The AmpliVue® Bordetella Assay is an in vitro diagnostic test for the qualitative detection of Bordetella pertussis nucleic acids isolated from nasopharyngeal swab specimens suspected of having respiratory tract infection attributable to Bordetella pertussis.

The AmpliVue® Bordetella Assay utilizes helicase-dependent amplification (HDA) of the insertion sequence IS481 and a selfcontained disposable amplification device that allows for manual evaluation of assay results. The IS48 sequence can also be found in strains of other organisms (i.e., B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the AmpliVue® Bordetella Assay do not prection and positive results do not rule out coinfection with other respiratory pathogens. Results from the AmpliVue® Bordetella Assay should be used in conjunction with information obtained during the patient's clinical evaluation as an aid in diagnosis infection and should not be used as the sole basis for treatment or other patient management decisions.

The AmpliVue® Bordetella Assay is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

Device Description

The AmpliVue® Bordetella Assay combines simple processing, an isothermal amplification technology named helicase-dependent amplification (HDA), and a selfcontained disposable amplicon detection device, for the detection of Bordetella pertussis from nasopharyngeal swabs.

Patient samples are collected using a nasopharyngeal swab and placed into a liguid medium. Fifty microliters (50 µL) of the sample are then transferred to a process buffer that is provided with the kit and mixed. The Process Buffer tubes are heated at 95 °C for 10 minutes. Fifty microliters (50 µL) of the Process Buffer containing sample is added to a reaction tube containing lyophilized mix of HDA reagents. Included in the reaction mix are the isothermal polymerase, helicase and single stranded binding protein. After completion of the HDA reaction the reaction tube is transferred to the amplicon cartridge containing the running buffer. The amplicon cartridge is closed and inserted into the detection chamber. The detection chamber is activated by depressing the detection chamber handle. Upon activation, the reservoir containing the running buffer and the 0.2 mL tube containing the amplicon is punctured and the solutions are wicked to the lateral flow strip.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Device Name: AmpliVue® Bordetella Assay

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the reported performance metrics of the clinical study and the analytical performance studies. The clinical study's primary metrics are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to a Composite Reference Method. The analytical studies establish limits of detection, inclusivity, and specificity.

Acceptance Criteria (Implicit)	Reported Device Performance (AmpliVue® Bordetella Assay)
Clinical Performance:
Positive Percent Agreement (PPA) with Composite Reference Method (CRM)	97.0% (64/66) with a 95% Confidence Interval of 89.6% to 99.2%
Negative Percent Agreement (NPA) with Composite Reference Method (CRM)	98.1% (755/770) with a 95% Confidence Interval of 96.8% to 98.8%
Analytical Performance:
Reproducibility Across Sites and Operators (for Low Positive, Moderate Positive, Negative, and Controls)	100% Agreement across 3 sites and between operators for BP Low Positive, BP Moderate Positive, BP Negative, BP Positive Control, and BP Negative Control samples. (95% CI: 95.9% to 100% for all categories).
Stability of samples (contrived 2x LOD) when stored at 2°C to 8°C for varying lengths of time (24, 48, 72 and 96-hours).	The contrived 2x LOD sample was stable when stored at 2℃ to 8°C.
Limit of Detection (LoD) for Bordetella pertussis (A639 and E431 strains)	A639: 3.93 CFU/assay (2,358 CFU/mL) E431: 1.27 CFU/assay (761 CFU/mL) The assay LOD for Bordetella pertussis is 3.93 CFU/assay or 2,358 CFU/mL.
Analytical Specificity (Cross-Reactivity) to rule out interference from common respiratory microorganisms at specified concentrations.	No cross-reactivity observed with 75 of 79 tested organisms at clinically relevant levels. Cross-reactivity observed with 1 of 4 Bordetella bronchiseptica strains (strain 4617) and 4 of 4 Bordetella holmesii strains (expected due to IS481 target). This finding is noted in the intended use and limitations.
Analytical Specificity (Interference) to rule out inhibition from various common substances potentially present in respiratory specimens.	No evidence of interference caused by 17 tested substances (e.g., cold medications, blood, nasal sprays) at medically relevant concentrations.
Analytical Reactivity (Inclusivity) for additional Bordetella pertussis strains at near LoD.	*All seven additional Bordetella pertussis* strains were detected** at 2,358 CFU/mL (near LoD level).
Compatibility with different transport media types.	Compatible with 8 different media types (Tris EDTA, Molecular Grade Water, Eswab, M4, M4-RT, M5, 0.85% Saline) for 4 Bordetella pertussis strains (A639, E431, BAA 1335, and 51445) at the assay LoD.

2. Sample size used for the test set and the data provenance

Sample Size (Clinical): 842 fresh nasopharyngeal swab specimens were initially collected. After removing 6 invalid specimens, 836 specimens were included in the final analysis.
Data Provenance: The specimens were collected from patients in the United States (at four locations). The study was prospective, collecting samples in Spring to Summer 2014 (April to August 2014) from patients suspected of having respiratory tract infection attributable to Bordetella pertussis.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The ground truth for the test set was established using a Composite Reference Method (CRM) involving molecular assays rather than human experts reviewing images or clinical data in an adjudicated manner.

The CRM included two manufacturer-validated, IS481-targeted real-time PCR assays (PCR1 and PCR2).
This was followed by bi-directional sequencing from PCR-positive specimens.

Therefore, the concept of "number of experts" and "qualifications of those experts" for establishing ground truth, as typically understood in fields like radiology or pathology, does not directly apply here. The ground truth was based on a laboratory-based molecular diagnostic gold standard.

4. Adjudication method (e.g., 2+1, 3+1, none) for the test set

The adjudication method was based on a Composite Reference Method (CRM):

Specimens were considered positive when bi-directional sequence sequencing results from either comparator PCR assay confirmed the presence of Bordetella pertussis amplicon.
Specimens were considered negative when neither comparator PCR assay produced Bordetella pertussis amplicon at the end of the 37 cycles.

This is a defined algorithmic (molecular) adjudication process rather than human expert consensus.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is an in vitro diagnostic assay for the qualitative detection of nucleic acids, not an imaging device or AI-assisted diagnostic tool that would involve human readers interpreting results.

6. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done

The clinical study evaluated the standalone performance of the AmpliVue Bordetella Assay. The device itself performs the detection and provides a result (visible lines in the cassette window indicating detection or no detection). While human observation is required to read the lines, the assay itself is an "algorithm only" in the sense that its output is directly analogous to a positive/negative result from a fully automated system. The performance metrics (PPA, NPA) reflect this standalone performance against the Composite Reference Method.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

The ground truth used was a Composite Reference Method (CRM) consisting of:

Two manufacturer-validated, IS481-targeted real-time PCR assays.
Bi-directional sequencing for confirmation of PCR-positive specimens.

This is a highly sensitive and specific molecular-based ground truth, often considered a "gold standard" for nucleic acid detection.

8. The sample size for the training set

The document does not report a sample size for a training set. This device uses Helicase-Dependent Amplification (HDA) with a lateral flow strip for detection. It is a molecular diagnostic assay that does not typically involve machine learning with distinct training and test sets in the same way an AI/CADe device would. The development of the assay (primers, probes, reaction conditions) would involve optimization, but this is not typically referred to as a "training set" in this context.

9. How the ground truth for the training set was established

As there is no explicit training set discussed in the context of machine learning, the question of how its ground truth was established is not applicable. The underlying components of the assay (primers, probes) are designed based on known biological sequences and Bordetella pertussis characteristics.

Summary

{0}------------------------------------------------

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

QUIDEL CORPORATION RONALD LOLLAR SENIOR DIRECTOR, CLINICAL AND REGULATORY AFFAIRS 2005 EAST STATE STREET SUITE 100 ATHENS OH 45701

December 10, 2014

Re: K143206

Trade/Device Name: Amplivue Bordetella Assay Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: II Product Code: OZZ Dated: November 6, 2014 Received: November 7, 2014

Dear Dr. Lollar:

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 medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

{1}------------------------------------------------

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

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 Industry and Consumer Education 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,

Uwe Scherf -S for

Sally Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

{2}------------------------------------------------

Indications for Use

510(k) Number (if known) K143206

Device Name Amplivue Bordetella Assay

Indications for Use (Describe)

The AmpliVue® Bordetella Assay is an in vitro diagnostic test for the qualitative detection of Bordetella pertussis nucleic acids isolated from nasopharyngeal swab speciments suspected of having respiratory tract infection attributable to Bordetella pertussis.

The AmpliVue® Bordetella Assay is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

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

☑ Prescription Use (Part 21 CER 801 Subpart D)
☐ Over-The-Counter Use (21 CER 801 Subpart C)

PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON A SEPARATE PAGE IF NEEDED.

FOR FDA USE ONLY

Concurrence of Center for Devices and Radiological Health (CDRH) (Signature)

{3}------------------------------------------------

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{4}------------------------------------------------

Applicant:

Quidel Corporation 12544 High Bluff Drive, Suite 200 San Diego, California 92130 Telephone: 858-552-7910 Fax: 858-646-8045

Contact Information:

Ronald H. Lollar, Senior Director Clinical and Regulatory Affairs 2005 East State Street, Suite 100 Athens, Ohio 45701 740-589-3300 – Corporate number 740-589-3373 – Desk phone 858-552-6451—Fax Ron.Lollar@quidel.com

Date of preparation of 510(k) summary:

November 05, 2014

A. 510(k) Number:

K143206

B. Purpose for Submission:

To obtain substantial equivalence for the AmpliVue Bordetella Assay

C. Measurand:

Insertion sequence IS481 of Bordetella pertussis

D. Type of Test:

Helicase-dependent amplification (HDA)

{5}------------------------------------------------

E. Applicant:

Quidel Corporation

F. Proprietary and Established Names:

AmpliVue Bordetella Assay

G. Regulatory Information:

Product Code	Classification	Regulation Section	Panel
OZZ – Bordetellapertussis DNAassay system	Class 2	21 CFR 866.3980 – Respiratoryviral panel multiplex nucleic acidassay	Microbiology(83)

H. Intended Use:

1. Intended Use(s):

The AmpliVue® Bordetella Assay is an in vitro diagnostic test for the qualitative detection of Bordetella pertussis nucleic acids isolated from nasopharyngeal swab specimens obtained from patients suspected of having respiratory tract infection attributable to Bordetella pertussis.

The AmpliVue® Bordetella Assay utilizes helicase-dependent amplification (HDA) of the insertion sequence IS481 and a self-contained disposable amplification detection device that allows for manual evaluation of assay results. The IS481 sequence can also be found in strains of other organisms (i.e., B. holmesii and B. bronchiseptica). B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

{6}------------------------------------------------

Negative results for the AmpliVue® Bordetella Assay do not preclude B. pertussis infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the AmpliVue® Bordetella Assay should be used in conjunction with information obtained during the patient's clinical evaluation as an aid in diagnosis of Bordetella pertussis infection and should not be used as the sole basis for treatment or other patient management decisions.

The AmpliVue Bordetella Assay is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

2. Indication(s) for Use:

Same as Intended Use.

3. Special conditions for use statement(s):

For in vitro diagnostic use only
For prescription use only

4. Special instrument requirements:

None

l. Device Description:

{7}------------------------------------------------

handle. Upon activation, the reservoir containing the running buffer and the 0.2 mL tube containing the amplicon is punctured and the solutions are wicked to the lateral flow strip.

Materials Provided:

. 16 Tests per Kit

Symbol	Component	Quantity	Storage
1	Detection Cassettes	16/kit	2° to 30°C
2	Process Buffer	16 tubes/kit 1.45mL	2° to 30°C
3	Reaction Tubes	16 tubes/kit	2° to 8°C
4	Amplicon Cartridge	16/kit	2° to 30°C

Materials required but not provided:

. External controls for Bordetella pertussis (e.g. Quidel Molecular Bordetella Control Set #M117, which contains positive and negative controls, serves as an external processing and extraction control)
Sterile DNAse-free filter-blocked or positive displacement micropipettor tips
Micropipettor ●
Stopwatch or timer
Scissors or a blade ●
Micro tube tray
Heat block capable of 95° C ± 2° C temperature ●
Heat block with heated lid capable of 64° ± 2° C temperature
. Thermometer

Substantial Equivalence Information: J.

1. Predicate device name(s):
  illumigene® Pertussis DNA Amplification Assay
1. Predicate 510(k) number(s): K133673

{8}------------------------------------------------

3. Comparison with predicate:

Similarities
Item	AmpliVue® Bordetella Assay	illumigene® Pertussis DNAAmplification Assay(K133673)
Intended Use	The AmpliVue® Bordetella Assay is anin vitro diagnostic test for thequalitative detection of Bordetella pertussis nucleic acids isolated fromnasopharyngeal swab specimensobtained from patients suspected ofhaving respiratory tract infectionattributable to Bordetella pertussis .The AmpliVue® Bordetella Assayutilizes helicase-dependentamplification (HDA) of the insertionsequence IS481 and a self-containeddisposable amplification detectiondevice that allows for manualevaluation of assay results. The IS481sequence can also be found in strainsof other organisms (i.e., B. holmesiiand B. bronchiseptica ). B. holmesiiinfection may cause clinical illnesssimilar to B. pertussis , and mixedoutbreaks involving both B. pertussisand B. holmesii infection have beenreported. Additional testing should beperformed if necessary to differentiateB. holmesii and B. pertussis . B.bronchiseptica is a rare cause ofinfection in humans. When clinicalfactors suggest that B. pertussis may	The illumigene® PertussisDNA Amplification Assay,performed on theillumipro-10™, is aqualitative in vitrodiagnostic test for thedirect detection ofBordetella pertussis inhuman nasopharyngealswab samples takenfrom patients suspectedof having respiratorytract infectionattributable to Bordetella pertussis .The illumigene Pertussisassay utilizes loop-mediated isothermalDNA amplification(LAMP) technology todetect Bordetella pertussis by targeting theIS481 insertional elementof the Bordetella pertussis genome. TheIS481 insertional elementcan also be found inBordetella holmesii and
Similarities
Item	AmpliVue® Bordetella Assay	illumigene® Pertussis DNAAmplification Assay(K133673)
	not be the cause of respiratoryinfection, other clinically appropriateinvestigation(s) should be carried outin accordance with publishedguidelines.Negative results for the AmpliVue®Bordetella Assay do not preclude B. pertussis infection and positive resultsdo not rule out co-infection with otherrespiratory pathogens. Results fromthe AmpliVue® Bordetella Assay shouldbe used in conjunction withinformation obtained during thepatient's clinical evaluation as an aid indiagnosis of Bordetella pertussisinfection and should not be used asthe sole basis for treatment or otherpatient management decisions.	Bordetella bronchisepticastrains. Respiratoryinfection withBordetella pertussis ,Bordetella holmesii orBordetellabronchiseptica mayyield positive testresults in IS481 assays.B. holmesii infection maycause clinical illnesssimilar to B. pertussis ,and mixed outbreaksinvolving both B.pertussis and B. holmesiiinfection have beenreported. Additionaltesting should beperformed if necessaryto differentiate B.holmesii and B. pertussis .B. bronchiseptica is a rarecause of infection inhumans. When clinicalfactors suggest that B.pertussis may not be thecause of respiratoryinfection, other clinicallyappropriateinvestigation(s) should
Similarities
Item	AmpliVue® Bordetella Assay	illumigene® Pertussis DNAAmplification Assay(K133673)
		be carried out inaccordance withpublished guidelines.
		Negative results for theillumigene PertussisDNA Amplification Assaydo not precludeBordetella pertussisinfection and positiveresults do not rule outco-infection with otherrespiratory pathogens.Results from theillumigene Pertussisassay should be used inconjunction withinformation obtainedduring the patient'sclinical evaluation as anaid in diagnosis ofBordetella pertussisinfection and should notbe used as the sole basisfor treatment or otherpatient managementdecisions.
		illumigene Pertussis isintended for use inhospital reference or
Similarities
Item	AmpliVue® Bordetella Assay	illumigene® Pertussis DNAAmplification Assay(K133673)
		state laboratory settings.The device is notintended for point-of-care use.
Sample Types	Nasopharyngeal swab specimensobtained from patients suspected ofhaving respiratory tract infectionattributable to Bordetella pertussis	Same
Sample HeatLysis	Manual	Same
TargetSequenceDetected	Bordetella pertussis IS481 insertionalelement	Same

{9}------------------------------------------------

AmpliVue® Bordetella Assay 11/05/2014 Page 6 of 19

{10}------------------------------------------------

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

Differences
Item	AmpliVue ® Bordetella Assay	illumigene® Pertussis DNAAmplification Assay(K133673)
DNA AmplificationTechnology	Helicase-dependentamplification (HDA); self-contained	Loop-Mediated IsothermalAmplification (LAMP); self-contained and automated
Self-ContainedSystem Assay aftersample preparation	No	Yes
Detection Technique	Manual	Automated
Instrument	None	illumipro-10™

{12}------------------------------------------------

Differences
Item	AmpliVue® Bordetella Assay	illumigene® Pertussis DNAAmplification Assay(K133673)
Testing Time	85 - 90 minutes	60 - 70 minutes

K. Standard/Guidance Document Referenced (if applicable):

Not applicable

L. Test Principle:

The AmpliVue® Bordetella Assay detects Bordetella pertussis DNA isolated from nasopharyngeal swab specimens obtained from symptomatic patients suspected of having respiratory tract infection attributable to Bordetella pertussis. The assay consists of three major steps: 1) specimen preparation, 2) isothermal Helicase-Dependent Amplification (HDA) of a target sequence of B. pertussis, and 3) detection of the amplified DNA by targetspecific hybridization probes via a colorimetric reaction on a lateral-flow strip which is embedded in a self-contained disposable cassette to prevent amplicon contamination.

Patient samples are collected using a nasopharyngeal swab and placed into a liquid medium. Fifty microliters (50 µL) of the sample are then transferred to a process buffer that is provided with the kit and mixed. The Process Buffer tubes are heated at 95 °C for 10 minutes. Fifty microliters (50 µL) of the Process Buffer containing sample is added to a reaction tube containing lyophilized mix of HDA reagents.

A HDA reaction is carried out in the Reaction Tube which contains lyophilized HDA reagents, dNTPs, primers and probes. Incubation at 64°C for 60 minutes results in isothermal amplification of the target sequence by B. pertussis specific primers. The amplified DNA is detected by a set of specific detection probes included in the Reaction Tube: B. pertussis target hybridizes to two specific probes labeled with Biotin (BioTEG) and 6-carboxyfluorescein (FAM). A competitive process control (PRC) is included in the Lysis Tube to monitor specimen processing and inhibitory substances in clinical samples, reagent failure or device failure. The PRC target is amplified and hybridizes to the PRC specific probes labeled with Biotin (BioTEG) and 2,4-dinitrophenyl (DNP).

