LIAISON PLEX Respiratory Flex Assay
Device Facts
| Record ID | K233410 |
|---|---|
| Device Name | LIAISON PLEX Respiratory Flex Assay |
| Applicant | Luminex Corporation |
| Product Code | QOF · Microbiology |
| Decision Date | Mar 1, 2024 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3981 |
| Device Class | Class 2 |
| Attributes | Real-World Evidence, Pediatric |
Real-World Evidence
| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
|---|---|---|---|---|---|
| K233410 · Mar 1, 2024 | LIAISON PLEX Respiratory Flex Assay | Luminex Corporation | Remnant clinical nasopharyngeal swab (NPS) specimens; Archived retrospective clinical NPS specimens | Clinical performance evaluation of the LIAISON PLEX Respiratory Flex Assay using prospectively collected and archived clinical specimens from patients with signs and symptoms of respiratory tract infection. | Prospective clinical study; Retrospective archived specimens; Clinical performance; Nasopharyngeal swabs |
Clinical Evidence
| Study Design | Population | Comparator | Key Endpoints |
|---|---|---|---|
| Prospective Clinical Study; Multi-site prospective clinical study; Follow-up/Duration: October 2022 to April 2023; Study Period: October 2022 - April 2023 | Pediatric and adult patients exhibiting clinical signs and symptoms of respiratory tract infections; Sample Size: 1843 evaluable specimens; Number of Sites: 6 | FDA-cleared molecular respiratory panels and PCR/Bi-Directional Sequencing | Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) |
| Archived Specimen Study; Retrospective evaluation of archived specimens; Follow-up/Duration: November 2013 through June 2023; Study Period: November 2013 - June 2023 | Patients with respiratory symptoms (archived specimens); Sample Size: 256; Number of Sites: 4 | FDA-cleared molecular respiratory panels and PCR/Bi-Directional Sequencing | Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) |
Indications for Use
The LIAISON PLEX Respiratory Flex (RSP Flex) Assay is a multiplexed qualitative test for the simultaneous in vitro detection and identification of multiple bacterial and viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of respiratory tract infection, including SARS-CoV-2. The test is performed on the automated LIAISON PLEX System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific nucleic acid gene sequences of the following organism types and subtypes: Viruses: Adenovirus, Human Coronavirus (HKU1, NL63, OC43, and 229E not differentiated), Human Enterovirus/Rhinovirus (not differentiated), Human Metapneumovirus, Influenza A, Influenza A (subtype H1), Influenza A (subtype H3), Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2). Bacteria: Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae. Nucleic acids from the bacterial and viral organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and identifying specific bacterial and viral nucleic acids from individuals exhibiting signs and symptoms of 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 patient management decisions. Negative results in the presence of a respiratory illness may be due to infection with pathogens that are not detected by this test or due to lower respiratory tract infection that is not detected by an NPS specimen. Conversely, positive results do not rule out infection or co-infection with organisms not detected by the LIAISON PLEX Respiratory Flex (RSP Flex) Assay. The agent(s) detected 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
The LIAISON PLEX Respiratory Flex Assay is an automated, multiplexed, qualitative in vitro diagnostic test for respiratory pathogens. Input: nasopharyngeal swab (NPS) specimens in transport media. Operation: The LIAISON PLEX System performs automated sample preparation (chemical/mechanical lysis, magnetic bead isolation), multiplex RT-PCR/PCR amplification, and microarray hybridization. Gold nanoparticle probes bind to target amplicons; silver enhancement allows light scatter measurement for detection. Output: Qualitative 'Detected' or 'Not Detected' results for 19 viral/bacterial targets. Used in clinical laboratories by trained personnel. Results aid diagnosis in conjunction with other clinical/epidemiological findings; not for sole diagnostic use. Benefits: Rapid, simultaneous identification of multiple respiratory pathogens to inform patient management.
Clinical Evidence
Prospective clinical study (n=1843) compared subject device to FDA-cleared molecular panels and validated sequencing methods. Performance metrics: PPA ranged 80.0-100% (B. parapertussis 80%, PIV-4 88.9%, others generally >90%); NPA ranged 95.7-100%. Retrospective study (n=256) and contrived study (n=300) supplemented data for rare pathogens. Results support substantial equivalence.
Technological Characteristics
Multiplexed nucleic acid test using RT-PCR and microarray hybridization. Materials: disposable cartridge with magnetic beads, gold nanoparticle probes. Energy: electrical (LIAISON PLEX System). Connectivity: standalone instrument with touchscreen UI. Software: preinstalled system software. Sterilization: N/A (disposable cartridge).
Indications for Use
Indicated for individuals with clinical signs and symptoms of respiratory tract infection, including SARS-CoV-2, to detect and identify multiple bacterial and viral nucleic acids in nasopharyngeal swabs (NPS).
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.
Predicate Devices
- BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031)
Submission Summary (Full Text)
{0}
FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY AND INSTRUMENT
# I Background Information:
A 510(k) Number
K233410
B Applicant
Luminex Corporation
C Proprietary and Established Names
LIAISON PLEX Respiratory Flex Assay
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QOF^{1} | 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 |
| OEM | Class II | 21 CFR 866.3980 – Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OOU | Class II | 21 CFR 866.3980 – Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OTG | Class II | 21 CFR 866.3980 – Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OZE | Class II | 21 CFR 866.3980 – Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OZX | Class II | 21 CFR 866.3980 – Respiratory viral panel | MI - Microbiology |
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
{1}
| | | multiplex nucleic acid assay | |
| --- | --- | --- | --- |
| OZY | Class II | 21 CFR 866.3980 – Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OZZ | Class II | 21 CFR 866.3980 – Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OCC | Class II | 21 CFR 866.3980 – Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| NSU | Class II | 21 CFR 862.2570 – Instrumentation for clinical multiplex test systems | CH - Clinical Chemistry |
¹Primary Product Code
## II Submission/Device Overview:
### A Purpose for Submission:
The purpose of this submission is to show that the LIAISON PLEX Respiratory Flex Assay is substantially equivalent to the BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031) and to obtain clearance for the LIAISON PLEX Respiratory Flex Assay.
### B Measurand:
Adenovirus, Human Metapneumovirus, Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza B, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4, Human Enterovirus/Rhinovirus (not differentiated), Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), Human Coronavirus (HKU1, NL63, OC43, and 229E not differentiated), Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae nucleic acid target sequences.
### C Type of Test:
A multiplexed nucleic acid test intended for use with the automated LIAISON PLEX instrument for the qualitative in vitro detection and identification of multiple respiratory pathogen nucleic acids in nasopharyngeal swabs (NPS) collected in BD Universal Transport Media (UVT) or Copan Universal Transport Media (UTM) and obtained from individuals with signs and symptoms of respiratory tract infections.
## III Intended Use/Indications for Use:
### A Intended Use(s):
See Indications for Use below.
### B Indication(s) for Use:
The LIAISON PLEX Respiratory Flex (RSP Flex) Assay is a multiplexed qualitative test for the simultaneous in vitro detection and identification of multiple bacterial and viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of
K233410 - Page 2 of 56
{2}
respiratory tract infection, including SARS-CoV-2. The test is performed on the automated LIAISON PLEX System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific nucleic acid gene sequences of the following organism types and subtypes:
# Viruses:
Adenovirus
Human Coronavirus (HKU1, NL63, OC43, and 229E not differentiated)
Human Enterovirus/Rhinovirus (not differentiated)
Human Metapneumovirus,
Influenza A
Influenza A (subtype H1)
Influenza A (subtype H3)
Influenza B
Parainfluenza 1
Parainfluenza 2
Parainfluenza 3
Parainfluenza 4
Respiratory Syncytial Virus
Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2)
# Bacteria:
Bordetella holmesii
Bordetella parapertussis
Bordetella pertussis
Chlamydia pneumoniae
Mycoplasma pneumoniae
Nucleic acids from the bacterial and viral organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and identifying specific bacterial and viral nucleic acids from individuals exhibiting signs and symptoms of 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 patient management decisions.
Negative results in the presence of a respiratory illness may be due to infection with pathogens that are not detected by this test or due to lower respiratory tract infection that is not detected by an NPS specimen. Conversely, positive results do not rule out infection or co-infection with organisms not detected by the LIAISON PLEX Respiratory Flex (RSP Flex) Assay. The agent(s) detected 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.
# C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
For in vitro diagnostic use only
# D Special Instrument Requirements:
For use with the LIAISON PLEX System, only.
K233410 - Page 3 of 56
{3}
# IV Device/System Characteristics:
## A Device Description:
The LIAISON PLEX Respiratory *Flex* Assay is performed on the LIAISON PLEX System. The LIAISON PLEX System consists of a touchscreen user interface that includes the software for running and analyzing assay results, one to six processing/imaging LIAISON PLEX modules, and a handheld barcode reader. Each LIAISON PLEX module processes one sample at a time under the control of the LIAISON PLEX System software. The LIAISON PLEX Respiratory *Flex* Assay components required to perform the test include the following single-use, disposables:
- LIAISON PLEX Respiratory *Flex* Assay Transfer Pipettes (or equivalent)
Prior to initiating a test on the LIAISON PLEX System, a 300 µL aliquot of NPS in Viral Transport Media (VTM) is pipetted by the user into the Sample Port within the Sample Prep Tray (recommended, but not required), followed by closing the Sample Port Closure. Next, the Sample ID barcode on the sample tube is scanned with the hand-held barcode reader, or the Sample ID is manually entered using the touchscreen keyboard. The user then scans the assay cartridge ID barcode with the hand-held barcode scanner. Lastly, the Respiratory *Flex* Assay Cartridge is inserted into the module by the user. The LIAISON PLEX System identifies the assay being run and automatically initiates the proper testing protocol to process the sample, analyze the data, and generate test results. Once the test is finished running (~ 2 hours), the user ejects the assay cartridge by selecting the green check mark or the eject icon on the touchscreen.
The Respiratory *Flex* Assay has 19 different reportable targets (organisms and influenza A subtypes H1 and H3). Reporting of these targets is based on detection of one or more of the nucleic acid targets. For each intended Respiratory *Flex* Assay target, four sequence components, referred to as oligonucleotides (Oligos), consisting of one or more Capture probe(s), Mediator probe(s), forward primer(s), and reverse primer(s), are required.
The LIAISON PLEX Respiratory *Flex* Assay can be run in Fixed mode, which will return the results for all target analytes. To allow testing flexibility, the LIAISON PLEX Respiratory *Flex* Assay can be run in *Flex* mode. This feature allows laboratories to generate custom panels, which include only a subset of analytes selected by the laboratory. No pre-set *Flex*-panels are defined, and laboratories can choose to create custom panels of specific targets for certain populations and/or seasons, if they desire to incorporate the *Flex* reporting feature. Target assays that are initially masked can later be unmasked using *Flex* credits when ordered by a physician. When *Flex* mode is implemented, all target assays are performed, however the raw data is not analyzed for masked targets until they are unmasked.
## Interpretation of Results
The Respiratory *Flex* Assay provides a qualitative result for the presence (Detected) or absence (Not Detected) of the Respiratory *Flex* target nucleic acid gene sequences. The image analysis of the Substrate provides light signal intensities from the target-specific capture spots as well as the internal processing controls, negative control, background, and imaging control spots. The mean signal intensity of a target, after background subtraction, is compared to the assay's signal detection threshold to make a determination. **Table 2** below lists the possible test results generated by the Respiratory *Flex* Assay representing identification of viral and bacterial nucleic acid sequences/targets.
K233410 - Page 4 of 56
{4}
**Table 2.** Respiratory *Flex* Assay Calls for Valid Tests
| Test Result Reported as “Detected” | Reported Target |
| --- | --- |
| **Viral Targets** | |
| Adenovirus (inclusive to A, B, C, D, E, and F) | Adenovirus |
| Human Metapneumovirus | Human Metapneumovirus |
| Human Parainfluenza Virus 1 | Human Parainfluenza Virus 1 |
| Human Parainfluenza Virus 2 | Human Parainfluenza Virus 2 |
| Human Parainfluenza Virus 3 | Human Parainfluenza Virus 3 |
| Human Parainfluenza Virus 4 | Human Parainfluenza Virus 4 |
| Human Coronavirus (inclusive to HKU1, NL63, OC43, and 229E) | Human Coronavirus |
| Influenza A* | Influenza A |
| Influenza A subtype H1** | Influenza A subtype H1 |
| Influenza A subtype H3** | Influenza A subtype H3 |
| Influenza B | Influenza B |
| Enterovirus | Enterovirus/Rhinovirus |
| Rhinovirus | |
| Respiratory Syncytial Virus (inclusive to RSV A and RSV B) | Respiratory Syncytial Virus |
| SARS-CoV-2 | SARS-CoV-2 |
| **Bacterial Targets** | |
| *Bordetella holmesii* | *Bordetella holmesii* |
| *Bordetella pertussis* (Toxin Promoter Region) | *Bordetella pertussis* |
| *Bordetella parapertussis* (IS1001) | *Bordetella parapertussis* |
| *Chlamydia pneumoniae* | *Chlamydia pneumoniae* |
| *Mycoplasma pneumoniae* | *Mycoplasma pneumoniae* |
| **Test Result Reported as “Not Detected”** | |
| All Analytes Not Detected | |
*Detection of Influenza A without an Influenza A/H1 or Influenza A/H3 subtype may occur at low titer of the virus in the specimen or may indicate a false positive due to contamination. The result could also indicate a novel Influenza A strain. In these cases, the sample should be retested. If an Influenza A detected result is obtained without detection of an Influenza A/H1 or A/H3 subtype upon retesting, contact local or state public health authorities for confirmatory testing.
**Detection of Influenza A/H1 or Influenza A/H3 subtypes without an Influenza A “Detected” result may occur at low titer of the virus in the specimen or may indicate a false positive due to contamination. The result could also indicate potential genetic mutations in the Matrix protein gene among circulating seasonal Influenza A viruses. In these cases, the sample should be retested. If an Influenza A/H1 or A/H3 subtype detected result is obtained again without detection of Influenza A upon repeat testing, further investigations may be warranted.
Reasons for invalid (no call) results, together with the appropriate recourse which should be taken by the user, are described in **Table 3**.
**Table 3.** LIAISON PLEX Respiratory *Flex* Assay Invalid Calls and Recourse
| Call | Reason | Recourse |
| --- | --- | --- |
| No Call | The hybridization internal control (IC) is not detected^{1} | Retest from the primary sample beginning with the assay package insert |
| | The amplification IC or extraction IC are not detected AND no DNA or RNA target pathogen is detected, respectively^{1} | |
K233410 - Page 5 of 56
{5}
| | Signal in regions of the microarray which do not contain capture oligos is too high | section Procedure, using a new cartridge |
| --- | --- | --- |
| | Signal in regions of the microarray containing oligomer spots to ensure proper stringency | |
| | The coefficient of variation of intensities for spots within at least one spot group is high | |
| | The overall signal across all spot groups, excluding Negative Control and Background, is too high | |
¹Additional information on the ICs (hybridization, amplification, and extraction) is provided in Section IV.C.Instrument Descriptive Information.5.Quality Control.Internal Controls, below.
### B Principle of Operation:
The Respiratory Flex Assay is a multiplexed molecular assay with automated nucleic acid isolation, amplification, and detection of unique genomic sequences of target pathogens. The Respiratory Flex Assay is performed using the LIAISON PLEX System, which is a bench-top sample-to-result molecular diagnostics workstation consisting of a touchscreen user interface that includes the software for running and analyzing assay results, one to six processing/imaging LIAISON PLEX modules, and a handheld barcode reader. The LIAISON PLEX System automates the Respiratory Flex Assay sample analysis steps, which occur within the cartridge, including: (1) Specimen Extraction – Chemical and mechanical RNA/DNA extraction from nasopharyngeal swab specimens obtained from symptomatic patients; (2) Target Amplification - Multiplex RT-PCR- and PCR-based amplification of the extracted nucleic acids to generate target-specific amplicons; (3) Hybridization - Amplicon hybridization to target specific capture DNA in a microarray format and mediator and gold-nanoparticle probe hybridization to captured amplicons. Silver enhancement of the gold nanoparticle probes bound at the capture sites results in gold-silver aggregates that are imaged optically with high efficiency by the LIAISON PLEX System. The user can monitor the status of the assay via the touch screen on the instrument, which displays the run time.
### Contamination Control
The Respiratory Flex Assay includes an Uracil DNA Glycosylase (UDG) enzyme-based strategy to eliminate amplicon contamination. Briefly, the lyophilized amplification master mix formulation contains deoxyuridine triphosphate (dUTP) in place of the standard deoxythymidine triphosphate (dTTP), and during the multiplexed RT-PCR step dUTP is incorporated into the amplicons. Prior to the start of an amplification step, the UDG enzyme renders any dUTP-containing previously generated amplicons non-amplifiable by selectively hydrolyzing at the uracil base, while not impacting the integrity of dTTP containing target RNA. The assay also uses a thermolabile version of UDG enzyme which is inactivated by heat prior to the RT step and does not interfere with the newly generated cDNA and/or the amplicon from the test. While the UDG-based strategy mitigates false positive risk due to lab-based carryover and cross-contamination, incomplete hydrolysis of uracil-containing amplicons may lead to amplification and detection of a contaminant. Additionally, this strategy does not address genomic contamination during the preparation of the samples. Strict adherence to the prescribed handling/preparation of samples and laboratory/system cleaning protocols and careful disposal of the used consumables can reduce the likelihood of contamination from user-based sources.
### End-Point Detection and Analysis
The target-specific amplicon is detected in an endpoint assay that utilizes a microarray format. For each of the bacterial or viral nucleic acid sequences/analytes detected by the Respiratory Flex Assay, two types of oligonucleotides are required for the endpoint gold nanoparticle probe-based detection: (1) Capture oligonucleotides (or captures) and (2) Mediator oligonucleotides (or mediators). The
K233410 - Page 6 of 56
{6}
Capture oligonucleotides are arrayed on the surface of a substrate (a microarray) within the test cartridge and are designed to specifically bind to one part of the analyte-specific target amplicon. The Mediator oligonucleotides bind to a different portion of the same amplicon and enable binding of gold nanoparticle probes. Notably, in a multiplexed detection system, numerous unique target-specific mediators can coexist and form unique hybridizations at the different captures on the microarray. Since all the mediators have a target specific region and a poly-A tail region, a single, universal gold nanoparticle poly-T probe is sufficient for target/mediator labeling. Silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency. Light scatter from the capture spots is imaged by the LIAISON PLEX System and intensities from the microarray spots are processed by a decision algorithm to make calls regarding the presence (Detected) or absence (Not Detected) of a nucleic acid sequence/analyte.
### C Instrument Description Information:
1. Instrument Name:
LIAISON PLEX System, software version 1.0.0.144.
2. Specimen Identification:
Specimen identification information is entered either manually or via barcode.
3. Specimen Sampling and Handling:
Nasopharyngeal swab (NPS) specimens collected in BD UVT or Copan UTM.
4. Calibration:
LIAISON PLEX modules are calibrated during the manufacturing process; calibration is not performed by the user.
5. Quality Control:
Internal Controls
The Assay contains three sets of internal controls to check to ensure performance of sample preparation amplification, and detection. They are described in more detail, below:
1. Extraction control. The extraction control verifies the presence of an amplicon for Bacteriophage MS2, which is added to the sample prior to the nucleic acid extraction step. The extracted control product is amplified and subsequently detected by unique spots on the hybridization array, thereby confirming successful nucleic acid extraction, Reverse Transcription, PCR amplification of RNA targets, and detection.
2. Amplification Control. The amplification control verifies the presence of an amplicon for a synthetic DNA oligonucleotide sequence in the lyophilized PCR master mix. The product is detected by a unique spot on the hybridization array, thereby confirming successful PCR amplification and detection of DNA targets.
3. Hybridization Control. The hybridization control target and mediator oligonucleotide are contained within the Sample Buffer and added to the post-amplification product prior to hybridization. The hybridization control is detected by a unique spot on the hybridization array, thereby confirming successful processing of hybridization and signal enhancement steps.
K233410 - Page 7 of 56
{7}
Internal controls results are reported as Pass, Fail, or N/A (see **Table 4** for detailed explanations of each control result). Internal controls must either (1) generate a signal above threshold in each internal reaction for the system to report a valid test result, or (2) the amplification or extraction control result can be below the signal threshold if a DNA or RNA target pathogen is detected, respectively.
**Table 4.** Interpretation of Internal Control Results for the LIAISON PLEX Respiratory *Flex* Assay
| Internal Control Result | Explanation | Suggested Action |
| --- | --- | --- |
| Pass | - The hybridization control was detected, indicating successful hybridization. - The amplification control was detected, indicating successful amplification. - The extraction control was detected, indicating successful extraction. | Review and report results |
| N/A | - The hybridization control was detected, indicating successful hybridization. - A DNA pathogen target was detected, indicating successful amplification. If a DNA pathogen target is detected, the amplification control result is ignored. - The extraction control was detected, indicating successful extraction. | Review and report results |
| N/A | - The hybridization control was detected, indicating successful hybridization. - The amplification control was detected, indicating successful amplification. - An RNA pathogen target was detected, indicating successful extraction. If an RNA pathogen target is detected, the extraction control result is ignored. | Review and report results |
| Fail | - The hybridization control was not detected, indicating hybridization was not successful. - The amplification control, or a DNA pathogen was detected, indicating successful amplification. - The extraction control, or an RNA pathogen was detected, indicating successful extraction. | Repeat test with a new cartridge |
| Fail | - The hybridization control was detected indicating successful hybridization. - The amplification control, or a DNA pathogen was not detected, indicating amplification was not successful. - The extraction control, or a RNA pathogen was detected, indicating successful extraction. | Repeat test with a new cartridge |
| Fail | - The hybridization control was detected indicating successful hybridization. | Repeat test with a new cartridge |
K233410 - Page 8 of 56
{8}
| | - The amplification control, or a DNA pathogen was detected, indicating successful amplification. - The extraction control, or a RNA pathogen, was not detected, indicating extraction was not successful. | |
| --- | --- | --- |
### External Controls
External controls are not provided with the Respiratory *Flex* Assay. However, five external control mixes (see **Table 5** below) were provided to the clinical study sites for daily testing during the prospective clinical study. External controls were tested on each day of testing, utilizing one external negative control and one of four external positive controls (tested on a rotating basis) representing all Respiratory *Flex* targets.
**Table 5.** External Controls Utilized in the Clinical Studies
| External Control | Expected Calls |
| --- | --- |
| Positive Run Control - Pool 1 | Adenovirus, *Chlamydia pneumoniae*, human coronavirus, hMPV, Influenza B, Parainfluenza 1, Parainfluenza 2, RSV |
| Positive Run Control - Pool 2 | *Bordetella holmesii*, *Bordetella pertussis*, Enterovirus/Rhinovirus, Influenza A, Influenza A (subtype H1), Influenza A (subtype H3), *Mycoplasma pneumoniae*, Parainfluenza 3, Parainfluenza 4 |
| Positive Run Control - Pool 3 | SARS-CoV-2 |
| Positive Run Control - Pool 4 | *Bordetella parapertussis*, *Bordetella pertussis* |
| Negative Run Control | NA |
The sponsor is also including the following in the product package insert, “*Positive and negative external controls should be tested with each new lot or shipment of reagents, or monthly, (whichever occurs first), or in accordance with updated local, regional, state, and/or federal guidelines. Verified negative nasopharyngeal swab (NPS) specimens can be used as the negative control. Previously characterized positive samples or verified negative NPS specimens spiked with well characterized organisms may be used as the external positive control. External controls should be used in accordance with laboratory protocols and in accordance with local, state, and federal accrediting organizations, as applicable.*”
## 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):
| Device & Predicate Device(s): | K233410 | DEN200031 |
| --- | --- | --- |
| Device Trade Name | LIAISON Plex Respiratory *Flex* Assay | BioFire Respiratory Panel 2.1 (RP2.1) |
| Regulation Number and Name | Same | 21 CFR 866.3981; Devices to detect and identify nucleic acid targets in respiratory samples from |
K233410 - Page 9 of 56
{9}
| | | microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-analyte test. |
| --- | --- | --- |
| **Product Code** | Same | QOF |
| **Intended Use/Indications For Use** | The LIAISON PLEX Respiratory *Flex* (RSP *Flex*) Assay is a multiplexed qualitative test for the simultaneous *in vitro* detection and identification of multiple bacterial and viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of respiratory tract infection, including SARS-CoV-2. The test is performed on the automated LIAISON PLEX System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific nucleic acid gene sequences of the following organism types and subtypes: *Viruses:* Adenovirus Human Coronavirus (HKU1, NL63, OC43, and 229E not differentiated) Human Enterovirus/Rhinovirus (not differentiated) Human Metapneumovirus, Influenza A Influenza A (subtype H1) Influenza A (subtype H3) Influenza B Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Parainfluenza 4 Respiratory Syncytial Virus Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) *Bacteria:* *Bordetella holmesii* *Bordetella parapertussis* *Bordetella pertussis* *Chlamydia pneumoniae* *Mycoplasma pneumoniae* Nucleic acids from the bacterial and viral organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and | 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 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 2 (SARS-CoV-2), - Human Metapneumovirus, - Human Rhinovirus/Enterovirus, - Influenza A, including subtypes H1, H3 and H1-2009, - 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 |
K233410 - Page 10 of 56
{10}
| | identifying specific bacterial and viral nucleic acids from individuals exhibiting signs and symptoms of 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 patient management decisions. Negative results in the presence of a respiratory illness may be due to infection with pathogens that are not detected by this test or due to lower respiratory tract infection that is not detected by an NPS specimen. Conversely, positive results do not rule out infection or co-infection with organisms not detected by the LIAISON PLEX Respiratory Flex (RSP Flex) Assay. The agent(s) detected 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. | 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 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 co-infection with other organisms. The agent(s) detected by the BioFire RP2.1 may not be the definite cause of disease. Additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient possible respiratory tract infection. |
| --- | --- | --- |
| Measurand | Same | Nucleic acids from target organisms |
| Sample Type | Same | Nasopharyngeal swab (NPS) |
| Instrumentation | LIAISON PLEX System | BioFire FilmArray 2.0 or BioFire FilmArray Torch Systems |
| Technological Principles | Highly multiplexed nucleic acid PCR and RT-PCR test with microarray detection. | Highly multiplexed nested nucleic acid amplification with melt analysis. |
| Internal Controls | Multiple internal controls contained in the cartridge monitor sample processing and RT and PCR functions. | Two controls are included in each reagent pouch to control for sample processing and both stages of PCR and melt analysis. |
| Automated Test Processes | Same | Nucleic Acid Extraction and Amplification, Detection and Results Interpretation |
| Bordetella Species Detected | - Bordetella parapertussis - Bordetella pertussis - Bordetella holmesii | - Bordetella parapertussis - Bordetella pertussis |
| Human Coronavirus Result Reporting | The human coronavirus target species (i.e., HKU1, OC43, 229E, NL63) are not differentiated. | Each target human coronavirus species (i.e., HKU1, OC43, 229E, NL63) is reported independently. |
| Influenza A Subtyping | Influenza A subtypes H1 and H3 detected/reported. | Influenza A subtypes H1, H1-2009, and H3 detected/reported. |
K233410 - Page 11 of 56
{11}
| **Time to Result** | ~ 2 hours | ~45 minutes |
| --- | --- | --- |
## VI Standards/Guidance Documents Referenced:
### Standards
- ISTA 3A. Packaged-Products for Parcel Delivery System Shipment 70 kg (150 lb) or Less. (2018).
- ITSA 7D. Temperature Test for Transport Packaging.
- CLSI EP07. Interference Testing in Clinical Chemistry; Third Edition.
- CLSI EP37. Supplemental Tables for Interference Testing in Clinical Chemistry; First Edition.
- CLSI EP25-A. Evaluation of Stability of *In Vitro* Diagnostic Reagents; Approved Guideline.
- CLSI EP17-A2. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition.
- CLSI EP05-A3. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition.
- CLSI EP12-A2. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline – Second Edition.
- CLSI EP24-A2. Assessment of the Diagnostic Accuracy of Laboratory Testing Using Receiver Operating Characteristic Curves; Approved Guideline – Second Edition.
- ISO 14971. Medical Devices – Application of Risk Management to Medical Devices. Third Edition (2019-12).
- ISO 23640. *In Vitro* Diagnostic Medical Devices – Evaluation of Stability of *In Vitro* Diagnostic Reagents.
- ISO 15223-1. Medical Devices – Symbols to be Used with Information to be Supplied by the Manufacturer-Part 1: General Requirements. Fourth Edition (2021-07).
- ISO 3864-1. Graphic Symbols – Safety Colors and Safety Signs – Part 1. Design Principles for Safety Signs and Safety Markings (2011).
- IEC 61010-1 Edition 3.1, Consolidated Version. Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use – Part 1: General Requirements, Including Corrigendum 1. (2017-01).
- IEC 61326-2-6 Edition 3.0. Electrical Equipment for Measurement Control and Laboratory Use – EMC Requirements – Part 2-6: Particular Requirements – *In Vitro* Diagnostic (IVD) Medical Equipment (2010-10).
- IEC 60601-1-2 Edition 4.0. Medical Electrical Equipment – Part 1-2: General Requirements for Basic Safety and Essential Performance – Collateral Standard: Electromagnetic Disturbances-Requirements and Tests (2014-02).
- IEC 61000-3-2. Electromagnetic Compatibility (EMC) – Part 3-2: Limits for Harmonic Current Emissions, Input Current Up to & Including 16A Per Phase (2014).
- IEC 61000-3-3. Electromagnetic Compatibility (EMC) – Part 3-3: Limits – Limitation of Voltage Changes, Voltage Fluctuations and Flicker in Public Low – Voltage Supply Systems for Equipment (2013).
### Special Controls
- Class II Special Controls as per 21 CFR 866.3981
### Guidance Documents
K233410 - Page 12 of 56
{12}
- Electronic Submission Template for Medical Device 510(k) Submissions - Guidance for Industry and Food and Drug Administration Staff (October 2, 2023).
- Respiratory Viral Panel Multiplex Nucleic Acid Assay - Class II Special Controls Guidance for Industry and FDA Staff (October 9, 2009).
- Content of Premarket Submissions for Device Software Functions - Guidance for Industry and Food and Drug Administration Staff (June 14, 2023).
- Cybersecurity in Medical Devices: Quality System Considerations and Content of Premarket Submissions - Guidance for Industry and Food and Drug Administration Staff (September 23, 2023).
- Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests - Guidance for Industry and FDA Staff (March 13, 2007).
# VII Performance Characteristics (if/when applicable):
# A Analytical Performance:
# 1. Precision/Reproducibility:
# a. Within-Laboratory Precision
Within-laboratory precision was evaluated at a single site using the Respiratory Flex Assay run on the LIAISON PLEX System. A total of three contrived panels containing known quantities of the target analytes were prepared in simulated NPS matrix, consisting of HeLa cells at a concentration of 2x10³ cells/mL in UTM. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Matrix Equivalency Study, later in this document. The viral and bacterial materials used to generate the positive panel members are denoted in Table 6. The contrived positive panels consisted of five representative target organisms co-spiked at a low positive concentration (1.5x LoD) and moderate concentration (5x LoD). A negative panel was also included in the study (see Table 7). The study was conducted with one operator and three cartridge lots over the course of 5 non-consecutive days on two LIAISON PLEX Systems. Each panel member was tested in triplicate once per day on 5 different days generating a total of 45 replicates per panel member (1 Site x 1 Operator x 3 Lots x 5 Days X 1 Run per Day x 3 Replicates per Run).
Table 6. Viral and Bacterial Strains Used in the Within-Laboratory Precision Study
| Description | Organism Type | Vendor | Catalog Number |
| --- | --- | --- | --- |
| *Bordetella pertussis* (9797) | Bacterium | ATCC | 9797 |
| Adenovirus (4E) | DNA virus | Zeptometrix | 0810070CF |
| Influenza B (Colorado/06/2017) | RNA virus | Zeptometrix | 0810573CF |
| hMPV (27A2) | RNA virus | Zeptometrix | 0810164CF |
| SARS-CoV-2 (USA-WA1/2020) | RNA virus | Zeptometrix | 0810587CFHI |
Table 7. Precision Study Sample Panel
| Panel ID | Description |
| --- | --- |
| 1 | *Bordetella pertussis*, Adenovirus, Influenza B, hMPV, SARS-CoV-2 (Low positive; all analytes at 1.5x LoD) |
| 2 | *Bordetella pertussis*, Adenovirus, Influenza B, hMPV, SARS-CoV-2 (Moderate positive; all analytes at 5x LoD) |
| 3 | Negative |
K233410 - Page 13 of 56
{13}
The qualitative results (i.e., % agreement with expected results) from the study are illustrated in **Table 8**.
**Table 8.** Within-Laboratory Precision Study – Qualitative Results
| Target | Panel ID | Panel Conc. | % Positive (pos n/ valid n) | % Agreement with Expected Results/ (95% CI) |
| --- | --- | --- | --- | --- |
| *Bordetella pertussis* | 1 | Low positive | 93.3% (42/45) | 93.3% (82.1-97.7%) |
| | 2 | Mod. positive | 100% (45/45) | 100% (92.1-100%) |
| | 3 | Negative | 0% (0/45) | 100% (92.1-100%) |
| Adenovirus | 1 | Low positive | 97.8% (44/45) | 97.8% (88.4-99.6%) |
| | 2 | Mod. positive | 97.8% (44/45) | 97.8% (88.4-99.6%) |
| | 3 | Negative | 0% (0/45) | 100% (92.1-100%) |
| Influenza B | 1 | Low positive | 100% (45/45) | 100% (92.1-100%) |
| | 2 | Mod. positive | 100% (45/45) | 100% (92.1-100%) |
| | 3 | Negative | 0% (0/45) | 100% (92.1-100%) |
| hMPV | 1 | Low positive | 100% (45/45) | 100% (92.1-100%) |
| | 2 | Mod. positive | 97.8% (44/45) | 97.8% (88.4-99.6%) |
| | 3 | Negative | 0% (0/45) | 100% (92.1-100%) |
| SARS-CoV-2 | 1 | Low positive | 100% (45/45) | 100% (92.1-100%) |
| | 2 | Mod. positive | 100% (45/45) | 100% (92.1-100%) |
| | 3 | Negative | 0% (0/45) | 100% (92.1-100%) |
*Note:* Results are shown only for the intended targets. Panel members co-spiked with 5 different targets are presented 5 times.
All low positive (1.5x LoD) panel members were positive >97.8%, except for *Bordetella pertussis*, which had a positivity of 93.3% (42/45). All moderate positive (5x LoD) panel members were 100% positive for the spiked target analytes, except for adenovirus and hMPV, which yielded 97.8% positivity (44/45). The negative panel member was 100% negative. There was no lot-to-lot variability observed in the study. The results of the study demonstrate acceptable assay variability.
# b. Reproducibility
A reproducibility study was conducted at three testing sites using the Respiratory *Flex* Assay run on the LIAISON PLEX System. The study incorporated potential sources of variation introduced by site (three testing sites), day (5 different days), operator (two operators per site), and instrument (six LIAISON PLEX Systems). One lot of Respiratory *Flex* Assay cartridges was tested at three sites by two operators per site over five days.
The same three contrived panels used to evaluate precision (see **Table 7**) were included in the reproducibility study. Three replicates of each panel member were tested by each operator at each site on all 5 days of testing generating a total of 90 replicates per panel member. The qualitative results of the study are illustrated in **Table 9**.
**Table 9.** Reproducibility Study – Qualitative Results
| Target | Panel ID | Panel Conc. | % Agreement with Expected Results | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Site 1 | Site 2 | Site 3 | Overall/ (95% CI) |
| *Bordetella pertussis* | 1 | Low positive | 93.3% (28/30) | 100% (30/30) | 96.7% (29/30) | 96.7% (87/90) (90.7-98.9%) |
K233410 - Page 14 of 56
{14}
| | 2 | Mod. positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| --- | --- | --- | --- | --- | --- | --- |
| | 3 | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| Adenovirus | 1 | Low positive | 96.7% (29/30) | 100% (30/30) | 96.7% (29/30) | 97.8% (88/90) (92.3-99.4%) |
| | 2 | Mod. positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| | 3 | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| Influenza B | 1 | Low positive | 93.3% (28/30) | 100% (30/30) | 96.7% (29/30) | 96.7% (87/90) (90.7-98.9%) |
| | 2 | Mod. positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| | 3 | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| hMPV | 1 | Low positive | 90.0% (27/30) | 100% (30/30) | 93.3% (28/30) | 94.4% (85/90) (87.6-97.6%) |
| | 2 | Mod. positive | 100% (30/30) | 93.3% (28/30) | 100% (30/30) | 97.8% (88/90) (92.3-99.4%) |
| | 3 | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| SARS-CoV-2 | 1 | Low positive | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | 98.9% (89/90) (94.0-99.8%) |
| | 2 | Mod. positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| | 3 | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
Mod – moderate; *Note:* Results are shown only for the intended targets. Panel members co-spiked with 5 different targets are presented 5 times.
The negative panel was negative 100% of the time. The moderate positive (5x LoD) target gave expected results 100% of the time for adenovirus, *Bordetella pertussis*, influenza B, and SARS-CoV-2. The moderate positive for human metapneumovirus yielded 97.8% (88/90) positive results.
For the low positive (1.5x LoD) target, all targets except for human metapneumovirus yielded at least 96.7% (87/90) positivity. The overall positivity for the human metapneumovirus low positive sample was 94.4% (85/90).
K233410 - Page 15 of 56
{15}
# 2. Linearity:
Not applicable; this is a qualitative assay.
# 3. Analytical Specificity/Interference:
# Analytical Reactivity (Inclusivity)
The inclusivity of the LIAISON PLEX Respiratory Flex Assay was evaluated using a combination of in silico analysis of publicly available sequence information and laboratory testing of contrived specimens containing viral and bacterial isolates that were selected to represent phylogenetic, geographic, and temporal diversity.
# a. Wet-Testing
This study was performed to determine the analytical reactivity of the Respiratory Flex Assay with clinically relevant strains, serotypes, or subtypes of the target species. The inclusivity panel was prepared by spiking various target microorganism strains/serotypes/subtypes encompassing temporal and geographical diversity into simulated NPS matrix at a concentration of ~3x LoD and testing in triplicate. Data supporting the use of simulated matrix can be found in section VII Performance Charactestics.B.2.Matrix Equivalency Study, later in this document. Strains that did not yield 100% reactivity at 3x LoD were reprepared at the same concentration and retested in triplicate. If 100% reactivity was obtained during retesting, reactivity was reached. In this situation, the original and retesting results were pooled for performance calculations. If less than 100% reactivity was observed during retesting, the strain was prepared at a higher concentration and tested until 100% reactivity was achieved. The strains evaluated and the lowest concentration that met the reactivity criteria outlined above are shown in Table 10 - Table 23, below.
Table 10. Inclusivity Testing – Adenovirus Results
| Strain | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- |
| | Copies/mL | xLoD | |
| A 31 | \( 5.28 \times 10^{3} \) | 3x | 100% (3/3) |
| B 3 | \( 2.06 \times 10^{3} \) | | 100% (3/3) |
| B 7A | \( 2.06 \times 10^{3} \) | | 100% (3/3) |
| B 21 | \( 2.06 \times 10^{3} \) | | 100% (3/3) |
| B 11 | \( 2.06 \times 10^{3} \) | | 100% (3/3) |
| B 14 | \( 2.06 \times 10^{3} \) | | 100% (3/3) |
| B 34 | \( 2.06 \times 10^{3} \) | | 83.3% (5/6)\( ^{1} \) |
| B 35 | \( 2.06 \times 10^{3} \) | | 100% (3/3) |
| C 1 | \( 3.35 \times 10^{3} \) | | 100% (3/3) |
| C 2 | \( 3.35 \times 10^{3} \) | | 100% (3/3) |
| C 5 | \( 3.35 \times 10^{3} \) | | 100% (3/3) |
| C 6 | \( 3.35 \times 10^{3} \) | | 100% (3/3) |
| D 26 | \( 2.24 \times 10^{3} \) | | 100% (3/3) |
| D 37 | \( 2.24 \times 10^{3} \) | | 100% (3/3) |
| E 4 | \( 1.06 \times 10^{3} \) | | 83.3% (5/6)\( ^{1} \) |
| F 40-Dugan | \( 1.45 \times 10^{3} \) | | 100% (3/3) |
| F 41-Tak | \( 1.45 \times 10^{3} \) | | 100% (3/3) |
\( ^{1} \) The original three replicates tested resulted in 66.7% (2/3) positivity for adenovirus. New test material was prepared and tested, resulting in 100% (3/3) positivity for adenovirus.
K233410 - Page 16 of 56
{16}
**Table 11.** Inclusivity Testing – *Bordetella holmesii* Results
| Strain | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- |
| | Copies/mL | xLoD | |
| FA061 | 2.19x10^{4} | 3x | 100% (3/3) |
| CDC F5101 [CDC 84-013939] | 2.19x10^{4} | | 83.3% (5/6)^{1} |
| CIP 104395 [G7702; 92A2997] | 2.19x10^{4} | | 80.0% (4/5)^{2} |
| CIP 104396 | 1.97x10^{5} | 27x^{3} | 100% (3/3) |
$^{1}$The original three replicates tested resulted in 66.7% (2/3) positivity for *B. holmesii*. New test material was prepared and tested, resulting in 100% (3/3) positivity for *B. holmesii*.
$^{2}$The original three replicates tested resulted in 33% (1/3) invalid replicates and 50% (1/2) positivity for *B. holmesii*. New test material was prepared and tested, resulting in 100% (3/3) positivity for *B. holmesii*.
$^{3}$Testing at lower concentrations (i.e., 3x and 9x LoD) failed to yield 100% detection. The lowest concentration that yielded 100% detection was 27x LoD.
**Table 12.** Inclusivity Testing – *Bordetella parapertussis* Results
| Strain | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- |
| | Copies/mL | xLoD | |
| NCTC 5952 [522] | 2.37x10^{3} | 3x | 100% (3/3) |
| 508 and 344 [NCTC10853] | | | 100% (3/3) |
| 517 | | | 100% (3/3) |
| 12822 | | | 100% (3/3) |
| 509 and 609 | | | 100% (3/3) |
| PT28G | | | 100% (3/3)^{1} |
| PT 26/28G | | | 100% (3/3)^{1} |
| C510 | | | 100% (3/3) |
$^{1}$Testing results in positivity for *B. parapertussis* and *B. pertussis*. CoAs from the vendor confirm that these are genetically modified strains, engineered to contain DNA sequences for both *B. parapertussis* and *B. pertussis* toxins.
**Table 13.** Inclusivity Testing – *Bordetella pertussis* Results
| Strain | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- |
| | Copies/mL | xLoD | |
| 18323 [NCTC 10739] | 1.14x10^{4} | 3x | 100% (3/3) |
| CNCTC Hp 12/63 [623] | | | 100% (3/3) |
| 10-536 | | | 100% (3/3) |
| 5 [17921] | | | 100% (3/3) |
| Tohama I | | | 100% (3/3) |
| MN2531 | | | 100% (3/3) |
| PT9/28G [W28] | | | 100% (3/3) |
| 589 | | | 100% (3/3) |
| F | | | 100% (3/3) |
**Table 14.** Inclusivity Testing – *Chlamydia pneumoniae* Results
| Strain | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- |
| | Copies/mL | xLoD | |
| CWL-029 | 1.71x10^{3} | 3x | 100% (3/3) |
| AR-39 | | | 100% (3/3) |
| J-21 | | | 100% (3/3) |
| 2023 | | | 100% (3/3) |
K233410 - Page 17 of 56
{17}
**Table 15. Inclusivity Testing – Human Coronavirus Results**
| Species/Strain | Concentration | | % Detected (# Detected/#Tested) | |
| --- | --- | --- | --- | --- |
| | | Copies/mL | | xLoD |
| HKU1 | CS-Lum2020-Resp-1146 | 5.0x10^{3} | 3x | 100% (3/3) |
| | CS-83254 | | | 100% (3/3) |
| NL63 | Source #: 0810228CF | 2.29x10^{2} | 3x | 100% (3/3) |
| | Source #: NR-470 | 6.88x10^{2} | 9x^{1} | 100% (3/3) |
| OC43 | Source #: 0810024CF | 2.84x10^{4} | 3x | 100% (3/3) |
| | Source #: VR-1558 | | | 100% (3/3) |
| 229E | Source #: 0810229CF | 1.20x10^{3} | 3x | 100% (3/3) |
| | Source #: VR-740 | 3.60x10^{3} | 9x^{1} | 100% (3/3) |
CS-Clinical sample
$^{1}$Testing at a lower concentration (i.e., 3x LoD) failed to yield 100% detection. The lowest concentration that yielded 100% detection was 9x LoD.
**Table 16. Inclusivity Testing – Enterovirus/Rhinovirus Results**
| Species/Strain | Concentration | | % Detected (# Detected/#Tested) | |
| --- | --- | --- | --- | --- |
| | | Copies/mL | | xLoD |
| Enterovirus A | Coxsackievirus A10 | 6.75x10^{4} | 3x | 100% (3/3) |
| | Coxsackievirus 71 | | | 100% (3/3) |
| Enterovirus B | Coxsackievirus A9 | | | 100% (3/3) |
| | Coxsackievirus B3 | | | 100% (3/3) |
| | Coxsackievirus B4 | | | 100% (3/3) |
| | Echovirus 6 | | | 100% (3/3) |
| | Echovirus 9 | | | 100% (3/3) |
| | Echovirus 11 | | | 100% (3/3) |
| | Echovirus 30 | | | 100% (3/3) |
| Enterovirus C | Coxsackievirus A21 | | | 100% (3/3) |
| | Coxsackievirus A24 | | | 100% (3/3) |
| Enterovirus D | 68 | | | 100% (3/3) |
| Rhinovirus A | 16 | 2.46x10^{4} | 3x | 100% (3/3) |
| | 2 | | | 100% (3/3) |
| | 34 | | | 100% (3/3) |
| | 57 | | | 100% (3/3) |
| | 7 | | | 100% (3/3) |
| | 77 | | | 100% (3/3) |
| | 85 | | | 100% (3/3) |
| Rhinovirus B | 14 | 2.45x10^{4} | 3x | 100% (3/3) |
| | 17 | | | 100% (3/3) |
| | 27 | | | 100% (3/3) |
| | 3 | | | 100% (3/3) |
| | 42 | | | 100% (3/3) |
| | 83 | | | 100% (3/3) |
| Rhinovirus C | CS-75029 (H7-3) | 5.75x10^{4} | 3x | 100% (3/3) |
| | CS-75466 (J3-3) | | | 100% (3/3) |
| | CS-NPS/UTU NEG 178 | | | 100% (3/3) |
| | CS-NEG004 (SAR-4) | | | 100% (3/3) |
CS-Clinical sample
K233410 - Page 18 of 56
{18}
**Table 17. Inclusivity Testing –Human Metapneumovirus Results**
| Strain | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- |
| | Copies/mL | xLoD | |
| hMPV-9 (Type A1) | 6.40x10^{3} | 3x | 100% (3/3) |
| hMPV-16 (Type A1) | | | 100% (3/3) |
| hMPV-20 (Type A2) | 6.12x10^{3} | 3x | 100% (3/3) |
| hMPV-27 (Type A2) | | | 100% (3/3) |
| hMPV-3 (Type B1) | 1.50x10^{4} | 3x | 100% (3/3) |
| hMPV-5 (Type B1) | | | 100% (3/3) |
| hMPV-4 (Type B2) | 4.50x10^{4} | 3x | 100% (3/3) |
| hMPV-8 (Type B2) | | | 100% (3/3) |
| hMPV-18 (Type B2) | | | 100% (3/3) |
**Table 18. Inclusivity Testing – Influenza A Results**
| Species/Strain | Concentration | % Detected (# Detected/#Tested) |
| --- | --- | --- |
| Copies/mL | xLoD |
| H1N1 | A/Wisconsin/588/2019 | 4.5x10^{3} | 3x | Matrix: 100% (3/3) |
| A/Hawaii/66/2019 X-345A | Subtype H1: 100% (3/3) |
| A/Indiana/02/2020 | Matrix: 100% (6/6) |
| A/Michigan/272/2017 | Subtype H1: 83.3% (5/6)^{1} |
| A/Idaho/07/2018 | Matrix: 100% (3/3) |
| A/Wisconsin/505/2018 | Subtype H1: 100% (3/3) |
| Guangdong-Maonan /SWL 1536/19 | Matrix: 100% (3/3) |
| Brisbane/02/18 | Subtype H1: 100% (3/3) |
| A/St.Petersburg/61/2015 | Matrix: 100% (6/6) |
| A/Bangladesh/3002/2015 | Subtype H1: 83.3% (5/6)^{1} |
| A/Denver/1/57 | Matrix: 100% (6/6) |
| New Caledonia/20/99 | Subtype H1: 83.3% (5/6)^{1} |
| PR/8/34 | 4.5x10^{3} | 3x | Matrix: 100% (3/3) |
| Singapore/63/04 | Subtype H1: 100% (3/3) |
| Solomon Islands/03/06 | Matrix: 100% (3/3) |
| Taiwan/42/06 | Subtype H1: 100% (3/3) |
| H1N1v | A/Ohio/09/2015 (Subtype Synthetic DNA) | Matrix: 100% (3/3) |
| A/Ohio/09/2015 | Subtype H1: 100% (3/3) |
| | 4.5x10^{3} | Matrix: 100% (3/3) | Subtype H1: 100% (3/3) |
| | | Matrix: 100% (3/3) | Subtype H1: 100% (3/3) |
| | | Matrix: 100% (3/3) | Subtype H1: 100% (3/3) |
| | | Matrix: 100% (3/3) | Subtype H1: 100% (3/3) |
| | | Matrix: 100% (3/3) | Subtype H1: 100% (3/3) |
| | | Matrix: 100% (3/3) | Subtype H1: 100% (3/3) |
| | | | Matrix: 100% (3/3) | Subtype H1: 100% (3/3) |
| | | | | Matrix: 100% (3/3) |
| | | | | Subtype H1: 100% (3/3) |
| | | | | Matrix: 0% (0/3)^{2} |
| | | | | Subtype H1: 100% (3/3) |
| | | | | Matrix: 100% (3/3) |
K233410 - Page 19 of 56
{19}
| Species/Strain | Concentration | | % Detected (# Detected/#Tested) | |
| --- | --- | --- | --- | --- |
| | | Copies/mL | | xLoD |
| | (Matrix Synthetic DNA) | | | Subtype H1: 0% (0/3)^{2} |
| H1N2 | A/swine/Ohio/09SW1484E/2009 | 4.5x10^{3} | | Matrix: 100% (3/3) |
| | | | | Subtype H1: 100% (3/3) |
| H1N2v | A/Minnesota/19/2011 | 4.5x10^{3} | | Matrix: 0% (0/3)^{2} |
| | (Subtype Synthetic DNA) | | | Subtype H1: 100% (3/3) |
| | A/Minnesota/19/2011 | | | Matrix: 100% (3/3) |
| | (Matrix Synthetic DNA) | | | Subtype H1: 0% (0/3)^{2} |
| H3N2 | A/Kansas/14/2017 NYMC X-327 | 5.89x10^{3} | 3x | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| | A/Texas/71/2017 | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| | A/Wisconsin/04/2018 | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| | A/Arizona/45/2018 | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| | A/Hong Kong/45/2019 | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| | A/Tasmania/503/2020 | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| | A/Delaware/01/2021 | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| | A/Singapore/INFIMH-16-0019/2016 | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| | /California/55/2020 | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 100% (3/3) |
| A/Alaska/232/2015 | Matrix: 100% (3/3) | | | |
| | Subtype H3: 100% (3/3) | | | |
| H3N2v | A/Hawaii/28/2020 | 5.89x10^{3} | | Matrix: 0% (0/3)^{2} |
| | (Subtype Synthetic DNA) | | | Subtype H3: 100% (3/3) |
| | A/Hawaii/28/2020 (Matrix Synthetic DNA) | | | Matrix: 100% (3/3) |
| | | | | Subtype H3: 0% (0/3)^{2} |
| H5N1 | A/Egypt/N03072/2010 | 5.89x10^{3} | | Matrix: 100% (3/3) |
| | | | | Subtype: 0% (0/3)^{2} |
| | A/Hubei/1/2010 | | | Matrix: 100% (3/3) |
| | | | | Subtype: 0% (0/3)^{2} |
| | A/Anhui/01/2005 | | | Matrix: 100% (3/3) |
| | | | | Subtype: 0% (0/3)^{2} |
| H7N2 | A/turkey/Virginia/4529/2002 | 5.89x10^{3} | | Matrix: 100% (3/3) |
| | | | | Subtype: 0% (0/3)^{2} |
| H7N7 | A/mallard/Netherlands/12/2000 | 5.89x10^{3} | | Matrix: 100% (3/3) |
| | | | | Subtype: 0% (0/3)^{2} |
| H9N2 | A/Hong Kong/33982/2009 | 5.89x10^{3} | | Matrix: 100% (3/3) |
| | | | | Subtype: 0% (0/3)^{2} |
$^{1}$The original three replicates tested resulted in 66.7% (2/3) positivity for the influenza A H1 subtype. New test material was prepared and tested, resulting in 100% (3/3) positivity for the influenza A H1 subtype.
$^{2}$No positivity was expected based on the strain and/or type of material being tested.
$^{3}$Testing at a lower concentration (i.e., 3x LoD) failed to yield 100% detection. The lowest concentration that yielded 100% detection was 9x LoD.
K233410 - Page 20 of 56
{20}
**Table 19. Inclusivity Testing – Influenza B Results**
| Lineage/Strain | Concentration | | % Detected (# Detected/#Tested) | |
| --- | --- | --- | --- | --- |
| | | Copies/mL | | xLoD |
| Victoria Lineage | B/Washington/02/2019 | 1.01x10^{3} | 3x | 100% (3/3) |
| | B/New Hampshire/01/2021 | | | 100% (3/3) |
| | B/Missouri/12/2018 (NA D197E) | | | 100% (3/3) |
| | B/Hawaii/01/2018 (NA D197N) | | | 100% (3/3) |
| | B/Michigan/01/2021 | | | 100% (3/3) |
| | B/Hong Kong/286/2017 | | | 100% (3/3) |
| | B/Colorado/6/2017 | | | 100% (3/3) |
| | B/Texas/43/2019 | | | 100% (3/3) |
| Yamagata Lineage | B/Wisconsin/1/10 | | | 100% (3/3) |
| | B/Florida/02/06 | | | 100% (3/3) |
| | B/Florida/07/04 | | | 100% (3/3) |
| | B/Phuket/3073/13 | | | 100% (3/3) |
| | B/Wisconsin/10/2016 (NA I221V) | | | 100% (3/3) |
| | B/Indiana/17/2017 (NA I221T) | | | 100% (3/3) |
| | B/Oklahoma/10/2018 (NA D197N) | | | 100% (3/3) |
**Table 20. Inclusivity Testing – *Mycoplasma pneumoniae* Results**
| Strain | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- |
| | Copies/mL | xLoD | |
| M129 | 3.89x10^{3} | 3x | 100% (3/3) |
| 15531-TTR | | | 100% (3/3) |
| Mac | | | 100% (3/3) |
| PI 1428 | | | 100% (3/3) |
| Bru | | | 100% (3/3) |
| M52 | | | 100% (3/3) |
| UTMB-10P | | | 100% (3/3) |
| Mutant 22 | | | 100% (3/3) |
| M129-B7 | | | 100% (3/3) |
**Table 21. Inclusivity Testing – Parainfluenza Results**
| Species/Strain | Concentration | | % Detected (# Detected/#Tested) | |
| --- | --- | --- | --- | --- |
| | | Copies/mL | | xLoD |
| PIV-1 | N/A | 2.28x10^{3} | 3x | 83.3% (5/6)^{1} |
| | C35 | | | 100% (3/3) |
| PIV-2 | N/A | 2.54x10^{4} | | 100% (3/3) |
| | Greer | | | 100% (3/3) |
| PIV-3 | N/A | 5.79x10^{3} | | 100% (3/3) |
| | ATCC-2011-5 | | | 83.3% (5/6)^{2} |
| | C243 | | | 100% (3/3) |
| | NIH 47885 | | | 100% (3/3) |
| PIV-4a | N/A | 1.73x10^{4} | | 100% (3/3) |
| | M-25 | | | 100% (3/3) |
K233410 - Page 21 of 56
{21}
| Species/Strain | Concentration | | % Detected (# Detected/#Tested) | |
| --- | --- | --- | --- | --- |
| | | Copies/mL | | xLoD |
| PIV-4b | N/A | | | 100% (3/3) |
| | CH 19503 | | | 100% (3/3) |
$^{1}$The original three replicates tested resulted in 66.7% (2/3) positivity for parainfluenza 1. New test material was prepared and tested, resulting in 100% (3/3) positivity for parainfluenza 1.
$^{2}$The original three replicates tested resulted in 66.7% (2/3) positivity for parainfluenza 3. New test material was prepared and tested, resulting in 100% (3/3) positivity for parainfluenza 3.
**Table 22.** Inclusivity Testing – RSV Results
| Species/Strain | Concentration | | % Detected (# Detected/#Tested) | |
| --- | --- | --- | --- | --- |
| | | Copies/mL | | xLoD |
| RSV-A | 2006 Isolate | 1.15x10^{4} | 3x | 100% (3/3) |
| | A2 | | | 100% (3/3) |
| | Long | | | 100% (3/3) |
| RSV-B | CH93(18)-18 | 4.82x10^{4} | | 100% (3/3) |
| | B WV/14617/85 | | | 100% (3/3) |
| | 18537 | | | 100% (3/3) |
| | B1 | | | 100% (3/3) |
**Table 23.** Inclusivity Testing – SARS-CoV-2 Results
| Strain | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- |
| | Copies/mL | xLoD | |
| B.1.617.1: Isolate: USA/CA-Stanford-15_S02/2021 (Kappa) | 7.20x10^{4} | 9x^{1} | 100% (3/3) |
| B.1.1.529 BA.1: Isolate: USA/MD-HP20874/2021 (Omicron) | 2.4x10^{4} | 3x | 100% (3/3) |
| Isolate: Italy-INMI1 | | | 100% (3/3) |
| Isolate: Hong Kong/VM20001061/2020 | | | 100% (3/3) |
| B.1_2020: Isolate: USA/NY-Wadsworth-103677-01/2020 | | | 100% (3/3) |
| B.1.1.7: Isolate: England/204820464/2020 (Alpha) | | | 100% (3/3) |
| B.1.1.7: Isolate: USA/CA_CDC_5574/2020 (Alpha) | | | 100% (3/3) |
| B.1.351: Isolate: South Africa/KRISP-K005325/2020 (Beta) | | | 100% (3/3) |
| P1: Isolate: Japan/TY7-503/2021 (Gamma) | | | 100% (3/3) |
| P2_2021: Isolate: NY-Wadsworth-21006055-01/2021 (Zeta) | | | 100% (3/3) |
| B.1.526_2021: Isolate: USA/NY-Wadsworth-21025952-01/2021 Isolate 1 (Lota) | | | 100% (3/3) |
| B.1.617.2: Isolate: USA/PHC658/2021 (Delta) | | | 100% (3/3) |
| USA-WA1/2020 | | | 100% (3/3) |
$^{1}$Testing at a lower concentration (i.e., 3x LoD) failed to yield 100% detection. The lowest concentration that yielded 100% detection was 9x LoD.
K233410 - Page 22 of 56
{22}
The results from this study demonstrate that the Respiratory *Flex* Assay is capable of detecting multiple clinically relevant strains of each target analyte.
### *In silico*
The inclusivity of the LIAISON PLEX Respiratory *Flex* Assay was evaluated using *in silico* analysis of the oligonucleotides (i.e., forward primer(s), reverse primer(s), capture and mediator probe(s)) for all assay targets in relation to sequences available in GISAID (for SARS-CoV-2, influenza A, influenza A/H1, influenza A/H3, and influenza B) and sequences in NCBI GenBank for all other target organisms.
