The QIAstat-Dx Respiratory Panel Plus is a multiplexed nucleic acid test intended for use with the QIAstat-Dx system for the simultaneous in vitro qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of respiratory tract infection, including SARS-CoV-2. The following organism types and subtypes are identified using the QIAstat-Dx Respiratory Panel Plus: Adenovirus, Human Coronavirus 229E, Human Coronavirus HKU1, Human Coronavirus NL63, Human Coronavirus OC43, Human Metapneumovirus, Influenza A, Influenza A H1, Influenza A H1N1 pdm09, Influenza B, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4, Respiratory Syncytial Virus, Human Rhinovirus/Enterovirus (not differentiated), Severe Acute Respiratory Syndrome Coronavirus (SARS-COV-2), Bordetella pertussis, Chlamydophila pneumoniae and Mycoplasma pneumoniae. Nucleic acids from viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and identifying specific viral and bacterial nucleic acids from individuals presenting with signs and symptoms of a respiratory infection aids in the diagnosis of respiratory infection, if used in conjunction with other clinical, epidemiological and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Negative results in the presence of a respiratory illness may be due to infection with pathogens that are not detected by the test or due to lower respiratory tract infection that is not detected by a NPS specimen. Conversely, positive results are indicative of the identified microorganism, but do not rule out co-infection with other pathogens not detected by the QIAstat-Dx Respiratory Panel Plus. The agent(s) detected by the QIAstat-Dx Respiratory Panel Plus may not be the definite cause of disease. The use of additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.
Device Story
Multiplexed nucleic acid test for respiratory pathogens; uses QIAstat-Dx Analyzer 1.0. Input: nasopharyngeal swab in Universal Transport Media. Process: automated cartridge-based system; pneumatic microfluidics; mechanical/chemical cell lysis; membrane-based nucleic acid purification; real-time multiplex PCR (rtPCR) in eight reaction chambers. Output: qualitative detection/identification of targets displayed on analyzer screen. Used in clinical settings by laboratory personnel. Results aid diagnosis in conjunction with clinical/epidemiological findings. Benefits: rapid (approx. 1 hour) simultaneous identification of multiple pathogens to inform patient management.
Clinical Evidence
Prospective clinical study (n=615) compared subject device to an FDA-cleared molecular respiratory panel. SARS-CoV-2 performance: PPA 96.8% (95% CI: 89.0%–99.6%), NPA 99.8% (95% CI: 99.0%–100.0%). Representative panel study (n=190) confirmed performance equivalency for original panel analytes (Influenza B, Coronavirus OC43, Parainfluenza 3, Rhinovirus/Enterovirus, Adenovirus, B. pertussis) compared to the original QIAstat-Dx Respiratory Panel.
Indicated for individuals with clinical signs/symptoms of respiratory tract infection. Uses nasopharyngeal swabs (NPS) for qualitative detection of viral/bacterial nucleic acids (Adenovirus, Coronaviruses, Metapneumovirus, Influenza A/B, Parainfluenza, RSV, Rhinovirus/Enterovirus, SARS-CoV-2, B. pertussis, C. pneumoniae, M. pneumoniae).
Regulatory Classification
Identification
A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) The intended use in the labeling required under § 809.10 of this chapter must include a description of the following: Analytes and targets the device detects and identifies, the specimen types tested, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the intended use locations including testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate.
(2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device.
(3) The labeling required under § 809.10(b) of this chapter must include:
(i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens;
(ii) Detailed descriptions of the performance characteristics of the device for each specimen type claimed in the intended use based on analytical studies including the following, as applicable: Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, precision, reproducibility, and clinical studies;
(iii) Detailed descriptions of the test procedure(s), the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing;
(iv) A warning statement that viral culture should not be attempted in cases of positive results for SARS-CoV-2 and/or any similar microbial agents unless a facility with an appropriate level of laboratory biosafety (
*e.g.,* BSL 3 and BSL 3+, etc.) is available to receive and culture specimens; and(v) A prominent statement that device performance has not been established for specimens collected from individuals not identified in the intended use population (
*e.g.,* when applicable, that device performance has not been established in individuals without signs or symptoms of respiratory infection).(vi) Limiting statements that indicate that:
(A) A negative test result does not preclude the possibility of infection;
(B) The test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician;
(C) There is a risk of incorrect results due to the presence of nucleic acid sequence variants in the targeted pathogens;
(D) That positive and negative predictive values are highly dependent on prevalence;
(E) Accurate results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe proper procedures in any one of these steps can lead to incorrect results; and
(F) When applicable (
*e.g.,* recommended by the Centers for Disease Control and Prevention, by current well-accepted clinical guidelines, or by published peer-reviewed literature), that the clinical performance may be affected by testing a specific clinical subpopulation or for a specific claimed specimen type.(4) Design verification and validation must include:
(i) Detailed documentation, including performance results, from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. The clinical study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained using a comparator that FDA has determined is appropriate. Detailed documentation must include the clinical study protocol (including a predefined statistical analysis plan), study report, testing results, and results of all statistical analyses.
(ii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel respiratory pathogen isolates or strains (
*e.g.,* regular review of published literature and periodic in silico analysis of target sequences to detect possible mismatches). All results of this protocol, including any findings, must be documented and must include any additional data analysis that is requested by FDA in response to any performance concerns identified under this section or identified by FDA during routine evaluation. Additionally, if requested by FDA, these evaluations must be submitted to FDA for FDA review within 48 hours of the request. Results that are reasonably interpreted to support the conclusion that novel respiratory pathogen strains or isolates impact the stated expected performance of the device must be sent to FDA immediately.(iii) A detailed description of the identity, phylogenetic relationship, and other recognized characterization of the respiratory pathogen(s) that the device is designed to detect. In addition, detailed documentation describing how to interpret the device results and other measures that might be needed for a laboratory diagnosis of respiratory infection.
(iv) A detailed device description, including device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including molecular target(s) for each analyte, design of target detection reagents, rationale for target selection, limiting factors of the device (
*e.g.,* saturation level of hybridization and maximum amplification and detection cycle number, etc.), internal and external controls, and computational path from collected raw data to reported result (*e.g.,* how collected raw signals are converted into a reported signal and result), as applicable.(v) A detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.
(vi) For devices intended for the detection and identification of microbial agents for which an FDA recommended reference panel is available, design verification and validation must include the performance results of an analytical study testing the FDA recommended reference panel of characterized samples. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens, the design verification and validation must include a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the Influenza A and B viruses that the device is designed to detect, a description of how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory identification of Influenza A or B virus and of specific Influenza A virus subtypes, and a description of the clinical and epidemiological parameters that are relevant to a patient case diagnosis of Influenza A or B and of specific Influenza A virus subtypes. An evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(5) When applicable, performance results of the analytical study testing the FDA recommended reference panel described in paragraph (b)(4)(vi) of this section must be included in the device's labeling under § 809.10(b) of this chapter.
(6) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens in addition to detection of SARS-CoV-2 and similar microbial agents, the required labeling under § 809.10(b) of this chapter must include the following:
(i) Where applicable, a limiting statement that performance characteristics for Influenza A were established when Influenza A/H3 and A/H1-2009 (or other pertinent Influenza A subtypes) were the predominant Influenza A viruses in circulation.
(ii) Where applicable, a warning statement that reads if infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to State or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
(iii) Where the device results interpretation involves combining the outputs of several targets to get the final results, such as a device that both detects Influenza A and differentiates all known Influenza A subtypes that are currently circulating, the device's labeling must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required followup actions or retesting in the case of an unusual or unexpected device result.
(iv) A limiting statement that if a specimen yields a positive result for Influenza A, but produces negative test results for all specific influenza A subtypes intended to be differentiated (
*i.e.,* H1-2009 and H3), this result requires notification of appropriate local, State, or Federal public health authorities to determine necessary measures for verification and to further determine whether the specimen represents a novel strain of Influenza A.(7) If one of the actions listed at section 564(b)(1)(A) through (D) of the Federal Food, Drug, and Cosmetic Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those influenza viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized influenza viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's website, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the website containing this information and must allow unrestricted viewing access.
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FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY AND INSTRUMENT
## I Background Information:
A 510(k) Number
K233100
B Applicant
QIAGEN GmbH
C Proprietary and Established Names
QIAstat-Dx Respiratory Panel Plus
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QOF | Class II | 21 CFR 866.3981 - Device To Detect And Identify Nucleic Acid Targets In Respiratory Specimens From Microbial Agents That Cause The SARS-Cov-2 Respiratory Infection And Other Microbial Agents When In A Multi-Target Test | MI - Microbiology |
## II Submission/Device Overview:
### A Purpose for Submission:
QIAGEN is formally submitting this Traditional 510(k) Premarket Notification for the QIAstat-Dx Respiratory Panel Plus, which is a modified version of the previously cleared QIAstat-Dx Respiratory Panel (K183597). The modified panel received an EUA (EUA200075) and this premarket notification is to receive 510(k) clearance of the EUA assay version.
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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B Measurand:
The QIAstat Respiratory Panel Plus detects and identifies nucleic acids from the following pathogens:
Severe Acute Respiratory Syndrome (SARS)-Coronavirus-2 (SARS-CoV-2) [RNA-dependent-RNA-polymerase gene (RdRp) & Envelope protein gene (E)]
Adenovirus [Hexon gene]
Human Coronavirus 229E [Membrane protein gene (M)]
Human Coronavirus HKU1 [Nucleocapsid protein gene (N)]
Human Coronavirus NL63 [Nucleocapsid protein gene (N)]
Human Coronavirus OC43 [Nucleocapsid protein gene (N)]
Human Metapneumovirus A+B [A/B Nucleoprotein gene (N)]
Influenza A [Matrix gene (M)]
Influenza A H1 [Hemagglutinin gene (HA)]
Influenza A H1N1 pdm09 [Neuraminidase gene (NA)]
Influenza A H3 [Hemagglutinin gene (HA)]
Influenza B [Nucleoprotein gene (M)]
Parainfluenza virus 1 [Hemagglutinin-neuraminidase glycoprotein gene (HN)]
Parainfluenza virus 2 [Hemagglutinin-neuraminidase glycoprotein gene (HN)]
Parainfluenza virus 3 [Phosphoprotein gene (P)]
Parainfluenza virus 4 [Nucleocapsid protein gene (N)]
Respiratory Syncytial Virus A+B [Matrix protein gene (M)]
Rhinovirus/Enterovirus* [5’-UTR region]
Bordetella pertussis [Transposase Insertion Sequence (IS481)]
Chlamydophila pneumoniae [Major outer membrane protein gene (ompA)]
Mycoplasma pneumoniae [Cytadhesin gene (P1)]
*Both Rhinovirus and Enterovirus are detected but not differentiated.
C Type of Test:
Multiplexed real-time polymerase chain reaction (RT-PCR) nucleic acid assay for use with the QIAstat-Dx System for the qualitative detection of viral and bacterial pathogens in individuals with signs and symptoms of respiratory tract infection.
III Intended Use/Indications for Use:
Intended Use(s):
The QIAstat-Dx Respiratory Panel Plus is a multiplexed nucleic acid test intended for use with the QIAstat-Dx system for the simultaneous in vitro qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of respiratory tract infection, including SARS-CoV-2.
The following organism types and subtypes are identified using the QIAstat-Dx Respiratory Panel Plus: Adenovirus, Human Coronavirus 229E, Human Coronavirus HKU1, Human Coronavirus NL63, Human Coronavirus OC43, Human Metapneumovirus, Influenza A, Influenza A H1, Influenza A H1N1 pdm09, Influenza A H3, Influenza B, Parainfluenza virus 1,
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Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4, Respiratory Syncytial Virus, Human Rhinovirus/Enterovirus (not differentiated), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), Bordetella pertussis, Chlamydophila pneumoniae and Mycoplasma pneumoniae.
Nucleic acids from viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and identifying specific viral and bacterial nucleic acids from individuals presenting with signs and symptoms of a respiratory infection aids in the diagnosis of respiratory infection, if used in conjunction with other clinical, epidemiological and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions.
Negative results in the presence of a respiratory illness may be due to infection with pathogens that are not detected by the test or due to lower respiratory tract infection that is not detected by a NPS specimen.
Conversely, positive results are indicative of the presence of the identified microorganism, but do not rule out co-infection with other pathogens not detected by the QIAstat-Dx Respiratory Panel Plus. The agent(s) detected by the QIAstat-Dx Respiratory Panel Plus may not be the definite cause of disease.
The use of additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.
A. Indication(s) for Use:
See Intended Use (above)
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 Respiratory Panel Plus is part of the QIAstat-Dx system and works only with the QIAstat-Dx Analyzer 1.0.
IV. Device/System Characteristics:
A. Device Description:
The QIAstat-Dx Respiratory Panel Plus is part of the QIAstat-Dx system and works with the QIAstat-Dx Analyzer 1.0. The QIAstat-Dx Respiratory Panel Plus Cartridge is a single-use
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cartridge that includes all reagents needed for nucleic acid extraction, nucleic acid amplification, and detection of bacteria and viruses (or their subtypes), including SARS-CoV-2, that cause respiratory symptoms. Testing requires a small specimen volume and minimal hands-on time, and the results are available in approximately one hour.
The QIAstat-Dx Respiratory Panel Plus is intended to be used with one (1) nasopharyngeal swab (NPS) eluted in Universal Transport Media (UTM), which is not provided with the QIAstat-Dx Respiratory Panel Plus.
The QIAstat-Dx Respiratory panel initially received FDA clearance in May 2019 (K183597). In March 2020, the QIAstat-Dx Respiratory SARS-CoV-2 Panel received EUA authorization which included the addition of SARS-CoV-2 reagents to an independent reaction chamber (i.e., not multiplexed with other analyte reagents) and a reduction in concentration of a lysis reagent. The QIAstat-Dx Respiratory Panel Plus is identical to the QIAstat-Dx Respiratory SARS-CoV-2 Panel. The other analyte reagents are unmodified from that cleared in K183597.
## B Principle of Operation:
The test starts automatically upon insertion of the test cartridge and runs for approximately 1 hour. 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. All other tested but not detected analytes are listed in green. 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").
All the reagents required for the complete execution of the test are pre-loaded and self-contained in the QIAstat-Dx Respiratory Panel Plus cartridge. 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.
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 (TC) to their intended destinations. Following the introduction of the sample from a disposable transfer pipette, the following assay steps occur automatically and sequentially:
- Resuspension of air-dried internal control and Proteinase K (ProtK) enzyme using provided buffer and mixing with the liquid sample (IC Cavity and ProtK Cavity).
- Cell lysis using mechanical (rotation) and chemical (chaotropic and isotonic) means (lysis chamber).
- Membrane based nucleic acid purification from lysate by:
- Mixing lysate with binding buffer and capturing on the membrane (purification chamber).
- First washing of membrane to remove bound proteins (purification chamber and waste chamber).
- Second washing of membrane to leave only bound nucleic acids (purification chamber and waste chamber).
- Rinsing of Transfer Chamber (TC) using the rinsing buffer before introduction of the eluate (TC).
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- Drying of membrane with bound nucleic acids with air flow generated by a high flow vacuum pump (purification chamber).
- Elution of nucleic acids with elution buffer (purification chamber and TC).
- Mixing of the purified nucleic acid (eluate) with lyophilized “Master Mix” reagents (dry chemistry container and TC).
- Sequential transfer of defined aliquots of mixed eluate/Master Mix from the Transfer Chamber to each of eight Reaction Chambers containing the specified, air dried primers and probes. Note that each target specific probe contains a specific fluorescent dye that permits differentiation of target specific amplification within each Reaction Chamber.
- Within each Reaction Chamber, real-time, multiplex PCR (“rtPCR”) testing is performed. Increase in fluorescence (indicative of detection of each target analyte) is detected directly within each Reaction Chamber.
- The detected signal per fluorescent marker per Reaction Chamber is then used by the system software to generate the assay result.
## C Instrument Description Information:
1. Instrument Name:
QIAstat-Dx Analyzer 1.0, with software version 1.4 or higher.
2. Specimen Identification:
The QIAstat-Dx Analyzer is a fully automated instrument that is bi-directionally interfaced and accepts orders from the LIS system. While test orders can be manually programmed through the attached instrument computer, a LIS-generate barcode can also be scanned to initiate testing.
3. Specimen Sampling and Handling:
Nasopharyngeal swab (NPS) specimens collected in transport medium.
4. Calibration:
The QIAstat-Dx Analyzer 1.0 is provided factory calibrated and does not require user calibration.
5. Quality Control:
Control Materials
### a) External Controls
External Controls are not provided with the QIAstat-Dx Respiratory Panel Plus. The following information on external control testing is included in the Instructions for Use,
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"External controls are not provided with the QIAstat-Dx Respiratory Panel Plus. Quality control requirements should be performed in conformance with local, state, and/or federal regulations or accreditation requirements and your laboratory's standard quality control procedures."
External controls for performance studies were commercially obtained: NATtrol ZeptoMetrix Respiratory Verification Panel (cat. id NATRVP-QIA) and ZeptoMetrix NATtrol SARS-CoV-2 (E/ORF1ab) recombinant (cat. id. 0831043). These were diluted with negative transport medium prior to use. The negative sample was prepared using transport medium only. The controls yielded expected results on all days of testing.
## b) Internal Controls
The QIAstat-Dx Respiratory Panel Cartridge includes an Internal Control (IC), which is a titered lyophilized MS2 bacteriophage that provides verification that all steps of the analysis process including sample resuspension, lysis, nucleic acid purification, reverse transcription, and PCR were successful. The results screen displays a message indicating that the internal controls "Passed" when the test was run successfully. An IC message of "Failed" indicates that the internal control was not amplified; 'Positive' test results are still reported as positive, but all 'Negative' results are invalid.
## V Substantial Equivalence Information:
### A Predicate Device Name(s):
BioFire Respiratory Panel 2.1 (RP2.1)
### B Predicate 510(k) Number(s):
DEN200031
### C. Comparison with Predicate(s):
| Characteristic | Subject Device | Predicate |
| --- | --- | --- |
| Name | QIAstat-Dx Respiratory Panel Plus | BioFire Respiratory Panel 2.1 (RP2.1) |
| De Novo/510(k) No. | K233100 | DEN200031 |
| Regulation | 21 CFR 866.3981 | 21 CFR 866.3981 |
| Product Code | QOF | QOF |
| Device Class | Class II | Class II |
| Similarities | | |
| Intended Use | The QIAstat-Dx Respiratory Panel Plus is a multiplexed nucleic acid test intended for use with the QIAstat-Dx system for the simultaneous in vitro qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with | The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BioFire FilmArray 2.0 or BioFire FilmArray Torch systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained |
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| Characteristic | Subject Device | Predicate |
| --- | --- | --- |
| | clinical signs and symptoms of respiratory tract infection, including SARS-CoV-2.
