ILLUMIGENE C. DIFFICILE, ILLUMIPRO-10 AND ILLUMIGENE C. DIFFICILE EXTERNAL CONTROL KIT, MODELS 280050, 610172, 279920

K100818 · Meridian Bioscience, Inc. · OMN · Jul 9, 2010 · Microbiology

Device Facts

• Record ID: K100818
• Device Name: ILLUMIGENE C. DIFFICILE, ILLUMIPRO-10 AND ILLUMIGENE C. DIFFICILE EXTERNAL CONTROL KIT, MODELS 280050, 610172, 279920
• Applicant: Meridian Bioscience, Inc.
• Product Code: OMN · Microbiology
• Decision Date: Jul 9, 2010
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.2660
• Device Class: Class 1
• Attributes: Pediatric

Indications for Use

The illumigene C. difficile DNA amplification assay, performed on the illumipro-10, is a qualitative in vitro diagnostic test for the direct detection of toxigenic C. difficile in human stool specimens suspected of having Clostridium difficile associated disease (CDAD). The illumigene C. difficile assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile PaLoc is a gene segment present in all known toxigenic C. difficile strains. The C. difficile PaLoc codes for both the Toxin A gene (tcdA) and the Toxin B gene (tcdB), has conserved border regions, and is found at the same site on the C. difficile genome for all toxigenic strains . The illumigene C. difficile assay detects the PaLoc by targeting a partial DNA fragment on the Toxin A gene. The tcdA target region was selected as an intact region remaining in all known A+B+ and A-B+ toxinotypes. illumigene C. difficile is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

Device Story

The illumigene C. difficile system is a molecular diagnostic test for detecting toxigenic C. difficile in human stool. The system consists of the illumigene DNA amplification test kit and the illumipro-10 automated isothermal amplification and detection system. Samples are prepared manually using a collection brush and extraction apparatus; target DNA is heat-extracted. The illumipro-10 performs loop-mediated isothermal amplification (LAMP) on the prepared sample. During amplification, Magnesium pyrophosphate precipitates; the system detects this via changes in light transmission. Results are reported as positive or negative. The device is operated by laboratory personnel in hospital or reference settings. The output provides a qualitative diagnostic result to assist clinicians in identifying CDAD, enabling timely patient management and treatment decisions.

Clinical Evidence

Clinical trial conducted at four independent sites with 697 patient samples compared to cytotoxic bacterial culture. Overall sensitivity was 95.2% (95% CI: 89.2% - 97.9%) and overall specificity was 95.3% (95% CI: 93.2% - 96.7%). No performance differences were observed based on patient age, sex, or geography.

Technological Characteristics

Uses loop-mediated isothermal amplification (LAMP) technology. Instrumentation: illumipro-10 automated system. Detection method: visible light transmission measurement of Magnesium pyrophosphate precipitation. Components: sample preparation apparatus, reaction buffer, test device, extraction tubes, and collection brushes. Qualitative diagnostic test.

Indications for Use

Indicated for qualitative in vitro detection of toxigenic C. difficile in human stool specimens from patients suspected of having Clostridium difficile-associated disease (CDAD). Intended for use in hospital, reference, or state laboratory settings; not for point-of-care use.

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.

Predicate Devices

• Cepheid® Xpert® C. difficile (K091109)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT COMBINATION TEMPLATE

A. 510(k) Number:
K100818

B. Purpose for Submission:
To obtain substantial equivalence for the Illumigene C. difficile assay

C. Measurand:
C. difficile toxin A gene

D. Type of Test:
Loop mediated isothermal amplification

E. Applicant:
Meridian Bioscience Inc.

F. Proprietary and Established Names:
Illumigene C. difficile Assay

G. Regulatory Information:
1. Regulation section:
21 CFR 866.2660 – Microorganism differentiation and identification device
2. Classification:
Class 1
3. Product code:
OMN – C. difficile nucleic acids

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4. Panel:

83 - Microbiology

H. Intended Use:

1. Intended use:

The illumigene C. difficile DNA amplification assay, performed on the illumipro-10, is a qualitative in vitro diagnostic test for the direct detection of toxigenic C. difficile in human stool specimens from patients suspected of having Clostridium difficile-associated disease (CDAD).