Sequence alignments were generated using a publicly available sequence alignment program. Sequences with full coverage of all four oligo-binding regions (forward primer, reverse primer, mediator probe and capture probe) were included in the analyses. Partial target sequences and sequences with ambiguous or degenerate bases in an oligo binding region were excluded. A match (i.e., predicted reactivity) was based on the following criteria for all organisms other than SARS-CoV-2: ≥90% homology between the oligo and reference sequences. If <90% homology was observed, a melting temperature (Tm) analysis was performed using a publicly available Tm calculator and applying assay specific conditions. The Tm analysis involved comparing the calculated mismatch Tm values against the assay's PCR annealing temperature for the primers, against the RT temperature for the RT primers, and against the target hybridization temperature for the capture and mediator probes. If the Tm of the mismatch oligo:sequence pair was above the assay's temperature for the pertinent RT-PCR step (i.e., annealing, RT, or target hybridization), then reactivity was predicted. The ≥90% homology threshold was chosen because the Respiratory *Flex* Assay's oligos are designed to have a much higher Tm than the annealing/RT temperature for primers and the hybridization temperature for the capture and mediator probes. As such, the longest oligos in the Respiratory *Flex* Assay are more tolerant to mismatches.
For SARS-CoV-2, the criteria were stricter due to the propensity of this organism to acquire point mutations. Specifically, 100% homology was expected between the oligo and reference sequences for at least one gene oligo set. If <100% homology was observed for all gene oligo sets, the same melting temperature analysis and reactivity criteria described above were employed.
For SARS-CoV-2, 5,622,325 sequences in GISAID (as of July 31, 2023) were included in the analysis. These sequences included all variants of concern or variants of interest defined as of July 31, 2023. The LIAISON PLEX Respiratory *Flex* Assay targets three SARS-CoV-2 gene regions. The Respiratory *Flex* Assay result logic states that if at least 1 of these targets is detected, SARS-CoV-2 is positive. This same result logic was implemented for the *in silico* inclusivity assessment. Of the sequences included in this evaluation, 99.94% (5,619,069/5,622,325) have *no* mismatch in at least one gene oligo set and thus are predicted to be detected by the Respiratory *Flex* Assay. Of the 0.04% (3,256/5,622,325) of sequences with mismatches in at least one oligo binding region in all 3 SARS-CoV-2 target genes, a Tm analysis revealed that amplification/hybridization were expected to occur. Thus, it is expected that 100% of SARS-CoV-2 sequences evaluated in this study will be detected by the assay.
For influenza A, influenza A H1, influenza A H3, and influenza B, sequences uploaded to GISAID between September 1, 2015, and July 7, 2023, were included in the analysis. The following number of sequences were included in the evaluation of influenza A, A H1, A H3, and influenza B: 112,056, 54,364, 104,428, 26,470. Of the 26,470 influenza B sequences,
K233410 - Page 23 of 56
{23}
66.1% (17,509/26,470) were Victoria lineage, 30.9% (8,167/26,470) were Yamagata lineage, and 3.0% (794/26,470) were of unknown/unclassified lineage. The influenza in silico inclusivity analysis results are shown in Table 24. Based on the reactivity criteria (>90% homology), 99.9% (112,034/112,056) of influenza A (matrix gene) sequences are expected to be detected, 98.9% (53,778/54,364) of influenza A H1 sequences are expected to be detected, 99.9% (104,315/104,428) of influenza A H3 sequences are expected to be detected, and 99.9% (26,433/26,470) of influenza B sequences are expected to be detected.
Table 24. Influenza In Silico Inclusivity Results
| Reportable Target | Target Gene | of Sequences in Alignment | # of Sequences with Percent Oligo Identify ≥90% |
| --- | --- | --- | --- |
| Influenza A | Matrix protein (MP) | 112,056 | 112,034^{1} |
| Influenza A H1 | HA | 54,364 | 53,778 |
| Influenza A H3 | HA | 104,428 | 104,315 |
| Influenza B | Non-structural protein (NS) | 26,470 | 26,433^{2} |
$^{1}$Analysis included influenza A subtype H0, H1, H3, H5, H7, H9, and H10 strains.
$^{2}$Analysis included 17,509 Victoria lineage strains, 8,167 Yamagata lineage strains, and 794 strains of unknown lineage.
For all other target organisms, in silico inclusivity analyses was performed using sequences available from the GenBank nt database as of July 7, 2023. The majority of sequences evaluated for each target organism are expected to be detected by the Respiratory Flex Assay.
### Cross-Reactivity
#### a. Cross-Reactivity Wet-Testing
##### i. Off-Panel Cross-Reactivity
This study evaluated the analytical specificity (cross-reactivity) of the Respiratory Flex Assay in the presence of non-targeted microorganisms that may be found in a respiratory tract clinical specimen. Sixty (60) non-target microorganisms (Table 25) were evaluated in the study. Panel members were composed of one individual non-target microorganism spiked into simulated NPS matrix at ≥10$^{5}$ TCID$_{50}$/mL (or equivalent) for viruses, ≥10$^{6}$ CFU/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. For Mycobacterium tuberculosis, genomic DNA was evaluated to minimize pathogenic exposure to the test operator. Additionally, DNA was evaluated for Bordetella parapertussis Bpp5, which was identified by in silico analysis to contain a sequence that was between 80.0% - 95.8% identical with the primers, mediators, or capture oligos of the Respiratory Flex Bordetella pertussis assay. Wet testing was performed with synthetic DNA consisting of the sequence for the matching region of the B. parapertussis genome to determine if cross-reactivity with the Bordetella pertussis assay occurred.
To evaluate cross-reactivity, each panel was evaluated in triplicate in the absence of the target organisms. No cross-reactivity was observed at the concentrations tested except for Mycoplasma genitalium at a concentration of 4x10$^{6}$ CCU/mL, which cross-reacted with the Mycoplasma pneumoniae assay in 50% of replicates (3/6). The concentration of
K233410 - Page 24 of 56
{24}
Mycoplasma genitalium was lowered to 1x10⁶ and 4x10⁵ CCU/mL and tested; cross-reactivity no longer occurred at either of these lower concentrations.
Table 25. Off-Panel Organisms Evaluated for Cross-Reactivity
| Organism | Conc./Unit | Organism | Conc./Unit |
| --- | --- | --- | --- |
| Acinetobacter baumannii | 1x10⁶ CFU/mL | Legionella pneumophila | 4x10⁵ CFU/mL |
| Aspergillus flavus | 1x10⁶ CFU/mL | Listeria innocua | 1x10⁶ CFU/mL |
| Aspergillus fumigatus | 4x10⁵ CFU/mL | Listeria monocytogenes | 1x10⁶ CFU/mL |
| Bordetella avium | 1x10⁶ CFU/mL | Measles | 1x10⁵ TCID₅₀/mL |
| Bordetella bronchiseptica | 1x10⁶ CFU/mL | MERS-CoV | NA² |
| Bordetella hinzii | 1x10⁶ CFU/mL | Moraxella catarrhalis | 1x10⁵ TCID₅₀/mL |
| Bordetella petrii | 1x10⁶ CFU/mL | Mumps Virus | 1x10⁵ TCID₅₀/mL |
| Bordetella trematum | 1x10⁶ CFU/mL | Mycobacterium tuberculosis (H37Rv gDNA) | 2.88 ng/uL |
| Bordetella parapertussis Bpp5 (synthetic DNA)¹ | 1x10⁶ copies/mL | Mycoplasma genitalium³ | 4x10⁶ CCU/mL |
| | | | 1x10⁶ CCU/mL |
| | | | 4x10⁵ CCU/mL |
| Candida albicans | 1x10⁶ CFU/mL | Mycoplasma hominis | 1x10⁶ CFU/mL |
| Candida glabrata | 1x10⁶ CFU/mL | Nasal Wash (pooled) | NA² |
| Chlamydia trachomatis Serovar D | 1x10⁶ IFU/mL | Neisseria elongata | 1x10⁶ CFU/mL |
| Coronavirus-SARS | NA² | Neisseria gonorrhoeae | 1x10⁶ CFU/mL |
| Corynebacterium diphtheriae | 1x10⁶ CFU/mL | Neisseria lactamica | 1x10⁶ CFU/mL |
| Corynebacterium pseudodiphtheriticum | 1x10⁶ CFU/mL | Neisseria meningitidis | 1x10⁶ CFU/mL |
| Corynebacterium striatum | 1x10⁶ CFU/mL | Neisseria mucosa | 1x10⁶ CFU/mL |
| Cytomegalovirus | 1x10⁵ TCID₅₀/mL | Neisseria sicca | 1x10⁶ CFU/mL |
| Epstein Barr Virus | 1x10⁵ copies/mL | Pneumocystis jiroveci | 1x10⁶ CFU/mL |
| Escherichia coli | 1x10⁶ CFU/mL | Proteus vulgaris | 1x10⁶ CFU/mL |
| Fluoribacter bozemanae | 4x10⁶ CFU/mL | Pseudomonas aeruginosa | 1x10⁶ CFU/mL |
| Fusobacterium necrophorum | 1x10⁶ CFU/mL | Serratia marcescens | 1x10⁶ CFU/mL |
| Haemophilus influenzae | 1x10⁶ CFU/mL | Staphylococcus aureus | 1x10⁶ CFU/mL |
| Haemophilus parainfluenzae | 1x10⁶ CFU/mL | Staphylococcus epidermidis | 1x10⁶ CFU/mL |
| Herpes Simplex Virus Type 1 | 1x10⁵ TCID₅₀/mL | Staphylococcus haemolyticus | 1x10⁶ CFU/mL |
| Klebsiella pneumoniae | 1x10⁶ CFU/mL | Streptococcus agalactiae | 1x10⁶ CFU/mL |
| Lactobacillus acidophilus | 1x10⁶ CFU/mL | Streptococcus pneumoniae | 1x10⁶ CFU/mL |
| Lactobacillus plantarum | 1x10⁶ CFU/mL | Streptococcus pyogenes | 1x10⁶ CFU/mL |
| Legionella anisa | 1x10⁶ CFU/mL | Streptococcus salivarius | 1x10⁶ CFU/mL |
| Legionella feeleii | 1x10⁶ CFU/mL | Ureaplasma urealyticum | 1x10⁶ CCU/mL |
| Legionella longbeachae | 1x10⁶ CFU/mL | Varicella-Zoster Virus | 2.34x10⁴ TCID₅₀/mL |
CFU = Colony Forming Units; CCU = Colony Changing Units; IFU = Inclusion Forming Units; TCID₅₀ = Median Tissue Culture Infectious Dose.
