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Antimicrobial Agents and Chemotherapy, July 2009, p. 2762-2772, Vol. 53, No. 7
0066-4804/09/$08.00+0 doi:10.1128/AAC.00130-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Ultrasensitive Genotypic Detection of Antiviral Resistance in Hepatitis B Virus Clinical Isolates
,
Jie Fang,1
Michael J. Wichroski,1
Steven M. Levine,1
Carl J. Baldick,1
Charles E. Mazzucco,1
Ann W. Walsh,1
Bernadette K. Kienzle,2
Ronald E. Rose,1
Kevin A. Pokornowski,1
Richard J. Colonno,1,
and
Daniel J. Tenney1*
Bristol-Myers Squibb Research and Development, Wallingford, Connecticut,1 Bristol-Myers Squibb Research and Development, Hopewell, New Jersey2
Received 28 January 2009/ Returned for modification 25 February 2009/ Accepted 30 April 2009
Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other L-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy.
* Corresponding author. Mailing address: Bristol-Myers Squibb Research and Development, Wallingford, CT. Phone: (203) 677-7846. Fax: (203) 677-6088. E-mail: Daniel.Tenney{at}bms.com
Published ahead of print on 11 May 2009.
Supplemental material for this article may be found at http://aac.asm.org/.
Present address: Presidio Pharmaceuticals, Inc., San Francisco, CA.
Antimicrobial Agents and Chemotherapy, July 2009, p. 2762-2772, Vol. 53, No. 7
0066-4804/09/$08.00+0 doi:10.1128/AAC.00130-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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