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Journal of Clinical Microbiology, October 2004, p. 4788-4795, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4788-4795.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Pyrosequencing for Detection of Lamivudine-Resistant Hepatitis B Virus

Anna Lindström,1* Jacob Odeberg,2 and Jan Albert1,{dagger}

Department of Laboratory Medicine, Division of Clinical Virology, Karolinska Institutet, Huddinge University Hospital, Huddinge/Stockholm,1 Department of Biotechnology, Royal Institute of Technology, Alba Nova University Centrum, Stockholm, Sweden2

Received 28 April 2003/ Returned for modification 24 June 2003/ Accepted 1 June 2004

Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.

* Corresponding author. Present address: Department of Virology, Swedish Institute for Infectious Disease Control, S-171 82 Solna, Sweden. Phone: 46 8 457 26 90. Fax: 46 8 33 72 72. E-mail: Anna.Lindstrom{at}smi.ki.se.

{dagger} Present address: Department of Virology, Swedish Institute for Infectious Disease Control, S-171 82 Solna, Sweden.

Journal of Clinical Microbiology, October 2004, p. 4788-4795, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4788-4795.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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