Quantification of HIV-1 proviral DNA by a standardized colorimetric PCR-based assay.
A simple method was developed for measuring human immunodeficiency virus type 1 (HIV-1) proviral DNA in mononuclear cells based on the commercially available Amplicor(TM) HIV-1 polymerase chain reaction (PCR) assay and the limiting dilution method. The lowest limit of detection was four proviral genomes per 10(6) cells. The accuracy was demonstrated by using serial dilutions of LAV-8E5 cells, and the interassay variability was 0.2 log. The technique was used to measure HIV-1 proviral DNA in the peripheral blood mononuclear cells (PBMC) of 18 antiretroviral drug-naive HIV-1-positive individuals before and 4 weeks after initiating double nucleoside therapy. The DNA proviral titers at baseline (median = 3.45, range = 2.11-4.7 log copies/10(6) cells) were 2.08 log greater than the infectivity titers, but there was a correlation between these two parameters (r = 0.63, P = 0.009). The mean decrease in the proviral DNA titer after 4 weeks of therapy was 0.31 log, whereas the decrease in the infectivity titer was 0.81 log and the decrease in the plasma RNA concentration was 1.29 log. The technique was also used to measure HIV-1 proviral DNA in the PBMC of 11 patients who had undetectable plasma HIV-1 RNA after being placed on combination antiretroviral therapy. Although proviral DNA remained detectable in all patients after 36 weeks of treatment, a gradual decline with an estimated half-life of 21-58 weeks was observed. The reliability of this simple and convenient colorimetric PCR-based technique indicates its suitability for assessing the effect of current antiretroviral regimens on the latent reservoirs of provirus.
Izopet
J
J
Laboratoire de Virologie, Hôpital Purpan, Toulouse, France. izopet@cict.fr
Tamalet
C
C
Pasquier
C
C
Sandres
K
K
Marchou
B
B
Massip
P
P
Puel
J
J
D000328:Adult; D019380:Anti-HIV Agents / Q000627:therapeutic use; D003124:Colorimetry / Q000379:methods*; D004279:DNA, Viral / Q000032:analysis*; D016049:Didanosine / Q000627:therapeutic use; D005069:Evaluation Studies as Topic; D005260:Female; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D006801:Humans; D019469:Indinavir / Q000627:therapeutic use; D019259:Lamivudine / Q000627:therapeutic use; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000097:blood; D019438:Ritonavir / Q000627:therapeutic use; D014158:Transcription, Genetic; D016047:Zalcitabine / Q000627:therapeutic use; D015215:Zidovudine / Q000627:therapeutic use
D019380:Anti-HIV Agents; D004279:DNA, Viral; D012367:RNA, Viral; D019259:Lamivudine; D015215:Zidovudine; D019469:Indinavir; D016047:Zalcitabine; D016049:Didanosine; D019438:Ritonavir