The QIAstat-Dx Gastrointestinal Panel 2 is a multiplexed nucleic acid test intended for use with the QIAstat-Dx Analyzer 1.0. for the simultaneous in vitro qualitative detection of nucleic acids from multiple viruses, bacteria. and parasites directly from preserved stool samples (Para-Pak C&S or FecalSwab) obtained from individuals with signs and/or symptoms of gastrointestinal infection. The following viruses, bacteria (including several diarrheagenic E. col/ Shigella pathotypes), and parasites are identified with the QIAstat-Dx Gastrointestinal Panel 2 :
• Adenovirus F40/F41
• Astrovirus
• Norovirus GI/GII
• Rotavirus A
• Campylobacter (C. jejuni, C. coli and C. upsaliensis)
• Shigella/Enteroinvasive Escherichia coli (EIEC)
• Enteropathogenic Escherichia coli (EPEC)
• Enterotoxigenic Escherichia coli (ETEC) lt/st
• Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2
(including specific identification of E. coli O157 serogroup within STEC)
• Salmonella
• Plesiomonas shigelloides
• Yersinia enterocolitica
• Cryptosporidium
• Cyclospora cayetanensis
• Entamoeba histolytica
• Giardia lamblia*
*(Also known as Giardia intestinalis and Giardia duodenalis)
Concomitant culture is necessary for organism recovery and further typing of bacterial agents.
The QIAstat-Dx Gastrointestinal Panel 2 is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness, in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule-out coinfection with organisms not detected by the QIAstat-Dx Gastrointestinal Panel 2. The organisms detected may not be the sole or definitive cause of the disease.
Negative QIAstat-Dx Gastrointestinal Panel 2 results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this assay test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Device Description

QIAstat-Dx is based on single-test cartridges with pre-packaged reagents including both wet and dry chemistry to handle the sample preparation and detection steps for the presence of a range of selected analytes by PCR technology. After insertion of the sample, the OIAstat-Dx assay cartridge is processed by the OIAstat-Dx Analyzer 1.0.

AI/ML Overview

Here's a summary of the acceptance criteria and study details for the QIAstat-Dx Gastrointestinal Panel 2, extracted from the provided text:

Acceptance Criteria and Device Performance for QIAstat-Dx Gastrointestinal Panel 2

The acceptance criteria for the QIAstat-Dx Gastrointestinal Panel 2 can be inferred from the performance metrics (Positive Percentage Agreement - PPA and Negative Percentage Agreement - NPA) reported for both prospective and retrospective clinical studies. While explicit 'acceptance criteria' values are not provided as a separate table, the reported performance demonstrates the device's ability to meet the necessary accuracy for clinical utility. The FDA's substantial equivalence determination implies these performance characteristics were found acceptable.

Implied Acceptance Criteria (based on reported performance):

High PPA: The device should accurately detect the target pathogens when they are present. Most reported PPA values are above 90%, with many at 100%. Even lower values like 75% for Giardia lamblia in FecalSwab are within a statistically acceptable range given the confidence intervals.
High NPA: The device should accurately report the absence of target pathogens when they are not present, minimizing false positives. Most reported NPA values are very high, often 99% or 100%.

Table of Acceptance Criteria (Implied) and Reported Device Performance

Given that specific numerical acceptance criteria (e.g., "PPA > X%") are not explicitly stated in the document, the table below showcases the reported clinical performance which serves as evidence of meeting the implicit acceptance criteria for reliable detection and non-detection of pathogens.

Analyte (Sample Type)	Implied Acceptance Criterion (High PPA/NPA)	Reported Performance (PPA)	95% Confidence Interval (PPA)	Reported Performance (NPA)	95% Confidence Interval (NPA)
Viruses
Adenovirus F40/F41 (FecalSwab)	High PPA, High NPA	83.3% (5/6)	43.7-97.0%	100.0% (1214/1214)	99.7-100.0%
Adenovirus F40/F41 (Para-Pak C&S)	High PPA, High NPA	50.0% (1/2)	9.5-90.6%	99.9% (703/704)	99.2-100.0%
Astrovirus (FecalSwab)	High PPA, High NPA	100.0% (3/3)	43.9-100.0%	100.0% (1219/1219)	99.7-100.0%
Astrovirus (Para-Pak C&S)	High PPA, High NPA	100.0% (6/6)	61.0-100.0%	100.0% (700/700)	99.5-100.0%
Norovirus GI/GII (FecalSwab)	High PPA, High NPA	93.9% (31/33)	80.4-98.3%	99.6% (493/495)	98.6-100.0%
Norovirus GI/GII (Para-Pak C&S)	High PPA, High NPA	77.8% (14/18)	54.8-91.0%	100.0% (399/399)	99.1-100.0%
Rotavirus A (FecalSwab)	High PPA, High NPA	91.3% (21/23)	73.2-97.6%	99.8% (1197/1199)	99.4-100.0%
Rotavirus A (Para-Pak C&S)	High PPA, High NPA	100.0% (3/3)	43.9-100.0%	99.9% (702/703)	99.2-100.0%
Bacteria
Campylobacter (FecalSwab)	High PPA, High NPA	97.0% (65/67)	89.8-99.2%	99.7% (1151/1155)	99.1-99.9%
Campylobacter (Para-Pak C&S)	High PPA, High NPA	96.8% (30/31)	83.8-99.4%	99.7% (675/677)	98.9-99.9%
Plesiomonas shigelloides (FecalSwab)	High PPA, High NPA	N/A (0/0)	N/A	99.8% (1220/1222)	99.4-100.0%
Plesiomonas shigelloides (Para-Pak C&S)	High PPA, High NPA	83.3% (5/6)	43.7-97.0%	99.7% (698/700)	99.0-99.9%
Salmonella (FecalSwab)	High PPA, High NPA	87.5% (14/16)	64.0-96.5%	100.0% (1206/1206)	99.7-100.0%
Salmonella (Para-Pak C&S)	High PPA, High NPA	95.0% (19/20)	76.4-99.1%	100.0% (688/688)	99.4-100.0%
Yersinia enterocolitica (FecalSwab)	High PPA, High NPA	93.8% (15/16)	71.7-99.0%	99.4% (1199/1206)	98.8-99.7%
Yersinia enterocolitica (Para-Pak C&S)	High PPA, High NPA	100.0% (3/3)	43.9-100.0%	99.3% (698/703)	98.4-99.7%
Diarrheagenic E. coli/Shigella
Enteropathogenic E. coli (EPEC) (Para-Pak C&S)	High PPA, High NPA	87.7% (57/65)	77.6-93.6%	100.0% (632/632)	99.4-100.0%
Enterotoxigenic E. coli (ETEC) lt/st (FecalSwab)	High PPA, High NPA	90.0% (9/10)	59.6-99.2%	99.3% (427/430)	98.0-99.8%
Enterotoxigenic E. coli (ETEC) lt/st (Para-Pak C&S)	High PPA, High NPA	90.0% (9/10)	59.6-99.2%	98.7% (390/395)	97.1-99.5%
Shiga-like toxin E. coli (STEC) stx1/stx2 (Para-Pak C&S)	High PPA, High NPA	83.3% (5/6)	43.6-97.0%	99.3% (397/400)	97.8-99.7%
E. coli O157 (Para-Pak C&S)	High PPA, High NPA	N/A (0/0)	N/A	100.0% (5/5)	56.6-100.0%
Shigella/Enteroinvasive E. coli (EIEC) (FecalSwab)	High PPA, High NPA	100.0% (10/10)	72.3-100.0%	100.0% (1212/1212)	99.7-100.0%
Shigella/Enteroinvasive E. coli (EIEC) (Para-Pak C&S)	High PPA, High NPA	100.0% (2/2)	34.2-100.0%	99.9% (703/704)	99.2-100.0%
Parasites
Cryptosporidium (FecalSwab)	High PPA, High NPA	50.0% (2/4)	15.0-85.0%	100.0% (1218/1218)	99.7-100.0%
Cryptosporidium (Para-Pak C&S)	High PPA, High NPA	100.0% (6/6)	61.0-100.0%	99.9% (699/700)	99.2-100.0%
Cyclospora cayetanensis (FecalSwab)	High PPA, High NPA	100.0% (3/3)	43.9-100.0%	100.0% (1219/1219)	99.7-100.0%
Cyclospora cayetanensis (Para-Pak C&S)	High PPA, High NPA	94.7% (18/19)	75.4-99.1%	100.0% (687/687)	99.4-100.0%
Entamoeba histolytica (FecalSwab)	High PPA, High NPA	N/A (0/0)	N/A	100.0% (1222/1222)	99.7-100.0%
Entamoeba histolytica (Para-Pak C&S)	High PPA, High NPA	N/A (0/0)	N/A	100.0% (706/706)	99.5-100.0%
Giardia lamblia (FecalSwab)	High PPA, High NPA	75.0% (6/8)	40.9-92.9%	98.4% (434/441)	96.8-99.2%
Giardia lamblia (Para-Pak C&S)	High PPA, High NPA	100.0% (1/1)	20.7-100.0%	100.0% (406/406)	99.1-100.0%

Study Details:

1. Sample sizes used for the test set and the data provenance:

Test Set (Clinical Study):
- Total Specimens: 2808
  - 1939 Prospective (1222 FecalSwab, 717 Para-Pak C&S)
  - 119 Prospective Archived (Norovirus GI/GII: 81, STEC: 18 plus 20 negative specimens where relevant)
  - 750 Retrospective Frozen Specimens
- Data Provenance: Multi-center international study conducted at thirteen clinical sites across 5 countries (4 sites in Europe and 9 sites in USA). Specimens were collected between May and July 2021.

2. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience):

The document does not specify the number or qualifications of experts used to establish the ground truth. Instead, it refers to the use of "one FDA-cleared test as comparator for most analytes" and a "composite comparator consisting of either three independent FDA-cleared test methods or two independent FDA-cleared tests methods and two validated PCR assays followed by bidirectional sequencing" for others. This implies that the ground truth was established through validated diagnostic methods rather than direct expert consensus on primary samples in many cases.

3. Adjudication method (e.g. 2+1, 3+1, none) for the test set:

For analytes where a composite comparator was used (Norovirus GI/GII, ETEC, STEC, and Giardia lamblia), the ground truth was determined by the majority of the three results.
- A positive composite comparator result: based on positive results for at least two comparator tests.
- A negative composite comparator result: based on negative results for at least two comparator tests.
For other analytes, where "one FDA-cleared test method" was used, the comparator's result directly served as the ground truth.
For cases with insufficient sample volume for complete composite comparator testing, a "worst-case model" was applied for PPA calculation.

4. If a muti-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

This information is not applicable as the device is an in vitro diagnostic (IVD) nucleic acid test for pathogen detection, not an AI-assisted diagnostic device interpreted by human readers for medical imaging or similar tasks. Therefore, an MRMC comparative effectiveness study involving human readers and AI assistance is not relevant to this device.

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

Yes, the performance presented (PPA and NPA) for the QIAstat-Dx Gastrointestinal Panel 2 is a standalone performance of the algorithm/device. The device automatically interprets test results and displays a summary, without a human interpretation loop for its core function.

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

The ground truth was established using FDA-cleared comparator methods and validated PCR assays followed by bidirectional sequencing. This acts as a robust diagnostic ground truth based on established molecular and clinical laboratory standards. Pathology or outcomes data were not explicitly mentioned as primary ground truth sources for individual pathogen detection in this context.

7. The sample size for the training set:

The document does not explicitly state a sample size for the training set. The clinical studies describe the evaluation of the device's performance using prospective, prospective archived, and retrospective samples, implying these are test sets rather than training sets. Analytical studies such as LoD and inclusivity also use specific strains and dilutions, but these are for analytical validation, not for training a machine learning model. For IVD devices like this, the "training set" concept (as understood in AI/ML) might be less direct, relying more on extensive analytical verification of probe/primer specificity and reactivity across a wide range of strains and concentrations, and then clinical validation.

8. How the ground truth for the training set was established:

As the training set size is not provided, the method for establishing its ground truth is also not detailed. However, for the analytical studies (which could be considered a form of "training/validation data generation" in a broader sense for IVDs), the ground truth for LoD and inclusivity was established by using culture isolates from commercial suppliers (ZeptoMetrix® and ATCC®) or clinical samples positive for target analytes. These were "prepared in human stool matrix" and tested at known concentrations validated by in-house developed and validated qPCR assays for molecular unit titers.

Summary

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May 31, 2024

Image /page/0/Picture/1 description: The image shows the logo for the U.S. Food and Drug Administration (FDA). On the left is the Department of Health and Human Services logo. To the right of that 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.

QIAGEN GmbH % Selina Salthouse Regulatory Affairs Manager QIAGEN Manchester Ltd CityLabs 2.0. 200 Hathersage Road Manchester, Manchester M13 0BH, United Kingdom

Re: K220062

Trade/Device Name: QIAstat-Dx Gastrointestinal Panel 2 Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal microorganism multiplex nucleic acid-based assay Regulatory Class: Class II Product Code: PCH Dated: April 4, 2023 Received: April 4, 2023

Dear Selina Salthouse:

We have reviewed your section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (the Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/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.

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

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Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review. the OS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about 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,

Noel J. Gerald -S

Noel J. Gerald, Ph.D. Branch Chief Bacterial Respiratory and Medical Countermeasures Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

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Indications for Use

510(k) Number (if known) K220062

Device Name QIAstat-Dx Gastrointestinal Panel 2

Indications for Use (Describe)

Pathogen	Para-Pak C&S	FecalSwab
• Adenovirus F40/F41	Reported	Reported
• Astrovirus	Reported	Reported
• Norovirus GI/GII	Reported	Reported
• Rotavirus A	Reported	Reported
• Campylobacter (C. jejuni, C. coli and C. upsaliensis)	Reported	Reported
• Shigella/Enteroinvasive Escherichia coli (EIEC)	Reported	Reported
• Enteropathogenic Escherichia coli (EPEC)	Reported	Not Reported
• Enterotoxigenic Escherichia coli (ETEC) lt/st	Reported	Reported
• Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2(including specific identification of E. coli O157 serogroup within STEC)	Reported	Not Reported
• Salmonella	Reported	Reported
• Plesiomonas shigelloides	Reported	Reported
• Yersinia enterocolitica	Reported	Reported
• Cryptosporidium	Reported	Reported
• Cyclospora cayetanensis	Reported	Reported
• Entamoeba histolytica	Reported	Reported
• Giardia lamblia*	Reported	Reported

*(Also known as Giardia intestinalis and Giardia duodenalis)

Concomitant culture is necessary for organism recovery and further typing of bacterial agents.

The QIAstat-Dx Gastrointestinal Panel 2 is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness, in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule-out coinfection with organisms not detected by the QIAstat-Dx Gastrointestinal Panel 2. The organisms detected may not be the sole or definitive cause of the disease.

Negative QIAstat-Dx Gastrointestinal Panel 2 results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this assay 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)

X Prescription Use (Part 21 CFR 801 Subpart D)

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

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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5.0 510(k) SUMMARY

General Information

Submitted by:	QIAGEN GmbHQiagen Strasse 1Hilden, 40724,Germany
Contact Person:	Selina SalthouseManager, Regulatory AffairsQIAGEN Manchester LtdCitylabs 2.0, 200 Hathersage RoadManchester, M13 0BH, UKPhone: +44 7823 862 321Fax: 44 161 204 1200Email: selina.salthouse@qiagen.com
Date Prepared:	May 30, 2024
Device Name:	QIAstat-Dx® Gastrointestinal Panel 2
Trade Name:	QIAstat-Dx® Gastrointestinal Panel 2
Common Name:	QIAstat-Dx® Gastrointestinal Panel 2
Classification:	21 CFR 866.3990 - Gastrointestinal Pathogen Panel MultiplexNucleic Acid-Based Assay System
Product Code:	PCH

Predicate Device

Manufacturer	Product Name	510(k) No.
BioFire Diagnostics LLC	FilmArray®Gastrointestinal (GI) Panel	K140407

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Device Description

Principle of Operation

The QIAstat-Dx Gastrointestinal Panel 2 is part of the QIAstat-Dx system and works with the QIAstat-Dx Analyzer 1.0.

The OIAstat-Dx Gastrointestinal Panel 2 is intended to be used with stool samples in Para-Pak C&S or FecalSwab transport media.

Once the cartridge has been inserted into the instrument, the test starts automatically and runs for approximately 78 minutes. When the test is finished, the cartridge is removed by the user and discarded. The QIAstat-Dx Analyzer 1.0 automatically interprets test results and displays a summary on the analyzer display screen. The results can be printed using a connected printer if needed. The detected analytes are displayed in red. For other analytes tested, they are displayed in green if not detected or in gray if not applicable or invalid. The analyzer will report if an error occurs during processing, in which case the test will fail and no results will be provided (screen will show "FAIL").

Sample Pre-treatment for PCR Inhibitors removal

Following insertion of the cartridge, the buffer located in Reservoir 1 is added inside the lysis chamber and homogenized with the sample using a rotor in the presence of silica beads. This enables separation of commonly found inhibitory substances in stool from the DNA/RNA by chemical means.

Resuspension of Internal Control (IC) and Proteinase K

Following sample pre-treatment, the IC and Proteinase K is resuspended with the buffer located in Reservoir 2 (resuspension buffer). The buffer from Reservoir 2 is added to the interconnected IC cavity and Proteinase K cavity and transferred repeatedly between the Transfer Chamber and the cavities to ensure resuspension. The resuspended IC and Proteinase K are transferred to the sample cavity.

Cell Lysis

Primary lysis of the cells and analytes present in a stool sample and IC occurs by a combination of chemical and mechanical processes using a rotor inside the lysis chamber in the presence of silica beads and a buffer that acts as a chemical agent in aiding the mechanical process. The fast movement of the rotor in the presence of the silica beads results in sample agitation, which creates turbulence and shear forces that favor the lysis of the cell wall.

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After mechanical lysis is completed, the primary lysate is transferred to the purification chamber through a frit with 2-5 um pore size. The second lysis buffer (from Reservoir 3) is added to the primary lysate to complete chemical lysis.

Purification

Binding reagent (from Reservoir 4) is added to the lysate in the purification chamber, and the mix is passed through the silica membrane. In this process, the DNA/RNA molecules stick to the membrane, and the remaining components of the lysate are delivered to the waste chamber. Then the membrane is washed with a first washing buffer (from Reservoir 5) to wash away proteins. This is followed by a second washing step with a second washing buffer (from Reservoir 6) to remove any remaining substances other than the nucleic acids. A subsequent drying step eliminates volatile substances from the silica membrane. Prior to the elution step, the Transfer Chamber is rinsed with the rinsing buffer (from Reservoir 7) in order to remove any potential inhibitors from previous processing steps. At the end of the process, the nucleic acids are released from the membrane using an elution buffer (from Reservoir 8). The eluate is collected in the Transfer Chamber.

Rehydration of Master Mix

A defined volume (135 µL) of the eluate is delivered to the reservoir of the Dry Chemistry Container (DCC) to rehydrate the Master Mix. Any remaining eluate is transferred to the Reservoir 7. The eluate/Master Mix solution is mixed by repeated transfer between the Transfer Chamber and the DCC.

Aliquoting and PCR

Defined aliquots (15 µL) of mixed eluate/Master Mix are sequentially transferred from the Transfer Chamber to each of eight Reaction Chambers containing the specified, air dried primers and probes.

Within each Reaction Chamber, a reverse-transcription step followed by real time, multiplex PCR ("rtPCR") is performed. Increase in fluorescence (indicative of detection of each target analyte) is detected directly within each Reaction Chamber.

The rtPCR process is conducted by two submodules of the QIAstat-Dx Analyzer 1.0: the Thermal Cycler and the qPCR Sensor.

Components Description

OIAstat-Dx Gastrointestinal Panel 2 Cartridge:

The QIAstat-Dx Gastrointestinal Panel 2 cartridge is a disposable plastic device that allows performing fully automated molecular assays. The main features of the OIAstat-Dx Gastrointestinal Panel 2 cartridge for the Gastrointestinal assay include the ability to test liquid samples and the capacity to store all necessary reagents within the cartridge needed for such testing. All sample preparation and assay steps will be performed within the cartridge.

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All the reagents required for the complete execution of the test are pre-loaded and selfcontained in the OIAstat-Dx Gastrointestinal Panel 2. The user does not need to manipulate any reagents. During the test, reagents are handled by pneumatically-operated microfluidics without any direct contact with the user or the analyzer actuators. This eliminates any possibility of exposure of the user or the analyzer to chemicals contained in the cartridge during the test and up to the disposal of used cartridges.

Reagents may be found in three different physical forms: liquid, air-dried on surfaces or lyophilized powder cake.

Within the cartridge, multiple steps are automatically performed in sequence by using pneumatic pressure and a multiport valve to transfer sample and fluids via the Transfer Chamber to their intended destinations.

QIAstat-Dx Analyzer 1.0

The QIAstat-Dx Analyzer 1.0 is the unit that hosts a cartridge and, on command from the user, is able to run predefined assay protocols. The software specific to this test is preloaded on the QIAstat-Dx Analyzer 1.0.

Other Materials

Each QIAstat-Dx Gastrointestinal Panel 2 cartridge will be used with a transfer pipette (provided with device). The stool sample from the patient will be collected in Para-Pak C&S or FecalSwab transport medium following the manufacturer's instructions for use (not provided with device).

QIAstat-Dx Analyzer 1.0 - the QIAstat-Dx Gastrointestinal Panel 2 cartridge can only be run on the QIAstat-Dx Analyzer 1.0.

Calibrators and/or Controls

Blank controls are not applicable to the device because it is a single test disposable cartridge. Negative and positive external controls are recommended by the company but not provided with the OIAstat-Dx Gastrointestinal Panel 2.

QIAGEN provides an IC within the QIAstat-Dx Gastrointestinal Panel 2 cartridge which provides a full process control covering lysis, nucleic acid purification, reverse transcription and DNA amplification. The IC is Schizosaccharomyces pombe. The IC is located in the IC cavity and is mixed with the sample during sample preparation and the eluate is mixed with the Master Mix, then aliquoted in all Reaction Chambers. The results screen displays a message indicating that the Internal Control "Passed" when the test is run successfully. An IC message of "Failed" indicates that the internal control was not amplified; 'Positive' test results are then reported as POSITIVE* (positive with warning), all 'Negative' results are invalid.

The OIAstat-Dx Analyzer 1.0 is provided factory calibrated and does not require user calibration. The OIAstat-Dx Analyzer 1.0 includes self-check controls to verify the performance of all sensors and actuators and will alert the user in case of failure.

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The RCA will provide the results to the Application Software (SW). The Application SW will store the information related to a given result in the database and will display a summary of detected analytes and the result for the IC. All POSITIVE analytes will be listed as "DETECTED PATHOGENS". The screen will also display the complete list of all "TESTED PATHOGENS", including positive, not applicable or invalid analytes. All results for tests executed on each QIAstat-Dx Analyzer 1.0 are stored and can be reviewed in a specific archive on each QIAstat-Dx Analyzer 1.0.

Specimen collection and transport materials

Stool samples are collected and resuspended in Para-Pak C&S or FecalSwab transport medium following the manufacturer's instructions (Para-Pak C&S (Meridian Bioscience) or FecalSwab (COPAN)).

Intended Use

The QIAstat-Dx® Gastrointestinal Panel 2 is a multiplexed nucleic acid test intended for use with the OIAstat-Dx Analyzer 1.0 for the simultaneous in vitro qualitative detection and identification of nucleic acids from multiple viruses, bacteria, and parasites directly from preserved stool samples (Para-Pak® C&S or FecalSwab™) obtained from individuals with signs and/or symptoms of gastrointestinal infection. The following viruses, bacteria (including several diarrheagenic E.coli/Shigella pathotypes), and parasites are identified with the QIAstat-Dx® Gastrointestinal Panel 2:

Pathogen	Para-Pak C&S	FecalSwab
Adenovirus F40/F41	✓	✓
Astrovirus	✓	✓
Norovirus GI/GII	✓	✓
Rotavirus A	✓	✓
Campylobacter (C. jejuni, C. coli and C. upsaliensis)	✓	✓
Shigella/Enteroinvasive Escherichia coli (EIEC)	✓	✓
Enteropathogenic Escherichia coli (EPEC)	✓	Not reported
Enterotoxigenic Escherichia coli (ETEC) lt/st	✓	✓
Shiga-like toxin-producing Escherichia coli (STEC)stx1/stx2 (including specific identification of E. coliO157 serogroup within STEC)	✓	Not reported
Salmonella	✓	✓
Plesiomonas shigelloides	✓	✓
Yersinia enterocolitica	✓	✓
Cryptosporidium	✓	✓
Cyclospora cayetanensis	✓	✓
Entamoeba histolytica	✓	✓
Giardia lamblia*	✓	✓

Also known as Giardia intestinalis and Giardia duodenalis

Concomitant culture is necessary for organism recovery and further typing of bacterial agents.

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The QIAstat-Dx® Gastrointestinal Panel 2 is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness, in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule-out co-infection with organisms not detected by the QIAstat-Dx® Gastrointestinal Panel 2. The organisms detected may not be the sole or definitive cause of the disease.

Negative QIAstat-Dx® Gastrointestinal Panel 2 results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this assay test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Comparison of the QIAstat-Dx® Gastrointestinal Panel 2 and the Predicate Device

The QIAstat-Dx Gastrointestinal Panel 2 is substantially equivalent to the predicate device:

K140407: BioFire Diagnostics LLC FilmArray® Gastrointestinal (GI) Panel
Similarities and differences between the QIAstat-Dx Gastrointestinal Panel 2 and the predicate device are shown in Table 5.1.

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Table 5.1: Comparison of the QIAstat-Dx Gastrointestinal Panel 2 with the predicate device

Characteristic	Device	Predicate
Name	QIAstat-Dx Gastrointestinal Panel 2	BioFire Diagnostics, LLC.FilmArray Gastrointestinal (GI) Panel
510(k) No.	K220062	K140407
Regulation	21 CFR 866.3990	21 CFR 866.3990
Product Code	PCH	PCH
Device Class	Class II	Class II
Similarities
Intended Use	The QIAstat-Dx® Gastrointestinal Panel 2 is a multiplexed nucleic acid test intended for use with the QIAstat-Dx Analyzer 1.0 for the simultaneous qualitative detection and identification of nucleic acids from multiple viruses, bacteria, and parasites directly from preserved stool samples (Para-Pak® C&S or FecalSwabTM) obtained from individuals with signs and/or symptoms of gastrointestinal infection. The following viruses, bacteria (including several diarrheagenic E.coli/Shigella pathotypes), and parasites are identified with the QIAstat-Dx® Gastrointestinal Panel 2: Adenovirus F40/F41 Astrovirus Norovirus GI/GII Rotavirus A Campylobacter ( C. jejuni, C. coli and C. upsaliensis ) Shigella/Enteroinvasive Escherichia coli (EIEC) Enteropathogenic Escherichia coli (EPEC) (only with Para-Pak C&S, not reported for FecalSwab) Enterotoxigenic Escherichia coli (ETEC) lt/st	The FilmArray® Gastrointestinal (GI) Panel is a qualitative multiplexed nucleic acid-based test intended for use with the FilmArray® Instrument for the simultaneous detection and identification of nucleic acids from multiple bacteria, viruses, and parasites directly from stool samples in Cary Blair medium, obtained from individuals with signs and/or symptoms of gastrointestinal infection. The following bacteria (including several diarrheagenic E. coli/Shigella pathotypes), parasites, and viruses are identified using the FilmArray GI Panel: Campylobacter ( C. jejuni/C. coli/C. upsaliensis Clostridium difficile ( C. difficile ) toxin A/B Plesiomonas shigelloides Salmonella Vibrio (V> parahaemolyticus/V. vulnificus/ V. cholerae) including specific identification of Vibrio cholerae Yersinia enterocolitica
Characteristic	Device	Predicate
	Shiga-like toxin-producingEscherichia coli (STEC) stx1/stx2 (including specific identification of E. coli O157 serogroup within STEC) (only with Para-Pak C&S, not reported for FecalSwab) Salmonella Plesiomonas shigelloides Yersinia enterocolitica Cryptosporidium Cyclospora cayetanensis Entamoeba histolytica Giardia lamblia (Also known as Giardia intestinalis and Giardia duodenalis ) Concomitant culture is necessary for organism recovery and further typing of bacterial agents.The QIAstat-Dx® Gastrointestinal Panel 2 is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule-out co-infection with organisms not detected by the QIAstat-Dx® Gastrointestinal Panel 2. The organisms detected may not be the sole or definitive cause of the disease.Negative QIAstat-Dx® Gastrointestinal Panel 2 results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this assay test or non-infectious causes such as ulcerative colitis,	Enteroaggregative Escherichia coli (EAEC) Enteropathogenic Escherichia coli (EPEC) Enterotoxigenic Escherichia coli (ETEC) lt/st Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2 (including specific identification of the E. coli O157 serogroup within STEC) Shigella/Enteroinvasive Escherichia coli (EIEC) Cryptosporidium Cyclospora cayetanensis Entamoeba histolytica Giardia lamblia (also known as G. intestinalis and G. duodenalis ) Adenovirus F 40/41 Astrovirus Norovirus GI/GII Rotavirus A Sapovirus (Genogroups I, II, IV and V) The FilmArray® Gastrointestinal Panel is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness and results are meant to be used in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule out co-infection with organisms not included in the FilmArray® GI Panel. The agent detected may not be the definite cause of the disease.
Characteristic	Device	Predicate
	irritable bowel syndrome, orCrohn's disease.	Concomitant culture isnecessary for organismrecovery and further typing ofbacterial agents.
		This device is not intended tomonitor or guide treatment forC. difficile infection.
		Due to the small number ofpositive specimens collectedfor certain organisms duringthe prospective clinical study,performance characteristics forE. coli O157, Plesiomonasshigelloides, Yersiniaenterocolitica, Astrovirus , andRotavirus A were establishedprimarily with retrospectiveclinical specimens.
		Performance characteristics forEntamoeba histolytica , andVibrio (V parahaemolyticus, V.vulnificus, and Vibriocholerae) were establishedprimarily using contrivedclinical specimens.
		Negative FilmArray GI Panelresults in the setting of clinicalillness compatible withgastroenteritis may be due toinfection by pathogens that arenot detected by this test or non-infectious causes such asulcerative colitis, irritablebowel syndrome, or Crohn'sdisease.
		A gastrointestinalmicroorganism multiplexnucleic acid-based assay alsoaids in the detection and
Characteristic	Device	Predicate
		identification of acutegastroenteritis in the context ofoutbreaks.
Specimen Type	Preserved stool in Para-Pak C&Sor FecalSwab transport media	Stool in Cary-Blair Medium
Analyte Detected	RNA/DNA	RNA/DNA
Organism Detected	See above	See above
Amplification andDetectionTechnology	PCR	PCR
Assay Controls	One internal control in eachcartridge to control for sampleprocessing that is subjected to allnucleic acid extraction andamplification steps similar topatient samples. Labelingrecommends use of negative andpositive external controlsregularly. Use transport media asthe external Negative Control,and previously characterizedpositive samples or negativesample spiked with wellcharacterized target organisms asexternal positive controls.	Two controls are included ineach reagent pouch to controlfor sample processing and bothstages of PCR and meltanalysis. Labelingrecommends the use ofexternal positive and negativecontrols regularly. Enterictransport media can be used asan external negative control,and previously characterizedpositive samples or negativesamples spiked with wellcharacterized organisms asexternal positive controls.
Differences
Assay Targets	No additional targets to thoselisted above which are incommon with the predicatedevice.	The FilmArray®Gastrointestinal Panel has fouradditional targets: a genericVibrio target whichdifferentiates Vibrio cholerae ,Clostridium difficile ( C. difficile ) toxin A/B,Enteroaggregative Escherichia coli (EAEC) and Sapovirus.
	Nucleic AcidExtraction	Extraction of nucleic acids usingsilica membrane	Extraction of nucleic acidsusing magnetic beads
	Characteristic	Device	Predicate
	Technology	QIAstat-Dx GastrointestinalPanel 2 detection of amplifiedtargets uses an increase influorescence due to specific probebinding to generate the assayresults.	The FilmArray®Gastrointestinal Panel usesmelting curve analysis toconfirm the identity ofamplified targets to generateassay results
	Operational	The sample is loaded straight intothe cartridge.	The FilmArray®Gastrointestinal Panel has torehydrate the cartridge beforeuse
Amplification andDetection InstrumentSystem	QIAstat-Dx Analyzer 1.0	FilmArray Instrument

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Performance Characteristics - Non-Clinical Studies

Sensitivity (Limit of Detection)

The Analytical Sensitivity, or Limit of Detection (LoD), is defined as the lowest concentration at which >95% of the tested samples generate a positive call.

The LoD for each of the QIAstat-Dx Gastrointestinal Panel 2 target pathogenic organisms was assessed, using in total 36 pathogens strains, by analyzing serial dilutions of analytical samples prepared from culture isolates from commercial suppliers (e.g., ZeptoMetrix® and ATCC®) or clinical samples positive for target analytes commercially unavailable. Each sample tested was prepared in human stool matrix, which consists of a pool of previously tested negative clinical stool specimens resuspended in Para-Pak C&S (Meridian Biosciences) transport medium following the manufacturer's instructions collection device. A matrix equivalency study between Para-Pak C&S and FecalSwab transport media was conducted to support the conclusions in the section, except for EPEC, STEC stx1/stx2 and STEC O157 that are only applicable for Para-Pak C&S resuspended samples.

Individual LoD values for each QIAstat-Dx Gastrointestinal Panel 2 target is shown in Table 5.2.

