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Journal of Clinical Microbiology, February 2005, p. 813-817, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.813-817.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Sensitivity and Specificity of the ViroSeq Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping System for Detection of HIV-1 Drug Resistance Mutations by Use of an ABI PRISM 3100 Genetic Analyzer

Susan H. Eshleman,^1* Gillian Crutcher,^2, Olga Petrauskene,³ Kevin Kunstman,⁴ Shawn P. Cunningham,¹ Christina Trevino,⁵ Cheryl Davis,⁶ John Kennedy,^7, Jeff Fairman,^8, Brian Foley,⁹ and JoAnn Kop^2,¶

The Johns Hopkins Medical Institutions, Baltimore, Maryland,¹ Celera Diagnostics, Alameda,² Applied Biosystems, Foster City,³ Stanford University, Palo Alto,⁵ PPGx, La Jolla,⁶ Clingenix, Sunnyvale, California,⁸ Northwestern University, Chicago, Illinois,⁴ Los Alamos National Laboratory, Los Alamos, New Mexico;,⁷ Pompano Beach, Florida⁹

Received 17 August 2004/ Returned for modification 23 September 2004/ Accepted 23 October 2004

The ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system is an integrated system for identification of drug resistance mutations in HIV-1 protease and reverse transcriptase (RT). Reagents are included for sample preparation, reverse transcription, PCR amplification, and sequencing. Software is provided to assemble and edit sequence data and to generate a drug resistance report. We determined the sensitivity and specificity of the ViroSeq system for mutation detection using an ABI PRISM 3100 genetic analyzer with a set of clinical samples and recombinant viruses. Twenty clinical plasma samples (viral loads, 1,800 to 10,500 copies/ml) were characterized by cloning and sequencing individual viral variants. Twelve recombinant-virus samples (viral loads, approximately 2,000 to 5,000 copies/ml) were also prepared. Eleven recombinant-virus samples contained drug resistance mutations as 40% mixtures. One recombinant-virus sample contained an insertion at codon 69 in RT (100% mutant). Plasma and recombinant-virus samples were analyzed using the ViroSeq system. Each sample was analyzed on three consecutive days at each of three testing laboratories. The sensitivity of mutation detection was 99.65% for the clinical plasma samples and 99.7% for the recombinant-virus preparations. The specificity of mutation detection was 99.95% for the clinical samples and 100% for the recombinant-virus mixtures. The base calling accuracy of the 3100 instrument was 99.91%. Mutations in clinical plasma samples and recombinant-virus samples were detected with high sensitivity and specificity, including mutations present as mixtures. This report supports the use of the ViroSeq system for identification of drug resistance mutations in HIV-1 protease and RT genes.

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* Corresponding author. Mailing address: The Johns Hopkins Medical Institutions, Ross Building 646, 720 Rutland Ave., Baltimore, MD 21205. Phone: (410) 614-4734. Fax: (410) 614-3548. E-mail: seshlem{at}jhmi.edu.

Present address: Chiron, Emeryville, Calif.

Deceased.

Present address: Juvaris BioTherapeutics, Inc., Pleasanton, Calif.

^¶ Present address: Arcturus, Mountain View, Calif.

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Journal of Clinical Microbiology, February 2005, p. 813-817, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.813-817.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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