The illumigene C. difficile assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile. The Clostridium difficile PaLoc is a gene segment present in all known toxigenic C. difficile strains. The C. difficile PaLoc codes for both the Toxin A gene (tcdA) and the Toxin B gene (tcdB), has conserved border regions, and is found at the same site on the C. difficile genome for all toxigenic strains. The illumigene C. difficile assay detects the PaLoc by targeting a partial DNA fragment on the Toxin A gene. The tcdA target region was selected as an intact region remaining in all known A+/B+ and A-/B+ toxinotypes.

illumigene C. difficile is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

2. Indication(s) for use:

The illumigene C. difficile DNA amplification assay, performed on the illumipro-10, is a qualitative in vitro diagnostic test for the direct detection of toxigenic C. difficile in human stool specimens from patients suspected of having Clostridium difficile-associated disease (CDAD).

The illumigene C. difficile assay utilizes loop-mediated isothermal amplification (LAMP) technology to detect the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile. The Clostridium difficile PaLoc is a gene segment present in all known toxigenic C. difficile strains. The C. difficile PaLoc codes for both the Toxin A gene (tcdA) and the Toxin B gene (tcdB), has conserved border regions, and is found at the same site on the C. difficile genome for all toxigenic strains. The illumigene C. difficile assay detects the PaLoc by targeting a partial DNA fragment on the Toxin A gene. The tcdA target region was selected as an intact region remaining in all known A+/B+ and A-/B+ toxinotypes.

illumigene C. difficile is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

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3. Special conditions for use statement:

For professional use

4. Special instrument requirements:

Illumipro -10 Automated Isothermal Amplification and Detection System

I. Device Description:

The illumigene Molecular Diagnostic Test System is comprised of the illumigene C. difficile DNA Amplification Test Kit, the illumigene C. difficile External Control Kit and the illumipro-10 Automated Isothermal Amplification and Detection System. Additional specialized laboratory equipment is not required for execution of illumigene Molecular Diagnostic Test System assays.

The illumigene C. difficile DNA amplification assay utilizes loop-mediated isothermal amplification (LAMP) technology to detect the presence of toxigenic C. difficile in patients suspected of having C. difficile associated disease (CDAD). Each illumigene C. difficile assay is completed using materials provided: illumigene Sample Preparation Apparatus, illumigene Reaction Buffer, illumigene C. difficile Test Device, Sample Collection Brush, and illumigene Extraction Tube. Samples are prepared using the Sample Collection Brush and the illumigene Sample Collection Apparatus, target DNA is heat extracted in the Extraction Tube and DNA amplification occurs in the illumigene C. difficile Test Device.

The illumipro-10 heats each illumigene C. difficile Test Device containing prepared samples, facilitating amplification of target DNA. When toxigenic C. difficile is present in the patient sample, a cytotoxin specific sequence is amplified and magnesium pyrophosphate is formed. As the LAMP reaction progresses, magnesium pyrophosphate forms a precipitate in the reaction mixture. The optics system of the illumipro-10 detects the change in light transmission through the reaction mixture created by the precipitating Magnesium pyrophosphate. Sample results are reported as Positive or Negative based on the detected change in transmission.

The illumigene C. difficile External Control Kit consists of a Positive Control Reagent and a Negative Control Reagent. External Control reagents are provided to aid the user in detection of reagent deterioration, adverse environmental or test conditions, or variance in operator performance that may lead to test errors. The illumigene C. difficile External Control Kit is required for routine Quality Control.

Final Component Summary

illumigene C. difficile DNA Amplification Assay

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- illumigene C. difficile Sample Preparation Apparatus: Sampling unit consisting of sample preparation chamber, dropper tip, cap and Sample Dilution Buffer (Phosphate Buffered Saline and formalin treated Staphylococcus aureus, with sodium azide (0.09%) as a preservative)

- illumigene Reaction Buffer: Tris-buffered solution containing sodium azide (0.09%) as a preservative

- illumigene C. difficile Test Device: Two separate chambers containing dry reagent lyospheres comprised of DNA polymerase, Deoxyribonucleoside Triphosphate (dNTPs), and either C. difficile specific primers (TEST Chamber) or S. aureus primers (CONTROL Chamber). The Control lyosphere contains quinoline yellow dye to differentiate it from the Test lyosphere

- Sample Collection Brushes: Tube cap fitted with stainless steel rod and nylon bristles

- illumigene Extraction Tubes: 1.5 mL polypropylene microcentrifuge tubes with caps; RNase, DNase and pyrogen-free

- illumigene C. difficile External Control Kit

- illumigene Positive Control: Tris-buffered solution containing non-infectious Plasmid DNA (S. aureus and C. difficile inserts) with azide (0.09%) as a preservative

- illumigene Negative Control: Tris-buffered solution containing non-infectious Plasmid DNA (S. aureus insert) with azide (0.09%) as a preservative

J. Substantial Equivalence Information:

1. Predicate device name: Cepheid Xpert C.difficile
2. Predicate 510(k) number: K091109
3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Sample matrix | Uniformed unpreserved stool samples | Same |
| Assay type | DNA amplification | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Assay targets a conserved DNA fragment on the toxin A (tcdA) gene in the pathogenicity locus of toxigenic C. difficile. | Assay targets the toxin B (tcdB) gene |
| Instrument | Assay uses the Illumipro-10 instrument | Assay uses the Cepheid GeneXpert Dx System |

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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Detection method | Assay uses loop mediated isothermal amplification (LAMP) technology and detects changes in turbidity by visible light transmission | Assay uses automated real time polymerase chain reaction (PCR) and detects fluorescence |
| Transport media | Sample in Cary Blair media is acceptable | Not for use with Cary Blair media |
| Extraction method | Manual | Self contained, automated |
| | | |

**K. Standard/Guidance Document Referenced (if applicable):**

| User Protocol for Evaluation of Qualitative Test Performance (EP 12-A2) |
| --- |
| User Verification of Performance for Precision and Trueness (IVD) (EP 15-A2) |
| Guidance for Industry and FDA Staff: Class II Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus RNA |
| Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices - Guidance for Industry and FDA Staff |
| Draft Guidance for Industry and FDA Staff: Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses |
| General Principles of Software Validation; Final Guidance for Industry and FDA Staff |

**L. Test Principle:**

The *illumigene C. difficile* Assay is based on loop-mediated isothermal amplification technology (LAMP). Loop-mediated amplification is accomplished by the use of specially designed primers that provide specific and continuous isothermal DNA amplification. Magnesium-pyrophosphate is produced as a by-product of LAMP amplification. The magnesium-pyrophosphate forms a white precipitate in the reaction solution, giving the reaction solution a turbid appearance. The presence of turbidity signifies a positive reaction while the absence of turbidity represents a negative reaction.

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# The Toxigenic C. difficile Pathogen Locus (PaLoc) and illumigene C. difficile Primer Regions:

The illumigene C. difficile assay contains primers that specifically amplify an ~204 base pair (bp) sequence within the 5' region of the toxin A (tcdA) gene that is part of the cytotoxin pathogen locus found in toxigenic C. difficile. This region is found in all cytotoxin positive (A+/B+ and A-/B+) strains characterized and is not present in cytotoxin negative strains (A-/B-).

The illumigene C. difficile assay detects the presence of toxigenic C. difficile in stool samples from patients suspected of having Clostridium difficile associated disease in the following manner:

Patient stool sample is collected using the sample brush portion of the illumigene Sample Collection Apparatus. The sample is added to the Sample Collection Apparatus containing Sample Dilution Buffer and formalin treated Staphylococcus aureus. S. aureus serves as an internal control for inhibition and system suitability.

The diluted sample is mixed, filtered through the Sample Collection Apparatus and dispensed drop-wise into an illumigene Extraction Tube. The Extraction Tube is heated to facilitate DNA extraction of the sample and the internal control material.

Extracted DNA is added to an illumigene Reaction Buffer tube, mixed and added to the illumigene C. difficile Test Device. The illumigene Test Device consists of two chambers, one for the sample (TEST) and one for the internal assay control (CONTROL). The TEST chamber contains a lyosphere comprised of DisplaceAce™ DNA polymerase, dNTPs, Bovine Serum Albumin, and illumigene C. difficile primers. The CONTROL chamber contains a lyosphere comprised of DisplaceAce™ DNA polymerase, dNTPs, Bovine Serum Albumin, yellow dye and illumigene Staphylococcus aureus internal control primers.

The Test Device is placed in the illumipro-10™ for amplification and detection. Amplification occurs when the illumigene C. difficile Test Device is incubated at 63 C. When toxigenic C. difficile is present in the patient sample, the cytotoxin specific sequence is amplified. Magnesium pyrophosphate, a by-product of DNA amplification, will reach saturation and precipitate producing a turbid solution in the TEST chamber of the Test Device. When sample preparation and the assay procedure have has been performed successfully and inhibition has not occurred, DNA from the internal control will amplify. Magnesium pyrophosphate will precipitate and form a turbid solution in the CONTROL chamber of the test device. Assay reactions take place in approximately 40 minutes.

Light transmission (650 ± 20 nm) through the TEST and CONTROL solutions is measured by the illumipro-10 at the on-set of the assay run and at completion. The illumipro-10 calculates the ratio between the initial absorbance reading and the final absorbance reading and compares the ratio to an established cut-off value. Valid test results will be reported for a change in absorbance above 90% in the CONTROL chamber. Positive test results will be reported for a change in absorbance above 90% in the TEST chamber and the CONTROL

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chamber.

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

Reproducibility studies were performed by three clinical sites using coded panels. The panels consisted of moderately positive (n=2), low positive (n=3), high negative (n=3) and negative (n=2) samples. The moderately positive and low positive samples were contrived from negative specimens spiked with toxinogenic C. difficile VPI 10463 to just above the LoD for the strain (weak positive) or below the limit of blank (high negative sample), as determined by replicate testing during manufacture of the samples. Negative samples were natural samples. Three lots of illumigene C. difficile and nine illumi pro-10 instruments were used in reproducibility studies. Positive and Negative Controls were tested with each panel. Each clinical site tested two panels each day for five days. At least two operators each day at each facility performed the testing to demonstrate reproducibility. Reproducibility of the assay is 100% for moderate positive, low positive and negative samples. Reproducibility is 91% for high negative samples.

Positive samples were contrived from negative specimens. The moderately positive and low positive samples were contrived from negative specimens spiked with toxinogenic C. difficile VPI 10463 to just above the LoD for the strain (weak positive) or below the limit of blank (high negative sample), as determined by replicate testing during manufacture of the samples. Negative samples were natural samples. Final concentrations of C. difficile in reproducibility panel samples were as follows: Moderate Positive: 200 CFU/test (Positive) Low Positive: 64 CFU/test (Positive) High Negative: 0.5 CFU/test (Negative)

The sources of variability examined in the study included operators (minimum 2 at each site), laboratories (3 sites), runs (Panels tested twice per day), test date (panels tested for five days), and illumi pro-10 instruments (9 instruments).

Precision (within-run and within-laboratory) was calculated following CLSI EP 15-A2. 95% confidence intervals were calculated using the Wilson method.

Reproducibility of the assay is 100% for moderate positive, low positive and negative samples. Reproducibility is 91% for high negative samples. Sample type and replicate data is as follows: Percent Agreement Positive Samples: 60/60 Replicates or 100% Percent Agreement Low Positive: 90/90 Replicates or 100% Percent Agreement High Negative: 82/90 Replicates or 91% Percent Agreement Negative: 59/59 Replicates or 100% (One negative sample produced an invalid result during testing. This value was not included in the calculations.)

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b. Linearity/assay reportable range:

N/A

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

Internal control is provided in the Sample Preparation Apparatus and the Assay Device. The Sample Preparation Apparatus contains Sample Dilution Buffer with formalin treated Staphylococcus aureus. The CONTROL Chamber of the Assay Device contains a lyosphere with S. aureus primers. The internal control has the same reactants to that of the test chamber except for the primer composition and a yellow dye. Internal Control components are intended to monitor sample preparation, polymerase inhibition, system suitability and reagent integrity.

illumigene External Control Reagents will be sold separately as adjunct reagents to the illumigene C. difficile Assay. Each External Control kit contains a Negative Control reagent and a Positive Control reagent. External Controls aid the user in detection of reagent deterioration, adverse environmental or test conditions or variance in operator performance that can cause test errors. External Controls are required for routine Quality Control.

Positive Control: Tris-buffered solution containing non-infectious Plasmid DNA (S. aureus and C. difficile inserts) at 200.0 fg/μL with sodium azide (0.09%) as a preservative.

Negative Control: Tris-buffered solution containing non-infectious Plasmid DNA (S. aureus insert) at 200.0 fg/μL with sodium azide (0.09%) as a preservative.

d. Detection limit:

Sensitivity studies were designed to determine, within 95% confidence intervals, the analytical limit of detection (LoD) of C. difficile diluted in a human stool matrix. The LoD is the lowest number of colony-forming units (CFUs) per test aliquot that can be distinguished from Native samples with a high degree of probability.

Seven different strains representing five toxinotypes of C. difficile were evaluated. Each bacterial strain was spiked into negative stool and then diluted serially. Twenty replicates of each dilution were individually processed and tested to give a stated probability of 95% for the LoD dilution. The results obtained with each individual strain are given in Tables 2 – 5 on the following pages. The LoD point estimates and 95% upper and lower confidence intervals for each toxinotype are summarized in Table 1 below.

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Table 1. 95% confidence intervals for illumigene C. difficile analytical LoD.

| Strain ID | Toxinotype | Phenotype | LoD/Test | Lower Limit (95% CI) | Upper Limit (95% CI) |
| --- | --- | --- | --- | --- | --- |
| VPI 10463 | 0 | A+/B+ | 4 CFU/test | 83.9% | 100% |
| 2007431 | III (NAP1) | A+/B+ | 32 CFU/test | 83.9% | 100% |
| BI8 | III | A+/B+ | 64 CFU/test | 76.4% | 99.1% |
| 2006240 | V (NAP7) | A+/B+ | 32 CFU/test | 83.9% | 100% |
| CF1 | VIII | A-/B+ | 64 CFU/test | 83.9% | 100% |
| 2007858 | IX/XXIII | A+/B+ | 32 CFU/test | 83.9% | 100% |
| 8864 | X | A-/B+ | 64 CFU/test | 83.9% | 100% |

e. Analytical specificity:

Interference Studies:

Potentially interfering substances were added to a natural negative and a contrived positive sample at final concentrations of 5% v/v or greater. The contrived positive samples were prepared from a pool of donor stools that were confirmed negative by toxinogenic culture. The sample was inoculated with C. difficile VPI 10463 to just above the limit of detection (18 CFU/test) for this organism.

Potentially interfering substances were added at final concentrations of 5% V/V or greater. Dilution Controls for each sample were prepared by adding a phosphate-buffered saline solution in place of the potentially interfering substance. Each sample was tested in triplicate. The following substances, at the specified saturated solvent/diluents concentrations, were included in the study: Barium sulfate (5 mg/mL), fecal fat (equivalent to 2.65 mg stearic plus 1.3 mg palmitic acids per mL), hemoglobin (as methemoglobin) (3.2 mg/mL), IgA (5 mg/mL), Imodium AD® (0.00667 mg/mL), Kaopectate® (0.87 mg/mL), Metronidazole (12.5 mg/mL), mucin (3.33 mg/mL) Mylanta® (4.2 mg/mL), Pepto-Bismol® (0.87 mg/mL), Prilosec® (0.5 mg/mL), Tagamet® (0.5 mg/mL), TUMS® (0.5 mg/mL), Vancomycin (12.5 mg/mL), white blood cells (5%V/V), whole blood (5% V/V).

Crossreactivity Studies:

Potentially crossreactive or interfering microorganisms were added to a natural negative and a contrived positive sample at concentrations of 1.2 X 10⁸/mL (bacteria and fungi) or at a minimum of 1 X 10⁵.⁰⁶ TCID₅₀/mL (viruses). The contrived positive sample was prepared from a pool of donor stools that were confirmed negative by toxinogenic culture. The sample was inoculated with toxinogenic C. difficile VPI 10463 to just above the limit of detection (18 CFU/test) for this strain.

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Potentially cross-reactive microorganisms were added at concentrations of $1.2 \times 10^{8} / \mathrm{mL}$ (bacteria and fungi) or at a minimum of $1 \times 10^{-5.06} / \mathrm{mL} \mathrm{TCID}_{50} / \mathrm{mL}$ (viruses). Dilution Controls for each sample were prepared by adding a phosphate-buffered saline solution in place of the potentially cross-reactive microorganisms. Each potential cross-reactive microorganism was tested in replicates of three. The following microorganisms were included in the study: Aeromonas hydrophila, Bacteroides fragilis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Candida albicans, Citrobacter freundii, Clostridium sordellii, Clostridium perfringens, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Escherichia coli O157:H7, Escherichia fergusonii, Escherichia hermannii, Helicobacter pylori, Klebsiella pneumoniae, Lactococcus lactis, Listeria monocytogenes, Peptostreptococcus anaerobius, Plesiomonas shigelloides, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella Groups B-E, Serratia liquefaciens, Serratia marcescens, Shigella boydii, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Vibrio parahaemolyticus, Yersinia enterocolitica, Adenovirus Types 40 and 41, Coxsackievirus, Echovirus, Rotavirus.

## Strain Reactivity Studies:

Aliquots from culture filtrates of each organism were first adjusted to MacFarland Standard 4 (approximately $1.2 \times 10^{9}$), then spiked into a natural negative stool matrix to a final concentration of (1) 64 CFU/test (the LoD at $95\%$ confidence as determined in the LoD study, and (2) at 100 times the LoD (to represent heavily infected samples).

Strain reactivity studies were performed by testing *C. difficile* stock cultures from different sources at 64 CFU/test and 640 CFU/test with *illumigene* *C. difficile*. Each sample was testing in triplicate. Strains and toxinotypes tested were as follows: Type 0 Strains: 10463, 2004111, 2004205, 2005070, 2005257, 2008029, 2008162, 2008341, 2008351, 2009066, 2009099, B1, G1, J7, K12, Y1; Type III Strains: 2004052, 2004118, 2007431, BI17, BI8; Type V Strains: 2005325, 2006240, 2008188, 2009018, 2009065, BK6; Type VIII Strains: 43598, 2008016, CF1; Type X Strains: 8864; Type XII Strains: 2007435; Type IX/XXIII Strains: 2007858; Unknown Strains: 2009132, 2009155, 2009277.

## f. Assay cut-off:

The assay has a fixed cut-off based on the measured change in light transmission at the assay endpoint. There are no range values for the cut-off. The *illumipro-10* measures the transmission of light through the Test Solution and the Control Solution at the start of the Assay Run and at the end point of the Assay Run, approximately 40 minutes for the *illumigene* *C. difficile* assay. The *illumipro-10* calculates the ratio of the Run End (Signal final or $S_f$) reads with the Run Start (Signal Initial or $S_i$) reads. Calculated $S_f: S_i$ ratios less than $90\%$ are reported as positive; Calculated $S_f: S_i$ ratios greater than or equal to $90\%$ are reported as negative. Fixed cut-off values

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were based on well characterized clinical specimens.

## 2. Comparison studies:

a. Method comparison with predicate device:

Only discrepant samples were tested with the predicate device, Cepheid® Xpert® C. difficile, and TECHLAB® C. DIFF CHEK™ - 60.

Of 5 false-negative samples, 2 were negative by an FDA-cleared molecular assay.

