Microbiology - Legionella
Legionellosis is diagnosed by a combination of culture, direct fluorescent antibody (DFA) staining, serum serologic testing (performed in Virology/Serology Unit) and other techniques in conjunction with the patient's clinical history. Culture is the recommended diagnostic procedure and should be attempted along with other methodologies. The Bacteriology Unit offers culture and DFA staining of clinical specimens and reference cultures to public and private health care providers. Urinary antigen detection and DNA probe procedures are not available in this Laboratory. Environmental specimens are not tested. Consultation and bench training are available upon request.
Recommended specimens for culture include respiratory tract secretions, tissues, fluids such as sputum, pleural fluid, transtracheal aspirates, bronchial washings and lung biopsies. Saline is not recommended to collect or dilute specimens for Legionella culture as it may inhibit growth; use sterile broth or sterile distilled water. If saline must be used to collect specimens, it may be centrifuged and the pellet resuspended in sterile distilled water or broth.
Collect specimens aseptically and place in a sterile screw-capped plastic centrifuge tube (such as those in SLPH sputum mailers); seal containers securely to prevent leakage. Flexible "in-house" suction tube collection cups are not acceptable for shipping specimens. DFA smears should be air dried, heat fixed, and 10% formalin fixed before mailing or packaging.
Each specimen must be clearly labeled with the patient´s name and accompanied by a completed DHHS form 4121. Two forms are required for paired specimens.
Lab records are computerized and the following data are required: patient name, patient Social Security Number (or assigned number), date of birth, patient Medicaid number (if applicable), submitter Federal Tax Number (Employer Identification Number), submitter return address, specimen collection date and specimen source. Without these data, specimen records cannot be entered into the computer, nor can a report of results be printed. Other data are helpful for epidemiological follow-up and for statistical purposes.
Note: CLIA regulations require the following information on all test requisitions:
- Patient name or identifier
- Name and address of submitting agency
- Test requested
- Date speicmen collected
NOTE: Specimens received without submitter return address are subject to rejection!
Isolated cultures for identification of Legionella sp. should be grown on charcoal yeast extract agar or chocolate slants or plates.
Clinical specimens for Legionella culture should be shipped refrigerated with cold packs in insulated containers. Specimens that must be held longer than three days should be frozen and shipped with dry ice or cold packs in insulated containers. Identification forms should be enclosed in sealed plastic bags to prevent wetting or contamination. Smears should be shipped in rigid slide mailers. Formalinized tissue for DFA staining should be shipped in screw-capped containers and should be labeled as formalinized specimens. Sputum mailers are available online at http://slphreporting.ncpublichealth.com/lims/ClinicalLims/Login.aspx.
Reference cultures of Legionella should be shipped in the Microbiology Reference Culture mailer or equivalent container that meets safety requirements.
Ship specimens as soon as possible after collection. When shipping by U.S. Mail, use first-class postage. Be sure to place return address on outside of container, regardless of shipping method and plainly label "Legionella" on the outside of the container. Prior to shipping large numbers of specimens, telephone the Microbiology Unit at (919) 733-7367.
Reporting Procedure and Interpretation
Legionella is identified in culture and smears by specific DFA staining. At least 33 species of legionellae have been described; approximately half of human infections are associated with L. pneumophila serogroup 1. This Laboratory examines smears for L.pneumophila serogroups 1-14 and for 25 other species. DFA staining is a presumptive test. Cross-reactivity may occur among legionellae.
Neither a negative DFA stain nor a negative culture rules out Legionella infection. Low numbers of organisms, improper specimen/smear handling and/or previous antimicrobial therapy can influence test results.
Legionella isolates requiring definitive identification are forwarded to the CDC.
Smears are reported according to the number of strongly fluorescing cells with typical morphology seen. The CDC criteria for reporting the results of DFA staining are as follows:
- Smears from lung tissue: 25 or more organisms per smear = DFA positive
- Smears from other respiratory specimens: five or more organisms per smear = DFA positive
If the number of fluorescing cells seen is fewer than the minimum needed for a positive DFA report, the number of cells seen is reported. The results of serologic and culture tests along with the patient´s clinical history may be useful in interpreting the DFA stain report. All positive DFAs are reported to Epidemiology.
Results of DFA examinations are available on the day of testing or the next work day, and can be acessed via the secure web page for results at https://slphreporting.ncpublichealth.com/lims and are followed by a computer-generated report. Positive cultures are reported by telephone and by mail as soon as growth is identified. Cultures are held for three weeks before reported as negative.
Reports are returned only to the submitting agency; the submitter is responsible for sending copies to any other agency. The submitting agency is responsible for maintaining reports in the patient´s file.
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