Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However, the emergence of lamivudine-resistant mutant virus, with amino acid substitution in the YMDD motif of DNA polymerase, has been reported. We report the emergence and takeover of YMDD mutant and re-takeover by wild type during and after long-term lamivudine therapy. YMDD mutants were detected in five patients who showed DNA breakthrough (HBV DNA becoming detectable after a period of DNA negativity), which occurred after 9 to 14 months of lamivudine therapy. Four of five mutants had amino acid sequence YIDD, and the remaining mutant had YVDD. Patients with high HBV-DNA titer and/or hepatitis B e antigen tended to develop breakthrough (P โซุโฌ .038). Using a sensitive and specific polymerase chain reaction (PCR)-based method developed in this study, the emergence of YMDD mutants was detected 1 to 4 months before DNA breakthrough, but not detected in any of the pretreatment sera. The mutants were predominant at breakthrough, but were replaced by wild-type virus 3 to 4 months after cessation of therapy in the two patients who discontinued therapy. One of these patients had a relapse of hepatitis. Mutant continued to replicate in the remaining three patients who continued to receive treatment, and relapse occurred in only one of these patients. Our results suggest that the replication of YMDD mutant viruses is less than wild type and is re-overtaken by wild type after cessation of therapy. Re-administration of lamivudine, possibly combined with other antiviral therapy, might be useful in some patients experiencing hepatitis with lamivudine-resistant variants. (HEPATOLOGY 1998;27: 1711-1716.)
Lamivudine, (-)-โค-L-2ะ,3ะ-dideoxy-3'-thiacytidine (3TC), is a potent inhibitor of RNA-dependent DNA polymerase of hepatitis B virus (HBV), as well as human immunodeficiency virus (HIV) reverse transcriptase. [1][2][3][4][5][6][7][8][9][10] The in vivo antiviral activity of lamivudine has been reported in animal studies as well as in humans. [11][12][13][14][15][16][17][18][19][20][21][22] However, the emergence of lamivudineresistant HBV strains was initially noticed in patients who received orthotopic liver transplantation and immunosuppressive therapy. [23][24][25][26][27][28] Such resistant viruses show a characteristic mutation of the 550th amino acid methionine in the YMDD motif of DNA polymerase to isoleucine (YIDD mutant) or valine (YVDD mutant). Honkoop et al. 29 recently showed the emergence of such mutant viruses in 5 of 14 (36%) immunocompetent patients who were treated with lamivudine over 26 weeks. However, in a larger placebo-controlled study in 358 Asian patients with HBV infection treated for 52 weeks, the rate of detection of lamivudine-resistant variants by polymerase chain reaction (PCR) was 19 of 134 (14%) in patients treated with 100 mg daily. 30 To our knowledge, there is no report that describes the presence of mutant viruses in the serum before administration of lamivudine. The low incidence and late emergence of YMDD mutant viruses suggest that such mutant strains originate from wild type by point mutation during the course of therapy. However, because the presence of mutant virus can be detected only by nucleotide sequence analysis, it has been difficult, until recently, to establish the exact time and mode of emergence of the mutant virus.
In this study, we developed a sensitive PCR-based assay to detect YMDD mutant and analyzed clinical samples from patients who received long-term lamivudine therapy. Furthermore, we also investigated whether the mutant viruses continued to replicate after cessation of therapy.
PATIENTS AND METHODS
Patients. We studied 20 consecutive Japanese adult patients with chronic hepatitis B who agreed to enter a long-term lamivudine (Glaxo-Wellcome, Greenford, UK) trial between September 1995 and July 1996 at the Department of Gastroenterology of Toranomon Hospital. The entry criteria included a positive test for HBV DNA by dot hybridization and elevated levels of alanine transaminase (ALT) (greater than twice the upper limit of normal value [50 IU/L]) within 3 months before the start of therapy. A needle biopsy was performed in each patient just before the trial, which confirmed the presence of chronic hepatitis. Individuals who had apparent cirrhosis or signs of hepatic decompensation were excluded from the study. Also excluded were patients positive for serum markers of hepatitis C virus and HIV. None of these patients received immunosuppressive or antiviral therapy at least 6 months before lamivudine therapy. None Abbreviations: HBV, hepatitis B virus; HIV, human immunodeficiency virus; PCR, polymerase chain reaction; ALT, alanine transaminase; Meq, genomic equivalents.