Sputum, lung fluid, pleural fluid, transtracheal aspirations, and bronchoalveolar lavage fluid are the respiratory specimens frequently used for the diagnosis of the condition. Blood is also used for culture.
Gram staining of the sputum or other respiratory specimens may show many leukocytes, but the organisms are rarely demonstrated. Bacteria, if found, are very poorly stained. Dieterle silver or Gimenez stain can be used to demonstrate bacteria.
Direct fluorescent antibody (DFA) test is a sensitive method to detect Legionella spp. in sputum and other specimens.
This test uses fluorescent-labeled monoclonal or polyclonal antibodies produced against Legionella species. This is a rapid test; result can be obtained within 2–4 hours. The test shows a high specificity of 96–99% using the monoclonal antibodies. However, this test has a low sensitivity.
The sensitivity of DFA depends on the presence of a large number of bacteria in specimen. Therefore, sensitivity is increased if samples from lower respiratory tract are used. Positive DFA test becomes negative in 4–6 days of treatment.
Isolation of Legionella spp. from sputum or other respiratory secretions (bronchoalveolar lavage, transtracheal aspiration, and bronchoscopic specimens) by culture is the definitive method for diagnosis of Legionella infection. Blood culture shows very low sensitivity.
BCYE agar is the medium most frequently used for culture of the bacteria. The bacteria produce small colonies after 3–5 days of incubation on BCYE agar at 35°C in the presence of 3–5% CO2. Bronchoalveolar lavage, transtracheal aspiration, and bronchoscopic specimens increase the sensitivity of the culture. However, blood culture shows very low sensitivity.
IFA test and ELISA are the most commonly used antibody-based serological tests for the diagnosis of Legionella infection. IFA titer of 1:256 or more on a single serum is diagnostic. A fourfold or greater increase in antibody titer between acute and convalescent serum to 1:128 or greater in titer is also con-sidered diagnostic of the disease.
ELISA, radio immunoassay (RIA), and the latex agglutina-tion test are used to detect Legionella lipopolysaccharide anti-gen in the urine and respiratory specimens. The antigens are demonstrated in more than two-thirds of the patients during 1–3 days of clinical disease. The urinary antigen detection is rel-atively rapid and simple and can even detect antigen excreted in patients who are on antibiotic therapy. Disadvantage of the test is that it can detect only L. pneumophila (serogroup 1 antigen). The antigens continue to be present in urine even for months after the infection has resolved.