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Chiron Diagnostics, 4560 Horton St., Emeryville, CA 94608-2916.
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-interferon (IFN) has been
approved by the US Food and Drug Administration (FDA); however, fewer
than half of HCV-infected individuals respond to treatment, and relapse
is common. No vaccine to prevent HCV infection is currently available.
Because HCV infection can have such serious consequences, because its
nonparenteral routes of transmission are poorly understood, and because
treatment is rarely efficacious, tests that can identify and monitor
HCV-infected patients are crucial for addressing this potentially
life-threatening viral disease.
The breakthrough for developing diagnostic tests for non-A, non-B
hepatitis came with the cloning of the HCV genome by Houghton and
colleagues (3). This work led to an antigen, with which
the first diagnostic tests were developed, and to elucidation of the
viral nucleic acid sequence from which numerous antigens, nucleic acid
probes, and phylogenetic relationships have been derived. It is now
known that HCV is an enveloped virus most closely related to the
pestiviruses and flaviviruses, and contains a single-stranded,
positive-sense RNA genome of 9.4 kb (Fig. 1
). Its one large open-reading frame encodes a polyprotein 3011
amino acids long, with structural genes at the 5' end and nonstructural
genes at the 3' end of the genome. As with many RNA viruses, HCV is
highly diverged—at least six major genotypes and multiple subtypes
have been described (reviewed in ref. 4). The distribution
of HCV genotypes varies worldwide. Genotypes 1–3 are the most
prevalent in the US; however, all six major genotypes have been noted
here. Although the 5'-untranslated region is the most highly conserved
among HCV genotypes, significant sequence variation recently has been
identified even in this region of the HCV genome (5). The
genetic heterogeneity of HCV has important implications for the
clinical utility of diagnostic tests.
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Today, several tests are used in the diagnosis and monitoring of patients infected with HCV. Serological tests such as enzyme-linked immunoassays (EIAs) are used primarily to screen blood donations and to diagnose HCV infection in symptomatic patients. RIBATM strip immunoblot assays (Chiron Diagnostics, Emeryville, CA), which are more specific and sensitive than EIAs, are particularly useful for confirmation of HCV infection. Tests for HCV RNA, including PCR-based assays and the branched-DNA (bDNA) assay (QuantiplexTM HCV RNA; Chiron Diagnostics), are used for therapeutic monitoring and prognostics. The development of these tests and their use in clinical practice are described in the following sections.
serological tests
The first set of clones obtained from the HCV genome was put to
use in developing a serological assay for HCV infection. A
first-generation EIA, in which a c100–3-human superoxide dismutase
recombinant protein was the source of antigen, became commercially
available in 1990. This assay was a remarkably effective means to
reduce the risk of HCV transmission through blood donations, preventing
an estimated 40 000 new HCV infections per year. The development of
second-generation EIAs that utilize multiple antigens and exhibit
greater sensitivity has led to the prevention of an additional
10 000–15 000 cases of HCV per year (6). Although the
newest generation of EIAs for HCV infection have greatly increased
sensitivities and specificities, these assays still have a relatively
high false-positive rate among low-risk populations. Consequently,
confirmatory serological tests often are used.
One confirmatory serological test is the RIBA HCV 2.0 strip immunoblot
assay (RIBA-2 SIA), used to identify immunoreactivity against specific
HCV antigens. With the four-antigen RIBA-2 SIA, detection of
immunoreactivity against two or more HCV antigens is considered
positive; failure to detect immunoreactivity is considered negative.
Immunoreactivity against only one HCV antigen is classified as an
indeterminate result, necessitating additional confirmatory tests.
Recently, a third-generation RIBA HCV 3.0 SIA was developed (RIBA-3
SIA), and is now in use in Europe (Fig. 2
). RIBA-3 SIA uses synthetic peptides from the c100 and C22
regions and recombinant proteins from the C33 and NS5 regions of the
HCV genome as sources of antigen. This third-generation test has led to
a substantial reduction in the number of patients' specimens
classified as indeterminate. In a recent study by Damen et al.
(7), 6.7% of HCV RNA-positive patients' specimens were
classified as indeterminate by RIBA-2 SIA, whereas only 0.5% were
classified as indeterminate by RIBA-3 SIA. Currently under development
at Chiron Diagnostics is a serotyping assay with the RIBA SIA format,
which is designed to detect type-specific epitopes of HCV
(8). This assay utilizes five synthetic peptides from the
NS4 region and three synthetic peptides from the core region of the HCV
genome, and can distinguish between HCV genotypes 1, 2, and 3.
