Abstract
Objectives
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.
Methods
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.
Results
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.
Conclusions
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.
Acknowledgements
Authors are grateful to CFAR-NIBSC for providing the “PCR Reference Kit/95 series (ARP 956)”; authors are grateful to Dott. Renato Pulvirenti and Dott. Alessandro Bellini (Abbott Molecular) for providing extraction and amplification kit supplies free of charge; authors are grateful to Dott. Stefano Belladonna (Abbott Molecular) for the technical support.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Research funding: This work has been supported by grant provided by the Strategic Research Program 2016 of the Polytechnic University of Marche.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: Authors state no conflict of interest.
Ethical approval: The local Institutional Review Board deemed the study exempt from review.
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2020-0142).
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