Rapid Testing

Duopath® Legionella

For the rapid detection of Legionella species and Legionella pneumophila in water and environmental samples.

EMD Cat. No. 1.04147.0002

25 tests

Introduction
Legionella are gram-negative, rod-shaped bacteria, which are present in nearly all aquatic biotopes of fresh water. They are frequently associated with Protozoans, e.g. amoebae, where they can grow intracellular.
In most cases Legionella is present in relatively low numbers in drinking water. Thus Legionella usually present no health risk to humans. However, special environmental conditions, e.g. water temperatures between 30°C and 45°C, longer water retention times (e.g, cooling towers, containments of hot water or water pipes which have not been unused for a longer periods) promote vigorous growth of these bacteria. Infections caused by Legionella occur via inhalation of Legionella-contaminated aerosols, e.g. while showering, in swimming- and bathing pools (especially whirlpools), as well as via air conditioning systems.
After inhalation Legionella is able to grow in pulmonary macrophages causing a serious, life-threatening and atypical Pneumonia, the so-called 'Legionnaires' Disease.
At least 42 different Legionella species are known. It is supposed that not only the well-known species Legionella pneumophila but all species can cause 'Legionnaires' Disease.
According to IS0 11731 [ed. 1998-5-01) and IS0 11731-2 (ed. 2004-05-01) the presence of all Legionella species (including Legionella pneumophila) in water samples must be tested. The presence of Legionella spp. in water samples is an indicator of concerning hygienic conditions in aquatic systems. The drastic increase of outbreaks of Pneumonia caused by Legionella spp. in public buildings demands reliable and rapid detection methods. Besides the traditional culture methods, immunological techniques are preferred by the users due to the higher sensitivity and specificity. It could be demonstrated recently [Helbig et al. Appl. Environ. Microbiol. 2006. Vol. 72: 4489-449 1) that Duopath® Legionella shows a significantly higher sensitivity rate than the widely used latex assay (93% vs. 32%) for the detection of Legionella spp.
Duopath® Legionella is a reliable, rapid, cost-effective immunological test based on the immune flow principle and is designed in such a way that time-consuming and personnel intensive working steps are avoided.

Mode of Action
Duopath® Legionella (1.04147) is an immunochromatographic rapid test based on gold-labelled antibodies. The test device has a circular sample port and an oval shaped test (Spec, L. pn) and control (C) window.

An isolated suspect colony is suspended and treated with polymyxin, disrupting the outer membrane and releasing the target antigens.
The sample is absorbed through the pad to the reaction zone containing colloidal, gold-labelled antibodies specific to Legionella
Any Legionella antigen present complexes with the gold-labelled antibody and migrates through the port until it encounters the binding zones in the test (Spec. L.pn) area.
The binding zones (Spec and L.pn) contain other anti-Legionella pneumophila antibodies. These immobilize the antigen Legionella at the test zone Spec and the antigen Legionella pneumophila at the test zones Spec and L.pn and form antigen-antibody complexes. NOTE: In rare cases, samples positive for L. pneumophila may form a red line only at the L.pn test zone.
The rest of the sample continues to migrate to another binding reagent zone within the control (C) zone, and also forms a further distinct red line (positive control). Regardless of whether any Legionella is present or not, this distinct red line is always formed in the control (C) zone, thus ensuring the test is working correctly.

Sample Material/Sample Enrichment

Inoculate 100 µl to 1000 ml of the water sample onto BCYE agar or GVPC agar
Filter 10 ml to 1000 ml of the water sample and treat accordingly
Place filter directly on BCYE agar or GVPC agar
Incubate for 10 days at 36 ± 2 °C
Allow to cool to room temperature

Experimental Proecedure and Evaluation
Sample Preparation
Prior to use, allow enriched sample and test devices to reach room temperature (+15 to +25°C)

Pick up 1-3 typical colonies (minimum diameter 1mm, maximum 3 mm) from the BCYE agar or GVPC agar with a wooden toothpick and dispense it well in 144µl of the 0.9%-NaCl / 1% Tween 20-Stock solution in an Eppendorf cup.
Add 16 µl Polymyxin stock solution and mix.
Incubate for 2-5 min at room temperature (+15 to +25 °C).
Heat up and incubate at 95°C for 5 min

Procedure

Remove the foil pouches from the required number of Duopath® Legionella devices. Place the test device(s) on a flat surface and label with appropriate sample identification. (Note: Perform the tests within a period of 2 hours after opening!)
Briefly shake the Eppendorf Cup with a Vortex mixer.
Using a micro pipette and disposable pipette tip, draw up 150µl and pipette it into the circular sample port on the test device.
Observe the test result 30 minutes after applying the sample to the device.

Interpretation of Results
The test can be regarded as working correctly if a distinct red line appears in the control zone (C) within 30 minutes.
A sample can be considered POSITIVE if at or prior to 30 minutes, red lines appear on both test (Spec and/or L.pn) and control (C) zones.
A sample can be considered NEGATIVE if no red line appears in the test (Spec and L.pn) zone but does appear distinctly in the control (C) zone 30 minutes after application of sample to the device.

Technical specifications
Detection limit
One colony (minimum diameter 1mm, maximum 3mm) can be regarded as being the lower detection limit.
Interferences
Results obtained to date on numerous water and environmental samples indicate that there is no interference of Duopath® Legionella with sample particles. The test has been developed based on using BCYE and GVPC Agar from Merck KGaA. Interference from other types of medium and other brands cannot be excluded.

Trouble-shooting

Problem	Measures
No line appears in either zone after 30 minute test period.	Re-run sample
Delay in sample reaching Nitrocellulose membrane	Touch sample pad with pipette tip

Precautions
Users of Duopath® Legionella must be familiar with the appropriate aseptic techniques for the isolation and identification of Legionella spp. Care must be taken when handling samples, enrichments and devices.

Disposal
Decontaminate Duopath® devices, enrichments, tubes, toothpicks, and pipettes by autoclave, bleach, etc. in accordance with local, state, and federal regulations.

Technical Assistance
For technical assistance, please contact us at 1-800-222-0342 or email us at emdinfo@emdchemicals.com

Ordering Information

Product	Catalog No.	Pack Size
Duopath® Legionella	1.04147.0002	25 tests
Bacillus cereus Selective Supplement (Polymyxin ß-sulfate)	1.09875.0001	16 vials
Sodium chloride	SX0420-1	5 kg
Tween® 20 (Polysorbate)	PX1296-1	1 L
Legionella CYE Agar base	1.10242.0500	500 g
Legionella BCYE α-Growth Supplement	1.10240.0001	5 vials
Legionella GVPC Selective Supplement	1.10241.0001	4 vials

Additional Required Materials and Instrumentation

Isolation media, e.g. 1.10242.0500 Legionella CYE Agar base; 1.10240 Legionella BCYE α-Growth Supplement; 1.10241 Legionella GVPC Selective Supplement;
Black nitrocellulose membrane filter (0.45 µm pore size)
0.9% NaCl / 1% Tween 20-Stock Solution: 1g Tween 20, PX1296-1. Tween 20 and 0.9g Sodium Chloride, SX0420-1. Sodium Chloride in 100 ml deionized water. Stock solution is stable for 6 months at 2-8°C.
Polymyxin - Stock Solution: 5mg Polymyxin-ß-sulfate, 1.09875. Supplement Bacillus cereus for 1 ml of deionized water. Stock solution is stable for 6 months at 2-8°C.
Incubators +35°C and +37°C
Distilled or deionized water
Autoclave
Water bath or incubator (95°C) for the denaturation of the sample
Micro pipettes and disposable tips for dispensing 150µl
Sterile foreceps
Wooden toothpicks or disposable loops

Duopath® Legionella:
Test result positive
for Legionella species

Duopath® Legionella:
Test result positive
for Legionella pneumophila