Abstract
Background/AimThe study aimed at developing a real-time quantitative PCR assay to monitor HBV serum virus load of chronic carriers enrolled in therapeutic trials.MethodQuantitative real-time PCR assay was carried out using SYBR-Green signal detection and primers specific to the S gene. Thermal cycling was performed in an ABi 5700 sequence detection system. The assay was calibrated against an international HBV DNA standard and inter- and intra-assay reproducibility determined. Levels of viral load were monitored for 1-year in lamivudine treated carriers. Correlation between HBV DNA levels and HBeAg sero-status was determined in untreated carriers.ResultsThe qPCR assay showed good intra- and inter-assay reproducibility over a wide dynamic range (1.5 × 103 to 1.5 × 108 copies/mL) and correlated well with those from a commercial assay (r = 0.91, (p < 0.001). Viral load levels dropped dramatically but temporarily during and after a short course of lamivudine therapy. HBV DNA was a more reliable indicator of the presence of virus than HBe antigen and was detected in 77.0% (161/209) of HBeAg negative and in all HBeAg positive carriers.ConclusionThis method is reliable, accurate, and reproducible. HBV DNA Quantification by qPCR can be used to monitor the efficacy of HBV therapy and useful in understanding the natural history of HBV in an endemic area.
Highlights
Hepatitis B virus (HBV) is the leading cause of viral hepatitis in humans worldwide
Infected carriers have a high risk of developing liver damage and hepatocellular carcinoma (HCC) and liver cancer is the commonest cause of death in adult males in The Gambia [4]
We describe the development and validation of a quantitative polymerase chain reaction (PCR) method to measure the concentration of HBV DNA in serum
Summary
Hepatitis B virus (HBV) is the leading cause of viral hepatitis in humans worldwide. Currently over two billion people have evidence of previous HBV infection and 350 million have become chronic carriers of the virus, 60 million of them residing in Africa [1]. Infected carriers have a high risk of developing liver damage and hepatocellular carcinoma (HCC) and liver cancer is the commonest cause of death in adult males in The Gambia [4]. The monitoring of hepatitis B virus DNA in serum is as important as serological markers in predicting the clinical outcome of infection. More recently molecular diagnostic methods have been used to quantify the levels of HBV DNA in serum as a marker of viral replicative activity [6]. More recently the development of real time PCR methodology has further improved the ease with which HBV DNA levels can be monitored and has increased the range over which such levels can be accurately quantified [16,17]
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