Selection and persistence of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 in patients starting and stopping non-nucleoside therapy.
BACKGROUND
Understanding the selection and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral therapy.


OBJECTIVE
To develop a high-throughput, real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay to quantify non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants K103N (AAT or AAC alleles) at frequencies as low as 0.1%, and to apply this to monitor these variants before, during, and after NNRTI therapy.


METHODS
HIV-1 RNA in longitudinal plasma samples obtained from patients starting and stopping NNRTI therapy was converted to cDNA and the target sequence region amplified and quantified by real-time PCR. Approximately 10 copies/reaction provided a template for a second round of PCR using primers that discriminated between the mutant and wild-type alleles. Amplification specificity was confirmed by thermal denaturation analysis.


RESULTS
Frequencies of 103N similar to assay background (0.029%) were observed in longitudinal samples from 9 of 12 treatment-naive patients; three patients had transient increases in 103N frequency to a range of 0.21-0.48%, which was 7-16.5 times assay background. Analysis of longitudinal plasma samples from six NNRTI-experienced patients showed three patterns: persistence of 103N variants after stopping NNRTI therapy, codon switching of 103N between AAC and AAT during NNRTI therapy, and decay of 103N variants to below assay background after cessation of NNRTI therapy.


CONCLUSIONS
Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1-100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable.
Palmer
Sarah
S
HIV Drug Resistance Program, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702-1201, USA. spalmer@ncifcrf.gov
Boltz
Valerie
V
Maldarelli
Frank
F
Kearney
Mary
M
Halvas
Elias K
EK
Rock
Diane
D
Falloon
Judith
J
Davey
Richard T
RT
Dewar
Robin L
RL
Metcalf
Julia A
JA
Mellors
John W
JW
Coffin
John M
JM
D000328:Adult; D000483:Alleles; D003588:Cytopathogenic Effect, Viral; D004334:Drug Administration Schedule; D024882:Drug Resistance, Viral / Q000235:genetics*; D005787:Gene Frequency; D015658:HIV Infections / Q000188:drug therapy* / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis / Q000097:blood*; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use*; D019562:Viral Load; D014766:Viremia
D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors