title: Determination of HIV-1 susceptibility to reverse transcriptase (RT) inhibitors by a quantitative cell-free RT assay.
abstract: BACKGROUND
  Mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene confer resistance to antiviral drugs acting on RT. Current methods employed to detect such resistance require time-consuming culture techniques during which selective pressures may affect the outcome of the test.
  
  
  OBJECTIVE
  We sought to determine whether drug-susceptible and drug-resistant HIV-1 derived from clinical specimens could be distinguished by the effects of the active form of the drug on the enzyme activity in a quantitative, cell-free RT assay.
  
  
  METHODS
  Polyethylene glycol (PEG)-precipitated virus was obtained from 7-day culture supernatants. RT activity in the lysed viral extracts was measured in the presence of increasing concentrations of the active form of the drug being tested. IC50 (50% inhibitory concentration) values were determined by application of the median effect equation.
  
  
  RESULTS
  Assays from nine post-nevirapine therapy isolates gave IC50 values at least 2 logs greater than pre-nevirapine isolates. The method also correctly distinguished between isolates sensitive and resistant to 2',3'-dideoxyinosine (ddI), but not between the ZDV-sensitive and ZDV-resistant isolates tested. The results agreed with data obtained by sequencing and by culture-based susceptibility assays.
authors.affiliation: NEN Life Science Products, Boston, MA 02118, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Greene
authors.forename: A J
authors.initials: AJ
authors.lastname: Japour
authors.forename: F
authors.initials: F
authors.lastname: Brewster
authors.forename: R A
authors.initials: RA
authors.lastname: Joseph
authors.forename: P H
authors.initials: PH
authors.lastname: Chung
authors.forename: P A
authors.initials: PA
authors.lastname: Kasila
authors.forename: P A
authors.initials: PA
authors.lastname: Chatis
mesh_terms: D002474:Cell-Free System; D016049:Didanosine / Q000494:pharmacology*; D004351:Drug Resistance; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D054303:HIV Reverse Transcriptase; D016049:Didanosine