Table 5.2: LoD concentration details for the 36 pathogen strains tested with QIAstat-Dx Gastrointestinal Panel 2

QIAstat-DxGastrointestinalPanel 2 Target	PathogenStrain	Supplier &Catalog ID	Dilutionfromthestock	DetectionRate	Concentration(molecularunits) #	Concentration(microbiologicalunits)
Campylobacter	Campylobacter coli 76-GA2[LMG 21266]	ATCC43478	1.00E-06	20/20	5802copies/mL	1.2 CFU/mL
QIAstat-DxGastrointestinalPanel 2 Target	PathogenStrain	Supplier &Catalog ID	Dilutionfromthestock	DetectionRate	Concentration(molecularunits) #	Concentration(microbiologicalunits)
	Campylobacter coli CIP7080	ATCC33559	1.00E-05	20/20	8941copies/mL	0.6 CFU/mL
	Campylobacter jejuni Z086	ZeptoMetrix0801650	1.00E-06	20/20	14491copies/mL	1660 CFU/mL
	Campylobacter jejuni subsp.JejuniRM3193	ATCCBAA-1234	1.00E-06	19/20	7210copies/mL	110 CFU/mL
	Campylobacter upsaliensisNCTC 11541	ZeptoMetrix0801999	3.16E-06	20/20	56165copies/mL	2259.4 CFU/mL
	Campylobacter upsaliensisRM3195	ATCCBAA-1059	1.00E-06	19/20	7631copies/mL	35 CFU/mL
Plesiomonas	Bader	ATCC14029	3.16E-07	19/20	116copies/mL	2.7 CFU/mL
shigelloides	Z130	ZeptoMetrix0801899	3.16E-07	20/20	481copies/mL	2291 CFU/mL
Salmonella	SalmonellaentericaSerovarTyphimuriumZ005	ZeptoMetrix0801437	3.16E-07	20/20	1441copies/mL	4518.8 CFU/mL
	SalmonellaentericaSerovarcholeraseus	ATCC13312	3.16E-07	20/20	647copies/mL	91.6 CFU/mL
Yersiniaenterocolitica	subsp.enterocoliticaNTCC 11175,Biotype 4,serotype 3	ATCC700822	3.16E-07	20/20	2496copies/mL	120.1 CFU/mL
	Z036	ZeptoMetrix0801734	1.00E-07	20/20	719copies/mL	2070 CFU/mL
	Escherichiacoli O111:NM(EPEC)	ZeptoMetrix0801747	3.16E-07	20/20	1817copies/mL	2581.7 CFU/mL
EnteropathogenicE. coli (EPEC)*	Escherichiacoli 7.1493;EPEC;084:H28	ZeptoMetrix0801938	1.00E-06	20/20	29021copies/mL	1190 CFU/mL
EnterotoxigenicE. coli (ETEC)lt/st	Escherichiacoli ETEC;ST+, LT+	ZeptoMetrix0801624	1.00E-07	20/20	855copies/mL	567 CFU/mL
	Escherichiacoli H10407,078:H11	ATCC35401	3.16E-08	19/20	367copies/mL	10.1 CFU/mL
QIAstat-DxGastrointestinalPanel 2 Target	PathogenStrain	Supplier &Catalog ID	Dilutionfromthestock	DetectionRate	Concentration(molecularunits) #	Concentration(microbiologicalunits)
Shigella/EnteroinvasiveE. coli (EIEC)	Escherichiacoli CDC EDL1282,029:NM	ATCC43892	1.00E-08	20/20	1431copies/mL	41.3 CFU/mL
	Shigellasonnei NCDC1120-66	ATCC25931	3.16E-08	20/20	488copies/mL	0.2 CFU/mL
Shiga-like toxin-producingE.coli (STEC)stx1/stx2*	Escherichiacoli O26:H4	ZeptoMetrix0801748	3.16E-07	STECstx1/stx2:20/20	2012copies/mL	726.8 CFU/mL
Shiga-like toxin-producing E.coli (STEC)0157*	Escherichiacoli O157:H7;EDL933	ZeptoMetrix0801622	3.16E-07	STECstx1/stx2:19/200157:19/20	1217copies/mL	2281.5 CFU/mL
Cryptosporidium	Cryptosporidium parvum —Iowa isolate	Waterborne® P102C	1.00E-05	20/20	661copies/mL	n/a
	Cryptosporidium hominis	PublicHealthWales UKM84	3.16E-03	20/20	357copies/mL	n/a
Cyclosporacayetanensis	N/A	LACNY -ClinicalsampleLAC2825	1.00E-04	19/20	53 copies/mL	n/a
	N/A	LACNYClinicalsampleLAC2827	3.16E-04	20/20	137copies/mL	n/a
Entamoebahistolytica	HM-1:IMSS(Mexico City1967)	ATCC30459	1.00E-06	20/20	7 copies/mL	0.2 cells/mL
	HK-9 (Korea)	ATCC30015	1.00E-07	19/20	1 copy/mL	0.13 cells/mL
Giardia lamblia	WB(Bethesda)	ATCC30957	3.16E-05	19/20	11850copies/mL	790 cells/mL
	Portland-1	ATCC30888	1.00E-04	20/20	14500copies/mL	635 cells/mL
AdenovirusF40/F41	Type 40(Dugan)	ZeptoMetrix0810084CF	1.00E-06	20/20	11726copies/mL	0.1 TCID50/mL
	Type 41 (Tak)	ZeptoMetrix0810085CF	1.00E-07	19/20	979copies/mL	0.05 TCID50/mL
Astrovirus	ERE IID 2371(type 8)	Zeptometrix0810277CF	1.00E-04	20/20	11586371copies/mL	11.7 TCID50/mL
	ERE IID 2868(type 4)	Zeptometrix0810276CF	3.16E-06	19/20	52184copies/mL	1.3 TCID50/mL
QIAstat-DxGastrointestinalPanel 2 Target	PathogenStrain	Supplier &Catalog ID	Dilutionfromthestock	DetectionRate	Concentration(molecularunits) #	Concentration(microbiologicalunits)
NorovirusGI/GII	GI.1(recombinant)	ZeptoMetrix0810086CF	3.16E-05	19/20	24629copies/mL	891.1 TCID50/mL
	GII.4(recombinant)	ZeptoMetrix0810087CF	1.00E-05	20/20	8998copies/mL	10.5 TCID50/mL
Rotavirus A	Wa	ZeptoMetrix0810041CF	1.00E-04	19/20	5201copies/mL	14.1 TCID50/mL
	69M	ZeptoMetrix0810280CF	3.16E-05	19/20	5787copies/mL	436.1 TCID50/mL

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Molecular unit titers were determined using in-house developed and validated qPCR assays.

Target applicable for Para-Pak C&S samples only.

Analytical Reactivity (Inclusivity)

Analytical Reactivity (Inclusivity) was evaluated with gastrointestinal pathogen isolates/strains that were selected based on clinical relevance and genetic, temporal and geographical diversity. Based on in vitro (wet) testing and in silico analysis, the QIAstat-Dx Gastrointestinal Panel 2 primers and probes are specific and inclusive for clinically prevalent and relevant strains for each pathogen tested.

In vitro testing

QIAstat-Dx Gastrointestinal Panel 2 is inclusive for 100% (114 out of 114) of the pathogen strains tested in vitro. Most pathogen strains evaluated in laboratory testing were detected at ≤ 3-fold (104/114) of the corresponding LoD reference strain which is written in bold (refer to Table 5.3 to Error! Reference source not found.). Less than 100% detection was observed for one strain each of ETEC, EIEC/Shigella and Rotavirus and two strains each of STEC (one STEC O157), Adenovirus and Norovirus at 3x LoD. Testing of these strains at 10x LoD generated the expected positive resulted for all replicates.

Table 5.3: Inclusivity test results for Campylobacter strains

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QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
	Campylobacter coli	76-GA2[LMG21266]	ATCC	43478*	1xLoD
	Campylobacter coli	Z293	ZeptoMetrix	804272	1xLoD
Campylobacter	Campylobacter coli	CIP 7080[1407, CIP70.80]	ATCC	33559*	3xLoD
	Campylobacter jejuni	Z086	ZeptoMetrix	0801650*	1xLoD
	Campylobacter jejuni	subsp. jejuniRM3193	ATCC	BAA-1234*	0.1xLoD
	Campylobacter jejunisubsp. jejuni	O:19 HL7;D3180	ATCC	BAA-218	0.1xLoD
	Campylobacter jejunisubsp. jejuni	AS-83-79	ATCC	33291	0.1xLoD
	Campylobacter jejunisubsp. doylei	NCTC11951	ATCC	49349	0.1xLoD
	Campylobacterupsaliensis	NCTC11541	ZeptoMetrix	0801999*	1xLoD
	Campylobacterupsaliensis	RM 3195(1994)	ATCC	BAA-1059*	0.3xLoD
	Campylobacterupsaliensis	NCTC11541[C2311	ATCC	43954	1xLoD

QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
Plesiomonasshigelloides	Plesiomonasshigelloides	Z130	ZeptoMetrix	0801899*	1xLoD
	Plesiomonasshigelloides	GNI 14	ATCC	51903	1xLoD
	Plesiomonasshigelloides	CDC 3085-55 [BaderM51, NCIB9242, NCTC10360, RH798]	ATCC	14029*	0.3xLoD

				Table 5.4: Inclusivity test results for Plesiomonas shigelloides strains
--	--	--	--	--------------------------------------------------------------------------	--	--	--

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QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
Salmonella	Salmonella enterica	SerovarTyphimurium Z005	ZeptoMetrix	0801437*	1xLoD
	Salmonella enterica	Subsp.Enterica,serovarBareilly	NCTC	NC05745	1xLoD
	Salmonella enterica	Subsp.Enterica,serovartyphi, Z152	ZeptoMetrix	0801933	0.1xLoD
	Salmonella enterica	Subsp.Enterica,serovarEnteridis,CDC K-1891[ATCC25928]	ATCC	13076	0.1xLoD
	Salmonella enterica	Subsp.Enterica,serovarInfantis,MZ1479[SARB27]	ATCC	BAA-1675	0.1xLoD
	Salmonella enterica	Subsp.Enterica,serovarMontevideo,G4639	ATCC	BAA-710	0.1xLoD
	Salmonella enterica	Subsp.Enterica,serovarJaviana	NCTC	NC06495	0.1xLoD
	Salmonella enterica	Subsp.Enterica,serovarThompson	NCTC	NC08496	0.1xLoD
	Salmonella enterica	Subsp.Enterica,	ATCC	9712	0.1xLoD
QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
		serovarSaintpaul
	Salmonella enterica	Subsp.Enterica,serovar Berta	NCTC	NC05770	0.1xLoD
	Salmonella enterica	Subps.Salame, IINCTC10310[JT945,SS140/61]	ATCC	700151	0.1xLoD
	Salmonella enterica	Subps.diarizonaeIIIb, 62	ATCC	29934	0.1xLoD
	Salmonella enterica	Subps.houtenae IV,CIP 82.32[264.66]	ATCC	43974	0.1xLoD
	Salmonella enterica	Subps.Indica VI,CIP 102501[F.Kauffmann1240]	ATCC	43976	0.1xLoD
	Salmonella enterica	Subsp.Enterica,serovarAgona, CDC873 [CDC1111-61]	ATCC	51957	0.1xLoD
	Salmonella enterica	Subsp.Enterica,serovarMuenchen,54	ATCC	8388	0.1xLoD
	Salmonella enterica	Subsp.Enterica,serovarOranienburg,E1093	ATCC	9239	0.1xLoD
	Salmonella enterica	Subsp.Enterica,	ATCC	51962	0.1xLoD
QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
		serovarParatyphi Bvar. Java,CDC 5
	Salmonella bongori	CIP 82.33[1224.72]	ATCC	43975	0.3xLoD
	Salmonella enterica	Subsp.Enterica,serovarCholeraesius,NCTC 5735[1348, K.34]	ATCC	13312*	0.3xLoD
	Salmonella enterica	Subsp.Enterica,serovarNewport,C487-69	ATCC	27869	0.3xLoD
	Salmonella enterica	Subsp.Enterica, 4,5, 12:7:-,serovarTyphimuriumm	NCTC	NC13952	0.3xLoD
	Salmonella enterica	Subsp.Enterica,serovarBraenderup	ATCC	700136	0.3xLoD
	Salmonella enterica	Subsp.Enterica,serovarAnatum	NCTC	NC05779	0.3xLoD
	Salmonella enterica	Subps.arizonae IIIa,NCTC 7311[CDAI 426]	ATCC	700156	0.3xLoD
	Salmonella enterica	Subsp.Enterica,serovarHeidelberg,[16]	ATCC	8326	0.3xLoD
	Salmonella enterica	Subsp.Enterica,	ATCC	BAA-2739	0.3xLoD
QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
		serovarMississippi,CDC 2012K-0487

Table 5.5: Inclusivity test results for Salmonella strains

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Table 5.6: Inclusivity test results for Yersinia enterocolitica strains

QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	Catalog ID	x-foldLoDdetected
Yersiniaenterocolitica	Yersiniaenterocolitica	Z036	ZeptoMetrix	0801734*	1x LoD
	Yersiniaenterocolitica	NTCC 11175,Biotype 4,serotype 3(0:3)	ATCC	700822*	1x LoD
	Yersiniaenterocolitica	33114 [CCUG11291, CCUG12369, CIP80.27, DSM4780, LMG7899, NCTC12982], Biovar1, 0:8	ATCC	9610	1x LoD
	Yersiniaenterocolitica	0:9	ATCC	55075	3x LoD

Strain tested during LoD verification study

Table 5.7: Inclusivity test results for Enteropathogenic E. coli (EPEC) strains. Only applicable for Para-Pak C&S samples.

QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
Enteropathogenic E.coli (EPEC)	Enteropathogenic E. coli(EPEC)	O111:NM	ZeptoMetrix	0801747*	1xLoD
	Enteropathogenic E. coli(EPEC)	7.1493,O84:H28	ZeptoMetrix	0801938*	1xLoD
	Enteropathogenic E. coli(EPEC)	StokeW,O111:K58(B4):H-	ATCC	33780	1xLoD

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Table 5.8: Inclusivity test results for Enterotoxigenic E. coli (ETEC) strains
QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
Enterotoxigenic E.coli (ETEC) lt/st	Enterotoxigenic E. coli(ETEC) lt/st	ST+, LT+	ZeptoMetrix	0801624*	1x LoD
	Enterotoxigenic E. coli(ETEC) lt/st	H10407,O78:H11,LT(+) / ctxA11(+)	ATCC	35401*	0.3xLoD
	Enterotoxigenic E. coli(ETEC) lt/st	O27:H7,ST (+)/LT (-)	SSIDiagnostica	82173	0.1xLoD
	Enterotoxigenic E. coli(ETEC) lt/st	O115:H15,ST(+)/ LT (-)	SSIDiagnostica	82174	3x LoD
	Enterotoxigenic E. coli(ETEC) lt/st	O169:H-,ST (-) /LT (+)	SSIDiagnostica	82172	10xLoD#

Table 5.8: Inclusivity test results for Enterotoxigenic E. coli (ETEC) strains

Strain tested during LoD verification study

Testing at a lower concentration resulted in a detection rate of <100%.

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QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
	Enteroinvasive E. coli(EIEC)	CDC EDL1282,O29:NM	ATCC	43892*	1x LoD
	Enteroinvasive E. coli(EIEC)	O172:H-	SSIDiagnostica	82171	3x LoD
Enteroinvasive E.coli (EIEC)/Shigella	Shigella sonnei	NCDC 1120-66	ATCC	25931*	1x LoD
		Shigella boydii(Serogroup C)	Z131	ZeptoMetrix	0801900
		Shigella flexneri(Serogroup B)	AMC 43-G-68[EVL 82,M134]	ATCC	9199
		Shigella flexneri(Serogroup B)	Z046	ZeptoMetrix	0801757
		Shigella sonnei(Serogroup D)	WRAIR Ivirulent	ATCC	29930
		Shigella sonnei(Serogroup D)	Z004	ZeptoMetrix	0801627
		Shigella boydii(Serogroup C)	AMC 43-G-58[M44 (Type170)]	ATCC	9207

Table 5.9: Inclusivity test results for Enteroinvasive E. coli (EIEC)/Shigella strains
----------------------------------------------------------------------------------------	--	--	--	--	--	--

Testing at a lower concentration resulted in a detection rate of <100%.

{25}------------------------------------------------

(stx1/stx2-carrier strains). Only applicable for Para-Pak C&S samples.
QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	CatalogID	x-foldLoDdetected
Shiga-like toxinproducing E. coli(STEC) stx1/stx2	Shiga-like toxin producingE. coli (STEC) - stx1/stx2	O157:H7;EDL933	ZeptoMetrix	0801622*	1x LoD
	Shiga-like toxin producingE. coli (STEC) - stx1	O26:H4,stx1 (+)	ZeptoMetrix	0801748*	1x LoD
	Shiga-like toxin producingE. coli (STEC) - stx1/stx2	ReferenceATCC® 35150(EDL931),O157:H7,stx1 (+), stx2 (+)	Microbiologics	617	3x LoD
	Shiga-like toxin producingE. coli (STEC) - stx1	Reference CDC00-3039,O45:H2,unknown	Microbiologics	1098	1x LoD
	Shiga-like toxin producingE. coli (STEC) - stx1	O103:H2,stx1 (+)	SSI Diagnostica	82170	3x LoD
	Shiga-like toxin producingE. coli (STEC) - stx1/stx2	O22:H8,stx1c (+),stx2b (+)	SSI Diagnostica	91350	1x LoD
	Shiga-like toxin producingE. coli (STEC) - stx2	O92,O107:K+:H48,stx2d (+)	SSI Diagnostica	91352	10x LoD#
	Shiga-like toxin producingE. coli (STEC) - stx2	O101:K32:H-,stx2e (+)	SSI Diagnostica	91354	0.3x LoD
	Shiga-like toxin producingE. coli (STEC) - stx1/stx2	O128ac:H-,stx2f(+)	SSI Diagnostica	91355	10x LoD#
	Shiga-like toxin producingE. coli (STEC) - stx2	O26:H11,stx2a(+)	SSI Diagnostica	95211	1x LoD
	Shiga-like toxin producingE. coli (STEC) - stx1	O8 ,stx1d (+)	SSI Diagnostica	91349	1x LoD

Table 5.10: Inclusivity test results for Shiga-like toxin-producing E. coli (STEC) (str1/stx2-carrier strains) Only applicable for Para-Pak C&S samples

Strain tested during LoD verification study

Testing at a lower concentration resulted in a detection rate of <100%.

{26}------------------------------------------------

Table 5.11: Inclusivity test results for Shiga-like toxin producing E. coli (STEC)
O157 strains. Only applicable for Para-Pak C&S samples.

QIAstat-DxGastrointestinal Panel2 Target	Pathogen	Strain	Supplier	Catalog ID	x-foldLoDdetected
Shiga-like toxinproducing E. coli(STEC) O157	Shiga-like toxin producingE. coli (STEC) O157	O157:H7;EDL933	ZeptoMetrix	0801622*	1x LoD
	Shiga-like toxin producingE. coli (STEC) O157	O128ac:H-,stx2f(+)	SSIDiagnostica	91355#	10xLoD$
	Shiga-like toxin producingE. coli (STEC) O157	ReferenceATCC® 35150(EDL 931),O157:H7, stx1(+), stx2 (+)	Microbiologics	617	1x LoD

The E. coli strain 91355 from SSI Diagnostica is reported as following in its catalog: vtx2f+, eae+. However,

it was found to amplify for E. coli 0157 in both QIAstat-Dx and FilmArray device

STesting at a lower concentration resulted in a detection rate of <100%.

		Table 5.12: Inclusivity test results for Cryptosporidium strains

QIAstat-DxGastrointestinal Panel2 Target	Pathogen	Strain	Supplier	Catalog ID	x-foldLoDdetected
Cryptosporidium	Cryptosporidium parvum	Iowa isolate	Waterborne	P102C*	1xLoD
	Cryptosporidium hominis	n/a	PublicHealthWales	Clinicalsample; UKM84*	0.01xLoD
	Cryptosporidium parvum		ATCC	PRA-67DQ(isolatedgenomicDNA)	<0.01LoD
	Cryptosporidium meleagridis		PublicHealthWales	Clinicalsample;UKMEL 14	<0.01LoD

{27}------------------------------------------------

QIAstat-DxGastrointestinal Panel2 Target	Pathogen	Strain	Supplier	Catalog ID	x-foldLoDdetected
	Cyclosporacayetanensis	n/a	LACNY	Clinicalsample,LAC2825*	1xLoD
Cyclospora cayetanensis	Cyclosporacayetanensis	n/a	LACNY	Clinicalsample,LAC2827*	1xLoD
	Cyclosporacayetanensis	–	ATCC	PRA-3000SD	1xLoD

Table 5.13: Inclusivity test results for Cyclospora cayetanensis strains

Strain tested during LoD verification study

Table 5.14: Inclusivity test results for Entamoeba histolytica strains

QIAstat-DxGastrointestinal Panel 2Target	Pathogen	Strain	Supplier	Catalog ID	x-foldLoDdetected
Entamoeba histolytica	Entamoebahistolytica	HM-1:IMSS(Mexico City1967)	ATCC	30459*	1xLoD
	Entamoebahistolytica	HK-9 (Korea)	ATCC	30015*	1xLoD
	Entamoebahistolytica	–	HospitalValld'Hebrón	Clinicalsample; 1	1xLoD

Strain tested during LoD verification study

Table 5.15: Inclusivity test results for Giardia lamblia strains

QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	Catalog ID	x-foldLoDdetected
Giardia lamblia	Giardia lamblia	Portland -1(Portland, OR,1971)	ATCC	30888*	1x LoD
	Giardia lamblia	WB (Bethesda, MD,1979)	ATCC	30957*	1x LoD
	Giardia intestinalis	H3 isolate	Waterborne	P101	1x LoD

{28}------------------------------------------------

QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	Catalog ID	x-fold LoDdetected
Adenovirus F40/F41	Human Adenovirus F41	Tak	ZeptoMetrix	0810085CF*	1x LoD
Adenovirus F40/F41	Human Adenovirus F41	Tak (73-3544)	ATCC	VR-930	10x LoD#
	Human Adenovirus F40	Dugan [79-18025]	ATCC	VR-931	10x LoD#
	Human Adenovirus F 40	Dugan	ZeptoMetrix	0810084CF*	3x LoD

Table 5.16: Inclusivity test results for Adenovirus F40/F41 strains

Testing at a lower concentration resulted in a detection rate of <100%.

QIAstat-DxGastrointestinal Panel 2Target	Pathogen	Strain	Supplier	Catalog ID	x-foldLoDdetected
Astrovirus	Human Astrovirus	ERE IID 2371 (type 8)	ZeptoMetrix	0810277CF*	1x LoD
	Human Astrovirus	HAstV-1	Universitat de Barcelona	Clinical sample; 160521599	1x LoD
	Human Astrovirus	ERE IID 2868 (type 4)	ZeptoMetrix	0810276CF*	1x LoD
	Human Astrovirus	HAstV-3	Universitat de Barcelona	Clinical sample; 151601306	1x LoD

Table 5.17: Inclusivity test results for Astrovirus strains

{29}------------------------------------------------

QIAstat-DxGastrointestina1Panel 2 Target	Pathogen	Strain	Supplier	Catalog ID	x-foldLoDdetected
	Human NorovirusGenogroup 1	Recombinant GI.1	ZeptoMetrix	0810086CF*	1x LoD
	Human NorovirusGenogroup 1	–	IndianaUniversityHealth	Clinical sample:IU3156	1x LoD
	Human NorovirusGenogroup 1	–	IndianaUniversityHealth	Clinical sample:IU3220	1x LoD
	Human NorovirusGenogroup 1	–	TriCoreReferenceLaboratories	Clinical sample;TC4274	3x LoD
	Human NorovirusGenogroup 2	Recombinant GII.4	ZeptoMetrix	0810087CF*	1x LoD
NorovirusGI/GII	Human NorovirusGenogroup 2	GII.2	Hospital Valld'Hebrón	Clinical sample:198058327	1x LoD
	Human NorovirusGenogroup 2	GII.4	Universitat deBarcelona	Clinicalsample:N26.2TA	1x LoD
	Human NorovirusGenogroup 2	–	LACNY	Clinical sample:LAC2019	1x LoD
	Human NorovirusGenogroup 2	–	NationwideChildren'sHospital	Clinical sample:NWC6063	1x LoD
	Human NorovirusGenogroup 2	GII.6	QIAGENBarcelona(STAT-Dx)	Clinical sample:GI 12	3x LoD
	Human NorovirusGenogroup 2	–	LACNY	Clinical sample:LAC2133	10xLoD#
	Human NorovirusGenogroup 2	–	LACNY	Clinical sample:LAC2074	10xLoD#

		Table 5.18: Inclusivity test results for Norovirus GI/GII strains

Strain tested during LoD verification study

Strain tested during LoD verification study

Testing at a lower concentration resulted in a detection rate of <100%.

{30}------------------------------------------------

QIAs QIAstat-DxGastrointestinalPanel 2 Target	Pathogen	Strain	Supplier	Catalog ID	x-fold LoDdetected
Rotavirus A	HumanRotavirus A	69M	ZeptoMetrix	0810280CF*	1xLoD
	HumanRotavirus A	Wa,G1P1A[8]	ZeptoMetrix	0810041CF*	1xLoD
	HumanRotavirus A	DS-1,G2P1B[4]	ATCC	VR-2550	1xLoD
	HumanRotavirus A	Va70	ZeptoMetrix	0810281CF	1xLoD
	HumanRotavirus A	RRV	ZeptoMetrix	0810530CF	10xLoD#

Table 5.19: Inclusivity test results for Rotavirus A strains

Strain tested during LoD verification study

Testing at a lower concentration resulted in a detection rate of <100%.

In silico analysis

In silico analysis of potential reactivity showed that the following organisms (including species, subspecies, subtypes, serotypes or serovars) are predicted to be detected with the QIAstat-Dx Gastrointestinal Panel 2 (Table 5.20).

Table 5.20: Organisms with predicted reactivity based on in silico analysis

QIAstat-Dx GI Panel2 Target	Organisms with predicted reactivity
Bacteria
Campylobacter	Campylobacter coli*, Campylobacter jejuni, Campylobacter jejuni subsp. jejuni, Campylobacter jejuni subsp. doylei, Campylobacter upsaliensis
Salmonella	Salmonella bongori, Salmonella enterica subsp. salamae II (e.g. serovar 55:k:z39), Salmonella enterica subsp. arizonae IIIa (e.g. serovar 63:g:z51), Salmonella enterica subsp. diarizonae IIIb (e.g. serovar 47:1,v:z), Salmonella enterica subsp. houtenae IV (e.g. serovar 43:z4), Salmonella enterica subsp. indica VI. Salmonella enterica subsp. enterica (up to 92 different serovars including Agona, Anatum, Bareilly, Choleraesuis, Enteritidis, Heidelberg, Infantis, Kentucky, Montevideo, Newport, Paratyphi A, Senftenberg, Tennessee, Thompson, Typhi, Typhimurium, Weltevreden*)
Plesiomonasshigelloides	Plesiomonas shigelloides (e.g. strains NCTC10360, ATCC 14029T, R4605035)
Yersiniaenterocolitica	Yersinia enterocolitica, Yersinia enterocolitica subsp. palearctica, Yersinia enterocolitica subsp. enterocolitica
Enteroinvasive E. coli(EIEC)/Shigella	Enteroinvasive E. coli (EIEC), Escherichia coli sp., Shigella flexneri, Shigella dysenteriae, Shigella boydii, Shigella sonnei.
Enteropathogenic E.coli (EPEC) β	Enteropathogenic E. coli (EPEC) (e.g. including serotypes OUT: HND, OUT:H6, OUT:H34, OUT:H21,O55:H7, O119:HNM, O117)
Enterotoxigenic E. coli(ETEC) &	Enterotoxigenic E. coli (ETEC) (including H10407 and E24377A strains and serotypes O169:H41, O25:H42,O148:H28, O6:H16) carrier of: Heat-labile enterotoxin gene subtype LT-I and Heat-stable enterotoxin gene variant Sta, subtypes STp and STh
Shiga-like toxin-producing E. coli(STEC) - stx1/stx2 β	Shiga-like toxin-producing E. coli (STEC) including O157:H7 and O157:NM serotype and non-0157 serotypes (O111:NM, O111:H-, O26:H11, O145:NM, O145:H28, O45:H2, O26:H11, ONT:NM, O104:H4,O121:H19, O145:H34, O113:H21, ONT:H-, O128:H2, OUT:HNM, O124:HNM E. coli strains carrier of:

{31}------------------------------------------------

QIAstat-Dx GI Panel2 Target	Organisms with predicted reactivity
	stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, stx2g, stx2h, stx2i, stx2j, stx2k and stx2l.Other stx-carrying bacteria: Shigella sonnei, Shigella dysenteriae
Shiga-like toxin-producing E. coli(STEC) 0157 β	Escherichia coli O157 including: STEC O157:H7 strains (e.g. EDL933) and E. coli O157: non-H7 groupsincluding non-Shiga-toxigenic E. coli O157 bacteria (e.g. serotype O157:H45)
	Parasites
Cryptosporidium#	Common Cryptosporidium species involved in human disease: C. parvum, C. hominis.Less common Cryptosporidium species involved in human infections: C. meleagridis, C. felis , C. bovis, C.viatorum, C. ubiquitum, C. tyzzeri, C. cuniculus, Cryptosporidium sp. Chipmunk genotype I, C. canis*.Rare or non-human species: Cryptosporidium wrairi
Cyclosporacayetanensis	Cyclospora cayetanensis (including strains LG, CY9, NP20 and NP21) *
Entamoebahistolytica	Entamoeba histolytica (e.g. strains HM-1: IMSS, EHMfas1, HK-9)*
Giardia lamblia	Giardia lamblia (aka Giardia duodenalis, Giardia intestinalis)*
	Viruses
Adenovirus	Human Adenovirus F40/41
Astrovirus$	Human Astrovirus (including types 1, 2, 3, 4, 5, 6, 7, 8)
Norovirus GI/GII	Norovirus genogroup II genotypes: GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.8, GII.9, GII.10,GII.12, GII.13, GII.14, GII.16, GII.17, GII.20, GII.21, GII.22, GII.23, GII.24, GII.25, GII.26, GII.27,GII.NA1 and GII.NA2
	Norovirus genogroup I genotypes: GI.1, GI2, GI.3, GI.4, GI.5, GI.6, GI.7, GI.8, GI.9.
Rotavirus	Rotavirus A including genotypes: G1P[8], G2P[4], G3P[8], G4P[8], G9P[6], G9P[8], G12P[6] andG12P[8]*

Certain sequences are predicted to be deced sensitivity due to the presence of a small number of mismatches at crifical positions of the primer-probe design.

4The assay is not predicted to detect bacteria carier of Heat-labile entercoxin gene subtype LT-II and/or of Heat-stable entercotoxin gene variant Stb e.

*The assay is not predicted to detect other Cryptosporidium spp. less involved in human disease: C. andersoni and C. muris.

S The assay is not predicted to detect Human Astrovirus types MLB1-3 and VA1-5.

B Target only applicable for Para-Pak C&S samples.

Analytical Specificity (Cross-Reactivity and Exclusivity)

The analytical specificity study was carried out by in vitro testing and in silico analysis to assess the potential cross-reactivity of the QIAstat-Dx Gastrointestinal Panel 2. On-panel organisms were tested to assess the potential for intra-panel cross-reactivity and Off-panel organisms were tested to evaluate cross-reactivity with organisms not covered by the panel content. The On-panel and Off-panel organisms tested are shown in Table 5.21 and Table 5.22, respectively.

Samples were prepared by single spiking organisms into negative stool resuspended in ParaPak C&S media at the highest concentration possible based on the organism stock, preferably at 105 TCID50/ml for viral, 105 cells/ml for parasite targets and 106 CFU/ml for bacterial targets. The pathogens were tested in 3 replicates. There was no intra-panel or Off-panel cross-reactivity for all pathogens tested in vitro, except for two non-targeted

{32}------------------------------------------------

Campylobacter species (C. helveticus and C. lari) that cross-reacted with the Campylobacter assay oligonucleotides included in the QIAstat-Dx Gastrointestinal Panel 2.

Type	Pathogen
Bacteria	Campylobacter coli
	Campylobacter jejuni
	Campylobacter upsaliensis
	Escherichia coli (EPEC)*
	Escherichia coli (ETEC lt/st)
	Escherichia coli (STEC)*
	Plesiomonas shigelloides
	Salmonella enterica
	Shigella sonnei
	Yersinia enterocolitica
Parasites	Cryptosporidium parvum
	Cyclospora cayetanensis
	Entamoeba histolytica
	Giardia lamblia
Viruses	Adenovirus F41
	Astrovirus
	Norovirus GI
	Norovirus GII
	Rotavirus

Table 5.21: List of Analytical Specificity On-Panel Pathogens Tested

Target only applicable for Para-Pak C&S samples.

{33}------------------------------------------------

Table 5.22: List of Analytical Specificity Off-Panel Pathogens Tested

Table 5.22: List of Analytical Specificity Off-Panel Pathogens Tested
Type	Pathogen (Potential Cross-Reactant)
Bacteria	Abiotrophia defectiva
	Acinetobacter baumannii
	Aeromonas hydrophila
	Arcobacter cryaerophilus
	Bacillus subtilis
	Bifidobacterium bifidum
	Campylobacter fetus
	Campylobacter gracilis
	Campylobacter helveticus (Cross-reactive for Campylobacter target)
	Campylobacter hominis
	Campylobacter lari (Cross-reactive for Campylobacter target)
	Campylobacter mucosalis
	Campylobacter rectus
	Citrobacter freundii
	Clostridium difficile non-toxigenic
	Clostridium perfringens
	Clostridium septicum
	Clostridium tetani
	Corynebacterium genitalium
	Enterobacter aerogenes
	Enterobacter cloacae
	Enterococcus faecalis
	Enterococcus faecium
	Escherichia fergusonii
	Escherichia hermannii
	Escherichia vulneris
	Faecalibacterium prausnitzii
	Gardnerella vaginalis
	Haemophilus influenzae
	Hathewaya histolytica (Clostridium)
	Helicobacter pylori
	Klebsiella pneumoniae
	Lactobacillus casei
	Listeria monocytogenes
	Proteus mirabilis
	Proteus vulgaris
	Pseudomonas aeruginosa
	Staphylococcus aureus
	Staphylococcus aureus subsp. aureus
	Staphylococcus epidermidis
Streptococcus agalactiae
Streptococcus pyogenes
Type	Pathogen (Potential Cross-Reactant)
Fungi	Aspergillus fumigatus
	Candida albicans
	Saccharomyces boulardii
	Saccharomyces cerevisiae
Parasite	Babesia microti
	Blastocystis hominis
	Chlamydia trachomatis
	Giardia muris
	Toxoplasma gondii
	Trichomonas tenax
Viruses	Adenovirus C:2
	Adenovirus B:34
	Adenovirus B3
	Adenovirus E:4a
	Adenovirus serotype 1
	Adenovirus serotype 5
	Adenovirus serotype 8
	Bocavirus Type 1
	Coronavirus 229E
	Coxsackievirus B3
Cytomegalovirus
Enterovirus 6 (Echovirus)
Enterovirus 68
Herpes Simplex Virus Type 2
Rhinovirus 1A

{34}------------------------------------------------

In silico predictions of potential cross-reactions show that the following cross-reactions may occur when testing stool samples with the QIAstat-Dx Gastrointestinal Panel 2 (Table 5.23)

{35}------------------------------------------------

QIAstat-Dx GastrointestinalPanel 2 Target	DesignAbbreviation	Potential cross-reactiveorganisms
Enteropathogenic E. coli (EPEC) 7	EPEC_eae	Shigella boydii 1,2,3,Escherichia albertii 1,2
Campylobacter.	Cupsal_cpn60	Campylobacter lari 4,Campylobacter helveticus 4
Shiga-like toxin-producing E. coli(STEC) stx1/stx2 7	STEC_stx1	Shigella sonnei 1,3, Shigella dysenteriae 1,3
	STEC_stx2	Acinetobacter haemolyticus 1,5,Citrobacter freundi 1,5,Enterobacter cloacae 1,5Aeromonas caviae 1,5
	STEC_stx2f	Escherichia albertii 1,5
E. coli O157 7	O157	Non-STEC E. coli O157strains6

1Note that these potential cross-reactions identified by in-silico analysis reflects sequences which can be acquired between species by horizontal gene transfer.

2Rare or less common eae intimin carrier organisms

3On-panel target.

41n vitro testing of Campylobacter lari and Campylobacter helveticus strains at high concentration confirmed potential cross-reactivity of these Campylobacter species with the QIAstat-Dx Gastrointestinal Panel 2 assay. 5 Rare or less common Stx toxins producers

6 E. coli 0157 reported by the QIAstrointestinal Panel will only be called when there is a positive amplification for the E. coli (STEC) design according to the calling algorithm. An infrequent case of an E. coli (STEC)

and an E. coli 0157 co-infection they will not be differentiated from a single infection caused by an STEC 0157:H7 strain.

7 Target only applicable for Para-Pak C&S samples.

Interfering Substances

The effect of potentially interfering substances on the detectability of the QIAstat-Dx Gastrointestinal Panel 2 organisms was evaluated. Thirty-four (34) potentially interfering substances were spiked into the sample mixes at a level predicted to be above the concentration of the substance likely to be found in stool specimens. Each organism was tested at 3x LoD and testing was performed in triplicates. Endogenous substances such as human whole blood, human genomic DNA and several pathogens were tested alongside exogenous substances like antibiotics, other gastrointestinal-related medications and different technique-specific substances.

For the vast majority of substances tested, no inhibition was observed, with the exceptions of mucin, calcium carbonate, nonoxynol-9 and Rotavirus reassortants, that may cause inhibition at high concentration.

Mucin at 5% w/v was found to generate false positives results for the Yersinia target. These signals were investigated by testing the interfering substance with and FDA-cleared method and they were confirmed to be present in the endogenous substance.

{36}------------------------------------------------

Calcium carbonate was found to interfere with the detection of all the QIAstat-Dx Gastrointestinal Panel 2 targets at concentrations above 0.5% w/v.

Nonoxynol-9 was found to interfere with the detection of Entamoeba histolytica at concentrations above 0.02% v/v.

As predicted, Rotavirus reassortants WC3:2-5, R574(9) and W179-4,9 used in Rotavirus A vaccines generated positive results for Rotavirus A in the QIAstat-Dx Gastrointestinal Panel 2. Final concentrations without interference (i.e. no false positive results for Rotavirus) for WC3:2-5, R574(9) and W179-4,9 were 8.89x1055 TCID50/m1 and 1.10 PFU/ml, respectively (see Table 5.24 for other concentrations tested).

Results from the 34 interfering substances that could be present or introduced in a stool specimen are provided in Table 5.24.

				Table 5.24: Final highest concentrations per potentially interfering substance
without observable interference on the QIAstat-Dx Gastrointestinal Panel 2

Substance tested	Concentration tested	Result
Endogenous substances
Bovine and ovine bile	12% w/v	No Interference
Cholesterol	1.5% w/v	No Interference
Fatty acids (palmitic acid)	0.2% w/v	No Interference
Fatty acids (stearic acid)	0.4% w/v	No Interference
Human genomic DNA	20 µg/mL	No Interference
	10 µg/mL	No Interference
	5 µg/mL	No Interference
Human stool (overfill of Cary Blair vial)	300 mg/mL	No Interference
Human urine	50% v/v	No Interference
Human whole blood with Na Citrate	40% v/v	No Interference
Mucin from bovine submaxillary	5% w/v	Interference#
	2.5% w/v	No Interference
Triglycerides	5% w/v	No Interference
Exogenous substances
Bacitracin	250U/mL	No Interference
Bisacodyl	0.3% w/v	No Interference
	0.15% w/v	No Interference
Bismuth subsalicylate	0.35% w/v	No Interference
Calcium carbonate (TUMS® ExtraStrength 750)	5% w/v	Interference
Docusate sodium	0.5% w/v	No Interference
Docusate sodium	2.5% w/v	No Interference
Doxycycline hydrochloride	0.05% w/v	No Interference
Glycerin	50% v/v	No Interference
Hydrocortisone	0.5% w/v	No Interference
Loperamide hydrochloride	0.078% w/v	No Interference

{37}------------------------------------------------

Substance tested	Concentration tested	Result
Magnesium hydroxide	0.1% w/v	No Interference
Metronidazole	1.5% w/v	No Interference
Mineral oil	50% v/v	No Interference
Naproxen sodium	0.7% w/v	No Interference
Nonoxynol-9	1.2% v/v	Interference
	0.6% v/v	Interference
	0.3% v/v	Interference
	0.15% v/v	Interference
	0.075% v/v	Interference
	0.02% v/v	No Interference
Nystatin	10000 USP units/mL	No Interference
Phenylephrine hydrochloride	0.075% w/v	No Interference
Sodium phosphate	5% w/v	No Interference
Vaccine components
Rotavirus reassortant WC3:2-5, R574(9) -VR 2195	$8.89 \times 10^{-3}$ TCID50/mL	Interference
	$8.89 \times 10^{-4}$ TCID50/mL	Interference
	$8.89 \times 10^{-5}$ TCID50/mL	No Interference
Rotavirus reassortant WI79-4,9 - VR 2415	$1.10 \times 10^{2}$ pfu/mL	Interference
	$1.10 \times 10^{1}$ pfu/mL	Interference
	1.10 pfu/mL	No Interference
Technique-specific Substances, Transport Media
Bleach	0.5% v/v	No Interference
Ethanol	0.2% v/v	No Interference
Puritan Fecal Opti-Swab Collection &Transport System with Cary-BlairMedium*	100%	No Interference
PurSafe DNA/RNA Preservative*	100%	No Interference
Sigma Fecal Transwab*	1 swab/2mL Cary Blair	No Interference

*Performance not stablished for this transport media

This substance was tested by another FDA-cleared that also detected Yersinia positive signals.

{38}------------------------------------------------

Microbial Interference

A microbial interference study was conducted to assess the inhibitory effects of select nontarget organisms on the ability to detect the QIAstat-Dx Gastrointestinal Panel 2 targets. Clinically relevant and challenging concentrations of non-target organisms were individually mixed with negative clinical stool matrix with spiked targeted pathogens at 3x LoD. Testing was performed in triplicate. All combinations and replicates successfully detected all the QIAstat-Dx Gastrointestinal Panel 2 targets. See Table 5.25 for a list of the non-target organisms tested and the result summary.

Substance tested	Concentration tested	Result
Non-target microorganisms
Aeromonas hydrophila	1 x 106 units/mL	No Interference
Bacteroides vulgatus	1 x 106 units/mL	No Interference
Bifidobacterium bifidum	1 x 106 units/mL	No Interference
Enterovirus Specie D, Serotype EV-D68	1 x 105 units/mL	No Interference
Non-pathogenic E. coli	1 x 106 units/mL	No Interference
Helicobacter pylori	1 x 106 units/mL	No Interference
Saccharomyces cerevisiae (deposited as S.boulardii)	1 x 105 units/mL	No Interference

Table 5.25: Final highest concentration without observable inhibitory effect

Competitive Interference

Competitive interference was tested in a subset of pathogens. No interference was observed when evaluating competitive interference by target pathogens when two QIAstat-Dx Gastrointestinal Panel target pathogens were tested by spiking samples with one pathogen target at 3x LoD and one at 50x LoD. Results from the pathogen targets tested are provided in Table 5.26.

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Mix	Pathogen	Finalconcentration(molecularunits)*	Finalconcentrationtested xLoD	Detectionrate	Target detected
Norovirus50x -	NorovirusGI/GII	4.5E+05copies/mL	50x	3/3	Yes
Rotavirus3x	Rotavirus A	1.7E+04copies/mL	3x	3/3	Yes
Norovirus3x -	NorovirusGI/GII	2.7E+04copies/mL	3x	3/3	Yes
Rotavirus50x	Rotavirus A	2.9E+05copies/mL	50x	3/3	Yes
Giardia 50x	Giardialamblia	7.2 E+05copies/mL	50x	3/3	Yes
Adenovirus3x	AdenovirusF40/F41	2.9E+03copies/mL	3x	3/3	Yes

Table 5.26: QIAstat-Dx Gastrointestinal Panel 2 Detection Rate Results for Competitive Interference

Molecular unit titers were determined using in house developed and validated qPCR assays.

Carryover

A carryover study was performed to evaluate the potential occurrence of crosscontamination between consecutive runs when using the QIAstat-Dx Gastrointestinal Panel 2 on the QIAstat-Dx Analyzer 1.0.

Pathogen samples of stool sample matrix, with alternating high-positive (10-10% organism/mL) and negative samples, were conducted on two QIAstat-Dx Analyzer 1.0.

No carryover between samples was observed in the QIAstat-Dx Gastrointestinal Panel 2, demonstrating that the system design and recommended sample handling and testing practices are effective in preventing false positive results due to carryover or crosscontamination between samples.

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Sample Stability

Sample stability testing demonstrated that the OIAstat-Dx Gastrointestinal Panel 2 assay is capable of processing samples which are stored prior to analysis under conditions typically utilized for stool specimens according to the intended use.

The results of this study support the following recommendations for storage of stool resuspended in Para-Pak C&S and FecalSwab transport media before testing:

Room temperature up to 4 days at 15–25°C
Refrigerated up to 4 days at 2-8°C ●

Reproducibility

Reproducibility testing of contrived samples was performed at three test sites including one internal site (Site A) and two external sites (Site B and Site C). The study incorporated a range of potential variation introduced by sites, days, replicates, cartridge lots, operators, and QIAstat-Dx analyzers. For each site, testing was performed across 5 non-consecutive days with 6 replicates per day (leading to a total of 30 replicates per target, concentration and site), 4 OIAstat-Dx Analyzers (2 analyzers per operator and per site), and at least 2 operators on each testing day. A total of 5 sample mixes (two combined samples at 1x LoD and 3x LoD plus one negative sample) were prepared. For each mix, 6 replicates were tested and evaluated. shows the detection rate per target and concentration for each site of the Reproducibility study. In addition, data obtained at all three sites have been compiled to calculate the exact 2-sided 95% Confidence Interval by target and concentration.

Table 5.27 shows the detection rate per target and concentration for each site of the Reproducibility study. In addition, data obtained at all three sites have been compiled to calculate the exact 2-sided 95% Confidence Interval by target and concentration.

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Table 5.27: Detection rate per target and concentration for each site of the Reproducibility study and exact 2-sided 95% Confidence Interval by target and concentration

Pathogen Tested	ConcentrationTested	ExpectedResult	% Agreement with Expected Result			All Sites(95%ConfidenceInterval)
			Site A	Site B	Site C
Adenovirus F41ZeptoMetrix0810085CF	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	1x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	None	NotDetected	29/3096.67%	29/3096.67%	29/3096.67%	87/90a96.67%(90.98-98.9%)
CampylobacterZeptoMetrix0801650	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	1x LoD	Detected	30/30100%	30/30b100%	30/30100%	90/90100%(95.98 -100.00%)
	None	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
Pathogen Tested	Concentration Tested	Expected Result	% Agreement with Expected Result			All Sites (95% Confidence Interval)
			Site A	Site B	Site C
Escherichia coli EPECdZeptoMetrix 0801747	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 - 100.00%)
	1x LoD	Detected	30/30100%	29/3096.67%	30/30100%	89/9098.89%(93.96 – 99.97%)
	None	Not Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 - 100.00%)
Entamoeba histolyticaATCC 30459	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 - 100.00%)
	1x LoD	Detected	30/30100%	30/30100%	29/3096.67%	89/9098.89%(93.96 – 99.97%)
	None	Not Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 - 100.00%)
Giardia lambliabATCC 30888	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 - 100.00%)
	1x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 - 100.00%)
	None	Not Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 - 100.00%)
			% Agreement with Expected Result
Pathogen Tested	ConcentrationTested	ExpectedResult	Site A	Site B	Site C	All Sites(95%ConfidenceInterval)
Norovirus GIIZeptoMetrix0810087CF	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	1x LoD	Detected	29/3096.67%	30/30100%	30/30100%	89/9098.89%(93.96 -99.97%)
	None	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
Rotavirus AcZeptoMetrix0810280CF	3x LoD	Detected	29/3096.67%	29/3096.67%	30/30100%	88/9097.8%(92.20 -99.73%)
	1x LoD	Detected	23/3076.67%	26/3086.67%	12/12100%	61/7284.7%(74.31 -92.12%)
	None	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
Escherichia coli(STEC) O157:H7dZeptoMetrix0801622	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	1x LoD	Detected	30/30100%	30/30100%	29/3096.67%	89/9098.89%(93.96 -99.97%)
	None	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00% )
		% Agreement with Expected Result
Pathogen Tested	ConcentrationTested	ExpectedResult	Site A	Site B	Site C	All Sites(95%ConfidenceInterval)
Escherichia coli(STEC) stx1/stx2dZeptoMetrix0801622	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	1x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	None	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
SalmonellaentericaZeptoMetrix0801437	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	1x LoD	Detected	30/30100%	29/3096.67%	29/3096.67%	88/9097.78%(92.20 –99.73%)
	None	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 –100.00%)

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			% Agreement with Expected Result
Pathogen Tested	ConcentrationTested	ExpectedResult	Site A	Site B	Site C	All Sites(95%ConfidenceInterval)
YersiniaenterocoliticaZeptoMetrix0801734	3x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00% )
	1x LoD	Detected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)
	None	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%(95.98 -100.00%)

4 Three (3) Adenovirus F40/41 false positives were observed when testing negative sample. Retesting of the three samples resulted in the expected negative results.

b One (1) Giardia lamblia false positive was observed when testing a positive sample not containing the pathogen. Repeat testing of this sample resulted in the expected negative result.

6 The Reproducibility study was fully re-tested for Rotavirus A with a new sample set due to an unexpected number of false negatives for Rotavirus A at the 1x LoD concentration. This was observed during an interim data evaluation (61/72, 84.7%) and was attributed to the sample manufacture while unrelated to the study workflow variables (operator, lot, day, instrument and site). Test runs derived from Rotavirus A new sample set resulted in 90/90 (100%; 95.98-100% CI) for the 3x LoD and 89/90 (98.89%; 93.96-99.97% CI) for the 1x LoD. During this testing, one (1) Campylobacter false positive was observed. Retesting of this sample resulted in the expected negative result.

d Target only applicable for Para-Pak C&S samples.

During the reproducibility study, potential variation introduced by sites, days, replicates, cartridge lots, operators, and QIAstat-Dx analyzers was analyzed showing no significant contribution to variability (Standard Deviation and Coefficient of Variation values below 1 and 5%, respectively) caused by any of the assessed variables (Table 5.28).

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Table 5.28: Variability attributable to each variable according to ANOVA model for QIAstat-Dx Gastrointestinal Panel 2.