Following completion of the HDA reaction, the Reaction Tube is transferred to a proprietary Cassette for detection. The Cassette is comprised of two components: 1) an

{13}------------------------------------------------

Amplicon Cartridge that holds the running buffer and the 0.2 mL Reaction Tube and 2) the Detection Chamber which houses the Amplicon Cartridge and an embedded vertical-flow DNA detection strip. The DNA detection strip is coated with anti-FAM and anti-DNP antibodies. Once the Cassette is closed, a razor blade and plastic pin located at the bottom of the Cassette opens the Reaction Tube and running buffer bulb, resulting in the release of their contents. The contents flows through a fiberglass paper connected to the DNA detection strip that is attached to a fiberglass pad pre-loaded with streptavidin-conjugated color particles. The B. pertussis amplicon with biotin- and FAM-labeled probes is captured by the anti-FAM antibodies at the test (T2) line, and the Process Control amplicon with biotin- and DNP-labeled probes is captured by the anti-DNP antibodies at the control (C) line. The streptavidin-conjugated color particles bind to the biotin in the probe-amplicon hybrid and the test results are displayed in the Cassette window as colored T2 and/or C lines that are visible to the naked eye.

Detection of B. pertussis is reported when the T2 line is visible through the detection window of the Cassette. No detection of B. pertussis is reported when only the C line is displayed. The assay is regarded as invalid when none of the lines are displayed.

M. Performance Characteristics:

1. Analytical performance:

a. Precision/Reproducibility:

Reproducibility

In order to confirm the reproducibility of the AmpliVue Bordetella Assay a blinded and randomized study panel containing Bordetella pertussis (BP) negative and positive samples (5x and 2x,) were tested at three (3) test sites (one in-house laboratory and two (2) clinical sites). Each site tested a reproducibility panel and Assay Controls for five (5) days in triplicate. Testing was done by two operators at each site. Each operator ran the panel once a day using one lot of AmpliVue Bordetella Assay. A total of four hundred fifty (450) specimens were tested (including controls). The AmpliVue Bordetella Assay generated reproducible results in this study.

	SITE
Category	Site #1		Site #2		Site #3		Overall PercentAgreement	95%ConfidenceInterval
	#expectedresults/#tested	%Agreement	#expectedresults/#tested	%Agreement	#expectedresults/#tested	%Agreement
BP Low Positive	30/30	100%	30/30	100%	30/30	100%	90/90	100%	95.9% to

{14}------------------------------------------------

AmpliVue Bordetella Assay 11/05/2014 Page 11 of 19

510(k) Summary

	SITE
	Site #1		Site #2		Site #3		Overall Percent		95%
Category	#expectedresults/#tested	%Agreement	#expectedresults/#tested	%Agreement	#expectedresults/#tested	%Agreement	Agreement		ConfidenceInterval
(4,716 cfu/mL)									100%
BP Moderate Positive(11,790 cfu/mL)	30/30	100%	30/30	100%	30/30	100%	90/90	100%	95.9% to100%
BP Negative	30/30	100%	30/30	100%	30/30	100%	90/90	100%	95.9% to100%
BP Positive Control	30/30	100%	30/30	100%	30/30	100%	90/90	100%	95.9% to100%
BP Negative Control	30/30	100%	30/30	100%	30/30	100%	90/90	100%	95.9% to100%

The results suggest that there are no significant differences between different users and different sites on different days. Reproducibility studies are acceptable.

b. Linearity/assay reportable range:
Not applicable – This assay is qualitative.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Traceability:

Not applicable. This assay is qualitative.

Specimen Stability:

A study was performed to determine the specimen stability using a contrived sample at 2x LOD. The contrived sample was stored at 2°C to 8°C for varying lengths of time (24, 48, 72 and 96-hours). The samples were brought to room temperature and tested with the AmpliVue Bordetella Assay.

Based on this study the contrived 2x LOD sample was stable when stored at 2℃ to 8°C

Controls:

{15}------------------------------------------------

Controls (Quidel Molecular Bordetella Control Set #M117, which contains positive and negative controls, serves as an external processing and extraction control) were run on the AmpliVue Bordetella Assay each day of testing. These controls are described as follows:

a. The process control is used to monitor sample processing, to detect HDA inhibitory specimens and to confirm the integrity of assay reagents and cassette detection. The process control is included in the Lysis Buffer tube.
b. The external positive control may be treated as a patient specimen. The control should be sampled and tested as if it were a swab specimen and processed as described in the Assay Procedure. The external positive control is intended to monitor substantial reagent and cassette failure.
The external negative control may be treated as a patient specimen. The ﻥ control should be sampled and tested as if it were a swab specimen and processed as described in the Assay Procedure. The external negative control is used to detect reagent or environmental contamination (or carry-over) by B. pertussis DNA or amplicon.

d. Detection limit:

The analytical sensitivity (limit of detection or LoD) of the AmpliVue "Bordetella Assay was determined using quantified (CFU/mL) cultures of two (2) Bordetella pertussis bacterial stocks, BP A639 and E431 serially diluted in negative nasal matrix. The LoD is defined as the lowest concentration at which 95% of all replicates tested positive.

The bacterial strains were freshly grown. The cell density of these bacterial suspensions was estimated using the OD600 reading. After a cell suspension of 0.25 McFarland units was established, the bacteria were serially diluted in PBS to densities ranging from 3x to 0.3x LoD levels based on preliminary studies.

Each test dilution was run as 20 replicates in the AmpliVue assay. The highest dilution where at least 19 of 20 replicates show detection of B. pertussis (95% positivity) was assigned the Limit of Detection of the strain. The CFU/mL was calculated based on the average bacterial plate count of the dilution.

{16}------------------------------------------------

510(k) Summary

BacterialStrain	ConcentrationCFU/mL	ConcentrationCFU/Assay
A639	2,358	3.93
E431	761	1.27

The assay LOD for Bordetella pertussis is 3.93 CFU/assay or 2,358 CFU/mL (sample input).

e. Analytical specificity:

Cross Reactivity:

A study was performed to evaluate the cross-reactivity of the AmpliVue "Bordetella Assay with seventy-nine (79) other microorganisms potentially found in specimens collected to test for Bordetella pertussis (BP) infection. Cross-reactive microorganism was tested at clinically relevant levels of viruses (10 fpfu/mL) and bacteria (10 cfu/mL) in the device. The organisms and their concentrations included in the cross-reactivity study are shown in the table below.

Organism	Test Concentration
Acinetobacter baumanii	2.90 x106 cfu/mL
Arcanobacterium haemolyticum	1.15 x106 cfu/mL
Bacteroides fragilis	1.19 x106 cfu/mL
Bordetella avium	3.85 x106 cfu/mL
Bordetella bronchiseptica	9.45 x106 cfu/mL
Bordetella bronchiseptica	1.17 x106 cfu/mL
Bordetella bronchiseptica (ATCC 4617)	7.74 x106 cfu/mL
Bordetella bronchiseptica	1.97 x106 cfu/mL
Bordetella hinzii	1.40 x106 cfu/mL
Bordetella holmesii (ZeptoMetrix	3.83 x106 cfu/mL
Bordetella holmesii (ATCC 51541)	4.10 x106 cfu/mL
Bordetella holmesii (ATCC 700053)	4.70 x106 cfu/mL
Bordetella holmesii (ATCC 700052)	4.00 x106 cfu/mL
Bordetella parapertussis A747	1.00 x106 cfu/mL
Bordetella petrii	6.26 x106 cfu/mL
Bordetella trematum	9.24 x106 cfu/mL
Burkholderia cenocepacia	2.35 x106 cfu/mL
Burkholderia cepacia	2.52 x106 cfu/mL
Burkholderia multivorans	1.95 x106 cfu/mL
Burkholderia thailandensis	3.95 x106 cfu/mL
Chlamydia trachomatis	7.83 x106 cfu/mL
Organism	Test Concentration
Chlamydophila pneumoniae	2.10 x106 DNA copies/mL
Corynebacterium diptheriae	4.00 x106 cfu/mL
Enterobacter aerogenes	1.31 x106 cfu/mL
Enterococcus faecalis	3.45 x106 cfu/mL
Escherichia coli	8.42 x106 cfu/mL
Fusobacterium necrophorum	3.10 x106 cfu/mL
Haemophilus influenzae	2.13 x106 cfu/mL
Klebsiella pneumoniae	1.61 x106 cfu/mL
Lactobacillus acidophilus	2.00 x106 cfu/mL
Lactobacillus plantarum	7.97 x106 cfu/mL
Legionella pneumophilia	1.76 x106 cfu/mL
Moraxella catarrhalis	9.90 x106 cfu/mL
Morganella morganii	1.57 x106 cfu/mL
Mycobacterium avium	1.84 x106 cfu/mL
Mycobacterium tuberculosis	1.80 x106 cfu/mL
Mycoplasma pneumoniae	3.16 x106 cfu/mL
Neisseria gonorrhoeae	2.45 x106 cfu/mL
Neisseria meningitidis	7.07 x106 cfu/mL
Neisseria mucosa	1.66 x106 cfu/mL
Parvimonas micra	1.55 x106 cfu/mL
Proteus mirabilis	1.06 x106 cfu/mL
Proteus vulgaris	3.40 x106 cfu/mL
Pseudomonas aeruginosa	2.60 x106 cfu/mL
Staphylococcus aureus (MRSA)	7.10 x106 cfu/mL
Staphylococcus epidermidis	2.14 x106 cfu/mL
Stenotrophomonas maltophilia	1.90 x106 cfu/mL
Streptococcus pneumoniae	1.00 x106 cfu/mL
Streptococcus pyogenes	1.29 x106 cfu/mL
Streptococcus salivarius	1.70 x106 cfu/mL
Candida albicans	3.00 x106 cfu/mL
Adenovirus 31	3.55 x105 TCID50/mL
Adenovirus 31	2.74 x107 DNA copies/mL
Coronavirus 229E	1.51 x106 TCID50/mL
Coronavirus NL63	1.41 x105 TCID50/mL
Coronavirus OC43	8.51 x106 TCID50/mL
Coxsackievirus B4	1.08 x105 TCID50/mL
Coxsackievirus B5/10/2006	1.02 x105 TCID50/mL
Echovirus 6	1.02 x106 TCID50/mL
Echovirus 7	1.05 x105 TCID50/mL
Echovirus 9	1.41 x105 TCID50/mL

{17}------------------------------------------------

{18}------------------------------------------------

Organism Test Concentration Echovirus 11 1.51 ×10° TCID50/mL 1.78 ×10° Enterovirus 70 TCID50/mL Enterovirus 71 4.17 ×10° TCID50/mL Epstein-Barr Virus 1.34 ×10° Virus particles/mL HSV Type 1 (Mclnytre) 6.65 ×10° TCID50/mL HSV Type 2 (G) 2.27 ×10° TCID50/mL 2.88 ×10° Influenza A/Mexico/4108/2009 Virus particles/mL Influenza B/Florida/04/2006 2.82 ×10° Virus particles/mL Measles virus 1.95 ×10° TCID50/mL Metapneumovirus A1 3.80 ×10° TCID50/mL 5.89 ×10° Mumps virus TCID50/mL 3.97 ×10° TCID50/mL Parainfluenza Type 1 (#2) Parainfluenza Type 2 (Greer) 3.15 ×10° TCID50/mL TCID50/mL Parainfluenza Type 3 (C234) 2.56 ×10° 1.37 ×106 Parainfluenza Type 4 (VR-1377) TCID50/mL Respiratory Syncytial Virus 1.15 ×106 TCID50/mL Rhinovirus 1A 1.26 ×10° TCID50/mL 1.70 x10° Varicella Zoster Virus DNA copies/mL

510(k) Summary

The Cross Reactivity study tested a panel of 79 microorganisms. This study determined that 1 of 4 Bordetella bronchiseptica strains (strain 4617) and 4 of 4 Bordetella holmesii strains tested were cross-reactive with the AmpliVue Bordetella Assay. These results can be expected as 5% of all Bordetella bronchiseptica strains and all Bordetella holmesii strains are known to carry the IS481 target sequence. These cross-reactive results are noted in the intended use and limitation sections.

Interference:

A study was conducted to determine if the AmpliVue "Bordetella assay is inhibited in the presence of a panel of seventeen (17) substances potentially present in specimens collected to test for Bordetella pertussis infection. Each of the potential interfering substances was tested in three replicates in the presence and absence of near LOD (2x) levels of B. pertussis in the AmpliVue Bordetella Assay. Substances were introduced into the assay at concentrations which were medically relevant.

Common Name	Test Concentration
Cepacol Sore Throat Lozenges	5% w/v
Halls Cherry Menthol-Lyptus Cough Drops	15% w/v
Children's Dimetapp	15% v/v
Chloraseptic Sore Throat Lozenges	10% w/v

{19}------------------------------------------------

510(k) Summary

Ricola Original Swiss Sugar-Free Herb Cough Suppressant Throat Drops	15% w/v
Sucrets Complete Lozenges - Vapor Cherry	5% w/v
Mucin (Bovine Submaxillary Gland, Type I-S)	5 mg/ml
Blood (human), EDTA anticoagulated	5% v/v
Neo-Synephrine	15% v/v
Afrin Nasal Spray Original	15% v/v
Zicam Non-Drowsy Allergy Relief Nasal Gel	5% v/v
Rite Aid Brand Saline Nasal Spray	15% v/v
Zanamivir (Relenza)	5 mg/ml
Tobramycin	4 µg/ml
Mupirocin	10 mg/ml
Oseltamivir Phosphate (Tamiflu)	10 mg/ml
Ricola Original Swiss Sugar Free Herb Cough Suppressant Throat Drops	15% w/v

There was no evidence of interference caused by the substances tested.

Analytical Reactivity (Inclusivity):

The reactivity of the AmpliVue " Bordetella assay was evaluated against an additional six (6) strains of Bordetella pertussis at concentrations near the level of detection (LoD) of the assay.

Each strain was tested as three replicates in the AmpliVue "Bordetella. The study was performed in multiple experiments of no more than 14 assays per experiment. For each experiment, three replicates of up to four strains were performed, along with a positive and a negative run control. All seven strains were detected by the AmpliVue `Bordetella Assay.

Bacterial Strain	ConcentrationCFU/mL	Strain Detected(Yes/No)
9797	2,358	Yes
9340	2,358	Yes
BAA-1335	2,358	Yes
BAA-589	2,358	Yes
51445	2,358	Yes
10380	2,358	Yes

f. Assay cut-off:

{20}------------------------------------------------

Not applicable.

1. Comparison studies:
- a. Method comparison with predicate device:

Not applicable

b. Matrix comparison:
The compatibility of eight different types of media with the AmpliVue Bordetella assay was determined using Validation Lot reagents and the proposed workflow. Four strains of pre-titered Bordetella pertussis stocks were tested at 1x LoD in the presence of eight different types of media (Tris EDTA, Molecular Grade Water, Eswab, M4, M4-RT, M5, 0.85% Saline).

For Bordetella pertussis, 4 of 4 strains (A639, E431, BAA 1335, and 51445) were determined to be compatible with the 8 different media types when tested at the assay LoD.

3. Clinical studies:

Clinical Sensitivity: a.
Performance characteristics of the AmpliVue® Bordetella Assay was established in the Spring to Summer of 2014 (April to August 2014) at four locations in the United States. Eight hundred forty two (842) fresh nasopharyngeal swab specimens were obtained from female and male patients suspected of having respiratory tract infection attributable to Bordetella pertussis from were collected and transported to each laboratory for testing with the AmpliVue® Bordetella Assay.

Clinical performance was based on comparison of the AmpliVue Bordetella Assay results to those obtained by Composite Reference Method that included two manufacturer validated, IS481-targeted real-time PCR assays (PCR1 and PCR2) followed by bi-directional sequencing from PCR positive specimens. The PCR1 and PCR2 assay protocols included 37 amplification cycles. Bi-directional sequencing was performed for all specimens producing amplicon prior to the end of 37-cycle amplification. Specimens were considered positive when bi-directional sequence sequencing results from either comparator PCR assay confirmed the presence of

{21}------------------------------------------------

Bordetella pertussis amplicon. Specimens were considered negative when neither comparator PCR assay produced Bordetella pertussis amplicon at the end of the 37cycles.

Eight hundred forty two (842) fresh nasopharyngeal swab specimens were tested as described above. Six (6) specimens (0.7%) were invalid (in both the initial and repeat test neither the T2 or control lines were detected) and have been removed from additional analysis. The table below details the comparison data of the AmpliVue Bordetella Assay and the Composite Reference Method for the remaining eight hundred thirty six (836) specimens.

Composite Reference Method versus AmpliVue Bordetella Assay
	Composite Reference Method
AmpliVue® Bordetella Assay	Positive	Negative	Total
Positive	64	15	79
Negative	2	755	757
Total	୧୧	770	836
95% Cl
Positive Percent Agreement	64/66	97.0%	89.6% to 99.2%
Negative Percent Agreement	755/770	98.1%	96.8% to 98.8%

b. Clinical specificity:
See Section 3a.
Other clinical supportive data (when a. and b. are not applicable): ﻥ
Not applicable
1. Clinical cut-off:
  Not applicable
1. Expected values:
  The prevalence of Bordetella pertussis detected with the AmpliVue® Bordetella Assay has been calculated for the combined sites based on the age of the patient. Six (6)

{22}------------------------------------------------

specimens (0.7%) were invalid (in both the initial and repeat test neither the T2 or control lines were detected) and have been removed from the Expected Values table. The table below presents the data for the remaining eight hundred forty two (842) specimens.

Combined Study – Expected Values (N=836)
Bordetella pertussis
Age	Total #	Total	Prevalence
		Positive
< 2 years	137	8	5.8%
3 to 12 years	274*	27	9.9%
13 to 21 years	145	30	20.7%
> 22 years	280**	14	5.0%

Four (4) specimens were invalid

** Two (2) specimens were invalid

N. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

Not applicable.

P. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10, 21 CFR 801.109, and the special controls.

Q. Conclusion:

The AmpliVue® Bordetella Assay is as safe and effective as the predicate device for the qualitative detection of Bordetella pertussis nucleic acids isolated from nasopharyngeal swab specimens obtained from patients suspected of having respiratory tract infection attributable to Bordetella pertussis.

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.