title: Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA.
abstract: Although a number of PCR-based quantitative assays for measuring HIV-1 persistence during suppressive antiretroviral therapy (ART) have been reported, a simple, sensitive, reproducible method is needed for application to large clinical trials. We developed novel quantitative PCR assays for cell-associated (CA) HIV-1 DNA and RNA, targeting a highly conserved region in HIV-1pol, with sensitivities of 3 to 5 copies/1 million cells. We evaluated the performance characteristics of the assays using peripheral blood mononuclear cells (PBMCs) from 5 viremic patients and 20 patients receiving effective ART. Total and resting CD4(+)T cells were isolated from a subset of patients and tested for comparison with PBMCs. The estimated standard deviations including interassay variability and intra-assay variability of the assays were modest, i.e., 0.15 and 0.10 log10copies/10(6)PBMCs, respectively, for CA HIV-1 DNA and 0.40 and 0.19 log10copies/10(6)PBMCs for CA HIV-1 RNA. Testing of longitudinally obtained PBMC samples showed little variation for either viremic patients (median fold differences of 0.80 and 0.88 for CA HIV-1 DNA and RNA, respectively) or virologically suppressed patients (median fold differences of 1.14 and 0.97, respectively). CA HIV-1 DNA and RNA levels were strongly correlated (r= 0.77 to 1;P= 0.0001 to 0.037) for assays performed using PBMCs from different sources (phlebotomy versus leukapheresis) or using total or resting CD4(+)T cells purified by either bead selection or flow cytometric sorting. Their sensitivity, reproducibility, and broad applicability to small numbers of mononuclear cells make these assays useful for observational and interventional studies that examine longitudinal changes in the numbers of HIV-1-infected cells and their levels of transcription.
authors.affiliation: University of Pittsburgh School of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA feh10@pitt.edu.
authors.forename: Feiyu
authors.initials: F
authors.lastname: Hong
authors.affiliation: Center for Biostatistics in AIDS Research, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
authors.forename: Evgenia
authors.initials: E
authors.lastname: Aga
authors.affiliation: University of Pittsburgh School of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA.
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Cillo
authors.affiliation: University of Pittsburgh School of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA.
authors.forename: Aarika L
authors.initials: AL
authors.lastname: Yates
authors.affiliation: University of Pittsburgh School of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Besson
authors.affiliation: University of Pittsburgh School of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Fyne
authors.affiliation: University of Pittsburgh School of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA.
authors.forename: Dianna L
authors.initials: DL
authors.lastname: Koontz
authors.affiliation: Rush Medical College, Department of Immunology and Microbiology, Chicago, Illinois, USA.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Jennings
authors.affiliation: Center for Biostatistics in AIDS Research, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
authors.forename: Lu
authors.initials: L
authors.lastname: Zheng
authors.affiliation: University of Pittsburgh School of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Mellors
mesh_terms: D000328:Adult; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005260:Female; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008137:Longitudinal Studies; D008297:Male; D008875:Middle Aged; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral