Keul-o-test

Clostridium Difficile Complete

KGST203
DIMDI Reg.-Nr.: DE/CA22/1116-134-IVD

Immunochromatographic rapid test for the detection of Clostridium Difficile Toxin A and Toxin B in feces

I- PRINCIPLE
Clostridium difficile is a major cause of antibiotic-associated diarrhea and pseudomembranous colitis (1). It is now one of the most commonly detected pathogens and an important cause of nosocomial infections in hospitals and nursing homes (2, 3).
The organism has been isolated from diverse natural habitats, including soils, hay, sand, dung from various large mammals (cows, donkeys and horses), and from dog, cat, rodent and human feces (4). C.difficile produces at least three potential virulence factors from which Toxin A and Toxin B are thought to be the most important in the pathogenesis of C.difficile associated diseases (5).
Toxin A is an enterotoxin which seems to interfere with the cytoskeleton of the intestinal epithelial cells, rendering them non functional while Toxin B is a cytotoxin that induces strong cytopathic effects in tissue cultures cell lines (6).
Since not all strains of Clostridium difficile produce toxins and approx. 2% of healthy adults as well as up to 50% of children younger than 2 years can be colonized with Clostridium difficile, the detection of the toxins (Toxin A and Toxin B) in stool samples of patients with diarrhea is more significant than culturing the bacteria.
The Keul-o-test Clostridium Difficile Complete is a lateral flow,  immunochromatographic rapid test for the qualitative detection of Clostridium difficile Toxin A and Toxin B in human feces.
The test device consists of a plastic housing containing two different sticks for the detection of C.difficile Toxin A or C.difficile Toxin B. A coloured anti C.difficile Toxin A or C.difficile Toxin B monoclonal antibody colloidal gold conjugate is placed at the left end of the membrane.
After collection in a tube containing the extraction solution, the feces sample is dissolved and few drops of this extract are added into each well ( =>) of the reaction device.
As the test sample flows through the absorbent device, the labelled antibody-dye conjugate binds to the Toxin A or Toxin B antigen (when present in the sample), forming an antibody antigen complex.
This complex binds to the polyclonal anti-Toxin A or Toxin B antibody in the positive reaction zone, producing a rose-pink coloured band.
In the absence of Toxin A or Toxin B, there is no line in the positive reaction zone. The reaction mixture continues flowing through the absorbent device, past the positive reaction zone and control zone.
Unbound conjugate binds to the reagent in the control zone producing a rose-pink coloured band demonstrating that the reagents are functioning correctly.

II- Keul-o-test Clostridium Difficile Complete KIT COMPONENTS 
Each kit contains everything needed to perform the tests.

• Keul-o-test Clostridium Difficile Complete reaction devices
• Plastic droppers
• Plastic tubes filled with 2 mL of extraction solution
• Sample applicators
• Instructions leaflet

III- STORAGE AND STABILITY

1. All Keul-o-test Clostridium Difficile Complete kit components should be stored in the sealed pouch at room temperature (between +4°C and +30°C).
2. Do not freeze the test kit.
3. The Keul-o-test Clostridium Difficile Complete kit is stable until the expiry date stated on the package label.

IV- PRECAUTIONS

1. This test is designed for in vitro diagnostic use and professional use only.
2. Read carefully instructions leaflet before using this test.
3. Do not use beyond the expiry date stated on the package label.
4. Do not use a test from a damaged protective wrapper.
5. All reagents and materials coming in contact with potential infectious specimens must be treated with appropriate disinfectants or autoclaved at 121°C for at least one hour.
6. Wear protective clothing such as laboratory coats and disposable gloves while assaying samples.
7. Do not eat, drink or smoke in the area where specimens and kit reagents are handled.
8. Avoid any contact between hands and eyes or nose during specimen collection and testing.

V- SPECIMEN COLLECTION AND PREPARATION

1)Preliminary notes
Stool specimen should be collected as soon as possible after onset of symptoms.
Diluted samples may be stored at +2°C to +8°C for 3 days without interference with assay performance.
For long term storage of undiluted specimens, storage at -20°C or colder is recommended. Repeated freezing and thawing of samples is not recommended and may cause erroneous results. 

Caution!
Do not collect specimens in containers having media, preservatives, animal serum or detergents, as any of these may interfere with the test.

2) Procedure

1. Label the plastic tube containing the extraction solution with patient's name or control number.
2. Open the tube and, using the sample applicator, transfer into it a stool sample:
   - volume of a pea, in case of solid stool.
   - 200 µl, in case of liquid stool.
3. Tighten the cap and mix the stool sample and the diluent by shaking well until the sample is dissolved.
4. Let the tube stand long enough for the large particles to settle to the bottom of the tube. Alternatively, centrifuge the tube at 500-1,000 RPM for 1 minute.

VI- ASSAY PROCEDURE

1. Bring all reagents at room temperature.
2. Remove the test device from the pouch by tearing along the split.
3. Open the plastic tube containing the extracted sample.
4. Using the plastic pipette, add 6 drops (200 µl) of the extracted solution into each of the sample wells (=>).
5. Read the test results 15 minutes after addition of the sample to the device.

VII- READING TEST RESULTS
possible results pattern

1. Negative
Only one coloured band appears in the control zone.
No band is visible in the test zone.

2. Positive
In addition to the control band, a clearly
distinguishable band also appears in the test zone.

3. Inconclusive
If there is no distinct coloured band visible in the control zone, the test is inconclusive. Repeat the test.

VIII- PERFORMANCES CHARACTERISTICS

 1. C. difficile  TOXIN A

a) Analytical sensitivity
The performance of the test has been assayed, using a range diluted solutions prepared from a commercially available purified toxin A antigen. The test is able to detect a concentration of  4 ng/mL.

b) Diagnostic sensitivity and specificity
A study was performed on 63 feces samples, using the Keul-o-test Clostridium Difficile A test in comparison with the cytotoxicity and cell culture methods.

The results are summarized in the table 1:

Table 1 : Evaluation results

All samples showed identical results using the cytotoxicity and the cell culture methods.
From the above table, the sensitivity of Keul-o-test Clostridium Difficile A test is 86.7% (13/15), compared to the cytotoxicity method and the  specificity is 91.7% (44/48), compared to the cytotoxicity method.

c) Cross reaction
Keul-o-test Clostridium Difficile A showed consistently negative results up to 500 ng/mL Toxin B.

2. C. difficile TOXIN B

a) Analytical sensitivity
The sensitivity of the test has been evaluated using a range of diluted solutions prepared from a commercially available purified C.D. Toxin B antigen. Under these conditions, the detection limit of the test has been found to be 5 ng/mL.

b) Specificity
A purified C.D.Toxin A antigen was used to determine the specificity of the test.
Keul-o-test Clostridium Difficile B showed consistently negative results up to 500 ng/mL Toxin A.

IX- LIMITATIONS 

1. The Keul-o-test Clostridium Difficile Complete is specifically designed to detect Toxin A or Toxin B antigen in the stool samples.
2. As for any in vitro diagnostic procedure, the physician should confirm the test results with other clinical methods.
3. A negative result does not generally exclude a C.difficile infection. It can be caused by proteolytic digestion of the toxins due to improper specimen storage. If a reasonable suspicion of an infection exists, another stool specimen should be investigated.
4. A positive result does not exclude the presence of other pathogens.
5. The presence of blood in the feces samples in significant quantity may lead to false positive results in limited cases.
6. Test and control lines colours may slightly change depending on the stool sample aspect. For example dark green lines (instead of pink lines) have been reported when assaying greenish or darkish stool samples. This stool coloration appears in case of treatment of iron deficiency with ferrous fumarate. The test result should be interpreted as usual, i.e. two lines for a positive result and one line for a negative result.

X- BIBLIOGRAPHY

1. Lierly, D,M, H.C. Krivan, and T.D. Wilkins. 1988. Clostridium difficile : its disease and toxins. Clin. Microbiol. Rev. 1:1-18.
2. Mulligan, M.E., L.R. Peterson, R.Y.Y. Kwok, C.R. Clabots, and D.N. Gerding. 1988. Immunoblots and plasmid fingerprints compared with serotyping and polyacrylamide gel electrophoresis for typing Clostridium difficile. J. Clin. Microbiol. 26 : 41-31.
3. Gilligan, P.H., L.R. McCarthy, and V.M. Genta. 1981. Relative frequency of Clostridium difficile in patients with diarrheal disease. J. Clin. Microbiol. 14 : 26-31.
4. George, W.L. 1989. Antimicrobial agent-associated diarrhea and colitis, p661-678. In S.M. Finegold and W.L. George (ed.), Anaerobic infections in humans. Academic Press, Inc. New York.
5. Sullivan, N.M., Pellet S., and T.D. Wilkins. 1982. Purification and characterization of toxins A and B of Clostridium difficile. Infect. Immun. 35 : 1032-1040.
6. Lyerly, D.M., D.E. Lockwood, S.H. Richardson, and T.D. Wilkins. 1982. Biological activities of toxins A and B of Clostridium difficile. Infect. Immun. 35 : 1147-1150.

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