The ARIES® C. difficile Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection of toxigenic Clostridium difficile (C. difficile) nucleic acid in unpreserved, unformed (liquid or soft) stool specimens obtained from patients suspected of having Clostridium difficile infection (CDI). The test targets the C. difficile toxin A gene (tcdA) and toxin B gene (tcdB) and is indicated for use as an aid in the diagnosis of C. difficile infection (CDI). The ARIES® C. difficile Assay is indicated for use with ARIES® Systems.

Device Description

The ARIES® C. difficile Assay is a polymerase chain reaction (PCR)-based qualitative in vitro diagnostic test system which consists of the ARIES® System or the ARIES® M1 System with their included ARIES® Software, a stool resuspension kit, an assay-specific cassette, and an assay-specific protocol file. The ARIES® C. difficile Assay cassette is a disposable, single-use cassette containing nucleic acid purification reagents, internal sample process control (SPC), and an assay-specific master mix capable of performing the designated assay on one sample. The ARIES® C. difficile Assay cassette directly detects toxigenic Clostridium difficile (C. difficile) from unformed stool specimens obtained from patients suspected of having Clostridium difficile infection. Specifically, the ARIES® C. difficile Assay cassette detects the C. difficile toxin A gene (tcdA) and toxin B gene (tcdB) and a DNA Sample Processing Control. Unpreserved raw stool is processed using the ARIES® Stool Resuspension Kit. The ARIES® Stool Resuspension Kit includes a flocked swab, a tube containing preprocessing beads, and Stool Resuspension Buffer. The preprocessing method involves transfer of a swab of stool specimen into a tube containing preprocessing beads and Stool Resuspension Buffer. The swab is mixed in the tube by vortexing and then centrifuged. Finally, the preprocessed sample is added to the ARIES® C. difficile Assay cassette. The specimen is lysed and nucleic acid is extracted using an ARIES® instrument. An extractable sample processing control (SPC) target is also present in the ARIES® C. difficile Assay cassette and is processed with the specimen. The SPC controls for recovery of extracted nucleic acid, for inhibitory substances and for PCR reagent and instrument integrity. The Ct value of the SPC is designed to verify nucleic acid extraction, to identify PCR inhibition, if any, and verify proper function of the extraction system and real-time instrument. The Tm value of the SPC is used as a reference for determining the Tm of the tcdA and tcdB targets (if present). The extracted nucleic acid and SPC are transferred via magnetic beads through the cassette to the ARIES® C. difficile Assay lyophilized PCR reagents in the PCR tube that contain primer pairs specific to tcdA, tcdB, and the SPC sequence. Each of the primer pairs is labeled with a distinct fluorophore and detected in distinct channels of an ARIES® System. PCR amplification is performed and assay fluorescence is monitored. Incorporation of a quencher-labeled nucleotide results in a decrease in fluorescence for the associated primer pair. Following amplification, the reaction is slowly heated to separate the fluorescent-labeled strand from the quencher-labeled strand, a process that results in an increase in the fluorescence signal. The reaction fluorescence is measured during this process and the temperature at which the change in fluorescence is the maximum Tm of the amplicon. The strands of the amplicons will separate at a specific melting temperature (Tm) and an increase in fluorescence is observed. The instrument fluorescence output is analyzed and test results are determined using the ARIES® System software and the ARIES® C. difficile Assay protocol and run files. ARIES® C. difficile Assay results may be reported from the ARIES Software or from the optional SYNCT® Software.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided FDA 510(k) summary for the ARIES® C. difficile Assay:

1. Table of Acceptance Criteria and Reported Device Performance

The FDA 510(k) summary does not explicitly state pre-defined acceptance criteria values for clinical performance as a single table. Instead, it presents the assay's performance metrics against a reference method and implicitly indicates that these results were considered "acceptable" for substantial equivalence. Based on the clinical performance section, we can infer the key performance metrics reported.

Metric	Acceptance Criteria (Inferred from reported performance deemed acceptable)	Reported Device Performance (ARIES® C. difficile Assay)
Positive Percent Agreement (PPA) vs. Direct Toxigenic Culture	High (e.g., typically >90%)	98.1% (Lower Bound 95% CI: 93.3%)
Negative Percent Agreement (NPA) vs. Direct Toxigenic Culture	High (e.g., typically >90%)	92.6% (Lower Bound 95% CI: 90.6%)
Sensitivity vs. Direct & Enriched Toxigenic Culture	High (e.g., typically >90%)	90.5% (Lower Bound 95% CI: 84.6%)
Specificity vs. Direct & Enriched Toxigenic Culture	High (e.g., typically >90%)	95.8% (Lower Bound 95% CI: 94.2%)
Overall Invalid Rate	Low	0.5% (after re-test)

2. Sample Size Used for the Test Set and Data Provenance

Sample Size:
- Clinical Study: 984 unique specimens were included in the data analysis. After exclusions and re-tests, 979 eligible specimens were used for performance calculations.
- Analytical Precision/Reproducibility:
  - Within-Laboratory: 252 samples (36 for each of 7 panel members).
  - Reproducibility (3 sites x 5 days x 6 panel members x 3 replicates): 270 samples (90 for each of 7 panel members, assuming negative also had 90 replicates).
- Limit of Detection (LoD): Not specified precisely, but implied to be multiple replicates at various dilutions.
- Analytical Reactivity: 15 distinct strains of toxigenic C. difficile (in addition to those in LoD).
- Analytical Specificity (Cross-Reactivity/Interference): 61 microorganisms/viruses + human DNA, each tested with three replicates of two C. difficile strains (BAA-1870 and BAA-1871) and three negative replicates.
- Carry-Over/Cross-Contamination: 30 high positive and 30 negative samples.
Data Provenance:
- Clinical Study: Prospective. The specimens were collected from 31-October-2016 to 21-February-2017 at four geographically distinct clinical sites within the United States. The specimens consisted of excess leftover de-identified, unpreserved, unformed stool specimens from patients suspected of having Clostridium difficile infection (CDI).

