Rapid Whole Genome Characterization of High-Risk Pathogens Using Long-Read Sequencing to Identify Potential Healthcare Transmission.

OBJECTIVE

Routine use of whole genome sequencing (WGS) has been shown to help identify transmission of pathogens causing healthcare-associated infections (HAIs). However, the current gold standard of short-read, Illumina-based WGS is labor and time-intensive. In light of recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing, we sought to establish a low resource utilization approach capable of providing accurate WGS-based comparisons of HAI pathogens within a time frame allowing for infection prevention and control (IPC) interventions.

METHODS

WGS was prospectively performed on antimicrobial-resistant pathogens at increased risk of potential healthcare transmission using the ONT MinION sequencer with R10.4.1 flow cells and Dorado basecalling algorithm. Potential transmission was assessed via Ridom SeqSphere+ for core genome multilocus sequence typing and MINTyper for reference-based core genome single nucleotide polymorphisms using previously published cut-off values. The accuracy of our ONT pipeline was determined relative to Illumina-based WGS data generated from the same genomic DNA sample.

RESULTS

Over a six-month period, 242 bacterial isolates from 216 patients were sequenced by a single operator. Compared to the Illumina gold-standard data, our ONT pipeline achieved a Q score of 60 for assembled genomes, even with a coverage rate of as low as 40X. The mean time from initiating DNA extraction to complete genetic analysis was 2 days (IQR 2-3.25 days). We identified five potential transmission clusters comprising 21 isolates (8.7% of all sequenced strains). Combining ONT WGS data with epidemiological data, >70% (15/21) of the isolates originated from patients with potential healthcare transmission links.

CONCLUSIONS

Via a stand-alone ONT pipeline, we detected potentially transmitted HAI pathogens rapidly and accurately, aligning closely with epidemiological data. Our low-resource method has the potential to assist in the efficient detection and deployment of preventative measures against HAI transmission.