Clostridium dificile GDH, Toxin A and Toxin B Combo Rapid Test

INTENDED USE

The Clostridium difficile GDH, Toxin A and Toxin B combo Rapid Test is a test for the detection of glutamate dehydrogenase antigen and toxins A and B. The test detects C. difficile antigen, glutamate dehydrogenase, as a screen for the presence of C. difficile, and confirms the presence of toxigenic C. difficile by detecting toxins A and B in stool specimens from people suspected of having C. difficile which is a difficult disease. The test will serve as an adjunct to the diagnosis of clostridium difficile. As with other C. difficile tests, the results should be considered in conjunction with the patient's medical history.

SUMMARY

Clostridium difficile is an anaerobic bacteria acting as an opportunistic pathogen: it grows in the intestine when the normal flora has been altered by treatment with antibiotics.^1,2,3 Toxinogenic strains of Clostridium difficile cause infections from mild-diarrhea to pseudomembranous colitis, potentially leading to death. Disease is caused by two toxins produced by toxinogenic strains of C.difficile: Toxin A (tissue-damaging enterotoxin) and Toxin B (cytotoxin). Some strains produce both toxins A and B, some others produce Toxin B only. The potential role of a third toxin in pathogenicity is still debated. The use of Glutamate Dehydrogenase (GDH) as an antigen marker of C.difficile proliferation has been shown to be very effective because all strains produce high amount of this enzyme.

Therefore, this product includes the detection of Clostridium difficile GDH and toxin A/B, which can detect Clostridium difficile more comprehensively.

TEST PROCEDURE

Bring tests, specimens, and/or controls to room temperature (15-30°C) before use.

1. Remove the test from its sealed pouch, and place it on a clean, level surface. Label the device with patient or control identification. For best results the assay should be performed within one hour.
2. SPECIMEN PREPARATION:

Unscrew the cap of the specimen collection tube, then randomly stab the sampling rod into the stool specimen in at least 3 different sites to collect approximately of feces (equivalent to 1/4 of a pea). Hold the Specimen collection tubes with extraction buffer vertically, insert the sampling rod, squeeze the bottom of the tube. Mix stool specimen with the buffer thoroughly by shaking the bottle for a few seconds.

3. ASSAY PROCEDURE:

• Tighten the cap onto the specimen collection tube, then shake the specimen collection tube vigorously to mix the specimen and the extraction buffer. Leave the tube alone for 2 minutes.
• Remove the small lid at the top.
• Hold the bottle in a vertical position over the sample well of the test card, deliver 3 drops(about 90μl) of diluted stool sample to the sample well (S) and start the timer.

Note: Avoid trapping air bubbles in the specimen well (S), and do not add any solution to the result area.

As the test begins to work, color will migrate across the result area in the center of the device.

• Wait for the colored band(s) to appear. Read the result between 5-10 minutes. A strong positive sample may show result earlier. Do not interpret the result after 15As the test begins to work, color will migrate across the result area in the center of the device

INTERPRETATION OF RESULTS

Clostridium dificile GDH

• Positive (+) : Two purple redbands appear. One is located in the detection area (T), the other is located in the quality control area (C). 

Note: The purple red band in the detection area (T) can show the phenomenon of dark and light color. However, during the specified observation time, regardless of the color of the band, even very weak band should be interpreted as a positive result.

• Negative (-) : Only a purple redband appears in the quality control area (C). No purple red bands were found in the detection area (T). A negative result indicates no Clostridium difficile
• Invalid:No purple red band in quality control area (C). Indicates incorrect operation or deterioration of the test. In this case, read the instructions carefully again and retest with a new test. If the problem persists, you should immediately stop using the batch number and contact your local supplier.

Clostridium difficile Toxin A and Toxin B

• Toxin APositive : The colored line in the control line region (C) appears, and a colored line appears in test line region T1. The result is positive for Clostridium difficile Toxin A and is indicative of Clostridium difficile 
• Toxin B Positive: The colored line in the control line region (C) appears, and a colored line appears in test line region T2. The result is positive for Clostridium difficile Toxin Band is indicative of Clostridium difficile infection.
• Toxin A&B Positive:The colored line in the control line region (C) appears, and two colored lines should appear in test line regions T1and T2. The color intensities of the lines do not have to match. The result is positive for Toxin A & Toxin B and is indicative of Clostridium difficile 
• Negative (-) : Only a purple redband appears in the quality control area (C). No purple red bands were found in the detection area (T1) and (T2). A negative result indicates no Clostridium difficile
• Invalid:No purple red band in quality control area (C). Indicates incorrect operation or deterioration of the test. In this case, read the instructions carefully again and retest with a new test. If the problem persists, you should immediately stop using the batch number and contact your local supplier.

• PERFORMANCE CHARACTERISTICS

  Clinical Sensitivity, Specificity and Accuracy

  The Clostridium difficile GDH, Toxin A and Toxin B combo Rapid Test used PCR as the reference method. The results showed that the Clostridium difficile GDH, Toxin A and Toxin B combo Rapid Test had high sensitivity and specificity.

  Table: Clostridium difficile GDH Antigen Rapid Test vs. RT-PCR  

  Method		RT-PCR		Total Results
  Clostridium difficile GDH Antigen Rapid Test	Results	Positive	Negative
  	Positive	120	3	123
  	Negative	6	312	318
  Total Result		126	315	441

  Relative Sensitivity: 95.24% (95%CI: 89.92%～98.23%)

  Relative Specificity: 99.05% (95%CI: 97.24%～99.80%) 

  Accuracy: 97.96% (95%CI: 96.16%～99.06%)

  Table: The Clostridium toxin A Antigen Rapid Test vs. RT-PCR  

  Method		RT-PCR		Total Results
  Clostridium toxin A Antigen Rapid Test	Results	Positive	Negative
  	Positive	111	5	116
  	Negative	9	109	118
  Total Result		120	114	234

  Relative Sensitivity: 92.50% (95%CI: 86.24%～96.51%)

  Relative Specificity: 95.61% (95%CI: 90.06%～98.56%) 

  Accuracy: 94.02% (95%CI: 90.17%～96.69%)

  Table: The Clostridium toxin B Antigen Rapid Test vs. RT-PCR  

  Method		RT-PCR		Total Results
  Clostridium toxin B Antigen Rapid Test	Results	Positive	Negative
  	Positive	99	4	103
  	Negative	4	114	118
  Total Result		103	118	221

  Relative Sensitivity: 96.12% (95%CI: 90.35%～98.93%)

  Relative Specificity: 96.61% (95%CI: 91.55%～99.07%) 

  Accuracy: 96.38% (95%CI: 87.43%～95.10%)

  Cross-Reactivity

  An evaluation was performed to determine the cross reactivity of Clostridium difficile GDH, Toxin A and Toxin B combo Rapid Test. No cross reactivity against gastrointestinal pathogens occasionally present as following:

   

  Campylobacter coli	Shigella dysenteriae	Salmonella enteritidis
  Campylobacter jejuni	Shigella flexneri	Salmonella paratyphi
  E.coli O157:H7	Shigella sonnei	Salmonella typhi
  H.pylori	Salmonella typhimurium	Staphylococcus aureus
  Listeria monocytogenes	Shigella boydii	Yersinia enterocolitica

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