ADVANCEMENT OF THE SCIENCE
FIGURE 1
Relationship of Clinical and Environmental Samples via Whole Genome Sequencing for Legionnaires’ Disease in New York During the 2019–2021 Outbreak
2 0 1 9
2 0 2 0
2 0 2 1
H ot T u b
P otentially Seeded Hot Tub in 2019 (Approximately 2 mi From Cooling Tower)
2– 5 SNP s Apart
3– 7 SNP s Apart
I ndu str i a l C ool i ng T ow er E
Cooling Tower (Approximately 2 mi From Hot Tub)
(L p1 P ositive 2019 and L p5 P ositive 2021)
L eg end: I nv esti g a ted C a se T y pes
L p1 Isolated (G enetically R elated) L p1 Isolates (Sputum, Not G enetically R elated) U AT+ (No L e g i o ne l l a Isolated From Sputum) U AT+ (No Sputum Collected)
Note. Figure 1 shows the relationship between environmental and clinical samples found to be genetically similar in 2019 and 2021 via whole genome sequencing. The strength of the relationship is described in single nucleotide polymorphisms (SNPs). Environmental samples from cooling tower E were genetically similar to four 2019 cases and separated by only 2–5 SNPs. A clinical sample and an environmental hot tub sample from 2021 were separated by only 3–7 SNPs in comparison with the environmental sample collected from cooling tower E in 2019. The cooling tower potentially seeded the hot tub in 2019. The hot tub was the likely point of exposure for the patient case in 2021. Lp = Legionella pneumophila ; UAT+ = urine antigen test positive; UAT- = urine antigen test negative.
become aerosolized. The three locations cho- sen were the trickling filter pump discharge, the primary aeration tank discharge, and the activated sludge discharge. CCHD sta had observed the trickling filter producing aerosols. The samples were collected by CCHD sta and shipped to four dierent laboratories: two con- tract laboratories, WC-BL, and WC-EBL. In 2019, the NYSDOH began request- ing access to LD case residences to sample their water systems for Legionella . Of the 17 requested home visits, 7 agreed to have their homes sampled. All homes used the chlori- nated public water system as a water source. In each home, a minimum of three samples were collected. In July 2021, two cases were identified within 2 mi of cooling tower E. Next, CCHD and NYSDOH sta made visits and chose
sample locations based on travel history of patient cases. One sample was collected from a hose in a bar basement used by a sta mem- ber at the bar. New samples were collected from cooling tower E and two other towers on the same property. The travel history of one case revealed a visit to a neighbor’s outdoor private hot tub. This private hot tub was also sampled by NYSDOH sta (water and swabs). Sample Collection and Processing Over the course of the investigation, a total of 74 environmental potable and nonpo- table samples were collected between 2019 and 2021. Samples were split and analyzed by multiple methods simultaneously. All 74 samples were analyzed by standard culture (ISO 11731). Of those samples, 45 were also analyzed by standard qPCR and 30 were
screened by rapid qPCR using the Spartan Cube (Table 2). Environmental water sam- ples were submitted to WC-EBL, WC-BL, and two contract laboratories to be analyzed per ISO 11731:2017 (International Organization for Standardization [ISO], 2017). The Spartan Cube is a 4-in. portable device that detects Legionella pneumophila DNA by qPCR in 45 min. This technology is designed to meet the objectives of ISO/Technical Stan- dard 12869:2019 (Ahmed et al., 2019; ISO, 2019). The Bureau of Water Supply Protec- tion used the Spartan Cube to screen samples and to generate fast actionable results. WC-BL extracted DNA from submitted water samples using the Lucigen MasterPure complete DNA purification kit and tested the DNA for the presence of Legionella by quanti- tative PCR (qPCR) assay as described by Naz-
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Volume 86 • Number 9
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