Direct Detection Methods

Several laboratory methods are used to detect Legio>■ nelta spp. directly in clinical specimens.

Stains

Because of their faint staining, Legionella spp. are not usually detectable directly in clinical material by Gram stain. Organisms can be observed on histologic nation of tissue sections using silver or Giemsa staiiis.

Antigens

One approach to direct detection of legionellae in clinical specimens is the direct immunofluorescent antibody (DFA) test of respiratory secretions. Polyclonal and monoclonal antisera conjugated with fluorescein are available from several commercial suppliers. Specimens are first tested with pools of antisera containing antibodies to several serotypes of L. pneumophila or several Legionella spp. Those that exhibit positive results are then reexamined with specific conjugated antisera. Qi reagent made by Genetic Systems (Seattle, Wash) iS monoclonal antibody directed against a cell wall prote: common to L. pneumophila. The manufacturer's directions should be followed explicitly, and material froitfi commercial systems should never be divided and use f separately. Laboratories should decide which serotypes to test for routinely based on the prevalence of isolate in their geographic area. The sensitivity of the DFA td| ranges from 25% to 75%, and its specificity is grfifljB than 95%.i3 If positive, organisms appear as brightjl fluorescent rods (Figure 37-1). Of importance, caitiff« always must be performed, because Legionella spp 01 serotypes not included in the antisera pool can be recovered.

Rapid detection of Legionella antigen in urint-^ other body fluids has been accomplished by radi.(| immunoassay (RIA), enzyme immunoassay (EIA), ao| immunochromatography; the RIA is no longer maflU| factored. Antigen may be present in the prodrou^

period and by 3 days after the onset of symptoms. A jpnajor drawback with the immunochromatographic urine antigen assay is that it only detects the presence of antigen of L. pneumophila serogroup I. One Legionella El (Biotest, Dreieich, Germany) is marketed by the manufacturer as for the detection of all serotypes of L pneumophila and some other species in that a broadly (Soss-reactive antibody is used. At this writing, the relative sensitivity of this test in detecting infections Caused by organisms other than L. pneumophila sero-gtoi7 l is unknown. Of note, a recent study demonstrated that the clinical utility for the diagnosis of legionnaires' disease of two EIAs differed depending on :he category of infection being investigated. Sensitivity (about 45%) for both EIAs were significantly lower for nosocomial cases than for either community-acquired or travel-associated ones.6 These assays have a sensitivity of 80% in their ability to detect infection caused by L. pneumophila serogroup 1 and are highly Specific, although nonspecific false-positive results do •occur. Boiling urine for 5 minutes and concentrating Urine by centrifugation help increase assay specificity »nd sensitivity, respectively. Of importance, because bacterial antigen may persist in urine for days to weeks after initiation of antibiotic therapy, these assays may be positive when other diagnostic tests are negative.

Nucleic Acid

Although not yet commercially available, direct detec-[on of Legionella nucleic acid by conventional and realtime polymerase chain reaction (PCR) has the potential to offer rapid results and increased sensitivity on respira-t0Ty samples over current methods13; of significance, pCR assays can detect all Legionella spp., not just L. pneumophila.

cultivation

Specimens for culture should be inoculated to two agar plates for recovery of Legionella, at least one of which is BCYE without inhibitory agents. This medium contains charcoal to detoxify the medium, remove carbon dioxide (C02), and modify the surface tension to allow the organisms to proliferate more easily. BCYE is also prepared with ACES buffer and the growth supplements cysteine (required by Legionella), yeast extract, a-ketoglutarate, and iron. A second medium, BCYE base with polymyxin B, anisomycin (to inhibit fungi), and cefamandole, is recommended for specimens, such as sputum, that are likely to be contaminated with other flora. These media are commercially available. Several other media, including a selective agar containing vancomycin and a differential agar containing bromthymol blue and bromcresol purple, are also available from Remel (Lenexa, Kan) and others. Specimens obtained from sterile body sites may be plated to two media without selective agents and perhaps also inoculated into the special blood culture broth without SPS. (Specimens should always be plated to standard media for recovery of pathogens other than Legionella that may be responsible for the disease.)

Plates are incubated in a candle jar at 35° to 37° C in a humid atmosphere. Only growth of L gormanii is stimulated by increased C02, so incubation in air is preferable to 5% to 10% C02, which may inhibit some legionellae. Within 3 to 4 days, colonies should be visible. Plates are held for a maximum of 2 weeks before discarding. Blood cultures in biphasic media should be held for 1 month. At 5 days, colonies are 3 to 4 mm in diameter, gray-white to blue-green, glistening, convex, and circular and may exhibit a cut-glass type of internal granular speckling (Figure 37-2). A Gram stain yields thin, gram-negative bacilli (Figure 37-3).

Figure 37-3 Gram stain of a colony of Legionella pneumophila showing thin, gram-negative bacilli (arrows).

Figure 37-3 Gram stain of a colony of Legionella pneumophila showing thin, gram-negative bacilli (arrows).

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