Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay
Device Facts
| Record ID | K222736 |
|---|---|
| Device Name | Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay |
| Applicant | Hologic, Inc. |
| Product Code | QOF · Microbiology |
| Decision Date | May 16, 2023 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3981 |
| Device Class | Class 2 |
| Attributes | Real-World Evidence |
Real-World Evidence
| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
|---|---|---|---|---|---|
| K222736 · May 16, 2023 | Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay | Hologic, Inc. | Archived clinical nasopharyngeal swab specimens | Retrospective clinical study using archived specimens to supplement prospective clinical performance data for low-prevalence pathogens (Flu B and RSV). | Retrospective study; Archived specimens; Clinical performance |
Clinical Evidence
| Study Design | Population | Comparator | Key Endpoints |
|---|---|---|---|
| Retrospective evaluation of archived specimens; Study Period: December 2019 - March 2020 | Patients with signs and symptoms of respiratory tract infections; Sample Size: 95; Number of Sites: 3 | U.S. FDA-cleared molecular Flu A/B/RSV assay | Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) |
Indications for Use
The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay is a fully automated multiplexed real-time polymerase chain reaction (RT-PCR) in vitro diagnostic test intended for the qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (Flu A), influenza B virus (Flu B), and respiratory syncytial virus (RSV). Nucleic acids are isolated and purified from nasopharyngeal (NP) specimens obtained from individuals exhibiting signs and symptoms of a respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza, and RSV can be similar. This assay is intended to aid in the differential diagnosis of SARS-CoV-2, Flu A, Flu B, and RSV infections in humans and is not intended to detect influenza C virus infections. Nucleic acids from the viral organisms identified by this test are generally detectable in NP specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory tract infection are indicative of the presence of the identified virus and aids in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results do not rule out coinfection with other organisms. The organism(s) detected by the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay may not be the definite cause of disease. Negative results do not preclude SARS-CoV-2, influenza A virus, influenza B virus, or RSV infections. This assay is designed for use on the Panther Fusion system. The Hologic RespDirect Collection Kit can be used to collect NP specimens for testing with the Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay. Additionally, other NP swabs (not provided with the Hologic RespDirect Collection Kit) may be used to collect NP specimens in 3mL of VTM or UTM.
Device Story
The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay is a multiplexed RT-PCR test performed on the fully automated Panther Fusion system. It processes nasopharyngeal (NP) swab specimens collected in transport media (VTM/UTM or Hologic RespDirect). The system performs automated sample lysis, nucleic acid capture using magnetic particles, elution, and multiplex RT-PCR amplification. It detects SARS-CoV-2 (ORF1ab), Flu A (Matrix), Flu B (Matrix), and RSV (Matrix) using specific fluorescent channels. The system compares fluorescence signals against predetermined cut-offs to provide qualitative results and cycle threshold (Ct) values. Used in clinical laboratories by trained personnel, the output aids healthcare providers in the differential diagnosis of respiratory infections. It benefits patients by providing rapid, simultaneous identification of multiple common respiratory viruses, facilitating appropriate clinical management.
Clinical Evidence
Prospective clinical study (n=1900) compared Panther Fusion assay to composite comparator algorithm (SARS-CoV-2) or FDA-cleared molecular assay (Flu A/B/RSV). SARS-CoV-2 PPA 96.9%, NPA 98.5%. Flu A PPA 98.4%, NPA 99.7%. Flu B and RSV performance supplemented by retrospective study (n=95). Analytical studies included precision, reproducibility, inclusivity, cross-reactivity, and interference testing.
Technological Characteristics
Multiplex real-time RT-PCR; automated nucleic acid extraction using magnetic particles; fluorescent-labeled probes (ROX, FAM, RED647, HEX, RED677 channels). Platform: Panther Fusion system (software v7.2.5). Reagents: Panther Fusion SARS-CoV-2/Flu A/B/RSV assay cartridge. Specimen collection: NP swabs in VTM/UTM or eSTM (RespDirect Kit).
Indications for Use
Indicated for qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV RNA in nasopharyngeal (NP) specimens from individuals with signs and symptoms of respiratory tract infection. For prescription use only.
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)
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FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY AND INSTRUMENT
## I Background Information:
A 510(k) Number
K222736
B Applicant
Hologic, Inc.
C Proprietary and Established Names
Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QOF | Class II | 21 CFR 866.3981 - Device To Detect And Identify Nucleic Acid Targets In Respiratory Specimens From Microbial Agents That Cause The SARS-Cov-2 Respiratory Infection And Other Microbial Agents When In A Multi-Target Test | MI - Microbiology |
| OOI | Class II | 21 CFR 862.2570 - Instrumentation for clinical multiplex test systems | CH - Clinical Chemistry |
## II Submission/Device Overview:
### A Purpose for Submission:
The purpose of this submission is to show that the Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay (for use on the Panther Fusion system) is substantially equivalent to the BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031) and to obtain clearance for the Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay.
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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B Measurand:
The Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay detects SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus RNA isolated from nasopharyngeal swab specimens from patients with signs and symptoms of respiratory infection.
C Type of Test:
This assay is a multiplex nucleic acid assay that detects and differentiates SARS-CoV-2, influenza A, influenza B, and RSV through nucleic acid extraction, amplification, and detection using real-time RT-PCR. All steps of the assay are automated, after the manual addition of sample into the sample lysis tube (SLT) and performed within the Panther and Panther Fusion system.
III Intended Use/Indications for Use:
A Intended Use(s):
See Indications for Use below.
B Indication(s) for Use:
Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay:
The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay is a fully automated multiplexed real-time polymerase chain reaction (RT-PCR) in vitro diagnostic test intended for the qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (Flu A), influenza B virus (Flu B), and respiratory syncytial virus (RSV). Nucleic acids are isolated and purified from nasopharyngeal (NP) specimens obtained from individuals exhibiting signs and symptoms of a respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza, and RSV can be similar. This assay is intended to aid in the differential diagnosis of SARS-CoV-2, Flu A, Flu B, and RSV infections in humans and is not intended to detect influenza C virus infections.
Nucleic acids from the viral organisms identified by this test are generally detectable in NP specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory tract infection are indicative of the presence of the identified virus and aids in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Positive results do not rule out coinfection with other organisms. The organism(s) detected by the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay may not be the definite cause of disease.
Negative results do not preclude SARS-CoV-2, influenza A virus, influenza B virus, or RSV infections. This assay is designed for use on the Panther Fusion system.
The Hologic RespDirect Collection Kit can be used to collect NP specimens for testing with the Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay. Additionally, other NP swabs (not provided with the Hologic RespDirect Collection Kit) may be used to collect NP specimens in 3mL of VTM or UTM.
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Ancillary Collection Kit:
RespDirect Collection Kit
The Hologic RespDirect Collection Kit is intended to be used for the collection of nasopharyngeal (NP) swab specimens (collected by a healthcare provider) for testing with the Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay.
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 Panther Fusion System, only.
IV Device/System Characteristics:
A Device Description:
Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay
The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay is a multiplex real-time reverse transcriptase PCR (RT-PCR) in vitro diagnostic test developed for use on the fully automated Panther Fusion system to detect and differentiate SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV) directly from nasopharyngeal swab specimens.
The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay cartridge contains the same sample preparation and PCR reaction chemistry as the previously cleared Panther Fusion Flu A/B/RSV assay (K171963). To accommodate addition of the SARS-CoV-2 reagents (primers/probes) to the multiplexed reagents, minor changes were made to the previously cleared analyte primer/probe concentrations and RFU cutoffs. Additionally, the fluorophore for Flu B was changed from ROX to RED647 to accommodate the addition of SARS-CoV-2 to the assay.
The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay involves the following steps:
a. Sample lysis - Prior to processing and testing on the Panther Fusion system, specimens are transferred to a Specimen Lysis Tube (SLT) containing specimen transport media (STM). Alternatively, samples can be collected with the RespDirect Collection kit which contains enhanced specimen transport media (eSTM). STM and eSTM lyse the cells, release target nucleic acid and protect them from degradation during storage.
b. Nucleic acid capture and elution - These steps take place in a single tube on the Panther Fusion system. The eluate is transferred to the Panther Fusion system reaction tube containing the assay reagents. The Internal Control-S (IC-S) is added to each test specimen and controls via the working Panther Fusion Capture Reagent-S (wFCR-S). The IC-S in the reagent is used to monitor specimen processing, amplification, and detection. Magnetic particles with covalently bound oligonucleotides mediate the nucleic acid capture. Capture oligonucleotides hybridize to total nucleic acid in the test specimen. Hybridized nucleic acid
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is then separated from the lysed specimen in a magnetic field. Wash and aspiration steps remove extraneous components debris from the reaction tube. The elution step elutes purified nucleic acid.
c. Elution transfer and multiplex RT-PCR - Eluted nucleic acid is transferred to a Panther Fusion reaction tube already containing oil and reconstituted master mix. A reverse transcriptase generates a DNA copy of the target sequence. Target specific forward and reverse primers and probes then amplify targets while simultaneously detecting and discriminating multiple target types via multiplex RT-PCR. The Panther Fusion system compares the fluorescence signal to a predetermined cut-off to produce a qualitative result for the presence or absence of the analyte. The positive result for each analyte will be accompanied by the cycle threshold (Ct value).
## Hologic RespDirect Collection Kit
An ancillary collection kit that consists of the RespDirect Swab, intended for collection of NP specimens, and the enhanced Direct Load Tube (eDLT), containing enhanced specimen transport media (eSTM). This transport media lyses cells, releasing target nucleic acids and protecting them from degradation during storage.
## B Principle of Operation:
The assay detects viral nucleic acids that have been extracted from a patient respiratory sample. A multiplex Real-time RT-PCR reaction is carried out under optimized conditions generating amplicons for SARS-CoV-2, influenza A, influenza B, and RSV. The Internal Control-S (IC-S) is added to each test specimen before processing to act as a control for specimen processing, amplification, and detection. Identification of SARS-CoV-2, influenza A, influenza B, RSV, and the IC-S occurs by the use of target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the viral genomes (Table 1).
