Authors
Grant, Robert M.Kuritzkes, Daniel R.
Johnson, Victoria A.
Mellors, John W.
Sullivan, John L.
Swanstrom, Ronald I.
D'Aquila, Richard T.
Van Gorder, Mark
Holodniy, Mark
Lloyd, Robert M. Jr.
Reid, Caroline
Morgan, Gillian F.
Winslow, Dean L.
UMass Chan Affiliations
Department of PediatricsDocument Type
Journal ArticlePublication Date
2003-04-01Keywords
Anti-HIV AgentsDrug Resistance, Viral
Gene Amplification
Genotype
HIV Infections
HIV Protease
HIV Reverse Transcriptase
HIV-1
Humans
Laboratories
Molecular Sequence Data
Mutation
RNA, Viral
*Reagent Kits, Diagnostic
Reproducibility of Results
Sequence Analysis, DNA
Software
Immunology and Infectious Disease
Pediatrics
Metadata
Show full item recordAbstract
Drug resistance and poor virological responses are associated with well-characterized mutations in the viral reading frames that encode the proteins that are targeted by currently available antiretroviral drugs. An integrated system was developed that includes target gene amplification, DNA sequencing chemistry (TRUGENE HIV-1 Genotyping Kit), and hardware and interpretative software (the OpenGene DNA Sequencing System) for detection of mutations in the human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequences. The integrated system incorporates reverse transcription-PCR from extracted HIV-1 RNA, a coupled amplification and sequencing step (CLIP), polyacrylamide gel electrophoresis, semiautomated analysis of data, and generation of an interpretative report. To assess the accuracy and robustness of the assay system, 270 coded plasma specimens derived from nine patients were sent to six laboratories for blinded analysis. All specimens contained HIV-1 subtype B viruses. Results of 270 independent assays were compared to "gold standard" consensus sequences of the virus populations determined by sequence analysis of 16 to 20 clones of viral DNA amplicons derived from two independent PCRs using primers not used in the kit. The accuracy of the integrated system for nucleotide base identification was 98.7%, and the accuracy for codon identification at 54 sites associated with drug resistance was 97.6%. In a separate analysis of plasma spiked with infectious molecular clones, the assay reproducibly detected all 72 different drug resistance mutations that were evaluated. There were no significant differences in accuracy between laboratories, between technologists, between kit lots, or between days. This integrated assay system for the detection of HIV-1 drug resistance mutations has a high degree of accuracy and reproducibility in several laboratories.Source
J Clin Microbiol. 2003 Apr;41(4):1586-93. doi: 10.1128/JCM.41.4.1586-1593.2003DOI
10.1128/JCM.41.4.1586-1593.2003Permanent Link to this Item
http://hdl.handle.net/20.500.14038/43447PubMed ID
12682149Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1128/JCM.41.4.1586-1593.2003