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Hepatitis C (HCV) is a major cause of chronic liver disease in the United States. Approximately 4 million people in the United States are currently infected and an estimated 40,000 new infections are suspected each year. The initial diagnosis of HCV infection is based on positive HCV antibody tests and the detection of HCV RNA by qualitative PCR.
The initial test for hepatitis C (HCV) infection is an immunoassay that detects antibodies to multiple HCV proteins. Supplemental or confirmatory testing is recommended for all reactive HCV antibody tests to determine the presence of active infection. Detection of HCV RNA in the blood by polymerase chain reaction (PCR) is indicative of active infection. PCR testing has been available since September 1995. In this assay, a DNA copy of viral RNA is synthesized by reverse transcription. This DNA molecule is amplified millions of times by PCR. Because of its high sensitivity, PCR can detect HCV infection much sooner than antibody tests. Most patients have detectable levels of HCV RNA in plasma within 1 to 2 weeks of exposure. HCV RNA detection precedes ALT elevation by 10 to 12 weeks and seroconversion by 10 to 24 weeks. The highest levels of circulating viral RNA are found during the early course of infection, suggesting that patients are most infectious during this time. Variation among HCV genotypes is less likely to affect PCR test performance than antibody tests, because PCR primers are based on the highly conserved untranslated region of the HCV genome.
Two types of PCR assays are available; qualitative and quantitative. In the past, qualitative PCR had a lower limit of detection (50 IU/mL) than quantitative Amplicor PCR (600 IU/mL) and was used both as a confirmatory test for anti-HCV and as an end-of-therapy assay. Quantitative HCV PCR was the preferred test for determining viral load prior to therapy, along with HCV genotype.
Once HCV infection has been established, quantitative HCV PCR (viral load) can provide prognostic information. Treatment response has been shown to be greater for patients with low levels of HCV RNA. Most HCV infected patients should have viral load testing performed prior to treatment unless there are clear contraindications to antiviral therapy. The HCV positive patient with negative viral load should have the test repeated in 3 to 4 months, because some patients with active infection have intermittently undetectable viral loads.
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Predictors of response to antiviral therapy include viral load of less than 2,000,000 IU/mL, genotype other than 1, shorter duration of infection, female gender, and low body weight.
With Pegylated IF/Ribivarin, a sustained virologic response is indicated by a 2 log drop in viral load in first 12 wks
If virus is not undetectable by 24 weeks, a patient will most likely relapse
Two patterns of no response have been seen:
Null response = don't see 2 log drop in first 12 weeks; stop therapy
Partial response = achieve 2 log drop in first 12 weeks, but viral load stabilizes & doesn't decrease further; stop Rx at 24 wks because patients won't become viral negative
Genotype 1 has 20% null responders
80% early response within 12 weeks
65% become viral negative
40=50% have a sustained viral response
Genotype 2 has 3% null responders
97% have early virologic response
90% have sustained viral response
The Roche Taqman real time quantitative PCR method, which has a lower limit of detection of 50 - 80 IU/ml (Hillyard, Valsamakis, 2005 Roche Molecular Advisory Board). This lower limit of detection obviates the need for qualitative PCR. The new method is also linear up to 200 million IU/mL in the upper range. With the switch from Amplicor to Taqman, 10% of patients that had undetectable HCV viral loads by the Amplicor method are positive by Taqman real time PCR. This change in viral status is due to the decrease in the lower limit of detection from 600 to 80 IU/mL. The specimen requirement for HCV RNA by PCR is one EDTA, ACD, or SST tube of blood. Tubes containing heparin cannot be used. Serum needs to be separated from cells within 6 hours of collection and refrigerated or frozen to avoid degradation of viral RNA. HCV PCR and genotype can usually be performed on residual specimen from anti-HCV testing. Client services can be contacted at 932-3850 to add an order for HCV RNA by PCR.
