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Clinical microbiological samples
As the clinical symptoms of infection with Legionella are indistinguishable from the
symptoms of other causes of pneumonia, specific diagnostic methods are needed to detect
Legionella. Often the identification of Legionnaires' disease is reliant upon clinicians
including Legionnaires' disease in the differential diagnosis and requesting the appropriate
investigations. The ability to detect Legionella in patients can be severely compromised
by antibiotic treatment regimes. It is very helpful in an investigation, therefore, if samples
are taken before an antibiotic treatment regime starts, although this is not always practical.
Samples which can be analysed by one or more of the techniques described below include sputum,
bronchial alveolar lavage, lung biopsy, urine or blood. More detail regarding the testing and
sensitivity/specificity can be found in [1]
Culture
Culture remains the gold standard for detection of Legionella in clinical samples.
Dependent on the clinical sample obtained some preparatory work may be required before samples
are plated onto a selective media containing appropriate antibiotics and incubated at
35-37ºC for 2 to 4 days, occasionally longer.
As with environmental samples, the advantages of this are that it is technologically simple,
requiring little specialist equipment, can detect most species at a high level of sensitivity and
is potentially quantifiable if required. It also produces material for further analysis and,
intuitively, positivity means the sample contains viable bacteria. The disadvantages are that it
is slower than other techniques and not always totally reliable, so negativity cannot be used to
completely rule out the presence of Legionella. Background flora can interfere with
growth and contamination which can lead to misidentification if laboratory is not experienced in
testing for Legionella.
Urinary antigen testing
This antigen-based immunoassay method is a very quick (15 min to 3 h), highly specific and
inexpensive test and has therefore found increasing favour over the years as the routine initial
test carried out in many diagnostic laboratories when Legionella infection is suspected.
The urinary antigen is detectable in the urine of most patients between one and three days after
the onset of symptoms and persists for some time even after other tests have become negative. The
major disadvantage is that it is specific only to L. pneumophila serogroup 1 antigen. It
is always recommended that borderline and positive test results are repeated preferably by a
reference laboratory both using the urinary antigen immunoassay and additional techniques if
available to provide confirmation.
Serology
Serology is usually the test of choice when urine is not available, as a follow up to the urinary
antigen test, when clinical diagnosis is made late in illness or when an unusual serogroup is
expected. The aim of a serology test is the diagnostic estimation of antibody levels in bodily
fluid, usually blood. Historically, a range of methods have been used, direct
fluorescence, indirect fluorescent antibody test (IFAT),
agglutination and enzyme-linked immunoassay (EIA); a range of
antigen preparations (heat/formalin killed - yolk-sac/ agar grown) and a range of manufacturers
(in-house/commercial) with limited validation data for commercial kits.
During an epidemiological investigation into an outbreak such tests can also identify Pontiac
fever cases, aid case finding and be used to determine exposure levels in more forensic studies.
Nucleic acid analysis
PCR is the most commonly used
DNA method for detection and
identification in clinical samples. There are a large number of both published methods and
commercially available kits able to identify Legionella using the PCR technique in clinical studies. One
advantage of PCR is that it is
quick. Samples can be analysed in hours to determine whether Legionella is present.
Another key benefit is that PCR
based investigation can be done on a range of clinical samples (Sputum, Bronchoalveolar lavage
(BAL), Biopsy, Post mortem), although preparation may be complex as contaminants which inhibit
the PCR need to be removed. One
disadvantage is that PCR does
not discriminate between living and dormant or dead cells, so a positive result is only an
indication of Legionella DNA.
The PCR can be optimised to
amplify a single target or multiple targets, with the multiple target approach having the power
to discriminate between species. Multiple targets is the basis of sequence based typing
(SBT) methods, but here the gene fragments are amplified separately and then the
DNA sequence is established.
This can be developed as a powerfully discriminatory technique to sub-species level. PCR can also be developed to be
quantifiable. PCR is now a
widely-used technique in microbial detection and molecular biology. As such, it has become
technically easier and less specialist in nature as techniques and hardware have improved. Whilst
not as technically easy as culture, it is certainly not as complex as some of the other
techniques described.
Other nucleic acid analysis techniques include pulse field gel electrophoresis
which is used to provide a whole genome "fingerprint" and in the future whole genome
sequencing.
- BARTRAM J. (2007) Legionella and the prevention of legionellosis WHO, Geneva ISBN 92 4
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