A clinical textbook

Hepatology 2018

11. Hepatitis C: diagnostic tests

Christian Lange and Christoph Sarrazin

Common symptoms of hepatitis C like fatigue, muscle ache, loss of appetite or nausea are non-specific and, in many cases, mild or not present. Consequently, hepatitis C is often diagnosed accidentally and, unfortunately, remains heavily under-diagnosed. It is estimated that only 30–50% of individuals infected with HCV are aware of their disease and can take advantage of treatment options and avoid the risk of further transmission of the virus (Deuffic-Burban 2010). Untreated hepatitis C advances to a chronic state in up to 80% of people, which leads to liver cirrhosis in 20–40% with an accompanying risk of hepatic decompensation, hepatocellular carcinoma and death (Nature Outlook 2011). In light of these facts, HCV diagnostics should be performed thoroughly in all patients presenting with increased aminotransferase levels, with chronic liver disease of unclear aetiology and with a history of enhanced risk of HCV transmission (i.e., past IV or nasal drug dependency, transfusion of blood or blood products before the year 1990, major surgery before 1990, needle stick injuries, non-sterile tattoos or piercings, enhanced risk of sexual transmission).

For the diagnosis of hepatitis C both serologic and nucleic acid-based molecular assays are available (Scott 2007). Serologic tests are sufficient when chronic hepatitis C is expected, with a sensitivity of more than 99% in the 3rd generation assays. Positive serologic results require HCV RNA or with slightly reduced sensitivity HCV core antigen measurement in order to differentiate between chronic hepatitis C and resolved HCV infection from the past. When acute hepatitis C is considered, serologic screening alone is insufficient because anti-HCV antibodies may develop late after transmission of the virus. In contrast, HCV RNA is detectable within a few days of infection, making nucleic acid-based tests mandatory in diagnosing acute hepatitis C. HCV RNA measurement may be for some DAA regimens furthermore important in the determination of treatment indication, duration and success (Sarrazin 2010). Quantitative HCV RNA measurement at baseline of antiviral therapy is crucial to determine treatment duration. Traditionally, it should be repeated 24 weeks after treatment completion to assess whether a sustained virologic response (SVR) has been achieved. However, as the probability of virologic relapse is similar after 12 and 24 weeks the new time point for assessment of final virologic treatment outcome is 12 weeks after the end-of-treatment (Yoshida 2014). Both qualitative and quantitative HCV RNA detection assays are available. Qualitative tests are highly sensitive and are used for diagnosing hepatitis C for the first time, for the screening of blood and organ donations and for confirming SVR after treatment completion. Quantitative HCV RNA detection assays offer the possibility of measuring the viral load exactly and are essential in treatment monitoring. Qualitative and quantitative HCV RNA assays have now been widely replaced by real-time PCR-based assays that can detect HCV RNA over a very wide range, from low levels of approximately 10 IU/mL up to 10 million IU/mL. In case of lack of availability or financial restrictions, HCV core antigen testing can be used to confirm ongoing HCV infection and to monitor treatment outcome.

After diagnosing hepatitis C, the HCV genotype should be determined by nucleic acid-based techniques in every patient considered for HCV therapy, because the currently recommended treatment schedules and durations as well as the additional use of ribavirin differ among HCV genotypes and subtypes.

Morphological methods like immunohistochemistry, in situ hybridisation or PCR from liver specimens play no relevant role in the diagnosis of hepatitis C because of their low sensitivity, poor specificity and low efficacy compared to serologic and nucleic acid-based approaches.

Serologic assays

In current clinical practice, antibodies against multiple HCV epitopes are detected by commercially available 2nd and 3rd generation enzyme-linked immunoassays (EIAs). In these tests, HCV-specific antibodies from serum samples are captured by recombinant HCV proteins and are then detected by secondary antibodies against IgG or IgM. These secondary antibodies are labelled with enzymes that catalyse the production of coloured, measurable compounds.

The first applied EIAs for the detection of HCV-specific antibodies were based on epitopes derived from the NS4 region (C-100) and had a sensitivity of 70–80% and a poor specificity (Scott 2007). C-100-directed antibodies occur approximately 16 weeks after viral transmission. 2nd generation EIAs additionally detect antibodies against epitopes derived from the core region (C-22), NS3 region (C-33) and NS4 region (C-100), which leads to an increased sensitivity of approximately 95% and to a lower rate of false positive results. With these assays HCV-specific antibodies can be detected approximately 10 weeks after HCV infection (Pawlotsky 2003). To narrow the diagnostic window from viral transmission to positive serological results, a 3rd generation EIA has been developed with an antigen from the NS5 region and/or the substitution of a highly immunogenic NS3 epitope. This innovation allows 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. Anti-HCV IgM detection is also not sufficient to 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.

The specificity of serologic HCV diagnostics is difficult to define since an appropriate gold standard is lacking. It is evident, however, that false positive results are more frequent in patients with rheumatoid factors and in populations with a low hepatitis C prevalence, i.e., in blood and organ donors. Although several immunoblots for the confirmation of positive HCV EIA results are available, these tests have lost their clinical importance since the development of highly sensitive methods for HCV RNA detection. Immunoblots are mandatory to make the exact identification of serologically false positive-tested individuals possible. Importantly, the sensitivity of immunoblotting is lower compared to EIAs, which bears the risk of false negatively classifying HCV-infected individuals.

False negative HCV antibody testing may occur in patients on hemodialysis or in severely immunosuppressed patients like in HIV infection or in hematological malignancies.

HCV core antigen assays

In principle, detection of the HCV core antigen in serum could be a cheaper alternative to nucleic acid testing for the diagnosis and management of hepatitis C. The first HCV core antigen detection system (trak-C, Ortho Clinical Diagnostics) became commercially available in the US and Europe several years ago. This HCV core antigen assay proved highly specific (99.5%), genotype independent, and had a low inter- and intra-assay variability (coefficient of variation 5–9%) (Veillon 2003). HCV core antigen is measurable 1–2 days after HCV RNA becomes detectable. The limit of detection is 1.5 pg/mL (approximately 10,000–50,000 IU/mL HCV RNA). In a study of anti-HCV antibody and HCV RNA positive patients presenting in an outpatient clinic, 6/139 people (4%) were HCV core antigen negative. In these patients, HCV RNA concentrations were 1300–58,000 IU/mL, highlighting the limitations of the HCV core antigen assay as confirmation of ongoing hepatitis C in anti-HCV positive patients. As a consequence, this first HCV core antigen assay was withdrawn from the market.

More recently, another quantitative HCV core antigen assay (Architect HCV Ag, Abbott Diagnostics), a further development of the previous assay, was approved by the EMA. This assay comprises 5 different antibodies to detect HCV core antigen, is highly specific (99.8%), equally effective for different HCV genotypes, and shows a relatively high sensitivity for determination of chronic hepatitis C (corresponding to 600-1000 IU/mL HCV RNA). However, HCV core antigen correlated well but not fully linearly with HCV RNA serum levels, and false negative results were obtained in patients with impaired immunity (Mederacke 2009, Medici 2011). Another study has shown that HCV core antigen quantification could be an alternative to HCV RNA quantification for on-treatment antiviral response monitoring (Vermehren 2012). Here, HCV core antigen below the limit of quantification at treatment week 1 was strongly predictive of RVR, whereas patients with a less than 1 log10 decline in HCV core antigen at treatment week 12 had a high probability of achieving non-response.

The new HCV core antigen assay could be a cheaper, though somewhat less sensitive, alternative for nucleic acid testing. For careful monitoring of older treatment modalities which depend on response-guided treatment algorithms, proper rules for the application of the HCV core antigen assay have not been developed. For highly effective all oral combination therapies without the need of on-treatment assessment of virologic response, the HCV core antigen assay can be an alternative for assessment of active HCV infection before initiation of antiviral therapy and for determination of viral eradication 12 weeks after the end-of-treatment, if an HCV RNA assay is not available or not affordable (EASL 2017).

Nucleic acid testing for HCV

Until 1997, HCV quantitative results from various HCV RNA detection systems did not represent the same concentration of HCV RNA in a clinical sample. Because of the importance of an exact HCV RNA determination for patient 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. Other limitations of earlier HCV RNA detection assays were the false negative results due to polymerase inhibition, for example by drug interference, false positive results due to sample contamination because the reaction tubes had to be opened frequently, or due to under- and over-quantification of samples of certain HCV genotypes (Morishima 2004, Pawlotsky 2003, Pawlotsky 1999). Currently, several HCV RNA assays are commercially available (Table 1).

Table 1. Commercially available HCV RNA detection assays
Assay Distributor Technology Approval status
Qualitative HCV RNA detection assays
Cobas Amplicor/Cobas® TaqMan qual Roche Molecular Systems PCR FDA, CE
Quantitative HCV RNA detection assays
HCV SuperQuant™ National Genetics Institute PCR
Cobas AmpliPrep/ High pure system /Cobas® TaqMan®+ Roche Molecular Systems Real-time PCR FDA, CE
Abbott RealTime™ HCV Abbott Diagnostics Real-time PCR FDA, CE
Artus HCV QS-RGQ assay Qiagen Real-time PCR CE
Versant™HCV 1.0 kPCR assay Siemens Real-time PCR CE
Veris HCV Assay Beckman Coulter Real-time PCR CE
Xpert HCV Viral Load Cepheid Real-time PCR
Aptima HCV Quant Dx Real Time TMA Hologic TMA CE
Cobas HCV Assay 4800, 6800, 8800 Roche Molecular Systems Real-time PCR

Qualitative assays for HCV RNA detection

Until recently, qualitative assays for HCV RNA had substantially lower limits of detection in comparison to quantitative HCV RNA assays. The costs of a qualitative assay are also lower compared to a quantitative assay. Therefore, qualitative HCV RNA tests are used for the first diagnosis of acute hepatitis C, in which HCV RNA concentrations are fluctuating and may be very low, as well as for confirmation of chronic hepatitis C infection in patients with positive HCV antibodies. In addition, they are used for the confirmation of virologic response during, at the end of, and after antiviral therapy, as well as in screening blood and organ donations for presence of HCV.

Qualitative RT-PCR

In reverse transcriptase-PCR- (RT-PCR-) based assays HCV RNA is used as a matrix for the synthesis of a single-stranded complementary cDNA by reverse transcriptase. The cDNA is then amplified by a DNA polymerase into multiple double-stranded DNA copies. Qualitative RT-PCR assays are expected to detect 50 HCV RNA IU/mL or less with equal sensitivity for all genotypes.

