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Address reprint requests and correspondence to Nizar N. Zein, MD, Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First St SW, Rochester, MN 55905
Serologic assays for diagnosis of hepatitis C infection may yield indeterminate results despite improvements in sensitivity and specificity through second- and third-generation assays. Direct detection of hepatitis C virus (HCV) RNA based on qualitative reverse transcription-polymerase chain reaction or transcription-mediated amplification allows diagnosis in the early stages of acute infection and in patients unable to mount an antibody response. Quantitative HCV RNA assays are useful for selecting appropriate antiviral therapies, but until recently they have lacked comparability between tests. More sensitive qualitative assays should be used for determining duration of treatment or recognizing a sustained virologic response to therapy. Hepatitis C virus genotyping can be performed from a limited sequence analysis of the viral genome by using various techniques. Although newer genotyping methods are relatively practicable and are satisfactory for the discrimination of the majority of genotypes, discrimination between subtypes can be challenging. Serologic typing of HCV lacks sensitivity and specificity compared with molecular-based techniques. Recent advances in serologic assays and nucleic acid detection techniques allow physicians to make accurate diagnoses, and these assays serve as important tools in treatment planning.
Hepatitis C virus (HCV) infection has reached epidemic proportions and become a major global health issue. Worldwide, the prevalence of chronic HCV infection is estimated to be 3%, varying from 0.1% to 5.0% depending on the geographic region studied.
In the United States alone, an estimated 30,000 persons are newly infected each year, and as many as 8000 to 10,000 deaths are attributed to HCV annually.
The actual incidence of new HCV infections is now declining as a result of routine screening of blood products, implementation of universal precautions, and increased awareness of the disease and its primary modes of transmission. However, owing to the slowly progressive nature of this disease and the large reservoir of previously infected individuals, the number of patients presenting with complications due to HCV infection and end-stage liver disease is expected to increase for the next 10 to 20 years. Hepatitis C virus has been strongly associated with the development of end-stage liver disease and hepatocellular carcinoma. In fact, HCV-associated end-stage liver disease is currently the most frequent indication for liver transplantation among adults.
Our current understanding of HCV pathogenicity has evolved over a relatively short time and has been accompanied by a rapid change in the diagnostic tests and treatments available to physicians involved in the care of HCV-infected patients. Therefore, the goals of this discussion are to review the currently available laboratory tests, to review the clinical implications of results obtained with these tests, and to present an algorithm summarizing our current recommendations for test utilization.
GENOMIC ORGANIZATION OF HCV
The basic organization of the HCV genome is illustrated in Figure 1. The original isolate (HCV-1) was a positive-sense RNA of 9401 nucleotides, containing a poly-A tail at the 3' end.
The sequence also contained a 5' untranslated region (UTR) of 341 nucleotides, a long open reading frame encoding for a polyprotein of 3011 amino acids, and a 3' UTR of approximately 27 nucleotides. This RNA structure is similar to that of the Flaviviridae, a family of arthropod-borne viruses. Consistent with the known functions of most flavivirus proteins, the 3 N-terminal HCV proteins likely represent structural proteins (C, E1, E2/NS1), while the 4 C-terminal proteins (NS2, NS3, NS4, NS5) appear to be functional and essential for viral replication.
Figure 1Genomic organization of hepatitis C virus (HCV). 5–1–1, c100, c33, c22, and NS5 are the capture antigens used for serologic diagnosis of HCV infection with the first-, second-, and third-generation assays. C = core; E = envelope; NS = nonstructural; UTR = untranslated region.
Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5’ untranslated region and poly(a) tails at the 3’ end.
found among all isolated strains of the virus. Comparison of published sequences of HCV has led to the identification of distinct genotypes that may differ from each other by as much as 33% over the entire viral genome.
Multiple classification systems evolved within a short time, and interpretation of the literature became more complex because of the various methods of classification (Table 1). A uniform classification system was agreed on in August 1994 at the Second International Conference of HCV and Related Viruses.
With this system, HCV was classified into 6 major genotypes comprising 11 subtypes. New genotypes (designated by an Arabic numeral) and subtypes (designated by a lowercase letter) can easily be added to the growing list in the order of their identification, thus minimizing the confusion that arose with the other methods of classification.
Table 1Classification Systems for Hepatitis C Virus Genotypes
Furthermore, the outcome of liver disease and the rates of response to interferon therapy alone or in combination with ribavirin may vary according to HCV genotype.
Previous work has also demonstrated that genetic variation associated with the various HCV genotypes can influence the results of serologic and virologic assays used for HCV detection and quantification.
DIAGNOSIS OF HCV
Serologic Assays
Cloning of the HCV genome led to the expression of the recombinant antigen c100–3 in yeast, which was eventually used to develop the first screening assay.
Since then, serologic assays have been improved, and newer generations have become available for the routine diagnosis of HCV infection.
First-Generation Assays.-The first-generation HCV antibody test approved by the US Food and Drug Administration (FDA) became commercially available in 1990. This was an enzyme immunoassay (EIA) that used a recombinant form of the c100–3 epitope derived from the nonstructural NS4 region of the virus (Figure 1). The first-generation EIA (EIA-1) was reactive in 80% to 90% of serum specimens obtained from patients with chronic posttransfusion non-A, non-B hepatitis but not from patients with acute or early infection with HCV.
Furthermore, screening in a low-risk population resulted in a high rate of false-positive results. These results led to introduction of a strip immunoassay (SIA) such as the recombinant immunoblot assay (Chiron Corporation, Emeryville, Calif). The SIA demonstrated a higher specificity and could be used as a confirmatory test in patients exhibiting positive EIA-1 results but could be adversely affected by the serum levels of circulating globulins, particularly in patients with autoimmune chronic active hepatitis.
As more reactive recombinant antigens were identified from more highly conserved regions of the HCV genome, second- and third-generation serologic assays were introduced and because of their improved sensitivity and specificity quickly replaced the first-generation assays. A review of first-generation assays is now of historical rather than practical importance.
Second-Generation Assays.-These assays include the second-generation EIA (EIA-2) and the second-generation SIA (SIA-2) approved by the FDA in 1992 and 1993, respectively. In addition to the 5–1–1 and c100–3 antigens, these assays incorporated the c22–3 antigen derived from the core region of the HCV genome and the c33c antigen derived from the nonstructural region NS3 (Figure 1).
Although the EIA-2 is a highly sensitive screening assay for the detection of HCV antibodies in sera and has almost eliminated the occurrence of posttransfusion hepatitis, caution must be used interpreting negative results obtained from certain patient groups, including those with a possibility of an acute infection, because seroconversion may require as long as 12 weeks after exposure to develop.
The SIA-2 is used primarily today to validate second-generation EIA results in low-risk populations such as organ, tissue, and blood donors. Current criteria for a positive SIA-2 require reactivity to at least 2 HCV antigens encoded by different parts of the HCV genome. A common problem is development of an “indeterminate” result defined as reactivity to a single antigen band or reactivity to 2 bands derived from the same coding region (ie, 5–1–1 and c100–3). A notable proportion of samples (5%-10%) repeatedly classed as reactive by the EIA-2 assay yield indeterminate results when evaluated by the SIA-2. Furthermore, numerous patients with indeterminate SIA-2 results do harbor detectable levels of HCV RNA as determined by reverse transcription-polymerase chain reaction (RT-PCR), thus confirming the presence of HCV viremia.
Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory.
Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22–3 reactivity and influence of HCV genotypes.
Of 720 EIA-2-positive samples tested in our diagnostic virology laboratory between January 1994 and January 1995, a total of 96 samples (13%) yielded indeterminate results. Of these indeterminate samples, 29 (30%) were HCV RNA positive by RT-PCR targeting the highly conserved 5' UTR of HCV. We also determined the HCV genotype distribution of SIA-2-indeterminate samples and found that it was markedly different from that of SIA-2-positive samples, suggesting HCV genotype does affect the reactivity of these assays. Hepatitis C virus genotypes infrequently encountered in the United States (including types 2a, 2c, 3a, 4a, and 5a) were more prevalent in samples yielding SIA-2-indeterminate results, suggesting that the serologic detection of antibodies associated with these genotypes may be less efficient than the detection of genotype 1-specific antibodies. These findings are consistent with our earlier report suggesting differences in serologic reactivity to HCV antigens between various HCV genotypes.
These genotype-dependent differences may have major implications for the use of the current HCV diagnostic tests, especially in populations with a high prevalence of HCV genotypes that are phylogenetically distant from HCV genotype 1a, the prototype sequence used in the development of these assays.
In summary, the second-generation serologic assays for HCV detection are highly sensitive and specific in immunocompetent hosts with chronic HCV infection. However, false-negative results can be obtained in immunocompromised and immunosuppressed patients as well as in acutely infected patients. In addition to the results in these patient groups, our studies suggest that all indeterminate SIA-2 results should be followed up with RT-PCR to determine the presence or absence of HCV RNA.
Third-Generation Assays.-Third-generation serologic assays (EIA-3 and SIA-3) were introduced in Europe more than 5 years ago but are not available commercially in the United States. In these assays, a recombinant NS5 antigen has been added to the 4 antigens used in second-generation assays (Figure 1). As a result, third-generation assays have a higher sensitivity and increased specificity compared with second-generation assays.
Chronic hepatitis C without anti-hepatitis C antibodies by second-generation assay: a clinicopathologic study and demonstration of the usefulness of a third-generation assay.
Clearly, the introduction of third-generation tests has not eliminated the need for the direct detection of HCV RNA in all individuals.
Hepatitis C RNA Detection: Qualitative Assays for HCV RNA
Because of the shortcomings of serologic assays for HCV antibodies, direct detection of HCV RNA by RT-PCR has become an essential tool in the diagnosis of HCV infection and the selection of patients for antiviral therapy. The advantages of direct detection of HCV RNA by RT-PCR include the early diagnosis of acute infection, the diagnosis of infection in patients unable to mount an antibody response (immunosuppressed patients, immunocompromised patients, and chronically ill patients such as those with chronic renal disease), and the confirmation of active infection in specific situations (patients with indeterminate antibody results and infants born to HCV-infected mothers). However, a lack of standardization among laboratories has hampered the direct comparison of various HCV RNA results because the sensitivity and specificity of RT-PCR detection of HCV RNA may vary considerably, depending on the choice of primers and methods used for sample collection and handling.
Most current laboratory protocols use primers specific for the 5' UTR of the HCV genome because 5' UTR represents the most highly conserved region among all HCV genotypes. Because of the extreme sensitivity of PCR and RT-PCR, these assays are prone to contamination from exogenous sources. Therefore, strict quality control measures are required in laboratories performing these tests.
Roche Diagnostics (Branchburg, NJ) introduced the Amplicor test for HCV RNA detection by RT-PCR several years ago. This test kit targeting the 5' UTR included both positive and negative controls as well as a modification (AmpErase) minimizing the possibility of carryover contamination, and it appeared to be reliable in the hands of experienced users.
Multicenter evaluation of the COBAS AMPLICOR HCV assay, an integrated PCR system for rapid detection of hepatitis C virus RNA in the diagnostic laboratory.
This assay, based on the original Amplicor assay, was modified to be run on the COBAS instrument, an automated platform designed to perform the amplification as well as to detect amplification products.
This assay also incorporated use of an internal control to identify processed specimens containing inhibitory substances that might interfere with the amplification process or to indicate any loss of target RNA that may have occurred during the sample extraction. The most recent modification of the Amplicor test resulted in version 2.0 of the COBAS Amplicor HCV test.
Second generation of the automated Cobas Amplicor HCV assay improves sensitivity of hepatitis C virus RNA detection and yields results that are more clinically relevant.
This assay includes several modifications intended to increase its sensitivity, including use of a larger sample volume and a more concentrated nucleic acid preparation, which should result in a 10-fold increase in the sensitivity of this assay compared with the previous version of the assay. Studies have suggested that the limit of sensitivity with use of this assay is approximately 100 copies of HCV RNA per milliliter, and the test inhibition rate is 3%.
Second generation of the automated Cobas Amplicor HCV assay improves sensitivity of hepatitis C virus RNA detection and yields results that are more clinically relevant.
Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics.
reported the analytical sensitivity of this assay to be 50 HCV IU/mL, meaning that samples containing 50 HCV IU/mL can be detected reliably 95% of the time. (The use of HCV international units is considered in the discussion of quantitative HCV RNA testing.) The introduction of standardized methods of HCV RNA detection, such as the Amplicor test and the semiautomated COBAS Amplicor tests, is likely to lead to more practical standardized qualitative HCV RNA testing that can be performed reliably by most clinical laboratories.
A relatively new alternative to the detection of HCV RNA by RT-PCR has recently become commercially available, a qualitative assay for the detection of HCV RNA relying on transcription-mediated amplification (TMA) technology (Bayer Diagnostics, Emeryville, Calif). Transcription-mediated amplification is a nucleic acid amplification method that relies on the enzymes reverse transcriptase and T7 RNA polymerase to generate detectable levels of RNA product from either an RNA or a DNA template. In this case, a region within the HCV 5' UTR is targeted along with an internal control transcript that is added to each sample. This nucleic acid amplification method is subject to some of the same limitations as RT-PCR, including the need for a highly conserved target, a purified nucleic acid sample free of inhibitory substances, and appropriate contamination control measures. Unlike RT-PCR, this assay, based on preliminary data from Bayer, has a sensitivity as low as 5 HCV IU/mL, at least 1 log10 lower than most RT-PCR-based assays.
Clinical laboratory evaluation of a new sensitive and specific assay for qualitative detection of hepatitis C virus RNA in clinical specimens [abstract].
Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays.
These preliminary results suggest that the TMA assay may become an important technique for the qualitative detection of extremely low levels of HCV RNA.
Hepatitis C RNA Titers: Quantitative Assays for HCV RNA
Currently, a number of assays are being used to determine viral load. However, the following discussion is limited to the 2 methods commonly used for the quantitative detection of HCV RNA.
The first of these methods is quantitative RT-PCR, which has been used in numerous laboratories during recent years. One of the main advantages of assays based on this method is their sensitivity. However, as with qualitative RT-PCR testing, standardization among laboratories has been lacking, which makes direct comparison of results obtained from these various assays difficult.
Quantitation of HCV-replication using one-step competitive reverse transcription-polymerase chain reaction and a solid phase, colorimetric detection method.
Assessment of hepatitis C virus RNA levels by quantitative competitive RNA polymerase chain reaction: high-titer viremia correlates with advanced stage of disease.
which uses RT-PCR techniques. Assay sensitivity is reported to be 100 RNA HCV copies/mL, with a linear quantitative range extending to 5 × 106 RNA HCV copies/mL, without the need to dilute the test sample. This dynamic range extends from approximately 40 to 2.0 × 106 HCV IU/mL.
The use of multiple amplification reactions with varying cycle numbers allows this assay to have a relatively large dynamic range as well as high level of sensitivity. These features have made this an attractive method for use in viral load testing, and the assay has become widely accepted in the last few years. It has also been used recently in several large multicenter clinical trials.
International Hepatitis Interventional Therapy Group
et al.
Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon a2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus.
The major drawback of using this assay is that it is not commercially available as a kit, and the user must use a central testing facility.
In addition to their commercially available qualitative RT-PCR assay, Roche Diagnostics has also developed a standardized competitive RT-PCR assay for HCV RNA quantification, the Amplicor HCV Monitor test version 1.0. However, since its introduction, this assay has been shown to have several shortcomings, including poor test reproducibility and differences in quantification associated with various HCV genotypes.
Comparison of plasma virus loads among individuals infected with hepatitis C virus (HCV) genotypes 1, 2, and 3 by quantiplex HCV RNA assay versions 1 and 2, Roche Monitor assay, and an in-house limiting dilution method.
The semiautomated COBAS Amplicor HCV Monitor test version 2.0 is a recently introduced modification of the original Amplicor Monitor test. Several modifications were incorporated in this second version in an effort to eliminate the HCV genotype-associated differences in quantification associated with the original assay in addition to conversion to the COBAS format. Preliminary studies have shown that the current assay is equally efficient in the quantification of both genotypes 1 and 2, suggesting that the differences in amplification efficiency noted in the previous version of the assay have been resolved.
Clinical evaluation of the automated COBAS AMPLICOR HCV MONITOR test version 2.0 for quantifying serum hepatitis C virus RNA and comparison to the quantiplex HCV version 2.0 test.
Clinical evaluation of the automated COBAS AMPLICOR HCV MONITOR test version 2.0 for quantifying serum hepatitis C virus RNA and comparison to the quantiplex HCV version 2.0 test.
HCV IU/mL. The sensitivity level, in combination with the relative ease of use of the COBAS system and the elimination of the problems associated with the original assay, has reportedly made the second version a rapid and reliable method for the quantification of HCV RNA in serum.
Clinical evaluation of the automated COBAS AMPLICOR HCV MONITOR test version 2.0 for quantifying serum hepatitis C virus RNA and comparison to the quantiplex HCV version 2.0 test.
However, the limited dynamic range of this assay may require sample dilution and repeat testing of many pretreatment samples to obtain a quantitative value, because many of these patients would be expected to have viral loads above the upper limit of this assay. In addition, given the modifications in the COBAS Amplicor HCV Monitor test version 2.0, which have directly influenced amplification efficiency of some HCV genotypes, results obtained from versions 1 and 2 of the Amplicor HCV Monitor test are not directly comparable. This assay has recently become available in the United States.
The second method widely used for HCV RNA quantification is the Quantiplex HCV RNA 2.0 assay or branched DNA (bDNA) assay (Bayer Diagnostics). This assay is based on a sandwich nucleic acid hybridization technique that uses synthetic oligonucleotide capture and amplifier probes to ultimately detect and quantify viral RNA in serum or plasma. The Quantiplex assay is less prone to run-to-run variability and the adverse effects of HCV sequence variability than early quantitative RT-PCR assays or the first-generation bDNA assay, and it has a larger dynamic than current RT-PCR-based assays.
Comparison of plasma virus loads among individuals infected with hepatitis C virus (HCV) genotypes 1, 2, and 3 by quantiplex HCV RNA assay versions 1 and 2, Roche Monitor assay, and an in-house limiting dilution method.
Clinical evaluation of the automated COBAS AMPLICOR HCV MONITOR test version 2.0 for quantifying serum hepatitis C virus RNA and comparison to the quantiplex HCV version 2.0 test.
Despite this shortcoming, the large dynamic range of this assay may prove to be more useful in the assessment of pretreatment viral load than the lower level of sensitivity provided by nucleic acid amplification-based methods, given the relatively high viral titers found in many pretreatment samples. The results of this assay are reported in megaequivalents of HCV RNA per milliliter (MEq/mL) where 1 MEq = 1 × 106 Eq/mL. The dynamic range of this assay extends from 0.2 to 120 MEq/mL, or approximately 3.2 × 104 to 1.9 × 107 HCV IU/mL using a conversion factor of 6.3 Eq/HCV IU. This conversion factor is based on previous studies
and information provided by Bayer Diagnostics (M. B. Topham, written communication, October 30, 2000).
The latest version of the bDNA assay, the Versant HCV RNA 3.0 assay (Bayer Diagnostics), is in a semiautomated test format that uses improved chemistry to provide greater sensitivity that should prove to be comparable to that of the Roche COBAS Amplicor HCV Monitor test version 2.0. Test results are calculated in HCV RNA copies per milliliter and HCV international units per milliliter rather than in megaequivalents per milliliter. The dynamic range of this assay is reported to extend from 2500 to 4.0 × 107 HCV RNA copies/mL or 521 to 8.3 × 106 HCV IU/mL. The Versant HCV RNA 3.0 assay is now available, and comparative studies are being conducted. However, this assay appears to incorporate high test sensitivity, a large dynamic range, and reporting in HCV international units per milliliter in a semiautomated test format that can be performed easily in most clinical laboratories.
The most recent versions of these commercially available quantitative HCV assays shift reporting of results to HCV international units per milliliter.
Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics.
The HCV international unit is a “virtual” unit that does not represent the actual number of virions in a given sample. Because no stable cell culture exists for HCV, quantification of HCV has not been standardized. Therefore, no uniform unit of measure has been used in the development of the various quantitative HCV assays, contributing to the wide range of quantitative values obtained from a single specimen as well as the confusion regarding assay-specific threshold values. In an effort to promote standardization of HCV RNA testing, the World Health Organization (WHO) International Standard for Nucleic Acid Amplification Technology Assays for HCV RNA (WHO Standard 96/790) was established.
A value of 100,000 HCV IU/mL was assigned to this WHO international standard based on a consensus of quantitative test values obtained from multiple testing laboratories that use a variety of quantitative testing methods and are located throughout the world. Unlike other reporting units that may vary widely depending on the definition used, the HCV international unit is a standardized unit of measure that will aid in the standardization of quantitative HCV testing.
Several studies have suggested that the duration of combined interferon-ribavirin therapy might be adjusted based on pretreatment viral load.
International Hepatitis Interventional Therapy Group
et al.
Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon a2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus.
However, differences in the methods and test reporting among the various assays-and even between versions of the same assay-have complicated the establishment of important clinical thresholds for each of the various assays as well as the direct comparison of quantitative test results obtained from the different assays. Recently, several investigators have addressed this issue by developing conversion factors useful for the comparison of data generated in the different quantitative assays.
recently suggested 800,000 HCV IU/mL as the decision threshold for tailoring the duration of interferon-ribavirin therapy in patients infected with HCV genotypes 1, 4, and 5 (genotypes 4 and 5 were included in this recommendation based on the absence of complete data regarding these genotypes) using either the Superquant assay, the Amplicor HCV Monitor test version 2.0, or the Quantiplex assay. Their conclusions were based on the results of previous studies
International Hepatitis Interventional Therapy Group
et al.
Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon a2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus.
Although quantitative assays have become an important tool in the management of HCV infection, it is important to note their limitations (Table 2). These quantitative assays have been developed for the accurate quantification of HCV in serum or plasma, and they do not have the sensitivity required for reliable detection of low-level viremia. Thus, the use of these assays should be restricted to the monitoring of viral load in patients with confirmed HCV infection and serve only as a supplement to qualitative HCV RNA testing. Complete viral clearance cannot be assessed reliably with the use of quantitative HCV RNA testing during treatment or during the posttreatment period because of the extremely low levels of virus that may be present in some of these patients. The use of highly sensitive qualitative assays is required for this purpose. In fact, recent evidence suggests that many of the currently used qualitative HCV RNA assays may not be adequate for the reliable detection of the extremely low levels of virus present in some of these patients.
Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays.
Ultracentrifugation and concentration of a large volume of serum for HCV RNA during treatment may predict sustained and relapse response in chronic HCV infection.
Because differences in geographic distribution, disease outcome, and response to therapy have been reported between infections with various HCV genotypes, reliable methods for genotype determination have become important in clinical testing.
Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22–3 reactivity and influence of HCV genotypes.
International Hepatitis Interventional Therapy Group
et al.
Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon a2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus.
genotypes 2 and 3 may have better response rates to interferonribavirin therapy, and patients infected with genotype 1 may benefit from an extended course of therapy.
International Hepatitis Interventional Therapy Group
et al.
Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon a2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus.
The standard for HCV genotyping involves direct sequencing of the entire HCV genome obtained from the patient, followed by a phylogenetic analysis of the viral sequence, but direct sequencing of the entire genome is impractical on a large scale. Alternatively, a limited sequence analysis of the HCV NS5, core, or 5' UTR has been used for the purpose of HCV genotyping, with generally comparable results.
However, direct sequencing of even a portion of the HCV genome may be impractical on a large scale because of the complexity of the procedure. Therefore, many of the methods that have been reported depend on the amplification of HCV RNA from clinical specimens, followed either by reamplification with type-specific primers
Detection of three types of hepatitis C virus in blood donors: investigation of type-specific differences in serologic reactivity and rate of alanine aminotransferase abnormalities.
A commercially available test kit for HCV genotyping, the INNO-LiPA HCV II, marketed in Europe and the United States (Innogenetics NV, Ghent, Belgium), is based on the hybridization of 5' UTR amplification products to a variety of genotype-specific hybridization probes attached to unique locations on a nitrocellulose membrane.
It is relatively easy to use and is compatible with the Amplicor assay.
Although all the genotyping methods described herein can identify the major genotypic groups, none is very efficient in the discrimination between subtypes, with the exception of direct nucleotide sequencing of variable regions of the viral genome such as the NS5 region.
However, because the products of highly sensitive detection assays can generally be used directly in genotyping procedures based on the 5' UTR without the need for the amplification of additional viral targets, there are obvious advantages to using this region for genotyping. Despite their inability to completely resolve all existing HCV genotypes and subtypes, methods targeting the 5' UTR provide a sensitive and efficient means of HCV genotyping in a clinical setting.
International Hepatitis Interventional Therapy Group
et al.
Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon a2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus.
Mapping of serotype-specific, immunodominant epitopes in the NS-4 region of hepatitis C virus (HCV): use of type-specific peptides to serologically differentiate infections with HCV types 1, 2, and 3.
Serologic typing has several advantages that make it suitable for large epidemiologic studies. These advantages include a reduced risk of sample cross-contamination as well as the relative simplicity of the assay and its ability to accommodate the relatively high throughput required for these types of studies. When compared with molecular-based techniques, however, serologic typing seems to lack both sensitivity and specificity. This has limited its usefulness.
The future of HCV serotyping in clinical practice remains unclear at this time.
CONCLUSIONS
A diagnostic approach that we have found useful in patients with suspected or known HCV infection is outlined in the algorithm presented in Figure 2. While some evidence supports the use of changes in HCV RNA titer during antiviral therapy to predict treatment outcome, data are insufficient to recommend this approach be used routinely. Our algorithm is based on the recommendations presented in the recently published international consensus statement on the diagnosis and treatment of HCV.
This approach must be modified according to the availability of these assays at different institutions. Also, if the initial evaluation is not conclusive and the level of suspicion is high, repeat testing may be necessary to rule out HCV infection.
Figure 2Algorithm for the diagnosis and monitoring of hepatitis C virus (HCV) infection. ALT = alanine aminotransferase; EIA = enzyme immunoassay; IFN = interferon; SIA = strip immunoassay.
*Testing by EIA-2 should be considered in addition to qualitative HCV RNA testing.
†If clinical suspicion is high, repetition of diagnostic assays is recommended in 3 to 6 months.
‡Hepatitis C virus genotyping, quantitative HCV RNA testing, and liver biopsy may be performed together as indicated by the dashed line to expedite the patient evaluation.
While the evolution of routine HCV RNA testing has altered use of HCV serologic assays, particularly the SIA-2 confirmatory tests, these assays play an important role in routine screening of low-risk populations such as healthy blood donors. Use of the EIA-2 and SIA-2 is required by the FDA for the screening of organ, tissue, and blood donors. In addition, the SIA-2 assays are useful in conjunction with other laboratory testing, including alanine aminotransferase levels and qualitative HCV RNA detection, to determine the importance of positive EIA-2 results (eg, to differentiate a false-positive EIA-2 result from a resolved HCV infection).
The recent approval of pegylated interferon by the FDA must also be taken into consideration with respect to this algorithm. While not yet approved for use in combination with ribavirin, the use of pegylated interferon monotherapy should be considered as an alternative to standard interferon monotherapy. With the future availability of new and more efficient antiviral therapies, new test algorithms may evolve.
Recent advances in serologic assays and nucleic acid detection techniques have allowed physicians who care for patients with liver diseases to obtain a more accurate diagnosis and to establish the presence of HCV RNA in HCV-infected patients. Hepatitis C virus nucleic acid quantification and genotyping assays can also serve as important tools in the management of patients undergoing antiviral therapy and may provide additional insight into the pathogenesis of liver disease associated with HCV.
REFERENCES
Benhamou J-P
Rodes J
Alter H
EASL International Consensus Conference on Hepatitis C, Paris, 26–28 February 1999: consensus statement.
Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5’ untranslated region and poly(a) tails at the 3’ end.
Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory.
Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22–3 reactivity and influence of HCV genotypes.
Chronic hepatitis C without anti-hepatitis C antibodies by second-generation assay: a clinicopathologic study and demonstration of the usefulness of a third-generation assay.
Multicenter evaluation of the COBAS AMPLICOR HCV assay, an integrated PCR system for rapid detection of hepatitis C virus RNA in the diagnostic laboratory.
Second generation of the automated Cobas Amplicor HCV assay improves sensitivity of hepatitis C virus RNA detection and yields results that are more clinically relevant.
Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics.
Clinical laboratory evaluation of a new sensitive and specific assay for qualitative detection of hepatitis C virus RNA in clinical specimens [abstract].
Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays.
Quantitation of HCV-replication using one-step competitive reverse transcription-polymerase chain reaction and a solid phase, colorimetric detection method.
Assessment of hepatitis C virus RNA levels by quantitative competitive RNA polymerase chain reaction: high-titer viremia correlates with advanced stage of disease.
International Hepatitis Interventional Therapy Group
et al.
Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon a2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus.
Comparison of plasma virus loads among individuals infected with hepatitis C virus (HCV) genotypes 1, 2, and 3 by quantiplex HCV RNA assay versions 1 and 2, Roche Monitor assay, and an in-house limiting dilution method.
Clinical evaluation of the automated COBAS AMPLICOR HCV MONITOR test version 2.0 for quantifying serum hepatitis C virus RNA and comparison to the quantiplex HCV version 2.0 test.
Ultracentrifugation and concentration of a large volume of serum for HCV RNA during treatment may predict sustained and relapse response in chronic HCV infection.
Detection of three types of hepatitis C virus in blood donors: investigation of type-specific differences in serologic reactivity and rate of alanine aminotransferase abnormalities.
Mapping of serotype-specific, immunodominant epitopes in the NS-4 region of hepatitis C virus (HCV): use of type-specific peptides to serologically differentiate infections with HCV types 1, 2, and 3.
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