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Despite the tremendous advances made in the technology for RIBA SIA, a substantial number of patients' specimens are classified as indeterminate. As proposed in an algorithm for diagnosis of HCV infection that incorporates sequential EIA and RIBA SIA testing (9), further clinical evaluation and detection of HCV RNA are recommended for resolving indeterminate RIBA SIA results.
assays for hcv viremia
Assays to detect and quantify HCV RNA increasingly are being used
to follow disease progression and to monitor therapeutic response in
HCV-infected individuals. Unlike serological tests, which yield insight
into the patient's immune response to HCV infection, HCV RNA assays
provide a direct measure of viral load. As eloquently stated by Nowak
and Bangham (10), "The abundance of virus—that is, the
virus load—is an important determinant of the outcome of
infection with many viruses: for instance... [in] HIV-1 and other
lentivirus infections, virus load is correlated with pathogenicity,
disease stage and progression of disease; in HTLV-1... ; in
HBV... ; in CMV infection... ; and in Lassa fever, mortality is
correlated with the level of viremia." Hence, viral load measurement
for chronic hepatitis C, like other viral diseases, provides unique
insight into the dynamics and outcome of HCV infection.
Two widely divergent strategies are used to detect and quantify HCV RNA
in clinical specimens (Fig. 3
). The first strategy, based on PCR techniques, is performed by
isolating the RNA from a specimen, reverse-transcribing it to generate
cDNAs, amplifying specific nucleic acid sequences by PCR, and then
using a variety of methods to detect the amplified sequences. Although
PCR-based assays are used to detect low quantities of virus (1000
equivalents per milliliter, or 1 MEq/L, 1 MEq being defined as the
amount of HCV RNA that generates light emission equivalent to that
generated by 106 copies of HCV RNA reference
standard), problems inherent to PCR itself lead to false-positive and
false-negative results (11) and to differences in PCR
results reported by different laboratories (12). The
second strategy for detecting and quantifying HCV RNA is based on bDNA
technology (Fig. 3
). Fundamentally different from target amplification
methods such as PCR, the bDNA assay directly measures nucleic acid
molecules through linear signal amplification. Using synthetic
oligonucleotide probes and bDNA molecules, the bDNA assay works by
anchoring the HCV RNA molecules to the surface of a microtiter well and
then boosting the signal through a series of hybridization steps. After
introduction of a chemiluminescent dioxetane substrate, which is
activated by alkaline phosphatase, the signal is quantified by photon
counting in a luminometer. Because as many as 18 bDNA molecules are
bound to each HCV RNA, as many as 810 separate alkaline phosphatase
molecules per HCV RNA can be hybridized, thus providing tremendous
enhancement of the signal.
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Recently, new oligonucleotide probes were designed to extend the sensitivity of the bDNA assay and to ensure that each of the major HCV genotypes was quantified equally (13). This improved bDNA assay, the Quantiplex HCV RNA 2.0, exhibits a specificity of 97% and sensitivity of 96% (95% confidence intervals, 91–98%). This assay is highly reproducible (CVs 21–24%) and has a linear dynamic quantification range exceeding 4 logs—from 0.2 to 120 MEq/mL. Moreover, the ability of the Quantiplex HCV RNA 2.0 to quantify equally the different HCV genotypes in clinical specimens has been documented (13). The sensitivity of the bDNA assay for HCV RNA quantification will be extended by the ongoing improvements in bDNA technology, which include the addition of preamplifier molecules (14) and the incorporation of novel nucleotides, isocytosine and isoguanosine, into oligonucleotide probes (Collins et al., submitted). When incorporated into bDNA assays for HIV RNA quantification, these advances have extended the detection limit of the bDNA assay to as low as 50 molecules/mL.
hcv rna assays as monitors of therapeutic response
With a reliable assay for HCV RNA quantification in hand, we can
now monitor changes in viral load in patients undergoing IFN
therapy. The goal of IFN treatment for HCV infection is to eliminate
or reduce the viral load of HCV, resulting in an improvement in hepatic
function, a cessation or slowing of progression to liver disease, and a
decrease in the infectivity of the patient. By evaluating a patient's
HCV RNA concentrations before therapy, as well as during and
posttherapy, a clinician may be able to determine if the patient is
responding to therapy and, if so, whether that response is likely to be
sustained.
