Organisms this product works with:
LEGIONELLA LATEX TEST
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 serotypes1,2.
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.
Oxoid Legionella Number |
Latex Kit/Serology % |
|
Legionella pneumophila serogroup 1 | 59/59 |
100 |
Legionella pneumophila serogroups 2–14 | 136/136 |
100 |
Other legionellae included in the kit | 51/54 |
94.4 |
Other legionellae not included in the kit | 60/60 |
100 |
Other organisms | 10/10 |
100 |
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.