The ProFlu+™ Assay is a multiplex Real Time 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. It is recommended that negative RSV results be confirmed by culture.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary.

If infections 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 (RSV) (Types A and B), and Internal Control. Nasopharyngeal swab specimens are collected from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium.

An Internal Control (IC) 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 Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS® easyMAGTM System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).

The purified nucleic acids are added to ProFlu+ Supermix along with enzymes included in the ProFlu+ Detection Kit. The ProFlu+ Supermix 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 ProFlu+ 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 SmartCycler instrument.

AI/ML Overview

Here's an analysis of the ProFlu+ Assay's acceptance criteria and the study data provided, structured according to your request:

Acceptance Criteria and Device Performance

The acceptance criteria are not explicitly stated in numerical thresholds in the provided document. However, the intent is to demonstrate substantial equivalence to predicate devices, implying that the performance should be comparable or superior. The reported device performance is presented in terms of Sensitivity and Specificity with 95% Confidence Intervals.

Table 1: Acceptance Criteria (Implied) and Reported Device Performance (Prospective Study)

Analyte	Implied Acceptance Criteria (High Sensitivity & Specificity for Diagnosis)	Reported Sensitivity (95% CI)	Reported Specificity (95% CI)
Influenza A	Comparable to predicate devices and aiding in diagnosis.	100% (97.1% - 100%)	92.6% (90.4% - 94.3%)
Influenza B	Comparable to predicate devices and aiding in diagnosis.	97.8% (88.7% - 99.6%)	98.6% (97.5% - 99.2%)
RSV	Comparable to predicate devices and aiding in diagnosis.	89.5% (75.9% - 95.8%)	94.9% (93.2% - 96.2%)

Table 2: Acceptance Criteria (Implied) and Reported Device Performance (Retrospective Study)

Analyte	Implied Acceptance Criteria (High Sensitivity & Specificity for Diagnosis)	Reported Sensitivity (95% CI)	Reported Specificity (95% CI)
Influenza A	Comparable to predicate devices and aiding in diagnosis.	100% (56.6% - 100%)	96.4% (87.7% - 99.0%)
Influenza B	Comparable to predicate devices and aiding in diagnosis.	89.5% (68.6% - 97.1%)	100% (91.4% - 100%)
RSV	Comparable to predicate devices and aiding in diagnosis.	100% (85.7% - 100%)	97.3% (86.2% - 99.5%)

Study Details:

Sample Sizes Used for the Test Set and Data Provenance:
- Prospective Study Test Set: 891 nasopharyngeal (NP) swab samples. After excluding 5 unresolved samples, 826 samples were used in the analysis (Note: The sum of total samples in individual analyte tables is 826).
- Retrospective Study Test Set: Not explicitly stated as a total, but individual analyte tables sum to 60 samples for Influenza A, 60 samples for Influenza B, and 60 samples for RSV (likely the same set of 60 samples analyzed for all three).
- Data Provenance:
  - Prospective Study: Conducted at 3 U.S. clinical laboratories during the 2006-2007 respiratory virus season (February - April).
  - Retrospective Study: Conducted at 1 U.S. site during the 2006-2007 respiratory virus season (February - April).
  - The samples were "collected for routine influenza or RSV testing by each site," indicating real-world clinical samples.
Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts:
- The document does not specify the number of experts or their qualifications for establishing the initial reference method (rapid culture/DFA).
- The ground truth for discrepant analysis was established using "RT-PCR with virus specific primers obtained from literature followed by sequencing." This implies a molecular biology expert, but no specific number or qualifications are given.
Adjudication Method for the Test Set:
- The primary reference method was rapid culture (shell vial) followed by direct fluorescent antibody (DFA) screening and identification.
- For samples where the ProFlu+ Assay and the reference method (culture/DFA) disagreed, discrepant analysis using RT-PCR with virus-specific primers followed by sequencing was performed. This acts as a tie-breaker or a higher-tier reference method for discordant results.
If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done:
- No. The study described is a direct comparison of the ProFlu+ assay against a reference method (culture/DFA and sequencing), not a multi-reader multi-case study comparing human readers with and without AI assistance. The ProFlu+ Assay is an in vitro diagnostic test, not an AI-assisted interpretation tool for human readers.
If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done:
- Yes, the performance data presented (Sensitivity, Specificity) represent the standalone performance of the ProFlu+ Assay diagnostic test itself, processing the samples and providing results without human interpretation influencing the diagnostic outcome beyond standard laboratory procedures (e.g., sample handling, instrument operation).
The Type of Ground Truth Used:
- The primary ground truth for the test set was established by a combination of methods:
  - Rapid culture (shell vial) and direct fluorescent antibody (DFA).
  - For discrepant results, the ground truth was re-established using RT-PCR with virus-specific primers followed by sequencing (which can be considered molecular pathology/genetic confirmation).
The Sample Size for the Training Set:
- The document does not provide details about a specific "training set" for the ProFlu+ Assay. This assay is a diagnostic test based on molecular biology principles (RT-PCR), not a machine learning or AI model that typically requires a separate training set. The "design" and "optimization" of the primers and probes would be done during assay development, but not in the same sense as training a predictive algorithm on labeled datasets.
How the Ground Truth for the Training Set Was Established:
- As noted above, the concept of a "training set" with ground truth in the context of an RT-PCR diagnostic assay like ProFlu+ is not directly applicable in the same way it would be for an AI algorithm. The development of the assay (e.g., selection of primer/probe sequences) would rely on known viral genetic sequences and established molecular biology techniques, rather than a "ground truth" derived from patient samples for algorithm training.

Summary

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ATTACHMENT 6

ProFlu+ Special 510(k) Submission

510(k) SUMMARY

April 8, 2008

CONTACT

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

NAME OF DEVICE

ProFlu+ Assay Trade Name: 21 CFR 866.3980 Regulation Number: Respiratory Viral Panel Multiplex Nuclcic Acid Assay Classification Name:

PREDICATE DEVICE

K073029 - ProFlu+ Assay, Prodesse, Inc. K063765 - ID-Tag Respiratory Virus Panel, Luminex Molecular Diagnostics, Inc.

INTENDED USE

If infections with a novel Influenza A virus is suspected based on current clinical and epidcmiological 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

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ATTACHMENT 6

PRODUCT DESCRIPTION

SUBSTANTIAL EQUIVALENCE

Clinical Performance

Performance characteristics of the ProFlu+ Assay were established during a prospective study at 3 U.S. clinical laboratories and a retrospective study at 1 U.S. site during the 2006-2007 respiratory virus season (February - April). Samples used for this study were nasopharyngeal (NP) swab specimens that were collected for routine influenza or RSV testing by each site.

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Real Time Solutions

ATTACHMENT 6

ProFlu+ Special 510(k) Submission

The reference method was rapid culture (shell vial) followed by direct fluorescent antibody (DFA) screening and identification.

A total of 891 NP swab samples were tested with the ProFlu+ Assay and by culture. Five (5) samples that initially gave unresolved results remained unresolved upon retesting with the ProFlu+ Assay and are not included in the analysis below. All 5 samples were culture negative.

A total of 23 samples were DFA Respiratory Virus Screen positive (screening reagent detects Influenza A and B, RSV, Parainfluenza 1, 2 and 3 and Adenovirus), but contained too few cells to obtain a specific positive identification. 21 of these 23 samples were also positive by the ProFlu+ Assay (9 Influenza A, 11 Influenza B and 1 RSV positives) and genetic sequencing analysis confirmed the identification of the specific virus. The other 2 DFA screen positive samples were negative by the ProFlu+ Assay and sequence analysis confirmed that they were negative for Influenza A, Influenza B and RSV; these 2 samples were considered true negatives. Discrepant analysis for samples where ProFlu+ Assay and culture results were in disagreement was performed using RT-PCR with virus specific primers obtained from literature followed by sequencing.

Results from Prospective Study:

Influenza A Comparison Results

		Reference Method
		Positive	Negative	Total	Comments
ProFlu+Assay	Positive	127	52a	179	Sensitivity 100% (97.1% - 100%) 95% CI
	Negative	0	647	647	Specificity 92.6% (90.4% - 94.3%) 95% CI
	Total	127	699	826

a Forty-three (43) samples positive for Influenza A by sequence analysis, 8 samples negative for Influenza A by sequence analysis, and 1 sample unavailable for sequence analysis.

Influenza B Comparison Results

		Reference Method
		Positive	Negative	Total	Comments
ProFlu+Assay	Positive	45	11a	56	Sensitivity 97.8% (88.7% - 99.6%)95% CI
	Negative	1b	769	770	Specificity 98.6% (97.5% - 99.2%)95% CI
	Total	46	780	826

a Eleven (11) samples positive for Influenza B by sequence analysis.

b One (1) sample negative for Influenza B by sequence analysis.

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Real Time Solutions

ATTACHMENT 6

RSV Comparison Results

	Reference Method
	Positive	Negative	Total
ProFlu+Assay	34a	40a	74	Sensitivity 89.5% (75.9% - 95.8%)95% CI
	4b	748	752	Specificity 94.9% (93.2% - 96.2%)95% CI
Total	38	788	826

a Thirty-four (34) samples positive for RSV by sequence analysis, 3 samples negative for RSV by sequence analysis, and 3 samples unavailable for sequence analysis.

andrylis, and o camples and railable for ooquence analysis and 3 samples negative for RSV by sequence analysis.

Results from Retrospective Study

	Reference Method
	Positive	Negative	Total	Comments
ProFlu+Assay	Positive	5	2a	7	Sensitivity 100% (56.6% - 100%) 95% CI
	Negative	0	53	53	Specificity 96.4% (87.7% - 99.0%) 95% CI
	Total	5	55	60

Influenza A Comparison Results

ª One (1) samples positive for Influenza A by sequence analysis and 1 sample negative for Influenza A by sequence analysis

Influenza B Comparison Results

	Reference Method
		Positive	Negative	Total	Comments
ProFlu+Assay	Positive	17	0	17	Sensitivity 89.5% (68.6% - 97.1%)95% CI
	Negative	2a	41	43	Specificity 100% (91.4% - 100%) 95%CI
	Total	19	41	60

a Two (2) samples positive for Influenza B by sequence analysis.

RSV Comparison Results

		Reference Method
		Positive	Negative	Total
ProFlu+Assay	Positive	23	1a	24	Sensitivity 100% (85.7% - 100%)95% CI
	Negative	0	36	36	Specificity 97.3% (86.2% - 99.5%)95% CI
	Total	23	37	60

a One sample positive for RSV by sequence analysis.

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ATTACHMENT 6

Reproducibility

The reproducibility of the ProFlu+ Assay was evaluated at 3 laboratory sites. Reproducibility was assessed using a panel of 10 simulated samples that included medium and low (near the assay limit of detection) Influenza A, Influenza B, or RSV positive and negative samples. Panels and controls were tested at each site by 2 operators for 5 days (10 samples and 5 controls X 2 operators X 5 days X 3 sites = 450). The overall percent agreement for the ProFlu+ Assay was 98%.

	Site 1			Site 2			Site 3			Total
PanelMember ID	Agreementwithexpected	AVECT	%CV	Agreementwithexpected	AVECT	%CV	Agreementwithexpected	AVECT	%CV	Agreementwithexpected	95%ConfidenceInterval
	result			result			result			result (%)
Negative (2PanelMembers)	20/20	30.5	3.2%	20/20	31.2	7.1%	19*/20	32.2	2.4%	59/60(98%)	91% - 100%
Influenza ALow Positive	10/10	36.0	3.3%	9/10	36.4	3.9%	7/10	37.8	5.3%	26/30(87%)	70% - 95%
Influenza AMediumPositive	10/10	32.6	1.4%	10/10	33.4	4.0%	10/10	33.0	2.5%	30/30(100%)	89% - 100%
Influenza BLow Positive	10/10	32.7	1.4%	10/10	32.6	1.4%	10/10	32.2	1.9%	30/30(100%)	89% - 100%
Influenza BMediumPositive	10/10	30.5	1.3%	10/10	30.1	0.7%	10/10	29.7	0.8%	30/30(100%)	89% - 100%
RSV A Lowpositive	8/10	30.1	8.3%	8/10	32.5	6.2%	8/10	30.7	6.8%	24/30(80%)	63% - 90%
RSV Amediumpositive	10/10	29.5	3.0%	10/10	29.5	3.0%	10/10	29.2	2.7%	30/30(100%)	89% - 100%
RSV B lowpositive	10/10	31.9	3.5%	10/10	32.3	5.5%	10/10	31.8	5.1%	30/30(100%)	89% - 100%
RSV Bmediumpositive	10/10	29.5	1.9%	10/10	29.5	4.0%	10/10	28.7	4.2%	30/30(100%)	89% - 100%
Influenza ARNA Control	10/10	33.5	1.6%	10/10	32.9	4.2%	10/10	34.4	0.9%	30/30(100%)	89% - 100%
Influenza BRNA Control	10/10	32.8	1.4%	10/10	32.1	3.1%	10/10	33.8	1.3%	30/30(100%)	89% - 100%
RSV A RNAControl	10/10	33.7	1.8%	10/10	32.3	3.1%	10/10	34.8	1.5%	30/30(100%)	89% - 100%
RSV B RNAControl	10/10	32.1	1.6%	10/10	31.9	4.3%	10/10	35.2	2.5%	30/30(100%)	89% - 100%
NegativeControl	10/10	28.9	4.0%	10/10	29.6	5.2%	10/10	30.2	1.4%	30/30(100%)	89% - 100%
TotalAgreementAll	148/150 (99%)			147/150 (98%)			144/150 (96%)			439/450(98%)	96% - 99%

I negative sample Unresolved (IC = FAIL). Cr values for Influenza A, Influenza B and RSV were negative, however.

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DEPARTMENT OF HEALTH & HUMAN SERVICES

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Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Karen Harrington, Ph.D. Manager, Clinical Affairs Prodesse, Inc. W229 N1870 Westwood Dr. Waukesha, WI 53186

MAY -2 2008

K081030 Re:

Trade/Device Name: ProFlu™ Plus Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: Class II Product Code: OCC Dated: April 25, 2008 Received: April 28, 2008

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 either class II (Special Controls) or class III (PMA), 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); 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 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.

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Page 2 -

This letter will allow you to begin marketing your device as described in your Section 510(k) premarket 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 Part 801), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at 240-276-0450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding postmarket surveillance, please contact CDRH's Office of Surveillance and Biometric's (OSB's) Division of Postmarket Surveillance at 240-276-3474. For questions regarding the reporting of device adverse events (Medical Device Reporting (MDR)). please contact the Division of Surveillance Systems at 240-276-3464. 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 (240) 276-3150 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely vours.

Sally attaym

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): K081030

Device Name: ProFlu+ Assay

Indication For Use:

The ProFlu+TM Assay is a multiplex Real Time 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 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)

Juh an

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

510(k) ======================================================================================================================================================================= KOSID30

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.