Legionnaires' Disease Outbreak Associated With a Hot Tub Display at the North Carolina Mountain State Fair, September 2019.

OBJECTIVES: On September 23, 2019, the North Carolina Division of Public Health identified a legionellosis increase in western North Carolina; most patients had recently attended the North Carolina Mountain State Fair. We conducted a source investigation. METHODS: Cases were fair attendees with laboratory-confirmed legionellosis and symptom onset within 2 to 14 days (Legionnaires' disease) or ≤3 days (Pontiac fever). We conducted a case-control study matching cases to non-ill fair attendees as control participants and an environmental investigation, and we performed laboratory testing (Legionella bacteria culture and polymerase chain reaction) of 27 environmental samples from fairgrounds and hot tubs and 14 specimens from case patients. We used multivariable unconditional logistic regression models to calculate adjusted odds ratios for potential Legionella exposure sources and risk factors. RESULTS: Of 136 people identified with fair-associated legionellosis, 98 (72%) were hospitalized and 4 (3%) died. Case patients were more likely than control participants to report walking by hot tub displays (adjusted odds ratio = 10.0; 95% CI, 4.2-24.1). Complete hot tub water treatment records were not kept, precluding evaluation of water maintenance conducted on display hot tubs. Legionella pneumophila sequence types (STs) were consistent among 10 typed clinical specimens (ST224) but distinct from the only positive environmental sample from the fair (ST7 and ST8). CONCLUSIONS: Hot tub displays were identified as the most likely outbreak source, making this the largest hot tub-associated Legionnaires' disease outbreak worldwide. Following the investigation, the North Carolina Division of Public Health and the Centers for Disease Control and Prevention released guidance on mitigating risk of Legionella exposure from hot tub displays. Results highlight the importance of properly maintaining equipment that aerosolizes water, including hot tubs intended for display purposes only.

Large Community Outbreak of Legionnaires Disease Potentially Associated with a Cooling Tower - Napa County, California, 2022.

Legionnaires disease is a serious infection acquired by inhalation of water droplets from human-made building water systems that contain Legionella bacteria. On July 11 and 12, 2022, Napa County Public Health (NCPH) in California received reports of three positive urinary antigen tests for Legionella pneumophila serogroup 1 in the town of Napa. By July 21, six Legionnaires disease cases had been confirmed among Napa County residents, compared with a baseline of one or two cases per year. NCPH requested assistance from the California Department of Public Health (CDPH) and CDC to aid in the investigations. Close temporal and geospatial clustering permitted a focused environmental sampling strategy of high-risk facilities which, coupled with whole genome sequencing results from samples and investigation of water system maintenance, facilitated potential linking of the outbreak with an environmental source. NCPH, with technical support from CDC and CDPH, instructed and monitored remediation practices for all environmental locations that tested positive for Legionella. The investigation response to this community outbreak illustrates the importance of interdisciplinary collaboration by public health agencies, laboratory support, timely communication with the public, and cooperation of managers of potentially implicated water systems. Timely identification of possible sources, sampling, and remediation of any facility testing positive for Legionella is crucial to interrupting further transmission.

Potential Association of Legionnaires' Disease with Hot Spring Water, Hot Springs National Park and Hot Springs, Arkansas, USA, 2018-2019.

Legionella pneumophila is the cause of Legionnaires' disease, a life-threatening pneumonia that occurs after inhalation of aerosolized water containing the bacteria. Legionella growth occurs in stagnant, warm-to-hot water (77°F-113°F) that is inadequately disinfected. Piped hot spring water in Hot Springs National Park, Arkansas, USA, has naturally high temperatures (>135°F) that prevent Legionella growth, and Legionnaires' disease has not previously been associated with the park or other hot springs in the United States. During 2018-2019, Legionnaires' disease occurred in 5 persons after they visited the park; 3 of these persons were potentially exposed in spa facilities that used untreated hot spring water. Environmental testing revealed Legionella bacteria in piped spring water, including 134°F stagnant pipe water. These findings underscore the importance of water management programs to reduce Legionella growth in plumbing through control activities such as maintaining hot water temperatures, reducing stored water age, and ensuring adequate water flow.

Comparative genome analysis reveals a complex population structure of Legionella pneumophila subspecies.

The majority of Legionnaires' disease (LD) cases are caused by Legionella pneumophila, a genetically heterogeneous species composed of at least 17 serogroups. Previously, it was demonstrated that L. pneumophila consists of three subspecies: pneumophila, fraseri and pascullei. During an LD outbreak investigation in 2012, we detected that representatives of both subspecies fraseri and pascullei colonized the same water system and that the outbreak-causing strain was a new member of the least represented subspecies pascullei. We used partial sequence based typing consensus patterns to mine an international database for additional representatives of fraseri and pascullei subspecies. As a result, we identified 46 sequence types (STs) belonging to subspecies fraseri and two STs belonging to subspecies pascullei. Moreover, a recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters revealed the presence of a fourth L. pneumophila subspecies that we have termed raphaeli. This subspecies consists of 15 STs. Comparative analysis was conducted using the genomes of multiple members of all four L. pneumophila subspecies. Whereas each subspecies forms a distinct phylogenetic clade within the L. pneumophila species, they share more average nucleotide identity with each other than with other Legionella species. Unique genes for each subspecies were identified and could be used for rapid subspecies detection. Improved taxonomic classification of L. pneumophila strains may help identify environmental niches and virulence attributes associated with these genetically distinct subspecies.

Distribution of Legionella and bacterial community composition among regionally diverse US cooling towers.

Cooling towers (CTs) are a leading source of outbreaks of Legionnaires' disease (LD), a severe form of pneumonia caused by inhalation of aerosols containing Legionella bacteria. Accordingly, proper maintenance of CTs is vital for the prevention of LD. The aim of this study was to determine the distribution of Legionella in a subset of regionally diverse US CTs and characterize the associated microbial communities. Between July and September of 2016, we obtained aliquots from water samples collected for routine Legionella testing from 196 CTs located in eight of the nine continental US climate regions. After screening for Legionella by PCR, positive samples were cultured and the resulting Legionella isolates were further characterized. Overall, 84% (164) were PCR-positive, including samples from every region studied. Of the PCR-positive samples, Legionella spp were isolated from 47% (78), L. pneumophila was isolated from 32% (53), and L. pneumophila serogroup 1 (Lp1) was isolated from 24% (40). Overall, 144 unique Legionella isolates were identified; 53% (76) of these were Legionella pneumophila. Of the 76 L. pneumophila isolates, 51% (39) were Lp1. Legionella were isolated from CTs in seven of the eight US regions examined. 16S rRNA amplicon sequencing was used to compare the bacterial communities of CT waters with and without detectable Legionella as well as the microbiomes of waters from different climate regions. Interestingly, the microbial communities were homogenous across climate regions. When a subset of seven CTs sampled in April and July were compared, there was no association with changes in corresponding CT microbiomes over time in the samples that became culture-positive for Legionella. Legionella species and Lp1 were detected frequently among the samples examined in this first large-scale study of Legionella in US CTs. Our findings highlight that, under the right conditions, there is the potential for CT-related LD outbreaks to occur throughout the US.

Lessons From an Outbreak of Legionnaires' Disease on a Hematology-Oncology Unit.

OBJECTIVES To define the scope of an outbreak of Legionnaires' disease (LD), to identify the source, and to stop transmission. DESIGN AND SETTING Epidemiologic investigation of an LD outbreak among patients and a visitor exposed to a newly constructed hematology-oncology unit. METHODS An LD case was defined as radiographically confirmed pneumonia in a person with positive urinary antigen testing and/or respiratory culture for Legionella and exposure to the hematology-oncology unit after February 20, 2014. Cases were classified as definitely or probably healthcare-associated based on whether they were exposed to the unit for all or part of the incubation period (2-10 days). We conducted an environmental assessment and collected water samples for culture. Clinical and environmental isolates were compared by monoclonal antibody (MAb) and sequence-based typing. RESULTS Over a 12-week period, 10 cases were identified, including 6 definite and 4 probable cases. Environmental sampling revealed Legionella pneumophila serogroup 1 (Lp1) in the potable water at 9 of 10 unit sites (90%), including all patient rooms tested. The 3 clinical isolates were identical to environmental isolates from the unit (MAb2-positive, sequence type ST36). No cases occurred with exposure after the implementation of water restrictions followed by point-of-use filters. CONCLUSIONS Contamination of the unit's potable water system with Lp1 strain ST36 was the likely source of this outbreak. Healthcare providers should routinely test patients who develop pneumonia at least 2 days after hospital admission for LD. A single case of LD that is definitely healthcare associated should prompt a full investigation. Infect Control Hosp Epidemiol 2017;38:306-313.

Legionnaires' Disease Outbreak at a Resort in Cozumel, Mexico.

Background. A Legionnaires' disease (LD) outbreak at a resort on Cozumel Island in Mexico was investigated by a joint Mexico-United States team in 2010. This is the first reported LD outbreak in Mexico, where LD is not a reportable disease. Methods. Reports of LD among travelers were solicited from US health departments and the European Working Group for Legionella Infections. Records from the resort and Cozumel Island health facilities were searched for possible LD cases. In April 2010, the resort was searched for possible Legionella exposure sources. The temperature and total chlorine of the water at 38 sites in the resort were measured, and samples from those sites were tested for Legionella. Results. Nine travelers became ill with laboratory-confirmed LD within 2 weeks of staying at the resort between May 2008 and April 2010. The resort and its potable water system were the only common exposures. No possible LD cases were identified among resort workers. Legionellae were found to have extensively colonized the resort's potable water system. Legionellae matching a case isolate were found in the resort's potable water system. Conclusions. Medical providers should test for LD when treating community-acquired pneumonia that is severe or affecting patients who traveled in the 2 weeks before the onset of symptoms. When an LD outbreak is detected, the source should be identified and then aggressively remediated. Because LD can occur in tropical and temperate areas, all countries should consider making LD a reportable disease if they have not already done so.

Legionella clemsonensis sp. nov.: a green fluorescing Legionella strain from a patient with pneumonia.

A novel Legionella species was identified based on sequencing, cellular fatty acid analysis, biochemical reactions, and biofilm characterization. Strain D5610 was originally isolated from the bronchial wash of a patient in Ohio, USA. The bacteria were gram-negative, rod-shaped, and exhibited green fluorescence under long wave UV light. Phylogenetic analysis and fatty acid composition revealed a distinct separation within the genus. The strain grows between 26-45°C and forms biofilms equivalent to L. pneumophila Philadelphia 1. These characteristics suggest that this isolate is a novel Legionella species, for which the name Legionella clemsonensis sp nov. is proposed.

Vital Signs: Deficiencies in Environmental Control Identified in Outbreaks of Legionnaires' Disease - North America, 2000-2014.

BACKGROUND: The number of reported cases of Legionnaires' disease, a severe pneumonia caused by the bacterium Legionella, is increasing in the United States. During 2000-2014, the rate of reported legionellosis cases increased from 0.42 to 1.62 per 100,000 persons; 4% of reported cases were outbreak-associated. Legionella is transmitted through aerosolization of contaminated water. A new industry standard for prevention of Legionella growth and transmission in water systems in buildings was published in 2015. CDC investigated outbreaks of Legionnaires' disease to identify gaps in building water system maintenance and guide prevention efforts. METHODS: Information from summaries of CDC Legionnaires' disease outbreak investigations during 2000-2014 was systematically abstracted, and water system maintenance deficiencies from land-based investigations were categorized as process failures, human errors, equipment failures, or unmanaged external changes. RESULTS: During 2000-2014, CDC participated in 38 field investigations of Legionnaires' disease. Among 27 land-based outbreaks, the median number of cases was 10 (range = 3-82) and median outbreak case fatality rate was 7% (range = 0%-80%). Sufficient information to evaluate maintenance deficiencies was available for 23 (85%) investigations. Of these, all had at least one deficiency; 11 (48%) had deficiencies in ≥2 categories. Fifteen cases (65%) were linked to process failures, 12 (52%) to human errors, eight (35%) to equipment failures, and eight (35%) to unmanaged external changes. CONCLUSIONS AND IMPLICATIONS FOR PUBLIC HEALTH PRACTICE: Multiple common preventable maintenance deficiencies were identified in association with disease outbreaks, highlighting the importance of comprehensive water management programs for water systems in buildings. Properly implemented programs, as described in the new industry standard, could reduce Legionella growth and transmission, preventing Legionnaires' disease outbreaks and reducing disease.

Three Genome Sequences of Legionella pneumophila subsp. pascullei Associated with Colonization of a Health Care Facility.

Here, we report the complete genome sequences of three Legionella pneumophila subsp. pascullei strains (including both serogroup 1 and 5 strains) that were found in the same health care facility in 1982 and 2012.

Genomic Resolution of Outbreak-Associated Legionella pneumophila Serogroup 1 Isolates from New York State.

UNLABELLED: A total of 30 Legionella pneumophila serogroup 1 isolates representing 10 separate legionellosis laboratory investigations ("outbreaks") that occurred in New York State between 2004 and 2012 were selected for evaluation of whole-genome sequencing (WGS) approaches for molecular subtyping of this organism. Clinical and environmental isolates were available for each outbreak and were initially examined by pulsed-field gel electrophoresis (PFGE). Sequence-based typing alleles were extracted from WGS data yielding complete sequence types (ST) for isolates representing 8 out of the 10 outbreaks evaluated in this study. Isolates from separate outbreaks sharing the same ST also contained the fewest differences in core genome single nucleotide polymorphisms (SNPs) and the greatest proportion of identical allele sequences in a whole-genome multilocus sequence typing (wgMLST) scheme. Both core SNP and wgMLST analyses distinguished isolates from separate outbreaks, including those from two outbreaks sharing indistinguishable PFGE profiles. Isolates from a hospital-associated outbreak spanning multiple years shared indistinguishable PFGE profiles but displayed differences in their genome sequences, suggesting the presence of multiple environmental sources. Finally, the rtx gene demonstrated differences in the repeat region sequence among ST1 isolates from different outbreaks, suggesting that variation in this gene may be useful for targeted molecular subtyping approaches for L. pneumophila This study demonstrates the utility of various genome sequence analysis approaches for L. pneumophila for environmental source attribution studies while furthering the understanding of Legionella ecology. IMPORTANCE: We demonstrate that whole-genome sequencing helps to improve resolution of Legionella pneumophila isolated during laboratory investigations of legionellosis compared to traditional subtyping methods. These data can be important in confirming the environmental sources of legionellosis outbreaks. Moreover, we evaluated various methods to analyze genome sequence data to help resolve outbreak-related isolates.

The importance of clinical surveillance in detecting legionnaires' disease outbreaks: a large outbreak in a hospital with a Legionella disinfection system-Pennsylvania, 2011-2012.

BACKGROUND: Healthcare-associated Legionnaires' disease (LD) is a preventable pneumonia with a 30% case fatality rate. The Centers for Disease Control and Prevention guidelines recommend a high index of suspicion for the diagnosis of healthcare-associated LD. We characterized an outbreak and evaluated contributing factors in a hospital using copper-silver ionization for prevention of Legionella growth in water. METHODS: Through medical records review at a large, urban tertiary care hospital in November 2012, we identified patients diagnosed with LD during 2011-2012. Laboratory-confirmed cases were categorized as definite, probable, and not healthcare associated based on time spent in the hospital during the incubation period. We performed an environmental assessment of the hospital, including collection of samples for Legionella culture. Clinical and environmental isolates were compared by genotyping. Copper and silver ion concentrations were measured in 11 water samples. RESULTS: We identified 5 definite and 17 probable healthcare-associated LD cases; 6 case patients died. Of 25 locations (mostly potable water) where environmental samples were obtained for Legionella-specific culture, all but 2 showed Legionella growth; 11 isolates were identical to 3 clinical isolates by sequence-based typing. Mean copper and silver concentrations were at or above the manufacturer's recommended target for Legionella control. Despite this, all samples where copper and silver concentrations were tested showed Legionella growth. CONCLUSIONS: This outbreak was linked to the hospital's potable water system and highlights the importance of maintaining a high index of suspicion for healthcare-associated LD, even in the setting of a long-term disinfection program.

Legionnaires' Disease Outbreak at a Long-Term Care Facility Caused by a Cooling Tower Using an Automated Disinfection System--Ohio, 2013.

On July 9, 2013, an outbreak of Legionnaires' disease (LD) was identified at Long-Term Care Facility A in central Ohio. This article describes the investigation of the outbreak and identification of the outbreak source, a cooling tower using an automated biocide delivery system. In total, 39 outbreak LD cases were identified; among these, six patients died. Water samples from a cooling tower were positive for Legionella pneumophila serogroup 1, reactive to monoclonal antibody 2, with matching sequence type to a patient isolate. An electronic control system turned off cooling tower pumps during low-demand periods, preventing delivery of disinfectant by a timed-release system, and leading to amplification of Legionella in the cooling tower. Guidelines for tower maintenance should address optimal disinfection when using automated systems.

Prevalence of sequence types among clinical and environmental isolates of Legionella pneumophila serogroup 1 in the United States from 1982 to 2012.

Since the establishment of sequence-based typing as the gold standard for DNA-based typing of Legionella pneumophila, the Legionella laboratory at the Centers for Disease Control and Prevention (CDC) has conducted routine sequence-based typing (SBT) analysis of all incoming L. pneumophila serogroup 1 (Lp1) isolates to identify potential links between cases and to better understand genetic diversity and clonal expansion among L. pneumophila bacteria. Retrospective genotyping of Lp1 isolates from sporadic cases and Legionnaires' disease (LD) outbreaks deposited into the CDC reference collection since 1982 has been completed. For this study, we compared the distribution of sequence types (STs) among Lp1 isolates implicated in 26 outbreaks in the United States, 571 clinical isolates from sporadic cases of LD in the United States, and 149 environmental isolates with no known association with LD. The Lp1 isolates under study had been deposited into our collection between 1982 and 2012. We identified 17 outbreak-associated STs, 153 sporadic STs, and 49 environmental STs. We observed that Lp1 STs from outbreaks and sporadic cases are more similar to each other than either group is to environmental STs. The most frequent ST for both sporadic and environmental isolates was ST1, accounting for 25% and 49% of the total number of isolates, respectively. The STs shared by both outbreak-associated and sporadic Lp1 included ST1, ST35, ST36, ST37, and ST222. The STs most commonly found in sporadic and outbreak-associated Lp1 populations may have an increased ability to cause disease and thus may require special attention when detected.

Identification of legionella in the environment.

Legionella is ubiquitous in freshwater systems worldwide and can also be found in soil. Legionellosis may be caused by inhalation of aerosolized water or soil particles containing Legionella. Isolation of Legionella from the environment is an essential step in outbreak investigation and may also be performed within the context of a hazard analysis and control risk management plan. Culture remains the gold standard for detection of Legionella in environmental samples. Specific properties of environmental sites that could be a source of Legionella contamination, collection of samples from such sites, and procedures for culture of these samples for Legionella are described in this chapter.

Survey of legionella species found in thai soil.

Members of the Gram-negative genus Legionella are typically found in freshwater environments, with the exception of L. longbeachae, which is present in composts and potting mixes. When contaminated aerosols are inhaled, legionellosis may result, typically as either the more serious pneumonia Legionnaires' disease or the less severe flu-like illness Pontiac fever. It is presumed that all species of the genus Legionella are capable of causing disease in humans. As a followup to a prior clinical study of legionellosis in rural Thailand, indigenous soil samples were collected proximal to cases' homes and workplaces and tested for the presence of legionellae by culture. We obtained 115 isolates from 22/39 soil samples and used sequence-based methods to identify 12 known species of Legionella represented by 87 isolates.

Accuracy and precision of Legionella isolation by US laboratories in the ELITE program pilot study.

A pilot study for the Environmental Legionella Isolation Techniques Evaluation (ELITE) Program, a proficiency testing scheme for US laboratories that culture Legionella from environmental samples, was conducted September 1, 2008 through March 31, 2009. Participants (n=20) processed panels consisting of six sample types: pure and mixed positive, pure and mixed negative, pure and mixed variable. The majority (93%) of all samples (n=286) were correctly characterized, with 88.5% of samples positive for Legionella and 100% of negative samples identified correctly. Variable samples were incorrectly identified as negative in 36.9% of reports. For all samples reported positive (n=128), participants underestimated the cfu/ml by a mean of 1.25 logs with standard deviation of 0.78 logs, standard error of 0.07 logs, and a range of 3.57 logs compared to the CDC re-test value. Centering results around the interlaboratory mean yielded a standard deviation of 0.65 logs, standard error of 0.06 logs, and a range of 3.22 logs. Sampling protocol, treatment regimen, culture procedure, and laboratory experience did not significantly affect the accuracy or precision of reported concentrations. Qualitative and quantitative results from the ELITE pilot study were similar to reports from a corresponding proficiency testing scheme available in the European Union, indicating these results are probably valid for most environmental laboratories worldwide. The large enumeration error observed suggests that the need for remediation of a water system should not be determined solely by the concentration of Legionella observed in a sample since that value is likely to underestimate the true level of contamination.

Virulence factors encoded by Legionella longbeachae identified on the basis of the genome sequence analysis of clinical isolate D-4968.

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.