Legiolert^*

Yes. Legiolert was granted NF Validation Certification by AFNOR in 2019 and is published as ASTM International Standard D8429-21 . It is also included in the UK Standing Committee of Analysts Blue Book: The determination of Legionella bacteria in waters and other environmental samples (2020) – Part 2 – Culture Methods for the detection and enumeration and is referenced as a bacterial enzyme method in ASHRAE Guideline 12 . 

Legiolert has also been extensively compared to traditional methods of Legionella detection in several independent peer-reviewed studies . Please contact IDEXX Water Technical Services at 1-800-321-0207 for the most up to date list of publications. 

Yes. The Legiolert test detects all serogroups of L. pneumophila. This was confirmed in the test’s ISO 13843 study, as well as an independent peer-reviewed study .⁵

Yes. Legiolert is a culture test and yields a viable isolate which can be serotyped and/or stored as necessary. IDEXX has developed and validated a procedure for serotyping directly from a positive well, which eliminates the need to subculture from positive wells. Please contact IDEXX Water Technical Services at 1-800-321-0207 for this procedure.

No. All microbiological methods have some rate of false positivity. Although the false positive rate of traditional spread plate methods of Legionella detection has not been well studied, false positives have been demonstrated in those methods.²⁶ In contrast, the Legiolert test’s rate of false positivity in environmental samples has been well studied, and has been demonstrated to be very low in several independent peer-reviewed studies , between 0 and 4%.^1-3,8-10,12 

Yes. Several CDC ELITE laboratories use the Legiolert test for routine monitoring of their customer’s buildings and fixtures. 

Legiolert cannot be used exclusively to perform the biannual performance test on the samples from the Wisconsin State Laboratory of Hygiene (ELITE samples). These samples include non-pneumophila Legionella, and Legiolert tests for all serogroups of L. pneumophila only. A method that tests for multiple species of Legionella must also be used. Legiolert results may be submitted to performance test, but the test used must be specified. Legiolert test results will be scored and reported. 

Additionally, CDC ELITE laboratories should not use Legiolert when a different method is mandated by law, for example, when complying with NYCRR Title 10 Part 4, which requires the use of ISO 11731.

The US CDC does not approve methods. No Legionella testing method is approved by the CDC. The “CDC Method,” while initially developed by the CDC, is not recommended or approved by the CDC. The CDC provides a Legionella toolkit with recommendations on test methods and laboratory considerations, but no specific method is recommended.²² 

The US EPA does not currently approve methods for the detection of Legionella, as testing for Legionella in water is not federally mandated in the United States.

Yes. The CDC leaves significant discretion around testing to each individual Water Management Team. While the CDC presents the option to consider testing for all species in its Legionella toolkit, it also highlights that non-pneumophila Legionella species are less associated with Legionnaires’ disease.²² 

The Legiolert test is designed specifically for potable and nonpotable water samples. 

Potable water is water that is fit for consumption and includes hot and cold water from kitchen or bathroom taps, showers, or water within a drinking water distribution system, among others.

Nonpotable water is not fit for consumption and includes water from cooling towers, pools, spas, decorative fountains, among others. 

Please contact IDEXX Water Technical Services at 1-800-321-0207 if you need assistance classifying your water sample.

Yes, the Legiolert potable and nonpotable protocols have each been designed for optimal performance with different types of water. Testing a nonpotable sample with the potable protocol may provide false-positive results due to bacteria other than L. pneumophila. Testing a potable sample with the nonpotable protocol may result in reduced L. pneumophila detection due to the pretreatment step and the smaller volume of sample tested.

Sodium thiosulfate must be utilized when collecting a chlorinated sample. Sodium thiosulfate does not need to be present when collecting a non-chlorinated sample, but if it is present, will not have an adverse impact on the Legiolert test results. 

Sample dilutions and QC/PTs must be prepared in sterile containers without sodium thiosulfate.
 

Both the 100 mL and 10 mL protocols have been validated to detect L. pneumophila in potable water. The 100 mL protocol has a lower limit of detection but requires the use of a supplement before the Legiolert reagent is added. Laboratories should choose between the 100 mL and 10 mL protocols based on their limit of detection and workflow requirements. 

The Legiolert test detects L. pneumophila in potable and nonpotable water samples. This test is based on a bacterial enzyme-detection technology that signals the presence of L. pneumophila through utilization of a substrate present in the Legiolert reagent. L. pneumophila cells grow rapidly and reproduce using the rich supply of amino acids, vitamins, and other nutrients present in the Legiolert reagent. Actively growing strains of L. pneumophila use the added substrate to produce a brown color indicator. 

L. pneumophila causes the overwhelming majority of Legionnaires’ disease worldwide. Waterborne, non-pneumophila Legionella causes less than 2% of Legionnaires’ disease in almost every country that tracks cases. A test that is more sensitive to L. pneumophila better protects public health than traditional methods of detecting Legionella. Not only do traditional, plate-based methods only recover certain Legionella species, they have demonstrated low recovery, low sensitivity, and a high rate of false negativity.^23-25

The detection limit of the Legiolert test depends on the protocol used:

100 mL protocol for potable water: <1 organism/100 mL

10 mL protocol for potable water: <10 organisms/100 mL

1 mL protocol for nonpotable water: <100 organisms/100 mL

Legiolert Supplement is only required to be used when testing an undiluted 100 mL potable water sample. With 100 mL potable samples, the hardness level must be checked, and the appropriate volume of supplement must be added. Note that supplement will be added regardless of the level of hardness; the results of the hardness testing determine how much supplement is added. Refer to the product insert for specific procedures. No sample types other than undiluted 100 mL potable samples require the use of Legiolert Supplement. 

The hardness of the water sample can impact the Legiolert test, the supplement adjusts hardness to ensure proper functioning of the test when using the undiluted 100 mL potable protocol.
 

Any wells with a brown color and/or turbidity are positive for L. pneumophila. No other colors are positive for L. pneumophila.

Potable water samples are incubated at 39°C (± 0.5°C) for 7 days. Nonpotable water samples are incubated at 37°C (± 0.5°C) for 7 days. Maintaining these temperatures is critical to the functioning and success of the test. L. pneumophila is more thermoresistant than other bacteria found in water, and the increased temperature acts as a selective agent. For this reason, trays cannot be removed completely from incubator prior to final read. 

Results can be read any time on the 7^th day.

No. Controlled, uninterrupted incubation temperature is a critical part of the Legiolert method. False-positive results due to bacteria other than L. pneumophila and/or reduced L. pneumophila detection can occur at temperatures other than 39°C (± 0.5°C) for potable samples and 37°C (± 0.5°C) for nonpotable samples. Because L. pneumophila is more thermoresistant than other bacteria, maintaining these temperatures is critical to ensuring the selectivity of the Legiolert test.

The Legiolert test requires a humidity level that ensures ≤15% sample volume loss in the Quanti-Tray/Legiolert wells. Appropriate humidity can typically be achieved by incubating Quanti-Tray/Legiolert in a container that traps moisture. For example, a large plastic bag could be used for several Quanti-Tray/Legiolert samples. Alternatively, humidity may be maintained by placing a pan of water on the bottom of the incubator so that it covers approximately 80% of the bottom interior surface. 

Yes, if the results are positive. An inoculated Legiolert test sample that is positive before 7 days is a confirmed positive test for L. pneumophila. However, for an accurate quantitative determination of L. pneumophila in a sample, the results should be read at 7 days. Do not remove trays completely from incubator prior to final read, as deviations in temperature during incubation may cause false positive results.

Yes, if the results are negative. If an inoculated Legiolert test sample is inadvertently incubated for more than 7 days, the lack of brown color and turbidity is a valid negative test result. However, brown color and/or turbidity that develops after 7 days is not a valid positive test result; the test should be repeated or verified.

Store the Legiolert test at 2–25°C away from light and humidity.

Check with your local, state, and/or federal authorities for proper disposal of biohazard materials at your facility.

L. pneumophila ATCC 33152 (WDCM 00107) or ATCC 33156 (WDCM 00180) are recommended positive control strains. Enterococcus faecalis ATCC 29212 (WDCM 00087) is a recommended negative control strain. Additionally, IDEXX offers IDEXX-QC Legionella pneumophila (catalog number WQC-LP). Please contact your IDEXX representative for ordering information. 

Yes, the Binder incubator available from IDEXX Water is able to handle the higher humidity required by the Legiolert Test. Appropriate humidity can typically be achieved by placing a pan of water on the bottom of the incubator so that it covers approximately 80% of the bottom interior surface. Appropriate humidity can also be achieved by incubating Quanti-Tray/Legiolert in a container that traps moisture. For example, a large plastic bag could be used for several Quanti-Tray/Legiolert samples.

Nonpotable water may contain high concentrations of bacteria other than L. pneumophila. The Legiolert test nonpotable protocol and the Legiolert Pretreatment have been optimized to prevent these non-target organisms from interfering with the test result.