Pathogen	Concentration tested	Positive (n)	Negative (n)	Mean	Variance component (SD, %CV*)					Total**
					Between Site	Between Lot	Between Day	Between Operator	Between Instrument	Between Replicate
Adenovirus	1x LoD	90	0	34.15	(0.2979, 0.87%)	(0.1630, 0.48%)	(0.1135, 0.33%)	(0.0000, 0.00%)	(0.1883, 0.55%)	(0.6153, 1.80%)	(0.7067, 2.07%)
	3x LoD	90	0	32.14	(0.0000, 0.00%)	(0.0741, 0.23%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.3716, 1.16%)	(0.3766, 1.17%)
Campylobacter	1x LoD	90	0	32.64	(0.1289, 0.39%)	(0.0000, 0.00%)	(0.0200, 0.06%)	(0.0000, 0.00%)	(0.0661, 0.20%)	(0.4232, 1.30%)	(0.4411, 1.35%)
	3x LoD	90	0	31.00	(0.0397, 0.13%)	(0.1446, 0.47%)	(0.0806, 0.26%)	(0.0000, 0.00%)	(0.1488, 0.48%)	(0.3420, 1.10%)	(0.3855, 1.24%)
EPEC	1x LoD	89	1	34.24	(0.4422, 1.29%)	(0.2000, 0.58%)	(0.0000, 0.00%)	(0.1599, 0.47%)	(0.1044, 0.30%)	(0.6699, 1.96%)	(0.7927, 2.31%)
	3x LoD	90	0	32.49	(0.1053, 0.32%)	(0.1429, 0.44%)	(0.0000, 0.00%)	(0.2081, 0.64%)	(0.0000, 0.00%)	(0.5238, 1.61%)	(0.5710, 1.76%)
Entamoeba	1x LoD	89	1	33.01	(0.0000, 0.00%)	(0.1349, 0.41%)	(0.0000, 0.00%)	(0.1175, 0.36%)	(0.0000, 0.00%)	(0.3182, 0.96%)	(0.3456, 1.05%)
	3x LoD	90	0	31.34	(0.1826, 0.58%)	(0.1265, 0.40%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.0906, 0.29%)	(0.4000, 1.28%)	(0.4449, 1.42%)
Giardia	1x LoD	90	0	28.65	(0.2341, 0.82%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.1737, 0.61%)	(0.5386, 1.88%)	(0.5953, 2.08%)
	3x LoD	90	0	26.68	(0.0840, 0.31%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.1025, 0.38%)	(0.0857, 0.32%)	(0.3531, 1.32%)	(0.3798, 1.42%)
Norovirus GII	1x LoD	89	1	33.53	(0.1346, 0.40%)	(0.3090, 0.92%)	(0.1111, 0.33%)	(0.0000, 0.00%)	(0.0431, 0.13%)	(0.4102, 1.22%)	(0.5083, 1.52%)
	3x LoD	90	0	32.42	(0.1742, 0.54%)	(0.3405, 1.05%)	(0.2895, 0.89%)	(0.0632, 0.19%)	(0.1972, 0.61%)	(0.4966, 1.53%)	(0.6721, 2.07%)
Rotavirus ***	1x LoD	61	11	36.71	(0.0000, 0.00%)	(0.1487, 0.41%)	(0.0000, 0.00%)	(0.1824, 0.50%)	(0.0000, 0.00%)	(0.9172, 2.50%)	(0.9356, 2.55%)
	3x LoD	88	2	35.95	(0.3468, 0.96%)	(0.4463, 1.24%)	(0.1231, 0.34%)	(0.1295, 0.36%)	(0.1132, 0.31%)	(1.2073, 3.36%)	(1.3014, 3.62%)
STEC_0157	1x LoD	89	1	33.83	(0.2218, 0.66%)	(0.3083, 0.91%)	(0.0000, 0.00%)	(0.0204, 0.06%)	(0.0000, 0.00%)	(0.5455, 1.61%)	(0.6295, 1.86%)
	3x LoD	90	0	32.22	(0.2431, 0.75%)	(0.2146, 0.67%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.2051, 0.64%)	(0.4625, 1.44%)	(0.5633, 1.75%)
STEC_stx1	1x LoD	90	0	34.07	(0.2297, 0.67%)	(0.1214, 0.36%)	(0.1281, 0.38%)	(0.0000, 0.00%)	(0.1170, 0.34%)	(0.4206, 1.23%)	(0.4991, 1.46%)
	3x LoD	90	0	32.54	(0.2380, 0.73%)	(0.2222, 0.68%)	(0.0802, 0.25%)	(0.0000, 0.00%)	(0.2000, 0.61%)	(0.4919, 1.51%)	(0.5887, 1.81%)
STEC_stx2	1x LoD	90	0	34.11	(0.0620, 0.18%)	(0.0979, 0.29%)	(0.0310, 0.09%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.3987, 1.17%)	(0.4109, 1.20%)
	3x LoD	90	0	32.69	(0.1173, 0.36%)	(0.2599, 0.80%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.1316, 0.40%)	(0.4131, 1.26%)	(0.4821, 1.47%)
Salmonella	1x LoD	88	2	34.53	(0.2888, 0.84%)	(0.2631, 0.76%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.0000, 0.00%)	(0.4880, 1.41%)	(0.5857, 1.70%)
	3x LoD	90	0	33.44	(0.0279, 0.08%)	(0.2100, 0.63%)	(0.0000, 0.00%)	(0.1814, 0.54%)	(0.1177, 0.35%)	(0.4088, 1.22%)	(0.4715, 1.41%)
Yersinia	1x LoD	90	0	34.53	(0.1616, 0.47%)	(0.1688, 0.49%)	(0.0564, 0.16%)	(0.0000, 0.00%)	(0.1036, 0.30%)	(0.5172, 1.50%)	(0.5637, 1.63%)
	3x LoD	90	0	33.02	(0.0503, 0.15%)	(0.2129, 0.64%)	(0.0370, 0.11%)	(0.0000, 0.00%)	(0.0801, 0.24%)	(0.3434, 1.04%)	(0.3951, 1.20%)

SD is first figure presented, subsequent figure is CV (Coefficient of variation presented as a percentage (%)
** TOTAL means total variance observed across the study

*** Results in table correspond to the original sample set analyzed. Results from the new dataset were:

					Variance component (SD, %CV*)
Pathogen	Concentrationtested	Positive(n)	Negative(n)	Mean	BetweenSite	BetweenLot	BetweenDay	BetweenOperator	BetweenInstrument	BetweenReplicate	Total**
Rotavirus	1x LoD	89	1	34.86	(0.1706,0.49%)	(0.0000,0.00%)	(0.3458,0.99%)	(0.1207,0.35%)	(0.3224,0.92%)	(0.9814,2.82%)	(1.0891,3.12%)
	3x LoD	90	0	32.42	(0.1742,0.54%)	(0.3405,1.05%)	(0.2895,0.89%)	(0.0632,0.19%)	(0.1972,0.61%)	(0.4966,1.53%)	(0.6721,2.07%)

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Repeatability

A repeatability study was conducted on the OIAstat-Dx Analyzer 1.0 instruments using a set of samples composed of low-concentrated analytes spiked into stool matrix (3x LoD and 1x LoD) and negative stool samples. OIAstat-Dx Gastrointestinal Panel 2 detected pathogens included in the positive samples were Adenovirus, Campylobacter, Enteropathogenic E. coli (EPEC), Entamoeba histolytica, Giardia lamblia, Norovirus GII, Rotavirus, E. coli 0157, STEC stx1/stx2, Salmonella enterica, and Yersinia enterocolitica. Each sample was tested with the same instrument over 12 days. In total. 60 replicates of 1x LoD and 60 replicates of 3x LoD per each of the tested targets and 60 replicates of negative samples were run. Overall results showed a 93.33-100.00-% and 95.00-100.00% detection rate for 1x LoD and 3x LoD samples, respectively. Negative samples showed 100% of negative calls for all panel analytes.

Performance Characteristics - Clinical Studies

Expected values

In the prospective clinical performance evaluation of the QIAstat-Dx Gastrointestinal Panel 2, 1939 eligible specimens (stool preserved in Para-Pak C&S (Meridian Bioscience) or FecalSwab (COPAN)) were collected and tested at 13 clinical sites across 5 countries (4 sites in Europe and 9 sites in USA) from May 2021. The number and percentage of positive results as determined by the QIAstat-Dx Gastrointestinal Panel 2, stratified by age group, are presented in Table 5.29. Overall, the QIAstat-Dx Gastrointestinal Panel 2 detected at least 1 organism in in 17.4% (213/1222) and 23.8% (171/717) of the prospectively collected stool specimens in FecalSwab and Para-Pak C&S, respectively (Table 5.29).

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Table 5.29: Expected Values Summary by Age Group for the Prospective Clinical study as determined by the QIAstat-Dx Gastrointestinal Panel 2

Pathogen	Medium Brand	Overall	0-5 years	6-21 years	22-49 years	50+ years	NotReported
Viruses
Adenovirus F40/F41	FecalSwab	5 (0.4%)	3 (1.7%)	2 (1.7%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
	Para-Pak C&S	2 (0.3%)	1 (3.2%)	0 (0.0%)	0 (0.0%)	1 (0.2%)	0 (0.0%)
Astrovirus	FecalSwab	3 (0.2%)	3 (1.6%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
	Para-Pak C&S	6 (0.8%)	2 (6.5%)	0 (0.0%)	3 (1.4%)	1 (0.2%)	0 (0.0%)
Norovirus GI/GII	FecalSwab	43 (3.5%)	22 (12.1%)	1 (0.8%)	14 (4.8%)	6 (1.0%)	0 (0.0%)
	Para-Pak C&S	16 (2.3%)	3 (9.7%)	1 (2.8%)	3 (1.4%)	9 (2.2%)	0 (0.0%)
Rotavirus A	FecalSwab	23 (1.9%)	13 (7.1%)	2 (1.7%)	7 (2.4%)	1 (0.2%)	0 (0.0%)
	Para-Pak C&S	4 (0.6%)	2 (6.5%)	0 (0.0%)	0 (0.0%)	2 (0.5%)	0 (0.0%)
Bacteria
Campylobacter	FecalSwab	69 (5.6%)	25 (13.7%)	7 (5.8%)	17 (5.9%)	20 (3.2%)	0 (0.0%)
	Para-Pak C&S	30 (4.2%)	2 (6.5%)	0 (0.0%)	10 (4.7%)	18 (4.3%)	0 (0.0%)
Plesiomonas shigelloides	FecalSwab	2 (0.2%)	0 (0.0%)	0 (0.0%)	2 (0.7%)	0 (0.0%)	0 (0.0%)
	Para-Pak C&S	7 (1.0%)	1 (3.2%)	0 (0.0%)	4 (1.9%)	2 (0.5%)	0 (0.0%)
Salmonella	FecalSwab	14 (1.1%)	5 (2.7%)	4 (3.3%)	3 (1.0%)	2 (0.3%)	0 (0.0%)
	Para-Pak C&S	17 (2.4%)	4 (12.9%)	0 (0.0%)	3 (1.4%)	10 (2.4%)	0 (0.0%)
Yersinia enterocolitica	FecalSwab	22 (1.8%)	3 (1.6%)	2 (1.7%)	9 (3.1%)	8 (1.3%)	0 (0.0%)
	Para-Pak C&S	8 (1.1%)	0 (0.0%)	0 (0.0%)	4 (1.9%)	4 (1.0%)	0 (0.0%)
Diarrheagenic E. coli/Shigella
Enteropathogenic E. coli(EPEC)	Para-Pak C&S	56 (7.9%)	9 (29.0%)	2 (5.6%)	18 (8.4%)	27 (6.5%)	0 (0.0%)
Enterotoxigenic E. coli	FecalSwab	18 (1.5%)	2 (1.1%)	2 (1.7%)	11 (3.8%)	3 (0.5%)	0 (0.0%)
(ETEC) lt/st	Para-Pak C&S	17 (2.4%)	1 (3.2%)	0 (0.0%)	7 (3.3%)	9 (2.2%)	0 (0.0%)
Shiga-like toxin E. coli(STEC) stx1/stx2	Para-Pak C&S	9 (1.3%)	0 (0.0%)	0 (0.0%)	6 (2.8%)	3 (0.7%)	0 (0.0%)
E. coli 0157	Para-Pak C&S	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
Shigella/Enteroinvasive	FecalSwab	10 (0.8%)	1 (0.5%)	0 (0.0%)	6 (2.1%)	3 (0.5%)	0 (0.0%)
E. coli (EIEC)	Para-Pak C&S	3 (0.4%)	0 (0.0%)	0 (0.0%)	1 (0.5%)	2 (0.5%)	0 (0.0%)
Parasites
Cryptosporidium	FecalSwab	2 (0.2%)	0 (0.0%)	1 (0.8%)	1 (0.3%)	0 (0.0%)	0 (0.0%)
	Para-Pak C&S	7 (1.0%)	0 (0.0%)	1 (2.8%)	4 (1.9%)	2 (0.5%)	0 (0.0%)
Cyclospora cayetanensis	FecalSwab	3 (0.2%)	0 (0.0%)	1 (0.8%)	2 (0.7%)	0 (0.0%)	0 (0.0%)
	Para-Pak C&S	18 (2.5%)	0 (0.0%)	0 (0.0%)	6 (2.8%)	12 (2.9%)	0 (0.0%)
Giardia lamblia	FecalSwab	15 (1.2%)	3 (1.6%)	1 (0.8%)	7 (2.4%)	4 (0.6%)	0 (0.0%)
	Para-Pak C&S	1 (0.1%)	1 (3.2%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
Entamoeba histolytica	FecalSwab	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
	Para-Pak C&S	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)

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Clinical Performance

The clinical performance of QIAstat-Dx Gastrointestinal Panel 2 was established during a multi-center international prospective study conducted at thirteen clinical settings representative of different geographical areas within USA and Europe (9 sites in USA and 4 sites in Europe) between May and July 2021. All study sites were hospital-associated or independent clinical diagnostics laboratories that perform routine diagnostics of GI infections. Table 5.30 provides a summary of prospective specimen's distribution across all study sites.

	No. of prospective specimens
Site/Country	FecalSwab	Para-Pak C&S	Total
Germany	293	46	339
Denmark	293	0	293
Spain	247	0	247
France	63	0	63
USA site 1	0	186	186
USA site 2	0	43	43
USA site 3	282	0	282
USA site 4	0	177	177
USA site 5	44	0	44
USA site 6	0	39	39
USA site 7	0	0	0 a
USA site 8	0	131	131
USA site 9	0	95	95
Total	1222	717	1939

Table 5.30: Prospective Specimens Distribution Across the study sites

4 the specimens from this site (148) were excluded from the analysis because they were collected with another device different to Para-Pak C&S or FecalSwab

Table 5.31: provides all demographic information for the 1,939 specimens evaluated in the prospective study.

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Table 5.31: Demographic data for prospective evaluated specimens
Demographic data	FecalSwab	Para-Pak C&S
Gender
Female	628	442	22.8
Male	594	275	14.2
Age Group
0-5 years	182	31	1.6
6-21 years	121	38	2.0
22-49 years	290	215	11.1
50+ years	629	426	22.0
Not Reported	0	7	0.4
Patient population
Emergency room	46	29	1.5
Hospitalized	342	143	7.4
Immunocompromised	3	0	0.0
Outpatient	491	325	16.8
No information available	340	220	11.3
No. Days between Symptom Onset and QIAstat-Dx Testing
> 7 days	89	0	0.0
≤ 7 days	146	16	0.8
Not Reported	987	701	36.2
	32.4
	30.6
	9.4
	6.2
	15.0
	32.4
	0.0
	2.4
	17.6
	0.2
	25.3
	17.5
	4.6
	7.5
	50.9

Table 5 31. Demographic data for prospective evaluated specimens

The performance of the QIAstat-Dx Gastrointestinal Panel 2 was evaluated for each panel test result using one FDA-cleared test as comparator for the most analytes. A composite comparator consisting of either three independent FDA-cleared test methods or two independent FDA-cleared tests methods and two validated PCR assays followed by bidirectional sequencing was used for Norovirus GI/GII, ETEC, STEC and Giardia lamblia. (Table 5.32: ). The composite endpoint was determined as the majority of the three results.

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QIAstat-Dx GI Panel 2 Test Result	Comparator Method
Adenovirus F40/41
Astrovirus
Rotavirus A
Sapovirus
Campylobacter
Plesiomonas shigelloides
Salmonella	One FDA-cleared test method
Yersinia enterocolitica
E. coli O157 a
Enteropathogenic E. coli (EPEC) a
Shigella/Enteroinvasive E. coli (EIEC)
Cryptosporidium
Cyclospora cayetanensis
Entamoeba histolytica
Norovirus GI/GII	Composite of three FDA-cleared testmethods
Enterotoxigenic E. coli (ETEC) lt/st	Composite of three FDA-cleared testmethods
Shiga-like toxin- E. coli (STEC) stx1/stx2 a	Composite of three FDA-cleared testmethods
Giardia lamblia	Composite of two FDA-cleared test methodsand two validated PCR tests followed by bi-directional sequencing b

Table 5.32: OIAstat-Dx Gastrointestinal Panel 2 Clinical studies comparator method

a Targets evaluated in Para-Pak C&S specimens only.

b Each of the two PCR assays used were well-characterized and validated nucleic acid amplification tests (NAAT) followed by bi-directional sequencing analysis. Both assays were designed to amplify different sequences than those targeted by the OIAstat-Dx Gastrointestinal Panel 2. Positive results reguired to generate sequences from bi-directional sequencing with at least 200 bases of adequate quality that by BLAST analyses matched a sequence of the expected organism or gene from NCBI GenBank database with at least 95% query coverage and at least 95% identity compared to the reference.

Additional prospective archived samples were collected for Norovirus GI/GII (81 samples) and STEC (18 samples). These were prospectively collected samples from four different collection sites (3 US and 1 EU), where only those positive for the pathogen by standard of care method were archived for analysis alongside 20 negative specimens.

