title: Standardization and performance evaluation of "modified" and "ultrasensitive" versions of the Abbott RealTime HIV-1 assay, adapted to quantify minimal residual viremia.
abstract: BACKGROUND
  Numerous studies investigating clinical significance of HIV-1 minimal residual viremia (MRV) suggest potential utility of assays more sensitive than those routinely used to monitor viral suppression. However currently available methods, based on different technologies, show great variation in detection limit and input plasma volume, and generally suffer from lack of standardization.
  
  
  OBJECTIVE
  In order to establish new tools suitable for routine quantification of minimal residual viremia in patients under virological suppression, some modifications were introduced into standard procedure of the Abbott RealTime HIV-1 assay leading to a "modified" and an "ultrasensitive" protocols.
  
  
  METHODS
  The following modifications were introduced: calibration curve extended towards low HIV-1 RNA concentration; 4 fold increased sample volume by concentrating starting material; reduced volume of internal control; adoption of "open-mode" software for quantification. Analytical performances were evaluated using the HIV-1 RNA Working Reagent 1 for NAT assays (NIBSC). Both tests were applied to clinical samples from virologically suppressed patients.
  
  
  RESULTS
  The "modified" and the "ultrasensitive" configurations of the assay reached a limit of detection of 18.8 (95% CI: 11.1-51.0 cp/mL) and 4.8 cp/mL (95% CI: 2.6-9.1 cp/mL), respectively, with high precision and accuracy. In clinical samples from virologically suppressed patients, "modified" and "ultrasensitive" protocols allowed to detect and quantify HIV RNA in 12.7% and 46.6%, respectively, of samples resulted "not-detectable", and in 70.0% and 69.5%, respectively, of samples "detected <40 cp/mL" in the standard assay.
  
  
  CONCLUSIONS
  The "modified" and "ultrasensitive" assays are precise and accurate, and easily adoptable in routine diagnostic laboratories for measuring MRV.
authors.affiliation: Laboratory of Virology, INMI L. Spallanzani, Via Portuense 292, 00149 Rome, Italy. alessandra.amendola@inmi.it
authors.forename: Alessandra
authors.initials: A
authors.lastname: Amendola
authors.forename: Maria
authors.initials: M
authors.lastname: Bloisi
authors.forename: Patrizia
authors.initials: P
authors.lastname: Marsella
authors.forename: Rosella
authors.initials: R
authors.lastname: Sabatini
authors.forename: Angela
authors.initials: A
authors.lastname: Bibbò
authors.forename: Claudio
authors.initials: C
authors.lastname: Angeletti
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Capobianchi
mesh_terms: D002138:Calibration; D019411:Clinical Laboratory Techniques; D003955:Diagnostic Tests, Routine / Q000379:methods* / Q000592:standards; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D012367:RNA, Viral / Q000097:blood; D011933:Reagent Kits, Diagnostic / Q000592:standards / Q000821:virology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load; D014766:Viremia / Q000175:diagnosis / Q000821:virology
chemical_list: D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic