Quantification of Human Immunodeficiency Virus Type 1 Proviral Load by a TaqMan Real-Time PCR Assay

Abstract

ABSTRACT 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.