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Journal of Clinical Microbiology, February 2001, p. 464-473, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.464-473.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Monitoring Resistance to Human Immunodeficiency Virus Type 1 Protease Inhibitors by Pyrosequencing

Deirdre O'Meara,1 Karin Wilbe,2 Thomas Leitner,2 Bo Hejdeman,3 Jan Albert,4 and Joakim Lundeberg1,*

Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm,1 Department of Clinical Virology, Swedish Institute for Infectious Disease Control/Karolinska Institute, S-171 82 Stockholm,2 Department of Dermatovenereology, Södersjukhuset, S-118 83 Stockholm,3 and Department of Clinical Virology (IMPI), Karolinska Institute, Huddinge University Hospital, S-141 86 Stockholm,4 Sweden

Received 31 July 2000/Returned for modification 26 September 2000/Accepted 25 October 2000

The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HIV-1-infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.

* Corresponding author. Mailing address: Department of Biotechnology, Royal Institute of Technology, KTH, Teknikringen 30, S-100 44 Stockholm, Sweden. Phone: 46 8 790 87 58. Fax: 46 8 24 54 52. E-mail: joakim.lundeberg{at}biochem.kth.se.

Journal of Clinical Microbiology, February 2001, p. 464-473, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.464-473.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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