Implementation of a high-throughput whole genome sequencing approach with the goal of maximizing efficiency and cost effectiveness to improve public health.

This manuscript describes the development of a streamlined, cost-effective laboratory workflow to meet the demands of increased whole genome sequence (WGS) capacity while achieving mandated quality metrics. From 2020 to 2021, the Wadsworth Center Bacteriology Laboratory (WCBL) used a streamlined workflow to sequence 5,743 genomes that contributed sequence data to nine different projects. The combined use of the QIAcube HT, Illumina DNA Prep using quarter volume reactions, and the NextSeq allowed the WCBL to process all samples that required WGS while also achieving a median turn-around time of 7 days (range 4 to 10 days) and meeting minimum sequence quality requirements. Public Health Laboratories should consider implementing these methods to aid in meeting testing requirements within budgetary restrictions. IMPORTANCE: Public Health Laboratories that implement whole genome sequencing (WGS) technologies may struggle to find the balance between sample volume and cost effectiveness. We present a method that allows for sequencing of a variety of bacterial isolates in a cost-effective manner. This report provides specific strategies to implement high-volume WGS, including an innovative, low-cost solution utilizing a novel quarter volume sequencing library preparation. The methods described support the use of high-throughput DNA extraction and WGS within budgetary constraints, strengthening public health responses to outbreaks and disease surveillance.

Comparative analysis of multiplexed PCR and short- and long-read whole genome sequencing to investigate a large Klebsiella pneumoniae outbreak in New York State.

In 2017, the New York State Department of Health investigated a large Klebsiella pneumoniae outbreak in a health care facility. A retrospective analysis was conducted to compare the use of multiple molecular typing methods for characterizing the outbreak. Forty-four isolates were characterized using the rapid real-time PCR OpGen Acuitas® AMR Gene Panel. Additionally, short-read whole genome sequencing (WGS) analysis was used to identify antimicrobial resistance (AMR) genes and assess isolate relatedness. Long-read Oxford Nanopore MinION WGS was used to characterize the plasmid content of a subset of isolates. All methods showed overall concordance, identifying four clusters, with a few discrepancies in the clustering of individual isolates. Though short- and long-read WGS results provided a more nuanced understanding of the molecular epidemiology of this outbreak, this study highlights the utility of the Acuitas® PCR-based approach, which can more easily be performed by health care facilities, for rapid clustering of patient isolates.

Laboratory diagnosis of bacterial meningitis by direct detection, serotyping and Next Generation Sequencing: How 10 years of testing in New York State has evolved to improve laboratory diagnosis and public health.

Since 2005, the Wadsworth Center (WC) has provided molecular testing on cerebrospinal fluid (CSF) and whole blood specimens in close collaboration with epidemiologists in New York State and New York City. In this study, we analyzed 10 years of data to demonstrate the significant value of utilizing molecular methods to assess patient specimens for etiologic agents of bacterial meningitis. A comprehensive molecular testing algorithm to detect and serotype/serogroup bacterial agents known to cause bacterial meningitis (Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae and Streptococcus agalactiae) has evolved, and retrospective specimen testing has been essential for each improvement. Over a ten-year span from 2010 to 2019 the WC received 831 specimens from 634 patients with suspected bacterial meningitis. Real-time PCR was positive for at least one of the agents in 223 (27%) specimens from 183 patients (29%). Of the 223 positives, 146 (66%) were further characterized by real-time PCR into serogroup/serotype. Additionally, examination of 131 paired specimens of CSF and whole blood from the same patients found better detection in CSF, but whole blood is a useful alternative for diagnosis when CSF is not available. For specimens initially PCR-negative, 16S rDNA Sanger sequencing was requested by the submitter for 146 cases resulting in the identification of bacterial agents in an additional 24 (16%) specimens. In a retrospective study, Next Generation Sequencing (NGS) was evaluated for the detection of pathogens in 53 previously tested PCR-negative CSF specimens and identified bacteria in 14 (26%) specimens. This molecular testing algorithm has provided clinicians a diagnosis when culture is negative with the potential to guide therapy. It has also aided public health in determining when antibiotic prophylaxis was needed, augmented surveillance data to yield a fuller picture of community prevalence, and highlighted gaps in the spectrum of agents that cause bacterial meningitis.

Legionnaires' Disease Outbreaks and Cooling Towers, New York City, New York, USA.

The incidence of Legionnaires' disease in the United States has been increasing since 2000. Outbreaks and clusters are associated with decorative, recreational, domestic, and industrial water systems, with the largest outbreaks being caused by cooling towers. Since 2006, 6 community-associated Legionnaires' disease outbreaks have occurred in New York City, resulting in 213 cases and 18 deaths. Three outbreaks occurred in 2015, including the largest on record (138 cases). Three outbreaks were linked to cooling towers by molecular comparison of human and environmental Legionella isolates, and the sources for the other 3 outbreaks were undetermined. The evolution of investigation methods and lessons learned from these outbreaks prompted enactment of a new comprehensive law governing the operation and maintenance of New York City cooling towers. Ongoing surveillance and program evaluation will determine if enforcement of the new cooling tower law reduces Legionnaires' disease incidence in New York City.