The Amplicor™ HCV 2.0 was an FDA- and CE-approved RT-PCR system for qualitative HCV RNA testing that allowed detection of HCV RNA concentrations down to 50 IU/mL of all genotypes (Table 1) (Nolte 2001). The DNA polymerase of Thermus thermophilus used in this assay provides both DNA polymerase and reverse transcriptase activity and allows HCV RNA amplification and detection in a single-step, single-tube procedure.

The below described Cobas Ampliprep / Cobas TaqMan system is available as a real-time PCR-based assay for the qualitative and highly sensitive detection of HCV genotype 1–6 samples, but also as a quantitative assay which allows precise quantification of HCV viral loads (see below).

Transcription-mediated amplification (TMA) of HCV RNA

TMA-based qualitative HCV RNA detection has a very high sensitivity (Hendricks 2003, Sarrazin 2002). TMA is performed in a single tube in three steps: target capture, target amplification and specific detection of target amplicons by a hybridisation protection assay. Two primers, one of which contains a T7 promoter, one T7 RNA polymerase and one reverse transcriptase, are necessary for this procedure. After RNA extraction from 500 µl serum, the T7 promoter-containing primer hybridises the viral RNA with the result of reverse transcriptase-mediated cDNA synthesis. The reverse transcriptase also provides an RNase activity that degrades the RNA of the resulting RNA/DNA hybrid strand. The second primer then binds to the cDNA that already contains the T7 promoter sequence from the first primer, and a DNA/DNA double-strand is synthesised by the reverse transcriptase. Next, the RNA polymerase recognises the T7 promoter and produces 100-1000 RNA transcripts, which are subsequently returned to the TMA cycle leading to exponential amplification of the target RNA. Within one hour, approximately 10 billion amplicons are produced. The RNA amplicons are detected by a hybridisation protection assay with amplicon-specific labelled DNA probes. The unhybridised DNA probes are degraded during a selection step and the labelled DNA is detected by chemiluminescence.

A commercially available TMA assay was the Versant™ HCV RNA Qualitative Assay. This system is accredited by the FDA and CE and provides an extremely high sensitivity, superior to RT-PCR-based qualitative HCV RNA detection assays (Hofmann 2005, Sarrazin 2001, Sarrazin 2000). The lower detection limit is 5-10 IU/mL with a sensitivity of 96-100%, and a specificity of more than 99.5%, independent of the HCV genotype.

More recently, a novel TMA-based assay, the Aptima HCV Quant Dx assay, has been approved which allows automated quantitative and qualitative HCV RNA measurement in a single step (Chevaliez 2017). This assay is highly sensitive with a lower limit of detection of 2.8 IU/mL. Furthermore, the Aptima HCV Quant Dx assay is highly specific and HCV RNA test results are highly concordant to the real-time PCR-based Abbott RealTime HCV and Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays.

Quantitative HCV RNA detection

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) (Table 1). 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. More recently, the Cobas® TaqMan® assay and the Abbott RealTime™ HCV test, both based on real-time PCR technology, have been introduced. The technical characteristics, detection limits and linear dynamic detection ranges of these systems are summarised below. Due to their very low detection limit and their broad and linear dynamic detection range, they have already widely replaced the previously used qualitative and quantitative HCV RNA assays.

Competitive PCR: Cobas® Amplicor™ HCV 2.0 monitor

The Cobas® Amplicor™ HCV 2.0 monitor was a semi-automated quantitative detection assay based on a competitive PCR technique. Quantification is achieved by the amplification of two templates in a single reaction tube, the target and the internal standard. The latter is an internal control RNA with nearly the same sequence as the target RNA with a clearly defined initial concentration. The internal control is amplified by the same primers as the HCV RNA. Comparison of the final amounts of both templates allows calculation of the initial amount of HCV RNA. The dynamic range of the Amplicor™ HCV 2.0 monitor assay is 500 to approximately 500,000 IU/mL with a specificity of almost 100%, independent of the HCV genotype (Konnick 2002, Lee 2000). For higher HCV RNA concentrations pre-dilution of the original sample is required.

Branched DNA hybridisation assay (Versant™ HCV RNA 1.0 quantitative assay)

Branched DNA hybridisation assay was based on signal amplification technology. After reverse transcription of the HCV RNA, the resulting single-stranded complementary DNA strands bind to immobilised captured oligonucleotides with a specific sequence from conserved regions of the HCV genome. In a second step, multiple oligonucleotides bind to the free ends of the bound DNA strands and are subsequently hybridised by multiple copies of an alkaline phosphatase-labelled DNA probe. Detection is achieved by incubating the alkaline phosphatase-bound complex with a chemiluminescent substrate (Sarrazin 2002). The Versant™ HCV RNA assay is at present the only FDA- and CE-approved HCV RNA quantification system based on a branched DNA technique. The lower detection limit of the current version 3.0 is 615 IU/mL and linear quantification is ensured between 615–8,000,000 IU/mL, independent of the HCV genotype (Morishima 2004). The bDNA assay only requires 50 µl serum for HCV RNA quantification and is currently the assay with the lowest sample input.

Real-time PCR-based HCV RNA detection assays

Real-time PCR technology provides optimal features for both HCV RNA detection and quantification because of its very low detection limit and broad dynamic range of linear amplification (Sarrazin 2006) (Figure 1). Distinctive for real-time PCR technology is the ability to simultaneously amplify and detect the target nucleic acid, allowing direct monitoring of the PCR process. RNA templates are first reverse-transcribed to generate complementary cDNA strands followed by a DNA polymerase-mediated cDNA amplification.

