Book on hepatitis from page 186 to 203

Book on hepatitis from page 186 to 203

186  Hepatology 2012
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Hepatitis D – Diagnosis and Treatment  187
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Hepatitis C: Diagnostic Tests  189
12. Hepatitis C: Diagnostic Tests
Christian Lange and Christoph Sarrazin
Common symptoms of hepatitis C like fatigue, muscle ache, loss of appetite or
nausea are unspecific 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 (McHutchison 2004). 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 etiology and with a
history of enhanced risk of HCV transmission (i.e., past IV or nasal drug
dependency, transmission 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 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 is furthermore essential in the determination of treatment indication,
duration and success  (Terrault 2005). The latter has to be confirmed at clearly
defined times during treatment to decide whether therapy should be continued or
not. It should be repeated 24 weeks after treatment completion to assess whether a
sustained virologic response (SVR) has been achieved. 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
190  Hepatology 2012
HCV RNA assays are now being 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.
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 specific
ribavirin doses differ among the genotypes.
Morphological methods like immunohistochemistry, in situ hybridization 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 3
rd
generation EIA has been completed by 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 rheuma-factors and in populations with a
low hepatitis C prevalence, for example 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 the false-negatively-classifying of HCV-infected individuals.
Hepatitis C: Diagnostic Tests  191
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.
However, the introduction of a reliable and sensitive HCV core antigen assay is
burdened with a number of difficulties like the development of specific monoclonal
antibodies recognizing all different HCV subtypes and the need for accumulation
and dissociation of HCV particles from immune complexes to increase sensitivity.
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 low 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). Taken
together, the new HCV core antigen assay could be a cheaper, though somewhat
less sensitive alternative for nucleic acid testing. 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.
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 load determination for management of
patients, 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 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
192  Hepatology 2012
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
Amplicor™ HCV 2.0  Roche Molecular Systems  PCR  FDA, CE
Versant™ HCV  Siemens Medical
Solutions Diagnostics
TMA  FDA, CE
Quantitative HCV RNA detection assays
Amplicor™
HCV Monitor 2.0  Roche Molecular Systems  PCR  CE
HCV SuperQuant™  National Genetics Institute  PCR
Versant™
HCV RNA 3.0  Siemens Medical
Solutions Diagnostics
bDNA  FDA, CE
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
Qualitative assays for HCV RNA detection
Until recently, qualitative assays for HCV RNA had substantially lower limits of
detection in comparison with 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 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 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.
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
hybridization protection assay. Two primers, one of which contains a T7 promoter,
Hepatitis C: Diagnostic Tests  193
one T7 RNA polymerase and one reverse transcriptase, are necessary for this
procedure. After RNA extraction from 500µl 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 recognizes 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 is 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.
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 summarized 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 is 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.
194  Hepatology 2012
Branched DNA hybridisation assay (Versant
®
HCV RNA 3.0
quantitative assay)
Branched DNA hybridisation assay is 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
Hepatitis C: Diagnostic Tests  195
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. For replacement of the
qualitative TMA and the quantitative bDNA-based assays, a real-time based PCR
test (Versant
®
kPCR Molecular System) is being developed.
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 fiber 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 recently released
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. However, a prototype second version of 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). The
second version CAP/CTM assay will be commercially available in 2012. 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.
196  Hepatology 2012
RealTime
HCV test
The CE-accredited RealTime HCV Test uses reporter-  and quencher-carrying
oligonucleotides specific for the 5’UTR as well. HCV RNA concentrations are
quantified by comparison with the amplification curves of a cDNA from the
hydroxypyruvate reductase gene from the pumpkin plant Curcurbita 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  (Vehrmeren 2008; Michelin 2007; Sabato 2007).
In a recent multi-centre study, its clinical utility to monitor antiviral therapy of
patients infected with HCV genotypes 1, 2 and 3 has been proven and the FDA
consequently 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 hybridization assay and the Versant
®
HCV RNA
Qualitative assay, respectively.
HCV genotyping
HCV is heterogeneous with an enormous genomic sequence variability, developed
due to a rapid replication cycle with the production of 10
12
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 characterized.
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 and ribavirin doses depend on the
HCV genotype, HCV genotyping is mandatory in every patient who considers
antiviral therapy  (Bowden 2006). For triple therapies with HCV protease inhibitors
and future multiple direct acting antiviral combination therapies, determination of
HCV subtypes is of importance because of significant different barriers to resistance
on the HCV subtype level.
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 analyzing 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).
Hepatitis C: Diagnostic Tests  197
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 indentifying genotypes 1-6 and more than 15 different subtypes and is currently the preferentially used 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. 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).
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 because of the exclusive
analyses of the 5’UTR (Pawlotsky 2003).
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 optimization  (Ciotti
2010).
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 newly developed 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 after viral transmission. In contrast,
198  Hepatology 2012
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 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%. 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 via 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 load determination before, during and after
antiviral therapy. When HCV RNA has been detected, exact genotyping and HCV
RNA load determination is necessary in every patient considered for antiviral
therapy. Exact subtyping might gain increased importance during therapies with
directly acting antiviral (DAA) agents because some subtypes behave differently
regarding the development of resistance. Low HCV RNA concentration (<600,000–
800,000 IU/ml) is a positive predictor of SVR. 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 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).
Dual combination therapy (PEG-IFN + ribavirin)
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. 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 should be extended to 72 weeks. 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
Hepatitis C: Diagnostic Tests  199
viral load) or extended to 36-48 weeks depending on the initial viral decline
(Manns 2006, Layden-Almer 2006).
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 levels 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.
Triple therapy (telaprevir or boceprevir + peginterferon/ribavirin)
Complex treatment algorithms have been introduced with the approval of triple-therapies that now include a HCV NS3 protease inhibitor for chronic HCV genotype
1 infection. These algorithms are  different for treatment with telaprevir or
boceprevir.
Shortening of treatment duration with boceprevir triple therapy to 28 weeks is
possible in treatment-naïve  patients who achieve an extended rapid virologic
response (eRVR) defined by undetectable HCV RNA levels at weeks 8 and 24 of
treatment. Stopping rules are based on detectable HCV RNA concentrations above
100 IU/ml at week 12 or detectable HCV RNA by a sensitive assay at week 24.
For telaprevir triple therapy, shortening of treatment duration to 24 weeks in
treatment-naïve and relapser patients is based on undetectable HCV RNA at weeks
4 and 12 of treatment. Stopping rules include detectable HCV RNA >1000 IU/ml at
week 4 or 12 and detectable HCV RNA at week 24.
Measurement of HCV RNA at additional time points during boceprevir-  or
telaprevir-based triple therapies is recommended to identify viral breakthrough due
to the emergence of HCV variants resistant to DAA agents.
SVR, defined as the absence of detectable HCV RNA 24 weeks after treatment
completion, should be assessed by an HCV RNA detection assay with a lower limit
of 50 IU/ml or less to evaluate long-lasting treatment success.
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.
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202  Hepatology 2012
13. Standard Therapy of Chronic Hepatitis C
Virus Infection
Markus Cornberg, Svenja Hardtke, Kerstin Port, Michael P. Manns, Heiner
Wedemeyer
Goal of antiviral therapy
Globally, there are approximately 130-170 million people chronically infected with
hepatitis C virus (HCV), in Europe 8-11 million (Cornberg 2011, Shepard 2005).
Despite the implementation of blood-donor screening in the early ‘90s, there is still
an anticipated increase of HCV-related cirrhosis, hepatic decompensation, and
hepatocellular carcinoma (HCC) over the course of the next decade (Davis 2010).
The goal of antiviral therapy is to cure hepatitis C via a sustained elimination of the
virus (2011). A sustained elimination of HCV is achieved if the HCV RNA remains
negative six months after the end of treatment (sustained virological response, SVR)
(Table 1). Follow-up studies document that more than 99% of patients who achieve
an SVR remain HCV RNA negative 4-5 years after the end of treatment and no
signs of hepatitis have been documented (Manns 2008, Swain 2010). Importantly,
long-term benefits of SVR  are  the reduction of HCV-related hepatocellular
carcinoma and overall mortality (Backus 2011, Veldt 2007). In 2011, the FDA
accepted SVR-12 as endpoint for future trials because HCV relapse usually occurs
within the first 12 weeks after the end of treatment.
In addition to liver disease, several extra hepatic manifestations, such as
cryoglobulinemia, non-Hodgkin lymphoma, membranoproliferative
glomerulonephritis or porphyria cutanea tarda have been reported in the natural
history of hepatitis C virus infection (HCV). Antiviral treatment may improve
symptoms even without achieving an SVR. On the other hand, antiviral therapy may
worsen extrahepatic manifestations (Pischke 2008, Zignego 2007).
Basic therapeutic concepts and medication
Before the identification of HCV as the infectious agent for non-A, non-B
hepatitis (Choo 1989), interferon α (IFN) led to a normalisation of transaminases
and an improvement of liver histology in some patients (Hoofnagle 1986). After the
identification of HCV it became possible to measure success of therapy as a long-
Standard Therapy of Chronic Hepatitis C Virus Infection  203
lasting disappearance of HCV RNA from serum, the SVR. Since then, SVR rates
have increased from 5-20% with IFN monotherapy up to 40-50% with the
combination of IFN + ribavirin (RBV)  (McHutchison 1998,  Poynard 1998).
Different HCV genotypes (HCV G) show different SVR rates. Patients with the
most frequent HCV G1 require a longer treatment duration and still get a lower
SVR compared to HCV G2 and HCV G3 (Figure 1). The development of pegylated
interferon α (PEG-IFN) improved the pharmacokinetics of IFN, allowing more
convenient dosing intervals and resulting in higher SVR, especially for HCV G1.
Two PEG-IFNs are available; PEG-IFN α-2b (PEG-Intron
®
, Merck) and PEG-IFN
α-2a (PEGASYS
®
, Roche). Although smaller trials from southern Europe have
suggested slightly higher SVR rates in patients treated with PEG-IFN α-2a (Ascione
2010, Rumi 2010), a large US multicentre study did not detect any significant
difference between the two PEG-IFNs + RBV regarding SVR (McHutchison
2009b).
The two PEG-IFNs do have different pharmacokinetic profiles due to their
different polyethylene glycol moieties. PEG-IFN α-2b is bound to a single linear 12-kDa polyethylene glycol molecule, whereas PEG-IFN α-2a is covalently attached to
a 40-kDa branched chain polyethylene glycol moiety. The distinct sizes of the PEG-IFN influence the volume of distribution. PEG-IFN α-2b is dosed according to body
weight (1.5 µg/kg once weekly), while the larger PEG-IFN α-2a is given in a fixed
dose of 180 µg once weekly (reviewed in (Cornberg 2002)) (Table 2). PEG-IFN α-2b may also be dosed at 1.0 µg/kg once patients become negative for HCV RNA
without major declines in SVR rates (Manns 2011a, McHutchison 2009b). RBV
should be administered according to the bodyweight of the patient. A retrospective
analysis of the large PEG-IFN α-2b + RBV pivotal trial revealed that the optimal
dose of RBV (Rebetol
®
, Merck) is at least 11 mg/kg (Manns 2001). A prospective,
multicentre, open-label, investigator-initiated study confirmed that PEG-IFN α-2b
plus weight-based RBV is more effective than flat-dose ribavirin, particularly in
HCV G1 patients (Jacobson 2007). A RBV dose of 15 mg/kg would be ideal,
although higher doses are associated with higher rates of anemia (Snoeck 2006).
When combined with PEG-IFN α-2a, a RBV dose of 1000 mg if <75 kg or 1200 mg
if ≥75 kg is recommended for HCV G1 patients while a flat dose of 800 mg RBV
was initially suggested for patients with HCV G2 and 3 (Hadziyannis 2004).
However, a weight-based dose of ribavirin (12-15 mg/kg) may be preferred,
especially in difficult to treat patients and in Response-guided Therapy (RGT)
treatment approaches (EASL 2011, Sarrazin 2010). In 2011, the first direct antiviral
agents (DAA) were approved for patients with HCV G1. Two inhibitors of the HCV
protease (PI),  boceprevir (Victrelis
®
, Merck) and telaprevir (Incivek
®
, Vertex;
Incivo
®
, Johnson & Johnson), improve SVR rates up to 75% in naïve HCV G1
patients (Jacobson 2011b, Poordad 2011b) and 29-88% in treatment experienced
HCV G1 (Bacon 2011, Zeuzem 2011). However, both PIs require combination with
PEG-IFN + RBV because monotherapy would result in rapid emergence of drug
resistance. Both boceprevir (BOC) and telaprevir (TLV) can be combined with
PEG-IFN α-2a or PEG-IFN α-2b.