Legionella – Respiratory PCR and Culture

Conformément au Règlement de l’Ontario 671/92 de la Loi sur les services en français, les renseignements d’analyses de laboratoire liés à la présente page ne sont offerts qu’en anglais parce qu’ils sont de nature scientifique ou technique et destinés uniquement à l’usage des fournisseurs de soins de santé qualifiés et non aux membres du public.

Background
This page provides testing information for Legionella species, the causative agent(s) of Legionnaires disease.

The available testing options for Legionnaires disease include PCR and culture (performed on lower respiratory specimens) and Urine Antigen Test (UAT) using urine specimen. This page is for the detection of Legionella species from respiratory specimens using PCR and culture methods.

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

Updates

Added best practice guidance on the recommended diagnostic tests for Legionnaire’s disease. Refer to the testing indication section below.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 12 janv. 2026.

Testing Indications

Best practice for the laboratory diagnosis of Legionnaires’ disease includes the collection and submission of both a lower respiratory specimen(s) and urine, concurrently. UAT allows for the rapid diagnosis of L. pneumophila Serogroup 1. Culture of respiratory specimens allows for the identification of additional species and serogroups. Respiratory specimens are essential, regardless of UAT results, for sequencing, a critical tool during outbreak investigations, in order to link cases to one another and to identify potential environmental sources.
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, particularly 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 twelve months.
Positive environmental tests for Legionella
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 bronchoalveolar lavage, bronchial wash, lung tissues, pleural fluid, sputum, etc. collected during the acute phase of illness.	1 mL of liquid specimen	Sterile container.
Legionella Isolate 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
Outbreak or investigation number if applicable
Indicate symptoms and date of onset

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 the required information may result in specimen rejection or testing delay. For additional information see: Criteria for Acceptance of Patient Specimens.

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 on ice packs. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Special Instructions

Specimens that are part of an outbreak should have the Public Health Unit’s assigned outbreak number on the label AND on the requisition.

For any cluster investigation, please contact PHO Laboratory Customer Service at 416-235-6556 or 1-877-604-4567 to obtain an investigation number prior to specimen submission. Outbreak and investigation numbers, as appropriate, MUST be documented on all specimens submitted related to the outbreak or investigation, as well as on the requisition.

NOTE: Respiratory specimens are required for molecular analyses (e.g. Sequence-Based Typing) and are therefore critical during outbreaks or cluster investigations. If submitting a urine specimen for UAT, include a respiratory specimen for PCR, culture and sequencing.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Legionella PCR testing is performed daily, Monday to Friday at PHO’s laboratory, Toronto and Kingston locations.

Legionella culture is performed only at PHO’s laboratory, Toronto location, Monday to Friday.

Turnaround time for PCR: Up to 4 days from receipt by PHO
Turnaround time for culture: Up to 15 days from receipt by PHO

Stat and Critical Specimens Testing

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

Test Methods

Legionella PCR:
All specimens are tested with a 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 Public Health Ontario for clinical use. It has not been licensed by Health Canada.

Sputum, lung tissues, and pleural fluid were not validated for this assay but will be tested and reported.

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

Legionella Culture:
Primary respiratory specimens or isolates from a pure culture are inoculated on Buffered Charcoal Yeast Extract (BCYE) agar, Buffered Polymyxin B, Anisomycin, and Vancomycin (BPAV) charcoal media and Blood Agar (BA) , then incubated at 35ºC-37ºC. Legionella species grow on BCYE and BPAV but not on BA. Colonies are examined microscopically for typical Legionella morphology. Isolates are identified to the genus, species, and serogroup levels as appropriate. Plates without growth are incubated for up to 14 days before final 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.
Detected.	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.	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 Legionellosis⁴.
Indeterminate.	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 organismis 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 speciesare 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: Laboratory Testing for Legionella)
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: Appendix 1: Case Definitions and Disease Specific Information Disease: Legionellosis