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Diagnostic Reagents

C. DIFFICLE TEST KIT

CODE: DR1107

Introduction
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.
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:

A comparison of methods for Isolation and identification of Clostridium difficile

Time
Oxoid Suggested Procedure
Conventional Laboratory Procedure
Faeces
Faeces
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)

Report
Negative Presumptive
Report
Positive
Report
48
hours
 
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

Negative
Report

Confirmed Positive Report
72
hours
Examine morphologically forClostridium difficile
Negative
Positive
Report
   
 
Gram Stain
Long Wave
UV light
G.L.C. (Approx
30 minutes)
Set up biochemical
Identification (24-48
Hours)
Read Results

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

For full procedure please see product insert.
Storage
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.

Precautions
The Positive Control contains inactivated Clostridium difficile but should be treated as a potentially infectious agent.
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.

Reference
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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