Unravelling the Mystery of Hepatitis C Serology

Dr. Sherker is a Gastroenterologist practicing in Quebec, Canada. This paper has been submitted as a contribution to the Canadian Hepatitis Education Council.

Patients with chronic hepatitis C virus (HCV) infection may come to medical attention because of symptoms or complications of their infection. More commonly however, they are identified on screening at the time of a blood donotion; upon the identification of a current or past risk factor for infection (as reviewed in Hepatitis Update Issue #1); or as part of the investigation of incidentally found abnormal liver enzymes. The virus in present in the serum of infected individuals in extremely low concentrations and to date the virus has not been imaged, even by electron microscopy. Unlike hepatitis B virus whose envelope protein (HBsAg) is detectable in the serum of carrier patients, the proteins of HCV are present in serum at levels well below the limits of detection. Thus, infection is inferred by the detection of host-generated antibodies to viral proteins.

This review will focus on the logical sequence and interpretation of available serological tests. Additionally, the techniques and indications for HCV RNA measurement, and the interpretation of the results of such testing will be discussed, along with the role of liver biopsy.

HEPATITIS C SEROLOGICAL TESTS

HCV is a single-stranded RNA virus related to the Flaviviridae. Its genome (genetic material) consists of approximately 9400 nucleotides and encodes a single large polyprotein which is then cleaved to yield the structural and non-structural proteins of the virus (see Figure 1).1 Despite the fact that there is a high degree of variability in the sequences of various HCV isolates and there are at least six major viral genotypes,2 genetic engineering techniques have been used to identify and synthetically produce a number of a number of proteins and synthetic peptides, the genetic and amino acid sequences of which are conserved across the different genotypes and various subspecies. These proteins and peptides have been used to develop immunoassays.

The first commercial HCV antibody test was an enzyme-linked immunosorbant assay (ELISA), in which the patients serum was tested for the presence of IgG antibody against the c100-3 antigen derived from the non-structural region of the virus.3¬†This was the first generation of HCV ELISA, called ELISA-1. The ELISA-a was first used to screen blood donors in May 1990 in Canada. This resulted in an immediate fall in the rates of post-transfusion non-A, non-B hepatitis. The test also proved useful in diagnosing patients with chronic liver disease, but was associated with a high rate of both false positive and false negative results. (The false positive rate was 5% to 25%. Overall, the correlation between the first generation ELISA and the more accurate later tests was 64% to 85%.) In part this was because using ELISA-1 there is a “window” period of up to six months between infection with the virus and seropositivity. Because of these shortcomings, the second generation ELISA-2 was developed, which incorporated another structural antigen from the core region of the virus, and expanded the non-structural epitope (antigenic region), resulting in improved sensitivity and specificity, and a shortening of the window period.4¬†ELISA-2 is widely used in Canadian hospitals, but ELISA-3, which incorporates an additional non-structural epitope is becoming available, and will further increase sensitivity and specificity.

The diagnosis of HCV infection has serious implications for patients. In individuals with elevated serum transaminases and a parenteral risk factor, a positive ELISA test is virtually diagnostic. However, in low risk populations a supplemental assay is necessary to confirm a positive ELISA assay. This may be a second ELISA (from a different manufacturer) but the most widely used supplemental assay is the recombinant immunoblot assay (RIBA). In this assay the patient’s serum is incubated with a nitroccellulose strip which has been coated with HCV antigens and a number of positive and negative controls. The strip is “developed” in a manner analogous to ELISA, with antibodies to human IgG. Positive reacting bands turn black. If no viral bands are positive the test is interpreted as negative. Two or more positive bands is read as a positive assay. A single band is indeterminate. The first generation RIBA was relatively insensitive and associated with frequent indeterminate results, and has been replaced with riba-2,5,6,7¬†which has four viral antigens (see Table 1). Indeterminate RIBA-2 assays still occur, but are much less frequent.8,9¬†In an indeterminate result, when the single positive RIBA band is 5-1-1 or c100-3, HCV RNA is almost never positive and patients tend to have normal transaminases and no risk factors. Isolated c33c occasionally occurs in true infections and a strong c22-3 is usually confirmed by HCV RNA testing.10¬†A new third generation RIBA appears to be more accurate than RIBA-2,10¬†but is not yet widely available in Canada.

