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11. Hepatitis C: diagnostic tests
https://t.me/medicina_free
Christian Lange and Christoph Sarrazin
Common symptoms of hepatitis C like fatigue, muscle ache, loss of
appetite or nausea are non-speciic and, in many cases, mild or not present.
Consequently, hepatitis C is oten diagnosed accidentally and, unfortu nately,
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 (Deuic-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 suicient
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 diferentiate between chronic hepatitis C and resolved HCV infection
from the past. When acute hepatitis C is considered, serologic screening
alone is insuicient because anti-HCV antibodies may develop late ater
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). Traditionally, HCV RNA measurement
should be repeated 24 weeks ater treatment completion to assess whether
a sustained virologic response (SVR) has been achieved. However, as
the probability of virologic relapse is similar ater 12 and 24 weeks the
new time point for assessment of inal virologic treatment outcome is 12
weeks ater 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 irst time,
for the screening of blood and organ donations and for conirming SV
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ater treatment completion. Quantitative HCV RNA detection assays ofer
the possibility of measuring the viral load exactly and may help treatment
monitoring. Qualitative and quantitative HCV RNA assays have now been
widely replaced by real-time PC-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 inancial restrictions, HCV
core antigen testing can be used to conirm ongoing HCV infection and to
monitor treatment outcome.
Ater diagnosing hepatitis C, traditionally the HCV genotype should be
determined by nucleic acid-based techniques in every patient considered
for HCV therapy. However, for selected easy to treat patients without
advanced cirrhosis and without previous treatment failure in the past
two years pangenotypic DAA regimens have been approved (velpatasvir/
sofosbuvir, glecaprevir/pibrentasvir) for whom the knowledge of HCV
genotype is not required. This may facilitate antiviral therapy especially in
highly endemic countries. In patients with cirrhosis the HCV genotype is
still required because for the use of Velpatasvir/Sofosbuvir in patients with
HCV genotype 3 the addition of ribavirin is recommended. In addition, for
patients with previous treatment failure to IFN-based therapies the HCV
genotype is also required for the use of glecaprevir/pibrentasvir as longer
treatment durations are recommended.
Morphological methods like immunohistochemistry, in situ
hybridisation or PC from liver specimens play no relevant role in the
diagnosis of hepatitis C because of their low sensitivity, poor speciicity and
low eicacy compared to serologic and nucleic acid-based approaches.
positive results. With these assays HCV-speciic antibodies can be detected
approximately 10 weeks ater 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 ater 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 suicient
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 speciicity of serologic HCV diagnostics is diicult to deine 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 conirmation 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 identiication 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.
Serologic assays
In current clin ical practice, antibodies against multiple HCV epitopes are
detected by commercially available 2nd and 3rd generation enzyme-linked
immunoassays (EIAs). In these tests, HCV-speciic 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 irst applied EIAs for the detection of HCV-speciic antibodies were
based on epitopes derived from the NS4 region (C-100) and had a sensitivity
of 70–80% and a poor speciicity (Scott 2007). C-100-directed antibodies
occur approximately 16 weeks ater 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
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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 irst 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 speciic
(99.5%), genotype independent, and had a low inter- and intra-assay
variability (coeicient of variation 5–9%) (Veillon 2003). HCV core antigen
is measurable 1–2 days ater 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 conirmation
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of ongoing hepatitis C in anti-HCV positive patients. As a consequence, this
irst 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 diferent antibodies
to detect HCV core antigen, is highly speciic (99.8%), equally efective
for diferent 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 quantiication could be an
alternative to HCV RNA quantiication for on-treatment antiviral response
monitoring (Vermehren 2012). Here, HCV core antigen below the limit of
quantiication at treatment week 1 was strongly predictive of VR, 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, but the clinical relevance of such regimens has
substantially declined. Yet, a recent large study has shown that for highly
efective 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 if an HCV RNA assay is not available or not afordable.,But
HCV RNA testing remains the gold standard to prove SV as three out of
148 patients with SV had a positive HCV core assay testing result (van
Tilborg 2018).
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-quantiication of samples
of certain HCV genotypes (Morishima 2004, Pawlotsky 2003, Pawlotsky
1999). Currently, several HCV RNA assays are commercially available
(Table 1). These assays are used in clinical practice to reliably quantify HCV
RNA concentration before, during and ater antiviral therapy in order
to identify candidates for antiviral therapy, to choose optimal treatment
regimens and durations and to prove treatment success.
However, with the implementation of highly efective and easy
antiviral regimens, current goals in the management of hepatitis C are
changing. While successful treatment of an individual patient is much
easier, current eforts aim to globally eliminate HCV infection. For this
purpose, easy diagnostic tools which can be widely distributed even in low
income countries to diagnose active HCV infection would be of high value.
