EntericBio Dx Assay

• K Number: K182703
• Device Name: EntericBio Dx Assay
• Manufacturer: Serosep, Ltd.
• Date Cleared: 2019-06-19 ( 265 days)
• Product Code: PCH , NSU , OOI
• Regulation Number: 866.3990
• Type: Traditional
• Panel: Microbiology
• Reference & Predicate Devices: K140407
• Predicate For: N/A

Intended Use

The EntericBio" Dx assay, performed on ABI 7500 Fast Dx real-time instrument, is an in vitro multiplexed nucleic acid test for the direct, simultaneous, qualitative detection and identification of multiple enteric pathogens in Cary-Blair preserved stool specimens from individuals with signs and symptoms of infectious colitis or gastroenteritis. The test is based on detection of nucleic acids from:

• . Salmonella enterica spp.
• Shigella spp./ Enteroinvasive E. coli (EIEC)
• Campylobacter spp. (jejuni, coli and lari)
• STEC (Shiga-like toxin-producing E. coli), stx1/stx2 genes
• Vibrio spp. (cholerae and parahaemolyticus)
• . Giardia lamblia (also known as G. intestinalis and G. duodenalis)
• Entamoeba histolytica

Testing is performed on Cary-Blair preserved diarrheal specimens from symptomatic patients with suspected acute gastroenteritis, enteritis or colitis of bacterial or parasitic origin. The test is performed directly on the specimen, utilizing real-time polymerase chain reaction (PCR) for the amplification of Salmonella-specific, Campylobacter-specific, Shigella/ EIEC-specific ipaH, stx1/stx2, Vibrio-specific, Entamoeba-specific and Giardia-specific gene sequences. The test utilizes fluorogenic sequence-specific hybridization probes for the detection of the amplified DNA.

This test is intended for use, in conjunction with clinical presentation, laboratory findings and epidemiological information, as an aid in the diagnosis of Salmonella, Shigella / EIEC, Shigalike toxin-producing E. coli, Campylobacter spp., Entamoeba histolytica and Giardia spp. infections in humans.

Results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results do not rule out co-infection with other organisms that are not detected by this test and may not be the sole or definitive cause of patient illness. Negative EntericBio" Dx assay results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Device Description

The EntericBio® Dx Assay provides PCR reagents to be used in conjunction with an automated pipetting system and the ABI 7500 Fast Dx instrument using standard filters. Results are interpreted using the EntericBio FastFinder plugin. The system provides automated, real-time amplification, detection and analysis and a user constructed template suitable for the EntericBio® Dx Assay.

The assay is composed of Stool Preparation Solution (SPS) tubes, PCR reagent strips containing lyophilized reagents, Resuspension Buffer (Negative Kit Control), Positive Kit Control containing DNA from all target analytes (with appropriate reconstitution buffer), and associated accessories,instruments and software for detection of bacterial and parasitic causes of gastroenteritis in humans.

The EntericBio" Dx assay detects target DNA from diarrheal Cary-Blair stool specimens from symptomatic individuals with suspected gastroenteritis or infectious colitis.

The assay works directly from a Cary-Blair preserved stool sample and does not require commercial nucleic acid extraction /purification. The PCR master mix with all the reagents required to perform each test is lyophilized into individual reaction wells on a strip. Each reaction well contains an Internal Amplification Control (IAC) to monitor for PCR inhibition.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study proving the device's performance, based on the provided FDA 510(k) summary for the Serosep, Ltd. EntericBio® Dx Assay:

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Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the performance targets outlined for the analytical and clinical studies. Due to the nature of this 510(k) summary, specific numerical acceptance thresholds for clinical performance (e.g., minimum sensitivity/specificity) are not explicitly stated, but rather the observed performance metrics are presented. The reproducibility study explicitly states acceptance criteria.

Table of Acceptance Criteria and Reported Device Performance

Category	Metric/Target	Acceptance Criteria (If explicitly stated)	Reported Device Performance (EntericBio® Dx Assay)
Analytical Performance
Reproducibility (Accuracy)	Agreement with expected results for target analytes (Moderate Positive)	100% agreement	Shigella sonnei: 100% (90/90)Vibrio parahaemolyticus: 100% (90/90)Giardia lamblia: 100% (90/90)
Reproducibility (Accuracy)	Agreement with expected results for target analytes (Low Positive)	≥95% agreement	Shigella sonnei: 100% (90/90)Vibrio parahaemolyticus: 100% (90/90)Giardia lamblia: 98% (89/90)
Reproducibility (Precision)	Cq Value Coefficient of Variation (%CV)	Not explicitly stated as an acceptance criterion for individual Cq values, but presented as a measure of precision.	Shigella sonnei: Moderate Positive (2.44% CV), Low Positive (1.97% CV)Vibrio parahaemolyticus: Moderate Positive (1.21% CV), Low Positive (1.82% CV)Giardia lamblia: Moderate Positive (3.53% CV), Low Positive (4.05% CV)
Limit of Detection (LoD)	Lowest concentration of analyte consistently detected	≥95% detection rate	Salmonella spp.: 8 x 10^4 CFU/mLShigella spp./EIEC: 1.25 x 10^4 - 1 x 10^4 CFU/mLCampylobacter spp.: 1 x 10^4 - 4 x 10^4 CFU/mLE. coli (STEC): 5 x 10^5 - 1 x 10^6 CFU/mLVibrio spp.: 1 x 10^4 CFU/mLGiardia lamblia: 25-100 cells/mLEntamoeba histolytica: 25-100 cells/mL
Analytical Reactivity	Detect all tested strains within target diversity	All organisms tested detectable over three replicates.	All 101 target organisms were detectable.
Analytical Exclusivity	No cross-reactivity with non-target organisms (or limited to closely related organisms with shared sequence similarity)	Not explicitly stated, but implies minimal/no false positives from unrelated organisms.	Cross-reactivity observed with Vibrio campbellii, V. fluvialis, V. furnissii, V. mimicus, V. fischeri, V. natriegens (all cross-reacted with Vibrio target). Other noted "cross-reactivity" issues were attributed to potential sample contamination or expected in-silico findings for some highly related organisms (e.g., Cryptosporidium viatorum/Shigella, Cryptosporidium cuniculus/Giardia).
Microbial Interference	No interference from high concentrations of common microorganisms.	No interference in presence of 3X LoD of each target analyte.	No interference observed with 11 tested microorganisms.
Interfering Substances	No interference from common exogenous/endogenous substances.	Detection of targets/IAC in presence of substances.	Most substances (21/23) showed no interference. False negative observed for V. parahaemolyticus with hemorrhoidal cream (1/3 replicates) and C. jejuni with tetracycline (2/3 replicates). IAC failures with >1% Benzalkonium chloride and >1% hemorrhoidal cream.
Competitive Inhibition	No competitive inhibition when multiple target analytes are present.	No competitive inhibition.	Competitive inhibition observed for Giardia lamblia in presence of Entamoeba histolytica. (Note: Not evaluated for Well B analytes: Vibrio and STEC).
Carry-over/Cross-Contamination	No carry-over/cross-contamination.	No carryover or cross-contamination.	None observed.
Specimen Stability	Determine recommended storage conditions.	Established storage conditions.	5 days at 2-8°C for Cary-Blair preserved stool.
Real Time Stability	Determine shelf life.	Established shelf life.	6 months at 2-8°C.
Clinical Performance
Overall Agreement (Positive)	Agreement with comparator method for target detection for Fresh, Archived, and Contrived samples. (Individual values depend on analyte)	Not explicitly stated, but implied to be high enough for substantial equivalence.	Salmonella: Fresh (92.3%), Select (90.0%), Frozen (85.7%). Campylobacter: Fresh (91.1%), Select (90.0%), Frozen (93.8%). Shigella/EIEC: Fresh (100%), Select (100%), Frozen (90.9%). STEC: Fresh (100%), Select (100%), Frozen (94.6%). Vibrio: Fresh (0.0% - due to low prevalence), Simulated (100%). Giardia: Fresh (85.7%), Select (100%), Frozen (100%). Entamoeba: Fresh (NA - no positives), Frozen (0.0% - no positives), Simulated (98.6%).
Overall Agreement (Negative)	Agreement with comparator method for non-detection for Fresh, Archived, and Contrived samples. (Individual values depend on analyte)	Not explicitly stated, but implied to be high enough for substantial equivalence.	Salmonella: Fresh (100%), Select (100%), Frozen (100%). Campylobacter: Fresh (99.7%), Select (100%), Frozen (100%). Shigella/EIEC: Fresh (100%), Select (100%), Frozen (100%). STEC: Fresh (99.9%), Select (100%), Frozen (100%). Vibrio: Fresh (100%), Simulated (100%). Giardia: Fresh (99.9%), Select (100%), Frozen (100%). Entamoeba: Fresh (100%), Frozen (100%), Simulated (100%).
Invalid Rate	Rate of invalid results.	Not explicitly stated, but low rate desirable.	Fresh: Initial 1.9% (resolved to 0.6%). Frozen: Initial 0.9% (resolved to 0.5%). Total: Initial 1.7% (resolved to 0.6%).
Indeterminate Rate	Rate of indeterminate results.	Not explicitly stated, but low rate desirable.	Fresh: Initial 0.1% (remained 0.1%). Frozen: Initial 0.9% (remained 0.9%). Total: Initial 0.2% (remained 0.2%).

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Study Details:

1. Sample Sizes Used for the Test Set and Data Provenance:

   • Reproducibility Study (Test Set): Contrived stool samples spiked with various combinations of Vibrio parahaemolyticus, Shigella sonnei, and Giardia lamblia. Each analyte evaluated at True Negative, Low Positive (1.5x LoD), and Moderate Positive (3x LoD).
     • Sample Size: For accuracy, 30 samples per concentration per site (90 total per concentration per analyte across 3 sites). For negative, 60 samples per site (180 total across 3 sites).
     • Data Provenance: In-house (simulating multi-site) from Serosep. Retrospective.
   • LoD Study (Test Set): Contrived samples with target analytes spiked into negative stool matrix.
     • Sample Size: 20 replicates for each sample type/concentration.
     • Data Provenance: In-house, retrospective.
   • Analytical Reactivity/Exclusivity/Interference/Competitive Inhibition/Carry-Over (Test Sets): Contrived samples spiked into negative stool matrix or genomic DNA/RNA.
     • Sample Size: Generally 3 replicates for analytical reactivity/exclusivity/interference/carry-over. Competitive inhibition tested in triplicate.
     • Data Provenance: In-house, retrospective.
   • Clinical Performance Study (Test Set):
     • Fresh Clinical Samples (Prospective): 1491 prospective, 32 select. Total 1523 enrolled, 1472 evaluable.
       • Data Provenance: Multi-site clinical study, including two (2) US clinical sites and one (1) non-US clinical site. Prospective collection from symptomatic patients.
     • Frozen Clinical Samples (Archived): 212 enrolled, 209 evaluable.
       • Data Provenance: Archived samples from unknown collection sites, selected based on previous positive testing. Retrospective.
     • Contrived Samples (Simulated): 310 evaluable samples.
       • Data Provenance: Prepared using residual fresh specimens that tested negative. Retrospective, in-house.

2. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts:

   • For the EntericBio® Dx Assay, the ground truth for the clinical study was established by comparator methods, not directly by human experts classifying images/data. The summary does not specify the number or qualifications of experts involved in running or interpreting the results of the FDA-cleared comparator assays, or for establishing the initial diagnosis for the archived samples. In diagnostics studies like this, the "ground truth" is typically another diagnostic method (often a laboratory reference method or a composite of multiple methods), which itself is run by trained laboratory personnel.

3. Adjudication Method for the Test Set:

   • For STEC and Campylobacter in fresh specimens, a composite comparator method was used: a specimen was positive if 2 out of 3 comparator assays were positive, and negative if 2 out of 3 were negative.
   • For all other target analytes and archived specimens, the comparator method consisted of one FDA-cleared assay.
   • This implies an adjudication method for the composite comparator (2/3 rule) but not for single comparator methods. There is no mention of human expert adjudication post-test to resolve discrepancies.

4. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

   • No, an MRMC comparative effectiveness study was not done. This device is a molecular diagnostic assay (PCR-based), not an AI-assisted diagnostic imaging device that involves human "readers" interpreting images. Therefore, the concept of human readers improving with AI assistance is not applicable here.

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

   • The EntericBio® Dx Assay is a standalone diagnostic test in the sense that its performance is evaluated directly against comparator methods. It does not inherently involve a "human-in-the-loop" decision-making process in the same way an AI-CAD (Computer-Aided Detection) system for radiology would. The device provides a result (positive/negative) based on its PCR analysis, and clinical interpretation occurs downstream by lab personnel and clinicians. The clinical performance tables (e.g., Table 12a-g) represent this standalone performance of the assay.

6. The type of ground truth used (expert consensus, pathology, outcomes data, etc.):

   • The ground truth for the clinical performance study was established by comparator methods, specifically:
     • For STEC and Campylobacter (fresh specimens): A composite comparator method of three FDA-cleared assays (2/3 rule).
     • For other targets (fresh) and all archived specimens: One FDA-cleared assay.
     • For contrived samples: The known spike concentration/strain was the ground truth.

7. The Sample Size for the Training Set:

   • This document is a 510(k) summary for an in vitro diagnostic (IVD) assay based on PCR, not a machine learning/AI model. Therefore, the concept of a "training set" for an algorithm is not directly applicable in the same way it would be for AI/ML device development. The device's design and optimization (analogous to "training") would have occurred during its development phase, using proprietary methods, and is not explicitly detailed as a distinct 'training set' of clinical samples in this regulatory submission.

8. How the Ground Truth for the Training Set was Established:

   • As explained above, this product is not an AI/ML algorithm requiring a "training set" in the conventional sense. The "ground truth" for the analytical studies (e.g., LoD, exclusivity) was based on precisely prepared samples with known concentrations and identities of microorganisms. For the development and validation of the molecular targets (primers/probes), ground truth would have been established through extensive molecular biology techniques, including sequencing and controlled lab cultures. These methods are inherent to the development of robust PCR assays.

Summary

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June 19, 2019

Serosep, Ltd. % Fran White MDC Associates, LLC 180 Cabot Street Beverly, Massachusetts 01915

Re: K182703

Trade/Device Name: EntericBio Dx Assay Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal microorganism multiplex nucleic acid-based assay Regulatory Class: Class II Product Code: PCH, OOI, NSU Dated: September 26, 2018 Received: September 27, 2018

Dear Fran White:

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. 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 located at https://www.accessdata.fda.gov/scripts/cdrh/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.

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

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801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-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 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

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

Sincerely,

for

Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health 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) K190121

Device Name IDS SHBG

Indications for Use (Describe)

The IDS SHBG assay is an in vitro diagnostic device intended for the quantitative determination of SHBG in human serum or plasma on the IDS System. Results are to be used as an aid in the diagnosis of androgen disorders

Type of Use (Select one or both, as applicable)
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X Prescription Use (Part 21 CFR 801 Subpart D)

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

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510(k) Summary

Date of Summary: June 18, 2019

Sponsor

Serosep, Ltd. Annacotty Business Park Annacotty, Limerick, Ireland

Correspondent

MDC Associates, Inc 180 Cabot Street Beverly, MA 01915 Contact : Fran White Phone : [phone] Fax : [phone] Email : [e-mail]

Device Trade or Proprietary Name

EntericBio® Dx Assay

Common Name

Gastrointestinal microorganism multiplex nucleic acid-based assay

Product Classification 866.3990

Classification

PCH, Class II

Substantial Equivalency

Serosep, Ltd. believes that the subject devices (new device) of this pre-submission document and subsequently a premarket notification submission (510k) is similar to other molecular devices currently marketed in the US. The device design, features and performance when compared to these devices is similar to BioFire, FilmArray Gastrointestinal (GI) Panel (K140407) in that the intended use, the targeted organism for detection, the analytes, technological principles, and specimen types are similar or the same.

There are differences between the subject new device and the predicate device. Because of these differences, the subject device features and suitability will be validated for its intended use using clinical specimens sourced from symptomatic patients or clinical library specimens and tested by the predicate device.

The similarities and differences between the subject device and the predicate device are summarized below.

