HCV diagnostic assays (tests) are important for four specific reasons: 1) to protect the blood supply; 2) to diagnose if an individual is acutely infected with HCV; 3) to determine if an individual is a chronic carrier; and 4) to evaluate the effect of anti-HCV therapies. There are currently three types of tests used for detection of HCV infection and quantification of virus: HCV enzyme immunoassay (EIA-2 and EIA-3); reverse immunoblot assay (RIBA-2 and RIBA-3), and polymerase chain reaction (PCR). Nonetheless, biopsy of liver tissue is still the gold standard for assessing the seriousness of hepatic disease in an individual with chronic HCV infection.

HCV EIA, RIBA, and Qualitative HCV RNA PCR

For most people, the diagnosis of HCV exposure is confirmed by detecting the presence of HCV antibodies (anti-HCV) in serum. Sensitive enzyme immunoassay (EIA) tests are commercially available for detecting anti-HCV. Three generations of these assays have been marketed to date: EIA-1, EIA-2, and EIA-3. The HCV EIA uses recombinant viral proteins that recognize epitopes of portions of the core and other viral proteins.

EIA-1 was developed in 1989 by Kuo and colleagues (Kuo 1989). It was an important breakthrough despite suboptimal sensitivity: only about 70­80% (Gretch 1997). In 1992, the much-improved EIA-2 was released, which recognized epitopes from the core (c22), NS3, and NS4 proteins. The newer test also offered much greater sensitivity and it remains the most commonly used assay -- outside blood donation centers -- for detecting anti-HCV. The newest HCV antibody assay, EIA-3, modified its NS3 and NS4 regions and now recognizes additional epitopes from the NS5 protein.

* Based upon detection of HCV RNA by PCR (Terrault 1999, based on Gretch 1997)
** Positive predictive value compared with RIBA

A major problem with the HCV EIA is the low positive predictive value and high false-positivity rate in low-prevalence populations (i.e., blood donors, those without known risk factors, or those with normal ALTs). In these populations, it is advised that a reactive (positive) HCV EIA be confirmed with a reverse immunoblot assay (RIBA-2 or -3), also known as a "Western blot." The newer RIBA assays detect antibodies to each of the HCV proteins (core, NS2 through 5) in a nitrocellular strip format.

Data from two studies have demonstrated that non-reactivity to RIBA-3 correlates well with an absence of HCV viremia (Uyttendaele 1994; Zein 1997). If, however, an individual's sample is found to be reactive or to have an indeterminate reaction to the RIBA, a qualitative² HCV RNA reverse-transcription polymerase chain reaction (PCR) assay is recommended to establish the presence or absence of HCV viremia (Terrault 1999).

In a high-risk population (those with elevated ALTs or a risk factor such as history of injection drug use, multiple sexual partners, or blood transfusion before 1992), a reactive HCV EIA-2 or -3 is often sufficient to confirm HCV infection. The next logical step would be either a qualitative HCV RNA PCR to differentiate acute versus chronic infection or a quantitative2 HCV RNA PCR (to establish baseline viral load) if the individual is considering anti-HCV treatment. PCR may be indicated for immunocompromised individuals (transplant patients, those with chronic renal failure or HIV infection). These individuals are often unable to develop an adequate antibody response, and PCR (qualitative or quantitative) may be necessary to detect HCV (Terrault 1999). Likewise, there is an increased rate of false-positive anti-HCV reactions in people with HIV infection (Zylberberg 1996). The current United States Public Health Service and Infectious Disease Society of America (USPHS/IDSA) Guidelines for the Prevention of Opportunistic Infections in Persons Infected with Human Immunodeficiency Virus recommends that positive screening antibody tests for HCV in people with HIV infection be confirmed with either the RIBA or HCV RNA PCR. Also, HIV-positive people with undetectable HCV antibodies, but evidence of unexplained chronic liver disease, should have an HCV RNA test performed (CDC 1999).

A qualitative HCV RNA PCR test is the best way to determine the presence of virus during the approximately two-month "window period" between HCV exposure and the presence of antibodies. HCV RNA is detectable by PCR within one to two weeks after exposure. Schreiber and colleagues conducted a study to accurately estimate the risk of transfusion-transmitted HIV, HTLV, HBV, and HCV from screened blood which was tested and determined antibody-negative (Schreiber 1996). Using data on 586,507 persons who had given blood more than once (a total of 2,318,356 blood donations), they calculated that the risk of a donor's being HCV RNA PCR- positive during the initial period before antibodies became detectable was 1 in 103,000 (range: 28,000­288,000). They concluded that screening blood for HCV with PCR would reduce the window of vulnerability by an estimated 59 days, and that the relative risk could be reduced by an additional 77%.

In 1999, Roth and colleagues confirmed these results and also demonstrated that PCR was suitable for rapid blood screening, testing 3,000 samples in seven to eight hours (Roth 1999). The authors make a case for routine HCV PCR screening in the blood-bank setting on the basis of improved safety as well as improved availability, with quarantine times for fresh-frozen plasma being reduced from six months after antibody testing to three to four weeks after PCR testing.

HCV RNA PCR qualitative and quantitative assays have not yet been approved by the FDA.³ Nevertheless, they are widely used in clinical practice and in treatment studies. They have become increasingly sensitive with a lower limit of detection of 10 to 1,000 genomic equivalents/mL. The major drawbacks of HCV RNA PCR assays are the wide variability and lack of standardization. Many studies have documented great variability in the detection of positive reference samples by labs using "in-house" PCR kits and labs using commercially available assays (Damen 1996). In 1997, a World Health Organization (WHO) international standard was established for HCV RNA nucleic acid testing (NAT) assays. This standard is used primarily in Europe; the U.S. FDA is currently working on its own standard.

Additionally, qualitative PCR is also an important tool for defining response to anti-HCV treatment with interferon (IFN) monotherapy or in combination with ribavirin (RBV). Below is a table explaining its clinical use:

HCV RNA PCR Quantitative Assays

HCV RNA PCR quantitative assays are used for two reasons: 1) To determine the amount of virus in an individual who is considering therapy; and 2) To observe the rate of decline of viral load during the first few weeks of treatment as a predictor of complete response (Zeuzem 1998). Although the amount of virus does not correlate with ALT levels (Ghany 1996; Zeuzem 1996) or liver histology (Lau 1993; Poynard 1997; De Moliner 1998), baseline viral loads have been shown to be a predictor of response to therapy (Davis 1997, 1998; Poynard 1998).

There are four commercially produced quantitative assays; however, the most sensitive assay, SuperQuant HCV assay, used in the interferon and ribavirin combination therapy registrational studies, is not yet commercially available. The table below, adapted from Terrault, is an analysis of all four assays based on limit of detection, units, and cost:

The Amplicor and Quantiplex assays use different types of amplification techniques to quantify viral RNA: the Amplicor uses target amplification of primers from most conserved region of the HCV genome (5' untranslated region [5'UTR]); and the Quantiplex uses signal amplification that captures and targets probes from the 5'UTR and core regions, amplifying the HCV RNA with synthetic, branched DNA oligonucleotides. Both have been compared in numerous studies (Gretch 1994; Tong 1998; Lunel 1999; Martinot-Peignoux 2000).

Martinot-Peignoux and colleagues recently published results from a study comparing three quantitative assays: Bayer's Quantiplex (bDNA) v2.0, Roche's new Cobas Amplicor HCV Monitor v2.0, and NGI's non-commercially available SuperQuant (Martinot-Peignoux 2000). Both the level and range of quantification were similar among the assays, and results correlated well among various HCV genotypes. The SuperQuant detected all 22 samples with fewer than two million copies of virus compared with 17 of 22 and 19 of 22 with the bDNA and COBAS assays, respectively (P>0.05). While the bDNA assay appears less likely to accurately detect low levels of virus, it is considered the best for obtaining high-end quantification values (i.e. >5 million copies) (Reichard 1998).

Because quantitative HCV RNA measurements are beneficial only for pretreatment evaluation and for on-treatment observation, the CDC has not recommended sequential HCV RNA monitoring for all patients (CDC1998).

HCV Genotype

Not all individuals with HCV have identical viruses. Many different genetic variations (genotypes) of the virus exist. There are at least 7 distinct genotypes and at least 30 subtypes of HCV. The majority of HCV-positive individuals in the U.S. and Europe are infected with genotype 1; genotype 1a is more common in the U.S., and 1b is more common in Europe. Below is a breakdown of genotype prevalence from two recently published studies:

Nucleotide sequencing and phylogenetic analysis of NS5B and E1 regions are considered the gold standard for determining HCV genotype, but cost prohibits these methods from being used clinically (Terrault 1999). The two commercial assays most commonly used are the INNO-LiPA HCV assay (Innogenetics, Zwijnaarde, Belgium) and the HCV serotyping assay (Abbott Laboratories).

Most natural history studies have demonstrated that HCV genotype in and of itself does not play a role in the clinical course of HCV. (For a detailed discussion, see the "Natural History of HCV" chapter.) Genotype can, however, be highly predictive of response to anti-HCV treatment. Recent clinical studies have demonstrated that sustained responses to treatment are significantly less likely for individuals with genotype 1 (Davis 1988, McHutchinson 1998; Poynard 1998). The accompanying chart details the sustained virologic response rates according to genotype for patients who received either 6 or 12 months of IFN alone or in combination with RBV in the U.S. and European IFN+RBV registrational studies:

The differences in the response rates between the genotype 1 group and the genotype 2/3 group -- regardless of ribavirin coadministration -- are dramatic.

Liver Biopsy

Liver biopsy is considered the gold standard for clinical assessment of individuals with chronic HCV. It is the only true way to determine the severity and activity of liver disease. Because HCV can be definitively confirmed with a qualitative HCV RNA PCR, a liver biopsy is most often reserved for those considering treatment in order to assess the grade and stage of hepatitis. A liver biopsy can also help rule out other forms of liver disease such as concurrent alcoholic liver disease, medication-induced liver injury, and iron overload.

A core liver biopsy is done in order to obtain intact tissue for a pathology reading. This procedure, with the use local anesthesia, involves the passage of a thin needle between the ribs through the skin to remove a tiny (1-inch long and 1/5-inch wide) piece of liver tissue. A liver biopsy is usually done in the hospital, and an individual may leave within three to six hours. The risk of complications from a liver biopsy -- primarily bleeding at the site of puncture into the liver -- is less than one percent. Approximately half of individuals have no pain from the biopsy, while others experience brief localized pain.

To avoid the risk of complications, some researchers have explored doing liver biopsies with imaging-guidance techniques such as ultrasonography (Papini 1991; Lindor 1996). Ultrasonography can aid in directing the needle away from large blood vessels, bile ducts, gallbladder, and colon, and thus potentially reduce complications. This procedure has gained some followers, yet it appears that most are skeptical about its necessity and concerned with the added cost (Smith 1999).

Recently, there has been a debate among clinicians about the need for liver biopsy in patients with HCV. Some believe that with the proper information from selected assays, they can accurately determine the histologic grade and stage of a patient's disease. An interesting study investigating clinicians' predictions of patients' liver histologies by surrogate markers was presented at the 1999 Digestive Disease Week annual meeting. Romagnuolo and colleagues from Canada studied 45 patients referred to their hospital for treatment (Romagnuolo 1999). All clinicians' predictions were within one point of the actual grade and stage. Thirty-five (66%) of the patients' inflammatory scores and 40 (75%) of the fibrosis scores were exactly predicted, including four cases of cirrhosis. Age >40, spider nevi (abnormal blood vessels on the skin of the abdomen), organomegaly (abnormal enlargement of liver and/or spleen), white blood cell count <4,000, ALT >120, bilirubin >20, albumin <3.5, and ferritin (an iron-protein complex) >200 were predictors of more severe inflammation. The same variables (except ferritin and ALT) with the addition of platelets >150,000 and prothrombin time >1.2 were significant predictors of fibrosis.

The Knodell scoring system (used mostly in the U.S.) is an important, yet complicated, tool for documenting histologic activity. It is used to grade the level and extent of inflammation, necrosis, and fibrosis of a liver biopsy. Biopsy specimens are graded in four categories:

1. Periportal +/- bridging hepatocellular necrosis I
2. Intralobular degeneration and hepatocellular necrosis
3. Portal inflammation
4. Fibrosis

A numeric score for each category is assigned to each liver biopsy specimen, and the combined score of the four categories form the HAI (Histology Activity Index) score for that biopsy specimen. Note: The score goes from 1 to 3, omitting 2 intentionally.

It is not crucial for individuals with HCV to know and memorize their HAI score. It can be useful, however, for them to understand the condition of their liver and to know the degree of inflammation, the stage of fibrosis, and if cirrhosis has occurred.

Fibrosis of the liver is the presence of scarring that results from the repair of hepatic tissue damage. In the case of HCV, the scarring is initiated by HCV-infected hepatocytes and the resultant inflammation. Liver fibrosis occurs slowly, first in the outer (portal) areas of the liver and then working its way in (bridging) to the central vein area.

Extensive fibrosis and deterioration of the liver's cellular architecture is called cirrhosis. Cirrhosis results when most normal liver cells have been replaced by scar tissue. It can greatly interfere with the liver's ability to perform many of its usual functions, including the production of proteins and enzymes, the regulation of cholesterol and storage of energy, and the metabolism of drugs and toxins. Cirrhosis can lead to internal bleeding, kidney failure, mental confusion, fluid accumulation, infection, and coma.

Below are photographs of liver biopsies documenting various histologic stages of HCV:

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1. Much of this chapter has been adapted from Norah Terrault's printed syllabus and lecture, Viral Diagnostic Studies, AASLD, Postgraduate Course, Dallas, 11/5/99.
   
2. A qualitative PCR tells you whether virus exists. A quantitative PCR tells you how much virus exists.
   
3. In May 2000, Roche Molecular Systems was granted priority review by the FDA for its two experimental qualitative HCV RN.