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Keywords:

  • Hepatitis C virus;
  • interferon alpha;
  • molecular biology;
  • real-time PCR;
  • ribavin;
  • serological tests

Abstract

  1. Top of page
  2. Abstract
  3. Antibodies to hepatitis C virus
  4. Hepatitis C virus RNA detection and quantification
  5. Analysis of the hepatitis C virus genome nucleotide sequence
  6. Using virological tools in practical management
  7. Conflicts of interest
  8. References

Chronic hepatitis C is a global health problem that may cause cirrhosis and progression to hepatocellular carcinoma. Currently available antiviral treatments are moderately effective. Several virological assays are available to help diagnose and manage patients infected with the hepatitis C virus (HCV). These include the anti-HCV antibody assays, measurement of HCV RNA viral load and HCV genotyping. HCV RNA can be assayed by two types of molecular biology-based techniques: target amplification as in polymerase chain reaction methods and signal amplification such as the branched DNA assay. Monitoring of viral kinetics during the early phases of antiviral treatment is crucial in making treatment decisions such as early stopping rules and also in optimizing the length of treatment. The HCV genotype can be determined by several methods. Whatever the method, pretreatment determination allows treatment length and ribavirin dose to be optimized and also offers prognostic information on treatment outcomes as certain genotypes respond more favourably to treatment. Thus, virological assays are indispensable in the diagnosis and management of individuals infected with the HCV.

Approximately 170 million people are chronically infected with the hepatitis C virus (HCV). HCV belongs to the Flaviviridae family, genus Hepacivirus. HCV is an enveloped RNA virus, with a genome of approximately 9500 nucleotides. The single positive-strand RNA genome contains a major open reading frame that encodes a large polyprotein of approximately 3000 amino acids. The polyprotein co- and post-translational processing yields a number of structural and non-structural proteins (1). HCV strains are classified into six major genotypes, and a putative seventh one has been identified recently (2, 3). The HCV genotypes also have distinct geographical distributions and the genotype is an essential prognosis marker of the likelihood of HCV eradication during antiviral treatment (4). Virological tools are needed to diagnose chronic HCV infections, but they have found their principal application in guiding treatment decisions and assessing the virological responses to therapy. Three HCV markers are useful in clinical practice, including total antibodies to HCV, HCV genotype and HCV RNA.

Antibodies to hepatitis C virus

  1. Top of page
  2. Abstract
  3. Antibodies to hepatitis C virus
  4. Hepatitis C virus RNA detection and quantification
  5. Analysis of the hepatitis C virus genome nucleotide sequence
  6. Using virological tools in practical management
  7. Conflicts of interest
  8. References

The detection of specific antibodies in body fluids is based on the use of sandwich enzyme immunoassays (EIAs). Recombinant antigens are used to capture circulating antibodies onto the wells of microtitre plates, microbeads or specific holders adapted to close automated devices. The presence of antibodies is revealed by anti-antibodies labelled with an enzyme that catalyses the transformation of a substrate into a coloured compound. The optical density (OD) ratio of the reaction (sample OD/internal control OD) is proportional to the amount of antigens or antibodies in the sample. EIAs are cheap, easy to use, can be fully automated and are well adapted to large volume testing.

The serological window, characterized by detectable HCV RNA in the absence of antibodies to HCV, has been estimated to be of approximately 60 days on average (5). Antibodies to HCV appear on average 2–8 weeks after the acute phase of infection and persist for life in patients who develop chronic HCV infection (Fig. 1). The presence of both antibodies to HCV and HCV RNA does not allow one to distinguish acute hepatitis C from an acute exacerbation of chronic hepatitis C or an acute hepatitis of another cause in a patient with chronic hepatitis C. However, the anti-HCV immunoglobulin G (IgG) avidity index within the first 8 days following the onset of clinical symptoms may be useful in identifying actual acute HCV infection cases (6).

image

Figure 1.  Kinetics of virological markers during chronic hepatitis C virus (HCV) infection. ULN, upper limit of normal values.