The following organism types and subtypes are identified using the QIAstat-Dx Respiratory Panel Plus: Adenovirus, Human Coronavirus 229E, Human Coronavirus HKU1, Human Coronavirus NL63, Human Coronavirus OC43, Human Metapneumovirus, Influenza A, Influenza A H1, Influenza A H1N1 pdm09, Influenza A H3, Influenza B, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4, Respiratory Syncytial Virus, Human Rhinovirus/Enterovirus (not differentiated), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), Bordetella pertussis, Chlamydophila pneumoniae and Mycoplasma pneumoniae.
Nucleic acids from viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and identifying specific viral and bacterial nucleic acids from individuals presenting with signs and symptoms of a respiratory infection aids in the diagnosis of respiratory infection, if used in conjunction with other clinical, epidemiological and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions.
Negative results in the presence of a respiratory illness may be due to infection with pathogens that are not detected by the test or due to lower respiratory tract infection that is not detected by a NPS specimen.
Conversely, positive results are indicative of the presence of the identified microorganism, but do not rule out co-infection with other pathogens not detected by the QIAstat-Dx Respiratory Panel Plus. The agent(s) detected by the QIAstat-Dx Respiratory Panel Plus may not be the definite cause of disease. | from individuals suspected of respiratory tract infections, including COVID-19.
The following organism types and subtypes are identified using the BioFire RP2.1:
• Adenovirus,
• Coronavirus 229E,
• Coronavirus HKU1,
• Coronavirus NL63,
• Coronavirus OC43,
• Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2),
• Human Metapneumovirus,
• Human Rhinovirus/Enterovirus,
• Influenza A, including subtypes H1, H1-2009, and H3,
• Influenza B,
• Parainfluenza Virus 1,
• Parainfluenza Virus 2,
• Parainfluenza Virus 3,
• Parainfluenza Virus 4,
• Respiratory Syncytial Virus,
• Bordetella parapertussis (IS1001),
• Bordetella pertussis (ptxP),
• Chlamydia pneumoniae, and
• Mycoplasma pneumoniae
Nucleic acids from the respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection is indicative of the presence of the identified microorganism and aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by an NPS specimen. Positive results do not rule out |
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VI Standards/Guidance Documents Referenced:
Standards
- CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition
- CLSI EP07 Interference Testing in Clinical Chemistry – Third Edition
- CLSI EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents – September 2009 / EN 23640:2015 In vitro diagnostic medical devices. Evaluation of stability of in vitro diagnostic reagents
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- CLSI EP12-A2 User Protocol for Evaluation and test Performance – Second Edition
- CLSI EP05 Evaluation of Precision of Quantitative Measurement procedures – Third Edition
- CLSI MM17 Validation and Verification of Multiplex Nucleic Acid Assays – Second Edition
- CLSI MM03 Molecular Diagnostic Methods for Infectious Diseases – Third Edition
- ICH GCP E6(R2) Good Clinical Practice
- HIPAA - Health Insurance Portability and Accountability Act of 1996
- IEC 62304 Edition 1.1 2015-06 Medical device software - Software life cycle processes
Special Controls
- Class II Special Controls as per 21 CFR 866.3981
FDA Guidance Documents
- Guidance for Test Developers and FDA Staff, Policy for Evaluating Impact of Viral mutations on COVID-19 tests (February 2021).
- Guidance for Industry and FDA Staff, Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays (October 2009).
- Guidance for Industry and Food and Drug Administration Staff, Highly Multiplexed Microbiological/Medical Countermeasure In Vitro Nucleic Acid Based Diagnostic Devices (August 2014).
- Guidance for Industry and Food and Drug Administration Staff, Content of Premarket Submissions for Management of Cybersecurity in Medical Devices (October 2014).
- Guidance for Industry and Food and Drug Administration Staff. Content of Premarket Submissions for Device Software Functions (June 2023).
VII Performance Characteristics:
A Analytical Performance:
1. Precision/Reproducibility:
a. Within-laboratory precision
Within-laboratory precision was examined at one site using the QIAStat-Dx Respiratory Panel Plus assay using contrived panels in simulated NPS matrix of SARS-CoV-2 and a subset of representative organisms present on the original panel (Table 1). Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Matrix Equivalency Study, later in this document. Positive panels consisted of combinations of the target analytes at low (1x LoD) and moderate concentrations (3x LoD). The testing was performed across 5 non-consecutive days using two operators and 3 reagent lots (1 lot per replicate). Each operator performed 3
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replicates per analyte at each sample concentration across 3 instruments each day for a total of 90 datapoints per analyte and concentration (2 operators x 3 instruments x 3 replicates x 5 days). The results are shown in Table 1.
Table 1. Within-laboratory Precision Study – Qualitative Results
| Analyte (Strain/Isolate) | Analyte Level | % Positive (pos n/ valid n) | % Agreement with Expected Results (95% CI) |
| --- | --- | --- | --- |
| SARS-CoV-2 (England/02/2020) | 1x LoD | 96.7% (87/90) | 96.7% (90.6 – 99.3%) |
| | 3x LoD | 100% (92/92) a | 100% (96.1-100%) |
| | Negative | 0% (90/90) | 100% (96.0- 100%) |
| Influenza B (Taiwan/62) | 1x LoD | 98.9% (89/90) | 98.9% (94.0-100%) |
| | 3x LoD | 98.9% (91/92) a | 98.9% (94.1-100%) |
| | Negative | 0% (90/90) | 100% (96.0-100%) |
| Human coronavirus HKU1 | 1x LoD | 100% (90/90) | 100% (96.0-100%) |
| | 3x LoD | 100% (92/92) a | 100% (96.1-100%) |
| | Negative | 0% (90/90) | 100% (96.0-100%) |
| Parainfluenza virus 3 (C-243) | 1x LoD | 97.8% (88/90) | 97.8% (92.2-99.7%) |
| | 3x LoD | 100% (92/92) a | 100% (96.1-100%) |
| | Negative | 0% (90/90) | 100% (96.0-100%) |
| Rhinovirus (A2) | 1x LoD | 100% (90/90) | 100% (96.0-100%) |
| | 3x LoD | 100% (92/92) a | 100% (96.1-100%) |
| | Negative | 0% (90/90) | 100% (96.0-100%) |
| Adenovirus (B3) | 1x LoD | 96.7% (87/90) | 96.7% (90.6-99.3)% |
| | 3x LoD | 100% (92/92) a | 100% (96.1-100%) |
| | Negative | 0% (90/90) | 100% (96.0-100%) |
| Mycoplasma pneumoniae (PI 1428) | 1x LoD | 95.6% (86/90) | 95.6% (89.0-98.8%) |
| | 3x LoD | 97.8% (90/92) a | 97.8% (92.4-99.7%) |
| | Negative | 0% (90/90) | 100% (96.0-100%) |
a Two additional runs were performed due to partially invalidated runs.
SARS-CoV-2 samples were detected within performance expectations across all concentrations. All negative and low positive samples for the remaining analytes exhibited expected performance, achieving a percent agreement of 100% and at least 95.6%, respectively. Moderate positive samples achieved 100% expected positivity except influenza B (98.9%) and M. pneumoniae (97.8%). Notably, two tests were unexpectedly negative for M. pneumoniae and one test for influenza B. Unexpected negatives occurred across two lots and was attributed to a processing error. Repeat testing using the same cartridge lots and samples produced expected results. Overall, all panels met the acceptability criteria.
The mean and variability analysis between days, instruments, replicates, operators, lots and the overall (total) variability for Ct values for the SARS-CoV-2 analyte is shown in Table 2. Overall, the percent coefficient of variation (% CV) ranged from 0.0 – 2.3% with the greatest factor of variability attributed to between replicate Ct-variability at the 1x LoD level with a standard deviation of 0.8 Ct-value and 2.21% CV.
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Assay variability for the subset of other analytes included in the study did not exceed those in the original 510(k) K183597 clearance (data not shown). Overall, Ct variability was low, and the study demonstrates assay variability within an acceptable range.
Table 2. Within-Laboratory Precision Study – SARS-CoV-2 Ct Signal Variability Analysis
| Analyte | Analyte Level | N | Mean Ct | Between Operators | | Between Instrument | | Between Lots | | Between Replicates | | Between Days | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| SARS-CoV-2 | 1x LoD | 90 | 36.3 | 0.13 | 0.37 | 0.25 | 0.69 | 0.00 | 0.00 | 0.80 | 2.21 | 0.00 | 0.00 | 0.83 | 2.30 |
| | 3x LoD | 92 | 34.7 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.67 | 1.92 | 0.23 | 0.67 | 0.68 | 2.01 |
b. Reproducibility
Reproducibility was assessed in K183597 and thus was not repeated here. The Reproducibility Study results can be found in the publicly available K183597 Decision Summary.
2. Linearity:
Not applicable. This is a qualitative assay.
3. Analytical Specificity/Interference:
Analytical Reactivity (Inclusivity)
The QIAstat-Dx Respiratory Panel Plus configuration of multiple viral and bacterial respiratory pathogens is the same as the previously cleared device (QIAstat-Dx Respiratory Panel, K183597), except for the addition of reagents for SARS-CoV-2 detection and a concentration change to a lysis reagent. Inclusivity wet-testing for the remaining on-panel analytes was performed previously in K183597 and not repeated here.
a. Wet-Testing
During Limit of Detection studies, five variants of SARS-CoV-2 were tested and successfully detected. Therefore, no additional inclusivity wet-testing was performed.
b. In silico Analysis
QIAGEN routinely evaluates the overall inclusivity of the entire set of SARS-CoV-2 available genomes, including all relevant variants and lineages. This includes (1) Inclusivity Analysis and mismatch detection among SARS-CoV-2 strains and (2) the same analysis focused on specific SARS-CoV-2 variants or lineages, evaluating the possible effect of detected mismatches on the QIAstat-Dx performance. Only critical mismatches were evaluated in the laboratory. Laboratory testing was performed using artificial genomic fragments corresponding to targeted regions and including the corresponding mismatch.
