Quantification of the HIV-1 total reservoir in the peripheral blood of naïve and treated patients by a standardised method derived from a commercial HIV-1 RNA quantification assay.

Abstract

HIV-1 DNA can persist in host cells, establishing a latent reservoir. This study was aimed to develop an extraction and amplification protocol for HIV-1 DNA quantification by modifying a quantitative commercial assay. HIV-1 DNA was extracted on an Abbott m2000sp instrument, using an open-mode protocol. Two calibrators, spiked with a plasmid containing HIV-1 genome (103 and 105 cps/mL), were extracted and amplified to generate a master calibration curve. Precision, accuracy, linear dynamic range, limit of quantification (LOQ) and limit of detection (LOD) were determined. A cohort of patients, naïve or chronically infected, was analysed. Calibration curve was obtained from 42 replicates of standards (stds); precision was calculated (coefficients of variability [CVs] below 10%); accuracy was higher than 90%. Linearity covered the entire range tested (10-104 copies per reaction), and LOD (95%) was 12 copies per reaction. HIV-1 DNA was significantly higher (p < 0.0001) in drug-naïve (62) than in chronically treated patients (50), and proviral loads correlated with lymphocytes (p = 0.0002) and CD4+ (p < 0.0001) counts only in naïve patients. Both groups displayed a significant inverse correlation between CD4+ nadir and proviral loads. A significant correlation (p < 0.0001) between viraemia and HIV-1 reservoir was disclosed. No significant difference was obtained from the comparison between proviral loads on whole blood and peripheral blood mononuclear cells (PBMCs) from the same patient. The novelty of our approach relies on the selection of appropriate reference standard extracted and amplified as clinical specimens avoiding any underestimation of the reservoir. Results confirm HIV-1 DNA as a marker of disease progression, supporting the relationship between the width of latent reservoir and the immunological status of the patient.