Legionellosis laboratory findings

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Overview

Laboratory abnormalities in Legionnaires' disease include leukocytosis with relative lymphopenia, hyponatremia, hypophosphatemia, and elevated levels of AST/ALT, CPK, ESR, CRP, LDH, and ferritin. Urine antigen testing in the first-line diagnostic method. Culture of the lower respiratory secretion is the gold standard for the detection of Legionella and diagnosis of Legionnaires' disease.

Indications to Test for Legionnaires' Disease

According to the Centers for Disease Control and Prevention (CDC), the following are indications to test for Legionnaires' disease:

• Patients who have failed outpatient antibiotic therapy
• Patients with severe pneumonia, in particular those requiring intensive care
• Immunocompromised host with pneumonia
• Patients with pneumonia in the setting of a legionellosis outbreak
• Patients with a travel history (Patients that have traveled away from their home within two weeks before the onset of illness).
• Patients suspected of healthcare-associated pneumonia

Laboratory Findings

Laboratory findings of Legionnaires' disease include:

• Leukocytosis
• Lymphopenia
• Hyponatremia
• Hypophosphatemia
• Elevated AST and ALT
• Elevated CPK
• Elevated ESR and CRP
• Elevated LDH
• Elevated ferritin

Diagnostic Studies

Test	Sensitivity (%)	Specificity (%)
Culture	20-80	100
Urine antigen	70-100	100
Paired serology	80-90	>99
Direct fluorescent antibody stain	25-75	≥95
PCR	unknown	unknown

Microscopy

Legionella pneumophila are small, Gram-negative coccobacilli which may be difficult to detect in specimens by Gram staining. The organism can also be detected by immunofluorescent microscopy with the use of direct fluorescent antibody.

Urine Antigen Test

The detection of soluble antigens (a component of the cell wall lipopolysaccharide) in the urine is the first-line diagnostic technique. The method is most accurate for detecting Lp1 MAb 3/1 subtypes. Sensitivity ranges from 56–99% and is lower in nosocomial infection and immunocompromised hosts. Urine antigen test may also be positive for Pontiac fever. IDSA/ATS guidelines recommend urinary antigen test for the following patients:

• Unresponsive to outpatient antibiotic therapy
• Severe pneumonia especially requiring intensive care
• Immunocompromised hosts
• Alcoholism
• Travelled within the past 2 week
• Age > 50 years
• In areas of an outbreak
• Suspected healthcare-associated pneumonia

Polymerase Chain Reaction

Nucleic acid amplification-based methods can be used to identify mip gene of Legionella in sputum, serum, and urine. Sensitivities range from 80–100% for lower respiratory tract secretion, 30–80% for serum, and 0–90% for urine samples.

Serology test

A 4-fold or greater rise in antibody titer between acute and convalescent sera may be diagnostic. However, seroconversion is not detectable until at least 3 weeks after infection and does not occur in up to a quarter of patients with culture-proven disease.

Culture

Isolation of Legionella from respiratory secretions, lung tissue, pleural fluid, or a normally sterile site is still an important method for diagnosis, despite the convenience and specificity of urinary antigen testing. Investigations of outbreaks of Legionnaires' disease rely on both clinical and environmental isolates. Clinical and environmental isolates can be compared using monoclonal antibody and molecular techniques. Because Legionella are commonly found in the environment, clinical isolates are necessary to interpret the findings of an environmental investigation.

Culture of samples (e.g., expectorated sputum, endotracheal aspirates, pleural fluid, blood, or tissue) remains the gold standard for detecting Legionnaires' disease. Unlike serology and urine antigen testing, the yield of cultures is independent of serotype and may be positive in cases of non-Legionella pneumophila species. The culture of non-respiratory samples is warranted when suspecting extrapulmonary infection. Buffered charcoal-yeast extract (BCYE) medium supplemented with 0.1% α-ketoglutaric acid is required for isolation and growth of Legionella.

CDC Recommendations for Specimen Collection

Environmental Specimen Collection

1. Collect water (1-liter samples, if possible) in sterile, screw-top bottles.
2. Collect culture swabs of internal surfaces of faucets, aerators, and shower heads in a sterile, screw-top container (e.g., 50 mL plastic centrifuge tube). Submerge each swab in approximately 5 mL of sample water taken from the same device from which the sample was obtained
3. Transport samples and process in a laboratory proficient at culturing water specimens for Legionella spp., as soon as possible after collection.
4. Test samples for the presence of Legionella spp. by using semiselective culture media using procedures specific to the cultivation and detection of Legionella spp.

Healthcare-associated Sites of Specimen Collection

Potable water system

• Incoming water main
• Water softener
• Holding tanks, cisterns
• Water heater tanks (at the inflows and outflows)

Potable water outlets, especially those in or near patient rooms

• Faucets or taps
• Showers

Cooling tower, evaporative condenser

• Makeup water (e.g., added to replace water lost because of evaporation, drift, leakage)
• Basin (i.e., area under the tower for collection of cooled water)
• Sump (i.e., section of basin from which cooled water returns to heat source)
• Heat sources (e.g., chillers)

Humidifiers ( e.g ., nebulizers)

• Bubblers for oxygen
• Water used for respiratory therapy equipment

Other sources

• Decorative fountains
• Irrigation equipment
• Fire sprinkler system (if recently used)
• Hot tubs