C. DIFFICILE TOXIN A + B FECAL ANTIGEN DETECTION MICROWELL ELISA KIT MODEL; CDIFF-96

K032897 · Ivd Research, Inc. · LLH · May 27, 2004 · Microbiology

Device Facts

• Record ID: K032897
• Device Name: C. DIFFICILE TOXIN A + B FECAL ANTIGEN DETECTION MICROWELL ELISA KIT MODEL; CDIFF-96
• Applicant: Ivd Research, Inc.
• Product Code: LLH · Microbiology
• Decision Date: May 27, 2004
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.2660
• Device Class: Class 1

Indications for Use

This microwell enzyme-linked immunoabsorbant assay (ELISA) detection kit (C. difficile Toxin A+B ELISA Kit) is an in vitro diagnostic (IVD) immunoassay intended for use as an aid in the diagnosis of C. difficile associated disease. The kit detects C. difficile toxin A and B in human feces using peroxidase as the indicator enzyme. The assay may be read visually or with an ELISA reader. This IVD C. difficile Toxin A+B ELISA Kit is intended to be used with human stools that are fresh, frozen or in Cary Blair transport media in a clinical laboratory use setting. The kit may also be used with IVD Research's Quick'N'Easy fecal dilution device.

Device Story

The C. difficile Toxin A+B Fecal Antigen Detection Microwell ELISA is an in vitro diagnostic immunoassay; detects Clostridium difficile toxins A and B in human fecal specimens. Principle of operation: microwell ELISA using peroxidase as an indicator enzyme. Input: human stool samples (fresh, frozen, or in Cary Blair transport media); optional use with Quick'N'Easy fecal dilution device. Output: visual color change or absorbance reading via ELISA reader. Used in clinical laboratory settings by laboratory personnel. Results aid clinicians in diagnosing C. difficile associated disease; facilitates timely patient management and treatment decisions.

Clinical Evidence

No clinical data provided in the document; summary focuses on regulatory clearance for an IVD immunoassay.

Technological Characteristics

Microwell ELISA format; rabbit polyclonal anti-Toxin A+B capture antibodies; peroxidase-labeled chicken anti-Toxin A+B conjugate; TMB chromogenic substrate. Detects Toxin A (≥2.0 ng/ml) and Toxin B (≥3.0 ng/ml). Manual or spectrophotometric readout. No specific material standards or connectivity features reported.

Indications for Use

Indicated for use as an aid in the diagnosis of C. difficile associated disease in patients providing human fecal samples (fresh, frozen, or in Cary Blair media). For professional use in clinical laboratory settings.

Regulatory Classification

Identification

A microorganism differentiation and identification device is a device intended for medical purposes that consists of one or more components, such as differential culture media, biochemical reagents, and paper discs or paper strips impregnated with test reagents, that are usually contained in individual compartments and used to differentiate and identify selected microorganisms. The device aids in the diagnosis of disease.

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
DEVICE ONLY TEMPLATE

A. 510(k) Number:
K032897

B. Analyte:
Clostridium difficile Toxin A & B

C. Type of Test:
Enzyme immunoassay

D. Applicant:
IVD Research Inc.

E. Proprietary and Established Names:
Clostridium difficile toxin A+B Fecal Antigen Detection Microwell ELISA

F. Regulatory Information:
1. Regulation section:
21 CFR Part 866.2660 Microorganism Differentiation and Identification Device
2. Classification:
Class I
3. Product Code:
LLH – Reagents, Clostridium difficile toxin
4. Panel:
83 (Microbiology)

G. Intended Use:
1. Intended use(s):
This microwell enzyme linked immunoabsorbant assay (ELISA) detection kit is an in vitro diagnostic (IVD) immunoassay for the detection of antigen to C. difficile A and B toxins in human feces using peroxidase as the indicator enzyme. The assay may be read visually or with and ELISA reader. This ELISA Kit is intended to be used with stools that are fresh, frozen or in Cary-Blair transport media. The assay is intended for use as an aid in diagnosis of C. difficile associated disease.

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2. **Indication(s) for use:**
This microwell enzyme-linked immunoabsorbant assay (ELISA) detection kit (C. difficile Toxin A+B ELISA Kit) is an in vitro diagnostic (IVD) immunoassay intended for use as an aid in the diagnosis of *C. difficile* associated disease. The kit detects *C. difficile* toxin A and B in human feces using peroxidase as the indicator enzyme. The assay may be read visually or with an ELISA reader. This IVD C. difficile Toxin A+B ELISA Kit is intended to be used with human stools that are fresh, frozen or in Cary Blair transport media in a clinical laboratory use setting. The kit may also be used with IVD Research's Quick'N'Easy fecal dilution device.

3. **Special condition for use statement(s):**
Prescription Use

4. **Special instrument Requirements:**
Not applicable

**H. Device Description:**
The kit consists of 96 test wells coated with rabbit anti-*C. difficile* Toxin A and B; conjugate consisting of peroxidase labeled chicken anti-Toxin A and B; positive and negative controls; sample diluent; wash buffer; color substrate, stop solution; transfer pipettes, procedure card; instructions for use and a plate cover.

**I. Substantial Equivalence Information:**

1. **Predicate device name(s):**
Meridian Premier Toxins A&B
Biostar *C. difficile* TOX A OIA

2. **Predicate K number(s):**
K926442
K991829

3. **Comparison with predicate:**

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate (s) |
| Intended Use | Detection of C. difficile Toxins A and B in fecal specimens | Detection of C. difficile Toxins A and B in fecal specimens |
| Technology | Enzyme immunoassay | Enzyme immunoassay |
| Material : device | Microwell | Microwell |
| Material: conjugate | Horseradish peroxidase conjugated to anti-toxins | Horse radish peroxidase conjugated to anti-toxins |
| Specimen type | Fresh human stool specimens or specimens in modified Cary-Blair | Fresh human stool specimens |

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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate (s) |
| Capture antibodies or molecules:device | chicken polyclonal anti-Toxin A+B and rabbit polyclonal anti-Toxin A +B | Biostar: rabbit antibody against Toxin A
Meridian: Mouse monoclonal anti-Toxin A and polyclonal goat anti-Toxin B |
| Antibodies: conjugate | Chicken polyclonal anti-Toxin A and B | Biostar: rabbit Toxin A antibody
Meridian: Polyclonal goat anti-Toxin A and anti-Toxin B |
| Sample volume | 100μl | same |

J. Standard/Guidance Document Referenced (if applicable):
CDRH Guidance Document for Industry and FDA Staff: “Review Criteria for assessment of laboratory tests directed at assisting in the diagnosis of C.difficile associated disease”

K. Test Principle:
The Clostridium difficile toxin A+B Fecal Antigen Detection Microwell ELISA test detects the presence of Toxin A and Toxin B in clinical stool specimens. The microwells are coated with rabbit polyclonal anti-Toxins A+B. A stool specimen is diluted in Sample Diluent or used directly if pre-diluted in modified Cary-Blair medium. The sample is added to a microwell allowing the toxins, if present, to bind to the immobilized antibodies. After washing to remove unbound components, a conjugate reagent containing anti-chicken polyclonal antibodies conjugated to peroxidase is added to each well. Unbound conjugate is removed by washing and a chromogenic substrate, tetramethylbenzidine (TMB) solution, is added to detect the presence of bound toxin. A stop reagent is added and the test results are read visually or spectrophotometrically. The presence of a yellow color indicates the presence of antigen to C. difficile A + B toxins.

L. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Reproducibility testing was conducted at three sites on three consecutive days with eleven blinded samples. The specimens included two negative specimens and nine positive specimens with varying levels of reactivity. The average inter-assay coefficient of variation (CV) range for the negative samples was .49-.62. The average inter-assay CV range for the positive samples was 0.2-.17.
b. Linearity/assay reportable range:
Not applicable
c. Traceability (controls, calibrators, or method):
Not applicable

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d. Detection limit:
The Clostridium difficile toxin A+B Fecal Antigen Detection
Microwell ELISA test kit detects Toxin A at levels of ≥2.0 ng/ml and Toxin B at levels of ≥3.0 ng/ml.

e. Analytical specificity:
Thirty (30) microorganisms were evaluated with the Clostridium difficile toxin A+B Fecal Antigen Detection assay. Bacteria isolates were tested at ≥10⁸ colony-forming units per ml (cfu/ml). No cross-reactivity was observed with all isolates except Clostridium sordellii. The following organisms were tested in the Clostridium difficile Toxin A+B Microwell assay.

