Anti-HCV screening assays
Enzyme immunoassay (EIA) is the most common method for detecting antibodies to HCV (anti-HCV). Three generations of anti-HCV screening assays have now been used in Australia. Improvements in the sensitivity of each successive generation of tests have been achieved by increasing the number of recombinant HCV antigens that are used, as well as modifying the other antigens present.
The drawback of the first generation tests was that they produced a high false-positive rate for anti-HCV in low-risk populations such as blood donors and people with no risk factors for HCV infection. They were also confounded by non-specific reactivity in patients with autoimmune diseases and hypergammaglobulinaemias. The sensitivity was also low because only one HCV antigen was included.
Supplemental assays
Supplemental assays for HCV antibodies are not widely used in Australian diagnostic laboratories. Such tests are designed to increase the specificity of serodiagnosis by detecting specific antibodies to individual HCV antigens.
All of the commercially available tests are expensive. Their cost effectiveness among various risk groups of patients has not been established. Some reference laboratories currently use these supplemental assays.
Sensitivity and specificity of serological assays
The overall sensitivity and specificity of second generation assays are both 95-98%. They may be increased somewhat by third generation tests which incorporate extra HCV antigens. The results obtained within each generation of tests are very similar, regardless of the commercial source of the test.
The results of screening tests can be divided into two sets based on the risk of infection:
- low-risk populations, including blood donors and individuals with no risk factors for HCV infection
- high-risk populations, including individuals with a risk factor(s) for HCV infection or documented liver disease presumed to be due to hepatitis C.
Low-risk populations
The first generation tests suggested that between 0.3% and 1.5% of blood donors world-wide were positive for anti-HCV. In Australia, 0.45% of blood donors in New South Wales were found to be anti-HCV positive. At first, HCV was only identified in 95-98% of the units of blood responsible for post-transfusion hepatitis C infections. This suggested that some infected units of blood were being missed.
Second generation assays detected one additional anti-HCV positive donor per 1000 tested. However, the introduction of these two generations of tests led to successive reductions in the incidence of post-transfusion hepatitis. The third generation tests are thought to detect a single additional infectious unit of blood for every 10 000 units screened.
Now that screening assays are more sensitive, blood banks are more concerned with eliminating false positive screening results because their primary aim is to supply blood for transfusion which is verifiably HCV negative. The major problem in low prevalence groups, like blood donors, has been that 30-50% of sera found to be repeatedly reactive in first generation EIA screening tests could not be confirmed as positive by a supplementary antibody assay. With second generation EIAs, 39-50% of screen positive sera were later found to be false positives after supplementary antibody tests and nucleic acid assay.
High-risk populations
The vast majority of infected high-risk individuals are detected by the serological screening tests. However, first generation EIAs were only able to detect seroconversion in 50% of patients at 4 months and in 90% of patients 6 months after primary HCV infection. This relatively late seroconversion to non-structural viral antigens meant that a diagnosis was delayed or missed if patients were tested at the onset of acute hepatitis or too soon after exposure (Fig. 1).
Fig. 1 A graphic representation of antibody titre to HCV antigens versus time
Antibodies to the C100 antigen appear after the response to recombinant structural antigens such as c22p (third generation tests) and c22-3 (second generation tests). Symptoms (*) develop in only 25% of cases.
ALT = alanine aminotransferase
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Second generation EIAs overcame the problem of late seroconversion to anti-HCV positive status in infected patients. Between 12% and 20% of patients with chronic HCV who were not detected with first generation assays were seropositive after second generation tests. This seroconversion was usually detected within 12 weeks. A further 20% of patients with cryptogenic liver disease were also found to be anti-HCV positive by the new assays. Based on limited data, third generation tests appear to detect seroconversion earlier.
A minority of infected high-risk individuals may not become anti-HCV positive.2 This includes both immunosuppressed patients with defective lymphocyte responses (who produce no antibody) and individuals infected with non-genotype 1 HCV. For example, second generation assays, which were based on genotype 1a or 1b antigens, can fail to detect other genotypes despite evidence of HCV RNA in the serum of patients with post-transfusion hepatitis. The sensitivity of all 3 generations of screening EIAs, in high-risk groups, is therefore slightly below that observed in low prevalence populations.
The Australian situation
In Australia, patients who have a positive screening test, are most likely to have HCV if they have one of the following features:
- a past risk factor for HCV infection (e.g. past injecting drug use, tattoos)
- abnormal physical findings (e.g. hepatomegaly, excess spider naevi)
- elevated alanine aminotransferase (ALT) levels
- a positive polymerase chain reaction (PCR) for HCV in the blood.3
The most prevalent genotypes are 1, 3 and 2. The currently used second and third generation screening tests do not appear to miss established infection in otherwise normal adults.4 However, most published Australian data include individuals derived from both high-risk (diagnostic) and low-risk (blood donor) populations and do not distinguish between the two groups. In addition, rare high-risk patients who fail to seroconvert to anti-HCV positive status do not appear in the test performance statistics.
Clinicians should be aware that screening tests alone do not absolutely exclude hepatitis C in patients who have a risk factor for HCV or evidence of hepatitis. For example, in migrants from countries outside the U.S.A., Canada and northern Europe, infections due to other HCV genotypes (genotype 4 – Middle East or genotype 6 – South-East Asia) should be considered when there is clinical or biochemical evidence of hepatitis, but currently used HCV genotype 1 based screening tests are negative.
Diagnostic testing strategies
Under a new National Health and Medical Research Council (NHMRC) strategy, a positive test must be confirmed before a report will be issued.5
In blood donors, the aim of testing is to establish, with the greatest possible certainty, which donors are not infected. The NHMRC strategy advises blood banks to retest all positive sera in duplicate using the same screening EIA. If the repeat tests are both negative, the donation is considered HCV negative.5 Clinical assessment will be required if the screening test is repeatedly positive.
For diagnostic laboratories, which are generally screening patients with liver disease or those who have a risk factor for HCV, the strategy aims to eliminate laboratory error and confirm the positive status of a reactive screening test (Fig. 2). Those sera which are positive in the first test are retested using a different EIA which contains a different range of recombinant HCV antigens. If the second test is positive, a positive result is issued to the clinician.
If there is a difference between the first and second test results, the serum is retested with the first EIA again. Where this repeat first test is no longer reactive, the screen is reported to the clinician as a true negative. However, if the repeat of the first EIA remains positive, the serum is referred to a reference laboratory for further testing.5
Fig. 2 Recommended NHMRC protocol for HCV serology in diagnostic laboratories5
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