Legionnaires' disease outbreak investigation toolbox

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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 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 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.

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