The quantitative COBAS-AM assay expands the range of patients for whom circulating virus levels can be measured to include patients who would have had undetectable virus levels by the Genostics assay. The COBAS-AM assay is an automated, PCR-based assay recently developed for quantitation of HBV DNA levels in serum. The assay is reproducible and has high degrees of accuracy and precision in both interassay and intra-assay comparisons. Noborg et al. (13
) previously reported on a similar comparison of the linearity, reproducibility, and precision of the COBAS-AM assay. They used the COBAS-AM assay to quantitate HBV DNA in clinical samples in which the levels were previously quantitated by the manual microwell plate version of the Amplicor HBV monitor assay. Our study evaluated 1,695 clinical samples in which the HBV DNA levels were previously quantitated by the Genostics assay.
The value of the COBAS-AM assay is that the linear range of detection extends from 102
), providing an opportunity for accurate evaluation of the HBV DNA concentrations in serum samples over a range not possible by the widely used Genostics assay. Although the reported LLOD of the Genostics assay is 1.6 pg/ml, the linear range of the assay has a lower limit of 10 or 20 pg/ml (1
No upper limit of the linear range has been reported for the Genostics assay, although the data presented here suggest that the linearity of the assay diminishes above 35 pg/ml (107 copies/ml). The plateau effect observed in the comparison of the two assays (Fig. ) suggests that the linearity of the Genostics assay does not extend beyond 107 copies/ml. There may be numerous causes for the inaccuracy of the Genostics assay; for example, since the assay does not require dilution of the serum samples, there could be insufficient denaturation of HBV DNA molecules, saturation of the column used for separation of unincorporated label, or an insufficient amount of probe.
With current antiviral therapy for chronic HBV infection, patients can experience decreases in serum HBV DNA levels to levels well below the LLOD of the Genostics assay (7
). With the advent of combination therapy, even greater decreases in serum HBV DNA levels may be achievable in many patients. The relevance of this enhanced suppression of viral replication to the clinical outcome is unknown, as studies in this field are only just beginning. Some information may come from studies that monitor the natural history of the disease, in which PCR-based assays for the measurement of HBV DNA levels have shown that viral replication can persist at low levels in patients who have attained HBe antigen seroconversion (7
). Similarly, very low levels of HBV DNA have been detected in patients with a phenotypic loss of HBe antigen due to infection with virus with precore mutations. Since the clinical courses of disease in these patient groups are quite distinct, the clinical relevance of detection of low-level viral replication without knowledge of the viral genotype is unclear.
Increasing HBV DNA levels may be detected earlier by a PCR-based assay than by the Genostics assay. In HBV-infected patients on antiviral therapy, a transient increase in viral replication indicated by an increase in serum HBV DNA levels might suggest a lack of compliance with therapy, while a persistent increase in serum HBV DNA levels might suggest the emergence of drug-resistant HBV mutants (15
). Thus, it may be relevant to continue to monitor HBV DNA levels in patients who are responding to antiviral therapy and whose HBV DNA levels have dropped below the LLOD of the Genostics assay (4
Real-time monitoring of patients with chronic HBV infection can be performed effectively by a PCR-based assay and can provide important virologic information. The COBAS-AM assay can be used effectively to monitor patients with high serum HBV DNA levels and will remain effective in the monitoring of those patients if their serum HBV DNA levels decrease to levels that might be undetectable by other commercially available assays.