A multicenter study evaluation of the digene hybrid capture II signal amplification technique for detection of hepatitis B virus DNA in serum samples and testing of EUROHEP standards

Abstract

We have evaluated the new Digene Hybrid Capture II HBV DNA Test (HCII HBV), which is a 96-well microtiter plate-based signal amplification assay. This test uses hybrid capture technology that specifically detects RNA-DNA hybrids. HCII HBV is able to quantify hepatitis B virus (HBV) DNA at between 1.4 x 10(5) and 1.7 x 10(9) HBV copies per ml in a standard format. By using a modified sample preparation method, which allows the input of 30-fold more serum for an ultrasensitive format, the sensitivity of the assay can be increased reproducibly to approximately 8,000 copies of HBV per ml. By using a combination of these two formats, the assay can quantify over a total range of 6 logs. In our multicenter evaluation study, the mean laboratory-to-laboratory coefficients of variation were 22, 7, and 12% at the three sites, respectively, with a combined specificity of 98.4%. The linearities of both the standard test and the ultrasensitive test were excellent, with Spearman correlation coefficients of 0.997 and 0.999, respectively. Furthermore, the intra-assay reproducibility for the standard assay gave coefficients of variation of from 13 to 33, 9 to 21, and 3 to 8% at the three sites, respectively. HCII HBV was shown to be genotype independent when the EUROHEP standards for genotypes A and D were used. This assay allows the accurate measurement of HBV DNA levels in serum and can be clinically used for the monitoring of responses to antiviral agents for patients chronically infected with HBV.

FIG. 1

(A) Correlation of HCS tube-based assay with HCII HBV. The calculated log₁₀ DNA concentrations were calculated for a set of 89 randomly selected clinical samples. Linear regression analysis showed a Spearman correlation of 0.986. (B) Comparison of HCS tube-based assay and HCII HBV as described by Bland and Altman (2). Only data for samples that were positive and above the detection level of the HCS tube-based assay are included. geq, genome equivalents.

FIG. 2

(A) Linear range of HCII HBV determined in the standard format. Five twofold serial dilutions were made from five different patients. Each sample was analyzed three times at the three different sites. The data for each site are marked with a different symbol. Linear regression was performed for input DNA concentrations. (B) Linear range of HCII HBV determined in the ultrasensitive format. Five twofold serial dilutions were made from three different patients. Each sample was analyzed three times at the three different sites. The data for each site are marked with a different symbol. Linear regression was performed for input DNA concentrations.

FIG. 3

Combined results of interlaboratory variation for quantitation of HBV DNA in 10 samples with viral loads of between 8.7 × 10⁶ and 4.4 × 10⁸ HBV DNA copies per ml. Each sample was tested singly on 3 consecutive days at each of the three sites. The data from the individual sites are depicted with different symbols.

FIG. 4

(A) Genotype-specific linear range of HCII HBV determined in the standard format. Serial dilutions were made from EUROHEP standards of genotypes A (⧫) and D (□). Each sample was analyzed at least three times. Linear regression was performed for input DNA concentrations. (B) Genotype-specific linear range of HCII HBV determined in the ultrasensitive format. Serial dilutions were made from EUROHEP standards of genotypes A (⧫) and D (□). Each sample was analyzed at least three times. Linear regression was performed for input DNA concentrations, geq, genome equivalents.