"
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Basic knowledge
Hepatitis C may be clinically silent for years and many people
have been infected with hepatitis C virus (HCV) for decades
without knowing it. Effective screening should focus on
populations at-risk for HCV infection. Hepatitis C is diagnosed by
simple blood tests (Dufour 2000) (Table 2.1):
Detection of HCV antibodies is done by enzyme immunoassay
(screening tests) and immunoblot (confirmation tests). A new
HCV rapid test device (OraQuick® HCV Rapid Antibody Test), was
approved recently in Europe for use with venous or fingerstick
blood, serum, plasma or oral fluid (Lee 2011). This may help
address the problem of under-diagnosis, by increasing testing
outside of traditional clinical settings. However, all these
techniques have a window-period limitation (due to the late
seroconversion), which can last 70-82 days, considerably
reducing their usefulness in the diagnosis of acute HCV
infection. Testing for anti-HCV may be performed at 18 months
of age or older (before this age there is a high rate of false
Hepatitis C may be clinically silent for years and many people
have been infected with hepatitis C virus (HCV) for decades
without knowing it. Effective screening should focus on
populations at-risk for HCV infection. Hepatitis C is diagnosed by
simple blood tests (Dufour 2000) (Table 2.1):
Detection of HCV antibodies is done by enzyme immunoassay
(screening tests) and immunoblot (confirmation tests). A new
HCV rapid test device (OraQuick® HCV Rapid Antibody Test), was
approved recently in Europe for use with venous or fingerstick
blood, serum, plasma or oral fluid (Lee 2011). This may help
address the problem of under-diagnosis, by increasing testing
outside of traditional clinical settings. However, all these
techniques have a window-period limitation (due to the late
seroconversion), which can last 70-82 days, considerably
reducing their usefulness in the diagnosis of acute HCV
infection. Testing for anti-HCV may be performed at 18 months
of age or older (before this age there is a high rate of false
positive results, due to passive antibodies transfer from the
mother).
Nucleic acid testing (NAT) – detection of presence and/or
amount (viral load – VL) of HCV RNA in the blood, reflects the
actual viral replication. These tests are the hallmark of HCV
diagnosis in both antibody-positive and negative patients, with
unexplained ALT elevations or liver disease documented by liver
biopsy (LB). A high VL is a negative predictor of therapeutic
success. Sequential VL measurements with the same method
during treatment (at weeks 4, 12, 24/48) and 6 month after
treatment completion inform response-guided therapy (RGT).
Table 2.1 – Blood tests for hepatitis C
Test/Type Application Comments
EIA
(enzyme immunoassay)
Indicates past or present
infection
Does not differentiate
between acute and
chronic infection
All positive EIA results should be checked with a supplemental HCV RNA
assay:
HCV RNA qualitative
(RT-PCR)
Detects virus as early as 1-2
weeks after infection.
Useful for reduction of
residual risk associated to
transfusions*
Presence of circulating
HCV RNA might be
intermittent
A single negative RT-PCR is not conclusive
HCV RNA quantitative
(Real-time PCR)
Determines concentration
of HCV RNA (VL)
Useful for assessing the
response to therapy
HCV RNA genotyping Groups isolates of HCV
based on major genetic
types and subtypes
Determines the length
of treatment and
prediction of SVR rate
* screening by Versant™ (Siemens Health Care Diagnostics) and Procleix™ HIV-
1/HCV assays (Gen-Probe).
Determination of HCV genotype. The molecular
characterization of genotypes and subtypes of HCV is
particularly important for the response to treatment and disease
prognosis (Scott 2007). There are 6 major genotypes of HCV and
more than 50 subtypes.
mother).
Nucleic acid testing (NAT) – detection of presence and/or
amount (viral load – VL) of HCV RNA in the blood, reflects the
actual viral replication. These tests are the hallmark of HCV
diagnosis in both antibody-positive and negative patients, with
unexplained ALT elevations or liver disease documented by liver
biopsy (LB). A high VL is a negative predictor of therapeutic
success. Sequential VL measurements with the same method
during treatment (at weeks 4, 12, 24/48) and 6 month after
treatment completion inform response-guided therapy (RGT).
Table 2.1 – Blood tests for hepatitis C
Test/Type Application Comments
EIA
(enzyme immunoassay)
Indicates past or present
infection
Does not differentiate
between acute and
chronic infection
All positive EIA results should be checked with a supplemental HCV RNA
assay:
HCV RNA qualitative
(RT-PCR)
Detects virus as early as 1-2
weeks after infection.
Useful for reduction of
residual risk associated to
transfusions*
Presence of circulating
HCV RNA might be
intermittent
A single negative RT-PCR is not conclusive
HCV RNA quantitative
(Real-time PCR)
Determines concentration
of HCV RNA (VL)
Useful for assessing the
response to therapy
HCV RNA genotyping Groups isolates of HCV
based on major genetic
types and subtypes
Determines the length
of treatment and
prediction of SVR rate
* screening by Versant™ (Siemens Health Care Diagnostics) and Procleix™ HIV-
1/HCV assays (Gen-Probe).
Determination of HCV genotype. The molecular
characterization of genotypes and subtypes of HCV is
particularly important for the response to treatment and disease
prognosis (Scott 2007). There are 6 major genotypes of HCV and
more than 50 subtypes.
Viral kinetics: methodology
Measuring VL at baseline, as well as early after treatment
initiation may help to predict response and determine the
optimal length of therapy. As shown in chapter 1, in order to
maximize treatment effectiveness, while minimizing toxicity,
the optimal duration of PegIFN/RBV therapy is determined by
viral genotype, with additional guidance provided by the ontreatment
response (RVR being an earlier predictor of treatment
success and EVR an accurate predictor of treatment failure).
Viral load monitoring
For HCV RNA measurement, different standardized
quantification assays, based on signal amplification [branched
DNA(bDNA) assay] and target amplification [reversetranscription
PCR (RT-PCR)] with different sensitivities are
commercially available. The development of a World Health
Organization HCV international unit (IU) standard has
contributed to a better accuracy and comparability of results
obtained by different assays. However, since standardization to
IU and the calibration of assay sensitivity are based on genotype
1a (deLeuw 2011), relative quantification results may vary
among assays. Using the WHO international standard, a VL of 2
millions copies/mL (the cut-off value predictive for therapeutic
success in early clinical trials with IFN) was found to correspond
to 800 000 IU/mL. Currently a cut-off of 400 000-800 000 IU/ml
separate low from high VL. Further studies have found that
patients with low baseline HCV RNA levels have a 15-39% better
response rate, a finding that is consistent across trials using
different formulations and dosages of IFN (Strader 2004).
Real-time PCR tests
Real-time PCR tests are faster and more cost-effective methods
that detect very low VL (10-15 IU/ml) (Vermehren 2008). Realtime
PCR can accurately quantify HCV RNA levels over a linear
range exceeding 6 logs (10 IU/mL to 100 million IU/mL) (Table
Measuring VL at baseline, as well as early after treatment
initiation may help to predict response and determine the
optimal length of therapy. As shown in chapter 1, in order to
maximize treatment effectiveness, while minimizing toxicity,
the optimal duration of PegIFN/RBV therapy is determined by
viral genotype, with additional guidance provided by the ontreatment
response (RVR being an earlier predictor of treatment
success and EVR an accurate predictor of treatment failure).
Viral load monitoring
For HCV RNA measurement, different standardized
quantification assays, based on signal amplification [branched
DNA(bDNA) assay] and target amplification [reversetranscription
PCR (RT-PCR)] with different sensitivities are
commercially available. The development of a World Health
Organization HCV international unit (IU) standard has
contributed to a better accuracy and comparability of results
obtained by different assays. However, since standardization to
IU and the calibration of assay sensitivity are based on genotype
1a (deLeuw 2011), relative quantification results may vary
among assays. Using the WHO international standard, a VL of 2
millions copies/mL (the cut-off value predictive for therapeutic
success in early clinical trials with IFN) was found to correspond
to 800 000 IU/mL. Currently a cut-off of 400 000-800 000 IU/ml
separate low from high VL. Further studies have found that
patients with low baseline HCV RNA levels have a 15-39% better
response rate, a finding that is consistent across trials using
different formulations and dosages of IFN (Strader 2004).
Real-time PCR tests
Real-time PCR tests are faster and more cost-effective methods
that detect very low VL (10-15 IU/ml) (Vermehren 2008). Realtime
PCR can accurately quantify HCV RNA levels over a linear
range exceeding 6 logs (10 IU/mL to 100 million IU/mL) (Table
2.2). Therefore, a single test result serves the purpose of both
quantitative and qualitative HCV NAT with similar sensitivity
(Ogawa 2010).
Table 2.2 – Currently available HCV RNA quantification methods*
Assay Manufacturer Method Conversion
Factor
(copies/mL)
Dynamic
Range
(IU/mL)
Cobas Taqman
HCV Test
Roche Molecular
Systems
Real time
PCR
3.4 43-69,000,000
Abbott Real-Time
PCR
Abbott Diagnostics Real time
PCR
3.4 12-100,000,000
LCx HCV RNA
Quantitative
Abbott Diagnostics RT-PCR 3.8 25-2,630,000
SuperQuant National Genetics
Institute
RT-PCR 3.4 30-1,470,000
Versant HCV RNA
3.0 Assay
Siemens Health
Care Diagnostics
bDNA 5.2 615-7,700,000
* According to data from Vermehren 2008 and Ghany 2009
Minimal residual viremia might be predictive of post-treatment
relapse (Matsuura 2009). Rules for treatment duration and early
discontinuation were mainly established with NAT assays with a
detection limit of 50 IU/ml. Lower detection limits (undetectable
VL defined as less than 15 IU/ml) did not significantly influence
the SVR rates after shortened treatment duration, for patients
with RVR (82% for G1 infected patients treated for 24 weeks and
95% for G2/3 infected patients treated for 16 weeks) (Sarrazin
2010).
The assay’s choice should be tailored to the dominant genotype
in the study population, as some assays have been reported to
substantially underestimate HCV RNA levels in certain
genotypes. The same assay should be used for all samples from a
single patient and, whenever possible, throughout the clinical
development program.
quantitative and qualitative HCV NAT with similar sensitivity
(Ogawa 2010).
Table 2.2 – Currently available HCV RNA quantification methods*
Assay Manufacturer Method Conversion
Factor
(copies/mL)
Dynamic
Range
(IU/mL)
Cobas Taqman
HCV Test
Roche Molecular
Systems
Real time
PCR
3.4 43-69,000,000
Abbott Real-Time
PCR
Abbott Diagnostics Real time
PCR
3.4 12-100,000,000
LCx HCV RNA
Quantitative
Abbott Diagnostics RT-PCR 3.8 25-2,630,000
SuperQuant National Genetics
Institute
RT-PCR 3.4 30-1,470,000
Versant HCV RNA
3.0 Assay
Siemens Health
Care Diagnostics
bDNA 5.2 615-7,700,000
* According to data from Vermehren 2008 and Ghany 2009
Minimal residual viremia might be predictive of post-treatment
relapse (Matsuura 2009). Rules for treatment duration and early
discontinuation were mainly established with NAT assays with a
detection limit of 50 IU/ml. Lower detection limits (undetectable
VL defined as less than 15 IU/ml) did not significantly influence
the SVR rates after shortened treatment duration, for patients
with RVR (82% for G1 infected patients treated for 24 weeks and
95% for G2/3 infected patients treated for 16 weeks) (Sarrazin
2010).
The assay’s choice should be tailored to the dominant genotype
in the study population, as some assays have been reported to
substantially underestimate HCV RNA levels in certain
genotypes. The same assay should be used for all samples from a
single patient and, whenever possible, throughout the clinical
development program.
HCV genotyping
HCV genotyping should be performed in all HCV-infected
persons prior to treatment initiation in order to plan for the
duration of therapy and to estimate the likelihood of response.
An assay based on viral population sequencing, reverse
hybridization or real-time PCR, which has been validated for
correct subtyping of at least subtypes 1a and 1b should be used.
Two commercial assays are frequently used for HCV genotypes:
– TruGene™ HCV Genotyping kit (Siemens Healthcare
Diagnostics Division, Tarrytown, NY), based on direct
sequence analysis of the 5’ UTR (untranslated region),
– Versant™ HCV Genotype Assay LiPA (version I; Siemens
Medical Solutions, Diagnostics Division, Fernwald,
Germany), based on reverse hybridization analysis with
genotype-specific oligonucleotide probes binding to the
5’ UTR. A second generation line probe assay (LiPA) contains
probes targeting both the 5’ UTR and the core regions of the
viral genome, improving the accuracy of discrimination
between subtypes 1a and 1b.
A new test which uses real-time PCR technology for HCV
genotyping has recently been developed by Abbott Molecular.
HCV resistance monitoring. Like HIV and HBV, HCV has a high
replication rate and replicates via an error-prone mechanism,
generating resistance variants. In the near future, HCV
resistance testing (Kieffer 2010) will most probably be part of the
clinical monitoring algorithms. Assays based on viral population
sequencing require a minimum VL of 1000 IU/mL and define the
most common mutation patterns, without detecting the lowfrequency
variants. Accurate determination of viral
genotype/subtype is critical for resistance testing during the
development of new direct-acting antivirals (DAAs) (Chevaliez
2009).
Pretreatment samples are analyzed to detect known or novel
predominant viral polymorphisms and to provide the
HCV genotyping should be performed in all HCV-infected
persons prior to treatment initiation in order to plan for the
duration of therapy and to estimate the likelihood of response.
An assay based on viral population sequencing, reverse
hybridization or real-time PCR, which has been validated for
correct subtyping of at least subtypes 1a and 1b should be used.
Two commercial assays are frequently used for HCV genotypes:
– TruGene™ HCV Genotyping kit (Siemens Healthcare
Diagnostics Division, Tarrytown, NY), based on direct
sequence analysis of the 5’ UTR (untranslated region),
– Versant™ HCV Genotype Assay LiPA (version I; Siemens
Medical Solutions, Diagnostics Division, Fernwald,
Germany), based on reverse hybridization analysis with
genotype-specific oligonucleotide probes binding to the
5’ UTR. A second generation line probe assay (LiPA) contains
probes targeting both the 5’ UTR and the core regions of the
viral genome, improving the accuracy of discrimination
between subtypes 1a and 1b.
A new test which uses real-time PCR technology for HCV
genotyping has recently been developed by Abbott Molecular.
HCV resistance monitoring. Like HIV and HBV, HCV has a high
replication rate and replicates via an error-prone mechanism,
generating resistance variants. In the near future, HCV
resistance testing (Kieffer 2010) will most probably be part of the
clinical monitoring algorithms. Assays based on viral population
sequencing require a minimum VL of 1000 IU/mL and define the
most common mutation patterns, without detecting the lowfrequency
variants. Accurate determination of viral
genotype/subtype is critical for resistance testing during the
development of new direct-acting antivirals (DAAs) (Chevaliez
2009).
Pretreatment samples are analyzed to detect known or novel
predominant viral polymorphisms and to provide the
comparator for mutations emerging at later time points, during
or after treatment. On-treatment viremic samples are analyzed
to determine specific changes associated with decreased
susceptibility and virologic failure. Post-treatment samples are
analyzed for persistence or loss of resistant variants and may
help distinguish between re-infection and relapse. More details
on the impact of resistance on HCV treatment are given in
chapter 4.
Assessment of hepatic fibrosis
CHC may progress to cirrhosis (in approximately 20% of patients,
with a mean duration of 20 years) and subsequently,
decompensation and complications, including HCC, develop in
about 30% of cases over a period of approximately 4 years
(DiBisceglie 2008). Histologically significant liver disease can be
also present in patients without symptoms and with normal ALT
levels. In theses cases, deferring treatment until liver function is
depressed (low albumin, altered PT) may decrease SVR rate and
increase the risk of AEs (Pradat 2002). Evaluation of liver fibrosis
is thus compulsory (Table 2.3).
Liver biopsy
Liver biopsy (LB) is the gold standard for (i) liver disease staging,
(ii) treatment decisions and (iii) prognostication, as it may reveal
advanced fibrosis or cirrhosis that necessitates surveillance for
HCC and/or screening for varices.
Before treatment LB is indicated for prognostic purposes and
guiding treatment decisions. If LB shows significant fibrosis
treatment should be initiated, otherwise, treatment can be
deferred (Afdhal 2009). Individualized treatment decisions are
based on the severity of liver disease. Treatment is indicated in
patients with compensated cirrhosis provided they do not have
contraindications to therapy.
or after treatment. On-treatment viremic samples are analyzed
to determine specific changes associated with decreased
susceptibility and virologic failure. Post-treatment samples are
analyzed for persistence or loss of resistant variants and may
help distinguish between re-infection and relapse. More details
on the impact of resistance on HCV treatment are given in
chapter 4.
Assessment of hepatic fibrosis
CHC may progress to cirrhosis (in approximately 20% of patients,
with a mean duration of 20 years) and subsequently,
decompensation and complications, including HCC, develop in
about 30% of cases over a period of approximately 4 years
(DiBisceglie 2008). Histologically significant liver disease can be
also present in patients without symptoms and with normal ALT
levels. In theses cases, deferring treatment until liver function is
depressed (low albumin, altered PT) may decrease SVR rate and
increase the risk of AEs (Pradat 2002). Evaluation of liver fibrosis
is thus compulsory (Table 2.3).
Liver biopsy
Liver biopsy (LB) is the gold standard for (i) liver disease staging,
(ii) treatment decisions and (iii) prognostication, as it may reveal
advanced fibrosis or cirrhosis that necessitates surveillance for
HCC and/or screening for varices.
Before treatment LB is indicated for prognostic purposes and
guiding treatment decisions. If LB shows significant fibrosis
treatment should be initiated, otherwise, treatment can be
deferred (Afdhal 2009). Individualized treatment decisions are
based on the severity of liver disease. Treatment is indicated in
patients with compensated cirrhosis provided they do not have
contraindications to therapy.
Post-treatment LB is essential to demonstrate regression of
cirrhosis after viral supression. It is not recommended for
assessment of the efficacy of therapeutic regimens, unless
hepatic safety issues impose it.
Table 2.3 – Liver fibrosis evaluation methods*
Methods Categories Classification Comments
1) Invasive Liver biopsy Percutaneous
Laparoscopic
Scoring systems are presented
in Table 2.4
Indirect markers Biomarker combinations or
composite indexes
Serum
biochemistry
Direct markers Reflect extra cellular matrix
removal/deposition, the
balance between hepatic
fibrogenesis and fibrolysis, or
cytokines (TGF-β 1† and PDGF†)
associated with fibrosis
Imaging
techniques
Elastography
Ultrasonography
CT, MRI, PET
Fibroscan® is the most used
technique
2) Non-invasive
Genetic
markers
Estimate the
transdifferentiation of hepatic
stellate cells to myofibroblasts
* According to data from Ahmad 2011.
† TGF-β1: transforming growth factors β1; PDGF: platelet derived growth factor
Different scoring systems (Table 2.4) have been defined in order
to classify the extent of necroinflammatory activity (grading)
and the extent of fibrosis (staging) in LB.
However, LB is invasive and has a number of drawbacks:
– substantial sampling error (extracts only 1/50,000 of the
liver)
– variability in interpretation
– potential serious adverse outcomes (bleeding)
– high cost (approximately $1000–$1500 per biopsy)
– low patient’s acceptability/reluctance to undergo repeated
biopsies
cirrhosis after viral supression. It is not recommended for
assessment of the efficacy of therapeutic regimens, unless
hepatic safety issues impose it.
Table 2.3 – Liver fibrosis evaluation methods*
Methods Categories Classification Comments
1) Invasive Liver biopsy Percutaneous
Laparoscopic
Scoring systems are presented
in Table 2.4
Indirect markers Biomarker combinations or
composite indexes
Serum
biochemistry
Direct markers Reflect extra cellular matrix
removal/deposition, the
balance between hepatic
fibrogenesis and fibrolysis, or
cytokines (TGF-β 1† and PDGF†)
associated with fibrosis
Imaging
techniques
Elastography
Ultrasonography
CT, MRI, PET
Fibroscan® is the most used
technique
2) Non-invasive
Genetic
markers
Estimate the
transdifferentiation of hepatic
stellate cells to myofibroblasts
* According to data from Ahmad 2011.
† TGF-β1: transforming growth factors β1; PDGF: platelet derived growth factor
Different scoring systems (Table 2.4) have been defined in order
to classify the extent of necroinflammatory activity (grading)
and the extent of fibrosis (staging) in LB.
However, LB is invasive and has a number of drawbacks:
– substantial sampling error (extracts only 1/50,000 of the
liver)
– variability in interpretation
– potential serious adverse outcomes (bleeding)
– high cost (approximately $1000–$1500 per biopsy)
– low patient’s acceptability/reluctance to undergo repeated
biopsies
Table 2.4 – Scoring systems for histological stage*
Stage IASL
(Desmet 1994 )
Batts-Ludwig
(Batts 1995)
Metavir
(Bedossa 1996)
Ishak
(Ishak 1995)
0 No fibrosis No fibrosis No fibrosis No fibrosis
1 Mild fibrosis Fibrous portal
expansion
Periportal
fibrotic
expansion
Fibrous expansion of
some portal areas
with or without short
fibrous septa
2 Moderate
fibrosis
Rare bridges or
septae
Periportal
septae
Fibrous expansion of
most portal areas
with or without short
fibrous septa
3 Severe fibrosis Numerous
bridges or
septae
Porto-central
septae
Fibrous expansion of
most portal areas
with occasional
portal to portal
bridging
4 Cirrhosis Cirrhosis Cirrhosis Fibrous expansion of
most portal areas
with marked
bridging
5 Marked bridging with
occasional nodules
6 Cirrhosis
* According to data from Ghany 2009.
Non invasive methods
Non-invasive assessment of liver fibrosis based on either
biochemical methods or imaging techniques have emerged over
the past ten years as an alternative to the systematic use of LB.
These methods are easy-to-do, reliable and can be repeated in
follow-up visits. However, these noninvasive tests are more
adequate for identifying patients with advanced
fibrosis/cirrhosis than in differentiating those with moderate
and mild fibrosis. According to the current recommendations,
these methods should not replace LB in routine clinical practice.
Transient elastography (FibroScan™) uses ultrasound and low
frequency elastic waves to measure liver elasticity/stiffness in
kilopascals (kPa).
Stage IASL
(Desmet 1994 )
Batts-Ludwig
(Batts 1995)
Metavir
(Bedossa 1996)
Ishak
(Ishak 1995)
0 No fibrosis No fibrosis No fibrosis No fibrosis
1 Mild fibrosis Fibrous portal
expansion
Periportal
fibrotic
expansion
Fibrous expansion of
some portal areas
with or without short
fibrous septa
2 Moderate
fibrosis
Rare bridges or
septae
Periportal
septae
Fibrous expansion of
most portal areas
with or without short
fibrous septa
3 Severe fibrosis Numerous
bridges or
septae
Porto-central
septae
Fibrous expansion of
most portal areas
with occasional
portal to portal
bridging
4 Cirrhosis Cirrhosis Cirrhosis Fibrous expansion of
most portal areas
with marked
bridging
5 Marked bridging with
occasional nodules
6 Cirrhosis
* According to data from Ghany 2009.
Non invasive methods
Non-invasive assessment of liver fibrosis based on either
biochemical methods or imaging techniques have emerged over
the past ten years as an alternative to the systematic use of LB.
These methods are easy-to-do, reliable and can be repeated in
follow-up visits. However, these noninvasive tests are more
adequate for identifying patients with advanced
fibrosis/cirrhosis than in differentiating those with moderate
and mild fibrosis. According to the current recommendations,
these methods should not replace LB in routine clinical practice.
Transient elastography (FibroScan™) uses ultrasound and low
frequency elastic waves to measure liver elasticity/stiffness in
kilopascals (kPa).
With a cutoff value of about 7-8 kPa, it can identify about 70% of
patients with histological signs of moderate to severe fibrosis.
With a cutoff of 14-15 kPa, it can identify about 85% of patients
with histological signs of cirrhosis.
Transient elastography is less reliable in ruling out moderate
fibrosis. The results are less certain in patients with a thick chest
wall, hepatic congestion of cardiac origin and acute
exacerbations of hepatitis. However, it has improved the ability
to define the extent of fibrosis without a LB, particularly when
combined with other noninvasive markers.
Biochemical scores are calculated based on panels of multiple
serum markers associated with hepatic fibrosis. Performance of
these measures appears similar in both HCV monoinfected and
HIV-HCV co-infected patients (Shaheen 2008). Several simple
tests are presented in Table 2.5.
Two tests have been specifically designed for HIV-HCV coinfection:
SHASTA index (includes hyaluronic acid, AST and
albumin) (Kelleher 2005) and FIB-4 (ALT and AST level, platelet
count and age) (Sterling 2006).