Table 1. Assay Primer and Probe Targets
| Analyte | Gene Targeted | Instrument Channel |
| --- | --- | --- |
| SARS-CoV-2 | ORF1ab | ROX |
| Influenza A Virus | Matrix | FAM |
| Respiratory Syncytial Virus A/B | Matrix | HEX |
| Influenza B Virus | Matrix | RED647 |
| Internal Control-S | Not applicable* | RED677 |
*Internal Control-S is a non-infectious synthetic nucleic acid sequence that is extracted and detected through targeted primers and probes.
## C Instrument Description Information:
1. Instrument Name: Panther System and Panther Fusion System, software version 7.2.5
2. Specimen Identification: Specimen identification is entered via barcode.
3. Specimen Sampling and Handling: Nasopharyngeal swab (NPS) specimens collected in transport media.
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4. Calibration:
Real Time Fluorometers (RTF) undergo a single calibration during manufacturing. No additional calibration is performed by the end user.
5. Quality Control:
The assay contains an internal control (IC-S) which is added to each test specimen via the working Panther Fusion Capture Reagent-S (wFCR-S). The IC-S is used to monitor specimen processing, amplification, and detection.
Two external controls are also included with this assay in a single use vial, the Panther Fusion SARS-CoV-2/Flu A/B/RSV Positive Control and the Panther Fusion Negative Control. The controls were validated in the analytical and clinical studies.
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): | K222736 | DEN200031 |
| --- | --- | --- |
| Device Trade Name | Panther Fusion SARS-CoV-2/Flu A/B/RSV assay | BioFire Respiratory Panel 2.1 (RP2.1) |
| Regulation Number/Name | Same | 21 CFR 866.3981; Multi-Target Respiratory Specimen Nucleic Acid Test Including SARS-CoV-2 And Other Microbial Agents |
| Product Code(s) | QOF, OOI | QOF |
| Prescription Use Only | Same | Yes |
| Intended Use | The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay is a fully automated multiplexed real-time polymerase chain reaction (RT-PCR) in vitro diagnostic test intended for the qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (Flu A), influenza B virus (Flu B), and respiratory syncytial virus (RSV). Nucleic acids are isolated and purified from | 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. |
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| | nasopharyngeal (NP) specimens obtained from individuals exhibiting signs and symptoms of a respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza, and RSV can be similar. This assay is intended to aid in the differential diagnosis of SARS-CoV-2, Flu A, Flu B, and RSV infections in humans and is not intended to detect influenza C virus infections.
Nucleic acids from the viral organisms identified by this test are generally detectable in NP specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory tract infection are indicative of the presence of the identified virus and aids in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Positive results do not rule out coinfection with other organisms. The organism(s) detected by the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay may not be the definite cause of disease.
Negative results do not preclude SARS-CoV-2, influenza A virus, influenza B virus, or RSV infections. This assay is designed for use on the Panther Fusion system. | The following organism types and subtypes are identified using the BioFire RP2.1:
• Adenovirus,
• Coronavirus 229E,
• Coronavirus HKU1,
• Coronavirus NL63,
• Coronavirus OC43,
• Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2),
• Human Metapneumovirus,
• Human Rhinovirus/Enterovirus,
• Influenza A, including subtypes H1, H1-2009, and H3,
• Influenza B,
• Parainfluenza Virus 1,
• Parainfluenza Virus 2,
• Parainfluenza Virus 3,
• Parainfluenza Virus 4,
• Respiratory Syncytial Virus,
• Bordetella parapertussis (IS1001),
• Bordetella pertussis (ptxP),
• Chlamydia pneumoniae, and
• Mycoplasma pneumoniae
Nucleic acids from the respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection is indicative of the presence of the identified microorganism and aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a respiratory illness may be due to |
| --- | --- | --- |
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| | The Hologic RespDirect Collection Kit can be used to collect NP specimens for testing with the Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay. Additionally, other NP swabs (not provided with the Hologic RespDirect Collection Kit) may be used to collect NP specimens in 3mL of VTM or UTM.
**Ancillary Collection Kit:**
**RespDirect Collection Kit**
The Hologic RespDirect Collection Kit is intended to be used for the collection of nasopharyngeal (NP) swab specimens (collected by a healthcare provider) for testing with the Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay. | 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 coinfection 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 with possible respiratory tract infection. |
| --- | --- | --- |
| **Intended User** | Same | Professional use |
| **Principle of Operation** | Same | Reverse transcriptase multiplexed polymerase chain reaction test |
| **Specimen Types** | Same | Nasopharyngeal swab (NPS) specimens |
| **Assay Controls** | Same | Internal and external controls |
| **Analyte Targets** | SARS-CoV-2, Flu A, Flu B, RSV (RSV A and RSV B) | Adenovirus, Coronaviruses (including 229E, HKU1, NL63, OC43), SARS-CoV-2, Human Metapneumovirus, Human Rhinovirus/Enterovirus, Influenza A (including subtypes H1, H1-2009, and H3), Influenza B, Parainfluenza Viruses (including 1, 2, 3, and 4), Respiratory Syncytial Virus (RSV), Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae |
| **Flu A Subtyping** | No | Yes |
| **Platform** | Automated nucleic acid amplification platform.
Uses Panther Fusion system for all steps including nucleic acid | Automated nucleic acid amplification platform.
Uses BioFire FilmArray 2.0 or BioFire FilmArray Torch |
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| | extraction, amplification, detection, and result processing. | systems including integrated sample preparation, amplification, detection, and analysis. |
| --- | --- | --- |
| Time to Obtain Test Results | ~ 2.5 hours | ~ 45 minutes |
VI Standards/Guidance Documents Referenced:
- Class II Special Controls as per 21 CFR 866.3981
- Guidance for Industry and Food and Drug Administration Staff, Recommended Content and Format of Non-Clinical Bench Performance Testing Information in Premarket Submissions (December 20, 2019)
- Guidance for Industry and Food and Drug Administration Staff, eCopy Program for Medical Device Submissions (April 27, 2020)
- Guidance for Industry and FDA Staff, Format for Traditional and Abbreviated 510(k)s (September 13, 2019)
- Guidance for Industry and Food and Drug Administration Staff, Refuse to Accept Policy for 510(k)s (April 21, 2022)
- Guidance for Industry and Food and Drug Administration Staff, The 510(k) Program: Evaluating Substantial Equivalence in Premarket Notifications [510(k)] (July 28, 2014)
- Guidance for Industry and FDA Staff, Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses (July 7, 2011)
VII Performance Characteristics (if/when applicable):
A Analytical Performance:
1. Precision/Reproducibility:
a. Within-Laboratory Precision
Within-laboratory precision was examined at one site using the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay. A total of 5 contrived panels containing known quantities of the target analytes were prepared in negative clinical NPS matrix. The viral materials used to generate the positive panel members are denoted in Table 2. The contrived positive panels consisted of combinations of the target analytes at low concentrations (2x LoD) and moderate concentrations (5x LoD) (Table 3). The study was conducted with two operators, three lots of reagents, and three Panther Fusion instruments over the course of 12 days. Each panel member was tested in duplicate by each operator on two different reagent lots on all 12 days generating a total of 96 replicates per panel member (2 operators x 2 replicates per test event x 2 lots x 12 days).
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Table 2. Viral Strains for Within-Laboratory Precision Study
| Description | Vendor | Catalog Number |
| --- | --- | --- |
| SARS-CoV-2 (USA-WA1/2020) | BEI | NR-52281 |
| Influenza A/H3N2/Kansas/14/17 | Zeptometrix | 0810586CF |
| Influenza B/Victoria/Washington/02/19 | Zeptometrix | 0810611CF |
| RSV B/ CH93(18)-18 | Zeptometrix | 0810040CF |
Table 3. Precision Study Sample Panel
| Panel ID | Description |
| --- | --- |
| 1 | Negative (unspiked) |
| 2 | SARS-CoV-2 (2x) / Flu A (2x) |
| 3 | Flu B (2x) / RSV (2x) |
| 4 | SARS-CoV-2 (5x) / Flu A (5x) |
| 5 | Flu B (5x) / RSV (5x) |
The qualitative (i.e., % agreement with expected results) and quantitative results from the study are illustrated in Table 4 and Table 5, respectively.
Table 4. Within-Laboratory Precision Study - Qualitative Results
| Target | Panel ID | Panel Conc. | % Positive (pos n/ valid n) | % Agreement with Expected Results/ (95% CI) |
| --- | --- | --- | --- | --- |
| SARS-CoV-2 | 2 | Low positive | 100% (96/96) | 100% (96.2-100%) |
| | 4 | Mod. positive | 100% (96/96) | 100% (96.2-100%) |
| | 1 | Negative | 1.0% (1/96) | 99.0% (94.3-99.8%) |
| Flu A | 2 | Low positive | 100% (96/96) | 100% (96.2-100%) |
| | 4 | Mod. positive | 100% (96/96) | 100% (96.2-100%) |
| | 1 | Negative | 0% (0/96) | 100% (96.2-100%) |
| Flu B | 3 | Low positive | 100% (96/96) | 100% (96.2-100%) |
| | 5 | Mod. positive | 100% (96/96) | 100% (96.2-100%) |
| | 1 | Negative | 0% (0/96) | 100% (96.2-100%) |
| RSV | 3 | Low positive | 100% (96/96) | 100% (96.2-100%) |
| | 5 | Mod. positive | 100% (96/96) | 100% (96.2-100%) |
| | 1 | Negative | 0% (0/96) | 100% (96.2-100%) |
Note: Results are shown only for the intended targets. Panel members co-spiked with two different targets are presented twice.
All low (2x) and moderate (5x) panel members were $100\%$ positive for the spiked target analytes. The negative panel member was $0.0\%$ positive for Flu A, Flu B, and RSV, while $1.0\%$ positive for SARS-CoV-2 (1/96) (Table 4, above).
Table 5. Within-Laboratory Precision Study - Ct Signal Variability Analysis Results
| Panel ID | Target | Mean (Ct) | Between Reagent Lots | | Between Instrument | | Between Operators | | Between Days | | Between Runs | | Within Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 (Negative) | IC | 33.7 | 0.19 | 0.57 | 0.08 | 0.23 | 0 | 0 | 0 | 0 | 0.21 | 0.62 | 0.29 | 0.86 | 0.42 | 1.23 |
| 2 (Low Pos) | Flu A | 35.1 | 0.33 | 0.93 | 0.06 | 0.17 | 0 | 0 | 0 | 0 | 0.30 | 0.85 | 0.56 | 1.59 | 0.72 | 2.04 |
| | SARS-CoV-2 | 35.9 | 0 | 0 | 0.13 | 0.36 | 0 | 0 | 0 | 0 | 0 | 0 | 0.60 | 1.67 | 0.61 | 1.71 |
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The mean and variability analysis between instruments, between reagent lots, between operators, between days, between runs, within runs, and overall (total) for Ct values is shown in Table 5. Overall %CV was ≤ 2.04%. The greatest source of variability was from SARS-CoV-2 in the Flu A and SARS-CoV-2 low positive panel (within-run % CV of 1.67%). Overall variability was low, and the study demonstrates assay variability within an acceptable range.
## b. Reproducibility
A reproducibility study was conducted at three testing sites using the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay. The study incorporated potential sources of variation introduced by site (three testing sites), day (5 different days), and operator (two operators per site). One lot of Panther Fusion SARS-CoV-2/Flu A/B/RSV assay reagents was tested at three sites by two operators per site on one Panther Fusion instrument per site over five days.
A total of 6 contrived panels containing known quantities of various Panther Fusion SARS-CoV-2/Flu A/B/RSV assay analytes were prepared in negative clinical NPS matrix. The same viral materials used in the precision study (see Table 2) were also included in the reproducibility study. The four contrived positive panels consisted of combinations of the target analytes at low concentrations (i.e., 1-2x LoD) and moderate concentrations (3-5x LoD) (Table 6). 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 and quantitative results of the study are illustrated in Table 7 and Table 8, respectively.
Table 6. Reproducibility Study Sample Panel
| Panel ID | Description |
| --- | --- |
| 1 | Negative (unspiked) |
| 2 | SARS-CoV-2 (1-2x) / Flu A (1-2x) |
| 3 | Flu B (1-2x) / RSV (1-2x) |
| 4 | SARS-CoV-2 (3-5x) / Flu A (3-5x) |
| 5 | Flu B (3-5x) / RSV (3-5x) |
Table 7. Reproducibility Study - Qualitative Results
| Target | Panel ID | Panel Conc. | % Agreement with Expected Results/ (95% CI) | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Site 1 | Site 2 | Site 3 | Overall |
| SARS-CoV-2 | 2 | Low positive | 100% | 100% | 100% | 100% |
| | | | (30/30) | (30/30) | (30/30) | (90/90) |
| | | | (88.7-100) | (88.7-100) | (88.7-100) | (95.9-100) |
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| | 4 | Mod. positive | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
| --- | --- | --- | --- | --- | --- | --- |
| | 1 | Negative | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
| Flu A | 2 | Low positive | 100% (30/30) (88.7-100) | 96.7% (29/30) (83.3-99.4) | 100% (30/30) (88.7-100) | 98.9% (89/90) (94.0-99.8) |
| | 4 | Mod. positive | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
| | 1 | Negative | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
| Flu B | 3 | Low positive | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
| | 5 | Mod. positive | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
| | 1 | Negative | 100% (30/30) (88.7-100) | 96.7% (29/30) (83.3-99.4) | 100% (30/30) (88.7-100) | 98.9% (89/90) (94.0-99.8) |
| RSV | 3 | Low positive | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
| | 5 | Mod. positive | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
| | 1 | Negative | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (30/30) (88.7-100) | 100% (90/90) (95.9-100) |
Mod = moderate; Note: Results are shown only for the intended targets. Panel members co-spiked with two different targets are presented twice
The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay demonstrated $100\%$ agreement for SARS-CoV-2, Flu A, Flu B, and RSV moderate positive panel members. For low positive panel members, the assay yielded $100\%$ agreement for SARS-CoV-2, Flu B, and RSV detection and $98.9\%$ agreement for Flu A detection (Table 7 above). A lower agreement for low positive panel members was expected, since the analyte concentration of their panel member analytes ranged between 1x and 2x the limit of detection, which is expected to yield $\geq 95\%$ detection rate. This performance is acceptable and demonstrates acceptable assay reproducibility.
Table 8. Reproducibility Study - Ct Signal Variability Analysis Results
| Target | Panel ID/conc. | Mean (Ct) | Between Sites | | Between Operators/Run1 | | Between Days | | Within Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| SARS-CoV-2 | 2-Low Pos | 35.53 | 0.24 | 0.68 | 0.18 | 0.50 | 0.19 | 0.52 | 0.49 | 1.38 | 0.60 | 1.70 |
| | 4-Mod Pos | 34.15 | 0.11 | 0.32 | 0.00 | 0.00 | 0.00 | 0.00 | 0.40 | 1.16 | 0.41 | 1.20 |
| Flu A | 2-Low Pos | 34.55 | 0.57 | 1.66 | 0.62 | 1.81 | 0.00 | 0.00 | 3.68 | 10.64 | 3.77 | 10.92 |
| | 4-Mod Pos | 33.55 | 0.09 | 0.27 | 0.03 | 0.10 | 0.17 | 0.49 | 0.48 | 1.42 | 0.51 | 1.53 |
| Flu B | 3-Low pos | 35.80 | 0.12 | 0.35 | 0.00 | 0.00 | 0.22 | 0.60 | 0.39 | 1.10 | 0.47 | 1.30 |
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| | 5-Mod Pos | 34.56 | 0.00 | 0.00 | 0.10 | 0.29 | 0.00 | 0.00 | 0.29 | 0.83 | 0.30 | 0.88 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| RSV | 3-Low pos | 35.78 | 0.07 | 0.20 | 0.23 | 0.65 | 0.14 | 0.39 | 0.59 | 1.64 | 0.65 | 1.82 |
| | 5-Mod Pos | 34.41 | 0.05 | 0.14 | 0.00 | 0.00 | 0.00 | 0.00 | 0.43 | 1.25 | 0.43 | 1.26 |
$\mathrm{Ct} =$ cycle threshold; $\mathrm{CV} =$ coefficient of variation; Mod $=$ moderate; Pos $=$ positive; SD $=$ standard deviation. Note: Variability from some factors may be numerically negative, which can occur if the variability due to those factors is very small. When this occurs, $\mathrm{SD} = 0$ and $\mathrm{CV} = 0\%$
$^{1}$ Between-Operator may be confounded with Between-Run; therefore, Between-Operator and Between-Run estimates are combined in Between-Operator/Run
The total signal variability, as measured by the standard deviation, was less than or equal to 0.65 across all target viruses and concentrations, with the exception of the Flu A low positive panel member (SD =3.77), which may be attributed to 1 false negative result (Table 8 above). For all positive panel members, the within-run factor (i.e., random error) was the largest contributor to total variability. These results indicate that the repeatability and reproducibility of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay on the Panther Fusion system are robust in NPS samples.
# 2. Linearity:
Not applicable; this is a qualitative assay.
# 3. Analytical Specificity/Interference:
# Analytical Reactivity (Inclusivity)
# a. Wet-Testing
This study was performed to determine the analytical reactivity of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay with clinically relevant strains, serotypes, or subtypes of the target species (i.e., SARS-CoV-2, Flu A, Flu B, and RSV). The inclusivity panel was prepared by spiking various SARS-CoV-2/Flu A/Flu B/RSV virus serotypes/subtypes/strains encompassing temporal and geographical diversity into negative clinical NP swab VTM/UTM matrix at a concentration of $\sim 3\mathrm{x}$ LoD and testing in triplicate. Strains that did not yield $100\%$ reactivity at $3\mathrm{x}$ LoD were prepared at higher concentrations and tested until the minimum concentration that achieved $100\%$ reactivity was reached. The strains evaluated and the lowest concentration that achieved $100\%$ reactivity are shown in Table 9, below.
Table 9. Inclusivity Wet-Testing Study Results
| Strain | Concentration (units/xLoD) | Result** | | | |
| --- | --- | --- | --- | --- | --- |
| | | SARS-CoV-2 | Flu A | Flu B | RSV |
| SARS-CoV-2 | | | | | |
| USA-WA1/2020* | 0.09 TCID50/mL (3x) | + | - | - | - |
| USA-CA1/2020 | 0.09 TCID50/mL (3x) | + | - | - | - |
| USA-AZ1/2020 | 0.09 TCID50/mL (3x) | + | - | - | - |
| USA-WI1/2020 | 0.09 TCID50/mL (3x) | + | - | - | - |
| USA/OR-OHSU-PHL00037/2021|B.1.1.7 | 0.09 TCID50/mL (3x) | + | - | - | - |
| Uganda/MUWRP-20200195568/2020|A.23.1 | 0.09 TCID50/mL (3x) | + | - | - | - |
| SA/PHC658/2021|B.1.617.2 | 0.09 TCID50/mL (3x) | + | - | - | - |
| USA/MD-HP05285/2021|B.1.617.2 | 0.09 TCID50/mL (3x) | + | - | - | - |
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| Strain | Concentration (units/xLoD) | Result** | | | |
| --- | --- | --- | --- | --- | --- |
| | | SARS-CoV-2 | Flu A | Flu B | RSV |
| USA/CA/VRLC009/2021|B.1.427 | 0.09 TCID_{50}/mL (3x) | + | - | - | - |
| USA/CA/VRLC012/2021|P.2 | 0.3 TCID_{50}/mL (10x) | + | - | - | - |
| USA/MD-HP03056/2021|B.1.525 | 0.3 TCID_{50}/mL (10x) | + | - | - | - |
| USA/CA-Stanford-16_S02/2021|B.1.617.1 | 0.09 TCID_{50}/mL (3x) | + | - | - | - |
| Peru/un-CDC-2-4069945/2021|C.37 | 0.09 TCID_{50}/mL (3x) | + | - | - | - |
| SA/MD-HP20874/2021|B.1.1.529 | 0.09 TCID_{50}/mL (3x) | + | - | - | - |
| USA/GA-EHC-2811C/2021|B.1.1.529 | 0.09 TCID_{50}/mL (3x) | + | - | - | - |
| **Influenza A H1N1** | | | | | |
| A/Brisbane/02/18* | 0.18 TCID_{50}/mL (3x) | - | + | - | - |
| A/Michigan/45/2015 | 0.18 TCID_{50}/mL (3x) | - | + | - | - |
| A/Christ Church/16/2010 | 180 TCID_{50}/mL (3000x)^{1} | - | + | - | - |
| A/Kentucky/2/06 | 0.6 TCID_{50}/mL(10x) | - | + | - | - |
| A/Solomon Islands/03/06 | 0.6 TCID_{50}/mL(10x) | - | + | - | - |
| A/Guangdong-maonan/1536/2019 | 180 TCID_{50}/mL (3000x)^{1} | - | + | - | - |
| A/Taiwan/42/2006 | 0.6 TCID_{50}/mL(10x) | - | + | - | - |
| A/Henan/8/05 | 0.6 TCID_{50}/mL(10x) | - | + | - | - |
| A/Hawaii/15/01 | 18 TCID_{50}/mL (300x)^{3} | - | + | - | - |
| A/California/07/2009 | 0.18 TCID_{50}/mL (3x) | - | + | - | - |
| A/Hawaii/66/2019 | 180 CEID_{50}/mL (NC) | - | + | - | - |
| A/Indiana/02/2020 | 60 CEID_{50}/mL (NC) | - | + | - | - |
| A/Michigan/45/2015 pdm09-like virus | 0.6 TCID_{50}/mL(10x) | - | + | - | - |
| **Influenza A H3N2** | | | | | |
| A/Kansas/14/17* | 0.33 TCID_{50}/mL (3.3x) | - | + | - | - |
| A/Arizona/45/2018 | 3.3 FFU/mL (NC) | - | + | - | - |
| A/New York/21/2020 | 3.3 FFU/mL (NC) | - | + | - | - |
| A/Hong Kong/45/2019 | 3.3 FFU/mL (NC) | - | + | - | - |
| A/Singapore/INFIMH-16-0019/2016 | 110 CEID_{50}/mL (NC) | - | + | - | - |
| A/Hong Kong/2671/2019 | 11 TCID_{50}/mL (110x)^{2} | - | + | - | - |
| A/Hiroshima/52/05 | 1.1 TCID_{50}/mL (11x) | - | + | - | - |
| A/Costa Rica/07/99 | 11 TCID_{50}/mL (110x)^{3} | - | + | - | - |
| A/Port Chalmers/1/73 | 1.1 TCID_{50}/mL (11x) | - | + | - | - |
| A/Brazil/113/99 | 1.1 TCID_{50}/mL (11x) | - | + | - | - |
| A/Perth/16/2009 | 0.33 TCID_{50}/mL (3.3x) | - | + | - | - |
| A/Texas/50/2012 | 0.33 TCID_{50}/mL (3.3x) | - | + | - | - |
| A/Hong Kong/4801/2014 | 1.1 TCID_{50}/mL (11x) | - | + | - | - |
| A/Indiana/08/2011 | 1.1 TCID_{50}/mL (11x) | - | + | - | - |
| **Influenza A H5N1** | | | | | |
| A/Hong Kong/486/97 | 0.01 ng/mL (NC) | - | + | - | - |
| **Influenza B Victoria** | | | | | |
| B/Washington/02/2019* | 0.09 TCID_{50}/mL (3x) | - | - | + | - |
| B/Colorado/06/2017 | 0.09 TCID_{50}/mL(3x) | - | - | + | - |
| B/Florida/78/2015 | 0.3 TCID_{50}/mL (10x) | - | - | + | - |
| B/Alabama/2/17 | 0.09 TCID_{50}/mL (3x) | - | - | + | - |
| B/Ohio/1/2005 | 0.3 TCID_{50}/mL (10x) | - | - | + | - |
| B/Michigan/09/2011 | 3 TCID_{50}/mL (100x)^{3} | - | - | + | - |
| B/Hawaii/01/2018 (NA D197N) | 0.9 TCID_{50}/mL (30x)^{1} | - | - | + | - |
| B/Brisbane/33/08 | 0.09 TCID_{50}/mL (3x) | - | - | + | - |
| **Flu B Yamagata** | | | | | |
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| Strain | Concentration (units/xLoD) | Result** | | | |
| --- | --- | --- | --- | --- | --- |
| | | SARS-CoV-2 | Flu A | Flu B | RSV |
| B/Phuket/3073/2013* | 0.006 TCID50/mL (2x) | - | - | + | - |
| B/Wisconsin/1/2010 | 2 TCID50/mL (666x)1 | - | - | + | - |
| B/U nah/9/14 | 0.006 TCID50/mL (2x) | - | - | + | - |
| B/St. Petersburg/04/06 | 0.06 TCID50/mL (2x) | - | - | + | - |
| B/Texas/81/2016 | 2 TCID50/mL (666x)1 | - | - | + | - |
| B/Indiana/17/2017 | 0.6 TCID50/mL (200x)1 | - | - | + | - |
| B/Oklahoma/10/2018 | 2 TCID50/mL (666x)1 | - | - | + | - |
| B/Massachusetts/02/2012 | 0.2 TCID50/mL (66x)4 | - | - | + | - |
| Flu B | | | | | |
| B/Lee/40 | 0.09 TCID50/mL (NC) | - | - | + | - |
| RSV A | | | | | |
| RSV-A/2006 Isolate* | 0.06 TCID50/mL (2x) | - | - | - | + |
| RSV A/4/2015 isolate #1 | 0.06 TCID50/mL (2x) | - | - | - | + |
| RSV A/A2 | 0.06 TCID50/mL (2x) | - | - | - | + |
| RSV A/12/2014 isolate #2 | 0.06 TCID50/mL (2x) | - | - | - | + |
| RSV B | | | | | |
| RSV-B/CH93(18)-18* | 0.3 TCID50/mL (10x) | - | - | - | + |
| RSV B/3/2015 isolate #1 | 0.09 TCID50/mL (3x) | - | - | - | + |
| RSV B/9320 | 0.09 TCID50/mL (3x) | - | - | - | + |
*Strain used to establish LoD and for which the concentration in multiples of the LoD (xLoD) was calculated.
**A positive symbol (+) indicates that reactivity was observed for 100% of replicates (3/3) while a negative symbol (-) indicates that reactivity was observed for 0% of replicates (0/3)
$^{1}$ In silico analysis showed 100% homology to amplification region. Virus stock degradation or error in TCID $_{50}$ /mL quantification may have impacted the nucleic acid target concentration at 100% detection.
$^{2}$ In silico analysis identified a single mismatch in the forward and reverse primers. Due to the location of these mismatches, amplification and detection are not expected to be impacted. Virus stock degradation or error in TCID $_{50}$ /mL quantification may have impacted the nucleic acid target concentration at 100% detection.
$^{3}$ Sequence of strain in targeted amplification regions are not available in NCBI or GISAID to further evaluate sensitivity.
$^{4}$ In silico analysis identified a single mismatch in the reverse primer. Due to the location of this mismatch, amplification and detection are not expected to be impacted. Virus stock degradation or error in TCID $_{50}$ /mL quantification may have impacted the nucleic acid target concentration at 100% detection.
NC - Not calculable
The results from this study demonstrate that the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay is capable of detecting multiple clinically relevant strains of SARS-CoV-2, Flu A, Flu B, and RSV.
# b. In silico
The inclusivity of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay was evaluated using in silico analysis of the forward primers, reverse primers, and probes for the SARS-CoV-2, Flu A, Flu B and RSV target systems in relation to sequences available in the NCBI and GISAID gene databases. For SARS-CoV-2, all available sequences up to June 25, 2022 from the GISAID and NCBI gene databases were evaluated. Due to the large size of the database (>9.3 million sequences) every 1 out of 10 sequences
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was randomly sampled from each database. For Flu A, Flu B, RSV A, and RSV B, all available sequences from GISAID and NCBI gene databases entered from January 1, 2015 to February 15, 2022 were evaluated. In total, 934,493 SARS-CoV-2 and 88,128 Flu A sequences were evaluated that contained both assay target regions, while an additional 31,801 Flu B sequences, 1,599 RSV A sequences, and 1,240 RSV B sequences containing the assay single target region were included in the analysis. The SARS-CoV-2 sequences included the following lineages and variants of concern (VOC) or variants of interest (VOI) that may have important epidemiological, immunological, or pathogenic properties from a public health perspective: Delta, Alpha, Omicron BA.1, Omicron BA.2, Omicron BA.4, Omicron BA.5, Gamma, Epsilon, Iota, Beta, Mu, Zeta, Kappa, Eta, Lamba, Theta and others.
Sequence alignments were generated using the multiple sequence alignment program MAFFT (Multiple Alignment using Fast Fourier Transform). All non-human isolates were removed from the analysis. Additionally, any sequence with missing or ambiguous sequence information for the target region was removed. Due to the dual amplification systems for SARS-CoV-2 and Flu A, only sequences with mismatches in both regions were assessed for potential impact to the inclusivity of the assay.
Based on the in silico analysis of GISAID and NCBI sequences available up to June 25, 2022 for SARS-CoV-2, the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay is predicted to detect all 934,493 SARS-CoV-2 sequences evaluated.
Based on in silico analysis of all sequences available from January 01, 2015 to February 15, 2022 in GISAID and NCBI databases, the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay is predicted to detect ≥99.998% of 88,128 Flu A, ≥99.94% of 31,801 Flu B, ≥98.12% of 1,599 RSV A, and ≥98.23% of 1,240 RSV B sequences evaluated.
## Exclusivity Testing
This study was performed to demonstrate that non-indicated influenza subtypes, specifically influenza C and D, are not detected by the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay. For this study, one Flu C strain (C/Taylor/1233/1947) and one Flu D strain (Bovine/Mississippi/C00046N/14) were diluted into negative clinical NP swab matrix at 1×10⁵ CEID₅₀/mL and 1×10⁴ TCID₅₀/mL concentrations, respectively. Seven replicates were tested for both strains and no positive results were obtained for any of the assay's target channels, demonstrating that Flu C and Flu D are not detected by the assay.
## Cross-Reactivity/Microbial Interference
### a. Wet-Testing
This study evaluated the analytical specificity (cross-reactivity) and sensitivity of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in the presence of non-targeted microorganisms that may be found in a respiratory tract clinical specimen. Forty-one (41) non-target microorganisms (Table 10) were evaluated in the study. Panel members were composed of 3 different non-target microorganisms spiked into negative clinical NP swab VTM/UTM matrix at 10³-10⁶ TCID₅₀/mL or copies/mL (for viruses), 10⁵-10⁸ CFU/mL or rRNA copies/mL (for bacteria) or 10⁶ CFU/mL (for fungi). To evaluate cross-reactivity, each panel was evaluated in triplicate in the absence of the target organisms. To evaluate
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microbial interference (negative effect on sensitivity), each panel was tested in triplicate in the presence of SARS-CoV-2, Flu A, Flu B, and RSV at 3x LoD. The organisms evaluated in the study are shown in the table below. No cross-reactivity or microbial interference was observed at the concentrations tested.
Table 10. Cross-Reactivity Study Microorganisms Evaluated By Wet-Testing
| Viruses | Conc.1 | Bacteria/Fungi | Conc.1 |
| --- | --- | --- | --- |
| Adenovirus 1 | 1x105TCID50/mL | Bordetella pertussis | 1x106CFU/mL |
| Adenovirus 7a | 1x105TCID50/mL | Candida albicans | 1x106CFU/mL |
| CMV Strain AD 169 | 1x104TCID50/mL | Chlamydophila pneumoniae | 1x106IFU/mL |
| Human coronavirus 229E | 1x104TCID50/mL | Corynebacterium diphtheriae | 1x106CFU/mL |
| Human coronavirus NL63 | 1x104TCID50/mL | Escherichia coli | 1x106CFU/mL |
| Human coronavirus OC43 | 1x105TCID50/mL | Haemophilus influenzae | 1x106CFU/mL |
| Epstein-Barr virus (EBV) | 1x106copies/mL | Lactobacillus plantarum | 1x106CFU/mL |
| Enterovirus (e.g., EV68) | 1x105TCID50/mL | Legionella pneumophila | 1x106CFU/mL |
| Human coronavirus HKU12 | 1x106copies/mL | Moraxella catarrhalis | 1x105CFU/mL |
| Human Metapneumovirus (hMPV) | 1x105TCID50/mL | Mycobacterium tuberculosis | 1x109rRNA copies/mL3 |
| HPIV-1 | 1x105TCID50/mL | Mycoplasma pneumoniae | 1x109rRNA copies/mL3 |
| HPIV-2 | 1x105TCID50/mL | Neisseria spp. | 1x106CFU/mL |
| HPIV-3 | 1x105TCID50/mL | Neisseria meningitides | 1x106CFU/mL |
| HPIV-4 | 1x104TCID50/mL | Neisseria mucosa | 1x106CFU/mL |
| Measles | 1x104TCID50/mL | Pneumocystis jirovecii | 1x106CFU/mL |
| MERS-Coronavirus | 5x104TCID50/mL | Pseudomonas aeruginosa | 1x106CFU/mL |
| Mumps virus | 1x105TCID50/mL | Staphylococcus aureus | 1x106CFU/mL |
| Rhinovirus 1A | 1x104TCID50/mL | Staphylococcus epidermidis | 1x106CFU/mL |
| SARS coronavirus 12 | 1x106copies/mL | Streptococcus pneumoniae | 1x106CFU/mL |
| Varicella Zoster Virus | 1x103TCID50/mL | Streptococcus pyogenes | 1x106CFU/mL |
| | | Streptococcus salivarius | 1x106CFU/mL |
$^{1}$ CFU = Colony Forming Units; IFU = Inclusion Forming Units; TCID $_{50}$ = Median Tissue Culture Infectious Dose.
2Cultured virus and whole genome purified nucleic acid for Human HKU1 and SARS-coronavirus were not readily available at the time of testing. HKU1 and SARS-coronavirus in vitro transcript (IVT) corresponding to the ORF1ab gene regions targeted by the assay were used to evaluate cross-reactivity and microbial interference.
3The $1 \times 10^{9}$ rRNA copies/mL concentration is equivalent to $2 \times 10^{5}$ CFU/mL.
# b. In silico
An in silico analysis was performed to demonstrate that the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay does not cross-react with closely related or commonly encountered microorganisms in a respiratory clinical specimen. A total of 545 individual sequences from GenBank, associated with 143 microorganisms, were analyzed with BLAST for homology to the primer and probe sequences for the SARS-CoV-2, Flu A, Flu B, RSV A, and RSV B targets included in the assay. A microorganism was determined to have no cross-reactivity when its sequence had $< 80\%$ homology with all of the assay's primers and probes. When $\geq 80\%$ homology was identified for a single primer or probe, proximity and mismatch analysis was used to demonstrate minimal likelihood of amplicon generation and/or detection. Specifically, microorganism sequences showing $\geq 80\%$
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homology to a single primer or probe and $< 50\%$ homology to the other primer/probe, were not expected to be amplified. For microorganism sequences showing $\geq 80\%$ homology to a single primer or probe and $\geq 50\%$ homology to the other primer/probe, proximity analysis was performed to determine the likelihood of amplification.
Seven organisms showed $\geq 80\%$ homology to one of the primers and $\geq 50\%$ homology to a second primer and probe. Six of these organisms were determined to be low risk and unlikely to cross-react due to proximity analysis. The remaining organism, SARS-1 coronavirus, was identified as having a small possibility of amplification due to a high level of homology to the primers and probe in the SARS-CoV-2 channel. However, mismatches at the $3^{\prime}$ end of the primers suggest that hybridization and amplification is unlikely. This observation was confirmed in a cross-reactivity wet-testing study performed at high concentrations (1.01x10 $^{6}$ copies/mL) of SARS-1 coronavirus, which demonstrated SARS-1 coronavirus does not cross react with any of the target assays.
A total of 29 organisms showed $\geq 80\%$ homology to one of the primers, $\geq 50\%$ homology to a second primer, but $< 50\%$ homology to a probe. Twenty-seven of these organisms were determined to be low risk and unlikely to interfere with assay analytes due to proximity, mismatches at the $3^{\prime}$ end of the primers, low homology to at least 1 primer, and/or $< 10$ continuous bases between the target and primers. Serratia marcescens was identified as having a possibility of low amplification without detection. To determine if S. marcescens cross-reacted with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay, wet-testing was performed at high concentrations (1x10 $^{6}$ CFU/mL) of S. marcescens. No cross-reactivity was observed at the concentrations tested.
# Interfering Substances
This study evaluated the performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in the presence of medications, over the counter products, and other potentially interfering substances found in a clinical respiratory specimen. Assay results were evaluated to determine if the presence of potentially interfering substances in target analyte negative or target analyte positive samples had an effect on assay performance. The analyte negative pools contained negative NP swab VTM/UTM clinical matrix and the potentially interfering substances, only. The analyte positive pools contained negative NP swab VTM/UTM clinical matrix, the potentially interfering substance(s), and one representative strain of each targeted analyte (i.e., SARS-CoV-2, Flu A, Flu B, and RSV B) spiked to a final testing concentration of $3\mathrm{x}$ LoD. Three replicates were tested per pool both in the presence and absence of the target analytes. The substances evaluated are denoted in Table 11 below. All target-spiked panels were $100\%$ positive, and all target-negative panels were $100\%$ negative, indicating that none of the evaluated substances interfered with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay.
Table 11. Interfering Substances Testing Panel
| Panel | Type | Potentially Interfering Substance | Active Ingredient(s) | Concentration1 |
| --- | --- | --- | --- | --- |
| 1 | Endogenous | Bovine submaxillary gland, type I-S | Purified mucin protein | 60 ug/mL |
| 2 | | Blood (human) | N/A | 2% v/v |
| 3 | | Neo-Synephrine | Phenylephrine | 15 % v/v |
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| | Nasal sprays or drops | Anefrin | Oxymetazoline | 15 % v/v |
| --- | --- | --- | --- | --- |
| 4 | | Saline | Sodium chloride | 15 % v/v |
| | | Ventolin HFA² | Albuterol | 45 ng/mL |
| 5 | Nasal corticosteroids | QVAR Beconase AQ² | Beclomethasone | 15 ng/mL |
| | | Dexacort² | Dexamethasone | 12 ug/mL |
| | | Nasacort | Triamcinolone | 5 % v/v |
| 6 | | Flonase | Fluticasone | 5 % v/v |
| | | Rhinocort | Budesonide | 5 % v/v |
| 7 | | Nasonex² | Mometasone | 0.5 ng/mL |
| | | AEROSPAN² | Flunisolide | 10 ug/mL |
| 8 | Nasal gel/homeopathic allergy relief medicine | Zicam (Allergy Relief) | Luffa opperculata, Galphimia, Glauca, Histaminum hydrochloricum, sulfur | 5 % v/v |
| 9 | Throat lozenges, oral anesthetic, and analgesic | Cepacol Extra Strength | Benzocaine, Menthol | 0.7 mg/mL |
| 10 | Anti-viral drugs | Relenza² | Zanamivir | 3.3 mg/mL |
| | | TamiFlu² | Oseltamivir | 400 ug/mL |
| | | Virazole² | Ribavirin | 10.5 ug/mL |
| 11 | Antibiotic, nasal ointment | Bactroban cream² | Mupirocin | 1.6 ug/mL |
| 12 | Antibacterial, systemic | Tobramycin² | Tobramycin | 33.1 ug/mL |
| 13 | Solvent Control | Water | N/A | 5% v/v |
| 14 | | Dimethyl Sulfoxide (DMSO) | N/A | 5%v/v |
| 15 | Control | None | N/A | N/A |
¹v/v: volume by volume
²Active ingredient tested, not substance
## Competitive Interference
The purpose of this study was to demonstrate that samples tested with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay that are co-infected with multiple types of targeted microorganisms do not inhibit the detection of either one (competitive interference). One representative strain of each targeted organism (SARS-CoV-2, Flu A, Flu B and RSV) was tested. Co-infection panels were made by spiking one target organism at high concentration (up to 10,000 TCID₅₀/mL) and another target organism at low concentration (~3x LoD) in negative clinical NP swab clinical matrix. If less than 100% positivity was observed for the low target, the high target was diluted down from 10,000 TCID₅₀/mL to a concentration where 100% positivity of the low target was achieved. The on-target analyte combinations evaluated and the results of this evaluation, are shown in Table 12.
