title: Establishing diagnostic cut-off criteria for the COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative test through validation against the Amplicor DNA test v1.5 for infant diagnosis using dried blood spots.
abstract: BACKGROUND
  As antibody testing cannot confirm HIV-1 infection in children less than 18 months of age, diagnosis in these children depends on nucleic acid testing. The COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) (CAP/CTM, Roche(®) Molecular Systems, Inc., Branchburg, NJ) HIV-1 Qualitative test is a total nucleic acid real-time PCR assay utilising whole EDTA blood or dried blood spots (DBS), which recently replaced the Roche(®) AMPLICOR(®) DNA test v1.5 (Amplicor) as the diagnostic HIV PCR assay in many South African laboratories. For the Amplicor assay, stringent diagnostic criteria were previously formulated for the local population, and a comparison reported the CAP/CTM's sensitivity at 99.7% and specificity at 100% for both sample types compared to these Amplicor criteria.
  
  
  OBJECTIVE
  To validate the assay prior to introduction in our laboratory and to define stringent diagnostic cut-off criteria.
  
  
  METHODS
  Whole EDTA blood samples from patients younger than 18 months sent for routine HIV-1 diagnosis were tested by Amplicor, and positive results were confirmed from DBS. CAP/CTM assays were subsequently performed from DBS.
  
  
  RESULTS
  The CAP/CTM had a sensitivity of 98.8% and a specificity of 97.1%, but a positive predictive value (PPV) of only 78.7% compared to the Amplicor assay. Samples positive by CAP/CTM but negative by Amplicor displayed poor amplification curves compared to concordant positive samples. Upon re-testing those with sufficient material available by CAP/CTM, all showed negative results.
  
  
  CONCLUSIONS
  The decreased PPV may either be due to false positive CAP/CTM results, or increased sensitivity compared to the Amplicor assay. Criteria were formulated for defining presumed false-positive results.
authors.affiliation: Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, Stellenbosch University and National Health Laboratory Service Tygerberg, P.O. Box 19063, Tygerberg 7505, South Africa. maritzj@sun.ac.za
authors.forename: Jean
authors.initials: J
authors.lastname: Maritz
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Preiser
authors.forename: Gert U
authors.initials: GU
authors.lastname: van Zyl
mesh_terms: D001800:Blood Specimen Collection / Q000379:methods*; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics*; D005260:Female; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D007223:Infant; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000592:standards*; D011237:Predictive Value of Tests; D012367:RNA, Viral / Q000097:blood; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity; D013019:South Africa; D019562:Viral Load
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic