Legionella spp. – Water
Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.
Background
This page provides environmental microbiology testing information for the Legionella spp. – Water analysis available at Public Health Ontario (PHO).
Update
This test information sheet now consists of additional details on the algorithm and interpretation of Legionella spp. water analysis.
Testing Indications
Acceptance/Rejection Criteria
Refer to the Public Health Inspector’s Guide to Environmental Microbiology Laboratory Testing.
Specimen Requirements
| Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
Legionella spp. – Water1 |
Water – potable (drinking water) or non-potable (e.g. whirlpools, cooling towers, etc.) |
Potable: 10 X 200 mL (to the fill line on the bottle; i.e. 1 L) 3 |
Requisition for Specimen Containers and Supplies form PHO laboratory water collection bottle kits (Cat# 300013) Sterile sampling bags with wire closure, record “Food Bag PT# 300240 B /250” on the comment section of the form |
Submission and Collection Notes
This analysis is available for investigations and/or outbreaks with a confirmed clinical case(s) of Legionella.
Use the Environmental Microbiology Investigation Requisition Form (07/2019 version, or newer) and ensure all mandatory fields on the form are completed, including Legionella as the confirmed etiological agent.
Two litres of water (potable or non-potable) are required for the analysis. This equates to 10 standard PHO laboratory water collection bottles filled to the 200 mL fill line. Five collection bottles (1 litre of water) will be used for PCR screening, and 5 collection bottles (1 litre of water) will be used for culture testing. The submitter must ensure that the 2 litres of water are collected aseptically, are homogeneous and collected under the same conditions. Only culture will be performed if less than 2 litres is received. All 10 collection bottles can be assigned one unique identifier.
Storage and Transport
Samples should be stored at refrigeration temperature (2 to 8°C) and shipped to PHO’s laboratory as soon as possible.
Special Instructions
For sampling instructions, refer to the Public Health Inspector’s Guide to Environmental Microbiology Laboratory Testing.
Shipping Instructions:
- Ship water samples in containers with hard walls and lids secured in the closed position.
- Shipping containers must be labelled with the submitting organization, unique identifier and contents e.g., WATER SAMPLES on the outside of the container.
- DO NOT SHIP WITH CLINICAL SPECIMENS. Shipping containers used for water samples should be dedicated to water samples.
- Ship water samples in an insulated container with sufficient cold packs to maintain a temperature of 2 to 8°C. If ice is used, contain the ice in a manner that does not allow water contact with the samples.
Test Frequency and Turnaround Time (TAT)
Legionella spp. testing is performed Monday to Friday at PHO’s Toronto laboratory. Upon receipt at PHO’s Toronto laboratory, the turnaround time for PCR testing is up to 2 business days, and culture is up to 14 business days. Additional time may be required for confirmation testing, where applicable.
PHO’s laboratory uses polymerase chain reaction (PCR) based testing as a screening method for the detection of Legionella in water samples by means of a commercial kit, designed to fully comply with ISO/TS 12869:2019. This PCR method detects three targets including: Legionella species, Legionella pneumophila, and Legionella pneumophila serogroup 1. The PCR results are reported as Detected, Not detected, or Indeterminate (invalid results), as appropriate.
PHO’s laboratory analyzes water samples using a modified Centers for Disease Control and Prevention culture methodology. The presence of viable Legionella spp. in water is determined by the culture method and the isolation of the bacterial filtrate on selective agar plates, followed by biochemical and serological identification. Results are reported as Detected with the specific Legionella spp. or Not detected, as appropriate.
Algorithm
Culture will be initiated for all PCR Detected or Indeterminate test results.
Interpretation
The following table provides possible test results with associated interpretations for Legionella PCR testing:
| Result | Interpretation | Comments |
|---|---|---|
| Not Detected | Legionella species DNA was not detected by PCR. | Culture will not be performed. |
| Indeterminate | Legionella PCR test results are not available and the laboratory is unable to report the PCR test result (no internal positive control detected and/or low or no target specific signal detected). Refer to culture. | Culture will be performed, refer to culture interpretation below. |
| Detected | Legionella species DNA was detected by PCR. Refer to culture. | Culture will be performed, refer to culture interpretation below. |
The following table provides possible test results with associated interpretations for Legionella culture testing:
| Result | Interpretation | Comments |
|---|---|---|
| Not Detected | Viable Legionella did not grow on the bacteriological medium. | Scenario 1: PCR is reported as ‘indeterminate’, and culture is reported as ‘not detected’; chemical and/or physical parameters may have interfered with testing and/or low or no target specific DNA was detected. Scenario 2: PCR is reported as ‘detected’ and culture is reported as ‘not detected’; PCR may have detected residual DNA of non-culturable or non-viable bacteria. For example, if the system had been treated with a biocide, bacterial death occurred during transport to laboratory, or disinfectant was added or present in the water sample. Note: the presence of other non-Legionella bacteria in the water may also mask the presence of the Legionella. |
| Detected | Viable Legionella did grow on the bacteriological medium. | Scenario 1: PCR is reported as ‘detected’ and culture is reported as ‘detected’; PCR detected DNA of Legionella bacteria, some or all of which were viable and able to grow in culture. Scenario 2: PCR is reported as ‘indeterminate’ and culture is reported as ‘detected’; PCR may have been inhibited or low levels of target specific DNA were present, but bacteria were viable and able to grow in culture. |
Results must be interpreted within the context of the clinical, environmental, epidemiological and laboratory information/data.
Reporting
Results are reported to the submitter (e.g., Board of Health) as indicated on the requisition.
References
- Public Health Ontario. Public Health Inspector’s Guide to Environmental Microbiology Laboratory Testing [Internet]. Evergreen ed. Toronto, ON: King’s Printer for Ontario, current version [cited December 29, 2023]. Available from: Public Health Inspectors Guide
- Biotecon Diagnostics. microproof® Legionella Quantification LyoKit – 5´Nuclease – . [Internet]. Germany, 01/2020 [cited December 29, 2023]. Available from https://cms.hygiena.com/sites/default/files/media/KIT230119-120_Legionella_Quantification_LyoKit.pdf
- U.S. Department of Health and Human Services. CDC Laboratory Guidance for Processing Environmental Samples. Atlanta, GA: Centers for Disease Control and Prevention (CDC), reviewed April 30, 2018 [cited December 29, 2023]. Available from https://www.cdc.gov/legionella/labs/procedures-manual.html
- Government of Canada. Mechanical Design 15161—2013: Control of Legionella in Mechanical Systems. [Internet]. Ottawa, ON, modified: August 31, 2022 [cited December 29, 2023]. Available from: https://www.tpsgc-pwgsc.gc.ca/biens-property/legionella/index-eng.html
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