Simplexa Flu A/B & RSV Direct Gen II, Simplexa Flu A/B & RSV Positive Control Pack
Device Facts
| Record ID | K201505 |
|---|---|
| Device Name | Simplexa Flu A/B & RSV Direct Gen II, Simplexa Flu A/B & RSV Positive Control Pack |
| Applicant | Diasorin Molecular, LLC |
| Product Code | OCC · Microbiology |
| Decision Date | Aug 22, 2020 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3980 |
| Device Class | Class 2 |
| Attributes | Pediatric |
Indications for Use
The DiaSorin Molecular Simplexa™ Flu A/B & RSV Direct Gen II assay is intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) RNA in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory tract infection in conjunction with clinical and epidemiological risk factors. This test is intended for use as an aid in the differential diagnosis of influenza A, influenza B, and RSV viral infections in humans. Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for treatment or other patient management decisions. Performance characteristics for influenza A were established with clinical specimens collected during the 2010/2011 influenza season when 2009 H1N1 influenza and H3N2 were the predominant influenza A viruses in circulation. 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 the 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. DiaSorin Molecular’s Simplexa™ Flu A/B & RSV Positive Control Pack is intended to be used as a control with the Simplexa™ Flu A/B & RSV Direct kit and the Simplexa™ Flu A/B & RSV Direct kit and the Simplexa™ Flu A/B & RSV Direct Gen II kit for use on the LIAISON® MDX instrument. This control is not intended for use with other assays or systems.
Device Story
The Simplexa Flu A/B & RSV Direct Gen II is an in vitro diagnostic assay for the qualitative detection and differentiation of influenza A, influenza B, and RSV RNA. The device utilizes nasopharyngeal swab samples processed on the LIAISON MDX instrument. It employs nucleic acid amplification technology to detect viral RNA. The system provides qualitative results to clinicians to aid in the differential diagnosis of respiratory viral infections. It is intended for use in clinical laboratory settings by trained personnel. The output assists healthcare providers in patient management and treatment decisions, though negative results do not rule out infection. The device includes a positive control pack for quality assurance.
Clinical Evidence
No clinical data provided in the document; the submission relies on substantial equivalence to a previously cleared predicate device.
Technological Characteristics
Real-time RT-PCR; targets Flu A (matrix), Flu B (matrix), RSV (M gene). Uses bi-functional fluorescent probe-primers. Instrument: LIAISON MDX with LIAISON MDX Studio Software. Consumable: Direct Amplification Disc (DAD) with microfluidic channels and laser-activated valves. Detection: 4-channel optical system (FAM, JOE, CR610, Q670). Sterilization: N/A (reagents). Connectivity: Standalone instrument.
Indications for Use
Indicated for human patients with signs and symptoms of respiratory tract infection to aid in the differential diagnosis of influenza A, influenza B, and RSV viral infections using nasopharyngeal swab specimens.
Regulatory Classification
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
- Simplexa Flu A/B & RSV Direct and Simplexa Flu A/B & RSV Positive Control Pack (K152408)
Related Devices
- K120413 — SIMPLEXA FLU A/B & RSV DIRECT, SIMPLEXA FLU A/B & RSV POSITIVE CONTROL PACK · Focus Diagnostics, Inc. · Jul 13, 2012
- K152408 — Simplexa Flu A/B & RSV Direct and Simplexa Flu A/B & RSV Positive Control Pack · Focus Diagnostics, Inc. · Sep 21, 2015
- K102170 — SIMPLEXA FLU A/B & RSV · Focus Diagnostics, Inc. · Nov 24, 2010
Submission Summary (Full Text)
{0}
FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
## I Background Information:
A 510(k) Number
K201505
B Applicant
DiaSorin Molecular LLC
C Proprietary and Established Names
Simplexa Flu A/B & RSV Direct Gen II, Simplexa Flu A/B & RSV Positive Control Pack
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| OCC | Class II | 21 CFR 866.3980 - Respiratory Viral Panel Multiplex Nucleic Acid Assay | MI - Microbiology |
| OOI | Class II | 21 CFR 862.2570 - Instrumentation for clinical multiplex test systems | CH - Clinical Chemistry |
## II Submission/Device Overview:
### A Purpose for Submission:
The submission contains data in support of modifications made to the Simplexa Flu A/B & RSV Direct K152408 (predicate). The changes were made to improve assay sensitivity for all three targets and to decrease reagent competition within the assay.
No design or formulation changes have been made to the Positive Control Pack.
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
{1}
K201505 - Page 2 of 26
# Measurand:
Viral RNA from Influenza A (Flu A), Influenza B (Flu B) and RSV
## B Type of Test:
Real-time RT-PCR system, qualitative
## III Intended Use/Indications for Use:
### A Intended Use(s):
See Indications for Use below.
### B Indication(s) for Use:
**Simplexa Flu A/B & RSV Direct Gen II**
The DiaSorin Molecular Simplexa™ Flu A/B & RSV Direct Gen II assay is intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) RNA in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory tract infection in conjunction with clinical and epidemiological risk factors. This test is intended for use as an aid in the differential diagnosis of influenza A, influenza B, and RSV viral infections in humans.
Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for treatment or other patient management decisions.
Performance characteristics for influenza A were established with clinical specimens collected during the 2010/2011 influenza season when 2009 H1N1 influenza and H3N2 were the predominant influenza A viruses in circulation. 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 the 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.
**Simplexa™ Flu A/B & RSV Positive Control Pack**
DiaSorin Molecular’s Simplexa™ Flu A/B & RSV Positive Control Pack is intended to be used as a control with the Simplexa™ Flu A/B & RSV Direct kit and the Simplexa™ Flu A/B & RSV Direct Gen II kit for use on the LIAISON® MDX instrument. This control is not intended for use with other assays or systems.
{2}
C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
Special Instrument Requirements:
LIAISON MDX instrument with LIAISON MDX Studio Software
IV Device/System Characteristics:
A Device Description:
The system consists of the Simplexa Flu A/B & RSV Direct Gen II assay reagents (supplied in a quantity for 24 reactions), the LIAISON MDX (with LIAISON MDX Studio Software), the Direct Amplification Disc and associated accessories. The LIAISON MDX instrument is a real-time Polymerase Chain Reaction (PCR) thermocycler used for the identification of nucleic acids from biological specimens. The Direct Amplification Disc (DAD) is compartmentalized into eight separate wedges and up to eight separate specimens or controls may be processed on each disc. Each wedge contains sample and reagent input wells, microfluidic channels and laser activated valves to control the fluid flow, and a reaction chamber. The user adds 50 μL of Reaction Mix to the reagent input well and 50 μL of unextracted specimen to the sample input well. The reverse transcription, amplification and detection are performed automatically by the instrument.
B Principle of Operation:
The Simplexa Flu A/B & RSV Direct Gen II assay uses bi-functional fluorescent probe-primers together with corresponding reverse primers to amplify Flu A, Flu B, RSV and internal control RNA. The assay targets conserved regions of influenza A viruses (matrix gene), influenza B viruses (matrix gene) and RSV (M gene) to identify these viruses in direct nasopharyngeal swabs.
The LIAISON MDX uses fluorescence to quantify the amount of targeted nucleic acid sequences in a sample. The LIAISON MDX has four optical detection channels, allowing for simultaneous detection of up to four targets. Each module is optimized for a specific detection dye based on its spectral characteristics. Each module contains a LED excitation source, filters, lenses and a fiber port. Fluorescence is collected at the fiber port and transmitted to a single photomultiplier detector. A sample is considered positive for a particular target if intensity of the optical reading (fluorescence) crosses a particular threshold before a predetermined cutoff cycle.
An RNA internal control is used to detect RT-PCR failure and/or inhibition.
C Instrument Description Information:
1. Instrument Name:
K201505 - Page 3 of 26
{3}
LIAISON MDX System
2. Specimen Identification:
Barcode scanner or manual entry
3. Specimen Sampling and Handling:
Uses direct nasopharyngeal specimens collected in Universal Transport Media (UTM)
4. Calibration:
Each reagent kit comes with a barcode card, which contains assay specific parameters and lot information. The barcode card is scanned prior to each run.
5. Quality Control:
The Simplexa™ Flu A/B & RSV Positive Control Pack may be used as an external positive control. Universal transport media (UTM) may be used as a negative external control (No Template Control).
V Substantial Equivalence Information:
A Predicate Device Name(s):
Simplexa Flu A/B & RSV Direct and Simplexa Flu A/B & RSV Positive Control Pack
B Predicate 510(k) Number(s):
K152408
C Comparison with Predicate(s):
| Device & Predicate Device(s): | K201505 | K152408 |
| --- | --- | --- |
| Device Trade Name | Simplexa Flu A/B & RSV Direct Gen II and Simplexa Flu A/B & RSV Positive Control Pack | Simplexa Flu A/B & RSV Direct and Simplexa Flu A/B & RSV Positive Control Pack |
| General Device Characteristic Similarities | | |
| Intended Use/Indications for Use | Simplexa™ Flu A/B & RSV Direct Gen II | Simplexa™ Flu A/B & RSV Direct |
K201505 - Page 4 of 26
{4}
K201505 - Page 5 of 26
| | The DiaSorin Molecular Simplexa™ Flu A/B & RSV Direct Gen II assay is intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) RNA in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory tract infection in conjunction with clinical and epidemiological risk factors. This test is intended for use as an aid in the differential diagnosis of influenza A, influenza B, and RSV viral infections in humans. Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for treatment or other patient management decisions. Performance characteristics for influenza A were established with clinical specimens collected during the 2010/2011 influenza season when 2009 H1N1 influenza and H3N2 were the predominant influenza A viruses in circulation. 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 the state or local health department for testing. Viral culture should not be | The Focus Diagnostics Simplexa™ Flu A/B & RSV Direct assay is intended for use on the 3M Integrated Cycler instrument for the in vitro qualitative detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) RNA in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory tract infection in conjunction with clinical and epidemiological risk factors. This test is intended for use as an 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 virus or RSV infection and should not be used as the sole basis for treatment or other patient management decisions. Performance characteristics for influenza A were established with clinical specimens collected during the 2010/2011 influenza season when 2009 H1N1 influenza and H3N2 were the predominant influenza A viruses in circulation. 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, |
| --- | --- | --- |
{5}
K201505 - Page 6 of 26
| | attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
Simplexa™ Flu A/B & RSV Positive Control Pack.
The DiaSorin Molecular Simplexa™ Flu A/B & RSV Positive Control Pack is intended to be used as a control with the Simplexa™ Flu A/B & RSV Direct kit and the Simplexa™ Flu A/B & RSV Direct Gen II kit for use on the LIAISON® MDX instrument. This control is not intended for use with other assays or systems. | specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to the 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.
Simplexa™ Flu A/B & RSV Positive Control Pack
Focus Diagnostics’ Simplexa™ Flu A/B & RSV Positive Control Pack is intended to be used as a control with the Simplexa™ Flu A/B & RSV Direct kit. This control is not intended for use with other assays or systems. |
| --- | --- | --- |
| Instrument | LIAISON MDX | Same |
| Measurand | Viral RNA | Same |
| Organisms Detected | Influenza A, Influenza B and RSV | Same |
| Genomic Targets | Influenza A matrix gene, Influenza B matrix gene, RSV M gene | Same |
| Technology | Real-time RT-PCR | Same |
| Quality Controls | Simplexa Flu A/B & RSV Positive Control Pack | Same |
| **Device Characteristics Differences** | | |
| Modifications were made to optimize the reaction mix formulation and concentrations of primer/probes and buffers. Updated cycling parameters (thresholds) and cutoff Ct values for all targets. Added Analytical Reactivity information for additional strains that can be detected with the kit. | | |
| Catalog No. (assay) | MOL2655 | MOL2650 |
{6}
VI Performance Characteristics:
A Analytical Performance:
1. Precision/Reproducibility:
The modified device was evaluated in a reproducibility study testing a panel of eight samples, representing a Low Positive (2x LoD) and a Moderate Positive (4x LoD) level for each virus. The panel members were prepared by diluting viral stocks of influenza A, influenza B and RSV strains (re-grown and titered) into pooled negative clinical matrix (NP swabs collected in UTM). The panel included a Positive Control and a negative sample consisting of UTM (no-template control).
Reproducibility Sample Panel
| | Panel Member | Level | Concentration | |
| --- | --- | --- | --- | --- |
| | | | TCID50/mL | Copies/mL |
| 1 | Influenza A/Hong Kong/8/68 (H3N2) | LP (2x LoD) | 4 | 4,858 |
| 2 | Influenza A/Hong Kong/8/68 (H3N2) | MP (4x LoD) | 8 | 9,716 |
| 3 | Influenza B/Massachusetts/02/2012 | LP (2x LoD) | 10 | 1,910 |
| 4 | Influenza B/Massachusetts/02/2012 | MP (4x LoD) | 20 | 3,820 |
| 5 | RSV-A2 | LP (2x LoD) | 20 | 1,858 |
| 6 | RSV-A2 | MP (4x LoD) | 40 | 3,716 |
| 7 | UTM (No Template Control | Neg | N/A | N/A |
| 8 | Positive Control | Pos | N/A | N/A |
The study was conducted at three distinct locations/work environments within the sponsor's facility to simulate three testing sites, where each site had two designated LIAISON MDX instruments and two dedicated operators. Each of the two operators used a unique lot of the reaction mix (for a total of two lots). Each panel member was tested in three replicates, in two runs per day, over five days, at three locations. A total of 90 measurements (3 replicates x 2 runs/day x 5 days x 3 sites) were generated for each panel member. The study design allowed for evaluation of multiple components of variance.
The data was assessed for (a) qualitative results, i.e., percent agreement with expected results, and (b) quantitative analysis of variance components, i.e., average Ct values and calculated SD and %CV.
Reproducibility Qualitative Results
| Panel Member | Site 1 | Site 2 | Site 3 | All Sites |
| --- | --- | --- | --- | --- |
| | Agreement with Expected Results | Agreement with Expected Results | Agreement with Expected Results | 95% CI |
| Flu A
Low Positive | 30/30
(100.0%) | 30/30
(100.0%) | 30/30
(100.0%) | 95.9% -100.0% |
K201505 - Page 7 of 26
{7}
K201505 - Page 8 of 26
| Flu A
Moderate Positive | 30/30
(100.0%) | 30/30
(100.0%) | 30/30
(100.0%) | 95.9% -100.0% |
| --- | --- | --- | --- | --- |
| Flu B
Low Positive | 30/30
(100.0%) | 30/30^{1}
(100.0%) | 30/30
(100.0%) | 95.9% -100.0% |
| Flu B
Moderate Positive | 30/30
(100.0%) | 30/30
(100.0%) | 90/90
(100.0%) | 95.9% -100.0% |
| RSV (A2)
Low Positive | 30/30
(100%) | 30/30
(100.0%) | 30/30
(100.0%) | 95.9% -100.0% |
| RSV (A2)
Moderate Positive | 30/30^{2}
(100.0%) | 30/30
(100.0%) | 30/30
(100.0%) | 95.9% -100.0% |
| Positive Control | 30/30
(100%) | 30/30
(100.0%) | 30/30
(100.0%) | 95.9% -100.0% |
| UTM | 30/30
(100.0%) | 30/30
(100.0%) | 30/30
(100.0%) | 95.9% -100.0% |
¹One false positive Flu A result observed when testing the Flu B Low Positive Sample
²One false positive Flu A result observed when testing the RSV Moderate Positive Sample
## Reproducibility Quantitative Results (by Site)
| Panel Member | Site 1 | | Site 2 | | Site 3 | | All Sites | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Avg. Ct | Total %CV | Avg. Ct | Total %CV | Avg. Ct | Total %CV | Avg. Ct | Total %CV | 95% CI |
| Flu A
Low Positive | 30.8 | 2.3% | 31.4 | 1.4% | 30.7 | 1.2% | 31.0 | 2.0% | 95.9% - 100.0% |
| Flu A
Moderate Positive | 29.8 | 2.5% | 30.1 | 3.4% | 29.6 | 1.6% | 29.8 | 2.6% | 95.9% - 100.0% |
| Flu B
Low Positive | 30.7 | 1.9% | 30.5 | 3.4% | 31.2 | 3.9% | 30.8 | 3.3% | 95.9% - 100.0% |
| Flu B
Moderate Positive | 29.8 | 1.4% | 29.5 | 1.9% | 29.8 | 2.1% | 29.7 | 1.9% | 95.9% - 100.0% |
| RSV (A2)
Low Positive | 29.7 | 2.3% | 29.6 | 1.8% | 29.8 | 2.1% | 29.7 | 2.1% | 95.9% - 100.0% |
| RSV (A2)
Moderate Positive | 29.1 | 1.7% | 28.7 | 1.5% | 28.7 | 1.5% | 28.8 | 1.7% | 95.9% - 100.0% |
| Positive Control | 25.3 | 0.8% | 24.4 | 4.7 | 25.0 | 2.9 | 24.9 | 3.4% | 95.9% - 100.0% |
| UTM | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | 0.0 | N/A | N/A |
## Reproducibility Variance Components Summary
| Panel Member | N | Mean Ct | Within Run | | Between Day | | Between Operator/Lot | | Between Site/Instruments | | Total Reproducibility | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Flu A
Low Positive | 90 | 31.0 | 0.50 | 1.6 | 0.00 | 0.0 | 0.21 | 0.7 | 0.34 | 1.1 | 0.64 | 2.1 |
{8}
The average Ct for the IC RNA, across all channels, for all samples tested, ranged from 27.6 to 28.0, with %CV range from 2.8% to 7.2%.
## 2. Linearity:
Not applicable (qualitative test)
## 3. Analytical Specificity/Cross-reactivity
The Simplexa Flu A/B & RSV Direct Gen II assay’s analytical specificity was evaluated in a study testing samples containing organisms that are present as normal flora in the nasopharyngeal passages as well as organisms that cause clinical symptoms similar to illness with influenza A, influenza B, and/or RSV infection. Thirty-two different bacteria and viruses were included in the study, with each organism tested in three replicates. No cross reactivity was observed with the organisms at the concentrations tested, as shown below.
Analytical Cross-reactivity for Simplexa Flu A/B & RSV Direct Gen II
| Organism | Tested Concentration | % Detection | | |
| --- | --- | --- | --- | --- |
| | | Flu A | Flu B | RSV |
| Adenovirus Type 1 | 1 x 10^{6} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Adenovirus Type 7A | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Bordetella pertussis A639 | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Chlamydia pneumoniae | 1 x 10^{6} IFU/mL | 0.0% | 0.0% | 0.0% |
| Cytomegalovirus (CMV) | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Coronavirus 229E | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Coronavirus OC43 | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Corynebacterium diphtheriae | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Enterovirus Type 71 | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
K201505 - Page 9 of 26
{9}
| Organism | Tested Concentration | % Detection | | |
| --- | --- | --- | --- | --- |
| | | Flu A | Flu B | RSV |
| Epstein-Barr Virus (EBV) | 1 x 10^{6} copies/mL | 0.0% | 0.0% | 0.0% |
| Escherichia coli O157-H7 | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Haemophilus influenzae | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Lactobacillus plantarum, 17-5 | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Legionella longbeachae | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Measles | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Metapneumovirus 9 | 1 x 10^{6} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Moraxella catarrhalis, NE1 | 1 x 10^{6} CFU/mL | 0.0% | 0.0% | 0.0% |
| Mumps | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Mycobacterium tuberculosis (genomic DNA) | 1 x 10^{7} copies/mL | 0.0% | 0.0% | 0.0% |
| Mycoplasma pneumoniae, M129 | 1 x 10^{6} CCU/mL | 0.0% | 0.0% | 0.0% |
| Neisseria elongata | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Neisseria meningitidis | 1 x 10^{6} CFU/mL | 0.0% | 0.0% | 0.0% |
| Parainfluenza 1 | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Parainfluenza 2 | 1 x 10^{6} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Parainfluenza 3 | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Pseudomonas aeruginosa | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Rhinovirus 1A | 1 x 10^{5} TCID_{50}/mL | 0.0% | 0.0% | 0.0% |
| Staphylococcus aureus, COL | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Staphylococcus epidermidis | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Streptococcus pneumoniae | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Streptococcus pyogenes, M1 | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
| Streptococcus salivarius | 1 x 10^{7} CFU/mL | 0.0% | 0.0% | 0.0% |
## 4. Interference
### Microbial Interference
The performance of the Simplexa Flu A/B & RSV Direct Gen II assay was evaluated for its ability to accurately detect influenza A, influenza B, and RSV in the presence of other clinically relevant pathogens. The same panel as above, consisting of 32 potentially inhibitory organisms, was individually spiked into a pool containing low concentrations (approximately $2 \times \mathrm{LoD}$) each of the influenza A, influenza B and RSV (A2) viruses. Samples were assayed in three replicates. No inhibition was observed for influenza A, influenza B, or RSV by the organisms at the concentrations shown below.
K201505 - Page 10 of 26
{10}
Microbial Interference for Simplexa Flu A/B & RSV Direct Gen II
| Organism | Tested Concentration | % Detection | | |
| --- | --- | --- | --- | --- |
| | | Flu A | Flu B | RSV |
| Adenovirus Type 1 | 1 x 10^{6} TCID_{50}/mL | 100% | 100% | 100% |
| Adenovirus Type 7A | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Bordetella pertussis A639 | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Chlamydia pneumoniae | 1 x 10^{6} IFU/mL | 100% | 100% | 100% |
| Cytomegalovirus (CMV) | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Coronavirus 229E | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Coronavirus OC43 | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Corynebacterium diphtheriae | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Enterovirus Type 71 | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Epstein-Barr Virus (EBV) | 1 x 10^{6} copies/mL | 100% | 100% | 100% |
| Escherichia coli O157-H7 | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Haemophilus influenzae | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Lactobacillus plantarum, 17-5 | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Legionella longbeachae | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Measles | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Metapneumovirus 9 | 1 x 10^{6} TCID_{50}/mL | 100% | 100% | 100% |
| Moraxella catarrhalis, NE1 | 1 x 10^{6} CFU/mL | 100% | 100% | 100% |
| Mumps | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Mycobacterium tuberculosis (genomic DNA) | 1 x 10^{7} copies/mL | 100% | 100% | 100% |
| Mycoplasma pneumoniae, M129 | 1 x 10^{6} CCU/mL | 100% | 100% | 100% |
| Neisseria elongata | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Neisseria meningitidis | 1 x 10^{6} CFU/mL | 100% | 100% | 100% |
| Parainfluenza 1 | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Parainfluenza 2 | 1 x 10^{6} TCID_{50}/mL | 100% | 100% | 100% |
| Parainfluenza 3 | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Pseudomonas aeruginosa | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Rhinovirus 1A | 1 x 10^{5} TCID_{50}/mL | 100% | 100% | 100% |
| Staphylococcus aureus, COL | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Staphylococcus epidermidis | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
| Streptococcus pneumoniae | 1 x 10^{6} CFU/mL | 100% | 100% | 100% |
| Streptococcus pyogenes, M1 | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
K201505 - Page 11 of 26
{11}
K201505 - Page 12 of 26
| Organism | Tested Concentration | % Detection | | |
| --- | --- | --- | --- | --- |
| | | Flu A | Flu B | RSV |
| Streptococcus salivarius | 1 x 10^{7} CFU/mL | 100% | 100% | 100% |
## Competitive Interference
The performance of the Simplexa Flu A/B & RSV Direct Gen II assay was evaluated for its ability to accurately detect influenza A, influenza B, and RSV in cases of co-infection with the assay's target organisms. A low-level sample (Baseline Sample) was contrived at 4x LoD for each target (influenza A, influenza B, RSV A, and RSV B), and a baseline Ct was determined for each sample, as shown below.
### Baseline Sample Concentrations and Ct Values
| Virus | LoD (TCID_{50}/mL) | Baseline Sample (4x LoD) | | Baseline Ct Value | Channel |
| --- | --- | --- | --- | --- | --- |
| | | TCID_{50}/mL | Copies/mL | | |
| Inluenza A/Hong Kong/8/68 | 2 | 8 | 9,716 | 29.6 | FAM |
| Influenza B/Massachusetts/02/2012 | 5 | 20 | 3,820 | 29.3 | JOE |
| RSV-A2 | 10 | 40 | 3,716 | 29.3 | CR610 |
| RSV-B CH93-18(18) | 20 | 80 | 25,968 | 28.8 | CR610 |
Each potential concomitant infecting virus was spiked into the Baseline Samples at approximately 10,000x LoD and assayed in triplicate. The following combinations were tested.
### Competitive Interference Test Panel
| Sample | High Positive (4x LoD) | Low Positive (10,000x LoD) |
| --- | --- | --- |
| 1 | Flu A | Flu B |
| 2 | Flu A | RSV A |
| 3 | Flu A | RSV B |
| 4 | Flu B | Flu A |
| 5 | Flu B | RSV A |
| 6 | Flu B | RSV B |
| 7 | RSV A | Flu A |
| 8 | RSV A | Flu B |
| 9 | RSV B | Flu A |
| 10 | RSV B | Flu B |
Because the study revealed interference of Flu A, when at concentrations of 10,000x LoD, additional testing was conducted with Flu A "interferent" samples contrived at 5,000x LoD and 1,000x LoD to determine the approximate concentration where no interference was observed with Flu B and RSV. The testing results from the study are summarized below.
{12}
Competitive Interference Results for Simplexa Flu A/B & RSV Direct Gen II
| Target Organism | Conc. TCID_{50}/mL | Baseline Avg. Ct | Competitive Strain | Conc. TCID_{50}/mL | No. Detected | Average Ct | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | | Flu A Ct | Flu B Ct | RSV Ct |
| Influenza A/Hong Kong/8/68 | 8 | 29.7 | Flu B | 5.00 x 10^{4} | 3/3 | 29.8 | 15.3 | N/A |
| | | | RSV A | 1.00 x 10^{5} | 3/3 | 29.1 | N/A | 18.3 |
| | | | RSV B | 1.00 x 10^{5} | 3/3 | 30.0 | N/A | 19.9 |
| | | | | 2.00 x 10^{5} | 3/3 | 31.0 | N/A | 18.7 |
| Influenza B/Massachusetts/02/2012 | 20 | 29.3 | Flu A | 2.00 x 10^{4} | 0/3 | 18.9 | Neg | N/A |
| | | | | 1.00 x 10^{4} | 0/3 | 20.1 | Neg | N/A |
| | | | | 2.00 x 10^{3} | 3/3 | 22.6 | 29.2 | N/A |
| | | | RSV A | 1.00 x 10^{5} | 3/3 | N/A | 29.7 | 18.0 |
| | | | RSV B | 2.00 x 10^{5} | 3/3 | N/A | 31.2 | 18.6 |
| | | | | 1.00 x 10^{5} | 3/3 | N/A | 30.7 | 18.6 |
| RSV A2 | 40 | 29.8 | Flu A | 2.00 x 10^{4} | 0/3 | 19.1 | N/A | Neg |
| | | | | 1.00 x 10^{4} | 2/3 | 20.4 | N/A | 34.2 |
| | | | | 2.00 x 10^{3} | 3/3 | 22.3 | N/A | 31.5 |
| | | | Flu B | 5.00 x 10^{4} | 3/3 | N/A | 15.2 | 34.1 |
| RSV B | 80 | 28.8 | Flu A | 2.00 x 10^{4} | 0/3 | 19.4 | N/A | Neg |
| | | | | 1.00 x 10^{4} | 0/3 | 19.9 | N/A | Neg |
| | | | | 2.00 x 10^{3} | 3/3 | 22.6 | N/A | 30.8 |
| | | | Flu B | 5.00 x 10^{4} | 3/3 | N/A | 15.2 | 30.3 |
| | | | | 2.50 x 10^{4} | 3/3 | N/A | 16.1 | 29.4 |
| | | | | 5.00 x 10^{3} | 3/3 | N/A | 18.7 | 28.3 |
The results indicate that the assay is subject to competitive inhibition in cases of co-infection, where high levels of influenza A (i.e., $>2.0 \times 10^{3} \mathrm{TCID}_{50} / \mathrm{mL}$) will likely suppress the detection of influenza B or RSV when those viruses are present at low levels. A limitation to the labeling has been to disclose this vulnerability of the assay to competitive inhibition from Flu A.
## Interfering Substances
The performance of the Simplexa™ Flu A/B & RSV Direct Gen II assay was evaluated with potentially interfering substances that may be present in nasopharyngeal passages. A total of 10 potentially interfering substances were individually spiked into a pooled nasopharyngeal swab matrix containing influenza A (Influenza A/Hong Kong/8/68), influenza B (Influenza B/Massachusetts/02/2012) and RSV (A2), each at a targeted concentration approximately $2 \times$ LoD; each sample was tested in three replicates. No interference was observed with the substances at the concentrations shown below.
K201505 - Page 13 of 26
{13}
Interference of Endogenous and Exogenous Substances on Simplexa Flu A/B & RSV Direct Gen II
| Potential Interferent | Active Ingredient | Interferent Concentration | % Detection | | |
| --- | --- | --- | --- | --- | --- |
| | | | Flu A | Flu B | RSV |
| Afrin Nasal Spray | Oxymetazoline | 15% (v/v) | 100% | 100% | 100% |
| Antibacterial, systemic | Tobramycin | 4 μg/mL | 100% | 100% | 100% |
| Antibiotic, nasal ointment | Mupirocin | 3.3 mg/mL | 100% | 100% | 100% |
| Blood | N/A | 2% (v/v) | 100% | 100% | 100% |
| Bovine Submaxillary Gland Type I-S | Purified Mucin Protein | 30 μg/mL | 100% | 100% | 100% |
| Nasal Corticosteroid - Beconase AQ | Beclomethasone | 5% (v/v) | 100% | 100% | 100% |
| Nasal Corticosteroid - Fluticasone | Fluticasone | 5% (v/v) | 100% | 100% | 100% |
| Relenza Antiviral Drug | Zanamivir | 3.3 mg/mL | 100% | 100% | 100% |
| Tamiflu Antiviral Drug | Oseltamivir | 1 μM | 100% | 100% | 100% |
| Zicam Nasal Gel | Luffa Opperculata, Galphimia glauca, histaminum hydrochloricum | 5% (v/v) | 100% | 100% | 100% |
5. Specimen Stability
Specimens Collected in UTM
Specimen stability was evaluated when samples were collected in UTM. Test samples were prepared at the concentrations relative to the LoD for each virus, ranging from approximately 2x LoD to 50x LoD, as shown below.
Specimen Stability Test Panel
| Sample | Flu A TCID_{50}/mL (copies/mL) | Flu B TCID_{50}/mL (copies/mL) | RSV TCID_{50}/mL (copies/mL) | No. of Reps. (per time point) |
| --- | --- | --- | --- | --- |
| 2x LoD | 4.3 (5,162) | 10.6 (2,029) | 21.3 (1,974) | 46 |
| 4x LoD | 8.0 (9,716) | 20.0 (3,820) | 40.0 (3,716) | 5 |
| 10x LoD | 20.0 (24,290) | 50.0 (9,550) | 100.0 (9,290) | 5 |
| 20x LoD | 40.0 (48,580) | 100.0 (19,100) | 200.0 (18,580) | 5 |
| 50x LoD | 100.0 (12,1450) | 250.0 (47,750) | 500.0 (46,450) | 5 |
The samples were aliquoted and stored at 2-8°C for 3 days and 7 days. After 7 days of storage in refrigerator, the samples were transferred to a freezer at ≤-70°C. Frozen samples were subjected to four freeze/thaw cycles and then were tested with the Simplexa Flu A/B & RSV Direct Gen II assay. The study utilized five LIAISON MDX instruments and one lot of
K201505 - Page 14 of 26
{14}
the Reaction Mix. At each study time-point, the test samples were removed from the storage conditions and equilibrated to room temperature prior to testing. The stability was evaluated by the change in Ct values from baseline Ct (at day 0).
For Flu A in UTM samples:
a. After storage at 2-8°C for 7 days, the observed average change from the baseline Ct, across all concentration levels, ranged from -0.1 to 0.1 Ct.
b. After 4 F/T cycles (7-day storage at 2-8°C followed by storage at -70°C), the observed average change from the baseline Ct, across all concentration levels, ranged from 0.1 to 0.6 Ct.
For Flu B in UTM samples:
a. after storage at 2-8°C for 7 days, the observed average change from the baseline Ct, across all concentration levels, ranged from -0.1 to 0.5 Ct.
b. After 4 F/T cycles (7-day storage at 2-8°C followed by storage at -70°C), the observed change from the baseline Ct, across all concentration levels, ranged from 0.0 to 0.3 Ct.
For RSV in UTM samples:
a. after storage at 2-8°C for 7 days, the observed average change from the baseline Ct, across all concentration levels, ranged from -0.1 to 0.1 Ct.
b. After 4 F/T cycles (7-day storage at 2-8°C followed by storage at -70°C), the observed average change from the baseline Ct, across all concentration levels, ranged from -0.3 to 0.1 Ct.
The results demonstrated acceptable stability of samples collected in UTM when stored at the above conditions for the time period under evaluation.
## Specimens Collected in Other Collection Media
Additionally, specimen stability was evaluated when samples were collected in three other collection media: Copan ESwab Liquid Amies, Remel M5, and Remel M6. The test samples were prepared by diluting viral stocks of Flu A, Flu B, and RSV into each of the selected media at concentrations ranging from approximately 2x LoD to 50x LoD. The samples were aliquoted and stored at 2-8°C for three days, then frozen at ≤-70°C, to evaluate three freeze/thaw cycles. The study utilized six LIAISON MDX instruments and one lot of the Reaction Mix. At each study time-point, the test samples were removed from the storage conditions and equilibrated to room temperature prior to testing.
Summary of results from the stability evaluation of samples collected in three other media, is shown below.
K201505 - Page 15 of 26
{15}
Summary of Sample Stability (other Media)
| | | Change in Average Ct Values, from the Baseline | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Flu A | | Flu B | | RSV | |
| | Conc. Level | After 3 Days at 2-8°C | After 3 F/T Cycles (at -70°C) | After 3 Days at 2-8°C | After 3 F/T Cycles (at -70°C) | After 3 Days at 2-8°C | After 3 F/T Cycles (at -70°C) |
| Liquid Amies | 2x LoD | 0.0 | 0.4 | -0.7 | 0.7 to 1.7 | 1.0 | 0.7 |
| | 4x LoD | 1.0 | 1.1 | -0.2 | 0.7 | 1.1 | 2.4 |
| | >4x LoD | 0.3 to 0.8 | 0.6 to 1.4 | 1.3 to 1.7 | 1.3 to 1.7 | 0.9 to 1.5 | 1.2 to 1.6 |
| Remel 5 | 2x LoD | 0.1 | 0.6 | -0.4 | -0.1 | 0.0 | 0.1 |
| | 4x LoD | 0.0 | 0.5 | 0.0 | 0.1 | -0.1 | 0.5 |
| | >4x LoD | 0.5 to 0.7 | 0.4 to 0.8 | 0.0 to 0.2 | 0.0 to 0.2 | -0.1 to 0.5 | -0.4 to 0.3 |
| Remel 6 | 2x LoD | 0.2 | 0.4 | 0.3 | 0.4 | 0.1 | 0.4 |
| | 4x LoD | 0.9 | 1.2 | -0.3 | -0.2 | 0.1 | 0.6 |
| | >4x LoD | 0.3-0.4 | 0.3 to 0.5 | -0.1 to 0.0 | -0.2 to 0.2 | -0.3 to 0.2 | -0.1 to 0.9 |
The results demonstrated acceptable stability of samples collected in the three types of media tested, when stored at the above conditions for the time period under evaluation.
The package insert includes the following storage and transport recommendations:
- Specimens collected in UTM should be transported on ice and may be stored at 2 to 8°C for up to seven (7) days.
- Specimens collected in Remel M5, Remel M6, or Copan ESwab™ (Liquid Amies) should be transported on ice and may be stored at 2 to 8°C for up to three (3) days.
6. Reaction Mix Open Vial Stability at Room Temperature
The stability of the Reaction Mix after opening was evaluated for room temperature stability at 30 and 60 minutes timepoints. The study was conducted using Positive Control neat and diluted 1:10 in UTM, testing two lots of reagents across two LIAISON MDX instruments. Each neat sample was tested in duplicate at each time point with each lot of reagents. The 1:10 diluted sample was tested in 12 replicates at each timepoint with each lot of reagents. All replicates were detected at all timepoints (0 (after preparation), 30 minutes and 60 minutes) and the observed change in Ct values was ≤ 1.2% for all three targets.
7. Quality Controls
External controls, the Simplexa™ Flu A/B & RSV Positive Control Pack, are available from DiaSorin and may be used to monitor the assay performance and integrity of the reagents over time.
Three lots of Positive Control were included in the evaluation of the Simplexa Flu A/B & RSV Direct Gen II assay. The summary of the QC results is presented below.
K201505 - Page 16 of 26
{16}
K201505 - Page 17 of 26
Summary of the QC Data Overall
| Target Analyte | Total N | Avg. Ct | SD | %CV |
| --- | --- | --- | --- | --- |
| Flu A | 169 | 25.5 | 0.6 | 2.33 |
| Flu B | 169 | 23.7 | 0.5 | 1.94 |
| RSV | 169 | 25.9 | 0.6 | 2.29 |
| IC | 169 | 28.7 | 1.0 | 3.52 |
8. Detection Limit:
The Limit of Detection (LoD) study was performed using six LIAISON MDX Instruments, two lots of Reaction Mix and two lots of Positive Control. A total of 67 runs were performed by two operators over three days. The test samples were prepared by diluting viral stocks of influenza A, influenza B, and RSV into pooled nasopharyngeal swab (NPS) specimens collected in UTM. Two strains of each virus were tested. The viral stocks were previously titered in $\mathrm{TCID}_{50} / \mathrm{mL}$ (Tissue Culture Infectious Dose at $50\%$ per mL) and in copies/mL using digital droplet PCR technique. Each of the virus strains was tested at four or more concentrations near the expected LoD during the initial screening, followed by confirmation testing at one or more concentrations with two lots of the Reaction Mix, in 20 replicates. The LoD was defined as the concentration of the virus that resulted in at least $95\%$ detection during confirmation testing for each of the virus strains. The confirmed LoD values are shown below.
Claimed LoD for Simplexa Flu A/B & RSV Direct Gen II
| Virus | TCID_{50}/mL | Copies/mL | No. Detected | Avg. Ct |
| --- | --- | --- | --- | --- |
| Influenza A/PR/8/34 | 0.05 | 825 | 19/20 | 33.7 |
| Influenza A/Hong Kong/8/68 | 2 | 2,429 | 20/20 | 32.8 |
| Influenza B/Malaysia/2506/2004 | 0.1 | 409 | 20/20 | 32.4 |
| Influenza B/Massachusetts/02/2012 | 5 | 955 | 20/20 | 32.0 |
| RSV-A2 | 10 | 929 | 20/20 | 31.9 |
| RSV-B CH93-18(18) | 20 | 6,492 | 20/20 | 30.5 |
9. Analytical Reactivity (Inclusivity)
The analytical reactivity of the Simplexa Flu A/B & RSV Direct Gen II with various strains of influenza A, influenza B and RSV, was evaluated by testing dilutions of quantified virus stocks prepared by spiking into pooled negative nasopharyngeal swab matrix (swabs collected in UTM) at concentrations near the LoD. A total of 65 Flu A strains, 25 Flu B strains and 11 RSV strains were tested in three replicates. Samples that were not detected in all three replicates at the initially contrived concentrations were retested at a higher concentration. The strains shown below were positive in all three replicates at the concentrations shown.
{17}
Simplexa Flu A/B & RSV Direct Gen II Analytical Reactivity with Flu A Strains
| Subtype | Organism | Tested Concentration^{1} |
| --- | --- | --- |
| (H1N1) pdm09 | A/California/4/2009 | 100 TCID_{50}/mL |
| (H1N1) pdm09 | A/California/7/2009 | 100 CEID_{50}/mL |
| (H1N1) pdm09 | A/California/12/2012 | 100 TCID_{50}/mL |
| (H1N1) pdm09 | A/Massachusetts/15/2013 | 1000 CEID_{50}/mL |
| (H1N1) pdm09 | A/Mexico/4108/2009 | 100 CEID_{50}/mL |
| (H1N1) pdm09 | A/New York/18/2009 | 100 CEID_{50}/mL |
| H1N1 | A/Brisbane/59/07 | 100 TCID_{50}/mL |
| H1N1 | A/Hawaii/15/2001 | 100 CEID_{50}/mL |
| H1N1 | A/New Caledonia/20/99 | 100 TCID_{50}/mL |
| H1N1 | A/Solomon Island/3/2006 | 100 TCID_{50}/mL |
| H1N1 | A/Taiwan/42/06 | 100 TCID_{50}/mL |
| H1N1 | A/WS/33 | 100 TCID_{50}/mL |
| H2N2 | A/Japan/305/57 | 0.326 ng/μL^{2} |
| H3N2 | A/Brisbane/10/07 | 100 TCID_{50}/mL |
| H3N2 | A/California/02/2014 | 100 CEID_{50}/mL |
| H3N2 | A/New York/55/2004 | 100 CEID_{50}/mL |
| H3N2 | A/Ohio/02/2012 | 200 CEID_{50}/mL |
| H3N2 | A/Port Chalmers/1/1973 | 100 TCID_{50}/mL |
| H3N2 | A/Rhode Island/01/2010 | 400 CEID_{50}/mL |
| H3N2 | A/Santiago/7981/2006 | 100 CEID_{50}/mL |
| H3N2 | A/Switzerland/9715293/2013 | 200 CEID_{50}/mL |
| H3N2 | A/Texas/50/2012 | 100 CEID_{50}/mL |
| H3N2 | A/Wisconsin/67/05 | 100 TCID_{50}/mL |
| H3N2v | A/Indiana/08/2011 | 100 CEID_{50}/mL |
| H3N2v | A/Minnesota/11/2010 | 100 CEID_{50}/mL |
| H5N1 | A/Egypt/N03072/2010(H5N1)-PR8-IDCDC-RG29 | 1:100,000 dilution |
| H5N1 | A/Hubei/1/2010(H5N1)-PR8-IDCDC-RG30 | 1:100,000 dilution |
| H5N1 | A/India/NIV/2006(H5N1)-PR8-IBCDC-RG7 | 1:100,000 dilution |
| H7N9 | A/Anhui/1/2013 | 1:100,000 dilution |
K201505 - Page 18 of 26
{18}
K201505 - Page 19 of 26
| Subtype | Organism | Tested Concentration^{1} |
| --- | --- | --- |
| H9N2 | A/Hong Kong/33982/2009(H9N2)-PR8-IDCDC_RG26 | 100 CEID_{50}/mL |
| H1N3 | A/shorebird/Delaware Bay/211/1994 | 100 CEID_{50}/mL |
| H1N8 | A/red knot/Delaware Bay/240/1994 | 200 CEID_{50}/mL |
| H3N6 | A/redhead/Alberta/192/2002 | 100 CEID_{50}/mL |
| H3N8 | A/duck/Chabarovsk/1610/1972 | 400 CEID_{50}/mL |
| H4N6 | A/duck/Czechoslovakia/1956 | 500 CEID_{50}/mL |
| H4N6 | A/red knot/Delaware/541/1988 | 200 CEID_{50}/mL |
| H5N1 | A/chicken/Vietnam/NCVD-016/2008(H5N1)-PR8-IDCDC-RG12 | 1:100,000 dilution |
| H5N2 | A/pheasant/New Jersey/1355/1998(H5N2)-PR8-IBCDC-4 | 1:100,000 dilution |
| H6N2 | A/turkey/Massachusetts/3740/1965 | 800 CEID_{50}/mL |
| H7N2 | A/turkey/Virginia/4529/2002 (H7N2)xPR8-IBCDC-5 | 1:100,000 dilution |
| H7N7 | A/mallard/Netherlands/12/2000(H7N7)/PR8-IBCDC-1 | 1:100,000 dilution |
| H10N7 | A/mallard/Illinois/10OS4334/2010 | 100 CEID_{50}/mL |
| H10N7 | A/chicken/Germany/N/49 | 100 CEID_{50}/mL |
| H10N1 | A/mallard/Wisconsin/4230/2009 | 100 CEID_{50}/mL |
| H10N8 | A/quail/Italy/1117/1965 | 100 CEID_{50}/mL |
| H11N9 | A/American green-winged teal/Mississippi/300/2010 | 100 CEID_{50}/mL |
| H12N5 | A/mallard/Wisconsin/4218/2009 | 100 CEID_{50}/mL |
| H12N6 | A/duck/Wisconsin/480/1979 | 100 CEID_{50}/mL |
| H13N6 | A/black-legged kittiwake/Quebec/02838-1/2009 | 200 CEID_{50}/mL |
| H16N3 | A/shorebird/Delaware/172/2006 | 400 CEID_{50}/mL |
| H1N1 | A/Swine/1976/31 | 100 TCID_{50}/mL |
| H1N1 | A/Swine/Iowa/15/30 | 100 TCID_{50}/mL |
| H1N2 | A/swine/Ohio/09SW1477/2009 | 100 TCID_{50}/mL |
| H3N2 | A/swine/Ohio/09SW83E/2009 | 400 CEID_{50}/mL |
| H3N2 | A/Singapore/INFIMH-16-0019/2016 | 100 CEID_{50}/mL |
| (H1N1) pdm09 | A/Michigan/45/2015 | 100 CEID_{50}/mL |
| H1N2 | A/Minnesota/19/2011 | 1000 CEID_{50}/mL |
{19}
| Subtype | Organism | Tested Concentration^{1} |
| --- | --- | --- |
| H3N2 | A/Hong Kong/4801/2014 | 200 CEID_{50}/mL |
| H3N2 | A/Kansas/14/2017 | 100 EID_{50}/mL |
| (H1N1) pdm09 | A/Brisbane/02/2018 | 100 EID_{50}/mL |
| (H1N1) pdm09 | A/Christ Church/16/2010 | 200 EID_{50}/mL |
| H3N2 | A/Hong Kong/2671/2019 | 50 EID_{50}/mL |
| (H1N1) pdm09 | A/Guangdong-Maonan/1536/2019 | 100 EID_{50}/mL |
| H3N2 | A/Perth/16/2009 | 50 EID_{50}/mL |
| (H1N1) pdm09 | A/NY/02/09 | 0.04 TCID_{50}/mL |
1 TCID<sub>50</sub>/mL = Tissue Culture Infectious Dose<br/>
CEID<sub>50</sub>/mL = Chicken Embryo Infectious Dose<br/>
EID<sub>50</sub>/mL = Egg Infectious Dose
2 Flu A/Japan/305/57 was in the form of unprotected genomic RNA stock and was prepared in Tris-EDTA (TE) buffer.
## Simplexa™ Flu A/B & RSV Direct Gen II Analytical Reactivity with Flu B Strains
| Lineage | Organism | Tested Concentration^{1} |
| --- | --- | --- |
| Victoria | B/Brisbane/33/2008 | 20 CEID_{50}/mL |
| Victoria | B/Brisbane/60/2008 | 20 CEID_{50}/mL |
| Victoria | B/Florida/02/2006 | 100 TCID_{50}/mL |
| Victoria | B/Lee/40 | 100 TCID_{50}/mL |
| Victoria | B/Nevada/03/2011 | 100 CEID_{50}/mL |
| Victoria | B/Texas/02/2013 | 100 TCID_{50}/mL |
| Victoria | B/Victoria/304/2006 | 50 CEID_{50}/mL |
| Yamagata | B/Christchurch/33/2004 | 100 CEID_{50}/mL |
| Yamagata | B/Florida/07/04 | 100 TCID_{50}/mL |
| Yamagata | B/Florida/04/2006 | 100 TCID_{50}/mL |
| Yamagata | B/Guangdong-Liwan/1133/2014 | 400 CEID_{50}/mL |
| Yamagata | B/Maryland/1/59 | 100 TCID_{50}/mL |
| Unknown | B/Great Lakes/1739/54 | 100 TCID_{50}/mL |
| Yamagata | B/Panama/45/90 | 100 TCID_{50}/mL |
| Yamagata | B/Phuket/3073/2013 | 200 CEID_{50}/mL |
| Yamagata | B/Utah/09/2014 | 100 CEID_{50}/mL |
K201505 - Page 20 of 26
{20}
| Lineage | Organism | Tested Concentration^{1} |
| --- | --- | --- |
| Yamagata | B/Wisconsin/01/2010 | 100 CEID_{50}/mL |
| Unknown | B/Allen/45 | 100 TCID_{50}/mL |
| Unknown | B/Hong Kong/5/72 | 100 TCID_{50}/mL |
| Unknown | B/Taiwan/2/62 | 100 TCID_{50}/mL |
| Victoria | B/Colorado/06/2017 | 20 TCID_{50}/mL |
| Victoria | B/Michigan/09/2011 | 5 EID_{50}/mL |
| Yamagata | B/Texas/81/2016 | 20 EID_{50}/mL |
| Victoria | B/Washington/02/2019 | 200 EID_{50}/mL |
| Yamagata | B/New Hampshire/01/2016 | 100 EID_{50}/mL |
1 TCID $_{50}$ /mL = Tissue Culture Infectious Dose
CEID $_{50}$ /mL = Chicken Embryo Infectious Dose
EID $_{50}$ /mL = Egg Infectious Dose
Simplexa Flu A/B & RSV Direct Gen II Analytical Reactivity with RSV Strains
| Organism | Tested Concentration^{1} |
| --- | --- |
| RSV ATCC-2012-10 | 100 PFU/mL |
| RSV A 1997/12-35 | 100 TCID_{50}/mL |
| RSV A 1998/12-21 | 100 TCID_{50}/mL |
| RSV A 1998/3-2 | 100 TCID_{50}/mL |
| RSV A 2000/3-4 | 100 TCID_{50}/mL |
| RSV A 2001/2-20 | 100 TCID_{50}/mL |
| RSV A 2001/3-12 | 100 TCID_{50}/mL |
| RSV A Long | 100 TCID_{50}/mL |
| RSV B 9320 | 100 TCID_{50}/mL |
| RSV B/Wash/18537/62 | 100 TCID_{50}/mL |
| RSV B/WV/14617/85 | 100 TCID_{50}/mL |
1 TCID $_{50}$ /mL = Tissue Culture Infectious Dose
PFU/mL = Plaque Forming Units
Additionally, testing of eight contemporary human influenza viruses was conducted to compare the reactivity of the modified device (Simplexa Flu A/B & RSV Direct Gen II) to the unmodified version (Simplexa Flu A/B & RSV Direct). The viruses, obtained from CDC, were diluted in pooled negative matrix and tested in three replicates, to determine the lowest concentration detectable by each version of the assay. The results are presented below.
K201505 - Page 21 of 26
{21}
| Type | Subtype/Lineage | Strain | Lowest Concentration detectable in 3/3 Replicates | |
| --- | --- | --- | --- | --- |
| | | | Simplexa Flu A/B & RSV Direct Gen II (modified)1 | Simplexa Flu A/B & RSV Direct (original) |
| A | (H1N1) pdm09 | A/Christ Church/16/2010 | 200 EID50/mL | 2,000 EID50/mL |
| A | H3N2 | A/Hong Kong/2671/2019 | 50 EID50/mL | 50 EID50/mL |
| A | (H1N1) pdm09 | A/Guangdong-Maonan/1536/2019 | 100 EID50/mL | 400 EID50/mL |
| A | H3N2 | A/Perth/16/2009 | 50 EID50/mL | 50 EID50/mL |
| B | Yamagata | B/Phuket/3073/2013b | 200 CEID50/mL | 1,000 CEID50/mL |
| B | Victoria | B/Michigan/09/2011 | 5 EID50/mL | 1,000 EID50/mL |
| B | Yamagata | B/Texas/81/2016 | 20 EID50/mL | 100 EID50/mL |
| B | Victoria | B/Washington/02/2019 | 200 EID50/mL | 4,000 EID50/mL |
$^{1}$ EID $_{50}$ /mL = Egg Infectious Dose at 50% per mL
CEID $_{50}$ /mL = Chicken Embryo Infectious Dose
Overall, the modified device demonstrated higher analytical reactivity with contemporary strains of human influenza viruses than the original device.
## 10. Assay Cut-Off:
The fluorescent thresholds and assay Ct cutoffs were adjusted to optimize the sensitivity and specificity of the Simplexa Flu A/B & RSV Direct Gen II based on the LoD data and results from testing of 880 No-Template-Control samples, using three lots of the Reaction Mix reagents, across 11 LIAISON MDX instruments. The overall positive and negative percent agreements obtained from the method comparison testing with clinical samples were determined to be satisfactory.
## 11. Carry-Over:
The potential for carry-over contamination using the LIAISON MDX instrument was previously evaluated and reviewed under K120413. The study was performed by testing high positive and negative samples in alternate positions on each disc. No carry-over contamination effect was seen in the Flu A, Flu B or RSV channels.
An additional study was conducted with the modified device to evaluate the risk for cross-contamination and to demonstrate the re-usability of the DAD consumable up to eight times.
In the first part of the study, two lots of DADs were evaluated, testing 10 DADs/lot and utilizing 12 LIAISON MDX instruments. Each DAD was used to test two wedges at a time
K201505 - Page 22 of 26
{22}
(alternating Positive Control and No Template Control) per run, for four runs per DAD, for a total of 80 measurements for each sample.
No carryover contamination was observed. The summary results are presented below.
