In the present paper, we have demonstrated that the analytical sensitivity of HCII HBV was approximately 10-fold higher than that of the HCS tube-based assay, with a detection limit of 200,000 copies of HBV DNA per ml. A simple high-speed centrifugation step further increased the sensitivity of HCII HBV down to 8,000 copies per ml. This step did not interfere with the binding of the RNA probe to the HBV DNA or the capture of the RNA-DNA hybrid to the 96-well capture plate and led to a more than 300-fold higher sensitivity than that obtained with the HCS tube-based assay. The detection limit thus obtained is more in the range of the detection limits of standard target amplification assays, like PCR. A great practical advantage of these two formats is that within a single plate the HBV DNA loads in samples with HBV DNA load differences of more than 6 logs can be simultaneously determined. Current commercially available PCR-based techniques can detect as few as 1,000 copies per ml if the EUROHEP panel is used as a standard, which is a narrower range of detection (11
). This indicates that with the new technology, the differences between standard hybridization assays, also called signal amplification assays and target amplification assays, like PCR or nucleic acid sequence-based amplification (NASBA), are greatly reduced.
Minor differences have been observed between the HCS tube-based assay and HCII HBV. The results obtained by HCII HBV were, on average, 0.25 log lower than those obtained by the HCS tube-based Assay. This difference might be obtained due to a slightly modified sample preparation step. The HCS tube-based assay uses a proteinase K digestion step, whereas HCII HBV does not. Since in HBV DNA the polymerase protein is covalently attached to the DNA, a proteinase K digestion step in the sample preparation procedure of the HCS tube-based assay might explain the observed difference (7
The lack of standardization has been a problem for nucleic acid detection assays, although in theory, molecular biology-based assays are ideal for standardization purposes (3
). With standard hybridization-based assays, sensitivity is generally the biggest problem. Although the basic PCR assay can be exquisitely sensitive, the first international quality control program for HBV has shown great differences in sensitivity and specificity among results from different laboratories (19
). This phenomenon is not specific for HBV DNA detection assays but applies to target amplification-based assays in general (6
). The lack of standardization has contributed to these differences in sensitivity. From the data obtained with the two EUROHEP reference samples for genotypes A and D (7
), it has also become clear that HCII HBV is not genotype dependent. This is in contrast to the Abbott Genostics assay, which is widely used in clinical studies worldwide. By that assay genotype differences can lead to a 35-fold differences in output signals (23
). Furthermore, the EUROHEP standard could be used to determine the sensitivity of the assay.
HCII HBV proved to be reproducible, with an overall CV of less then 13% and a maximum CV of 33%. This indicates that the results of tests with a single sample will typically vary by a factor 2. When the three different laboratories tested the same samples in a test of linearity for both the standard and the ultrasensitive formats, the results that were obtained did not differ by more than a factor of 2. This implies that results from different laboratories may be compared if the results are within these limits. Additional sample volume in the preparation step, as has also been shown for signal and target amplification-based assays targeted at human immunodeficiency virus type 1 (5
) or hepatitis C virus (14
), will probably further increase the sensitivity of the test.
Determination of the serum HBV DNA level has been shown to be useful for monitoring of the effect of antiviral treatment and for patient management. Furthermore, the emergence of resistance to antiviral drugs can be determined by the detection of increases in viral loads during treatment (9
). Typically, signal amplification-based assays are less sensitive, which may not allow the detection of early elevations in viral loads until they reach 106
HBV DNA copies per ml (23
). Standard antiviral treatment with nucleoside analogues will reduce HBV DNA levels within a few weeks to a level not detectable by these assays (9
). HCII HBV increases the sensitivity approximately 300-fold. Limited information is available on what levels will be reached during antiviral treatment or what HBV DNA levels are present in asymptomatic carriers. Only Niitsuma et al. (17
) found that an HBV DNA level of approximately 10,000 HBV DNA copies per ml should be reached after successful antiviral treatment. Below this level, no hepatitis occurred in their study group. This is a detection level just above the cutoff of the ultrasensitive format of HCII HBV.
In summary, the performance characteristics of HCII HBV indicate that this assay is a reliable tool for the accurate measurement of HBV DNA levels in serum and therefore could be used to monitor the effects of antiviral therapy. In its ultrasensitive format, the assay has a sensitivity which is just above the range of the currently available PCR-based assays (1