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Further Information related to  DR1107

CODE: DR1107

Clostridium difficile is recognised as a major cause of infectious diarrhoea in hospital patients. Infection with the organism is commonly responsible for pseudomembranous colitis and antibiotic associated diarrhoea, as well as post-operative diarrhoea1. The isolation and identification of Clostridium difficile is important in establishing the aetiology of these pathologies.
Methods for the selective isolation of Clostridium difficile have been described 2 and the rate of isolation is increased by culturing on selective media7. However, subsequent identification of the organism has required the use of gas liquid chromatography since conventional biochemical tests are often of little value. Although detection of Clostridium difficile cytotoxin is widely used in diagnosis, the association between the presence of cytotoxin and the symptoms of infection is inconsistent 3,4.

A comparison of methods for Isolation and identification of Clostridium difficile

Oxoid Suggested Procedure
Conventional Laboratory Procedure
24 hours

Selective enrichment broth
(e.g. GCC) 37°C

Alcohol shock Procedure
Enrichment broth 37°C, 18-24 hours

Test with Oxoid C.difficile latex (2 mins)

Negative Presumptive
Subculture onto selective Medium (Clostridium Difficle agar base + selective supplements - SR0096 or SR0173) 37°C
Anaerobically, 24-48 hours
Subculture onto selective medium (CCFA)

Test with Oxoid C.difficile latex (2 mins)

Incubate at 37°C
Anaerobically 48-72 hours


Confirmed Positive Report
Examine morphologically forClostridium difficile
Gram Stain
Long Wave
UV light
G.L.C. (Approx
30 minutes)
Set up biochemical
Identification (24-48
Read Results

Intended Use
The Oxoid C. difficile Test Kit is a rapid and simple latex agglutination test for the early identification of Clostridium difficile and is ideally suited to screening selective and enrichment broth cultures. The test can also be used for the identification of Clostridium difficile colonies from selective solid media 2,6. The recommended procedure for the identification of Clostridium difficile using this kit is compared with a typical procedure below:

The advantages of using the Oxoid C. difficile Test Kit are:
1. Earlier identification of Clostridium difficile in primary selective cultures allowing presumptive positive identification overnight, and confirmed positive results within two days. Identification by anaerobic culture and confirmatory tests requires 3-6 days.
2. The high predictive value of a negative result allows early elimination of negative selective cultures.
3. Reduction in the required number of subcultures.
4. No requirement for extended anaerobic subcultures of 48-72 hours.
5. No requirement for expensive equipment.

Principle of the Test
Latex particles are coated with IgG antibodes specific for Clostridium difficile cell wall antigens. When mixed on a reaction card with selective or enrichment broth containing the organism, or with a suspension of Clostridium difficile colonies from solid media, the latex particles agglutinate in large visible clumps within 2 minutes.

Components of the Kit
C.difficile Reagent: Latex particles coated with rabbit IgG against Clostridium difficile; preserved with 0.02% merthiolate (2.5 ml)
Positive Control: Inactivated C. difficile (0.5 ml)
0.85% isotonic saline, preserved with 0. 1 % sodium azide (5 ml)
Disposable reaction cards
Disposable mixing sticks

Materials required but not provided
Pasteur pipettes

The Oxoid C. difficile Test Kit should be stored at 2- 8°C. Do not freeze. The kit should not be used after the expiry date printed on the outside of the carton.

1. Oxoid C. difficile Test Kit is for in vitro diagnostic use only.
2. The Positive Control contains inactivated Clostridium difficile but should be treated as a potentially infectious agent.
3. Do not mouth pipette.
4. Do not cross contaminate solutions or samples.
5. Do not use kit after expiry date printed on outside of carton.
6. Do not use kit if the Positive Control fails to agglutinate the C. difficile Reagent.
7. Ensure the reaction card is clean and dry before use.
8. Discarded reagents and materials must be autoclaved before disposal.
9. The isotonic saline is preserved with sodium azide which can form potentially explosive compounds on contact with copper and lead plumbing. On disposal of reagent, flush with copious quantities of water to prevent azide build up.

Method for Use
Allow the Oxoid C. difficile Test Kit reagents to reach room temperature before use.

The following controls should be examined each day the kit is used.
1. Reagent Control
Add 1 drop of the Oxoid C. difficile Latex Reagent to 1 drop of saline in the same circle on a reaction card, mix and observe for agglutination. No agglutination should occur. If this control shows agglutination the Latex Reagent is probably contaminated and should be discarded.
2. Positive Control
Gently mix the Positive Control suspension by inverting. Place one drop on the reaction card. Add 1 drop of Oxoid C. difficile Latex Reagent to the same circle and mix with a mixing stick. After two minutes observe for agglutination, indicating a positive reaction.

Assay Procedure
A. Method for Identification from Solid Media
1. Place a reaction card on the work bench.
2. Add 1 drop of saline within one circle on the reaction card.
3. Using a mixing stick or inoculating loop, emulsify the suspect colony in the drop of saline to produce a heavy smooth suspension. Suspensions should be made from colonies with morphologies typical of Clostridium difficile.
4. Observe the suspension for any agglutination or clumping, which would indicate autoagglutination. If the suspension remains
smooth proceed to Step 5. (See Limitations of Use, point l.)
5. Mix the C.difficile Latex Reagent gently by inverting and add a drop to the saline suspension. DO NOT ALLOW THE DROPPER TO TOUCH THE ORGANISM SUSPENSION. Mix the Reagent and organism suspension with a clean mixing stick for 30 seconds and examine for agglutination within a maximum of 2 minutes. DO NOT ROCK THE REACTION CARD.
NOTE: It is advisable to mix the Latex Reagent and suspension and then observe the reaction on the bench after 2 minutes, rather than rocking the reaction card.
6. After reading, discard the used reaction cards into suitable disinfectant.

B. Method for Testing Broths
Selective or non-selective broths can be tested. However, the performance characteristics reported below are achieved with selective enrichment broth which has been shown to increase isolation rates and therefore improve test performance.
1. Mix the broth culture by gently inverting and remove 1 drop of culture using a Pasteur pipette.
2. Place a single drop of the broth culture within one circle on the test card.
3. Mix the Oxoid C.difficile Latex Reagent by gently inverting and add 1 drop to the broth on the card. DO NOT ALLOW THE DROPPER TO TOUCH THE DROP OF BROTH CULTURE. Mix thoroughly using a clean mixing stick or inoculating loop.
4. Examine for agglutination after 2 minutes. DO NOT ROCK THE REACTION CARD.
NOTE: It is advisable to mix the Latex Reagent and sample and then to observe for agglutination without rocking the reaction card.
5. After reading, dispose of the reaction cards in disinfectant.

Agglutination within 2 minutes is a positive result and indicates the presence of Clostridium difficile. Care must be taken when testing broth cultures that adherence of latex to particulate matter is not
interpreted as agglutination.
When testing broth cultures, cross reaction may occur with Clostridium sordellii, Clostridium glycollicum and Clostridium bifermentans. Positive reactions should be confirmed by performing the Oxoid C. difficile test on typical colonies isolated on Clostridium difficile Selective Agar. No agglutination within two minutes indicates a negative result.

Limitations of Use
1. In general and when using the Oxoid C. difficile Test Kit, identification of Clostridium difficile should be performed on selective cultures since this increases the isolation rate.
2. Culture-derived suspensions which autoagglutinate cannot be tested with the Oxoid C. difficile Test Kit.
3. Cross reaction may occur with Clostridium sordellii, Clostridium glycollicum and Clostridium bifermentans present in broth cultures. Positive results should be confirmed by subculturing onto C. difficile Selective Agar and retesting with the Oxoid C. difficile Test Kit. The cross reacting organisms rarely grow on C. difficile Selective Agar and are clearly distinguishable from Clostridium difficile
when cultured on Clostridium Difficile agar base + selective supplements - SR0096 or SR0173 (CCFA) by their production of a lecithinase, which causes zones of precipitation around the colonies.
4. The pathogenic significance of any isolate should be assessed in the context of the patient’s clinical condition.

Performance Characteristics
A total of 329 stool sample broth cultures were analysed at an independent laboratory using three different methods7. Selective enrichment broth cultures were tested for Clostridium difficile by:
1. Oxoid C. difficile Test Kit latex agglutination (2 minutes duration)
2. Gas liquid chromatography (GLC) (approximately 30 minutes duration)
3. 48-72 hour anaerobic subculture on selective agar followed by morphological examination.

There are limitations to using either GLC or culture as definitive reference tests. GLC is less sensitive than culture and culture in turn will not detect Clostridium difficile present in low numbers. This occasionally occurs due to overgrowth by other organisms such as Lactobacilli.
Test performances are compared below:

Oxoid C.difficile Test Kit
v Culture
Oxoid C.difficile Test Kit
GLC v Culture
Sensitivity (%)
Specificity (%)

The predictive value of a negative result with Oxoid C. difficile Test Kit is >96% when compared with either GLC or culture. Oxoid C. difficile Test Kit is thus ideally suited as a rapid and low cost
screening test.

1. Larson, H.E., Price, A.B., Honour, P. and Borriello, S.P. (1978) Lancet 8, 1063 1066
2. George, W.L., Sutter, VL, Citron, D. and Finegold, S.M. (1979) J.Clin. Microbiol. 9, 214 219
3. Riley, TV., Bowman, R.A. and Carroll, S.M. (1983) Med. J. Aust i, 166 169
4. Haslam, S.C., Ketley, J.M., Mitchell, T.J., Stephen, J, Burdon, D.W. and Candy, D.C.A. (1986) J. Med. Microbiol. 21, 293 297
5. Mollby, R, Nord, C.E. and Aronsson, B. (1980) Scand. J. Infect. Dis. 22 (Suppl), 30 36
6. Carroll, S.M., Bowman, R.A. and Riley, TV. (1982) Pathol. 15, 165 167
7. Bowman, R.A., Arrow, S.A. and Riley, T.V. (1986) J. Clin. Pathol. 39, 212 214
8. Chang, T.W. and Gorbach, S. L. (1982) J. Clin. Microbiol. 15, 465 467

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