Table 2.5 – Simple biochemical scores
Test Markers Interpretation
AAR
(Williams 1998)
AST to ALT *ratio AST/ALT ≥ 1: significant
cirrhosis
APRI
(Wai 2003)
AST-platelet ratio APRI < 0.5: no/minimal fibrosis
APRI > 1.5: significant fibrosis
Fibrosis Index (FI)
(Ohta 2006)
Platlet count and serum
albumin
FI < 2.1: no/ minimal fibrosis
FI ≥ 2.1: significant fibrosis
FI ≥ 3.3: cirrhosis
* AST: aspartate aminotransferase; ALT: alanine aminotransferase
Several composite tests based on mathematical algorithms have
been introduced in practice (Table 2.6).
patients with histological signs of moderate to severe fibrosis.
With a cutoff of 14-15 kPa, it can identify about 85% of patients
with histological signs of cirrhosis.
Transient elastography is less reliable in ruling out moderate
fibrosis. The results are less certain in patients with a thick chest
wall, hepatic congestion of cardiac origin and acute
exacerbations of hepatitis. However, it has improved the ability
to define the extent of fibrosis without a LB, particularly when
combined with other noninvasive markers.
Biochemical scores are calculated based on panels of multiple
serum markers associated with hepatic fibrosis. Performance of
these measures appears similar in both HCV monoinfected and
HIV-HCV co-infected patients (Shaheen 2008). Several simple
tests are presented in Table 2.5.
Two tests have been specifically designed for HIV-HCV coinfection:
SHASTA index (includes hyaluronic acid, AST and
albumin) (Kelleher 2005) and FIB-4 (ALT and AST level, platelet
count and age) (Sterling 2006).
Table 2.5 – Simple biochemical scores
Test Markers Interpretation
AAR
(Williams 1998)
AST to ALT *ratio AST/ALT ≥ 1: significant
cirrhosis
APRI
(Wai 2003)
AST-platelet ratio APRI < 0.5: no/minimal fibrosis
APRI > 1.5: significant fibrosis
Fibrosis Index (FI)
(Ohta 2006)
Platlet count and serum
albumin
FI < 2.1: no/ minimal fibrosis
FI ≥ 2.1: significant fibrosis
FI ≥ 3.3: cirrhosis
* AST: aspartate aminotransferase; ALT: alanine aminotransferase
Several composite tests based on mathematical algorithms have
been introduced in practice (Table 2.6).
Table 2.6 – Composite biochemical scores
Test Markers Interpretation
FibroTest™
(Imbert-Bismut 2001)
alpha-2-macroglobulin,
apolipoprotein A1,
haptoglobin, GGT,
bilirubin
Result is provided as a score of 0
to 1, proportional to the severity
of the fibrosis, with conversion
to the METAVIR system (from F0
to F4).
HepaScore™
(Adams 2005)
alpha 2 -macroglobulin
GGT*, bilirubin,
hyaluronic acid, age,
gender
A HepaScore <0.55 is considered
“negative” and indicates a
METAVIR score of F0 or F1.
A HepaScore ≥0.55 is considered
“positive” and indicates a
METAVIR score of F2 to F4.
FibroMeter™
(Cales 2005)
alpha2–macroglobulin,
hyaluronic acid,
platelets, prothrombin
index, AST, urea, age,
gender
FibroMeter™ has two main
diagnostic targets (fibrosis stage
and area of fibrosis), being
adapted for special explicit
causes†.
* GGT: γ-glutamyltranspeptidase
† chronic viral hepatitis B or C, alcoholic liver disease and non-alcoholic fatty
liver disease
FibroTest™ (Biopredictive, Paris, France) identifies about 70% of
patients with histological signs of moderate to severe fibrosis
and about 90% of patients with histological signs of cirrhosis,
using the manufacturers’ recommended cutoff values. The
FibroTest™ together with FibroScan™ have excellent utility for
the identification of HCV-related cirrhosis, but lesser accuracy
for earlier stages (Shaheen 2007).
All these tests are based on routine biochemistry blood assays
and can be influenced by intercurrent conditions. At the same
time, these scores may fluctuate or revert to lower classes after
initial worsening and a dynamic overview is more valuable than
a single determination.
In Europe, the typical approach is to perform a blood test such as
one of the commercially available assays, followed by transient
elastography. If both tests have concordant results on the
disease stage, no biopsy is needed; if there is discordance, biopsy
is performed. New fibrosis indexes combining the biochemical
Test Markers Interpretation
FibroTest™
(Imbert-Bismut 2001)
alpha-2-macroglobulin,
apolipoprotein A1,
haptoglobin, GGT,
bilirubin
Result is provided as a score of 0
to 1, proportional to the severity
of the fibrosis, with conversion
to the METAVIR system (from F0
to F4).
HepaScore™
(Adams 2005)
alpha 2 -macroglobulin
GGT*, bilirubin,
hyaluronic acid, age,
gender
A HepaScore <0.55 is considered
“negative” and indicates a
METAVIR score of F0 or F1.
A HepaScore ≥0.55 is considered
“positive” and indicates a
METAVIR score of F2 to F4.
FibroMeter™
(Cales 2005)
alpha2–macroglobulin,
hyaluronic acid,
platelets, prothrombin
index, AST, urea, age,
gender
FibroMeter™ has two main
diagnostic targets (fibrosis stage
and area of fibrosis), being
adapted for special explicit
causes†.
* GGT: γ-glutamyltranspeptidase
† chronic viral hepatitis B or C, alcoholic liver disease and non-alcoholic fatty
liver disease
FibroTest™ (Biopredictive, Paris, France) identifies about 70% of
patients with histological signs of moderate to severe fibrosis
and about 90% of patients with histological signs of cirrhosis,
using the manufacturers’ recommended cutoff values. The
FibroTest™ together with FibroScan™ have excellent utility for
the identification of HCV-related cirrhosis, but lesser accuracy
for earlier stages (Shaheen 2007).
All these tests are based on routine biochemistry blood assays
and can be influenced by intercurrent conditions. At the same
time, these scores may fluctuate or revert to lower classes after
initial worsening and a dynamic overview is more valuable than
a single determination.
In Europe, the typical approach is to perform a blood test such as
one of the commercially available assays, followed by transient
elastography. If both tests have concordant results on the
disease stage, no biopsy is needed; if there is discordance, biopsy
is performed. New fibrosis indexes combining the biochemical
scores and Fibroscan™ are being developed in order to provide a
more accurate fibrosis stage classification (Boursier 2011).
Correlation between biochemical, histological and
virological markers and HCV treatment
Patients should have serum transaminases (ALT and AST) levels
monitored at one month, and then every 3 months, following
initiation of therapy. Mild to moderate fluctuations in liver
enzyme levels are common in persons with chronic HCV
infection, and in the absence of signs and/or symptoms of liver
disease they do not require interruption of antiviral therapy.
Significant elevation in liver enzymes levels – more than 5 times
the upper limit of normal – should prompt careful evaluation for
liver insufficiency and for alternative causes of liver injury.
Eventually, withdrawal of antiviral treatment may be required.
A high baseline VL correlates with higher fibrosis and necrosisinflammation
scores (Mallet 2008). In patients with histologically
proven cirrhosis without esophageal varices, successful
treatment, as defined by a SVR, is associated with a reduction in
decompensation, occurrence of HCC and mortality (Bruno 2007).
The Child-Pugh (CP) classification of patients with HCV-induced
cirrhosis is used in predicting the likelihood of SVR rate after
antiviral therapy (AISF 2009):
– Patients with “histologically proven” cirrhosis without
esophageal varices (Child class A5 to 6), identified by stages
5 and 6 of Ishak’s score and stage 4 of the Metavir and
Knodell scores. Presumed SVR rate is 25% in HCV G1 and
75% in non-G1 infected patients.
– Patients with “compensated” cirrhosis with or without
esophageal varices (including Child class B7). Recognized
SVR rate is <15% in HCV G1 and <60% in non-G1 infected
patients.
– Patients with “decompensated” cirrhosis (Child class B8 or
more) defined by any evidence of previous decompensation
(ascites, esophageal bleeding, portal encephalopathy,
more accurate fibrosis stage classification (Boursier 2011).
Correlation between biochemical, histological and
virological markers and HCV treatment
Patients should have serum transaminases (ALT and AST) levels
monitored at one month, and then every 3 months, following
initiation of therapy. Mild to moderate fluctuations in liver
enzyme levels are common in persons with chronic HCV
infection, and in the absence of signs and/or symptoms of liver
disease they do not require interruption of antiviral therapy.
Significant elevation in liver enzymes levels – more than 5 times
the upper limit of normal – should prompt careful evaluation for
liver insufficiency and for alternative causes of liver injury.
Eventually, withdrawal of antiviral treatment may be required.
A high baseline VL correlates with higher fibrosis and necrosisinflammation
scores (Mallet 2008). In patients with histologically
proven cirrhosis without esophageal varices, successful
treatment, as defined by a SVR, is associated with a reduction in
decompensation, occurrence of HCC and mortality (Bruno 2007).
The Child-Pugh (CP) classification of patients with HCV-induced
cirrhosis is used in predicting the likelihood of SVR rate after
antiviral therapy (AISF 2009):
– Patients with “histologically proven” cirrhosis without
esophageal varices (Child class A5 to 6), identified by stages
5 and 6 of Ishak’s score and stage 4 of the Metavir and
Knodell scores. Presumed SVR rate is 25% in HCV G1 and
75% in non-G1 infected patients.
– Patients with “compensated” cirrhosis with or without
esophageal varices (including Child class B7). Recognized
SVR rate is <15% in HCV G1 and <60% in non-G1 infected
patients.
– Patients with “decompensated” cirrhosis (Child class B8 or
more) defined by any evidence of previous decompensation
(ascites, esophageal bleeding, portal encephalopathy,
jaundice). Assumed SVR rate is <7% in HCV G1 and <40% in
non-G1 infected patients.
The progression of fibrosis and other HCV-associated
histopathologic changes may also be related to coagulationcascade
activity and hepatic accumulation of iron, which have
been associated with mutations in factor V and
hemochromatosis genes, respectively.
The HIV-HCV coinfection is a particularly challenging situation.
The severity of liver disease must be routinely assessed in these
patients in order to initiate treatment before progression of liver
disease. An important number of coinfected patients are referred
to hepatology clinics only when they have hepatic
decompensation, at which time the HCV treatment options are
limited.
Drug-induced liver injuries (DILI) following antiretroviral
therapy pose significant problems in HIV/HCV co-infection,
especially in persons with advanced liver disease and cirrhosis.
Dose modifications or even avoidance of liver-metabolized
antiretroviral drugs may be required in patients with CP class B
and C disease. Overall, in the absence of clinically significant
fibrosis, it seems worthwhile to defer treatment. However, it is
equally important to apply the results of the clinical studies on a
case by case basis, weighing the treatment response rate and the
long-term outcomes.
Outlook
Nucleic acid testing, genotyping and assessment of the level of
hepatic fibrosis are invaluable tools in the diagnosis of HCV
infection, treatment guidance and monitoring.
Although LB is still considered the gold standard for the
progression of hepatic fibrosis in chronic hepatitis C, a series of
non-invasive radiological and serum-based markers are being
investigated for their diagnostic accuracy. New real-time PCR
tests are faster and more cost-effective methods for the
non-G1 infected patients.
The progression of fibrosis and other HCV-associated
histopathologic changes may also be related to coagulationcascade
activity and hepatic accumulation of iron, which have
been associated with mutations in factor V and
hemochromatosis genes, respectively.
The HIV-HCV coinfection is a particularly challenging situation.
The severity of liver disease must be routinely assessed in these
patients in order to initiate treatment before progression of liver
disease. An important number of coinfected patients are referred
to hepatology clinics only when they have hepatic
decompensation, at which time the HCV treatment options are
limited.
Drug-induced liver injuries (DILI) following antiretroviral
therapy pose significant problems in HIV/HCV co-infection,
especially in persons with advanced liver disease and cirrhosis.
Dose modifications or even avoidance of liver-metabolized
antiretroviral drugs may be required in patients with CP class B
and C disease. Overall, in the absence of clinically significant
fibrosis, it seems worthwhile to defer treatment. However, it is
equally important to apply the results of the clinical studies on a
case by case basis, weighing the treatment response rate and the
long-term outcomes.
Outlook
Nucleic acid testing, genotyping and assessment of the level of
hepatic fibrosis are invaluable tools in the diagnosis of HCV
infection, treatment guidance and monitoring.
Although LB is still considered the gold standard for the
progression of hepatic fibrosis in chronic hepatitis C, a series of
non-invasive radiological and serum-based markers are being
investigated for their diagnostic accuracy. New real-time PCR
tests are faster and more cost-effective methods for the
assessment viral kinetics. Virological end points are surrogate
references for assessing the efficiency of HCV treatments, but
many randomized trials on similar drug classes have established
their value in correctly evaluating the clinical outcome.
However, biochemical and histological improvements can be
attained even in patients who fail to eradicate HCV infection.
Obtaining data on the long-term clinical outcomes in patients
included in previous treatment trials is logistically difficult, due
to relatively high dropout rates and to interferences of retreatment
regimens. Cumulative meta-analysis may be relevant
for the planning of future clinical trials.
Links
– Centers for Disease Control and Prevention. Guidelines
for laboratory testing and result reporting of antibody to
hepatitis C virus. MMWR Recomm Rep 2003;52(RR-3):1-13.
http://www.ncbi.nlm.nih.gov/pubmed/12585742
– World Health Organization. Hepatitis C 2002.
http://www.who.int/csr/disease/hepatitis/Hepc.pdf
– Short guide to Hepatitis C. By Mauss, Berg, Rockstroh,
Sarrazin, Wedemeyer, et al. Flying Publisher 2011, 128 pages,
www.goo.gl/7aTq4
references for assessing the efficiency of HCV treatments, but
many randomized trials on similar drug classes have established
their value in correctly evaluating the clinical outcome.
However, biochemical and histological improvements can be
attained even in patients who fail to eradicate HCV infection.
Obtaining data on the long-term clinical outcomes in patients
included in previous treatment trials is logistically difficult, due
to relatively high dropout rates and to interferences of retreatment
regimens. Cumulative meta-analysis may be relevant
for the planning of future clinical trials.
Links
– Centers for Disease Control and Prevention. Guidelines
for laboratory testing and result reporting of antibody to
hepatitis C virus. MMWR Recomm Rep 2003;52(RR-3):1-13.
http://www.ncbi.nlm.nih.gov/pubmed/12585742
– World Health Organization. Hepatitis C 2002.
http://www.who.int/csr/disease/hepatitis/Hepc.pdf
– Short guide to Hepatitis C. By Mauss, Berg, Rockstroh,
Sarrazin, Wedemeyer, et al. Flying Publisher 2011, 128 pages,
www.goo.gl/7aTq4
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