QIAstat-Dx Gastrointestinal Panel 2

K220062 · QIAGEN GmbH · PCH · May 31, 2024 · Microbiology

Device Facts

• Record ID: K220062
• Device Name: QIAstat-Dx Gastrointestinal Panel 2
• Applicant: QIAGEN GmbH
• Product Code: PCH · Microbiology
• Decision Date: May 31, 2024
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3990
• Device Class: Class 2
• Attributes: Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K220062 · May 31, 2024	QIAstat-Dx Gastrointestinal Panel 2	QIAGEN GmbH	Prospective clinical study of routine diagnostic stool specimens	The clinical performance of the QIAstat-Dx Gastrointestinal Panel 2 was established using prospectively collected stool specimens from patients presenting with clinical indications of gastrointestinal infection at routine clinical diagnostic laboratories.	Prospective clinical study; Routine clinical diagnostics; Clinical performance

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Prospective Clinical Study of QIAstat-Dx Gastrointestinal Panel 2; Multi-center international prospective study; Follow-up/Duration: May to July 2021; Study Period: May to July 2021	Patients with clinical indications of diarrhea caused by gastrointestinal infection; Sample Size: 1939 prospectively collected evaluable stool specimens; Number of Sites: 13 clinical settings (9 in US, 4 in Europe)	FDA-cleared molecular assays or composite of FDA-cleared molecular assays/sequencing	Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to reference methods

Indications for 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 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 : 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 Story

The QIAstat-Dx Gastrointestinal Panel 2 is a single-use, fully automated molecular diagnostic cartridge used with the QIAstat-Dx Analyzer 1.0. It processes preserved stool samples (Para-Pak C&S or FecalSwab) to detect nucleic acids from 20+ gastrointestinal pathogens. The cartridge integrates all reagents for sample preparation, nucleic acid extraction, and multiplex real-time PCR amplification. Operation involves inserting the cartridge into the analyzer, which performs automated lysis, purification, and rtPCR. Results are displayed on the analyzer screen in approximately 78 minutes. The device aids clinicians in diagnosing gastrointestinal illness by identifying specific pathogens, though it does not rule out co-infections or non-infectious causes. It is intended for use in clinical laboratory settings.

Clinical Evidence

Prospective multi-center study (13 sites, 1939 specimens) compared QIAstat-Dx GI Panel 2 against FDA-cleared comparators or composite reference methods. Performance metrics (PPA/NPA) calculated for each analyte. PPA generally >90% for most targets; NPA >99%. Study included prospective archived and retrospective samples to supplement low-prevalence targets. Bench testing confirmed LoD, inclusivity, specificity, and interference resistance.

Technological Characteristics

The device uses a disposable, single-use plastic cartridge containing pre-loaded reagents. It employs chemical and mechanical lysis (silica beads/rotor), silica membrane-based nucleic acid purification, and multiplex real-time PCR (rtPCR) amplification. The system is closed, utilizing pneumatically operated microfluidics. It is designed for use exclusively with the QIAstat-Dx Analyzer 1.0. The internal control is Schizosaccharomyces pombe.

Indications for Use

Indicated for individuals with signs/symptoms of gastrointestinal infection. Detects nucleic acids from viruses (Adenovirus F40/F41, Astrovirus, Norovirus GI/GII, Rotavirus A), bacteria (Campylobacter, Shigella/EIEC, EPEC, ETEC, STEC, Salmonella, Plesiomonas shigelloides, Yersinia enterocolitica), and parasites (Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia) in preserved stool samples.

Regulatory Classification

Identification

A gastrointestinal microorganism multiplex nucleic acid-based assay is a qualitative in 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.

Special Controls

*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).

Predicate Devices

• FilmArray® Gastrointestinal (GI) Panel (K140407)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY

# I Background Information:

A 510(k) Number

K220062

B Applicant

QIAGEN GmbH

C Proprietary and Established Names

QIAstat-Dx Gastrointestinal Panel 2

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PCH | Class II | 21 CFR 866.3990 - Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assay | MI - Microbiology |

# II Submission/Device Overview:

A multiplexed nucleic acid-based test intended for use with QIAstat-Dx system for the qualitative in vitro detection and identification of multiple bacteria, viruses, and parasites. The QIAstat-Dx Gastrointestinal Panel 2 assay is performed directly from stool samples collected in Para-Pak C&S (Meridian Bioscience) and FecalSwab (COPAN) transport media.

A Purpose for Submission:

To obtain a substantial equivalence determination for the QIAstat-Dx Gastrointestinal Panel 2 microorganism multiplex nucleic acid-based assay.

B Measurand:

Targeted nucleic acid sequences of the following gastrointestinal microorganisms:

- Adenovirus F40/F41
- Astrovirus

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- 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

### C Type of Test:

A multiplex nucleic acid-based test intended for use with the QIAstat-Dx system for the qualitative in vitro detection and identification of multiple bacteria, viruses, and parasites in preserved stool samples collected from individuals suspected of gastrointestinal infection.

### III Intended Use/Indications for Use:

### A Intended Use(s):

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 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 |

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| 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) | ✓ | 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. 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 test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

### B Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

For in vitro Diagnostic Use Only

### C Special Instrument Requirements:

The QIAstat-Dx Gastrointestinal Panel 2 is intended for use only with the QIAstat-Dx Analyzer 1.0.

### IV Device/System Characteristics:

### A Device Description:

The QIAstat-Dx Gastrointestinal Panel 2 assay is run on the QIAstat-Dx Analyzer 1.0. The QIAstat-Dx Gastrointestinal Panel 2 cartridge is a single-use cartridge that includes all reagents needed for nucleic acid extraction, nucleic acid amplification, and detection of bacteria, viruses or parasites associated with gastrointestinal infection. Testing requires a 200

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μL specimen volume and minimal hands-on time, and the results are available in approximately 78 minutes.

# **B 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 QIAstat-Dx Gastrointestinal Panel 2 is intended to be used with stool samples collected 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. The QIAstat-Dx Analyzer 1.0 automatically interprets test results and displays a summary on the analyzer display screen.

# *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.

After mechanical lysis is completed, the primary lysate is transferred to the purification chamber through a frit with 2-5 μm 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

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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

QIAstat-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 QIAstat-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.

All the reagents required for the complete execution of the test are pre-loaded and self-contained in the QIAstat-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

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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 pre-loaded 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 Cary-Blair 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 but not provided with the QIAstat-Dx Gastrointestinal Panel 2.

QIAGEN provides an internal control ('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 QIAstat-Dx Analyzer 1.0 is provided factory calibrated and does not require user calibration. The QIAstat-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.

### **V Substantial Equivalence Information:**

#### **A Predicate Device Name(s):**

FilmArray Gastrointestinal (GI) Panel

#### **B Predicate 510(k) Number(s):**

K140407

#### **C Comparison with Predicate(s):**

| Device & Predicate Device(s): | K220062 | K140407 |
| --- | --- | --- |
| Device Trade Name | QIAstat-Dx Gastrointestinal Panel 2 | FilmArray Gastrointestinal (GI) Panel |
| General Device Characteristics Similarities | | |

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| Device & Predicate Device(s): | K220062 | K140407 |
| --- | --- | --- |
| Pathogens Detected | Adenovirus F40/F41, Astrovirus Norovirus GI/GII Rotavirus A Campylobacter (C. jejuni, C. coli, C. upsaliensis), Shigella/Enteroinvasive E. coli (EIEC) Enteropathogenic E. coli (EPEC) Enterotoxigenic E. coli (ETEC) Shiga-like toxin-producing E.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 | Same plus additional pathogens detected (See below for differences ) |
| Analyte | DNA/RNA | DNA/RNA |
| Amplification and Detection Technology | PCR | PCR |
| Technological Principles | Multiplex nucleic acid test | Same (See below for differences) |
| Intended Use/Indications For 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 and FecalSwab) obtained from individuals with signs and/or symptoms of gastrointestinal infection. The following viruses, bacteria (including | 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), |

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| Device & Predicate Device(s): | K220062 | K140407 |
| --- | --- | --- |
| | 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 • Shiga-like toxin-producing Escherichia 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 G. intestinalis and G. 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 | 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 • 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, |

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| Device & Predicate Device(s): | K220062 | K140407 |
| --- | --- | --- |
| | 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. | 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. Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor or guide treatment for C. difficile infection. Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for E. coli O157, Plesiomonas shigelloides, Yersinia enterocolitica, Astrovirus, and Rotavirus A were established primarily with retrospective clinical specimens. Performance characteristics for Entamoeba histolytica, and Vibrio (V parahaemolyticus, V. vulnificus, and Vibrio cholerae) were established primarily using contrived clinical specimens. Negative FilmArray GI Panel results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks. |
| Assay Controls | One internal control in each cartridge to control for sample | Two controls are included in each reagent pouch to control for |

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| Device & Predicate Device(s): | K220062 | K140407 |
| --- | --- | --- |
| | processing that is subjected to all nucleic acid extraction and amplification steps similar to patient samples. External controls are not provided with the QIAstat-Dx Gastrointestinal Panel 2. Labeling recommends use of negative and positive external controls regularly. Use transport media as the external Negative Control, and previously characterized positive samples or negative sample spiked with well characterized target organisms as external positive controls. | sample processing and both stages of PCR and melt analysis. Labeling recommends the use of external positive and negative controls regularly. Enteric transport media can be used as an external negative control, and previously characterized positive samples or negative samples spiked with well characterized organisms as external positive controls. |
| **General Device Characteristic Differences** | | |
| Assay Targets | No additional targets to those listed above which are in common with the predicate device. | The FilmArray Gastrointestinal Panel has four additional targets: *Clostridium difficile* (C. difficile) toxin A/B, *Enteroaggregative Escherichia coli* (EAEC), Sapovirus and a generic *Vibrio* target which differentiates *Vibrio cholerae*. |
| Specimen Types | Human stool sample collected in Para-Pak C&S and FecalSwab transport media. | Human stool sample collected in Cary Blair transport media |
| Nucleic Acid Extraction | Extraction of nucleic acids using silica membrane | Extraction of nucleic acids using magnetic beads |
| Technology | QIAstat-Dx Gastrointestinal Panel 2 detection of amplified targets uses an increase in fluorescence due to specific probe binding to generate the assay results. | The FilmArray Gastrointestinal Panel uses melting curve analysis to confirm the identity of amplified targets to generate assay results. |
| Amplification and Detection Instrument System | QIAstat-Dx Analyzer 1.0 | FilmArray Instrument |

# VI Standards/Guidance Documents Referenced:

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None

## VII Performance Characteristics (if/when applicable):

### A Analytical Performance:

#### 1. Reproducibility/Repeatability:

##### *Reproducibility Study*

Reproducibility testing evaluating 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 factors that may introduce variability in assay results, including sites, days, replicates, cartridge lots, operators, and QIAstat-Dx analyzers. For each site, testing was performed across five non-consecutive days with two replicates per day (total of 30 replicates per target, concentration, and site), four QIAstat-Dx Analyzers (two analyzers per operator and per site), and at least two operators on each testing day. A total of five sample mixes (including evaluation of analyte levels corresponding to 1x LoD and 3x LoD as well as negative samples containing no analyte) were prepared in a matrix consisting of pooled stool in Para-Pak C&S transport medium. For each mix, six replicates were tested and evaluated, and data obtained at all three sites were compiled to calculate the two-sided 95% Confidence Interval by target and concentration.

The percent agreement with expected results for all analytes was $\geq 95\%$ for samples tested at 1x and 3x LoD, meeting the acceptance criteria for the study. All analytes evaluated were detected within performance expectations for qualitative agreement across all concentrations tested. The Reproducibility Study site-to-site qualitative results (agreements with expected results) are presented in Table 1 below.

**Table 1: Reproducibility Study, Qualitative Results**

| Pathogen Tested | Concentration Tested | Expected Result | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Site A | Site B | Site C | All Sites (95% Confidence Interval) |
| Adenovirus F41 ZeptoMetrix 0810085CF | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | None | Not Detected | 29/30 100% | 29/30 100% | 29/30 100% | 87/90^{1} 96.7% (90.98 – 98.9%) |

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| Pathogen Tested | Concentration Tested | Expected Result | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Site A | Site B | Site C | All Sites (95% Confidence Interval) |
| *Campylobacter* ZeptoMetrix 801650 | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| *Escherichia coli* EPEC^{4} ZeptoMetrix 801747 | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 29/30 96.67% | 30/30 100% | 89/90 98.89% (93.96 – 99.97%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| *Entamoeba histolytica* ATCC 30459 | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 30/30 100% | 29/30 96.67% | 89/90 98.89% (93.96 – 99.97%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| *Giardia lamblia*^{2} ATCC 30888 | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |

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| Pathogen Tested | Concentration Tested | Expected Result | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Site A | Site B | Site C | All Sites (95% Confidence Interval) |
| Norovirus GII ZeptoMetrix 0810087CF | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 29/30 96.67% | 30/30 100% | 30/30 100% | 89/90 98.89% (93.96 – 99.97%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| Rotavirus A^{3} ZeptoMetrix 0810280CF | 3x LoD | Detected | 29/30 96.67% | 29/30 96.67% | 30/30 100% | 88/90 97.8% (92.20 – 99.73%) |
| | 1x LoD | Detected | 23/30 76.67% | 26/30 86.67% | 12/12 100% | 61/72 84.7% (74.31 – 92.12%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| *Escherichia coli* (STEC) O157:H7^{4} ZeptoMetrix 0801622 | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 30/30 100% | 29/30 96.67% | 89/90 98.89% (93.96 – 99.97%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| *Escherichia coli* (STEC)^{4} *stx1/stx2 ZeptoMetrix 0801622 | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |

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| Pathogen Tested | Concentration Tested | Expected Result | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Site A | Site B | Site C | All Sites (95% Confidence Interval) |
| *Salmonella enterica* ZeptoMetrix 0801437 | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 29/30 96.67% | 29/30 96.67% | 88/90 97.78% (92.20 - 99.73%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 – 100%) |
| *Yersinia enterocolitica* Zeptometrix 0801734 | 3x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | 1x LoD | Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 - 100%) |
| | None | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | 90/90 100% (95.98 – 100%) |

¹ Three (3) Adenovirus F40/41 false positives were observed when testing negative sample. Retesting of the three samples resulted in the expected negative results.

² 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.

³ 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 with during an interim data evaluation (61/72, 84.7%) and that was attributed to the sample manufacture and 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.

⁴ Only applicable for Para-Pak C&S samples

During the study, there were three false positive results for Adenovirus F40/41 in negative (non-spiked) samples as well as one false positive result for Campylobacter and one false positive result for Giardia lamblia in samples containing other analytes. Repeat testing of the same five samples yielded the expected negative result for each applicable analyte. The investigation into the unexpected false positive results showed that these events were observed in five different analyzers. For the sample that was false positive for Campylobacter, the sample tested immediately prior was also positive for Campylobacter. For the other four false positive samples, the samples run immediately prior were not positive for the applicable pathogen suggesting that these unexpected results were likely not due to inter-run carryover.

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A total of six cartridge failures (6/645, 0.93%) were observed during the study. Failures included cartridge errors (5) and an instrument error (1). No invalid results due to internal control failure were observed.

An additional analysis of variation was conducted across specific potential sources of variability such as sites, days, replicates, cartridge lots, operators, and QIAstat-Dx analyzers. Results from the analysis showed no significant contribution to variability (Standard Deviation and Coefficient of Variation values below 1 and 5%, respectively) caused by any of the assessed variables. Overall, Ct variability was low, and the study demonstrates assay variability within an acceptable range. Results are presented in Table 2 below.

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**Table 2: Reproducibility Study, Numerical Analysis of Variability**

| Pathogen | Concentration tested | Positive (n) | Negative (n) | Mean | Variance component (SD, %CV*) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | Between Site | Between Lot | Between Day | Between Operator | Between Instrument | Between Replicate | Total** |
| 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 | 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 | 33.85 | (0.8986, 2.65%) | (0.2050, 0.61%) | (0.5187, 1.53%) | (0.0000, 0.00%) | (0.2847, 0.84%) | (1.3126, 3.88%) | (1.6070, 4.75%) |
| STEC_O157 | 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 for Rotavirus. Results from the new dataset were:

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| | | | | | Variance component (SD, %CV*) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Pathogen | Concentration tested | Positive (n) | Negative (n) | Mean | Between Site | Between Lot | Between Day | Between Operator | Between Instrument | Between Replicate | 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%) |

# Repeatability Study:

To further assess the precision of the QIAstat-Dx Gastrointestinal Panel 2, an in-house repeatability study was conducted using a set of samples containing analyte concentrations of 3X LoD and 1X LoD as well as negative samples prepared in stool matrix in Para-Pak C&S transport medium. Specifically, pathogens included in the positive samples were Adenovirus, Campylobacter, Entamoeba histolytica, Giardia lamblia, Norovirus GII, Rotavirus, Salmonella enterica, and Yersinia enterocolitica, in addition to Enteropathogenic E. coli (EPEC), STEC stx1/stx2, and E. coli O157 which are only applicable for Para-Pak C&S samples. Each panel member was tested with the same instrument over 12 days. In total, 60 replicates at 1x LoD and 60 replicates at 3x LoD per each of the tested targets and 60 negative samples were run in the study. Overall results showed a 93.33-100% and 95.00-100% detection rate for 1x LoD and 3x LoD samples, respectively. Negative samples showed 100% of negative calls for all panel analytes. One failed run due to a cartridge error occurred during the study (1/426, 0.23%). Testing results met the study acceptance criteria demonstrating acceptable repeatability of the QIAstat-Dx Gastrointestinal Panel 2.

# 2. Linearity:

Not applicable.

# 3. Analytical Specificity/Interference:

# Analytical Specificity/Cross-Reactivity

The Analytical Specificity study was performed to demonstrate the absence of cross-reactivity between the QIAstat-Dx Gastrointestinal Panel 2 assay on-panel microorganisms and viruses (intra-panel cross-reactivity) and between on-panel and off-panel microorganisms/viruses (i.e., organisms not covered by the panel and therefore not intended to be detected by the QIAstat-Dx Gastrointestinal Panel 2).

Samples were prepared by single spiking microorganisms/viruses at high concentrations into negative stool resuspended in Para-Pak C&S transport medium. Sample concentrations tested were 1.0E+06 CFU/mL for bacteria, 1.0E+05 TCID50/mL for viruses and 1.0E+05 cells/mL for parasites, or the highest available concentration based on the pathogen stock. Testing was conducted in triplicate.

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Study results indicated no false positive results for on-panel or off-panel analytes with the exception of two non-targeted Campylobacter species (C. helveticus and C. lari) that cross-reacted with the Campylobacter assay oligonucleotides included in the QIAstat-Dx Gastrointestinal Panel 2. The on-panel and off-panel microorganisms/viruses evaluated in the study are presented in Tables 3 and 4 respectively.

Table 3: List of Analytical Specificity On-Panel Pathogens Tested

| Type | Pathogen | Concentration Tested |
| --- | --- | --- |
| Bacteria | Campylobacter coli | 1.0E+06 CFU/mL |
| | Campylobacter jejuni | 1.0E+06 CFU/mL |
| | Campylobacter upsaliensis | 1.0E+06 CFU/mL |
| | Escherichia coli (EPEC) | 1.0E+06 CFU/mL |
| | Escherichia coli (ETEC lt/st) | 1.0E+06 CFU/mL |
| | Escherichia coli (STEC) | 1.0E+06 CFU/mL |
| | Plesiomonas shigelloides | 1.0E+06 CFU/mL |
| | Salmonella enterica | 1.0E+06 CFU/mL |
| | Shigella sonnei | 1.0E+06 CFU/mL |
| | Yersinia enterocolitica | 1.0E+06 CFU/mL |
| Parasites | Cryptosporidium parvum | 1.0E+05 oocysts/mL |
| | Cyclospora cayetanensis | 1.0E+05 genome copies/mL |
| | Entamoeba histolytica | 1.0E+05 cells/mL |
| | Giardia lamblia | 1.0E+05 cells/mL |
| Viruses | Adenovirus F41 | 1.0E+05 TCID_{50}/mL |
| | Astrovirus | 1.0E+05 copies/mL |
| | Norovirus GI | 1.0E+05 TCID_{50}/mL |
| | Norovirus GII | 1.0E+05 TCID_{50}/mL |
| | Rotavirus | 1.0E+05 U/mL |

Table 4: List of Analytical Specificity Off-Panel Pathogens Tested

| Type | Pathogen (Potential Cross-Reactant) | Concentration Tested |
| --- | --- | --- |
| Bacteria | Abiotrophia defectiva | 1.0E+06 cells/mL |
| | Acinetobacter baumannii | 1.0E+06 CFU/mL |
| | Aeromonas hydrophila | 1.0E+06 CFU/mL |
| | Arcobacter cryaerophilus | 1.0E+06 cells/mL |
| | Bacillus subtilis | 1.0E+06 CFU/mL |
| | Bifidobacterium bifidum | 1.0E+06 CFU/mL |
| | Campylobacter fetus | 1.0E+06 cells/mL |
| | Campylobacter gracilis | 1.0E+06 CFU/mL |
| | Campylobacter helveticus* | 1.0E+06 CFU/mL |
| | Campylobacter hominis | 1.0E+06 cells/mL |
| | Campylobacter lari* | 1.0E+06 CFU/mL |
| | Campylobacter mucosalis | 1.0E+06 CFU/mL |
| | Campylobacter rectus | 1.0E+06 cells/mL |
| | Chlamydia trachomatis | 1.0E+06 cells/mL |
| | Citrobacter freundii | 1.0E+06 CFU/mL |

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| | Clostridium difficile non-toxigenic | 1.0E+06 CFU/mL |
| --- | --- | --- |
| | Clostridium perfringens | 1.0E+06 cells/mL |
| | Clostridium septicum | 1.0E+06 CFU/mL |
| | Clostridium tetani | 1.0E+06 CFU/mL |
| | Corynebacterium genitalium | 1.0E+06 cells/mL |
| | Enterobacter aerogenes | 1.0E+06 CFU/mL |
| | Enterobacter cloacae | 1.0E+06 CFU/mL |
| | Enterococcus faecalis | 1.0E+06 cells/mL |
| | Enterococcus faecium | 1.0E+06 CFU/mL |
| | Escherichia fergusonii | 1.0E+06 CFU/mL |
| | Escherichia hermannii | 1.0E+06 cells/mL |
| | Escherichia vulneris | 1.0E+06 CFU/mL |
| | Faecalibacterium prausnitzii | 1.0E+06 CFU/mL |
| | Gardnerella vaginalis | 1.0E+06 cells/mL |
| | Haemophilus influenzae | 1.0E+06 CFU/mL |
| | Hathewaya histolytica (Clostridium) | 1.0E+06 CFU/mL |
| | Helicobacter pylori | 1.0E+06 cells/mL |
| | Klebsiella pneumoniae | 1.0E+06 CFU/mL |
| | Lactobacillus casei | 1.0E+06 CFU/mL |
| | Listeria monocytogenes | 1.0E+06 cells/mL |
| | Proteus mirabilis | 1.0E+06 CFU/mL |
| | Proteus vulgaris | 1.0E+06 CFU/mL |
| | Pseudomonas aeruginosa | 1.0E+06 cells/mL |
| | Staphylococcus aureus | 1.0E+06 CFU/mL |
| | Staphylococcus aureus subsp. aureus | 1.0E+06 CFU/mL |
| | Staphylococcus epidermidis | 1.0E+06 cells/mL |
| | Streptococcus agalactiae | 1.0E+06 CFU/mL |
| | Streptococcus pyogenes | 1.0E+06 CFU/mL |
| Fungi | Aspergillus fumigatus | 1.0E+05 cells/mL |
| | Candida albicans | 1.0E+05 CFU/mL |
| | Saccharomyces boulardii | 1.0E+05 CFU/mL |
| | Saccharomyces cerevisiae | 1.0E+05 copies/mL |
| Parasite | Babesia microti | N/A** |
| | Blastocystis hominis | 1.0E+05 cells/mL |
| | Giardia muris | 1.0E+05 cysts/mL |
| | Toxoplasma gondii | 1.0E+05 cells/mL |
| | Trichomonas tenax | 1.0E+05 cells/mL |
| Viruses | Adenovirus C:2 | 1.0E+05 TCID_{50}/mL |
| | Adenovirus B:34 | 1.0E+05 TCID_{50}/mL |
| | Adenovirus B3 | 1.0E+05 TCID_{50}/mL |
| | Adenovirus E:4a | 1.0E+05 TCID_{50}/mL |
| | Adenovirus serotype 1 | 1.0E+05 TCID_{50}/mL |
| | Adenovirus serotype 5 | 1.0E+05 TCID_{50}/mL |
| | Adenovirus serotype 8 | 1.0E+05 TCID_{50}/mL |

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| | Bocavirus Type 1 | 1.0E+05 copies/mL |
| --- | --- | --- |
| | Coronavirus 229E | 1.0E+05 U/mL |
| | Coxsackievirus B3 | 1.0E+05 TCID_{50}/mL |
| | Cytomegalovirus | 1.0E+05 TCID_{50}/mL |
| | Enterovirus 6 (Echovirus) | 1.0E+05 U/mL |
| | Enterovirus 68 | 1.0E+05 TCID_{50}/mL |
| | Herpes Simplex Virus Type 2 | 1.0E+05 PFU/mL |
| | Rhinovirus 1A | 1.0E+05 TCID_{50}/mL |

*Positive results generated for Campylobacter

**The stock concentration for Babesia microti is expressed as 32 % parasitemia (Number of infected cells/Total cells*100)

In silico analysis of potential cross-reactivity predicted that the following microorganisms may cross-react with QIAstat-Dx Gastrointestinal Panel 2 targets (Table 5).

Table 5: Potential Cross-Reactivity Based On in silico Analysis

| QIAstat-Dx Gastrointestinal Panel 2 Target | Potential cross-reactive microorganisms |
| --- | --- |
| Enteropathogenic E. coli (EPEC) | Shigella boydii^{1,2,3}, Escherichia albertii^{1,2} |
| Campylobacter. | Campylobacter lari^{4}, Campylobacter helveticus^{4} |
| Shiga-like toxin-producing E. coli (STEC) stx1/stx2 | Shigella sonnei^{1,3}, Shigella Dysenteriae^{1,3}, Acinetobacter haemolyticus^{1,5}, Citrobacter freundii^{1,5}, Enterobacter cloacae^{1,5}, Aeromonas caviae^{1,5}, Escherichia albertii^{1,5} |
| E. coli O157 | Non-STEC E.coli O157 strains^{6} |

$^{1}$Predicted cross-reactivity identified by in silico analysis reflects sequences that can be acquired between species by horizontal gene transfer.

$^{2}$Rare or less common eae intimin carrier organisms

$^{3}$On-panel target.

$^{4}$In 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 O157 will only be reported by the QIAstat-Dx Gastrointestinal Panel 2 assay 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 O157 co-infection will not be differentiated from a single infection caused by a STEC O157:H7 strain.

### Interfering Substances Study:

The effect of potentially interfering substances on the performance of the QIAstat-Dx Gastrointestinal Panel 2 was evaluated. Thirty-five (35) potentially interfering substances were spiked into the sample mixes at a level predicted to be above the concentration of the substance

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likely to be found in stool specimens. Substances included endogenous, exogenous as well as technique-specific substances.

Testing included samples containing negative clinical stool matrix in Para-Pak C&S medium with and without the addition of each potentially interfering substance. Samples containing organism mixes with one strain for each targeted pathogen were tested at a concentration of 3x LoD. Testing was performed in triplicates. Additionally, for endogenous substances, negative specimens (stool matrix in Para-Pak C&S medium matrix with no organism mix) were spiked with only the test substance to evaluate the potential for false positive results due to the test substance itself.

For the majority of substances tested, no interference was observed, with the exceptions of mucin, calcium carbonate, nonoxynol-9, and Rotavirus reassortants which demonstrated interference at high concentrations.

The mucin at 5% w/v sample generated unexpected false positives results for the Yersinia target. These results were investigated by testing the interfering substance with an FDA-cleared method that also detected Yersinia in the sample, demonstrating that the unexpected positive results were not due to mucin interference.

Calcium carbonate at concentrations above 0.5% w/v was found to generate false negative results for all the QIAstat-Dx Gastrointestinal Panel 2 targets and the internal control.

Nonoxynol-9 at concentrations above 0.02% v/v was found to generate false negative results for detection of Entamoeba histolytica.

As expected, Rotavirus reassortants WC3:2-5, R574(9) and WI79-4,9 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 WI79-4,9 were 8.89x10⁻⁵ TCID₅₀/mL and 1.10 PFU/mL, respectively.

The strains used for sample mixes tested in the study are presented in Table 6. Results from testing the 35 endogenous and exogenous substances evaluated are provided in Table 7.

Table 6: Interfering Substances Study, Microorganisms/Viruses Evaluated

| Mix | Target | Strain | Supplier | Catalog ID | Final Concentration tested (3x LoD) |
| --- | --- | --- | --- | --- | --- |
| 1 | Giardia lamblia | Portland-1 | ATCC | 30888 | 1.91E+03 cells/mL |
| | Enteropathogenic E. coli (EPEC) | B171-8 (O111:NM) | ZeptoMetrix | 0801747 | 7.75E+03 CFU/mL |
| | Yersinia enterocolitica | Z036 | ZeptoMetrix | 0801734 | 6.21E+03 CFU/mL |
| | Salmonella | Salmonella enterica Serovar Typhimurium; Z005 | ZeptoMetrix | 0801437 | 1.36E+04 CFU/mL |

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| Mix | Target | Strain | Supplier | Catalog ID | Final Concentration tested (3x LoD) |
| --- | --- | --- | --- | --- | --- |
| | Adenovirus F40/F41 | Adenovirus Type 41 (Tak) | ZeptoMetrix | 0810085CF | 1.50E-01 TCID_{50}/mL |
| 2 | Norovirus GI/GII | Norovirus GII-recombinant | ZeptoMetrix | 0810087CF | 3.15E+01 TCID_{50}/mL |
| | *Entamoeba histolytica* | HM-1:IMSS | ATCC | 30459 | 6.60E-01 cells/mL |
| | *Campylobacter*. | *Campylobacter jejuni* Z086 | ZeptoMetrix | 0801650 | 4.98E+03 CFU/mL |
| | Shiga-like toxin-producing *E. coli* (STEC) O157 | O157:H7; EDL933 | ZeptoMetrix | 0801622 | 6.84E+03 CFU/mL |
| 3 | *Cryptosporidium* | *Cryptosporidium parvum*, Iowa isolate | Waterborne | P102C | 1.88E+03 oocysts/mL |
| | *Plesiomonas shigelloides* | Z130 | ZeptoMetrix | 0801899 | 6.87E+03 CFU/mL |
| | Rotavirus A | 69M | ZeptoMetrix | 0810280CF | 1.31E+03 U/mL |
| 4 | Astrovirus | (Type 8) ERE IID 2371 | ZeptoMetrix | 0810277CF | 3.51E+01 U/mL |
| | *Cyclospora cayetanensis* | n/a | LACNY | LAC2825 | 1.37E+04 copies/mL |
| | Enterotoxigenic *E. coli* (ETEC) *It/st* | ETEC; ST+, LT+ | ZeptoMetrix | 0801624 | 1.70E+03 CFU/mL |
| | Enteroinvasive *E. coli* (EIEC)/*Shigella* | CDC EDL 1282; Escherichia coli serotype O29:NM | ATCC | 43892 | 1.24E+02 CFU/mL |
| 5 | Negative (IC) | n/a | n/a | n/a | n/a |

Table 7: Interfering Substances Study Results

| 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 5 ug/mL | No Interference No Interference |
| Human stool (overfill of transport medium vial) | 300 mg/mL | No Interference |
| Human urine | 50% v/v | No Interference |
| Human whole blood with Na Citrate | 40% v/v | No Interference |

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| Substance tested | Concentration tested | Result |
| --- | --- | --- |
| Mucin from bovine submaxillary | 5% w/v 2.5% w/v | **Interference**^{1} No Interference |
| Triglycerides | 5% w/v | No Interference |
| **Exogenous substances** | | |
| Bacitracin | 250U/mL | No Interference |
| Bisacodyl | 0.3% w/v | No Interference |
| Bismuth subsalicylate | 0.35% w/v | No Interference |
| Calcium carbonate (TUMS Extra Strength 750) | 5%w/v 0.5% w/v | **Interference** 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 |
| 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 0.6% v/v 0.3% v/v 0.15% v/v 0.075% v/v 0.02% v/v | **Interference** **Interference** **Interference** **Interference** **Interference** 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 x 10^{-3} TCID_{50}/mL 8.89 x 10^{-4} TCID_{50}/mL 8.89 x 10^{-5} TCID_{50}/mL | **Interference** **Interference** No Interference |
| Rotavirus reassortant WI79-4,9 - VR 2415 | 1.10 x 10^{2} pfu/mL 1.10 x 10^{1} pfu/mL 1.10 pfu/mL | **Interference** **Interference** No Interference |
| **Technique-specific Substances, Transport Media** | | |
| Bleach | 0.5% v/v | No Interference |
| Ethanol | 0.2% v/v | No Interference |

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| Substance tested | Concentration tested | Result |
| --- | --- | --- |
| Puritan Fecal Opti-Swab Collection &Transport System with Cary-Blair Medium^{2} | 100% | No Interference |
| Puritan PurSafe DNA/RNA Preservative^{2} | 100% | No Interference |
| Sigma Fecal Transwab^{2} | 1 swab/2mL Cary Blair | No Interference |

$^{1}$This substance was tested by another FDA-cleared test that also detected Yersinia positive signals.

$^{2}$Note: Performance has not established for these transport media

### Microbial Interference:

A microbial interference study was conducted to assess whether non-target microorganisms/viruses can interfere with the detection of panel targets. Clinically relevant and challenging concentrations of non-target organisms (1.0E+06 x 10$^{6}$ CFU/mL for bacteria, 1.0E +05 cells/mL for yeast and 1.0E+05 TCID$_{50}$/mL for viruses) were individually co-spiked with each of the targeted pathogens (Table 6 above) at 3x LoD. Samples were prepared in negative stool matrix in Para-Pak C&S transport medium. Testing was performed in triplicate. Microbial interference was not observed as all combinations and replicates tested successfully detected all the QIAstat-Dx Gastrointestinal Panel 2 targets. Results from the study are presented in Table 8.

Table 8: Microbial Interference Testing Results

| Non-Target Microrganism/Virus Tested | Concentration tested | Result |
| --- | --- | --- |
| Aeromonas hydrophila | 1.0E+06 units/mL | No Interference |
| Bacteroides vulgatus | 1.0E+06 units/mL | No Interference |
| Bifidobacterium bifidum | 1.0E+06 units/mL | No Interference |
| Enterovirus Species D, Serotype EV-D68 | 1.0E+05 units/mL | No Interference |
| Non-pathogenic E. coli | 1.0E+06 units/mL | No Interference |
| Helicobacter pylori | 1.0E+06 units/mL | No Interference |
| Saccharomyces cerevisiae (deposited as S. boulardii) | 1.0E+05 units/mL | No Interference |

### Competitive Interference

The potential for competitive interference was evaluated for a subset of analytes detected by the QIAstat-Dx Gastrointestinal Panel 2. The samples were prepared as a combination of two targeted pathogens in stool matrix (negative pooled stools resuspended in Para-Pak C&S transport medium) with one pathogen spiked at a high concentration (50x LoD) and the other spiked at a low concentration (3x LoD). Testing was conducted in triplicate. No false positives or false negative results were observed during the study. Results are presented in Table 9.

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**Table 9: QIAstat-Dx Gastrointestinal Panel 2 Detection Rate Results for Competitive Interference**

| Mix | Pathogen | Concentration tested x LoD | Concentration tested | Detection rate | Coinfection Detected |
| --- | --- | --- | --- | --- | --- |
| Norovirus 50x - Rotavirus 3x | Norovirus GI/GII | 50x | 4.5E+05 copies/mL | 3/3 | Yes |
| | Rotavirus A | 3x | 1.7E+04 copies/mL | 3/3 | Yes |
| Norovirus 3x - Rotavirus 50x | Norovirus GI/GII | 3x | 2.7E+04 copies/mL | 3/3 | Yes |
| | Rotavirus A | 50x | 2.9E+05 copies/mL | 3/3 | Yes |
| Giardia 50x - Adenovirus 3x | Giardia lamblia | 50x | 7.2 E+05 copies/mL | 3/3 | Yes |
| | Adenovirus F40/F41 | 3x | 2.9E+03 copies/mL | 3/3 | Yes |

\*Target only applicable for Para-Pak C&S samples

### *Analytical Reactivity (Inclusivity)*

Analytical Reactivity (Inclusivity) testing was conducted to assess the ability of the QIAstat-Dx Gastrointestinal panel 2 to detect gastrointestinal pathogen isolates/strains that were selected based on clinical relevance as well as temporal, geographical and phylogenetic diversity. Samples were prepared with pathogen mixes in negative stool matrix collected in Para-Pak C&S transport medium. Based on the previously established LoD for each assay target, three serial log dilutions were tested with four replicates per concentration and per strain/isolate. The lowest concentration for which all four replicates were detected was identified and two more replicates were tested for a total of six sample replicates. Most pathogen strains evaluated (104/114) were detected at $\leq 3$-fold of the corresponding LoD reference strain. 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 when tested at a concentration of $< 3x$ LoD. Testing of these strains at 10x LoD generated the expected positive resulted for all replicates. For each strain evaluated in the study, the lowest concentration (based on a factor of LoD) that generated positive results for 6/6 replicates are presented in tables 10-26 below. LoD reference strains are shown in bold for each targeted analyte.

An additional evaluation of inclusivity for the QIAstat-Dx GI Panel 2 targets was conducted using *in silico* analysis of bacterial, viral, and parasite strains with available sequences in the database. All single primers and probes used for detection of each assay target included in the QIAstat-Dx Gastrointestinal Panel 2 were analyzed to predict the reactivity of the panel using BLAST. Further analysis of the predicted reactivity was performed through alignment

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analysis including both entire genome sequences and specific target gene sequences available from the identified BLAST accession hits. Results from in silico analysis are presented in Table 27 below.

Based on the results observed for inclusivity in vitro (wet) testing and in silico analysis, the QIAstat-Dx GI Panel 2 primers and probes are inclusive for each targeted analyte for clinically relevant strains, including species, subspecies, subtypes, serotypes or serovars, as applicable.

Table 10: Inclusivity, Campylobacter strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Factor of LoD |
| --- | --- | --- | --- | --- | --- |
| Campylobacter | Campylobacter coli | 76-GA2 [LMG 21266] | ATCC | 43478^{1} | 1x LoD |
| | Campylobacter coli | Z293 | ZeptoMetrix | 0804272 | 1x LoD |
| | Campylobacter coli | CIP 7080 [1407, CIP 70.80] | ATCC | 335591 | 3x LoD |
| | Campylobacter jejuni | Z086 | ZeptoMetrix | 0801650^{1} | 1x LoD |
| | Campylobacter jejuni | subsp. jejuni RM3193 | ATCC | BAA-1234* | 0.1x LoD |
| | Campylobacter jejuni subsp. jejuni | O:19 HL7; D3180 | ATCC | BAA-218 | 0.1x LoD |
| | Campylobacter jejuni subsp. jejuni | AS-83-79 | ATCC | 33291 | 0.1x LoD |
| | Campylobacter jejuni subsp. doylei | NCTC 11951 | ATCC | 49349 | 0.1x LoD |
| | Campylobacter upsaliensis | NCTC 11541 | ZeptoMetrix | 0801999^{1} | 1x LoD |
| | Campylobacter upsaliensis | RM 3195 (1994) | ATCC | BAA-1059 | 0.3x LoD |
| | Campylobacter upsaliensis | NCTC 11541 [C231] | ATCC | 43954 | 1x LoD |

$^{1}$Strain tested during LoD verification study.

Table 11: Inclusivity, Plesiomonas shigelloides strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Factor of LoD |
| --- | --- | --- | --- | --- | --- |
| Plesiomonas shigelloides | Plesiomonas shigelloides | Z130 | ZeptoMetrix | 0801899^{1} | 1x LoD |
| | Plesiomonas shigelloides | GNI 14 | ATCC | 51903 | 1x LoD |

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| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Factor of LoD |
| --- | --- | --- | --- | --- | --- |
| | Plesiomonas shigelloides | CDC 3085-55 [Bader M51, NCIB 9242, NCTC 10360, RH 798] | ATCC | 14029¹ | 0.3x LoD |

¹Strain tested during LoD verification study.

Table 12: Inclusivity, Salmonella strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Factor of LoD |
| --- | --- | --- | --- | --- | --- |
| Salmonella | Salmonella enterica | Serovar Typhimurium Z005 | ZeptoMetrix | 0801437¹ | 1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Bareilly | NCTC | NC05745 | 1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar typhi, Z152 | ZeptoMetrix | 0801933 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Enteridis, CDC K-1891 [ATCC 25928] | ATCC | 13076 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Infantis, MZ1479 [SARB27] | ATCC | BAA-1675 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Montevideo, G4639 | ATCC | BAA-710 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Javiana | NCTC | NC06495 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Thompson | NCTC | NC08496 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Saintpaul | ATCC | 9712 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Berta | NCTC | NC05770 | 0.1x LoD |
| | Salmonella enterica | Subsp. Salame, II NCTC 10310 [JT945, SS140/61] | ATCC | 700151 | 0.1x LoD |
| | Salmonella enterica | Subsp. diarizonae IIIb, 62 | ATCC | 29934 | 0.1x LoD |
| | Salmonella enterica | Subsp. houtenae IV, CIP 82.32 [264.66] | ATCC | 43974 | 0.1x LoD |
| | Salmonella enterica | Subsp. Indica VI, CIP 102501 [F. Kauffmann 1240] | ATCC | 43976 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Agona, CDC 873 [CDC 1111-61] | ATCC | 51957 | 0.1x LoD |

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| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Factor of LoD |
| --- | --- | --- | --- | --- | --- |
| | Salmonella enterica | Subsp. Enterica, serovar Muenchen, 54 | ATCC | 8388 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Oranienburg, E1093 | ATCC | 9239 | 0.1x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Paratyphi B var. Java, CDC 5 | ATCC | 51962 | 0.1x LoD |
| | Salmonella bongori | CIP 82.33 [1224.72] | ATCC | 43975 | 0.3x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Choleraesius, NCTC 5735 [1348, K.34] | ATCC | 13312^{1} | 0.3x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Newport, C487-69 | ATCC | 27869 | 0.3x LoD |
| | Salmonella enterica | Subsp. Enterica, 4, 5, 12:7 serovar Typhimurium | NCTC | NC13952 | 0.3x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Braenderup | ATCC | 700136 | 0.3x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Anatum | NCTC | NC05779 | 0.3x LoD |
| | Salmonella enterica | Subsp. arizonae IIIa, NCTC 7311 [CDAI 426] | ATCC | 700156 | 0.3x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Heidelberg, [16] | ATCC | 8326 | 0.3x LoD |
| | Salmonella enterica | Subsp. Enterica, serovar Mississippi, CDC 2012K-0487 | ATCC | BAA-2739 | 0.3x LoD |

$^{1}$ Strain tested during LoD verification study

Table 13: Inclusivity, Yersinia enterocolitica strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Yersinia enterocolitica | Yersinia enterocolitica | Z036 | ZeptoMetrix | 0801734^{1} | 1x LoD |
| | Yersinia enterocolitica | NTCC 11175, Biotype 4, serotype 3 (O:3) | ATCC | 700822* | 1x LoD |
| | Yersinia enterocolitica | 33114 [CCUG 11291, CCUG 12369, CIP 80.27, DSM 4780, LMG 7899, NCTC 12982], Biovar 1, O:8 | ATCC | 9610 | 1x LoD |
| | Yersinia enterocolitica | 0:9 | ATCC | 55075 | 3x LoD |

