Legionella – Respiratory–PCR and Culture

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 PCR and Culture testing information for Legionella species, the causative agent(s) of Legionnaires' disease.

The following testing options are also available for Legionella: Legionella Urine Antigen Test (urine specimen) and Environmental Legionella Test.

For information regarding other testing options, refer to the following PHO webpages:

This page is for information specific to Respiratory Legionella Specimens.

*Uncontrolled print copy. Valid only on day of print: 01 May 2024.

Testing Indications

Listed below are some of the indications that warrant testing patients with pneumonia for Legionnaires’ disease:²

Patients who have failed outpatient antibiotic treatment for community-acquired pneumonia
Patients with severe pneumonia, in particular those requiring intensive care
Immunocompromised patients with pneumonia
Patients with a travel history (patients who have traveled away from their home overnight within 14 days before symptom onset)
Hospitalized patients with healthcare-associated pneumonia (pneumonia with onset ≥48 hours after admission) at risk for Legionnaires’ disease
Patients with an overnight stay in a healthcare facility within 14 days before symptom onset
Patients with an epidemiologic link to a setting with a confirmed source of Legionella or that has been associated with at least one laboratory-confirmed case of Legionnaires’ disease

Testing for healthcare-associated Legionnaires’ disease is especially important if any of the following are identified in a healthcare facility:²

Other patients with healthcare-associated Legionnaires’ disease diagnosed in the past 12 months
Legionella detection in environmental samples during routine monitoring or an investigation.
Current changes in water quality that may lead to Legionella growth (e.g., changes in water temperature, chlorine levels or construction)

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Legionella PCR	General Test Requisition	Lower respiratory tract specimens such as BAL, bronchial wash, lung tissues, pleural fluid, sputum, etc. collected during the acute phase of illness.	1 ml of liquid specimen	Sterile container
Legionella Culture Identification and Serogrouping	General Test Requisition	Pure viable cultured isolate	N/A	Culture plate with selective media for Legionella

Submission and Collection Notes

Complete all fields of the requisition form, including:

Test(s) requests and indications for testing such as travel and healthcare exposure
Patient setting/population/source
Symptoms and date of onset if diagnostic testing is required.

Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier, such as the patient’s date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.

Timing of Specimen Collection

Specimens should be collected during the acute phase of illness.

Storage and Transport

Specimens should be stored at 2-8°C following collection and shipped to PHO’s laboratory on ice packs. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Special Instructions

For specimens that are part of an outbreak ensure the Public Health Unit assigned outbreak number is indicated on the requisition. For any cluster investigation, please contact PHO’s laboratory Customer Service at 416-235-6556/1-877-604-4567 regarding assigning an investigation number prior to specimen submission

To facilitate the linkage or collation of the specimen associated to the investigation or outbreak, ensure Outbreak or Investigation number is properly indicated on all related specimens submitted.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Legionella PCR testing is performed daily, Monday to Friday at Public Health Ontario’s laboratory- Toronto and Kingston sites. Legionella culture is only performed at Public Health Ontario’s laboratory-Toronto, Monday to Friday.

Turnaround time is up to 4 days from receipt by PHO’s laboratory for PCR.
Turnaround time is up to 15 days from receipt by PHO’s laboratory for culture.

Stat and Critical Specimens Testing

STAT testing is not available; however, for urgent specimens (i.e. critical) please notify customer service to ensure the specimen is on the next available run.

Test Methods

Legionella PCR :
All specimens are tested with real-time assay that detects two targets: Legionella pneumophila and Legionella species. The Legionella species target has been shown to cross react with Stenotrophomonas maltophilia and Moraxella catarrhalis.No such cross reaction has been found with the L. pneumophila specific target. Results should be interpreted in the clinical context of the patient.

This assay was developed by the Center for Disease Control and Prevention, Respiratory Diseases Branch¹ and verified at the Public Health Ontario’s laboratory for clinical use. It has not been cleared or approved by Health Canada.

Sputum, lung tissues, pleural fluid were not validated for this assay but may be tested and reported with a disclaimer.

All specimens for which Legionella is detected by PCR are subsequently cultured.

Legionella Culture:
Primary respiratory specimen or isolate from a pure culture submitted is sub-cultured onto Buffered Charcoal Yeast Extract (BCYE) agar, Buffered Polymyxin B, Anisomycin, and Vancomycin (BPAV) Charcoal media and Blood Agar (BA) which are incubated at 35ºC-37 ºC. Legionella species grow on BCYE and BPAV but not on BA. Growth is observed under the microscope for typical Legionella colonies. Presumptive Legionella colonies are subjected to slide agglutination testing (SAT) to determine species and serogroup, as appropriate. Any isolate that did not react on SAT is subjected to Legionella PCR testing for confirmation. Culture positive specimens are identified to the genus, species and serogroup levels as applicable. Cultures are incubated up to 14 days before reporting.

Interpretation

The following table provides possible test results with associated interpretations for Legionella PCR:

Result	Interpretation	Comments
Not detected.	Legionella species including Legionella pneumophila NOT DETECTED.
Detected.	Legionella species DETECTED, Legionella pneumophila DETECTED.	Culture Report to follow
Legionella species DETECTED, Legionella pneumophila NOT DETECTED.	Culture Report to follow Note: A patient with clinically compatible signs and symptoms and demonstration of Legionella spp. DNA by PCR in a respiratory tract specimen should be considered a probable case of Legionellosis.
Indeterminate.	Indeterminate for both Legionella species and Legionella pneumophila.	Note: Please resubmit specimen if clinically indicated.
Indeterminate for Legionella species, and negative for Legionella pneumophila.	Note: Please resubmit specimen if clinically indicated. A patient with clinically compatible signs and symptoms, and demonstration of Legionella spp. DNA by PCR in a respiratory tract specimen should be considered a probable case of Legionellosis.4.
Indeterminate for Legionella pneumophila, and negative for Legionella species.	Please resubmit specimen if clinically indicated.
Inhibited.	PCR results not available due to presence of inhibitory substance in specimen.

Legionella Culture is reported as:

“Legionella species not isolated” – if there is no growth of Legionella species
“Legionella species isolated, Legionella [species name], serogroup (if applicable to the species) – if Legionella is isolated

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Specimens for which Legionella species is detected are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

G. Yang, R. Benson, T. Pelish, E Brown, JM Winchell, B Fields. Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region. Available from: https://pubmed.ncbi.nlm.nih.gov/19438641/
Center for Disease Control. Legionella (Legionnaire’s Disease and Pontiac Fever). Available from: https://www.cdc.gov/legionella/clinicians/diagnostic-testing.html)
Ontario Public Health Standards: Requirements for Programs, Services and Accountability Infectious Disease Protocol. Appendix 1: Case Definitions and Disease Specific Information Disease: Legionellosis . Available from: https://files.ontario.ca/moh-ophs-legionellosis-en-2022.pdf