Laboratory Testing for Legionella

Key points

  • Clinical laboratories can provide diagnostic testing for Legionella infections using urinary antigen testing (UAT), culture, or molecular methods.
  • UAT typically detects only one type of Legionella, although infections with other types occur.
  • ÐÇ¿ÕÓéÀÖ¹ÙÍø recommends Legionella UAT paired with lower respiratory specimen testing by culture or molecular methods for diagnosis of Legionnaires' disease.
  • Learn the advantages and disadvantage of each diagnostic test.
Laboratorian working with an environmental sample in the Biological Safety Cabinet.

Preferred methods

The preferred diagnostic tests for Legionnaires' disease are both of the following paired together:

  • Culture or molecular testing of lower respiratory specimens to detect a range of Legionella types
  • Legionella UAT for Legionella pneumophila serogroup 1

Lower respiratory specimens include sputum, bronchoalveolar lavage, tracheal aspirate, and lung tissue.

Importance of testing a lower respiratory specimen

Collecting and testing lower respiratory specimens is important to enable detection of all species and serogroups of Legionella. Both culture and many molecular tests can detect Legionella species and serogroups that the UAT doesn't. UAT typically only detects L. pneumophila serogroup 1. Additionally, molecular tests may be more sensitive than culture for detecting Legionella infection after the start of antibiotic treatment.

Culture allows for comparison of clinical and environmental isolates during an outbreak investigation.

Keep Reading Investigation Guidance: Legionella Testing and Specimen Collection

Benefits of the urinary antigen test

The UAT is the most commonly used laboratory test for diagnosis of Legionnaires’ disease. It detects a piece of the Legionella bacterium (known as an antigen) in urine. It can detect Legionella infection in some cases for days to weeks (or longer, under rare circumstances) after treatment.

Lab methods

Sensitivity and specificity

Sensitivity varies depending on the following:

  • Quality and timing of clinical specimen collection
  • Technical skill of the laboratory worker performing the test
This table provides general ranges for the sensitivity and specificity of each diagnostic test.
Test Sensitivity (%) Specificity (%)
Culture 20–80 100
Urinary antigen for L. pneumophila serogroup 1A (Lp1) 70–100 95–100
Polymerase Chain Reaction (PCR)B 95–99 >99
Direct Fluorescent Antibody (DFA) Stain 25–75 >95
Paired serologyC 80–90 >99

Advantages and disadvantages

This table describes advantages and disadvantages for each diagnostic test.
Test Advantages Disadvantages
Nucleic Acid Amplification Test
  • Can be performed on pathologic specimens (usually lung tissue)
  • Rapid and sensitive
  • Some tests detect species and serogroups other than Lp1
  • Confirmatory laboratory evidence
  • Assays vary by laboratoryD and commercial availability may be limited in the United StatesE
Culture
  • Detects all species and serogroups
  • Yields clinical and environmental isolates that can be compared
  • Confirmatory laboratory evidence
  • Slow (up to 14 days to grow)
  • Sensitivity is highly variable
  • Affected by appropriate antibiotic treatment
  • Requires specialized media, which some laboratories may not have readily available and can be difficult to obtain
Urinary Antigen
  • Rapid
  • Confirmatory laboratory evidence
  • Can only be used to detect L. pneumophila serogroup 1A (Lp1)
  • Does not allow for molecular comparison to clinical or environmental isolates
DFA
  • Can be performed on pathologic specimens (usually lung tissue)
  • May detect species and serogroups other than Lp1
  • Supportive laboratory evidence
  • Technically difficult
  • Reagents may be difficult to obtain
Serology
  • May detect species and serogroups other than Lp1
  • Confirmatory laboratory evidence for Lp1; supportive for other types of Legionella
  • Must have paired sera collected at acute onset to 2 weeks after symptoms appear and 3 to 6 weeks later C,F
  • Single results cannot be interpreted as indicative of Legionnaires’ disease
  • Not timely for clinical decision making

Specimen collection

Best practice is to obtain both lower respiratory specimens and urine concurrently.

It's ideal to obtain the lower respiratory specimens prior to antibiotic administration. However, antibiotic treatment shouldn't be delayed to facilitate this process. Lower respiratory specimen collection can occur after antibiotic administration begins.

Submitting specimens

Public health department laboratory staff can forward approved specimens or isolates to ÐÇ¿ÕÓéÀÖ¹ÙÍø or the for specialized testing.

Keep Reading Specimen Collection and Submission

Bacteria detection

Incorrect rejection criteria

Laboratories sometimes reject lower respiratory specimens during a work-up for pneumonia based on specimen quality. Issues can include a lack of white blood cells in the sample or contamination with other bacteria.

However, laboratories shouldn't reject lower respiratory specimens for these reasons when working-up Legionnaires' disease because Legionella can often be recovered1. Sputum produced by patients with Legionnaires' disease may not have many white blood cells2. Contaminating bacteria don't negatively impact isolation of Legionella on selective media (e.g., Buffered Charcoal Yeast Extract [BCYE] agar plus antibiotics)3.

Resources


Laboratories are able to test their proficiency at isolating Legionella from simulated environmental samples. This program issues documentation to those who pass the proficiency test.


This reference center accepts testing requests from governmental public health or environmental laboratories. It performs Legionella testing on clinical specimens and isolates, as well as environmental isolates and outbreak samples.

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Content Source:
National Center for Immunization and Respiratory Diseases; National Center for Environmental Health
  1. Cross reactions with other species and serogroups have been documented.
  2. Avni T, Bieber A, Green H, et al. . J Clin Micro. 2016;54(2):401–11.
  3. ÐÇ¿ÕÓéÀÖ¹ÙÍø labs don't perform serologic testing for legionellosis diagnosis due to inherent challenges in obtaining appropriate specimens. It's important to note that because paired sera are required, results are delayed and thus may not be useful for acute case diagnosis or during active outbreak investigations.
  4. Laboratories can develop, adopt, or use existing assays (known as laboratory developed tests) for in vitro diagnostic purposes. Labs do this by establishing and validating test performance under the Clinical Laboratory Improvement Amendments of 1988 (CLIA) requirements or equivalent regulations.
  5. Currently the U.S. Food and Drug Administration has cleared at least one nucleic acid-based molecular assay for Legionella pneumophila detection.
  6. McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR. . N Engl J Med. 1977;297(22):1197–203.
  1. Ito A, Ishida T, Tachibana H, Nakanishi Y, Kawataki M, Yamazaki A, Washio Y. . J Clin Microbiol. 2024;62(4):e0166523.
  2. Murdoch DR. . Clin Infect Dis. 2003;36:64–9.
  3. Mercante JW, Winchell JM. . Clin Microbiol Rev. 2015;28:95–133.