Table 12. Competitive Interference Study Sample Panel Composition & Study Results
| Target 1 (Low Conc.) | | Target 2 (High Conc.) | | % Detected | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Virus | Conc. | Virus | Conc. | SARS-CoV-2 | Flu A | Flu B | RSV |
| | | Flu A | 1x10⁴ TCID₅₀/mL | 100% | 100% | 0% | 0% |
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| Target 1 (Low Conc.) | Target 2 (High Conc.) | % Detected |
| --- | --- | --- |
| Virus | Conc. | Virus | Conc. | SARS-CoV-2 | Flu A | Flu B | RSV |
| SARS-CoV-2 | 0.09 TCID50/mL (3x LoD) | (H3N2) | (>90,000x LoD) | (3/3) | (3/3) | (0/3) | (0/3) |
| Flu B (Victoria) | 1x104TCID50/mL (>300,000x LoD) | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) |
| RSV-A | 1x104TCID50/mL (500,000x LoD) | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| RSV-B | 1x104TCID50/mL (>300,000x LoD) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| 1x103TCID50/mL (>30,000x LoD) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| 100 TCID50/mL (>3000x LoD) | 66.7% (2/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| 30 TCID50/mL (1000x LoD) | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| |
| Flu A | 0.33 TCID50/mL (3.3x LoD) | SARS-CoV-2 | 1x104TCID50/mL (>300,000x LoD) | 100% (3/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) |
| 1x103TCID50/mL (>30,000x LoD) | 100% (3/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) |
| 1x102TCID50/mL (>3000x LoD) | 100% (3/3) | 100% (3/3) | 0% (0/3) | 0% (0/3) |
| Flu B | 1x104TCID50/mL (>300,000x LoD) | 0% (0/3) | 100% (3/3) | 100% (3/3) | 0% (0/3) |
| RSV-A | 1x104TCID50/mL (500,000x LoD) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) |
| RSV-B | 1x104TCID50/mL (>300,000x LoD) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| 1x103TCID50/mL (>30,000x LoD) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| 100 TCID50/mL (>3000x LoD) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| 30 TCID50/mL (1000x LoD) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) |
| |
| Flu B | 0.09 TCID50/mL (3x LoD) | SARS-CoV-2 | 1x104TCID50/mL (>300,000x LoD) | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) |
| Flu A | 1x104TCID50/mL (>90,000x LoD) | 0% (0/3) | 100% (3/3) | 100% (3/3) | 0% (0/3) |
| RSV-A | 1x104TCID50/mL (500,000x LoD) | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3) |
| RSV-B | 1x104TCID50/mL (>300,000xLoD) | 0% (0/3) | 0% (0/3) | 66.7% (2/3) | 100% (3/3) |
| 1x103TCID50/mL (>30,000x LoD) | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3) |
| |
| RSV-A | 0.06 TCID50/mL (2x LoD) | SARS-CoV-2 | 1x104TCID50/mL (>300,000x LoD) | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| Flu A | 1x104TCID50/mL (>90,000x LoD) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) |
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| Target 1 (Low Conc.) | | Target 2 (High Conc.) | | % Detected | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Virus | Conc. | Virus | Conc. | SARS-CoV-2 | Flu A | Flu B | RSV |
| | | Flu B | 1x104TCID50/mL (>300,000x LoD) | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3) |
| | | | | | | | |
| RSV-B | 0.09 TCID50/mL (3x LoD) | SARS-CoV-2 | 1x104TCID50/mL (>300,000x LoD) | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| | | Flu A | 1x104TCID50/mL (>90,000x LoD) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) |
| | | Flu B | 1x104TCID50/mL (>300,000x LoD) | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3) |
Interference was observed for the following co-infections:
- SARS-CoV-2 (low concentration) in the presence of RSV B (high concentrations of $1 \times 10^{4}$ , $1 \times 10^{3}$ , and $1 \times 10^{2} \mathrm{TCID}_{50} / \mathrm{mL}$ ). Interference was no longer observed at an RSV B concentration of $30 \mathrm{TCID}_{50} / \mathrm{mL}$ .
- Flu A (low concentration) in the presence of SARS-CoV-2 (high concentrations of $1 \times 10^{4}$ and $1 \times 10^{3} \mathrm{TCID}_{50} / \mathrm{mL}$ ). Interference was no longer observed at a SARS-CoV-2 concentration of $1 \times 10^{2} \mathrm{TCID}_{50} / \mathrm{mL}$ .
- Flu A (low concentration) in the presence of RSV B (high concentrations of $1 \times 10^{4}$ , $1 \times 10^{3}$ , and $1 \times 10^{2} \mathrm{TCID}_{50} / \mathrm{mL}$ ). Interference was no longer observed at an RSV B concentration of $30 \mathrm{TCID}_{50} / \mathrm{mL}$ .
- Flu B (low concentration) in the presence of RSV B (high concentration of $1 \times 10^{4}$ $\mathrm{TCID}_{50} / \mathrm{mL}$ ). Interference was no longer observed at an RSV B concentration of $1 \times 10^{3}$ $\mathrm{TCID}_{50} / \mathrm{mL}$ .
4. Assay Reportable Range:
Not applicable; this is a qualitative assay.
5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
a. Controls
The assay contains an internal control (IC-S) added to each test specimen and external positive and negative controls. For more information, see Section IV.C.5. Quality Control, above.
b. Sample Stability
Stability studies have been performed to support the following claims:
Primary Specimen (NPS storage in VTM/UTM)
Specimens can be stored in VTM/UTM (prior to transfer to the Panther Fusion Specimen Lysis Tube (SLT)) under the following conditions:
- Refrigerated $(2 - 8^{\circ}\mathrm{C})$ for up to 96 hours before transfer to the Panther Fusion Specimen Lysis Tube or,
- Frozen at $-70^{\circ}\mathrm{C}$ . Samples may be freeze/thawed up to 3 times prior to testing.
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# Processed Specimen (NPS storage in a Panther Fusion SLT)
Once a patient specimen in VTM/UTM is transferred to the Panther Fusion SLT, samples can be stored under the following conditions:
- Room temperature (15-30°C) for up to 6 days or,
- Refrigerated (2-8°C) for up to 3 months or,
- Frozen at -20°C for up to 3 months. Freeze/thaw cycles should be minimized due to potential for sample degradation.
- Frozen at -70°C for up to 3 months. Freeze/thaw cycles should be minimized due to potential for sample degradation.
# RespDirect Collection Kit (eSTM)
A NPS specimen collected with the RespDirect Collection Kit can be stored under the following conditions:
- Room temperature (15-30°C) for up to 6 days or,
- Refrigerated (2-8°C) for up to 3 months or,
- Frozen at -20°C for up to 3 months. Samples may be freeze/thawed up to 3 times prior to testing or,
- Frozen at -70°C for up to 3 months. Samples may be freeze/thawed up to 3 times prior to testing.
## c. Kit Stability
### Panther Fusion SARS-CoV-2/Flu A/B/RSV Reagents
Stability of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay cartridge and positive control were evaluated. Stability of universal Panther Fusion respiratory assay fluids (i.e., Panther Fusion Capture Reagents-S, Panther Fusion Enhancer Reagent-S, Panther Fusion Internal Control-S, Panther Fusion Elution Buffer, Panther Fusion Oil, Panther Fusion Reconstitution Buffer 1, and the negative control) were not assessed, as stability for these reagents has been previously established for Panther Fusion respiratory assays, including the Panther Fusion Flu A/B/RSV assay (K171963). Two types of stability studies were performed: Shelf-Life Stability and In-Use (or On-Board) Stability. The study data supports the storage conditions for the Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay Cartridge and Positive Control illustrated in Table 13.
Table 13. Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay Specific Reagent Storage Conditions
| Reagents | Unopened Storage | On-Board/Open Stability | Opened Storage |
| --- | --- | --- | --- |
| Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay Cartridge | 2°C to 8°C | 60 days | 2°C to 8°C |
| Panther Fusion SARS-CoV-2/Flu A/B/RSV Positive Control | 2°C to 8°C | Single use vial | NA* |
*NA-Not Applicable, as controls are single use.
### RespDirect Collection Kit
Stability of the RespDirect Collection Kit was evaluated by performing a Shelf-Life Study. The study data supports the storage conditions indicated in Table 14.
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Table 14. RespDirect Collection Kit Storage Conditions
| Reagents | Unopened Storage | On-Board/Open Stability | Opened Storage |
| --- | --- | --- | --- |
| RespDirect Collection Kit | 15°C to 30°C | NA* | NA* |
*NA-Not Applicable
d. Shipping Stability
Panther Fusion SARS-CoV-2/Flu A/B/RSV Reagents
The purpose of this study was to demonstrate that exposure to extreme hot or cold temperatures potentially encountered by the Panther Fusion SARS-CoV-/Flu A/B/RSV assay components during shipment would not impact the performance of the assay. One lot of cartridges and positive controls for the Panther Fusion SARS-CoV-/Flu A/B/RSV assay were tested in this study. Similar to the Kit Stability Study (see Section c. Kit Stability, Panther Fusion SARS-CoV-2/Flu A/B/RSV Reagents, above), stability of the universal Panther Fusion respiratory assay fluids were not assessed, as stability for these reagents has previously been shown. Components were exposed to the extreme temperatures in their final container closure systems.
The Panther Fusion SARS-CoV-/Flu A/B/RSV assay cartridges and positive controls were cycled between the extreme low temperature (-40 ±3°C), room temperature (28 ±2°C), and extreme high temperature (55 ±5°C). One stress cycle consisted of incubation for at least 9 hours at extreme high temperatures and then at least 15 hours at room-temperature, followed by at least 9 hours at extreme low temperature, followed by at least 15 hour incubation at room temperature (1 cycle = approx. 48 hours). A total of 5 cycles were performed to evaluate the worst case scenario of shipment to customers.
