Quantification of total HIV-1 DNA in buffy coat cells, feasibility and potential added value for clinical follow-up of HIV-1 infected patients on ART

Abstract

Background: Successfully treated HIV-1 infected patients have a sustained undetectable viral RNA load. In these cases the total HIV-1 DNA load may constitute a valuable tool to further follow the overall viral burden. The value of this marker outside of cure research has been rarely studied.

Objectives: To develop a quantitative (q)PCR for total HIV-1 DNA quantification in buffy coat cells and to evaluate the value of this parameter in clinical follow-up.

Study design: A qPCR using primers and a probe in the conserved HIV-1 LTR region was adapted for use on DNA extracted from buffy coat cells. Sensitivity, accuracy and reproducibility were evaluated using 8E5 cells and samples from naive and treatment experienced patients. The clinical value of DNA load analysis was assessed by testing 119 longitudinal samples from 9 patients before and after ART initiation and 249 cross sectional samples from therapy-experienced patients.

Results: Inter- and intra-assay coefficients of variability were 5.56 and 5.94 (%CV). HIV-1 DNA was detected in 249 of the 263 (94.7%) patients on ART for at least 5 months (median: 53 months; IQR: 28-84 months). The HIV-1 DNA load varied between 0.60 and 3.37 copies/10⁶ blood cells and showed significant correlation with the pre-ART CD4⁺ T-cell count nadir and peak viral RNA load. ART initiation resulted in a slow and limited decline of the total HIV-1 DNA concentration.

Conclusions: Quantification of total HIV-1 DNA from buffy coat cells is feasible, sensitive and reliable. Although determination of the on-therapy HIV-1 DNA load may be informative, regular testing has limited clinical value because of the very slow evolution.

Keywords: DNA-load; HIV-1; HIV-1 DNA quantification; Provirus.