Carryover Contamination Summary Results
| Sample | Flu A (FAM) | | Flu B (JOE) | | RSV (CFR610) | | IC (Q670) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | % Detection | Mean Ct | % Detection | Mean Ct | % Detection | Mean Ct | % Detection | Mean Ct |
| PC | 100% (80/80) | 25.1 | 100% (80/80) | 23.7 | 100% (80/80) | 26.0 | 98.8% (79/80)* | 29.0 |
| NTC | 0% (0/80) | N/A | 0% (0/80) | N/A | 0% (0/80) | N/A | 0% (0/80) | 28.8 |
*One PC replicate did not detect IC. IC detection is not required if Flu A, Flu B or RSV is detected
In the second part of the study, four DADs were tested using Positive Control (PC) and Universal Transport Media (UTM) as a No Template Control (NTC). Since each Disc can accommodate up to eight samples, the purpose of the study was to demonstrate that each disc can be used up to eight times, without a risk of contamination. Each wedge of the DAD was run one wedge at a time per run for a total of eight runs per DAD and 16 replicates of each sample.
No carryover contamination was observed. The summary results are presented below.
DAD Re-usability Study Results
| Sample | Flu A (FAM) | | Flu B (JOE) | | RSV (CFR610) | | IC (Q670) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | % Detection | Mean Ct | % Detection | Mean Ct | % Detection | Mean Ct | % Detection | Mean Ct |
| PC | 100% (16/16) | 25.2 | 100% (16/16) | 23.6 | 100% (16/16) | 25.5 | 100% (16/16) | 28.2 |
| NTC | 0% (0/16) | N/A | 0% (0/16) | N/A | 0% (0/16) | N/A | 100% (16/16) | 27.8 |
# B Comparison Studies:
# 1. Method Comparison with Predicate Device:
A clinical agreement study was performed using retrospective pre-selected positive and negative leftover nasopharyngeal swab (NPS) specimens collected in $3\mathrm{mL}$ Universal Transport Media (UTM) from patients with signs and symptoms of respiratory tract infection. The specimens were collected at three collection sites at two geographical locations. A total of 240 pre-selected positive and negative nasopharyngeal swabs, collected between January 24, 2018 and December 15, 2019, were included in the evaluation. The specimens were shipped on dry ice and stored at $\leq -70^{\circ}\mathrm{C}$ until testing. All specimens were pre-selected for positive and negative status based on routine laboratory results at the collection sites. The specimens were re-blinded and randomized prior to distributing them to the internal clinical testing site at DiaSorin Molecular. Each specimen was tested using the predicate device, Simplexa Flu A/B & RSV Direct, and the modified device, Simplexa Flu
K201505 - Page 23 of 26
{23}
A/B & RSV Direct Gen II. Any sample with discordant results was tested with another FDA cleared molecular assay. Six LIAISON MDX instruments were used in the evaluation.
The following tables show the results from the method comparison testing.
Influenza A
| Simplexa Flu A/B & RSV Direct Gen II (modified) | Simplexa Flu A/B & RSV Direct (predicate) | | Total |
| --- | --- | --- | --- |
| | Detected | Not Detected | |
| Detected | 55 | 4^{a} | 59 |
| Not Detected | 0 | 181 | 181 |
| Total | 55 | 185 | 240 |
| PPA = 100.0% (55/55)
95% CI: 93.5% to 100% | NPA = 97.8% (181/185)
95% CI: 94.6% to 99.2% | | |
a All four samples negative by the predicate and positive by the modified assay, were positive by another FDA cleared NAAT.
Influenza B
| Simplexa Flu A/B & RSV Direct Gen II (modified) | Simplexa Flu A/B & RSV Direct (predicate) | | Total |
| --- | --- | --- | --- |
| | Detected | Not Detected | |
| Detected | 58 | 4^{a} | 62 |
| Not Detected | 0 | 178 | 178 |
| Total | 58 | 182 | 240 |
| PPA = 100.0% (58/58)
95% CI: 93.8% to 100% | NPA = 97.8% (178/182)
95% CI: 94.5% to 99.1% | | |
a Of the four samples negative by the predicate and positive by the modified assay, one was positive by another FDA cleared NAAT.
K201505 - Page 24 of 26
{24}
RSV
| Simplexa Flu A/B & RSV Direct Gen II (modified) | Simplexa Flu A/B & RSV Direct (predicate) | | Total |
| --- | --- | --- | --- |
| | Detected | Not Detected | |
| Detected | 52 | 6^{a} | 58 |
| Not Detected | 0 | 182 | 182 |
| Total | 52 | 188 | 240 |
| PPA = 100.0% (52/52)
95% CI: 93.1% to 100% | NPA = 96.8% (182/188)
95% CI: 93.2% to 98.5% | | |
*All six samples negative by the predicate and positive by the modified assay, were positive by another FDA cleared NAAT.
2. Matrix Comparison:
Not applicable.
C Clinical Studies:
1. Clinical Sensitivity and Specificity:
The clinical performance of the Simplexa Flu A/B & RSV Direct (unmodified device) was established in comparison to viral culture, during a clinical study conducted between September 2010 and March 2011 (see K120413).
Clinical Performance of the Simplexa Flu A/B & RSV Direct Prospective Data (K120413)
| | Sensitivity | 95% CI | Specificity | 95% CI |
| --- | --- | --- | --- | --- |
| Flu A | 97.1%
(66/68) | 89.9% to 99.2% | 97.9%
(639/653) | 96.4% to 98.7% |
| Flu B | 100.0%
(21/21) | 84.5% to 100.0% | 99.9%
(697/698) | 99.2% to 100.0% |
| RSV | 97.6%
(82/84) | 91.7% to 99.4% | 94.4%*
(592/627) | 92.3% to 96.0% |
*Of the 45 samples positive by the Simplexa Flu A/B & RSV Direct and negative by culture for RSV, 35 samples were positive for RSV by another molecular assay.
2. Other Clinical Supportive Data:
See Comparison Studies, section B above.
K201505 - Page 25 of 26
{25}
D Clinical Cut-Off:
Not applicable
E Expected Values/Reference Range:
The original Simplexa Flu A/B & RSV Direct was evaluated during the 2010/2011 influenza season and that data was previously presented in the Decision Summary for K120413. The same data is shown below, for completeness of the record.
The positivity rates observed for influenza A, influenza B and RSV during the clinical study conducted between September 2010 and March 2011 are shown below. The observed prevalence (as determined by reference method) is included.
Positivity Rates for Simplexa Flu A/B & RSV Direct (2010-2011) (K120413)
| Australia (n=330) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Analyte | Total (Prevalence) | < 5 years (n=63) | 5-21 years (n=106) | 22-60 years (n=147) | > 60 years (n=14) |
| Influenza A | 2.7% (9/330) | 0 | 5 | 4 | 0 |
| Influenza B | 2.9% (7/330) | 3 | 2 | 2 | 0 |
| Respiratory Syncytial Virus | 0.7% (1/330) | 0 | 0 | 1 | 0 |
| Ohio (n=245) | | | | | |
| Analyte | Total (Prevalence) | < 5 years (n=195) | 5-21 years (n=48) | 22-60 years (n=2) | > 60 years (n=0) |
| Influenza A | 11.4% (28/245) | 14 | 14 | 0 | 0 |
| Influenza B | 1.2% (3/245) | 1 | 2 | 0 | 0 |
| Respiratory Syncytial Virus | 29.8% (73/245) | 70 | 3 | 0 | 0 |
| Virginia (n=147) | | | | | |
| Analyte | Total (Prevalence) | < 5 years (n=69) | 5-21 years (n=69) | 22-60 years (n=9) | > 60 years (n=0) |
| Influenza A | 21% (31/147) | 10 | 20 | 1 | 0 |
| Influenza B | 7.5% (11/147) | 1 | 10 | 0 | 0 |
| Respiratory Syncytial Virus | 7.5% (11/147) | 9 | 2 | 0 | 0 |
VII Proposed Labeling:
The labeling is compliant with 21 CFR 809.10 and is sufficient.
VIII Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
K201505 - Page 26 of 26
