Great Basin Toxigenic C. difficile Direct Test (CDF2)

• K Number: K232092
• Device Name: Great Basin Toxigenic C. difficile Direct Test (CDF2)
• Manufacturer: Vela Operations USA
• Date Cleared: 2023-11-14 ( 124 days)
• Product Code: OZN
• Regulation Number: 866.3130

Intended Use

The Great Basin Toxigenic C. difficile Direct Test, performed on the Great Basin PA500 Analyzer, is a qualitative in vitro diagnostic test for the detection of toxigenic Clostridioides difficile in unformed (liquid or soft) stool samples collected from patients suspected of having C. difficile infection (CDI). The automated assay utilizes polymerase chain reaction (PCR) to detect a conserved region of the toxin gene (tcdB) associated with toxin-producing C. difficile. The Toxigenic C. difficile Direct Test is intended for use as an aid in the diagnosis of CDI in conjunction with clinical and epidemiological risk factors.

Device Description

The Great Basin Toxigenic C. difficile Direct Test (CDF2) performed on the PA500 Analyzer utilizes automated, hot-start PCR amplification technology to amplify specific nucleic acid sequences that are then detected using hybridization probes immobilized on a modified silicon chip surface, in a single-use, self-contained test cartridge. A swab volume of the specimen (raw stool) is first processed using the Sample Preparation Device (SPD). During processing through the SPD the specimen is infused with a synthetic bacterial sample processing control (SPC). An aliquot (250 µL) of the eluate obtained from the SPD, containing diluted specimen mixed with SPC, is loaded into the sample port of the CDF2 Test Cartridge. Genomic DNA is extracted from microbial cells and diluted to reduce potential inhibitors of PCR. During the PCR process, biotin-labeled primers direct the amplification of specific nucleic acid sequences within a conserved region of the C. difficile Toxin B (tcdB) gene. Following PCR, biotin-labeled, amplified target DNA sequences are hybridized to sequence specific probes immobilized on the silicon chip surface and incubated with antibody conjugated to the horseradish peroxidase enzyme (HRP). The unbound conjugate is washed away, and then tetramethylbenzidine (TMB) is added to produce a colored precipitate at the location of the probe/target sequence complex. The resulting signal is detected by the automated PA500 Optical Reader within the PA500 Analyzer System. The SPC undergoes the same extraction, and detection steps as the sample in order to monitor for inhibitory substances, as well as process inefficiency due to instrument or reagent failure. No operator intervention is required once the sample is loaded into the sample port and the Toxigenic C. difficile Direct Test cartridge is loaded into the PA500 Analyzer. The Great Basin System is a fully automated in vitro diagnostic system that includes: the PA500 Analyzer, single-use SPDs, single-use Toxigenic C. difficile Direct Test Cartridges, and the PA500 Data Analysis Software Program. The PA500 Analyzer is designed to perform automated sample preparation, target amplification via PCR, chip-based (optical) target detection, and integrated data analysis in less than two hours.

Predicate Devices

K172569

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description details a standard PCR-based in vitro diagnostic test with automated sample processing, amplification, detection, and data analysis. There is no mention of AI or ML algorithms being used for interpretation or decision-making. The data analysis software performs integrated analysis, which in this context likely refers to standard signal processing and thresholding for positive/negative determination, not AI/ML.

Therapeutic

No
This device is an in vitro diagnostic test designed to detect toxigenic Clostridioides difficile, which aids in the diagnosis of C. difficile infection. It does not directly treat or prevent a disease or condition.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the device "is a qualitative in vitro diagnostic test" and "is intended for use as an aid in the diagnosis of CDI."

SaMD (Software as a Medical Device)

No

The device is an in vitro diagnostic system that includes hardware components (PA500 Analyzer, SPDs, Test Cartridges) in addition to the software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The "Intended Use / Indications for Use" section explicitly states: "The Great Basin Toxigenic C. difficile Direct Test, performed on the Great Basin PA500 Analyzer, is a qualitative in vitro diagnostic test for the detection of toxigenic Clostridioides difficile in unformed (liquid or soft) stool samples collected from patients suspected of having C. difficile infection (CDI)."

Furthermore, the "Device Description" refers to the system as a "fully automated in vitro diagnostic system".

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Great Basin Toxigenic C. difficile Direct Test, performed on the Great Basin PA500 Analyzer, is a qualitative in vitro diagnostic test for the detection of toxigenic Clostridioides difficile in unformed (liquid or soft) stool samples collected from patients suspected of having C. difficile infection (CDI). The automated assay utilizes polymerase chain reaction (PCR) to detect a conserved region of the toxin gene (tcdB) associated with toxin-producing C. difficile.

The Toxigenic C. difficile Direct Test is intended for use as an aid in the diagnosis of CDI in conjunction with clinical and epidemiological risk factors.

Product codes (comma separated list FDA assigned to the subject device)

OZN

Device Description

The Great Basin Toxigenic C. difficile Direct Test (CDF2) performed on the PA500 Analyzer utilizes automated, hot-start PCR amplification technology to amplify specific nucleic acid sequences that are then detected using hybridization probes immobilized on a modified silicon chip surface, in a single-use, self-contained test cartridge.

A swab volume of the specimen (raw stool) is first processed using the Sample Preparation Device (SPD). During processing through the SPD the specimen is infused with a synthetic bacterial sample processing control (SPC). An aliquot (250 µL) of the eluate obtained from the SPD, containing diluted specimen mixed with SPC, is loaded into the sample port of the CDF2 Test Cartridge.

Genomic DNA is extracted from microbial cells and diluted to reduce potential inhibitors of PCR. During the PCR process, biotin-labeled primers direct the amplification of specific nucleic acid sequences within a conserved region of the C. difficile Toxin B (tcdB) gene.

Following PCR, biotin-labeled, amplified target DNA sequences are hybridized to sequence specific probes immobilized on the silicon chip surface and incubated with antibody conjugated to the horseradish peroxidase enzyme (HRP). The unbound conjugate is washed away, and then tetramethylbenzidine (TMB) is added to produce a colored precipitate at the location of the probe/target sequence complex.

The resulting signal is detected by the automated PA500 Optical Reader within the PA500 Analyzer System. The SPC undergoes the same extraction, and detection steps as the sample in order to monitor for inhibitory substances, as well as process inefficiency due to instrument or reagent failure. No operator intervention is required once the sample is loaded into the sample port and the Toxigenic C. difficile Direct Test cartridge is loaded into the PA500 Analyzer.

The Great Basin System is a fully automated in vitro diagnostic system that includes: the PA500 Analyzer, single-use SPDs, single-use Toxigenic C. difficile Direct Test Cartridges, and the PA500 Data Analysis Software Program. The PA500 Analyzer is designed to perform automated sample preparation, target amplification via PCR, chip-based (optical) target detection, and integrated data analysis in less than two hours.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

≥ 2 years of age

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Description of the test set: Prospective multi-center method comparison study using excess remnants of unpreserved stool specimens from symptomatic individuals suspected of C. difficile infection (CDI).
Sample size: 829 specimens.
Data source: Three geographically diverse U.S. clinical study sites (Midwest, West) from June 2022 - February 2023.
Annotation protocol: Specimens processed according to routine standard of care testing. All specimens enrolled under an Institutional Review Board (IRB) approved protocol and testing was performed by trained laboratory personnel.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Studies:

• Specimen Stability: A room temperature and refrigerated Specimen Stability study was conducted. C. difficile ATCC 43255 cells were spiked into pooled clinical negative stool matrix. Tested in triplicate. Results showed C. difficile target stable up to and beyond 72 hours when stored refrigerated (2° - 8°C) and stable for low positive samples up to 24 hours at room temperature (18° - 22°C).
• Analytical Sensitivity (LoD): Measured for two toxigenic Clostridioides difficile strains (ATCC 43255 and ATCC BAA-1805). Strains were serially diluted and plated to determine CFU/mL, then diluted into pooled clinical negative stool. Probit analysis used to calculate theoretical LoD at 95% agreement, confirmed with additional testing.
  • Key results: LoD for ATCC 43255 was 2.6 x 10^4 CFU/mL, and for ATCC BAA-1805 was 7.1 x 10^4 CFU/mL.
• Analytical Reactivity (Inclusivity): Evaluated by testing 22 additional toxigenic Clostridioides difficile strains covering six unique toxinotypes. Strains were diluted into pooled clinical negative stool to 2X LoD and tested in triplicate.
  • Key results: All 22 strains tested were detected, with every replicate correctly identified, indicating detection of multiple strains and toxinotypes.
• Analytical Specificity (Exclusivity): Evaluated potential cross-reactivity to non-target organisms (83 microorganisms found in stool: bacteria, fungi/yeast, parasites, viruses, human genomic DNA). Contrived samples spiked into pooled clinical negative stool matrix. Each organism tested in triplicate.
  • Key results: All exclusivity organisms gave the expected 'NEGATIVE, Toxin NOT DETECTED' result, with one exception (Salmonella enterica ATCC 6962), which upon retesting yielded the expected 'Negative' result. No cross-reactivity observed.
• Microbial Interference (Competitive Inhibition): Evaluated interference from 82 microorganisms pooled into 17 unique pools. Each non-target organism spiked into pooled negative clinical stool matrix at input concentration from Exclusivity Study. Microbial Interference pool contrived to 2X LoD C. difficile ATCC 43255 and tested in triplicate.
  • Key results: All Microbial Interference pool replicates gave the expected 'POSITIVE, Toxin DETECTED' result (100% agreement), indicating no interference or competitive inhibition.
• Interfering Substances (Chemical Interference): Evaluated potential endogenous chemical interference from 30 substances common in stool or present in diarrhea patients. Each substance tested in background of 2X LoD contrived positive stools (two C. difficile strains) and true negative stool, in triplicate.
  • Key results: No CDF2 interference observed for any endogenous or exogenous substances at concentrations listed.
• Reproducibility: Between-site study conducted at 1 internal and 2 external sites. Included multiple operators (n=6), non-consecutive testing days (n=5), multiple Analyzers (n=24), and cartridge lots (n=2). Panel consisted of 5 members: 2 toxigenic C. difficile strains (ATCC 43255 and ATCC BAA-1805) at 1.5X LoD and 3X LoD, and a negative sample. Tested in triplicate.
  • Sample size: 360 positive results (4 positive samples x 3 replicates x 6 operators x 5 days), 90 negative results (1 negative sample x 3 replicates x 6 operators x 5 days).
  • Key results: Overall agreement for positive results was 99.4% (358/360) [98.0 - 99.9% CI]. Overall agreement for negative results was 98.9% (89/90) [94.0 - 100% CI]. Acceptable reproducibility demonstrated.

Clinical Studies:

• Prospective Multi-center Method Comparison Study: Compared performance of Toxigenic C. difficile Direct Test (CDF2) to an FDA-cleared molecular reference.
  • Sample size: 829 specimens.
  • Key results (All Sites):
    • Positive Percent Agreement (PPA): 95.8% (138/144) [91.2 - 98.5% CI]
    • Negative Percent Agreement (NPA): 98.5% (675/685) [97.3 - 99.3% CI]
    • Prevalence: 17.4%
    • Positive Predictive Value (PPV): 93.2% (87.9% - 96.7%)
    • Negative Predictive Value (NPV): 99.1% (98.1% - 99.7%)
• Discrepant Resolution Testing: Discrepant results (CDF2 vs. molecular reference) investigated using a third molecular test.
  • Key results: 5 of 6 false negative specimens tested negative on second FDA-cleared test (concordant with CDF2). 1 remaining false negative tested positive on BD Max (concordant with reference). All 10 CDF2 false positive results tested negative on second FDA-cleared test (concordant with reference).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Clinical Performance (All Sites):
Positive Percent Agreement (PPA): 95.8% (91.2 - 98.5) (138/144)
Negative Percent Agreement (NPA): 98.5% (97.3 - 99.3) (675/685)
Prevalence: 17.4%
Positive Predictive Value (PPV): 93.2% (87.9% - 96.7%)
Negative Predictive Value (NPV): 99.1% (98.1% - 99.7%)

Reproducibility:
Overall agreement of positive results: 99.4% (358/360) [98.0 - 99.9% CI]
Overall agreement of negative results: 98.9% (89/90) [94.0 - 100% CI]

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Meridian Bioscience® GenePOC® CDiff Assay (K172569)

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

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.

Summary

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November 14, 2023

Vela Operations USA Larry Rea Chief Operating Officer 2441 South 3850 West Salt Lake City, Utah 84120

Re: K232092

Trade/Device Name: Great Basin Toxigenic C. difficile Direct Test (CDF2) Regulation Number: 21 CFR 866.3130 Regulation Name: Clostridium Difficile Toxin Gene Amplification Assay Regulatory Class: Class II Product Code: OZN Dated: July 5, 2023 Received: July 13, 2023

Dear Larry Rea:

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 (the 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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrb/cfdocs/cfpmn/pmn.cfm identifies.combination product submissions. 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.

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

1

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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 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 Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely.

Natasha Griffin -S

0.B.O. Ribhi Shawar, Ph.D. (ABMM) Branch Chief General Bacteriology and Antimicrobial Susceptibility Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K232092

Device Name

Great Basin Toxigenic C. difficile Direct Test (CDF2)

Indications for Use (Describe)

The Great Basin Toxigenic C. difficile Direct Test, performed on the Great Basin PA500 Analyzer, is a qualitative in vitro diagnostic test for the detection of toxigenic Clostridioides difficile in unformed (liquid or soft) stool samples collected from patients suspected of having C. difficile infection (CDI). The automated assay utilizes polymerase chain reaction (PCR) to detect a conserved region of the toxin gene (tcdB) associated with toxin-producing C. difficile.

The Toxigenic C. difficile Direct Test is intended for use as an aid in the diagnosis of CDI in conjunction with clinical and epidemiological risk factors

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 - TOXIGENIC C. DIFFICILE DIRECT TEST

A. Submitted By

Vela Operations USA 2441 South 3850 West Salt Lake City, Utah 84120

Contact Information

Larry Rea Chief Operating Officer Vela Operations USA (DBA Great Basin Scientific)

B. Name of Device

Proprietary Name: Great Basin Toxigenic C. difficile Direct Test Common or Usual Names: Toxigenic C. difficile Direct Test CDF2

C. Requlatory Information:

• a. Requlation: 21 CFR 866.3130 - Clostridium difficile toxin gene amplification assay
• b. Classification: Class II (Toxigenic C. difficile Direct Test; non-exempt) Class II (PA500 Analyzer System)
• Microbiology (83) c. Panel:
• OZN C. difficile Toxin Gene Amplification Assay Product Code: ರ

D. Intended use(s)/Indications for Use

The Great Basin Toxigenic C. difficile Direct Test, performed on the Great Basin PA500 Analyzer, is a qualitative in vitro diagnostic test for the detection of toxigenic Clostridioides difficile in unformed (liguid or soft) stool samples collected from patients suspected of having C. difficile infection (CDI). The automated assay utilizes polymerase chain reaction (PCR) to detect a conserved region of the toxin gene (tcdB) associated with toxin producing C. difficile.

The Toxigenic C. difficile Direct Test is intended for use as an aid in the diagnosis of CDI in conjunction with clinical and epidemiological risk factors.

E. Device Description

Test Principle:

The Great Basin Toxigenic C. difficile Direct Test (CDF2) performed on the PA500 Analyzer utilizes automated, hot-start PCR amplification technology to amplify specific nucleic acid sequences that are then detected using hybridization probes immobilized on a modified silicon chip surface, in a single-use, self-contained test cartridge.

A swab volume of the specimen (raw stool) is first processed using the Sample Preparation Device (SPD). During processing through the SPD the specimen is infused with a synthetic bacterial sample processing control (SPC). An aliquot (250 µL) of the eluate obtained from the SPD, containing diluted specimen mixed with SPC, is loaded into the sample port of the CDF2 Test Cartridge.

Genomic DNA is extracted from microbial cells and diluted to reduce potential inhibitors of PCR. During the PCR process, biotin-labeled primers direct the amplification of specific nucleic acid sequences within a conserved region of the C. difficile Toxin B (tcdB) gene.

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Image /page/4/Picture/2 description: The image shows a pattern of black ellipses arranged in a triangular shape. The ellipses are arranged in rows, with each row containing fewer ellipses than the row below it. The bottom row has two ellipses, the next row has two ellipses, the next row has two ellipses, and the top row has two ellipses. The ellipses are all the same size and shape, and they are all oriented in the same direction.

Following PCR, biotin-labeled, amplified target DNA sequences are hybridized to sequence specific probes immobilized on the silicon chip surface and incubated with antibody conjugated to the horseradish peroxidase enzyme (HRP). The unbound conjugate is washed away, and then tetramethylbenzidine (TMB) is added to produce a colored precipitate at the location of the probe/target sequence complex.

The resulting signal is detected by the automated PA500 Optical Reader within the PA500 Analyzer System. The SPC undergoes the same extraction, and detection steps as the sample in order to monitor for inhibitory substances, as well as process inefficiency due to instrument or reagent failure. No operator intervention is required once the sample is loaded into the sample port and the Toxigenic C. difficile Direct Test cartridge is loaded into the PA500 Analyzer.

Test Device:

The Great Basin System is a fully automated in vitro diagnostic system that includes: the PA500 Analyzer, single-use SPDs, single-use Toxigenic C. difficile Direct Test Cartridges, and the PA500 Data Analysis Software Program. The PA500 Analyzer is designed to perform automated sample preparation, target amplification via PCR, chip-based (optical) target detection, and integrated data analysis in less than two hours. The PA500 Analyzer has been granted 510(k) clearance for the Portrait Toxigenic C. difficile Assay (DEN120013/K113358), Portrait GBS Assay (K143312), Staph ID/R Blood Culture Panel (K152470), Shiga Toxin Direct Test (K152955), Stool Bacterial Pathogens Panel (K163571), and the Bordetella Direct Test (K170284).

F. Substantial Equivalence Information

A comparison of the Toxigenic C. difficile Direct Test (CDF2) and the predicate device, Meridian Bioscience® GenePOC® CDiff Assay (K172569), is provided in Table 1.

Similarities
Features/
Characteristics	Great Basin Toxigenic C. difficile
Direct Test (CDF2)	Predicate Device
GenePOC® CDiff Assay (K172569)
Manufacturer	Vela Operations USA	Meridian Bioscience
Trade Name	Great Basin Toxigenic C. difficile Direct
Test	GenePOC® CDiff Assay
510(k) Number	K232092	K172569
Classification	II	II
Product Code	OZN	OZN
Intended Use/
Indications for Use	The Great Basin Toxigenic C. difficile
Direct Test, performed on the Great
Basin PA500 Analyzer, is a qualitative in
vitro diagnostic test for the detection of
toxigenic Clostridioides difficile in
unformed (liquid or soft) stool samples
collected from patients suspected of
having C. difficile infection (CDI). The
automated assay utilizes polymerase
chain reaction (PCR) to detect a
conserved region of the toxin gene
( tcdB ) associated with toxin-producing
C. difficile .

The Toxigenic C. difficile Direct Test is
intended for use as an aid in the
diagnosis of CDI in conjunction with
clinical and epidemiological risk factors. | The GenePOC CDiff assay performed
on the revogene instrument is a
qualitative in vitro diagnostic test that
utilizes automated sample processing
and real-time polymerase chain reaction
(PCR) to detect the toxin B ( tcdB ) gene
of toxigenic Clostridium difficile ( C.
difficile ) in unformed (liquid or soft) stool
specimens obtained from patients
suspected of having C. difficile infection
(CDI). The GenePOC CDiff assay is
intended to aid in the
diagnosis of CDI. |
| Qualitative/
Quantitative | Qualitative | Qualitative |

Table 1. Predicate Comparison

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Image /page/5/Picture/2 description: The image shows a series of black ellipses arranged in a pattern. The ellipses are organized in rows, with each row containing a decreasing number of ellipses as one moves upwards. The bottom row has two ellipses, the next row has two, then two, then one, and finally one. The ellipses are all oriented horizontally and appear to be of similar size and shape.

Instrument	PA500 Analyzer	revogene®
Specimen Type	Unformed (liquid or soft) stool
specimens	Same
Assay Target	toxin B gene (tcdB)	Same
Sample Lysis and
DNA Extraction	Automated sample lysis and DNA
extraction in a self-contained cartridge	Same
Amplification
Technology	Multiplex polymerase chain reaction
(PCR)	Same
Result
Interpretation	Automated	Same
Test Principle	Automated and integrated sample
preparation, nucleic acid amplification,
and detection of the target sequence in
a single-use, disposable test cartridge	Same
Controls	Integrated Sample Processing Control
(SPC) to control for adequate
processing of the target bacteria and
monitor the PCR amplification reaction
for the presence of inhibitors as well as
microfluidic cartridge, instrument or
reagent failure.

Fiducial and Hybridization Controls (HC) to control for cartridge selection,
cartridge integrity, and to verify
detection chemistry | Integrated Processing Control
(PrC) to verify sample processing and
amplification steps. The PrC allows for
the verification of potential
inhibitor substances as well as
microfluidic, instrument or reagent
failure. |

Differences
Features/
Characteristics	Great Basin Toxigenic C. difficile
Direct Test (CDF2)	Predicate Device
GenePOC® CDiff Assay (K172569)
Detection Probes	Hybridization probes	TaqMan probes
Detection
Technology	Colorimetric target specific hybridization
to probe on a chip surface, optical
reader, automated software with built-in
result interpretation.	Real-time PCR (fluorescent signals) and
embedded detection algorithm
Sample Input	1 Swab Volume	1 Disposable Transfer Loop Volume
(5 uL)
Samples per Run	1	Up to 8
Time to Result	~2 hours	~70 min

G. Performance Summary - Analytical Studies

a. Specimen Stability

A room temperature and refrigerated Specimen Stability study was conducted to determine the allowable time and temperature storage conditions for clinical specimens. The study was conducted using fresh, never frozen C. difficile ATCC 43255 cells spiked into a pooled, clinical negative stool matrix at a concentration of approximately 2.5X LoD. A true negative sample was also evaluated. Stability samples were tested in triplicate in a time course study. The Specimen Stability Study demonstrated that the C. difficile target remains stable up to and beyond 72 hours when specimens are stored refrigerated (2° - 8°C). The results also demonstrate that the CDF2 test performance is consistent and stable for low positive samples stored up to 24 hours at room temperature (18° - 22°C).

b. Analytical Sensitivity

The limit of detection (LoD) of the Toxigenic C. difficile Direct Test was measured for two (2) toxigenic Clostridioides difficile (tcdB+) strains: Clostridioides difficile ATCC 43255 (Toxinotype

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0. tcdA+, tcdB+) and Clostridioides difficile ATCC BAA-1805 (Toxinotype IIIb, NAP1/027, tcdA+, tcdB+).

For limit of detection (LoD) assessment, C. difficile strains were freshly cultured in liquid medium, serially diluted, and plated on agar to determine the cell concentration (i.e., CFU/mL). Enriched broths were then diluted into a pooled clinical negative stool to create a dilution series that was utilized for preliminary range finding on the CDF2 test. Results of the preliminary range finding were analyzed via Probit analysis to calculate the theoretical LoD at 95% agreement. The Probit estimated LoD was then confirmed with an additional round of testing from frozen cultures. The LoD for each strain tested is provided in Table 2.

Table 2. Toxigenic C. difficile Direct Test Limit of Detection

| Strain | Limit of Detection
(LoD) |
|---------------------------------------------|-----------------------------|
| Clostridioides difficile
(ATCC 43255) | $2.6 x 10^4$ CFU/mL |
| Clostridioides difficile
(ATCC BAA-1805) | $7.1 x 10^4$ CFU/mL |

c. Analytical Reactivity (Inclusivity)

The Analytical Reactivity of the Toxiqenic C. difficile Direct Test was evaluated by testing an additional twenty-two (22) toxigenic Clostridioides difficile strains covering six (6) unique toxinotypes. C. difficile strains were grown as described in the LoD studies, diluted into pooled, clinical negative stool to a concentration of 2X LoD (9.7 x 104 CFU/mL) and tested in triplicate. All additional strains tested were detected in CDF2.

The CDF2 test correctly identified every replicate of all 22 strains evaluated, indicating that the test can detect multiple strains and toxinotypes of Clostridioides difficile strains that harbor the tcdB toxin gene. Results of Analytical Reactivity (Inclusivity) testing are provided in Table 3.

Table 3. Clostridioides difficile Strains Tested for Analytical Reactivity (Inclusivity)

| Clostridioides

difficile Strain	Toxinotype, Toxin
ATCC 9689
ATCC 17857
ATCC 17858
ATCC 43594
ATCC 43596
ATCC 43599
ATCC 43600
ATCC 51695	0, A+, B+
ATCC 700792
ATCC BAA-1382
ATCC BAA-1804
ATCC BAA-1808
ATCC BAA-1871
ATCC BAA-1872
ATCC BAA-1874
ATCC BAA-2156
ATCC BAA-1803	IIIc, NAP1, A+, B+
ATCC BAA-1875	V, A+, B+
ATCC 43598	VIII, A-, B+
ATCC BAA-1812	XII, A+, B+
ATCC BAA-1814	XXII, A+, B+
ATCC BAA-2155

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Image /page/7/Picture/2 description: The image shows a pattern of black ovals arranged in a triangular shape. The ovals are stacked on top of each other, with the largest ovals at the bottom and the smallest ovals at the top. The ovals are all the same color and shape, and they are evenly spaced apart. There are 2 ovals on the bottom row, and the number of ovals decreases by one with each row.

d. Analytical Specificity (Exclusivity)

The potential for cross-reactivity to non-target organisms was evaluated in an Exclusivity Study, by testing microorganisms commonly found in stool, with the CDF2 test. The study included 83 microorganisms phylogenetically related to C. difficile, as well as other bacteria, fungi/yeast, parasites, viruses, and human genomic DNA (69 unique bacterial strains, 1 yeast, 3 parasites, 9 viruses, and human genomic DNA). Genomic DNA was tested in place of whole organism, for those isolates that were classified as Biosafety Level III, or unable to be cultured via standard clinical microbiology techniques.

Contrived samples were formulated for Exclusivity testing by spiking previously frozen, enumerated cell culture stocks, commercially sourced genomic templates, or commercially sourced viruses into pooled, clinical negative stool matrix. Apart from a single strain (Clostridium butyricum ATCC 19398 tested at an input of 1.0 x 108 CFU/mL), all bacterial and yeast strains were tested at concentrations ≥ 1x10° CFU/mL. Genomic templates and viral strains were tested at ≥ 1x10° copies/mL or ≥ 1x106 TCIDs0mL, respectively. Each organism was tested in triplicate.

All Exclusivity organisms (see Table 4) gave the expected 'NEGATIVE, Toxin NOT DETECTED' result indicating no cross-reactivity was observed in CDF2. The one exception was Salmonella enterica ATCC 6962 which was "Detected" in the CDF2 test in a single replicate. Suspecting contamination, the strain was re-grown, and testing was repeated. Upon retesting all replicates of Salmonella enterica ATCC 6962 yielded the expected 'Negative' result.

Species	Strain ID	Input Tested
Bacteria and Fungi
Abiotrophia defectiva	ATCC 49176	4.64 x 108 CFU/mL
Acinetobacter baumannii	ATCC19606	1.39 x 108 CFU/mL
Aeromonas hydrophila	ATCC 35654	9.70 x 107 CFU/mL
Alcaligenes faecalis	ATCC 15554	6.20 x 107 CFU/mL
Bacillus cereus	ATCC 14579	6.10 x 106 CFU/mL
Bacteroides fragilis	ATCC 23745	1.16 x 108 CFU/mL
Campylobacter coli	ATCC 49941	4.84 x 108 CFU/mL
Campylobacter fetus	ATCC 25936	3.30 x 106 CFU/mL
Campylobacter jejuni	ATCC 49943	1.50 x 109 CFU/mL
Candida albicans	ATCC 14053	1.40 x 106 CFU/mL
Citrobacter freundii	ATCC 8090	7.40 x 107 CFU/mL
Clostridioides difficile (tcdA-, tcdB-)	ATCC 43593	3.00 x 107 CFU/mL
Clostridioides difficile (tcdA-, tcdB-)	ATCC 43601	2.20 x 107 CFU/mL
Clostridium bifermentans	ATCC 638	1.61 x 107 CFU/mL
Clostridium butyricum	ATCC 19398	1.00 x 105 CFU/mL
Clostridium novyi	ATCC 19402	≥ 1 x 106 CFU/mL
Clostridium perfringens	ATCC 13124	1.10 x 107 CFU/mL
Clostridium scindens	ATCC 35704	7.02 x 107 CFU/mL
Clostridium septicum	ATCC 12464	≥ 1 x 106 CFU/mL
Clostridium sporogenes	ATCC 15579	5.80 x 106 CFU/mL
Edwardsiella tarda	ATCC 15947	1.10 x 108 CFU/mL
Enterobacter aerogenes	ATCC 15038	1.20 x 109 CFU/mL
Enterobacter cloacae	ATCC 13047	1.40 x 108 CFU/mL
Enterococcus faecalis	ATCC 29212	2.14 x 108 CFU/mL
Enterococcus faecalis (vanB)	ATCC 51299	5.65 x 107 CFU/mL
Enterococcus faecium	ATCC 19434	7.10 x 107 CFU/mL
Escherichia coli	ATCC 4157	6.80 x 107 CFU/mL
Escherichia coli (stx1+, stx2+, eae+)	ATCC BAA-2221	4.70 x 107 CFU/mL
Escherichia coli (stx1+, stx2+, O157:H7)	ATCC 43895	8.60 x 106 CFU/mL
Escherichia coli (stx2+)	ATCC BAA-2326	5.90 x 107 CFU/mL
Escherichia fergusonii	ATCC 35469	3.20 x 107 CFU/mL
Escherichia hermanii	ATCC 33650	5.60 x 106 CFU/mL
Flavonifractor plautii (Clostridium orbiscindens)	ATCC 49531	4.00 x 106 CFU/mL
Hathewaya histolytica (Clostridium histolyticum)	ATCC 19401	4.00 x 107 CFU/mL
Helicobacter pylori	ATCC 43504	2.55 x 107 CFU/mL
Klebsiella oxytoca	ATCC 49131	1.21 x 108 CFU/mL
Klebsiella pneumoniae	ATCC 13883	6.70 x 107 CFU/mL
Lactobacillus acidophilus	ATCC 4356	1.70 x 107 CFU/mL
Species	Strain ID	Input Tested
Lactobacillus lactis	ATCC 49032	3.64 x 108 CFU/mL
Listeria monocytogenes	ATCC 19115	1.34 x 108 CFU/mL
Paeniclostridium (Clostridium) sordellii	ATCC 9714	6.40 x 107 CFU/mL
Peptostreptococcus anaerobius	ATCC 27337	1.40 x 107 CFU/mL
Plesiomonas shigelloides	ATCC 51903	8.00 x 106 CFU/mL
Porphyromonas asaccharolytica	ATCC 27908	1.32 x 108 CFU/mL
Prevotella melaninogenica	ATCC 700524	2.25 x 106 CFU/mL
Proteus mirabilis	ATCC 25933	2.14 x 108 CFU/mL
Proteus vulgaris	ATCC 6896	4.90 x 107 CFU/mL
Providencia alcalifaciens	ATCC 9886	1.25 x 108 CFU/mL
Providencia rettgeri	ATCC 9250	1.25 x 108 CFU/mL
Pseudomonas aeruginosa	ATCC 10145	9.60 x 107 CFU/mL
Pseudomonas fluorescens	ATCC 13525	2.18 x 107 CFU/mL
Salmonella enterica subsp. Arizonae	ATCC 13314	7.98 x 108 CFU/mL
Salmonella enterica subsp. enterica	ATCC 13312	1.38 x 108 CFU/mL
Salmonella enterica subsp. enterica	ATCC 29628	4.20 x 107 CFU/mL
Salmonella enterica subsp. enterica		1.37 x 108 CFU/mL
Salmonella enterica subsp. enterica	ATCC 6962	1.40 x 108 CFU/mL
Salmonella enterica subsp. enterica	ATCC 6539	2.50 x 106 CFU/mL
Salmonella enterica subsp. enterica	ATCC 13311	1.37 x 108 CFU/mL
Serratia liquefaciens	ATCC 35551	1.90 x 108 CFU/mL
Serratia marcescens	ATCC 13880	1.12 x 108 CFU/mL
Shigella boydii	ATCC 29928	9.00 x 107 CFU/mL
Shigella dysenteriae	ATCC 9361	1.84 x 107 CFU/mL
Shigella flexneri	ATCC 25929	7.20 x 107 CFU/mL
Shigella sonnei	ATCC 25931	6.00 x 107 CFU/mL
Staphylococcus aureus	ATCC 43300	1.20 x 108 CFU/mL
Staphylococcus aureus	ATCC 12598	1.76 x 107 CFU/mL
Staphylococcus epidermidis	ATCC 12228	5.80 x 107 CFU/mL
Streptococcus agalactiae	ATCC 13813	1.20 x 106 CFU/mL
Streptococcus dysgalactiae	ATCC 43078	≥ 1 x 106 CFU/mL
Vibrio parahaemolyticus	ATCC 17802	1.71 x 107 CFU/mL
Yersinia enterocolitica	ATCC 49397	1.82 x 108 CFU/mL
Viruses and Parasites
Adenovirus type 40	ATCC VR-931D	1.67 x 1010 copies/mL
Adenovirus type 41	ATCC VR-930D	1.34 x 1013 copies/mL
Coxsackievirus B2	ATCC VR-29	1.10 x 107 TCID50/mL
Cryptosporidium parvum	ATCC PRA-67D	1.11 x 108 copies/mL
Echovirus 4	ATCC VR-1734D	4.25 x 109 copies/mL
Entamoeba histolytica	ATCC 30459DQ	5.70 x 107 copies/mL
Enterovirus 71	ATCC VR-1775DQ	4.80 x 107 copies/mL
Giardia intestinalis	ATCC 50803D	1.52 x 107 copies/mL
Herpesvirus 5 (Cytomegalovirus)	ATCC VR-538D	2.64 x 109 copies/mL
Norovirus GI	ATCC VR-3234SD	4.70 x 107 copies/mL
Norovirus GII	ATCC VR-3235SD	4.80 x 107 copies/mL
Rotavirus A	ATCC VR-2018DQ	5.30 x 107 copies/mL
Human genomic DNA	Promega G147A	4.99 x 106 copies/mL

Table 4. Analytical Specificity (Exclusivity) Study Results

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Image /page/8/Picture/2 description: The image shows a collection of black oval shapes arranged in a pyramid-like structure. The base of the pyramid consists of two ovals, followed by a layer of four ovals, then a layer of two ovals. The ovals are solid black and appear to be floating against a white background. The arrangement is symmetrical, with the ovals evenly spaced apart.

e. Microbial Interference (Competitive Inhibition)

The CDF2 test was evaluated for interference from mixed microbial populations using 82 microorqanisms tested in the Exclusivity Study. Potentially interfering microorganisms were pooled into seventeen (17) unique pools, each containing 3-5 non-target organisms (Table 5).

Test samples were contrived for Microbial Interference testing by first spiking previously frozen cultures of each non-target organism into pooled, negative clinical stool matrix. Each non-target organism was spiked into the Microbial Interference pool at the same input concentration tested in the Analytical Specificity (Exclusivity) Study (see Table 4 above). The Microbial Interference pool was then contrived to a final concentration of 2X LoD C. difficile ATCC 43255 (5.2 x 104 CFU/mL) with previously frozen cell stocks and tested in triplicate. Details of the Microbial Interference Study pools are shown in Table 5.

All Microbial Interference pool replicates gave the expected 'POSITIVE, Toxin DETECTED' result (100% agreement) indicating no interference or competitive inhibition from non-target microbes was observed.

9

	A

Potentially Interfering Microbe
Species	Strain ID	Pool
Aeromonas hydrophila	ATCC 35654
Bacteroides fragilis	ATCC 23745
Campylobacter coli	ATCC 49941	1
Campylobacter fetus	ATCC 25936
Campylobacter jejuni	ATCC 49943
Citrobacter freundii	ATCC 8090
Clostridioides difficile	ATCC 43593
Clostridioides difficile	ATCC 43601	2
Clostridium perfringens	ATCC 13124
Paeniclostridium (Clostridium) sordellii	ATCC 9714
Enterobacter cloacae	ATCC 13047
Enterococcus faecalis	ATCC 29212
Enterococcus faecium	ATCC 19434	3
Escherichia coli	ATCC 4157
Escherichia coli (O157:H7)	ATCC 43895
Escherichia fergusonii	ATCC 35469
Escherichia hermanii	ATCC 33650
Helicobacter pylori	ATCC 43504	4
Klebsiella pneumoniae	ATCC 13883
Peptostreptococcus anaerobius	ATCC 27337
Salmonella enterica	ATCC 13312
Salmonella enterica	ATCC 29628
Salmonella enterica	ATCC 6962	5
Salmonella enterica	ATCC 6539
Salmonella enterica	ATCC 13311
Serratia liquefaciens	ATCC 35551
Serratia marcescens	ATCC 13880
Shigella boydii	ATCC 29928	6
Shigella flexneri	ATCC 25929
Shigella sonnei	ATCC 25931
Candida albicans	ATCC 14053
Staphylococcus aureus	ATCC 43300
Staphylococcus aureus	ATCC 12598	7
Staphylococcus epidermidis	ATCC 12228
Yersinia enterocolitica	ATCC 49397
Klebsiella oxytoca	ATCC 49131
Proteus mirabilis	ATCC 25933
Salmonella enterica	ATCC 13314	8
Shigella dysenteriae	ATCC 9361
Streptococcus agalactiae	ATCC 13813
Acinetobacter baumannii	ATCC 19606
Bacillus cereus	ATCC 14579
Edwardsiella tarda	ATCC 15947	9
Enterobacter aerogenes	ATCC 15038
Pseudomonas fluorescens	ATCC 13525
Abiotrophia defectiva	ATCC 49176
Alcaligenes faecalis	ATCC 15554
Clostridium bifermentans	ATCC 638	10
Clostridium butyricum	ATCC 19398
Clostridium novyi	ATCC 19402
Hathewaya histolytica (Clostridium histolyticum)	ATCC 19401
Lactobacillus lactis	ATCC 49032
Listeria monocytogenes	ATCC 19115	11
Pseudomonas aeruginosa	ATCC 10145
Vibrio parahaemolyticus	ATCC 17802
Clostridium sporogenes	ATCC 15579
Lactobacillus acidophilus	ATCC 4356
Plesiomonas shigelloides	ATCC 51903	12
Proteus vulgaris	ATCC 6896

Table 5. Microbial Interference (Competitive Inhibition) Study Results

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Image /page/10/Picture/2 description: The image shows a pattern of black ellipses arranged in a triangular shape. The ellipses are oriented horizontally and decrease in size as they move upwards in the triangle. The base of the triangle consists of two large ellipses, while the top consists of two small ellipses.

Flavonifractor plautii (Clostridium orbiscindens)	ATCC 49531
Providencia alcalifaciens	ATCC 9886
Salmonella enterica	ATCC 6962	13
Escherichia coli	ATCC BAA-2221
Escherichia coli	ATCC BAA-2326
Adenovirus type 40	ATCC VR-931D
Enterovirus 71	ATCC VR-1775DQ
Norovirus GI	ATCC VR-3234SD	14
Norovirus GII	ATCC VR-3235SD
Rotavirus A	ATCC VR-2018DQ
Clostridium scindens	ATCC 35704
Clostridium septicum	ATCC 12464
Enterococcus faecalis (vanB)	ATCC 51299	15
Porphyromonas asaccharolytica	ATCC 27908
Prevotella melaninogenica	ATCC 700524
Adenovirus type 41	ATCC VR-930D
Coxsackievirus B2	ATCC VR-29
Herpesvirus 5 (Cytomegalovirus)	ATCC VR-538D	16
Echovirus 4	ATCC VR-1734D
Giardia intestinalis	ATCC 50803D
Cryptosporidium parvum	ATCC PRA-67D
Entamoeba histolytica	ATCC 30459DQ	17
Human aDNA	Promega G147A

Interfering Substances (Chemical Interference) f.

The CDF2 test was evaluated for potential endogenous Chemical Interference against a panel of thirty (30) substances that are common stool contaminants, or likely present in patients with diarrhea. Each substance was tested in the background of 2X LoD contrived positive stools and true negative stool. Two (2) C. difficile strains were evaluated in the Interfering Substances study at 2X LoD, ATCC 43255 (5.2 x 104 CFU/mL) and ATCC BAA-1805 (1.4 x 105 CFU/mL). The same clinical negative (i.e., un-spiked) stool matrix was also tested as a true negative to evaluate potential interference with the internal assay control in the absence of target analyte.

Each substance was tested in triplicate against both C. difficile strains and a negative sample for a total of nine (9) CDF2 tests per substance. During testing if any substance resulted in less than 100% agreement, the input concentration of the potentially interfering substance was reduced by 10-20% and re-tested stepwise until 100% agreement was achieved. The highest, non-inhibitory input is reported. No CDF2 interference was observed for any of the endogenous or exogenous substances at the concentrations listed in Table 6.

Table 6. Interfering Substances Study Results

| Interfering Substance | Input Tested | C. difficile
ATCC 43255
2X LoD | C. difficile
ATCC BAA-1805
2X LoD | Negative |
|--------------------------------------|--------------|--------------------------------------|-----------------------------------------|-------------------------|
| (Brand Evaluated) | | POSITIVE CDF2 Result | | NEGATIVE
CDF2 Result |
| Bismuth subsalicylate | 50% v/v | 3/3 | 3/3 | 3/3 |
| Liquid glycerin suppository | 50% v/v | 3/3 | 3/3 | 3/3 |
| Water-based personal lubricant | 50% v/v | 3/3 | 3/3 | 3/3 |
| Silicon-based personal lubricant | 50% v/v | 3/3 | 3/3 | 3/3 |
| Benzalkonium chloride wipe solution | 50% v/v | 3/3 | 3/3 | 3/3 |
| Aloe leaf juice flushable wipes | 50% v/v | 3/3 | 3/3 | 3/3 |
| Nonoxynol-9 condom lubricant | 40% w/v | 3/3 | 3/3 | 3/3 |
| Hemorrhoidal cream | 20% w/v | 3/3 | 3/3 | 3/3 |
| Hydrocortisone cream | 30% w/v | 3/3 | 3/3 | 3/3 |
| Miconazole nitrate anti-fungal cream | 30% w/v | 3/3 | 3/3 | 3/3 |
| Petroleum jelly | 30% w/v | 3/3 | 3/3 | 3/3 |
| Antacid tablet | 38% w/v | 3/3 | 3/3 | 3/3 |
| Loperamide anti-diarrheal | 50% v/v | 3/3 | 2/3 | 3/3 |
| Loperamide anti-diarrheal | 44% v/v | Not Tested | 3/3 | Not Tested |
| Senna glycoside natural laxative | 17% w/v | 3/3 | 3/3 | 3/3 |
| NSAID, Naproxen Sodium | 10% w/v | 3/3 | 3/3 | 3/3 |

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Image /page/11/Picture/2 description: The image shows a series of black oval shapes arranged in a triangular pattern. The ovals are of varying sizes, with the largest ones at the bottom and the smallest ones at the top. The ovals are arranged in rows, with each row containing a different number of ovals. The overall effect is one of a simple, abstract design.

| Interfering Substance
(Brand Evaluated) | Input Tested | C. difficile
ATCC 43255
2X LoD | C. difficile
ATCC BAA-1805
2X LoD | Negative |
|--------------------------------------------|--------------|--------------------------------------|-----------------------------------------|-------------------------|
| | | POSITIVE CDF2 Result | | NEGATIVE
CDF2 Result |
| Mineral oil | 50% v/v | 3/3 | 3/3 | 3/3 |
| Biotin supplement | 51% w/v | 3/3 | 3/3 | 3/3 |
| Barium sulfate | 25% w/v | 3/3 | 3/3 | 3/3 |
| Stearic acid | 1.1% w/v | 2/3 | 3/3 | 3/3 |
| | 1% w/v | 3/3 | Not Tested | Not Tested |
| Palmitic acid | 1.5% w/v | 3/3 | 3/3 | 3/3 |
| Whole Blood | 40% v/v | 3/3 | 3/3 | 3/3 |
| Biotin | 0.5 w/v | 3/3 | 3/3 | 3/3 |
| Nonoxynol-9 | 48% v/v | 3/3 | 2/3 | 3/3 |
| | 42% v/v | Not Tested | 3/3 | Not Tested |
| Mesalazine | 2.5% w/v | 3/3 | 3/3 | 3/3 |
| Mucin from Porcine Stomach | 2.5% w/v | 3/3 | 3/3 | 3/3 |
| Topical Antibiotic | 50% w/v | 3/3 | 3/3 | 3/3 |
| Nystatin | 10% w/v | 3/3 | 3/3 | 3/3 |
| Metronidazole | 0.5% w/v | 3/3 | 3/3 | 3/3 |
| Erythromycin | 10% w/v | 3/3 | 3/3 | 3/3 |
| | 5% w/v | 0/3 | 3/3 | 3/3 |
| Vancomycin (100 mg/mL in DMSO) | 0.4% w/v | 3/3 | Not Tested | Not Tested |

g. Reproducibility

A between-site Reproducibility Study was conducted at one (1) internal and two (2) external sites. The study incorporated several variables including multiple operators (n=6) across the three (3) study sites, multiple non-consecutive testing days (n=5), multiple Analyzers (n=24), and cartridge lots (n=2).

The Reproducibility Panel consisted of five (5) panel members. Each panel contained two (2) toxigenic Clostridioides difficile strains: ATCC 43255 (toxinotype 0, ribotype 087) and ATCC BAA-1805 (toxinotype IIIb, NAP1/ribotype 027). Each strain was evaluated at both low positive (1.5X LoD) and moderate positive (3X LoD) concentrations. A single negative sample was also included in the panel. Contrived Reproducibility Panel samples were prepared by spiking previously enumerated and frozen Clostridioides difficile cultures into pooled negative clinical stool matrix. The contrived panels were frozen, shipped to sites frozen, and thawed at the test site prior to testing on the Toxigenic C. difficile Direct Test.

Results of the Reproducibility Study are provided by site in Table 7.

| | Agreement
(Actual/Expected Outcomes) | | | | |
|-------------------------------|-----------------------------------------|------------------------------|------------------------------|------------------------------|-------------------------------|
| C. difficile Strain | ATCC 43255 | | ATCC BAA-1805 | | N/A |
| Concentration (LoD) | 1.5X | 3X | 1.5X | 3X | Negative |
| Clinical Site 1 | 100%
(30/30) | 100%
(30/30) | 100%
(30/30*) | 100%
(30/30*) | 100%
(30/30) |
| Clinical Site 2 | 93.3%
(28/30) | 100%
(30/30) | 100%
(30/30) | 100%
(30/30) | 96.7%
(29/30) |
| Internal Site | 100%
(30/30) | 100%
(30/30) | 100%
(30/30) | 100%
(30/30) | 100%
(30/30) |
| Total (All Sites)
[95% CI] | 97.8%
(88/90)
[92.2-99.7%] | 100%
(90/90)
[96-100%] | 100%
(90/90)
[96-100%] | 100%
(90/90)
[96-100%] | 98.9%
(89/90)
[94-100%] |

Table 7. Reproducibility Study Results

• This dataset contained a single (1) 'Invalid' result that was resolved upon retest.

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Image /page/12/Picture/2 description: The image shows a series of black ovals arranged in a pyramid-like structure. The ovals are stacked on top of each other, with the largest ovals at the bottom and the smallest ovals at the top. The ovals are all the same shape, but they vary in size. The image is simple and abstract.

Analysis of Positive Results: A total of four (4) positive samples were tested in triplicate by each of the 6 operators over 5 days. Therefore, the study contains a total of 60 positive results per operator and 360 total positives.

Two (2) low positive (test concentration = 1.5X LoD) Reproducibility panel samples yielded 'NEGATIVE' results in a single replicate. Both false negative replicates are assumed to be due to assay variability and stochastic sampling of the contrived sample for this analyte at concentrations very near the limit of detection. Although both false negatives occurred at Site 2, there was no commonality between the operator or the test day. Even with these two missed replicates the overall reproducibility for this analyte was still acceptable (≥ 95%).

Analysis of Negative Results: To assess negative results, aliquots of the same pooled negative matrix that was used to contrive the panel were tested. A single (1) neqative sample was included in each panel, tested in triplicate by each of 6 operators over 5 days each, therefore a total of 15 negative results per operator and 90 total negative results in the study.

A single negative Reproducibility panel sample yielded a 'POSITIVE' result in a single replicate. The false positive was most likely caused by unintentional sample contamination by the operator.

Overall agreement for positive and negative Reproducibility panel samples are provided in Table 8. The agreement of positive results for toxigenic C. difficile was 98.9% at low positive concentrations (1.5X LoD) and 100% at moderate positive concentrations (3X LoD). The overall agreement of positive results was 99.4% [Clgs 98.0 - 99.9%]. The overall agreement of negative results was 98.9% [Clgs 94.0 - 100%]. Given the number of variables included in this study (cartridge lots, analyzers, operators, sites, days) the study demonstrated acceptable reproducibility of the Toxigenic C. difficile Direct Test.

| Analyte | Concentration | Agreement
(Actual/Expected Outcomes)
[95% CI] | Agreement
(Actual/Expected Outcomes)
[95% CI] |
|-------------------------------------|---------------|-----------------------------------------------------|-----------------------------------------------------|
| Clostridioides difficile
(tcdB+) | 1.5X LoD | 98.9%
(178/180)
[96-99.9%] | 99.4%
(358/360)
[98-99.9%] |
| Clostridioides difficile
(tcdB+) | 3X LoD | 100%
(180/180)
[98-100%] | |
| Negative | N/A | 98.9%
(89/90)
[94-100%] | 98.9%
(89/90)
[94-100%] |

Table 8. Summary of Reproducibility Results

Performance Data - Clinical Studies H.

A prospective multi-center method comparison study was conducted to compare the performance of the Toxigenic C. difficile Direct Test (CDF2) to an FDA-cleared molecular reference. The study was conducted at three, geographically diverse, U.S. clinical study sites (Midwest, West) from June 2022 - February 2023.

Specimens enrolled in the study were excess remnants of unpreserved stool specimens that were prospectively collected from symptomatic individuals ≥ 2 years of age suspected of C. difficile infection (CDI), processed according to routine standard of care testing, and would have otherwise been discarded. All specimens were enrolled under an Institutional Review Board (IRB) approved protocol and testing was performed by trained laboratory personnel.

A total of 829 specimens met the inclusion criteria and were included in the final dataset included specimens collected from 431 females (52%), 393 males (47.4%), and 5 (0.6%) individuals of undeclared or unknown gender. The age distribution of study participants is shown in Table 9.

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Table 9. Study Population Distribution by Age Group

| Age Group
(Years) | Number of
Specimens |
|----------------------|------------------------|
| C. difficile
Direct
Test | Positive | 71 | 7 | 78 |
| | Negative | 2 | 320 | 322 |
| | Total | 73 | 327 | 400 |

Site 2		Molecular Comparator
		Positive	Negative	Total
Toxigenic
C. difficile
Direct
Test	Positive	60	2	62
	Negative	4	318	322
	Total	64	320	384

PPA = 93.8% (84.8% - 98.3%)
NPA = 99.4% (97.8% - 99.9%)
Prevalence = 16.7%
PPV = 96.8% (88.8% - 99.6%)
NPV = 98.8% (96.9% - 99.7%)

PPA = 100.0% (59.0% - 100.0%) NPA = 97.4% (86.2% - 99.9%)

Prevalence = 15.6%

PPA = 97.3% (90.5% - 99.7%) NPA = 97.9% (95.6% - 99.1%)

PPV = 91.0% (82.4% - 96.3%) NPV = 99.4% (97.8% - 99.9%)

Prevalence = 18.3%

Site 3		Molecular Comparator
		Positive	Negative	Total
Toxigenic
C. difficile
Direct
Test	Positive	7	1	8
	Negative	0	37	37
	Total	7	38	45

	All Sites	Molecular Comparator
		Positive	Negative	Total
Toxigenic
C. difficile
Direct
Test	Positive	138	10	148
	Negative	6	675	681
	Total	144	685	829

PPV = 87.5% (47.4% - 99.7%)	NPV = 97.8% (88.2% - 99.9%)
PPA = 95.8% (91.2% - 98.5%)	NPA = 98.5% (97.3% - 99.3%)

Prevalence = 17.4%
PPV = 93.2% (87.9% - 96.7%)
NPV = 99.1% (98.1% - 99.7%)

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Discrepant results were defined as results obtained from the CDF2 test that were not with the standard of care molecular reference method. Specimens with discrepant results were investigated by testing the specimens on a third molecular test.

As shown in Table 11, five (5) of six (6) false negative specimens also tested negative when evaluated with a second FDA-cleared molecular C. difficile test and are therefore concordant with the CDF2 result. The remaining false negative (1) specimen tested positive on the BD Max and found to be in concordance with the reference test.

All ten (10) CDF2 false positive results tested negative when evaluated with a second FDA-cleared molecular C. difficile test and found to be in concordance with the reference test.

Results of discrepant resolution testing are summarized in Table 11.

Table 11. Prospective Study Discrepant Testing Results

| Resolved
False Negatives | Resolved
False Positives |
|-----------------------------|-----------------------------|
| 5/6 | 0/10 |

Established clinical performance, positive (PPA) and negative (NPA) percent agreement along with 95% Confidence Intervals, of the Toxigenic C. difficile Direct Test is shown in Table 12. Overall, the results from the clinical evaluation demonstrated acceptable performance of the Great Basin Toxigenic C. difficile Direct Test and support the Intended Use of this product.

Table 12. Summary Table - Clinical Performance of the Toxigenic C. difficile Direct Test

| | n | PPA
(95% CI) | NPA
(95% CI) |
|--------------------------------------------------------|-----|-----------------------------------|-----------------------------------|
| Toxigenic
C. difficile Direct Test
(CDF2) | 829 | 95.8%
(91.2 - 98.5)
138/144 | 98.5%
(97.3 - 99.3)
675/685 |

l. Overall Conclusion

The information submitted in this 510(k) pre-market notification is complete and supports substantial equivalence.