Legionnaires' disease outbreak investigation toolbox

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Environmental sampling in an outbreak

Sampling is essential in an outbreak situation and an outbreak control team generally needs to sample the environment for Legionella for two reasons. Firstly to identify the source, and secondly to assess the effectiveness of remedial action. Sampling should be targeted towards high risk processes or equipment first, followed by sampling of lower risk processes. It is important to continually assess potential sources and redirect the numbers and focus of the sampling as the investigation develops and results and information become available. It is important to note that sampling staff will require training before an investigation.

Reliably detecting the presence of Legionella bacteria is technically difficult and requires specialised and accredited laboratory facilities. The interpretation of results is also difficult; a negative result is no guarantee that Legionella bacteria are not present. Conversely, a positive result may not indicate a failure of controls, as Legionella are present in almost all natural water sources. Interpretation would ideally be made by an environmental microbiologist.

Choosing sampling points and number of samples

Generally, any water source that may produce aerosols should be considered a potential source for the transmission of Legionellae.
  • Review epidemiological information to decide where to focus the initial environmental investigations and control measures.
  • The number and types of sites that should be tested to detect Legionellae must be determined on an individual system basis. This requires detailed knowledge of the design of the water system to be examined and all equipment that utilises water or generates aerosols. It is also important to have a thorough understanding of the ecology of the organism prior to taking any sample.
  • Examples of sampling sites with suggested number and volume of samples to be taken can be found here. Although sites suggested are within a hospital, they can be applied more generally to investigations in other establishments.
  • In outbreak investigations there may be no information available on the design of the system or of any previous risk assessments. Therefore one would need to conduct a dynamic risk assessment to support the outbreak investigation and the health and safety interests of sampling staff.
  • Establish which outlets the patient(s) has been exposed to.
  • Ensure traceability and continuity of evidence. Sampler should use tamper proof bags etc.

Sampling methodologies

Particular areas might have larger numbers of certain equipment/machines that might cause concern to the outbreak control team such as spa pools at hotels and respiratory devices in hospitals. If resources permit, the domestic water systems of the case should be sampled so as to eliminate such potential sources [1]. Click here for further guidance on methodologies.

Collecting environmental samples [2]

Two primary sample types - water samples and swabs of point-of-use devices or system surfaces - should be collected when sampling for Legionellae.

  • Water samples: collection of at least 1 litre of water allows the sample to be concentrated. If the water source has recently been treated with an oxidizing biocide, such as chlorine or bromine, sodium thiosulfate must be added to each 1litre sample in sufficient quantities to neutralise any disinfectant present. Depending on the reason for sampling, the sample may be taken as a first flush (i.e. no disinfection). This is appropriate for most occasions and will represent the worst case. After disinfection, the sample will be taken from a running outlet representing the circulating system.

  • Swabs: during outbreak investigations, swabs should be taken in conjunction with water samples from sites where biofilms are likely to form. These swabs can be taken from various points within plumbing systems, from surfaces such as biofilms, and from areas that are difficult to reach, such as within the jets of hot tubs, thermostatic mixer valves or showers. The swabs can be submerged in a small volume of water taken at the same time, or in Pages's saline, to prevent drying during transportation to the laboratory.

All samples should be transported to the laboratory in dark, within insulated containers to protect them from extreme temperatures and from light.

Contextual information should be recorded to help interpret the results, usually summarised on the laboratory sample request form below, but individual outbreaks might require additional information for operational reasons.

Sample request forms - minimum information

Each laboratory will have its own sample request forms and these should be used to provide continuity of evidence. As a minimum, the following information should be included on the request form:

  • the site and sample point
  • the sample references and date
  • the reason for sampling
  • the temperature of the sample source (e.g. the temperature of a hot-water system at one minute after turning on tap, and at two minutes after turning on cold tap)
  • any biocide used
    • the timing of the dosage in relation to sampling
    • the concentration detected at the time of sampling
  • any other risk factors of importance (e.g. closed system opened for maintenance)
  • risk of nutrient present, such as in plastics manufacturing plants
  • any cases associated with the site.

  1. LEE J. V. & JOSEPH C. ON BEHALF OF THE PHLS ATYPICAL PNEUMONIA WORKING GROUP (2002) Guidelines for investigating single cases of legionnaires' disease Communicable Disease and Public Health 5(2), pp.157-62 http pdf
  2. BARTRAM J. (2007) Legionella and the prevention of legionellosis WHO, Geneva ISBN 92 4 156297 http pdf