Development Of A Rapid Method For Quatitation Of Lamivudine-resistant Mutations In Hepatitis B Virus And Its Application

It is estimated that 350 million indivudials are chronically infected with hepatitis B virus (HBV).HBV is the leading cause of liver cirrhosis and hepatocellular carcinoma(HCC)and is responsible for 60～80%of HCC cases.Many antiviral agents were used for treatment of HBV infection.Lamivudine is one of the most effective antiviral agents for patients with chronic hepatitis B and advanced liver diseases.However,long-term monotherapy commonly does not result in sustained suppression of viral replication and is associated with the emergence of resistant mutations.Specific mutations that result in replacement of methionnine(M)in the tyrosine-methionine-aspartate-aspartate(YMDD)motif of the HBV reverse transcriptase(rt)by valine(V),isoleucine(I),or serine(S)confer resistance to lamivudine and lead to virological breakthrough.In the present study,we established a real-time PCR assay with selective primers and TaqMan probe to detect the percentage of lamivudine-resistant mutants in total HBV without the need of external DNA standards.This percentage was calculated as the PCR efficiency raised to the differences between threshold cycle number(△Ct)of mutant and control reactions.Clones of the HBV polymerase gene containing the different YMDD variants were diluted in series and tested.Serum samples from 145 lamivudine-treated and 98 untreated patients with chronic hepatitis B virus infection were analyzed using this method and compared with DNA sequencing. 268 patients with chronic hepatitis B using Lamivudine were analyzed by this method to see the ratio of total virus mutantion and the duration of lamivudine therapy.Additional 162 patients with HBeAg-negative chronic hepatitis B were monitored for YMDD mutation during their treatment using this method.98 were treated with lamivudine alone and 64 were treated with lamivudine and interferon alpha 2b sequential treatmen.Treatment efficacy and YMDD mutations were compared between the two groups.Activation of T lymphocytes and histology improvement were also analyzed.As little as 0.1%mutant plasmids in 106～109 copies per milliliter of wild-type plasmids were detected.Among the 145 patients treated with lamivudine,42 of them had mutants with percentages of 5～100%.In six discordant results between real-time PCR and DNA sequencing, real-time PCR detected mutants with percentages of 5～20%,which were concordant with the result of subclone sequencing.Five of 95 lamividine-untreated patients had mutants of 10～20% in wild-type virus populations.Compared to DNA sequencing,real-time PCR was fast and cost-effective.After 48 weeks of treatment,the treatment efficacy has no significant difference between lamivudine monotherapy group and sequential treatment group.However,The percentage of patients with lamivudine-resistant mutations was significantly higher with lamivudine monotherapy(22.45%)than with sequential therapy(P =0.00).This real-time PCR is a rapid and sensitive method for detection of YMDD mutants in patients with HBV infection.It can be used for monitoring of lamivudine-resistant mutations in patients with chronic hepatitis B on lamivudine therapy.