The present study demonstrates that the Cobas-AM test has high sensitivity and reproducibility. The virus in all samples with an input above 400 copies/ml and in 83% of samples with input of 100 copies/ml was detected. The CV ranged from 8.8 to 36.3%, and the most variation was observed for samples with very low HBV DNA concentrations.
In comparison with the MWP-AM (7
), the automation of amplification, detection, and calculation reduced the hands-on time and risk for technical or computational errors. The sensitivity was higher than that for MWP-AM, but the detection range was narrower as the upper limit of detection was reduced from 107
copies/ml. Thus, the range of detection is particularly suitable for measuring low-level viremia, as seen in most HBeAg-negative patients (13
) and during therapy. Recently, new antiviral therapies, such as lamivudine, have been introduced (8
). The advantage of such antiviral treatment is that it is given orally and has few side effects. On the other hand, viremia tends to reappear at discontinuation of therapy, and breakthrough due to drug resistance has recently been described (1
). Therefore, in the future combination therapy may prove the most effective strategy, and as in the management of human immunodeficiency virus, monitoring viral load with highly sensitive methods may prove essential for control of HBV infection (2
). As regards treatment with interferon, which probably acts through both immunomodulation and antiviral effects, the value of monitoring HBV DNA remains to be demonstrated. In the present study, monitoring of a patient consecutively treated with interferon and lamivudine showed that the (transient) response to interferon (including ALT normalization and HBeAg seroconversion) was accompanied by a reduction of viral load to 6 × 104
copies/ml, and the clinical response to lamivudine included reduction of viral levels to around 700 copies/ml during treatment. Of interest, the ALT peaks, both the flare during interferon treatment and the flare at the relapse after interferon treatment, were preceded by increasing HBV DNA levels, indicating that HBV DNA monitoring may be useful in identifying and (when further antiviral drugs emerge) reducing the hepatocellular damage caused by viral activations and treatment breakthroughs. Further investigations are needed to find out if suppression of viremia below a certain limit is enough or if eradication of viral replication is required for preventing further liver damage and minimizing the risk for viral resistance and/or reactivation of hepatitis when therapy is discontinued.
There was a good correlation between results from Cobas-AM and those from MWP-AM. Analysis of 153 clinical samples by both methods showed a regression coefficient of 1.17 and a mean ratio of 0.97 for individual log HBV DNA results measured by Cobas and results measured by MWP. Thus, the ratio for Cobas:MWP results was approximately 1:1, indicating that results of the tests are interchangeable. The MWP-AM has the advantage of a broader detection range, allowing quantification of virus at relatively high levels as well. However, our data indicate that for samples with HBV DNA levels close to or above 107 copies/ml the levels are underestimated by MWP-AM, unless they are diluted in negative serum prior to analysis (not shown).
In HBeAg-positive samples, the median HBV DNA concentration was 108.6 copies/ml, i.e., far above the upper limit of detection by Cobas-AM. Therefore, predilution is required for analysis of serum samples from HBeAg-positive carriers (and HBeAg-negative carriers if they are suspected to be highly viremic). Our data indicate that the most suitable predilution for HBeAg-positive samples is 1:105. This moves the detection range of Cobas-AM from 102 to 105 copies/ml to 107 to 1010 copies/ml and should allow detection and quantification of more than 90% of HBeAg-positive samples, less than 5% of which would be categorized as containing more than 1010 copies/ml.
In summary, HBV DNA quantification by Cobas-AM is highly sensitive and reproducible but has a range of detection that requires predilution of high-titer samples. It should be particularly suitable for monitoring response to antiviral therapy.