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The Oxoid Legionella Latex Test is a latex agglutination test for the identification of predominant Legionella species grown on plate media from patients with suspected Legionellosis or from environmental sources. The Oxoid Legionella Latex Test allows a separate identification of Legionella pneumophila serogroup 1 and serogroups 2–14 and detection of seven other Legionella species which have been implicated in human disease.

Introduction
Legionnaires’ disease, named after the outbreak in 1976 at the American Legion Convention in Philadelphia, is caused by Legionella pneumophila and other Legionella species. It is characterised as an acute febrile respiratory illness ranging in severity from mild illness to fatal pneumonia. Since that time, it has been recognised that the disease occurs in both epidemic and endemic form and that the sporadic cases are not readily differentiated from other respiratory infections by clinical symptoms.
It is estimated that worldwide about 25,000 cases of Legionella infections occur 1 annually. Known risk factors include immunosuppression, cigarette smoking,
alcohol consumption and concomitant pulmonary disease. The mortality rate, which can be as high as 25% in untreated immunosuppressed patients, can be
lowered if the disease is diagnosed rapidly and appropriate antimicrobial therapy started earlier.
Legionnella pneumophila has been shown to be a major cause of both pneumonia and an acute self limiting febrile disease called Pontiac Fever. Legionella pneumophila strains and other Legionella species are isolated from patients with pneumonia and from the environment (mainly water).
Rare isolations have also been made in cases other than pneumonia, such as wound abscesses. The major reservoir of Legionella species appears to be fresh
water sites, air-conditioning units and various water plumbing fixtures.
Legionella pneumophila is the most common cause of Legionnaires’ disease. At present, 14 different serotypes exist of which Legionella pneumophila serogroup 1 accounts for 90% of cases.
The Oxoid Legionella Latex Test uses antibody sensitised blue latex particles which will agglutinate in the presence of specific Legionella cell wall antigens to form visible clumps. This provides a fast and simple screening procedure for predominant pathogenic Legionella species and serotypes^1,2.

Components of the Kit
DR0801 Legionella pneumophila serogroup 1 Test Reagent
Consists of blue latex particles sensitised with specific rabbit antibody reactive with Legionella pneumophila serogroup 1 antigen. Each kit contains sufficient reagent for 50 tests.
DR0802 Legionella pneumophila serogroups 2–14 Test Reagent
Consists of blue latex particles sensitised with specific rabbit antibody reactive with Legionella pneumophila serogroups 2–14 antigen. Each kit contains sufficient reagent for 50 tests.
DR0803 Legionella species Test Reagent
Consist of blue latex particles sensitised with specific rabbit antibody reactive with the following species and serotypes:
Legionella longbeachae 1 and 2
Legionella bozemanii 1 and 2
Legionella dumoffii
Legionella gormanii
Legionella jordanis
Legionella micdadei
Legionella anisa
Each kit contains sufficient reagent for 50 tests.
DR0804 Positive Control Suspension
A polyvalent suspension of Legionella cells in buffer, sufficient for 25 tests.
DR0805 Negative Control Suspension
A suspension of Legionella spiritensis cells in buffer non-reactive with the test reagents, sufficient for 25 tests.
DR0806 Control Latex
Consists of blue latex particles sensitised with non-reactive rabbit globulin. Each kit contains sufficient reagent for 50 tests.
DR0807 Suspension Buffer
A phosphate buffered saline solution. pH 7.3.
DR500 Reaction Cards
There are 50 disposable reaction cards provided in the kit.
Instruction Leaflet.

Materials Required
The following materials are required but not provided in this kit:
Microbiological loop and bunsen burner.
0.85% saline (for optional tube method).
Suitable laboratory disinfectant e.g. Sodium hypochlorite solution >1.3% w/v.

Precautions
This product is for in vitro diagnostic use only.
Do not freeze.
Reagents contain 0.095% sodium azide as a preservative.
Sodium azide may react with lead or copper plumbing to produce metal azides which are explosive by contact detonation. To prevent azide accumulation in plumbing flush with copious amounts of water immediately after waste disposal.
Specimen materials may contain pathogenic organisms, handle with appropriate precautions.
Aerosol formation should be avoided, particular care should be taken during vortexing.

