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

doi:10.1128/JCM.39.2.464-473.2001

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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 immunodeficiencyvirus type 1 (HIV-1) replication during treatment with antiretroviraldrugs. Sequencing to determine the resistance profiles of thesevariants has become increasingly important in the clinical managementof HIV-1 patients, both in the initial design of a therapeuticplan and in selecting a salvage regimen. Here we have developeda pyrosequencing assay for the rapid characterization of resistanceto HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primerswere designed and were evaluated on the MN strain and on viralDNA from peripheral blood mononuclear cells from eight untreatedHIV-1-infected individuals. The method had a limit of detectionof 20 to 25% for minor sequence variants. Pattern recognition(i.e., comparing actual sequence data with expected wild-typeand mutant sequence patterns) simplified the identification ofminor sequence variants. This real-time pyrosequencing methodwas applied in a longitudinal study monitoring the developmentof PI resistance in plasma samples obtained from four patientsover a 2 1/2-year period. Pyrosequencing identified eight primaryPI resistance mutations as well as several secondary mutations.This sequencing approach allows parallel analysis of 96 reactionsin 1 h, facilitating the monitoring of drug resistance in eightpatients simultaneously and, in combination with viral load analysis,should be a useful tool in the future to monitor HIV-1 duringtherapy.

^* Corresponding author. Mailing address: Department of Biotechnology, Royal Institute of Technology, KTH, Teknikringen 30, S-10044 Stockholm, Sweden. Phone: 46 8 790 87 58. Fax: 46 8 24 54 52.E-mail: joakim.lundeberg{at}biochem.kth.se.

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