title: A qualitative PCR minipool strategy to screen for virologic failure and antiretroviral drug resistance in South African patients on first-line antiretroviral therapy.
abstract: BACKGROUND
  The high cost of commercial HIV-1 viral load tests for monitoring of patients on antiretroviral treatment limits their use in resource-constrained settings. Commercial genotypic antiretroviral resistance testing is even more costly, yet it provides important benefits.
  
  
  OBJECTIVE
  We sought to determine the sensitivity and negative predictive value of a qualitative PCR targeting partial reverse transcriptase for detection of virologic failure when 5 patient specimens are pooled.
  
  
  METHODS
  A total of 300 South African routine patient samples were included and tested in 60 pools of 5 samples each. A qualitative nested PCR was optimised for testing pools and individual samples from positive pools. All positive samples were sequenced to detect drug resistance-associated mutations. Results were compared to those of conventional viral load monitoring.
  
  
  RESULTS
  Twenty-two of 60 pools tested positive. Individual testing yielded 29 positive individual samples. Twenty-six patients had viral loads of above 1000 copies/ml. The pooling algorithm detected 24 of those 26 patients, resulting in a negative predictive value of 99.3%, and a positive predictive value of 89.7%. The sensitivity for detecting patients failing therapy was 92%, with a specificity of 98.9%. Of the patients failing first-line ART, 83.3% had NRTI and 91.7% NNRTI resistance mutations.
  
  
  CONCLUSIONS
  The pooled testing algorithm presented here required 43% fewer assays than conventional viral load testing. In addition to offering a potential cost saving over individual viral load testing, it also provided drug resistance information which is not available routinely in resourced-limited settings.
authors.affiliation: Division of Medical Virology, Department of Pathology, National Health Laboratory Service (NHLS), Tygerberg and Stellenbosch University, Cape Town, Western Cape, South Africa. Electronic address: howard.newman@nhls.ac.za.
authors.forename: Howard
authors.initials: H
authors.lastname: Newman
authors.affiliation: Division of Medical Virology, Department of Pathology, National Health Laboratory Service (NHLS), Tygerberg and Stellenbosch University, Cape Town, Western Cape, South Africa; Medical Mission Institute and University of Würzburg, Würzburg, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Breunig
authors.affiliation: Division of Medical Virology, Department of Pathology, National Health Laboratory Service (NHLS), Tygerberg and Stellenbosch University, Cape Town, Western Cape, South Africa.
authors.forename: Gert
authors.initials: G
authors.lastname: van Zyl
authors.affiliation: Medical Mission Institute and University of Würzburg, Würzburg, Germany.
authors.forename: August
authors.initials: A
authors.lastname: Stich
authors.affiliation: Division of Medical Virology, Department of Pathology, National Health Laboratory Service (NHLS), Tygerberg and Stellenbosch University, Cape Town, Western Cape, South Africa.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Preiser
mesh_terms: D019380:Anti-HIV Agents / Q000627:therapeutic use*; D023241:Antiretroviral Therapy, Highly Active; D001483:Base Sequence; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D015658:HIV Infections / Q000188:drug therapy* / Q000821:virology; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use; D017423:Sequence Analysis, RNA; D013019:South Africa; D019562:Viral Load
chemical_list: D019380:Anti-HIV Agents; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors
keywords: ART monitoring; Cost effectiveness; HIV drug resistance; Pooled testing; Resource limited setting