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E. COLI 0157 LATEX TEST

A latex agglutination test for the identification of Escherichia Coli serogroup 0157.
Certain strains of Escherichia coli have been implicated in cases of haemorrhagic colitis (HC) and haemolytic ureamic syndrome (HUS).
It has been shown that these strains produce a vero-cytotoxin (VT). The Escherichia coli serotype most frequently isolated from HC and HUS cases is 0157:H7. Isolation of this serotype from a diarrhoeal stool particularly with the presence of blood is indicative of a vero-cytotoxin producing strain^{1,2,3,4,5,6,7,8}.
The Oxoid E. coli 0157 latex test will demonstrate by slide agglutination Escherichia coli strains possessing the 0157 serogroup antigen. The test is best used in
conjunction with Sorbitol MacConkey Agar (Oxoid CM0813). Escherichia coli 0157:H7 strains do not ferment Sorbitol and therefore give colourless colonies on this medium. The majority of Escherichia coli isolates do ferment Sorbitol and give characteristic pink Sorbitol MacConkey Agar should be used as the primary screen. Non-sorbitol fermenting colonies can then be tested with the latexreagents, to determine whether the isolate belongs to the 0157 serogroup and therefore a potential VT producing strain.

Components of the Kit DR0620
DR0621 Test Latex
Consists of blue latex particles sensitised with specific rabbit antibody reactive with the 0157 somatic antigen. Each kit contains sufficient reagent for 100 tests.
DR0622 Control Latex
Consists of blue latex particles sensitised with pre-immune rabbit globulin. Each kit contains sufficient reagent for 100 tests.
DR0623 Positive Control Suspension
A suspension of inactivated Escherichia coli 0157 cells in buffer.Sufficient for 25 tests.
DR0624 Negative Control Suspension
A suspension of Escherichia coli 0116 cells in buffer. Sufficient for 25 tests.
DR0500 Reaction Cards
There are 35 disposable reaction cards provided in the kit.
Instruction leaflet.

Materials required but not provided.
Microbiological loop and bunsen burner.
0.85% saline.
Suitable disinfectant e.g. sodium hypochlorite solution >1.3% w/w.

Precautions
This product is for in vitro diagnostic use only.
Do not freeze.
Reagents with different lot numbers should not be interchanged.
Reagents contain 0.1% 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.

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 must cause visible agglutination with the latex reagent within one minute.
The negative control suspension should cause 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 specimen on the reaction card.
Ensure that the caps are securely fitted after each use to prevent contamination and drying out of the reagent.
After use return the kit to the refrigerator ensuring that the bottle is stored in an upright position.

Culture Material
Non-sorbitol fermenting (NSF) colonies may be taken from Sorbitol MacConkey Agar (Oxoid CM0813) or alternatively NSF isolates may be inoculated onto nonselective media such as Nutrient Agar for testing. It is necessary to test up to 10 separate NSF colonies to ensure a high probability of detecting any 0157 strains which may be in a mixed culture with NSF Escherichia coli of other serotypes. The use of the control latex will ensure that the isolate is not an autoagglutinating strain.

Test 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. Dispense 1 drop of the test latex onto a circle on the reaction card. Place it close to the edge of the circle.
3. Add some loopfuls or a pasteur pipette drop of saline to the circle. Ensure that the latex and saline do not mix at this stage.
4. Using a loop pick off a portion of the colony to be tested and carefully emulsify in the saline drop. Ensure that the resulting suspension is smooth.
5. Mix the test latex and together and spread to cover the reaction area using the loop. Flame the loop.
6. Rock the card in a circular motion observing for agglutination. Do not rock the card for more than 1 minute and do not use a magnifying glass.
7. If no agglutination occurs then proceed to test other NSF colonies if these are present.
8. If agglutination with the test reagent does occur, then it is necessary to test a further portion of the colony to ensure that the isolate is not an auto-agglutinating strain.
9. When finished dispose of the reaction card into disinfectant.

Interpretation of Test Results
Agglutination of the test latex within one minute is a positive result. This indicates the presence of Escherichia coli Serogroup 0157. No agglutination occurring within one
minute is a negative result. This indicates the absence of Escherichia coli Serogroup 0157.
Results cannot be interpreted if there is agglutination of both the test and control latex.
Some strains of Escherichia coli are difficult to emulsify in saline and may give a stringy type reaction with both the test and the control reagents. This does not look like true agglutination and should be ignored. If this stringiness is found to be too severe for a correct judgement to be made then the colony should be suspended in 0.3 ml of saline. Allow the lumps to settle and retest this smooth supernatant.

Limitations of the Test
If a positive result is obtained on a colony of unknown species, then biochemical tests should be performed to confirm that the organism is an Escherichia coli strain. Neither the Sorbitol MacConkey Agar nor the Escherichia coli 0157 latex test will directly confirm the isolate a toxin-producing strain.
Other serotypes have been found which produce the vero-cytotoxin. Some strains of Escherichia hermanii may cross react with Escherichia coli 0157 sera and the latex test due to a shared antigen (borczyk Refe et al.)¹. Escherichia hermanii may be differentiated from Escherichia coli as they ferment cellobiose, grow in the presence of KCN and produce a yellow pigment, which may be delayed¹⁰.

Performance Characteristics
In clinical comparisons of the performance of the Oxoid E. coli 0157 latex test and commercially antisera, results demonstrated 99.3% (151/152) agreement between the two methods⁹. The sensitivity of the Oxoid tests was 100% (49/49), and the specificity was 99.0% (101/103). The discrepant sample was further tested and found to be positive for Escherichia coli 0157. This was in agreement with the Oxoid test result.
The Oxoid reagents did not show crossreactivity with other E. coli strains or nonsorbitol fermenting organisms. The Oxoid test was performed directly from a variety of culture media. None of these media were found to cause interference with the latex test.

References
1. Borczyk A., Lior H., Crebin B (1987) Int. J Food. Microbial. 4, 347-349.
2. Konowalchuk, J., Speirs, J. and Stavric S. (1977) Infect. Immun 18, 775-779.
3. Scotland S., Day N and Rowe B. (1980) FEMS. Microbiol. Lett, 7, 15-17.
4. Centres for Disease Control (1982) Morbid Mortal Wkly 31 580-585
5. Karmali M., Still, B., Petrick M., and Lim C. (1983), Lancet I, 619-620.
6. Johnson, W., Liroh, H., and Bezanson, (1983) Lancet I, 76.
7. March S., and Ratnam (1986) J. Clin. Microbiol. 23, - 869-872.
8. Krishnan, C., Fitzgerald V., Dakin S., and Behme R. (1987) J. Clin. Microbial. 25, 1043-1047.
9. P.A. Chapman (1989) Evaluation of Commercial latex slide test for identifying Escherichia coli O157.42:1109-1110.