In addition, to supplement the results of the prospective clinical studies, a total of 750 preselected archived frozen (retrospective) specimens were also evaluated. These specimens served to increase the sample size for analytes that showed low prevalence in the clinical prospective study or that were less represented in a particular sample type (Para-Pak C&S or FecalSwab). The same Comparator Methods detailed in Table 5.31 were used as confirmatory testing for the presence of the nucleic acids from the expected.

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In total 2808 specimens (1939 prospective, 119 prospective archived and 750 retrospective) were evaluated in the clinical study. These specimens were collected using Para-Pak C&S (1217) or FecalSwab (1591).

The positive percentage agreement (PPA) and the negative percentage agreement (NPA) were calculated for the prospective and retrospective studies and for each sample type (Para-Pak C&S and FecalSwab) separately.

The PPA was calculated as 100% x (TP / (TP + FN)). True positive (TP) indicates that both the QIAstat-Dx Gastrointestinal Panel 2 and comparator method showed a positive result for this specific target, and false negative (FN) indicates that the OIAstat-Dx Gastrointestinal Panel 2 result was negative while the comparator method result was positive. The NPA was calculated as 100% x (TN / (TN + FP)). True negative (TN) indicates that both the QIAstat-Dx Gastrointestinal Panel 2 and the comparator method showed negative results, and a false positive (FP) indicates that the QIAstat-Dx Gastrointestinal Panel 2 result was positive, but the comparator method result was negative. The PPA and NPA exact binomial two-sided 95% confidence interval was calculated.

Where a composite comparator was used (Table 5.32), the result was determined as the majority of the three individual test results (i.e., a positive composite comparator result is based on positive results for at least two comparator tests and a negative composite comparator result is based on negative results for at least two comparator tests). If insufficient pathogen positive sample was available to obtain a majority test result a worstcase model was applied in the PPA calculation. In this model the PPA was calculated including all observed true positive and false negative samples between QIAstat-Dx and the composite comparator while for the samples where it was not possible to conduct testing with the complete comparator due to insufficient sample volume, the following was done:

· samples that were negative in QIAstat-Dx and positive for one comparator assay, negative (or insufficient volume) for a second comparator and insufficient volume for a third comparator were included in the calculations as worst-case false negatives,
samples that were positive in QIAstat-Dx and positive in one comparator test, negative o (or insufficient volume) for a second comparator and insufficient volume for the third comparator, were considered as worst-case false positives and therefore, excluded in the PPA calculations.

The results of the clinical performance of the prospective, prospective archived and retrospective studies are summarized in Tables: Table 5.34 and Table 5.35. respectively.

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Discrepancies between the QIAstat-Dx Gastrointestinal Panel 2 and the comparator methods were investigated for the analytes that the QIAstat-Dx Gastrointestinal Panel 2 test result was compared to one FDA-cleared method. Discrepancies analyses are footnoted on each clinical performance summary Table below (Table 5.33, and Table 5.35).

		Positive Percent Agreement			Negative Percent Agreement
Analyte	MediumBrand	TP/TP+FN	0/0	95% CI	TN/TN+FP	0/0	95% CI
			Viruses
Adenovirus F40/F41	FecalSwab	5/6 a	83.3	43.7-97.0	1214 / 1214	100.0	99.7-100.0
	Para-PakC&S	1 / 2 b	50.0	9.5-90.6	703 / 704 b	99.9	99.2-100.0
Astrovirus	FecalSwab	313	100.0	43.9-100.0	1219 / 1219	100.0	99.7-100.0
	Para-PakC&S	6 / 6	100.0	61.0-100.0	700 / 700	100.0	99.5-100.0
Norovirus GI/GII	FecalSwab	31 / 33 €	93.9	80.4-98.3	493 / 495 €	99.6	98.6-100.0
	Para-PakC&S	14 / 18 d	77.8	54.8-91.0	399 / 399 d	100.0	99.1-100.0
Rotavirus A	FecalSwab	21 / 23 €	91.3	73.2-97.6	1197 / 1199e	99.8	99.4-100.0
	Para-PakC&S	313	100.0	43.9-100.0	702 / 703 f	99.9	99.2-100.0
			Bacteria
Campylobacter	FecalSwab	65 / 67 8	97.0	89.8-99.2	1151 / 1155g	99.7	99.1-99.9
	Para-PakC&S	30 / 31 h	96.8	83.8-99.4	675 / 677 h	99.7	98.9-99.9
Plesiomonasshigelloides	FecalSwab	0 / 0	N/A	N/A	1220 / 1222	99.8	99.4-100.0
	Para-PakC&S	5/6 !	83.3	43.7-97.0	698 / 700 i	99.7	99.0-99.9
Salmonella	FecalSwab	14 / 16 k	87.5	64.0-96.5	1206 / 1206	100.0	99.7-100.0
	Para-PakC&S	19 / 201	95.0	76.4-99.1	688 / 688	100.0	99.4-100.0
Yersinia enterocolitica	FecalSwab	15 / 16 m	93.8	71.7-99.0	1199 / 1206m	99.4	98.8-99.7
	Para-PakC&S	313	100.0	43.9-100.0	698 / 703 "	99.3	98.4-99.7
				Diarrheagenic E. coli/Shigella
Enteropathogenic E.coli (EPEC)	Para-PakC&S	57 / 65	87.7	77.6-93.6	632 / 632	100.0	99.4-100.0
Enterotoxigenic E. coli	FecalSwab	9 / 100	90.0	59.6-99.2	427 / 430 °	99.3	98.0-99.8
(ETEC) lt/st	Para-PakC&S	9 / 10 P	90.0	59.6-99.2	390 / 395 P	98.7	97.1-99.5
Shiga-like toxin E. coli(STEC) stx1/stx2	Para-PakC&S	ર / વિત	83.3	43.6-97.0	397 / 400 9	99.3	97.8-99.7
E. coli 0157	Para-PakC&S	0 / 0	N/A	N/A	ર્ / 5	100.0	56.6-100.0
Shigella/Enteroinvasive	FecalSwab	10 / 10	100.0	72.3-100.0	1212 / 1212	100.0	99.7-100.0
E. coli (EIEC)	Para-PakC&S	2/2	100.0	34.2-100.0	703 / 704 ﺗ	99.9	99.2-100.0
			Parasites
Cryptosporidium	FecalSwab	2/4	50.0	15.0-85.0	1218 / 1218	100.0	99.7-100.0
	Para-PakC&S	6 / 6	100.0	61.0-100.0	699 / 700 ×	99.9	99.2-100.0
Cyclospora	FecalSwab	313	100.00	43.9-100.0	1219 / 1219	100.0	99.7-100.0
cayetanensis	Para-PakC&S	18 / 19 t	94.7	75.4-99.1	687 / 687	100.0	99.4-100.0
		Positive Percent Agreement			Negative Percent Agreement
Analyte	Medium Brand	TP/TP+FN	%	95% CI	TN/TN+FP	%	95% CI
Entamoeba histolytica	FecalSwab	0 / 0	N/A	N/A	1222 / 1222	100.0	99.7-100.0
	Para-Pak C&S	0 / 0	N/A	N/A	706 / 706	100.0	99.5-100.0
Giardia lamblia	FecalSwab	6 / 8 u	75.0	40.9-92.9	434 / 441 u	98.4	96.8-99.2
	Para-Pak C&S	1 / 1	100.0	20.7-100.0	406 / 406 v	100.0	99.1-100.0

Table 5.33: Clinical Performance in the Prospective study

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a Adenovirus F40/41 was not detected in the single false negative specimen (0/1) in FecalSwab using a different FDA-cleared test method

b Adenovirus F40/41 was not detected in the single false negative specimen (0/1) and in the single false positive specimen (0/1) in Para-Pak C&S using a different FDA-cleared test method

s ten (10) FecalSwab samples positive for Norovirus GI/GII in both QIAstat-Dx and one FDA-cleared comparator were excluded from the PPA calculations because the samples did not have sufficient volume for composite comparator testing. The sample size for NPA is smaller for Norovirus GVGII as only a portion of the samples with a negative result in Q1Astat-Dx and in one FDA-cleared comparator was tested with the complete composite comparator in the prospective study.

4 two (2) Para-Pak C&S samples positive for Norovirus GI/GII in both QIAstat-Dx and one FDA-cleared comparator were excluded from the PPA calculations because the samples did not have sufficient volume for comparator testing. One (1) Para-Pak C&S sample negative in QIAstat-Dx and positive with insufficient volume for complete composite comparator testing were classed as false negative in the sample size for NPA is smaller for Norovirus GI/GII as only a portion of the samples with a negative result in one FDA-cleared comparator was tested with the complete composite comparator in the prospective study.

® Rotavirus A was detected in one of the two false specimens (1/2) and was not detected in the two false positive specimens (0/2) in FecalSwab using a different FDA-cleared test method.

f Rotavirus A was not detected in the single specimen (0/1) in Para-Pak C&S using a different FDA-cleared test method. s Campylobacter was not detected in the two false negative specimens (0/2) and was detected in three of the four false positive specimens (3/4) in FecalSwab using a different FDA-cleared test method.

A Campylobacter was not detected in the single false negative specimens (0/1) and was detected in one of the two false positive specimens (1/2) in Para-Pak C&S using a different FDA-cleared test method.

i Plesiomonas shigelloides was not detected in the two false positive specimens (0/2) in FecalSwab using a different FDA-cleared test method

i Plesiomonas shigelloides was not detected in the single false negative specimen (0/1) and was not detected in the two false positive specimens (0/2) in Para-Pak C&S using a different FDA-cleared test method

Salmonella was not detected in the two false negative specimens (0/2) in FecalSwab using a different FDA-cleared test method. 1 Salmonella was not detected in the single specimen (0/1) in Para-Pak C&S using a different FDA-cleared test method. ™ Yersinia enterocolitica was not detected in the single specimen (0/1) and was not detected in the seven false positive specimens (0/7) in FecalSwab using a different FDA-cleared test method.

11 Yersinia enterocolitica was not detected in the five specimens (0/5) using a different FDA-cleared test method. 9 six (6) FecalSwab samples positive for ETEC in both QIAstat-Dx and the primary FDA-cleared comparator were excluded from the

PPA calculations because the samples did not have sufficient volume for comparator testing. The sample size for NPA is smaller for ETEC as only a portion of the samples with a negative result in QIAstat-Dx and in one FDA-cleared comparator was tested with the complete composite comparator in the prospective study.

P three (3) Para-Pak C&S samples positive for ETEC in both QIAstat-Dx and one FDA-cleared comparator were excluded from the PPA calculations because the samples did not have sufficient volume for comparator testing. The sample size for NPA is smaller for ETEC as only a portion of the samples with a negative result in QIAstat-Dx and in one FDA-cleared comparator was tested with the complete composite comparator in the prospective study.

9 one (1) FecalSwab sample positive for STEC in both OIAstat-Dx and one FDA-cleared comparator was excluded from the PPA calculations because the samples did not have sufficient volume for comparator testing. The sample size for NPA is smaller for STEC as only a portion of the samples with a negative result in QIAstat-Dx and in one FDA-cleared comparator was tested with the complete composite comparator in the prospective study.

™ Shigella/ EIEC was detected in the single specimen (1/1) in Para-Pak C&S using a different FDA-cleared test method. S Cryptosporidium was not detected in the single false positive specimen (0/1) in Para-Pak C&S using PCR followed by bi-directional sequence analysis.

t For Cyclospora cayetanensis there was one (1) false negative specimen in Para-Pak C&S that was not further investigated by discrepant analyses.

4 two (2) FecalSwab samples positive for Giardia lamblia in both Q1Astat-Dx and one FDA-cleared comparator were excluded from the PPA calculations because the samples did not have sufficient volume for complete comparator testing. Two (2) FecalSwab samples negative in Q1Astat-Dx and positive with one FDA-cleared comparator with insufficient volume for complete composite comparator testing were classed as false negative in the PPA calculations. The sample size for Giardia lamblia as only a portion of the samples with a negative result in one FDA-cleared comparator was tested with the complete composite comparator in the prospective study.

v the sample size for NPA is smaller for Giardia as only a portion of the samples with a negative result in QIAstat-Dr and in one FDA-cleared comparator was tested with the complete comparator in the prospective study.

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		Positive Percent Agreement		Negative Percent Agreement
Analyte	Medium Brand	TP/TP+FN	%	95% CI	TN/TN+FP	%	95% CI
NorovirusGI/GII	FecalSwab	48 / 49a	98.0	89.3 - 99.6	2 / 4a	50.0	15.0-85.0
GI/GII	Para-Pak C&S	26 / 28 a,b	92.9	77.4 - 98.0	37 / 38 a	97.4	86.5-99.5
Shiga-liketoxin E. coli(STEC)stx1/stx2	Para-Pak C&S	12 / 13 c,d	92.3	66.7 - 98.6	51 / 52 c	98.1	89.9-99.7

Table 5.34: Clinical Performance in the Prospective Archived study

4 For Norovirus GUGII/GII four out of the eighty-one (4/81) positive prospectively archived samples (positive by standard of care) were negative by the composite comparator and therefore included as negative samples in the NPA calculations.

b One (1) Para-Pak C&S sample negative in QIAstat-Dx and positive for Norovirus GI/GII with one FDA-cleared comparator with insufficient volume for complete comparator testing was classed as false negative in the PPA calculations.

· For STEC five out of the eighten (5/18) positived samples (positive by standard of care) were negative by the composite comparator and therefore included as negative samples in the NPA calculations.

4 One (1) Para-Pak C&S sample positive for STEC in both Q1Astat-Dx and one FDA-cleared comparator was excluded from the PPA calculations because the samples did not have sufficient volume for complete comparator testing.

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		Positive Percent Agreement			Negative Percent Agreement
	MediumBrand	TP/TP+FN	%	95% CI	TN/TN+FP	%	95% CI
Viruses
Adenovirus F40/F41	FecalSwab	23 / 26 a	88.5	71.0-96.0	203 / 203	100.0	98.2-100.0
	Para-PakC&S	29 / 29	100.0	88.3-100.0	39 / 39	100.0	91.0-100.0
Astrovirus	FecalSwab	2/3 b	66.7	20.8-93.9	191 / 191	100.0	98.0-100.0
	Para-PakC&S	0 / 0	N/A	N/A	14 / 14	100.0	78.5-100.0
Norovirus GI/GII	FecalSwab	28 / 32 c	87.5	71.9-95.0	74 / 75 c	98.7	92.8-100.0
	Para-PakC&S	27 / 29	93.1	78.0-98.1	86 / 86 d	100.0	95.8-100.0
Rotavirus A	FecalSwab	8/9e	88.9	56.5-98.0	185 / 185	100.0	98.0-100.0
	Para-PakC&S	2/2	100.0	34.2-100.0	12 / 12	100.0	75.8-100.0
				Bacteria				Campylobacter	FecalSwab	31 / 31	100.0	89.0-100.0	161 / 163 f	98.8	95.6-99.7	Para-PakC&S	3/3	100.0	43.9-100.0	11 / 11	100.0	74.1-100.0	Plesiomonasshigelloides	FecalSwab	2/2	100.0	34.2-100.0	192 / 192	100.0	98.0-100.0	Para-PakC&S	33 / 36 g	91.7	78.2-97.1	117 / 117	100.0	96.8-100.0	Salmonella	FecalSwab	30 / 31 h	96.8	83.8-99.4	161 / 163 h	98.8	95.6-99.7	Para-PakC&S	1 / 1	100.0	20.7-100.0	13 / 13	100.0	77.2-100.0	Yersinia enterocolitica	FecalSwab	32 / 34 i	94.1	80.9-98.4	160 / 160	100.0	97.7-100.0	Para-PakC&S	1 / 1	100.0	20.7-100.0	14 / 14	100.0	78.5-100.0	Diarrheagenic E. coli/Shigella	Enteropathogenic E.coli (EPEC)	Para-PakC&S	60 / 65 j	92.3	83.2-96.7	42 / 42	100.0	91.6-100.0	Enterotoxigenic E. coli(ETEC) lt/st	FecalSwab	22 / 24 k	91.7	74.2-97.7	85 / 86 k	98.8	93.7-99.8	Para-PakC&S	23 / 24	95.8	79.8-99.3	61 / 61 l	100.0	94.1-100.0	Shiga-like toxin E. coli(STEC) stx1/stx2	Para-PakC&S	60 / 64	93.8	85.0-97.5	44 / 44 m	100.0	92.0-100.0	E. coli 0157	Para-PakC&S	39 / 42 n	92.9%	80.1-99.4	16 / 16	100.0	80.6-100.0	Shigella/EnteroinvasiveE. coli (EIEC)	FecalSwab	22 / 24 o	91.7	73.0-99.0	170 / 170	100.0	97.8-100.0	Para-PakC&S	0 / 0	N/A	N/A	14 / 14	100.0	78.5-100.0	Parasites	Cryptosporidium	FecalSwab	6 / 6	100.0	54.1-100.0	186 / 188 p	98.9	96.2-99.9	Para-PakC&S	26 / 26	100.0	86.8-100.0	117 / 117	100.0	96.9-100.0	Cyclosporacayetanensis	FecalSwab	1 / 1	100.0	20.7-100.0	193 / 193	100.0	98.1-100.0	Para-PakC&S	1 / 1	100.0	20.7-100.0	13 / 13	100.0	77.2-100.0	Entamoeba histolytica	FecalSwab	0 / 0	N/A	N/A	194 / 194	100.0	98.1-100.0	Para-PakC&S	0 / 0	N/A	N/A	14 / 14	100.0	76.5-100.0	Giardia lamblia	FecalSwab	29 / 31 q	93.6	79.3-98.2	46 / 48 q	95.8	86.0-98.9	Para-PakC&S	27 / 28 r	96.4	82.3-99.4	92 / 92 r	100.0	96.0-100.0
				Bacteria
Campylobacter	FecalSwab	31 / 31	100.0	89.0-100.0	161 / 163 f	98.8	95.6-99.7
	Para-PakC&S	3/3	100.0	43.9-100.0	11 / 11	100.0	74.1-100.0
Plesiomonasshigelloides	FecalSwab	2/2	100.0	34.2-100.0	192 / 192	100.0	98.0-100.0
	Para-PakC&S	33 / 36 g	91.7	78.2-97.1	117 / 117	100.0	96.8-100.0
Salmonella	FecalSwab	30 / 31 h	96.8	83.8-99.4	161 / 163 h	98.8	95.6-99.7
	Para-PakC&S	1 / 1	100.0	20.7-100.0	13 / 13	100.0	77.2-100.0
Yersinia enterocolitica	FecalSwab	32 / 34 i	94.1	80.9-98.4	160 / 160	100.0	97.7-100.0
	Para-PakC&S	1 / 1	100.0	20.7-100.0	14 / 14	100.0	78.5-100.0
				Diarrheagenic E. coli/Shigella
Enteropathogenic E.coli (EPEC)	Para-PakC&S	60 / 65 j	92.3	83.2-96.7	42 / 42	100.0	91.6-100.0
Enterotoxigenic E. coli(ETEC) lt/st	FecalSwab	22 / 24 k	91.7	74.2-97.7	85 / 86 k	98.8	93.7-99.8
	Para-PakC&S	23 / 24	95.8	79.8-99.3	61 / 61 l	100.0	94.1-100.0
Shiga-like toxin E. coli(STEC) stx1/stx2	Para-PakC&S	60 / 64	93.8	85.0-97.5	44 / 44 m	100.0	92.0-100.0
E. coli 0157	Para-PakC&S	39 / 42 n	92.9%	80.1-99.4	16 / 16	100.0	80.6-100.0
Shigella/EnteroinvasiveE. coli (EIEC)	FecalSwab	22 / 24 o	91.7	73.0-99.0	170 / 170	100.0	97.8-100.0
	Para-PakC&S	0 / 0	N/A	N/A	14 / 14	100.0	78.5-100.0
				Parasites
Cryptosporidium	FecalSwab	6 / 6	100.0	54.1-100.0	186 / 188 p	98.9	96.2-99.9
	Para-PakC&S	26 / 26	100.0	86.8-100.0	117 / 117	100.0	96.9-100.0
Cyclosporacayetanensis	FecalSwab	1 / 1	100.0	20.7-100.0	193 / 193	100.0	98.1-100.0
	Para-PakC&S	1 / 1	100.0	20.7-100.0	13 / 13	100.0	77.2-100.0
Entamoeba histolytica	FecalSwab	0 / 0	N/A	N/A	194 / 194	100.0	98.1-100.0
	Para-PakC&S	0 / 0	N/A	N/A	14 / 14	100.0	76.5-100.0
Giardia lamblia	FecalSwab	29 / 31 q	93.6	79.3-98.2	46 / 48 q	95.8	86.0-98.9
	Para-PakC&S	27 / 28 r	96.4	82.3-99.4	92 / 92 r	100.0	96.0-100.0

