LEGIONELLA URINARY ANTIGEN TEST

Legionella pneumophila is an important cause of both community-acquired "atypical" and nosocomial pneumonias, and it remains an elusive organism to diagnose. This nutritionally fastidious, gram-negative aerobic bacillus takes its genus name from an outbreak at the American Legion convention in Philadelphia in 1976. At least forty species have since been described; however, 90% of human disease is caused by L. pneumophila and of these, >80% of disease is due to L. pneumophila serogroup 1.

A prospective US multicenter study in 1990 ranked Legionella as the third (6.7%) most common cause of community acquired pneumonia (CAP); S. pneumoniae was number one at 15%. The disease spectrum for Legionella pneumonia can be quite broad, from subclinical to life-threatening. Mortality for L. pneumonia CAP may reach 10% and for nosocomial disease, mortality may be as high as 30-50%. High-risk populations include elderly males, cigarette smokers, immunosuppressed patients, and those with underlying pulmonary disease (attack rates may be up to 30% in exposed high-risk populations). Sources of nosocomial outbreaks are usually linked to water, including baths and showers, air conditioning systems, NG tubes, humidifiers and respiratory equipment. Rapid diagnosis and appropriate antimicrobial therapy may lower mortality. While there are small numbers of Legionella naturally occurring in the environment, this number must be significantly amplified in order to cause pathology in humans. Most commonly, this amplification occurs in warm water containing algae, inside which bacteria grow. This algal growth mimics the growth in human white blood cells, particularly monocytes. It is because of this intracellular existence that Legionella is best treated with antibiotics--such as erythromycin or fluoroquinolones -- which are concentrated inside cells. Legionella control in the environment primarily rests on control of the growth of the host algae.

The fastidious intracellular nature of this organism makes diagnosis difficult. Current methods used include:

• Direct methods: culture of body fluids and tissues or direct fluorescent antibody (DFA) testing. The characteristic lack of sputum production in patients with Legionnaires disease makes specimen collection difficult, this often necessitates more invasive procedures. Other difficulties include culturing this fastidious organism in the lab and the time taken for positive growth (up to two weeks).
• Antibody detection techniques include the indirect fluorescent antibody (IFA) test and enzyme linked immunosorbent assays (ELISA). A particular limitation of the IFA test is that it cannot be performed on sputum because the antibody may stick to bacteria that comprise our normal flora.

None of these techniques have optimal sensitivities (Table 1) and delayed results decrease their clinical usefulness. If culture of Legionella is desired, please inform the Clinical Microbiology Lab in advance.

Table 1.

Antigen Detection Method

The knowledge that Legionella antigens are polysaccharides and are excreted in the urine provides an attractive alternative for diagnosis. The polysaccharide antigens, with a molecular weight of 8,000-10,000 can be detected in the urine as early as 1-3 days after the onset of symptoms and may persist for up to one year. Over 80% of patients with L. pneumophila serogroup1 will excrete this antigen and it is detectable by ELISA, RIA and PCR. Urine is readily available and easily obtained, making it an ideal specimen to perform diagnostic testing.

Therefore, the Legionella DFA test will now be replaced by the Binax NOW Legionella Urinary Antigen Test, an immunochromatographic membrane assay for the rapid qualitative detection of L. pneumophila serogroup 1, available in the Yale-New Haven Hospital Clinical Microbiology Laboratory. We are currently concentrating urine for this assay, using polyacrylamide gels. Rabbit anti-Legionella pneumophila serogroup1 is conjugated to colloidal gold particles for visualization and adsorbed onto a nitrocellulose membrane. Goat anti-rabbit IgG is adsorbed onto the same membrane as a second stripe. The resulting conjugate pad and the striped membrane are combined to construct the test strip and are incubated with the prepared urine specimen. This new technology is in the lateral flow membrane format (ICT) and provides results within 1-2 hours.

Both the sensitivity and specificity for detection of L. pneumophila serogroup1 (as determined by a 300 sample retrospective study) are 95% with respective confidence intervals of 88.7%- 98.4% and 91.0%- 97.6%.

To send a urine specimen for Legionella pneumophila type 1 to the Yale-New Haven Hospital Clinical Microbiology Laboratory, we require 5-10 ml of urine in a clean, leakproof container. The test is available from 6am - 12pm 7 days a week. The Clinical Microbiology laboratory will accept a maximum of one specimen every three days. Because antigen can be excreted in the urine for days to weeks after infection, the urine antigen assay should be utilized only for diagnosis and not as a "test of cure."

Please call the Clinical Microbiology Laboratory at 688-2460 with any questions.

References

1. Fang GD, et al: New and emerging etiologies for community-acquired pneumonia with implications for therapy: A prospective multicenter study of 359 cases. Medicine 69:307-316, 1990.

2. Kashuba AM, Ballow CH: Legionella Urinary Antigen Testing: Potential Impact on Diagnosis and Antibiotic Therapy. Diagn Microbiol Infect Dis 24:129-139 1996.

3. Konaman EW, et al: Color Atlas and Textbook of Diagnostic Microbiology, Fifth Ed., Lippincott 1997.

4. WHO, Epidemiology prevention and control of legionellosis: Memorandum from a WHO meeting. Bull WHO 68: 155-164, 1990.

Marissa Wilck, M.D.
Stephen Edberg, Ph.D.