← Product Code [LLH](/submissions/MI/subpart-c%E2%80%94microbiology-devices/LLH) · K033479

# PROSPECT CLOSTRIDIUM DIFFICILE TOXIN A/B MICROPLATE ASSAY (K033479)

_Remel, Inc. · LLH · Feb 26, 2004 · Microbiology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/MI/subpart-c%E2%80%94microbiology-devices/LLH/K033479

## Device Facts

- **Applicant:** Remel, Inc.
- **Product Code:** [LLH](/submissions/MI/subpart-c%E2%80%94microbiology-devices/LLH.md)
- **Decision Date:** Feb 26, 2004
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.2660
- **Device Class:** Class 1
- **Review Panel:** Microbiology

## Indications for Use

Remel’s ProSpecT® Clostridium difficile Toxin A/B Microplate Assay is a qualitative enzyme immunoassay (EIA) for the detection of C. difficile Toxin A and B in human fecal specimens from patients suspected of having Clostridium difficile disease. The test is intended for use as an aid in diagnosis of Clostridium difficile associated disease (CDAD).

## Device Story

Qualitative enzyme immunoassay (EIA) for detection of C. difficile Toxin A and B in human fecal specimens; utilizes 96-well microplate coated with mouse anti-Toxin A and rabbit anti-Toxin B antibodies. Stool specimen diluted in sample diluent or modified Cary-Blair medium added to wells; toxins bind to immobilized antibodies. After washing, peroxidase-labeled conjugate (goat anti-Toxin A-HRP and rabbit anti-Toxin B-HRP) added; unbound conjugate washed away. Chromagenic substrate added; stop reagent applied. Results read visually or spectrophotometrically; yellow color indicates presence of toxin. Used in clinical laboratory settings by trained personnel to aid in CDAD diagnosis; output informs clinical decision-making regarding patient treatment for antibiotic-associated diarrhea or pseudomembranous colitis.

## Clinical Evidence

Clinical trial at three US sites compared the device to the cellular cytotoxicity assay (CTA). Dual wavelength (450/620-650 nm) results: sensitivity 90.3% (n=165, 95% CI 84.7-94.4%), specificity 96.2% (n=599, 95% CI 94.3-97.5%). Visual interpretation: sensitivity 85.0% (n=100, 95% CI 76.5-91.4%), specificity 95.5% (n=486, 95% CI 93.2-97.1%). Reproducibility testing (n=288) showed intra-assay CV of 7.7% and inter-assay CV of 18.9%. Analytical sensitivity: >0.20 ng/ml for Toxin A and ≥0.61 ng/ml for Toxin B.

## Technological Characteristics

Enzyme immunoassay (EIA) using microwell strips coated with mouse monoclonal anti-Toxin A and rabbit anti-Toxin B antibodies. Conjugate uses horseradish peroxidase (HRP) labeled antibodies. Detection via chromagenic substrate. Manual or spectrophotometric readout. No electronic connectivity or software algorithms.

## Regulatory 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.

## Predicate Devices

- Meridian Premier Toxins A&B ([K993914](/device/K993914.md))
- Wampole C. difficile TOX A/B II ([K003306](/device/K003306.md)/[K971182](/device/K971182.md))

## Submission Summary (Full Text)

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

A. 510(k) Number:
K033479

B. Analyte:
Clostridium difficile Toxin A & B

C. Type of Test:
Enzyme immunoassay, Qualitative

D. Applicant:
Remel Inc.

E. Proprietary and Established Names:
ProSpect® Clostridium difficile toxin A/B Microplate Assay

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):
Remel’s ProSpecT® Clostridium difficile Toxin A/B Microplate Assay is a qualitative enzyme immunoassay (EIA) for the detection of C. difficile Toxin A and B in human fecal specimens from patients suspected of having Clostridium difficile disease. The test is intended for use as an aid in diagnosis of Clostridium difficile associated disease (CDAD).
2. Indication(s) for use:
Remel’s ProSpecT® Clostridium difficile Toxin A/B Microplate Assay is a qualitative enzyme immunoassay (EIA) for the detection of C. difficile Toxin A and B in human fecal specimens from patients suspected of having Clostridium difficile disease. The test is intended for use as an aid in diagnosis of Clostridium difficile associated disease (CDAD)

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3. Special condition for use statement(s):
The product is for prescription use
4. Special instrument Requirements:
Not applicable

