Simplexa COVID-19/ Flu A/B & RSV Direct (MOL4450); Simplexa COVID-19/ Flu A/B & RSV Positive Control Pack (MOL4460)
Device Facts
| Record ID | K252387 |
|---|---|
| Device Name | Simplexa COVID-19/ Flu A/B & RSV Direct (MOL4450); Simplexa COVID-19/ Flu A/B & RSV Positive Control Pack (MOL4460) |
| Applicant | Diasorin Molecular, LLC |
| Product Code | QOF · Microbiology |
| Decision Date | Oct 30, 2025 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3981 |
| Device Class | Class 2 |
Indications for Use
The Simplexa™ COVID-19 / Flu A/B & RSV Direct is a real-time RT-PCR assay intended for use on the LIAISON® MDX instrument for the simultaneous in vitro qualitative detection and differentiation of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A (Flu A) virus, influenza B (Flu B) virus and respiratory syncytial virus (RSV) in nasopharyngeal swab and anterior nasal swab specimens from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar. The Simplexa™ COVID-19 / Flu A/B & RSV Direct assay is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B and RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection. This test is not intended to detect influenza C virus infections. Positive results are indicative of the presence of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Simplexa™ COVID-19 / Flu A/B & RSV Direct real-time RT-PCR assay may not be the definite cause of the disease. Negative results do not preclude SARS-CoV-2, influenza A, influenza B, or RSV infection and should not be used as the sole basis for patient management decisions.
Device Story
The Simplexa COVID-19/Flu A/B & RSV Direct is a qualitative, multiplex RT-PCR assay for the LIAISON MDX instrument. It processes nasopharyngeal or anterior nasal swabs in transport media using a Direct Amplification Disc (DAD) format, enabling sample-to-answer processing without separate nucleic acid extraction. The system uses fluorescent dye-labeled probes and primers to target specific viral genomic regions (SARS-CoV-2 S/ORF7a, Flu A matrix, Flu B matrix/nucleoprotein, RSV M/G). An encapsulated MS2 bacteriophage RNA internal control is co-amplified to monitor for inhibition or process failure. The LIAISON MDX instrument performs thermal cycling and real-time fluorescence detection. The software automates signal detection, amplification curve analysis, and qualitative result interpretation. Results aid clinicians in the differential diagnosis of respiratory infections when used with other clinical and epidemiological data. The device is intended for use in clinical laboratory settings.
Clinical Evidence
Prospective clinical study across 9 sites (1,401 NPS, 978 NS specimens). Comparator: FDA-cleared molecular assay. PPA/NPA for targets ranged from 88.1% to 100%. Specifically, Flu A PPA 96.5-98.0%, Flu B PPA 96.6-97.8%, RSV PPA 88.1-93.1%, and COVID-19 PPA 93.8-97.7%. Reproducibility assessed across 3 sites with 99.9% total agreement.
Technological Characteristics
Multiplex real-time RT-PCR; TaqMan probe chemistry; direct amplification (no extraction). Targets: SARS-CoV-2 (S, ORF1ab), Flu A (Matrix), Flu B (Matrix, nucleoprotein), RSV (M, G). Internal control: Bacteriophage MS2. Instrument: LIAISON MDX (benchtop thermocycler). Connectivity: USB to PC with LIAISON MDX Studio software. Consumable: Direct Amplification Disc (DAD).
Indications for Use
Indicated for individuals with signs and symptoms of respiratory tract infection to detect and differentiate SARS-CoV-2, influenza A, influenza B, and RSV RNA in nasopharyngeal and anterior nasal swab specimens. 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
- Panther Fusion® SARS-CoV-2/Flu A/B/RSV (K242465)
Related Devices
- K241806 — Applied Biosystems TaqPath COVID-19, Flu A, Flu B, RSV Select Panel · Life Technologies Corporation · Jan 8, 2025
- K242465 — Panther Fusion SARS-CoV-2/Flu A/B/RSV assay · Hologic, Inc. · Nov 15, 2024
- K243406 — cobas liat SARS-CoV-2, Influenza A/B & RSV nucleic acid test · Roche Molecular Systems, Inc. · Apr 25, 2025
Submission Summary (Full Text)
{0}
FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY AND INSTRUMENT
## I Background Information:
A 510(k) Number
K252387
B Applicant
Diasorin Molecular, LLC
C Proprietary and Established Names
Simplexa COVID-19/ Flu A/B & RSV Direct (MOL4450); Simplexa COVID-19/ Flu A/B & RSV Positive Control Pack (MOL4460)
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QOF | Class II | 21 CFR 866.3981 - Device To Detect And Identify Nucleic Acid Targets In Respiratory Specimens From Microbial Agents That Cause The Sars-Cov-2 Respiratory Infection And Other Microbial Agents When In A Multi-Target Test | MI - Microbiology |
## II Submission/Device Overview:
A Purpose for Submission:
The purpose of this submission is to show that the Simplexa COVID-19/ Flu A/B & RSV Direct (MOL4450) test is substantially equivalent to the FDA cleared Panther Fusion SARS-CoV-2/Flu A/B/RSV assay (K242465).
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
http://www.fda.gov
{1}
B Measurand:
- Influenza A RNA
- Influenza B RNA
- Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA
- Respiratory Syncytial Virus (RSV) RNA
C Type of Test:
Qualitative RT-PCR
III Intended Use/Indications for Use:
A Intended Use(s):
See Indications for Use below.
B Indication(s) for Use:
The Simplexa COVID-19 / Flu A/B & RSV Direct is a real-time RT-PCR assay intended for use on the LIAISON MDX instrument for the simultaneous in vitro qualitative detection and differentiation of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A (Flu A) virus, influenza B (Flu B) virus and respiratory syncytial virus (RSV) in nasopharyngeal swab and anterior nasal swab specimens from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar.
The Simplexa COVID-19 / Flu A/B & RSV Direct assay is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B and RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection. This test is not intended to detect influenza C virus infections.
Positive results are indicative of the presence of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Simplexa COVID-19 / Flu A/B & RSV Direct real-time RT-PCR assay may not be the definite cause of the disease. Negative results do not preclude SARS-CoV-2, influenza A, influenza B, or RSV infection and should not be used as the sole basis for patient management decisions.
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 LIAISON MDX Instrument.
K252387 - Page 2 of 29
{2}
K252387 - Page 3 of 29
## IV Device/System Characteristics:
### A Device Description:
The Simplexa COVID-19 & Flu A/B & RSV Direct assay is a qualitative, multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the simultaneous detection and differentiation of RNA from SARS-CoV-2, Influenza A, Influenza B, and Respiratory Syncytial Virus (RSV) in nasopharyngeal swabs (NPS) and anterior nasal swabs (ANS) in UTM/UVT and M4RT specimen transport media. The assay is performed on the LIAISON MDX Instrument using a Direct Amplification Disc (DAD) format, enabling sample-to-answer processing without separate nucleic acid extraction.
The LIAISON MDX Instrument is a benchtop real-time PCR thermocycler that utilizes a self-contained, single-use direct amplification disc (DAD) to process samples. It performs thermal cycling and real-time fluorescence detection using optical detection modules, each with specific excitation and emission wavelengths. The instrument includes a laser enclosed in a laser product housing, with integrated hardware and software interlocks to ensure user safety. It is operated via a USB connection to a dedicated computer running the LIAISON MDX Studio software.
The assay kit includes single-use reaction mix vials, a positive control pack with inactivated viral particles in transport media, and the Direct Amplification Disc consumable, which supports up to eight simultaneous reactions.
The assay format is designed for direct amplification, with 24 single-use reaction mix vials per kit. The required sample volume input is 50 µL. The reaction mix is provided in single-use vials and includes DNA polymerase, reverse transcriptase, RNase inhibitor, primers, probes, and encapsulated RNA templates. The buffer component in the reaction mix maintains optimal pH and ionic strength to support enzyme activity and amplification efficiency throughout the RT-PCR process.
The assay includes an encapsulated RNA internal control (RNA IC) in each reaction to monitor for potential RT-PCR inhibition or process failure. The RNA IC is derived from *bacteriophage MS2*. This non-target RNA is co-amplified with the assay's viral targets and detected independently using post-amplification melting curve analysis. The presence of the RNA IC in a negative specimen confirms that the amplification process functioned as expected, while its absence—along with no target detection—results in an invalid outcome. Detection of the RNA IC is not required in the Positive Control but is expected in the No Template Control (NTC) to verify assay validity.
