title: New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma.
abstract: More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 10(6) per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.
authors.affiliation: HIV Drug Resistance Program, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702-1201, USA. spalmer@ncifcrf.gov
authors.forename: Sarah
authors.initials: S
authors.lastname: Palmer
authors.forename: Ann P
authors.initials: AP
authors.lastname: Wiegand
authors.forename: Frank
authors.initials: F
authors.lastname: Maldarelli
authors.forename: Holly
authors.initials: H
authors.lastname: Bazmi
authors.forename: JoAnn M
authors.initials: JM
authors.lastname: Mican
authors.forename: Michael
authors.initials: M
authors.lastname: Polis
authors.forename: Robin L
authors.initials: RL
authors.lastname: Dewar
authors.forename: Angeline
authors.initials: A
authors.lastname: Planta
authors.forename: Shuying
authors.initials: S
authors.lastname: Liu
authors.forename: Julia A
authors.initials: JA
authors.lastname: Metcalf
authors.forename: John W
authors.initials: JW
authors.lastname: Mellors
authors.forename: John M
authors.initials: JM
authors.lastname: Coffin
mesh_terms: D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014766:Viremia / Q000821:virology
chemical_list: D012367:RNA, Viral