Hepatitis C: diagnosis and monitoring

The diagnostic blood tests for hepatitis C virus (HCV) are the serological assays, which detect antibodies to HCV, and the molecular assays which detect or quantify HCV RNA. Screening tests for antibodies are first done using serological tests such as enzyme immunoassay. The molecular assays can be used to confirm the diagnosis or monitor the response to antiviral therapy. Detection of HCV RNA in patient serum provides evidence of active HCV infection. New assays can identify different HCV genotypes. All of these assays have limitations which affect their utility as diagnostic tests.

Advances in viral diagnosis have significantly reduced the risk of post-transfusion hepatitis C in developed countries. The first serological assays were developed after the RNA sequence of hepatitis C virus (HCV) was identified in 1989.¹ Molecular biology techniques have since been used to detect and quantitate viral nucleic acid.

Various algorithms help the physician correctly identify patients infected with hepatitis C, evaluate them for the presence of significant liver disease and monitor their response to antiviral therapy. Interpreting hepatitis C tests depends on an awareness of the risk factors (Table 1) in conjunction with liver function test results.

Infectious HCV particles (virions) are less than 80 nm in diameter, have a lipid envelope and are strongly associated with the lipoprotein fraction of human serum. Each virion contains a single RNA molecule.

The virus circulates in the blood as a population composed of a master sequence and a large number of minor variants. This occurs because of random mutations during viral replication and also the selection pressure exerted by the host's immune response. This mixed population of viral particles is referred to as a quasispecies and is the basis of the variation found in the HCV genome. Such intrinsic variability may explain why chronic HCV develops in over 80% of acutely infected people. Comparison of HCV master sequences from around the world has led to subclassification of the virus into 6 distinct genotypes.

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).

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

Molecular assays for viral RNA can be used to assess indeterminant EIA results. They can be divided into two distinct categories. The first and most common are qualitative tests which detect minute amounts of viral RNA in the serum, body fluids and tissues. These assays are based on the polymerase chain reation (PCR) (see `Diagnostic tests: DNA i. approach and techniques.' Aust Prescr 1995;18:45-8). A positive result confirms the diagnosis of HCV infection; however, a negative result does not exclude infection.

Quantitative tests, including quantitative PCR and branch-chain DNA assays, have also been developed to measure HCV RNA levels in serum and other body fluids. These assays presently lack the sensitivity of the qualitative tests, but hold promise as a means of tailoring antiviral treatment schedules to pretreatment viraemia and for monitoring HCV replication during antiviral therapy.⁶

Qualitative tests

Detection of HCV RNA using reverse transcription polymerase chain reaction (RT-PCR) has become an increasingly important tool. The presence of HCV RNA in the serum differentiates current from past infection and can act as a marker of response to antiviral therapy. As most anti-HCV positive patients develop chronic hepatitis C, the NHMRC strategy suggests that testing for HCV RNA is not mandatory to confirm the presence of viraemia.⁵

Indications for qualitative PCR include:

• identification of viraemia to confirm active replication of HCV in people with equivocal or indeterminant serological tests (includes newborns with passively acquired maternal antibody)
• early diagnosis of acute HCV infection
• monitoring during trials of treatments for HCV
• defining the route of transmission in epidemiological studies
• assessment of infectivity risk.²

When attempting to resolve equivocal and indeterminant serological profiles, the results of qualitative PCR assays vary with the characteristics of the population under test. For example, among screen-positive blood donors in the U.S.A., only 25% are positive by HCV PCR. The misdiagnosis of HCV on a single blood test should be avoided, especially in low-risk patients.

Quantitative tests

Before quantitative tests can replace qualitative tests, they must reach a higher degree of sensitivity.⁶ Ideally, they should help us to answer these questions:

• do HCV RNA levels predict the outcome of treated and untreated disease?
• what is the lowest level of HCV RNA indicative of hepatic viral replication and RNA clearance during therapy?
• does the pretreatment RNA level predict clinically validated criteria of therapeutic response?

At this stage, we know that HCV RNA levels are relatively stable in untreated patients with chronic HCV and that interferon therapy reduces the viraemia in a significant proportion of treated patients.

Genotype assays

There is a high degree of variability among hepatitis C sequences obtained from different geographical sources and risk groups. These genotypes may have clinical significance. For example, some studies have shown that genotype 1b is relatively resistant to interferon therapy and may be more likely to lead to cirrhosis and hepatocellular carcinoma.⁷

Genotyping has been used extensively in epidemiological studies. At present, its clinical utility as a predictor of the outcome of HCV disease is being evaluated in both treated and untreated patients.

Diagnostic research will focus on several areas. One priority is the identification and production of better recombinant antigens to improve the detection of anti-HCV antibodies and enhance the specificity of both screening tests and supplementary assays. In addition, quantitative antibody assays may have a role in monitoring disease activity and response to therapy.

Improvements in the quantitative HCV RNA assays will be essential in the therapeutic monitoring of chronically infected patients and those undertaking antiviral therapy. The ability to detect and characterise genetic variants of HCV quickly will also become increasingly important. Variants that influence the virulence of HCV, the natural history of chronic hepatitis C or confer resistance to new treatments may have prognostic significance.

In Australian diagnostic laboratories, an anti-HCV screening test is reported as positive only after the patient's serum is found to be reactive in two different assay systems. While HCV RNA is likely to be present in the serum of high-risk patients who are anti-HCV positive, viral RNA is less likely to be detected in the serum of low-risk anti-HCV positive patients. Medium- to long-term follow-up, with repeated testing for evidence of viral RNA, may be required for those who do not have a risk factor for HCV infection, but repeatedly test positive on HCV screening assays. Misdiagnosis of HCV on the basis of a single positive screening test should be avoided, particularly in low-risk patients. Quantitative assays and HCV genotyping may eventually provide data which predict the long-term risks and outcomes in both treated and untreated HCV infection.

Further reading

Farrell GC. Chronic viral hepatitis. Med J Aust 1998;168:619-26.

The following statements are either true or false.

1. The polymerase chain reaction which is negative for viral RNA does not always exclude infection because low level or intermittent viraemia may go undetected in chronically infected patients.

2. Most patients infected with hepatitis C clear the infection within a few weeks.

Answers to self-test questions

1. True

2. False