PROGRESS IN DIAGNOSIS OF HEPATITIS C VIRUS: ANTIBODY ASSAYS

• Introduction
• Table 1. Complications of hepatitis C virus infection
• EIA screening tests
• Table 2. Major HCV Antigens used in three generations of EIA test kits
• Supplementary or confirmatory tests
• Table 3. Comparison of two generations of HCV RIBA
• CDC Test Algorithm for Asymptomatic Patients
• Figure 1. Hepatitis C Virus Infection Testing Algorithm for Asymptomatic Persons (Ref. 1)
• Clinical Virology Laboratory Test Policy
• Table 4. Interpretation of Hepatitis C antibody assays
• Limitations of HCV diagnosis
• Conclusions

Hepatitis C virus (HCV) is the main cause of parenterally transmitted non-A, non-B hepatitis and is the most common chronic blood-borne infection in the U.S. (1). It is estimated that approximately 4 million Americans have been infected with HCV. Up to 80% of HCV-infected persons are chronically infected and are at risk for developing cirrhosis and hepatocellular carcinoma, as well as other HCV-associated diseases (Table 1). At present, HCV-associated end-stage liver disease is the most frequent indication for liver transplantation in adults.

Table 1. Complications of hepatitis C virus infection

Hepatic
Acute hepatitis
Chronic active hepatitis
Cirrhosis
End-stage chronic liver disease
Hepatocellular carcinoma
Extra-hepatic
Essential mixed cryoglobulinemia
Membranoproliferative glomerulonephritis
Porphyria cutanea tarda
Aplastic anemia
Possible associations
Sjogren syndrome
Lichen planus
Autoimmune thyroiditis
Mooren corneal ulcers
Idiopathic pulmonary fibrosis

Recent studies have shown that intravenous-drug use currently accounts for 60% of HCV transmission in the U.S. (1). Other risk factors include blood transfusion (primarily before 1990), health care workers involved in patient care or clinical laboratory work, hemodialysis, hemophilia, household or sexual exposure to a hepatitis case, and multiple sexual partners. For 10%, the route of transmission is unidentified.

Since most HCV infected persons are aged 30-49 years, the morbidity and mortality due to HCV could increase substantially during the next 10-20 years as this group of infected persons reaches an age at which complications from chronic liver disease usually occur.

Laboratory diagnosis is essential to identify patients for antiviral therapy and to prevent transmission to others (1,2). Laboratory diagnosis has made tremendous progress since 1988 when viral sequences were first cloned from the serum of an experimentally infected chimpanzee (3). HCV is now classified in the Flaviviridae family, genus pestivirus. There are three structural proteins, [core or nucleocapsid (C) and envelope (E1 and E2/NS1)], and six nonstructural proteins (NS2, NS3, NS4a, NS4b, NS5 and NS5b). To date, 6 genotypes and over 90 subtypes have been identified.

EIA screening tests

The first generation EIA screening test for HCV (EIA-1) in 1990 used a single initial clone (c100-3) as antigen. Since then tremendous progress has been made both in test sensitivity and in reducing the time to detection of seroconversion (4), as shown in Table 2. The third generation EIA uses recombinant antigens from 4 different regions of the viral polyprotein. However, specificity remains a problem. The positive predictive value of the EIA is very high in high-risk groups; however, in low-risk groups, such as healthy blood donors, many positive EIAs are in fact false positives. As nonspecific reactions are reduced with the antigens used in EIA-1 and EIA-2, new antigens being added (e.g. NS5) result in a new group of patients with false-positive results.

Table 2. Major HCV Antigens used in three generations of EIA test kits

In the U.S., EIA-3 was not actually approved as a third generation test, but rather as an improved second generation test. Consequently, laboratories do not have to convert to EIA-3, but still may use EIA-2. Diagnostic tests were also developed based on genotype 1, which predominates in the U.S. but not necessarily in other parts of the world. While third generation EIA and RIBA have improved detection of non-1 genotypes, their reactivity is still less than with type 1.

Supplementary or confirmatory tests

False-positive reactions in HCV EIAs occur due to contamination of recombinant HCV proteins with vector proteins or fusion proteins, or to homologies with a variety of other proteins. Consequently, a recombinant immunoblot assay (RIBA) was developed as a supplementary test to improve accuracy. In this assay, patient serum is reacted with HCV proteins applied to nitrocellulose strips. Two immunoglobulin standards are provided to assess anti-HCV band intensity. Intensity equivalent to the level I standard is required (i.e. 1+) and bands from at least two different regions of the HCV genome must be present at 1+ intensity for a RIBA to be considered positive. However, since the same proteins used in the EIA are used in the RIBA, this may be a less than ideal confirmatory assay. If there are no risk factors and reactivity on RIBA is weak, further investigation with detection of HCV RNA may be indicated.

The recently approved RIBA-3 resolves many of the RIBA-2 indeterminate samples due to the substitution of recombinant proteins with synthetic peptides (Table 3). The value of the newly added NS5 protein is uncertain since potential gains in sensitivity may be at the expense of a new cohort of false positives (5).

Table 3. Comparison of two generations of HCV RIBA

Not all infected persons will have a positive RIBA. The EIA becomes positive earlier than RIBA, and sequential testing may be needed. Non-1 genotypes may give indeterminate results on RIBA and immunocompromised hosts may not be able to effectively mount an antibody response. An alternative confirmatory test is the detection of HCV RNA in serum or plasma. These tests will be discussed in a future issue of Lab News.

CDC Test Algorithm for Asymptomatic Patients

The Centers for Disease Control has published an algorithm for hepatitis C infection testing for asymptomatic persons (Figure 1). Unfortunately, the laboratory often does not know whether a patient is asymptomatic or has risk factors for HCV. In most hospital and commercial diagnostic laboratories, a RIBA is not automatically performed when a positive EIA is obtained, but only on request of the physician.

Figure 1. Hepatitis C Virus Infection Testing Algorithm for Asymptomatic Persons (Ref. 1)

Clinical Virology Laboratory Test Policy

From two separate evaluations in our laboratory in 1997 and 1998, it was determined that a high positive anti-HCV EIA result was associated with a positive RIBA in 97% of samples. Thus, a RIBA is automatically performed only on low positive ELISAs.

The Clinical Virology Laboratory at Yale New Haven Hospital currently performs EIA-3 on all samples submitted for HCV antibody testing. If the EIA is positive, the sample is retested in duplicate. High positive EIAs are then reported with a comment (Table 4). If the patient is asymptomatic or does not have risk factors, this can still represent a false positive result and the responsible clinician should call the laboratory to request a RIBA.

Table 4. Interpretation of Hepatitis C antibody assays

Limitations of HCV diagnosis

Despite the progress made in diagnostic testing, limitations exist. A seronegative window of up to 20 weeks persists with EIA-3. In some populations, including health-care workers, the rate of false positivity for anti-HCV is at least 50% and supplemental assays should be used to assess validity of the repeatedly positive EIA results (6). Anti-HCV antibodies do not distinguish past from present infection. Antibody without RNA may indicate that the carrier state was not established and the patient has cleared the virus. Alternatively, the level of HCV RNA circulating in the blood fluctuates and may be below the limits of detection at the time of testing. Furthermore, approximately 5% of HCV infections are antibody-negative, but HCV RNA is detected.

Conclusions

Tremendous strides have been made in the accurate diagnosis of hepatitis C infection in the past decade. However, the interpretation of a positive EIA remains strongly dependent upon the risk factors and symptoms of the patient. Clinicians must notify the laboratory and request a RIBA in low risk or asymptomatic patients. HCV RNA tests are very useful in clarifying equivocal antibody results and in detecting antibody-negative infected patients. Accurate diagnosis is essential for prevention and control of HCV infection and liver disease.

References

1. CDC. Recommendations for Prevention and Control of Hepatitis C virus (HCV) Infection and HCV-related Chronic Disease. MMWR 47: RR-19, October 16, 1998.

2. McHutchinson JG , et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 339:1485-1492, 1998.

3. Choo QL, Kuo G, Weiner J et al. 1989. Isolation of a cDNA clone derived from a blood borne non-A non-B viral hepatitis genome. Science 244:359-362.

4. Vrielink H, et al. 1997. Performance of three generations of anti-hepatitis C virus enzyme-linked immunosorbent assays in donors and patients. Transfusion 37:845-849.

5. Uyttendaele S, et al. 1994. Evaluation of third-generation screening and confirmatory assays for HCV antibodies. Vox Sang 66:122-129.

6. CDC. Recommendations for follow-up of health-care workers after occupational exposure to hepatitis C virus. MMWR 46:603-606, July 4, 1997.

Marie Louise Landry, M.D.