All runs tested gave the expected result for spiked and un-spiked samples. This study demonstrates that Panther Fusion SARS-CoV-/Flu A/B/RSV assay cartridge and positive control are not altered by exposure to extreme hot or cold temperatures that may be encountered during shipping.
RespDirect Collection Kit
The purpose of this study was to demonstrate that exposure to extreme hot or cold temperatures potentially encountered by the RespDirect Collection Kit during shipment would not impact performance of the RespDirect when tested with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay. One RespDirect Collection Kit lot was tested in this study. For this study, RespDirect Collection Kits were exposed to extreme temperatures in their final container closure systems.
RespDirect Collection Kits were cycled between the extreme low temperature (-70°C, ranging from -65°C to -85°C), room temperature (28 ±2°C), and extreme high temperature (55 ±5°C). One stress cycle consisted of incubation for at least 24 hours at extreme low temperatures and then at least 4 hours at room-temperature, followed by 24 hours at extreme high temperatures and then 4 hours at room-temperature (1 cycle = approx. 56 hours). A total of 5 cycles were performed to evaluate the worst case scenario of shipment to customers.
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All runs tested gave the expected result for spiked and un-spiked samples, except for one spiked sample at 3x LoD that was negative for SARS-CoV-2. This study demonstrates that the RespDirect Collection Kit is not altered by exposure to extreme hot or cold temperatures that may be encountered during shipping.
# 6. Detection Limit:
Processed Specimens (i.e., NP Swab Specimen in VTM/UTM Diluted into Panther Fusion Specimen Lysis Tube, containing STM)
The analytical sensitivity (limit of detection or LoD) of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay was determined by testing processed negative clinical NP swab matrix spiked with multiple concentrations of the WHO International Standard for SARS-CoV-2 (NIBSC, 20/146) or viral cultures of SARS-CoV-2 (1 strain), Influenza A (one H1N1 and one H3N2 strain), Influenza B (one Victoria lineage and one Yamagata lineage), and RSV (one RSV A and one RSV B strain). To estimate the LoD, a minimum of 24 replicates were tested per concentration with each of three reagent lots. Using the estimated LoD study results, the LoD for each target was further estimated by Probit analysis for each reagent lot and was confirmed with an additional 24 replicates using a single reagent lot. The concentration with $\geq 95\%$ detection was determined to be the final, confirmed LoD for each assay target. Confirmed LoD concentrations for each analyte are summarized in Table 15.
Table 15. Confirmatory LoD Study Results
| Target | Strain | LoD Conc. | Percent Positivity for each Target Analyte | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | SARS-CoV-2 | Flu A | Flu B | RSV |
| SARS-CoV-2 | USA-WA1/2020 | 0.03 TCID50/mL | 95.8% (23/24) | 0% (0/24) | 0% (0/24) | 0% (0/24) |
| | WHO International Standard (NIBSC, 20/146) | 47.20 IU/mL | 95.8% (23/24) | 0% (0/24) | 0% (0/24) | 0% (0/24) |
| Flu A | Brisbane/02/18 (H1N1) | 0.06 TCID50/mL | 0% (0/24) | 95.8% (23/24) | 0% (0/24) | 0% (0/24) |
| | Kansas/14/17 (H3N2) | 0.1 TCID50/mL | 0% (0/24) | 100% (24/24) | 0% (0/24) | 0% (0/24) |
| Flu B | Phuket/3073/13 (Yamagata) | 0.003 TCID50/mL | 0% (0/24) | 0% (0/24) | 95.8% (23/24) | 0% (0/24) |
| | Washington/02/19 (Victoria) | 0.03 TCID50/mL | 0% (0/24) | 0% (0/24) | 100% (24/24) | 0% (0/24) |
| RSV | RSV A | 0.03 TCID50/mL | 0% (0/24) | 0% (0/24) | 100% (24/24) | 0% (0/24) |
| | RSV B | 0.03 TCID50/mL | 0% (0/24) | 0% (0/24) | 100% (24/24) | 0% (0/24) |
The LoD for co-analyte spiked samples was also evaluated and shown to be equivalent to single analyte spiked samples.
# RespDirect Collection Kit (eSTM)
The LoD of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay was assessed with the RespDirect Collection Kit (containing eSTM). For this study, negative clinical eSTM matrix spiked with WHO International Standard for SARS-CoV-2 (NIBSC, 20/146) or viral cultures of Influenza A (one H3N2 strain), Influenza B (one Victoria lineage), and RSV (one RSV A
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and one RSV B strain) were evaluated. Thirty (30) replicates were tested for each concentration/RespDirect Collection Kit workflow. The RespDirect Collection Kit can be handled according to the following workflows described in the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay IFU: (1) directly load tube on the Panther Fusion Instrument or, (2) vortex the tube for 10 minutes at 1,800 rpm on a multi-tube vortexer prior to loading on the Panther Fusion Instrument or, (3) vortex tube for 15 seconds using a standard bench top vortexer prior to loading on the Panther Fusion Instrument. The concentration with ≥95% detection was determined to be the final, confirmed LoD for each of the assay targets. The RespDirect Collection Kit workflows yielded equivalent LoDs for each assay target. The LoD concentrations for each analyte are outlined below.
- SARS-CoV-2, WHO International Standard (NIBSC, 20/146): 98.6 IU/mL
- Influenza A (H3N2): 0.11 TCID₅₀/mL
- Influenza B (Victoria lineage): 0.03 TCID₅₀/mL
- RSV A: 0.03 TCID₅₀/mL
- RSV B: 0.05 TCID₅₀/mL
The LoD for co-analyte spiked samples was also evaluated and shown to be equivalent to single analyte spiked samples.
7. Assay Cut-Off:
The relative fluorescence units (RFU) range is the difference between maximum and minimum fluorescent signal seen in a sample during amplification. For Panther Fusion SARS-CoV-2/Flu A/B/RSV positive samples, the amplification curve rises above the background fluorescence RFU resulting in a high RFU range for the intended targets. The IC resembles a positive target and also has a high RFU range. For the Panther Fusion SARS-CoV-2/Flu A/B/RSV negative samples, the amplification curve remains flat resulting in a low RFU range for the intended targets. This difference in RFU range is the primary criteria used to distinguish positive and negative specimens. RFU range thresholds were set at 500 for SARS-CoV-2, 1,000 for Flu A, 1000 for Flu B, and 775 for RSV for determination of positivity in their specific channels (ROX, FAM, RED647, HEX, respectively) and 1000 RFU range for IC for determination of validity in its specific channel (RED677). The RFU range thresholds were set based on data from multiple RFU readings from known positive samples. The cycle number at which the amplification curve crosses target specific assigned RFU range threshold value is called Ct. Target specific Ct will be generated only for positive valid reaction for that target.
8. Accuracy (Instrument):
Not applicable
9. Carry-Over:
RespDirect Collection Kit
Carry-over/cross-contamination with processed specimens (i.e., NP specimens in VTM/UTM diluted into STM) was previously evaluated in K171963 and found acceptable. Therefore, carry-over/cross-contamination with processed specimens was not evaluated with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay.
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The purpose of this study was to demonstrate that no carry-over/cross-contamination is observed for NP specimens collected in eSTM using the RespDirect Collection Kit when tested with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay.
Carry-over or cross-contamination in the RespDirect Collection Kit was assessed with pooled negative clinical matrix collected in RespDirect Collection Kits. Pooled negative clinical matrix were used to prepare negative and high-titer positive panels. A high-titer positive panel was prepared by spiking one strain of Flu A (H3N2, Kansas/14/17) in negative clinical matrix at a concentration of $1 \times 10^{4} \mathrm{TCID}_{50} / \mathrm{mL}$ . Non-spiked negative clinical matrix was used as the negative panel. Panels were tested in a checkboard pattern by alternating high-titer positive and negative samples over 5 total runs on each of two Panther Fusion instruments. Each run included 30 positive and 30 negative reactions (60 total reactions). A baseline run of 100 negative reactions was run prior to the checkerboard pattern runs. One lot of Panther Fusion SARS-CoV-2 /Flu A/B/RSV assay cartridges was used. No carry-over was observed.
# B Comparison Studies:
1. Method Comparison with Predicate Device: Not applicable
2. Matrix Comparison:
# a. Transport Media Equivalency Study
The purpose of this study was to demonstrate equivalency between viral transport media types indicated for use with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay. The following transport media types were included in the study: Remel Micro Test M4RT, Remel Micro Test M5, Remel Micro Test M6, BD Universal Viral Transport Media, Copan Universal Transport Medium, and Hardy Diagnostics Viral Transport Media. To demonstrate equivalency between the viral transport medias, contrived positive samples were created by spiking one strain of SARS-CoV-2, Flu A H3N2, Flu B Victoria, and RSV B into either true negative clinical NP swab matrix or simulated NP swab matrix (i.e., transport media spiked with HeLa cells at a concentration of $2 \times 10^{4}$ cells/mL) at 3 concentrations (0.5x, 1x, and 5x LoD). Flu A, Flu B, and RSV B were co-spiked while SARS-CoV-2 was evaluated independently. Contrived positives generated with true clinical NP matrix were included to demonstrate equivalency between clinical NP matrix and simulated NP matrix, supporting the use of simulated viral transport media in the study. Twenty (20) replicates per target concentration were tested for each transport media type and analyte concentration. The results are shown in Table 16 and support that the Panther Fusion SARS-CoV-2/Flu A/B RSV assay can be used with the evaluated viral transport media types.
