collected from 15 of the initially investigated LD cases over the course of the investigation (Table 1). Of the 15 clinical samples with available sputum, 8 were positive for Lp1 via culture (Table 1). Lp1 isolates from 5 of the 8 culture-positive sputum samples were found to be genetically related (Figure 1). Of the 74 total environmental samples col- lected, 13 cultured positive for Lp1: 7 cooling tower basins, 2 cooling tower swabs, 1 hot tub water sample, 2 hot tub swab samples, and 1 wastewater treatment plant sample (Table 3). Two cooling tower basin samples (i.e., two follow-up samples of cooling tower E) cul- tured positive for Lp2 through Lp15. A total of 32 cooling tower samples, 22 basin water samples, and 10 cooling tower swab samples were collected and analyzed (Table 4). In total, 11 cooling towers were sampled, some on multiple occasions. A total of 29 water sam- ples were collected from LD case residences. Neither Legionella species nor L. pneumophila were detected in any of the home samples via culture (Table 4). Legionella species DNA was detected via standard qPCR in 12 of 29 sam- ples from case residences submitted for qPCR analysis (Table 4). Other environmental sources sampled included a decorative fountain, a swab of a humidifier, a basement hose spigot, and a baseball field irrigation system. A decorative fountain in the center of town was positive for Legionella species via standard PCR but negative for any species of Legionella via cul- ture (Table 4). Three wastewater treatment plant samples were analyzed. Of the four lab- oratories processing these split samples, only WC-BL was able to detect the presence of Lp1 DNA and isolate colonies for the trickling fil- ter pump discharge sample (Table 4). In 2019, whole genome sequencing deter- mined that there was a genetic link between a cooling tower E sample Lp1 isolate and isolates recovered from four nearby clinical cases (Figure 1). This cooling tower E was one of three year-round towers located on the grounds of a heat treatment and processing plant located within 2 mi of four case resi- dences. More than 50 gallons of iron sludge was removed from the basin of cooling tower E. After the sludge removal, water quality analyses still revealed a range of 1.0–2.0 ppm of iron present in the basin water. The hose sample from the bar basement sampled in June 2021 was negative for any
TABLE 3
Water Samples Positive for Legionella pneumophila Serogroup 1 by Culture in New York During the 2019–2021 Outbreak
Sample Type
# of Samples
# of Culture Positive Samples (Lp1)
22 10 29
7 2 0 0 1 2 0 0 1 0
Cooling tower basin* Cooling tower swab Residence potable water
Residence potable swab (e.g., humidifier)
1 2 3 1 1 3 2
Hot tub water Hot tub swab
Decorative fountain
Decorative fountain swab Wastewater treatment plant
Other
Total
74
13
* A total of 13 samples tested positive for Legionella pneumophila serogroup 1 (Lp1). An additional two cooling tower basin samples tested positive for Lp5.
arian et al. (2008) and Weiss et al. (2017). Samples that were positive for L. pneumophila serogroup 1 (Lp1) DNA were cultured on selective media for up to 10 days at 37 °C, as previously described (Nazarian et al., 2008). Simultaneously, the Lp1 PCR-positive samples were also analyzed using a modified IDEXX Legiolert method. Briefly, 200 µl of the sample was added to 200 µl of IDEXX pretreat- ment and incubated at room temperature for 60 s. After the incubation, 200 µl of the sample was transferred to a bottle containing IDEXX Legiolert media, which was then transferred into a Quanti-tray, sealed, and incubated at 37 °C for up to 10 days. During incubation, the Quanti-trays were monitored for any wells that turn brown or turbid; then the media in these wells were removed and cultured on selec- tive media as described above. Legionella -like bluish-gray colonies were confirmed by mul- tiplex real-time PCR and underwent whole genome sequencing and analysis using the Legionella Clustering (LegioCluster) pipeline as described by Haas et al. (2021), Lapierre et al. (2017), and Wells et al. (2018). Wind Direction Analysis Wind direction data were obtained from the nearest New York State Mesonet (NYS Mesonet) weather station. The local station, located
approximately 3.5 km northwest of the town, provides averages from all sensors in 5-min, hourly, and daily increments (NYS Mesonet, 2024). Hourly averages of wind direction col- lected from a sonic anemometer at 10 m above the ground from December 2017 to August 2022 were requested. Exposure periods were the 12 days between 14 days and 2 days prior to the symptom onset date for each LD case. Wind direction was grouped by degrees into cardinal wind directions, and the hourly frequency of each cardinal direction occur- rence was then calculated for each estimated exposure period. Frequencies of wind direc- tion from west-southwest, southwest, and south-southwest were summed to represent the frequency of the optimal wind direc- tion. Optimal wind direction was estimated based on the location of cooling tower E and its proximity to the residences of cases with genetically similar clinical samples. Data cleaning and grouping of wind directions was performed in Python 3.10, and figures were generated using Microsoft Excel.
Results
Clinical and Environmental Samples A total of 17 cases were UAT positive and not ruled out due to travel history. Sputum was
19
May 2024 • our6*l o/ 6=2ro6me6;*l e*l;1
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