Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay.
Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log(10). Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10(6) PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10(6) PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (P = 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5' end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4(+) cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.
Désiré
N
N
Service de Microbiologie, Hôpital Rothschild, 33 Boulevard de Picpus, 75571 Paris Cedex 12, France. ndesire@infobiogen.fr
Dehée
A
A
Schneider
V
V
Jacomet
C
C
Goujon
C
C
Girard
P M
PM
Rozenbaum
W
W
Nicolas
J C
JC
D019380:Anti-HIV Agents / Q000627:therapeutic use; D001483:Base Sequence; D004279:DNA, Viral / Q000097:blood*; D004359:Drug Therapy, Combination; D005724:Ganglia / Q000821:virology; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses; D012367:RNA, Viral / Q000097:blood; D012007:Rectum / Q000821:virology; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism; D019562:Viral Load
D019380:Anti-HIV Agents; D004279:DNA, Viral; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D019914:Taq Polymerase