K Number

Device Name

EntericBio Dx Assay

Manufacturer

Serosep, Ltd.

Date Cleared

2019-06-19

(265 days)

Product Code

PCH NSU OOI

Regulation Number

866.3990

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.

More Information

Contact

Type

Center

Panel

Date Received

Product Code

Subsequent Product Codes

Applicant Name (Manufacturer)

Device Name

Decision

Street 1

Street 2

City

State

Country Code

ZIP

Postal Code

Class Advise Committee

SSP Indicator

Third Party Reviewed

Expedited Review

Predicate Devices

K140407

Reference Devices

K140407

AI/ML (Artificial Intelligence / Machine Learning)

No
The document describes a standard real-time PCR assay for detecting specific nucleic acid sequences. The analysis and interpretation are based on standard PCR principles (amplification and detection of fluorescent signals) and a "user constructed template suitable for the EntericBio® Dx Assay," which suggests rule-based or threshold-based analysis, not AI/ML. There is no mention of AI, ML, or related concepts like neural networks, deep learning, or algorithms that learn from data to improve performance.

Therapeutic

No
This device is an in vitro diagnostic (IVD) test used to detect and identify multiple enteric pathogens, which aids in diagnosis. It does not provide therapy or treatment.

Diagnostic

Yes

Explanation: The "Intended Use / Indications for Use" section explicitly states that the device is intended "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." This clearly indicates its role in the diagnostic process.

SaMD (Software as a Medical Device)

The device description explicitly states it includes "PCR reagents," "Stool Preparation Solution (SPS) tubes," "PCR reagent strips containing lyophilized reagents," "Resuspension Buffer (Negative Kit Control)," "Positive Kit Control," and "associated accessories, instruments and software." This indicates it is a system that includes physical components (reagents, tubes, strips) in addition to software.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the EntericBio® Dx assay is an "in vitro multiplexed nucleic acid test" for the detection and identification of enteric pathogens in stool specimens. This directly aligns with the definition of an in vitro diagnostic device, which is used to examine specimens taken from the human body to provide information for diagnosis, treatment, or prevention of disease.
Specimen Type: The test is performed on "Cary-Blair preserved stool specimens," which are specimens taken from the human body.
Purpose: The test is intended "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." This clearly indicates a diagnostic purpose.
Device Description: The "Device Description" further reinforces this by describing the components used to perform the test on human specimens for the detection of bacterial and parasitic causes of gastroenteritis.

The entire description and intended use clearly fall under the scope of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

. 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.

Product codes

PCH, OOI, NSU

Device Description

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Stool specimens/feces

Indicated Patient Age Range

Not Found

Intended User / Care Setting

CLIA certified clinical laboratories

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

Not Found

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

The clinical performance was evaluated using:

Fresh, prospectively-collected, Cary-Blair preserved fecal specimens: 1491 samples were collected, with 1472 evaluable samples after exclusions.
Fresh, selected Cary-Blair preserved fecal specimens: 32 samples were collected.
Frozen, well characterized Cary-Blair preserved fecal specimens: 212 samples were collected, with 209 evaluable samples after exclusions. These were archived specimens based on previously testing positive for desired target analytes by routine diagnostic methods.
Spiked/Contrived Cary-Blair preserved fecal samples: 310 samples were prepared from residual fresh specimens that had 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.

Comparator Method:
For STEC and Campylobacter target analytes in fresh specimens, a composite comparator method of three FDA-cleared assays 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 the remaining target analytes and archived specimens, the comparator method consisted of one FDA-cleared assay.

Annotation Protocol:

Archived specimens: Distributed across three testing sites and randomized so users were blinded to expected results.
Contrived samples: Randomized with negative specimens so that users were blinded to the expected result.

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

Study Type: Analytical and Clinical Performance Studies.

