K Number
K163085
Date Cleared
2017-02-14

(103 days)

Product Code
Regulation Number
866.3130
Reference & Predicate Devices
Predicate For
N/A
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdpartyExpeditedreview
Intended Use

Simplexa C. difficile Direct: The Focus Diagnostics Simplexa™ C. difficile Direct assay is intended for use on the Integrated Cycler instrument for the detection of Clostridium difficile toxin B gene (tcdB) present in liquid or unformed stool samples from individuals suspected of C. difficile infection (CDI). This test aids in the diagnosis of illness resulting from CDI. The assay is for professional and prescription use only.

Simplexa C. difficile Positive Control Pack: The Simplexa™ C. difficile Positive Control Pack is intended to be used as a control with the Simplexa™ C. difficile Direct kit. This control is not intended for use with other assays or systems.

Device Description

The Simplexa™ C. difficile Direct assay system is a real-time PCR system that enables the direct amplification and detection of toxigenic C. difficile DNA from unprocessed liquid or unformed stool specimens that have not undergone nucleic acid extraction. The system consists of the Simplexa™ C. difficile Direct assay, the Integrated Cycler (with Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories. In the Simplexa™ C. difficile Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify C. difficile and DNA internal control targets. The assay targets a sequence which is in a well conserved region of C. difficile toxin B gene (todB). A DNA internal control is used to detect PCR failure and/or inhibition

AI/ML Overview

Acceptance Criteria and Device Performance for Simplexa C. difficile Direct

1. Table of Acceptance Criteria and Reported Device Performance

The provided document describes the performance of the Simplexa C. difficile Direct assay in comparison to various gold standards and other NAATs. The acceptance criteria are implicit in the presented tables.

Acceptance Criteria for Simplexa C. difficile Direct (versus Combined Culture + Toxin Assay):

MetricAcceptance Criteria (Implied)Reported Device Performance95% Confidence Interval (CI)
SensitivityNot explicitly stated85.9% (336/391)82.1% to 89.0%
SpecificityNot explicitly stated95.1% (1849/1945)94.0% to 95.9%
PPVNot explicitly stated77.8% (336/432)73.6% to 81.4%
NPVNot explicitly stated97.1% (1849/1904)96.3% to 97.8%

Additional Performance (versus other FDA-cleared NAATs for reference):

ComparisonMetricReported Device Performance95% Confidence Interval (CI)
Simplexa vs NAAT-1Positive Agreement93.4% (114/122)87.6% to 96.6%
Negative Agreement96.6% (683/707)95.0% to 97.7%
Simplexa vs NAAT-2Positive Agreement93.9% (138/147)88.8% to 96.7%
Negative Agreement94.0% (591/629)91.8% to 95.6%
Simplexa vs NAAT-3Positive Agreement84.8% (112/132)77.8% to 90.0%
Negative Agreement99.2% (584/589)98.0% to 99.6%

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

The primary clinical performance study (method comparison) used 2351 samples prospectively collected.

  • Data Provenance: Samples were prospectively collected from five (5) geographically diverse sites in the USA between December 3, 2015, and June 10, 2016.
  • Evaluable Samples: 2330 samples were evaluable on Simplexa C. difficile Direct & the Direct Culture method, and 2336 were evaluable on Simplexa C. difficile Direct & the Combination of Direct/Enriched Culture methods.
  • Invalid Rate: The initial invalid rate was 0.64% (15/2351), reduced to a final invalid rate of 0.13% (3/2351) after repeat testing.

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

The document does not explicitly state the "number of experts" or their "qualifications" involved in establishing the ground truth for the clinical sample test set. However, the ground truth was based on Direct Culture + Toxin Assay and Combined Culture (Direct & Enriched) + Toxin Assay, with confirmatory testing using FDA cleared nucleic acid amplification test (NAAT) results for discrepant analysis. This implies that the ground truth was established by laboratory methods and not by individual expert interpretation.

4. Adjudication Method for the Test Set

For discrepant samples in the clinical study, discrepant analysis was performed using FDA cleared nucleic acid amplification test (NAAT) results provided by the sites.

  • For the "Simplexa C. difficile Direct versus Direct Toxigenic Culture Method" table:
    • 116/163 discrepant Simplexa-positive/Culture-negative samples were positive, and 46/163 were indeterminate for C. difficile Toxin B gene DNA when tested using an FDA cleared molecular test.
    • 8/12 discrepant Simplexa-negative/Culture-positive samples were negative, and 4/12 were positive for C. difficile Toxin B gene DNA when tested using an FDA cleared molecular test.
  • For the "Simplexa C. difficile Direct versus Combined Direct and Enriched Toxigenic Culture Methods" table:
    • 59/96 discrepant Simplexa-positive/Culture-negative samples were positive, and 37/96 were negative for C. difficile Toxin B gene DNA when tested using an FDA cleared molecular test.
    • 43/55 discrepant Simplexa-negative/Culture-positive samples were negative for C. difficile Toxin B gene DNA when tested using an FDA cleared molecular test.

This indicates an adjudication process involving a third, independent, FDA-cleared NAAT for resolving discrepancies between the investigational device and the culture methods.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, an MRMC comparative effectiveness study was not explicitly described in the provided text. The study focuses on the performance of the analytical device rather than human reader interpretation with or without AI assistance.

6. If a Standalone Study (Algorithm Only Without Human-in-the-loop Performance) Was Done

Yes, the studies presented are standalone performance evaluations of the Simplexa C. difficile Direct assay. The assay is an automated real-time PCR system designed for direct detection of C. difficile toxin B gene, meaning its performance is measured independently of human interpretation of its results, although trained lab personnel would operate the instrument.

7. The Type of Ground Truth Used

The ground truth for the clinical performance study was established using:

  • Direct Culture + Toxin Assay
  • Combined Culture (Direct & Enriched) + Toxin Assay
  • FDA cleared molecular tests (NAATs) for discrepant analysis.

This represents a composite reference standard based on microbiological culture techniques and molecular detection for toxigenic C. difficile, with additional molecular confirmation for discordant results.

8. The Sample Size for the Training Set

The document does not specify a separate "training set" sample size. The studies described are performance evaluations of the device, implying that the device's development and internal optimization would have occurred prior to these validation studies. Therefore, information regarding a training set is not provided as this is a diagnostic assay, not a machine learning algorithm that requires a separate training and test set in the same way.

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

As no specific training set is mentioned for the device (which is a molecular diagnostic assay), the method for establishing ground truth for such a set is not applicable or described in this document. The focus is on the analytical and clinical validation of the completed product.

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Image /page/0/Picture/1 description: The image shows the logo for the Department of Health & Human Services - USA. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter. Inside the circle is a stylized image of three human profiles facing to the right, with a flowing, ribbon-like design connecting them.

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

February 14, 2017 FOCUS DIAGNOSTICS, INC.: DBA DIASORIN MOLECULAR LLC IRENE M. GUZMAN SENIOR REGULATORY AFFAIRS SPECIALIST 11331 VALLEY VIEW STREET CYPRESS CA 90630

Re: K163085 Trade/Device Name: Simplexa C. difficile Direct: Simplexa C. difficile Positive Control Pack Regulation Number: 21 CFR 866.3130 Regulation Name: Clostridium difficile toxin gene amplification assay Regulatory Class: II Product Code: OZN Dated: January 12, 2017 Received: January 17, 2017

Dear Ms. Guzman:

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

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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

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

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

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

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

Sincerely yours,

Ribhi Shawar -A

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

Enclosure

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Indications for Use

510(k) Number (if known) K163085

Device Name

Simplexa C. difficile Direct Simplexa C. difficile Positive Control Pack

Indications for Use (Describe)

Simplexa C. difficile Direct

The Focus Diagnostics Simplexa™ C. difficile Direct assay is intended for use on the Integrated Cycler instrument for the detection of Clostridium difficile toxin B gene (tcdB) present in liquid or unformed stool samples from individuals suspected of C. difficile infection (CDI). This test aids in the diagnosis of illness resulting from CDI. The assay is for professional and prescription use only.

Simplexa C. difficile Positive Control Pack

The Simplexa™ C. difficile Positive Control Pack is intended to be used as a control with the Simplexa™ C. difficile Direct kit.

This control is not intended for use with other assays or systems.

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

X Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

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Image /page/3/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo consists of the word "FOCUS" in bold, sans-serif font, with a curved, swooping line extending from the top left of the "F" and arching over the word. Below "FOCUS" is the word "Diagnostics" in a smaller, sans-serif font, underlined with a thin line.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017 Page 1 of 20

ApplicantFocus Diagnostics, Inc.DBA DiaSorin Molecular LLC11331 Valley View StreetCypress, California 90630USA
Establishment Registration No.2023365
Contact PersonIrene M. Guzmantel 562.240.6133fax 562.240.6529irene.guzman@diasorin.com
Summary DateFebruary 10, 2017
Proprietary NameSimplexa™ C. difficile Direct andSimplexa™ C. difficile Positive Control Pack
Generic NameC. difficile RT PCR
Classification21 CFR 866.3130 Clostridium difficile toxin gene amplification assayClass II Special Controls
Predicate DeviceBD MAX CDIFF ASSAY (K130470)

Intended Use

Simplexa™ C. difficile Direct (MOL2950)

The Focus Diagnostics Simplexa™ C. difficile Direct assay is intended for use on the Integrated Cycler instrument for the detection of Clostridium difficile toxin B gene (tcdB) present in liquid or unformed stool samples from individuals suspected of C. difficile infection (CDI). This test aids in the diagnosis of illness resulting from CDI.

The assay is for professional and prescription use only.

Simplexa™ C. difficile Positive Control Pack (MOL2960)

The Simplexa™ C. difficile Positive Control Pack is intended to be used as a control with the Simplexa™ C. difficile Direct kit.

This control is not intended for use with other assays or systems.

Device Description

The Simplexa™ C. difficile Direct assay system is a real-time PCR system that enables the direct amplification and detection of toxigenic C. difficile DNA from unprocessed liquid or unformed stool specimens that have not undergone nucleic acid extraction. The system consists of the Simplexa™ C. difficile Direct assay, the Integrated Cycler (with Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ C. difficile Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify C. difficile and DNA internal control targets. The assay targets a sequence which is in a well conserved region of C. difficile toxin B gene (todB). A DNA internal control is used to detect PCR failure and/or inhibition

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Image /page/4/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo has the word "FOCUS" in large, bold, sans-serif font. To the left of the word is a curved, black shape. Below the word "FOCUS" is a horizontal line, and below that is the word "Diagnostics" in a smaller, sans-serif font.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 2 of 20

Predicate Device Information

ItemDevicePredicate
NameSimplexa™ C. difficile DirectBD MAX™ Cdiff(K130470)
Intended UseThe Focus Diagnostics Simplexa™ C. difficile Direct assay is intended for useon the Integrated Cycler instrument for the detection of Clostridium difficile toxinB gene (tcdB) present in liquid or unformed stool samples from individualssuspected of C. difficile infection (CDI). This test aids in the diagnosis of illnessresulting from CDI.The BD MAX™ Cdiff Assay performed on the BD MAX™ System is an automated invitro diagnostic test for the direct, qualitative detection of the Clostridium difficiletoxin B gene (tcdB) in human liquid or soft stool specimens from patients suspectedof having C. difficile infection (CDI). The test, performed directly on the specimen,utilizes real-time polymerase chain reaction (PCR) for the amplification of C. difficiletoxin B gene DNA and fluorogenic target-specific hybridization probes for thedetection of the amplified DNA. The BD MAX™ Cdiff Assay is intended to aid in thediagnosis of CDI.
Assay TargetsC. difficile toxin B gene (tcdB)C. difficile toxin B gene (tcdB)
Sample TypesLiquid or unformed stoolLiquid or soft stool specimen
Extraction MethodsAutomatedAutomated
Assay MethodologyThe Simplexa C. difficile Direct assay incorporates direct, qualitative detectionof toxigenic C. difficile DNA from clinical specimens in human stool specimensusing the Integrated Cycler. The assay utilizes real-time PCR technology withfluorescently labeled, bifunctional primer-probes that amplify and detect aconserved region of the toxin B (tcdB) gene.The BD MAX™ Cdiff Assay performed on the BD MAX™ System is an automated invitro diagnostic test for the direct, qualitative detection of the Clostridium difficiletoxin B gene (tcdB) in human liquid or soft stool specimens from patients suspectedof having C. difficile infection (CDI). The test, performed directly on the specimen,utilizes real-time polymerase chain reaction (PCR) for the amplification of C. difficiletoxin B gene DNA and fluorogenic target-specific hybridization probes for thedetection of the amplified DNA. The BD MAX™ Cdiff Assay is intended to aid in thediagnosis of CDI.
DetectionTechniquesReal time PCR with bifunctional fluorescent primer-probes using the IntegratedCycler.The test, performed directly on the specimen, utilizes real-time polymerase chainreaction (PCR) for the amplification of C. difficile toxin B gene DNA and fluorogenictarget- specific hybridization probes for the detection of the amplified DNA.
Limit of DetectionC. difficile Bacterial StrainLoD Concentration (CFU/mL)ATCC 432550.95NAP1A0.43C. difficile Strain Toxinotype LoD in CFU per-loopATCC® 43255 0 265 (95% CI: 140, 502)ATCC® 9689 0 156 (95% CI: 82. 298)ATCC® BAA-i1805 III 205 (95% CI: 102, 412)ATCC® 43598 VIII 125 (95% CI: 66, 235)
ItemDevicePredicate
NameSimplexa™ C. difficile DirectBD MAX™ Cdiff(K130470)
ReproducibilityLow Positive 100% (90/90)Medium Positive 100% (89/89)High Negative 98.9% (89/90)No Template Control (NTC) 98.9% (89/90)ATCC 43255 – LP*54.4% (49/90) Positive95% CI: 44.2 to 64.3%ATCC 43255 – LP**100.0% (90/90) Positive95% CI: 95.9 to 100%ATCC 43255 – MP100.0% (90/90) Positive95% CI: 95.9 to 100.0%Negative100% 100.0% (90/90) Negative95% CI: 95.9 to 100.0%Positive Control100.0% (90/90) Positive95% CI: 95.9 to 100.0%NAP1A — LP98.9% (89/90) Positive95% CI: 94.0 to 99.8%NAP1A – MP100.0% (90/90) Positive95% CI: 95.9 to 100.0%*Lower percent detection of C. difficile is related to qualified underperformance ofmatrix during original build as determined in LoD study.**Reformulated with a different matrixFor Site-to-Site Reproducibility, the overall percent agreement was 100% for MP, LPand Neg categories, with 92.2% and 50.0% negative agreement for HN1:100 andHN1:10 categories, respectively. For Lot-to- Lot Reproducibility, the overall percentagreement was 100% for MP, LP and Neg categories, with 96.7% and 64.4%negative agreement for HN1:100 and HN1:10 categories, respectively. SecondDerivative Peak Abscissa (SDPA), an internal criteria used to determine a final assayresult, was selected as an additional means of assessing assay reproducibility.

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Image /page/5/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo consists of the word "FOCUS" in bold, sans-serif font, with a curved graphic element to the left of the word. Below "FOCUS" is the word "Diagnostics" in a smaller, non-bold font, underlined with a thin line. The entire logo is in black and white.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960

February 10, 2017

Page 3 of 20

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510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017 Page 4 of 20

METHOD COMPARISON

Two thousand three hundred and fifty one (2351) samples were prospectively collected from five (5) geographically diverse sites between December 3, 2015 and June 10, 2016 from patients with signs and symptoms of C. difficile infection. Of the 2351 specimens, 2330 were evaluable on Simplexa™ C. difficile Direct & the Direct Culture method and 2336 were evaluable on Simplexa™ C. difficile Direct & the Combination of Direct/Enriched Culture methods. Of the combination culture evaluable specimens, 1232 (52.7%) were collected from females and 1104 (47.3%) were collected from males. The patient ages ranged from less than one year old to 106 years old, and 2198 (94.1%) were 22 years old or older.

Samples were tested on Simplexa™ C. difficile Direct at the collection sites and the comparator culture method was performed at one (1) central laboratory. The initial invalid rate of the clinical prospective study using Simplexa™ C. difficile Direct was 0.64% (15/2351 samples). After repeat testing, 3/15 samples remained invalid, thus resulting in a final invalid rate of 0.13% (3/2351 samples). Discrepant analysis was performed using FDA cleared nucleic acid amplification test (NAAT) results provided by the sites.

Positive Controls and No Template Controls were tested daily at each site during the clinical studies. A total of 458 Positive Controls and 459 No Template Controls were tested across all sites and produced valid expected initial results for 456 (99.6%) Positive Controls and 451 (98.3%) No Template Controls. After QC re-testing, 100% of the unexpected QC results were resolved by repeat.

Direct Culture + Toxin Assay
Simplexa ResultsDetectedNot DetectedTotal
Detected265163a428
Not Detected12b18901902
Total27720532330c
Positive Agreement95.7% (265/277)95% CI: 92.6% to 97.5%Negative Agreement92.1% (1890/2053)95% CI: 90.8% to 93.2%

Simplexa™ C. difficile Direct versus Direct Toxigenic Culture Method

a 116/163 discrepant samples were positive, 46/163 was indeterminate for C. difficile Toxin B gene DNA when tested using a FDA cleared molecular test.

8/12 discrepant samples were negative and 4/12 were positive for C. difficile Toxin B gene DNA when tested using a FDA cleared molecular test.

· Direct culture results were not available from 6 specimens due to plating errors. Overall results were obtained by enriched culture for the 6 samples and are tabulated in the Combined Culture.

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Image /page/7/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo consists of the word "FOCUS" in bold, sans-serif font, with the word "Diagnostics" in a smaller, sans-serif font underneath. A curved, black swoosh is located to the left of the word "FOCUS", adding a dynamic element to the design. A horizontal line is placed above the word "Diagnostics".

K163085

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017 Page 5 of 20

Simplexa™ C. difficile Direct versus Combined Direct and Enriched Toxigenic Culture Methods

Combined Culture (Direct & Enriched) + Toxin Assay
SimplexaDetectedNot DetectedTotal
Detected33696a432
Not Detected55b18491904
Total39119452336
Sensitivity85.9%(336/391)95% CI: 82.1% to 89.0%Specificity95.1%(1849/1945)95% CI: 94.0% to 95.9%
PPV77.8%(336/432)95% CI: 73.6% to 81.4%NPV97.1%(1849/1904)95% CI: 96.3% to 97.8%

4 59/96 discrepant samples were positive and 37/96 were negative for C. difficile Toxin B gene DNA when tested using a FDA cleared molecular test.

® 43/55 discrepant samples were negative for C. difficile Toxin B gene DNA when tested using a FDA cleared molecular test.

In addition, samples from the multi-site investigational study were also tested with three (3) commercially available FDA cleared molecular NAAT assays, and the performance of the Simplexa™ C. difficile Direct assay was compared to the cleared assays. A total of 2326 specimens were tested across 5 external sites. The Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) was calculated for the Simplexa™ C. difficile Direct versus each of the NAAT comparators.

Simplexa™ C. difficile Direct versus Nucleic Acid Amplification Test 1 (NAAT-1)

NAAT1
Simplexa™DetectedNot DetectedTotal
Detected11424138
Not Detected8683691
Total122707829
Positive Agreement93.4% (114/122)95% CI: 87.6% to 96.6%Negative Agreement96.6% (683/707)95% CI: 95.0% to 97.7%

Simplexa™ C. difficile Direct versus Nucleic Acid Amplification Test 2 (NAAT-2)

NAAT-2
Simplexa™DetectedNot DetectedTotal
Detected13838176
Not Detected9591600
Total147629776
Positive Agreement93.9% (138/147)95% CI: 88.8% to 96.7%Negative Agreement94.0% (591/629)95% CI: 91.8% to 95.6%

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Image /page/8/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo features the word "FOCUS" in bold, sans-serif font, with a curved, swooping line extending from the left side of the "F" and arching over the word. Below "FOCUS" is the word "Diagnostics" in a smaller, sans-serif font, underlined with a thin line. The overall design is clean and professional.

K163085

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017 Page 6 of 20

NAAT-3
Simplexa™DetectedNot DetectedTotal
Detected1125117
Not Detected20584604
Total132589721
Positive Agreement84.8% (112/132)95% CI: 77.8% to 90.0%Negative Agreement99.2% (584/589)95% CI: 98.0% to 99.6%

Simplexa™ C. difficile Direct versus Nucleic Acid Amplification Test 3 (NAAT-3)

REPRODUCIBILITY

Reproducibility for Simplexa™ C. difficile Direct was performed at three sites on at least five non-consecutive days per site. A total of seven sample panel members were tested to evaluate reproducibility of the assay.

The panel included a positive control (PC), a negative control (TE-stool matrix without analyte) and four contrived samples spiked into a negative stool - TE matrix. The four contrived samples consisted of a low positive (LP) and a medium positive (MP) for C. difficile. Each contrived sample was prepared by spiking a specific concentration of the strain(s) into negative stool - TE matrix. Combined results for all sites are presented in the table below. The invalid rate for the reproducibility study using Simplexa™ C. difficile Direct was 0.24% (2/819 samples). After repeat testing, 0/2 samples remained invalid, thus resulting in a final invalid rate of 0.0% (0/819 samples).

Site - 1Site - 2Site - 3Total %
Sample%AgreementwithExpectedResultsAvgCtTotal%CV%AgreementwithExpectedResultsAvgCtTotal%CV%AgreementwithExpectedResultsAvgCtTotal%CVAgreementwithExpectedResults95% CI
ATCC43255 — LP100.0%(30/30)38.01.5100.0%(30/30)38.11.4100.0%(30/30)37.51.4100.0%(90/90)95.9% to 100.0%
ATCC43255 - MP100.0%(30/30)37.31.7100.0%(30/30)37.11.3100.0%(30/30)37.01.5100.0%(90/90)95.9% to 100.0%
Negative1100.0%(30/30)N/AN/A100.0%(30/30)N/AN/A100.0%(30/30)N/AN/A100.0%(90/90)95.9% to 100.0%
PositiveControl100.0%(30/30)31.71.7100.0%(30/30)31.41.6100.0%(30/30)30.91.4100.0%(90/90)95.9% to 100.0%
NAP1A - LP100.0%(30/30)39.62.6100.0%(30/30)39.12.096.7%(29/30)39.41.998.9%(89/90)94.0% to 99.8%
NAP1A -MP100.0%(30/30)36.41.3100.0%(30/30)36.31.0100.0%(30/30)36.61.4100.0%(90/90)95.9% to 100.0%

1Expected results of negative sample is "Negative" for C. difficile.

ANALYTICAL SENSITIVITY/LIMIT OF DETECTION

The Limit of Detection (LoD) for Simplexa™ C. difficile Direct was determined using a panel consisting of negative stool-TE matrix spiked with 2 strains of C. difficile bacterial stock. The stock was serially diluted. A total of 32 replicates were tested. The lowest concentration level of C. difficile where at least 95% of the replicates were detected as "C. difficile" was determined to be the LoD of the strain.

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K163085

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017 Page 7 of 20

The following table shows Limit of Detection (LoD) value in terms of CFU/mL for two C. difficile bacterial strains.

C. difficile Bacterial StrainLoD Concentration (CFU/mL)
ATCC 432550.95
NAP1A0.43

ANALYTICAL REACTIVITY

Simplexa™ C. difficile Direct was evaluated for analytical reactivity to 32 C. difficile strains not tested in the Limit of Detection (LoD) study.

Analytical reactivity samples were prepared by spiking one of the 32 regrown and re-titered strains into negative stool-TE matrix at 2-4 X LoD. Each sample was tested in triplicate. The strain was initially spiked at a 2-4 X LoD. If at least one of three replicates was negative for C. difficile, a higher concentration was tested until all three replicates were detected.

All 32 strains were detected positive for C. difficile at or below 512 CFU/mL

StrainConcentration(CFU/mL)ToxinotypeToxinRibotype
Clostridium difficile ATCC 17857160A+B+001
Clostridium difficile ATCC 1785880A+B+054
Clostridium difficile ATCC 43594160A+B+005
Clostridium difficile ATCC 4359640A+B+012
Clostridium difficile ATCC 435984VIIIA-B+017
Clostridium difficile ATCC 43599320A+B+001
Clostridium difficile ATCC 4360040A+B+014
Clostridium difficile ATCC 5169540A+B+001
Clostridium difficile ATCC 700792160A+B+005
Clostridium difficile ATCC 968940A+B+001
Clostridium difficile BAA-138240A+B+012
Clostridium difficile BAA-18058IllbA+B+027
Clostridium difficile BAA-1814256XXIIA+B+251
Clostridium difficile BAA-18704IllbA+B+027
Clostridium difficile BAA-187140A+B+001
Clostridium difficile BAA-1872320A+B+207

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510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017 Page 8 of 20

StrainConcentration(CFU/mL)ToxinotypeToxinRibotype
Clostridium difficile BAA-187340A+B+053
Clostridium difficile BAA-18745120A+B+002
Clostridium difficile BAA-187516VA+B+078
Clostridium difficile CCUG 8864 (CCUG 20309)4XA-B+Unknown
Clostridium difficile IS814IIIA+B+034
Clostridium difficile R18804IA+B+086
Clostridium difficile R77714VIIIA-B+110
Clostridium difficile R836640A+B+001
Clostridium difficile R86374IXA+B+019
Clostridium difficile R93674XIIIA+B+070
Clostridium difficile R93854XVA+B+122
Clostridium difficile R104564IVA+B+058
Clostridium difficile R107254VA+B+078
Clostridium difficile R108424VIA+B+045
Clostridium difficile R108704XIVA+B+111
Clostridium difficile R124254IIA+B+103

Cross Reactivity (Analytical Specificity)

The cross-reactivity study for Simplexa™ C. difficile Direct was evaluated using 127 organisms for potential cross reactivity.

The samples were prepared at one internal site. Cross-reactivity samples were prepared by spiking each of the 127 organisms at clinically relevant concentrations into negative stool-TE matrix. Clinically relevant concentration is defined as ≥10° CFU/mL for bacteria and fungus and ≥10° TCIDsymL (PFU/mL) for viruses. If TCIDsymL (PFU/mL) or CFU/mL were not available, clinically relevant concentrations were prepared using industry accepted units. After preparation, all the samples were stored at -70 °C or below until testing. Each cross-reactivity sample was tested at least in triplicate. If at least one of the three signals was detected, an additional five replicates of the same cross reactant was tested. Baseline samples were tested in five replicates per instrument with four baseline references based on batch testing of organisms.

OrganismTestedConcentrationC. difficile QualitativeResults: % Detection(# Detected/#Tested)
Abiotrophia defectiva1.00 X 106 CFU/mL0.0% (0/3)
Acinetobacter baumannii1.00 X 106 CFU/mL0.0% (0/3)
Acinetobacter Iwoffi1.00 X 106 CFU/mL0.0% (0/3)
Adenovirus 11.00 X 105 TCID50/mL0.0% (0/3)
Aeromonas hydrophila1.00 X 106 CFU/mL0.0% (0/3)
Alcaligenes faecalis subsp. Faecalis1.00 X 106 CFU/mL0.0% (0/3)
Anaerococcus tetradius1.00 X 106 CFU/mL0.0% (0/3)
Bacillus cereus ATCC 117781.00 X 106 CFU/mL0.0% (0/3)
Bacillus cereus ATCC 134721.00 X 106 CFU/mL0.0% (0/3)
Bacteroides caccae1.00 X 106 CFU/mL0.0% (0/3)
OrganismTestedConcentrationC. difficile QualitativeResults: % Detection (#Detected/#Tested)
Bacteroides fragilis1.00 X 106 CFU/mL0.0% (0/3)
Bacteroides merdae1.00 X 106 CFU/mL0.0% (0/3)
Bacteroides stercoris1.00 X 106 CFU/mL0.0% (0/3)
Bifidobacterium adolescentis1.00 X 106 CFU/mL0.0% (0/3)
Bifidobacterium longum1.00 X 106 CFU/mL0.0% (0/3)
Campylobacter coli1.00 X 106 CFU/mL0.0% (0/3)
Campylobacter jejuni11.00 X 106 CFU/mL0.0% (0/3)
Candida albicans1.00 X 106 CFU/mL0.0% (0/3)
Candida catenulate1.00 X 106 CFU/mL0.0% (0/3)
Cedecea davisae1.00 X 106 CFU/mL0.0% (0/3)
Chlamydia trachomatis1.00 X 106 IFU/mL0.0%(0/3)
Citrobacter amalonaticus1.00 X 106 CFU/mL0.0% (0/3)
Citrobacter freundii1.00 X 106 CFU/mL0.0% (0/3)
Citrobacter koseri1.00 X 106 CFU/mL0.0% (0/3)
Citrobacter sedlakii1.00 X 106 CFU/mL0.0% (0/3)
Clostridium beijerinckii1.00 X 106 CFU/mL0.0% (0/3)
Clostridium bifermentans1.00 X 106 CFU/mL0.0% (0/3)
Clostridium bolteae1.00 X 106 CFU/mL0.0% (0/3)
Clostridium butyricum1.00 X 106 CFU/mL0.0% (0/3)
Clostridium chauvoei1.00 X 106 CFU/mL0.0% (0/3)
Clostridium difficile ATCC 435931.00 X 106 CFU/mL0.0% (0/3)
Clostridium difficile ATCC 436011.00 X 106 CFU/mL0.0% (0/3)
Clostridium fallax1.00 X 106 CFU/mL0.0% (0/3)
Clostridium haemolyticum1.00 X 106 CFU/mL0.0% (0/3)
Clostridium histolyticum1.00 X 106 CFU/mL0.0% (0/3)
Clostridium innocuum1.00 X 106 CFU/mL0.0% (0/3)
Clostridium methylpentosum1.00 X 106 CFU/mL0.0% (0/3)
Clostridium nexile1.00 X 106 CFU/mL0.0% (0/3)
Clostridium novyi1.00 X 106 CFU/mL12.5% (1/8)
Clostridium paraputrificum1.00 X 106 CFU/mL0.0% (0/3)
Clostridium perfringens1.00 X 106 CFU/mL0.0% (0/3)
Clostridium ramosum1.00 X 106 CFU/mL0.0% (0/3)
Clostridium scindens1.00 X 106 CFU/mL0.0% (0/3)
Clostridium sepiticum1.00 X 106 cells/mL0.0% (0/3)
Clostridium sordellii1.00 X 106 CFU/mL0.0% (0/3)
Clostridium sphenoides1.00 X 106 CFU/mL0.0% (0/3)
Clostridium spiroforme1.00 X 106 CFU/mL0.0% (0/3)
Clostridium sporogenes1.00 X 106 CFU/mL0.0% (0/3)
Clostridium symbiosum1.00 X 106 CFU/mL0.0% (0/3)
Clostridium tertium1.00 X 106 CFU/mL0.0% (0/3)
Clostridium tetani1.00 X 106 CFU/mL0.0% (0/3)
Collinsella aerofaciens1.00 X 106 CFU/mL0.0% (0/3)
OrganismTestedConcentrationC. difficile QualitativeResults: % Detection (#Detected/#Tested)
Corynebacterium genitalium1.00 X 106 CFU/mL0.0% (0/3)
Coxsackievirus A101.00 X 105 TCID50/mL0.0% (0/3)
Cytomegalovirus AD-1691.00 X 105 TCID50/mL0.0% (0/3)
Echovirus 111.00 X 105 TCID50/mL0.0% (0/3)
Edwardsiella tarda1.00 X 106 CFU/mL0.0% (0/3)
Eggerthella lenta1.00 X 106 CFU/mL0.0% (0/3)
Enterobacter aerogenes1.00 X 106 CFU/mL0.0% (0/3)
Enterobacter cloacae1.00 X 106 CFU/mL0.0% (0/3)
Enterococcus casseliflavus1.00 X 106 CFU/mL0.0% (0/3)
Enterococcus cecorum1.00 X 106 CFU/mL0.0% (0/3)
Enterococcus dispar1.00 X 106 CFU/mL0.0% (0/3)
Enterococcus faecalis vanB1.00 X 106 CFU/mL0.0% (0/3)
Enterococcus faecium vanA1.00 X 106 CFU/mL0.0% (0/3)
Enterococcus gallinarum vanC1.00 X 106 CFU/mL0.0% (0/3)
Enterococcus hirae1.00 X 106 CFU/mL0.0% (0/3)
Enterococcus raffinosus1.00 X 106 CFU/mL0.0% (0/3)
Enterovirus 711.00 X 106 TCID50/mL0.0% (0/3)
Escherichia coli ATCC 117751.00 X 106 CFU/mL0.0% (0/3)
Escherichia coli ATCC 235111.00 X 106 CFU/mL0.0% (0/3)
Escherichia coli ATCC 259221.00 X 106 CFU/mL0.0% (0/3)
Escherichia coli O157:H7 (ATCC 700927)1.00 X 106 CFU/mL0.0% (0/3)
Escherichia fergusonii1.00 X 106 CFU/mL0.0% (0/3)
Escherichia hermannii1.00 X 106 CFU/mL0.0% (0/3)
Flavonifractor plautii (deposited as Clostridium orbiscindens)1.00 X 106 CFU/mL0.0% (0/3)
Fusobacterium varium1.00 X 106 CFU/mL0.0% (0/3)
Gardnerella vaginalis1.00 X 106 CFU/mL0.0% (0/3)
Gemella morbillorum1.00 X 106 CFU/mL0.0% (0/3)
Hafnia alvei1.00 X 106 CFU/mL0.0% (0/3)
Helicobacter fennelliae1.00 X 106 CFU/mL0.0% (0/3)
Helicobacter pylori1.00 X 106 CFU/mL0.0% (0/3)
Klebsiella oxytoca1.00 X 106 CFU/mL0.0% (0/3)
Klebsiella pneumoniae subsp. Pneumoniae1.00 X 106 CFU/mL0.0% (0/3)
Lactobacillus acidophilus1.00 X 106 CFU/mL0.0% (0/3)
Lactobacillus reuteri1.00 X 106 CFU/mL0.0% (0/3)
Lactococcus lactis1.00 X 106 CFU/mL0.0% (0/3)
Leminorella grimontii1.00 X 106 CFU/mL0.0% (0/3)
Listeria grayi1.00 X 106 CFU/mL0.0% (0/3)
Listeria innocua1.00 X 106 CFU/mL0.0% (0/3)
Listeria monocytogenes1.00 X 106 CFU/mL0.0% (0/3)
Norovirus G21.00 X 105 copies/mL0.0% (0/3)
Peptoniphilus asaccharolyticus1.00 X 106 CFU/mL0.0% (0/3)
OrganismTestedConcentrationC. difficile QualitativeResults: % Detection (#Detected/#Tested)
Plesiomonas shigelloides1.00 X 106 CFU/mL0.0% (0/3)
Porphyromonas asaccharolytica1.00 X 106 CFU/mL0.0% (0/3)
Prevotella melaninogenica1.00 X 106 CFU/mL0.0% (0/3)
Proteus mirabilis1.00 X 106 CFU/mL0.0% (0/3)
Proteus penneri1.00 X 106 CFU/mL0.0% (0/3)
Providencia alcalifaciens1.00 X 106 CFU/mL0.0% (0/3)
Providencia rettgeri1.00 X 106 CFU/mL0.0% (0/3)
Providencia stuartii1.00 X 106 CFU/mL0.0% (0/3)
Pseudomonas aeruginosa1.00 X 106 CFU/mL0.0% (0/3)
Pseudomonas putida1.00 X 106 CFU/mL0.0% (0/3)
Rotavirus, strain Wa1.00 X 105 TCID50/mL0.0% (0/3)
Salmonella enterica subsp. Arizonae1.00 X 106 CFU/mL0.0% (0/3)
Salmonella enterica subsp. Enterica serovar Choleraesuis1.00 X 106 CFU/mL0.0% (0/3)
Salmonella enterica subsp. Enterica serovar Typhimurium1.00 X 106 CFU/mL0.0% (0/3)
Serratia liquefaciens1.00 X 106 CFU/mL0.0% (0/3)
Serratia marcescens subsp. marcescens1.00 X 106 CFU/mL0.0% (0/3)
Shigella boydii1.00 X 106 CFU/mL0.0% (0/3)
Shigella dysenteriae1.00 X 106 CFU/mL0.0% (0/3)
Shigella sonnei1.00 X 106 CFU/mL0.0% (0/3)
Staphylococcus aureus1.00 X 106 CFU/mL0.0% (0/3)
Staphylococcus epidermidis1.00 X 106 CFU/mL0.0% (0/3)
Stenotrophomonas maltophilia1.00 X 106 CFU/mL0.0% (0/3)
Streptococcus agalactiae1.00 X 106 CFU/mL0.0% (0/3)
Streptococcus dysgalactiae1.00 X 106 CFU/mL0.0% (0/3)
Streptococcus intermedius1.00 X 106 CFU/mL0.0% (0/3)
Streptococcus uberis1.00 X 106 CFU/mL0.0% (0/3)
Trabulsiella guamensis1.00 X 106 CFU/mL0.0% (0/3)
Veillonella parvula1.00 X 106 CFU/mL0.0% (0/3)
Vibrio cholerae1.00 X 106 CFU/mL0.0% (0/3)
Vibrio parahaemolyticus1.00 X 106 CFU/mL0.0% (0/3)
White Blood Cells (Human)1.00 X 106 WBC/mL0.0% (0/3)
Yersinia bercovieri1.00 X 106 CFU/mL0.0% (0/3)
Yersinia rohdei1.00 X 106 CFU/mL0.0% (0/3)

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510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 9 of 20

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510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950
Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 10 of 20

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510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 11 of 20

1 Campylobacter jejuni was tested during validation. However, it is unknown if subspecies jejuni was tested. In silico NCBI BLAST analysis was performed for C. jejuni subsp jejuni and it did not show cross reactivity with the Simplexa ™ C. difficile Direct kit based on in silico analysis.

Note: Clostridium botulinum, Desulfovibrio piger, and Rumin were not available for testing. In silico NCBI BLAST analysis was performed for all three organisms. Clostridium, Desulfovibrio piger, and Ruminococcus bromii do not
show cross reactivity with the Simplexa™ C. difficile Direct

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510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017 Page 12 of 20

INTERFERENCE

A total of 33 substances (endogenous & exogenous) were tested for potential interference with Simplexa™ C. difficile Direct using two bacterial strains of C. difficile - ATCC 43255 & NAP1A.

The following table summarizes the qualitative results for all 33 potentially interfering substances.

InterferingSubstanceActive IngredientInterferingSubstanceConcentrationC. difficile QualitativeDetection: %Detection(#Detected/#Tested)
ATCC 43255NAP1A
AfrinOxymetazoline hydrochloride0.05%10% (w/v)100.0%(3/3)100.0%(3/3)
Antacid and Anti-gas genericAluminum hydroxide/Magnesiumhydroxide0.1 mg/mL100.0%(3/3)100.0%(3/3)
Anusol Plus (Pramoxine HCL)Pramoxine hydrochloride 1% andzinc sulfate monohydrate 0.5%10% (w/v)100.0%(3/3)100.0%(3/3)
Barium SulfateBarium sulfate (98% forsuspension)5 mg/mL100.0% (3/3)100.0% (3/3)
Benzalkonium (Towlettes)Benzalkonium chloride, Ethanol10% (v/v)100.0% (3/3)100.0% (3/3)
BloodGlucose, Hormones, Enzymes,lons, Iron, etc.5% (v/v)100.0% (3/3)100.0% (3/3)
Colace¹Ducosate sodium USP 100 mg2.5% (w/v)62.5% (5/8)87.5% (7/8)
DramamineDimenhydrinate10% (w/v)100.0% (3/3)100.0% (3/3)
Dulcolax²Bisacodyl USP 5 mg10% (w/v) forATCC 43255 &0.3125% (w/v) forNAP1A100.0% (3/3)100.0% (3/3)
Gynol II, Nonoxynol 9Nonoxynol 9 (4.0%)10% (w/v)100.0% (3/3)100.0% (3/3)
Hydrocortisone creamHydrocortisone2% (w/v)100.0% (3/3)100.0% (3/3)
ImodiumLoperamide hydrochloride0.005 mg/mL100.0% (3/3)100.0% (3/3)
KY JellyGlycerin2% (w/v)100.0% (3/3)100.0% (3/3)
MesalazineMesalazine rectal suspension(4 g/60 mL)10% (w/v)100.0% (3/3)100.0% (3/3)
MetronidazoleMetronidazole14 mg/mL100.0% (3/3)100.0% (3/3)
Milk of MagnesiaMagnesium hydroxide0.2 mg/mL62.5% (5/8)62.5% (5/8)
Mineral OilMineral oil2% (w/v)100.0% (3/3)100.0% (3/3)
InterferingSubstanceActive IngredientInterferingSubstanceConcentrationC. difficile QualitativeDetection: %Detection(#Detected/#Tested)
ATCC 43255NAP1A
Monistat 7Miconazole nitrate 2% (100 mg)10% (w/v)100.0% (3/3)100.0% (3/3)
MucinImmunoglobulins, Lysozyme,Polymers, etc.3 mg/mL100.0% (3/3)100.0% (3/3)
Naproxen3Naproxen sodium7 mg/mL100.0% (3/3)100.0% (3/3)
NystatinNystatin10000 USPunits/ml100.0% (3/3)100.0% (3/3)
Palmitic AcidPalmitic acid2 mg/mL100.0% (3/3)100.0% (3/3)
Pepto-BismolBismuth subsalicylate0.175 mg/mL100.0% (3/3)100.0% (3/3)
Preparation HPhenylephrine2% (w/v)100.0% (3/3)100.0% (3/3)
SennosidesSennosides0.1 mg/mL100.0% (3/3)100.0% (3/3)
SPF 30 SunscreenAvobenzone 3%, homosalate 8%,octisalate 5%, octocrylene 4%, andoxybenzone 4%1% (w/v)100.0% (3/3)100.0% (3/3)
SPF 50 SunscreenAvobenzone 3%, homosalate 13%,octisalate 5%, octocrylene 7%, andoxybenzone 4%1% (w/v)100.0% (3/3)100.0% (3/3)
Stearic AcidStearic acid4 mg/mL100.0% (3/3)100.0% (3/3)
TumsCalcium Carbonate0.1 mg/mL100.0% (3/3)100.0% (3/3)
VagisilBenzocaine USP (20% - Externalanalgesic), Resorcinol (3% -External analgesic)10% (w/v)100.0% (3/3)100.0% (3/3)
VancomycinVancomycin1.4 mg/mL100.0% (3/3)100.0% (3/3)
VaselineWhite petrolatum USP (100%)10% (w/v)100.0% (3/3)100.0% (3/3)
Witch hazelWitch hazelLiquid from 1 wipe100.0% (3/3)100.0% (3/3)

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510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

1 Colace tested at two higher concentrations (10% and 5% w/v) produced "Invalid" results in all replicates.

² Dulcolax also tested at 10%, 5%, 2.5%, and 1.25% w/v produced "Invalid" results for all replicates of strain NAP1A. Dulcolax at 0.625% w/v produced "Invalid" results in five out of eight replicates and a "Not Detected" result in one replicate of strain NAP1A.

3 Naproxen also tested at 14 mg/mL produced "Not Detected" results in seven out of eight replicates of strains NAP1A and ATCC 43255.

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Image /page/16/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo consists of the word "FOCUS" in bold, black capital letters, with the word "Diagnostics" in a smaller font size underneath it. There is a curved, black shape to the left of the word "FOCUS", which appears to be an abstract design element. A horizontal line is placed underneath the word "FOCUS".

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017 Page 14 of 20

INHIBITION BY OTHER MICROORGANISMS

Simplexa™ C. difficile Direct was evaluated by testing the ability C. difficile when potentially inhibitory organisms are present.

130 organisms were evaluated for potential interference when the concentration of C. difficile in the sample was at a low/medium concentration (~2-4 X LoD) using two bacterial strains of C. difficile (NAP1A & ATCC 43255). A total of 127 organisms were tested during this study, and in silico NCBI BLAST analysis was performed for 3 organisms to determine microbial interference.

Microbial inhibition samples were prepared by spiking each of the 127 organisms at clinically relevant concentrations into neqative stool-TE matrix. If TCIDsomL (PFU/mL were not available, clinically relevant concentrations were prepared using industry accepted units. Each microbial inhibition sample was tested in at least in triplicate. If at least one of the three signals was not detected, an additional five replicates of the same inhibitor was tested.

All replicates of baseline sample and microbial inhibition samples were positive for C. difficile. No evidence of microbial inhibition was observed for the 127 organisms at the tested concentrations. Below are the results of the study.

OrganismTestedConcentrationC. difficile Strain:ATCC 43255C. difficile StrainNAP1A
C. difficileQualitative Results:% Detection (#Detected/#Tested)C. difficileQualitative Results:% Detection (#Detected/#Tested)
BaselineNot Applicable100.0% (5/5)100.0% (5/5)
Abiotrophia defectiva1.00 X 106 CFU/mL100.0% (3/3)75.0% (6/8)
Acinetobacter baumannii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Acinetobacter Iwoffi1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Adenovirus 11.00 X 105TCID50/mL100.0% (3/3)100.0% (3/3)
Aeromonas hydrophila1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Alcaligenes faecalis subsp. Faecalis1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Anaerococcus tetradius1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Bacillus cereus ATCC 117781.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Bacillus cereus ATCC 134721.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Bacteroides caccae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Bacteroides fragilis1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Bacteroides merdae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Bacteroides stercoris1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
OrganismTestedConcentrationC. difficile Strain:ATCC 43255C. difficileQualitative Results:% Detection (#Detected/#Tested)C. difficile StrainNAP1AC. difficileQualitative Results:% Detection (#Detected/#Tested)
Bifidobacterium adolescentis1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Bifidobacterium longum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Campylobacter coli1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Campylobacter jejuni11.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Candida albicans1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Candida catenulate1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Cedecea davisae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Chlamydia trachomatis1.00 X 106 IFU/mL100.0% (3/3)100.0% (3/3)
Citrobacter amalonaticus1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Citrobacter freundii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Citrobacter koseri1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Citrobacter sedlakii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium beijerinckii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium bifermentans1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium bolteae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium butyricum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium chauvoei1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium difficile ATCC 435931.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium difficile ATCC 436011.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium fallax1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium haemolyticum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium histolyticum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium innocuum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
OrganismTestedConcentrationC. difficile Strain:ATCC 43255C. difficileQualitative Results:% Detection (#Detected/#Tested)C. difficile Strain:NAP1AC. difficileQualitative Results:% Detection (#Detected/#Tested)
Clostridium methylpentosum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium nexile1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium novyi1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium paraputrificum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium perfringens1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium ramosum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium scindens1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium sepiticum1.00 X 106 cells/mL100.0% (3/3)87.5% (7/8)
Clostridium sordellii1.00 X 106 CFU/mL100.0% (3/3)87.5% (7/8)
Clostridium sphenoides1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium spiroforme1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium sporogenes1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium symbiosum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium tertium1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Clostridium tetani1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Collinsella aerofaciens1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Corynebacterium genitalium1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Coxsackievirus A101.00 X 105TCID50/mL100.0% (3/3)100.0% (3/3)
Cytomegalovirus1.00 X 105TCID50/mL100.0% (3/3)100.0% (3/3)
Echovirus 111.00 X 105TCID50/mL100.0% (3/3)100.0% (3/3)
Edwardsiella tarda1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Eggerthella lenta1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
OrganismTestedConcentrationC. difficile Strain:ATCC 43255C. difficile StrainNAP1A
C. difficileQualitative Results:% Detection (#Detected/#Tested)C. difficileQualitative Results:% Detection (#Detected/#Tested)
Enterobacter aerogenes Z0521.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterobacter cloacae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterococcus casseliflavus1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterococcus cecorum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterococcus dispar1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterococcus faecalis1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterococcus faecium1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterococcus gallinarum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterococcus hirae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterococcus raffinosus1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Enterovirus 711.00 X 106TCID50/mL100.0% (3/3)100.0% (3/3)
Escherichia coli ATCC 117751.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Escherichia coli ATCC 23511 O16:K1L:NM1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Escherichia coli ATCC 259221.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Escherichia coli O157H71.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Escherichia fergusonii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Escherichia hermannii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Flavonifractor plautii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Fusobacterium varium1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Gardnerella vaginalis1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Gemella morbillorum1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Hafnia alvei1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Helicobacter fennelliae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
OrganismTestedConcentrationC. difficile Strain:ATCC 43255C. difficile StrainNAP1A
C. difficileQualitative Results:% Detection (#Detected/#Tested)C. difficileQualitative Results:% Detection (#Detected/#Tested)
Helicobacter pylori1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Klebsiella oxytoca1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Klebsiella pneumoniae subsp. Pneumoniae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Lactobacillus acidophilus1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Lactobacillus reuteri1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Lactococcus lactis1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Leminorella grimontii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Listeria grayi1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Listeria innocua1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Listeria monocytogenes1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Norovirus G21.00 X 106 copies/mL100.0% (3/3)100.0% (3/3)
Peptoniphilus asaccharolyticus1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Peptostreptococcus anaerobius1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Plesiomonas shigelloides1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Porphyromonas asaccharolytica1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Prevotella melaninogenica1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Proteus mirabilis Z0501.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Proteus penneri1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Providencia alcalifaciens1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Providencia rettgeri1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Providencia stuartii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Pseudomonas aeruginosa1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Pseudomonas putida1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
TestedC. difficile Strain:ATCC 43255C. difficile StrainNAP1A
OrganismConcentrationC. difficileQualitative Results:% Detection (#Detected/#Tested)C. difficileQualitative Results:% Detection (#Detected/#Tested)
Rotavirus Strain Wa1.00 X 106TCID50/mL100.0% (3/3)100.0% (3/3)
Salmonella enterica subsp. Arizonae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Salmonella enterica subsp. Enterica1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Salmonella enterica subsp. Enterica serovarTyphim1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Serratia liquefaciens1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Serratia marcescens subsp. marcescens1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Shigella boydii1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Shigella dysenteriae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Shigella sonnei1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Staphylococcus aureus (MRSA), ATCC 7006991.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Staphylococcus epidermidis (MRSE), ATCC298871.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Stenotrophomonas maltophilia1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Streptococcus agalactiae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Streptococcus dysgalactiae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Streptococcus intermedius1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Streptococcus uberis1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Trabulsiella guamensis1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Veillonella parvula1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Vibrio cholerae1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
Vibrio parahaemolyticus1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
White Blood Cells (Human)1.00 X 106 WBC/mL100.0% (3/3)100.0% (3/3)
Yersinia bercovieri1.00 X 106 CFU/mL100.0% (3/3)100.0% (3/3)
OrganismTestedConcentrationC. difficile Strain:ATCC 43255C. difficile StrainNAP1A
Yersinia rohdei1.00 X 106 CFU/mLC. difficileQualitative Results:% Detection (#Detected/#Tested)100.0% (3/3)C. difficileQualitative Results:% Detection (#Detected/#Tested)100.0% (3/3)

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Image /page/17/Picture/0 description: The image shows the logo for Focus Diagnostics. The word "FOCUS" is in large, bold, sans-serif font. Above the word "FOCUS" is a curved, black shape that resembles a check mark. Below the word "FOCUS" is the word "Diagnostics" in a smaller font, underlined with a thin black line.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950
Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 15 of 20

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Image /page/18/Picture/0 description: The image shows the logo for Focus Diagnostics. The word "FOCUS" is in large, bold, sans-serif font. Above and to the left of the word "FOCUS" is a curved, black shape. Below the word "FOCUS" is the word "Diagnostics" in a smaller, sans-serif font, underlined with a thin black line.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 16 of 20

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Image /page/19/Picture/0 description: The image shows the logo for Focus Diagnostics. The word "FOCUS" is in large, bold, sans-serif font. A curved, black shape is to the left of the word, resembling a stylized check mark or swoosh. Below "FOCUS" is the word "Diagnostics" in a smaller, sans-serif font, underlined with a thin black line.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 17 of 20

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Image /page/20/Picture/0 description: The image shows the logo for Focus Diagnostics. The word "FOCUS" is in large, bold, sans-serif font. Above the word "FOCUS" is a curved, black shape that resembles a check mark. Below the word "FOCUS" is the word "Diagnostics" in a smaller, sans-serif font, underlined with a thin black line.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950
Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 18 of 20

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Image /page/21/Picture/0 description: The image shows the logo for Focus Diagnostics. The word "FOCUS" is in large, bold, sans-serif font. Above and to the left of the word is a curved, black shape. Below the word "FOCUS" is a horizontal line, and below that is the word "Diagnostics" in a smaller, sans-serif font.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960 February 10, 2017

Page 19 of 20

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Image /page/22/Picture/0 description: The image shows the logo for Focus Diagnostics. The logo consists of the word "FOCUS" in bold, sans-serif font, with the word "Diagnostics" in a smaller, non-bold font underneath. A curved, black shape is to the left of the word "FOCUS", adding a visual element to the logo.

510(k) Summary

Simplexa™ C. difficile Direct Catalog No. MOL2950 Simplexa™ C. difficile Positive Control Pack Catalog No. MOL2960

February 10, 2017 Page 20 of 20

1 Campylobacter jejuni was tested during validation. However, it is unknown if subspecies jejuni was tested. In silico NCBI BLAST analysis was performed for C. jejuni subsp jejuni and it did not show cross reactivity with the Simplexa™ C. difficile Direct kit based on in silico analysis.

§ 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.