Table 16. Transport Media Equivalency Study Results
| Target | Media | Concentration | % Positivity (n pos/n Tested) |
| --- | --- | --- | --- |
| None, Negative Samples | Remel M4RT | Negative | 0% (0/20) |
| | BD UVT | | 0% (0/20) |
| | Copan UTM | | 0% (0/20) |
| | Hardy VTM | | 0% (0/20) |
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| | NP Samples^{1} | | 0% (0/20) |
| --- | --- | --- | --- |
| | Remel M5 | | 0% (0/20) |
| | Remel M6 | | 0% (0/20) |
| SARS-CoV-2 | Remel M4RT | 0.5x LoD | 90% (18/20) |
| | 1x LoD | **100% (20/20)** |
| | 5x LoD | 100% (20/20) |
| BD UVT | 0.5x LoD | **95% (19/20)** |
| | 1x LoD | 100% (20/20) |
| | 5x LoD | 100% (20/20) |
| Copan UTM | 0.5x LoD | 80% (16/20) |
| | 1x LoD | **95% (19/20)** |
| | 5x LoD | 100% (20/20) |
| Hardy VTM | 0.5x LoD | 80% (16/20) |
| | 1x LoD | **100% (20/20)** |
| | 5x LoD | 100% (20/20) |
| NP Samples | 0.5x LoD | **95% (19/20)** |
| | 1x LoD | 100% (20/20) |
| | 5x LoD | 100% (20/20) |
| Remel M5 | 0.5x LoD | 90% (18/20) |
| | 1x LoD | **100% (20/20)** |
| | 5x LoD | 100% (20/20) |
| | 0.5x LoD | 95% (19/20) |
| Flu A H3N2 | Remel M4RT | 0.5x LoD | **95% (19/20)** |
| | 1x LoD | 95% (19/20) |
| | 5x LoD | 100% (20/20) |
| BD UVT | 0.5x LoD | 85% (17/20) |
| | 1x LoD | **100% (20/20)** |
| | 5x LoD | 100% (20/20) |
| Copan UTM | 0.5x LoD | 75% (15/20) |
| | 1x LoD | **100% (20/20)** |
| | 5x LoD | 100% (20/20) |
| Hardy VTM | 0.5x LoD | 90% (18/20) |
| | 1x LoD | **100% (20/20)** |
| | 5x LoD | 100% (20/20) |
| NP Samples | 0.5x LoD | 85% (17/20) |
| | 1x LoD | **100% (20/20)** |
| | 5x LoD | 100% (20/20) |
| Remel M5 | 0.5x LoD | **95% (19/20)** |
| | 1x LoD | 100% (20/20) |
| | 5x LoD | 100% (20/20) |
| | 0.5x LoD | 70% (14/20) |
| Flu B Victoria | Remel M4RT | 0.5x LoD | **95% (19/20)** |
| | 1x LoD | 100% (20/20) |
| | 5x LoD | 100% (20/20) |
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| | Copan UTM | 0.5x LoD | 100% (20/20) |
| --- | --- | --- | --- |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | Hardy VTM | 0.5x LoD | 100% (20/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | NP Samples | 0.5x LoD | 100% (20/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | Remel M5 | 0.5x LoD | 100% (20/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | Remel M6 | 0.5x LoD | 95% (19/20) |
| | | 1x LoD | 95% (19/20) |
| 5x LoD | | 100% (20/20) | |
| RSV B | Remel M4RT | 0.5x LoD | 90% (18/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | BD UVT | 0.5x LoD | 95% (19/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | Copan UTM | 0.5x LoD | 95% (19/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | Hardy VTM | 0.5x LoD | 95% (19/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | NP Samples | 0.5x LoD | 95% (19/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | Remel M5 | 0.5x LoD | 90% (18/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
| | Remel M6 | 0.5x LoD | 90% (18/20) |
| | | 1x LoD | 100% (20/20) |
| | | 5x LoD | 100% (20/20) |
$^{1}$ NP Samples = Contrived positive samples generated with true negative clinical NP swab matrix
b. Collection Device Equivalency – RespDirect Collection Kit (eSTM) vs. VTM/UTM The purpose of this study was to demonstrate equivalency between two collection devices: (1) NP specimens collected into VTM/UTM and (2) NP specimens collected with the RespDirect Collection Kit (eSTM). For this study, contrived positive panels were prepared by adding one strain each of SARS-CoV-2 (WHO International Standard), Flu A (H3N2), Flu B (Victoria), and RSV B into paired, negative clinical NP eSTM and negative clinical NP VTM/UTM matrix collected from individuals with signs and symptoms of respiratory infection. Contrived panels were generated at both 2x and 5x LoD. SARS-CoV-2 was co-spiked with Flu A, Flu B, and RSV and was also tested independently. In total, 100 SARS-CoV-2 positives and 50 positives each for Flu A, Flu B, and RSV were evaluated. Additionally, 181 negative samples, consisting of negative
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clinical NP matrix, only, were included in the study. Results are shown in Table 17, below and demonstrate that the two collection devices are equivalent.
Table 17. Collection Device Equivalency Study Results
| Analyte | Sample Concentration | N per Collection Device | VTM/UTM % Positive (N Pos/N Tested) | RespDirect % Positive (N Pos/N Tested) |
| --- | --- | --- | --- | --- |
| None (Negative Samples) | NA | 181 | 0% (0/181) | 0% (0/181) |
| SARS-CoV-2 | 2x | 50 | 100% (50/50) | 98% (49/50) |
| | 5x | 50 | 100% (50/50) | 100% (50/50) |
| Flu A (H3N2) | 2x | 25 | 100% (25/25) | 100% (25/25) |
| | 5x | 25 | 100% (25/25) | 100% (25/25) |
| Flu B (Victoria) | 2x | 25 | 100% (25/25) | 100% (25/25) |
| | 5x | 25 | 100% (25/25) | 100% (25/25) |
| RSV B | 2x | 25 | 100% (25/25) | 100% (25/25) |
| | 5x | 25 | 100% (25/25) | 100% (25/25) |
## C Clinical Studies:
### 1. Clinical Sensitivity:
#### Prospective Study
The clinical performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay was established in a multi-center study conducted with residual (leftover) and de-identified nasopharyngeal swab (NPS) specimens in VTM or UTM that were prospectively collected from patients with signs and symptoms of respiratory tract infections during periods of the 2020-2022 respiratory illness seasons. NPS specimens, from five geographically diverse clinical sites in the U.S., were enrolled and tested with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay at three U.S. testing sites.
The Panther Fusion SARS-CoV-2/Flu A/B/RSV assay was evaluated for SARS-CoV-2 performance by comparing the candidate device testing results to a composite comparator algorithm (CCA) consisting of two highly sensitive U.S. FDA EUA SARS-CoV-2 molecular tests and a validated PCR followed by bi-directional sequencing (PCR/BDS) assay. A final CCA result was assigned when two of the three composite comparator assays were in concordance. The comparator method utilized to establish performance for the Flu A, Flu B, and RSV targets was a U.S. FDA-cleared molecular Flu A/B/RSV assay. All comparator testing was performed in accordance with the respective package inserts.
The Panther Fusion SARS-CoV-2/Flu A/B/RSV was evaluated with prospectively (i.e., all comers between two time points that met the clinical study inclusion criteria) collected specimens. Both Category I specimens (i.e., prospectively collected, tested fresh) and Category II specimens (i.e., prospectively collected, tested frozen) were included in the study.
A total of 1949 residual NPS specimens were acquired and enrolled for the prospective clinical study. Of these 1949 specimens, 1056 were collected between January 2022 and
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March 2022, while the remaining 893 were collected between November 2020 and March 2021. Forty-five (45) of these specimens were withdrawn from the study because of a reception delay that resulted in frozen specimens being received thawed $(n = 44)$ or a specimen not being handled according to the package insert $(n = 1)$ . In addition, another 4 specimens were withdrawn because they yielded invalid results with the investigational device upon retesting $(n = 4)$ , bringing the total number of withdrawn samples to forty-nine. The initial prospective clinical study invalid rate was $0.6\%$ (12/1904), and $0.2\%$ (4/1904) following retesting.
This left 1900 prospective specimens enrolled in the evaluable population. For the SARS-CoV-2 target, 13 specimens were excluded from analysis due to unknown infection status obtained from the CCA tests. This left a total of 1887 prospective samples evaluated for SARS-CoV-2, of which 790 (41.9%) were tested fresh and 1097 (58.1%) which were tested frozen with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay. For the Flu A, Flu B, and RSV targets, 63 specimens were excluded from analysis due to obtaining an invalid result from the comparator test. This left a total of 1837 valid prospective specimens evaluated for Flu A, Flu B, and RSV, of which 798 (43.4%) were tested fresh and 1039 (56.6%) were tested frozen with the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay. Table 18 below provides a summary of demographic information for the 1900 specimens included in the prospective clinical study.
Table 18. Demographic Data for Prospectively Collected Specimens
| | | Overall | Site 1 | Site 2 | Site 3 | Site 4 | Site 5 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sex | Male | 850 (44.7%) | 99 | 209 | 316 | 216 | 10 |
| | Female | 1049 (55.2%) | 82 | 200 | 527 | 228 | 12 |
| | Unknown | 1 (0.1%) | 0 | 0 | 1 | 0 | 0 |
| Age | <5 years | 388 (20.4%) | 86 | 252 | 42 | 5 | 3 |
| | 5-21 years | 435 (22.9%) | 77 | 156 | 173 | 17 | 12 |
| | 22-40 years | 372 (19.6%) | 8 | 1 | 286 | 70 | 7 |
| | 41-60 years | 326 (17.2%) | 3 | 0 | 212 | 111 | 0 |
| | >60 years | 379 (19.9%) | 7 | 0 | 131 | 241 | 0 |
| | Total | 1900 | 181 | 409 | 844 | 444 | 22 |
A summary of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay prospective clinical study performance is provided in Table 19. Positive Percent Agreement (PPA) was calculated as $100\% \times (\mathrm{TP} / (\mathrm{TP} + \mathrm{FN}))$ . True positive (TP) indicates that both the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay and the comparator method had a positive result for the specific analyte, and false negative (FN) indicates that the Panther Fusion SARS-CoV-2/Flu A/B/RSV was negative while the comparator result was positive. Negative Percent Agreement (NPA) was calculated as $100\% \times (\mathrm{TN} / (\mathrm{TN} + \mathrm{FP}))$ . True negative (TN) indicates that both the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay and the comparator method had negative results, and false positive (FP) indicates that the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay was positive while the comparator result was negative. Specimens that obtained discordant results underwent additional testing with a U.S. FDA EUA SARS-CoV-2/Flu A/B/RSV molecular test, when sufficient sample volume remained.
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Table 19. Panther Fusion SARS-CoV-2/Flu A/B/RSV Assay Performance
| Analyte | Positive Percent Agreement | | | Negative Percent Agreement | | | |
| --- | --- |…
