Validation and Application of Long-Read Whole-Genome Sequencing for Antimicrobial Resistance Gene Detection and Antimicrobial Susceptibility Testing.
Antimicrob Agents Chemother
; 67(1): e0107222, 2023 01 24.
Article
em En
| MEDLINE
| ID: mdl-36533931
ABSTRACT
Next-generation sequencing applications are increasingly used for detection and characterization of antimicrobial-resistant pathogens in clinical settings. Oxford Nanopore Technologies (ONT) sequencing offers advantages for clinical use compared with other sequencing methodologies because it enables real-time basecalling, produces long sequencing reads that increase the ability to correctly assemble DNA fragments, provides short turnaround times, and requires relatively uncomplicated sample preparation. A drawback of ONT sequencing, however, is its lower per-read accuracy than short-read sequencing. We sought to identify best practices in ONT sequencing protocols. As some variability in sequencing results may be introduced by the DNA extraction methodology, we tested three DNA extraction kits across three independent laboratories using a representative set of six bacterial isolates to investigate accuracy and reproducibility of ONT technology. All DNA extraction techniques showed comparable performance; however, the DNeasy PowerSoil Pro kit had the highest sequencing yield. This kit was subsequently applied to 42 sequentially collected bacterial isolates from blood cultures to assess Ares Genetics's pipelines for predictive whole-genome sequencing antimicrobial susceptibility testing (WGS-AST) performance compared to phenotypic triplicate broth microdilution results. WGS-AST results ranged across the organisms and resulted in an overall categorical agreement of 95% for penicillins, 82.4% for cephalosporins, 76.7% for carbapenems, 86.9% for fluoroquinolones, and 96.2% for aminoglycosides. Very major errors/major errors were 0%/16.7% (penicillins), 11.7%/3.6% (cephalosporins), 0%/24.4% (carbapenems), 2.5%/7.7% (fluoroquinolones), and 0%/4.1% (aminoglycosides), respectively. This work showed that, although additional refinements are necessary, ONT sequencing demonstrates potential as a method to perform WGS-AST on cultured isolates for patient care.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Farmacorresistência Bacteriana
/
Antibacterianos
Tipo de estudo:
Diagnostic_studies
/
Guideline
Limite:
Humans
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article