THE VIRUS

The HCV virion was fully sequenced by the Chiron Corporation in 1988 and found to be an enveloped, 9.5 kilobase, single stranded RNA virus. It has been classified in the family Flaviviridae and in the putative genus Hepacivirus. The genome has been divided into seven areas, the core region that encodes the capsid C protein, the E1 and E2 regions that encode two envelope proteins, gp33 and gp72 and several nonstructural protein regions, NS2, NS3, NS4 and NS5, that encode six proteins. HCV RNA sequences show significant genetic variability and six genotypes have been identified each containing different subtypes. In order of decreasing prevalence, genotypes 1, 3, 2 and 5 are found in Australia. Immunity to one subtype does not confer immunity to other subtypes.

LABORATORY TESTS

Laboratory tests are aimed at detecting specific antibody (Serology) or the presence of virus (Molecular assays). Antibody appears during or soon after the acute phase but viraemia, which commences earlier, may continue indefinitely. Once positive, antibody persists in the serum indefinitely in untreated patients.

Serology (anti-HCV)

Serology comprises screening enzyme immunoassays (EIA) and confirmatory recombinant immunoblot assays (RIBA). In the first year after virus exposure, > 99.5% of individuals with intact immune system are anti-HCV positive with both screening and confirmatory assays.

The detection of anti-HCV has evolved from first to third generation tests, with the more recent tests detecting antibody sooner and with greater specificity than the earlier tests. All tests employ a combination of synthetic or recombinant DNA-derived HCV peptides. All the third generation tests employ a mixture of HCV peptides (usually the core, NS3, NS4 and NS5).

Serum with a reactive EIA is tested by a second assay (with different antigens) to confirm the initial result. If reactive in both assays, the specimen is considered positive for anti-HCV. Discrepant results in the two EIA’s are followed-up by confirmatory RIBA, in which antibodies to each individual recombinant protein are detected. Each generation of RIBA (RIBA-1, RIBA-2, RIBA-3) corresponds with each generation of screening assay. RIBA are considered positive if antibodies to two proteins from different regions of the genome are present.

Molecular assays

PCR aids in the diagnosis of acute HCV and determines quantitative levels of virus.

1) QUALITATIVE HCV PCR is extremely sensitive and specific. It can detect as few as 10⁴ copies/mL. However false negatives and false positives can occur. At the present time a negative PCR should not be regarded as infallible evidence that the individual is non-infectious.

2) QUANTITATIVE ASSAYS FOR HCV RNA are used to monitor response to antiviral therapy. The level of pretreatment viraemia may be a determinant of interferon responsiveness. About 33% of patients will become HCV PCR negative at 3 months which is an indication of good therapeutic response. Approximately 50% will relapse when therapy is stopped.

3) GENOTYPING is performed using PCR techniques. Different HCV genotypes vary in response to interferon.

CHOOSING THE APPROPRIATE HCV TEST/S

1. Screening

• Asymptomatic patients from epidemiological risk groups (eg. past or present IVDU, blood product receipt prior to 1990, tattoos, body piercing, prisoners, long-term sexual partners of anti-HCV positive people) = anti-HCV

Note. A positive anti-HCV test provides good evidence of past or current infection but does not distinguish between the two. There is a significant window period during acute infection when the patient is viraemic but antibody negative. Seroconversion window period is 6 to 8 weeks with anti-HCV. In immunocompromised patients, this window period may extend for months or years. PCR may be required to make the diagnosis in these cases. Viraemia window period is days to weeks with PCR.

• Occupational exposure = anti-HCV (+/- PCR – discuss with clinical microbiologist on 83822317).

It is necessary to a repeat a negative anti-HCV test at intervals of 3 or 6 months.

2. HCV testing on patients with clinical hepatitis

• Anti-HCV

If negative – retest after 3 - 6 months

Note. Few patients present with acute HCV

3. HCV testing of infants

Transplacental anti-HCV remains detectable for 6 months or longer.

• One month to 12 months of age - HCV PCR on serum is clear evidence of infection.

• > 12 months - anti-HCV (a positive result is a strong indication that the infant is infected).

4) Further investigation of asymptomatic anti-HCV positive patients

• Is HCV still active?

Qualitative PCR. A positive PCR on serum is clear evidence of current virus replication (a negative result may mean low level viral turnover).

• Does the patient have significant liver disease?

This is difficult to answer. Patients with normal serum ALT can have cirrhosis on liver biopsy. It is usual to retest ALT after 3-6 months before contemplating liver biopsy.

5) Monitoring anti-viral therapy

• Monitor quantitative HCV RNA serum levels during interferon therapy.

One approach is testing a sample for a baseline prior to initiation of therapy, at 3 months after treatment has begun, at the end of therapy (at 3 months, 6 months or 12 months) and then 1 - 3 months following completion of therapy.

Cossart Y. E. Laboratory Investigation of Hepatitis C: a review. Pathology 1999; 31: 102 –108.

Cook L. Hepatitis C Virus Diagnosis and Therapeutic Monitoring: Methods and Interpretation. Clinical Microbiology Newsletter May 1, 1999; 21, No. 9: 67 – 73.

Farrell G.C. Chronic Viral Hepatitis. Medical Journal of Australia 15 June 1998; 168: 619 – 623.