Figure 1. Detection limits and linear dynamic ranges of commercially available HCV RNA detection assays

DNA detection simultaneous to amplification is preferentially achieved by the use of target sequence-specific oligonucleotides linked to two different molecules, a fluorescent reporter molecule and a quenching molecule. These probes bind the target cDNA between the two PCR primers and are degraded or released by the DNA polymerase during DNA synthesis. In case of degradation the reporter and quencher molecules are released and separated, which results in the emission of an increased fluorescence signal from the reporter. Different variations of this principle of reporter and quencher are used by the different commercially available assays. The fluorescence signal, intensified during each round of amplification, is proportional to the amount of RNA in the starting sample. Quantification in absolute numbers is achieved by comparing the kinetics of the target amplification with the amplification kinetics of an internal control of a defined initial concentration.

Highly effective and almost completely automated real-time PCR-based systems for HCV RNA measurement have been introduced.

All commercially available HCV RNA assays are calibrated to the WHO standard based on HCV genotype 1. Significant differences between different RT-PCR assays and other quantitative HCV RNA tests have been reported – in the case of the real-time PCR-based assays a slight under-quantification by one assay and a slight over-quantification by the other, in comparison to the WHO standard by Cobas® TaqMan®. In addition, it has been shown that results may vary significantly between assays with different HCV genotypes despite standardisation to IU (Chevaliez 2007, Vehrmeren 2008).

Cobas® TaqMan® HCV test

The FDA- and CE-accredited Cobas® TaqMan® (CTM) assay uses reporter- and quencher-carrying oligonucleotides specific to the 5’ UTR of the HCV genome and to the template of the internal control, a synthetic RNA for binding the same primers as for HCV RNA. Reverse transcription and cDNA amplification is performed by the Z05 DNA polymerase. For HCV RNA extraction from serum or plasma samples, a Cobas® TaqMan® assay was developed either in combination with the fully automated Cobas® AmpliPrep (CAP) instrument using magnetic particles, or in combination with manual HCV RNA extraction with glass fibre columns using the High Pure System (HPS) viral nucleic acid kit. The current versions of both combinations have a lower detection limit of approximately 10 IU/mL and a linear amplification range of HCV RNA from approximately 40 to 10,000,000 IU/mL. Samples from HCV genotypes 2-5 have been shown to be under-quantified by the first version of the HPS-based Cobas® TaqMan® assay. The second version of this assay has now demonstrated equal quantification of all HCV genotypes (Colucci 2007). For the Cobas® AmpliPrep/Cobas® TaqMan® (CAP/CTM) assay, significant under-quantification of HCV genotype 4 samples has been shown. In the meanwhile, a second version CAP/CTM assay (CAP/CTM HCV Test, v2.0) was evaluated. Based on a dual-probe design, this assay was able to accurately quantify HCV RNA samples from patients infected with all HCV genotypes, including HCV genotype 4 transcripts with rare sequence variants that had been under-quantified by the first generation assay (Vermehren 2011). Furthermore, this assay has a lower limit of detection and quantification of approximately 15 IU/mL across all HCV genotypes, and a linear amplification range of HCV RNA from approximately 15 to 10,000,000 IU/mL (Zitzer 2013). Taken together, the Cobas® TaqMan® assay makes both highly sensitive qualitative and linear quantitative HCV RNA detection feasible with excellent performance in one system with complete automation.

RealTime HCV test

The CE-accredited RealTime HCV test also uses reporter- and quencher-carrying oligonucleotides specific for the 5’UTR. HCV RNA concentrations are quantified by comparison with the amplification curves of a cDNA from the hydroxypyruvate reductase gene from the pumpkin plant Cucurbita pepo, which is used as an internal standard. This internal standard is amplified with different primers from those of the HCV RNA, which may be the reason for the linear quantification of very low HCV RNA concentrations. The RealTime HCV test provides a lower detection limit of approximately 10 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, Vehrmeren 2008). In a multi-centre study, its clinical utility to monitor antiviral therapy of patients infected with HCV genotypes 1, 2 and 3 was proven and the FDA approved the RealTime HCV test (Vermehren 2011). In this study, highly concordant baseline HCV RNA levels as well as highly concordant data on rapid and early virologic response were obtained compared to reference tests for quantitative and qualitative HCV RNA measurement, the Versant® HCV Quantitative 3.0 branched DNA hybridisation assay and the Versant® HCV RNA Qualitative assay.

Artus hepatitis C virus QS-RGQ assay

Qiagen has developed a novel real-time based HCV RNA assay, the artus HCV QS-RGQ assay. The artus HCV RNA assay has a lower limit of quantification of 30 IU/mL and a linear range of quantification up to 108 IU/mL. Compared to the Cobas® TaqMan® assay, the artus HCV assay had a slightly lower sensitivity (Paba 2012).

Versant HCV 1.0 kPCR assay

For replacement of the qualitative TMA and the quantitative bDNA-based assays, a real-time-based PCR test (Versant® kPCR Molecular System) has been introduced recently. While little is known for the use of this assay in response-guided conventional dual and triple therapies in HCV genotype 1-infected patients, a limitation of this assay seems to be a substantial underquantification of HCV RNA concentrations in certain HCV subtypes (2a, 3a, 4a) (Kessler 2013).

Cepheid Xpert HCV Viral Load Assay and Beckman Coulter DxN Veris HCV Assay

In the meanwhile, Cepheid has developed the RT-PCR-based Xpert HCV Viral Load assay, which – according to the manufacturer’s instructions – quantifies viral load in a linear range from 10 to 100,000,000 IU/mL for HCV genotypes 1-6, with a lower limit of detection of 4.0 IU/mL. These excellent performance characteristics have been confirmed in an independent validation study (Mc Hugh 2017). Of note, the Xpert HCV Viral Load assay is also suitable to detect and quantify HCV RNA from finger-stick capillary blood samples (Grebely 2017). Another RT-PCR-based assay is the Beckman Coulter DxN Veris HCV Assay.

Futhermore, the novel TMA-based Aptima HCV Quant Dx assay has been approved which allows highly specific and sensitive HCV RNA detection and quantification (details above).

HCV genotyping

HCV is heterogeneous with an enormous genomic sequence variability due to a rapid replication cycle with the production of 1012 virions per day and the 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 characterised. These genotypes vary in approximately 30% of their RNA sequence with a median variability of approximately 33%. HCV subtypes are defined by differences in their RNA sequence of approximately 10%. Within one subtype, numerous quasispecies exist and may emerge during treatment with specific antivirals. These quasispecies are defined by a sequence variability of less than 10% (Simmonds 2005). Because the currently recommended treatment durations can depend on the HCV genotype, HCV genotyping is mandatory in every patient who considers antiviral therapy (Lange 2014). For DAA-based therapies, determination of HCV genotypes and even subtypes is important because of significantly distinct barriers to resistance on the HCV subtype level. Furthermore, rarely viral recombinants exist of different HCV sub- or genotypes. The most frequent viral chimera is the so named St. Petersburg variant consisting of a HCV genotype 2k/1b recombinant. Proper diagnosis by routine HCV genotyping assays and treatment of viral chimeras may be challenging (see below). However, the importance for HCV genotyping may decline with the availability of highly and broadly effective all oral combination therapies in the future.

Both direct sequence analysis and reverse hybridisation technology allow HCV genotyping. Initial assays were designed to analyse exclusively the 5’ untranslated region (5’UTR), which is burdened with a high rate of misclassification especially on the subtype level. Current assays were improved by additionally analysing the coding regions, in particular the genes encoding the non-structural protein NS5B and core protein, both of which provide non-overlapping sequence differences between the genotypes and subtypes (Bowden 2006).

Reverse hybridising assay (Versant® HCV Genotype 2.0 System (LiPA))

In reverse hybridising, biotinylated cDNA clones from HCV RNA are produced by reverse transcriptase and then transferred and hybridised to immobilised oligonucleotides specific to different genotypes and subtypes. After removing unbound DNA by a washing step, the biotinylated DNA fragments can be detected by chemical linkage to coloured probes.

The Versant® HCV Genotype 2.0 System is suitable for identifying 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 to differentiate the genotype 1 subtypes. In a study evaluating the specificity of the Versant® HCV Genotype 2.0 System, 96.8% of all genotype 1 samples and 64.7% of all genotype samples were correctly subtyped. No misclassifications at the genotype level were observed. Difficulties in subtyping occurred in particular in genotypes 2 and 4. Importantly, none of the misclassifications would have had clinical consequences, which qualifies the Versant® HCV Genotype 2.0 System as highly suitable for clinical decision-making (Bouchardeau 2007).

However, the recent discovery of intergenotypic chimeras, which cannot be classified accurately by the current version of the LiPA assay, has shown that exclusive usage of the LiPA-assay for HCV genotyping can in rare cases result in the selection of inadequate all-oral treatment regimens (details below).

Direct sequence analysis (Trugene® HCV 5’NC genotyping kit)

The TruGene® 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 (approx. 20% misclassifications according to a recent study) because of the exclusive analyses of the 5’UTR (Sarrazin 2015).

Real-time PCR technology (RealTime™ HCV Genotype II assay)

The current RealTime HCV Genotype II assay is based on real-time PCR technology, which is less time consuming than direct sequencing. Preliminary data revealed 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. Nevertheless, single genotype 2, 3, 4, and 6 isolates were misclassified at the genotype level, indicating a need for assay optimisation (Ciotti 2010). A more recent study has shown that the RealTime HCV Genotype II assay fails to correctly classify HCV genotype 6n and 6e genotypes (Yang 2014), Furthermore, misclassifications on the subtype level have been reported for HCV genotype 1a/1b (Liu 2015). The diagnostic performance of this assay for viral recombinants is unclear but theoretically, due to amplification of areas in the structural and non-structural HCV genome, viral chimeras may be recognised.

Cobas® HCV genotyping test

The Cobas® HCV genotyping test is a novel PCR-based assay using genotype-specific primers for three different regions of the HCV genome (Stelzl 2016). Compared to direct sequencing analysis, the Cobas® HCV genotyping test produced concordant results in 95.7% for genotyping of HCV genotype 2-6 and in 99.2% for subtyping of HCV genotype 1a/1b. No misgenotyping was observed (Nieto-Aponte 2016).

