Clinical validation of a quantitative HIV-1 DNA droplet digital PCR assay: Applications for detecting occult HIV-1 infection and monitoring cell-associated HIV-1 dynamics across different subtypes in HIV-1 prevention and cure trials

Laura Powell; Adit Dhummakupt; Lilly Siems; Dolly Singh; Yann Le Duff; Priyanka Uprety; Cheryl Jennings; Joseph Szewczyk; Ya Chen; Eleni Nastouli; Deborah Persaud

doi:10.1016/j.jcv.2021.104822

Clinical validation of a quantitative HIV-1 DNA droplet digital PCR assay: Applications for detecting occult HIV-1 infection and monitoring cell-associated HIV-1 dynamics across different subtypes in HIV-1 prevention and cure trials

J Clin Virol. 2021 Jun:139:104822. doi: 10.1016/j.jcv.2021.104822. Epub 2021 Apr 14.

Authors

Affiliations

¹ Johns Hopkins University, School of Medicine, Department of Pediatrics, Division of Infectious Diseases, Baltimore, MD, United States.
² Center for AIDS Reagents, National Institute for Biological Standards and Controls, England, UK.
³ Department of Pathology and Laboratory Medicine, Robert Wood Johnson University Hospital, Rutgers University, New Brunswick, NJ, United States.
⁴ Rush University Medical Center, Department of Molecular Pathogens and Immunity, Chicago, IL, United States.
⁵ Department of Population, Policy and Practice, UCL Great Ormond Street Institute of Child Health and Francis Crick Institute, London, UK.
⁶ Johns Hopkins University, School of Medicine, Department of Pediatrics, Division of Infectious Diseases, Baltimore, MD, United States; Departments of Molecular Microbiology and Immunology and International Health, Johns Hopkins Bloomberg School of Public Health, United States. Electronic address: dpers@jhmi.edu.

Abstract

Background: In HIV-1-exposed infants, nucleic acid testing (NAT) is required to diagnose infection since passively transferred maternal antibodies preclude antibody testing. The sensitivity of clinical NAT assays is lowered with infant antiretroviral prophylaxis and, with empiric very early antiretroviral treatment of high-risk infants, thereby impacting early infant diagnosis. Similarly, adult HIV-1 infections acquired under pre-exposure prophylaxis may occur at low levels, with undetectable plasma viremia and indeterminate antibody tests, for which HIV-1 DNA testing maybe a useful adjunct. Cell-associated HIV-1 DNA concentrations are also used to monitor HIV-1 persistence in viral reservoirs with relevance to HIV-1 cure therapeutics, particularly in perinatal infections.

Objectives: We clinically validated an HIV-1 DNA quantitative assay using droplet digital PCR (ddPCR), across different HIV-1 subtypes.

Study design: The analytical sensitivity and specificity of an HIV-1 DNA ddPCR assay was determined using serial dilutions of a plasmid containing HIV-1 LTR-gag spiked into peripheral blood mononuclear cells (PBMCs), with MOLT-4 cells or PBMCs infected with different HIV-1 subtypes (A, B and C), and U1 cells spiked into PBMCs. Inter- and intra-run variability were used to determine assay precision.

Results: The HIV-1 LTR-gag ddPCR assay was reliable and reproducible, and exhibited high analytical specificity with sensitivity to near single copy level, across multiple HIV-1 subtypes, and a limit of detection of 4.09 copies/million PBMCs.

Conclusions: This assay has applications for detecting occult HIV-1-infection in the setting of combination and long-acting regimens used for HIV-1 prevention, across different HIV-1 subtypes, in infants and adults, and in HIV-1 cure interventions.

Keywords: HIV-1; Methods; ddPCR.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Anti-Retroviral Agents / therapeutic use
DNA, Viral / genetics
HIV Infections* / diagnosis
HIV Infections* / drug therapy
HIV-1* / genetics
Humans
Leukocytes, Mononuclear
Polymerase Chain Reaction
Sensitivity and Specificity
Viral Load

Substances

Anti-Retroviral Agents
DNA, Viral

Abstract

Publication types

MeSH terms

Substances

Grants and funding