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Environmental samples
Two types of environmental sample are routinely taken, water samples and swabs. Either can be
used for microbiological analysis, although the water samples may need concentrating before
analysis to improve the sensitivity of detection.
When investigating the environment it is essential that samples are taken
from appropriate representative places. Potential sampling strategies are
discussed elsewhere in the toolbox. More detail regarding the testing and sensitivity/specificity
can be found in [1]
Culture
Culture remains the gold standard for detection of Legionella. Samples are plated onto a
selective media containing appropriate antibiotics and incubated at 35-37ºC for 3 to 10
days. 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 quantifiable. It also
produces material for further analysis and a positive result means the sample contains viable
bacteria. The disadvantages are that it is slow and not always totally reliable, so a negative
result 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 the
laboratory is not experienced in testing for Legionella.
- : ISO 11731 parts 1 & 2
- Water quality -- Detection and enumeration of Legionella
Antigen-based methods
There are a range of antigen detection methods based on detection of cell wall components
specific to Legionella species. Some of these are commercially available and others are
in-house laboratory methods. These methods include direct immunofluorescence, indirect
immunofluorescence, immunodiffusion and slide agglutination. Some are applicable to environmental
samples directly and others require growth first. The commercially available kits tend to be
specific for Legionella pneumophila but do have the advantage that they can be simple to
use and do not necessarily require specialist equipment to analyse the samples.
Fluorescence staining
Whilst immunofluorescence techniques target cell wall components, it is also possible to develop
techniques to stain internal cell components such as DNA. For DNA this technique is called FISH (Fluorescence In
Situ Hybridisation), where DNA probes linked to fluorophores bind specifically to target
DNA. This technique is used
largely as a research tool as it requires specialist equipment and expertise and therefore
doesn't lend itself readily to routine sample analysis. However when combined with culture growth
it has been developed commercially to speed up analysis and can identify Legionella
pneumophila and other Legionella within 3 days but only to genus level.
Nucleic acid analysis
The most commonly used DNA
method used for detection and identification is polymerase chain reaction (PCR).
This involves the amplification of DNA using primer DNA molecules specific to the DNA target of interest. There are a large number of both published
methods and commercially available kits able to identify Legionella using the
PCR technique. One advantage of
PCR is that it is quick;
samples can be analysed in hours to determine whether Legionella is present. 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. 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.
The disadvantages are that PCR
does not discriminate between living and dormant or dead cells so a positive result is only an
indication of Legionella DNA. Preparation may also be more complex as contaminants which
inhibit the PCR need to be
removed but there are many commercially available DNA purification matrices which work well. Additional steps can
therefore be easily incorporated into an existing sample concentration methodology.
Other nucleic acid analysis techniques include pulse field gel electrophoresis
which is used and whole genome sequencing which could be used in the future.
Both of these techniques are powerful discriminatory techniques for identifying down to the
species level and beyond. Both however are also technically complex and in the case of whole
genome sequencing yet to be a viable option for the vast majority of even reference laboratories.
- BARTRAM J. (2007) Legionella and the prevention of legionellosis WHO, Geneva ISBN 92 4
156297 http pdf
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