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The significance of the presence of anti-HCV IgM during HCV infection is unclear. Anti-HCV IgM have been reported in 50–93% of patients with acute hepatitis C and 50–70% of patients with chronic hepatitis C (7–9). Therefore, anti-HCV IgM cannot be used as a reliable marker of acute HCV infection, and IgM assays have not been used in clinical practice. However, the serial measurements of the anti-HCV IgM titres based on at least three determinations from the fifth to the 15th day from the onset of the symptoms may identify patients with acute hepatitis C (10).

The HCV genotype can be determined by means of a competitive ELISA assay using genotype-specific antigens (11). This assay allows identification of the six HCV genotypes (1–6) but not the subtype.

Hepatitis C virus RNA detection and quantification

  1. Top of page
  2. Abstract
  3. Antibodies to hepatitis C virus
  4. Hepatitis C virus RNA detection and quantification
  5. Analysis of the hepatitis C virus genome nucleotide sequence
  6. Using virological tools in practical management
  7. Conflicts of interest
  8. References

Hepatitis C virus RNA detection and quantification can be achieved using two types of molecular biology-based techniques, including target amplification [such as polymerase chain reaction (PCR)] and signal amplification (such as the branched DNA assay). Whatever the technique used, HCV RNA international units per millilitre (IU/ml) must be preferred to any other quantitative unit and should now be implemented in all commercial quantitative assays. Conversion factors can be used to establish a relationship between the IUs and the non-standardized copies (12). It is clear that use of the standardized IU to express viral load will allow comparisons to be made between different studies, whereas the former systems expressing viral load as copies/ml were only adequate to compare results within an individual study.

The classical techniques for viral genome detection and quantification are now progressively being replaced by real-time PCR assays in most virology laboratories (Table 1). Real-time PCR techniques have a broad dynamic range of quantification, well suited to the clinical needs (upper range of quantification: 7–8 log10 IU/ml). Real-time PCR is more sensitive than classical PCR, with lower limits of detection of the order of 10–15 IU/ml. Real-time PCR assays do not yield false-positive results due to carryover contaminations, and they can be fully automated. Therefore, real-time PCR has become the technique of choice to detect and quantify HCV RNA in clinical practice.

Table 1.   Commercially available real-time polymerase chain reaction assays for hepatitis C virus RNA detection and quantification
AssayManufacturerMethodLower limit of detection (IU/ml)Dynamic range (IU/ml)
  1. HCV, hepatitis C virus; PCR, polymerase chain reaction.

Cobas Taqman® HCVRoche Molecular SystemsReal-time PCR on Cobas Taqman® after automated extraction (Cobas Ampliprep®)1546–69 000 000
Abbott Real-time HCVAbbott DiagnosticReal-time PCR on m2000RT after automated extraction (m2000SP)12–3012–100 000 000 

Two real-time PCR platforms are currently available for detection and quantification of HCV RNA: the Cobas Taqman® (Roche Molecular Systems, Pleasanton, CA, USA) platform, which can be used together with automated sample preparation with the Cobas AmpliPrep® system (CAP–CTM; Roche Molecular System), and the Abbott platform (Abbott Diagnostics, Chicago, IL, USA), which uses the m2000RT amplification platform together with the m2000SP device for sample preparation. Another assay, developed by Siemens Medical Solutions Diagnostics (Tarrytown, NY, USA), will be available soon (Table 1). The intrinsic performances of available tests differ. Indeed, approximately 15% of HCV genotype 2 and 30% of HCV genotype 4 samples are substantially underestimated in the CAP–CTM, most likely because of nucleotide mismatches, whereas this problem has not been found with the Abbott assay (13 and S Chevaliez, M Bouvier-Alias and JM Pawlotsky, unpublished results).

Hepatitis C virus RNA detection and quantification is useful in clinical practice to: (i) diagnose chronic HCV infection; (ii) identify patients who need antiviral therapy; and (iii) monitor the virological responses to antiviral therapy (12).