Implications for diagnosing and managing acute and chronic hepatitis C

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 lower detection limit of 50 IU/mL or less are required, for example TMA, qualitative RT-PCR or the newer real-time PCR systems. Testing for anti-HCV alone is insufficient for the diagnosis of acute hepatitis C because HCV specific antibodies appear only weeks (up to 6 months) after viral transmission. In contrast, measurable HCV RNA serum concentrations emerge within the first days after infection. However, 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 clarify this because its presence is common in both situations. In rare cases and especially in association with low amounts of inoculum, HCV infection may be only associated with transient HCV RNA detectability or exclusively by markers of innate immune response (Heller 2013).

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%. False negative results may occur rarely in immunosuppressed patients (i.e., HIV) and in patients on dialysis. 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. The latter cannot be distinguished by HCV antibody tests from rarely occurring false positive serological results, the exact incidence of which is unknown. Serological false positive results can be identified by the additional performance of an immunoblot assay. Many years after disease resolution, anti-HCV antibodies may become undetectable on commercial assays in some patients.

Diagnostic tests in the management of hepatitis C therapy

The current treatment recommendations for acute and chronic hepatitis C are based on HCV genotyping and on HCV RNA determination before, (during) and after antiviral therapy. When HCV RNA has been detected, exact genotyping and HCV RNA determination is necessary in every patient considered for antiviral therapy. Exact subtyping appears to be highly important for therapies with some directly acting antiviral (DAA) agents because some subtypes (especially HCV genotype 1a vs. 1b) behave differently regarding treatment response and the development of resistance. In this regard, it is important that conventional genotyping (based on reverse hybridisation) can miss the detection of intergenotypic chimeras and misclassify them as easy-to-treat HCV genotype 2 strains (details below). Low HCV RNA concentration (<600,000–800,000 IU/mL) is a positive predictor of SVR for some treatment regimens, including dual combination therapy with PEG-IFN and ribavirin, conventional triple therapies with one DAA in combination with pegylated interferon and ribavirin, and all-oral therapy with grazoprevir, elbasvir and ribavirin in patients infected with HCV genotype 1a or 4 (Sarrazin 2010, Komatsu 2016). Furthermore, for treatment-naïve, non-cirrhotic patients shortening treatment duration with the all-oral, interferon-free combination therapy of sofosbuvir and ledipasvir to 8 weeks is possible based on a new baseline viral load cut-off of 6 million IU/mL according to the EMA and FDA labels. Genotyping is mandatory for the selection of the optimal treatment regimen and duration of therapy, since many DAA agents are selectively effective for only some HCV genotypes (Lange 2014).

Dual combination therapy (PEG-IFN + ribavirin)

Here we summarise treatment algorithms for the previous standard of care, a dual combination of PEG-IFN-α and ribavirin, because in some countries with limited access to DAAs such conventional therapies may still be used.

For HCV genotype 1 (and 4) treatment can be shortened to 24 weeks in patients with low baseline viral load (<600,000–800,000 IU/mL) and rapid virologic response (RVR) with undetectable HCV RNA at week 4 of treatment (Sarrazin 2010). In slow responders with a 2 log10 decline but still detectable HCV RNA levels at week 12 and undetectable HCV RNA at week 24, treatment could be extended to 72 weeks, but treatment with DAAs would certainly be the preferred strategy in these patients (Sarrazin 2010). In patients with complete early virologic response with undetectable HCV RNA at week 12 (cEVR), standard treatment is continued to 48 weeks. Genotypes 5 and 6 are treated the same as genotype 1-infected patients due to the lack of adequate clinical trials, whereas genotypes 2 and 3 generally allow treatment duration of 24 weeks, which may be shortened to 16 weeks (depending on RVR and [low] baseline viral load) or extended to 36-48 weeks depending on the initial viral decline (Sarrazin 2010).

Independent of the HCV genotype, proof of HCV RNA decrease is necessary to identify patients with little chance of achieving SVR. HCV RNA needs to be quantified before and 12 weeks after treatment initiation and antiviral therapy should be discontinued if a decrease of less than 2 log10 HCV RNA is observed (negative predictive value 88-100%). In a second step, HCV RNA should be tested with highly sensitive assays after 24 weeks of treatment because patients with detectable HCV RNA at this time point only have a 1-2% chance of achieving SVR.

Sofosbuvir-based triple therapies (sofosbuvir + PEG-IFN + ribavirin)

All patients on sofosbuvir-based combination therapies with PEG-IFN α and ribavirin achieved undetectable HCV RNA concentrations on antiviral therapy and no response-guided therapy approaches have been developed (Jacobson 2013, Lawitz 2013, Lawitz 2013). Therefore, on-treatment monitoring of HCV RNA is not necessary for determination of treatment duration or early stopping rules. However, HCV RNA measurement during treatment may be useful for assessment of adherence and motivation of patients.

All-oral IFN-free therapies

So far, no response-guided treatment-algorithms have been established for approved all-oral DAA combination therapies.

Furthermore, it was shown in a large retrospective analysis of the ION studies, that the initial viral load decline during sofosbuvir and ledipasvir therapy had no relevant impact on treatment outcome in general (Welzel 2014). In another study, no correlation between early viral kinetics and outcome of treatment with paritaprevir/r, ombitasvir and dasabuvir was observed (Sulkowski 2014). Another study has shown that on-treatment HCV RNA levels ≥45 IU/mL (assessed with the Cobas® TaqMan® assay) were associated with high relapse rates in HCV genotype 3 patients who were treated with sofosbuvir and ribavirin (Massoumy 2016). However, this was not the case in patients treated with more potent regimens such as sofosbuvir and daclatasvir.

