Puritan Enviromax and ESK for Surface Sampling

Legionella Detection and Identification Methods

Lab M - Neogen - Legionella media

Key Points

  • Gram -ve non-spore forming rod
  • Require L-cysteine
  • Motile by means of polar flagella
  • Aerobic
  • Optimum growth temperature 36°C
  • Intracellular pathogen of macrophages

Legionella1 Legionella pneumophila infects the lungs and causes a sometimes fatal form of pneumonia. It is the causative organism of Legionaires Disease and Pontiac Fever. Legionnaires' disease can have symptoms like many other forms of pneumonia: a high fever, chills and a cough. Some people may also suffer from muscle aches and headaches. These symptoms usually begin 2 to 14 days after being exposed to the bacteria.

Classically Legionella spp. are found in cooling towers, air conditioners, spa equipment, fountains, humidifiers and showers, hot and cold water systems, oil/water emulsions used for lubricating lathes, misting devices, decorative fountains and water features, dentistry tools, TMV's (thermostatic mixing valves). The mode of transmission is through inhalation of airborne droplets.Legionella detection and identification methods fall into 2 categories: those methods that are aimed at the clinical diagnosis of disease and those aimed at monitoring and identifying risk in water systems. These methods are listed separately.

Prevention of Legionnaires' disease depends on good maintenance of possible sources, including regular cleaning and disinfection and the application of other physical (temperature - keeping hot water at 60°C and cold below 20°C) or chemical measures (biocide).

There have been reports in several journals of viable but non-culturable [VBNC] Legionella. In some instances it would appear that these are associated with free-living amoebae - a reported consequence of this is significantly increased resistance to biocides. Particular care must be taken when choosing the optimal test method for evaluating such samples.

There are many contract testing companies that specialise in Legionella management and testing of water.

Detection Techniques

For clinical samples, culture of the causative organism is regarded as essential for definitive proof of diagnosis (some patients do not produce antibodies) and allows for subgrouping and molecular typing (epidemiological data).

Typically a charcoal yeast extract (CYE) agar or buffered charcoal yeast extract (BCYE) agar is used. Incubation is at 35-37°C for 3 to 10 days in a moist atmosphere.

Legionella is typically isolated from water by filtration followed by heat or acid treatment then plating onto BCYE with glycine vancomycin polymyxin cyclohexamide (GVPC) agar. An alternative media, GVPN replaces the potentially hazardous cyclohexamide with natamycin. Or MWY (BCYE with glycine, polymxin B, anisomycin and vancomycin) contains dyes that will colour the target colonies aiding in their identification. Incubation is for up to 10 days at 36°C.

As an alternative to growth on agar there are a variety of technologies which may provide rapid results e.g.: antibody/antigen interactions using direct or indirect fluorescent immunoassay methods; molecular methods such as PCR/nucleic acid. Sometimes combinations of these techniques are used to further enhance the speed to result. In some situations these may be the method of choice if VBNC legionella are suspected in the sample.

PCR can eliminate the need for identification in the event of a positive result and may also provide quantitative information.

Identification/Confirmation

Legionella spp. have an absolute growth requirement for L-cysteine and without this amino acid they are unable to grow, colonies with typical morphology on selective media are subcultured onto BCYE and BCYE without L-cysteine. Those isolates that grow on BCYE but fail to grow on this media may be presumptively identified as Legionella spp. Quicker identification can be carried out using latex agglutination, direct immunofluoresence or molecular techniques.

Immunological methods such as latex agglutination allows rapid elimination of negative samples, positives can then be checked out with more targeted tests.

Molecular methods using PCR or nucleic acid techniques are routinely used as confirmatory tests.

Comments

There are 8000 - 18,000 cases of Legionaires disease requiring hospitalization annually in the USA - many more infections are not reported or are undiagnosed. In the UK 542 cases were provisionally reported for 2006 [figures do not include Pontiac fever].

AFNOR is currently developing the AFNOR Validation Mark for water. The examination of the first certification, for a Legionella PCR Kit, commenced at the beginning of 2007.

Supplier Information

Molecular Detection

Identification

Culture Media

Serological Confirmation

QC Organisms

Separation - FlowCytometry - Immunomagnetic

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