Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B

Abstract

Background: The Amplicor™ HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates.

Objective: The performance of the Amplicor™ HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor™ HBV Monoitor assay was compared to the Digene Hybrid Capture™ System HBV DNA assay for the quantitation of HBV in patient sera.

Study design: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays.

Results: The detection limit was found to be 10³ copies/ml with the Amplicor™ PCR assay compared to 10⁶ to 10⁷ copies/ml with the Digene™ hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor™ PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10³ and 10⁷ copies/ml and all of them tested below the detection limit with the hybridization assay.

Conclusion: The Amplicor™ HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.

Introduction

The level of hepatitis B virus (HBV) DNA in serum or plasma probably reflects accurately the replicative activity of HBV and has been shown useful in the assessment, management, and antiviral treatment of patients with chronic HBV infection (Perrillo et al., 1990, Pastore et al., 1992, Lok, 1994). Molecular assays for detecting serum HBV DNA have been used since the beginning of the 1980s. Early hybridization assays employed radioactive probes for membrane hybridization (Lieberman et al., 1983, Scotto et al., 1983) or liquid hybridization (Kuhns et al., 1989, Jalava et al., 1992) techniques. More recently, a commercial non-radioactive hybridization assay that uses RNA to bind single-stranded DNA followed by capture of hybrids by an antibody and measurement by chemiluminescence (Barlet et al., 1996, De Lamballerie et al., 1995) and the branched DNA technology (Urdea et al., 1987, Hendricks et al., 1995) were described. Introduction of polymerase chain reaction (PCR) for detection of serum HBV DNA resulted in a significant improvement of sensitivity (Kaneko et al., 1989, Heermann et al., 1995, Erhardt et al., 1996). In-house assays have been described that employed radioactive (Zaaijer et al., 1994) or non-radioactive (Lehtovaara et al., 1993, Ranki et al., 1995) hybridization techniques.

Recently, a commercially available PCR assay that utilizes DNA hybridization of amplified product followed by colorimetric detection on a microwell plate has been introduced. In this study, the performance of the new PCR assay was evaluated using HBV DNA standards and routine clinical sera and was compared with a commercially available non-radioactive hybridization assay.