Examples of viral load changes in patients treated with IFN are
shown in Fig. 4
(15). Fig. 4A
depicts the parallel changes in HCV
RNA concentrations and serum alanine aminotransferase (ALT)
concentrations in a patient who responded to IFN therapy. In this
patient, both HCV RNA and serum ALT decreased on initiation of therapy
and remained low for at least 6 months after cessation of therapy. The
profile of a nonresponding patient (Fig. 4B
) shows that both HCV RNA
and serum ALT decreased on initiation of therapy but then increased
somewhat erratically during and after cessation of therapy. Notice in
this patient that the HCV RNA values never fell below the
quantification limit of the bDNA assay, and that increases in HCV RNA
concentrations consistently preceded increases in serum ALT.
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These two examples of responding and nonresponding patients are fairly
straightforward—both patients showed changes in serum ALT and HCV RNA
that were roughly parallel. However, this is not the case for other
HCV-infected patients treated with IFN. Changes in serum ALT and HCV
RNA concentrations for three patients undergoing IFN therapy are
illustrated in Fig. 5
. These patients were all classified as sustained responders on
the basis of normalization of their ALT values. However, examination of
the HCV RNA load of these patients made it apparent that none of these
patients had a virological response, even during therapy.
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These examples illustrate the clinical importance of HCV RNA measurement. Even though serum ALT is a routine and relatively inexpensive test, it is a nonspecific measure of HCV disease. By contrast, HCV RNA quantification provides a direct measure of the virus and can provide valuable insight into viral dynamics during therapeutic monitoring.
hcv rna assays as predictors of therapeutic response
Currently, IFN is the only FDA-approved treatment for HCV
infection. Unfortunately, IFN treatment is expensive and plagued by
unpleasant side effects. Moreover, sustained response to IFN
treatment occurs in <20% of HCV-infected patients. It therefore would
be useful to clinicians and patients to be able to predict the
likelihood of a patient's response to IFN treatment. Several
studies have shown that HCV RNA concentrations are predictive of
response to IFN therapy (see, e.g., refs. 16–18). The
continuum of response to interferon according to HCV RNA
concentrations, shown in Fig. 6
, was constructed from results of several different studies
(19). In Fig. 6
, each point includes all of the points
below it. For example, there is a 38% chance of sustained response for
all of the patients who have an HCV RNA load 
1 MEq/mL. Once these
studies have been confirmed, this kind of information could be used to
set expectations for patients undergoing IFN therapy.
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Knowledge of the HCV RNA viral load also may be useful in timing IFN
therapy. In chronically infected patients, HCV RNA concentrations are
typically quite stable. However, HCV RNA quantities do fluctuate in
some patients, and may be particularly unstable in patients after
IFN therapy (20). In retreatment studies, as in studies
of IFN treatment in therapy-naive patients, those patients with the
lowest pretreatment HCV RNA concentrations appeared to be the most
likely to respond to IFN therapy (21)(22).
Thus, it may be possible to monitor HCV RNA load in those patients with
a poor response to an initial course of IFN therapy, then recommence
IFN therapy at a time when HCV RNA concentrations are low, when the
patient has the best chance of responding to the treatment.
In conclusion, serological assays for HCV infection play a critical role in preventing transmission of HCV through the blood supply, in diagnosing HCV infection, and in confirming an individual's infection status. With a quantitative assay for HCV RNA, clinicians may follow viral load throughout the course of disease, select and adjust treatment protocols, and evaluate the efficacy of therapeutic regimes. In combination with serotyping assays, measurement of HCV RNA concentrations may be used to predict therapeutic outcomes, so that the patient and physician can make more informed decisions about treatment. Finally, these serological and virological assays provide the basis for studies on the epidemiology, natural history, progression, and treatment of HCV infection.
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Footnotes |
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1 Nonstandard abbreviations: HCV, hepatitis C virus; IFN, -interferon; EIA, enzyme-linked immunoassay; bDNA, branched DNA; MEq, megaequivalent; and ALT, alanine aminotransferase.
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References |
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therapy is associated with more favorable long-term response in patients with chronic hepatitis C. J Med Virol 1995;46:109-115.
[Web of Science][Medline]
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