New combination test for hepatitis C virus genotype and viral load determination using Amplicor GT HCV MONITOR test v2.0

Abstract

Aim: To develop a new sensitive and inexpensive hepatitis C virus (HCV) combination test (HCV Guideline test) that enables the determination of HCV genotypes 1, 2 and 3, and simultaneous determination of HCV viral load using commercial Amplicor GT HCV MONITOR test v2.0 (microwell version).

Methods: The HCV Guideline test used the PCR product generated in commercial Amplicor GT HCV Monitor test v2.0 for viral load measurement using microwell plate version of Amplicor HCV Monitor and also captured on separate plates containing capture probes and competitive oligonucleotide probes specific for HCV genotypes 1, 2 and 3, The HCV genotype was subsequently determined using the biotin-labeled PCR product and five biotin-labeled HCV-specific probes.

Results: The sensitivity of the HCV Guideline test was 0.5 KIU/mL. Specificity of the HCV Guideline test was confirmed by direct sequencing of HCV core region and molecular evolutionary analyses based on a panel of 31 samples. The comparison of the HCV Guideline test and an in-house HCV core genotyping assay using 252 samples from chronic hepatitis C patients indicated concordant results for 97.2% of samples (59.5% genotype 1, 33.7% genotype 2, 6.0% genotype 3, and 0.8% mixed genotypes). Similarly, the HCV Guideline test showed concordance with a serological test, and the serological test failed to assign any serotype in 12.7% of the samples, indicating a better sensitivity of the HCV Guideline test.

Conclusion: Clinically, both viral load and genotypes (1, 2 and 3) have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C based on guidelines and they are, in normal circumstances, performed as separate stand-alone assays. The HCV Guideline test is a useful method for screening large cohorts in a routine clinical setting for determining the treatment regimen and for predicting the outcome of antiviral therapy of chronic hepatitis C.

Figure 1

HCV Guideline test flow.

Figure 2

Strategy of HCV Guideline test. Step 1: The PCR product (here genotype 1) was hybridized to the specific capture probe, immobilized on a micro-plate. Other PCR products (e.g. of genotype 2) did not hybridize to the immobilized probe and were captured by the competitive oligonucleotide probes. The genotype specific-hybridized PCR product is retained on the micro-plate, while other PCR products were washed out from the plate. Step 2: Additional universal biotin-labeled-probes hybridized to the specific PCR product resulting in signal amplification. Step 3: Biotin reacted with streptavidine which was conjugated to the horseradish-peroxidase (HRP) enzyme. The color reaction was measured after the addition of enzyme specific substrates.

Figure 3

Specificity of HCV Guideline test. 36 anti-HCV-positive samples and 36 anti-HCV-negative samples were used to determine the specificity of the HCV Guideline test. All the anti-HCV-positive samples showed an absorbance greater than 0.2, whereas all anti-HCV-negative samples showed an absorbance below 0.2.