Legionella Investigations

Legionella spp. testing supports investigations related to confirmed clinical cases of legionellosis. Public Health Inspectors can review requirements and guidelines before submitting samples to PHO for Legionella testing.

Microbiological Analysis

PHO Laboratory performs environmental testing for Legionella for investigations of confirmed culture clinical cases or outbreaks of legionellosis. Clinical case information must be provided to the laboratory when environmental testing is requested and prior to sample collection. Sample collection requirements, sample handling, shipping conditions, test information including testing frequency, turnaround times and reporting limits are dependent on the specific matrix. [Click on individual testing links for test directory details - Legionella (water) and Legionella (swab).]

The WHO outlines Legionella infection by category; community acquired, travel associated and nosocomial infection in relation to environmental sampling sites:¹⁰

Category	Community Acquired	Travel Associated	Nosocomial
Modes of transmission	Inhalation of contaminated aerosol	Inhalation of contaminated aerosol	Inhalation of contaminated aerosol, aspiration, wound infection
Sources of Legionella	Cooling towers; hot and cold-water systems; spa pools, thermal pools, springs; humidifiers; domestic plumbing; potting mixes and compost	Cooling towers; hot and cold-water systems; spa pools, thermal springs and pools; humidifiers	Cooling towers; hot and cold-water systems; spa pools, natural pools, thermal springs; respiratory therapy equipment; medical treatment
Resevoir of Legionella	Industrial sites, shopping centres, restaurants, clubs, leisure centres, sports clubs, private residences	Hotels, cruise ships, camp sites, shopping centres, restaurants, clubs, leisure centres, sports clubs	Hospitals, medical equipment

Sample Preparation and Collection

Instructions

To identify the source of the Legionella, selection of sampling sites for the investigation should consider both environmental and epidemiological factors. The CDC provides an example of the Legionella Environmental Assessment Form which can be used to document details on the water systems, and information on developing a sampling plan.
Legionella outbreaks occur because of aerosolization and inhalation of high numbers of Legionella. Investigators should try to determine where in a water system there is stagnation of water and subsequent aerosolization of stagnant water; for example any dead ends in a water system may allow Legionella to grow to high numbers. L. pneumophila survived and multiplied in water at temperatures between 20 ° C and 50 ° C, with an optimal temperature range of 32–42 ° C.
Samples from locations based on the environmental assessment and most likely to be positive and associated with the clinical case exposure history should be collected and submitted.

The WHO also provided a list of environmental sampling sites which was adapted from Barbaree JM, et al. 1987. This information can be found on the table below and it can be used as a guide for selection of sampling sites, but it is not a list of mandatory sampling sites.

Environmental Sites	Examples
Potable water outside or on the boundary of the health-care facility property	Treatment plant (raw and refined water), guardhouse or other facility if water is not fed from health‑care facility, fire hydrants
General potable water system for healthcare facility	Incoming water pipe(s), water softener (pre and post), preheater (discharge side), primary heater (discharge side), circulating pumps, holding tanks (cold water, discharge side), expansion tank for hot water, back drain on sprinkler system(s), fire line where it branches off main system, water used for respiratory therapy equipment, outlets in patients’ rooms
Air compressor system	Vacuum water source. Positive pressure equipment side: Condensate from tank(s), water separator(s) directly off compressors, water source(s) near air intake(s), air samples where patients were ill with legionellosis
Potable water final distribution outlets	Haemodialysis water source, before or after demineralizer
Intensive care units	Respiratory therapy (patient’s room), cardiac, services with different geographical locations, ice-maker (entry water) and ice
Air-conditioning system	Air handling unit serving area where disease occurred. Cooling towers: return from heat exchanger to water (spray/trough and gutter) distribution or pond (sump), water supply
Hot tubs	Pool and balance tank (if fitted), jets and pipes (swab)
Other	Decorative fountain, creeks, ponds, sites of stagnant water

Considerations

Ideally, sampling should be performed before disinfection of the water system.
Dead-ends in the water system may be disconnected where pipes are capped instead of completely removed. Any fixtures and water lines that are not used on a regular basis may allow Legionella to grow to high numbers.
Random sampling may delay the identification of the source of the outbreak. The most obvious sampling sites would be water sources in close proximity to the suspected clinical case exposure.

The table below describes types of environmental sampling sites with typical outbreak incubation period pattern and implicated Legionella species:

Sampling Site	Disease Outbreaks	Commonly Implicated Legionella species
Cooling water systems	Rapid onset over wide area, resolve within incubation period	Predominantly L. pneumophila sg 1
Hot and cold-water systems	Low numbers of cases over prolonged periods	L. pneumophila sg 1, 2, 4, 6, 12, L. micdadei, L. bozemanii, L. feeleii and others
Hot tubs, natural spa pools, thermal springs	Rapid onset confined to users and those in close proximity	L. pneumophila sg 1, L. micdadei, L. gormanii, L. anisa
Humidifiers, respiratory equipment	Low numbers over prolonged periods. Rapid onset confined to users and those in close proximity	L. pneumophila sg 1, 3, and others
Potting mixes, compost	Low numbers of cases over prolonged periods	Exclusively L. longbeachae

Containers for Sampling

Instructions

PHO Laboratory water collection bottles and environmental swabs are available from the PHO Laboratory warehouse and can be ordered by calling the PHO Laboratory Customer Service Centre at 416-235-6556 or toll free 1- 877-604-4567. PHO Laboratory water collection bottles contain sodium thiosulfate which is a dechlorination agent used to remove any residual halogen such as chlorine and prevents continuation of bacterial action during sample transit. The swab vial contains neutralizing solution as transport medium. This medium can neutralize quaternary ammonium compounds and phenolic disinfectants. [Click on individual testing links for test directory - Legionella (water) Legionella (swab)
Prior to collecting environmental samples from sources other than water or environmental swabs, contact the environmental laboratory through PHO Laboratory Customer Service Centre at 416-235-6556 or toll free 1-877-604-4567.
Detailed instructions are also available in the reference document CDC Sampling Procedure and Potential Sampling Sites.

Sample Preparation

The following steps are recommended to organize and minimize sample collection time:

Investigate before sampling to determine a sampling plan. Swab samples should be collected first to capture any biofilm from the shower head or aerator. Then collect a water sample slowly into the PHO Laboratory water bottle to collect any dislodged material.
Ensure all materials, including appropriate personal protective equipment, are available prior to collection.
Label sample containers using a permanent marker.
Complete requisition(s) before or after, never during, sample collection.
After collection, double check to ensure the sample unique identifier on the sample container and requisition are legible and match.

Sample Collection

Sample Collection Type	Swabs	Water
Instructions	Use one swab per site, e.g., jets or shower head. Refer to the test directory link for sample collection details - Legionella (swab). Complete all sections of the Environmental Microbiology Investigation Requisition with appropriate information to ensure accurate tracking of sampling and complete all fields as outlined on page 2 of the form.	Using aseptic technique, collect the water sample in PHO Laboratory water collection bottles. For both potable and non-potable water, collect two litres of water. Ten standard PHO Laboratory water bottles filled to the 200 mL fill line will satisfy this requirement. Complete all sections of the Environmental Microbiology Investigation Requisition form with appropriate information to ensure accurate tracking of sampling and complete all fields as outlined on page 2 of the form. Samples should be stored at refrigeration temperature (2 to 8 °C) and shipped to the PHO Laboratory as soon as possible as outline in the test directory for - Legionella (water).
Considerations	Leaking swab vials will be rejected by the laboratory. Ensure the vial is closed tight to prevent leakage. Absence of a unique identifier linking sample(s) to a sampling location on a requisition will result in rejection of sample(s) by the laboratory.	For potable water systems, e.g., taps or showers, pre-flush samples should be collected to represent the water held in the tap or fitting. Do not allow the faucets to run before sampling. Turn water on and allow the water to run slowly into the sample bottle to minimize aerosol production. Removing the aerator from the faucet can also help to minimize aerosol production. Use of Personal Protective Equipment (PPE) should be guided by local board of health internal health and safety guidelines.

Laboratory Acceptance Criteria

The accuracy of the test results may be affected by improper collection, handling and/or shipping. Legionella samples that do not meet the acceptance criteria may be rejected by the laboratory and a new sample may be re-submitted with a newly completed requisition. The acceptance criteria are outlined in the table below.

Description	Acceptance Reason
Submitter	Sample must be received from an authorized board of health submitter.
Sample Type	Swabs and water will be accepted for testing to support investigations of confirmed clinical cases or an outbreak investigation of legionellosis.
Sample Requisition	The requisition must be completed when it is received at the laboratory including date and location of collection. If a sample is received without a requisition it will not be processed; however, the sample will be held for 24 hours. The requisition must have a unique identifier that matches the identifier on the water collection bottle or swab collection vial.
Sample Collection	PHO Laboratory water collection bottles and environmental swabs are available from the PHO Laboratory warehouse and can be ordered by calling PHO Laboratory Customer Service Centre at 416-235-6556 or toll free 1-877-604-4567. Unapproved containers will not be accepted.
Sample Transport	The sample container must be secure to avoid leaks during transport. The sample temperature must meet the requirements listed below when received at the laboratory: Samples should be shipped with cold packs to maintain a temperature between 2.0 to 8.0 °C when received at the laboratory. Swabs received frozen or greater than room temperature may be subjected to cancellation.

Reporting and Interpretation of Test Results

Several factors must be considered when interpreting Legionella results, such as sampling points, sampling in relation to water system treatment (use of biocide or thermal shock), conditions to support microbial growth such as temperature and stagnation of water; and any delay or temperature changes in shipping conditions. Collection of water and swabs can be used to describe the colonization of the organism within the system at the time of collection only, recognizing that conditions can change over time. The following table describes the reporting and acceptable limits for Legionella.

Testing Type	Reporting Limit	Acceptable Limit
Legionella (water)	Detected / Not detected	Not detected
Legionella (swab)	Detected / Not detected	Not detected

Legionella is ubiquitous to the environment – water, soil and sediment; and can grow in a wide range of temperatures. If Legionella was isolated from an environmental sampling site in connection to an outbreak investigation and is a match to the clinical case, remediation is required and consultation with PHO Laboratory microbiologist is recommended. If Legionella was isolated and is not a match to the clinical case, remediation may be required and PHO Laboratory microbiologist is available for consultation.

Remedial action may be performed immediately if there is strong epidemiological information to suggest potential Legionella exposure to a susceptible population. Post remediation samples may be collected and submitted to verify if a previous laboratory confirmed positive source has been effectively treated after remedial action.

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Updated 22 Nov 2019