Storage
This kit must be stored at 2–8°C. Under these conditions the reagents will retain their reactivity until the expiry date shown on the kit box.

Control Procedures
The control suspensions provided should be used to check the correct working of the latex reagents each day before routine tests are performed.
The positive control suspension (DR0804) must show agglutination with the latex reagent within one minute. The negative control suspension (DR0805) must show no agglutination within one minute.
Do not use the test if reactions with the control suspensions are incorrect.

Important Procedure Notes
Do not allow the reagents to become contaminated by letting the dropper tip touch the specimens on the reaction card. Ensure that the caps are securely fitted after use to prevent contamination and drying out of reagents. After use, return the kit to the refrigerator ensuring that the bottles are stored in an upright position.

Specimen Collection and Preparation
Isolates derived from environmental and clinical samples may be cultured on standard non-selective or selective
Legionella culture media. Typical isolation schemes are given in references 3 and 4.
Legionella species on primary isolation have an absolute requirement for L-cysteine hydrochloride. To ensure that an isolate is a Legionella it is necessary to show that it cannot grow on any medium which does not contain L-cysteine hydrochloride. This confirmation may be performed prior to or after the latex test.
The following Oxoid media and supplements may be used for the culture of Legionella before performing the latex test.
BCYE (CM0655 + SR0110), BPMA (CM0655 + SR0110 + SR0111), MWY (CM0655 + SR0110 + SR0118), GVPC (CM0655 + SR0110
+ SR0152).
For further details of these products please consult your local Oxoid agent.
Cultures may be tested at any stage of growth providing that the colonies are of sufficient size. Older cultures, however, may produce stringy reactions making
interpretation more difficult.

Test Methods
There are two test methods which may be used. Both direct and tube methods give reliable results. If an isolate has a stringy consistency it is recommended that the tube method is used.
(a) Direct Test
1. Bring the latex reagents to room temperature. Make sure the latex suspensions are mixed by vigorous shaking. Expel any latex from the dropper pipette for complete mixing.
2. Dispense 1 drop of each of the latex reagents onto 4 circles on the reaction card. Place them close to the edge of the circle.
3. Add 1 drop of diluent buffer suspension to each of the 4 test circles. Ensure that the latex and buffer do not mix at this stage.
4. Using a loop, pick off a colony of at least 1 mm (use 2 or more if the colonies are smaller) and carefully emulsify in the buffer. For optimal results ensure that the suspension is smooth. Repeat for similar colonies with each of the other reagents.
5. Mix the latex reagents and suspensions together and spread to cover the reaction areas using the loop. Flame the loop.
6. Gently rock the card in a circular motion and look for agglutination. Do not rock the card for more than 1 minute and do not use a magnifying glass to aid reading the result.
7. When finished, dispose of the reaction card into a suitable disinfectant.
8. Recap the bottles and return to the refrigerator.
(b) Tube Method
1. Bring the latex reagents to room temperature. Make sure the latex suspensions are mixed by vigorous shaking. Expel any latex from the dropper pipette for complete mixing.
2. Label test tubes appropriately and dispense 0.4 ml of 0.85% saline into each tube.
3. Select 4–10 colonies of a similar colonial appearance with a loop and emulsify in the saline.
4. Vortex the cell suspension for 5 seconds. (Refer to Precautions Section.)
5. Dispense 1 drop of each latex reagent (3 test reagents and the control reagent) onto 4 circles on the reaction card. Place them close to the edge of the circle.
6. Using a Pasteur pipette add 1 drop of cell suspension to each of the 4 circles, and mix this into the latex reagents. Spread to cover the reaction areas.
7. Gently rock the card in a circular motion and look for agglutination. Do not rock the card for more than 1 minute and do not use a magnifying glass to aid reading of the result.
8. When finished, dispose of the reaction card into a suitable disinfectant.
9. Recap the bottles and return to the refrigerator.

Reading and Interpretation of Results
Positive Results
A result is positive if agglutination of the blue latex particles occurs within 1 minute and with no agglutination in the control circle. A positive reaction indicates that antigens to that serogroup of Legionella species have been detected in the sample.
Negative Results
A negative result is obtained if no agglutination occurs and a smooth blue suspension remains after 1 minute in the test circles.
Uninterpretable Result
The test is uninterpretable if the control reagent shows agglutination. This indicates that the culture causes autoagglutination.
Granular or Stringy Reactions
Occasional granular or stringy reactions may be seen due to the particulate nature of the test material. When such reactions are seen to occur they should be interpreted using the following criteria:
The result is positive when there is noticeable clearing of the blue background in the test reagents.

