Hepatitis C is often diagnosed accidentally and, unfortunately, remains heavily underdiagnosed. HCV diagnostics should be performed thoroughly in all patients presenting with increased aminotransferase levels, with chronic liver disease of unclear etiology and with a history of enhanced risk of HCV transmission.
With 2nd generation enzyme-linked immunoassays (EIAs), HCV-specific antibodies can be detected approximately 10 weeks after infection (Pawlotsky 2003b). To narrow the diagnostic window from viral transmission to positive serological results, a 3rd generation EIA has been introduced that includes an antigen from the NS5 region and/or the substitution of a highly immunogenic NS3 epitope, allowing the detection of anti-HCV antibodies approximately four to six weeks after infection with a sensitivity of more than 99% (Colin 2001). Anti-HCV IgM measurement can narrow the diagnostic window in only a minority of patients and cannot discriminate between acute and chronic hepatitis C.
False-positive results are more frequent in patients with rheuma factors and in populations with a low hepatitis C prevalence, for example in blood and organ donors. False-negative HCV antibody testing may occur in patients on hemodialysis or in severely immunosuppressed patients or in hematological malignancies.
One quantitative HCV core antigen assay (Architect HCV Ag, Abbott Diagnostics) has been approved so far. This assay comprises 5 different antibodies, is highly specific (99.8%) and shows somewhat less sensitivity for determination of chronic hepatitis C as HCV RNA measurement (Morota 2009). False-negative results are obtained in patients with impaired immunity (Mederacke 2009, Medici 2011). For careful monitoring of treatment with standard dual combination therapies or directly acting antiviral agents, prospective studies have to be performed to determine proper rules and time points for response-guided treatment algorithms.
Because of the importance of an exact HCV RNA load determination for therapeutic management, the World Health Organization (WHO) established the HCV RNA international standard based on international units (IU) which is used in all clinically applied HCV RNA tests. Currently, several HCV RNA assays are commercially available.
Qualitative HCV RNA tests include the qualitative RT-PCR, of which the Amplicor™ HCV 2.0 (Roche, USA) is an FDA- and CE-approved RT-PCR system for qualitative HCV RNA testing that allows detection of HCV RNA concentrations down to 50 IU/ml of all HCV genotypes (Nolte 2001).
Transcription-mediated amplification- (TMA)-based qualitative HCV RNA detection has a very high sensitivity (lower limit of detection 5-10 IU/ml) (Sarrazin 2002, Hendricks 2003). A commercially available TMA assay is the Versant™ HCV RNA Qualitative Assay (Siemens, Germany). This system is accredited by FDA and CE and provides an extremely high sensitivity, superior to RT-PCR-based qualitative HCV RNA detection assays (Sarrazin 2000, Sarrazin 2001, Hofmann 2005).
HCV RNA quantification can be achieved either by target amplification techniques (competitive and real-time PCR) or by signal amplification techniques (branched DNA (bDNA) assay). Several FDA- and CE-approved standardised systems are commercially available. The Cobas Amplicor™ HCV Monitor is based on a competitive PCR technique whereas the Versant™ HCV RNA Assay is based on a bDNA technique. Both have restricted lower limits of detection (500-615 IU/ml). More recently, the Cobas TaqMan assay and the Abbott RealTime™ HCV test, both based on real-time PCR technology, have been introduced and now replace the qualitative and quantitative methods.
All commercially available HCV RNA assays are calibrated to the WHO standard based on HCV genotype 1. It has been shown that results may vary significantly between assays with different HCV genotypes despite standardisation (Chevaliez 2007, Vehrmeren 2008). The Cobas TaqMan assay makes both highly sensitive qualitative (limit of detection approx. 10 IU/ml) and linear quantitative HCV RNA detection (35-107 IU/ml) feasible with high specificity and excellent performance in one system with complete automation. The Abbott RealTime™ HCV Test provides a lower limit of detection of 12 IU/ml, a specificity of more than 99.5% and a linear amplification range from 12 to 10,000,000 IU/ml independent of the HCV genotype (Michelin 2007, Sabato 2007, Schutten 2007, Vermehren 2008).