3. Number of Experts Used to Establish Ground Truth for the Test Set and Qualifications of Experts

The document does not explicitly state the "number of experts" or their specific "qualifications" for establishing the ground truth using the reference method.

Ground Truth Method: The reference method for the clinical study was "direct and enriched toxigenic culture." This is a laboratory-based method.
Location of Ground Truth Establishment: "Reference method testing was performed at a centralized testing facility." It is implied that qualified laboratory personnel perform these culture tests, but specific number or qualifications are not provided.

4. Adjudication Method for the Test Set

The document does not explicitly describe an adjudication method (like 2+1, 3+1, none) for discordant results between the ARIES® C. difficile Assay and the reference method.

However, for discordant cases, further characterization was performed:

For specimens negative by direct toxigenic culture but positive by ARIES® C. difficile Assay (False Positives based on direct culture), enriched toxigenic culture was used, and for some, bi-directional sequencing with analytically validated primers was performed.
For specimens positive by direct toxigenic culture but negative by ARIES® C. difficile Assay (False Negatives based on direct culture), bi-directional sequencing with analytically validated primers was used.
Similar follow-up testing (bi-directional sequencing) was done for discords when comparing to "Direct and Enriched Toxigenic Culture."

This suggests a form of further characterization for discordant results rather than an adjudication panel of experts re-reading primary data.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done

No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done. This device is an in vitro diagnostic (IVD) assay, not an AI imaging or diagnostic algorithm designed to assist human readers. Its performance is evaluated stand-alone against a reference laboratory method.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the clinical performance study evaluated the ARIES® C. difficile Assay as a standalone algorithm/device. The assay generates a qualitative result (POSITIVE, NEGATIVE, or INVALID) based on its internal processing and software analysis, without human interpretation of raw data for diagnosis. The ARIES® System software automatically determines and reports results.

7. The Type of Ground Truth Used

The primary ground truth used for the clinical performance study was direct and enriched toxigenic culture.

Direct Toxigenic Culture: This involves culturing the C. difficile from the stool sample directly to identify toxigenic strains.
Enriched Toxigenic Culture: This involves an enrichment step before culturing to increase the yield of C. difficile, potentially improving sensitivity.

For discordant results, further analysis using bi-directional sequencing with analytically validated primers was employed.

8. The Sample Size for the Training Set

The document does not specify a distinct training set sample size. For IVD submissions like this one, it's common that assay parameters (like Ct cut-offs and Tm windows) are established during "internal optimization studies" and "internal verification studies." The document explicitly states:

"Initial Assay Protocol File parameters were set during internal optimization studies."
"The final Assay Protocol File parameters were then established during internal verification studies."

The sizes of these internal optimization and verification studies are not provided but would have involved various analytical samples (LoD, inclusivity, exclusivity, etc.) rather than a dedicated "training set" of clinical samples in the same way an AI model might be trained. The clinical trial data (979 samples) served as a test set for validation, not for training the assay's parameters.

9. How the Ground Truth for the Training Set Was Established

Since no distinct "training set" of clinical samples is explicitly described for algorithm development in the AI sense, the ground truth establishment for the internal optimization and verification studies likely involved:

Carefully characterized reference strains of C. difficile (toxigenic and non-toxigenic).
Spiked stool matrixes with known concentrations of C. difficile to determine detection limits and reactivity.
Potentially, a smaller set of pre-characterized clinical samples to refine initial assay parameters.

Again, these studies would rely on established microbiological and molecular biology techniques (culture, PCR, sequencing) to define the presence/absence and characteristics of C. difficile in the samples used for internal development.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

July 21, 2017

Luminex Corporation Wendy Ricker Manager, Regulatory Affairs 12212 Technology Blvd. Austin, Texas 78727

Re: K171441

Trade/Device Name: ARIES C. difficile Assay Complete Kit, ARIES C. difficile Assay Protocol File Kit, ARIES C. difficile Assay Kit (24 cassettes), ARIES Stool Resuspension Kit Regulation Number: 21 CFR 866.3130 Regulation Name: Clostridium difficile toxin gene amplification assay Regulatory Class: Class II Product Code: OZN, OOI Dated: May 15, 2017 Received: May 16, 2017

Dear Wendy Ricker:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply

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with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and Part 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerelv.

Ribhi Shawar -S For

Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K171441

Device Name ARIES C difficile Assay

Indications for Use (Describe)

The ARIES C. difficile Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection of toxigenic Clostridium difficile (C. difficile) nucleic acid in unpreserved, unformed (liquid or soft) stool specimens obtained from patients suspected of having Clostridium difficile infection (CDI). The test targets the C. difficile toxin A gene (tcdA) and toxin B gene (tcdB) and is indicated for use as an aid in the diagnosis of C. difficile infection (CDI).

The ARIES C. difficile Assay is indicated for use with ARIES Systems.

Type of Use (Select one or both, as applicable)
-------------------------------------------------

Prescription Use (Part 21 CFR 801 Subpart D)	☑
Over-The-Counter Use (21 CFR 801 Subpart C)	☐

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510(k) Summary

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

A. 510(k) Number:

K171441

B. Purpose for Submission:

Traditional 510(k), New Device

C. Measurand:

Targets DNA sequences of the toxin A (tcdA), and toxin B (tcdB) genes within the Pathogenicity Locus (PaLoc) of toxigenic strains of Clostridium difficile.

