LEGIONELLA - World Health Organization

Method
Sens t v ty
(%)
PCR • Rapid
Respiratory
tract specimen
85–92 94–99
Urine, serum 33–70 98–98
Spec f c ty
(%) Comments References
• Diagnostic
validity of positive
results without
confirmation by
other methods
remains unclear
• Detects all
Legionella species
• Not commercially
available
LEGIONELLA AND THE PREVENTION OF LEGIONELLOSIS
Fields, Benson & Besser,
2002; Lück, Helbig &
Schuppler, 2002; Uldum
& Molbak, 2002; van der
Zee et al., 2002; Roig &
Rello, 2003
BAL = bronchoalveolar lavage; DFA = direct immunofluoresence assay; L.p.sg1 = Legionella pneumophila
serogroup 1; PCR = polymerase chain reaction
11.2.1 Diagnosing legionellosis using culture media
Before the development of an in vitro medium that could sustain legionellae (Feeley et al., 1978;
Feeley et al., 1979), legionellae could only be grown by isolating them in guinea pigs or hen eggs
(McDade et al., 1977; Morris et al., 1979). Currently, the preferred technique for checking
other diagnostic methods is to grow the bacteria on direct culture.
Primary isolation of Legionella spp. is carried out using a defined Legionella agar medium
containing L-cysteine, such as buffered charcoal yeast extract (BCYE) agar. Supplements that
reduce the background competing bacterial flora and yeasts may be added to increase selectivity
of the media. These supplements include BCYE-agar with anisomycin (Dournon, 1988),
BMPAα medium with buffered cefamandole, polymixin B, or anisomycin agar (Edelstein,
1981). It is best to use both selective and nonselective agars, because cefamandole may inhibit
some Legionella species (Edelstein, 1981). Supplemented BCYE medium is the most commonly
used. This medium can be easily prepared by any large clinical microbiological laboratory
and can be made in a semiselective form. However, supplements need to be added carefully
so that they are not overheated. The quality of each batch of the media (i.e. each flask) must be
checked using Legionella strains that have not been adapted to laboratory media by successive
subculture. This is because laboratory strains adapt to laboratory media and are less sensitive
to poor-quality media than fresh isolates of Legionella from clinical and environmental samples.
Culture yield is greatest in highly experienced laboratories using multiple media and preplating
specimen decontamination. Culture plates are incubated at 36+/– 1 °C for up to
14 days and are examined every two or three days. Even the detection of one or a few colonies
is sufficient to confirm the diagnosis. The appearance of colonies may be delayed if patients
have received appropriate antibiotics, and if the specimen is contaminated with other microorganisms
or another species (Stout & Yu, 1997; Lück, Helbig & Schuppler, 2002).