sasaki N, Hino K, Ishiwata C, Kako M, Ujiie N, et al. Transmission of hepatitis C virus from mothers to infants. N Engl J Med 1994;330: 744-750.

32. Zanetti AR, Tanzi E, Newell ML. Mother-to-infant transmission of hepatitis C virus. J Hepatol 1999;31 (Suppl 1): 96-100.

33. Wejstal R, Widell A, Mansson AS, Hermodsson S, Norkrans G. Mother-to-infant transmission of hepatitis C virus. Ann Intern Med 1992;117: 887-890.

34. Hoofnagle JH. Therapy for acute hepatitis C. N Engl J Med 2001;345:1495-1497.

35.Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL, Jr., Haussinger D, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347: 975-982.

36. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, 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: 958-965.

37. Hadziyannis SJ, Cheinquer H, Morgan T, Diago M, Jensen DM, Sette H, Ramadori G, 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.

38. Davis GL, Wong JB, McHutchison JG, Manns MP, Harvey J, Albrecht J. Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C. Hepatology 2003;38: 645-652.