NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2)
Device Facts
| Record ID | K231758 |
|---|---|
| Device Name | NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2) |
| Applicant | Luminex Molecular Diagnostics, Inc. |
| Product Code | QOF · Microbiology |
| Decision Date | Mar 11, 2024 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3981 |
| Device Class | Class 2 |
| Attributes | Real-World Evidence, Pediatric |
Real-World Evidence
| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
|---|---|---|---|---|---|
| K231758 · Mar 11, 2024 | NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2) | Luminex Molecular Diagnostics, Inc. | Archived clinical nasopharyngeal swab specimens | Archived clinical specimens were used to supplement the clinical performance evaluation for analytes with low prevalence in the prospective clinical study. | Archived clinical specimens; Clinical performance; Respiratory pathogens |
Clinical Evidence
| Study Design | Population | Comparator | Key Endpoints |
|---|---|---|---|
| Archived Clinical Specimen Study; Retrospective analysis of preselected, archived clinical specimens | Patients exhibiting signs and symptoms of respiratory tract infection; Sample Size: 308; Number of Sites: 6 | PCR/bidirectional sequencing | Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) |
Indications for Use
The NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2) is a multiplexed polymerase chain reaction (PCR) test intended for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19. The following organism types and subtypes are identified and differentiated using the NxTAG RPP v2: Viral Targets: Influenza A, Influenza A H1, Influenza A H1pdm09, Influenza B, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B, SARS-CoV-2, Coronavirus 229E, Coronavirus OC43, Coronavirus NL63, Coronavirus HKU1, Human Metapneumovirus, Rhinovirus, Adenovirus, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4 Bacterial Targets: Chlamydia pneumoniae, Mycoplasma pneumoniae Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in nasopharyngeal specimens during the acute phase of infection. The detection of specific viral and bacterial nucleic acids from individuals exhibiting signs and or symptoms of respiratory infection are indicative of the identified microorganism 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 management decisions. Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the NxTAG RPP v2 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. The NxTAG® Respiratory Pathogen Panel v2 is indicated for use with the Luminex® MAGPIX® Instrument and xPONENT® and SYNCTTM software.
Device Story
NxTAG RPP v2 is a multiplexed RT-PCR assay for qualitative detection of 21 respiratory pathogens in nasopharyngeal swabs. Samples undergo nucleic acid extraction (bioMérieux easyMAG/EMAG); extracted nucleic acids are added to 96-well plates containing lyophilized PCR primers, hybridization beads, and amplification enzymes. Following thermocycling, amplified products hybridize to tagged beads; the MAGPIX instrument performs automated bead sorting and fluorescence reading. Data are analyzed via SYNCT software to report 'detected', 'not detected', or 'invalid' results. Used in clinical laboratories by trained personnel. Output aids diagnosis when combined with clinical/epidemiological data. Benefits include rapid, simultaneous identification of multiple pathogens, facilitating patient management and infection control.
Clinical Evidence
Prospective clinical study (n=1820) and pre-selected specimen study (n=308) compared NxTAG RPP v2 to FDA-cleared molecular assays and PCR/sequencing. Contrived specimens (n=199) supplemented low-prevalence targets. Performance metrics (PPA/NPA) were calculated for all 21 targets. Overall PPA/NPA were high, supporting diagnostic accuracy for respiratory tract infection detection.
Technological Characteristics
Multiplex RT-PCR assay using universal tag sorting system. Materials: Lyophilized Bead Reagents (LBRs), MagPlex microspheres. Energy: Thermal cycler for amplification, MAGPIX for fluorescence detection. Connectivity: Standalone instrument (MAGPIX) with xPONENT/SYNCT software. Sterilization: N/A (reagents). Software: Rule-based qualitative calling algorithm.
Indications for Use
Indicated for individuals with signs/symptoms of respiratory tract infection (including COVID-19) for qualitative detection of 21 viral/bacterial pathogens in nasopharyngeal swabs. 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
K231758
B Applicant
Luminex Molecular Diagnostics, Inc.
C Proprietary and Established Names
NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2)
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:
To obtain a substantial equivalence determination for the NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2).
B Measurand:
The NxTAG RPP v2 detects and identifies nucleic acids from the following pathogens:
Adenovirus, Coronavirus 229E, Coronavirus OC43, Coronavirus NL63, Coronavirus HKU1,
Human Metapneumovirus, Influenza A virus with subtyping of Influenza A H1, Influenza A
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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H1pdm09 and Influenza A H3 (reported separately), Influenza B virus, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B, Rhinovirus/Enterovirus, Severe Acute Respiratory Syndrome (SARS)-Coronavirus-2, *Chlamydia pneumoniae* and *Mycoplasma pneumoniae*.
### C Type of Test:
Multiplex nucleic acid assay for use with the MAGPIX Instrument for the qualitative detection of viral and/or bacterial pathogens in nasopharyngeal swab specimens from patients with signs and symptoms of respiratory tract infection.
### III Intended Use/Indications for Use:
#### A Intended Use(s):
See Indications for Use below.
#### B Indication(s) for Use:
The NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2) is a multiplexed polymerase chain reaction (PCR) test intended for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19.
The following organism types and subtypes are identified and differentiated using the NxTAG RPP v2:
| Viral Targets | |
| --- | --- |
| Influenza A | Coronavirus NL63 |
| Influenza A H1 | Coronavirus HKU1 |
| Influenza A H1pdm09 | Human Metapneumovirus |
| Influenza A H3 | Rhinovirus/Enterovirus |
| Influenza B | Adenovirus |
| Respiratory Syncytial Virus A | Parainfluenza virus 1 |
| Respiratory Syncytial Virus B | Parainfluenza virus 2 |
| SARS-CoV-2 | Parainfluenza virus 3 |
| Coronavirus 229E | Parainfluenza virus 4 |
| Coronavirus OC43 | |
| Bacterial Targets | |
| *Chlamydia pneumoniae* | *Mycoplasma pneumoniae* |
Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in nasopharyngeal 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 are indicative of the presence of the identified microorganism and aids in diagnosis if used in conjunction with other clinical and epidemiological information,
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and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the NxTAG RPP v2 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.
The NxTAG Respiratory Pathogen Panel v2 is indicated for use with the Luminex MAGPIX Instrument and xPONENT and SYNCT software.
# C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
For in vitro diagnostic use
# D Special Instrument Requirements:
Luminex MAGPIX Instrument
bioMérieux NUCLISENS easyMAG Instrument or EMAG Instrument
IVD-Labeled Thermal Cycler
# IV Device/System Characteristics:
# A Device Description:
The NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2) is a qualitative reverse-transcription polymerase chain reaction (RT-PCR) in vitro diagnostic assay for the simultaneous detection and discrimination of 21 viral and bacterial respiratory pathogens in nasopharyngeal swab specimens from patients with signs and symptoms of respiratory tract infection. The assay uses the proprietary Luminex bead hybridization tag sorting system to discriminate amplified products and is performed on the Luminex MAGPIX Instrument equipped with xPONENT software following nucleic acid extraction and PCR amplification using off-the-shelf instrument systems. Signals obtained with the MAGPIX Instrument are analyzed using the NxTAG RPP v2 Assay File for SYNCT Software.
The NxTAG RPP v2 differs from the original NxTAG Respiratory Pathogen Panel (NxTAG RPP; K152386/K193167) as shown in Table 1. Other minor differences include changes to the fluorescence thresholds for certain assays and the Internal Control.
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Table 1. Changes to the NxTAG RPP implemented for the NxTAG RPP v2
| Target Analyte | Description of Change for NxTAG RPP v2 |
| --- | --- |
| Influenza A | Modified primers for improved inclusivity |
| Influenza A H1 | Differentiation of subtypes H1 and H1pdm09 |
| Influenza A H3 | Modified primers for improved inclusivity |
| Human Bocavirus | Results not reported |
| Human Metapneumovirus | New target region for improved inclusivity |
| Rhinovirus/Enterovirus | Modified formulation for improved specificity |
| SARS-CoV-2 | New analyte |
### B Principle of Operation:
Nucleic acids are extracted for analysis from nasopharyngeal swab samples in transport medium using either the IVD-labeled bioMérieux NucliSENS easyMAG or EMAG extraction systems. The extracted nucleic acids are added to 96-well plates containing lyophilized PCR primers, hybridization beads and amplification enzymes. Following rehydration, the plates are sealed and loaded into a thermocycler for reverse transcription and PCR amplification. The amplified PCR products hybridize to the tagged beads in near real-time and upon completion of the reaction, the sealed plate is loaded into the MAGPIX Instrument for automated bead sorting and fluorescence reading using xPONENT 4.3u2 software. The data are analyzed using the NxTAG RPP v2 Assay File for SYNCT 1.1u2 software and results for each analyte are reported as “detected”, “not detected” or “invalid”. Details of the method of result interpretation for influenza A and its associated subtypes are provided in Table 2.
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**Table 2.** Method of result interpretation for influenza A
| Final Result | NxTAG RPP v2 Analyte | | | | Applicable Footnotes |
| --- | --- | --- | --- | --- | --- |
| | Influenza A | Influenza A H1 | Influenza A H1pdm09 | Influenza A H3 | |
| Influenza A Not Detected | Negative | Negative | Negative | Negative | -- |
| Influenza A H1 | Positive | Positive | Negative | Negative | -- |
| | Negative | Positive | Negative | Negative | 1 |
| Influenza A 2009 H1N1 | Positive | Negative | Positive | Negative | -- |
| | Negative | Negative | Positive | Negative | 1 |
| Influenza A H1 and Influenza A H1pdm09 | Positive | Positive | Positive | Negative | 2 |
| | Negative | Positive | Positive | Negative | 1, 2 |
| Influenza A H3 | Positive | Negative | Negative | Positive | -- |
| | Negative | Negative | Negative | Positive | 1 |
| Influenza A H3 and Influenza A H1 | Positive | Positive | Negative | Positive | 2 |
| | Negative | Positive | Negative | Positive | 1, 2 |
| Influenza A H3 and Influenza A H1pdm09 | Positive | Negative | Positive | Positive | 2 |
| | Negative | Negative | Positive | Positive | 1, 2 |
| Influenza A H1, Influenza A H1pdm09 and Influenza A H3 | Positive | Positive | Positive | Positive | 2 |
| | Negative | Positive | Positive | Positive | 1 |
| Influenza A (no sub-type detected) | Positive | Negative | Negative | Negative | 3 |
$^{1}$ Detection of influenza A H1, A H1pdm09 or A H3 subtypes without a corresponding positive result for influenza A may occur at low target levels, be due to contamination or be indicative of a mutation in the matrix gene target region used for detection of influenza A. Retesting is recommended prior to reporting test results if clinically indicated or for epidemiological investigation. Further investigation may be warranted if the same result is generated upon retesting.
