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Journal of Clinical Microbiology, July 1999, p. 2291-2296, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Worldwide Evaluation of DNA Sequencing Approaches for Identification of Drug Resistance Mutations in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Rob Schuurman,1,dagger ,* Lisa Demeter,2,Dagger Patricia Reichelderfer,3,Dagger Jolanda Tijnagel,1,dagger Tom de Groot,1,dagger and Charles Boucher1,dagger

Department of Virology, University Hospital Utrecht, Utrecht, The Netherlands1; Infectious Diseases Unit, University of Rochester School of Medicine and Dentistry, Rochester, New York2; and National Institute of Child Health and Human Development, Bethesda, Maryland3

Received 29 December 1998/Returned for modification 12 February 1999/Accepted 15 April 1999

A panel (ENVA-1) of well-defined blinded samples containing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was analyzed by automated DNA sequencing in 23 laboratories worldwide. Drug resistance mutations at codons 41, 215, and 184 were present in the panel samples at different ratios to the wild type. The presence of mutant genotypes was determined qualitatively and quantitatively. All laboratories reported the presence of sequence heterogeneities at codons 41, 215, and 184 in one or more of the panel samples, though not all reported the correct codon genotypes. Two laboratories reported a mutant genotype in samples containing only the wild type, whereas two and three laboratories failed to detect the mutant genotypes at codons 41 and 215, respectively, in a completely mutant DNA population. Mutations present at relative concentrations of 25% of the total DNA population were successfully identified by 13 of 23, 10 of 23, and 16 of 23 labs for codons 41, 215, and 184Val, respectively. For more than 80% of those laboratories that qualitatively detected the presence of a mutation correctly, the estimated wild type/mutant ratio was less than 25% different from the input ratio in those samples containing 25 to 50% or 75% mutant input. This first multicenter study on the quality of DNA sequencing approaches for identifying HIV-1 drug resistance mutations revealed large interlaboratory differences in the quality of the results. The application of these procedures in their current state would in several cases lead to inaccurate or even incorrect diagnostic results. Therefore, proper quality control and standardization are urgently needed.

* Corresponding author. Mailing address: Department of Virology, University Hospital Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Phone: 31 30 250 6526. Fax: 31 30 250 5426. E-mail: r.schuurman{at}lab.azu.nl.

dagger On behalf of the European Network for the Virological Evaluation of International Trials for New Anti-HIV Therapies.

Dagger On behalf of the Sequencing Working Group.

Journal of Clinical Microbiology, July 1999, p. 2291-2296, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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