Hepatitis A (HAV):
Immune status: To determine if some one is immune to HAV either due to vaccination or previous infection the anti-HAV total is our diagnostic test. This assay measures both IgG and IgM antibodies and reflects, "total immunity". We do not offer a test that measures only HAV IgG.
Acute infection: To determine if a person has had an acute or recent infection with HAV anti HAV IgM should be ordered. Note that the IgM antibody may remain detectable for more than 6 months so a positive test may represent a "recent" rather than an acute infection. Occasionally acute EBV, parvovirus B19, and hepatitis E virus infections can produce an immune response that will cross-react with the HAV IgM test. Recommendations from the National Microbiology Laboratory (NML) suggest that prior screening with HAV total antibodies will reduce the possibility of potential false positive reactions. Patients with acute HAV will also have IgG in addition to IgM by the time they present and these other acute viral infections do not appear to cross-react with the anti-HAV total assay to the same extent. Upon this recommendation we will now be performing anti-HAV total assay prior to HAV IgM testing and will not proceed with HAV IgM testing if the total antibodies are negative.
Occasionally we have situations where a patient's serum is above the positive cut off point in our test however the "level of positivity" or the amount that the sample tests positive is a "low positive". This often represents a false positive and further confirmation testing by the National Microbiology Laboratory will be performed.Hepatitis B (HBV):
We offer assays that can determine the presence of Hepatitis B surface antigen (HBsAg). Antibodies directed at HBsAg (anti HBs) and IgG antibodies to hepatitis B virus core antigen (anti-HBc).
Chronic infection: To determine whether a patient is a chronic HBV carrier HBsAg should be ordered. A positive HBsAg detected on two separate occasions at least 6 months apart suggest that patient is a chronic carrier of HBV. Occasionally we will have patient with an indeterminate HBsAg where the sample tests in our indeterminate range. This may reflect waning HBsAg levels during seroconversion or may reflect a false positive result. An anti-HBc may help in differentiating theses situations; If the anti-core is negative it is unlikely that the patient has HBV and the indeterminate HBsAg most likely represents a false positive result. The samples will be referred to the NML for further testing consisting of a different assay to look for the presence of HBsAg and qualitative PCR to determine the presence of HBV viral DNA. The results of these tests may take up to 6 weeks to return.
Patients with chronic infection will need further work up to determine if treatment is warranted, consisting of HBeAg and anti-HBe and HBV viral load. These tests are also valuable in following people who are on treatment. These tests are sent to the NML and take up to 6 weeks for results to return. Because these test are not routine, these tests will only be processed if the ordering physician is a Gastroenterologist or infectious diseases physician.
Immunity: Immunity to HBV either through vaccination or from resolved infection can be determined by testing for anti-HBs. A level of >10 IU indicates immunity, a level of <10 IU suggests that the patient lacks immunity and is susceptible to infection. Immunized individuals who have been shown to have at least one titre of greater or equal to 10 IU are considered to have lifelong immunity, even if subsequent titres fall below this level. Very rarely a patient may have both HBsAg and anti-HBs. This likely reflects a mutant HBV that has changes in its surface antigen composition.
Anti HBc: The utility of anti HBc is limited to special circumstances. It is routinely used in the transplant program for screening donors and recipients. In addition, an anti-HBc can be used to differentiate immunity based on vaccination vs infection; Anti-HBc are only present in someone who has had HBV infection and will be absent in someone who has anti-Hbs due to vaccination. It can also be helpful in differentiating ongoing HBV infection in the presence of a HBV mutant that is not picked up by our HBsAg test. All of these circumstances are rare; therefore anti-HBc will only be performed on consultation with a Microbiologist.Hepatitis C (HCV):
We offer anti-HCV as a screening test, qualitative PCR as a confirmatory test, recombinant immunoblot assay (RIBA) as a supplemental test, HCV genotyping and HCV quantitative (viral Load) PCR.
HCV screening: If the anti-HCV antibodies are positive confirmation testing using qualitative PCR is used to confirm whether or not the person has active infection. A negative HCV PCR can represent three possibilities;- the positive anti-HCV represents a false positive result;
- the person has spontaneously cleared the virus (this can occur in 15% of people infected with HCV) or
- 3a negative PCR may reflect a very low level of infection which is below the limit of detection of our PCR test (the lower limit of detection in the assay we are using is 50 genome copies/ml).
To distinguish between past infection and a false positive screening test, a RIBA is performed. This test is essentially the same as a western blot in HIV testing that looks for the presence of antibodies directed at specific HCV antigens in the patient's serum (figure 1). The presence of ≥2 of four bands suggests the screening test is correct and that the patient has had a previous infection. A RIBA test with no bands is negative which means that the screening test was a false positive and the patient has not been infected with HCV. A RIBA test that has only one band is considered indeterminate. This may reflect a window period where the HCV RNA disappears and seroconversion is occurring or may represent a false positive. The patient should be retested in 3 months. If the patient remains indeterminate the specimen can be repeated to more times and if they remain inderetminate it is unlikely that the person has HCV and the positive anti-HCV and indeterminate RIBA are falsely positive. Because the person will likely always remain indeterminate repeat testing should not be performed unless there is history of a new exposure.
Figure 1. HCV RIBA testing
The first strip illustrated the positions of the HCV proteins on the blot. The remaining 4 blots are examples of negative, positive and indeterminate results (respectively).
From a laboratory perspective it may be difficult to distinguish between patients in whom the virus has been cleared and those with a level of viral replication below the limit of detection. In this circumstance the PCR should be repeated, particularly if there is any clinical evidence to suggest they have active disease (e.g. an elevation in liver function tests). Although there is the slim possibility that there is a low level of replication, these patients do not progress to get severe liver disease and do not need treatment.
HCV PCR (qualitative): In addition to its utility as a confirmatory test in screening, the qualitative PCR is also useful in monitoring patients who are on treatment. Studies have demonstrated that a person who has a positive PCR test at 24 weeks of treatment will not respond to treatment. This allows someone to stop a therapy that is every expensive and may have significant side effects rather than continuing on for the complete 48 week course.
HCV genotyping: Treatment of HCV infection is tailored for patients depending on their genotype and viral load testing. Patients with genotype 2 or 3 require only 24 weeks of treatment compared with 48 weeks in patients with genotypes 1, 4 or 5. Currently genotyping is done using a line probe assay. Occasionally it is unable to give a distinct genotype. When this occurs the sample will be referred to the NML for sequence based testing to determine the genotype.
HCV viral load testing: Quantitative PCR to determine the viral load in patients with HCV infection is an important tool in the management of HCV viral infection. The lower limit of detection of this assay is 600 copies/ml. In studies examining the viral kinetics in patients being treated with pegylated interferon and ribavirin there appears to be a biphasic response to therapy. In the initial phase the treatment produces a steep decline in the HCV-RNA levels in the first 24-48 hours due to the blockade of virus production and release. This reduction can be up to a 4-log decease in virus. The second elimination phase is slower and correlates better with patients who will go on to have a sustained virologic response. This stage reflects whether the immune mediated clearance of virus-infected cells. There is a third phase present in some patients receiving ribavirn in combination with interferon compared with those receiving peg-interferon alone. This stage was felt to represent enhanced degradation of infected cells.
Studies using HCV viral load monitoring have shown that it can be predict which patients will go on to have a sustained virologic response. Patients who had a greater than 2 log drop in their viral load compared to base line (defined as an early virologic response (EVR) have a positive predictive value of about 80% (i.e. 80% will go on to have a sustained virologic response which is defined as qualitative PCR negative 6 months after completion of therapy). If a patient fails to achieve this drop by 12 weeks of therapy they will not respond. This allows the patient to stop therapy thereby saving money (the cost of treatment is about 1800.00 a month) and alleviate potential side effects of treatment. This is mainly for patients with genotypes 1,4 or 5. Genotypes 2 or 3 respond well (95% of patients will have EVR) and have a shorter duration of total therapy (24 weeks) testing at 12 weeks is not cost effective.
Due to the expense of these tests they will only be conducted when ordered by a gastroenterologist, transplant surgeon or infectious disease physician, to ensure that those who are going to be treated will have priority.
The following table provides different possible results and an interpretation of these results as a quick reference guide:
| Result | Interpretation |
HAV |
|
| Anti-HAV IgM positive | Suggestive of acute or recent infection or vaccination. Clinical correlation required (HAV IgM can last up to 6 months) |
| Anti-HAV IgM "low positive" | A "low positive" and may reflect a false positive result. Occasionally acute EBV, parvovirus B19, and hepatitis E virus can produce an immune response that will cross react with the HAV IgM test. Specimen is being referred to the National Microbiology Laboratory for Confirmation. Clinical correlation is required. |
| Anti-HAV IgM indeterminate | Early acute or convalescent or false positive result. Specimen is being referred to the National Microbiology Laboratory for Confirmation. |
| Anti-HAV IgM negative | Results suggest no evidence of acute infection |
| HAV total antibodies positive. | Suggests immunity from prior infection or vaccination. Clinical correlation required. |
| HAV total antibodies negative | No evidence of immunity to HAV |
| HAV IgM not performed | The anti-HAV total Abs are negative. Occasionally acute EBV, parvovirus B19, and hepatitis E virus can produce an immune response that will cross react with the HAV IgM test. Recommendations from the NML suggest that prior screening with HAV total antibodies will reduce the possibility of potential false positive reactions as these acute viral infections do not appear to cross react with the anti-HAV total Ab assay to the same extent. |
| Result | Interpretation |
HBV |
|
| HBsAg positive | Acute or chronic infection. Repeat testing in 3-6 month would be helpful in defining whether infection is chronic (HbsAg positivity persists). |
| HBsAg indeterminate | Indeterminate HBV surface antigen (HbsAg), which is of uncertain significance at this time. This may represent waning HbsAg levels in the process of seroconversion or a false positive result. Sample sent to National Microbiology Laboratory for Confirmation testing. |
| HBsAg negative | No evidence of HBV infection |
| HBsAg negative, anti-Hbs positve, anti-HBcAb positive. | Hepatitis B surface antigen (HbsAg) negative in the presence of anti-HBV core antibodies and anti HBs. This pattern suggests resolved HBV infection |
| Anti-HBs (immune) | An antibody level of ≥10 IU indicates immunity to HBV from previous vaccination or infection |
| Anti-Hbs (non-immune) | An antibody level of <10 IU indicates is not immune and is susceptible to HBV. (unless previously known to have titre of >/ 10 IU in past) |
| Anti-HBs (immune) and Anti-HBc negative | Presence of anti-HBs in the absence of anti - HBV core is suggestive of immunity from previous vaccination |
| Result | Interpretation |
HBV |
|
| Anti-HCV negative | Unlikely to have HCV infection. Occasionally immunosuppressed individuals, particularly HIV patients will not have any serologic evidence of infection but will be PCR positive. Clinical correlation is required. |
| HCV-RNA (PCR) positive | Active HCV infection |
| Anti-HCV positive HCV PCR negative HCV - RIBA positive |
Past infection with resolution and viral clearance or current infection with viral RNA that is below the limit of detection. If clinically warranted, send a repeat specimen for HCV-RNA testing in one month |
| Anti-HCV positive HCV PCR negative HCV - RIBA indeterminate |
Window period during seroconversion or a false positive. Patient should have repeat testing in 3 months |
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