$^{1}$ Strain tested during LoD verification study

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Table 14: Inclusivity, Enteropathogenic E. coli (EPEC) strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Enteropathogenic E. coli (EPEC) | Enteropathogenic E. coli (EPEC) | O111:NM | ZeptoMetrix | 0801747^{1} | 1x LoD |
| | Enteropathogenic E. coli (EPEC) | 7.1493,O84:H28 | ZeptoMetrix | 0801938 | 1x LoD |
| | Enteropathogenic E. coli (EPEC) | Stoke W,O111:K58(B4):H- | ATCC | 33780 | 1x LoD |

$^{1}$ Strain tested during LoD verification study

Table 15: Inclusivity, Enterotoxigenic E. coli (ETEC) strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Enterotoxigenic E. coli (ETEC) lt/st | Enterotoxigenic E. coli (ETEC) lt/st | ST+, LT+ | ZeptoMetrix | 0801624^{1} | 1x LoD |
| | Enterotoxigenic E. coli (ETEC) lt/st | H10407,O78:H11,LT(+)/ctx A11(+) | ATCC | 35401^{1} | 0.3x LoD |
| | Enterotoxigenic E. coli (ETEC) lt/st | O27:H7,ST (+)/ LT (-) | SSI Diagnostica | 82173 | 0.1x LoD |
| | Enterotoxigenic E. coli (ETEC) lt/st | O115:H15,ST (+)/ LT (-) | SSI Diagnostica | 82174 | 3x LoD |
| | Enterotoxigenic E. coli (ETEC) lt/st | O169:H-,ST (-)/LT (+) | SSI Diagnostica | 82172 | 10x LoD^{2} |

$^{1}$Strain tested during LoD verification study

$^{2}$Testing at a lower concentration resulted in a detection rate of <100%

Table 16: Inclusivity, Enteroinvasive E. coli (EIEC)/Shigella strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Enteroinvasive E. coli (EIEC)/Shigella | Enteroinvasive E. coli (EIEC) | CDC EDL 1282, O29:NM | ATCC | 43892^{1} | 1x LoD |
| | Enteroinvasive E. coli (EIEC) | O172:H- | SSI Diagnostica | 82171 | 3x LoD |
| | Shigella sonnei | NCDC 1120-66 | ATCC | 25931^{1} | 1x LoD |
| | Shigella boydii (Serogroup C) | Z131 | ZeptoMetrix | 0801900 | 1x LoD |
| | Shigella flexneri (Serogroup B) | AMC 43-G-68 [EVL 82, M134] | ATCC | 9199 | 1x LoD |
| | Shigella flexneri (Serogroup B) | Z046 | ZeptoMetrix | 0801757 | 1x LoD |
| | Shigella sonnei (Serogroup D) | WRAIR I virulent | ATCC | 29930 | 1x LoD |

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| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| | Shigella sonnei (Serogroup D) | Z004 | ZeptoMetrix | 0801627 | 3x LoD |
| | Shigella boydii (Serogroup C) | AMC 43-G-58 [M44 (Type 170)] | ATCC | 9207 | 10x LoD^{2} |

$^{1}$Strain tested during LoD verification study

$^{2}$Testing at a lower concentration resulted in a detection rate of <100%

Table 17: Inclusivity, Shiga-like toxin-producing E. coli (STEC) (stx2-carrier strains)

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Shiga-like toxin producing E. coli (STEC) - stx1/stx2 | Shiga-like toxin producing E. coli (STEC) - stx1/stx2 | O157:H7; EDL933 | ZeptoMetrix | 0801622^{1} | 1x LoD |
| | Shiga-like toxin producing E. coli (STEC) - stx1 | O26:H4,stx1 (+) | ZeptoMetrix | 0801748^{1} | 1x LoD |
| | Shiga-like toxin producing E. coli (STEC) - stx1/stx2 | Reference ATCC® 35150 (EDL 931),O157:H7,stx1 (+), stx2 (+) | Microbiologics | 617 | 3x LoD |
| | Shiga-like toxin producing E. coli (STEC) - stx1 | Reference CDC 00-3039,O45:H2,unknown | Microbiologics | 1098 | 1x LoD |
| | Shiga-like toxin producing E. coli (STEC) - stx1 | O103:H2,stx1 (+) | SSI Diagnostica | 82170 | 3x LoD |
| | Shiga-like toxin producing E. coli (STEC) - stx1/stx2 | O22:H8,stx1c (+), stx2b (+) | SSI Diagnostica | 91350 | 1x LoD |
| | Shiga-like toxin producing E. coli (STEC) - stx2 | O92,O107:K+:H48, stx2d (+) | SSI Diagnostica | 91352 | 10x LoD^{2} |
| | Shiga-like toxin producing E. coli (STEC) - stx2 | O101:K32:H-,stx2e (+) | SSI Diagnostica | 91354 | 0.3x LoD |
| | Shiga-like toxin producing E. coli (STEC) - stx1/stx2 | O128ac:H-,stx2f (+) | SSI Diagnostica | 91355 | 10x LoD^{2} |
| | Shiga-like toxin producing E. coli (STEC) - stx2 | O26:H11,stx2a (+) | SSI Diagnostica | 95211 | 1x LoD |
| | Shiga-like toxin producing E. coli (STEC) - stx1 | O8 ,stx1d (+) | SSI Diagnostica | 91349 | 1x LoD |

$^{1}$Strain tested during LoD verification study

$^{2}$Testing at a lower concentration resulted in a detection rate of <100%

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Table 18: Inclusivity, Shiga-like toxin producing E. coli (STEC) O157 strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalogue ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Shiga-like toxin producing E. coli (STEC) O157 | Shiga-like toxin producing E. coli (STEC) - O157 | O157:H7; EDL933 | ZeptoMetrix | 0801622^{1} | 1x LoD |
| | Shiga-like toxin producing E. coli (STEC) O157 | O128ac:H-,stx2f (+) | SSI Diagnostica | 91355^{2} | 10x LoD^{3} |
| | Shiga-like toxin producing E. coli (STEC) O157 | Reference ATCC 35150 (EDL 931), O157:H7, stx1 (+), stx2 (+) | Microbiologics | 617 | 1x LoD |

$^{1}$Strain tested during LoD verification study

$^{2}$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 O157 in both QIAstat-Dx and FilmArray device

$^{3}$Testing at a lower concentration resulted in a detection rate of <100%

Table 19: Inclusivity, Cryptosporidium strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Cryptosporidium | Cryptosporidium parvum | Iowa isolate | Waterborne | P102C^{1} | 1x LoD |
| | Cryptosporidium hominis | n/a | Public Health Wales | Clinical sample; UKM 84^{1} | 0.01x LoD |
| | Cryptosporidium parvum | – | ATCC | PRA-67DQ (isolated genomic DNA) | <0.01 LoD |
| | Cryptosporidium meleagridis | – | Public Health Wales | Clinical sample; UKMEL 14 | <0.01 LoD |
| | | | | | |

$^{1}$ Strain tested during LoD verification study

Table 20: Inclusivity, Cyclospora cayetanensis strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalogue ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Cyclospora cayetanensis | Cyclospora cayetanensis | n/a | Clinical sample | LAC2825^{1} | 1x LoD |
| | Cyclospora cayetanensis | n/a | Clinical sample | LAC2827^{1} | 1x LoD |
| | Cyclospora cayetanensis | – | ATCC | PRA-3000SD | 1x LoD |

$^{1}$ Strain tested during LoD verification study

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**Table 21: Inclusivity, *Entamoeba histolytica* strains**

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalogue ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| *Entamoeba histolytica* | *Entamoeba histolytica* | HM-1:IMSS (Mexico City 1967) | ATCC | 30459^{1} | 1x LoD |
| | *Entamoeba histolytica* | HK-9 (Korea) | ATCC | 30015^{1} | 1x LoD |
| | *Entamoeba histolytica* | – | Vall d'Hebrón | Clinical sample | 1x LoD |

$^{1}$ Strain tested during LoD verification study

**Table 22: Inclusivity, *Giardia lamblia* strains**

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalogue ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| *Giardia lamblia* | *Giardia lamblia* | Portland -1 (Portland, OR, 1971) | ATCC | 30888^{1} | 1x LoD |
| | *Giardia lamblia* | WB (Bethesda, MD, 1979) | ATCC | 30957^{1} | 1x LoD |
| | *Giardia intestinalis* | H3 isolate | Waterborne | P101 | 1x LoD |

$^{1}$ Strain tested during LoD verification study

**Table 23: Inclusivity, Adenovirus F40/F41 strains**

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Adenovirus F40/F41 | Human Adenovirus F41 | Tak | ZeptoMetrix | 0810085CF^{1} | 1x LoD |
| | Human Adenovirus F41 | Tak (73-3544) | ATCC | VR-930 | 10x LoD^{2} |
| | Human Adenovirus F40 | Dugan [79-18025] | ATCC | VR-931 | 10x LoD^{2} |
| | Human Adenovirus Type 40 | Dugan | ZeptoMetrix | 0810084CF^{1} | 3x LoD |

$^{1}$Strain tested during LoD verification study

$^{2}$Testing at a lower concentration resulted in a detection rate of <100%

**Table 24: Inclusivity, Astrovirus strains**

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Astrovirus | Human Astrovirus | ERE IID 2371 (type 8) | ZeptoMetrix | 0810277CF^{1} | 1x LoD |
| | Human Astrovirus | HAstV-1 | Universitat de Barcelona | Clinical sample; 160521599 | 1x LoD |

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| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| | Human Astrovirus | ERE IID 2868 (type 4) | ZeptoMetrix | 0810276CF^{1} | 1x LoD |
| | Human Astrovirus | HAstV-3 | Universitat de Barcelona | Clinical sample; 151601306 | 1x LoD |

$^{1}$ Strain tested during LoD verification study

Table 25: Inclusivity, Norovirus GI/GII strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Norovirus GI/GII | Human Norovirus Genogroup 1 | Recombinant GI.1 | ZeptoMetrix | 0810086CF^{1} | 1x LoD |
| | Human Norovirus Genogroup 1 | – | Indiana University Health | Clinical sample; IU3156 | 1x LoD |
| | Human Norovirus Genogroup 1 | – | Indiana University Health | Clinical sample; IU3220 | 1x LoD |
| | Human Norovirus Genogroup 1 | – | TriCore Reference Laboratories | Clinical sample; TC4274 | 3x LoD |
| | Human Norovirus Genogroup 2 | Recombinant GII.4 | ZeptoMetrix | 0810087CF^{1} | 1x LoD |
| | Human Norovirus Genogroup 2 | GII.2 | Vall d'Hebrón | Clinical sample; 198058327 | 1x LoD |
| | Human Norovirus Genogroup 2 | GII.4 | Universitat de Barcelona | Clinical sample; N26.2TA | 1x LoD |
| | Human Norovirus Genogroup 2 | – | Lacny Hospital | Clinical sample; LAC2019 | 1x LoD |
| | Human Norovirus Genogroup 2 | – | Nationwide Children's Hospital | Clinical sample; NWC6063 | 1x LoD |
| | Human Norovirus Genogroup 2 | GII.6 | QIAGEN Barcelona (STAT-Dx) | Clinical sample; GI 12 | 3x LoD |
| | Human Norovirus Genogroup 2 | – | Lacny Hospital | Clinical sample; LAC2133 | 10x LoD^{2} |
| | Human Norovirus Genogroup 2 | – | Lacny Hospital | Clinical sample; LAC2074 | 10x LoD^{2} |

$^{1}$Strain tested during LoD verification study

$^{2}$Testing at a lower concentration resulted in a detection rate of <100%

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Table 26: Inclusivity, Rotavirus A strains

| QIAstat-Dx target | Pathogen | Strain | Supplier | Catalog ID | Times LoD |
| --- | --- | --- | --- | --- | --- |
| Rotavirus A | Human Rotavirus A | 69M | ZeptoMetrix | 0810280CF^{1} | 1x LoD |
| | Human Rotavirus A | Wa, G1P1A[8] | ZeptoMetrix | 0810041CF^{1} | 1x LoD |
| | Human Rotavirus A | DS-1, G2P1B[4] | ATCC | VR-2550 | 1x LoD |
| | Human Rotavirus A | Va70 | ZeptoMetrix | 0810281CF | 1x LoD |
| | Human Rotavirus A | RRV | ZeptoMetrix | 0810530CF | 10x LoD^{2} |

$^{1}$Strain tested during LoD verification study

$^{2}$Testing at a lower concentration resulted in a detection rate of <100%

Table 27: Microorganisms/Viruses with Predicted Reactivity Based on In silico Analysis

| QIAstat-Dx GI Panel 2 Target | Microorganisms/viruses 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:l,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*) |
| *Plesiomonas shigelloides* | *Plesiomonas shigelloides* (e.g. strains NCTC10360, ATCC 14029T, R4605035) |
| *Yersinia enterocolitica* | *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) ^{δ} | 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-O157 serotypes (O111:NM, O111:H-, O26:H11, O145:NM, O145:H28, O45:H2, |

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| QIAstat-Dx GI Panel 2 Target | Microorganisms/viruses with predicted reactivity |
| --- | --- |
| | 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: *stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, 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) O157^{β} | *Escherichia coli* O157 including: STEC O157:H7 strains (e.g. EDL933) and *E. coli* O157: non-H7 groups including non-Shiga-toxigenic *E. coli* O157 bacteria (e.g. serotype O157:H45) |
| **Parasites** | |
| *Cryptosporidium*^{δ} | 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* |
| *Cyclospora cayetanensis* | *Cyclospora cayetanensis* (including strains LG, CY9, NP20 and NP21) * |
| *Entamoeba histolytica* | *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]* and G12P[8]* |

* Certain sequences are predicted to be detected with reduced sensitivity due to the presence of a reduced number of mismatches at critical positions of the primer-probe design.

&The assay is not predicted to detect bacteria carrier of Heat-labile enterotoxin gene subtype LT-II and/or of Heat-stable enterotoxin gene variant Stb e.

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

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

β Only applicable for samples with Para-Pak C&S collection device

### 4. Assay Reportable Range:

Not applicable

### 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

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### *Sample Stability*

A study was conducted to evaluate the stability of stool specimens collected in Para-Pak C&S or FecalSwab transport media that are stored under claimed storage conditions (i.e., up to four days at room temperature (15°C to 25°C) or four days at refrigerated conditions (2°C to 8°C)). After storage under each storage condition, samples were tested with QIAstat-Dx Gastrointestinal Panel 2. For Para-Pak C&S, at least one strai…