### B Principle of Operation:
The Simplexa COVID-19 / Flu A/B & RSV Direct assay targets specific genomic regions for each virus: the S and ORF7a regions of SARS-CoV-2, the matrix gene of Influenza A, the matrix and nucleoprotein genes of Influenza B, and the M and G genes of RSV. An encapsulated RNA internal control (RNA IC) derived from *bacteriophage MS2* is included in each reaction to monitor for RT-PCR inhibition or failure. The RNA IC is co-amplified with the assay targets and detected
{3}
independently using post-amplification melting curve analysis. It is assigned to a dedicated fluorescent channel and does not interfere with target detection. Its presence in a negative specimen confirms that the amplification process functioned as expected, while its absence—along with no target detection—results in an invalid outcome. Detection of the RNA IC is not required in the Positive Control but is expected in the No Template Control (NTC) to verify assay validity. The assay employs fluorescent dye-labeled probes and primers specific to each target, and the LIAISON MDX software automates signal detection, amplification curve analysis, and qualitative result interpretation.
## C Instrument Description Information:
1. Instrument Name:
The LIAISON MDX instrument using LIAISON MDX Studio Software version 2.4.4.0 or higher
2. Specimen Identification:
Barcodes on the reagent kit and the barcode card contain the assay definition protocol and parameters for identification for the test and specimen type.
3. Specimen Sampling and Handling:
Nasopharyngeal and anterior nasal swab specimens collected in Universal Transport Medium (UTM), BD Universal Viral Transport (UVT) or M4RT.
4. Calibration:
The user does not perform calibration of the LIAISON MDX instrument.
5. Quality Control:
The RNA Internal Control (RNA-IC) is derived from bacteriophage MS2. The RNA-IC is included in each reaction to monitor for RT-PCR inhibition or failure. In addition, the test utilizes external controls – the Simplexa COVID-19 / Flu A/B & RSV Positive Control Pack (REF MOL4460) and No Template Control, provided separately.
## V Substantial Equivalence Information:
A Predicate Device Name(s):
Panther Fusion SARS-CoV-2/Flu A/B/RSV assay
B Predicate 510(k) Number(s):
K242465
C Comparison with Predicate(s):
K252387 - Page 4 of 29
{4}
K252387 - Page 5 of 29
| Device & Predicate Device(s): | K252387 | K242465 |
| --- | --- | --- |
| Device Trade Name | Simplexa COVID-19 & Flu A/B & RSV Direct | Panther Fusion SARS-CoV-2/Flu A/B/RSV |
| General Device Characteristic Similarities | K252387 | K242465 |
| Product Code | Same | QOF |
| Regulation | Same | 21 CFR 866.3981 |
| Organisms Detected | Same | Influenza A, Influenza B, Sars-CoV-2, Respiratory Syncytial Virus |
| Measurand | Same | Nucleic acid from Organisms detected |
| Intended Use/Indications For Use | The Simplexa COVID-19 / Flu A/B & RSV Direct is a real-time RT-PCR assay intended for use on the LIAISON MDX instrument for the simultaneous in vitro qualitative detection and differentiation of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A (Flu A) virus, influenza B (Flu B) virus and respiratory syncytial virus (RSV) in nasopharyngeal swab and anterior nasal swab specimens from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar.
The Simplexa COVID-19 / Flu A/B & RSV Direct assay is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B and RSV infections if | 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) swab specimens and anterior nasal (AN) swab 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 |
{5}
K252387 - Page 6 of 29
| | used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection. This test is not intended to detect influenza C virus infections.
Positive results are indicative of the presence of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Simplexa COVID-19 / Flu A/B & RSV Direct real-time RT-PCR assay may not be the definite cause of the disease. Negative results do not preclude SARS-CoV-2, influenza A, influenza B, or RSV infection and should not be used as the sole basis for patient management decisions. | 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 and AN swab 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. |
| --- | --- | --- |
| Specimen Types | NPS/ANS swabs in UVT/UTM/M4RT | NP/AN swabs in VTM/UTM/eSTM |
| Internal Control | MS2 phage (bacteriophage) | Internal Control-S (IC-S) synthetic RNA |
| Positive Control | Manual single-use vial | PRD-07401, system-integrated control |
{6}
| General Device Characteristic Differences | K252387 | K242465 |
| --- | --- | --- |
| Technology | RT-PCR (direct amplification) | RT-PCR (automated extraction + amplification) |
VI Standards/Guidance Documents Referenced:
1. 21 CFR 866.3981 - Special Controls
2. AAMI. Principles for medical device security – Risk Management. AAMI document TIR57:2016. Association for the Advancement of Medical Instrumentation; 2016.
3. AAMI. Principles for medical device security – Postmarket risk management for device manufacturers. AAMI document TIR97:2019. Association for the Advancement of Medical Instrumentation; 2019.
4. CLSI. Information Technology Security of In Vitro Diagnostic Instruments and Software Systems; Approved Standard – Second Edition. CLSI document AUTO11-A2. Wayne, PA: Clinical Laboratory Standards Institute; 2014.
5. CLSI. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition. CLSI document EP05-A3. Wayne, PA: Clinical Laboratory Standards Institute; 2019.
6. CLSI. Interference Testing in Clinical Chemistry. 3rd Ed. CLSI Document EP07. Wayne, PA: Clinical Laboratory Standards Institute; 2018.
7. CLSI. Evaluation of Qualitative, Binary Output Examination Performance; Approved Guideline – Third Edition. CLSI document EP12. Wayne, PA: Clinical Laboratory Standards Institute; 2023.
8. CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition. CLSI document EP17-A2. Wayne, PA: Clinical Laboratory Standards Institute; 2012.
9. CLSI. Assessment of the Diagnostic Accuracy of Laboratory Tests Using Receiver Operating Characteristic Curves; Approved Guideline – Second Edition. CLSI document EP24-A2. Wayne, PA: Clinical Laboratory Standards Institute; 2011.
10. CLSI. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline. CLSI document EP25-A. Wayne, PA: Clinical Laboratory Standards Institute; 2009.
11. CLSI. Collection Transport Preparation and Storage of Specimens for Molecular Methods. 2nd Edition. CLSI Document MM13. Wayne, PA: Clinical Laboratory Standards Institute; 2020.
12. CLSI. Verification and Validation of Multiplex Nucleic Acid Assays. 2nd Edition. CLSI Document MM17. Wayne, PA: Clinical Laboratory Standards Institute; 2018.
13. ISTA. Packaged-Products for Parcel Delivery System Shipment 70 kg (150 lb) or Less. ISTA Document 3A. International Safe Transit Association. 2018.
14. IEC 62366-1 Edition 1.1 2020-06 Consolidated Version; Medical devices – Part 1: Application of usability engineering to medical devices
15. IEC 61010-1 Edition 3.1 2017-01 Consolidated Version; Safety requirements for electrical equipment for measurement, control, and laboratory use – Part 1: General requirements
16. IEC 60601-1-2 Edition 4.1 2020-09 Consolidated Version; Medical electrical equipment – Part 1-2: General requirements for basic safety and essential performance – Collateral Standard: Electromagnetic disturbances – Requirements and tests
K252387 - Page 7 of 29
{7}
17. IEC 61326-1 Edition 3.0 2020-10; Electrical equipment for measurement, control and laboratory use – EMC requirements – Part 1: General requirements
18. IEC 61326-2 Edition 3.0 2020-10; Electrical equipment for measurement, control and laboratory use – EMC requirements – Part 2-6: Particular requirements – In vitro diagnostic (IVD) medical equipment
19. IEC 62304 Edition 1.1 2015-06 Consolidated Version; Medical device software – Software life cycle processes
20. IEC TR 60878 Ed. 4.0 2022-11; Graphical symbols for electrical equipment in medical practice [Including: Corrigendum 1 (2023)]
21. IEC TR 80001-2-2:2012. Application of risk management for IT Networks incorporating medical devices – Part 2-2: Guidance for the disclosure and communication of medical device security needs, risks and controls
22. IEC TR 80001-2-8 Edition 1.0 206-05; Application of risk management for IT – networks incorporating medical devices – Part 2-8: Application guidance – Guidance on standards for establishing the security capabilities identified in IEC TR 80001-2-2
23. ISO 14971:2019 Medical Devices – Application of risk management to medical devices
24. ISO 15223-1: 2021-07 – Medical Devices- Symbols to be used with information to be supplied by the manufacturer – Part 1: General requirements
25. UL ANSI 2900-1 First Edition 2017; Standard for Safety, Standard for Software Cybersecurity Network-Connectable Products, Part 1: General Requirements
26. UL ANSI 2900-2-1 First Edition 2017; Standard for Safety, Software Cybersecurity for Network-Connectable Products, Part 2-1: Particular Requirements for Network Connectable Components of Healthcare and Wellness Systems
K252387 - Page 8 of 29
{8}
VII Performance Characteristics (if/when applicable):
# A Analytical Performance:
# 1. Precision/Reproducibility:
# a) Within-Laboratory Precision
Within-laboratory precision of the Simplexa COVID-19/FLUA/B & RSV Direct assay was evaluated at a single site by testing a contrived panel (Table 1) in duplicate each day across six non-consecutive days using a single instrument. The panel consisted of ten members (live virus for influenza A, influenza B and RSV, and inactivated virus for SARS-CoV-2) prepared by spiking organisms in negative pooled NPS matrix. Three reagent lots were used in this study with 8 replicates of each panel member tested with each reagent lot. Results were generated from a total of 24 replicates of each panel member (2 runs/day x 2 replicate/run x 6 days).