D. Type of Test:

Qualitative Real Time Polymerase Chain Reaction (PCR)

E. Applicant:

Wendy Ricker Luminex Corporation 12212 Technology Blvd Austin, TX 78727 Tel: (608) 203-8936

F. Proprietary and Established Names:

ARIES® C. difficile Assay

G. Regulatory Information:

ProductCode	Classification	Regulation Section	Panel
OZN	II	21 CFR 866.3130— C. difficile toxin geneamplification assay	Microbiology(83)

H. Intended Use:

1. Intended use(s):

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The test targets the C. difficile toxin A gene (tcdA) and toxin B gene (tcdB) and is indicated for use as an aid in the diagnosis of C. difficile infection (CDI).

The ARIES® C. difficile Assay is indicated for use with ARIES® Systems.

1. Indication(s) for use:
  Same as intended use.
1. Special conditions for use statement(s):
  For prescription use only.
1. Special instrument requirements:
  For use with ARIES® Systems.

l. Device Description:

Unpreserved raw stool is processed using the ARIES® Stool Resuspension Kit. The ARIES® Stool Resuspension Kit includes a flocked swab, a tube containing preprocessing beads, and Stool Resuspension Buffer. The preprocessing method involves transfer of a swab of stool specimen into a tube containing preprocessing beads and Stool Resuspension Buffer. The swab is mixed in the tube by vortexing and then centrifuged. Finally, the preprocessed sample is added to the ARIES® C. difficile Assay cassette.

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The specimen is lysed and nucleic acid is extracted using an ARIES® instrument. An extractable sample processing control (SPC) target is also present in the ARIES® C. difficile Assay cassette and is processed with the specimen. The SPC controls for recovery of extracted nucleic acid, for inhibitory substances and for PCR reagent and instrument integrity. The Ct value of the SPC is designed to verify nucleic acid extraction, to identify PCR inhibition, if any, and verify proper function of the extraction system and real-time instrument. The Tm value of the SPC is used as a reference for determining the Tm of the tcdA and tcdB targets (if present).

The extracted nucleic acid and SPC are transferred via magnetic beads through the cassette to the ARIES® C. difficile Assay lyophilized PCR reagents in the PCR tube that contain primer pairs specific to tcdA, tcdB, and the SPC sequence. Each of the primer pairs is labeled with a distinct fluorophore and detected in distinct channels of an ARIES® System. PCR amplification is performed and assay fluorescence is monitored. Incorporation of a quencher-labeled nucleotide results in a decrease in fluorescence for the associated primer pair. Following amplification, the reaction is slowly heated to separate the fluorescent-labeled strand from the quencher-labeled strand, a process that results in an increase in the fluorescence signal. The reaction fluorescence is measured during this process and the temperature at which the change in fluorescence is the maximum Tm of the amplicon. The strands of the amplicons will separate at a specific melting temperature (Tm) and an increase in fluorescence is observed. The instrument fluorescence output is analyzed and test results are determined using the ARIES® System software and the ARIES® C. difficile Assay protocol and run files. ARIES® C. difficile Assay results may be reported from the ARIES Software or from the optional SYNCT® Software.

J. Substantial Equivalence Information:

1. Predicate device name(s):
  Quidel Molecular Direct C. difficile Assay
Predicate 510(k) number(s): 2.
K123998
Comparison with predicate: 3.
The following tables compare Luminex's ARIES® C. difficile Assay to Quidel's Molecular Direct C. difficile Assay (K123998).

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Similarities
Attribute	New Device	Predicate Device (K123998)
Intended Use	The ARIES® C. difficile Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection of toxigenic Clostridium difficile ( C. difficile ) nucleic acid in unpreserved, unformed (liquid or soft) stool specimens obtained from patients suspected of having Clostridium difficile infection (CDI).The test targets the C. difficile toxin A gene ( tcdA ) and toxin B gene ( tcdB ) and is indicated for use as an aid in the diagnosis of C. difficile infection (CDI).The ARIES® C. difficile Assay is indicated for use with ARIES® Systems.	The Quidel Molecular Direct C. difficile Assay is a qualitative, multiplexed in vitro diagnostic test for the direct detection of toxin A gene ( tcdA ) or toxin B gene ( tcdB ) sequences of toxigenic strains of Clostridium difficile from unformed (liquid or soft) stool specimens collected from patients suspected of having Clostridium difficile-Associated Disease (CDAD).The Quidel Molecular Direct C. difficile Assay is a real-time PCR test and utilizes proprietary sample preparation with fluorescently labeled primers and probes. The assay can be performed using either the Life Technologies QuantStudio® Dx; the Applied Biosystems 7500 Fast Dx, or the Cepheid SmartCycler II, to detect the toxin gene sequences associated with toxin-producing C. difficile strains.The assay is intended to be performed directly on CDAD-suspected stool specimens, and is indicated from use as an aid in the diagnosis of CDAD.
Analyte	Toxin A gene ( tcdA ) and Toxin B gene ( tcdB )	Toxin A gene ( tcdA ) and Toxin B gene ( tcdB )
Sample type	Unpreserved, unformed stool (liquid or soft)	Unpreserved, unformed stool (liquid or soft)
Assay format	Real-time PCR	Real-time PCR
Assay results	Qualitative	Qualitative
Automated	Yes	Yes
Differences
Attribute	New Device	Predicate Device (K123998)
Detection method	Pairs of fluorescently-labeled primers with quencher labeled nucleotides. Measures decrease in assay fluorescence with each PCR cycle.	PCR with fluorescently labeled probes; detection based on Taqman chemistry. Measures an increase in assay fluorescence with each cycle.
Test Container	Disposable single-use cassette	Manual amplification set-up in PCR microfuge tubes or plates with wells
Instrument	ARIES® System, ARIES® M1 System	QuantStudio®Dx; the Applied Biosystems 7500 Fast Dx or the Cepheid SmartCycler II

Table 11.1: Similarities between New Device and Predicate

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Table 11.2: Differences between New Device and Predicate

K. Standards/Guidance Documents Referenced:

FDA Guidance Class II Special Controls Guideline Document: Toxin Gene Amplification Assays for the Detection of Clostridium difficile.

L. Test Principle:

The ARIES® C. difficile Assay chemistry is based on an expanded genetic alphabet technology, consisting of synthetic DNA base pair 2'-deoxy-5-methyl-isocytidine (iC): 2'deoxyisoguanosine (iG). The isobases (iC and iG) pair specifically with each other and not with natural nucleotides. In addition, isobases are efficiently incorporated during PCR. During PCR amplification, a quencher-modified iGTP is incorporated by the polymerase opposite an iC and a fluorophore reporter attached to a PCR primer. If the target is present and is amplified, assay fluorescence decreases with every cycle as amplification product accumulates. The decrease in assay fluorescence is monitored in real time using an ARIES® System. Following PCR, the amplification products are thermally denatured and assay fluorescence is monitored. The strands of the amplification products are separated and assay fluorescence increases, thus enabling determination of the melting temperature (Tm) of the amplicon.

M. Performance Characteristics:

1. Analytical performance:
- a. Precision/Reproducibility:

Precision

Within Laboratory Precision/Repeatability of the ARIES® C. difficile Assay was evaluated by two operators performing testing across multiple ARIES® instruments using one lot of ARIES® C. difficile Assay Cassettes. Testing was performed on 12 days and included analysis of a total of 252 samples. A

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repeatability panel was prepared, containing moderate positive, low positive and high negative/low positive samples, independently for two toxigenic C. difficile strains as well as a negative sample. The results of the repeatability study are presented in Table 11.3.

Target Type	Expected Positivity	Agreement withExpected	95% ConfidenceInterval
BAA-1870 Moderate Positive	100%	100% (36/36)	90.4 – 100%
BAA-1870 Low Positive	Approximately 95%	100% (36/36)	90.4 – 100%
BAA-1870 High Negative/LowPositive	20% – 80%	66.7% (24/36)	50.3 – 79.8%
BAA-1871 Moderate Positive	100%	100% (36/36)	90.4 – 100%
BAA-1871 Low Positive	Approximately 95%	97.2% (35/36)	85.8 – 99.5%
BAA-1871 High Negative/LowPositive	20% – 80%	25.0% (9/36)	13.7-41.1%
C. difficile Negative	100%	100% (36/36)	90.4% to 100%

Table 11.3: ARIES® C. difficile Assay Within Laboratory Precision/Repeatability Resultsa

An overall invalid rate of 0.8% (2/254) was observed.

b 2/36 samples produced Invalid results on initial testing; both reported as Positive upon repeat

Reproducibility

Reproducibility of the ARIES® C. difficile Assay was evaluated by testing of one lot of ARIES® C. difficile Assay Cassettes on two ARIES® Instruments by two operators at each of three sites on 5 non-consecutive days. A reproducibility panel was prepared, containing moderate positive, low positive and high negative/low positive samples, independently for two toxigenic C. difficile strains as well as a negative sample. The reproducibility panels were created by an independent operator and blinded. The results of the reproducibility study are presented in Tables 11.4 and Table 11.5.

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	Site 1		Site 2		Site 3
	Agreement with		Agreement with		Agreement with
	expected results		expected results		expected results
BAA-1870 Moderate
Positive	30/30	100%	30/30	100%	30/30	100%
BAA-1870 Low Positive	30/30	100%	30/30	100%	30/30	100%
BAA-1870 High
Negative/Low Positive	28/30	93.3%	21/30	70%	27/30	90%
BAA-1871 Moderate
Positive	30/30	100%	30/30	100%	30/30b	100%
BAA-1871 Low Positive	30/30	100%	30/30	100%	29/30	96.7%
BAA-1871 High
Negative/Low Positive	23/30	76.7%	28/30	93.3%	24/30	80%
C. difficile Negative	30/30	100%	29/30b	96.7%	30/30	100%

Table 11.4: ARIES® C. difficile Assay Site to Site Reproducibility Results®
-----------------------------------------------------------------------------	--	--	--

4 The expected results for the reproducibility panel targets are 100% for Moderate Positive, 95% for Low Positive, and 20-80% for High Negative/Low Positive for BAA-1871. Negative target is expected to be 0% positive.