In total, 8,118,241 available genomes between January 1, 2020 and June 5, 2022 from around the globe were analyzed for inclusivity. Of them, 7,932,071 (97.71%) presented
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no evidence of mismatches in the targeted regions. The remainder of genomes (2.01%) presented mismatches among the sequence in the binding region, but only 19,045 (0.23%) presented a mismatch in a critical position (i.e., near the 3'-end of any primer) with a frequency >0.2%. Genetic patterns included among those sequences were evaluated in laboratory testing, with no loss of performance at LoD level. Consequently, 100% of sequences analyzed were predicted to be detected.
Variants of Concern, of Interest and Under Investigation (VOCs, VOIs, VUI respectively) were also evaluated from the 8,118,241 sequences. After classifying all previously analyzed genomes into lineages based on GISAID and PANGO classifications, the following variants were included: Alpha (9 clades), Beta (5 clades), Gamma (23 clades), Lambda (2 clades), Mu (4 clades), Delta (242 clades), Omicron (73 clades). As a result, all variants and lineages described are 100% predicted to be detected.
For the rest of the analytes detected by the QIAstat-Dx Respiratory Panel Plus, an updated in silico analysis was performed to confirm previous device inclusivity results. All single primers and probes corresponding to targets included in the QIAstat-Dx Respiratory Panel Plus (non-SARS-CoV-2 targets) were analyzed using BLAST.
All QIAstat-Dx Respiratory Panel Plus primers and probes are predicted to be inclusive for all clinical prevalent and relevant strains for each pathogen against all available sequences in the NCBI data base.
QIAGEN has created a dedicated Post Market Surveillance (PMS) team with the objective of periodically analyzing all new genomic sequences of SARS-CoV-2 to detect any mutation pattern that may affect the QIAstat-Dx assay performance and create an action plan accordingly.
## Cross-Reactivity (Exclusivity) and Competitive/Microbial Interference
The potential for cross-reactivity between all QIAstat-Dx Respiratory Panel Plus on-panel organisms (including SARS-CoV-2) and various on-panel or off-panel organisms that can occur in clinical respiratory specimens was evaluated with a combination of laboratory (in vitro) wet testing and in silico characterization. For laboratory testing, on-panel (intra-panel cross-reactivity) and off-panel tested organisms (not covered by the panel content and, therefore, not intended to be detected by the QIAstat-Dx Respiratory Panel Plus) complement the organisms evaluated in the previously cleared device (QIAstat-Dx Respiratory Panel), and additional organisms were selected to assess the specificity of the new SARS-CoV-2 assays included in the Panel. Additionally, all single primers and probes for the SARS-CoV-2 detection were analyzed in silico to complement the overall specificity prediction of the device.
## a. Wet-Testing
i. On-Panel Cross-Reactivity Testing (Analytical Specificity)
Potential intra-panel cross-reactivity was evaluated with twenty-two (22) on-panel microorganisms (Table 3). The on-panel organisms were evaluated by spiking each independently into simulated NPS matrix at >10⁵ TCID₅₀/mL (or equivalent) for viruses and >10⁶ CFU/mL (or equivalent) for bacteria, or the highest concentration
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available. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2. Matrix Equivalency Study, later in this document. To evaluate potential intra-panel cross-reactivity, each organism was evaluated in triplicate. No intra-panel cross-reactivity was observed. The results of testing are shown in Table 3.
Table 3. On-Panel Organisms Tested for QIAstat-Dx Respiratory Panel Plus
| Organism (Strain) | Isolate ID | Concentration Tested | Detection rate (positive replicates) | Cross-Reactivity Detected |
| --- | --- | --- | --- | --- |
| Mycoplasma pneumoniae (UTMB-10P) | ATCC 49894 | 1.00E+05 CCU/mL | 3/3 | None |
| Bordetella pertussis (E431) | ZeptoMetrix 0801460 | 1.00E+06 CFU/mL | 3/3a | None |
| Chlamydia pneumoniae (TWAR strain TW-183) | ATCC VR-2282 | 1.00E+06 IFU/mL | 3/3b | None |
| Influenza A H1N1 (A/New Jersey/8/76) | ATCC VR-897 | 1.00E+05 CEID50/mL | 3/3 | None |
| Influenza A H3N2 (A/Switzerland/971529/2013) | ATCC VR-1837 | 1.00E+05 TCID50/mL | 3/3 | None |
| Influenza A/2009/H1N1 (A/California/07/2009 NYMC X) | ATCC VR-1884 | 1.00E+05 CEID50/mL | 3/3 | None |
| Influenza B (B/Florida/4/2006) | ATCC VR-1804 | 1.00E+05 CEID50/mL | 3/3 | None |
| Human Coronavirus 229E | ZeptoMetrix 0810229CFHI | 1.00E+04 U/mL | 3/3 | None |
| Human Coronavirus OC43 | ZeptoMetrix 0810024CFHI | 1.00E+04 U/mL | 3/3 | None |
| Human Coronavirus NL63 | BEI NR-470 | 1.00E+05 copies/mL | 3/3 | None |
| Human Coronavirus HKU1 (Clinical sample) | Stat-Dx S506 | 1.00E+05 copies/mL | 3/3 | None |
| Parainfluenza Virus 1 (C35) | ATCC VR-94 | 1.00E+05 TCID50/mL | 3/3c | None |
| Parainfluenza Virus 2 (Greer) | ATCC VR-92 | 1.00E+05 TCID50/mL | 3/3 | None |
| Parainfluenza Virus 3 (C 243) | ATCC VR-93 | 1.00E+05 CEID50/mL | 3/3 | None |
| Parainfluenza Virus 4 | ZeptoMetrix 0810060BCFHI | 1.00E+05 U/mL | 3/3 | None |
| RSV (A2) | ATCC VR-1540 | 1.00E+05 CFU/mL | 3/3 | None |
| hMPV (IA10-2003) | ZeptoMetrix 0810161CFHI | 1.00E+05 U/mL | 3/3c | None |
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# ii. Off-Panel Cross-Reactivity Testing (Analytical Specificity)
This study evaluated the analytical specificity (cross-reactivity) of the QIAstat-Dx Respiratory Panel Plus in the presence of non-targeted microorganisms that may be found in a respiratory tract clinical specimen. Forty-nine (49) non-target microorganisms (Table 4) were evaluated in the study. Panel members were composed of one individual non-target microorganism spiked into simulated NPS matrix at $>10^{5}\mathrm{TCID}_{50} / \mathrm{mL}$ (or equivalent) for viruses, $>10^{6}\mathrm{CFU} / \mathrm{mL}$ (or equivalent) for bacteria/fungi, or the highest concentration available. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Matrix Equivalency Study, later in this document.
To evaluate cross-reactivity, each microorganism was evaluated in triplicate in the absence of the target organisms. No cross-reactivity was observed at the concentrations tested except for Bordetella bronchiseptica at a concentration of $1 \times 10^{6} \mathrm{CFU/mL}$ and Bordetella holmesii at a concentration of $1 \times 10^{5} \mathrm{CFU/mL}$ , which both cross-reacted with the Bordetella pertussis primer/probes in $100\%$ of the replicates tested for each bacterium. As described for the previously cleared QIAstat-Dx Respiratory Panel (K183597), the targeted gene for Bordetella pertussis (IS481) is a mobile transposon also present in B. holmesii (detected in 6/6 replicates) and some clusters of B. bronchiseptica (detected in 3/3 replicates), so observed cross-reaction with B. pertussis was expected in both cases. A limitation to this affect has been included in the Instructions for Use). The results are summarized in Table 4.
Table 4. Off-Panel Organisms Tested for QIAstat-Dx Respiratory Panel Plus
| Organism | Isolate ID | Concentration Tested | Detection rate (#Detected/#Tested) | Cross-Reactivity Detected |
| --- | --- | --- | --- | --- |
| Acinetobacter calcoaceticus | ZeptoMetrix 0804096 | 1.00E+06 CFU/mL | 0/5a | None |
| Bordetella avium | ZeptoMetrix 0804316 | 1.00E+06 CFU/mL | 0/3 | None |
| C. licheniformis | ZeptoMetrix 0804320 | 1.00E+06 CFU/mL | 0/3 | None |
| D. ruminantium | ZeptoMetrix 0804321 | 1.00E+06 CFU/mL | 0/3 | None |
| E. rhamnosus | ZeptoMetrix 0804322 | 1.00E+06 CFU/mL | 0/3 | None |
| F. rhamnosus | ZeptoMetrix 0804323 | 1.00E+06 CFU/mL | 0/3 | None |
| G. rhamnosus | ZeptoMetrix 0804324 | 1.00E+06 CFU/mL | 0/3 | None |
| H. rhamnosus | ZeptoMetrix 0804325 | 1.00E+06 CFU/mL | 0/3 | None |
| I. rhamnosus | ZeptoMetrix 0804326 | 1.00E+06 CFU/mL | 0/3 | None |
| J. rhamnosus | ZeptoMetrix 0804327 | 1.00E+06 CFU/mL | 0/3 | None |
a B. pertussis initial testing resulted in only two positive replicates with unexpected high Ct value. Investigation concluded that testing was performed with the incorrect dilution and it was repeated with the correct dilution, obtaining expected results.
b Two cartridges resulted in Positive with Warning (IC fail) and were repeated to confirm obtained results.
One cartridge fail was repeated.
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| Bordetella bronchiseptica | ATCC 4617 | 1.00E+06 CFU/mL | 3/3 | Bordetella pertussis^{b} |
| --- | --- | --- | --- | --- |
| Bordetella hinzii | Vircell MC089 | 1.00E+06 copies/mL | 0/6 | None |
| Bordetella holmesii | ATCC 51541 | 1.00E+05 CFU/mL | 6/6 | Bordetella pertussis^{b} |
| Corynebacterium diphteriae | ATCC 11913 | 5.00E+03 CFU/mL | 0/3 | None |
| Enterobacter aerogenes | ATCC 13048 | 1.00E+06 CFU/mL | 0/6 | None |
| Haemophilus influenzae | ATCC 49766 | 1.00E+06 CFU/mL | 0/6 | None |
| Haemophilus influenzae | ATCC 8142 | 1.00E+06 CFU/mL | 0/3 | None |
| Haemophilus influenzae | ATCC 10211 | 1.00E+06 CFU/mL | 0/3 | None |
| Klebsiella oxytoca | ATCC 700324 | 1.00E+06 CFU/mL | 0/6 | None |
| Lactobacillus acidophilus | ATCC 4356 | 1.00E+06 CFU/mL | 0/6^{c} | None |
| Legionella feeleii | Vircell MC092 | 1.00E+06 copies/mL | 0/6 | None |
| Legionella micdadei | ZeptoMetrix 0801576 | 1.00E+06 CFU/mL | 0/3 | None |
| Legionella pneumophila | ATCC 33152 | 1.00E+05 CFU/mL | 0/5^{c} | None |
| Legionella pneumophila | ZeptoMetrix 0801645 | 1.00E+06 CFU/mL | 0/3 | None |
| Legionella pneumophila | ATCC 33156 | 1.00E+06 CFU/mL | 0/3 | None |
| Mycobacterium tuberculosis | ATCC 25177DQ | 1.00E+06 copies/mL | 0/3 | None |
| Mycoplasma genitalium | ZeptoMetrix 0804094-I | 1.00E+06 CCU/mL | 0/3 | None |
| Mycoplasma hominis | ATCC 27545 | 1.00E+05 CFU/mL | 1/9 | None^{d} |
| Mycoplasma orale | ATCC 23714 | 1.00E+06 CFU/mL | 0/3 | None |
| Neisseria elongata | ZeptoMetrix 0801510 | 1.00E+06 CFU/mL | 0/3 | None |
| Neisseria gonorrhoeae | ZeptoMetrix 0801482 | 1.00E+06 CFU/mL | 0/3 | None |
| Serratia marcescens | ATCC 14756 | 1.00E+06 CFU/mL | 0/6 | None |
| Staphylococcus aureus | ATCC CRM6538 | 1.00E+06 CFU/mL | 0/3 | None |
| Staphylococcus epidermidis | ATCC 14990 | 1.00E+06 CFU/mL | 0/3 | None |
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| Stenotrophomonas maltophilia | ATCC 13637 | 1.00E+06 CFU/mL | 0/6 | None |
| --- | --- | --- | --- | --- |
| Streptococcus pneumoniae | ZeptoMetrix 0801439 | 1.00E+06 CFU/mL | 0/3 | None |
| Streptococcus salivarius | ATCC 13419 | 1.00E+06 CFU/mL | 0/6^{c} | None |
| Streptococcus salivarius | ZeptoMetrix 0801896 | 1.00E+06 CFU/mL | 0/3 | None |
| Ureaplasma urealyticum | ATCC 27618 | 1.00E+06 CFU/mL | 0/3 | None |
| Escherichia coli (0157) | ZeptoMetrix 0801622 | 1.00E+06 CFU/mL | 0/6 | None |
| Klebsiella pneumoniae | ATCC 13883 | 1.00E+06 CFU/mL | 0/3 | None |
| Moraxella catarrhalis | ATCC 25240 | 1.00E+06 CFU/mL | 0/6^{c} | None |
| Neisseria meningitidis | ATCC 35561 | 1.00E+06 CFU/mL | 0/5^{a} | None |
| Proteus mirabilis | ZeptoMetrix 0801544 | 1.00E+06 CFU/mL | 0/3 | None |
| Pseudomonas aeruginosa | ATCC 15442 | 1.00E+06 CFU/mL | 0/3 | None |
| Staphylococcus epidermidis | ATCC 12228 | 1.00E+06 CFU/mL | 0/3 | None |
| Streptococcus pyogenes | ATCC 14289 | 1.00E+06 CFU/mL | 2/6 | None^{d} |
| Streptococcus agalactiae | ZeptoMetrix 0801545 | 1.00E+06 CFU/mL | 0/3 | None |
| Bocavirus | University of Kansas Medical Center, Kansas, USA (Clinical Sample) | 1.00E+05 copies/mL | 0/6 | None |
| Cytomegalovirus | ZeptoMetrix 0810499CFHI | 1.00E+05 U/mL | 0/3 | None |
| Epstein-Barr Virus | ATCC VR-1492PQ | 1.00E+05 copies/mL | 0/3 | None |
| Measles Virus | ATCC VR-24 | 1.00E+06 TCID_{50}/mL | 0/3 | None |
| MERS Coronavirus | Vircell MC121 | 1.00E+05 copies/mL | 0/3 | None |
| Mumps | ATCC VR-106 | 1.00E+04 TCID_{50}/mL | 0/3 | None |
| Herpes Simplex Virus 1 | ATCC VR-1778 | 1.00E+05 PFU/mL | 0/3^{c} | None |
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| | ATCC VR-1789 | 1.00E+05 PFU/mL | 0/3 | None |
| --- | --- | --- | --- | --- |
| Herpes Simplex Virus 2 | ATCC VR-1779 | 1.00E+05 PFU/mL | 0/5^{a} | None |
| | ATCC VR-734 | 1.00E+05 PFU/mL | 0/3 | None |
| Candida albicans | ATCC CRM-10231 | 1.00E+06 CFU/mL | 0/3 | None |
| Aspergillus flavus | ZeptoMetrix 0801598 | 1.00E+06 CFU/mL | 0/3 | None |
| Aspergillus fumigatus | ZeptoMetrix 0801716 | 1.00E+06 CFU/mL | 1/6 | None^{d} |
| Cryptococcus neoformans | ATCC 32045 | 1.00E+06 CFU/mL | 0/6 | None |
a. Two reliability errors were repeated. Repeats were valid and no cross-reactivity was observed (Reliability errors is a summary term for a class of possible errors that occur during the execution of the cartridge on the instrument).
b. As described for the previously cleared device, targeted gene for Bordetella pertussis (IS481) is a mobile transposon also present in B. holmesii (detected in 6/6 replicates) and some clusters of B. bronchiseptica (detected in 3/3 replicates), so observed cross-reaction with B. pertussis was expected in both cases.
c. One cartridge fail was repeated.
d. B. pertussis was detected with large Ct value (low concentration) for A. fumigatus (1/6 replicates), Mycoplasma hominis (1/9 replicates) and S. pyogenes (2/6 replicates). Proper investigation confirmed B. pertussis contamination in all cases. In silico analysis confirmed lack of cross-reactivity of B. pertussis assay with all three organisms.
### iii. Competitive Interference
A competitive interference study was done to determine if the presence of a subset of representative on-panel organisms at clinically relevant and challenging concentrations (1.00E+06 CFU/mL for bacteria, 1.00E+05 PFU/mL for viruses) would impede the detection of SARS-CoV-2 at low concentrations (3x LoD) in simulated NPS matrix. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Matrix Equivalency Study, later in this document. Testing was performed in triplicate with SARS-CoV-2 detection in all samples and replicates (Table 5).
Table 5. On-panel Targets and SARS-CoV-2 Competitive Interference Study Results
| High Concentration Target Organism | Strain/Isolate | Source/ Catalog # | # SARS-CoV-2 detected/ valid runs |
| --- | --- | --- | --- |
| Human Coronavirus | 229E | ZeptoMetrix 0810229CFHI | 3/3 |
| Human Coronavirus | OC43 | ZeptoMetrix 0810024CFHI | 3/3 |
| Adenovirus A12 | Huie | ATCC VR-863 | 3/3 |
| Parainfluenza Virus 3 | C243 | ATCC VR-93 | 3/3 |
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| Bordetella pertussis | 18323 | ATCC 9797 | 3/3 |
| --- | --- | --- | --- |
| Enterovirus D68 | US/IL/14-18952 | ATCC VR-1824 | 3/3 |
| Rhinovirus | 2060 (Type 1A) | ATCC VR-1559 | 3/3 |
| Echovirus 6a | D-1 (Cox) | ATCC VR-241 | 3/3 |
| RSV | Long | ATCC VR-26 | 3/3 |
| hMPV | IA10-2003 | ZeptoMetrix 0810161CFHI | 3/3 |
| Influenza A H1N1 | New Cal/20/99 | ZeptoMetrix 0810036CFHI | 3/3 |
a Echovirus is an Enterovirus and expected to report a positive Rhinovirus/Enterovirus result, accordingly.
# iv. Microbial Interference
A microbial interference study was conducted to assess the inhibitory effects of select non-target organisms on the ability to detect SARS-CoV-2. Clinically relevant and challenging concentrations (1.00E+06 CFU/mL for bacteria/fungi, 1.00E+05 PFU/mL for viruses unless otherwise noted) of non-target organisms were individually mixed with SARS-CoV-2 at 3x LoD in simulated NPS matrix. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Matrix Equivalency Study, later in this document. Testing was performed in triplicate with two additional tests performed if SARS-CoV-2 was not detected in any one of the original three replicates. All combinations and replicates successfully detected SARS-CoV-2 except for two samples containing Legionella pneumophila, one Streptococcus salivarius sample, and one S. epidermidis sample (Table 6). For these, additional replicates successfully detected SARS-CoV-2. Where available, at least one additional strain of L. pneumophila or S. salivarius was also tested in triplicate with all samples successfully detecting SARS-CoV-2. See Table 6 for a list of the strains tested and the result summary.
Table 6. Microbial Interference Study Results
| Non-Target Organism | Strain/Isolate | Source/ Catalog # | # SARS-CoV-2 detected/ valid runs |
| --- | --- | --- | --- |
| Staphylococcus aureusa | FDA 209 | ATCC CRM-6538 | 3/3 |
| Streptococcus pneumoniae | Z022 19F | ZeptoMetrix 0801439 | 3/3 |
| Streptococcus salivarius | C699 [S30D] | ATCC 13419 | 3/3 |
| Streptococcus salivarius | Z127 | ZeptoMetrix 0801896 | 4/5 |
| Haemophilus influenzae | AMC 36-A-7 | ATCC 8142 | 3/3 |
| Haemophilus influenzae | AMC 36-A-1 | ATCC 10211 | 3/3 |
| Candida albicans | CBS 562 | ATCC 18804 | 3/3 |
| Herpes Simplex Virus 1 | ATCC-2011-9 | ATCC VR-1789 | 3/3 |
| Staphylococcus epidermidis | Fussel | ATCC 14990 | 4/5 |
| Pseudomonas aeruginosa | PRD-10 | ATCC 15442 | 3/3 |
| Legionella pneumophila | Philadelphiab | ZeptoMetrix 0801645 | 3/5 |
| Legionella pneumophila | Philadelphia-1b | ATCC 33152 | 3/3 |
| Legionella pneumophila | Los Angeles-1 | ATCC 33156 | 3/3 |
| Neisseria meningitidisa | serogroup A | ATCC 13077 | 3/3 |
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b. In silico
The QIAstat-Dx Respiratory Panel Plus differs from the previously cleared device (QIAstat-Dx Respiratory Panel, K183597) by the addition of primers and probes for the detection of SARS-CoV-2 and a decrease in the concentration of a lysis reagent. Evaluation of the two SARS-CoV-2 assays was carried out to corroborate the lack of cross-reactivity.
Exclusivity BLAST-based in silico screening was performed on April 20th 2022, including all available sequences in the GenBank database. BLAST homology characterization was performed excluding SARS-CoV-2 sequences from the analysis to optimize BLAST results to non-target organisms. Results are summarized in Table 7.
Table 7. Summary of BLAST Homology Analysis of the SARS-CoV-2 Assays
| Target gene | Sequence type | BLAST hits | >70% homology sequences | Final sequences^a | SARS-CoV-2 related sequences | Other Organisms |
| --- | --- | --- | --- | --- | --- | --- |
| RdRp (Orf1ab) gene | Forward | 8651 | 1439 | 35 | 14 Synthetic constructs or cloning vectors. 9 SARS-CoV-2-related coronavirus | 12 Bat Coronaviruses |
| | Probe | 5503 | 1405 | | | |
| | Reverse | 6774 | 1046 | | | |
| E gene | Forward | 5462 | 1880 | 130 | 14 Synthetic constructs or cloning vectors. 11 SARS-CoV-2 related coronavirus | 9 Pangolin coronavirus and 96 Bat coronaviruses |
| | Probe | 5034 | 811 | | | |
| | Reverse | 3477 | 630 | | | |
<a>^a</a>Final sequences matching Forward primer, Probe and Reverse primer with right amplicon size.
Only Thirty-five (35) and 130 evaluated sequences (RdRp and E genes, respectively) presented possible cross-amplification. After removing synthetic constructs, recombinant, cloning vectors, or SARS-CoV-2 related coronavirus, only potential nonspecific signals could be produced by a cross-reaction of the SARS-CoV-2 assays with a few coronaviruses specific for bats and/or pangolins. Since these organisms are not known to currently circulate in the human population, the risk of a false positive SARS-CoV-2 result is low. A limitation on potential cross-reactivity of bat and pangolin sequences has
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been added to the Instructions for Use. No GenBank sequences corresponding to human-infective pathogens were predicted to be positive for the primer/probe sets included in the QIAstat-Dx system to detect SARS-CoV-2.
# Interfering Substances
The performance of the QIAStat-Dx Respiratory Panel Plus for the detection of SARS-CoV-2 was evaluated in the presence of medically and/or physiologically relevant concentrations of potentially interfering substances that may be present in an NPS specimen. In addition, several technique-specific substances (e.g., disinfectants and various brands of swabs or VTM transport mediums) were also tested as potential sources of assay interference. Samples were tested in triplicate with and without addition of the potentially inhibitory substance in simulated NPS matrix using SARS-CoV-2 (NIBSC cat no. 20/146) at 3x LoD. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Matrix Equivalency Study, later in this document. No interference was observed at the concentrations listed for any of the substance tested (Table 8).
Table 8. Potential Interfering Substances Concentrations and Results
| Substance Type | Substance / Pathogen | Concentration tested | Positive Samples | Negative Samples |
| --- | --- | --- | --- | --- |
| Endogenous Substances | Human genomic DNA 200 ng/μL | 20 ng/μL | 3/3 | 0/3 |
| | Human Blood (+Na Citrate) | 1 % | 3/3 | 0/3 |
| | Mucin from bovine submaxillary glands | 1 % | 3/3 | 0/4 |
| Exogenous substances | Tobramycin (systemic antibiotic) | 0.6 mg/ml | 3/3 | N/A |
| | Mupirocin | 2 % | 3/3 | N/A |
| | OCEAN Saline Nasal Spray | 1 % | 3/3 | N/A |
| | Afrin Original Pump Mist | 1 % | 3/3 | N/A |
| | Chiroflu Influenza Vaccine (surface antigen inactivated) | 1.00E-05 % | 3/3 | N/A |
| | Analgesic ointment (Vicks VapoRub) | 1 % | 3/3 | N/A |
| | Petroleum Jelly (Vaseline) | 1 % | 3/3 | N/A |
| Technique Specific Substances | Bleach | 5 % | 5/5* | N/A |
| | Meliseptol Wipes (disinfecting wipes) | 2.50E-04 in2/μl | 3/3 | N/A |
| | DNAZap | 1 % | 3/3 | N/A |
| | ProtectRNA RNase Inhibitor 500x Concentrate | 1 % | 3/3 | N/A |
| | Ethanol | 5 % | 3/3 | N/A |
| | Swab Copan 168C (rayon/twisted aluminum shaft) | 1 swab/ml | 3/3 | N/A |
| | Swab Copan FloQ (flocked nylon/plastic shaft) | 1 swab/ml | 3/3 | N/A |
| | Swab Copan 175KS01 (polyester/aluminum shaft) | 1 swab/ml | 3/3 | N/A |
| | Swab Puritan | 1 swab/ml | 3/3 | N/A |
| | VTM Remel M4-RT | 100 % | 3/3 | N/A |
| | VTM Sigma Virocult | 100 % | 3/3 | N/A |
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| Substance Type | Substance / Pathogen | Concentration tested | Positive Samples | Negative Samples |
| --- | --- | --- | --- | --- |
| | VTM-RT | 100 % | 3/3 | N/A |
| | BD Universal Viral Transport | 100 % | 3/3 | N/A |
| | DeltaSwab Virus | 100 % | 3/3 | N/A |
*Two additional cartridges were run in error, results from these runs were valid and are reported here.
All other targets of the QIAstat-Dx Respiratory Panel Plus have been evaluated in interfering substance studies during the previous device clearance with the results remaining applicable (see QIAstat-Dx Respiratory Panel, K183597). The interference of SARS-CoV-2 detection with a live, intranasal influenza vaccine (i.e., FluMist or Fluenz) was not assessed. Results from the previously cleared device on other panel analytes determined that the live intranasally administered influenza vaccine did not interfere at concentrations of 0.000001% v/v or less, however, interference was noted at a ten-fold higher concentration (A limitation to this effect has been added to the Instructions for Use).
4. Assay Reportable Range:
Not applicable; this is a qualitative assay.
5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
a. Controls
This assay contains an internal control within each cartridge. External positive and negative controls are not provided with the assay but were run during the performance studies. A description of the external controls used in these studies is presented under section External Controls, earlier in this document.
b. Sample Stability
Sample stability testing demonstrated that NPS specimens in Universal Transport Medium (UTM) may be stored at the conditions listed below.
- Room temperature up to 4 hours at 15–25°C
- Refrigerated up to 3 days at approximately 4°C
- Frozen up to 14 days at –20°C
c. Cartridge Package Open stability
In a study comprising a subset of panel analytes, including SARS-CoV-2, it was determined that after the cartridge package is opened, sample should be introduced into the QIAstat-Dx Respiratory Panel Plus Cartridge and loaded into the QIAstat-Dx Analyzer 1.0 within 120 minutes.
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# 6. Detection Limit:
A limit of detection study (LoD) was performed to evaluate the analytical sensitivity of the SARS-CoV-2 assays of the QIAstat-Dx Respiratory Panel Plus. In addition, a subset of the original panel analytes (QIAstat-Dx Respiratory Panel, K183597) was tested side-by-side with the candidate device and the previously cleared device (QIAstat-Dx Respiratory Panel, K183597) to demonstrate that the addition of the SARS-CoV-2 assays and a minor modification to the lysis buffer does not impact the performance of other analyte assays in other reaction chambers.
## a. Limit of Detection for SARS-CoV-2
For this study, five (5) SARS-CoV-2 strains (Table 9) were evaluated individually by testing serial dilutions prepared in NPS matrix. Testing was broken into two parts: preliminary and confirmatory LoD testing. For the preliminary LoD study, a serial dilution series consisting of four concentrations was tested in replicates of four per dilution. The preliminary LoD for each target was defined as the lowest concentration at which 100% of replicates were positive for SARS-CoV-2. The confirmed LoD was established by testing twenty (20) replicates at the concentration determined from the preliminary LoD for each strain. The LoD for each strain was confirmed if ≥ 95% of the replicates were positive. To further confirm the LoD, at least one dilution below the LoD was also tested for each strain in twenty (20) replicates and was required to result in less than 95% positivity. The confirmed LoD for SARS-CoV-2 is summarized in Table 10.
Table 9. SARS-CoV-2 strains used in the Limit of Detection Study
| Strain Information | Source | Catalog ID | Lot | Stock concentration | Live/inactivated |
| --- | --- | --- | --- | --- | --- |
| n/a | STAT-Dx Life, S.L. – Clinical sample | 243 | n/a | 1,40E+07 copies/mL^{a} | Clinical sample, no inactivation |
| USA-WA1-2020 | ZeptoMetrix | 0810587CFH | 324163 | 4,25E+10 copies/mL^{a} | SARS-CoV-2 (Isolate: USA-WA1/2020) Culture Fluid (Heat Inactivated) |
| England/02/2020 | NIBSC | First WHO International Standard for SARS-CoV-2 RNA 20/146 | n/a | 1,40E+07 copies/mL^{a} | The first WHO International Standard for SARS-CoV-2 RNA for Nucleic acid Amplification Technique (NAT)-based assays consists of acid-heat inactivated England/02/2020 isolate of SARS-CoV-2. |
| n/a | Vall d’Hebron hospital – Clinical sample | S1229 | n/a | 4,60E+06 copies/mL^{a} | Clinical sample, no inactivation |
| n/a | Vall d’Hebron hospital – Clinical sample | S1231 | n/a | 1,90E+07 copies/mL^{a} | Clinical sample, no inactivation |
a Titer determined in molecular units (copies/mL) by in-house developed and validated qPCR assay.
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Table 10. Confirmatory LoD Results for SARS-CoV-2
| Strain | Concentration (copies/mL)b | Detection rate | Mean Ct SARS-CoV-2 |
| --- | --- | --- | --- |
| STAT-Dx Life, S.L. - Clinical sample (ID 243) | 1,000 | 20/20 (100%) | 35.2 |
| | 600 | 20/20a (100%) | 35.8 |
| | 500 | 17/20 (85%) | 35.7 |
| | 316 | 17/20 (85%) | 36.0 |
| | 60 | 6/20 (30%) | 36.2 |
| USA-WA1-2020, Zeptometrix | 3,160 | 19/20 (95%) | 35.5 |
| | 316 | 1/20 (5%) | 35.7 |
| England/02/2020, NIBSC | 316 | 19/20a (95%) | 35.9 |
| | 100 | 11/20 (55%) | 36.0 |
| Vall d'Hebron hospital - Clinical Sample (ID S1229) | 19,000 | 20/20 (100%) | 33.9 |
| | 6,000 | 18/20a (90%) | 34.9 |
| | 1,900 | 17/20 (85%) | 34.9 |
| Vall d'Hebron hospital - Clinical sample (ID S1231) | 19,000 | 20/20a (100%) | 35.4 |
| | 6,000 | 14/20 (70%) | 35.0 |
| | 1,900 | 10/20 (50%) | 35.4 |
a One "failed" (invalid) result was obtained. Valid result was obtained upon retesting. b Titer determined in molecular units (copies/mL) by in-house developed and validated qPCR assay.
# b. LoD Comparison Study
A subset of six (6) representative analytes (one from each Reaction Chamber) as listed in Table 11 was included in the LoD comparison study to demonstrate that the addition of SARS-CoV-2 assays and changes to the lysis reagent concentration do not impact the performance of assays in other chambers. Twenty (20) replicates of the subset of original panel analytes were prepared as co-formulated samples using pooled negative clinical matrix at the LoD concentrations established for the previously cleared device (QIAstat-Dx Respiratory Panel, K183597). Additionally, twenty (20) replicates were tested at $0.1\mathrm{x}$ LoD to further verify LoD equivalency. The panel of representative analytes was tested in parallel with the previously cleared device (QIAstat-Dx Respiratory Panel, K183597) and the candidate device. The expected detection rates for both devices were $\geq 95\%$ of replicates tested $(\geq 19 / 20)$ at the LoD concentration, and $< 95\%$ $(\leq 18 / 20)$ at the $0.1\mathrm{x}$ LoD concentration. As summarized in Table 12, the LoD for a subset of original panel analytes was confirmed on both devices in side-by-side testing, which confirmed that the addition of the SARS-CoV-2 assays and changes to the lysis reagent do not impact the performance of assays in other chambers. Moreover, the LoDs as determined in K183597 for single-spiked analytes are equivalent to the LoDs determined for individual analytes under multi-spiked conditions. Multi-spiked LoD studies were not repeated here.
Table 11. Representative strains used in the Limit of Detection Comparison Study
| Target Organism/ Analyte | Strain information | Source | Catalogue ID | Lot | Stock concentration |
| --- | --- | --- | --- | --- | --- |
| Influenza B | B/Taiwan/2/62 | ATCC | VR-295 | 60517845 | 1.58E+07 CEID50/mL |
| E. coli | B/Taiwan/2/62 | ATCC | VR-295 | 60517845 | 1.58E+07 CEID50/mL |
| E. coli | B/Taiwan/2/62 | ATCC | VR-295 | 60517845 | 1.58E+07 CEID50/mL |
a One "failed" (invalid) result was obtained. Valid result was obtained upon retesting. b Titer determined in molecular units (copies/mL) by in-house developed and validated qPCR assay.
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| Coronavirus HKU1 | n/a | STAT-Dx Life, S.L – Clinical sample | n/a | S510 | 4.00E+08 copies/mLa |
| --- | --- | --- | --- | --- | --- |
| Parainfluenza virus 3 | C 243 | ATCC | VR-93 | 59380357 | 2.32E+06 TCID50/mL |
| Rhinovirus | HGP (Rhinovirus A2) | ATCC | VR-482 | 58524058 | 2.81E+07 TCID50/mL |
| Adenovirus | GB (Adenovirus B3) | ATCC | VR-3 | 59490909 | 1.58E+10 TCID50/mL |
| Mycoplasma pneumoniae | PI 1428 | ATCC | 29085 | 70010561 | 1.00E+06 CCU/mL |
a Titer determined in molecular units (copies/mL) by in-house developed and validated qPCR assay.
Table 12. Summary of LoD Comparison Study with a Subset of Panel Analytes
| | | | QIAstat-Dx Respiratory Panel Plus (# Detected/# Tested, Mean Ct) | | QIAstat-Dx Respiratory Panel (# Detected/#Tested, Mean Ct) | |
| --- | --- | --- | --- | --- | --- | --- |
| Target Organism/ Analyte | Isolate | LoD Concentration | 1x LoD | 0.1x LoD | 1x LoD | 0.1x LoD |
| Influenza B | B/Taiwan/2/62 | 5000.0 CEID50/mL | 20/20 (28.3) | 15/20 (30.8) | 20/20 (27.7) | 17/20 (30.7) |
| Coronavirus HKU1 | n/a | 4.00E+04 copies/mLa | 20/20 (33.8) | 4/20 (34.2) | 20/20 (33.4) | 5/20 (34.1) |
| Parainfluenza Virus 3 | C 243 | 2.3 TCID50/mL | 20/20 (32.9) | 7/20 (32.1) | 20/20 (33.1) | 12/20 (33.0) |
| Rhinovirus | HGP (Rhinovirus A2) | 8.9 TCID50/mL | 20/20 (32.3) | 7/20 (31.7) | 20/20 (31.8) | 5/20 (31.9) |
| Adenovirus | GB (Adenovirus B3) | 4993.0 TCID50/mL | 20/20 (32.1) | 8/20 (34.1) | 20/20 (32.4) | 10/20 (33.8) |
| Mycoplasma pneumoniae | PI 1428 | 1.0 CCU/mL | 20/20 (33.4) | 9/20 (34.9) | 20/20 (33.1) | 8/20 (35.0) |
a Titer determined in molecular units (copies/mL) by in-house developed and validated qPCR assay.
# 7. Assay Cut-Off:
The QIAStat Dx Respiratory Panel Plus includes defined Ct value cutoffs for SARS-CoV-2, which were established by testing two SARS-CoV-2 isolates. Ct cutoffs for other assays
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present on the panel and the internal control assay were established in the original K183597 clearance studies and no changes were made with addition of SARS-CoV-2 detection. The Ct cutoffs are included as automatic calculations in the assay definition file (ADF).
8. Accuracy (Instrument):
Not Applicable
9. Carry-Over:
A carryover study was performed using the QIAstat-Dx Respiratory Panel Plus to evaluate the potential occurrence of cross-contamination between consecutive runs and carryover between cartridge chambers on the QIAstat-Dx Analyzer 1.0. Two panels consisting of high concentrations of influenza A, parainfluenza virus 3, and *M. pneumoniae* (panel A) or coronavirus NL63, RSV, and SARS-CoV-2 (panel B) in simulated NPS matrix were each tested multiple times between runs of negative (no analyte) cartridges for a total of 62 runs across several days. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Matrix Equivalency Study, later in this document. No carryover between cartridges or chambers within the cartridges was observed.
B Comparison Studies:
1. Method Comparison with Predicate Device: Not Applicable
2. Matrix Equivalency Study:
Equivalent performance of simulated NPS matrix and natural clinical NPS matrix was established by testing SARS-CoV-2 isolate England/02/2020 at either 1x LoD or 0.1x LoD concentrations, co-spiked with a subset of on-panel organisms at a low concentration (1x LoD). SARS-CoV-2 was also tested alone. Testing of 20 replicates per each combination was performed with a SARS-CoV-2 detection rate of ≥ 95% for all samples at 1x LoD. Below the LoD testing performed as expected and, thus, overall matrix equivalency was established.
Additionally, this study demonstrates that the LoDs of single-spiked SARS-CoV-2 samples are equivalent to the LoDs determined for SARS-CoV-2 under multi-spiked conditions.
C Clinical Studies:
1. Clinical Performance
The clinical performance of the SARS-CoV-2 assays in the QIAstat-Dx Respiratory Panel Plus was established through a multi-center prospective (i.e., all comers) clinical study conducted at five (5) geographically diverse study sites in the U.S. [(1) Northridge, CA; (2) Redondo Beach, CA; (3) Nashville, TN; and (4) Easley, SC; and (5) Milwaukee, WI]. Each site was tasked with prospective sample collection and testing using the candidate device and the comparator method. Site 5 was further tasked with discordant testing.
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Note: Clinical performance for the non-SARS-CoV-2 analytes was established in K183597 and not repeated here.
During this study, nasopharyngeal swab (NPS) specimens were prospectively collected in UTM from individuals with signs and symptoms of respiratory infection, from February to May 2023 and during February 2024.
The clinical performance of the SARS-CoV-2 assays in the QIAstat-Dx Respiratory Panel Plus was established by comparing results to an FDA-cleared molecular respiratory panel under 21 CFR 866.3981 that includes SARS-CoV-2.
A total of 616 prospective NPS specimens were enrolled and tested in this clinical study. One specimen was excluded due to failure to meet the inclusion criteria. Overall, 615 evaluable specimens were included in the performance analysis.
Study demographics are summarized in Table 13.
Table 13. Prospective Specimens: Demographic Summary of Evaluable Subjects
| Sex | Total | |
| --- | --- | --- |
| | N | Percentage |
| Female | 391 | 63.58 |
| Male | 224 | 36.42 |
| All | 615 | 100.00 |
| Age Group | Total | |
| | N | Percentage |
| <5 | 23 | 3.74 |
| 5 - 9 | 23 | 3.74 |
| 10 - 13 | 4 | 0.65 |
| 14 - 18 | 31 | 5.04 |
| 19 - 29 | 208 | 33.82 |
| 30 - 49 | 206 | 33.50 |
| 50 - 69 | 102 | 16.59 |
| 70 - 85 | 18 | 2.93 |
| All | 615 | 100.00 |
| Race | Total | |
| | N | Percentage |
| American Indian or Alaskan Native | 2 | 0.33 |
| American Indian or Alaskan Native, Other | 1 | 0.16 |
| Asian | 19 | 3.09 |
| Asian, White | 3 | 0.49 |
| Black or African American | 56 | 9.11 |
| Black or African American, Native Hawaiian or Other Pacific Islander, White | 1 | 0.16 |
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| Black or African American, White | 1 | 0.16 |
| --- | --- | --- |
| Native Hawaiian or Other Pacific Islander | 1 | 0.16 |
| Native Hawaiian or Other Pacific Islander, White | 1 | 0.16 |
| Not Reported | 85 | 13.82 |
| Other | 51 | 8.29 |
| White | 391 | 63.58 |
| White, Other | 3 | 0.49 |
| All | 615 | 100.00 |
| SARS-CoV-2 Vaccination Status | Total | |
| | N | Percentage |
| Not vaccinated | 132 | 21.46 |
| Prefer not to say/No information | 69 | 11.22 |
| Vaccinated | 414 | 67.32 |
| All | 615 | 100.00 |
The overall performance of the QIAstat-Dx Respiratory Panel Plus SARS-CoV-2 assays only is shown in Table 14.
Table 14. QIAstat-Dx Respiratory Panel Plus Prospective Clinical Performance Summary for SARS-CoV-2 Assays
| Target | Positive Percent Agreement (PPA) | | | Negative Percent Agreement (NPA) | | |
| --- | --- | --- | --- | --- | --- | --- |
| | TP/ (TP+FN) | % | 95% CI | TN/ (TN+FP) | % | 95% CI |
| SARS-CoV-2 | 61 / 631 | 96.8% | 89.0%-99.6% | 551 / 5522 | 99.8% | 99.0%-100.0% |
TP-True Positive, FP-False Positive, TN-True Negative, FN-False Negative
1The two samples with false negative SARS-CoV-2 results by the QIAstat-Dx Respiratory Panel Plus were both positive by two FDA-EUA molecular SARS-CoV-2 assays.
2The single sample with a false positive SARS-CoV-2 result by the QIAstat-Dx Respiratory Panel Plus was positive by two FDA-EUA molecular SARS-CoV-2 assays.
The PPA for SARS-CoV-2 is $96.8\%$ with a two-sided $95\%$ CI of $89.0\% - 99.6\%$ . The NPA for SARS-CoV-2 is $99.8\%$ with a two-sided $95\%$ CI of $99.0\% - 100\%$ .
# Co-infection Summary
There were two specimens that yielded a co-infection including SARS-CoV-2 by the QIAstat-Dx Respiratory Panel Plus. One was positive for Human Metapneumovirus and the second was positive for Rhinovirus/Enterovirus. Both results were true positive based on the comparator result.
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Representative Panel Assays Equivalency
To confirm the clinical performance of the original panel assays and to demonstrate clinical performance equivalency between the two devices (the previously cleared QIAstat-Dx Respiratory Panel and the candidate device, QIAstat-Dx Respiratory Panel Plus), a subset of original panel analytes was chosen as a representative panel of analytes for a comparison study. The representative panel covers all target analyte types (i.e., RNA and DNA viruses and a bacterium). Additionally, the panel was constructed by choosing one analyte from each Reaction Chamber within the test cartridge. The final list includes the following organisms:
1. Influenza B
2. Coronavirus OC43
3. Parainfluenza virus 3
4. Rhinovirus/enterovirus
5. Adenovirus
6. Bordetella pertussis
A total of 190 de-identified clinical NPS specimens, both positive and negative as per Standard of Care, were obtained and tested for the representative panel analytes. Each specimen was run in parallel on the QIAstat-Dx Respiratory Panel and the QIAstat-Dx Respiratory Panel Plus. Performance estimates were calculated using the comparator method (QIAstat-Dx Respiratory Panel) as the reference result. The performance data, calculated in positive percent (PPA) and negative percent agreement (NPA) are summarized in Table 15.
Clinical performance of the QIAstat-Dx Respiratory Panel Plus demonstrated that the addition of SARS-CoV-2 and a decrease in the lysis reagent concentration had no impact to the performance of the original panel assays.
Table 15. PPA and NPA of the QIAstat-Dx Respiratory Panel Plus with Representative Panel Analytes
| Grouping Variable(s) | | Proportion | | Two-Sided 95% Confidence Limit | |
| --- | --- | --- | --- | --- | --- |
| Analysis | Pathogen | Fraction^{a} | Percentage | Lower | Upper |
| PPA | Influenza B | 20 / 20 | 100.0% | 83.2% | 100.0% |
| | Coronavirus OC43 | 22 / 22 | 100.0% | 84.6% | 100.0% |
| | Parainfluenza virus 3 | 24 / 24 | 100.0% | 85.8% | 100.0% |
| | Rhinovirus / Enterovirus | 43 / 43 | 100.0% | 91.8% | 100.0% |
| | Adenovirus | 38 / 40 | 95.0% | 83.1% | 99.4% |
| | Bordetella pertussis | 24 / 24 | 100.0% | 85.8% | 100.0% |
| NPA | Influenza B | 167 / 168 | 99.4% | 96.7% | 100.0% |
| | Coronavirus OC43 | 166 / 166 | 100.0% | 97.8% | 100.0% |
| | Parainfluenza virus 3 | 163 / 164 | 99.4% | 96.7% | 100.0% |
| | Rhinovirus / Enterovirus | 144 / 145 | 99.3% | 96.2% | 100.0% |
| | Adenovirus | 144 / 148 | 97.3% | 93.2% | 99.3% |
| | Bordetella pertussis | 163 / 164 | 99.4% | 96.7% | 100.0% |
The performance of the QIAstat-Dx Respiratory Panel Plus was determined by comparing against results obtained from the QIAstat-Dx Respiratory Panel.
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2. Clinical Specificity:
See section “Clinical Performance” above.
3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
Not Applicable
C Clinical Cut-Off:
Not Applicable
D Expected Values/Reference Range:
Table 16. Summary of Expected Values of SARS-CoV-2 by age based on QIAstat-Dx Respiratory Panel Plus Result
| | Overall (n=615) | | 2-21 yrs. (n=108) | | 22-49 yrs. (n=387) | | >49 yrs. (n=120) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Analyte | N Positive | % Positive | N Positive | % Positive | N Positive | % Positive | N Positive | % Positive |
| SARS-CoV-2 | 62 | 10.1 | 2 | 1.8 | 36 | 9.3 | 24 | 20.0 |
E Other Supportive Instrument Performance Characteristics Data:
Not applicable.
VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.
IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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