Organism
Bacteriodes fragilis
Campylobacter coli
Campylobacter jejuni
Campylobacter fetus
Candida albicans

Organism
Clostridium haemolyticum
Clostridium perfringens
Clostridium septicum
Clostridium sordelleii
Clostridium sporogenes
Clostridium novyi
Citrobacter braakii
Enterobacter cloacae
Enterococcus faecalis
Escherichia coli
Escherichia hermanii
Helicobacter cinaedi
Klebsiella pneumoniae
Proteus vulgaris
Pseudomonas aeruginosa
Salmonella choleraesuis (typhimurium)
Salmonella hadar
Salmonella infantis
Salmonella enteritidis
Serratia liquefaciens
Shigella dysenteriae
Shigella flexneri
Shigella sonnei
Staphylococcus aureus
Yersinia enterocolitica

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f. Assay cut-off:
The assay was determined to detect Toxin A at levels of ≥ 2.0 ng/ml and Toxin B at ≥ 3.0 ng/ml. Concentrations of purified toxins were assigned from serial dilution results obtained through either gold standard testing or by testing in a predicate device. Concentration of toxin testing close to the assay cut off was determined. The toxins were also titrated beyond the assay cut off on the test device. The last dilution remaining at or above the assay cut off was defined as the endpoint dilution. The highest negative and the lowest positive sample toxin concentrations were plotted versus OD values were used to calculate the concentration at the OD cut off assay value.

2. Comparison studies:
a. Method comparison with predicate device:
See below studies 3, 4 (Part B), and 5.
b. Matrix comparison:
Not applicable

3. Clinical studies:
a. Clinical sensitivity:
Sensitivity/Specificity

Study #1 (Toxin B Cell Culture)
A total of 69 stools were tested against a cytotoxin B cell culture procedure. The following results were obtained.

| | Cyto B + | Cyto B - |
| --- | --- | --- |
| IVD
ELISA + | 14 | 2 |
| IVD
ELISA - | 4 | 49 |

Sensitivity: 78% (14/18)
Specificity: 96% (49/51)
95% CI = 52% to 94%
95% CI = 87% to 100%

Study #2 (In House Study)
A total of 24 stools in Cary Blair transport media (5 positive, 19 negative) were tested in this ELISA. In addition, 14 fresh/frozen stools (6 positive, 8 negative) were diluted using the Quick'N'Easy Fecal Sample Prep device and tested in the ELISA. The following results were obtained.

| | ELISA/OIA + | ELISA/OIA - |
| --- | --- | --- |
| IVD
ELISA + | 11 | 0 |
| IVD
ELISA - | 0 | 27 |

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Positive Agreement = 100% (11/11)
Negative Agreement = 100% (27/27)

## Study #3 (Mid-West Clinical Lab)

This study compared 53 fresh samples versus another commercial A+B ELISA. The IVD ELISA had a positive agreement of 100% (2/2) and a negative agreement of 98% (50/51).

## Study #4 (Reference #17)

A total of 311 stools were tested against culture and another commercial toxin A+B ELISA kit.

The following results were obtained.

| | Culture + | Culture – |
| --- | --- | --- |
| IVD ELISA + | 49 | 8 |
| IVD ELISA - | 33 | 221 |

Sensitivity: 60% (49/82)
Specificity: 97% (221/229)
95% CI = 48% to 70%
95% CI = 93% to 99%

The other commercial ELISA showed a sensitivity of 66% (54/82) and a specificity of 98% (225/229) on the same set of samples.

Between the two ELISA’s there was a positive agreement of 91% (49/54) and a negative agreement of 98% (221/225).

## Study #5 (East Coast Hospital Lab)

This study compared 82 fresh or frozen samples versus another commercial Toxin A Only Optical Immunoassay (OIA). The IVD ELISA had a positive agreement of 79% (27/34) and a negative agreement of 94% (45/48).

b. Clinical specificity:
Refer to (a) above

c. Other clinical supportive data (when a and b are not applicable):
Not applicable

4. Clinical cut-off:
See assay cut-off above

5. Expected values/Reference range:

A positive reaction indicates that the patient is shedding detectable amounts of C. difficile antigen. The frequency of C. difficile disease is dependent on various factors such as the type of institution, patient population and potential outbreak status. Asymptomatic carrier rates have been reported from a low of 2% in Sweden to a high of 15% in Japan.

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Hospitalized patients taking certain antibiotics are at high risk of acquiring C. difficile with infection rates of 21% being reported in one study. A recent article in Journal of Clinical Microbiology (ref. #18) provides a good overview of testing for C. difficile. Further information on C. difficile and antibiotic colitis can also be found in the Manual of Clinical Microbiology, ASM Press, 7th Edition.

**M. Conclusion:**

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.