Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a standard multiplexed nucleic acid test and an automated system for processing samples. There is no mention of AI, ML, or any computational methods beyond standard data analysis for interpreting the results of the molecular assays. The performance studies focus on analytical and clinical validation of the molecular test itself.

Therapeutic

No

This device is an in vitro diagnostic test designed to detect and identify pathogens from patient samples, aiding in diagnosis rather than providing direct treatment or therapy.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the BioCode RPP is a "qualitative multiplexed nucleic acid-based in vitro diagnostic test." It further clarifies that its purpose is the "detection and identification of nucleic acids from multiple viruses and bacteria extracted from nasopharyngeal swab (NPS) samples" to aid "in the diagnosis of respiratory infection."

SaMD (Software as a Medical Device)

No

The device is an in vitro diagnostic test that requires the use of the BioCode MDx 3000 Instrument, which is a hardware system for processing samples and performing optical detection.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The "Intended Use / Indications for Use" section explicitly states:

"The BioCode Respiratory Pathogen Panel (RPP) is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with BioCode MDx 3000 Instrument."

This statement directly identifies the device as an in vitro diagnostic test.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The BioCode Respiratory Pathogen Panel (RPP) is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with BioCode MDx 3000 Instrument. The BioCode of the simultaneous detection and identification of nucleic acids from multiple viruses and bacteria extracted from nasopharyngeal swab (NPS) samples obtained from individuals with signs and/or symptoms of respiratory tract infection. The following pathogens and subtypes are identified using the BioCode RPP:

Adenovirus
Coronavirus (229E, OC43, HKU1, and NL63)
Human Metapneumovirus A/B
Influenza A, including subtypes H1, H1 2009 Pandemic, and H3
Influenza B
Parainfluenza Virus 1
Parainfluenza Virus 2
Parainfluenza Virus 3
Parainfluenza Virus 4
Respiratory Syncytial Virus A/B
Rhinovirus/Enterovirus
Bordetella pertussis
Chlamydia pneumoniae
Mycoplasma pneumoniae

The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection 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 a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the BioCode RPP 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.

Due to the genetic similarity between Human Rhinovirus, the BioCode RPP cannot differentiate them. A positive BioCode RPP Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required. The BioCode RPP detects Human Rhinovirus with reduced sensitivity. If a more accurate Rhinovirus result is required, it is recommended that specimens found to be negative for Human Rhinovirus/Enterovirus after examination using BioCode RPP be confirmed by an alternate method (e.g. FDA cleared molecular tests).

Performance characteristics for Influenza A were established when Influenza A H1 2009 Pandemic and A H3 were the predominant Influenza A viruses in circulation. Performance of detecting Influenza A may vary if other Influenza A strains are circulating or a novel Influenza A virus emerges.

Product codes

OCC, OZE, OEP, OEM, OOU, OTG, OZX, OZY, OZZ, NSU

Device Description

The BioCode® Respiratory Pathogen Panel is a respiratory pathogen multiplex nucleic acid test designed for use with the BioCode® MDx-3000 system. The BioCode® MDx-3000 is an automated system that integrates PCR amplification, target capture, signal generation and optical detection for multiple viral and bacterial pathogens from a single nasopharyngeal swab specimen collected in transport media. Specimens are processed and nucleic acids extracted with the NucliSens easyMAG or Roche MagNA Pure 96 automated systems. Once the PCR plate is set up and sealed, all other operations are automated on MDx-3000. The BioCode® RPP simultaneously tests for 17 pathogens and/or subtypes (see table below) from nasopharyngeal swab specimens collected in UTM or VTM. Results from the BioCode RPP test are available within about 5 hours, including off-board nucleic acids extraction.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

nasopharyngeal swab (NPS) samples

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Clinical Performance Study:

Sample Size: 2649 residual NPS specimens (1401 fresh and 1248 frozen specimens).
Data Source: Prospectively collected residual (leftover) and de-identified nasopharyngeal swab (NPS) specimens in VTM or UTM from patients suspected of respiratory tract infections at five geographically diverse clinical sites in the U.S.
Annotation Protocol: Specimens were tested with an FDA-cleared molecular multiplexed respiratory pathogen panel as the Standard of Care (SOC). Discrepant results between the BioCode RPP and the comparator test were investigated using independent molecular tests, including analytically validated PCR followed by bi-directional sequencing assays and alternate NAATs.

Testing of Preselected Archived Specimens:

Sample Size: 165 clinical specimens (archived NPS in VTM or UTM).
Data Source: Specimens previously tested positive for specific pathogens (coronavirus 229E, coronavirus HKU1, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 4, Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae) or negative in previous laboratory testing.
Annotation Protocol: The performance was evaluated by comparing BioCode RPP results with those from an FDA-cleared molecular multiplexed respiratory pathogen panel (the same as used in the prospective study). Specimens were randomized and blinded to the testing users.

Testing of Contrived Specimens:

Sample Size: 110 samples, including 100 positives.
Data Source: Contrived clinical specimens prepared using 50 unique natural NPS in VTM or UTM specimens that were previously tested negative for all BioCode RPP analytes. Spiked at concentrations of 2X LOD or greater using different strains for each pathogen (Chlamydia pneumoniae and Influenza A H1).
Annotation Protocol: Samples were randomized and tested at one of the five testing sites that participated in the prospective clinical study.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Clinical Performance (Prospective Study)

Study Type: Multi-center prospective clinical study.
Sample Size: 2649.
Key Results:
- Overall success rate for initial specimen testing: 98.8% (2618/2649).
- Final validity rate: 99.9% (2647/2649).
- Positive Agreement (PA) and Negative Agreement (NA) highlights:
  - Adenovirus: PA 87.2%, NA 98.4%.
  - Bordetella pertussis: PA 100%, NA 99.3%.
  - Chlamydia pneumoniae: PA 100%, NA 100%.
  - Coronavirus: PA 83.5%, NA 99.1%.
  - Human Metapneumovirus: PA 95.1%, NA 99.3%.
  - Human Rhinovirus/Enterovirus: PA 80.8%, NA 98.4% (Note: BioCode RPP cannot differentiate Human Rhinovirus/Enterovirus due to genetic similarity; detects Human Rhinovirus with reduced sensitivity).
  - Influenza A: PA 96.4%, NA 99.0%.
  - Influenza A H1 2009pdm: PA 98.1%, NA 99.7%.
  - Influenza A H3: PA 93.6%, NA 99.6%.
  - Influenza B: PA 94.4%, NA 99.5%.
  - Mycoplasma pneumoniae: PA 100%, NA 99.2%.
  - Parainfluenza Virus 1: PA 88.2%, NA 100%.
  - Parainfluenza Virus 2: PA 83.3%, NA 99.9%.
  - Parainfluenza Virus 3: PA 96.7%, NA 99.3%.
  - Parainfluenza Virus 4: PA 88.9%, NA 99.9%.
  - Respiratory Syncytial Virus: PA 98.0%, NA 99.1%.
- Mixed Infections: 193/2649 (7.3%) samples had mixed infections detected by BioCode RPP.

Clinical Performance (Archived Specimens)

Study Type: Evaluation of preselected archived retrospective specimens.
Sample Size: 165.
Key Results:
- Positive Agreement (PA) and Negative Agreement (NA) highlights:
  - Adenovirus: PA 100%, NA 98.1%.
  - Bordetella pertussis: PA 100%, NA 92.9%.
  - Chlamydia pneumoniae: PA 100%, NA 100%.
  - Coronavirus: PA 88.1%, NA 93.4%.
  - Human Metapneumovirus: PA 100%, NA 100%.
  - Human Rhinovirus/Enterovirus: PA 69.6%, NA 99.3%.
  - Influenza B: PA 66.7%, NA 100%.
  - Mycoplasma pneumoniae: PA 100%, NA 96.8%.
  - Parainfluenza Virus 1: PA 92.3%, NA 100%.
  - Parainfluenza Virus 2: PA 95.0%, NA 99.3%.
  - Parainfluenza Virus 3: PA 100%, NA 100%.
  - Parainfluenza Virus 4: PA 93.3%, NA 100%.
  - Respiratory Syncytial Virus: PA 91.7%, NA 99.3%.

Testing of Contrived Specimens

Study Type: Analytical study using contrived specimens.
Sample Size: 110 (100 positives + negative samples).
Key Results: All contrived positive samples for Chlamydia pneumoniae (50/50 samples) and Influenza A H1N1 (50/50 samples) were detected (100% PA) at concentrations of 2x LoD or greater. All negative controls were correctly identified (100% NA).

Reproducibility Study

Study Type: Multi-site reproducibility study (intra-assay, inter-assay, day-to-day, site-to-site).
Sample Size: Not explicitly stated as a single number, but involves multiple runs across 3 sites with easyMAG and 1 site with MP96, 2 operators, 1 instrument per site for 5 days (10 runs per site), with 6 contrived positive samples and 1 negative sample, each extracted in triplicate and assayed in singlet.
Key Results: Acceptance criteria were met for all analytes at 1.5x LoD and 3x LoD, with most showing 100% agreement with expected results across all sites. Examples: Adenovirus 100% PA at both LoD levels, Influenza A/H3 98.9% PA at 1.5x LoD. All None (no analyte) samples showed very high to 100% Negative Agreement.

Multiple vs. Single Spiked Samples

Study Type: Analytical study.
Key Results: All organisms were detected within 3x LoD for both single and multiple spiked samples, indicating equivalency for use in other bench studies.

Limit of Detection (LoD)

Study Type: Analytical study.
Key Results: LoD for each organism was determined, and LoDs were comparable (within 3-fold) for each extraction system (easyMAG and MP96). Defined as the lowest concentration with >=95% detection of 20 replicates.

Analytical Reactivity/Inclusivity

Study Type: Analytical study and in silico analysis.
Key Results: The BioCode RPP showed good inclusivity for the targeted organisms and for related organisms with similar genetic lineage/sequence. Detected various strains/serotypes of Influenza A, Influenza B, Respiratory Syncytial Virus, Human Metapneumovirus, Parainfluenza Virus (1-4), Adenovirus, Coronavirus, Human Rhinovirus, Enterovirus, Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae at or above 3x LoD (with some exceptions discussed where higher concentrations were needed or in silico analysis was performed).

Zoonotic Influenza A Analytical Reactivity/Inclusivity Testing

Study Type: Analytical testing and in silico analysis.
Key Results: Nonseasonal influenza A strains (H2N2, H5N2, H7N9, and H5N1), except for H3N2v, were detected as Influenza A (no subtype). H3N2v was detected as Influenza A/H3 at higher concentrations and Influenza A (no subtype) at the lowest tested concentration. In silico analysis predicted detection of H5N8, H1N2v, and H1N1 swine influenza as Influenza A (no subtype) or Influenza A/H1 (with reduced sensitivity for A/H1).

Analytical Specificity/Cross Reactivity

Study Type: Analytical study and in silico analysis.
Key Results: No cross-reactivity was observed with most on-panel or off-panel microorganisms at the concentrations tested.
- Exceptions:
  - Rhinovirus/Enterovirus assay may react with other Enterovirus species (Coxsackievirus and Echoviruses).
  - Bordetella pertussis assay may react with Bordetella holmesii and Bordetella bronchiseptica based on in silico analysis. One instance of cross-reactivity with Bordetella holmesii was confirmed via empirical testing.
- SARS-CoV, MERS-CoV were tested and showed no cross-reactivity. One SARS-CoV sample was inhibitory.

Interfering Substances/Microbes

Study Type: Analytical study.
Key Results: None of the tested substances or microbial interferents were shown to interfere with BioCode® RPP at the concentrations tested, except for mucin at higher concentrations (0.6%) which could lead to loss of signal with easyMAG extraction. Flu Mist was reactive with the BioCode RPP assay.

Competitive Inhibition

Study Type: Analytical study.
Key Results: No competitive inhibition was observed, meaning that the presence of one target at a high concentration did not prevent the detection of two other targets at low concentrations in mixed samples.

Cross Contamination/Sample Carryover

Study Type: Referenced prior studies (K180041, K190585) for the BioCode® MDx-3000 system.
Key Results: No additional testing performed for BioCode® RPP as the system-level carry-over studies were already completed and deemed sufficient.

Specimen Stability

Study Type: Analytical study.
Key Results: All replicates were valid and detected for all tested storage conditions for NPS in transport media (room temp for 8 and 12 hours, refrigerated 4°C for 5, 7, and 10 days, frozen -80°C for 30, 60, and 90 days with 2 freeze/thaw cycles) and for purified nucleic acids (refrigerated 4°C for 8 and 12 hours, frozen -80°C for 30, 60, and 90 days with 2 freeze/thaw cycles).

Matrix Equivalency: Simulated Nasopharyngeal Swabs (sNPS) vs Native Nasopharyngeal Swabs (NPS)

Study Type: Analytical study.
Key Results: No matrix effect was observed. All replicates for contrived samples in both simulated and natural matrices were valid and detected for expected targets using both easyMAG and MagNA Pure 96 extraction systems.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Key metrics presented are Positive Agreement (PA) and Negative Agreement (NA).