§ 1/30 samples was reported as Invalid on initial testing; reported Toxigenic C. difficile Positive upon repeat

	Agreement with
	expected		95% Confidence
	results		Interval
BAA-1870 Moderate Positive	90/90	100.0%	95.9 - 100.0%
BAA-1870 Low Positive	90/90	100.0%	95.9 - 100.0%
BAA-1870 High Negative/Low Positive	76/90	84.4%	75.6 - 90.5%
BAA-1871 Moderate Positive	90/90	100.0%	95.9 - 100.0%
BAA-1871 Low Positive	89/90	98.9%	94.0 - 99.8%
BAA-1871 High Negative/Low Positive	75/90	83.3%	74.3 - 89.6%
C. difficile Negative	89/90	98.9%	94.0 - 99.8%

Table 11.5: Reproducibility Panel Total Results

8 The expected results for the reproducibility panel targets are 100% for Moderate Positive, 95% for Low Positive, and 20-80% for High Negative/Low Positive for BAA-1871. Negative target is expected to be 0% positive.

b 1/90 replicates produced a false positive result. The SPC was observed to be positive in all replicates.

b. Linearity/assay reportable range:
Not applicable. The ARIES® C. difficile Assay is a qualitative assay.
C. Traceability, Stability, Expected values (controls, calibrators, or methods):
Controls:

Process Control

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Each ARIES® C. difficile Assay cassette contains a Sample Processing Control (SPC), which is processed with the sample and analyzed during the amplification reaction. The SPC verifies nucleic acid extraction, and proper reagent, cassette, ARIES® System, and assay protocol performance. The SPC has known melting temperature (Tm) and Ct ranges. Each time an assay is run, the system measures the melting temperature and fluorescence intensity of the SPC to ensure the thermal and optical subsystems have remained in calibration.

External Controls

External controls should be tested according to guidelines or requirements of local, provincial and/or federal regulations or accreditation organizations. A reference toxigenic C. difficile strain or well characterized toxigenic C. difficile clinical isolates may be used as positive controls. A non-toxigenic strain of C. difficile may be used as a Negative Control. Alternatively, clinical specimens known to be positive or negative for toxigenic C. difficile may be used as Positive and Negative External Controls, respectively. The ARIES® C. difficile Assay Cassette Kit does not include external positive and negative controls.

Stability:

Specimen Stability

Fresh specimen stability was determined for toxigenic C. difficile in a negative clinical stool pool at 2 – 8°C. This was assessed by testing six replicates of each of three C. difficile target concentrations and the negative clinical pool across five different time points. The C. difficile concentrations tested included a moderate positive, low positive and high negative/low positive. The results of the study demonstrated that specimens for the ARIES® C. difficile Assay are stable when stored at 2 – 8°C for up to 7 days.

Room temperature specimen stability was determined for toxigenic C. difficile in a negative clinical stool pool at 15 – 30°C. This was assessed by testing six replicates of each of three C. difficile target concentrations and the negative clinical stool pool across four different time points. The C. difficile concentrations tested included a moderate positive, a low positive, and a high negative/low positive. The results of the study demonstrated that specimens for the ARIES® C. difficile Assay are stable when stored at 15 – 30°C for up to 4 hours.

Frozen specimen stability was determined for toxigenic C. difficile in a negative clinical stool pool at -65 to -95°C. This was assessed by testing six replicates of each of three C. difficile target concentrations and the negative clinical stool pool across multiple time points. The C. difficile concentrations tested included a moderate positive, low positive and high negative/low positive. The results of

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Luminex.

the study demonstrated that specimens for the ARIES® C. difficile Assay are stable when stored at -65 to -95°C for up to 3 months.

Freeze thaw specimen stability was determined for specimens after cycles of freezing and thawing. This was assessed by testing three C. difficile contrived target concentrations in negative stool matrix across five freeze thaw cycles. The concentrations used for testing were high positive, moderate positive, and low positive. The results of the study demonstrated that specimens for the ARIES® C. difficile Assay are stable when subjected to up to five freeze thaw cycles.

Shelf-Life Stability

Real time stability was determined for the ARIES® C. difficile Assay Cassette in order to establish a shelf life. This was assessed by testing at multiple time points six toxigenic C. difficile positive and negative replicates with each of three different lots of ARIES® C. difficile Assay Cassettes that had been stored at two different temperatures (4°C and room temperature). All targets for all lots and all storage temperatures gave the expected results. No trend of increased invalid rate was observed over the course of the study and the overall invalid rate was 1.2%. The expected results were observed upon retesting samples with initial Invalid results. Based on this study, a shelf life of 12 months has been assigned to the ARIES® C. difficile Assay.

Open Box stability was determined for the ARIES® C. difficile Assay Cassettes after they were removed from their individual pouches. Cassettes were removed from their pouches and placed on a laboratory bench where they were exposed to ambient temperatures, humidity, and light for up to 10 hours. Data were collected for toxigenic C. difficile Positive and C. difficile Negative (ARIES® Stool Resuspension Buffer) samples at five time points. Three lots of cassettes were used to assess open box stability. At the end of 10 hours, all three lots of cassettes produced expected results. An overall invalid rate of 1.6% was observed over the course of the study. The expected results were observed upon retesting samples with initial Invalid results. Therefore the ARIES® C. difficile Assay Cassettes are stable in ambient laboratory conditions for up to 10 hours after they have been removed from the storage pouch.

d. Detection Limit:
The limit of detection of the ARIES C. difficile Assay was determined by testing dilutions of enumerated stocks of toxigenic C. difficile in stool matrix. The Limit of Detection (LoD) was defined as the lowest concentration tested at which ≥95% of assay replicates produced positive results

The confirmed reportable LoD concentrations of each C. difficile strain were determined by colony counting and are presented in Table 11.6.

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			LOD Concentration (CFU) ᵃ
C. difficileStrain	Toxinotype	tcdA/tcdBGenes	Per mL Stool	Per cassette
ATCC® 43598	VIII	-/+	139.9	4.7
ATCC® BAA-1812	XII	+/+	110.4	3.7
ATCC® BAA-1803	IIIc ᵇ	+/+	18.6	0.6
ATCC® BAA-1870	IIIb ᵇ	+/+	19.2	0.6
ATCC® BAA-1871	0	+/+	31.2	1.0

Table 11.6. LoD Results for C. difficile strains

a The number of cfu at LoD concentration was determined by colony counting.

ಿ Outbreak-associated Pulsed Field Gel Electrophoresis type NAP1

Analytical Reactivity (Inclusivity) e.
Analytical reactivity performance characteristics for the ARIES® C. difficile Assay were assessed by testing 15 strains of toxigenic C. difficile in addition to those included in the LoD Study (1 each of toxinotypes V and XXII and 13 toxinotype 0) and an in-silico analysis of C. difficile toxinotype X. All strains positive for tcdA and/or tcdB were detected and in-silico analysis suggests that strains of toxinotype X will also be detected. In total, analytical testing demonstrated the ability of the ARIES C. difficile Assay to detect seven different toxinotypes (0, V, Illc (Nap1), Illb, VIII, XII, XXII).
Analytical specificity: f.

Cross-Reactivity and Microbial Interference:

A study was performed to evaluate cross reactivity and interference of the ARIES® C. difficile Assay with 61 microorganisms and viruses that might be present in the sample matrix, in addition to human DNA (Table 11.7). Interference was evaluated by testing three replicates each of C. difficile strains BAA-1870 and BAA-1871 (both tcdA*/tcdB*)in the presence of each potentially interfering species. To assess the potential for cross-reaction, toxigenic C. difficile Negative replicates (Negative Stool Matrix) were also tested. Testing of potentially cross-reactive or interfering species was performed at a concentration of ≥10 cfu/mL of Resuspension Buffer for bacteria and yeast and ≥10 TCID50/mL for viruses, or the highest available concentration. Human DNA was tested at 5μg/mL of Stool Resuspension Buffer. The results of the study are presented in Table 11.7.

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On initial testing, 1/3 replicates for C. bifermentans and E. coli were reported as positive for toxigenic C. difficile. Upon repeat testing of both organisms, 3/3 replicates gave negative results with the ARIES C. difficile Assay.

Invalid results were obtained on initial testing of A. baumannii (3/3 replicates) and S. marcescens (2/3 replicates) at >10' CFU/mL. Both produced negative results when retested at 10° CFU/mL.

One organism, C. botulinium, that was not available for laboratory testing was evaluated by in-silico analysis, on the basis of which, it is not expected to cross react or interfere with the ARIES® C. difficile Assay.

Table 11.7: Cross Reacting Organisms Tested
Organism Name	Test Concentration(per mL StoolResuspension Buffer)
Abiotrophia defectiva	5.80 x 107 cfu/mL
Acinetobacter baumannii	1.0 x 106 cfu/mL
Adenovirus Type 7A	5.12 x 106 TCID50/mL
Aeromonas hydrophila	7.5 x 107 cfu/mL
Alcaligenes faecalis subsp. faecalis	1.1 x 109 cfu/mL
Bacillus cereus	4.87 x 106 cfu/mL
Bacteroides fragilis	2.39 x 108 cfu/mL
Campylobacter coli	2.55 x 107 cfu/mL
Campylobacter jejuni	1.44 x 106 cfu/mL
Candida albicans	1.33 x 107 cfu/mL
Citrobacter freundii	1.45 x 108 cfu/mL
Clostridium bifermentans	5.25 x 107 cfu/mL
Clostridium butyricum	2.24 x 107 cfu/mL
Clostridium haemolyticumb	1.29 x 105 cfu/mL
Clostridium perfringens	5.30 x 106 cfu/mL
Clostridium scindens	8.90 x 107 cfu/mL
Clostridium septicum	8.10 x 106 cfu/mL
Clostridium sordellii	1.64 x 106 cfu/mL
Clostridium sporogenes	5.15 x 107 cfu/mL
Clostridium novyi	1.40 x 108 cells/mL
Coxsackievirus (Type A16)	2.04 x 106 TCID50/mL
Cytomegalovirus (Type AD-169)	5.74 x 105 TCID50/mL
Echovirus Type 11	2.94 x 106 TCID50/mL
Edwardsiella tarda	4.42 x 107 cfu/mL
Enterobacter aerogenes	8.75 x 108 cfu/mL
Enterobacter cloacae	2.99 x 108 cfu/mL
Enterococcus faecalis vanB	4.95 x 107 cfu/mL
Enterovirus (Type 71)b	2.08 x 104 TCID50/mL
Escherichia coli (026:H4)	1.80 x 108 cfu/mL
Organism Name	Test Concentration(per mL StoolResuspension Buffer)
Escherichia coli (O157:H7)	2.05 x 108 cfu/mL
Flavonifactor plautii a	3.07 x 107 cfu/mL
Helicobacter pylori	9.80 x 106 cfu/mL
Human genomic DNA	5 µg/mL
Klebsiella oxytoca	5.20 x 108 cfu/mL
Lactobacillus acidophilus	1.25 x 107 cfu/mL
Listeria monocytogenes	4.65 x 108 cfu/mL
Non-toxigenic Clostridium difficile strain 43593	5.05 x 106 cfu/mL
Non-toxigenic Clostridium difficile strain 43601	1.01 x 107 cfu/mL
Non-toxigenic Clostridium difficile strain 43602	3.17 x 106 cfu/mL
Non-toxigenic Clostridium difficile strain 43603	4.05 x 106 cfu/mL
Norovirus Group I	4.26 x 106 TCID50/mL
Norovirus Group II	4.26 x 106 TCID50/mL
Peptostreptococcus anaerobius	2.29 x 106 cfu/mL
Plesiomonas shigelloides	1.52 x 108 cfu/mL
Porphyromonas asaccharolytica	3.70 x 106 cfu/mL
Prevotella melaninogenica	2.05 x 106 cfu/mL
Proteus mirabilis	1.42 x 108 cfu/mL
Providencia alcalifaciens	2.07 x 108 cfu/mL
Pseudomonas aeruginosa	1.97 x 108 cfu/mL
Rotavirusb	8.49 x 103 TCID50/mL
Salmonella enterica (typhimurium)	5.95 x 108 cfu/mL
Salmonella enterica subsp. arizonae	5.80 x 108 cfu/mL
Salmonella enterica subsp. enterica	2.60 x 108 cfu/mL
Serratia liquefaciens	5.45 x 108 cfu/mL
Serratia marcescens	1.00 x 106 cfu/mL
Shigella boydii	2.32 x 108 cfu/mL
Shigella dysentariae	1.59 x 108 cfu/mL
Shigella sonnei	1.15 x 108 cfu/mL
Staphylococcus aureus	5.45 x 108 cfu/mL
Staphylococcus epidermidis	1.45 x 108 cfu/mL
Streptococcus agalactiae	8.25 x 107 cfu/mL
Vibrio parahaemolyticus	1.07 x 108 cfu/mL

Table 11.7. Cross Reacting Organisms Tested

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ªSame species as Clostridium orbisceindens.

b Tested at the highest available concentration provided by Zeptometrix Corporation.

Interfering Substances:

The effect of potential interfering substances on the ARIES® C. difficile Assay was evaluated by spiking prepared solutions of potential interfering substances, presented in Table 11.8, into stool matrix with and without toxigenic C. difficile. For each potentially interfering substance, three replicates of each of two strains of toxigenic C. difficile (both tcdA*/tcdB*) were tested, in addition to three C.

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difficile negative samples. A total of 14 interfering substances were tested for inhibitory effects on the ARIES® C. difficile Assay. False negative results were obtained in the presence of mucin at 3.5% w/v, although no interference was observed when mucin was tested at 0.35% w/v. In the presence of all other substances tested, C. difficile was detected in 100% of replicates.

Interfering Substance	Concentration of InterferingSubstance in Stool
Barium sulfate	1.3% w/v
Fecal fat (Triglyceride)	20.0% w/va
Fecal fat (Cholesterol)	4.9% w/v
Hemoglobin (tarry stool)	12.5% w/v
Hydrocortisone Cream	2.0% w/v
Imodium	0.63% w/vb
Kaopectate	0.1% w/v
Metronidazole	140.0 mg/mLa
Moist towelettes (BenzalkoniumChloride)	10.0% v/v
Mucin	0.35% w/vc
Pepto-Bismol	0.1% w/va
Preparation H	2.0% w/vd
Vagisil anti-itch cream	2.0% w/v
Whole Blood	20% v/v

Table 11.8. Interfering Substances Tested

a 1/3 replicates for BAA-1871 was negative for tcdA

2/3 replicates for BAA-1870 were negative for tcdA

° False negative results may be observed in the presence of mucin at concentrations >0.35%

d 2/3 replicates for both BAA-1870 and BAA-1871 were negative for tcdA

Carry-Over/Cross-Contamination:

Carry-over and cross contamination for the ARIES® C. difficile Assay was assessed by testing 30 high positive C. difficile samples and 30 C. difficile negative samples (Negative Stool Matrix). Samples were tested in an alternating pattern with high positive samples run adjacent to negative samples across 11 consecutive runs. No carry-over or cross contamination was observed. The overall percent agreement was 100% for positive and negative samples.

g. Assay cut-off
For the ARIES® C. difficile Assay, each target (tcdA and tcdB) has a Ct cut-off, Tm window, and Tm Peak Threshold. In addition, the internal sample processing control (SPC) also has a corresponding Ct cut-off, Tm window, and Tm Peak Threshold. Collectively, the cut-off values compose the assay protocol file parameters, which are used to determine the assay result for the detection target as POSITIVE, NEGATIVE, or INVALID. These values are hard-coded into the

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ARIES® C. difficile Assay protocol file and are not modifiable. The Assay Protocol File parameters were determined, and their performance in the ARIES® C. difficile Assay evaluated according to the following general procedure:

. Initial Assay Protocol File parameters were set during internal optimization studies
The final Assay Protocol File parameters were then established during internal verification studies
. The selected Assay Protocol File parameter values were utilized in the determination of assay performance in the multi-site clinical trial conducted for the ARIES C. difficile Assay

The specific assay parameters for the ARIES® C. difficile Assay are considered confidential and proprietary.

1. Clinical Performance:
  Performance of the ARIES® C. difficile Assay was evaluated prospectively from 31-October-2016 to 21-February-2017 at four geographically distinct clinical sites within the United States using the ARIES® System. Specimens for the clinical study consisted of excess leftover de-identified, unpreserved, unformed stool specimens from patients suspected of having Clostridium difficile infection (CDI). All eligible leftover stool specimens were tested with a reference method (direct and enriched toxigenic culture) and ARIES® C. difficile Assay and the results compared. Reference method testing was performed at a centralized testing facility while ARIES® C. difficile Assay testing was performed at each clinical site on their own clinical specimens.

