Legionnaires' disease outbreak investigation toolbox

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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 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.

  1. BARTRAM J. (2007) Legionella and the prevention of legionellosis WHO, Geneva ISBN 92 4 156297 http pdf