Analytical Performance:

Reproducibility:
- Sample Size: Not explicitly stated as a single number, but involved contrived stool samples spiked with various concentrations of Vibrio parahaemolyticus, Shigella sonnei, and Giardia lamblia (3 concentrations each: True Negative, Low Positive, Moderate Positive). Tested using three instruments and two operators for five non-consecutive days.
- Key Results:
  - Moderate positive concentration (3x LoD) showed 100% agreement with expected results for all targets.
  - Low positive concentration (1.5x LoD) showed 100% agreement for Shigella sonnei and Vibrio parahaemolyticus, and 98% for Giardia lamblia (89/90 detections).
  - Cq reproducibility showed low %CV values (generally below 5%) across sites and concentrations.
Limit of Detection (LoD):
- Sample Size: 20 replicate EntericBio® Stool Preparation Solution (SPS) were tested from each sample using three independently manufactured lots.
- Key Results: LoD was determined for each target organism, ranging from 1x 10^4 CFU/mL to 1x 10^6 CFU/mL for bacteria, and 25 to 100 cells/mL for parasites.
Fresh vs. Frozen Specimen Stability:
- Sample Size: Sixty contrived samples prepared for each target analyte.
- Key Results: 100% agreement between fresh and frozen samples for Salmonella, Shigella, and STEC. 1% v/v) and Hemorrhoidal cream (>1% w/v). False negative for V. parahaemolyticus in 1/3 replicates with hemorrhoidal cream. False negatives for C. jejuni in 2/3 replicates with tetracycline (1.6% w/v).
Competitive Inhibition:
- Key Results: Competitive inhibition observed for Giardia lamblia in the presence of both low or high concentrations of Entamoeba histolytica.
Carry-Over and Cross Contamination:
- Sample Size: 30 samples per experiment (15 positive, 15 negative) for two target analytes (Shigella sonnei, Giardia lamblia), over three experiments.
- Key Results: No carryover or cross-contamination.
Specimen Stability:
- Key Results: Recommended storage for Cary-Blair preserved stool is 5 days at 2-8°C.
Real Time Stability:
- Key Results: Shelf life determined to be six (6) months at 2-8°C.

Clinical Performance:

Study Type: Multi-site clinical study (2 US, 1 non-US).
Sample Size: 1523 fresh samples (1472 evaluable); 212 frozen archived specimens (209 evaluable); 310 contrived samples (310 evaluable).
Key Results (Summary of % Agreement - Positive and Negative, using 95% CI):
- Salmonella: Fresh All-Comers (Positive: 92.3% (24/26); Negative: 100% (1446/1446)), Select (Positive: 90.0% (9/10); Negative: 100% (22/22)), Frozen Archived (Positive: 85.7% (12/14); Negative: 100% (195/195)), Simulated (Negative: 99.7% (309/310)).
- Campylobacter: Fresh All-Comers (Positive: 93.8% (15/16); Negative: 100% (1456/1456)), Select (NA), Frozen Archived (Positive: 93.8% (15/16); Negative: 100% (193/193)), Simulated (Negative: 100% (310/310)).
- Shigella/EIEC: Fresh All-Comers (Positive: 100% (14/14); Negative: 100% (1458/1458)), Select (Positive: 100% (3/3); Negative: 100% (29/29)), Frozen Archived (Positive: 90.9% (10/11); Negative: 100% (198/198)).
- STEC: Fresh All-Comers (Positive: 100% (8/8); Negative: 99.9% (1462/1464)), Select (Positive: 100% (3/3); Negative: 100% (29/29)), Frozen Archived (Positive: 94.6% (70/74); Negative: 100% (135/135)), Simulated (Negative: 100% (310/310)).
- Vibrio: Fresh All-Comers (Positive: 0.0% (0/3); Negative: 100% (1469/1469)), Simulated (Positive: 100% (100/100); Negative: 100% (210/210)). Low prevalence in fresh/frozen specimens, supplemented with simulated.
- Giardia lamblia: Fresh All-Comers (Positive: 85.7% (12/14); Negative: 99.9% (1457/1458)), Select (Positive: 100% (3/3); Negative: 100% (29/29)), Frozen Archived (Positive: 100% (29/29); Negative: 100% (180/180)).
- Entamoeba histolytica: Frozen Archived (Positive: 0.0% (0/2); Negative: 100% (207/207)), Simulated (Positive: 98.6% (74/75); Negative: 100% (235/235)). Low prevalence in fresh/frozen specimens, supplemented with simulated.
Multi-detections (Co-infections):
- EntericBio® Dx assay: 3 specimens (0.20% of fresh) reported multi-detections, all concordant with comparator method(s).
- Comparator method(s): 5 specimens (0.33% of fresh) reported multi-detections. EntericBio® Dx assay did not detect a second target in 2 discordant specimens.
Invalid/Indeterminate Results:
- Initial Invalid Rate: 1.9% for fresh, 0.9% for retrospective.
- Final Invalid Rate: 0.6% for fresh, 0.5% for retrospective.
- Initial Indeterminate Rate: 0.1% for fresh, 0.9% for retrospective.
- Final Indeterminate Rate: 0.1% for fresh, 0.9% for retrospective.
- Total Non-reportable (Invalid + Indeterminate): Initial 1.9% (fresh), 1.9% (retrospective). Final 0.7% (fresh), 1.4% (retrospective).

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

The document presents " % Agreement" which can be interpreted as Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) when comparing against a comparator method assumed to be the reference standard.

Selected Metrics from Clinical Performance Tables:

Salmonella:

Fresh All-Comers: PPA = 92.3% (24/26); NPA = 100% (1446/1446)
Select: PPA = 90.0% (9/10); NPA = 100% (22/22)
Frozen Archived: PPA = 85.7% (12/14); NPA = 100% (195/195)
Simulated: NPA = 99.7% (309/310)

Campylobacter:

Fresh All-Comers: PPA = 93.8% (15/16); NPA = 100% (1456/1456)
Frozen Archived: PPA = 93.8% (15/16); NPA = 100% (193/193)
Simulated: NPA = 100% (310/310)

Shigella/EIEC:

Fresh All-Comers: PPA = 100% (14/14); NPA = 100% (1458/1458)
Select: PPA = 100% (3/3); NPA = 100% (29/29)
Frozen Archived: PPA = 90.9% (10/11); NPA = 100% (198/198)

STEC:

Fresh All-Comers: PPA = 100% (8/8); NPA = 99.9% (1462/1464)
Select: PPA = 100% (3/3); NPA = 100% (29/29)
Frozen Archived: PPA = 94.6% (70/74); NPA = 100% (135/135)
Simulated: NPA = 100% (310/310)

Vibrio:

Fresh All-Comers: PPA = 0.0% (0/3); NPA = 100% (1469/1469)
Simulated: PPA = 100% (100/100); NPA = 100% (210/210)

Giardia lamblia:

Fresh All-Comers: PPA = 85.7% (12/14); NPA = 99.9% (1457/1458)
Select: PPA = 100% (3/3); NPA = 100% (29/29)
Frozen Archived: PPA = 100% (29/29); NPA = 100% (180/180)
Simulated: NPA = 100% (310/310)

Entamoeba histolytica:

Frozen Archived: PPA = 0.0% (0/2); NPA = 100% (207/207)
Simulated: PPA = 98.6% (74/75); NPA = 100% (235/235)

Predicate Device(s)

K140407

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

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).

Summary

Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

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

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 (DICE@fda.hhs.gov) or phone (1-800-638-2041 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

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)
-------------------------------------------------	--

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 : (978) 705 5011 Fax : (978) 927 1308 Email : fran@mdcassoc.com

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.

Element	Subject (New) Device	Proposed Predicate Device	Discussion
Device Name	EntericBio® Dx Assay	FilmArray Gastrointestinal (GI) Panel	New vs. Predicate Device
FDA Device
Premarket
Notification	New device	K140407	New vs Predicate Device
FDA Device
Classification	Class II, 21 CFR 866.3990 –
Gastrointestinal microorganism
multiplex nucleic acid-based assay,
Microbiology (83 Panel)	Class II, 21 CFR 866.3990 –
Gastrointestinal microorganism
multiplex 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 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 BioFire FilmArray Gastrointestinal
Panel is designed to be used with the
FilmArray® instrument. The FilmArray
Gl pouch contains freeze-dried
reagents to perform nucleic acid
purification and nested, multiplex PCR
with DNA melt analysis.	Different: New device differs from
the predicate device in that the
realtime PCR assay kits are
designed to be used with an
automated pipetting station to
accelerate sample preparation,
and a commercially available, FDA-
cleared PCR instrument to
measure the fluorescent probe
signal generated during
amplification that are analyzed by
the EntericBio automated analysis
and interpretation software.
Targets for
Detection	Salmonella enterica spp.
Shigella spp./ Enteroinvasive E. coli
(EIEC)
Campylobacter spp. (jejuni, coli and lari)	Clostridium difficile ( C. difficile ) toxin
A/B, Campylobacter spp. ( C. jejuni, C.
coli and C. upsaliensis ), Plesiomonas
shigelloides, Salmonella spp., Vibrio
spp., Yersinia enterocolitica.	Similar, except the predicate
device is additionally cleared for
detection of a range of other
nucleic acid targets from bacteria,
parasites and viruses.
Element	Subject (New) Device	Proposed Predicate Device	Discussion
	STEC (Shiga toxin-producing E. coli),
stx1/stx2 genes
Vibrio spp. (cholerae and
parahaemolyticus )
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 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 the following organisms: (see
organisms above).	The FilmArray Gastrointestinal Panel
(GI) is intendent for use with the
FilmArray® instrument for the
qualitative in vitro detection and
identification of multiple bacteria,
viruses and parasites. The FilmArray GI
Panel is performed directly from stool
specimens in Cary-Blair transport
media. The following pathogen types,
subtypes and toxin genes are
identified using the FilmArray GI Panel:
(see organisms above).	Similar except for the additional
organisms detected by the
predicate device.
Analyte	DNA/RNA from Cary-Blair preserved
fecal specimens	DNA/ RNA from Cary-Blair preserved
fecal specimens	Same
Technological
Principles	Multiplex nucleic acid PCR	Multiplex nucleic acid PCR	Same
Specimen
Types	Human stool (Cary-Blair preserved)	Human stool (Cary-Blair preserved)	Same
Element	Subject (New) Device	Proposed Predicate Device	Discussion
Controls	Internal Amplification Control for each
sample. Kit positive and negative
controls are processed with each batch
of samples.	Two controls are included in each
reagent pouch to control for sample
processing and both stages of PCR and
melt analysis.	Similar: The new device IAC is
lyophilized within the PCR mix
whereas the predicate device has
two controls included in each
reagent pouch.
Substantial equivalence will be
demonstrated in clinical testing
using human specimens and
compared to the predicate device.
PCR Sample
Preparation/
Extraction	Sample processed directly following
heat treatment of specimen in a SPS
tube.	Sample processing is automated in the
FilmArray® instrument. The sample is
lysed by a combination of mechanical
(bead beating) and chemical means
and the liberated nucleic acid is
captured, washed and eluted using
magnetic bead technology.	EntericBio® Dx kit provides
reagents and procedure for DNA
testing without extraction of stool
specimens.
Substantial equivalence will be
demonstrated in clinical testing
using human specimens and
compared to the predicate device.
Technological
Principles	Real-time multiplex RT-PCR based on
the hydrolysis probe reagent chemistry.	Nested multiplex RT-PCR followed by
high resolution melting analysis to
confirm identity of the PCR product.	Different: Both devices use
multiplex real-time PCR however
the new device uses hydrolysis
probe reagent chemistry
compared to melting analysis in
the predicate device.
Substantial equivalence will be
demonstrated in clinical testing
using human specimens and
compared to the predicate device.
Element	Subject (New) Device	Proposed Predicate Device	Discussion
Detection
Methodology/
Platform	The Applied Biosystems 7500 Fast Dx
Real-Time PCR instrument is a real-time
nucleic acid amplification and five color
fluorescence detection system for use
with the EntericBio® Dx Assay. Results
are analyzed and interpreted using the
EntericBio® FastFinder plugin.	Detection and interpretation is
automated on the FilmArray®
instrument by analysis of the specific
PCR product melts (melting
temperature).	Different: The detection system
and analysis differ between the
two devices.
Substantial equivalence will be
demonstrated in clinical testing
using human specimens and
compared to the predicate device.
Device Format	The EntericBio® Dx Assay kits provide a
PCR master mix with all the reagents
required to perform each test which are
lyophilized into individual reaction
wells. Each reaction well contains an
Internal Amplification Control (IAC) to
monitor for PCR inhibition. A synthetic
Positive Control (containing target
sequences) is provided with each kit to
monitor the thermal cycling steps and
reagent integrity during amplification
and detection process.	The FilmArray Gl panel provides all the
reagents lyophilized into a disposable
pouch. Each pouch contains two
controls which monitor sample
processing and both stages of PCR and
melt analysis.	Assay specific requirements