A total of 1021 stool specimens from subjects suspected of having CDI were collected from four geographically diverse locations within the United States. Of these 1021 specimens, 37 were excluded from the study based on inclusion/exclusion criteria leaving a total of 984 unique specimens that met the predetermined inclusion criteria and that were included in the data analysis. These 984 specimens were enrolled in the study and tested for toxigenic C. difficile by both the reference method of direct and enriched toxigenic culture and the ARIES® C. difficile Assay. There were 28 specimens (28/984, 2.8%) that were re-tested with ARIES® C. difficile Assay because they yielded initial invalid results due to run failure or instrument error. An additional 15 specimens were re-tested with the ARIES® C. difficile Assay because of either sample mix-up (N=5) or improper sample storage or processing (N=10). Thirty-eight (38) of the 43 specimens that were re-run generated valid ARIES® C. difficile Assay results (i.e. positive or negative) after re-test. Five (5)

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specimens remained invalid by ARIES® C. difficile Assay upon re-test for an overall invalid rate after repeat testing of 0.5% (5/984).

For the 979 eligible specimens that were included in the device performance calculations, positive percent agreement of the ARIES® C. difficile Assay for toxigenic C. difficile against direct toxigenic culture was 98.1% (103/105) with a lower bound 95% confidence interval of 93.3%. When compared to direct and enriched toxigenic culture, clinical sensitivity of the ARIES® C. difficile Assay for toxigenic C. difficile was 90.5% (133/147) with a lower bound 95% confidence interval of 84.6%. Negative percent agreement of the ARIES® C. difficile Assay for toxigenic C. difficile in comparison to direct toxigenic culture was 92.6% (809/874, Lower Bound 95% Cl, 90.6%), while clinical specificity in comparison to direct and enriched culture was 95.8% (797/832, Lower Bound 95% Cl, 94.2%).

Table 11.9. ARIES® C. difficile Assay Performance Compared to Direct Culture (N=979)

ARIES® C. difficile Assay	Direct Toxigenic Culture
	Positive	Negative	TOTAL
Positive	103	652	168
Negative	21	809	811
TOTAL	105	874	9793
		95% CI
Positive PercentAgreement	98.1%	93.3% - 99.5%
Negative PercentAgreement	92.6%	90.6% - 94.1%

2 One of the ARIES® C. difficile Assay negative specimens that was positive by direct toxigenic culture (i.e. False Negative) was C. difficile negative by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the ARIES® C. difficile Assay.

4 Of the 65 ARIES® C. difficile Assay positive specimens that were negative by direct toxigenic culture (i.e. False Positive), 30 were positive by enriched toxigenic culture. An additional 15 specimens were positive by bidirectional sequencing analytically validated primers that targeted genomic regions distinct from the ARIES® C. difficile Assay.

3 Five (5) specimens generated invalid results by the ARIES C. difficile Assay after allowable re-run. Four (4) of these were negative and one (1) was positive by direct toxigenic culture. All of these 5 specimens were excluded from the device performance calculations.

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ARIES® C. difficile Assay	Direct and Enriched Toxigenic Culture
	Positive	Negative	TOTAL
Positive	133	352	168
Negative	141	797	811
TOTAL	147	832	9793
		95% CI
Sensitivity	90.5%	84.6% - 94.2%
Specificity	95.8%	94.2% - 97.0%

Table 11.10. ARIES® C. difficile Assay Performance Compared to Direct and Enriched Toxigenic Culture (N=979)

4 Thirteen (13) of the ARIES® C. difficile Assay negative specimens that were positive by direct and enriched toxigenic culture (i.e. False Negative) were C. difficile negative by bi-directional sequencing analysis using analytically validated primers that targeted genomic regions distinct from the ARIES® C. difficile Assay ² Fifteen (15) of the ARIES® C. difficile Assay positive specimens that were negative by enriched toxigenic culture (i.e. False Positive) were positive by bi-directional sequencing analytically validated primers that

targeted genomic regions distinct from the ARIES® C. difficile Assay.

3 Five (5) specimens generated invalid results by the ARIES °C. difficile Assay after allowable re-run. Four (4) of these were negative and one (1) was positive by direct and enriched toxigenic culture. All of these 5 specimens were excluded from the device performance calculations.

The study results demonstrate that the diagnostic accuracy of the ARIES® C. difficile Assay is acceptable for the detection of C. difficile in unpreserved, soft or liquid stool specimens from patients suspected of having Clostridium difficile infection (CDI).

Expected values/Reference range: 3.

The prevalence of toxigenic C. difficile observed during a multi-center clinical trial using the ARIES C. difficile Assay was estimated as 17.2% (168/979). Of the patient populations included in the study, the majority of patients were senior adults (≥60 years) and the prevalence of C. difficile in this age group was found to be 18.1% (88/487). The second largest age group was adults (age 22 to <60 years) and the prevalence was found to be 16.1% (74/460). The next age group was adolescents (12 to <22 years) and the prevalence was found to be 22.2% (6/27). The remaining age group included 4 children (2 to <12 years) and one infant (<2 years) where the prevalence was found to be 0%.

N. Proposed Labeling:

The labeling provided in the submission satisfies the requirements of 21 CFR 809.10.

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O. Conclusion:

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

Regulation Number and Section

§ 866.3130 Clostridium difficile toxin gene amplification assay.

(a)
Identification. AClostridium difficile toxin gene amplification assay is a device that consists of reagents for the amplification and detection of target sequences inClostridium difficile toxin genes in fecal specimens from patients suspected of havingClostridium difficile infection (CDI). The detection of clostridial toxin genes, in conjunction with other laboratory tests, aids in the clinical laboratory diagnosis of CDI caused byClostridium difficile. (b)
Classification. Class II (special controls). The special controls are set forth in FDA's guideline document entitled: “Class II Special Controls Guideline: Toxin Gene Amplification Assays for the Detection ofClostridium difficile; Guideline for Industry and Food and Drug Administration Staff.” See § 866.1(e) for information on obtaining this document.