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Element	Subject (New) Device	Proposed Predicate Device	Discussion
Device Name	EntericBio® Dx Assay	FilmArray Gastrointestinal (GI) Panel	New vs. Predicate Device
FDA DevicePremarketNotification	New device	K140407	New vs Predicate Device
FDA DeviceClassification	Class II, 21 CFR 866.3990 –Gastrointestinal microorganismmultiplex nucleic acid-based assay,Microbiology (83 Panel)	Class II, 21 CFR 866.3990 –Gastrointestinal microorganismmultiplex nucleic acid-based assay,Microbiology (83 Panel)	Same
Type of Test	Qualitative nucleic acid test	Qualitative nucleic acid test	Same
Users	CLIA certified clinical laboratories	CLIA certified clinical laboratories	Same
Assay Method	The EntericBio® Dx Assayprovides PCR reagents to be used inconjunction with an automatedpipetting system and the ABI 7500 FastDx instrument using standard filters.Results are interpreted using theEntericBio FastFinder plugin. The systemprovides automated, real-timeamplification, detection and analysisand a user constructed templatesuitable for the EntericBio® Dx Assay.	The BioFire FilmArray GastrointestinalPanel is designed to be used with theFilmArray® instrument. The FilmArrayGl pouch contains freeze-driedreagents to perform nucleic acidpurification and nested, multiplex PCRwith DNA melt analysis.	Different: New device differs fromthe predicate device in that therealtime PCR assay kits aredesigned to be used with anautomated pipetting station toaccelerate sample preparation,and a commercially available, FDA-cleared PCR instrument tomeasure the fluorescent probesignal generated duringamplification that are analyzed bythe EntericBio automated analysisand interpretation software.
Targets forDetection	Salmonella enterica spp.Shigella spp./ Enteroinvasive E. coli(EIEC)Campylobacter spp. (jejuni, coli and lari)	Clostridium difficile ( C. difficile ) toxinA/B, Campylobacter spp. ( C. jejuni, C.coli and C. upsaliensis ), Plesiomonasshigelloides, Salmonella spp., Vibriospp., Yersinia enterocolitica.	Similar, except the predicatedevice is additionally cleared fordetection of a range of othernucleic acid targets from bacteria,parasites and viruses.
Element	Subject (New) Device	Proposed Predicate Device	Discussion
	STEC (Shiga toxin-producing E. coli),stx1/stx2 genesVibrio spp. (cholerae andparahaemolyticus )Giardia lamblia (also known as G. intestinalis and G. duodenalis )Entamoeba histolytica	Enteriaggregative E. coli ,Enteropathogenic E. coli ,Enterotoxigenic E. coli LT/ST toxins,Shiga-like toxin-producing E. coli ,Shigella/ Enteroinvasive E. coli ,Cryptosporidium spp. , Cyclospora cayetanensis , Entamoeba histolytica ,Giardia lamblia , Adenovirus F40/41,Astrovirus, Norovirus GI/GII, Rotavirus A, Sapovirus (GI, GII, GIV and GV).
Intended Use	The EntericBio® Dx Assay performed onABI 7500 Fast Dx real-time instrument,is an in vitro multiplexed nucleic acidtest for the direct, simultaneousqualitative detection and identificationof multiple enteric pathogens in Cary-Blair preserved stool specimens fromindividuals with signs and symptoms ofinfectious colitis or gastroenteritis. Thetest is based on detection of nucleicacids from the following organisms: (seeorganisms above).	The FilmArray Gastrointestinal Panel(GI) is intendent for use with theFilmArray® instrument for thequalitative in vitro detection andidentification of multiple bacteria,viruses and parasites. The FilmArray GIPanel is performed directly from stoolspecimens in Cary-Blair transportmedia. The following pathogen types,subtypes and toxin genes areidentified using the FilmArray GI Panel:(see organisms above).	Similar except for the additionalorganisms detected by thepredicate device.
Analyte	DNA/RNA from Cary-Blair preservedfecal specimens	DNA/ RNA from Cary-Blair preservedfecal specimens	Same
TechnologicalPrinciples	Multiplex nucleic acid PCR	Multiplex nucleic acid PCR	Same
SpecimenTypes	Human stool (Cary-Blair preserved)	Human stool (Cary-Blair preserved)	Same
Element	Subject (New) Device	Proposed Predicate Device	Discussion
Controls	Internal Amplification Control for eachsample. Kit positive and negativecontrols are processed with each batchof samples.	Two controls are included in eachreagent pouch to control for sampleprocessing and both stages of PCR andmelt analysis.	Similar: The new device IAC islyophilized within the PCR mixwhereas the predicate device hastwo controls included in eachreagent pouch.Substantial equivalence will bedemonstrated in clinical testingusing human specimens andcompared to the predicate device.
PCR SamplePreparation/Extraction	Sample processed directly followingheat treatment of specimen in a SPStube.	Sample processing is automated in theFilmArray® instrument. The sample islysed by a combination of mechanical(bead beating) and chemical meansand the liberated nucleic acid iscaptured, washed and eluted usingmagnetic bead technology.	EntericBio® Dx kit providesreagents and procedure for DNAtesting without extraction of stoolspecimens.Substantial equivalence will bedemonstrated in clinical testingusing human specimens andcompared to the predicate device.
TechnologicalPrinciples	Real-time multiplex RT-PCR based onthe hydrolysis probe reagent chemistry.	Nested multiplex RT-PCR followed byhigh resolution melting analysis toconfirm identity of the PCR product.	Different: Both devices usemultiplex real-time PCR howeverthe new device uses hydrolysisprobe reagent chemistrycompared to melting analysis inthe predicate device.Substantial equivalence will bedemonstrated in clinical testingusing human specimens andcompared to the predicate device.
Element	Subject (New) Device	Proposed Predicate Device	Discussion
DetectionMethodology/Platform	The Applied Biosystems 7500 Fast DxReal-Time PCR instrument is a real-timenucleic acid amplification and five colorfluorescence detection system for usewith the EntericBio® Dx Assay. Resultsare analyzed and interpreted using theEntericBio® FastFinder plugin.	Detection and interpretation isautomated on the FilmArray®instrument by analysis of the specificPCR product melts (meltingtemperature).	Different: The detection systemand analysis differ between thetwo devices.Substantial equivalence will bedemonstrated in clinical testingusing human specimens andcompared to the predicate device.
Device Format	The EntericBio® Dx Assay kits provide aPCR master mix with all the reagentsrequired to perform each test which arelyophilized into individual reactionwells. Each reaction well contains anInternal Amplification Control (IAC) tomonitor for PCR inhibition. A syntheticPositive Control (containing targetsequences) is provided with each kit tomonitor the thermal cycling steps andreagent integrity during amplificationand detection process.	The FilmArray Gl panel provides all thereagents lyophilized into a disposablepouch. Each pouch contains twocontrols which monitor sampleprocessing and both stages of PCR andmelt analysis.	Assay specific requirements

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Intended Use

The EntericBio" Dx assay, performed on ABI 7500 Fast Dx real-time instrument, is an in vitro multiplexed nucleic acid test for the direct, simultaneous, qualitative detection and identification of multiple enteric pathogens in Cary-Blair preserved stool specimens from individuals with signs and symptoms of infectious colitis or gastroenteritis. The test is based on detection of nucleic acids from:

• . Salmonella enterica spp.
• Shigella spp./ Enteroinvasive E. coli (EIEC)
• Campylobacter spp. (jejuni, coli and lari)
• STEC (Shiga-like toxin-producing E. coli), stx1/stx2 genes
• Vibrio spp. (cholerae and parahaemolyticus)
• . Giardia lamblia (also known as G. intestinalis and G. duodenalis)
• Entamoeba histolytica

Testing is performed on Cary-Blair preserved diarrheal specimens from symptomatic patients with suspected acute gastroenteritis, enteritis or colitis of bacterial or parasitic origin. The test is performed directly on the specimen, utilizing real-time polymerase chain reaction (PCR) for the amplification of Salmonella-specific, Campylobacter-specific, Shigella/ EIEC-specific ipaH, stx1/stx2, Vibrio-specific, Entamoeba-specific and Giardia-specific gene sequences. The test utilizes fluorogenic sequence-specific hybridization probes for the detection of the amplified DNA.

This test is intended for use, in conjunction with clinical presentation, laboratory findings and epidemiological information, as an aid in the diagnosis of Salmonella, Shigella / EIEC, Shigalike toxin-producing E. coli, Campylobacter spp., Entamoeba histolytica and Giardia spp. infections in humans.

Results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results do not rule out co-infection with other organisms that are not detected by this test and may not be the sole or definitive cause of patient illness. Negative EntericBio" Dx assay results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Methodology

The EntericBio" Dx assay is a molecular in vitro diagnostic test for direct, qualitative detection and identification of the following enteric organisms, associated with human gastroenteritis, directly, from Cary-Blair preserved fecal specimens:

• . Salmonella enterica spp.,
• Shigella spp./ Enteroinvasive E. coli,
• Campylobacter spp. (jejuni, coli and lari),
• Vibrio spp. (cholerae and parahaemolyticus), ●

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• STEC(Shiga-like toxin-producing E. coli) stx1/stx2 genes,
• Giardia lamblia (also known as G. intestinalis and G. duodenalis),
• Entamoeba histolytica

The assay is composed of Stool Preparation Solution (SPS) tubes, PCR reagent strips containing lyophilized reagents, Resuspension Buffer (Negative Kit Control), Positive Kit Control containing DNA from all target analytes (with appropriate reconstitution buffer), and associated accessories,instruments and software for detection of bacterial and parasitic causes of gastroenteritis in humans.

The EntericBio" Dx assay detects target DNA from diarrheal Cary-Blair stool specimens from symptomatic individuals with suspected gastroenteritis or infectious colitis.

The assay works directly from a Cary-Blair preserved stool sample and does not require commercial nucleic acid extraction /purification. The PCR master mix with all the reagents required to perform each test is lyophilized into individual reaction wells on a strip. Each reaction well contains an Internal Amplification Control (IAC) to monitor for PCR inhibition.

Performance Data:

Analytical Performance

Reproducibility

A reproducibility study was performed to determine the inter-site and overall reproducibility of the EntericBio® Dx assay. Reproducibility testing was performed in-house, simulating a multi-site study by using three instruments (hereinafter 'in-house sites') and panels of contrived stool samples, each spiked with various combinations of Vibrio parahaemolyticus, Shigella sonnei and Giardia lamblia. These three target analytes were representative of each of the three component multiplex assays and each analyte was evaluated at three different concentrations (True Negative, Low Positive and Moderate Positive) in three independently manufactured batches of EntericBio® Dx Assay. Reproducibility panels were tested at each of the in-house sites by two different operators for five non-consecutive days.

The acceptance criteria for this study were that the moderate positive target concentration (3x LoD) must show 100% agreement with the expected result for each target analyte and the low target concentration (1.5x LoD) must show ≥95% agreement with the expected result for each target analyte.

All targets showed 100% agreement with the expected result for moderate positive concentrations tested across the in-house sites. Shigella sonnei and Vibrio parahaemolyticus samples showed 100% agreement with the expected result for low positive concentrations tested the in-house sites. Giardia lamblia at the low positive concentration was observed at 98% relative to the expected result. All acceptance criteria for each target analyte at each concentration tested across the in-house sites were met, as shown in Tables 1-2 below.