Table 1. Precision/Reproducibility Study Sample Panel
| Target | Concentration | Concentration (cps/mL) |
| --- | --- | --- |
| Positive Control (PC): β-PL inactivated Flu A Strain A/California/7/2009, β-PL inactivated Flu B Strain B/Malaysia/2506/2004, SARS-nCOV-2 USA/WA1/2020, β-PL Inactivated RSV Strain A2 | 5x LoD | Influenza A, Influenza B, SARS-CoV-2: 2500 cps/mL RSV: 5000 cps/mL |
| No Template Control (NTC) – Pooled Negative Human Nasopharyngeal Matrix | N/A | N/A |
| Influenza A/Victoria/4897/2022 (H1N1) | Low (2x LoD) | 1000 cps/mL |
| Influenza A/Victoria/4897/2022 (H1N1) | Moderate (5x LoD) | 2500 cps/mL |
| Influenza B/Austria/1359417/2021 | Low (2x LoD) | 1000 cps/mL |
| Influenza B/Austria/1359417/2021 | Moderate (5x LoD) | 2500 cps/mL |
| RSV, B CH93(18)-18 | Low (2x LoD) | 2000 cps/mL |
| RSV, B CH93(18)-18 | Moderate (5x LoD) | 5000 cps/mL |
| SARS-CoV-2, Lineage BA.2.3; Omicron Variant | Low (2x LoD) | 1000 cps/mL |
| SARS-CoV-2, Lineage BA.2.3; Omicron Variant | Moderate (5x LoD) | 2500 cps/mL |
The qualitative results (i.e., % agreement with expected results) from the study are illustrated in Table 2.
Table 2. Within-Laboratory Precision Study - Qualitative Results
| Target | Level | Positive Results | Total Results | % Expected Result | Two-sided 95% CI Lower Bound | Two-sided 95% CI Upper Bound |
| --- | --- | --- | --- | --- | --- | --- |
| Influenza A | 2x LoD | 24 | 24 | 100 | 86.2 | 100 |
| | 5x LoD | 24 | 24 | 100 | 86.2 | 100 |
| Influenza B | 2x LoD | 24 | 24 | 100 | 86.2 | 100 |
K252387 - Page 9 of 29
{9}
| Target | Level | Positive Results | Total Results | % Expected Result | Two-sided 95% CI Lower Bound | Two-sided 95% CI Upper Bound |
| --- | --- | --- | --- | --- | --- | --- |
| | 5x LoD | 24 | 24 | 100 | 86.2 | 100 |
| RSV A | 2x LoD | 24 | 24 | 100 | 86.2 | 100 |
| | 5x LoD | 24 | 24 | 100 | 86.2 | 100 |
| SARS-CoV-2 | 2x LoD | 24 | 24 | 100 | 86.2 | 100 |
| | 5x LoD | 24 | 24 | 100 | 86.2 | 100 |
| PC | 5x LoD | 96 | 96 | 100 | 86.2 | 100 |
| No Template Control | Blank | 0 | 24 | 100 | 86.2 | 100 |
All positive panel members exhibited a detection rate of $100\%$ . The negative panel was negative $100\%$ of the time. There was no lot-to-lot variability observed in the study. The results of the study demonstrate acceptable assay variability.
The means, standard deviations, and coefficients of variation $(\%)$ for cycle threshold (Ct) values by target analyte and expected concentration (Positive Panel Members) are shown in Table 3.
Table 3. Precision - standard deviations and coefficients of variation of Ct values
| Target | Level | Hit rate % | Mean Ct | Inter-Day | | Inter-Lot | | Inter-Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| Influenza A | 2x LoD | 100 | 31.1 | 0.4 | 1.4 | 0.4 | 1.4 | 0.7 | 2.1 | 0.7 | 2.3 |
| | 5x LoD | 100 | 30.4 | 0.5 | 1.6 | 0.5 | 1.6 | 0.4 | 1.3 | 0.6 | 1.9 |
| Influenza B | 2x LoD | 100 | 32.3 | 0.6 | 1.8 | 0.6 | 1.8 | 0.4 | 1.2 | 0.7 | 2.1 |
| | 5x LoD | 100 | 31.7 | 0.3 | 0.8 | 0.3 | 0.8 | 0.4 | 1.4 | 0.4 | 1.4 |
| RSV A | 2x LoD | 100 | 30.6 | 0.3 | 1.0 | 0.3 | 1.0 | 0.5 | 1.7 | 0.5 | 1.7 |
| | 5x LoD | 100 | 29.7 | 0.5 | 1.5 | 0.5 | 1.6 | 1.1 | 3.7 | 1.0 | 3.5 |
| SARS-CoV-2 | 2x LoD | 100 | 29.9 | 0.2 | 0.6 | 0.2 | 0.6 | 0.4 | 1.2 | 0.3 | 1.2 |
| | 5x LoD | 100 | 29.4 | 0.3 | 1.1 | 0.4 | 1.3 | 0.4 | 1.4 | 0.5 | 1.6 |
Note: SD= standard deviation, CV= coefficient of variation, Ct= cycle threshold. LoD limit of detection
# b) Reproducibility
A reproducibility study was conducted with the Simplexa COVID-19/FLUA/B & RSV Direct assay at three clinical sites by a total of six operators (two operators at each site). Each operator tested the contrived panel used to evaluate precision (Table 1) in triplicate each day across five non-consecutive days. The panel consisted of ten members (live
K252387 - Page 10 of 29
{10}
virus for influenza A, influenza B and RSV, and inactivated virus for SARS-CoV-2) prepared by spiking organisms in negative pooled NPS matrix. Six instruments (two per site) and one reagent lot were used in this study. Results were generated from a total of 90 replicates of each panel member (3 sites x 5 days x 2 operators/site x 3 replicates/operator/day). The qualitative results of the study are illustrated in Table 4.
Table 4. Reproducibility Study- Qualitative Results
| Target | Level | % Agreement with Expected Results (n Detected/ N Tested) | | | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | Site 3 | Overall (95% CI) |
| NA | Negative | 100% (30/30) | 100% (30/30) | 100% (30/300) | 100% (90/90) (95.9-100%) |
| PC | 5x LoD | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| Influenza A | 2x LoD | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| Influenza B | | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| RSV | | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| SARS-CoV-2 | | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| Influenza A | 5x LoD | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| Influenza B | | 100% (30/30) | 100% (30/30) | 100% (29/30) | 98.9% (89/90) (94-99.8%) |
| RSV | | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
| SARS-CoV-2 | | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9-100%) |
The Simplexa COVID-19/FLUA/B & RSV Direct assay results demonstrated acceptable site-to-site, instrument-to-instrument, day-to-day, and between operator variation for the $\sim 2\mathrm{x}$ LoD, and $\sim 5\mathrm{x}$ LoD panel members (Table 5).
Table 5. Reproducibility - Standard Deviations and Coefficients of Variation of Ct Values
| Target | Level | (n/N)a | Mean Ct | Within Run (Repeatability) | | Site | | Operator | | Day | | Instrument | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Influenza A | 2x LoD | 90/90 | 33.2 | 0.76 | 2.3 | 0.50 | 1.5 | 0.51 | 1.5 | 0.85 | 2.6 | 0.6 | 1.8 | 0.99 | 3.0 |
| | 5x LoD | 90/90 | 32.5 | 0.45 | 1.4 | 0.65 | 2.0 | 0.59 | 1.8 | 0.65 | 2.0 | ncb | ncb | 0.69 | 2.1 |
| Influenza B | 2x LoD | 90/90 | 33.0 | 0.94 | 2.9 | 0.51 | 1.5 | 0.47 | 1.4 | 0.78 | 2.4 | 0.55 | 1.7 | 1.12 | 3.4 |
K252387 - Page 11 of 29
{11}
| Target | Level | (n/N)a | Mean Ct | Within Run (Repeatability) | | Site | | Operator | | Day | | Instrument | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| | 5x LoD | 89/90 | 31.9 | 0.61 | 1.9 | 0.10 | 0.3 | 0.11 | 0.4 | 0.59 | 1.8 | 0.41 | 1.3 | 0.75 | 2.4 |
| RSV | 2x LoD | 90/90 | 29.7 | 0.84 | 2.8 | 0.16 | 0.5 | 0.22 | 0.7 | 0.58 | 1.9 | 0.40 | 1.4 | 0.94 | 3.1 |
| | 5x LoD | 90/90 | 28.8 | 0.78 | 2.7 | 0.21 | 0.7 | 0.23 | 0.8 | 0.51 | 1.8 | 0.35 | 1.2 | 0.87 | 3.0 |
| SARS-CoV-2 | 2x LoD | 90/90 | 30.7 | 0.44 | 1.4 | 0.42 | 1.4 | 0.38 | 1.2 | 0.49 | 1.6 | 0.34 | 1.1 | 0.59 | 1.9 |
| | 5x LoD | 90/90 | 30.0 | 0.41 | 1.4 | 0.45 | 1.5 | 0.43 | 1.4 | 0.49 | 1.6 | 0.35 | 1.2 | 0.56 | 1.9 |
Ct = cycle threshold, LoD = limit of detection, SD = standard deviation, CV (%) = percent coefficient of variation, nc = not calculable.
${}^{a}$ n is the number of tests which matched the expected results. N is the total number of valid tests for the panel member.
b Between instrument SD and $\% \mathrm{CV}$ could not be calculated for Influenza A $(\sim 5\mathrm{x}$ LoD) as all replicates were tested on a single instrument per site per operator
# 2. Linearity:
Not applicable; this is a qualitative assay.
# 3. Analytical Specificity/Interference:
The inclusivity of the Simplexa COVID-19/FLUA/B & RSV Direct assay was evaluated using a combination of in silico analysis of publicly available sequence information and laboratory testing of contrived specimens containing viral isolates that were selected to represent phylogenetic, geographic, and temporal diversity.
# Analytical Reactivity (Inclusivity)
# a) Wet-Testing
This study was performed to determine the analytical reactivity of the Simplexa COVID-19/FLUA/B & RSV Direct test with clinically relevant strains, serotypes, or subtypes of the target specie. The inclusivity panel was prepared by spiking various target microorganisms into pooled negative NPS matrix. Each strain was tested with 3 replicates near LoD starting at $3\mathrm{x}$ LoD. The concentration which showed a $100\%$ hit rate is shown in Table 6 through Table 9.
Table 6. Strains of Influenza A Evaluated for Inclusivity
| Subtype | Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| H1N1 | A/New Caledonia/20/99 | Microbiologics | Custom growth - J2019F | 1500 | 3x | 100% (3/3) |
| | A/Puerto Rico/8/34 | Virapur | Custom growth - I1508A | 1500 | 3x | |
| | A/Brisbane/59/07 | ZeptoMetrix | 0810244CF | 1500 | 3x | 100% (3/3) |
| | A/California/07/09 | ZeptoMetrix | 0810165CF | 1500 | 3x | 100% (3/3) |
K252387 - Page 12 of 29
{12}
K252387 - Page 13 of 29
| Subtype | Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected
(# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| H1N1
pdm09 | A/Guangdong-Maonan/SWL1536/2019 (H1N1) | Microbiologics | Lot: B2214X | 1500 | 3x | 100% (3/3) |
| | A/Brisbane/02/18 (H1N1) (CDC Panel) | Microbiologics | Lot: D1929A | 1500 | 3x | 100% (3/3) |
| | A/Nebraska/14/2018 (H1N1) | Microbiologics | Lot: D2013A | 1500 | 3x | 100% (3/3) |
| | A/Victoria/2570/2019 (H1N1) | Microbiologics | Lot: B2214P | 2000 | 4x | 100% (3/3) |
| | A/Mexico/4108/2009 (H1N1) | ZeptoMetrix | 0810166CF | 1500 | 3x | 100% (3/3) |
| | A/Sydney/05/2021 (H1N1) | Microbiologics | Lot: E2305A | 1500 | 3x | 100% (3/3) |
| | A/New York/02/09 (H1N1) | ZeptoMetrix | 0810109CF N | 1500 | 3x | 100% (3/3) |
| H3N2 | A/Perth/16/2009 (H3N2) (CDC Panel) | Virapur | L1909A | 2250 | 3x | 100% (3/3) |
| | A/Hong Kong/2671/2019 (H3N2) (CDC Panel) | Microbiologics | Lot: B2214V | 2250 | 3x | 100% (3/3) |
| | A/Kansas/14/2017 (H3N2) (CDC Panel) | Microbiologics | Lot: D1929B | 2250 | 3x | 100% (3/3) |
| | A/Cambodia/e0826360/2020 (H3N2) | Microbiologics | Lot: I2107G | 2250 | 3x | 100% (3/3) |
| | A/Darwin/6/2021 (H3N2) | Microbiologics | Lot: L2216B | 2250 | 3x | 100% (3/3) |
| | A/Tasmania/503/2020 (H3N2) | Microbiologics | Lot: B2221J | 2250 | 3x | 100% (3/3) |
| | A/Switzerland/9715293/13 (H3N2) | ZeptoMetrix | 0810511CF | 2250 | 3x | 100% (3/3) |
| | A/Texas/50/2012 (H3N2) | Microbiologics | Lot: B2322D | 2250 | 3x | 100% (3/3) |
| | A/Thailand/08/2022 (H3N2) | Microbiologics | Lot: C2419C | 2250 | 3x | 100% (3/3) |
| | A/Singapore/INFIMH-16-0019/2016 (H3N2) (CDC Panel) | Microbiologics | Lot: B1913A | 2250 | 3x | 100% (3/3) |
| | A/Hong Kong/4801/14 (H3N2) | Microbiologics | Lot: B1913E | 2250 | 3x | 100% (3/3) |
| | A/Massachusetts/18/2022 (H3N2) | Microbiologics | Lot: C2429E | 2250 | 3x | 100% (3/3) |
| | A/Hong Kong/8/1968 (H3N2) | Virapur | Lot: L1503A | 2250 | 3x | 100% (3/3) |
| | A/Port Chalmers 1/1973 (H3N2) | Microbiologics | Lot: K1516D | 2250 | 3x | 100% (3/3) |
| H5 | Influenza A H5N1 Gentaur | AffiGen | AFG-CHK-0591 | 1500 | 3x | 100% (3/3) |
| | A/Anhui/01/2005 (H5N1) | IRR | FR-735 | 1500 | 3x | 100% (3/3) |
| | A/Egypt/N03072/2010 (H5N1) | IRR | FR-1065 | 1500 | 3x | 100% (3/3) |
{13}
Table 7. Strains of Influenza B Evaluated for Inclusivity
| Subtype | Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| Victoria | B/Brisbane/60/2008 (Victoria) (CDC Panel) | Virapur | Lot: E1703B | 1500 | 3x | 100% (3/3) |
| | B/Colorado/06/2017 (Victoria) (CDC Panel) | Microbiologics | Lot: L2212B | 1500 | 3x | 100% (3/3) |
| | B/Brisbane/33/08 (Victoria) | ZeptoMetrix | 0810253 CF | 1500 | 3x | 100% (3/3) |
| | B/Texas/2/13 (Victoria) (CDC Panel) | ZeptoMetrix | 0810527 CF | 1500 | 3x | 100% (3/3) |
| | B/Florida/02/06 (Victoria) | ZeptoMetrix | 0810037 CF | 1500 | 3x | 100% (3/3) |
| | B/Malaysia/2506/2 004 (Victoria) | Virapur | Lot: G1626A | 1500 | 3x | 100% (3/3) |
| | B/Alabama/2/17 (Victoria) | ZeptoMetrix | 0810572 CF | 2500 | 5x | 100% (3/3) |
| | B/Maryland/01/1959 (Victoria) | Microbiologics | Lot: D1915A | 1500 | 3x | 100% (3/3) |
| | B/Washington 02/2019 (Victoria) (CDC Panel) | Microbiologics | Lot: D2013B | 1500 | 3x | 100% (3/3) |
| Yamagata | B/Florida/07/04 (Yamagata) | ZeptoMetrix | 0810256 CF | 1500 | 3x | 100% (3/3) |
| | B/Massachusetts/2/2012 (Yamagata) | Microbiologics | Lot: C2207E | 1500 | 3x | 100% (3/3) |
| | B/Utah/9/14 (Yamagata) | ZeptoMetrix | 0810516 CF | 1500 | 3x | 100% (3/3) |
| | B/Florida/04/06 (Yamagata) | ZeptoMetrix | 0810255 CF | 1500 | 3x | 100% (3/3) |
| | B/Panama/45/90 (Yamagata) | ZeptoMetrix | 0810259 CF | 1500 | 3x | 100% (3/3) |
Table 8. Strains of RSV Evaluated for Inclusivity
| Subtype | Strain/ Isolate | Source | Catalogue No. | Concentration | | % Detected (# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Cp/mL | xLoD | |
| A | 2006 Isolate | ZeptoMetrix | 0810040ACF | 3000 | 3x | 100% (3/3) |
| | 3/2015 Isolate 3 | ZeptoMetrix | 0810482CF | 4000 | 4x | 100% (3/3) |
| | A2 | Microbiolog ics | Lot: K2115I | 3000 | 3x | 100% (3/3) |
K252387 - Page 14 of 29
{14}
Table 9. Strains of SARS-CoV-2 Evaluated for Inclusivity
| Strain/Isolate | Source | Catalogue No. | Concentration | | % Detected(# Detected/#Tested) |
| --- | --- | --- | --- | --- | --- |
| | | | Cp/mL | xLoD | |
| Hong Kong/VM20001061/2020 | Virapur | Lot: F2016D | 1500 | 3x | 100% (3/3) |
| England/204820464/2020 | Microbiologics | Lot: D2112H | 1500 | 3x | 100% (3/3) |
| South Africa/KRISP-EC-K005325/2020 | Microbiologics | Lot: D2126L | 1500 | 3x | 100% (3/3) |
| hCoV-19/Japan/TY7-503/2021 | Microbiologics | Lot: G2126B | 1500 | 3x | 100% (3/3) |
| hCoV-19/USAIPHC 658/2021 Delta | Microbiologics | Lot: G2126D | 1500 | 3x | 100% (3/3) |
| Omicron B.1.1.529 | Microbiologics | Lot: C2330F | 1500 | 3x | 100% (3/3) |
| USA-WI1/2020 | Microbiologics | Lot: J2026G | 1500 | 3x | 100% (3/3) |
| B.1.617.1; Kappa Variant USA/CA-Stanford-15_S02/2021 | ZeptoMetrix | 081 0623CFH1 | 1500 | 3x | 100% (3/3) |
| Iota, (B.1.526_2021) NY-Wadsworth-21025952-01/2021 | ZeptoMetrix | 081 061 9CFHI | 1500 | 3x | 100% (3/3) |
| Zeta, (P2_2021) NY-Wadsworth-21006055-01/2021 | ZeptoMetrix | 0810618CFH1 | 1500 | 3x | 100% (3/3) |
| USA/CA3/2020 | ZeptoMetrix | 0810040ACF | 1500 | 3x | 100% (3/3) |
| Lineage B Isolate Germany/BavPat1/2020 | ZeptoMetrix | 081 0482CF | 1500 | 3x | 100% (3/3) |
| Epsilon, (B.1.429) USA/CA/VRLC014/2021 | Microbiologics | Lot: F2321F | 1500 | 3x | 100% (3/3) |
| Delta (AY4.2) hCoV-19/USA/VAFBCH_675/2021 | Microbiologics | Lot: F2321H | 1500 | 3x | 100% (3/3) |
| Lambda (C.37) Peru/un-CDC-2-4069945/2021 | Microbiologics | Lot: I2120F | 1500 | 3x | 100% (3/3) |
| Omicron B.1.1.529 Lineage BQ.1.1 Isolate hCoV-19/USA/MDHP388 61/2022 | Microbiologics | Lot: C2221L | 1500 | 3x | 100% (3/3) |
| Omicron XBB hCoV19/USA/CAStan dford-109_S21/2022 | Microbiologics | Lot: D2310F | 1500 | 3x | 100% (3/3) |
K252387 - Page 15 of 29
{15}
b) In Silico
SARS-CoV-2 In Silico Analysis
The inclusivity of the Simplexa COVID-19/FLUA/B & RSV Direct assay was evaluated using in silico analysis of the forward primers, reverse primers, and probes for the SARS-CoV-2 target in relation to all sequences available in the GISAID gene databases. In silico analysis on May 5th, 2025, of the dual target SARS-CoV-2 primer and probe binding regions indicate 99.99% detection of all available complete and high-coverage sequences in GISAID (>5.3M sequences, <0.003% predicted failed detections) databases. In silico analysis indicated inclusivity to all known SARS-CoV-2 variants.
Cross-Reactivity and Microbial Interference
Cross-reactivity and microbial interference of the Simplexa COVID-19/FLUA/B & RSV Direct assay was evaluated by testing a panel of bacteria, fungi, and viruses commonly found in the respiratory tract for cross-reactivity and interference. The effect of non-target microorganisms on the performance of Simplexa COVID-19/FLUA/B & RSV Direct assay was tested by introducing non-target microorganisms into pooled negative NPS matrix spiked with and without SARS-CoV-2, influenza A, influenza B or RSV viruses at 3x LoD. Three (3) replicates in target positive background and three (3) replicates in target negative background were tested for each non-target microorganism. Bacteria and fungi were spiked at 1.0e+6 units/mL and viruses at 1.0e+5 units/mL, or the highest concentration possible. As summarized in Table 10, no cross-reactivity or microbial interference was observed at the concentrations tested, and no invalid results were obtained.
Table 10. Cross-reactivity and Microbial Interference Results for Non-Target Microorganisms
| Microorganism | Concentration | Negative Sample (Specificity Test) | | | | Positive Sample (Interference Test) | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Target Result: n Detected/N Tested | | | | Target Result: n Detected/N Tested | | | |
| | | SARS-CoV-2 | Influenza A | Influenza B | RSV | SARS-CoV-2 | Influenza A | Influenza B | RSV |
| Adenovirus 1 | 1E5 TCID50/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Adenovirus 7A | 1E5 TCID50/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Aspergillus fumigatus | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Bordetella pertussis | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
K252387 - Page 16 of 29
{16}
K252387 - Page 17 of 29
| Bordetella parapertussis E595 | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Candida albicans | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Chlamydophila pneumoniae | 1E6 cps/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Coronavirus 229E | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Coronavirus NL63 | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Coronavirus OC43 | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Coronavirus HKU1 | 1E5 cps/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Corynebacterium diphtheriae | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Cytomegalovirus (CMV) | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Enterovirus | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Epstein-Barr Virus (EBV) | 1E5 cps/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Escherichia coli | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Fusobacterium necrophorum | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Haemophilus influenzae | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Lactobacillus plantarum | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Legionella pneumophila | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Measles | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| MERS-coronavirus | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Metapneumovirus | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Moraxella catarrhalis | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Mumps | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Mycobacterium tuberculosis (genomic DNA) | 1E6 cps/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Mycoplasma pneumoniae | 1E6 cps/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Mycoplasma genitalium | 1E6 CCU/mL | | | | | | | | |
| Neisseria gonorrhoeae | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Neisseria meningitidis | 1E6 CFU/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Parainfluenza virus 1 | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Parainfluenza virus 2 | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Parainfluenza virus 3 | 1E5 TCID_{50}/mL | 0/3 | 0/3 | 0/3 | 0/3 | 3/3 | 3/3 | 3/3 | 3/3 |
{17}
CFU = Colony Forming Units; cps = copies; TCID50 = Median Tissue Culture Infectious Dose; CCU = Color Changing Unit; n.a. = not applicable.
# Interfering Substances
An analytical study was performed to assess the potential inhibitory effects of endogenous and exogenous substances that may be commonly found in NPS and NS specimens. All substances in Table 11 were evaluated in the presence and absence of SARS-CoV-2, influenza A, influenza B or RSV viruses at $3\mathrm{x}$ LoD in pooled negative NPS matrix. Testing for each condition was performed in replicates of ten. The results are shown in Table 11 and Table 12.
None of the evaluated substances, at the concentrations tested, interfered with detection of the candidate assay targets.
Table 11. Substances Evaluated for Interference for Negative Samples
| Potentially Interfering Substance | Active Ingredient | Interferent Concentration* | Flu A | Flu B | SARS-CoV-2 | RSV | Internal Control |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) |
| Afrin Nasal spray | Oxymetazoline | 15% (v/v) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| Antibacterial, systemic | Tobramycin | 4 μg/mL | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| Antibiotic, nasal ointment | Mupirocin | 6.6 mg/mL | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| Whole Blood | N/A | 2% (v/v) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
* In the presence of SARS-CoV-2, influenza A, influenza B or RSV viruses at 3x LoD in pooled negative NPS matrix. Testing for each condition was performed in replicates of ten. The results are shown in Table 11 and Table 12.
