Development and Application of Performance Assessment Criteria for Next-Generation Sequencing-Based HIV Drug Resistance Assays
Michael G. Becker,
Dun Liang,
Breanna Cooper,
Yan Le,
Tracy Taylor,
Emma R. Lee,
Sutan Wu,
Paul Sandstrom,
Hezhao Ji
Affiliations
Michael G. Becker
National HIV and Retrovirology Laboratories, National Microbiology Laboratory at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada
Dun Liang
ViroDx Clinical Diagnostics Laboratory, St. Louis, MO 63017, USA
Breanna Cooper
ViroDx Clinical Diagnostics Laboratory, St. Louis, MO 63017, USA
Yan Le
ViroDx Clinical Diagnostics Laboratory, St. Louis, MO 63017, USA
Tracy Taylor
National HIV and Retrovirology Laboratories, National Microbiology Laboratory at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada
Emma R. Lee
National HIV and Retrovirology Laboratories, National Microbiology Laboratory at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada
Sutan Wu
SutanStats, St. Louis, MO 63017, USA
Paul Sandstrom
National HIV and Retrovirology Laboratories, National Microbiology Laboratory at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada
Hezhao Ji
National HIV and Retrovirology Laboratories, National Microbiology Laboratory at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada
Next-generation sequencing (NGS)-based HIV drug resistance (HIVDR) assays outperform conventional Sanger sequencing in scalability, sensitivity, and quantitative detection of minority resistance variants. Thus far, HIVDR assays have been applied primarily in research but rarely in clinical settings. One main obstacle is the lack of standardized validation and performance evaluation systems that allow regulatory agencies to benchmark and accredit new assays for clinical use. By revisiting the existing principles for molecular assay validation, here we propose a new validation and performance evaluation system that helps to both qualitatively and quantitatively assess the performance of an NGS-based HIVDR assay. To accomplish this, we constructed a 70-specimen proficiency test panel that includes plasmid mixtures at known ratios, viral RNA from infectious clones, and anonymized clinical specimens. We developed assessment criteria and benchmarks for NGS-based HIVDR assays and used these to assess data from five separate MiSeq runs performed in two experienced HIVDR laboratories. This proposed platform may help to pave the way for the standardization of NGS HIVDR assay validation and performance evaluation strategies for accreditation and quality assurance purposes in both research and clinical settings.