HCV RNA MEASUREMENT

Although HCV antibody assays are extremely useful in diagnosing HCV infection, the detection of HCV antibodies in serum does not unequivocally establish viremia. Additionally, even with the latest generation tests, there still are patients who have indeterminate serological results. The detection of HCV RNA in serum is the “gold standard” for detection of viremia. HCV RNA detection allows HCV infection to be diagnosed in early acute disease prior to antibody detection or aminotransferase elevation, or in rare (particularly immunocompromised) individuals who fail to generate specific antibodies in the face of chronic infection.11Additionally, RNA testing establishes the presence or absence of virus in anti-body-positive patients and has proved useful in establishing perinatal transmission of HCV from infected mothers to their newborns.12

Because of the extremely low concentration of virus found in HCV infected individuals, viral RNA cannot be detected directly in serum. HCV must first be amplified using the extremely sensitive reverse transcriptase/polymerase chain reaction (TR/PCR) technique. This specialized technique is expensive and the reaction conditions used have not been standardized. Results obtained on identical samples may vary significantly between different laboratories.13 The strict conditions required for sample handling are not practical for application of this technique in hospital diagnostic laboratories and the results obtained by the standard RT/PCR assay are not quantitative.

For this reason, commercial HCV RNA kits have been developed for use in diagnostic laboratories. The Quantiplex7 assay is based on branched DNA technology for amplification of the signal obtained from a single viral genome. It is somewhat less sensitive than RT/PCR but has the advantages of reproducibility, authormatability, rapidity and quantitation although it is costly.14,15,16 Another commercial quantitative HCV RNA assay, Amplicor7, has been developed based on a modified TR/PCR protocol which minimizes the risk of false-postive results.

CLINICAL USE OF HEPATITIS C TESTING

The ELISA-2 test is the standard test used by most laboratories to diagnose hepatitis C infection. Testing is indicated in patients who have identifiable risk factors for hepatitis C (see Hepatitis Update #1), have unexplained elevated liver enzymes, or chronic liver disease of unknown etiology. Since hepatitis C is a common disease, hepatitis C antibody testing should be a part of the initial work-up of all such patients.

According to the LCDC, specific testing algorithms should be followed by all laboratories undertaking hepatitis C testing. The recommended algorithms are shown in Figure 2. These algorithms call for a positive ELISA to be repeated in duplicate with another ELISA. Strictly speaking, this should be an ELISA from a different manufacturer. Few commercial laboratories are yet this stringent in their testing. In practice, a strongly positive ELISA-2 in high titre is almost universally associated with a positive HCV RNA in serum. Futhermore, if associated with elevated liver enzymes and a history of a risk factor for hepatitis C, no further testing is really necessary. If RIBA testing is performed, a positive RIBA (two or more positive bands) also indicates active infection. HCV RNA is almost always positive.

HCV RNA testing is thus not required for the majority of patients who are anti-HCV positive. However, there are some patients for whom HCV RNA testing may be extremely helpful:

1. Anti-HCV positive patinets with normal liver enzymes. These are usually patients in whocm anti-HCV screening was performed (e.g. for in vitro fertilization or Red Cross or Hospital look-back programs after blood transfusion). There are several possible interpretations of these results. This could be a false positive, or it could be a true positive (i.e. the patient is truly infected) but there is no, or minal, hepatic inflammation present. Alternatively, anti-HCV could be positive in someone who initially developed hepatitis C infection, but subsequently cleared the virus.

A negative HCV RNA in these circumstances makes it unlikely that the patient is truly infected. However, it should be noted that even the HCV RNA tests are not infallible. The current tests have a lower limit of detection of about 105 viral genomes. Thus low titres of HCV may not be detected in these assays. Furthermore, the long term prognosis of anti-HCV-positive, HCV RNA-negative patients is not known. It is, at least, theoretically possible that the virus is dormant and can reactivate at a later stage. Such patients need to be monitored indefinitely, given our current state of knowledge.

2. Anti-HCV-negative patients with liver disease which resembles viral hepatitis, and in whom no other case has been found. Many of these patients will be immunocompromised, such as the patients in renal failure, HIV-positive patients, patients with various forms of cancer or those on steroids. Before undertaking HCV RNA testing, similar diseases such as hepatitis B, drug-induced hepatitis and autoimmune hepatitis type 1 and 2 (anti-smooth muscle antibody-positive and anti-liver kidney microsomal antigen-positive, respectively) should be excluded.

3. ACUTE HEPATITIS C PATIENTS Anti-HCV testing in patients acutely infected with hepatitis C is usually negative until several weeks after infection. Thus, in a symptomatic patient HCV RNA testing may be useful to make the diagnosis, but there is little point in undertaking HCV RNA testing unless treatment is planned. As yet, treatment for acute hepatitis C is not standard practice. However, there have been reports of enhanced clearance of the virus after treatment with interferon in doses of three to ten million units daily, given for the duration of elevated enzymes.17 If treatment is not to be given, anti-HCV testing three to six weeks later will indicate whether seroconversion has occurred.

4. ALTHOUGH NOT RECOMMENDED FOR THE ASSESSMENT OF PATIENTS, HCV RNA TESTING MAY PREDICT WHICH PATIENTS ARE LIKELY TO RESPOND TO THERAPY. The lower the HCV RNA, the more likely the response. Patients who fail to lose RNA during therapy are highly unlikely to experience a sustained remission. 14,15,18,19 HCV RNA monitoring is not recommended for routine monitoring of patients undergoing interferon alpha treatment.

5. ALTHOUGH NOT RECOMMENDED FOR ROUTINE USE, HCV RNA may predict transmission of hepatitis C from a pregnant mother to her infant. If the maternal HCV RNA titre is below 107 genomes/mL, transmission is unlikely.20

6. HCV GENOME ANALYSIS CAN BE USED TO DOCUMENT PERSON-PERSON TRANSMISSION. This is an expensive and labour-intensive process, and is not widely available. Essentially the technique uses genetic engineering techniques to document that the genetic sequence of the virus in both infected individual is the same.

ANTI-HCV TESTING AND AUTOIMMUNE LIVER DISEASE

The frequency of autoimmune phenomena is higher with chronic hepatitis C than with other forms of viral hepatitis.21,22,23,24 This, coupled with the finding that autoimmune hepatitis some times causes a false positive anti-HCV result may make it difficult to distinguish these two diseases. Smooth muscle antibodies (SMA) are found in 11% of patients with chronic hepatitis C.21 Antinuclear antibodies (ANA) are present in 32%.21 The titres of these autoantibodies are usually low (<1/160).25 Furthermore, ANA and SMA are seldom present together in chronic hepatitis C. However, both diseases may coexist and an HCV RNA assay can be helpful. Other common autoantibodies include antithyroid antibodies and rheumatoid factor. Anti-liver kidney microsomal antibodies (anti-LKM) and anti-DNA antibodies are rare.

Patients with autoimmune hepatitis may have false-positive anti-HCV tests, Elisa-2 is falsely positive in 40% of patients with autoimmune hepatitis.25 This is usually associated with hyper-gammaglobulinemia.26 The RIBA-2 assay is usually negative, and the HCV RNA is also negative. ELISA-3 is more specific, reducing the frequency of false positives in autoimmune hepatitis to 20%,25 unfortunately, this comes at the expense of a loss of sensitivity. In autoimmune hepatitis ELISA-3 detects true hepatitis C infection in only 57% of cases.

In patients with no risk factor for chronic hepatitis C, autoimmune hepatitis should be suspected and autoantibody testing performed. However, in patients with a clear risk factor for hepatitis C the diagnosis is so likely to be chronic hepatitis C that autoimmune testing is not required, unless some factor in the history suggests another diagnosis. Table 2 provides suggestions for the further management of patients with overlapping features of chronic hepatitis C and autoimmune hepatitis.

THE ROLE OF LIVER BIOPSY

Hepatitis C can cause a spectrum of liver pathology ranging from mild nonspecific changes to end-stage liver disease including cirrhosis and hepatocellular carcinoma.27 There are no pathognomonic features of hepatitis C, but the biopsy may be helpful in ruling out additional pathologic conditions. Although nonspecific, portal lymphoid follicles, intrahepatic bile duct damage and steatosis are typical in chronic hepatitis C. Serum aminotransferases are a poor predictor of histopathology and even patients with normal transaminases may have advanced chronic liver disease. Patients with cirrhosis are less likely to respond to interferon-alpha therapy. The liver biopsy may provide useful staging information in hepatitis C and in the absence of contraindications in considered mandatory before instituting interferon-alpha treatment.28

Since hepatitis C can be associated with normal liver enzymes, and since other liver diseases (fatty liver, alcoholic liver disease), which can contribute to the elevation of the transaminases are relatively common, a biopsy is necessary to confirm the presence of viral hepatitis. In addition, the presence of fibrosis indicates the potential of the disease in this patient to progress to further damage, and is an additional argument in favour of treating the disease.

Suggested testing algorithms for screening of individuals with risk factors for HCV infection as well as blood, organ and sperm donors (A); patients presenting with accute hepatitis (B); and, patients with chronic liver disease (C). Adapted from ref. 29

Suggested testing algorithms for screening of individuals with risk factors for HCV infection as well as blood, organ and sperm donors (A); patients presenting with acute hepatitis (B); and, patients with chronic liver disease(C). Adapted from ref.29.
Averell Sherker, MD


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