K252387 - Page 18 of 29
{18}
Table 12. Substances Evaluated for Interference for Positive Samples
| Potentially Interfering Substance | Active Ingredient | Interferent Concentration* | Flu A | Flu B | SARS-CoV-2 | RSV | Internal Control |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) |
| Afrin Nasal spray | Oxymetazoline | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Antibacterial, systemic | Tobramycin | 4 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Antibiotic, nasal ointment | Mupirocin | 6.6 mg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Whole Blood | N/A | 2% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Cold Eeze (Throat lozenges, Oral anesthetic and analgesic) | N/A | 1.25% (w/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Nasal corticosteroid (Beconase AQ) | Beclomethasone | 5% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Cold Cough | Cyclosporine | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Coughing | Cyclosporine | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Coughing | Cyclosporine | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% |
| Coughing | Cyclophosphamide | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% |
*μg/mL = Micrograms/milliliter, mg/mL = Milligrams/milliliter, μM = Micromolar, v/v = Volume per Volume, w/v = Weight/Volume
K252387 - Page 19 of 29
{19}
| Potentially Interfering Substance | Active Ingredient | Interferent Concentration* | Flu A | Flu B | SARS-CoV-2 | RSV | Internal Control |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) | % Detection (#Detected/#Tested) |
| Nasal corticosteroid (Flonase) | Fluticasone | 5% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Relenza Antiviral Drug | Zanamivir | 3.3 mg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Tamiflu Antiviral drug | Oseltamivir | 1 μM | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Zicam Nasal Gel | Luffa operculata, Galphimia glauca, histaminum hydrochloricum | 5% (w/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Zicam Nasal Spray (Homeopathic allergy relief medicine) | N/A | 10% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Bovine submaxillary gland mucin, type I-S | Purified Mucin Protein | 2.5 mg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Antiviral drug | Remdesivir | 10 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Human Leukocytes | N/Aa | 5% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
*μg/mL = Micrograms/milliliter, mg/mL = Milligrams/milliliter, μM = Micromolar, v/v = Volume per Volume, w/v = Weight/Volume
## COMPETITIVE INTERFERENCE
### Competitive Interference (Co-Infection)
To assess potential competitive interference between the viral targets, Samples were prepared by spiking one (1) assay target analyte at a low concentration (3x LoD) into negative NPS matrix in the presence of a high concentration (up to 2000x LoD) of one (1) of the other three (3) assay target analytes. Three replicates were tested with one viral target at 3x LoD which was mixed with another target at high concentration. As shown in Table 13, none of the targets present at high concentration interfered with the detection of other viral targets at low concentration levels.
Table 13. Competitive Interference (Co-infection) Study Results
| Baseline | Competitive Interferent | Flu A Valid Positive Results/ Total # of Valid Results | Flu B Valid Positive Results/ Total # of Valid Results | SARS-CoV-2 Valid Positive Results/ Total # of Valid Results | RSV Valid Positive Results/ Total # of Valid Results |
| --- | --- | --- | --- | --- | --- |
| Influenza A/Victoria/4897/2022 (H1N1) | Influenza B 2000x LoD (1E6 cps/mL) | 100% (3/3) | 100% (3/3) | 0% (0/3) | 0% (0/3) |
| | SARS-CoV-2 | 100% | 0% | 100% | 0% |
K252387 - Page 20 of 29
{20}
| 3x LoD | 2000x LoD (1E6 cps/mL) | (3/3) | (0/3) | (3/3) | (0/3) |
| --- | --- | --- | --- | --- | --- |
| | RSV 1000x LoD (1E6 cps/mL) | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| Influenza B/Austria/1359417/2 021 3x LoD | Influenza A 2000x LoD (1E6 cps/mL) | 100% (3/3) | 100% (3/3) | 0% (0/3) | 0% (0/3) |
| | SARS-CoV-2 2000x LoD (1E6 cps/mL) | 0% (0/3) | 100% (3/3) | 100% (3/3) | 0% (0/3) |
| | RSV B 1000x LoD (1E6 cps/mL) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) |
| SARS-CoV-2, Lineage BA.2.3 3x LoD | Influenza A 2000x LoD (1E6 cps/mL) | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) |
| | Influenza B 2000x LoD (1E6 cps/mL) | 0% (0/3) | 100% (3/3) | 100% (3/3) | 0% (0/3) |
| | RSV B 1000x LoD (1E6 cps/mL) | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3) |
| RSV, B CH93(18)-18 3x LoD | Influenza A 2000x LoD (1E6 cps/mL) | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |
| | Influenza B 2000x LoD (1E6 cps/mL) | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3) |
| | SARS-CoV-2 2000x LoD (1E6 cps/mL) | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3) |
# 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 (RNA-IC) added to each reaction 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 sample stability claims:
Primary Specimen (NPS and NS specimens collected in Universal Transport Medium (UTM), BD Universal Viral Transport (UVT) or M4RT)
Stored at $2 - 8^{\circ}\mathrm{C}$ for up to 7 days
- $\leq -70^{\circ}\mathrm{C}$ for up to 3 months.
up to three freeze/thaw cycles at $\leq -70^{\circ}\mathrm{C}$
K252387 - Page 21 of 29
{21}
c) Kit Stability
The Simplexa COVID-19/FLUA/B & RSV Direct assay Reaction Mix and the Simplexa COVID-19/FLUA/B & RSV Direct assay Positive Control
- 30 minutes post-thaw at room temperature.
- shelf life of 6 months when stored at −20°C ±10°C.
d) Shipping Stability
The Simplexa COVID-19/FLUA/B & RSV Direct assay Reaction Mix and the Simplexa COVID-19/FLUA/B & RSV Direct assay Positive Control
The shipping validation study for the Simplexa COVID-19/Flu A/B & RSV Direct Kit and its associated Positive Control Pack was conducted to demonstrate that the product maintains its integrity and performance following international shipping conditions. A total of twelve reaction kits and ten positive control packs were shipped from DiaSorin Molecular’s manufacturing facility in Cypress, CA to the FedEx Packaging Lab in Collierville, TN, where they were subjected to the 72-hour HEAT profile simulating worst-case summer shipping conditions. The kits were then returned to the manufacturing site for inspection and testing. Temperature monitoring confirmed that internal temperatures remained below −10°C throughout the 122-hour shipping duration, with no significant excursions. Upon receipt, all kits were visually inspected and confirmed to be free of physical damage, with all liquid components remaining frozen and intact.
Post-shipping analytical performance testing was conducted using the LIAISON MDX platform and included 30 replicates of the shipped reaction mix tested with both non-template controls (NTCs) and positive controls (PCs), as well as 30 replicates of the shipped positive control tested with reaction mix that were not exposed to the simulated shipping condition. Comparator kits from the same lot stored at −20°C ± 10°C served as controls. All NTC replicates were valid for the internal control, with no detection of Flu A, Flu B, SARS-CoV-2, or RSV in the respective channels, except for one Flu B detection, which triggered extended testing per protocol. The additional 17 NTC replicates yielded no further detections. All PC replicates demonstrated 100% detection across all targets. Result of the study These support the use of the validated shipping configuration for both domestic and international distribution and demonstrate that the shipping process preserves product integrity and performance.
6. Detection Limit:
The purpose of this study was to determine the limit of detection (LoD) of the Simplexa COVID-19/FLUA/B & RSV Direct assay for the detection of influenza A, influenza B, SARS-CoV-2 and RSV as single analytes in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens. The study assessed two active strains of influenza A (influenza A H1N1, Victoria/4897/2022 and influenza A H3N2, Darwin/9/21), two active strains of influenza B (influenza B Victoria, Austria/1359417/2021 and influenza B Yamagata, Phuket/3073/2013), two inactivated strains of SARS-CoV-2 (USA-WA1/2020 and Omicron
K252387 - Page 22 of 29
{22}
BA.2.3) and two active strains of RSV (RSV A 1/2015, RSV B CH93(18)-18). Each organism was individually spiked into pooled negative clinical NPS or NS matrix at the appropriate final concentration for testing per the instructions for use for the Simplexa COVID-19/FLUA/B & RSV Direct assay. Each virus was tested in a range finding study that included five concentrations, where each concentration was tested with six replicates. The lowest concentration with 100% detection was estimated to be the LoD. The estimated LoD for each strain was verified by testing 60 replicates at the estimated LoD concentration and demonstrating that at least 57/60 replicates were positive. If the criteria of at least 95% of positive replicates were not met, testing was repeated with a higher concentration until at least 95% of replicates gave a positive result.
The LoD of the Simplexa COVID-19/FLUA/B & RSV Direct test for influenza A, influenza B, SARS-CoV-2 and RSV in NPS and ANS specimen are summarized in Table 14.
Table 14: Confirmed LoD for the Simplex COVID-19/FLUA/B & RSV Direct Assay
| Target | Confirmed LoD (cps/mL) NPS | Confirmed LoD (cps/mL) ANS |
| --- | --- | --- |
| Influenza A Victoria/4897/2022 (H1N1) | 500 | 750 |
| Influenza A Darwin/9/21 (H3N2) | 750 | 750 |
| Influenza B Austria/1359417/2021 (Victoria) | 500 | 500 |
| Influenza B Phuket/3073/2013 (Yamagata) | 500 | 500 |
| SARS-CoV-2 USA/WA 1/2020 | 500 | 500 |
| SARS-CoV-2 Lineage BA.2.3; Omicron Variant | 500 | 500 |
| Respiratory syncytial virus (RSV) A 1/2015 Isolate | 1000 | 2000 |
| Respiratory syncytial virus (RSV) B CH93(18)-18 | 1000 | 1000 |
7. Assay Cut-Off:
Data from several analytical studies were used to analyze the fluorescence signal distribution and set cutoffs for all organisms to maximize sensitivity and specificity.
8. Accuracy (Instrument):
Not applicable.
9. Carry-Over:
The carry-over and cross-contamination rate for the Simplexa COVID-19/FLUA/B & RSV Direct assay was determined by testing 28 samples of pooled negative NPS matrix and 28 high positive SARS-CoV-2 samples at a concentration of 1E6 cps/mL in an alternating sequence across seven LIAISON MDX instruments. All 28 replicates of the negative sample were negative, resulting in a cross-contamination rate of 0%.
An additional study was conducted to evaluate the risk for cross-contamination and to demonstrate the re-usability of the DAD consumable up to eight times. A total of 18 DADs were tested by loading fluorescent dyes, FAM and CFR610 alternatively into the eight wells
K252387 - Page 23 of 29
{23}
of each disc to detect potential cross-contamination. No carryover contamination events were observed across any of the 18 discs.
## B Comparison Studies:
1. Method Comparison with Predicate Device:
Not applicable.
2. Media Equivalency Study:
Equivalency between NPS stabilized in UTM and M4RT was evaluated. Pooled negative clinical NPS matrix was spiked with RSV, influenza A, SARS-CoV-2 or influenza B. For each target and media type, 30 samples were tested at 2x LoD, 10 at 5x LoD, and 10 negative samples. All replicates tested were positive for the respective viral target for both matrices with 100% hit rate.
## C Clinical Studies:
1. Clinical Sensitivity:
The clinical performance of the Simplexa COVID-19/ Flu A/B & RSV Direct assay was evaluated versus a FDA 510(k) cleared comparator in NPS and NS specimens from individuals experiencing signs and symptoms of respiratory viral infection. Fresh specimens were collected at nine (9) collection sites during the 2024-2025 respiratory viral season and tested with the Simplexa COVID-19/ Flu A/B & RSV Direct assay at five (5) testing sites.
A total of 1,401 NPS specimens and 978 NS specimens were enrolled for the prospective clinical study and tested with the Simplexa COVID-19/ Flu A/B & RSV Direct assay and the comparator method. 526 NPS specimens and NS specimens were collected in pair from the same subject. Forty (40) NPS specimens and forty-six (46) NS specimens were not evaluable due to protocol deviations, sample handling issues and/or invalid reference testing result. The Simplexa COVID-19/ Flu A/B & RSV Direct assay demonstrated adequate clinical performance. The Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) point estimates between the Simplexa COVID-19/ Flu A/B & RSV Direct assay and the comparator for the different target pathogens in NS and, NPS samples are summarized in Tables 15 and 16. The performance of the Simplexa COVID-19/ Flu A/B & RSV Direct assay in unpaired NS and NPS samples is summarized in Table 17. Samples that produced a discordant call between the Simplexa COVID-19/ Flu A/B & RSV Direct assay and the comparator test were further tested with a second FDA-cleared molecular assay and/or PCR/bidirectional sequencing (BDS). Results from discordant sample testing are presented as footnotes below the table but were not used in performance calculations
Demographics of subjects that were included in the final performance analysis are summarized in Table 18.
Table 15: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of Prospective Data Set (NPS)
K252387 - Page 24 of 29
{24}
K252387 - Page 25 of 29
| Pathogen Target | Positive Percent Agreement | | | Negative Percent Agreement | | |
| --- | --- | --- | --- | --- | --- | --- |
| | TP / (TP+FN) | PPA (%) | 95% CI | TN / (TN+FP) | NPA (%) | 95% CI |
| Flu A | 362/375^{a} | 96.5% | 94.2%-98.0% | 971/978^{b} | 99.3% | 98.5%-99.7% |
| Flu B | 45/46^{c} | 97.8% | 88.7%-99.6% | 1305/1307^{d} | 99.8% | 99.4%-100.0% |
| RSV | 95/102^{e} | 93.1% | 86.5%-96.6% | 1238/1244^{f} | 99.5% | 99.0%-99.8% |
| SARS-CoV-2 | 61/65^{g} | 93.8% | 85.2%-97.6% | 1281/1288^{h} | 99.5% | 98.9%-99.7% |
aFive (5) of the thirteen (13) Flu A False Negative specimens were negative by PCR/BDS. One (1) additional specimen was negative by Standard of Care.
bThree (3) of the seven (7) Flu A False Positive specimens were positive by PCR/BDS. Two (2) additional specimens were positive by Standard of Care.
cThe one (1) Flu B False Negative specimen was negative by PCR/BDS.
dBoth of the two (2) Flu B False Positive specimens were positive by PCR/BDS and one (1) specimen was also positive by Standard of Care.
eThree (3) of the seven (7) RSV False Negative specimens were negative by PCR/BDS. Three (3) additional specimens were negative by Standard of Care.
fFive (5) of the six (6) RSV False Positive specimens were positive by PCR/BDS.
gTwo (2) of the four (4) SARS-CoV-2False Negative specimens were negative by PCR/BDS.
hFour (4) of the seven (7) COVID-19 False Positive specimens were positive by PCR/BDS.
Table 16: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of Prospective Data Set (ANS)
| Pathogen Target | Positive Percent Agreement | | | Negative Percent Agreement | | |
| --- | --- | --- | --- | --- | --- | --- |
| | TP / (TP+FN) | PPA (%) | 95% CI | TN / (TN+FP) | NPA (%) | 95% CI |
| Flu A | 250/255^{a} | 98.0% | 95.5%-99.2% | 662/675^{b} | 98.1% | 96.7%-98.9% |
| Flu B | 28/29^{c} | 96.6% | 82.8%-99.4% | 899/901^{d} | 99.8% | 99.2%-99.9% |
| RSV | 52/59^{e} | 88.1% | 77.5%-94.1% | 863/865^{f} | 99.8% | 99.2%-99.9% |
| SARS-CoV-2 | 42/43^{g} | 97.7% | 87.9%-99.6% | 882/887^{h} | 99.4% | 98.7%-99.8% |
aFour (4) of the five (5) Flu A False Negative specimens were negative by PCR/BDS. The other specimen was negative by Standard of Care.
bAll thirteen (13) Flu A False Positive specimens were positive by PCR/BDS.
cThe one (1) Flu B False Negative specimen was negative by Standard of Care.
dThe two (2) Flu B False Positive specimens were negative by PCR/BDS.
eTwo (2) of the seven (7) RSV False Negative specimens were negative by PCR/BDS.
fOne (1) of the two (2) RSV False Positive specimens was positive by PCR/BDS.
gThe SARS-CoV-2 False Negative specimen was negative by PCR/BDS.
hTwo (2) of the five (5) SARS-CoV-2 False Positive specimens were positive by PCR/BDS.
Table 17: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of Prospective Data Set (Unpaired)
| Target | Sample Type | TP/(TP+FN) | PPA | 95%CI | TN/(TN+FP) | NPA | 95%CI |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Flu A | NS | 94/96 | 97.90% | 92.7%-99.4% | 301/308 | 97.70% | 95.4%-98.9% |
| | NPS | 215/224 | 96.00% | 92.5%-97.9% | 620/626 | 99.00% | 97.9%-99.6% |
| | Total | 309/320^{a} | 96.60% | 94.0%-98.1% | 921/934^{b} | 98.60% | 97.6%-99.2% |
{25}
Table 18: General Demographic Details of the Prospective Study Population
| | NPS (N = 1361) | NS (N=932) |
| --- | --- | --- |
| Sex | | |
| Male | 669 (49.2%) | 440 (47.2%) |
| Female | 691 (50.8%) | 492 (52.8%) |
| Unknown | 1 (0.1%) | 0 (0.0%) |
| Total | 1361 (100%) | 932 (100%) |
| Age | | |
| <= 5 | 508 (37.3%) | 242 (26.0%) |
| 6 - 18 | 486 (35.7%) | 417 (44.7%) |
| 19 - 14 | 153 (11.2%) | 137 (14.7%) |
| 41 - 60 | 102 (7.5%) | 94 (10.1%) |
| 61 + | 106 (7.8%) | 42 (4.5%) |
| Unknown | 6 (0.4%) | 0 (0.0%) |
| Total | 1361 (100%) | 932 (100%) |
| Subject Location | | |
| ER | 692 (50.8%) | 176 (18.9%) |
| ICU | 1 (0.1%) | 754 (80.9%) |
| (80.9%) | 84 (6.2%) | 0 (0.0%) |
| Outpatient | 584 (42.9%) | 0 (0.0%) |
${}^{a}$ Five (5) of the eleven (11) Flu A False Negative specimens were negative by PCR/BDS.
${}^{\mathrm{b}}$ Nine (9) of the thirteen (13) Flu A False Positive specimens were positive by PCR/BDS. Three (3) additional specimens were positive by Standard of Care.
The one (1) Flu B False Negative specimen was negative by PCR/BDS.
Four (4) of the seven (7) RSV False Negative specimens were negative by PCR/BDS. One (1) additional specimen was negative by Standard of Care.
${}^{\mathrm{c}}$ Five (5) of the six (6) RSV False Positive specimens were positive by PCR/BDS.
Two (2) of the four (4) SARS-CoV-2 False Negative specimens were negative by PCR/BDS.
One (1) of the four (4) SARS-CoV-2 False Positive specimens was positive by PCR/BDS.
K252387 - Page 26 of 29
{26}
| Unknown | 0 (0.0%) | 2 (0.2%) |
| --- | --- | --- |
| Total | 1361 (100%) | 932 (100%) |
2. Clinical Specificity:
See section VII. Performance Characteristics. C. Clinical Studies 1. Clinical Sensitivity, above.
3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
D Clinical Cut-Off:
Not applicable.
E Expected Values/Reference Range:
The Simplexa COVID-19/ Flu A/B & RSV Direct assay prospective clinical studies included a total of 1,401 prospectively collected NPS specimens and 978 NS specimens during the 2024-2025 respiratory viral season. Forty NPS specimens and forty-six NS specimens were not evaluable due to protocol deviations, sample handling issues and/or invalid reference testing result. The number and percentage of cases positive for SARS-CoV-2, influenza A, influenza B, and RSV, as determined by the Simplexa COVID-19/ Flu A/B & RSV Direct assay from NPS samples, are presented in Tables 19 - 20, stratified by collection site and age. Tables 21 -22 below shows the expected values for NS specimens, as determined by the Simplexa COVID-19/ Flu A/B & RSV Direct assay, stratified by the collection site and patient's age.
Table 19. Simplexa COVID-19/FLU A/B & RSV Direct Expected Values for Prospective NPS Specimens by Site (N=1353) $^{1}$
| | Site 01 (N = 189) | | Site 02 (N = 127) | | Site 03 (N = 27) | | Site 04 (N = 9) | | Site 06 (N = 141) | | Site 09 (N = 308) | | Site 10 (N = 144) | | Site 11 (N = 398) | | Site 13 (N = 10) | | Total (N = 1353) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) |
| Flu A | 58 | 30.7% (58/189) | 24 | 18.9% (24/127) | 18 | 66.7% (18/27) | 2 | 22.2% (2/9) | 45 | 31.9% (45/141) | 79 | 25.6% (79/308) | 14 | 9.7% (14/144) | 128 | 32.2% (128/398) | 1 | 10.0% (1/10) | 369 | 27.3% (369/1353) |
| Flu B | 7 | 3.7% (7/189) | 11 | 8.7% (11/127) | 0 | 0.0% (0/27) | 0 | 0.0% (0/9) | 3 | 2.1% (3/141) | 0 | 0.0% (0/308) | 17 | 11.8% (17/144) | 9 | 2.3% (9/398) | 0 | 0.0% (0/10) | 47 | 3.5% (47/1353) |
| RSV | 2 | 1.1% (2/184) | 7 | 5.5% (7/127) | 1 | 3.7% (1/27) | 0 | 0.0% (0/9) | 13 | 9.2% (13/141) | 12 | 3.9% (12/307) | 20 | 13.9% (20/144) | 46 | 11.6% (46/397) | 0 | 0.0% (0/10) | 101 | 7.5% (101/1346) |
| SARS-CoV-2 | 5 | 2.6% (5/189) | 5 | 3.9% (5/127) | 1 | 3.7% (1/27) | 0 | 0.0% (0/9) | 8 | 5.7% (8/141) | 21 | 6.8% (21/308) | 2 | 1.4% (2/144) | 25 | 6.3% (25/398) | 1 | 10.0% (1/10) | 68 | 5.0% (68/1353) |
1Eight (8) NPS specimens returned invalid results on the Simplexa COVID-19/ FLU A/B & RSV Direct assay and are excluded from this analysis.
Table 20. Simplexa COVID-19/FLU A/B & RSV Direct Expected Values for Prospective NPS Specimens by Age (N=1353) $^{1}$
| | <=5 Years (N=506) | 6-18 Years (N=483) | 19-40 Years (N=153) | 41-60 Years (N=100) | 61+ Year (N=105) | Unknown (N=6) | Overall (N=1353) |
| --- | --- | --- | --- | --- | --- | --- | --- |
K252387 - Page 27 of 29
{27}
| Target | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Flu A | 135 | 26.7% (135/506) | 153 | 31.7% (153/483) | 35 | 22.9% (35/153) | 26 | 26.0% (26/100) | 19 | 18.1% (19/105) | 1 | 16.7% (1/6) | 369 | 27.3% (369/1353) |
| Flu B | 12 | 2.4% (12/506) | 33 | 6.8% (33/483) | 2 | 1.3% (2/153) | 0 | 0.0% (0/100) | 0 | 0.0% (0/105) | 0 | 0.0% (0/6) | 47 | 3.5% (47/1353) |
| RSV | 73 | 14.5% (73/505) | 17 | 3.5% (17/479) | 3 | 2.0% (3/152) | 2 | 2.0% (2/100) | 5 | 4.8% (5/104) | 1 | 16.7% (1/6) | 101 | 7.5% (101/1346) |
| SARS-CoV-2 | 27 | 5.3% (27/506) | 16 | 3.3% (16/483) | 6 | 3.9% (6/153) | 6 | 6.0% (6/100) | 12 | 11.4% (12/105) | 1 | 16.7% (1/6) | 68 | 5.0% (68/1353) |
$^{1}$ Eight (8) NPS specimens returned invalid results on the Simplexa COVID-19 / FLU A/B & RSV Direct assay and are excluded from this analysis.
Table 21. Simplexa COVID-19/FLU A/B & RSV Direct Expected Values for Prospective NS Specimens by Site $(\mathrm{N} = 930)^{1}$
| | Site 01 (N = 190) | | Site 02 (N = 161) | | Site 03 (N = 139) | | Site 04 (N = 68) | | Site 06 (N = 149) | | Site 09 (N = 3) | | Site 13 (N = 220) | | Total (N = 930) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) |
| Flu A | 58 | 30.5% (58/190) | 36 | 22.4% (36/161) | 55 | 39.6% (55/139) | 11 | 16.2% (11/68) | 52 | 34.9% (52/149) | 0 | 0.0% (0/3) | 51 | 23.2% (51/220) | 263 | 28.3% (263/930) |
| Flu B | 8 | 4.2% (8/190) | 15 | 9.3% (15/161) | 1 | 0.7% (1/139) | 0 | 0.0% (0/68) | 3 | 2.0% (3/149) | 0 | 0.0% (0/3) | 3 | 1.4% (3/220) | 30 | 3.2% (30/930) |
| RSV | 3 | 1.6% (3/190) | 10 | 6.3% (10/160) | 7 | 5.0% (7/139) | 1 | 1.5% (1/68) | 15 | 10.3% (15/145) | 0 | 0.0% (0/3) | 18 | 8.2% (18/219) | 54 | 5.8% (54/924) |
| SARS-CoV-2 | 4 | 2.1% (4/190) | 5 | 3.1% (5/161) | 8 | 5.8% (8/139) | 8 | 11.8% (8/68) | 10 | 6.7% (10/149) | 0 | 0.0% (0/3) | 12 | 5.5% (12/220) | 47 | 5.1% (47/930) |
$^{1}$ Two (2) NS specimens returned invalid results on the Simplexa COVID-19 / FLU A/B & RSV Direct assay and are excluded from this analysis.
Table 22. Simplexa COVID-19/FLU A/B & RSV Direct Expected Values for Prospective NS Specimens by Age $(\mathrm{N} = 930)^{1}$
| | <=5 Years (N=241) | | 6-18 Years (N=416) | | 19-40 Years (N=137) | | 41-60 Years (N=94) | | 61+ Year (N=42) | | Overall (N=930) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) | #Pos | (%) |
| Flu A | 83 | 34.4% (83/241) | 133 | 32.0% (133/416) | 28 | 20.4% (28/137) | 12 | 12.8% (12/94) | 7 | 16.7% (7/42) | 263 | 28.3% (263/930) |
| Flu B | 2 | 0.8% (2/241) | 24 | 5.8% (24/416) | 2 | 1.5% (2/137) | 2 | 2.1% (2/94) | 0 | 0.0% (0/42) | 30 | 3.2% (30/930) |
| RSV | 28 | 11.7% (28/240) | 18 | 4.4% (18/413) | 2 | 1.5% (2/135) | 1 | 1.1% (1/94) | 5 | 11.9% (5/42) | 54 | 5.8% (54/924) |
| SARS-CoV-2 | 9 | 3.7% (9/241) | 16 | 3.8% (16/416) | 9 | 6.6% (9/137) | 8 | 8.5% (8/94) | 5 | 11.9% (5/42) | 47 | 5.1% (47/930) |
$^{1}$ Two (2) NS specimens returned invalid results on the Simplexa COVID-19 / FLU A/B & RSV Direct assay and are excluded from this analysis.
# F Other Supportive Instrument Performance Characteristics Data:
Not applicable.
# VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.
K252387 - Page 28 of 29
{28}
IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
K252387 - Page 29 of 29