It is important to know that viral load monitoring during the approval studies of sofosbuvir-based IFN-free regimens has been performed with the HPS-based Cobas® TaqMan® assay. However, if on-treatment viral load monitoring is performed with other assays (e.g., the RealTime HCV test), positive HCV RNA detection below the limit of quantification (i.e., <12 IU/mL positive) has been observed on an IFN-free regimen without any negative impact on treatment outcome, despite detectable residual HCV RNA until the end-of-treatment in individual patients (Cloherty 2015). Another study has performed repetitive early HCV RNA measurements in 11 HCV genotype 1 patients who were treated with combination therapy of paritaprevir/r, ombitasvir, dasabuvir and ribavirin for 12 weeks (Sarrazin 2015). HCV RNA quantification results were compared for the RealTime HCV (ART) and the High-Pure-System/Cobas® TaqMan® (HPS) assays. On-treatment HCV RNA was detectable in a relevant number of samples when assessed by the ART but not when assessed by the HPS assay, while the converse has rarely been reported. However, residual HCV RNA detection even at late points of antiviral therapy did not correlate with treatment failure in this study. More data are required to fully understand this phenomenon. However, for the time being it is very important not to consider these test results as treatment failure but to continue antiviral therapy for the originally planned duration in such a scenario. Further studies should also better define whether very early HCV RNA kinetics may have an impact on treatment outcome and on determination of optimal treatment duration of such potent all-oral regimens: it has been shown that the usage of different assays for HCV RNA quantification may have a profound impact on results of on-treatment HCV viral load monitoring. For example, a recent study showed that the Roche / High-Pure-System Cobas® TaqMan® V2 measures HCV RNA levels that are 0.46 log IU/mL higher than those determined by the Abbott RealTime HCV test before and during all-oral therapy with paritaprevir, ombitasvir and dasabuvir (Wiesmann 2014).

A viral load <6 million IU/mL at baseline with sofosbuvir plus ledipasvir allows shortening treatment duration from 12 weeks to 8 weeks, according to the ledipasvir label in the US. These recommendations were derived from on-treatment monitoring with the HPS-based Cobas® TaqMan® assay. Therefore, when using other commercially available assays such as the Cobas AmpliPrep Cobas® TaqMan® assay or the RealTime HCV test for viral load quantification, rates of patients with a viral load <6 million IU/mL may be much higher. In fact, a recent study has shown that HCV-RNA levels were significantly higher when measured with the Cobas AmpliPrep Cobas® TaqMan® assay versus the RealTime HCV assay in the same sample. According to this study, treatment-naïve, non-cirrhotic HCV genotype 1 patients, 95% or 78% had HCV-RNA viral load <6 million IU/mL, when measured with the RealTime HCV assay compared to the Cobas AmpliPrep Cobas® TaqMan® assay, respectively (Vermehren 2016). It may therefore be relevant to assess whether the recommendation to shorten treatment with sofosbuvir and ledipasvir in patients with viral load <6 million IU/mL is valid for test results from assays other than the HPS-based Cobas® TaqMan®. Calculation of conversion factors revealed a viral load cut-off between 2 and 3 million IU/mL as suitable for RealTime HCV and Cobas Ampliprep Cobas® TaqMan® (Fevery 2014, Kessler 2015). However, data from recent real-world studies showed that application of the 6 Million IU/mL HCV RNA cut-off rule based on different commercially available assays was associated with high SVR rates (97-98%) (Kowdley 2016).

It is important to note that neither baseline viral load nor on-treatment viral kinetics play a role in determining treatment durations with the newer DAA-combination regimens sofosbuvir plus velpatasvir, sofosbuvir plus velpatasvir plus voxilaprevir, or glecaprevir plus pibrentasvir. In contrast, HCV genotype, failure of previous DAA therapy, and the presence of liver cirrhosis or decompensated liver cirrhosis are important determinants of required treatment durations with these regimens (see Chapter 12 for details). An important exception is therapy with grazoprevir plus elbasvir of patients infected with HCV genotype 1a or 4. In these patients, treatment extension to 16 weeks (versus 12 weeks) and additional administration of ribavirin is required in case of a baseline antiviral load >800,000 IU/mL, at least in the absence of reliable resistance testing (Zeuzem 2017). In contrast, no treatment extension is required in patients infected with HCV genotype 1b or in patients with HCV genotype 1a in whom baseline resistance mutations have been excluded.

Detection of intergenotypic recombinant strains (chimeras)

Recent reports have described the occurrence of intergenotypic recombinant strains (chimeras), in which the 5´ part of the genome corresponded to HCV genotype 2 sequences and the 3`part to HCV genotype 1 sequences (the recombination breakpoint was located between NS2 and NS3). Of note, the widely used INNO-LiPA 2.0 assay has classified these variants as HCV genotype 2 isolates, though they clinical behave like HCV genotype 1 isolates (i.e. lower responsiveness to sofosbuvir/ribavirin, but not to currently preferred sofosbuvir / velpatasvir and glecaprevir / pibrentasvir therapy than one would expect for HCV genotype 2). Intergenotypic chimeras, which were misclassified as HCV genotype 2 isolates, were observed in 2,5% of all HCV genotype 2 isolates, based on genotyping results using the INNO-LiPA (Hedskog 2015). Of note, in some geographic regions (e.g. Georgia), such chimeras may occur much more frequently (Karchava 2015). Due to migration, high frequencies of HCV genotype 2k/1b chimeras have been observed in cohorts from Israel and Germany as well (14% and 25% of HCV genotype “2” samples, respectively) (Susser 2017). A correct clinical classification of these variants as viral recombinants can be achieved by sequencing the 5’ NTR or part of the structural HCV genes together with an area within the non-structural genes (NS3, NS5A or NS5B).

