Anti-hepatitis C virus antibody detection
The most common screening test for HCV is an immunoassay [enzyme immunoassay (EIA), microparticle EIA, chemiluminescence immunoassay (CIA)] that detects anti-HCV antibodies. These assays have many advantages for diagnosis: they are easy to use, automation is simple, have a low variability and are inexpensive.
Nowadays, the detection of antibodies directed against various HCV epitopes in plasma or serum is based on the use of third-generation EIAs. These assays detect antibodies to recombinant antigens from core (c22) and non-structural proteins 3 (c33), 4 (c100, c200) and 5. The specificity and sensitivity of third-generation immunoassays in patients with chronic liver disease were found to be >98 and >97% respectively (15–17). The mean time to seroconversion is shortened by 2–3 weeks with third-generation compared with second-generation assays with the detection of HCV-specific antibodies approximately 4–6 weeks after infection (18). Anti-HCV-IgM detection cannot discriminate between acute and chronic hepatitis C because some chronically infected patients produce anti-HCV IgM intermittently and not all patients respond to acute HCV infection by producing anti-HCV IgM. Anti-HCV antibodies may become undetectable via commercial assays in some patients many years after successful treatment.
Detection and quantification of hepatitis C virus RNA
There are a number of different commercial assays approved by the FDA and EMEA for the determination of HCV RNA (19–24).
Cobas® Amplicor HCV version 2.0 (Roche Molecular Diagnostics, Pleasonton, CA, USA) based on a standard reverse transcription-polymerase chain reaction (RT-PCR) is available for the qualitative measurement of HCV RNA. The lowest detection limit is 50 IU/ml whatever the HCV genotype (19).
The versant HCV qualitative assay (Siemens Healthcare Diagnostics, Deerfield, IL, USA) based on transcription-mediated amplification is also a highly sensitive test for a qualitative HCV RNA measurement with the lower detection limit of 5–10 IU/ml whatever the HCV genotype (25).
The versant HCV quantitative Test (Siemens Healthcare Diagnostics) is a quantitative HCV RNA assay based on signal amplification by branched DNA (bDNA). The bDNA assay version 3.0 is standardized for IU, and the assay has been reported to be linear over its entire dynamic range from the lower detection limit of 615 IU/ml to 8 million IU/ml whatever the HCV genotype (22).
The COBAS® Amplicor HCV Monitor 2.0 (CAM, Roche Molecular Diagnostics) is a standard RT-PCR-based assay with a linear detection range of 500–500 000 IU/ml, whatever the HCV genotype. For higher HCV RNA concentrations, predilution of the original sample is required (21).
Currently, two real-time PCR assays are available: the COBAS® Ampliprep/Cobas® TaqMan® assay (CAP/CTM, Roche Molecular Diagnostics) and the real-time HCV assay (also named AccuGene® HCV, Abbott Molecular Inc., Des Plaines, IL, USA). These assays have the advantage of having a broad dynamic range of amplification, thus improving the limits of detection (LOD) to ≤10 IU/ml, and linear quantification up to 107–108 IU/ml (26, 27). Four different modes of results are possible with the Abbott HCV assay: (a) undetectable (below the LOD of the assay), (b) possible detection but below 12 IU/ml, (c) positive above 12 IU/ml with an exact HCV RNA concentration or (d) positive above 8.0 log10 IU/ml (which represents the upper limit of quantification).
The results of the Roche CAP/CTM are reported in four stages: (a) undetectable (below the detection limit), (b) positive but unquantifiable (<15 IU/ml), (c) detectable and quantifiable (>15 IU/ml), reported as an exact HCV RNA concentration in IU/ml above 30–40 IU/ml, or (d) detectable, quantifiable but above the upper limit (>6.9 × 107 IU/ml) (28, 29).
It has also been shown that the results of assays can vary significantly with different HCV genotypes despite IU standardization. Generally, the HCV RNA levels in genotype 1 samples measured with the Roche CAP/CTM are higher than those obtained by the Abbott real-time HCV assay and the Siemens bDNA assay (approximately 0.5 log10 IU/ml), while the HCV RNA concentrations in samples with genotype 4 are lower (26, 29–31).