Of 27 false-positive samples, 15 were positive by an FDA-cleared molecular assay

Of the 12 remaining false-positive samples, 8 were positive by an FDA-cleared assay for C. difficile Common Antigen.

b. Matrix comparison:

N/A

## 3. Clinical studies:

a. Clinical Sensitivity:

Samples utilized in this study were stool specimens from patients suspected of having C. difficile associated disease. No restrictions with regard to age, gender or geographical location were included. All samples were left-over samples. The consistency of each sample was categorized as solid, semisolid, bloody, and watery with no solids or in transport medium. A total of 697 qualified patient samples were used in the study. One sample was obtained per patient. Two tests were performed with each sample (cytotoxic bacterial culture and illumigene C. difficile assay). Performance characteristics of the illumigene C. difficile assay were determined by comparison to cytotoxic bacterial culture. Four independent clinical test sites located in the Midwestern and Southern regions of the United States and the manufacturer evaluated a total of 697 qualified patient samples. Samples were collected from 274 (39.3%) males and 419 (60.1%) females. In the case of 4 (0.6%) of the patients, sex was not known. The age groups of patients range from 2 years of age to 96 years. No differences in test performance were observed based on patient age, sex, or geographical location.

Overall sensitivity was determined to be 95.2% (95% CI: 89.2% - 97.9%); overall specificity was determined to be 95.3% (95% CI: 93.2% - 96.7%).

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Overall performance data
Cytotoxic bacterial
culture
illumigene C. difficile

| | Positive | Negative | Total |
| --- | --- | --- | --- |
| Positive | 99 | 5 | 104 |
| Negative | 27 | 546 | 573 |
| Total | 126 | 551 | 677 |
| | | | 95% CI |
| Sensitivity | 99/104 | 95.2% | 89.2 - 97.9% |
| Specificity | 546/573 | 95.3% | 93.2 - 96.7% |
| Correlation | 645/677 | 95.3% | 93.4 - 96.6% |

b. Clinical specificity:
See 3(a) above

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

4. Clinical cut-off:
N/A

5. Expected values/Reference range:
Samples used in this study were left-over samples for which a physician had ordered C. difficile testing. Observed C. difficile prevalence rate was approximately 15%.

N. Instrument Name:
The illumipro-10

O. System Descriptions:

1. Modes of Operation:
The illumipro-10 is a menu driven laboratory instrument with two independent sample processing blocks: Block A and Block B. Each illumipro-10 Block is capable of batch processing up to five closed illumigene test devices.
The illumipro-10 operates in four basic modes: ASSAY, RESULTS, SERVICE, and SYSTEM. Assay Selection and Sample Amplification occur in the ASSAY mode; Test Results are managed in the RESULTS mode; Basic instrument set-up is performed in the SYSTEM mode; and Optical performance verification is completed in the SERVICE

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mode.

2. Software:

FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:

Yes ☐ X ☐ or No ☐

3. Specimen Identification:

Samples are identified by position. Default Sample Identification is based on Block and Well position (e.g. Block A, Well 1). The user may input Sample Identification information using the keypad, the barcode scanner or the optional external keyboard.

4. Specimen Sampling and Handling:

Specimen Sampling and Handling is performed external to the illumipro-10. Prepared Samples in closed illumigene Test Devices are placed in the illumipro-10 for amplification and detection. The illumipro-10 has no direct contact with samples. Closed illumigene Test Devices are discarded at the end of the assay to reduce the likelihood of contamination of the illumipro-10 or the workspace.

5. Calibration:

The illumipro-10 was designed to be a self-monitoring instrument. Calibration by the end user is not required.

6. Quality Control:

Quality Control requirements for the illumipro-10 are limited to verification of optical performance and routine surface cleaning/decontamination. Optics system verification is performed by the user at installation and at 30 day intervals thereafter. Optics Verification Standards consist of ten red acrylic pieces molded and polished to replicate the dimensions of each illumigene Test Device chamber. The verification standards act like a high pass optical filter with the pass wavelength of $650\mathrm{nm}$ extending into the infrared range. Light transmission through the verification standard is used to confirm proper performance of the optics system. Failed optics verification testing for an instrument block will disable the block until the error is resolved. The illumipro-10 completes a Power-On Self Test (POST) at each power-on. POST testing confirms that the Hardware and Software elements of the system are performing as expected. The illumipro-10 reports an error if POST failures are obtained; the instrument is disabled until the error is resolved.

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P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

N/A

Q. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

R. Conclusion:

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

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