Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on standard molecular diagnostic techniques (real-time PCR) and do not mention any AI or ML components in the analysis or interpretation of results.

Therapeutic

No.
This device is an in vitro diagnostic (IVD) test, designed to detect and identify specific Shiga toxin genes and the E.coli O157 gene cluster from stool specimens. Its intended use is as an aid in detection, not for treatment or other patient management decisions, which is characteristic of a therapeutic device.

Diagnostic

Yes
The device is described as "an aid in detection of specific Shiga-toxin expressing strains of E. coli ('STEC') from patients with diarrhea," and its results are to be used "in conjunction with clinical presentation, laboratory findings and epidemiological risk factors." This indicates its role in the diagnostic process.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the PanNAT System consists of an instrument with onboard software and a touchscreen, and the instrument includes hardware components such as a pneumatic subsystem, thermal control unit, and fluorescent optical detectors. The software is part of a larger hardware system.

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 test is "qualitative, in vitro nucleic acid amplification-based" and is used for the "simultaneous detection and identification of the stx1 and stx2 Shiga toxin genes and the O-antigen gene cluster of E.coli O157 (fc/)". It also specifies that testing is performed on "stool specimens from individuals with signs and symptoms of gastrointestinal infection" and is intended "as an aid in detection of specific Shiga-toxin expressing strains of E. coli ("STEC") from patients with diarrhea." This clearly describes a test performed outside the body on a biological sample to provide information for diagnosis.
Device Description: The description details a system that processes a biological sample (stool) in a cartridge to perform DNA purification, amplification, and detection. This is characteristic of an in vitro diagnostic device.
Performance Studies: The document includes detailed performance studies (Analytical Sensitivity, Specificity, Precision, etc.) which are standard requirements for demonstrating the performance of an IVD.
Predicate Device: The mention of a "Predicate Device(s)" with a K number (K140111; BD Max Enteric Bacterial Panel) is a strong indicator that this device is being submitted for regulatory review as an IVD, as predicate devices are used for comparison in the regulatory process for new IVDs.

All these elements align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Micronics PanNAT STEC Test is a qualitative, in vitro nucleic acid amplification-based test intended for the simultaneous detection and identification of the stx1 and stx2 Shiga toxin genes and the O-antigen gene cluster of E.coli O157 (fcl). Testing is performed in a unitized cartridge on the PanNAT System on soft to diarrheal unpreserved or Cary-Blair preserved stool specimens from individuals with signs and symptoms of gastrointestinal infection. The PanNAT STEC Test is intended for use, in conjunction with clinical presentation, laboratory findings and epidemiological risk factors, as an aid in detection of specific Shiga-toxin expressing strains of E. coli ("STEC") from patients with diarrhea.

The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Positive PanNAT STEC Test results do not rule out the potential for coinfection with other pathogens that are not detected by this device and may not be indicative of the sole or definitive cause of patient illness. Negative PanNAT STEC Test results in the context 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 (comma separated list FDA assigned to the subject device)

PCI, PCH, OOI

Device Description

The PanNAT System consists of the instrument with onboard software and 9-inch touchscreen used to process PanNAT STEC Test cartridges. The instrument automates all steps of the assay after sample addition to the test cartridge and insertion into the instrument; including DNA purification, nucleic acid amplification and detection of the target nucleic acid sequences using qualitative real-time PCR.

The system is a portable device that is powered by an external mains supply with a voltage range of 100-240VAC and a frequency range of 50-60 Hz. An onboard battery is included to provide power to the instrument for up to one hour in the event that mains power supply is interrupted. The instrument includes a pneumatic subsystem, a thermal control unit, fluorescent optical detectors, and software needed to process a test cartridge,

The PanNAT STEC Test cartridge is a unique, single use, disposable device in which all test reagents and controls are incorporated and all steps of the assay are performed. The PanNAT STEC Test cartridge uses a PanNAT Sample Transfer Pack accessory that contains a flocked swab, a prefilled Sample Buffer Tube and an Adaptor Cap.

There are three integrated controls for the PanNAT STEC Test: an endogenous human DNA internal process control that is coextracted and coamplified with the target nucleic acids, a negative control, and a positive control. These controls are performed automatically and do not require any action from the operator. External quality controls for the PanNAT STEC Test are also commercially available. Alternatively, laboratories may prepare their own controls.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

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

Not Found

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

A total of 1331 prospective specimens (1231 Cary-Blair preserved and 100 unpreserved) from a range of age groups were enrolled in the clinical evaluation. Of these, 30 were removed from the analysis due to pre- or post-analytical errors. Of the remaining 1301 specimens, 7.6% (99/1301) were initially invalid. Specimens with initial invalid results were retested. On retest, 78 were valid resulting in a repeat invalidity rate of 1.6% (21/1301). The number of prospective specimens that qualified for the agreement analysis was 1280 (1184 Cary-Blair preserved and 96 unpreserved).

Additional testing was also performed using unpreserved, retrospective frozen specimens. The comparator method for the retrospective study was alternative PCR (nested)/bidirectional sequencing. Of the 114 specimens initially enrolled in the study, eight (8) were excluded from the analysis of performance because they did not yield sufficient DNA for comparator testing (7) or previous enrollment (1). On initial testing 8/106 (7.5%) retrospective specimens produced invalid results, all of which were retested and three (3) of which remained invalid for a final invalid rate of 2.8% (3/106). The retrospective study data set therefore contained 103 evaluable results.
Additionally, 16 contrived unpreserved specimens, each spiked with an enumerated stock of a different strain of Shiga-toxin producing E. coli or E. coli 0157:H7 to a final concentration of 5 x 10^6 CFU/mL, were used. The contrived specimens were frozen and tested at the clinical sites interspersed with the retrospective specimens.

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

Reproducibility/Precision Study:

Study Type: Reproducibility/Precision
Sample Size: 90 samples (10 for each of 9 panel members), tested in triplicate over a minimum of 6 days at each site. Total (n) = 773.
Key Results:
- Overall %Agreement: 97.8% (Total 756/773 agreed).
- Site A %Agreement: 98.8%
- Site B %Agreement: 99.2%
- Site C %Agreement: 95.3%
- For Panel Member #8 (All Low concentration for stx1, stx2, O157), overall agreement was 88.6%. At Site C, this panel member showed 76.7% agreement, with individual target detection ranging from 86.6%-96.7%.
- Mean cycle numbers (and CV) were reported for different analytes across sites and concentrations, showing consistent performance.

Analytical Sensitivity/Limit of Detection (LoD):

Study Type: Analytical Sensitivity
Sample Size: Not explicitly stated as a single number, refers to dilutions of E. coli strains and replicates (28 replicates for confirmation).
Key Results:
- In unpreserved stool specimens, the LoD for EDL933 and 3007-85 (stx1, stx2, and 0157) is 1.00E+06 CFU/mL.
- In stool specimens preserved in Cary-Blair media, the LoD for EDL933 and 3007-85 (stx1, stx2 and O157) is 1.75E+06 CFU/mL.

Analytical Reactivity:

Study Type: Analytical Reactivity
Sample Size: 26 strains (7 O157, 12 non-O157, 6 un-typeable (O), 1 control).
Key Results:
- Detected stx1 and stx2 in O157, non-O157, and un-typeable O strains.
- Detected at least 2 subtypes of each toxin gene.
- For stx1: 92% agreement (24/26 strains). False negatives were stx1d variants due to probe-binding region mismatches.
- For stx2: 96% agreement (25/26 strains). False negative was an stx2 variant with extensive mismatches.
- For O157 O-antigen (fcl): 100% agreement (26/26 strains).

Analytical Specificity - Cross Reactivity:

Study Type: Analytical Specificity (Cross Reactivity)
Sample Size: 37 bacterial strains, 8 viruses, 1 yeast, 3 parasites (tested in triplicate).
Key Results: No cross-reactivity was observed for any tested analytes, except for Shigella dysenteriae, which is expected due to sequence homology with the stx1 target.

Analytical Specificity - Microbial Interference:

Study Type: Analytical Specificity (Microbial Interference)
Sample Size: 49 non-target organisms (same as cross-reactivity study).
Key Results: No competitive inhibition of PanNAT STEC Test analytes was demonstrated.

Analytical Specificity - Interfering Substances:

Study Type: Analytical Specificity (Interfering Substances)
Sample Size: 25 endogenous and exogenous substances (tested with 3 replicates per condition).
Key Results:
- Gaviscon® and Imodium AD® each caused one false positive for stx1+/stx2+/0157+ in the presence of an stx1+/stx2+/0157+ strain.
- Stearic acid caused one false negative for stx1-/stx2-/0157-.
- No interference for other substances.

Carry Over / Cross Contamination:

Study Type: Carry Over / Cross Contamination
Sample Size: Alternating series of contrived high positive and negative stool samples, tested five times on three instruments.
Key Results: No carryover or cross-contamination observed. All positive samples reported as stx1+/stx2+/0157+. 14 of 15 negative samples reported as stx1-/stx2-/0157-, one resulted in an invalid.

Clinical Performance Studies:

Study Type: Prospective and Retrospective Specimen Testing, supplemented with Contrived Specimens.
Sample Size:
- Prospective: 1280 evaluable specimens (1184 Cary-Blair preserved, 96 unpreserved).
- Retrospective: 103 evaluable specimens.
- Contrived: 16 specimens.
Key Results (PPA and NPA): See "Key Metrics" section for detailed PPA and NPA.

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

Prospective Study (Preserved Specimens):

stx1: PPA 100.0% (4/4), NPA 99.3% (1172/1180)
stx2: PPA 100.0% (5/5), NPA 99.4% (1172/1179)
O157: PPA 100.0% (3/3), NPA 99.8% (1179/1181)

Prospective Study (Unpreserved Specimens):

stx1: NPA 100.0% (96/96) (no positive specimens obtained)
stx2: PPA 100.0% (2/2), NPA 98.9% (93/94)
O157: PPA 100.0% (2/2), NPA 100.0% (96/94) [Typo in source document 96/94, assuming 94/94]

Retrospective Study (Unpreserved Specimens):

stx1: PPA 88.5% (23/26), NPA 97.4% (75/77)
stx2: PPA 90.0% (27/30), NPA 98.6% (72/73)
O157: PPA 89.7% (26/29), NPA 98.6% (72/73)

Contrived Study (Unpreserved Specimens):

stx1: PPA 100.0% (12/12), NPA 100.0% (4/4)
stx2: PPA 100.0% (8/8), NPA 100.0% (8/8)
O157: PPA 100.0% (2/2), NPA 100.0% (14/14)

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

BD Max Enteric Bacterial Panel (K140111)

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

Not Found

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

Not Found

Regulation Number and Section

§ 866.3990 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

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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 1, 2018

Micronics, Inc Karen Hedine President 8464 154th Ave NE Redmond, Washington 98052

Re: K173330

Trade/Device Name: PanNAT STEC Test Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal microorganism multiplex nucleic acid-based assay Regulatory Class: Class II Product Code: PCI, PCH, OOI Dated: April 27, 2018 Received: May 1, 2018

Dear Karen Hedine:

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

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

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

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Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

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/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). 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 (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Ribhi Shawar -S

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

Enclosure

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Indications for Use

510(k) Number (if known) K173330

Device Name PanNAT STEC Test

Indications for Use (Describe)

The Micronics PanNAT STEC Test is a qualitative, in vitro nucleic acid amplification-based tor the simultaneous detection and identification of the stx1 and stx2 Shiga toxin genes and the O-antigen gene cluster of E.coli O157 (fc/). Testing is performed in a unitized cartridge on the PanNAT System on soft to diarrheal unpreserved or Cary-Blair preserved stool specimens from individuals with signs and symptoms of gastrointestinal infection. The PanNAT STEC Test is intended for use, in conjunction with clinical presentation, laboratory findings and epidemiological risk factors, as an aid in detection of specific Shiga-toxin expressing strains of E. coli ("STEC") from patients with diarrhea.

The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Positive PanNAT STEC Test results do not rule out the potential for coinfection with other pathogens that are not detected by this device and may not be indicative of the sole or definitive cause of patient illness. Negative PanNAT STEC Test results in the context 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.

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

	☑ Prescription Use (Part 21 CFR 801 Subpart D)
	☐ Over-The-Counter Use (21 CFR 801 Subpart C)

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5. 510(k) Summary

5.1 Applicant

Micronics, Inc. 8463 154th Avenue NE Redmond. WA 98052 Phone: (425) 895-9197 Fax: (425) 895-1183

Contact Person:

Karen Hedine President Phone: (425) 896-3026 Fax: (425) 895-1183 Email: khedine@micronics.net

Consultant:

Gail E. Radcliffe, PhD 2311 Fairbanks St. West Boylston, MA 01583 Phone: (508) 835-1688 Email: gradcliffe@RMDCI.com

Date of Preparation: May 22, 2018

5.2 Device

Trade Name: PanNAT® STEC Test Common name: Gastrointestinal Bacterial Panel Multiplex Nucleic Acid-Based Assay System Regulation number: 866.3990, Gastrointestinal microorganism multiplex nucleic acidbased assay Product Code: PCI, PCH, OOI Class: 2

5.3 Predicate Device

BD Max Enteric Bacterial Panel (K140111)

5.4 Intended Use

The Micronics PanNAT STEC Test is a qualitative, in vitro nucleic acid amplificationbased test intended for the simultaneous detection and identification of the stx2 Shiga toxin genes and the O-antigen gene cluster of E.coli O157 (fcl). Testing is performed in a unitized cartridge on the PanNAT System on soft to diarrheal unpreserved or Cary-Blair preserved stool specimens from individuals with signs and symptoms of

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gastrointestinal infection. The PanNAT STEC Test is intended for use, in conjunction with clinical presentation, laboratory findings and epidemiological risk factors, as an aid in detection of specific Shiga-toxin expressing strains of E. coli ("STEC") from patients with diarrhea.

The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Positive PanNAT STEC Test results do not rule out the potential for coinfection with other pathogens that are not detected by this device and may not be indicative of the sole or definitive cause of patient illness. Negative PanNAT STEC Test results in the context 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.

5.5 Device Description

The PanNAT System consists of the instrument with onboard software and 9-inch touchscreen used to process PanNAT STEC Test cartridges. The instrument automates all steps of the assay after sample addition to the test cartridge and insertion into the instrument; including DNA purification, nucleic acid amplification and detection of the target nucleic acid sequences using qualitative real-time PCR.

The system is a portable device that is powered by an external mains supply with a voltage range of 100-240VAC and a frequency range of 50-60 Hz. An onboard battery is included to provide power to the instrument for up to one hour in the event that mains power supply is interrupted. The instrument includes a pneumatic subsystem, a thermal control unit, fluorescent optical detectors, and software needed to process a test cartridge,

The PanNAT STEC Test cartridge is a unique, single use, disposable device in which all test reagents and controls are incorporated and all steps of the assay are performed. The PanNAT STEC Test cartridge uses a PanNAT Sample Transfer Pack accessory that contains a flocked swab, a prefilled Sample Buffer Tube and an Adaptor Cap.

There are three integrated controls for the PanNAT STEC Test: an endogenous human DNA internal process control that is coextracted and coamplified with the target nucleic acids, a negative control, and a positive control. These controls are performed automatically and do not require any action from the operator. External quality controls for the PanNAT STEC Test are also commercially available. Alternatively, laboratories may prepare their own controls.

5.6 Principle of Operation

Using the swab from the Sample Transfer Pack, the operator transfers a specimen (either soft to diarrheal unpreserved stool specimen or Cary-Blair preserved stool specimen) to the Sample Buffer Tube, which is then sealed using the Adaptor Cap. The Sample Buffer Tube is then attached to the sample inlet port of the cartridge by means of the attached Adaptor Cap; fully containing all fluids within the cartridge-tube assembly. Once the test cartridge with attached Sample Buffer Tube is inserted into the instrument, the cartridge barcode is scanned and the test starts automatically.

Utilizing proprietary microfluidic design, and under control of the instrument, sample and reagents are moved through the cartridge to complete all of the steps of the procedure.

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DNA is extracted from lysed cells, captured on silica membranes, and eluted to accomplish purification. DNA sequences specific to stx1, stx2 and O157, together with the internal process control, are amplified and then detected by fluorescent probe hybridization for qualitative real-time PCR detection.

When the test is completed, sample results are displayed on the screen and stored in the instrument database. Test results can be exported to a USB flash drive or printer.

5.7 Substantial Equivalence

A comparison of similarities and differences between the PanNAT STEC Test and the predicate device is provided in Table 5-1.

Features	PanNAT STEC Test	Predicate: BD Max™ Enteric Bacterial Panel (K140111)
Similarities
Intended Use	The Micronics PanNAT STEC Test is a qualitative, in vitro nucleic acid amplification-based test intended for the simultaneous detection and identification of the stx1 and stx2 Shiga toxin genes and the O-antigen gene cluster of E.coli O157 ( fcl ). Testing is performed in a unitized cartridge on the PanNAT System on soft to diarrheal unpreserved or Cary-Blair preserved stool specimens from individuals with signs and symptoms of gastrointestinal infection. The PanNAT STEC Test is intended for use in conjunction with clinical presentation, laboratory findings and epidemiological risk factors, as an aid in detection of specific Shiga-toxin expressing strains of E. coli (“STEC”) from patients with diarrhea.

The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Positive PanNAT STEC Test results do not rule out the potential for coinfection with other pathogens that are not detected by this device and may not be indicative of the sole or definitive cause of patient illness Negative PanNAT STEC Test results in the context 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 | The BD MAX™ Enteric Bacterial Panel performed on the BD MAX™ System is an automated in vitro diagnostic test for the direct qualitative detection and differentiation of enteric bacterial pathogens. The BD MAX™ Enteric Bacterial Panel detects nucleic acids from:
Salmonella spp.Campylobacter ssp. (jejuni and coli)Shigella spp./Enteroinvasive E. coli (EIEC)Shiga Toxin 1 ( stx1 )/Shiga toxin 2 ( stx2 ) genes (found in Siga toxin-producing E. coli as well as Shigella dysenteriae , which can possess a Shiga toxin gene ( stx ) that is identical to the stx1 gene of STEC.Testing is performed on unpreserved soft to diarrheal unpreserved stool specimens or Cary Blair preserved stool symptomatic patients with suspected acute gastroenteritis, enteritis or colitis. The test is performed directly on the specimen, utilizing real-time polymerase chain reaction (PCR) for the amplification of SpaO , a Campylobacter specific tuf gene sequence, ipaH and stx1/stx2 . The test utilizes fluorogenic sequence-specific hybridization probes for detection of the amplified DNA.

This test is intended for use, in conjunction with clinical presentation, laboratory findings and epidemiological |

Table 5-1. Substantial Equivalence Table Comparing PanNAT STEC Test to Predicate

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| | colitis, irritable bowel syndrome, or
Crohn's disease. | information, as an aid in the differential diagnosis of Salmonella, Shigella/EIEC, Campylobacter and Shiga toxin-producing E. coli (STEC) infections.

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 definitive cause of patient illness. Negative 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. |
|------------------------|-----------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Specimen Type | Unpreserved and Cary-Blair preserved stool | Same |
| Assay Format | Real-time PCR amplification:
Fluorogenic target-specific hybridization | Same |
| Detection of stx1/stx2 | Presence of stx1 and stx2 genes specific to Shiga toxin-producing organisms | Same |
| Sample Preparation | Automated by the PanNAT System | Automated by the BD MAXTM System |
| Detection probes | Fluorescent Probes
MGB PleiadesTM Probe | Fluorescent Probes
TaqMan® Probe |
| Assay controls | Internal Process Control and Integrated positive and negative controls | Sample Processing Control (SPC) |
| | Differences | |
| Detection of E. coli | fcl gene for E. coli O157 | ipaH gene for E coli (EIEC) |
| Analysis platform | PanNAT® System | BD MAXTM System |

5.8 Analytical Performance Studies

Precision/ Reproducibility

Reproducibility of the PanNAT STEC Test was evaluated at three sites (2 external and 1 internal) utilizing 3 test cartridge lots, 6 operators (2 per site), and 13 PanNAT instruments. A total of 90 samples, 10 for each of 9 panel members were evaluated in triplicate over a minimum of 6 days at each site.

The coded 9-member panel consisted of E. coli strains containing a single analyte at low or moderate concentrations of stxl, stx2 or 0157, a strain positive for all analytes at low or moderate concentrations and a strain negative for all 3 analytes.

Agreement results by panel member by site are shown in Table 5-2. Each site used a different lot of cartridges; therefore lot-to-lot variability is demonstrated by site. In Table 5-3, agreement for each analyte is shown by concentration and by site/lot.

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| Analyte
Panel | Panel 1
All Neg | Panel 2
Low stx1 | Panel 3
Med stx1 | Panel 4
Low stx2 | Panel 5
Med stx2 | Panel 6
Low O157 | Panel 7
Med O157 | Panel 8
All Low | Panel 9
All Med | Total |
|------------------|--- | Overall | | %Agmt | 100.00% | (95% CI) | Total (n) | 83 | Agmt (n) | 83 | Site A | | % Agmt | 100.0% | (95% CI) | Total (n) | 28 | Agmt (n) | 28 | Site B | | % Agmt | 100.0% | (95% CI) | Total (n) | 29 | Agmt (n) | 29 | Site C | | % Agmt | 100.0% | (95% CI) | Total (n) | 26 | Agmt (n) | 26 -----------------|---------------------|---------------------|---------------------|---------------------|---------------------|---------------------|--------------------|--------------------|-------|
| | | | | | | | | |
| 97.60% | 98.8% | 97.70% | 100.00% | 98.90% | 100.00% | 88.60% | 98.90% | 97.8% |
| (95.6,100) | (91.8,99.4) | (93.4,99.8) | (91.9, 99.4) | (95.8, 100) | (93.8, 99.8) | (95.8, 100) | (80.3, 93.7) | (93.8, 99.8) | |
| 85 | 82 | 86 | 87 | 88 | 87 | 88 | 87 | 773 |
| 83 | 81 | 84 | 87 | 87 | 87 | 78 | 86 | 756 |
| | | | | | | | | |
| 100.0% | 100.0% | 100.0% | 100.0% | 100.0% | 100.0% | 93.3% | 96.3% | 98.8% |
| (87.9,100) | (87.9,100) | (87.5,100) | (87.5,100) | (87.9,100) | (88.6,100) | (88.6,100) | (78.7,98.2) | (81.7,99.3) | |
| 28 | 27 | 27 | 28 | 30 | 30 | 30 | 27 | 255 |
| 28 | 27 | 27 | 28 | 30 | 30 | 28 | 26 | 252 |
| | | | | | | | | |
| 100.0% | 96.7% | 100.0% | 100.0% | 100.0% | 100.0% | 96.4% | 100.0% | 99.2% |
| (88.3,100) | (88.3,100) | (83.3,99.4) | (88.3,100) | (88.3,100) | (88.3,100) | (87.9,100) | (82.3,99.4) | (88.6,100) | |
| 29 | 30 | 29 | 29 | 29 | 28 | 28 | 30 | 261 |
| 29 | 29 | 29 | 29 | 29 | 28 | 27 | 30 | 259 |
| | | | | | | | | |
| 92.90% | 100.0% | 93.30% | 100.0% | 96.60% | 100.0% | 76.70%* | 100.0% | 95.3% |
| (87.1, 100) | (77.3, 98.0) | (87.1, 100) | (78.7, 98.2) | (88.6, 100) | (82.8, 99.4) | (88.3, 100) | (59.1, 88.2) | (88.6, 100) | |
| 28 | 25 | 30 | 30 | 29 | 29 | 30 | 30 | 257 |
| 26 | 25 | 28 | 30 | 28 | 29 | 23 | 30 | 245 |

Table 5-2 Agreement hy Panel Member by Site

*83/90 possible results for Panel Member #8 were concordant (92% [80.0, 100]). The individual test results for this panel member at Site C were: stx 1: 93.3% (80, 100), 28/30 concordant results; stx 2: 86.6% (70, 90), 26/30 concordant results; fcl: 96.7% (80, 100), 29/30 concordant results. For all of the seven Panel Member #8 test results that did not demonstrate with the expected result, only one target was missed at each test point. None of these missed 2 targets and none missed 3 targets. Further, all three target concentrations in Panel Member #8 were at the LoD of the assay (1.00E+06 CFUmL) and as such are not expected to be detected 100% of the time. When looking at the individual targets, the detection rate ranged from 86.6%-96.7%.

Table 5-3. Agreement by Concentration, Analyte and Site/Lot for Positive Panel Members

	Analyte	stx1			stx2			O157
		Neg	Low	Med	Neg	Low	Med	Neg	Low	Med
Total^	%Agmt	100.0%	97.1%	98.8%	100.0%	96.0%	100.0%	100.0%	97.2%	99.4%
	(95% CI)	(98.9,100)	(93.4,98.8)	(95.8,99.7)	(98.9,100)	(91.9,98.0)	(97.8,100)	(98.9,100)	(93.5,98.8)	(96.8,99.9)
	Total (n)	348	173	169	342	174	174	340	176	174
	Agmt (n)	348	168	167	342	167	174	340	171	173
Site A	%Agmt	100.0%	100.0%	98.1%	100.0%	100.0%	100.0%	100.0%	96.7%	98.2%
Lot 1	(95% CI)	(96.8,100)	(93.8,100)	(90.2,99.7)	(96.8,100)	(93.7,100)	(93.5,100)	(96.6,100)	(88.6,99.1)	(90.7,99.7)
	Total (n)	115	58	54	115	57	55	110	60	57
	Agmt (n)	115	58	53	115	57	55	110	58	56
Site B	%Agmt	100.0%	98.2%	98.3%	100.0%	98.2%	100.0%	100.0%	98.2%	100.0%
Lot 2	(95% CI)	(96.8,100)	(90.7,99.7)	(91.1,99.7)	(96.8,100)	(90.7,99.7)	(93.9,100)	(96.8,100)	(90.7,99.7)	(93.8,100)
	Total (n)	115	57	60	116	57	59	117	57	58
	Agmt (n)	115	56	59	116	56	59	117	56	58
Site C	%Agmt	100.0%	93.1%	100.0%	100.0%	90.0%	100.0%	100.0%	96.6%	100.0%
Lot 3	(95% CI)	(96.9,100)	(83.6,97.3)	(93.5,100)	(96.7,100)	(79.8,95.3)	(94.0,100)	(96.7,100)	(88.5,99.1)	(93.9,100)
	Total (n)	118	58	55	111	60	60	113	59	59
	Agmt (n)	118	54	55	111	54	60	113	57	59

Total^: total combined across sites. stx1 neg: panel members 4, 5, stx1 10w: panel 2 ,8, stx1 medium: panel members 3, 9, st:2 neg: panels 2, 3, 6, 7, st:2 low: panel members 4, 8, stx2 medium: panel members 5,9, 0157 neg: panels 2, 3, 4, 5. 0157 low: panels 6, 8, 0157 medium: panel members 7, 9 *95% Cl: 95% confidence intervals calculated utilizing Wilson's Method.

For evaluation of the quantitative results, all negative samples in panel member 1 reported "0" and were therefore in 100% agreement. For all positive samples with a valid result, the mean, standard deviation and coefficient of variation (%CV) are reported.

To further evaluate performance by Site/Lot the mean, standard deviation, and coefficient of variation of cycle number were calculated. These parameters are calculated excluding samples that were expected to yield a positive result but where the PanNAT STEC Test cycle number was 0 (Table 5-4).

8

Site	Analyte:	stx 1		stx 2		O157		Process Control 1
		Low	Med	Low	Med	Low	Med
Site A
Lot 1	n	58	53	57	55	58	56	84
	Mean (std)	36.2 (0.8)	34.8 (0.9)	35.5 (1.0)	33.6 (1.1)	35.4 (0.8)	34.0 (1.0)	28.6 (1.0)
	CV	2.2%	2.6%	2.7%	3.2%	2.1%	3.0%	3.6%
Site B
Lot 2	n	56	59	56	59	56	58	87
	Mean (std)	36.0 (0.8)	34.9 (0.7)	35.4 (1.0)	33.9 (0.8)	35.6 (0.8)	34.2 (0.8)	28.7 (1.0)
	CV	2.2%	1.9%	2.7%	2.3%	2.1%	2.4%	3.4%
Site C
Lot 3	n	54	55	54	60	57	59	78
	Mean (std)	35.3 (0.7)	34.0 (0.7)	35.1 (0.9)	33.5 (0.8)	35.4 (1.0)	33.9 (0.7)	28.9 (0.9)
	CV	2.0%	2.2%	2.7%	2.5%	2.7%	2.0%	3.0%

Table 5-4. Reproducibility Cycle Numbers by Site, Analyte and Concentration

In negative panel members

n=number of observations, mean = mean cycle number, std = standard deviation for mean cycle number, CV=percent coefficient of variation.

Controls

The PanNAT STEC Test has an internal process control, a negative control, and a positive control, integrated within the cartridge. External controls are not provided, but are commercially available. Alternatively, a strain of E. coli that is positive for stx2 and fcl, such as strain EDL933 (ATCC® #43895™) may be used as an external positive control. A known negative sample such as a suspension of human A549 cells (ATCC® CCL-185TM) may be used as an external negative control.

Specimen Stability

The recommended storage time and temperature for Cary-Blair preserved and unpreserved stool specimens is:

. Refrigerated storage (2-8℃) for up to 7 days
To assess the stability of PanNAT STEC Test targets, testing was performed on both Cary-Blair preserved and unpreserved specimens at 3X LoD. The study tested the 0157 STEC reference strain (ATCC #43895), which contains all three PanNAT STEC Test targets. Specimens were stored at 2-8℃ and tested at intervals over the course of the study. Five replicates per specimen type per time point were tested. Results demonstrated that both specimen types were stable for 7 days under these conditions for all PanNAT STEC Test analytes.

Analytical Sensitivity/ Limit of Detection (LoD)

An estimation of the LoD for seven E. coli strains with different combinations of the stx1, stx2 and 0157 was performed by testing dilutions of each strain with the PanNAT STEC Test. Unpreserved and preserved specimens were tested at different concentrations. The estimated LoD was the lowest concentration at which all replicates were positive. The estimated LoD was then confirmed with strains EDL933 (stx1+, stx2+, 0157+) and 3007-85 (stx1+, stx2+, 0157-) tested in replicates of 28 at the estimated LoD in both preserved and unpreserved specimen types. To be considered confirmed, ≥95% of replicates had to produce positive results at the LoD target level.

Results:

9

In unpreserved stool specimens, the LoD for EDL933 and 3007-85 (stxl, stx2, and 0157) is 1.00E+06 CFU/mL.
In stool specimens preserved in Cary-Blair media, the LoD for EDL933 and 3007-85 (stx1, stx2 and O157) is 1.75E+06 CFU/mL.

Analytical Reactivity

A collection of well-characterized O157 and non-O157 strains was tested using the PanNAT STEC Test. The presence of the two Shiga toxin genes, stx1 and stx2, was assessed and measured in seven (7) 0157 strains, twelve (12) non-0157 strains, six (6) un-typeable (0) strains, and in a single O-antigen negative laboratory strain as a control (K12).

A summary of results is listed below:

The PanNAT STEC Test detected stx1 and stx2 in O157, non-O157 serotype strains . and un-typeable O strains.
The PanNAT STEC Test detected at least 2 subtypes of each toxin gene.
. For stxl, the PanNAT STEC Test generated the expected results for 24/26 strains (92%), including strains with subtypes stx1a and stx1c. The 2 strains that gave false negative results (TW07740, TW07754) were determined to be stx1d variants. The PanNAT STEC Test only weakly detects stxld due to nucleotide mismatches in the probe-binding region.
For stx2, the PanNAT STEC Test generated the expected results for 25/26 strains (96%), including strains with subtypes stx2a and stx2c/d. The strain that gave false negative results (TW07731) was determined to be an stx2 variant that closely resembles stx2b. The PanNAT STEC Test cannot detect this variant due to extensive nucleotide mismatches in the primer and probe binding regions.
. For the 0157 O-antigen (fcl) gene cluster, the PanNAT STEC Test generated the expected results in 26/26 strains (100%).

Analytical Specificity - Cross Reactivity

Thirty-seven (37) unique bacterial strains, eight (8) viruses, one (1) yeast organism, and three (3) parasites were spiked into a STEC negative pool matrix and evaluated for cross reactivity (Table 5-5).

10

| Organism | Strain ID/Catalog # | Input Tested | % Agreement
with Expected
Result
(stx1-/stx2-/
0157-) |
|---------------------------------|-----------------------------------------------------|---------------------|-------------------------------------------------------------------|
| Bacteria | | | |
| Acinetobacter baumannii | ZeptoMetrix 307-0294 0801597 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Aeromonas hydrophila | ZeptoMetrix Z161 0804098 and ATCC 35654 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Bacillus cereus | ZeptoMetrix ZO91 0801823 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Bacteroides fragilis | ZeptoMetrix Z029 0801583 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Campylobacter jejuni | ZeptoMetrix Z086 0801650 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Citrobacter koseri | ZeptoMetrix Z039 0801745 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Clostridium difficile | ZeptoMetrix NAP1 0801619 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Clostridium parabotulinum | ATCC 17863 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Clostridium perfringens | ZeptoMetrix TypeA 0801585 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Enterobacter cloacae | ZeptoMetrix Z101 0801830 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Enterococcus faecalis | ZeptoMetrix VRE 0801693 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Enterococcus faecium | ZeptoMetrix VRE, vanA 0801892 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli K12 | ATCC 29425 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli O111a (EAEC) | ATCC 29552 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli O29:NM (EIEC) | STEC Center O29:NM TW01095 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli O111a:2 (EPEC) | STEC Center O111a:2 TW07886 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli 08:H19 (ETEC) | STEC Center O8:H19 TW09068 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia hermannii | ATCC 33650 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Hafnia alvei | ATCC 13337 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Klebsiella pneumoniae | ZeptoMetrix Z026 0801506 | 1.00E+06 CFU/mL | 2/2^ (100%) |
| Listeria monocytogenes | ZeptoMetrix serotype 1/2b 0801534 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Morganella morganii | ATCC 25830 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Plesiomonas shigelloides | ZeptoMetrix Z130 0801899 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Proteus mirabilis | ZeptoMetrix Z050 0801544 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Pseudomonas aeruginosa | ZeptoMetrix clinical isolate 0801519 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Salmonella enterica | ATCC 35664 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Serratia marcescens | ZeptoMetrix Z053 0801723 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Shigella dysenteriae | ATCC 13313 | 1.00E+06 CFU/mL | 3/3^ (100%) |
| Shigella flexneri | ZeptoMetrix Z046 0801757 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Shigella sonnei | ZeptoMetrix clinical isolate 0801627 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Staphylococcus aureus | ZeptoMetrix Z020 0801531 | 1.00E+06 CFU/mL | 2/2^ (100%) |
| Staphylococcus epidermidis | MSSE:HER1292 0801689 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Streptococcus agalactiae | ZeptoMetrix Z019 0801545 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Streptococcus dysgalactiae | ZeptoMetrix Z068 0801516 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Vibrio spp | ATCC 11985 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Yersinia enterocolitica | ZeptoMetrix Z036 0801734 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Yersinia pseudotuberculosis | ATCC 4284 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Viruses | | | |
| Adenovirus 18 | ZeptoMetrix D.C.(VR-19) VPL-030 | 1.00E+05 PFU/mL | 3/3 (100%) |
| Adenovirus 40 | ZeptoMetrix Dugan HEK293A 0810084CF | 1.00E+05 PFU/mL | 3/3 (100%) |
| Coxsackie B4 | ZeptoMetrix LLC-mk2 0810075CF | 1.00E+05 PFU/mL | 3/3 (100%) |
| Cytomegalovirus | ZeptoMetrix AD-169 MRC-5 0810003CF | 1.00E+05 PFU/mL | 3/3 (100%) |
| Echovirus 11 | ZeptoMetrix LLC-MK2 0810023CF | 1.00E+05 PFU/mL | 3/3 (100%) |
| Enterovirus 68 | ZeptoMetrix FO2-3607 corn (VR-1197) Hela
VPL-030 | 1.00E+05 PFU/mL | 3/3 (100%) |
| Norovirus (recombinant) | ZeptoMetrix Vero Group 10810086CF | 1.00E+05 PFU/mL | 3/3 (100%) |
| Rotavirus | ZeptoMetrix WA MA-104 MRC-5 0810041CF | 1.00E+05 PFU/mL | 3/3 (100%) |
| Yeast and Parasites | | | |
| Candida albicans | ZeptoMetrix Z006 0801504 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Cryptosporidium parvum | Waterborne Iowa Isolate | 1.00E+06 Oocysts/mL | 3/3 (100%) |
| Entamoeba histolytica (lysate) | ZeptoMetrix DS4-868 BacT-035R05 | 1.00E+06 Cysts/mL | 3/3 (100%) |
| Giardia lamblia | Waterborne H3 isolate | 1.00E+06 Cysts/mL | 3/3 (100%) |

Table 5-5. Summary of Cross Reactivity Data

11

Indicates that one of the replicates was "INCOMPLETE" and was removed from analysis.

S. dysenteriae detection is expected.

Analytical Specificity – Microbial Interference

For microbial interference, the same 49 non-target organisms were spiked into Shiga toxin negative pooled, unpreserved matrix containing 1X LoD of the reference strain E. coli O157:H7 (EDL933 ATCC #43895), which is positive for target analytes stx1, stx2 and O157 (Table 5-6).

Table 5-6. Summary of Microbial Interference Data

| Organism | Strain ID/Catalog # | Input Tested
CFU/mL | % Agreement
with Expected
Result
(stx1+ / stx2+ /
0157+) |
|---------------------------------|-----------------------------------------|------------------------|----------------------------------------------------------------------|
| Bacteria | | | |
| Acinetobacter baumannii | ZeptoMetrix 307-0294 0801597 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Aeromonas hydrophilia | ZeptoMetrix Z161 0804098 and ATCC 35654 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Bacillus cereus | ZeptoMetrix ZO91 0801823 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Bacteroides fragilis | ZeptoMetrix Z029 0801583 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Campylobacter jejuni | ZeptoMetrix Z086 0801650 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Citrobacter koceri | ZeptoMetrix Z039 0801745 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Clostridium difficile | ZeptoMetrix NAP1 0801619 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Clostridium parabotulinum | ATCC 17863 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Clostridium perfringens | ZeptoMetrix TypeA 0801585 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Enterobacter cloacae | ZeptoMetrix Z101 0801830 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Enterococcus faecalis | ZeptoMetrix VRE 0801693 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Enterococcus faecium | ZeptoMetrix VRE, vanA 0801892 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli K12 | ATCC 29425 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli O111a (EAEC) | ATCC 29552 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli O29:NM (EIEC) | STEC Center O29:NM TW01095 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli O111a:2 (EPEC) | STEC Center O111a:2 TW07886 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia coli 08:H19 (ETEC) | STEC Center O8:H19 TW09068 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Escherichia hermannii | ATCC 33650 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Hafnia alvei | ATCC 13337 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Klebsiella pneumoniae | ZeptoMetrix Z026 0801506 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Listeria monocytogenes | ZeptoMetrix serotype 1/2b 0801534 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Morganella morganii | ATCC 25830 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Plesiomonas shigelloides | ZeptoMetrix Z130 0801899 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Proteus mirabilis | ZeptoMetrix Z050 0801544 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Pseudomonas aeruginosa | ZeptoMetrix clinical isolate 0801519 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Salmonella enterica | ATCC 35664 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Serratia marcescens | ZeptoMetrix Z053 0801723 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Shigella dysenteriae | ATCC 13313 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Shigella flexneri | ZeptoMetrix Z046 0801757 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Shigella sonnei | ZeptoMetrix clinical isolate 0801627 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Staphylococcus aureus | ZeptoMetrix Z020 0801531 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Staphylococcus epidermidis | ZeptoMetrix MSSE:HER1292 0801689 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Streptococcus agalactiae | ZeptoMetrix Z019 0801545 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Streptococcus dysgalactiae | ZeptoMetrix Z068 0801516 | 1.00E+06 CFU/mL | 3/3 (100%) |
| Vibrio spp | ATCC 11985 | 1.00E+06 CFU/mL | 3/3 (100%) |

12

Yersinia enterocolitica	ZeptoMetrix Z036 0801734	1.00E+06 CFU/mL	3/3 (100%)
Yersinia pseudotuberculosis	ATCC 4284	1.00E+06 CFU/mL	3/3 (100%)
Viruses
Adenovirus 18	ZeptoMetrix D.C.(VR-19) VPL-030	1.00E+05 PFU/mL	3/3 (100%)
Adenovirus 40	ZeptoMetrix Dugan HEK293A 0810084CF	1.00E+05 PFU/mL	3/3 (100%)
Coxsackie B4	ZeptoMetrix LLC-mk2 0810075CF	1.00E+05 PFU/mL	3/3 (100%)
Cytomegalovirus	ZeptoMetrix AD-169 MRC-5 0810003CF	1.00E+05 PFU/mL	3/3 (100%)
Echovirus 11	ZeptoMetrix LLC-MK2 0810023CF	1.00E+05 PFU/mL	3/3 (100%)
Enterovirus 68	ZeptoMetrix FO2-3607 corn (VR-1197) Hela
VPL-030	1.00E+05 PFU/mL	3/3 (100%)
Norovirus (recombinant)	ZeptoMetrix Vero Group 10810086CF	1.00E+05 PFU/mL	3/3 (100%)
Rotavirus	ZeptoMetrix WA MA-104 MRC-5 0810041CF	1.00E+05 PFU/mL	3/3 (100%)
Yeast and Parasites
Candida albicans	ZeptoMetrix Z006 0801504	1.00E+06 CFU/mL	2/2^ (100%)
Cryptosporidium parvum	Waterborne lowa Isolate	1.00E+06 Oocysts/mL	3/3 (100%)
Entamoeba histolytica (lysate)	ZeptoMetrix DS4-868 BacT-035R05	1.00E+06 Cysts/mL	3/3 (100%)
Giardia lamblia	Waterborne H3 isolate	1.00E+06 Cysts/mL	3/3 (100%)

^ Indicates that one of the replicates was "INCOMPLETE" and was removed from analysis.

The analytical reactivity studies demonstrated no cross reactivity for any analytes tested except for Shigella dysenteriae, which is expected due to sequence homology between the S. dysenteriae stxA gene and the stxl region targeted by the PanNAT STEC Test. No competitive inhibition of PanNAT STEC Test analytes was demonstrated in any of the samples tested.

Analytical Specificity - Interfering Substances

The PanNAT STEC Test was evaluated for potential interference from 25 endogenous and exogenous substances that are common stool contaminants, or likely to be present in patients with diarrhea (Table 5-7). All substances were tested in unpreserved stool specimens in the presence and absence of two stx1+/stx2+/0157+ strains of E. coli, with three replicates per condition. In the presence of an stx 1+/stx2+/0157+ strain, Gaviscon® and Imodium AD® each gave a stx1+/stx2+/0157result on one of three replicates, while stearic acid gave an stxl-/stx2-/0157- result on one of three replicates. No interference was demonstrated for any of the remaining substances tested.

| Interfering

Substance	Concentration tested
Whole Blood (EDTA)	40% v/v
Mucin	3.5% w/v (35mg/mL)
Cholesterol	4.80% w/v (48mg/mL)
Human genomic DNA	10 ug/mL
Bile	25% w/v (250mg/mL)
Kaopectate®	3.5% v/v
Pepto-Bismol®	5% v/v
Imodium AD®	5% v/v

Table 5-7. List of Interferents Tested and Concentrations

13

Petroleum Jelly (Vaseline®)	50% w/v (500mg/mL)
Baby Wipes	50% v/v
Hydrocortisone Cream	50% w/v (500mg/mL)
Diaper Rash Cream	50% w/v (500mg/mL)
Stearic Acid	4.80% w/v (48mg/mL)
Palmitic Acid	4.80% w/v (48mg/mL)
Gaviscon®	10% w/v (100mg/mL)
Milk of Magnesia®	10% w/v (100mg/mL)
Aleve®	0.5 mg/mL
Preparation H®	9.5% w/v (95mg/mL)
Mineral oil	50% w/v (500mg/mL)
Amoxicillin	5% w/v (50mg/mL)
Nystatin	0.43% w/v (4.28mg/mL)
Metronidazole	5% w/v (50mg/mL)
Ciprofloxacin	1.25% w/v (12.5mg/mL)
Sennica laxatives	1% w/v (10mg/mL)
Diaper Eluent	25% v/v

Carry Over / Cross Contamination

The potential for false positive results with the PanNAT STEC Test due to run-to-run contamination was evaluated by testing an alternating series of contrived high positive stool samples and clinically negative stool samples in direct succession five (5) times on three (3) PanNAT instruments.

All positive samples reported stx1+/stx2+/0157+ results, and 14 of the 15 negative samples reported stx1-/stx2-/0157- results. One of the negative samples yielded an invalid result (negative sample, instrument 3, round 5). No carryover or cross contamination was observed.

Assay Cut-Off

The PanNAT STEC Test detects Shiga toxin 1 (stxr}, Shiga toxin 2 (stx2), and E. coli 0157. For each target, the valid cycle number range is 16 to 39. Cycle numbers are automatically calculated by the PanNAT system software. Each of the target analytes (stx1, stx2, 0157), as well as the Process Control, must have a cycle number within the valid range in order to be reported as "Positive," otherwise it is reported as "Negative." Cycle number cutoffs were verified on a panel of seven (7) STEC strains of known genotypes, and subsequently validated in the multi-site clinical study.

5.9 Clinical Performance Studies

Prospective and Retrospective Specimen Testing

Performance characteristics of the PanNAT STEC Test were determined on stool samples prospectively collected at six (6) clinical sites and on frozen, retrospective samples. Prospective data were collected from pediatric and adult patients as part of routine patient

14

care. Results for standard of care testing (comparator method) were obtained using multiple methods, including a test for Shiga toxins 1 and 2 and a test for E.coli O157. A presumptive result for 0157 on specimens that tested positive with sorbitol on MacConkey agar was confirmed by latex agglutination and biochemical analysis.

For prospective specimens (both unpreserved and Cary-Blair preserved), the leftover, deidentified specimens were tested by two comparator methods: 1) an immunoassay for detection of Shiga toxins 1 and 2 following enrichment culture, and 2) a SMAC plate for presumptive detection of 0157. Discordant analyses were performed by an independent molecular reference laboratory on discrepant samples using alternative PCR and bidirectional (BDS) sequencing methods.

A total of 1331 prospective specimens (1231 Cary-Blair preserved and 100 unpreserved) from a range of age groups (Table 5-8) were enrolled in the clinical evaluation. Of these, 30 were removed from the analysis due to pre- or post-analytical errors. Of the remaining 1301 specimens, 7.6% (99/1301) were initially invalid. Specimens with initial invalid results were retested. On retest, 78 were valid resulting in a repeat invalidity rate of 1.6% (21/1301). The number of prospective specimens that qualified for the agreement analysis was 1280 (1184 Cary-Blair preserved and 96 unpreserved) (Table 5-9).

	Total Results	Valid Results		Invalid Results
Age Group	n	n	%	n	%
stx1 Only	100%
(4/4)	Not applicable	25.0%
(1/4)	100%
(7/7)
stx2 Only	100%
(2/2)	Not applicable	0.0%
(0/2)	100%
(3/3)
O157 Only	Not applicable	Not applicable	Not applicable	100%
(1/1)
stx1 + stx2	Not applicable	Not applicable	Not applicable	100%
(4/4)
stx1 + O157	Not applicable	Not applicable	0.0%
(0/1)	Not applicable
stx2 + O157	100%
(3/3)	100%
(2/2)	85.7%
(6/7)	Not applicable
stx1 + stx2 +
O157	Not applicable	Not applicable	95.2%
(20/21)	100%
(1/1)
Negative	98.7%
(1160/1175)2	98.9%
(93/94)3	98.5%
(67/68)4	Not applicable

1 Analyte(s) present as determined by the applicable comparator method for the study 2 Of the 15 specimens with false positive results by the PanNAT STEC Test, 8 were positive for str.1, 5 for stx2 and 2 for both st:2

and 0157

3 1/94 negative specimens was positive by the PanNAT STEC Test for stx2

4 1/68 negative specimens was positive by the PanNAT STEC Test for stx1, stx2 and 0157

5.10 Conclusion

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.