Jean-Michel Pawlotsky, MD, PhD

NIH Consensus Development Conference on
Management of Hepatitis C: 2002 

Bethesda, Maryland

June 10-12, 2002

Two categories of tests are used in the management of hepatitis C virus (HCV)-infected patients: (i) indirect tests that detect antibodies to HCV (anti-HCV); (ii) direct tests that detect, quantify, or characterize viral particle components, such as HCV RNA or core antigen. Direct and indirect virological tests play a crucial role in the diagnosis of infection, therapeutic choices, and assessment of the virological response to therapy.

Indirect Tests

Anti-HCV detection. Anti-HCV is typically detected using second- or third-generation enzyme immunoassays (EIAs) that detect mixtures of antibodies directed to various HCV epitopes. The specificity of currently available EIAs for anti-HCV is higher than 99 percent.

Their sensitivity is more difficult to determine in the absence of a more sensitive gold standard. EIAs for anti-HCV detect antibodies in more than 99 percent of immunocompetent patients with detectable HCV RNA. EIAs are sometimes negative despite the presence of active HCVreplication in hemodialysis patients or patients with profound immunodeficiencies. Immunoblot tests have been used in the past as confirmatory assays. Given the good performance of the current anti-HCV EIAs, immunoblot tests no longer have utility in the clinical virology setting.

They are still useful in the blood bank setting, where the positive predictive value of a positive EIA result is significantly lower than in the diagnostic setting.

Serological determination of HCV genotype. HCV genotype can be determined by detection of type-specific antibodies using a competitive EIA (so-called “serotyping”). This assay provides interpretable results in approximately 90 percent of immunocompetent patients with chronic hepatitis C. Its sensitivity is lower in hemodialysis or immunodepressed patients.

The assay identifies the type (1 to 6) but not the subtype of HCV. Concordance with molecular assays is in the order of 95 percent. Currently, no serotyping assay is FDA-approved.

Available Tests

Direct Tests

Qualitative detection of HCV RNA. Qualitative (i.e., nonquantitative) HCV RNA detection assays are useful because they are significantly more sensitive than most available quantitative assays. The qualitative assays are based on the principle of target amplification using either polymerase chain reaction (PCR) or transcription-mediated amplification (TMA). The lower detection cutoffs of the corresponding commercial assays are 50 HCV RNA international units (IU)/ml and 10 IU/ml, respectively. Their specificity is of the order of 98–99 percent. The PCR assay is FDA-approved.

Viral level quantification. HCV RNA level can be quantified by means of target amplification techniques (PCR or TMA) or signal amplification techniques (“branched DNA”  assay). The lower detection cutoffs of the current assays vary between 30 IU/ml and 615 IU/ml, and the upper limit of linear quantification between 500,000 IU/ml and 7,700,000 IU/ml. Samples with a viral level higher than the upper limit of an assay should be retested after 1/10 or 1/100 dilution. Quantification is independent of the HCV genotype. The international unit, recently defined with reference to the WHO HCV RNA standard, should be used in any HCV RNA quantitative assay in order to compare results given by different assays and to apply global recommendations. Variations of less than 0.5 logs (i.e., of less than threefold) should not be taken into account as they may relate to the intrinsic variability of the assays. No HCV RNA quantification assay is approved currently in the United States, but several are likely to be in the future.

Molecular determination of HCV genotype (genotyping). The gold standard for genotyping is direct sequencing of the NS5B or E1 regions. In clinical practice, HCV genotype can be determined by direct sequence analysis, reverse hybridization onto genotype-specific oligonucleotide probes, or restriction fragment length polymorphism analysis after PCR amplification of the 5’ noncoding region. Typing errors are uncommon, but subtyping errors may occur in 10–25 percent of cases. These errors may be related to the region studied (5’ noncoding) rather than the technique used. Subtyping errors have few clinical consequences because only thegenotype is useful for clinical decisions. No genotyping assay is currently approved in the United States.

Detection and quantification of total HCV core antigen. Total HCV core antigen can be detected and quantified by means of EIA assay. The HCV core antigen titer (in pg/ml) correlates closely with HCV RNA level, and thus can be used as an indirect marker of viral replication. However, the current version of the assay does not detect HCV core antigen when HCV RNA is below approximately 20,000 IU/ml. This assay is not FDA-approved.

