The results of our study have shown a very good correlation among the cross-linking, hybrid-capture amplification, and bDNA assays in the detection and quantification of HBV DNA over a wide range of HBV DNA levels. The cross-linking and hybrid-capture amplification assays tend to detect HBV DNA in more samples than the bDNA assay, particularly over the low-level viremia range. This potentially increases the sensitivity of identification of persistent low-grade viremia in patients receiving antiviral treatments, but the clinical significance of this finding requires further investigation. Discordant results do occur within the quoted detection range of the three assays. The reasons for the discrepancy are not entirely clear. It could be related to assay-specific inhibition, a difference in the target probe sequence, or nonspecific hybridization.
The cross-linking and hybrid-capture amplification assays have the advantage over the bDNA assay that they require less time to complete. The cross-linking and hybrid-capture amplification assays take approximately 5 and 3 h, respectively, while the bDNA assay requires overnight incubation. The higher mean within-run CV in both the cross-linking and bDNA assays in our study compared with that reported previously might be related to the smaller number of replicates performed in our study (16
). Although no samples required retesting in the hybrid-capture amplification assay, we are still concerned about the reliability of validation by the use of calibrators alone, as recommended by the manufacturer. It has been shown that the within-run CV of the hybrid-capture amplification assay ranged from 5 to 21% among three different centers (7
Three of the 96 (3%) samples in our study had HBV DNA levels exceeding the upper detection limit of the hybrid-capture amplification assay, which was about 1/2 log unit lower than that of the cross-linking and bDNA assays. The failure to quantify HBV DNA in the very high level viremic patients may potentially limit the use of the hybrid-capture amplification assay in predicting high-risk patients who may need higher doses or a longer duration of antiviral treatment.
HBV DNA remained undetectable in 28% of serum samples by all three of the assays in our study. Whether this indicates very low levels of virus or complete clearance of HBV DNA is not clear. Our previous study has shown that approximately 50% of patients who had undetectable HBV DNA levels by the bDNA assay were in fact PCR positive by our in-house PCR assay (3
). Although PCR is highly sensitive and can detect HBV DNA levels as low as 100 copies/ml, this very low level of viremia is of doubtful clinical significance. Moreover, PCR assays are prone to contamination, giving rise to false-positive results (20
). A quantitative PCR assay with a sensitivity of 103
viral copies/ml has recently been introduced (13
). Whether it is useful and reliable in the monitoring of response to antiviral treatments requires further evaluation.
In summary, we found that the cross-linking assay and the hybrid-capture amplification assay are at least as sensitive as the bDNA assay for the detection and quantification of HBV DNA. These two new assays required shorter procedure times than the bDNA assay. Based on these results, the cross-linking and hybrid-capture amplification assays can be recommended for monitoring HBV DNA levels in antiviral therapies.