Appendix C: Legionella test methods

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Personnel must take all necessary health and safety precautions including the use of personal protective equipment (PPE) when taking water samples for bacterial testing. Refer to the Occupational Safety and Health Agency (OSHA) Technical Manual Section III Capter VI Legionaire's Disease for guidance on require PPE.

A testing program for Legionella is not a substitute for sound engineering practice. Very often, reliance on negative test results provides a false sense of security. Test results are from a specific area and time, whereas conditions in water systems can vary quickly and dramatically. Systems that do not have a regimented inspection and cleaning or a water treatment system are always at risk.

There is no simple correlation between the presence of the organism in a water source and the risk of infection. The bacteria are often present in water systems without being associated with any known case of Legionnaires' disease. Also, the risk of illness depends on many factors other than exposure, including host susceptibility, strain virulence, and efficiency of aerosolization.

However, testing can be useful when carried out for a specific purpose such as verifying the effectiveness of a water treatment method or tracing the source of an outbreak.

Legionella culture test (swab or bulk water collection)

The gold standard for Legionella testing is culturing according to ISO/TS 11731:2004 Water Quality—Detection and Enumeration of Legionella—Part 2: Direct Membrane Filtration Method for Waters with Low Bacterial Counts. This form of testing is widely accepted as the most reliable diagnostic test for Legionella bacteria, including the strain that causes Legionnaire's disease. It is also accepted by the US Centre for Disease Control.

However, this method has some drawbacks; the main one is that species of Legionella are slow-growing and culture results may only be available after 7–14 days. Initial outgrowth may be slow, and genus-level identification normally requires at least one round of subculture involving an additional period of incubation. The presence of naturally occurring bacterial or fungal co-contaminants may necessitate additional subculture or may mask the presence of Legionella altogether.

Samples for this method are collected either using a swab or by collection of approximately 500–1,000 mL of water in a container. Specialist laboratories need to be consulted prior to collection to ensure that proper sampling techniques are followed, samples are handled and transported properly, and that sample collection bottles are appropriate.

Culture test methods can allow for the quantification of total bacteria, Legionella bacteria, as well as specific serotypes including Legionella pneumophila serotype 1, the causative organism in many outbreaks.

Test results are generally reported as Non-Legionella pneumophila Legionella (NLPL) species and Legionella pneumophila serogroup 1 (LPSG1) species. Units for both types of test results are colony forming units per millilitre, or cfu/mL.

Dipslide test

This method of testing is recommended by the U.K. Health and Safety Executive (HSE). For cooling towers, HSE recommends weekly dipslide testing, with action limits ranging from 10,000 to 100,000 cfu/mL. At 10,000 cfu/mL, HSE suggests a review of operations. When the higher action limit of 100,000 cfu/mL is reached, HSE recommends corrective action such as cleaning and disinfection.

The dipslide test is a surrogate test for Legionella, as it measures only total bacteria count (TBC). The test simply warns that the TBC of a water source has attained a level showing that the nutrients and temperature conditions may be conducive to Legionellagrowth.

The use of commercially available dipslides makes this method easy and relatively inexpensive. Sampling involves the use of a kit provided by specialist suppliers, and collection of a bulk water sample on a media. Bacteria levels are determined visually, and the results are available within 24–72 hours.

Used dipslide tests should be sterilized before disposal by soaking for at least 60 minutes in a 5% bleach solution.

Polymerase chain reaction test

The polymerase chain reaction (PCR) test is based on analysis of the Desoxyribonucleic Acid (DNA) of the Legionella bacteria. Different types of PCR tests can produce qualitative as well as quantitative results in a very short period of time.

A very large number of PCR-based analyses for Legionellaas a whole and L. pneumophila as a specific target have been developed, including a recent ISO standard (ISO/TS 12869:2012 Water Quality—Detection and Quantification of Legionella spp. and/or Legionella pneumophila by Concentration and Genic Amplification by Quantitative Polymerase Chain Reaction (qPCR)). They have the immediate advantage over culture techniques in that they can return results in 1–2 days. PCR-based tests for Legionella can be broken down into three major categories.

The first category is simple PCR (also referred to as conventional PCR), a presence/absence indication of Legionella DNA, and/or DNA of specific Legionellatypes. Simple PCR techniques can include nested forms that use targets within targets to focus on specific DNA markers, or multiplex forms that can simultaneously detect multiple targets.

The second category is quantitative PCR (qPCR), which can provide information on the quantities of Legionella DNA present, as well as quantities of DNA of selected individual species or other subtypes (e.g., L. pneumophila as a whole, or L. pneumophila serogroup 1). Modifications to the standard qPCR assay, such as the inclusion of hydrolysis probes, can significantly enhance the analytical specificity of the procedure. Quantitative information on the gene target can, in turn, be used to predict the amount of Legionella in the sample in genome equivalents per millilitre (GE/mL), an approximation of the total Legionellacell population.

A third approach is “viability PCR,” where sample pretreatments with DNA-destroying agents such as ethidium or propidium azide can be used to eliminate DNA that is not protected within living cells. These pre-treatment techniques can then be combined with simple PCR or qPCR to confirm the presence or estimate of the quantity of DNA associated with probable living inoculum of Legionella.

An issue with the simple PCR test is that it may detect the residual DNA of dead cells. This may be of advantage in cases where significant Legionella inoculum has been killed by disinfectant in water being transported to the lab, or has been killed by a biocide application before the source of an outbreak has been traced. It becomes a disadvantage in situations where a few clinically insignificant dead cells may give the same reading (“present” as opposed to “absent”) as a high inoculum of hazardous living material.

Comparison of culture-based testing versus polymerase chain reaction testing

A summary comparison of the culture-based testing and analyses method versus the PCR testing and analyses method is shown in Table A1 below:

Table summary

This table shows a summary comparison of the culture-based testing and analyses method versus the PCR testing and analyses method.

Table A1: Comparison of test methods
Advantages of culture-based analyses Disadvantages of culture-based analyses
  • Quantitative results (cfu/mL) allow interpretation of data as it relates to source strength.
  • Serotyping of bacteria allows comparison with the organism serotype found in the infected patients.
  • Application of biocides to the water source may prevent detection.
  • Analysis takes 7 to 10 days.
  • Thiosulfate or metabisulfite must be added to samples to prevent chlorine in the water from killing bacteria during shipping.
Advantages of polymerase chain reaction analyses Disadvantages of polymerase chain reaction analyses
  • Analysis can be performed in 1 to 2 days.
  • Quantitative results if the qPCR method is used (genome equivalents/mL).
  • Treating the water with biocides prior to sampling does not prevent detection of Legionella.
  • Costs comparable to culture-based analyses.
  • Identification to species level can be accomplished, but serotyping of organisms is not possible.

Note that the limitation to detecting L. pneumophila serogroup 1, by far the most medically important serogroup, has been overcome by development of a specific detection procedure.

Reference Source: Sporometrics

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