Analysis of the hepatitis C virus genome nucleotide sequence

  1. Top of page
  2. Abstract
  3. Antibodies to hepatitis C virus
  4. Hepatitis C virus RNA detection and quantification
  5. Analysis of the hepatitis C virus genome nucleotide sequence
  6. Using virological tools in practical management
  7. Conflicts of interest
  8. References

Viral genome sequence analysis is generally based on direct sequencing (so-called population sequencing), which provides the full sequence of the analysed fragment, or reverse hybridization, which identifies specific nucleotides or motifs at given positions. In practice, signature sequences are used to classify HCV strains into phylogenetic groups of clinical interest, i.e. the six HCV genotypes (1–6). In the future, with the advent of novel specific HCV inhibitors, subtype determination may be useful, as well as the identification of amino acid substitutions known to confer viral resistance to these molecules.

The reference method for HCV genotype determination is phylogenetic analysis of sequences generated after PCR amplification of a portion of the viral genome relative to reference sequences. Direct sequence analysis is the gold standard for genomic sequence analysis. However, it only identifies viral variants representing at least 20–25% of the circulating viral populations. The Trugene® 5′NC HCV Genotyping kit (Siemens Medical Solutions Diagnostics) has been developed for HCV genotype determination by direct sequencing of a portion of the 5′ non-coding region of the viral genome.

Reverse hybridization of PCR amplicons to membrane-bound probes is more sensitive than direct sequence analysis to detect minor variants representing as few as 5% of the entire viral population (15). Line probe assays (INNO-LiPA, Innogenetics, Gent, Belgium) use a series of short immobilized oligonucleotide probes to discriminate among different PCR fragments. The most recent version of the line probe assay for HCV genotype determination (Versant® HCV Genotype 2.0 Assay, Siemens Medical Solutions Diagnostics) carries consistently improved accuracy for HCV genotype 1 subtype and HCV genotype 6 determination compared with the previous assays (15–18).

Using virological tools in practical management

  1. Top of page
  2. Abstract
  3. Antibodies to hepatitis C virus
  4. Hepatitis C virus RNA detection and quantification
  5. Analysis of the hepatitis C virus genome nucleotide sequence
  6. Using virological tools in practical management
  7. Conflicts of interest
  8. References

Serological and molecular markers are used in clinical practice to diagnose chronic hepatitis C, guide treatment decisions and monitor the antiviral efficacy of treatment. These will be discussed in order below.

To diagnose HCV infection, anti-HCV, HCV RNA and serum liver chemistry values are commonly used. The last is not necessary from a virological point of view because up to 40% of viraemic patients can have a normal aminotransferase value on a single determination. The persistence of HCV RNA for more than 6 months defines chronic HCV infection. In patients with clinical and/or biological signs of chronic liver disease, chronic hepatitis C is diagnosed by the simultaneous presence of antibodies to HCV and HCV RNA. Detectable HCV replication in the absence of antibodies to HCV is exceptional with current anti-HCV EIAs. It is almost exclusively observed in profoundly immunosuppressed patients such as those who are HIV-positive, on haemodialysis or agammaglobulinaemic (19, 20).

The current standard treatment for chronic hepatitis C is the combination of pegylated interferon α-2a or -2b and ribavirin (21). Critical clinical decisions on whether to start treatment or not, how to monitor efficacy both on treatment and afterwards, when to stop prematurely and whether or not to consider retreatment all depend on the availability of information provided by the virological tools. The efficacy endpoint of chronic hepatitis C treatment is universally accepted as the sustained virological response (SVR), defined by an undetectable HCV RNA in serum with a sensitive assay (lower limit of detection of 50 IU/ml or less) 24 weeks after the end of treatment. Other surrogate markers of treatment response such as aminotransferase normalization have been shown to be sometimes discordant from the virological response, i.e. liver chemistry values may normalize despite ongoing viraemia. Therefore, economically disadvantaged places in the world where HCV RNA cannot be routinely measured face a huge and probably insurmountable obstacle to antiviral treatment. Because the cost of the antiviral therapy, even cheap generic non-pegylated interferon α, far outweighs the cost of HCV RNA assays, it makes sense in those places to factor in the cost of two or three HCV RNA assays into the treatment protocol, and arrange to ship the sera to another laboratory for the virological tests.