$^{2}$ Co-detection of multiple influenza A subtypes (H1 and/or H1pdm09 and/or H3) may be due to coinfection, false-positive results due to contamination or the presence of a multi-valent influenza virus vaccine in the sample. Retesting is recommended prior to reporting test results. If the same result is obtained upon retest, the presence of multiple subtypes should be confirmed by alternative FDA-cleared methods.
$^{3}$ Detection of influenza A without a corresponding positive result for an influenza A subtype may occur at low target levels, be due to contamination or be indicative of the presence of a novel influenza A strain. If a specimen is confirmed positive for influenza A on retesting but produces negative test results for A H1, A H1pdm09 and A H3, the appropriate local, state, or federal public health authorities should be notified to determine necessary measures for verification and to determine whether the specimen contains a novel strain of Influenza A.
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# C Instrument Description Information:
# 1. Instrument Name:
Luminex MAGPIX Instrument
bioMérieux NUCLISENS easyMAG Instrument or EMAG Instrument
IVD-labeled thermal cycler
# 2. Specimen Identification:
Specimen identification numbers may be entered into the SYNCT Software manually or using the optional barcode reader.
# 3. Specimen Sampling and Handling:
The NxTAG RPP v2 assay is intended for use with nasopharyngeal swabs collected in Universal Transport Medium (UTM, Copan Diagnostics) or MicroTest M4RT medium (Remel) using a nylon flocked or polyester swab. Specimens may be stored for up to 7 days at 2-8 °C or up to 12 months at ≤ -70 °C.
The respective locations of Positive and Negative External Controls and patient samples on the MAGPIX assay plate must be defined in the SYNCT Software. For controls, the expected results for each analyte must also be assigned. Each sample and control within a run must have a unique identifier. The locations of the External Controls should be selected to enable correct orientation of the MAGPIX plate.
The SYNCT software may be used to select which test results to report for each sample prior to analysis based on the test order. Masked results will not be reported. A default Test Panel that includes all available analytes is provided but, for convenience, users also have the option to create custom Test Panels from within the list of available NxTAG RPP v2 target analytes. Targets that were previously masked, may be unmasked by reanalyzing the data in the SYNCT software upon receipt of an appropriate test order.
# 4. Calibration:
Calibration of the MAGPIX Instrument using the MAGPIX Calibration Kit is required at least weekly to normalize the settings for the classification and reporter optical channels. Following calibration, the MAGPIX Performance Verification Kit must be used to verify the integrity of the system. Daily use of the MAGPIX Performance Verification Kit to verify the calibration and optical integrity of the MAGPIX system and the fluidics channels is also recommended. The MAGPIX Calibration Kit and MAGPIX Performance Verification Kit are provided separately from the NxTAG RPP v2.
# 5. Quality Control:
Prior to processing, bacteriophage MS2 is added to each patient sample or External Control as an Internal Control to monitor the integrity of nucleic acid extraction, reverse transcription, PCR amplification and detection. External Positive Controls for use with the
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NxTAG RPP v2 assay are not provided with the assay kit but are recommended for use in accordance with local, state and/or federal guidelines and good laboratory practices.
Luminex recommends inclusion of a Negative Extraction Control (transport medium) to monitor for contamination. Optionally, a Negative Amplification Control comprised of RNase-free water may be included on each amplification plate to monitor for contamination downstream from the nucleic acid extraction process. The Negative Extraction Control and Negative Amplification Control should produce negative test results for all analytes.
If the External Positive Control, Negative Amplification Control and Negative Extraction Control do not produce the expected results, patient results should not be reported.
## 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): | K231758 | DEN200031 |
| --- | --- | --- |
| Device Trade Name | NxTAG Respiratory Pathogen Panel v2 | BioFire Respiratory Panel 2.1 |
| **General Device Characteristic Similarities** | | |
| Intended Use/Indications For Use | The NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2) is a multiplexed polymerase chain reaction (PCR) test intended for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19. The following organism types and subtypes are | The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BioFire FilmArray 2.0 or BioFire FilmArray Torch systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19. The following organism types and subtypes are |
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| | identified and differentiated using the NxTAG RPP v2: *Viral targets:* Influenza A Influenza A H1 Influenza A H1pdm09 Influenza A H3 Influenza B Respiratory Syncytial Virus A Respiratory Syncytial Virus B SARS-CoV-2 Coronavirus 229E Coronavirus OC43 Coronavirus NL63 Coronavirus HKU1 Human Metapneumovirus Rhinovirus/Enterovirus Adenovirus Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Parainfluenza Virus 4 *Bacterial targets:* *Chlamydia pneumoniae* *Mycoplasma pneumoniae* Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in nasopharyngeal 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 | 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* (prxP) 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 |
| --- | --- | --- |
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| | respiratory infection are indicative of the presence of the identified microorganism 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. Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the NxTAG RPP v2 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. The NxTAG Respiratory Pathogen Panel v2 is indicated for use with the Luminex MAGPIX Instrument and xPONENT and SYNCT software. | the identified microorganism and aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by an NPS specimen. Positive results do not rule out 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. |
| --- | --- | --- |
| Specimen Type | Same | Nasopharyngeal swabs |
| Patient Population | Individuals with signs and symptoms of respiratory tract infection, including COVID-19 | Individuals suspected of respiratory tract infections, including COVID-19 |
| Organisms Detected | Same except for: | *Viruses:* Adenovirus |
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| | a) differentiation of Respiratory Syncytial Virus A and B b) omission of assays for *Bordetella pertussis* and *Bordetella parapertussis* | Coronavirus 229E Coronavirus HKU1 Coronavirus NL63 Coronavirus OC43 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Human Metapneumovirus Human Rhinovirus/Enterovirus Influenza A Virus Subtypes: H1, H3, H1-2009 Influenza B Virus Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Parainfluenza Virus 4 Respiratory Syncytial Virus *Bacteria:* *Bordetella parapertussis* *Bordetella pertussis* *Chlamydia pneumoniae* *Mycoplasma pneumoniae* |
| --- | --- | --- |
| Technology | Same | PCR amplification |
| **General Device Characteristic Differences** | | |
| System | Separate instruments for nucleic acid extraction, PCR amplification and detection | Integrated nucleic acid extraction, amplification and detection in a sealed vessel. |
| Assay Read | MAGPIX Instrument | BIOFIRE FilmArray 2.0 or BIOFIRE FilmArray Torch Systems |
| Detection | Hybridization of amplified products with fluorescently labeled beads, sorting of tagged products | Array-based melt curve analysis |
### VI Standards/Guidance Documents Referenced:
CLSI. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline. 3rd ed. CLSI document EP05-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
CLSI. Interference Testing in Clinical Chemistry. 3rd ed. CLSI guideline EP07. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
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CLSI. *User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline*. 2$^{nd}$ ed. CLSI document EP12-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
CLSI. *Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline*. 2$^{nd}$ ed. CLSI document EP17-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
CLSI. *Assessment of the Diagnostic Accuracy of Laboratory Tests Using Receiver Operating Characteristic Curves; Approved Guideline*. 2$^{nd}$ ed. CLSI document EP24-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2011.
CLSI. *Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline*. CLSI document EP25-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
CLSI. *Supplementary Tables for Interference Testing in Clinical Chemistry*. 1$^{st}$ ed. CLSI supplement EP37. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
ISO 14971: 2019. Medical devices – Application of risk management to medical devices.
BS EN ISO 23640: 2015. *In vitro* diagnostic medical devices – Evaluation of stability of *in vitro* diagnostic reagents.
CLSI. *Molecular Diagnostic Methods for Infectious Diseases*. 3$^{rd}$ ed. CLSI report MM03. Wayne, PA: Clinical and Laboratory Standards Institute; 2015.
CLSI. *Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline*. 2$^{nd}$ ed. CLSI document MM09-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
CLSI. *Validation and Verification of Multiplex Nucleic Acid Assays*. 2$^{nd}$ ed. CLSI guideline MM17. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
CLSI. *Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline*. CLSI document MM18-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
## VII Performance Characteristics (if/when applicable):
### A Analytical Performance:
#### 1. Precision/Reproducibility:
##### *Lot-to-Lot and Within-Run Precision*
The lot-to-lot precision of the NxTAG RPP v2 was evaluated using three lots of assay kits, each containing unique lots of critical raw materials/sub-components. The panel of samples for analysis comprised simulated nasopharyngeal swab matrix that was spiked with different combinations of all the NxTAG RPP v2 analytes, as well as a negative sample (**Table 3**). Please refer to **Section VII A (6)** for data supporting use of simulated nasopharyngeal swab matrix for Analytical Studies, as well as testing of samples containing multiple analytes.
Each positive panel member was prepared at both low and moderate target levels (1.5X [low] and 5X LoD [moderate], respectively, except where noted). Each panel member at each
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concentration was tested a total of 10 times with each lot of reagents (total 30 replicates per panel member per target level). There was close to 100% positive and negative agreement with the expected results for each panel member at each target level with all three lots of reagents (Table 4). These results are acceptable.
Table 3. Panel members used to evaluate lot-to-lot assay precision
| Sample | Analyte | Strain/Isolate | Source | Catalogue # |
| --- | --- | --- | --- | --- |
| 1 | Influenza A H1pdm09 (subtype) ^{1,2,3} | A/NY/02/09 | ZeptoMetrix | 0810109CFN |
| | Respiratory Syncytial Virus A | A2 | ATCC | VR-1540 |
| | Rhinovirus | 50-525-CV54 | ATCC | VR-1195 |
| 2 | Influenza A H3 (subtype) ^{1,4} | Wisconsin/67/05 | ZeptoMetrix | 0810252CF |
| | Respiratory Syncytial Virus B | 18537 | ATCC | VR-1580 |
| 3 | Influenza B | B/Florida/02/06 | ZeptoMetrix | 0810037CF |
| | Parainfluenza Virus 3 | C 243 | ATCC | VR-93 |
| | *Mycoplasma pneumoniae*^{5} | M129 | ZeptoMetrix | 0801579 |
| 4 | SARS-CoV-2 | USA-WA1/2020 | ATCC | VR-1986HK |
| | Human Metapneumovirus | hMPV-3, Type B1, Peru2-2002 | ZeptoMetrix | 0810156CF |
| | Adenovirus B ^{2} | Type 14, 2006 isolate | ZeptoMetrix | 0810108CF |
| 5 | Influenza A H3 (matrix) | Wisconsin/67/05 | ZeptoMetrix | 0810252CF |
| | Coronavirus NL63 | -- | ZeptoMetrix | 0810228CF |
| | Coronavirus HKU1 | Genotype B | Clinical | -- |
| 6 | Influenza A H1 (subtype) ^{1,6} | A/Brisbane/59/07 | ZeptoMetrix | 0810244CF |
| | Parainfluenza Virus 1 | -- | ZeptoMetrix | 0810014CF |
| | *Chlamydia pneumoniae* | TW-183 | ATCC | VR-2282 |
| 7 | Parainfluenza Virus 2 | Greer | ATCC | VR-92 |
| | Parainfluenza Virus 4B | CH 19503 | ATCC | VR-1377 |
| | Coronavirus 229E | -- | ATCC | VR-740 |
| 8 | Parainfluenza Virus 4A | -- | ZeptoMetrix | 0810060CF |
| | Coronavirus OC43 | Betacoronavirus 1 | ATCC | VR-1558 |
| 9 | Negative | -- | -- | -- |
Samples 1-8 were formulated at 1.5X (Low) and 5X LoD (Moderate), except where noted.
$^{1}$ Formulated based on the LoD for the applicable influenza A subtype.
$^{2}$ Formulated at approximately 3X and 9X LoD.
$^{3}$ Expected results at the Low target level based on the respective analytical sensitivities of the H1pdm09 subtyping and influenza A assays: H1pdm09 subtype "positive", Influenza A "positive".
$^{4}$ Expected results at the Low target level based on the respective analytical sensitivities of the H3 subtyping and influenza A assays: H3 subtype "positive", Influenza A "positive" or "negative".
$^{5}$ Formulated at approximately 2X and 7X LoD.
$^{6}$ Expected results at the Low target level based on the respective analytical sensitivities of the H1 subtyping and influenza A assays: H1 subtype "positive", Influenza A "positive".
Table 4. Lot-to-lot agreement with expected results
| Sample | NxTAG RPP v2 Analyte | Level | Percent Agreement | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Lot 1 | Lot 2 | Lot 3 | Total |
| 1 | Influenza A H1pdm09 (subtype) ^{1,2} | Low ^{3} | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | | Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | Respiratory Syncytial Virus A | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | | Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | Rhinovirus/Enterovirus | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | | Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | All Other | Low | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) |
| | | Mod | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) |
| 2 | Influenza A H3 (subtype) ^{1} | Low ^{4} | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | | Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | Respiratory Syncytial Virus B | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | | Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| | All Other | Low | 100 (190/190) | 100 (190/190) | 100 (190/190) | 100 (570/570) |
| | | Mod | 100 (190/190) | 100 (190/190) | 100 (190/190) | 100 (570/570) |
| 3 | Influenza B | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
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| Sample | NxTAG RPP v2 Analyte | Level | Percent Agreement | |
| --- | --- | --- | --- | --- |
| Lot 1 | Lot 2 | Lot 3 | Total |
| | Parainfluenza virus 3 | Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| *Mycoplasma pneumoniae*^{5} | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| All Other | Low | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) | 100 (1080/1080) |
| Mod | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) |
| 4 | SARS-CoV-2 | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Human Metapneumovirus | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Adenovirus^{2} | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| All Other | Low | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) | 100 (1080/1080) |
| Mod | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) |
| 5 | Influenza A (matrix) | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Coronavirus NL63 | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Coronavirus HKU1 | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| All Other | Low | 100 (180/180) | 99.4 (179/180) | 100 (180/180) | 99.8 (539/540) | 99.9 (1079/1080) |
| Mod | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) |
| 6 | Influenza A H1 (subtype)^{1} | Low^{6} | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Parainfluenza virus 1 | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| *Chlamydia pneumoniae* | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| All Other | Low | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) | 99.9 (1079/1080) |
| Mod | 100 (180/180) | 99.4 (179/180) | 100 (180/180) | 99.8 (539/540) |
| 7 | Parainfluenza virus 2 | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Parainfluenza virus 4 | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Coronavirus 229E | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| All Other | Low | 100 (180/180) | 100 (180/180) | 99.4 (179/180) | 99.8 (539/540) | 99.9 (1079/1080) |
| Mod | 100 (180/180) | 100 (180/180) | 100 (180/180) | 100 (540/540) |
| 8 | Parainfluenza virus 4 | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| Coronavirus OC43 | Low | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) | 100 (60/60) |
| Mod | 100 (10/10) | 100 (10/10) | 100 (10/10) | 100 (30/30) |
| All Other | Low | 100 (190/190) | 100 (190/190) | 100 (190/190) | 100 (570/570) | 100 (1140/1140) |
| Mod | 100 (190/190) | 100 (190/190) | 100 (190/190) | 100 (570/570) |
| 9 | Negative | N/A | 99.0 (208/210) | 100 (210/210) | 100 (210/210) | 99.7 (628/630) | |
N/A: Not applicable; Low: 1.5X LoD; Mod (Moderate): 5X LoD, except where noted
$^{1}$ Formulated based on the LoD for the applicable influenza A subtype.
$^{2}$ Formulated at approximately 3X (Low) and 9X LoD (Moderate).
$^{3}$ Expected results at the Low target level based on the respective analytical sensitivities of the H1pdm09 subtyping and influenza A assays: H1pdm09 subtype 'positive', Influenza A 'positive'.
$^{4}$ Expected results at the Low target level based on the respective analytical sensitivities of the H3 subtyping and influenza A assays: H3 subtype 'positive', Influenza A 'positive' or 'negative'; agreement for Influenza A is included under 'All Other' (n = 10 per lot per level).
$^{5}$ Formulated at approximately 2X (Low) and 7X LoD (Moderate).
$^{6}$ Expected results at the Low target level based on the respective analytical sensitivities of the H1 subtyping and influenza A assays: H1 subtype 'positive', Influenza A 'positive'.
### Site-to-Site and Within-Site (Operator-to-Operator) Reproducibility
The site-to-site and within site (operator-to-operator) reproducibility of the NxTAG RPP v2 was evaluated using a panel of samples that were tested by two operators at each of three study sites in replicates of four on five non-consecutive days, using a single reagent lot and unique set of critical equipment at each site (2 operators x 3 sites x 4 replicates x 5 days x 1
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lot = 120 replicates per panel member). The panel of samples for analysis comprised simulated nasopharyngeal swab matrix that was spiked with different combinations of a subset of 11 of the NxTAG RPP v2 target analytes, as well as a negative sample (Table 5). Each positive panel member was prepared at both low and moderate target levels (1.5X and 5X LoD, respectively, except where noted). Positive and negative agreement for each panel member within and between study sites and operators was close to 100% (Table 6). These results are acceptable.
Table 5. Panel members used to evaluate site-to-site and within-site reproducibility
| Sample | Analyte | Strain/Isolate | Source | Catalogue # |
| --- | --- | --- | --- | --- |
| 1 | Influenza A H1pdm09 (subtype) ^{1,2,3} | A/NY/02/09 | ZeptoMetrix | 0810109CFN |
| | Respiratory Syncytial Virus A | A2 | ATCC | VR-1540 |
| | Rhinovirus | 50-525-CV54 | ATCC | VR-1195 |
| 2 | Influenza A H3 (subtype) ^{1,4} | Wisconsin/67/05 | ZeptoMetrix | 0810252CF |
| | Respiratory Syncytial Virus B | 18537 | ATCC | VR-1580 |
| 3 | Influenza B | B/Florida/02/06 | ZeptoMetrix | 0810037CF |
| | Parainfluenza Virus 3 | C 243 | ATCC | VR-93 |
| | *Mycoplasma pneumoniae*^{5} | M129 | ZeptoMetrix | 0801579 |
| 4 | SARS-CoV-2 | USA-WA1/2020 | ATCC | VR-1986HK |
| | Human Metapneumovirus | hMPV-3, Type B1, Peru2-2002 | ZeptoMetrix | 0810156CF |
| | Adenovirus B ^{2} | Type 14, 2006 isolate | ZeptoMetrix | 0810108CF |
| 5 | Negative | -- | -- | -- |
Samples 1-4 were formulated at both 1.5X (Low) and 5X LoD (Moderate), except where noted
$^{1}$ Formulated based on the LoD for the applicable influenza A subtype.
$^{2}$ Formulated at approximately 3X (Low) and 9X LoD (Moderate).
$^{3}$ Expected results based on the relative analytical sensitivities of the H1pdm09 subtyping and influenza A assays: H1pdm09 subtype "positive", Influenza A "positive".
$^{4}$ Expected results based on the relative analytical sensitivities of the H3 subtyping and influenza A assays: H3 subtype "positive", Influenza A "positive" or "negative".
$^{5}$ Formulated at approximately 2X (Low) and 7X LoD (Moderate).
Table 6. Site-to-site agreement with expected results
| Sample | NxTAG RPP v2 Analyte | Panel Level | Percent Agreement | Total |
| --- | --- | --- | --- | --- |
| Site 1 | Site 2 | Site 3 |
| Operator 1 | Operator 2 | Operator 1 | Operator 2 | Operator 1 | Operator 2 |
| 1 | Influenza A H1pdm09 (subtype) ^{1,2,3} | Low | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (120/120) |
| 100 (40/40) | 100 (40/40) | 100 (40/40) |
| Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (120/120) |
| 100 (40/40) | 100 (40/40) | 100 (40/40) |
| Respiratory Syncytial Virus A | Low | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (120/120) |
| 100 (40/40) | 100 (40/40) | 100 (40/40) |
| Rhinovirus/Enterovirus | Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (120/120) |
| 100 (40/40) | 100 (40/40) | 100 (40/40) |
| Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (120/120) |
| 100 (40/40) | 100 (40/40) | 100 (40/40) |
| All Other | Low | 99.7 (359/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (2159/2160) |
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| Sample | NxTAG RPP v2 Analyte | Panel Level | Percent Agreement | Total |
| --- | --- | --- | --- | --- |
| Site 1 | Site 2 | Site 3 |
| Operator 1 | Operator 2 | Operator 1 | Operator 2 | Operator 1 | Operator 2 |
| | | | **99.9** (719/720) | **100** (720/720) | **100** (720/720) | |
| | | Mod | 99.7 (359/360) | 99.7 (359/360) | 100 (360/360) | 100 (360/360) | 99.7 (359/360) | 100 (360/360) | **99.9** (2157/2160) |
| | | | **99.7** (718/720) | **100** (720/720) | **99.9** (719/720) | |
| **2** | Influenza A H3 (subtype)^{1,4} | Low | 95.0 (19/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **99.2** (119/120) |
| **97.5** (39/40) | **100** (40/40) | **100** (40/40) | |
| Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| **100** (40/40) | **100** (40/40) | **100** (40/40) | |
| Respiratory Syncytial Virus B | Low | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| **100** (40/40) | **100** (40/40) | **100** (40/40) | |
| Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| **100** (40/40) | **100** (40/40) | **100** (40/40) | |
| All Other | Low | 99.5 (378/380) | 99.7 (379/380) | 100 (380/380) | 100 (380/380) | 100 (380/380) | 100 (380/380) | **99.9** (2277/2280) |
| **99.6** (757/760) | **100** (760/760) | **100** (760/760) | |
| Mod | 99.7 (379/380) | 99.5 (378/380) | 100 (380/380) | 100 (380/380) | 100 (380/380) | 100 (380/380) | **99.9** (2277/2280) |
| **99.6** (757/760) | **100** (760/760) | **100** (760/760) | |
| **3** | Influenza B | Low | 95.0 (19/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **99.2** (119/120) |
| **97.5** (39/40) | **100** (40/40) | **100** (40/40) | |
| Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| **100** (40/40) | **100** (40/40) | **100** (40/40) | |
| Parainfluenza virus 3 | Low | 95.0 (19/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **99.2** (119/120) |
| **97.5** (39/40) | **100** (40/40) | **100** (40/40) | |
| Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| **100** (40/40) | **100** (40/40) | **100** (40/40) | |
| *Mycoplasma pneumoniae*^{5} | Low | 95.0 (19/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **99.2** (119/120) |
| **97.5** (39/40) | **100** (40/40) | **100** (40/40) | |
| Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| **100** (40/40) | **100** (40/40) | **100** (40/40) | |
| All Other (Negative) | Low | 99.5 (358/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | **99.9** (2158/2160) |
| **99.7** (718/720) | **100** (720/720) | **100** (720/720) | |
| Mod | 99.7 (359/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | **100** (2159/2160) |
| **99.9** (719/720) | **100** (720/720) | **100** (720/720) | |
| **4** | SARS-CoV-2 | Low | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| **100** (40/40) | **100** (40/40) | **100** (40/40) | |
| Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
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| Sample | NxTAG RPP v2 Analyte | Panel Level | Percent Agreement | | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Site 1 | | Site 2 | | Site 3 | | |
| | | | Operator 1 | Operator 2 | Operator 1 | Operator 2 | Operator 1 | Operator 2 | |
| | | | **100** (40/40) | | **100** (40/40) | | **100** (40/40) | | |
| | Human Metapneumovirus | Low | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| | | | **100** (40/40) | | **100** (40/40) | | **100** (40/40) | | |
| | | Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| | | | **100** (40/40) | | **100** (40/40) | | **100** (40/40) | | |
| | Adenovirus^{2} | Low | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| | | | **100** (40/40) | | **100** (40/40) | | **100** (40/40) | | |
| | | Mod | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | 100 (20/20) | **100** (120/120) |
| | | | **100** (40/40) | | **100** (40/40) | | **100** (40/40) | | |
| | All Other (Negative) | Low | 99.5 (358/360) | 99.5 (358/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | **99.8** (2156/2160) |
| | | | **99.5** (716/720) | | **100** (720/720) | | **100** (720/720) | | |
| | | Mod | 99.7 (359/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | 100 (360/360) | **100** (2159/2160) |
| | | | **99.9** (719/720) | | **100** (720/720) | | **100** (720/720) | | |
| 5 | Negative | N/A | 100 (420/420) | 100 (420/420) | 100 (420/420) | 100 (420/420) | 100 (420/420) | 100 (420/420) | **100** (2520/2520) |
| | | | **100** (840/840) | | **100** (840/840) | | **100** (840/840) | | |
Low: 1.5X LoD; Mod (Moderate): 5X LoD, except where noted
Neg.: Negative; N/A: Not applicable
$^{1}$ Formulated based on the LoD for the applicable influenza A subtype.
$^{2}$ Formulated at approximately 3X (Low) and 9X LoD (Moderate).
$^{3}$ Expected results based on the respective analytical sensitivities of the H1pdm09 subtyping and influenza A assays: H1pdm09 subtype 'positive', Influenza A 'positive'.
$^{4}$ Expected results based on the relative analytical sensitivities of the H3 subtyping and influenza A assays: H3 subtype 'positive', Influenza A 'positive' or 'negative'; agreement for Influenza A is included under 'All Other' (n = 40 per site).
$^{5}$ Formulated at approximately 2X (Low) and 7X LoD (Moderate).
## 2. Linearity:
Not applicable.
## 3. Analytical Specificity/Interference:
### *In silico Analysis*
An *in silico* BLAST analysis was performed to evaluate the potential for cross-reaction of the NxTAG RPP v2 oligonucleotides with nucleic acids from on- and off-panel species. The analysis was performed with all the primers and probes in the assay mixture against sequences in the GenBank nt sequence database as of April 9, 2023. Prediction of potential cross-reaction/interference was based on the percentage homology with the non-target sequences, the relative locations of potential hybridization (amplicon length), the location of any mismatches and predicted T$_{m}$. Based on this analysis, the NxTAG RPP v2 SARS-CoV-2 primers and probe were determined to cross-react with some bat coronavirus and bat-SARS-like coronavirus strains. In addition, the Coronavirus 229E primers and probe are predicted to cross-react with 229E-like coronavirus sequences from bats. This observation was confirmed empirically using synthetic template. The potential for cross-reaction of the Coronavirus
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NL63 primers and probes with bat-coronaviruses was demonstrated in silico but was not confirmed through functional testing. Overall, in silico analysis demonstrated low likelihood for cross-reaction of the NxTAG RPP v2 assay primers and probes with non-target nucleic acids. However, the potential for cross-reaction with some bat SARS-like and 229E-like coronavirus strains is noted as a Limitation in the device labeling.
# Laboratory Testing of Analytical Specificity
The analytical specificity of the NxTAG RPP v2 was evaluated by testing high concentrations of potentially cross-reactive on- and off-panel bacteria, fungi and viruses, in addition to pooled nasal wash. No unexpected cross-reaction was observed except with Enterovirus 68 (Species D, US/IL/14-18952), which produced false-positive results for Influenza A H3 (Tables 7 and 8). This is noted as a Limitation in the device labeling.
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Table 7. Off-panel bacteria, fungi and viruses evaluated for potential cross-reaction
| Species | Strain/Isolate | Source/Catalogue No. | Concentration Per mL | Cross-reaction |
| --- | --- | --- | --- | --- |
| Bordetella parapertussis | A747 | Zeptometrix 0801641 | 1.00 x 10⁶ CFU | None¹ |
| Bordetella pertussis | A639 | Zeptometrix 0801459 | 1.00 x 10⁶ CFU | None |
| Chlamydia trachomatis | Z054; D-UW3; Serovar D | Zeptometrix 0801775 | 1.00 x 10⁶ IFU | None |
| Corynebacterium diphtheriae | Z116 | Zeptometrix 0801882 | 1.00 x 10⁶ CFU | None |
| Corynebacterium striatum | FS1 | Zeptometrix BAA-1851 | 1.00 x 10⁶ CFU | None |
| Escherichia coli | O157:H7; EDL 933 | Zeptometrix 0801622 | 1.00 x 10⁶ CFU | None |
| Fusobacterium necrophorum | Z239 | Zeptometrix 0804189 | 1.00 x 10⁶ CFU | None |
| Haemophilus influenzae | Type b; MinnA | Zeptometrix 0801680 | 1.00 x 10⁶ CFU | None |
| Klebsiella pneumoniae | Z135; IMP-13, CTX-M | Zeptometrix 0801904 | 1.00 x 10⁶ CFU | None |
| Lactobacillus acidophilus | Z048 | Zeptometrix 0801540 | 1.00 x 10⁶ CFU | None |
| Lactobacillus plantarum | Z524 | Zeptometrix 0804436 | 1.00 x 10⁶ CFU | None |
| Legionella (Tatlockia) micdadei | Tatlock | Zeptometrix 0801576 | 1.00 x 10⁶ CFU | None |
| Legionella pneumophila | Philadelphia | Zeptometrix 0801645 | 1.00 x 10⁶ CFU | None |
| Moraxella catarrhalis | Strain NE 11 | Zeptometrix 0801509 | 1.00 x 10⁶ CFU | None |
| Mycobacterium tuberculosis | H37Rx-1 | Zeptometrix 0801660 | 1.00 x 10⁶ CFU | None |
| Mycoplasma genitalium | UMTB-10G | ATCC 49899 | 1.00 x 10⁵ CCU² | None |
| Mycoplasma hominis | Z317 | Zeptometrix 0804011 | 1.00 x 10⁶ CCU | None |
| Neisseria elongate | Z071 | Zeptometrix 0801510 | 1.00 x 10⁶ CFU | None |
| Neisseria gonorrhoeae | Z017 | Zeptometrix 0801482 | 1.00 x 10⁶ CFU | None |
| Neisseria meningitidis | Serotype A | Zeptometrix 0801511 | 1.00 x 10⁶ CFU | None |
| Pseudomonas aeruginosa | Clinical isolate | Zeptometrix 0801519 | 1.00 x 10⁶ CFU | None |
| Serratia marcescens | Z053 | Zeptometrix 0801723 | 1.00 x 10⁶ CFU | None |
| Staphylococcus aureus | 102-04 | Zeptometrix BAA-1765 | 1.00 x 10⁶ CFU | None |
| Staphylococcus epidermidis | MRSE, RP62A | Zeptometrix 0801651 | 1.00 x 10⁶ CFU | None |
| Streptococcus agalactiae | Z019 | Zeptometrix 0801545 | 1.00 x 10⁶ CFU | None |
| Streptococcus pneumoniae | Z022 | Zeptometrix 0801439 | 1.00 x 10⁶ CFU | None |
| Streptococcus pyogenes | Z018 | Zeptometrix 0801512 | 1.00 x 10⁶ CFU | None |
| Streptococcus salivarius | Z127 | Zeptometrix 0801896 | 1.00 x 10⁶ CFU | None |
| Cytomegalovirus | Merlin | Zeptometrix 0810500CF | 1.00 x 10⁵ TCID₅₀ | None |
| Epstein Barr Virus | B95-8 | Zeptometrix 0810008CF | 1.00 x 10⁵ copies | None |
| Herpes Simplex virus Type 1 | Macintyre | Zeptometrix 0810005CF | 1.00 x 10⁵ TCID₅₀ | None |
| Human Bocavirus | -- | Clinical specimen | 1.00 x 10⁵ copies | None |
| Measles Virus | -- | Zeptometrix 0810025CF | 1.00 x 10⁵ TCID₅₀ | None |
| MERS-coronavirus | Florida/USA-2_Saudi_Arabia_2014 | Zeptometrix 0810575CFHI | 1.00 x 10⁵ TCID₅₀ | None |
| Mumps Virus | -- | Zeptometrix 0810079CF | 1.00 x 10⁵ TCID₅₀ | None |
| SARS-coronavirus | 2003-00592 | Zeptometrix NATSARS-ST | 3.01 x 10⁵ copies | None |
| Varicella Zoster Virus | Ellen | Zeptometrix 0810171CF | 1.00 x 10⁵ TCID₅₀ | None |
| Aspergillus flavus | Z013 | Zeptometrix 0801598 | 1.00 x 10⁶ CFU | None |
| Aspergillus fumigatus | QM 1981 | ATCC 1022 | 1.00 x 10⁶ CFU | None |
| Candida albicans | Z006 | Zeptometrix 0801504 | 1.00 x 10⁶ CFU | None |
| Pneumocystis carinii | -- | ATCC PRA-159 | 1.00 x 10⁶ nuclei | None |
| Pooled nasal wash | -- | -- | -- | None |
CCU: Color Changing Units; CFU: Colony Forming Units; IFU: Inclusion Forming Units; TCID₅₀: 50% Tissue Culture Infectious Dose
¹ On initial testing, 1/3 replicates gave a positive result for Rhinovirus/Enterovirus; upon repeat testing of the same samples, 3/3 replicates produced negative results for all analytes. A further 3/3 replicates prepared in negative nasopharyngeal swab matrix also produced negative results for all analytes.
² Approximate concentration based on supplier Certificate of Analysis.
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Table 8. On-panel viruses and bacteria evaluated for potential cross-reaction
| Analyte | Strain/Isolate | Source/Catalogue No. | Concentration per mL | Cross-reaction |
| --- | --- | --- | --- | --- |
| Adenovirus B | Type 14, 2006 isolate | Zeptometrix 0810300CF | 1.00 x 10^{5} TCID_{50} | None |
| Coronavirus 229E | -- | ATCC VR-740 | 1.00 x 10^{5} TCID_{50} | None |
| Coronavirus HKU1 | Genotype A | Clinical | 1.00 x 10^{6} copies | None |
| Coronavirus NL63 | -- | Zeptometrix 0810228CF | 1.00 x 10^{5} TCID_{50} | None |
| Coronavirus OC43 | -- | ATCC VR-1558 | 1.00 x 10^{5} TCID_{50} | None |
| Enterovirus | Species D, Type 68, 2014 Isolate 1 | Zeptometrix 0810300CF | 1.00 x 10^{5} TCID_{50} | None |
| Enterovirus | Species D, Type 68, 2007 Isolate | Zeptometrix 0810237CF | 1.00 x 10^{5} TCID_{50} | None |
| Enterovirus | Species D, Type 68, Fermon | ATCC VR-1826 | 1.00 x 10^{5} TCID_{50} | None |
| Enterovirus | Species D, Type 68, US/IL/14-18952 | ATCC VR-1824 | 1.00 x 10^{5} TCID_{50} | Influenza A H3^{1} |
| Enterovirus | Species D, Type 68, US/KY/14-18953 | ATCC VR-1825 | 1.00 x 10^{5} TCID_{50} | None |
| Enterovirus | Species D, Type 68, US/MO/14-18947 | ATCC VR-1823 | 1.00 x 10^{5} TCID_{50} | None |
| Human Metapneumovirus | 16, Type A1, IA10-2003 | Zeptometrix 0810161CF | 1.00 x 10^{5} TCID_{50} | None |
| Human Metapneumovirus | 3, Type B1, Peru2-2002 | Zeptometrix 0810156CF | 3.89 x 10^{4} TCID_{50}^{2} | None |
| Influenza A H1pdm09 | A/NY/02/09 | Zeptometrix 0810109CFN | 1.00 x 10^{5} TCID_{50} | None |
| Influenza A H1 | A/Brisbane/59/07 | Zeptometrix 0810244CF | 1.00 x 10^{5} TCID_{50} | None |
| Influenza A H3 | A/Wisconsin/67/05 | Zeptometrix 0810252CF | 1.00 x 10^{5} TCID_{50} | None |
| Influenza B | B/Bangladesh/5972/2007 | IRR FR-450 | 1.00 x 10^{5} TCID_{50} | None |
| Influenza B | B/Brigit (B/Russia/69) | ATCC VR-786 | 1.00 x 10^{5} CEID_{50} | None |
| Influenza B | B/Brisbane/3/2007 | IRR FR-18 | 1.00 x 10^{5} CEID_{50} | None |
| Influenza B | B/Brisbane/33/2008 | Zeptometrix 0810253CF | 1.00 x 10^{5} TCID_{50} | None |
| Influenza B | B/GreLakes/1739/1954 | BEI NR-3179 | 1.00 x 10^{5} CEID_{50} | None |
| Influenza B | B/Hong Kong/259/2010 | IRR FR-663 | 1.00 x 10^{5} CEID_{50} | None |
| Influenza B | B/Hong Kong/5/72 | ATCC VR-823 | 1.00 x 10^{5} CEID_{50} | None |
| Influenza B | B/Hubei-Wujiagang/158/2009 | IRR FR-469 | 1.00 x 10^{5} CEID_{50} | None |
| Influenza B | B/Massachusetts/2/12 | Zeptometrix 0810239CF | 1.00 x 10^{5} TCID_{50} | None |
| Influenza B | B/New Jersey/1/2012 | IRR FR-1270 | 1.00 x 10^{5} TCID_{50} | None |
| Influenza B | B/Russia/69 | ATCC VR-790 | 1.00 x 10^{5} CEID_{50} | None |
| Influenza B | B/Texas/02/2013 | IRR FR-1302 | 1.00 x 10^{5} TCID_{50} | None |
| Influenza B | B/Wisconsin/1/2010 | Zeptometrix 0810241CF | 1.00 x 10^{5} TCID_{50} | None |
| Influenza B | B/Florida/02/06 | Zeptometrix 0810037CF | 1.00 x 10^{5} TCID_{50} | None^{3} |
| Parainfluenza virus 1 | Type 1 | Zeptometrix 0810014CF | 1.00 x 10^{5} TCID_{50} | None |
| Parainfluenza virus 2 | Greer | ATCC VR-92 | 1.00 x 10^{5} TCID_{50} | None |
| Parainfluenza virus 3 | C 243 | ATCC VR-93 | 1.00 x 10^{5} TCID_{50} | None |
| Parainfluenza virus 4A | -- | Zeptometrix 0810060CF | 1.00 10^{5} TCID_{50} | None |
| Parainfluenza virus 4B | CH 19503 | ATCC VR-1377 | 1.00 x 10^{5} TCID_{50} | None |
| Respiratory Syncytial Virus A | A2 | ATCC VR-1540 | 1.00 x 10^{5} PFU | None |
| Respiratory Syncytial Virus B | 18537 | ATCC VR-1580 | 7.00 x 10^{4} PFU | None |
| Rhinovirus | Type 85, 50-525-CV54 [V-192-001-021] | ATCC VR-1195 | 1.00 x 10^{5} TCID_{50} | None |
| SARS-CoV-2 | USA-WA-1/2020 | ATCC VR-1986HK | 1.00 x 10^{7} copies | None |
| Chlamydia pneumoniae | TW-183 | ATCC VR-2282 | 1.00 x 10^{6} IFU | None |
| Mycoplasma pneumoniae | M129 | Zeptometrix 0801579 | 1.00 x 10^{6} CCU | None |
CCU: Color Changing Units; CEID$_{50}$; 50% Chicken Embryo Infectious Dose; IFU: Inclusion Forming Units; PFU: Plaque Forming Units; TCID$_{50}$; 50% Tissue Culture Infectious Dose
$^{1}$ Cross-reaction observed at levels ≥ 1.00 x 10$^{5}$ TCID$_{50}$/mL; no cross-reaction observed at 1.00 x 10$^{2}$ TCID$_{50}$/mL; this is noted as a Limitation in the device labeling.
$^{2}$ Highest attainable concentration.
$^{3}$ 3/9 replicates produced false-positive results for Coronavirus 229E due to suspected contamination of the viral stock; positive results were not obtained with any other strains of Influenza B.
### Off-Panel Microbial Interference
The potential for interference with the NxTAG RPP by off-panel microorganisms that may be present in nasopharyngeal swab specimens was evaluated by testing 11 potentially cross-reactive species (Table 9) at high concentration in the presence of low-moderate levels of 22 on-panel target analytes, including different subtypes of influenza A. Testing was performed with combinations of two or three off-panel analytes at high concentration in the presence of different combinations of three on-panel analytes, each at ~3-6X LoD. The expected positive
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results were obtained for each of the on-panel analytes, with no evidence of interference from the off-panel species.
Table 9. Off-panel species evaluated for potential interference with the NxTAG RPP v2
| Species/ | Strain | Source/Catalogue No. | Concentration per mL | Interference |
| --- | --- | --- | --- | --- |
| Bordetella pertussis | A639 | Zeptometrix 0801459 | 1.00 x 10⁶ CFU | None |
| Corynebacterium diphtheriae | Z116 | Zeptometrix 0801882 | 1.00 x 10⁶ CFU | None ¹ |
| Haemophilus influenzae | Type b; MinnA | Zeptometrix 0801680 | 1.00 x 10⁶ CFU | None |
| Moraxella catarrhalis | Strain NE 11 | Zeptometrix 0801509 | 1.00 x 10⁶ CFU | None |
| Neisseria meningitidis | Serotype A | Zeptometrix 0801511 | 1.00 x 10⁶ CFU | None |
| Pseudomonas aeruginosa | Clinical | Zeptometrix 0801519 | 1.00 x 10⁶ CFU | None |
| Staphylococcus aureus | 102-04 | Zeptometrix BAA-1765 | 1.00 x 10⁶ CFU | None |
| Streptococcus pneumoniae | Z022 | Zeptometrix 0801439 | 1.00 x 10⁶ CFU | None |
| Cytomegalovirus | Merlin | Zeptometrix 0810500CF | 1.00 x 10⁵ TCID₅₀ | None |
| Measles Virus | -- | Zeptometrix 0810025CF | 1.00 x 10⁵ TCID₅₀ | None |
| Mumps Virus | -- | Zeptometrix 0810079CF | 1.00 x 10⁵ TCID₅₀ | None |
CFU: Colony Forming Units; TCID₅₀: 50% Tissue Culture Infectious Dose
¹ On initial testing, 1/3 samples containing high levels of Corynebacterium diphtheriae produced false negative results for Coronavirus NL63 and Coronavirus HKU1 but produced the expected positive results for both analytes upon repeat.
### On-Panel Competitive Interference
The potential for competitive interference between viral and bacterial species targeted by the NxTAG RPP v2 assay was evaluated by testing combinations of on panel analytes at asymmetric concentrations. Analytes at low-moderate concentration (3-6X LoD) were combined into four groups (Table 10) that were each tested in the presence of a single high concentration analyte (Table 11) in simulated nasopharyngeal swab matrix. In all cases (n = 3 ea.), each analyte was successfully detected irrespective of the concentration tested, demonstrating the absence of assay interference.
Table 10. Combinations of on-panel analytes at 3-6X LoD used to evaluate potential competitive interference
| Sample | Analyte | Strain | Source/Catalogue No. |
| --- | --- | --- | --- |
| 1 | Influenza A H1pdm09 (subtype) ¹ | A/NY/02/09 | Zeptometrix 0810109CFN |
| | Respiratory Syncytial Virus A | A2 | ATCC VR-1540 |
| | Rhinovirus | 50-525-CV 54 | ATCC VR-1195 |
| 2 | Respiratory Syncytial Virus B | 18537 | ATCC VR-1580 |
| | Influenza A H3 (subtype) ¹ | A/Wisconsin/67/05 | Zeptometrix 0810252CF |
| 3 | Influenza B | B/Florida/02/06 | Zeptometrix 0810037CF |
| | Parainfluenza virus 3 | C 243 | ATCC VR-93 |
| | Mycoplasma pneumoniae | M129 | Zeptometrix 0801579 |
| 4 | Adenovirus B | Type 14 | Zeptometrix 0810108CF |
| | Human Metapneumovirus B1 | hMPV-3 | Zeptometrix 0810156CF |
| | SARS-CoV-2 | USA-WA1/2020 | ATCC VR-1986HK |
CCU: Color Changing Units; PFU: Plaque Forming Units; TCID₅₀: 50% Tissue Culture Infectious Dose
¹ Target level based on the LoD for the applicable subtype.
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**Table 11.** On-panel analytes tested at high concentration to evaluate potential competitive interference
| Analyte | Strain | Source/Catalogue No. | Concentration per mL |
| --- | --- | --- | --- |
| Adenovirus B | Type 14 | Zeptometrix 0810108CF | 1.00 x 10^{5} TCID_{50} |
| Coronavirus NL63 | -- | Zeptometrix 0810228CF | 1.00 x 10^{5} TCID_{50} |
| Influenza A H1pdm09 | A/NY/02/09 | Zeptometrix 0810109CFN | 1.00 x 10^{5} TCID_{50} |
| Influenza A H3 | A/Wisconsin/67/05 | Zeptometrix 0810252CF | 1.00 x 10^{5} TCID_{50} |
| Influenza B | B/Florida/02/06 | Zeptometrix 0810037CF | 1.00 x 10^{5} TCID_{50} |
| Human Metapneumovirus | hMPV-16, Type A1, IA10-2003 | Zeptometrix 0810161CF | 1.00 x 10^{5} TCID_{50} |
| Human Metapneumovirus | hMPV-3, Type B1, Peru2-2002 | Zeptometrix 0810156CF | 3.50 x 10^{4} TCID_{50} |
| Human Metapneumovirus | hMPV-5, Type B1, Peru3-2003 G gene | Zeptometrix 0810158CF | 1.00 x 10^{5} TCID_{50} |
| Respiratory Syncytial Virus B | B/18537 | ATCC VR-1580 | 6.30 x 10^{4} PFU |
| Respiratory Syncytial Virus B | B/WV/14617/85 | ATCC VR-1400 | 1.00 x 10^{5} TCID_{50} |
| Rhinovirus | 50-525-CV 54 [V-192-001-021] | ATCC VR-1195 | 1.00 x 10^{5} TCID_{50} |
| SARS-CoV-2 | USA-WA1/2020 | VR-1986HK | 1.00 x 10^{7} copies |
PFU: Plaque Forming Units; TCID$_{50}$: 50% Tissue Culture Infectious Dose
Because there is limited information with respect to co-infections involving SARS-CoV-2, additional evaluation of the potential for competitive interference was performed with low concentrations of SARS-CoV-2 (3X LoD) in the presence of high concentrations of 13 on-panel analytes that were not included in **Table 11 (Table 12)**. In all cases (n = 3 ea.), positive results were reported for both SARS-CoV-2 and the high concentration analyte, demonstrating the absence of competitive interference.
**Table 12.** On-panel analytes tested at high concentration to evaluate potential competitive interference with the detection of SARS-CoV-2
| Analyte | Strain | Source/Catalogue No. | Concentration per mL |
| --- | --- | --- | --- |
| Coronavirus 229E | -- | ATCC VR-740 | 1.00 x 10^{5} TCID_{50} |
| Coronavirus OC43 | Betacoronavirus 1 | ATCC VR-1558 | 1.00 x 10^{5} TCID_{50} |
| Coronavirus HKU1 | Clinical specimen | -- | 1.00 x 10^{6} copies |
| Enterovirus | Type 68, 2007 isolate | Zeptometrix 0810237CF | 1.00 x 10^{5} PFU |
| Influenza A H1 | A/Brisbane/59/07 | Zeptometrix 0810244CF | 1.00 x 10^{5} TCID_{50} |
| Parainfluenza virus 1 | Type 1 | Zeptometrix 0810014CF | 1.00 x 10^{5} TCID_{50} |
| Parainfluenza virus 2 | Greer | ATCC VR-92 | 1.00 x 10^{5} TCID_{50} |
| Parainfluenza virus 3 | C 243 | ATCC VR-93 | 1.00 x 10^{5} TCID_{50} |
| Parainfluenza virus 4A | Type 4A | Zeptometrix 0810060CF | 1.00 x 10^{5} TCID_{50} |
| Parainfluenza virus 4B | CH 19503 | ATCC VR-1377 | 1.00 x 10^{5} TCID_{50} |
| Respiratory Syncytial Virus A | A2 | ATCC VR-1540 | 1.00 x 10^{5} PFU |
| *Chlamydia pneumoniae* | TW-183 | ATCC VR-2282 | 1.00 x 10^{6} IFU |
| *Mycoplasma pneumoniae* | M129 | Zeptometrix 0801579 | 1.00 x 10^{6} CCU |
CCU: Color Changing Units; IFU: Inclusion Forming Units; PFU: Plaque Forming Units; TCID$_{50}$: 50% Tissue Culture Infectious Dose
### *Interfering Substances*
Testing was conducted to evaluate the potential for interference with the NxTAG RPP v2 assay by endogenous and exogenous substances that may be present in nasopharyngeal swab specimens. Eight multi-analyte samples (**Table 13**) comprised of simulated nasopharyngeal swab matrix containing on-panel analytes at 3-6X LoD were tested in triplicate in the presence of 22 potentially interfering substances (**Table 14**). Each substance was also added to negative simulated nasopharyngeal matrix to evaluate the potential for false positive results.
The expected results were obtained in all cases, except in the presence of menthol and FluMist influenza vaccine. Menthol 1% (w/v) was shown to interfere with the detection of Coronavirus
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OC43, although no interference was observed at lower concentration (0.5% w/v). Positive results for Influenza A, Influenza A H1pdm09, Influenza A H3 and Influenza B were obtained in the presence of FluMist because the vaccine contains attenuated strains of these analytes that are detectable at the levels used in this study. The potential for false results with menthol and FluMist is noted in the Limitations section of the device labeling.
Table 13. Multi-analyte panels used to evaluate the potential for assay interference by endogenous and exogenous substances
| Sample | Analyte^{1} | Strain | Source | Catalogue No. |
| --- | --- | --- | --- | --- |
| 1 | Influenza A H1pdm09 (subtype)^{2} | A/NY/02/09 | Zeptometrix | 0810109CFN |
| | Respiratory Syncytial Virus A | A2 | ATCC | VR-1540 |
| | Rhinovirus | 50-525-CV54 | ATCC | VR-1195 |
| 2 | Influenza A H3 (subtype)^{2} | A/Wisconsin/67/05 | Zeptometrix | 0810252CF |
| | Respiratory Syncytial Virus B | CH 19503 | ATCC | VR-1377 |
| 3 | Influenza B | B/Florida/02/06 | Zeptometrix | 0810037CF |
| | Parainfluenza virus 3 | C 243 | ATCC | VR-93 |
| | Mycoplasma pneumoniae | M129 | Zeptometrix | 0801579 |
| 4 | SARS-CoV-2 | USA-WA1/2020 | ATCC | VR-1986HK |
| | Human Metapneumovirus B1 | hMPV-3 | Zeptometrix | 0810156CF |
| | Adenovirus B | Type 14 | Zeptometrix | 0810108CF |
| 5 | Influenza A H3 (matrix) | A/Wisconsin/67/05 | Zeptometrix | 0810252CF |
| | Coronavirus NL63 | -- | Zeptometrix | 0810228CF |
| | Coronavirus HKU1 | Clinical specimen | -- | -- |
| 6 | Influenza H1 (subtype)^{2} | A/Brisbane/59/07 | Zeptometrix | 0810244CF |
| | Parainfluenza virus 1 | -- | Zeptometrix | 0810014CF |
| | Chlamydia pneumoniae | TW-183 | ATCC | VR-2282 |
| 7 | Parainfluenza virus 2 | Greer | ATCC | VR-92 |
| | Parainfluenza virus 4B | CH 19503 | ATCC | VR-1377 |
| | Coronavirus 229E | -- | Zeptometrix | 0810228CF |
| 8 | Parainfluenza virus 4A | -- | Zeptometrix | 0810060CF |
| | Coronavirus OC43 | Betacoronavirus 1 | ATCC | VR-1558 |
| 9 | Negative | -- | -- | -- |
$^{1}$ Each analyte was tested at 3-6X LoD.
$^{2}$ Target level based on the LoD for the applicable subtype.
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Table 14. Endogenous and exogenous substances evaluated for potential interference with the NxTAG RPP v2
| Source | Substance | Active Ingredient | Concentration | Interference |
| --- | --- | --- | --- | --- |
| Endogenous | Human whole blood | -- | 5% (v/v) | None^{1} |
| | Human Genomic DNA | -- | 20 μg/mL | None^{2} |
| | Leukocytes | | 1.00 x 10^{3} cells/μL | None |
| | Mucin | -- | 100 μg/mL | None |
| Exogenous | Beclomethasone dipropionate | -- | 25 μg/mL | None |
| | Benzocaine | | 10% (w/v) | None |
| | Budesonide | -- | 6.30 x 10^{-3} μg/mL | None |
| | Dexamethasone | -- | 12 μg/mL | None |
| | Early Defense Nasal Spray | Zinc | 5% (v/v) | None |
| | FluMist | Influenza A H1N1 Influenza A H3N2 Influenza B (Yamagata) Influenza B (Victoria) | 0.5% (v/v) | Yes^{3} |
| | Flunisolide | -- | 5 μg/mL | None |
| | Fluticasone propionate | -- | 1.26 x 10^{-3} μg/mL | None |
| | Menthol | -- | 1% (w/v) | Yes^{4} |
| | | | 0.5% (w/v) | None |
| | Mometasone furoate | -- | 4.50 x 10^{-4} μg/mL | None |
| | Mupirocin | -- | 1.5 mg/mL | None |
| | Drixoral | Oxymetaxoline | 10% (v/v) | None |
| | | | 15% (v/v) | None |
| | Phenylephrine | -- | 0.03 μg/mL | None |
| | Salinex | Sodium chloride | 1% (v/v) | None |
| | | | 15% (v/v) | None |
| | Tobramycin | -- | 33 μg/mL | None |
| | | | 600 μg/mL | None |
| | Triamcinolone acetonide | -- | 22 μg/mL | None |
| | Zanamivir | | 100 μg/mL | None |
| | ZICAM Allergy Relief | Galphimia glauca, Histaminum hydrochloricum, Luffa operculata, Sulfur | 1% (v/v) | None |
| | | | 5% (v/v) | None |
$^{1}$ On initial testing, 1/3 replicates with Sample 5 from Table 13 produced false negative results for Coronavirus NL63 and Coronavirus HKU1 and the result was confirmed upon retesting; however, upon testing with new samples 3/3 replicates gave the expected positive results.
$^{2}$ On initial testing, 1/3 replicates with Sample 2 from Table 13 produced a false negative result for RSV B; upon re-testing of the same sample, the expected result was obtained.
$^{3}$ All samples containing FluMist produced positive results for influenza A, influenza A H1pdm09, influenza A H3 and influenza B; this is noted as a Limitation in the device labeling.
$^{4}$ 3/3 replicates in the presence of 1% (w/v) menthol produced false negative results for Coronavirus OC43; interference by menthol was alleviated at lower concentration (0.5% w/v); this is noted as a Limitation in the device labeling.
### 4. Assay Reportable Range:
Not applicable.
### 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
#### Controls
Bacteriophage MS2 is added to each sample to be tested with the NxTAG RPP v2 prior to nucleic acid extraction as an internal control to monitor process and reagent integrity. Failure to detect the MS2 control (in conjunction with failure to detect any of the target analytes) is indicative of failure of one or more processes: nucleic acid extraction, reverse transcription PCR amplification and/or detection.
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### *External Controls*
External Positive and Negative Controls are not provided with in the NxTAG RPP v2 assay kit but are recommended for inclusion in every assay run in accordance with good laboratory practice and applicable local, state and federal accrediting organizations. External Controls will be reported as “Pass”, “Fail” or “Invalid” in accordance with the expected values entered by the user in the SYNCT Software.
External Positive and Negative Controls were included in each run of the NxTAG RPP v2 assay in the Clinical Studies described in **Section VII C**. On each day of testing, the External Positive and Negative Controls were required to produce the expected results for the results obtained with clinical specimens to be considered acceptable.
### *Specimen Stability*
To evaluate the stability of the NxTAG RPP v2 target analytes in nasopharyngeal swab matrix, separate studies were performed with samples prepared in Universal Transport Medium (UTM, Copan Diagnostics) and MicroTest M4RT (M4RT, Remel). All the analytes targeted by the NxTAG RPP v2 assay were included in the analysis of performance with UTM, whereas only a representative subset was included in evaluating the stability of specimens prepared in M4RT (**Table 15**). The positive samples for testing were prepared at two concentrations (1.5X [Low] and 5X LoD [Moderate], unless otherwise noted in **Table 15**) and stored at 2-8 °C or -70 ± 5 °C prior to testing. The results of the study support the claimed stability of the target analytes in nasopharyngeal swab matrix collected in UTM or M4RT for up to 7 days at 2-8 °C or 12 months at -70 ± 5 °C.
Additional testing also demonstrated the stability of extracted nucleic acid after storage at 2-8 °C for up to 4 hours or -70 ± 5 °C for up to 12 months.
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**Table 15.** Multi-analyte samples used to evaluate specimen stability
| Sample | Analyte | Strain | Source | Catalogue No. |
| --- | --- | --- | --- | --- |
| 1^{1} | Influenza A H1pdm09 (subtype)^{2, 3} | A/NY/02/09 | Zeptometrix | 0810109CFN |
| | Respiratory Syncytial Virus A | A2 | ATCC | VR-1540 |
| | Rhinovirus | 50-525-CV54 | ATCC | VR-1195 |
| 2^{1} | Influenza A H3 (subtype)^{2} | A/Wisconsin/67/05 | Zeptometrix | 0810252CF |
| | Respiratory Syncytial Virus B | CH 19503 | ATCC | VR-1377 |
| 3^{1} | Influenza B^{2} | B/Florida/02/06 | Zeptometrix | 0810037CF |
| | Parainfluenza virus 3 | C 243 | ATCC | VR-93 |
| | *Mycoplasma pneumoniae*^{4} | M129 | Zeptometrix | 0801579 |
| 4^{1} | SARS-CoV-2 | USA-WA1/2020 | ATCC | VR-1986HK |
| | Human Metapneumovirus B1 | hMPV-3 | Zeptometrix | 0810156CF |
| | Adenovirus B^{2, 3} | Type 14 | Zeptometrix | 0810108CF |
| 5 | Influenza A H3 (matrix) | A/Wisconsin/67/05 | Zeptometrix | 0810252CF |
| | Coronavirus NL63 | -- | Zeptometrix | 0810228CF |
| | Coronavirus HKU1 | Clinical specimen | -- | -- |
| 6 | Influenza H1 (subtype)^{2} | A/Brisbane/59/07 | Zeptometrix | 0810244CF |
| | Parainfluenza virus 1 | -- | Zeptometrix | 0810014CF |
| | *Chlamydia pneumoniae* | TW-183 | ATCC | VR-2282 |
| 7 | Parainfluenza virus 2 | Greer | ATCC | VR-92 |
| | Parainfluenza virus 4B | CH 19503 | ATCC | VR-1377 |
| | Coronavirus 229E | -- | Zeptometrix | 0810228CF |
| 8 | Parainfluenza virus 4A | -- | Zeptometrix | 0810060CF |
| | Coronavirus OC43 | Betacoronavirus 1 | ATCC | VR-1558 |
| 9^{1} | Negative | -- | -- | -- |
$^{1}$ Panel member included in evaluation of the stability of specimens in M4RT medium.
$^{2}$ Target level based on the LoD for the applicable strain/subtype.
$^{3}$ Formulated at approximately 3X (Low) and 9X LoD (Moderate).
$^{4}$ Formulated at approximately 2X (Low) and 7X LoD (Moderate).
### *Reagent Stability*
A Real-time Stability Study to evaluate the stability of the NxTAG RPP v2 reagents under the recommended storage conditions of 2-8 °C is currently on-going. The study includes three independent kit lots that were manufactured with unique lots of critical reagents. Each kit lot will be tested at 3-month intervals over the course of the study using a panel of Positive Controls that collectively include target nucleic acids for each assay on the panel, in addition to the MS2 bacteriophage Internal Control (n = 3 replicates per assay per kit lot at each test point). To-date, testing has been completed that supports the stability of NxTAG RPP v2 kits for up to 12 months from the date of manufacture when stored under the recommended conditions.
### *In-use Reagent Stability*
Lyophilized NxTAG RPP v2 assay reagents are supplied in foil-sealed 8-well strips that are packaged in sealed pouches of 96 reactions and stored at 2-8 °C. Testing was performed to demonstrate the stability of the prefilled wells after removal from the original packaging, prior to sample addition. Additional testing was also conducted to support resealing of the original reagent pouch and storage of unused foil-sealed wells if less than a full 96 well plate of reactions is consumed. Under all conditions tested, the expected results were obtained, supporting the in-use stability of NxTAG RPP v2 reagents in accordance with the Instructions For Use.
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# 6. Detection Limit:
# Limit of Detection
The analytical sensitivity of the NxTAG RPP v2 was evaluated by testing contrived specimens that were prepared by adding quantified, diluted stocks of representative viral and bacterial strains/isolates of each target analyte to pooled negative nasopharyngeal swab matrix in Universal Transport Medium (UTM) that was processed using the bioMérieux EMAG Extraction System. Each analyte was tested individually in a three-fold dilution series to estimate the limit of detection (LoD) which was then confirmed by testing an additional 20 replicates at or near the concentration that yielded 3/3 positive replicates in the preliminary titration. The confirmed LoD for each strain/isolate was defined as the lowest concentration that yielded ≥ 19/20 positive replicates (i.e., ≥ 95% proportion positive) (Table 16).
Table 16. Analytical sensitivity of the NxTAG RPP v2 with representative strains/isolates of the target analytes (tested individually using the EMAG Extraction System)
| NxTAG RPP v2 Analyte | Strain/Isolate Information | Source | Catalogue No. | LoD Concentration per mL | |
| --- | --- | --- | --- | --- | --- |
| | | | | Copies^{1} | Supplier Units |
| Adenovirus | Type 14, Species B, 2006 isolate | ZeptoMetrix | 0810108CF | 1,420 | 0.144 TCID_{50} |
| | Type 1, Species C | ZeptoMetrix | 0810050CF | 20,100 | 92.8 TCID_{50} |
| | Type 4, Species E | ZeptoMetrix | 0810070CF | 7,330 | 0.191 TCID_{50} |
| Coronavirus 229E | -- | ATCC | VR-740 | 381 | 0.122 TCID_{50} |
| Coronavirus HKU1 | Genotype A | Clinical | 68972 | 4,180 | -- |
| Coronavirus NL63 | -- | ZeptoMetrix | 0810228CF | 100 | 0.00645 TCID_{50} |
| Coronavirus OC43 | -- | ATCC | VR-1558 | 4,550 | 0.0732 TCID_{50} |
| Human Metapneumovirus | hMPV-16, Type A1, IA10-2003 | ZeptoMetrix | 0810161CF | 71.5 | 0.0576 TCID_{50} |
| | hMPV-3, Type B1, Peru2-2002 | ZeptoMetrix | 0810156CF | 262 | 0.0178 TCID_{50} |
| Influenza A (matrix) | A/Brisbane/59/07 (H1) | ZeptoMetrix | 0810244CF | 119 | 0.0283 TCID_{50} |
| | A/NY/02/09 (H1pdm09) | ZeptoMetrix | 0810109CFN | 328 | 0.0374 TCID_{50} |
| | A/Wisconsin/67/05 (H3) | ZeptoMetrix | 0810252CF | 168 | 0.645 TCID_{50} |
| Influenza A H1 (subtype) | A/Brisbane/59/07 | ZeptoMetrix | 0810244CF | 1,600 | 0.382 TCID_{50} |
| Influenza A H1pdm09 (subtype) | A/NY/02/09 | ZeptoMetrix | 0810109CFN | 984 | 0.112 TCID_{50} |
| Influenza A H3 (subtype) | A/Wisconsin/67/05 | ZeptoMetrix | 0810252CF | 56 | 0.215 TCID_{50} |
| Influenza B | B/Florida/02/06 | ZeptoMetrix | 0810037CF | 63.3 | 0.967 TCID_{50} |
| Parainfluenza Virus | Serotype 1, N/A | ZeptoMetrix | 0810014CF | 692 | 0.764 TCID_{50} |
| | Serotype 2, Greer | ATCC | VR-92 | 345 | 0.732 TCID_{50} |
| | Serotype 3, C 243 | ATCC | VR-93 | 1,010 | 110 TCID_{50} |
| | Serotype 4A | ZeptoMetrix | 0810060CF | 16,900 | 0.858 TCID_{50} |
| | Serotype 4B, CH 19503 | ATCC | VR-1377 | 7,150 | 59.9 TCID_{50} |
| Respiratory Syncytial Virus A | A2 | ATCC | VR-1540 | 4,970 | 37.7 PFU |
| Respiratory Syncytial Virus B | 18537 | ATCC | VR-1580 | 7,205 | 0.320 PFU |
| Rhinovirus/Enterovirus | Rhinovirus, 50-525-CV54 (V-192-001-021) | ATCC | VR-1195 | 1,536 | 68.7 TCID_{50} |
| | Enterovirus Type 68, 2007 Isolate | ZeptoMetrix | 0810237CF | 3,526 | 2.30 TCID_{50} |
| SARS-CoV-2 | USA-WA1/2020 | ATCC | VR-1986HK | 500 | 7.68 TCID_{50} |
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| NxTAG RPP v2 Analyte | Strain/Isolate Information | Source | Catalogue No. | LoD Concentration per mL | |
| --- | --- | --- | --- | --- | --- |
| | | | | Copies^{1} | Supplier Units |
| *Chlamydia pneumoniae* | TW-183 | ATCC | VR-2282 | 238 | 37.4 IFU |
| *Mycoplasma pneumoniae* | M129 | ZeptoMetrix | 0801579 | 3,230 | 55.6 CCU |
CCU: Color Changing Units; Copies: copies of nucleic acid target as determined by quantitative PCR; IFU: Inclusion Forming Units; PFU: Plaque Forming Units; TCID$_{50}$: 50% Tissue Culture Infectious Dose
Limit of Detection with Multi-analyte Samples and Simulated Nasopharyngeal Swab Matrix
Additional testing was performed to evaluate the analytical sensitivity of the NxTAG RPP v2 assay with samples containing multiple target analytes and to evaluate use of simulated nasopharyngeal swab matrix in analytical studies. Samples containing combinations of two or three target species at concentrations equivalent to 1-2X the confirmed single target LoD in either negative clinical or simulated nasopharyngeal swab matrix were tested in parallel. The analyte combinations evaluated are shown in Table 17. Twenty replicates of each analyte combination were included in the study and all analytes were successfully detected (≥ 95% proportion positive), demonstrating the absence of interference from the presence of multiple analytes at levels close to the LoD of assay and supporting use simulated nasopharyngeal swab matrix in other analytical studies to evaluate device performance.
Table 17. Multi-analyte samples used to evaluate the analytical sensitivity
| Sample | Analyte | Strain | Source | Catalogue No. | % Detected (n = 20) | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | | NCM | NSM |
| 1 | Influenza A H1pdm09 (subtype)^{1,2} | A/NY/02/09 | ZeptoMetrix | 0810109CFN | 100 | 100 |
| | Respiratory Syncytial Virus A | A2 | ATCC | VR-1540 | 95 | 100 |
| | Rhinovirus | 50-525-CV54 | ATCC | VR-1195 | 100 | 100 |
| 2 | Influenza A H3 (subtype)^{1} | A/Wisconsin/67/05 | ZeptoMetrix | 0810252CF | 100 | 100 |
| | Respiratory Syncytial Virus B | 18537 | ATCC | VR-1580 | 100 | 100 |
| 3 | Influenza B | B/Florida/02/06 | ZeptoMetrix | 0810037CF | 100 | 95 |
| | Parainfluenza Virus 3 | C 243 | ATCC | VR-93 | 100 | 100 |
| | *M. pneumoniae*^{2} | M129 | ZeptoMetrix | 0801579 | 100 | 100 |
| 4 | SARS-CoV-2 | USA-WA1/2020 | ATCC | VR-1986HK | 100 | 100 |
| | Human Metapneumovirus | hMPV-3, Type B1, Peru2-2002 | ZeptoMetrix | 0810156CF | 100 | 95 |
| | Adenovirus B^{2} | Type 14, 2006 isolate | ZeptoMetrix | 0810108CF | 100 | 100 |
| 5 | Influenza A H3 (matrix) | A/Wisconsin/67/05 | ZeptoMetrix | 0810252CF | 100 | 100 |
| | Coronavirus NL63 | NL63 | ZeptoMetrix | 0810228CF | 100 | 100 |
| | Coronavirus HKU1 | Genotype B | Clinical | HKU1-20210301 | 100 | 95 |
| 6 | Influenza A H1 (subtype)^{1} | A/Brisbane/59/07 | ZeptoMetrix | 0810244CF | 100 | 95 |
| | Parainfluenza Virus 1 | -- | ZeptoMetrix | 0810014CF | 100 | 100 |
| | *C. pneumoniae* | TW-183 | ATCC | VR-2282 | 100 | 100 |
| 7 | Parainfluenza Virus 2 | Greer | ATCC | VR-92 | 95 | 95 |
| | Parainfluenza Virus 4B | CH 19503 | ATCC | VR-1377 | 100 | 100 |
| | Coronavirus 229E | -- | ATCC | VR-740 | 100 | 95 |
| 8 | Parainfluenza Virus 4A | -- | ZeptoMetrix | 0810060CF | 100 | 100 |
| | Coronavirus OC43 | -- | ATCC | VR-1558 | 100 | 100 |
NCM: Nasopharyngeal Clinical Matrix; NSM: Nasopharyngeal Simulated Matrix
$^{1}$ Target level based on the LoD for the influenza A subtype.
$^{2}$ Formulated at 1-2X LoD; all other analytes tested at 1X LoD.
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### *Comparison of Alternative Methods of Nucleic Acid Extraction*
The analytical sensitivity of the NxTAG RPP v2 assay was established using samples that were processed using the bioMérieux EMAG Extraction System and NUCLISENS extraction reagents. Additional testing was performed to evaluate the analytical sensitivity of the assay with…