Resistance testing during DAA therapies

As described more in detail in chapter 13, HCV variants resistant to DAAs can emerge during antiviral therapy and result in treatment failure. Resistance testing prior to antiviral therapy can help select the optimal treatment regimen for individual patients (Schneider 2014). For example, before initiating simeprevir-based triple therapy, patients should be screened for the presence of the frequent Q80K variant in NS3, because in HCV genotype 1a patients with Q80K variants, the addition of simeprevir did not improve SVR rates (Jacobson 2013). The presence of resistance variants at baseline of IFN-free therapy with a first generation NS3 plus NS5A inhibitor like daclatasvir plus asunaprevir or daclatasvir plus simeprevir also strongly reduced the chance of achieving an SVR (approx. 40% vs. 84% in patients without resistance variants) (Manns 2014, Zeuzem 2015).

Furthermore, it was shown that the presence of NS5A resistance-associated substitutions (RAS) at baseline with sofosbuvir + ledipasvir resulted in reduced SVR rates, especially in patients who were treated for only 8 weeks instead of 12 weeks, or in patients with previous failure to antiviral therapy (Sarrazin 2016). Similar results have been obtained for combination regimens of sofosbuvir plus another NS5A inhibitor like daclatasvir or velpatasvir. Furthermore, baseline NS5A RAS negatively impact on outcome of treatment with grazoprevir, elbasvir and ribavirin in patients infected with HCV genotypes 1a and 4. The underlying principle seems to be the combination of several negative treatment predictors. While the importance of the pre-existence of RAS alone is limited a combination of RAS plus another stress factor like cirrhosis or shortened treatment duration is associated with markedly reduced SVR rates (Sarrazin 2015).

A report has also described a variant in the HCV NS5B polymerase (C316N), which, if detectable at baseline, was associated with lower SVR rates after treatment with sofosbuvir in combination with ribavirin with and without interferon alfa (Vermehren 2015). Of note, the C316N variant was detected almost exclusively in baseline serum samples of HCV genotype 1b patients compared to HCV genotype 1a patients. For combination regimens of sofosbuvir with another highly active DAA like ledipasvir no importance of C316N variant was observed (Sarrazin 2014).

Baseline resistance variants were detected in 20,5% (NS3), 11,9% (NS5A) and 22,1% (NS5B) of patients infected with HCV genotype 1 infection (Dietz 2015). Yet, it has been shown, that baseline resistance testing allows a selection of approved interferon-free regimens for which 98,6% and 100% of HCV genotype 1b and 1a patients are wildtype, respectively. Even more important, resistance testing allows selection of appropriate re-treatment regimens after failure of IFN-free all oral combination therapies. A recent study has identified RAS in 90% and 39% in NS3, NS5A or NS5B in HCV genotype 1 and genotype 3 patients, respectively, who had experienced treatment-failure after prior IFN-free therapy (Vermehren 2016). Re-treatment was performed with DAA combinations for which no RAS were detected and resulted in SVR in approx. 90% of patients. In addition, an association of a major NS5A RAS (Y93N) with the presence of the beneficial IL28B (IFN-L3) CC genotype was reported. This observation explains the unexpected low SVR rates in patients with IL28B CC genotype after several IFN-free DAA combination regimens (Peiffer 2016).

The so far largest study of the emergence of RAS after DAA failure has shown that after simeprevir or paritaprevir failure, R155K and D168E/V in NS3 are typically observed, whereas Q80K/R is a typical RAS selected after treatment with simeprevir. Typical RAS after failure with NS5A inhibitors were Y93H and L31M in NS5A. L159F and S282T RAS in NS5B were observed in patients with failure of sofosbuvir-containing regimens (Dietz 2017).

However, it is important to note that so far no relevant impact of the presence of RAS on treatment outcome with the newest DAA combinations glecaprevir / pribrentasvir or sofosbuvir / velpatasvir / voxilaprevir has been shown (Krishnan 2017; Bourlière 2017). This applies also for re-treatment of patients with prior DAA-failure with sofosbuvir / velpatasvir / voxilaprevir.

Commercially available assays for resistance testing are available in the US and are currently being established in other countries. However, currently no validated and standardised assay for HCV resistance testing is available and correspondingly results of resistance testing in different experienced laboratories will vary substantially. In summary, resistance testing should be performed – if possible – before treatment of HCV genotype 1a or 4 patients with grazoprevir, elbasvir and ribavirin (the presence of NS5A RAS requires extended treatment duration of 16 weeks), before treatment of HCV genotype 3 patients with sofosbuvir and velpatasvir (if NS5A RAVS are detected, patients should be treated with additional ribavirin), before treatment with simeprevir (which should be avoided in the presence of NS3 Q80K variants), and perhaps in selected cases before re-treatment after failure of IFN-free DAA combination therapies (EASL 2016).

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