National Hepatitis C Testing Policy May 2007
8. Diagnostic strategies
- Exposure to HCV is determined by testing for HCV antibodies (anti-HCV) in serum or plasma.
- A sample not reactive in the screening immunoassay can be generally regarded as anti-HCV negative.
- A sample reactive in the screening immunoassay should be subject to a minimum of one alternative supplemental immunoassay to confirm the result.
- A sample reactive in two immunoassays with different antigen specificity can be reported as anti-HCV positive.
- Current HCV infection is usually determined by qualitative testing for HCV RNA.
- Qualitative HCV RNA testing should be a standard component of the diagnostic work-up of all anti-HCV positive individuals.
- The major role of HCV genotype and viral load testing is in guiding treatment dose and duration.
Laboratory investigations are usually directed towards answering one or more of the following questions:
- Has the patient been infected with HCV? This is usually determined by testing for HCV antibodies (anti-HCV).
- Does the patient have current infection? This is usually determined by testing for HCV RNA.
- What is the current level of virus replication? Viral load prior to treatment can be a prognostic indicator of long-term response to interferon-based therapy and can also be used to monitor response while on therapy.
- What is the infecting virus genotype? Virus genotype is a key predictor of treatment response and can be used to guide treatment dose and duration of therapy.
- What testing should be performed for epidemiological surveillance?
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8.1. Anti-HCV testing. Has the patient been infected with HCV?Exposure to HCV is usually assessed by performing an immunoassay for the detection of antibody to HCV (anti-HCV) in serum or plasma. The presence of true antibody shows that the patient has been infected with the virus but does not indicate whether the infection is acute, resolved or chronic. In a majority of cases (65–85%) an individual with genuine antibody to HCV will be chronically infected. The absence of anti-HCV usually means the patient has never been infected, although antibody may become undetectable years after the infection has been cleared. Furthermore, antibody may not be detectable very early in the course of infection (the so-called window period) or in people who are immunosuppressed, for example people with HIV infection and people undergoing therapies such as chemotherapy and dialysis.
Approaches to confirmatory testingIn Australia the practice for confirming the presence of true anti-HCV has been strongly influenced by the protocols set out in A Strategy for the Detection and Management of Hepatitis C in Australia.5 This document stipulated that all samples initially reactive in one immunoassay should be retested with a second independent immunoassay to confirm specificity. A sample is not considered to be positive for anti-HCV until the supplemental immunoassay has been performed and found to be reactive. The algorithm has created interpretative difficulties with samples that are reactive in the initial immunoassay but not the secondary one. Furthermore, samples that have low reactivity for technical reasons may also show low reactivity in a second enzyme immunoassay because some combinations of immunoassays can share false reactivity.6 Reasons for low or indeterminate activity include:
- Adventitious reactivity to the recombinant antigens
- Early period of seroconversion before antibody response has fully developed
- Immunosuppression by disease or therapy
- Waning antibody levels in resolved infection
- Maternal antibody passively transferred from an anti-HCV positive mother to her infant
- Testing with another immunoassay that uses antigen combinations that differ from those used in the first assay.
- Referral to a reference laboratory for advice or for further antibody testing or assessment by a qualitative test for HCV RNA.
Note: In the case of initially reactive results, it is important to follow the manufacturer's instructions on the package insert. These instructions are assessed by the Therapeutic Goods Administration as part of the evaluation of the test kit's performance. Responsibility for any deviation from the instructions rests with the user of the test kit. Non-compliance is considered a failure to perform the test as intended. Consequently, the in-vitro Diagnostic Device (IVD) will become an in-house IVD with the associated regulatory implications. For more information, refer to Chapter 9 Quality Assuring hepatitis C Testing.
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Appropriate combinations of immunoassaysHCV recombinant antigens have been based predominantly on expression of clones from HCV genotype 1. As a result, the same or very similar antigens are used by a number of immunoassays designed with different test formats. The National Serology Reference Laboratory, Australia (NRL) has identified the immunoassays that share common false reactivity, that is, false positive results as defined in the NHMRC guidelines. The reliability of the results of these assays used in combination and their ability to yield specific confirmatory results should be assessed in an ongoing manner. Using this information, the NRL can advise which combinations of immunoassay kits are likely to provide the most reliable results. However, as new generation and modified assays for anti-HCV become available, they too will need to be evaluated as to their compatibility with other immunoassays.
Discordant resultsBefore results are reported pathologists and medical laboratory scientists should be confident that a discordant result is not due to non-specific reactivity because of technical limitations of the initial assay. Otherwise, further testing should be done, such as repeating the initial assay, using a third immunoassay or immunoblot (perhaps by referring to a reference laboratory) or performing a qualitative HCV RNA test.
NotificationsIn Australia, it has been recognised that laboratory notifications of infectious diseases are a cornerstone of our disease surveillance system. Hepatitis C is a notifiable disease and it is important that notifications are reported to the respective State or Territory Health Department. Data is collated into the National Notifiable Diseases Surveillance System and then published in the Communicable Diseases Intelligence. (Refer to Chapter 5)
Determining anti-HCV positive samplesThe following minimum practices should be adopted by all pathology services in determining whether a sample is anti-HCV positive:
- A sample non-reactive on a single immunoassay screen can be confidently reported as anti-HCV negative.
- Assays should be performed in accordance with the manufacturer's instructions. Samples testing positive by one method should be subject to a minimum of one alternative supplemental immunoassay to enhance the positive predictive power of the assigned result.
- A sample that is reactive on two separate immunoassays based on different antigens and different immunoassay formats has a very high probability of containing true hepatitis C antibodies and can be reported as positive.
- When seeking to confirm a result, laboratories should use only appropriate pairings of first and supplemental immunoassays, as recommended by the NRL or other reputable sources such as the scientific literature.
- Test results should not be reported until these minimum steps to confirm anti-HCV have been taken.
- If further information is not available from the requesting practitioner, then the interpretive comment should indicate that this result may be owing to non-specific reactivity or due to recent or distant past HCV infection. Testing a second, follow-up sample is recommended to exclude recent infection.
- Perform HCV-RNA testing. If HCV RNA is detected, then this may indicate a possible recent infection. Note that if the sample tested is not a dedicated aliquot (i.e. collected for the purpose of performing NAT), contamination during antibody testing may contribute to a false positive HCV RNA result. It is recommended that a second sample be collected in 4 weeks to confirm seroconversion and allow notification as new infection. If a discordant sample is negative by qualitative HCV RNA testing, a repeat sample should still be requested for anti-HCV and/or HCV RNA testing to determine the true HCV infection status.
- If none of the above procedures is possible in the testing laboratory or if none is appropriate, the sample should be referred to a reference laboratory for further investigation.
8.2 . HCV RNA testing. Does the patient have current infection?Between 15 and 35% of people infected with HCV will spontaneously resolve their infection within the first year of initial exposure to the virus. The predictors of this spontaneous resolution are not yet clear. This specific subpopulation may be anti-HCV reactive but HCV RNA negative. A single negative test for HCV RNA does not, however, definitively exclude the presence of infection. Some patients have levels of virus around the limit of detection of the test and may be intermittently negative, or their viraemia may be suppressed by antiviral therapy. Others may have a negative result owing to suboptimal specimen storage and transport conditions. A positive result from an appropriate sample in a qualitative HCV RNA test provides a high degree of certainty that the patient is currently infected but it does not confirm chronic infection unless the HCV RNA is shown to persist for at least 6 months.
Qualitative HCV RNA testing should be a standard component of the diagnostic work-up of all individuals who are anti-HCV reactive.
8.3. Viral load measurement. What is the current level of virus replication?Measurement of the level of virus replication or viral load has become an important tool for clinical management of patients with HCV infection. The initial level of virus is a predictor of response to antiviral therapy and long-term response to therapy can be predicted by viral dynamics on therapy. In particular, for patients infected with HCV genotype 1, non-response can be predicted by viral load measurement at week 12 of therapy.
A number of commercial HCV RNA quantification assays are registered on the Australian Register of Therapeutic Goods (ARTG). These may be used for measuring levels of HCV RNA. They include target amplification assays, predominantly PCR based and signal amplification assays which rely on hybridisation of HCV RNA.
8.4 . Virus characterisation. What is the infecting virus genotype?Based largely on sequence divergence, HCV can be divided into six genotypes (HCV 1-6). In Australia, the predominant genotypes are 1 (around 55%) and 3 (around 35%). It has been shown that genotype is a major predictor of response to interferon-based therapy. A number of methods have been described to determine genotype and most are based on sequence variation in the conserved 5' untranslated region (5' UTR) and include direct sequencing after PCR amplification and a reverse phase hybridisation assay. Genotyping should only be carried out using test kits registered by the TGA.
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8.5. Epidemiological surveillanceData from the assays discussed, in particular immunoassay data, are regularly analysed to assess the extent and distribution of HCV infection in the community. For this purpose, it is critical that each confirmed positive result is classified as:
- A repeat sample from a known infected individual who has already been entered on the appropriate database.
The first test on an individual who has not previously been tested, that is, a newly notified individual for whom the date of acquisition of infection is not necessarily known.
A repeat test on an individual who previously tested negative, that is, a new infection or seroconversion where the time boundaries of the transmission event may be estimated. The Communicable Diseases Network of Australia defines a new infection as being a positive anti-HCV test in a patient known to have had a negative test within the preceding 24 months.
5National Health and Medical Research Council 1997, A Strategy for the Detection and Management of Hepatitis C in Australia, NHMRC, Canberra.
6Most low level activity in immunoassays is false reactivity but not always.
7In the IVD Policy to be introduced in 2007, any changes in using commercial tests outside the manufacturer's instructions will render the test an "in-house test" which will require validation of the test to the level required by the IVD Policy Rules.