A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs.

Antimicrobial Agents and Chemotherapy
K HertogsR Pauwels

Abstract

Combination therapy with protease (PR) and reverse transcriptase (RT) inhibitors can efficiently suppress human immunodeficiency virus (HIV) replication, but the emergence of drug-resistant variants correlates strongly with therapeutic failure. Here we describe a new method for high-throughput analysis of clinical samples that permits the simultaneous detection of HIV type 1 (HIV-1) phenotypic resistance to both RT and PR inhibitors by means of recombinant virus assay technology. HIV-1 RNA is extracted from plasma samples, and a 2.2-kb fragment containing the entire HIV-1 PR- and RT-coding sequence is amplified by nested reverse transcription-PCR. The pool of PR-RT-coding sequences is then cotransfected into CD4+ T lymphocytes (MT4) with the pGEMT3deltaPRT plasmid from which most of the PR (codons 10 to 99) and RT (codons 1 to 482) sequences are deleted. Homologous recombination leads to the generation of chimeric viruses containing PR- and RT-coding sequences derived from HIV-1 RNA in plasma. The susceptibilities of the chimeric viruses to all currently available RT and/or PR inhibitors is determined by an MT4 cell-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based cell viability assay in an automated system th...Continue Reading

References

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Citations

May 24, 2000·Journal of Clinical Pathology·M Helbert, J Breuer
Jun 16, 2001·Antimicrobial Agents and Chemotherapy·J LennerstrandB A Larder
Apr 18, 2002·Antimicrobial Agents and Chemotherapy·Raymond F SchinaziSusan Erickson-Viitanen
Oct 31, 1998·Antimicrobial Agents and Chemotherapy·D Boden, M Markowitz
Feb 19, 2002·Antimicrobial Agents and Chemotherapy·Mark SaleDaniel S Stein
Feb 19, 2002·Antimicrobial Agents and Chemotherapy·P Richard Harrigan, Brendan A Larder
Nov 19, 2002·Antimicrobial Agents and Chemotherapy·Valery FikkertMyriam Witvrouw
Oct 9, 2002·Journal of Virological Methods·Mutsunori IgaHirohito Tsubouchi
Aug 26, 2003·Antimicrobial Agents and Chemotherapy·Luis M F GonzalezAmilcar Tanuri
Apr 5, 2002·Clinical Microbiology Reviews·Robert W Shafer
Jun 4, 2011·Journal of Acquired Immune Deficiency Syndromes : JAIDS·Lotke TambuyzerJohan Vingerhoets
Aug 10, 2011·Antimicrobial Agents and Chemotherapy·Martin StürmerMargriet van Houtte
Oct 14, 2011·The Journal of Antimicrobial Chemotherapy·Maria José BuzonJavier Martinez-Picado
Oct 15, 2011·Journal of Acquired Immune Deficiency Syndromes : JAIDS·Rima KulkarniKirsten L White

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