Figure 3.1 - Detection limits and linear dynamic ranges of commercially available HCV RNA detection assays.
HCV is heterogeneous with an enormous genomic sequence variability due to its rapid replication cycle producing 1012 virions a day and low fidelity of the HCV RNA polymerase. Six genotypes (1-6), multiple subtypes (a, b, c…) and most recently a seventh HCV genotype have been characterized. Within one subtype, numerous quasispecies exist and may emerge during treatment with specific antivirals. Because the currently recommended treatment durations and ribavirin doses depend on the HCV genotype, HCV genotyping is mandatory in every patient considering antiviral therapy (Bowden 2006). With the new oral treatment modalities and those to come, HCV subtype determination will help to reveal possible barriers to resistance. Both direct sequence analysis and reverse hybridisation technology allow HCV genotyping.
The VersantTM HCV Genotype 2.0 System is suitable for indentifying genotypes 1-6 and more than 15 different subtypes and is currently the preferred assay for HCV genotyping. By simultaneous analyses of the 5’UTR and core region, a high specificity is achieved especially to differentiate the genotype 1 subtypes (1a versus 1b). The TruGene direct sequence assay determines the HCV genotype and subtype by direct analysis of the nucleotide sequence of the 5’UTR region. Incorrect genotyping rarely occurs with this assay. However, the accuracy of subtyping is poor. The current RealTime™ HCV Genotype II assay is based on real-time PCR technology, which is less time-consuming than direct sequencing. Preliminary data reveal a 96% concordance at the genotype level and a 93% concordance on the genotype 1 subtype level when compared to direct sequencing of the NS5B and 5’UTR regions.
Diagnosing acute hepatitis C
When acute hepatitis C is suspected, the presence of both anti-HCV antibodies and HCV RNA should be tested. For HCV RNA detection, sensitive qualitative techniques with a detection limit of 50 IU/ml or less are required, for example TMA, qualitative RT-PCR or the newly developed real-time PCR systems. HCV RNA may fluctuate during acute hepatitis C, making a second HCV RNA test necessary several weeks later in all negatively tested patients with a suspicion of acute hepatitis C. When HCV RNA is detected in seronegative patients, acute hepatitis C is very likely. When patients are positive for both anti-HCV antibodies and HCV RNA, it may be difficult to discriminate between acute and acutely exacerbated chronic hepatitis C. Anti-HCV IgM detection will not suffice because its presence is common in both situations.
Diagnosing chronic hepatitis C
Chronic hepatitis C should be considered in every patient presenting with clinical, morphological or biological signs of chronic liver disease. When chronic hepatitis C is suspected, screening for HCV antibodies by 2nd or 3rd generation EIAs is adequate because their sensitivity is >99%. When anti-HCV antibodies are detected, the presence of HCV RNA has to be determined in order to discriminate between chronic hepatitis C and resolved HCV infection.
Exact HCV subtyping may gain increased importance in the future use of direct-acting antiviral agents (DAA) because some HCV subtypes behave differently regarding antiviral activity and the development of resistance. Low HCV RNA concentration (<600,000-800,000 IU/ml) at baseline is a positive predictor of a sustained virological response (SVR). Genotyping is mandatory for the selection of the optimal treatment regimen and duration of therapy, since many DAA agents are effective for only some HCV genotypes (Lange 2010), and since treatment durations generally can be shorter for patients infected with HCV genotypes 2 or 3 compared to patients infected with genotypes 1 or 4 (Manns 2006). Due to the differences in HCV RNA concentrations of up to a factor of 4 between the different commercially available assays, despite standardisation of the results to IU, and due to intra- and interassay variability of up to a factor of 2, it is recommended to always use the same assay in a given patient before, during and after treatment and to repeat HCV RNA measurements at baseline in cases with HCV RNA concentrations between 400,000 and 1,000,000 IU/ml. Furthermore, the new stopping rules for boceprevir and telaprevir triple therapies based on viral cut-offs of 100 and 1000 IU/ml respectively, were assessed by the Cobas® TaqMan® assay and no comparative data with other HCV RNA assays are available yet.