Practical Use of Virological Tests

The phrase “HCV RNA detection by means of a sensitive technique” used in this presentation refers to a technique with a lower limit of detection of 50 IU/ml or less. Furthermore, in discussing HCV RNA quantitation, it is assumed that the results are within the limits of its range of linear quantification of the assay.

Diagnosis of HCV Infection

Acute hepatitis C. During acute hepatitis of unknown origin, anti-HCV should be tested by EIA and HCV RNA by a sensitive HCV RNA technique. The presence of HCV RNA without anti-HCV is strongly indicative of acute hepatitis C, a diagnosis that can be confirmed by subsequent seroconversion. In the absence of both markers, acute hepatitis C is unlikely. In the presence of both, it is difficult to differentiate acute hepatitis C from an acute exacerbation of chronic hepatitis C or from acute hepatitis of other cause in a patient with chronic hepatitis C.

Chronic hepatitis C. In a patient with chronic liver disease, the diagnosis of chronic hepatitis C can be made based on detection of both anti-HCV and HCV RNA using a sensitive technique. The lack of anti-HCV in the presence of HCV RNA is uncommon in  immunocompetent patients with chronic hepatitis C. It can occur (although rarely with the current EIAs) in hemodialysis or profoundly immunodeficient patients.

Mother-to-infant transmission. The diagnosis of HCV infection in a baby born to an HCV-infected mother should be based on the detection of HCV RNA with a sensitive technique rather than anti-HCV, because antibodies are passively transferred in utero and remain detectable for several months to more than a year after delivery regardless of whether transmission occurs. The optimal timing for HCV RNA testing for diagnosis is not known. Appropriate times are 6 to 12 months after birth.

Diagnosis of infection after an occupational exposure. HCV RNA is detectable in serum within one to two weeks after an accidental parenteral exposure. The diagnosis of acute infection should be based on detection of HCV RNA by a sensitive technique. This testing can be performed at any time after the first week after exposure, but antiviral treatment is not an emergency in this setting and can be initiated after appearance of serum aminotransferase elevations or clinical symptoms appear.

Prognosis of HCV-Related Disease

No virologic test (including viral load and genotype) correlates with the severity of liver injury or fibrosis, or predicts the natural course or outcome of disease or presence of extra-hepatic disease. Virologic tests are not helpful as prognostic markers.

Antiviral Treatment of HCV Infection

Decision to treat. Only patients with detectable HCV RNA should be considered for treatment. HCV genotype determination should be performed before treatment as results may help in the decision to treat as well as in determining the duration of treatment. Thus, because of the high rates of response and need for 24 weeks of therapy only in patients with HCV genotypes 2 and 3, many investigators recommend therapy to all such patients provided there are no contraindications. Because response rates are only 40–45 percent and therapy must be given for 48 weeks in patients with genotype 1, the benefits of therapy must be balanced against its risks and cost. In this context, the assessment of the natural prognosis of infection by liver biopsy examination may help in making the decision to treat. In the absence of sufficient information, the same applies to genotypes 4, 5, and 6.

Virologic followup and assessment of response. Measurement of HCV RNA levels before treatment and again at 12 weeks has been proposed as an appropriate approach to monitoring patients with chronic hepatitis C who are treated with peginterferon and ribavirin. This is particularly true for patients with genotype 1. In patients infected with genotypes 2, 3, 4, 5, and 6, monitoring of HCV RNA levels may be less important, and there is little data supporting its usefulness. The basis for this will be discussed later in this conference. In all patients, however, the virological response should be assessed by testing for HCV RNA by a sensitive technique at the end of therapy. The presence of HCV RNA at the end of treatment is highly predictive of a relapse when therapy is stopped. The absence of HCV RNA at the end of  treatment indicates virological response and should lead to retesting for HCV RNA by a sensitive method 24 weeks later to document that the virological response is sustained.

Followup of Untreated Patients

Repeat virological testing is not necessary in untreated patients, as results have no prognostic value.

References

LINK TO FULL-TEXT ARTICLE
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