Verigene Respiratory Pathogens Flex Nucleic Acid Test (RP Flex)
Device Facts
| Record ID | K143653 |
|---|---|
| Device Name | Verigene Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) |
| Applicant | Nanosphere, Inc. |
| Product Code | OCC · Microbiology |
| Decision Date | Sep 4, 2015 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3980 |
| Device Class | Class 2 |
Intended Use
The Verigene® Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) is a multiplexed qualitative test intended for the simultaneous detection and identification of multiple viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infection. The test is performed on the automated Verigene System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and microarray hybridization to detect gene sequences of the following organism types and subtypes: Adenovirus, Human Metapneumovirus, Influenza A, Influenza A (subtype H1), Influenza A (subtype H3), Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B, Rhinovirus, Bordetella parapertussis/bronchiseptica, Bordetella holmesii, and Bordetella pertussis. Detecting and identifying specific viral and bacterial nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory infection with other clinical and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or patient management decisions. Negative results in the presence of a respiratory illness do not preclude respiratory infection and may be due to infection with pathogens that are not detected by this test or lower respiratory tract infection that is not detected by an NPS specimen. Conversely, positive results do not rule-out infection with organisms not detected by RP Flex. The agent(s) detected may not be the definite cause of disease. The use of additional laboratory testing and clinical presentation may be necessary to establish a final diagnosis of respiratory infection. Clinical evaluation indicates a lower sensitivity specific to RP Flex for the detection of Rhinovirus. If infection with Rhinovirus is suspected, negative samples should be confirmed using an alternative method. Performance characteristics for Influenza A were established when Influenza A/H1 (2009 Pandemic) and A/H3 were the predominant Influenza A viruses in circulation. RP Flex may not detect novel Influenza A strains. 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 used specifically for novel virulent influenza viruses and sent to appropriate health authorities for testing. Viral culture should not be attempted in these cases unless a biosafety level (BSL) 3+ facility is available to receive and culture specimens.
Device Story
The Verigene RP Flex is a sample-to-result, automated molecular diagnostics workstation. It accepts nasopharyngeal swabs (NPS) as input. The system performs automated nucleic acid extraction (cell lysis, magnetic bead-based isolation), target amplification (RT-PCR), and microarray hybridization. Detection utilizes gold nanoparticle probes; silver enhancement creates gold-silver aggregates that scatter light. The Verigene Reader images light scatter from capture spots on the microarray to determine the presence or absence of specific analytes. Used in clinical laboratory settings by trained personnel. Output is a qualitative result (Detected/Not Detected) for 16 respiratory pathogens. Results aid clinicians in diagnosing respiratory infections alongside other clinical/laboratory findings, potentially improving patient management by identifying specific viral or bacterial causes.
Clinical Evidence
Clinical performance was evaluated in a multi-site prospective study (n=3266 specimens). Comparator methods included an FDA-cleared molecular respiratory panel and PCR with bi-directional sequencing. Performance was stratified by specimen type (fresh/frozen prospective, selected, contrived). Results showed high positive and negative percent agreement across the 16 target analytes. Analytical testing included LoD, inclusivity (136 strains), exclusivity (107 organisms), and interference studies.
Technological Characteristics
Molecular assay using microarray format. Employs Capture and Mediator oligonucleotides with gold nanoparticle probe-based endpoint detection and silver enhancement. Automated system (Verigene Reader and Processor SP) performs extraction, amplification, and hybridization. Connectivity via barcode scanner and touch-screen interface. Single-use disposable test consumables and cartridges.
Indications for Use
Indicated for individuals suspected of respiratory tract infection exhibiting signs and symptoms. Uses nasopharyngeal swabs (NPS) for simultaneous qualitative detection of viral and bacterial nucleic acids. Not for sole diagnostic use.
Regulatory Classification
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.
Special Controls
*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.
Predicate Devices
- FilmArray Respiratory Panel (RP) System (K143080, K123620, K120267, K110764, and K103175)
Related Devices
- K233410 — LIAISON PLEX Respiratory Flex Assay · Luminex Corporation · Mar 1, 2024
- K103209 — VERIGENE RESPIRATORY VIRUS NUCLEIC ACID TEST +(RVNAT+) · Nanosphere, Inc. · Jan 10, 2011
- K083088 — VERIGENE RESPIRATORY VIRUS NUCLEIC ACID TEST · Nanosphere, Inc. · May 1, 2009
- K192485 — BioCode Respiratory Pathogen Panel (RPP) · Applied BioCode, Inc. · Dec 23, 2019
- K092566 — VERIGENE RESPIRATORY VIRUS NUCLEIC ACID TEST ON THE VERIGENE SP SYSTEM (RVNATSP) · Nanosphere, Inc. · Oct 9, 2009
Submission Summary (Full Text)
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### DEPARTMENT OF HEALTH & HUMAN SERVICES
Public Health Service
Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002
September 4, 2015
Nanosphere, Inc. c/o Fran White MDC Associates, LLC. 180 Cabot Street Beverly, MA 01915
Re: K143653
Trade/Device Name: Verigene® Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory Viral Panel Multiplex Nucleic Acid Assay Regulatory Class: II Product Code: OCC, OEM, OEP, OOU, OZE, OZZ, OOI Dated: July 27, 2015 Received: July 28, 2015
Dear Ms. White:
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. 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 Parts 801 and 809); medical device reporting (reporting of
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medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Small Manufacturers. International and Consumer Assistance at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.
Sincerely yours,
Tamara V. Feldblyum -S for
Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
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# Indications for Use
510(k) Number (if known) K143653
#### Device Name
Verigene® Respiratory Pathogens Flex Nucleic Acid Test (RP Flex)
#### Indications for Use (Describe)
The Verigene® Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) is a multiplexed qualitative test intended for the simultaneous detection and identification of multiple viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infection. The test is performed on the automated Verigene System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and microarray hybridization to detect gene sequences of the following organism types and subtypes:
| Viruses | Bacteria |
|-------------------------------|-----------------------------------------|
| Adenovirus | Bordetella parapertussis/bronchiseptica |
| Human Metapneumovirus | Bordetella holmesii |
| Influenza A | Bordetella pertussis |
| Influenza A (Subtype H1) | |
| Influenza A (Subtype H3) | |
| Influenza B | |
| Parainfluenza 1 | |
| Parainfluenza 2 | |
| Parainfluenza 3 | |
| Parainfluenza 4 | |
| Respiratory Syncytial Virus A | |
| Respiratory Syncytial Virus B | |
| Rhinovirus | |
Detecting and identifying specific viral and bacterial nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory infection with other clinical and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or patient management decisions.
Negative results in the presence of a respiratory illness do not preclude respiratory infection and may be due to infection with pathogens that are not detected by this test or lower respiratory tract infection that is not detected by an NPS specimen. Conversely, positive results do not rule-out infection with organisms not detected by RP Flex. The agent(s) detected may not be the definite cause of disease. The use of additional laboratory testing and clinical presentation may be necessary to establish a final diagnosis of respiratory infection.
Clinical evaluation indicates a lower sensitivity specific to RP Flex for the detection of Rhinovirus. If infection with Rhinovirus is suspected, negative samples should be confirmed using an alternative method.
Performance characteristics for Influenza A were established when Influenza A/H1 (2009 Pandemic) and A/H3 were the predominant Influenza A viruses in circulation. RP Flex may not detect novel Influenza A strains. 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 used specifically for novel virulent influenza viruses and sent to appropriate health authorities for testing. Viral culture should not be attempted in these cases unless a biosafety level (BSL) 3+ facility is available to receive and culture specimens.
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X Prescription Use (Part 21 CFR 801 Subpart D)
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#### 510(K) Summary 1.
### 510(k) Number:
Verigene® Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) K143653:
## Summary Preparation Date:
August 18, 2015
### Submitted by:
Nanosphere, Inc. 4088 Commercial Avenue Northbrook, IL 60062 Phone: 847-400-9000 Fax: 847-400-9199
## Contact:
Fran White MDC Associates
### Proprietary Names:
For the instrument: Verigene® System For the assay: Verigene® Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) Verigene® RP Flex
#### Common Names:
For the instrument:
Bench-top molecular diagnostics workstation
For the assay:
Respiratory Pathogens Nucleic Acid Test Respiratory Pathogens Flex Nucleic Acid Test Respiratory Pathogens identification and differentiation system Respiratory assay Respiratory test Verigene RP Flex RP Flex
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#### Regulatory Information:
Regulation section:
866.3980 - Respiratory Viral Panel Multiplex Nucleic Acid Assay
Classification:
Class II
Panel:
Microbiology (83)
Product Code(s):
- OCC Respiratory Virus Panel Nucleic Acid Assay System
- Human Metapneumovirus (hMPV) RNA Assay System OEM
- OEP Influenza A Virus Subtype Differentiation Nucleic Acid Assay
- OOI Real Time Nucleic Acid Amplification System
- OOU Parainfluenza Multiplex Nucleic Acid Assay
- OZE Influenza A and Influenza B Multiplex Nucleic Acid Assay
- Bordetella Pertussis DNA Assay System OZZ
## Predicate Devices:
FilmArray Respiratory Panel (RP) System (K143080, K123620, K120267, K110764, and K103175) (BioFire Diagnostics, Inc.)
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## Intended Use:
The Verigene® Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) is a multiplexed qualitative test intended for the simultaneous detection and identification of multiple viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infection. The test is performed on the automated Verigene System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and microarray hybridization to detect gene sequences of the following organism types and subtypes:
| Viruses | Bacteria |
|-------------------------------|-----------------------------------------|
| Adenovirus | Bordetella parapertussis/bronchiseptica |
| Human Metapneumovirus | Bordetella holmesii |
| Influenza A | Bordetella pertussis |
| Influenza A (subtype H1) | |
| Influenza A (subtype H3) | |
| Influenza B | |
| Parainfluenza 1 | |
| Parainfluenza 2 | |
| Parainfluenza 3 | |
| Parainfluenza 4 | |
| Respiratory Syncytial Virus A | |
| Respiratory Syncytial Virus B | |
| Rhinovirus | |
Detecting and identifying specific viral and bacterial nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory infection, if used in conjunction with other clinical and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or patient management decisions.
Negative results in the presence of a respiratory illness do not preclude respiratory infection and may be due to infection with pathogens that are not detected by this test or lower respiratory tract infection that is not detected by an NPS specimen. Conversely, positive results do not rule-out infection or co-infection with organisms not detected by RP Flex. The agent(s) detected may not be the definite cause of disease. The use of additional laboratory testing and clinical presentation may be necessary to establish a final diagnosis of respiratory infection.
Clinical evaluation indicates a lower sensitivity specific to RP Flex for the detection of Rhinovirus. If infection with Rhinovirus is suspected, negative samples should be confirmed using an alternative method.
Performance characteristics for Influenza A were established when Influenza A/H1 (2009 Pandemic) and A/H3 were the predominant Influenza A viruses in circulation. RP Flex may not detect novel Influenza A strains. 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 used
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specifically for novel virulent influenza viruses and sent to appropriate health authorities for testing. Viral culture should not be attempted in these cases unless a biosafety level (BSL) 3+ facility is available to receive and culture specimens.
## Technological Characteristics:
The Verigene Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) is a molecular assay that relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial or viral nucleic acid sequences detected by RP Flex, unique Capture and Mediator oligonucleotides are used, with gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides are covalently bound to the microarray substrate and hybridize to a specific portion of the nucleic acid targets. The Mediator oligonucleotides have a region that binds to a different portion of the same nucleic acid targets and also have a sequence that allows binding of a gold nanoparticle probe. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency and provide accurate detection of target capture.
The RP Flex test is performed on the Verigene System, a "sample-to-result," fully automated, bench-top molecular diagnostics workstation. The System enables automated nucleic acid extraction from nasopharyngeal swabs (NPS) and detection of analyte-specific target nucleic acids. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP.
The Reader is the Verigene System's user interface and serves as the central control unit for all aspects of test processing, automated imaging, and result generation using a touch-screen control panel and a barcode scanner. The Verigene Processor SP executes the test procedure, automating the steps of (1) Sample Preparation and Target Amplification – cell lysis and magnetic bead-based bacterial and viral nucleic acid isolation and amplification, and (2) Hybridization- detection and identification of analyte-specific nucleic acid in a microarray format by using gold nanoparticle probe-based technology. Once the specimen is loaded by the operator, all other fluid transfer steps are performed by an automated pipette that transfers reagents between wells of the trays and finally loads the specimen into the Test Cartridge for hybridization. Single-use disposable test consumables and a self-contained Verigene Test Cartridge are used for each sample tested with the RP Flex assay.
To obtain the test results after test processing is complete, the user removes the Test Cartridge from the Processor SP, and inserts the substrate holder into the Verigene Reader for analysis. Light scatter from the capture spots is imaged by the Verigene Reader and intensities from the microarray spots are used to make a determination regarding the presence (Detected) or absence (Not Detected) of a targeted nucleic acid sequence/analyte. This determination is made by means of software-based decision algorithm resident in the Verigene Reader.
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### Performance Data - Analytical Testing
#### Analytical Sensitivity / Limit of Detection (LoD)
Limit of Detection (LoD) of the Verigene RP Flex test was determined for twenty-eight (28) strains of respiratory pathogens, representing all sixteen (16) Verigene RP Flex reportable target analytes. The LoD was defined as the concentration at which the test produces a positive result greater than or equal to 95% of the time. Serial dilutions of the strains were tested and the initial tentative LoD confirmed with 20 replicates. To ensure the accuracy of the LoD determination, if the initial detection rate was 100%, an additional 20 replicates were performed at the next lower concentration until ≤95% was achieved. The confirmed LoDs for the twenty-eight (28) strains tested and the corresponding LoDs for the RP Flex test reportable targets are shown in the table below.
| Viral Species and<br>Bacterial Genus | Viral Strains and<br>Bacterial Species | LoD |
|--------------------------------------|----------------------------------------|------------------------------|
| Adenovirus | C (AdV-1) | $1.2\times10^{1}$ TCID50/mL |
| Adenovirus | B (AdV-3) | $1.1\times10^{0}$ TCID50/mL |
| Adenovirus | E (AdV-4) | $4.1\times10^{-2}$ TCID50/mL |
| Human<br>Metapneumovirus | Metapneumovirus 9 (A1) | $3.0\times10^{1}$ TCID50/mL |
| Human<br>Metapneumovirus | Metapneumovirus 27 (A2) | $1.1\times10^{0}$ TCID50/mL |
| Human<br>Metapneumovirus | Metapneumovirus 3 (B1) | $1.0\times10^{1}$ TCID50/mL |
| Human<br>Metapneumovirus | Metapneumovirus 8 (B2) | $3.3\times10^{0}$ TCID50/mL |
| Influenza A | Brisbane/59/2007 (H1N1) | $3.0\times10^{1}$ TCID50/mL |
| Influenza A | California/04/2009pdm09 (H1N1) | $1.0\times10^{1}$ TCID50/mL |
| Influenza A | Port Chalmers/1/73 (H3N2) | $3.3\times10^{0}$ TCID50/mL |
| Influenza A | Victoria/361/2011 (H3N2) | $3.7\times10^{1}$ TCID50/mL |
| Influenza A | Wisconsin/67/05 (H3N2) | $3.3\times10^{0}$ TCID50/mL |
| Influenza B | Brisbane/60/2008 | $1.2\times10^{1}$ TCID50/mL |
| Influenza B | Florida/02/2006 | $3.0\times10^{1}$ TCID50/mL |
| Influenza B | Massachusetts/02/2012 | $1.2\times10^{1}$ TCID50/mL |
| Parainfluenza | Parainfluenza 1 | $9.0\times10^{1}$ TCID50/mL |
| Parainfluenza | Parainfluenza 2 | $1.0\times10^{1}$ TCID50/mL |
| Parainfluenza | Parainfluenza 3 | $3.3\times10^{0}$ TCID50/mL |
| Parainfluenza | Parainfluenza 4a | $2.7\times10^{2}$ TCID50/mL |
| Rhinovirus | A (Rhinovirus 39) | $1.0\times10^{1}$ TCID50/mL |
| Rhinovirus | B (Rhinovirus 14) | $9.0\times10^{1}$ TCID50/mL |
| Rhinovirus | C (Rhinovirus C41) | $2.4\times10^{3}$ PFU/mL |
| Respiratory<br>Syncytial Virus | RSV A (A2) | $3.3\times10^{0}$ TCID50/mL |
| Respiratory<br>Syncytial Virus | RSV B (Wash/18537/62) | $3.7\times10^{1}$ TCID50/mL |
| Bordetella | parapertussis | $2.4\times10^{3}$ CFU/mL |
| Bordetella | bronchiseptica | $2.4\times10^{3}$ CFU/mL |
| Bordetella | holmesii | $2.4\times10^{3}$ CFU/mL |
#### Table 1: Limit of Detection (LoD)
CONFIDENTIAL AND PROPRIETARY Nanosphere, Inc.
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#### Analytical Reactivity (Inclusivity)
The analytical reactivity (inclusivity) of the RP Flex test was demonstrated with a comprehensive panel of one-hundred and eight (108) strains representing temporal, evolutionary, and geographic diversity for each of the RP Flex panel organisms. Together with the twentyeight (28) strains evaluated as part of the Limit of Detection Study, a total of one-hundred and thirty-six (136) strains were evaluated for analytical inclusivity to RP Flex through empirical testing.
The organisms in the inclusivity panel were prepared in Simulated NPS. Thirteen (13) strains of Influenza A (subtypes H2N2, H2N3, H5N1, H5N3, H7N2, H7N7, H7N9, H9N2 & H10N7) were prepared and tested at a BSL 3 laboratory. Each sample was tested with the RP Flex in triplicate at an initial concentration 3-fold higher than the LoD determined for each analyte. In cases where the expected targets were not detected in one or more replicates, concentrations at a 3-fold higher level were evaluated.
RP Flex demonstrated analytical reactivity to all one-hundred and eight (108) strains tested, with some strains requiring higher titers for detection. The individual strains and concentrations at which positive test results were obtained for all three (3) replicates are presented by target organism in the tables below.
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## Adenovirus Inclusivity Results
| Adenovirus<br>Species | Serotype | Strain # | Source | Concentration<br>(TCID50/mL) | Multiples of<br>LoD |
|-----------------------|----------|-----------|-------------|------------------------------|---------------------|
| A | 31 | 0810073CF | Zeptometrix | $1.1\times10^0$ | 1x |
| B1 | 7 | VR-7 | ATCC | $3.3\times10^0$ | 3x |
| B1 | 21 | VR-1099 | ATCC | $3.3\times10^0$ | 3x |
| B2 | 11 | VR-12 | ATCC | $3.3\times10^0$ | 3x |
| B2 | 14 | 0810108CF | Zeptometrix | $3.3\times10^0$ | 3x |
| B2 | 34 | VR-716 | ATCC | $3.3\times10^0$ | 3x |
| B2 | 35 | VR-718 | ATCC | $1.0\times10^1$ | 9x |
| C | 2 | 111010 | TriCore | $3.3\times10^0$ | 3x |
| C | 5* | 0810020CF | Zeptometrix | $8.1\times10^2$ | 729x |
| C | 6* | 0810111CF | Zeptometrix | $2.7\times10^2$ | 243x |
| D | 26 | 0810117CF | Zeptometrix | $1.1\times10^0$ | 1x |
| D | 37 | 0810119CF | Zeptometrix | $1.1\times10^0$ | 1x |
| F | 40 | 0810084CF | Zeptometrix | $1.1\times10^0$ | 1x |
| F | 41 | 0810085CF | Zeptometrix | $1.1\times10^0$ | 1x |
Table 2: Adenovirus Inclusivity Results
* Based on in silico analysis, the oligonucleotide identities of all the tested Adenovirus C subtypes have very similar ranges. Based on the investigation of viral stocks titers using a quantitative TagMan real-time PCR developed at Nanosphere that is specific for all Adenovirus species (note: the TaqMan assay are not the same primers used in the RP Flex), it appears that the amplifiable genome equivalents available in these two adenovirus viral stocks are significantly reduced comparing to that of the other adenovirus stocks tested in the study.
#### Influenza A Inclusivity Results
| Table 3: | Influenza A Inclusivity Results |
|----------|---------------------------------|
|----------|---------------------------------|
| Influenza A<br>Subtype | Strain | Source | Influenza A | | A/H1 or A/H3 | |
|------------------------|---------------------------|--------------------|------------------------------|--------------------|------------------------------|---------------------|
| | | | Concentration<br>(TCID50/mL) | Multiple of<br>LoD | Concentration<br>(TCID50/mL) | Multiples of<br>LoD |
| H1N1 | A/California/07/2009pdm09 | IRR | $9.0\times10^1$ | 3x | $9.0\times10^1$ | 9x |
| | A/New Caledonia/20/99 | Zeptometrix | $9.0\times10^1$ | 3x | $9.0\times10^1$ | 9x |
| | A/New Jersey/8/76 | TriCore | $2.7\times10^2$ | 9x | $3.0\times10^1$ | 3x |
| | A/NWS/33 | TriCore | $3.0\times10^1$ | 1x | $3.0\times10^1$ | 3x |
| | A/PR/8/34 | Charles River Labs | $3.0\times10^1$ | 1x | $3.0\times10^1$ | 3x |
| | A1/Denver/1/57 | TriCore | $3.0\times10^1$ | 1x | $3.0\times10^1$ | 3x |
| | A1/FM/1/47 | TriCore | $3.0\times10^1$ | 1x | $3.0\times10^1$ | 3x |
| | A/ Solomon Islands/3/2006 | Zeptometrix | $3.0\times10^1$ | 1x | $3.0\times10^1$ | 3x |
| | A/Hawaii/15/2001 | IRR | $2.7\times10^2$ | 9x | $2.7\times10^2$ | 27x |
| H3N2 | A/ Aichi/ 68 | Charles River Labs | $1.0\times10^1$ | <1x | $1.0\times10^1$ | 3x |
| | A/ Hong Kong/ 8/ 68 | Charles River Labs | $3.0\times10^1$ | 1x | $1.0\times10^1$ | 3x |
| | A/ Victoria/ 3/ 75* | Charles River Labs | $2.4\times10^3$ | 81x | $2.4\times10^3$ | 729x |
| | A/Ohio/02/2012 | IRR | $2.7\times10^2$ | 9x | $2.7\times10^2$ | 81x |
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| Influenza A<br>Subtype | Strain | Source | Influenza A | | A/H1 or A/H3 | |
|------------------------|------------------------------------------|--------|------------------------------|--------------------|------------------------------|---------------------|
| | | | Concentration<br>(TCID50/mL) | Multiple of<br>LoD | Concentration<br>(TCID50/mL) | Multiples of<br>LoD |
| | A/Indiana/08/2011 | IRR | $1.0\times10^1$ | <1x | $1.0\times10^1$ | 3x |
| H3N2v | A/Minnesota/11/2010** | IRR | $2.4\times10^3$ | 81x | $9.0\times10^1$ | 27x |
| | A/Indiana/10/2011 | IRR | $1.0\times10^1$ | <1x | $3.0\times10^1$ | 9x |
| H2N2 | Japan/305/1957 | MRI | $9.0\times10^1$ | 3x | - | - |
| H2N3 | Mallard/Albert79/03 | MRI | $9.0\times10^1$ | 3x | - | - |
| H5N1 | A/Duck/Hunan/795/02 | MRI | $9.0\times10^1$ | 3x | - | - |
| | A/Chicken/Korea/IS/2006 | MRI | $9.0\times10^1$ | 3x | - | - |
| | A/Scaly-breasted Munia/<br>HongKong/2006 | MRI | $9.0\times10^1$ | 3x | - | - |
| H5N3 | A/Duck/Singapore/645/1997 | MRI | $8.1\times10^2$ | 27x | - | - |
| H7N2 | A/New York/107/2003 | MRI | $9.0\times10^1$ | 3x | - | - |
| H7N7 | A/Netherlands/219/2003 | MRI | $2.7\times10^2$ | 9x | - | - |
| | Equine-1/Prague/1956 | MRI | $9.0\times10^1$ | 3x | - | - |
| H7N9 | Anhui/01/2013 | MRI | $9.0\times10^1$ | 3x | - | - |
| H9N2 | Hong Kong/1073/99 | MRI | $9.0\times10^1$ | 3x | - | - |
| | Chicken/Hong Kong/G9/97 | MRI | $9.0\times10^1$ | 3x | - | - |
| H10N7 | Chick/Germany/n/1949 | MRI | $9.0\times10^1$ | 3x | - | - |
彩 Based on in silico analysis, the oligonucleotide identities of all the tested Influenza A/H3N2 strains have very similar ranges. Based on the investigation of viral stocks titers using a quantitative TaqMan real-time PCR developed at Nanosphere that is specific for Influenza A/H3 strains (note: the primers for the TaqMan assay are not the same primers used in the RP Flex), it appears that the amplifiable genome equivalents available in this Influenza A/H3N2 viral stock are significantly reduced comparing to that of the other Influenza A/H3N2 stocks tested in the study.
** Based on in silico analysis, the oligonucleotide identities to this strain have slightly lower ranges than the other two H3N2v strains tested.
#### Influenza B Inclusivity Results
Table 4: Influenza B Inclusivity Results
| Type | Strain | Source | Concentration<br>(TCID50/mL) | Multiples of<br>LoD |
|-------------|----------------------|-------------------|------------------------------|---------------------|
| Influenza B | B/ Allen/45 | TriCore | $9.0\times10^1$ | 3x |
| | B/Florida/07/2004 | TriCore | $9.0\times10^1$ | 3x |
| | B/GL/1739/54 | TriCore | $9.0\times10^1$ | 3x |
| | B/Hong Kong/5/72 | ATCC | $9.0\times10^1$ | 3x |
| | B/Malaysia/2506/2004 | TriCore | $9.0\times10^1$ | 3x |
| | B/Maryland/1/59 | TriCore | $9.0\times10^1$ | 3x |
| | B/Taiwan/2/62 | TriCore | $9.0\times10^1$ | 3x |
| | B/Wisconsin/01/2010 | IRR | $9.0\times10^1$ | 3x |
| | B/ Lee/40 | Charles River Lab | $9.0\times10^1$ | 3x |
| | B/Florida/04/2006 | Zeptometrix | $9.0\times10^1$ | 3x |
{12}------------------------------------------------
### Human Metapneumovirus Inclusivity Results
| Subtype | Strain | Source | Concentration<br>(TCID50/mL) | Multiples of<br>LoD |
|---------|--------|-----------------------|------------------------------|---------------------|
| hMPV A1 | 16 | Zeptometrix 0810161CF | $9.0\times10^1$ | 3x |
| hMPV A2 | 20 | Zeptometrix 0810163CF | $9.0\times10^1$ | 3x |
| hMPV B1 | 5 | Zeptometrix 0810158CF | $9.0\times10^1$ | 3x |
| hMPV B2 | 4 | Zeptometrix 0810157CF | $9.0\times10^1$ | 3x |
| | 18 | Zeptometrix 0810162CF | $9.0\times10^1$ | 3x |
#### Table 5: Human Metapneumovirus Inclusivity Results
#### Parainfluenza 1-4 Inclusivity Results
#### Parainfluenza 1-4 Inclusivity Results Table 6:
| Type | Source/Strain | Concentration<br>(TCID50/mL) | Multiples of<br>LoD |
|-----------------|------------------------|------------------------------|---------------------|
| Parainfluenza 1 | Zeptometrix 0810014CF | $2.7\times10^2$ | 3x |
| Parainfluenza 2 | Zeptometrix 0810015CF | $3.0\times10^1$ | 3x |
| Parainfluenza 3 | ATCC VR-93* | $2.7\times10^2$ | 81x |
| | BEI NR-3233 | $3.0\times10^1$ | 9x |
| | TriCore (ATCC VR-1782) | $9.0\times10^1$ | 27x |
| Parainfluenza 4 | Zeptometrix 0810060CF | $8.1\times10^2$ | 3x |
| | VR-1377 | $8.1\times10^2$ | 3x |
| | Zeptometrix 0810060BCF | $8.1\times10^2$ | 3x |
* For Parainfluenza 3, the extracted eluate from the three strains tested in the inclusivity study were each evaluated with PCR/bi-directional sequencing, and the sequence information were used to assess the homology to the RP Flex oligos. Based on the in silico analysis, the three strains have the identical homology to the RP Flex oligos, indicating that the apparent difference in sensitivity was not due to sequence diversity in the gene targeted by the RP Flex. The apparent variation in the sensitivity of the RP Flex test for these strains is likely attributable to inconsistencies in the quantification of the viral stocks.
## RSV Inclusivity Results
#### Table 7: RSV Inclusivity Results
| Subtype | Source/Strain | Concentration<br>(TCID50/mL) | Multiples of<br>LoD |
|-------------------------------|------------------------|------------------------------|---------------------|
| Respiratory Syncytial Virus A | ATCC VR-26 | $1.0\times10^1$ | 3x |
| | Zeptometrix 0810040ACF | $1.0\times10^1$ | 3x |
| Respiratory Syncytial Virus B | Zeptometrix 0810040CF | $1.1\times10^0$ | 3x |
| | ATCC VR-1400 | $1.1\times10^0$ | 3x |
| | ATCC VR-955 | $3.3 \times10^0$ | 9x |
{13}------------------------------------------------
## Rhinovirus A and B Inclusivity Results
| Rhinovirus Species | Strain | Source | Concentration<br>(TCID50/mL) | Multiples of<br>LoD |
|--------------------|--------|------------------------|------------------------------|---------------------|
| Rhinovirus A | 1 | Zeptometrix 0810012CFN | $2.7\times10^2$ | 3x |
| | 2 | ATCC VR-482 | $2.7\times10^2$ | 3x |
| | 7 | ATCC VR-1601 | $2.7\times10^2$ | 3x |
| | 16 | ATCC VR-283 | $2.7\times10^2$ | 3x |
| | 34 | ATCC VR-507 | $2.7\times10^2$ | 3x |
| | 57 | ATCC VR-1600 | $2.7\times10^2$ | 3x |
| | 77 | ATCC VR-1187 | $2.7\times10^2$ | 3x |
| | 85 | ATCC VR-1195 | $2.7\times10^2$ | 3x |
| Rhinovirus B | 3 | ATCC VR-483 | $2.7\times10^2$ | 3x |
| | 17 | ATCC VR-1663 | $2.7\times10^2$ | 3x |
| | 27 | ATCC VR-1137 | $2.7\times10^2$ | 3x |
| | 42 | ATCC VR-338 | $2.7\times10^2$ | 3x |
| | 83 | ATCC VR-1193 | $2.7\times10^2$ | 3x |
#### Rhinovirus A and B Inclusivity Results Table 8:
### Rhinovirus C Inclusivity Results
Table 9: Rhinovirus C Inclusivity Results
| Rhinovirus Species | Strain | Source | Concentration<br>(PFU/mL)* | Multiples of<br>LoD |
|--------------------|--------|------------|----------------------------|---------------------|
| Rhinovirus C | C2 | UW-Madison | $7.3\times10^3$ | 3x |
| Rhinovirus C | C15 | UW-Madison | $7.3\times10^3$ | 3x |
* As there is no susceptible cell line to grow Rhinovirus C, the strains were cloned into a plasmid vector and transfected into WisL cells (primary human lung fibroblasts). All were sequenced to confirm identity. The titers were established by qPCR using serial dilutions of Rhinovirus 16 as a surrogate to provide actual PFU/mL values for the standard curve. Therefore, it has been assumed that Rhinovirus 16 has similar virulence rates to Rhinovirus C.
{14}------------------------------------------------
## Bordetella Species Inclusivity Results
| Bordetella Species | Source | RP Flex<br>Target | Concentration<br>(CFU/mL) | Multiples of<br>LoD |
|--------------------|------------------------|------------------------------------------------|---------------------------|---------------------|
| B. pertussis | ATCC 51445 | B. pertussis | $2.4\times10^3$ | 3x |
| | ATCC 10380 | | $2.4\times10^3$ | 3x |
| | ATCC 9340 | | $2.4\times10^3$ | 3x |
| | ATCC BAA-589 | | $2.4\times10^3$ | 3x |
| | ATCC BAA-1335 | | $2.4\times10^3$ | 3x |
| | ATCC 53894 | | $2.4\times10^3$ | 3x |
| | ATCC 9306 | | $2.4\times10^3$ | 3x |
| | ATCC 8467 | | $7.3\times10^3$ | 9x |
| | ATCC 15237 | Bordetella<br>Parapertussis/<br>bronchiseptica | $7.3\times10^3$ | 3x |
| | ATCC 9305 | | $7.3\times10^3$ | 3x |
| B. parapertussis | ATCC BAA-587 | | $7.3\times10^3$ | 3x |
| | ATCC 15989 | | $7.3\times10^3$ | 3x |
| | Zeptometrix<br>0801461 | | $2.2\times10^4$ | 9x |
| | | ATCC 4617 | | $7.3\times10^3$ |
| | | ATCC 7773 | | $7.3\times10^3$ |
| | ATCC 785 | Bordetella<br>Parapertussis/<br>bronchiseptica | $7.3\times10^3$ | 3x |
| B. bronchiseptica | ATCC 14064 | | $7.3\times10^3$ | 3x |
| | ATCC 10580 | | $7.3\times10^3$ | 3x |
| | ATCC 19395 | | $7.3\times10^3$ | 3x |
| | Zeptometrix<br>0801464 | | $2.2\times10^4$ | 9x |
| | B. holmesii | | ATCC 700053 | B. holmesii |
| | | | ATCC 700052 | |
# Table 10: Bordetella Species Inclusivity
# Analytical Specificity (Exclusivity)
One hundred and seven (107) organisms (tables below), consisting of forty-six (46) bacterial/fungal strains (tested at 1×106 CFU/mL), twenty-six (26) viruses, twenty-two in-panel tested in the LoD study, and thirteen (13) additional influenza A virus strains with other hemagglutinin (HA) types were tested with RP Flex to determine analytical specificity (exclusivity).
The viral and bacterial/fungal samples were contrived in Simulated NPS at high concentrations (1×105 TCID50/mL for viral targets and at 1×106 CFU/mL for bacterial and fungal targets, except for Mumps virus which was tested at the highest available concentration of 1.60×100 TCID50/mL). Four (4) organisms which were not available as titered stocks were evaluated using genomic DNA at 1×10 copies/mL. All samples were tested in triplicate with the RP Flex.
{15}------------------------------------------------
# Bacterial and Fungal Organisms Tested for RP Flex Analytical Specificity
| | Table 11: Bacterial and Fungal Organisms Tested for Analytical Specificity | | | | | |
|--|-----------------------------------------------------------------------------|--|--|--|--|--|
|--|-----------------------------------------------------------------------------|--|--|--|--|--|
| Genus | Species | Strain Number |
|-------------------------|------------------------------|---------------------------------|
| Acinetobacter | baumannii | ATCC 19606 |
| Bordetella | avium | ATCC 35086 |
| Bordetella | hinzii | ATCC 51784 |
| Bordetella | petrii | ATCC BAA-461 |
| Bordetella | trematum | ATCC 700309 |
| Candida | albicans | ATCC 18804 |
| Candida | glabrata | ATCC 38326 |
| Chlamydophila | pneumoniae | ATCC VR-1360 |
| Chlamydia | trachomatis Serovar D | ATCC VR-885 |
| Corynebacterium | pseudodiphtheriticum | ATCC 10700 |
| Corynebacterium | diphtheriae | ATCC 14779 |
| Corynebacterium | striatum | ATCC BAA-1293 |
| Escherichia | coli | ATCC 25922 |
| Haemophilus | influenzae | ATCC 49144 |
| Haemophilus | parainfluenzae | ATCC 9796 |
| Klebsiella | pneumoniae subsp. pneumoniae | ATCC 13883 |
| Lactobacillus | acidophilus | Zeptometrix 0801540 |
| Lactobacillus | plantarum | ATCC BAA-793 |
| Legionella | pneumophilia | ATCC 33152 |
| Legionella | longbechiae | ATCC 33462 |
| Legionella | micdadei | ATCC 33204 |
| Listeria | innocua | ATCC 51742 |
| Listeria | monocytogenes serotype 4b | ATCC 19115 |
| Moraxella (Branhamella) | catarrhalis | ATCC 43617 |
| Mycobacterium | tuberculosis | ATCC BAA-2237D-2ª |
| Mycoplasma | genitalium | ATCC 49123ª |
| Mycoplasma | hominis | ATCC 27545-TTR |
| Mycoplasma | pneumoniae | ATCC 15531-TTR |
| Neisseria | elongata subsp. elongata | ATCC 25295 |
| Neisseria | gonorrhoeae | ATCC 31426 |
| Neisseria | meningitidis | ATCC 53415D-5ª |
| Neisseria | lactamica | ATCC 23970 |
| Neisseria | mucosa | ATCC 49233 |
| Neisseria | sicca | ATCC 29256 |
| Pneumocystis | jirovecii | Erasme-Belgium-Clinical Sample* |
| Proteus | vulgaris | ATCC 6380 |
| Pseudomonas | aeruginosa | ATCC 27853 |
| Serratia | marcescens | ATCC 29021 |
| Staphylococcus | aureus subsp. aureus | ATCC 12600 |
| Staphylococcus | epidermidis | ATCC 12228 |
| Staphylococcus | haemolyticus | ATCC 29970 |
| Streptococcus | agalactiae | ATCC 12386 |
| Streptococcus | pneumoniae | ATCC 6303 |
| Streptococcus | pyogenes | ATCC 14289 |
| Streptococcus | salivarius | ATCC 13419 |
| Ureaplasma | urealyticum | ATCC 27618ª |
ª Genomic DNA tested at 1×106 copies/mL
{16}------------------------------------------------
# Viral Organisms Tested for RP Flex Analytical Specificity
| Virus Name | Type | Source/Strain Number |
|------------------------|----------------------------|-----------------------|
| Bocavirus | - | Clinical Sample |
| Coronavirus | 229E | Zeptometrix 0810229CF |
| Coronavirus | NL63 | Zeptometrix 0810228CF |
| Coronavirus | OC43 | Zeptometrix 0810024CF |
| Coronavirus | HKU1 | LIJ-Clinical Sample |
| Cytomegalovirus | - | ATCC VR-977 |
| Enterovirus A | Type 71 | Zeptometrix 0810047CF |
| Enterovirus A | Coxsackievirus A2 | ATCC VR-1550 |
| Enterovirus A | Coxsackievirus A10 | Zeptometrix 0810106CF |
| Enterovirus B | Coxsackievirus A9 | Zeptometrix 0810017CF |
| Enterovirus B | Coxsackievirus B4 | ATCC VR-184 |
| Enterovirus B | Coxsackievirus B5 | ATCC VR-185 |
| Enterovirus B | Echovirus 6 | Zeptometrix 0810076CF |
| Enterovirus B | Echovirus 9 | Zeptometrix 0810077CF |
| Enterovirus B | Echovirus 11 | Zeptometrix 0810023CF |
| Enterovirus B | Echovirus 30 | Zeptometrix 0810078CF |
| Enterovirus C | Coxsackievirus A21 | Zeptometrix 0810235CF |
| Enterovirus C | Coxsackievirus A24* | ATCC VR-1662 |
| Enterovirus C | Poliovirus 2 (attenuated)* | ATCC VR-301 |
| Enterovirus C | Poliovirus 3 (attenuated)* | ATCC VR-193 |
| Enterovirus D | Type 68* | ATCC VR-561 |
| Epstein Barr Virus | - | Zeptometrix 0810008CF |
| Herpes Simplex virus | Type 1 | Zeptometrix 0810005CF |
| Measles | - | ATCC VR-24 |
| Mumps virus | - | ATCC VR-106 |
| Varicella-Zoster virus | - | Zeptometrix 0810026CF |
## Table 12: Viral Organisms Tested for Analytical Specificity
In-Panel RP Flex Organisms (Viruses and Bacteria) and Additional Influenza A Virus Strains with Other Hemagglutinin (HA) Types Tested for Analytical Specificity
| Table 13: In-Panel Organisms Tested for Analytical Specificity | |
|-----------------------------------------------------------------|--|
| | |
| Bacteria/Virus Name | Type | Source/Strain Number |
|-------------------------------------|-----------------|-----------------------|
| Adenovirus A | Type 31 | Zeptometrix ×810073CF |
| Adenovirus D | Type 26 | Zeptometrix 0810117CF |
| Adenovirus D | Type 37 | Zeptometrix 0810119CF |
| Adenovirus F | Type 40 | Zeptometrix 0810084CF |
| Adenovirus F | Type 41 | Zeptometrix 0810085CF |
| Adenovirus E | Type 4 | Zeptometrix 0810070CF |
| Bordetella holmesii | - | ATCC 51541 |
| Bordetella pertussis | - | ATCC 9797 |
| Influenza A /Brisbane/59/2007 | H1N1 | TriCore |
| Influenza A /Wisconsin/67/05 | H3N2 | Zeptometrix N/A |
| Influenza A/California/04/2009pdm09 | H1N1 - pandemic | TriCore |
| Influenza A/Victoria/361/2011 | H3N2 | Zeptometrix 0810240CF |
| Influenza A | H2N2a | Japan/305/1957 |
| Influenza A | H5N1a | A/Duck/Hunan/795/02 |
CONFIDENTIAL AND PROPRIETARY
Nanosphere, Inc.
{17}------------------------------------------------
| Bacteria/Virus Name | Type | Source/Strain Number |
|------------------------------|---------|------------------------------------|
| Influenza A | H5N1a | A/Chicken/Korea/IS/2006 |
| Influenza A | H5N1a | Scaly-breasted Munia/HongKong/2006 |
| Influenza A | H7N2a | New York/107/2003 |
| Influenza A | H7N7a | Netherlands/219/2003 |
| Influenza A | H7N9a | Anhui/01/2013 |
| Influenza A | H9N2a | Hong Kong/1073/99 |
| Influenza A | H2N3a | Mallard/Albert79/03 |
| Influenza A | H5N3a | Duck/Singapore/645/1997 |
| Influenza A | H7N7a | Equine-1/Prague/1956 |
| Influenza A | H9N2a | Chicken/Hong Kong/G9/97 |
| Influenza A | H10N7a | Chick/Germany/n/1949 |
| Influenza B /Florida/02/2006 | - | TriCore |
| Metapneumovirus 9 | Type A1 | TriCore |
| Metapneumovirus 8 | Type B2 | TriCore |
| Parainfluenza 1 | - | TriCore VR-94 |
| Parainfluenza 2 | - | TriCore VR-92 |
| Parainfluenza 3 | - | Zeptometrix 0810016CF |
| Parainfluenza 4a | - | TriCore VR-1378 |
| Respiratory Syncytial Virus | Type A2 | TriCore VR-1540 |
| Respiratory Syncytial Virus | Type B | TriCore VR-1580 |
| Rhinovirus 14 | Type B | TriCore |
4 Prepared and tested at a BSL 3 laboratory.
All of the organisms tested vielded the expected "Not Detected" results at the concentrations tested with the exception of the enteroviruses marked with an asterisk and Pneumocystis jirovecii (from a clinical sample) marked with an asterisk, which gave "Rhinovirus detected" results in some of the replicates.
Based on in silico analyses, a number of Enterovirus strains have a relatively high homology to RP Flex Rhinovirus oligos, with some percent identities to Rhinovirus RP Flex oligos of 84%. As a result, some cross- reactivity at high titer was expected.
In silico analysis also determined that Pneumocystis jirovecii sequences have a maximum Oligo Identity to RP Flex targets of 67% and therefore Pneumocystis jirovecii was not predicted to be cross-reactive to RP Flex Rhinovirus probes. Extracted nucleic acids from all Rhinovirus positive tests of the Pneumocystis jirovecii positive clinical sample were evaluated with an analytically validated PCR/BDS Rhinovirus assay. PCR/BDS test results confirmed the presence of Rhinovirus in all samples, indicating that the Pneumocystis jirovecii positive clinical sample also contains Rhinovirus nucleic acids.
{18}------------------------------------------------
#### Interference (Exogenous and Endogenous Substances)
#### Microbial Interference
Three (3) representative target organisms detected by RP Flex, Adenovirus 3 (B), Influenza A (H1N1), and Bordetella pertussis were evaluated at 3x their respective LoD for potential interference in the presence of seven (7) potentially interfering microorganisms not detected by RP Flex: Staphylococcus aureus, Neisseria meningitidis, Corynebacterium diphtheria, Haemophilus influenza, Streptococcus pneumoniae, Mycoplasma pneumoniae, and cytomegalovirus. These seven (7) microorganisms represent the most prevalent microorganisms known to be present in the human upper respiratory tract and therefore the most likely to be encountered in NPS specimens. These normal flora organisms were tested at a concentration of 1×10° CFU/mL with the exception of Mycoplasma pneumoniae, which was tested at 1×10° CCU/mL, and Neisseria meningitidis, which was tested at 1×10° genomic copies/mL and cytomegalovirus, which was tested at 1×10° PFU/mL. No interference was observed with the RP Flex test for any of these samples tested.
#### Exogenous and Endogenous Substances
A comprehensive interfering substances study was performed to assess the potential effects of endogenous and exogenous substances that can commonly be found in clinical upper respiratory specimens. Three (3) representative target organisms detected by RP Flex, Adenovirus 3 (B), Influenza A (H1N1), and Bordetella pertussis were evaluated at 3x their respective LoD for potential interference in the presence of thirty-six (36) potentially interfering exogenous substances (table below). Two (2) endogenous substances were also included, human blood and human DNA. None of the substances at the concentrations tested showed any inhibitory effect on the detection of target respiratory pathogens using the RP Flex test.
| Interfering Substances Tested | |
|--------------------------------------|-------------------------------------|
| Wal-Four® Nasal Spray | Staphylococcus aureus |
| Anefrin Nasal Spray | Neisseria meningitidis |
| Saline Nasal Spray | Corynebacterium diphtheriae |
| Similasan Sinus Relief | Haemophilus influenzae |
| Anbesol® (Anesthetic) | Streptococcus pneumoniae |
| Qvar® (Nasal corticosteroid) | Mycoplasma pneumoniae |
| Dexacort® (Nasal corticosteroid) | Cytomegalovirus |
| AeroBid® (Nasal corticosteroid) | Mucin, bovine submaxillary Type I-S |
| Triamcinolone (Nasal corticosteroid) | Mucin, porcine stomach Type II |
| Pulmicort® (Nasal corticosteroid) | Mucin, porcine stomach Type III |
| Flocon® (Nasal corticosteroid) | BD Universal Viral Transport Media |
#### Table 14: Interfering Substances
{19}------------------------------------------------
| Interfering Substances Tested | |
|----------------------------------|------------------------------------------------------|
| Flonase® (Nasal corticosteroid) | Remel M4® |
| Veramyst® (Fluticasone furoate) | Remel M4-RT® |
| Tobramycin (systemic antibiotic) | Remel M5® |
| Relenza™ (Anti-viral) | Remel M6™ |
| Tamiflu® (Anti-viral) | BD Liquid Amies |
| Sulfur (Boiron®) | Remel Regan Lowe Semi-Solid Transport Media |
| Galphimia Glauca (Boiron®) | Copan ClassiqSwabs (Aluminum, rayon tipped, sterile) |
| Histaminum Hydrochloricum | Copan FloqSwabs (Nylon® ,regular, sterile) |
| Mupirocin (antibiotic) | Ethyl Alcohol, Absolute 200 Proof |
| Menthol | Acetonitrile |
| Human Blood | FluMist® Influenza Vaccine Live, Intranasal |
| Human DNA | |
## Competitive Interference
In order to assess potential competitive inhibition for RP Flex, binary combinations of all test panel organisms representing all possible dual infections, were evaluated. Contrived samples were prepared in negative simulated NPS matrix, with one panel organism present at a Low Positive titer (3x LoD) and a second organism present at a High Positive titer (1×10) TCIDsomL for viruses, 1×10° CFU/mL for bacteria). The performance of Verigene RP Flex was evaluated with each of the one-hundred and eighty-two (182) unique sample combinations tested in replicates of three (3). No evidence of competitive inhibition was observed at the titers tested.
## Carryover and Cross-Contamination
The potential for carryover and cross-contamination on the Verigene system was assessed by alternately testing three (3) high positive respiratory pathogen samples; Adenovirus 3 (B), Influenza A (H1N1) (both at 1×10° TCID50/mL), and Bordetella pertussis (at 1×10° CFU/mL), followed by testing a negative NPS sample. The high-titer sample was alternated with the negative sample five (5) times on six (6) unique Processor SPs. No carryover or crosscontamination was observed.
## Specimen Stability
Fourteen (14) viral and bacterial strains in pooled Negative Clinical NPS were evaluated at Low Positive (2x LoD) and Moderate Positive (5x LoD) concentrations. Samples were stored at various temperature conditions and tested at defined timepoints in triplicate. The results of this stability study support the stability claim for RP Flex testing of clinical NPS specimens preserved in UTM at the following storage conditions: 4 hours at 20-25°C, 72 hours at 2-8°C, and 30 days at <- 70°C.
{20}------------------------------------------------
#### Precision
The Precision Study involved the testing of a representative test panel daily by two (2) operators for twelve (12) non-consecutive days for a total of forty-eight (48) tests per panel sample. The Precision Study used three (3) lots of each of the consumables (cartridges, extraction trays and amplification trays). All precision testing was performed at a single laboratory site with one (1) Verigene reader and twelve (12) Verigene Processor SPs. The test panel, representing all the RP Flex analytes except for B. parapertussis and B. bronchiseptica, consisted of two (2) negative samples (one negative simulated NPS matrix and one Staphylococcus aureus spiked in negative simulated NPS matrix), as well as seven (7) positive mixed samples at two (2) different concentrations for a total of sixteen (16) unique samples. Samples were prepared by spiking previously characterized and quantified organism stocks into simulated NPS matrix at Moderate Positive (5x LoD) and Low Positive (2x LoD) concentrations.
The results of the precision study are summarized below. which provides the percent agreement between the expected results and the obtained results for each sample tested.
| Verigene RP Flex Target | Positive Percent Agreement (95% CI) | | Negative Percent Agreement*<br>(95% CI) |
|-------------------------|-------------------------------------|------------------------------|-----------------------------------------|
| | Low | Moderate | |
| Parainfluenza 1 | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 100%<br>671/671<br>(99.4-100) |
| Parainfluenza 2 | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 100%<br>671/671<br>(99.4-100) |
| Parainfluenza 3 | 100%<br>48/48<br>(92.6-100) | 95.8<br>46/48<br>(86.0-98.8) | 100%<br>671/671<br>(99.4-100) |
| Parainfluenza 4 | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 99.9%<br>670/671<br>(99.2-100) |
| RSV A | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 100%<br>671/671<br>(99.4-100) |
| RSV B | 93.8%<br>45/48<br>(83.2-97.9) | 100%<br>48/48<br>(92.6-100) | 100%<br>671/671<br>(99.4-100) |
| Influenza A | 100%<br>96/96<br>(96.2-100) | 100%<br>96/96<br>(96.2-100) | 100%<br>575/575<br>(99.3-100) |
| Influenza A/H1 | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 100%<br>671/671<br>(92.4-100) |
| Influenza A/H3 | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 100%<br>671/671<br>(92.4-100) |
| Influenza B | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 100%<br>671/671<br>(92.4-100) |
| | Table 15: Precision Study | |
|--|-----------------------------|--|
|--|-----------------------------|--|
CONFIDENTIAL AND PROPRIETARY Nanosphere, Inc.
Page 17 of 27
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| Verigene RP Flex Target | Positive Percent Agreement (95% CI) | | Negative Percent Agreement* (95% CI) |
|-------------------------|-------------------------------------|-------------------------------|--------------------------------------|
| | Low | Moderate | |
| Rhinovirus | 97.9%<br>47/48<br>(89.1-99.6) | 100%<br>48/48<br>(92.6-100) | 99.9%<br>670/671<br>(99.2-100) |
| hMPV | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 100%<br>671/671<br>(92.4-100) |
| Adenovirus | 100%<br>48/48<br>(92.6-100) | 100%<br>48/48<br>(92.6-100) | 99.7%<br>669/671<br>(98.9-99.9) |
| B. pertussis | 100%<br>48/48<br>(92.6-100) | 97.9%<br>46/47<br>(88.9-99.6) | 100%<br>672/672<br>(99.4-100) |
| B. holmesii | 100%<br>47/48<br>(89.1-99.6) | 100%<br>47/47<br>(92.4-100) | 100%<br>672/672<br>(99.4-100) |
* Negative Percent Agreement (NPA) was determined with all samples that did not contain the target analyte.
## Reproducibility
The Reproducibility Study involved the testing of a representative test panel daily by two (2) operators for five (5) non-consecutive days at three (3) sites for a total of ninety (90) tests per sample. The test panel, representing all the RP Flex analytes except for B. parapertussis and B. bronchiseptica, consisted of two (2) negative samples (one negative simulated NPS matrix and one Staphylococcus aureus spiked in negative simulated NPS matrix), as well as seven (7) positive mixed samples at two (2) different concentrations for a total of sixteen (16) unique samples. Samples were prepared by spiking previously characterized and quantified organism stocks into simulated NPS matrix at Moderate Positive (5x LoD) and Low Positive (2x LoD) concentrations.
The results of the reproducibility study are summarized below, which provides the percent agreement between the expected results and the obtained results for each sample tested.
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| Table 16: Reproducibilitv Study | |
|-----------------------------------|--|
| | |
| Verigene RP Flex Target | Positive Percent Agreement (95% CI) | Negative Percent Agreement* | | | |
|-------------------------|-------------------------------------|-----------------------------|--------------|--|--|
| | Low | Moderate | (95% CI) | | |
| | 100% | 100% | 100% | | |
| Parainfluenza 1 | 90/90 | 90/90 | 1258/1258 | | |
| | (96.2-100) | (96.2-100) | (99.7-100) | | |
| | 100% | 100% | 99.8% | | |
| Parainfluenza 2 | 89/89 | 90/90 | 1256/1259 | | |
| | (95.9-100) | (96.2-100) | (99.3-99.9) | | |
| | 100% | 100% | 100% | | |
| Parainfluenza 3 | 90/90 | 90/90 | 1258/1258 | | |
| | (96.2-100) | (96.2-100) | (99.7-100) | | |
| | 100% | 100% | 100% | | |
| Parainfluenza 4 | 90/90 | 89/89 | 1259/1259 | | |
| | (96.2-100) | (95.9-100) | (99.7-100) | | |
| | 98.9% | 97.8% | 100% | | |
| RSV A | 89/90 | 88/90 | 1258/1258 | | |
| | (94.0-99.8) | (92.3-99.4) | (99.7-100) | | |
| | 100% | 100% | 99.9% | | |
| RSV B | 90/90 | 90/90 | 1257/1258 | | |
| | (96.2-100) | (96.2-100) | (99.6-100) | | |
| | 99.4 | 100% | 100% | | |
| Influenza A | 179/179 | 180/180 | 1079/1079 | | |
| | (97.9-100) | (97.9-100) | (99.6-100) | | |
| | 100% | 100% | 99.8% | | |
| Influenza A/H1 | 90/90 | 90/90 | 1256/1258 | | |
| | (96.2-100) | (96.2-100) | (99.4-100) | | |
| | 98.9% | 100% | 99.6% | | |
| Influenza A/H3 | 88/89 | 90/90 | 1254/1259 | | |
| | (93.9-99.8) | (96.2-100) | (99.1-99.8) | | |
| | 100% | 100% | 99.8% | | |
| Influenza B | 90/90 | 90/90 | 1255/1258 | | |
| | (96.2-100) | (96.2-100) | (99.3-99.9) | | |
| | 100% | 100% | 99.9% | | |
| Rhinovirus | 90/90 | 90/90 | 1257/1258 | | |
| | (96.2-100) | (96.2-100) | (99.6-100) | | |
| | 100% | 100% | 99.9% | | |
| hMPV | 90/90 | 89/89 | 1258/1259 | | |
| | (96.2-100) | (95.9-100) | (99.6-100) | | |
| | 100% | 100% | 99.8% | | |
| Adenovirus | 90/90 | 90/90 | 1255/1258 | | |
| | (96.2-100) | (96.2-100) | (99.3 -99.9) | | |
| | 100% | 100% | 99.8% | | |
| Bordetella para/bronch | 90/90 | 90/90 | 1256/1258 | | |
| | (96.2-100) | (96.2-100) | (99.4-100) | | |
| | 96.7% | 100% | 99.9% | | |
| B. pertussis | 87/90 | 90/90 | 1257/1258 | | |
| | (90.7-98.9) | (96.2-100) | (99.6-100) | | |
| | 100% | 100% | 99.9% | | |
| B. holmesii | 90/90 | 90/90 | 1257/1258 | | |
| | (96.2-100) | (96.2-100) | (99.6-100) | | |
* Negative Percent Agreement (NPA) was determined with all samples that did not contain the target analyte.
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## Performance Data - Clinical Testing
The clinical performance characteristics of the RP Flex test were determined by comparing viral and bacterial test results to an FDA-cleared molecular respiratory panel and/or PCR amplification followed by confirmatory bi-directional sequencing in a multi-site prospective investigation study at six (6) external clinical study sites. Subjects included individuals whose routine care called for respiratory pathogen testing.
A total of 3299 specimens were enrolled, of which 18 specimens were excluded from the study due to protocol violations, and 15 specimens which yielded a final "No Call" result were excluded from the performance analyses. Therefore, a total of 3266 specimens were included in the performance analyses. Enrolled specimens included 1082 prospectively-collected fresh specimens (of which 1069 were included in the performance analyses), 1330 prospectivelycollected frozen specimens (of which 1317 were included in the performance analyses), 526 retrospectively-collected frozen specimens (of which 520 were included in the performance analyses), and 361 contrived frozen specimens (of which 360 were included in the performance analyses). The following table provides a summary of demographic information for the 2412 prospectively-collected specimens in the enrolled dataset.
| | Prospective Fresh | | Prospective Frozen | | Combined | |
|--------------|---------------------|------------|---------------------|------------|---------------------|------------|
| Age Range | No. of<br>Specimens | Percentage | No. of<br>Specimens | Percentage | No. of<br>Specimens | Percentage |
| 0-1 | 151 | 14.0% | 165 | 12.4% | 316 | 13.1% |
| >1-5 | 176 | 16.3% | 382 | 28.7% | 558 | 23.1% |
| >5-12 | 73 | 6.7% | 98 | 7.4% | 171 | 7.1% |
| >12-21 | 74 | 6.8% | 67 | 5.0% | 141 | 5.8% |
| >21-65 | 426 | 39.4% | 275 | 20.7% | 701 | 29.1% |
| >65 | 163 | 15.1% | 155 | 11.7% | 318 | 13.2% |
| Not Provided | 19 | 1.8% | 188 | 14.1% | 207 | 8.6% |
| Total | 1082 | 100% | 1330 | 100% | 2412 | 100% |
| | Table 17: Prospective Clinical Studies | | |
|--|------------------------------------------|--|--|
|--|------------------------------------------|--|--|
The comparator methods were a composite of an FDA-cleared molecular respiratory panel and analytically validated PCR with bi-directional sequencing. The tables below contain the clinical performance data generated during the RP Flex test clinical studies, stratified by specimen type, which, as previously described, were categorized as fresh and frozen prospectively collected specimens, frozen selected specimens and frozen contrived specimens.
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## Table 18: Results Stratified by Target Analyte – Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza B, Respiratory Syncytial Virus (RSV)A, Respiratory Syncytial Virus (RSV)B
| | Specimen Type | | n= | % Agreement (95% CI) | | | Specimen Type | | n= | % Agreement (95% CI) | |
|----------------------------|----------------------------|-----------|------|-----------------------------------|-------------------------------------|----------------------------|----------------------------|-----------|------|---------------------------------|-------------------------------------|
| | | | | Positive | Negative | | | | | Positive | Negative |
| Influenza A | Prospectively<br>Collected | Fresh | 1049 | 100%<br>12/12<br>(75.7 - 100) | 99.4%<br>1030/1037<br>(98.6 - 99.7) | Influenza A subtype H1 | Prospectively<br>Collected | Fresh | 1048 | -<br>- | 99.8%<br>1046/1048<br>(99.3 - 99.9) |
| | | Frozen | 1144 | 97.9%<br>46/47<br>(88.9 - 99.6) | 99.4%<br>1091/1097<br>(98.8 – 99.7) | | | Frozen | 1144 | 97.8%<br>45/46<br>(88.7 - 99.6) | 99.6%<br>1092/1096<br>(99.1 - 99.9) |
| | | All | 2193 | 98.3%<br>58/59<br>(91.0-99.7) | 99.4%<br>2121/2134<br>(99.0 - 99.6) | |…