Clinical Performance (Prospective Study):

Adenovirus: PA 87.2% (95% CI: 78.0%, 92.9%), NA 98.4% (95% CI: 97.8%, 98.8%)
Bordetella pertussis: PA 100% (95% CI: 34.2%, 100%), NA 99.3% (95% CI: 98.9%, 99.5%)
Chlamydia pneumoniae: PA 100% (95% CI: 51.0%, 100%), NA 100% (95% CI: 99.8%, 100%)
Coronavirus: PA 83.5% (95% CI: 76.2%, 88.8%), NA 99.1% (95% CI: 98.7%, 99.4%)
Human Metapneumovirus: PA 95.1% (95% CI: 90.2%, 97.6%), NA 99.3% (95% CI: 98.9%, 99.6%)
Human Rhinovirus/Enterovirus: PA 80.8% (95% CI: 77.0%, 84.1%), NA 98.4% (95% CI: 97.8%, 98.9%)
Influenza A: PA 96.4% (95% CI: 93.0%, 98.2%), NA 99.0% (95% CI: 98.5%, 99.3%)
Influenza A H1: PA N/A+ (No positive reference results recorded), NA 100% (95% CI: 99.9%, 100%)
Influenza A H1 2009pdm: PA 98.1% (95% CI: 90.1%, 99.7%), NA 99.7% (95% CI: 99.3%, 99.8%)
Influenza A H3: PA 93.6% (95% CI: 88.7%, 96.5%), NA 99.6% (95% CI: 99.3%, 99.8%)
Influenza B: PA 94.4% (95% CI: 84.9%, 98.1%), NA 99.5% (95% CI: 99.1%, 99.7%)
Mycoplasma pneumoniae: PA 100% (95% CI: 82.4%, 100%), NA 99.2% (95% CI: 98.8%, 99.5%)
Parainfluenza Virus 1: PA 88.2% (95% CI: 65.7%, 96.7%), NA 100% (95% CI: 99.9%, 100%)
Parainfluenza Virus 2: PA 83.3% (95% CI: 55.2%, 95.3%), NA 99.9% (95% CI: 99.7%, 100%)
Parainfluenza Virus 3: PA 96.7% (95% CI: 91.9%, 98.7%), NA 99.3% (95% CI: 98.9%, 99.6%)
Parainfluenza Virus 4: PA 88.9% (95% CI: 67.2%, 96.9%), NA 99.9% (95% CI: 99.7%, 100%)
Respiratory Syncytial Virus: PA 98.0% (95% CI: 95.1%, 99.2%), NA 99.1% (95% CI: 98.7%, 99.4%)

Preselected Archived Specimens:

Adenovirus: PA 100% (95% CI: 64.6%, 100%), NA 98.1% (95% CI: 94.6%, 99.4%)
Bordetella pertussis: PA 100% (95% CI: 72.2%, 100%), NA 92.9% (95% CI: 87.7%, 96.0%)
Chlamydia pneumoniae: PA 100% (95% CI: 72.2%, 100%), NA 100% (95% CI: 97.6%, 100%)
Coronavirus: PA 88.1% (95% CI: 77.5%, 94.1%), NA 93.4% (95% CI: 87.0%, 96.8%)
Human Metapneumovirus: PA 100% (95% CI: 51.0%, 100%), NA 100% (95% CI: 97.7%, 100%)
Human Rhinovirus/Enterovirus: PA 69.6% (95% CI: 49.1%, 84.4%), NA 99.3% (95% CI: 96.1%, 99.9%)
Influenza A: PA N/A+, NA 100% (95% CI: 97.7%, 100%)
Influenza A H1: PA N/A+, NA 100% (95% CI: 97.7%, 100%)
Influenza A H1 2009pdm: PA N/A+, NA 100% (95% CI: 97.7%, 100%)
Influenza A H3: PA N/A+, NA 100% (95% CI: 97.7%, 100%)
Influenza B: PA 66.7% (95% CI: 20.8%, 93.9%), NA 100% (95% CI: 97.7%, 100%)
Mycoplasma pneumoniae: PA 100% (95% CI: 64.6%, 100%), NA 96.8% (95% CI: 92.8%, 98.6%)
Parainfluenza Virus 1: PA 92.3% (95% CI: 66.7%, 98.6%), NA 100% (95% CI: 97.5%, 100%)
Parainfluenza Virus 2: PA 95% (95% CI: 76.4%, 99.1%), NA 99.3% (95% CI: 96.2%, 99.9%)
Parainfluenza Virus 3: PA 100% (95% CI: 20.7%, 100%), NA 100% (95% CI: 97.7%, 100%)
Parainfluenza Virus 4: PA 93.3% (95% CI: 70.2%, 98.8%), NA 100% (95% CI: 97.5%, 100%)
Respiratory Syncytial Virus: PA 91.7% (95% CI: 64.6%, 98.5%), NA 99.3% (95% CI: 96.4%, 99.9%)

Predicate Device(s)

K170604

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.

(a)
Identification. A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B;
(2) Influenza A subtype H1 and Influenza A subtype H3;
(3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B;
(4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus;
(5) Human Metapneumovirus;
(6) Rhinovirus; and
(7) Adenovirus.
(b)
Classification. Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;”
(2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and
(3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.

Summary

0

December 23, 2019

Image /page/0/Picture/1 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

Applied BioCode, Inc. Robert Tullio Regulatory Consultant 10020 Pioneer Blvd Suite 102 Santa Fe Springs, California 90670

Re: K192485

Trade/Device Name: BioCode Respiratory Pathogen Panel (RPP) Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: Class II Product Code: OCC, OZE, OEP, OEM, OOU, OTG, OZX, OZY, OZZ, NSU Dated: December 5, 2019 Received: December 6, 2019

Dear Robert Tullio:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmp/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see

1

https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Tamara Feldblyum, Ph.D. Chief Viral Respiratory and STI Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K192485

Device Name BioCode Respiratory Pathogen Panel (RPP)

Indications for Use (Describe)

The BioCode Respiratory Pathogen Panel (RPP) is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with BioCode MDx 3000 Instrument. The BioCode of the simultaneous detection and identification of nucleic acids from multiple viruses and bacteria extracted from nasopharyngeal swab (NPS) samples obtained from individuals with signs and/or symptoms of respiratory tract infection. The following pathogens and subtypes are identified using the BioCode RPP:

Adenovirus
· Coronavirus (229E, OC43, HKU1, and NL63)
Human Metapneumovirus A/B
· Influenza A, including subtypes H1, H1 2009 Pandemic, and H3
Influenza B
Parainfluenza Virus 1
Parainfluenza Virus 2
· Parainfluenza Virus 3
· Parainfluenza Virus 4
· Respiratory Syncytial Virus A/B
Rhinovirus/Enterovirus
· Bordetella pertussis
Chlamydia pneumoniae
Mycoplasma pneumoniae

The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection 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 a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the BioCode RPP 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.

Due to the genetic similarity between Human Rhinovirus, the BioCode RPP cannot differentiate them. A positive BioCode RPP Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required. The BioCode RPP detects Human Rhinovirus with reduced sensitivity. If a more accurate Rhinovirus result is required, it is recommended that specimens found to be negative for Human Rhinovirus/Enterovirus after examination using BioCode RPP be confirmed by an alternate method (e.g. FDA cleared molecular tests).

Performance characteristics for Influenza A were established when Influenza A H1 2009 Pandemic and A H3 were the predominant Influenza A viruses in circulation. Performance of detecting Influenza A may vary if other Influenza A strains are circulating or a novel Influenza A virus emerges.

3

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.

Type of Use (Select one or both, as applicable)
-------------------------------------------------	--

X Prescription Use (Part 21 CFR 801 Subpart D)

| | Over-The-Counter Use (21 CFR 801 Subpart C)

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1.0 510(k) SUMMARY

Introduction: According to the requirements of 21 CFR 807.92, the following provides information to understand the basis for a determination of substantial equivalence.

Submitted by:

Applied BioCode®, Inc. 10020 Pioneer Blvd. Suite 102 Santa Fe Springs, CA 90670

Contact:

Robert Di Tullio Regulatory Consultant rditullio@apbiocode.com Telephone: 310 801 1235 Fax: 323 372 3816

Date Submitted: September 9, 2019

Trade Name: BioCode® Respiratory Pathogen Panel (RPP)

Classification Name and Regulation Number:

Respiratory Viral Panel Multiplex Nucleic Acid Assay (21 CFR 866.3980)

Predicate Device:

K170604 – BioFire FilmArray Respiratory Pathogen Panel 2 (RP2)

Intended Use:

BioCode® Respiratory Pathogen Panel (RPP)

The BioCode® Respiratory Pathogen Panel (RPP) is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with BioCode MDx-3000 Instrument. The BioCode RPP is capable of the simultaneous detection and identification of nucleic acids from multiple viruses and bacteria extracted from nasopharyngeal swab (NPS) samples obtained from individuals with signs and/or symptoms of respiratory tract infection. The following pathogens and subtypes are identified using the BioCode RPP:

Adenovirus
Coronavirus (229E, OC43, HKU1, and NL63)
Human Metapneumovirus A/B
Influenza A, including subtypes H1, H1 2009 Pandemic, and H3
Influenza B
Parainfluenza 1
Parainfluenza 2
Parainfluenza 3
Parainfluenza 4

5

Respiratory Syncytial Virus A/B
Rhinovirus/Enterovirus
Bordetella pertussis
Chlamydia pneumoniae
. Mycoplasma pneumoniae

The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection 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 a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the BioCode RPP 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.

Due to the genetic similarity between Human Rhinovirus and Enterovirus, the BioCode RPP cannot differentiate them. A positive BioCode RPP Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required. The BioCode RPP detects Human Rhinovirus/Enterovirus with reduced sensitivity. If a more accurate HRV/EV result is required, it is recommended that specimens found to be negative for Human Rhinovirus/Enterovirus after examination using BioCode RPP be confirmed by an alternate method (e.g. FDA cleared molecular tests).

Performance characteristics for Influenza A were established when Influenza A H1 2009 Pandemic and A H3 were the predominant Influenza A viruses in circulation. Performance of detecting Influenza A may vary if other Influenza A strains are circulating or a novel Influenza A virus emerges. 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 virulenza 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.

6

Device Description:

The BioCode® Respiratory Pathogen Panel is a respiratory pathogen multiplex nucleic acid test designed for use with the BioCode® MDx-3000 system. The BioCode® MDx-3000 is an automated system that integrates PCR amplification, target capture, signal generation and optical detection for multiple viral and bacterial pathogens from a single nasopharyngeal swab specimen collected in transport media. Specimens are processed and nucleic acids extracted with the NucliSens easyMAG or Roche MagNA Pure 96 automated systems. Once the PCR plate is set up and sealed, all other operations are automated on MDx-3000. The BioCode® RPP simultaneously tests for 17 pathogens and/or subtypes (see table below) from nasopharyngeal swab specimens collected in UTM or VTM. Results from the BioCode RPP test are available within about 5 hours, including off-board nucleic acids extraction.

Viruses	Viruses
Influenza A	Respiratory Syncytial Virus A and B
• Subtype H1	Human Metapneumovirus A and B
• Subtype H1 2009pdm	Rhinovirus/Enterovirus
• Subtype H3	Coronavirus (229E, OC43, HKU1, and NL63)
Influenza B	Adenovirus
Parainfluenza 1	Bacteria
Parainfluenza 2	Mycoplasma pneumoniae
Parainfluenza 3	Chlamydia pneumoniae
Parainfluenza 4	Bordetella pertussis
	Internal Control (MS2)

Device Comparison:

Comparison of the BioCode RPP with the Predicate Device

Characteristic	Proposed Device	Predicate
Name	BioCode® Respiratory Pathogen
Panel (RPP)	BioFire FilmArray Respiratory
Pathogen Panel 2 (RP2)
Common Name	Respiratory Pathogen Panel
Multiplex Nucleic acid assay	Respiratory Viral Panel
Multiplex Nucleic acid assay
510(k) No.	K192485	K170604
Regulation	21CFR 866.3980	21CFR 866.3980
Product Code	OCC, OZE, OEM, OOU, OEP,
OTG, OZX, OZY, OZZ, NSU	OCC, OEM, OOU, OEP, OTG,
OZX, OZY, OZZ, OOI
Device Class	II	II

Similarities

7

Premarket Notification 510(k)

Intended Use
	The BioCode® Respiratory Pathogen Panel (RPP) is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with BioCode MDx-3000 Instrument. The BioCode RPP is capable of the simultaneous detection and identification of nucleic acids from multiple viruses and bacteria extracted from nasopharyngeal swab (NPS) samples obtained from individuals with signs and/or symptoms of respiratory tract infection. The following pathogens and subtypes are identified using the BioCode RPP:
• Adenovirus
• Coronavirus (229E, OC43, HKU1, and NL63)
• Human Metapneumovirus A/B
• Influenza A, including subtypes H1, H1 2009 Pandemic, and H3
• Influenza B
• Parainfluenza 1
• Parainfluenza 2
• Parainfluenza 3
• Parainfluenza 4
• Respiratory Syncytial Virus A/B
• Rhinovirus/Enterovirus
• Bordetella pertussis
• Chlamydia pneumoniae
• Mycoplasma pneumoniae	The FilmArray Respiratory Panel 2 (RP2) is a multiplexed nucleic acid test intended for use with FilmArray® 2.0 or 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. The following organism types and subtypes are identified using the FilmArray RP2:
• Adenovirus
• Coronavirus 229E
• Coronavirus HKU1
• Coronavirus NL63
• Coronavirus OC43
• Human Metapneumovirus
• Human Rhinovirus/Enterovirus
• Influenza A, including subtypes H1, H1-2009, and H3
• Influenza B
• Parainfluenza Virus 1
• Parainfluenza Virus 2
• Parainfluenza Virus 3
• Parainfluenza Virus 4
• Respiratory Syncytial Virus
• Bordetella parapertussis (IS1001)
• Bordetella pertussis (ptxP)
• Chlamydia pneumoniae
• Mycoplasma pneumoniae
	The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection 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 a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the BioCode RPP may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence and radiography)	The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection 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 a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the FilmArray RP2 may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and)
	may be necessary when evaluating a
patient with possible respiratory tract
infection.
Due to the genetic similarity between
Human Rhinovirus and Enterovirus, the
BioCode RPP cannot differentiate them. A
positive BioCode RPP
Rhinovirus/Enterovirus result should be
followed up using an alternate method
(e.g., cell culture or sequence analysis) if
differentiation is required. The BioCode
RPP detects Human Rhinovirus/Enterovirus
with reduced sensitivity. If a more accurate
HRV/EV result is required, it is
recommended that specimens found to be
negative for Human Rhinovirus/Enterovirus
after examination using BioCode RPP be
confirmed by an alternate method (e.g.
FDA cleared molecular tests).
Performance characteristics for Influenza A
were established when Influenza A H1
2009 Pandemic and A H3 were the
predominant Influenza A viruses in
circulation. Performance of detecting
Influenza A may vary if other Influenza A
strains are circulating or a novel Influenza A
virus emerges. 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.	radiography) may be necessary when
evaluating a patient with possible
respiratory tract infection.
Due to the genetic similarity between
Human Rhinovirus and Enterovirus, the
FilmArray RP2 cannot reliably differentiate
them. A positive FilmArray RP2
Rhinovirus/Enterovirus result should be
followed up using an alternate method (e.g.,
cell culture or sequence analysis) if
differentiation is required.
Performance characteristics for Influenza A
were established when Influenza A H1-2009,
A H1, and A H3 were the predominant
Influenza A viruses in circulation.
Performance of detecting Influenza A may
vary if other Influenza A strains are
circulating or a novel Influenza A virus
emerges. 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.
Sample Type	Nasopharyngeal swab in Viral or Universal
Transport Media	Same
Controls	Externally Sourced	Same
Differences
Methodology	Multiplex RT-PCR in a single reaction and
probe hybridization followed by
fluorescence detection and decoding of
barcoded magnetic beads (BMB) that
capture biotinylated products with
streptavidin conjugate	Nested multiplex PCR executed in two
stages. First, a single, large volume, highly
multiplexed reverse transcription PCR (RT-
PCR) reaction. Second, nested PCR, is
performed in singleplex fashion in each well
of the array. Followed by fluorescent
detection of images of the PCR reactions.

8

9

Summary of Performance Characteristics of the BioCode RPP®

Clinical Performance

Study Overview

The clinical performance of the BioCode RPP was established in a multi-center study conducted during periods of the 2017-2019 respiratory illness seasons. Residual (leftover) and de-identified nasopharyngeal swab (NPS) specimens in VTM or UTM that were prospectively collected from patients suspected of respiratory tract infections at five geographically diverse clinical sites in the U.S. were enrolled and tested with the BioCode RPP at five testing sites during the prospective clinical study. The enrolled prospective specimens were tested freshly with an FDA-cleared molecular multiplexed respiratory pathogen panel as part of the Stand of Care (SOC), and were either tested freshly with the BioCode RPP (i.e., specimens that were stored in a 2-8°C refrigerator for no more than 7 days), or stored frozen and then thawed and tested with the BioCode RPP at a testing site at a later date (i.e., specimens that were initially stored in a 2-8°C refrigerator but were not able to be tested by the BioCode RPP within 7 days from specimen collection).

