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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

  • Laura Di Sante , Andrea Costantini , Sara Caucci , Alice Corsi , Lucia Brescini , Stefano Menzo and Patrizia Bagnarelli EMAIL logo
Published/Copyright: April 28, 2020
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 59 Issue 3

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.

Keywords: calibration curve; HIV-1 infection; HIV-1 total DNA; HIV reservoir; nadir T CD4+ cell; naïve; proviral load; qPCR
Corresponding author: Prof. Patrizia Bagnarelli, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Via Tronto 10/A, 60131 Ancona, Italy,

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.

  1. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  2. Research funding: This work has been supported by grant provided by the Strategic Research Program 2016 of the Polytechnic University of Marche.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: Authors state no conflict of interest.

  6. 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).

Received: 2020-02-11
Accepted: 2020-04-05
Published Online: 2020-04-28
Published in Print: 2021-02-23

©2020 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2020-0142
Keywords for this article
calibration curve; HIV-1 infection; HIV-1 total DNA; HIV reservoir; nadir T CD4+ cell; naïve; proviral load; qPCR

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Complex analytical procedures in diagnostic laboratories and the IVDR
  4. Review
  5. Re-examining ferritin-bound iron: current and developing clinical tools
  6. Opinion Papers
  7. Designing a diagnostic Total Testing Process as a base for supporting diagnostic stewardship
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  9. EFLM Papers
  10. The Academy of the European Federation of Clinical Chemistry and Laboratory Medicine and the European Register of Specialists in Laboratory Medicine: guide to the Academy and the Register, version 4 – 2020
  11. A proposed Common Training Framework for Specialists in Laboratory Medicine under EU Directive 2013/55/EC (The Recognition of Professional Qualifications)
  12. Guidelines and Recommendations
  13. High-sensitivity cardiac troponin I and T methods for the early detection of myocardial injury in patients on chemotherapy
  14. General Clinical Chemistry and Laboratory Medicine
  15. Measurement of serum 17-hydroxyprogesterone using isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method and evaluation of the performance for three routine methods
  16. Evaluation of carbohydrate-deficient transferrin measurements on the V8 capillary electrophoresis system and comparison with the IFCC approved HPLC reference method and N-Latex immunonephelometric assay
  17. The clinical relevance of anti-dsDNA antibodies determined by the Elia™ dsDNA assay in patients with negative indirect immunofluorescence on the HEp-2 cell
  18. A hierarchical bivariate meta-analysis of diagnostic test accuracy to provide direct comparisons of immunoassays vs. indirect immunofluorescence for initial screening of connective tissue diseases
  19. Individual uromodulin serum concentration is independent of glomerular filtration rate in healthy kidney donors
  20. Comparison of iohexol plasma clearance formulas vs. inulin urinary clearance for measuring glomerular filtration rate
  21. Overweight-obesity is associated with decreased vitamin K2 levels in hemodialysis patients
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  23. Evaluating the performance of an updated high-sensitivity troponin T assay with increased tolerance to biotin
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