title: Rapid screening of phenotypic resistance to nevirapine by direct analysis of HIV type 1 reverse transcriptase activity in plasma.
abstract: Drug susceptibility testing for the clinical management of human immunodeficiency virus type 1 (HIV-1)-infected persons is often curtailed because such testing is expensive and time consuming. We describe a non-culture-based phenotypic assay for the rapid analysis of HIV-1 resistance to nevirapine. The assay measures the susceptibility of plasma reverse transcriptase (RT) activity to inhibition by nevirapine by using the PCR-based Amp-RT assay. Assay validation was made using two reference wild-type (WT) and six other nevirapine-resistant (>100-fold) HIV-1 isolates. Amp-RT IC50 values were found to correlate with those obtained by a conventional replication-based assay. The results also indicated that 50 microM nevirapine can be used in a single screening test to detect nevirapine resistance. Analysis of virus mixtures showed a detection threshold of 10% of nevirapine-resistant HIV-1 in a background of WT virus. To evaluate the assay on clinical samples, 30 plasma specimens collected longitudinally from 4 patients before and after treatment with nevirapine were analyzed, and results were compared with codon 181 genotypes. Preteatment samples and those obtained during the first 6 days of therapy (n = 21) were sensitive to nevirapine, and none had detectable Y181C mutation. Phenotypic resistance was seen in eight samples obtained after 1 week of treatment and was correlated with detection of the Y181C mutation. An increase in the level of phenotypic resistance was seen over time. These data validate this rapid and simple assay for monitoring phenotypic resistance to nevirapine.
authors.affiliation: HIV and Retrovirology Branch, Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
authors.forename: G
authors.initials: G
authors.lastname: Vázquez-Rosales
authors.forename: J G
authors.initials: JG
authors.lastname: García Lerma
authors.forename: S
authors.initials: S
authors.lastname: Yamamoto
authors.forename: W M
authors.initials: WM
authors.lastname: Switzer
authors.forename: D
authors.initials: D
authors.lastname: Havlir
authors.forename: T M
authors.initials: TM
authors.lastname: Folks
authors.forename: D D
authors.initials: DD
authors.lastname: Richman
authors.forename: W
authors.initials: W
authors.lastname: Heneine
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D003062:Codon; D004352:Drug Resistance, Microbial / Q000235:genetics; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000097:blood; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D006801:Humans; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D019829:Nevirapine / Q000494:pharmacology* / Q000627:therapeutic use; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000097:blood; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D010948:Viral Plaque Assay; D014776:Virus Cultivation
chemical_list: D019380:Anti-HIV Agents; D003062:Codon; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine; D054303:HIV Reverse Transcriptase