(124 days)
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 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.
The Great Basin Toxigenic C. difficile Direct Test (CDF2) is a qualitative in vitro diagnostic test designed to detect toxigenic Clostridioides difficile in unformed stool samples from patients suspected of CDI. The device utilizes PCR to detect a conserved region of the toxin B gene (tcdB).
The study that proves the device meets the acceptance criteria is a prospective multi-center method comparison study (section H. Performance Data - Clinical Studies, page 12).
Here's a breakdown of the requested information:
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are not explicitly stated as numerical targets in the provided text. However, the study aims to demonstrate "acceptable performance" and states that the "information submitted...supports substantial equivalence." Based on the clinical performance, the implicit acceptance criteria would involve high positive and negative percent agreements with an FDA-cleared molecular reference.
| Metric | Acceptance Criteria (Implicit) | Reported Device Performance (All Sites Combined) |
|---|---|---|
| Positive Percent Agreement (PPA) | High agreement with reference | 95.8% (91.2% - 98.5% CI) |
| Negative Percent Agreement (NPA) | High agreement with reference | 98.5% (97.3% - 99.3% CI) |
2. Sample Size Used for the Test Set and Data Provenance
- Sample Size (Clinical Test Set): A total of 829 specimens were included in the final dataset (page 12).
- Data Provenance: The study was conducted at three geographically diverse U.S. clinical study sites (Midwest, West). The data is prospective, as specimens were collected from symptomatic individuals suspected of CDI between June 2022 and February 2023 (page 12). The specimens were "excess remnants of unpreserved stool specimens...processed according to routine standard of care testing, and would have otherwise been discarded" (page 12).
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications
The ground truth for the clinical test set was established using an FDA-cleared molecular reference (page 12), not human experts. Discrepant results were investigated by testing on a third molecular test (page 14). Therefore, no human experts were directly involved in establishing the primary ground truth for the clinical samples.
4. Adjudication Method for the Test Set
The adjudication method for discrepant results was comparison to a third FDA-cleared molecular C. difficile test (page 14). If the CDF2 test result differed from the initial molecular comparator, the specimen was re-tested using a third molecular test to resolve the discrepancy.
5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study was done
No, an MRMC comparative effectiveness study was not done. The study compares the device performance against a molecular reference, not human readers.
6. If a Standalone Study (algorithm only without human-in-the-loop performance) was done
Yes, this was a standalone study. The device is an automated in vitro diagnostic test where "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" (page 4). The performance metrics (PPA, NPA) directly reflect the device's accuracy against a molecular comparator without human interpretation of the results.
7. The Type of Ground Truth Used
The primary ground truth for the clinical study was an FDA-cleared molecular reference (page 12). For discrepant samples, a third FDA-cleared molecular C. difficile test was used for resolution (page 14).
8. The Sample Size for the Training Set
The document does not explicitly state a separate training set or its size for the algorithm itself. The studies described are primarily analytical (LoD, inclusivity, exclusivity, interference, reproducibility) and a clinical validation study. These types of in vitro diagnostic device submissions typically focus on analytical and clinical performance rather than a distinct "training set" for an AI algorithm in the same sense as image recognition or similar machine learning applications. The device's "automated data analysis software program" (page 4) is part of its design, and its performance is evaluated through the described analytical and clinical studies.
9. How the Ground Truth for the Training Set Was Established
As no specific "training set" is described for an AI algorithm, the method for establishing its ground truth is not detailed. However, the performance is established through rigorous analytical studies using characterized microbial strains and spiked samples (e.g., cell cultures with known CFU/mL) and clinical specimens compared to a molecular reference.
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Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.
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).
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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 (DICE@fda.hhs.gov) or phone (1-800-638-2041 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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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. difficileDirect Test (CDF2) | Predicate DeviceGenePOC® CDiff Assay (K172569) |
| Manufacturer | Vela Operations USA | Meridian Bioscience |
| Trade Name | Great Basin Toxigenic C. difficile DirectTest | 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. difficileDirect Test, performed on the GreatBasin PA500 Analyzer, is a qualitative invitro diagnostic test for the detection oftoxigenic Clostridioides difficile inunformed (liquid or soft) stool samplescollected from patients suspected ofhaving C. difficile infection (CDI). Theautomated assay utilizes polymerasechain reaction (PCR) to detect aconserved region of the toxin gene( tcdB ) associated with toxin-producingC. difficile .The Toxigenic C. difficile Direct Test isintended for use as an aid in thediagnosis of CDI in conjunction withclinical and epidemiological risk factors. | The GenePOC CDiff assay performedon the revogene instrument is aqualitative in vitro diagnostic test thatutilizes automated sample processingand real-time polymerase chain reaction(PCR) to detect the toxin B ( tcdB ) geneof toxigenic Clostridium difficile ( C.difficile ) in unformed (liquid or soft) stoolspecimens obtained from patientssuspected of having C. difficile infection(CDI). The GenePOC CDiff assay isintended to aid in thediagnosis of CDI. |
| Qualitative/Quantitative | Qualitative | Qualitative |
Table 1. Predicate Comparison
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| Instrument | PA500 Analyzer | revogene® |
|---|---|---|
| Specimen Type | Unformed (liquid or soft) stoolspecimens | Same |
| Assay Target | toxin B gene (tcdB) | Same |
| Sample Lysis andDNA Extraction | Automated sample lysis and DNAextraction in a self-contained cartridge | Same |
| AmplificationTechnology | Multiplex polymerase chain reaction(PCR) | Same |
| ResultInterpretation | Automated | Same |
| Test Principle | Automated and integrated samplepreparation, nucleic acid amplification,and detection of the target sequence ina single-use, disposable test cartridge | Same |
| Controls | Integrated Sample Processing Control(SPC) to control for adequateprocessing of the target bacteria andmonitor the PCR amplification reactionfor the presence of inhibitors as well asmicrofluidic cartridge, instrument orreagent failure.Fiducial and Hybridization Controls (HC) to control for cartridge selection,cartridge integrity, and to verifydetection chemistry | Integrated Processing Control(PrC) to verify sample processing andamplification steps. The PrC allows forthe verification of potentialinhibitor substances as well asmicrofluidic, instrument or reagentfailure. |
| Differences | ||
|---|---|---|
| Features/Characteristics | Great Basin Toxigenic C. difficileDirect Test (CDF2) | Predicate DeviceGenePOC® CDiff Assay (K172569) |
| Detection Probes | Hybridization probes | TaqMan probes |
| DetectionTechnology | Colorimetric target specific hybridizationto probe on a chip surface, opticalreader, automated software with built-inresult interpretation. | Real-time PCR (fluorescent signals) andembedded 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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- 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)
| Clostridioidesdifficile 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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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.
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| 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. difficileATCC 432552X LoD | C. difficileATCC BAA-18052X LoD | Negative |
|---|---|---|---|---|
| (Brand Evaluated) | POSITIVE CDF2 Result | NEGATIVECDF2 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. difficileATCC 432552X LoD | C. difficileATCC BAA-18052X LoD | Negative |
|---|---|---|---|---|
| POSITIVE CDF2 Result | NEGATIVECDF2 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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Image /page/13/Picture/2 description: The image shows a stack of black oval shapes. The ovals are arranged in a pyramid shape, with the largest ovals at the bottom and the smallest ovals at the top. There are two ovals in each row, and the rows are stacked on top of each other.
Table 9. Study Population Distribution by Age Group
| Age Group(Years) | Number ofSpecimens |
|---|---|
| <2 | Excluded |
| 2-12 | 37 (4.5%) |
| 13-18 | 32 (3.9%) |
| 19-64 | 442 (53.3%) |
| 65-88 | 299 (36.1%) |
| ≥ 89 | 19 (2.3%) |
| Total | 829 (100.0%) |
Results of the prospective method comparison study were analyzed for concordance by site and in total using a 2x2 table. The positive percent agreement (PPA), negative percent agreement (NPA), disease prevalence, positive predictive value (PPV), negative predictive value (NPV) along with the 95% confidence intervals (CI) were then calculated. Results of Prospective Specimen testing by site and in total are shown in Table 10.
| Table 10. Prospective Study Results | |
|---|---|
| ------------------------------------- | -- |
| Site 1 | Molecular Comparator | |||
|---|---|---|---|---|
| Positive | Negative | Total | ||
| ToxigenicC. difficileDirectTest | Positive | 71 | 7 | 78 |
| Negative | 2 | 320 | 322 | |
| Total | 73 | 327 | 400 |
| Site 2 | Molecular Comparator | |||
|---|---|---|---|---|
| Positive | Negative | Total | ||
| ToxigenicC. difficileDirectTest | 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 | ||
| ToxigenicC. difficileDirectTest | Positive | 7 | 1 | 8 |
| Negative | 0 | 37 | 37 | |
| Total | 7 | 38 | 45 |
| All Sites | Molecular Comparator | |||
|---|---|---|---|---|
| Positive | Negative | Total | ||
| ToxigenicC. difficileDirectTest | 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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Image /page/14/Picture/2 description: The image shows a series of black oval shapes arranged in a step-like 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 a way that creates a sense of depth and perspective. The image is simple and minimalist, with a focus on the shapes and their arrangement.
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
| ResolvedFalse Negatives | ResolvedFalse 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) | |
|---|---|---|---|
| ToxigenicC. 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.
§ 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.