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Organism Tested	ConcentrationTested	FastFinderExpectedResult	% Agreement with Expected Result			All
			Serosep1	Serosep2	Serosep3
Shigella sonneiDSM 5570	ModeratePositive3x LoD	Positive	30/30100%	30/30100%	30/30100%	90/90100%
	Low Positive1.5x LoD	Positive	30/30100%	30/30100%	30/30100%	90/90100%
	True Negative	Negative	60/60100%	60/60100%	59/59*100%	179/179100%
VibrioparahaemolyticusCCUG 14474	ModeratePositive3x LoD	Positive	30/30100%	30/30100%	30/30100%	90/90100%
	Low Positive1.5x LoD	Positive	30/30100%	30/30100%	30/30100%	90/90100%
	True Negative	Negative	60/60100%	60/60100%	59/59*100%	179/179100%
Giardia lambliaP101	ModeratePositive3x LoD	Positive	30/30100%	30/30100%	30/30100%	90/90100%
	Low Positive1.5x LoD	Positive	30/30100%	30/30100%	29/3095%	89/9098%
	True Negative	Negative	60/60100%	60/60100%	59/59*100%	179/179100%

Table 1: Reproducibility study results

• One True Negative sample was invalid on the EntericBio FastFinder plugin and subsequently removed from study

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Organism Tested	Assay	ConcentrationTested	Test Site	Cq Reproducibility
				Mean(Cq)	STDEV	%CV
Shigella sonneiDSM 5570	Well A	ModeratePositive	Serosep 1	27.10	$\pm$ 0.68	2.51
			Serosep 2	26.84	$\pm$ 0.47	1.74
			Serosep 3	27.01	$\pm$ 0.79	2.91
		3x LoD	All Sites	26.98	$\pm$ 0.66	2.44
		Low Positive	Serosep 1	29.07	$\pm$ 0.57	1.97
			Serosep 2	28.88	$\pm$ 0.53	1.85
			Serosep 3	28.95	$\pm$ 0.57	1.98
		1.5x LoD	All Sites	28.97	$\pm$ 0.57	1.97
VibrioparahaemolyticusCCUG 14474	Well B	ModeratePositive	Serosep 1	31.73	$\pm$ 0.42	1.34
			Serosep 2	31.87	$\pm$ 0.24	0.74
			Serosep 3	31.74	$\pm$ 0.44	1.40
		3x LoD	All Sites	31.79	$\pm$ 0.38	1.21
		Low Positive	Serosep 1	33.50	$\pm$ 0.48	1.44
			Serosep 2	33.34	$\pm$ 0.50	1.51
			Serosep 3	33.30	$\pm$ 0.79	2.37
		1.5x LoD	All Sites	33.38	$\pm$ 0.61	1.82
Giardia lambliaP101	Well C	ModeratePositive	Serosep 1	32.96	$\pm$ 0.85	2.58
			Serosep 2	33.04	$\pm$ 1.36	4.12
			Serosep 3	33.30	$\pm$ 1.27	3.81
		3x LoD	All Sites	33.10	$\pm$ 1.17	3.53
		Low Positive	Serosep 1	33.98	$\pm$ 1.18	3.49
			Serosep 2	34.40	$\pm$ 1.38	4.01
			Serosep 3	33.11	$\pm$ 1.26	3.80
		1.5x LoD	All Sites	33.85	$\pm$ 1.37	4.05

Table 2: Summary of the Reproducibility of the Cq Values

Limit of Detection (LoD)

The analytical sensitivity (limit of detection or LoD) of the EntericBio® Dx Assay was determined using contrived samples with target analytes spiked into a negative stool matrix (Cary-Blair preserved stool) at three concentrations: greater than estimated LoD (High), estimated LoD (Medium) and less than LoD (Low). A total of 20 replicate EntericBio® Stool Preparation Solution (SPS) were tested from each sample using three independently manufactured lots of EntericBio® Dx assay. The LoD is defined as the lowest concentration of analyte that can be consistently detected ≥95% of the time. A minimum of two strains were tested for each EntericBio® Dx target organism and toxin gene. Table 3 lists the LoD determined for each target organism.

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Organism	Strain	LoD
Salmonella spp.	Salmonella enterica serovar Enteritidis DSM 17420	$8 x 10^4$ CFU/mL
	Salmonella enterica serovar Typhi NCTC 10787	$8 x 10^4$ CFU/mL
Shigellaspp./EIEC	Shigella sonnei DSM 5570	1.25 x $10^4$ CFU/mL
	E. coli (ipaH) DSM 9029	1 x $10^4$ CFU/mL
Campylobacterspp.	Campylobacter jejuni ATCC 33560	4 x $10^4$ CFU/mL
	Campylobacter coli DSM 4689	4 x $10^4$ CFU/mL
	Campylobacter lari DSM 11375	1 x $10^4$ CFU/mL
Escherichia coli(STEC)	E. coli (stx1) NVRL 15x23 RE-008 (0111:H-)	5 x $10^5$ CFU/mL
	E. coli (stx2) NVRL 15x24 RE-006 (O26:H11)	1 x $10^6$ CFU/mL
Vibrio spp.	Vibrio parahaemolyticus CCUG 14474	1 x $10^4$ CFU/mL
	Vibrio cholerae NCTC 3661	1 x $10^4$ CFU/mL
Giardia lamblia	Giardia intestinalis (WB) ATCC 30957	25 cells/mL
	Giardia intestinalis (New Orleans) ATCC 50137	100 cells/mL
Entamoebahistolytica	Entamoeba histolytica (HM-1: IMSS) ATCC 30459	25 cells/mL
	Entamoeba histolytica (HK-9) ATCC 30015	100 cells/mL

Table 3: LoD determined using the EntericBio® Dx Assay

Fresh vs. Frozen

The Fresh versus Frozen Specimen Stability study was performed to support the inclusion of frozen, retrospective specimens and contrived samples in the clinical and analytical studies of the EntericBio® Dx assay; the test is not intended for use on frozen specimens.

Sixty contrived samples were prepared for each EntericBio® Dx target analyte and tested at Time 0 (TO). These samples were subsequently frozen at -20°C for 3 months before re-testing. Contrived samples were prepared in a negative stool matrix (Cary-Blair preserved stool) with target analytes spiked at three concentrations (5X, 2X and 1X LoD).

Agreement between detection of fresh and frozen samples was 100% for Salmonella, Shigella and STEC and <80% for four analytes (Campylobacter, Vibrio, Giardia and Entamoeba). Additional testing was performed using frozen clinical specimens from the prospective study to further support the inclusion of these four analytes in clinical studies.

Analytical Reactivity/Inclusivity

The analytical reactivity (Inclusivity) of the EntericBio® Dx Assay was determined using a panel of 101 target organisms (Table 4 below) which represents the diversity of the EntericBio® Dx target analytes. Fifteen of these organisms were also evaluated in the LoD determination study.

Table 4: EntericBio® target analytes used in this study

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Organism	Supplier	Catalogue Number
Salmonella enterica serovar Enteritidis	DSMZ1	DSM 17420
Salmonella enterica serovar Typhi	NCTC2	NCTC 10787
Salmonella enterica subsp. enterica I serovarCholerasuis	ATCC3	ATCC 7001
Salmonella enterica subsp. enterica I serovarParatyphi B	ATCC	ATCC 8759
Salmonella enterica subsp. enterica I serovarParatyphi A	ATCC	ATCC 9281
Salmonella enterica subsp. enterica I serovarParatyphi C	ATCC	ATCC 13428
Salmonella enterica subsp. enterica I serovarTyphimurium	DSMZ	DSM 101475
Salmonella enterica subsp. enterica I serovarDublin	DSMZ	DSM 102345
Salmonella enterica subsp. enterica I serovarAgona	DSMZ	DSM 102864
Salmonella enterica subsp. enterica I serovarHeidelberg	DSMZ	DSM 9379
Salmonella enterica subsp. enterica I serovarInfantis	NCTC	NCTC 10679
Salmonella enterica subsp. enterica I serovarThompson	NCTC	NCTC 2252
Salmonella enterica subsp. enterica I serovarOranienburg	NCTC	NCTC 5743
Salmonella enterica subsp. enterica I serovarBareilly	NCTC	NCTC 5745
Salmonella enterica subsp. enterica I serovarMontevideo	NCTC	NCTC 5747
Salmonella enterica subsp. enterica I serovarBraenderup	NCTC	NCTC 5750
Salmonella enterica subsp. enterica I serovarMuenchen	NCTC	NCTC 5755
Salmonella enterica subsp. enterica I serovarSaintpaul	NCTC	NCTC 6022
Salmonella enterica subsp. enterica I serovarMississippi	NCTC	NCTC 6487
Salmonella enterica subsp. enterica I serovarJaviana	NCTC	NCTC 6495
Salmonella enterica subsp. enterica I serovarNewport	NCTC	NCTC 6704
Salmonella enterica subsp. enterica I serovarSchwarzengrund	NCTC	NCTC 6756
Salmonella enterica subsp. enterica I serovarHadar	NCTC	NCTC 9877
Salmonella enterica subsp. enterica I serovar 4,[5] 12:i:-	NSSLRL5	NSSLRL Ms170397
Salmonella enterica subsp. II (salame)	DSMZ	DSM 9220
Organism	Supplier	Catalogue Number
Salmonella enterica subsp. Illa (arizonae)	DSMZ	DSM 9386
Salmonella enterica subsp. Illb (diarizonae)	DSMZ	DSM 14847
Salmonella enterica subsp. IV (houtenae)	DSMZ	DSM 9221
Salmonella enterica subsp. VI (indica)	DSMZ	DSM 14848
Shigella sonnei	DSMZ	DSM 5570
Shigella sonnei	DSMZ	DSM 25715
Shigella sonnei	ATCC	ATCC 11060
Shigella sonnei	ATCC	ATCC 25931
Shigella sonnei	ATCC	ATCC 9290
Shigella flexneri (serotype 2a)	DSMZ	DSM 4782
Shigella flexneri (serotype 2a)	ATCC	ATCC 700930
Shigella flexneri (serotype 1a)	ATCC	ATCC 9199
Shigella flexneri (serotype 2b)	ATCC	ATCC 12022
Shigella flexneri (serotype 6)	ATCC	ATCC 12025
Shigella boydii (serotype 2)	DSMZ	DSM 7532
Shigella boydii (serotype 1)	ATCC	ATCC 9207
Shigella boydii (serotype 20)	ATCC	ATCC BAA-1247
Shigella boydii (serotype 10)	ATCC	ATCC 12030
Shigella boydii (serotype 4)	ATCC	ATCC 9210
Shigella dysenteriae (serotype 1)	NCTC	NCTC 4837
Shigella dysenteriae (serotype 2)	NCTC	NCTC 5109
Shigella dysenteriae (serotype 7)	NCTC	NCTC 9763
Shigella dysenteriae (serotype 3)	NCTC	NCTC 6340
Shigella dysenteriae (serotype 9)	NCTC	NCTC 9347
Escherichia coli EIEC (serotype O28ac:H-)	DSMZ	DSM 9025
Escherichia coli EIEC (serotype O29:H10)	DSMZ	DSM 9026
Escherichia coli EIEC (serotype 0136:H-)	DSMZ	DSM 9032
Escherichia coli EIEC (serotype 0124:H30)	DSMZ	DSM 9031
Escherichia coli EIEC (serotype O144:H-) (ipaH)8	DSMZ	DSM 9029
Campylobacter jejuni	ATCC	ATCC 33560
Campylobacter jejuni subsp. jejuni	DSMZ	DSM 104743
Campylobacter jejuni subsp. jejuni	DSMZ	DSM 27585
Campylobacter jejuni subsp. doylei	DSMZ	DSM 104768
Campylobacter jejuni subsp. doylei	NCTC	NCTC 12208
Campylobacter coli	DSMZ	DSM 110395
Campylobacter coli	DSMZ	DSM 24155
Campylobacter coli	DSMZ	DSM 24106
Campylobacter coli	DSMZ	DSM 24206
Campylobacter coli	DSMZ	DSM 4689
Campylobacter lari	DSMZ	DSM 11375
Campylobacter lari	NCTC	NCTC 12892
Campylobacter lari	NCTC	NCTC 12893
Campylobacter lari	NCTC	NCTC 12894
Campylobacter lari	NCTC	NCTC 12895
Vibrio parahaemolyticus	DSM	DSM 101031
Organism	Supplier	Catalogue Number
Vibrio parahaemolyticus	DSM	DSM 11058
Vibrio parahaemolyticus	DSM	DSM 15477
Vibrio parahaemolyticus	DSM	DSM 27657
Vibrio parahaemolyticus	CCUG6	CCUG 14474
Vibrio cholerae (0:1 Ogawa classical)	NCTC	NCTC 3661
Vibrio cholerae 0:1 Biotype El Tor	NCTC	NCTC 8457
Vibrio cholerae 0:1 Ogawa	NCTC	NCTC 8021
Vibrio cholerae non-O:1, non-0139 (0:3)	NCTC	NCTC 11502
Escherichia coli 0157 (stx2)	NVRL4	NVRL 17X01 RE-001
Escherichia coli 0157 (stx1 & stx2)	NVRL	NVRL17X04 RE-002
Escherichia coli 0157 (stx2)	NVRL	NVRL 17X09 RE-003
Escherichia coli O157 (stx1 & stx2)	NVRL	NVRL 17X15 RE-004
Escherichia coli 0157 (stx2)	NVRL	NVRL 17S110 RE-005
Escherichia coli 0103:H2 (stx1)	NVRL	NVRL 17X128 RE-010
Escherichia coli 0111:H8 (stx1)	NVRL	NVRL 13S5371 RE-009
Escherichia coli O121:H19 (stx2)	NVRL	NVRL 15X18 RE-007
Escherichia coli O157:NM	NVRL	NVRL 06-CC3 RE-011
Escherichia coli O157:H7 (stx1 & stx2)	NCTC	NCTC 12079 NVRLRE-012
Escherichia coli O157:H- (stx2)	NCTC	NCTC 12080
Escherichia coli 0111:H- (stx1)	NVRL	NVRL 15x23 RE-008
Escherichia coli O26:H11 (stx2)	NVRL	NVRL 15x24 RE-006
Escherichia coli 0113 7	N/A	N/A
Escherichia coli 045 7	N/A	N/A
Escherichia coli O104 7	N/A	N/A
Escherichia coli 0145 7	N/A	N/A
Giardia intestinalis (WB)	ATCC	ATCC 30957
Giardia intestinalis (New-Orleans-1)	ATCC	ATCC 50137
Giardia intestinalis GS Assemblage B	ATCC	ATCC 50581
Giardia intestinalis Portland 1	ATCC	ATCC 30888
Giardia intestinalis Mario	ATCC	ATCC PRA-244
Entamoeba histolytica (HM-1: IMSS)	ATCC	ATCC 30459
Entamoeba histolytica (HK-9)	ATCC	ATCC 30015
Entamoeba histolytica HB-301:NIH	ATCC	ATCC 30190
Entamoeba histolytica HU-21:AMC	ATCC	ATCC 30457
Entamoeba histolytica IP:1182;2	ATCC	ATCC PRA-357

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1 DSMZ - Deutsche Sammlung von Mikroorganismen und Zenllkulturen

2 NCTC - National Collection of Type Cultures, a Culture Collection of Public Health England

3 ATCC – American Type Culture Collection

4 NVRL - National VTEC Reference Laboratory, Ireland

• 5 NSSLRL- National Salmonella, Shigella and Listeria Reference Lab, Galway, Ireland
• 6 CCUG Culture Collection University of Gothenburg
• 7 Inclusivity predicted based on in-silico analysis

8 EIEC strain which also generates positive result for Shigella/EIEC (ipaH)

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Testing was performed using contrived samples with target analytes spiked into a negative stool matrix (Cary-Blair preserved stool) at a concentration of approximately three times the respective LoD, where possible.

All organisms tested were detectable over three replicates demonstrating the inclusivity of the EntericBio® Dx assay.

Analytical Exclusivity

The analytical reactivity (Exclusivity) of the EntericBio® Dx Assay was determined using a comprehensive panel of 133 bacteria, viruses and parasites (Table 5 below). The panel of organisms tested consisted of organisms closely related to the EntericBio® Dx assay targets and organisms likely to be found in human feces.

Organism	Organism	Organism	Organism
Yersinia aldovae	Cryptosporidiummeleagridis	Citrobacter koseri	Pseudomonas putida1
Yersinia bercovieri	Cryptosporidium Skunkgenotype1	Clostridium difficile	Rahnella aquatilis
Yersiniaentomophaga	Cryptosporidiumubiquitum1	Clostridium perfringens	Rotavirus A1
Yersinia frederiksenii	Cryptosporidiumviatorum1, 2	Clostridium sordelli	Ruminococcus gauvreauii
Yersinia intermedia	Escherichia vulneris	Cronobacter sakazakii	Saccharomyces cerevisiae
Yersinia kristensenii	Cryptosporidiumcuniculus1,3	Dientamoeba fragilis	Sapovirus1
Yersinia massiliensis	Cryptosporidium Horsegenotype1	Edwardsiella tarda	Serratia liquefaciens
Yersinia mollaretti	Cryptosporidium felis1	Encephalitozoon cuniculi1	Serratia marcescens
Yersinia nurmii	Cryptosporidium canis1	Enterobacter aerogenes	Serratia odoriferae
Yersinia pekkaneneii	Cryptosporidium xiaoi1, 4	Enterobacter cloacae	Serratia rubidaea
Yersiniapseudotuberculosis	Cryptosporidiumandersoni1	Enterococcus faecalis	Staphylococcus aureus
Yersinia rodhei	Cryptosporidium baileyi1	Enterococcus faecium	Staphylococcus epidermidis
Yersinia ruckeri	Entamoeba dispar5	Eubacterium rectale1, 6	Stenotrophomonasmaltophilia
Yersinia similis	Entamoebamoshkovoskii	Ewingella americana	Streptococcus agalactiae
Vibrio alginolyticus	Entamoeba invadens	Fusobacteriumgonidiaformans	Streptococcus bovis
Vibrio fluvialis	Aeromonas hydrophilia	Fusobacterium nucleatum	Streptococcus equinus
Organism	Organism	Organism	Organism
Vibrio furnissii	Adenovirus F401	Fusobacterium varium	Toxoplasma gondii
Vibrio mimicus	Alcaligenes faecalis	Hafnia alvei	Campylobacter concisus1
Vibrio harvei	Anaerococcushydrogenalis	Klebsiella oxytoca	Campylobacter curvus1
Vibrio fischeri1	Anaerostipes hadrus7	Klebsiella pneumoniae	Campylobacter fetus subsp.Fetus
Vibrio damsela	Arcobacter butzleri	Lactobacillus acidophilus	Campylobacter fetus subsp.Venerealis
Griomontia hollisae	Astrovirus1	Lactobacillus lactis	Campylobacter gracilis
Vibrio diazotrophicus	Bacillus cereus	Listeria monocytogenes	Campylobacter helveticus1
Vibrio proteolyticus	Bacillus subtilis subspsubtilis	Morganella morganii	Campylobacter hominis
Vibrio natrigens	Bacillus subtilis subspspizizenii	Neisseria gonorrhoeae	Campylobacterhyointestinalis
Vibrio pelagius1	Bacteroides fragilis	Norovirus GGI1	Campylobacter mucosalis1
Vibrio campbellii	Bacteroidesthetaiotaomicron	Norovirus GGII1	Campylobacter rectus1
Vibrio vulnificus8	Bifidobacterium breve	Plesiomonas shigelloides	Campylobacter showae
Escherichia coli nontoxigenic	Bifidobacterium longum	Prevotella melaninogenica	Campylobacter sputorum1,9
Escherichia coli(Enteropathogenic)	Blastocystis hominis	Proteus mirabilis	Campylobacter ureolyticus
Escherichia coli(Enterotoxigenic)	Candida albicans	Proteus vulgaris	Salmonella bongori
Escherichiahermannii	Citrobacteramalonaticus1	Providencia stuartii	Salmonella subterranea
Escherichia blattae	Citrobacter freundii	Pseudomonas aeruginosa	Campylobacter upsaliensis

Table 5: Microorganisms used in this study

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Escherichia fergusonii

1 Organisms for which testing was performed using genomic DNA

3 Cryptosporidium viatorum: Shiqella detected in 3/3 replicates. Bi-directional sequencing demonstrated the presence of Shigella DNA suggesting possible sample contamination with Shigella. In silico analysis indicated no cross-reactivity should occur.

3 Cryptosporidium cuniculus: Giardia detected in 2/3 replicates. Bi-directional sequencing of amplicon failed and thus it could not be confirmed empirically if the sample was contaminated with Giardia or if cross reactivity-occurred. In silicated no crossreactivity should occur.

4 Cryptosporidium xiaoi: Campylobacter detected in 3/3 replicates. Bi-directional sequencing demonstrated the presence of Campylobacter DNA suggesting possible sample contamination with Compylobacter. In silico analysis indicated no cross-reactivity should occur.

5 Entamoeba dispar: IAC failures were observed for 5/6 replicates after repeat testing. In silico analysis demology for the primers and predicts amplification to occur and competitively inhibit the IAC, but the probe has several to produce a detected signal.

6 Eubacterium rectale: Campylobacter detected in 3/3 replicates. Bi-directional sequencing demonstrated the presence of Campylobacter and Vibrio DNA suggesting possible sample contamination with Vibrio and Campylobacter. In silico analysis indicated no cross-reactivity should occur.

7 Anaerostipes hadrus: Campylobacter detected in 2/3 replicates. Bi-directional sequencing demonstrated the presence of Campylobacter DNA suggesting possible sample contamination with Campylobocter. In silico analysis indicated no cross-reactivity should occur. 8 Vibrio vulnificus: Evaluated by in silico analysis indicated no cross-reactivity should occur.

9 Campylobacter sputorum: Vibrio detected in 1/3 replicates. Bi-directional sequencing demonstrated the presence of Vibrio DNA suggesting possible sample contamination with Vibrio. In silico analysis indicated no cross-reactivity should occur.

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Testing was performed using contrived samples with target analytes spiked into a negative stool matrix (Cary-Blair preserved stool) at a concentration of 10° CFU or cells/mL. Genomic DNA/RNA was tested at a concentration of 106 genomic equivalents per reaction where possible.

The non-target organisms which were shown to cross react were all closely related to their respective target organism and shared significant sequence similarity and are presented below (Table 6).

Table 6: Non-target organisms for which cross reactivity was observed with the EntericBio® Dx assay

Cross Reacting Organisms	Target Analyte
Vibrio campbellii	Vibrio
Vibrio fluvialis	Vibrio
Vibrio furnissii	Vibrio
Vibrio mimicus	Vibrio
Vibrio fischeri	Vibrio
Vibrio natriegens	Vibrio

Microbial Interference

A study was performed to evaluate the performance of the EntericBio® Dx Assay in the presence of high concentrations of eleven microorganisms that are commonly found in fecal specimens (Table 7). Each potentially interfering microorganism was tested in the presence of 3X LoD of each target analyte in the EntericBio® Dx assay. No interference was observed between the potentially interfering organisms and the IAC or any of the EntericBio® Dx target analytes tested.

Substance	Source/ID	Concentration tested
Aeromonas hydrophila	DSM 17695	106 CFU/mL
Bacteroides fragilis	DSM 2151	106 CFU/mL
Staphylococcus aureus	DSM 20231	106 CFU/mL
Escherichia coli	DSM 30083	106 CFU/mL
Enterococcus faecalis	DSM 20478	106 CFU/mL
Clostridium perfringens	DSM 798	106 CFU/mL
Saccharomyces cerevisiae	DSM 1848	106 CFU/mL
Blastocystis hominis	Clinical specimen	Not quantifiable
Pseudomonas aeruginosa	DSM 50071	106 CFU/mL
Klebsiella oxytoca	DSM 5175	106 CFU/mL
Candida albicans	DSM 1577	106 CFU/mL

Table 7: Potentially interfering microorganisms tested in this study

DSMZ - Deutsche Sammlung von Mikroorganismen und Zenllkulturen

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Potentially Interfering Substances

A study was performed to evaluate the performance of the EntericBio® Dx Assay in the presence of 23 potentially interfering/cross-reactive substances, at high, but clinically relevant levels, that might be present in fecal specimens. Each substance was tested in the presence of each target analyte detected by the assay at low positive concentrations. Each substance was also evaluated in negative stool samples (without target organisms) where results demonstrated IAC failure in the presence of Benzalkonium chloride at a concentration of >1% v/v and Hemorrhoidal cream at a concentration of >1% w/v.

Overall, the presence of 21/23 potentially interfering substances had no effect on the detection of the target analytes or the EntericBio® Dx assay internal control at the concentrations listed in Table 8 below. A false negative result for V. parahaemolyticus was observed for 1/3 sample replicates containing hemorrhoidal cream (1% w/v) and false negative results for C. jejuni were observed for 2/3 sample replicates containing tetracycline (1.6% w/v).

Substance	Passing Concentration
Amoxicillin	1% w/v3
Benzalkonium Chloride	0.15% v/v3
Ceftriaxone	1% w/v
Cholesterol	7% w/v
Ciprofloxacin	5.4% w/v
Erythromycin	1.5% w/v
Hemorrhoidal cream	1% w/v1,3
Human DNA	0.1% v/v
Hydrocortisone	50% w/v
Laxative	5% v/v
Loperamide Hydrochloride	0.5% w/v3
Lubricant	50% w/v
Magnesium Hydroxide	0.5% w/v3
Metronidazole	6% w/v
Mucin	10% w/v
Naproxen sodium (NSAID)	10% w/v
Nystatin Cream	50% w/v
Sudocrem	50% w/v
Sulfamethoxazole	4% w/v3
Tetracycline	0.8% w/v2,3
Trimethoprim	1.6% w/v
Vagisil	50% w/v
Whole Human Blood	5% v/v3

Table 8: Highest concentration of each substance for which most EntericBio® Dx target analytes were detected (Substances for which Interference was observed are bolded)

1 V. parahaemolyticus failed in 1/3 replicates at this concentration

² C. jejuni was only tested at 1.6% w/v and 2/3 replicates failed at this concentration

3 Interference for detection of one or more targeted organisms was observed for samples with higher than the listed concentration

Competitive Inhibition

This study was performed to evaluate the performance of the EntericBio® Dx Assay when challenged with combinations of target analytes in order to determine the potential for

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competitive interference in patient specimens with mixed infections. The combinations of analytes tested were selected based on the frequency of co-infections reported in the literature. This study was performed using Cary Blair preserved negative stool specimens spiked with dual EntericBio® Dx target analyte combinations. Binary combinations of target analytes were spiked at both high and low concentrations and tested in triplicate (Table 9).

The potential for competitive inhibition was not evaluated for analytes detected in Well B (Vibrio cholerae/parahaemolyticus and STEC)

Based on the results data, competitive inhibition was observed for Giardia lamblia in the presence of both low or high concentrations of Entamoeba histolytica.

Table 9: Combinations of Entericbio " Dx Target Analytes Evaluated For Competitive Inhibition

EntericBio® Dx Target Analyte Combinations
Salmonella High (300X LoD)	& Campylobacter Low (3X LoD)
Salmonella Low (18X LoD)	& Campylobacter High (50X LoD)
Shigella High (200X LoD)	& Giardia Low (3X LoD)
Shigella Low (12X LoD)	& Giardia High (50X LoD)
Entamoeba High (50X LoD)	& Cryptosporidium Low (10X LoD)
Entamoeba Low (3X LoD)	& Cryptosporidium High (50X LoD)
Cryptosporidium High (50X LoD)	& Giardia Low (3X LoD)
Cryptosporidium Low (10X LoD)	& Giardia High (50X LoD)
Campylobacter High (50X LoD)	& Giardia Low (3X LoD)
Campylobacter Low (3X LoD)	& Giardia High (50X LoD)
Campylobacter High (50X LoD)	& Cryptosporidium Low (10X LoD)
Campylobacter Low (3X LoD)	& Cryptosporidium High (50X LoD)
Salmonella High (300X LoD)	& Cryptosporidium Low (10X LoD)
Salmonella Low (12X LoD)	& Cryptosporidium High (50X LoD)
Entamoeba High (50X LoD)	& Giardia Low (3X LoD)
Entamoeba Low (3X LoD)	& Giardia High (50X LoD)

Carry-Over and Cross Contamination

The Cross-Contamination (Carryover) study was performed to investigate the potential for carryover and cross-contamination of the EntericBio® Dx assay on the EntericBio® Workstation between and within experiments. Samples with a high concentration of target organism(s) were processed in alternating sequence with negative samples. Contrived samples were prepared in a negative stool matrix (Cary-Blair preserved stool) with two representative target analytes (one bacterial (Shigella sonnei) and one parasitic target (Giardia lamblia)) spiked at a high concentration (800x and 1000x LoD respectively). Two representative negative samples were prepared from an uninoculated negative stool matrix. The study consisted of three separate experiments for each target analyte and each experiment contained 30 samples (15 positive and 15 negative).

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No carryover or cross-contamination occurred with the EntericBio® Dx assay on the EntericBio® Workstation between or within each of the assay experiments.

Specimen Stability

The specimen stability study was performed to determine the recommended specimen storage conditions for use with the EntericBio® Dx assay.

The recommended storage conditions for Cary-Blair preserved stool is 5 days at 2-8°C.

Real Time Stability

The objective of this study is to determine the shelf life in real-time of the EntericBio® Dx assay and its constituent components.

The shelf life was determined to be six (6) months at 2-8°C.

Clinical Performance

Performance characteristics of the EntericBio" Dx assay were established in a multi-site clinical study, including three (3) distinct study sites - two (2) US clinical sites and one (1) non-US clinical site.

The performance of the EntericBio" Dx assay was assessed by testing 4 specimen types:

• 1. Fresh, prospectively-collected, Cary-Blair preserved fecal specimens from patients presenting with symptoms of gastrointestinal infection;
• 2. Fresh, selected Cary-Blair preserved fecal specimens from patients presenting with symptoms of gastrointestinal infection. Specimens were selected based on the results obtained with Standard of Care methods in use at the study sites;
• 3. Frozen, well characterized Cary-Blair preserved fecal specimens from patients presenting with symptoms of gastrointestinal infection, known to be positive for selected target analytes;
• 4. Spiked/Contrived Cary-Blair preserved fecal samples for lower prevalence targets

The performance of the EntericBio" Dx assay in fresh and archived specimens was evaluated by comparing the test result for each target analyte with the appropriate comparator method(s).

For STEC and Campylobacter target analytes, a composite comparator method of three FDAcleared assays was used for fresh specimens. A specimen was characterised as positive if 2 out of 3 comparator assays were positive and a specimen was characterized as negative if 2 out of 3 comparator assays were negative. For the remaining target analytes and archived specimens, the comparator method consisted of one, FDA-cleared assay.

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A total of 1523 fresh samples (1491 prospective, 32 select) were enrolled during the clinical trial. Of the 1491 prospective samples, 19 were excluded from the study, giving 1472 evaluable samples. The reasons for exclusion included invalid test result for the EntericBio Dx assay (n=9) or by the comparator method (n=6), Indeterminate result by the EntericBio Dx assay (n=1), specimen not tested with comparator method within sample stability (n=2), duplicate specimen from previously enrolled patient (n=1).

Table 10 provides a summary of demographic information for the fresh specimens enrolled into the clinical study.

Gender	Number of specimens (%)
Male	636 (41.8%)
Female	880 (57.8%)
N/A	7 (0.4%)
Age Group	Number of specimens (%)
<1 year	11 (0.7%)
1-5 years	65 (4.3%)
6-12 years	27 (1.8%)
13-21 years	62 (4.1%)
22-65 years	863 (56.7%)
+ 65 years	489 (32.1%)
N/A	6 (0.4%)
Patient Status	Number of specimens (%)
Outpatient	692 (45.4%)
In-patient	700 (46.0%)
Emergency Care	94 (6.2%)
Long term care	22 (1.4%)
N/A	15 (1.0%)
Total	1523

Table 10: Demographic Summary for the fresh specimens (n=1523) enrolled into the clinical study

Several target analytes had a low prevalence in the fresh clinical samples. To supplement the results of the fresh testing, 212 frozen, retrospective specimens were included in the

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study. These specimens were selected and archived based on previously testing positive for the desired target analytes by routine diagnostic methods used by the collection sites.

Of the 212 archived specimens enrolled into the frozen clinical study, 3 samples were excluded from the study, giving 209 evaluable samples. The reason for exclusion was invalid test result for the EntericBio Dx assay (n=1) and Indeterminate result by the EntericBio "Dx assay (n=2).

The archived specimens were distributed across the three testing sites and were randomized such that the users performing the testing were blinded as to the expected test result.

Table 11 provides a summary of demographic information for the archived specimens enrolled into the clinical study.

Table 11: Demographic Summary for the archived specimens (n=212) enrolled into the
clinical study

Gender	Number of specimens (%)
Male	105 (49.5%)
Female	103 (48.6%)
N/A	4 (1.9%)
Age Group	Number of specimens (%)
<1 year	5 (2.4%)
1-5 years	51 (24.1%)
6-12 years	21 (9.9%)
13-21 years	14 (6.6%)
22-65 years	96 (45.3%)
+ 65 years	21 (9.9%)
N/A	4 (1.9%)
Patient Status	Number of specimens (%)
Outpatient	136 (64.2%)
In-patient	42 (19.8%)
Emergency Care	27 (12.7%)
Long term care	1 (0.5%)
N/A	6 (2.8%)
Total	212

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Since prevalence of some target analytes (Vibrio and Entamoeba histolytica) was very low, both prospective and retrospective specimens did not yield adequate specimen numbers to demonstrate sufficient performance with the EntericBio" Dx assay. To supplement the fresh and frozen specimen data, contrived samples (n=310) were prepared and tested with the EntericBio" Dx assay. Contrived samples were prepared for each target analyte using unique fecal matrix from residual fresh specimens which had previously tested negative for all target analytes. Positive specimens were spiked at various levels, using multiple strains for each organism. Contrived samples were randomized with negative specimens so that the users were blinded to the expected result.

Of the 310 contrived samples prepared for the contrived sample study, there were no samples that were excluded from the study, giving 310 evaluable samples.

The performance of the EntericBio Dx assay in contrived samples was evaluated by comparing the test result for each target analyte with the expected sample result, based on the organism/ strain used for spiking.

The results of the EntericBio® Dx assay testing are presented in Tables 12a-12g below. Clinical study results have been stratified by the specimen type.

Table 12a: Summary of the Clinical Performance for Salmonella
Specimen Type		n=	% Agreement (95% CI)
			Positive	Negative
Salmonella	Clinical Specimens	Fresh	All-Comers	1472	92.3%24/261(75.9-97.9)	100%1446/1446(99.7-100)
			Select	32	90.0%9/102(59.6-98.2)	100%22/22(85.1-100)
		Frozen	Archived	209	85.7%12/143(60.1-96.0)	100%195/195(98.1-100)
			Simulated	310	NA	99.7%309/310(98.2-99.9)

12/2 Salmonella FN observed were negative for Salmonella when tested with an alternative FDA cleared PCR assay

21/1 Salmonella FN observed was positive for Salmonella when tested with an alternative FDA cleared PCR assay

31/2 Salmonella FN observed was negative for Salmonella when tested with an alternative FDA cleared PCR assay. 1/2 Salmonella FN observed was positive for Salmonella when tested with an alternative FDA cleared PCR assay.

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Specimen Type		n=	% Agreement (95% CI)
Campylobacter	Clinical Specimens		Positive	Negative
		Fresh	All-Comers	1472
			Select	32
Frozen	Archived		209	93.8%15/164(71.7-98.9)
	Simulated	310	NA	100%310/310(98.8-100)

41/1 Campylobacter FN observed were negative for Campylobacter when tested with an alternative FDA cleared PCR assay

Table 12c: Summary of the Clinical Performance for Shigella/EIEC
Shigella/EIEC	Clinical Specimens	Specimen Type	n=	% Agreement (95% CI)
				Positive	Negative
	Fresh	All-Comers	1472	100%14/14(78.5-100)	100%1458/1458(99.7-100)
		Select	32	100%3/3(43.9-100)	100%29/29(88.3-100)
		Archived	209	90.9%10/115(62.3-98.4)	100%198/198(98.1-100)
	Frozen

³1/1 Shigella/EIEC FN observed was negative for Shigella/EIEC when tested with an alternative FDA cleared PCR assay

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Table 12d: Summary of the Clinical Performance for STEC
	Specimen Type	n=	% Agreement (95% CI)
			Positive	Negative
STEC	Clinical Specimens Fresh	All-Comers	1472	100%8/8(67.6-100)	99.9%1462/1464(99.5-99.9)
		Select	32	100%3/3(43.9-100)	100%29/29(88.3-100)
	Frozen	Archived	209	94.6%70-746(86.9-97.9)	100%135/135(92.7-100)
		Simulated	310	NA	100%310/310(98.8-100)

َ4/4 STEC FN observed were negative for STEC when tested with an alternative FDA cleared PCR assay

Table 12e: Summary of the Clinical Performance for Vibrio
Vibrio	Clinical Specimens	Specimen Type		n=	% Agreement (95% CI)
					Positive	Negative
	Fresh	All-Comers		1472	0.0%0/37(0.0-56.1)	100%1469/1469(99.7-100)
		Select		32	NA	100%32/32(89.3-100)
	Frozen	Archived		209	NA	100%209/209(98.2-100)
			Simulated	310	100%100/100(96.3-100)	100%210/210(98.2-100)

'3/3 Vibrio FN observed were negative for Vibrio when tested with an alternative FDA cleared PCR assay

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Table 12f: Summary of the Clinical Performance for Giardia lamblia
Giardia lamblia	Clinical Specimens	Specimen Type	n=	% Agreement (95% CI)
				Positive	Negative
	Fresh	All-Comers	1472	85.7%12/148(60.1-96.0)	99.9%1457/1458(99.6-99.9)
		Select	32	100%3/3(43.9-100)	100%29/29(88.3-100)
	Frozen	Archived	209	100%29/29(88.3-100)	100%180/180(97.9-100)
		Simulated	310	NA	100%310/310(98.8-100)

82/2 Giardia FN observed were negative for Giardia when tested with an alternative FDA cleared PCR assay

Table 12g: Summary of the Clinical Performance for Entamoeba histolytica
Entamoeba histolytica	Clinical Specimens	Specimen Type	n=	% Agreement (95% CI)
				Positive	Negative
Fresh	All-Comers	1472	NA	100%1472/1472(99.7-100)
	Select	32	NA	100%32/32(89.3-100)
Frozen	Archived	209	0.0%0/29(0.0-65.8)	100%207/207(98.2-100)
	Simulated	310	98.6%74/75(92.3-99.8)	100%235/235(98.4-100)

92/2 Entamoeba FN observed were positive for Entamoeba when tested with an alternative FDA cleared PCR

The EntericBio" Dx assay reported multiple organism detections (co-infections) for a total of 3 specimens. This represents 0.20% of all fresh specimens tested (3/1504). All multiple detections contained two target analytes and all were concordant with the comparator method(s) used for the respective target analytes. The summary of the multi-detections reported by the EntericBio Dx assay is presented in Table 13.

The comparator assay(s) reported multiple organism detections (co-infections) for a total of 5 specimens. This represents 0.33% of all fresh specimens tested (5/1504). All multiple detections contained two target analytes. From the 5 samples with reported co-detections, the EntericBio® Dx assay did not detect a second target analyte in 2 specimens and none of the discordant specimens reported as co-infections were confirmed with an alternative FDA-

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cleared assay. The summary of the multi-detections reported by the comparator assay is presented in Table 14.

Analyte_1	Analyte_2	Prevalence		Number of Discrepantspecimens (FP byEntericBio)	DiscrepantAnalyte(s)
Campylobacter	Giardia	1	0.07	0	N/A
Shigella	Giardia	1	0.07	0	N/A
Campylobacter	STEC	1	0.07	0	N/A
	Total	3	0.20

Table 13: Distinct Multi-detections detected in fresh clinical specimens (n=1,504) by the EntericBio" Dx assay

Table 14: Distinct Multi-detections detected in fresh clinical specimens (n=1,504) by the
comparator methods

Analyte_1	Analyte_2	Prevalence		Number ofDiscrepantspecimens (FN byEntericBio)	DiscrepantAnalyte(s)
Campylobacter	Giardia	2	0.13	1a	Giardiaa
Shigella	Giardia	2	0.13	1b	Giardiab
Campylobacter	STEC	1	0.07	0	N/A
Total		5	0.33

a Sample was negative with EntericBio® Dx and an alternative FDA-cleared assay

b Sample was negative with EntericBio® Dx and an alternative FDA-cleared assay

Of the 1482 prospective (fresh) specimens initially evaluated with EntericBio" Dx assay, 28 specimens (1.9%) were initially reported as Invalid. Following a repeat test, 19 out of 28 invalid specimens generated valid results. Repeat testing was not performed for 3 of the 28 invalid specimens and these specimens remained as Invalid. None of the 32 select (fresh) specimens were initially reported as invalid. Of the 212 retrospective specimens initially evaluated with EntericBio" Dx assay, 2 specimens (0.9%) were initially reported as Invalid. Following a repeat test, 1 out of 2 invalid specimens was resolved. Repeat testing was not performed for the other specimen which remained invalid. The total numbers provided in Table 15 are based on compliant specimens and EntericBio® Dx assay results.

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Table 15: Summary of Invalid results observed during clinical trial with the EntericBio Dx assay

	Initial Invalid			Final Invalid
	Count	Percent	95% CI	Count	Percent	95% CI
Prospective (Fresh)	28/1482	1.9%	1.3-2.7%	9*/1482	0.6%	0.3-1.2%
Select (Fresh)	0/32	0.0%	0.0-10.7%	0/32	0.0%	0.0-10.7%
Total (Fresh)	28/1514	1.9%	1.3-2.7%	9*/1514	0.6%	0.3-1.1%
Retrospective (Frozen)	2/212	0.9%	0.3-3.4%	1**/212	0.5%	0.1-2.6%
Total (All)	30/1726	1.7%	1.2-2.5%	10/1726	0.6%	0.3-1.2%

*19/28 initial invalids for fresh specimens were resolved upon repeat; 6/28 were invalid upon repeat and 3/28 were not repeated **1/2 initial Invalid results was resolved upon repeat and 1/2 was not repeated

Of the 1482 prospective (fresh) specimens initially evaluated with EntericBio® Dx assay, 1 specimen (0.1%) was initially reported as Indeterminate. Repeat testing was not performed and the specimen remained as Indeterminate.

None of the 32 select (fresh) specimens initially reported as Indeterminate. Of the 212 retrospective specimens initially evaluated with EntericBio® Dx assay, 2 specimens (0.9%) were initially reported as Indeterminate. Repeat testing for these 2 specimens was not performed and the specimens remained as Indeterminate. The total numbers provided in Table 16 are based on compliant specimens and EntericBio® Dx assay results.

Table 16: Summary of Indeterminate results observed during clinical trial with the EntericBio® Dx assay

	Initial Indeterminate			Final Indeterminate
	Count	Percent	95% CI	Count	Percent	95% CI
Prospective (Fresh)	1/1482	0.1%	0.0-0.4%	1*/1482	0.1%	0.0-0.4%
Select (Fresh)	0/32	0.0%	0.0-10.7%	0/32	0.0%	0.0-10.7%
Total (Fresh)	1/1514	0.1%	0.0-0.4%	1*/1514	0.1%	0.0-0.4%
Retrospective (Frozen)	2/212	0.9%	0.3-3.4%	2**/212	0.9%	0.3-3.4%
Total (All)	3/1726	0.2%	0.1-0.5%	3/1726	0.2%	0.1-0.5%

*1/1 initial indeterminate result was not repeated

**2/2 initial indeterminate results were not repeated

Of the 1482 prospective (fresh) specimens initially evaluated with EntericBio® Dx assay, 29 specimens (2.0%) were initially Non-reportable (Invalid and Indeterminate combined). Following a repeat test, 19 out of 29 Non-reportable results were resolved. Repeat testing was not performed for 4 of the 29 non-reportable results and the specimens remained as Non-reportable. None of the 32 select (fresh) specimens were Non-reportable.

Of the 212 retrospective specimens initially evaluated with EntericBio® Dx assay, 4 specimens (1.9%) were initially Non-reportable. Following a repeat test, 1 out of 4 Non-reportable results was resolved. Repeat testing was not performed for 3 of the 4 specimens with non-reportable

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results, and the specimens remained as Non-reportable.

The total numbers provided in Table 17 are based on compliant specimens and EntericBio® Dx assay results.

		Table 17: Summary of all Non-reportable results observed during clinical trial with the
EntericBio® Dx assay
	Initial All Non-reportable			Final All Non-reportable
	Count	Percent	95% CI	Count	Percent	95% CI
Prospective (Fresh)	29/1482	2.0%	1.4-2.8%	10/1482	0.7%	0.4-1.2%
Select (Fresh)	0/32	0.0%	0.0-10.7%	0/32	0.0%	0.0-10.7%
Total (Fresh)	29/1514	1.9%	1.3-2.7%	10*/1514	0.7%	0.4-1.2%
Retrospective (Frozen)	4/212	1.9%	0.7-4.8%	3**/212	1.4%	0.5-4.1%
Total (All)	33/1726	1.9%	1.4-2.7%	13/1726	0.8%	0.4-1.3%

*19/29 initial non-reportable results for fresh specimens were resolved upon reportable upon repeat and 4/29 were not repeated

**1/4 initial non-reportable results was resolved upon repeat and 3/4 were not repeated

Statement of Safety and Effectiveness

The data presented clearly demonstrates the safety and efficacy of the EntericBio" Dx assay as compared to the reference method when the product's Instructions for Use are followed.

Regulation Number and Section

§ 866.3990 Gastrointestinal microorganism multiplex nucleic acid-based assay.

(a)
Identification. A gastrointestinal microorganism multiplex nucleic acid-based assay is a qualitativein vitro diagnostic device intended to simultaneously detect and identify multiple gastrointestinal microbial nucleic acids extracted from human stool specimens. The device detects specific nucleic acid sequences for organism identification as well as for determining the presence of toxin genes. The detection and identification of a specific gastrointestinal microbial nucleic acid from individuals exhibiting signs and symptoms of gastrointestinal infection aids in the diagnosis of gastrointestinal infection when used in conjunction with clinical evaluation and other laboratory findings. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.(b)
Classification. Class II (special controls). The special controls are set forth in FDA's guideline document entitled: “Class II Special Controls Guideline: Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assays for Detection and Identification of Microorganisms and Toxin Genes from Human Stool Specimens.” For availability of the guideline document, see § 866.1(e).