Laura Powell1, Adit Dhummakupt1, Lilly Siems1, Dolly Singh1, Yann Le Duff2, Priyanka Uprety3, Cheryl Jennings4, Joseph Szewczyk1, Ya Chen1, Eleni Nastouli5, Deborah Persaud6. 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. Crown
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. Crown
Authors: T M Folks; K A Clouse; J Justement; A Rabson; E Duh; J H Kehrl; A S Fauci Journal: Proc Natl Acad Sci U S A Date: 1989-04 Impact factor: 11.205
Authors: Michael C Sneller; Erin D Huiting; Katherine E Clarridge; Catherine Seamon; Jana Blazkova; Jesse S Justement; Victoria Shi; Emily J Whitehead; Rachel F Schneck; Michael Proschan; Susan Moir; Anthony S Fauci; Tae-Wook Chun Journal: J Infect Dis Date: 2020-10-13 Impact factor: 5.226
Authors: Benjamin J Hindson; Kevin D Ness; Donald A Masquelier; Phillip Belgrader; Nicholas J Heredia; Anthony J Makarewicz; Isaac J Bright; Michael Y Lucero; Amy L Hiddessen; Tina C Legler; Tyler K Kitano; Michael R Hodel; Jonathan F Petersen; Paul W Wyatt; Erin R Steenblock; Pallavi H Shah; Luc J Bousse; Camille B Troup; Jeffrey C Mellen; Dean K Wittmann; Nicholas G Erndt; Thomas H Cauley; Ryan T Koehler; Austin P So; Simant Dube; Klint A Rose; Luz Montesclaros; Shenglong Wang; David P Stumbo; Shawn P Hodges; Steven Romine; Fred P Milanovich; Helen E White; John F Regan; George A Karlin-Neumann; Christopher M Hindson; Serge Saxonov; Bill W Colston Journal: Anal Chem Date: 2011-10-28 Impact factor: 6.986
Authors: Jori Symons; Abha Chopra; Eva Malatinkova; Ward De Spiegelaere; Shay Leary; Don Cooper; Chike O Abana; Ajantha Rhodes; Simin D Rezaei; Linos Vandekerckhove; Simon Mallal; Sharon R Lewin; Paul U Cameron Journal: Retrovirology Date: 2017-01-13 Impact factor: 4.602
Authors: Mohamed Abdel-Mohsen; Douglas Richman; Robert F Siliciano; Michel C Nussenzweig; Bonnie J Howell; Javier Martinez-Picado; Nicolas Chomont; Katharine J Bar; Xu G Yu; Mathias Lichterfeld; Jose Alcami; Daria Hazuda; Frederic Bushman; Janet D Siliciano; Michael R Betts; Adam M Spivak; Vicente Planelles; Beatrice H Hahn; Davey M Smith; Ya-Chi Ho; Maria J Buzon; Christian Gaebler; Mirko Paiardini; Qingsheng Li; Jacob D Estes; Thomas J Hope; Jay Kostman; Karam Mounzer; Marina Caskey; Lawrence Fox; Ian Frank; James L Riley; Pablo Tebas; Luis J Montaner Journal: Nat Med Date: 2020-09-07 Impact factor: 87.241
Authors: Matías Moragas; Maximiliano Distefano; Debora Mecikovsky; Solange Arazi Caillaud; Carolina Cernadas; Rosa Bologna; Paula Aulicino; Andrea Mangano Journal: PLoS One Date: 2018-10-23 Impact factor: 3.240