Hepatitis Virus Characterisation

HCV RNA Qualitative assay

As there is no suitable method for direct detection of viral antigens, assays have been developed to detect the presence of HCV RNA in blood. The commercial kit from Roche, the HCV AMPLICOR had been used since 1994 and was superceded by the semi-automated HCV COBAS AMPLICOR test. The test is specific and the limit of detection is 50 International Units/mL.

HCV Genotype Assay

HCV is an RNA virus with considerable heterogeneity. Using sequence analysis and phylogenetics, HCV can be classified into sets of distinct genotypes which, in some instances, can be further divided into subtypes. At VIDRL, genotyping is done using the Bayer line probe assay, LiPA 11, which has been designed to discriminate genotypes and subtypes based on the small but characteristic differences in sequence in the 5’untranslated region of the virus. This assay can detect the main genotypes present but does not detect the newly discovered genotypes found mainly in South East Asia, genotypes 7, 8 and 9. In Australia, the main genotypes are genotype 1 and 3 as shown in the graph below.

hcv_genotypes

Determination of HCV genotype is a clinically useful tool as it is a major predictor of treatment outcome. Those patients with genotype 1 or 4 need 12 months treatment for optimal response whereas those with genotypes 2 or 3 can achieve the best result after only 6 months treatment. Due to the large throughput of genotyping data in our laboratory, we have created a database of results from samples which have usual banding patterns on the LiPA. By sequencing in the HCV core region after PCR amplification, we have been able to establish the true genotype status of these samples. This page describes the unusual banding pattern data.

HCV quantification (viral load) test

This test measures the amount of virus present in the blood and is also a useful predictor of response to therapy. A correlation has been established with pre-treatment levels of virus and response to treatment, with those patients with lower pre-treatment levels having a more favourable response. More recently, it has been shown that viral load testing at week 12 of therapy can also help in predicting the patient’s longer-term response to therapy. Patients without a drop in viral load of greater than 2 logs from screening to week 12 most likely will not achieve a sustained response and therapy should be interrupted. The test used at VIDRL is the Bayer HCV 3.0 bDNA assay which has a dynamic range of 3,200 – 40,000,000 copies/mL or 615 – 7,692,310 IU/mL. See graphs below for distribution of levels for samples tested at VIDRL.

hcv_vload

HCV Viral Load Distribution Jan 2005 – July 2007 (n=4020)

[HCV] IU/mL Observed Expected* [HCV] copies/mL
cutoff Log10IU % Cumulative % Cumulative cutoff Log10
<=1000 3.00 0.7 0.7 0.1 0.1 <=5200 3.72
2138 3.33 1.4 2.1 0.3 0.5 11117 4.05
4677 3.67 1.5 3.6 1.0 1.4 24322 4.39
10000 4.00 2.6 6.2 2.3 3.7 52000 4.72
21380 4.66 3.7 9.9 4.6 8.3 111174 5.05
46774 4.67 5.6 15.5 8.0 16.3 243222 5.39
100000 5.00 9.2 24.7 11.8 28.1 520000 5.72
213796 5.33 12.7 37.4 14.8 43.0 1111740 6.05
467735 5.67 15.4 52.8 15.9 58.9 2432223 6.39
1000000 6.00 17.2 70.0 14.6 73.5 5200000 6.72
2137962 6.33 16.1 86.1 11.4 84.8 11117403 7.05
4677351 6.67 10.0 96.1 7.6 92.4 24322227 7.39
10000000 7.00 3.9 100.0 4.3 96.7 52000000 7.72
21379621 7.33 0.0 100.0 2.1 98.7 111174029 8.05
46773514 7.67 0.0 100.0 0.9 99.6 243222273 8.39
100000000 8.00 0.0 100.0 0.4 100.0 520000000 8.72
*for Lognormal distribution
 

The Bayer Versant HCV RNA 3.0 Assay Kit was used to obtain the above HCV Load results. The lower limit of the assay is 615 IU/mL (3200 copies/mL). The upper limit of the assay is 7,692,310 IU/mL (40,000,000 copies/mL). Results from patients on treatment were removed prior to analysis (where this information was available). The red line curve is the best fit for a Lognormal distribution. The blue bars represent the actual distribution of HCV load values. From the table, about half the patients tested had a pre-treatment load of approximately 500,000 IU/mL (2,500,000 copies/mL).