Abstract
The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) established a robust proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. While many of the lessons learned during the development of such programs may also apply to next generation sequencing (NGS)-based HIVDR assays, challenges remain for the ongoing evaluation of NGS-based testing. These challenges include a proper assessment of assay accuracy and the reproducibility of low abundance variant detection, intra- and inter-assay performance comparisons among laboratories using lab-defined tests, and different data analysis pipelines designed for NGS. In collaboration with the World Health Organization (WHO) Global HIVDR Laboratory Network and the Public Health Agency of Canada, the Rush VQA program distributed archived proficiency testing panels to ten laboratories to evaluate internally developed NGS assays. Consensus FASTA files were submitted using 5%, 10%, and 20% variant detection thresholds, and scored based on the same criteria used for SS. This small study showed that the SS External Quality Assurance (EQA) approach can be used as a transitional strategy for using NGS to generate SS-like data and for ongoing performance while using NGS data from the same quality control materials to further evaluate NGS assay performance.
Highlights
The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) program was operated by Rush University Medical Center from 1993–2019 (Rush VQA).The main objective of this program was to provide a comprehensive quality assurance program for laboratories supporting NIAID-sponsored clinical trials
This testing helped to set the groundwork for the future analysis of laboratory performance for Sanger sequencing (SS) HIV-1 drug resistance (HIVDR) testing, which included the receipt and handling of specimens, the extraction, amplification and sequencing of the panel specimens and the reporting of amino acid substitutions associated with HIVDR
In the early years of testing, the majority of the laboratories that participated in the VQA HIVDR external quality assurance (EQA) program used research use only versions of manufactured kits that were eventually approved by the U.S Food and
Summary
The data were ignored for proficiency testing scoring New technologies, such as generation sequencing (NGS), offer a more quantitative way to evaluate HIV drug resistance, but the sensitivity of these assays and the clinical relevance of low abundant variants (LAVs) is not yet defined. In 2017, the Rush VQA, in collaboration with the WHO Global HIV-1 Drug Resistance laboratory network [30] and the Public Health Agency of Canada [31], invited laboratories that had developed HIVDR testing using NGS to participate in a pilot study, aiming to explore the feasibility of assessing NGS HIVDR data using Sanger sequencing (SS)-based EQA scoring strategies as a transitional approach for switching to NGS technologies while simultaneously collecting data for more in-depth analyses of NGS data and laboratory performance
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