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.

Device Description

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.

AI/ML Overview

{
  "1. A table of acceptance criteria and the reported device performance": {
    "Influenza A": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "98.3% (58/59) (91.0-99.7)",
        "Negative Percent Agreement (95% CI)": "99.4% (2121/2134) (99.0-99.6)"
      }
    },
    "Influenza A subtype H1": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "97.8% (45/46) (88.7-99.6)",
        "Negative Percent Agreement (95% CI)": "99.7% (2138/2144) (99.4-99.9)"
      }
    },
    "Influenza A subtype H3": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "100% (13/13) (77.2-100)",
        "Negative Percent Agreement (95% CI)": "99.8% (2173/2177) (99.5-99.9)"
      }
    },
    "Influenza B": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "98.0% (49/50) (89.5-99.6)",
        "Negative Percent Agreement (95% CI)": "99.6% (2139/2147) (99.3-99.8)"
      }
    },
    "RSV A": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "100% (17/17) (81.6-100)",
        "Negative Percent Agreement (95% CI)": "99.9% (2150/2153) (99.6-99.9)"
      }
    },
    "RSV B": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "100% (173/173) (97.8-100)",
        "Negative Percent Agreement (95% CI)": "98.8% (1973/1997) (98.2-99.2)"
      }
    },
    "Parainfluenza 1": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "90.0% (27/30) (74.4-96.5)",
        "Negative Percent Agreement (95% CI)": "99.9% (2165/2167) (99.7-100)"
      }
    },
    "Parainfluenza 2": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "92.3% (12/13) (66.7-98.6)",
        "Negative Percent Agreement (95% CI)": "99.9% (2181/2184) (99.6-99.9)"
      }
    },
    "Parainfluenza 3": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "82.4% (14/17) (59.0-93.8)",
        "Negative Percent Agreement (95% CI)": "99.9% (2177/2180) (99.6-99.9)"
      }
    },
    "Parainfluenza 4": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "79.2% (19/24) (59.3-90.8)",
        "Negative Percent Agreement (95% CI)": "99.8% (2169/2173) (99.5-99.9)"
      }
    },
    "Adenovirus": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "86.0% (49/57) (74.7-92.7)",
        "Negative Percent Agreement (95% CI)": "97.2% (2081/2140) (96.5-97.9)"
      }
    },
    "Human Metapneumovirus (hMPV)": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "100% (46/46) (92.3-100)",
        "Negative Percent Agreement (95% CI)": "99.7% (2145/2151) (99.4-99.9)"
      }
    },
    "Rhinovirus": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "81.9% (407/497) (78.3-85.0)",
        "Negative Percent Agreement (95% CI)": "97.1% (1578/1625) (96.2-97.8)"
      }
    },
    "Bordetella parapertussis/bronchiseptica": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "100% (2/2) (34.2-100)",
        "Negative Percent Agreement (95% CI)": "99.9% (2290/2291) (99.8-100)"
      }
    },
    "Bordetella pertussis": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "100% (8/8) (67.6-100)",
        "Negative Percent Agreement (95% CI)": "99.9% (2187/2189) (99.7-100)"
      }
    },
    "Bordetella holmesii": {
      "Performance Criteria": "Positive Agreement ≥ 90%, Negative Agreement ≥ 95%",
      "Reported Performance (All specimens)": {
        "Positive Percent Agreement (95% CI)": "100% (1/1) (20.6-100)",
        "Negative Percent Agreement (95% CI)": "100% (2305/2305) (99.8-100)"
      }
    }
  },
  "2. Sample sized used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)": {
    "Total specimens enrolled": 3299,
    "Specimens included in performance analysis": 3266,
    "Specimen types and provenance": {
      "Prospectively-collected fresh specimens": {
        "Count": 1069,
        "Type": "Prospective"
      },
      "Prospectively-collected frozen specimens": {
        "Count": 1317,
        "Type": "Prospective"
      },
      "Retrospectively-collected frozen specimens": {
        "Count": 520,
        "Type": "Retrospective"
      },
      "Contrived frozen specimens": {
        "Count": 360,
        "Type": "Contrived"
      }
    },
    "Country of Origin": "Not explicitly stated, but implies U.S. due to FDA submission."
  },
  "3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)": "Not specified in the provided text. The ground truth was established by comparing to an FDA-cleared molecular respiratory panel and/or PCR amplification followed by confirmatory bi-directional sequencing.",
  "4. Adjudication method (e.g. 2+1, 3+1, none) for the test set": "The method of adjudication for discrepancies between the predicate and PCR/sequencing results is not explicitly detailed. The comparison was made against a 'composite of an FDA-cleared molecular respiratory panel and analytically validated PCR with bi-directional sequencing', implying a reference standard was used for ground truth, but the steps for resolving conflicting results among these methods are not described.",
  "5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance": "Not applicable. This is a standalone diagnostic device, not an AI-assisted human reader study.",
  "6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done": "Yes, a standalone performance study was conducted. The clinical performance characteristics were determined by comparing the device's results to a composite reference method (FDA-cleared molecular respiratory panel and/or PCR amplification with bi-directional sequencing). The device is described as an 'automated Verigene System' which implies a standalone algorithm making the detection calls.",
  "7. The type of ground truth used (expert concensus, pathology, outcomes data, etc)": "A composite reference standard was used as ground truth: an FDA-cleared molecular respiratory panel and/or PCR amplification followed by confirmatory bi-directional sequencing.",
  "8. The sample size for the training set": "The document does not explicitly state a 'training set' size for a machine-learning model. This is a molecular diagnostic assay, and its development would typically involve analytical testing (LoD, inclusivity, exclusivity, etc.) rather than a 'training set' in the AI sense. The analytical inclusivity study used a comprehensive panel of 108 strains, and additional 28 strains from the LoD study, making a total of 136 strains for analytical inclusivity tests.",
  "9. How the ground truth for the training set was established": "For analytical studies (LoD, inclusivity, exclusivity), the 'ground truth' was based on known concentrations of characterized viral and bacterial strains (TCID50/mL or CFU/mL) in simulated NPS. For inclusivity, identification of the strains was confirmed from their source (e.g., ATCC, Zeptometrix, IRR) and in some cases, further confirmed by quantitative TaqMan real-time PCR or PCR/bi-directional sequencing."
}

Summary

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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)

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

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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:

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.

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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:

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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 andBacterial Genus	Viral Strains andBacterial 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
HumanMetapneumovirus	Metapneumovirus 9 (A1)	$3.0\times10^{1}$ TCID50/mL
HumanMetapneumovirus	Metapneumovirus 27 (A2)	$1.1\times10^{0}$ TCID50/mL
HumanMetapneumovirus	Metapneumovirus 3 (B1)	$1.0\times10^{1}$ TCID50/mL
HumanMetapneumovirus	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
RespiratorySyncytial Virus	RSV A (A2)	$3.3\times10^{0}$ TCID50/mL
RespiratorySyncytial 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

AdenovirusSpecies	Serotype	Strain #	Source	Concentration(TCID50/mL)	Multiples ofLoD
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 ASubtype	Strain	Source	Influenza A		A/H1 or A/H3
			Concentration(TCID50/mL)	Multiple ofLoD	Concentration(TCID50/mL)	Multiples ofLoD
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

{11}------------------------------------------------

Influenza ASubtype	Strain	Source	Influenza A		A/H1 or A/H3
			Concentration(TCID50/mL)	Multiple ofLoD	Concentration(TCID50/mL)	Multiples ofLoD
	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/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(TCID50/mL)	Multiples ofLoD
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(TCID50/mL)	Multiples ofLoD
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(TCID50/mL)	Multiples ofLoD
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(TCID50/mL)	Multiples ofLoD
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(TCID50/mL)	Multiples ofLoD
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(PFU/mL)*	Multiples ofLoD
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 FlexTarget	Concentration(CFU/mL)	Multiples ofLoD
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	BordetellaParapertussis/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
	Zeptometrix0801461		$2.2\times10^4$	9x
		ATCC 4617		$7.3\times10^3$
		ATCC 7773		$7.3\times10^3$
	ATCC 785	BordetellaParapertussis/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
	Zeptometrix0801464		$2.2\times10^4$	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*(95% CI)
	Low	Moderate
Parainfluenza 1	100%48/48(92.6-100)	100%48/48(92.6-100)	100%671/671(99.4-100)
Parainfluenza 2	100%48/48(92.6-100)	100%48/48(92.6-100)	100%671/671(99.4-100)
Parainfluenza 3	100%48/48(92.6-100)	95.846/48(86.0-98.8)	100%671/671(99.4-100)
Parainfluenza 4	100%48/48(92.6-100)	100%48/48(92.6-100)	99.9%670/671(99.2-100)
RSV A	100%48/48(92.6-100)	100%48/48(92.6-100)	100%671/671(99.4-100)
RSV B	93.8%45/48(83.2-97.9)	100%48/48(92.6-100)	100%671/671(99.4-100)
Influenza A	100%96/96(96.2-100)	100%96/96(96.2-100)	100%575/575(99.3-100)
Influenza A/H1	100%48/48(92.6-100)	100%48/48(92.6-100)	100%671/671(92.4-100)
Influenza A/H3	100%48/48(92.6-100)	100%48/48(92.6-100)	100%671/671(92.4-100)
Influenza B	100%48/48(92.6-100)	100%48/48(92.6-100)	100%671/671(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%47/48(89.1-99.6)	100%48/48(92.6-100)	99.9%670/671(99.2-100)
hMPV	100%48/48(92.6-100)	100%48/48(92.6-100)	100%671/671(92.4-100)
Adenovirus	100%48/48(92.6-100)	100%48/48(92.6-100)	99.7%669/671(98.9-99.9)
B. pertussis	100%48/48(92.6-100)	97.9%46/47(88.9-99.6)	100%672/672(99.4-100)
B. holmesii	100%47/48(89.1-99.6)	100%47/47(92.4-100)	100%672/672(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. ofSpecimens	Percentage	No. ofSpecimens	Percentage	No. ofSpecimens	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	ProspectivelyCollected	Fresh	1049	100%12/12(75.7 - 100)	99.4%1030/1037(98.6 - 99.7)	Influenza A subtype H1	ProspectivelyCollected	Fresh	1048	--	99.8%1046/1048(99.3 - 99.9)
		Frozen	1144	97.9%46/47(88.9 - 99.6)	99.4%1091/1097(98.8 – 99.7)			Frozen	1144	97.8%45/46(88.7 - 99.6)	99.6%1092/1096(99.1 - 99.9)
		All	2193	98.3%58/59(91.0-99.7)	99.4%2121/2134(99.0 - 99.6)			All	2190	97.8%45/46(88.7-99.6)	99.7%2138/2144(99.4-99.9)
		Selected	513	99.2%122/123(95.5 - 99.9)	99.5%387/390(97.8 - 99.7)			Selected	512	97.6%40/41(87.4 - 99.6)	99.6%469/471(98.5 - 99.9)
	Contrived		360	--	100%360/360(98.9 - 100)		Contrived		360	--	100%360/360(98.9 - 100)
Influenza A subtype H3	ProspectivelyCollected	Fresh	1048	100%12/12(75.7 - 100)	99.6%1032/1036(99.0 - 99.8)	Influenza B	ProspectivelyCollected	Fresh	1052	98.0%49/50(89.5 - 99.6)	99.3%995/1002(98.6 - 99.7)
		Frozen	1142	100%1/1(20.6 - 100)	100%1141/1141(99.7 – 100)			Frozen	1145	--	99.9%1144/1145(99.5 - 100)
		All	2190	100%13/13(77.2 - 100)	99.8%2173/2177(99.5-99.9)			All	2197	98.0%49/50(89.5 - 99.6)	99.6%2139/2147(99.3 - 99.8)
		Selected	512	100%82/82(95.5-100)	99.5%428/430(98.3 - 99.9)			Selected	516	100%26/26(87.1 - 100)	99.6%488/490(98.5 - 99.9)
	Contrived		360	--	100%360/360(98.9-100)		Contrived		360	--	100%360/360(98.9-100)
RSV A	ProspectivelyCollected	Fresh	1049	100%11/11(74.1-100)	99.8%1036/1038(99.3-99.9)	RSV B	ProspectivelyCollected	Fresh	1049	100%8/8(67.6 - 100)	99.6%1037/1041(96.7 - 98.6)
		Frozen	1121	100%6/6(61.0-100)	99.9%1114/1115(99.5-100)			Frozen	1121	100%165/165(97.7 - 100)	97.9%936/956(96.8 - 98.6)
		All	2170	100%17/17(81.6-100)	99.9%2150/2153(99.6-99.9)			All	2170	100%173/173(97.8 - 100)	98.8%1973/1997(98.2 - 99.2)
		Selected	498	94.8%55/58(85.9-98.2)	99.3%437/440(98.0-99.8)			Selected	498	100%23/23(85.7 - 100)	98.5%468/475(97.0 - 99.3)
	Contrived		360	--	100%360/360(98.9-100)		Contrived		360	--	99.7%359/360(98.4 - 99.9)
	Specimen Type		n=	% Agreement (95% CI)			Specimen Type		n=	% Agreement (95% CI)
				Positive	Negative					Positive	Negative
ProspectivelyCollected	Parainfluenza 1	Fresh	1052	-	100%1052/1052(99.6 - 100)	ProspectivelyCollected	Parainfluenza 2	Fresh	1052	100%11/11(74.1 - 100)	99.7%1038/1041(99.2 - 99.9)
		Frozen	1145	90.0%27/30(74.4 - 96.5)	99.8%1113/1115(99.3 - 99.9)			Frozen	1145	50.0%1/2(9.5 - 90.5)	100%1143/1143(99.7 - 100)
		All	2197	90.0%27/30(74.4 - 96.5)	99.9%2165/2167(99.7 - 100)			All	2197	92.3%12/13(66.7 - 98.6)	99.9%2181/2184(99.6 - 99.9)
		Selected	516	100%50/50(92.9 - 100)	100%466/466(99.2 - 100)			Selected	516	100%28/28(87.9 - 100)	99.8%487/488(98.8 - 100)
		Contrived	360	-	100%360/360(98.9 - 100)			Contrived	360	-	99.7%359/360(98.4 - 99.9)
ProspectivelyCollected	Parainfluenza 3	Fresh	1052	83.3%10/12(55.2 - 95.3)	99.7%1037/1040(99.2 - 99.9)	ProspectivelyCollected	Parainfluenza 4	Fresh	1052	100%3/3(43.8 - 100)	100%1049/1049(99.6 - 100)
		Frozen	1145	80.0%4/5(37.5 - 96.4)	100%1140/1140(99.7 - 100)			Frozen	1145	76.2%16/21(54.9 - 89.4)	99.6%1120/1124(99.1 - 99.9)
		All	2197	82.4%14/17(59.0 - 93.8)	99.9%2177/2180(99.6 - 99.9)			All	2197	79.2%19/24(59.3 - 90.8)	99.8%2169/2173(99.5 - 99.9)
		Selected	516	100%31/31(89.0 - 100)	100%485/485(99.2 - 100)			Selected	516	100%41/41(91.4 - 100)	99.6%473/475(98.5 - 99.9)
		Contrived	360	-	100%360/360(98.9 - 100)			Contrived	360	-	100%360/360(98.9 - 100)

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Table 19: Results Stratified by Target Analyte –Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4

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	Specimen Type	n=	% Agreement (95% CI)			Specimen Type	n=	% Agreement (95% CI)
			Positive	Negative				Positive	Negative
AdenovirusProspectivelyCollected	Fresh	1052	91.7%22/24(74.1 – 97.7)	98.2%1009/1028(97.1 – 98.8)	hMPVProspectivelyCollected	Fresh	1052	100%10/10(72.2 – 100)	99.5%1037/1042h(98.9 – 99.8)
	Frozen	1145	81.8%27/33(65.6 – 91.4)	96.4%1072/1112(95.1 – 97.3)		Frozen	1145	100%36/36(90.4 – 100)	99.9%1108/1109i(99.5 – 100)
	All	2197	86.0%49/57(74.7 – 92.7)	97.2%2081/2140(96.5 – 97.9)		All	2197	100%46/46(92.3 – 100)	99.7%2145/2151(99.4 – 99.9)
	Selected	516	97.4%38/39(86.8 – 99.5)	98.3%469/477(96.7 – 99.1)		Selected	516	92.6%25/27g(76.6 – 97.9)	99.8%488/489j(98.8 – 100)
	Contrived	360	–	99.4%358/360(98.0 – 99.8)		Contrived	360	–	99.4%358/360(98.0 – 99.8)
RhinovirusProspectivelyCollected	Fresh	1000	85.9%214/249(81.1 – 89.7)	95.7%719/751(94.1 – 97.0)
	Frozen	1122	77.8%193/248(72.2 – 82.5)	98.3%859/874o(97.2 – 99.0)
	All	2122	81.9%407/497(78.3 – 85.0)	97.1%1578/1625(96.2 – 97.8)
	Selected	509	80.0%28/35(64.1 – 90.0)	98.3%466/474(96.7 – 99.1)
	Contrived	360	–	99.7%359/360(98.4 – 99.9)

Table 20: Results Stratified by Target Analyte –Adenovirus, human Metapneumovirus (hMPV), Rhinovirus

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Table 21: Results Stratified by Target Analyte –Bordetella parapertussis/bronchiseptica, Bordetella pertussis, Bordetella holmesii

	SpecimenType	n=	% Agreement (95% CI)			SpecimenType	n=	% Agreement (95% CI)
			Positive	Negative				Positive	Negative
Bordetella parapertussis/bronchisepticaProspectivelyCollected	Fresh	1041	100%2/2(34.2 - 100)	100%1039/1039(99.6 - 100)	Bordetella pertussisProspectivelyCollected	Fresh	1052	100%1/1(20.6 - 100)	99.9%1050/1051(99.5 - 100)
	Frozen	1255	-	99.9%1254/1255(99.5 - 100)		Frozen	1145	100%7/7(64.6 - 100)	99.9%1137/1138(99.5 - 100)
	All	2296	100%2/2(34.2 - 100)	99.9%2290/2291(99.8 - 100)		All	2197	100%8/8(67.6 - 100)	99.9%2187/2189(99.7 - 100)
	Selected	463	71.4%5/7(35.9 - 91.8)	99.8%455/456(98.8 - 100)		Selected	516	96.6%28/29(82.8 - 99.4)	100%487/487(99.2 - 100)
	Contrived	360	100%104/104(96.4 - 100)	100%256/256(98.5 - 100)		Contrived	360	-	100%360/360(98.9 - 100)
Bordetella holmesiiProspectivelyCollected	Fresh	1043	100%1/1(20.6 - 100)	100%1042/1042(99.6 - 100)
	Frozen	1263	-	100%1263/1263(99.7 - 100)
	All	2306	100%1/1(20.6 - 100)	100%2305/2305(99.8 - 100)
	Selected	490	50%1/2(9.4 - 90.1)	100%488/488(99.2 - 100)
	Contrived	360	100%56/56(93.6 - 100)	100%304/304(98.6 - 100)

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Substantial Equivalence

The Verigene Respiratory Pathogens Flex Nucleic Acid test (RP Flex) has been shown to be substantially equivalent to the BioFire FilmArray Respiratory Panel (RP) System. The RP Flex test has similar intended use and indications, technological characteristics, and performance characteristics. Performance data demonstrate that the RP Flex test performs comparably to the predicate device. Thus, the RP Flex test is substantially equivalent to the predicate device.

	Similarities
Element	New Device:Verigene Respiratory Pathogens Flex NucleicAcid Test (RP Flex)K143653	Predicate:FilmArray® Respiratory Panel (RP)K143080/K123620/K120267/K110764/K103175
Intended Use	The Verigene® Respiratory Pathogens FlexNucleic Acid Test (RP Flex) is a multiplexedqualitative test intended for the simultaneousdetection and identification of multiple viraland bacterial nucleic acids in nasopharyngealswabs (NPS) obtained from individualssuspected of respiratory tract infection. Thetest is performed on the automated VerigeneSystem utilizing reverse transcription (RT),polymerase chain reaction (PCR), andmicroarray hybridization to detect genesequences of the following organism types andsubtypes: Adenovirus, HumanMetapneumovirus, Influenza A, Influenza A(subtype H1), Influenza A (subtype H3),Influenza B, Parainfluenza 1, Parainfluenza 2,Parainfluenza 3, Parainfluenza 4, RespiratorySyncytial Virus A, Respiratory SyncytialVirus B, Rhinovirus, Bordetellaparapertussis/bronchiseptica, Bordetellaholmesii, and Bordetella pertussis.Detecting and identifying specific viral andbacterial nucleic acids from individualsexhibiting signs and symptoms of respiratoryinfection aids in the diagnosis of respiratoryinfection, if used in conjunction with otherclinical and laboratory findings. The results ofthis test should not be used as the sole basisfor diagnosis, treatment, or patientmanagement decisions.	The FilmArray® Respiratory Panel (RP) is amultiplexed nucleic acid test intended for usewith the FilmArray Instrument for thesimultaneous qualitative detection andidentification of multiple respiratory viral andbacterial nucleic acids in nasopharyngeal swabs(NPS) obtained from individuals suspected ofrespiratory tract infections. The followingorganism types and subtypes are identified usingthe FilmArray RP: Adenovirus, Coronavirus229E, Coronavirus HKU1, Coronavirus NL63,Coronavirus OC43, Human Metapneumovirus,Influenza A, Influenza A subtype H1, InfluenzaA subtype H3, Influenza A subtype H1-2009,Influenza B, Parainfluenza Virus 1,Parainfluenza Virus 2, Parainfluenza Virus 3,Parainfluenza Virus 4, HumanRhinovirus/Enterovirus, Respiratory SyncytialVirus, Bordetella pertussis, Chlamydophilapneumoniae, and Mycoplasma pneumoniae.The detection and identification of specific viraland bacterial nucleic acids from individualsexhibiting signs and symptoms of a respiratoryinfection aids in the diagnosis of respiratoryinfection if used in conjunction with otherclinical and epidemiological information. Theresults of this test should not be used as the solebasis for diagnosis, treatment, or othermanagement decisions.
	Negative results in the presence of arespiratory illness do not preclude respiratoryinfection and may be due to infection withpathogens that are not detected by this test orlower respiratory tract infection that is notdetected by an NPS specimen. Conversely,	Negative results in the setting of a respiratoryillness may be due to infection with pathogensthat are not detected by this test or, lowerrespiratory tract infection that is not detected bya nasopharyngeal swab specimen. Positiveresults do not rule out co-infection with otherorganisms: the agent(s) detected by the Film
Similarities
Element	New Device:Verigene Respiratory Pathogens Flex NucleicAcid Test (RP Flex)K143653	Predicate:FilmArray® Respiratory Panel (RP)K143080/K123620/K120267/K110764/K103175
	infection with organisms not detected by RPFlex. The agent(s) detected may not be thedefinite cause of disease. The use ofadditional laboratory testing and clinicalpresentation may be necessary to establish afinal diagnosis of respiratory infection.Clinical evaluation indicates a lowersensitivity specific to RP Flex for thedetection of rhinovirus. If infection withRhinovirus is suspected, negative samplesshould be confirmed using an alternativemethod.Performance characteristics for Influenza Awere established when Influenza A/H1 (2009Pandemic) and A/H3 were the predominantInfluenza A viruses in circulation. RP Flexmay not detect novel Influenza A strains. Ifinfection with a novel Influenza A virus issuspected based on current clinical andepidemiological screening criteriarecommended by public health authorities,specimens should be collected withappropriate infection control precautions usedspecifically for novel virulent influenzaviruses and sent to appropriate healthauthorities for testing. Viral culture shouldnot be attempted in these cases unless abiosafety level (BSL) 3+ facility is availableto receive and culture specimens.	disease. Additional laboratory testing (e.g.bacterial and viral culture, immunofluorescence,and radiography) may be necessary whenevaluating a patient with possible respiratorytract infection.Due to the small number of positive specimenscollected for certain organisms during theprospective clinical study, performancecharacteristics for Bordetella pertussis ,Coronavirus 229E, Coronavirus OC43, InfluenzaA H1, Influenza A H3, Influenza A H1-2009,Influenza B, Mycoplasma pneumoniae ,Parainfluenza Virus 1, Parainfluenza.Due to the genetic similarity between HumanRhinovirus and Enterovirus, the FilmArray RPcannot reliably differentiate them. A positiveFilmArray RP Rhinovirus/Enterovirus resultshould be followed-up using an alternate method(e.g., cell culture or sequence analysis).The FilmArray RP assay for Coronavirus OC43may cross-react with some isolates ofCoronavirus HKU1. A dual positive result maybe due to cross-reactivity or may indicate a co-infection.Performance characteristics for Influenza A wereestablished when Influenza A H1-2009, A H1,and A H3 were the predominant Influenza Aviruses in circulation. Performance of detectingInfluenza A may vary if other Influenza Astrains are circulating or a novel Influenza Avirus emerges. If infection with a novelInfluenza A virus is suspected based on currentclinical and epidemiological screening criteriarecommended by public health authorities,specimens should be collected with appropriateinfection control precautions for novel virulentInfluenza viruses and sent to state or local healthdepartments for testing. Viral culture should notbe attempted in these cases unless a BSL 3+facility is available to receive and culturespecimens.
SpecimenType	Nasopharyngeal swabs (NPS)	Nasopharyngeal swabs (NPS)
Nucleic AcidAmplification	Multiplexed RT-PCR	Multiplexed RT-PCR
Organisms/NA	Adenovirus, Human Metapneumovirus,	Adenovirus, Coronavirus 229E, Coronavirus
Similarities
Element	New Device:Verigene Respiratory Pathogens Flex NucleicAcid Test (RP Flex)K143653	Predicate:FilmArray® Respiratory Panel (RP)K143080/K123620/K120267/K110764/K103175
TargetsDetected	Influenza A, Influenza A (subtype H1),Influenza A (subtype H3), Influenza B,Parainfluenza 1, Parainfluenza 2,Parainfluenza 3, Parainfluenza 4, RespiratorySyncytial Virus A, Respiratory SyncytialVirus B., Rhinovirus, Bordetellaparapertussis/bronchiseptica, Bordetellaholmesii, Bordetella pertussis	HKU1, Coronavirus NL63, Coronavirus OC43,Human Metapneumovirus, Influenza A,Influenza A subtype H1, Influenza A subtypeH3, Influenza A subtype H1-2009, Influenza B,Parainfluenza Virus 1, Parainfluenza Virus 2,Parainfluenza Virus 3, Parainfluenza Virus 4,Human Rhinovirus/Enterovirus, RespiratorySyncytial Virus, Bordetella pertussis,Chlamydophila pneumoniae, and Mycoplasma

Table 22: Substantial Equivalence

CONFIDENTIAL AND PROPRIETARY Nanosphere, Inc.

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{30}------------------------------------------------

Differences
Element	New Device:Verigene Respiratory Pathogens Flex NucleicAcid test (RP Flex)K143653	Predicate:FilmArray® Respiratory Panel (RP)K123620/K120267/K110764/K103175
Time to Result	About 2 hours	About 1 hour
DetectionMethod	Multiplexed RT-PCR followed by a goldnanoparticle probe-based detection ofmicrobial-specific DNA in a microarrayformat	Nested multiplex RT-PCR followed by highresolution melting analysis to confirm identity ofamplified product
OpticalDetection	Light Scattering	Fluorescence

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.