¹A portion of the B. parapertussis Bpp5 genome was identified by in-silico analysis as potentially cross-reactive with B. pertussis. Synthetic DNA was tested that matched the region of high homology in the assay. Testing was included in the off-panel cross-reactivity study since the targeted sequence was not expected to be detected as B. parapertussis by the assay.
²No concentration provided by the supplier.
K233410 - Page 25 of 56
{25}
$^{3}$At 4x10$^{6}$ CCU/mL, *Mycoplasma genitalium* was cross-reactive with the *Mycoplasma pneumoniae* assay in 50.0% (3/6) of replicates. The *Mycoplasma genitalium* concentration was lowered to 1x10$^{6}$ CCU/mL and 4x10$^{5}$ CCU/mL and had 0% (0/3) positivity for both concentrations.
# ii. On-Panel Cross-Reactivity
Potential intra-panel cross-reactivity was evaluated with twenty-eight (28) on-panel microorganisms (**Table 26**). The on-panel organisms were evaluated by spiking each independently into simulated NPS matrix at ≥10$^{5}$ TCID$_{50}$/mL (or equivalent) for viruses and ≥10$^{6}$ CFU/mL (or equivalent) for bacteria, 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 potential intra-panel cross-reactivity, each panel was evaluated in triplicate. The results of testing are shown in **Table 26**.
**Table 26.** On-Panel Organisms Evaluated for Cross-Reactivity
| Organism | Conc./Unit | Expected Target Positivity | Unexpected Target Positivity |
| --- | --- | --- | --- |
| Adenovirus | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| *Bordetella holmesii* | 1x10^{6} CFU/mL | 100% (3/3) | 0% (0/3) |
| *Bordetella parapertussis* | 1x10^{6} CFU/mL | 100% (3/3) | 0% (0/3) |
| *Bordetella pertussis* | 1x10^{6} CFU/mL | 100% (3/3) | 0% (0/3) |
| *Chlamydia pneumoniae* | 1x10^{6} IFU/mL | 100% (3/3) | 0% (0/3) |
| Human Coronavirus 229E | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Human Coronavirus HKU1 (Lum2020-Resp-1528) | 6.62x10^{4} copies/mL | 100% (3/3) | 0% (0/3) |
| Human Coronavirus NL63 | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Human Coronavirus OC43 | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Echovirus (Enterovirus/Rhinovirus) | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Human Metapneumovirus | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Influenza B (Washington/02/2019/Victoria Lineage) | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Influenza B (Phuket/3073/13/Yamagata Lineage) | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| *Mycoplasma pneumoniae* | 1x10^{6} CCU/mL | 100% (3/3) | 0% (0/3) |
| Parainfluenza 1 | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Parainfluenza 2 | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Parainfluenza 3 | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Parainfluenza 4 | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
K233410 - Page 26 of 56
{26}
| RSV A | 1x10^{5} PFU/mL | 100% (3/3) | 0% (0/3) |
| --- | --- | --- | --- |
| RSV B | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
| Influenza A H1N1 | 1x10^{5} TCID_{50}/mL | Matrix: 100% (3/3) Subtype H1: 100% (3/3) | 0% (0/3) |
| Influenza A H3N2 | 1x10^{5} TCID_{50}/mL | Matrix: 100% (3/3) Subtype H3: 100% (3/3) | 0% (0/3) |
| Influenza A H5N1^{1} | 2x10^{7} copies/mL | Matrix: 100% (3/3) | 0% (0/3) |
| Influenza A H7N2^{1} | 6x10^{6} copies/mL | Matrix: 100% (3/3) | 0% (0/3) |
| Influenza A H7N7^{1} | 2x10^{7} copies/mL | Matrix: 100% (3/3) | 0% (0/3) |
| Influenza A H9N2^{1} | 4x10^{8} copies/mL | Matrix: 100% (3/3) | 0% (0/3) |
| Influenza A H1N2^{2} | 3x10^{7} copies/mL | Matrix: 100% (3/3) Subtype H1: 100% (3/3) | 0% (0/3) |
| SARS-CoV-2 | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 0% (0/3) |
CFU = Colony Forming Units; IFU = Inclusion Forming Units; TCID50 = Median Tissue Culture Infectious Dose; PFU = Plaque Forming Units
$^{1}$These influenza A non-subtype H1/H3 strains are expected to be inclusive to the influenza A matrix target, only (i.e., are expected to be reported as influenza A positive, subtype H1/subtype H3 negative). All were negative for both subtype H1 and subtype H3, as anticipated.
$^{2}$This influenza A H1N2 strain is expected to be inclusive to the influenza A matrix target and influenza A subtype H1 target. This strain was positive for both the influenza A matrix target and influenza A subtype H1 target, as expected.
b. *In silico*
*In silico* analysis of assay specificity/exclusivity was performed by conducting a BLAST comparison of the assay's oligos sequences to the GenBank nt sequence database, as of July 14, 2023. Sequences for 83 off-panel organisms (68 bacteria/fungi and 15 viruses) that can be found in a respiratory specimen were included. Additionally, sequences for all on-panel organisms were included to evaluate intra-panel cross-reactivity. A summary of the results from the analysis is provided in **Table 27**. The LIAISON PLEX Respiratory *Flex* assays were shown to be specific for their respective analytes with the following exceptions, which are noted in the device labeling:
- Cross-reaction of the Adenovirus assays with closely related Adenovirus G (serotype 52) strains.
- Cross-reaction of the SARS-CoV-2 assays with closely related bat and pangolin coronavirus sequences;
- Cross-reaction of the *B. parapertussis* assay with strains of *B. bronchiseptica* that carry IS1001;
- Cross-reaction of the influenza A H1 subtyping assay with 3 swine H3N2 strains and 1 avian H6N1 strain;
K233410 - Page 27 of 56
{27}
- Cross-reaction of the influenza A H3 subtyping assay with 59 swine H1N1 and swine H1N2 strains, 1 duck H5N2 strain, 1 ostrich H7N1 strain, 1 avian H7N9 strain, 1 avian H8N4 strain, and 1 avian H11N9 strain.
Table 27. Organisms Predicted by In Silico Analyses to Cross-React with the Respiratory Flex Assay.
| Assay | Predicted Cross-Reaction |
| --- | --- |
| Adenovirus | Adenovirus G (serotype 52) - strains |
| SARS-CoV-2 | Bat coronavirus and Bat SARS-like coronavirus (accessions MG772933, MG772934, and MN996532) |
| Bordetella parapertussis | Bordetella bronchiseptica containing IS1001 element (accessions JX013523 to JX013527 and CP022962) |
| Influenza A H1 | H5N1 (accession CY110922)^{1}; swine H3N2 (accessions KM110061, KM110062, KM110063, and OM935891); avian H6N1 (accession OP888980) |
| Influenza A H3 | swine H1N1 and swine H1N2 – 59 strains; duck H5N2 (accession OK103962); ostrich H7N1 (accession AF202244); avian H7N9 (accession KP413675); avian H8N4 (accession OK103964); avian H11N9 (accession OK103956) |
$^{1}$This H5N1 human strain sequence is a chimeric sequence containing H1N1 sequence fragments. Therefore, detection of this sequence by the H1 oligos is not considered a cross-reaction.
### Microbial Interference
To determine if non-target organisms can interfere with detection of on-panel organisms in the same sample, a microbial interference study was conducted. The study evaluated 16 non-target organisms spiked into simulated NPS matrix at ≥10$^{5}$ TCID$_{50}$/mL for viruses and ≥10$^{6}$ CFU/mL 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. In addition to each non-target organism, each sample was co-spiked with five representative on-panel targets (i.e., B. pertussis, adenovirus, influenza B, hMPV, and SARS-CoV-2) at 3x LoD. To evaluate microbial interference, testing was performed in triplicate. If on-panel organisms were detected at <100%, samples were reprepared at the same concentration and retested in triplicate. If 100% detection of the on-panel targets was obtained during retesting, no additional testing was performed. When this situation was encountered, the original and retesting results were pooled for performance calculations. Results of the study are shown in Table 28. No microbial interference was observed except for Streptococcus pyogenes at 1x10$^{6}$ CFU/mL and Legionella pneumophila at 4x10$^{5}$ CFU/mL, which interfered with detection of adenovirus in 16.7% (1/6) of replicates.
Table 28. Microbial Interference Study Results
| Off-Panel Organism | Conc./Unit | % Positivity (# Detected/# Tested) | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Adenovirus | B. pertussis | Flu B | hMPV | SARS-CoV-2 |
| Candida albicans | 1x10^{6} CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Corynebacterium diphtheriae | 1x10^{6} CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
K233410 - Page 28 of 56
{28}
| Cytomegalovirus | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| --- | --- | --- | --- | --- | --- | --- |
| *Haemophilus influenzae* | 1x10^{6} CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Herpes Simplex Virus 1 | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| MERS | NA | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| *Neisseria meningitidis* | 1x10^{6} CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| *Staphylococcus aureus* | 1x10^{6} CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| *Pseudomonas aeruginosa* | 1x10^{6} CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| *Streptococcus pneumoniae* | 1x10^{6} CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| *Streptococcus pyogenes* | 1x10^{6} CFU/mL | 83.3% (5/6)^{1} | 100% (6/6) | 100% (6/6) | 100% (6/6) | 100% (6/6) |
| SARS | NA | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| *Legionella pneumophila* | 4x10^{5} CFU/mL | 83.3% (5/6)^{1} | 100% (6/6) | 100% (6/6) | 100% (6/6) | 100% (6/6) |
| Measles | 1x10^{5} TCID_{50}/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| *Moraxella c…