Limitations
1. The latex agglutination test is presumptively diagnostic. Confirm positive results using biochemical tests⁵.
2. A negative latex agglutination test does not mean that the culture is not a Legionella species. It only indicates that the culture is not Legionella pneumophila
serogroups 1 through to 14 nor Legionella longbeachae 1 and 2, Legionella bozemanii 1 and 2, Legionella dumoffii, Legionella gormanii, Legionella jordanis, Legionella micdadei, Legionella anisa⁶.
3. A cross reaction may occur between Legionella pneumophila serogroup 1 and serogroup 9 due to naturally occurring group antigens. If both the Legionella pneumophila serogroup 1 and 2–14 reagents agglutinate with the isolate then this cross reaction should be suspected.
4. Cross reactions with the Legionella Species Test Reagent have been reported to occur occasionally with certain serotypes of other Legionellae (e.g. Legionella parisiensis, Legionella sainthelensi, Legionella steigerwaltii, Legionella wadsworthii, Legionella santicrucis, Legionella tusconensis, Legionella gratiana, Legionella cincinatiensis⁶.)
5. The test is designed to differentiate between different species and serotypes of Legionella. Cultures should be confirmed as Gram-negative rods which do not grow on cysteine-deficient media.

Performance Characteristics
The reagents in the Oxoid Agglutination Legionella Test Kit have been tested for cross-reactivity against a panel of organisms listed below. No cross-reactivity was observed with any of the organisms:
Legionella cherri
Legionella birminghamensis
Legionella rubrilucens
Legionella maceachernii
Legionella oakridgensis
Legionella erythra
Legionella feelei
Legionella fairfieldensis
Legionella brunensis
Legionella spiritensis
Pseudomonas fluorescens
Pseudomonas cepacia
Pseudomonas aeruginosa
Aeromonas hydrophila
Legionella hackeliae
Legionella israeliensis
Legionella jamestowniensis
Legionella quinlivanii
Legionella moravica
Stenotrophomonas maltophilia
Bacillus subtilis
Citrobacter freundii
Escherichia coli
Serratia marcescens
The Oxoid Legionella Latex Test was evaluated in a clinical and an environmental laboratory. A total of 40 clinical isolates and 279 environmental isolates were tested, covering Legionella pneumophila serogroups 1–14 and non-pneumophila Legionella. Each isolate was confirmed by serology. The performance of the Oxoid kit was also compared against other commercially available Legionella Latex Reagent Kits. The results of the trial are summarised⁷.

The overall sensitivity of the Oxoid Legionella Latex Kit was 99%.
The overall specificity of the Oxoid Legionella Latex Kit was 100%.

WARNING: This product contains sodium azide. Harmful if swallowed.

References
1. Sedgwick A.K., & Tilton R.C. (1983) J. Clin. Microbiol. 17: 365-368.
2. Ciesielski C.A., Blaser M.J. & Wang W.L. (1986) Infect. Immun. 51: 397-404.
3. Dennis P.J.L. (1988) Isolation of Legionella from Environmental Specimens p.31-44. In Harrison T.G. and Taylor A.G. (eds). A Laboratory Manual for Legionella: John Wiley & Sons Ltd. Chichester, UK.
4. Dournon E. (1988) Isolation of Legionellae from Clinical Specimen p.13-30. In Harrison T.G. and Taylor A.G. (eds). A Laboratory Manual for Legionella. John Wiley & Sons Ltd., Chichester, U.K.
5. Cowan and Steel's Manual for the Identification of Medical Bacteria 3rd Ed. Barrow, G.I., and Feltham R.K.A., (eds) (1993) p161-163. University Press, London.
6. Harrison T.G. and Taylor A.G. (1988). Identification of Legionella by Serological Methods. In Harrison T.G. and Taylor A.G. (eds). A Laboratory Manual for Legionella: John Wiley & Sons Ltd., Chichester, UK.
7. Data on file Oxoid Ltd.