The results of this study indicate that the accurate determination of N
in serum or plasma samples containing HBV particles is not a trivial task. We confirm previous reports (3
) that different hybridization assays generate highly divergent results. Butterworth et al. (3
) described an in-house dot blot assay by which the values obtained for an HBV DNA-positive serum sample were 120 times lower than those obtained by the bDNA assay. The values obtained by the widely used Genostics liquid hybridization assay were also 100 times lower than those obtained in that particular study. The results of our own (K.-H.H. and W.H.G.) in-house dot blot hybridization assay for R1 and R2 (25
) had to be corrected by use of a factor of 8 in light of our new data. This assay had a design similar to that of the dot blot assay from laboratory 5, which was not included for determination of the final result in phase 2. Virion-derived DNA does not behave, in most hybridization assays, like the cloned HBV DNA which is used as a reference sample. The attempt to use cloned HBV DNA encapsidated within a bacteriophage as a more realistic reference sample for the Genostics assay (14
) also failed. Virion-derived HBV DNA contains a covalently linked protein. This protein remains bound to the DNA if it is not removed by digestion with protease. It significantly enhances the binding of HBV DNA to glass fibers in low-salt buffers and causes incomplete elution from purification columns. Furthermore, it causes extraction of HBV DNA in the phenol phase unless it is completely removed (9
). Plasmid DNA into which HBV DNA is inserted is protein-free. Thus, calibration of assays of virion-derived DNA extracts with cloned DNA or consideration cloned DNA as a gold standard for the determination of virion-derived DNA is not justified unless the validity of the assumption has been proven. To overcome this problem, four participants in our study determined N
in extracts from virions by limiting-dilution assays which were able to recognize one molecule. For this type of assay no reference sample is required. Laboratory 1 initially had difficulties completely recovering virion-derived HBV DNA from the samples, probably because undiluted plasma exhausted the capacity of proteinase K digestion under the conditions that were used. Incomplete recovery of HBV DNA from virions also seems to have occurred in the Eurohep quality control trial of the HBV PCR (17
). The 39 participants in that trial received coded dilution series made from R1 or from purified cloned DNA and seven positive serum samples. Virus in one of these serum samples was missed by nine participants, who reported otherwise faultless results. These were considered “moderate performers.” Six of the 10 “good performers” who detected all seven positive serum samples were able to detect 300 Eurohep Units/ml, but none of the moderate performers was able to do so. Even 30,000 Eurohep Units/ml was not detected by six of the nine moderate performers. In contrast, 300 and 30,000 copies of cloned DNA were detected with equal success rates by the good and the moderate performers. In theory, detection of HBV DNA in dilution of R1 should have been easier than detection of DNA in the samples with cloned HBV DNA because, as shown in this report, they contained 2.7 times more HBV DNA than anticipated by the data from phase 1. The incomplete extraction could not be recognized because no virion-based reference sample with known N
was available to the participants at that time.
The consistent PCR results for R1 and R2 obtained by three laboratories that used four different methods of lysis or extraction and four different primer sets for PCR suggest that, within the statistical confidence intervals, the GMVs of N for R1 and R2 are close to the true value of N, although the possibility that minor amounts of HBV DNA were lost before PCR cannot be excluded. The fact that two quite different commercially available hybridization assays yielded concordant values which were well within the 95% CI of the limiting dilution assays suggests that these two assays detect cloned and virion-derived DNA equally well, at least within the accuracies of the limiting-dilution assays.
The numbers of HBV genomes in R1 and R2 are typical for low-level or asymptomatic HBV carriers with high concentrations of HBsAg and HBeAg. By immune electron microscopy, similar plasma samples were found to contain 2 × 1010
HBV-like particles per ml which were coimmunoprecipitated with the known number of 20-nm HBsAg particles in these samples (20a
). The higher value for particle numbers than for genome numbers found in this study may be explained by the similar but nonidentical plasma samples, the potential overrepresentation of HBV particles in HBsAg immune complexes, and the existence of a variable proportion of empty HBV particles which may account for more than half of the particles (23a
). Although not directly comparable to this study, our previous studies on particle numbers indicate that the virion-associated genome numbers found in this study are not unexpectedly high. For standardization of HBV DNA assays, the use of particle counts is, however, not technically feasible. Infectivity titers in HBeAg-positive samples from chimpanzee virus carriers are about 108
infectious doses/ml or greater (20
). This implies, in agreement with previous studies (1
), that in such HBV carriers almost every 10th virion is infectious, although variations in this ratio are likely to occur in individual carriers. Our data also imply that virions are not heavily aggregated in such samples because N
was found to be the same irrespective of whether the limiting-dilution assay was done with plasma dilutions or dilutions of the DNA extract (10a
This study shows that the results of the widely used Genostics assay are very precise, but they must be multiplied by a factor of 28 if the genotype of HBV DNA is not known. The correction factor suggests that the total viral release in a carrier with a high level of viremia is not
virions/day, as determined by Genostics, but is closer to 1013
). It appears that the Genostics assay reacted better with genotype D than with genotype A by a factor of 1.5. A genotype effect was also found by laboratory 5, but in laboratory 5 genotype A was better detected than genotype D by a factor of 2. In light of these observations it is highly advisable to evaluate a new method for the quantitative determination of HBV DNA first with R1 and R2 or similarly designed reference samples infected with different genotypes. Only if samples with virions of different genotypes yield consistent results that are comparable to those obtained with cloned DNA may these less practical reference samples be replaced by samples containing cloned HBV DNA of one genotype. Assays showing significant differences by genotype are not suitable for use for routine diagnosis in the laboratory because genotyping will usually not be done. If such assays are used, the calibrations obtained with R1 and R2 may not be valid for genotypes (15
) which are prevalent in East Asia (genotypes B and C), Africa (genotype E), and South America (genotype F). Depending on the genome region of the PCR amplification or hybridization probe, different results may be obtained if the HBV carrier contains genomes with large deletions. This was, however, not the case for R1 or R2 because PCRs covering either the X or S gene region yielded identical results.
The use of reference samples R1 and R2 has been essential for clarifying the divergences between various assays and for quality control trials. They do not satisfy the requirements of a World Health Organization-approved international standard concerning volume and lyophilization. It is planned that such a standard be derived by diluting R1 in HBV-negative cryoglobulin supernatant and freeze-drying it for that purpose (19a
). R1 and R2 remain available for special studies.