A waiver of the informed consent requirement was obtained from the Institutional Review Boards (IRBs) at each specimen enrollment site for the use of residual NPS in VTM or UTM specimens.

The following information was recorded on the Case Report Form (CRF) for each subject from whom a specimen was enrolled:

· Age and sex
Date and time of specimen collection
Standard of care (SOC) comparator test result
Specimen storage status, i.e., fresh or frozen

A total of 2654 residual NPS specimens in VTM that were prospectively collected at the five clinical sites from August 2017 to May 2019 were enrolled initially for the clinical study. Five specimens were withdrawn from the clinical study due to incomplete data collection and testing, resulted in a total of 2649 prospective specimens (1401 fresh and 1248 frozen specimens) that were included in the prospective clinical study.

The prospective specimens enrolled for evaluation were tested at the five testing sites by trained laboratory personnel. DNA/RNA was extracted using either the BioMerieux NucliSENS easyMAG system or Roche MagNA Pure 96 system. After extraction, the samples were tested using the BioCode RPP on the BioCode MDx-3000 System according to the instructions for use.

Characteristic	Prospective Study Specimens
Total Specimens	2649
Gender (n/N (%))
Male	1346/2649 (50.8%)

Demographics - Prospective Samples

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Characteristic	Prospective Study Specimens
Female	1303/2649 (49.2%)
Age Category (n/N (%))
0-5 yrs	1004/2649 (37.9%)
6-21 yrs	609/2649 (23.0%)
22-59 yrs	531/2649 (20.0%)
60+ yrs	505/2649 (19.1%)
Status (n/N (%))a
Inpatient	1555/2646 (58.8%)
Outpatient	1091/2646 (41.2%)

a - Inpatient/outpatient status was unavailable for 3 specimens.

		Storage Method		Extraction Method
Site	Samples Tested	Fresh	Frozen	easyMAG	MagNA Pure 96
Site 01	530	250	280	530	0
Site 02	419	182	237	0	419
Site 03	600	366	234	600	0
Site 04	550	300	250	550	0
Site 05	550	303	247	0	550
Total:	2649	1401	1248	1680	969

Prospective Sample Type and Test Method Breakdown

Performance of the BioCode RPP was evaluated by comparing the BioCode RPP test results with those from an FDA-cleared molecular multiplexed respiratory pathogen panel. Positive agreement was calculated as TP/(TP + FN). TP = true positive by both the comparator test and BioCode RPP; FN = false negative or negative by BioCode RPP only. Negative agreement was calculated as TN/(TN + FP). TN = true negative or negative by both the comparator test and BioCode RPP; FP = false positive or positive by BioCode RPP only. The two-sided 95% confidence interval was calculated with Score method (per CLSI EP12-A2).

Samples for which false positive and/or false negative results (i.e., discrepant results) were obtained when comparing the BioCode RPP results to the comparator test results were further investigated. The discrepancy investigation was mainly conducted by performing independent molecular tests, including analytically validated PCR followed by bi-directional sequencing assays and alternate NAATs.

Of the 2649 specimens included in the prospective clinical study, two specimens, one fresh and one frozen specimen, obtained a final "invalid" result from the BioCode RPP, and were excluded from the performance analyses for all analytes. In addition, three specimens, one fresh and two frozen specimens, obtained a final influenza A "indeterminate" result by the BioCode RPP, and two specimens, one fresh and one frozen, obtained an influenza A "equivocal" result from the comparator test. They were included in the performance analyses for all analytes but excluded from the performance

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calculations for Flu A and Flu A subtypes. Furthermore, two frozen specimens obtained a valid influenza A result from the comparator test without the accompanying Flu A subtyping results. They were included in the performance analyses for all analytes but excluded from the performance calculations for Flu A subtypes.

The Prospective study results stratified by storage condition are presented in the table below.

			Positive Agreement		Negative Agreement
Target	Storage	(n)	PA (%)	95% Cl	NA (%)	95% Cl
Adenovirusa	Fresh	1400	31/40 (77.5%)	(62.5%, 87.7%)	1340/1360 (98.5%)	(97.7%, 99.0%)
	Frozen	1247	37/38 (97.4%)	(86.5%, 99.5%)	1188/1209 (98.3%)	(97.4%, 98.9%)
	Total	2647	68/78 (87.2%)	(78.0%, 92.9%)	2528/2569 (98.4%)	(97.8%, 98.8%)
Bordetella pertussisb	Fresh	1400	1/1 (100%)	(20.7%, 100%)	1387/1399 (99.1%)	(98.5%, 99.5%)
	Frozen	1247	1/1 (100%)	(20.7%, 100%)	1239/1246 (99.4%)	(98.8%, 99.7%)
	Total	2647	2/2 (100%)	(34.2%, 100%)	2626/2645 (99.3%)	(98.9%, 99.5%)
Chlamydia pneumoniaec	Fresh	1400	2/2 (100%)	(34.2%, 100%)	1397/1398 (99.9%)	(99.6%, 100%)
	Frozen	1247	2/2 (100%)	(34.2%, 100%)	1245/1245 (100%)	(99.7%, 100%)
	Total	2647	4/4 (100%)	(51.0%, 100%)	2642/2643 (100%)	(99.8%, 100%)
Coronavirusd	Fresh	1400	35/50 (70%)	(56.2%, 80.9%)	1338/1350 (99.1%)	(98.5%, 99.5%)
	Frozen	1247	76/83 (91.6%)	(83.6%, 95.9%)	1154/1164 (99.1%)	(98.4%, 99.5%)
	Total	2647	111/133 (83.5%)	(76.2%, 88.8%)	2492/2514 (99.1%)	(98.7%, 99.4%)
Human Metapneumoviruse	Fresh	1400	89/93 (95.7%)	(89.5%, 98.3%)	1299/1307 (99.4%)	(98.8%, 99.7%)
	Frozen	1247	46/49 (93.9%)	(83.5%, 97.9%)	1189/1198 (99.2%)	(98.6%, 99.6%)
	Total	2647	135/142 (95.1%)	(90.2%, 97.6%)	2488/2505 (99.3%)	(98.9%, 99.6%)
Human Rhinovirus/Enterovirusf	Fresh	1400	221/261 (84.7%)	(79.8%, 88.5%)	1119/1139 (98.2%)	(97.3%, 98.9%)
	Frozen	1247	162/213 (76.1%)	(69.9%, 81.3%)	1020/1034 (98.6%)	(97.7%, 99.2%)
	Total	2647	383/474 (80.8%)	(77.0%, 84.1%)	2139/2173 (98.4%)	(97.8%, 98.9%)
Influenza Ag	Fresh	1398	115/120 (95.8%)	(90.6%, 98.2%)	1265/1278 (99.0%)	(98.3%, 99.4%)
	Frozen	1244	98/101 (97.0%)	(91.6%, 99.0%)	1131/1143 (99.0%)	(98.2%, 99.4%)
	Total	2642	213/221 (96.4%)	(93.0%, 98.2%)	2396/2421 (99.0%)	(98.5%, 99.3%)
Influenza A H1	Fresh	1398	N/A+	N/A+	1398/1398 (100%)	(99.7%, 100%)
	Frozen	1242	N/A+	N/A+	1242/1242 (100%)	(99.7%, 100%)
	Total	2640	N/A+	N/A+	2640/2640 (100%)	(99.9%, 100%)
Influenza A H1 2009pdmh	Fresh	1398	29/30 (96.7%)	(83.3%, 99.4%)	1365/1368 (99.8%)	(99.4%, 99.9%)
	Frozen	1242	23/23 (100%)	(85.7%, 100%)	1213/1219 (99.5%)	(98.9%, 99.8%)
	Total	2640	52/53 (98.1%)	(90.1%, 99.7%)	2578/2587 (99.7%)	(99.3%, 99.8%)
			Positive Agreement		Negative Agreement
Target	Storage	(n)	PA (%)	95% CI	NA (%)	95% CI
Influenza A H3	Fresh	1398	82/88 (93.2%)	(85.9%, 96.8%)	1306/1310 (99.7%)	(99.2%, 99.9%)
	Frozen	1242	65/69 (94.2%)	(86.0%, 97.7%)	1168/1173 (99.6%)	(99.0%, 99.8%)
	Total	2640	147/157 (93.6%)	(88.7%, 96.5%)	2474/2483 (99.6%)	(99.3%, 99.8%)
Influenza B	Fresh	1400	7/7 (100%)	(64.6%, 100%)	1388/1393 (99.6%)	(99.2%, 99.8%)
	Frozen	1247	44/47 (93.6%)	(82.8%, 97.8%)	1191/1200 (99.2%)	(98.6%, 99.6%)
	Total	2647	51/54 (94.4%)	(84.9%, 98.1%)	2579/2593 (99.5%)	(99.1%, 99.7%)
Mycoplasma pneumoniae	Fresh	1400	8/8 (100%)	(67.6%, 100%)	1381/1392 (99.2%)	(98.6%, 99.6%)
	Frozen	1247	10/10 (100%)	(72.2%, 100%)	1228/1237 (99.3%)	(98.6%, 99.6%)
	Total	2647	18/18 (100%)	(82.4%, 100%)	2609/2629 (99.2%)	(98.8%, 99.5%)
Parainfluenza Virus 1	Fresh	1400	4/4 (100%)	(51.0%, 100%)	1396/1396 (100%)	(99.7%, 100%)
	Frozen	1247	11/13 (84.6%)	(57.8%, 95.7%)	1234/1234 (100%)	(99.7%, 100%)
	Total	2647	15/17 (88.2%)	(65.7%, 96.7%)	2630/2630 (100%)	(99.9%, 100%)
Parainfluenza Virus 2	Fresh	1400	2/3 (66.7%)	(20.8%, 93.9%)	1396/1397 (99.9%)	(99.6%, 100%)
	Frozen	1247	8/9 (88.9%)	(56.5%, 98.0%)	1236/1238 (99.8%)	(99.4%, 100%)
	Total	2647	10/12 (83.3%)	(55.2%, 95.3%)	2632/2635 (99.9%)	(99.7%, 100%)
Parainfluenza Virus 3	Fresh	1400	77/79 (97.5%)	(91.2%, 99.3%)	1312/1321 (99.3%)	(98.7%, 99.6%)
	Frozen	1247	41/43 (95.3%)	(84.5%, 98.7%)	1196/1204 (99.3%)	(98.7%, 99.7%)
	Total	2647	118/122 (96.7%)	(91.9%, 98.7%)	2508/2525 (99.3%)	(98.9%, 99.6%)
Parainfluenza Virus 4	Fresh	1400	1/1 (100%)	(20.7%, 100%)	1399/1399 (100%)	(99.7%, 100%)
	Frozen	1247	15/17 (88.2%)	(65.7%, 96.7%)	1228/1230 (99.8%)	(99.4%, 100%)
	Total	2647	16/18 (88.9%)	(67.2%, 96.9%)	2627/2629 (99.9%)	(99.7%, 100%)
Respiratory Syncytial Virus	Fresh	1400	91/93 (97.8%)	(92.5%, 99.4%)	1293/1307 (98.9%)	(98.2%, 99.4%)
	Frozen	1247	109/111 (98.2%)	(93.7%, 99.5%)	1129/1136 (99.4%)	(98.7%, 99.7%)
	Total	2647	200/204 (98.0%)	(95.1%, 99.2%)	2422/2443 (99.1%)	(98.7%, 99.4%)

Table. Summary of Clinical Study results: Prospective specimens stratified by storage condition.

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No positive reference results recorded

a – Adenovirus: The 10 FNs were not detected by PCR/bi-directional sequencing or alternative NAAT. Of 41 FPs, 37 were not detected and 4 were indeterminate by PCR/bi-directional sequencing.

b – Bordetella pertussis: Of 19 FPs, 4 were detectional sequencing, 3 were indeterminate and 12 were not detected by PCR/bi-directional sequencing.

c – Chlamydia pneumoniae: The 1 FP was detected by PCR/bi-directional sequencing.

d – Coronavirus: Of 22 FNs, 5 were detectional sequencing. 2 were not detected by PCR/bi-directional sequencing but detected by alternative NAAT. 12 were not detected by either alternative NAAT or PCR/bi-directional sequencing. 3 were not detected by PCR/bi-directional sequencing and were not tested by alternative NAAT. The 22 FPs were not detectional sequencing.

e – Human Metapneumovirus: Of 7 FNs, 3 were detected by PCR/bi-directional sequencing. Of 17 FPs, 7 were detected while 10 were not detected by PCR/bi-directional sequencing.

f – Human Rhinovirus/Enterovirus: Of 91 FNs, 26 were indeterminate by PCR/bi-directional sequencing. 25 were not detected by PCR/bi-directional sequencing but were detected by alternative NAAT. 11 were not detectional

13

sequencing or by alternative NAAT. 27 were not detectional sequencing and had insufficient volume for alternative NAAT. Of 34 FPs, 8 were detected and 26 were not detected by PCR/bi-directional sequencing.

g – Influenza A: Of the 8 FNs, 7 were not detectional sequencing. 1 had insufficient volume for follow-up testing. Of 25 FPS, 5 were detected by PCR/bi-directional sequencing, 19 were not detectional sequencing, and 1 had insufficient volume for follow-up testing.

h – Influenza A H1 2009pdm: The 1 FN had insufficient volume for follow-up testing. Of 9 FPs, 4 were detectional sequencing, and 5 were not detected by PCR/bi-directional sequencing.

i – Influenza A H3: Of 10 FNs, 7 were not detected by PCR/bi-directional sequencing. 2 had insufficient volume for followup testing. Of 9 FPs, 3 were detected, 1 was indeterminate, and 4 were not detectional sequencing. 1 had insufficient volume for follow-up testing.

j – Influenza B: The 3 FNs were not detected by PCR/bi-directional sequencing. Of 14 FPs, 1 was detected by PCR/bidirectional sequencing, and 1 had insufficient volume for follow-up testing.

k – Mycoplasma pneumonioe: Of 20 FPs, 7 were detected, and 1 was invalid by PCR/bi-directional sequencing. 2 had insufficient volume for follow-up testing.

I – Parainfluenza Virus 1: The 2 FNs were not detected by PCR/bi-directional sequencing.

m – Parainfluenza Virus 2: Of 2 FNs, 1 was detected by PCR/bi-directional sequencing. The 3 FPs were detected by PCR/bi-directional sequencing.

n – Parainfluenza Virus 3: Of 4 FNs, 2 were detected, and 1 was indeterminate, by PCR/bi-directional sequencing. Of 17 FPs, 8 were detected, 8 were not detected, and 1 was indeterminate, by PCR/bi-directional sequencing.

o – Parainfluenza Virus 4: The 2 FNs were detected by PCR/bi-directional sequencing. The 2 FPs were detectional sequencing.

p – Respiratory Syncytial Virus: The 4 FNs were not detectional sequencing. Of 21 FPs, 18 were not detected, 2 were detected, and 1 was indeterminate, by PCR/bi-directional sequencing.

Prospective prevalence as detected by BioCode® RPP, stratified by age or study site, are presented in tables below.

Table. Prospective prevalence detected by BioCode® RPP stratified by Age

| Analyte | Overall
(N=2647) | ≤5 yrs
(N=1004) | 6-21 yrs
(N=609) | 22-59 yrs
(N=531) | 60+ yrs
(N=503) |
|------------------------------|---------------------|--------------------|---------------------|----------------------|--------------------|
| Adenovirus | 109 (4.1%) | 80 (8.0%) | 20 (3.3%) | 5 (0.9%) | 4 (0.8%) |
| Bordetella pertussis | 21 (0.8%) | 8 (0.8%) | 12 (2.0%) | 0 (0.0%) | 1 (0.2%) |
| Chlamydia pneumoniae | 5 (0.2%) | 1 (0.1%) | 3 (0.5%) | 1 (0.2%) | 0 (0.0%) |
| Coronavirus | 133 (5.0%) | 63 (6.3%) | 27 (4.4%) | 19 (3.6%) | 24 (4.8%) |
| Human Metapneumovirus | 152 (5.7%) | 94 (9.4%) | 26 (4.3%) | 15 (2.8%) | 17 (3.4%) |
| Human Rhinovirus/Enterovirus | 417 (15.8%) | 234 (23.3%) | 101 (16.6%) | 51 (9.6%) | 31 (6.2%) |
| Influenza A | 238 (9.0%) | 71 (7.1%) | 84 (13.8%) | 47 (8.9%) | 36 (7.2%) |
| Influenza A H1 | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) |
| Influenza A H1 2009pdm | 62 (2.3%) | 24 (2.4%) | 15 (2.5%) | 15 (2.8%) | 8 (1.6%) |
| Influenza A H3 | 157 (5.9%) | 45 (4.5%) | 61 (10.0%) | 26 (4.9%) | 25 (5.0%) |
| Influenza B | 65 (2.5%) | 13 (1.3%) | 26 (4.3%) | 15 (2.8%) | 11 (2.2%) |
| Mycoplasma pneumoniae | 38 (1.4%) | 9 (0.9%) | 21 (3.4%) | 6 (1.1%) | 2 (0.4%) |
| Parainfluenza Virus 1 | 15 (0.6%) | 4 (0.4%) | 1 (0.2%) | 4 (0.8%) | 6 (1.2%) |
| Parainfluenza Virus 2 | 13 (0.5%) | 6 (0.6%) | 4 (0.7%) | 3 (0.6%) | 0 (0.0%) |
| Parainfluenza Virus 3 | 135 (5.1%) | 74 (7.4%) | 21 (3.4%) | 21 (4.0%) | 19 (3.8%) |

14

| Analyte | Overall
(N=2647) | ≤5 yrs
(N=1004) | 6-21 yrs
(N=609) | 22-59 yrs
(N=531) | 60+ yrs
(N=503) |
|-----------------------------|---------------------|--------------------|---------------------|----------------------|--------------------|
| Parainfluenza Virus 4 | 18 (0.7%) | 12 (1.2%) | 1 (0.2%) | 2 (0.4%) | 3 (0.6%) |
| Respiratory Syncytial Virus | 221 (8.3%) | 156 (15.5%) | 30 (4.9%) | 19 (3.6%) | 16 (3.2%) |

Table. Prospective prevalence detected by BioCode® RPP stratified by site.

| Analyte | Overall
(N=2647) | Site 1
(N=529) | Site 2
(N=419) | Site 3
(N=599) | Site 4
(N=550) | Site 5
(N=550) |
|------------------------------|---------------------|-------------------|-------------------|-------------------|-------------------|-------------------|
| Adenovirus | 109 (4.1%) | 8 (1.5%) | 27 (6.4%) | 21 (3.5%) | 17 (3.1%) | 36 (6.5%) |
| Bordetella pertussis | 21 (0.8%) | 0 (0.0%) | 19 (4.5%) | 1 (0.2%) | 0 (0.0%) | 1 (0.2%) |
| Chlamydia pneumoniae | 5 (0.2%) | 0 (0.0%) | 3 (0.7%) | 0 (0.0%) | 1 (0.2%) | 1 (0.2%) |
| Coronavirus | 133 (5.0%) | 36 (6.8%) | 21 (5.0%) | 14 (2.3%) | 26 (4.7%) | 36 (6.5%) |
| Human Metapneumovirus | 152 (5.7%) | 18 (3.4%) | 22 (5.3%) | 26 (4.3%) | 38 (6.9%) | 48 (8.7%) |
| Human Rhinovirus/Enterovirus | 417 (15.8%) | 48 (9.1%) | 83 (19.8%) | 65 (10.9%) | 125 (22.7%) | 96 (17.5%) |
| Influenza A | 238 (9.0%) | 49 (9.3%) | 50 (11.9%) | 35 (5.8%) | 41 (7.5%) | 63 (11.5%) |
| Influenza A H1 | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) |
| Influenza A H1 2009pdm | 62 (2.3%) | 13 (2.5%) | 7 (1.7%) | 11 (1.8%) | 22 (4.0%) | 9 (1.6%) |
| Influenza A H3 | 157 (5.9%) | 32 (6.0%) | 40 (9.5%) | 19 (3.2%) | 17 (3.1%) | 49 (8.9%) |
| Influenza B | 65 (2.5%) | 12 (2.3%) | 32 (7.6%) | 15 (2.5%) | 2 (0.4%) | 4 (0.7%) |
| Mycoplasma pneumoniae | 38 (1.4%) | 4 (0.8%) | 15 (3.6%) | 5 (0.8%) | 3 (0.5%) | 11 (2.0%) |
| Parainfluenza Virus 1 | 15 (0.6%) | 4 (0.8%) | 0 (0.0%) | 11 (1.8%) | 0 (0.0%) | 0 (0.0%) |
| Parainfluenza Virus 2 | 13 (0.5%) | 0 (0.0%) | 0 (0.0%) | 3 (0.5%) | 8 (1.5%) | 2 (0.4%) |
| Parainfluenza Virus 3 | 135 (5.1%) | 12 (2.3%) | 28 (6.7%) | 41 (6.8%) | 9 (1.6%) | 45 (8.2%) |
| Parainfluenza Virus 4 | 18 (0.7%) | 0 (0.0%) | 1 (0.2%) | 9 (1.5%) | 5 (0.9%) | 3 (0.5%) |
| Respiratory Syncytial Virus | 221 (8.3%) | 21 (4.0%) | 35 (8.4%) | 25 (4.2%) | 49 (8.9%) | 91 (16.5%) |

The overall success rate for initial specimen testing in the prospective study was 98.8% (2618/2649) (95% Cl: 98.3% - 99.2%); 31 tests were unsuccessful (26 tests with an invalid result and 5 tests due to low BMB count/instrument error). Upon a single retest per the instructions for use, 29 of the 31 initially unsuccessful specimens generated a valid result. The final validity rate was 99.9% (2647/2649) (95% Cl: 99.7%-100%).

15

There were 193 samples with mixed infections by the BioCode® RPP in the prospective clinical study (193/2649 or 7.3%). The distribution, prevalence and most common co-infections combinations detected by BioCode RPP in the prospective clinical study are summarized in the tables below.

Table. Distribution of co-infection combinations detected by BioCode® RPP from prospective clinical study

| Analytes Detected

Simultaneously	Number of Specimen
2	168 (87.0%)
3	24 (12.4%)
5	1 (0.5%)
Total Co-Infections	193

Table. Prevalence of targets in co-infection combinations detected by BioCode® RPP from prospective clinical study

Analyte	Prevalence in Co-Infections (N=193)
Adenovirus	51 (26.4%)
Bordetella pertussis	14 (7.3%)
Chlamydia pneumoniae	1 (0.5%)
Coronavirus	47 (24.4%)
Human Metapneumovirus	35 (18.1%)
Human Rhinovirus/Enterovirus	101 (52.3%)
Influenza A	37 (19.2%)
Influenza B	8 (4.1%)
Mycoplasma pneumoniae	7 (3.6%)
Parainfluenza Virus 1	2 (1.0%)
Parainfluenza Virus 2	4 (2.1%)
Parainfluenza Virus 3	39 (20.2%)
Parainfluenza Virus 4	8 (4.1%)
Respiratory Syncytial Virus	59 (30.1%)

Table. Most prevalent multiple detection combinations (5 or more instances) detected by BioCode® RPP from prospective clinical study.

Co-Infection Combination	Number of Specimen
Human Rhinovirus/Enterovirus + Respiratory Syncytial Virus	17
Adenovirus + Human Rhinovirus/Enterovirus	13
Human Metapneumovirus + Human Rhinovirus/Enterovirus	13
Human Rhinovirus/Enterovirus + Influenza A	11
Adenovirus + Respiratory Syncytial Virus	10
Human Rhinovirus/Enterovirus + Parainfluenza Virus 3	10
Coronavirus + Human Rhinovirus/Enterovirus	8
Coronavirus + Respiratory Syncytial Virus	7
Bordetella pertussis + Human Rhinovirus/Enterovirus	6

16

Co-Infection Combination	Number of Specimen
Adenovirus + Parainfluenza Virus 3	5
Coronavirus + Human Metapneumovirus	5
Coronavirus + Influenza A	5

Testing of Preselected Archived Specimens

Some of the pathogens on the BioCode RPP were of low prevalence and were not encountered in sufficiently large numbers during the prospective study to adequately demonstrate system performance. To supplement the results of the prospective clinical study, an evaluation of preselected archived retrospective specimens was performed. These specimens were archived NPS in VTM or UTM specimens that were selected because they had previously tested positive for one of the following pathogens at the source laboratory: coronavirus 229E, coronavirus HKU1, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 4, Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae, or had been negative in previous laboratory testing.

A total of 165 clinical specimens were enrolled for testing in this retrospective study. The specimens were randomized such that the users performing the BioCode RPP assay were blinded to the expected test result and shipped to one of two of the testing sites participated in the prospective clinical study for testing.

A summary of the demographic information of the tested samples is provided in the following table:

Characteristic	Archived Study Specimens
Total Specimens	165
Gender (n/N (%))
Male	96/165 (58.2%)
Female	69/165 (41.8%)
Age Category (n/N (%))
0-5 yrs	77/165 (46.7%)
6-21 yrs	58/165 (35.2%)
22-59 yrs	15/165 (9.1%)
60+ yrs	15/165 (9.1%)

Demographics of Archived Specimens

The performance of the BioCode RPP was evaluated by comparing the BioCode RPP test results with those from an FDA-cleared molecular multiplexed respiratory pathogen panel, the same panel test as the one used as the comparator in the prospective clinical study. The BioCode RPP retrospective performance data expressed as positive percent and negative percent agreements against the comparator method are presented by pathogen in the table below.

17

Target	(n)	PA (%)	95% CI	NA (%)	95% CI
Adenovirusa	165	7/7 (100%)	(64.6%, 100%)	155/158 (98.1%)	(94.6%, 99.4%)
Bordetella pertussisb	165	10/10 (100%)	(72.2%, 100%)	144/155 (92.9%)	(87.7%, 96.0%)
Chlamydia pneumoniae	165	10/10 (100%)	(72.2%, 100%)	155/155 (100%)	(97.6%, 100%)
Coronavirusc	165	52/59 (88.1%)	(77.5%, 94.1%)	99/106 (93.4%)	(87.0%, 96.8%)
Human Metapneumovirus	165	4/4 (100%)	(51.0%, 100%)	161/161 (100%)	(97.7%, 100%)
Human Rhinovirus/Enterovirusd	165	16/23 (69.6%)	(49.1%, 84.4%)	141/142 (99.3%)	(96.1%, 99.9%)
Influenza A	165	N/A+	N/A+	165/165 (100%)	(97.7%, 100%)
Influenza A H1	165	N/A+	N/A+	165/165 (100%)	(97.7%, 100%)
Influenza A H1 2009pdm	165	N/A+	N/A+	165/165 (100%)	(97.7%, 100%)
Influenza A H3	165	N/A+	N/A+	165/165 (100%)	(97.7%, 100%)
Influenza Be	165	2/3 (66.7%)	(20.8%, 93.9%)	162/162 (100%)	(97.7%, 100%)
Mycoplasma pneumoniaef	165	7/7 (100%)	(64.6%, 100%)	153/158 (96.8%)	(92.8%, 98.6%)
Parainfluenza Virus 1g	165	12/13 (92.3%)	(66.7%, 98.6%)	152/152 (100%)	(97.5%, 100%)
Parainfluenza Virus 2h	165	19/20 (95%)	(76.4%, 99.1%)	144/145 (99.3%)	(96.2%, 99.9%)
Parainfluenza Virus 3	165	1/1 (100%)	(20.7%, 100%)	164/164 (100%)	(97.7%, 100%)
Parainfluenza Virus 4i	165	14/15 (93.3%)	(70.2%, 98.8%)	150/150 (100%)	(97.5%, 100%)
Respiratory Syncytial Virusj	165	11/12 (91.7%)	(64.6%, 98.5%)	152/153 (99.3%)	(96.4%, 99.9%)

Table. Results from Archived Specimens tested by the BioCode® RPP with easyMAG extraction system.

No positive reference results recorded

a - Adenovirus: Of 3 FPs, 1 was detected by PCR/bi-directional sequencing and 2 were not detected by PCR/bi-directional sequencing.

b - Bordetella pertussis: Of 11 FPs, 7 were detected by PCR/bi-directional sequencing and 4 were not detected by PCR/bidirectional sequencing.

c - Coronavirus: Of 7 FNs, 4 were detected by PCR/bi-directional sequencing while 3 were not detectional sequencing. The 7 FPs were not detected by PCR/bi-directional sequencing. All had low MFls (Chlamydia pneumoniae | AR39 | ATCC VR-
53592 | 16.7 CFU/mL | 20/20 | 33.3 CFU/mL | 20/20 |
| Mycoplasma
pneumoniae | M129 | Zeptometrix
0801579 | 15.0 CCU/mL | 20/20 | 15.0 CCU/mL | 20/20 |

Table. Limit of Detection by extraction system

27

Coronavirus HKU1 clinical sample quantified (copies/mL) with Applied BioCode validated SYBR assay using an IVT RNA a. standard.

Conclusion: LoDs were comparable (within 3-fold) for each extraction system.

Analytical Reactivity/Inclusivity

A study was performed to verify Analytical Reactivity/Inclusivity of the BioCode® Respiratory Pathogen Panel (RPP). Different strains, serotypes were selected that represent various temporal, geographic, and genetic diversity for each analyte. This study tested a panel of titered stocks of relevant organisms diluted in simulated NPS in UTM matrix starting at 3x LoD. Samples not detected at 3x LoD, were tested at higher concentrations. For Influenza B strains were grouped into Yamagata, Victoria or unknown lineages. Yamagata strains were tested according to the protocol at 3x and higher of the Yamagata strain LoD (LoD - 0.01 TCIDsomL Flu B/Florida/4/2006). However, for Victoria strains, the validation LoD result was higher than expected (LoD-48.6 TCID50/mL for Flu B /Hong Kong/S/1972) which could be related to difference in titration from vendor sources. Therefore, for Victoria strains, testing for inclusivity was performed starting at 0.03x and 0.3x of the Victoria strain of unknown lineages of influenza B were tested at 3x LoD of Yamagata strain. Each sample was extracted in triplicate on the easyMAG and tested with the RPP on the BioCode® MDx-3000 system according to the instructions for use.

Assay reactivity for less common strains or serotypes that could not be tested due to unavailability was predicted using in silico analysis.

Results: The organisms listed below were detected at the concentrations indicated. See Table below.

| Organism/
Typea | Strain/Location/Year Detected | Multiple of
LoD
Detected |
|---------------------------|---------------- | Influenza A H1N1 | Solomon Island/03/06 | | Singapore/63/04 | | PR/8/34 | | A/Brisbane/59/2007 | | A/Taiwan/42/06 | | A/New jersey/8/76b | Organism/
Typea | Strain/Location/Year Detected | Multiple of
LoD
Detected |
| | A/Denver/1/1957 | | A/FM/1/47 | | A/Weiss/43c | | A/Beijing/262/95d | | A/Mal/302/54 | Influenza A H1N2 | Recombinant; A/NWS/1934 (HA) x
A/Rockefeller Institute/5/1957
(NA)e | BEI | NR-3682 | 135 CEID50/mL | | NY/01/09 | | NY/02/09 | | NY/03/09 | | A/Houston/3H/20 (H1N1)pdm09f | | Influenza (Canada/6294/09) | | Influenza (Mexico/4108/09) | Influenza A H1N1
pdm09 | California/04/09, cell isolateg | | A/Christ Church/16/2010h | | A/Brisbane/02/2018 | | A/Wisconsin/15/2009j | | A/Texas/50/12 | | A/Brisbane/10/2007 | | A/Port Chalmers/1/73 | Influenza A H3N2 | A/Victoria/3/1975 | Organism/
Typea | Strain/Location/Year Detected | Multiple of
LoD
Detected |
| | A/Victoria/361/2011l | | A/Victoria/3/75 | | A/Uruguay/716/07m | | A/HK/H090-756-V1(0)/2009n | | A/Hong Kong/8/68 | | A/Switzerland/9715293/ 13 | | A/Aichi/2/68 | | MRC-2 | | A/Perth/16/2009 | | A/Kansas/14/2017o | Vendor | Catalog # | Concentration
-----------------------------------------------------------------------------|-------------|------------|---------------------------|--------------------------------|
| Zeptometrix | 0810036CFN | 45 TCID50/mL | 3x |
| Zeptometrix | 0810246CF | 45 TCID50/mL | 3x |
| Zeptometrix | 0810245CF | 45 TCID50/mL | 3x |
| Zeptometrix | 0810244CF | 45 TCID50/mL | 3x |
| Zeptometrix | 0810247CF | 45 TCID50/mL | 3x |
| ATCC | VR-897 | 7.5x103 CEID50/mL | 500xb |
| Vendor | Catalog # | Concentration
| VIRAPUR | NA | 45 TCID50/mL | 3x |
| ATCC | VR-97 | 45 CEID50/mL | 3x |
| ATCC | VR-96 | 1.5x104 CEID50/mL | 1000xc |
| BEI | NR-12277 | 450 CEID50/mL | 30xd |
| ATCC | VR-98 | 45 CEID50/mL | 3x |
Kilbourne F64,
| 5xe |
| Zeptometrix | 0810248CF | 1.2 TCID50/mL | 3x |
| Zeptometrix | 0810109CFN | 1.2 TCID50/mL | 3x |
| Zeptometrix | 0810249CF | 1.2 TCID50/mL | 3x |
09
| BEI | NR-20340 | 1.2 TCID50/mL | 3x |
A H1N1pdm
| Zeptometrix | 0810109CFJ | 1.2 TCID50/mL | 3x |
A H1N1pdm
| Zeptometrix | 0810166CF | 1.2 TCID50/mL | 3x |
| BEI | NR-13658 | 40 TCID50/mL | 100xg |
| CDC | N/A | 400 EID50/mL | 1000xh |
| CDC | N/A | 40 EID50/mL | 100xi |
| BEI | NR-42007 | 12 CEID50/mL | 3x |
| Zeptometrix | 0810238CF | 12 TCID50/mL | 3x |
| Zeptometrix | 0810138CF | 12 TCID50/mL | 3x |
| ATCC | VR-810 | 200 CEID50/mL | 50xk |
| VIRAPUR | NA | 12 TCID50/mL | 3x |
| Vendor | Catalog # | Concentration
| BEI | NR-44022 | 12 CEID50/mL | 3x |
| ATCC | VR-822 | 12 CEID50/mL | 3x |
| BEI | NR-42003 | 40 TCID50/mL | 10x |
| BEI | NR-44344 | 12 TCID50/mL | 3x |
| Zeptometrix | 0810250CF | 12 TCID50/mL | 3x |
| VIRAPUR | NA | 12 TCID50/mL | 3x |
| ATCC | VR-547 | 12 TCID50/mL | 3x |
| ATCC | VR-777 | 12 TCID50/mL | 3x |
| CDC | N/A | 40 EID50/mL | 10x |
| CDC | N/A | 8000 EID50/mL | 2000xo |

Table. Influenza A isolates tested during the inclusivity study

28

29

a - In silico analysis predicts detection of Influenza A H2N3, H5N2, H5N3, H7N7, H7N9, H3N2, H3N5, H3N7, H3N8 as Influenza A. However, predicted reactivities of the subtyping assays for these influenza A strains of animal origin are variable.

b – Influenza A H1N1 – [A/New jersey/8/76]. Detected as Flu A (no subtype) at 3x LoD. Detected as dual positive A/H1 and A/H1pdm09 at 500x LoD.

c – Influenza A H1N1 [A/Weiss/43]. Detected as Flu A (no subtype) at 100x LoD. In silico analysis showed several mismatches in the forward primer for the Flu A/H1 subtyping assay which may account for the observed lower sensitivity of the Flu A/H1 subtyping assay for this strain.

d – Influenza A H1N1 [A/Beijing/262/95]. Virus obtained through BEI Resources. NIAID. NH: Influenza A. A/Beijing/262/95 (H1N1), NR-12277. Detected as Flu A (no subtype) at 10x LoD. In silico analysis showed a G-A mismatch in the 3' terminal position in the forward primer for the Flu A/H1 subtyping assay which may account for the observed lower sensitivity of the Flu A/H1 subtyping assay for this strain.

e - Influenza A H1N2 [Recombinant Virus obtained through BEI Resources, NIAID, NH: Influenza A, Kilbourne F64, A/NWS/1934 (HA) x A/Rockefeller Institute/5/1957 (NA) (H1N2), NR-3682.Detected as Flu A (no subtype) at 3x LoD.

f - Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Houston/3H/2009 (H1N1)pdm09, NR-20340. g – Virus obtained through BEI Resources, NIAID, NH: Influenza A, A/California/04/09, cell isolate (H1N1) pdm09, NR-13658. In silico analysis of a partial sequence of this strain for the Flu A/H1pdm09 subtyping assay does nalytical reactivity. Titering inconsistency from the vendor rather than reduced reactivity due to assay design is suggested. h - Virus obtained through the CDC Influenza Divis of partial sequences of this strain for the Flu A/H1pdm09 HA subtyping assay and the Flu A matrix gene assay does not predict reactivity. Titering inconsistency from the source laboratory (EIDsp/mL vs. TCID-n/mL) rather than reduced reactivity due to assay design is suggested.

i - Virus obtained through the CDC Influenza Division. In silico analysis did not reveal any critical mismatch in the Flu A/H1pdm09 HA subtyping assay primers and probe binding regions or any mismatch in the FluA matrix gene assay primers and probe binding regions that would predict reactivity. Titering inconsistency from the source laboratory (EID50/mL vs. TCID50/mL) rather than reduced reactivity due to assay design is suggested.

j – Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Wisconsin/15/2009 (H3N2), NR-42007.

k - In silico analysis revealed a few non-critical mismatches in the FluA/H3 subtyping HA assay probe binding region that could contribute to the observed lower reactivity. However, titering inconsistency from the than reduced reactivity due to assay design is suspected.

30

l - Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Victoria/361/2011 (H3N2), NR-44022 m - Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/Uruguay/716/07 (H3N2), NR-42003 n - Virus obtained through BEI Resources, NIAID, NIH: Influenza A, A/HK/H090-756-V1(0)/2009 (H3N2), NR-44344 o – Virus obtained through the CDC Influenza Division. This strain was detected by BioCode RPP as Flu A (no subtype) at 50xLoD. In silico analysis did not reveal any mismatch in the Flu A matrix assay primers and probe binding regions but showed 3 mismatches in the Flu A/H3 subtyping HA assay primers binding regions that could predict reactivity, 1 mismatch at the 9th position from the 3' end of the reverse primer (mismatch at the 18th position from the 3' end of the reverse primer (mismatch at the 20th position from the 3' end of the reverse primer (mismatch #3). Wet testing data suggested that mismatch #1 is likely the root cause for the observed significant reduction in analytical reactivity for this strain. For patient samples contain a Flu A/H3 strain that harbors mismations, the BioCode RPP will likely report a Flu A (no subtype detected) result. Although estimated prevalence of a sequence variant based solely on in silico analysis may not accurately reflect the actual prevalence variant in circulation during an influenza season, based on an in silico analysis, of all the Flu A/H3 strains isolated in 2019 with published HA sequences, 73.8% of the strains harbor mismatch #1.

| Organism/Lineage | Location/Strain/ Year | Vendor | Catalog # | Concentration
Detected | Multiple of
LoD
Detecteda |
|-------------------------|--------------------------|-------------|-----------|---------------------------|---------------------------------|
| | B/Malaysia/2506/2004d | BEI | NR-9723 | 1.458
TCID50/mL | 0.03x |
| | B/Malaysia/2506/2004 | Zeptometrix | 0810258CF | 1.458
TCID50/mL | 0.03x |
| | B/Ohio/01/2005e | BEI | NR-41801 | 14.58
CEID50/mL | 0.3x |
| Influenza B (Victoriab) | B/Brisbane/33/2008f | BEI | NR-42006 | 14.58
CEID50/mL | 0.3x |
| | B/Nevada/03/2011g | BEI | NR-44023 | 1.458
CEID50/mL | 0.03x |
| | B/Michigan/09/2011 | CDC | N/A | 14.58 EID50/mL | 0.3x |
| | B/Colorado/06/2017 | CDC | N/A | 14.58 EID50/mL | 0.3x |
| | B/Texas/06/2011h | BEI | NR-44024 | 50 TCID50/mL | 5000xh |
| | B/Sydney/507/2006i | BEI | NR-36526 | 8.0 TCID50/mL | 800xi |
| | B/Wisconsin/1/10 | Zeptometrix | 0810241CF | 0.03 TCID50/mL | 3x |
| Influenza B (Yamagata) | B/Massachusetts/2/12 | Zeptometrix | 0810239CF | 0.03 TCID50/mL | 3x |
| | B/Christchurch/33/2004j | BEI | NR-36536 | 0.03 TCID50/mL | 3x |
| | B/New Hampshire/01/2016k | CDC | N/A | 10 EID50/mL | 1000xk |
| | B/Phuket/3073/2013l | CDC | N/A | 5 EID50/mL | 500xl |
| | B/lee/1940 | Zeptometrix | 0810257CF | 0.03 TCID50/mL | 3x |

		Table. Influenza B isolates tested during the inclusivity study

31

| Organism/Lineage | Location/Strain/ Year | Vendor | Catalog # | Concentration
Detected | Multiple of
LoD
Detecteda |
|-----------------------------------|-----------------------|--------|-----------|---------------------------|---------------------------------|
| Influenza B (unknown
lineagec) | B/Taiwan/2/1962 | ATCC | VR-295 | 5.0 CEID50/mL | 500xm |
| Influenza B (unknown
lineagec) | B/Allen/45 | ATCC | VR-102 | 0.05 CEID50/mL | 5x |
| Influenza B (unknown
lineagec) | B/Brigit | ATCC | VR-786 | 0.03 TCID50/mL | 3x |

a - If either FluB assay has MFI above the cutoff, the software will report as Detected for Influenza B

b - For Victoria lineage strains testing started at 0.03x LoD rather than 3x LoD.

c - Strains of unknown lineages were assayed starting at 3x LoD of Yamagata strain.

d - Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Malaysia/2506/2004, NR-9723.

e - Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Ohio/01/2005, NR-41801.

f - Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Brisbane/33/2008, NR-42006.

g - Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Nevada/03/2011, NR-44023.

h - Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Texas/06/2011, NR-44024. In silico analysis does not predict reduced analytical reactivity. Titering inconsistency from the vendor reactivity due to assay design is suggested.

i - Virus obtained through BEI Resources, NIAID, NH: Influenza B, B/Sydney/507/2006, NR-36526. In silico analysis does not predict reduced analytical reactivity. Titering inconsistency from the vendor reactivity due to assay design is suggested.

j - Virus obtained through BEI Resources, NIAID, NIH: Influenza B, B/Christchurch/33/2004, NR-36536.

k - Virus obtained through the CDC Influenza Division. In silico analysis did not reveal any critical mismatch in the Flu B NS1 assay primers and probe binding regions that would predict reactivity. And In silico analysis did not reveal any mismatch in the Flu B matrix assay primers and probe binding regions. Titering inconsistency from the source laboratory (EID50/mL vs. TCID50/mL) rather than reduced reactivity due to assay design is suggested.

l - Virus obtained through the CDC Influenza Divis did not reveal any critical mismatch in the Flu B NS1 assay primers and probe binding regions that would predict reduced analytical reactivity. And In silico analysis did not reveal any mismatch in the Flu B matrix assay primers and probe binding regions. Titering inconsistency from the source laboratory (EID50/mL vs. TCID50/mL) rather than reduced reactivity due to assay design is suggested.

m - In silico analysis could not be performed due to unavailability of sequence information for this strain. Titering inconsistency from the vendor rather than reduced reactivity due to assay design is suspected.

32

| Organism/Type | Strain/Location/Year | Vendor | Catalog # | Concentration
Detected | Multiple of
LoD
Detected |
|---------------------------------------|-----------------------------|-------------|-----------|---------------------------|--------------------------------|
| Respiratory Syncytial
Virus Type A | TN/1998/3-2a | BEI | NR-28529 | 0.99 TCID50/mL | 3x |
| | TN/2000/3-4b | BEI | NR-28530 | 3.3 TCID50/mL | 10x |
| | TN/98/12-21c | BEI | NR-28528 | 0.99 TCID50/mL | 3x |
| | Long/Maryland/1956 | ATCC | VR-26 | 3.3 TCID50/mL | 10x |
| | 9320/Massachusetts/1977 | ATCC | VR-955 | 0.99 TCID50/mL | 3x |
| Respiratory Syncytial
Virus Type B | B1d | BEI | NR-4052 | 3.3 TCID50/mL | 10x |
| | WV/14617/85 | ATCC | VR-1400 | 0.99 TCID50/mL | 3x |
| | 18537/Washington
DC/1962 | ATCC | VR-1580 | 0.99 TCID50/mL | 3x |
| | CH93(18)18 | Zeptometrix | 0810040CF | 0.99 TCID50/mL | 3x |

Table. Respiratory Syncytial Virus isolates tested during the inclusivity study

a - Virus obtained through BEI Resources, NIAID, NIH: RSV, TN/1998/3-2, NR-28529

b - Virus obtained through BEI Resources, NIAID, NIH: RSV, TN/2000/3-4, NR-28530

c - Virus obtained through BEI Resources, NIAID, NIH: RSV, TN/98/12-21, NR-28528

d - Virus obtained through BEI Resources, NIAID, NIH: RSV, B1, NR-4052

Table. Human Metapneumovirus isolates tested during the inclusivity study

| Genotype | Serotype | Location/
Year | Vendor | Catalog # | Concentration
Detected | Multiple
of LoD
Detecteda |
|-------------------------------------|----------|-------------------|-------------|-----------|---------------------------|---------------------------------|
| Human
Metapneumovirus
Type A1 | 9 | lowa3/2002 | Zeptometrix | 0810160CF | 45 TCID50/mL | 3x |
| Human
Metapneumovirus
Type A2 | 27 | lowa27/2004 | Zeptometrix | 0810164CF | 45 TCID50/mL | 3x |
| Human
Metapneumovirus
Type B1 | 3 | Peru2/2002 | Zeptometrix | 0810156CF | 45 TCID50/mL | 3x |
| Human
Metapneumovirus
Type B1 | 5 | Peru3/2003 | Zeptometrix | 0810158CF | 45 TCID50/mL | 3x |
| Human
Metapneumovirus
Type B2 | 4 | Peru1/2002 | Zeptometrix | 0810157CF | 45 TCID50/mL | 3x |
| Human
Metapneumovirus
Type B2 | 8 | Peru6/2003 | Zeptometrix | 0810159CF | 45 TCID50/mL | 3x |
| Human
Metapneumovirus
Type B2 | 18 | lowa18/2003 | Zeptometrix | 0810162CF | 45 TCID50/mL | 3x |
| Human
Metapneumovirus
Type B2 | Unknown | TN/91-316b | BEI | NR-22232 | 45 TCID50/mL | 3x |

a - If either assay has MFI above the cutoff, the software will report as Detected for Human Metapneumovirus

b - Virus obtained through BEI Resources, NIAID, NIH: Human Metapneumovirus, TN/91-316, NR-22232

33

| Organism/Subtype | Strain/Location/Year | Vendor | Catalog # | Concentration
Detected | Multiple of
LoD
Detected |
|------------------------|--------------------------------|-------------|------------|---------------------------|--------------------------------|
| Parainfluenza Virus 1 | FRA/27344044/2007a | BEI | NR-48681 | 27 TCID50/mL | 3x |
| Parainfluenza Virus 1 | FRA/29221106/2009b | BEI | NR-48680 | 27 TCID50/mL | 3x |
| Parainfluenza Virus 1 | Unknown | Zeptometrix | 0810014CF | 27 TCID50/mL | 3x |
| Parainfluenza Virus 2 | Greerc | BEI | NR-3229 | 5.4 TCID50/mL | 3x |
| | Unknown | Zeptometrix | 0810015CF | 5.4 TCID50/mL | 3x |
| Parainfluenza Virus 3 | NIH 47885,
Wash/47885/57d | BEI | NR-3233 | 45 TCID50/mL | 3x |
| | C243/Washington
DC/1957 | ATCC | VR-93 | 45 TCID50/mL | 3x |
| Parainfluenza Virus 4a | M-25/1958e | BEI | NR-3237 | 27 TCID50/mL | 3x |
| Parainfluenza Virus 4b | CH-19503/Washington
DC/1962 | ATCC | VR-1377 | 27 TCID50/mL | 3x |
| | Unknown | Zeptometrix | 0810060BCF | 27 TCID50/mL | 3x |

Table. Parainfluenza Virus (1-4) isolates tested during inclusivity.

a - Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 1, HIPIV1/FRA/27344044/2007, NR-48681

b - Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 1, HPIV1/FRA/29221106/2009, NR-48680

c - Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 2, Greer, NR-3229

d - Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 3, NIH 47885, NR-3233

e - Virus obtained through BEI Resources, NIAID, NIH: Parainfluenza Virus 4a, M-25, NR-3237

Table. Adenovirus isolates tested during the inclusivity study

| Speciesb | Serotype | Strain/Location/Year | Vendor | Catalog # | Concentration
Detected | Multiple of
LoD
Detectedc |
|---------------|----------|----------------------------|-------------|-----------|---------------------------|---------------------------------|
| Adenovirus Aa | 31 | Unknown | Zeptometrix | 0810073CF | 18 TCID50/mL | 3x |
| | 12 | Huie/Massachusetts | ATCC | VR-863 | 18 TCID50/mL | 3x |
| | 18 | Washington DC/1954 | ATCC | VR-19 | 18 TCID50/mL | 3x |
| Adenovirus B | 3 | GB/Maryland/1953 | ATCC | VR-3 | 3.6 TCID50/mL | 3x |
| | 14 | Unknown | Zeptometrix | 0810108CF | 3.6 TCID50/mL | 3x |
| | 16 | CH.79/Saudi
Arabia/1955 | ATCC | VR-17 | 3.6 TCID50/mL | 3x |
| | 35 | Holden | ATCC | VR-718 | 3.6 TCID50/mL | 3x |
| Adenovirus C | 1 | Unknown | Zeptometrix | 0810050CF | 18 TCID50/mL | 3x |
| | 5 | Unknown | Zeptometrix | 0810020CF | 18 TCID50/mL | 3x |

34

| Speciesb | Serotype | Strain/Location/Year | Vendor | Catalog # | Concentration
Detected | Multiple of
LoD
Detectedc |
|---------------|----------|--------------------------------------|-------------|-----------|---------------------------|---------------------------------|
| Adenovirus Da | 6 | Tonsil 99/Washington
DC | ATCC | VR-6 | 18 TCID50/mL | 3x |
| | 8 | Unknown | Zeptometrix | 0810069CF | 18 TCID50/mL | 3x |
| | 17 | CH. 22/Saudi
Arabia/1955 | ATCC | VR-1836 | 18 TCID50/mL | 3x |
| | 20 | Unknown | Zeptometrix | 0810115CF | 18 TCID50/mL | 3x |
| | 26 | Unknown | Zeptometrix | 0810117CF | 18 TCID50/mL | 3x |
| | 37 | Unknown | Zeptometrix | 0810119CF | 18 TCID50/mL | 3x |
| Adenovirus Fa | 40 | Unknown | Zeptometrix | 0810084CF | 18 TCID50/mL | 3x |
| | 41 | Tak/73-
3544/Netherlands/19
73 | ATCC | VR-930 | 18 TCID50/mL | 3x |

a – Adenovirus C LoD used as LoD was not determined for Species A, D or F.

b – In silico analysis predicts detection of Adenovirus species E serotypes (Adenovirus 4 strain was tested as a part of LoD studies as well.

c – If either AdV assay has MFI above the cutoff the software will report as Detected for Adenovirus.

| Organism | Location/Year | Vendor | Catalog # | Concentration
Detected | Multiple
of LoD
Detected |
|------------------|----------------|----------------------|-----------|---------------------------|--------------------------------|
| Coronavirus 229E | Unknown | ATCC | VR-740 | 1.8 TCID50/mL | 3x |
| Coronavirus NL63 | Amsterdam/2003 | BEIb | NR-470 | 0.12 TCID50/mL | 3x |
| Coronavirus OC43 | Unknown | ATCC | VR-1558 | 0.12 TCID50/mL | 3x |
| HKU1a | Unknown | Clinical Sample 5016 | Unknown | 1.51x105 copies/mL | 3x |
| | Unknown | Clinical Sample 5036 | Unknown | 1.51x105 copies/mL | 3x |
| | Unknown | Clinical Sample 5037 | Unknown | 5.02x105 copies/mL | 10x |

Table. Coronavirus isolates tested during the inclusivity study

a - Coronavirus HKU1 clinical samples titered with Applied BioCode validated SYBR assay using an IVT standard.

b - Virus obtained through BEI Resources, NIAID, NIH: Human Coronavirus NL63, NR-470

35

| Organism/
Species | Serotype/Strain/Isolate | Vendor | Catalog # | Concentration
Detected | Multiple of
LoD
Detected |
|----------------------|-----------------------------|-------------|-----------|---------------------------|--------------------------------|
| Rhinovirus A | Serotype 7/[68-CV11] | ATCC | VR-1601 | 3.6 TCID50/mL | 3x |
| | Serotype 16 [1A] | Zeptometrix | 0810285CF | 3.6 TCID50/mL | 3x |
| | Serotype 16 [11757/DC/1960] | ATCC | VR-283 | 3.6 TCID50/mL | 3x |
| | Serotype 34 [137-3] | ATCC | VR-1365 | 3.6 TCID50/mL | 3x |
| | Serotype 57 [Ch47} | ATCC | VR-1600 | 3.6 TCID50/mL | 3x |
| | Serotype 77 [130-63] | ATCC | VR-1187 | 3.6 TCID50/mL | 3x |
| | Serotype 80 | Zeptometrix | 0810288CF | 3.6 TCID50/mL | 3x |
| | Serotype 85 [50-525-CV54] | ATCC | VR-1195 | 3.6 TCID50/mL | 3x |
| | Serotype 95 [SF-998} | ATCC | VR-1301 | 3.6 TCID50/mL | 3x |
| Rhinovirus B | Serotype 3 [FEB] | ATCC | VR-483 | 3.6 TCID50/mL | 3x |
| | Serotype 14 | Zeptometrix | 0810284CF | 3.6 TCID50/mL | 3x |
| | Serotype 42 | Zeptometrix | 0810286CF | 3.6 TCID50/mL | 3x |
| | Serotype 70 | Zeptometrix | 0810287CF | 3.6 TCID50/mL | 3x |
| Enterovirus 71 | EV 71 | Zeptometrix | 0810236CF | 9.0 TCID50/mL | 3x |

Table. Human Rhinovirus and Enterovirus isolates tested during the inclusivity study

Table. Bordetella pertussis isolates tested during the inclusivity study

| Organism | Strain/Isolate | Vendor | Catalog # | Concentration
Detected | Multiple
of LoD
Detected |
|----------------------|---------------------|--------|-----------|---------------------------|--------------------------------|
| Bordetella pertussis | F | ATCC | 8467 | 45 CFU/mL | 3x |
| | 5[17921] | ATCC | 9340 | 45 CFU/mL | 3x |
| | 10-536 | ATCC | 10380 | 45 CFU/mL | 3x |
| | CNCTC Hp 12/63[623] | ATCC | 51445 | 45 CFU/mL | 3x |
| | Tohama 1 | ATCC | BAA-589 | 45 CFU/mL | 3x |
| | MN2531 | ATCC | BAA-1335 | 45 CFU/mL | 3x |

	Table. Mycoplasma pneumoniae isolates tested during the inclusivity study
--	----------------------------------------------------------------------------------	--

| Organism | Strain/Isolate | Vendor | Catalog # | Concentration
Detected | Multiple
of LoD
Detected |
|-----------------------|----------------|--------|-----------|---------------------------|--------------------------------|
| Mycoplasma pneumoniae | M129-B7 | ATCC | 29342 | 45 CFU/mL | 3x |
| | PI 1428 | ATCC | 29085 | 45 CCU/mL | 3x |
| | Mac | ATCC | 15492 | 45 CFU/mL | 3x |
| | UTMB-10P | ATCC | 49894 | 45 CCU/mL | 3x |

36

| Organism | Strain/Isolate | Vendor | Catalog # | Concentration
Detected | Multiple
of LoD
Detected |
|----------------------|----------------|--------|-----------|---------------------------|--------------------------------|
| Chlamydia pneumoniae | CM-1 | ATCC | VR-1360 | 50.1 CFU/mL | 3x |
| | CWL-029 | ATCC | VR-1310 | 50.1 CFU/mL | 3x |

	Table. Chlamydia pneumoniae isolates tested during the inclusivity study

Conclusion: This inclusivity testing suggests that the BioCode RPP is analytically reactive for the targeted organisms. BioCode RPP assay displayed good inclusivity for related organisms with similar genetic lineage/sequence.

Zoonotic Influenza A Analytical Reactivity/Inclusivity Testing

Due to public health concerns related to zoonotic transmission of influenza A viruses to humans (primarily swine and avian lineages), serial dilutions of the following isolates of swine and avian influenza A viruses (viral nucleic acids) were also tested assessing analytical reactivity:

A/Japan/305/57 (H2N2)
· A/duck/Pennsylvania/10218/1984 (H5N2)
· A/turkey/Wisconsin/1/1966 (H9N2)
A/Anhui/1/2013 (H7N9)
· A/Hubei/1/2010 (H5N1)
A/Minnesota/11/2010 (H3N2v)

Consistent with the in silico predictions of analytical reactivity to zoonotic influenza A viruses, each nonseasonal influenza A strain tested (H2N2, H5N2, H7N9, and H5N1), except for the H3N2v strain, was detected as Influenza A (no subtype) at various concentrations tested (i.e., detected by the BioCode RPP Influenza A matrix assay only). For the H3N2v strain, it was detected as Influenza A/H3 at higher concentrations but was detected as Influenza A (no subtype) at the lowest concentration tested.

Analytical testing could not be performed to assess reactivity to H5N8, H1N2v and H1N1 swine viruses due to the lack of availability of such zoonotic influenza A strains for wet testing. Based on an in silico analysis, H5N8 (KP739416), H1N2v (MK239077), and H1N1 swine (KP822962.1) were predicted to be detected as Influenza A (no subtype), Influenza A/H1 (with reduced sensitivity for the A/H1 assay), and Influenza A (no subtype), respectively.

Analytical Specificity/Cross Reactivity

A study was performed to verify that the BioCode® Respiratory Pathogen Panel (RPP) does not detect DNA or RNA from off-panel organisms commonly found in respiratory specimens or from organisms that can cause similar clinical symptoms. In addition, on-panel organisms were tested at high concentrations to ensure there is no cross-reactivity with other on-panel targets. This study tested a panel of titered stocks of relevant organisms. Organisms that were not available for wet testing were analyzed in silico comparing the whole organism sequence against all primers to assess potential for cross reactivity. Analysis was conducted using BLASTn and Primer-BLAST programs. All testing was performed at Applied BioCode®, Inc. Microorganisms were tested at 10° CFU/mL for bacteria or fungi and 10° TCIDso/mL for

37

virus or higher when possible. Stocks were combined with simulated NPS in UTM matrix at the of extraction. Each organism was extracted in triplicate on the EasyMag and assayed in singlet with the RPP on the BioCode® MDx-3000 system according to the instructions for use. For each concentration tested, the number of replicates that gave valid results per the Interpretation Algorithm was determined. If any replicates were detected, testing was repeated from 5 additional extractions assayed in singlet. If detected after repeat with 5 additional replicates, serial dilutions were performed to determine the lower limit.

| Organism | Vendor | Catalog # | Titer tested | Cross-
reactivity
(Y/N) |
|-------------------------------------------------------------------|-------------|-----------|------------------------|-------------------------------|
| Acinetobacter baumannii | Zeptometrix | 801597 | 9.67 x 106 CFU/mL | N |
| Aspergillus flavus | Zeptometrix | 801598 | 1.72 x 106 CFU/mL | N |
| Bordetella bronchiseptica | Zeptometrix | 801649 | 6.68 x 107 CFU/mL | N |
| Bordetella holmesii | Zeptometrix | 801464 | 3.83 x 109 CFU/mL | Y
a |
| Bordetella parapertussis | Zeptometrix | 8011461 | 1.00 x 106 CFU/mL | N |
| Burkholderia cepacia | Zeptometrix | 801584 | 4.13 x 107 CFU/mL | N |
| Candida albicans | Zeptometrix | 801504 | 1.96 x 106 CFU/mL | N |
| Candida glabrata | Zeptometrix | 801535 | 1.73 x 107 CFU/mL | N |
| Corynebacterium diphtheriae | Zeptometrix | 801882 | 4.57 x 106 CFU/mL | N |
| Haemophilus influenzae | Zeptometrix | 801679 | 2.40 x 106 CFU/mL | N |
| Klebsiella pneumoniae | Zeptometrix | 801506 | 5.10 x 107 CFU/mL | N |
| Lactobacillus plantarum | Zeptometrix | 801507 | 1.80 x 107 CFU/mL | N |
| Legionella longbeachae | Zeptometrix | 8101577 | 1.93 x 107 CFU/mL | N |
| Legionella micdadei | Zeptometrix | 801576 | 2.70 x 107 CFU/mL | N |
| Legionella pneumophila | Zeptometrix | 801645 | 3.17 x 107 CFU/mL | N |
| Moraxella catarrhalis | Zeptometrix | 801509 | 1.13 x 106 CFU/mL | N |
| Mycobacterium tuberculosis | Zeptometrix | 801660 | 7.23 x 106 CFU/mL | N |
| Mycoplasma genitalium | ATCC | 33530 | 1.00 x 103 CFU/mL | N |
| Mycoplasma hominis | ATCC | 23114 | 2.7 x 104 CFU/mL | N |
| Neisseria elongata | Zeptometrix | 801510 | 1.74 x 107 CFU/mL | N |
| Neisseria gonorrhoeae | Zeptometrix | 801482 | 1.26 x 107 CFU/mL | N |
| Neisseria meningitidis | Zeptometrix | 801511 | 2.55 x 106 CFU/mL | N |
| Neisseria sicca | Zeptometrix | 801754 | 1.02 x 106 CFU/mL | N |
| Proteus vulgaris | Zeptometrix | 801898 | 4.13 x 107 CFU/mL | N |
| Pseudomonas aeruginosa | ATCC | 39324 | 2.11 x 106 CFU/mL | N |
| Serratia marcescens | Zeptometrix | 801723 | 2.06 x 107 CFU/mL | N |
| Staphylococcus haemolyticus | Zeptometrix | 801591 | 8.50 x 106 CFU/mL | N |
| Streptococcus agalactiae | Zeptometrix | 801545 | 3.73 x 106 CFU/mL | N |
| Streptococcus dysgalactiae | Zeptometrix | 801516 | 1.23 x 106 CFU/mL | N |
| Streptococcus intermedius | Zeptometrix | 801895 | 5.07 x 106 CFU/mL | N |
| Streptococcus mitis | Zeptometrix | 801695 | 5.73 x 106 CFU/mL | N |
| Streptococcus pneumoniae | Zeptometrix | 801439 | 4.17 x 106 CFU/mL | N |
| Ureaplasma urealyticum | ATCC | 27618 | 1.00 x 107 CFU/mL | N |
| Organism | Vendor | Catalog # | Titer tested | Cross-
reactivity
(Y/N) |
| SARS-CoV, formaldehyde- and UV-inactivated,
purified (vaccine) | BEI | NR-3883 | 1:100 Dilution | Na |
| MERS-CoV genomic RNA | BEI | NR-45843 | 1.01 x 107 Copies/mL | N |
| MERS-CoV EMC/2012, Heat-Inactivated | BEI | NR-50171 | 2 x 105 TCID50/ml | N |
| Coxsackievirus A10 | Zeptometrix | 0810106CF | 1.05 x 103 TCID50/mL | Yb |
| Coxsackievirus A21 | Zeptometrix | 0810235CF | ≤ 1.03 x 102 TCID50/mL | Yb |
| Coxsackievirus A24 | ATCC | VR-583 | 1.14 x 101 TCID50/mL | Yb |
| Coxsackievirus B2 | ATCC | VR-29 | 5.62 x 103 TCID50/mL | Yb |
| Coxsackievirus B3 | Zeptometrix | 0810074CF | 1.76 x 103 TCID50/mL | Yb |
| Coxsackievirus B4 | Zeptometrix | 0810075CF | 1.36 x 104 TCID50/mL | Yb |
| Coxsackievirus B5 | Zeptometrix | 0810019CF | ≤ 5.89 x 102 TCID50/mL | Yb |
| Coxsackievirus A9 | Zeptometrix | 0810017CF | 1.38 x 103 TCID50/mL | Yb |
| Cytomegalovirus | Zeptometrix | 0810003CF | 4.17 x 104 TCID50/mL | N |
| Echovirus 11 | Zeptometrix | 0810023CF | 1.68 x 103 TCID50/mL | Yc |
| Echovirus 30 | Zeptometrix | 0810078CF | ≤ 1.95 x 102 TCID50/mL | Yc |
| Echovirus 6 | Zeptometrix | 0810076CF | 1.09 x 104 TCID50/mL | Yc |
| Echovirus 9 | Zeptometrix | 0810077CF | 1.07 x 101 TCID50/mL | Yc |
| Epstein-Barr Virus | Zeptometrix | 0810008CF | 3.43 x 106 TCID50/mL | N |
| Herpes Simplex Virus Type 1 | Zeptometrix | 0810187CF | 9.12 x 106 TCID50/mL | N |
| Measles Virus | Zeptometrix | 0810025CF | 1.31 x 105 TCID50/mL | N |
| Mumps Virus | Zeptometrix | 0810176CF | 1.89 x 105 TCID50/mL | N |

Table. Off-panel bacteria and fungi analyzed for analytical specificity (Cross reactivity)

38

a - Bordetella holmesii was detected by the Bordetella pertussis (BP) assay with 2 of 3 replicates down to 3.83 x 100 CFU/mL.

Table. Off-panel Viruses analyzed for analytical specificity (Cross reactivity)

a – Inhibitory (no RNA-IC detected) at 1:10 dilution

b – The Coxsackieviruses assayed here were detected by the Human Rhinovirus/Enterovirus (HRV) assay in at least 1 or the 3 replicates down to the concentrations indicated.

c – The Echoviruses assayed here were detected by the Human Rhinovirus/Enterovirus (HRV) assay in at least 1 or the 3 replicates down to the concentrations indicated.

				Table. On-panel organisms analyzed for analytical specificity (Cross reactivity).

Organism	Vendor	Catalog #	Titer tested	Cross-
reactivity
(Y/N)
Influenza A H1N1/New Caledonia/20/99	Zeptometrix	0810036CF	1.15 x 105 TCID50/mL	N
Influenza A H1N1 /NWS/33	ATCC	VR-219	7.40 x 105 TCID50/mL	N
Influenza A H1N1 pdm09/California/07/09	Zeptometrix	0810165CF	1.31 x 105 TCID50/mL	N
Influenza A H3N2 /Wisconsin/67/05a	Zeptometrix	0810252CF	1.08 x 105 TCID50/mL	N
Influenza A H3N2/Alice	ATCC	VR-776	1.43 x 106 TCID50/mL	N
Influenza B/Florida/4/2006 (Yamagata)	Zeptometrix	0810255CF	1.08 x 105 TCID50/mL	N
Influenza B/Hong Kong/S/1972 (Victoria)	ATCC	VR-823	8.57 x 105 TCID50/mL	N
Respiratory Syncytial Virus (Type A)	Zeptometrix	0810040ACF	4.57 x 105 TCID50/mL	N
Human Metapneumovirus 16 (Type A1)	Zeptometrix	0810161CF	8.51 x 105 TCID50/mL	N
Parainfluenza Virus 1	ATCC	VR-94	1.60 x 105 TCID50/mL	N

39

| Organism | Vendor | Catalog # | Titer tested | Cross-
reactivity
(Y/N) |
|----------------------------------|-------------|---------------------|----------------------|-------------------------------|
| Parainfluenza Virus 2 | ATCC | VR-92 | 1.35 x 105 TCID50/mL | N |
| Parainfluenza Virus 3 | Zeptometrix | 0810016CF | 3.39 x 105 TCID50/mL | N |
| Parainfluenza Virus 4a | Zeptometrix | 0810060CF | 1.13 x 105 TCID50/mL | N |
| Adenovirus Species B Serotype 7A | Zeptometrix | 0810021CF | 5.83 x 105 TCID50/mL | N |
| Adenovirus Species C Serotype 2 | ATCC | AV-846 | 2.81 x 105 TCID50/mL | N |
| Adenovirus Species E Serotype 4 | Zeptometrix | 0810070CF | 1.08 x 105 TCID50/mL | N |
| Coronavirus 229E | Zeptometrix | 0810229CF | 1.09 x 105 TCID50/mL | N |
| Coronavirus HKU1 | N/A | Clinical
samplea | 1.92 x 105 TCID50/mL | N |
| Coronavirus NL63 | Zeptometrix | 0810228CF | 1.08 x 105 TCID50/mL | N |
| Coronavirus OC43 | Zeptometrix | 0810024CF | 1.08 x 105 TCID50/mL | N |
| Human Rhinovirus Type A1 | Zeptometrix | 0810012CF | 1.05 x 105 TCID50/mL | N |
| Enterovirus D68 | Zeptometrix | 0810300CF | 1.08 x 105 TCID50/mL | N |
| Bordetella pertussis | Zeptometrix | 801459 | 3.86 x 107 CFU/mL | N |
| Mycoplasma pneumoniae | Zeptometrix | 801579 | 1.06 x 106 CCU/mL | N |
| Chlamydia pneumoniae (AR-39) | ATCC | 53592 | 1.24 x 106 CFU/mL | N |
| Chlamydia pneumoniae (CWL-029) | ATCC | VR-1310 | 1.00 x 106 CFU/mL | N |

a - Coronavirus HKU1 clinical samples titered with Applied BioCode validated SYBR assay using an IVT RNA standard.

Conclusion: Cross reactivity was not observed with the on-panel or off-panel microorganisms at the concentrations tested in this study except the following:

Empirical testing and in silico sequence analysis indicate that the Rhinovirus/Enterovirus assay (HRV) may also react with other Enterovirus species (i.e., Coxsackievirus and Echoviruses; see table for testing results).
In silico sequence analysis indicate that the Bordetella pertussis assay (BP) may react with Bordetella holmesii and Bordetella bronchiseptica.

Interfering Substances/Microbes

A study was performed to demonstrate the accuracy of the BioCode® Respiratory Pathogen Panel on the BioCode® MDx-3000 in the presence of potentially interfering substances or microorganisms. Each member of the interfering substance panel was added to contrived samples in simulated NPS (sNPS) in UTM matrix containing representative members of the BioCode RPP at 3x LoD and a negative sample comprised of only sNPS in UTM matrix. Each sample was tested with and without potentially interfering substances. Each sample was extracted in triplicate on both the easyMAG and MP96 extraction systems and tested singly with the RPP on the BioCode® MDx-3000 system. Concentrations of interferents were determined by reviewing results of previous RP clinical trials. Substances that produce interference at the original test concentration were tested at lower concentrations.

Sample Name	Organism	Source
RPP A	Adenovirus B Serotype 7A	Zeptometrix 0810060CF
	Mycoplasma pneumoniae	Zeptometrix 801579

Table . Contrived samples (3x LoD in sNPS)

40

	Influenza A H3N2 A/Wisconsin/67/2005	Zeptometrix 0810252CF
	Respiratory Syncytial Virus (Type A)	Zeptometrix 0810040ACF
RPP B	Influenza A H1N1/California/07/09	Zeptometrix 0810165CF
	Human Metapneumovirus (16; type A1)	Zeptometrix 0810161CF
	Parainfluenza Virus 3	Zeptometrix 0810016CF
RPP C	Coronavirus NL63	Zeptometrix 0810228CF
	Influenza B/Florida/4/2006	Zeptometrix 0810255CF
HRV	Human Rhinovirus	Zeptometrix 0810012CF

Results: All targets of Samples RPP A, RPP C and HRV were detected (3/3) at the concentrations below, suggesting no interference from these potential interferents at the concentrations tested.

| Microbial Interferent | Brand/Source | Concentration | Interference
Yes (Y) or No (N) |
|--------------------------|--------------|-------------------|-----------------------------------|
| Streptococcus pneumoniae | Zeptometrix | 1 X 106 CFU/mL | N |
| Haemophilus influenzae | Zeptometrix | 1 X 106 CFU/mL | N |
| Neisseria meningitidis | Zeptometrix | 1 X 106 CFU/mL | N |
| Staphylococcus aureus | ATCC | 1 X 106 CFU/mL | N |
| Cytomegalovirus | Zeptometrix | 1 X 105 TCID50/mL | N |

Table: Non-microbial Interfering substances tested for BioCode® RPP assay

| Substance Interferent | Brand/Source | Concentration | Interference
Yes (Y) or No (N) |
|------------------------------------------|--------------------|---------------|-----------------------------------|
| Genomic DNA | Promega | 10 ng/μl | N |
| Mucin (MagNA Pure 96) | Sigma | 0.6% W/V | N |
| Mucin (EasyMAG)ª | Sigma | 0.5% W/V | N |
| Human Blood | Poplar Health | 1% V/V | N |
| Zanamivir | APExBIO | 550 ng/mL | N |
| Oseltamivir | APExBIO | 142 ng/mL | N |
| Nasal spray | Equate | 1% V/V | N |
| Nasal decongestant spray | Bayer | 1% V/V | N |
| Nasal Allergy spray (Fluticasone) | Equate | 1.5% V/V | N |
| Petroleum Jelly | Equate | 1% W/V | N |
| Analgesic Ointment | Vicks | 1% W/V | N |
| Mupirocin | Alfa Aesar™ | 2% W/V | N |
| Tobramycin | MP Biomedicals,LLC | 0.6 mg/mL | N |
| Bleach (10%) | VWR | 5% V/V | N |
| Disinfecting wipes | Clorox | 50% V/V | N |
| Ethanol (70%) | LabChem | 7% V/V | N |
| Remel M4 Media | Remel | 90% V/V | N |
| Remel M4-RT Media | Remel | 90% V/V | N |
| Remel M5 Media | Remel | 90% V/V | N |
| Remel M6 Media | Remel | 90% V/V | N |
| Copan FloQ (Flocked nylon/plastic shaft) | Copan | 1 swab | N |

41

| Substance Interferent | Brand/Source | Concentration | Interference
Yes (Y) or No (N) |
|----------------------------------------------|---------------|---------------|-----------------------------------|
| Copan 168C (rayon/twisted aluminum
shaft) | Copan | 1 swab | N |
| Polyester / Aluminum shaft swab | Puritan/Copan | 1 swab | N |
| DNAzap | Invitrogen | 1% V/V | N |
| RnaseOut | Invitrogen | 1% V/V | N |

a - It was observed that mucin at higher concentration (0.6%) led to loss of signal for some targets (loss of analyte detection) when extracted with NucliSENS easyMAG.

Table: Nasal influenza vaccine (FluMist) tested for BioCode® RPP assay

FluMist® 2010-2011 (V/V%)	Influenza A			Influenza B
	H1	H1N1-2009	H3
10%	-	+	+	+
1%	-	+	+	+
0.1%	-	+	+	+
0.01%	-	+	+	+
0.001%	-	+	+	+
0.0001%	-	+	+a	+
0.00001%	-	-	-	+a
0.000001%	-	-	-	-

a - 2/3 replicates detected

Conclusion: None of the substances were shown to interfere with BioCode® RPP at the concentrations tested. However, it was observed that mucin at higher concentration (0.6%) could lead to loss of signal for some targets (loss of analyte detection) when extracted with easyMAG. The effect of mucin was dependent on the concentration in the sample tested.

Flu Mist was evaluated to be reactive as predicted with the BioCode RPP assay, therefore recent administration or contamination of specimens by Flu vaccine prior to NPS collection could lead to false detection by BioCode® RPP.

Competitive Inhibition

A study was performed to evaluate the potential for inhibition in samples with mixed infections. Targets were spiked into simulated NPS in UTM matrix with one target at high concentration (≥10°CFU/mL for bacteria and ≥105 units/mL for viruses) and two targets at low concentration (≤3x LoD). Common coinfections were determined by reviewing results of previous Respiratory Panel clinical trials from 510k summaries, publications/posters and internal clinical sample testing. Each sample was extracted in triplicate on the easyMAG and each extraction tested in singlet with the RPP on the BioCode® MDx-3000 system.

Results: Results are shown in the table below.

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| Panel
Designation | Viral/Bacteria Strain | Source | Level | Titer Tested | Result (n of
3 Detected) |
|----------------------------------------|------------------------------------------|---------------------------|-------|-----------------|-----------------------------|
| Competitive
Inhibition
Sample 1 | Adenovirus species C Serotype 2 | ATCC VR-846 | High | 1x105 TCID50/mL | 3/3 |
| | Respiratory syncytial virus Type A | Zeptometrix
0810040ACF | Low | 0.99 TCID50/mL | 3/3 |
| | Influenza A H3N2
A/Wisconsin/67/05a | Zeptometrix
0810252CF | Low | 12 TCID50/mL | 3/3 |
| Competitive
Inhibition
Sample 2 | Respiratory syncytial virus Type A | Zeptometrix
0810040ACF | High | 1x105 TCID50/mL | 3/3 |
| | Influenza A H3N2
A/Wisconsin/67/05a | Zeptometrix
0810252CF | Low | 12 TCID50/mL | 3/3 |
| | Adenovirus species C Serotype 2 | ATCC VR-846 | Low | 18 TCID50/mL | 3/3 |
| Competitive
Inhibition
Sample 3 | Influenza A H3N2
A/Wisconsin/67/05a | Zeptometrix
0810252CF | High | 1x105 TCID50/mL | 3/3 |
| | Adenovirus species C Serotype 2 | ATCC VR-846 | Low | 18 TCID50/mL | 3/3 |
| | Respiratory syncytial virus Type A | Zeptometrix
0810040ACF | Low | 0.99 TCID50/mL | 3/3 |
| Competitive
Inhibition
Sample 4 | Coronavirus OC43 | Zeptometrix
0810024CF | High | 1x105 TCID50/mL | 3/3 |
| | Human Metapneumovirus | Zeptometrix
0810161CF | Low | 45 TCID50/mL | 3/3 |
| | Bordetella pertussis | Zeptometrix
801459 | Low | 45 CFU/mL | 3/3 |
| Competitive
Inhibition
Sample 5 | Human Metapneumovirus | Zeptometrix
0810161CF | High | 1x105 TCID50/mL | 3/3 |
| | Bordetella pertussis | Zeptometrix
801459 | Low | 45 CFU/mL | 3/3 |
| | Coronavirus OC43 | Zeptometrix
0810024CF | Low | 0.12 TCID50/mL | 3/3 |
| Competitive
Inhibition
Sample 6 | Bordetella pertussis | Zeptometrix
801459 | High | 1x106 CFU/mL | 3/3 |
| | Coronavirus OC43 | Zeptometrix
0810024CF | Low | 0.12 TCID50/mL | 3/3 |
| | Human Metapneumovirus | Zeptometrix
0810161CF | Low | 45 TCID50/mL | 3/3 |
| Competitive
Inhibition
Sample 7 | Influenza A H1N1 pdm
California/07/09 | Zeptometrix
0810165CF | High | 1x105 TCID50/mL | 3/3 |
| | Parainfluenza Virus 3 | Zeptometrix
0810016CF | Low | 45 TCID50/mL | 3/3 |
| Competitive
Inhibition
Sample 8 | Human Rhinovirus type A | Zeptometrix
0810012CFN | Low | 3.6 TCID50/mL | 3/3 |
| | Parainfluenza Virus 3 | Zeptometrix
0810016CF | High | 1x105 TCID50/mL | 3/3 |
| Panel
Designation | Viral/Bacteria Strain | Source | Level | Titer Tested | Result (n of
3 Detected) |
| Competitive
Inhibition
Sample 9 | Influenza A H1N1 pdm
California/07/09 | Zeptometrix
0810165CF | Low | 1.2 TCID50/mL | 3/3 |
| | Human Rhinovirus type A | Zeptometrix
0810012CFN | High | 1x105 TCID50/mL | 3/3 |
| | Influenza A H1N1 pdm
California/07/09 | Zeptometrix
0810165CF | Low | 1.2 TCID50/mL | 3/3 |
| | Parainfluenza Virus 3 | Zeptometrix
0810016CF | Low | 45 TCID50/mL | 3/3 |
| Competitive
Inhibition
Sample 10 | Mycoplasma pneumoniae | Zeptometrix
801579 | High | 1x106 CCU/mL | 3/3 |
| | Coronavirus NL63 | Zeptometrix
0810228CF | Low | 1.2 TCID50/mL | 3/3 |
| | Influenza B/Florida/4/2006 | Zeptometrix
0810255CF | Low | 0.04 TCID50/mL | 3/3 |
| Competitive
Inhibition
Sample 11 | Coronavirus NL63 | Zeptometrix
0810228CF | High | 1x105 TCID50/mL | 3/3 |
| | Influenza B/Florida/4/2006 | Zeptometrix
0810255CF | Low | 0.04 TCID50/mL | 3/3 |
| | Mycoplasma pneumoniae | Zeptometrix
801579 | Low | 45 CCU/mL | 3/3 |
| Competitive
Inhibition
Sample 12 | Influenza B/Florida/4/2006 | Zeptometrix
0810255CF | High | 1x105 TCID50/mL | 3/3 |
| | Mycoplasma pneumoniae | Zeptometrix
801579 | Low | 45 CCU/mL | 3/3 |
| | Coronavirus NL63 | Zeptometrix
0810228CF | Low | 1.2 TCID50/mL | 3/3 |

Table. Competitive inhibition testing results

43

Conclusion: All replicates from each pooled sample were valid and detected. No competitive inhibition was observed at the concentrations tested.

Cross Contamination/Sample Carryover

Carry-over contamination studies have been performed for the BioCode® MDx-3000 system in conjunction with the easyMAG (K180041) and MagNA Pure 96 systems (K190585). Since this study is not assay-specific, no additional testing was performed for BioCode® RPP.

Specimen Stability

A study was performed to assess the Specimen Stability limitations for the optimal performance of the BioCode® Respiratory Pathogen Panel (RPP) on the BioCode® MDx-3000. Representative organisms from the BioCode® RPP were spiked into prescreened negative natural NPS in UTM matrix at 3x LoD and assayed with 3 extractions on the easyMAG at each timepoint 0). This study assessed the following storage conditions:

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Nasopharyngeal Swabs in transport media
Storage temp	Target (storage time)	Test time points
Room Temperature 25°C	8 hours	8,12 hours
Refrigerated 4°C	7 days	5, 7, 10 days
Fresh Vs Frozen (-70°C)	90 days (2x freeze/thaw)	30, 60, 90 days
Purified nucleic acids
Storage temp	Target (storage time)	Test time points
Refrigerated 4°C	8 hours	8, 12 hours
Fresh Vs Frozen (-70°C)	90 days (2x freeze/thaw)	30, 60, 90 days

Table. Specimen stability conditions

Table. Contrived samples (3x LoD in prescreened negative natural matrix)

Sample Name	Organism	Source
RP A	Adenovirus B Serotype 7a	Zeptometrix 0810021CF
RP A	Mycoplasma pneumoniae	Zeptometrix 0801579
RP A	Influenza A H3N2 A/Wisconsin/67/2005	Zeptometrix 0810252CF
RP B	Respiratory Syncytial Virus (Type A)	Zeptometrix 0810040ACF
RP B	H1N1pdm California/07/09	Zeptometrix 0810165CF
RP B	Human Metapneumovirus (16; type A1)	Zeptometrix 0810161CF
RP C	Parainfluenza Virus 3	Zeptometrix 0810016CF
RP C	Coronavirus NL63	Zeptometrix 0810228CF
RP C	Flu B/Florida/4/2006	Zeptometrix 0810255CF

Results: All replicates were Valid and detected for all conditions assayed. Summary results are shown in the table below.

45

Premarket Notification 510(k)

			Sample A			Sample B			Sample C
Sample Type	Temp	Time
(Hours
or
Days)	ADV	MPN	Flu A
H3N2	RSV	Flu A
H1N1
2009
pdm	hMPV	PIV3	NL63	Flu B
Fresh (baseline)	N/A	0	7/7	7/7	7/7	7/7	7/7	7/7	7/7	7/7	7/7
Spiked NPS	Room Temp	8 Hour	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
		12 Hour	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
	4ºC	5 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
		7 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
		10 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
	-80°C	30 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
		60 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
		90 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Extracted Nucleic
acid	4ºC	8 Hour	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
		12 Hour	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
	-80°C
(2x Freeze/thaw)	30 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
			60 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
			90 Day	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3

Table. Qualitative results from Specimen Stability study

Conclusions: All replicates were Valid and detected for each specimen stability condition.

Matrix equivalency: Simulated Nasopharyngeal Swabs (sNPS) vs Native Nasopharyngeal Swabs (NPS)

Analytical validation studies are mostly performed with contrived specimens (i.e., spiking known concentrations of pathogens into negative NPS in UTM or VTM matrix). This would require a large volume of negative natural NPS to be collected and screened, while typically only between 1.0 to 2.0 mL per donor could be obtained after NPS collection and screening to confirm negative status. This makes collection from donors burdensome. An analytical study was performed to assess the performance equivalency of testing a simulated NPS (sNPS) in UTM matrix compared to testing natural NPS in UTM using the BioCode RPP, with both the NucliSENS easyMAG and the MagNA Pure 96 extraction systems. The sNPS in UTM matrix consists of 2x103 HeLa cells/mL diluted in UTM.

Samples were contrived in negative natural NPS in UTM matrix with matrix with multispiked pathogens at close to LoD levels (approximately 1.5x to 15.0xLoD). Each sample was extracted in guadruplicate on both the easyMAG and the MagNA Pure 96 extraction system and tested singly with the BioCode RPP on the BioCode MDx-3000 system.

Results: All four replicates of pathogens in three pools were Valid and detected. No unexpected targets were detected in the sNPS.

46

Pools	Organism	Source	Concentration	Multiple of LoD tested		Detected (N of 4)
				easyMAG	MagNA Pure 96	easyMAG		MagNA Pure 96
						Natural NPS	sNPS	Natural NPS	sNPS
Pool A	Adenovirus E Serotype 4	Zeptometrix 0810070CF	0.6 TCID50/mL	15x LoD	15x LoD	4/4	4/4	4/4	4/4
	Chlamydia pneumoniae	ATCC 53592	75.0 CFU/mL	4.5x LoD	2.3x LoD	4/4	4/4	4/4	4/4
	Influenza A H3N2 A/Wisconsin/67/2005	Zeptometrix 0810252CF	6.0 TCID50/mL	1.5x LoD	4.6x LoD	4/4	4/4	4/4	4/4
Pool B	Influenza A H1N1/New Caledonia/ 20/99	Zeptometrix 0810036CF	67.5 TCID50/mL	4.5x LoD	13.5x LoD	4/4	4/4	4/4	4/4
	Respiratory Syncytial Virus (Type A)	Zeptometrix 0810040ACF	1.5 TCID50/mL	4.5x LoD	4.5x LoD	4/4	4/4	4/4	4/4
	Human Metapneumovirus (16; type A1)	Zeptometrix 0810161CF	67.5 TCID50/mL	4.5x LoD	4.5x LoD	4/4	4/4	4/4	4/4
Pool C	Influenza B/Florida/4/2006	Zeptometrix 0810255CF	0.06 TCID50/mL	6x LoD	6x LoD	4/4	4/4	4/4	4/4
	Coronavirus OC43	Zeptometrix 0810024CF	0.06 TCID50/mL	1.5x LoD	6x LoD	4/4	4/4	4/4	4/4
	Parainfluenza Virus 4	Zeptometrix 0810060CF	13.5 TCID50/mL	1.5x LoD	1.5x LoD	4/4	4/4	4/4	4/4
Negative Control		N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A

Table. Results of natural NPS Vs sNPS comparison

Conclusions: No matrix effect was observed in this study. All replicates for each contrived sample assayed with the simulated matrix (sNPS- HeLa cells in UTM) and natural matrix were valid and detected for expected targets per the algorithm using both extraction systems easyMAG and MagNA Pure 96 extraction systems.