scholarly journals Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1456
Author(s):  
Cheryl Jennings ◽  
Neil T. Parkin ◽  
Daniel J. Zaccaro ◽  
Rupert Capina ◽  
Paul Sandstrom ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Quality Assurance ◽  
Next Generation Sequencing ◽  
Proficiency Testing ◽  
External Quality Assurance ◽  
External Quality ◽  
Assay Performance ◽  
Variant Detection ◽  
Generation Sequencing ◽  
Hiv 1

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.

External Quality Assurance Elispot Assay Proficiency Testing in HIV-1 Clinical Trials in Kenya

2014 ◽  
Vol 30 (S1) ◽  
pp. A176-A177
Author(s):  
Robert Kipyegon Langat ◽  
Jackton Indangasi ◽  
Simon Ogola ◽  
Bashir Farah ◽  
Peter Hayes ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Quality Assurance ◽  
Proficiency Testing ◽  
Elispot Assay ◽  
External Quality Assurance ◽  
External Quality ◽  
Hiv 1

Routine drug resistance testing in HIV-1 proviral DNA, using an automated next- generation sequencing assay

2019 ◽  
Vol 121 ◽  
pp. 104207 ◽  
Author(s):  
Enagnon Kazali Alidjinou ◽  
Pauline Coulon ◽  
Christophe Hallaert ◽  
Olivier Robineau ◽  
Agnès Meybeck ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Resistance Testing ◽  
Next Generation ◽  
Proviral Dna ◽  
Drug Resistance Testing ◽  
Generation Sequencing ◽  
Hiv 1

scholarly journals Performance of a high-throughput next-generation sequencing method for analysis of HIV drug resistance and viral load

2020 ◽  
Vol 75 (12) ◽  
pp. 3510-3516 ◽  
Author(s):  
Jessica M Fogel ◽  
David Bonsall ◽  
Vanessa Cummings ◽  
Rory Bowden ◽  
Tanya Golubchik ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Viral Load ◽  
Next Generation Sequencing ◽  
High Throughput ◽  
Whole Genome ◽  
Hiv Viral Load ◽  
Viral Loads ◽  
Viral Load Assay ◽  
Generation Sequencing ◽  
Hiv 1

Abstract Objectives To evaluate the performance of a high-throughput research assay for HIV drug resistance testing based on whole genome next-generation sequencing (NGS) that also quantifies HIV viral load. Methods Plasma samples (n = 145) were obtained from HIV-positive MSM (HPTN 078). Samples were analysed using clinical assays (the ViroSeq HIV-1 Genotyping System and the Abbott RealTime HIV-1 Viral Load assay) and a research assay based on whole-genome NGS (veSEQ-HIV). Results HIV protease and reverse transcriptase sequences (n = 142) and integrase sequences (n = 138) were obtained using ViroSeq. Sequences from all three regions were obtained for 100 (70.4%) of the 142 samples using veSEQ-HIV; results were obtained more frequently for samples with higher viral loads (93.5% for 93 samples with >5000 copies/mL; 50.0% for 26 samples with 1000–5000 copies/mL; 0% for 23 samples with <1000 copies/mL). For samples with results from both methods, drug resistance mutations (DRMs) were detected in 33 samples using ViroSeq and 42 samples using veSEQ-HIV (detection threshold: 5.0%). Overall, 146 major DRMs were detected; 107 were detected by both methods, 37 were detected by veSEQ-HIV only (frequency range: 5.0%–30.6%) and two were detected by ViroSeq only. HIV viral loads estimated by veSEQ-HIV strongly correlated with results from the Abbott RealTime Viral Load assay (R2 = 0.85; n = 142). Conclusions The NGS-based veSEQ-HIV method provided results for most samples with higher viral loads, was accurate for detecting major DRMs, and detected mutations at lower levels compared with a method based on population sequencing. The veSEQ-HIV method also provided HIV viral load data.

scholarly journals Development of an international external quality assurance program for HIV-1 incidence using the Limiting Antigen Avidity assay

PLoS ONE ◽  
2019 ◽  
Vol 14 (9) ◽  
pp. e0222290
Author(s):  
Sheila M. Keating ◽  
Wes Rountree ◽  
Eduard Grebe ◽  
Andrea L. Pappas ◽  
Mars Stone ◽  
...  
Keyword(s):  
Quality Assurance ◽  
External Quality Assurance ◽  
Quality Assurance Program ◽  
External Quality ◽  
Avidity Assay ◽  
Hiv 1

scholarly journals External Quality Assurance Program for PCR Amplification of Genomic DNA: An Italian Experience

2003 ◽  
Vol 49 (5) ◽  
pp. 782-791 ◽  
Author(s):  
Claudia Casini Raggi ◽  
Pamela Pinzani ◽  
Angelo Paradiso ◽  
Mario Pazzagli ◽  
Claudio Orlando
Keyword(s):  
Quality Assurance ◽  
Dna Extraction ◽  
Proficiency Testing ◽  
Pcr Amplification ◽  
Nucleic Acid Amplification ◽  
External Quality Assurance ◽  
Quality Assurance Program ◽  
External Quality ◽  
Consensus Values ◽  
Interpretation Of Results

Abstract Background: External quality assurance (EQA) programs for diagnostic tests based on nucleic acid amplification have not been widely implemented in clinical laboratories and remain limited to few tests. Development of specific EQA programs based on application-based proficiency testing for any diagnostic molecular target is challenging. Development of EQA trials based on methodologic proficiency testing and directed to the evaluation of analytical aspects common to the majority of PCR-based tests may be valuable. Methods: We developed an EQA program for evaluation of DNA extraction and amplification and analysis of products after PCR. Participants received a package containing primers and reference materials to evaluate three specific controls for, respectively, DNA extraction (quality and quantity), PCR performance (specificity and efficiency), and interpretation of results after electrophoresis. Each participant was asked to return to the organizers a form with their numerical results and an aliquot of all amplified samples for joint evaluation. Results: Results varied in all phases of the experimental procedure: preamplification, amplification, and post-PCR interpretation. To give a general estimation on the quality of performances for each laboratory, we designed a score scheme in which the results of any specific action were evaluated on the basis of the distribution around the median consensus values. The maximum possible score was 84. On the basis of total score obtained by each laboratory, we created a qualitative ranking list that provided the final interpretation of results as excellent (>63 points; n = 4 laboratories), good (53–63 points; n = 13), sufficient (42–52 points; n = 15), poor (31–41 points; n = 3), and not acceptable (<31 points; n = 4). Conclusions: This survey demonstrates the importance of EQA trials based on methodologic proficiency testing directed to evaluation of analytical aspects common to the majority of PCR-based tests.

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