Hence, several point-of-care assays which can rapidly and cheaply detect
HCV RNA or HCV core antigen in a small drop of blood are in clinical
evaluation, with which diagnostic sensitivities and speciicities of >95%
can be achieved.
Table 1. Commercially available HCV RNA detection assays
Assay Distributor Technology Approval status
Qualitative HCV RNA detection assays
Cobas Amplicor/
Cobas® TaqMan qual
Quantitative HCV RNA detection assays
Cobas AmpliPrep/
High pure system /
®
Taq M a n
Cobas
Abbott RealTime™
HCV
Artus HCV QS-RGQ
assay
Versant™HCV 1.0
kPCR assay
Veris HCV Assay Beckman Coulter Real-time PCR CE
Xpert HCV Viral
Load
Aptima HCV Quant
Dx Real Time TMA
Cobas HCV Assay
4800, 6800, 8800
®+
Roche Molecular
Systems
Roche Molecular
Systems
Abbott Diagnostics Real-time PCR FDA, CE
Qiagen Real-time PCR CE
Siemens Real-time PCR CE
Cepheid Real-time PCR CE
Hologic TMA CE
Roche Molecular
Systems
PCR FDA, CE
Real-time PCR FDA, CE
Real-time PCR CE, FDA
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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 irst diagnosis of acute hepatitis
C, in which HCV RNA concentrations are luctuating and may be very low,
as well as for conirmation of chronic hepatitis C infection in patients with
positive HCV antibodies. In addition, they are used for the conirmation of
virologic response during, at the end of, and ater antiviral therapy, as well as
in screening blood and organ donations for presence of HCV.
Qualitative RT-PCR
In reverse transcriptase-PC- (T-PC-) 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 ampliied by a DNA polymerase
into multiple double-stranded DNA copies. Qualitative T-PC 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 T-PC 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
ampliication and detection in a single-step, single-tube procedure.
The below described Cobas Ampliprep / Cobas TaqMan system is
available as a real-time PC-based assay for the qualitative and highly
sensitive detection of HCV genotype 1–6 samples, but also as a quantitative
assay which allows precise quantiication 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 ampliication and speciic 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. Ater RNA extraction from
500 l 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 irst primer,
and a DNA/DNA double-strand is synthesised by the reverse transcriptase.
Next, the RNA polymerase recognises the T7 promoter and produces 1001000 RNA transcripts, which are subsequently returned to the TMA cycle
leading to exponential ampliication 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-speciic 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 T-PC-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
speciicity of more than 99.5%, independent of the HCV genotype.
More recently, a novel TMA-based assay, t he Ap tima 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 speciic and HCV RNA test results
are highly concordant to the real-time PC-based Abbott RealTime HCV and
Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays.
Quantitative HCV RNA detection
HCV RNA quantiication can be achieved either by target ampliication
techniques (competitive and real-time PCR) or by signal ampliication
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 PC 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 PC technology, have been introduced. The technical
characteristics, detection limits a nd 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.
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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 PC technique.
Quantiication is achieved by the ampliication 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
deined initial concentration. The internal control is ampliied by the same
primers as the HCV RNA. Comparison of the inal 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 speciicity 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 ampliication
technology. Ater reverse transcription of the HCV RNA, the resulting
single-stranded complementary DNA strands bind to immobilised
captured oligonucleotides with a speciic 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 quantiication
system based on a branched DNA technique. The lower detection limit of
the current version 3.0 is 615 IU/mL and linear quantiication is ensured
between 615–8,000,000 IU/mL, independent of the HCV genotype
(Morishima 2004). The bDNA assay only requires 50 l serum for HCV RNA
quantiication and is currently the assay with the lowest sample input.
Real-time PCR-based HCV RNA detection assays
Real-time PC technology provides optimal features for both HCV
RNA detection and quantiication because of its very low detection limit
and broad dynamic range of linear ampliication (Sarrazin 2006) (Figure
1). Distinctive for real-time PC technology is the ability to simultaneously
amplify and detect the target nucleic acid, allowing direct monitoring of
the PC process. RNA templates are irst reverse-transcribed to generate
complementary cDNA strands followed by a DNA polymerase-mediated
cDNA ampliication.
Figure 1. Detection limits and linear dynamic ranges of commercially available HCV RNA
detection assays
DNA detection simultaneous to ampliication is preferentially achieved
by the use of target sequence-speciic oligonucleotides linked to two
diferent molecules, a luorescent reporter molecule and a quenching
molecule. These probes bind the target cDNA between the two PC 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 luorescence
signal from the reporter. Diferent variations of this principle of reporter
and quencher are used by the diferent commercially available assays.
The luorescence signal, intensiied during each round of ampliication, is
proportional to the amount of RNA in the starting sample. Quantiication
in absolute numbers is achieved by comparing the kinetics of the target
ampliication with the ampliication kinetics of an internal control of a
deined initial concentration.
Highly efective and almost completely automated real-time PC-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. Signiicant diferences between
diferent T-PC assays and other quantitative HCV RNA tests have been
reported – in the case of the real-time PC-based assays a slight underquantiication by one assay and a slight over-quantiication by the other,
in comparison to the WHO standard by Cobas® TaqMan®. In addition, it
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has been shown that results may vary signiicantly between assays with
diferent HCV genotypes despite standardisation to IU (Chevaliez 2007,
Vehrmeren 2008).
Cobas® TaqMan® HCV test
The FDA- and CE-accredited Cobas® TaqMan® (CTM) assay uses reporterand quencher-carrying oligonucleotides speciic to the 5’ UT 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
ampliication 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 ibre 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 ampliication
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-quantiied by the
irst version of the HPS-based Cobas® TaqMan® assay. The second version of
this assay has now demonstrated equal quantiication of all HCV genotypes
(Colucci 2007). For the Cobas® AmpliPrep/Cobas® TaqMan® (CAP/CTM)
assay, sign iicant under-quantiication 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-quantiied by the irst generation assay
(Vermehren 2011). Furthermore, this assay has a lower limit of detection
and quantiication of approximately 15 IU/mL across all HCV genotypes,
and a linear ampliication 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.
Yet, the Cobas® TaqMan® assay has been replaced by Cobas 6800/8800
HCV and Cobas 4800 HCV assays for high and medium-throughput
demands, respectively. It was shown that the Cobas 6800/8800 HCV
and Cobas 4800 HCV assays were able to quantify HCV genotype 1 RNA
concentrations in a linear range from lower limits of detection of 8.2 IU/ml
and 11.7 IU/ml, respectively. The concordances using a cutof of 6 million IU/
ml was at least 90% with previously established assays (Vermehren 2017).
RealTime HCV test
The CE-accredited RealTime HCV test also uses reporter- and quenchercarrying oligonucleotides speciic for the 5’UTR. HCV RNA concentrations
are quantiied by comparison with the ampliication 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
ampliied with diferent primers from those of the HCV RNA, which
may be the reason for the linear quantiication of very low HCV RNA
concentrations. The RealTime HCV test provides a lower detection limit
of approximately 10 IU/mL, a speciicity of more than 99.5% and a linear
ampliication range from 12 to 10,000,000 IU/mL independent of the
HCV genotype (Michelin 2007, Sabato 2007, Vehrmeren 2008). In a multicentre 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-GQ assay. The artus HCV RNA assay has a lower limit of
quantiication of 30 IU/mL and a linear range of quantiication 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 bDNAbased assays, a real-time-based PC test (Versant® kPC 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
underquantiication of HCV RNA concentrations in certain HCV subtypes
(2a, 3a, 4a) (Kessler 2013).
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Cepheid Xpert HCV Viral Load Assay and Beckman Coulter
DxN Veris HCV Assay
In the meanwhile, Cepheid has developed the T-PC-based Xpert HCV
Viral Load assay, which – according to the manufacturer’s instructions
– quantiies 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 conirmed 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 ingerstick capillary blood samples (Grebely 2017). Another T-PC-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 speciic and sensitive HCV RNA
detection and quantiication (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 idelity 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 deined by diferences in their RNA sequence of
approximately 10%. Within one subtype, numerous quasispecies exist and
may emerge during treatment with speciic antivirals. These quasispecies
are deined 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 several DAA-based therapies,
determination of HCV genotypes and even subtypes is important because
of signiicantly distinct barriers to resistance on the HCV subtype level.
Furthermore, rarely viral recombinants exist of diferent 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
declined with the availability of highly and broadly efective all oral
combination therapies. In fact for treatment-naïve patients without liver
cirrhosis antiviral therapy with velpatasvir/sofosbuvir for 12 weeks or
glecaprevir/pibrentasvir for 8 weeks is possible without the need for HCV
genotyping (Feld 2015, Zeuzem 2018).
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
misclassiication 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 diferences between the
genotypes and subtypes (Bowden 2006).
Reverse hybridising assay
®
(Versant
In reverse hybridising, biotinylated cDNA clones from HCV RNA are
produced by reverse transcriptase and then transferred and hybridised to
immobilised oligonucleotides speciic to diferent genotypes and subtypes.
Ater 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 diferent subtypes and is currently the
preferred assay for HCV genotyping. By simultaneous analyses of the 5’
UT and core region, a high speciicity is achieved to diferentiate the
genotype 1 subtypes. In a study evaluating the speciicity 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 misclassiications at
the genotype level were observed. Diiculties in subtyping occurred in
particular in genotypes 2 and 4. Importantly, none of the misclassiications
would have had clinical consequences, which qualiies 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 classiied 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).
