Legionella Latex Test

(1) Note: L. pneumophila serogroup 16 is now mentioned in the Manual of Clinical Microbiology. Serogroup 16 should cross-react with serogroup 6, so theoretically the 2-15 latex reagent should also agglutinate with serogroup 16 isolates.

The kit is intended for professional use only, to be used for industrial diagnostics only and not for use in clinical testing.

For Test Reagent 1, latex particles are coated with polyclonal rabbit antibodies raised against L. pneumophila serogroup 1. For Test Reagent 2-15, latex particles are coated with polyclonal rabbit antibodies raised against serogroups 2-15. For Test Reagent Species, latex particles are coated with polyclonal rabbit antibodies raised against 10 commonly isolated Legionella species (see table below). When these latex particles are mixed with a suspension containing the appropriate Legionella bacteria or heat killed antigens from the relevant Legionella bacteria, an immunochemical reaction takes place causing the latex particles to agglutinate into aggregates which are easily visible to the naked eye. The latex reagent coated with serogroup 1 antibodies will only agglutinate in the presence of serogroup 1 antigens. The latex reagent coated with serogroup 2-15 antibodies will only agglutinate in the presence of antigens from any one of the serogroups 2-15. The latex reagent coated with Legionella species antibodies will agglutinate with the 10 relevant Legionella species (see table below).

Reactions of Test Reagent 1 with serogroup 1 antigens are generally stronger and faster than those between Test Reagent 2-15 and serogroup 2-15 antigens. This is because of the dilution effect of blending 15 different antisera during manufacture of Test Reagent 2-15. The Legionella species latex reacts in a similar manner to the Legionella pneumophila serogroup 1 latex.

Kit contents (50 tests)

• Test Reagent 1 (M45a, 2.5 mL, blue cap): latex particles coated with rabbit polyclonal antibodies to L. pneumophila serogroup 1. Preserved with 0.099% sodium azide.
• Test Reagent 2-15 (M45b, 2.5 mL, yellow cap): latex particles coated with polyclonal rabbit antibodies to L. pneumophila serogroups 2-15. Preserved with 0.099% sodium azide.
• Positive control (M45c, 1.0 mL, black cap): suspension of inactivated Legionella antigens reactive with Test Reagents 1, 2-15 and Species. Preserved with 0.099% sodium azide.
• Test Reagent Species (M45d, 2.5 mL, red cap): latex particles coated with polyclonal rabbit antibodies to 10 commonly isolated Legionella species (see table above). Preserved with 0.099% sodium azide.
• 0.85% isotonic saline (M40, 2 x 5.0 mL, white cap). Preserved with 0.099% sodium azide.
• Disposable agglutination slides (25 pcs).
• Disposable mixing sticks (3 x 25 pcs).

Additional materials required (not supplied in the kit)

• Bacteriological loops.
• Legionella selective medium.
• Glass tubes for boiling.
• 0.85% isotonic saline.

The kit should be stored at 2-8 °C when not in use. The kit should not be used after the expiry date printed on the box.

Before using this product, refer to Precautions and Limitations. Please note, the controls specified in the Quality control section should be performed each time the kit is used.

Specimens

Colonies grown on selective agar plates can be tested with this kit. The morphology of the colonies tested should resemble that of Legionella. Colonies should be at least 3 days old.

Test procedure

For the positive control protocol, see the Quality control section.

1. Dispense 1 drop of isotonic saline (M40) on to each of three circles of the agglutination slide.
2. Using an inoculating loop, remove 3-4 Legionella-like colonies from the selective agar plate and make a thick even smear on the slide alongside each drop of saline.
3. Mix the colonies with the 0.85% isotonic saline and emulsify to form a smooth heavy suspension, spreading the liquid over the entire surface of the circle.
4. If the suspension remains smooth, proceed to step 7. If the suspension is "stringy" or "granular" (often as a result of old and/or mucoid cultures), proceed as follows:
5. Dispense 0.5 mL 0.85% isotonic saline into a glass tube. Prepare a homogeneous turbid suspension of organisms taken from the selective agar plate.
6. Boil the suspension for 5 minutes. Allow to cool to room temperature. Place 30 µL boiled suspension on to each of three circles of an agglutination slide.
7. Gently mix each test latex reagent (M45a, M45b and M45d) to ensure a homogeneous suspension.
8. Add one drop of Test Reagent 1 to one of the bacterial suspensions, one drop of Test Reagent 2-15 to the second suspension and one drop of Test Reagent Species to the third suspension. Do not allow the reagent dropper to touch the suspension.
9. Mix the reagent and suspension using a new mixing stick for each combination. Spread the liquid over the entire area of the circle.
10. Rock the slide gently for 2 minutes and observe for agglutination.
11. Discard the used mixing sticks and slides into a suitable disinfectant.

Interpretation of results

An agglutination reaction is indicated by visible aggregation of the latex particles. The Legionella Latex Test results should be interpreted as follows:

(2) A non-specific agglutination pattern does not preclude the presence of Legionella bacteria but results have to be interpreted as inconclusive.

Safety

1. The kit is intended for professional use only, to be used for industrial diagnostics only and not for use in clinical testing.
2. Sodium azide, which is used as a preservative in the kit reagents, can react with lead or copper plumbing to form potentially explosive metal azides. Dispose by flushing with a large volume of water to prevent azide build-up.
3. Appropriate precautions should be taken when handling or disposing of potential pathogens. Decontamination of infectious material can be achieved with sodium hypochlorite at a final concentration of 3% for 30 minutes. Liquid waste containing acid must be neutralised before treatment.
4. The positive control has been inactivated during the manufacturing process. However, it should be handled as though potentially infectious.

Procedural

1. The Legionella Latex Test should be used according to the kit instructions.
2. Allow all reagents to reach room temperature before use.
3. Do not dilute any of the kit reagents.
4. Do not intermix reagents from different batches of kits.
5. Do not freeze any of the kit reagents.
6. Do not allow the latex reagent dropper to touch the positive control or bacterial samples.
7. Be careful only to record agglutination. Reactions that are "curdy" or "stringy" may not be true agglutination.
8. Ensure the agglutination slide is clean and dry prior to use.

1. Results should be interpreted in the context of all available laboratory information.
2. Stringy reactions on the slide may not be true positive reactions and further tests are required.
3. Old and/or mucoid cultures may not give a smooth suspension in saline and may give atypical agglutination. These should be pre-treated by boiling as described in the method above.
4. The Legionella Latex Test is intended for the identification of L. pneumophila serotypes 1-15 and detection of a range of 10 Legionella species following culture on selective agar plates. Colonies giving positive results should be confirmed as Legionella spp. by their inability to grow on Legionella selective culture media deficient in cysteine.
5. Media from different manufacturers may elicit slow antigen expression. Users should test their media with known strains to ensure the test performs as intended before testing unknown isolates.

The following controls should be performed each time the kit is used.

1. Reagent control: Gently mix the test latex reagents (M45a, M45b, M45d) and add one drop of each reagent to separate circles on the agglutination slide. Add one drop of saline solution (M40) to each drop of latex reagent. Using a different mixing stick for each circle, mix the latex reagent and saline thoroughly, spreading the liquid over the entire area of the circle. Rock the slide gently for 2 minutes and observe for agglutination. If any agglutination is seen, at least one of the reagents is contaminated and a fresh kit should be used.
2. Positive control: Add one drop of positive control (M45c) to one circle on the agglutination slide. Gently mix Test Reagent 1 (M45a) and add 1 drop to the same circle. Do not allow the dropper to touch the positive control. Mix with a mixing stick, rock the slide gently for 2 minutes and observe for agglutination. A positive result, indicated by agglutination, should be seen. Repeat this process using Test Reagent 2-15 (M45b) and Test Reagent Species (M45d). Again, a positive result should be seen. If agglutination is not seen with any of the three latex reagents, a fresh kit should be used.

NOTE: The reaction strength with the positive control may not be the same.

Dispose of according to any local, national, or regional regulations.