← Product Code [OCC](/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC) · K073029

# PROFLU+ ASSAY (K073029)

_Prodesse, Inc. · OCC · Jan 4, 2008 · Microbiology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC/K073029

## Device Facts

- **Applicant:** Prodesse, Inc.
- **Product Code:** [OCC](/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC.md)
- **Decision Date:** Jan 4, 2008
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.3980
- **Device Class:** Class 2
- **Review Panel:** Microbiology

## Indications for Use

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. A negative test is presumptive and it is recommended these results be confirmed by cell culture. 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. 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 Story

ProFlu+ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test; detects/discriminates Influenza A, Influenza B, and RSV nucleic acids. Input: nasopharyngeal swab specimens collected in viral transport medium. Process: nucleic acid isolation/purification via Roche MagNA Pure LC Instrument/Kit; addition of ProFlu+ Supermix (primers/probes); reverse transcription and amplification on Cepheid SmartCycler II instrument. Principle: Taqman chemistry; 5'-3' exonuclease activity cleaves dual-labeled probes; generates fluorescent signal proportional to amplification products. Output: real-time fluorescent intensity monitored by SmartCycler. Used in clinical laboratories; operated by trained laboratory personnel. Results aid clinicians in differential diagnosis of respiratory viral infections; negative results are presumptive and require confirmation by cell culture.

## Clinical Evidence

Prospective (n=891) and retrospective (n=60) clinical studies compared ProFlu+ Assay to rapid culture (shell vial) and DFA. Prospective study sensitivity: Influenza A 100%, Influenza B 97.8%, RSV 89.5%. Specificity: Influenza A 92.6%, Influenza B 98.6%, RSV 94.9%. Retrospective study sensitivity: Influenza A 100%, Influenza B 89.5%, RSV 100%. Specificity: Influenza A 96.4%, Influenza B 100%, RSV 97.3%. Reproducibility study (n=450) showed 98% overall agreement.

## Technological Characteristics

Multiplex Real Time RT-PCR assay; Taqman chemistry; uses oligonucleotide primers/probes targeting conserved viral genetic regions. Instrumentation: Roche MagNA Pure LC (extraction), Cepheid SmartCycler II (amplification/detection). Detection channels: FAM (Flu A), TET (RSV A/B), Texas Red (Flu B), Cy5 (Internal Control). Software: Cepheid SmartCycler Dx version 1.7b. Reagents include M-MLV reverse transcriptase, RNase inhibitor, and Taq DNA polymerase.

## Regulatory 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.

## Special Controls

*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.

## Predicate Devices

- xTAG™ RVP (Respiratory Viral Panel) (k063765)

## Submission Summary (Full Text)

> This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com.
>
> Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/).

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
k073029

B. Purpose for Submission:
New device

C. Measurand:
Respiratory specimen virus nucleic acid (RNA or DNA) target sequences. Viruses targeted have been associated with respiratory infections in adults and/or children. Viral types detected:
Influenza A, Influenza B, Respiratory Syncytial Virus Type A, Respiratory Syncytial Virus Type B

D. Type of Test:
Multiplex nucleic acid assay, qualitative determination of 4 respiratory virus types (Influenza A, Influenza B, Respiratory Syncytial Virus Type A, Respiratory Syncytial Virus Type B) in nasopharyngeal swabs using nucleic acid isolation, amplification and detection on the Cepheid SmartCycler II Real Time Instrument with Dx Software version 1.7b, which generates signals based on the acquisition of spectrofluorometric data.

E. Applicant:
Prodesse Incorporated

F. Proprietary and Established Names:
ProFlu™ Plus

G. Regulatory Information:
1. Regulation section:
21 CFR 866.3980, Respiratory viral panel multiplex nucleic acid assay
2. Classification:
Class II
3. Product code:
OCC
4. Panel:
Microbiology (83)

H. Intended Use:
1. Intended use(s):
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

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viral infections in humans and is not intended to detect Influenza C.

A negative test is presumptive and it is recommended these results be confirmed by cell culture. 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.

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.

2. Indication(s) for use:
Same as Intended Use.
3. Special conditions for use statement(s):
For prescription use only
4. Special instrument requirements:
Cepheid SmartCycler II Real Time Instrument

I. 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:

1. Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium (refer to Materials Required but not Provided).
2. Add an Internal Control (IC) to every sample to monitor for inhibitors present in the specimens.
3. Perform isolation and purification of nucleic acids using the MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche).
4. Add purified nucleic acids 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 (see Table below).

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5. 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 chemistry, which utilizes the  $5^{\prime} - 3^{\prime}$  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 real-time instrument.

|  Analyte | Gene Targeted | Probe Fluorophore | Absorbance Peak | Emission Peak | Instrument Channel  |
| --- | --- | --- | --- | --- | --- |
|  Influenza A Virus | Matrix | FAM | 495 nm | 520 nm | FAM  |
|  Respiratory Syncytial Virus A | Polymerase | Cal Orange 560 | 540 nm | 561 nm | TET  |
|  Respiratory Syncytial Virus B | Polymerase | Cal Orange 560 | 540 nm | 561 nm | TET  |
|  Influenza B Virus | Non-structural NS1 and NS2 | Cal Red 610 | 595 nm | 615 nm | Texas Red  |
|  Internal Control | NA | Quasar 670 | 647 nm | 667 nm | Cy5  |

# Materials Provided

ProFlu+ Assay consists of two separate boxes: Detection Kit (Cat. # H44VK00) and Control Kit (Cat. # H44VK55).

Box 1: Detection Kit (Cat. # H44VK00)

|  Reagents | Description | Quantity/ Tube | Cap Color | Cat. # | Reactions/ Tube  |
| --- | --- | --- | --- | --- | --- |
|  ProFlu+ Supermix | Taq DNA polymerase 5 oligonucleotide primer pairs 5 oligonucleotide probes Buffer containing dNTPs (dATP, dCTP, dGTP, dTTP), MgCl2and stabilizers Bovine serum albumin | 1030 μL | Brown | HSM77 | 50 (2 tubes provided)  |
|  M-MLV Reverse Transcriptase | 10U/μL | 30 μL | Red | GLS26 | 100  |
|  RNase Inhibitor | 40U/μL | 100 μL | Blue | GLS27 | 100  |
|  Internal RNA Control III | Non-infectious in vitro transcribed RNA | 30 μL | Yellow | GCT12 | 100  |

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Box 2: Control Kit (Cat. # H44VK55)

|  Reagents | Description | Quantit y/ Tube | Cap Color | Cat. # | Control s/ Tube  |
| --- | --- | --- | --- | --- | --- |
|  Influenza A RNA Control III | Non-infectious in vitro transcribed RNA of specific viral sequences | 300 μL | White | HCT 75 | 15  |
|  Influenza B RNA Control III | Non-infectious in vitro transcribed RNA of specific viral sequences | 300 μL | Green | HCT 76 | 15  |
|  RSV A RNA Control III | Non-infectious in vitro transcribed RNA of specific viral sequences | 300 μL | Purple | HCT 77 | 15  |
|  RSV B RNA Control III | Non-infectious in vitro transcribed RNA of specific viral sequences | 300 μL | Clear | HCT 78 | 15  |

## Materials Required But Not Provided

### Plasticware and consumables

☐ Polyester, rayon or nylon tipped nasopharyngeal swabs
☐ RNase/DNase-free 1.5 mL polypropylene microcentrifuge tubes
☐ Sterile RNase/DNase-free filter or positive displacement micropipettor tips
☐ MagNA Pure LC System Disposables (Reagent Tubs, Reaction Tips, Tip Trays, Cartridges)
☐ Cepheid PCR reaction tubes, 25μL
☐ Parafilm® M or MagNA Pure LC Cartridge Seal

### Reagents

☐ Roche MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Cat. # 03038505001) for 192 isolations
☐ Micro Test™ M4 Viral Transport Medium (Remel, Inc. Cat. # 12500) or BD Universal Viral Transport medium (UTM' Becton, Dickinson and Co. Cat. # 220220)
☐ Molecular Grade Water (RNase/DNase Free)
☐ Extraction Control (e.g. previously characterized positive sample)

### Equipment

☐ – 70°C Freezer
☐ Roche MagNA Pure LC System with software version 3.0.11
☐ Cepheid SmartCycler II Real Time Instrument with Dx Software version 1.7b
☐ Micropipettors (range between 1-10 μL, 10-200 μL and 100-1000 μL)
☐ Mini-centrifuge with adapter for Cepheid Reaction Tubes
☐ Cepheid cooling block

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# Interpretation of Specimen Results

The SmartCycler Dx software automatically determines the specimen results. The interpretation of the assay specimen results is as follows:

|  Sample ID | Assay Result | IC Result | Warning/Error Code | Influenza A Result | RSV Result | Influenza B Result | Interpretation of Results  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Sample ID | Negative | Pass |  | NEG | NEG | NEG | Influenza A, B and RSV nucleic acid not detected  |
|  Sample ID | Positive | NA* |  | POS | NEG | NEG | Influenza A nucleic acid detected  |
|  Sample ID | Positive | NA* |  | NEG | POS | NEG | RSV nucleic acid detected  |
|  Sample ID | Positive | NA* |  | NEG | NEG | POS | Influenza B nucleic acid detected  |
|  Sample ID | Positive | NA* |  | POS | POS | NEG | Influenza A and RSV nucleic acid detected  |
|  Sample ID | Positive | NA* |  | POS | NEG | POS | Influenza A and Influenza B nucleic acid detected  |
|  Sample ID | Positive | NA* |  | NEG | POS | POS | RSV and Influenza B nucleic acid detected  |
|  Sample ID | Unresolved | Fail |  | NEG | NEG | NEG | Unresolved – PCR inhibition or reagent failure  |
|  Sample ID | ND³ | ND | 3079² | ND | ND | ND | Not Determined – error code 3079  |
|  Sample ID | Invalid |  | 4098³ | ND | ND | ND | Not determined – error code 4098  |

¹ Columns and data not used for interpretation are not included
² Error Code 3079: Warning/Error Code 3079 is periodically observed with Influenza A positives (Influenza A Positive Control, Influenza A positive NP swab samples). Warning/Error Code 3079 occurs when the fluorescence (RFU) signal is too high. In this case, all results for that sample are reported by the Dx software as ND (Not Determined). If a Ct value ≥ 13 is reported in the Influenza A, RSV, and/or Influenza B Ct columns, the sample results can be recorded as POS for the specific analyte(s).
³ An Invalid assay run will display Error Code 4098
* Detection of the Internal Control in the Cy5 detection channel is not required for positive result. High viral load can lead to reduced or absent Internal Control signal.

# J. Substantial Equivalence Information:

1. Predicate device name(s):
xTAG™ RVP (Respiratory Viral Panel)
Common Name: Respiratory Viral Panel (RVP) Multiplex Nucleic Acid Detection Assay

2. Predicate 510(k) number(s):
k063765

3. Comparison with predicate:
Both assays detect Influenza A and B, and respiratory syncytial virus using

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nucleic acid amplification techniques. Both assays use nasal pharyngeal swabs as the collection device and the MagNA Pure LC system for nucleic acid isolation. The detection system with both assays involves spectrophotometric detection. The assays differ in that the predicate also detects Influenza A subtypes H1 and H3, Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus, Rhinovirus, and Adenovirus.

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

- Special controls guidance documents will be promulgated
- Guidance on Class II Special Controls Guidance Document: Reagents for Detection of Specific Novel Influenza A Viruses (March 2006)
- Guidance on In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path (April 2006)
- Guidance on Informed Consent for In Vitro Diagnostic Device Studies Leftover Human Specimens that are Not Individually Identifiable (April 2006)
- Draft Guidance on Nucleic Acid Based In Vitro Diagnostic Devices for Detection of Microbial Pathogens (Dec 2005) – http://www.fda.gov/cdrh/oivd/guidance/1560.html
- Software Guidance for the content of premarket submissions for software contained in medical devices (May 2005) – http://www.fda.gov/cdrh/ode/guidance/337.html
- General Guidance on Software Validation (Jan 2002) – http://www.fda.gov/cdrh/comp/guidance/938.html
- CLSI EP17-A: Guidance for Protocols for Determination of Limits of Detection and Limits of Quantitations (Vol. 2, No. 34) (Oct 2004).
- CLSI MM13-A: Guidance for the Collection, Transport, Preparation and Storage of Specimens for Molecular Methods (Vol. 25, No. 31) (Dec 2005).
- CLSI EP7-A2: Guidance for Interference Testing in Clinical Chemistry (Vol. 25, No.27 Second Ed) (Nov 2005).
- CLSI EP12-A: Guidance for User Protocol for Evaluation of Qualitative Test Performance (Vol. 22, No. 14) (Sept 2002).
- CLSI MM6-A: Guidance for the Quantitative Molecular Methods for Infectious Diseases (Vol. 23, No.28) (Oct 2003).
- CLSI EP5-A2: Guidance for Evaluation of Precision Performance of Quantitative Measurement Methods (Vol. 24, No. 25 Second Ed.) (Aug 2004).

## L. Test Principle:

See I

## M. Performance Characteristics (if/when applicable):

### Expected Values

The prevalence of Influenza and RSV varies each year with epidemics occurring during the fall and winter months in the US. Variables that affect the rate of positivity observed in respiratory testing include: the efficiency and timing of specimen collection, handling and transport of the specimen, the time of year, age of the patient, and local disease prevalence. During the 2006-2007 U.S. respiratory season, the combined prevalence of Influenza A and Influenza B was 13.2% and in 2005-2006 the combined prevalence was 12.1%. The prevalence of RSV during the 2005-2006 season was 16.2%. In the 2007

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ProFlu+ multi-center clinical study (samples collected between February and April), the prevalence as observed with culture of Influenza A was 15.8%, Influenza B was 5.4% and RSV was 4.2%. As influenza and RSV seasons overlap, dual positive infections can occur. During this study, culture and the ProFlu+ Assay each detected one Influenza A and RSV dual-positive (although not the same sample) and ProFlu+ detected one Influenza A and Influenza B dual-positive out of the 901 total samples included in the study. Because the incidence of a triple infection of Influenza A, Influenza B, and RSV is low, it is recommended that the samples undergo repeat testing if nucleic acids from all three analytes are detected in a single sample.

## 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.

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 positive, 11 Influenza B positive and 1 RSV positive) 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.

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# 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.

RSV Comparison Results

|   | Reference Method |   |   |   |   |
| --- | --- | --- | --- | --- | --- |
|   |   |  Positive | Negative | Total |   |
|  ProFlu+Assay | Positive | 34a | 40a | 74 | Sensitivity 89.5% (75.9% - 95.8%) 95% CI  |
|   |  Negative | 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.
${}^{b}$  One (1) sample positive for RSV by sequence analysis and 3 samples negative for RSV by sequence analysis.

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# Results from Retrospective Study

Influenza A Comparison Results

|   | 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 |   |

${}^{a}$  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.

# 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  $98\%$ .

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|  Panel Member ID | Site 1 |   |   | Site 2 |   |   | Site 3 |   |   | Total Agreement with expected result (%) | 95% Confidence Interval  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Agreement with expected result | AVE C_T | %CV | Agreement with expected result | AVE C_T | %CV | Agreement with expected result | AVE C_T | %CV  |   |   |
|  Negative (2 Panel Members) | 20/20 | 30.5 | 3.2% | 20/20 | 31.2 | 7.1% | 19*/20 | 32.2 | 2.4% | 59/60 (98%) | 91% - 100%  |
|  Influenza A Low 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 A Medium Positive | 10/10 | 32.6 | 1.4% | 10/10 | 33.4 | 4.0% | 10/10 | 33 | 2.5% | 30/30 (100%) | 89% - 100%  |
|  Influenza B Low 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 B Medium Positive | 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 Low positive | 8/10 | 30.1 | 8.3% | 8/10 | 32.5 | 6.2% | 8/10 | 30.7 | 6.8% | 24/30 (80%) | 63% - 90%  |
|  RSV A medium positive | 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 low positive | 10/10 | 31.9 | 3.5% | 10/10 | 32.3 | 5.5% | 10/10 | 31.8 | 5.1% | 30/30 (100%) | 89% - 100%  |
|  RSV B medium positive | 10/10 | 29.5 | 1.9% | 10/10 | 29.5 | 4.0% | 10/10 | 28.7 | 4.2% | 30/30 (100%) | 89% - 100%  |
|  Influenza A RNA 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 B RNA 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 RNA Control | 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 RNA Control | 10/10 | 32.1 | 1.6% | 10/10 | 31.9 | 4.3% | 10/10 | 35.2 | 2.5% | 30/30 (100%) | 89% - 100%  |
|  Negative Control | 10/10 | 28.9 | 4.0% | 10/10 | 29.6 | 5.2% | 10/10 | 30.2 | 1.4% | 30/30 (100%) | 89% - 100%  |
|  Total Agreement All | 148/150 (99%) |   |   | 147/150 (98%) |   |   | 144/150 (96%) |   |   | 439/450 (98%) | 96% - 99%  |

* 1 negative sample Unresolved (IC = FAIL). C_T values for Influenza A, Influenza B and RSV were negative, however.

## Analytical Sensitivity

The analytical sensitivity (limit of detection or LoD) of the ProFlu+ Assay was determined using quantified (TCID₅₀/mL) cultures of 4 Influenza A (2 H1N1 and 2 H3N2), 2 Influenza B, 2 Respiratory Syncytial Virus Type A, and 2 Respiratory Syncytial Virus Type B strains serially diluted in nasopharyngeal clinical matrix. Each viral strain was extracted using the Roche MagNA Pure LC and tested in replicates of 20 per concentration of virus.

Analytical sensitivity (LoD) as defined as the lowest concentration at which ≥ 95% of all

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replicates tested positive, ranged from  $10^{2} - 10^{-1}$  TCID $_{50}$ /mL.

|  Viral Strain | LoD Concentration  |
| --- | --- |
|  Influenza A/Port Chalmers/1/73 (H3N2) | 101 TCID50/mL  |
|  Influenza A/CA/7/04 (H3N2) | 100 TCID50/mL  |
|  Influenza A/New Caledonia/12/99 (H1N1) | 102 TCID50/mL  |
|  Influenza A/WS/33 (H1N1) | 100 TCID50/mL  |
|  Influenza B/Lee/40 | 101 TCID50/mL  |
|  Influenza B/Wisconsin/2/06 | 100 TCID50/mL  |
|  Respiratory Syncytial Virus Type A Strain Long | 100 TCID50/mL  |
|  Respiratory Syncytial Virus Type A Strain A-2 | 101 TCID50/mL  |
|  Respiratory Syncytial Virus Type B Strain Wildtype B-1 | 10-1 TCID50/mL  |
|  Respiratory Syncytial Virus Type B Strain Wash/18537/62 | 101 TCID50/mL  |

# Reactivity

The reactivity of the ProFlu+ Assay was evaluated against multiple strains of Influenza A (H1N1, H3N2, and H5N1 subtypes), Influenza B, and Respiratory Syncytial Viruses. The panel consisted of 12 Influenza A subtype H1N1, 13 Influenza A subtype H3N2, 2 Influenza A subtype H5N1, 11 Influenza B, and 5 Respiratory Syncytial Virus strains. Each viral strain was extracted using the Roche MagNA Pure LC and tested in triplicate. All viral cultures of the panel were detected by the ProFlu+ Assay.

|  Viral Strain | Concentration | Influenza A (FAM) | RSV (TET) | Influenza B (Tex Red)  |
| --- | --- | --- | --- | --- |
|  Influenza A/Fujian/156/00 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Hawaii/15/01 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Kentucky/2/06 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Jiangxi/160/05 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Henan/8/05 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Taiwan/42/06 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Virginia/1/06 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Hong Kong/2652/06 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/New Caledonia/12/99(H1N1) | 102TCID50/mL | + | - | -  |