HCV Genotype 2.0 System (LiPA))
Direct sequence analysis
®
(Tr ugen e
The TruGene® assay determines the HCV genotype and subtype by
direct analysis of the nucleotide sequence of the 5’UT region. Incorrect
HCV 5’NC genotyping kit)
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genotyping rarely occurs with this assay. However, the accuracy of
subtyping is poor (approx. 20% misclassiications according to a recent
study) because of the exclusive analyses of the 5’UT (Sarrazin 2015).
Real-time PCR technology
(RealTime™ HCV Genotype II assay)
The current RealTime HCV Genotype II assay is based on real-time
PC 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’UT regions. Nevertheless, single genotype
2, 3, 4, and 6 isolates were misclassiied 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, misclassiications
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 ampliication of areas in the structural and nonstructural HCV genome, viral chimeras may be recognised.
Cobas® HCV genotyping test
The Cobas® HCV genotyping test is a novel PC-based assay using
genotype-speciic primers for three diferent 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).
PC systems. Testing for anti-HCV alone is insuicient for the diagnosis of
acute hepatitis C because HCV speciic antibodies appear only weeks (up to
6 months) ater viral transmission. In contrast, measurable HCV RNA serum
concentrations emerge within the irst days ater infection. However, HCV
RNA may luctuate during acute hepatitis C, making a second HCV RNA test
necessary several week s 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 diicult 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 (or alternatively HCV core antigen, details above)
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 identiied by the additional performance of an immunoblot assay.
Many years ater disease resolution, anti-HCV antibodies may become
undetectable on commercial assays in some patients.
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 T-PC or the newer real-time
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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 ater antiviral therapy. When HCV RNA has been detected,
exact genotyping and HCV RNA determination is recommended in patients
considered for antiviral therapy. Exact geno- and subtyping appears to
be highly important for therapies in pre-treated patients, patients with
cirrhosis and for some directly acting antiviral (DAA) agents because some
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subtypes (especially HCV genotype 1a vs. 1b) behave diferently 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 SV
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 treatmentnaïve, non-cirrhotic patients shortening treatment duration with the alloral, interferon-free combination therapy of sofosbuvir and ledipasvir to 8
weeks is possible based on a new baseline viral load cut-of 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
some DAA regimens are selectively efective for only some HCV genotypes
(Lange 2014).
Interferon-based therapies
Here we summarise treatment algorithms for the previously used
interferon-based therapies, which might be of (historical) interest for some
readers.
For HCV genotype 1 (and 4) treatment could be shortened to 24 weeks
in patients with low baseline viral load (<600,000–800,000 IU/mL) and
rapid virologic response (VR) 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 was extended to 72 weeks (Sarrazin 2010). In patients
with complete early virologic response with undetectable HCV RNA at
week 12 (cEVR), standard treatment was continued to 48 weeks. Genotypes
5 and 6 were treated the same as genotype 1-infected patients due to the
lack of adequate clinical trials, whereas genotypes 2 and 3 generally
allowed treatment duration of 24 weeks, which was shortened to 16 weeks
(depending on V 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 was
necessary to identify patients with little chance of achieving SVR. HCV RNA
needs to be quantiied before and 12 weeks ater treatment initiation and
antiviral therapy was usually discontinued if a decrease of less than 2 log
10
HCV RNA was observed (negative predictive value 88-100%). 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 was not necessary for determination of treatment duration or
early stopping rules.
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 quantiication (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 genot ype
1 patients who were treated with combination therapy of paritaprevir/r,
ombitasvir, dasabuvir and ribavirin for 12 weeks (Sarrazin 2015). HCV RNA
quantiication results were compared for the RealTime HCV (AT) 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
AT 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 deine whether very
early HCV RNA kinetics may have an impact on treatment outcome and
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on determination of optimal treatment duration of such potent all-oral
regimens: it has been shown that the usage of diferent assays for HCV RNA
quantiication 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 quantiication, 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 signiicantly 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-of 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-of
rule based on diferent commercially available assays was associated with
high SV 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 elbasv ir of pat ient s
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 classiied 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 misclassiied 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, Hostager 2019). A cor rect clinical classiication of these var iants
as viral recombinants can be achieved by sequencing the 5’ NT or part of
the structural HCV genes together with an area within the non-structural
genes (NS3, NS5A or NS5B). In this regard, a recent study has shown that
the real-time-PC-based assays Cobas HCV GT and Abbott RealTime HCV
Genotype II Assay) could classify HCV 2/1 chimeras correctly in 90% and
65% of all cases, respectively, whereas the Versant HCV Genotype 2.0 (LiPA
2.0) hybridization assay failed to identify chimeras in 100% of cases (Peifer
2019).
Resistance testing during DAA therapies
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 SV rates (Jacobson 2013). The
presence of resistance variants at baseline of IFN-free therapy with a irst
generation NS3 plus NS5A inhibitor like daclatasvir plus asunaprevir or
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