Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations

Quantitative detection of hepatitis B virus (HBV) in serum or plasma is of significance for monitoring of therapy and establishment of the prognosis o f the disease, as well as for infectivity assessment and quality control of the diagnosis. Unfortunately, various commercially available test kits for HBV DNA yielded conflicting quantitative results, with differences of up t o a factor of 120, The Eurohep Pathobiology Group has established two refer ence samples of plasma from HBV carriers and determined as accurately as po ssible the number of HBV DNA molecules in these samples. Plasma donations f rom two single highly viremic carriers of HBV genotype A (HBV surface antig en subtype adw2) and genotype D (ayw2/3), respectively, were collected, and coded dilutions of these samples were analyzed by members of the Eurohep P athobiology Group. Quantitative results from the seven laboratories reporti ng consistent results were initially divergent. Limiting dilution and neste d PCR assays suffered from incomplete DNA extraction. Hybridization assays used inaccurately quantitated cloned DNA as a reference. Two hybridization assays could not be calibrated directly with cloned HBV DNA, because virion -derived DNA reacted much less efficiently. After identification and elimin ation of these problems, limiting-dilution assays from three laboratories a nd hybridization assays from two producers generated consistent and concord ant results: 2.7 x 10(9) HBV DNA molecules/ml (range, 2.1 x 10(9) to 3.4 x 10(9) HBV DNA molecules/ml) in the plasma from the carrier of genotype A an d 2.6 X 10(9) HBV DNA molecules/ml (range, 2.1 x 10(9) to 3.0 x 10(9) HBV D NA molecules/ml) in the plasma from the carrier of genotype D. The two Euro hep reference plasma samples have already been used for the standardization of test kits and in quality control trials, and the plasma from the carrie r of genotype A will probably be the basis of a World Health Organisation r eference sample.