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Journal of Clinical Microbiology, January 1999, p. 68-73, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Quantitative Detection of Hepatitis B Virus DNA in Two International Reference Plasma Preparations

Klaus-Hinrich Heermann,1 Wolfram H. Gerlich,2,* Michael Chudy,3 Stephan Schaefer,2 Reiner Thomssen,1 and The Eurohep Pathobiology Groupdagger

Division of Medical Microbiology and National Reference Laboratory for Viral Hepatitis, University of Göttingen, D 37075 Göttingen,1 Institute of Medical Virology, Justus Liebig University Giessen, D 35392 Giessen,2 and Paul Ehrlich Institute, D 63225 Langen,3 Germany

Received 30 June 1998/Returned for modification 2 September 1998/Accepted 9 October 1998

Quantitative detection of hepatitis B virus (HBV) in serum or plasma is of significance for monitoring of therapy and establishment of the prognosis of 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 to a factor of 120. The Eurohep Pathobiology Group has established two reference samples of plasma from HBV carriers and determined as accurately as possible the number of HBV DNA molecules in these samples. Plasma donations from two single highly viremic carriers of HBV genotype A (HBV surface antigen subtype adw2) and genotype D (ayw2/3), respectively, were collected, and coded dilutions of these samples were analyzed by members of the Eurohep Pathobiology Group. Quantitative results from the seven laboratories reporting consistent results were initially divergent. Limiting dilution and nested 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 elimination of these problems, limiting-dilution assays from three laboratories and hybridization assays from two producers generated consistent and concordant results: 2.7 × 109 HBV DNA molecules/ml (range, 2.1 × 109 to 3.4 × 109 HBV DNA molecules/ml) in the plasma from the carrier of genotype A and 2.6 × 109 HBV DNA molecules/ml (range, 2.1 × 109 to 3.0 × 109 HBV DNA molecules/ml in the plasma from the carrier of genotype D. The two Eurohep reference plasma samples have already been used for the standardization of test kits and in quality control trials, and the plasma from the carrier of genotype A will probably be the basis of a World Health Organization reference sample.

* Corresponding author. Mailing address: Institute of Medical Virology, Justus-Liebig-University Giessen, Frankfurter Str. 107, D 35392 Giessen, Germany. Phone: 49-641-99 41200. Fax: 49-641-99 41209. E-mail: Wolfram.H.Gerlich{at}viro.med.uni-giessen.de.

dagger Members of the Eurohep Pathobiology group were G. Gerken and K. H. Meyer zum Büschenfelde (coordinators) and F. Bonino, C. Brechot, M. C. Carneiro de Moura, W. H. Gerlich, H. C. Thomas, and S. Schalm (head).

Journal of Clinical Microbiology, January 1999, p. 68-73, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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