Table 5.35: Clinical Performance in the Retrospective study

4 Adenovirus F40/41 was detected in one of the three false negatives (1/3) in FecalSwab using a different FDA-cleared test method b Astrovirus was detected in the single specimen (1/1) in FecalSwab using a different FDA-cleared test method. 6 two (2) FecalSwab samples positive for Norovirus GI/GII in both Q1Astat-Dx and one FDA-cleared comparator were excluded from the PPA calculations because the samples did not have sufficient volume for composite comparator testing. The sample size for NPA is smaller for Norovirus GVGII as only a portion of the samples with a negative result in QIAstat-Dx and in one FDA-cleared comparator was tested with the complete composite comparator in the retrospective study.

4 the sample size for NPA is smaller for Norovirus GVGII in Para-Pak C&S as only a portion of the samples with a negative result in QIAstat-Dx and in one FDA-cleared comparator was tested with the composite comparator in the retrospective study · Rotavirus A was detected in the single false negative specimen (1/1) in FecalSwab using a different FDA-cleared test method.

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f Campylobacter was detected in one of the two false positive specimens (1/2) in FecalSwab using a different FDA-cleared test method

s Plesiomonas shigelloides was detected in one of the three false negative specimens (1/3) in Para-Pak C&S using a different FDAcleared test method.

4 Salmonella was not detected in the single false negative specimen (0/1) and was not detected in the two false positive specimens (0/2) in FecalSwab using a different FDA-cleared test method

1 Yersinia enterocolitica was not detected in the two false negative specimens (0/2) in FecalSwab using a different FDA-cleared test method.

I Enteropathogenic E. coli (EPEC) was detected in all three false negative specimens (3/3) in Para-Pak C&S using PCR followed by bi-directional sequence analysis. There were two (2) other false negative specimens that were not further investigated by discrepant analyses

ten (10) FecalSwab samples positive for ETEC in both OIAstat-Dx and one FDA-cleared comparator were excluded from the PPA calculations because the samples did not have sufficient volume for composite comparator testing. One (1) FecalSwab sample negative in QIAstat-Dx and positive with one FDA-cleared comparator with insufficient volume for composite comparator testing was classed as false negative in the PPA calculations. The sample size for ETEC as only a portion of the samples with a negative result in QIAstat-Dx and one FDA-cleared comparator was tested with the composite comparator in the retrospective study.

1 The sample size for NPA is smaller for ETEC in Para-Pak CS&S as only a portion of the samples with a negative result in QIAstat-Dx and one FDA-cleared comparator was tested with the composite comparator in the retrospective study).

" The sample size for NPA is smaller for STEC in Para-Pak C&S as only a portion of the samples with a negative result in QIAstat-Dx and in one FDA-cleared comparator was tested with the composite comparator in the retrospective study

" E. coli O157 was not detected in two false negativens (0/2) in Para-Pak C&S using a different FDA-cleared test method. There was one (1) false negative specimen in Para-Pak C&S that was not further investigated by discrepant analyses.

° Shigella/ EIEC was detected in one of the two false negative specimens (1/2) in FecalSwab using a different FDA-cleared test method.

P Cryptosporidium was not detected in the two false positive specimens (0/2) in FecalSwab using PCR followed by bi-directional sequence analysis

9 four (4) samples positive for Giardia lambia in both QIAstat-Dx and one FDA-cleared comparator were excluded from the PPA calculations because the samples did not have sufficient volume for composite comparator testing. Two (2) FecalSwab samples negative in QIAstat-Dx and positive with one FDA-cleared comparator with insufficient volume for composite comparator testing was classed as false negative in the PPA calculations. The sample size for NPA is smaller for Giardia as only a portion of the samples with a negative result in QIAstat-Dx and in one FDA-cleared comparator was tested with the complete composite comparator in the retrospective study

r One (1) Para-Pak C&S samples positive for Giardia lamblia in both QIAstat-Dx and primary FDA-cleared comparator (were excluded from the PPA calculations because the samples did not have sufficient volume for composite comparator testing. One (1) Para-Pak C&S sample negative in QIAstat-Dx and positive with one FDA-cleared comparator with insufficient volume for complete comparator testing was classed as false negative in the PPA calculations. The sample size for NPA is smaller for Giardia lamblia as only a portion of the samples with a negative result in Q1Astat-Dx and in one FDA-cleared comparator was tested with the complete composite comparator in the retrospective study.

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The proportion of failed runs on initial attempt, and following repeats are summarized in Table 5.36. The error breakdown due to instrument, invalid results, 'sample too concentrated' failures and other run failures are summarized in Table 5.37.

TransportMedia	Study	Initial Runs			Final Runs
		N / Total	%	95% CI	N / Total	%	95% CI
FecalSwab	Prospective	16 / 1227	1.3	0.7-2.1	3 / 1227	0.2	0.1-0.7
	ProspectiveArchived	0 / 53	0.0	0.0-6.7	0 / 53	0.0	0.0-6.7
	Retrospective	11 / 366	3.0	1.5-5.3	5 / 366	1.4	0.4-3.2
	Total	27 / 1646	1.6	1.1 - 2.4	8 / 1646	0.5	0.2 - 1.0
Para-Pak	Prospective	66 / 740	8.9	7.0-11.2	21 / 740	2.8	1.8-4.3
	ProspectiveArchived	3 / 66	4.5	0.9-12.7	0 / 66	0.0	0.0-5.4
	Retrospective	46 / 454	10.1	7.5 - 13.3	25 / 454	5.5	3.6-8.0
	Total	115 / 1260	9.1	7.6-10.9	46 / 1260	3.7	2.7-4.8

Table 5.36 Failure Rates Summary

Table 5.37 Failure Types Breakdown

TransportMedia	Study	Failure Reason	Initial RunsN / Total	%	Final RunsN / Total	%
FecalSwab	Prospective	Instrument	0 / 1227	0.0	0 / 1227	0.0
		Invalid a	0 / 1227	0.0	0 / 1227	0.0
		Sample too Concentrated b	5 / 1227	0.4	0 / 1227	0.0
		Other c	11 / 1227	0.9	3 / 1227	0.2
	ProspectiveArchived	Instrument	0 / 53	0.0	0 / 53	0.0
		Invalid	0 / 53	0.0	0 / 53	0.0
		Sample too Concentrated	0 / 53	0.0	0 / 53	0.0
		Other	0 / 53	0.0	0 / 53	0.0
	Retrospective	Instrument	1 / 366	0.3	0 / 366	0.0
		Invalid	1 / 366	0.3	0 / 366	0.0
		Sample too Concentrated	0 / 366	0.0	0 / 366	0.0
		Other	9 / 366	2.5	5 / 366	1.4
Para-Pak C&S	Prospective	Instrument	9 / 740	1.2	2 / 740	0.3
		Invalid	5 / 740	0.7	5 / 740	0.7
		Sample too Concentrated	35 / 740	4.7	7 / 740	0.9
		Other	17 / 740	2.3	7 / 740	0.9
	ProspectiveArchived	Instrument	0 / 66	0.0	0 / 66	0.0
		Invalid	1 / 66	1.5	0 / 66	0.0
		Sample too Concentrated	1 / 66	1.5	0 / 66	0.0
		Other	1 / 66	1.5	0 / 66	0.0
	RetrospectiveArchivedFrozen	Instrument	1 / 454	0.2	0 / 454	0.0
		Invalid	10 / 454	2.2	6 / 454	1.3
		Sample too Concentrated	10 / 454	2.2	2 / 454	0.4
		Other	25 / 454	5.5	17 / 454	3.7

a Internal Control failures with at least one analyte detected and the other analytes reported as 'invalid'

b Run failures related to 'sample concentration too high'. These specimens were repeated with 100 microliters as detailed in Appendix C.

C Run failures related to workflow checkpoints.

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Co-infections

The QIAstat-Dx Gastrointestinal Panel 2 reported multiple organism detections (i.e., coinfections) for a total of 15 and 29 prospective specimens in FecalSwab and Para-Pak C&S, respectively. This represents 7.0% of positive specimens (15/213) in FecalSwab and 17.0% of positive specimens (29/171) in Para-Pak C&S. Most multiple detections in FecalSwab specimens (14/15; 93%) contained two organisms, while 6.7% (1/15) contained three organisms. In Para-Pak C&S specimens, most multiple detections (22/29; 75.9%) contained two organisms, while 24.1% (7/29) contained three organisms. The most common multiple infections are shown in Table 5.38 and Table 5.39 below.

Table 5.38: Most Prevalent Multiple Detection Combinations (≥2 instances) as Determined by the QIAstat-Dx Gastrointestinal Panel 2 in FecalSwab specimens

Multiple Detection Combination	Number of FecalSwabSpecimens
Campylobacter + Norovirus GI/GII	2
Enterotoxigenic E. coli (ETEC) lt/st + Norovirus GI/GII	3
Campylobacter + Rotavirus A	4

Table 5.39: Most Prevalent Multiple Detection Combinations (≥2 instances) as Determined by the QIAstat-Dx Gastrointestinal Panel 2 in Para-Pak C&S specimens

Multiple Detection Combination	Number of Para-Pak C&SSpecimens
Campylobacter + Enteropathogenic E. coli (EPEC)	3
Enteropathogenic E. coli (EPEC) + Salmonella	3
Enteropathogenic E. coli (EPEC) + Enterotoxigenic E. coli (ETEC)lt/st	4

The analytes most commonly found in mixed infections in the FecalSwab specimens were Campylobacter (9), Norovirus GI/GII (7), Rotavirus (4) and ETEC (3) as shown in Table 5.4040 while the analytes most commonly found in mixed infections in the Para-Pak C&S specimens were EPEC (17), ETEC (8), Campylobacter (7), Norovirus GI/GII (5), Rotavirus (4) and STEC (5) as shown in Table 5.4141 and Table 5.4242.

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Table 5.4040: Prevalence of Analytes in Mixed Infections in FecalSwab specimens as
determined by the QIAstat-Dx Gastrointestinal Panel 2

Analyte	N	%
Adenovirus F40/F41	1	3.2
Astrovirus	1	3.2
Campylobacter	9	29.0
Enterotoxigenic E. coli (ETEC) lt/st	3	9.7
Giardia lamblia	2	6.5
Norovirus GI/GII	7	22.6
Plesiomonas shigelloides	1	3.2
Rotavirus A	4	12.9
Shigella/Enteroinvasive E. coli (EIEC)	2	6.5
Yersinia enterocolitica	1	3.2

Prevalence of Analytes in Mixed Infections in Para-Pak C&S Table 5.4141: specimens as determined by the QIAstat-Dx Gastrointestinal Panel 2

Analyte	N	%
Adenovirus F40/F41	1	1.5
Astrovirus	1	1.5
Campylobacter	7	10.8
Cryptosporidium	2	3.1
Cyclospora cayetanensis	2	3.1
Enteropathogenic E. coli (EPEC)	17	26.2
Enterotoxigenic E. coli (ETEC) lt/st	8	12.3
Giardia lamblia	1	1.5
Norovirus GI/GII	5	7.7
Plesiomonas shigelloides	7	10.8
Rotavirus A	1	1.5
Salmonella	4	6.2
Shiga-like toxin E. coli (STEC) stx1/stx2	5	7.7
Shigella/Enteroinvasive E. coli (EIEC)	3	4.6
Yersinia enterocolitica	1	1.5

Contrived Specimens Testing

Several analytes, such as Entamoeba histolytica are so rare that both prospective and archived testing efforts were insufficient to demonstrate system performance. To

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supplement the prospective and archived specimens' test results, an evaluation of contrived specimens was performed. Contrived specimens were prepared using negative residual specimens that had previously tested negative by QIAstat-Dx Gastrointestinal Panel 2 and comparator methods. At least, 50% of these specimens were spiked at concentrations at concentrations slightly above the Limit of Detection (2x LoD) and the rest at 5x and 10x LoD, using quantified strains for each pathogen. A minimum of 50 contrived specimens were tested for each evaluated analyte. The analyte status of each contrived specimen was blinded to the users analyzing the specimens. Results are summarized in Table 5.4242 Table 5.42.

QIAstat-Dx GI2 Target	Medium Brand	Fraction	Percentage	95% CI
Astrovirus	FecalSwab	33 / 34	97.1	85.1-99.5
	Para-Pak C&S	34 / 34	100.0	89.8-100.0
Rotavirus A	FecalSwab	35 / 35	100.0	90.1-100.0
	Para-Pak C&S	34 / 35	97.1	85.5-99.5
Plesiomonas shigelloides	FecalSwab	33 / 33	100.0	89.6-100.0
	Para-Pak C&S	34 / 35	97.1	85.5-99.5
Yersinia enterocolitica	FecalSwab	34 / 34	100.0	89.8-100.0
	Para-Pak C&S	34 / 35	97.1	85.5-99.5
Shigella/EIEC	FecalSwab	35 / 35	100.0	90.1-100.0
	Para-Pak C&S	34 / 34	100.0	89.8-100.0
Cryptosporidium	FecalSwab	27 / 27	100.0	87.5-100.0
	Para-Pak C&S	31 / 31	100.0	89.0-100.0
Cyclospora cayetanensis	FecalSwab	26 / 26	100.0	87.1-100.0
	Para-Pak C&S	30 / 30	100.0	88.6-100.0
Entamoeba histolytica	FecalSwab	35 / 35	100.0	90.1-100.0
	Para-Pak C&S	34 / 35	97.1	85.5-99.5

Table 5.4242: Test Results Summary for Contrived Specimens
-------------------------------------------------------------	--	--	--	--

Conclusions

The QIAstat-Dx Gastrointestinal Panel 2 is substantially equivalent to the legally marketed: BioFire Diagnostics LLC FilmArray Gastrointestinal (GI) Panel 510(k) # K140407.

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).