Establishing a Neisseria gonorrhoeae whole-genome sequencing external quality assessment for the Antimicrobial Resistance Laboratory Network (AR Lab Network) regional laboratories.

Drug-resistant Neisseria gonorrhoeae (GC) is classified by the Centers for Disease Control and Prevention (CDC) as an urgent threat. Effective surveillance of antimicrobial resistance and strain types through whole-genome sequencing (WGS) is essential to preserve existing antibiotics for treatment. To ensure consistent, high-quality data, we implemented an External Quality Assessment (EQA) program within the Antimicrobial Resistance Laboratory Network in fall 2020. This biannual program distributes five blinded GC reference isolates in triplicate to four regional laboratories for antimicrobial susceptibility testing and WGS. Data were transferred to the CDC for taxonomic classification, assembly, phylogenetics, and quality evaluation. During fall 2020 (pilot)-spring (Cycle 1) 2023, five laboratories (four external; one at CDC) participated in a pilot and five EQA cycles. In the pilot cycle, all laboratories met all quality metrics. However, in 2021 Cycle 1, one laboratory scored below passing (<80%), highlighting the need for ongoing assessment. Issues were due to low coverage and poor-quality assemblies. Multilocus sequence typing and single-nucleotide polymorphism distances identified sample-swapping errors. In 2022 Cycle 1, two laboratories scored <80% due to sample handling issues and low sequence quality. After identifying the root cause and implementing corrective actions, all laboratories achieved 100% pass rates in 2022 Cycle 2 and 2023 Cycle 1. The EQAs identified shortcomings in WGS and sample management workflows not evident in routine processing and supported continual improvement of the participating laboratories and WGS quality management. This approach supports effective surveillance of drug-resistant GC, which informs the development of CDC's sexually transmitted infection treatment guidelines.IMPORTANCEThe External Quality Assessment (EQA) program for whole-genome sequencing (WGS) of Neisseria gonorrhoeae (GC) is necessary for robust surveillance of antimicrobial resistance (AR)-associated genomic markers and strain prevalence. By ensuring high-quality, consistent WGS data across laboratories participating in genomic surveillance, this program addresses issues identified in sequencing workflows and sample management that were not apparent in routine processing. Eight sequence quality metrics and alignment of phenotypic and genotypic data enhance data reliability, supporting the Centers for Disease Control and Prevention's efforts to monitor and control drug-resistant GC. The successful implementation of corrective actions after issues discovered in some EQA cycles demonstrates the program's effectiveness in improving laboratory performance. This program contributes to the fidelity of and capacity for genomic surveillance, identification of AR genomic variants, and public health strategies to reduce GC infections.