## H. Device Description:

The kit consists of a 96 well microplate coated with mouse anti-Toxin A and rabbit anti-Toxin B; conjugate consisting of peroxidase labeled goat anti-Toxin A and rabbit anti-Toxin 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
Wampole C. difficile TOX A/B II
2. Predicate K number(s):
K993914
K003306/K971182
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  |
|  Differences  |   |   |
|  Item | Device | Predicate (s)  |
|  Capture antibodies or molecules:device | Mouse monoclonal anti-Toxin A and rabbit anti-Toxin B | Wampole: Polyclonal goat antibody against Toxins A and B
Meridian: Mouse monoclonal anti-Toxin A and polyclonal goat anti-Toxin B  |
|  Antibodies: conjugate | Goat anti-Toxin A and rabbit anti-Toxin B | Wampole: Toxin A monoclonal mouse antibody and Toxin B polyclonal goat antibody  |

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|   |  | Meridian: Polyclonal goat anti-Toxin A and anti-Toxin B  |
| --- | --- | --- |
|  Sample volume | 200μl | 50μl  |

## 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 ProSpecT® Clostridium difficile Toxin A/B test detects the presence of Toxin A and Toxin B in clinical stool specimens through the use of specific antibodies. Microwell strips are coated with mouse monoclonal anti-Toxin A and rabbit anti-Toxin B antibodies. 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 goat anti-Toxin A-HRP and rabbit anti-Toxin B-HRP is added to each well. Unbound conjugate is removed by washing and a chromagenic substrate 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 toxin.

## L. Performance Characteristics (if/when applicable):

### 1. Analytical performance:

#### a. Precision/Reproducibility:

Reproducibility testing was conducted at three sites on three separate days with four blinded samples. Each site tested eight replicate wells of each specimen on each day of testing (n=288). The specimens included one negative specimen and three positive specimens with varying levels of reactivity. The average intra-assay coefficient of variation (CV) for a mid-range sample was 7.7%. The average inter-assay CV for a mid-range sample was 18.9%.

#### b. Linearity/assay reportable range:

Not applicable

#### c. Traceability (controls, calibrators, or method):

Not applicable

#### d. Detection limit:

The ProSpecT® Clostridium difficile Toxin A/B Microplate Assay detects Toxin A at levels of ≥0.20 ng/ml and Toxin B at levels of ≥0.61 ng/ml.

#### e. Analytical specificity:

Forty (40) microorganisms were evaluated with the ProSpecT® Clostridium difficile Toxin A/B Microplate Assay. Bacteria and yeast isolates were tested at ≥10⁸ colony-forming units per ml (cfu/ml). Viral isolates were tested at concentrations of 10⁴ TCID₅₀/ml (tissue culture infectious dose per milliliter). No cross-reactivity was observed. There was no cross-reactivity observed with Clostridium sordellii ATCC®

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9714. However, published literature indicates that certain strains of  $C$  sordellii can produce toxins which may be cross-reactive with antibodies to  $C$  difficile Toxins A and B. The following organisms were tested in the ProSpecT® Clostridium difficile Toxin A/B Microplate Assay.

|  Organism | ATCC® designation  |
| --- | --- |
|  Aeromonas hydrophila | 35654  |
|  Bacillus cereus | 11778  |
|  Bacillus subtilis | 6633  |
|  Bacteriodes fragilis | 25285  |
|  Campylobacter coli | 33559  |
|  Campylobacter jejuni | 33291  |
|  Candida albicans | 10231  |
|  Organism | ATCC® designation  |
| --- | --- |
|  Clostridium beijerinckii (butyricum) | 8260  |
|  Clostridium difficile (non-toxigenic) | 700057  |
|  Clostridium haemolyticum | 9650  |
|  Clostridium histolyticum | 19401  |
|  Clostridium novyi (toxin A) | 19402  |
|  Clostridium perfringens (Type A) | 13124  |
|  Clostridium septicum | 12464  |
|  Clostridium sordellii | 9714  |
|  Clostridium sporogenes | 19404  |
|  Clostridium tetani | 19406  |
|  Enterobacter aerogenes | 35028  |
|  Enterobacter cloacae | 13047  |
|  Enterococcus faecalis | 19433  |
|  Escherichia coli | 11229  |
|  Klebsiella pneumoniae | 13882  |
|  Peptostreptococcus anaerobius | 27337  |
|  Porphyromonas asaccharolytica | 25260  |
|  Proteus vulgaris | 49132  |
|  Pseudomonas aeruginosa | 27853  |
|  Salmonella choleraesuis (typhimurium) | 23852  |
|  Serratia liquefaciens | 27592  |
|  Shigella dysenteriae | 11835  |
|  Shigella flexneri | 12022  |
|  Shigella sonnei | 25931  |
|  Staphylococcus aureus | 25923  |
|  Staphylococcus aureus (Cowan) | 12598  |
|  Staphylococcus epidermidis | 12228  |
|  Vibrio cholerae | 9459  |
|  Vibrio parahaemolyticus | 17802  |
|  Yersinia enterocolitica | 23715  |
|  Adenovirus type 40 | VR-930  |

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Adenovirus type 41

VR-931

Rotavirus (Complement fixation antigen)

## Interfering Substances

The following substances were tested with the ProSpecT® Clostridium difficile Toxin A/B Microplate Assay: Vancomycin (12.5 mg/ml), Metronidazole (12.5 mg/ml), blood, mucous, fecal fat, and the following over-the-counter anti-diarrheal products: Pepto-Bismol®, Imodium® A-D, and Kaopectate® (active ingredients: bismuth subsalicylate, loperamide HCl, and attapugite respectively). No interference with positive or negative specimens was observed.

f. Assay cut-off:

The assay was determined to detect Toxin A at levels of ≥ 0.20 ng/ml and Toxin B at ≥ 0.61 ng/ml. Concentrations of purified toxins were assigned from titration results obtained using 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. Toxin concentrations were plotted versus OD values. Then an equation that best represented the curve was used to calculate the concentration at the OD cut off assay value

2. Comparison studies:

a. Method comparison with predicate device: Not applicable
b. Matrix comparison: Not applicable

3. Clinical studies:

a. Clinical sensitivity:

Sensitivity/Specificity

Compared to Tissue Culture Cytotoxicity Assay

Testing was conducted at three laboratories in North America. For all specimens evaluated, the overall sensitivity of the ProSpecT® Clostridium difficile Toxin A/B Microplate Assay when compared to Tissue Culture Cytotoxicity Assay (CTA) was 90.3% (149/165) and the overall specificity when compared to CTA was 96.2% (576/599).

|  OVERALL | CTA Results  |   |   |
| --- | --- | --- | --- |
|   |   |  + | -  |
|  ProSpecT® EIA Results | + | 149 | 23  |
|   |  - | 16 | 576  |
|  Total |   | 165 | 599  |

90.3% Sensitivity (149/165); 95% CI = 84.7% - 94.4%

96.2% Specificity (576/599); 95% CI = 94.3% - 97.5%

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Visual Interpretation of Test

Visual read data was collected at two of the three laboratories for a total of 586 specimens. The overall sensitivity when compared to CTA was 85.0% and the overall specificity was 95.5%. The visual read results were in 99.0% (580/586) agreement with the spectrophotometric results obtained for each specimen.

|   | CTA Results  |   |   |
| --- | --- | --- | --- |
|   |   |  + | -  |
|  ProSpecT® EIA Visual Results | + | 85 | 22  |
|   |  - | 15 | 464  |
|  Total |   | 100 | 486  |

85.0% Sensitivity: (85/100); 95% CI = 76.5% - 91.4%

95.5% Specificity: (464/486); 95% CI = 93.2% - 97.1%

The ProSpecT® Clostridium difficile Toxin A/B Microplate Assay was also compared to two commercially available Enzyme Immunoassays (predicate devices). The performance of the ProSpecT® Clostridium difficile Toxin A/B Microplate Assay and the predicate devices when compared to a CTA (using the same specimens) are as follows:

|   | Performance versus CTA  |   |   |   |
| --- | --- | --- | --- | --- |
|  EIA | Sensitivity |   | Specificity  |   |
|   |  # | % | # | %  |
|  ProSpecT® | 33/40 | 82.5 | 263/268 | 98.1  |
|  Predicate 1 | 33/40 | 82.5 | 260/268 | 97.0  |
|  ProSpecT® | 115/124 | 92.7 | 302/320 | 94.3  |
| --- | --- | --- | --- | --- |
|  Predicate 2 | 98/124 | 79.0 | 309/320 | 96.5  |

b. Clinical specificity:

Refer to (a) above

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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:
Expected values were established from the literature.

C difficile colitis occurs at a much higher frequency in patients who are hospitalized and is the fourth most common nosocomial disease reported to the Centers for Disease Control and Prevention. C. difficile is responsible for 20-30% of antibiotic-associated diarrhea and more than 90% of pseudomembranous colitis. The incidence rate of nosocomial CDAD may vary with hospital populations and is influenced by the presence of predisposing factors, such as increased patient age, type and duration of antimicrobial therapy, severity of underlying illness(es), and length of hospital stay. C. difficile is found in 3-5% of healthy adults and up to 50% of infants and young adults asymptotically carry both the bacteria and its toxins.

M. Conclusion:
In clinical settings, the ProsPecT® Clostridium difficile Toxin A/B Microplate assay is substantially equivalent in performance to the predicate devices for the detection of Clostridium difficile Toxins A & B from human fecal specimens.

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**Source:** [https://fda.innolitics.com/submissions/MI/subpart-c%E2%80%94microbiology-devices/LLH/K033479](https://fda.innolitics.com/submissions/MI/subpart-c%E2%80%94microbiology-devices/LLH/K033479)

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