Intended Use

. 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

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), ●

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.

| Organism Tested | Concentration
Tested | FastFinder
Expected
Result | % Agreement with Expected Result | | | All |
|------------------------------------------|--------------------------------|----------------------------------|----------------------------------|---------------|----------------|-----------------|
| | | | Serosep
1 | Serosep
2 | Serosep
3 | |
| Shigella sonnei
DSM 5570 | Moderate
Positive
3x LoD | Positive | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100% |
| | Low Positive
1.5x LoD | Positive | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100% |
| | True Negative | Negative | 60/60
100% | 60/60
100% | 59/59*
100% | 179/179
100% |
| Vibrio
parahaemolyticus
CCUG 14474 | Moderate
Positive
3x LoD | Positive | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100% |
| | Low Positive
1.5x LoD | Positive | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100% |
| | True Negative | Negative | 60/60
100% | 60/60
100% | 59/59*
100% | 179/179
100% |
| Giardia lamblia
P101 | Moderate
Positive
3x LoD | Positive | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100% |
| | Low Positive
1.5x LoD | Positive | 30/30
100% | 30/30
100% | 29/30
95% | 89/90
98% |
| | True Negative | Negative | 60/60
100% | 60/60
100% | 59/59*
100% | 179/179
100% |

Table 1: Reproducibility study results

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

| Organism Tested Tested | Test Site | Cq Reproducibility | |------------------------------------------| | (Cq) | STDEV | %CV |
| Shigella sonnei
DSM 5570 | Well A | Moderate
parahaemolyticus
CCUG 14474 | Well B | Moderate
Positive | Serosep 1 | 31.73 | | | | | | | | Giardia lamblia
P101 | Well C | Moderate
| |
--------|-------------------------|-----------|--------------------|------------|------|
| | | | Mean
/> | $\pm$ 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 |
| $\pm$ 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 |
/> | $\pm$ 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.

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
Shigella
spp./EIEC	Shigella sonnei DSM 5570	1.25 x $10^4$ CFU/mL
	E. coli (ipaH) DSM 9029	1 x $10^4$ CFU/mL
Campylobacter
spp.	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
Entamoeba
histolytica	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 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

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).

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;
1. 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;
1. Frozen, well characterized Cary-Blair preserved fecal specimens from patients presenting with symptoms of gastrointestinal infection, known to be positive for selected target analytes;
1. 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.

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 (%)
Campylobacter	Clinical Specimens


Frozen	Archived
15/164
(71.7-98.9)
	Simulated
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

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

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-

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 Discrepant
specimens (FP by
EntericBio) | Discrepant
Analyte(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 of
Discrepant
specimens (FN by
EntericBio) | Discrepant
Analyte(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.

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

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.