The decision to treat chronic hepatitis C depends on multiple parameters including a precise assessment of the severity of liver disease, the presence of absolute or relative contra-indications to therapy and the patient's willingness to be treated.

Hepatitis C virus genotype determination should be systematically determined before treatment, as it determines the indication, the duration of treatment, the dose of ribavirin and the virological monitoring procedure (Fig. 2) (4). Genotype 2- and 3-infected patients require 24 weeks of treatment and a low dose of ribavirin, i.e. 800 mg daily. In contrast, genotypes 1-, 4-, 5- and 6-infected patients require 48 weeks of treatment and a higher, body weight-based dose of ribavirin, i.e. 1000–1400 mg daily (21, 22).

image

Figure 2.  Algorithms for the use of hepatitis C virus (HCV) virological tools in the treatment of chronic hepatitis C, according to the HCV genotype.

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Monitoring of HCV RNA levels is recommended to tailor treatment to the actual virological response. A sensitive assay with a broad range of quantification should be used. A real-time PCR assay should ideally be used. In HCV genotype 1-infected patients, the HCV RNA level should be measured before therapy and 12 weeks after its initiation (Fig. 2). The lack of a 12-week early virological response (EVR) (i.e. no change or an HCV RNA decrease of <2 log10 at week 12) indicates that the patient has virtually no chance of achieving an SVR and should stop treatment. In contrast, treatment must be continued when a 2 log10 drop in HCV RNA level has been observed at week 12, until week 48 if HCV RNA is undetectable, and perhaps prolonged until week 72 if HCV RNA is still detectable at week 12 (23, 24). This highlights an important distinction in the week-12 EVR group: those who achieve negativity of HCV RNA have a much higher chance of achieving SVR (approximately 70%) compared with those who have detectable HCV RNA but >2 log10 drop from baseline (the ‘partial EVR’), who only have an approximately 25–30% chance of SVR with the standard 48-week treatment course. Thus, the latter group should be considered for prolonged therapy up to 72 weeks.

Conversely, recent studies have suggested that the patients who achieve a rapid virological response, defined by an undetectable HCV RNA (<50 IU/ml) at week 4 of therapy, could benefit from a shorter treatment duration, i.e. 24 weeks in patients infected with HCV genotype 1, 4, 5 or 6 and 12–16 weeks in those infected with HCV genotype 2 or 3 (25–31). These results, however, need confirmation and new algorithms should be drawn to tailor treatment duration to the virological response at week 4 without losing a chance of viral eradication (32).

The SVR corresponds to a cure of infection in more than 99% of cases. In the medium-term future, triple combination therapy with pegylated interferon α, ribavirin and a specific HCV inhibitor will likely become the standard treatment of chronic hepatitis C. The SVR will remain the endpoint of therapy. On-treatment monitoring and the corresponding decision algorithms will need to be established.

Some patients with chronic HCV infection will not be treated at the current time, for a variety of reasons such as mild disease, relative or absolute contra-indication or unwillingness to undergo therapy. Many such patients wish to know their level of viraemia. However, we strongly discourage this practice, as the HCV RNA level has no prognostic value. The level of HCV replication does not correlate with the severity of liver disease, nor with the risk of liver disease progression to cirrhosis or HCC. Repeated aminotransferase level assessments are recommended. Assessment of liver inflammation and fibrosis by means of liver biopsy or non-invasive serological or elasticity-echography testing is needed in the case of persistent or intermittent elevations of aminotransferase levels (21).

References

  1. Top of page
  2. Abstract
  3. Antibodies to hepatitis C virus
  4. Hepatitis C virus RNA detection and quantification
  5. Analysis of the hepatitis C virus genome nucleotide sequence
  6. Using virological tools in practical management
  7. Conflicts of interest
  8. References
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