{11}

|  Viral Strain | Concentration | Influenza A (FAM) | RSV (TET) | Influenza B (Tex Red)  |
| --- | --- | --- | --- | --- |
|  Influenza A/WS/33 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/PR/8 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Brazil/1137/99 (H1N1) | 102TCID50/mL | + | - | -  |
|  Influenza A/Fuijan/411/02 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/New York/55/2004 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/California/07/04 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Victoria/512/05 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Hong Kong/218/06 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Anhui/1239/05 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Hong Kong/2831/05 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Hiroshima/52/05 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Kentucky/03/06 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Port Chalmers/1/73 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Bahamas/2686/99 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/Brazil/02/99 (H3N2) | 103TCID50/mL | + | - | -  |
|  Influenza A/Costa Rica/07/99 (H3N2) | 102TCID50/mL | + | - | -  |
|  Influenza A/HK 486 (H5N1) | 0.14 ng/μL* | + | - | -  |
|  Influenza A/VN 1203 (H5N1) | 0.27 ng/μL** | + | - | -  |
|  Influenza B/Florida/7/04 | 102TCID50/mL | - | - | +  |
|  Influenza B/Hawaii/11/05 | 102TCID50/mL | - | - | +  |
|  Influenza B/Michigan/2/06 | 102TCID50/mL | - | - | +  |
|  Influenza B/Wisconsin/2/06 | 102TCID50/mL | - | - | +  |
|  Influenza B/Hawaii/33/04 | 102TCID50/mL | - | - | +  |
|  Influenza B/Ohio/1/05 | 102TCID50/mL | - | - | +  |
|  Influenza B/Florida/2/06 | 102TCID50/mL | - | - | +  |
|  Influenza B/St Petersburg/14/06 | 102TCID50/mL | - | - | +  |
|  Influenza B/Michigan/4/06 | 102TCID50/mL | - | - | +  |
|  Influenza B/Lee/40 | 102TCID50/mL | - | - | +  |
|  Influenza B/Malaysia/2506/04 | 102TCID50/mL | - | - | +  |
|  RSV A Strain A2 | 102TCID50/mL | - | + | -  |
|  RSV A Strain Long | 102TCID50/mL | - | + | -  |
|  RSV B Strain Wildtype B-1 | 102TCID50/mL | - | + | -  |
|  RSV B Strain Wash/18537/62 | 102TCID50/mL | - | + | -  |
|  RSV B Strain 9320 | 102TCID50/mL | - | + | -  |

*estimated concentration  ${2.8} \times  {10}^{5}{\mathrm{{TCID}}}_{50}/\mathrm{{mL}}$
**estimated concentration  $7.5 \times 10^{4} \mathrm{TCID}_{50} / \mathrm{mL}$

NOTE: Although the ProFlu+ Assay has been shown to detect cultured avian influenza viruses, including avian Influenza A subtype H5N1 virus, the performance characteristics of this test with specimens from humans infected with H5N1 or other avian influenza viruses are unknown.

{12}

# Analytical Specificity

The analytical specificity of the ProFlu+ Assay was evaluated by testing a panel of 50 cultures consisting of 22 viral,
27 bacterial, and 1 yeast strain representing common respiratory pathogens or flora commonly present in nasopharynx. Bacteria and yeast were tested at concentrations of  $10^{6}$  to  $10^{7}$  CFU/mL. Viruses were tested at concentrations of  $10^{3}$  to  $10^{6}$  TCID $_{50}$ /mL. Samples were extracted using the Roche MagNA Pure LC and tested in triplicate. Analytical specificity of the ProFlu+ Assay was  $100\%$ .

|  Strains | Concentration | Influenza A (FAM) | RSV (TET) | Influenza B (Tex Red)  |
| --- | --- | --- | --- | --- |
|  Influenza A/Port Chalmers | 104TCID50/mL | + | - | -  |
|  Influenza B/Wisconsin | 104TCID50/mL | - | - | +  |
|  RSV A/Strain Long | 104TCID50/mL | - | + | -  |
|  RSV B Strain Wash | 104TCID50/mL | - | + | -  |
|  Adenovirus 1/Adenoid 71 | 106TCID50/mL | - | - | -  |
|  Coronavirus 229E | 106TCID50/mL | - | - | -  |
|  Coxsackievirus B4 | 104TCID50/mL | - | - | -  |
|  Coxsackievirus B5/10/2006 | 105TCID50/mL | - | - | -  |
|  Cytomegalovirus | 104TCID50/mL | - | - | -  |
|  Echovirus 2 | 106TCID50/mL | - | - | -  |
|  Echovirus 3 | 104TCID50/mL | - | - | -  |
|  Echovirus 6 | 105TCID50/mL | - | - | -  |
|  Echovirus 11 | 105TCID50/mL | - | - | -  |
|  Enterovirus 68 | 103TCID50/mL | - | - | -  |
|  Enterovirus 70 | 103TCID50/mL | - | - | -  |
|  HSV Type 1 MacInnytre strain | 105TCID50/mL | - | - | -  |
|  HSV Type 2 G strain | 104TCID50/mL | - | - | -  |
|  Human Rhinovirus 39 | 103TCID50/mL | - | - | -  |
|  Human Rhinovirus | 104TCID50/mL | - | - | -  |
|  Measles/7/2000 | 104TCID50/mL | - | - | -  |
|  Mumps virus | 104TCID50/mL | - | - | -  |
|  Parainfluenza Type 1 | 104TCID50/mL | - | - | -  |
|  Parainfluenza Type 2 | 105TCID50/mL | - | - | -  |
|  Parainfluenza Type 3 | 105TCID50/mL | - | - | -  |
|  Parainfluenza Type 4 | 104TCID50/mL | - | - | -  |
|  Varicella Zoster | 104TCID50/mL | - | - | -  |
|  Bordetella pertussis | 106CFU/mL | - | - | -  |
|  Bordetella | 107CFU/mL | - | - | -  |

{13}

|  Strains | Concentration | Influenza A (FAM) | RSV (TET) | Influenza B (Tex Red)  |
| --- | --- | --- | --- | --- |
|  bronchiseptica |  |  |  |   |
|  Chlamydophila pneumoniae | 10^{4} TCID_{50}/mL | - | - | -  |
|  Chlamydia trachomatis | 10^{4}TCID_{50}/mL | - | - | -  |
|  Legionella pneumophila | 10^{6} CFU/mL | - | - | -  |
|  Mycobacterium intracellulare | 10^{7} CFU/mL | - | - | -  |
|  Mycobacterium tuberculosis | 10^{7} CFU/mL | - | - | -  |
|  Mycobacterium avium | 10^{7} CFU/mL | - | - | -  |
|  Haemophilus influenzae | 10^{6} CFU/mL | - | - | -  |
|  Pseudomonas aeruginosa | 10^{6} CFU/mL | - | - | -  |
|  Proteus vulgaris | 10^{6} CFU/mL | - | - | -  |
|  Proteus mirabilis | 10^{6} CFU/mL | - | - | -  |
|  Neisseria gonorrhoeae | 10^{6} CFU/mL | - | - | -  |
|  Neisseria menigitidis | 10^{6} CFU/mL | - | - | -  |
|  Neisseria mucosa | 10^{7} CFU/mL | - | - | -  |
|  Klebsiella pneumoniae | 10^{6} CFU/mL | - | - | -  |
|  Escherichia coli | 10^{6} CFU/mL | - | - | -  |
|  Moraxella catarrhalis | 10^{7} CFU/mL | - | - | -  |
|  Corynebacterium diphtheriae | 10^{7} CFU/mL | - | - | -  |
|  Lactobacillus plantarum | 10^{6} CFU/mL | - | - | -  |
|  Streptococcus pneumoniae | 10^{6} CFU/mL | - | - | -  |
|  Streptococcus pyogenes | 10^{6} CFU/mL | - | - | -  |
|  Streptococcus salivarius | 10^{6} CFU/mL | - | - | -  |
|  Staphylococcus epidermidis | 10^{6} CFU/mL | - | - | -  |
|  Staphylococcus aureus | 10^{6} CFU/mL | - | - | -  |
|  Candida albicans | 10^{6} CFU/mL | - | - | -  |

{14}

15

# Competitive Inhibition

Competitive inhibition of the ProFlu+ Assay was evaluated using simulated samples with varying concentrations of Influenza A Virus (LoD to 3 logs above LoD) and Respiratory Syncytial Virus A (LoD to 4 logs above LoD) in a single sample. Samples were extracted using the Roche MagNA Pure LC and tested in triplicate. The presence of both Influenza A Virus and Respiratory Syncytial Virus A at varying concentrations in a single sample had no effect on the analytical sensitivity (limit of detection or LoD) of the ProFlu+ Assay.

# Carry-over/Contamination

In an internal study there was no evidence of carry-over/cross contamination with the ProFlu+ Assay using the Roche MagNA Pure LC automated nucleic acid extraction instrument.

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

---

**Source:** [https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC/K073029](https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC/K073029)

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