The Prodesse ProFlu™+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus. Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A. Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

Negative results do not preclude influenza or RSV virus infection and should not be used as the sole basis for treatment or other management decisions. Conversely, positive results do not rule-out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing and clinical presentation must be considered in order to obtain the final diagnosis of respiratory viral infection.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation (2006 - 2007 respiratory season). Performance characteristics for Influenza A were confirmed when Influenza A/H1, Influenza A/H3, and Influenza A/2009 H1N1 were the predominant Influenza A viruses in circulation (2008 and 2009). When other Influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

Device Description

The ProFlu+ Assay enables detection and discrimination of Influenza A Virus, Influenza B Virus, RSV and universal internal control nucleic acid. Nasopharyngeal swab specimens are collected from patients with signs and symptoms of a respiratory infection using a polyester, rayon or nylon tipped swab and placed into viral transport medium. A Universal Internal Control (UIC) is added to each sample prior to nucleic acid isolation to monitor for inhibitors present in the specimens. The isolation and purification of the nucleic acids is performed using either a MagNA Pure LC Instrument (Roche) and the MagNA Purc Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS® easyMAG™ System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux). The purified nucleic acids are added to Influenza B/RSV Mix along with enzymes included in the ProFlu+ Assay Kit. The Influenza A/Influenza B/RSV Mix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end. Reverse transcription of the RNA in the sample into complementary DNA (cDNA) and subsequent amplification of DNA is performed in a Cepheid SmartCycler® II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFity- Assay is based on Tagman chemistry, which utilizes the 5 - 3 exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the SmartCyclerII instrument.

AI/ML Overview

The provided document describes a 510(k) premarket notification for a modified in vitro diagnostic device, the Prodesse ProFlu™+ Assay. As such, the information typically associated with acceptance criteria and a detailed study proving device performance against those criteria in the context of AI/ML or image processing devices is not present. This document focuses on demonstrating substantial equivalence to a predicate device, rather than proving performance against specific acceptance criteria with detailed statistical results.

However, I can extract the relevant information based on the prompt's request, interpreting "acceptance criteria" as the claimed performance or non-inferiority that the modification verification studies aimed to confirm.

Here's a breakdown of the requested information, acknowledging the limitations of a 510(k) submission for a molecular diagnostic device:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state numerical "acceptance criteria" in the format typically seen for sensitivity/specificity for algorithms. Instead, the modifications were verified to ensure the fundamental scientific technology and clinical performance remained unchanged from the predicate device. The "acceptance criteria" were met if these aspects were confirmed.

Aspect Tested	Acceptance Criteria (Implied)	Reported Device Performance
Universal Internal Control (UIC) Impact	- Ability to detect target organisms at LOD.- Clinical performance remains consistent with the current ProFlu+ Assay.	- The UIC did not affect the ability of the ProFlu+ Assay to detect target organisms at the limit of detection (LOD) as evinced by results of Analytical Sensitivity, IC Interference, Extractor Equivalency, and Sample Stability studies.- A retrospective clinical comparison study demonstrated the modified ProFlu+ Assay with UIC continues to meet the performance claims for the current ProFlu+ Assay.
Modified Positive Controls	- Stability claims are met.- Continued ability to monitor for global assay failures.	- Stability studies demonstrated current stability claims are met.- Clinical validation of the modified positive controls demonstrated their continued ability to monitor for global assay failures.
Influenza A H3N2v and H7N9 Reactivity	- Ability to detect specific strains (A/Indiana/10/2011 (H3N2v) and A/Anhui/1/2013 (H7N9)).	- Results of the Reactivity Study demonstrated the ability of the ProFlu+ Assay to detect A/Indiana/10/2011 (H3N2v) and A/Anhui/1/2013 (H7N9) nucleic acids at concentrations near the limit of detection of the assay.
Increased Freeze-Thaw Cycles (M-MLV RT & RNase II)	- Assay performance is not affected by 10 freeze-thaw cycles.	- Stability studies demonstrated that ProFlu+ Assay performance was not affected when the MMLV Reverse Transcriptase and the RNase Inhibitor II underwent 10 freeze-thaw cycles.

2. Sample Size Used for the Test Set and Data Provenance

The document mentions a "retrospective clinical comparison study" for the UIC impact. However, the specific sample size used for this test set is not provided. The geographic provenance (e.g., country of origin) of the data is also not specified. The study is described as "retrospective."

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

For this type of molecular diagnostic device, ground truth is typically established by laboratory testing methods (e.g., gold standard PCR, sequencing, or culture) rather than expert human interpretation of images or clinical data, especially for a retrospective study focused on assay performance. Therefore, the concept of "number of experts" and their "qualifications" for establishing ground truth as one might consider for imaging devices does not directly apply in this context. The "ground truth" would be the result of a reference laboratory method.

4. Adjudication Method for the Test Set

Given that ground truth is likely based on objective laboratory methods, an "adjudication method" involving multiple human readers (e.g., 2+1, 3+1) is not applicable here.

5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study was Done

No, a Multi Reader Multi Case (MRMC) comparative effectiveness study was not done. This type of study is relevant for evaluating the impact of AI assistance on human reader performance, which is not applicable to a non-AI molecular diagnostic assay validation.

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

The ProFlu™+ Assay is a molecular diagnostic test. The entire assay system (reagents, instrumentation, and protocol) constitutes the "algorithm" in a broad sense. The verification studies assess the performance of this system directly. Therefore, the "standalone" performance is what was evaluated in the analytical and clinical studies described, as there isn't a separate "human-in-the-loop" component in the direct interpretation of the PCR results for this device. The user performs the test and interprets the results based on predefined thresholds, but the core detection is algorithmic.

7. The Type of Ground Truth Used

The ground truth for the verification studies would likely be established through:

Reference molecular methods: Such as a validated laboratory-developed test (LDT), sequencing, or other nucleic acid amplification tests (NAATs) that are considered the gold standard for detecting the target viruses.
Viral culture: For confirmation of viable virus.
Analytical spiking: For analytical sensitivity and reactivity studies, where known concentrations of target nucleic acids are used.

While the document doesn't explicitly state the exact "ground truth" method for the clinical comparison, for a molecular diagnostic, it would invariably involve a highly accurate reference laboratory test.

8. The Sample Size for the Training Set

This submission describes modifications to an existing device and its verification, not the development of a de novo algorithm requiring a "training set" in the context of machine learning. Therefore, the concept of a "training set" does not apply here. The initial development of the predicate ProFlu+ Assay would have involved studies to establish its design parameters.

9. How the Ground Truth for the Training Set Was Established

As noted in point 8, the concept of a "training set" is not relevant to this type of device modification submission.

Summary

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K132129

Page 1 of 3 8/6/2013

Attachment D 510(k) SUMMARY

CONTACT

Emily Ziegler Scientist I Gen-Probe Prodesse, Inc. 20925 Crossroads Circle Waukesha, W153186

4UG 0.9 2013

NAME OF DEVICE

Trade Name: Regulation Number: Product Code: Classification Name: Prodesse ProFluTM+ Assay 21 CFR 866.3980 OCC, 001 Nucleic acid amplification assay for detection and differentiation of Influenza A, Influenza B, and RSV

PREDICATE DEVICE

K110968, ProFlu™+ Assay .

INTENDED USE

The Prodesse ProFlu™+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A, Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

PRODUCT DESCRIPTION

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A Universal Internal Control (UIC) is added to each sample prior to nucleic acid isolation to monitor for inhibitors present in the specimens. The isolation and purification of the nucleic acids is performed using either a MagNA Pure LC Instrument (Roche) and the MagNA Purc Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS® easyMAG™ System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).

The purified nucleic acids are added to Influenza B/RSV Mix along with enzymes included in the ProFlu+ Assay Kit. The Influenza A/Influenza B/RSV Mix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end (see table below).

Analyte	Gene Targeted	ProbeFluorophore	AbsorbancePeak	EmissionPeak	InstrumentChannel
Influenza A Virus	Matrix	FAM	495 nm	520 nm	FAM
RespiratorySyncytial Virus A	Polymerase	CAL FluorOrange 560	540 nm	561 nm	TET
RespiratorySyncytial Virus B	Polymerase	CAL FluorOrange 560	540 nm	561 nm	TET
Influenza B Virus	Non-structuralNS1 and NS2	CAL Fluor Red610	595 nm	615 nm	Texas Red
Universal InternalControl	NA	Quasar 670	647 nm	667 nm	Cy5

Reverse transcription of the RNA in the sample into complementary DNA (cDNA) and subsequent amplification of DNA is performed in a Cepheid SmartCycler® II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFity- Assay is based on Tagman chemistry, which utilizes the 5 - 3 exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the SmartCyclerII instrument.

DEVICE COMPARISON

The modified ProFlu+ Assay differs from the current kit in the following ways:

Outsourcing of control stock manufacturing leading to a change in control vector; .
Universal Internal Control, consisting of an RNA in vitro transcript and a DNA plasmid, . incorporated into the kit;
Two modified positive controls replacing the four current positive controls. In addition, the 1:10 . dilution step performed by customers has been removed;
. Additional reactivity for Influenza A/Indiana/10/2011 (H3N2v) and Influenza A/Anhui/1/2013 (H7N9).
e Increase the number of allowable freeze-thaw cycles for the M-MLV Reverse Transcriptase and RNase Inhibitor II from five (5) to ten (10).

The labeling was updated accordingly to incorporate the modifications listed above.

SUBSTANTIAL EQUIVALENCE

1. The Intended Use and Warnings or Precautions of the modified device as described in the labeling have not changed.
  Confidential to Gen-Probe Prodesse, Inc.

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The modifications detailed in the table below had not had any effect or caused any changes to the 2. fundamental scientific technology of the device.

Modification	Potential Impact of Modification	Verification/Validation Result
Outsourcing of controls leading tominor changes in sequenceIncorporation of a UniversalInternal Control, containing bothRNA and DNA internal controlsequences.	Modification of the internal controlmay affect the ability of the device todetect the target organisms.Additionally, it may change theclinical performance of the ProFlu+Assay.	The UIC did not affect the ability of theProFlu+ Assay to detect targetorganisms at the limit of detection asevinced by the results of AnalyticalSensitivity, IC Interference, ExtractorEquivalency, and Sample Stabilitystudies. Additionally, the results of aretrospective clinical comparison studydemonstrated the modified ProFlu+Assay with UIC continues to meet theperformance claims for the currentProFlu+ Assay.
Outsourcing of controls leading tominor changes in sequenceTwo modified positive controls(Inf. A/Inf. B/RSV A Control andRSV B Control) replacing currentfour positive controls.Modified positive controlsprovided "at use" concentration,no dilution is necessary.	Changes in the sequences, format(pooled vs. individual), orconcentration may affect theperformance of the modified positivecontrols in terms of stability orability to detect global assay failures.	Stability studies demonstrated currentstability claims are met. Clinicalvalidation of the modified positivecontrols demonstrated their continuedability to monitor for global assayfailures.
Influenza A H3N2v and H7N9*Reactivity Claims	NA	Results of the Reactivity Studydemonstrated the ability of the ProFlu+Assay to detect A/Indiana/10/2011(H3N2v) and A/Anhui/1/2013 (H7N9)nucleic acids at concentrations near thelimit of detection of the assay.
Increase the number of freeze-thaw cycles for the M-MLVReverse Transcriptase and RNaseInhibitor II from five (5) to ten(10)	Increasing the number of freeze-thawcycles for the MMLV ReverseTranscriptase and RNase Inhibitor IImay affect the assay performance.	Stability studies demonstrated thatProFlu+ Assay performance was notaffected when the MMLV ReverseTranscriptase and the RNase InhibitorII underwent 10 freeze-thaw cycles.

Although this test has been shown to detect A/Anhui/1/2013 H7N9 RNA and influenza A/ Indiana/10/2011 (H3N2v) virus cultured from positive human respiratory specimens, the performance characteristics of this device with clinical specimens that are positive for H7N9 or H3N2v influenza viruses have not been established. The Prodesse ProFlu™+ Assay can distinguish between influenza A and B viruses, but it cannot differentiate influenza A subtypes.

Verification and validation studies performed demonstrated that all clinical and analytical 3. performance/functionality remains unchanged from the previous device.
The appropriate Design Control activities were performed; 4.
- A Risk Analysis was performed and did not raise any new concerns of safety and efficacy a. associated with the modifications.
- A declaration of conformity with design controls has been submitted. b.

The modified ProFlu+ Assay is substantially equivalent to the current legally marketed device, ProFlu+ Assay.

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Image /page/3/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized eagle with three tail feathers, representing the department's mission to protect the health of all Americans and provide essential human services. The eagle is positioned within a circle, and the words "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" are arranged around the circle's perimeter.

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

Emily Ziegler Scientist I Gen-Probe Prodesse, Inc. 20925 Crossroads Circle Waukesha, WI 53186

August 9, 2013

Re: K132129

Trade/Device Name: ProFlu™+ Assay Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory Virus Panel Multiplex Nucleic *Acid Assay Regulatory Class: Class II Product Code: OCC, OOI Dated: July 9, 2013 Received: July 10, 2013

Dear Ms. Ziegler:

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 Part 801); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set

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Page 2 - Emily Ziegler

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 regulation (21 CFR Part 801), please contact the Division of Small Manufacturers, International and Consumer Assistance at its tollfree 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" (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/Resourcesfor You/Industry/default.htm.

Sincerely vours.

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

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Indication for Use

510(k) Number (if known): K132129 · Device Name: ProFluTM+ Assay

Indications for Use:

The ProFlu™+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus. Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A. Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

Prescription UseX(21 CFR Part 801 Subpart D)	And/Or	Over the Counter Use(21 CFR Part 801 Subpart C)
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(PLEASE DO NOT WRITE BELOW THIS LINE; CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of Center for Devices and Radiological Health (CDRH)

Tamara V. Feldblyum -S 2013.08.08 12:31:20 -04'00'

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.