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.

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 ruleout 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 differentiation of Influenza A Virus. Influenza B Virus, Respiratory Syncytial Virus (Types A and B), and Internal Control.

An overview of the procedure is as follows:

Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium.
Add an Internal Control (IC) to every sample to monitor for inhibitors present in the specimens.
Perform isolation and purification of nucleic acids using a MagNA Pure LC System (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAG System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).
Add purified nucleic acids to Influenza A/Influenza B/RSV Mix along with enzymes included in the ProFlu+ Detection 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 and a quencher (see table below).
Perform reverse transcription of RNA into complementary DNA (cDNA) and subsequent amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFlu+ Assay is based on Taqman reagent 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 realtime instrument.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study details for the Gen-Probe Prodesse, Inc. ProFlu+ Assay, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria in terms of numerical thresholds for Agreement percentages. However, the study aims to demonstrate substantial equivalence by showing high agreement rates with the original ProFlu+ Assay. The reported performance is presented as Percent Positive Agreement (PPA) and Percent Negative Agreement (PNA) with 95% Confidence Intervals (CI).

Analyte	Acceptance Criteria (Implied)	Reported Device Performance (New ProFlu+ Assay vs. Current ProFlu+ Assay)
Influenza A	High agreement with the current ProFlu+ Assay	Percent Positive Agreement: 100% (93.98%-100%) 95% CI Percent Negative Agreement: 99.4% (96.80%-99.90%) 95% CI
Influenza B	High agreement with the current ProFlu+ Assay	Percent Positive Agreement: 100% (78.47% - 100%) 95% CI Percent Negative Agreement: 100% (98.28% - 100%) 95% CI
RSV	High agreement with the current ProFlu+ Assay	Percent Positive Agreement: 100% (90.11% - 100%) 95% CI Percent Negative Agreement: 99.0% (96.39%-99.72%) 95% CI

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

Analyte	Sample Size (Total)	Positive Samples	Negative Samples	Data Provenance
Influenza A	233	60 (determined by original ProFlu+)	173 (determined by original ProFlu+)	Prospectively collected archived samples from respiratory season years 2008 and 2009, collected at two clinical study sites (Columbus, OH and Albuquerque, NM), USA.
Influenza B	233	14 (determined by original ProFlu+)	219 (determined by original ProFlu+)	Prospectively collected archived samples from respiratory season years 2008 and 2009, collected at two clinical study sites (Columbus, OH and Albuquerque, NM), USA.
RSV	233	35 (determined by original ProFlu+)	198 (determined by original ProFlu+)	Prospectively collected archived samples from respiratory season years 2008 and 2009, collected at two clinical study sites (Columbus, OH and Albuquerque, NM), USA.

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

The document does not explicitly mention "experts" in the traditional sense (e.g., radiologists, pathologists) for establishing ground truth for individual cases. The ground truth for the comparison study was based on a reference standard as described below.

4. Adjudication Method for the Test Set

The primary reference for the comparison study was the original ProFlu+ Assay.

"True" influenza A, influenza B or RSV positives were considered as any sample that tested positive for the respective analyte by the original ProFlu+ Assay.
"True" Influenza A, Influenza B or RSV negatives were considered as any sample that tested negative by the original ProFlu+ Assay.

For discordant results (where the "New" ProFlu+ Assay differed from the "Current" ProFlu+ Assay), bidirectional sequencing was used as an adjudicator:

For Influenza A: One sample was negative by the current ProFlu+ but positive by the new ProFlu+. Sequencing confirmed it was positive for Influenza A.
For RSV: Two samples were negative by the current ProFlu+ but positive by the new ProFlu+. Sequencing confirmed they were positive for RSV.

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

This is not applicable. The ProFlu+ Assay is an in vitro diagnostic test (a lab assay), not an AI-powered image analysis or diagnostic tool that assists human readers. Therefore, an MRMC study or an assessment of human reader improvement with AI assistance would not be conducted for this type of device.

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

This is not applicable in the context of "algorithm only" as typically referred to for AI products. The ProFlu+ Assay is a PCR-based diagnostic test, and its performance is evaluated as a standalone test in the laboratory, without direct human cognitive input into the result generation process itself. The "New ProFlu+ Assay" was compared directly against the "Current ProFlu+ Assay" as standalone tests.

7. The Type of Ground Truth Used

The ground truth for the comparison study was established using a combination of a pre-existing validated assay (the original ProFlu+ Assay) as the primary reference, and bidirectional sequencing for resolving discordant results.

Specifically:

Initial determination: The results from the original ProFlu+ Assay served as the provisional "true" positive or negative status.
Adjudication for discrepant results: Bidirectional sequencing (targeting a different gene or region of the same gene) was used to confirm the presence of the virus in samples where the new and original assays disagreed. This sequencing acts as a higher-level confirmatory test.

8. The Sample Size for the Training Set

The document does not explicitly describe a "training set" in the context of machine learning (AI). This device is a diagnostic assay, not an AI model requiring a training phase with labeled data in the same way. The "reformulated supermix" implies a development and optimization process, but the specific data used for that internal development (if analogous to training data) is not detailed. The numbers provided (233 samples per analyte) refer to the clinical comparison study data.

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

As noted above, the concept of a "training set" with ground truth in the AI/machine learning sense is not directly applicable here. The assay's development (including the reformulated supermix) would have involved extensive R&D and validation using characterized samples, but this information is not provided in a format that aligns with AI training set descriptions.

Summary

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Gen-Probe Prodesse, Inc. ProFlu+ Assay 510(k) Submission K110968

Page 1 of 4 Date: June 20, 2011

Attachment D 510(k) SUMMARY

CONTACT

JUN 27 2011

Karen Harrington Gen-Probe Prodesse, Inc. W229 N1870 Westwood Dr. Waukesha, WI 53186

NAME OF DEVICE

Trade Name:	ProFlu+ TM Assay
Regulation Number:	21 CFR 866.3980
Classification Name:	Respiratory viral panel multiplex nucleic acid assay

PREDICATE DEVICE

K063765, K081483, K091677 ID Tag Respiratory Virus Panel, Luminex Molecular ● Diagnostics
. K073029, K081030, K092500 - ProFlu+ Assay, Gen-Probe Prodesse, Inc.

INTENDED USE

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

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Gen-Probe Prodesse, Inc. ProFlu+ Assay 510(k) Submission K110968

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.

PRODUCT DESCRIPTION

The ProFlu+ Assay enables detection and differentiation of Influenza A Virus. Influenza B Virus, Respiratory Syncytial Virus (Types A and B), and Internal Control.

An overview of the procedure is as follows:

1. Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium.
1. Add an Internal Control (IC) to every sample to monitor for inhibitors present in the specimens.
1. Perform isolation and purification of nucleic acids using a MagNA Pure LC System (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAG System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).
1. Add purified nucleic acids to Influenza A/Influenza B/RSV Mix along with enzymes included in the ProFlu+ Detection 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 and a quencher (see table below).
1. Perform reverse transcription of RNA into complementary DNA (cDNA) and subsequent amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFlu+ Assay is based on Taqman reagent 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 realtime instrument.

Analyte	Gene Targeted	Probe Fluorophore	AbsorbancePeak	EmissionPeak	InstrumentChannel
Influenza A Virus	Matrix	FAM	495 nm	520 nm	FAM
Respiratory SyncytialVirus A	Polymerase	CAL FluorOrange 560	540 nm	561 nm	TET
Respiratory SyncytialVirus 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
Internal Control	NA	Quasar 670	647 nm	667 nm	Cy5

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SUBSTANTIAL EQUIVALENCE

Clinical Comparison Study

The ProFlu+ Assay's supermix was reformulated and performance characteristics were established by comparing the reformulated assay to the original ProFlu+ Assay. All samples positive for IA, IB or RSV using either the current ProFlu+ Assay and/or the reformulated "New" ProFlu+ Assay were confirmed using bidirectional sequencing. The sequencing assays targeted either a different gene than the ProFlu+ Assay or targeted a different region of the same gene as the ProFlu+ Assay. Prospectively collected archived samples from respiratory season years 2008 and 2009 that were collected at two clinical study sites (Columbus, OH and Albuquerque, NM) were used for this study.

"True" influenza A. influenza B or RSV positives were considered as any sample that tested positive for the respective analyte by the original ProFlu+ Assay. "True" Influenza A, Influenza B or RSV negatives were considered as any sample that tested negative by the original ProFlu+ Assay.

Influenza A Comparison Results

	Current ProFlu+ Assay
	Positive	Negative	Total	Comments
New ProFlu+AssayPositive	60	1*	61	Percent Positive Agreement 100%(93.98%-100%) 95% CI
New ProFlu+AssayNegative	0	172	172	Percent Negative Agreement 99.4%(96.80%-99.90%) 95% CI
Total	60	173	233

Sample was positive for Influenza A using bi-directional sequencing.

Influenza B Comparison Results

		Current ProFlu+ Assay
		Positive	Negative	Total	Comments
New ProFlu+Assay	Positive	14	0	14	Percent Positive Agreement 100%(78.47% - 100%) 95% CI
	Negative	0	219	219	Percent Negative Agreement 100%(98.28% - 100%) 95% CI
	Total	14	219	233

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Gen-Probe Prodesse, Inc. ProFlu+ Assay 510(k) Submission K110968

. .

RSV Comparison Results

	Current ProFlu+ Assay
	Positive	Negative	Total	Comments
New ProFlu+AssayPositive	35	2*	37	Percent Positive Agreement 100%(90.11% - 100%) 95% CI
New ProFlu+AssayNegative	0	.196	196	Percent Negative Agreement 99.0%(96.39%-99.72%) 95% CI
Total	35	198	233

Two samples positive for RSV using bi-directional sequencing.

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/4/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular border with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" arranged around the top half of the circle. Inside the circle is a stylized image of an abstract human figure, with three faces overlapping and merging together, resembling a bird in flight.

Public Health Service

Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993

Gen-Probe Prodesse, Inc. c/o Karen Harrington, Ph.D. Manager, Clinical Affairs W229 N1870 Westwood Drive Waukesha, Wisconsin 53186

Re: K110968

JUN 2 7 2011

Trade/Device Name: ProFlu+"M assay Regulation Number: 21 CFR§ 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: Class II Product Code: OCC, OOI . Dated: April 4, 2011 Received: April 6, 2011

Dear Dr. Harrington:

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.

If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. 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); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket

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Page 2 - Karen Harrington

notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. 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/ReportalProblem/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/cdrh/industry/support/index.html.

Sincerely yours,

Huyatagmas

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Indication for Use

510(k) Number (if known): K110968

Device Name: ProFlu+TM Assay

Indication 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.

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.

Prescription Use X (21 CFR Part 801 Subpart D) And/Or

Over the Counter Use (21 CFR Part 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE; CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD)

Uwe Schlf

Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) K110968 .

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.